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.
Highlights
Recurrent pregnancy loss (RPL) is defined as having at least two consecutive embryo miscarriages within the first or early second trimester of pregnancy [1]
ROC Analysis ROC analysis results are shown in Figure 3, for either achieving a pregnancy without taking into account a possible subsequent miscarriage, and for undergoing a recurrent miscarriage associated with a male factor and without the female factors mentioned previously
In a previous paper a descriptive study was performed on different groups of patients discussing the relationship about the different profiles of alkaline and neutral Comet assay regarding the aetiology of DNA breaks [41]
Summary
Recurrent pregnancy loss (RPL) is defined as having at least two consecutive embryo miscarriages within the first or early second trimester of pregnancy [1]. Taking into account that sperm cells and oocytes provide half of the nuclear embryo DNA, it may be assumed that both males and females could be involved in either infertility or RPL [3,4]. It has been described that the male factor may be involved in RPL when poor semen parameters, Y chromosome microdeletions, or a higher percentage of sperm aneuploidies detected by FISH are found [10,11,12,13,14,15,16,17,18]. The paternal effect in these cases is being underestimated, and only a few recent reports provide data suggesting the possible relation of the sperm DNA status in the aetiology of RPL [12,19]. Some studies have shown that sperm DNA fragmentation (SDF) is increased in semen samples from RPL couples by using Sperm
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