http://informahealthcare.com/aan ISSN: 1939-6368 (print), 1939-6376 (electronic) Syst Biol Reprod Med, Early Online: 1–7 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/19396368.2014.973123
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The relationship between serum anti-Mu¨llerian hormone levels and the follicular arrest for women with polycystic ovary syndrome Jian Li1,2, Rong Li1*, Hua Yu2, Shuyun Zhao1, Yang Yu1, and Jie Qiao1 Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital No 49, Beijing, China and 2Center for Reproductive Medicine, Dalian Obstetric and Gynecology Hospital No 1, Dalian, China
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Abstract
Keywords
The aim of this study was to ascertain whether higher levels of serum anti-Mu¨llerian hormone (AMH) are associated with the ovarian follicular arrest in women with polycystic ovary syndrome (PCOS). This prospective study compared AMH levels between serum and dominant follicular fluid (FF) in ovulatory polycystic ovary (PCO) women and anovulatory (menstrual cycle 60 days.) PCOS women. All 102 women provided a baseline hormone profile and underwent controlled ovarian hyperstimulation (COH). The anovulatory PCO women had a similar body mass index (BMI), antral follicle count (AFC), and baseline serum AMH levels as the ovulatory PCO women except that their median luteinizing hormone (LH; 10.0 mIU/ml), testosterone (T) (0.61 ng/l), and androstenedione (A) (3.47 ng/l) levels were significantly higher than ovulatory PCO women (4.9 mIU/m; 0.43 ng/l and 2.09 ng/l, respectively). The ovarian response to gonadotropin stimulation during COH including serum AMH on the day of HCG administration and dominant FF AMH at 36 hours after HCG administration, total follicle stimulating hormone (FSH) dose administrated, peak E2, (estrogen) levels and number of occytes retrieved were all similar between women with anovulatory and ovulatory PCO. Using multiple regression analysis it was found that an important independent determinant affecting AMH was AFC, as opposed to LH and T. Logistic regression analysis showed that the two most important factors affecting ovulation were serum LH and T, whereas serum AMH and AFC were not selected for inclusion in the model. The reduction in AMH during COH occurs as a consequence of dominant follicles with a corresponding reduction in small antral follicle number. Elevated serum AMH levels in PCO women seem to be related only to follicular excess and not follicular arrest.
Anti-Mu¨llerian hormone, follicular arrest, LH, polycystic ovary syndrome, testosterone History Received 21 March 2014 Revised 31 July 2014 Accepted 2 August 2014 Published online 20 October 2014
Abbreviations: AMH: anti-Mu¨llerian hormone; PCOS: polycystic ovary syndrome; FF: follicular fluid; COH: controlled ovarian hyperstimulation; BMI: body mass index; AFC: antral follicle count; LH: luteinizing hormone; T: testosterone; A: androstenedione; FSH: follicle stimulating hormone; E2: estrogen; PCOM: polycystic ovarian morphology; MIS: Mu¨llerian inhibitory substance; IVF: in vitro fertilization; GnRHa: gonadotropin-releasing hormone agonist
Introduction Reproductive dysfunction presenting as polycystic ovary syndrome (PCOS) is primarily due to disrupted follicular development, including an excess of small follicles and the selective disruption of dominant follicles, i.e., follicular arrest. It has been demonstrated that increased levels of anti-Mu¨llerian hormone (AMH) are associated with follicular excess from polycystic ovarian morphology (PCOM) in women with PCOS [Chen et al. 2008; Laven et al. 2004; Pigny et al. 2006; Pigny et al. 2003]. AMH, also known as Mu¨llerian inhibitory substance (MIS), is primarily produced *Address correspondence to Rong Li, Center for Reproductive Medicine, Department of Obstetrics and Gynecology , Peking University Third Hospital, No 49, North Huayuan Road, Haidian District, Beijing, China 100191. Tel/Fax: +86-10-82265080. E-mail: [email protected]
by granulosa cells of secondary, preantral, and small antral follicles [Baarends et al. 1995; Weenen et al. 2004]. AMH has been proposed as a marker of ovarian reserve [La Marca et al. 2006; van Rooij et al. 2002]. A role for AMH in the regulation of follicle selection and maturation has also been hypothesized in some studies [Baarends et al. 1995; Pigny et al. 2003; Skalba et al. 2008]. In contrast to an excess of small follicles, follicular arrest does not always occur in patients with PCOS and some of them do even ovulate monthly [Jonard and Dewailly 2004]. It is not clear whether elevated serum AMH inhibits growing follicles (follicular arrest) in women with PCOS. If it is indeed the case, one would expect that the AMH levels in PCO women with anovulation, i.e., follicular arrest, would be higher than in PCO women with ovulation, i.e., without follicular arrest. In this study, our aim was to compare clinical
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Table 1. Comparison of baseline clinical and biochemical features on cycle day 2 among three groups of subjects (median and range). Control group (n ¼ 35) Age (years) BMI (kg/m2) AFC E2 (pg/l) FSH (mIU/ml) LH (mIU/ml) LH/FSH Androstenedione (ng/l) Testosterone (ng/l) AMH(ng/ml)
34 21.4 8 32 8.3 4.1 0.48 2.18 0.4 0 2.52
Ovulatory PCO group (n ¼ 29)
(27–39) (17.8–24.2) (2–16) (20–65) (5.5–16.1) (2.2–9.6) (0.14–0.99) (1.37–2.95) (0.37–0.55) (0.22–10.8)
Anovulatory PCO group (n ¼ 38)
32(25–37) 23.9 (19.0–28.6)a 22 (20–40)a 35 (20–115) 6.4 (3.0–8.8)a 4.9 (1.9–14) 0.69 (0.33–2.27) 2.09 (1.46–3.78) 0.43 (0.20–0.67) 5.87 (2.34–15.61)a
33 23.6 24 35 6.6 10.0 1.20 3.47 0.61 7.99
(26–39) (19.2–30.5)a (22–40)a (20–94) (3.7–15.5)a (2.8–16.8)a,b (0.64–2.67)a,b (1.52–6.10)a,b (0.23–1.60)a,b (3.13–14.80)a
: compared with control group, p50.05; b: compared with ovulatory PCO group, p50.05.
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a
(A) levels in the anovulatory PCO group were significantly higher than in the ovulatory PCO and control groups. In the anovulatory PCO group, 13/38 (34.2%) subjects had elevated T levels (40.8 ng/ml) and 19/38 (50%) subjects had elevated A levels (43.1 ng/ml), whereas in the ovulatory PCO group none of the subjects had elevated T levels, and 4/29 (13.8%) subjects had elevated A levels. Ovarian response during COH
Figure 1. Box and whisker plots show comparison of serum anti-Mu¨llerian hormone (AMH) levels on cycle day 2 (red, also UP) and day HCG (green, also DOWN), respectively, among three groups. Horizontal lines inside boxes represented the median, whereas the upper and lower limits of boxes and whickers represent, respectively, the 75th–25th percentiles and the 95th–5th percentiles.
and biochemical features between women with the ultrasound feature of PCO with anovulation and women with the ultrasound feature of PCO but with ovulation to ascertain if higher AMH levels or other factors such as high testosterone (T) are associated with the ovarian follicular arrest in women with the ultrasound feature of PCO.
Results Baseline clinical and biochemical features The clinical characteristics, baseline endocrine, and ultrasound data in the three groups (control, anovulatory PCO, and ovulatory PCO) are compared in Table 1. Body mass index (BMI), AFC, and serum AMH levels on day 2 of the cycle were similar between the anovulatory PCO group and the ovulatory PCO group, although the results in the control group were significantly (p50.01) lower than the PCO groups (Figure 1). The luteinizing hormone (LH), LH/follicle stimulating hormone (FSH) ratio, and T and androstenedione
The endocrine and clinical data of ovarian response to gonadotropin stimulation during controlled ovarian hyperstimulation (COH) is shown in Table 2. Following ovarian stimulation, serum AMH levels on the day of HCG administration were significantly (p50.001) lower than the baseline levels in each group. The serum AMH levels on the day of HCG administration in the anovulatory PCO group and ovulatory PCO group were both significantly (p50.001) higher than that of the control group, but there was no difference in the results between the anovulatory PCO group and ovulatory PCO groups (Figure 1). The decrease in serum AMH levels from cycle day 2 to the day of HCG administration was positively and significantly (p50.05) correlated with the number of oocytes retrieved in all three groups (control group r ¼ 0.780; ovulatory PCO group r ¼ 0.529, and anovulatory PCO group r ¼ 0.457). The increase in serum estrogen (E2) levels from day 2 to the day of HCG administration was also positively and significantly (p50.05) associated with the number of oocytes retrieved in each group. In addition, there was a significant (p50.01) positive correlation between the decrease in serum AMH levels and the increase in serum E2 levels in all three groups (control group r ¼ 0.649; ovulatory PCO group r ¼ 0.722, and anovulatory PCO group r ¼ 0.634). The follicular fluid (FF) AMH levels in the anovulatory PCO and ovulatory PCO groups were significantly (p50.05 and p50.001, respectively) higher than those of the control group, but there was no difference between the anovulatory PCO group and the ovulatory PCO group (Table 2). Correlations of factors affecting serum AMH Logistic regression analysis was performed to examine the possible factors affecting anovulation, including AMH, LH,
AMH level predicts PCO patients’ follicle excessive growth
DOI: 10.3109/19396368.2014.973123
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Table 2. Comparison of ovarian response during controlled ovarian hyperstimulation among three groups of subjects (median and range). Control group (n ¼ 35) Number of oocytes E2 on day hCG (pg/l) AMH on day hCG (ng/ml) Dominant FF AMH (ng/g) FSH dose (ampoule)
Ovulatory PCO group (n ¼ 29)
Anovulatory PCO group (n ¼ 38)
a
14 (3–28) 3145 (937–7398) 0.79 (0.03–2.88) 31.7 (8.1–178.0) 28 (20–60)
22 (14–38)a 4472 (1543–8000) 2.90 (1.13–7.00)a 64.1 (19.6–281.0)a 19 (14–29)a
20 (8–40) 2839 (862–8946) 2.45 (0.67–9.23)a 54.8 (22.3–203.3)a 18 (8–32)a
a
Compared with control group, p50.05; bCompared with ovulatory PCO group, p50.05.
Table 3. Logistic regression statistics of variables affecting anovulation. Independent variables
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Step 1
a
Step 2a
Step 3a
Step 4a
AMH LH Testosterone AFC Androstenedione Constant AMH LH Testosterone Androstenedione Constant LH Testosterone Androstenedione Constant LH Testosterone Constant
B
S.E.
Wald
Sig.
Exp(B)
0.052 0.211 0.593 0.018 0.139 3.718 0.024 0.214 0.620 0.129 3.884 0.219 0.660 0.125 3.777 0.216 0.915 3.057
0.166 0.120 0.714 0.064 0.187 1.830 0.132 0.120 0.709 0.184 1.741 0.117 0.679 0.185 1.636 0.114 0.585 1.180
0.098 3.108 0.690 0.081 0.554 4.127 0.033 3.197 0.763 0.490 4.977 3.539 0.945 0.460 5.333 3.614 2.447 6.706
0.755 0.078 0.406 0.776 0.457 0.042 0.857 0.074 0.382 0.484 0.026 0.060 0.331 0.498 0.021 0.057 0.118 0.010
1.053 1.235 1.809 0.982 1.149 0.024 1.024 1.239 1.858 1.138 0.021 1.245 1.934 1.133 0.023 1.242 2.496 0.047
a
Variable(s) entered on Step 1: AMH, LH, testosterone, AFC, androstenedione.
T, androstenedione, and AFC. Only baseline serum LH and T were found to be significantly correlated to anovulation, whereas baseline serum AMH, A, and AFC were not selected for inclusion in the model (Table 3). Spearman correlation analysis showed that only early AFC was significantly correlated with the baseline levels of serum AMH in each group. When the two PCO groups were combined, a significant correlation was observed between AMH and LH or T. In the control group, there was a significantly negative correlation with age, follicle stimulating hormone (FSH) levels, and total FSH dose administered. Furthermore, no relationship was found between baseline serum AMH and the other factors (Table 4). Multiple regression analysis was performed in the control group and the combined PCO group respectively, using AMH as the dependent variable and AFC, FSH, LH, T, and the total FSH dose as independent variables (Table 4). The only factor significantly correlated to baseline serum AMH levels (p50.05; p50.001) in the control group and combined PCO group was AFC, whereas other factors including baseline serum T, FSH, LH, and total FSH dose did not. The AMH levels were not significantly different between the pregnancy groups of the control group and the combined PCO group respectively (Table 5).
Discussion In this study, we have compared the baseline hormone profile and correlated the results to AMH levels, and the ovarian
Table 4. Spearman correlation analysis of variables affecting serum AMH levels. Control group
Age BMI AFC E2 FSH LH Androstenedione Testosterone FSH dose
Combined PCO group
Correlation
p
Correlation
p
0.46 0.16 0.77 0.37 0.50 0.40 0.25 0.23 0.72
50.05 NS 50.001 NS 50.05 NS NS NS 50.001
0.02 0.04 0.54 0.12 0.13 0.32 0.26 0.41 0.16
NS NS 50.001 NS NS 50.05 NS 50.01 NS
response to gonadotropin stimulation between women with ovulatory and anovulatory PCO undergoing IVF treatment. The baseline serum AMH levels were similar in ovulatory PCO women and anovulatory PCO women, but two to three times higher than in the control. However, we found that the baseline serum T, A, and LH concentrations in anovulatory PCO women were significantly higher than ovulatory PCO women and the control. This suggests that high T, A, and LH levels interfered with the final stage of follicular development leading to anovulation, consistent with the finding of previous studies [Dumesic et al. 2007; Yang et al. 2010]. Our observation that AMH was similar between ovulatory and anovulatory PCO women appears to be different from several previous studies [Diamanti-Kandarakis et al.
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Table 5. The relationship between pregnancy and serum AMH levels. Control group AMH levels (ng/ml)
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Pregnancy Yes No
1.13 ± 1.14 0.86 ± 0.74
Combined PCO group p
AMH levels (ng/ml)
p
0.551
3.40 ± 2.91 3.11 ± 1.76
0.650
2009; Homburg et al. 2013; Piouka et al. 2009; Thomson et al. 2009] which reported that AMH levels correlated with the severity of the symptoms of PCO, with apparently higher levels of AMH associated with menstrual irregularity and hyperandrogenism. A possible explanation for the observation in these previous reports is that AFC is an important confounding variable of the serum AMH result. Different follicle counts or other confounding variables may contribute to the difference in AMH levels between the groups. It is therefore uncertain if amenorrhea or hyperandrogenism is primarily a consequence of high serum AMH levels. In our study, there was no difference in follicle number between the two PCO groups, which helped to clarify the association between AMH levels and anovulation. Jonard and Dewailly [2004] reviewed the pathophysiological mechanism of follicular arrest, concluding that intraovarian hyperandrogenism may be the main culprit for the follicular arrest. However, it is unclear whether higher AMH serum levels are related to elevated androgen concentrations which in turn cause the disruption of folliculogenisis. Our results are in accordance with previous studies that showed a significantly positive correlation between AMH and T or LH [Homburg et al. 2013; Laven et al. 2004; Pigny et al. 2006; Skalba et al. 2011]. However, none of these studies performed multiple regression analysis. In the present study, multiple regression analysis showed that the most important factor affecting serum AMH levels in the model was AFC, whereas LH and T were not independent determinants in both the control group and combined PCO group (ovulatory and anovulatory PCO). This is in agreement with the findings of Pigny et al. [2003] in PCO women, confirming that the increase of serum AMH was primarily a consequence of an excess count of small antral follicles. In comparison, the difference in results may also be due to different study design. Some studies combined non-PCOS and PCOS women together [Eldar-Geva et al. 2005; Piouka et al. 2009] or combined different cohorts with different serum AMH levels [Laven et al. 2004], that may yield a biased association between AMH and androgen in these combined groups. However, using logistic regression in the PCO group, we found that LH and T appeared to be the two most important factors affecting ovulation, whereas AMH and AFC did not appear to predict anovulation. This further suggests that AMH and AFC are not correlated with features characteristic for PCO such as anovulation or hyperandrogenism. It has been speculated that the excess amount of AMH in PCO could be involved in follicular arrest through aromatase inhibition by interacting negatively with FSH [Catteau-Jonard et al. 2007; Durlinger et al. 2001; Franks et al. 2008;
Homburg et al. 2013]. However, the interaction between serum AMH and FSH in the selection failure of a dominant follicle remains unclear. Serum AMH has been considered a more sensitive marker of ovarian reserve than basal FSH [van Houten et al. 2010]. We observed a negative correlation between baseline AMH and FSH levels in the control group, but not in the PCO group. This was because both AMH and FSH reflect the number of small antral follicles in control women, whereas FSH is less sensitive in predicting ovarian reserve in PCO women. However, multiple regression analysis confirmed no correlation between AMH and FSH serum levels in each group, in agreement with previous observation in PCOS women [Laven et al. 2004; Pigny et al. 2003]. It was reported that serum AMH levels did not significantly change along with serum FSH levels that fluctuate throughout the menstrual cycle [La Marca et al. 2004; La Marca et al. 2006]. FSH either produced endogenously or administered did not appear to exert an effect on serum AMH levels when only one dominant follicle emerged [van Rooij et al. 2002; Wachs et al. 2007]. However serum AMH levels did change when there was multiple follicle development during COH [Fanchin et al. 2003; La Marca et al. 2004] and androgens might play a role in AMH regulation in women [Weintraub et al. 2014]. The decrease in serum AMH levels may be the result of increasing numbers of small follicles developing into large mature follicles, which lose their AMH expression. AMH is mainly expressed in secondary, preantral, and small antral follicles, with expression gradually weakening with follicular growth [Baarends et al. 1995; Weenen et al. 2004]. During COH, serum AMH levels decreased significantly while multiple dominant follicles emerged. However, serum AMH on the day of HCG administration and dominant FF AMH at 36 hours after HCG administration in anovulatory and ovulatory PCO women were still two to three times higher than control women. Similarly, the baseline serum AMH levels in women with PCO were two to three times higher than in control women. It seems that the high levels of AMH observed in PCO women in the late follicular phase (day of HCG administration) is not the reason for follicular arrest (the disruption of follicular development and maturation), whereas it predicts an excessive number of small antral follicles in PCO women which were not detected by ultrasound on the day of HCG administration, compared to control women. It has been observed that serum AMH levels remain rather constant in early and late follicular phases of a natural cycle [La Marca et al. 2004; La Marca et al. 2006; van Houten et al. 2010], indicating that serum AMH levels might not influence follicular maturation. It appears that serum AMH levels at various stages of a natural or stimulated cycle are positively correlated with the numbers of small antral follicles in the ovary. In this respect, the level of serum AMH may be considered as a surrogate of early antral follicle number in the ovary for the definition of PCOS in situations where accurate ultrasonographic data is not available [Dewailly et al. 2011; Pigny et al. 2006]. During COH the change in serum AMH and E2 levels was positively associated with dominant follicular counts (the number of oocytes retrieved) [Fanchin et al. 2003; La Marca et al. 2004]. This suggested that as multiple small antral
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DOI: 10.3109/19396368.2014.973123
AMH level predicts PCO patients’ follicle excessive growth
follicles grow into dominant follicles, AMH levels decrease, but at the same time E2 levels increase. Significant correlation was found between the decrease of serum AMH levels and the increase of E2 levels which parallel the emergence of multiple dominant follicles during COH, suggesting the change of serum AMH and E2 levels as result of follicular growth might not influence follicular development. The AMH levels had no significant difference between pregnancy and the control group and the combined PCO group, respectively (Table 5). In summary, the reduction in AMH during COH occurs as a consequence of dominant follicles with a corresponding reduction in the number of small antral follicles, but is not due to other factors such as high androgen. Elevated serum AMH levels in PCO women seem to be related only to follicular excess but not follicular arrest.
in this group did not take into consideration whether or not they had hyperandrogenaemia.
Materials and Methods The human tissues collection and study procedure have been approved by the Institutional Review Board at Peking University Third Hospital in the present study. The patients involved in the present study were informed of all details of the procedure, including sample utility and research destination, and signed an in vitro informed consent document voluntarily. Subjects This prospective study was conducted in the Peking University Third Hospital. Informed consent was obtained from all participants. Three groups of subjects, all undergoing in vitro fertilization (IVF) treatments, were recruited. Exclusion criteria in the study included a history of hypothalamus-pituitary diseases, abnormal thyroid function, endometriosis, previous ovarian surgery, and steroid hormone treatment. Control group (n ¼ 35) Women in the control group had tubal infertility, regular menstrual periods (25–35 days), normal uterus and ovarian morphology, and normal endocrine profile including FSH, LH, T, A, E2, testosterone, androstenedione, estrogen, and prolactin. Ovulatory PCO group (n ¼ 29) All women in this group exhibited ultrasound morphology typical of PCO, with 12 or more follicles measuring 2–9 mm in diameter in at least one ovary. Specifically, all women in this group were ovulatory. Serum androgen levels were not a criteria for inclusion in this group.
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Ovulation stimulation treatment All subjects underwent controlled ovarian hyperstimulation (COH) with gonadotropin-releasing hormone agonist (GnRHa) according to the short down-regulation protocol. GnRHa (Diphereline 0.1 mg; Beaufour Ipsen, France) was administered from day 2 of menstruation or progestin-induced withdrawal bleeding (cycle day 2). Ovarian stimulation started on day 3 with 150 450 IU daily of rFSH (Gonal-F 75 IU Per ampoule; Serono, Italy) according to patients’ ages and antral follicle count (AFC). The antral follicle count (follicle diameter 2 9 mm) was recorded on cycle day 2. Transvaginal ultrasound for follicular tracking and blood sampling for E2 levels were performed every 2 3 days. The FSH dose was adjusted based on E2 levels and follicular development. HCG (Profasi; Serono) 10,000 IU was administered when two or more dominant follicles reached 18 mm in diameter and oocyte retrieval was scheduled to take place 36 h after HCG administration. Sample collection Blood samples were collected from all patients on day 2 of menstruation or withdrawal bleeding and on day of HCG injection (day HCG). Serum samples were obtained after centrifuging the blood samples at 3000 rpm for 3 min, then stored at 80 C. The FF was aspirated gently from the first dominant follicle (18–20 mm in diameter) during oocyte retrieval. After the oocyte was isolated, FF was centrifuged at 3000 rpm for 15 min, and the supernatant stored at 80 C. Hormone assays AMH measurement was performed with the use of ELISA assay (Immunotach Beckman Coulter Laboratories, Brea, CA, American). Inter- and intra-assay coefficients of variation were as 59% and 56%, respectively. The determination range for AMH was 0.05–15 ng/ml. Serum E2, FSH, LH, T, and A concentrations were determined by using enzyme-amplified time-resolved fluorescence technology (DPC 1000 automatic analyzer, Los Angeles, CA, American). To make allowance for the variation in FF volume, FF hormone levels were adjusted to the relative content of per gram protein [Fanchin et al. 2005; Franchimont et al. 1989]. FF protein contents were determined by using routine biuret reaction method (BCA protein assay reagent kit, Pierce Rockford, IL, American). FF AMH levels were expressed as per gram of protein. Statistical analysis
Anovulatory PCO group (n ¼ 38) The definition of anovulatory was the menstrual cycle 60 days. All the women in this group were anovulatory with ultrasound morphology typical of PCO, with 12 or more follicles measuring 2–9 mm in diameter in at least one ovary. They did therefore fulfill the diagnostic criteria for PCOS [Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group 2004]. Similarly, the selection of women
Statistical analysis was performed with the use of SPSS10.0 Software System (SPSS Inc., Chicago, IL, USA). Descriptive statistics were generated to enable comparisons between groups. Continuous variables were checked for normality and means were presented with SD, or medians and ranges, as appropriate. Comparisons were performed using Student t test or ANOVA if the data distribution was normal. Mann-whitney U test and Kruskal-Wallis test were used if the data were not
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normally distributed. The relationship between AMH and other variables was analyzed by using spearman correlation analysis. Multiple regression analysis was used to control potential confounding variables. Logistic regression analysis was performed with ovulation or not as the dependent variable and AMH, AFC, LH, T, and A as covariates variable. A p value of 50.05 was considered statistically significant.
Declaration of interest
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This study was supported by National ‘‘Twelfth Five-Year’’ Plan for Science & Technology Support 2012BAI32B01, National Science Fund 81300482 & 81170538, and Beijing Municipal Natural Science Foundation 7142166. The authors declare that they have no competing interests.
Author contributions Carried out the data collection, performed the statistical analysis: JL; Supervised the analysis, helped in the study coordination and the data collection: RL; Took part in the discussion and data analysis: HY, SZ; Participated in the design of the study and drafted the manuscript: JQ. All authors read and approved the final manuscript.
References Baarends, W.M., Uilenbroek, J.T., Kramer, P., Hoogerbrugge, J.W., van Leeuwen, E.C., Themmen, A.P., et al. (1995) Anti-mullerian hormone and anti-mullerian hormone type II receptor messenger ribonucleic acid expression in rat ovaries during postnatal development, the estrous cycle, and gonadotropin-induced follicle growth. Endocrinology 136:4951–62. Catteau-Jonard, S., Pigny, P., Reyss, A.C., Decanter, C., Poncelet, E., and Dewailly, D. (2007) Changes in serum anti-mullerian hormone level during low-dose recombinant follicular-stimulating hormone therapy for anovulation in polycystic ovary syndrome. J Clin Endocrinol Metab 92:4138–43. Chen, M.J., Yang, W.S., Chen, C.L., Wu, M.Y., Yang, Y.S., and Ho, H.N. (2008) The relationship between anti-Mullerian hormone, androgen and insulin resistance on the number of antral follicles in women with polycystic ovary syndrome. Hum Reprod 23:952–7. Dewailly, D., Gronier, H., Poncelet, E., Robin, G., Leroy, M., Pigny, P., et al. (2011) Diagnosis of polycystic ovary syndrome (PCOS): Revisiting the threshold values of follicle count on ultrasound and of the serum AMH level for the definition of polycystic ovaries. Hum Reprod 26:3123–9. Diamanti-Kandarakis, E., Piouka, A., Livadas, S., Piperi, C., Katsikis, I., Papavassiliou, A.G., et al. (2009) Anti-mullerian hormone is associated with advanced glycosylated end products in lean women with polycystic ovary syndrome. Eur J Endocrinol 160:847–53. Dumesic, D.A., Abbott, D.H., and Padmanabhan, V. (2007) Polycystic ovary syndrome and its developmental origins. Rev Endocr Metab Disord 8:127–41. Durlinger, A.L., Gruijters, M.J., Kramer, P., Karels, B., Kumar, T.R., Matzuk, M.M., et al. (2001) Anti-Mullerian hormone attenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology 142:4891–9. Eldar-Geva, T., Margalioth, E.J., Gal, M., Ben-Chetrit, A., Algur, N., Zylber-Haran, E., et al. (2005) Serum anti-Mullerian hormone levels during controlled ovarian hyperstimulation in women with polycystic ovaries with and without hyperandrogenism. Hum Reprod 20: 1814–19. Fanchin, R., Louafi, N., Mendez Lozano, D.H., Frydman, N., Frydman, R., and Taieb, J. (2005) Per-follicle measurements indicate that anti-mullerian hormone secretion is modulated by the extent of follicular development and luteinization and may reflect qualitatively the ovarian follicular status. Fertil Steril 84:167–73.
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Fanchin, R., Schonauer, L.M., Righini, C., Frydman, N., Frydman, R., and Taieb, J. (2003) Serum anti-Mullerian hormone dynamics during controlled ovarian hyperstimulation. Hum Reprod 18:328–32. Franchimont, P., Hazee-Hagelstein, M.T., Hazout, A., Frydman, R., Schatz, B., and Demerle, F. (1989) Correlation between follicular fluid content and the results of in vitro fertilization and embryo transfer. I. Sex steroids. Fertil Steril 52:1006–11. Franks, S., Stark, J., and Hardy, K. (2008) Follicle dynamics and anovulation in polycystic ovary syndrome. Hum Reprod Update 14: 367–78. Homburg, R., Ray, A., Bhide, P., Gudi, A., Shah, A., Timms, P., et al. (2013) The relationship of serum anti-Mullerian hormone with polycystic ovarian morphology and polycystic ovary syndrome: A prospective cohort study. Hum Reprod 28:1077–83. Jonard, S. and Dewailly, D. (2004) The follicular excess in polycystic ovaries, due to intra-ovarian hyperandrogenism, may be the main culprit for the follicular arrest. Hum Reprod Update 10:107–17. La Marca, A., Malmusi, S., Giulini, S., Tamaro, L.F., Orvieto, R., Levratti, P., et al. (2004) Anti-Mullerian hormone plasma levels in spontaneous menstrual cycle and during treatment with FSH to induce ovulation. Hum Reprod 19:2738–41. La Marca, A., Stabile, G., Artenisio, A.C., and Volpe, A. (2006) Serum anti-Mullerian hormone throughout the human menstrual cycle. Hum Reprod 21:3103–7. Laven, J.S., Mulders, A.G., Visser, J.A., Themmen, A.P., De Jong, F.H., and Fauser, B.C. (2004) Anti-Mullerian hormone serum concentrations in normoovulatory and anovulatory women of reproductive age. J Clin Endocrinol Metab 89:318–23. Pigny, P., Jonard, S., Robert, Y., and Dewailly, D. (2006) Serum antiMullerian hormone as a surrogate for antral follicle count for definition of the polycystic ovary syndrome. J Clin Endocrinol Metab 91:941–5. Pigny, P., Merlen, E., Robert, Y., Cortet-Rudelli, C., Decanter, C., Jonard, S., et al. (2003) Elevated serum level of anti-mullerian hormone in patients with polycystic ovary syndrome: Relationship to the ovarian follicle excess and to the follicular arrest. J Clin Endocrinol Metab 88:5957–62. Piouka, A., Farmakiotis, D., Katsikis, I., Macut, D., Gerou, S., and Panidis, D. (2009) Anti-Mullerian hormone levels reflect severity of PCOS but are negatively influenced by obesity: Relationship with increased luteinizing hormone levels. Am J Physiol Endocrinol Metab 296:E238–43. Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group (2004) Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 81:19–25. Skalba, P., Cygal, A., and Dabkowska-Huc, A. (2008) The influence of anti-Mullerian hormone on folliculogenesis. Ginekol Pol 79: 137–40. Skalba, P., Cygal, A., Madej, P., Dabkowska-Huc, A., Sikora, J., Martirosian, G., et al. (2011) Is the plasma anti-Mullerian hormone (AMH) level associated with body weight and metabolic, and hormonal disturbances in women with and without polycystic ovary syndrome? Eur J Obstet Gynecol Reprod Biol 158:254–9. Thomson, R.L., Buckley, J.D., Moran, L.J., Noakes, M., Clifton, P.M., Norman, R.J., et al. (2009) The effect of weight loss on antiMullerian hormone levels in overweight and obese women with polycystic ovary syndrome and reproductive impairment. Hum Reprod 24:1976–81. van Houten, E.L., Themmen, A.P., and Visser, J.A. (2010) AntiMullerian hormone (AMH): Regulator and marker of ovarian function. Ann Endocrinol (Paris) 71:191–7. van Rooij, I.A., Broekmans, F.J., te Velde, E.R., Fauser, B.C., Bancsi, L.F., de Jong, F.H., et al. (2002) Serum anti-Mullerian hormone levels: A novel measure of ovarian reserve. Hum Reprod 17:3065–71. Wachs, D.S., Coffler, M.S., Malcom, P.J., and Chang, R.J. (2007) Serum anti-mullerian hormone concentrations are not altered by acute administration of follicle stimulating hormone in polycystic ovary syndrome and normal women. J Clin Endocrinol Metab 92: 1871–4. Weenen, C., Laven, J.S., Von Bergh, A.R., Cranfield, M., Groome, N.P., Visser, J.A., et al. (2004) Anti-Mullerian hormone expression pattern in the human ovary: Potential implications for initial and cyclic follicle recruitment. Mol Hum Reprod 10:77–83.
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Weintraub, A., Margalioth, E.J., Chetrit, A.B., Gal, M., Goldberg, D., Alerhand, S., et al. (2014) The dynamics of serum anti-Mullerianhormone levels during controlled ovarian hyperstimulation with GnRH-antagonist short protocol in polycystic ovary syndrome and low responders. Eur J Obstet Gynecol Reprod Biol 176:163–7.
Yang, J.L., Zhang, C.P., Li, L., Huang, L., Ji, S.Y., Lu, C.L., et al. (2010) Testosterone induces redistribution of forkhead box-3a and downregulation of growth and differentiation factor 9 messenger ribonucleic acid expression at early stage of mouse folliculogenesis. Endocrinology 151:774–82.
CLINICAL CORNER: COMMUNICATION
The relationship between serum anti-Mu¨llerian hormone levels and the follicular arrest for women with polycystic ovary syndrome Jian Li1,2, Rong Li1*, Hua Yu2, Shuyun Zhao1, Yang Yu1, and Jie Qiao1 Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital No 49, Beijing, China and 2Center for Reproductive Medicine, Dalian Obstetric and Gynecology Hospital No 1, Dalian, China
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Abstract
Keywords
The aim of this study was to ascertain whether higher levels of serum anti-Mu¨llerian hormone (AMH) are associated with the ovarian follicular arrest in women with polycystic ovary syndrome (PCOS). This prospective study compared AMH levels between serum and dominant follicular fluid (FF) in ovulatory polycystic ovary (PCO) women and anovulatory (menstrual cycle 60 days.) PCOS women. All 102 women provided a baseline hormone profile and underwent controlled ovarian hyperstimulation (COH). The anovulatory PCO women had a similar body mass index (BMI), antral follicle count (AFC), and baseline serum AMH levels as the ovulatory PCO women except that their median luteinizing hormone (LH; 10.0 mIU/ml), testosterone (T) (0.61 ng/l), and androstenedione (A) (3.47 ng/l) levels were significantly higher than ovulatory PCO women (4.9 mIU/m; 0.43 ng/l and 2.09 ng/l, respectively). The ovarian response to gonadotropin stimulation during COH including serum AMH on the day of HCG administration and dominant FF AMH at 36 hours after HCG administration, total follicle stimulating hormone (FSH) dose administrated, peak E2, (estrogen) levels and number of occytes retrieved were all similar between women with anovulatory and ovulatory PCO. Using multiple regression analysis it was found that an important independent determinant affecting AMH was AFC, as opposed to LH and T. Logistic regression analysis showed that the two most important factors affecting ovulation were serum LH and T, whereas serum AMH and AFC were not selected for inclusion in the model. The reduction in AMH during COH occurs as a consequence of dominant follicles with a corresponding reduction in small antral follicle number. Elevated serum AMH levels in PCO women seem to be related only to follicular excess and not follicular arrest.
Anti-Mu¨llerian hormone, follicular arrest, LH, polycystic ovary syndrome, testosterone History Received 21 March 2014 Revised 31 July 2014 Accepted 2 August 2014 Published online 20 October 2014
Abbreviations: AMH: anti-Mu¨llerian hormone; PCOS: polycystic ovary syndrome; FF: follicular fluid; COH: controlled ovarian hyperstimulation; BMI: body mass index; AFC: antral follicle count; LH: luteinizing hormone; T: testosterone; A: androstenedione; FSH: follicle stimulating hormone; E2: estrogen; PCOM: polycystic ovarian morphology; MIS: Mu¨llerian inhibitory substance; IVF: in vitro fertilization; GnRHa: gonadotropin-releasing hormone agonist
Introduction Reproductive dysfunction presenting as polycystic ovary syndrome (PCOS) is primarily due to disrupted follicular development, including an excess of small follicles and the selective disruption of dominant follicles, i.e., follicular arrest. It has been demonstrated that increased levels of anti-Mu¨llerian hormone (AMH) are associated with follicular excess from polycystic ovarian morphology (PCOM) in women with PCOS [Chen et al. 2008; Laven et al. 2004; Pigny et al. 2006; Pigny et al. 2003]. AMH, also known as Mu¨llerian inhibitory substance (MIS), is primarily produced *Address correspondence to Rong Li, Center for Reproductive Medicine, Department of Obstetrics and Gynecology , Peking University Third Hospital, No 49, North Huayuan Road, Haidian District, Beijing, China 100191. Tel/Fax: +86-10-82265080. E-mail: [email protected]
by granulosa cells of secondary, preantral, and small antral follicles [Baarends et al. 1995; Weenen et al. 2004]. AMH has been proposed as a marker of ovarian reserve [La Marca et al. 2006; van Rooij et al. 2002]. A role for AMH in the regulation of follicle selection and maturation has also been hypothesized in some studies [Baarends et al. 1995; Pigny et al. 2003; Skalba et al. 2008]. In contrast to an excess of small follicles, follicular arrest does not always occur in patients with PCOS and some of them do even ovulate monthly [Jonard and Dewailly 2004]. It is not clear whether elevated serum AMH inhibits growing follicles (follicular arrest) in women with PCOS. If it is indeed the case, one would expect that the AMH levels in PCO women with anovulation, i.e., follicular arrest, would be higher than in PCO women with ovulation, i.e., without follicular arrest. In this study, our aim was to compare clinical
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Table 1. Comparison of baseline clinical and biochemical features on cycle day 2 among three groups of subjects (median and range). Control group (n ¼ 35) Age (years) BMI (kg/m2) AFC E2 (pg/l) FSH (mIU/ml) LH (mIU/ml) LH/FSH Androstenedione (ng/l) Testosterone (ng/l) AMH(ng/ml)
34 21.4 8 32 8.3 4.1 0.48 2.18 0.4 0 2.52
Ovulatory PCO group (n ¼ 29)
(27–39) (17.8–24.2) (2–16) (20–65) (5.5–16.1) (2.2–9.6) (0.14–0.99) (1.37–2.95) (0.37–0.55) (0.22–10.8)
Anovulatory PCO group (n ¼ 38)
32(25–37) 23.9 (19.0–28.6)a 22 (20–40)a 35 (20–115) 6.4 (3.0–8.8)a 4.9 (1.9–14) 0.69 (0.33–2.27) 2.09 (1.46–3.78) 0.43 (0.20–0.67) 5.87 (2.34–15.61)a
33 23.6 24 35 6.6 10.0 1.20 3.47 0.61 7.99
(26–39) (19.2–30.5)a (22–40)a (20–94) (3.7–15.5)a (2.8–16.8)a,b (0.64–2.67)a,b (1.52–6.10)a,b (0.23–1.60)a,b (3.13–14.80)a
: compared with control group, p50.05; b: compared with ovulatory PCO group, p50.05.
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a
(A) levels in the anovulatory PCO group were significantly higher than in the ovulatory PCO and control groups. In the anovulatory PCO group, 13/38 (34.2%) subjects had elevated T levels (40.8 ng/ml) and 19/38 (50%) subjects had elevated A levels (43.1 ng/ml), whereas in the ovulatory PCO group none of the subjects had elevated T levels, and 4/29 (13.8%) subjects had elevated A levels. Ovarian response during COH
Figure 1. Box and whisker plots show comparison of serum anti-Mu¨llerian hormone (AMH) levels on cycle day 2 (red, also UP) and day HCG (green, also DOWN), respectively, among three groups. Horizontal lines inside boxes represented the median, whereas the upper and lower limits of boxes and whickers represent, respectively, the 75th–25th percentiles and the 95th–5th percentiles.
and biochemical features between women with the ultrasound feature of PCO with anovulation and women with the ultrasound feature of PCO but with ovulation to ascertain if higher AMH levels or other factors such as high testosterone (T) are associated with the ovarian follicular arrest in women with the ultrasound feature of PCO.
Results Baseline clinical and biochemical features The clinical characteristics, baseline endocrine, and ultrasound data in the three groups (control, anovulatory PCO, and ovulatory PCO) are compared in Table 1. Body mass index (BMI), AFC, and serum AMH levels on day 2 of the cycle were similar between the anovulatory PCO group and the ovulatory PCO group, although the results in the control group were significantly (p50.01) lower than the PCO groups (Figure 1). The luteinizing hormone (LH), LH/follicle stimulating hormone (FSH) ratio, and T and androstenedione
The endocrine and clinical data of ovarian response to gonadotropin stimulation during controlled ovarian hyperstimulation (COH) is shown in Table 2. Following ovarian stimulation, serum AMH levels on the day of HCG administration were significantly (p50.001) lower than the baseline levels in each group. The serum AMH levels on the day of HCG administration in the anovulatory PCO group and ovulatory PCO group were both significantly (p50.001) higher than that of the control group, but there was no difference in the results between the anovulatory PCO group and ovulatory PCO groups (Figure 1). The decrease in serum AMH levels from cycle day 2 to the day of HCG administration was positively and significantly (p50.05) correlated with the number of oocytes retrieved in all three groups (control group r ¼ 0.780; ovulatory PCO group r ¼ 0.529, and anovulatory PCO group r ¼ 0.457). The increase in serum estrogen (E2) levels from day 2 to the day of HCG administration was also positively and significantly (p50.05) associated with the number of oocytes retrieved in each group. In addition, there was a significant (p50.01) positive correlation between the decrease in serum AMH levels and the increase in serum E2 levels in all three groups (control group r ¼ 0.649; ovulatory PCO group r ¼ 0.722, and anovulatory PCO group r ¼ 0.634). The follicular fluid (FF) AMH levels in the anovulatory PCO and ovulatory PCO groups were significantly (p50.05 and p50.001, respectively) higher than those of the control group, but there was no difference between the anovulatory PCO group and the ovulatory PCO group (Table 2). Correlations of factors affecting serum AMH Logistic regression analysis was performed to examine the possible factors affecting anovulation, including AMH, LH,
AMH level predicts PCO patients’ follicle excessive growth
DOI: 10.3109/19396368.2014.973123
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Table 2. Comparison of ovarian response during controlled ovarian hyperstimulation among three groups of subjects (median and range). Control group (n ¼ 35) Number of oocytes E2 on day hCG (pg/l) AMH on day hCG (ng/ml) Dominant FF AMH (ng/g) FSH dose (ampoule)
Ovulatory PCO group (n ¼ 29)
Anovulatory PCO group (n ¼ 38)
a
14 (3–28) 3145 (937–7398) 0.79 (0.03–2.88) 31.7 (8.1–178.0) 28 (20–60)
22 (14–38)a 4472 (1543–8000) 2.90 (1.13–7.00)a 64.1 (19.6–281.0)a 19 (14–29)a
20 (8–40) 2839 (862–8946) 2.45 (0.67–9.23)a 54.8 (22.3–203.3)a 18 (8–32)a
a
Compared with control group, p50.05; bCompared with ovulatory PCO group, p50.05.
Table 3. Logistic regression statistics of variables affecting anovulation. Independent variables
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Step 1
a
Step 2a
Step 3a
Step 4a
AMH LH Testosterone AFC Androstenedione Constant AMH LH Testosterone Androstenedione Constant LH Testosterone Androstenedione Constant LH Testosterone Constant
B
S.E.
Wald
Sig.
Exp(B)
0.052 0.211 0.593 0.018 0.139 3.718 0.024 0.214 0.620 0.129 3.884 0.219 0.660 0.125 3.777 0.216 0.915 3.057
0.166 0.120 0.714 0.064 0.187 1.830 0.132 0.120 0.709 0.184 1.741 0.117 0.679 0.185 1.636 0.114 0.585 1.180
0.098 3.108 0.690 0.081 0.554 4.127 0.033 3.197 0.763 0.490 4.977 3.539 0.945 0.460 5.333 3.614 2.447 6.706
0.755 0.078 0.406 0.776 0.457 0.042 0.857 0.074 0.382 0.484 0.026 0.060 0.331 0.498 0.021 0.057 0.118 0.010
1.053 1.235 1.809 0.982 1.149 0.024 1.024 1.239 1.858 1.138 0.021 1.245 1.934 1.133 0.023 1.242 2.496 0.047
a
Variable(s) entered on Step 1: AMH, LH, testosterone, AFC, androstenedione.
T, androstenedione, and AFC. Only baseline serum LH and T were found to be significantly correlated to anovulation, whereas baseline serum AMH, A, and AFC were not selected for inclusion in the model (Table 3). Spearman correlation analysis showed that only early AFC was significantly correlated with the baseline levels of serum AMH in each group. When the two PCO groups were combined, a significant correlation was observed between AMH and LH or T. In the control group, there was a significantly negative correlation with age, follicle stimulating hormone (FSH) levels, and total FSH dose administered. Furthermore, no relationship was found between baseline serum AMH and the other factors (Table 4). Multiple regression analysis was performed in the control group and the combined PCO group respectively, using AMH as the dependent variable and AFC, FSH, LH, T, and the total FSH dose as independent variables (Table 4). The only factor significantly correlated to baseline serum AMH levels (p50.05; p50.001) in the control group and combined PCO group was AFC, whereas other factors including baseline serum T, FSH, LH, and total FSH dose did not. The AMH levels were not significantly different between the pregnancy groups of the control group and the combined PCO group respectively (Table 5).
Discussion In this study, we have compared the baseline hormone profile and correlated the results to AMH levels, and the ovarian
Table 4. Spearman correlation analysis of variables affecting serum AMH levels. Control group
Age BMI AFC E2 FSH LH Androstenedione Testosterone FSH dose
Combined PCO group
Correlation
p
Correlation
p
0.46 0.16 0.77 0.37 0.50 0.40 0.25 0.23 0.72
50.05 NS 50.001 NS 50.05 NS NS NS 50.001
0.02 0.04 0.54 0.12 0.13 0.32 0.26 0.41 0.16
NS NS 50.001 NS NS 50.05 NS 50.01 NS
response to gonadotropin stimulation between women with ovulatory and anovulatory PCO undergoing IVF treatment. The baseline serum AMH levels were similar in ovulatory PCO women and anovulatory PCO women, but two to three times higher than in the control. However, we found that the baseline serum T, A, and LH concentrations in anovulatory PCO women were significantly higher than ovulatory PCO women and the control. This suggests that high T, A, and LH levels interfered with the final stage of follicular development leading to anovulation, consistent with the finding of previous studies [Dumesic et al. 2007; Yang et al. 2010]. Our observation that AMH was similar between ovulatory and anovulatory PCO women appears to be different from several previous studies [Diamanti-Kandarakis et al.
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Table 5. The relationship between pregnancy and serum AMH levels. Control group AMH levels (ng/ml)
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Pregnancy Yes No
1.13 ± 1.14 0.86 ± 0.74
Combined PCO group p
AMH levels (ng/ml)
p
0.551
3.40 ± 2.91 3.11 ± 1.76
0.650
2009; Homburg et al. 2013; Piouka et al. 2009; Thomson et al. 2009] which reported that AMH levels correlated with the severity of the symptoms of PCO, with apparently higher levels of AMH associated with menstrual irregularity and hyperandrogenism. A possible explanation for the observation in these previous reports is that AFC is an important confounding variable of the serum AMH result. Different follicle counts or other confounding variables may contribute to the difference in AMH levels between the groups. It is therefore uncertain if amenorrhea or hyperandrogenism is primarily a consequence of high serum AMH levels. In our study, there was no difference in follicle number between the two PCO groups, which helped to clarify the association between AMH levels and anovulation. Jonard and Dewailly [2004] reviewed the pathophysiological mechanism of follicular arrest, concluding that intraovarian hyperandrogenism may be the main culprit for the follicular arrest. However, it is unclear whether higher AMH serum levels are related to elevated androgen concentrations which in turn cause the disruption of folliculogenisis. Our results are in accordance with previous studies that showed a significantly positive correlation between AMH and T or LH [Homburg et al. 2013; Laven et al. 2004; Pigny et al. 2006; Skalba et al. 2011]. However, none of these studies performed multiple regression analysis. In the present study, multiple regression analysis showed that the most important factor affecting serum AMH levels in the model was AFC, whereas LH and T were not independent determinants in both the control group and combined PCO group (ovulatory and anovulatory PCO). This is in agreement with the findings of Pigny et al. [2003] in PCO women, confirming that the increase of serum AMH was primarily a consequence of an excess count of small antral follicles. In comparison, the difference in results may also be due to different study design. Some studies combined non-PCOS and PCOS women together [Eldar-Geva et al. 2005; Piouka et al. 2009] or combined different cohorts with different serum AMH levels [Laven et al. 2004], that may yield a biased association between AMH and androgen in these combined groups. However, using logistic regression in the PCO group, we found that LH and T appeared to be the two most important factors affecting ovulation, whereas AMH and AFC did not appear to predict anovulation. This further suggests that AMH and AFC are not correlated with features characteristic for PCO such as anovulation or hyperandrogenism. It has been speculated that the excess amount of AMH in PCO could be involved in follicular arrest through aromatase inhibition by interacting negatively with FSH [Catteau-Jonard et al. 2007; Durlinger et al. 2001; Franks et al. 2008;
Homburg et al. 2013]. However, the interaction between serum AMH and FSH in the selection failure of a dominant follicle remains unclear. Serum AMH has been considered a more sensitive marker of ovarian reserve than basal FSH [van Houten et al. 2010]. We observed a negative correlation between baseline AMH and FSH levels in the control group, but not in the PCO group. This was because both AMH and FSH reflect the number of small antral follicles in control women, whereas FSH is less sensitive in predicting ovarian reserve in PCO women. However, multiple regression analysis confirmed no correlation between AMH and FSH serum levels in each group, in agreement with previous observation in PCOS women [Laven et al. 2004; Pigny et al. 2003]. It was reported that serum AMH levels did not significantly change along with serum FSH levels that fluctuate throughout the menstrual cycle [La Marca et al. 2004; La Marca et al. 2006]. FSH either produced endogenously or administered did not appear to exert an effect on serum AMH levels when only one dominant follicle emerged [van Rooij et al. 2002; Wachs et al. 2007]. However serum AMH levels did change when there was multiple follicle development during COH [Fanchin et al. 2003; La Marca et al. 2004] and androgens might play a role in AMH regulation in women [Weintraub et al. 2014]. The decrease in serum AMH levels may be the result of increasing numbers of small follicles developing into large mature follicles, which lose their AMH expression. AMH is mainly expressed in secondary, preantral, and small antral follicles, with expression gradually weakening with follicular growth [Baarends et al. 1995; Weenen et al. 2004]. During COH, serum AMH levels decreased significantly while multiple dominant follicles emerged. However, serum AMH on the day of HCG administration and dominant FF AMH at 36 hours after HCG administration in anovulatory and ovulatory PCO women were still two to three times higher than control women. Similarly, the baseline serum AMH levels in women with PCO were two to three times higher than in control women. It seems that the high levels of AMH observed in PCO women in the late follicular phase (day of HCG administration) is not the reason for follicular arrest (the disruption of follicular development and maturation), whereas it predicts an excessive number of small antral follicles in PCO women which were not detected by ultrasound on the day of HCG administration, compared to control women. It has been observed that serum AMH levels remain rather constant in early and late follicular phases of a natural cycle [La Marca et al. 2004; La Marca et al. 2006; van Houten et al. 2010], indicating that serum AMH levels might not influence follicular maturation. It appears that serum AMH levels at various stages of a natural or stimulated cycle are positively correlated with the numbers of small antral follicles in the ovary. In this respect, the level of serum AMH may be considered as a surrogate of early antral follicle number in the ovary for the definition of PCOS in situations where accurate ultrasonographic data is not available [Dewailly et al. 2011; Pigny et al. 2006]. During COH the change in serum AMH and E2 levels was positively associated with dominant follicular counts (the number of oocytes retrieved) [Fanchin et al. 2003; La Marca et al. 2004]. This suggested that as multiple small antral
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DOI: 10.3109/19396368.2014.973123
AMH level predicts PCO patients’ follicle excessive growth
follicles grow into dominant follicles, AMH levels decrease, but at the same time E2 levels increase. Significant correlation was found between the decrease of serum AMH levels and the increase of E2 levels which parallel the emergence of multiple dominant follicles during COH, suggesting the change of serum AMH and E2 levels as result of follicular growth might not influence follicular development. The AMH levels had no significant difference between pregnancy and the control group and the combined PCO group, respectively (Table 5). In summary, the reduction in AMH during COH occurs as a consequence of dominant follicles with a corresponding reduction in the number of small antral follicles, but is not due to other factors such as high androgen. Elevated serum AMH levels in PCO women seem to be related only to follicular excess but not follicular arrest.
in this group did not take into consideration whether or not they had hyperandrogenaemia.
Materials and Methods The human tissues collection and study procedure have been approved by the Institutional Review Board at Peking University Third Hospital in the present study. The patients involved in the present study were informed of all details of the procedure, including sample utility and research destination, and signed an in vitro informed consent document voluntarily. Subjects This prospective study was conducted in the Peking University Third Hospital. Informed consent was obtained from all participants. Three groups of subjects, all undergoing in vitro fertilization (IVF) treatments, were recruited. Exclusion criteria in the study included a history of hypothalamus-pituitary diseases, abnormal thyroid function, endometriosis, previous ovarian surgery, and steroid hormone treatment. Control group (n ¼ 35) Women in the control group had tubal infertility, regular menstrual periods (25–35 days), normal uterus and ovarian morphology, and normal endocrine profile including FSH, LH, T, A, E2, testosterone, androstenedione, estrogen, and prolactin. Ovulatory PCO group (n ¼ 29) All women in this group exhibited ultrasound morphology typical of PCO, with 12 or more follicles measuring 2–9 mm in diameter in at least one ovary. Specifically, all women in this group were ovulatory. Serum androgen levels were not a criteria for inclusion in this group.
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Ovulation stimulation treatment All subjects underwent controlled ovarian hyperstimulation (COH) with gonadotropin-releasing hormone agonist (GnRHa) according to the short down-regulation protocol. GnRHa (Diphereline 0.1 mg; Beaufour Ipsen, France) was administered from day 2 of menstruation or progestin-induced withdrawal bleeding (cycle day 2). Ovarian stimulation started on day 3 with 150 450 IU daily of rFSH (Gonal-F 75 IU Per ampoule; Serono, Italy) according to patients’ ages and antral follicle count (AFC). The antral follicle count (follicle diameter 2 9 mm) was recorded on cycle day 2. Transvaginal ultrasound for follicular tracking and blood sampling for E2 levels were performed every 2 3 days. The FSH dose was adjusted based on E2 levels and follicular development. HCG (Profasi; Serono) 10,000 IU was administered when two or more dominant follicles reached 18 mm in diameter and oocyte retrieval was scheduled to take place 36 h after HCG administration. Sample collection Blood samples were collected from all patients on day 2 of menstruation or withdrawal bleeding and on day of HCG injection (day HCG). Serum samples were obtained after centrifuging the blood samples at 3000 rpm for 3 min, then stored at 80 C. The FF was aspirated gently from the first dominant follicle (18–20 mm in diameter) during oocyte retrieval. After the oocyte was isolated, FF was centrifuged at 3000 rpm for 15 min, and the supernatant stored at 80 C. Hormone assays AMH measurement was performed with the use of ELISA assay (Immunotach Beckman Coulter Laboratories, Brea, CA, American). Inter- and intra-assay coefficients of variation were as 59% and 56%, respectively. The determination range for AMH was 0.05–15 ng/ml. Serum E2, FSH, LH, T, and A concentrations were determined by using enzyme-amplified time-resolved fluorescence technology (DPC 1000 automatic analyzer, Los Angeles, CA, American). To make allowance for the variation in FF volume, FF hormone levels were adjusted to the relative content of per gram protein [Fanchin et al. 2005; Franchimont et al. 1989]. FF protein contents were determined by using routine biuret reaction method (BCA protein assay reagent kit, Pierce Rockford, IL, American). FF AMH levels were expressed as per gram of protein. Statistical analysis
Anovulatory PCO group (n ¼ 38) The definition of anovulatory was the menstrual cycle 60 days. All the women in this group were anovulatory with ultrasound morphology typical of PCO, with 12 or more follicles measuring 2–9 mm in diameter in at least one ovary. They did therefore fulfill the diagnostic criteria for PCOS [Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group 2004]. Similarly, the selection of women
Statistical analysis was performed with the use of SPSS10.0 Software System (SPSS Inc., Chicago, IL, USA). Descriptive statistics were generated to enable comparisons between groups. Continuous variables were checked for normality and means were presented with SD, or medians and ranges, as appropriate. Comparisons were performed using Student t test or ANOVA if the data distribution was normal. Mann-whitney U test and Kruskal-Wallis test were used if the data were not
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J. Li et al.
normally distributed. The relationship between AMH and other variables was analyzed by using spearman correlation analysis. Multiple regression analysis was used to control potential confounding variables. Logistic regression analysis was performed with ovulation or not as the dependent variable and AMH, AFC, LH, T, and A as covariates variable. A p value of 50.05 was considered statistically significant.
Declaration of interest
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This study was supported by National ‘‘Twelfth Five-Year’’ Plan for Science & Technology Support 2012BAI32B01, National Science Fund 81300482 & 81170538, and Beijing Municipal Natural Science Foundation 7142166. The authors declare that they have no competing interests.
Author contributions Carried out the data collection, performed the statistical analysis: JL; Supervised the analysis, helped in the study coordination and the data collection: RL; Took part in the discussion and data analysis: HY, SZ; Participated in the design of the study and drafted the manuscript: JQ. All authors read and approved the final manuscript.
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DOI: 10.3109/19396368.2014.973123
AMH level predicts PCO patients’ follicle excessive growth
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