Research Article

Time-dependent Deterioration of Sperm Motility and DNA Integrity During Prolonged Semen Exposure

DOI:

10.3791/71515

⸱

August 7th, 2026

 ,  ,  ,  ,  ,  , 
1University Hospitals Fertility Center

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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.

Protocol

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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.

Results

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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). Initial semen parameter results of the group yielded an average semen volume of 3.7 mL, average concentration of 42.2 million/mL, average motility of 46.9%, average normal morphology of 4%, and negative white blood cells for all samples by qualitative test (Table 1).

A progressive decline in sperm motility was observed over time. Compared with baseline (0 h), average sperm motility decreased by 7.4% (range 3–16%) at 1 h, 14.5% (range 4–31%) at 2 h, and 18.7% (range 8–37%) at 4 h. Sperm motility declined progressively over time, from 58.6% at baseline to 42.5% after 4 h of exposure (Figure 1). Overall, the reduction in motility across time points was statistically significant (p = 0.0001585). Concurrently, SDF increased significantly with prolonged incubation in seminal plasma. Mean SDF increased from 15.6% at baseline to 21.1% at 1 h, 27.1% at 2 h, and 31.7% at 4 h. Relative to baseline, SDF increased by 5.5 percentage points (range 4–8 percentage points) at 1 h, 11.5 percentage points (range 8–16 percentage points) at 2 h, and 16.1 percentage points (range 13–22 percentage points) at 4 h. Statistically significant increases in SDF were detected at all subsequent time points compared with baseline, including 1 h (p = 0.021129), 2 h (p < 0.00001), and 4 h (p = 0.000018) (Table 2).

DATA AVAILABILITY
The patient-level motility and SDF data used for the analysis are provided in Supplementary Table S1.

figure-results-1
Figure 1. Change in sperm motility over time after ejaculation. Sperm motility was assessed at baseline and after 1 h, 2 h, and 4 h of semen exposure. Motility declined progressively with increasing exposure time. Please click here to view a larger version of this figure.

ParameterAverageStandard deviation
Patient age35.4 (range, 24–45 years)6.22
Semen volume3.7 mL2.18
Concentration42.2 million/mL33.45
Motility46.9%18.77
White blood cellsNegative (all)—
Morphology4.0%0.77

Table 1: Baseline semen characteristics of the study samples. Semen parameters were recorded after liquefaction and before time-dependent assessment of sperm DNA fragmentation and motility.

Time0 h1 h2 h4 h
Average sperm motility reduction from 0 h*—-7.4%-14.5%-18.7%
Average SDF15.6%21.1%27.1%31.7%
Standard deviation5.28%5.27%5.31%4.80%
P-value vs. 0 h—p = 0.021129p < 0.00001p = 0.000018
*Repeated-measures ANOVA with Tukey HSD post hoc comparisons.

Table 2: Time-dependent changes in sperm DNA fragmentation and motility. Sperm DNA fragmentation and motility were assessed at baseline and after 1 h, 2 h, and 4 h of semen exposure. Values are presented as reported by the authors, with statistical comparisons performed against baseline.

Supplementary Table S1. Patient-level sperm motility difference and sperm DNA fragmentation index values. Values are expressed as proportions; multiply by 100 to convert to percentages. Please click here to download this file.

Discussion

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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 DNA fragmentation6,8,13. The results of this study may inform semen handling in ART cycles.

First, it may be worthwhile for clinics to add this test to the traditional semen analysis parameters to ensure the sperm DNA quality before utilizing the sperm in ART cycles. Optimizing sperm quality may be associated with improved outcomes, such as higher numbers of frozen embryos, increased euploidy rates, and higher rates of positive pregnancies and live births, as reported in previous studies16.

Second, these results provide insight into the effects of home semen collection on ART outcomes. During the COVID-19 pandemic, many clinics began to utilize at-home semen collection as a safer option for patients15,17. As practices began to make this shift, patients were afforded the opportunity not only to reduce COVID exposure but also to perform the collection in the privacy of their own homes. However, our findings suggest that these practice shifts may involve tradeoffs related to sperm quality and potentially ART outcomes.

Previous studies on at-home collection kits have shown large fluctuations of semen analysis results for men with varying male factor diagnoses, and men who typically produce “normal” semen samples18. This fluctuation may be due to the stagnation of sperm during the transport process from home to clinic, especially when shipping semen samples overnight in some cases. Other studies support our result that SDF rapidly increases in the first 3–4 h of incubation in 5% CO2 at 37 °C after sperm preparation either in frozen/thawed sperm19 or fresh sperm20, suggesting that sperm should be used as quickly as possible after sperm preparation for ART.

Kerdtawee et al. performed a meta-analysis of the effect of sperm collection location on semen parameters and fertility outcomes. While they did not show any significant differences between at-home and clinic-collected samples, they did note significant heterogeneity across the studies analyzed, most of which were observational studies16. Importantly, these studies also did not have a controlled timeline of when samples collected at home should be returned to the clinics and analyzed. The data analyzed in this study, however, enabled a controlled analysis of DNA fragmentation across several time points, yielding greater precision and consistency in experimental results.

While at-home collection may be an option for some patients, understanding the rate of SDF may help develop guidelines to identify patients for whom home collection may be detrimental to ART outcomes. A systematic review and meta-analysis by Christoforaki et al. found that oxidative reduction potential in seminal fluid negatively affects male infertility21. Parameters analyzed include sperm concentration, count, motility and morphology, all of which were unfavorably affected by higher oxidative reduction potential, demonstrating that the quality of sperm may be influenced by oxidative stress.

Strengths of this study include its novelty, as few studies have assessed time-dependent SDF changes during prolonged exposure to seminal fluid. This study was also performed in a controlled setting with a relatively homogenous group of men without underlying seminal pathology. However, this study was limited to a small sample size of 14 patients. Further experiments should be completed to maximize confidence in the collected data points and conclusive remarks.

Further research should also investigate variables that could affect SDF rates, specifically to better understand the factors that contribute to maintaining sperm quality. Some factors to consider include, but are not limited to, handling media utilized during sperm preparation, temperature of transport conditions from home to clinic, cryopreservation methods, and underlying conditions of male patients. Gaining comprehensive insight into the factors that contribute to SDF will ultimately allow clinics to optimize their guidelines and better inform their decisions when establishing protocols for treating patients receiving ART.

The observed deterioration in sperm quality demonstrated a clear time-dependent pattern, characterized by decreasing motility and increasing DNA fragmentation during prolonged exposure to seminal fluid. Notably, the most pronounced deterioration occurred after 2–4 h of storage, when SDF exceeded 30%, and motility declined by nearly one-fifth from baseline. These findings indicate that delays between ejaculation and semen processing may negatively affect sperm functional integrity and support the importance of minimizing transport and handling times for samples collected off-site for assisted reproductive technology procedures. Andrology labs may consider establishing and communicating an acceptable time threshold to patients for receiving semen from off-site collection, especially in the setting of preparing for IUI or IVF. Time from ejaculation to semen analysis and/or preparation should be considered regarding SDF, especially when collection occurs off-site from the andrology lab for ART.

Because of the limitations of this study's small sample size, future studies should evaluate the effect of prolonged semen exposure on SDF across a broader range of semen quality categories, including samples from men with abnormal semen parameters. In addition, prospective randomized controlled trials are needed to determine whether time-dependent increases in SDF translate into clinically significant differences in ART outcomes, including fertilization, embryo development, implantation, pregnancy, miscarriage, and live birth rates.

Disclosures

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The authors have no conflicts of interest to declare.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
ANOVA CalculatorStatistics KingdomStatistical analysis
https://www.statskingdom.com/anova_manova.html
Halosperm G2HalotechHT-HSG2In vitro diagnostic kit for measuring sperm DNA fragmentation
https://halotechdna.com/en/producto/halosperm-g2/
MicrocellVitrolife15423Disposable counting chamber for semen analysis
https://www.vitrolife.com/products/sperm-processing/microcell--accessories/
Quality Control for WBC/pH Test StripsMedical Electronic SystemsA-CA-01752-00Positive 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 StripsMedical Electronic Systems0700Determine leukocytes (WBCs) in semen
https://mes-global.com/products/semen-analysis-validation-kits/qwikcheck-test-kits/

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MedicineEncyclopedia of Experiments Biological TechniquesDNA DamageDNA FragmentationSemen Analysis

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