A homozygous FANCM mutation underlies a familial case of non-syndromic primary ovarian insufficiency.

Primary Ovarian Insufficiency (POI) affects ~1% of women under forty. Exome sequencing of two Finnish sisters with non-syndromic POI revealed a homozygous mutation in FANCM, leading to a truncated protein (p.Gln1701*). FANCM is a DNA-damage response gene whose heterozygous mutations predispose to breast cancer. Compared to the mother's cells, the patients' lymphocytes displayed higher levels of basal and mitomycin C (MMC)-induced chromosomal abnormalities. Their lymphoblasts were hypersensitive to MMC and MMC-induced monoubiquitination of FANCD2 was impaired. Genetic complementation of patient's cells with wild-type FANCM improved their resistance to MMC re-establishing FANCD2 monoubiquitination. FANCM was more strongly expressed in human fetal germ cells than in somatic cells. FANCM protein was preferentially expressed along the chromosomes in pachytene cells, which undergo meiotic recombination. This mutation may provoke meiotic defects leading to a depleted follicular stock, as in Fancm-/- mice. Our findings document the first Mendelian phenotype due to a biallelic FANCM mutation.

A novel action of follicle-stimulating hormone in the ovary promotes estradiol production without inducing excessive follicular growth before puberty.

In cyclic females, FSH stimulates ovarian estradiol (E2) production and follicular growth up to the terminal stage. A transient elevation in circulating FSH and E2 levels occurs shortly after birth. But what could be the action of FSH on the ovary during this period, and in particular how it stimulates ovarian steroidogenesis without supporting terminal follicular maturation is intriguing. By experimentally manipulating FSH levels, we demonstrate in mice that the mid-infantile elevation in FSH is mandatory for E2 production by the immature ovary, but that it does not stimulate follicle growth. Importantly, FSH increases aromatase expression to stimulate E2 synthesis, however it becomes unable to induce cyclin D2, a major driver of granulosa cell proliferation. Besides, although FSH prematurely induces luteinizing hormone (LH) receptor expression in granulosa cells, LH pathway is not functional in these cells to induce their terminal differentiation. In line with these results, supplying infantile mice with a superovulation regimen exacerbates E2 production, but it does not stimulate the growth of follicles and it does not induce ovulation. Overall, our findings unveil a regulation whereby high postnatal FSH concentrations ensure the supply of E2 required for programming adult reproductive function without inducing follicular maturation before puberty.

Membrane associated cancer-oocyte neoantigen SAS1B/ovastacin is a candidate immunotherapeutic target for uterine tumors.

The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation. Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus. A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes. Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement. A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death. The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathway, and tumor cell growth arrest induced by antibody-toxin conjugates suggest therapeutic approaches that would selectively target tumors while limiting adverse drug effects in healthy cells. The SAS1B metalloproteinase is proposed as a prototype cancer-oocyte tumor surface neoantigen for development of targeted immunotherapeutics with limited on-target/off tumor effects predicted to be restricted to the population of growing oocytes.

The ovarian reserve in mammals: a functional and evolutionary perspective.

The constitution and the control of the ovarian reserve is of importance in mammals and women. In particular, the number of primordial follicles at puberty is positively correlated with the number of growing follicles and their response to gonadotropin treatments. The size of this ovarian reserve depends on genes involved in germ cell proliferation and differentiation, sexual differentiation, meiosis, germ cell degeneration, formation of primordial follicles, and on a potential mechanism of self-renewal of germ stem cells. In this review, we present the state of the art of the knowledge of genes and factors involved in all these processes. We first focus on the almost 70 genes identified mainly by mouse invalidation models, then we discuss the most plausible hypothesis concerning the possibility of the existence of germ cell self-renewal by neo-oogenesis in animal species and human, with a special interest for the role of corresponding genes in evolutionary distinct model species. All of the genes pointed out here are candidates susceptible to explain fertility defects such as the premature ovarian failure in human.

Kit ligand and the somatostatin receptor antagonist, BIM-23627, stimulate in vitro resting follicle growth in the neonatal mouse ovary.

In the mammalian ovary, kit ligand (KL), coded by a cAMP-stimulatable gene, is a protein that promotes initiation of follicle growth. The neuropeptide somatostatin (SST) is a small peptide that inhibits cAMP generation in many cell types. Consequently, SST receptor agonists might alter KL production and subsequent follicle growth. The present study was undertaken to look for the existence of a functional SST system in the mouse ovary, to test the effects of the SST receptor 2 (SSTR-2) antagonist BIM-23627 on in vitro folliculogenesis, and to compare them with those of KL, which was demonstrated to stimulate follicle growth in the neonatal rat ovary. Pairs of ovaries from 5-d-old mice were incubated in vitro during 15 d in the presence of either KL or BIM-23627. For every mouse, one ovary was cultured in culture medium (control), and the other ovary was cultured in the presence of either KL or BIM-23627. After 5, 10, and 15 d culture, the ovaries were histologically assessed for the content of primordial, primary, and secondary follicles. The SSTR-2 and -5, but not SST, were identified at the transcriptional and translational (mainly in granulosa cells) levels. Both KL and BIM-23627 triggered a reduction of the percentages of primordial follicles and an increase of the percentages of primary and secondary follicles when compared with control ovaries from the same animal. In conclusion, extraovarian SST, acting through its receptors 2 and 5 present on granulosa cells, may be involved in mouse folliculogenesis by reducing recruitment of resting follicles.

The antral follicle count: practical recommendations for better standardization.

OBJECTIVE: To provide recommendations for the standardized use of the Antral follicle count (AFC) which is used to predict ovarian response to gonadotrophin stimulation during assisted reproductive technology treatment. However, the nature of the follicles that are visualized by ultrasound and the competence of the oocytes held within are largely unknown. In addition, there is considerable variability in the clinical definitions and technical methods used to count and measure antral follicles in both published studies and clinical practice. DESIGN AND SETTING: In December 2007, specialist reproductive medicine clinicians and scientists attended a workshop in an effort to address these issues. Literature concerning the physiology and measurement of ovarian antral follicles was reviewed, clinical and technical considerations regarding antral follicle measurement were discussed, and an operational definition of AFC was developed. PATIENT(S): None. INTERVENTION(S): None. OUTCOME MEASURES: Simple recommendations were established for the standardization of AFC assessment in routine clinical practice. The basic clinical and technical requirements required for AFC evaluation were agreed upon, and a systematic method of measuring and counting antral follicles in routine practice was proposed. CONCLUSION(S): The use of a standardized approach according to the practical recommendations for antral follicle counting as presented is encouraged in future clinical trials and routine practice. The authors also advocate a systematic evaluation of these recommendations as standardized study data become available.

Estimating human ovarian non-growing follicle number: the application of modern stereology techniques to an old problem.

BACKGROUND Previous published reports on the number of non-growing follicles (NGFs) in the human ovary have employed model-based methods for number estimates. These methods are time-intensive, and require correction factors and assumptions that ultimately limit their accuracy. Here, we describe the modification, application and validation of a modern fractionator/optical disector technique for the estimation of human ovarian NGF number. METHODS Forty-eight pairs of normal human ovaries were collected from women (age 8-51 years) undergoing elective bilateral oophorectomy, organ donation, or from autopsy. After gross pathologic examination, systematic random sampling was utilized to obtain tissue for analysis by the fractionator/optical disector method. The precision of individual NGF counts was determined by calculating the observed coefficient of error (OCE). Intra-observer variability and variation in NGF number between ovaries within a pair were also determined. RESULTS The mean OCE was 16.6% with larger variations observed at lower follicle counts. In recount experiments of the same ovary, NGF number estimates varied by 15-29%, except at very low follicle counts where variation was greater, but absolute differences were small. There was no significant difference in NGF number between ovaries within a pair (Wilcoxon signed rank test, P = 0.81). CONCLUSIONS Modern stereology methods provide an unbiased, efficient method for estimating NGF number in the human ovary. Both ovaries within a pair contain similar numbers of NGFs.

Anti-Müllerian hormone concentrations in the follicular fluid of the preovulatory follicle are predictive of the implantation potential of the ensuing embryo obtained by in vitro fertilization.

CONTEXT: The strong relationship between serum anti-Müllerian hormone (AMH) levels and the number of antral follicles supports the use of AMH measurements as a quantitative marker of the ovarian follicular status. Yet, it still is unclear whether the aptitude of an individual follicle to produce AMH reflects its reproductive competence. OBJECTIVE: This study examined the possible relationship between serum or follicular fluid (FF) AMH concentrations and the fate of the ensuing oocytes and embryos obtained by in vitro fertilization-embryo transfer conducted in monodominant follicle cycles. DESIGN AND SETTING: We conducted a prospective study at the University of Paris XI, Assistance Publique-Hôpitaux de Paris, Institut National de la Santé et de la Recherche Médicale U782. PATIENTS: Patients included 118 infertile in vitro fertilization-embryo transfer candidates. INTERVENTIONS: Concentrations of AMH, progesterone, and estradiol were measured in the serum on cycle d 3 and on the day of oocyte pickup (dOPU), and in FF. Cycles were sorted into three sets of three distinct groups according to whether serum d 3, serum dOPU, and FF AMH concentrations were 30th centile or below (low AMH), between the 31st and the 70th centiles (average AMH), or above the 70th centile (high AMH) of measurements. MAIN OUTCOME MEASURE: Clinical pregnancy and embryo implantation rates were assessed. RESULTS: Clinical pregnancy rates (5.7, 20.0, and 39.5%, respectively; P < 0.002) and embryo implantation rates (11.8, 30.8, and 65.4, respectively; P <0.001) were markedly different among the low, moderate, and high FF AMH groups but not among the serum (d 3 or dOPU) AMH groups. Fertilization rates and embryo morphology remained similar irrespective of AMH concentrations in the serum or in FF. Incidentally, FF AMH concentrations were negatively correlated with FF progesterone (r = -0.27; P <0.003) and FF estradiol (r = -0.21; P <0.02) concentrations. CONCLUSIONS: Concentrations of AMH in the FF, but not in the serum, constitute a useful follicular marker of embryo implantation and are negatively related to FF progesterone and estradiol concentrations.

Oocyte bone morphogenetic protein 15, but not growth differentiation factor 9, is increased during gonadotropin-induced follicular development in the immature mouse and is associated with cumulus oophorus expansion.

Bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9 are oocyte-secreted growth factors that are critical local regulators of ovarian function and may be involved in preovulatory cumulus expansion. As cumulus expansion occurs in response to the ovulatory surge, the present study was designed: 1) to investigate whether GDF9 and BMP15 are regulated by gonadotropins in the mouse ovary; and 2) to visualize changes in both GDF9 and BMP15 immunostaining in response to gonadotropins. Immature 21-day-old mice were sequentially treated with recombinant human FSH (r-hFSH), 5 IU daily, at Days 21, 22, and 23 of life, then injected with 5 IU hCG at Day 24 of life. In response to r-hFSH, steady-state Bmp15 mRNA expression levels increased in both total ovaries and cumulus-oocyte complexes, whereas Gdf 9 mRNA levels did not. In addition, BMP15 protein levels increased in total ovaries. The GDF9 immunostaining was exclusively seen in growing oocytes in both control and gonadotropin-treated mice, whereas that of BMP15, which was also primarily seen in growing oocytes, exhibited important changes in response to gonadotropins. Strong BMP15 immunostaining was observed in the follicular fluid of atretic antral follicles after FSH treatment and in expanded, but not in compact, cumulus cells after hCG. The present results show for the first time that BMP15 levels increase during gonadotropin-induced follicular development, in parallel with oocyte maturation, and that this local factor is likely involved in cumulus expansion as previously suggested by studies in Bmp15-null mice.

Sequential gonadotropin treatment of immature mice leads to amplification of transforming growth factor beta action, via upregulation of receptor-type 1, Smad 2 and 4, and downregulation of Smad 6.

The present study was designed to establish the cellular localization and expression of transforming growth factor beta (TGFbeta) signaling pathway components, including TGFbeta1 and beta2; TGFbeta receptors type I (TbetaRI) and II (TbetaRII); and Smads 2, 3, 4, and 6 during gonadotropin-induced follicular maturation and ovulation in the mouse ovary. Immature 21-day-old mice were sequentially treated with recombinant human FSH, 5 IU daily for 3 days, and hCG once at Day 24 of life. Immunohistochemical experiments revealed a TGFbeta1 staining in granulosa cells (GC) and theca interna cells (TIC) as well as in oocytes, whereas that of TGFbeta2 was mainly localized in oocytes and GC. Strong immunostaining for both TbetaRI and -RII was observed in the TIC and, to a lesser extent, in GC. Whereas oocytes did not exhibit any staining for TbetaRII, their TbetaRI immunostaining was strong. Smads were detected in oocytes, GC, and luteal cells and in a lesser amount in TIC; the immunostaining for Smad 4 was the strongest. Western blotting and reverse transcription-polymerase chain reaction analyses indicated that, in response to gonadotropins, TGFbeta2, TbetaRI, Smad 2 and Smad 4 mRNA and protein levels increased, while those of Smad 6 decreased in ovarian homogenates. In conclusion, these results show that, in a model of immature mouse exposed to a sequential gonadotropin treatment, FSH and LH increased the expression of the TGFbeta signaling system through the increase of TGFbeta2, TbetaRI, stimulatory Smad 2, and common Smad 4 expression, which occurred concomitantly with a decrease of the inhibitory Smad 6 expression.

[Invasive hydatiform mole in a postmenopausal woman]. / Môle hydatiforme invasive chez une patiente ménopausée.

Gestational trophoblastic disease occurs rarely in postmenopausal women. We report the case of a 51 year-old postmenopausal woman with an invasive complete mole. Invasive mole should be distinguished from choriocarcinoma, by a thorough sampling showing infiltrative molar villi associated with a prominent trophoblastic proliferation. Gestational trophoblastic diseases in postmenopausal women can represent malignant changes of trophoblastic remnants of a prior pregnancy after a period of latency or correspond to a possible current pregnancy as demonstrated by an ovarian corpus luteum of pregnancy in our patient. The unusual finding in our case is that the gestational trophoblastic disease follows a pregnancy occurring after a biologically confirmed menopause.

Expression of tumor necrosis factor-alpha-related apoptosis-inducing ligand and its receptors in rat testis during development.

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor-alpha family of cytokines that is known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, the presence of TRAIL and its receptors was investigated in the rat testis during development. TRAIL and its receptors were immunolocalized to the different testicular cell types. TRAIL and its receptors were also identified in the rat testis in terms of protein and mRNA. Our immunohistochemical studies indicate that TRAIL, DR5/TRAIL-R2, and DcR2-TRAIL-R4 are detected in Leydig cells, whereas ligand and all receptors are localized in germ cells. TRAIL was permanently immunodetected in germ cells from the fetal stage to adulthood, whereas its receptors were immunolocalized exclusively in postmeiotic germ cells. The expression of TRAIL and receptor mRNAs was consistent with the immunodetection of TRAIL and receptor proteins. Indeed, TRAIL ligand mRNA was also identified in the rat testis from the fetal stage to adulthood. The mRNAs of the death receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2, were weakly detected during the perinatal period and increased from the pubertal stage to adulthood. The mRNAs of the decoy receptors, DcR1 and DcR2, were present in the rat testis at all ages studied, but the DcR2/TRAIL-R4 mRNa level was higher from the pubertal period to adulthood. Together, the present findings demonstrate that 1) TRAIL and its receptors are expressed in the testis during normal development, and 2) TRAIL protein is present in the different germ cell types, whereas its receptors were predominantly detected in the postmeiotic germ cells.