Comet Assay

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between 'desirable' and 'essential' information: 'essential' information refers to the precise details that are necessary to assess the quality of the experimental work, whereas 'desirable' information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers.

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.

Methods for quantifying DNA damage, as well as repair of that damage, in a high-throughput format are lacking. Single cell gel electrophoresis (SCGE; comet assay) is a widely-used method due to its technical simplicity and sensitivity, but the standard comet assay has limitations in reproducibility and throughput. We have advanced the SCGE assay by creating a 96-well hardware platform coupled with dedicated data processing software (CometChip Platform). Based on the original cometchip approach, the CometChip Platform increases capacity ~200 times over the traditional slide-based SCGE protocol, with excellent reproducibility. We tested this platform in several applications, demonstrating a broad range of potential uses including the routine identification of DNA damaging agents, using a 74-compound library provided by the National Toxicology Program. Additionally, we demonstrated how this tool can be used to evaluate human populations by analysis of peripheral blood mononuclear cells to characterize susceptibility to genotoxic exposures, with implications for epidemiological studies. In summary, we demonstrated a high level of reproducibility and quantitative capacity for the CometChip Platform, making it suitable for high-throughput screening to identify and characterize genotoxic agents in large compound libraries, as well as for human epidemiological studies of genetic diversity relating to DNA damage and repair.

Study questionCan measurement of double-stranded sperm DNA fragmentation (dsSDF) via a neutral comet assay predict the probability of live birth following IVF?Summary answerIn a multicentre IVF cohort, dsSDF measured by a neutral comet assay was a strong, independent predictor of live birth.What is known alreadyWhile much of the focus has traditionally been on female factors, emerging research highlights sperm DNA fragmentation as a significant contributor to reproductive outcomes. Over the past decade, studies have shown that different types of sperm DNA damage can affect reproduction differently, with single-stranded breaks being closely linked to reduced spontaneous conception rates, while double-stranded breaks are linked to higher miscarriage rates.Study design, size, durationProspective cohort study including a total of 302 males from three European IVF clinics, over a 3-year study period (March 2021-October 2024), with 126 healthy sperm donors with confirmed live birth serving as controls.Participants/materials, setting, methodsdsSDF was quantified with a neutral comet assay, expressed as Average Comet Score (ACS) and Incidence of Damage (IOD). The primary outcome was live birth per initiated cycle. Associations were evaluated using multivariable logistic regression, adjusting for female and male age (and centre in sensitivity analyses).Main results and the role of chanceAcross the cohort, 30% of couples achieved a live birth. Higher dsSDF was associated with reduced odds of live birth, and this association remained statistically significant after adjustment for female age, male age, and recruitment site. Both ACS and IOD were independently predictive of live birth in adjusted models. For ACS, each 1-point increase was associated with 16% lower odds of live birth (OR = 0.84, 95% CI 0.72-0.97; P = 0.026). For IOD, each 1-point increase corresponded to 5% lower odds of live birth (OR = 0.95, 95% CI 0.90-0.99; P = 0.025). As expected, female age remained a strong inverse predictor of live birth across models (OR = 0.86, 95% CI 0.78-0.94; P Limitations, reasons for cautionThis study examined couples undergoing their first or only IVF cycle and did not include couples with repeat IVF failures. Limitations include potential centre-level confounding, which may benefit from mixed-effects modelling. We did not collect or adjust for several cycle-level covariates that influence live birth (e.g. IVF vs ICSI, number of oocytes retrieved, embryo transfer strategy, use of preimplantation genetic testing for aneuploidy, stimulation protocol), so residual confounding is possible.Wider implications of the findingsThese results support dsSDF as a clinically relevant biomarker that complements conventional semen parameters.Study funding/competing interest(s)The study was part-funded using an unrestricted medical educational grant provided by Merck Serono Limited (0111897641) to the Liverpool Women's Hospital. T.C.B.M., S.H.M., and E.B. were funded in part by UKRI SIP FMI and Peace Plus HF-TIC grants with Ulster University. L.R., A.S., M.L., C.J.L., L.P., and T.C.B.M. are employed at Examen Lab LTD. A.J.D. is a recipient of Merck Serono Limited (0111897641) grant to the Liverpool Women's Hospital. P.H. has received unrestricted research grants from Merck and Gedeon Richter Nordics and honoraria for lectures from Merck, Gedeon Richter, and IBSA. The remaining authors have nothing to disclose.Trial registration numberN/A.