USING SPERM DOUBLE-STRANDED DNA TO PREDICT EMBRYO PLOIDY

Abstract

To demonstrate how the presence of double-stranded DNA breaks (dsDNA) in spermatozoa can be used to predict embryo ploidy. Over the last 12 months, a prospective pilot study was carried out on semen samples to assess dsDNA levels. These samples were also concurrently assessed to determine total DNA damage. A correlation between the two assays was established. Couples were divided into two groups according to the adopted dsDNA threshold, and clinical outcomes were recorded and compared. Consenting men had their ejaculates screened for dsDNA rates utilizing an in-house protocol of the neutral Comet assay, assessing at least 200 spermatozoa. Concurrently, samples were assessed by terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) with a commercial kit, screening at least 500 spermatozoa. ICSI was performed in the standard fashion. Resulting blastocysts were biopsied on day 5 or 6 post-insemination for preimplantation genetic testing for aneuploidy (PGT-A) using next-generation sequencing. The initial pilot study reported an average total DNA fragmentation by TUNEL of 11.3±6% and a dsDNA average of 2.2±3% by neutral Comet assay. The results of TUNEL and dsDNA presented with a linear correlation (R2=0.96; P<0.05), and dsDNA values were extrapolated. After establishing a threshold of 3%, 231 couples treated in 381 ICSI/PGT-A cycles were compared according to above or below threshold. A total of 173 couples (maternal age, 37.2±4 years; paternal age, 38.9±5 years) with normal dsDNA levels underwent 296 ICSI cycles, yielding a fertilization rate of 76.9%; 1,562 embryos were screened by PGT-A. This resulted in 574 normal (36.7%), 827 abnormal (52.9%), and 161 mosaic embryos (10.3%). There have been 171 frozen embryo transfers (FET) of euploid blastocysts thus far, with a 51.6% implantation rate and a 55.6% clinical pregnancy rate (CPR). A total of 48 couples with abnormal dsDNA levels underwent 85 ICSI cycles. They had a comparable maternal age of 38.0±5 years, but a much older male partner (43.2±8 years; P<0.0001). They achieved a fertilization rate of 72.7% (P<0.01), with 375 embryos biopsied for PGT-A. A total of 115 were euploid (30.7%; P<0.05), 222 were aneuploid (59.2%; P<0.05), and 38 were mosaic (10.1%). Interestingly, FET cycles from these cases trended toward lower implantation (43.9%) and CPRs (47.4%). This study confirmed the relevance of dsDNA on structural chromosomal abnormalities and therefore the proportion of euploid embryos. Examining dsDNA of the male gamete can provide important information on the prospects of successful embryo implantation.