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INTRODUCTION Prenatal administration of synthetic corticosteroids has been the standard procedure in cases of preterm delivery since 1972, when Liggins and Howie [1] demonstrated that steroid treatment has beneficial effects on the incidence of respiratory distress syndrome (RDS) and neonatal mortality associated with premature birth before 34 weeks’ gestation. A Cochrane Database review published in 2006 summarized 21 studies including 3885 mothers and 4269 infants and confirmed significant reductions in the risks of mortality, RDS and intraventricular haemorrhage (IVH) in preterm infants of 31, 44 and 46%, respectively, after a single course of steroids [2]. Steroids given prenatally also likely decrease the risks of necrotizing enterocolitis (NEC), neonatal ICU (NICU) admission and infection in newborns during the first 48 h of life, as well as the need for respiratory support. Antenatal steroids have not been reported to affect birth weight, delay foetal central nervous system development or increase the risk of maternal death, intra-amniotic infection or puerperal sepsis [2].

ICU) admission and infection in newborns during the first 48 h of life, as well as the need for respiratory support. Antenatal steroids have not been reported to affect birth weight, delay foetal central nervous system development or increase the risk of maternal death, intra-amniotic infection or puerperal sepsis [2]. Neonatal respiratory outcomes differ among ethnic groups independent of birth weight and gestational age [3]. Haas et al. [4▪▪] showed that genetic polymorphisms in maternal and foetal genotypes for drug-metabolizing enzymes and steroid pathway genes were independently associated with neonatal RDS after treatment with betamethasone for preterm labour. The respiratory outcome severity, including bronchopulmonary dysplasia (BPD) and the need for respiratory support or surfactant use, may be associated with single maternal and foetal nucleotide polymorphisms in key betamethasone pathways [5▪▪]. Box 1 no caption available

Neonatal respiratory outcomes differ among ethnic groups independent of birth weight and gestational age [3]. Haas et al. [4▪▪] showed that genetic polymorphisms in maternal and foetal genotypes for drug-metabolizing enzymes and steroid pathway genes were independently associated with neonatal RDS after treatment with betamethasone for preterm labour. The respiratory outcome severity, including bronchopulmonary dysplasia (BPD) and the need for respiratory support or surfactant use, may be associated with single maternal and foetal nucleotide polymorphisms in key betamethasone pathways [5▪▪]. Box 1 no caption available The mechanism of steroid action is complex and affects not only foetal lung maturation [6,7] but also regulation of foetal growth, organ system maturation and the functions of the immune system and sympathetic nervous system [8]. Alteration of the hypothalamic-pituitary-adrenocortical (HPA) axis is a primary consequence observed in the offspring [9]. Steroids play important roles in foetal brain development, altering neuronal migration, synaptic plasticity and neurotransmitter activity [10]. Synthetic corticosteroids given prenatally are not readily metabolized by placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) and cross the placenta more easily; their influence on the foetal brain may be more pronounced than natural glucocorticoids [11]. Ma et al.[12▪] showed increased expression of 11β-HSD2 in pregnant women with diet-treated GDM, which may limit excessive exposure of the foetus to glucocorticoids.

ogenase type 2 (11β-HSD2) and cross the placenta more easily; their influence on the foetal brain may be more pronounced than natural glucocorticoids [11]. Ma et al.[12▪] showed increased expression of 11β-HSD2 in pregnant women with diet-treated GDM, which may limit excessive exposure of the foetus to glucocorticoids. In 68% of cases, steroid administration transiently decreases vascular resistance in the placental vessels, and the effects last up to 3 days [13]. Clinically, a transient reduction in foetal movements is observed [14]. Foetal exposure to betamethasone also reduces foetal heart rate variability and breathing activity and increases foetal quiescence [15]. Although the effects of antenatal corticosteroids on lung maturation appear to be dose dependent, the biological effects and optimal dosage regimen of antenatal glucocorticoids remain under investigation. The short-term and long-term effects on other systems, particularly the central nervous system, and neurodevelopmental outcomes after different doses and repeat courses require further research. The type of steroid, the dosage and the timing of exposure determine the magnitude of the effects of antenatal steroid treatment on the foetus and mother.

effects on other systems, particularly the central nervous system, and neurodevelopmental outcomes after different doses and repeat courses require further research. The type of steroid, the dosage and the timing of exposure determine the magnitude of the effects of antenatal steroid treatment on the foetus and mother. STEROID TYPES According to the current standard of practice, women at risk of preterm delivery at 24–34 weeks’ gestation receive either a single course of betamethasone intramuscularly in two 12-mg doses at a 24-h interval or dexamethasone in four 6-mg doses at 12-h intervals [16,17]. Although dexamethasone is characterized by a greater affinity for glucocorticoid receptors, betamethasone has a longer half-life [18]. Dexamethasone and betamethasone similarly reduce the risk of perinatal death and alter biophysical activity, although dexamethasone more effectively decreases the incidence of IVH [18]. However, betamethasone is less frequently associated with adverse neurological outcomes [19]. Unlike betamethasone acetate, Jobe et al.[20] showed that a single dose of betamethasone phosphate did not induce foetal lung maturation in sheep. To maintain maximal occupancy of glucocorticoid receptors in tissues, a 1 : 1 mixture of betamethasone phosphate and betamethasone acetate is optimal. The current data are insufficient to recommend one steroid over the other, although a recent study by Remesal et al.[21] suggests that betamethasone might be a better choice than dexamethasone due to stronger inhibition of the expression of phospholipase A2 through the reduction of tumour necrosis factor α in the lungs of newborn rats.

are insufficient to recommend one steroid over the other, although a recent study by Remesal et al.[21] suggests that betamethasone might be a better choice than dexamethasone due to stronger inhibition of the expression of phospholipase A2 through the reduction of tumour necrosis factor α in the lungs of newborn rats. DOSAGE REGIMEN The guideline-recommended regimen (two 12-mg doses separated by 24 h) was developed to mimic the natural secretion of steroid hormones in preterm infants, which allows for 75% saturation of steroid receptors in foetal tissues [22]. The 24-h dosage interval was selected arbitrarily [18]. Prior to the second dose, the cord blood levels of betamethasone decrease to those observed in untreated children [23]. The more frequent administration of 12 mg or lower doses may allow the steroids to reach the foetus in a nearly constant and even manner [24▪▪]. The total dose of 24 mg was based on animal studies and is sufficient to achieve the steroid concentrations observed in infants after birth during normal physiological stress [25]. Giamfi et al.[26] showed that maternal serum and cord blood betamethasone concentrations did not differ between twin and singleton pregnancies.

total dose of 24 mg was based on animal studies and is sufficient to achieve the steroid concentrations observed in infants after birth during normal physiological stress [25]. Giamfi et al.[26] showed that maternal serum and cord blood betamethasone concentrations did not differ between twin and singleton pregnancies. The superiority of the generally established regimen over the alternatives has not been demonstrated. Pharmacokinetic data show that betamethasone levels in maternal blood reach the lowest level by 12 h postadministration [23]. Khandelwal et al.[24▪▪] conducted a randomized, noninferiority open trial comparing the effects of two betamethasone regimens: 12 mg over 12-h versus 24-h dosing intervals at 23–34 weeks’ gestation. There were no differences in the RDS incidence, although an increased NEC incidence was observed with 12-h dosing (P = 0.03). If all the women had received the 24-h dosing schedule, approximately 12% of the neonates would not have received the complete course before delivery. In maternal serum, betamethasone levels decreased by more than 50% within 6 h after administration [23]. Our study compared the effects of two betamethasone dosage regimens: six 4-mg doses at 8-h intervals versus two 12-mg doses at a 24-h interval [27▪▪]. The incidences of moderate or severe RDS, IVH and NEC, neonatal infection and anaemia did not differ between groups. Only mild RDS occurred more often in the 4-mg dose group (17.8 versus 15.3%; P = 0.055). The higher single steroid dose most commonly caused maternal anaemia and a significant increase in leukocytosis. The administration of a total dose of 24 mg is most likely the most important for maximal neonatal benefits, but a lower single steroid dose may be useful to reduce maternal side effects, including those in patients with prepregnancy diabetes [28,29].

caused maternal anaemia and a significant increase in leukocytosis. The administration of a total dose of 24 mg is most likely the most important for maximal neonatal benefits, but a lower single steroid dose may be useful to reduce maternal side effects, including those in patients with prepregnancy diabetes [28,29]. DURATION OF STEROID ACTION There are two important aspects concerning the duration of steroid action: the interval between the administration and the start of protection from neonatal complications and the duration of the positive effect. Throughout treatment, foetal serum betamethasone levels are approximately one-third of maternal levels [23]. Hormones are eliminated from the maternal and foetal circulation after 48 h, and the clinical benefit of these compounds is greatly reduced after 7 days [30]. The optimum benefits of glucocorticoid use are achieved 24 h after treatment initiation [16]. Antenatal steroids are most effective when the delivery occurs within 7 days after dose completion, when the reduction in the RDS risk is nearly 50% [2]. Antenatal steroids can prevent RDS for up to 14 days, and there is no linear association between the treatment-to-delivery interval and RDS occurrence [30,31].

[16]. Antenatal steroids are most effective when the delivery occurs within 7 days after dose completion, when the reduction in the RDS risk is nearly 50% [2]. Antenatal steroids can prevent RDS for up to 14 days, and there is no linear association between the treatment-to-delivery interval and RDS occurrence [30,31]. EXPOSURE TIME The efficacy of antenatal steroid treatment depends on gestational age. Mori et al.[32] estimated the effects of steroids in 11 067 children born at 22–33 weeks’ gestation. Antenatal steroids reduced the RDS risk by 9% in the entire cohort, but the effect was significant in infants born at 24–29 weeks’ gestation, reducing the RDS incidence by nearly 20%. Similarly, steroid treatment significantly reduced the risk of IVH in neonates born at 24–29 weeks’ gestation. In a randomized study of 320 patients, Porto et al.[33] demonstrated that steroid administration at gestational weeks 34–36 did not reduce the RDS incidence in newborn infants. Recently, in neonates born at gestational ages of 34 weeks or later, Kamath-Rayne et al.[34▪] confirmed that antenatal corticosteroid treatment following the determination of foetal lung immaturity did not reduce respiratory morbidity. Similar results were shown by Yinon et al.[35▪], who found no differences in RDS incidence between late premature infants treated with steroids and untreated infants, although the neonates given steroids prenatally less frequently presented composite respiratory morbidity (8.4 versus 21%; P = 0.02) or required ventilator support (8.4 versus 20%; P = 0.03). The Royal College of Obstetricians and Gynaecologists recommends a single course of steroids in cases of elective caesarean section prior to 39 weeks’ gestation [16]. Stutchfield et al.[36] analysed the course of the neonatal period for 998 infants delivered by caesarean after 37 weeks’ gestation; steroids were given to 503 women prior to caesarean delivery. NICU admission due to respiratory distress was necessary in 35 children (0.051% with steroids versus 0.024% untreated). To prevent the admission of one neonate to a special care unit, more than 400 children were required to receive unnecessary steroids.

ion; steroids were given to 503 women prior to caesarean delivery. NICU admission due to respiratory distress was necessary in 35 children (0.051% with steroids versus 0.024% untreated). To prevent the admission of one neonate to a special care unit, more than 400 children were required to receive unnecessary steroids. PARTIAL COURSE OF STEROIDS There is evidence that incomplete treatment with betamethasone or dexamethasone provides some benefits in terms of decreasing morbidity [25]. The smaller benefits attributed to incomplete steroid courses could be due to inadequate doses and shorter durations of foetal exposure. Chien et al.[37] presented data concerning antenatal steroid administration to 11 440 infants in Canada. Only 30% of children completed the antenatal steroid course. Infants who received a complete steroid course exhibited a significant reduction in RDS risk when born before 24 weeks’ or at 24–34 weeks’ gestation. A partial steroid treatment course reduced the incidence of IVH (grades III and IV) and mortality among infants born at 24–35 weeks’ gestation but had no significant effect on RDS. The incidence of NEC was similar in infants receiving a partial or complete course of steroids. Similarly, an analysis by Wong et al.[38] confirmed that hospital mortality was significantly worse without steroids (30 versus 20%; P < 0.001) in extremely premature neonates. Those with no steroid coverage were more likely to have NEC and Grade 3 or 4 IVH.

n infants receiving a partial or complete course of steroids. Similarly, an analysis by Wong et al.[38] confirmed that hospital mortality was significantly worse without steroids (30 versus 20%; P < 0.001) in extremely premature neonates. Those with no steroid coverage were more likely to have NEC and Grade 3 or 4 IVH. MULTIPLE STEROID DOSES According to the American College of Obstetricians and Gynecologists (ACOG), a repeated course of steroids is acceptable if previous treatment was completed over 14 days prior, but only before 34 weeks’ gestation [17]. According to British recommendations, antenatal steroid treatment may be repeated before 34 weeks’ gestation if the first dose was completed 7 days prior and before 26 weeks’ gestation [16]. Crowther et al.[39] summarized the results of eight studies and 3206 children concerning repeat steroid treatment after a 7-day interval. RDS was observed slightly more rarely [relative risk 0.83; 95% confidence interval (CI) 0.75–0.91]. Garite et al.[40] reported a significantly lower risk of neonatal composite morbidity only in infants born 2–7 days after a repeated standard course of betamethasone for deliveries prior to 34 weeks’ gestation (39.3 versus 69.8%; P = 0.035).

htly more rarely [relative risk 0.83; 95% confidence interval (CI) 0.75–0.91]. Garite et al.[40] reported a significantly lower risk of neonatal composite morbidity only in infants born 2–7 days after a repeated standard course of betamethasone for deliveries prior to 34 weeks’ gestation (39.3 versus 69.8%; P = 0.035). Exposure to multiple doses is associated with more profound consequences. Kanagawa et al.[41] compared the effects of repeated doses of dexamethasone on neurogenesis in neonatal rats and showed significant dose-dependent decreases in the number of bromodeoxyuridine-labelled cells in the cortex and the subgranular and subventricular zones. In humans, French et al.[42] reported that serial steroid courses between 24 and 33 weeks’ gestation decreased birth weight and head circumference by 9 and 4%, respectively. A smaller head at birth might be an effect of decreased neurogenesis. Repeated corticosteroid courses were associated with increased rates of aggressive, destructive, distractible and hyperkinetic behaviour at the ages 3 and 6 years [43]. The MACS (Multiple Courses of Antenatal Corticosteroids for Preterm Birth) trial, which included 1858 women at 25–32 weeks’ gestation who did not deliver within 14–21 days after an initial course, showed that multiple antenatal corticosteroid courses (every 14 days) did not improve neonatal outcomes, RDS, BPD, IVH (grades III and IV), cystic periventricular leukomalacia (PVL) or NEC [44]. According to the secondary analysis of the MACS trial, repeated corticosteroid courses were associated with decreased neonatal birth weight (–33.50 g; P = 0.045), length (–0.339 cm, P = 0.019) and head circumference (–0.296 cm, P < 0.001) [45▪▪]. There was a trend towards an incremental decrease in birth weight, length and head circumference for each additional course of antenatal corticosteroids. According to the results of the ACTORDS (Antenatal Collaborative Trial of Repeat Doses of Prenatal Steroids) trial, infants exposed to weekly doses of repeat antenatal corticosteroids presented postnatal growth acceleration 3–5 weeks after birth, which is similar to the catch-up growth observed in intrauterine growth restricted infants [46▪]. Compared with a single course of antenatal glucocorticoids, exposure to repeated doses of antenatal betamethasone was not associated with adverse effects on lung function or altered bone mass in early school-age children [47,48].

s similar to the catch-up growth observed in intrauterine growth restricted infants [46▪]. Compared with a single course of antenatal glucocorticoids, exposure to repeated doses of antenatal betamethasone was not associated with adverse effects on lung function or altered bone mass in early school-age children [47,48]. However, multiple steroid cycles may cause adrenal insufficiency or osteoporosis in mothers [49].

s similar to the catch-up growth observed in intrauterine growth restricted infants [46▪]. Compared with a single course of antenatal glucocorticoids, exposure to repeated doses of antenatal betamethasone was not associated with adverse effects on lung function or altered bone mass in early school-age children [47,48]. However, multiple steroid cycles may cause adrenal insufficiency or osteoporosis in mothers [49]. LONG-TERM EFFECTS OF ANTENATAL STEROIDS Animal and human data strongly suggest that steroids given prenatally influence and programme the HPA axis, with consequences in the postnatal period [50,51]. Acute suppression of foetal cortisol synthesis and an increase in cortisol bioactivity occur in response to synthetic steroids given prenatally [52]. The suppression of endogenous cortisol production persists in preterm infants and returns to normal after the first week of life [52]. Although baseline levels of cortisol normalize, suppression of the cortisol response to painful stimuli persists even at 4–6 weeks postbirth in premature infants [53,54]. Among healthy full-term infants given steroids prenatally, a pronounced cortisol response to painful stimuli is observed despite normal baseline levels [55]. In a cross-sectional study, Alexander et al.[56▪▪] assessed cortisol secretion patterns in response to a standardized laboratory stressor in 209 6 to 11-year-old children born at term and exposed to antenatal steroids, and they demonstrated significantly increased cortisol reactivity compared with controls (P < 0.001). This finding was independent of the specific synthetic glucocorticoid used and was more pronounced in girls. Erni et al.[57▪] examined psychobiological stress reactivity to a standardized psychosocial stress test in 115 healthy children at 10 years of age and found that it differed in those exposed to prenatal steroids. Animal studies have shown that exposure to elevated glucocorticoid levels during pregnancy is associated with adult-onset diseases, including elevated blood pressure, impaired cardiac and vascular function and altered metabolic function [8].

years of age and found that it differed in those exposed to prenatal steroids. Animal studies have shown that exposure to elevated glucocorticoid levels during pregnancy is associated with adult-onset diseases, including elevated blood pressure, impaired cardiac and vascular function and altered metabolic function [8]. CONCLUSION A total dose of 24 mg of betamethasone or dexamethasone appears to be sufficient to achieve the steroid concentrations observed in infants after birth during normal physiological stress. A dosage interval of 24 h for betamethasone administration was selected arbitrarily. The administration of 12 mg of betamethasone at shorter intervals may allow more children to be treated with a complete steroid course. A lower single dose of 6 or even 4 mg appears to be equally effective for the foetus as the standard 12-mg dose and is less toxic to the mother; it may therefore be useful for reducing maternal side effects. Multiple pregnancies do not require dose increases over 24 mg due to similar maternal serum and cord blood concentrations of betamethasone in twin and singleton pregnancies. The short-term and long-term effects of the dosage regimen on both the pregnant mother and the foetus require further investigation. Acknowledgements None. Conflicts of interest There are no conflicts of interest. REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as:▪ of special interest ▪▪ of outstanding interest

INTRODUCTION: ANTIMÜLLERIAN HORMONE, OVARIAN RESERVE, AND WOMEN'S HEALTH Appropriate assessment of ovarian reserve with serum antimüllerian hormone (AMH) testing has the potential to improve the medical care provided to women in a variety of ways [1▪,2,3]. However, ovarian reserve assessment, the quantitative and qualitative characterization of a woman's supply of oocytes, is exceptionally complicated to approach because of the lack of consensus in terminology, differences in clinical study designs, and variability in testing methodology in clinical research [4▪▪]. Ovarian reserve assessment has been difficult to obtain in routine clinical practice as no biomarker with sufficient clinical accuracy has been easily available. Rapidly increasing numbers of clinical publications confirm serum AMH as a clinically useful, widely available, primarily quantitative, measure of ovarian reserve that is more accurate than serum follicle-stimulating hormone (FSH) alone. AMH is a powerful clinical biomarker that helps improve the management of infertility treatment, planning of future pregnancy, menopause prediction, monitoring of ovarian damage from medications and procedures, and detection of ovary-related diseases such as polycystic ovary syndrome (PCOS) and premature or primary ovarian insufficiency (POI; Fig. 1  ).

r that helps improve the management of infertility treatment, planning of future pregnancy, menopause prediction, monitoring of ovarian damage from medications and procedures, and detection of ovary-related diseases such as polycystic ovary syndrome (PCOS) and premature or primary ovarian insufficiency (POI; Fig. 1  ). FIGURE 1 Age-specific AMH and associated conditions. Displayed at the top (yellow) are conditions associated with high age-specific AMH. At the bottom (pink), conditions associated with low age-specific AMH are listed. AMH, antimullerian hormone; PCOS, polycystic ovary syndrome; POI, premature/primary ovarian insufficiency; POF, premature ovarian failure. Box 1 no caption available However, it is now clearly important for clinicians to understand that serum AMH results can be misleading if appropriate steps are not taken to account for sources of variability in serum AMH results in clinical practice and ensure patients understand that information from AMH testing is directional, not definitive. Key practical steps are provided here for consideration to help ensure the appropriate use of AMH testing in clinical practice.

s are not taken to account for sources of variability in serum AMH results in clinical practice and ensure patients understand that information from AMH testing is directional, not definitive. Key practical steps are provided here for consideration to help ensure the appropriate use of AMH testing in clinical practice. ANTIMÜLLERIAN HORMONE BIOLOGY AND PHYSIOLOGY At the cellular level, AMH is thought to restrict the growth of ovarian follicles in response to serum FSH and also inhibit estradiol (E2) secretion [1▪,2]. AMH is produced by the granulosa cells surrounding each oocyte in the developing follicles, with rapid reduction in both AMH gene and protein expression observed in follicles reaching 8 mm and above in diameter [5▪]. The decline in AMH expression in larger follicles appears to be because of, at least in part, a reduction in the AMH gene promoter activity through E2 receptor beta [6]. One study demonstrates that reduced AMH levels, averaged from large follicles retrieved during in-vitro fertilization (IVF), were associated with higher IVF success rates [7], suggesting this follicular AMH reduction is a desired physiologic process.

ction in the AMH gene promoter activity through E2 receptor beta [6]. One study demonstrates that reduced AMH levels, averaged from large follicles retrieved during in-vitro fertilization (IVF), were associated with higher IVF success rates [7], suggesting this follicular AMH reduction is a desired physiologic process. Using population average serum AMH levels (Fig. 2), the literature appears to agree that values decline yearly at a fairly consistent rate after 25 years of age until below the clinical detection limit by 50 years of age [8,9▪▪,10,11,12▪▪,13]. However, the pattern of rise prior to age 25 has conflicting representation in the literature, with one study showing peaks and troughs [14] and another showing a gradual rise from birth to 15 years of age then decline [15▪▪] (Fig. 2). Therefore, clinicians should currently be more cautious about interpreting the rates of change in serum AMH in women below 25 years of age. Additionally, there are few data available to determine whether the pattern of decline in AMH values within an individual woman matches the pattern in the population average.

. 2). Therefore, clinicians should currently be more cautious about interpreting the rates of change in serum AMH in women below 25 years of age. Additionally, there are few data available to determine whether the pattern of decline in AMH values within an individual woman matches the pattern in the population average. FIGURE 2 Population average serum AMH levels during a woman's lifetime. A model of a population average serum AMH level in women, plotted by year of life in green, demonstrates the rising serum AMH with two peaks and troughs prior to maximal value at 25 years of age followed by an almost linear decline (specific AMH values are not included on the Y-axis to avoid potential misapplication of laboratory-specific values). Although the literature generally agrees with the pattern of decline after 25 years of age, conflicting patterns of rise are described at younger ages (red dashed line and question mark). Caution is required with clinical interpretation of serum AMH in women below 25 years of age until further studies are reported. Further study is also required to determine how the pattern of decline in serum AMH within individuals relates to the decline of the population average display the same pattern of decline as demonstrated by the population average. AMH, antimüllerian hormone. Reproduced with permission [14].

urther studies are reported. Further study is also required to determine how the pattern of decline in serum AMH within individuals relates to the decline of the population average display the same pattern of decline as demonstrated by the population average. AMH, antimüllerian hormone. Reproduced with permission [14]. OOCYTE QUANTITY AND QUALITY, FERTILITY TREATMENT, AND NATURAL FERTILITY It is well established that AMH testing effectively improves the management of ovarian stimulation in assisted reproductive technology (ART) therapy by identifying women likely to respond either poorly (with cycle cancelation/low oocyte yield) or excessively [with ovarian hyperstimulation syndrome (OHSS)/high oocyte yield] [1▪]. Recent research has targeted developing specific ovarian stimulation protocols individualized by AMH results [16▪▪,17▪]. Other recent studies focus on refining information such as demonstrating that an AMH value from a single point in time will remain predictive for approximately 12 months [18], how certain stimulation protocols affect different patient phenotypes such as PCOS vs. low responders [19▪▪,20], and prospectively demonstrating that application of AMH testing can improve expectation management and possibly treatment costs [21].

le point in time will remain predictive for approximately 12 months [18], how certain stimulation protocols affect different patient phenotypes such as PCOS vs. low responders [19▪▪,20], and prospectively demonstrating that application of AMH testing can improve expectation management and possibly treatment costs [21]. The value of serum AMH, independent of age, to predict live-birth rate or oocyte quality has remained controversial, with some studies continuing to show no association, whereas other studies demonstrate a small but useful association [22▪,23–28,29▪▪]. This controversy may be due in part to differences in AMH testing methodology and study design masking the association with serum AMH and oocyte quality. Ultimately, as a recent meta-analysis concludes, it is probable that AMH has an association with oocyte quality, but this association is likely weak to moderate at best independent of a woman's age [30▪▪]. AMH may best improve the oocyte quality prediction when incorporated in a multivariate, algorithmic approach [29▪▪]. However, as there continues to be evidence that live births are possible with very low AMH levels, AMH values alone should not be used to withhold care [31▪].

at best independent of a woman's age [30▪▪]. AMH may best improve the oocyte quality prediction when incorporated in a multivariate, algorithmic approach [29▪▪]. However, as there continues to be evidence that live births are possible with very low AMH levels, AMH values alone should not be used to withhold care [31▪]. In terms of natural fertility or time to conception, recent reports demonstrate conflicting data [32,33,34▪], which perhaps may be because of small sample sizes or AMH only being helpful when below a certain threshold or in a certain population of patients. Currently, however, the available data suggest that serum AMH levels in isolation cannot be used to counsel a patient whether or not natural conception is possible. MENOPAUSE Menopause is complex and no single test currently can definitively predict the onset with a high degree of accuracy. However, independent studies from a variety of sources for over a decade demonstrate serum AMH improves the prediction of menopause [1▪]. Serum AMH was recently added as a marker for menopausal staging because it declines much earlier than other signs of menopause such as increasing serum FSH or irregular menses [35▪]. Recently, serum AMH was shown to improve the prediction of menopause onset more than maternal age [36▪]. Furthermore, different models using AMH to predict a higher chance of early or late onset of menopause relative to the expected average have now been cross-validated [37▪▪].

sing serum FSH or irregular menses [35▪]. Recently, serum AMH was shown to improve the prediction of menopause onset more than maternal age [36▪]. Furthermore, different models using AMH to predict a higher chance of early or late onset of menopause relative to the expected average have now been cross-validated [37▪▪]. There are a number of variables which, if combined with serum AMH algorithmically, may increase the accuracy of menopause predictions. For example, one cross-sectional study of 2635 women demonstrated that for women with serum AMH in the lower 5th percentile for their age, expected age of menopause was 43.4 years of age for obese, nonsmokers versus 37.6 in thin women who smoked [38▪]. In women with serum AMH in the upper 95th percentile for age, predicted age of menopause increased considerably but was again affected by body weight and smoking status (55.1 years of age in obese, nonsmokers versus 52.4 years of age in underweight smokers). Ethnicity also may influence serum AMH [39]. Interestingly, a recent study in 44 Japanese women demonstrated after AMH became undetectable, menopause onset was within 3 years [40], instead of 5 years shown by a study of large U.S. and European populations [41], although the study design and assay performance may contribute to these observational differences.

39]. Interestingly, a recent study in 44 Japanese women demonstrated after AMH became undetectable, menopause onset was within 3 years [40], instead of 5 years shown by a study of large U.S. and European populations [41], although the study design and assay performance may contribute to these observational differences. Although the relationship of AMH to the timing of menopause onset is highly statistically significant in the studies cited, the confidence intervals related to the predicted age of menopause remain wide at present. Thus, currently the present clinical interpretation is qualitative: women with a serum AMH very low or very high for their age are more likely to go into menopause earlier or later than average, respectively. Notably, for women with very low age-specific serum AMH who are not ready to attempt conception naturally or with donor sperm, oocyte cryopreservation, no longer considered experimental [42▪▪], is now available as an alternative to help ensure the future ability to have children. Although serum AMH testing clearly provides helpful directional information in decision making, prior to ordering AMH testing, a clinician should consider verifying whether their patient considers qualitative information sufficiently meaningful to use.

an alternative to help ensure the future ability to have children. Although serum AMH testing clearly provides helpful directional information in decision making, prior to ordering AMH testing, a clinician should consider verifying whether their patient considers qualitative information sufficiently meaningful to use. POLYCYSTIC OVARY SYNDROME The relationship of AMH with PCOS is complicated by the diagnosis itself being subject to a debate primarily centered, ironically, over whether polycystic ovaries are included (Rotterdam criteria) or are not included [National Institutes of Health (NIH) criteria] in the diagnosis of the disorder. Numerous studies demonstrate that PCOS is significantly associated with elevated serum AMH both with the NIH criteria [43▪,44▪] and with the Rotterdam criteria [44▪,45▪▪,46▪▪,47–49]. One study demonstrated that in women presenting for fertility evaluation with serum AMH elevated, high, and very high, the frequency of PCOS diagnosis was 51.6% (n = 84), 97% (n = 30), and 100% (n = 20), respectively [50▪]. As serum AMH correlates well with the polycystic appearance of ovaries on ultrasound, several studies are proposing to set thresholds by AMH as an alternative to ultrasound [44▪,46▪▪,48]. Recent evidence in 463 PCOS women suggests that serum AMH may provide insight into the subphenotypes of PCOS with higher serum AMH predicting longer menstrual cycle length, higher luteinizing hormone (LH) levels, and hirsuitism [51▪▪]. Clearly, markedly elevated age-specific AMH correlates with the clinical diagnosis and severity of PCOS and may eventually be adopted as a diagnostic criteria for PCOS.

he subphenotypes of PCOS with higher serum AMH predicting longer menstrual cycle length, higher luteinizing hormone (LH) levels, and hirsuitism [51▪▪]. Clearly, markedly elevated age-specific AMH correlates with the clinical diagnosis and severity of PCOS and may eventually be adopted as a diagnostic criteria for PCOS. DIMINISHED OVARIAN RESERVE, PREMATURE OR PRIMARY OVARIAN INSUFFICIENCY, AND PREMATURE OVARIAN FAILURE Not surprisingly, numerous studies demonstrate serum AMH is dramatically lower in women symptomatic from ovarian insufficiency [47,52▪,53,54], although one study shows that serum LH levels may be better at identifying the onset of menstrual irregularities [55]. It seems unlikely that defects in the AMH molecule itself are a concern as a cause of POI as suggested by a recent study of 211 idiopathic POI women and 233 controls, which demonstrated no identifiable genetic differences in the AMH or the AMH receptor genes [56]. An area of current controversy is whether serum AMH has a clinically useful association with the number of CGG repeats in the fragile X gene, FMR1, which is known to have 55 or greater CGG repeats much more frequently in women with POI than normal controls [52▪,57]. An association of repeat length and serum AMH in women with fertility issues has been described by primarily one group [58–62], but this was not observed in another recent study of women not selected for fertility issues [63].

or greater CGG repeats much more frequently in women with POI than normal controls [52▪,57]. An association of repeat length and serum AMH in women with fertility issues has been described by primarily one group [58–62], but this was not observed in another recent study of women not selected for fertility issues [63]. As AMH is clearly the earliest and most accurate serum biomarker reflecting decline in the quantitative aspects of ovarian reserve, it may be the earliest clinical means of detecting women likely to develop diminished ovarian reserve (DOR), POI, or premature ovarian failure (POF) prior to becoming symptomatic [52▪]. In fact, a recent U.S. study of 5354 women presenting to fertility centers from 30 different states demonstrated that in women with reassuring early follicular serum FSH, serum AMH values concerning for low ovarian reserve were identified in 20% of women under 35 years of age and in over 30% of women by 40 years of age (Fig. 3) [12▪▪]. Therefore, serum AMH testing can be an important means to identify many patients in routine practice at risk for an accelerated decline in ovarian reserve.

rum AMH values concerning for low ovarian reserve were identified in 20% of women under 35 years of age and in over 30% of women by 40 years of age (Fig. 3) [12▪▪]. Therefore, serum AMH testing can be an important means to identify many patients in routine practice at risk for an accelerated decline in ovarian reserve. FIGURE 3 Frequency of discordance by age of serum FSH and AMH during estradiol-confirmed menstrual cycle days 2–4 in 5354 women from U.S. fertility centers. Discordance was defined when one test was ‘concerning’ and the other test was ‘reassuring’. Cutpoints for serum AMH were less than 0.8 ng/ml (concerning) and at least 0.8 ng/ml (reassuring), and for serum FSH were at least 10 IU/l (concerning) and less than 10 IU/l (reassuring). Y-axis displays the frequency of discordance of women in each of four age groups shown the X-axis. Note: AMH values are not standardized across laboratories. These data using cutpoints were calibrated to number of oocytes retrieved through ovarian stimulation and are specific to Ref. [12▪▪]. AMH, antimüllerian hormone; FSH, follicle-stimulating hormone. Modified with permission [12▪▪].

r age groups shown the X-axis. Note: AMH values are not standardized across laboratories. These data using cutpoints were calibrated to number of oocytes retrieved through ovarian stimulation and are specific to Ref. [12▪▪]. AMH, antimüllerian hormone; FSH, follicle-stimulating hormone. Modified with permission [12▪▪]. ASSOCIATION WITH OTHER DISORDERS: AUTOIMMUNITY, GRANULOSA CELL TUMORS, AND ENDOMETRIOSIS Substantial evidence from a number of sources demonstrates serum AMH levels are associated with other diseases. For example, AMH is significantly lower in autoimmune disorders [64] such as lupus [65▪▪,66] and Crohn's disease [67▪]. Elevated AMH levels can also be useful in postmenopausal women as a strong indicator of granulosa cell tumors (GCTs) and for monitoring for the recurrence of GCTs, though in asymptomatic, premenopausal women, elevated AMH is too nonspecific for clinical utility as a screening test for these tumors [68,69]. The understanding of AMH and endometriosis continue to develop, with a recent report indicating AMH may actually play a role in endometriosis [70]. Although some consider endometrioma removal as important for improving fertility [71], clear evidence demonstrates serum AMH is lowered by ovarian surgeries such as removing cysts and endometriomas [72▪,73▪▪,74–76].

continue to develop, with a recent report indicating AMH may actually play a role in endometriosis [70]. Although some consider endometrioma removal as important for improving fertility [71], clear evidence demonstrates serum AMH is lowered by ovarian surgeries such as removing cysts and endometriomas [72▪,73▪▪,74–76]. MONITORING OVARIAN DAMAGE WITH MEDICAL THERAPIES AND SURGICAL PROCEDURES Medical and surgical treatments are now being assessed and monitored for ovarian damage by utilization of ovarian reserve markers such as AMH. Numerous cancer therapy publications have demonstrated the dramatic effects of various chemotherapeutics in reducing serum AMH levels. Some of these studies have demonstrated that AMH testing can improve treatment selection by identifying which therapies are most toxic to the ovary and which patients are most at risk for postchemotherapy ovarian insufficiency [77▪,78▪▪,79–84]. Furthermore, numerous reports are confirming that after some ovarian-related surgeries such as removal of benign cysts and endometriomas, significant reductions in AMH are observed which may persist [72▪,73▪▪,74–76] with a recent study demonstrating similar reductions with benign cysts and endometriomas and more severe reductions with bilateral procedures [85]. Interestingly, one report shows only transient reductions in AMH in 22 PCOS patients undergoing the more minor procedure of ovarian puncture [86]. Given this data, a clinician and patient must carefully consider the possible negative impact of common ovarian surgeries on ovarian reserve. The ability of serum AMH levels to indicate and predict damage to the ovary confirm it is important to measure serum AMH before and after medical and surgical treatments to help plan treatment approaches, counsel the patient, and monitor ovarian reserve status.

ive impact of common ovarian surgeries on ovarian reserve. The ability of serum AMH levels to indicate and predict damage to the ovary confirm it is important to measure serum AMH before and after medical and surgical treatments to help plan treatment approaches, counsel the patient, and monitor ovarian reserve status. ALGORITHMS AND MULTIVARIATE COMBINATIONS OF TESTS WITH ANTIMÜLLERIAN HORMONE Multivariate, algorithmic approaches allow optimization of combinations of clinical variables to predict outcome. The reality is clinicians, if not provided with a validated algorithm, are forced in daily practice to weigh AMH with the provided variables such as age, BMI, medical history, antral follicle count (AFC), and serum FSH to the best of their ability without the benefit of mathematical optimization and large datasets. A number of recent studies demonstrate benefit by combining AMH with other variables in predicting the outcomes such as menopause [37▪▪,38▪], live birth [33], or response to ovarian stimulation protocols in ART [29▪▪,87]. However, other studies have not found benefit in combining multiple tests, including a recent study combining 28 databases [88▪]. It should be noted that the differences in AMH assay materials and laboratory performance make combining different AMH datasets complex and likely would reduce the associations with AMH and outcome. Therefore, although a multivariate approach is likely to dramatically improve the accuracy of decision making, it will require minimization of variability in AMH assay, laboratory performance, patient population definition, and study design.

atasets complex and likely would reduce the associations with AMH and outcome. Therefore, although a multivariate approach is likely to dramatically improve the accuracy of decision making, it will require minimization of variability in AMH assay, laboratory performance, patient population definition, and study design. THE CHALLENGE OF VARIABILITY IN ANTIMÜLLERIAN HORMONE RESULTS: BIOLOGY, EXPOSURE, AND LABORATORY Perhaps the most important clinical advance in the medical literature related to AMH is the recent recognition of the significant variability in AMH results which must be taken into account for appropriate interpretation in clinical care. As described below, there are now three recognized sources for this variability (Fig. 4): biological fluctuation within certain individuals; exposure to medications (such as contraceptive hormones) and certain ovarian surgeries; and laboratory-specific AMH values as each laboratory provides its own value ranges and calibration. That said, the variability should be contextualized with the fact that AMH is still the best available serum marker of quantitative aspects of ovarian reserve. The practical steps outlined below mitigate the issues of variability. FIGURE 4 Variability by source possibly affecting a reported AMH result. Upper panel demonstrates the potential effect of a single source variable. Lower panel demonstrates how multiple sources of variability can be additive. AMH, antimüllerian hormone. Reproduced with permission from [3].

THE CHALLENGE OF VARIABILITY IN ANTIMÜLLERIAN HORMONE RESULTS: BIOLOGY, EXPOSURE, AND LABORATORY Perhaps the most important clinical advance in the medical literature related to AMH is the recent recognition of the significant variability in AMH results which must be taken into account for appropriate interpretation in clinical care. As described below, there are now three recognized sources for this variability (Fig. 4): biological fluctuation within certain individuals; exposure to medications (such as contraceptive hormones) and certain ovarian surgeries; and laboratory-specific AMH values as each laboratory provides its own value ranges and calibration. That said, the variability should be contextualized with the fact that AMH is still the best available serum marker of quantitative aspects of ovarian reserve. The practical steps outlined below mitigate the issues of variability. FIGURE 4 Variability by source possibly affecting a reported AMH result. Upper panel demonstrates the potential effect of a single source variable. Lower panel demonstrates how multiple sources of variability can be additive. AMH, antimüllerian hormone. Reproduced with permission from [3]. BIOLOGICAL FLUCTUATION IN SERUM ANTIMÜLLERIAN HORMONE VALUES One of the most attractive aspects of serum AMH measurements, unlike serum FSH testing, is the lack of large changes in the average values at the population level during the menstrual cycle [89]. However, this led to the misconception that individuals do not show any fluctuation across the menstrual cycle. Fluctuation in serum AMH levels can be clearly observed by examining the graphs in previous studies which display individual patient data [89,90]. A recent study measuring AMH multiple times with the same menstrual cycle of 44 normally cycling healthy women demonstrated AMH values (ng/ml) within four individuals (9%) ranging approximately: 0.4–1.9, 1.9–4.2, 0.4–1.4, and 2.3–4.4 [91▪▪]. In another study, seven of 12 women re-measured during the same menstrual cycle were clinically reclassified [92▪]. Therefore, the clinician should avoid using AMH as the only marker of ovarian reserve, counsel patients that occasionally results can fluctuate, and consider re-testing if the AMH value is not consistent with the clinical picture.

12 women re-measured during the same menstrual cycle were clinically reclassified [92▪]. Therefore, the clinician should avoid using AMH as the only marker of ovarian reserve, counsel patients that occasionally results can fluctuate, and consider re-testing if the AMH value is not consistent with the clinical picture. EXPOSURE TO CONTRACEPTIVE MEDICATIONS, PREGNANCY, METHOTREXATE, AND DEHYDROEPIANDROSTERONE Another attractive aspect of serum AMH measurements is that they are not affected by contraceptive medications to the same degree as serum FSH, which propagated the unfortunate misunderstanding that AMH was not affected by contraceptives. Numerous studies have now confirmed that hormonal contraceptives often lower serum AMH [93–96]. A well designed prospective study of 44 women demonstrated serum AMH was lowered by an average of approximately 30% within two menstrual cycles of starting the contraceptive regardless of the route [97▪▪]. This was expected as other scenarios which disrupt the hormonal milieu (such as pregnancy) affect serum AMH levels. One study of 554 women in pregnancy demonstrated an approximately 50% lowering of serum AMH with each trimester and recovery after delivery [98▪▪]. The limited recent data on methotrexate use do not demonstrate an effect on serum AMH [99], consistent with the prior observations [100]. A number of fertility centers administer dehydroepiandrosterone (DHEA) to poor responders to ovarian stimulation in order to improve AMH values and follicular responsiveness [101]; however, the number of treated patients is small and poorly controlled, and in sum, current data do not support a clear effect by this medication [102,103].

lity centers administer dehydroepiandrosterone (DHEA) to poor responders to ovarian stimulation in order to improve AMH values and follicular responsiveness [101]; however, the number of treated patients is small and poorly controlled, and in sum, current data do not support a clear effect by this medication [102,103]. ANTIMÜLLERIAN HORMONE ASSAYS: VALUES ARE LABORATORY SPECIFIC The major challenge for the clinician attempting to apply serum AMH values to clinical care is the fact that each laboratory provides their own value ranges which may be clinically significantly different. Currently, there is no international reference standard for AMH measurement. A major recent advance for improved patient care is simply the recognition in the clinical literature that AMH values are currently laboratory specific and generally cannot be ‘mixed and matched’ [9▪▪,104–110]. Previously, clinicians were misguided into thinking they could simply apply the values they received locally using reported cutpoints in AMH studies done elsewhere. This is largely because of the mistaken assumptions propagated in the clinical literature such as: high correlations between AMH assays meant results were interchangeable between laboratories with simple factoring; there is only one kit (i.e. the Beckman Gen II AMH assay) in use when in fact there are several kits in use; and laboratories using the same kit would supply the same numerical result. In fact, virtually every study comparing different AMH assay systems provides different conversion methods, none of which are simple factoring but usually involve linear equations [104–108,110]. Currently, no fewer than four kits are commercially available [111,112], with more on the way. Current publications describe the findings from seven different AMH kits used over the last 3–5 years. In addition, the Beckman Gen II assay itself has undergone two major methodology changes within 18 months (Beckman Product Notifications November 2012 and June 2013) [113], and not surprisingly, a systematic shift in calibration of the assay has just been reported in a cohort of 10 981 patients [9▪▪]. Furthermore, when 10 laboratories using the same Beckman Gen II assay were compared, there was a 40% variation in the values obtained [109]. In addition, specimen handling protocols affect the serum AMH values [107].

stematic shift in calibration of the assay has just been reported in a cohort of 10 981 patients [9▪▪]. Furthermore, when 10 laboratories using the same Beckman Gen II assay were compared, there was a 40% variation in the values obtained [109]. In addition, specimen handling protocols affect the serum AMH values [107]. It is also critical to understand how a laboratory calibrates their clinical cutpoints by patient population and clinical outcome; otherwise, accurate interpretation of the result will be difficult. A critical, additional mistake likely to be made in the future is concluding that FDA clearance of an AMH assay or production of an international standard will mean laboratories will report the same absolute value or calibrate clinical cutpoints the same way. FSH testing provides a recent history lesson demonstrating that this is not true. In fact, current FSH testing remains so controversial that no universally defined cutpoints are agreed upon despite multiple FDA cleared platforms and international reference standards [4▪▪]. Fortunately, to overcome this challenge, a clinician can follow a few practical steps outlined below.

ing that this is not true. In fact, current FSH testing remains so controversial that no universally defined cutpoints are agreed upon despite multiple FDA cleared platforms and international reference standards [4▪▪]. Fortunately, to overcome this challenge, a clinician can follow a few practical steps outlined below. PRACTICAL METHODS TO MINIMIZE THE VARIABILITY IN ANTIMÜLLERIAN HORMONE RESULTS AND MAXIMIZE THE CLINICAL UTILITY Although the challenges facing the clinician applying laboratory results from ovarian testing such as AMH can seem daunting, by following a few practical steps in a checklist format, a clinician can overcome these challenges (Table 1). First and foremost, do not ‘mix and match’ the AMH values from different laboratories, but identify a reliable, single source of AMH testing which calibrates its testing to the clinical outcomes of interest and commits to updating the clinician should any changes occur in the calibration of the results. Medical insurance companies can present a challenge by restricting the testing services to a laboratory with which a clinician has no familiarity, but a clinician now has numerous publications as outlined in this review to demonstrate the medical risks this restrictive practice poses. Second, avoid using a serum AMH measurement alone to assess ovarian reserve, but incorporate other markers such as AFC and early follicular phase serum FSH. Third, identify whether the patient has a medical condition, has taken medications (e.g. hormonal contraceptive or chemotherapy), or had ovarian surgeries (e.g. cyst or endometrioma removal) that affect the AMH levels. Fourth, counsel the patient prior to testing about the qualitative nature of the information and that a single test result may change substantially in a certain subset of individuals because of biological fluctuations. Fifth, consider retesting if AMH results are clinically inconsistent, or if, based upon the testing results, life-changing decisions are to be made.

out the qualitative nature of the information and that a single test result may change substantially in a certain subset of individuals because of biological fluctuations. Fifth, consider retesting if AMH results are clinically inconsistent, or if, based upon the testing results, life-changing decisions are to be made. CONCLUSION Hundreds of clinical studies confirm that adding serum AMH testing to a complete ovarian assessment provides a powerful tool to help provide better healthcare for women. The benefits of this testing can optimize fertility treatments; help lead to earlier diagnoses of PCOS, POI, POF, and certain autoimmune conditions; provide the opportunity for better planning for procreation and menopause; and allow for better medical decision-making by monitoring ovarian damage from exposures to medical or surgical therapies. Although challenges with variability in AMH results make the provided practical steps a prerequisite for appropriate interpretation of the testing, the clinical benefits of testing more than justify this additional effort. Acknowledgements The authors would like to thank Dr Eric Widra for a thorough and helpful review of this manuscript. Funding for this review: None. Conflicts of interest B.L. – ReproSource, Inc; V.L.B. – no relevant conflicts of interest. REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as:▪ of special interest ▪▪ of outstanding interest Table 1 Checklist to maximize the clinical utility of serum AMH testing

Conflicts of interest B.L. – ReproSource, Inc; V.L.B. – no relevant conflicts of interest. REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as:▪ of special interest ▪▪ of outstanding interest Table 1 Checklist to maximize the clinical utility of serum AMH testing 1. Use one laboratory, calibrated to outcomes Avoid ‘mixing and matching’ AMH values from different laboratories and identify a reliable, single source for testing which both calibrates the results to the clinical outcomes of interest and commits to updating the clinician if calibration of the results changes. 2. Utilize more than one ovarian reserve test Avoid using a single serum AMH measurement alone to assess ovarian reserve, and incorporate other markers such as antral follicle count (AFC) and/or early follicular phase serum FSH. 3. Identify exposures Identify whether the patient has taken medications (e.g. hormonal contraceptives and chemotherapy) or had surgery (ovarian cyst or endometrioma removal) that affects the AMH levels. 4. Counsel patient Prior to testing, verify the patient understands the directional nature of the information being provided by AMH testing, and that, in a subset of women, the test result may change substantially with biologic fluctuations. 5. Consider retesting If testing results lead to life-changing decisions or if the results are inconsistent with the clinical scenario, consider retesting. Many improvements to the management of women's health are possible through appropriate AMH testing. However, variability in AMH results can lead to clinically significant variability in AMH results, making careful approach to the interpretation essential. With the above simple steps, a clinician can rapidly minimize the risks for incorrect interpretation.

f women's health are possible through appropriate AMH testing. However, variability in AMH results can lead to clinically significant variability in AMH results, making careful approach to the interpretation essential. With the above simple steps, a clinician can rapidly minimize the risks for incorrect interpretation. AMH, antimüllerian hormone; FSH, follicle stimulating hormone.

INTRODUCTION Preterm birth, defined as birth before 37 weeks of gestation, is a major cause of neonatal and infant mortality [1▪▪,2]. In Europe, about 75% of all neonatal deaths and 60% of all infant deaths occur to infants born preterm [1▪▪]. Although survival of preterm infants has increased significantly in the past decade, these infants remain at higher risks of long-term motor and cognitive impairments as well as of chronic disease and mortality later in life than infants born at term [3,4]. Initiatives to prevent preterm births have had limited success [5,6]. In countries with comparable levels of development and healthcare systems, preterm birth rates vary markedly – a range from 5 to 10% among live births in Europe [7▪▪,8,9▪▪]. Why these disparities exist is poorly understood, yet this knowledge is invaluable for orienting health policy and prevention initiatives. This review thus seeks to identify the most likely sources of heterogeneity in preterm birth rates, which could explain differences between European countries. Drawing on the most recent literature and in the light of data from the 2013 European Perinatal Health Report [1▪▪], our review focuses on population characteristics, reproductive policies as well as medical practices, which may affect preterm birth rates. Box 1 no caption available

In countries with comparable levels of development and healthcare systems, preterm birth rates vary markedly – a range from 5 to 10% among live births in Europe [7▪▪,8,9▪▪]. Why these disparities exist is poorly understood, yet this knowledge is invaluable for orienting health policy and prevention initiatives. This review thus seeks to identify the most likely sources of heterogeneity in preterm birth rates, which could explain differences between European countries. Drawing on the most recent literature and in the light of data from the 2013 European Perinatal Health Report [1▪▪], our review focuses on population characteristics, reproductive policies as well as medical practices, which may affect preterm birth rates. Box 1 no caption available SEARCH STRATEGY AND SOURCES We searched PubMed for publications between 2011 and 2014, which focused on explaining differences in preterm birth rates between countries in Europe. Because we could not identify recent studies looking at this issue, we enlarged our search to studies from other high-income countries, including Australia, Canada, Japan, and the United States. Our assumption is that results from these contexts are relevant to European populations. We also extended our review to include studies that have evaluated the impact of specific risk factors on population-level preterm birth rates or trends in preterm birth rates within countries. Last, we used data from the Euro-Peristat project, which aims to monitor perinatal health using a recommended set of national-level indicators derived from routine systems [1▪▪]. These data illustrate the variability in specific risk factors for preterm birth across Europe and the extent to which preterm birth rate variations across countries may reflect differences in their prevalence. The 2013 Euro-Peristat report presented 2010 data from 29 countries on the preterm birth rate and factors affecting preterm birth risk such as: multiple births, maternal age, prepregnancy BMI, smoking during pregnancy, and migration status, which we compiled for this review (Table 1).

t differences in their prevalence. The 2013 Euro-Peristat report presented 2010 data from 29 countries on the preterm birth rate and factors affecting preterm birth risk such as: multiple births, maternal age, prepregnancy BMI, smoking during pregnancy, and migration status, which we compiled for this review (Table 1). PRETERM BIRTH RATES IN EUROPE In Europe, preterm birth rates for live births varied in 2010 between 5.2–5.9% in Iceland, Finland, Lithuania, Estonia, Latvia, Sweden, and Ireland and 8.2–10.4% in Belgium, Austria, Germany, Romania, Hungary, and Cyprus as illustrated in Fig. 1 and Table 1. This corresponds to a 50% excess in countries with higher vs. lower rates and corresponds to a 3 percentage-point absolute difference (Fig. 1). Although overall rates have increased in general, as reported by a World Health Organization (WHO) study of preterm birth in 64 countries [8], trends are heterogeneous and, in particular, rates of singleton preterm birth have been stable or declined in about half of European countries over the past 15 years [9▪▪]. FIGURE 1 Rates of preterm birth (PTB) among live births in Europe in 2010.

PRETERM BIRTH RATES IN EUROPE In Europe, preterm birth rates for live births varied in 2010 between 5.2–5.9% in Iceland, Finland, Lithuania, Estonia, Latvia, Sweden, and Ireland and 8.2–10.4% in Belgium, Austria, Germany, Romania, Hungary, and Cyprus as illustrated in Fig. 1 and Table 1. This corresponds to a 50% excess in countries with higher vs. lower rates and corresponds to a 3 percentage-point absolute difference (Fig. 1). Although overall rates have increased in general, as reported by a World Health Organization (WHO) study of preterm birth in 64 countries [8], trends are heterogeneous and, in particular, rates of singleton preterm birth have been stable or declined in about half of European countries over the past 15 years [9▪▪]. FIGURE 1 Rates of preterm birth (PTB) among live births in Europe in 2010. MEASUREMENT Measurement of gestational age is a potential source of variation between countries [10]. Timing of the first day of the mother's last menstrual period (LMP) or biometric measures from ultrasound (US) can be used to establish the first day of the pregnancy. The method of determining gestational age influences estimates of the preterm birth rate [5]. US dating tends to shift all pregnancies toward earlier gestational ages [10,11▪] mainly because LMP dating assumes that all women have a 28-day cycle, whereas in reality, average cycle length is slightly longer [12]. However, US removes errors in gestational age estimation and these corrections reduce the preterm birth rate because errors have more influence at the extremes of the distribution. The algorithms used to derive gestational age when LMP and US are both available will also affect the preterm birth rate [10]. Another potential source of variation between countries may be the references for US dating, as these are not standardized [13]. Finally, population characteristics influence gestational age measurement and vary across healthcare systems; socially disadvantaged women have less accurate dates [10,14,15▪], which may reflect difficulties in accessing prenatal care

en countries may be the references for US dating, as these are not standardized [13]. Finally, population characteristics influence gestational age measurement and vary across healthcare systems; socially disadvantaged women have less accurate dates [10,14,15▪], which may reflect difficulties in accessing prenatal care In Europe, prenatal care starting in the first trimester is the norm and the ‘best obstetric estimate’ is the standard for pregnancy dating, although information on how this estimate is derived is not available in international databases [1▪▪,11▪,16▪▪]. Some routine data systems, such as in Norway and Sweden, record both LMP and the US estimate. In the United States, official preterm estimates are mainly based on LMP, but the clinical/obstetrical estimate is also recorded [11▪,17,18]. The use of LMP vs. clinical estimates explains half of the difference between United States and Canadian rates (12.3 vs. 7.6%, respectively in 2002) [19]. We could not find recent European studies about how gestational age measurement affects the preterm birth rate. Differences in the registration of births and deaths at 22 and 23 weeks of gestation are highly problematic for international comparisons of perinatal and infant mortality [20,21▪], but their effect on overall preterm birth rates is probably small: in 2010, only 0.1% of live births in the countries included in Table 1 were born at 22–23 weeks [1▪▪]. These differences will, however, have a larger impact on comparisons of very preterm birth rates.

parisons of perinatal and infant mortality [20,21▪], but their effect on overall preterm birth rates is probably small: in 2010, only 0.1% of live births in the countries included in Table 1 were born at 22–23 weeks [1▪▪]. These differences will, however, have a larger impact on comparisons of very preterm birth rates. MULTIPLE PREGNANCIES Increasing multiple birth rates, starting in the 1980s, have contributed to overall rises in preterm birth rates [22,23]. In 2010, preterm birth rates for multiples in Europe ranged between 39.6 and 66.0%, in contrast with between 4.1 and 7.6% for singletons [1▪▪]. Multiple birth rates vary from about 2 to 4% of all births, as shown in Table 1.

rates, starting in the 1980s, have contributed to overall rises in preterm birth rates [22,23]. In 2010, preterm birth rates for multiples in Europe ranged between 39.6 and 66.0%, in contrast with between 4.1 and 7.6% for singletons [1▪▪]. Multiple birth rates vary from about 2 to 4% of all births, as shown in Table 1. Variation in multiple birth rates is related to the proportion of older mothers who have more spontaneous multiple pregnancies and a greater demand for fertility treatments. It is also related to subfertility treatment policies and practices (in-vitro fertilization, ovulation induction and inseminations), which differ across high-income countries [24,25▪,26▪▪]. For instance, elective single embryo transfer (eSET) has been extensively promoted by several countries including Belgium, Sweden, Finland, and Australia [24,25▪,27]. In contrast, in Italy, the law requires transfer of all fertilized embryos in each cycle, although it limits the number of fertilized embryos to three [28]. Recent studies comparing use of eSET across countries showed a clear impact on multiple births [25▪,26▪▪]. eSET policies in Slovenia were credited with the stabilization of the proportion of assisted reproductive technology (ART) very preterm twins in past years after a 27-fold increase from 1987 to 2010 [29].

ee [28]. Recent studies comparing use of eSET across countries showed a clear impact on multiple births [25▪,26▪▪]. eSET policies in Slovenia were credited with the stabilization of the proportion of assisted reproductive technology (ART) very preterm twins in past years after a 27-fold increase from 1987 to 2010 [29]. One source of heterogeneity between countries could thus be multiple births. To assess their contribution, we recomputed preterm birth rates assuming that all countries had the same multiple birth rate (set at the European average of 3.2%), as shown in Table 1. Substantial variability persists after this adjustment, although standardized rates are over half a percentage point lower in some countries. Larger declines occur more often in countries with high rates. CHARACTERISTICS OF THE POPULATION OF CHILDBEARING WOMEN Maternal characteristics associated with preterm delivery risk include age, socioeconomic status, migration status, BMI, smoking, drug use and alcohol consumption, occupational exposure, short interpregnancy intervals, previous preterm birth, preexisting medical conditions, ART use, and previous induced abortions [30,31▪▪,32▪▪,33–36]. It is hard to obtain European-level data on the prevalence of many of these risk factors, but as shown in Table 1, those available in the Euro-Peristat project clearly differ between countries, including maternal age, migrant status, smoking, and BMI. Articles included in our review addressed maternal age, social status, migration, smoking, obesity, diet, and previous induced abortion.

f these risk factors, but as shown in Table 1, those available in the Euro-Peristat project clearly differ between countries, including maternal age, migrant status, smoking, and BMI. Articles included in our review addressed maternal age, social status, migration, smoking, obesity, diet, and previous induced abortion. In 2010, the proportion of mothers 35 years of age and older in European countries ranged between 11 and 35% (Table 1); given that older women face higher risks of preterm birth, this could be one explanation for country-level differences. Auger et al.[37▪▪] tested the hypothesis that advancing maternal age may be a cause of rising preterm birth rates. In a study comparing singleton births in Denmark and Quebec, where preterm birth rates rose over the past 15 years, they found that rates had increased the most among women aged 20–29 years and stayed stable or decreased for women 35 and older. Paradoxically, the increase in the proportions of older mothers appeared to favor more stable rates over time in these countries.

uebec, where preterm birth rates rose over the past 15 years, they found that rates had increased the most among women aged 20–29 years and stayed stable or decreased for women 35 and older. Paradoxically, the increase in the proportions of older mothers appeared to favor more stable rates over time in these countries. Recent studies explored the relationship between preterm birth and disadvantaged socioeconomic circumstances [38–41]. Two studies found that social disadvantage was more strongly associated with very preterm than moderate preterm birth [42▪,43▪]. The 2010 WHO Multicountry study also found that less educated mothers had fewer provider-initiated preterm deliveries [44▪▪]. In northern England, although overall preterm birth rates stayed the same between 1960 and 2000, rates increased in the most deprived areas and decreased in less deprived areas resulting in widened social inequalities [45▪]. In Iceland, the 2008 economic crisis was associated with increases in the risk of low birth weight, but no change in preterm birth [46]. These studies illustrate the complexity of assessing the importance of social conditions in cross-national studies, both because of the variation across population sub-groups and the dependence on other contextual factors.

astasis of oviductal tumors resemble the relatively indolent behavior characteristic of so-called Type I ovarian carcinomas in humans, for which endometrioid carcinoma is a prototype. This model emphasizes that importance of cellular context and the need to further understand the cell of origin for ovarian cancer [63]. Genomic landscape of high-grade serous ovarian carcinoma: the role of TP53 One of the hallmarks of HGSOC is the universal presence of mutations in the TP53 tumor suppressor gene [4,64,65▪▪,66▪▪]. The most common site of mutation of TP53 is the DNA-binding domain, but mutations in other regions have been identified [64]. Mutation of TP53 is the first known molecular event in the transformation of fallopian tube secretory cells, and can be identified in early tumor precursors [17]. Recent studies indicate that stabilizing TP53 missense mutations, but not loss of endogenous wildtype TP53, promote secretory cell survival and cell–cell aggregation under anchorage independent growth conditions. This mutant-mediated autocrine matrix deposition leads to the formation of cell clusters with mesothelial-intercalation capacity which is likely necessary for peritoneal dissemination [67▪]. Interestingly, it appears that the most common TP53 missense mutations, including R273H, R175H, and R248Q, express a large number and high amounts of shorter p53 protein isoforms that are translated from the mutated full-length p53 mRNA. These shorter isoforms, like Δ160p53, exhibit all the gain-of-function properties attributed to the mutant protein, including enhanced cell survival, proliferation, adhesion, and invasion [68▪]. These data suggest that early mutation of TP53 is necessary for HGSOC initiation. For these reasons, mutant p53 has re-emerged as an appealing therapeutic target in HGSOC. Small molecules that sculpt the mutant protein into a more wildtype confirmation are being evaluated in preclinical and clinical trials [69]. In addition, perturbation of pathways, like the mevalonate pathway, that lead to degradation of mutant p53 are being exploited for therapeutic gains [70].

s associated with increases in the risk of low birth weight, but no change in preterm birth [46]. These studies illustrate the complexity of assessing the importance of social conditions in cross-national studies, both because of the variation across population sub-groups and the dependence on other contextual factors. Migrant flows between European member states and from non-European countries have been increasing and migrant status has been identified as a risk factor for preterm birth [47▪,48,49▪]. In 2010, foreign born mothers represented between 0.0 (Poland) and 66.0% (Luxembourg) of childbearing women (Table 1). However, associations with preterm birth depend on preterm birth subtype (spontaneous vs. nonspontaneous), region of origin, reference groups used for comparison, reasons for migration (refugee, economic migrants), and length of residence [50,51▪▪,52]. A review by Urquia et al.[53] showed that adverse pregnancy outcomes in Europe were different depending on maternal country of origin. In another study, eastern European migrants had better perinatal health outcomes than United States born women even with later entry into prenatal care or less education, which may be explained by the healthy migrant effect [54]. However, in Sorbye et al.'s study of migrant women in Norway between 1999 and 2009, both spontaneous and nonspontaneous preterm birth rates were higher among immigrants than among Norwegian-born women. For migrants, provider-initiated preterm deliveries increased with increased length of residence, whereas spontaneous preterm deliveries remained unchanged [51▪▪].

women in Norway between 1999 and 2009, both spontaneous and nonspontaneous preterm birth rates were higher among immigrants than among Norwegian-born women. For migrants, provider-initiated preterm deliveries increased with increased length of residence, whereas spontaneous preterm deliveries remained unchanged [51▪▪]. Behavioral risk factors mediate the relationship between sociodemographic characteristics and preterm birth. A systematic review published in 2010 summarized the epidemiologic evidence on behavioral factors, including tobacco, alcohol, and illicit drug use, and physical, sexual, and occupational activity. The authors concluded that with the exception of tobacco, which was consistently but weakly associated with preterm birth, evidence for a causal role for other factors was slight [30]. A recent national French study added new results by showing that cannabis consumption increased spontaneous preterm birth risks; however, only 1.2% of women reported smoking during pregnancy [55].

ch was consistently but weakly associated with preterm birth, evidence for a causal role for other factors was slight [30]. A recent national French study added new results by showing that cannabis consumption increased spontaneous preterm birth risks; however, only 1.2% of women reported smoking during pregnancy [55]. Prenatal smoking rates vary across Europe, from 5 to 19% of women in the countries that could provide these data (Table 1). Smoking was found to explain differences in preterm birth rates between socioeconomic groups, about one-third of the variation in Finland from 1987 to 2010 [56▪]. However, in another international study, the effect was not as large across Europe [57]. A study from Belgium reported reductions in the risk of preterm birth subsequent to the introduction of smoking bans in 2007 and 2010 [58], raising the question of exposure to second-hand smoke [59,60▪]; however, other factors may have contributed to these observed effects.

e effect was not as large across Europe [57]. A study from Belgium reported reductions in the risk of preterm birth subsequent to the introduction of smoking bans in 2007 and 2010 [58], raising the question of exposure to second-hand smoke [59,60▪]; however, other factors may have contributed to these observed effects. Recent studies advanced our knowledge of the impact of maternal BMI on preterm birth, another maternal characteristic that varies in Europe (Table 1). Cnattingius et al.[31▪▪] found a dose–response relationship between maternal overweight and indicated preterm birth in a large population-based study from Sweden and also showed that obese women were at increased risk for extremely preterm delivery following premature rupture of membranes and spontaneous labor. This latter finding has been confirmed in other populations [61▪▪,62]. In a study that looked at more refined BMI categories including severe (<16 kg/m2), moderate (16–16.99 kg/m2), and mild thinness (17–18.49 kg/m2), Lynch et al.[61▪▪] showed that women at the lower extremes of BMI were at increased risk for both spontaneous preterm labor and medically indicated delivery.

ions [61▪▪,62]. In a study that looked at more refined BMI categories including severe (<16 kg/m2), moderate (16–16.99 kg/m2), and mild thinness (17–18.49 kg/m2), Lynch et al.[61▪▪] showed that women at the lower extremes of BMI were at increased risk for both spontaneous preterm labor and medically indicated delivery. Bloomfield [63], based on a review of epidemiological and experimental studies, posited an important role for poor maternal nutrition in the association between extreme BMIs and prematurity. Other studies also explored dietary risk factors for preterm birth, such as artificially sweetened drinks, which were responsible for increased preterm birth risk in two large cohort studies [64,65]. Further, probiotics, vitamin D, and vitamin C supplementation may reduce preterm birth risk by preventing genital infections, but more research is needed [66▪].

factors for preterm birth, such as artificially sweetened drinks, which were responsible for increased preterm birth risk in two large cohort studies [64,65]. Further, probiotics, vitamin D, and vitamin C supplementation may reduce preterm birth risk by preventing genital infections, but more research is needed [66▪]. Recent studies examined the contribution of previous induced abortion to preterm birth rate [35,67▪▪]. The EuroPOP study had shown that induced abortions were associated with preterm birth rates [68]. In Scotland, using data from the 1980s to 2000, this association was found to weaken over time and disappeared altogether by 2000, maybe because of changes in abortion methods [68]. However, a study from Finland showed no statistically significant difference in preterm birth by abortion method (4.0% in the medical group vs. 4.9% in the surgical group) [69]. In parts of Eastern Europe where there is a history of abortion being used as contraception, variations in the prevalence of induced abortion may impact on differences in preterm birth rates.

ally significant difference in preterm birth by abortion method (4.0% in the medical group vs. 4.9% in the surgical group) [69]. In parts of Eastern Europe where there is a history of abortion being used as contraception, variations in the prevalence of induced abortion may impact on differences in preterm birth rates. VARIATION OWING TO INDICATED PRETERM BIRTH There is strong evidence that preterm birth rates in high-income countries are affected by obstetric practices related to indicated preterm births. Indicated singleton late preterm births have been identified as the main driver of North American preterm birth rates as opposed to changes in women's risk profiles [70–73]. Vanderweele et al.[74] showed that in the United States, although overall preterm births increased from 11.2 to 12.8% between 1989 and 2004, medically induced rates increased 94% from 3.4 to 6.6% and spontaneous rates declined by 21%, from 7.8 to 6.2%.

birth rates as opposed to changes in women's risk profiles [70–73]. Vanderweele et al.[74] showed that in the United States, although overall preterm births increased from 11.2 to 12.8% between 1989 and 2004, medically induced rates increased 94% from 3.4 to 6.6% and spontaneous rates declined by 21%, from 7.8 to 6.2%. In Europe, Zeitlin et al.[9▪▪] showed that both spontaneous and induced preterm deliveries contributed to increasing preterm birth trends between 1996 and 2008; the contribution of each subgroup varied across countries, especially for singletons. In 2008, rates of nonspontaneous singleton preterm births ranged from 1.1 to 3.0%, whereas spontaneous onset preterm births ranged from 2.8 to 4.8%. For multiples, the rates of nonspontaneous preterm birth ranged from 12.0 to 34.4%, and spontaneous onset births from 15.1 to 38.2% [9▪▪]. In Scotland, for instance, between 1989 and 2004, nonspontaneous onset deliveries increased by almost 50% and spontaneous deliveries by 10% [75]. In other European countries, however, nonspontaneous onset preterm births have not increased over past decades.

34.4%, and spontaneous onset births from 15.1 to 38.2% [9▪▪]. In Scotland, for instance, between 1989 and 2004, nonspontaneous onset deliveries increased by almost 50% and spontaneous deliveries by 10% [75]. In other European countries, however, nonspontaneous onset preterm births have not increased over past decades. Previous obstetric history and delivery mode are strong predictors of both spontaneous and indicated preterm delivery [32▪▪,76▪▪], but women's risk profiles can influence preterm birth subtypes in different ways. An Australian study, using population-based data from 1984 to 2006, showed that over time the population-attributable fraction associated with women's preexisting medical conditions and pregnancy complications increased, for both indicated and spontaneous preterm deliveries. The proportion of women with more than one medical condition increased from 4.9 to 19% in spontaneous preterm births and from 10.4 to 25.8% in medically indicated preterm deliveries [76▪▪].

men's preexisting medical conditions and pregnancy complications increased, for both indicated and spontaneous preterm deliveries. The proportion of women with more than one medical condition increased from 4.9 to 19% in spontaneous preterm births and from 10.4 to 25.8% in medically indicated preterm deliveries [76▪▪]. Provider-initiated preterm births aim to improve the health of the child, and especially to reduce the risk of stillbirth; however, they are controversial, as evidence of the benefits to the child of early extraction are not always conclusive and countries have more or less interventionist policies. Variations in gestational age patterns of cesarean delivery rates in Europe were recently described; these suggest wide variations in clinical practice by gestational age and highlight areas where consensus on best practices is lacking [77▪]. Further research should analyze the extent to which increases in indicated preterm births have affected not only preterm birth rates but also perinatal mortality. ENVIRONMENTAL FACTORS Pregnant women are exposed to a myriad of environmental factors and this field of research is expanding [4]. Patel et al.[78] used United States national survey data from 2000 to 2006 and looked at 201 different environment factors (i.e., amount of chemical compound in tap water sources of participants) including the number one suspect in terms of adverse health outcomes, Bisphenol A (BPA), which proved to be associated with preterm birth. BPA may represent an important health threat because of its toxicity and high prevalence in everyday products.

.e., amount of chemical compound in tap water sources of participants) including the number one suspect in terms of adverse health outcomes, Bisphenol A (BPA), which proved to be associated with preterm birth. BPA may represent an important health threat because of its toxicity and high prevalence in everyday products. Air pollution has also been linked in several recent studies to preterm birth. Air pollution exposures differ across Europe and vary over time [79▪▪]. For instance, urban population exposure to fine particulate matter has decreased between 2002 and 2011 in most countries except in central and eastern European countries where it increased dramatically [79▪▪]. Fine particulate matter may induce systemic inflammation, which could influence the duration of pregnancy [80▪]. Dadvand et al.'s [81▪] is the first study to report on the association between PPROM and PM2.5 and to report an increased risk of up to 50% in premature rupture of membranes associated with air pollution exposure. The negative impact of air pollution on gestational age was confirmed in Stieb et al.'s [82] 2012 meta-analysis, although there was a wide heterogeneity in study design and measures of exposure. More research on the physiological mechanisms through which air pollution influences gestational length is needed and clinical data are lacking from many observational studies.

age was confirmed in Stieb et al.'s [82] 2012 meta-analysis, although there was a wide heterogeneity in study design and measures of exposure. More research on the physiological mechanisms through which air pollution influences gestational length is needed and clinical data are lacking from many observational studies. Other environmental factors such as temperature [83,84▪▪,85,86] and UV light-induced vitamin D deficiency [87] have been explored, but it is unknown whether these could contribute to variations in preterm birth across countries.

age was confirmed in Stieb et al.'s [82] 2012 meta-analysis, although there was a wide heterogeneity in study design and measures of exposure. More research on the physiological mechanisms through which air pollution influences gestational length is needed and clinical data are lacking from many observational studies. Other environmental factors such as temperature [83,84▪▪,85,86] and UV light-induced vitamin D deficiency [87] have been explored, but it is unknown whether these could contribute to variations in preterm birth across countries. INTEGRATED APPROACHES Several recent studies tackled the larger question of how multiple population risk factors and medical practices explained preterm rate variations across countries or time. Zeitlin et al.[88▪▪] compared singleton preterm birth rates, based on obstetric estimates of gestational age, in France and the United States in 1995, 1998, and 2003; although many risk factors were different – in the United States, there were more teen pregnancies and women with insufficient prenatal care, but fewer smokers – adjustment for these factors did not reduce the constant excess risk of 70% in the United States (8.4% in the United States vs. 4.9% in France in 2003). Differences in rates could not be explained by obstetric interventions either: although preterm births associated with cesarean and induction were higher in absolute terms in the United States, spontaneous preterm birth rates were also elevated and the proportion of preterm births linked to these obstetrical interventions was the same. Garn et al.[89▪▪] compared maternal social and lifestyle characteristics, including stressful life events in Canada and the United States in 2005–2006 (preterm birth rates: 4.9 vs. 7.6%, respectively). Risk factors for preterm birth differed across countries and after adjustment, women in the United States still had a higher risk [89▪▪]. These results reinforce conclusions from a study which found that half of the increase in preterm birth rates from 1989 to 2004 (10.6–12.5%) in the United States remained unexplained after taking into account the contribution of maternal age, maternal race, maternal education, ART, multiple births, stillbirths averted, marital status, pregnancy intention, barriers to prenatal care initiation, as well as nonmedically indicated cesarean delivery and labor induction [7▪▪].

States remained unexplained after taking into account the contribution of maternal age, maternal race, maternal education, ART, multiple births, stillbirths averted, marital status, pregnancy intention, barriers to prenatal care initiation, as well as nonmedically indicated cesarean delivery and labor induction [7▪▪]. These studies illustrate the complexity of understanding the drivers of a country's preterm birth rate and pinpointing those that ‘explain’ the difference between countries. Multiple risk factors impact on preterm birth and studies in this review underscored the interdependence between them. Data on the whole range of key exposures are unlikely to be included in any one database and studies that combine databases face issues related to the comparability of data definitions [89▪▪]. Further, many risk factors interact with the type of preterm birth, that is spontaneous vs. indicated and differing approaches to indicated preterm births by country mean that common relationships may be obscured.

se and studies that combine databases face issues related to the comparability of data definitions [89▪▪]. Further, many risk factors interact with the type of preterm birth, that is spontaneous vs. indicated and differing approaches to indicated preterm births by country mean that common relationships may be obscured. CONCLUSION Among the multiple factors that emerged from this review of recent studies on preterm birth variations and trends within and between high-income countries, medical practices and policies related to subfertility treatments and indicated preterm deliveries had a clear impact on country-level preterm birth rates and trends. Understanding how some countries have maintained stable indicated preterm birth rates, whereas others have not – as well as the impact of these variations on child health – is an important research area. United States and Canadian studies showed that measurement of gestational age can have a large impact on the preterm birth rate estimate. Although this is unlikely to be a large contributor to European differences, we do not know whether gestational age determination differs across countries and it is important to rule out measurement artifacts. Finally, studies confirmed the role of many potentially modifiable population factors – BMI, smoking, and environmental exposures – in determining preterm birth risk. These factors likely interact and are associated with more general health and social policies that promote healthy childbearing. More knowledge about how these contribute to low and stable preterm birth risk would be enormously useful for shaping future policy.

vironmental exposures – in determining preterm birth risk. These factors likely interact and are associated with more general health and social policies that promote healthy childbearing. More knowledge about how these contribute to low and stable preterm birth risk would be enormously useful for shaping future policy. Acknowledgements This article drew on the work of the many people who contributed to the European Perinatal Health Report: The health and care of pregnant women and babies in Europe in 2010. They include statisticians, researchers, clinicians, administrators and others from each of the collaborating countries who compiled and submitted aggregated data for their countries to Euro-Peristat. They are too numerous to list here, but their names can found in Appendix A1 of the European Health Report at www.europeristat.com. The authors would like to thank them for their contributions. Thank you to Dr Michael Kramer who provided valuable comments for this paper. Financial support and sponsorship The Euro-Peristat project was funded by a grant from the European Commission (2010 13 01). The funding agency was not involved in the study. Conflicts of interest There are no conflicts of interest. REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as:▪ of special interest ▪▪ of outstanding interest Table 1 Preterm birth rates and prevalence of maternal risk factors in European countries in 2010

Conflicts of interest There are no conflicts of interest. REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as:▪ of special interest ▪▪ of outstanding interest Table 1 Preterm birth rates and prevalence of maternal risk factors in European countries in 2010 Country Live births (N) PTBa(%) Multiple births (%) Stand PTBb (%) <20 years of age (%) >35 years of age (%) Foreign bornc (%) Smoking during pregnancy (%) BMI <18.5 (%) BMI ≥30 (%) Austria 78698 8.4 3.5 8.3 3.2 19.7 29.3 BE: Brussels 24860 8.4 4.5 7.8 2.0 23.2 66.2 5.7 10.4 BE: Flanders 69637 7.9 3.8 7.7 1.8 14.3 22.4 5.3 12.4 BE: Wallonia 38228 8.3 3.3 8.3 3.8 16.0 25.2 7.1 13.6 Cyprus (2007) 8575 10.4 5.4 9.2 1.9 15.5 32.7 Czech Republic 116399 8.1 4.1 7.7 2.9 15.4 2.6 6.2 Denmark 63273 6.4 4.1 6.1 1.4 20.9 15.2 12.8 6.8 12.6 Estonia 15816 5.6 2.9 5.8 2.3 20.7 24.9 7.8 Finland 61191 5.7 3.1 5.7 2.3 18.0 6.2 1.0 3.6 12.1 France 14761 6.5 3.0 6.7 2.5 19.2 18.3 17.1 8.3 9.9 Germany 635561 8.4 3.7 8.1 2.1 23.6 16.9 8.5 3.6 13.7 Hungary 90322 8.9 NA NA 5.9 17.5 NA Iceland 4886 5.2 2.8 5.4 3.1 19.1 12.1 Ireland 75243 5.7 3.4 5.7 2.7 27.9 24.6 Italy 544991 7.3 3.2 7.4 1.4 34.7 19.0 Latvia 19139 5.8 2.5 6.1 5.9 14.7 30.2 Lithuania 30831 5.4 2.6 5.7 3.8 14.9 12.8 4.5 Luxembourg 6519 8.1 3.6 8.0 1.8 23.3 66.0 12.5 Malta 4018 7.2 4.0 6.9 6.5 15.5 9.2 5.2 12.7 Netherlands 177817 7.5 3.4 7.4 1.4 21.6 21.1 6.2 Norway 62678 6.2 3.3 6.2 2.2 19.5 24.8 7.6 4.1 12.2 Poland 413295 6.6 2.7 6.8 4.5 11.8 0.04 12.3 8.7 7.1 Portugal 101463 7.7 3.0 7.8 4.0 21.7 19.0 Romania 212199 8.2 1.8 8.7 10.6 10.9 NA Slovakia 55645 7.1 2.9 7.3 7.3 12.6 NA Slovenia 22298 7.2 3.7 7.1 1.2 15.4 NA 4.7 9.0 Spain 398914 8.0 4.2 7.5 2.5 29.5 23.6 14.4d Sweden 114706 5.9 2.8 6.1 1.6 22.5 24.4 4.9 2.5 12.6 Switzerland 79931 7.1 3.7 6.9 1.1 25.8 41.1 UK: England & Wales 718266 7.0 3.1 7.1 5.7 19.7 25.2 14.0e UK: Northern Ireland 25586 7.1 3.1 7.2 5.1 19.9 13.5 15.0 UK: Scotland 57151 7.0 3.1 7.1 6.4 19.9 13.9 19.0 2.6 20.7 United Kingdom 799 082 5.7 19.7 24.0 12.0 Total 4252575 Source: European Perinatal Health Report. The health and care of pregnant women and babies in Europe in 2010 [1▪▪].

7.0 3.1 7.1 5.7 19.7 25.2 14.0e UK: Northern Ireland 25586 7.1 3.1 7.2 5.1 19.9 13.5 15.0 UK: Scotland 57151 7.0 3.1 7.1 6.4 19.9 13.9 19.0 2.6 20.7 United Kingdom 799 082 5.7 19.7 24.0 12.0 Total 4252575 Source: European Perinatal Health Report. The health and care of pregnant women and babies in Europe in 2010 [1▪▪]. aPTB: preterm birth rate, defined as birth before 37 completed weeks of gestation. bStand. PTB: standardized preterm birth rate – adjusted on the prevalence of multiple births. cMothers born outside of the host country or of foreign nationality at birth (in Italy, Latvia, Lithuania, Malta, Poland) or ethnicity (in Denmark, Germany, Estonia) if data were unavailable. dData are from Catalonia. eAverage rate for UK: England (12.0%) and UK: Wales (16.0%).

INTRODUCTION Worldwide, approximately 240 000 women are diagnosed with ovarian cancer each year, and 140 200 are expected to succumb to the disease in 2016 [1,2]. This case-to-fatality ratio is nearly three times that of breast cancer, and makes ovarian cancer the most deadly gynecologic malignancy in developed countries. Patients with stage III or IV disease have a dismal 25% 5-year survival rate [2]. However, despite its aggressive clinical course, the American Cancer Society expects the number of ovarian cancer survivors to increase by 45 000 over the next decade [3].

cer the most deadly gynecologic malignancy in developed countries. Patients with stage III or IV disease have a dismal 25% 5-year survival rate [2]. However, despite its aggressive clinical course, the American Cancer Society expects the number of ovarian cancer survivors to increase by 45 000 over the next decade [3]. Ovarian cancer is a nonspecific term for a variety of tumors that involve the ovary. Ovarian cancers can be classified into three large groups: epithelial, germ cell, and specialized stromal cell tumors. The vast majority of ovarian cancers are epithelial ovarian cancers (EOCs). EOC can be further subdivided into various histological subtypes that fall into two main groups: Type I and Type II tumors. Type I tumors include low-grade serous, mucinous, endometrioid, clear cell carcinomas and tend to grow more slowly, often from an identifiable precursor. In contrast, Type II tumors are characterized by high-grade and rapidly progressive disease. High-grade serous ovarian carcinoma (HGSOC) is the most common Type II tumor, accounting for almost 75% of all EOCs. Unfortunately, it is also one of the most aggressive. There are currently no robust methods for early detection of HGSOC. As a result, the majority of women are diagnosed when the cancer has already metastasized to other tissues, usually within the peritoneal cavity. The lack of specific symptoms, even when the disease has spread to the peritoneum, contributes to delayed diagnosis and poor survival rates.

ods for early detection of HGSOC. As a result, the majority of women are diagnosed when the cancer has already metastasized to other tissues, usually within the peritoneal cavity. The lack of specific symptoms, even when the disease has spread to the peritoneum, contributes to delayed diagnosis and poor survival rates. The post-TCGA (The Cancer Genome Atlas) landscape for HGSOC is marked by surprisingly few recurrent somatic mutations [4]. Instead, this disease exhibits a complex genomic terrain, marked by copy number alterations that are so widespread that few other cancer types mirror its complexity. Intertumoral and intratumoral heterogeneity in HGSOC further decrease the likelihood of finding a single therapy that will prove beneficial for the majority of patients. Thus, HGSOC will require individualized therapy in which we unbraid a tumor's genomic profile to identify altered genes or pathways that offer an opportunity for therapeutic intervention. Box 1 no caption available

The post-TCGA (The Cancer Genome Atlas) landscape for HGSOC is marked by surprisingly few recurrent somatic mutations [4]. Instead, this disease exhibits a complex genomic terrain, marked by copy number alterations that are so widespread that few other cancer types mirror its complexity. Intertumoral and intratumoral heterogeneity in HGSOC further decrease the likelihood of finding a single therapy that will prove beneficial for the majority of patients. Thus, HGSOC will require individualized therapy in which we unbraid a tumor's genomic profile to identify altered genes or pathways that offer an opportunity for therapeutic intervention. Box 1 no caption available Pathogenesis of epithelial ovarian cancer Originally, the ovary was thought to be the primary site of HGSOC tumorigenesis and the ovarian surface epithelium (OSE) represented the cell of origin. The ‘incessant ovulation’ hypothesis suggested that HGSOC developed because of repetitive injury to the OSE with each ovulatory cycle [5]. It was thought that this repetitive injury causes increased inflammation and changes in hormone levels, leading to DNA damage produced by oxidative stress [5]. Incessant ovulation, through a rupture and repair mechanism, along with the normal proliferation of the OSE, was thought to drive metaplastic changes toward a more Müllerian-type epithelium. If this Müllerian-type epithelium harbored unresolved DNA damage, it would represent a prime target for neoplastic transformation [6]. Although the OSE model could account for a number of important features associated with ovarian cancer, particularly Type I tumors, it fails to present a path toward understanding of Type II tumors. Perhaps most importantly, attempts to reproducibly identify precursor lesions for HGSOC in the OSE have been largely unsuccessful.

the OSE model could account for a number of important features associated with ovarian cancer, particularly Type I tumors, it fails to present a path toward understanding of Type II tumors. Perhaps most importantly, attempts to reproducibly identify precursor lesions for HGSOC in the OSE have been largely unsuccessful. The cloning of the BRCA1 and BRCA2 genes quickly led to the practice of risk-reducing bilateral salpingo-oophorectomies in mutation carriers to reduce the risk of developing ovarian cancer [7▪]. These specimens afforded pathologists the opportunity to examine these tissues for occult cancers. Some of the earliest studies suggesting that the fallopian tube epithelium plays a much larger role in the development of ovarian cancer were reported by Piek et al.[8,9]. Subsequent studies confirmed the paradoxical observation that in the search for early ovarian cancers, most lesions were identified in the fallopian tube [10–15]. The development of a pathology protocol, called the SEE-FIM (Sectioning and Extensively Examining the FIMbriated end) protocol, to systematically evaluate the fallopian tubes of BRCA mutation carriers led to the reproducible identification of early serous carcinomas in the distal end of the fallopian tube. The vast majority of cases was localized to the fimbria and included serous tubal intraepithelial carcinoma (STIC) [16–18]. No intraepithelial or invasive serous carcinomas were identified in the ovaries of these samples [18,19]. Like the foci of invasive HGSOC, the STIC lesions were proliferative, as measured by Ki67 immunohistochemistry (IHC) and stained strongly for p53. More importantly, DNA sequencing revealed that the majority of STIC lesions harbor the same TP53 mutation as the concurrent HGSOC [20,21], indicative of their clonal nature.

Like the foci of invasive HGSOC, the STIC lesions were proliferative, as measured by Ki67 immunohistochemistry (IHC) and stained strongly for p53. More importantly, DNA sequencing revealed that the majority of STIC lesions harbor the same TP53 mutation as the concurrent HGSOC [20,21], indicative of their clonal nature. Further examination of the fallopian tubes identified short stretches of benign-appearing secretory cells that stained strongly for p53 and γ-H2AX, a marker of DNA damage. These foci of p53-positive cells harbored TP53 mutations but were not proliferative [17]. These patches were called ‘p53 signatures’ based on the requisite p53 IHC necessary to identify the otherwise benign looking cells. Importantly, the ‘p53 signature’, the STIC lesion, and HGSOC from the same patient harbor the same TP53 mutation [17], implying a clonal relationship between the nonproliferative ‘p53 signature’, the intraepithelial lesion, and the invasive cancer (Fig. 1).

IHC necessary to identify the otherwise benign looking cells. Importantly, the ‘p53 signature’, the STIC lesion, and HGSOC from the same patient harbor the same TP53 mutation [17], implying a clonal relationship between the nonproliferative ‘p53 signature’, the intraepithelial lesion, and the invasive cancer (Fig. 1). FIGURE 1 Pathological and genomic features of high-grade serous ovarian carcinomas (HGSOCs). The majority of HGSOCs emerge from the fallopian tube epithelium through a series of precursor lesions that target the secretory cell. Normal fallopian tube epithelium contains both secretory and ciliated cells and is typically immunonegative for p53. The benign ‘p53 signature’ is composed entirely of secretory cells that exhibit strong p53 expression and evidence of DNA damage but are not proliferative. With progression to a serous tubal intraepithelial carcinoma or ‘STIC’, there is acquisition of nuclear pleomorphism, mitoses, and loss of polarity. Invasive HGSOC shares all these properties and clinical symptoms typically emerge with advanced disease [22].

ression and evidence of DNA damage but are not proliferative. With progression to a serous tubal intraepithelial carcinoma or ‘STIC’, there is acquisition of nuclear pleomorphism, mitoses, and loss of polarity. Invasive HGSOC shares all these properties and clinical symptoms typically emerge with advanced disease [22]. What percentage of HGSOCs arises from the fallopian tube? Studies that implement the SEE-FIM protocol report that approximately 50–60% of HGSOCs are associated with a STIC lesion in the fallopian tube (Table 1). A number of explanations have been offered to explain why the association between HGSOC and STIC is not higher. These include insufficient sampling of tissue blocks [50,51], interobserver variability [52–54], consumption of precursors by the invasive carcinoma, and the high frequency of p53-negative STIC lesions [55]. It is also possible that extrauterine Müllerian epithelium [56] or derivatives of the OSE harbor precursor lesions. However, until reproducible precursors are identified at these sites, their contributions remain unclear. Resolving whether all HGSOCs arise from the fallopian tube or other sites remains to be determined and will likely require additional shared common resources and specimen banks [57▪].

OSE harbor precursor lesions. However, until reproducible precursors are identified at these sites, their contributions remain unclear. Resolving whether all HGSOCs arise from the fallopian tube or other sites remains to be determined and will likely require additional shared common resources and specimen banks [57▪]. The fallopian tube paradigm for HGSOC pathogenesis has motivated the development of new, robust, and tractable experimental model systems that focus on the fallopian tube as the site of origin. In particular, several mouse models were created by genetically manipulating murine oviductal cells [58–62]. Some of these models have recapitulated the development of tubal precursor lesions [58,60] and demonstrated that salpingectomy blocks tumor development [58,61]. More recently, Cho and colleagues developed a mouse in which the Ovgp1 promoter controls expression of a tamoxifen-regulated Cre recombinase in oviductal epithelium – the murine equivalent of human fallopian tube epithelium [59]. Deletion of Apc and Pten in this model was compared with a model in which an adenovirus expressing Cre was injected into the ovarian bursa to target the OSE. Tumors that emerged from the fallopian tube more closely resembled human endometrioid ovarian cancers than those from the OSE. The slow progression and late metastasis of oviductal tumors resemble the relatively indolent behavior characteristic of so-called Type I ovarian carcinomas in humans, for which endometrioid carcinoma is a prototype. This model emphasizes that importance of cellular context and the need to further understand the cell of origin for ovarian cancer [63].

peutic target in HGSOC. Small molecules that sculpt the mutant protein into a more wildtype confirmation are being evaluated in preclinical and clinical trials [69]. In addition, perturbation of pathways, like the mevalonate pathway, that lead to degradation of mutant p53 are being exploited for therapeutic gains [70]. Drugging BRCA in high-grade serous ovarian carcinoma Despite the high frequency of TP53 mutations observed in the development of HGSOC, TCGA data suggest that recurrent mutations in other genes are relatively uncommon, with the exception of BRCA1 and BRCA2[4]. BRCA1 and BRCA2 are proteins that play a critical role in maintaining the integrity of the genome by orchestrating DNA repair through homologous recombination. Homologous recombination is a high-fidelity process and is considered to be an error-free mechanism of repairing double-stranded breaks (DSBs) because it uses the sister chromatid as a template for repairs. This mechanism is in contrast to the other major pathway, known as nonhomologous DNA end joining (NHEJ), which simply ligates DSB ends without a template and is more error-prone. Double-stranded DNA breaks occur most frequently during DNA replication, especially when the replication machinery encounters a single-stranded break (SSB), ultimately leading to genomic instability and cell death if unrepaired. Mutations in BRCA1 and BRCA2 cause homologous recombination deficiency (HRD), making cells rely much more heavily on the NHEJ pathway to repair DSB. Although germline and somatic mutations in the BRCA genes account for approximately 15–20% of all HGSOCs, dysfunction in the BRCA network and homologous recombination appears to be more widespread, with approximately 50% of HGSOC harboring alterations in genes involved in homologous recombination [4,65▪▪,71–73]. For instance, the promoter of BRCA1 can be highly methylated, resulting in loss of gene expression and mimicking the BRCA1 mutant phenotype [4]. In addition to the BRCA genes, there are several inherited DNA repair genes that likely contribute to HRD when mutated. These include genes in the Fanconi anemia complex, the RAD51 paralogs (RAD51B, RAD51C, and RAD51D), BRIP1, BARD1, PALB2, as well as RAD50, CHEK2, ATR, and ATM[74–76]. These alterations collectively display HRD and are often described as having a ‘BRCAness’ phenotype [77,78] because of the genomic instability associated with BRCA dysfunction [65▪▪,79–81].

nemia complex, the RAD51 paralogs (RAD51B, RAD51C, and RAD51D), BRIP1, BARD1, PALB2, as well as RAD50, CHEK2, ATR, and ATM[74–76]. These alterations collectively display HRD and are often described as having a ‘BRCAness’ phenotype [77,78] because of the genomic instability associated with BRCA dysfunction [65▪▪,79–81]. Traditionally, ovarian cancers have been treated with cytotoxic agents, typically platinum-based chemotherapy, regardless of histological subtype. In fact, there are only two FDA approved targeted agents for use in ovarian cancer. The first is bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF). This antiangiogenic therapy was approved for use in recurrent, platinum-resistant ovarian cancer [82–84]. The second is olaparib, a poly-ribose polymerase (PARP) inhibitor. Olaparib was approved in 2014 for use in patients with BRCA mutations and recurrent disease [85,86]. The success of PARP inhibition is grounded in the idea that loss of PARP1 function in the setting of HRD (i.e., BRCA1/2 mutation) causes an increase in DNA aberrations, not all of which could be repaired due to HRD, resulting in cell death via synthetic lethality [86–89]. Synthetic lethality occurs when there is an inactivation of two genes or pathways, neither of which produces lethal effects on its own, but when combined cause cell death. There are a number of mechanisms that may underlie PARP–BRCA synthetic lethality. First, PARP-1 is involved in the repair of single strand breaks (SSBs), which, in the presence of a PARP inhibitor, may persist and cause collapse of replication forks leading to DSBs. Because BRCA defective cancer cells lack homologous recombination, the resulting DSBs would be selectively toxic to the cancer cells. Another mechanism involves PARP trapping. PARP inhibitors trap PARP-1 onto SSBs that form spontaneously or during base excision repair. Trapped PARP-1 can pose an obstacle to replication that would require homologous recombination to resolve [90]. Interestingly, despite the selective activity of PARP inhibitors in BRCA mutant tumors, more patients responded to PARP inhibitor therapy than those individuals with confirmed BRCA mutations [91].

ion repair. Trapped PARP-1 can pose an obstacle to replication that would require homologous recombination to resolve [90]. Interestingly, despite the selective activity of PARP inhibitors in BRCA mutant tumors, more patients responded to PARP inhibitor therapy than those individuals with confirmed BRCA mutations [91]. In fact, a recently published phase III clinical trial using the PARP inhibitor niraparib as maintenance therapy for patients with platinum-sensitive, recurrent ovarian cancer, demonstrated significantly prolonged progression free survival of patients regardless of their BRCA or HRD status [92▪▪]. These observations suggest that PARP inhibitors may have a broader role in ovarian cancer therapy. To date in the United States, only olaparib is FDA approved, although rucaparib was recently given breakthrough status by the FDA and others are expected to follow in the near future [93,94]. Currently, several clinical trials are progressing using different PARP inhibitors alone, or in combination with other drugs [95,96]. Studies like these show that PARP inhibitors have the potential to change the course of therapy for many individuals with ovarian cancer.

e expected to follow in the near future [93,94]. Currently, several clinical trials are progressing using different PARP inhibitors alone, or in combination with other drugs [95,96]. Studies like these show that PARP inhibitors have the potential to change the course of therapy for many individuals with ovarian cancer. CCNE1: a unique opportunity in high-grade serous ovarian carcinoma HGSOC is characterized by obligatory mutation of the TP53 gene, mutations in the homologous recombination DNA repair pathway, and widespread copy number alterations [4]. One of the most common copy number alterations in ovarian cancer is the amplification of the 19q12 locus. The Bowtell laboratory used a systematic knockdown of genes within the 19q12 amplicon to map CCNE1 as a key driver of the 19q12 amplicon [97]. CCNE1 encodes Cyclin E1, and it is amplified in a number of solid tumors (Fig. 2) and in approximately 20% of HGSOC cases (Fig. 1) [4,65▪▪]. Cyclin E1 protein levels vary during the cell cycle and play a major role in the G1-S phase transition by binding and activating the cyclin-dependent kinase 2 (CDK2) [98]. Aberrant Cyclin E1 expression is known to trigger unscheduled DNA replication, centrosome amplification, and chromosomal instability [99,100,101▪,102]. Importantly, CCNE1 amplification is associated with primary or refractory chemoresistant ovarian cancer [103] and poor overall survival [104,105]. Interestingly, amplification of CCNE1 and increased Cyclin E1 protein can be detected in STIC lesions, indicating that dysregulation of CCNE1 is an early event in the development of HGSOC [100,101▪,106].

on is associated with primary or refractory chemoresistant ovarian cancer [103] and poor overall survival [104,105]. Interestingly, amplification of CCNE1 and increased Cyclin E1 protein can be detected in STIC lesions, indicating that dysregulation of CCNE1 is an early event in the development of HGSOC [100,101▪,106]. FIGURE 2 Amplification of CCNE1 across human cancers. The cbioportal (http://www.cbioportal.org) was queried for ‘CCNE1: AMP’ and the resulting bar graph was limited to tumors with at least 4% amplification.

on is associated with primary or refractory chemoresistant ovarian cancer [103] and poor overall survival [104,105]. Interestingly, amplification of CCNE1 and increased Cyclin E1 protein can be detected in STIC lesions, indicating that dysregulation of CCNE1 is an early event in the development of HGSOC [100,101▪,106]. FIGURE 2 Amplification of CCNE1 across human cancers. The cbioportal (http://www.cbioportal.org) was queried for ‘CCNE1: AMP’ and the resulting bar graph was limited to tumors with at least 4% amplification. Protein abundance of Cyclin E1 is controlled at several levels, including by ubiquitin-mediated proteolysis by E3 ligases FBXW7 and PARK2, both of which are frequently deleted in human tumors [107], and by PP2A-B55β, a phosphatase that also controls Cyclin E1 turnover [108]. Proteolytic cleavage of Cyclin E1 to low-molecular weight (LMW) isoforms by the elastase family of serine proteases enhances transformation [109,110] and increased expression of LMW isoforms is associated with poor outcome in breast cancer [111]. We recently showed that induced expression of CCNE1 in fallopian tube secretory epithelial cells harboring a TP53 missense mutation leads to increase proliferation, colony formation, loss of contact inhibition, centrosome amplification, and modest anchorage independent growth [100,101▪]. As expected, we detected increased DNA damage in these cells as measured by phosphorylation of histone H2AX and increased comet tails [100]. Expression analysis of these CCNE1-overexpressing cells revealed that they upregulate key factors involved in homologous recombination and replication fork protection. Most notable was the upregulation of BRCA1, FANCD2, CDC25C, BLM, and XRCC2 (a RAD51 paralog). Amazingly, a synthetic lethal screen identified many of the same proteins as essential in CCNE1-amplified HGSOC cell lines [112]. These findings strongly suggest that the chromosomal instability generated by defects in the homologous recombination pathway and amplification of CCNE1 cannot coexist within the same cell and at least one of these pathways must be functional for survival of the cell. It also suggests that inhibition of DNA repair and replication fork protection pathways may be a viable therapeutics strategy in CCNE1-amplified tumors.

us recombination pathway and amplification of CCNE1 cannot coexist within the same cell and at least one of these pathways must be functional for survival of the cell. It also suggests that inhibition of DNA repair and replication fork protection pathways may be a viable therapeutics strategy in CCNE1-amplified tumors. Although CCNE1-amplified tumors represent a subset of HGSOC that deserve clinical attention, there are currently no targeted therapies for these tumors. The most obvious target is CDK2, the kinase partner of Cyclin E [113,114,115▪▪]. In fact, the development of small molecule inhibitors of CDKs has been an intense area of research [116] given their central role as regulators of cell division. Unfortunately, most compounds are not CDK2-specific and target multiple CDKs, eliciting dose-limiting toxicities that have slowed further clinical development [112,115▪▪,117]. However, the recent impressive findings with the CDK4/6 inhibitor palbociclib, targeted to Cyclin D1 and estrogen receptor-positive breast cancer [118], have renewed interest in the field [119–123]. In particular, a recent high-throughput compound screen in CCNE1-amplified ovarian cancer cell lines was performed to identify selective synergistic drug combinations with dinaciclib, a CDK1/2 inhibitor in clinical development. A synergistic therapeutic effect was elicited when dinaciclib was combined with an AKT2 inhibitor [115▪▪]. AKT2 and CCNE1 both reside on chromosome 19 and analysis of genomic data from TCGA demonstrated coamplification of CCNE1 and AKT2 in HGSOC. This finding suggests a specific dependency of CCNE1-amplified tumors for AKT activity, and points to a novel combination of dinaciclib and AKT inhibitors that may selectively target patients with CCNE1-amplified HGSOC, and possibly other solid tumors.

from TCGA demonstrated coamplification of CCNE1 and AKT2 in HGSOC. This finding suggests a specific dependency of CCNE1-amplified tumors for AKT activity, and points to a novel combination of dinaciclib and AKT inhibitors that may selectively target patients with CCNE1-amplified HGSOC, and possibly other solid tumors. CONCLUSION There is now significant clinical and experimental evidence pointing to the fallopian tube as the site of origin for a majority of HGSOCs. Next generation sequencing efforts have provided us with a panoramic view of HGSOCs and have revealed significant genomic heterogeneity. Alterations in the BRCA and CCNE1 pathways represent two distinct genotypes that exhibit unique vulnerabilities in DNA repair. The emergence of PARP inhibitors will change the clinical management of patients with BRCA mutant tumors as well as patients with tumors that are not HRD. Therapeutic approaches for CCNE1-amplified tumors are evolving and will likely exploit their dependency on homologous recombination and replication fork protection pathways. Acknowledgements We would like to thank Michael Cooper (www.cooper247.com) for assistance with graphic illustration. Financial support and sponsorship This work was supported by grants from the National Cancer Institute at the NIH P50-CA083636, NIH U01-CA152990, the CDMRP-OCRP (W81XWH-15-1-0160), the Dr Miriam and Sheldon G. Adelson Medical Research Foundation, and the Honorable Tina Brozman Foundation. Conflicts of interest R.D. serves on the Scientific Advisory Board of Siamab Therapeutics, Inc.

Financial support and sponsorship This work was supported by grants from the National Cancer Institute at the NIH P50-CA083636, NIH U01-CA152990, the CDMRP-OCRP (W81XWH-15-1-0160), the Dr Miriam and Sheldon G. Adelson Medical Research Foundation, and the Honorable Tina Brozman Foundation. Conflicts of interest R.D. serves on the Scientific Advisory Board of Siamab Therapeutics, Inc. REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as:▪ of special interest ▪▪ of outstanding interest Table 1 Incidents of tubal precursors in HGSOC

Financial support and sponsorship This work was supported by grants from the National Cancer Institute at the NIH P50-CA083636, NIH U01-CA152990, the CDMRP-OCRP (W81XWH-15-1-0160), the Dr Miriam and Sheldon G. Adelson Medical Research Foundation, and the Honorable Tina Brozman Foundation. Conflicts of interest R.D. serves on the Scientific Advisory Board of Siamab Therapeutics, Inc. REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as:▪ of special interest ▪▪ of outstanding interest Table 1 Incidents of tubal precursors in HGSOC Author % STIC in HGSOCa # STIC # HGSOC SEE-FIM Notes Leeper et al. [12] 60 3 5 No Powell et al. [13] 57 4 7 No Carcangiu et al. [23] 50 3 6 No Finch et al. [14] 86 6 7 No Medeiros et al. [18] 100 5 5 Yes Callahan et al. [24] 100 7 7 No Kindelberger et al. [20] 48 20 42 Yes Carlson et al. [25] 40 18 45 Some 47% with SEE-FIM, 35% without SEE-FIM Hirst et al. [26] 80 4 5 Yes Jarboe et al. [27] 23 5 22 Yes Roh et al. [28] 35 30 87 Yes Maeda et al. [29] 47 7 15 Yes Przybycin et al. [30] 59 24 41 Yes Leonhardt et al. [31] 33 3 9 Yes Manchanda et al. [32] 71 10 14 No Diniz et al. [33] 71 24 34 Some Powell et al. [34] 50 5 10 No Seidman et al. [35] 56 5 9 Some Tang et al. [36] 19 6 32 Yes Gao et al. [37] 92 107 116 Yes Lee et al. [38] 32 6 19 No Reitsma et al. [39] 75 3 4 Some Cases after 2006 are SEE-FIM Conner et al. [40] 74 14 19 Yes Koc et al. [41] 36 9 25 Yes Mingels et al. [42] 43 23 54 Yes Sherman et al. [43] 16 4 25 No Gilks et al. [44] 95 20 21 Yes Munakata and Yamamoto [45] 22 5 23 Some Only 10% SEE-FIM Seidman [46] 40 81 202 Some 1991–2007 no SEE-FIM, 2007–2011 half SEE-FIM Malmberg et al. [47] 61 8 13 No Mittal et al. [48] 22 7 32 Yes Zakhour et al. [49▪] 64 9 14 Some HGSOC, high-grade serous ovarian carcinoma; SEE-FIM, Sectioning and Extensively Examining the FIMbriated end; STIC, serous tubal intraepithelial carcinoma.

an [46] 40 81 202 Some 1991–2007 no SEE-FIM, 2007–2011 half SEE-FIM Malmberg et al. [47] 61 8 13 No Mittal et al. [48] 22 7 32 Yes Zakhour et al. [49▪] 64 9 14 Some HGSOC, high-grade serous ovarian carcinoma; SEE-FIM, Sectioning and Extensively Examining the FIMbriated end; STIC, serous tubal intraepithelial carcinoma. aValues are in %.