Abstract
The impact of ejaculatory abstinence on semen parameters using in-office semen analyses has been well-established; however, their variability has not been evaluated in men using mail-in semen analysis kits. Our study aims to describe how the sperm parameters using mail-in semen analysis tests change with abstinence and validate their equivalence to those seen with in-office semen analysis tests. We retrospectively reviewed the semen analysis results of men using mail-in semen analysis tests provided by Give Legacy, Inc (Legacy) facilities from 2019 to 2021. We collected their demographic information, abstinence duration, and semen parameters (conventional and kinematic) from their records. Semen samples were categorized as normozoospermic and oligozoospermic based on concentration. The shape of the relationship between abstinence duration and semen parameters was assessed via generalized additive models. We have collected 3,469 unique samples provided by 2,609 (75%) normozoospermic men and 860 (25%) oligozoospermic from all over the United States. In normozoospermic men, longer periods of sexual abstinence were linked to higher levels of sperm concentration, total sperm count, and total motile sperm. However, there was a decline in both total and progressive motility. Conversely, in oligozoospermic men, extended periods of abstinence led to a rapid decline in total motile sperm, as well as total and progressive motility. There was no significant correlation observed between sexual abstinence and variations in sperm morphology. Our study shows that variability of sperm parameters with abstinence, as measured through mail-in semen analysis tests, is comparable to the patterns observed with conventional in-office sperm testing.
Introduction
Infertility is a global health concern that affects couples of reproductive age worldwide. Male factors solely account for 20% of infertility cases and contribute overall to 50% of the total (Agarwal et al., 2015). A semen analysis test has been traditionally regarded as the cornerstone for evaluating male infertility, in which quantitative and qualitative characteristics of sperm are used to predict the possibility of conception (Barratt, 2007). Semen parameters are dynamic variables that change across time, geography, and individuals. Semen analysis results also exhibit variance within the same individual as they are highly affected by lifestyle options, environmental exposures, and health status (du Plessis et al., 2014).
Another confounding factor that significantly contributes to the variability of semen analysis results is the duration of ejaculatory abstinence or frequency of ejaculations (Pellestor et al., 1994). Given the complex physiological events and changes that occur during spermatogenesis and epididymal maturation, spermatozoa that spend variable lengths of time in the epididymis are expected to have different structural and functional characteristics upon ejaculation (Gervasi & Visconti, 2017). The impact of ejaculatory abstinence on semen parameters has been extensively evaluated by previous studies (Keihani et al., 2017; Sokol et al., 2021). Longer abstinence periods are reported to yield more favorable sperm count and concentration with a noticeable deterioration in sperm motility and morphology with prolonged periods (Sokol et al., 2021). All previous studies utilized in-office semen analysis tests where presenting patients provide fresh samples that are analyzed within 1 hr at traditional Andrology labs (Ayad et al., 2018a). Their findings have limited generalizability to men who perform at-home, mail-in semen analysis tests, as the samples are collected at home and shipped overnight to the lab for analysis.
Despite the increasing popularity and convenience of home-based semen testing kits, no research study to date has evaluated the variability of sperm parameters with abstinence in this population of men. Our study aims to explore this association in men using a mail-in semen analysis test and validate their comparability to the measurements seen with traditional in-office analysis.
Materials and Method
Approval from our institutional review board was obtained for this study (STUDY#20220802). We collected data from GiveLegacy Inc, a fertility company established in 2018 and headquartered in Boston, Massachusetts. They specialize in providing convenient mail-in fertility testing services such as semen analysis and sperm storage. The mail-in testing kits used in this study were previously validated (Aznavour et al., 2022; Navarrete et al., 2022). GiveLegacy research activities are available through Western Institutional Review Board Copernicus Group IRB approval (Study # 1317234) for this retrospective study.
We retrospectively reviewed the records of men who requested semen analysis from 2019 to 2021. The need for informed consent was waived as only retrospective de-identified data was used. Comprehensive semen analysis was performed in all subjects as part of their fertility workup. The abstinence period, age, body mass index (BMI), smoking status, race/ethnicity, geographic location, and sperm analysis results were documented for each individual. The abstinence period was recorded by the client on Legacy’s secure website. Analyzed semen parameters included sperm concentration, total sperm count, total sperm motility, progressive motility, total motile sperm count, and sperm morphology. All semen samples were self-collected at home. A fixed volume of pre-packaged transport buffer media was added to the produced sample to preserve the quality and viability of the ejaculated sperm. This is the same buffer media used for sperm preservation during assisted reproductive technology (ART) procedures. The samples were shipped overnight and the majority reached the lab within 24 hours. Sperm kinematic parameters including the amplitude of lateral head displacement (ALH), linearity (LIN), average path velocity (VAP), and beat cross frequency (BCF) were also measured with the advent of the Ceros CASA system (Hamilton Thorne Research, Beverly, MA). Results were interpreted using the World Health Organization (WHO) guidelines for semen analysis (“WHO Laboratory Manual for the Examination and Processing of Human Semen,” n.d.). Men with a sperm concentration less than 15 million/mL were considered oligozoospermic, whereas those with values above 15 million/mL were considered normozoospermic. Subjects that were less than 18 years old, azoospermic, or found to have missing values in their semen analysis results were excluded from the study.
Sperm Processing
Semen samples were collected by masturbation into sterile containers mixed with transportation media (mHTF Vitrolife) using at-home semen collection kits. Ejaculated samples were shipped overnight to the main Legacy laboratory in San Antonio, Texas for sperm analysis. Ejaculates were liquefied and subsequently processed by centrifugation, suspended, and washed one time in mHTF to remove seminal plasma, immature germ cells, and non-sperm cells. Ten microliters of the purified sperm were diluted in Origio sperm wash (cat# 84055060) to follow sperm motility assessment and morphology.
Semen Volume
The semen collection cup used by Legacy is the standard ISO-certified sterile cup used at the physical traditional Andrology labs. The cup is graduated up to 30 mL with anti-leakage properties. Along with the collection cup, each semen kit semen contains a standard 10 mL vial of a commercial Semen Transport Media that was approved and validated to preserve the quality of the sperm during the 24 hr overnight transport through FedEx mail. After producing the sample via masturbation, the patient would add the whole 10 mL transport media and seal the cup. The cup is placed in the mail-in padded kit with the multiple long-durability ice packs and mailed to the CLIA-certified Legacy Labs. All kits have a temperature indicator sticker that would allow the detection of extreme temperatures during transport. Any kit that arrives after 24 hr is discarded and the patient is notified to send a new complementary kit.
Similarly, kits with signs of damage during shipping, ice packs melting, or extreme temperatures are also discarded. Upon arrival, the andrology lab technician would record the total volume of the cup and then subtract 10 mL to calculate the original semen volume produced. This will allow the estimation of the actual ejaculated seminal volume, and subsequently calculate the concentration and total motile sperm count. For any discrepancy less than 1 mL, the technician will calculate the mean between the two values. For any discrepancy of more than 1 mL, the patient would be asked to repeat a complementary test.
Motility Assessment
Sperm suspensions (5 μl) were loaded into one pre-warmed chamber slide (depth, 20 μm; Cell vision, The Netherlands) and placed onto a microscope stage at 37°C. Sperm movements were examined using the CASA system. The default settings include the following: One-second tracks were captured using the following settings: 60 frames per second, 60 frames acquired, minimum contrast = 80, minimum size = 3 pixels, default cell size = 6 pixels, default cell intensity = 160, slow cells counted as motile, low VAP cutoff =10 μm/s, low straight line velovity (VSL) cutoff = 0 μm/s, minimum intensity gate = 0.18, maximum intensity gate = 1.21, minimum size gate = 0.56 pixels, maximum size gate = 2.63 pixels, minimum elongation gate = 0 pixels, and maximum elongation gate = 99 pixels. The percentage of motility was calculated by dividing non-progressive motility over progressive motility.
Sperm Morphology
For the Diff-Quik staining kit method, the WHO manual guidelines were followed (“WHO Laboratory Manual for the Examination and Processing of Human Semen,” n.d.). Ten microliters of semen was smeared on a slide, which was fixed by immersion in triarylmethane fixative for 15 seconds after complete air drying. The smears were then consecutively stained by solution 1 (10 seconds), then air-dried and stained by solution 2 (5 seconds). Finally, the slides were washed in running tap water to remove the excess stain (10 to 15 times). The stained slides were scored under Ceros CASA system and DIMENSIONS II Software (Hamilton Thorne, Beverly, MA) at 100 x magnification with oil immersion (Leica Microsystems) within 5 hours of their preparation.
Statistical Analysis
All analyses were conducted in R (version 4.1.2). For descriptive tables, participants were grouped by days of abstinence into four groups (1–2, 3–5, 6–8, and 9–10 days). Differences between these groups were assessed via Kruskal-Wallis tests for continuous variables and chi-squared tests for categorical variables.
For statistical modeling, as each semen parameter was positively skewed they were log(x+1) transformed prior to analysis. The relationship between transformed semen parameters and the number of days of abstinence was assessed via generalized additive models (GAMs). Specifically, the shape of the relationship was modeled using thin plate regression splines (Wood, 2003). Separate smooth functions were included for the normozoospermic and oligozoospermic groups. Estimated spline functions are plotted with 95% credible intervals and differences in semen parameters between the normozoospermic and oligozoospermic groups are assessed at different days of abstinence. Age, BMI, and smoking status were included as covariates, with age and BMI also modeled via thin plate regression splines.
The values of some variables were missing in a number of participants (presented in Supplemental Table 1). Missing data were imputed via multivariable imputation by chained equations using predictive mean matching including all other covariates and (transformed) semen parameters in the imputation model for a given variable. Twenty imputed data sets were created using the mice package for R (van Buuren & Groothuis-Oudshoorn, 2011). Each imputed data set was analyzed via GAMs, which were fit via Bayesian estimation via the brms package for R (Bürkner, 2018). The prior distributions on model parameters were vague on the scale of analysis. For the analysis of each imputed dataset there were 2,000 posterior samples from 4 parallel chains with the first 1,000 discarded as warm up, leaving 4,000 post-warm-up samples. Results were pooled across the 20 imputed datasets by combining the posterior samples before calculating posterior quantities (e.g., posterior mean, 95% credible interval).
Results
A total of 3,469 unique semen samples were retrospectively reviewed and included in the analysis. Our cohort included 2,609 (75%) normozoospermic and 860 (25%) oligozoospermic men that requested semen analysis for fertility evaluation from 2019 to 2021. The mean age of the whole cohort was 34.9 ± 7.3 years and the mean length of ejaculatory abstinence 3.8 ± 1.4 days. The demographic characteristics of the enrolled subjects including their racial/ethnic background, BMI, and geographic location based on place of residence are illustrated in Table 1 and reflect the diversity of our population.
Demographic Characteristics of the Study Cohort
The mean age of the normozoospermic group was 34.9 ± 6.8 with a mean abstinence duration of 4.0 ± 1.4 days. Table 2 shows a progressive increase in sperm concentration (million/mL), sperm count (million), and total motile sperm (million) with increasing abstinence periods subsequently peaking after 6–8 days at 118.8 ± 107.9, 302.5 ± 237.3, and 121.0 ± 148.6, respectively (p < .001). While shorter abstinence periods were associated with lower sperm count and concentrations, their values were still considered within the normal reference range. On the contrary, prolonged abstinence durations were associated with a significant decrease in total and progressive sperm motility (p < .001; Table 1). The spline functions affirm these relationships between different semen parameters and period of ejaculatory abstinence in normozoospermic men (Figure 1). Progressive improvement in sperm concentration, count, and total motile sperm is seen after 2 to 3 days of abstinence (Supplemental Table 2). A steady deterioration in total and progressive sperm motility is revealed with increasing abstinence periods. The decrease in percentage of normal sperm morphology with increasing abstinence in normozoopsermic men was insignificant (Table 2) and reflected in the spline functions (Figure 1).
Variation of Semen Parameters Among Different Abstinence Categories in Normozoospermic Group
Note. BMI = Body Mass Index; VAP = average path velocity; ALH = amplitude of lateral head displacement; LIN = linearity; BCF = beat cross frequency.
Variation of Semen Parmeters With Abstinence in Normozoospermic (Left) and Oligozoospermic (Right) Men Using Thin Plate Regression Spline Models
The mean age of the oligozoospermic group was 35.0 ± 7.7 with a mean abstinence duration of 3.6 ± 1.4 days. There was no significant variation in the total sperm count, concentration, and percentage of normal sperm morphology among different abstinence categories in the oligozoospermic group (Table 3). However, a progressive decrease in total motile sperm count and total and progressive sperm motility was significantly associated with increasing abstinence durations. According to the modeled relationship in Figure 1, an increase in abstinence is associated with a decline in total motile sperm and progressive motility in oligozoospormic men. The percentage of normal sperm morphology is seemingly unaltered by prolonged abstinence periods (Figure 1). No improvement in sperm parameters was observed in oligozoospermic men with increasing abstinence (Supplemental Table 3).
Variation of Semen Parameters Among Different Abstinence Categories in Oligozoospermic Group
Note. BMI = Body Mass Index; VAP = average path velocity; ALH = amplitude of lateral head displacement; LIN = linearity; BCF = beat cross frequency.
Rapid and objective analyses of semen samples by the CASA system enabled the measurement of sperm kinematic parameters (VAP, LIN, ALH, and BCF). These variables provide a profound characterization of the movement characteristics of sperm cells. A decrease in all kinematic parameters was noted with longer abstinence durations in both groups; however, this variability did not reach statistical significance. Similarly, significant variation in kinematic parameters could not be appreciated with increasing abstinence duration in the spline function models illustrated in Supplemental Figure S1.
Discussion
To our knowledge, this is the first study to utilize at-home, mail-in sperm testing to evaluate the impact of abstinence on semen parameters. This approach provides a unique perspective on the role of ejaculatory abstinence in this specific population of men. Our study shows the variability of semen parameters with abstinence as measured through mailed samples is similar to the patterns seen with traditional in-office collection methods.
Utilizing a mail-in semen analysis system can be challenging as it requires tight control over the conditions to which the samples are exposed during transportation and the time elapsed since specimen collection. Introducing at-home semen collection kits offers several advantages to individuals seeking fertility testing. Mail-in tests improve overall convenience and accessibility to fertility care as individuals can collect semen samples in the privacy of their own home, especially if they live in remote areas or have limited access to medical facilities (Bradshaw et al., 2020). It also provides increased privacy for individuals looking to get evaluated for infertility compared to in-office testing (Gonzalez et al., 2021). Furthermore, this system allows for better standardization by centralizing the analysis procedures in one location. This minimizes the inter-observer variability that is usually seen with in-office semen analysis (Masterson & Patel, 2021).
Total sperm count and concentration are closely correlated with the spermatogenic activity in testes and their optimization is traditionally believed to improve fertility potential (Kumar & Singh, 2015). The progressive increase in sperm count, concentration, and total motile sperm with prolonged abstinence is a well-established observation in normozoospermic men that was demonstrated by our study as well as others (Agarwal et al., 2016; Chen et al., 2022).
Sperm motility is acquired during epididymal maturation and is regarded as an absolute prerequisite for the transit and penetration of oocyte (Gervasi & Visconti, 2017). In our study, CASA results showed significantly higher percentages of total and progressive sperm motility after short periods of abstinence when compared with longer abstinence periods. Other studies have also reported that sperm motility is optimal before 3 days of abstinence and progressively worsens afterward in normozoospermic men (Bahadur et al., 2016; Elzanaty et al., 2005). Deterioration in sperm motility with abstinence can be attributed to the longer time spent in the epididymis. This includes prolonged exposure to enzymes and motility-inhibiting factors from degrading cells, limited energy reserves, increased heat stress, and elevated levels of oxidative stress (Alahmar, 2019).
The sperm kinematic parameters as measured by CASA showed generally higher VAP, ALH, LIN, and BCF with shorter abstinence periods in both normozoospermic and oligozoospermic groups; however, their variation was not statistically significant. This might be due to the relatively high amount of missing values of kinematic parameters from our dataset. Despite employing an imputation approach to handle missing data, we cannot accurately determine the shape of the relationship. A smaller study using CASA revealed significantly higher VAP, LIN, and BCF with shorter abstinence periods with no differences detected in ALH (Ayad et al., 2018b). Similarly, another study by Elzanaty el al. demonstrated that men who abstained for shorter periods had higher values for VSL and LIN with no significant differences in VAP and VCL between the abstinence groups (Elzanaty et al., 2005). Whether optimizing sperm velocities and movement is clinically advantageous and improves fertility outcomes is yet to be elucidated with more research in the future.
Several studies have evaluated the impact of sexual abstinence on sperm morphology; however, the results have been conflicting. The CASA results in our study demonstrate minimal variation of sperm morphology with abstinence, as corroborated by other studies (Lehavi et al., 2014). On the contrary, one report suggests significant improvement in the sperm morphology of oligozoospermic men when measured manually after very short abstinence periods (Bahadur et al., 2016). It is noteworthy that using the CASA system in our study enabled a more objective and accurate assessment of sperm morphology with high reproducibility of results in comparison to the manual visual assessment employed by previous studies (Welliver et al., 2016).
Our results confirm prior work demonstrating the lack of benefit in increasing abstinence intervals in oligozoospermic males (Levitas et al., 2005). Similar to our findings, a large cohort study in oligozoospermic men revealed worsened semen quality marked by a higher percentage of normal tail morphology and decreased sperm motility after 2 days of abstinence (Keihani et al., 2017). With the exception of semen volume, no clear benefits and improvements in semen parameters were observed with abstinence in oligozoospermic men (Levitas et al., 2005).
The mechanisms by which abstinence impacts sperm parameters have not been fully determined yet. Generally, unejaculated spermatozoa that remain in the epididymis for long periods of time are subjected to significant oxidative stress due to increased levels of reactive oxygen species (ROS) and depletion of sperm antioxidant capacity (Ko et al., 2014). Such exposure can potentially lead to sperm DNA and membrane damage. Oligozoospermic people appear to be more susceptible to oxidative damage caused by long abstinence periods. This has been supported by several reports that have identified higher levels of ROS and sperm DNA damage in the semen of infertile men (Agarwal et al., 2014).
The major strength of this first study is using a robust dataset of sperm parameters that correspond to a relatively large cohort of normozoospermic and oligozoospermic men from all over the country using mail-in semen kits. Our data also included information on patient-specific factors such as age, BMI, and smoking status that were integrated in our analysis to adjust for their potential impact on semen characteristics. Furthermore, our study used a different approach compared with most other studies. We employed a generalized additive models (GAM) approach which treated the duration of ejaculatory abstinence as a continuous variable. Most previous studies divided the abstinence duration into arbitrary categories. This introduces the potential for bias and residual confounding that can result from dichotomizing a continuous variable (Royston et al., 2006). Our study had several limitations. For example, the retrospective nature of our study and the self-reported abstinence periods introduced potential response and recall bias. In addition, our analysis was restricted to conventional and kinematic sperm characteristics and did not include any functional sperm parameters such as DNA fragmentation and ROS levels. We were not able to report semen volume in this study due to sample dilution prior to transit, however, studies that analyzed fresh samples have consistently demonstrated increased overall semen volume after 3 to 5 days of abstinence (De Jonge et al., 2004; Lehavi et al., 2014). We did not establish any correlations between abstinence periods and fertility outcomes, such as pregnancy rates as this data is unavailable to us. Finally, this study did not account for the intra-variability of sperm parameters as all semen samples were provided by unique individuals.
Conclusion
Our study provides a meaningful insight into the variability of sperm parameters with abstinence in a group of men using mail-in semen analysis tests. We show that variation in semen parameters with abstinence using mail-in fertility testing is similar to that seen in men using in-office semen analysis. In normozoospermic men, increased abstinence periods were associated with increased sperm concentration, total sperm count, and total motile sperm, whereas total and progressive motility declined. In oligozpoospermic men, prolonged abstinence was associated with a rapid deterioration in total motile sperm and total and progressive motility. Variations in sperm morphology were not significantly associated with sexual abstinence.
Supplemental Material
sj-docx-1-jmh-10.1177_15579883231197910 – Supplemental material for The Variability of Semen Parameters With Sexual Abstinence Using Mail-in Sperm Testing Is Similar to That Seen With Traditional In-Office Semen Analysis
Supplemental material, sj-docx-1-jmh-10.1177_15579883231197910 for The Variability of Semen Parameters With Sexual Abstinence Using Mail-in Sperm Testing Is Similar to That Seen With Traditional In-Office Semen Analysis by Jad Badreddine, Stephen Rhodes, Nicholas Sellke, Felipe Navarrete, Stephanie Keller, Vrushab Gowda, Paul H. G. Simon and Ramy Abou Ghayda in American Journal of Men's Health
Footnotes
Author Contribution
All listed authors contributed to this manuscript through data collection, analysis, interpretation, writing, and reviewing the manuscript. Manuscript writing/revision: J.B., N.S., and R.A.G.; data collection/management: F.N. and S.K.; data analysis: S.R.; study conception/design: R.A.G., V.G., and P.H.G.S.
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The authors J.B., S.R., and N.S. have nothing to disclose. R.A.G., V.G., and P.H.G.S. are the Chief Medical Officer, Head of Regulatory Affairs, and VP of Operations at GiveLegacy, Inc, respectively. F.N. is a former research associate at GiveLegacy, Inc. The authors declare that they have no competing interests regarding the research described in this article. They did not receive any additional funding, or financial or non-financial gains associated with this research. GiveLegacy, Inc. did not intervene nor influence the design, outcomes, materials, methods, or results of this study. There is no direct or indirect gain for GiveLegacy, Inc. in relation to the publication of this study.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
Ethics Statement
This study has been reviewed and approved by the institutional review board at University Hospitals Cleveland Medical Center (STUDY#20220802). The need for informed consent was waived as only retrospective de-identified data were used. We collected data from GiveLegacy Inc. GiveLegacy research activities are available through Western Institutional Review Board Copernicus Group IRB approval (study # 1317234) for this retrospective study.
Supplemental Material
Supplemental material for this article is available online.
References
Supplementary Material
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