Semen parameters after SARS‐CoV‐2 infection: A literature review

Abstract Background and Aims The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is known to affect multiple organs by binding to angiotensin‐converting enzyme 2 receptors and might therefore affect male fertility. This review aims to collect all original articles on the effects of SARS‐CoV‐2 infection on male fertility, including the duration of time after infection required for these effects to begin to manifest and recommend how clinicians should approach cases with a recent illness. Methods This review was developed according to the preferred reporting items for systematic reviews and meta‐analyses guidelines. The search string was applied to four online databases—namely Pubmed, Embase, Medline, and the Cochrane COVID‐19 Register—and screened using the online tool Covidence.org. Articles were eligible for inclusion if they were cohort studies involving a healthy male population diagnosed with COVID‐19, each of whom had semen samples collected before and after the infection or two different semen samples collected after the diagnosis. Results Nine cohort studies were eventually included. Five articles had pre‐ and post‐COVID‐19 data while four had two sets of post‐COVID‐19 data. The three largest studies found a statistically significant decrease in all semen parameters when waiting less than 3 months from diagnosis before sample collection, and no significant differences in results when the ejaculate was analyzed more than 3 months after recovery. One study compared the COVID‐19 patients with a control group and found a significant decrease in semen parameters in the COVID‐19 group. Conclusion Spermatogenesis seems to be affected by SARS‐CoV‐2 infection, but the impact tends to reverse within 3–4 months. It is still unclear why male fertility is affected by SARS‐CoV‐2 infection, and it might be the result of several different components. Clinicians should consider recent SARS‐CoV‐2 infection as a possible reason for the low semen quality of patients' semen samples, and might therefore need to collect new samples after 4 months before further treatment.


| INTRODUCTION
Over 2 years have passed since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered. 1 In just a few months, it spread to the entire world, resulting in the death of around 5½ million people, and to date, almost 3 billion cases of coronavirus 2019 (COVID-19) have been reported. 2 As a result of the pandemic caused by the virus, researchers all over the whole started exploring various ways the disease affected people and could be transmitted. It was discovered that people reacted very differently to infection with the virus-some people got critically ill and others were just asymptomatic carriers. 3 Parallel with research efforts, some countries shut down noncritical hospital wards, including fertility clinics, 4 to limit the spread of the virus. 5 At the same time, the European Society of Human Reproduction and Embryology started collecting relevant research and knowledge of the area to provide guidelines for fertility clinics. 6 It became clear at the outbreak of the virus that it affects more than just the respiratory system. 3 Being similar to viruses that caused past pandemics such as the SARS and the Middle East respiratory syndrome, researchers already had hypotheses about how this novel SARS-CoV-2 could affect reproduction. 3 Acute illnesses with febrilia might affect spermatogenesis for a limited time, 7 which means that since SARS-CoV-2 infection often presents with this symptom, 3 this effect might also occur in this case.
Another theory builds on the fact that the SARS-CoV-2 viral genome enters the host cell via spike proteins, binding to the angiotensinconverting enzyme 2 (ACE2) receptor which is located in different cells in the body, including the lungs just like the former coronaviruses. 4 ACE2 is also expressed selectively in Leydig cells in the male genitals, suggesting that it can affect male reproduction. 4 Even if reversible, spermatogenesis takes about 74 days, giving a window of about 3 months within which a man could briefly fulfill the criteria for assisted reproductive treatment (ART) by falling under the reference values established by the World Health Organization (WHO). 8 Earlier reviews mostly address if COVID-19 affects male fertility or can be found in semen.
To our knowledge, no review has yet collected studies that compare changes in an individual's fertility status in connection to COVID-19.
The aim of this review is to evaluate how SARS-CoV-2 infection affects semen quality and male fertility. Further, this review aims to assess how long the semen quality might be affected if a connection is found between the virus and semen quality.
Clinicians planning ART for patients who are or have been SARS-CoV-2-positive will find this knowledge relevant. searched. An advanced search was first made on pubmed.org, resulting in the following search string comprising of keywords from the main aim of this review: ((((corona virus 2019) OR (sars-cov-2)) OR (covid19)) AND ((((semen) OR (sperm)) OR (spermatozoa)) OR (spermatid))). Afterward, the same words were used to conduct an expanded search on Embase and Medline including both keywords and terms. Lastly, the search string from PubMed was used in the Cochrane COVID-19 register. All four searches were made on the same day in September 2021.

| Criteria
To get an idea of how COVID-19 affects semen quality, we focused on publications about men with data on their semen quality before infection.
Therefore, all included articles were cohort studies involving male populations diagnosed with COVID-19, each of whom underwent preinfection and postinfection semen quality analyses. Because of the limited number of studies on COVID-19 and male fertility, the search string could not be too specific or we would have missed some articles.
Only a few articles were found during the full-text screening, as a result of which the inclusion criteria above were expanded. Studies with no data before COVID-19 infection but with more than one analyzed semen sample from each patient after the diagnosis of COVID-19 were included. This made it possible to observe the potential changes in semen quality over time after infection.

| Screening
The results from the searches were screened using the online tool Covidence.org, which removed duplicates automatically. Title and abstract screening were conducted by one investigator twice, with an interval of a couple of days in between, without knowing the results from the first time to make sure that no article was overlooked. The full-text screening was conducted by one investigator, with support from another investigator if the relevance of an article to the review was unclear. The final articles were all read by two investigators.

| Search results
As illustrated in Figure 1, half of the studies identified were removed as duplicates, leaving 394 articles for the title and abstract screening. Nine studies satisfied the aims and criteria and were therefore included in the final review.

| Data extraction
The main semen parameters analyzed by most of the articles were used to create Tables 1 and 2.
Where the articles used the same measures, it was possible to compare the results directly.
If an article presented no statistical data, an effort was made to find the original individual data in the article or as supplementary data in the study. We used GraphPad Prism 9.3.1 to perform statistical analysis on these data. Paired Student's t-test was used to compare results within groups. Data are presented as means ± standard deviations. QQ plot was used to check for normality.
Where original data were not found, the results were not included in the tables, but their conclusions were still included in the comparison.
When additional results were found relevant, they were included and commented on.

| Included articles
Five articles had pre-and post-COVID-19 infection data and four articles had more than one set of post-COVID-19 infection data but no pre-COVID-19 data. The two groups of articles were used to create Table 1 and Table 2, respectively. Parameters in Tables 1 and 2 include semen volume, sperm concentration, total motility, and progressive motility. These parameters are illustrated in Figure 2A-D, which uses data from Tables 1 and 2.
One article in each group only had individual patient data available in the article 9 or as supplementary data, 14 and analytical data could then be determined and included.
It was not possible to find the original data on patients in the last article 17 in the group with two semen samples collected after COVID-19 infection. Because of the different focus, the results were F I G U R E 1 Preferred reporting items for systematic reviews and meta-analysis flowchart illustrating the process of selecting the articles.   Note: Studies with data before and after covid-19, before = before covid19, and after = after covid19.
Results that are in bold are statistically significant (p < 0.05). Data are presented as mean value ± standard deviation unless specified otherwise.

Note:
Maleki et al. 17 presented no data and were not included in the table.
Studies with two semen samples after the positive covid test.
Results that are in bold are statistically significant (p < 0.05), and data are presented as mean value ± standard deviation unless specified otherwise.
a Result presented as median with an interquartile range.
only presented as a figure, making it difficult to compare the outcomes directly in Table 2. 3.2 | The five articles with pre-and post-COVID-19 data In four of the five articles with pre-and post-COVID-19 data the patients were either found via the database of a fertility clinic or via data available otherwise from being examined previously for their fertility status. [10][11][12][13] The last article by Ma et al. 9 focused on a single sample from each COVID-19 patient compared to a control group, and, in addition, three patients had data from previous semen analyses. 9 Two of these three patients presented with two semen analyses before SARS-CoV-2 infection but with no exact date of sample collection. With the knowledge that events and lifestyle changes could affect semen quality, [18][19][20][21] it was assumed that the most accurate to compare with, was the most recent one. 9 In three articles, 10-12 the number of participants was between 21 and 29, while in the article by Erbay et al., 13 there were 69 patients, divided into 26 with mild symptoms and 43 with moderate symptoms.
In these four articles, 10-13 the criteria for exclusion included any known event that might lead to reduced fertility, like previous urogenital infection, testicular diseases, azoospermia, or oligozoospermia. One article mentioned hormonal drug use as an exclusion criterion, 13 and one described no exclusion criteria at all. 9 Lastly, one article excluded patients who had been sick with a non-COVID-19 febrile illness in the last 3 months. 12 When looking at the duration of time between pre-and post-COVID-19 semen sample analyses, two articles mentioned that the data were obtained within a year or 2, 10,13 while the rest were unclear. Two articles described the authors to have waited at least 3 months after confirmation of patients' COVID-19 infection to collect their semen samples. 10,12 A third article waited 3 months after the patients were proven to have recovered, according to their nasopharyngeal swab sample tests, before semen sampling. 13 Gul et al. 10 found no statistical difference between the semen parameters before and after COVID-19 infection, and the result did not change when the hospitalization time and medication were considered. 10 Pazir et al. 12 adopted almost the same time interval until testing as Gul et al., 10 and there was a significant decrease in total motility (p = 0.01) and total motile sperm count (p = 0.02). 12 In addition, Pazir et al. 12  Comparing the results to the WHO 8 reference in Figure 2, we see that both the progressive and total motility were above normal before COVID-19 infection but fell below the "cut-off" after the infection. 11 Erbay et al. 13 stood out by dividing the patients into groups according to their symptoms. In the group with mild symptoms, progressive and total motility decreased significantly (p = 0.002 in both), while in the group with moderate symptoms, all parameters decreased significantly (p < 0.05). 13 In addition to these results, which are illustrated in Table 1, the group with mild symptoms showed a significant decrease in vitality (p = 0.03), while the moderate symptom group showed a decrease in both the total sperm number and vitality (p = 0.001 for both). 13 The article by Ma et al. 9 was the one with only three patients, and the authors found no significance in the changes in semen parameters before and after the COVID-19 tests.

| The four articles with two sets of post-COVID-19 test data
In three [15][16][17]  All four articles excluded patients with known infertility or other factors that are known to increase the risk of infertility, like earlier cryptorchidism or scrotal surgery, abnormal secondary sexual characteristics and small testicular size, or a history of mumps or sexually transmitted infections. Two articles only included patients with proven fertility, like men whose wives had given birth to healthy children within the previous two years. 15,17 Two articles included COVID-19 negative control groups. 16,17 The time between diagnosis and first and second semen sample collection differed in all of the articles. found that the progressive motility and total sperm motility of the first sample after COVID-19 diagnosis were below normal, but both parameters increased significantly (p < 0.0001 for both) on Day 120 and reached the normal range (see Figure 2). 15 Furthermore, the sperm morphology improved significantly (p = 0.0002) on Day 120 but did not reach the normal value according to the WHO criteria. 8 Guo et al. 16 Table 2, the total sperm count and motile sperm count increased significantly (p = 0.0029 and p = 0.0391, respectively). 16 The article by Maleki  the group with fever, whereas they found a significant decrease in total motility in the group without fever (p = 0.03). 12 The article did not describe the reference value used as a cut-off for temperature, the temperature range within the group with fever, and the lengths of febrile episodes. 12 These results could be explained by the possibility of coincidence in them, because there were only 12 patients in each group or because the fever was not high enough to lead to the affection of germ cells and inhibition of spermatogenesis. 23 The study by Maleki In the study by Koç and Keseroglu,11 in which only 9.5% of the patients presented with fever, a statistically significant decrease was found in semen volume (p = 0.005), progressive motility (p < 0.001), and total motility (p = 0.001) in the samples collected after positive COVID-19 diagnosis, compared to those collected before. In the study by Erbay et al., 13 69.3% and 72.1% of patients had a fever in the groups with mild symptoms and moderate symptoms respectively, and significant decreases in all parameters were found in the group with moderate symptoms whereas there were significant decreases only in progressive and total motility in the mild group. 13 The article by Guo et al. 16  Erbay et al., 13 some parameters seem to take longer to recover after being affected by COVID-19, e.g. the number of motile sperm cells.
The group of articles with more than one semen sample collected after a positive COVID-19 diagnosis seems to support this conclusion. Maleki and Tartibian 17 took less than two months after the patient's discharge from the hospital before collection of the first sample after COVID-19 diagnosis, and they found no significant differences in the pre-and post-COVID-19 semen parameters in the COVID-19 group, but, compared with the control group, the values were significantly lower in the COVID-19 group. 17 Falahieh et al. 15 and Gou et al. 16 collected the first samples less than three months after diagnosis and the second samples more than three months after diagnosis. There was an increase in each parameter from the first sample to the second in both studies and statistically significant increases in progressive and total motility in Falahieh et al. 15 and in sperm concentration in Guo et al. 16 Besides showing that SARS-CoV-2 infection affects spermatogenesis, these data tend to suggest that the damage is reversible.
After more or less three months, which is the average duration for spermatogenesis, the semen parameters return to normal.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
All the main data and materials are available in the article. Additional data are available upon request.

TRANSPARENCY STATEMENT
Jens Fedder affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.