Double Stranded Sperm DNA Breaks, Measured by Comet Assay, Are Associated with Unexplained Recurrent Miscarriage in Couples without a Female Factor

Abstract

It is known that sperm samples from recurrent pregnancy loss (RPL) couples have an increase in their sperm DNA fragmentation (SDF), but no studies have been performed in order to identify differences between single stranded SDF (ssSDF) and double stranded SDF (dsSDF) in these patients. This could be relevant because the type of DNA damage could have different effects. Semen samples were classified attending their clinical status: 25 fertile donors and 20 RPL patients with at least two unexplained first trimester miscarriages. SDF was analysed using alkaline and neutral Comet assay, SCD test and pulsed-field gel electrophoresis (PFGE), and ROC analysis including data from 105 more infertile patients (n = 150) was performed to establish predictive threshold values. SDF for alkaline and neutral Comet, and the SCD test was analysed in these categories of individuals. Data revealed the presence of two subgroups within fertile donors. The values obtained were 21.10±9.13, 23.35±10.45 and 12.31±4.31, respectively, for fertile donors with low values for both ssSDF and dsSDF; 27.86±12.64, 80.69±12.67 and 12.43±5.22, for fertile donors with low ssSDF and high dsSDF; and 33.61±15.50, 84.64±11.28 and 19.28±6.05, for unexplained RPL patients, also showing a low ssSDF and high dsSDF profile. This latter profile was seen in 85% of unexplained RPL and 33% of fertile donors, suggesting that it may be associated to a male risk factor for undergoing RPL. ROC analysis regarding recurrent miscarriage set the cut-off value at 77.50% of dsDNA SDF. PFGE for low ssSDF and high dsSDF profile samples and positive controls treated with DNase, to induce dsDNA breaks, showed a more intense band of about 48 kb, which fits the toroid model of DNA compaction in sperm, pointing out that some nuclease activity may be affecting their sperm DNA in RPL patients. This work identifies a very specific SDF profile related to the paternal risk of having RPL.

DNA fragmentation in brighter sperm predicts male fertility independently from age and semen parameters

“…Fertile men with high values of sDF (mainly composed of the dimmer fraction) resemble the subgroup of fertile men recently identified by Ribas-Maynou et al (34) showing high percentages of sperm with double-strand sDF as detected by neutral comet assay. According to the same authors, DNA damage in those spermatozoa derives mainly from nuclease activity (35).…”

Histone retention, protein carbonylation, and lipid peroxidation in spermatozoa: Possible role in recurrent pregnancy loss

“…The deleterious effect of fragmented paternal DNA on embryonic viability and progression of pregnancy has been experimentally shown in mouse models [Ahmadi and Ng 1999] and similar results were also observed in human patients who opt for assisted reproductive technologies such as IVF and ICSI [Lin et al 2008;Muriel et al 2006;Robinson et al 2012;Tandara et al 2014;Tesarik et al 2004;Wdowiak et al 2015;Zhao et al 2014;Zini et al 2008]. In idiopathic cases of recurrent miscarriages too, the higher rates of sperm DNA fragmentation have been implicated, in the absence of any detectable female factor abnormality [Absalan et al 2012;Brahem et al 2011;Carrell et al 2003;Kumar et al 2012;Ramasamy et al 2015;Ribas-Maynou et al 2012;Zidi-Jrah et al 2016]. In congruence, studies have also reported interrelations between sperm chromatin alterations and early embryo post-implantation development in couples undergoing ART [ (Borini et al 2006;Seli et al 2004].…”

Paternal Obesity, Interventions, and Mechanistic Pathways to Impaired Health in Offspring

“…1). These molecular perturbations in spermatozoa are independently associated with reduced pregnancy rates and an increased risk of miscarriage [3]. A small number of clinical studies have also implicated male obesity as a negative factor for pregnancy success, with decreased pregnancy rates and an increase in pregnancy loss in couples undergoing assisted reproductive technologies (ART) [4].…”