Comparison of Three Different Techniques of Human Sperm DNA Isolation for Methylation Assay

Summary： Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method （method A）, traditional phenol-chloroform method （method B）, and TianGen kit method （method C）. Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C （P〈0.01）. PCR results revealed that method A was more reliable in amplifying DEAD-box polypeptide 4 （DDX4） and copy number variations （CNVs） than methods B and C, which generated false-positive errors. The results of sperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reli- able results and could be an optimal technique for extracting sperm DNA for methylation assay.

Expression and Significance of NF-κB, IL-1β and COX-2 in the Murine Model of Estrogen-dependent Experimental Vulvovaginal Candidiasis

Objective To investigate the possible role of estrogen in the pathogenesis of vulvovaginal candidiasis（VVC）. Methods Estrogen-dependent experimental murine model of C. albicans vaginal infection was established by injecting subcutaneously with estradiol benzoate and then 5 × 10^6 stationary-phase C. albicans blastoconidia was inoculated intravaginally to mice （group El）, and other 3 groups were set up.＂ estrogen-treated but not infected （group E）; estrogen-untreated but infected （group I）; normal control （group C）. The dynamic change of colony-forming unit （CFU） of cervivovaginal lavage fluid was observed. Vaginal tissues at different time points （d 2, d 4, d 7 and d 14） after inoculation of C. albicans were obtained. In situ hybridization staining was used to detect expression of cyclooxygenase-2 （COX-2） mRNA and expression of nuclear factor-κB （NF-κB） was examined by immuohistochemistry. ELISA was applied to determine the interlenkin-1β （IL-1β） level. Results The constitutional high level expression of COX-2 mRNA in the vaginal tissue of group E was significantly higher than that in group C on d 4 and d 7 （P〈0. 01）, and the optical density （OD） in group EI was higher than that in the other 3 groups （P〈0.05）. There were higher IL-1β levels in vaginal tissues from d 4 to d 7 postinoculation in group EI and group I than group C （P〈0.01）. Furthermore, IL-1β in group EI was markedly elevated on d 4 and d 7 compared with group I （P〈0.01）. Compared with group C, the expression of NF-κB in group E was increased obviously on d 4 （P〈0.01）, and there was significant difference between group E1 and group I on d 4 and d 7 （P〈0.01）. Conclusions In the murine model of estrogen-dependent experimental WC, estrogen promotes the infection establishment by up-regulating expression of COX-2 via activating NF-κB signal pathway, and the high expression of COX-2 promoted by the interaction of IL-1β and NF-κB after infection formation was involved in persistence of infection.

Endothelial Nitric Oxide Synthase Traffic Inducer in the Umbilical Vessels of the Patients with Pre-eclampsia

The expression of endothelial nitric oxide synthase traffic inducer （NOSTRIN） was examined in the umbilical vessels of the patients with pre-eclampsia （PE） to explore its possible role in the pathogenesis of PE. The NOSTR1N rnRNA in umbilical tissues was determined by RT-PCR. The eNOS activity in umbilical vessels was spectrophotometrically detected. NO2 /NO3, the stable metabolic end products of NO, was measured by using nitrate reductase. RT-PCR showed that the expression level of NOSTRIN was significantly higher in women with PE than in the normal group （P〈0.01）. The activity of eNOS was significantly decreased in PE group [（12.83±3.61） U/mg] than in normal group [（21.72±3.83） U/mg] （P〈0.01）. The level of NO2-/NO3- in PE patients （27.53±7.48） pmol/mg was significantly lower than that of normal group （54.27±9.53） μmol/mg （P〈0.01）. The significant negative correlation existed between the expression of NOSTRIN and the activity of eNOS in umbilical vessels of women with PE （r=-0.58, P〈0.01）. It was concluded that the level of NOSTR1N expression was increased in umbilical vessel of women with PE, indicating that it may be involved in the pathogenesis of PE.

Expression of MCMV M83 mRNA in Infected Testes and Its Relationship with Testicular Lesions

Objective To investigate the expression of routine cytomegalovirus （MCMV） M83 mRNA in mouse testes at the different periods of MCMV infection and its relationship with the severity of testicular lesions Methods Used digoxin-labeled MCMV mRNA oligonucleotide probe, the mouse testicular tissues at the different periods following MCMV inoculation were detected by in situ hybridization（ISH）. The average absorbency （A） of ISH positive signals represented the resticular MCMV load. The expression of MCMV M83 mRNA in the infected testes and its relation with the severity of testicular lesions were analyzed. Results At different periods after MCMV inoculation, the expression of MCMV M83 mRNA was detectable in the testicular tissues. The ISH positive signals were mainly located in cytoplasm of the testicular interstitial cells. The average A values at d 9 after MCMV infection were the highest （comparison between the experimental groups, P〈O. O01）. There were pathological inJTammatory changes to varying degrees in seminiferous epithelia and interstitial cells at the diJferent periods of infection, especially during d 6 to d 9. Conclusion In testicular MCMV infection, the levels of MCMV M83 mRNA were related with the severity of testicular lesions, suggesting the dynamic detection of MCMV M83 mRNA could evaluate the prognosis of MCMV infection.

Enhancing Antitumor by Immunization with Fusion of Dendritic Cells and Engineered Tumor Cells

A novel approach for a dentritic cells (DCs) based tumor vaccine was developed for the formation of hybrid engineered J558 after fusion with DCs. To make the hybrid tumor vaccine generate more efficient specific CTL cytotoxicity against wild type tumor cells, we genetically engineered tumor cells with mIL 12 gene prior to the cell fusion. mIL 12 was detected at 870±60 pg/(10 5 cells/ml) in the culture supernatants and the fusion ratio was about 30 % by the co focal microscopic analysis. Vaccination of mice with DCs fused with engineered J558 induced more efficient tumor specific CTL cytotoxicity against wild type tumor cells in vitro and with efficient antitumor immunity in vivo . These results suggest that this approach of using DCs fused with engineered tumor cells could be applied in clinical settings of DCs based cancer vaccines.

Development of Mutiplex Polymerase Chain Reaction for the Detection and Differentiation of Enteric Adenoviruses in Stool Samples

To find a technique of detecting and differentiating enteric adenoviruses (EAds) in clinical samples, a novel PCR approach was developed. EAds were able to be detected by use of a pair of subgroup F general primers (P1 and P2), and they were also be able to be differentiated from each other in the presence of another adenovirus type 40 (Ad40) specific primer (P3) in the same tube. Our results showed that there was one band for Ad41 and two bands for Ad40, respectively, on running-gel after PCR performance. PCR was performed on 40 specimens in parallel directly with dot-hybridization assay on the same diluted stool samples. 20 of 40 specimens were positive by hybridization (of them 12 were Ad41 and 8 were Ad40), whereas 26 were positive by PCR performance on the same samples with Ad41 18 and Ad40 8 positive as well. Our study indicated that this novel method could be used in clinical laboratory or in epidemic investigation for Eads

Effect of rhTNF-α on Mitochondrial Transmembrane Potential and Motility of Human Sperm in vitro by Flow Cytometry and Computer Aided of Semen Analysis

To evaluate effect of recombined human tumor necrosis factor （rhTNF- α） on mitochondrial transmembrane potential and motility of human sperm in vitro Methods Semen samples for study were obtained from 40 health men （average age 26 ± 1.2 years） with normal semen analysis. Sperm suspension with computer aided of semen analysis （CASA） technique; 2） were stained in the presence of 10 μg/ml Rh123 and PI, mitochondrial transmembrane potential of those was analyzed by flow cytometry （FCM）. Results Significant differences were found between experimental groups and control groups on viability, straight line velocity, curvilinear velocity, average path velocity, progressive motility of human sperm and number of sperm with normal mitochondrial transmembrane potential （P〈0.01） expect final concentration 30 pg/ml group （P〉0. 05）. Sperm motility lowed with increasing rhTNF-α concentration and incubating time （P〈0. 01）. Number of sperm with normal mitochondrial transmembrane potential decreased with increasing rhTNF-α concentration and incubating time （P〈0.01）. Conclusion rh TNF-α can decrease human sperm motility function in vitro, which can interfere the function of human sperm mitochondrial transmembrane potential and may inhibit sperm mitochondrial enzymatic activities.

Effects of Murine Cytomegalovirus Infection on Sperm Viability in Mice

In order to explore the effects of testicular infection of murine cytomegalovirus （MCMV） on mature sperm viability at different periods following MCMV inoculation in mice, 91 BALB/c mice without MCMV infection were randomly divided into two groups： an experimental group （n= 56） and a control group （n=35）. The mice in the experimental group were treated by inoculating MCMV intratesticularly, while those in the controlled group were directly inoculated with DMEM without MCMV. The mice in both groups were sacrificed separately on the day 1,1.5, 2, 4, 6, 9 and 14 post-inoculation （D1, 1.5, 2, 4, 6, 9 and 14 PI）. The MCMV M83 mRNA gene was detected in the testis by in situ hybridization （ISH） with MCMV late-mRNA probe labeled with digoxin. Sperm viability of mature sperm in the epididymis cauda was measured. The results demonstrated the positive signal of ISH of MCMV was found mainly in the cytoplasm of the testicular interstitial cells and spermatogenic cells in the experimental group. Compared with that in the controlled group, the sperm viability in the experimental group was decreased significantly on D1 PI and D1.5 PI （P〈 0.05）. No statistically significant difference in the sperm viability was found after D2 PI between two groups （P〉0.05）. This suggested that sperm viability in mice might be descended significantly shortly after MCMV infection and might return to normal with time, indicating that MCMV acute infection might temporarily degrade sperm quality and influence procreation transiently.

Histopathologic changes of hypothalamus and pituitary in a rat model of polycystic ovary syndrome

Objective:To investigate the histopathologic changes of hypothalamus and pituitary in a rat model of polycystic ovary syndrome(PCOS). Methods:Rat model of PCOS was established in 6 immature female SD rats and another 6 immature rats treated with placebo were as control.The tissues of hypothalamus and pituitary were obtained and observed by light microscope and transmission electron microscope. Results:Light microscopy revealed little difference in morphology of neurons in arcuate nucleus or basophilic cells in pituitary between PCOS rats and normal rats.Electron microscopic examination showed that,compared with those in normal rats,GnRH neurons in PCOS rats were larger and fuller,with Golgi complex and mitochondria increased. The mitochondria were small,round and swelling.More high-density secretory granules and bright vesicles were observed in the cytoplasm.The Golgi complex near nucleus in pituitary gonadotropin cell in PCOS rats was fractured and expanded,and there were increased number mitochondria and different sizes of the higher electron density secretory granules in the cytoplasm. Conclusion:The morphological alterations in hypothalamus and pituitary could play a very important role in the development of PCOS.

Intracytoplasmic sperm injection in the treatment of male infertility due to obstructive or non-obstructive azoospermia

Objective: To evaluate the effects of intracytoplasmic sperm injection (ICSI) ontreatment of infertility due to obstructive and non-obstructive azoospermia..Methods: A retrospective analysis of fertilization, cleavage, embryo implantationand pregnancy rates was done in 158 ICSI cycles including 112 obstructive azoospermiaand 46 non-obstructive azoospermia. Ovarian hyperstimulation and ICSI procedureswere performed by conventional protocol. The sperm was collected by percutaneous epi-didymal sperm aspiration (PESA) or testicular sperm extraction (TESE).Results:The fertilization rate (73.1% vs. 67.0%), cleavage rate (88.6% vs. 86.3%), embryo implantation rate (20.7% vs. 11.4%), clinical pregnancy rate per trans-fer cycle (35.7% vs. 19.6%) were obtained for obstructive and non-obstructiveazoospermia, respectively.Conclusion: The results revealed that in the cases of obstructive azoospermia, ferti-lization rate, embryo implantation rate and clinical pregnancy rate were significantlyhigher than those of non-obstructive azoospermia. But there was no significant differ-ence of the cleavage rate between two groups.

Morphology of Rat Testis Preserved in Three Different Fixatives

Histopathological examination of testes is important in assessing spermatogenesis and testicular function. Modified Davidson＇s fluid （mDF） has been proposed as a superior substitute for Bouin＇s fluid （BF） for fixation of adult animal testes. Besides, 4% paraformaldehyde （PFA） has been commonly used to fix testes with convenience. We compared the morphology of the rat testis fixed in 4% PFA, mDF, or BF using hematoxylin and eosin （HE）-stained sections. Fixation in 4% PFA resulted in obvious tissue shrinkage artifacts, especially between seminiferous epithelium cells. Shrinkage artifacts were also observed in the central area of the testes fixed in BF. Use of mDF did not cause shrinkage artifacts between seminiferous tubules, though a small amount can be observed in seminiferous tubules between germ cells. Clarity of nuclear detail in testes fixed in mDF and BF is better compared to 4% PFA. Our study demonstrated that fixation in mDF provided better morphologic details in the rat testis as compared with 4% PFA and BF.

Urodynamic Characteristics of Awake Rats under Retrained versusFreely Moving Condition：Using a Novel Model

Urodynamic investigation in conscious rats is widely employed to explore functional bladder disorders of various etiologies and pathogeneses. Rats can be placed in restraining cages or wide cabinets where they are allowed to move freely during cystometry. However, the requirements of special devices hampered the application of urodynamic test in freely moving rats, and whether the restraint has any effects on urodynamic parameters in conscious rats remains obscure. In the present study, we described a novel approach for urodynamic investigation in both restrained and freely moving conscious rats. In addition, we for the first time systematically compared the urodynamic parameters of rats in the two conditions. With the current method, we successfully recorded stable and repeatable intravesical pressure traces and collected expected reliable data, which supported the idea that the restraint does not affect the activity of the micturition reflex in rats, provided sufficient and appropriate measures could be applied during cystometry. Fewer technique problems were encountered during urodynamic examination in restrained rats than in freely moving ones. Taken together, conscious cystometry in rats placed in restraining cages with proper managements is a reliable and practical approach for evaluating the detrusor activity and bladder function.

Concentration Changes of Medroxyprogesterone Acetate in Serum and Milk in Lactating Woman Who Used Depo Geston~

Objective To determine the concentration changes of medroxyprogesterone acetate （MPA） in serum and milk in the Chinese lactating women who used Depo Geston Methods Ten postpartum and breastfeeding women received an injectable contraceptive of Depo Geston （depo medroxyprogesterone acetate, DMPA, 150 mg/ampoule）. Serum and milk samples were collected in the 1^st, 2^st, 4^th, 6^th, 8^th 10^th and 12^th week after contraceptive injection. The concentrations of medroxyprogesterone acetate （MPA） in serum and milk were determined by radioimmunoassay. Results The highest MPA concentration in serum was observed at the 1^st week and then the level of serum MPA decreased obviously at the 2^na and 4^th week. After the 4^th week, the decline of serum MPA level slowed down. The concentration of milk MPA reached the highest level at 1^th week. But at the 2^nd week the average level of MPA in milk dropped by half The MPA concentration in the milk was changed in the range of 5.2-8.3 ng/mL in the following 10 weeks. During observation, the average ratios of concentration and area under curve （AUC） in milk/serum were both 0.55. There was a wide variation in MPA concentrations and ratios of milk/serum between subjects. Conclusion MPA concentrations maintained a certaim level at the 12^th week after DMPA injection. However, whether mather＇s intake of DMPA during feeding period has effect on children＇s growth, espcially pubertal growth, deserves further studies.

Screening on the Pharmacodynemic Active Parts of Protecting Liver of Peristrope japonica (thunb.)Bremek

The pharmacodynamic active parts of protecting liver of Peristrope japonica (thunb.)Bremek were identified. Rat acute liver injury model was induced by D-galactosamine (D-GlaN). The active parts were identified on the whole extraction and 4 fractions. The results showed that the pharmacodynamic active parts of Peristrope japonica were the n-BuOH fraction.

Investigation of the First Spermatorrhea Age of Males in Jiangsu Province, China

Background: The growth of adolescents is affected by economic development and education levels. We investigated the first spermatorrhea age of adolescents in urban area and in rural area of Jiangsu province, China. Methods: The data were obtained through anonymous questionnaire by using cross-sectional methods. From May 2016 to March 2017 in Jiangsu province, China, a total of 2131 Han nationality students were enrolled in cluster. The survey was contained date of birth, age, grade, age of first spermatorrhea and how long ago of the first spermatorrhea, all data were analyzed by Prism 8.0. Results: The questionnaire accounted for response was 97.3%, and the effective rate was 96.3%. The mean age of first spermatorrhea was (13.7 ± 1.5) years old, which included 13.6 ± 1.4 years in urban and 13.9 ± 1.5 years in rural. The first spermatorrhea occurred in the fifth and sixth grade primary school, and 1, 2, 3 grades of junior high school were 3.5%, 13%, 27.4%, 28.5% and 15.1%, respectively. There was 62.3% of first spermatorrhea occurred in summer. The rate of spermatorrhea with natural occurrence accounted for 95.1%, and with active accounted for 4.9%. Conclusions: Compared with rural area, the age of the first spermatorrhea of males in urban area is significant earlier. The adolescent’s first spermatozoa were mostly occurred in summer. The relevant physical and reproductive education should be started from the third grade of primary school. The reproductive health education should be carried out when the youth are 7 years old.

Evidence for the involvement of NGF in human sperm motility

Motility is an important physiological characteristic of a mature sperm. Nerve growth factor (NGF) is a key protein for the survival, maintenance and development of the central and peripheral nervous systems. It has been shown that NGF and its receptors TrkA and p75 are widely expressed in the testis, accessory reproductive organ, and the epididymal sperms. These observation have shifted the attention to the role of NGF on male reproductive physiology. In the present study, we found that NGF remarkably increased testicular coefficient in rats subjected to unpredictable chronic mild stress. Furthermore, we investigated the role of NGF on human sperm motility in vitro by CASA. The results showed that the parameters of sperm motility after NGF treatment had significantly increased, the means of VAP, VSL, VCL, BCF and LIN were significantly increased 32% than those of NGF absence, the means of MAD, STR, ALH and WOB had no notable difference. In addition, NGF promotes the sperm motility in a time- and dose-dependent manner. Taken together, our findings suggest that NGF plays a promoted role in sperm motility.

AN ANALYSIS OF EFFECTS OF FETAL ORDER AND PARENTAL AGE ON RETINOBLASTOMA

Seventy-eight cases of retinoblastoma (Rb) diagnosed at the Eye ENT Hospital, Shanghai Medical University from 1953 to 1985 were studied. The results of multiple logistic regression analysis and conditional probability model showed that high fetal order had a moderate risk (OR=1.28, P<0.05) of developing unilateral Rb. Both unilateral and bilateral cases were independent of parental ages, and no interaction could be found between fetal order and paternal of maternal age; also, the effect of fetal order was not confounded by parental ages.

Identification of two compound heterozygous GNRHR mutations in two siblings with congenital hypogonadotropic hypogonadism

DearEditor,Congenital hypogonadotropic hypogonadism(CHH)is a rare genetic disease characterized by gonadotropin-releasing hormone(GnRH)deficiency or insufficiency and normal hypothalamic-pituitary structures,resulting in defective sexual maturation or/and infertility.!CHH presents as a variety of nonreproductive phenotypes,such as renal hypoplasia,dental hypoplasia,or oculomotor abnormalities.

Urgent Need for Contraceptive Education and Services in Chinese Unmarried Undergraduates:A Multi-campus Survey

In order to ascertain prevalence rate of premarital sexual intercourse,unintended pregnancy and abortion,and evaluate associated factors of unintended pregnancy among undergraduates from all over China,the representative sample of unmarried undergraduates was obtained by using a multi-stage,stratified,probability cluster design,and data were collected by using a survey questionnaire.62 326 available responders were gained.11.6% of them acknowledged having experiences of premarital sexual intercourse（standardized prevalence rate of sexual intercourse was 13.8%）.31.5% of students active in premarital sex acknowledged undergoing unintended pregnancy.76.2% of pregnant students selected abortion to end it.Of students active in premarital sex,46.2% used contraception at the first sexual intercourse,28.2% replied ＂always＂ using contraception in sexual intercourse.The rate of using condoms,oral contraceptives（OCs）,and withdrawal among students who had used contraception was 52.0%,31.0%,and 27.2% respectively.＂No preparation for sex＂（40.3%）,＂pleasure decrement＂（32.1%）,＂won＇t-be-pregnancy in occasional sexual intercourse＂（30.2%） were their common excuses for using no contraception.The identified risk factors for unintended pregnancy among students active in premarital sex by multivariate analysis were as follows：having no steady lover [having no steady lover vs having a steady lover：odds ratio（OR）,1.875;95% confidence interval（CI）,1.629-2.158],unaware of the course of conception（unaware vs aware：OR,2.023;95% CI,1.811-2.260）,considering abortion not endanger women＇s physical and mental health（no endangerment vs endangerment：OR,2.659;95% CI,2.265-3.121）,nonuse of contraception（never use vs always use：OR,1.682;95% CI,1.295-2.185）.Medical students were not less likely to experience an unintended pregnancy than nonmedical students（OR,1.111;95% CI,0.906-1.287）.The substantial proportion of unintended pregnancy among undergraduates indicates a need for convenient and targeted contraceptive education and services.

Association between MTHFR A1298C Polymorphism and Male Infertility：A Meta-analysis

There have been several epidemiological studies evaluating the potential association between the methylenetetrahydrofolate reductase（MTHFR） A1298 C polymorphism and the risk of male infertility.However,the results obtained were inconsistent.Therefore,we performed a meta-analysis to further examine the association between the MTHFR A1298 C polymorphism and male infertility.A comprehensive search was conducted to identify all eligible studies from the online literature databases published prior to January 15 th,2016.A total of 20 studies with 4293 cases and 4507 controls were included.An odds ratio（OR） and a 95% confidence interval（95% CI） were calculated to assess the strength of the association.A cumulative meta-analysis,sensitivity analysis and assessment of the publication bias were also performed in this study.The results showed that in the overall analysis,the association between the MTHFR A1298 C polymorphism and male infertility was not significant.A stratified analysis by ethnicity revealed a significant increase in the risk of male infertility in the Asian population with the MTHFR A1298 C polymorphism（especially in the heterozygote model：OR=1.20,95% CI=1.01–1.44,P=0.994;the dominant model：OR=1.23,95% CI=1.04–1.45,P=0.996;and the allele model：OR=1.20,95% CI=1.04–1.39,P=0.985） but not in the Caucasian population.In the stratified analyses,no significant association was observed between the different types of male infertility.This meta-analysis suggests the MTHFR A1298 C polymorphism may be a potential risk factor for male infertility,especially in the Asian population.