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Recent studies have confirmed that, in addition to sperm DNA, environmental exposure factors such as parental diet structure and stress state affect early embryonic development and offspring growth, thus leading to cross-generational inheritance of acquired traits. Many studies also show that environmental stressors can change the expression level of sperm small non-coding RNAs (sncRNAs). Furthermore, as the carrier of paternal genetic information transmission and epigenetic marker, sncRNAs are directly or indirectly involved in epigenetic regulation, mediating inter-generational inheritance of acquired traits. This review focuses on the two sncRNAs derived from microRNA (miRNA) and tRNA (tsRNA) in sperm epigenetics research.

Infertility affects approximately 20% of childbearing couples in the world, and azoospermia accounts for 10－15% of the causes of male infertility. The use of fresh or frozen-thawed testicular sperm for intracytoplasmic sperm injection (ICSI) has become a main method for azoospermia patients to realize their dream for reproduction. However, testicular spermatozoa are not further matured in the epididymis and therefore have an obviously lower anti-freezing ability than ejaculated sperm. The viability and retrieval rate of sperm are low after freeze-thaw with the conventional method of cryopreservation. Since the first live birth with frozen-thawed testicular spermatozoa, continuous improvement has been made in the methods of testicular sperm cryopreservation and increased the viability and retrieval rate of spermatozoa after freeze-thaw. This review focuses on the methods of testicular sperm cryopreservation in the past 20 years to provide a theoretical basis for the development of assisted reproductive technology.

Objective: To explore the correlation of Mycoplasma genitalium (MG) infection with male infertility., Methods: Totally, 27 314 males with infertility and 200 fertile sperm donors underwent MG and routine seminal examinations. The infertile men were divided into azoospermia, oligozoospermia, asthenozoospermia, oligoasthenospermia, and normal semen quality groups based on the results of seminal examination, the 27 286 of them with age data into eight age groups (<21, 21－25, 26－30, 31－35, 36－40, 41－45, 46－50 and ≥51 years old), and the 9 058 with definite diagnosis into primary and secondary infertility groups. Fifty-six cases of MG infection among the infertile males were treated with antibiotics for 2 weeks and examined for changes of the semen parameters., Results: Compared with the normal controls, the oligozoospermia patients showed a significantly higher rate of MG infection (0.50% vs 3.62%, P = 0.024), the highest in the ≥51 yr group (3.68%, P = 0.021), followed by the 21－25 yr group (3.00%, P = 0.048), and so did the infertile males (3.64%, P = 0.011), the men with primary infertility (3.73%, P = 0.010) and those with secondary infertility (3.57%, P = 0.015). MG infection was found to be associated with oligozoospermia (OR = 7.471, 95% CI: 1.001－55.784), primary infertility (OR = 7.704, 95% CI: 1.073－55.309) and secondary infertility (OR = 7.362, 95% CI: 1.026－52.837) but not with the age of the patients. Both sperm concentration and sperm count were significantly lower in the infected men before treatment than in the non-infection group after treatment (P < 0.05)., Conclusions: MG infection is related to male infertility and reduces the semen volume and sperm concentration, but does not affect sperm motility.

Objective: To investigate whether in vitro culture medium (IVCM) for sparse spermatozoa can improve human sperm motility for the purpose of helping clinicians, laboratorians and patients choose a better strategy of assisted reproduction., Methods: Semen samples were obtained from 178 males for routine semen examination from March to August 2016, including 151 cases of asthenozoospermia and 27 cases of normal sperm motility. A total of 200 μl was collected from each sample and divided into two equal portions and equal volumes of IVCM (experimental group) and F10 (1×) (control group) were added to the two portions, respectively, followed by 30-minute incubation at 37℃ in an incubator with 5% CO2. Sperm concentration, motility and viability and the percentages of progressively motile, non-progressively motile and immotile sperm were recorded before and after incubation., Results: After activated with IVCM, neither the samples with asthenozoospermia nor those with normal sperm motility showed any statistically significant difference in sperm viability from the baseline or the control group (P>0.05). The rates of progressively and non-progressively motile sperm from the asthenozoospermia males were increased by 14.02% and 4.86% respectively, while that of immotile sperm decreased by 19.01% in the experimental group (P >0.01), and similar results were observed in the semen samples from the men with normal sperm motility. The percentage of reduced immotile viable sperm was positively correlated with that of immotile viable sperm in both the asthenozoospermia patients (r = 0.260, P <0.01) and the men with normal sperm motility (r = 0.679, P <0.01)., Conclusions: IVCM can increase sperm motility without affecting sperm viability in men with either asthenozoospermia or normal sperm motility. The larger the proportion of immotile viable sperm, the higher the percentages of progressively and non-progressively motile sperm in the semen after IVCM activation, and this correlation is more significant in men with normal sperm motility than in asthenozoospermia patients.

Objective: To investigate the pregnancy outcomes of assisted reproductive technology (ART) with cryopreserved donor sperm and the safety of the offspring thus conceived., Methods: The Human Sperm Bank of CITIC Xiangya Hospital provided cryopreserved donor semen to 31 reproductive centers in China between January 2006 and December 2012, with which 50247 ART cycles were accomplished. We compared the rates of birth defects and spontaneous abortion of intracervical insemination (ICI), intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI)., Results: A total of 39 047 ART cycles were performed by artificial insemination with cryopreserved donor sperm, including 36 674 cycles of ICI and 2 372 cycles of IUI. Among the 8 612 clinical pregnancies achieved by ICI, there were 917 cases of spontaneous abortion (at <28 gestational wk) (10.6%) and 6133 live births, with 43 cases of birth defect (0.70%). Of the 547 clinical pregnancies achieved by IUI, there were 41 cases of spontaneous abortion (7.5%) and 426 live births, with 2 cases of birth defect (0.47%). Totally, 11 200 cycles of IVF and ICSI were accomplished with cryopreserved donor sperm. Of the 5 860 clinical pregnancies achieved by IVF, there were 456 cases of spontaneous abortion (7.8%) and 5089 live births, with 55 cases of birth defect (1.08%). Among the 350 clinical pregnancies achieved by ICSI, there were 30 cases of spontaneous abortion (8.6%) and 229 live births, with 3 cases of birth defect (1.31%). The birth defect rate of ART with cryopreserved donor sperm was significantly lower than that published by the Chinese Ministry of Health (0.86% vs 1.53%,P<0.01)., Conclusions: The safety of the offspring conceived by ART with cryopreserved donor sperm is controllable.

Objective: To establish a long-term proliferation culture system for mouse spermatogonial stem cells., Methods: Testis tissues were obtained from 30 newborn male ICR mice on postnatal day 2-6. Testis cell suspension was collected by two-step enzymatic digestion prior to culture. The dissociated cells were aliquoted into tissue culture plates and cultivated with a modified system composed of serum-free defined medium on mouse embryonic fibroblasts (MEF) feeders. Their proliferation was determined by the BrdU incorporation test and the cultured cells identified by alkaline phosphatase (AP) activity, immunofluorescence staining and RT-PCR assay., Results: The cultures remained in a steady state and continued to generate germ cell colonies. The undifferentiated state was confirmed by strong positivity for AP activity, immunofluorescent staining of GFRalpha-1+ /Oct-4+ /VASA+ /SCP3- and GFRalpha-1+ /Oct-4+/SCP3- at the gene expression levels., Conclusion: Mouse spermatogonial stem cells could be expanded in our defined culture system and passaged steadily in vitro. The harvested cells remained in an undifferentiated state, which has provided a good platform for the study of spermatogenesis in vitro.

Objective: To separate the low abundance protein and establish the 2-DE synthetic map of total protein of human normal spermatozoa by using the 2-DE technology., Methods: All the needed human spermatozoa were collected and mixed, and proteins were extracted at one time with the method of urea/thiourea and ultra-sound. 0.8 mg, 0.6 mg, 0.5 mg, 0.3 mg sperm protein extracts were separated with 2-DE. Analyzed with MALDI-TOF-MS, PI and MW of 2 spots were obtained. Then set the 2 spots as the referent spots, different maps were compared and analyzed. At last, a synthetic map enriched with low abundance protein was obtained., Results: 1,080 +/- 23 protein spots have been separated on the 2-DE map with standard 0.5 mg loading amount and a synthetic map A was constructed which consist of 889 matched protein spots on the all maps with 0.5 mg loading amount. 381, 50 and 32 new spots were detected individually on the maps with 0.8 mg, 0.6 mg and 0.3 mg protein loading amount. A synthetic map with 1,352 protein spots was obtained., Conclusion: Low abundance protein was separated and a synthetic map enriched with low abundant protein was obtained by changing the protein loading amount.

Objective: To identify the resource suspected sperm of donor in human sperm bank and apply the parentage testing between the donor and his offspring., Methods: We took the 6 semen specimen of the donor involved and correspondently suspected semen as well as the semen of one volunteer and peripheral blood of his offspring. All specimens were amplified by PCR, and DNA fingerprint was detected by PAGE electrophoresis., Results: By DNA fingerprinting we discovered that the 5 suspected sperm samples came from corresponding donors and the other sample from another. The volunteer and his offspring were identified as consanguinity., Conclusion: We can identify the difference between sperm of donors and the suspected sperm of donors, and sperm of donors and the peripheral blood of their offspring exactly by DNA fingerprinting.

To understand molecular mechanism of spermatogenesis, two ESTs BG720564 and AI700454, were found from SPAG4 (sperm antigen 4), a gene related to dense fiber protein of outer membrane of the human sperm and mouse spermatocytes gene AK006225. The gap was filled by polymerase chain reaction, and a 1252 bp fragment was obtained. This 1252 bp fragment was named TSARG4 (testis and spermatogenesis related gene 4 (GenBank accession number AF401350). Its opening reading frame was 94-1233 bp, as was proved by RT-PCR in human testis. TSARG4 gene was located in 20q11.2, and the putative protein was 379 amino acid with a theoretical molecular weight of 43 081 and isoelectric point of 8.61. The homologies of amino acid sequences were 74% between TSARG4 and AK006225 gene and 45% between TSARG4 and SPAG4 gene, respectively. The human TSARG4 mRNA is expressed in a wide range of adult tissues, including testis, whereas the homologous mouse spermatocytes gene AK006225 is expressed specifically in the testis.