The Impact of Seasonal Variation on Clinical Pregnancy and Live Birth Rates in Assisted Reproductive Technology: A Retrospective Cohort Study in Hainan

Objective:To investigate the influence of season on live birth and clinical pregnancy rates,as well as assisted reproductive technology(ART)outcomes,in the Hainan region.Methods:Patients were categorized into four groups based on the dates of artificial insemination and transplantation:spring,summer,autumn,or winter.The main outcome measures were clinical pregnancy rates and live birth rates.Secondary outcomes included body mass index(BMI),oocyte number,two pronuclei(2PN)cleavage rate,total gonadotropin(Gn)dosage and days,age,2PN fertilization rate,sperm concentration,sperm PR rate,anti-Müllerian hormone(AMH),and endometrial thickness.Outpatient semen quality indicators included sperm PR rate,total sperm count,sperm concentration,and total sperm motility.Results:This retrospective cohort study analyzed 2,016 artificial insemination cycles and 1,783 ovarian retrieval cycles from January 2017 to October 2022,and assessed the semen quality of 6,651 outpatients from May 2017 to October 2022.In artificial insemination cycles,sperm PR rate and clinical pregnancy rate were highest in winter,with a statistically significant difference between groups(P<0.05).Clinical pregnancy rate was influenced by both age and sperm PR rate(P<0.05).In ovarian retrieval cycles,the winter group had significantly higher clinical pregnancy,2PN fertilization,and 2PN cleavage rates than the other groups.The autumn group had higher live birth rates,though not significantly different.Additionally,winter months showed higher total sperm concentration and total sperm number compared to other seasons.Conclusion:Seasonality affected clinical pregnancy and live birth rates in artificial insemination cycles but not in ovarian retrieval cycles in the Hainan region.These findings suggest that while there is no need to choose a specific season for ovarian retrieval cycles,artificial insemination in winter may be preferable for patients.

Heterotopic pregnancy after assisted reproductive techniques with favorable outcome of the intrauterine pregnancy:A case report

BACKGROUND Heterotopic pregnancy(HP)is a rare condition in which both ectopic and intrauterine pregnancies occur.HP is uncommon after natural conception but has recently received more attention due to the widespread use of assisted reproductive techniques(ART)such as ovulation promotion therapy.CASE SUMMARY Here,we describe a case of HP that occurred after ART with concurrent tubal and intrauterine singleton pregnancies.This was treated successfully with surgery to preserve the intrauterine pregnancy,resulting in the birth of a low-weight premature infant.This case report aims to increase awareness of the possibility of HP during routine first-trimester ultrasound examinations,especially in pregnancies resulting from ART and even if multiple intrauterine pregnancies are present.CONCLUSION This case alerts us to the importance of comprehensive data collection during regular consultations.It is important for us to remind ourselves of the possibility of HP in all patients presenting after ART,especially in women with an established and stable intrauterine pregnancy that complain of constant abdominal discomfort and also in women with an unusually raised human chorionic gonadotropin level compared with simplex intrauterine pregnancy.This will allow symptomatic and timeous treatment of patients with better results.

MiR-142-3p Regulates ILC1s by Targeting HMGB1 via the NF-κB Pathway in a Mouse Model of Early Pregnancy Loss

Objective Innate lymphoid cells(ILCs)are a class of newly discovered immunocytes.Group 1 ILCs(ILC1s)are identified in the decidua of humans and mice.High mobility group box 1(HMGB1)is predicted to be one of the target genes of miR-142-3p,which is closely related to pregnancy-related diseases.Furthermore,miR-142-3p and HMGB1 are involved in regulating the NF-κB signaling pathway.This study aimed to examine the regulatory effect of miR-142-3p on ILC1s and the underlying mechanism involving HMGB1 and the NF-κB signaling pathway.Methods Mouse models of normal pregnancy and abortion were constructed,and the alterations of ILC1s,miR-142-3p,ILC1 transcription factor(T-bet),and pro-inflammatory cytokines of ILC1s(TNF-α,IFN-γand IL-2)were detected in mice from different groups.The targeting regulation of HMGB1 by miR-142-3p in ILC1s,and the expression of HMGB1 in normal pregnant mice and abortive mice were investigated.In addition,the regulatory effects of miR-142-3p and HMGB1 on ILC1s were detected in vitro by CCK-8,Annexin-V/PI,ELISA,and RT-PCR,respectively.Furthermore,changes of the NF-κB signaling pathway in ILC1s were examined in the different groups.For the in vivo studies,miR-142-3p-Agomir was injected in the uterus of abortive mice to evaluate the abortion rate and alterations of ILC1s at the maternal-fetal interface,and further detect the expression of HMGB1,pro-inflammatory cytokines,and the NF-κB signaling pathway.Results The number of ILC1s was significantly increased,the level of HMGB1 was significantly upregulated,and that of miR-142-3p was considerably downregulated in the abortive mice as compared with the normal pregnant mice(all P<0.05).In addition,miR-142-3p was found to drastically inhibit the activation of the NF-κB signaling pathway(P<0.05).The number of ILC1s and the levels of pro-inflammatory cytokines were significantly downregulated and the activation of the NF-κB signaling pathway was inhibited in the miR-142-3p Agomir group(all P<0.05).Conclusion miR-142-3p can regulate ILC1s by targeting HMGB1 via the NF-κB signaling pathway,and attenuate the inflammation at the maternal-fetal interface in abortive mice.

Two-sample Mendelian randomization analysis of causal relationship between eczema and autoimmune diseases

Objective:The causal relationship between eczema and autoimmune diseases has not been previously reported.This study aims to evaluate the causal relationship between eczema and autoimmune diseases.Methods:The two‐sample Mendelian randomization(MR)method was used to assess the causal effect of eczema on autoimmune diseases.Summary data from the Genome-Wide Association Study Catalog(GWAS)were obtained from the Integrative Epidemiology Unit(IEU)database.For eczema and autoimmune diseases,genetic instrument variants(GIVs)were identified according to the significant difference(P<5×10−8).Causal effect estimates were generated using the inverse‐variance weighted(IVW)method.MR Egger,maximum likelihood,MR-PRESSO,and MR-RAPS methods were used for alternative analyses.Sensitivity tests,including heterogeneity,horizontal pleiotropy,and leave-one-out analyses,were performed.Finally,reverse causality was assessed.Results:Genetic susceptibility to eczema was associated with an increased risk of Crohn’s disease(OR=1.444,95%CI 1.199 to 1.738,P<0.001)and ulcerative colitis(OR=1.002,95%CI 1.001 to 1.003,P=0.002).However,no causal relationship was found for the other 6 autoimmune diseases,including systemic lupus erythematosus(SLE)(OR=0.932,P=0.401),bullous pemphigoid(BP)(OR=1.191,P=0.642),vitiligo(OR=1.000,P=0.327),multiple sclerosis(MS)(OR=1.000,P=0.965),ankylosing spondylitis(AS)(OR=1.001,P=0.121),rheumatoid arthritis(RA)(OR=1.000,P=0.460).Additionally,no reverse causal relationship was found between autoimmune diseases and eczema.Conclusion:Eczema is associated with an increased risk of Crohn’s disease and ulcerative colitis.No causal relationship is found between eczema and SLE,MS,AS,RA,BP,or vitiligo.

Ultra-conservative noncoding RNA uc.243 confers chemo-resistance by facilitating the efflux of the chemotherapeutic drug in ovarian cancer

Background:Despite improvements in objective response rates to cisplatin-based combination chemotherapy,the majority of advanced ovarian cancer remains suboptimal,resulting in poor survival.it has been found that non-coding RNAs(ncRNAs)not only participate in the transmission of signals between various cells but also participate in tumor immunity and anti-tumor immune responses,thereby regulating tumor occurrence and development.However,the function and detailed mechanism of ultraconserved RNA(ucRNA)in ovarian cancer chemoresistance is still unclear.Methods:Western blotting assay,Quantitative real-time PCR analysis(qPCR),and Kaplan-Meier Plotter analysis were performed to analyze the expression and prognosis of uc.243 in ovarian carcinoma.Cytotoxicity assay and Annexin V assay were performed to analyze the function of uc.243 in cisplatin resistance in ovarian cancer cells.RNA pull-down and qPCR experiments were performed to explore the molecular mechanism of uc.243 enhancing cisplatin resistance in ovarian cancer cells.Results:Herein,we found that uc.243 was remarkably upregulated and correlated with patient survival in chemoresistance ovarian cancer patients compared with chemo-sensitive ovarian cancer.Functional experiment displayed that uc.243 induced cisplatin resistance on ovarian cancer cells by facilitating the efflux of cisplatin(CDDP);but inhibiting the expression of uc.243 significantly reverses this function.Mechanistically,uc.243 can inhibit the binding of RNA binding protein DGCR8 microprocessor complex subunit to pri-miR-155,thereby inhibiting the cleavage of pri-miR-155 and decrease in mature miR-155,subsequently upregulates the expression of ATP binding cassette subfamily B member(ABCB1,ABCC2).Conclusion:Our research findings indicate that uc.243 can induce chemotherapy resistance in ovarian cancer,suggesting that it may become a new prognostic biomarker for malignant ovarian cancer.

Reproductive Outcomes in Women with Prior Cesarean Section Undergoing In Vitro Fertilization:A Retrospective Case-control Study

The impact of prior cesarean section（CS） on the pregnancy and neonatal outcomes of in vitro fertilization and embryo transfer（IVF-ET） was investigated. A retrospective analysis was performed on 144 patients with prior CS between January 2013 and December 2015. The pregnancy, delivery, and neonatal outcomes of patients who had previous CS delivery and received IVF-ET were analyzed. The control group comprised 166 patients who had only previous vaginal delivery（VD） and received IVF-ET during the same period. The results showed that the basal follicle stimulating hormone level, estradiol level on human chorionic gonadotropin（h CG） day, gonadotrophin dosage, duration of stimulation, retrieved oocytes, fertilization rate, high-quality embryo rate, multiple birth rate, abortion rate and ectopic pregnancy rate had no significant difference between the two groups（P〉0.05）. The pregnancy rate（40.28% vs. 54.22%） and implantation rate（24.01% vs. 34.67%） were significantly lower（P〈0.05）, and the ratio of embryo difficulty transfer（9/144 vs. 0/166） was significantly higher in CS group than in VD group. The risk of pernicious placenta previa and postpartum hemorrhage in twin deliveries was significantly increased in CS group as compared with that in VD group（P〈0.05）, and gestational age and neonatal birth weight were significantly reduced in twin deliveries as compared with singleton deliveries in both groups（P〈0.05）. It was suggested that the existence of CS scar may impact embryo implantation and clinical pregnancy outcome, and increase the difficulty of ET. We should limit the number of transfer embryos to avoid multiple pregnancies and strengthen gestational supervision in patients with cesarean scar.

Melatonin and its protective role against male reproductive toxicity induced by heavy metals,environmental pollutants,and chemotherapy:A review

Melatonin,as a ubiquitous indoleamine hormone,is synthesized primarily by the pineal gland.It has diverse biological effects through quite complex mechanisms.More recently,studies have focused on the mechanism of melatonin in anti-reproductive toxicity/damage.Since melatonin possesses strong antioxidant and anti-apoptotic properties,researchers have examined its potential role in protecting against male reproductive toxicity/damage,which may be induced by chemotherapy or environmental toxicants and can lead to male infertility.In this article,recent progress regarding the protective effects of melatonin on male reproductive toxicity/damage is reviewed.

Influence of Acupuncture on Idiopathic Male Infertility in Assisted Reproductive Technology

The clinical effects of acupuncture on idio pathic male infertility in sperm parameter and on therapeutic results in assiste d reproductive technology were investigated. 22 patients failed in intracytoplas mic sperm injection (ICSI) with idiopathic male infertility were treated with ac upuncture twice weekly for 8 weeks, followed by ICSI treatment again. The sperm concentration, motility, morphology, fertilization rates and embryo quality were observed. Quick sperm motility after acupuncture(18.3 %±9.6 %) was significa ntly improved as compared with that before treatment (11.0 %±7.5 %, P <0.01) . The normal sperm ratio was increased after acupuncture (21.1 %±10.4 % vs 16.2 %± 8.2 %, P <0.05). The fertilization rates after acupuncture (66.2 %) were obviously higher than that before treatment (40.2 %, P <0.01). There was no significant difference in sperm concentration and general sperm motility between before and after acupuncture. The embryo quality after acupuncture was improved , but the difference between them was not significant ( P >0.05). Acupuncture can improve sperm quality and fertilization rates in assisted reproductive techn ology.

Expression of Attractin in Male Reproductive Tract of Human and Mice and Its Correlation with Male Reproduction

The expression of Attractin mRNA and protein in testis and semen of human and male mice was investigated. Human testis and semen samples were all collected from Reproductive Center of Reumin Hospital, Wuhan University in December, 2012. Testis samples were collected from 7 cases of obstructive azoospermias when they were subjected to diagnosed testis biopsy, and 30 nor- mal human semen samples were obtained from those cases of semen analysis. Adult mice testis tis- sues were obtained from 10 2-month-old male BALB/c mice, and 60 male mice at different ages were classified into 10 groups （day 1, 5, 10, 15, 21, 28, 35, 42, 56, and 120 respectively, n=6 each）. The expression of Attractin mRNA and protein in testis was detected by RT-PCR and Western blotting re- spectively. Human semen samples were centrifuged into sperm plasma （SP） and sperm extract （SE）, and mice sperm samples were collected from the epididymis of 10 adult male BALB/c mice. Western blotting was used to determine the Attractin protein expression level. Attractin mRNA and protein were expressed in the testis of both patients with obstructive azoospermias and adult Bcl/B mice. Quantitative RT-PCR revealed that no Attractin mRNA was detectable in day 1 male BALB/c mice group. The Attractin mRNA and protein levels were low on the day 10, and increased with age until day 56. On the day 120, the expression levels of Attractin were decreased. As for human semen sam- pies, Attractin protein was expressed in both SP and SE, but didn＇t exist in samples from the epidi- dymis of male BALB/c mice. It was suggested that Attractin acted as a novel active substance and was involved in male reproduction in both human and BALB/c mice, but it exerted a different ex- pression profile in different mammal species.

Cluster analysis reveals a homogeneous subgroup of PCOS women with metabolic disturbance associated with adverse reproductive outcomes

Background:Polycystic ovarian syndrome(PCOS)is a heterogeneous and complex reproductive endocrinological disease that could lead to infertility.There were many attempts to classify PCOS but it remains unclear whether there is a specific subgroup of PCOS that is associated with the best or worst reproductive outcomes of assisted reproductive techniques(ART).Methods:Infertile PCOS patients who underwent their first cycle of in vitro fertilization(IVF)in West China Second University Hospital,Sichuan University from January 2019 to December 2021 were included.Basic clinical and laboratory information of each individual were extracted.Unsupervised cluster analysis was performed.Controlled ovarian stimulation parameters and reproductive outcomes were collected and compared between the different clusters of PCOS.Results:Our analysis clustered women with PCOS into"reproductive","metabolic",and"balanced"clusters based on nine traits.Reproductive group was characterized by high levels of testosterone(T),sex hormone-binding globulin(SHBG),follicular stimulation hormone(FSH),luteinizing hormone(LH),and anti-Müllerian hormone(AMH).Metabolic group was characterized by high levels of body mass index(BMI),fasting insulin,and fasting glucose.Balanced group was characterized by low levels of the aforementioned reproductive and metabolic parameters,except for SHBG.Compared with PCOS patients in reproductive and balanced clusters,those in metabolic cluster had lower rates of good quality day 3 embryo and blastocyst formation.Moreover,PCOS patients in the reproductive cluster had greater fresh embryo transfer(ET)cancelation rate and clinical pregnancy rate after fresh ET than metabolic cluster(odds ratio[OR]=3.37,95%confidence interval[CI]:1.77-6.44,and OR=6.19,95%CI:1.58-24.24,respectively).And compared with PCOS of metabolic cluster,PCOS of balanced cluster also had higher chance for fresh ET cancelation(OR=2.83,95%CI:1.26-6.35).Conclusion:Our study suggested that PCOS patients in metabolic cluster may be associated with adverse reproductive outcomes and might need individualized treatment and careful monitoring before and during ART.

Antioxidant procyanidin B2 protects oocytes against cryoinjuries via mitochondria regulated cortical tension

Background:Irreversible cryodamage caused by oocyte vitrification limited its wild application in female fertility preservation.Antioxidants were always used to antagonist the oxidative stress caused by vitrification.However,the comprehensive mechanism underlying the protective role of antioxidants has not been studied.Procyanidin B2(PCB2)is a potent natural antioxidant and its functions in response to vitrification are still unknown.In this study,the effects of PCB2 on vitrified-thawed oocytes and subsequent embryo development were explored,and the mechanisms underlying the protective role of PCB2 were systematically elucidated.Results:Vitrification induced a marked decline in oocyte quality,while PCB2 could improve oocyte viability and further development after parthenogenetic activation.A subsequent study indicated that PCB2 effectively attenuated vitrification-induced oxidative stress,rescued mitochondrial dysfunction,and improved cell viability.Moreover,PCB2 also acts as a cortical tension regulator apart from strong antioxidant properties.Increased cortical tension caused by PCB2 would maintain normal spindle morphology and promote migration,ensure correct meiosis progression and finally reduce the aneuploidy rate in vitrified oocytes.Further study reveals that ATP biosynthesis plays a crucial role in cortical tension regulation,and PCB2 effectively increased the cortical tension through the electron transfer chain pathway.Additionally,PCB2 would elevate the cortical tension in embryo cells at morula and blastocyst stages and further improve blastocyst quality.What's more,targeted metabolomics shows that PCB2 has a beneficial effect on blastocyst formation by mediating saccharides and amino acids metabolism.Conclusions:Antioxidant PCB2 exhibits multi-protective roles in response to vitrification stimuli through mitochondria-mediated cortical tension regulation.

Research progress of natural products in treating polycystic ovary syndrome

Polycystic ovary syndrome(PCOS)is a common metabolic disorder reproductive endocrine disease in puberty and women of childbearing age.At present,metformin is mainly used in clinical to treat PCOS.From recent researches,many natural products,such as flavonoids,alkaloids,cumarins,etc,showed curing effects on PCOS.These have been attracting more and more attention.By searching the researches from database of CNKI,PubMed etc,this paper aims at providing a reference for PCOS drug research and development.

Application of next-generation sequencing in thalassemia screening:A systematic review and meta-analysis

Objective:To evaluate the value of next-generation sequencing(NGS)in the prevention and management of thalassemia.Methods:A systematic search was performed in eight databases including China Biomedical Literature Database,Chinese National Knowledge Infrastructure,Chinese Scientific Journals Database,Wanfang database,PubMed,EMBASE,Web of Science,and Cochrane Library from the inception to 1 June 2022.Stata 17.0 and Review Manager 5.4 were used for the meta-analysis.Results:Nine studies containing 14794 participants were included in the meta-analysis.Compared with the routine genetic testing(including Gap-PCR and reverse dot blot),NGS had higher detection rates in screening thalassemia(RR 1.22,95%CI 1.13-1.31,P<0.01),particularly for theα-thalassaemia mutation carriers(RR 1.24,95%CI 1.07-1.44,P<0.01).However,no significant difference was found in the screening ofβ-thalassemia(RR 1.10,95%CI 0.99-1.23,P>0.05).Conclusions:Compared with routine genetic testing,NGS had a higher detection rate in general,particularly in the detection ofα-thalassemia.

Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells （ESCs）, which may be an important source ofhistocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines （hPES-1 and hPES-2） from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA- 1-60, and TRA- 1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies （EBs） of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers （hPES-2 did not）. Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat （STR） results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

MIF May Participate in Pathogenesis of Polycystic Ovary Syndrome in Rats through MAPK Signalling pathway

The polycystic ovary syndrome (PCOS) model was established in fats and correlation between the expression of macrophage migration inhibitory factor (MIF) and cytokinesis with the MAPK signalling pathway in the rat ovary was measured. The PCOS model in rats was established by dehydroepiandrosterone (DHEA).Thirty sexually immature female Sprague-Dawley rats were randomly and equally assigned to three groups:control group,PCOS group,and PCOS with high-fat diet (HFD) group.Serum hormones were assayed by radioimmunoassay (RIA).The ovaries'were immunohistochemically stained with MIF,and the expression of MIF,p-JNK and p-p38 was detected by Western blotting in ovaries.The serum testosterone level,LH concentration,LH/FSH ratio,fasting insulin level and HOMA IR index in the PCOS group (6.077±0.478,13.809±1.701,1.820±0.404,10.83±1.123 and 1.8692±0.1096)and PCOS with HFD group (6.075±0.439,14.075±1.927,1.779±0.277,10.20±1.377 and 1.7736±0.6851)were significantly higher than those in the control group (4.949±0.337, 2.458±0.509,1.239±0.038,9.53±0.548 and 1.5329±0.7363),but there was no significant difference between the PCOS group and PCOS with HFD group.The expression levels of MIF,p-JNK,and p-p38 in the PCOS group (0.4048±0.013,0.6233±0.093 and 0.7987±0.061)and PCOS withHFD group (0.1929±0.012,0.3346±0.103 and 0.3468±0.031)were obviously higher than those in control group (0.2492±0.013, 0.3271±0.093 and 0.3393±0.061),but no Significant difference was observed between PCOS group and PCOS with HFD group.It was suggested that MIF may participate in the pathogenesis of PCOS through the MAPK signalling pathway in PCOS rats induced by DHEA.

A new potential risk factor in patients with erectile dysfunction and premature ejaculation： folate deficiency

We investigated serum folic acid （FA） levels in patients with erectile dysfunction （ED） and/or premature ejaculation （PE）. Fasting serum samples were obtained from 42 patients with ED, 36 with PE, 25 ED patients with PE, and 30 healthy men; the mean intravaginal ejaculation latency time （IELT） was measured during a 4 weeks baseline period. Levels of sex hormones （follicle-stimulating hormone, luteinizing hormone, total testosterone）, homocysteine （Hcys）, and FA were measured using chemiluminescent immunoassays. The sexual functions of PE patients and normal control men were evaluated using the Chinese Index of Premature Ejaculation （CIPE）. The abridged Intemational Index of Erectile Function-5 （IIEF-5） questionnaire was used to gauge erectile quality for ED patients and for normal controls. Serum FA concentrations were lower in ED （7.61 ~ 3.97 ng ml-1）, PE （9.37 ＋ 3.40 ng ml-1）, and ED/PE （8.84 ＋ 4.28 ng m1-1） patients than in healthy men （12.23 ~ 5.76 ng ml-z, P〈 0.05）. No significant differences in sex hormone levels were found between patients with sexual dysfunction and healthy controls （P 〉 0.05）. There were positive correlations between serum FA concentrations and ClPE scores （r= 0.530, P〈 0.01）, IIEF-5 scores （r= 0.589, P〈 0.01）, and IELT （r= 0.445, P〈 0.01）; negative correlations with Hcys concentrations （r= -0.487, P〈 0.01） were found in all participants. These findings showed a strong relationship between serum FA levels and sexual dysfunction, possibly due to an effect of FA on the metabolism of nitric oxide, Hcys, and 5-hydroxytryptamine.

Apoptosis and Expression of Protein TRAIL in Granulosa Cells of Rats with Polycystic Ovarian Syndrome

The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in apoptosis of granulosa cells were explored. By using sodium prasterone sulfate rat PCOS model was induced. The apoptosis of granulosa cells in ovaries of rats was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL), and the expression of TRAIL protein and mRNA in granulosa cells was detected by using immunhistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) respectively. The apoptotic rate and the expression of protein TRAIL in granulosa cells were significantly higher in antral follicles from the PCOS rats than in those from the control rats (P<0.01, P<0.05). There was no significant difference in apoptotic rate and the expression of TRAIL protein in granulosa cells of preantral follicles between the PCOS rats and the control rats (P>0.05). No apoptosis and the expression of TRAIL protein in granulosa cells of primordial follicles were found in the two groups. The expression of TRAIL mRNA was significantly stronger in granulosa cells from the PCOS rats than in those from the con- trol rats (P<0.01). It was suggested that the apoptotic rate in granulosa cells was significantly higher in antral follicle from the PCOS rats than in those from the control rats. TRAIL played a role in regu- lating the apoptosis of granulosa cells in PCOS rats.

Prevalent false positives of azoospermia factor a （AZFa） microdeletions caused by single-nucleotide polymorphism rs72609647 in the sY84 screening of male infertility

Multiplex polymerase chain reaction （PCR） has been widely used to detect Y-chromosome micredeletions, which is one of the major causes of male infertility. Both the European Academy of Andrology （EAA） and the European Molecular Genetics Quality Network （EMQN） have recommended the use of sY84 and sY86 markers for the detection of azoospermia factor a （AZFa） microdeletion during DNA testing for male infertility. In this study, a large-scale analysis of AZF microdeletion in a total of 630 Chinese males, including healthy semen donors （n=200）, infertile males with normal sperm count （n=226） and patients with either nonobstructive azoospermia or severe oligozoospermia （n=204）, was performed. A series of nine sequence-tagged site （STS） markers from the AZF region of the Y chromosome was used to detect microdeletions. All primers were designed based on the recommendations of the National Center for Biotechnology Information. An unusually high incidence （73/630, 11.6%） of sY84-absent but sY86-present genotypes was observed in the AZFa microdeletion screening. Sequencing the sY84-flanking region revealed a total of 73 patients with sY84-absent but sY86-present genotypes have a T-to-G transversion at the fifth base from the 5＇ end of the reverse sY84 primer. These prevalent false positives, which were not only observed in infertile men, but also observed in donors, resulted from a single-nucleotide polymorphism （SNP） named rs72609647 in the targeting sequence of the reverse sY84 primer. Our study suggests that a pre-screening of existence of rs72609647 polymorphism can prevent the frequent false positive results of AZFa microdeletions detection in the infertile Chinese males. Given the SNP rs72609647 was recently found in a deep sequencing of a Chinese individual, the current EAA and EMQN standards may need to be scrutinized among different populations to avoid the potential genetic variations in the primer binding sequences.

Up-regulated Expression of Tim-3/Gal-9 at Maternal-fetal Interface in Pregnant Woman with Recurrent Spontaneous Abortion

The relationship between T cell immunoglobulin domain and mucin domain protein 3（Tim-3）/Galectin（Gal）-9 pathway and recurrent spontaneous abortion（RSA） was studied. Thirty-one pregnant women with RSA and 27 normal early gravidas were investigated to detect the levels of Tim-3 and Gal-9 in villi and deciduas by Western blotting. Meanwhile, the concentration of interleukin（IL）-4 and IL-12 in peripheral blood plasma was determined by ELISA in 25 healthy fertile non-pregnant controls, the normal early gravidas and pregnant women with RSA mentioned above, respectively. It was found that the relative expression levels of Tim-3 and Gal-9 in villi and deciduas were significantly increased in pregnant women with RSA as compared with those in the normal early gravidas. The concentration of IL-4 in peripheral blood plasma of pregnant women with RSA was lower than that of the normal early gravidas（P〈0.05） and healthy fertile non-pregnant controls（P〈0.05）, but that of IL-2 in pregnant women with RSA was significantly higher than that of the normal early gravidas（P〈0.05） and healthy fertile non-pregnant controls（P〈0.05）. It was suggested that the overexpression of Tim-3/Gal-9 pathway may be related to the pathogenesis of RSA.

Clinical Observation on Treatment of 2,062 Cases of Immune Infertility with Integration of Traditional Chinese Medicine and Western Medicine

To study the therapeutic effect of integrated traditional Chinese medicine and western medicine on female immune infertility. 3,496 women suffering from primary or secondary infertility had their ASAb, EMAb,AOAb and ACAb level tested, with the positive rate of 23.11%, 34.95%, 20.77% and 30.41% respectively.2,062 positive cases were periodically treated with the Chinese drug Xiaokangwan (消抗丸) plus dexamethasone, vitamin E and vitamin C for 2 periods as a course of treatment. At the end of a treatment course, the rate for the antibodies to turn negative reached over 85% and the average pregnant rate reached 36.66%. The treatment of immune infertility with the integrated approach can reduce or eliminate the influence of antibodies in the serum of patients on various links of pregnancy, thus reaching the goal of curing infertility.