Primordial Follicle Recruitment Research Articles

Expression of bone morphogenetic protein-15 in human oocyte and cumulus granulosa cells primed with recombinant follicle-stimulating hormone followed by human chorionic gonadotropin

Bone morphogenetic protein-15 is expressed in oocytes and cumulus granulosa cells. It may play an important role in the hCG-induced oocyte final maturation and corpus luteum (CL) formation.

Ovarian follicle development in Booroola sheep exhibiting impaired bone morphogenetic protein signalling pathway

The role of bone morphogenetic proteins (BMPs) in the regulation of ovarian function has been extensively investigated but the mechanism of regulation is not well understood. The aim of this study was to investigate the effect of mutation in the BMP receptor in Booroola sheep on the number of primordial follicles and rate of follicle recruitment in comparison with that in normal merino sheep in vivo. Whole sheep ovaries at the time of birth, 1.5 and 5 years old were collected and processed for the follicle quantification, using computerised stereological methods and statistical analyses. At birth, the total number of primordial follicles in Booroola sheep was significantly lower than in merino sheep. At 1.5 and 5 years, a reversed pattern in favour of Booroola ewes was seen with significantly more primordial follicles than merino. In parallel, the rate of primordial follicle recruitment to developing cohort was substantially lower in Booroola ewes with only 51 and 66% of primordial follicle consumption at 1.5 and 5 years respectively compared to 92 and 97% in merino ewes. On other hand, the mean numbers of developing primary follicles were smaller in Booroola sheep at the time of birth, yet, Booroola ewes possess more primary follicles than merino at 1.5 years. These findings suggest that attenuation of the intraovarian signalling pathway of BMPs may in fact be a successful means of rationalising follicle consumption, preventing unnecessary loss of follicles from the initial primordial follicle pool, hence increasing reproductive longevity and fertility.

509. REGULATING MURINE FOLLICLE DEVELOPMENT BY STIMULATION OF THE JAK2/STAT3 SIGNAL TRANSDUCTION PATHWAY

Follicular development requires the recruitment of primordial follicles into the growing follicle pool following initiation of multiple cytokine signalling pathways. Suppression of follicular development is thought to be key to maintaining the population of primordial follicles and allowing for controlled release of these follicles throughout the reproductive lifespan of the female. However, little is known of the processes and signalling molecules that suppress primordial follicle activation and early follicle growth. Our group has identified significant upregulation of the Janus Kinase 2 (JAK2)/ Signal Transducer and Activator of Transcription 3 (STAT3) signalling pathway inhibitor the Suppressor of Cytokine Signalling 4 (SOCS4) that coincides with the initial wave of follicular activation in theneonatal mouse ovary. Further studies by our group have localised the SOCS4 protein to the granulosa cells of activating and growing follicles, suggesting SOCS4 expression may be linked to follicular activation. We have focused on examining protein localisation and gene expression patterns of the eight SOCS family members CIS and SOCS1-7. We have recently demonstrated that co-culture of neonatal ovaries with Kit Ligand (KL) for 2 days increases the mRNA levels of all SOCS genes. We also demonstrated the co-localisation of SOCS2 proteins with the KL receptor c-kit in the mural granulosa cells of antral, and large pre-antral follicles suggesting a significant role for SOCS2 in the later stages of follicular development. We have also shown that culturing ovaries with the potent JAK2 inhibitor AG490 substantially reduces mRNA levels of all SOCS and STAT genes that we have so far measured. We hypothesise a significant role for JAK2/STAT3 signalling in promoting the activation and early growth of ovarian follicles. Our investigations have identified significant roles for JAK2/STAT3 and the SOCS family in the regulation of ovarian follicle development.

The Role of BMP Signalling Pathway in the Regulation of Ovarian Follicle Development.

Bone morphogenetic proteins (BMPs) regulation of ovarian function has been investigated in several animal models, demonstrating their role in primordial germ cell formation, primordial-to-primary follicle recruitment, follicle development, steroidogenesis and ovulation. However, the mechanism by which BMPs regulate individual processes of ovarian function is not well understood. The aim of this study was to investigate the effect of the BMP receptor mutation in Booroola sheep on primordial follicle formation and rate of recruitment in comparison to that in normal merino sheep in vivo. Whole ovaries from Booroola and merino sheep at birth, 1.5 years and 5 years of age were obtained by bilateral ovariectomy and processed for the quantification of primordial and primary follicles using computerised stereological methods (Stereo Investigator Software Version 7, MicroBrightField, Inc.) and JMP 6 statistical package (SAS Institute Inc, 2005). Regardless of animal strain, the overall absolute number of primordial and primary follicles per ovary of newborn sheep was considerably higher than in the 1.5 and 5-year old ewes. Log transformed data analyses revealed that the total number of primordial follicles in newborn Booroola sheep was significantly (P=0.0065) lower than in merino of the same age. At 1.5 years a trend showing a considerable reversal in the number of primordial follicles was observed in Booroola ewes, which then significantly (p<0.0036) increased at 5 years of age. The rate of primordial follicle recruitment was substantially lower in Booroola ewes compared with that in merino, as reflected by the difference in the absolute number of primordial follicles remaining at 1.5 and 5 years of age. Immediately after birth, the mean total number of primordial follicles in Booroola strain was only 36% of that present in the merino strain. This ratio became 207% and 367% at 1.5 and 5 years, respectively. The ratios of primordial follicle consumption in Booroola ewes were 51% and 66% up to the age of 1.5 and 5 years respectively compared to 92% and 97% of primordial follicles have been used in merino ewes. On the other hand, the mean numbers of primary follicles per ovary in newborn animals was similar in both sheep strains. However, at 1.5 years, Booroola ewes possess more primary follicles than merino, and the absolute number of primary follicles present at 1.5 years was equal to 26% of the number present at birth, compared with only 8% of primary follicles present in merino ewes. At 5 years of age the primary follicles were not counted in either animal strain. These findings suggest that attenuation of the intraovarian signaling pathway of BMPs may in fact be a successful means of rationalizing follicle consumption, preventing unnecessary loss of follicles from the initial follicle pool, hence increasing reproductive age and fertility.

Photoperiod-dependent modulation of anti-Müllerian hormone in female Siberian hamsters, Phodopus sungorus

Fertility and fecundity decline with advancing age in female mammals, but reproductive aging was decelerated in Siberian hamsters (Phodopus sungorus) raised in a short-day (SD) photoperiod. Litter success was significantly improved in older hamsters when reared in SD and the number of primordial follicles was twice that of females held in long days (LD). Because anti-Müllerian hormone (AMH) appears to inhibit the recruitment of primordial follicles in mice, we sought to determine whether the expression patterns of AMH differ in the ovaries and serum of hamsters raised in SD versus LD. Ovaries of SD female hamsters are characterized by a paucity of follicular development beyond the secondary stage and are endowed with an abundance of large eosinophilic cells, which may derive from granulosa cells of oocyte-depleted follicles. In ovaries from 10-week-old SD hamsters, we found that the so-called 'hypertrophied granulosa cells' were immunoreactive for AMH, as were granulosa cells within healthy-appearing primary and secondary follicles. Conversely, ovaries from age-matched LD animals lack the highly eosinophilic cells present in SD ovaries. Therefore, AMH staining in LD was limited to primary and secondary follicles that are comparable in number to those found in SD ovaries. The substantially greater AMH expression in SD ovaries probably reflects the abundance of hypertrophied granulosa cells in SD ovaries and their relative absence in LD ovaries. The modulation of ovarian AMH by day length is a strong mechanistic candidate for the preservation of primordial follicles in female hamsters raised in a SD photoperiod.

Impact of gonadotrophin stimulation for assisted reproductive technology on ovarian ageing and menopause

This study investigated whether menopausal age is inversely related to the number of assisted reproduction cycles and/or ampoules of FSH used, as an indication of the possibility that gonadotrophin treatment accelerates follicular depletion. A questionnaire addressing reproductive health, history and treatment was mailed to women who had been treated for infertility at Bourn Hall Clinic, Cambridge, between 1981 and 1994. Additional data were extracted from clinical records. Of 739 mailings, 199 responses were obtained and 101 provided full sets of data. No significant associations by multiple regression analysis were found between total number of ampoules of FSH, number of assisted reproduction cycles or pregnancies and menopausal age; the mean of about 50 years is similar to values reported in other menopause studies. No relationship was found between the severity of menopausal symptoms and the number of assisted reproduction cycles or any other covariate. The only significant relationship found was an association between number of IVF cycles and increasing menopausal age ( P = 0.001); this may be due to women with later menopause being self-selected for more IVF treatment. This study provides reassuring evidence that ovarian stimulation does not compromise reproductive potential by hastening menopause, an observation consistent with experimental studies showing that primordial follicle recruitment is independent of gonadotrophin stimulation.

Implications of Polycystic Ovary Syndrome on Oocyte Development

Human follicle development requires the recruitment of primordial follicles into a cohort of growing follicles from which one follicle is selected to ovulate a mature oocyte. During this developmental process, complex endocrine and intraovarian paracrine signals create a changing intrafollicular hormonal milieu. With this microenvironment, appropriate cumulus cell-oocyte signaling governs oocyte developmental competence, defined as the ability of the oocyte to complete meiosis and undergo fertilization, embryogenesis, and term development. Many of these mechanisms are perturbed in polycystic ovary syndrome (PCOS), a heterogeneous syndrome characterized by ovarian hyperandrogenism, hyperinsulinemia from insulin resistance, and reduced fecundity. In addition to these endocrinopathies, PCOS also is characterized by paracrine dysregulation of follicle development by intraovarian proteins of the transforming growth factor-beta family. Consequently, PCOS patients undergoing ovarian stimulation for in vitro fertilization are at increased risks of impaired oocyte developmental competence, implantation failure, and pregnancy loss. Recent data demonstrate links between endocrine/paracrine factors and oocyte gene expression in PCOS and suggest that new clinical strategies to optimize developmental competence of PCOS oocytes should target correction of the entire follicle growth and oocyte development process.

AMH TYPE II RECEPTOR DEFICIENT MICE DISPLAY A MORE SEVERE OVARIAN PHENOTYPE THAN AMH DEFICIENT MICE

Anti-Mullerian hormone (AMH) is a member of the transforming growth factor beta family and signals through a specific type II receptor (AMHRII). Previously we have shown that in ovaries of AMH-deficient mice more primordial follicles are recruited, resulting in a larger pool of growing follicles. As a consequence of the increased rate of recruitment, the primordial follicle pool is depleted at an earlier age than in wild type mice. Similar to AMH, the AMHRII is expressed in granulosa cells of healthy preantral and small antral follicles. Furthermore, AMHRII is expressed in theca cells of these follicles, and expression in the pregranulosa cells of primordial follicles is suggested. To determine the role of the type II receptor in ovarian function, we have analyzed the follicle pool of AMHRII-deficient mice. In 13-month-old AMHRII null mice, the primordial follicle pool was completely depleted and as a consequence significantly less growing follicles (10-fold) were present compared to wild type mice. Ovaries of 4-month-old AMHRII null mice displayed a 3-fold increase in number of small growing follicles whereas the number of primordial follicles was decreased compared to wild type mice. These results suggest that primordial follicle recruitment is enhanced in AMHRII null mice, resembling the AMH null ovarian phenotype. However, the decrease in number of primordial follicles was more pronounced in AMHRII- than AMH-deficient mice at 4 months of age. Furthermore, whereas in AMH-deficient mice the number of primordial follicles did not differ between wildtype and null mice at 25 days of age, ovaries of AMHRII-deficient mice contained significantly less primordial follicles. This suggests that AMHRII may have an AMHindependent role in the ovary. In conclusion, AMHRII-deficient mice display a more severe ovarian phenotype than AMH-deficient mice. Studies are ongoing to determine whether enhanced recruitment alone is sufficient to explain the ovarian phenotype of the AMHRII null mice, or whether other mechanisms also contribute to the smaller primordial pool in AMHRII null mice. (poster)

A polymorphism in the AMH type II receptor gene is associated with age at menopause in interaction with parity

Anti-Müllerian hormone (AMH) inhibits primordial follicle recruitment in the mouse ovary. We hypothesize that in women AMH signaling also regulates the usage of the primordial follicle pool and hence influences the onset of menopause. Since age at menopause has a strong genetic component, we investigated the role of AMH signaling using a candidate gene approach. In two large population-based cohorts of Dutch post-menopausal women (n = 2381 and n = 248), we examined the association between two polymorphisms, one in the AMH gene and one in the AMH type II receptor (AMHR2) gene, and natural age at menopause. The AMH Ile(49)Ser polymorphism (rs10407022) was not associated with age at menopause in either cohort. In the Rotterdam cohort, the AMHR2 -482 A > G polymorphism (rs2002555) was associated with age at menopause in interaction with the number of offspring (P = 0.001). Nulliparous women homozygous for the G-allele entered menopause 2.6 years earlier compared with nulliparous women homozygous for the A-allele (P = 0.005). In the LASA cohort, women with the G/G genotype tended to enter menopause 2.8 years earlier compared with the A/A genotype (P = 0.063). The observed association of the AMHR2 -482 A > G polymorphism with natural age at menopause suggests a role for AMH signaling in the usage of the primordial follicle pool in women.

Anti-Müllerian hormone and anti-Müllerian hormone type II receptor polymorphisms are associated with follicular phase estradiol levels in normo-ovulatory women

In mice, anti-Müllerian hormone (AMH) inhibits primordial follicle recruitment and decreases FSH sensitivity. Little is known about the role of AMH in human ovarian physiology. We hypothesize that in women AMH has a similar role in ovarian function as in mice and investigated this using a genetic approach. The association of the AMH Ile(49)Ser and the AMH type II receptor (AMHR2) -482 A > G polymorphisms with menstrual cycle characteristics was studied in a Dutch (n = 32) and a German (n = 21) cohort of normo-ovulatory women. Carriers of the AMH Ser(49) allele had higher serum estradiol (E(2)) levels on menstrual cycle day 3 when compared with non-carriers in the Dutch cohort (P = 0.012) and in the combined Dutch and German cohort (P = 0.03). Carriers of the AMHR2 -482G allele also had higher follicular phase E(2) levels when compared with non-carriers in the Dutch cohort (P = 0.028), the German cohort (P = 0.048) and hence also the combined cohort (P = 0.012). Women carrying both AMH Ser(49) and AMHR2 -482G alleles had highest E(2) levels (P = 0.001). For both polymorphisms no association with serum AMH or FSH levels was observed. Polymorphisms in the AMH and AMHR2 genes are associated with follicular phase E(2) levels, suggesting a role for AMH in the regulation of FSH sensitivity in the human ovary.

Increased Oocyte Degeneration and Follicular Atresia during the Estrous Cycle in Anti-Müllerian Hormone Null Mice

Anti-Müllerian hormone (AMH) plays an important role in folliculogenesis. AMH null mice display an increased recruitment of primordial follicles. Nevertheless, these mice do not have proportionally more preovulatory follicles. Therefore, AMH null mice provide an interesting genetic model to study the regulation of species-specific number of preovulatory follicles. We studied the follicle pool throughout the estrous cycle at 4 months of age. Analysis of the follicle pool revealed that AMH null mice have an increased and earlier cyclic recruitment of growing follicles despite a blunted FSH surge at estrus. However, FSH levels at estrus were apparently too low to support growth to the preovulatory stage because an increased level of atresia was observed, which neutralized the increased cyclic recruitment. When AMH null mice were subjected to a superovulation scheme, the rise in FSH levels resulted in the rescue of the recruited cohort of growing follicles. Analysis of the follicle pool also revealed that the increased recruitment of primordial follicles in AMH null mice was neutralized by an increased loss of follicles during the transition from small preantral to large preantral follicle. This major loss of follicles was not completely reflected by a corresponding augmentation of atresia but did correspond with an increased number of oocyte remnants observed in AMH null mice. We conclude that a combination of increased oocyte degeneration and increased follicular atresia neutralizes the increased initial and cyclic recruitment in AMH null mice to a normal number of preovulatory follicles.

KIT/KIT Ligand in Mammalian Oogenesis and Folliculogenesis: Roles in Rabbit and Murine Ovarian Follicle Activation and Oocyte Growth1

In rodent ovaries Kit ligand (KITL) and its receptor KIT have diverse roles, including the promotion of primordial follicle activation, oocyte growth, and follicle survival. Studies were undertaken to determine whether KITL and KIT carry out similar activities in rabbits. KitlandKitmRNA and protein were localized to oocytes and granulosa cells, respectively, in the rabbit ovary. Ovarian cortical explants from juvenile rabbits and neonatal mouse ovaries were subsequently cultured with recombinant mouse KITL and/or KITL neutralizing antibody. Indices of follicle growth initiation were compared with controls and between treatment groups for each species. Recombinant mouse KITL had no stimulatory effect on primordial follicle recruitment in cultured rabbit ovarian explants. However, the mean diameter of oocytes from primordial, early primary, primary, and growing primary follicles increased significantly in recombinant mouse KITL-treated explants compared with untreated tissues. In contrast, recombinant mouse KITL promoted both primordial follicle activation and an increase in the diameter of oocytes from primordial and early primary follicles in the mouse, and these effects were inhibited by coculture with KITL-neutralizing antibody. Recombinant mouse KITL had no effect on follicle survival for either species. These data demonstrate that KITL promotes the growth of rabbit and mouse oocytes and stimulates primordial follicle activation in the mouse but not in the rabbit. We propose that the physiologic roles of KITL and KIT may differ between species, and this has important implications for the design of in vitro culture systems for folliculogenesis in mammals, including the human.

Reduced recruitment and survival of primordial and growing follicles in GH receptor-deficient mice

GH influences female fertility. The goal of the present study was to obtain more insight into the effect of loss of GH signalling, as observed in humans suffering from Laron syndrome, on ovarian function. Therefore, serial paraffin sections of ovaries of untreated and IGF-I-treated female GH receptor knock-out (GHR/GHBP-KO) mice were examined to determine the follicular reserve and the percentage of follicular atresia in each ovary. Our observations demonstrate that the amount of primordial follicles was significantly elevated in GHR/GHBP-KO mice, while the numbers of primary, preantral and antral follicles were lower compared with wild-type values. The reduced number of healthy growing follicles in GHR/GHBP-KO mice was accompanied by a significant increase in the percentage of atretic follicles. IGF-I treatment of GHR/GHBP-KO mice for 14 days resulted in a reduced number of primordial follicles, an increased number of healthy antral follicles, and a decreased percentage of atretic follicles. The results of the present study suggest that GH may play a role, either directly or indirectly, via for instance IGF-I, in the recruitment of primordial follicles into the growing pool. Furthermore, GH seems to protect antral follicles, directly or indirectly from undergoing atresia.

Identification and characterization of a novel Mt-retrotransposon highly represented in the female mouse germline

The control of primordial follicle recruitment into the growing follicle population is a major limiting process in female reproduction. In order to gain insight into the molecular processes occurring at the time of primordial follicle activation, a subtractive hybridization analysis was performed between cDNAs prepared from temporally distinct mouse neonatal ovarian tissues that differed according to the state of primordial follicle activation. One highly represented clone associated with activation was an Mt retrotransposon-like sequence designated Mtfull, which was subsequently cloned and determined to be novel and restricted in expression to the ovary. The polyadenylated 1684-bp sequence has long terminal repeats, is predicted to be noncoding, and is the predominant Mti-related sequence present in the mouse ovary. In situ hybridization further localized Mtfull expression to the oocyte and confirmed that expression is concomitant with follicle activation. Together with in silico data, we predict Mtfull plays an essential role in folliculogenesis through regulation of gene expression.

Anti-Müllerian hormone: a new marker for ovarian function

Anti-Müllerian hormone (AMH) is a member of the transforming growth factor beta family of growth and differentiation factors. In the ovary, AMH has an inhibitory effect on primordial follicle recruitment as well as on the responsiveness of growing follicles to follicle-stimulating hormone (FSH). The ovary-specific expression pattern in granulosa cells of growing nonselected follicles makes AMH an ideal marker for the size of the ovarian follicle pool. This review summarizes recent findings concerning AMH and its role as a marker for the quantitative aspect of ovarian reserve as well as ovarian dysfunction.

TGFβ signalling in the development of ovarian function

Ovarian development begins back in the embryo with the formation of primordial germ cells and their subsequent migration and colonisation of the genital ridges. Once the ovary has been defined structurally, the primordial germ cells transform into oocytes and become housed in structures called follicles (in this case, primordial follicles), a procedure that, in most mammals, occurs either shortly before or during the first few days after birth. The growth and differentiation of follicles from the primordial population is termed folliculogenesis. Primordial follicles give rise to primary follicles that transform into preantral follicles, then antral follicles (secondary follicles) and, finally (preovulatory) Graafian follicles (tertiary follicles) in a co-ordinated series of transitions regulated by hormones and local intraovarian factors. Members of the transforming growth factor-beta (TGFbeta) superfamily have been shown to play important roles in this developmental process starting with the specification of primordial germ cells by the bone morphogenetic proteins through to the recruitment of primordial follicles by anti-Mullerian hormone and, potentially, growth and differentiation factor-9 (GDF9) and, finally, their transformation into preantral and antral follicles in response to activin and TGF-beta. Developmental and mutant mouse models have been used to show the importance of this family of growth factors in establishing the first wave of folliculogenesis.

Anti-Mullerian Hormone: Its Role in Follicular Growth Initiation and Survival and as an Ovarian Reserve Marker

In this paper the role in the ovary of anti-Müllerian hormone (AMH), a member of the transforming growth factor-beta family of growth and differentiation factors, is reviewed. AMH has an inhibitory effect on primordial follicle recruitment and may also inhibit follicle-stimulating homone-dependent selection of follicles for dominance. In addition to its functional role in the ovary, AMH in serum is an excellent candidate marker as an indication of the ovarian reserve, not only in infertility clinic patients but also in women during and after cancer treatment.

Expression of Sox8, Sf1, Gata4, Wt1, Dax1, and Fog2 in the mouse ovarian follicle: Implications for the regulation of Amh expression

Anti-Mullerian hormone (Amh) is expressed in the granulosa cells of growing and preovulatory follicles in the mouse ovary where it acts to decrease responsiveness to follicle stimulating hormone (FSH) and plays a role in inhibiting primordial follicle recruitment. Recently, co-culture of isolated oocytes and granulosa cells has demonstrated that Amh expression is up-regulated in the presence of oocytes and, at preantral stages, this effect is dependent upon close contact. In Sertoli cells, Amh expression is regulated by several transcription factors including SOX9, SF1, GATA4, WT1, and DAX1, which, with the exception of SOX9, are also expressed in granulosa cells where GATA4 is known to up-regulate Amh expression antagonised by FOG2. Here, we demonstrate that Sox8, which is closely related to Sox9 and encodes the protein SOX8 which can transactivate Amh, is expressed in the postnatal mouse ovarian follicle but is not co-expressed with Amh in the granulosa cells of preantral follicles. Sox8 expression was found only in the oocytes of preantral follicles and in the oocytes, cumulus cells, and mural granulosa cells of preovulatory follicles. Also, increased expression of Amh in granulosa cells co-cultured with oocytes was not associated with increased mRNA levels of the transcription factors Sf1, Gata4, Wt1, Dax1, or Fog2. These findings reveal Sox8 expression in the ovarian follicle and show that oocyte regulation of Amh expression is not due to oocyte regulation of Sf1, Gata4, Wt1, Dax1, or Fog2 expression in granulosa cells.

Stockpiling of Transitional and Classic Primary Follicles in Ovaries of Women with Polycystic Ovary Syndrome

Recently, we proposed an oocyte-growth differentiation factor-9 hypothesis that predicts alterations in the initial stages of folliculogenesis in polycystic ovary syndrome (PCOS) ovaries. Here, we test this hypothesis by scoring the composition of follicles in normal and PCOS ovaries. Follicles were classified as primordial, transitional primary, classic primary, secondary, and Graafian. A total of 2274 follicles were scored. The total number of growing follicles was significantly greater in PCOS ovaries than normal, but the number of nongrowing primordial follicles did not differ. Consequently, the increase in growing follicles in PCOS cannot be explained by increased primordial follicle recruitment. Differential counts showed that the number of growing follicles at each stage of development was significantly greater: PCOS had 2.7-fold more primary, 1.8-fold more secondary, and 2-fold more Graafian follicles than normal. The greatest effect was on the classic primary follicles where the number was almost 5-fold greater in PCOS ovaries. The absence of apoptosis in normal and PCOS preantral follicles argues that the increase in growing follicles in PCOS cannot be explained by changes in atresia. We conclude, therefore, that primary follicle growth is abnormally slow in PCOS and the dynamics are reflected in a stockpiling of classic primary follicles.

Anti-Müllerian hormone expression pattern in the human ovary: potential implications for initial and cyclic follicle recruitment.

Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-beta superfamily, which plays an important role in both ovarian primordial follicle recruitment and dominant follicle selection in mice. However, the role of AMH in folliculogenesis in humans has not been investigated in detail. In the present study, AMH expression was assessed using immunohistochemistry in ovarian sections, obtained from healthy regularly cycling women. To this end, a novel monoclonal antibody to human AMH was developed. AMH expression was not observed in primordial follicles, whereas 74% of the primary follicles showed at least a weak signal in the granulosa cells. The highest level of AMH expression was present in the granulosa cells of secondary, preantral and small antral follicles <or=4 mm in diameter. In larger (4-8 mm) antral follicles, AMH expression gradually disappeared. In conclusion, in the human AMH expression follows a similar pattern as compared to the mouse and rat, suggesting an important role of AMH in folliculogenesis.