Early apoptotic changes in human spermatozoa and their relationships with conventional semen parameters and sperm DNA fragmentation

Aim： To investigate whether early apoptotic changes in spermatozoa can be significant markers for sperm quality. Methods： Two early apoptotic changes in the semen of 56 men were assessed using Annexin V （AN）/propidium iodide （PI） staining for phosphatidylserine externalization and JC-1 staining for mitochondrial membrane potential （MMP）. The results were compared with conventional semen parameters and DNA fragmentation identified using the TUNEL assay. Results： The different labeling patterns in the bivariate Annexin V/PI analysis identified four distinctive spermatozoa populations. The percentage of AN^-/PI^- spermatozoa positively correlated with conventional semen parameters and MMP, but negatively correlated with TUNEL （＋） spermatozoa. As for the AN^-/PI^＋ fraction, we found an opposite result in comparison to AN^-/PI^- spermatozoa. The level of early apoptotic AN^＋/PI^- spermatozoa negatively correlated with MMP and sperm motility. The level of late apoptotic AN^＋/PI^＋ spermatozoa negatively correlated with conventional semen parameters and MMP, and positively correlated with TUNEL （＋） spermatozoa. MMP positively correlated with conventional semen parameters, but negatively correlated with TUNEL （＋） spermatozoa. Conclusion： Although early apoptotic AN^＋/PI^- spermatozoa only negatively correlates with sperm motility, the differences in proportion of each subpopulation of spermatozoa （especially, the percentage of AN^-/PI^- spermatozoa）, and decreased MMP might be significant markers for diagnosing male infertility. They possibly bring additional information to predict the outcome of in vitro fertilization. （Asian J Androl 2008 Mar; 10： 227-235）

Ileal pouch anal anastomosis with modified double-stapled mucosectomy-the experience in China

AIM:To investigate the feasibility and long-term functional outcome of ileal pouch-anal anastomosis with modified double-stapled mucosectomy.METHODS:From January 2002 to March 2011,fourtyfive patients underwent ileal pouch anal anastomosis with modified double-stapled mucosectomy technique and the clinical data obtained for these patients were reviewed.RESULTS:Patients with ulcerative colitis(n = 29) and familial adenomatous polyposis(n = 16) underwent ileal pouch-anal anastomosis with modified doublestapled mucosectomy.Twenty-eight patients underwent one-stage restorative proctocolectomy,ileal pouch anal anastomosis,protective ileostomy and the ileostomy was closed 4-12 mo postoperatively.Two-stage procedures were performed in seventeen urgent patients,proctectomy and ileal pouch anal anastomosis were completed after previous colectomy with ileostomy.Morbidity within the first 30 d of surgery occurred in 10(22.2%) patients,all of them could be treated conservatively.During the median follow-up of 65 mo,mild to moderate anastomotic narrowing was occurred in 4 patients,one patient developed persistent anastomotic stricture and need surgical intervention.Thirtyfive percent of patients developed at least 1 episode of pouchitis.There was no incontinence in our patients,the median functional Oresland score was 6,3 and 2 after 1 year,2.5 years and 5 years respectively.Nearly half patients(44.4%) reported "moderate functioning",37.7% reported "good functioning",whereas in 17.7% of patients "poor functioning" was observed after 1 year.Five years later,79.2% of patients with good function,16.7% with moderate function,only 4.2% of patients with poor function.CONCLUSION:The results of ileal pouch anal anastomosis with modified double-stapled mucosectomy technique are promising,with a low complication rate and good long-term functional results.

Outcomes of Preimplantation Genetic Diagnosis Cycles by Fluorescent In situ Hybridization of Infertile Males with Nonmosaic 47,XYY Syndrome

Background： The 47,XYY syndrome could result in fertility problems. However, seldom studies reported comprehensive researches on the embryonic development and pregnancy outcomes of these patients. This study aimed to evaluate the clinical outcomes of nonmosaic 47,XYY patients performed with fluorescent in situ hybridization （FISH） and preimplantation genetic diagnosis （PGD） treatment. Methods： This was a retrospective study. Between January 2012 and May 2017, 51 infertile males with nonmosaic 47,XYY syndrome underwent FISH-PGD were included in the study. According to sex chromosomal FISH results, embryos were classified as normal signal, no nuclei fixed, no signal in fixed nuclei, suspensive signal, and abnormal signal groups, respectively. The incidence of each group, the fixation rate, and hybridization rate were calculated. Embryonic development and pregnancy outcomes were also analyzed. The measurement data were analyzed with Student＇s t-test. The comparison of categorical data was analyzed with the Chi-square test and Fisher＇s exact test when expected cell count was 〈5. Results： The 53 PGD cycles with 433 embryos were analyzed. The fixation rate was 89.6%, while the hybridization rate was 96.4%. There were 283 embryos with two sex chromosomal signals with clear diagnosis （65.4%）. The numbers of no nuclei fixed, no signal in fixed nuclei, suspensive signal, and abnormal signal groups were 45 （10.4%）, 14 （3.2%）, 24 （5.5%）, and 67 （15.5%）, respectively. Embryos with abnormal signals were abandoned. The number of good-quality embryos was 210 （57.4%）, including implanted embryos on day 4/day 5 a.ld cryopreserved. The rates of good-quality embryos in the no nuclei fixed （22.2%）, no signal in fixed nuclei （28.6%）, and suspensive signal groups （33.3%） were comparable （P 〉 0.05）, and were significantly lower than the normal signal group （66.4%, P 〈 0.001 ）. The clinical pregnancy rates of fresh and frozen embryos transferred cycles were 70.6% and 85.7%, respectively. Conclusions： Among embryos with a clear diagnosis of sex chromosome, about one-fifth showed abnormal signals. Embryos with two sex chromosomal signals are more likely to develop into good-quality ones. The application of the PGD by FISH may help to improve the clinical outcomes.

Decline of semen quality among Chinese sperm bank donors within 7 years （2008-2014）

Semen from 5210 sperm bank donors was analyzed and trends in semen quality were evaluated at Shandong Human Sperm Bank between 2008 and 2014. After 2-7 days of abstinence, semen samples were collected. Measurements of semen volume, sperm concentration, sperm forward motility, and total sperm count were performed. There were significant declining trends in semen volume, sperm concentration, sperm forward motility, and total sperm count. Our results indicate that the quality of semen in this cohort of sperm donors had decreased during the study period.

A new TEX11 mutation causes azoospermia and testicular meiotic arrest

There are many unknown genetic factors that lead to infertility in nonobstructive azoospermia men.Here,we performed whole-exome sequencing in blood samples obtained from 40 azoospermia patients with meiotic arrest and found a novel c.151_154del(p.D51fs)frame-shift mutation in exon 3 of the testis expressed 11(TEX11)gene in one patient.Sanger sequencing analysis of the patient and 288 fertile men was performed to validate the mutation.Immunohistochemical analysis showed TEX11 expression in late-pachytene spermatocytes and in round spermatids in fertile human testes.In contrast,testes of the patient with TEX11 mutation underwent meiotic arrest and lacked TEX11 expression.Western blotting of human embryonic kidney(HEK293)cells transfected with a vector for the p.D51fs TEX11 variant detected no TEX11 expression.In conclusion,we identified a novel frame-shift mutation in the TEX11 gene in an azoospermia patient,emphasizing that this gene should be included in genetic screening panels for the clinical evaluation of azoospermia patients.

Predictive significance of serum inhibin B on testicular haploid gamete retrieval outcomes in nonobstructive azoospermic men

The purpose of this study was to determine the diagnostic accuracy of serum inhibin B(INHB)as a predictor of the retrieval outcome of testicular haploid gametes(spermatids and testicular spermatozoa)in nonobstructive azoospermic men.Serum hormone levels,testicular volume,and histological evaluation were performed in 403 Chin ese non obstructive azoospermic men.Testicular haploid gamete was successfully retrieved in 213 of 403 patients(52.85%).The haploid gamete group always had higher INHB levels than the non-haploid gamete group.According to the receiver operating characteristic(ROC)curve analysis,INHB was a good predictor of testicular haploid gamete retrieval outcome in all patients(sensitivity:77.93%and specificity:91.58%)and patients with normal follicle-stimulating hormone(FSH;sensitivity:88.52%and specificity:70.83%).The area under the ROC curve(AUC)of INHB was similar to that of FSH in all patients or patients with normal FSH.In patients with elevated FSH,INHB was superior to FSH in predicting the presence of haploid gamete(AUC:0.73 vs 0.55,P<0.05),with a sensitivity of 60.00%and a specificity of 80.28%.It con eluded that serum INHB as an effective marker for spermatoge nesis was a sign ificant predictor of testicular haploid gamete retrieval outcomes in nonobstructive azoospermic men.Especially,INHB is superior to FSH in predicting the presenee of haploid gamete in the patients with elevated FSH.

Mutational analysis of the GATA4 gene in Chinese men with nonobstructive azoospermia

As a crucial transcription factor for spermatogenesis,GATA-binding protein 4(GATA4)plays important roles in the functioning of Sertoli and Leydig cells.Conditional knockout of GATA4 in mice results in age-dependent testicular atrophy and loss of fertility.However,whether GATA4 is associated with human azoospermia has not been reported.Herein,we analyzed the GATA4 gene by direct sequencing of samples obtained from 184 Chinese men with idiopathic nonobstructive azoospermia(NOA).We identified a missense mutation(c.191G>A,p.G64E),nine single-nucleotide polymorphisms(SNPs),and one rare variant(c.^(*)84C>T)in the 3′untranslated region(UTR).Functional studies demonstrated that the p.G64E mutation did not affect transactivation ability of GATA4 for spermatogenesis-related genes(claudin-11 and steroidogenic acute regulatory protein,Star),and the 3′UTR rare variant c.^(*)84C>T did not generate microRNA-binding sites to repress GATA4 expression.To our knowledge,this is the first report to investigate the association between GATA4 and azoospermia;our results indicate that mutations in GATA4 may not be pathogenic for NOA in Chinese men.