The activin-follistatin system and in vitro early follicle development in goats

The aim of the present study was to investigate the effects of activin-A and follistatin on in vitro primordial and primary follicle development in goats. To study primordial follicle development (experiment 1), pieces of ovarian cortex were cultured in vitro for 5 days in minimal essential medium (MEM) supplemented with activin-A (0, 10 or 100 ng/ml), follistatin (0, 10 or 100 ng/ml) or combinations of the two. After culture, the numbers of primordial follicles and more advanced follicle stages were calculated and compared with those in non-cultured tissue. Protein and mRNA expression of activin-A, follistatin, Kit ligand (KL), growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in non-cultured and cultured follicles were studied by immunohistochemistry and PCR. To evaluate primary follicle growth (experiment 2), freshly isolated follicles were cultured for 6 days in MEM plus 100 ng/ml activin-A, 100 ng/ml follistatin or 100 ng/ml activin-A plus 200 ng/ml follistatin. Morphology, follicle and oocyte diameters in cultured tissue and isolated follicles before and after culture were assessed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) reactions were performed to study DNA fragmentation in follicles. In experiment 1, it was found that goat primordial follicles were activated to develop into more advanced stages, i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin, KL, GDF-9 and BMP-15. Fewer than 30% of the atretic follicles in cultured cortical tissue had TUNEL-positive (oocyte or granulosa) cells. Activin-A did not affect the occurrence of TUNEL-positive cells in follicles within cortical tissue. In experiment 2, addition of activin-A to cultured isolated primary follicles significantly stimulated their growth, the effect being counteracted by follistatin. Absence of such a neutralizing effect of follistatin in the cultures with ovarian cortical tissue can be due to lower dose of follistatin used and incomplete blockage of activin in these experiments. In contrast to cortical enclosed atretic follicles, all atretic follicles that had arisen in cultures with isolated primary follicles had TUNEL-positive cells, which points to differences between isolated and ovarian tissue-enclosed follicles with regard to the followed pathways leading to their degeneration. In summary, this in vitro study has demonstrated that cultured goat primordial follicles are activated to grow and develop into intermediate and primary follicles. During in vitro culture, the follicles maintain their ability to express activin-A, follistatin, KL, GDF-9 and BMP-15. The in vitro growth and survival of activated follicles enclosed in cortical tissue and the in vitro growth of isolated primary follicles are stimulated by activin-A.     

The primordial to primary follicle transition

The mechanisms that regulate the gradual exit of ovarian follicles from the non-growing, primordial pool are very poorly understood. A better understanding of the signals that initiate follicular growth in mammals, and of the conditions necessary for sustained growth of early preantral follicles in vitro, could have practical implications for contraception, alleviation of infertility, and regulation of the rate of follicle depletion (menopause). Our laboratory has developed two experimental systems that can be used to study factors involved in the activation of primordial follicles. In the first experimental system, small pieces of ovarian cortex, containing mostly primordial follicles, are isolated from fetal ovaries of cattle or baboons and cultured in serum-free medium. Under these conditions most primordial follicles become activated between 12 and 24 h of culture; their granulosa cells change shape, from flattened to cuboidal, and begin to express proliferating cell nuclear antigen (PCNA). During 7 days in culture, the newly-formed primary follicles and their oocytes increase significantly in diameter. This wholesale 'spontaneous' activation in serum-free medium is quite different from the much more gradual exit of primordial follicles from the resting pool that occurs in vivo and suggests that primordial follicles in vivo may be subject to a tonic inhibition of growth initiation or, alternatively, that some aspect(s) of the environment in vitro stimulates growth initiation. Recently we developed a second experimental system for studying activation of primordial follicles. Pieces of ovarian cortex from bovine or baboon fetuses were grafted beneath the developing chorioallantoic membrane (CAM) of 6-day-old chick embryos, a site known to support xenografted tissues. The cortical pieces were rapidly vascularized and histological analysis of pieces recovered after 2, 4, 7, or 10 days 'in ovo' revealed no increase in the number of primary follicles and maintenance of original numbers of primordial follicles. Therefore, grafting ovarian cortical pieces beneath the chick CAM provides an experimental system in which follicles remain at the primordial stage in a readily accessible environment and which, thus, may be used to study potential regulators of the initiation of follicle growth. The results suggest that vascularization of isolated pieces of ovarian cortex provides conditions that maintain follicular quiescence, whereas culture in vitro allows unrestrained activation of primordial follicles. Future studies with and comparisons of the in vitro and in ovo models may provide new insight into the mechanisms that regulate the primordial to primary follicle transition.     

Growth differentiation factor-9 promotes the growth, development, and survival of human ovarian follicles in organ culture.

Growth differentiation factor-9 (GDF-9) is a growth factor and a member of the TGFbeta superfamily that is secreted by oocytes in growing ovarian follicles. In the current study we cultured human ovarian follicles within slices of ovarian cortical tissue in the presence and absence of recombinant rat GDF-9. Ovarian tissue was obtained by biopsy during cesarean section (31 women) or gynecological laparoscopy (3 women). The mean age (+/-SD) of the women who donated tissue was 32.8 +/- 5.0 yr (range, 19-41 yr). In these biopsies a total of 900 follicles were analyzed. A significantly higher proportion of cultured primordial follicles showed growth initiation and reached the secondary stage of development in the presence of GDF-9. In serum-free cultures after 7 d, 53% of the follicles had reached the secondary stage with GDF-9 vs. 31% in the control group (P < 0.01). Follicle viability was also improved in the presence of GDF-9 after 7 d in culture (74% GDF-9 vs. 48% control), which resulted in a smaller reduction in follicle numbers due to atresia. Treatment with GDF-9 in vitro promoted the survival and progression of human follicular development to the secondary stage. This oocyte-derived factor may be essential for the development of somatic cells in early human follicles and useful in designing culture conditions for maturation of follicles and oocytes in vitro.     

In vitro development of oocytes from porcine and bovine primary follicles

A limiting factor to realising the full potential of many of the new reproductive techniques is the lack of availability of fertile oocytes. Methods for maturing oocytes in vitro (IVM) have been developed to address this problem but the success rate and quality of embryos produced by IVM is variable. The variation in success may be due to the poor quality of oocytes that are being selected for maturation, since these would be taken from developed antral follicles. To attempt to eliminate this variation and increase the numbers produced, it may be better to use the large source of oocytes from preantral and primordial follicles by developing systems for in vitro growth (IVG). In vitro systems that utilise early growing follicles as a source of oocytes have been developed for laboratory species and these have been successful in producing live young. If successful, IVG in association with IVM would supercede existing technology for assisted reproduction in both humans and animals by making it possible to develop the desired number of high quality oocytes from small amounts of ovarian tissue. However, developing IVG systems for species with follicles that develop over several months presents enormous technical challenges. We have developed systems that permit the growth of individual porcine and bovine preantral follicles for periods of up to 20 days. Porcine follicles grown in micro-wells show a higher rate of survival if grown in the presence of serum than follicles grown under serum free conditions. Oocytes recovered from in vitro grown porcine follicles are capable of reaching metaphase II after in vitro maturation. A similar system has been developed for bovine follicles and survival rate is high under serum free conditions but as yet no oocytes from in vitro grown oocytes have been capable of completing meiotic maturation.     

Disordered follicle development in ovaries of prenatally androgenized ewes

Exposure to excess androgens in utero induces irreversible changes in gonadotrophin secretion and results in disrupted reproductive endocrine and ovarian function in adulthood, in a manner reminiscent of the common clinical endocrinopathy of polycystic ovary syndrome (PCOS). We have recently identified an abnormality in early follicle development in PCOS which we suggested might be an androgenic effect. We propose that altered ovarian function in androgenized ewes is due to prenatal androgens not only causing an abnormality of gonadotrophin secretion, but also exerting a direct effect on the early stages of folliculogenesis. Therefore, in this study, we explored the possible differences between small preantral follicles in the ovarian cortex of androgenized female lambs with those of normal lambs. At 8 months of age, small ovarian cortical biopsies (approximately 5 mm3) were obtained at laparotomy from nine female lambs that had been exposed to androgens in utero from embryonic days 30 to 90 of a 147-day pregnancy, and 11 control female lambs. Further, ovarian tissue was obtained at 20 months of age from ten androgenized and nine control animals. Tissue was either fixed immediately for histology or cultured for up to 15 days prior to fixing. The number of follicles in haematoxylin and eosin-stained sections was counted and recorded along with the stage of development. Before culture, the total follicle density (follicles/mm3 tissue) was not statistically significantly different between the two types of ovary at either 8 or 20 months of age. Furthermore, there were no statistically significant differences in the density of follicles at each stage of development. However, there was a lower percentage of primordial follicles, but a higher percentage of primary follicles, in biopsies taken at 8 months from androgenized lambs when compared with controls. At 20 months, the proportions of follicles at the primordial and primary stages were not significantly different between the two groups, but this was mainly attributable to an increase in the proportion of growing follicles in biopsies from control animals. Culture of ovarian cortex from 8-month-old lambs resulted in a progressive increase in the proportion of growing follicles when compared with tissue fixed on the day of surgery. However, there was no difference between androgenized and control tissue in the percentage of growing follicles. The increase in the proportion of growing follicles in the cortex of androgenized animals is reminiscent of similar observations in human polycystic ovaries and suggests that excess exposure to androgen in early life plays a part in the accelerated progression of follicle development from the primordial to the primary stage in polycystic ovaries.     

Expression of gap junctional proteins connexin 43, 32, and 26 throughout follicular development and atresia in cows.

Detection of connexin (Cx) proteins has been used as an indicator of the presence of structural and functional gap junctions in tissues. To examine the role of gap junctions during follicular growth and atresia, the presence of three major connexins, Cx43, Cx32, and Cx26, was evaluated in bovine ovaries by using immunohistochemistry and Western immunoblot analysis. Cx43 was not present in primordial follicles, but was present in granulosa cells of primary/secondary and antral follicles. Cx43 also was present on the borders between granulosa cells and the oocyte. Expression of Cx43 increased in healthy developing antral follicles, but decreased during follicular atresia. Cx32 was not present in healthy follicles but was present in granulosa cells of atretic antral, and especially small antral follicles. Cx26 was present in the oocyte of primordial and primary/secondary follicles, and in the granulosa and/or thecal cell layers of healthy antral follicles. The percentage of healthy antral follicles that expressed Cx26 also increased during follicular development, but decreased during atresia. Cx32 and Cx26 also were detected in ovarian blood vessels and in stromal tissues adjacent to the tunica albuginea in some ovaries. The pattern of expression of these Cx indicates that gap junctional proteins may be involved in the control of follicular growth and atresia in cows.     

Regulation of ovarian primordial follicle assembly and development by estrogen and progesterone: endocrine model of follicle assembly

The assembly of the developmentally arrested primordial follicle and the subsequent transition of the primordial follicle to the primary follicle are critical processes in normal ovarian physiology that remain to be elucidated. Ovarian follicles do not proliferate and the primordial follicles present in the neonate represent the total number of gametes available to a female throughout her reproductive life. The primordial follicles are oocytes surrounded by less differentiated squamous granulosa cells and are derived from oocyte nests, and primary follicles are oocytes surrounded by a single layer of cuboidal granulosa cells that have initiated follicle development. Abnormalities in primordial follicle assembly, arrest, and development (i.e. primordial to primary follicle transition) can cause pathological conditions such as premature ovarian failure. In this study newborn rat ovaries were cultured for 7 d. The rate of primordial follicle assembly in vivo was identical with the rate in vitro. Interestingly, the rate of primordial follicle transition to the primary follicle was found to be 3 times greater in culture. This abnormal rate of primary follicle development in culture suggests the primordial follicle does not arrest in development as observed in vivo. To investigate this phenomena newborn rat ovaries were cultured in the presence of progesterone, estradiol or calf serum. Estradiol, progesterone, or calf serum significantly reduced the level of initial primordial to primary follicle transition. Approximately 60% of follicles make the primordial to primary follicle transition in control ovaries and about 30% in treated ovaries. Steroids and calf serum had no effect on the primordial to primary follicle transition in ovaries collected and cultured from postnatal 4-d-old rats, suggesting the effects observed are restricted to the initial wave of primordial to primary follicle transition. Interestingly, progesterone was also found to significantly reduce the rate of primordial follicle assembly. All viable oocytes assembled into primordial follicles in control ovaries and approximately 40% remained unassembled in progesterone-treated ovaries. Progesterone was also found to reduce primordial follicle assembly in vivo with 10% of the total follicles remaining unassembled in progesterone injected neonatal animals. Analysis of cellular apoptosis demonstrated that progesterone inhibited the coordinated oocyte apoptosis required for primordial follicle assembly. The hypothesis developed is that high levels of maternal and fetal steroids prevent premature primordial follicle assembly and primordial to primary follicle transition in the embryo. After birth steroid levels fall dramatically and the primordial follicles are free to assemble and initiate development. These observations suggest a novel role for steroids and the maternal-fetal endocrine unit in the control of ovarian primordial follicle assembly and early follicular development.     

Follicles are found in the ovaries of adolescent girls with Turner's syndrome

Infertility caused by ovarian failure is a characteristic feature in Turner's syndrome. Spontaneous pregnancies are seen in 2-5% of these women, and up to 30% have at least some pubertal development, indicating the presence of follicles in their ovaries in adolescence. It has not been clear at which age the follicles disappear. We analyzed the numbers and densities of follicles in ovarian cortical tissue from nine adolescent girls with Turner's syndrome who came to our clinics after having been informed about the study, with an aim to preserve ovarian tissue for possible infertility treatment later in life. A quarter to one whole ovary was laparoscopically removed for the procedure. Follicles were seen in the biopsy tissue in eight of nine subjects from whom ovarian tissue was laparoscopically obtained, the highest numbers being seen in the youngest girls and in those with mosaicism. In one 17-yr-old girl, no ovarian tissue was found. Follicle density was correlated with serum levels of FSH; individuals with the lowest FSH levels had the highest follicle density. One to 190 follicles were counted in the approximately 0.1-2.0 mm(3) of tissue analyzed, giving a density of 1.5-499 follicles/mm(3) of ovarian cortical tissue. Girls up to the age of 17 had primordial follicles in their ovaries. Three girls, two aged 15 yr and one aged 19, had only secondary follicles, with many being atretic. Our finding that adolescent girls with Turner's syndrome still have follicles in their ovarian cortical tissue raises the possibility of future fertility through cryopreservation of ovarian tissue. However, before such procedures can be recommended for clinical management, it is essential that future studies be performed to determine whether the oocytes retrieved from girls with Turner's syndrome have a normal chromosomal complement.     

Early postnatal methoxychlor exposure inhibits folliculogenesis and stimulates anti-Mullerian hormone production in the rat ovary

Methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl) ethane; MXC] is a chlorinated hydrocarbon pesticide commonly used in the United States as a replacement for DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane]. While MXC is a weak estrogenic compound, its more active, major metabolite [2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane; HPTE] shows estrogenic, anti-estrogenic, or anti-androgenic properties depending on the receptor subtype with which it interacts. Anti-Mullerian hormone (AMH) is a paracrine factor that suppresses initial follicle recruitment in the ovary. Studies have shown the effects of exposure to MXC on adult ovarian morphology and function. However, the effect of exposure to MXC at an early postnatal stage on pre-pubertal follicular development and ovarian AMH production has not been studied. Around postnatal day (P) 4, most of the primordial follicular assembly in rats is complete, and a large number of primordial follicles transition into the primary follicle stage, a process that is inhibited by estrogen. The objective of this study was to examine the effect of early postnatal (P3-P10) MXC exposure on ovarian morphology and size, follicle number, and AMH production in the pre-pubertal (P20) rat ovary and to investigate the effect of HPTE on AMH production in immature rat granulosa cells in vitro. Female rats were injected (s.c.) daily with vehicle (control) or 1, 10, 50, 100, or 500 mg MXC/kg per day (referred to here as 1MXC, 10MXC, and so forth.) between P3 and P10. On P20, uterine and ovarian weights were determined, ovarian histology was examined, and follicles were counted and classified into primordial, primary, secondary, pre-antral, or antral stages using the two largest serial sections at the center of the ovary. Ovarian AMH production was examined using immunohistochemistry and western blot analysis. The effect of HPTE (0.5-25 microM) on AMH production in cultured immature rat granulosa cells was determined by western blot analysis. Ovarian weight was reduced by 50, 100, and 500MXC (P < 0.01). MXC treatment inhibited folliculogenesis. Both 100 and 500MXC had a reduced number of antral follicles (P < 0.05) with a concomitant increase in pre-antral follicles (P < 0.05). Follicle numbers were not significantly affected by 1, 10, or 50MXC. Total follicle number and the number of primordial, primary, or secondary stage follicles were not significantly different in all treatment groups. Immunohistochemistry showed that MXC-treated ovaries had more AMH-positive follicles with stronger AMH immunostaining. Western blot analysis showed that AMH production was 1.6 +/- 0.2, 1.85 +/- 0.6, and 2.2 +/- 0.5 times higher in the 50, 100, and 500MXC ovaries as compared with the control ovaries respectively (P < 0.05). Granulosa cells treated with 1 or 5 microM HPTE had significantly greater AMH production (P < 0.05). These results demonstrate that MXC inhibits early ovarian development and stimulates AMH production directly in the rat ovary. In addition, HPTE was shown to stimulate AMH production in rat granulosa cells. Endocrine disruptors are widespread in the environment, and MXC represents a model endocrine disruptor due to the multiple actions of its metabolites. This study confirms that the endocrine disruptor MXC inhibits follicular development and demonstrates for the first time that MXC and HPTE directly stimulate AMH production in the ovary. This novel finding suggests that elevated AMH may play a role in MXC's inhibitory effect in the ovary.     

Early human preantral follicles have relaxin and relaxin receptor (LGR7), and relaxin promotes their development

The regulatory mechanisms of early follicle development are not clearly understood. Although relaxin is a peptide that controls cell proliferation and differentiation in many tissues, its role in human follicular development is unclear. In this study we cultured slices of human ovarian cortical tissue in the presence and absence of recombinant human relaxin. Ovarian tissue was obtained by biopsy during gynecological laparotomy or laparoscopy (14 women; mean age +/- sem, 29.0 +/- 6.1 yr; range, 17-37 yr). A significantly higher proportion of secondary follicles (14.5% vs. 5.0% in the control group; P < 0.01) and a significantly decreased proportion of primordial follicles (30.1% vs. 47.4% in the control group; P < 0.05) were found in tissues cultured with relaxin for 7 d. Immunocytochemical studies with the anti-C-peptide of prorelaxin and antirelaxin antibodies revealed the localization of relaxin in the oocyte and in flat pregranulosa and granulosa cells of primordial, primary, and secondary follicles. The presence of the relaxin receptor LGR7 was observed in flat pregranulosa and granulosa cells of primordial, primary, and secondary follicles by immunocytochemical and in situ hybridization analyses. These results suggest that relaxin plays a role through its receptor during the early stage of follicle development.     

L’oncofertilité : vers une nouvelle spécialité ?

Although treatment and survival are the primary focus of health-care patients, with cancer survivors living longer it is now appropriate to consider their quality of life after treatment, including the possibility of becoming parents. There are several options for fertility preservation in cancer patients. Even though most of them are still experimental and their efficacy and reliability have not been determined, especially in women. The most successful alternative for female survivors is embryo cryopreservation, an approach not suitable for many single or virgin women or even possible for prepubertal girls. Reports of live birth after transplantation of human ovarian tissue have reinforced the clinical potential of ovarian tissue banking for fertility preservation. Many exciting studies are underway to improve the efficacy and solve the problems with current fertility preservation strategies, especially for in vitro culture of cryopreserved tissue or follicles. Continuous efforts to improve current strategies and to develop new strategies will benefit many women and children who are facing premature ovarian failure and sterility.     

Kit-ligand/stem cell factor induces primordial follicle development and initiates folliculogenesis.

Initiation of folliculogenesis through the induction of primordial follicle development in the ovary has an important role in determining the fertility and reproductive fitness of most mammalian species. The factors that control this critical process are largely unknown. The hypothesis tested in the current study was that kit-ligand/stem cell factor (KL) promotes the initiation and progression of primordial follicle development in the ovary. Ovaries from 4-day-old rats were maintained in organ culture for 5 and 14 days and treated with no factor (control), recombinant kit-ligand (KL), or gonadotropins (FSH and hCG). Follicles in ovarian sections were counted and histologically classified as primordial (stage 0), early primary (stage 1), primary (stage 2), transitional (stage 3), or preantral (stage 4). Fresh ovaries from 4-day-old rats contained 68% primordial follicles (stage 0) and 32% developing follicles (stages 1-4) per section. After 5 and 14 days in culture, section from control ovaries contained approximately 41% and 55%, respectively, developing follicles (stage 1-4) per section due to spontaneous development of primordial follicles. Spontaneous primordial follicle development was completely blocked by ACK-2, a c-kit antibody that blocks KL actions. This observation suggests that endogenous KL is necessary for primordial follicle development in vitro. After 14 days of KL treatment, sections from ovaries contained 17% primordial follicles (stage 0) and 83% developing follicles (stage 1-4) per section demonstrating a dramatic induction of primordial follicle development by KL. Gonadotropins (FSH and hCG) did not induce primordial follicle development but did increase the percentage of preantral follicles (stage 4) per section. This small increase in preantral follicles in response to gonadotropins was blocked by ACK-2 suggesting that KL may in part mediate gonadotropin actions after the initiation of primordial follicle development. Ovaries contained an average of 309+/-10 follicles per section. The total number of follicles per section did not significantly vary between treatments suggesting that the effects of KL were not due to an alteration in follicle number (i.e. survival). KL appears to be one of the first factors identified to be involved in the promotion of primordial follicle development. Results suggest that KL is necessary and sufficient to induce primordial follicle development and initiate folliculogenesis.     

In vitro growth and ovulation of follicles from ovaries of estrogen receptor (ER){alpha} and ER{beta} null mice indicate a role for ER{beta} in follicular maturation

Both estrogen receptor (ER) alpha and beta are expressed within the ovary and lack of either of these receptors affects ovarian function. In this study, the role of ERalpha and ERbeta in folliculogenesis and ovulation was further analyzed. Evaluation of ovarian follicle populations in wild-type and ERbeta knockout (betaERKO) ovaries revealed reduced late antral growth and ovulatory capacity of betaERKO follicles, indicated by reduced numbers of large antral follicles and corpora lutea and increased atresia of large antral follicles. An in vitro culture system was used to study growth, rupture, and luteinization of wild-type, ERalpha knockout (alphaERKO) and betaERKO ovarian follicles. alphaERKO follicles exhibited wild-type-like growth and ovulation rates but an increased capacity to synthesize estradiol. In contrast, betaERKO follicles showed a significant lack of progression from early antral to large antral stage, decreased estradiol production, and reduced ovulation. Expression patterns of several genes involved in follicle maturation and ovulation were analyzed in follicles grown in vitro. Ar, Pgr, and Has2 mRNA expression levels were the same among the three genotypes. However, betaERKO follicles showed reduced expression of Cyp19 mRNA during follicle maturation and reduced Lhcgr and Ptgs2 mRNA expression after human chorionic gonadotropin stimulus. Luteinization occurs normally in alphaERKO and betaERKO follicles, shown by increased progesterone secretion and increased cdkn1b mRNA expression after human chorionic gonadotropin. Collectively, these data indicate that ERbeta, but not ERalpha, plays a direct role in folliculogenesis. ERbeta appears to facilitate follicle maturation from the early antral to the preovulatory stage.     

Anti‐Müllerian hormone (AMH) in female reproduction: is measurement of circulating AMH a useful tool?

Anti-Müllerian hormone (AMH) is a dimeric glycoprotein, a member of the transforming growth factor (TGF) superfamily. It is produced exclusively in the gonads and is involved in the regulation of follicular growth and development. In the ovary AMH is produced by the granulosa cells of early developing follicles and seems to be able to inhibit the initiation of primordial follicle growth and FSH-induced follicle growth. As AMH is largely expressed throughout folliculogenesis, from the primary follicular stage towards the antral stage, serum levels of AMH may represent both the quantity and quality of the ovarian follicle pool. Compared to other ovarian tests, AMH seems to be the best marker reflecting the decline of reproductive age. AMH measurement could be useful in the prediction of the menopausal transition. It could also be used to predict poor ovarian response and possibly the prognosis of in vitro fertilization (IVF) cycles. AMH has been shown to be a good surrogate marker for polycystic ovary syndrome (PCOS). Finally, its use as a marker for granulosa cell tumours has been proposed. A clearer understanding of its role in ovarian physiology may help clinicians to find a role for AMH measurement in the field of reproductive medicine.     

Stage-specific expression of androgen receptor, follicle-stimulating hormone receptor, and anti-Müllerian hormone type II receptor in single, isolated, human preantral follicles: relevance to polycystic ovaries

CONTEXT: Recent evidence indicates that the increase in follicle numbers seen in polycystic ovary syndrome occurs early in folliculogenesis, with androgens being a likely causative candidate. In primates and sheep, androgen excess in utero results in ovarian changes similar to those in polycystic ovary syndrome. There is also increasing interest in the role of anti-Müllerian hormone (AMH) in early folliculogenesis because AMH knockout mice have an early depletion of their stock of primordial follicles. Initiation and early folliculogenesis may therefore be under negative control by AMH and positive control by androgens. OBJECTIVE: Because AMH signals exclusively through its type II receptor (AMHRII), the aim of this study was to determine and colocalize the stage-specific expression of AMHRII, androgen receptor (AR), and FSH receptor (FSHR) mRNA in individual, well-characterized preantral follicles. METHOD: Follicles were isolated from human ovarian cortex obtained from either oophorectomies or cortical biopsies at cesarean section. Expression of AR, FSHR, and AMHRII mRNA was determined using a nested RT-PCR protocol. RESULTS: AR mRNA was not detected in any primordial follicles but was from the transitional stage onward. The number of AR-positive follicles increased at each progressive growth stage. The expression of AR preceded that of FSHR, and only a small percentage of primary follicles expressed FSHR. AMHRII expression was rarely detected. CONCLUSIONS: This is the first study to identify the expression of AR in human transitional follicles. Results suggest a role for androgens in promoting early follicle growth and challenging the hypothesis that AMH exerts a direct, inhibitory effect on follicles at this stage.     

Prolonged survival in culture of preantral follicles from polycystic ovaries

CONTEXT: In polycystic ovary syndrome (PCOS), an increased proportion of follicles leave the primordial (resting) pool and initiate growth. However, there is little evidence for a reduced reproductive life span (early menopause) in women with PCOS, suggesting that the dynamics of follicle growth, and of follicle loss by atresia, is altered in PCOS. OBJECTIVE: The aim of this study was to investigate the possibility that loss of preantral follicles by atresia is reduced in PCOS, leading to prolonged follicle survival. DESIGN: We compared follicle growth in normal and polycystic ovaries using cultures of small ovarian biopsies. SETTING: Tissue samples were obtained at routine laparoscopy from 12 patients with anovulatory PCOS and 16 controls and processed in an ovarian physiology laboratory. MAIN OUTCOME MEASURES: We performed morphometric analysis of follicle population in tissue fixed at time of biopsy (d 0) or after 5, 10, or 15 d in culture. Analyses included assessment of follicle and oocyte diameter, number and proportion of primordial and growing follicles, and number and proportion of atretic follicles. RESULTS: In tissue fixed on d 0, the proportion of healthy growing follicles was, as expected, greater in ovaries from PCOS patients than in normal ovaries (64 vs. 28%; P = 0.0005), but there were no differences between PCOS and normal tissue during culture. The rate of atresia throughout the period of culture in follicles was, however, significantly lower in PCOS tissue (P < 0.0001). After culture, 80% of follicles in normal ovarian tissue were atretic compared with 53% in PCOS biopsies. CONCLUSION: Follicles from polycystic ovaries demonstrate a decreased rate of atresia in culture, suggesting a mechanism for maintaining a larger follicle pool throughout reproductive life.     

In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYP17 in ovaries of immature rats

Growth differentiation factor (GDF)-9 is a cystine knot-containing hormone of the transforming growth factor-beta superfamily produced by the oocyte. In GDF-9 null mice, follicle development is arrested at the primary stage and GDF-9 treatment in vitro enhances preantral follicle growth. Immature female rats were treated with recombinant GDF-9 for 7 or 10 days. At 10 days, treatment with GDF-9 augmented ovarian weights, concomitant with an increase in the number of primary and small preantral follicles by 30 and 60%, respectively. Furthermore, the number of primordial follicles was decreased by 29%, but the number of large preantral follicles was not affected. In contrast, treatment with FSH increased the number of small and large preantral follicles by 36 and 177% but did not influence the number of primary and primordial follicles. Immunoblot analysis showed an increase of CYP17, a theca cell marker, in the ovarian homogenate after treatment with GDF-9 but not FSH. The present results indicate that in vivo treatment with GDF-9 enhances the progression of primordial and primary follicles into small preantral follicles. Thus, GDF-9 treatment could provide an alternative approach to stimulate early follicle development in addition to the widely used FSH that acts mainly on the development of more advanced follicles.     

Serum anti-mullerian hormone levels reflect the size of the primordial follicle pool in mice

Reproductive aging is the decline of female fertility with age. It is caused by the decrease in the number of growing follicles, resulting from primordial follicle pool depletion. Recently, we have shown that anti-Müllerian hormone (AMH) is produced by growing follicles, and studies in women indicate that serum AMH levels decrease with age and correlate with antral follicle count. However, whether serum AMH levels correlate directly with the size of the primordial follicle pool cannot be determined in women. In this work, we describe studies in mice in which we determined the dynamics of ovarian follicles during aging. Furthermore, we describe the development of a mouse AMH ELISA, allowing us to measure AMH levels in mice, for the first time. We observed that serum AMH levels decline with increasing age, whereas expression of AMH in individual growing follicles, studied by immunohistochemistry, did not change with age. Thus, the decline in serum AMH correlates directly with the decline in the number of growing follicles (r = 0.86, P < 0.0001). We observed that the number of growing follicles correlated with the number of primordial follicles (r = 0.93, P < 0.0001). Similarly, we found a strong correlation between AMH levels and number of primordial follicles (r = 0.83, P < 0.0001). In conclusion, serum AMH levels reflect the size of the primordial follicle pool in aging mice. Therefore, AMH is an excellent marker to assess the quantitative aspect of ovarian reserve, which may be useful for women at risk for early ovarian aging such as survivors of childhood cancers.