Genetic Study Of Premature Ovarian Failure And Safety Study Of PTEN Inhibitor On Activation Of Primordial Follicles In Ovary

Chapter Ⅰ. Genetic Study of Premature Ovarian Failure Section Ⅰ. Karyotype Analysis of281Chinese Han Women with Premature Ovarian FailureOBJECTIVE:The purpose of the present study is to analyze the chromosome karyotypes of premature ovarian failure (POF) in Chinese Han women and to study the association between abnormal type and site of chromosome with pathogenesis of POF.METHODS:Peripheral blood was taken from281Chinese Han POF patients for lymphocytes cultivation and karyotype analysis was performed by G-banding technique. A minimum of30cells per patient were analyzed. Informed written consents were obtained from all participants.RESULTS:(DAmong the281Chinese Han POF patients, there were235cases with normal chromosomal karyotype (83.6%,235/281); And46patients were identified with abnormal chromosomal karyotype (16.4%,46/281).(2)Among the abnormal karyotypes, there were34cases involved with X chromosome only,6subjects with autosomes only,4patients with chromosome translocation between X chromosome and autosome,1with46, XY and1with45, X/46,XY. X chromosome is the most common involved.(3)There were19cases with breakage, rearrangement or deletion in X chromosome, especially in Xq13-27.(4)The incidence of abnormal chromosome karyotype in POF patients with primary amenorrhea is26.3%(15/57), significantly higher than that in POF subjects with secondary amenorrhea (13.8%,31/224). CONCLUSIONS:(1) Chromosomal abnormality is one of the most important etiologies of POF.(2)X chromosome is the most common involved in the abnormal chromosome karyotype, especially in Xq13-27.(3)It is very necessary to take chromosome karyotype examination in patients with POF especially patients with primary amenorrhea. Section II. Mutational Analysis of PTEN in Chinese Women with Premature Ovarian FailureOBJECTIVE:PTEN (phosphatase and tensin homolog deleted on chromosome10), originally identified as a tumor suppressor gene, has been recently demonstrated to play a role in the activation of primordial follicles. Lack of expression of the Pten gene in murine oocytes leads to premature ovarian failure (POF) due to accelerated depletion of the follicular pool. Mutational analysis of the PTEN gene in Chinese POF patients has not been done. The purpose of the present study is to investigate whether mutations in the PTEN gene may contribute to the etiology of Chinese idiopathic POF.METHODS:We recruited161Chinese women with secondary amenorrhea associated with POF based on the phenotype of Pten knock-out mice and sequenced the PTEN gene on an automated sequencer.RESULTS:Three known single-nucleotide polymorphisms (SNPs) were identified (rs11202592in the5’ UTR region, rs34051577in intron4, and rs17849090in exon7). No plausible pathogenic mutations were detected. No additional SNPs or mutations were found in exons of the PTEN gene.CONCLUSIONS:Our findings suggest that the PTEN gene may not be a common cause of Chinese women with POF. Future studies in large cohorts from different ethnic populations are warranted. Section Ⅲ. Mutational analysis of SKP2and P27in Chinese Han women with premature ovarian failureOBJECTIVE:P27and SKP2, a major regulator of P27, play a crucial role in ovarian function in mice. Both p27-deficient and.Skp2-deficient female mice developed premature ovarian failure (POF). Mutational analysis of the SKP2gene in POF patients has not been done. The purpose of the present study is to investigate whether mutations in the SKP2and P27gene may contribute to the etiology of Chinese idiopathic POF.METHODS:We recruited200Chinese Han POF women and sequenced both the SKP2and P27gene on an automated sequencer. The coding region of SKP2and P27were examined in200Chinese women with POF, compared with200control volunteers.RESULTS:One known single nucleotide polymorphism (SNP), rs61755301(c.846A>G) in exon7of SKP2and one known SNP (rs16908375, c.165G>A) in exon1of P27were identified respectively. Comparisons of genotype and allelic frequencies between POF cases and the general population showed no significant differences of the two SNPs. No additional SNPs or mutations were found.CONCLUSIONS:The present study is the first to discover variants occurring in SKP2in association with POF. The results suggested that mutations in SKP2and P27are not common in Chinese Han women with POF. Section Ⅳ. Genome-wide Association Study of Chinese Premature Ovarian FailureOBJECTIVE:Premature ovarian failure (POF) is a complex heritable disorder known to be caused by chromosomal abnormalities and to date a limited number of known mutations. We sought to identify additional genetic loci associated with POF by performing the first large-scale genome-wide association study (GWAS).METHODS:GWAS, using Affymetrix SNP6.0chip, was conducted in an initial discovery set of391well-documented Chinese Han POF patients, compared with895unrelated Chinese female controls. A replication study on the most significant loci was then performed in an independent set of400cases and800controls.RESULTS:This GWAS, involving by far the largest sample of POF cases accumulated to date, revealed heretofore unrecognized association between POF and a novel genetic locus or region of unknown nature on8q22.3. Replication of eight single-nucleotide polymorphisms (SNPs)(rs10464815, rs10808365, rs3847152, rs3847153, rs3847154, rs3843552,rs10955242, rs3843555)(P≤3.86x10-6) was confirmed in verification sets. No specific candidate gene was found in the immediate region of8q22.3.CONCLUSIONS:Suggestive significant associations were observed at8q22.3. We speculate existence of a long-distance regulatory region that has relevance to the control of ovarian differentiation or oogenesis. Chapter Ⅱ. Safety Study of PTEN Inhibitor on Activation of Primordial FolliclesOBJECTIVE:Primordial ovarian follicles, which are often present in the ovaries of premature ovarian failure (POF) patients or are cryopreserved from the ovaries of young cancer patients who are undergoing gonadotoxic anticancer therapies, cannot be used to generate mature oocytes for in vitro fertilization (IVF). There has been very little success in triggering growth of primordial follicles to obtain fertilizable oocytes due to the poor understanding of the biology of primordial follicle activation. It was recently reported that PTEN (Phosphatase and tensin homolog deleted on chromosome10) prevents primordial follicle activation in mice, and deletion of Pten from the oocytes of primordial follicles leads to follicular activation. Consequently, the PTEN inhibitor has been successfully used in vitro to activate primordial follicles in both mouse and human ovaries. These results suggest that PTEN inhibitors could be used in ovarian culture medium to trigger the activation of primordial follicle. The purpose of the present study is to study the efficacy and safety of the use of PTEN inhibitor.METHODS:Primordial follicles were activated from neonatal mouse ovaries by transient treatment with a PTEN inhibitor bpV(HOpic). These ovaries were then transplanted under the kidney capsules of recipient mice to generate mature oocytes. The mature oocytes were fertilized in vitro and progeny mice were obtained after embryo transfer.RESULTS:Long-term monitoring up to the second generation of progeny mice showed that the mice were reproductively active and were free from any overt signs or symptoms of chronic illnesses.CONCLUSIONS:Our results indicate that the use of PTEN inhibitors could be a safe and effective way of generating mature human oocytes for use in novel IVF techniques. Chapter Ⅲ. PrognisticValue of Basal Serum Testosterone Levels in Women with Diminished Ovarian Reserve in Assisted Reproductive TechnologyOBJECTIVE:To evaluate basal testosterone (T) levels during follicular phase of the menstrual cycle as a predictor in women with diminished ovarian reserve for ovarian response and in vitro fertilization (IVF) outcome.METHODS:We analyzed data retrospectively from hospital-based IVF center including one hundred and eighty seven Chinese Han women with diminished ovarian reserve. We studied the association of basal T levels with ovarian response and IVF outcome.RESULTS:Basal T levels were significantly different between pregnant and non-pregnant women. A testosterone level of47.85ng/dl was shown to predict pregnancy outcome with a sensitivity of52.8%and specificity of65.3%; and the basal T was correlated with the numbers of large follicles (>14mm) on HCG day. Significantly negative correlations were observed between basal T, days of stimulation and total dose of gonadotropins after adjusting for confounding factors.CONCLUSIONS:In women with diminished ovarian reserve, lower level of basal T was associated with potential ovarian poor response. Basal T level could be used as a predictor for pregnancy outcome in IVF.