PATL2 regulated the apoptosis of ovarian granulosa cells in patients with PCOS.

AIM: PCOS often showed abnormal follicular development. Previous studies have found that the increased apoptosis of granulosa cells (GCs) is one of the key factors leading to follicular dysplasia. It has been found that the decrease or deletion of PATL2 function can significantly inhibit the development and maturation of human oocytes. We found that PATL2 was also expressed in human ovarian GCs, suggesting that PATL2 may be involved in the regulation of related biological events in GCs. This study aims to explore the function of PATL2 on regulation of GCs apoptosis, and the potential role of PATL2 in the development of PCOS-related abnormal follicles. MATERIALS AND METHODS: The follicular GCs of PCOS patients and normal ovulating female patients were collected. Moreover, human granular cell line (KGN) was used for in vitro experiments. RESULTS: (1) The maturation rate and fertilization rate of oocytes in the PCOS group were significantly lower than those in the normal control group (p＜0.05). (2) Flow cytometry and TUNEL staining showed that the apoptosis level of GCs in the PCOS group was significantly increased. (3) Immunofluorescence and Western Blot showed that the PATL2 expression level of GCs in the PCOS group was significantly reduced. (4) Knocking down the expression of PATL2 by siRNA significantly prevented the apoptosis of GCs. CONCLUSIONS: Reduced PATL2 could resulted in the increased apoptosis level of ovarian GCs, which might be closely related to the occurrence and development of abnormal follicles in PCOS.

The phenotype of a pig with monosomy X resembling Turner syndrome symptoms: a case report.

The partial or complete loss of one X chromosome in humans causes Turner syndrome (TS), which is accompanied by a range of physical and reproductive pathologies. This article reports similarities between the phenotype of a pig with monosomy X and the symptoms of TS in humans. Born as the offspring of a male pig carrying a mutation in an X-chromosomal gene, ornithine transcarbamylase (OTC), the female pig (37,XO) was raised to the age of 36 months. This X-monosomic pig presented with abnormal physical characteristics including short stature, micrognathia, and skeletal abnormalities in the limbs. Furthermore, the female did not exhibit an estrous cycle, even after reaching the age of sexual maturity, and showed no ovarian endocrine activity except for an irregular increase in blood 17ß-estradiol levels, which was seemingly attributable to sporadic follicular development. An autopsy at 36 months revealed an undeveloped reproductive tract with ovaries that lacked follicles. These data demonstrated that the growth processes and anatomical and physiological characteristics of an X-monosomic pig closely resembled those of a human with TS.

Novel mutations in ZP1, ZP2, and ZP3 cause female infertility due to abnormal zona pellucida formation.

The human zona pellucida (ZP) is an extracellular glycoprotein matrix composed of ZP1, ZP2, ZP3, and ZP4 surrounding the oocyte, and it plays an important role in sperm-egg interactions during fertilization. Structural and functional changes in the ZP can influence the process of fertilization and lead to female infertility. Previous studies have identified mutations in ZP1, ZP2, and ZP3 that lead to female infertility caused by oocyte degeneration, empty follicle syndrome, or in vitro fertilization failure. Here we describe seven patients from six independent families who had several abnormal oocytes or suffered from empty follicle syndrome, similar to the previously reported phenotypes. By whole-exome sequencing and Sanger sequencing, we identified several novel mutations in these patients. These included three homozygous mutations in ZP1 (c.1708G > A, p.Val570Met; c.1228C > T, p.Arg410Trp; c.507del, p.His170Ilefs*52), two mutations in a compound heterozygous state in ZP1 (c.1430 + 1G > T, p.Cys478X and c.1775-8T > C, p.Asp592Glyfs*29), a homozygous mutation in ZP2 (c.1115G > C, p.Cys372Ser), and a heterozygous mutation in ZP3 (c.763C > G, p.Arg255Gly). In addition, studies in CHO cells showed that the mutations in ZP1, ZP2, and ZP3 might affect the corresponding protein expression, secretion, and interaction, thus providing a mechanistic explanation for the phenotypes. Our study expands the spectrum of ZP gene mutations and phenotypes, and provides a further understanding of the pathogenic mechanism of ZP gene mutations in vitro.

JNK/STAT signalling pathway is involved in fluoride-induced follicular developmental dysplasia in female mice.

Excessive fluoride (F) intake decreases the development of potential oocytes by inducing oxidative stress and apoptosis in female mice in our previous study. This study aims to investigate the underlying mechanisms of F-induced follicular developmental dysplasia. Pathomorphological changes in the ovary tissues were observed under light and transmission electron microscopes. DNA damage and proliferation in granulosa cells were analysed by TUNEL staining and BrdU measurement. The protein expression of cell proliferation related regulatory factors including JNK, STAT3, STAT5, CDK2, CDK4, PCNA and Ki67 in the ovary tissues was measured by immunohistochemistry and Western blot analyses. Results indicated that the structure of granulosa cells in the ovary was seriously damaged by excessive F, evident by the swollen endoplasmic reticulum, mitochondria with vacuoles and nucleus shrinkage. F treatment also considerably enhanced the apoptosis and inhibited the proliferation of granulosa cells. The number of granulosa cells around the oocyte decreased after F treatment. The expression levels of STAT3, CDK2, CDK4 and Ki67 in the ovary tissues were up-regulated, and STAT5 and PCNA did not change significantly after F treatment, whereas JNK expression was down-regulated with increasing F dose. In summary, changes in the expression levels of JNK, STAT3, STAT5, CDK2, CDK4, PCNA and Ki67 in the JNK/STAT signalling pathway are involved in F-induced follicular dysplasia in the ovary.

Ovotesticular Disorder of Sex Development Presenting as an Acute Scrotum.

Hermaphroditism is known as ovotesticular disorder of sex development. A 14-year-old boy was admitted with right acute scrotum. Exploration revealed tunica rupture and hematoma, with no viable tissue. After 1 month, he was admitted again with left hemiscrotal pain. Microscopic examination of the left gonad demonstrated foci of hemorrhagic cysts, primordial follicles, and regions of seminiferous tubules. We preserved a testicular tissue and the ovarian part was extracted completely. Long-term follow-up with his hormonal profile is reported. This is a case of ovotesticular disorder presented with acute scrotum and we also tried to reduce long-term hormone therapy, with preservation of testicular part.

Mutation in the protease cleavage site of GDF9 increases ovulation rate and litter size in heterozygous ewes and causes infertility in homozygous ewes.

Litter size (LS) in sheep is determined mainly by ovulation rate (OR). Several polymorphisms have been identified in the growth differentiation factor 9 (GDF9) gene that result in an increase in OR and prolificacy of sheep. Screening the databank of the Brazilian Sheep Breeders Association for triplet delivery, we identified flocks of prolific Ile de France ewes. After resequencing of GDF9, a point mutation (c.943C>T) was identified, resulting in a non-conservative amino acid change (p.Arg315Cys) in the cleavage site of the propeptide. This new allele was called Vacaria (FecG(v) ). A flock of half-sib ewes was evaluated for OR in the first three breeding seasons, and Vacaria heterozygotes had higher OR (P < 0.001), averaging 2.1 ± 0.1 when compared to 1.2 ± 0.1 in wild-type ewes. The OR was also influenced by age, increasing in the second and third breeding seasons (P < 0.001). In flocks segregating this allele, the LS was higher in mutant sheep (P < 0.001), averaging 1.61 ± 0.07 in heterozygotes and 1.29 ± 0.03 in wild-type ewes. Analysis of homozygote reproductive tract morphology revealed uterine and ovarian hypoplasia. Ovarian follicles continue to develop up to small antral stages, although with abnormal oocyte morphology and altered arrangement of granulosa cells. After the collapse of the oocyte in most follicles, the remaining cells formed clusters that persisted in the ovary. This SNP is useful to improve selection for dam prolificacy and also as a model to investigate GDF9 post-translation processing and the fate of the follicular cells that remain after the oocyte demise.

Follicular ring sign: a simple sonographic sign for early diagnosis of ovarian torsion.

The follicular ring sign is a novel, easily identifiable sonographic finding of ovarian torsion consisting of hyperechoic rings around the antral follicles. In a retrospective analysis of 15 consecutive patients with torsion undergoing surgery, the follicular ring sign was frequently present, starting early in the disease course. This sign could be valuable for accurate and early diagnosis of torsion in the absence of specific conventional sonographic features.

The prevalence of genuine empty follicle syndrome.

OBJECTIVE: To describe the prevalence of "genuine" empty follicle syndrome (EFS) and "false" EFS at assisted reproductive technology (ART). DESIGN: Retrospective cohort. SETTING: Large private fertility center. PATIENT(S): A total of 12,359 patients who underwent ART between 2004 and 2009. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The failure to recover an oocyte during oocyte retrieval at ART, with and without a detectable serum ß-hCG on the day of retrieval. RESULT(S): Two cases of genuine EFS and nine cases of false EFS were identified in the cohort examined. The prevalence of genuine EFS was 0.016%, and the prevalence of false EFS was 0.072%. Only two out of 11 cases of EFS were considered genuine. CONCLUSION(S): Genuine EFS is a rare occurrence. Because this syndrome tends to recur with dismal pregnancy rates at ART, continued identification and further investigation of the syndrome are needed.

Human ovarian follicular development: from activation of resting follicles to preovulatory maturation.

By integrating morphometrical and endocrinological data, as well as biological effects of various molecules synthesized by the human follicle, we propose a dynamic view of the follicle growth within the human ovary. Folliculogenesis starts with entry of resting follicles into the growth phase, a process where the kit system plays a key role. Several months are required for a new growing follicle to reach the preantral stage (0.15mm), then 70 additional days to reach the size of 2mm. Early growing follicle growth is regulated by subtle interactions between follicle-stimulating hormone (FSH) and local factors produced by theca and granulosa cells (GCs), as well as the oocyte. From the time they enter the selectable stage during the late luteal phase, follicles become sensitive to cyclic changes of FSH in terms of granulosa cell proliferation. During the early follicular phase, the early selected follicle grows very quickly and estradiol is present in the follicular fluid. However, the total steroid production remains moderate. From the mid-follicular phase, the preovulatory follicle synthesizes high quantities of estradiol, then after the mid-cycle gonadotropin surge, very large amounts of progesterone. At this stage of development, the responsiveness of the follicle to gonadotropins is maximum, especially to luteinizing hormone (LH) that triggers granulosa wall dissociation and cumulus expansion as well as oocyte nuclear maturation. Thus, as the follicle develops, its responsiveness to gonadotropins progressively increases under the control of local factors acting in an autocrine/paracrine fashion.

Recent developments in identifying genetic determinants of premature ovarian failure.

Premature ovarian failure (POF) is defined as cessation of menstruation and associated elevation of gonadotropin levels (FSH >40 IU/l) as a result of decreased ovarian function prior to the age of 40. An estimated 1% of the population is affected before age 40, with 0.1% affected prior to age 30. Although the causes for POF are many, the majority of POF cases have idiopathic etiologies. In an effort to investigate potential mechanisms of the disease, genetic determinants of POF have received particular attention in recent years. Transgenic mouse models have been instrumental in the discovery of novel genetic determinants of gonadal development and failure and have informed research identifying mutations in women with POF. Here, we review recent developments in identifying genetic determinants of POF.

Selective expression of KrasG12D in granulosa cells of the mouse ovary causes defects in follicle development and ovulation.

Activation of the RAS family of small G-proteins is essential for follicle stimulating hormone-induced signaling events and the regulation of target genes in cultured granulosa cells. To analyze the functions of RAS protein in granulosa cells during ovarian follicular development in vivo, we generated conditional knock-in mouse models in which the granulosa cells express a constitutively active KrasG12D. The KrasG12D mutant mice were subfertile and exhibited signs of premature ovarian failure. The mutant ovaries contained numerous abnormal follicle-like structures that were devoid of mitotic and apoptotic cells and cells expressing granulosa cell-specific marker genes. Follicles that proceeded to the antral stage failed to ovulate and expressed reduced levels of ovulation-related genes. The human chorionic gonadotropin-stimulated phosphorylation of ERK1/2 was markedly reduced in mutant cells. Reduced ERK1/2 phosphorylation was due, in part, to increased expression of MKP3, an ERK1/2-specific phosphatase. By contrast, elevated levels of phospho-AKT were evident in granulosa cells of immature KrasG12D mice, even in the absence of hormone treatments, and were associated with the progressive decline of FOXO1 in the abnormal follicle-like structures. Thus, inappropriate activation of KRAS in granulosa cells blocks the granulosa cell differentiation pathway, leading to the persistence of abnormal non-mitotic, non-apoptotic cells rather than tumorigenic cells. Moreover, those follicles that reach the antral stage exhibit impaired responses to hormones, leading to ovulation failure. Transient but not sustained activation of RAS in granulosa cells is therefore crucial for directing normal follicle development and initiating the ovulation process.

Maternal exposure to polycyclic aromatic hydrocarbons diminishes murine ovarian reserve via induction of Harakiri.

Maternal smoking during pregnancy is associated with a variety of adverse neonatal outcomes including altered reproductive performance. Herein we provide molecular evidence for a pathway involved in the elimination of the female germline due to prepregnancy and/or lactational exposure to polycyclic aromatic hydrocarbons (PAHs), environmental toxicants found in cigarette smoke. We show that ovaries of offspring born to mice exposed to PAHs contained only a third of the ovarian follicle pool compared with offspring of unexposed female mice. Activation of the cell death pathway in immature follicles of exposed females was mediated by the aryl hydrocarbon receptor (Ahr), as ovarian reserve was fully rescued by maternal cotreatment with the Ahr antagonist, resveratrol, or by inactivation of the Ahr gene. Furthermore, in response to PAHs, Ahr-mediated activation of the harakiri, BCL2 interacting protein (contains only BH3 domain), was necessary for execution of cell death. This pathway appeared to be conserved between mouse and human, as xenotransplanted human ovarian cortex exposed to PAHs responded by activation of the identical cell death cascade. Our data indicate that maternal exposure to PAHs prior to pregnancy and/or during lactation compromises ovarian reserve of female offspring, raising the concern about the transgenerational impact of maternal smoking on ovarian function in the human.

Retrieval of zona-free immature oocytes in a woman with recurrent empty follicle syndrome: a case report.

BACKGROUND: Different ovulation trigger methods such as gonadotropin releasing hormone-agonist (GnRH-a) and recombinant human chorionic gonadotropin (r-hCG) plus rescue oocyte retrieval might reveal oocytes in patients with recurrent empty follicle syndrome. CASE: Endogenous luteinizing hormone was triggered with a GnRH-a (Buserelin [Suprefact pro-injection, Aventis-Pharma, Turkey], 250 microg subcutaneously) in a GnRH antagonist (Cetrorelix [Cetrotide 0.25, SeronoTurkey], 0.25 mg/d, starting on day 6), down-regulated cycle. At the first scheduled retrieval, 3 cumulus-oocytecorona complexes were recovered from the left ovary. During chemical denudation with hyaluronidase, 2 of them underwent lysis. The third was a zona-free, germinalvesicle-stage oocyte after mechanical denudation. Oocyte pickup was stopped, and recombinant human chorionic gonadotropin (hCG) (250 microg subcutaneously) was injected. Five cumulus-oocyte-corona complexes were retrieved from the right ovary 24 hours after rescue with recombinant hCG. Only mechanical denudation was done, and 4 zona-free oocytes with germinal vesicle breakdown were seen. All oocytes underwent intracytoplasmic sperm injection, and none of them were fertilized. CONCLUSION: Oocyte maturation defects should be included in etiologic mechanisms for counseling patients with empty follicle syndrome.

[Premature ovarian failure: present aspects]. / Falência ovariana prematura: aspectos atuais.

Premature ovarian failure occurs in approximately 1:1000 women before 30 years, 1:250 by 35 years and 1:100 by the age of 40. It is characterized by primary or secondary amenorrhea and cannot be considered as definitive because spontaneous conception may occur in 5 to 10% of cases. In 95% of cases, premature ovarian failure is sporadic. The known causes of premature ovarian failure include chromosomal defects, autoimmune diseases, exposure to radiation or chemotherapy, surgical procedures, and certain drugs. Frequently, however, the etiology is not clear and these cases are considered to be idiopathic. Premature ovarian failure is defined by gonadal failure and high serum follicle-stimulating hormone (FSH) levels. Clinical approach includes emotional support, hormonal therapy with estrogens and progesterone or progestogens, infertility treatment, and prevention of osteoporosis and potential cardiovascular risk.

Falência ovariana prematura: aspectos atuais / Premature ovarian failure: present aspects

A falência ovariana prematura (FOP) acomete aproximadamente 1:1000 mulheres antes dos 30 anos, 1:250 em torno dos 35 anos e de 1:100 aos 40 anos. Manifesta-se como amenorréia primária ou amenorréia secundária, não podendo ser considerada definitiva em todas as pacientes, uma vez que a concepção espontânea pode ocorrer em até 5-10 por cento das FOP. Na maioria dos casos apresenta-se na forma esporádica, pois apenas 5 por cento apresentam história familial. Entre as causas conhecidas estão as alterações cromossômicas, dos genes ligados ao cromossomo X e cromossomos autossômicos, doenças autoimunes, alterações tóxicas e iatrogênicas. Com relativa freqüência, a causa etiológica não é obtida, sendo então denominada de idiopática. O diagnóstico da FOP é feito baseado na história clínica e níveis elevados do hormônio folículo estimulante (FSH), sendo posteriormente investigadas as causas mais específicas. O manejo clínico visa o suporte emocional, o tratamento hormonal com estrogênios e progestogênios, a abordagem da infertilidade e a prevenção de co-morbidades como a osteoporose e potencial maior risco cardiovascular.

Premature ovarian failure occurs in approximately 1:1000 women before 30 years, 1:250 by 35 years and 1:100 by the age of 40. It is characterized by primary or secondary amenorrhea and cannot be considered as definitive because spontaneous conception may occur in 5 to 10 percent of cases. In 95 percent of cases, premature ovarian failure is sporadic. The known causes of premature ovarian failure include chromosomal defects, autoimmune diseases, exposure to radiation or chemotherapy, surgical procedures, and certain drugs. Frequently, however, the etiology is not clear and these cases are considered to be idiopathic. Premature ovarian failure is defined by gonadal failure and high serum follicle-stimulating hormone (FSH) levels. Clinical approach includes emotional support, hormonal therapy with estrogens and progesterone or progestogens, infertility treatment, and prevention of osteoporosis and potential cardiovascular risk.

Meiotic abnormalities in in vitro-matured marmoset monkey (Callithrix jacchus) oocytes: development of a non-human primate model to investigate causal factors.

BACKGROUND: Meiotic abnormalities are thought to be a major causal factor of low embryo development rates, for embryos developed from in vitro-matured oocytes. A new non-human primate model, in the common marmoset, is being developed to facilitate investigation of the mechanisms involved. METHODS: Oocytes were dissected from antral follicles from three size classes. They were allowed to mature in vitro for only 24 h, in order to focus the investigation on the rapidly maturing oocytes. Chromosome spreads were visualized with Giemsa staining, and spindles /chromosomes with fluorescently labelled anti-alpha-tubulin antibody combined with a DNA fluorochrome. RESULTS: 40% of the oocytes had reached metaphase II (MII) after 24 h. Of the MII oocytes selected for karyotyping, readable chromosomal spreads were obtained from 64%. Overall, 63% of these presented a normal haploid chromosome number of 23,X, with all abnormal karyotypes occurring in the oocytes from small follicles. For another group of MII oocytes, where meiotic spindles were visualized, only half of the MII oocytes displayed well-formed spindles and apparently correct chromosomal alignment. CONCLUSIONS: This work provides the first information on the normal and aneuploid MII meiotic chromosome sets for the marmoset oocyte, and demonstrates a high rate of chromosomal and spindle abnormality among rapidly maturing oocytes from small antral follicles.

Subfertility and defective folliculogenesis in female mice lacking androgen receptor.

The roles of the androgen receptor (AR) in female fertility and ovarian function remain largely unknown. Here we report on the generation of female mice lacking AR (AR(-/-)) and the resulting influences on the reproductive system. Female AR(-/-) mice appear normal but show longer estrous cycles and reduced fertility. The ovaries from sexually mature AR(-/-) females exhibited a marked reduction in the number of corpora lutea. After superovulation treatment, the AR(-/-) ovaries produced fewer oocytes and also showed fewer corpora lutea. During the periovulatory period, an intensive granulosa apoptosis event occurs in the AR(-/-) preovulatory follicles, concurrent with the down-regulation of p21 and progesterone receptor expression. Furthermore, the defective conformation of the cumulus cell-oocyte complex from the AR(-/-) females implies a lower fertilization capability of the AR(-/-) oocytes. In addition to insufficient progesterone production, the diminished endometrial growth in uteri in response to exogenous gonadotropins indicates that AR(-/-) females exhibit a luteal phase defect. Taken together, these data provide in vivo evidence showing that AR plays an important role in female reproduction.

Mutations in the genes for oocyte-derived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep (Ovis aries).

Belclare and Cambridge are prolific sheep breeds, the origins of which involved selecting ewes with exceptionally high litter size records from commercial flocks. The variation in ovulation rate in both breeds is consistent with segregation of a gene (or genes) with a large effect on this trait. Sterile ewes, due to a failure of normal ovarian follicle development, occur in both breeds. New naturally occurring mutations in genes for the oocyte-derived growth factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are described. These mutations are associated with increased ovulation rate in heterozygous carriers and sterility in homozygous carriers in both breeds. This is the first time that a mutation in the gene for GDF9 has been found that causes increased ovulation rate and infertility in a manner similar to inactivating mutations in BMP15, and shows that GDF9 is essential for normal folliculogenesis in sheep. Furthermore, it is shown, for the first time in any species, that individuals with mutations in both GDF9 and BMP15 have a greater ovulation rate than sheep with either of the mutations separately.

Histochemical study of the extracellular matrix components in the follicular wall of induced polycystic ovaries

Numerous models have been developed to study polycystic ovarian syndrome in rats. In the present study, the syndrome was induced by exposure to constant light. The histological structure and differential distribution of extracellular matrix (ECM) fibers as well as the glycosaminoglycans (GAGs) content and composition of the ovarian follicular wall of rats with polycystic syndrome were evaluated. Histochemical differences were observed in the graunlosa and theca externa of follicular cysts when compared to normal preovulatory follicles. The colagen content of the theca externa of follicular cysts, quantified by the picrosirius method, was higher than in the controls. The neural carbohydrate and acidic GAC levels were lower in the granulosa and higher in the theca externa of cyst follicles than in control ovaries. Histomorphometrically, the follicular diameter was both a convenient and appropriate measurement for describing the cyst status; there were no differences in the thickness of each follicular layer. In conclusion, differences in the components of ECM were observed in the follicular wall of ovarian cysts compared eith normal preovulatory follicles. Howere, sinde these changes did not occur uniformly in all layers of the follicular wall, their role in cyst development remains to be established.