Impact of Apoptosis and DNA Damage on Sperm Chromatin Decondensation Following Intracytoplasmic Injection

Abstract

There is a likelihood that some sperm selected for intracytoplasmic sperm injection (ICSI), despite appearing normal, contain fragmented DNA, thus affecting the outcome of the procedure. DNA damage is assumed to result from apoptotic events. Magnetic cell sorting (MACS) using paramagnetic annexin V-conjugated microbeads is an effective technique for the elimination of apoptotic spermatozoa. The procedure delivers 2 sperm fractions: annexin-negative (non-apoptotic) and annexin-positive (apoptotic). We have previously reported that MACS can be used as a sperm preparation technique to enhance the sperm quality and oocyte penetration. Our objective was to investigate if the incidence of early fertilization is impacted by apoptosis and DNA damage and whether it can be increased by MACS. Prospective-controlled study. Semen samples (n=18) were collected from healthy donors and subjected to density gradient centrifugation. Spermatozoa were re-suspended in human tubal fluid media with 10% synthetic serum supplement and divided into 2 aliquots. The first aliquot was kept as a control (aliquot A) while the second was further divided by MACS into annexin-negative (non-apoptotic, aliquot B) and annexin-positive (apoptotic, aliquot C). Sperm DNA fragmentation was assessed using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL)-coupled flow cytometry. Hamster oocyte-ICSI (Ahmadi and Ng, 1996) was performed to evaluate the sperm chromatin decondensation (SCD) as a sign of early fertilization. To perform the assay, spermatozoa were separately injected inside hamster oocytes cytoplasm using micromanipulator. Sperm nuclear changes were monitored 18 hours following injection using bisbenzamide (Hoechst 33258) fluorochrome. Data were analyzed using one-way analysis of variance (ANOVA) with repeated measures and Pearson’s correlation test. The percentages (%) of spermatozoa with DNA fragmentation as well as the % of spermatozoa showing chromatin decondensation following ICSI are presented in the table. Annexin-negative sperm separated by MACS had significantly lower % of DNA fragmentation compared to the annexin-positive and the control. The % of spermatozoa showing chromatin decondensation following ICSI was significantly higher in annexin-negative sperm compared to the annexin-positive sperm but almost identically equal to the controls. No statistically significant correlations were detected between the % of DNA fragmented sperm and the % of spermatozoa showing chromatin decondensation following ICSI. Tabled 1 Our data supports the association of apoptosis with DNA fragmentation in human spermatozoa. Apoptosis and sperm DNA fragmentation do not seem to impact early stages of fertilization. Although, MACS can be used to isolate spermatozoa with compromised genomic integrity, it does not increase the incidence of SCD following ICSI.