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Research Article
Sperm DNA fragmentation increased and motility declined significantly with prolonged semen exposure after ejaculation. In 14 normal samples, DNA fragmentation increased by 5.5%, 11.5%, and 16.1% at 1 h, 2 h, and 4 h, respectively, while motility decreased. These findings support minimizing delays for off-site semen collection and processing.
Sperm DNA fragmentation is known to be detrimental to sperm quality, which can negatively impact assisted reproductive technology (ART) cycles. Examining sperm DNA fragmentation (SDF) in seminal fluid over time can provide insight into the effects of prolonged semen exposure on sperm DNA integrity. In this preliminary study, an SDF assay was performed at various time points to simulate off-site semen collection conditions using the sperm chromatin dispersion assay on 14 normal semen samples. Statistical analysis of DNA fragmentation percentages over the duration of the assay showed that prolonged stagnation of seminal fluid from ejaculation to semen analysis significantly increased DNA fragmentation. Average sperm motility was significantly reduced by 7.4% at 1 h, 14.5% at 2 h, and 18.7% at 4 h, and average SDF was significantly increased by 5.5% at 1 h, 11.5% at 2 h, and 16.1% at 4 h after semen collection. This decrease in sperm quality over time may inform fertility clinics’ protocols when considering sperm preparation for ART cycles, especially when semen collection occurs off-site from the andrology lab. Laboratories may consider establishing an acceptable time threshold for receiving semen from off-site collection.
The World Health Organization (WHO) identifies one in six people or 17.5% of the world population as experiencing infertility at some point in their lifetime1. Approximately 20% of infertility cases have a sole diagnosis of male factor infertility, with an additional 30–40% of cases having male factor as a secondary diagnosis2. Even in healthy men, seminal parameters can vary greatly from individual to individual, including fluctuations in concentration, motility, morphology, and vitality3. Thus, it is important to understand the specific factors that compromise male fertility to provide proper treatment in a clinical setting.
A developing area of research is the effect of sperm DNA integrity on assisted reproductive technology (ART) cycles4,5,6,7,8,9,10,11,12,13. The WHO laboratory manual for the examination and processing of human semen, 5th edition, states that sperm DNA fragmentation (SDF) (either via single or double-stranded breaks) may affect embryo development, implantation and pregnancy outcomes14. Muriel et al. performed an analysis of SDF and found that increased SDF yielded impaired fertilization rates, embryo quality, and implantation rates in ART cycles10. However, a better understanding of what causes DNA fragmentation is needed.
Previous studies report that oxidative stress can lead to decreased sperm motility, impaired enzymatic production and function, and, most significantly, DNA damage8. Such DNA damage may include, but is not limited to, gene deletions, mistranslations, and SDF. SDF assays are currently used to assess sperm DNA integrity. Although studies have correlated cryopreservation, handling media, and oxidative stress with SDF, little is known about SDF over the period from ejaculation to analysis4,5. This is a critical time in sperm lifespan, given the increased prevalence of home sperm collections for sperm preparation in ART cycles15. Home collections have become increasingly popular during the COVID-19 pandemic, as they limit direct contact with the patient and increase comfort and convenience. However, there are no specific guidelines in place that suggest an optimal time from ejaculation to analysis for home collections. Thus, the primary objective of our study was to understand whether SDF changes over time in stagnant seminal fluid and whether such changes may be relevant to ART sample handling.
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The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of University Hospitals (STUDY20201371). Informed consent was obtained from all subjects involved in the study. Fourteen semen samples were selected from patients in a single academic fertility center in Northeast Ohio. All samples analyzed in this experiment were collected on-site at our facility. Sperm chromatin dispersion testing was performed on semen samples by a fully trained andrologist, in accordance with the manufacturer’s instructions, to minimize interpersonal variation. With this kit, intact, unfixed, fresh sperm were immersed in an inert agarose microgel on a pretreated slide and then cooled at 4 °C for 5 min to solidify the microgel. An initial acid treatment was used to denature DNA in sperm cells with fragmented DNA, and a lysis buffer solution was applied to remove nuclear proteins. After washing with distilled water, slides were dehydrated in graded ethanol solutions and air-dried. When visualized under light microscopy after staining with the kit-provided staining solutions, sperm without fragmentation demonstrated nucleoids with a big dispersion halo (bigger than 1/3 of the diameter of the core), whereas sperm with fragmentation showed a small halo (less than 1/3 of the diameter of the core) or the absence of halo. The percentage of sperm with DNA fragmentation was calculated as the number of fragmented and degraded sperm and expressed as a percentage. A total of 300 sperm were counted on a slide and were categorized as either having a big or medium dispersion halo or as the absence of a dispersion halo. This count was replicated twice to assess technical variability, and the two counts were averaged. The diagnostic sensitivity and specificity of the kit are 93%, and both positive and negative controls were included in each experiment. Semen samples were assessed at 0, 1, 2, and 4 h (0 h was defined as 15 min after the liquefaction period at 37 °C) and were maintained at room temperature (22–25 °C).
An additional motility analysis was performed on each sample at 0, 1, 2, and 4 h. The motility assessment was performed by an experienced andrologist via a counting protocol using a disposable counting chamber. Three hundred motile sperm were counted per grid, and the count was replicated twice. An average concentration of motile sperm was calculated based on the two replicated counts.
Statistical analyses were performed using repeated-measures ANOVA to compare each time group. When a significant overall effect was detected, Tukey’s honestly significant difference (HSD) test was applied for post hoc pairwise comparisons. A p-value < 0.05 was considered statistically significant. Detailed information on materials is summarized in the Table of Materials.
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To assess the impact of prolonged semen exposure on sperm motility and DNA fragmentation, a total of 14 normozoospermic patient samples were analyzed at four time points from collection. The samples analyzed were considered “normal” in the semen analysis parameters according to WHO 5th edition14. All samples were collected on-site at our Fertility Center to optimize the accuracy of the analysis parameters at each time point. The average age of patients was 35.4 years (24–45 years). Init...
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In the samples included in this experiment, SDF increased over time, while motility decreased over the same period. This suggests that sperm DNA integrity may decrease when seminal fluid remains stagnant for longer durations between ejaculation and analysis or processing. Increasing the time that samples remained stagnant before analysis may have increased exposure to conditions associated with oxidative stress. As previously discussed, oxidative stress can be detrimental to sperm DNA quality, leading to high levels of D...
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The authors have no conflicts of interest to declare.
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| ANOVA Calculator | Statistics Kingdom | Statistical analysis https://www.statskingdom.com/anova_manova.html | |
| Halosperm G2 | Halotech | HT-HSG2 | In vitro diagnostic kit for measuring sperm DNA fragmentation https://halotechdna.com/en/producto/halosperm-g2/ |
| Microcell | Vitrolife | 15423 | Disposable counting chamber for semen analysis https://www.vitrolife.com/products/sperm-processing/microcell--accessories/ |
| Quality Control for WBC/pH Test Strips | Medical Electronic Systems | A-CA-01752-00 | Positive and negative control for QwikCheck WBC test strips https://mes-global.com/products/semen-analysis-validation-kits/qwikcheck-test-kits/qwikcheck-wbc-ph-test-strips-quality-control/ |
| QwikCheck WBC/pH Test Reagent Strips | Medical Electronic Systems | 0700 | Determine leukocytes (WBCs) in semen https://mes-global.com/products/semen-analysis-validation-kits/qwikcheck-test-kits/ |
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