Departments of 1 Obstetrics and Gynecology and Reproductive Medicine, Clamart, France, and 2 Biochemistry and 3 Hormonology, Paris and Clamart, France
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology and Reproductive Medicine, Hôpital Antoine Béclère, 157, rue de la Porte de Trivaux, 92141, Clamart, France. e-mail: renato.fanchin{at}abc.ap-hop-paris.fr
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Abstract |
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Key words: anti-Müllerian hormone/antral follicles/endocrine profile/Müllerian-inhibiting substance (MIS)/ovarian reserve
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Introduction |
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Basic research data obtained from the adult ovary indicate that AMH is likely to be involved in the regulation of follicular steroidogenesis. Experiments conducted in animals suggest that AMH reduces aromatase activity and the number of LH receptors in FSH-stimulated granulosa cells (Josso et al., 1998), and also influences testosterone production by theca cells (Ingraham et al., 2000
). In addition, growing evidence indicates that AMH mainly is expressed in pre-antral and early antral follicles (Baarends et al., 1995
) and has either direct or indirect roles in various phases of folliculogenesis, from the primordial (Durlinger et al., 2002
) to FSH-sensitive (Durlinger et al., 2001
; McGee et al., 2001
) follicular stages, probably via specific AMH type II receptors that are expressed in granulosa and theca cells. This suggests that, in contrast to other hormonal markers of ovarian function, AMH secretion might concurrently reflect the activity of pre-antral and early antral follicles, which makes it a promising parameter in the evaluation of ovarian follicular reserve.
Recent clinical evidence is in agreement with this hypothesis. One group (de Vet et al., 2002) demonstrated that serum AMH levels on cycle day 3 decrease progressively along with age and become undetectable after menopause. This suggests that peripheral AMH levels are a valuable parameter to monitor the relative follicular exhaustion due to ovarian ageing. Consistently, others (Seifer et al., 2002
) showed that day 3 AMH levels are positively related with the number of oocytes retrieved after controlled ovarian hyperstimulation (COH). Taken together, these results indicate that circulating AMH levels reflect the number of selectable follicles during the early follicular phase. Indeed, the early antral follicle count has been shown to reliably predict the fertility potential of women (Reuss et al., 1996
) and their responsiveness to COS (Chang et al., 1998
; László et al., 2002
).
However, the question of whether serum AMH measurements on cycle day 3 reflect ovarian follicular status better than the usual hormonal parameters remains unanswered. Hence, the present investigation was designed to weigh the relationship between early antral follicles and AMH levels against that between early antral follicles and other putative markers of ovarian function and fertility potential, including serum levels of inhibin B, estradiol (E2), FSH and LH on cycle day 3.
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Materials and methods |
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Study protocol
On day 3 of the menstrual cycle, each woman underwent blood sampling by venipuncture for measurement of serum levels of AMH, inhibin B, E2, FSH and LH at approximately 09:00 h. Later in the morning, ovarian ultrasound scanning (Figure 1) was performed using a 4.57.2 MHz multi-frequency transvaginal probe (Siemens Elegra; Siemens S.A.S., Saint-Denis, France) by one operator (R.F.), who was blinded to the results of the hormone assays. The objective of the ultrasound examination was to evaluate the number and sizes of early antral follicles and to calculate the mean ovarian volume. The sum of all follicles measuring 210 mm in mean diameter (mean of two orthogonal diameters) in both ovaries was considered. In an attempt to optimise the reliability of ovarian follicular assessment, the ultrasound scanner used was equipped with a tissue harmonic imaging system (Thomas and Rubin, 1998); this allowed improved image resolution and adequate recognition of follicular borders. Ovarian volumes, calculated according to the formula for an ellipsoid (0.526xlengthx heightxwidth) (Sharara and McClamrock, 1999
), corresponded to the mean volume for both ovaries. Intra-analysis coefficients of variation (CV) for follicular and ovarian measurements were <5%, and their lower limit of detection was 0.1 mm.
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Statistical analysis
The measure of central tendency used was the median, and data variability was expressed by the ranges. Relationship between two different continuous variables was assessed by correlation. The Fisher r to z-test was used to determine if the coefficient of correlation (r) was significantly different from zero. Comparison of strength of correlations was performed using the Hotellings t-test (Hotelling, 1940), which assesses the difference between coefficients of correlation for correlated samples that share a common variable. A P-value < 0.05 was considered statistically significant.
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Results |
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Discussion |
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These results not only corroborate but also expand clinical data reported previously by other investigators (de Vet et al., 2002; Seifer et al., 2002
). First, the age-related decrease in AMH levels (de Vet et al., 2002
) probably results from the relative follicular attrition that characterizes the decline of ovarian function, with a noticeable reduction in the number of early antral follicles (Reuss et al., 1996
). The negative relationship between day 3 serum AMH levels and the womens age seen in the present study supports this hypothesis. Second, the reported correlation between serum AMH levels on cycle day 3 and the number of oocytes retrieved after COS (Seifer et al., 2002
) is in keeping with the relationship between AMH and the number of early antral follicles shown in the present study. Indeed, the number of early antral follicles is a putative predictor of COS outcome (Chang et al., 1998
; László et al., 2002
). Third, the correlation between serum AMH levels and early antral follicle count confirms preliminary data reported by others (de Vet et al., 2002
), though the latter authors observed similar coefficients of correlation for this relationship (0.71) as in the present study (0.74).
Nonetheless, the central result of the present investigation was that AMH correlated more intensely with early antral follicular counts than did the other hormonal markers of follicular status and development that were monitored. The reasons for this striking phenomenon are unclear, but it may be related to possible different regulation of AMH as compared with inhibin B, E2 and FSH. During the lutealfollicular transition, the secretion of inhibin B (Welt et al., 1997) and E2 (Mais et al., 1987
) by the early antral follicles modulates their own stimulation by FSH. This implies that inhibin B and E2 levels depend not only on the bulk of active granulosa cells available, as represented by follicular number and sizes, but also on their stimulation by FSH. Although little is known about FSH effects on AMH expression during the early follicular phase, it can be presumed that this hormone is less FSH-sensitive than inhibin B and E2. Indeed, AMH is also secreted by follicles that are barely sensitive to FSH, such as pre-antral follicles (Baarends et al., 1995
). Therefore, AMH may represent a more independent and reliable marker of early antral follicle activity than inhibin B and E2, and FSH on cycle day 3. Further studies are required to investigate whether AMH measurements on day 3 reflect not only the activity of early antral follicles but also that of follicles during earlier stages of folliculogenesis.
Nevertheless, a negative relationship was observed between FSH and AMH levels. Whereas these data challenge the hypothesis of a stimulating role of FSH on granulosa cell production of AMH, they do not rule out its potential inhibiting action on AMH production. Indeed, the results of studies conducted in rats treated with GnRH antagonist and FSH have indicated that FSH inhibits AMH and its type II receptor expression in pre-antral and early antral follicles (Baarends et al., 1995). However, since in the present series AMH and FSH levels were both correlated with a third common parameter (early antral follicular count), their mutual correlation might be merely indirect and not causative. Likewise, the correlation observed between AMH and inhibin B levels may not correspond to a direct relationship between both hormones.
Another issue deserving consideration is the observed lack of influence of endogenous LH concentrations on peripheral AMH levels. Indeed, the present data agree with previous results obtained in normo-ovulating and polycystic ovary syndrome (PCOS) patients (Cook et al., 2002). In PCOS, basal AMH levels are significantly higher as compared with normal controls (Cook et al., 2002
). Although these results may be attributed to the large number of antral follicles that characterizes PCOS, they do not permit the exclusion of a possible influence of LH on AMH regulation. Experiments conducted in rats and pigs have shown that AMH inhibits the number of LH receptors in FSH-stimulated granulosa cells (Josso et al., 1998
, 2001). Further studies involving pituitary-desensitized women treated with controlled FSH and/or LH doses will undoubtedly contribute to clarifying the possible regulation of AMH by pituitary gonadotrophins.
Similarly, conclusive data on AMH secretion patterns during more advanced stages of the follicular phase and during the luteal phase of the menstrual cycle are still lacking. A preliminary study showed that serum AMH levels are slightly and significantly higher at mid-cycle as compared with early follicular and mid-luteal phase values in women (Cook et al., 2000). However, these data did not detail the possible relationship between AMH production and eventual follicular development and/or corpus luteum activity. Also, they contrast with in-vitro studies that demonstrated a progressive decrease of AMH expression during the final steps of follicular maturation from pre-antral to pre-ovulatory follicles (Baarends et al., 1995
). Hence, further characterization of subtle changes in serum AMH levels throughout the menstrual cycle is required. Likewise, investigation of possible effects of COS with exogenous gonadotrophins on peripheral AMH levels may provide further insights into AMH regulation and its pathophysiological role in adult women.
In conclusion, the present results indicate not only that serum AMH levels are strongly correlated with ovarian follicular status during the early follicular phase, but also that this relationship is more strict than that obtained with other hormonal markers of the follicular functioning. This leads to the conclusion that serum AMH measurements on cycle day 3 are a better predictor of the number of early antral follicles than conventional hormone measurements. However, further studies aimed at weighing the predictability of AMH levels against conventional day 3 inhibin B, E2 and FSH measurements on COS and assisted reproductive technology outcomes are needed to challenge this conclusion. Moreover, uncertainties persist with respect to the control of granulosa cell AMH production and its physiological role during folliculogenesis. The understanding of these key issues will be helpful to refine future clinical applications of AMH measurements in evaluating the fertility potential of women and monitoring infertility treatments.
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References |
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Chang, M.Y., Chiang, C.H., Hsieh, T.T., Soong, Y.K. and Hsu, K.H. (1998) Use of the antral follicle count to predict the outcome of assisted reproductive technologies. Fertil. Steril., 69, 505510.[CrossRef][ISI][Medline]
Cook, C.L., Siow, Y., Taylor, S. and Fallat, M.E. (2000) Serum mullerian-inhibiting substance levels during normal menstrual cycles. Fertil. Steril., 73, 859861.[CrossRef][ISI][Medline]
Cook, C.L., Siow, Y., Brenner, A.G. and Fallat, M.E. (2002) Relationship between serum müllerian-inhibiting substance and other reproductive hormones in untreated women with polycystic ovary syndrome and normal women. Fertil. Steril., 77, 141146.[CrossRef][ISI][Medline]
de Vet, A., Laven, J.S., de Jong, F.H., Themmen, A.P. and Fauser, B.C. (2002) Antimullerian hormone serum levels: a putative marker for ovarian aging. Fertil. Steril., 77, 357362.[CrossRef][ISI][Medline]
Durlinger, A.L., Gruijters, M.J., Kramer, P., Karels, B., Kumar, T.R., Matzuk, M.M., Rose, U.M., de Jong, F.H., Uilenbroek, J.T., Grootegoed, J.A. and Themmen, A.P. (2001) Anti-Mullerian hormone attenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology, 142, 48914899.
Durlinger, A.L., Gruijters, M.J., Kramer, P., Karels, B., Ingraham, H.A., Nachtigal, M.W., Uilenbroek, J.T., Grootegoed, J.A. and Themmen, A.P. (2002) Anti-Mullerian hormone inhibits initiation of primordial follicle growth in the mouse ovary. Endocrinology, 143, 10761084.
Groome, N.P., Illingworth, P.J., OBrien, M., Pai, R., Rodger, F.E., Mather, J.P. and McNeilly, A.S. (1996) Measurement of dimeric inhibin B throughout the human menstrual cycle. J. Clin. Endocrinol. Metab., 81, 14011405.[Abstract]
Hotelling, H. (1940) The selection of variates for use in prediction with some comments on the problem of nuisance parameters. Ann. Math. Stat., 11, 271283.
Hudson, P.L., Dougas, I., Donahoe, P.K., Cate, R.L., Epstein, J., Pepinsky, R.B. and MacLaughlin, D.T. (1990) An immunoassay to detect human mullerian inhibiting substance in males and females during normal development. J. Clin. Endocrinol. Metab., 70, 1622.[Abstract]
Ingraham. H.A., Hirokawa, Y., Roberts, L.M., Mellon, S.H., McGee, E., Nachtigal, M.W. and Visser, J.A. (2000) Autocrine and paracrine Mullerian inhibiting substance hormone signaling in reproduction. Recent Prog. Horm. Res., 55, 5367.[ISI][Medline]
Josso, N., Racine, C., di Clemente, N., Rey, R. and Xavier, F. (1998) The role of anti-Mullerian hormone in gonadal development. Mol. Cell. Endocrinol., 145, 37.[CrossRef][ISI][Medline]
Josso, N., di Clemente, N. and Gouedard, L. (2001) Anti-Mullerian hormone and its receptors. Mol. Cell. Endocrinol., 179, 2532.[CrossRef][ISI][Medline]
László, F.J.M.M., Bancsi, L.F., Broekmans, F.J., Eijkemans, M.J., de Jong, F.H., Habbema, J.D. and te Velde, E.R. (2002) Predictors of poor ovarian response in in vitro fertilization: a prospective study comparing basal markers of ovarian reserve. Fertil. Steril., 77, 328336.[CrossRef][ISI][Medline]
Lee, M.M. and Donahoe, P.K. (1993) Mullerian inhibiting substance: a gonadal hormone with multiple functions. Endocr. Rev., 14, 152164.[Abstract]
Long, W.Q., Ranchin, V., Pautie, P., Belville, C., Denizot, P., Cailla, H., Lhomme, C., Picard, J.Y., Bidart, J.M. and Rey, R. (2000) Detection of minimal levels of serum anti-Mullerian hormone during follow-up of patients with ovarian granulosa cell tumor by means of a highly sensitive enzyme-linked immunosorbent assay. J. Clin. Endocrinol. Metab., 85, 540544.
Mais, V., Cetel, N.S., Muse, K.N., Quigley, M.E., Reid, R.L. and Yen, S.S. (1987) Hormonal dynamics during luteal-follicular transition. J. Clin. Endocrinol. Metab., 64, 11091114.[Abstract]
McGee, E.A., Smith, R., Spears, N., Nachtigal, M.W., Ingraham, H. and Hsueh, A.J. (2001) Mullerian inhibitory substance induces growth of rat preantral ovarian follicles. Biol. Reprod., 64, 293298.
Pepinsky, R.B., Sinclair, L.K., Chow, E.P., Mattaliano, R.J., Manganaro, T.F., Donahoe, P.K. and Cate, R.L. (1988) Proteolytic processing of Mullerian inhibiting substance produces a transforming growth factor--like fragment. J. Biol. Chem., 263, 1896118964.
Rajpert-De Meyts, E., Jorgensen, N., Graem, N., Muller, J., Cate, R.L. and Skakkebaek, N.E. (1999) Expression of anti-Mullerian hormone during normal and pathological gonadal development: association with differentiation of Sertoli and granulosa cells. J. Clin. Endocrinol. Metab., 84, 38363844.
Reuss, M.L., Kline, J., Santos, R., Levin, B. and Timor-Tritsch, I. (1996) Age and the ovarian follicle pool assessed with transvaginal ultrasonography. Am. J. Obstet. Gynecol., 174, 624627.[ISI][Medline]
Seifer, D.B., MacLaughlin, D.T., Christian, B.P., Feng, B. and Shelden, R.M. (2002) Early follicular serum mullerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil. Steril., 77, 468471.[CrossRef][ISI][Medline]
Sharara, F.I. and McClamrock, H.D. (1999) The effect of aging on ovarian volume measurements in infertile women. Obstet. Gynecol., 94, 5760.
Thomas, J.D. and Rubin, D.N. (1998) Tissue harmonic imaging: why does it work? J. Am. Soc. Echocardiogr., 11, 803808.[ISI][Medline]
Vigier, B., Picard, J.Y., Tran, D., Legeai, L. and Josso, N. (1984) Production of anti-Müllerian hormone: another homology between Sertoli and granulosa cells. Endocrinology, 114, 13151320.[Abstract]
Welt, C.K., Martin, K.A., Taylor, A.E., Lambert-Messerlian, G.M., Crowley, W.F., Jr, Smith, J.A., Schoenfeld, D.A. and Hall, J.E. (1997) Frequency modulation of follicle-stimulating hormone (FSH) during the luteal-follicular transition: evidence for FSH control of inhibin B in normal women. J. Clin. Endocrinol. Metab., 82, 26452652.
Submitted on July 5, 2002; accepted on September 25, 2002.