NLRP4E regulates actin cap formation through SRC and CDC42 during oocyte meiosis.

BACKGROUND: Members of the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing (NLRP) family regulate various physiological and pathological processes. However, none have been shown to regulate actin cap formation or spindle translocation during the asymmetric division of oocyte meiosis I. NLRP4E has been reported as a candidate protein in female fertility, but its function is unknown. METHODS: Immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were employed to examine the localization and expression levels of NLRP4E and related proteins in mouse oocytes. small interfering RNA (siRNA) and antibody transfection were used to knock down NLRP4E and other proteins. Immunoprecipitation (IP)-mass spectrometry was used to identify the potential proteins interacting with NLRP4E. Coimmunoprecipitation (Co-IP) was used to verify the protein interactions. Wild type (WT) or mutant NLRP4E messenger RNA (mRNA) was injected into oocytes for rescue experiments. In vitro phosphorylation was employed to examine the activation of steroid receptor coactivator (SRC) by NLRP4E. RESULTS: NLRP4E was more predominant within oocytes compared with other NLRP4 members. NLRP4E knockdown significantly inhibited actin cap formation and spindle translocation toward the cap region, resulting in the failure of polar body extrusion at the end of meiosis I. Mechanistically, GRIN1, and GANO1 activated NLRP4E by phosphorylation at Ser429 and Thr430; p-NLRP4E is translocated and is accumulated in the actin cap region during spindle translocation. Next, we found that p-NLRP4E directly phosphorylated SRC at Tyr418, while p-SRC negatively regulated p-CDC42-S71, an inactive form of CDC42 that promotes actin cap formation and spindle translocation in the GTP-bound form. CONCLUSIONS: NLRP4E activated by GRIN1 and GANO1 regulates actin cap formation and spindle translocation toward the cap region through upregulation of p-SRC-Tyr418 and downregulation of p-CDC42-S71 during meiosis I.

Acute exposure of triclocarban affects early embryo development in mouse through disrupting maternal-to-zygotic transition and epigenetic modifications.

Triclocarban (TCC) is a broad-spectrum antibacterial agent used globally, and high concentrations of this harmful chemical exist in the environment. The human body is directly exposed to TCC through skin contact. Moreover, TCC is also absorbed through diet and inhaled through breathing, which results in its accumulation in the body. The safety profile of TCC and its potential impact on human health are still not completely clear; therefore, it becomes imperative to evaluate the reproductive toxicity of TCC. Here, we explored the effect of TCC on the early embryonic development of mice and its associated mechanisms. We found that acute exposure of TCC affected the early embryonic development of mice in a dose-dependent manner. Approximately 7600 differentially expressed genes (DEGs) were obtained by sequencing the transcriptome of 2-cell mouse embryos; of these, 3157 genes were upregulated and 4443 genes were downregulated in the TCC-treated embryos. GO and KEGG analysis revealed that the enriched genes were mainly involved in redox processes, RNA synthesis, DNA damage, apoptosis, mitochondria, endoplasmic reticulum, Golgi apparatus, cytoskeleton, peroxisome, RNA polymerase, and other components or processes. Moreover, the Venn analysis showed that the zygotic genome activation (ZGA) was affected and the degradation of maternal effector genes was inhibited. TCC induced changes in the epigenetic modification of 2-cell embryos. The level of DNA methylation increased significantly. Further, the levels of H3K27ac, H3K9ac, and H3K27me3 histone modifications decreased significantly, whereas those of H3K4me3 and H3K9me3 modifications increased significantly. Additionally, TCC induced oxidative stress and DNA damage in the 2-cell embryos. In conclusion, acute exposure of TCC affected early embryo development, destroyed early embryo gene expression, interfered with ZGA and maternal gene degradation, induced changes in epigenetic modification of early embryos, and led to oxidative stress and DNA damage in mouse early embryos.

Resilience of infertile families undergoing in vitro fertilization: An application of the double ABC-X model.

BACKGROUND: Family resilience plays a crucial role in protecting the mental health and family stability of infertile patients. However, information associated with infertile families resilience is scarce. The double ABC-X model provides a roadmap for this, helps organize knowledge, and lays the foundation for knowledge development. AIMS: To describe the current situation of family resilience of infertile women, and to test the predictive theoretical model of family resilience based on infertility stigma, individual resilience, coping style, and posttraumatic growth. DESIGN: A cross-sectional study. METHODS: A convenience sample of 372 infertile women undergoing in vitro fertilization were recruited between April and August 2020. The Chinese-Family Resilience Assessment Scale, Infertility Stigma Scale, Simplified Coping Style Questionnaire, Chinese version of Connor-Davidson Resilience Scale, and Chinese version of Post Traumatic Growth Inventory were used to measure family resilience, infertility stigma, individual resilience, coping style, and posttraumatic growth. Structural equation models were used to analyze the relationship among these variables. RESULTS: The results showed that family resilience was related to infertility stigma, positive coping, and individual resilience. Moreover, the path analysis indicated that positive coping and individual resilience mediated the effects of infertility stigma on family resilience. CONCLUSIONS: A high level of stigma among infertile women should be identified. Interventions for targeting positive coping and individual resilience might ultimately increase their family resilience.

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.

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.

Association of single nucleotide polymorphisms in the USF1, GTF2A1L and OR2W3 genes with non-obstructive azoospermia in the Chinese population.

PURPOSE: To research the association between the single nucleotide polymorphisms (SNPs) of three spermatogenesis-related genes (USF1, GTF2A1L and OR2W3) and non-obstruction azoospermia (NOA). METHODS: We investigated 361 NOA cases and 368 controls from the Chinese Han population, and we used Sequenom iplex technology to analyze the candidate 9 SNPs from the USF1, GTF2A1L and OR2W3 genes. RESULTS: In this study, we found that the variant rs2516838 of USF1 was associated with NOA susceptibility (P = 0.020, OR = 1.436), and the haplotype TCG of the variants rs1556259, rs2516838, and rs2774276 of USF1 conferred an increased risk of NOA (P = 0.019, OR = 1.436). Furthermore, we found that the rs11204546 genotype of OR2W3 and the rs11677854 genotype of GTF2A1L were correlated with the FSH level in the patients (P = 0.004 and P = 0.018, respectively). CONCLUSIONS: Our results provided a new insight into susceptibility of USF1 variant with male infertility. Clinically, the SNPs (rs11204546 of OR2W3 and rs11677854 of GTF2A1L ) might be additional valuable molecular predictive markers for assessing the treatment of NOA patients.

CREM variants rs4934540 and rs2295415 conferred susceptibility to nonobstructive azoospermia risk in the Chinese population.

To evaluate the association of variants related to spermatogenesis with susceptibility to Chinese idiopathic nonobstructive azoospermia (NOA), seventeen tag single-nucleotide polymorphisms (SNPs) in CREM, ACT, KIF17b, and SPAG8 were analyzed in 361 NOA patients and 368 controls by Sequenom iplex technology. The results showed that two CREM SNPs, rs4934540 and rs22954152, were significantly associated with NOA and played protective roles against the disease (P value with Bonferroni correction = 0.00017, odds ratio [OR] = 0.624 and P = 0.012, OR = 0.686, respectively). Haplotype analysis of CREM gene variants suggested that haplotype CGTG of the SNPs, rs4934540, rs2295415, rs11592356, and rs1148247, exhibited significant protective effect against the occurrence of NOA (P = 0.001, OR = 0.659). The haplotype TATG conferred a significantly increased risk of NOA (P = 0.011, OR = 1.317). Furthermore, making use of quantitative RT-PCR, we demonstrated that relative mRNA expression of CREM in NOA patients with maturation arrest was only one-third of that in the controls with normal spermatogenesis (P < 0.0001). Our findings indicated that the polymorphisms of CREM gene were associated with NOA in the Chinese population and low CREM expression might be involved in the pathogenesis of spermatogenesis maturation arrest.

Genetic study of Hormad1 and Hormad2 with non-obstructive azoospermia patients in the male Chinese population.

PURPOSE: To evaluate the association of the Hormad1 and Hormad2 single nucleotide polymorphisms (SNPs) variants with non-obstructive azoospermia (NOA) in the Chinese population. METHODS: In the present study, we assessed 10 single nucleotide polymorphisms (SNPs) of Hormad1 and Hormad2 using Sequenom iplex technology in 361 NOA cases and 368 normal controls from Chinese population. RESULTS: We observed no statistical differences in the distribution of allele frequencies. Further genetic model analysis and haplotype analysis also showed no significant difference between the two groups. However, we found that genotype distribution of rs718772 of Hormad2 was significantly different between the larger testis group (average testis volume ≥10 ml) and the small testis group (average testis volume <10 ml) in the NOA patients (P = 0.035). CONCLUSIONS: In conclusion, Hormad1 and Hormad2 might not be the susceptible genes for the non-obstructive azoospermia in our study population. However, rs718772 of Hormad2 variant might be associated with testis development in NOA patients.

HTR1B regulates mitochondrial homeostasis and mitophagy by activating the ERK/ MAPK signalling pathway during human embryonic arrest.

Background: Previous studies have shown that serotonin and its receptors are widely distributed in mammalian reproductive tisssues and play an important role in embryonic development. However, the specific effects of the serotonergic system on embryonic arrest (EA) and the underlying mechanism require further investigation. Methods: Chorionic villi were collected from patients with EA and healthy pregnant women. Western blotting (WB) and immunohistochemistry (IHC) were used to detect serotonin receptor 1B (HTR1B) levels and evaluate mitochondrial function. Additionally, HTR-8/SVneo cells were transfected with an HTR1B overexpression plasmid. Quantitative real-time polymerase chain reaction(qRT-PCR), Cell Counting Kit-8 (CCK-8), and wound healing assays were utilized to evaluate mitophagy level, cell proliferation and cell migration, respectively. Results: We discovered elevated HTR1B levels in the chorionic villi of the patients with EA compared to controls. Concurrently, we observed enhanced levels of nucleus-encoded proteins including mitofilin, succinate dehydrogenase complex subunit A (SDHA), and cytochrome c oxidase subunit 4 (COXIV), along with the mitochondrial fusion protein optic atrophy 1(OPA1), fission proteins mitochondrial fission protein 1(FIS1) and mitochondrial fission factor (MFF) in the EA group. Additionally, there was an excessive mitophagy levels in EA group. Furthermore, a notable activation of mitogen-activated protein kinase (MAPK) signaling pathway proteins including extracellular regulating kinase (ERK), c-Jun N-terminal kinase (JNK), and P38 was observed in the EA group. By overexpressing HTR1B in HTR-8/SVneo cells, we observed a significant reduction in cell proliferation and migration. HTR1B overexpression also caused an increase in levels of SDHA and FIS1, as well as an upregulation of mitophagy. Notably, the ERK inhibitor U0126 effectively mitigated these effects. Conclusion: These findings show that HTR1B influences mitochondrial homeostasis, promoting excessive mitophagy and impairing cell proliferation and migration by activating the MAPK signalling pathway during post-implantation EA. Therefore, HTR1B may serve as a potential therapeutic target for patients with EA.

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.

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.

[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.

Recent Insights into Noncoding RNAs in Primary Ovarian Insufficiency: Focus on Mechanisms and Treatments.

CONTEXT: Primary ovarian insufficiency (POI) is a heterogeneous disease with an unknown underlying trigger or root cause. Recently many studies evaluated noncoding RNAs (ncRNAs), especially microRNAs (miRNAs), long noncoding RNA (lncRNAs), circular RNAs (circRNAs), and small interfering RNAs (siRNAs) for their associations with POI. EVIDENCE ACQUISITION: In this review, we outline the biogenesis of various ncRNAs relevant to POI and summarize the evidence for their roles in the regulation of disease occurrence and progression. Articles from 2003 to 2022 were selected for relevance, validity, and quality from results obtained in PubMed and Google Scholar using the following search terms: noncoding RNAs; primary ovarian insufficiency; premature ovarian failure; noncoding RNAs and primary ovarian insufficiency/premature ovarian failure; miRNAs and primary ovarian insufficiency/premature ovarian failure; lncRNAs and primary ovarian insufficiency/premature ovarian failure; siRNAs and primary ovarian insufficiency/premature ovarian failure; circRNAs and primary ovarian insufficiency/premature ovarian failure; pathophysiology; and potential treatment. All articles were independently screened for eligibility by the authors. EVIDENCE SYNTHESIS: This review summarizes the biological functions and synthesis of miRNAs, lncRNAs, siRNAs, and circRNAs in POI and discusses the findings of clinical and in vitro and in vivo studies. Although there is variability in the findings of individual studies, overall the available literature justifies the conclusion that dysregulated ncRNAs play significant roles in POI. CONCLUSION: The potential of ncRNAs in the treatment of POI requires further investigation, as ncRNAs derived from mesenchymal stem cell-secreted exosomes play pivotal roles and have considerable therapeutic potential in a multitude of diseases.

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.

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.

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.

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.

[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 recurrent miscarriages and insulin resistance: a meta analysis].

OBJECTIVE: To evaluate the association between recurrent miscarriages and insulin resistance. METHODS: The case-control studies on the association between recurrent spontaneous abortion and insulin resistance from June 1996 to April 2012 were collected from Medline, Elsevier, Chinese Journal Full-text Database, Chinese Biological Medicine Database, data base of Wanfang, Springer link and EMBASE. RevMan 5.1 software was used for Meta analysis. RESULTS: According to the included criteria, 7 clinical trials were finally selected. Total 467 cases with recurrent pregnancy loss were enrolled in study group, while 413 women with no history of abnormal pregnancies were enrolled in control group. No significant difference was found in average age and body mass index between the two groups (P > 0.05). Meta analysis results showed that the level of fasting glucose was no statistical difference between study group and control group (WMD = 2.27, 95%CI: -1.11 to 5.65, P > 0.05); fasting insulin level was higher 2.05 mU/L in study group than that of in control group, the difference was statistically significant (WMD = 2.05, 95%CI: 1.03 to 3.08, P < 0.01). Case number of study group on Homa-insulin resistance > 4.5 was more than that of control group (OR = 3.36, 95%CI: 1.72 to 6.57, P < 0.01). Case number of study group on glucose/insulin ratio < 4.5 was more than that of the control group, statistical difference was found (OR = 3.37, 95%CI: 1.90 to 5.99, P < 0.01). CONCLUSION: Insulin resistance is associated with the susceptibility to recurrent miscarriages, and it may contribute to the occurrence of recurrent miscarriages.

[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.