Systematic review and meta-analysis of COVID-19 association with human semen quality: Changes in semen parameters pre- and post-COVID

Abstract

Coronavirus disease (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may infect the cells through binding with the angiotensin-converting enzyme 2 (ACE2) receptors that can also be found in the male reproductive organs. Relevant studies were searched in medical databases and gray literature using controlled search terms including COVID-19, SARS-CoV-2, semen, and sperm, among other related keywords. The authors of this systematic review assessed the eligibility of selected articles, followed by analysing the quality and reliability of the studies using the Cochrane Risk of Bias in Nonrandomized Studies of Exposure (ROBINS-E) tool. Collected data were processed using RevMan5 software, with a random effects model for meta-analysis. Combined mean difference and confidence intervals of semen parameters were obtained by using inverse variance method for assigning the weight of each included article. Seven studies with complete pre- and post-COVID-19 semen analysis data of the same individuals were included in the systematic review. Compiled data from a total of 153 participants revealed that COVID-19 is associated with semen quality impairment, with meta-analysis showing significant decrease of sperm concentration and progressive motility. However, the risk of bias analyses considered all the included studies as “very high risk.” This study revealed the susceptibility of semen parameter impairments in association with COVID-19. However, this finding is limited by the small number of high-quality studies and the lack of control for other confounding variables of semen quality and male fertility.

Graphical abstract

Keywords

andrology / COVID-19 / infertility / male infertility / semen parameters / semen quality

1 INTRODUCTION

A novel coronavirus was initially discovered at the end of 2019. It rapidly spread, followed by an increasing number of cases all over the world. The disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was declared a global pandemic in the year 2020[1,2].

SARS-CoV-2 mainly affects the respiratory system by damaging the lung epithelial cells and its vasculature, which results in diffuse alveolar damage and, ultimately, respiratory insufficiency[3]. SARS-CoV-2 enters the cell through binding with the angiotensin-converting enzyme 2 (ACE2) receptors[4]. Considering the abundance of ACE2 receptors expressed in the male reproductive organs, some even hypothesized that COVID-19 could be sexually transmitted and negatively affect male fertility. Numerous cohorts and case series investigating this matter have been published with an overall mixed result. Several studies have detected the presence of SARS-CoV-2 in testicular tissues and semen[4–6]. At the same time, others have found contradicting evidence[7–9]. Several prospective cohort studies have observed a mixed result of abnormal levels of sex hormone and sperm quality in men with a history of SARS-CoV-2 infection[10,11].

The probable mechanisms of SARS-CoV-2 affecting the male reproductive system are direct viral invasion of the testicular germ cells, disruption of the endocrine system and axes, and secondary damage from systemic inflammation as a response to the viral infection.

There are case reports on patients with acute COVID-19 who complained of testicular pain with evidence of pathologies within the testicular tissue[12,13]. Six out of 34 Chinese men suffered from scrotal discomfort around the time of COVID-19 diagnosis confirmation[14]. They did not find any evidence of SARS-CoV-2 with reverse transcription-polymerase chain reaction (RT-PCR) from the semen samples, making the sign of scrotal pain remain unclear. A post-mortem study conducted by Yang et al. observed significant pathological injury to Sertoli cells and seminiferous tubules, reduction of Leydig cells, and mild inflammatory interstitium[6]. In the same study, SARS-CoV-2 virus was detected by RT-PCR in one out of 12 samples. However, the authors argued that this detection was most likely derived from blood rather than from testicular tissue, considering the testicular tissue sample contained very limited seminiferous tubules.

Given that SARS-CoV-2 is present in the testis, there is a chance that the testis-blood barrier will be breached, and with fewer germ cells, the current evidence and findings are inconclusive as to whether COVID-19 can be transmitted sexually and what impact this might have on male reproduction. A systematic review by Gonzales et al. found that only one out of eight studies successfully detected seminal SARS-CoV-2[15,16]. Positive SARS-CoV-2 was obtained from 6 (15.8%) of 15 (39.5%) patients during the acute stage of infection. However, there are several limitations to this study, only qualitative RT-PCR was used to analyze the semen samples for SARS-CoV-2, both the limits of detection and the threshold values could not be specified, there is no information regarding the PCR primer used, and the PCR primers used in the literature are heterogeneous. However, viral infection is not the only factor that determines whether the virus may exist in the testis. High blood viral loads, local inflammation, hyperpyrexia, and a defective testis-blood barrier are other conditions where the virus may infiltrate and alter testicular tissue.

Previous investigations of patients infected by the SARS-CoV-1 showed disruption of hormone levels: increased serum levels of prolactin, follicle-stimulating hormone (FSH), and luteinizing hormone (LH); decreased levels of oestradiol, progesterone, and thyroid-stimulating hormone[17]. Considering the viral genomic similarity between SARS-CoV-1 and SARS-CoV-2, it has been hypothesized that SARS-CoV-2 infection may also cause endocrine disturbances[18].

Findings from a cross-sectional study performed by Ma et al. were in line with the mentioned hypothesis[10]. Higher serum LH and a lower ratio of testosterone to LH were observed in 119 men with a history of COVID-19 compared to 273 age-matched controls. Leydig cells are the major source of testosterone in the adult male. The damaged Leydig cells produced less androgen, which creates feedback to the pituitary to produce more LH. Such findings suggest the negative impact of SAR-CoV-2 on the hypothalamus-pituitary-gonadal axis, while others thought the hypogonadism was immune-mediated.

While acute COVID-19 in men is associated with an initial reduction in testosterone levels, evidence of long-term effects on male reproductive health remains limited. The damage to male reproductive organs may stem from secondary effects of systemic inflammation, such as fever, metabolic syndrome, altered body composition, and overall malaise[19]. During infection, disruption of homeostasis leads to the overproduction of reactive oxygen species, which facilitates viral replication and induces cellular apoptosis, potentially harming the male reproductive system[20]. Fever, a common symptom of SARS-CoV-2 infection, can be particularly detrimental to spermatogenesis. The ideal temperature for sperm production ranges from 29°C to 36°C, and prolonged fever can raise testicular temperature, leading to germ cell degeneration[21]. For instance, a cohort study involving 18 men with COVID-19 and fever observed significant declines in semen quality across various parameters, including sperm concentration, total sperm count, and motility[8].

Patients with severe COVID-19 have high levels of circulating pro-inflammatory cytokines, which contribute to the development of cytokine storms[22]. Cytokines such as interleukin-6 and tumor necrosis factor-alpha disrupt the integrity of the testis-blood barrier, which allows them to enter the testicular tissue inducing orchitis[23,24]. Orchitis damages the seminiferous epithelium, spermatogonia stem cells, and homeostasis of the testis microenvironment.

With the COVID-19 pandemic and its possible pathophysiological involvement in the male urogenital system, a comprehensive review of the literature is needed to research the causes and understand the implications of the involvement and effect of SARS-CoV-2 on semen parameters.

There were rapid publications of systematic reviews investigating the effect of COVID-19 on the male reproductive system. The main topic of those reviews followed the trend of published articles where earlier (April 2020–July 2020) studies concentrated on detecting SARS-CoV-2 in semen and testicular tissue, while later (August 2020–April 2021) studies explored the quality of sperm, male hormone levels, and fertility. Earlier systematic reviews published by Gonzales et al. and Sharma et al. investigated articles reviewing the presence of SARS-CoV-2 virus in semen samples[15,25]. Later reviews like those published by Tiwari et al., He et al., and Tur-Kaspa et al. analyzed the impact of COVID-19 on sperm parameters, male hormone level, and fertility[26–28]. However, none of the published systematic reviews specifically include studies that have pre- and post-COVID-19 sperm parameter values. The authors considered this to be a significant limitation of the mentioned published reviews, knowing that semen parameters are subjected to inherent intraindividual variability over time[29–31]. Furthermore, none of the systematic reviews mentioned above conducted or attempted to report the risk of bias assessment of the included studies, which raises concern about the overall rigor and transparency of the systematic review findings.

The lack of a comprehensive quality assessment or risk of bias assessment in a systematic review has nega-tive consequences which can limit the overall reporting and interpretation of the study results[32,33]. To control for potential biases and confounding in a systematic review, the authors often attempt to minimize bias in the protocol development stage by setting certain inclusion and exclusion criteria and further assessing in the quality assessment stage[32,34]. However, most of the systematic reviews studying the effect of COVID-19 on male urogenital health did not set strict inclusion criteria. For example, there was no consensus regarding the time of semen sample collection, and the method for analyzing semen samples was not specifically mentioned, resulting in high heterogeneity.

The primary rationale for conducting this systematic review is the significant increase in research and publications concerning SARS-CoV-2 and COVID-19, which necessitates a comprehensive evaluation of the impact of these studies on understanding the effects of the virus on sperm quality. However, most of the research is constrained by a small sample size, limited duration, and could be of questionable quality. Assuming the continuous update and publication of new studies with larger sample size and longer follow-up time, it is essential to include newer studies in an updated systema-tic review. Moreover, none of the published systematic reviews included studies that specifically analyzed before and after COVID-19 semen parameters of the same individual.

Given this background of the potential involvement of SARS-CoV-2 in affecting male reproductive functions and male health and the ongoing concern of deterioration of semen quality on a global scale, the authors conducted a comprehensive systematic review to study the potential changes in semen parameters of the same individual. This is achieved by specifically including pre-post studies or quasi-experimental studies to evaluate the comparison of semen parameters of the same patient before and after COVID-19, which will give a better representation of the real impact of SARS-CoV-2 infection on semen quality as measured by total sperm count, sperm concentration, motility, vitality, and morphology.

2 METHODS

This systematic review was conducted following the guidelines and methods of the “Cochrane Handbook for Systematic Reviews of Interventions (second Edition)”[34]. The protocol of this review and its manuscript is reported following the recommendations of the “Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocol” (PRISMA-P) and Statement, the protocol is registered on PROSPERO (CRD42022356886)[35,36]. Ethical approval is not required because the study does not involve direct patient contact or data regarding the study participants' individual information. All the data were extracted from published articles and preprints from peer-reviewed literature.

The review included both prospective and retrospective studies that provided information on the presence of SARS-CoV-2 and changes in semen parameters. Reviews, commentaries, and overviews were excluded, although their references were screened to identify relevant studies. Given the current pandemic and the limited number of studies available, preprints and unpublished studies were also included and thoroughly analyzed.

Only studies published in English after December 2019 were included in this review, with no restrictions on the study location. Eligible studies reported data on males over 18 with a confirmed SARS-CoV-2 infection, defined by positive RT-PCR results. Participants with a history of urological conditions that could impair semen parameters, such as anatomical abnormalities, inflammatory diseases, endocrine or hormonal imbalances, or prior surgeries affecting fertility, were excluded to minimize confounding factors.

The exposure of interest was defined as patients with or without clinically recognizable COVID-19 symptoms who tested positive for SARS-CoV-2 via nucleic acid amplification tests (NAAT). If NAATs were unavailable, participants who tested positive on antigen tests were included with additional remarks due to concerns about false results. Patients were considered to have recovered from COVID-19 if diagnosed more than 30 days prior, with resolved symptoms, and/or negative NAAT results.

Recognizing that treatment plans for severe COVID-19 cases, ICU admission, and prolonged bed rest due to ventilation might influence semen parameters, data on the severity of participants' illness and proxies for severe cases were carefully recorded. Studies including participants with conditions like varicocele, cryptorchidism, urogenital inflammation, retrograde ejaculation, and other urologic dysfunctions were eva-luated cautiously, given the potential impact of these factors on sperm parameters, complicating the determination of whether changes were due to SARS-CoV-2 infection.

The primary outcome was the change in sperm parameters of the same individual before and after SARS-CoV-2 infection. All authors were contacted for clarifications on missing or insufficient data, but no responses have been received to date.

To be included in this systematic review, studies had to adhere to the “WHO Laboratory Manual for the Examination and Processing of Human Semen,” with both the 5th and 6th editions considered. Data extracted from the studies included total sperm count, semen concentration, volume, motility, vitality, and morphology. Eligible studies collected semen samples both prior to and during/after COVID-19.

The WHO manual recommends 2–7 days of abstinence before semen sample collection to minimize variability in sperm output. Studies reporting abstinence information were noted. Databases such as PubMed, Ovid, Web of Science, Scopus, Europe PMC, and medRxiv were searched for articles published from November 1, 2019, to December 31, 2021. A broad search strategy was used to ensure comprehensive coverage, with terms focusing on COVID-19 and semen. Search terms included: (“COVID-19”[MeSH Terms] OR “COVID-19”[Text Word] OR “SARS-CoV-2”[Text Word] OR “sars-cov-2”[MeSH Terms] OR “Severe Acute Respiratory Syndrome Coronavirus 2”[Text Word] OR “coronavirus”[MeSH Terms]) AND (“semen”[MeSH Terms] OR “seminal”[Text Word] OR “semen*”[Text Word] OR “sperm”[Text Word]). The search strategy is displayed in Table 1.

Duplicates and irrelevant articles were removed, and inclusion was determined after a thorough full-text review by the authors. Study quality was assessed using the “Risk of Bias in Nonrandomized Studies of Exposure (ROBINS-E)” tool[43]. Relevant data, including author details, publication date, study location, design, sample size, and participant demographics, were extracted and summarized. Continuous data on semen parameters before and after COVID-19 were analyzed using RevMan v.5.4 software, applying a random effects model due to high heterogeneity. The combined mean differences and confidence intervals were calculated using the inverse variance method, with summary statistics including means and standard deviations.

3 RESULTS

The initial electronic database search yielded 2366 records. After removing duplicates, 1376 records were screened by title and abstract. Of these, 51 full-text articles were further assessed for eligibility. Most were excluded due to a lack of semen analysis data or failure to compare pre- and post-COVID-19 semen parameters in the same individuals. Additionally, two studies were excluded due to participants' prior urological conditions, which could confound semen analysis results. Ultimately, seven studies met the inclusion criteria, as summarized in Figure 1.

Table 2 provides an overview of these seven studies, detailing their methodologies and populations. The publications span from 2019, at the onset of the pandemic, to 2022. Two of the included studies did not specifically report their study design[10,37], the remaining articles were case reports (n = 2)[38,41], cohort studies (n = 2)[40,42], and cross-sectional study (n = 1)[39]. Across these studies, demographic data and medical histories were collected both prospectively and retrospectively, with semen samples gathered prospectively pre- and post-COVID-19.

The location of the included studies is very limited, three out of seven studies are from Turkey[37,39,40], two from China[10,42], and two from the United States[38,41]. There have been studies that reported variability and significant regional differences among men in semen quality, which raises concerns about the generalizability and comparability of the included studies[44]. Another additional factor that may influence the regional variability of semen parameters is seasonal change. Previous studies reported in their observational study that seasonal factor plays an important part in influencing sperm concentrations and total sperm counts, with the tendency that the sperm concentrations and sperm count tend to be highest in colder seasons and lowest in summer[45].

Being mindful of the nature of most COVID-19 studies and the incapability of randomizing study participants to the exposure, in this case, SARS-CoV-2 infection. Most studies have set relatively strict inclusion and exclusion criteria to control for potential confounding factors. The inclusion and exclusion criteria for most studies are specified in the methods section. Two case reports did not specify inclusion and exclusion criteria but briefly provided the patient's history and health status. Age and SARS-CoV-2 infection were two of the most basic requirements and criteria for selecting the study participants. Prior semen sample collection preceding SARS-CoV-2 infection is another consideration for inclusion in most of the included studies and case reports. Two studies did not specifically require previous semen sample collection before COVID-19 as a priori, previous semen samples were collected for other purposes, the study's main objective did not specifically aim to evaluate changes in semen parameters of the same individual and instead included and presented the same individuals' semen parameters in a subgroup as additional findings[10,42].

Included study participants were predominantly adult males with partners seeking fertility consults. This is to be expected because the likelihood of collecting semen samples is not a routine or common practice in the urology clinic unless there is a need for evaluating the patients' fertility. In all included studies, only one study reported the recruitment method, which is a convenient sampling strategy. Based on reading the study setting and methods section, the authors postulated that the likely recruitment and sampling method is non-probability convenience sampling, which purposively recruited and collected the study participants who had their semen parameters checked before COVID-19 and asked to be included in the study to recollect their semen samples after being diagnosed with COVID-19.

Based on the above considerations, it could be speculated that semen parameters remain affected for up to 74–90 days post-SARS-CoV-2 infection[46]. Most studies collected post-COVID-19 semen samples after the patients had fully recovered, confirmed by RT-PCR or other means. Only one case report attempted to collect the semen sample during the active infection phase[38].

Erbay et al. reported a statistically significant decrease in semen volume after being infected with COVID-19[37]. Seckin et al. observed similar findings of a decreased semen volume within weeks after COVID-19 diagnosis, but it returned to normal values 3 months following recovery[41]. Other included studies did not observe a significant change in semen volume (see Table 3).

Compared to semen volume, semen concentration appears to be more greatly influenced by SARS-CoV-2 infection. Five studies reported a decrease in semen concentration, one of which is statistically significant[10,37,40–42] (see Table 4).

Three studies reported a decrease in total sperm count, two of which are statistically significant[10,37,40]. Two of the case reports observed similar findings, which is a decrease in total sperm count after SARS-CoV-2 infection[38,41]. Two studies reported a slight increase in total sperm count, but both results are not statistically significant[39,42] (see Table 5).

Sperm motility was reported slightly differently in some of the studies, five studies presented and reported motility as the total motility and progressive motility[37–41], whereas two other studies further delineated the sperm motility parameter as non-progressive motility[10,42]. Both of which are acceptable and in consonance with the WHO manual of reporting. Five studies reported a decrease in total motility and progressive motility[37,38,40–42], two of which are statistically significant with a p-value less than 0.05[37,40]. Gul et al. reported an increase in sperm motility but is not statistically significant[39], whereas Ma et al. concluded that the change in sperm motility was inconclusive[10].

The 6th edition of the WHO manual focuses on the total sperm count, motility, and morphology. In the case when motility is found to be abnormal, the examination of sperm vitality is required. None of the included studies presented sperm morphology and only one study reported sperm vitality by Erbay et al. reported a decrease in sperm vitality (p = 0.03) in both mild and moderate COVID-19 groups[37] (see Tables 6 and 7). None of the included studies presented the data for sperm morphology.

Following SARS-CoV-2 infection, semen volume decreased by 0.5 mL (95% confidence intervals [CI]: 0.34–0.45, p = 0.79), sperm concentration decreased by 4.77% (95% CI: 0.37–9.17, p = 0.03), total sperm count decreased by 14.48 million per mL (95% CI: 4.64–33.60, p = 0.14), total motility decreased by 6.84% (95% CI: 4.64–33.60, p = 0.17), lastly, progressive motility decreased by 4.65% (95% CI: 0.15–9.15, p = 0.04). Visual inspection of the forest plots shows that almost all studies had overlapping 95% CIs with the null value, indicating statistically insignificant results[47]. A commonly used cut-off point of I² over 50% is considered to be of “high heterogeneity,” suggesting variability and inconsistency in study results[48] (see Figures 2–6). A summary of the risk of bias assessment of each study is presented in Figure 7.

4 DISCUSSION

The current unprecedented COVID-19 pandemic and the overall decline in semen quality raised interest in the evaluation of the SARS-CoV-2 virus effects on the male urogenital system particularly changes in semen parameters of men who have previously been infected with COVID-19. Semen parameters were initially thought to have no other clinical purpose other than being a carrier of paternal DNA, without having a substantial impact on embryonic development. Researchers have recently understood that the complex epigenetic changes during spermatogenesis under certain exposures may lead to a variety of problems ranging from decreased sperm motility to embryo lethality[49,50]. Historically, analysis of semen parameters is only used for routine evaluation of male infertility, but studies have suggested that semen parameters may play a bigger role as one of the fundamental biomarkers for men's health[51,52].

We learned from the previous 2003 SARS outbreak that SARS-CoV-1 has been associated with orchitis, testicular damage, germ cell destruction, and subsequently, impaired semen parameters in infected men[21]. However, the investigations on SARS-CoV-2 and its presence in the seminal fluid and its effect on semen parameters have yielded some inconsistent results. Currently, a number of studies have been conducted in an attempt to find the presence of SARS-CoV-2 in semen samples of men who have been previously infected with SARS, coupled with studies to find out about its potential to be transmitted via sexual intercourse through the seminal fluid and potential changes in semen parameters after positive COVID-19 diagnosis[7,50,53]. The hypothesis that supports this pathophysiologic mechanism is correlated to genetic similarities of SARS-CoV-2 to SARS-CoV-1 and its affinity to ACE-2 receptors, which can be found in testicular tissues[21,53]. Shen et al. reported that ACE-2 expression was shown to be higher in the gonads of young adult males, implying that young males of reproductive age are potentially more susceptible to impaired spermatogenesis due to SARS-CoV-2 infection, further emphasizing the need for this systematic review[54]. The age distribution of the study populations in the included studies ranges from 20 to 50 years old, with the exception of the case report by Gharagozloo et al. which recruited a study participant older than 50 years of age[38]. The results of the included studies were insufficient to validate the claims that the semen parameters of young adult males are more likely to be affected by COVID-19, due to the lack of age-group trend of semen parameters impairment. Further work needs further analysis to confirm whether the degree of semen parameters impairment is associated with age.

In a recently published trial with 120 post-COVID-19 cohorts, Donders et al. reported compelling evidence that the SARS-CoV-2 virus is not transmissible through the seminal fluid through sexual intercourse following COVID-19 convalescence, none of the sperm samples contained viral RNA which is vital for transmission of viral disease, they discovered a significant decrease in sperm concentration, the total sperm count and both total and progressive motility of the spermatozoa following SARS-CoV-2 infection of 120 study participants[53]. The most detrimental change can be observed during the first month following SARS-CoV-2 infection, the changes in semen parameters became less obvious or slightly recovered in the subsequent month and almost returned to their normal value two months after infection. Based on the conclusion of the latter study, one can assume that the time of semen sample collection after contracting COVID-19 plays an important role when interpreting the results of the included studies in this systematic review.

Although the exposure of this study, which is COVID-19 diagnosis, can be established by a single measurement, time-varying confounding needs to be taken into account. Most of the included studies collected the second semen sample after the patients tested negative and were proven to have recovered from COVID-19, the average reported time of the second semen sample collection is three months post-COVID-19. With the exception of the two case reports where they collected multiple semen samples during SARS-CoV-2 infection and after the patient fully recovered, Wang et al. reported semen sample collection less than one month after recovery from COVID-19 and the second semen sample collection four months after recovery from COVID-19[42]. The rationale that the study authors provided was because of the duration of the spermatogenesis process which can last up to 74 days, suggesting that the detrimental effects on semen could still be observable within this time period. Other than the relatively small sample size, perhaps the time of semen sample collection could be used as justification for why some of the changes in semen parameters were not statistically significant, referring to the cohort study conducted by Donders et al., the recovery rate of the semen parameters of infected individuals differs, but most are likely to recover within 3 months [53].

Other than the time of semen sample collection, the number of semen sample collections should also be taken into consideration. Semen quality is influenced by variables that can be challenging or even impossible to change. For instance, recent febrile illness, fixed sperm production by the testes, and secretions of the accessory organ. Other factors that could influence the interpretation of semen quality, such as days of abstinence prior to semen sample collection, are required to be documented and taken into consideration when attempting to understand the results.

The case report by Gharagozloo et al. was unique because they collected multiple semen samples throughout SARS-CoV-2 infection phases to clearly describe fluctuations in semen quality[38]. This case reported semen parameters during the short period of COVID-19 symptomatic infection, in which the total sperm count of the patient was rendered to zero or a state of azoospermia, along with the reduction in sperm motility and sperm concentration and progressed to recovery at 3-months post-COVID-19. They estimated that the viral incubation period might be earlier considering the duration of time needed for spermatogenesis. It was further postulated that the likely pathophysiologic mechanism for the decline in sperm motility is due to the direct effect of oxidative stress of SARS-CoV-2, causing apoptosis[38,42].

Remarkably, the presence of fever and the severity of COVID-19 symptoms do not appear to have a positive correlation with reduced semen parameters. Despite having a relatively large sample size, the study by Donders et al. has several limitations, such as the lack of comparisons within the same individuals prior to the SARS-CoV-2 infection[53]. In this systematic review, the study authors specifically searched for studies that included pre- and post-COVID-19 of the same individual as a comparison, in order to minimize potential biases and confounding factors that might arise due to lack of randomization. Erbay et al. was the only study that divided and presented the study participants into subgroups based on the presence of fever and COVID-19 severity, the authors concluded that fever is not associated with impaired semen parameters[37].

Based on the literature search conducted for this systematic review, the main consensus of the included articles is that SARS-CoV-2 infection can indeed influence semen parameters. Initially, the study authors did not plan to perform a meta-analysis due to concerns about the quality of the included studies and heterogeneity. Comprehension and interpretation of meta-analysis results require an understanding of the technical concept of heterogeneity. Even in the case where meta-analysis is conducted rigorously if the included studies are highly heterogeneous, the results may be less interpretable or useful[55].

Multiple studies included in systematic reviews will always differ in some manner, but studies are truly heterogeneous when their target population, measurement methods, and timing of outcome measurements differ[55]. One of the concerns for potential heterogeneity is differences in the time of semen sample collection, COVID-19 severity, study designs, semen sample collection and analysis method, and the demographic characteristics of the study participants. However, after the initial screening and after an in-depth reading of all the included articles, in terms of the outcome measurement, all semen samples were collected and analyzed in accordance with the 5th edition of the WHO manual, apart from a case report which will not be included in the meta-analysis. Furthermore, COVID-19 severity was consistent across studies, all study participants were presented with mild or moderate COVID-19 symptoms and none of the included articles recruited patients with severe COVID-19. The included studies are comparable, and meta-analysis might be feasible excluding the two case reports. This leaves the time of semen sample collection as the biggest concern for a potential source of heterogeneity, most samples were collected within three months after the patient had recovered from COVID-19, however, we have learned from a previous cohort study by Donders et al. that changes in semen parameters can be substantial and can recover with time after recovery from COVID-19[53].

Based on the meta-analysis, the results of sperm concentration and progressive motility were the only results with p-values that were less than 0.05 and thus considered to be statistically significant, no statistical significance was observed among the results of semen volume, total sperm count, and total motility. By inspecting the forest plot, the study by Gul et al. stood out among the others, the authors reported an increase in all semen parameters after contracting COVID-19, however, the results were not statistically significant and there were no correlations between the change in semen parameters and the medication used or hospitalization status[39]. The potential explanation for the increase could be due to non-differential measurement error.

The leading hypothesis and reasonable assumption for how SARS-CoV-2 that cannot be identified in all seminal fluid samples of the collected samples can potentially lead to testicular damage are likely due to the excessive inflammatory cytokines produced as a consequence of the body's response to the infection, which may disrupt the blood testicular barrier and negatively impair Sertoli and Leydig cell functions[6,21,53]. The inflammatory process characterized by fever, as one of the cardinal signs in most infectious diseases, can cause potential damage to the germ cells by the mechanism of leukocyte infiltration and subsequently affect the Leydig cells which cause a decrease in testosterone level[56]. Other potential semen parameters impairment mechanisms might be due to COVID-19 medications. Temiz et al. have conducted a prospective study to investigate the association of hydroxychloroquine, azithromycin, and impaired semen parameters, the study found that sperm morphology was impaired, but through statistical analysis, the study authors determined that the morphological abnormality was caused by fever[57].

One of the limitations of this study is the relatively high risk of bias in the domains. In systematic reviews, we often cannot control for bias, and can only conduct a risk of bias assessment to evaluate and compensate for how bias that arises from the study design flaws could have an impact on the study results, “once it's in a study, you can't fix it,” we can only control for these factors in the study protocol or through careful interpretation of the results. However, despite the relatively high risk of bias, the results of this study remain valuable, especially concerning the pandemic and the health implications that COVID-19 has on male health.

The findings of this systematic review revealed a significant association between SARS-CoV-2 infection and susceptibility to semen quality impairments, particularly sperm concentration and progressive motility. It is still unclear whether the deterioration of semen parameters or spermatogenic dysfunction is only caused by COVID-19. Other important variables such as age group, existing urogenital abnormalities, COVID-19 severity, given COVID-19 medication and vaccinations should be investigated further as potential contributing factors. Future studies with larger sample sizes, long-term follow-ups with multiple semen sample collections, and higher-quality study designs are necessary to validate the direct causality of SARS-CoV-2 infection on semen quality and its subsequent effects on male fertility.

In the clinical setting, patients seeking fertility consultation should be educated on the potential temporary decrease of semen quality during SARS-CoV-2 infection. Furthermore, therapeutic strategies such as dietary supplements, and lifestyle modification should be further investigated to aid these patients for early restoration and recovery of the semen parameters.

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