Persistent Male Genital Tract Inammation and Semen Impairment: A Long-Term Effect of SARS-Cov-2 Infection

Background: Recent reports evidenced an impairment of semen parameters in men affected by coronavirus disease 2019 (COVID-19). In particular, our group previously reported that 1 over 4 COVID-19 healed men were found to be crypto- / azoo-spermic. Moreover, most patients had elevated IL-8 semen levels at sperm analysis. The aim of our study was to assess semen parameters and inammation by evaluating a panel of sperm cytokine levels (IL-1, IL-4, IL-6, IL-8, IL-17, INF gamma, TNF-alpha) on average 1 month after the second SARS-CoV-2 negative nasopharyngeal swab and 3 months later. Methods: Ten men who showed normozoospermia (n=3), oligozoospermia (n=3) or crypto/azoospermia (n=4) 1 month after healing from COVID-19 in our previous study, were re-called and re-evaluated 3 months after the rst semen analysis. Semen parameters were evaluated according to WHO manual and seminal plasma cytokine levels by an ELISA method. Results: At 3-months follow-up, 8 men showed an overall increase of semen parameters compared to levels assessed after 1 month. In particular, of the 4 crypto-/azoo-spermic men 1 month after healing, 2 resulted oligozoospermic, 1 normozoospermic and only 1 remained azoospermic. Two of the 3 oligozoospermic men turned normozoozpermic. Semen cytokine levels were remarkably high one month after healing and remained elevated after 3 months, with the exception of IL-6. Conclusions: This is the rst longitudinal, prospective study comparing semen parameters and semen inammatory markers one and three months after recovering from COVID-19. Our data indicate an overall tendency to an improvement of semen parameters although a genital tract inammatory condition appears to persist at least 3 months after COVID-19 recovery. This condition could have an impact on male fertility requiring a careful follow up of these patients.


Introduction
Male reproductive system is vulnerable to several viral infections, including those from ZIKA, Mumps, hepatitis B and C virus or human immunode ciency virus, which exert different impacts on male fertility [1]. The main types of male gonadal cells -spermatogonia, Leydig and Sertoli cells -express Angiotensin-Converting Enzyme 2 (ACE2) receptor and transmembrane serine protease 2 (TMPRSS2) on their membrane [2]. Several reports suggest a role for ACE2 and TMPRSS2 as the cellular receptor for SARS-CoV-2, allowing the access of the virus to the male reproductive organs, with a consequent risk of testicular impairment in men with Covid-19 [3].
In a recent series of 34 Chinese men (median age: 37) recovering from COVID-19, SARS-CoV-2 was not detected in the semen 1 month after COVID-19 diagnosis [4]. However, subjects with a moderate infection from Covid-19 showed an impairment of sperm quality, even if SARS-CoV-2 RNA is not detected in semen [5].
Gonadal impairment could be due either to a direct effect of SARS-CoV-2 on testicular cells and/or to the high in ammatory response consequent to the disease [3]. In particular, the COVID-19 cytokine storm may produce a widespread tissue damage, resulting in multi-organ failure with the potential involvement of the reproductive system as well 3,6 .
We previously demonstrated that at least one-fourth of men, recently recovered from COVID- 19, presented oligo/crypto/azoospermia and such condition was related to the severity of the illness 7 . In addition, most of these men, showed elevated semen levels of IL-8, a surrogate marker of male genital tract in ammation 7 . Alterations of semen quality in patients healed from COVID-19 have been demonstrated in other studies 8,9 . Even if some evidence indicates the loss of testicular architecture at post-mortem examination of COVID-19 severe cases, data regarding the long-term impact of COVID-19 on male reproductive function and semen quality after recovery are lacking 3,10 .
Aim of the present study was the evaluation of semen parameters (Sperm Concentration, Total Spermatozoa Number, Progressive Motility) and semen in ammatory cytokine levels (IL-1, IL-4, IL-6, IL-8, IL-17, INF gamma, TNF-alpha), in 10 sexually active men one and 3 months after recovery from COVID-19.

Study Design
A prospective cohort study was designed following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines. Current study was approved by Ethics Commettee Area Vasta Centro -based at AOU Careggi in June 2020, under the code 17104. Subsequently It was registered in clinicaltrials.gov with the identi er NCT04446169. It was designed and conducted according to the Declaration of Helsinki. All enrolled participants signed an informed consent. We re-contacted 10 subjects, crypto / azoospermic (n = 4), normozoospermic (n = 3) and oligozoospermic (n = 3) evaluated by spermiogram performed on average 1 months after recovery from COVID-19 according to our previous study7 and offered them a reassessment of sperm parameters 3 months after the rst one. Everyone agreed to participate in the study.
Data recording, specimen collection and SARS-CoV-2 RNA detection Clinical data were collected according to the previously reported scheme 7 . (aggiungere come sono stati raccolte le cose)

Semen Analysis
Semen was collected after a period of 2-7 days of abstinence. WHO guidelines 12 were strictly followed for semen analysis. Sperm concentration was determined using an improved Neubauer hemocytometer. The percentages of progressive, non-progressive and immotile spermatozoa were evaluated on 200 spermatozoa/sample by phase contrast microscopy. Sperm morphology was assessed after Diff-Quick staining on 200 spermatozoa at 1000x bright eld microscopy. Semen leukocytes were quanti ed by counting the number of round cells/ml and evaluating the percentage of leukocytes after May-Grunwald staining of the sample.

Immunoplex Assay Semen Plasma
Frozen-thawed samples of seminal plasma were centrifuged at 800 g for 10 minutes to remove particulates. Detection and quanti cation of cytokines was performed using the Milliplex® Map kit Human Cytokine/Chemokine/Growth Factor Panel A Magnetic Bead Panel (Merck Kgaa, Darnstadt, Germany) following the manufacturer's protocols. The plate was analyzed with the Luminex 200 MAGPIX® and data were then analyzed using a 5-parameter logistic curve-tting method for calculating analyte concentrations in samples.

Statistical Analysis
Continuous and categorical variables were reported as median (interquartile range) and number (percentage), accordingly. Patients were then divided according to their semen analysis results, thus normo-, oligo-and azoo-spermic. Statistical comparisons between groups were conducted with Mann-Whitney u-test and univariate ANOVA for continuous variables, and with χ2 and Fisher's Exact test for categorical variables, as appropriate according to sample size. Intragroup comparison from baseline to follow-up were done with Wilcoxon test for continuous variables. Statistical signi cance was set with a p-value<0.05. All statistical analyses were performed using IBM SPSS version 20.0 (SPSS Inc, Chicago, IL, USA).

Results
Patient characteristics and the received treatments during COVID-19 infection are reported in Table 1 and 2, respectively. Table 3 reports semen parameters and seminal plasma IL-8 levels 1 month and 3 months after recovery. Levels of the other evaluated cytokines, 1 and 3 months after recovery, are reported in Table 4. In the latter table, semen levels of cytokines in healthy controls and in patients with chronic prostatitis according to a previous study where cytokine levels were measured in the same laboratory 13 are shown for comparison.  Non-Hospitalized (n, %)   Eight out of the 10 patients showed an increase in sperm count (Figure 1, panels A-B and Table 3) after three months. Interestingly, 1 normozoospermic patient at the time of recovery (#2) tested oligozoospermic 3 months later. He is a young (32-years-old), healthy man, who was treated with remdesivir, hydroxychloroquine and haeparin during hospitalization. One oligozoospermic patient (#6), who was treated with remdesivir and hydroxychloroquine during hospitalization, showed a small increase in sperm concentration (from 1.1 to 7.6 x10 6 /mL).
Progressive motility (Figure 1 values above the 5th percentile of WHO reference [12], at 3 months follow up. None of the enrolled patients required new medications or hospitalization during the follow-up period. IL-8 levels were above the cut-off value of 3.8 ng/ml 13 in all enrolled patients at the time of recovery (Table 3). Three months later, the levels of IL-8 were still high, with the exception of 1 patient (#1) ( Figure  2). Overall, we demonstrate a halving of IL-8 levels between rst (median: 95) and second (median: 9,) semen analysis, although more than 3 months after healing from COVID-19, IL-8 levels remained more than double of cut-off values (8) in the majority of patients.
Semen cytokines were all elevated 1 month after recovery and were persistently high after 3 months, with the exception of IL-6 ( Figure 3). Mean cytokines concentrations in post COVID-patients were all higher when compared with healthy population 13 both 1 month after recovery and after further 3 months (Table   4). Interestingly, the mean levels of INFγ and TNFα were signi cantly higher as compared to those reported in men with proven prostatitis 13 (Table 4).

Discussion
Our study indicates that alterations of semen parameters observed shortly after recovering from COVID-19 may be transient as most patients show an improvement of semen quality after three months although an in ammatory status of the male genital tract seems to persist.
Several viruses, including HBV, HCV, HPV, HSV, Mumps, may cause an impairment of testicular function and can be detected in semen 14 . Some viruses such as MuV, HIV and SARS-CoV can affect testicular cells, resulting in severe orchitis, which can compromise male fertility 15 . During the very rst weeks after recovery from Covid-19, the majority of men show high semen levels of IL-8, and at least one-fourth of these men, show alterations of semen parameters 7 . A recently published systematic review based on data collected from 70 studies con rms these data 16 . In particular, the Authors suggest that COVID-19 may cause an in ammatory condition of the testis which is correlated to the severity of the disease 16 .
SARS-CoV-2 is very contagious and has already infected a higher proportion of young men during the second pandemic wave, as compared with the rst one 17 . Since the highest expression of ACE2 in testicular cells occurs at about thirty years of age, the interaction between SARS-CoV-2 and ACE2 receptor and TMPRSS may cause severe damage to the testis in young males, rising concerns about male fertility 18 . Impaired gonadal function can be due to in ammation an autoimmune response 12 as well as high fever and medications during the course of the disease 19 . In particular, a signi cant increase of semen levels of in ammatory cytokines, such as IL-6 and TNF-α was observed in COVID-19 patients 1 .
Furthermore, evidence suggests that both testicular function and semen quality may be reduced following severe COVID-19, due to several aspects of the disease including high fever and medications, indicating the need for andrological evaluation of recovered men 7,9 . Such a recommendation is also indicated in the o cial website of the government of Hubei Province which posted a bulletin inviting all men recovered from SARS-CoV-2 to undergo fertility checks, suggesting a big concern for a possible link between this novel disease and the male reproductive system 20 .
In the present study, we report a longitudinal prospective case series of sexually active men based on two consecutive sperm analysis performed after healing from COVID-19 and 3 months later, after a complete new cycle of spermatogenesis. Overall, 3 out of 7 (43%) oligo-crypto-azoospermic patients at the time of recovery, showed an improvement of testicular function with an increase of sperm number, 3 months after healing, suggesting that the detrimental effects of COVID-19 could be temporary. However, in few cases, no improvement in sperm number has been achieved. Since semen quality of these men before COVID-19 was not available, whether or not persistence of crypto/azoospermia is due to the illness cannot be ascertained. However, 6 out of 7 patients (86%) reported paternity before COVID-19 7 .
In most patients, an improvement of sperm progressive motility was observed, but values still remained below the 5th percentile of the WHO reference values in 6 of them.
As previously reported, IL-8 is a cytokine associated with in ammation of prostate, seminal vesicles and epididymis 13 . Semen IL-8 concentrations 1 month after recovery from COVID-19 were related with severity of the illness, including the need of hospitalization, intensive care, oxygen therapy and invasive ventilation 7 . Interestingly, semen IL-8 levels remain high (more than double of the cut-off value of 3.8 ng/ml) in most of the enrolled patients also 3 months after COVID-19 healing, suggesting the persistence of an in ammatory condition in the male genital tract that may require further assessment. Similarly, semen levels of 7 cytokines which are part of the SARS-CoV-2 cytokine storm 21 resulted elevated both one and 3 months after COVID-19 recovery in all patients recruited in this study, with the exception of IL-6 which decreased signi cantly after 3 months. In particular, levels of some cytokines (IL-8, IL-1, IL-6, INF gamma and TNF-alpha), resulted higher respect to those found in patients affected by prostatitis (Table   4) 13 , indicating that the in ammatory status of the male genital tract of patients recovering from COVID-19 may be similar or even more compromised.
A limitation of our study regards the lack of semen parameters before COVID-19 diagnosis. Another limitation regards the small cohort of evaluated patients. However, our intent was to determine whether those patients that showed alterations of semen quality 1 month after healing from COVID-19 7 , could recover after 3 months, representing an entire spermatogenetic cycle. Our study has also strengths. To our knowledge, this is the rst study comparing semen parameters in healed men from COVID-19 at 1 and 3 months after recovery, although in a small number of subjects. In addition, we evaluated a wide panel of seminal plasma cytokines involved in male genital tract in ammation.
In conclusion, our study demonstrates that, despite an overall amelioration of semen quality, COVID-19 induced in ammation in the male reproductive tract may persist 3 months after recovery, with a possible detrimental effect on male fertility. Further studies are needed to understand the link between the cytokine storm during COVID-19 and the damage to reproductive system in order to minimize the potential