Multigenerational paternal obesity enhances the susceptibility to male subfertility in offspring via Wt1 N6-methyladenosine modification.

There is strong evidence that obesity is a risk factor for poor semen quality. However, the effects of multigenerational paternal obesity on the susceptibility to cadmium (a reproductive toxicant)-induced spermatogenesis disorders in offspring remain unknown. Here, we show that, in mice, spermatogenesis and retinoic acid levels become progressively lower as the number of generations exposed to a high-fat diet increase. Furthermore, exposing several generations of mice to a high fat diet results in a decrease in the expression of Wt1, a transcription factor upstream of the enzymes that synthesize retinoic acid. These effects can be rescued by injecting adeno-associated virus 9-Wt1 into the mouse testes of the offspring. Additionally, multigenerational paternal high-fat diet progressively increases METTL3 and Wt1 N6-methyladenosine levels in the testes of offspring mice. Mechanistically, treating the fathers with STM2457, a METTL3 inhibitor, restores obesity-reduced sperm count, and decreases Wt1 N6-methyladenosine level in the mouse testes of the offspring. A case-controlled study shows that human donors who are overweight or obese exhibit elevated N6-methyladenosine levels in sperm and decreased sperm concentration. Collectively, these results indicate that multigenerational paternal obesity enhances the susceptibility of the offspring to spermatogenesis disorders by increasing METTL3-mediated Wt1 N6-methyladenosine modification.

NLRC5 potentiates anti-tumor CD8+ T cells responses by activating interferon-ß in endometrial cancer.

OBJECTIVES: NLR family CARD domain containing 5 (NLRC5) could promote major histocompatibility complex class I (MHC-I)-dependent CD8+ T cell-mediated anticancer immunity. In this study, the immunosurveillance role and underlying mechanisms of NLRC5 in endometrial cancer (EC) were characterized. METHODS: CD8+ T cells were separated from healthy women's peripheral blood by using magnetic beads. The effect of NLRC5 and interferon-ß (IFN-ß) on immunosurveillance of EC were examined through a mouse tumor model and a CD8+ T cell-EC cell coculture system after NLRC5 overexpression and IFN-ß overexpression or depletion. The effect of NLRC5 on IFN-ß expression was examined with gain- and loss-of-function experiments. RESULTS: NLRC5 overexpression in the EC cell and CD8+ T cell coculture system inhibited EC cell proliferation and migration and promoted EC cell apoptosis and CD8+ T cell proliferation. In vivo, NLRC5 overexpression increased the proportion of CD8+ T cells and inhibited EC progression. Furthermore, IFN-ß overexpression in the EC cell and CD8+ T cell coculture system activated CD8+ T cell proliferation; however, genetic depletion of IFN-ß exerted the opposite effects. In addition, NLRC5 could negatively regulate IFN-ß expression in EC cells. Mechanistically, NLRC5 potentiated the antitumor responses of CD8+ T cells to EC by activating IFN-ß. CONCLUSIONS: Taken together, our findings demonstrated that NLRC5 potentiates anti-tumor CD8+ T cells responses by activating interferon-ß in EC, suggesting that genetically escalated NLRC5 and IFN-ß may act as potential candidates for the clinical translation of adjuvant immunotherapies to patients with EC.

METTL3 facilitates immunosurveillance by inhibiting YTHDF2-mediated NLRC5 mRNA degradation in endometrial cancer.

BACKGROUND: N6-methyladenosine (m6A) methylation is the most abundant chemical posttranscriptional modification of mRNA, and it is associated with the regulation of the immune response to tumors. However, the function of m6A modification in the immune response to endometrial cancer (EC) remains unknown. Our study investigated the immunological role of methyltransferase-like 3 (METTL3) in EC and the underlying molecular mechanism. METHODS: We investigated the correlation between the expression of METTL3 and CD8 by using an endometrial tissue microarray cohort. Next, we investigated the role and mechanism of METTL3 in the immune response to EC using a mouse tumor model and a CD8+ T cell-EC cell coculture system after METTL3 overexpression or depletion. Additionally, RNA immunoprecipitation (RIP), methylated RIP, and RNA stability experiments were used to investigate the mechanism underlying the function of METTL3 in immunosurveillance of EC. RESULTS: METTL3 levels were downregulated in EC patients, low levels of METTL3 were correlated with poor prognosis in EC patients. There was a positive correlation between METTL3 expression and CD8 expression. Overexpression of METTL3 in the EC cell and CD8+ T cell coculture system inhibited EC cell proliferation, migration, and promoted CD8+ T-cell proliferation, and in vivo, METTL3 overexpression increased CD8+ T cell proportions and inhibited EC progression; however, genetic depletion of METTL3 exerted the opposite effects. NLR family CARD domain-containing 5 (NLRC5) was identified as a target of METTL3-mediated m6A modification. The degradation of NLRC5 was increased by YTH domain-containing family 2 (YTHDF2). CONCLUSIONS: Overall, METTL3, YTHDF2, and NLRC5 have potential to be the diagnostic and prognostic biomarkers for EC. METTL3 facilitated the m6A modifications of NLRC5 and inhibited its degradation through a YTHDF2-dependent mechanism in EC. Genetic overexpression of METTL3 attenuated the immune evasion of EC by promoting NLRC5-mediated immunosurveillance, suggesting that the METTL3/YTHDF2/NLRC5 axis is a promising target of immunotherapy in EC.

Role of EGFR expressed on the granulosa cells in the pathogenesis of polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is one of the most common endocrinological disorders affecting between 6 to 20% of reproductive aged women. However, the etiology of PCOS is still unclear. Epidermal growth factor receptor (EGFR) plays a critical role in the growth and development of ovarian follicles. In our previous study, we showed that the expression level of EGFR was significantly higher in the cumulus granulosa cells from women with PCOS than that of normal women, suggesting that EGFR may play a potential role in the pathogenesis of PCOS. The present study further evaluated the association between EGFR and PCOS through both in clinical observation and animal experiments. We firstly validated the differential expression of EGFR in cumulus granulosa cells between PCOS patients and normal subjects by qRT-PCR and immunofluorescence staining. Then we generated a mouse model (n=20) of PCOS by injecting dehydroepiandrosterone (DHEA). The PCOS mice were then injected with an E corpus GFR inhibitor (AG1478) (n=10), which significantly improved the sex hormone levels in the estrous cycle stage, and the serum levels of LH, FSH and testosterone were compared with the PCOS mice without EGFR inhibitor treatment (n=10). Decreasing the expression level of EGFR in the PCOS mice also improved the ovulatory function of their ovaries which was indicated by the multifarious follicle stage in these mice as compared with the PCOS mice without EGFR inhibitor treatment. Also, the number of corpopa lutea were higher in the control group and the EGFR inhibitor treated group than in the PCOS group. The sex hormone levels and reproductive function were not significantly different between the control mice and the PCOS mice treated with the EGFR inhibitor. Our results demonstrated that EGF/EGFR signaling affected the proliferation of cumulus granulosa cells, oocyte maturation and meiosis, and played a potential role in the pathogenesis of PCOS. Therefore, the selective inhibition of EGFR may serve as a novel strategy for the clinical management of PCOS.

NLRC5 Might Promote Endometrial Cancer Progression by Inducing PD-L1 Expression.

Aims: The NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5 (NLRC5) was dysregulated in endometrial cancer (EC). However, the potential regulatory mechanisms of NLRC5 in EC remained unclear. We aimed to explore whether NLRC5 could regulate the programmed cell death protein ligand 1 (PD-L1) in EC. We also investigated the related molecular which led to the inactivation of NLRC5 in EC. Methods: The expressions of NLRC5 and PD-L1 in endometrium tissue microarray were detected by immunohistochemistry. Pearson's correlation analysis was performed to detect the expression correlation between NLRC5 and PD-L1. Immunofluorescence staining, western blotting, and quantitative real-time PCR (qRT-PCR) were used to detect the role of NLRC5 in PD-L1 in EC cell lines. The somatic mutation in EC patients was detected by whole-exome sequencing (WGS). Results: NLRC5 was downregulated in the endometrium of EC patients when compared to those in the normal endometrium. The level of PD-L1 in the endometrium of EC patients was higher when compared to those in the normal endometrium. There was a negative expression correlation between NLRC5 and PD-L1. NLRC5 could promote the expression of PD-L1 in EC cell lines. The mutations of ANKRD20A2, C2orf42, ADGRB3, AVPR2, GOLGA6C, and IPPK may lead to the downregulation of NLRC5 in EC patients. Conclusion: NLRC5 could inhibit the activation of PD-L1 in EC. Mutations of ANKRD20A2, C2orf42, ADGRB3, AVPR2, GOLGA6C, and IPPK may lead to the downregulation of NLRC5 in EC patients. Future study should investigate the mechanism of NLRC5 in PD-L1, as well as the mechanism of ANKRD20A2, C2orf42, ADGRB3, AVPR2, GOLGA6C, and IPPK in NLRC5.

Association analysis between the tag single nucleotide polymorphisms of DENND1A and the risk of polycystic ovary syndrome in Chinese Han women.

BACKGROUND: The DENND1A gene is one of the most important sites associated with polycystic ovary syndrome (PCOS). We attempted to analyze the correlation between five single nucleotide polymorphisms (SNPs) in the DENND1A gene and the development of PCOS. METHODS: A total of 346 PCOS patients and 225 normal ovulatory women were involved in the case-control study. Clinical variables and hormones were recorded. According to the Hap Map database, five tagging SNPs (rs2479106, rs2768819, rs2670139, rs2536951 and rs2479102) in the DENND1A gene were identified. The TaqMan probe and the PCR-RFLP (restriction fragment length polymorphism) methods were used for revealing these genotypes. TaqMan Genotype Software was used to analyze the alleles of the five SNPs. RESULTS: Linkage disequilibrium and the gene frequency analysis demonstrated that the CCGGG haplotype might increase the risk of PCOS (P = 0.038, OR = 1.89, 95% CI = 1.027-3.481). Significant differences were found in genotypic and allelic distributions at the rs2536951 and rs2479102 loci between PCOS women and controls (P <  0.001). The LH levels and LH/FSH ratios were higher in PCOS patients than in the control group. A detailed analysis revealed that for the rs2479106 locus, these two values were significantly different in the control subjects who had AA, AG and GG genotypes (P = 0.013 and P = 0.007, respectively), and for the rs2468819 locus, these two values were significantly different among the PCOS patients with AA, AG and GG genotypes (P = 0.013 and 0.002, respectively). CONCLUSIONS: The tagging SNPs rs2479106 and rs2468819 in the DENND1A gene are associated with PCOS in the Chinese population, whereas rs2670139, rs2536951 and rs2479102 are not correlated with PCOS in the same population.

Delivery of folic acid-modified liposomal curcumin for targeted cervical carcinoma therapy.

Introduction: In this study, novel folic acid (FA)-modified curcumin (CUR) liposomes (LPs) were developed and evaluated for their antitumor activity in vitro and in vivo. Methods: Characterization of the LPs, including transmission electron microscopy, morphology, particle size, and zeta potential studies, was carried out. Drug entrapment efficiency, drug-loading capacity, and release properties in vitro were tested. The in vitro growth inhibition activity, cellular uptake efficiency, and cell apoptosis of FA-modified CUR LPs were also investigated by a cervical cancer HeLa cell model. Results: The optimized distearoyl-l-a-phosphatidylethanolamine (DSPE)-PEG2000-FA-LPs/CUR formed spherical vesicles of nanometer sizes and had particle sizes of 112.3±4.6 nm, polydispersity index of 0.19±0.03, and zeta potential of -15.3±1.4 mV. In addition, the EE% and DL% of (DSPE)-PEG2000-FA-LPs/CUR were 87.6% and 7.9%, respectively. Compared with the free drug, FA-modified CUR LPs had sustained-release properties in vitro. In vivo, a strong green fluorescence was observed in the cytoplasmic region after incubation of (DSPE)-PEG2000-FA-LPs/CUR for 2 hrs. Conclusion: (DSPE)-PEG2000-FA-LPs/CUR showed a superior antiproliferative effect on HeLa cells and had a better antitumor effect in vivo than the non-modified LPs. These results indicated that (DSPE)-PEG2000-FA-LPs/CUR was a promising candidate for antitumor drug delivery.

Stimulative role of ST6GALNAC1 in proliferation, migration and invasion of ovarian cancer stem cells via the Akt signaling pathway.

BACKGROUND: Ovarian cancer is known as one of the most common cancers in the world among women. ST6GALNAC1 is highly expressed in cancer stem cells (CSCs), which correlates to high tumor-initiating, self-renewal and differentiation abilities. This present study aims to investigate how ST6GALNAC1 affects ovarian cancer stem cells (OCSCs). METHODS: In order to identify the differentially expressed genes related to ovarian cancer, microarray-based gene expression profiling of ovarian cancer was used, and ST6GALANC1 was one of the identified targets. After that, levels of ST6GALNAC1 in OCSCs and ovarian cancer cells were examined. Subsequently, an Akt signaling pathway inhibitor LY294002 was introduced into the cluster of differentiation 90+ (CD90+) stem cells, and cell proliferation, migration and invasion, levels of CXCL16, EGFR, CD44, Nanog and Oct4, as well as tumorigenicity of OCSCs were examined. RESULTS: By using a comprehensive microarray analysis, it was determined that ST6GALNAC1 was highly expressed in ovarian cancer and it regulated the Akt signaling pathway. High levels of ST6GALNAC1 were observed in OCSCs and ovarian cancer cells. Silencing ST6GALNAC1 was shown to be able to reduce cell proliferation, migration, invasion, self-renewal ability, tumorigenicity of OCSCs. In accordance with these results, the effects of ST6GALNAC1 in OCSCs were dependent on the Akt signaling pathway. CONCLUSIONS: When taken together, our findings defined the potential stimulative roles of ST6GALNAC1 in ovarian cancer and OCSCs, which relied on the Akt signaling pathway.

HMGA2 gene silencing reduces epithelial-mesenchymal transition and lymph node metastasis in cervical cancer through inhibiting the ATR/Chk1 signaling pathway.

Many cervical cancer (CC) patients suffer from cancer invasion and lymph node metastasis, resulting in poor therapeutic outcome. Evidence has indicated the involvement of misexpressed high-mobility group AT-hook 2 (HMGA2) in poor survival of cancer patients. This study hereby aims to investigate the role of HMGA2 in CC cell biological functions via the ATR/Chk1 signaling pathway. The cell line with the highest HMGA2 expression was selected to establish cell lines with wild-type and stable HMGA2 silencing. The underlying regulatory mechanisms of HMGA2 in CC cells were analyzed with the treatment of the ATR/Chk1 signaling pathway activator, inhibitor, shRNA against HMGA2 or pcDNA-HMGA2 plasmids, followed by quantification of expression levels of ATR, Chk1, Bcl-2, Bax, MMP-2, MMP-9, E-cadherin and N-cadherin. CC cell apoptosis, proliferation, migration, invasion and lymph node metastasis in nude mice were evaluated. The HeLa cell line with the highest HMGA2 expression was selected. HMGA2 inhibited the activation of the ATR/Chk1 signaling pathway. Notably, HMGA2 silencing or inhibition of the ATR/Chk1 signaling pathway inhibited epithelial mesenchymal transition (EMT), CC cell proliferation, invasion, migration, tumorigenicity and lymph node metastasis while promoting apoptosis, indicated by reduced expression of Bcl-2, MMP-2, MMP-9 and N-cadherin, with increased expression of E-cadherin and Bax. Collectively, our study provides evidence that HMGA2 gene silencing inhibits the activation of the ATR/Chk1 signaling pathway, whereby repressing EMT, proliferation, migration and invasion of CC cells and lymph node metastasis, and promoting CC cell apoptosis.

Guggulsterone-induced apoptosis in cholangiocarcinoma cells through ROS/JNK signaling pathway.

Cholangiocarcinoma (CCA), the most common biliary tract malignancy, is arising from the bile duct epithelium with the global significantly increased morbidity and mortality. Here, we showed the effect of guggulsterone, a steroid found in the resin of the guggul plant, on human HuCC-T1 and RBE CCA cells. Exposure to various concentrations of guggulsterone for multiple action time resulted in significant apoptosis in the CCA cells via activating both extrinsic and intrinsic pathways. Furthermore, we demonstrated that the apoptosis of CCA cells was induced by Reactive oxygen species (ROS) mediated JNK signaling pathway. Consistently, inhibition of JNK activity, overexpression of JBD, its binding protein or reduction of ROS by overexpression of catalase, all decreased apoptotic cells. Our results also revealed that guggulsterone-induced apoptosis was coupled with endoplasmic reticulum stress (ERS) in CHOP-dependent pathway. Downregulation of CHOP instead of other ERS markers could inhibit CCA cell apoptosis. Taken together, our results showed that guggulsterone could induce apoptosis of human CCA cells through ROS/JNK signaling pathway, indicating that guggulsterone could be important for the clinical therapy of CCA.

[Effect of domestic highly purified urinary follicle stimulating hormone on outcomes of in vitro fertilization-embryo transfer in controlled ovarian stimulation].

OBJECTIVE: To investigate the effect of domestic urine-derived high-purity follicle- stimulating hormone (HP-FSH, Lishenbao) on the outcome of in vitro fertilization(IVF) embryo transfer (ET) in controlled ovarian stimulation (COS). METHODS: From 1 September 2010 to 31 March 2011, total of 3178 infertility patients from 14 Reproductive Center with IVF or intracytoplasmic sperm injection (ICSI) indications who accepted first IVF or ICSI cycle were studied retrospectively. Their causes of infertility include all infertility factors except ovulatory dysfunction infertility and uterine factor infertility. The only long luteal phase gonadotropin-releasing hormone agonist (GnRH-a) protocol was included. Patients were divided into 2 groups according to the type of follicle-stimulating hormone (FSH) agents used: 1932 cases in HP-FSH group and 1246 cases in recombinant FSH (rFSH)group. Patients in both groups were combined with human menopausal gonadotropin (hMG) at doses of 150 U when follicle with diameter reached to 14-16 mm. When 3 dominate follicle with diameter reached 18 mm, hCG at dose of 5000 to 10 000 U or recombinant hCG at dose of 250 µg was administered by intramuscular injection. After 34 to 36 hours, oocytes were obtained guided by ultrasound, then IVF-ET were underwent in their Reproductive Center. The primary endpoint was comparison of live birth rate between the two groups. The secondary endpoints were comparisons of clinical pregnancy rate, miscarriage rate, and implantation rate, as well as COS and IVF outcome between the two groups. RESULTS: (1) There were significantly differences in baseline characteristics of the patients between two groups. The mean age was elder(32 ± 4 versus 30 ± 4, P < 0.01) , the infertility duration was longer (5 ± 4 versus 5 ± 3, P < 0.01) , and antral follicle count (AFC) was less (11 ± 5 versus 13 ± 7, P < 0.01) in patients of HP-FSH group compared with those in patients of rFSH group. (2) As compared with rFSH, the total doses of gonadotropin needed was (2348 ± 1011) U in HP-FSH group versus (2022 ± 659) U in rFSH group, the number of oocytes 13 ± 6 in HP-FSH group and 14 ± 7 in rFSH group, the rate of embryo frozen cycle of 66.30% (1281/1932) in HP-FSH group and 74.88% (933/1246) in rFSH group, which all reached statistical difference (P < 0.01). However, there were no significant different implantation rate [30.49% (1111/3644) versus 32.45% (737/2271)] between two groups. The other clinical parameters did not show significant difference, including clinical pregnancy rate per started cycle [41.61% (804/1932) versus 41.97% (523/1246) ] , clinical pregnancy rate per ET cycle[46.58% (804/1726) versus 48.47% (523/1079)], live birth rate per started cycle[34.21% (661/1932) versus 34.19% (426/1246)], live birth rate per ET cycle [38.30% (661/1726) versus 39.48% (426/1079)], miscarriage rate[13.6% (109/804) versus 16.4% (86/523)], and moderate/severe ovarian hyperstimulation syndrome (OHSS) rate [5.80% (112/1932) versus 7.78% (97/1246)](P > 0.05).(3) Treatment cost: the cost of gonadotropins needed for the patients in HP-FSH group was lower than that in rFSH group (4005 ± 1650 versus 6482 ± 2095, P < 0.01). CONCLUSION: In IVF/ICSI treatment cycles, domestic HP-FSH has similar live birth rate and lower financial burden when compared with rFSH.

Microsatellite polymorphism in the fibrillin 3 gene and susceptibility to PCOS: a case-control study and meta-analysis.

The D19S884 marker at the fibrillin 3 gene has been analysed as a candidate location for polycystic ovary syndrome (PCOS) mainly in Caucasian descendants. A case-control study was performed with 272 PCOS women and 271 controls to test the hypothesis that variants in the D19S884 marker increase susceptibility to PCOS in Chinese women and a meta-analysis was undertaken to clarify whether there is an allele consistently contributing to the susceptibility. The association analysis showed that PCOS women were significantly different from controls in the distribution of D19S884 allele frequencies. Instead of the well-known A8 allele, the most common allele in Chinese population was proved to be A7, and the allele frequencies of A7 were statistically different between cases and controls (P=0.037). The meta-analysis of A8 and A7 only identified A8 as a significant allelic association at the D19S884 marker in all combined samples (A8: OR 1.391, 95% CI 1.169-1.654; A7: OR 1.154, 95% CI 0.894-1.490). In conclusion, the association study showed a potential association of the D19S884 marker with PCOS in Chinese Han women and the meta-analysis identified that A8 may increase susceptibility to PCOS. Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age, and it affects an estimated 15% of women worldwide based on the Rotterdam criteria. Many studies in Caucasian descendants suggested that variants of the D19S884 marker at the fibrillin 3 gene are associated with the risk of this syndrome. Here we performed a case-control study with 272 PCOS women and 271 controls to investigate whether variants in the D19S884 marker increase susceptibility to PCOS in Chinese women. We also carried out a meta-analysis of some relevant studies to find a more reliable result. Our association analysis showed that PCOS women were significantly different from controls in the distribution of D19S884 allele frequencies, and instead of the well-known A8 (the letter 'A' represents 'allele'), the most common allele in Chinese population was proved to be A7, whose allele frequencies were statistically different between cases and controls. The meta-analysis of A8 and A7 only identified A8 as a significant allelic association at the D19S884 marker in all combined samples. In conclusion, our association study showed a potential association of the D19S884 marker with PCOS in Chinese Han women and the meta-analysis identified that A8 may increase susceptibility to PCOS.

PRM1 variant rs35576928 (Arg>Ser) is associated with defective spermatogenesis in the Chinese Han population.

Protamine genes play important roles in DNA packaging within the sperm nucleus. In order to evaluate the association of PRM1, PRM2, KIT and KITLG variants with susceptibility to severely defective spermatogenesis, 309 male infertility patients (199 cases with non-obstructive azoospermia and 110 cases with severe oligozoospermia) and 377 controls were recruited in the Chinese Han population. This study genotyped 38 single-nucleotide polymorphisms (SNP) in PRM1, PRM2, KIT and KITLG using Sequenom iplex. The results showed that PRM1 variant rs35576928 (p.R34S) was significantly associated with severe oligozoospermia and played a protective role against the disease (P=0.0079, Bonferroni correction, OR 0.426). The dominant model (variant-containing genotypes) of the SNP was confirmed to protect against the occurrence of oligozoospermia (P=0.0078, Bonferroni correction, OR 0.387). Haplotype analysis of PRM1 and PRM2 in combination exhibited that haplotype TACCGGC exhibited a significant protective effect against the occurrence of oligozoospermia when compared with controls (P=0.002, Bonferroni correction, OR 0.602). Haplotype TACCTGC was strongly associated with risk of the clinical phenotype severe oligozoospermia (P=0.002, Bonferroni correction, OR 2.716). The findings indicated that PRM1 variant rs35576928 (p.R34S) was associated with severely defective spermatogenesis in the Chinese Han population. Male spermatogenic failure may be associated with gene variants. We demonstrated whether such genetic variation of PRM1 and PRM2 affected clinicopathological characteristics and conferred susceptibility to this entity. In this study, we found that PRM1 variant rs35576928 (Arg>Ser) played a protective role against severe oligozoospermia. The dominant model analysis (variant-containing genotypes) confirmed that the SNP was a risk factor of a spermatogenesis defect. Haplotype analysis of PRM1 and PRM2 showed that TACCGGC was a common factor protecting against severe oligozoospermia, while the haplotype TACCTGC was strongly associated with the risk of the severe oligozoospmeria. Our findings indicate that the PRM1 variant rs35576928 (Arg>Ser) is associated with spermatogenesis defect in the Chinese Han population.

[Abnormalities of meiotic recombination in Han Chinese azoospermic patients].

OBJECTIVE: To analyze defective homologous chromosomal recombination in Han Chinese azoospermic patients. METHODS: Testicular biopsy samples from 7 healthy controls and 7 Han Chinese azoospermic patients including 2 obstructive azoospermia (OA group) and 5 non-obstructive azoospermia (NOA group) were analyzed. Immunofluorescence staining was performed to categorize early stage cells at meiosis prophase and to analyze chromosome pairing and recombination of pachytene spermatocyte. Newly developed meiotic proteins antibodies (anti-SCP3, anti-synaptonemal complex proteins 3, anti-MLH1, anti-Mut-L Homolog 1, anti-CREST, chromosome centromere antibody) were used to identify synaptonemal complex (anti-SCP3), recombination sites (anti-MLH1) and centromere (anti-CREST), respectively. Staging of spermatocyte was determined according to SCP3 formation progression. Qualitative data were compared by a Chi-square test, and ANOVA was used to analyze quantitative data. RESULTS: Respectively, 2346 and 2932 spermatocytes were categorized in the controls and azoospermic patients. The proportions of zygotene cells in both OA group and NOA group were significantly higher than that of the control group. Investigation of 1967 pachytene cells from the controls and 354 pachytene cells from azoospermic patients indicated that the mean MLH1 foci per pachytene cell of NOA group was statistically lower than that of the controls. Compared with the controls, incomplete synaptonemal complexes cells (containing gap and/or split) were significantly increased in the NOA group. CONCLUSION: Delayed meiosis prophase is relatively common in azoospermic patients, and changes in quantity and distribution of recombination foci may be the cause for spermatogenesis arrest in Han Chinese population.

Association between polymorphisms of the CYP11A1 gene and polycystic ovary syndrome in Chinese women.

The cholesterol side chain cleavage enzyme (CYP11A1) gene plays an important part in the synthesis of sex hormones and has been reported to be involved in the pathogenesis of polycystic ovary syndrome. A case-control study including 314 PCOS patients and 314 controls was conducted to assess the association of the SNPs rs4077582 and rs11632698 in CYP11A1 with PCOS using the polymerase chain reaction-restriction fragment length polymorphism method. Thereafter, 100 DNA samples were re-genotyped by direct sequencing for confirmation. The genotypic distribution of rs4077582 in women with PCOS differed from that in controls (P = 0.002). No such distributional difference was found in rs11632698 (P = 0.912). Data from our previous study of these two SNPs in another population including 290 PCOS patients and 344 controls was combined with the current data. Combined analysis (a total of 1262 participants, including 604 PCOS patients and 658 control women) showed a much more significant difference in the genotypic distribution of rs4077582 between PCOS and controls (P < 0.001). The T allele was more prevalent in PCOS patients (Odds ratio = 1.314; 95 % CI 1.122-1.540). The testosterone levels among the three genotypes for rs4077582 were different in the control group, as were the LH levels and the LH/FSH ratio. Therefore, SNP rs4077582 in CYP11A1 is strongly associated with susceptibility to PCOS and may alter the testosterone levels by the regulation of LH in different genotypes. No association was observed in rs11632698.

[Clinical application of in vitro maturation of human immature oocytes for infertile women with polycystic ovary syndrome].

OBJECTIVE: To investigate clinical effect and safety of in vitro maturation (IVM) of human immature oocytes in infertile women with polycystic ovary syndrome by comparing with conventional in vitro fertilization(IVF)and intracytoplasmic sperm injection(ICSI). METHODS: From Jan. 2003 to Dec. 2009, 157 infertile women with PCOS underwent 162 cycles IVM in Center for Reproductive Medicine, the First Affiliated Hospital of Anhui Medical University. In the mean time, 109 patients with PCOS underwent 114 IVF/ICSI cycles as control group 1 and 106 patients with other factors underwent 106 IVF/ICSI cycles as control group 2. Treatment and outcome of pregnancy and infant were compared among those 3 groups. RESULTS: No statistically significant difference were found in terms of the positive rate of hCG in urine [35.7% (56/157), 42.2% (46/109), 44.3% (47/106)], the rate of clinical pregnancy [29.3% (46/157), 37.6% (41/109), 41.5% (44/106)], the rate of entopic pregnancy [1.9% (3/157), 1.8% (2/109), 0.9% (1/106)], the rate of miscarriage [18.6% (8/43), 12.8% (5/39), 20.9% (9/43)] and the rate of live-birth [22.3% (35/157), 31.2% (34/109), 32.1% (34/106)] among three groups (IVM group, control group 1, control group 2, P > 0.05). The rate of preterm labor, low weight newborn, mean birth weight, ratio of male to female did not show significantly difference among 3 groups (P > 0.05). The average control ovarian stimulation was 6 days, the median dose of gonadotropin (Gn) was 675 IU, and the total hospital cost was (8392 ± 1328) RMB in IVM group, which were statistically lower than those in the other two control groups (P < 0.01). The rate of multiple pregnancy was 4.7% (2/43) and ovarian hyperstimulation syndrome (OHSS) 0 in IVM group, which were significantly lower than those in the other control group (P < 0.01). CONCLUSION: In vitro maturation is an effective treatment in infertile women with PCOS, it could obtain the similar pregnancy outcome and reduce total cost, the dosage of gonadotropin-releasing hormone and rate of OHSS compared with conventional IVF/ICSI.

SNP rs2470152 in CYP19 is correlated to aromatase activity in Chinese polycystic ovary syndrome patients.

CYP19 encodes aromatase, a key enzyme essential for estrogen biosynthesis. Single nucleotide polymorphism (SNP) rs2470152 in CYP19 is associated with serum estradiol (E2) level and the E2/T (estradiol/testosterone) ratio. A case­control study including 661 individuals [364 polycystic ovary syndrome (PCOS) patients and 297 controls] was conducted to assess the association of SNP rs2470152 with PCOS. The subjects were genotyped using the polymerase chain reaction-restriction fragment length polymorphism method. Hormone levels were analyzed among various genotypes. The genotypic distributions of rs2470152 did not differ in PCOS patients when compared to the controls. However, differences in the E2/T ratio were detected, exhibiting a lower ratio in the heterozygous TC genotype in PCOS patients (p=0.01036) and controls (p=0.000). Testosterone levels also differed between the three genotypes of PCOS patients (p=0.00625), with a higher level in the TC genotype. Therefore, rs2470152 in CYP19 was not a major etiological factor for PCOS; however, the heterozygous TC genotype may inhibit aromatase activity, resulting in hyperandrogenism, particularly in PCOS patients.

Polymorphisms of the HSD17B6 and HSD17B5 genes in Chinese women with polycystic ovary syndrome.

OBJECTIVES: To investigate the polymorphisms of the 17ß-hydroxysteroid dehydrogenase type 5 and type 6 (HSD17B5 and HSD17B6) genes in Chinese women with polycystic ovary syndrome (PCOS). METHODS: Two hundred twenty-two PCOS patients and 283 controls were studied. Menarche age was recorded. Body mass indices (BMI) were calculated. Blood samples were obtained for single nucleotide polymorphism (SNP) analyses and hormone measurements. Genotyping of HSD17B6 and HSD17B5 in cases and controls was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: The SNP rs898611 of the HSD17B6 gene (TT, CT, CC) in women with PCOS (0.680, 0.270, 0.050, respectively) did not differ from those in controls (0.700, 0.258, 0.042, respectively), and the SNP rs3763676 of the HSD17B5 gene (AA, AG, GG) was rare in Chinese women. Total testosterone and other reproductive hormones, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), LH/FSH, and estradiol (E(2)), were also similar among the different genotypes of the HSD17B6 in the PCOS subjects and the controls, whereas BMI was different in the three genotypes of the HSD17B6 in PCOS subjects. CONCLUSIONS: Our data suggest that there is no association of HSD17B6 and HSD17B5 variants with the occurrence of PCOS in the Chinese population, but the polymorphism of SNP rs898611 is associated with BMI in PCOS patients.

[Polymorphism of CYP11A1 gene in Chinese patients with polycystic ovarian syndrome].

OBJECTIVE: To investigate the association between polymorphism of cytochrome P450 subfamily XIA polypeptide 1 (CYP11A1) gene and polycystic ovarian syndrome (PCOS) in Chinese population. METHODS: From May 2005 to Dec. 2008, 290 PCOS cases treated in the First affiliated hospital of Anhui Medical University matched with 344 reproductive women as controls were enrolled in this study. Genotypes of 7 tagging single nucleotide polymorphisms (tSNP, rs12438594, rs4077582, rs9806234, rs16968477, rs4887139, rs1843090, rs11632698) covering CYP11A1 gene (r(2) > or = 0.8) and 3 microsatellite markers (D15S1547, D16S520, D15S1546) were chosed from the phase II database of Han population in HapMap data. Genotype and frequency of allele were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and haplotype of gene polymorphism were analyzed in 290 PCOS cases and 344 controls. RESULTS: Among 7 tSNPs and 3 microsatellite markers, the frequency of rs4077582, D15S1547, D15S1546 and rs11632698 between two groups reached statistical difference (P = 0.010, 0.044, 0.018 and 0.026). The allele frequency of rs4077582, rs4887139, rs1843090, D15S1547 and D16S520 showed significant difference between two groups (P = 0.002, 0.048, 0.030, 0.001 and 0.024). Among 5 haplotype of CYP11A1 (ACGCA13/6/9AG, ACGTA16/6/11AA, GCACG12/8/8AA, GTACA14/4/7GG, GTGCA13/6/7AG), the frequency of GTACA14/4/7GG and ACGCA13/6/9AG were 7.8% (45/580) and 25.3% (147/580) in PCOS group and 11.9% and 19.6% in control group, which showed statistical difference (P < 0.05). There was no significant difference in the level of serum androgen at difference genotype from rs4077582 between two groups (P > 0.05). CONCLUSION: The polymorphism of CYP11A1 gene was associated with PCOS, however, the relationship between gene sequence covered by tSNP/microsatellite markers and hyperandrogenism of PCOS should be further investigated.

Association between CYP19 gene SNP rs2414096 polymorphism and polycystic ovary syndrome in Chinese women.

BACKGROUND: Several studies have reported the association of the SNP rs2414096 in the CYP19 gene with hyperandrogenism, which is one of the clinical manifestations of polycystic ovary syndrome (PCOS). These studies suggest that SNP rs2414096 may be involved in the etiopathogenisis of PCOS. To investigate whetherthe CYP19 gene SNP rs2414096 polymorphism is associated with the susceptibility to PCOS, we designed a case-controlled association study including 684 individuals. METHODS: A case-controlled association study including 684 individuals (386 PCOS patients and 298 controls) was performed to assess the association of SNP rs2414096 with PCOS. Genotyping of SNP rs2414096 was conducted by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that was performed on genomic DNA isolated from blood leucocytes. Results were analyzed in respect to clinical test results. RESULTS: The genotypic distributions of rs2414096 (GG, AG, AA) in the CYP19 gene (GG, AG, AA) in women with PCOS (0.363, 0.474, 0.163, respectively) were significantly different from that in controls (0.242, 0.500, 0.258, respectively) (P = 0.001). E2/T was different between the AA and GG genotypes. Age at menarche (AAM) and FSH were also significantly different among the GG, AG, and AA genotypes in women with PCOS (P = 0.0391 and 0.0118, respectively). No differences were observed in body mass index (BMI) and other serum hormone concentrations among the three genotypes, either in the PCOS patients or controls. CONCLUSIONS: Our data suggest that SNP rs2414096 in the CYP19 gene is associated with susceptibility to PCOS.