Evaluation of Sperm DNA Fragmentation Using Two Different Methods: TUNEL via Fluorescence Microscopy, and Flow Cytometry.

: Sperm DNA fragmentation refers to any break in one or both of the strands of DNA in the head of a sperm. The most widely used methodologies for assessing sperm DNA fragmentation are the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion assay (SCD), the single-cell gel electrophoresis assay (SCGE-comet), and the terminal-deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay. The aim of this study was to compare the efficiency and sensitivity of the analysis of sperm DNA fragmentation using TUNEL via fluorescence microscopy, and flow cytometry.

The human embryo following biopsy on day 5 versus day 3: viability, ultrastructure and spindle/chromosome configurations.

Research Question: Are there any differences in viability, spindle abnormalities and mitochondrial and other organelle structures amongst embryos biopsied on day 3 versus day 5 before and after vitrification?

Design: A total of 240 day 3 biopsied embryos that developed to blastocysts but were rejected for transfer following preimplantation genetic testing for monogenic/single gene defects (PGT-M) (n = 115) or for aneuploidies (PGT-A) (n = 125) were divided into two groups: (i) 120 blastocysts treated for viability, spindle/chromosome configuration (SCC) analysis and transmission electron microscopy (TEM) analysis (fresh n = 20, n = 20, n = 20 and following vitrification/warming n = 20, n = 20, n = 20); (ii) 120 embryos were re-biopsied at the blastocyst stage and treated for viability, SCC and TEM analysis (fresh n = 20, n = 20, n = 20 and following vitrification/warming n = 20, n = 20, n = 20). Also, 60 vitrified blastocysts biopsied only on day 5 that were rejected for transfer following PGT-M (n = 6) or PGT-A (n = 54) were treated following warming for viability (n = 20), SCC (n = 20) and TEM analysis (n = 20).

Results: No differences were observed in SCC and ultrastructure between embryos biopsied on day 5 and day 3 but following vitrification higher numbers of abnormal spindles, distension of mitochondria, multivesicular bodies, lipofuscin droplets, altered cell junctions and occasionally excessive accumulation of glycogen granules were evident.