Journal of Obstetrics and Gynaecology, November 2013; 33: 857–861 © 2013 Informa UK, Ltd. ISSN 0144-3615 print/ISSN 1364-6893 online DOI: 10.3109/01443615.2013.831050
GYNAECOLOGY
Can anti-Müllerian hormone be predictive of spontaneous onset of pregnancy in women with unexplained infertility? L. Casadei, C. Manicuti, F. Puca, A. Madrigale, E. Emidi & E. Piccione
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Infertility Center, Section of Gynecology and Obstetrics, Academic Department of Biomedicine and Prevention and Clinical Department of Surgery, Tor Vergata University Hospital, Rome, Italy
The objective of the study was to assess anti-Müllerian hormone’s (AMH) role in predicting spontaneous onset of pregnancy. This observational cohort study included 83 women with unexplained infertility and normal or low ovarian reserve. Serum AMH, FSH, LH, 17β-oestradiol, inhibin B levels were measured and the number of early antral follicles (2–9 mm) was evaluated on days 2–5 of the cycle. Spearman’s correlation was used for comparison of strength of correlation. The diagnostic power of AMH in predicting spontaneous pregnancy was evaluated by receiver operating characteristic (ROC) curves. Markers of ovarian reserve in pregnant women and women without pregnancy were similar. In the entire study population, any markers (AMH, FSH, AFC, age), correlated with each other, but no marker was correlated with pregnancy. The area under the ROC curve for AMH reached a value of 0.385 0.07 (0.25–0.52, 95% confidence interval, CI); for FSH 0.415 0.08 (0.25–0.58, 95% CI); for AFC 0.418 0.08 (0.26–0.57, 95% CI), for age 0.496 0.08 (0.34–0.65, 95% CI). The study did not find a predictive role for AMH in predicting spontaneous onset of pregnancy. Even when AMH levels are very low, a spontaneous pregnancy may still occur. Keywords: Anti-Müllerian hormone (AMH), antral follicles, ovarian reserve, spontaneous pregnancy, unexplained infertility
Introduction In modern society, because of economic, social and professional reasons, women now more often decide to become pregnant in their 40s, when fertility is greatly reduced. Moreover, many women are affected by premature ovarian failure (POF) because of congenital conditions, gonad toxic therapies (chemotherapy, radiotherapy), pelvic surgery, endometriosis and smoking (Hehenkamp et al. 2007; Lie Fong et al. 2009; Younis 2011; Ferraretti et al. 2011), so it is necessary to identify the marker that can predict ovarian failure and allow women to plan pregnancy. In recent years, scientific literature considered the antiMüllerian hormone (AMH), also called Müllerian inhibiting substance, the best endocrine marker to assess ovarian reserve (Fiçicioğlu et al. 2006; van Disseldorp et al. 2010). AMH is a dimeric glycoprotein member of the transforming growth-factor-beta superfamily; it is produced by the granulosa cells and seems to have a regulator paracrine role of early follicular
growth. Serum levels of AMH grow in childhood up to puberty, then decrease in reproductive age and become undetectable in menopause (Lambalk et al. 2009). In women, AMH is produced by the preantral and small antral follicles with diameters 10 mm (Andersen et al. 2010). Recent studies demonstrate that AMH and the antral follicle count (AFC) predict realistically ovarian responsiveness to gonadotrophin hyperstimulation in women who underwent assisted reproductive technologies (ART) (Nardo et al. 2009; Jayaprakasan et al. 2010). AMH presents some advantages with respect to the other conventional markers of ovarian reserve. The blood sampling to assess plasmatic levels of AMH is less invasive and better tolerated than transvaginal ultrasonography. Moreover, the synthesis of AMH is FSH-independent, so it is possible to measure serum levels any day of the menstrual cycle, in pregnancy, amenorrhoea and under oral contraceptive and GnRH agonist pituitary downregulation (La Marca et al. 2006a,b; Mohamed et al. 2006; Somunkiran et al. 2007; Streuli et al. 2008; van Disseldorp et al. 2010; Andersen et al. 2010). Despite evidence of AMH’s role as a predictor of ovarian reserve and response to ovarian hyperstimulation in women undergoing ART (Smeenk et al. 2007), cut-off to identify the onset of spontaneous pregnancy or after ART is not yet established. Until 2007, no pregnancy had been described in women with serum AMH levels 1.1 ng/ml (Hazout et al. 2004) and 1.4 ng/ml (Fréour et al. 2007). Fraisse et al. (2008) published a case report of two spontaneous pregnancies obtained with serum AMH levels 0.4 ng/ml, and Tocci et al. (2009) reported the case of pregnancy in a woman undergoing ART with serum AMH levels 0.5 ng/ml. The aim of the present study was to verify whether the AMH level could predict the spontaneous onset of pregnancy in women with unexplained infertility.
Materials and methods This study included 83 women with unexplained infertility, selected from a larger group of infertile women who were referred to the Infertility Center, Section of Gynecology, Tor Vergata University Hospital, Rome, between May 2009 and September 2011. The study was carried out in accordance with the Helsinki Declaration for Medical Research involving Human Subjects and
Correspondence: L. Casadei, Section of Gynecology, Academic Department of Biomedicine and Prevention and Clinical Department of Surgery, Tor Vergata University Hospital, Viale Oxford 81, 00133 Rome, Italy. E-mail: [email protected]
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it was approved by the local ethical committee (Prot. No. 72/11). The patients gave their written consent. Inclusion criteria were: (1) unexplained infertility, that is the lack of pregnancy after 1 year of unprotected sexual intercourse in women without apparent disorder of fertility; (2) normal or low ovarian reserve; (3) both ovaries present; (4) regular menstrual cycles. The exclusion criteria were: (1) polycystic ovarian syndrome (PCOS), according to the Rotterdam criteria (Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group 2004); (2) congenital adrenal hyperplasia; (3) androgensecreting tumours; (4) Cushing syndrome; (5) male infertility; (6) tubal pathologies; (7) anovulation; (8) hyperprolactinaemia; (9) hypothalamic amenorrhoea; (10) previous ovarian surgery; (11) ovarian tumours; (12) anatomical abnormalities of the uterine cavity; (13) intraperitoneal adhesions; (14) endometriosis and other pelvic pathologies; (15) thyroid dysfunction and other endocrinological disorders such as diabetes mellitus; (16) recurrent pregnancy loss; (17) autoimmune diseases. Ultrasonographic evidence of ovulation was performed by a series of transvaginal ultrasound monitoring. All patients underwent hysterosalpingography and laparoscopy or only laparoscopy, to exclude tubal occlusions and other pelvic diseases. Partners were evaluated with at least two semen analysis determined normal according to the 2000 World Health Organization criteria. Patients were evaluated with a sonographic screening performed by a 6.5 MHz transvaginal transducer (Hitachi H21 HiVision, Hitachi Medical Corporation, Tokyo, Japan) in the early follicular phase (days 2–5 of cycle). The objectives were: evaluate the number and size of the early antral follicles and calculate the mean ovarian volume. The sum of all follicles of 2–9 mm in mean diameter in both ovaries was considered. Ovarian volume was calculated by the formula V 0.526 length height width. In all women, anti-Müllerian hormone (AMH); follicle-stimulating hormone (FSH); luteinising hormone (LH); 17β-oestradiol (E2); prolactin (PRL); total testosterone (TT) and free testosterone (FT), measurements on the 2nd–5th day of the cycle were made after a spontaneous bleed. AMH concentration were measured using the enzyme immunoassay AMH-EIA (reference A11893) provided by Immunotech (Beckman Coulter, Marseille, France). The sensitivity of the method defined as the lowest AMH concentration, significantly different from the zero calibrator, was 1 pM or 0.14 ng/ml. Intraand interassay coefficients of variation were 12.3% and 14.2%, respectively. Conversion factor: 1 pmol/l ng/ml 7.143. Our aims were to evaluate the spontaneous onset of pregnancy without medical therapy during 6 months of the diagnostic work-up in our infertility centre, and compare the main indicators of ovarian reserve, i.e. AMH, FSH, AFC, age, with the spontaneous onset of pregnancy. After this
observational and diagnostic period, the couples without pregnancy underwent ART. Statistical analysis was performed by SPSS software (v. 14.0 SPSS, Inc., Chicago, IL). Comparison of strength of correlations was performed using the Spearman’ correlation. Receiver operating characteristic (ROC) curves were constructed to examine the diagnostic test performance. Comparisons between the groups were performed by multivariate general linear model-based one-way analysis of variance (ANOVA). Bonferroni’s post-hoc test was applied whenever appropriate. The changing frequencies between the groups were analysed using the χ2-test or Fisher’s exact test. Statistical significance was defined as p 0.05.
Results After the diagnostic procedure, this study considered 83 women with unexplained infertility out of 425 infertile couples examined. For all women, the mean standard deviation (SD) and the range (min–max) of the parameters considered were: age 35.9 5.4 years (range 21–48 years); BMI 23.25 3.9 kg/m2 (range 16.9–41.4 kg/m2); duration of infertility 2.8 2.4 years (range 0.5–12 years); AFC 6.2 4.2 follicles (range 1–15); AMH 12.54 10.49 pmol/l (range 0.1–52.2 pmol/l); FSH 8.4 4.2 mIU/ ml (0.7–23 mIU/ml). The women were divided into two groups: group A included women who achieved spontaneous pregnancy and group B included women without pregnancy. Between the two groups, there were no statistically significant differences in the parameters considered (Table I). Table II shows the clinical, hormonal and ultrasonographic features in women divided according to the value of AMH or 5.35 pmol/l (0.75 ng/ml), which has been suggested as a cut-off for poor ovarian response in women undergoing ART (La Marca et al. 2007). Table II shows that there were no differences in the pregnancy rate between the two groups, however there were statistically significant differences in age, serum FSH levels and AFC. Of all 83 women, 14 achieved a spontaneous pregnancy (17%); five of these women had serum AMH 2.85 pmol/l ( 0.4 ng/ml). Table III shows the clinical, hormonal and ultrasonographic features and outcome of pregnancy in pregnant women with serum AMH 2.85 pmol/l. In the entire study population, any markers of ovarian reserve (AMH, FSH, AFC, age) correlated with each other; AMH levels were positively correlated with AFC (r 0.63, p 0.001) and negatively correlated with age (r 0.30, p 0.006) and FSH levels (r 0.47, p 0.001) but no marker was correlated with the pregnancy (Table IV). Using the ROC curves, the diagnostic potency of the AMH assay in predicting spontaneous onset of pregnancy was tested.
Table I. Clinical, hormonal and ultrasonographic features in all women, in pregnant and non-pregnant women. All women Mean SD
Range
Women (n) 83 Age (years) 21–48 35.9 5.4 BMI (kg/m2) 16.9–41.4 23.25 3.9 AMH (pmol/l) 12.54 10.49 0.1–52.2 FSH (mIU/ml) 0.7–23 8.4 4.2 Follicles 2–9 mm 1–15 6.2 4.2 Primary infertility (n) 54 Duration of infertility (years) 0.5–12 2.8 2.4 NS, not significant.
Pregnant women Mean SD 14 37.5 4.8 24.8 5.7 7.98 6.37 9.9 5.4 5.1 3.4 7 2.1 1.5
Range
Non-pregnant women Mean SD
Range
69 29–47 21–48 35.5 5.4 18.7–41.4 16.9–33.6 22.9 3.4 0.27–23.46 13.47 10.94 0.1–52.2 3.74–21.7 0.75–23 8.1 3.9 1–12 1–15 6.4 3.4 47 0.5–6 0.5–12 2.9 2.5
p value NS NS NS NS NS NS NS
Anti-Müllerian hormone and pregnancy 859 Table II. Clinical, hormonal and ultrasonographic features in women divided according to serum AMH. AMH 5.35 pmol/l Mean SD Women (n) Age (years) BMI (kg/m2) AMH (pmol/l) FSH (mIU/ml) Follicles 2–9 mm Primary infertility (n) Duration on infertility (years) Pregnancy Abortion Full-term pregnancy
27 38.8 4.3 24 3.6 2.1 1.52 11.1 5 3.7 2.1 17 3.1 2.5 5 2 3
Range 30–47 17.9–33.6 0.1–5 4.9–23 1–9.5 0.5–10
AMH 5.35 pmol/l Mean SD 56 34.4 5.2 22.9 4.1 17.57 9.15 7.1 3 7.4 3.4 37 2.6 2.3 9 0 9
Range
p value
21–48 16.9 –41.4 5.7–52.2 0.7–21.6 1–15
0.001 NS 0.001 0.001 0.001 NS NS NS NS NS
0.5–12
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NS, not significant.
As shown in Figure 1, the area under the ROC curve for AMH reached a value of 0.385 0.07 (0.25–0.52, 95% confidence interval, CI); for FSH 0.415 0.08 (0.25–0.58, 95% CI); for AFC 0.418 0.08 (0.26–0.57, 95% CI); for age 0.496 0.08 (0.34–0.65, 95% CI). Two threshold values of serum AMH level were analysed in terms of specificity and sensitivity from the ROC curve data. With a cut-off value of 2.85 pmol/l was obtained a specificity of 23.2% and a sensitivity of 78.6% and with a cut-off value of 5.35 pmol/l was obtained a specificity of 33.3% and a sensitivity of 64.3%.
Discussion In the last decade, AMH has been studied as an indicator of ovarian reserve. Many studies (Nardo et al. 2009; Jayaprakasan et al. 2010) demonstrated that antral follicle count and AMH values are the best indicators of ovarian reserve, because these markers allow the identification of women poorly responsive to ovarian stimulation. Some authors, in women undergoing ART, established an AMH cut-off of 5.35 pmol/l (0.75 ng/ml) (La Marca et al. 2007) or 5.0 pmol/l (Nelson et al. 2009) in predicting ovarian response to hyperstimulation. Women with AMH above 5.0 pmol/l who underwent in-vitro fertilisation had a significantly higher ongoing pregnancy rate than women with a serum AMH below 5.0 pmol/l but this cut-off is not specific as a predictor of ongoing pregnancy (Kini et al. 2010). Several studies in women undergoing ART, showed a strong positive correlation between serum AMH levels and the number of retrieved oocytes, oocyte quality and embryo morphology but some of these concluded that AMH level had little value for predicting pregnancy (Fiçicioğlu et al. 2006; Smeenk et al. 2007). In recent years, AMH has rapidly become the main test for ovarian reserve, but it is still uncertain how exactly it should be used. The question is whether we are causing too much unnecessary distress, or if low AMH should be used as a guide to IVF only. This study is useful because women with low AMH and
unexplained infertility are often compelled to do IVF. But it is not always necessary. In a recent study (Steiner et al. 2011) conducted on 100 women aged 30–44 years without a history of infertility, AMH seemed to qualify as an ovarian reserve test with significant predictive value for pregnancy, independent of the woman’s age. On the other hand, it has been reported, in 416 women without a history of infertility divided into four groups according to their age-specific AMH levels, a non-significant correlation between AMH and the number of pregnancies, abortions and deliveries (La Marca et al. 2012). To our knowledge, no study has been published about the correlation of AMH and the onset of spontaneous pregnancy in women with unexplained infertility. This is the first study that attempts to verify whether AMH is predictive of the onset of pregnancy and our preliminary data have shown, using a ROC curve, that AMH serum levels have no predictive role of spontaneous onset of pregnancy. On the other hand, in scientific literature, only two spontaneous pregnancies achieved in two women with serum AMH levels 0.4 ng/ml, are described (Fraisse et al. 2008), moreover it was not reported whether they were miscarriages or full-term pregnancies. In the present study, there were two abortions of five pregnancies obtained in women with AMH 2.85 pmol/l ( 0.4 ng/ml). In 2011, a consensus conference (Ferraretti et al. 2011) established diagnostic criteria for the definition of Poor Ovarian Response (POR). The minimal criteria required are the presence of at least two of the following three features: (1) advanced maternal age ( 40 years) or any other risk factor for POR; (2) a previous POR ( 3 oocytes with a conventional stimulation protocol); (3) an abnormal ovarian reserve test (i.e. AFC 5–7 follicles or AMH 0.5–1.1 ng/ml). However, POR does not necessarily mean sterility because each definition used in predicting the onset of pregnancy has no absolute value and also poor responders could become pregnant (Ferraretti et al. 2011). So, even in the case of POR, careful counselling for the infertile
Table III. Clinical, hormonal and ultrasonographic features and outcome of pregnancy in women with serum AMH 2.85 pmol/l. Case no. 1 2 3 4 5
Age (years)
Duration and type of infertility
AMH (pmol/l)
FSH (mIU/ml)
Follicles 2–9 mm (mean) (n)
Outcome of pregnancy
37 38 44 47 40
4 years, primary 1 year, primary 6 years, primary 1.5 years, secondary 3 years, secondary
2.85 2.85 1.92 1.14 0.80
13.43 4.88 12 21.67 20.91
2 6 2 3 1
Miscarriage at 5 weeks of pregnancy Full-term pregnancy, caesarean section Full-term pregnancy, caesarean section Miscarriage at 7 weeks of pregnancy Full-term pregnancy, caesarean section
860
L. Casadei et al. Table IV. Correlation between antral follicular count (AFC) and principal biochemical markers (all women). Age Age AMH FSH AFC Pregnancy
0.30 (p 0.006) 0.33 (p 0.002) 0.34 (p 0.002) 0.14 (p NS)
AMH
FSH
AFC
Pregnancy
0.30 (p 0.006)
0.33 (p 0.002) 0.47 (p 0.001)
0.34 (p 0.002) 0.63 (p 0.001) 0.60 (p 0.001)
0.14 (p NS) 0.20 (p NS) 0.16 (p NS) 0.13 (p NS)
0.47 (p 0.001) 0.63 (p 0.001) 0.20 (p NS)
0.6 (p 0.001) 0.16 (p NS)
0.13 (p NS)
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Values reported as Spearman’s index. NS, not significant.
couple is crucial to explain that reproductive potential is greatly diminished, but pregnancy is still possible (Weghofer et al. 2011). Management of women with POR presents clinical and ethical issues and also an economic burden. In some countries, fertility treatments are offered by the national health system, for which ART is already limited by age and reserved for women with reasonable chances of pregnancy. In the current study, it seems that no marker of ovarian reserve (AFC, AMH, FSH) has a predictive value of spontaneous onset of pregnancy. The limit of this study is the small sample size that limits actual predictive conclusions, and, given the understandably low pregnancy rate, our paper is only a little bigger than a case series, and a much larger study should be made in order to validate our preliminary data. Despite this limitation, the data reported in this study performed on women with unexplained infertility during a waiting period of 6 months without therapy, show that even when AMH levels are extremely low, the pregnancy can still occur. In other words, a reduced number of follicles and extremely low serum AMH levels do not seem to indicate reduced oocyte quality. Any link between AMH and
egg quality is still unknown and few studies have been able to address this complex question sufficiently. Oocyte quality, fertilisation, implantation and ongoing pregnancy depend on many factors, not only ovarian reserve. However, AMH the best ovarian reserve marker, seems not to predict the onset of pregnancy. However, now there are numerous reports of pregnancies in women with AMH levels below the detection limit and noone can be counted out based upon an AMH level. Perhaps the probability of conception is low, but there is still some chance, no matter how small. The woman needs to have this probability put into proper perspective by the consultant. In conclusion, AMH serum levels are normal in women with hypogonadotropic amenorrhoea, are low or undetectable in premature ovarian failure (La Marca et al. 2006a) but are high in PCOS women (La Marca et al. 2006a; Casadei et al. 2013), so AMH serum levels together with AFC, FSH serum levels and age of the woman, can help gynaecologists to find therapeutic strategies and appropriate ovarian hyperstimulation protocols in women undergoing ART. These approaches could potentially result in reduced complications and failures. It is clearly demonstrated that a single AMH
Figure 1. ROC curves for AMH, FSH, AFC and age for the prediction of spontaneous onset of pregnancy in women with unexplained infertility.
Anti-Müllerian hormone and pregnancy 861 assay may be used to individualise treatment strategies for ART (Nelson et al. 2009). Chronological age is a determinant of oocyte developmental potential, and serum AMH levels are useful to identify ovarian ageing and the prognosis in women of advanced reproductive age, in order to evaluate the waiting time before undergoing ART. Our study suggests that after a little waiting time, even in women of advanced reproductive age, when AMH levels are very low, the pregnancy can still occur, therefore some attempts at ART should not be denied. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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GYNAECOLOGY
Can anti-Müllerian hormone be predictive of spontaneous onset of pregnancy in women with unexplained infertility? L. Casadei, C. Manicuti, F. Puca, A. Madrigale, E. Emidi & E. Piccione
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Infertility Center, Section of Gynecology and Obstetrics, Academic Department of Biomedicine and Prevention and Clinical Department of Surgery, Tor Vergata University Hospital, Rome, Italy
The objective of the study was to assess anti-Müllerian hormone’s (AMH) role in predicting spontaneous onset of pregnancy. This observational cohort study included 83 women with unexplained infertility and normal or low ovarian reserve. Serum AMH, FSH, LH, 17β-oestradiol, inhibin B levels were measured and the number of early antral follicles (2–9 mm) was evaluated on days 2–5 of the cycle. Spearman’s correlation was used for comparison of strength of correlation. The diagnostic power of AMH in predicting spontaneous pregnancy was evaluated by receiver operating characteristic (ROC) curves. Markers of ovarian reserve in pregnant women and women without pregnancy were similar. In the entire study population, any markers (AMH, FSH, AFC, age), correlated with each other, but no marker was correlated with pregnancy. The area under the ROC curve for AMH reached a value of 0.385 0.07 (0.25–0.52, 95% confidence interval, CI); for FSH 0.415 0.08 (0.25–0.58, 95% CI); for AFC 0.418 0.08 (0.26–0.57, 95% CI), for age 0.496 0.08 (0.34–0.65, 95% CI). The study did not find a predictive role for AMH in predicting spontaneous onset of pregnancy. Even when AMH levels are very low, a spontaneous pregnancy may still occur. Keywords: Anti-Müllerian hormone (AMH), antral follicles, ovarian reserve, spontaneous pregnancy, unexplained infertility
Introduction In modern society, because of economic, social and professional reasons, women now more often decide to become pregnant in their 40s, when fertility is greatly reduced. Moreover, many women are affected by premature ovarian failure (POF) because of congenital conditions, gonad toxic therapies (chemotherapy, radiotherapy), pelvic surgery, endometriosis and smoking (Hehenkamp et al. 2007; Lie Fong et al. 2009; Younis 2011; Ferraretti et al. 2011), so it is necessary to identify the marker that can predict ovarian failure and allow women to plan pregnancy. In recent years, scientific literature considered the antiMüllerian hormone (AMH), also called Müllerian inhibiting substance, the best endocrine marker to assess ovarian reserve (Fiçicioğlu et al. 2006; van Disseldorp et al. 2010). AMH is a dimeric glycoprotein member of the transforming growth-factor-beta superfamily; it is produced by the granulosa cells and seems to have a regulator paracrine role of early follicular
growth. Serum levels of AMH grow in childhood up to puberty, then decrease in reproductive age and become undetectable in menopause (Lambalk et al. 2009). In women, AMH is produced by the preantral and small antral follicles with diameters 10 mm (Andersen et al. 2010). Recent studies demonstrate that AMH and the antral follicle count (AFC) predict realistically ovarian responsiveness to gonadotrophin hyperstimulation in women who underwent assisted reproductive technologies (ART) (Nardo et al. 2009; Jayaprakasan et al. 2010). AMH presents some advantages with respect to the other conventional markers of ovarian reserve. The blood sampling to assess plasmatic levels of AMH is less invasive and better tolerated than transvaginal ultrasonography. Moreover, the synthesis of AMH is FSH-independent, so it is possible to measure serum levels any day of the menstrual cycle, in pregnancy, amenorrhoea and under oral contraceptive and GnRH agonist pituitary downregulation (La Marca et al. 2006a,b; Mohamed et al. 2006; Somunkiran et al. 2007; Streuli et al. 2008; van Disseldorp et al. 2010; Andersen et al. 2010). Despite evidence of AMH’s role as a predictor of ovarian reserve and response to ovarian hyperstimulation in women undergoing ART (Smeenk et al. 2007), cut-off to identify the onset of spontaneous pregnancy or after ART is not yet established. Until 2007, no pregnancy had been described in women with serum AMH levels 1.1 ng/ml (Hazout et al. 2004) and 1.4 ng/ml (Fréour et al. 2007). Fraisse et al. (2008) published a case report of two spontaneous pregnancies obtained with serum AMH levels 0.4 ng/ml, and Tocci et al. (2009) reported the case of pregnancy in a woman undergoing ART with serum AMH levels 0.5 ng/ml. The aim of the present study was to verify whether the AMH level could predict the spontaneous onset of pregnancy in women with unexplained infertility.
Materials and methods This study included 83 women with unexplained infertility, selected from a larger group of infertile women who were referred to the Infertility Center, Section of Gynecology, Tor Vergata University Hospital, Rome, between May 2009 and September 2011. The study was carried out in accordance with the Helsinki Declaration for Medical Research involving Human Subjects and
Correspondence: L. Casadei, Section of Gynecology, Academic Department of Biomedicine and Prevention and Clinical Department of Surgery, Tor Vergata University Hospital, Viale Oxford 81, 00133 Rome, Italy. E-mail: [email protected]
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it was approved by the local ethical committee (Prot. No. 72/11). The patients gave their written consent. Inclusion criteria were: (1) unexplained infertility, that is the lack of pregnancy after 1 year of unprotected sexual intercourse in women without apparent disorder of fertility; (2) normal or low ovarian reserve; (3) both ovaries present; (4) regular menstrual cycles. The exclusion criteria were: (1) polycystic ovarian syndrome (PCOS), according to the Rotterdam criteria (Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group 2004); (2) congenital adrenal hyperplasia; (3) androgensecreting tumours; (4) Cushing syndrome; (5) male infertility; (6) tubal pathologies; (7) anovulation; (8) hyperprolactinaemia; (9) hypothalamic amenorrhoea; (10) previous ovarian surgery; (11) ovarian tumours; (12) anatomical abnormalities of the uterine cavity; (13) intraperitoneal adhesions; (14) endometriosis and other pelvic pathologies; (15) thyroid dysfunction and other endocrinological disorders such as diabetes mellitus; (16) recurrent pregnancy loss; (17) autoimmune diseases. Ultrasonographic evidence of ovulation was performed by a series of transvaginal ultrasound monitoring. All patients underwent hysterosalpingography and laparoscopy or only laparoscopy, to exclude tubal occlusions and other pelvic diseases. Partners were evaluated with at least two semen analysis determined normal according to the 2000 World Health Organization criteria. Patients were evaluated with a sonographic screening performed by a 6.5 MHz transvaginal transducer (Hitachi H21 HiVision, Hitachi Medical Corporation, Tokyo, Japan) in the early follicular phase (days 2–5 of cycle). The objectives were: evaluate the number and size of the early antral follicles and calculate the mean ovarian volume. The sum of all follicles of 2–9 mm in mean diameter in both ovaries was considered. Ovarian volume was calculated by the formula V 0.526 length height width. In all women, anti-Müllerian hormone (AMH); follicle-stimulating hormone (FSH); luteinising hormone (LH); 17β-oestradiol (E2); prolactin (PRL); total testosterone (TT) and free testosterone (FT), measurements on the 2nd–5th day of the cycle were made after a spontaneous bleed. AMH concentration were measured using the enzyme immunoassay AMH-EIA (reference A11893) provided by Immunotech (Beckman Coulter, Marseille, France). The sensitivity of the method defined as the lowest AMH concentration, significantly different from the zero calibrator, was 1 pM or 0.14 ng/ml. Intraand interassay coefficients of variation were 12.3% and 14.2%, respectively. Conversion factor: 1 pmol/l ng/ml 7.143. Our aims were to evaluate the spontaneous onset of pregnancy without medical therapy during 6 months of the diagnostic work-up in our infertility centre, and compare the main indicators of ovarian reserve, i.e. AMH, FSH, AFC, age, with the spontaneous onset of pregnancy. After this
observational and diagnostic period, the couples without pregnancy underwent ART. Statistical analysis was performed by SPSS software (v. 14.0 SPSS, Inc., Chicago, IL). Comparison of strength of correlations was performed using the Spearman’ correlation. Receiver operating characteristic (ROC) curves were constructed to examine the diagnostic test performance. Comparisons between the groups were performed by multivariate general linear model-based one-way analysis of variance (ANOVA). Bonferroni’s post-hoc test was applied whenever appropriate. The changing frequencies between the groups were analysed using the χ2-test or Fisher’s exact test. Statistical significance was defined as p 0.05.
Results After the diagnostic procedure, this study considered 83 women with unexplained infertility out of 425 infertile couples examined. For all women, the mean standard deviation (SD) and the range (min–max) of the parameters considered were: age 35.9 5.4 years (range 21–48 years); BMI 23.25 3.9 kg/m2 (range 16.9–41.4 kg/m2); duration of infertility 2.8 2.4 years (range 0.5–12 years); AFC 6.2 4.2 follicles (range 1–15); AMH 12.54 10.49 pmol/l (range 0.1–52.2 pmol/l); FSH 8.4 4.2 mIU/ ml (0.7–23 mIU/ml). The women were divided into two groups: group A included women who achieved spontaneous pregnancy and group B included women without pregnancy. Between the two groups, there were no statistically significant differences in the parameters considered (Table I). Table II shows the clinical, hormonal and ultrasonographic features in women divided according to the value of AMH or 5.35 pmol/l (0.75 ng/ml), which has been suggested as a cut-off for poor ovarian response in women undergoing ART (La Marca et al. 2007). Table II shows that there were no differences in the pregnancy rate between the two groups, however there were statistically significant differences in age, serum FSH levels and AFC. Of all 83 women, 14 achieved a spontaneous pregnancy (17%); five of these women had serum AMH 2.85 pmol/l ( 0.4 ng/ml). Table III shows the clinical, hormonal and ultrasonographic features and outcome of pregnancy in pregnant women with serum AMH 2.85 pmol/l. In the entire study population, any markers of ovarian reserve (AMH, FSH, AFC, age) correlated with each other; AMH levels were positively correlated with AFC (r 0.63, p 0.001) and negatively correlated with age (r 0.30, p 0.006) and FSH levels (r 0.47, p 0.001) but no marker was correlated with the pregnancy (Table IV). Using the ROC curves, the diagnostic potency of the AMH assay in predicting spontaneous onset of pregnancy was tested.
Table I. Clinical, hormonal and ultrasonographic features in all women, in pregnant and non-pregnant women. All women Mean SD
Range
Women (n) 83 Age (years) 21–48 35.9 5.4 BMI (kg/m2) 16.9–41.4 23.25 3.9 AMH (pmol/l) 12.54 10.49 0.1–52.2 FSH (mIU/ml) 0.7–23 8.4 4.2 Follicles 2–9 mm 1–15 6.2 4.2 Primary infertility (n) 54 Duration of infertility (years) 0.5–12 2.8 2.4 NS, not significant.
Pregnant women Mean SD 14 37.5 4.8 24.8 5.7 7.98 6.37 9.9 5.4 5.1 3.4 7 2.1 1.5
Range
Non-pregnant women Mean SD
Range
69 29–47 21–48 35.5 5.4 18.7–41.4 16.9–33.6 22.9 3.4 0.27–23.46 13.47 10.94 0.1–52.2 3.74–21.7 0.75–23 8.1 3.9 1–12 1–15 6.4 3.4 47 0.5–6 0.5–12 2.9 2.5
p value NS NS NS NS NS NS NS
Anti-Müllerian hormone and pregnancy 859 Table II. Clinical, hormonal and ultrasonographic features in women divided according to serum AMH. AMH 5.35 pmol/l Mean SD Women (n) Age (years) BMI (kg/m2) AMH (pmol/l) FSH (mIU/ml) Follicles 2–9 mm Primary infertility (n) Duration on infertility (years) Pregnancy Abortion Full-term pregnancy
27 38.8 4.3 24 3.6 2.1 1.52 11.1 5 3.7 2.1 17 3.1 2.5 5 2 3
Range 30–47 17.9–33.6 0.1–5 4.9–23 1–9.5 0.5–10
AMH 5.35 pmol/l Mean SD 56 34.4 5.2 22.9 4.1 17.57 9.15 7.1 3 7.4 3.4 37 2.6 2.3 9 0 9
Range
p value
21–48 16.9 –41.4 5.7–52.2 0.7–21.6 1–15
0.001 NS 0.001 0.001 0.001 NS NS NS NS NS
0.5–12
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NS, not significant.
As shown in Figure 1, the area under the ROC curve for AMH reached a value of 0.385 0.07 (0.25–0.52, 95% confidence interval, CI); for FSH 0.415 0.08 (0.25–0.58, 95% CI); for AFC 0.418 0.08 (0.26–0.57, 95% CI); for age 0.496 0.08 (0.34–0.65, 95% CI). Two threshold values of serum AMH level were analysed in terms of specificity and sensitivity from the ROC curve data. With a cut-off value of 2.85 pmol/l was obtained a specificity of 23.2% and a sensitivity of 78.6% and with a cut-off value of 5.35 pmol/l was obtained a specificity of 33.3% and a sensitivity of 64.3%.
Discussion In the last decade, AMH has been studied as an indicator of ovarian reserve. Many studies (Nardo et al. 2009; Jayaprakasan et al. 2010) demonstrated that antral follicle count and AMH values are the best indicators of ovarian reserve, because these markers allow the identification of women poorly responsive to ovarian stimulation. Some authors, in women undergoing ART, established an AMH cut-off of 5.35 pmol/l (0.75 ng/ml) (La Marca et al. 2007) or 5.0 pmol/l (Nelson et al. 2009) in predicting ovarian response to hyperstimulation. Women with AMH above 5.0 pmol/l who underwent in-vitro fertilisation had a significantly higher ongoing pregnancy rate than women with a serum AMH below 5.0 pmol/l but this cut-off is not specific as a predictor of ongoing pregnancy (Kini et al. 2010). Several studies in women undergoing ART, showed a strong positive correlation between serum AMH levels and the number of retrieved oocytes, oocyte quality and embryo morphology but some of these concluded that AMH level had little value for predicting pregnancy (Fiçicioğlu et al. 2006; Smeenk et al. 2007). In recent years, AMH has rapidly become the main test for ovarian reserve, but it is still uncertain how exactly it should be used. The question is whether we are causing too much unnecessary distress, or if low AMH should be used as a guide to IVF only. This study is useful because women with low AMH and
unexplained infertility are often compelled to do IVF. But it is not always necessary. In a recent study (Steiner et al. 2011) conducted on 100 women aged 30–44 years without a history of infertility, AMH seemed to qualify as an ovarian reserve test with significant predictive value for pregnancy, independent of the woman’s age. On the other hand, it has been reported, in 416 women without a history of infertility divided into four groups according to their age-specific AMH levels, a non-significant correlation between AMH and the number of pregnancies, abortions and deliveries (La Marca et al. 2012). To our knowledge, no study has been published about the correlation of AMH and the onset of spontaneous pregnancy in women with unexplained infertility. This is the first study that attempts to verify whether AMH is predictive of the onset of pregnancy and our preliminary data have shown, using a ROC curve, that AMH serum levels have no predictive role of spontaneous onset of pregnancy. On the other hand, in scientific literature, only two spontaneous pregnancies achieved in two women with serum AMH levels 0.4 ng/ml, are described (Fraisse et al. 2008), moreover it was not reported whether they were miscarriages or full-term pregnancies. In the present study, there were two abortions of five pregnancies obtained in women with AMH 2.85 pmol/l ( 0.4 ng/ml). In 2011, a consensus conference (Ferraretti et al. 2011) established diagnostic criteria for the definition of Poor Ovarian Response (POR). The minimal criteria required are the presence of at least two of the following three features: (1) advanced maternal age ( 40 years) or any other risk factor for POR; (2) a previous POR ( 3 oocytes with a conventional stimulation protocol); (3) an abnormal ovarian reserve test (i.e. AFC 5–7 follicles or AMH 0.5–1.1 ng/ml). However, POR does not necessarily mean sterility because each definition used in predicting the onset of pregnancy has no absolute value and also poor responders could become pregnant (Ferraretti et al. 2011). So, even in the case of POR, careful counselling for the infertile
Table III. Clinical, hormonal and ultrasonographic features and outcome of pregnancy in women with serum AMH 2.85 pmol/l. Case no. 1 2 3 4 5
Age (years)
Duration and type of infertility
AMH (pmol/l)
FSH (mIU/ml)
Follicles 2–9 mm (mean) (n)
Outcome of pregnancy
37 38 44 47 40
4 years, primary 1 year, primary 6 years, primary 1.5 years, secondary 3 years, secondary
2.85 2.85 1.92 1.14 0.80
13.43 4.88 12 21.67 20.91
2 6 2 3 1
Miscarriage at 5 weeks of pregnancy Full-term pregnancy, caesarean section Full-term pregnancy, caesarean section Miscarriage at 7 weeks of pregnancy Full-term pregnancy, caesarean section
860
L. Casadei et al. Table IV. Correlation between antral follicular count (AFC) and principal biochemical markers (all women). Age Age AMH FSH AFC Pregnancy
0.30 (p 0.006) 0.33 (p 0.002) 0.34 (p 0.002) 0.14 (p NS)
AMH
FSH
AFC
Pregnancy
0.30 (p 0.006)
0.33 (p 0.002) 0.47 (p 0.001)
0.34 (p 0.002) 0.63 (p 0.001) 0.60 (p 0.001)
0.14 (p NS) 0.20 (p NS) 0.16 (p NS) 0.13 (p NS)
0.47 (p 0.001) 0.63 (p 0.001) 0.20 (p NS)
0.6 (p 0.001) 0.16 (p NS)
0.13 (p NS)
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Values reported as Spearman’s index. NS, not significant.
couple is crucial to explain that reproductive potential is greatly diminished, but pregnancy is still possible (Weghofer et al. 2011). Management of women with POR presents clinical and ethical issues and also an economic burden. In some countries, fertility treatments are offered by the national health system, for which ART is already limited by age and reserved for women with reasonable chances of pregnancy. In the current study, it seems that no marker of ovarian reserve (AFC, AMH, FSH) has a predictive value of spontaneous onset of pregnancy. The limit of this study is the small sample size that limits actual predictive conclusions, and, given the understandably low pregnancy rate, our paper is only a little bigger than a case series, and a much larger study should be made in order to validate our preliminary data. Despite this limitation, the data reported in this study performed on women with unexplained infertility during a waiting period of 6 months without therapy, show that even when AMH levels are extremely low, the pregnancy can still occur. In other words, a reduced number of follicles and extremely low serum AMH levels do not seem to indicate reduced oocyte quality. Any link between AMH and
egg quality is still unknown and few studies have been able to address this complex question sufficiently. Oocyte quality, fertilisation, implantation and ongoing pregnancy depend on many factors, not only ovarian reserve. However, AMH the best ovarian reserve marker, seems not to predict the onset of pregnancy. However, now there are numerous reports of pregnancies in women with AMH levels below the detection limit and noone can be counted out based upon an AMH level. Perhaps the probability of conception is low, but there is still some chance, no matter how small. The woman needs to have this probability put into proper perspective by the consultant. In conclusion, AMH serum levels are normal in women with hypogonadotropic amenorrhoea, are low or undetectable in premature ovarian failure (La Marca et al. 2006a) but are high in PCOS women (La Marca et al. 2006a; Casadei et al. 2013), so AMH serum levels together with AFC, FSH serum levels and age of the woman, can help gynaecologists to find therapeutic strategies and appropriate ovarian hyperstimulation protocols in women undergoing ART. These approaches could potentially result in reduced complications and failures. It is clearly demonstrated that a single AMH
Figure 1. ROC curves for AMH, FSH, AFC and age for the prediction of spontaneous onset of pregnancy in women with unexplained infertility.
Anti-Müllerian hormone and pregnancy 861 assay may be used to individualise treatment strategies for ART (Nelson et al. 2009). Chronological age is a determinant of oocyte developmental potential, and serum AMH levels are useful to identify ovarian ageing and the prognosis in women of advanced reproductive age, in order to evaluate the waiting time before undergoing ART. Our study suggests that after a little waiting time, even in women of advanced reproductive age, when AMH levels are very low, the pregnancy can still occur, therefore some attempts at ART should not be denied. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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