The Clinical Significance Of Sperm DNA Fragmentation: Clinical And Experimental Study

Sperm DNA fragmentation (SDF) has been recognized as an independent biological and clinical parameter to represent male fertility. High SDF results in low capability to be father by natural intercourse or by intrauterine insemination. But the negative effect of SDF on clinical results and safety of offspring from in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) still remains to be confirmed. Such debate is so much concerned in the era when IVF and ICSI become common alternatives to treat male infertility with high prevalence of high DNA damaged sperm. In the present study, firstly, we investigated the effect of SDF on IVF and ICSI results, then, we explored the effect of SDF on genetic and epigenetic mutation in mid-gestation mouse derived from ICSI, and finally, we evaluated whether di-2-ethylhexyl phthalate, an important environmental pollute, has reproductive toxicological consequence by its effect on SDF.Part I Effects of SDF on clinical results of IVF and ICSIConsecutive 3350 couples underwent their first IVF or ICSI cycles between April 2009 and December 2012 were respectively analyzed. Standard leuteal long down-regulation protocol (LP) or short flare-up protocol (SP) was used in all treatment cycles. SP was adopted in one of the following conditions indicating reduced ovarian reserve:[1] the women’s basal FSH>10 IU/L; [2] the number of antral ovarian follicle< 6; and [3] the female’s age≥38 years. SDF was assessed by sperm chromatin dispersion 1-2 months before treatment. All cycles were divided into one of 4 groups:LP/IVF, LP/ICSI, SP/IVF and SP/ICSI. The cutoffs of SDF for analysis were set to 20%,30% and 40%, respectively. The relationships of SDF and fertilization rate, cleavage rate, early embryo score, implantation rate (IR), clinical pregnancy rate (CPR) and early abortion rate were analyzed. Our results showed that [1] A significant decrease in IR and a non-significant trend towards lower CPR were observed in LP/IVF group when SDF was≥20% or 30%; [2] A significant increased risk of early miscarriage was observed when SDF was≥40% in LP/IVF group; [3] SDF had no effect on IR and CPR in LP/ICSI group; [4] High SDF decreased significantly IR and CPR with different thresholds of 20%,30%, and 40% in SP/IVF and SP/ICSI group; and [5] A negative effect of SDF on fertilization and early embryo quality was observed in SP/ICSI group, but not in LP/IVF, LP/ICSI and SP/IVF group. Our results suggested that SDF has negative effect on clinical recults of IVF and ICSI, especially in women with reduced ovarian reserve. Oocyte quality is an important determinant on the effect of SDF on IVF and ICSI results.Part II Effects of SDF on genomic mutation frequency and DNA methylation patterns in the resulting mouse offspring conceived through ICSIThe abnormalities in male gamete inherent in the infertile male have been suggested to be important contributing factors of the safety of assisted reproductive technology (ART). In the present study, we investigated the possibility whether the midgestation mouse fetuses derived from ICSI using DNA damaged sperm could have increased genomic mutation. We used a mutation detecting system-Big Blue(?) transgenic mice, which contain an integrated transgene (lambda bacteriophage shuttle vector), making it possible to detect rare mutations within a background of largely non-mutated copies of the bacterial lacI gene. In addition, we also investigated whether sperm DNA damage could alter DNA methylation patterns of two imprinted genes, H19 and Snrpn in offspring. B6D2F, (C57BL/6×DBA/2) strain mice and Big Blue(?) transgenic mice were used as oocyte and sperm donors. Spermatozoa were frozen-thawed repeatedly and then DNA damage extent was evaluated. Midgestation mouse fetuses were produced and collected, followed by DNA extraction, λ packaging, lacI mutant plaques plating, and finally mutation frequency (MF) was calculated. The MF detected in the mouse fetuses(B6D2F1 oocyte×Big Blue(?) sperm) represented the point mutation derived from paternal genome, MF in DNA damaged group (1.45±0.34) ×10-5 was significantly increased compared with control group (1.03±0.09) ×10-5 (P=0.0049); The MF detected in the mouse fetuses (Big Blue(?) oocyte×B6D2F1 sperm) represented the point mutation derived from maternal genome, MF in DNA damaged group (1.64±0.22) ×10-5 was also elevated significantly compared with control group (0.98±0.12) ×10-5 (P=0.0008).On the other hand, methylation status of DMRs of H19 and Snrpn in these fetuses was established by DNA methylation analysis. For H19 DMR, there was a substantial increase in methylation level compared with control (69.9% vs.52.5%, P=0.0375), showing aberrant hypermethylation of the H19 DMR. Oppositely, a considerably reduction of methylation level was observed in Snrpn DMR in experimental fetuses comparing with control ones, also reaching statistically significance (31.5% vs.53.0%, P=0.0451). Thus, DNA damaged group exhibited abnormal methylation status at these same loci, demonstrating epimutations in fetus at both two different imprinted genes. In conclusion, based on the analyses of transgenic mutation detecting system and methylation patterns of imprinted genes, our study indicated that sperm DNA damage could pose significant risk to F1 offspring with respect to maintenance of genetic and epigenetic integrity.Part Ⅲ Effects of Di-(2-ethylhexyl)-phthalate exposure on sperm DNA damage, fertilization and embryonic developmentThe present study was undertaken to determine the reproductive hazards of Di-(2-ethylhexyl)-phthalate (DEHP) on mouse spermatozoa and embryos in vitro and genomic changes in vivo. Direct low-level DEHP exposure (1 μg/ml) on spermatozoa and embryos was investigated by in vitro fertilization (IVF) process, culture of preimplantation embryos in DEHP-supplemented medium and embryo transfer to achieve full term development. Big Blue(?)ransgenic mouse model was employed to evaluate the mutagenesis of testicular genome with in vivo exposure concentration of DEHP (500 mg/kg/day). Generally, DEHP-treated spermatozoa (1 μg/ml,30min) presented reduced fertilization ability (P< 0.05) and the resultant embryos had decreased developmental potential compared to DMSO controls (P< 0.05). Meanwhile, the transferred 2-cell stage embryos derived from treated spermatozoa also exhibited decreased birth rate than that of control (P< 0.05). When fertilized oocytes or 2-cell stage embryos were recovered by in vivo fertilization (without treatment) and then exposed to DEHP, the subsequent development proceed to blastocysts was different, fertilized oocytes were significantly affected (P< 0.05) whereas developmental progression of 2-cell stage embryos was similar to controls (P > 0.05). Testes of the Big Blue(?) transgenic mice treated with DEHP for 4 weeks indicated an approximately 3-fold increase in genomic DNA mutation frequency compared with controls (P< 0.05). We next investigated whether DEHP-induced reproductive hazards was associated with sperm DNA lesion using SCD assay. Both the spermatozoa exposed to DEHP in vitro and in vivo displayed an increase in average percentage of DNA fragmented sperm after treatment compared with that of control groups, but did not reach a significant difference (P> 0.05). In conclusion, these findings unveiled the hazardous effects of direct low-level exposure of DEHP on spermatozoa’s fertilization ability as well as embryonic development, and proved that in vivo DEHP exposure posed mutagenic risks in the reproductive organ-at least in testes, are of great concern to human male reproductive health, and the mechanism DEHP exerts reproductive hazard may not through the pathway of damaging sperm DNA.