1 Institute of Obstetrics and Gynecology, Policlinico of Modena, University of Modena and Reggio Emilia, 2 Institute of Obstetrics and Gynecology, Policlinico Le Scotte, University of Siena, Italy and 3 Institute of Obstetrics and Gynecology, Rabin Medical Center, University of Tel Aviv, Israel
4 To whom correspondence should be addressed at: Institute of Obstetrics and Gynecology, Policlinico di Modena, Largo del Pozzo, 41100 Modena, Italy. Email: antlamarca{at}libero.it
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Abstract |
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Key words: anti-Müllerian hormone/early puerperium/estradiol/FSH/pregnancy
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Introduction |
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In contrast to other members of the family, which are widely expressed and have a varied repertoire of biological activities depending on the cellular context, AMH is expressed only in the gonads and exerts inhibitory effects on the development and function of reproductive organs. The most striking effect of AMH is its capacity to induce regression of the Müllerian ducts, the anlage of the female internal reproductive organs. In the absence of AMH, Müllerian ducts of both sexes develop into uterus, Fallopian tubes and the upper part of the vagina (Munsterberg and Lovell-Badge, 1991). In females, AMH is expressed by the ovary (Hirobe et al., 1994
). AMH is secreted by the granulosa cells of ovarian follicles (Vigier et al., 1984
) and appears to regulate early follicle development (Durlinger et al., 2002
). AMH seems to affect the transition from resting primordial follicles into growing follicles (Durlinger et al., 1999
).
AMH is detected in serum from women of reproductive age and its levels vary slightly with the menstrual cycle, reaching the peak value in the late follicular phase (Hudson et al., 1990; Josso et al., 1990
; Cook et al., 2000
). Serum AMH levels have been shown to decrease over time in young normo-ovulatory women (de Vet et al., 2002
), and to correlate with age, FSH and the number of antral follicles. In a recent study, a group of women was studied twice and the interval between the two visits ranged from 1.1 to 7.3 years. A reduction in mean AMH levels of
38% was observed (de Vet et al., 2002
). Therefore, AMH might represent a sensitive marker for ovarian ageing (de Vet et al., 2002
; Fanchin et al., 2003
; Laven et al., 2004
).
Currently there are no data on AMH modifications during human pregnancy. The present study evaluated serum AMH levels throughout gestation and after delivery in healthy pregnant women. Moreover, pregnancy offers the opportunity to study the relationship between AMH and FSH.
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Materials and methods |
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The groups analysed were (respectively: number of women and range of age): A, as internal controls n=15, 1832 years, without ongoing pregnancy; B, n=27, 1935 years, studied in the first trimester of pregnancy; C, n=21, 1734 years, studied in the second trimester; D, n=13, 2037 years, studied in the third trimester; E, n=8, 2035 years, studied in the early puerperium.
A medical questionnaire and blood samples were obtained for AMH, FSH and E2 determination. Blood samples were obtained between 09:00 and 11:00 after a light breakfast. Plasma FSH and E2 levels were assayed by double-antibody radioimmunoassay using commercial kits from Radim (Italy) for FSH and from Sorin (Italy) for E2. Samples were assayed in duplicate at two dilutions. Samples from a given subject were analysed for each hormone in the same assay to avoid inter-assay variation. Quality control pools at low, normal and high FSH and E2 concentrations were present in each assay. The detection limit of the assay was 0.18 IU/l for FSH and 5 pg/ml for E2. Intra- and inter-assay variations were 6.2 and 6.5% for FSH and 4.2 and 4.9% for E2.
Serum AMH was measured using the AMH enzyme-linked immunosorbent assay kit (Immunotech, France). Briefly, 25 µl of each serum sample was incubated in duplicate on a polystyrene plaque pre-coated with a monoclonal anti-AMH antibody. After 1 h incubation, a second monoclonal anti-AMH antibody, coupled to biotin, was added, together with a streptavidinhorseradish peroxidase complex. After addition of TMB substrate, the resulting colour reaction was quantified using an MRX spectrophotometer at 450 nm. A preparation of purified recombinant human AMH was used to construct a standard curve. The limit of sensitivity of the assay was 0.7 pmol/l (0.1 ng/ml); inter- and intra-assay coefficients of variation were 8.7 and 5.3% respectively for a serum AMH concentration of 35 pmol/l, and 7.8 and 4.9% for a serum AMH concentration of 1100 pmol/l. No cross-reaction was observed with pure transforming growth factor-.
Results are expressed as means and SD. Comparisons between the two groups were calculated by analysis of variance and by MannWhitney U-test. Correlations between different parameters were determined by using bivariate correlation statistics and are expressed as Spearman correlation coefficients. Statistical analysis was performed by the software Statsoft. Statistical significance was set at P<0.05. On the basis of our results we calculated that 7500 patients should be recruited to obtain a significant difference (alpha 90%) in AMH levels during the three trimesters.
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Results |
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Discussion |
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Maternal circulating AMH levels are similar to those found in the follicular phase of non-pregnant women. No significant changes were found 23 days after the delivery. The lower but not statistically significant levels of AMH during the third trimester and during the puerperium might be due to small differences in age between the five groups. Indeed it has been demonstrated that AMH levels decreased over time in normo-ovulatory women (de Vet et al., 2002; Laven et al., 2004
).
Our results may indicate that placenta does not produce and secrete AMH in the maternal circulation. Indeed it has been shown that AMH is expressed only in the gonads (Teixeira et al., 2001). However, appropriate studies on possible placental AMH production are needed. Until then it cannot be excluded that both the ovary and placenta contribute to AMH levels found in pregnant women. Recently it has been reported that there might be an estrogen-responsive element in the promoter area of the AMH gene (Chen et al., 2003
). Since estrogen levels are much higher during pregnancy than in the normal cycle, estrogens might therefore inhibit expression of the AMH during pregnancy. The lack of AMH decline after delivery argues against the placental production and the effect of high levels of estradiol on AMH gene expression.
The findings of our study suggest that initial follicle recruitment is not abolished during pregnancy. It is thought that prolonged elevation of circulating progesterone during pregnancy may reduce follicular recruitment. Early studies in pregnant mice indicated that fewer follicles start growth per unit time with a consequent conservation of follicle reserve in ageing animals (Lapolt et al., 1998).
Epidemiological studies indicate that women with increased parity show a delay in menopausal onset (Whelan et al., 1990; Cramer et al., 1995
). However, considering that AMH is solely produced by granulosa cells of preantral and small antral follicles (Durlinger et al., 2002
), we should conclude that initial non-cycle recruitment of follicles is not abolished during pregnancy.
Furthermore, our study provided information regarding the relationship between FSH and AMH. Studies conducted in rats have shown that FSH inhibits AMH secretion (Baarends et al., 1995) and, as reported by other independent studies, a physiological link between FSH and AMH has been proposed (Seifer et al., 2002
; Van Rooij et al., 2002
; Fanchin et al., 2003
). We found that the deep suppression of pituitary FSH secretion observed during pregnancy is not followed by modification in AMH serum levels. This observation should exclude a direct role of FSH on AMH production. Probably the relationship between FSH and AMH found in non-pregnant women is consequent to their mutual correlation with a third parameter (the early antral follicular count). However, pregnancy is a complex situation that could limit the interpretation of data.
The present study on AMH serum levels during pregnancy has obtained information on ovarian activity and on the possible relationship between FSH and AMH.
We hypothesize that the profile of the new marker of ovarian activity, AMH, may indicate that initial non-cyclic ovarian follicular activity during pregnancy is not abolished. Moreover, FSH does not seem to play a direct role on AMH synthesis and secretion.
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References |
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Submitted on August 13, 2004; resubmitted on November 12, 2004; accepted on January 21, 2005.