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Institute for Experimental Endocrinology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Max Rubner Center (MRC) for Cardiovascular Metabolic Renal Research, D-10115 Berlin, Germany
Institute for Experimental Endocrinology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Max Rubner Center (MRC) for Cardiovascular Metabolic Renal Research, D-10115 Berlin, Germany
Institute for Experimental Endocrinology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Max Rubner Center (MRC) for Cardiovascular Metabolic Renal Research, D-10115 Berlin, Germany
Das Kinderwunsch Institut Schenk GmbH, Am Sendergrund 11, A-8143 Dobl, AustriaDepartment of Obstetrics and Gynecology, Medical University of Graz, Auenbruggerplatz 14, 8036 Graz, Austria
Institute for Experimental Endocrinology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Max Rubner Center (MRC) for Cardiovascular Metabolic Renal Research, D-10115 Berlin, Germany
Antibodies to SARS-CoV-2 are directly detectable in seminal plasma
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SARS-CoV-2 antibodies correlate positively between seminal plasma and serum
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Sperm parameters were unaffected by seminal plasma SARS-CoV-2 antibodies
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Vaccination-induced SARS-CoV-2 antibodies were not related to sperm pathology
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SARS-CoV-2 antibodies in seminal plasma suggest passage across blood-testis barrier
Abstract
There is public concern that COVID-19 vaccination and SARS-CoV-2 antibodies negatively affect male fertility. However, evidence for the presence of SARS-CoV-2 antibodies in seminal plasma is lacking. We examined whether antibodies were detectable in seminal plasma after COVID-19 vaccination in 86 men via a direct antibody measurement and by quantification of their neutralizing activity. The results show the presence of SARS-CoV-2 antibodies in seminal plasma with a strong correlation to the serum antibodies, increasing with the number of vaccinations. Further, the antibody titers are correlating with the neutralization activity. The SARS-CoV-2 vaccination parameters showed no association with markers of sperm quality. Conclusively, this study indicates substantial levels of antibodies in seminal plasma after COVID-19 vaccination that correlate with serum antibody titers, but do not associate with sperm quality.
There is public concern about COVID-19 vaccination negatively affecting male fertility [1]. In the case of a SARS-CoV-2 infection, the notion was supported in post-mortem analyses involving autopsy [2], and in clinical trials with COVID-19 patients [3]. The abnormalities in seminal plasma (SP) appeared to correlate with the concentration of serum antibodies (Ab) to SARS-CoV-2 following infection [4]. However, a potential presence of vaccination-induced SARS-CoV-2 Ab in SP has not yet been reported, and there is no data indicating a potential relevance of such Ab to poor sperm quality. In healthy men, the blood-testis barrier (BTB) separates the basal and apical compartments of the seminiferous epithelium and protects developing germ cells from potentially harmful constituents circulating in blood. SARS-CoV-2 has been shown to overcome this barrier, likely due to high local expression of angiotensin-converting enzyme 2 (ACE2) [5], and downregulation of a number of proteins, leading to an impairment of spermatogenesis and sperm motility [2]. Our aim was to investigate whether Ab to SARS-CoV-2 are directly detectable in seminal plasma and whether their titers are associated with serum concentrations and parameters of sperm quality.
Methods
Study design
In this observational case-control study, vaccination-induced Ab and their neutralization activity against S-Protein were analyzed in paired serum and SP samples from 86 adult men (n=43 vaccinated; n=43 non-vaccinated). The analysis of the SARS-CoV-2 vaccination parameters was carried out by researchers blinded to clinical information in a laboratory in Berlin, Germany. The samples and fertility data were collected by the fertility center Das Kinderwunsch Institut Schenk GmbH (Dobl, Austria) and stored under standardized conditions at Biobank Graz, Medical University of Graz, Austria (Cohort 5001_12, KIWI Collection) [6]. The recommendations of the Declaration of Helsinki were followed, all participants gave written informed consent before analysis, and the protocol had been approved by the Ethics Committee of the Medical University of Graz, Austria (approval number: 34-186ex21/22; 23-Feb-2022).
Quantification of SARS-CoV-2 Antibodies and their neutralization activity
Antibody titers were determined with a sensitive binding assay using the S1 domain of SARS-CoV-2 fused to firefly luciferase. Briefly, serum and SP were incubated with a fusion protein containing secreted firefly luciferase in frame to the S1-protein of SARS-CoV-2. Samples were incubated overnight at 4°C, and the immune complexes formed (SARS-CoV-2-aAb bound to Luciferase-S1 fusion protein) were precipitated with protein A-sepharose, washed and analysed for luciferase activity in a luminometer. Luminescence corresponding to SARS-CoV-2 Ab concentration in the original sample was recorded as relative light units (RLU) and analysed in relation to negative control signals. Results were quantified as binding index (BI) (fold over control). Inter- and intra-assay CV using a positive sample as standard were below 15% and 11%.
In order to confirm validity, neutralizing activity to the binding of spike protein to ACE2 (SPIA, Spike Protein Inhibition Assay, product code: DKO205/RUO, ids Holdings PLC) was determined [7]. Briefly, neutralizing Ab against SARS-CoV-2 were measured in serum and SP by a competitive method. The interference of the recombinant spike protein with the SARS-CoV-2 receptor ACE2 was quantified. The measurement range extends from 0-100%. Inter- and intra-assay CV using a control sample as standard were below 10% and 20%.
Determination of sperm quality parameters
Sperm quality parameters were determined at the fertility center. Briefly, men were instructed to abstain for 2-7 days prior to semen collection. Samples were collected at home and transported immediately to the clinics or donated directly in the clinics, and processed within 20 min of reception. The native semen samples were analyzed macroscopically and microscopically, morphology was assessed using the Sperm Morpho Slide (Vitromed GmbH, Langenfeld, Germany), and a spermiogram was recorded according to current WHO guidelines by a validated methodology [8].
Statistics
Distribution of numerical variables was investigated applying the Shapiro-Wilk-Test. Pairwise comparisons were conducted applying Wilcoxon rank sum test, and categorical variables were compared using Fisher's exact test and Pearson's Chi-squared test to detect differences in serum and SP markers. Spearman`s rank correlation was used to detect correlations between continuous variables. All statistical analyses were two-sided, and p-values below 0.05 were classified as statistically significant. Statistical analyses were performed using R on R-Studio (version 1.02.5042).
Results
Descriptive patient data and variables of sperm quality were compared between vaccinated and non-vaccinated subjects (Supplementary Table 1). The groups did not differ in age (p=0.41), body mass index (BMI) (p=0.95) or smoking habits (p=0.52). The overall median of age and BMI was at 38.2 years and 25.4 kg/m² (Supplementary Table 1). On average, vaccinated males had higher concentrations of SARS-CoV-2 Ab in serum and seminal plasma compared with unvaccinated participants (Supplementary Figure 1A,Figure 1A). Neutralizing activity was higher in serum and SP samples of vaccinated men as compared to the non-vaccinated (Supplementary Figure 1B,Figure 1B).
Figure 1SARS-CoV-2 antibodies and their neutralizing activity in serum and seminal plasma. A Comparison of SARS-CoV-2 antibody concentrations in seminal plasma of vaccinated and non-vaccinated subjects. B Comparison of neutralizing activity in seminal plasma of vaccinated and non-vaccinated subjects. Wilcoxon-Rank-sum test was used to calculate the p-value. C Correlation of seminal plasma SARS-CoV-2 antibodies and serum SARS-CoV-2 antibodies. D Correlation of neutralizing activity and SARS-CoV-2 antibodies in seminal plasma. Correlations were analyzed applying the Spearman` rank test. E Comparison of serum SARS-CoV-2 antibody titers based on number of vaccinations. F Comparison of seminal plasma SARS-CoV-2 antibody titers based on number of vaccinations. Kruskal-Wallis-Test was used to calculate the p-value for difference. BI; binding index.
Serum and SP Ab titers to SARS-CoV-2 correlated positively with neutralizing activity of serum and SP (Supplementary Figure 1C,Figure 1D). A significant correlation was observed between SARS-CoV-2 Ab titers in serum and in SP (Figure 1C). SARS-CoV-2 Ab titers in serum and SP increased with number of vaccinations (Figure 1E,F), independent of vaccine type or vaccine combination used (Supplementary Figure 1A,B). SARS-CoV-2 Ab were detectable in all groups of sperm pathology and showed no significant difference in titers (Figure 2A). There was no significant correlation of SARS-CoV-2 Ab titers in SP and their neutralizing activity with sperm concentration, morphology, motility or TMMSC (Figure 2B,C).
Figure 2SARS-CoV-2 antibody titers in seminal plasma and their association to sperm parameters. A SARS-CoV-2 antibody titers in seminal plasma according to sperm pathology. Kruskal-Wallis-Test was used to calculate the p-value for difference. B and C Correlation of the SARS-CoV-2 antibodies and neutralizing activity in seminal plasma with different sperm parameters. Correlations were analyzed applying the Spearman` rank test. BI; binding index, TMMSC; total motile morphologically normal sperm count.
This study describes the detection of SARS-CoV-2 Ab in SP and their neutralizing activity. Further, the potential association of the SARS-CoV-2 vaccination parameters to sperm parameters was analyzed. Antibodies in SP correlated significantly to serum Ab, and sperm pathology was not associated with the presence of Ab, indicating Ab passage through the BTB in fertile and non-fertile men. Seminal plasma Ab titers increased with the number of vaccinations, similar to serum Ab and irrespective of vaccine or vaccine combination chosen, suggesting a general positive association between serum and SP Ab. In serum and SP, Ab concentration and neutralizing activity showed a significant correlation. An analysis of SP Ab to SARS-CoV-2 with different sperm parameters yielded no significant associations, in agreement with studies analyzing serum Ab and sperm parameters after vaccination [9, 10], independent of vaccine (mRNA or viral vector) or combinations thereof [11].
The direct detection of SARS-CoV-2 Ab in SP constitutes a relevant finding with regard to current concerns of a potential interaction of SARS-CoV-2 vaccination with male fertility and reproduction. The lack of associations between Ab titers in SP with any of the established sperm quality parameters provides further assurance for the vaccine safety in regard to fertility. However, the consistent and concordant presence of the Ab in blood and SP irrespective of vaccination choice or underlying pathology challenges our understanding of the role of the BTB for protecting sperm from humoral immune responses, due to vaccination, infections or in autoimmune diseases. The current and highly variant immune status concerning SARS-CoV-2 provides a particular wide and promising research opportunity to better characterize the general mechanisms of immunoglobulin passage into SP. The absence of associations between SARS-CoV-2 Ab in SP with sperm parameters may be due to a lack of relevant autoantigens in SP, but this assuring finding should not be extrapolated to all infection- and immunization-induced Ab or pathogenic autoantibodies.
The strength of our study is the direct detection of SARS-CoV-2 Ab in paired samples of serum and SP, with no apparent effects on sperm quality. Limitations of the study include the single time point of analysis without further follow up, the differences in vaccination mode between participants, and the lack of information on vaccination side effects.
In conclusion, this study detected vaccine-induced antibodies to SARS-CoV-2 in SP that do not appear to affect sperm quality.
Acknowledgements: The authors would like to acknowledge the study participants and physicians collecting the samples. We thank our colleagues Gabriele Boehm, Vartitér Seher and Anja Fischbach for helpful technical support in the laboratory analyses.
Conflict of Interest Disclosures: The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication.
Data Sharing Policy: Anonymised data will be made available upon reasonable request from the corresponding author.
Author Contributions: L. Schomburg had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Concept and design: Chillon, Schomburg, Schenk
Acquisition, analysis, or interpretation of data: Chillon, Demircan, Weiss, Schomburg
Drafting of the manuscript: Chillon, Demircan, Weiss, Schomburg
Critical revision of the manuscript for important intellectual content: All authors
Statistical analysis: Chillon, Demircan
Obtained funding: Schomburg
Administrative, technical, or material support: Weiss, Minich,
Supervision: Schomburg, Schenk, Weiss
Funding/Support: This study was funded by the German Federal Ministry for Economic Affairs and Energy (BMWi, ZIM program, project #KK5051601BM0 to LS).
Role of the Funder/Sponsor: The funder had no role in study design, data analysis, decision to publish, or preparation of the manuscript.
Conflict of Interest Disclosures: The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication.
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