Improving native human sperm freezing protection by using a modified vitrification method / 亚洲男科学杂志(英文版)

Slow freezing is the most commonly used technique for the cryopreservation of spermatozoa in clinical practice. However, it has been shown to have a negative impact on sperm function and structure. Vitrification as a successful alternative method has been proved to have better protective effects on human embryos, but vitrification of spermatozoa is still subject to low recovery rates. In this study, a modified vitrification method for native spermatozoa was developed. A total of 28 semen samples were included; each sample was divided into three equal parts and assigned to fresh, slow freezing, and vitrification groups. Sperm vitality, motility, morphology, DNA integrity, and acrosome reaction were assessed for each of the groups. The results showed that vitrification achieves better results for several sperm protection parameters than slow freezing; vitrification achieves a higher recovery rate (P < 0.05), motility (P <0.05), morphology (P <0.05), and curve line velocity (P <0.05) than slow freezing. Furthermore, DNA fragmentation was decreased (P <0.05) and better acrosome protection (P <0.05) was exhibited in the spermatozoa after vitrification. Principal component analysis of all sperm parameters revealed that the vitrification cluster was closer to the fresh cluster, indicating that spermatozoa are better preserved through vitrification. In conclusion, while both slow freezing and vitrification have negative effects on sperm function and structure, the vitrification protocol described here had a relatively better recovery rate (65.8%) and showed improved preservation of several sperm quality parameters compared with slow freezing.

Influence of sperm morphology on pregnancy outcome and offspring in / 亚洲男科学杂志(英文版)

Sperm morphology was once believed as one of the most predictive indicators of pregnancy outcome in assisted reproductive technology (ART). However, the impact of teratozoospermia on in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) outcomes and its offspring remains inconclusive. In order to evaluate the influence of teratozoospermia on pregnancy outcome and newborn status after IVF and ICSI, a retrospective study was conducted. This was a matched case-control study that included 2202 IVF cycles and 2574 ICSI cycles and was conducted at the Reproductive and Genetic Hospital of CITIC-Xiangya in Changsha, China, from June 2013 to June 2018. Patients were divided into two groups based on sperm morphology: teratozoospermia and normal sperm group. The pregnancy outcome and newborn outcome were analyzed. The results indicated that couples with teratozoospermia had a significantly lower optimal embryo rate compared to those with normal sperm morphology in IVF (P = 0.007), while there were no statistically significant differences between the two groups in terms of the fertilization rate, cleavage rate, implantation rate, and pregnancy rate (all P > 0.05). Additionally, teratozoospermia was associated with lower infant birth weight in multiple births after IVF. With regard to ICSI, there was no significant difference in both pregnancy outcome and newborn outcome between the teratozoospermia and normal groups (both P > 0.05). Furthermore, no increase in the risk of birth defects occurred in the teratozoospermia group after IVF/ICSI. Consequently, we believe that teratozoospermia has limited predictive value for pregnancy outcomes in IVF/ICSI, and has little impact on the resulting offspring if multiple pregnancy is avoided.

In vitro culture medium for sparse spermatozoa improves human sperm motility / 中华男科学杂志

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.

Safety of the offspring conceived by assisted reproductive technology with cryopreserved donor sperm / 中华男科学杂志

<p><b>Objective</b>To investigate the pregnancy outcomes of assisted reproductive technology (ART) with cryopreserved donor sperm and the safety of the offspring thus conceived.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>The safety of the offspring conceived by ART with cryopreserved donor sperm is controllable.</p>

Establishment of a long-term culture system for mouse spermatogonial stem cells in vitro / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish a long-term proliferation culture system for mouse spermatogonial stem cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Establishment of a high-resolution 2-D reference map of human spermatozoal proteins from 12 fertile sperm-bank donors / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To extend the analysis of the proteome of human spermatozoa and establish a 2-D gel electrophoresis (2-DE) reference map of human spermatozoal proteins in a pH range of 3.5-9.0.</p><p><b>METHODS</b>In order to reveal more protein spots, immobilized pH gradient strips (24 cm) of broad range of pH 3-10 and the narrower range of pH 6-9, as well as different overlapping narrow range pH immobilized pH gradient (IPG) strips, including 3.5-4.5, 4.0-5.0, 4.5-5.5, 5.0-6.0 and 5.5-6.7, were used. After 2-DE, several visually identical spots between the different pH range 2-D gel pairs were cut from the gels and confirmed by mass spectrometry and used as landmarks for computer analysis.</p><p><b>RESULTS</b>The 2-D reference map with pH value from 3.5 to 9.0 was synthesized by using the ImageMaster analysis software. The overlapping spots were excluded, so that every spot was counted only once. A total of 3872 different protein spots were identified from the reference map, an approximately 3-fold increase compared to the broad range pH 3-10 IPG strip (1306 spots).</p><p><b>CONCLUSION</b>The present 2-D pattern is a high resolution 2-D reference map for human fertile spermatozoal protein spots. A comprehensive knowledge of the protein composition of human spermatozoa is very meaningful in studying dysregulation of male fertility.</p>

Establishment of the 2-D synthetic map of total protein of normal human spermatozoa enriched with low abundance protein / 中华男科学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Low abundance protein was separated and a synthetic map enriched with low abundant protein was obtained by changing the protein loading amount.</p>