P-619: Characterization of four sperm cell populations resolved and flow cytometry sorted by the Sperm Chromatin Structure Assay (SCSA®)

Abstract

1) characterize by light microscope image analysis and neutral pH Comet analysis the four SCSA derived sperm populations consisting of: Main (no DNA damage detected), Moderate (moderately damaged sperm) and High (highly damaged sperm) DNA Fragmentation Index (% DFI) and High DNA Stainability (HDS is a measure of immature chromatin). 2) Estimate the selection frequency of the moderate DFI populations for ICSI patients in comparison to the more natural selection of sperm by the eggs for the routine IVF patients. Retrospective. ART patients undergoing routine IVF or ICSI 1) SCSA- sort the four sperm populations for: a) computer image analysis of Feulgen stained sperm for morphology and morphometry, and b) neutral pH Comet- defined DNA strand breaks. 2) Statistical frequency selection estimation of the Moderate DFI sperm population by ICSI technicians vs more natural selection of sperm by eggs in routine IVF resulting in pregnancy/miscarriage. The morphology/morphometry features of the Moderate DFI sperm population were equally normal as the Main population sperm but one-half were Comet positive. The High DFI sperm population showed cell morphology/morphometry features with considerable signs of necrosis/apoptosis and were 100% Comet positive. Semen samples derived from the routine IVF pregnancies had significantly lower % moderate DFI values in comparison to the % moderate DFI population pregnancies from the ICSI pregnancies (P<0.001). Sperm with normal morphology/morphometry may have single and double strand DNA breaks. The SCSA detects both single-strand DNA breaks (moderate DFI and neutral Comet negative) and 100% sperm with single and double strand DNA breaks (neutral Comet positive). Since one- half of the Moderate DFI sperm have DNA double strand breaks identified by the neutral pH Comet assay but with normal morphology/morphometry, the Moderate DFI population is likely to be picked out as frequently for ICSI injection as the main population without SCSA-identifiable DNA damage. The high DFI population is likely detected as abnormal and not used for ICSI. To date, six ICSI studies have shown a relationship between elevated sperm DNA fragmentation and increased miscarriage rates. These Moderate DFI sperm may be responsible for decreased pregnancy and increased spontaneous abortion rates, as well as the possibility for increased birth defects seen at birth or latent genetic defects seen in later life.