Genome-wide DNA methylation profiles and small noncoding RNA signatures in sperm with a high DNA fragmentation index.

BACKGROUND: A growing number of studies have reported that sperm DNA fragmentation (SDF) is associated with male infertility. However, no studies have compared genome-wide DNA methylation profiles and sncRNA signatures between sperm with high and low sperm DNA fragmentation indices (DFIs). METHODS: Whole-genome bisulfite sequencing (WGBS) was performed on sperm samples from a weak group (DFI ≥ 30%, n = 6) and normal group (DFI ≤ 15%, n = 7). Small noncoding RNA (sncRNA) deep sequencing was conducted for sperm samples from the weak (DFI ≥ 30%, n = 13) and normal (DFI ≤ 15%, n = 17) groups. RESULTS: A total of 4939 differentially methylated regions (DMRs) were identified in the weak group sperm samples relative to normal group sperm samples, with 2072 (41.95%) of them located in promoter regions. The percentages of hypermethylated DMRs were higher than those of hypomethylated DMRs in all seven examined gene annotation groups. Hypermethylated DMRs were significantly enriched in terms associated with neurons and microtubules. Compared with the normal group, the global DNA methylation level of the weak group sperm showed a downward trend, with lower correlation for methylation in the weak group sperm; therefore, the chromosomes of high-DFI sperm may be loose. On average, 40.5% of sncRNAs were annotated as rsRNAs, 19.3% as tsRNAs, 10.4% as yRNAs, and 7.1% as miRNAs. A total of 27 miRNAs, 151 tsRNAs, and 70 rsRNAs were differentially expressed between the two groups of sperm samples. Finally, 7 sncRNAs were identified as candidate sperm quality biomarkers, and the target genes of the differentially expressed miRNAs are involved in nervous system development. CONCLUSION: Our findings suggest that genome-wide DNA methylation profiles and sncRNA signatures are significantly altered in high-DFI sperm. Our study provides potential biomarkers for sperm quality.

Effects of low lead exposure on sperm quality and sperm DNA methylation in adult men.

INSTRUCTION: Lead (Pb) exposure is a risk factor for male infertility, but the epigenetic changes in sperm DNAattributable to lead exposure is poorly defined. METHODS: In this study, we investigated whether low Pb exposure (< 10 µg/dL) affects the sperm quality. Blood, urine, and semen samples of 297 men of childbearing age were analyzed for all relevant parameters. Based on the blood Pb level (BLL), participants were allocated to RL (0-2.5 µg/dL), RM (2.5-5 µg/dL), and RH (5-10 µg/dL) groups. The 5-methylcytosine and 5-hydroxymethylcytosine patterns in the sperm DNA were identified using methylated DNA immunoprecipitation and hydroxymethylated DNA immunoprecipitation sequencing. RESULTS: The non-progressive motility (NP) was significantly increased and associated with global hypomethylation of sperm DNA in the RH group compared with the RL group, indicating that aberrant sperm methylation due to low Pb exposure is possibly associated with reduced sperm motility. The hypomethylated promoter regions were primarily enriched in the calcium (Ca) homeostasis pathway. Further, the interaction between Ca and Pb was associated with sperm rapid progressive motility and asthenospermia risk, although no significant methylation abnormality was observed in those with BLL < 5 µg/dL. When BLL was > 5 µg/dL or when predicting NP, no significant Pb-Ca interaction was observed. DISCUSSION: Overall, our results indicate that aberrant DNA methylation of the Ca homeostasis pathway, induced by low Pb exposure, is the potential cause for reduced sperm velocity.

Long non­coding RNA PCAT6 promotes the development of osteosarcoma by increasing MDM2 expression.

Osteosarcoma is a severe malignant tumor. Several studies indicated that lncRNA prostate cancer­associated transcript 6 (PCAT6) promoted the development of multiple types of cancers. Studies have also revealed that MDM2 could aggravate tumor symptoms inhibiting P53 expression. However, whether lncRNA PCAT6 could affect the proliferation and metastasis of osteosarcoma cells by regulating P53 expression is unclear. The present study established lncRNA PCAT6­overexpressing osteosarcoma cells. Cell Counting Kit­8, wound healing and Transwell assays were performed to detect the change in proliferation, migration and invasion of these cells, respectively. Subsequently, E3 ubiquitin­protein ligase Mdm2 (MDM2), P53 and P21 expression were determined using western blotting. Finally, MDM2 expression was inhibited and the proliferation, migration and invasion of these cells was determined again. The present study found that the proliferation, migration and invasion of osteosarcoma cells increased following overexpression of lncRNA PCAT6. MDM2 expression was upregulated while the levels of P53 and P21 decreased following overexpression of lncRNA PCAT6. However, the proliferation, migration and invasion of osteosarcoma cells were inhibited following MDM2 knockdown. Additionally, P53 and P21 was rescued following MDM2 knockdown. To conclude, lncRNA PCAT6 promoted the proliferation, migration and invasion of osteosarcoma cells by promoting the expression of MDM2 and suppressing the expression of P53 and P21.

A Diagnostic Model to Improve the Predictability of Natural Pregnancy Potential in Patients with Oligoasthenospermia.

BACKGROUND Oligoasthenospermia is one of the major reasons for male infertility in clinical practice. Nevertheless, some patients with oligoasthenospermia show normal fertility. Currently, there is a lack of an effective method to distinguish patients with oligoasthenospermia showing normal fertility from those who lack natural fertility and should participate in in vitro fertilization and assisted reproduction. MATERIAL AND METHODS In this study, we collected semen and blood samples from 153 males of Shui nationality at reproductive age in Guizhou Province, southwest China. We measured the routine parameters for semen and some serological indicators. A clinical diagnosis model was then constructed to evaluate the fertility potential of oligoasthenospermia patients using a logistic stepwise regression method, which was then visualized with a nomogram. RESULTS Our results showed that this model could effectively assess the natural pregnancy potential of patients with oligoasthenospermia, and its sensitivity and specificity were superior to those of a traditional model that used only sperm motility and count to assess male fertility potential (area under the curve=0.7626 vs. 0.6677). Additionally, we evaluated the clinical net benefit for patients with oligoasthenospermia at different risk scores in our model using decision curve analysis. The results showed that the net benefit was obtained at scores ranging from 0.1 to 0.6. CONCLUSIONS This comprehensive clinical prediction model can be used to determine whether infertile oligoasthenospermia patients lack natural fertility.

System analysis of teratozoospermia mRNA profile based on integrated bioinformatics tools.

mRNA has an important role in spermatogenesis and the maintenance of fertility, and may act as a potential biomarker for the clinical diagnosis of infertility. In the present study, potential biomarkers associated with teratozoospermia were screened through systemic bioinformatics analysis. Initially, genome­wide expression profiles were downloaded from the Gene Expression Omnibus and primary analysis was conducted using R software, which included preprocessing of raw microarray data, transformation between probe ID and gene symbol and identification of differentially expressed genes. Subsequently, a functional enrichment analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery to investigate the biological processes involved in the development of teratozoospermia. Finally, a protein­protein interaction network of notable differentially expressed genes was constructed and cross­analysis performed for multiple datasets, to obtain a potential biomarker for teratozoospermia. It was observed that G protein subunit ß 3, G protein subunit α o1 and G protein subunit g transducin 1 were upregulated and enriched using Kyoto Encyclopedia of Genes and Genomes (KEGG) in the network and in cross analysis. Furthermore, ribosomal protein S3 (RPS3), RPS5, RPS6, RPS16 and RPS23 were downregulated and enriched using KEGG in teratozoospermia. In conclusion, the results of the present study identified several mRNAs involved in sperm morphological development, which may aid in the understanding and treatment of infertility.

Reproductive effects of cadmium on sperm function and early embryonic development in vitro.

Cadmium is a major environmental toxicant that is released into the atmosphere, water and soil in the form of cadmium oxide, cadmium chloride, or cadmium sulfide via industrial activities, such as the manufacturing of batteries and pigments, metal smelting and refining and municipal waste incineration. In the present study, we investigated the effects of cadmium exposure on sperm quality parameters, fertilization capacity and early embryonic development. Our study showed that in vitro incubation of human or mouse sperms with cadmium for a long time (up to 24 hours) could significantly decreased sperm motility in a concentration- and time-dependent manner. Exposure to cadmium in the environment for a short term (30 min) did not affect sperm motility but significantly reduced in vitro fertilization rate. We also evaluated the effects of cadmium at concentrations of 0.625 µg/ml, and 1.25 µg/ml on early embryonic development in vitro and observed that the blastocyst formation rate dramatically decreased with increasing cadmium concentration. This finding emphasizes the hazardous effects of cadmium on sperm quality as well as on natural embryo development and raises greater concerns regarding cadmium pollution.

Correction: Decreased Sperm Motility Retarded ICSI Fertilization Rate in Severe Oligozoospermia but Good-Quality Embryo Transfer Had Achieved the Prospective Clinical Outcomes.

[This corrects the article DOI: 10.1371/journal.pone.0163524.].

Lectin binding of human sperm associates with DEFB126 mutation and serves as a potential biomarker for subfertility.

Coating on the sperm surface, glycocalyx, plays a key role in sperm motility, maturation and fertilization. A comprehensive profile of sperm surface glycans will greatly facilitate both basic researches and clinical studies. Because of the capability of recognizing different glycan moieties, lectins are widely used in glycobiology. However, lacking high-throughput technology, limited lectins have been reported for analyzing the glycan of human sperm. In this study, we employed a lectin microarray for profiling the surface glycans of human sperm, on which 54 out of 91 lectins showed positive binding. Based on this technique, we compared lectin binding profiling of sperm with homozygous DEFB126 mutation (del/del) with that of wild type (wt/wt). DEFB126 was reported to contribute to the sialylation on sperm surface and its homozygous mutation was related to male subfertility. Six lectins (Jacalin/AIA, GHA, ACL, MPL, VVL and ABA) were found to develop lower binding affinity to sperm with del/del. Further validation showed that these lectins, especially ABA and MPL, can be potential biomarkers for clinical diagnosis of subfertility due to the mutation of DEFB126. Our research provides insight into the detection of some unexplained male subfertility, and the lectin microarray is generally applicable for infertility/subfertility sperm biomarker discovery.

ICSI treatment of severe male infertility can achieve prospective embryo quality compared with IVF of fertile donor sperm on sibling oocytes.

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.

Another functional frame-shift polymorphism of DEFB126 (rs11467497) associated with male infertility.

DEFB126 rs140685149 mutation was shown to cause sperm dysfunction and subfertility. Indel rs11467497 is another 4-nucleotide frame-shift mutation (151bp upstream of rs140685149) that leads to the premature termination of translation and the expression of peptide truncated at the carboxyl terminus. In the present study, we performed a comprehensive association study to check the contribution of rs140685149 and rs11467497 to male infertility. Our results confirmed the previous findings that there was no association between rs140685149 and sperm motility. In contrast, we found a significant association of another indel rs11467497 with male infertility. Moreover, rs11467497 was shown to be associated with higher number of round cells in the infertile males with low sperm motility. Surprisingly, the two mutations commonly existed in the sperm donors (n = 672), suggesting a potential application of the two indels in the screening for eligible sperm donors. Western blotting assays showed the sperms with rs140685149 2-nt deletion tended to have unstable DEFB126 protein in contrast of no DEFB126 protein expressed in the sperms with rs11467497 4-nt deletion, suggesting a more severe consequence caused by rs11467497 mutation. In conclusion, our study presented a significant contribution of another functional frame-shift polymorphism of DEFB126 (rs11467497) to male infertility.

Meta-analyses of 4 CFTR variants associated with the risk of the congenital bilateral absence of the vas deferens.

AIMS: The aim of our study was to evaluate the relationship between four CFTR variations and the congenital bilateral absence of the vas deferens (CBAVD). METHODS: A systematic search was performed in the literature databases for the case-control studies of CFTR variations with the risk of CBAVD. A total of 29 studies among 1139 controls and 1562 CBAVD patients were gathered for the meta-analyses of three commonly tecsted variations (5T, ΔF508 and M470V) with CBAVD. RESULTS: Our meta-analyses observed significant associations between CBAVD and all the three variations, including 5T (P < 0.001, OR = 8.35, 95% CI = 6.68-10.43), M470V (P = 0.027, OR = 0.74, 95% CI = 0.60-0.91) and ΔF508 (P < 0.001, OR = 22.20, 95% CI = 7.49-65.79). CONCLUSION: In the current study, we demonstrated a significant association between CFTR variations and CBAVD. Our results showed that the 5T variation was a risk factor of CBAVD in French, Spanish, Japanese, Chinese, Iranian, Indian, Mexican and Egyptian populations. CFTR ΔF508 was another important risk factor in Caucasians, including Slovenians, Canadians, Iranians, and Egyptians. In addition, M470V was a protective factor among French, Chinese, Italian and Iranian populations.

Reproductive effects of two neonicotinoid insecticides on mouse sperm function and early embryonic development in vitro.

Acetamiprid (ACE) and imidacloprid (IMI) are two major members in the family of neonicotinoid pesticides, which are synthesized with a higher selectivity to insects. The present study determined and compared in vitro effects of ACE, IMI and nicotine on mammalian reproduction by using an integrated testing strategy for reproductive toxicology, which covered sperm quality, sperm penetration into oocytes and preimplantation embryonic development. Direct chemical exposure (500 µM or 5 mM) on spermatozoa during capacitation was performed, and in vitro fertilization (IVF) process, zygotes and 2-cell embryos were respectively incubated with chemical-supplemented medium until blastocyst formation to evaluate the reproductive toxicity of these chemicals and monitor the stages mainly affected. Generally, treatment of 500 µM or 5 mM chemicals for 30 min did not change sperm motility and DNA integrity significantly but the fertilization ability in in vitro fertilization (IVF) process, indicating that IVF process could detect and distinguish subtle effect of spermatozoa exposed to different chemicals. Culture experiment in the presence of chemicals in medium showed that fertilization process and zygotes are adversely affected by direct exposure of chemicals (P<0.05), in an order of nicotine>IMI>ACE, whereas developmental progression of 2-cell stage embryos was similar to controls (P>0.05). These findings unveiled the hazardous effects of neonicotinoid pesticides exposure on mammalian sperm fertilization ability as well as embryonic development, raising the concerns that neonicotinoid pesticides may pose reproductive risks on human reproductive health, especially in professional populations.

[Quantitative analysis by real-time elastosonography for the differential diagnosis of azoospermia: preliminary application].

OBJECTIVE: To evaluate the quantitative analysis by real-time elastosonography in the differential diagnosis of obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). METHODS: We evaluated the elastosonographic images of 200 cases of OA, 300 cases of NOA and 100 normal healthy controls, calculated the strain ratio of the testis to the scrotal skin and the median strain ratio among the three groups, and analyzed the best cut-off point for differentiating OA and NOA by the receiver operation characteristic (ROC) curve. RESULTS: The median strain ratio of NOA was 0.49 +/- 0.43, while that of OA was 0.35 +/- 0.31, with significant difference between the two groups (Z = - 19.173, P = 0.000 < 0.017). According to the results of ROC curve analysis, the area under the curve was 0.857 +/- 0.012 and the best cut-off point for differentiating OA and NOA was 0.395 (sensitivity = 84.5%, specificity = 74.5%, accuracy = 80.5%). CONCLUSION: Quantitative analysis by real-time elastosonography is a new valuable technique for the differential diagnosis of azoospermia.

A sperm viability test using SYBR-14/propidium iodide flow cytometry as a tool for rapid screening of primary ciliary dyskinesia patients and for choosing sperm sources for intracytoplasmic sperm injection.

Spermatozoa viability tests based on dual-color flow cytometry after staining with Sybr-14/propidium iodide were performed on 44 men with complete asthenospermia for primary ciliary dyskinesia (PCD) screening, and seven were identified with PCD by electron microscopy of ultrastructural ciliary defects. Six PCD patients underwent eight intracytoplasmic sperm injection therapy cycles using ejaculated sperm or testicular sperm, obtaining a mean fertilization rate of 46.6%, with three healthy babies born and one in utero at the time of writing.

[Ultrasonographic features of epididymides in obstructive azoospermia].

OBJECTIVE: To investigate the ultrasonographic features of epididymides in congenital obstructive azoospermia (COA) and acquired obstructive azoospermia (AOA). METHODS: A total of 211 infertile men with obstructive azoospermia were observed by scrotal ultrasonography, and the features of the epididymal ultrasonograms were compared between COA and AOA. RESULTS: COA exhibited significantly higher rates of ectasia in the epididymal head, cord-like changes, abrupt tapering and absence of the epididymal body and tail than AOA (P < 0.05), while AOA showed markedly higher rates of epididymal body and tail duct ectasia and epididymal inflammatory mass than COA (P < 0.01). Tubular ectasia of the epididymal duct in the head, body and tail were markedly higher in the COA (14 [5.9%], 41 [17.2%] and 20 [8.4%] cases in 236 epididymides) than in the AOA (P < 0.05). Retiform ectasia were markedly higher in the AOA (119 [64.0%], 142 [76.3%] and 109 [58.6%] cases in 186 epididymides) than in the COA (P < 0.05), with statistically significant differences between the two groups (P < 0.05). Ultrasonographically, the epididymides of the COA patients were characterized by irregular ectasia of the epididymal tube with decreased and unclear wall echoes (P < 0.05), and those of the AOA patients by regular ectasia with enhanced wall echoes (P < 0.01). CONCLUSION: The ultrasonographic epididymal features of COA are obviously different from those of AOA, which is of important clinical application value for distinguishing the two conditions from each other.

[The detection of mutations in VHL gene from a single cell in a patient with von Hippel-Lindau disease].

OBJECTIVE: To explore a technology for diagnosing VHL mutations from a single cell and provide experimental evidences for the feasibility of applying technology in detecting genetic mutations from a single cell. METHODS: After whole genome amplification (WGA) based on multiple displacement amplication (MDA) for a single cell, we did regular PCR following sequencing and detected the genotypes using the real time PCR based on TaqMan probes. We detected VHL mutations by the different terminal fluorescent changing. RESULTS: The rate of amplification for single cell based on MDA was 90.91%. The rate of contamination was 0. After sequencing, the allele drop out (ADO) rate of heterozygotes was 26.67%(8/30); combined with the different terminal fluorescent changing, the rate of ADO of heterozygotes was 16.67%. CONCLUSION: WGA based on MDA for a single cell followed by regular PCR with sequencing and real time PCR can specially and accurately detect the VHL genotypes of single cells.

[Male infertility update].

Exact etiological factors cannot be found in more than 70% of male infertility. Although the causes of some male infertility are known, the pathogenesis is not yet clear. Therefore, the diagnosis and treatment of male infertility are particularly complicated. Some breakthroughs have been made in the treatment of refractory male infertility since the development of ICSI in 1992. However, traditional treatment should also be taken into account for individual male infertility. The standardization of diagnosis and treatment and the improvement of the success rate and safety of assistant reproductive technology will be the dominant factors in the development of male infertility researches.

[Clinical analysis of serum endothelin in type 2 diabetic patients with vascular complications].

OBJECTIVE: To explore the clinical significance of changes in urine microalbumin (UM) and endothelin (ET) in different courses of type 2 diabetes. METHODS: A clinical analyses of 30 type 2 diabetic patients with vascular complications (group A) was conducted in comparison with type 2 diabetic patients without vascular complications (group B) and patients with impaired glucose tolerance (IGT, group C). With 30 healthy subjects with family history of type 2 diabetes (group D) and 30 healthy subjects without such family history (group E) as controls, UM and ET were determined in all the subjects for a statistical analysis. RESULTS: Significant differences in UM content was noted between group A and the other 4 groups (P<0.05), while the content did not differ significantly between the latter 4 groups (P>0.05). ET content was significantly higher in group A than in the other 4 groups (P<0.01), and was the lowest in group E (P<0.01). CONCLUSIONS: Type 2 diabetic patients sustain extensively impaired endothelium function, which is exacerbated with the progression of the disease courses. Synchronized changes between vascular complications and impaired endothelium function indicates that vascular endothelial injury relates to vascular complications and the progression of diabetes.

[Relationship between serum vWF and PAF in type 2 diabetic patients and diabetic nephropathy].

OBJECTIVE: To observe the changes in urine albumin (UALB), von Willebrand factor (vWF) and platelet-activating factor (PAF) in different courses of type 2 diabetes. METHODS: Levels of UALB, vWF and PAF were determined in 30 type 2 diabetic nephropathy patients(group A), type 2 diabetic patients without DNP(group B, n=30), patients with impaired glucose tolerance (group C, n=30), the first-degree relatives of type 2 diabetic patients with normal glucose tolerance(group D, n=30) and 30 normal glucose tolerance subjects without family history of type 2 diabetes(group E). RESULTS: UALB and PAF contents were significantly higher in group A than in the other groups (P<0.01), while between the latter 4 groups, the contents showed no significant differences (P>0.05). Serum vWF level was significantly higher in group A than those in the other groups (P<0.05). CONCLUSIONS: Renal dysfunction and platelet activation are present in type 2 diabetes with microvascular complications, and the synchronized changes between renal dysfunction and platelet activation indicate the involvement of elevated PAF in vascular injury.

[Impact of high response of ovary to gonadotropin stimulation on the outcome of in vitro fertilization and embryo transfer or intracytoplasmic sperm injection].

OBJECTIVE: To evaluate the impact of elevated peak estradiol (E(2)) levels and a high number of retrieved oocytes on implantation and pregnancy rate in patients undergoing in vitro fertilization-embryo transfer (IVF-ET) and intracytoplasmic sperm injection (ICSI). METHODS: Retrospectively analyzed 474 infertile women undergoing 510 cycles for IVF-ET/ICSI treatment during the period of March 1999 to December 2000. Using a standard long protocol/flare-up protocol [(gonadotropic hormone releasing hormone agonist/high pure follicle-stimulating hormone (FSH-HP)/human chorionic gonadotropin (hCG)] for ovarian stimulation. High responders were defined as those who had peak E(2) levels of > 11 010 pmol/L on the day of hCG administration (n = 160) or > 15 retrieved oocytes (n = 148). Normal responders were defined as those who had peak E(2) levels of