Huangtao Guan

Cell-Free Seminal mRNA and MicroRNA Exist in Different Forms

Background The great interest in cell-free mRNA, microRNA (miRNA) as molecular biomarkers for clinical applications, and as ‘signaling’ molecules for intercellular communication highlights the need to reveal their physical nature. Here this issue was explored in human cell-free seminal mRNA (cfs-mRNA) and miRNA (cfs-miRNA). Methodology/Principal Findings Selected male reproductive organ-specific mRNAs, miRNAs, and piRNAs were quantified by quantitative real-time PCR in all experiments. While the stability of cfs-miRNA assessed by time-course analysis (up to 24 h at room temperature) was similar with cfs-mRNA, the reductive changes between cfs-miRNA and cfs-mRNA after filtration and Triton X-100 treatment on seminal plasma were very different, implying their different physical nature. Seminal microvesicles (SMVs) were then recovered and proportions of cfs-mRNA and cfs-miRNA within SMVs were quantified. The amounts of SMVs- sequestered cfs-mRNAs almost were the same as total cfs-mRNA, and were highly variable depending on the different sizes of SMVs. But most of cfs-miRNA was independent of SMVs and existed in the supernatant. The possible form of cfs-miRNA in the supernatant was further explored by filtration and protease K digestion. It passed through the 0.10-µm pore, but was degraded dramatically after intense protease K digestion. Conclusions/Significance The predominant cfs-mRNA is contained in SMVs, while most cfs-miRNA is bound with protein complexes. Our data explained the stability of extracellular RNAs in human semen, and shed light on their origins and potential functions in male reproduction, and strategy of developing them as biomarkers of male reproductive system.

Inhibition of human sperm function and mouse fertilization in vitro by an antibody against cation channel of sperm 1: the contraceptive potential of its transmembrane domains and pore region

OBJECTIVE To explore the contraceptive potential of the CatSper1 transmembrane domains and pore region in vitro. DESIGN In vitro study with human sperm and mouse fertilization. SETTING Andrology laboratory of an academic research center. PATIENT(S) AND ANIMAL(S) Normozoospermia and viripotent BALB/c mice. INTERVENTION(S) The specific binding of an anti-CatSper1 IgG antibody (H-300) to CatSper1 was confirmed by Western blot and immunofluorescence. Sperm from humans and mice were incubated with H-300. MAIN OUTCOME MEASURE(S) The effects of H-300 on human sperm progressive motility, abnormal acrosome, hyperactivated motility, and mouse in vitro fertilization rates were analyzed. RESULT(S) A significant decline in sperm progressive motility was observed after 1, 2, and 4 hours of incubation with H-300; the change was mainly ascribed to the decline of fast progressive motility. Significant inhibition of the hyperactivated motility was observed after 5 hours of incubation with H-300. The incubation of mouse sperm with H-300 before insemination reduced the in vitro fertilization rate to 28% of control levels (72% inhibition). CONCLUSION(S) CatSper1 may be a potential target for immunocontraception, and the antibody may be a tool to study the function of ion channels in sperm in which relatively fewer methods can be applied.

Characterization of a piRNA binding protein Miwi in mouse oocytes

Argonaute proteins and Piwi proteins bind with microRNA (mRNA) and Piwi-interacting RNA (piRNA), respectively, to form functional complexes. Piwi proteins are mostly restricted to germ cells and stem cells, and the Piwi-piRNA pathway is required for normal spermatogenesis. Although piRNAs were also recently identified in mammalian oocytes, expression of Piwi proteins in the ovary has not been well characterized. Previous studies did not detect mRNA of Miwi, a murine homologue of Piwi proteins, in total RNA of mouse ovary tissue. We demonstrated herein the presence of Miwi in murine oocytes. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunofluorescence based on quantum dots immune labeling technique were used to investigate the expression profile of Miwi in oocytes of adult and neonatal females at 0, 1, 2, 3, and 4 weeks postpartum. Although RT-PCR was negative in total RNA of the adult ovary, both RT-PCR and Western blot detected Miwi in oocytes of adult mice, and ovaries of neonatal females. Miwi transcript and protein peaked at 1 and 2 weeks postpartum, respectively. Miwi mRNA was detectable in newborn mouse ovaries, implying its transcription was initiated at least in the primordial follicle. Its protein was strong in late primary and secondary follicles, but appeared to decrease as maturation proceeded. The exclusion of anti-Miwi immunofluorescence from some cytoplasmic granules was observed. Given that diverse biologic and molecular functions have been revealed for the Piwi-piRNA pathway in germline cells of many species, Miwi might be an important functional protein in murine folliculogenesis.

Evaluation of semen quality in 1808 university students, from Wuhan, Central China.

The aim of this study was to evaluate the semen quality of university students in Wuhan, the largest city in the world in terms of the number of university students. All student sperm donors recorded in the Hubei Province Human Sperm Bank from 1 March 2010 to 31 December 2013 were screened. At last, a total of 3616 semen samples from 1808 university student sperm donors were eligible and retrospectively analyzed. Each donor′s semen parameters were averaged over two samples and compared with the World Health Organization criteria, and a generalized linear regression model was used to examine several determinants of semen quality. We found that the mean and median values were 3.0 ml and 2.8 ml for semen volume, 50.2 × 10 6 ml−1 and 50.0 × 10 6 ml−1 for sperm concentration, 148.1 × 10 6 and 142.1 × 10 6 for total sperm count, and 58.6% and 60.0% for total sperm motility. About 85.0% of donors had parameters that were all normal. Season and duration of abstinence were critical factors affecting semen quality. We also found a decrease in sperm concentration during the 4 years observation; however, this may not be a strong evidence to confirm the declining trend of semen quality. In conclusion, semen quality of university students in Wuhan was not optimal and should be paid high attention, long-term observation and further study should be carried out to confirm the present situation.

Variability in the morphologic assessment of human sperm: use of the strict criteria recommended by the World Health Organization in 2010.

OBJECTIVE To determine the variability in the recognition of normal sperm and various sperm defects using the strict criteria recommended by the World Health Organization (5th edition, 2010). DESIGN Sperm morphologic assessment by three experienced evaluators. SETTING Image processing laboratory and reproduction research institute. PATIENT(S) Semen donors from a sperm bank. INTERVENTION(S) The morphology of 5,296 sperm was evaluated using statistical analyses of variability. MAIN OUTCOME MEASURE(S) The proportion and coefficients of variation (CVs) of normal sperm, defects of specific parts, and the categories of defects were measured. The degree of agreement between any two of the three evaluators was calculated. The multiple anomalies index, teratozoospermia index, sperm deformity index, and the CVs were also measured. RESULT(S) The CVs of normal sperm, multiple anomalies index, teratozoospermia index, and sperm deformity index were 4.80%, 4.14%, 5.75%, and 6.81%, respectively. A broader range (4.80%-132.97%) of CVs was observed for the recognition of various defects. The coefficients of the degree of agreement concerning specific morphologic parts of sperm varied (0.387-0.607), with lower relative values for the head and mid-piece than for the tail and cytoplasm. CONCLUSION(S) The sperm head is more difficult to evaluate than the other parts using the criteria recommended by the World Health Organization in 2010. The degree of agreement concerning specific parts and various defects varied in broad ranges. A stricter definition for each defect is needed.

Relationship between donor sperm parameters and pregnancy outcome after intrauterine insemination: analysis of 2821 cycles in 1355 couples.

The aim of this study was to investigate whether sperm parameters can affect the pregnancy outcome of artificial intrauterine insemination with cryopreserved donor spermatozoon (AID). A total of 1355 couples received 2821 AID treatment cycles in the Reproductive Medicine Center of the Tongji Medical College between January 2010 and December 2013, and the data were collected and retrospectively analysed. The relationship between pre‐freezing, post‐thawing as well as optimised sperm parameters and AID pregnancy outcome was investigated. Clinical pregnancy rate and cumulated pregnancy rate were also calculated. A total of 728 cycles from 2821 treatment cycles achieved pregnancies, and cumulated pregnancy rate was 25.81%. Pre‐freezing progressive sperm motility in pregnant cycles was higher than that in nonpregnant cycles (P = 0.001); logistic regression analysis also indicated that pre‐freezing progressive sperm motility was the only parameter affecting pregnancy outcome (P = 0.0001). Our study also showed that the cumulated pregnancy rate increased progressively and reached a plateau after the fifth cycle. In conclusion, pre‐freezing progressive sperm motility should be a valuable predictor for AID pregnancy outcome. Female fertility factors should be considered, or IVF/ICSI should be recommended when couples received more than 5 AID cycles without pregnancy.

The plasma miR-125a, miR-361 and miR-133a are promising novel biomarkers for Late-Onset Hypogonadism.

Circulating miRNAs have been shown to serve as diagnostic/prognostic biomarkers in cancers and other diseases. However, the role of plasma miRNAs in Late-onset hypogonadism (LOH) diagnosis is still unknown. Using Illumina HiSeq2000 sequencing at discovery phase, and then two-step validated by reverse transcriptase polymerase chain reaction (RT-PCR) assays in verification phases. We verified that the expression levels of miR-125a-5p, miR-361-5p and miR-133a-3p were significantly altered in LOH group compared to the control group. The area under the receiver operating characteristic (ROC) curve (AUC) is 0.682, 0.698 and 0.765, respectively. The combination of three miRNAs showed a larger AUC (0.835) that was more efficient for the diagnosis of LOH. Among three miRNAs, miR-133a-3p had the best diagnostic value for LOH with 68.2% sensitivity and 77.3% specificity. Regression analyses show that miR-133a-3p level was negatively associated with the ageing males’ symptoms (AMS) scale. However, miR-361-5p level was positively associated with serum testosterone concentrations. In summary, plasma miRNAs are differentially expressed between LOH and healthy controls. We validated three miRNAs that could act as novel biomarkers for diagnosis of LOH. These miRNAs may be involved in the development of LOH. However, further large and functional studies are warranted to confirm our findings.

The rs5934505 single nucleotide polymorphism (SNP) is associated with low testosterone and late-onset hypogonadism, but the rs10822184 SNP is associated with overweight and obesity in a Chinese Han population: a case-control study.

Low testosterone is associated with late‐onset hypogonadism (LOH) and obesity. Recently, studies have shown that four single nucleotide polymorphisms (SNPs), rs12150660, rs727428, rs5934505, and rs10822184, are associated with testosterone levels in populations of European descent. Therefore, we investigated whether the SNP loci are related to low testosterone, LOH, or obesity in a Chinese Han population. Ruling out co‐morbidities, DNA was prepared from 409 men (aged 40–65 years) with low serum testosterone (defined as total testosterone <11.6 nmol/L) and 1 : 1 normal controls (matched age, body mass index (BMI), and the same living area) who were selected from 6898 males. According to the same standards, 310 men with LOH and 1 : 1 normal controls were selected from 6898 males. Excluding the cases with an unreliable sequencing result, genetic analyses were performed. The minor allele frequencies of the SNP loci rs12150660, rs727428, rs5934505, and rs10822184 were 0.1%, 44.6%, 18.7%, and 38.9%, respectively. rs5934505 was associated with the serum total testosterone and calculated free testosterone (CFT) levels (p = 0.045 and p = 0.021). rs5934505 (C>T) was associated with an increased risk of low total testosterone, low CFT, and LOH and adjusted for other factors, with an odds ratio (OR) of 2.01 (1.34–3.01), 2.14 (1.42–3.20), and 1.64 (1.04–2.58). rs10822184 was significantly correlated with weight and BMI (p = 0.035 and p = 0.027). rs10822184 (T>C) was associated with an increased risk of overweight and obesity. We adjusted for other factors, with odds ratios (ORs) of 1.94 (1.36–2.78) and 1.56 (1.00–2.43). In summary, our study provided convincing evidence that rs5934505 (C>T) was associated with the risk of low testosterone and LOH in Chinese populations. We were the first to find that rs10822184 (T>C) was significantly correlated with the risk of overweight and obesity in Chinese populations. However, further large and functional studies are warranted to confirm our findings.

Decline in semen concentration of healthy Chinese adults: evidence from 9357 participants from 2010 to 2015

The present study aims to analyze sperm concentration trends among young and healthy Chinese adults in Wuhan, Central China, from 2010 to 2015. Semen analysis data from 9357 participants were collected and analyzed using a general linear model and the Cochran–Armitage trend test. A significant decline was observed in sperm concentration (β [standard deviation]: −1.53 [0.16]; P < 0.001). In addition, a decline in sperm density was observed by stratifying student versus nonstudent sperm donors and by analyzing the year of birth or birth year cohort of the participants. Furthermore, the percentage of participants with sperm densities of over 40 × 106 ml−1 significantly decreased with year. Notably, a dramatic decline in sperm density was recorded over the first 5 years of study. This research reported a decline in sperm concentration among young adults in Wuhan, Central China, in 2010–2015.

Prevalence and risk factors of lower urinary tract symptoms in Chinese adult men: a multicentre cross-sectional study

There has been no previous population-based study reporting the prevalence and risk factors of male lower urinary tract symptoms (LUTS) among men in mainland China. This cross-sectional study was conducted from 2013 to 2014 in three representative provinces of China: Guangdong, Hubei and Jiangsu. 3250 individuals participated in the interviews, which involved a questionnaire covering sociodemographic characteristics, lifestyle, dietary patterns and the International Prostate Symptom Score (IPSS). Blood was collected for lipids, glucose, insulin and reproductive hormone tests. The incidences of LUTS and its obstructive and irritative symptoms were calculated. Risk factors for LUTS were identified using multivariable logistic regression analysis. The prevalence of moderate to severe LUTS and its obstructive and irritative symptoms was 14.3%, 13.1% and 16.1%, respectively, and increased with age. The prevalence in Guangdong was much lower than that in Hubei and Jiangsu in different ages. Increased fasting plasma glucose and decreased HDL-C levels were associated with an increased risk of moderate to severe LUTS (OR = 1.30, 95% CI: 1.02–1.65 and OR = 2.06, 95% CI: 1.08–3.94, respectively). Free testosterone < 0.22 ng/ml decreased the risk of moderate to severe LUTS and obstructive and irritative symptoms by about 20–30%. An inadequate daily intake of vegetables, fruit and water significantly increased the risk of LUTS by 1.3–to 2.0 times. In conclusion, the prevalence of LUTS in Chinese men is high and increases with age. Dietary patterns may be critical for the development of LUTS. Thus, dietary modifications could be a useful strategy for preventing the development of LUTS.