Nuclear DNA fragmentation negatively affects zona binding competence of Y bearing mouse spermatozoa.

PURPOSE: To investigate the influence of sperm DNA integrity on the zona binding ability of mouse spermatozoa in relation to their sex chromosomal constitution. METHOD(S): In this prospective experimental study, the sperm DNA fragmentation was induced by exposing testicular area of Swiss Albino mice (Mus musculus) to different doses of γ-radiation (0, 2.5, 5.0 and 10.0 Gy). Sperm DNA fragmentation was quantified by single cell gel electrophoresis (comet assay). In vitro sperm zona binding assay was performed and the numbers of zona bound X and Y bearing spermatozoa were determined using fluorescence in situ hybridization (FISH). RESULT(S): The assessment of zona pellucida bound X and Y-bearing spermatozoa using fluorescence in situ hybridization has revealed a unique binding pattern. The number of zona bound Y-spermatozoa declined significantly (P < 0.01 to 0.0001) with increase in the DNA damage. The skewed binding pattern of X and Y-bearing sperm was strongly correlated with the extent of sperm DNA damage. CONCLUSION(S): The zona pellucida may have a role in preventing DNA damaged mouse sperm binding especially towards Y-bearing sperm. However, the exact mechanism behind this observation needs to be elucidated further.

Stage-specific response in early mouse embryos exposed to prednisolone in vitro.

Corticosteroids are increasingly being used during the peri-implantation period to treat women with repeated IVF failure and recurrent miscarriage. However, the direct effects of prednisolone (PRDL), one of the commonly used corticosteroids on early embryo development is not understood. To mimic the possible clinical scenario and to understand the embryonic response to direct PRDL exposure, this pilot study was conducted in a mouse model. Cleavage stage embryos exposed to 3 and 30 µM PRDL in vitro were assessed for peri-implantation developmental potential, genetic integrity, inner cell mass (ICM) proliferation and pluripotency markers in the proliferated ICM cells. Exposure to 30 µM PRDL delayed the embryonic progression beyond compaction (P < 0.05) in comparison to vehicle control and, had reduced total cell number (P < 0.001) than all other groups. In addition, 30 µM PRDL exposure resulted in poor hatching potential (P < 0.05) and increased apoptosis in blastocysts (P < 0.05) compared to 3 µM PRDL. On the other hand, completely formed ICM outgrowths were significantly higher (P < 0.05) in 3 µM PRDL compared to control. However, no significant differences were observed in the expression of pluripotency genes. In conclusion, the trend observed in embryos exposed to PRDL in vitro provides important information concerning the use of this drug when treating patients at the peri-implantation phase of IVF cycles. However, the clinical value of this observation on human embryo development needs further research.

In vitro matured oocytes are more susceptible than in vivo matured oocytes to mock ICSI induced functional and genetic changes.

BACKGROUND: Concerns regarding the safety of ICSI have been intensified recently due to increased risk of birth defects in ICSI born children. Although fertilization rate is significantly higher in ICSI cycles, studies have failed to demonstrate the benefits of ICSI in improving the pregnancy rate. Poor technical skill, and suboptimal in vitro conditions may account for the ICSI results however, there is no report on the effects of oocyte manipulations on the ICSI outcome. OBJECTIVE: The present study elucidates the influence of mock ICSI on the functional and genetic integrity of the mouse oocytes. METHODS: Reactive Oxygen Species (ROS) level, mitochondrial status, and phosphorylation of H2AX were assessed in the in vivo matured and IVM oocytes subjected to mock ICSI. RESULTS: A significant increase in ROS level was observed in both in vivo matured and IVM oocytes subjected to mock ICSI (P<0.05-0.001) whereas unique mitochondrial distribution pattern was found only in IVM oocytes (P<0.01-0.001). Importantly, differential H2AX phosphorylation was observed in both in vivo matured and IVM oocytes subjected to mock ICSI (P <0.001). CONCLUSION: The data from this study suggests that mock ICSI can alter genetic and functional integrity in oocytes and IVM oocytes are more vulnerable to mock ICSI induced changes.

The extent of paternal sperm DNA damage influences early post-natal survival of first generation mouse offspring.

OBJECTIVES: To study the post-natal characteristics and the survival of offspring derived from DNA damaged sperm. STUDY DESIGN: This experimental prospective study was conducted on Swiss Albino mice (Mus musculus). Sperm DNA damage was induced by different doses of γ-irradiation in male mice who were then mated with healthy female mice. The post-natal characteristics including the survival of first generation offspring were studied and then correlated with the amount of paternal sperm DNA damage. RESULTS: A significant reduction of survival in the early post-natal period was observed in the first generation offspring derived from the DNA damaged sperm, and a strong association was observed between the extent of sperm DNA damage and the survival of the offspring. CONCLUSION: The DNA damage load in sperm at the time of fertilization influences early post-natal survival of the mouse offspring.

Frozen-thawed spermatozoa from oligozoospermic ejaculates are susceptible to in situ DNA fragmentation in polyvinylpyrrolidone-based sperm-immobilization medium.

OBJECTIVE: To elucidate the effect of sperm immobilization media that are and are not based on polyvinylpyrrolidone (PVP) on the DNA integrity of fresh and frozen-thawed spermatozoa during standard intracytoplasmic sperm injection (ICSI) conditions. DESIGN: Experimental prospective study. SETTING: Embryology research laboratory. PATIENT(S): Forty-six ejaculates from normozoospermic and oligozoospermic men. INTERVENTION(S): Assessment of sperm DNA fragmentation by single-cell gel electrophoresis assay. MAIN OUTCOME MEASURE(S): DNA integrity of fresh and frozen-thawed spermatozoa from normozoospermic and oligozoospermic ejaculates exposed to PVP-based and non-PVP-based media. RESULT(S): Exposure of fresh and frozen thawed spermatozoa from normozoospermic and oligozoospermic ejaculates to PVP-based medium in an ICSI dish for 30 minutes statistically significantly increased the DNA fragmentation. In contrast, the extent of DNA fragmentation in non-PVP-based medium did not statistically significantly differ from control. CONCLUSION(S): A PVP-based medium can induce a statistically significant amount of sperm DNA fragmentation in an ICSI dish, and frozen-thawed sperm from oligozoospermic ejaculates are more susceptible to in situ DNA fragmentation.