Reasearch On Molecular Mechanism Of Environment & Genetic Factors On Male Infertility

In sexual reproduction, embryo is originated from the fusion of spermatozoa and oocyte. As size disparity between sperm and oocyte and the characteristic transformation of sperm during its development, it had been widely accepted that sperm only supply its DNA in fertilization. With the deep discovery of sperm reproduction functions, there are evidences indicated important and indispensable role of sperm DNA and RNA in regulating sperm-oocyte fusion and early embryogenesis. We proceeded following researches to discover the effects of sperm DNA and RNA on male reproduction and outcomes of Assisted Reproductive Technology(ART):Sperm DNA integrity was measured using sperm chromatin structure assay (SCSA) with flow cytometry. The degree of DNA fragmentation and immature spermatozoa ware expressed by DNA Fragmentation index (DFI) and high DNA stainability (HDS) respectively. Our results showed the patients consuming≥20 cigarettes/day or smoking≥10 years have few total semen volume, lower percentage of a+b grade sperm and higher abnormal morphology sperm (p< 0.05) compared to other patients. Smokers'spermatozoa have a significantly higher DFI and HDS than non-smokers (p < 0.05). So the study suggested that cigarettes/day≥20 or duration of smoking≥10 years may have deleterious effects on semen volume, sperm vitality, morphology. Smoking decrease sperm DNA integrity and nuclear maturation.Then we focused on the effects of sperm DNA on ART treatment outcomes. The patients were grouped as following criteria:group 1, DFI<27% & HDS≤15%;group 2 DFI>27% & HDS≤15%;group3,DFI≤27% & HDS>15% and group 4,DFI>27% & HDS>15%.In intra uteral insemination(IUI)cycles, pregnancy rate of group2 and group3 showed tendency of decline but no statistical significance.ROC curve indicated the incidence of pregnancy <5% when DFI>20.6% or HDS>35.5%. The patients with DFI>27% had lower good embryo rate and higher embryo fragmentation on day3 in IVF cycles, but no decrease in fertilization rate, viable embryo rate and pregnancy rate in fresh embryo transfer cycles. While, the patients with HDS>15% had decreased fertilization rate but no difference in viable embryo rate, good embryo rate and pregnancy rate in fresh embryo transfer cycles compared to other groups. An interesting phenomena was no patients got pregnancy with sperm DFI>27% and HDS>15% in IUI, IVF and ICSI treatment which indicated that the combination of DFI and HDS will be a better predicting indication for male infertility. Through the study on human spermatozoa RT-PCR, we have found a novel sperm-specific gene hssp411 mRNA had two isoforms, and the two isoforms had diversity expression. About 90% samples had expression of isoform5 but the left 10% only had isoform4; on the other hand, the mRNA expression of isoform5 was much higher than isoform4. The ssp411 expression vectors were constructed successfully in vivo and we discovered only isoform5 could translation to protein althouth both isoforms had mRNA expression. So we speculated that hssp411 may be a novel molecular marker for diagnosis of male infertility.primary ciliary dyskinesia(PCD)is a phenotypically and genetically heterogeneous disorder with ciliary swing dysfunction. The PCD patients was extremely or completely asthenospermia and so they need ICSI to get their offsprings. However, the efficiency of ICSI on PCD patients was unpredictable. In our study, we diagnosed 7 PCD male who had abnormal ciliary ultrastructure and we discovered a point mutation in DNAHA gene,248315G→C among 5 patients.There was no published data on this mutation in PCD.Six patients of the 7 PCD patients proceeded totally 8 cycles of ICSI.4 cycles used ejaculated sperm,3 cycles used sperm originated from TESA and 1 cycles used both. We got 73 mature oocytes totally and average fertilization rate was 46.6%(34/73).All cycles gained at least 1 viable embryo but only 5 had good embryos, among which,3 got clinical pregnant, we concluded that ICSI was the only efficient method for PCD males and the higher viability of sperm may predicting better outcomes of ICSI; sperm viability test could be a screen for PCD male patients and might be developed as a regular evaluating test for predicting outcomes of ICSI for PCD patients.