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Introduction Uterine fibroids are a common condition affecting women in their reproductive and post-reproductive years, with an estimated lifetime incidence of 50 % in white women and 80 % in black women. Complementary and alternative medicine (CAM) is broadly defined as systems of medicine that fall outside of mainstream care and are external to the politically dominant health system practices [1]. In this definition, both complementary and alternative medicines are used interchangeably. The National Centre for Complementary and Integrative Health (NCCIH) expands this definition, stating that ‘complementary medicine is used together with conventional medicine and alternative medicine is used in place of conventional medicine’ [2]. The boundaries between CAM and conventional medicine are not absolute, and specific CAM practices may, over time, become widely accepted. To confuse the matter further, certain therapies that are considered as CAM in the West are part of conventional medicine in the East. For example, acupuncture and Chinese herbal medicine are part of the conventional medical system in China. For purposes of this article, we will discuss only those therapies that are CAM as defined in the West. Recent data shows that patient choice is integrative in that they are more likely to use both conventional medicine and CAM. Women are more likely to use CAM than men [3]. CAM use is generally greater in white and middle-class women with a higher education compared to black women or women living in areas of depravity [4].

Recent data shows that patient choice is integrative in that they are more likely to use both conventional medicine and CAM. Women are more likely to use CAM than men [3]. CAM use is generally greater in white and middle-class women with a higher education compared to black women or women living in areas of depravity [4]. A trial of 150,000 men and women registered with a Dutch health insurer reported that those who used integrated CAM/conventional clinic services are more likely to live longer. Interestingly, the health care cost to the integrated clinics was lower than non-integrated clinics, suggesting a role for an integrated service model in the prevention of long-term health problems [5]. Aetiopathogenesis Related to CAM Diet Dietary therapy is one of the top motivations a woman has for visiting a CAM practitioner [6]. The incidence of uterine fibroids has been shown to be greater in populations who consume more red meats such as beef and ham and alcohol. Women who drink a beer a day or more increase the risk of developing uterine fibroids by more than 50 % [7]. On the other hand, dairy consumption appears to reduce the risk of fibroids, thus suggesting a role for calcium, magnesium and phosphorus in the pathogenesis of fibroids; both calcium and butyric acid inhibit cell proliferation [8]. A diet low in fruits and vegetables has been associated with an increased risk of developing fibroids [9]. Moreover, women who consume more citrus fruits are less likely to develop fibroids, possibly due to the anti-proliferative effect of flavonoids [9].

The incidence of uterine fibroids has been shown to be greater in populations who consume more red meats such as beef and ham and alcohol. Women who drink a beer a day or more increase the risk of developing uterine fibroids by more than 50 % [7]. On the other hand, dairy consumption appears to reduce the risk of fibroids, thus suggesting a role for calcium, magnesium and phosphorus in the pathogenesis of fibroids; both calcium and butyric acid inhibit cell proliferation [8]. A diet low in fruits and vegetables has been associated with an increased risk of developing fibroids [9]. Moreover, women who consume more citrus fruits are less likely to develop fibroids, possibly due to the anti-proliferative effect of flavonoids [9]. Isoflavones Isoflavones are a branch of flavanoids and are mostly produced by the monophyletic family, Fabaceae, to which Chinese herbal medicine (CHM) such as Puerariae Lobata Radix (Gegen), and foods such as soyabeans, and other legumes belong. Isoflavone consumption differs; in the West, 2 mg per day is usual [10]. In the East, up to 50 mg per day can be consumed [11]. There is also a significant difference between herb and food isoflavone content. The main dietary source of isoflavones is soyabean; one half a cup will yield around 47 mg of isoflavones [12]. The yield from Radix Paeoniae Rubra (Chi Shao) however is about 30 times higher [12].

up to 50 mg per day can be consumed [11]. There is also a significant difference between herb and food isoflavone content. The main dietary source of isoflavones is soyabean; one half a cup will yield around 47 mg of isoflavones [12]. The yield from Radix Paeoniae Rubra (Chi Shao) however is about 30 times higher [12]. Isoflavones contain very small amounts of phytoestrogens that are weakly estrogenic compounds. Moreover, they are both estrogenic and anti-estrogenic depending on cell type and also have anti-oxidant properties, and perhaps, this may explain why they have not been shown to have an association or causation with uterine fibroids [13]. The flavones apigenin and luteolin can induce inhibition of uterine fibroid growth by promoting apoptosis, and quercetin, the main fruit flavonoid and anti-oxidant, also displays pro-apoptic effect on tumour cells [14, 15]. The ability of quercetin to prevent growths in human cancer cell lines with virtually no side effects to normal cells has made it an attractive candidate for further investigation [16].

moting apoptosis, and quercetin, the main fruit flavonoid and anti-oxidant, also displays pro-apoptic effect on tumour cells [14, 15]. The ability of quercetin to prevent growths in human cancer cell lines with virtually no side effects to normal cells has made it an attractive candidate for further investigation [16]. Vitamin D is associated with uterine fibroids and is obtained through diet and sun exposure. Dietary sources of vitamin D include fatty fish like salmon and tuna and fortified milk. The recommended daily allowance is 20 ng/ml (600 IU) of vitamin D a day for people under 70 and over 12 months [17]. In rats, high doses of vitamin D3 shrank uterine fibroids by as much as 75 %, suggesting a role for vitamin D3. Although the doses were comparatively much larger than the recommended daily intake for humans, they were still within the limits considered safe [18]. Serum vitamin D levels have been correlated with fibroid size; deficiency correlated with the largest fibroids, whereas the highest serum vitamin D levels correlated with the smallest fibroids. Total fibroid volume correlated inversely with vitamin D in African American women. An inverse correlation was also observed in Caucasians but was not significant [19••]. Vitamin A has also been investigated, and a dose-dependent relationship was observed between vitamin A and the formation of uterine fibroids [20]. Animal sources of vitamin A appear to be primarily responsible and not sources derived from fruit or vegetables [9]. The anti-oxidant properties of vitamins C and E have not shown any association however [9].

d, and a dose-dependent relationship was observed between vitamin A and the formation of uterine fibroids [20]. Animal sources of vitamin A appear to be primarily responsible and not sources derived from fruit or vegetables [9]. The anti-oxidant properties of vitamins C and E have not shown any association however [9]. Stress Stress is a threat to homeostasis. Chronic life stress is characterised by reward eating (consumption of high-energy dense and palatable foods), elevation of cortisol, and long-term weight gain correlates with the incidence of uterine fibroids [21–24]. Increases in cortisol and insulin may be a natural somatic protective response to stress, wherein the stress response both causes and is caused by a threat to homeostasis; i.e. the mechanistic trail may be convoluted. For example, stress increases activities associated with pleasure such as reward eating in order to inhibit the hypothalamic-pituitary-adrenal axis (HPA) as protective mechanism [25]. The consequent chronic suppression of cortisol levels may eventually cause insulin resistance, which in turn may result in the development of obesity, hypertension and atherosclerosis; all of which are implicated in fibroid growth [26].

inhibit the hypothalamic-pituitary-adrenal axis (HPA) as protective mechanism [25]. The consequent chronic suppression of cortisol levels may eventually cause insulin resistance, which in turn may result in the development of obesity, hypertension and atherosclerosis; all of which are implicated in fibroid growth [26]. Hypertension Evidence suggests that hypertension is involved in the pathogenesis of fibroids and precedes the development of fibroids [27]. Hypertension is significantly more likely in women with fibroids than without, and the risk of fibroid growth increases with blood pressure, in both users and non-users of hypertensive medication. For every diastolic increase of 10 mmHG, the risk of fibroid growth increases by 8 and 10 %, respectively [28, 29]. Faerstein et al. postulated that elevated blood pressure may cause smooth muscle injury and/or secretion of cytokines similarly to that found in the pathogenesis of atherosclerosis [28]. Atherosclerosis Atherosclerosis and uterine fibroids are both smooth muscle, monoclonal growths which may in part explain their association. High-density lipids (HDLs) which are protective of atherosclerotic changes are lower in women with fibroids than in women without, and thicker carotid intima-media have been shown to be positively associated with uterine fibroids [28, 30•].

smooth muscle, monoclonal growths which may in part explain their association. High-density lipids (HDLs) which are protective of atherosclerotic changes are lower in women with fibroids than in women without, and thicker carotid intima-media have been shown to be positively associated with uterine fibroids [28, 30•]. Obesity The risk of developing a fibroid is between twofold and threefold in obese patients [31]. Elevated BMI and obesity correlate with patients who have both fibroids and hypertension. However, obesity does not correlate with the number of fibroids within the uterus, indicating that in addition to gonadotropins, the growth of uterine fibroids may involve other factors [32–34]. Adipokines and Renin-Aldosterone-Angiotensin System Both adipokines and the renin-aldosterone-angiotensin system (RAS) are expressed in the reproductive system, and increase in expression may be implicated in the pathogenesis of uterine growths and their recurrence [35–37]. The presence of uterine fibroids is associated with significantly decreased levels of the adiponectin [38]. Although adiponectin is exclusively secreted by adipose tissue, yet its expression is inversely correlated with body fat. Low adiponectin levels are implicated in the development of atherosclerosis, obesity and insulin resistance [39].

fibroids is associated with significantly decreased levels of the adiponectin [38]. Although adiponectin is exclusively secreted by adipose tissue, yet its expression is inversely correlated with body fat. Low adiponectin levels are implicated in the development of atherosclerosis, obesity and insulin resistance [39]. CAM Therapies for Management of Uterine Fibroids Dietary Therapy Diets related to the pathogenesis of fibroids such as red meats and high-energy dense foods should be avoided, whereas diets which prevent fibroid pathogenesis such as flavonoids, oily fish, green vegetables, citrus fruits, soya and broad beans should be promoted. These modifications not only have direct effects on fibroids but on related mechanisms. For example, hypertension is implicated in pathogenesis and reducing sodium levels can reduce blood pressure. A treatment strategy that excludes red meat, fat and animal fat, and restricts diet to fruits vegetables and poultry, and reduces sodium intake can thus reduce blood pressure and may also exert a protective effect on fibroid growth [40]. A suggested dietary therapy is outlined in Table 1. Whilst the recommendations are based on direct and indirect pathogenesis associations, they have not been subjected to large-scale trials.Table 1 Dietary therapy and advice

t vasodilatation in coronary arterioles through a range of neuromodulators including endothelial nitric oxide synthase (eNOS) and angiotensin II [55, 56]. Interestingly, it also inhibits vascular smooth muscle cell proliferation and decreases intimal thickening through mechanisms involving MAPK signalling pathway [57]. Green Tea (Camellia sinensis) Twenty-five percent of green tea is comprised of the flavanols and catechins, and epigallocatechin-3-gallate (EGCG) is extracted from this group. A small randomised controlled trial (n = 39) found that the flavanol EGCG significantly reduced volume of uterine fibroids and improved symptoms of anaemia and blood loss [58•]. The reduction of fibroid volume is attributed in part to the inhibitory action of EGCG on catechol-O-methyltransferase (COMT). In comparison to surrounding myometrial tissue, COMT is elevated in uterine fibroids and involved in their pathogenesis [59]. A common genetic variation of COMT is also implicated in cardiovascular disease and high blood pressure [60, 61]. Furthermore, a reduction of atherosclerotic lesions induced by COMT in animals implies a relationship between EGCG and COMT in the treatment of this disease in humans [62]. Acupuncture TCM is a system of medicine with several therapies, of which TCM acupuncture is the most popular in literature. In the 1950s, electro-acupuncture was introduced to TCM. There is an abundance of evidence that acupuncture modulates a wide range of endocrinological, neurohormonal, immunological, paracrine and autocrine factors [63–65].

CAM Therapies for Management of Uterine Fibroids Dietary Therapy Diets related to the pathogenesis of fibroids such as red meats and high-energy dense foods should be avoided, whereas diets which prevent fibroid pathogenesis such as flavonoids, oily fish, green vegetables, citrus fruits, soya and broad beans should be promoted. These modifications not only have direct effects on fibroids but on related mechanisms. For example, hypertension is implicated in pathogenesis and reducing sodium levels can reduce blood pressure. A treatment strategy that excludes red meat, fat and animal fat, and restricts diet to fruits vegetables and poultry, and reduces sodium intake can thus reduce blood pressure and may also exert a protective effect on fibroid growth [40]. A suggested dietary therapy is outlined in Table 1. Whilst the recommendations are based on direct and indirect pathogenesis associations, they have not been subjected to large-scale trials.Table 1 Dietary therapy and advice Dietary therapy and lifestyle advice Inclusionary foods and drinks Exclusionary foods and drinks Supplements include Lifestyle options include Fish Omega 3 and 6 Mindfulness meditation Salmon Vitamin D Yoga Tuna Vitamin A Tai Chi Mackerel Vitamin C Regular exercise Other oily fish Vitamin E Breathing exercises Iron supplements including Dietary support Meats Chicken Beef Turkey Ham Other white meat Lamb Other red meat Vegetables Soyabeans Chips/crisps Fava beansa Potatoes Green vegetables Rice Other legumes Sweets and chocolate Fruit Apples Oranges Tangerines Other citrus fruits Fluids Green tea Vegetable juice Alcohol/beer Unsweetened fruit juice Sweetened juice Milk (semi-skinned) High-sugar drinks

r white meat Lamb Other red meat Vegetables Soyabeans Chips/crisps Fava beansa Potatoes Green vegetables Rice Other legumes Sweets and chocolate Fruit Apples Oranges Tangerines Other citrus fruits Fluids Green tea Vegetable juice Alcohol/beer Unsweetened fruit juice Sweetened juice Milk (semi-skinned) High-sugar drinks aNot to be consumed by people with the hereditable condition of Favism Herbal Medicine The first record of CHM in gynaecology (the treatment of fertility) was written in 200 AD. However, the modern construction of a herbal formula is still based on the traditional criteria insofar as the phytokinetics and dynamics of the herbs is concerned. Integrating a medical system can prove beneficial to patients, for example, using clomiphene citrate with a traditional formula to treat anovulatory infertility arising from polycystic ovarian syndrome (PCOS) can achieve better results than using either CHM or clomiphene citrate alone [41].

s of the herbs is concerned. Integrating a medical system can prove beneficial to patients, for example, using clomiphene citrate with a traditional formula to treat anovulatory infertility arising from polycystic ovarian syndrome (PCOS) can achieve better results than using either CHM or clomiphene citrate alone [41]. Gui Zhi Fu Ling Tang Ramulus cinnomomi and Poriae cocos decoction The most commonly used traditional Chinese medicine (TCM) formula to treat uterine fibroids is Gui Zhi Fu Ling Tang (GFLT) [42]. This formula is effective in the treatment of dysmenorrhea either as a stand-alone treatment or in combination with progesterone receptor modulator such as mifepristone. The combination GFLT mifepristone therapy was shown to be more effective than using mifepristone alone [43•]. Three of the herbs used are known to be anti-proliferative and are involved in tumour cell apoptosis and induction of follistatin, Mu Dan Pi (Cortex Moutan), Chi Shao (Radix Paeoniae Rubra), and Tao Ren (Semen Persicae) [44–46]. Danshen Gegen decoction (Salviae Miltiorrhizae Radix et Rhizome and Purariae Lobatae Radix decoction) The CHM formula Danshen Gegen decoction (DGD) has a long history of use and recently has been investigated for its anti-atherosclerotic effects. Investigative results into DGD indicate that it positively modulates key early events in atherosclerosis [47–49]. The main active components in Danshen and Gegen, tanshinones and genistein, respectively, are most likely responsible for the effects of DGD, although other factors in the formula may also be involved.

cts. Investigative results into DGD indicate that it positively modulates key early events in atherosclerosis [47–49]. The main active components in Danshen and Gegen, tanshinones and genistein, respectively, are most likely responsible for the effects of DGD, although other factors in the formula may also be involved. Genistein The isoflavone genistein has a number of properties involved in the inhibition atherosclerosis. Genistein is an anti-inflammatory and modulates vascular inflammation [50]. Genistein also inhibits tyrosine kinases, enzymes involved in cellular growth and proliferation signal cascade; blocks platelet aggregation; and modulates genes related to cell cycle and apoptosis [51]. In addition, genistein also modulates nuclear factor-kappa B (NFkB). As a result, genistein may also be able to mediate uterine fibroid growth [52]. Tanshinones The flavonoid tanshinone IIA is derived from Danshen (Salviae Miltiorrhizae Radix et Rhizome), a herb used in the treatment of hypertension, cardiovascular disease and hypertension, cancer and liver damage [53]. Tanshinone IIA attenuates atherosclerosis [54]. Its effects include downregulation of adhesion molecules, improvement of microcirculation by inducing endothelium-dependent vasodilatation in coronary arterioles through a range of neuromodulators including endothelial nitric oxide synthase (eNOS) and angiotensin II [55, 56]. Interestingly, it also inhibits vascular smooth muscle cell proliferation and decreases intimal thickening through mechanisms involving MAPK signalling pathway [57].

m of medicine with several therapies, of which TCM acupuncture is the most popular in literature. In the 1950s, electro-acupuncture was introduced to TCM. There is an abundance of evidence that acupuncture modulates a wide range of endocrinological, neurohormonal, immunological, paracrine and autocrine factors [63–65]. Acupuncture acts on a variety of therapeutic targets associated in the pathogenesis and symptomatology of fibroids. It is effective treatment for dysfunctional bleeding and chronic pelvic inflammatory disease and dysmenorrhoea [66–68] (see Table 2 for acupuncture points used in the treatment of heavy periods).Table 2 Acupuncture points: heavy periods Acupuncture points for heavy periods resulting from blood stasis Channel name and number Pinyin Actions associated with traditional use Gall bladder 34 Yang Ling Quan Moves blood Ren 6 Qihai Moves blood Spleen 10 Xuehai Invigorates blood Bladder 17 Geshu Invigorates blood Stomach 29 Guilai Invigorates blood in the lower abdomen Liver 3 Taichong Invigorates liver blood Spleen 6 Sanyinjiao Invigorates liver blood Liver 8 Qu Quan Nourishes blood Stomach 36 Zusanli Nourishes blood Bladder 20 Pishu Nourishes blood Penetrating vessel spleen 4 + pericardium 6 Gongsun + Neiguan, respectively Regulates blood Maciocia [69] and Deadman et al. [70]

domen Liver 3 Taichong Invigorates liver blood Spleen 6 Sanyinjiao Invigorates liver blood Liver 8 Qu Quan Nourishes blood Stomach 36 Zusanli Nourishes blood Bladder 20 Pishu Nourishes blood Penetrating vessel spleen 4 + pericardium 6 Gongsun + Neiguan, respectively Regulates blood Maciocia [69] and Deadman et al. [70] Stener-Victorin suggested that stimulation of afferent nerve fibres may be one of the mechanisms that acupuncture exerts its effects. Such stimulation would inhibit sympathetic outflow at the spinal level. Furthermore, acupuncture also causes secretion of sensory neurotransmitters, such as substance P and calcitonin gene-related peptide (CGRP), which may effect neuronal transmission. Nerve impulses (action potentials) usually travel away from the nerve cell body, along the axon, to the axon terminals, from which the impulses are conveyed (to a subsequent neuron). This usual direction of travel is termed orthodromic. However, because acupuncture induces the secretion of sensory neuromodulators, an effect of acupuncture may induce nerve impulses to travel in the opposite direction, that is, away from the axon terminals towards the cell body. This opposite direction of travel is termed anti-dromic and may be another mechanism by which acupuncture modulates the central nervous system [71, 72] (see Fig. 1).Fig. 1 Nerve impulses (also known as an action potentials) usually travel from the cell body to the axon terminals (orthodromic). In some cases, nerve impulses travel from the axon terminals towards the cell body (antidromic)

ther mechanism by which acupuncture modulates the central nervous system [71, 72] (see Fig. 1).Fig. 1 Nerve impulses (also known as an action potentials) usually travel from the cell body to the axon terminals (orthodromic). In some cases, nerve impulses travel from the axon terminals towards the cell body (antidromic) Whether these effects seen in mixed populations translates into improvements in bleeding and pain symptoms associated with fibroids per se remains to be seen. Acupuncture treatments are effective in improving outcomes in overweight and obese patients. Results from animal experiments have found that acupuncture treatments reduce appetite and affect satiety at the level of the hypothalamus, thus implicating a role of energy homeostasis neurohormones such as leptin in the management of appetite by acupuncture [73]. This effect may also be a result of the pro-inflammatory cytokine downregulation involved in acupuncture treatments [74]. Both manual acupuncture and low-frequency electro-acupuncture (LFEA) can lower leptin and raise adiponectin secretion [75, 76]. Acupuncture and Infertility The role of fibroids in causation of infertility is controversial. Fibroids in certain locations and sizes have been shown to lower success rates. On the other hand, irrespective of cause, infertility and its treatments result in significant stress and anxiety to patients. Women are more likely to experience guilt and hostility than fertile women, and treatment can lead to clinical anxiety and depression [77].

ons and sizes have been shown to lower success rates. On the other hand, irrespective of cause, infertility and its treatments result in significant stress and anxiety to patients. Women are more likely to experience guilt and hostility than fertile women, and treatment can lead to clinical anxiety and depression [77]. Fertility-related stress may also negatively affect IVF treatment [78•]. Trials have also shown that acupuncture significantly alleviates depression, anxiety and stress by modulating both specific and non-specific neurological signalling and also neuromodulators such as cortisol, prolactin, epinephrine and beta endorphin [79, 80]. The effect of acupuncture treatments for IVF/ICSI has been examined during the last decade. Whilst some trials have found significant increases in clinical pregnancy and live birth rates when acupuncture is integrated with IVF, the results of meta-analyses are inconclusive due to methodological differences and heterogeneity of populations studied [81–84]. In addition, placebo acupuncture may not be inert, which may explain some of the differences between placebo-controlled trials involving acupuncture [85]. A variety of mechanisms have been proposed to explain the anti-fertility effects of uterine fibroids. Of particular interest to CAM is an increase in myometrial contractility observed with fibroids during implantation and increase in conception rates following myomectomy in women with two or more peristaltic movements in 3 min [86].

echanisms have been proposed to explain the anti-fertility effects of uterine fibroids. Of particular interest to CAM is an increase in myometrial contractility observed with fibroids during implantation and increase in conception rates following myomectomy in women with two or more peristaltic movements in 3 min [86]. A potential candidate marker for effectiveness of acupuncture therapy is the vasoactive modulator, CGRP, which is upregulated during the window of implantation [87]. CGRP inhibits angiotensin II and relaxes smooth muscle in the uterus [88]. Modulation of CGRP by acupuncture may inhibit uterine contractions associated with fibroids, and a trial investigating this is underway at our centre. Suggested Approach to the Management of Uterine Fibroids An integrated approach using both CM and CAM is essential in providing holistic and safe and effective treatment [89, 90]. Following referral, a detailed history is taken including lifestyle, diet, family history, and signs and symptoms relating to TCM. The results of any previous investigations and treatments including CM are also reviewed. It is best practice that the diagnosis of fibroids is established using conventional medicine.

90]. Following referral, a detailed history is taken including lifestyle, diet, family history, and signs and symptoms relating to TCM. The results of any previous investigations and treatments including CM are also reviewed. It is best practice that the diagnosis of fibroids is established using conventional medicine. Therapeutic options depend on the outcome of consultation, investigations, interdisciplinary discussions and patient choice. CAM treatment may involve herbal, acupuncture and dietary/lifestyle choices. Insofar as CAM treatments are selected, CHM is the treatment of choice to reduce volume of uterine fibroids, and, for management of chronic conditions, whereas acupuncture is more efficient in the management of acute conditions, probably due to its modulation of neurohormones such as beta endorphins, leptin and other biochemicals. The increased frequency of acupuncture treatments potentiates the effects of acupuncture, and so, the therapy can also be used in the treatment of chronic conditions. During the course of treatments, other problems may arise and it is important to treat what is presented by the patient at that time. Conclusion A variety of CAM therapies are available, and there is an urgent need to subject them to academic rigour. A clear understanding of underlying mechanistic pathways and patient’s symptoms and needs is crucial in planning individualised therapies. An integrated CM CAM model is likely to yield better patient outcomes and reduced health care costs. This article is part of the Topical Collection on Uterine Fibroids

Conclusion A variety of CAM therapies are available, and there is an urgent need to subject them to academic rigour. A clear understanding of underlying mechanistic pathways and patient’s symptoms and needs is crucial in planning individualised therapies. An integrated CM CAM model is likely to yield better patient outcomes and reduced health care costs. This article is part of the Topical Collection on Uterine Fibroids Open access for this article was funded by King's College London. Compliance with Ethical Standards Conflict of Interest Nick Dalton-Brewer declares no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

Introduction Fibroids are the most common uterine tumour in the reproductive age group affecting 20–50 % of these women, and hence, their relation with infertility although controversial is always a great concern to the clinician as well as the patient [1, 2••]. Mechanisms of Infertility Fibroids vary to a great extent in terms of their size, location and number and so does the mechanism by which they may cause infertility. Physical Factors Given their size and location, it is unsurprising that simple physical impedance to the transport of sperm, egg or embryo has been proffered as a mechanism to explain the anti-fertility effects of fibroids. However, the microscopic size of the gametes and both the bilateralism and the resilience of the reproductive system suggest that this by itself is unlikely to be sole mechanism in the vast majority of cases. Alteration of Uterine Contractions Uterine contractions increase in frequency in the early follicular phase from the fundus to cervix whereas in peri-ovulatory and luteal phase, their direction is reversed from the cervix to fundus [3]. Fibroids are also known to influence the contractility of the myometrium and induce a chronic inflammatory reaction, both of which may hinder implantation [4–7, 8•]. Some studies have reported increase in myometrial peristalsis in patients with intramural and submucosal fibroids when compared with healthy controls during the mid-luteal cycle phase, although there was a decrease in the peri-ovulatory phase [4–6].

nflammatory reaction, both of which may hinder implantation [4–7, 8•]. Some studies have reported increase in myometrial peristalsis in patients with intramural and submucosal fibroids when compared with healthy controls during the mid-luteal cycle phase, although there was a decrease in the peri-ovulatory phase [4–6]. Yoshino et al. using Cine mode, MRI demonstrated accelerated mid-luteal uterine peristalsis (defined as ≥2 peristaltic movements in 3 min) in the presence of intramural fibroids and achieved 40 % pregnancy rate in this population over 1 year following restoration of normal peristalsis by myomectomy [7]. Yoshino also reported another prospective study (n = 95) where they looked at the impact of uterine peristaltic movements due to fibroids on outcome of non-IVF fertility treatment. Thirty-four per cent women in the low-frequency group achieved pregnancy, compared with none (0 %) in the high-frequency group (P < 0.005) [8•]. Many underlying mechanisms have been suggested for increase in myometrial contractility like excess of cytokines, growth factors, neurotensin, neuropeptides, enkephalin and oxytocin modulators in the fibroid capsule [9].

Yoshino et al. using Cine mode, MRI demonstrated accelerated mid-luteal uterine peristalsis (defined as ≥2 peristaltic movements in 3 min) in the presence of intramural fibroids and achieved 40 % pregnancy rate in this population over 1 year following restoration of normal peristalsis by myomectomy [7]. Yoshino also reported another prospective study (n = 95) where they looked at the impact of uterine peristaltic movements due to fibroids on outcome of non-IVF fertility treatment. Thirty-four per cent women in the low-frequency group achieved pregnancy, compared with none (0 %) in the high-frequency group (P < 0.005) [8•]. Many underlying mechanisms have been suggested for increase in myometrial contractility like excess of cytokines, growth factors, neurotensin, neuropeptides, enkephalin and oxytocin modulators in the fibroid capsule [9]. Cytokine Factors Certain early pregnancy intrauterine cytokines are thought to be responsible for implantation and early embryonic development. Ben-Nagi et al. reported significant reduction in levels of certain cytokines mainly IL10 and glycodelin in the mid-luteal uterine washings of women with submucosal fibroids [10••]. Glycodelin is a progesterone-regulated glycoprotein secreted into uterine luminal cavity by secretory/decidualized endometrial glands and has properties like promoting angiogenesis and suppressing natural killer (NK) cells.

IL10 and glycodelin in the mid-luteal uterine washings of women with submucosal fibroids [10••]. Glycodelin is a progesterone-regulated glycoprotein secreted into uterine luminal cavity by secretory/decidualized endometrial glands and has properties like promoting angiogenesis and suppressing natural killer (NK) cells. Genetic Endometrial HOXA10, HOXA11 and BTEB1 gene expression has been shown to modulate endometrial receptivity. The reduction or absence of HOXA10 in the uterine endometrium leads to infertility due to the inability of the embryo to implant [11•]. Rackow et al. demonstrated a significant reduction in concentration of these genes during follicular phase in infertile women with submucosal fibroids (FIGO L0 to L2). Interestingly, the reduction was present throughout the uterine cavity and not just in the endometrium overlying the fibroid. There was no significant decrease for intramural (IM) fibroids, although a trend to lower levels was noted [5]. On the other hand, Matsusaki et al. was able to demonstrate a significant decrease in HOXA10 concentrations during luteal phase in infertile women with intramural fibroids compared to healthy patient controls [12]. The downregulation of endometrial HOXA 1 gene expression results in defective decidualization possibly mediated via secretion of transforming growth factor beta3 (TGF-β3) [13]. Alizadeh reported increase in endometrial HOXA1 gene expression following myomectomy [14].

Genetic Endometrial HOXA10, HOXA11 and BTEB1 gene expression has been shown to modulate endometrial receptivity. The reduction or absence of HOXA10 in the uterine endometrium leads to infertility due to the inability of the embryo to implant [11•]. Rackow et al. demonstrated a significant reduction in concentration of these genes during follicular phase in infertile women with submucosal fibroids (FIGO L0 to L2). Interestingly, the reduction was present throughout the uterine cavity and not just in the endometrium overlying the fibroid. There was no significant decrease for intramural (IM) fibroids, although a trend to lower levels was noted [5]. On the other hand, Matsusaki et al. was able to demonstrate a significant decrease in HOXA10 concentrations during luteal phase in infertile women with intramural fibroids compared to healthy patient controls [12]. The downregulation of endometrial HOXA 1 gene expression results in defective decidualization possibly mediated via secretion of transforming growth factor beta3 (TGF-β3) [13]. Alizadeh reported increase in endometrial HOXA1 gene expression following myomectomy [14]. Alterations in the Endo-myometrial Junctional (EMJ) Zone The EMJ which represents the inner 1/3rd of the myometrium abutting the endometrium contributes macrophages and uterine natural killer (uNK) cells which are essential for the process of endometrial decidualization in the mid-luteal window of implantation. In women with uterine fibroids, Kitaya et al. found significant reduction in concentrations of both macrophages and uNK cells in the EMJ, thus, negatively affecting implantation [15]. Also, it is possible that the presence of intramural or submucosal fibroids physically disrupts the EMJ and alters the steroid receptors, leading to implantation failure [16].

et al. found significant reduction in concentrations of both macrophages and uNK cells in the EMJ, thus, negatively affecting implantation [15]. Also, it is possible that the presence of intramural or submucosal fibroids physically disrupts the EMJ and alters the steroid receptors, leading to implantation failure [16]. Infertility and Reproductive Outcomes The evidence base in relation to fibroids and infertility is complex, with an overrepresentation of observational data and a lack of well-designed controlled trials. Moreover, the heterogeneity in patient populations and fibroid disease and multifactorial aetiology of infertility mean that it is often difficult to plan and successfully execute large scale multi-centre randomised controlled trials. So far, we have explored biological plausibility by which fibroids may cause infertility. In this section, we will explore the evidence base for harm and treatment benefit. Evidence of Harm: Does Presence of Uterine Fibroids Reduce Implantation Rates? It is generally accepted that submucous fibroids have a negative impact on fertility and early pregnancy by the virtue of their involvement in the endometrial cavity. A systematic review by Pritts et al. concluded that submucosal fibroids (FIGO L0 to L2) which cause distortion of the uterine cavity resulted in the decreased rates of clinical pregnancy, implantation and ongoing pregnancy/live birth, as well as an increased rate of spontaneous miscarriage [17••].

e endometrial cavity. A systematic review by Pritts et al. concluded that submucosal fibroids (FIGO L0 to L2) which cause distortion of the uterine cavity resulted in the decreased rates of clinical pregnancy, implantation and ongoing pregnancy/live birth, as well as an increased rate of spontaneous miscarriage [17••]. In contrast to this, there is a considerable controversy regarding fibroids that do not cause distortion of the uterine cavity. The review by Pritts et al. found that women with fibroids with no submucosal involvement, i.e. pure intramural fibroids (FIGO L3 to L4), had decreased rates of implantation and ongoing pregnancy/live birth, and an increased rate of spontaneous miscarriage when compared with controls without fibroids. One weakness of Pritts’ review is that most of the studies included did not use a formal means such as hysteroscopy or saline sonography to exclude the involvement of the uterine cavity, i.e. there may be an ascertainment bias and overestimation of effect size in that some of the cases deemed as intramural and may have an undiagnosed submucosal component [17••]. What is clear from the review is that there was no evidence to suggest that subserosal (FIGO L5 to L7) fibroids decreased any measure of fertility.

there may be an ascertainment bias and overestimation of effect size in that some of the cases deemed as intramural and may have an undiagnosed submucosal component [17••]. What is clear from the review is that there was no evidence to suggest that subserosal (FIGO L5 to L7) fibroids decreased any measure of fertility. A synthesis of available evidence shows a 21 % reduction in live birth rates following in vitro fertilization (IVF) in women with non-cavity distorting intramural fibroids, when compared with non-fibroid controls [18••]. The group whilst acknowledging the inherent weakness of the review owing to the heterogeneity of patient populations highlighted the relatively lower chance of achieving a live birth, when compared with clinical pregnancy rate, and attributed this to increased rates of miscarriage and premature birth [18••]. A major confounding factor in infertility success is access to health care. There is a strong residual effect in that women who receive treatment early are most likely to have successful outcomes, whereas those who suffer long duration of infertility are the residuals whose prognosis is worse irrespective of treatment.

A major confounding factor in infertility success is access to health care. There is a strong residual effect in that women who receive treatment early are most likely to have successful outcomes, whereas those who suffer long duration of infertility are the residuals whose prognosis is worse irrespective of treatment. There is plethora of evidence that, Afro-Caribbean women, in whom fibroids are more common and more severe, have poor access to health care compared to Caucasian women, and therefore under-represented in ART databases [19]. Feinberg et al. examined the disparity in outcomes of the first non-donor IVF cycles between African-American patients and Caucasian patients, in the Department of Defence population, which is an equal access to care setting [20]. Fibroids were approximately three times as common in African-American as opposed to Caucasian women (30.8 versus 10.7 %). Women were offered routine saline sonography prior to IVF, and those with fibroids larger than 3 cm or submucosal component offered surgery. Although, the study could neither ascertain the staging of fibroids at baseline scan nor the proportion of patients who underwent surgery, African-American women were found to have statistically significant higher rates of miscarriage, when compared to Caucasian patients and fibroids were thought to be a contributing factor for this variation [20]. The reproductive outcomes between the two groups were similar when adjusted for fibroids. In both groups of women, the presence of fibroids at baseline scan reduced IVF implantation and live birth rates by 18 % (95 % CI 2–31 %) and 27 % (95 % CI 4–44 %), respectively [20].

a contributing factor for this variation [20]. The reproductive outcomes between the two groups were similar when adjusted for fibroids. In both groups of women, the presence of fibroids at baseline scan reduced IVF implantation and live birth rates by 18 % (95 % CI 2–31 %) and 27 % (95 % CI 4–44 %), respectively [20]. Evidence of Treatment Benefit: Does Treatment of Uterine Fibroids (For Example Myomectomy) Improve Fertility Rates and Outcomes? There are many case series reporting the benefits of myomectomy. For example, Babaknia et al. in 1978 reported 38 % term pregnancy rate following myomectomy in 34 women with otherwise unexplained infertility [21•].

enefit: Does Treatment of Uterine Fibroids (For Example Myomectomy) Improve Fertility Rates and Outcomes? There are many case series reporting the benefits of myomectomy. For example, Babaknia et al. in 1978 reported 38 % term pregnancy rate following myomectomy in 34 women with otherwise unexplained infertility [21•]. Casini et al. reported the only RCT published to date, in the group of women with fibroids but otherwise unexplained infertility [22••]. All women except for those whose fibroids were purely subserous, i.e. no intramural component (n = 11), were included and randomised (total randomised n = 170) to undergo myomectomy or not and spontaneous conception rates observed over 12 months following surgery. All women undergoing myomectomy, which was carried out either hysteroscopically or by laparotomy, reported increased pregnancy rates irrespective of baseline fibroid staging [22••]. Statistically, significant increase was, however, only observed in women with submucosal fibroid [pregnancy rates myomectomy versus no myomectomy; submucosal group = 43.3 versus 27.2 %; intramural with submucosal component = 40 versus 15 %; all submucosal = 21/52 (40.4) versus 9/42 (21.4 %)]. The pregnancy rates of the 11 women excluded on basis of pure subserous staging was 63.6 % [22••].

with submucosal fibroid [pregnancy rates myomectomy versus no myomectomy; submucosal group = 43.3 versus 27.2 %; intramural with submucosal component = 40 versus 15 %; all submucosal = 21/52 (40.4) versus 9/42 (21.4 %)]. The pregnancy rates of the 11 women excluded on basis of pure subserous staging was 63.6 % [22••]. Whilst there are many non-randomised controlled trials in published literature, their common fallacy is the choice of inappropriate controls, i.e. to give a valid answer, both the treatment and control arm should suffer disease in question of uterine fibroids. It is not correct to compare women who undergo myomectomy with infertile controls who do not have fibroids at all. In absence of appropriate controls, evidence from studies where patients serve as their own internal controls, i.e. a before and after effect, is acceptable, although not without its own methodological problems. A review of literature reveals only one such adequately controlled trial, albeit non-randomised, which investigated the treatment effect of myomectomy prior to IVF [23]. All the patients selected had between one and five fibroids, with one measuring at least 5 cm and all without a submucosal component. The study established the beneficial effects of pre-IVF myomectomy, as shown by the 25 % delivery rate in the myomectomy group, when compared to the 12 % delivery rate in the no surgery group.

e patients selected had between one and five fibroids, with one measuring at least 5 cm and all without a submucosal component. The study established the beneficial effects of pre-IVF myomectomy, as shown by the 25 % delivery rate in the myomectomy group, when compared to the 12 % delivery rate in the no surgery group. A Cochrane review of three RCT’s [22••, 24, 25] concluded that there is insufficient evidence to recommend a myomectomy for the purpose of improving fertility outcomes in the case of intramural or subserosal fibroids [26••]. In summary, the published literature make clear divisions between the location of fibroids and the benefit of myomectomy on reproductive outcomes, both in terms of spontaneous pregnancies as well as IVF outcomes. The consensus based on clinical experience would appear to imply very little causation linking subserosal fibroids and infertility. Therefore, unless there were other indications, a myomectomy to remove subserosal fibroids for infertility is not evidence based. Submucosal fibroids, on the other hand, are shown to lower fertility rates and studies have demonstrated by removing such fibroids; there is an improvement in both conception and live birth rates. With regard to intramural fibroids, both the evidence and consensus for myomectomy, purely for infertility, is weak. Given the risk of significant morbidity of surgery including that of postoperative adhesion formation, particularly those performed through posterior uterine incisions, [27] further research is outstanding and cases have to be managed on an individual basis.

nd consensus for myomectomy, purely for infertility, is weak. Given the risk of significant morbidity of surgery including that of postoperative adhesion formation, particularly those performed through posterior uterine incisions, [27] further research is outstanding and cases have to be managed on an individual basis. A Pragmatic Approach to Management Attribution is the exercise of determining a causal association between a finding and a symptom, i.e. the exercise of establishing causation. In vast majority of cases, it is difficult or even impossible to ascertain causation with absolute certainty, and therefore, the attribution exercise should also include a means of determining the strength/likelihood of the causal association, so a treatment effect can be estimated. Accurate fibroid mapping, i.e. description of size, location and nature of fibroids, using ultrasound scan is a critical step in such an assessment. Saline infusion sonography (SIS) can be used to rule out submucosal involvement, and MRI reserved for complex cases or to differentiate from adenomyosis.

A Pragmatic Approach to Management Attribution is the exercise of determining a causal association between a finding and a symptom, i.e. the exercise of establishing causation. In vast majority of cases, it is difficult or even impossible to ascertain causation with absolute certainty, and therefore, the attribution exercise should also include a means of determining the strength/likelihood of the causal association, so a treatment effect can be estimated. Accurate fibroid mapping, i.e. description of size, location and nature of fibroids, using ultrasound scan is a critical step in such an assessment. Saline infusion sonography (SIS) can be used to rule out submucosal involvement, and MRI reserved for complex cases or to differentiate from adenomyosis. All patients should complete preliminary investigations to ascertain causation of infertility; the important domains include assessment of ovarian reserve and ovulation, as well as seminal fluid analysis. Tubal patency tests are invasive, and in the presence of fibroids are inaccurate [28]. Accordingly, patency should be assessed opportunistically at time of myomectomy, or if indicated, by HSG or HyCoSy as appropriate. An overview of the investigations and treatment of infertility is outlined in Fig. 1 (adapted with permission from WILEY-TOG article) [28].Fig. 1 Overview of investigations and treatment of infertility (adapted with permission from Yalandu and Narvekar, Wiley publishing) [28]

indicated, by HSG or HyCoSy as appropriate. An overview of the investigations and treatment of infertility is outlined in Fig. 1 (adapted with permission from WILEY-TOG article) [28].Fig. 1 Overview of investigations and treatment of infertility (adapted with permission from Yalandu and Narvekar, Wiley publishing) [28] The two critical factors which help assess the need for treatment are (a) the absence or presence of other causal aetiologies and (b) the overall chance of conception, whether natural or otherwise. For example, if fibroids are a part of a multi-factorial aetiology, it is difficult to determine which aetiology is most causative in infertility. On the other hand, if the aetiology is otherwise unexplained, then it is appropriate to consider treatment either surgical or medical for the fibroids. The overall chance of conception is also an important factor in decision making. For example, the removal of submucous or large intramural fibroids is likely to be successful in a woman age <40 with otherwise unexplained infertility, as opposed to a woman age 40 or more with low/poor ovarian reserve, where myomectomy irrespective of size and location of fibroids is unlikely to be of major benefit.

For example, the removal of submucous or large intramural fibroids is likely to be successful in a woman age <40 with otherwise unexplained infertility, as opposed to a woman age 40 or more with low/poor ovarian reserve, where myomectomy irrespective of size and location of fibroids is unlikely to be of major benefit. Surgical Treatments Hysteroscopic Myomectomy If the fibroids are predominately located within the cavity (FIGO L0, L1), hysteroscopic myomectomy would appear to help restore cavity dimensions and subsequently improve fertility outcomes. The risk of endometrial damage and intrauterine adhesions, and its subsequent effect on conception and pregnancy outcomes, has to be discussed with patient during pre-operative counselling. Intrauterine adhesions have been reported to occur in up to 7.5 % of hysteroscopic myomectomies [29]. Valle et al. showed that increasing severity of intrauterine adhesions correlated with corresponding decrease in reproductive outcomes. This ranged from a term pregnancy rate of 81.3 % in patients with mild disease to 31.9 % in patients with severe disease [29].

occur in up to 7.5 % of hysteroscopic myomectomies [29]. Valle et al. showed that increasing severity of intrauterine adhesions correlated with corresponding decrease in reproductive outcomes. This ranged from a term pregnancy rate of 81.3 % in patients with mild disease to 31.9 % in patients with severe disease [29]. FIGO L2 fibroids, whereby less than 50 % of the fibroid is located with the cavity, are more difficult to resect and may require a two-stage procedure, especially if larger than 3 cm. Camanni et al. demonstrated that hysteroscopic approach is suitable for fibroids measuring up to 5 cm in diameter [30]. One has to exercise extreme caution in assessing what is feasible technically and what is best for the management of patient’s symptoms. Whilst it is perfectly reasonable to perform resection of large L2 fibroids, albeit in multiple procedures, for the management of severe menstrual symptoms, the risk of endometrial damage and adhesions may negate any fertility benefits. As such, for infertility, it may be prudent to remove such fibroids by laparoscopy, although it does increase the risks associated with a full thickness myometrial incision such as uterine rupture in the future pregnancy and labour.

mptoms, the risk of endometrial damage and adhesions may negate any fertility benefits. As such, for infertility, it may be prudent to remove such fibroids by laparoscopy, although it does increase the risks associated with a full thickness myometrial incision such as uterine rupture in the future pregnancy and labour. Laparoscopic Versus Laparotomy All fibroids FIGO L3 and above (and large L2 as outlined above) are best removed by laparoscopy or laparotomy. The improvement in reproductive outcomes appears to be similar by both the approaches. Combined data of 267 women from two RCTs comparing laparoscopic myomectomy and abdominal myomectomy demonstrated similar reproductive outcomes in both groups [24]. In the first study of 131 patients undergoing myomectomy for infertility and at least 1 fibroid > 5 cm, pregnancy rates were similar in the laparoscopy and laparotomy groups (53.6 versus 55.9 %).Febrile morbidity was reduced in the laparoscopy group (26.2 versus 12.1 %), when compared with laparotomy as well as a smaller mean drop in haemoglobin and a shorter inpatient stay [24]. In the second study involving 132 women with fibroids, whilst cumulative outcomes within the first 12 months following surgery were similar (cumulative pregnancy rate 52.9 versus 38.2 %), the per cycle outcomes such as pregnancy rate per cycle (6.5 versus 3.9 %) and time to the first pregnancy (WMD = 1 month) were significantly higher in the laparoscopic compared to the laparotomy group [25].

tcomes within the first 12 months following surgery were similar (cumulative pregnancy rate 52.9 versus 38.2 %), the per cycle outcomes such as pregnancy rate per cycle (6.5 versus 3.9 %) and time to the first pregnancy (WMD = 1 month) were significantly higher in the laparoscopic compared to the laparotomy group [25]. Medical Treatments Medical treatments such as combined oral contraceptive pill (COCPs), progesterone only-pill (POP) and levonorgestrel intrauterine system (LNG-IUS), whilst useful in managing menstrual and pain symptoms, are contraceptive and therefore not applicable to the infertile women. Other medical treatments such as mefenamic and tranexamic acid can be safely prescribed [31]. Ulipristal acetate (UPA), a selective progesterone receptor modulator is now approved and licensed for the medical treatment of uterine fibroids in many countries. UPA has been shown to improve menstrual symptoms and lead to regression in fibroid size [32]. The regressive effects are maintained for 6 months, primarily because the compound increases apoptosis of leiomyoma cells [33•]. This phenomenon has allowed for intermittent dosing, and UPA is now licensed accordingly [34]. The maximum duration of therapy is 3 months, and the recommended interval between therapies has to be a minimum of two washout-menstrual cycles, which also allows for any endometrial changes, the so-called PAEC to revert to normal.

henomenon has allowed for intermittent dosing, and UPA is now licensed accordingly [34]. The maximum duration of therapy is 3 months, and the recommended interval between therapies has to be a minimum of two washout-menstrual cycles, which also allows for any endometrial changes, the so-called PAEC to revert to normal. UPA is marketed in strengths of up to 100 mg for emergency contraception [35]; however, contraceptive effects of a daily 5 mg dose are unknown, and therefore, patients should be advised to use alternate contraception such as condoms during therapy in order to avoid any teratogenicity. Any conception benefits of UPA, resulting from fibroid regression, have to be evaluated following end of therapy or in the washout cycles if prescribed intermittent dosing. Luyckx et al. reported the first series of 18 such pregnancies in 52 women participating from a single centre in Pearl II and Pearl III studies. Thirty-seven women were treated with one-off 3-month UPA therapy (Pearl II, Pearl III) and 15 with intermittent therapy lasting a total of 6 to 12 months (Pearl III extension). Of the 21 women who wished to conceive after completion of UPA therapy, 19 underwent myomectomy, and 2 were not. Seventy-one per cent (15/71) women conceived for a total of 18 times, 12 of which were spontaneous and a further 6 achieved with IVF [36]. There were a total of 13 live births (1 twin) and 6 miscarriages. The two women, who did not undergo myomectomy, had a total of three pregnancies between them, but only one live birth [36].

one per cent (15/71) women conceived for a total of 18 times, 12 of which were spontaneous and a further 6 achieved with IVF [36]. There were a total of 13 live births (1 twin) and 6 miscarriages. The two women, who did not undergo myomectomy, had a total of three pregnancies between them, but only one live birth [36]. Whilst the data shows feasibility and safety of conception after UPA therapy, it also highlights the high miscarriage rate in the presence of fibroids despite reduction in size (2/3 versus 4/15 conceptions in women who did not undergo myomectomy versus those who did) and therefore the superiority of myomectomy over reductive therapies.

hows feasibility and safety of conception after UPA therapy, it also highlights the high miscarriage rate in the presence of fibroids despite reduction in size (2/3 versus 4/15 conceptions in women who did not undergo myomectomy versus those who did) and therefore the superiority of myomectomy over reductive therapies. Uterine Artery Embolization Uterine artery embolization was first described in 1995 by Ravina as an alternative radiological treatment option for women with large fibroids no longer desiring their fertility [37]. MRI imaging shows a transient ischemia within the body of the uterus and the endometrium typically lasting for up to 72 after the uterine artery embolization (UAE) procedure. This ischemic change is intended to be irreversible within fibroid tissue only, and temporary within healthy uterine muscle and endometrium, but nevertheless, raises concerns regarding its effect on whole uterine and endometrial function. Also, the uterine and ovarian artery has been shown to anastomose on angiography, in at least one side in approximately 46 % of women. Therefore, inadvertent embolization of ovarian tissue may result in premature ovarian insufficiency and failure especially in older women or those with low baseline ovarian reserve. Reassuringly, the reported incidence of amenorrhea in the under 40 age group is less than 1 %.

st one side in approximately 46 % of women. Therefore, inadvertent embolization of ovarian tissue may result in premature ovarian insufficiency and failure especially in older women or those with low baseline ovarian reserve. Reassuringly, the reported incidence of amenorrhea in the under 40 age group is less than 1 %. Mara et al. conducted an RCT evaluating UAE versus abdominal myomectomy in an infertile population [38]. The pregnancy rates were 50 and 78 % in the UAE and myomectomy arms, respectively. They recruited young patients below age 35 [mean age 32 (SD ±4.1) years] which may explain the high conception rates overall. Also, the latency period, i.e. time to conception was longer for UAE (mean = 18 months) compared with myomectomy (mean = 13 months). The re-intervention rates were higher (19 out of 58) in the UAE arm, as has been observed in other studies [39]. Following a systematic review of the published literature, Homer et al. reported a 35.2 % rate of miscarriage in UAE conceptions as compared to 16.5 %in fibroid-containing pregnancies (odds ratio [OR] 2.8; 95 % confidence interval [CI] 2.0–3.8) matched for age and fibroid location [40]. There was a higher incidence of caesarean section and PPH in the UAE pregnancies, whereas rates for preterm delivery and malpresentation were similar in the two groups. Given the current evidence base, UAE is not a treatment of first choice for women with infertility or those desirous of future fertility. Instead, it is to be reserved for poor surgical candidates.

Following a systematic review of the published literature, Homer et al. reported a 35.2 % rate of miscarriage in UAE conceptions as compared to 16.5 %in fibroid-containing pregnancies (odds ratio [OR] 2.8; 95 % confidence interval [CI] 2.0–3.8) matched for age and fibroid location [40]. There was a higher incidence of caesarean section and PPH in the UAE pregnancies, whereas rates for preterm delivery and malpresentation were similar in the two groups. Given the current evidence base, UAE is not a treatment of first choice for women with infertility or those desirous of future fertility. Instead, it is to be reserved for poor surgical candidates. Magnetic Resonance-Guided Focused Ultrasound Surgery Another alternative treatment modality which has demonstrated encouraging preliminary results is the use of magnetic resonance-guided focused ultrasound surgery (MRgFUS). This treatment involves the application of MRI-directed beams of ultrasound capable of heating an area of fibroid tissue to up to 70 °C and causing destruction through coagulative necrosis. Rabinovici et al. reviewed all pregnancies reported to the FDA MAUDE (manufacturer and user facility device experience) database following MRgFUS. In total, 54 pregnancies were reported in 51 women with a mean age at MRgFUS of 37.2 years and mean time to conception of 8 months [41, 42]. The miscarriage rate was 28 %. The preliminary experience is encouraging, with a high rate of delivered and ongoing pregnancies.

lity device experience) database following MRgFUS. In total, 54 pregnancies were reported in 51 women with a mean age at MRgFUS of 37.2 years and mean time to conception of 8 months [41, 42]. The miscarriage rate was 28 %. The preliminary experience is encouraging, with a high rate of delivered and ongoing pregnancies. Conclusion The evidence regarding effect of fibroids on infertility and reproductive outcomes is weak and mostly inconclusive. In infertile women, appropriate evaluation and classification of fibroids, particularly those involving or suspected to be involving the endometrial cavity is essential. Submucosal fibroids (FIGO L0-L2) should be treated hysteroscopically (or laparoscopic for large L2) to improve conception rates. The management of intramural fibroids should be individualised on a case to case basis, whereas subserosal fibroid are unlikely to have any major impact on fertility. Conservative treatment measures (Medical, UAE and MrgRUS) should not be routinely offered to women who wish to maintain or improve their fertility due to lack of data on their safety and effectiveness. This article is part of the Topical Collection on Uterine Fibroids Compliance with Ethical Standards Conflict of Interest P. Purohit and K. Vigneswaran declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

Introduction Uterine fibroids (leiomyomata uteri) are benign solid tumors that affect the majority of women in the USA by age 50 [1]. While often asymptomatic, fibroids can result in abnormal uterine bleeding, pelvic pressure, subfertility, dyspareunia, and other symptoms. Uterine fibroids are the leading indication for hysterectomy in the USA, Europe, and other countries [2–4]. While treatment options (hysterectomy, myomectomy, uterine artery embolization) exist, they typically involve major surgery and inpatient admission, require incisions and general anesthesia, and can be associated with significant adverse events and prolong the return to the activities of daily living. Hysteroscopic myomectomy is limited to smaller submucous fibroids, with some requiring staggered treatment with multiple partial resections to avoid inadvertent serosal injury along with fluid overload and other complications [5]. A 2013 study indicated that nearly 80% of women expressed an interest in minimally invasive options for fibroid treatment and 51% reported a desire to conserve their uteri; of particular note, 25% delayed treatment up to 5 years [6]. The situation is similar in Europe, as Downes and colleagues found that nearly 33% of Italian women with symptomatic fibroids, and almost a quarter of fibroid patients in the UK, waited over 5 years before obtaining treatment [4].

ire to conserve their uteri; of particular note, 25% delayed treatment up to 5 years [6]. The situation is similar in Europe, as Downes and colleagues found that nearly 33% of Italian women with symptomatic fibroids, and almost a quarter of fibroid patients in the UK, waited over 5 years before obtaining treatment [4]. In response to the desire for novel, minimally invasive solutions for women with fibroids who desire uterine conservation, there has been a growing interest in the use of radiofrequency (RF) energy to ablate uterine leiomyomata. The successful application of RF ablation to solid tumors of the liver and other organs has affirmed the validity of this approach [7–9]. The Sonata® System (Gynesonics; Redwood City, CA), previously known as VizAblate™, integrates radiofrequency ablation for the treatment of fibroids with intrauterine sonography for real-time imaging within a single medical device [10]. It is CE-marked (Conformité Européene)in the European Union and is currentlyunder investigation in the USA. Because it is placed transcervically, it is incisionless and does not require general anesthesia. In particular, Sonata enables the outpatient treatment of a wide range of uterine fibroid types. While Sonata can treat FIGO (Fédération Internationale de Gynécologie et d'Obstétrique) type 1 and type 2 fibroids, it can also ablate fibroids that are not treatable with hysteroscopic methods (e.g., FIGO types 3, 4, 5, 6 and types 2–5 [transmural]).

s the outpatient treatment of a wide range of uterine fibroid types. While Sonata can treat FIGO (Fédération Internationale de Gynécologie et d'Obstétrique) type 1 and type 2 fibroids, it can also ablate fibroids that are not treatable with hysteroscopic methods (e.g., FIGO types 3, 4, 5, 6 and types 2–5 [transmural]). This paper will provide an overview of transcervical radiofrequency ablation (RFA) for uterine fibroids, review the current evidence base for the Sonata System, and offer a perspective on its potential role in the armamentarium of general and specialized obstetrician-gynecologists who manage the many women with symptomatic uterine leiomyomata. Transcervical Radiofrequency Ablation of Uterine Fibroids As a hyperthermic energy source, radiofrequency energy heats soft tissue to effect coagulative necrosis. The zone of coagulative necrosis undergoes granulation and other inflammatory responses, resulting in fibrosis and volume reduction.

This paper will provide an overview of transcervical radiofrequency ablation (RFA) for uterine fibroids, review the current evidence base for the Sonata System, and offer a perspective on its potential role in the armamentarium of general and specialized obstetrician-gynecologists who manage the many women with symptomatic uterine leiomyomata. Transcervical Radiofrequency Ablation of Uterine Fibroids As a hyperthermic energy source, radiofrequency energy heats soft tissue to effect coagulative necrosis. The zone of coagulative necrosis undergoes granulation and other inflammatory responses, resulting in fibrosis and volume reduction. While radiofrequency ablation of uterine fibroids dates to the early 1990s, these earlier efforts did not involve concurrent imaging in order to match the volume of ablated tissue to that of the targeted fibroid nor was the energy delivered transcervically [11–14]. The use of concurrent sonography has enabled a volumetric approach to RFA, which allows the operator to minimize the number of ablations necessary to ablate most or all of the targeted fibroid. And while earlier devices for radiofrequency ablation required either laparotomy or laparoscopy, sonographic guidance has enabled a transcervical approach, obviating the need for surgical incisions [15–20].

allows the operator to minimize the number of ablations necessary to ablate most or all of the targeted fibroid. And while earlier devices for radiofrequency ablation required either laparotomy or laparoscopy, sonographic guidance has enabled a transcervical approach, obviating the need for surgical incisions [15–20]. Unlike the situation with malignancies, it is not necessary to ablate 100% or more of a fibroid’s volume to provide sustained clinical benefit [21]. Nonetheless, it is preferable to ablate as much of a fibroid as is safely possible, as early clinical evidence suggests a higher efficacy with increasing percentages of ablated fibroid volume [21–23]. Furthermore, fibroids that have been sufficiently ablated are rendered largely or entirely necrotic, and while some tissue may remain in situ, patients can realize symptom relief. When thermal energy is delivered transcervically, it is important to ensure that heat is not transferred to the uterine serosa and beyond, as that may be adhesiogenic and risk thermal injury to adjacent organs such as the bowel and the bladder. Graphical guidance systems can be helpful, both to assist with fibroid targeting and to deliver energy within the serosal margin [24]. Overview of the Sonata System The Sonata System consists of a reusable intrauterine ultrasound (IUUS) probe and a single-use disposable RFA handpiece with proprietary Graphical Guidance Software (GGS) for diagnosis and targeting. These components are integrated to provide the gynecologist with a real-time image-guided treatment system.

onata System The Sonata System consists of a reusable intrauterine ultrasound (IUUS) probe and a single-use disposable RFA handpiece with proprietary Graphical Guidance Software (GGS) for diagnosis and targeting. These components are integrated to provide the gynecologist with a real-time image-guided treatment system. The IUUS probe is used to identify fibroids from within the uterine cavity and guide deployment of an introducer and needle electrodes into one or more targeted fibroids. The intrauterine sonography image offers a unique high-resolution perspective of the uterus and nearby structures, including the bowel, the bladder, and the adnexae. The IUUS probe image is curvilinear, penetrates more than 9 cm, has a transmit frequency of 4.8–9.0 Mhz, and provides a 90° field of view. In conventional transvaginal sonography, the endometrial stripe is a useful landmark. But with intrauterine sonography, the IUUS probe resides within the endometrial cavity proper. The imaging plane is always directed at a 90° sector to the sonography probe, so there is a single imaging plane rather than the sagittal and coronal planes that make transvaginal sonography perhaps more challenging.

andmark. But with intrauterine sonography, the IUUS probe resides within the endometrial cavity proper. The imaging plane is always directed at a 90° sector to the sonography probe, so there is a single imaging plane rather than the sagittal and coronal planes that make transvaginal sonography perhaps more challenging. The RFA handpiece is a single-use component that contains an introducer and needle electrode array. The RFA handpiece snaps together with the IUUS probe to form and integrate into a single treatment device (Fig. 1) that contains all the controls for the physician to place and size the ablation. Mechanical stops and lockouts within the RFA handpiece provide definitive Mechanical stops and lockouts within the RFA handpiece provide definitive physical endpoints, ensuring the ablation is properly located and sized as selected by the physician using the included graphical software.Fig. 1 The Sonata treatment device (combination of IUUS probe and RFA handpiece)

ndpiece provide definitive Mechanical stops and lockouts within the RFA handpiece provide definitive physical endpoints, ensuring the ablation is properly located and sized as selected by the physician using the included graphical software.Fig. 1 The Sonata treatment device (combination of IUUS probe and RFA handpiece) The Sonata Graphical Guidance Software, also known as the SMART Guide™, is a real-time graphical overlay on the ultrasound display. The SMART Guide allows the operating gynecologist to visually select the deployment length, width, and position of the ablation guides (thus setting the mechanical stops for the introducer and needle electrodes) before any hardware has been inserted into the fibroid target. By displaying the ellipsoidal region where the ablation will take place (ablation zone), along with a surrounding ellipsoid (thermal safety border) where tissue temperatures will be elevated, the SMART Guide may provide a safer and more accurate fibroid ablation. These ellipsoidal guides were validated in more than 4000 ablations in bovine muscle and human extirpated uteri, both ex vivo, as well as in vivo during laparotomy (during early preclinical testing in which serosal temperatures were monitored with direct placement of thermocouples and concurrent infrared camera measurements of surface temperatures).

alidated in more than 4000 ablations in bovine muscle and human extirpated uteri, both ex vivo, as well as in vivo during laparotomy (during early preclinical testing in which serosal temperatures were monitored with direct placement of thermocouples and concurrent infrared camera measurements of surface temperatures). Sonographically, the serosa appears as a hyperechoic structure. By using the SMART Guide, an ablation is positioned in order to encompass as much of the fibroid as possible while keeping thermal energy within the uterine serosal margin (Fig. 2). Once the desired ablation size is selected and safe placement of the needle electrodes is confirmed by rotating the IUUS probe in multiple planes, therapeutic RF energy is delivered to the fibroid according to a fixed treatment cycle that is dependent on ablation size. The system is designed to modulate power (up to 150 W) to keep temperatures at the needle electrode tips around 105 °C, and the time at temperature (2–7 min) is automatically set based on the ablation size that the gynecologist has selected via the SMART Guide. The Sonata System can create a continuous range of ablation sizes up to 4.0 cm wide and up to 5.0 cm long. Multiple ablations may be created within a single fibroid.Fig. 2 Transcervical RF ablation with the Sonata System, demonstrating the SMART Guide that delineates the ablation zone (red ellipsoid) and thermal safety border (green ellipsoid. Note explanation for each panel within the Figure)

cm wide and up to 5.0 cm long. Multiple ablations may be created within a single fibroid.Fig. 2 Transcervical RF ablation with the Sonata System, demonstrating the SMART Guide that delineates the ablation zone (red ellipsoid) and thermal safety border (green ellipsoid. Note explanation for each panel within the Figure) Unlike operative hysteroscopy or transcervical fibroid morcellation, only a small amount of hypotonic solution is instilled within the endometrial cavity. This is performed for acoustic coupling of the IUUS probe rather than for significant uterine distension. The diameter of the treatment device, which is comprised of both the IUUS probe and RFA handpiece attached to one another, is 8.3 mm, compatible with a cervical dilatation of 27 French. The Sonata System, with its IUUS probe coupled to another device (in this case, a RFA handpiece), forms a platform in which other potential devices may be attached to the IUUS probe and provide additional functionality beyond ablation. Clinical Evidence There is a growing evidence base relative to transcervical, intrauterine sonography-guided RFA of fibroids with the Sonata System.

The Sonata System, with its IUUS probe coupled to another device (in this case, a RFA handpiece), forms a platform in which other potential devices may be attached to the IUUS probe and provide additional functionality beyond ablation. Clinical Evidence There is a growing evidence base relative to transcervical, intrauterine sonography-guided RFA of fibroids with the Sonata System. Ablation Data After Immediate and Delayed Hysterectomy A single-site cohort study was performed involving 19 women (20 fibroids) undergoing either immediate (n = 12) or delayed (n = 7) hysterectomy after transcervical RF ablation with the VizAblate System (as Sonata was formerly known) [24]. The study was intended to demonstrate the ability of the device to ablate the majority of fibroid volume in fibroids up to 5.0 cm in diameter while avoiding the uterine serosa. Hysterectomies were performed via laparotomy in all cases, with the delayed cohort having their hysterectomies 2 weeks after transcervical RF fibroid ablation. All patients in the delayed hysterectomy group received conscious sedation during their transcervical ablation procedures, whereas those in the immediate hysterectomy group were managed with epidural anesthesia. Ablation percentages in ablated fibroids were ascertained after hysterectomy by staining the extirpated uteri with the viability stain triphenyltetrazolium chloride (TTC) to quantify fibroid ablation dimensions and assess the serosa for thermal injury. There were 17 fibroids ≤5.0 cm in diameter among the 19 patients, in which a median 75% of fibroid volume was ablated (mean 67.2 ± 27.0%; range 15–100%). No uteri were found on either gross visualization at hysterectomy or upon histopathologic evaluation to have evidence for uterine serosal injury.

for thermal injury. There were 17 fibroids ≤5.0 cm in diameter among the 19 patients, in which a median 75% of fibroid volume was ablated (mean 67.2 ± 27.0%; range 15–100%). No uteri were found on either gross visualization at hysterectomy or upon histopathologic evaluation to have evidence for uterine serosal injury. European Clinical Trial Data: the Fibroid Ablation Study-EU Trial The Fibroid Ablation Study-EU (FAST-EU) Trial was a multicenter, prospective trial in the Netherlands, the UK, and Mexico, that examined the clinical effects of the VizAblate System in a cohort of women with symptomatic fibroids [15, 16]. Each patient served as her own control, and an independent core imaging laboratory was used to ensure quality control and standardized interpretation of baseline and post-ablation magnetic resonance images (MRI). Patients had to have up to five treatable fibroids from 1 to 5 cm in diameter, not desire future fertility, and were excluded for the presence of type 0 myomata, ovulatory dysfunction, coagulopathy, and adenomyosis. All patients had to have at least one fibroid that indented the endometrial cavity (FIGO types 1 and 2), a menstrual pictogram (MP) score of at least 120 and a baseline score of 20 or greater on the Symptom Severity Score (SSS) subscale of the Uterine Fibroid Symptom-Quality of Life (UFS-QOL) questionnaire. Anesthesia was per physician and patient choice.

fibroid that indented the endometrial cavity (FIGO types 1 and 2), a menstrual pictogram (MP) score of at least 120 and a baseline score of 20 or greater on the Symptom Severity Score (SSS) subscale of the Uterine Fibroid Symptom-Quality of Life (UFS-QOL) questionnaire. Anesthesia was per physician and patient choice. The primary endpoint of the FAST-EU trial was the percentage change in perfused volume of ablated fibroids at 3 months as determined by contrast-enhanced MRI. Reduction in perfused fibroid volume is a representation of how much of the ablated fibroid is devascularized after ablation and is believed to correlate with treatment success and durability of symptom improvement [21]. Other endpoints, which were reached at 6 months, included reduction in the MP score (which represents the degree of menstrual blood loss), overall safety, improvements in the SSS and health-related quality of life (HRQOL) subscales of the UFS-QOL, patient satisfaction, anesthesia regimen, and recovery pain. A total of 50 women (92 fibroids) were treated with transcervical RFA under intrauterine sonography guidance. All patients had undergone baseline transvaginal sonography, hysteroscopy, or hysterosonography, as well as contrast-enhanced MRI. Of the 92 fibroids treated, 56 (60.9%) were either a type 1 (n = 14) or a type 2 (n = 42) fibroid, which is consistent with the trial inclusion requirement for the presence of at least one indenting myoma. Ablated fibroid diameters, as determined by baseline MRI, ranged from 1.1–6.9 cm.

s well as contrast-enhanced MRI. Of the 92 fibroids treated, 56 (60.9%) were either a type 1 (n = 14) or a type 2 (n = 42) fibroid, which is consistent with the trial inclusion requirement for the presence of at least one indenting myoma. Ablated fibroid diameters, as determined by baseline MRI, ranged from 1.1–6.9 cm. At 3 months, there was a 76.9% median reduction in perfused fibroid volume and a 62.5% median decrease in mean total fibroid volume (P < 0.001). By 12 months, among 28 patients who provided their consent, contrast-enhance MRI revealed a 73.3% median reduction in perfused fibroid volume and a 73.3% median reduction in total fibroid volume (P < 0.001).

edian reduction in perfused fibroid volume and a 62.5% median decrease in mean total fibroid volume (P < 0.001). By 12 months, among 28 patients who provided their consent, contrast-enhance MRI revealed a 73.3% median reduction in perfused fibroid volume and a 73.3% median reduction in total fibroid volume (P < 0.001). With regard to menstrual bleeding, by 3 months, 89.8% of patients reported a reduction in menstrual bleeding, with the mean MP score decreasing consistently through 12 months (Table 1). Lukes and colleagues have determined that a reduction in menstrual bleeding ≥22% is considered to be clinically meaningful to women with abnormal uterine bleeding [25]. In the FAST-EU trial, most patients (57.1–72.9%, based on the time point) realized more than a 50% reduction in their MP scores during the study, while 75.5% of patients achieved a clinically meaningful reduction in their MP scores by 3 months. Similarly, there were significant improvements (all P < 0.001 from baseline) in the SSS and HRQOL subscales through 12 months (Table 1). A 10-point reduction in SSS is considered to represent a moderate effect size [26]. Patients in the FAST-EU trial realized a mean 35.3-point reduction in their SSS scores at 12 months, with as many as 86% of patients at a single time point achieving at least a 10-point reduction.Table 1 Median percentage improvement in patient-reported outcomes from baseline 3 months (%) 6 months (%) 12 months (%) MP 56.9 68.6 72.3 SSS 52.5 66.7 62.5 HRQOL 123 118 127

With regard to menstrual bleeding, by 3 months, 89.8% of patients reported a reduction in menstrual bleeding, with the mean MP score decreasing consistently through 12 months (Table 1). Lukes and colleagues have determined that a reduction in menstrual bleeding ≥22% is considered to be clinically meaningful to women with abnormal uterine bleeding [25]. In the FAST-EU trial, most patients (57.1–72.9%, based on the time point) realized more than a 50% reduction in their MP scores during the study, while 75.5% of patients achieved a clinically meaningful reduction in their MP scores by 3 months. Similarly, there were significant improvements (all P < 0.001 from baseline) in the SSS and HRQOL subscales through 12 months (Table 1). A 10-point reduction in SSS is considered to represent a moderate effect size [26]. Patients in the FAST-EU trial realized a mean 35.3-point reduction in their SSS scores at 12 months, with as many as 86% of patients at a single time point achieving at least a 10-point reduction.Table 1 Median percentage improvement in patient-reported outcomes from baseline 3 months (%) 6 months (%) 12 months (%) MP 56.9 68.6 72.3 SSS 52.5 66.7 62.5 HRQOL 123 118 127 MP menstrual pictogram, SSS Symptom Severity Score, HRQOL Health-related Quality of Life (the last two are subscales of the Uterine Fibroid Symptom-Quality of Life questionnaire)

With regard to menstrual bleeding, by 3 months, 89.8% of patients reported a reduction in menstrual bleeding, with the mean MP score decreasing consistently through 12 months (Table 1). Lukes and colleagues have determined that a reduction in menstrual bleeding ≥22% is considered to be clinically meaningful to women with abnormal uterine bleeding [25]. In the FAST-EU trial, most patients (57.1–72.9%, based on the time point) realized more than a 50% reduction in their MP scores during the study, while 75.5% of patients achieved a clinically meaningful reduction in their MP scores by 3 months. Similarly, there were significant improvements (all P < 0.001 from baseline) in the SSS and HRQOL subscales through 12 months (Table 1). A 10-point reduction in SSS is considered to represent a moderate effect size [26]. Patients in the FAST-EU trial realized a mean 35.3-point reduction in their SSS scores at 12 months, with as many as 86% of patients at a single time point achieving at least a 10-point reduction.Table 1 Median percentage improvement in patient-reported outcomes from baseline 3 months (%) 6 months (%) 12 months (%) MP 56.9 68.6 72.3 SSS 52.5 66.7 62.5 HRQOL 123 118 127 MP menstrual pictogram, SSS Symptom Severity Score, HRQOL Health-related Quality of Life (the last two are subscales of the Uterine Fibroid Symptom-Quality of Life questionnaire) Patients returned to normal activity in a median 4.0 days (mean 4.4 ± 3.1 days) and there was an overall satisfaction rate of 87.8%. Two patients were admitted overnight for observation, one with lower abdominal pain believed secondary to cystitis and the other for bradycardia after treatment received under general anesthesia. The most frequent adverse events consisted of dysmenorrhea (12%), abnormal uterine bleeding above baseline (12%), pelvic pain/cramping (8%), and cystitis (4%). One patient had a fibroid expulsion that was free of sequelae. Four patients underwent elective reintervention between 7 and 12 months post-ablation, two with hysteroscopic myomectomy, one with balloon thermal endometrial ablation, and one with total abdominal hysterectomy.

pelvic pain/cramping (8%), and cystitis (4%). One patient had a fibroid expulsion that was free of sequelae. Four patients underwent elective reintervention between 7 and 12 months post-ablation, two with hysteroscopic myomectomy, one with balloon thermal endometrial ablation, and one with total abdominal hysterectomy. There was a single pregnancy, which was diagnosed 6 months after ablation when the patient presented with 3 months of amenorrhea [27]. This resulted in a live-born singleton pregnancy delivered by elective repeat Cesarean section at term, with an uncomplicated perinatal course. Future Research Efforts The Sonata System is currently being evaluated as part of an FDA-approved investigational device exemption (IDE) trial (SONATA: Sonography-Guided Transcervical Ablation of Uterine Fibroids; ClinicalTrials.gov Identifier: NCT02228174) involving clinical sites in the USA, Mexico, and Europe [28]. This trial aims to show the safety and efficacy of transcervical RFA of uterine fibroids associated with heavy menstrual bleeding.

(IDE) trial (SONATA: Sonography-Guided Transcervical Ablation of Uterine Fibroids; ClinicalTrials.gov Identifier: NCT02228174) involving clinical sites in the USA, Mexico, and Europe [28]. This trial aims to show the safety and efficacy of transcervical RFA of uterine fibroids associated with heavy menstrual bleeding. As noted, successful pregnancy after treatment with the Sonata System (formerly VizAblate) has been reported. There is a growing evidence base concerning the potential for women who have undergone hyperthermic ablation with either focused ultrasound, microwaves, or radiofrequency energy to experience normal fertility and fecundity [20, 27, 29–36]. Nonetheless, radiofrequency ablation of uterine fibroids in women who desire fertility remains investigational in the USA, although treatment in women desiring pregnancy has been approved by the US Food and Drug Administration in the case of MR-guided focused ultrasound (MRgFUS), another hyperthermic ablation modality. In order to examine whether transcervical RFA of uterine fibroids with the Sonata System conserves the structure of the myometrium, baseline and 12-month post-ablation MR images will be examined as part of the ongoing pivotal trial with regard to myometrial thickness and integrity. Additional clinical trials are planned to provide further evidence regarding the role, if any, of transcervical RF ablation in women who desire fecundity.

he myometrium, baseline and 12-month post-ablation MR images will be examined as part of the ongoing pivotal trial with regard to myometrial thickness and integrity. Additional clinical trials are planned to provide further evidence regarding the role, if any, of transcervical RF ablation in women who desire fecundity. Discussion and Perspective While hysterectomy and other more invasive options remain prevalent, there remains a need for less invasive fibroid therapy that is incisionless, preserves the uterus, and can treat fibroids that are not limited to the narrow range of fibroid types (FIGO types 0 and 1 and smaller type 2 fibroids) amenable to transcervical treatment with a hysteroscope. It is established that hysteroscopic management of type 2 fibroids may be particularly challenging; hysteroscopic resection of type 2 fibroids may have a 50% probability of requiring at least one additional attempt at complete removal [37, 38].

r type 2 fibroids) amenable to transcervical treatment with a hysteroscope. It is established that hysteroscopic management of type 2 fibroids may be particularly challenging; hysteroscopic resection of type 2 fibroids may have a 50% probability of requiring at least one additional attempt at complete removal [37, 38]. The Sonata System, because it has an integrated intrauterine sonography probe, is not limited to submucous fibroids visible with hysteroscopic methods. The only fibroids that would not be ablated with Sonata are pedunculated, namely FIGO type 0 and type 7 myomata. Ablation of a type 7 fibroid (pedunculated subserous myoma) would require ablation outside of the uterine serosa. Type 0 (intracavitary/pedunculated submucous) myomata are generally amenable to hysteroscopic resection [39, 40]. Unlike uterine artery embolization of submucous myomata, which is more commonly associated with bulk fibroid expulsion, hyperthermic ablation technologies such as MRgFUS and RFA tend to result in a gradual sloughing of fibroid tissue from the endometrial cavity, with bulk expulsion being much less common [16, 39, 41].

Unlike uterine artery embolization of submucous myomata, which is more commonly associated with bulk fibroid expulsion, hyperthermic ablation technologies such as MRgFUS and RFA tend to result in a gradual sloughing of fibroid tissue from the endometrial cavity, with bulk expulsion being much less common [16, 39, 41]. Because the imaging and treatment components of the Sonata System are integrated into a single handheld device, the operator only manages one device and one image, rather than coordinate multiple devices (e.g., laparoscope, RF device, sonography probe) and images (video, sonography). Intrauterine sonography provides a different imaging perspective from that of transvaginal sonography. As the curvilinear IUUS probe always maintains the same relationship to the portion of the uterus being imaged (i.e., there are no separate coronal and sagittal planes depending on how the sonography probe is angled), the imaging plane remains consistent while scanning the entire circumference of the uterus. The uterine serosa is an important landmark, appearing as a hyperechoic border, and demarcates the limit beyond which the SMART Guide’s thermal safety border must not pass.

lanes depending on how the sonography probe is angled), the imaging plane remains consistent while scanning the entire circumference of the uterus. The uterine serosa is an important landmark, appearing as a hyperechoic border, and demarcates the limit beyond which the SMART Guide’s thermal safety border must not pass. The unique perspective and high-resolution imaging afforded by the IUUS probe enables precise targeting and RF ablation of a wide range of fibroid types, while the SMART Guide provides a real-time visual display (ablation zone) of where the ablation will occur along with the constraint of the thermal safety border. Because the mechanical stops within the RFA handpiece limit needle electrode deployment based on the ablation size graphically chosen by the operator, no manual measurements are needed to ascertain where the needle electrodes should be deployed. The system automatically calculates the time at temperature (105 °C) based on the chosen ablation size and will stop the delivery of RF energy when the necessary time at temperature has elapsed (2–7 min).

sen by the operator, no manual measurements are needed to ascertain where the needle electrodes should be deployed. The system automatically calculates the time at temperature (105 °C) based on the chosen ablation size and will stop the delivery of RF energy when the necessary time at temperature has elapsed (2–7 min). Radiofrequency ablation of uterine fibroids with the Sonata System is a promising treatment approach that is currently investigational in the USA and available under CE mark in Europe. As a transcervically delivered fibroid treatment, the uterine serosa is not compromised. Unlike hysteroscopic resection, tissue is not morcellated nor is significant and prolonged uterine distention necessary. There is evidence in the literature for ablation of fibroids up to 6.9 cm with the Sonata System, and fibroids larger than 5.0 cm (the maximum deployment length of the needle electrodes) may be approached with multiple ablations [10, 16]. Neoadjuvant treatment with gonadotropin-releasing hormone agonists or selective progesterone receptor modulators, which medically reduce fibroid volume, may be a complementary approach to enable the optimal ablation of larger fibroids.

length of the needle electrodes) may be approached with multiple ablations [10, 16]. Neoadjuvant treatment with gonadotropin-releasing hormone agonists or selective progesterone receptor modulators, which medically reduce fibroid volume, may be a complementary approach to enable the optimal ablation of larger fibroids. In an era in which physicians are increasingly performing more invasive hysterectomies and myomectomies in the wake of the US Food and Drug Administration guidance regarding laparoscopic power morcellation, it is important that women continue to have options for uterine fibroid therapy that are less invasive and preserve their choices with regard to uterine conservation [42]. The Sonata System, by virtue of being a transcervical procedure that has a wider range of treatable fibroid types than existing transcervical options, could significantly change the current paradigm in which women either lose their uteri or potentially undergo surgical and radiologic procedures that have significant drawbacks in terms of invasiveness and recovery time or require multiple treatment sessions.

treatable fibroid types than existing transcervical options, could significantly change the current paradigm in which women either lose their uteri or potentially undergo surgical and radiologic procedures that have significant drawbacks in terms of invasiveness and recovery time or require multiple treatment sessions. Conclusions Because it is incisionless, does not require general anesthesia, can ablate most types of uterine fibroids, and preserves the uterus, the Sonata System represents an exciting new technology for the treatment of uterine fibroids. This has been supported to date by the results of the FAST-EU trial in Europe and Mexico, in which there were significant reductions in fibroid volume and fibroid-associated symptoms. The ongoing pivotal IDE trial (SONATA) will provide further evidence regarding clinical outcomes and safety in a mostly US-based patient cohort. This article is part of the Topical Collection on Emerging and Pipeline Technologies in Ob/Gyn Compliance with Ethical Standards Conflict of Interest D. B. Toub declares personal fees from Gynesonics and patent US 8,992,427 B2 issued. Due to the nature of this section and the anticipation of bias from the author as it pertains to his article topic, the article has been reviewed by Dr. James Greenberg of Brigham and Women’s Hospital and by Dr. Stephanie Morris of Newton-Wellesley Hospital. Both reviewers do not have any conflict of interest with the product reviewed in the article, and they assert that the article is factual and free of excessive bias toward the subject.

icle has been reviewed by Dr. James Greenberg of Brigham and Women’s Hospital and by Dr. Stephanie Morris of Newton-Wellesley Hospital. Both reviewers do not have any conflict of interest with the product reviewed in the article, and they assert that the article is factual and free of excessive bias toward the subject. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

Introduction There is a growing body of evidence regarding the safe and effective use of barbed sutures or Knotless Tissue Control Devices in general and minimally invasive gynecological procedures. Barbed sutures, which allow consistent tension control over the suture line and avoid the need for knots, were first used in gynecologic surgery by Greenberg and Einarsson in 2008 [1•]. These devices are effective for reducing procedural time, achieving superior hemostasis and comparable wound approximation to traditional sutures. Ethicon has a portfolio of two technologies in the market: STRATAFIX™ Spiral and STRATAFIX™ SYMMETRIC Knotless Tissue Control Devices. These devices eliminate the need to tie surgical knots and can reduce operating room time and potential knot-related complications. Additionally, by eliminating knots, the STRATAFIX™ Knotless Tissue Control Devices reduce suturing difficulty, especially in minimally invasive gynecological procedures. The barbs on these devices aid in achieving an intimate closure and enhanced tissue engagement with each pass.

d potential knot-related complications. Additionally, by eliminating knots, the STRATAFIX™ Knotless Tissue Control Devices reduce suturing difficulty, especially in minimally invasive gynecological procedures. The barbs on these devices aid in achieving an intimate closure and enhanced tissue engagement with each pass. A variety of studies have proven that barbed devices such as STRATAFIX™ Spiral have similar or improved outcomes as compared to traditional sutures. The safety and effectiveness have been established in dermal closure of non-emergent Pfannenstiel incisions [2, 3], closure of the uterus in laparoscopic myomectomies [4, 5, 6, 7], vaginal cuff closure in total laparoscopic hysterectomies [8, 9], and in robotic sacrocolpopexy [10]. Isolated cases of bowel obstruction have been associated with the use of barbed sutures during laparoscopic surgery.

rgent Pfannenstiel incisions [2, 3], closure of the uterus in laparoscopic myomectomies [4, 5, 6, 7], vaginal cuff closure in total laparoscopic hysterectomies [8, 9], and in robotic sacrocolpopexy [10]. Isolated cases of bowel obstruction have been associated with the use of barbed sutures during laparoscopic surgery. The purpose of this paper is to provide an overview of the design and performance characteristics of the new knotless tissue control device STRATAFIX™ SYMMETRIC PDS™ Plus. This device is the first barbed suture that can be used for the closure of high tension areas, like fascia. This device has a novel barb design (anchors) to facilitate high strength soft tissue approximation along with the performance and absorption characteristics of PDS™ Plus Suture. The added features and benefits of the anchors may increase the efficiency of tissue approximation while ensuring the appropriate wound support during healing. As with PDS™ Plus Antibacterial (polydioxanone) Suture, STRATAFIX™ SYMMETRIC PDS™ Plus Device is used in many surgical applications such as fascial closure where an absorbable suture offering extended wound support (up to 6 weeks) is required [11•, 12•, 13].

while ensuring the appropriate wound support during healing. As with PDS™ Plus Antibacterial (polydioxanone) Suture, STRATAFIX™ SYMMETRIC PDS™ Plus Device is used in many surgical applications such as fascial closure where an absorbable suture offering extended wound support (up to 6 weeks) is required [11•, 12•, 13]. Device Design and Characteristics STRATAFIX™ SYMMETRIC PDS™ Plus Device is a novel, absorbable, antibacterial, Knotless Tissue Control Device developed to facilitate soft tissue approximation by providing the performance characteristics and wound holding security of conventional PDS™ Plus Suture with added features that offer increased efficiency and control. The device is composed of polydioxanone, which is identical in composition to the absorbable polymer material that is used to produce PDS™ Plus Suture. The STRATAFIX™ SYMMETRIC PDS™ Plus Device is illustrated in Fig. 1. It features a solid core with a series of unidirectional anchors evenly spaced down the length of the device in pairs symmetrically orientated 180° from each other. The strength and integrity of the core are maintained as the anchors are integrally formed onto the core.Fig. 1 Example of a STRATAFIX™ SYMMETRIC PDS™ Plus Device

ures a solid core with a series of unidirectional anchors evenly spaced down the length of the device in pairs symmetrically orientated 180° from each other. The strength and integrity of the core are maintained as the anchors are integrally formed onto the core.Fig. 1 Example of a STRATAFIX™ SYMMETRIC PDS™ Plus Device The size and spacing of the anchors is uniquely designed to provide maximum holding in soft tissue where PDS™ Plus Suture is commonly used, such as fascia. The anchors are also designed to provide tactile feedback during passage through tissue, which helps the surgeon apply the desired tension. Each pass of the device maintains the tissue approximation without the need for an assistant to hold tension or “follow the suture” as is necessary when sewing with a continuous or running technique using a conventional suture. The distal end of the device has a unique feature described as a fixation tab that anchors the first pass into tissue. The fixation tab eliminates the need for a surgical knot to anchor the proximal end of the incision. The device is implanted using a continuous suturing pattern and requires only minimal changes in the technique used to implant conventional sutures. The only significant technique differences are at the ends of the suture line, as described in the next section.

surgical knot to anchor the proximal end of the incision. The device is implanted using a continuous suturing pattern and requires only minimal changes in the technique used to implant conventional sutures. The only significant technique differences are at the ends of the suture line, as described in the next section. This new device is also treated with IRGACARE ® MP (triclosan) which inhibits bacterial colonization of the device as it does with traditional PDS™ Plus Suture [14]. STRATAFIX™ SYMMETRIC PDS™ Plus Devices have been proven in vitro to kill bacteria on the device that are commonly associated with surgical site infections and to create zones of inhibition [15].

ACARE ® MP (triclosan) which inhibits bacterial colonization of the device as it does with traditional PDS™ Plus Suture [14]. STRATAFIX™ SYMMETRIC PDS™ Plus Devices have been proven in vitro to kill bacteria on the device that are commonly associated with surgical site infections and to create zones of inhibition [15]. Description of Intra-operative Usage STRATAFIX™ SYMMETRIC PDS™ Plus Devices are designed to be used in continuous suture patterns without anchoring knots at the beginning or end of the closure line. The basic steps are illustrated in Fig. 2a–e. To begin the closure, take the first pass directly above or adjacent to the apex in a direction away from the incision (2a). Pull the device through the tissue to gently seat the fixation tab. The fixation tab should be seated above the tissue plane and be visible (2b). Moving toward the apex of the incision, take a pass in the intact tissue perpendicular to the initial pass to lock-in the fixation tab. Multiple passes are acceptable (2c). Proceed with a continuous suturing pattern to close the incision, taking apposing bites on either side of the wound in standard fashion. To achieve the desired approximation and tension, gently pull on the device with each tissue passage (2d). Over tightening of any suture can lead to ischemia and necrosis. To complete and secure the closure, take two passes in the reverse direction across the incision. Finally, gently pull on the free end of the device and cut flush with the surface of the tissue (2e).Fig. 2 Illustrations of the basic steps to close an incision using STRATAFIX SYMMETRIC PDS™ Plus devices

ia and necrosis. To complete and secure the closure, take two passes in the reverse direction across the incision. Finally, gently pull on the free end of the device and cut flush with the surface of the tissue (2e).Fig. 2 Illustrations of the basic steps to close an incision using STRATAFIX SYMMETRIC PDS™ Plus devices This technique provides a balanced closure for STRATAFIX™ SYMMETRIC PDS™ Plus Devices from end to end. Locking the fixation tab into tissue at the proximal end mimics the pair of reverse passes at the distal end. The unidirectional anchors lock into tissue with each pass along the length of the closure to achieve intimate and secure approximation. Device Performance and Evaluation Tensile Strength Suture strength is characterized by the maximum tensile load of a simple knot throw as per USP requirements [16]. It is well established that the knot is the weakest point in an implanted suture [17]. Since the STRATAFIX™ SYMMETRIC PDS™ Plus Devices require no knots to approximate tissue, the maximum tensile load of the core for the STRATAFIX™ SYMMETRIC PDS™ Plus Devices was compared to the maximum tensile load of equivalent sized PDS™ Plus Suture knots. The tensile strength of the STRATAFIX™ SYMMETRIC PDS™ Plus Devices was evaluated using a standard Tensile Testing method. A 4-in.-long specimen was fixed at a set gauge length using steel-faced pneumatic clamps. Specimens were loaded at a constant strain rate until rupture using a calibrated INSTRON™ Mechanical Testing Unit.

nsile strength of the STRATAFIX™ SYMMETRIC PDS™ Plus Devices was evaluated using a standard Tensile Testing method. A 4-in.-long specimen was fixed at a set gauge length using steel-faced pneumatic clamps. Specimens were loaded at a constant strain rate until rupture using a calibrated INSTRON™ Mechanical Testing Unit. The tensile strength of the PDS™ PLUS Suture was evaluated using the same method described for STRATAFIX™ SYMMETRIC PDS™ Plus Devices with the exception of a simple knot introduced into the center of the specimen length prior to loading into the INSTRON™. This testing methodology is established in the reference standard 881 for Suture Tensile Strength issued by the US Pharmacopeia [16]. Twenty samples per group were tested. The maximum tensile loads of the STRATAFIX™ SYMMETRIC PDS™ Plus Devices are statistically significantly higher than the equivalent size PDS™ Plus Suture knots at the 95% confidence interval as determined using a t test. The maximum average tensile for size 1 STRATAFIX™ SYMMETRIC PDS™ Plus Devices was 19.16 +/− 0.6 lbs as compared to PDS™ Plus suture at 14.28 +/− 0.7 lbs. Size 0 averaged 15.42 +/− 0.5 lbs as compared to PDS™ Plus suture at 10.14 +/− 0.7 lbs. Size 2/0 averaged 10.72 +/− 0.8 lbs. as compared to PDS™ Plus suture at 7.32 +/− 0.7 lbs and size 3/0 devices averaged 7.35 +/− 05 lbs as compared to PDS™ Plus suture at 5.84 +/− 0.4 lbs.

ed to PDS™ Plus suture at 14.28 +/− 0.7 lbs. Size 0 averaged 15.42 +/− 0.5 lbs as compared to PDS™ Plus suture at 10.14 +/− 0.7 lbs. Size 2/0 averaged 10.72 +/− 0.8 lbs. as compared to PDS™ Plus suture at 7.32 +/− 0.7 lbs and size 3/0 devices averaged 7.35 +/− 05 lbs as compared to PDS™ Plus suture at 5.84 +/− 0.4 lbs. Fixation Tab Mass Comparison to Conventional Suture Knot Tower Conventional suturing technique requires surgical knots to secure the proximal and distal ends of a continuous suture pattern and for each stitch of an interrupted suture pattern. Knots add foreign body mass to the implanted suture. The fixation tab of the STRATAFIX™ SYMMETRIC PDS™ Plus Device that secures the proximal end of the closure adds significantly less mass than traditional knots. The mass of a Size 1 STRATAFIX™ SYMMETRIC PDS™ Plus Device fixation tab was compared to a 5-throw surgical knot tower of Size 1 PDS™ Suture. In order to obtain the knot towers, five throw knots were tied around a cylinder and the knot tower was cut away from the cylinder. The loop below the knot tower would be entirely within tissue in clinical use. The amount of suture comprising this loop is dependent on many variables such as needle geometry and tissue thickness. Therefore, the mass from the loop below the knot tower was not included in the measurements; only the knot tower itself and the short tag ends were weighed making it a conservative value. The fixation tabs were simply cut from the distal end of the STRATAFIX™ SYMMETRIC PDS™ Plus Device.

e geometry and tissue thickness. Therefore, the mass from the loop below the knot tower was not included in the measurements; only the knot tower itself and the short tag ends were weighed making it a conservative value. The fixation tabs were simply cut from the distal end of the STRATAFIX™ SYMMETRIC PDS™ Plus Device. Ten samples per group were evaluated. The mass of the fixation tab is approximately one third of a 5-throw knot tower of the equivalent size suture. The STRATAFIX™ SYMMETRIC PDS™ Plus fixation tab averaged 0.00496 g as compared to PDS™ Plus 5 throw not tower of 0.01839 g. A t test was used to determine the difference is statistically significant at the 95% confidence interval. Initiation Stitch Strength A functional test was developed to mimic the motion and mechanics of the surgeon during initial placement of the STRATAFIX™ SYMMETRIC PDS™ Plus Device into tissue by measuring initiation stitch strength. This method includes seating the fixation tab into intact tissue as shown in Fig. 2a–c and then making a single point of closure within the incision. Axial load is applied directly to the device using the remaining length to determine initiation stitch strength. For control specimens prepared using PDS™ Plus suture, the initiation stitch is created by applying a 5-throw surgical knot to make a single point of closure within the incision. The remaining length of suture is used for axial loading.

y to the device using the remaining length to determine initiation stitch strength. For control specimens prepared using PDS™ Plus suture, the initiation stitch is created by applying a 5-throw surgical knot to make a single point of closure within the incision. The remaining length of suture is used for axial loading. Testing was conducted for Sizes 0 and 1 STRATAFIX™ SYMMETRIC PDS™ Plus Devices and PDS™ Plus suture in porcine midline abdominal (fascia) tissue. Cadaveric porcine tissue specimens were prepared by separating the subcutaneous fat and skin layer using a scalpel and retaining the muscle layer with midline fascia for applying the devices. Two small incisions (∼1.5 in. each) were made in the fascia midline to allow for four test locations per tissue specimen. The tissue beyond the incision ends was left intact in order to simulate an “apex” location for the initiation stitch. The tissue specimen was secured in a proprietary fixture. Axial load was applied to the free end of the device or suture (loaded perpendicular to the fixed tissue plane) until a device or tissue failure was noted by the test operator. This set-up simulates a surgeon pulling ‘up’ on the free device or suture end after initiation into the tissue. The free end of the device or suture is fixed into the upper grip. Using a calibrated INSTRON™ Mechanical Testing Unit, specimens were loaded and pulled at a constant strain rate until rupture. Note: the fixture was weighted down to apply resistance to the linear load.

ce or suture end after initiation into the tissue. The free end of the device or suture is fixed into the upper grip. Using a calibrated INSTRON™ Mechanical Testing Unit, specimens were loaded and pulled at a constant strain rate until rupture. Note: the fixture was weighted down to apply resistance to the linear load. Ten samples per group were tested. The average maximum initiation strength of STRATAFIX™ SYMMETRIC PDS™ Plus Size 1 devices was 15.16 +/− 1.96 lbs as compared to 13.61 +/− 1.17 lbs for PDS™ Plus suture. For size 0, the average maximum initiation strength of STRATAFIX™ SYMMETRIC PDS™ Plus Size 0 devices was 13.89 +/− 0.4 lbs as compared to 8.34 +/− 1.62 lbs for PDS™ Plus suture. The STRATAFIX™ SYMMETRIC PDS™ Plus Device achieved statistically higher maximum initiation stitch strength for both sizes 0 and 1 relative to the 5-throw PDS™ Plus knot. Regarding the failure modes for STRATAFIX™ SYMMETRIC PDS™ Plus Device, the size 1 devices have a stronger, more robust core than the size 0 devices and thus experienced more fixation tab failures. For PDS™ Plus, failure occurred primarily at the surgical knot, regardless of size. Surgical knots introduce stress concentrations into the strand, which greatly reduce their inherent tensile strength. Tissue tearing may occur in this ex vivo model and was noted to occur for PDS™ in the same measured range as device failure.

S™ Plus, failure occurred primarily at the surgical knot, regardless of size. Surgical knots introduce stress concentrations into the strand, which greatly reduce their inherent tensile strength. Tissue tearing may occur in this ex vivo model and was noted to occur for PDS™ in the same measured range as device failure. The results of measuring initiation stitch strength are valuable in verifying the integrity of the STRATAFIX™ SYMMETRIC PDS™ Plus device. The fixation tab’s placement into intact tissue positively impacts the performance of the device when beginning an incision closure by counteracting the direct and upward loading on the device to ensure secure initiationTable 1 Maximum Wound Holding Strength of STRATAFIX™ SYMMETRIC PDS™ Plus Devices and PDS™ Plus Loop, PDS™ Plus Suture and Coated VICRYL™ Plus Suture n = 10 samples per group Size Avg. Maximum Wound Holding Strength (lbs) +/− Std Dev. STRATAFIX™ SYMMETRIC PDS™ Plus Devices (95% CI) Avg. Maximum Wound Holding Strength (lbs) +/− Std Dev. of PDS™ Plus Loop or PDS™ Plus Suture (95% CI) Avg. Maximum Wound Holding Strength (lbs) +/− Std Dev. of Coated VICRYL™ Plus Suture p value 2–0 Subcutaneous tissue 90.06 +/− 16.38 (73.68, 106.44) 86.32 +/− 18.62 (67.70, 104.94) Not tested 0.64 0 Abdominal wall tissue 129.50 +/− 17.88 (111.62, 147.38) 109.79 +/− 31.84 (77.95, 141.63) 93.42+/−22.02 (63.68, 128.34) 0.009 .

Maximum Wound Holding Strength (lbs) +/− Std Dev. of Coated VICRYL™ Plus Suture p value 2–0 Subcutaneous tissue 90.06 +/− 16.38 (73.68, 106.44) 86.32 +/− 18.62 (67.70, 104.94) Not tested 0.64 0 Abdominal wall tissue 129.50 +/− 17.88 (111.62, 147.38) 109.79 +/− 31.84 (77.95, 141.63) 93.42+/−22.02 (63.68, 128.34) 0.009 . Wound Holding Strength Table 1 Wound closure integrity is a critical performance parameter during the wound healing process. The ability of any wound closure device to maintain wound approximation during healing is a function of device strength and placement technique, tissue integrity, and peripheral loading of the incision site. The factors inherent to the device are controlled primarily during application at the time of surgery. Tissue health and loading are unique to each patient and can be challenging to predict and mitigate post-surgery. In order to assess wound holding performance under simulated physiologic load, a porcine survival model was developed. The robust porcine abdominal tissue is a well-established model that mimics high tension surgical closures in the human abdominal wall and fascia tissue layers. The model allows for device application along the linea alba, a secure fixture of the ex vivo test specimen, and stable axial tensile strain until rupture of the incision site. Benchtop assessments were validated against data collected using a human cadaver specimen in order to ensure holding strength values are on a comparable scale.

for device application along the linea alba, a secure fixture of the ex vivo test specimen, and stable axial tensile strain until rupture of the incision site. Benchtop assessments were validated against data collected using a human cadaver specimen in order to ensure holding strength values are on a comparable scale. Fresh cadaveric porcine tissue was procured from a local vendor. Specimens were full thickness ventral slabs large enough to dissect and re-approximate; typically10 × 10 in. in size. Slab thickness varied between animals yet linea alba thickness was consistently 0.4–0.6 cm. A scalpel was used to separate the abdominal wall tissue from the subcutaneous layer (and skin layer). The abdominal wall tissue specimen was incised along the linea alba with a scalpel, creating a 10 cm length incision with intact tissue beyond the apexes. A stainless steel template was used to mark the area intended for closure. Lateral spacing between bites was 1 cm and medial to the incision was 1 cm from incision edge. This spacing represents the natural curvature and length of a surgical needle used for abdominal closures. Size 0 devices were used in this tissue layer.

tainless steel template was used to mark the area intended for closure. Lateral spacing between bites was 1 cm and medial to the incision was 1 cm from incision edge. This spacing represents the natural curvature and length of a surgical needle used for abdominal closures. Size 0 devices were used in this tissue layer. The subcutaneous tissue specimen was incised to create a similar 10 cm incision with intact tissue beyond the apexes. The same marking template was used on these specimens. Size 2–0 devices were used in this tissue layer. The STRATAFIX™ SYMMETRIC PDS™ Plus Devices were applied in the prepared porcine tissue following the steps outlined in the Intra-Operative Usage description. The fixation tab was seated into intact tissue beyond the proximal incision apex and secured with a single pass. The device was applied in a continuous pattern to close the (marked) 10 cm incision. At the distal apex, the device was passed back over the incision closure two times, creating a “double reverse” locking stitch as shown in Fig. 2e. The device was trimmed at the level of the tissue plane to complete the closure. Size 0 PDS™ Plus Looped Suture closures were made in the abdominal wall tissue using a continuous pattern following the same 1 cm spacing scheme and secured on the proximal end by the suture loop and by a 5-throw knots on the distal end; two surgeon’s knots and an extra throw. The PDS™ Plus Suture Size 2–0 devices used in the subcutaneous tissue were not looped and were instead secured on either incision end using 5-throw knots as described.

1 cm spacing scheme and secured on the proximal end by the suture loop and by a 5-throw knots on the distal end; two surgeon’s knots and an extra throw. The PDS™ Plus Suture Size 2–0 devices used in the subcutaneous tissue were not looped and were instead secured on either incision end using 5-throw knots as described. Size 0 Coated VICRYL™ Plus Antibacterial (polyglactin 910) Suture closures were made in the abdominal wall tissue using a continuous pattern following the same 1 cm spacing scheme and secured on the proximal and distal ends using 5-throw knots as described. These incision closures are intended to mimic high-tension abdominal wall closures over time. These closures are appropriate for suture sizes 2–0 and larger and are compatible with either continuous or interrupted suture closure techniques. Using a fabricated test fixture, specimens were loaded axially on an INSTRON™ Mechanical Testing Unit) at a constant strain rate until tissue or device failure was observed by the operator. Clamp distance was set at the same distance medial to the incision closure for each specimen. The maximum load at failure and the failure description were recorded for each specimen. Ten samples per group were tested.

l Testing Unit) at a constant strain rate until tissue or device failure was observed by the operator. Clamp distance was set at the same distance medial to the incision closure for each specimen. The maximum load at failure and the failure description were recorded for each specimen. Ten samples per group were tested. As with the maximum initiation strength test, the failure modes were also assessed in the wound holding strength testing. In subcutaneous tissue, six samples of size 2/0 STRATAFIX™ SYMMETRIC PDS™ Plus failured by suture break and the remainder by tissue gapping. PDS™ Plus sutures had nine knot failures and one tissue failure. In abdominal wall tissue, size 0 STRATAFIX™ SYMMETRIC PDS™ Plus Devices all had tissue failure whereas the PDS™ Plus sutures all failed for suture break. Size 0 VICRYL™ Plus Suture had six failures for tissue gapping, three for tissue failure and one for suture break. A one-way ANOVA was used to determine if the difference is statistically significant at the 95% confidence interval for the Size 0 devices in abdominal tissue. The STRATAFIX™ SYMMETRIC PDS™ Plus Device displayed statistically superior maximum tissue holding forces to both Coated VICRYL™ Plus Suture and PDS™ Plus Looped Suture. The STRATAFIX™ SYMMETRIC PDS™ Plus Device also demonstrated a lower (14%) coefficient of variation (COV) than the comparators (24% VICRYL™ and 31% PDS™ Loop). The relatively low COV of the STRATAFIX™ SYMMETRIC PDS™ Plus Device may be due to there being only one failure mode. This indicates consistency between samples.

The STRATAFIX™ SYMMETRIC PDS™ Plus Device also demonstrated a lower (14%) coefficient of variation (COV) than the comparators (24% VICRYL™ and 31% PDS™ Loop). The relatively low COV of the STRATAFIX™ SYMMETRIC PDS™ Plus Device may be due to there being only one failure mode. This indicates consistency between samples. A t test was used to determine if the difference is statistically significant at the 95% confidence interval for the Size 2–0 devices in subcutaneous tissue. The STRATAFIX™ SYMMETRIC PDS™ Plus Device displayed statistically equivalent maximum tissue holding forces compared to PDS™ Plus Suture size 2–0. These results are valuable to validate the performance of STRATAFIX™ SYMMETRIC PDS™ Plus Devices when used in various tissue closures including those under significant loading. The wound holding capabilities are demonstrated to be as good as or better than conventional devices routinely used in soft tissue approximation.

ese results are valuable to validate the performance of STRATAFIX™ SYMMETRIC PDS™ Plus Devices when used in various tissue closures including those under significant loading. The wound holding capabilities are demonstrated to be as good as or better than conventional devices routinely used in soft tissue approximation. Conclusions STRATAFIX™ SYMMETRIC PDS™ Plus Device is an absorbable, antibacterial, knotless tissue control device developed to facilitate soft tissue approximation by providing the performance characteristics and wound holding security of conventional PDS™ Plus Suture with added features that offer increased efficiency and control. The devices have increased tensile strength relative to the knot tensile strength of PDS™ Plus Sutures with comparable and sometimes superior wound holding strength in fascia. The advantages that these features provide over conventional suture are relevant and beneficial for obstetric/gynecologic surgeries. Further studies are needed to provide outcomes regarding safety, effectiveness, and adverse events. This article is part of the Topical Collection on Emerging and Pipeline Technologies in Ob/Gyn Compliance with Ethical Standards Conflict of Interest Jesse G. Nawrocki, Heather Nonnenmann, Mark Mooney, Nadia Sutton, and Niels-Derrek Schmitz all declare to be salaried employees with stock options of Johnson & Johnson – Ethicon.

This article is part of the Topical Collection on Emerging and Pipeline Technologies in Ob/Gyn Compliance with Ethical Standards Conflict of Interest Jesse G. Nawrocki, Heather Nonnenmann, Mark Mooney, Nadia Sutton, and Niels-Derrek Schmitz all declare to be salaried employees with stock options of Johnson & Johnson – Ethicon. Due to the nature of this section and the anticipation of bias from the author as it pertains to his article topic, the article has been reviewed by Dr. Jon I. Einarsson of Brigham and Women’s Hospital and by Dr. Mark Levie of Montefiore Medical Center. Both reviewers do not have any conflict of interest with the product reviewed in the article, and they assert that the article is factual and free of excessive bias toward the subject. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

Introduction Uterine fibroids (leiomyomas) are the most common gynaecological tumours affecting premenopausal women [1]. They are benign, hormonally dependent tumours arising from the myometrial layer of the uterus [2•]. Prevalence of fibroids ranges between 3.3 and 77% [3, 4] and varies with age and ethnicity [5]. Although most women with fibroids are asymptomatic, fibroids are a major source of gynaecological morbidity and have a substantial impact on women’s health and quality of life [6]. The most common symptoms are menorrhagia (heavy menstrual bleeding) and dysmenorrhoea (menstrual pain). The current management of fibroids includes medical therapy (e.g. ulipristal acetate), minimally invasive procedures (e.g. uterine artery embolisation) and surgery (e.g. myomectomy, hysterectomy). Despite the range of treatment options, surgery remains the mainstay of therapy. Fibroid tissue is comprised mainly of extracellular matrix and characterised by a low mitotic index and is usually considered slow growing [7]. However, some fibroids can grow very large—up to 30 cm3 [1].

Although most women with fibroids are asymptomatic, fibroids are a major source of gynaecological morbidity and have a substantial impact on women’s health and quality of life [6]. The most common symptoms are menorrhagia (heavy menstrual bleeding) and dysmenorrhoea (menstrual pain). The current management of fibroids includes medical therapy (e.g. ulipristal acetate), minimally invasive procedures (e.g. uterine artery embolisation) and surgery (e.g. myomectomy, hysterectomy). Despite the range of treatment options, surgery remains the mainstay of therapy. Fibroid tissue is comprised mainly of extracellular matrix and characterised by a low mitotic index and is usually considered slow growing [7]. However, some fibroids can grow very large—up to 30 cm3 [1]. It is well known that fibroids are sensitive to oestrogens, which may act to grow or maintain their size [8]. Oestrogen, progesterone and epidermal growth factor (EGF) are considered essential to the formation of fibroids [1]. Due to their hormonal nature, it was originally thought that uterine fibroids would develop after puberty, continue to grow in a consistent manner during reproductive life and shrink after the menopause. Despite recent advances in imaging, our understanding of the natural life cycle of fibroids remains poor, which makes counselling difficult.

ture, it was originally thought that uterine fibroids would develop after puberty, continue to grow in a consistent manner during reproductive life and shrink after the menopause. Despite recent advances in imaging, our understanding of the natural life cycle of fibroids remains poor, which makes counselling difficult. Radiological Life Cycle of Fibroids in the Inter-Gravid State Variability in Growth of Fibroids in the Inter-Gravid State It was originally thought that fibroids had a linear growth during a woman’s reproductive life. However, there is evolving evidence that this is not always the case and typically the life cycle of a fibroid may involve growth as well as regression. Indeed, Peddada et al. tracked the growth of 262 fibroids (range 1.0 to 13.0cms) in 72 premenopausal women by performing four MRI scans over a period of 12 months [9]. They found a median growth rate for fibroids was 9.0% per 6 months (range − 89.0% to + 138.0%). Spontaneous regression, defined by a reduction in fibroid volume of greater than 20%, occurred in 7.0% of fibroids. Rapid growth, defined by an increase in volume of greater than 20%, occurred in 34.0% of fibroids. The authors also found that different fibroids within individuals grow at different rates, despite a uniform hormonal milieu [9].

defined by a reduction in fibroid volume of greater than 20%, occurred in 7.0% of fibroids. Rapid growth, defined by an increase in volume of greater than 20%, occurred in 34.0% of fibroids. The authors also found that different fibroids within individuals grow at different rates, despite a uniform hormonal milieu [9]. These findings were in agreement with another study by Mavrelos et al., who used ultrasound as their imaging modality [10••]. One hundred twenty-two women underwent a minimum of two transvaginal ultrasound assessments, all performed by a single operator over a nine-year period. The median scan interval between the initial and final ultrasound was 21.5 months (range 8–90 months). Fibroids were measured in three perpendicular planes and the position of every fibroid was recorded. The authors concluded that fibroids underwent both growth and regression despite the maintenance of a stable hormonal environment in premenopausal women. The volume of the largest fibroid increased by a median of 35.0% a year. Whilst, 21.3% (26/122) fibroids appeared to have regressed spontaneously [10••]. In a separate study by Baird et al., MRI assessments of 101 fibroids were made in 18 black and 18 white women at enrolment, 3, 6 and 12 months interval [11]. The median growth rate of fibroids was found to be 7.0% per 3 months. Growth spurts, defined as a greater than or equal to 30% increase over 3 months, were found in 36.6% (37/101) of fibroids. Conversely, shrinkage spurts were seen in 1.0% (1/101). 62.4% (63/101) of fibroids did not demonstrate a growth or shrinkage spurt [11].

n growth rate of fibroids was found to be 7.0% per 3 months. Growth spurts, defined as a greater than or equal to 30% increase over 3 months, were found in 36.6% (37/101) of fibroids. Conversely, shrinkage spurts were seen in 1.0% (1/101). 62.4% (63/101) of fibroids did not demonstrate a growth or shrinkage spurt [11]. Tsuda et al. proposed the use of fibroid vascularity with transvaginal and transabdominal ultrasound as a predictor of fibroid growth [8]. The authors measured fibroid volume and attempted to identify a leiomyoma artery for each fibroid. The participants were reassessed 3 monthly for a year. Fibroid volume increased in 46.2% (24/52) of patients with a leiomyoma artery, compared with 6.1% (3/49) when a leiomyoma artery was not identified (n = 52, P < 0.001). The authors concluded that the presence of leiomyoma artery can be a useful predictor of fibroid growth [8].

ants were reassessed 3 monthly for a year. Fibroid volume increased in 46.2% (24/52) of patients with a leiomyoma artery, compared with 6.1% (3/49) when a leiomyoma artery was not identified (n = 52, P < 0.001). The authors concluded that the presence of leiomyoma artery can be a useful predictor of fibroid growth [8]. Factors Affecting Fibroid Growth Starting Volume of Fibroid Mavrelos et al. found that the size of fibroids at presentation significantly influenced their subsequent growth rates [10••]. Small fibroids, defined by a mean diameter of less than 20 mm, increased in size by a median of 51.3% (IQR, 9.3–210.3) per year, whilst large fibroids (diameter of more than 50 mm) increased by a median of 40.7% (IQR, 14.1–67.0) per year. Intermediate fibroids (between 20 and 50 mm) only increased in size by a median 16.8% (IQR, − 7.4–63.0) per year (P = 0.007). Fibroids classified as either small or large demonstrated a growth rate nearly three times more than fibroids classed as intermediate in size at presentation [10••]. Similarly, Baird et al. found that the size of the fibroid at initial assessment was a significant variable in predicting growth rates [11]. However, the authors found that large fibroids (≥ 50 mm) were significantly less likely to have short-term variability in growth when compared to small fibroids (< 50 mm) (P < 0.001) [11].

, Baird et al. found that the size of the fibroid at initial assessment was a significant variable in predicting growth rates [11]. However, the authors found that large fibroids (≥ 50 mm) were significantly less likely to have short-term variability in growth when compared to small fibroids (< 50 mm) (P < 0.001) [11]. Peddada et al. did not find a significant difference in the growth rates of fibroids when fibroids < 14.0 cm3 were compared with larger fibroids (P = 0.48) [12]. Similarly, Tsuda et al. had the same conclusion when comparing fibroids larger or smaller than 31.9 cm3 [8]. Type of Fibroid Tsuda et al. found that submucous fibroids were less likely to increase in size. 15.4% (2/13) of submucous fibroids increased in size per year on ultrasound, compared with 27.4% (17/62) of intramural fibroids and 30.7% (8/26) subserous fibroids (P < 0.05). This was independent of whether a leiomyoma artery was identified [8]. Conversely, Mavrelos et al. found that intramural fibroids grew the fastest, followed by subserous then submucous fibroids [10••]. Intramural fibroids increased in volume by a median of 53.2% (IQR, 11.2–217.0) per year, subserous fibroid increased by 25.1% (IQR, 1.1–87.1) per year and submucous fibroids increased by 22.8% (IQR, − 11.7 to 48.3) per year (P = 0.012). However, due to a disproportionate number of small intramural fibroids, the authors concluded that fibroid growth rate appeared to be independently influenced by the fibroid size, rather than a true reflection of the effect of growth rate on fibroid position [10••].

by 22.8% (IQR, − 11.7 to 48.3) per year (P = 0.012). However, due to a disproportionate number of small intramural fibroids, the authors concluded that fibroid growth rate appeared to be independently influenced by the fibroid size, rather than a true reflection of the effect of growth rate on fibroid position [10••]. Age Fibroid growth tends to decline with age. Mavrelos et al. found that women less than or equal to 35 years old had a median fibroid of growth rate of 69.1% (IQR, 8.3–185.1) per year, whilst women more than 35 years of age had a median fibroid growth rate of 29.8% (IQR, 0.0–78.7) (P = 0.113) [10••]. A similar decline was also reported by Peddada et al.; however, this did not occur for all races [9]. In their study, only white women ≥ 45 years of age demonstrated a significant decline in growth rate, − 19.57% (95% CI, − 30.48 to 6.94) per 6 months when compared white women < 35 years old (P = 0.04). For black women ≥ 45 years of age, there was no significant difference in fibroid growth rate compared to black women who were < 35 years old, − 3.38% (95% CI, 17.31 12.90) (P = 0.67). Furthermore, fibroids from white women ≥ 45 years of age grew more slowly than those from older black of the same age (2% vs. 15% growth rate, respectively) [9].

of age, there was no significant difference in fibroid growth rate compared to black women who were < 35 years old, − 3.38% (95% CI, 17.31 12.90) (P = 0.67). Furthermore, fibroids from white women ≥ 45 years of age grew more slowly than those from older black of the same age (2% vs. 15% growth rate, respectively) [9]. Number of Fibroids Peddada et al. found that women with solitary fibroids appeared to have a faster growth rate, 33.75% (95% CI, 7.58–61.17) per 6 months when compared to women with multiple fibroids (P = 0.06) [9]. However, only 6.9% (5/72) of women had solitary fibroids in this study. The influence of the number of fibroids on growth rates was not commented on other authors [10••, 11]. Radiological Life Cycle of Fibroids in the Gravid State The incidence of fibroids in pregnancy is estimated at 0.1 to 10.7% [13, 14, 15••]. The conventional belief is that fibroids grow in a consistent manner during pregnancy due to a number of factors: the increasing steroid hormone levels, hypertrophy and oedema and stretching of the myometrium. Conversely, expansion of the amniotic cavity and ischemia-driven degenerative changes may lead to shrinkage or disappearance of fibroids. A recent systematic review concluded that fibroids do appear to have a linear growth pattern in the first trimester of pregnancy. However, the growth patterns in later gestations remain contentious with inconsistent evidence [16]. There are also increasing reports that fibroids often remain unchanged or even decrease in size during pregnancy [17, 18].

ded that fibroids do appear to have a linear growth pattern in the first trimester of pregnancy. However, the growth patterns in later gestations remain contentious with inconsistent evidence [16]. There are also increasing reports that fibroids often remain unchanged or even decrease in size during pregnancy [17, 18]. Non-linear Growth of Fibroids in Pregnancy Ciavenetti et al. performed prospective scans on 109 women at four intervals, before pregnancy (scan 1), 7–8 weeks (scan2), 10–13 weeks (scan 3) and 20–22 weeks (scan 4) [15••]. The authors found that of the fibroids measuring 10 to 50 mm at scan 1, 76.9% (110/143) increased in volume between scan 1 and 2, 72.0% (103/143) increased between scan 2 and 3 and 40.6% (58/143) increased between scan 3 and 4. The median growth rate was 122% between scan 1 and 2, 108% between scan 2 and 3 and 25% between scan 3 and 4. The authors concluded that most fibroids increase in size in the first 7 weeks of pregnancy; however, growth during pregnancy was non-linear as growth in the later trimesters was slower [15••].

en scan 3 and 4. The median growth rate was 122% between scan 1 and 2, 108% between scan 2 and 3 and 25% between scan 3 and 4. The authors concluded that most fibroids increase in size in the first 7 weeks of pregnancy; however, growth during pregnancy was non-linear as growth in the later trimesters was slower [15••]. Further suggestions of non-linear fibroid growth in pregnancy was found by De Vivo et al. who performed three ultrasound scans at 12 (scan 1), 21 (scan 2) and 33 weeks (scan 3) gestation [13]. The mean (± standard deviation) fibroid volume was 17.04 cm3 (± 29.1) at scan 1, 38.8cm3 (± 67.8) at scan 2 and 42.1 cm3 (± 69.0) at scan 3 (P < 0.05). The authors concluded that fibroids increased in size throughout pregnancy but growth rates were significantly greater in the first half of pregnancy as compared with the second half of pregnancy [13]. In support of rapid fibroid growth in early pregnancy, Benaglia et al. compared fibroid growth rates between a pregnant cohort (n = 46) and a non-pregnant control cohort (n = 41), following in vitro fertilisation (IVF) treatment [19]. Ultrasound assessments for fibroid volume and diameter were performed at the start of IVF stimulation and 5 weeks after embryo transfer in both cohorts. The median change of the diameter of all fibroids in pregnant women was + 34% and in non-pregnant women was + 2% (P < 0.001). The median change in volume of all fibroids in pregnant women was + 140% and in non-pregnant women 0% (P < 0.001). The authors concluded that fibroids more than double in size within 7 weeks of pregnancy [19].

ge of the diameter of all fibroids in pregnant women was + 34% and in non-pregnant women was + 2% (P < 0.001). The median change in volume of all fibroids in pregnant women was + 140% and in non-pregnant women 0% (P < 0.001). The authors concluded that fibroids more than double in size within 7 weeks of pregnancy [19]. Strobelt et al. performed regular ultrasound fibroid assessments on 134 pregnant women from 16 weeks gestation until term [20]. The authors found that overall, fibroid growth was only found in 14.9% (20/134) of pregnant women and the remaining 85.1% of women had no change in fibroid size after 16 weeks of pregnancy [20]. Factors Affecting Fibroid Growth in Pregnancy Starting Size of Fibroid Similar to non-pregnant women, the initial size of fibroid appears to influence growth rates. Ciavenetti et al. found in a logistic regression analysis that the odds ratio (OR) for increase in fibroid volume between scan 1 and 2 was 0.97 (95% CI 0.95–1.0) (P = 0 .04), between scan 2 and 3 [OR 0.93 (95% CI 0.89–0.97)] (P < .001) and between scan 3 and 4 [OR = 0.95 (95% CI 0.92–0.99)] (P < .001) [15]. The authors concluded that fibroids with smaller starting volumes were more likely to increase in size than larger ones during pregnancy [15].

d 2 was 0.97 (95% CI 0.95–1.0) (P = 0 .04), between scan 2 and 3 [OR 0.93 (95% CI 0.89–0.97)] (P < .001) and between scan 3 and 4 [OR = 0.95 (95% CI 0.92–0.99)] (P < .001) [15]. The authors concluded that fibroids with smaller starting volumes were more likely to increase in size than larger ones during pregnancy [15]. Strobelt et al. found that after 16 weeks gestation, 75.0% (69/92) fibroids less than 5 cm in diameter resolved (could not be identified or distinguished from surrounding myometrium) during pregnancy, compared with only 35.7% (15/42) of fibroids greater than 5 cm in diameter (P < 0.001) [20]. The authors concluded that this may be a result of crowding of the pelvis in advanced gestations or due to changes in the fibroid echogenicity and stretching of the myometrium in advanced gestations. Other authors had found that there was no correlation between initial fibroid volume and fibroid growth during pregnancy [13].

Strobelt et al. found that after 16 weeks gestation, 75.0% (69/92) fibroids less than 5 cm in diameter resolved (could not be identified or distinguished from surrounding myometrium) during pregnancy, compared with only 35.7% (15/42) of fibroids greater than 5 cm in diameter (P < 0.001) [20]. The authors concluded that this may be a result of crowding of the pelvis in advanced gestations or due to changes in the fibroid echogenicity and stretching of the myometrium in advanced gestations. Other authors had found that there was no correlation between initial fibroid volume and fibroid growth during pregnancy [13]. Laughlin et al. performed a cohort study, using transvaginal ultrasound assessment in early pregnancy and at 3–6 months post-partum on 171 women, with a single fibroid [21]. 36.0% (62/171) of fibroids had resolved beyond the authors’ ability to detect them at the post-partum ultrasound examination. 109/171 fibroids remained detectable on the post-partum ultrasound. However, 79.0% of these fibroids were significantly smaller than on the original early pregnancy scan (median reduction in diameter of 0.5 cm (P < 0.001)). Infarction of uterine fibroids secondary to uterine involution in the post-partum period is thought to facilitate this complete regression of small fibroids in the post-partum period [22]. This is further supported by epidemiologic data that suggest that parity is protective against incidence and further development of fibroids [23].

terine fibroids secondary to uterine involution in the post-partum period is thought to facilitate this complete regression of small fibroids in the post-partum period [22]. This is further supported by epidemiologic data that suggest that parity is protective against incidence and further development of fibroids [23]. Number of Fibroids Strobelt et al. reported that women with solitary fibroids were more likely to resolve when compared to women with multiple fibroids, 73.7% (70/95) and 35.9% (14/ 39), respectively (P < 0.001) [20]. There was no correlation with pain which may indicate fibroid degeneration/necrosis during pregnancy that could perhaps explain the apparent disappearance. Other studies did not replicate these findings [13, 15••, 19].

Number of Fibroids Strobelt et al. reported that women with solitary fibroids were more likely to resolve when compared to women with multiple fibroids, 73.7% (70/95) and 35.9% (14/ 39), respectively (P < 0.001) [20]. There was no correlation with pain which may indicate fibroid degeneration/necrosis during pregnancy that could perhaps explain the apparent disappearance. Other studies did not replicate these findings [13, 15••, 19]. Conclusions Despite fibroids being the most common uterine tumour, their life cycle remains poorly understood. The original teaching dictates that fibroids grow in a linear pattern, starting in puberty and continues through life until the hormonal milieu of the woman changes dramatically at menopause when shrinkage was expected. A review of current evidence suggests that fibroid growth is variable and can range from 18 to 120% per year. Fibroids also undergo spontaneous regression, growth and shrinkage spurts outside of menopause, despite a stable hormonal environment. There is conflicting evidence regarding factors that affect fibroid growth. Many studies have investigated the impact of size at presentation; however, there is no agreement as to whether smaller or larger fibroids grew faster. With regard to the position of fibroids in relation to the uterine cavity, submucous fibroids were least likely to increase in size. Whilst reduced growth rates have been reported with increasing age, this trend was not statistically significant especially in white women.

whether smaller or larger fibroids grew faster. With regard to the position of fibroids in relation to the uterine cavity, submucous fibroids were least likely to increase in size. Whilst reduced growth rates have been reported with increasing age, this trend was not statistically significant especially in white women. Recent evidence would suggest a non-linear growth pattern during pregnancy with during pregnancy with significantly significantly increased growth rates in the first half of pregnancy, especially in the first trimester. The starting volumes of fibroids have been found to affect fibroid growth; however, there was no consistent evidence of their effects. Solitary fibroids in pregnancy were more likely to resolve when compared to multiple fibroids. It would be ideal if we were able to counsel women regarding the specific timing of various medical and surgical interventions and their outcomes in pregnancy. However, this would require further research into the life cycle of fibroids and further understanding of growth variables, both in the inter-gravid and gravid states. Recent evidence has challenged traditional views and provided valuable insight into the variability of the radiological life cycle of fibroids, but more evidence is needed so that gynaecological and obstetric treatments can be optimised. This article is part of the Topical Collection on Uterine Fibroids

It would be ideal if we were able to counsel women regarding the specific timing of various medical and surgical interventions and their outcomes in pregnancy. However, this would require further research into the life cycle of fibroids and further understanding of growth variables, both in the inter-gravid and gravid states. Recent evidence has challenged traditional views and provided valuable insight into the variability of the radiological life cycle of fibroids, but more evidence is needed so that gynaecological and obstetric treatments can be optimised. This article is part of the Topical Collection on Uterine Fibroids Compliance with Ethical Standards Conflict of Interest Wee Liak Hoo is a co-investigator for LIBERTY 1—An International Phase 3 Randomized, Double-Blind, Placebo-Controlled Efficacy and Safety Study to Evaluate Relugolix Co-Administered with and without Low-Dose Estradiol and Norethindrone Acetate in Women with Heavy Menstrual Bleeding Associated with Uterine Fibroids. Srirupa Ghosh, Joel Naftalin and Rachel Imrie declare no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

Introduction Endometriosis is a common estrogen (E)-dependent gynecological disorder, characterized by presence of tissue resembling endometrium tissue outside the uterine cavity. Typical locations for the ectopic deposits are on the pelvic peritoneum, the ovaries, uterosacral ligaments, pouch of Douglas, and the rectovaginal septum. Endometriosis has been associated with chronic pelvic pain, reduced fertility, and severe dysmenorrhea [1, 2]. Definitive diagnosis can only be established through surgery, and, though other classifications exist, the disease is most commonly staged using the revised American Fertility Society (rAFS) classification, based on the total surface size of the lesions, presence of adhesions, and ovarian lesions [3]. Because endometriosis is E-dependent, it occurs almost exclusively in women of reproductive age [4]. Because of the need for surgical diagnosis, the exact population prevalence is unknown. Based on community prevalence rates of pelvic pain and infertility, the prevalence of endometriosis is estimated to be about 5–10 % in premenopausal women [5], increasing to 35–50 % in women undergoing laparoscopy for pelvic pain and infertility [1, 6].

or surgical diagnosis, the exact population prevalence is unknown. Based on community prevalence rates of pelvic pain and infertility, the prevalence of endometriosis is estimated to be about 5–10 % in premenopausal women [5], increasing to 35–50 % in women undergoing laparoscopy for pelvic pain and infertility [1, 6]. The etiology of endometriosis remains unclear. The most widely accepted mechanism for development of the peritoneal endometriotic lesions is via retrograde menstruation [7]. However, menstrual debris is present in the peritoneal cavity of up to 90 % of the menstruating women [8]. Possible explanations for adhesion and growth of endometriotic lesions in only some women include increased exposure to menstrual debris (e.g., increased menstrual flow, shorter cycle length), abnormal eutopic endometrium, altered peritoneal environment, reduced immune surveillance, and increased angiogenic capacity [9–11]. It is possible that several, if not all, of these factors play a role to an extent, and/or that specific subtypes of disease are due to specific underlying biological pathways; research continues to elucidate these mechanisms.

eritoneal environment, reduced immune surveillance, and increased angiogenic capacity [9–11]. It is possible that several, if not all, of these factors play a role to an extent, and/or that specific subtypes of disease are due to specific underlying biological pathways; research continues to elucidate these mechanisms. One approach to study the underlying biological pathways leading to a complex disease such as endometriosis (the development of which is determined by multiple genetic and environmental factors) is to study the effect of genetic variants on disease causation. The involvement of genetic factors in the development of endometriosis is supported by different studies [12–14]. Twin studies have shown increased concordance in monozygotic twins when compared to dizygotic twins [15, 16]; the largest such study carried out to date among Australian 3,096 female twins concluded that about 51 % of the variation in endometriosis risk is heritable [16]. In addition to human studies, familial aggregation of spontaneous (i.e., non-induced) endometriosis also has been shown in nonhuman primates such as the rhesus macaque [17].

study carried out to date among Australian 3,096 female twins concluded that about 51 % of the variation in endometriosis risk is heritable [16]. In addition to human studies, familial aggregation of spontaneous (i.e., non-induced) endometriosis also has been shown in nonhuman primates such as the rhesus macaque [17]. Study designs to search for genes underlying endometriosis can be separated into hypothesis-based and hypothesis-free approaches. Hypothesis-based “candidate gene” studies typically rely on prior biological hypotheses/knowledge of the disease or of the approximate genomic location of a disease-predisposing variant derived from a hypothesis-free study. Hypothesis-free studies search the entire genome to identify disease-predisposing variants, without knowledge of their functional relevance. This review summarizes the evidence from the most recent studies investigating the genetic variation contributing to endometriosis. Of particular focus will be the results from large-scale collaborative genome-wide approaches, which have started to provide new insights into potential pathways leading to endometriosis.

This review summarizes the evidence from the most recent studies investigating the genetic variation contributing to endometriosis. Of particular focus will be the results from large-scale collaborative genome-wide approaches, which have started to provide new insights into potential pathways leading to endometriosis. Hypothesis-Based Research: Candidate Gene Association Studies Historically, the search of genes contributing to susceptibility of many complex diseases such as endometriosis began with hypothesis-driven “candidate gene association” studies (CGASs). Although hypothesis-free approaches have since taken off, candidate gene studies based on a biological “hunch” alone are still commonplace. CGASs are based on the a priori selection of genes with inferred biological function and association of variants in these genes with disease risk. With limited budgets and faced with over 20,000 genes in the human genome, the approach seems an attractive one. However, CGASs are inherently limited by current knowledge of biological mechanisms underlying the studied phenotype, which is attenuated in diseases where the underlying mechanisms are not very well understood, such as in endometriosis. Furthermore, to fully test the involvement of a biological pathway, one would need to investigate all genes making up the pathway (as well as factors regulating their expression), in a large sample of cases and controls; a costly approach that has, to our knowledge, never been adopted. Therefore, the probability of success of a CGAS as commonly conducted, based on the testing of a few, selected, variants in a handful of genes, is extremely low. This probability may be greatly improved if evidence of the likely genomic location of a disease-predisposing variant, derived from a whole genome linkage study for example, is included, although this approach has its own drawbacks (described later in this article).

ed, variants in a handful of genes, is extremely low. This probability may be greatly improved if evidence of the likely genomic location of a disease-predisposing variant, derived from a whole genome linkage study for example, is included, although this approach has its own drawbacks (described later in this article). Association studies these days typically focus on the most abundant genetic variants in the genome: single nucleotide polymorphisms (SNPs), which comprise over 90 % of all common genetic variation in the human genome [18]. SNPs are changes to the DNA at one nucleotide base pair, and can have two identities, or “alleles.” Association studies compare the frequency of the alleles of a genetic variant between cases and controls, and can be of direct and indirect design. Direct association studies aim to test specific genetic variants by genotyping these, whereas indirect association studies type a set of common (population allele frequency > 0.05) SNPs carefully selected on the basis of local genomic “linkage disequilibrium” (LD). LD is the nonrandom association of genetic variants in a population. This phenomenon means, in simple terms, that an SNP can predict the status of a genetic variant nearby because of common ancestry of that particular genomic segment. The International HapMap Project [19] has mapped LD patterns across the human genome in a number of ethnic populations, which has enabled the investigation of whole sections of the genome for their association with many common complex diseases; by testing those SNPs that are highly predictive of the status of other SNPs in the region (termed “tagSNPs”), all common genetic variation present in the population can be tested with very high coverage. Genome-wide association studies (GWASs) are of indirect design; they utilize LD by typically genotyping ~500,000–1,000,000 SNPs that tag a large proportion of the roughly 10 million common SNPs present in the human genome. Most CGASs of endometriosis have been of direct design (i.e., a limited number of specific genetic variants were genotyped and tested for association, although indirect tag approaches have also been used [20]). For a detailed review of the design of CGASs and GWASs and their underlying principles, see Zondervan & Cardon [21].

ASs of endometriosis have been of direct design (i.e., a limited number of specific genetic variants were genotyped and tested for association, although indirect tag approaches have also been used [20]). For a detailed review of the design of CGASs and GWASs and their underlying principles, see Zondervan & Cardon [21]. We previously published a review of CGASs published up to April 1, 2008 [20]. For this paper, we identified CGASs of endometriosis published from April 1, 2008 to April 1, 2012, conducting a systematic literature search in PubMed for English language publications, using the terms “endometriosis” with “genetics” or “genes” or “polymorphisms.” An overview of these recent CGASs is presented in Table 1. Variants from 73 candidate genes were tested (Table 1), involved in sex steroid biosynthesis and signaling pathways, adhesion molecules and matrix enzymes, immunological mechanisms, inflammatory pathways, estradiol metabolism, growth factor systems, cell cycle regulation, oncogenes, apoptosis, and angiogenic factors [22].Table 1 Candidate gene association studies of endometriosis published from April 2008 to April 2012, and total evidence for these from all studies published until 2012

mechanisms, inflammatory pathways, estradiol metabolism, growth factor systems, cell cycle regulation, oncogenes, apoptosis, and angiogenic factors [22].Table 1 Candidate gene association studies of endometriosis published from April 2008 to April 2012, and total evidence for these from all studies published until 2012 Candidate gene** Chr Number of studies with association 2008–2012* Significant (n cases, n controls) Nonsignificant (n cases, n controls) #Sign/Total up to 2012, n/n*** Adhesion molecules and matrix enzymes CDH1 16q22 Indian (715, 500) [73] – 2/2 Japanese (511, 498) [74] ICAM1 19p13 Iranian (84, 120) [75] Korean (390, 351) [76] 2/5 MMP2 16q12 Estonian (150, 199) [77] – 2/3 MMP9 20q11-13 Estonian (150, 199) [77] – 1/2 MUC2 11p15 Taiwainese (195, 196) [78] – 1/1 MUC4 3q29 Taiwainese (140, 150) [79] – 1/1 Apoptosis, cell-cycle, DNA repair, and oncogenes AKT1 14q32 – Indian (30, 30) [80] 0/1 APE1 14q11-12 – Turkish (52, 101) [81] 0/1 CDKN1A 6p21 – Taiwainese (180, 330) [82] 0/2 CDKN1B 12p13 Brazilian (104, 109) [83] – 1/1 HOGG1 3p26 – Turkish (52, 101) [81] 0/1 PI3KCA 3q26 – Indian (30, 30) [80] 0/1 TP53 17p13 Mexican (151, 204) [84] Brazilian (19, 19) [86] 4/9 Caucasian, Asian (749, 857)a [85•], Taiwainese (180, 330) [82] XPD 19q13 – Turkish (52, 101) [81] 0/1 XPG 13q33 – Turkish (52, 101) [81] 0/1 XRCC1 19q13 – Turkish (52, 101) [81] 0/1 XRCC3 14q32 – Turkish (52, 101) [81] 0/1 Cytokines/inflammation C3 19p13 Puerto Rican (214, 147) [87] – 1/1 CTLA4 2q33 – Brazilian (177, 172) [88] 0/2 DR4/5 8p21 – Korean (138, 214) [89] 0/1 FCRL3 1q21-22 Brazilian (167, 167) [90] – 0/1 IFNG 12q24 Korean (622, 442) [91] – 3/3 Indian (302, 324) [92] IL10 1q31-32 Danish (100, 358) [93] – 5/6 Taiwainese (196, 397) [94] Chinese (214, 160) [95] IL16 15q26 Chinese (230, 203) [96] – 1/1 IL18 11q22 Turkish (135, 84) [97] – 1/2 IL1B 2q13-21 – Turkish (118, 78) [98] 0/4 Taiwainese (196, 397) [94] IL2RB 22q13 – Korean (237, 164) [99] 1/2 IL6 7p21-15 – Taiwainese (196, 397) [94] 0/5 Korean (390, 351) [76] LOXL4 10q24 Puerto Rican (214, 147) [87] – 1/1 NFKB1 4q24 Chinese (206, 365) [100] – 1/1 PTGS2 1q25 – Taiwainese (196, 397) [94] 0/1 PTPN22 1q13 Brazilian (140,180) [101] Caucasian (789, 351) [103] 3/4 Polish (171, 310) [102] TCRB 7q34 – Italian (70, 120) [104] 0/1 TNFA 6p21 Indian (245, 85) [105] Korean (105, 101) [106] 1/2 TNFR1 12p13 – Korean (105, 101) [106] 0/1 TNFR2 1p36 – Korean (105, 101) [106] 0/2 TNFRSF11B 8q24 – Korean (138, 214) [89] 0/1 TNFSF13B 13q32-34 Dutch (87, 107) [107] –

sian (789, 351) [103] 3/4 Polish (171, 310) [102] TCRB 7q34 – Italian (70, 120) [104] 0/1 TNFA 6p21 Indian (245, 85) [105] Korean (105, 101) [106] 1/2 TNFR1 12p13 – Korean (105, 101) [106] 0/1 TNFR2 1p36 – Korean (105, 101) [106] 0/2 TNFRSF11B 8q24 – Korean (138, 214) [89] 0/1 TNFSF13B 13q32-34 Dutch (87, 107) [107] – 1/1 TRAIL 8p22-21 – Korean (138, 214) [89] 0/1 Estradiol metabolism COMT 22q11 – Brazilian (198, 168) [108] 0/5 Caucasian (256, 567) [109] HSD17B1 17q11-21 Estonian (150, 199) [110] Caucasian (256, 567) [109] 3/4 HSD17B2 16q24 – Caucasian (256, 567) [109] 0/1 Growth factor systems BNC2 9p22.2 – Caucasian (789, 351) [111] 0/1 FGF1 5q31 – Chinese (421, 421) [112] 0/1 FGF2 4q26 Chinese (421, 421) [112] – 1/1 IGF1 12q23 – Korean (128, 108) [113] 0/1 IGF1R 15q26 – Korean (128, 108) [113] 0/1 IGF2 11p15 Korean (128, 108) [113] – 1/1 TGFB1 19q13 – Korean (485, 352) [114] 1/4 Korean (131, 107) [115] VEGF 6p21-12 Turkish (98, 94) [116] Australian (958, 959)c [26•] 10/12 Chinese (344, 360) [117] Caucasian (186, 180) [118] Estonian (150, 199) [110] Japanese, Australian, Spanish, Chinese, Estonian (1785, 1879)b [25•] Hormone receptors ESR1 6q24-27 Chinese (214, 160) [95] Caucasian (256, 567) [109] 7/12 Estonian (150, 199) [110] ESR2 14q21-22 Brazilian (108, 210) [119] Caucasian (256, 567) [109] 4/6 Brazilian (136, 209) [120] Estonian (150, 199) [110] PGR 11q22-23 Caucasian (348, 5820)d [121•] Caucasian (256, 567) [109] 6/9 Human leukocyte antigen system and immune components CCL21 9p13 Caucasian (789, 351) [103] – 1/1 CD40 20q12-13 – Caucasian (789, 351) [103] 0/1 HLA region 6p21 – Japanese (89, 136) [122] 0/1 HLA-DRB1 6p21 Caucasian (789, 351) [103] – 1/2 IRF5 7q32 – Caucasian (789, 351) [103] 0/1 STAT4 2q32 – Caucasian (789, 351) [103] 0/1 TRAF1-C5 9q33-34 – Caucasian (789, 351) [103] 0/1 VDR 12q13 – Brazilian (132, 195) [123] 0/1 Other enzymes and metabolic systems eNOS 7q36 Korean (299, 459) [124] Indian (232, 210) [125] 2/4 IRS2 13q34 Turkish (135, 135) [126] – 1/1 LHB 19q13 Brazilian (110, 209) [127] – 1/3 SERPINE 7q21-22 Brazilian (140, 148) [128] Italian (368, 329) [129] 2/3 Steroid-synthesizing enzymes, detoxifying enzymes, and receptors CYP17A1 10q24 Turkish (46, 39) [130] Caucasian (256, 567) [109] 1/9 Italian (104, 86) [131] CYP19A1 15q21 Caucasian (256, 567) [109] – 4/6 Italian (104, 86) [131] CYP1A1 15q24 – Caucasian (256, 567) [109] 1/7 CYP1A2 15q24 – Caucasian (256, 567) [109] 0/1 CYP2C19 10q24 Turkish (50, 50) [132] Turkish (46, 39) [130] 1/2 GSTM1 1p13 Ira

Turkish (46, 39) [130] Caucasian (256, 567) [109] 1/9 Italian (104, 86) [131] CYP19A1 15q21 Caucasian (256, 567) [109] – 4/6 Italian (104, 86) [131] CYP1A1 15q24 – Caucasian (256, 567) [109] 1/7 CYP1A2 15q24 – Caucasian (256, 567) [109] 0/1 CYP2C19 10q24 Turkish (50, 50) [132] Turkish (46, 39) [130] 1/2 GSTM1 1p13 Ira nian (120, 200) [133] – 7/13 GSTP1 11q13 – Korean (260, 164) [134] 1/3 PPARG 3p25 Korean (446, 427) [135] – 3/3 Chr chromosome *When more than one polymorphism was investigated in a study, the result is indicated as significant if one or more of the variants were reported as significant by the authors. ** Candidate gene categories are as suggested by Falconer et al. [22]. *** Total number of candidate studies is summed together with data from our review in 2008 [20] aLiterature-based meta-analysis of P53 codon 72 polymorphism from six published studies of Asian and Caucasian ancestry. The study showed significant association of the polymorphism with endometriosis only in the Asian patients [85•] bLiterature-based meta-analysis of five polymorphisms in the VEGF gene from 11 studies from Chinese, Indian, Korean, Japanese, Spanish, Turkish, Estonian, and Australian patients. The study identified one significant polymorphism with endometriosis from the overall meta-analysis [25•]

aLiterature-based meta-analysis of P53 codon 72 polymorphism from six published studies of Asian and Caucasian ancestry. The study showed significant association of the polymorphism with endometriosis only in the Asian patients [85•] bLiterature-based meta-analysis of five polymorphisms in the VEGF gene from 11 studies from Chinese, Indian, Korean, Japanese, Spanish, Turkish, Estonian, and Australian patients. The study identified one significant polymorphism with endometriosis from the overall meta-analysis [25•] cGenotyped and tested the association of four VEGF polymorphisms with endometriosis in a large Australian sample. Then, performed a meta-analysis with four additional published studies and showed no evidence for association neither in the genotyped association analysis they have performed, nor in the meta-analysis for any of the four VEGF polymorphisms [26•] dAn international ovarian cancer consortium, including eight ovarian cancer case-controls studies from Australia, Europe, and the United States, investigated the association of polymorphisms in PGR with endometriosis in 348 endometriosis patients and 5812 control patients [121•]

cGenotyped and tested the association of four VEGF polymorphisms with endometriosis in a large Australian sample. Then, performed a meta-analysis with four additional published studies and showed no evidence for association neither in the genotyped association analysis they have performed, nor in the meta-analysis for any of the four VEGF polymorphisms [26•] dAn international ovarian cancer consortium, including eight ovarian cancer case-controls studies from Australia, Europe, and the United States, investigated the association of polymorphisms in PGR with endometriosis in 348 endometriosis patients and 5812 control patients [121•] For evidence of association to be credible, it is paramount that the observed associations should be replicated in an independent sample from the same underlying ethnic population [21, 23]. Generally, many reported associations do not show significant association in independent studies. This can be due to many factors, such as differences in disease definition, inadequate control selection, low coverage of the candidate genes, small sample size of the initial dataset (creating a false-positive result) or of the replication dataset (creating a false-negative result), or population differences between the discovery and the replication datasets. Moreover, to avoid failure to replicate due to Winner’s curse (the statistical phenomenon in which the effect size [the odds ratio] of the association in the discovery dataset is larger than in any subsequent replication dataset), the replication studies need to be larger in sample size and have greater power than the discovery study to detect the effect of the putative association [24].

tistical phenomenon in which the effect size [the odds ratio] of the association in the discovery dataset is larger than in any subsequent replication dataset), the replication studies need to be larger in sample size and have greater power than the discovery study to detect the effect of the putative association [24]. As can be seen from the results in Table 1, no candidate gene has been robustly associated with endometriosis across studies. One of the most frequently investigated and plausible genes, encoding for vascular endothelial growth factor (VEGF), probably shows the most suggestive evidence of association. A literature-based meta-analysis of five polymorphisms from 11 studies including 1785 cases and 1879 control patients of Chinese, Indian, Korean, Japanese, Spanish, Turkish, Estonian, and Australian origin suggested one significantly associated polymorphism (+936T/C; TT+TC vs CC: P = 0.02) [25•], although no such association was found in a large study of 958 Australian cases and 959 control patients (P = 0.31) [26•]. Similarly, our previous review of 76 studies published until April 1, 2008 concluded that none provided clear support for any genetic variant to be robustly associated with endometriosis, and that some reported results may represent true associations, but that given the small effect sizes expected, large replication datasets or meta-analyses are required. In a large GWAS of 3,194 cases and 7,060 control patients of European ancestry, all candidate genes from the 2008 Montgomery et al. review [20] were investigated for nominal evidence of association [27•]; the only gene with a nominal P < 10−3 for SNPs in the GWAS data was the gene encoding the progesterone receptor (PGR) on chromosome 11, but the result for the SNP in this gene was not significant in the replication stage. This does not necessarily mean that none of these candidate genes are involved in the causation of (subtypes) of endometriosis; it also could mean that even in a large study of all types of endometriosis, power is lacking to detect their effect.

ult for the SNP in this gene was not significant in the replication stage. This does not necessarily mean that none of these candidate genes are involved in the causation of (subtypes) of endometriosis; it also could mean that even in a large study of all types of endometriosis, power is lacking to detect their effect. As can be seen in Table 1, the recent CGASs were conducted in ethnically diverse populations. Because different populations may have different genetic contributions to a disease, and LD patterns may vary between ethnic populations, a genetic variant identified as causal in one population may not be acting as a susceptibility variant in another. Therefore, care should be taking in interpreting and comparing findings in terms of “replication.”

ay have different genetic contributions to a disease, and LD patterns may vary between ethnic populations, a genetic variant identified as causal in one population may not be acting as a susceptibility variant in another. Therefore, care should be taking in interpreting and comparing findings in terms of “replication.” Hypothesis-Free Research: Whole Genome Linkage Studies and Their Follow-up One of the two hypothesis-free approaches to investigate underlying genetic etiology of a disease is linkage mapping, which adopts genetic investigation of families with multiple affected individuals. This methodology became popular in the 1980s after the discovery of highly variable genetic markers (“microsatellites”), and works by 1) genotyping about 400 microsatellites across the genome; 2) comparing whether the allelic status of these variants are shared between affected people in a family; 3) summing this evidence of sharing across multiple families; 4) calculating whether the amount of sharing is more than expected by chance based on simple Mendelian laws of inheritance; and 5) identifying those genomic regions where excess sharing is statistically significant. The linkage approach was highly successful in identifying genetic variants responsible for rare, monogenic disorders. Such disorders show clear Mendelian segregation patterns through families [28, 29], and the presence of the causal genetic variant determines whether or not the disease in question will develop. The genetic causes of many of these monogenic diseases could be ‘mapped’ by investigating a handful of extended families with many affected members.

ar Mendelian segregation patterns through families [28, 29], and the presence of the causal genetic variant determines whether or not the disease in question will develop. The genetic causes of many of these monogenic diseases could be ‘mapped’ by investigating a handful of extended families with many affected members. Mendelian disorders are very different from complex diseases such as endometriosis, in which one genetic variant only carries susceptibility to disease (not 100 % risk). Many more families with multiple affected women are required to conduct a linkage study of endometriosis. Linkage studies are fundamentally different from association studies; the two approaches are complementary. In contrast to association studies, which focus on SNPs common in the general population, linkage studies are designed to detect disease-causing variants responsible for disease in a family, but that are otherwise rare in the general population. Moreover, because linkage studies use recombination events in families, the resolution of the approach is very large compared to association studies: a significantly linked region typically extends across 10–50 Mb and contains hundreds of genes, in contrast to the high resolution of association studies which (depending on the local LD structure) are able to pinpoint a signal to within 10–500 Kb.

solution of the approach is very large compared to association studies: a significantly linked region typically extends across 10–50 Mb and contains hundreds of genes, in contrast to the high resolution of association studies which (depending on the local LD structure) are able to pinpoint a signal to within 10–500 Kb. The largest genome linkage scan to date was conducted by the International Endogene Study (consisting of 1,176 affected sib-pairs from Australian and United Kingdom families), which identified a region of significant linkage on chromosome 10q26 [30]. In a subsequent analysis involving a subset of 248 families with 3 or more affected members, a second region was found on chromosome 7p13-15 likely due to one or more rare variants following near-Mendelian patterns of inheritance [31]. In a recent study, the linkage peak on chromosome 10q26 region was extensively genotyped using 11,984 SNPs in 1,144 familial cases and 1,190 control patients. The study identified three independent signals in the region, at 96.59 Mb (rs11592737), 105.63 Mb (rs1253130), and 124.25 Mb (rs2250804), with nominally significant evidence of association. However, only rs11592737 in the cytochrome P450 subfamily C (CYP2C19) gene replicated (P = 0.04) in an independent sample of 2,079 cases and 7,060 control patients [32•]. CYP2C19 is a plausible candidate for endometriosis because it is involved in the metabolism of drugs and E including conversion of E2 to estrone (E1), and the production of E1 and E2 2a- and 16a-hydroxylation metabolites [33, 34]. Future studies should follow up by investigating novel rare genetic variants in this region and conducting gene expression analyses to further understand the role of CYP2C19 in endometriosis.

nd E including conversion of E2 to estrone (E1), and the production of E1 and E2 2a- and 16a-hydroxylation metabolites [33, 34]. Future studies should follow up by investigating novel rare genetic variants in this region and conducting gene expression analyses to further understand the role of CYP2C19 in endometriosis. As a first approach to follow-up the significant linkage peak on chromosome 7 region, the coding regions and upstream regulatory regions of three promising candidate genes (INHBA, SFRP4, and HOXA10 with known roles in endometrial development) located within or close to the linkage signal were sequenced to identify potential causal polymorphisms. Sequencing, rather than genotyping, was chosen because the signal was likely due to one or more novel rare variants not present on any genotyping array. Sequencing was conducted in 47 cases from the 15 families contributing most to the linkage signal, and 11 variants were found. The minor allele frequencies (MAFs) of observed variants were compared with MAFs from two publicly available reference populations of European ancestry: 60 individuals in HapMap and 150 individuals in the 1000 Genomes Project. Five of the 11 variants were common (MAF > 0.05); the remaining six variants were rare and unlikely to be individually or cumulatively responsible for the linkage signal. These results indicate that the coding regions of these three genes do not harbor rare mutations responsible for linkage to endometriosis in these families [35]. However, this does not exclude the possibility that variants responsible for the linkage signal exist in noncoding regions of these three genes, or that they are located in other genes in this region.

ions of these three genes do not harbor rare mutations responsible for linkage to endometriosis in these families [35]. However, this does not exclude the possibility that variants responsible for the linkage signal exist in noncoding regions of these three genes, or that they are located in other genes in this region. Hypothesis-Free Research: Genome-wide Association Studies GWASs are based on the premise that common diseases such as endometriosis are caused by genetic variants that are common themselves (Common disease-common variant hypothesis [CDCV]). The first and seminal papers using GWAS methodology successfully were published in 2005–2007 [36–38], and since then the method has taken off, resulting in the detection of many common genetic variants associated with complex diseases. The NHGRI (National Human Genome Research Institute) GWA Catalog (www.genome.gov/GWAStudies) [39] counted the number of published significant genome-wide associations (P ≤ 5 × 10−8) up to June 2011 at 1,449 for 237 traits. Key developments that enabled GWASs were (1) the documentation of hundreds of thousands of common SNPs, and the LD patterns between them in different populations in the human genome by the HapMap Consortium [19]; and 2) the development of high-throughput genomics platforms capable of genotyping over 1 million SNPs in one assay at ever decreasing cost.

Ss were (1) the documentation of hundreds of thousands of common SNPs, and the LD patterns between them in different populations in the human genome by the HapMap Consortium [19]; and 2) the development of high-throughput genomics platforms capable of genotyping over 1 million SNPs in one assay at ever decreasing cost. The first GWASs of endometriosis were published in 2010 and 2011: two in women of Japanese ancestry [40•, 41] and one in women of European ancestry [27•]. Only two [27•, 40•] reported genome-wide significant signals, which were replicated (Table 2). The first Japanese endometriosis GWAS included 1,423 cases and 1,318 control patients in the discovery sample, with cases a mixture of surgically confirmed and clinically diagnosed women. After the application of SNP quality control criteria, 460,945 SNPs were included in the discovery analysis. A replication analysis was conducted in an independent set of 484 cases and 3,974 control patients, in which the top 100 most significant SNPs from the discovery set were genotyped. A significant association for one SNP was found, rs10965235, in CDKN2BAS on chromosome 9p21 (P = 6.79 × 10−6, odds ratio (OR) = 1.56 [1.29–1.89], P = 4.89 × 10−4). Combining the discovery and replication samples provided a genome-wide significant result for rs10965235 (P = 5.57 × 10−12, OR = 1.44 [1.30–1.59]). Rs10965235 is located in intron 6 of CDKN2BAS, which encodes for the cyclin-dependent kinase inhibitor 2B antisense RNA (discussed further in this article). Uno et al. [40•] observed a second interesting potential association with rs16826658, only providing suggestive evidence, on chromosome 1p36 in a region close to WNT4 (combined datasets: P = 1.66 × 10−6, OR = 1.20 [1.11–1.29]). WNT4 encodes for wingless-type MMTV integration site family, member 4. Both these variants on chromosome 9p21 and 1p36 are novel susceptibility loci for endometriosis in the Japanese population [40•], and are discussed further in this article.Table 2 Summary of significant genetic variants discovered by GWASs of endometriosis

codes for wingless-type MMTV integration site family, member 4. Both these variants on chromosome 9p21 and 1p36 are novel susceptibility loci for endometriosis in the Japanese population [40•], and are discussed further in this article.Table 2 Summary of significant genetic variants discovered by GWASs of endometriosis Study Novel loci Top associated SNP Position Chr Population Sample size (Ca:Co), n:n Rep Biological explanation Uno et al. [40•] CDKN2BAS rs10965235 Genic 9 Japanese 1,423: 1,318 Yes Silencing of tumor suppressor gene, CDKN2A, by CDKN2BAS may have an important role in the development of endometriosis Painter et al. [27•] and Uno et al. [40•] WNT4 rs16826658 Genic 1 Japanese 1,423: 1,318 Yes Plays a role in development of the female reproductive tract, ovarian follicle development and steroidogenesis Caucasian 3,194: 7,060 Painter et al. [27•] mir-148a, NFE2L3, HOXA11 rs12700667 Intergenic 7 Caucasian 3,194: 7,060 Yes Variant associated with WHR as a measure of fat distribution. Fat distribution is also hormonally regulated; suggestive pleiotropic locus GWAS genome-wide association studies; SNP single nucleotide polymorphism; Ch chromosome; Ca cases; Co controls; Rep replication; WHR Waist to hip ratio; Ref reference

,194: 7,060 Yes Variant associated with WHR as a measure of fat distribution. Fat distribution is also hormonally regulated; suggestive pleiotropic locus GWAS genome-wide association studies; SNP single nucleotide polymorphism; Ch chromosome; Ca cases; Co controls; Rep replication; WHR Waist to hip ratio; Ref reference A second, independent, Japanese GWAS was published recently, comprising a meta-analysis of two GWAS on two case–control datasets. After quality control, 282,828 SNPs were tested for association in 696 endometriosis cases (not all surgically confirmed) and 825 control patients. Limited by their sample size, their aim was to detect potential common susceptibility loci with large effect on the disease. The study did not reveal any significant susceptibility loci for endometriosis, which is likely to be due to the small sample size of their dataset [41].

ally confirmed) and 825 control patients. Limited by their sample size, their aim was to detect potential common susceptibility loci with large effect on the disease. The study did not reveal any significant susceptibility loci for endometriosis, which is likely to be due to the small sample size of their dataset [41]. The largest GWAS on endometriosis to date was performed in women of European ancestry by the International EndoGene Consortium (IEC) [27•]. The study involved 3,194 surgically confirmed endometriosis cases and 7,060 control patients from Australia and the United Kingdom. Disease severity was assessed retrospectively from surgical records using the rAFS classification system and grouped into two phenotypes: stage A (stage I or II disease or some ovarian disease with a few adhesions; n = 1,686, 52.7 %) or stage B (stage III or IV disease; n = 1,364, 42.7 %), or unknown (n = 144, 4.6 %). After quality control, analyses were performed using 504,723 SNPs. Analyzing all SNPs combined, Painter et al. [27•] showed a significantly increased genetic loading among 1,364 cases with stage B endometriosis compared to 1,666 with stage A disease (proportion of endometriosis variation explained by common SNPs: 0.34 [SD: 0.04] vs 0.15 [SD: 0.15] respectively; P = 1.8 × 10−3). Because of this result, two GWA analyses were performed, using (1) 3,194 “all” endometriosis cases and (2) 1,364 stage B cases. For “all” endometriosis, the strongest signal observed was rs12700667 in an intergenic region on chromosome 7p15.2 (P = 2.6 × 10−7, OR = 1.22 [1.13–1.32]), which was considerably stronger when limiting cases to those with stage B endometriosis (P = 1.5 × 10−9, OR = 1.38 [1.24–1.53]). A second strong association was found for rs1250248 (2q35) within FN1 (P = 3.2 × 10−8). In the replication phase, 70 SNPs that produced nominal evidence of association with “all” or stage B were genotyped in an independent dataset comprising 2,392 self-reported surgically confirmed cases and 2,271 control patients from the Nurses’ Health Study I and II in the USA. The association on 7p15.2 with rs12700667 was replicated (P = 1.2 × 10−3, OR = 1.17 [1.06–1.28]). However, there was no evidence for replication of rs12540248 (FN1) or association with the remaining SNPs. Combined analysis of all 5,586 cases and 9,331 control patients from Australian, UK and US datasets further confirmed association between “all” endometriosis and 7p15.2 (rs12700667, P = 1.4 × 10−9, OR = 1.20 [1.13–1.27]).

there was no evidence for replication of rs12540248 (FN1) or association with the remaining SNPs. Combined analysis of all 5,586 cases and 9,331 control patients from Australian, UK and US datasets further confirmed association between “all” endometriosis and 7p15.2 (rs12700667, P = 1.4 × 10−9, OR = 1.20 [1.13–1.27]). Of note is that the estimated percentage of “all” endometriosis variance explained by rs12700667 was only 0.69 % of the estimated 51 % heritability. Rs12700667 is located in a roughly 924-kb intergenic region containing at least one noncoding RNA (AK057379), predicted transcripts and regulatory elements, and a micro RNA (miRNA [hsa-mir-148a]) about 88 kb upstream (discussed further in this article). Painter et al. [27•] also investigated the associations reported by Uno et al. [40•] in Japanese women. They found no evidence for association with rs10965235 on chromosome 9p21 (rs10965235 is monomorphic in individuals of European descent, reflecting the different genetic (ancestral) backgrounds between the studies), nor with any SNPs in LD with rs10965235. However, there was evidence for replication of rs7521902 on 1p36, close to WNT4 gene, with the strongest signal for stage B endometriosis (P = 7.5 × 10−6, OR = 1.25, 95 % CI 1.13–1.38); meta-analysis of the evidence from “any” endometriosis (as severity of disease was not assessed in the Japanese GWAS) combining the three GWAS datasets resulted in a genome-wide significant P value of 4.2 × 10−8 (OR = 1.19, 95 % CI 1.12–1.27).

al for stage B endometriosis (P = 7.5 × 10−6, OR = 1.25, 95 % CI 1.13–1.38); meta-analysis of the evidence from “any” endometriosis (as severity of disease was not assessed in the Japanese GWAS) combining the three GWAS datasets resulted in a genome-wide significant P value of 4.2 × 10−8 (OR = 1.19, 95 % CI 1.12–1.27). GWASs for endometriosis, to date, have been conducted on samples of relatively modest sizes compared to other conditions, but these first studies have shown that there are no common variants with large effects. ORs for replicated, genome-wide significant signals are all lower than 1.5, an observation that mirrors results from the many GWAS performed on complex diseases to date. The results from Painter et al. [27•] suggest that future larger studies enriched for surgically confirmed rAFS stage III/IV cases will be better powered to identify risk loci of endometriosis, but further work, requiring larger samples, needs to be conducted to assess whether specific severe subtypes such as deep infiltrating endometriosis [42] or rectovaginal disease [43] are genetically heterogeneous from each other, or indeed if rAFS stage III/IV is genetically heterogenous with respect to presence of endometriomas or scarring/adhesions. Furthermore, the implicated variants from GWASs are likely to be in LD with the actual disease-causing variants, and further in-depth work to explore genetic variation in the regions, as well as their influence on gene expression and downstream biological pathways, now needs to be conducted. The variants themselves are of small effect and explain only a very small proportion of disease prevalence; hence, they are unsuitable individually to serve as diagnostic markers. However, they provide important data to define the underlying pathways contributing to the disease, can be used to define genetically heterogeneous subtypes that are likely to respond differently to treatments, and thus, through the identification of differential pathways, help in the development of diagnostic tests and future treatments.

e important data to define the underlying pathways contributing to the disease, can be used to define genetically heterogeneous subtypes that are likely to respond differently to treatments, and thus, through the identification of differential pathways, help in the development of diagnostic tests and future treatments. Genes Implicated by GWAS of Endometriosis to Date Although there was no evidence of a signal in women of European ancestry [27], the GWAS by Uno et al. [40•] clearly implicated CDKN2BAS as potentially involved in endometriosis in Japanese women (Table 2). CDKN2BAS is an interesting candidate because it is expressed in the uterus and regulates the expression of CDKN2B (p15), CDKN2A (p16), and ARF (p14), which are tumor suppressor genes [44–46]. CDKN2A is a cell-cycle–dependent kinase inhibitor and acts as a negative cell-cycle regulator [47, 48]. Inactivation of CDKN2A has been reported in a subset of endometrial carcinomas [49–52]. Moreover, hypermethylation of the CDKN2A promoter region has been observed in endometriosis [52], and loss of heterozygosity on the CDKN2A locus has been found in endometriosis, suggesting that CDKN2A might play a role in the regulation of endometrial cell growth [53]. The evidence from the literature suggests that silencing of tumor suppressor genes such as CDKN2A by CDKN2BAS might have an important role in the development of endometriosis [53].

e CDKN2A locus has been found in endometriosis, suggesting that CDKN2A might play a role in the regulation of endometrial cell growth [53]. The evidence from the literature suggests that silencing of tumor suppressor genes such as CDKN2A by CDKN2BAS might have an important role in the development of endometriosis [53]. The second novel genetic variant, which was supported by evidence from GWASs in both women of Japanese and European ancestry, is located near WNT4, with evidence from the latter mainly limited to rAFS stage III/IV disease. WNT4 is known to play an important role in the development of the female genital tract from the Müllerian duct that develops into the fallopian tubes and uterus; the loss of WNT4 in knockout mice was shown to lead to complete absence of the Müllerian duct [54, 55]. Moreover, WNT4 is expressed in normal peritoneum, suggesting that endometriosis may arise through metaplasia using developmental pathways involved in the development of the female genital tract [54]. Furthermore, it is possible that genetic variants in WNT4 might contribute to endometriosis susceptibility through abnormal cell growth in female genital tract. With all the evidence from the literature, WNT4, through its prominent role in development of the female reproductive tract [55], ovarian follicle development, and steroidogenesis [56], might play a critical role in development of endometriosis.

iosis susceptibility through abnormal cell growth in female genital tract. With all the evidence from the literature, WNT4, through its prominent role in development of the female reproductive tract [55], ovarian follicle development, and steroidogenesis [56], might play a critical role in development of endometriosis. The third novel genetic variant found only in the GWASs of women of European ancestry [27•], rs12700667, is located in an intergenic region on chromosome 7p15.2. This locus was not reported among the top 100 signals in the Japanese GWAS by Uno et al. [40•]; however, given their sample size they would have had only 13 % power to detect it [27•]. Chromosome 7p15.2 contains multiple genomic features that may be involved in endometriosis development. The region contains a number of expressed sequence tags (ESTs) and a miRNA (has-mir-148a), suggesting a role in gene expression regulation. Further downstream, there are several genes including NFE2L3 (highly expressed in placenta) and two endometriosis candidate genes, HOXA10 and HOXA11, which are transcription factors that play a role in uterine development. Another interesting aspect about this variant comes from a large GWAS (n = 77,167) investigating the genetic variants associated with fat distribution as measured by waist-to-hip ratio adjusted for the effect of body mass index (WHRadjBMI) [57]. Interestingly, rs12700667 is positioned at the same locus as one of 13 genome-wide significant loci they reported as associated with reduced WHRadjBMI, a result that is currently further being explored. As endometriosis and fat distribution are both hormonally dependent, pleiotropic genetic loci acting on both traits may well exist.

rs12700667 is positioned at the same locus as one of 13 genome-wide significant loci they reported as associated with reduced WHRadjBMI, a result that is currently further being explored. As endometriosis and fat distribution are both hormonally dependent, pleiotropic genetic loci acting on both traits may well exist. Challenges and Future Directions The first GWASs of endometriosis have identified three genetic loci highly likely to be involved in the pathogenesis of endometriosis in women of Japanese and European ancestry. Although they have identified the loci, they have not pinpointed the actual genetic variants that are causal to endometriosis. Furthermore, they explain only a fraction of the heritability of endometriosis. Therefore, directions for further investigation include the following:The GWASs of endometriosis to date are relatively crude, in that they generally only describe associations with endometriosis, without regard for subtypes or differences in symptomatology (pain and infertility). Only the IEC GWAS [27•] considered stage B versus stage A endometriosis, showing that the two are genetically different in etiology. Future studies need to include cases who are phenotyped in much greater detail to enable distinction between endometriosis subtypes. For these studies to have sufficient power, however, even larger sample sizes are required than used to date.

A endometriosis, showing that the two are genetically different in etiology. Future studies need to include cases who are phenotyped in much greater detail to enable distinction between endometriosis subtypes. For these studies to have sufficient power, however, even larger sample sizes are required than used to date. Of the 1,449 novel loci identified by GWASs up to June 2011 [39], the number of loci identified per complex trait varies greatly. Visscher et al. [58] showed that the number of discovered variants is strongly correlated with the sample size of different studies. The sample sizes of the endometriosis GWASs are at the lower end of those that have been successful in identifying loci involved in the etiology of a complex trait. This means that increasing the discovery sample size will increase the number of discovered variants, which also was suggested by prediction analyses based on all genotyped SNPs in the GWAS of the IEC [27•]. When individual causal variants only explain a small amount of variation, then the power to detect them is low in studies with small sample sizes. Therefore, there is a need for larger endometriosis GWASs in different populations, and meta-analyses of GWASs, to detect additional common causal variants with modest effect on risk. As mentioned before, the power of these studies can be increased by focusing on more severe subtypes, and in general by ensuring detailed and accurate clinical phenotyping allowing the investigation of such subtypes.

ns, and meta-analyses of GWASs, to detect additional common causal variants with modest effect on risk. As mentioned before, the power of these studies can be increased by focusing on more severe subtypes, and in general by ensuring detailed and accurate clinical phenotyping allowing the investigation of such subtypes. To maximize information gained from GWASs, dense genotyping platforms containing at least 500,000 SNPs selected on the basis of LD should be utilized in GWASs, and imputation approaches should be used to estimate non-genotyped common variants (eg, using 1000 Genomes project data [http://www.1000genomes.org]). To follow up on initial GWA scans and to further improve the coverage, custom-made gene arrays could be designed including dense SNP sets focused on implicated genes (although, if budget allows, sequencing of these may be preferable); these could be supplemented by genes robustly implicated in hypothesized related common disease such as gynecological cancers [59, 60], migraine [61] or complex traits such as age of menarche [62] and body mass index (BMI) [63]. The use of whole exome genotyping arrays, including carefully selected sets of common and low-frequency variants in exons, also could be a promising approach to identify additional signals.

ch as gynecological cancers [59, 60], migraine [61] or complex traits such as age of menarche [62] and body mass index (BMI) [63]. The use of whole exome genotyping arrays, including carefully selected sets of common and low-frequency variants in exons, also could be a promising approach to identify additional signals. Although GWASs are unbiased by prior biological knowledge or genomic location, they are limited with regard to their ability to only assess the effect of common SNPs (MAF > ~0.05) in the general population. It is possible that some of the unexplained genetic variation may be due to rarer variants (MAF < 0.5 %), either single-site or structural, that are not captured by current GWA genotyping arrays [64, 65]. Indeed, the effects of structural variation are generally missed out in GWAS (although some of the GWA arrays contain selected probes to test for common copy number variation [CNV]). Rare and structural variants can be investigated through exome or whole-genome sequencing; however, these studies are still challenging because of their cost, with bioinformatic methodology to accurately call structural variants in particular still under development.

bes to test for common copy number variation [CNV]). Rare and structural variants can be investigated through exome or whole-genome sequencing; however, these studies are still challenging because of their cost, with bioinformatic methodology to accurately call structural variants in particular still under development. Preliminary studies have suggested the role of gene–gene and gene–environment interactions in the development of endometriosis [66, 67]. There is no doubt that such interactions play a role in the development of many complex traits, but to robustly ascertain them without strong prior hypotheses, even larger sample sizes are generally required than advocated for GWAS. A biological route through which environmental factors could impact on the influence of genes on disease is through epigenetic modification of DNA (mainly methylation). There is considerable interest in epigenetic variants as contributors to the unexplained genetic variation of endometriosis [68]. One method through which the role of epigenetic variants in the development of endometriosis could be tested is by using a twin cohort where age-specific endometriosis concordance rates are obtained from monozygotic and dizygotic twins. If the monozygotic twin concordance rates for endometriosis increase with age, this could provide evidence for the potential role of epigenetic factors [69]. However, a major issue for endometriosis could be age-specific confounding by patterns of diagnosis. Platforms to detect genome-wide DNA methylation are being developed, and although currently still based on selected loci, are increasingly providing exciting new avenues for exploring the effect of epigenetic changes on complex disease such as endometriosis.

s could be age-specific confounding by patterns of diagnosis. Platforms to detect genome-wide DNA methylation are being developed, and although currently still based on selected loci, are increasingly providing exciting new avenues for exploring the effect of epigenetic changes on complex disease such as endometriosis. Although the study of the genetics of endometriosis is starting to reap fruits, it is still faced with many challenges inherent to the complexities of the disease. This includes the general lack of correlation between disease stage and pain severity; much greater phenotypic detail of cases included in genetic studies is needed to start distinguishing between potential subtypes of disease and symptomatologies. A particular problem is the need for a surgical diagnosis, which hampers population-based research. A complementary avenue may be to study the disease in animal models, in particular nonhuman primates such as the rhesus macaque [70] or the baboon [71, 72], with the rhesus macaque a promising model for the study of heritable, spontaneously occurring endometriosis [17]. Work on the genetics of endometriosis in the rhesus macaque, comparing human and rhesus macaque genomes, is currently underway.

ular nonhuman primates such as the rhesus macaque [70] or the baboon [71, 72], with the rhesus macaque a promising model for the study of heritable, spontaneously occurring endometriosis [17]. Work on the genetics of endometriosis in the rhesus macaque, comparing human and rhesus macaque genomes, is currently underway. Conclusions There is mounting evidence for genetic variants contributing to endometriosis susceptibility. To date, candidate gene studies have not provided robust, replicated genetic variants associated with endometriosis and they have low a-priori chance of success when based solely on biological hypotheses. Linkage studies in pedigrees have found some regions of interest, which are likely to harbor variants implicated in familial endometriosis; however, due to the methodology, the regions identified are large and contain many genes of potential interest. These regions require further investigations to elucidate the susceptibility variants. The much-anticipated initial GWAS results identified three regions showing robust association with endometriosis in women of Japanese and European ancestry. Although they are by far the largest studies of endometriosis to date, they were of relatively small sample size compared to GWASs of other traits, and generally lacked detailed clinical information that would have allowed investigation of more etiologically homogeneous subphenotypes of endometriosis. Such information needs to be collected systematically for future analyses, and well-designed and sufficiently powered GWASs of thousands of cases and controls need to be conducted to identify novel genetic variants associated with endometriosis. These larger studies also will allow for better investigation of potential gene–gene and gene–environment interactions. Moreover, there are upcoming opportunities to study the impact of environment on genetic factors influencing endometriosis, through epigenetic studies, with the development of platforms to detect epigenetic changes genome-wide. Identification of robust, replicated genetic/epigenetic variants of endometriosis will pave the way to functional studies to better understand the underlying mechanisms of endometriosis, leading to better diagnosis and treatment of the disease. Complimentary research in animal models, particularly nonhuman primate models such as the rhesus macaque and the baboon, which can develop endometriosis spontaneously, should help to further elucidate the genetics of this complex condition.

sms of endometriosis, leading to better diagnosis and treatment of the disease. Complimentary research in animal models, particularly nonhuman primate models such as the rhesus macaque and the baboon, which can develop endometriosis spontaneously, should help to further elucidate the genetics of this complex condition. Disclosures Dr. N. Rahmioglu: none; Dr. S. A. Missmer has received grants from the National Institutes of Health of the United States (RO1 HD052473; RO1 HD057210); Dr Grant W. Montgomery is supported by the Australian National Health and Medical Research Council Fellowship scheme (339446, 619667) and received NHMRC grants (496610, APP1026033). Dr. Krina T. Zondervan is supported by a Wellcome Trust Career Development Award (WT085235/Z/08/Z); has received a grant from the Wellcome Trust (WT084766/Z/08/Z); has served as a board member and consultant for Bayer Schering Ltd.; and has received travel expense compensation from Abbott Endocrine Ltd. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

Introduction Hydatidiform mole (HM) is an aberrant human pregnancy with abnormal embryonic development. It occurs once in every 600 pregnancies in Western countries [1] but at higher rates in the Middle East, Latin America, Africa, and the Far East [2–4]. Sporadic moles have a multifactorial etiology involving various combinations of several environmental and genetic factors. Among women with one HM, 10 % to 20 % have other forms of reproductive loss, mainly as spontaneous abortions [5–8]. Because the frequency of two reproductive losses (one HM and one spontaneous abortion) in these patients (10–20 %) is 2–4 times higher than the frequency of two spontaneous abortions in the general population (2–5 %) [9–11], it is believed that some of these patients have genetic predisposition to recurrent reproductive loss.

the frequency of two reproductive losses (one HM and one spontaneous abortion) in these patients (10–20 %) is 2–4 times higher than the frequency of two spontaneous abortions in the general population (2–5 %) [9–11], it is believed that some of these patients have genetic predisposition to recurrent reproductive loss. Recurrent hydatidiform moles are defined by the occurrence of at least two molar pregnancies in the same patient. The earliest report of RHMs available to us through PubMed search is by Mack and Catherwood in 1930 [12]. In this paper, the authors describe one case of 10 RHMs, review the literature for cases of RHMs, and cite a report in 1912 by Essen-Moeller of a patient with 18 RHMs. The frequency of RHMs varies among populations and countries. In Western countries, studies by several groups from various countries have shown that 1 % to 2 % of patients with a prior mole have a second one [5, 13, 14]. However, higher frequencies of RHMs are reported from the Middle and Far East; in these regions, the frequency of RHMs ranges from 2.5 % up to 9.4 % [6, 15–18]. In rare cases, RHMs have been seen in related women from the same family, and these cases are termed familial cases of RHMs. Such cases are considered very rare, and their frequency is not known.

are reported from the Middle and Far East; in these regions, the frequency of RHMs ranges from 2.5 % up to 9.4 % [6, 15–18]. In rare cases, RHMs have been seen in related women from the same family, and these cases are termed familial cases of RHMs. Such cases are considered very rare, and their frequency is not known. At the clinical level, patients with RHMs do not have any particular feature that distinguishes them from those with nonrecurrent sporadic moles [19], which highlights the importance of DNA testing to determine patients who are at risk for mole recurrence. Also, current data indicate that determining the parental contribution to the molar tissues can help detect patients at higher risk for mole recurrence. In this review, we summarize known data about RHMs and highlight the benefits of DNA testing.

of DNA testing to determine patients who are at risk for mole recurrence. Also, current data indicate that determining the parental contribution to the molar tissues can help detect patients at higher risk for mole recurrence. In this review, we summarize known data about RHMs and highlight the benefits of DNA testing. NLRP7 NLRP7, a nucleotide oligomerization domain (NOD)-like receptor, pyrin containing 7, maps to 19q13.4 and is the first identified causative gene for RHMs [20]. Studies from various groups and populations concur that NLRP7 is a major gene for this condition and is mutated in 48–80 % of patients with at least two HMs, depending on patients’ ascertainment criteria and populations [21–24]. To date, 47 mutations in NLRP7 have been reported in patients with two defective alleles (Fig. 1a) ([25] and http://fmf.igh.cnrs.fr/ISSAID/infevers/). These mutations include stop codons, small deletions or insertions (less than 20-bp), splice mutations, large deletions or insertions, and complex rearrangements. In addition to these mutations, two protein-truncating mutations, a stop codon, L823X [21], and a deletion of 60-kb extending from intron 8 of NLRP7 to intron 11 of NLRP2 [26] and approximately 17 missenses have also been seen as single heterozygous mutations or variants in patients with recurrent and sporadic moles (Fig. 1a) [26–31]. However, the pathological significance of these single mutations or variants is still the subject of debate, and more data are needed to reach a conclusion on their potential involvement in the causation or genetic susceptibility for moles. NLRP7 transcripts have been identified in several human tissues, including endometrium, placenta, hematopoietic cells, all oocytes stages, and preimplantation embryos. NLRP7 transcripts decrease after fertilization and during preimplantation development to reach their lowest level at day 3 of embryonic development, which corresponds to the blastocyst stage, and then increase sharply from day 3 to day 5, which coincides with the transcriptional activation of the embryonic genome.Fig. 1 Schematic representations of NLRP7 and KHDC3L protein structures with identified mutations and non-synonymous variants in patients with hydatidiform moles and reproductive loss. (a) NLRP7 protein structure with its domains. PYD = pyrin domain; NACHT == domain present in NAIP, CIITA, HET-E, and TP1 family proteins; ATP = 5′-triphosphate binding motif; LRR = leucine-rich repeats.

ures with identified mutations and non-synonymous variants in patients with hydatidiform moles and reproductive loss. (a) NLRP7 protein structure with its domains. PYD = pyrin domain; NACHT == domain present in NAIP, CIITA, HET-E, and TP1 family proteins; ATP = 5′-triphosphate binding motif; LRR = leucine-rich repeats. The ATP binding domain is a small motif of 8 amino acids and starts at position 178. (b) KHDC3L protein structure with identified mutations and non-synonymous variants. KH stands for K homology domain. Mutation nomenclature is according to the Human Genome Variation Society guidelines (http://www.hgvs.org/mutnomen/recs.html). Mutations found in patients with two defective alleles are in red. Non-synonymous variants (NSVs) found only in patients in heterozygous state and not in controls are in blue. NSVs found in patients and in subjects from the general population are in black. Mutations found in patients who had at least one live birth are underlined Functional Roles of NLRP7 NLRP7 codes for 1037 amino acid protein (including all coding exons of all splice isoforms) and has three main domains: pyrin, NACHT (i.e., found in the NAIP, CIITA, HET-E, and TP1 family proteins) and 10 leucine-rich repeats (LRR). NLRP7 is a member of the NLR family of proteins with a role in inflammation and apoptosis. Below, we outline known roles of NLRP7 in various cellular models and discuss their potential involvement in the pathophysiology of recurrent moles.

NAIP, CIITA, HET-E, and TP1 family proteins) and 10 leucine-rich repeats (LRR). NLRP7 is a member of the NLR family of proteins with a role in inflammation and apoptosis. Below, we outline known roles of NLRP7 in various cellular models and discuss their potential involvement in the pathophysiology of recurrent moles. Overexpressed NLRP7 Downregulates Intracellular IL-1β Emerging data from three different groups about the role of NLRP7 indicate that its overexpression in transient transfections downregulates the production of IL-1β, an important mediator of the inflammatory response. The first study by Kinoshita et al. demonstrated that overexpressed NLRP7 interacts with overexpressed pro-IL-1β and pro-caspase-1 and downregulates caspase-1-dependent IL-1β secretion in HEK293 cells by inhibiting pro-IL-1β processing [32]. Another study by Messaed et al. confirmed the inhibitory effect of overexpressed NLRP7 on IL-1β, but showed that NLRP7 acts primarily on pro-IL-1β and inhibits its intracellular synthesis [33•]. In addition, this study showed that NLRP7 inhibitory function is mediated concomitantly by its three domains, and mostly by the LRR. Although the precise mechanism by which NLRP7 downregulates intracellular IL-1β (pro- or mature) is not fully understood, NLRP7 has been shown to interact physically with IL-1β, caspase-1, and ASC, with the latter mediated by the pyrin domain [32, 34].

mediated concomitantly by its three domains, and mostly by the LRR. Although the precise mechanism by which NLRP7 downregulates intracellular IL-1β (pro- or mature) is not fully understood, NLRP7 has been shown to interact physically with IL-1β, caspase-1, and ASC, with the latter mediated by the pyrin domain [32, 34]. Physiological Level of NLRP7 Inhibits IL-1β Secretion in Monocytic Cells Using an ex vivo cellular model, Messaed et al. also looked at the consequences of NLRP7 mutations on IL-1β secretion by peripheral blood mononuclear cells (PBMCs) from patients with NLRP7 mutations [33•]. They showed that patient cells secrete lower levels of IL-1β than control cells despite the fact that these same cells have normal or slightly higher amounts of intracellular pro-IL-1β synthesis, indicating NLRP7’s role in IL-1β secretion into the extracellular milieu. These findings are in line with those obtained by Kinoshita et al. in stable transfections of THP-1 cells (of human monocytic origin), where expressing an N-terminal 35-kDa NLPR7 fragment, which mimics some protein-truncating mutations observed in patients with RHMs, reduced IL-1β secretion. This finding was also confirmed in a third cellular model described by Khare et al., who demonstrated that NLRP7 knockdown using small interfering RNA in macrophages significantly impairs IL-1β release upon stimulation with microbial acylated lipopeptides [34]. Within monocytic cells, NLRP7 co-localizes with the Golgi and microtubule-organizing center and associates with microtubules. This suggests that NLRP7 mutations may decrease cytokine secretion by affecting the structure of cytoskeletal microtubules, either directly or indirectly, and impairing the trafficking of IL-1β-containing vesicles [33•]. This suggestion is further supported by the fact that treating hematopoietic cells with nocodazole, a microtubule depolymerizing agent, fragmented NLRP7’s signal [33•].

affecting the structure of cytoskeletal microtubules, either directly or indirectly, and impairing the trafficking of IL-1β-containing vesicles [33•]. This suggestion is further supported by the fact that treating hematopoietic cells with nocodazole, a microtubule depolymerizing agent, fragmented NLRP7’s signal [33•]. An Interesting Emerging Role for NLRP7 in Trophoblast Differentiation Another novel and interesting role for NLRP7 was recently demonstrated by Mahadevan et al. In this study, the authors showed that NLRP7 knockdown in human embryonic stem cells led to an earlier expression of two trophoblast differentiation markers, GCM1 and INSL4, suggesting that NLRP7 loss of function accelerates trophoblast differentiation [35•]. Another interesting finding in this study was that NLRP7 knockdown increased the level of human chorionic gonadotropin (hCG), known to be very high in patients with molar pregnancies. This new role of NLRP7 is very important in view of the fact that hydatidiform mole is characterized by hyperproliferation of the trophoblast and production of high levels of hCG.

as that NLRP7 knockdown increased the level of human chorionic gonadotropin (hCG), known to be very high in patients with molar pregnancies. This new role of NLRP7 is very important in view of the fact that hydatidiform mole is characterized by hyperproliferation of the trophoblast and production of high levels of hCG. Possible Roles of NLRP7 in the Pathology of Moles The known functions of NLRP7 in inflammatory signalling of hematopoietic cells raise questions as to whether NLRP7’s role in IL-1β production may be the cause of the early embryonic development arrest observed in molar pregnancies. Available data do indicate some connection between IL-1β, ovulation, and oocyte maturation. For instance, in several mammalian species, intra-follicular injection of IL-1β increases the rate of ovulation, but decreases the quality of the oocytes and, consequently, the rate of normal embryonic development [36, 37]. However, this role for IL-1β in oocytes is in contradiction with data on cells from patients with NLRP7 mutations, which secrete lower amounts of IL-1β. In addition, mice lacking IL-1β [38] or Type 1 IL-1 receptor (Il1r1) [39] are fertile, indicating that the lack of IL-1β signalling does not significantly affect fertility and embryo viability in mice. In addition to its role in IL-1β secretion, NLRP7 has been shown to promote cellular proliferation and invasion in testicular and endometrial cancer, respectively.

1 IL-1 receptor (Il1r1) [39] are fertile, indicating that the lack of IL-1β signalling does not significantly affect fertility and embryo viability in mice. In addition to its role in IL-1β secretion, NLRP7 has been shown to promote cellular proliferation and invasion in testicular and endometrial cancer, respectively. In conclusion, we believe that, individually, none of the above-described roles of NLRP7 may explain the pathology of moles or recapitulate all of their features, as it is impossible to model a pregnancy in any cellular assay. Perhaps a combination of the above-described functions, with some acting in the oocytes and affecting the differentiation and proliferation of embryonic and trophoblastic tissues, and others acting in hematopoietic inflammatory cells present in the endometrium and downregulating the maternal immune response, together contribute to the three fundamental aspects of moles: retained human pregnancies with no embryo and excessive trophoblastic proliferation. We believe that the role of NLRP7 in downregulating maternal inflammation (intra- or extracellular) and the inability of patients to spontaneously eliminate these unviable pregnancies is a fundamental aspect of this disease that distinguishes it from all other forms of early foetal loss. Indeed, it is the retention of these early arrested pregnancies that has homogenized and distinguished this category of foetal loss from all other forms of early spontaneous abortions and has consequently facilitated the identification of two of its causative genes.

guishes it from all other forms of early foetal loss. Indeed, it is the retention of these early arrested pregnancies that has homogenized and distinguished this category of foetal loss from all other forms of early spontaneous abortions and has consequently facilitated the identification of two of its causative genes. KHDC3L KHDC3L (KH domain containing 3-like), which was identified in 2011, is a second recessive gene responsible for RHMs [40•]. KHDC3L maps to chromosome 6, and available data indicate that this gene is a minor gene for RHMs, accounting for 10–14 % of patients who do not have mutations in NLRP7. To date, four mutations in KHDC3L have been reported in patients with two defective alleles (Fig. 1b) [40•, 41]. KHDC3L transcripts have been identified in several human tissues, including all oocytes stages, preimplantation embryos, and hematopoietic cells. KHDC3L codes for a small protein of 217 amino acids belonging to the KHDC1 (KH homology domain containing 1) protein family, members of which contain an atypical KH domain that does not bind RNA as opposed to proteins with canonical KH domain. In humans, this family includes KHDC3L, KHDC1, DPPA5 (developmental pluripotency associated 5), and OOEP (oocyte-expressed protein) [42]. Expression of KHDC3L is highest in oocytes at the germinal vesicle stage and then decreases during preimplantation development and becomes undetectable at the blastocyst stage [40•], similar to the expression prolife of NLRP7 [43]. In addition, KHDC3L co-localizes with NLRP7 to the microtubule organizing center and the Golgi apparatus in lymphoblastoid cell lines [41], which suggests that the two genes may have similar or overlapping functions in oocyte and early embryonic development.

stage [40•], similar to the expression prolife of NLRP7 [43]. In addition, KHDC3L co-localizes with NLRP7 to the microtubule organizing center and the Golgi apparatus in lymphoblastoid cell lines [41], which suggests that the two genes may have similar or overlapping functions in oocyte and early embryonic development. RHMs Caused by Mutations in NLRP7 or KHDC3L are Mostly Diploid Biparental Common sporadic nonrecurrent CHMs are mostly diploid androgenetic. Among them, approximately 80 % are monospermic and the remaining are dispermic. Common sporadic PHMs are mostly triploid dispermic. Deviations from these common genotypes, such as monospermic triploidy, digynic triploidy, triandric tetraploidy, biparental tetraploidy, and biparental diploidy, have also been reported among both complete and partial moles but account for a minority of cases, estimated at about 8 % of common moles [44, 45]. This is not the case, however for molar tissues from patients with NLRP7 or KHDC3L mutations. In patients with two NLRP7 defective alleles, the parental contribution to approximately 81 HM tissues has been reported, and all were found to be diploid biparental with the exception of two, which were found to be triploid dispermic [26] and triploid digynic [46] (Table 1). The same applies to patients with two KHDC3L defective alleles. Among these patients, the parental contributions to nine HM tissues have been determined, and all of them were found to be diploid biparental [23, 41, 47, 48] with the exception of one HM tissue that was found to be triploid digynic [46] (Table 1).Table 1 Summary of molar genotypes from patients with NLRP7 and KHDC3L mutations

ese patients, the parental contributions to nine HM tissues have been determined, and all of them were found to be diploid biparental [23, 41, 47, 48] with the exception of one HM tissue that was found to be triploid digynic [46] (Table 1).Table 1 Summary of molar genotypes from patients with NLRP7 and KHDC3L mutations Diploid biparental Diploid androgenetic Triploid dispermic Triploid digynic References NLRP7 mutations 2 defective alleles 81 (98 %) 0 (0 %) 1 (1 %) 1 (1 %) [21, 23, 26, 27, 30, 46, 55, 65–72] 1 defective allele 4 (33 %) 5 (42 %) 3 (25 %) 0 (0 %) [21, 26, 27, 30, 73] KHDC3L mutations 2 defective alleles 8 (100 %) 0 (0 %) 0 (0 %) 0 (0 %) [23, 41, 47] Among patients with a single heterozygous NLRP7 mutation or very rare variants not seen in controls, parental contribution to 15 HM tissues has been reported. Of these, four were found to be diploid biparental [26], seven were found to be diploid androgenetic monospermic [21, 27, 30], and four were found to be triploid dispermic [22] (Table 1). With respect to KHDC3L, no molar tissues from patients with single heterozygous variants have been characterized.

has been reported. Of these, four were found to be diploid biparental [26], seven were found to be diploid androgenetic monospermic [21, 27, 30], and four were found to be triploid dispermic [22] (Table 1). With respect to KHDC3L, no molar tissues from patients with single heterozygous variants have been characterized. Genomic Imprinting in Diploid Biparental Moles Altered DNA Methylation at Imprinted Genes in the Conceptions of Patients with KHDC3L or NLRP7 Mutations Genomic imprinting refers to epigenetic modifications such as DNA methylation, histone modification, or/and chromatin remodeling that lead to the expression of only one of the two parental copies of a gene. The involvement of genomic imprinting in the pathology of hydatidiform moles emerged soon after the demonstration that sporadic complete moles are androgenetic, which made them an important experimental tool in characterizing the expression and/or methylation of imprinted genes [49–51]. Later, the identification of recurrent familial moles that have the same histopathological features as the sporadic androgenetic moles [52] and the finding that these moles are diploid biparental [53] entertained the plausible and interesting idea that the causative gene for recurrent moles would be responsible for setting or maintaining the maternal imprints in the oocytes. To date, four studies have examined the DNA methylation of imprinted genes in a total of eight diploid biparental hydatidiform moles from patients with two defective alleles in KHDC3L or NLRP7 [23, 47, 54, 55]. The first study demonstrated, in one diploid biparental CHM from a patient with two KHDC3L defective alleles, the loss of methylation marks at six of seven analyzed differentially methylated regions (DMR) that are normally maternally methylated, and the gain of methylation marks on one paternally methylated DMR (NESP55) that acquires its methylation at the blastocyst stage (Table 2). In contrast, the methylation at the H19 DMR, which is normally established in the male germ line, was normal. Two additional diploid biparental moles from the same patient were later studied, but unfortunately at different DMRs, and their analysis showed the same trend of abnormal methylation with the exception of one gene, PEG10, which preserved its normal methylation on the maternal allele (Table 2) [23].

m line, was normal. Two additional diploid biparental moles from the same patient were later studied, but unfortunately at different DMRs, and their analysis showed the same trend of abnormal methylation with the exception of one gene, PEG10, which preserved its normal methylation on the maternal allele (Table 2) [23]. Other studies also examined the methylation status of DMRs in moles from patients with two NLRP7 defective alleles and reported abnormal loss and gain of methylation at some of them [23, 54, 55]. In one of these studies, single nucleotide polymorphisms were used to distinguish parental alleles at some imprinted genes and showed that the abnormal methylation, indeed, affected the maternal alleles [54] (Table 2). In conclusion, these data demonstrated the presence of imprinting abnormalities in diploid biparental moles from patients with KHDC3L or NLRP7 mutations and indicated that these abnormalities may have occurred during oogenesis and/or early embryogenesis. Because NLRP7 and KHDC3L proteins do not have DNA binding domains or any domain that is found in DNA methyltransferases, it was not clear whether these methylation defects play a primary causal role in the oocytes leading to the formation of moles or whether they are a secondary consequence of abnormal postzygotic development.Table 2 Recapitulation of methylation analysis data in diploid biparental molar tissues from patients with NLRP7 or KHDC3L mutations DMR Chr KHDC3L

Other studies also examined the methylation status of DMRs in moles from patients with two NLRP7 defective alleles and reported abnormal loss and gain of methylation at some of them [23, 54, 55]. In one of these studies, single nucleotide polymorphisms were used to distinguish parental alleles at some imprinted genes and showed that the abnormal methylation, indeed, affected the maternal alleles [54] (Table 2). In conclusion, these data demonstrated the presence of imprinting abnormalities in diploid biparental moles from patients with KHDC3L or NLRP7 mutations and indicated that these abnormalities may have occurred during oogenesis and/or early embryogenesis. Because NLRP7 and KHDC3L proteins do not have DNA binding domains or any domain that is found in DNA methyltransferases, it was not clear whether these methylation defects play a primary causal role in the oocytes leading to the formation of moles or whether they are a secondary consequence of abnormal postzygotic development.Table 2 Recapitulation of methylation analysis data in diploid biparental molar tissues from patients with NLRP7 or KHDC3L mutations DMR Chr KHDC3L NLRP7 Conclusion Reference [47] [23] [54] [55] [23] Patient ID L1 4 & 6 HM70 & HM73 S4 Number of HMs (n) n = 1 n = 2 n = 2 n = 2 n = 1 Maternal methylated KCNQ1OT1 a 11 − − − − − − − − − − − − − − − SNRPN b 15 − − − −, − − − − − − − − PEG1 7 − − − − − − PEG3 19 − − − − − − − − − − − − GNAS-1A a 20 − − − − − − − − − GNAS-AS 20 − − − Complex Inconcl. GNAS-XLαS b 20 Normal Normal Normal ZAC a 6 − − − − − − − − − PEG10 a 7 Normal, − − Normal Normal Paternally methylated

NLRP7 Conclusion Reference [47] [23] [54] [55] [23] Patient ID L1 4 & 6 HM70 & HM73 S4 Number of HMs (n) n = 1 n = 2 n = 2 n = 2 n = 1 Maternal methylated KCNQ1OT1 a 11 − − − − − − − − − − − − − − − SNRPN b 15 − − − −, − − − − − − − − PEG1 7 − − − − − − PEG3 19 − − − − − − − − − − − − GNAS-1A a 20 − − − − − − − − − GNAS-AS 20 − − − Complex Inconcl. GNAS-XLαS b 20 Normal Normal Normal ZAC a 6 − − − − − − − − − PEG10 a 7 Normal, − − Normal Normal Paternally methylated H19 a 11 Normal +, ++ Normal Inconcl. GNAS-NESP55 b 20 +++ +++ +++ +++ c +++ +++ Chr, chromosome; a primary imprint; b secondary imprint ; c gain of methylation at this locus was found in the two diploid biparental moles as well as in one normal term placenta and in one androgenetic mole; Inconcl., inconclusive. Different results on two HM tissues are separated by a comma

GNAS-NESP55 b 20 +++ +++ +++ +++ c +++ +++ Chr, chromosome; a primary imprint; b secondary imprint ; c gain of methylation at this locus was found in the two diploid biparental moles as well as in one normal term placenta and in one androgenetic mole; Inconcl., inconclusive. Different results on two HM tissues are separated by a comma Altered DNA Methylation Beyond Non-Imprinted Genes To investigate the role of NLRP7 in establishing methylation marks at imprinted genes, Mahadevan et al. recently examined the consequences of NLRP7 knockdown on the DNA methylation of imprinted genes during the differentiation of human embryonic stem cells (hESCs) into trophoblast cells [35•]. However, they did not observe any DNA methylation changes at imprinted DMRs, including those that were previously shown to be abnormally methylated in diploid biparental molar tissues. They explained their findings by the known high degree of epigenetic stability and resistance of hESC lines to perturbations in DNA methylation at imprinted loci [56]. Conversely, they found that NLRP7 knockdown altered the DNA methylation of many non-imprinted CpGs. Another interesting study showed that the DNA methylation of a total 131 imprinted and non-imprinted loci were altered in blood DNA of an individual with multiple anomalies born to a mother with a single heterozygous NLRP7 mutation (A719V) [57]. It would have been interesting in this study to have determined if the mutation in the mother occurred de novo or if it was inherited, and from which of her parents. In addition, it is not clear whether the abnormal child inherited his mother’s mutation. Surprisingly, comparing the abnormally methylated genes from the studies by Mahadevan et al. and Beygo et al. [35•, 57] did not reveal any common gene with altered methylation, which raise questions about the specificity and significance of these findings and their relation to NLRP7 mutations that remain to be clarified in future studies.

ing the abnormally methylated genes from the studies by Mahadevan et al. and Beygo et al. [35•, 57] did not reveal any common gene with altered methylation, which raise questions about the specificity and significance of these findings and their relation to NLRP7 mutations that remain to be clarified in future studies. Altered Expression of CDKN1C in the Conceptions of Patients with NLRP7 Mutations In line with the above data, one study demonstrated the underexpression of p57KIP2, the product of the paternally imprinted, maternally expressed gene CDKN1C in the cytotrophoblast and villous stroma of a series of diploid biparental CHMs [58]. p57KIP2 is the protein coded by a cyclin-dependent kinase inhibitor. CDKN1C deficiency in mice leads to altered cellular proliferation and differentiation, resulting in a variety of developmental defects [59]. Although CDKN1C is paternally methylated in the cytotrophoblast and villous stroma of normal first-trimester placenta, its expression has been shown to depend on the maternal methylation of KvDMR1, a CpG island located at the promoter of KCNQ1OT1 believed to control the imprinted expression of CDKN1C during embryonic development [60, 61]. The same is observed in humans, where the loss of maternal methylation marks at KvDMR1 leads to the silencing of CDKN1C in patients with Beckwith-Wiedemann syndrome [62], a pediatric overgrowth disorder in which the placenta share some histopathological features with PHMs.

CDKN1C during embryonic development [60, 61]. The same is observed in humans, where the loss of maternal methylation marks at KvDMR1 leads to the silencing of CDKN1C in patients with Beckwith-Wiedemann syndrome [62], a pediatric overgrowth disorder in which the placenta share some histopathological features with PHMs. Despite the complexity of the methylation and imprinting data and the variations between studies and samples, the common findings were the lack of DNA maternal methylation marks at several maternally imprinted, paternally expressed genes and the unspecific/stochastic extension of methylation abnormalities to non-imprinted genes. We believe that further studies are needed to delineate the exact roles of NLRP7 and KHDC3L genes in the DNA methylation of imprinted and non-imprinted genes.

on marks at several maternally imprinted, paternally expressed genes and the unspecific/stochastic extension of methylation abnormalities to non-imprinted genes. We believe that further studies are needed to delineate the exact roles of NLRP7 and KHDC3L genes in the DNA methylation of imprinted and non-imprinted genes. Conclusions NLRP7 and KHDC3L DNA Testing Because of the high rate of NLRP7 mutations in patients with RHMs, which seems to vary with populations, patients with at least two HMs (complete or partial) should be first offered NLRP7 DNA testing that is now available in many clinical and research laboratories, including ours. Methods currently in use rely on PCR amplification of the 11 exons of NLRP7 from genomic DNA, followed by direct sequencing of the PCR products in the two directions and the comparison of the sequences with the reference sequence NM_001127255.1. This analysis is highly sensitive in identifying point mutations within the coding region, small deletions and insertions affecting the amplified regions, and DNA changes at the invariant splice sites. However, this method is not reliable to identify deep intronic single-nucleotide changes affecting the splicing of the gene or regulatory sequences, large deletions, insertions, and complex rearrangements.

on, small deletions and insertions affecting the amplified regions, and DNA changes at the invariant splice sites. However, this method is not reliable to identify deep intronic single-nucleotide changes affecting the splicing of the gene or regulatory sequences, large deletions, insertions, and complex rearrangements. Patients without NLRP7 mutations should be screened for KHDC3L mutations, which account for up to 14 % of cases who are NLRP7-negative [40•, 41, 63]. Similarly, for KHDC3L, currently methods rely on PCR amplification of its 3 exons from genomic DNA, followed by direct sequencing of the PCR products in the two directions and the comparison of the sequences with the reference sequence NM_001017361. Because of the causal involvement of this gene in a minority of cases, only some of the laboratories that offer NLRP7 testing are currently systematically sequencing KHDC3L for all patients who are NLRP7-negative. The identification of two defective alleles in either gene allows to confirm a genetic defect underlying mole recurrence and to counsel the patients accordingly based on available data in the field.

ratories that offer NLRP7 testing are currently systematically sequencing KHDC3L for all patients who are NLRP7-negative. The identification of two defective alleles in either gene allows to confirm a genetic defect underlying mole recurrence and to counsel the patients accordingly based on available data in the field. Prognosis for Future Pregnancies The goal of patients seeking DNA testing is to ascertain their chances of conceiving healthy babies and their risk for mole recurrence and malignant sequelae. Studies from various groups have shown that the chances of a normal live birth are very low in women with two defective alleles in NLRP7. Among reported patients from our group, only 3 out of 43 (7 %) had normal live births, which accounted for 1.5 % of their pregnancies. No other cases of live births have been reported by other groups in patients with RHMs and NLRP7 or KHDC3L mutations, with the exception of a recently described case of one live birth to a patient with two defective alleles in NLRP7 [35•]. In the few reported patients with two KHDC3L defective alleles, no live birth has been reported. Based on available data, both genes NLRP7 and KHDC3L are required in the oocytes. Therefore, theoretically, ovum donation is expected to improve patients’ reproductive outcomes. Thus far, few patients with mutations in NLRP7 have tried ovum donation, and three had normal live births ([64•] and Slim et al. in preparation), which provides some hope for patients despite the elevated cost of such procedure and its inaccessibility for many of them.

pected to improve patients’ reproductive outcomes. Thus far, few patients with mutations in NLRP7 have tried ovum donation, and three had normal live births ([64•] and Slim et al. in preparation), which provides some hope for patients despite the elevated cost of such procedure and its inaccessibility for many of them. For patients with RHMs and no mutations in either gene, we believe that moles in these patients are most likely to be of a genetic etiology caused by undetected mutations in either NLRP7 or KHDC3L or by mutations in non-identified genes. Based on our current understanding of moles and their mode of formation, the best help that can be offered to these patients is to review the histopathology of their moles and determine the parental contribution to the molar genomes. Based on the results of this analysis, the patients may be classified into two categories:Patients with at least two diploid biparental moles that fit the histological classification of complete molar pregnancies can be counselled in the same way as patients with two NLRP7 or KHDC3L mutations despite the lack of identified mutations. In patients where the histopathological re-evaluation disagrees with the diagnosis of moles, such cases can be counselled similarly to patients with recurrent spontaneous abortions.

lar pregnancies can be counselled in the same way as patients with two NLRP7 or KHDC3L mutations despite the lack of identified mutations. In patients where the histopathological re-evaluation disagrees with the diagnosis of moles, such cases can be counselled similarly to patients with recurrent spontaneous abortions. Patients with androgenetic or triploid dispermic moles have higher chances of having live births from their own oocytes than patients with recurrent diploid biparental moles. Because androgenetic and triploid dispermic moles are caused by errors that occur either at the time of fertilization or very early in the zygote, these patients may benefit from in vitro fertilization followed by preimplantation genetic screening (PGS) to select for diploid embryos to be transferred to the patients. This option may not completely prevent having additional moles, but may help to maximize the patients’ chances of having normal pregnancies, which should be monitored according to standard prenatal care for women with recurrent reproductive loss. A chart summarizing our suggested approach for DNA testing and genetic counselling of patients with RHMs is provided in Fig. 2.Fig 2 Suggested screening recommendation for DNA testing and genetic counselling of patients with recurrent hydatidiform moles. Patients with at least two HMs should be offered DNA testing first for NLRP7, in which mutations are found in 48–80 % of such patients. Among those with two mutated alleles, up to 7 % may have normal live birth (LB) from their own oocytes in 1.5 % of their pregnancies. To date, three cases of successful ovum donation have been observed in such patients. Patients without NLRP7 mutations should be tested for KHDC3L, in which mutations are found in 10–14 % of such patients. For patients with no mutations in either gene, we propose to re-examine the histopathology of their moles and determine the parental contribution to them. Patients with confirmed complete moles that are diploid biparental can be counselled in the same way as patients with mutations in NLRP7 or KHDC3L. Those with androgenetic or triploid dispermic moles have higher chances of live births from their own oocytes and may be benefit from in vitro fertilization (IVF) and preimplantation genetic screening (PGS) for aneuploidies

loid biparental can be counselled in the same way as patients with mutations in NLRP7 or KHDC3L. Those with androgenetic or triploid dispermic moles have higher chances of live births from their own oocytes and may be benefit from in vitro fertilization (IVF) and preimplantation genetic screening (PGS) for aneuploidies Risk for Malignant Degeneration With respect to the risk for malignant degeneration of moles in patients with mutations in NLRP7 and KHDC3L, we do not have accurate statistics with regard to their risk as compared to patients with sporadic common moles. However, available data on our cases and on those reported by other groups indicate that despite their higher risk for mole recurrence, patients with mutations in either gene are at least not at higher risk for choriocarcinoma. However, future studies are needed to delineate their risk for the less severe neoplastic degeneration and requirement of chemotherapy. Acknowledgment Ngoc Minh Phuong Nguyen was supported by the Réseau Québécois en Reproduction, the Alexander McFee McGill Faculty of Medicine Fellowships, and the Canadian Institute for Health Research for RS (MOP-102469). Compliance with Ethics Guidelines ᅟ Conflict of Interest Ngoc Minh Phuong Nguyen and Rima Slim declare that they have no conflict of interest Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

Introduction Gestational trophoblastic neoplasia (GTN) is highly chemosensitive and has a high cure rate. Since the introduction of chemotherapy, reliable measurement of human chorionic gonadotropin (hCG) levels, and individualized risk-based therapy into the management of GTN, almost all low-risk and more than 80 % of high-risk GTN cases are curable [1]. However, approximately 25 % of high-risk GTN patients developed resistance to chemotherapy or relapsed after completion of initial therapy, which often necessitate salvage combination chemotherapy [2•]. On the other end of the spectrum, a proportion of patients with gestational trophoblastic disease (GTD) have persistently low levels of hCG, without clinical or radiological evidence of disease, a condition called quiescent GTD [1]. Recently, measurement of hyperglycosylated hCG has been proposed for the management of patients with quiescent GTD [3]. Although representing a small proportion of GTD cases, the management of patients with chemoresistant and quiescent GTD often poses challenges to medical practitioners. Chemoresistant Gestational Trophoblastic Disease Chemoresistant GTN occurs when there is a plateau or an increase in hCG levels, with or without development of new metastases, often while the patient is receiving therapy. Relapsed GTN occurs when there are at least two elevated levels of hCG in the absence of pregnancy after achieving a period of normal hCG values with treatment. Drug resistance and relapse are known to occur in around 3 % of low-risk GTN and 7-10 % of high-risk GTN cases [4].

ten while the patient is receiving therapy. Relapsed GTN occurs when there are at least two elevated levels of hCG in the absence of pregnancy after achieving a period of normal hCG values with treatment. Drug resistance and relapse are known to occur in around 3 % of low-risk GTN and 7-10 % of high-risk GTN cases [4]. Risk factors that predispose a patient to drug resistance and relapse include number of consolidation courses administered, clinicopathologic diagnosis of choriocarcinoma, initial hCG level, extent of disease (brain, liver, and gastrointestinal metastases have a worse prognosis), and higher World Health Organization (WHO) risk scores [5, 6]. Most of these patients are salvageable by further chemotherapy; however, 20 % of patients will eventually become resistant to treatment and die. The overall 5-year survival was more than 90 % for patients with relapsed GTN, which was nearly 100 % for low-risk GTN and about 85 % for high-risk GTN [7]. The prognosis for patients with chemoresistant GTN is worse than for those with relapsed GTN[7]. In a series of 81 patients, the long-term serologic complete remission rate reported was 52.6 % in chemoresistant GTN and 76.7 % in relapsed GTN [8].

h was nearly 100 % for low-risk GTN and about 85 % for high-risk GTN [7]. The prognosis for patients with chemoresistant GTN is worse than for those with relapsed GTN[7]. In a series of 81 patients, the long-term serologic complete remission rate reported was 52.6 % in chemoresistant GTN and 76.7 % in relapsed GTN [8]. Management For low-risk GTN patients who had been resistant to the single agent methotrexate, actinomycin-D is commonly used, followed by MAC (methotrexate, actinomycin-D, cyclophosphamide) or EMA-CO (etoposide, methotrexate, actinomycin-D, cyclophosphamide, vincristine) if further salvage therapy is needed [2•]. Methotrexate resistance was reported in about a third of patients with GTD where change of treatment was required. In a retrospective study of 485 patients who developed GTN after a hydatidiform mole, 150 (31 %) patients developed resistance to methotrexate [9]. Those patients who developed methotrexate resistance or toxicity at a relatively low hCG level (≤100 IU/L) were often cured with actinomycin-D, with a reported response rate of 87 %, while patients with hCG greater than 100 IU/L were salvaged with combination chemotherapy with EMA-CO, with a reported response rate of 99 % [9]. In the United Kingdom, actinomycin-D will replace methotrexate if hCG is less than 300 IU/l, while combined chemotherapy such as EMA-CO will be administered if hCG is higher than 300 IU/l [10]. In our centre in Hong Kong, actinomycin-D can be added for patients with low-risk GTN who do not respond to methotrexate.

[9]. In the United Kingdom, actinomycin-D will replace methotrexate if hCG is less than 300 IU/l, while combined chemotherapy such as EMA-CO will be administered if hCG is higher than 300 IU/l [10]. In our centre in Hong Kong, actinomycin-D can be added for patients with low-risk GTN who do not respond to methotrexate. For high-risk GTN patients who are resistant to first-line chemotherapy or have relapse, salvage combined chemotherapy with or without surgery will be required [11]. Various salvage regimens are used worldwide (Table 1); however, it is unclear which regimens are the most effective and the least toxic. Table 2 summarizes the salvage chemotherapy regimens reported in the literature (only publications after year 2000 are included) for chemoresistant or relapsed GTN. The favored regimen is EMA-EP (etoposide, methotrexate, actinomycin-D, etoposide, cisplatin) [2•].Table 1 Salvage chemotherapy for resistant or relapsed gestational trophoblastic neoplasia Chemotherapy Regimens EMA-EP Etoposide, Methotrexate, Actinomycin-D, Etoposide, Cisplatin BEP Bleomycin, Etoposide, Cisplatin TP/TE Paclitaxel, Cisplatin / Paclitaxel, Etoposide FA 5-Fluorouracil, Actinomycin-D FAEV Floxuridine, Actinomycin-D, Etoposide, Vincristine MBE Methotrexate, Bleomycin, Etoposide VIP/ICE Ifosfamide, Cisplatin, Etoposide Table 2 Retrospective studies of salvage chemotherapy for resistant or relapsed gestational trophoblastic neoplasia

, Cisplatin / Paclitaxel, Etoposide FA 5-Fluorouracil, Actinomycin-D FAEV Floxuridine, Actinomycin-D, Etoposide, Vincristine MBE Methotrexate, Bleomycin, Etoposide VIP/ICE Ifosfamide, Cisplatin, Etoposide Table 2 Retrospective studies of salvage chemotherapy for resistant or relapsed gestational trophoblastic neoplasia Authors, Year (Ref) Primary Therapy Salvage Therapy Participants Outcomes Toxicity Remarks Newland et al. 2000 [47] EMA-CO EMA-EP 34 resistant/ relapsed 22 Non-assessable (hCG levels approaching normal) 12 Assessable: CR 75 %, OS 88 % ≥G3 neutropenia 68 % ≥G3 thrombocytopenia 40 % Matsui et al. 2002 [48] MEA / EMA-CO FA 10 resistant/ relapsed OS 80 % G4 leukocytopenia 6 % G4 thrombocytopenia 4 % Mean FU 10 yr 1 had adjuvant surgery Xiang et al. 2004 [49] N/A EMA-EP 12 resistant 3 PSTT CR 11/15 (73 %) PR 3/15 (20 %) N/A Ngan et al. 2006 [12] CHAMOC / MA MBE A: 8 resistant B: 8 relapsed C: 4 ultra-high-risk A: CR 88 %, 5-yr DFS 75 % B: CR 88 % C: CR 75 % Overall CR 85 % ≥G3 neutropenia 12 (60 %) ≥G3 thrombocytopenia 4 (20 %) 1 had adjuvant surgery 6 had brain radiation 1 had liver radiation Wang et al. 2006 [50] Various MEF 9 resistant/ relapsed CR 7/9 (78 %) OS 8/9 (96 %) ≥G3 neutropenia 26 % ≥G3 thrombocytopenia 5 % Mean FU 37 mth Mao et at. 2007 [51] EMA-CO EMA-EP A: 11 resistant B: 7 relapsed A: CR 9/11 (82 %) B: CR 3/7 (43 %) Overall CR 12/18 (67 %) ≥G3 neutropenia 28 % ≥G3 thrombocytopenia 3 % G3 hepatotixicity 3 % 8 had adjuvant surgery Wan et al. 2007 [52] Various FAEV 11 resistant CR 7/11 (64 %) N/A Lu et al. 2008 [53] EMA-CO EMA -EP 10 resistant 3 relapsed CR 11/13 (85 %) N/A 5 had adjuvant surgery / brain radiation Wang et al. 2008 [54] Various TP/TE A: 16 relapsed (6 with failed cisplatin based regimen) B: 8 toxicities with previous therapy A: Response rate 50 %: CR 3 (19 %), PR 5 (31 %) OS 44 % (70 % if 6 with failed cisplatin based regimen excluded) B: CR 2, PR 2, Non-assessable 4 OS 75 % ≥G3 neutropenia 10 (42 %) ≥G3 thrombocytopenia 3 (13 %) G2 renal toxicity 1 (4 %) Discontinued due to G3 neuropathy 1 (4 %) A: Median FU 24 mth B: Median FU 19 mth Zhao et al. 2009 [55] FA BEP 12 resistant CR 10/12 (83 %) N/A Feng et al. 2011 [56] Various: mainly FAV, EMA-CO, 5-FU FAEV 91 resistant/ relapsed CR 55/91 (60 %) NR 29/91 (32 %) 3-yr OS 75 % ≥G3 neutropenia 24 (26.4 %) Febrile neutropenia 6 (6.6 %) ≥G3 thrombocytopenia 3 (3.3 %) Discontinued due to toxicity 7 (7.7 %) 23 of 55 SCR had adjuvant surgery Lurain et al.

0/12 (83 %) N/A Feng et al. 2011 [56] Various: mainly FAV, EMA-CO, 5-FU FAEV 91 resistant/ relapsed CR 55/91 (60 %) NR 29/91 (32 %) 3-yr OS 75 % ≥G3 neutropenia 24 (26.4 %) Febrile neutropenia 6 (6.6 %) ≥G3 thrombocytopenia 3 (3.3 %) Discontinued due to toxicity 7 (7.7 %) 23 of 55 SCR had adjuvant surgery Lurain et al. 2012 [11] EMA-CO Various: EMA-EP; BEP; VIP; ICE; TP/TE 28 resistant CR 82 % N/A 11 had adjuvant surgery 4 had brain radiation Manopunya et at. 2012 [57] Various FA 5 resistant (≥3 prior chemotherapy regimens) CR 1/5 (20 %) G4 neutropenia 25 % G3 diarrhoea 8 % G2/3 mucositis 92 % EMA-CO: etoposide, methotrexate, actinomycin-D, cyclophosphamide, vincristine; FA: 5-fluorouracil, actinomycin-D; CHAMOC: cyclophosphamide, hydroxyurea, actinomycin-D, methotrexate with folinic acid and vincristine; MA: methotrexate, actinomycin-D; MEA: methotrexate, etoposide, actinomycin-D; FAV: 5-fluorouracil, actinomycin-D, vincristine; 5-FU: 5-fluorouracil; EMA-EP: etoposide, methotrexate, actinomycin-D, etoposide, cisplatin; EP: etoposide, cisplatin; MEF: methotrexate, etoposide, 5-fluorouracil; BEP: bleomycin, etoposide, cisplatin; VIP: vincristine, ifosfamide, cisplatin; ICE: ifosfamide, cisplatin, etoposide; TP/TE: paclitaxel, cisplatin/paclitaxel, etoposide; MBE: methotrexate, bleomycin, etoposide; FAEV: floxuridine, actinomycin-D, etoposide, vincristine; PSTT: placental site trophoblastic tumour; hCG: human chorionic gonadotrophin; CR: complete response; OS: overall survival; PR: partial response; NR: no response; DFS: disease free survival; N/A: not available; FU: follow-up

: methotrexate, bleomycin, etoposide; FAEV: floxuridine, actinomycin-D, etoposide, vincristine; PSTT: placental site trophoblastic tumour; hCG: human chorionic gonadotrophin; CR: complete response; OS: overall survival; PR: partial response; NR: no response; DFS: disease free survival; N/A: not available; FU: follow-up Direct comparisons between the chemotherapy regimens in terms of outcomes and toxicity are inappropriate because the patients included were heterogeneous. Factors that influenced the salvage rate included type and number of previous chemotherapy regimens administered, use of adjuvant surgery or radiotherapy and WHO risk scores. Furthermore, the rates of toxicity reported are affected by the different measures used to counteract the toxic effects. For example, the threshold for administration of granulocyte-colony stimulating factor (G-CSF) will affect the rate of neutropenia reported. The FIGO Cancer Report 2012 on trophoblastic disease suggested the use of EMA-EP protocol for patients resistant to EMA-CO or who have a recurrence after previous multiagent chemotherapy [1]. Alternatively, EMA (etoposide, methotrexate, actinomycin-D) may be used with cisplatin and doxorubicin. For EMA-EP resistant cases, TP/TE (paclitaxel, cisplatin / paclitaxel, etoposide) or paclitaxel and 5-fluorouracil, or ICE (ifosfamide, cisplatin, etoposide), or BEP (bleomycin, etoposide, cisplatin) have been used.

Alternatively, EMA (etoposide, methotrexate, actinomycin-D) may be used with cisplatin and doxorubicin. For EMA-EP resistant cases, TP/TE (paclitaxel, cisplatin / paclitaxel, etoposide) or paclitaxel and 5-fluorouracil, or ICE (ifosfamide, cisplatin, etoposide), or BEP (bleomycin, etoposide, cisplatin) have been used. In our centre in Hong Kong, CHAMOC (cyclophosphamide, hydroxyurea, actinomycin-D, methotrexate with folinic acid and vincristine) has been used as first-line therapy for high-risk GTN, while MBE (methotrexate, bleomycin, etoposide) is used as second-line therapy. Our centre reported use of MBE in 16 patients who developed drug resistance to combination chemotherapy or relapsed after combination therapies, with a response rate of 88 % and 5-year disease free survival of 63 % [12]. The use of newer chemotherapy agents such as paclitaxel [13] and gemcitabine [14, 15], or high-dose chemotherapy with or without autologous bone marrow transplantation or peripheral stem cell support [16-19], may be considered in the management of selected high-risk patients. The major dose-limiting factor in most of these combined chemotherapy regimens is myelosuppression; therefore, most patients will require G-CSF and transfusion support to prevent treatment delays or dose reductions.

eripheral stem cell support [16-19], may be considered in the management of selected high-risk patients. The major dose-limiting factor in most of these combined chemotherapy regimens is myelosuppression; therefore, most patients will require G-CSF and transfusion support to prevent treatment delays or dose reductions. Role of Surgery Although GTN is chemosensitive, surgery may be required and can result in a cure in selected patients with chemoresistant or persistent foci of disease in the uterus or metastatic sites. During the course of treatment, about half of high-risk patients will require some form of surgical procedure to achieve cure [20]. The Charing Cross Gestational Trophoblastic Disease Center, UK, reported using hysterectomy in 9 of 20 patients who developed resistance to EMA-CO after other chemotherapy [21]. The Sheffield Trophoblastic Disease Center, UK, reported that 9 (75 %) of 12 patients who underwent hysterectomy for chemoresistant uterine disease had a complete clinical response to surgery [22].

ter, UK, reported using hysterectomy in 9 of 20 patients who developed resistance to EMA-CO after other chemotherapy [21]. The Sheffield Trophoblastic Disease Center, UK, reported that 9 (75 %) of 12 patients who underwent hysterectomy for chemoresistant uterine disease had a complete clinical response to surgery [22]. The John I. Brewer Trophoblastic Disease Centre, USA, reported on a series of 50 patients with high-risk GTN treated with EMA-CO as primary or secondary therapy where 24 (48 %) had adjuvant surgical treatments, and 21 (87.5 %) were cured [23]. In a study by Lehman et al. involving 33 patients with chemorefractory GTD who underwent salvage surgery, 42 % had serologic complete response. 30 % had partial response, and 27 % had no response [24]. Initial salvage procedures included 29 hysterectomies, 4 thoracotomies, and 1 nephrectomy (in conjunction with a hysterectomy). A second salvage surgery was also performed in 4 patients, with all achieving complete response. Factors that have been found to influence the therapeutic response to surgical interventions included age, type of antecedent pregnancy, preoperative hCG level, time from diagnosis to surgery, number of preoperative disease sites, preoperative WHO risk score, and histologic type [24, 25].

The John I. Brewer Trophoblastic Disease Centre, USA, reported on a series of 50 patients with high-risk GTN treated with EMA-CO as primary or secondary therapy where 24 (48 %) had adjuvant surgical treatments, and 21 (87.5 %) were cured [23]. In a study by Lehman et al. involving 33 patients with chemorefractory GTD who underwent salvage surgery, 42 % had serologic complete response. 30 % had partial response, and 27 % had no response [24]. Initial salvage procedures included 29 hysterectomies, 4 thoracotomies, and 1 nephrectomy (in conjunction with a hysterectomy). A second salvage surgery was also performed in 4 patients, with all achieving complete response. Factors that have been found to influence the therapeutic response to surgical interventions included age, type of antecedent pregnancy, preoperative hCG level, time from diagnosis to surgery, number of preoperative disease sites, preoperative WHO risk score, and histologic type [24, 25]. In cases where drug resistance is related to pulmonary metastasis and the lung lesion is amenable to operation, thoracotomy and lung resection, with a reported remission rate of up to 90 %, may be considered [26]. Criteria that have been used in patient selection for pulmonary resection included solitary pulmonary nodule, no evidence of other metastatic sites or uterine disease, and hCG level <1000 IU/L. Mutch et al. reported that 4 of 9 patients (44 %) who underwent thoracotomy with pulmonary wedge resection of resistant choriocarcinoma survived [27]. Patients with rapid regression of hCG within one to two weeks of surgical resection usually have a favorable outcome.

sites or uterine disease, and hCG level <1000 IU/L. Mutch et al. reported that 4 of 9 patients (44 %) who underwent thoracotomy with pulmonary wedge resection of resistant choriocarcinoma survived [27]. Patients with rapid regression of hCG within one to two weeks of surgical resection usually have a favorable outcome. Quiescent Gestational Trophoblastic Disease Quiescent, or inactive, GTD occurs in a proportion of patients where there is a persistently low level of hCG in the absence of any clinical or radiological evidence of GTN. Usually the hCG level is in the range of 50–100 mIU/mL and remains static for at least 3 months [1, 28]. It is associated with prior history of GTD or spontaneous abortion, and does not respond to therapy [29-31]. This condition is thought to occur when a small focus of (or maybe individual) dispersed, differentiated syncytiotrophoblast cells are present. These slow-growing syncytiotrophoblast cells produce small stable amounts of hCG and do not usually progress to invasive disease as long as the cytotrophoblast, or intermediate cells, are absent [32]. These syncytiotrophoblast cells do not respond to chemotherapy, and surgery does not result in normalization of hCG [31].

ese slow-growing syncytiotrophoblast cells produce small stable amounts of hCG and do not usually progress to invasive disease as long as the cytotrophoblast, or intermediate cells, are absent [32]. These syncytiotrophoblast cells do not respond to chemotherapy, and surgery does not result in normalization of hCG [31]. False-Positive hCG Quiescent GTD must be differentiated from false-positive hCG test results, or so-called “phantom hCG”. Approximately 2 % of women of reproductive age will have a low level of hCG (<300 mIU/mL) detectable by conventional hCG test without the presence of trophoblasts [33]. The false-positive results have led to some women being diagnosed with GTD and undergoing various diagnostic procedures, chemotherapy, hysterectomy, and other surgical procedures before it is established that the results are spurious. This false-positive test result occurs as a result of the presence of nonspecific heterophile antibodies in the patient’s serum, which binds the animal antibodies used in the hCG assay [34]. False-positive hCG can be determined by 1) urine hCG assay, which will be negative because heterophile antibody is not excreted in the urine due to the large molecule size; 2) serial dilution of the sample, as hCG levels are unaffected by dilution; 3) use of different commercial assays that will often result in a significant fluctuation in the hCG level [35].

) urine hCG assay, which will be negative because heterophile antibody is not excreted in the urine due to the large molecule size; 2) serial dilution of the sample, as hCG levels are unaffected by dilution; 3) use of different commercial assays that will often result in a significant fluctuation in the hCG level [35]. False-positive hCG test results can also be found in 1 % of perimenopausal and 7 % of postmenopausal women. These false-positive results are due to raised levels of pituitary follicle-stimulating hormone and luteinizing hormone, as well as a benign low level of pituitary hCG secretion [36]. In the case of pituitary hCG, the production can be inhibited with oral contraceptive pills [37]. Treatment is not required in false-positive hCG tests, because an abnormal trophoblast is absent. Hyperglycosylated hCG Hypergylcosylated hCG (hCG-H) measurement has been proposed for the management of patients with quiescent GTD [3]. hCG-H is a glycoprotein produced by cytotrophoblast cells and is associated with trophoblast invasion, growth of cytotrophoblast cells, and overall promotion of placental implantation [32, 38-40]. It is a promoter of choriocarcinoma growth and tumorigenesis, and is the main form of hCG produced in active choriocarcinoma and gestational trophoblastic neoplasm [41].

blast cells and is associated with trophoblast invasion, growth of cytotrophoblast cells, and overall promotion of placental implantation [32, 38-40]. It is a promoter of choriocarcinoma growth and tumorigenesis, and is the main form of hCG produced in active choriocarcinoma and gestational trophoblastic neoplasm [41]. The USA hCG Reference Service has demonstrated that the proportion of hCG-H (hCG-H / total hCG) is a 100 % sensitive marker for distinguishing active GTN/choriocarcinoma from quiescent GTD and suggested its use as a marker to identify active trophoblastic malignancy [42]. In their study comparing the proportion of hCG-H in 82 women with GTN (including 30 with histologic choriocarcinoma), 26 with resolving hydatidiform mole and 69 with quiescent GTD, the proportion of hCG-H was found to be significantly higher in GTN/choriocarcinoma cases than those with resolving hydatidiform mole or quiescent GTD [42].

mparing the proportion of hCG-H in 82 women with GTN (including 30 with histologic choriocarcinoma), 26 with resolving hydatidiform mole and 69 with quiescent GTD, the proportion of hCG-H was found to be significantly higher in GTN/choriocarcinoma cases than those with resolving hydatidiform mole or quiescent GTD [42]. Quiescent GTD is likely the most common cause for persistently low hCG levels outside of pregnancy in women of reproductive age. hCG-H has been found to be undetectable or at very low levels in patients with quiescent GTD, and therefore is useful for their identification. In a report of 133 patients diagnosed with quiescent GTD, all had low hCG levels persisting for 3 months or longer and a history of GTD [3]. Of these patients, 127 (95 %) had undetectable hCG-H, and 6 had low positive hCG-H, accounting for 4–27 % of serum total hCG concentration. In this condition, chemotherapy was ineffective because the tissue in quiescent GTD is not growing, and in most cases hCG returned to normal within 6 months. Thus, it was suggested that when hCG-H is undetectable, even with persistently low hCG levels, intervention is not needed. Meanwhile, if hCG-H becomes detectable, then this may indicate clinically relevant disease and therapy may be required.

GTD is not growing, and in most cases hCG returned to normal within 6 months. Thus, it was suggested that when hCG-H is undetectable, even with persistently low hCG levels, intervention is not needed. Meanwhile, if hCG-H becomes detectable, then this may indicate clinically relevant disease and therapy may be required. Approximately 20 % of patients with quiescent GTD will start to produce increased hCG after a period of several weeks to years [1, 42]. During the quiescent period, the hCG-H is undetectable, but once the hCG rises, a significant proportion is hCG-H; this is often noted before the appearance of clinically detectable disease. Furthermore, hCG-H were able to first detect active disease 0.5 to 11 months prior to rapidly rising hCG or clinically detectable tumour [42]. Hence, hCG-H has been suggested as a marker for the early detection of new or recurrent GTN/choriocarcinoma. Management The International Society for the Study of Trophoblastic Disease 2001 recommended that in the management of patients with quiescent GTD, false-positive hCG results should be ruled out and that investigations for evidence of disease should be performed. Immediate chemotherapy or surgery should be avoided, and long-tem monitoring with serial hCG while avoiding pregnancy should be advised [43]. In the event that there is significant rise in hCG or presence of overt clinical disease, then treatment should be instigated promptly.

r evidence of disease should be performed. Immediate chemotherapy or surgery should be avoided, and long-tem monitoring with serial hCG while avoiding pregnancy should be advised [43]. In the event that there is significant rise in hCG or presence of overt clinical disease, then treatment should be instigated promptly. Recently, Agarwal et al. at the Charing Cross Gestational Trophoblastic Disease Center reported on 76 patients from a cohort of 13,960 with hydatidiform moles who had persistently elevated but declining hCG levels 6 months after evacuation [44•]. In this study, 66 (87 %) patients were treated expectantly, where 65 (98 %) had spontaneous resolution of hCG to normal and 1 had persistently elevated hCG due to chronic renal failure, but she remains healthy. The duration for hCG to return to normal was within 8 months of evacuation for 44 (67 %) patients, in the next 4 months for 15 (23 %) patients, and longer than 1 year for 6 (9 %) patients. The remaining 10 (13 %) were treated with chemotherapy, and hCG returned to normal in 8 (80 %) of these patients, but remained slightly raised, though asymptomatic, for 2 (20 %) patients. Argarwal concluded that surveillance for more than 6 months is safe for women with persistently high but falling hCG levels, because a declining trend represents spontaneous, although slow, regression of residual molar tissue.

of these patients, but remained slightly raised, though asymptomatic, for 2 (20 %) patients. Argarwal concluded that surveillance for more than 6 months is safe for women with persistently high but falling hCG levels, because a declining trend represents spontaneous, although slow, regression of residual molar tissue. The findings of the study by Argawal et al. are reassuring; however, how should a decision on whether a patient be observed or treated with chemotherapy be made? The investigators suggested a cut-off hCG level of 345 IU/L at 6 months – which was the median hCG value of patients who responded to chemotherapy in their cohort – for initiating chemotherapy [44•]. Meanwhile, Cole et al. suggested that in quiescent GTD, chemotherapy should be initiated only when hCG begins to rise and is >3000 IU/L, because chemotherapy would probably be ineffective below this level [3]. However, adopting this approach could lead to progression of disease to a more advanced stage, including development of distant metastases, which is associated with unfavorable prognosis and survival [45].

hen hCG begins to rise and is >3000 IU/L, because chemotherapy would probably be ineffective below this level [3]. However, adopting this approach could lead to progression of disease to a more advanced stage, including development of distant metastases, which is associated with unfavorable prognosis and survival [45]. Utilization of hCG-H could improve the management of quiescent GTD and help to identify the characteristics of the condition before starting treatment. However, this approach is only feasible if the hCG-H assay is readily available and affordable. Furthermore, its use needs to be validated by gestational trophoblastic disease management centres [46]. Therefore, it is essential that treatment be individualized, and preferably patients with GTN should be managed in centres with dedicated specialists. Centres with many patients should collaborate to enable data collection and establishment of reasonable hCG cut-off values, and ultimately improved management of this small but intriguing group of patients. Conclusion Ideally, patients with chemoresistant, or quiescent, GTD should be managed at a Trophoblastic Centre. A single-center, randomized controlled trial comparing interventions for chemoresistant, or quiescent, GTN will be very challenging, given the small numbers of patients with these conditions. Therefore, international multi-center collaboration is required to provide the high-quality evidence required to determine the most effective treatment.

zed controlled trial comparing interventions for chemoresistant, or quiescent, GTN will be very challenging, given the small numbers of patients with these conditions. Therefore, international multi-center collaboration is required to provide the high-quality evidence required to determine the most effective treatment. Compliance with Ethics Guidelines Conflict of Interest Siew-Fei Ngu and Karen K.L. Chan declare that they have no conflict of interest Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

Introduction Despite improvement in modern female contraceptive methods and the ability to rapidly introduce new effective methods to developing countries, there are unmet needs for family planning in women 15 to 49 years [1–3]. Achieving the desired number of children is beneficial to women, families, and society. In order to meet the unmet need for women, countries need to make available modern contraceptives, provide high quality services, and improve access to such services. In addition, development of new methods of contraception involving men should be a priority. Male contraceptive methods available to the public include the condom which has a high user failure rate [4] and vasectomy which is considered irreversible and involves a minor surgical procedure.

prove access to such services. In addition, development of new methods of contraception involving men should be a priority. Male contraceptive methods available to the public include the condom which has a high user failure rate [4] and vasectomy which is considered irreversible and involves a minor surgical procedure. The mechanisms of action of hormonal male contraception is based on the suppression of the secretion and production of the gonadotropins, both luteinizing and follicle stimulating hormone (LH, FSH), from the pituitary by exogenous sex steroids (androgens with or without progestins) or gonadotropin releasing hormone (GnRH) analogues. Gonadotropin suppression results in marked decrease in intratesticular testosterone and suppression of spermatogenesis. This profound suppression of sperm output inhibits fertility in men providing an effective male-directed contraceptive method. Sexual function, male characteristics, and effects on nonreproductive organs are maintained by the androgen (usually testosterone) that is part of the contraceptive regimen. The current goal is to identify a hormonal male contraceptive that is effective, reversible, safe, acceptable, affordable, and available. The focus has been on the development of a long-acting hormonal delivery system such as testosterone undecanoate intramuscular injections (IM) or user-friendly provider-independent combinations of androgens and progestins such as transdermal applications of combined testosterone and nestorone gels or novel androgens that have activity of both hormones [5–8].

a long-acting hormonal delivery system such as testosterone undecanoate intramuscular injections (IM) or user-friendly provider-independent combinations of androgens and progestins such as transdermal applications of combined testosterone and nestorone gels or novel androgens that have activity of both hormones [5–8]. Early Studies Demonstrating Efficacy of Hormonal Male Contraception (Table 1) Beginning in the early 1970s, the National Institutes of Health (NIH) in the USA began male hormonal contraceptive clinical trials using testosterone enanthate, an intramuscular (IM) injectable preparation available in many countries since the 1950s with good safety profile. These early studies showed when small numbers of healthy male volunteers were administered testosterone enanthate IM for several months, sperm concentration was markedly suppressed to very low levels (oligozoospermia, low sperm concentration arbitrarily defined as <5 or <3 million/ml in studies in the 1990s and <1 million/ml in more recent studies, and azoospermia, no sperm cells in ejaculate) in a high proportion of men [9–12]. The World Health Organization (WHO) undertook two multicenter, multinational studies involving larger numbers of healthy men in the 1990s that established the efficacy of male hormonal contraception when sperm output was suppressed to very low levels. The first study recruited 271 healthy couples from ten centers in seven countries in four continents [13]. Testosterone enanthate was administered IM every week for ≤6 months until the men achieved azoospermia (Table 1). After azoospermia was reached, 157 couples used the weekly testosterone enanthate injections as their only method of contraception, and 119 couples completed the efficacy of 12 months. There was one pregnancy in 1486 months of efficacy evaluation (Pearl Index 0.8 [95 % confidence interval (CI) 0.02–4.5] per 100 person-years; Pearl Index is the most common measure of the effectiveness of contraceptive methods and is based on the number of pregnancies, the number of women, and time exposed to the method) [13]. The second study followed using the same study design except the couples were eligible for entry into the efficacy phase when the male partner developed severe oligozoospermia (initially arbitrarily defined as <5 million/ml ejaculate, later revised as <3 million/ml). The study recruited 399 couples from three countries in Asia, and six countries in Australia, Europe, and North America.

ples were eligible for entry into the efficacy phase when the male partner developed severe oligozoospermia (initially arbitrarily defined as <5 million/ml ejaculate, later revised as <3 million/ml). The study recruited 399 couples from three countries in Asia, and six countries in Australia, Europe, and North America. Three hundred and fifty-seven men (89 %) completed the efficacy phase with eight men (2.2 %) failing to reach the threshold for oligozoospermia (Table 1). After 180 person-years of exposure, five pregnancies occurred with three occurring when sperm concentration was >4 million/ml, and the threshold criteria for entry into efficacy phase was lowered to <3 million/ml. No pregnancy occurred in the azoospermic group, and four occurred in the oligozoospermic group. The overall pregnancy rate was 1.4 (95 % CI 0.4 to 3.7) per 100 person-years. In these two studies, the azoospermia rate was higher in Asian men, with non-Asian less likely to achieve suppression to severe oligozoospermia. The study also demonstrated that the pregnancy rate in the couples in the efficacy phase was related to the sperm output of the male partner. These two early landmark studies provided strong evidence that when spermatogenesis is suppressed to yield very low sperm output by the administration of exogenous androgens, contraceptive efficacy can be as high as female contraceptive methods [14].Table 1 Contraceptive efficacy in hormonal male contraception clinical trials

wo early landmark studies provided strong evidence that when spermatogenesis is suppressed to yield very low sperm output by the administration of exogenous androgens, contraceptive efficacy can be as high as female contraceptive methods [14].Table 1 Contraceptive efficacy in hormonal male contraception clinical trials Study reference Sperm concentration threshold million/ml Drugs Number enrolled Number completing suppression phase (6 m) Number reaching threshold Number entering efficacy Number completing efficacy Number with sperm rebound Years of exposure to risk of pregnancy Pregnancies/failure ratea WHO 1990 [13] Azoospermia TE 200 mg/week 271 225 (83 %) 157 (70 %) 157 119 21 (1.4 %) 123.8 1 0.8 (0.0–45.)a WHO 1996 [14] <3 (reduced form <5) TE 200 mg/week 399 357 (89 %) 349 (97.8 %) 268 209 4 (0.2 %) 279.9 (230.4 azoospermia) 4 1.4 (0.4–3.7)a McLachlan et al 2000 [15] <1 T implants 800 or 1200 mg/4 months 36 29 21 (72 %) 16 17 4 17.8 0 Turner et al 2003 [19] <1 T implants 800 mg/4–6 months DMPA 300 mg/3 months 55 55 53 (94 %) 51 30 0 with T implants/4 months 35.5 0 (0–8)a Gu et al 2003 [27] <3 TU 1000 mg loading 500 mg/month 308 308 299 (97.1 %) 296 280 6 (2.3 %) 143 0 1 in sperm rebound 2.3 (0.5–4.2)a Gu et al 2009 [28••] <1 TU 1000 mg loading 500 mg/month 1045 898 855 (95.2 %) 855 733 10 (1.3 %) 1554.1 9 1.1 (0.4–1.8)a WHO/CONRAD 2015 [29•] <1 TU 1000 mg and Net–EN 200 mg/8 weeks 320 283 274 (95.9 %) 266 111b 6 ? 4 1.57 (0.59–4.14) 2.18 (0.82–5.80)a

Gu et al 2003 [27] <3 TU 1000 mg loading 500 mg/month 308 308 299 (97.1 %) 296 280 6 (2.3 %) 143 0 1 in sperm rebound 2.3 (0.5–4.2)a Gu et al 2009 [28••] <1 TU 1000 mg loading 500 mg/month 1045 898 855 (95.2 %) 855 733 10 (1.3 %) 1554.1 9 1.1 (0.4–1.8)a WHO/CONRAD 2015 [29•] <1 TU 1000 mg and Net–EN 200 mg/8 weeks 320 283 274 (95.9 %) 266 111b 6 ? 4 1.57 (0.59–4.14) 2.18 (0.82–5.80)a T testosterone, TE testosterone enanthate, TU testosterone undecanoate, DMPA depo-medroxyprogesterone acetate, NET-EN norethisterone enanthate aPearl rate (95 % confidence interval) per 100 couple-year bTrial terminated before planned end of study These two large-scale studies were confirmed by a smaller contraceptive efficacy study using testosterone implants. In the first single center study, the investigators implanted 800- or 1200-mg testosterone implants into the abdominal wall with or without a 5-alpha-reductase inhibitor to suppress intratesticular dihydrotestosterone levels. The rationale of adding a 5-alpha-reductase inhibitor was to completely suppress the conversion of testosterone to the active androgen dihydrotestosterone in the testis and more effective suppression of spermatogenesis. When the sperm concentration reached <1 million/ml in the male partner, the 16 couples were allowed to enter the efficacy phase with 16. No pregnancy occurred during 214 months of exposure [15].

n of testosterone to the active androgen dihydrotestosterone in the testis and more effective suppression of spermatogenesis. When the sperm concentration reached <1 million/ml in the male partner, the 16 couples were allowed to enter the efficacy phase with 16. No pregnancy occurred during 214 months of exposure [15]. Development of Androgens, Progestins, and GnRH Antagonists as Potential Male Contraceptives Studies that followed examined adding progestins and GnRH analogues to the androgen (testosterone) to increase the rate of suppression of spermatogenesis to severe oligozoospermia to over 90 % in men. Progestins including depot medroxyprogesterone acetate, levonorgestrel pills and implants, desogestrel pills, etonogestrel implants, oral cyproterone acetate pills, and norethindrone enanthate injectables have been used in combination with testosterone implants, injectables, and transdermal preparations. These small contraceptive clinical trials showed that addition of a progestin increased the proportion of men whose spermatogenesis was suppressed to produce very low sperm output (<1 million/ml) [16–18]. A two-center study employed a combination of testosterone implants 800 mg every 4 months together with depot medroxyprogesterone acetate 300 mg IM every 3 months. Efficacy phase of the study began when the male partner had two consecutive monthly semen samples with sperm concentration <1 million/ml. Fifty-five couples enrolled in the study, and 51 couples entered the efficacy phase. There was no pregnancy reported during the 425 months of exposure. The contraceptive failure rate was zero (95 % CI 0 to 8) per 100 person-years (Table 1) [19].

consecutive monthly semen samples with sperm concentration <1 million/ml. Fifty-five couples enrolled in the study, and 51 couples entered the efficacy phase. There was no pregnancy reported during the 425 months of exposure. The contraceptive failure rate was zero (95 % CI 0 to 8) per 100 person-years (Table 1) [19]. GnRH agonists downregulate gonadotropin receptor actions that result in initial elevation but subsequent marked suppression of the gonadotropins. GnRH antagonists act by competitive binding to receptors and reduced both LH and FSH to very low levels. GnRH agonists were not effective suppressors of sperm output [20, 21], whereas the antagonists were very effective. Unfortunately, clinical studies showed that GnRH antagonists required frequent subcutaneous injections and were expensive to produce. Some of the antagonists caused a histamine-like acute reaction on the skin [22, 23]. Subsequent short-term studies showed that the suppression of spermatogenesis by GnRH antagonists plus an androgen can be maintained by administration of the androgen alone [24, 25]. Because GnRH antagonists required frequent injections, orally bioavailable GnRH antagonists are being developed but not yet available for testing as a contraceptive agent.

wed that the suppression of spermatogenesis by GnRH antagonists plus an androgen can be maintained by administration of the androgen alone [24, 25]. Because GnRH antagonists required frequent injections, orally bioavailable GnRH antagonists are being developed but not yet available for testing as a contraceptive agent. More Recent Contraceptive Efficacy Trials with Injectable Testosterone Undecanoate and Norethisterone Enanthate (Table 1) New testosterone delivery systems have been developed for testosterone replacement in hypogonadal men. Testosterone undecanoate administered as IM injection was studied as a potential long-acting hormonal male contraceptive agent [26]. Chinese investigators studied spermatogenic suppression by testosterone undecanoate administered as a monthly injection to 308 volunteers. During a 6-month suppression phase, 9 out of 308 men did not reach a sperm concentration of <3 million/ml (failure of suppression 2.9 %) and 296 men entered the efficacy phase of 6 months. There were no pregnancies when the men had azoospermia or severe oligozoospermia. Sperm rebound to levels above threshold during treatment occurred in 2.3 %, and one pregnancy occurred during sperm rebound [27]. Based on these encouraging results, these investigators conducted a phase 3 mutlicenter contraceptive efficacy study in 1045 men in ten centers in China. Testosterone undecanoate 500 mg was administered as a monthly injection after an initial loading dose of testosterone undecanoate 1000 mg. During the suppression phase of 6 months, 43/898 participants (4.8 %) did not reach sperm concentration of <1 million/ml. Eight hundred fifty-five couples entered efficacy phase of 24 months, and 733 completed the study. During treatment period, 1.3 % of subjects had a sperm rebound during treatment. During the efficacy phase, nine pregnancies occurred of which six might have been due to sperm rebound. There were 1554 months of exposure, and the failure rates were 1.0 % (95 % CI 0.3 to 1.7 %) and 1.1 (95 % CI 0.4 to1.8) at the end of 12 and 24 months, respectively. The study provided substantial evidence that testosterone undecanoate alone may provide a safe, effective, reversible contraceptive formulation for Asian men [28••].

1554 months of exposure, and the failure rates were 1.0 % (95 % CI 0.3 to 1.7 %) and 1.1 (95 % CI 0.4 to1.8) at the end of 12 and 24 months, respectively. The study provided substantial evidence that testosterone undecanoate alone may provide a safe, effective, reversible contraceptive formulation for Asian men [28••]. Because of the finding in non-Asian men that the suppression of spermatogenesis may not be as efficacious using androgen alone, the World Health Organization (WHO) together with the Contraceptive Research and Development Program (CONRAD) at Eastern Virginia Medical School led a multicenter, multinational study that recruited 320 men who were given a combination of testosterone undecanoate (1000 mg) and a progestin norethisterone enanthate (200 mg) every 8 weeks with a suppression phase of up to 6 months and an efficacy phase (no other method of contraception for the couple) for 12 months. These 320 participants received at least one injection, and 95.9 % reached the efficacy eligibility of sperm concentration <1 million/ml. Four pregnancies occurred in 266 couples participating in the study during the efficacy phase giving a contraceptive failure rate of 1.5 (95 % CI 0.59 to 4.14 ) per 100 users. Common adverse events included acne, pain at injection site, and mood changes which led to the WHO Human Reproduction Program Research Project Review Panel to recommend halting further injections before the planned end of study. Despite the early termination, this study provided additional evidence that an androgen plus a progestin achieved suppression of spermatogenesis to very low levels in over 95 % of men and a failure rate of 1.5 % consistent with the results of the early studies [29•]

g further injections before the planned end of study. Despite the early termination, this study provided additional evidence that an androgen plus a progestin achieved suppression of spermatogenesis to very low levels in over 95 % of men and a failure rate of 1.5 % consistent with the results of the early studies [29•] Reported Adverse Events from Male Hormonal Contraception Clinical Trials (Table 2) Table 2 Adverse events reported in larger scale male contraceptive clinical trials (over 50 men per group) Clinical trial Adverse events (% of enrolled subjects) N Pain/problems with injection/implant Acne Weight gain Mood changesg Libido changesh Behavior changei Fatigue Testosterone only TE [13] 271 7.5 29.5 4.4 1.5 4.4 2.6 8.1 TE [14]a 399 5.3 2.0 0.5 1.0 0.8 0.8 0 TU 2003 [27] 308 + 6.8 + 0 + 0 0 TU 2009 [28••]b 1045 3.9 7.4 + 0.8 + ? 0 Testosterone + Progestin T implants DMPA [19]a,c 55 10.9 ? + 1.2 + d ? + d TU+ Etonogestrel implants [42•]e Active 297 0.6 26 24 19 13 ? + Placebo 52 2 10 10 10 0 ? ? TU + NET-EN [29•]f 320 23.1 45.9 3.8 24.4 42.2 5.7 1.6 TE testosterone enanthate, TU testosterone undecanoate, DMPA depot medroxyprogesterone acetate, NET-EN norethisterone enanthate aOnly adverse events leading to discontinuations reported (italics) bCough after injections in 2.1 %; facial swelling/rash 0.8 % cTwo serious adverse events: myotonic dystrophy during recovery phase and multiple congenital malformations (Vater Anomalad) in one twin conceived during recovery phase. Both considered not related

aOnly adverse events leading to discontinuations reported (italics) bCough after injections in 2.1 %; facial swelling/rash 0.8 % cTwo serious adverse events: myotonic dystrophy during recovery phase and multiple congenital malformations (Vater Anomalad) in one twin conceived during recovery phase. Both considered not related dSymptoms of hypogonadism (lethargy and sexual dysfunction) occurred in men during month 5 or 6 after T implants because of inadequate testosterone release from the pellets after 4 months. This necessitated a protocol amendment eNight sweating was reported in 27 % of men in the active group versus 8 % in the placebo group fMost of the adverse events are mild or moderate, and many occurred in one center: acne (90 % mild), mood changes (includes 16.9 % emotional changes, 97 % mild; 4.7 % mood swings; 84 % mild; and 2.8 % depression, five subjects had moderate and two subjects had severe depressive mood/depression), injection site pain (93 % mild), libido changes (mainly increase; 88 % mild), and behavior changes (83 % mild) gMood changes include emotional disorder, mood swings, altered mood, and depression hLibido changes include increase or decrease libido iBehavior changes include aggression or hostility

fMost of the adverse events are mild or moderate, and many occurred in one center: acne (90 % mild), mood changes (includes 16.9 % emotional changes, 97 % mild; 4.7 % mood swings; 84 % mild; and 2.8 % depression, five subjects had moderate and two subjects had severe depressive mood/depression), injection site pain (93 % mild), libido changes (mainly increase; 88 % mild), and behavior changes (83 % mild) gMood changes include emotional disorder, mood swings, altered mood, and depression hLibido changes include increase or decrease libido iBehavior changes include aggression or hostility Testosterone and its esters are used as replacement therapy for hypogonadal men, and their side effects are well-known [30–32]. The common side effects are acne, oiliness of skin, weight gain, decrease in high-density lipoprotein (HDL) cholesterol, increases in hematocrit and hemoglobin, and sleep-related breathing disorders. These adverse effects may be dependent on the dose and methods of testosterone administration. The potential long-term effects on the prostate are not known. It is unlikely that testosterone replacement causes benign prostatic hyperplasia or prostate cancer [32]. Because of the standard of care requiring monitoring of testosterone treatment with digital rectal examination and serum prostate-specific antigen concentrations in many clinical trials, this may result in more prostate biopsies [32]. Recent controversy on testosterone therapy and cardiovascular disease stemmed from controversial epidemiological studies [33–35] and meta-analyses [36, 37] where some of the studies showed that testosterone therapy may be associated with increases in cardiovascular disease risk including myocardial infarction and stroke and others demonstrated opposite results. The United States Food and Drug Administration and many experts in the field have expressed their opinion that the evidence for or against cardiovascular risk is inadequate and long-term adequately powered safety studies would be required.

uding myocardial infarction and stroke and others demonstrated opposite results. The United States Food and Drug Administration and many experts in the field have expressed their opinion that the evidence for or against cardiovascular risk is inadequate and long-term adequately powered safety studies would be required. In the larger scale contraceptive clinical trials using testosterone enanthate 200 mg every week, which is a supra-physiological dose, the reported common adverse events include pain at injection site, acne, weight gain, fatigue, and mood changes including aggressiveness leading to discontinuation in 5.5, 3.3, 0.7, 0.3, and 1.4 % of men, respectively. Increased or decreased libido and disturbed sleep occurred in a small number of subjects [38]. In the more recent contraceptive clinical trials using testosterone undecanoate (1000 mg loading and then 500 mg every week) in over 1000 men, the most common reported side effects were pain at site of injection (3.9 %), acne (7.4 %), coughing after injection (2.1 %), and mood or behavior changes (0.8 %) [28••] (Table 2).

the more recent contraceptive clinical trials using testosterone undecanoate (1000 mg loading and then 500 mg every week) in over 1000 men, the most common reported side effects were pain at site of injection (3.9 %), acne (7.4 %), coughing after injection (2.1 %), and mood or behavior changes (0.8 %) [28••] (Table 2). There are few clinical trials utilizing combined testosterone plus a progestin with large number of subjects. When testosterone implants were administered with depot medroxyprogesterone acetate, discontinuations were due to mainly to problems of implants (extrusion, pain, androgen deficiency due to inadequate testosterone delivery by implants, mood fluctuation, or study-unrelated personal reasons) [19]. In the most recent contraceptive efficacy study where testosterone undecanoate 1000 mg was combined with norethisterone enanthate 200 mg every 8 weeks, there were adverse events in the 320 participants including the following: acne, injection site pain, increased or decreased libido, mood changes including mood swings and depression, aggression and hostility, and muscle and skeletal pain. One center reported many of the adverse events. This study was stopped prematurely because of these adverse events [29•]. In these studies, changes in sexual function or mood were not prospectively monitored at the sites, and there was no placebo group. In studies where sexual function and mood were monitored prospectively by a diary [39], no significant negative changes in sexual function were found [40, 41•].

these adverse events [29•]. In these studies, changes in sexual function or mood were not prospectively monitored at the sites, and there was no placebo group. In studies where sexual function and mood were monitored prospectively by a diary [39], no significant negative changes in sexual function were found [40, 41•]. In the placebo-controlled randomized clinical trial using injectable testosterone decanoate and etonogestrel implants which were highly effective in suppressing spermatogenesis, adverse events that were reported to occur more frequently in the active treatment groups include acne, increase in body weight, libido change, mood changes, and night sweating [42•] (Table 2). There is no uniform requirement of reporting or coding of adverse events in these contraceptive studies, as the earlier studies reported adverse events leading to discontinuation, whereas the more current reports contained all adverse events. Laboratory tests to evaluate safety outcomes showed no significant changes in renal or liver function. As anticipated with testosterone treatment, significant increases in hematocrit and hemoglobin are observed in most studies, but discontinuation due to increases in red cell indices is infrequent. High-density lipoprotein cholesterol levels are also decreased by testosterone or testosterone plus progestin, but the clinical significance of small decreases in high-density lipoprotein cholesterol is not known because of the complexities of lipoprotein metabolism and the relationship with risk of cardiovascular disease [43, 44].

lipoprotein cholesterol levels are also decreased by testosterone or testosterone plus progestin, but the clinical significance of small decreases in high-density lipoprotein cholesterol is not known because of the complexities of lipoprotein metabolism and the relationship with risk of cardiovascular disease [43, 44]. Factors Affecting Suppression of Spermatogenesis and Reversibility of Hormonal Male Contraception In an integrated analysis where semen analyses and clinical data were available from 1549 healthy male volunteers who participated in many hormonal male contraception clinical trials, using androgens or androgens plus progestins showed that addition of a progestin increased the rate and extent of suppression. In response to male hormonal contraceptive agents, non-Asian men suppressed spermatogenesis faster initially, but eventually, they achieved a less complete suppression of sperm output [45•]. This study confirmed the earlier reports that Asian men had more complete suppression of sperm production than non-Asian men [13, 14]. The reason for this difference is not known, but several mechanisms have been proposed. There are differences in testicular histomorphology and testosterone metabolism between East-Asian and Caucasian men. However, whether this contributes significantly to varying degrees of sperm suppression in response to exogenous hormonal male contraception is not clear [46]. Because of these differences between Asian and non-Asian men, the path of male hormonal contraceptive development has focused on androgens plus progestins.

men. However, whether this contributes significantly to varying degrees of sperm suppression in response to exogenous hormonal male contraception is not clear [46]. Because of these differences between Asian and non-Asian men, the path of male hormonal contraceptive development has focused on androgens plus progestins. Male hormonal agents are frequently used in female contraception, known to suppress gonadotropins, and reversibility has been assumed. The same integrated analysis examined whether all men recovered from male hormonal contraceptive agents that suppressed sperm output to very low regions. The study showed that the median time to recovery to 10 million/ml was 3 months and to 20 million/ml was 3.4 months which is the anticipated time to recovery. These sperm concentrations are compatible with male fertility. The probability of recovery to 20 million with 12 months is 90 and 100 % within 24 months. The recovery rates were slower with longer duration of treatment, use of longer acting hormonal preparations, and non-Asian ethnicity. The recovery rates were higher with older age, men who suppressed spermatogenesis faster, higher baseline sperm concentration, and lower baseline serum luteinizing hormone, but these effect sizes were very small [47••, 48]. The conclusion from this study was that all men would recover after hormonal male contraception indicating that efforts to develop this reversible and efficacious method of contraception should continue.

perm concentration, and lower baseline serum luteinizing hormone, but these effect sizes were very small [47••, 48]. The conclusion from this study was that all men would recover after hormonal male contraception indicating that efforts to develop this reversible and efficacious method of contraception should continue. Newer Agents Used for Hormonal Male Contraception The focus of hormonal male contraception development in recent years has been the development of novel androgens that may be more potent than testosterone in the suppression of gonadotropins and with fewer potential side effects. 7-Alpha-methyl-19-nortestosterone (MENT) cannot be 5-alpha reduced to dihydrotestosterone like steroids and may have less stimulating effects on the prostate gland. In rats, MENT is ten times more potent than testosterone in the muscle and only four times more potent in the prostate gland suggesting less simulating effects on the prostate compared to testosterone [49]. This was confirmed in monkeys [50]. Initial studies using MENT as an implant in men showed very effective suppression of spermatogenesis with four MENT implants (releasing a total of 1600 mcg/day MENT) [51]. MENT implants are under development to optimize the delivery rate as a 1-year implant by the Population Council [52].

was confirmed in monkeys [50]. Initial studies using MENT as an implant in men showed very effective suppression of spermatogenesis with four MENT implants (releasing a total of 1600 mcg/day MENT) [51]. MENT implants are under development to optimize the delivery rate as a 1-year implant by the Population Council [52]. Another androgen, dimethandrolone (DMA, 7-alpha,11-beta-dimethyl-19-nortestosterone) undecanoate, is being developed as an oral preparation as well as an intramuscular injection. In the body, DMA undecanoate (DMAU) is converted to DMA which is the active entity. In in vitro studies, DMA showed dose-dependent androgenic and progestational activities. The dual activities on the androgen and progesterone receptors may create a single agent capable of suppressing spermatogenesis while maintaining androgenic activity in men [53]. DMAU has antifertility effects in male rabbits [54]. A single-dose escalation study in healthy male volunteers showed that DMAU appeared to be safe and well-tolerated. DMAU has to be administered with a fatty meal to enhance absorption from the gastrointestinal tract. After administration of a single dose of DMAU powder (at a dose >200 mg/day), significant suppression of serum gonadotropins and testosterone occurred which had not been reported with other hormonal agents [55]. Repeat daily dosing of DMAU over 28 days is currently in progress where the goals are to study safety and tolerability as well as the extent of suppression of gonadotropins. It is also being formulated as an injection by the National Institute of Child Health and Human Development (NICHD), National Institutes of Health, USA, for hormonal male contraception studies. NICHD is also developing levonorgestrel butanoate as a long-acting progestin injection for male and female contraception.

ins. It is also being formulated as an injection by the National Institute of Child Health and Human Development (NICHD), National Institutes of Health, USA, for hormonal male contraception studies. NICHD is also developing levonorgestrel butanoate as a long-acting progestin injection for male and female contraception. Nestorone (16-methylene-17alpha-acetoxy-19-norpregn-4-ene-3,20-dione) is a potent progestin that has minimal androgen or estrogen activity. The Population Council is developing nestorone for both female and male contraception [52, 56, 57]. Nestorone applied transdermally has been used in combination with testosterone gel for hormonal contraception clinical trials in men. Nestorone and testosterone gels when applied on the skin suppressed both gonadotropins (LH and FSH) to very low levels in healthy male volunteers [58]. In a 6-month study, a combined gel (testosterone 100 mg/day and nestorone 8 mg/day on skin) suppressed sperm output to <1 million/ml in 89 % of men compared to 23 % of those who applied testosterone gel alone. There were minimal adverse effects [41•, 59]. Based on the studies, a contraceptive efficacy study is planned for 2016 using a combined nestorone and testosterone gel as a provider independent daily application on the skin.

ut to <1 million/ml in 89 % of men compared to 23 % of those who applied testosterone gel alone. There were minimal adverse effects [41•, 59]. Based on the studies, a contraceptive efficacy study is planned for 2016 using a combined nestorone and testosterone gel as a provider independent daily application on the skin. Men Will Use Male Contraception and Their Partners Approve Development of Male Contraception Acceptability of male contraception was studied with interviews in seven cities in Asia, North and South America, and Europe that were conducting male hormonal contraceptive studies supported by the World Health Organization. The study planned to gauge the male users’ evaluation of the hormonal method compared to other male methods and whether these hormonal methods modify sexual function and behavior [60]. The study showed that a daily pill or a monthly injectable might have the highest acceptability; monthly interviews appeared to be useful in assessing any sexual dysfunction associated with the new method. Other studies of men participating in clinical trials using various nonvalidated questionnaires showed that in some studies over 61 % of participants found testosterone undecanoate injectable method excellent or good [61], while other studies showed that transdermal application is acceptable in about 56 % of men [62, 63]. In the monthly injections of testosterone undecanoate clinical trial in China [27], most participants (male and female), providers, and policy makers thought that a new hormonal method would be acceptable if proven to be safe and effective. In the 308 men who were receiving the injections, 72.3 % thought monthly injections and semen analyses, and the need to use another contraceptive method during the period of sperm suppression were inconveniences that might reduce the acceptability of the method [64]. Future studies of acceptability of male contraceptive method will require the development of validated questionnaires. If the surveys are conducted in participants in male contraceptive trials, the data collected may be biased and are not reflective of the general population.

educe the acceptability of the method [64]. Future studies of acceptability of male contraceptive method will require the development of validated questionnaires. If the surveys are conducted in participants in male contraceptive trials, the data collected may be biased and are not reflective of the general population. Structured interviews using questionnaires in four cities (Edinburgh, Cape Town, Shanghai, and Hong Kong) in three countries, UK, South Africa, and China, showed that 44 to 83 % of men would use a male contraceptive pill. A pill was preferred to an injectable. Acceptability was variable and influenced by knowledge of other contraceptive methods and cultural background [65]. In another study that surveyed over 9000 men (18 to 50 years) in nine countries and four continents showed that in 55 to 81.5 % of couples, both partners were involved in selecting the method of contraception. From 28.5 to 71.4 % of men surveyed expressed willingness to use a new male method of contraception with wide variation among different racial and ethnic groups [66•]. In a study in England (n = 380), both men and women had favorable attitudes to a male contraceptive pill. Women had a more positive attitude to the new male pill but had less trust that men would use it effectively. Men in a stable relationship had a more positive attitude toward a male pill than those in casual relationships [67]. The attitude of female partners was also studied in 1894 women attending family planning clinics in Edinburgh, Cape Town, Shanghai, and Hong Kong. Over 90 % of women in Edinburgh and Cape Town considered a male pill a good idea, whereas in Hong Kong and Shanghai, the percent were lower at 71 and 87 %, respectively. Only 2 % of women would not trust their partner to use a male pill [68].

ing family planning clinics in Edinburgh, Cape Town, Shanghai, and Hong Kong. Over 90 % of women in Edinburgh and Cape Town considered a male pill a good idea, whereas in Hong Kong and Shanghai, the percent were lower at 71 and 87 %, respectively. Only 2 % of women would not trust their partner to use a male pill [68]. These studies in men and women provided evidence that if a new male contraceptive method is available, over 50 % of men may try to use it. Couples in stable relationships trust each other, and the female partner would trust the male taking a male contraceptive pill.

ing family planning clinics in Edinburgh, Cape Town, Shanghai, and Hong Kong. Over 90 % of women in Edinburgh and Cape Town considered a male pill a good idea, whereas in Hong Kong and Shanghai, the percent were lower at 71 and 87 %, respectively. Only 2 % of women would not trust their partner to use a male pill [68]. These studies in men and women provided evidence that if a new male contraceptive method is available, over 50 % of men may try to use it. Couples in stable relationships trust each other, and the female partner would trust the male taking a male contraceptive pill. Challenges to Bring Male Hormonal Methods to Become Available to Men Male contraception can fill an unmet need in contraception, and based on published surveys, this would be acceptable to many men and their partners. In addition, new male methods may relieve the contraceptive burden of women. Because the required visits to the clinic and interviews with physicians or providers knowledgeable in reproduction may make men more aware of their reproductive health. Since the 1970s, hormonal male contraceptive trials have been conducted in many parts of the world. Large-scale studies from 1990s and those from more recent times showed that hormonal contraception with androgens alone is very efficacious in Asian men and regimens with androgens and progestins are efficacious in all men. The possibility of escape from suppression of sperm production in men by an androgen alone regimen may be higher than in androgen and progestin combinations. The use of two agents for male contraception is more complex as the bioavailability and pharmacokinetics of the two entities should be complementary to each other. Mismatching injectable testosterone long-acting injections with a slower increase in serum testosterone level with shorter acting injectable progestins that have a rapid burst effect may result in inadequate androgen effects and symptoms of hypogonadism in the presence of high progestin levels. The current goals of clinical investigation are to find different combinations of androgens and progestins that have similar pharmacokinetics with additive action on suppression of spermatogenesis and minimal adverse effects. Newer hormonal steroids that have both androgenic and progestational activity are being developed in addition to DMAU. The selected agents must be safe without major adverse effects and can be delivered in a user friendly method, e.g., daily pill or transdermal gels or three monthly or yearly injections, and if possible without surgical intervention. The steady delivery of hormonal steroids for male contraception has been perceived to have less adverse events than those that provide serum peaks and troughs of the androgen or the progestin.

hod, e.g., daily pill or transdermal gels or three monthly or yearly injections, and if possible without surgical intervention. The steady delivery of hormonal steroids for male contraception has been perceived to have less adverse events than those that provide serum peaks and troughs of the androgen or the progestin. The goal is to develop a number of male hormonal contraceptives such as long-acting injectables, implants, pills, and transdermal gels to meet the preferences of men and their partners. The economic aspects of male hormonal contraception have been studied. Assuming a market size of 10 million men in the USA and 50 million men worldwide, the market value of a new male contraceptive method is estimated to be worth 40 to 200 billion dollars [69]. The NICHD currently supports the development of novel targets for male contraception as well as funding to male hormonal contraceptive clinical trials. The Population Council’s contraceptive development focuses on Nestorone and MENT.

new male contraceptive method is estimated to be worth 40 to 200 billion dollars [69]. The NICHD currently supports the development of novel targets for male contraception as well as funding to male hormonal contraceptive clinical trials. The Population Council’s contraceptive development focuses on Nestorone and MENT. The pharmaceutical industry has withdrawn support in research and development of contraception. This lack of industry support despite a large potential market is disappointing but may be due to several concerns including that men will not use the prescribed contraception method consistently, cost of male hormonal methods and differential pricing for developed and developing countries, and potential unknown long term side effects. Thus, government and academia will need to collaborate with community advocates to generate interest in smaller, specialty pharmaceutical companies that are interested in reproductive health. Advocacy from public interest groups will also help to push the agenda forward for male contraceptive development.

side effects. Thus, government and academia will need to collaborate with community advocates to generate interest in smaller, specialty pharmaceutical companies that are interested in reproductive health. Advocacy from public interest groups will also help to push the agenda forward for male contraceptive development. Conclusion Large-scale clinical trials in men proved that when sperm output is suppressed to a very low level, the contraceptive efficacy is very high. Androgens plus a progestin will most likely be the first hormonal contraception method for men. The matching of a progestin to testosterone may be more complex, and a balance between the two steroids may be necessary to produce a viable, safe, and reversible male hormonal contraception. Surveys in men and women in the reproductive age group suggest that many men and women would accept male hormonal methods as a contraceptive option. Partnership between government, nongovernment agencies, academia, and industry will identify the best combinations of steroids to bring forward, conduct clinical trials, and submit reports to regulatory agencies for approval, and ensure accessibility of these approved agents by all men. This article is part of the Topical Collection on Family Planning

Conclusion Large-scale clinical trials in men proved that when sperm output is suppressed to a very low level, the contraceptive efficacy is very high. Androgens plus a progestin will most likely be the first hormonal contraception method for men. The matching of a progestin to testosterone may be more complex, and a balance between the two steroids may be necessary to produce a viable, safe, and reversible male hormonal contraception. Surveys in men and women in the reproductive age group suggest that many men and women would accept male hormonal methods as a contraceptive option. Partnership between government, nongovernment agencies, academia, and industry will identify the best combinations of steroids to bring forward, conduct clinical trials, and submit reports to regulatory agencies for approval, and ensure accessibility of these approved agents by all men. This article is part of the Topical Collection on Family Planning Drs. Wang and Swerdloff are supported by research contracts from the NICHD NO1 Contract HHSN275220130024I and research support from Clarus Therapeutics, Lipocine, Besins Healthcare International. Dr. Wang is supported by the National Center for Advancing Translational Sciences through UCLA CTSI Grant UL1TR000124 and a grant from UNDP/UNFPA/UNICEF/WHO/World Bank Special Programme of Research, Development, and Research Training in Human Reproduction.

Clarus Therapeutics, Lipocine, Besins Healthcare International. Dr. Wang is supported by the National Center for Advancing Translational Sciences through UCLA CTSI Grant UL1TR000124 and a grant from UNDP/UNFPA/UNICEF/WHO/World Bank Special Programme of Research, Development, and Research Training in Human Reproduction. Compliance with Ethical Standards Conflict of Interest Christina Wang declares grants from UNDP/UNFPA/UNICEF/WHO/World Bank Special Programme of Research, Development, and Research Training in Human Reproduction, and grants from NIH, NICHD; she also declares grants from Clarus Therapeutics, Antares, and Besins Healthcare International; she declares a grant and personal fees from Lipocine (temporary advisor) and personal fees from TesoRX (temporary advisor). Mario P. R. Festin declares no conflict of interest. Ronald S. Swerdloff declares grants from NIH, NICHD, and grants from Clarus Therapeutics, Besins Healthcare International, and Antares; he declares a grant and personal fees from Lipocine and personal fees from TesoRX. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

Introduction Over the last decade, prescription opioid misuse and illicit heroin use have skyrocketed, and overdose deaths have quadrupled [1]. From 2002 to 2013, the largest increase in heroin use was among women [2]. A 2014 SAMHSA report showed female treatment admissions for opioid pain relievers as the primary substance of abuse outnumber male admissions in all age categories [3]. The rate of opioid use during pregnancy is approximately 5.6 per 1000 live births [4], with one study reporting greater than 85 % of pregnancies in women with opioid use disorder were unintended [5]. Given the high rates of opioid use among US women of childbearing age, and high incidence of unintended pregnancy in those with opioid use disorder, obstetric providers will need to be prepared to care for this growing demographic. Despite the increasing acceptance of substance use disorder as a chronic illness, only 11 % of the 24 million Americans with substance use disorder receive treatment [6], and stigma remains a significant barrier to treatment. For pregnant women, this is especially potent. Barriers to care include lack of access to gender-specific care, limited child-care availability at treatment facilities [7], few providers with obstetrics and addiction treatment expertise [8], increased social stigma, and fear of criminal or child welfare consequences. Only 19 states have funded drug treatment programs specifically designed for pregnant women, and 12 states provide pregnant women with priority access to treatment programs.

few providers with obstetrics and addiction treatment expertise [8], increased social stigma, and fear of criminal or child welfare consequences. Only 19 states have funded drug treatment programs specifically designed for pregnant women, and 12 states provide pregnant women with priority access to treatment programs. State policies regarding the response to and reporting of substance use disorder in pregnancy vary wildly. Recently in some states, policymakers have debated legislation criminalizing pregnant women with substance use disorders. In 2014, Tennessee became the first state to pass a law criminalizing illicit drug use during pregnancy.1 Eighteen states require health care professionals to report suspected prenatal use of illicit substances, four require health care professionals to test for prenatal drug exposure if substance use disorders are suspected, and 18 states consider substance abuse during pregnancy to be child abuse under civil child-welfare status [9]. Low-income women and women of color are at higher risk for barriers to appropriate care of substance use disorders during pregnancy, in part explaining the poor perinatal outcomes associated with this population [10]. One study found a clear association between little or no prenatal care and opioid use, with a cohort of postpartum patients reporting external locus of control, fear of being reported to the police, and disbelief in the efficacy of care as factors [11].

the poor perinatal outcomes associated with this population [10]. One study found a clear association between little or no prenatal care and opioid use, with a cohort of postpartum patients reporting external locus of control, fear of being reported to the police, and disbelief in the efficacy of care as factors [11]. In the context of rising opioid deaths and increased attention from policymakers, the Department of Health and Human Services has suggested three strategies to reduce deaths: safe prescribing, expansion of medication treatment, and expansion of naloxone access. In the following review, we will address pharmacologic treatment options for pregnant women with opioid use disorder, with special consideration of mood disorders, trauma history, and breastfeeding. Should Pregnant Women with Opioid Use Disorder Undergo “Detoxification”? Opioid withdrawal (detoxification) in pregnancy is complex and has both risks associated with withdrawal and of relapse for the mother and the fetus. Current practice recommendations are to avoid opioid withdrawal (detoxification) as the benefits of opioid agonist treatment (OAT) for the mother and the fetus exceed the risks [12]. This recommendation was originally based on a few case studies from the 1970s associating opioid withdrawal with fetal death, miscarriage, and preterm labor [13–15].

ions are to avoid opioid withdrawal (detoxification) as the benefits of opioid agonist treatment (OAT) for the mother and the fetus exceed the risks [12]. This recommendation was originally based on a few case studies from the 1970s associating opioid withdrawal with fetal death, miscarriage, and preterm labor [13–15]. Retrospective studies have attempted to evaluate the risk of fetal loss with maternal opioid withdrawal. Luty et al. concluded opioid withdrawal in the first trimester may be associated with miscarriage but that it was likely safer in the second and third trimesters [16]. Several studies evaluating opioid withdrawal in pregnancy have not shown significant risk of fetal loss, often with miscarriage rates comparable to population norms [17, 18, 19•, 20]. Unfortunately, these studies are underpowered and contain confounders making the risk of pregnancy loss secondary to opioid withdraw difficult to quantify. Maternal opioid withdrawal does decrease incidence and duration of neonatal abstinence syndrome [17, 18]. Despite recent studies suggesting fetal safety of medically assisted opioid withdrawal in pregnancy, abstinence of maternal drug use was low and maternal relapse rates range from 59 to 99 % [18, 19•]. We believe opioid withdrawal is an inferior option compared to OAT for pregnant women, and risk of maternal relapse, with inherent overdose mortality risk, outweighs the potential reduction in NAS. In the general population, OAT is the standard of care for opioid use disorder; a standard which should not be altered by pregnancy.

opioid withdrawal is an inferior option compared to OAT for pregnant women, and risk of maternal relapse, with inherent overdose mortality risk, outweighs the potential reduction in NAS. In the general population, OAT is the standard of care for opioid use disorder; a standard which should not be altered by pregnancy. Treatment Options for Pregnant Women: Opioid Agonist Treatment OAT is the first-line recommendation for pregnant women with opioid use disorders. The goals of treatment are to manage withdrawal, reduce cravings, and provide opioid blockade (preventing euphoria from illicit use). The goals of OAT in pregnancy are to prevent illicit opioid use which can increase the risk of fetal growth restriction, abruptio placentae, fetal death, preterm labor, and intrauterine passage of meconium. OAT has been shown to increase adherence to prenatal care, reduce illicit drug use, reduce infection exposure secondary to IVDU, such as HIV, HCV, HCB, improve maternal nutrition, and improve infant birth weight [21].

wth restriction, abruptio placentae, fetal death, preterm labor, and intrauterine passage of meconium. OAT has been shown to increase adherence to prenatal care, reduce illicit drug use, reduce infection exposure secondary to IVDU, such as HIV, HCV, HCB, improve maternal nutrition, and improve infant birth weight [21]. Neonatal abstinence syndrome (NAS) is an expected and treatable condition due to prenatal opioid exposure. Forty to 80 % of exposed neonates develop withdrawal requiring hospitalization and pharmacotherapy. Historically, methadone has been the gold standard OAT choice for pregnant women. Emerging evidence supports the use of buprenorphine as an effective therapeutic option in pregnancy and shows improved neonatal outcomes. A 2010 multicenter randomized clinical trial showed buprenorphine-exposed neonates required, on average, 89 % less morphine to treat NAS, a 43 % shorter hospital stay, and a 58 % shorter duration of medical treatment for NAS compared to methadone-exposed newborns. These results support the use of buprenorphine as a potential first-line medication for pregnant opioid-dependent women [22••].

d neonates required, on average, 89 % less morphine to treat NAS, a 43 % shorter hospital stay, and a 58 % shorter duration of medical treatment for NAS compared to methadone-exposed newborns. These results support the use of buprenorphine as a potential first-line medication for pregnant opioid-dependent women [22••]. The advantages of buprenorphine over methadone include a lower risk of overdose, fewer drug interactions, office-based treatment delivery, and shorter NAS course. The disadvantages compared with methadone include potential hepatic dysfunction, lack of long-term data on consequences of fetal exposure for the infant, potential limited efficacy in patients with high opioid debt, requirement of moderate withdrawal symptoms prior to initiation to avoid iatrogenic withdrawal, and an increased risk of diversion (i.e., sharing or sale) [21]. To date, studies have not assessed the impact of maternal opioid addiction severity on newborn outcomes and maternal long-term recovery. For the provider, the choice of OAT type to initiate must be individualized and is often restricted by availability and access to services. We recommend, whenever possible, to optimize patient autonomy in this decision. Despite buprenorphine’s neonatal advantages, it will not be effective for all women. We suggest the best neonatal outcomes will be achieved by providing the most appropriate and effective treatment for the mother.

ty and access to services. We recommend, whenever possible, to optimize patient autonomy in this decision. Despite buprenorphine’s neonatal advantages, it will not be effective for all women. We suggest the best neonatal outcomes will be achieved by providing the most appropriate and effective treatment for the mother. Overdose and the Use of Naloxone in Pregnancy Currently, opioid overdose is a leading cause of death in Americans 25–45 years of age. Education and expansion of access to naloxone is one evidence-based strategy to address this issue. No study has looked at pregnancy outcomes in patients treated with naloxone for acute opioid overdose, but the use of naloxone for resuscitation in overdose should not be withheld out of concern for the developing fetus. Despite potential negative effects of acute opioid withdrawal on the fetus, including stillbirth, fetal distress, and premature labor [18], prevention of maternal death from opioid overdose is the priority. Extended Release Naltrexone Use in Pregnancy Naltrexone (pregnancy category C) is a non-selective opioid receptor antagonist which has shown promise in treating opioid use disorders by decreasing drug-seeking behaviors, drug cravings, and increasing treatment retention [23–25].

Overdose and the Use of Naloxone in Pregnancy Currently, opioid overdose is a leading cause of death in Americans 25–45 years of age. Education and expansion of access to naloxone is one evidence-based strategy to address this issue. No study has looked at pregnancy outcomes in patients treated with naloxone for acute opioid overdose, but the use of naloxone for resuscitation in overdose should not be withheld out of concern for the developing fetus. Despite potential negative effects of acute opioid withdrawal on the fetus, including stillbirth, fetal distress, and premature labor [18], prevention of maternal death from opioid overdose is the priority. Extended Release Naltrexone Use in Pregnancy Naltrexone (pregnancy category C) is a non-selective opioid receptor antagonist which has shown promise in treating opioid use disorders by decreasing drug-seeking behaviors, drug cravings, and increasing treatment retention [23–25]. Naltrexone has the potential to treat select opioid-dependent pregnant women while eliminating the risk of neonatal abstinence syndrome; however, limited human data are available on naltrexone’s safety and efficacy in pregnancy. Animal studies using 175 times the recommended human dosing have not shown teratogenic effects [26]. Opioid receptors in fetal mammalian tissue exposed to exogenous opioids or opioid antagonists may alter neurobiology and growth [27]. Studies of human-equivalent dosing of implantable naltrexone in rodents show no changes in offspring’s brain morphometry. Behavioral changes in these naltrexone-exposed offspring suggest possible alterations in morphine receptor sensitivity which may persist [28]. Case reports of depot naltrexone exposure in humans have not shown teratogenic effects [29], and in comparison with MMT, it shows lower PTD rates, higher birth weights, and improved APGAR scores. No significant differences in gestational age at birth or birth weight have been noted between non-exposed human neonates and naltrexone implant exposed [30]. Naltrexone does not demonstrate tolerance mitigating the need for increase doses in pregnancy [31].

r PTD rates, higher birth weights, and improved APGAR scores. No significant differences in gestational age at birth or birth weight have been noted between non-exposed human neonates and naltrexone implant exposed [30]. Naltrexone does not demonstrate tolerance mitigating the need for increase doses in pregnancy [31]. Induction of depot naltrexone during pregnancy is a complicated proposal and is not currently recommended. OAT remains the standard of care in pregnancy due to improved adherence to addiction treatment services, prenatal care, and in-hospital delivery [19•]. However, women who are stable on extended release naltrexone who become pregnant, based on human and animal data available to date, could be reasonably continued on naltrexone through pregnancy. Individualized treatment plans weighing the risk of altering therapy (stopping naltrexone) and potential destabilization of maternal recovery must be considered. The risk of relapse and possible overdose with such an alteration in addiction treatment may significantly outweigh the naltrexone exposure risk to the fetus alone. Future research on in utero exposure and perioperative pain management of extended release naltrexone are needed [32•].

ternal recovery must be considered. The risk of relapse and possible overdose with such an alteration in addiction treatment may significantly outweigh the naltrexone exposure risk to the fetus alone. Future research on in utero exposure and perioperative pain management of extended release naltrexone are needed [32•]. Special Considerations: Trauma, Mood Disorders, Breastfeeding Trauma and Substance Use Disorder in Women Prior traumatic experiences from exposure to physical, sexual, and emotional abuse are common among women with substance use disorder, with estimates ranging from 50 to 80 % [33–35]. Pregnancy can be a vulnerable and triggering time for women with trauma histories. A recent qualitative study by Torchalla et al. of pregnant and newly parenting women with substance use disorder in Vancouver identified that all women reported childhood adversity and 77 % reported prior sexual abuse [36]. This study identified themes of continued adverse experiences persisting into adulthood and high rates of intimate partner violence during pregnancy. Women expressed concern over the possible trans-generational effects of their trauma on their infants, but the majority of women questioned had not received any trauma-specific services, and many expressed ambivalence or reluctance in participating in trauma counseling.

es of intimate partner violence during pregnancy. Women expressed concern over the possible trans-generational effects of their trauma on their infants, but the majority of women questioned had not received any trauma-specific services, and many expressed ambivalence or reluctance in participating in trauma counseling. Trauma-informed care, or the principle that service delivery is designed with an understanding of the impact of victimization and trauma, has become more prevalent as a method to care for women in gender-specific substance use treatment programs [37, 38]. Yet, a recent secondary database review from publicly available data sets by Terplan et al. examined 13,000 addiction treatment facilities in the USA and found that trauma-related services were selectively available in urban centers and states with larger populations, and a large percentage of women in treatment programs reported unmet needs [39•]. A case study by Goodman et al. examined the complexities of providing obstetric care for women with comorbid PTSD and substance use and stressed the importance of early screening and a coordinated multidisciplinary approach to improving outcomes for the substance-exposed mother-infant dyad [40]. Mood Disorders in Women with Opioid Use Disorders According to the National Survey of Substance Abuse Treatment Services, 45 % of Americans seeking treatment for substance use disorders have co-occurring mental health disorders, specifically depression and anxiety [41].

Trauma-informed care, or the principle that service delivery is designed with an understanding of the impact of victimization and trauma, has become more prevalent as a method to care for women in gender-specific substance use treatment programs [37, 38]. Yet, a recent secondary database review from publicly available data sets by Terplan et al. examined 13,000 addiction treatment facilities in the USA and found that trauma-related services were selectively available in urban centers and states with larger populations, and a large percentage of women in treatment programs reported unmet needs [39•]. A case study by Goodman et al. examined the complexities of providing obstetric care for women with comorbid PTSD and substance use and stressed the importance of early screening and a coordinated multidisciplinary approach to improving outcomes for the substance-exposed mother-infant dyad [40]. Mood Disorders in Women with Opioid Use Disorders According to the National Survey of Substance Abuse Treatment Services, 45 % of Americans seeking treatment for substance use disorders have co-occurring mental health disorders, specifically depression and anxiety [41]. Infants of untreated, depressed mothers demonstrate reduced scores for motor adaptation and self-regulation, have higher arousal scores, are more difficult to console, and can exhibit developmental delay and poor neonatal attachment [42–44].

Mood Disorders in Women with Opioid Use Disorders According to the National Survey of Substance Abuse Treatment Services, 45 % of Americans seeking treatment for substance use disorders have co-occurring mental health disorders, specifically depression and anxiety [41]. Infants of untreated, depressed mothers demonstrate reduced scores for motor adaptation and self-regulation, have higher arousal scores, are more difficult to console, and can exhibit developmental delay and poor neonatal attachment [42–44]. Several well-conducted reviews demonstrate the safety of selective serotonin reuptake inhibitors (SSRIs) in pregnancy, with the exception of paroxetine, which may increase the risk of cardiac defects [27]. The most common and the best established adverse effect of SSRIs is poor neonatal adaptation (PNA); PNA is prolonged when SSRIs are combined with benzodiazepines [45]. In addition, SSRIs in combination with opioids have been shown to worsen severity of NAS [46, 47]. The decision of when and how to treat perinatal depression is a complex risk-benefit algorithm. Mild to moderate depression should probably be treated with cognitive behavioral therapy or interpersonal therapy (if available). The studies supporting this are small but CBT and IPT are proven effective treatments for depression [48].

of when and how to treat perinatal depression is a complex risk-benefit algorithm. Mild to moderate depression should probably be treated with cognitive behavioral therapy or interpersonal therapy (if available). The studies supporting this are small but CBT and IPT are proven effective treatments for depression [48]. For anxiety disorders, SSRIs should be considered the first-line pharmacotherapy. Benzodiazepines are relatively contraindicated in women with opioid use disorders. For patients on OAT, benzodiazepines exacerbate the sedating effect of methadone and increase accidental injury, especially in women prescribed with buprenorphine [49]. Neonates exposed in utero to both OAT and benzodiazepines experience prolonged NAS [46]. The authors caution that benzodiazepine use disorder and benzodiazepine diversion are growing public health concerns and recommend vigilance in prescribing practice.

l injury, especially in women prescribed with buprenorphine [49]. Neonates exposed in utero to both OAT and benzodiazepines experience prolonged NAS [46]. The authors caution that benzodiazepine use disorder and benzodiazepine diversion are growing public health concerns and recommend vigilance in prescribing practice. Breastfeeding and OAT in Pregnancy Breastfeeding is recommended for women on OAT. Methadone and buprenorphine are both lactation category C and transferred via the breast milk but in amounts <1 % of the maternal dose [50–53]. Published guidelines from the American Academy of Pediatrics (AAP), the American College of Obstetricians and Gynecologists (ACOG), and the Academy of Breastfeeding Medicine (ABM) all support breastfeeding for women stabilized on OAT [54–56]. The 2015 ABM statement includes criteria for encouraging breastfeeding among mothers with the following: (1) compliance with a substance use disorder treatment program, (2) consistent prenatal care, (3) negative urine toxicology screen at the time of delivery, and (4) negative urine screens for 30–90 days prior to delivery [54]. One institution expanded on the ABM criteria by (1) defining consistent prenatal care as attendance of at least 50 % of scheduled visits, including two visits within the last 2 months, and (2) creating a “4-week guideline” of no positive urine drug screens for breastfeeding initiation, leading to subsequent improved breastfeeding rates [57]. The use of prenatal urine drug screens as a predictor for postpartum recovery remains unknown despite these recommendations.

two visits within the last 2 months, and (2) creating a “4-week guideline” of no positive urine drug screens for breastfeeding initiation, leading to subsequent improved breastfeeding rates [57]. The use of prenatal urine drug screens as a predictor for postpartum recovery remains unknown despite these recommendations. For women on OAT, breastfeeding can be a complex issue. Despite AAP, ACOG, and ABM statements endorsing the benefits of breastfeeding on OAT and its safety with coexisting hepatitis C status, policies differ among institutions. Policies may reflect provider fear of maternal relapse, concern for concurrent psychiatric medications, and how these may complicate the course of NAS [58–60]. However, numerous studies have shown a strong association between breastfeeding and improved NAS symptoms, with reduction in need for pharmacotherapy and shortened hospitalizations [61–64]. In addition, there are significant maternal benefits including reinforcing sobriety, enhancing maternal self-esteem, and the promoting mother-infant bonding. Previous studies have not differentiated between direct breastfeeding and pumped breast milk, nor mixed feeding versus exclusive breastfeeding. The benefit is likely related to maternal presence at the bedside, skin-to-skin contact, and engagement in care.

maternal self-esteem, and the promoting mother-infant bonding. Previous studies have not differentiated between direct breastfeeding and pumped breast milk, nor mixed feeding versus exclusive breastfeeding. The benefit is likely related to maternal presence at the bedside, skin-to-skin contact, and engagement in care. Conclusion Over the past 10 years, there has been a surge of illicit opioid use in the USA. Pregnant women with opioid use disorder are at an increased risk for negative birth outcomes. Limited access to care, social stigma, inadequate insurance coverage, and fear of legal consequences are some of the many barriers to care for pregnant women. The current standard of care for opioid use disorder in pregnancy is an opioid agonist treatment. Assisted opioid withdrawal is not recommended due to the high risk of relapse. Extended release naltrexone is a viable option for carefully selected women. With further study, the use of buprenorphine + naloxone (dual-therapy) may replace treatment with buprenorphine (mono-therapy) in pregnancy. Opioid use disorder treatment should be gender specific, and prenatal care programs should incorporate trauma-informed care and provide mental health services. Initiation of pharmacologic treatment for mental health disorders must consider maternal efficacy, maternal and fetal safety, and risk of NAS potentiation. Breastfeeding for women on OAT is safe and should be supported to decrease onset, duration, and severity of NAS.

a-informed care and provide mental health services. Initiation of pharmacologic treatment for mental health disorders must consider maternal efficacy, maternal and fetal safety, and risk of NAS potentiation. Breastfeeding for women on OAT is safe and should be supported to decrease onset, duration, and severity of NAS. In our current healthcare system, the expansion of safe, effective, and dignified multidisciplinary treatment options for pregnant women with opioid use disorders is greatly needed. Expansion of access to care for pregnant women can be achieved by increasing the number of buprenorphine-waivered obstetric providers and expanding comprehensive obstetric and addiction medicine models of care. Obstetric providers must strive to decrease the stigma associated with the disease, reduce the barriers to care, and implement comprehensive programs to meet the rising demand. Our recommendations for health care systems to provide to pregnant women with opioid use disorder can be found in Table 1.Table 1 Recommendations for health care systems to provide to pregnant women with opioid use disorder o Access to opioid agonist treatment options Methadone or buprenorphine o Access to obstetric care Recovery-affirming and trauma-informed Comprehensive obstetric and addiction medicine services Group prenatal care as an option o Access to psychiatry consultation: assessment and treatment options for co-occurring disorders o Access to behavioral health counseling: weekly individual or group counseling o Resource guides for community-based relapse prevention

Recovery-affirming and trauma-informed Comprehensive obstetric and addiction medicine services Group prenatal care as an option o Access to psychiatry consultation: assessment and treatment options for co-occurring disorders o Access to behavioral health counseling: weekly individual or group counseling o Resource guides for community-based relapse prevention Mutual aid support groups Mothers-in-recovery groups o Development of enhanced postpartum care: program development to intensify recovery support potentially utilizing peer supports Close follow-up (<2 weeks from delivery) Allow for multiple postpartum visits Consider visits every 2 weeks for 3–6 visits Breastfeeding/lactation support Screening/treatment for postpartum depression Transition to a primary care provider familiar with opioid use disorder and its treatment 1 Due to opposition from health care professionals and patient advocacy groups, this law is currently being repealed. An erratum to this article can be found at http://dx.doi.org/10.1007/s13669-016-0176-9. Compliance with Ethical Standards Conflict of Interest Kelley A. Saia, Davida Schiff, Elisha M. Wachman, Annmarie Vilkins, Michelle Sia, Jordana Price, Tirah Samura, Justin DeAngelis, Clark V. Jackson, Sawyer F. Emmer, and Daniel Shaw declare that they have no conflict of interest. Pooja Mehta and Sarah Bagley declare funding from the ASAM-Millennium Research Institute research fellowship award, unrelated to this work. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.