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.

MicroRNA-218 is upregulated in gastric cancer after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy and increases chemosensitivity to cisplatin

AIM: To investigate the molecular mechanisms of miRNA in advanced gastric cancers(AGCs) before and after cytoreductive surgery(CRS) + hyperthermic intraperitoneal chemotherapy(HIPEC). METHODS: A miRNA microarray containing human mature and precursor miRNA sequences was used to compare expression profiles in serum samples of 5 patientswith AGC before and after CRS + HIPEC. The upregulation of miR-218 was confirmed by real-time reverse transcription polymerase chain reaction and its expression was analyzed in SGC7901 gastric cancer cells. RESULTS: miRNA microarray chip analysis found that the level of miR-218 expression was upregulated more than 8 fold after CRS + HIPEC. Furthermore, miR-218 increased gastric cancer cell chemosensitivity to cisplatin in vitro and inhibited gastric cell tumor growth in nude mice in vivo(0.5 vs 0.78, P < 0.05).CONCLUSION: Our results indicated that targeting miR-218 may provide a strategy for blocking the development of gastric cancer and reverse the multi-drug resistance of gastric cell lines.

Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF

The aim of this study was to investigate whether the sperm chromatin structure assay （SCSA） results after swim-up are related to fertilization rates, embryo quality and pregnancy rates following in vitrofertilization （IVF）. A total of 223 couples undergoing IVF in our hospital from October 2008 to September 2009 were included in this study. Data on the IVF process and sperm chromatin structure assay results were collected. Fertilization rate, embryo quality and IVF success rates of different DNA fragmentation index （DFI） subgroups and high DNA stainability （HDS） subgroups were compared. There were no significant differences in fertilization rate, clinical pregnancy or delivery rates between the DFI and HDS subgroups. However, the group with abnormal DFI had a lower good embryo rate. So, we concluded that the SCSA variables, either DFI or HDS after swim-up preparation, were not valuable in predicting fertilization failure or pregnancy rate, but an abnormal DFI meant a lower good embryo rate following IVF.

Proteomic analysis of seminal plasma from asthenozoospermia patients reveals proteins that affect oxidative stress responses and semen quality

Asthenozoospermia （AS） is a common cause of human male infertility. In one study, more than 80% of the samples from infertile men had reduced sperm motility. Seminal plasma is a mixture of secretions from the testis, epididymis and several male accessory glands, including the prostate, seminal vesicles and Cowper＇s gland. Studies have shown that seminal plasma contains proteins that are important for sperm motility. To further explore the pathophysiological character of AS, we separated the seminal plasma proteins from AS patients and healthy donors using sodium dodecyl sulfate polyacrylamide gel electrophoresis and in-gel digestion, and then subjected the proteins to liquid chromatography-mass spectrometry （LC-MS/MS） analysis. A total of 741 proteins were identified in the seminal plasma, with a false discovery rate of 3.3%. Using spectral counting, we found that 45 proteins were threefold upregulated and 56 proteins were threefold downregulated in the AS group when compared with the control. Most of these proteins originated from the epididymis and prostate. This study identified a rich source of biomarker candidates for male infertility and indicates that functional abnormalities of the epididymis and prostate can contribute to AS. We identified D J-1--a protein that has been shown elsewhere to be involved in the control of oxidative stress （OS）-as a downregulated protein in AS seminal plasma. The levels of D J-1 in AS seminal plasma were about half of those in the control samples. In addition, the levels of reactive oxygen species were 3.3-fold higher in the AS samples than in the controls. Taken together, these data suggest that downregulation of DJ-1 is involved in OS in semen, and therefore affects the quality of the semen.

Malignant adenomyoepithelioma of the breast:Two case reports and review of the literature

BACKGROUND Malignant adenomyoepithelioma(AME)of the breast is a rare tumor in which malignancy can arise from either epithelial or myoepithelial components,or from both cell types.The incidence and prognosis of malignant AME of the breast are difficult to assess due to its rarity.Therefore,the optimal treatment for this disease is still controversial.CASE SUMMARY We present two middle-aged women(48 and 56 years old)with malignant AME of the breast.Core needle biopsy was performed before surgery.However,breast adenoma and malignant tumors were observed.The preoperative diagnosis of malignant AME of the breast is still challenging for pathologists and clinicians.Both patients underwent mastectomy and sentinel lymph node biopsy,both of which were negative,followed by adjuvant chemotherapy.CONCLUSION The follow-up duration of the two patients was two years and four months,respectively.No signs of relapse or metastasis have been observed thus far.

The PIWI-specific insertion module helps load longer piRNAs for translational activation essential for male fertility

PIWI-clade proteins harness pi RNAs of 24–33 nt in length.Of great puzzles are how PIWI-clade proteins incorporate pi RNAs of different sizes and whether the size matters to PIWI/pi RNA function.Here we report that a PIWI-Ins module unique in PIWIclade proteins helps define the length of pi RNAs.Deletion of PIWI-Ins in Miwi shifts MIWI to load with shorter pi RNAs and causes spermiogenic failure in mice,demonstrating the functional importance of this regulatory module.Mechanistically,we show that longer pi RNAs provide additional complementarity to target m RNAs,thereby enhancing the assembly of the MIWI/e IF3f/Hu R super-complex for translational activation.Importantly,we identify a c.1108C>T(p.R370W)mutation of HIWI(human PIWIL1)in infertile men and demonstrate in Miwi knock-in mice that this genetic mutation impairs male fertility by altering the property of PIWI-Ins in selecting longer pi RNAs.These findings reveal a critical role of PIWI-Ins-ensured longer pi RNAs in fine-tuning MIWI/pi RNA targeting capacity,proven essential for spermatid development and male fertility.

Effect of advanced paternal age on reproductive outcomes in IVF cycles of non-male-factor infertility: a retrospective cohort study

Advanced paternal age has been overlooked,and its effect on fertility remains controversial.Previous studies have focused mainly on intracytoplasmic sperm injection(ICSI)cycles in men with oligozoospermia.However,few studies have reported on men with semen parameters within reference ranges.Therefore,we conducted a retrospective cohort study analyzing the reproductive outcomes of couples with non-male-factor infertility undergoing in vitro fertilization(IVF)cycles.In total,381 cycles included were subgrouped according to paternal age(<35-year-old,35–39-year-old,or≥40-year-old),and maternal age was limited to under 35 years.Data on embryo quality and clinical outcomes were analyzed.The results showed that fertilization and high-quality embryo rates were not significantly different(all P>0.05).The pregnancy rate was not significantly different in the 35–39-year-old group(42.0%;P>0.05),but was significantly lower in the≥40-year-old group(26.1%;P<0.05)than that in the<35-year-old group(40.3%).Similarly,the implantation rate significantly decreased in the≥40-year-old group(18.8%)compared with that in the<35-year-old group(31.1%)and 35–39-year-old group(30.0%)(both P<0.05).The live birth rate(30.6%,21.7%,and 19.6%)was not significantly different across the paternal age subgroups(<35-year-old,35–39-year-old,and≥40-year-old,respectively;all P>0.05),but showed a declining trend.The miscarriage rate significantly increased in the 35–39-year-old group(44.8%)compared with that in the<35-year-old group(21.0%;P<0.05).No abnormality in newborn birth weight was found.The results indicated that paternal age over 40 years is a key risk factor that influences the assisted reproductive technology success rate even with good semen parameters,although it has no impact on embryo development.

Association of symptomatic late-onset hypogonadism and lower urinary tract symptoms in aging males: a community-based study

Objective:Testosterone deficiency may be a risk factor for lower urinary tract symptoms(LUTS),and there may be a causal ink between the emergence of LUTS and the incidence of late-onset hypogonadism(LOH).We perfomed an epidemiologic study to investigate the association between symptomatic late-onset hypogonadism(SLOH)and LUTS in middle-aged and elderly rural Chinese males.Methods:A total of 965 men completed a questionnaire and underwent a detailed physical examination.The Aging Males'Symptoms(AMS)scale was used to assess SLOH,and the International Prostate Symptom Score(IPSS)questionnaire was used to assess LUTS.Serum reproductive hormone levels of testosterone,sex hormone-binding globulin(SHBG)and luteinizing hormone(LH)were measured.Results:A total of 965 males(mean age:56.34±8.85,range:40-80 years)were recruited for the present study.A total of 20.93%(202/965)were diagnosed with SLOH.A total of 93.16%(899/965)had mild LUTS,5.18%(50/965)had moderate LUTS,and 1.66%(16/965)had severe LUTS.Among SLOH patients,13.40%(27/202)and 3.90%(8/202)had moderate and severe LUTS,respectively.Patients with severe LUTS had increased SHBG and LH compared with those with mild and moderate LUTS(P<0.01).Correlation analysis revealed that the AMS total score was positively correlated with the IPSS score(P<0.05).The prevalence of SLOH was significantly increased with LUTS severity In addition to the known effect of age,the results of mutiple regression analysis also showed that serum LH or SHBG appeared to have a weak link with SLOH and LUTS that requires etiological and biological clarification in our future study.Conclusion:In this cross-sectional analysis of SLOH and LUTS,LUTS severity was significantly associated with hypogonadism symptoms.Additionally,the prevalence of SLOH advanced with increasing LUTS severity.Serum SHBG or LH showed a positive correlation with SLOH and LUTS.

严重男性不育症ICSI治疗能获得与同源卵子供精IVF预期的胚胎结局

Azoospermia, cryptozoospermia and necrospermia can markedly decrease the ability of males to achieve pregnancy in fertile females. However, patients with these severe conditions still have the option to be treated by intracytoplasmic sperm injection （ICSI） to become biological fathers. This study analyzed the fertilization ability and the developmental viabilities of the derived embryos after ICSI treatment of the sperm from these patients compared with in vitro fertilization （IVF） treatment of the proven-fertile donor sperm on sibling oocytes as a control. On the day of oocyte retrieval, the number of sperm suitable for ICSI collected from two ejaculates or testicular sperm extraction was lower than the oocytes, and therefore, excess sibling oocytes were treated by IVF with donor sperm. From 72 couples （73 cycles）, 1117 metaphase Ⅱ oocytes were divided into 512 for ICSI and 605 for IVF. Compared with the control, husbands＇ sperm produced a lower fertilization rate in nonobstructive azoospermia （65.4% vs 83.2%; P〈 0.001）, crytozoospermia （68.8% vs 75.5%; P〈 0.05） and necrospermia （65.0% vs 85.2%; P〈 0.05）. The zygotes derived in nonobstructive azoospermia had a lower cleavage rate （96.4% vs 99.4%; P 〈 0.05）, but the rate of resultant good-quality embryos was not different. Analysis of the rates of cleaved and good-quality embryos in crytozoospermia and necrospermia did not exhibit a significant difference from the control. In conclusion, although the sperm from severe male infertility reduced the fertilization ability, the derived embryos had potential developmental viabilities that might be predictive for the expected clinical outcomes.

Prohibitin(PHB)interacts with AKT in mitochondria to coordinately modulate sperm motility

Prohibitin(PHB),an evoluti on arily con served mitochondrial inner membra ne protein,is highly expressed in cells that require strong mitoch on drial function.Recently,we dem on strated that the deleti on of Phb in spermatocytes results in impaired mitochondrial function.In addition,PHB expression in the mitochondrial sheath of human sperm has a significantly negative correlation with mitochondrial reactive oxygen species levels,but a positive one with mitochondrial membrane potential and sperm motility.These results suggest that mitochondrial PHB expression plays a role in sperm motility.However,the mechanism of PHB-mediated regulation of sperm motility remai ns unk nown.Here,we dem on strate for the first time that PHB interacts with protei n kinase B(AKT)and exists in a complex with phospho-PHB(pT258)and phospho-AKT in the mitochondrial sheath of murine sperm,as determined using colocalization and coimmunoprecipitation assays.After blocking AKT activity using wortmannin(a phosphatidylinositol 3-kinase[PI3K]inhibitor),murine sperm have significantly(P<0.05)decreased levels of phospho-PHB(pT258)and the total and progressive motility.Furthermore,significantly(P<0.05)lower levels of phospho-PI3K P85 subunit a+γ(pY199 and pY46)and phospho-AKT(pS473;pT308)are found in sperm from infertile asthenospermic and oligoasthenospermic men compared with no rmospermic subjects,which suggest a reduced activity of the PI3K/AKT pathway in these in fertile subjects.Importantly,these sperm from infertile subjects also have a significantly(P<0.05)lower level of phospho-PHB(pT258).Collectively,our findings suggest that the interaction of PHB with AKT in the mitochondrial sheath is critical for sperm motility,where PHB phosphorylation(pT258)level and PI3K/AKT activity are key regulatory factors.

Sperm-specific protein ACTL7A as a biomarker for fertilization outcomes of assisted reproductive technology

Obtaining high-quality embryos is one of the key factors to improve the clinical pregnancy rate of assisted reproductive technologies(ART).So far,the clinical evaluation of embryo quality depends on embryo morphology.However,the clinical pregnancy rate is still low.Therefore,new indicators are needed to further improve the evaluation of embryo quality.Several studies have shown that the decrease of sperm-specific protein actin-like 7A(ACTL7A)leaded to low fertilization rate,poor embryo development,and even infertility.The aim of this study was to study whether the different expression levels of ACTL7A on sperm can be used as a biomarker for predicting embryo quality.In this study,excluding the factors of severe female infertility,a total of 281 sperm samples were collected to compare the ACTL7A expression levels of sperms with high and low effective embryo rates and analyze the correlation between protein levels and in-vitro fertilization(IVF)laboratory outcomes.Our results indicated that the ACTL7A levels were significantly reduced in sperm samples presenting poor embryo quality.Furthermore,the protein levels showed a significant correlation with fertilization outcomes of ART.ACTL7A has the potential to be a biomarker for predicting success rate of fertilization and effective embryo and the possibility of embryo arrest.In conclusion,sperm-specific protein ACTL7A has a strong correlation with IVF laboratory outcomes and plays important roles in fertilization and embryo development.

Effects of Cryopreservation on Human Sperm Glycocalyx

Background:To study the effects of cryopreservation on human sperm glycocalyx.Methods:The lectin binding profilings of sperm after freeze-thaw were compared by lectin microarray.Results:CryoSperm^(TM) and direct fumigation were confirmed to be the optimized cryoprotectant and method by comparing the sperm recovery rate.In 91 lectins,33 lectins were significantly changed after sperm cryopreservation.Among them,9 lectins greatly decreased and 24 lectins mainly increased.The binding signals of MAA,PSA,ABA,and AIA were verified by FACS,and the results were consistent with that of lectin microarray.Conclusions:Sperm glycocalyx had significant changes after cryopreservation.The sialic acid,playing an important role in protecting sperm,was greatly lost,which exposed the inner carbohydrates.Thus,the glycocalyx damage due to the cryopreservation might be one of the reasons for low sperm fertility.