Study of Apoptosis by DNA Fragmentation and Phosphatidylserine Externalization, and of Meiotic Segregation in Ejaculated Sperm of Chromosomal Translocation Carrier Patients.

OBJECTIVE: In order to try to explain the infertility of the chromosomal translocation carrier patients, we have compared the expression of two markers of apoptosis (Phosphatidylserine externalization and DNA fragmentation) in sperm of these men with sperm of semen donor (control samples), and studied the meiotic segregation in ejaculated spermatozoa of translocation carriers.
DESIGN: Controlled comparative study.
MATERIALS AND METHODS: Twenty semen samples of translocation carriers, divided into reciprocal (n=14) and Robertsonian (n=6) were compared with semen samples of donors (n=20). According to the initial
sperm concentration, different tests were applied: Annexin V (AN) Binding Assay combined to the use of vital staining by Propidium Iodide (PI) in order to detect the translocation of Phosphatidylserine (PS) to the outer leaflet of the plasma membrane; Terminal deoxynucleotidy transferasemediated
dUTP-biotin Nick End Labelling (TUNEL) in order to measure DNA fragmentation; Fluorescence In Situ Hybridization (FISH) to analyze the meiotic segregation. Values were compared by the Mann-Whittney test
(pRESULTS: Higher values of sperm concentration and forward motility were found in control samples (n=20) compared with samples of translocation carriers (n=20). The results of the Annexin V Binding assay in sperm of patients with chromosomal translocation (reciprocal translocation group, n=11; Robertsonian translocation group, n=6), showed a significantly increased proportion of spermatozoa with externalized PS (AN+PI-: viable spermatozoa with externalized PS, apoptosis-live and AN+PI+: dead spermatozoa with externalized PS, apoptosis-dead) and a lower percentage of intact spermatozoa (AN-PI-: spermatozoa without any staining), than the control group (n=20). The rates of DNA fragmentation investigated by TUNEL reaction were higher in samples of translocation carriers (n=14), than in donors (n=20). They were no significant differences in the results of the Annexin V Binding Assay and TUNEL reaction between the reciprocal and Robertsonian group. The results of the FISH test showed that the
proportion of gametes produced by alternate(n=7; from 33.0 to 58.8%) and adjacent I (n=7 from 30.0 to 43.8%) segregation were predominant in the reciprocal translocation group, and showed a majority of normal meiotic segregation form in the Robertsonian group (n=5; from 76.0 to 88.6%).
CONCLUSION: In contrast to the theoretical meiotic segregation, our data show significantly higher rates of normal or balanced spermatozoa. As PS externalization and DNA fragmentation are higher in ejaculated spermatozoa of translocation carriers, and as concentration and forward motility are higher in sperm of donors, this suggests a biological process excluding unbalanced gametes. As has been suggested in animals, apoptosis may be involved in this mechanism. In order to confirm and explain the involvment of this process, specific markers (Fas, mitochondrial potential, caspases)should be studied in a larger patient group.