1 Division of Reproductive Medicine, Department of Obstetrics and Gynecology, 2 Center for Clinical Decision Sciences, Department of Public Health, 3 Department of Internal Medicine, Erasmus MC, Rotterdam and 4 Department of Reproductive Medicine, University Medical Center, Utrecht, The Netherlands
5 To whom correspondence should be addressed at: Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Erasmus MC, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Email: j.laven{at}erasmusmc.nl
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Abstract |
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Key words: anti-Müllerian hormone (AMH)/anovulation/infertility/ovarian ageing/PCOS
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Introduction |
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Ovaries of AMH knock-out mice as well as female mice heterozygous for the AMH deletion showed an accelerated exhaustion of the primordial follicle stock (Durlinger et al., 1999), suggesting important roles for AMH in depletion of the primordial follicle pool. Moreover, AMH was able to inhibit the initiation of primordial follicle growth in cultured neonatal mouse ovaries (Durlinger et al., 2002
), and AMH has been shown to inhibit FSH-induced follicle growth in female mice (Durlinger et al., 2001
). Recent data suggest that AMH expression in the human ovary is similar to that observed in mouse and rat (Weenen et al., 2004
), suggesting important roles for AMH in the regulation of human early follicle growth as well.
AMH serum levels decline with increasing age in normo-ovulatory women (de Vet et al., 2002) and are strongly correlated with the number of antral follicles. Hence AMH may be used as a marker for ovarian ageing (de Vet et al., 2002
; van Rooij et al., 2002
; Fanchin et al., 2003a
). In fact, poor response during ovarian stimulation for IVF [indicative of ovarian ageing (Beckers et al., 2002
)] has been shown to be associated with reduced early follicular phase AMH serum concentrations (van Rooij et al., 2002
; Seifer et al., 2002
; Fanchin et al.
, 2003b
).
Chronic anovulation is a common cause of infertility and it is diagnosed in 2025% of couples with fertility problems (ESHRE Capri Workshop Group, 1995
; Laven et al., 2002
). Most of these women present with irregular menstrual cycles and normal serum FSH concentrations [World Health Organization (WHO) group 2] (Rowe et al., 2000
). Recent data have shown that serum levels of AMH are elevated in WHO 2 and polycystic ovary syndrome (PCOS) patients (Cook et al., 2002
; Pigny et al., 2003
; Laven et al., 2004
). Moreover, it seems that AMH levels correlate well with the extent of ovarian dysfunction in anovulatory women (Laven et al., 2004
). Finally, the decline in AMH serum levels with increasing age in this cross-sectional data set differs when comparing anovulatory women and normo-ovulatory controls (Laven et al., 2004
).
Since AMH constitutes an important regulator of primordial follicle pool depletion (Durlinger et al., 1999), an increased intra-ovarian AMH production may slow down the process of depletion of the primordial follicle pool. Due to retarded exhaustion of the primordial stock of follicles, the age of menopause might be delayed in these anovulatory women. The current longitudinal cohort study was designed to investigate whether the decrease in AMH serum concentrations over time is different comparing women with WHO 2 anovulation (including PCOS) and ovulatory controls.
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Materials and methods |
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The control group consisted of 41 normo-ovulatory women, as described before (de Vet et al., 2002). All control women participated in previous studies between 1993 and 1999 (van Santbrink et al., 1995
; Schipper et al., 1998
; Hohmann et al., 2001
). Inclusion criteria were regular menstrual cycle (2630 days), 2036 years of age, body mass index (BMI) (1926 kg/m2), absence of endocrine disorders or any other relevant disease, and no use of medications or oral contraceptives during the 3 months prior to the start of the study.
For the anovulatory patients, repetitive standardized screening [clinical investigation, fasting blood withdrawal and transvaginal sonography (TVS)] was performed on a random day between 9 and 11 a.m., as previously described (Imani et al., 1998). For each individual anovulatory patient, the length of the interval between visits is dependent on the time between each step of the treatment regimen (Imani et al., 1998
). For the normo-ovulatory controls, repetitive TVS and blood sampling were performed during the early follicular phase (cycle day 3, 4 or 5) (de Vet et al., 2002
). For each control, the interval length between visits is dependent on the time between participation in both studies (de Vet et al., 2002
).
Blood samples were obtained by venepuncture and processed within 2 h after withdrawal, as described previously. Serum was stored at 20°C until assayed. The hormone assays used have all been described elsewhere (Imani et al., 1998; de Vet et al., 2002
). Serum AMH levels were measured by using an ultrasensitive enzyme-linked immunosorbent assay (Immunotech-Coulter, Marseilles, France) as described elsewhere (Long et al., 2000
). This assay uses the same components as the normal assay, but some procedural adaptations result in increased sensitivity, making it possible to determine lower serum concentrations of AMH as they exist in women. Intra- and inter-assay coefficients of variation were <5 and <15% for LH, <3 and <8% for FSH, <8 and <11% for androstenedione (AD), <3 and <5% for testosterone, <5 and <7% for estradiol (E2), <4 and <5% for sex hormone-binding globulin (SHBG), <9 and <15% for inhibin B, and <5 and <8% for AMH, respectively (Imani et al., 1998
; de Vet et al., 2002
).
Results are presented as the mean±SD if distributed normally, or otherwise as the median and range. To assess differences between groups, MannWhitney or KruskalWallis tests were used. Associations between different parameters were assessed by Spearman's rank correlation coefficient. To establish whether variables changed over time, the Wilcoxon matched pairs signed rank sum test was used. To determine the rate of change over time, regression analysis was used. After log transformation, the ratio (value visit 2:visit 1) of variables was plotted against the time interval between visit 1 and 2. The relative decline per year was introduced in the present analysis since ovarian follicle depletion occurs at a constant rate of proportional decline for women under 38 years of age (Faddy et al., 1992). A possible difference in rate of decline between WHO 2 and controls was tested for in the analysis. All regression analyses were corrected for age. Data were analysed using the commercially available software package SPSS (Chicago, IL). A P-value of 0.05 was chosen as the threshold level for statistical significance.
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Results |
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Discussion |
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PCO differ from normal ovaries in that follicle development is arrested at the stage where dominant follicle selection would have taken place under normal conditions (Pache et al., 1992a; van Santbrink et al., 1995
; Fauser and Van Heusden, 1997
). Upon histological examination, it has been shown that the number of developing and subsequently atretic follicles was doubled compared with normo-ovulatory controls (Hughesdon, 1982
; Webber et al., 2003
). Moreover, the number of primordial follicles per section did not differ between women with and without PCO (Hughesdon, 1982
; Webber et al., 2003
). However, since the total ovarian volume is increased in PCO, it might be speculated that the primordial follicle pool is enlarged in these women. Indeed, recent histological studies using more sophisticated morphometric techniques suggest that the increased density of small pre-antral follicles in PCO possibly could result from a higher initial population of primordial follicles (Webber et al., 2003
). Alternatively, the rate of follicle depletion in women with PCO may also vary. At present, evidence regarding dissimilarities involved in regulation of ovarian ageing in women with and without PCO is lacking. However, data currently available suggest that the intrinsic ovarian abnormality associated with abberant follicular dynamics in the PCO might cause a reduced rate of atresia (Webber et al., 2003
).
Menopause represents the clinical hallmark of follicle pool exhaustion and the definitive end of reproductive life. In addition, the commencement of menopause at an earlier age is associated with an earlier initiation of subfertility, sterility and transition to cycle irregularity, and vice versa (te Velde and Pearson, 2002). For normo-ovulatory women, it has been demonstrated that menstrual cycle irregularities associated with increasing age are dependent on the number of remaining follicles (Richardson et al., 1987
). The basis of ovarian ageing in women is depletion of the primordial follicle pool (Richardson et al., 1987
; Nikolaou and Templeton, 2003
). Critical aspects involved in the process of ovarian ageing are the number of primordial follicles present in the initial stock and the factors that regulate the rate of loss of this stockpile (Wise et al., 1996
). It seems likely that the ovary is the predominant pacemaker in reproductive ageing (te Velde et al., 1998
; te Velde and Pearson, 2002
). Studies in mice suggested an important role for AMH in depletion of the primordial follicle pool (Durlinger et al., 1999
, Durlinger et al., 2002
). AMH, produced in growing ovarian follicles, has been shown subsequently to be an excellent marker for ovarian ageing (de Vet et al., 2002
; Seifer et al., 2002
; Fanchin et al., 2003a
). Recently, AMH levels were found to be elevated in anovulatory and PCOS patients compared with normal controls (Cook et al., 2002
; Pigny et al., 2003
; Laven et al., 2004
).
In anovulatory women for whom the number of all classes of follicles including the total number of primordial follicles seems to be increased (Pache et al., 1992b; Webber et al., 2003
), the age-related menstrual cycle irregularities (Kok et al., 2003
) and follicle pool exhaustion might occur later. Moreover, it may be speculated that the process of ovarian ageing is indeed delayed in women with PCO, since levels of AMH, an important inhibitor of primordial follicle pool depletion (Durlinger et al., 1999
), are increased. Indeed, cross-sectional data have suggested that women with PCOS may reach menopause at a later age (Dahlgren et al., 1992
). Furthermore, it has been reported previously that cycle irregularities improve with increasing age (Dahlgren et al., 1992
; Elting et al., 2000
; Bili et al., 2001
), possibly associated with a decrease in the follicle cohort size (Elting et al., 2003
). Although no information is available regarding the age of menopause in women with WHO 2 anovulation, it has been shown in a cross-sectional study that advanced age is associated with lower LH and androgen levels in this group (Bili et al., 2001
), as could be confirmed for the androgens in the current longitudinal study. Both oocyte quantity and quality dictate the subsequent reproductive events including decrease of fertility, increased abortion rate, the end of fertility, the beginning of cycle irregularity and, when almost no follicles are left, the occurrence of menopause (te Velde et al., 1998
). As a consequence, it might be hypothesized that women with WHO 2 anovulatory infertility when compared with normo-ovulatory controls still might be able to conceive at an advanced age. However, from this point of view, oocyte quality is not taken into account. Finally, the possibility that a deviant AMH synthesis or receptor is causally related to PCOS cannot be ruled out at this stage.
In summary, the current longitudinal study confirms that AMH serum levels are elevated in anovulatory women presenting with PCO, and demonstrates for the first time that the decline in AMH with age is less pronounced compared with controls. Considering important and well-documented roles of intra-ovarian AMH in the pace of follicle pool depletion and resulting female reproductive ageing, it may be proposed that the reproductive life span is extended in PCOS. Nevertheless, most women included in the present analysis have not yet reached the age of menopause. In order to substantiate further a delayed exhaustion of the primordial stock in women with WHO 2 anovulation, collection of additional follow-up data is required.
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References |
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---|
Balen AH, Laven JS, Tan SL and Dewailly D (2003) Ultrasound assessment of the polycystic ovary: international consensus definitions. Hum Reprod Update 9, 505514.
Beckers NG, Macklon NS, Eijkemans MJ and Fauser BC (2002) Women with regular menstrual cycles and a poor response to ovarian hyperstimulation for in vitro fertilization exhibit follicular phase characteristics suggestive of ovarian aging. Fertil Steril 78, 291297.[CrossRef][Medline]
Bili H, Laven J, Imani B, Eijkemans MJ and Fauser BC (2001) Age-related differences in features associated with polycystic ovary syndrome in normogonadotrophic oligo-amenorrhoeic infertile women of reproductive years. Eur J Endocrinol 145, 749755.[Medline]
Cate RL, Mattaliano RJ, Hession C, Tizard R, Farber NM, Cheung A, Ninfa EG, Frey AZ, Gash DJ, Chow EP et al. (1986) Isolation of the bovine and human genes for Mullerian inhibiting substance and expression of the human gene in animal cells. Cell, 4568545698.
Cook CL, Siow Y, Brenner AG and Fallat ME (2002) Relationship between serum mullerian-inhibiting substance and other reproductive hormones in untreated women with polycystic ovary syndrome and normal women. Fertil Steril 77, 141146.[CrossRef][Medline]
Dahlgren E, Johansson S, Lindstedt G, Knutsson F, Oden A, Janson PO, Mattson LA, Crona N and Lundberg PA (1992) Women with polycystic ovary syndrome wedge resected in 1956 to 1965: a long-term follow-up focusing on natural history and circulating hormones. Fertil Steril 57, 505513.[Medline]
de Vet A, Laven JS, de Jong FH, Themmen AP and Fauser BC (2002) Antimullerian hormone serum levels: a putative marker for ovarian aging. Fertil Steril 77, 357362.[CrossRef][Medline]
Durlinger AL, Kramer P, Karels B, de Jong FH, Uilenbroek JT, Grootegoed JA and Themmen AP (1999) Control of primordial follicle recruitment by anti-Mullerian hormone in the mouse ovary. Endocrinology 140, 57895796.
Durlinger AL, Gruijters MJ, Kramer P, Karels B, Kumar TR, Matzuk MM, Rose UM, de Jong FH, Uilenbroek JT, Grootegoed JA et al. (2001) Anti-Mullerian hormone attenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology 142, 48914899.
Durlinger AL, Gruijters MJ, Kramer P, Karels B, Ingraham HA, Nachtigal MW, Uilenbroek JT, Grootegoed JA and Themmen AP (2002) Anti-Mullerian hormone inhibits initiation of primordial follicle growth in the mouse ovary. Endocrinology 143, 10761084.
Elting MW, Korsen TJ, Rekers-Mombarg LT and Schoemaker J (2000) Women with polycystic ovary syndrome gain regular menstrual cycles when ageing. Hum Reprod 15, 2428.
Elting MW, Kwee J, Korsen TJ, Rekers-Mombarg LT and Schoemaker J (2003) Aging women with polycystic ovary syndrome who achieve regular menstrual cycles have a smaller follicle cohort than those who continue to have irregular cycles. Fertil Steril 79, 11541160.[CrossRef][Medline]
ESHRE Capri Workshop Group (1995) Anovulatory infertility. Hum Reprod 10, 15491553.[Abstract]
Faddy MJ, Gosden RG, Gougeon A, Richardson SJ and Nelson JF (1992) Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod 7, 13421346.[Abstract]
Fanchin R, Schonauer LM, Righini C, Guibourdenche J, Frydman R and Taieb J (2003a) Serum anti-Mullerian hormone is more strongly related to ovarian follicular status than serum inhibin B, estradiol, FSH, LH on day 3. Hum Reprod 18, 323327.
Fanchin R, Schonauer LM, Righini C, Frydman N, Frydman R and Taieb J (2003b) Serum anti-Mullerian hormone dynamics during controlled ovarian hyperstimulation. Hum Reprod 18, 328332.
Fauser BC and Van Heusden AM (1997) Manipulation of human ovarian function: physiological concepts and clinical consequences. Endocr Rev 18, 71106.
Hohmann FP, Laven JS, de Jong FH, Eijkemans MJ and Fauser BC (2001) Low-dose exogenous FSH initiated during the early, mid or late follicular phase can induce multiple dominant follicle development. Hum Reprod 16, 846854.
Hughesdon PE (1982) Morphology and morphogenesis of the Stein-Leventhal ovary and of so-called hyperthecosis. Obstet Gynecol Surv 37, 5977.[Medline]
Imani B, Eijkemans MJ, te Velde ER, Habbema JD and Fauser BC (1998) Predictors of patients remaining anovulatory during clomiphene citrate induction of ovulation in normogonadotropic oligoamenorrheic infertility. J Clin Endocrinol Metab 83, 23612365.
Imani B, Eijkemans MJ, te Velde E, Habbema JD and Fauser BC (1999) Predictors of chances to conceive in ovulatory patients during clomiphene citrate induction of ovulation in normogonadotropic oligoamenorrheic infertility. J Clin Endocrinol Metab 84, 16171622.
Josso N, Cate RL, Picard JY, Vigier B, di Clemente N, Wilson C, Imbeaud S, Pepinsky RB, Guerrier D, Boussin L et al. (1993) Anti-mullerian hormone: the Jost factor. Recent Prog Horm Res 48, 159.[Medline]
Jost A (1947) Recherches sur la differenciation sexuelle de l'embryon de lapin. Arch Anat Microsc Morphol Exp 36, 271315.
Kok HS, van Asselt KM, van der Schouw YT, Grobbee DE, te Velde ER, Pearson PL and Peeters PH (2003) Subfertility reflects accelerated ovarian ageing. Hum Reprod 18, 644648.
Laven JS, Imani B, Eijkemans MJ and Fauser BC (2002) New approach to polycystic ovary syndrome and other forms of anovulatory infertility. Obstet Gynecol Surv 57, 755767.[CrossRef][Medline]
Laven JS, Mulders AG, Visser JA, Themmen AP, de Jong FH and Fauser BC (2004) Anti-Mullerian hormone (AMH) serum concentrations in normo-ovulatory and anovulatory women. J Clin Endocrinol Metab 89, 318323.
Lee MM and Donahoe PK (1993) Mullerian inhibiting substance: a gonadal hormone with multiple functions. Endocr Rev 14, 152164.[Abstract]
Lee MM, Donahoe PK, Hasegawa T, Silverman B, Crist GB, Best S, Hasegawa Y, Noto RA, Schoenfeld D and MacLaughlin DT (1996) Mullerian inhibiting substance in humans: normal levels from infancy to adulthood. J Clin Endocrinol Metab 81, 571576.[Abstract]
Long WQ, Ranchin V, Pautier P, Belville C, Denizot P, Cailla H, Lhomme C, Picard JY, Bidart JM 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.
Mulders AG, Eijkemans MJ, Imani B and Fauser BC (2003a) Prediction of chances for success or complications in gonadotrophin ovulation induction in normogonadotrophic anovulatory infertility. Reprod Biomed Online 7, 170178.[Medline]
Mulders AG, Laven JS, Imani B, Eijkemans MJ and Fauser BC (2003b) IVF outcome in anovulatory infertility (WHO group 2)including polycystic ovary syndromefollowing previous unsuccessful ovulation induction. Reprod Biomed Online 7, 5058.[Medline]
Nikolaou D and Templeton A (2003) Early ovarian ageing: a hypothesis: detection and clinical relevance. Hum Reprod 18, 11371139.
Pache TD, Hop WC, de Jong FH, Leerentveld RA, van Geldorp H, Van de Kamp TM, Gooren LJ and Fauser BC (1992a) 17 beta-oestradiol, androstenedione and inhibin levels in fluid from individual follicles of normal and polycystic ovaries, and in ovaries from androgen treated female to male transsexuals. Clin Endocrinol 36, 565571.[Medline]
Pache TD, Wladimiroff JW, Hop WC and Fauser BC (1992b) How to discriminate between normal and polycystic ovaries: transvaginal US study. Radiology 183, 421423.[Abstract]
Pigny P, Merlen E, Robert Y, Cortet-Rudelli C, Decanter C, Jonard S and Dewailly D (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, 59575962.
Rajpert-De Meyts E, Jorgensen N, Graem N, Muller J, Cate RL and Skakkebaek NE (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.
Richardson SJ, Senikas V and Nelson JF (1987) Follicular depletion during the menopausal transition: evidence for accelerated loss and ultimate exhaustion. J Clin Endocrinol Metab 65, 12311237.[Abstract]
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 (PCOS). Hum Reprod 19, 4147.
Rowe PJ, Comhaire FH, Hargreave TB and Mellows H (2000) Female partner. In Rowe PJ, Comhaire FH, Hargreave TB, and Mellows H (eds), WHO Manual for the Standardized Investigation and Diagnosis of the Infertile Couple. Press Syndicate of the University of Cambridge, Cambridge, pp. 4067.
Schipper I, de Jong FH and Fauser BC (1998) Lack of correlation between maximum early follicular phase serum follicle stimulating hormone concentrations and menstrual cycle characteristics in women under the age of 35 years. Hum Reprod 13, 14421448.[Abstract]
Seifer DB, MacLaughlin DT, Christian BP, Feng B and Shelden RM (2002) Early follicular serum mullerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil Steril 77, 468471.[CrossRef][Medline]
te Velde ER and Pearson PL (2002) The variability of female reproductive ageing. Hum Reprod Update 8, 141154.
te Velde ER, Scheffer GJ, Dorland M, Broekmans FJ and Fauser BC (1998) Developmental and endocrine aspects of normal ovarian aging. Mol Cell Endocrinol 145, 6773.[CrossRef][Medline]
van Rooij IA, Broekmans FJ, te Velde ER, Fauser BC, Bancsi LF, de Jong FH and Themmen AP (2002) Serum anti-Mullerian hormone levels: a novel measure of ovarian reserve. Hum Reprod 17, 30653071.
van Santbrink EJ, Hop WC, van Dessel TJ, de Jong FH and Fauser BC (1995) Decremental follicle-stimulating hormone and dominant follicle development during the normal menstrual cycle. Fertil Steril 64, 3743.[Medline]
van Santbrink EJ, Hop WC and Fauser BC (1997) Classification of normogonadotropic infertility: polycystic ovaries diagnosed by ultrasound versus endocrine characteristics of polycystic ovary syndrome. Fertil Steril 67, 452458.[Medline]
Webber LJ, Stubbs S, Stark J, Trew GH, Margara R, Hardy K and Franks S (2003) Formation and early development of follicles in the polycystic ovary. Lancet 362, 10171021.[CrossRef][Medline]
Weenen C, Laven JS, von Bergh AR, Cranfield M, Groome NP, Visser J, Kramer P, Fauser BC and Themmen AP (2004) Anti-Mullerian hormone (AMH) expression pattern in the human ovary: potential implications for initial and cyclic recruitment. Mol Hum Reprod 10, 7783.
Wise PM, Krajnak KM and Kashon ML (1996) Menopause: the aging of multiple pacemakers. Science 273, 6770.[Abstract]
Submitted on February 23, 2004; accepted on May 25, 2004.