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Semen impairment and occurrence
of SARS-CoV-2 virus in semen after
recovery from COVID-19
M. Gacci
1,
*, M. Coppi
2,3
, E. Baldi
2,4
, A. Sebastianelli
1
, C. Zaccaro
1
,
S. Morselli
1
, A. Pecoraro
1
, A. Manera
1
, R. Nicoletti
1
, A. Liaci
1
,
C. Bisegna
1
, L. Gemma
1
, S. Giancane
1
, S. Pollini
2,3
, A. Antonelli
2,3
,
F. Lagi
2,5
, S. Marchiani
4
, S. Dabizzi
4
, S. Degl’Innocenti F. Annunziato
2
,
M. Maggi
6
, L. Vignozzi
4
, A. Bartoloni
2,5
, G.M. Rossolini
2,3
, and
S. Serni
1,2
1
Department of Minimally Invasive and Robotic Urologic Surgery and Kidney Transplantation, Careggi University Hospital (AOUC),
University of Florence, 50134 Florence, Italy
2
Department of Experimental and Clinical Medicine, University of Florence, 50134 Florence,
Italy
3
Clinical Microbiology and Virology Unit, Florence Careggi University Hospital, 50134 Florence, Italy
4
Unit of Andrology, Female
Endocrinology and Gender Incongruence, AOUC and Center of Excellence DeNothe, University of Florence, 50134 Florence, Italy
5
Infectious and Tropical Diseases Unit, Careggi University Hospital, 50134 Florence, Italy
6
Endocrinology Unit and Department of
Experimental, Clinical and Biomedical Sciences, University of Florence, 50134 Florence, Italy
*Correspondence address. Department of Minimally Invasive and Robotic Urologic Surgery and kidney transplantation, AOUC, University
of Florence, Largo Brambilla 3, 50134 Florence, Italy. Tel. þ39 3396640070; E-mail: maurogacci@yahoo.it
Submitted on October 8, 2020; resubmitted on December 28, 2020; editorial decision on January 18, 2021
STUDY QUESTION: How is the semen quality of sexually active men following recovery from severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) infection?
SUMMARY ANSWER: Twenty-five percent of the men with recent SARS-Cov-2 infections and proven healing were oligo-crypto-
azoospermic, despite the absence of virus RNA in semen.
WHAT IS KNOWN ALREADY: The presence of SARS-CoV-2 in human semen and its role in virus contagion and semen quality
after recovery from coronavirus disease 2019 (COVID-19) is still unclear. So far, studies evaluating semen quality and the occurrence of
SARS-CoV-2 in semen of infected or proven recovered men are scarce and included a limited number of participants.
STUDY DESIGN, SIZE, DURATION: A prospective cross-sectional study on 43 sexually active men who were known to have
recovered from SARS-CoV2 was performed. Four biological fluid samples, namely saliva, pre-ejaculation urine, semen, and post-ejaculation
urine, were tested for the SARS-CoV-2 genome. Female partners were retested if any specimen was found to be SARS-CoV-2 positive.
Routine semen analysis and quantification of semen leukocytes and interleukin-8 (IL-8) levels were performed.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Questionnaires including International Index of Erectile Function and Male
Sexual Health Questionnaire Short Form were administered to all subjects. The occurrence of virus RNA was evaluated in all the biological
fluids collected by RT-PCR. Semen parameters were evaluated according to the World Health Organization manual edition V. Semen
IL-8 levels were evaluated by a two-step ELISA method.
MAIN RESULTS AND THE ROLE OF CHANCE: After recovery from COVID-19, 25% of the men studied were oligo-
crypto-azoospermic. Of the 11 men with semen impairment, 8 were azoospermic and 3 were oligospermic. A total of 33 patients (76.7%)
showed pathological levels of IL-8 in semen. Oligo-crypto-azoospermia was significantly related to COVID-19 severity (P<0.001). Three patients
(7%) tested positive for at least one sample (one saliva; one pre-ejaculation urine; one semen and one post-ejaculation urine), so the next day
new nasopharyngeal swabs were collected. The results from these three patients and their partners were all negative for SARS-CoV-2.
LIMITATIONS, REASONS FOR CAUTION: Although crypto-azoospermia was found in a high percentage of men who had recovered
from COVID-19, clearly exceeding the percentage found in the general population, the previous semen quality of these men was unknown
nor is it known whether a recovery of testicular function was occurring. The low number of enrolled patients may limit the statistical
power of study.
V
CThe Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved.
For permissions, please email: journals.permissions@oup.com
Human Reproduction, Vol.0, No.0, pp. 1–10, 2021
Advance Access Publication on February 1, 2021 doi:10.1093/humrep/deab026
ORIGINAL ARTICLE Andrology
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WIDER IMPLICATIONS OF THE FINDINGS: SARS-CoV-2 can be detected in saliva, urine, and semen in a small percentage of men
who recovered from COVID-19. One-quarter of men who recovered from COVID-19 demonstrated oligo-crypto-azoospermia indicating
that an assessment of semen quality should be recommended for men of reproductive age who are affected by COVID-19.
STUDY FUNDING/COMPETING INTEREST(S): None.
TRIAL REGISTRATION NUMBER: N/A.
Key words: COVID-19 / coronavirus disease 2019 / semen / oligo-crypto-azoospermia / fertility / sexual transmission / SARS-CoV-2 /
severe acute respiratory syndrome coronavirus 2
Introduction
The world is currently experiencing the outbreak of coronavirus dis-
ease (COVID-19) infection caused by severe acute respiratory syn-
drome coronavirus 2 (SARS-CoV-2). The virus exhibits a strong
infectivity with a low virulence compared to previous coronavirus
strains, and a higher fatality rate in men than in women (COVID-19
Dashboard by the Center for Systems Science and Engineering (CSSE)
at Johns Hopkins University (JHU), 2020). Widespread contact tracing
followed by hospital isolation or quarantine is recommended to
contain the outbreak (Swerdlow and Finelli, 2020).
Human-to-human transmission of SARS-CoV-2 usually occurs within
households, including relatives and friends who are in intimate contact
with symptomatic or incubating patients (Pan et al., 2020). In some
patients, blood samples showed positive RT-PCR test results, suggest-
ing that infection may be occasionally systemic (Wang et al., 2020).
Respiratory droplets and physical contact of contaminated surfaces are
the confirmed transmission modality of SARS-CoV-2 (Rothe et al.,
2020), even if the virus has also been detected in saliva (To et al.,
2020), teardrops, urine, and stool (Peng et al., 2020).
The occurrence of viral genomes in semen of infected men, previ-
ously not identified as being sexually transmitted, has been reported
forEbolaandZika(Feldmann, 2018). The high expression of
angiotensin-converting enzyme (ACE) and transmembrane serine
protease 2 (TMPRSS2) in somatic and germ cells of the testis suggests
that SARS-CoV-2 virus may localize in the gonad (Wang and Xu,
2020). A recent study (Rastrelli et al., 2020) demonstrated the devel-
opment of hypergonadotrophic hypogonadism in patients with active
COVID-19 infection, suggesting an impairment of Leydig cell function,
although whether this impairment is related to localization of the virus
in the testis is unknown. However, in view of the essential role
played by testosterone in spermatogenesis (Smith and Walker, 2014),
alterations of semen quality in patients with COVID-19 can be hypoth-
esized. In addition, semen parameters may be compromised by the in-
flammatory condition occurring in men with COVID-19.
Few studies have evaluated the presence of the virus in human se-
men. Paoli et al. reported the absence of SARS-CoV-2 RNA in semen
and urine samples 13 days after COVID-19 diagnosis and 4 days after
the second negative nasopharyngeal (NP) swab in a single patient who
tested positive for SARS-CoV-2 through a NP-swab (PaoLi et al.,
2020). Li et al. (2020) demonstrated that the semen of six patients
tested positive for SARS-CoV-2, with four of these patients at the
acute stage of infection and two at 2 and 3days after clinical recovery,
respectively. Pan et al. (2020) did not find viral RNA in any of 34 se-
men samples from males with active infection. Similarly, Song et al.
(2020) demonstrated no detectable SARS-CoV-2 RNA in semen
samples collected from 12 patients during the recovery phase: after
1–16 days from the second negative NP swab and 14–42 days after
acute infection (Song et al., 2020). Only two studies (Guo et al.,2020;
Ma et al., 2020) evaluated semen quality in a small cohort of men dur-
ing infection or in the recovery phase of COVID-19.
In the present study, we investigated the direct (RNA) presence of
SARS-CoV-2 in saliva, urine, and semen; evaluated semen parameters;
and investigated semen IL-8 levels as a surrogate marker of the male
genital tract inflammatory condition (Lotti et al.,2011
) of 43 sexually
active men with laboratory evidence of recovery from COVID-19.
Materials and methods
Study population and design
This prospective cohort study was conducted following the
Strengthening the Reporting of Observational Studies in Epidemiology
(STROBE) guidelines. All male patients with SARS-CoV-2 infection,
confirmed using molecular methods on respiratory specimens, were
identified. Sexually active men aged 18–65 years with proven recovery
from SARS-CoV2 infection (two consecutive negative NP-swabs for
SARS-CoV2 RNA) were eligible for the study (World Health
Organization, 2020). Men without sexual activity or with ejaculatory
disorders, being treated with prostatic surgery or alpha-blockers and
those with an inability to express informed consent were excluded
from the study. All the partners of the enrolled patients were tested
for SARS-CoV-2 positivity (NP swabs) at the time of the original diag-
nosis of the men and retested according to National Health Care
System criteria until NP-swab double negativity, as suggested by
World Health Organization (WHO) on clinical management of
COVID-19 (WHO, 2020).
Moreover, if one or more specimens collected from the patients en-
rolled during the study were positive, partners were retested.
Ethics
The study was carried out with the approval of the local Ethics
Committee (Rif: CEAVC17104) and registered on clinicaltrial.gov (Rif:
NCT04446169), in compliance with the Declaration of Helsinki. All
enrolled men provided written informed consent.
Clinical data and specimen collection
Clinical data included patient demographics, comorbidities, medica-
tions, hospitalization time and features (including intensive care need),
and laboratory tests and treatments (including oxygen therapy).
Moreover, patients were asked to fill out questionnaires, including
2Gacci et al.
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International Index of Erectile Function (IIEF-5) and Male Sexual Health
Questionnaire Short Form (MSHQ-SF), in order to assess a compre-
hensive urological, sexual and reproductive anamnesis. Data on
COVID-19 status were recorded for patients’ partners.
Four biological fluid samples (saliva, pre-ejaculation urine, sperm
obtained with masturbation, and first fraction post-ejaculation urine)
were collected in four sterile jars to be tested for SARS-CoV-2.
After liquefaction of semen and assessment of volume, semen samples
were divided into two aliquots, one for evaluation of the presence of
SARS-CoV-2 virus by RT-PCR and one for semen analysis.
Detection of SARS-CoV-2 in biological
specimens
All samples were processed on the same day as collection or stored at
80C until further analysis. Nucleic acids from samples were extracted
with the Microlab Nimbus IVD system (Seegene Inc, Seoul, South
Korea) using the Starmag Universal Cartridge and amplified with the
multiplex RT-PCR Allplex
TM
2019-nCoV assay (Seegene Inc), targeting
RdrP, E and N genes, according to the manufacturers’ instructions.
Semen analysis and IL-8 evaluation
Semen analysis was carried out according to WHO guidelines (WHO,
2010). Quantification of leukocytes in semen was performed by count-
ing the number of round cells per milliliter using an improved
Neubauer hemocytometer and evaluating the percentage of leukocytes
and immature germ cells after May-Grunwald staining of the sample.
For IL-8 evaluation, semen plasma aliquots were stored frozen and
IL-8 levels were quantified by conventional two-step ELISA using a hu-
man IL-8 ELISA kit (BD Bioscience, San Diego, CA, USA), according
to the manufacturer’s instructions.
Statistics
Patients were divided into different groups for comparisons according
to hospitalization, intensity of treatment, and semen parameters.
Differences were tested with Independent Sample Student’s t-test,
Mann–Whitney u-test, and univariate analysis of variance (ANOVA)
for continuous variables, and with v
2
and Fisher’s Exact Test for cate-
gorical variables according to sample size. A logistic regression was
carried out including significant factors to better define the risk of sex-
ual transmission and to identify the main determinants of impairment
of semen quality. A value of P<0.05 was considered to be significant.
All statistical analyses were performed using IBM SPSS version 20.0
(SPSS Inc, Chicago, IL, USA).
Results
Patient characteristics and clinical features
From 326 male patients with a positive NP-swab, 179 were between
18 and 65 years of age and were eligible for inclusion, among these,
43 agreed to participate in the study (Fig. 1). All enrolled patients
previously affected by COVID-19 had a negative result from at least
two consecutive NP swabs before sample collection. Clinical features,
including urological and andrological medical history, are summarized
in Table I.
Timelines from the first positive NP swab to sample collection
are reported in Fig. 2. In particular, median proven healing time (time
from first positive to second consecutive negative NP-swab) was
31 days (range: 3–65), whereas median SARS-CoV-2-free time
(time between second negative NP-swab and sample collection) was
35 days (r: 24–43).
Twelve patients (27.9%) were not hospitalized, 26 (60.5%) were
hospitalized in internal medicine unit, and 5 (11.6%) were admitted to
intensive care unit (Table II). Overall 19 patients (44.2%) did not re-
quire oxygen therapy, 10 (23.2%) were treated with low flow O
2
ther-
apy, whereas 14 (32.6%) necessitated high flow/invasive O
2
therapy.
The sexual habits of enrolled men are reported in Table II. Six patients
(13.9%) were without a fixed partner, while 37 (86.1%) had a stable re-
lationship. Fourteen out of 37 stable female partners (37.8%) presented
a positive NP swab for SARS-CoV-2 at the time of partner diagnosis
(Table II) and four were positive at the time of enrollment of men, with
two subsequent negative results for the two NP swabs performed
according to National guidelines (WHO, 2020). Five of the 11 stable
partners of nonhospitalized (45.5%), 8 of the 22 of the hospitalized men
(34.8%) and one of the four stable partners of men requiring intensive
care support (20%) tested positive at the time of partner diagnosis.
Twenty-nine male patients with a stable partner (78.4%) did not use
condom: 10 of these female partners (34.5%) had a positive NP swab.
In this subpopulation of stable partners having sex without condom,
multivariate analysis did not show a difference in the number of sexual
intercourse events per month between female partners with positive
versus negative NP swab (median: 5 vs. 4, P¼0.470; data not shown).
SARS-CoV-2 detection in collected
specimens
A total of 170 samples collected from 43 enrolled patients were tested
by RT-PCR, namely saliva, urine sample collected before ejaculation,
and semen and urine sample collected after ejaculation. Two samples
from two different patients yielded inconclusive results. Forty patients
(93%) were negative for SARS-CoV-2 RNA in any tested sample.
Three patients (7%) tested positive in at least one sample: one (patient
A) in saliva (collected 62 days after the second negative swab), which
was positive for all target genes; one (patient B) in pre-ejaculation
urines (44 days after the second negative swab), which were positive
only for the N-gene (Threshold Cycle (Ct)¼37.04); one (patient C) in
semen, where all target genes were detected, and in post-ejaculation
urines where only the E- and N-genes were detected (Ct ¼34.99 and
37.16, respectively) (21 days after the second negative swab). Patients
A and C needed intensive care support during COVID-19 infection.
These three patients and their partners were retested for SARS-CoV-2
RNA with NP-swabs and all samples tested negative.
Patient A was rehospitalized for sepsis resulting from bacterial infec-
tion and was treated with antibiotics, with subsequent healing. Patient
B was asymptomatic, with no signs or symptoms of recurrence of
COVID-19 and decided independently to undergo self-quarantine.
Patient C reported having unprotected oral, vaginal, and anal sex with
his stable partner after recovering from COVID-19. Therefore, they
were further investigated using a urethral swab (patient) and pharyn-
geal, vaginal, and rectal swabs (partner): all samples tested negative.
COVID-19: effect on semen quality 3
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Figure 1. Flowchart of screening, eligibility, and the inclusion process for the study sample of men who recovered from
COVID-19. Age is in years. CODIV-19: coronavirus disease 2019, SARS-CoV-2: severe acute respiratory syndrome coronavirus.
4Gacci et al.
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Semen parameters
Data for parental and fertility status of the included patients are
reported in Table II. Five of the 12 (41.6%) nonhospitalized, 5 of the
26 (19.2%) of the hospitalized and one of the five men (20%) requiring
intensive care support had no children, with no significant difference
between groups (P¼0.152).
Semen analysis demonstrated that eight patients (18.6%) were azoo-
spermic and three were oligospermic with less than 2 million/mL
(7.0%) spermatozoa: overall, 25.6% of patients were oligo-crypto-
azoospermic. The occurrence of azoospermia was highly related to
the severity of the illness: the condition was found in four out of five
patients admitted to the intensive care unit, in three of the 26 hospital-
ized in the medicine department and only in one among the 12 nonho-
spitalized (P<0.001). No relation was found between occurrence of
azoospermia and severity of oxygen therapy (P¼0.417). Semen
parameters are reported in Table III.
The presence of leukocytes in semen was found in 16 patients
(37.2%) and their occurrence was higher in men admitted to the inten-
sive care unit as compared to non-hospitalized patients or those ad-
mitted to the medicine department, but was not related to the
intensity of oxygen therapy (36.8% in men without O
2
vs. 30.0% with
low flow O
2
and 42.9% with high flow/invasive O
2
).
Thirty-three patients (76.7%) showed pathological levels of semen
IL-8 (sIL-8) (i.e., >3.8 ng/mL) (Hofny et al.,2011): among them, 7 out
of 12, (58.3%) were not hospitalized during the illness, 21 of 26
(80.8%) were hospitalized in the medicine department, and 5 (100.0%)
were admitted to the intensive care unit (P¼0.142) (Fig. 3A).
Pathological levels of sIL-8 were related to the severity of oxygen ther-
apy (r¼0.356; P¼0.050). As shown in Fig. 3B,highlevelsofsIL-8
were found in 12 out of 19 (63.2%) men without oxygen therapy, 9
out of 10 (90.0%) with low flow O
2
-therapy and 12 out of 14 (85.7%)
necessitating high flow/invasive O
2
therapy.
In univariate analysis, age, hospitalization and sIL-8 were all signifi-
cant items for azoospermia (Supplementary Table SI). In multivariate
analysis, hospitalization (not recovered vs. hospitalized vs. intensive
care unit) was the main determinant of crypto-azoospermia
(P¼0.039) (data not shown).
Discussion
In our sample, the age range was from 30 to 64 years. This study
demonstrates that young and sexually active men with proven recovery
................................................................................................................................................................................................. ...........................
Table I Comparison of clinical, social, and uro-andrological characteristics of the study sample (N ¼43) stratified by clinical
management of COVID-19.
Nonhospitalized
(N 512; 27.9%)
Hospitalized
(without ICU)
(N 526; 60.5%)
ICU
(N 55; 11.6%)
Pvalue
a
DEMOGRAPHICS
Age (years) (median, IQR) 44 (33–49) 52 (48–58) 59 (56–59) 0.003
BMI (kg/m
2
) (median, IQR) 26.1 (23.7–27.9) 26.9 (23.6–31.0) 27.4 (26.3–28.7) 0.380
Smoking status Never (n, %) 9 (75.0%) 22 (84.6%) 4 (80.0%) 0.850
Current (n, %) 2 (16.7%) 1 (3.8%) 0 (0.0%) 0.157
Former (n, %) 1 (8.3%) 3 (11.5%) 1 (20.0%) 0.401
Period between last negative
oropharyngeal swab and
sample collection (days) (median, IQR)
30 (23–39) 37 (26–49) 24 (23–32) 0.558
ANDROLOGICAL FEATURES
Previous orchitis/genital infection (n, %) 2 (16.7%) 3 (11.5%) 0 (0.0%) 0.814
Previous parotitis (n, %) 4 (33.3%) 3 (11.5%) 1 (20.0%) 0.209
Previous surgically treated varicocele (n, %) 2 (16.7%) 2 (7.7%) 0 (0.0%) 0.747
IIEF-5
b
(median, IQR) 25 (24–25) 24 (21–25) 22 (22–23) 0.389
MSHQ
c
erection scale (
1–4
) (median, IQR) 19 (17–20) 19 (17–20) 19 (17–20) 0.487
MSHQ
c
ejaculation scale (
5–12
) (median, IQR) 37 (33–38) 36 (35–38) 34 (32–39) 0.165
MSHQ
c
satisfaction scale (
13–18
) (median, IQR) 25 (23–25) 26 (24–30) 28 (26–29) 0.433
MSHQ
c
sexual activity (19) (median, IQR) 3 (2–4) 3 (2–4) 4 (3–5) 0.554
MSHQ
c
sexual activity (20, 21) (median, IQR) 8 (6–8) 8 (7–8) 8 (8–8) 0.179
MSHQ
c
sexual desire (22–25) (median, IQR) 16 (14–16) 16 (16–17) 17 (15–18) 0.353
a
Calculated using Chi Quadro or Fischer test for categorical variables or ANOVA test for continuous variables.
b
International Index of Erectile Function Questionnaire.
c
Male Sexual Health Questionnaire.
COVID-19: coronavirus disease 2019; IQR: interquartile range, ICU: intensive care unit.
COVID-19: effect on semen quality 5
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from COVID-19 have a significant risk of developing oligo-crypto-
azoospermia.
Prolonged viral shedding from the nasopharynx in convalescent
patients, up to at least 3 weeks after recovery from symptoms, has
been previously reported (Young et al.,2020). According to the
European Centre for Disease Prevention and Control (ECDC) we
considered as healed from SARS-CoV-2 infection those men with two
upper respiratory tract samples that were negative for SARS-CoV-2
RNA, and showing clinical resolution of symptoms (https://www.
ecdc.europa.eu/sites/default/files/documents/COVID-19-Discharge-
criteria.pdf). However, in our population, one patient died from
COVID-19 after clinical relapse and four tested positive again for
SARS-CoV-2 infection by NP swab during the screening phase (from
March 2020 to May 2020). Moreover, three men presented with per-
sistence of SARS-COV-2 RNA in biological fluids 3–9 weeks after dou-
ble negative NP swabs.
The occurrence of the virus in saliva, urine, and semen of asymp-
tomatic patients several weeks after double negative NP swabs was
demonstrated in a very small percentage of men in our cohort; never-
theless previous studies (Guo et al., 2020;Ma et al.,2020;Pan et al.,
2020) did not report occurrence of virus in semen of healed patients
or in their recovery phase. Interestingly, none of the partners of these
three newly positive patients tested positive for SARS-CoV-2 including
the patient with a positive test result for semen. Overall, these studies
suggest that the occurrence of virus in semen is a rare event. Further
studies will be required to determine the risk of SARS-CoV-2 trans-
mission by sexual intercourse.
The occurrence in the male reproductive system, in particular testis,
has been described for several viral strains. The HIV and mumps virus
might lead to male infertility as a result of direct damage to the testicu-
lar structure (Masarani et al.,2006;Garolla et al.,2013). Abnormal
sperm parameters have been observed in other viral infections such as
herpes simplex virus, hepatitis virus B, or hepatitis virus C (Garolla
et al.,2013). Recent findings seem to support an influence of SARS-
CoV-2 infection on male sex hormones, with an increase in plasma
levels of LH and a marked decrease in testosterone and FSH (Ma
et al.,2020).
In our study, we found that one out of four (11/43, 25.5%) of
COVID-19 healed patients were oligo-crypto-azoospermic, a percent-
age clearly exceeding that reported for the general population (about
1% for azoospermia, Jarow et al., 1989; 3% for oligozoospermia,
Ombelet et al., 2009). Interestingly, all azoospermic patients reported
a previous unimpaired fertility status (five had one child, two had two
children, and one had three children), and only one out of three oligo-
crypto-azoospermic men reported no parenthood (while the other
two each had two children). Occurrence of azoospermia could be re-
lated to antibiotics or other drugs used for the treatment of patients
during COVID-19. Indeed, antibiotics, antiviral drugs, chloroquine,
Figure 2. Timeline in days of the period between the first positive nasopharyngeal swab for COVID-19 and sample collection.
Urine pre: urine collected before semen collection; urine post: urine collected after semen collection. Timeline (days) from first positive nasopharyn-
geal swab for COVID-19 and the sample collection for each patient. In the first column, the origin of the samples is reported. White box: not
hospitalized patient; ‘H’: patient hospitalized in medicine units; ‘I’: patient hospitalized in intensive care unit. Red squares indicate the period between
the first positive nasopharyngeal swab for COVID-19 and the second negative nasopharyngeal swab, while the green ones represent the period
between the second negative nasopharyngeal swab and the date of sample collection. The symbols ‘þ’ and ‘’ indicate a positive or a negative
nasopharyngeal swab result for SARS-CoV-2 RNA. ‘S’: azoospermic; ‘S*’: oligospermic; 䊉: sample collection time; #: median proven healing time:
31 days (range 3–65); §: median SARS-CoV-2-free time: 35 days (range: 24–43).
6Gacci et al.
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corticosteroids, and immunomodulators might affect male fertility sta-
tus (Semet et al.,2017). However, a contribution to impairment of
testicular function from SARS-COV-2 infection cannot be excluded,
considering that, on average, the time from recovery was around
30 days, which is below the length of a complete cycle of human sper-
matogenesis. The virus may localize in the testis owing to the elevated
expression of ACE and TMPRSS2 in the organ, and COVID-19
patients with active infection show low testosterone levels (Rastrelli
et al., 2020;Wang et al.,2020). Moreover, although antibiotics have
long been suspected of contributing to male infertility (Semet et al.,
2017), limited data support this hypothesis (Samplaski and Nangia,
2015). Semen quality could also be affected by febrile illness, even if
limited data are currently available. In particular, semen concentration
was significantly influenced by fever occurring during the meiosis
and postmeiotic period, with a mean decrease of 32.6% and 35%,
respectively (Carlsen et al.,2003). Moreover, a large variation in sperm
concentration was observed, and semen recovery was obtained
almost 60days after temperature normalization (MacLeod, 1951).
................................................................................................................................................................................................. ...........................
Table II Comparison of relationship and parental status of the study sample (N ¼43) stratified by clinical management
of COVID-19.
Nonhospitalized
(N 512; 27.9%)
Hospitalized
(without ICU)
(N 526; 60.5%)
ICU
(N 55; 11.6%)
Pvalue
a
RELATIONSHIPS AND PARENTHOOD
Long-term female partner (n, %) 11 (91.7%) 22 (84.6%) 4 (80.0%) 0.838
Long-term partner Past COVID-19
infection at time
of partner
diagnosis (n, %)
5 (45.5%) 8 (34.8%) 1 (20.0%) 0.714
Positive for
COVID-19 at
enrollment (n, %)
2 (18.2%) 2 (9.0%) 0 0.710
Patients without children 5 (41.6%) 5 (19.2%) 1 (20.0%) 0.152
Number of children (median, IQR) 2 (0–2) 2 (1–2) 1 (1–1) 0.472
Number of sexual relationships
during the last 3 months
None (n, %) 2 (16.7%) 6 (23.1%) 1 (20.0%) 0.979
1–4 per month (n, %) 9 (75.0%) 16 (61.5%) 3 (60.0%) 0.648
4–8 per month (n, %) 1 (8.3%) 3 (11.5%) 1 (20.0%) 0.635
>8 per month (n, %) 0 (0.0%) 1 (3.9%) 0 (0.0%) 0.958
Problems having a baby (n, %) 3 (25.0%) 4 (16.0%) 1 (20.0%) 0.851
Abortion by the long-term partner (n, %) 3 (25.0%) 6 (23.1%) 0 (0.0%) 0.447
a
Calculated using Chi Quadro or Fischer test for categorical variables or ANOVA test for continuous variables.
................................................................................................................................................................................................. ...........................
Table III Comparison of semen analysis of the study sample (N ¼43) stratified by clinical management of COVID-19.
Nonhospitalized
(N 512; 27.9%)
Hospitalized (without ICU)
(N 526; 60.5%)
ICU
(N 55; 11.6%)
Pvalue
a
SEMEN ANALYSIS
Collected semen volume (mL) (median, IQR) 2.5 (1.5–3.5) 2.0 (0.8–2.5) 1.5 (1–2) 0.778
Total sperm number (millions) (median, IQR) 133.25 (50.5–244.1) 38.1 (5.5–123.8) 0.0 (0.0–7.0) 0.021
Sperm concentration (millions/mL) (median, IQR) 65.8 (23.8–71.0) 17.8 (5.5–70.0) 0.0 (0.0–3.5) 0.215
Progressive motility (%) (median, IQR) 36.0 (26.0–58.0) 25.0 (12.0–42.0) 27.0 (27.0–27.0) 0.154
Time from collection to analysis
b
(minutes) (median, IQR) 210 (168–270) 240 (180–300) 340 (120–270) 0.558
Vitality (live sperm, %) (median, IQR) 59 (47–81) 49 (31–67) 0 (0–9) 0.003
Sperm morphology (%) (median, IQR) 2 (2–5) 3 (1–4) 0 (0–0) 0.332
pH (median, IQR) 7.6 (7.4–7.6) 7.3 (7.2–7.6) 7.6 (7.6–7.6) 0.598
Leucocytes (10
6
/mL) (median, IQR) 0.0 (0.0–0.24) 0.0 (0.0–0.1) 0.3 (0.1–1.7) 0.324
Interleukin-8 (ng/mL) (median, IQR) 6 (3–10) 7 (5–11) 37 (13–78) <0.001
a
Calculated using Chi Quadro or Fischer test for categorical variables or ANOVA test for continuous variables.
b
Time needed to carry the samples to laboratory.
COVID-19: effect on semen quality 7
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In our cohort of patients, one crypto-azoospermic semen was ana-
lyzed 4weeks after temperature normalization and one oligospermic
after 6weeks, while the other nine oligo-crypto-azoospermic patients
were tested at least 8 weeks after COVID-19 clinical healing.
Moreover, 8 out of 11 men with semen impairment were crypto-
azoospermics and three were oligospermics. Thus, the impact of fever
on semen quality seems negligible.
Furthermore, the virus can have direct (viral replication and dissemi-
nation in the testis) and indirect (fever and immunopathology) effects
on testicular function (Carlsen et al.,2003). Leukocytospermia is asso-
ciated with male accessory gland infection (MAGI). However, the
pathophysiology of leukocytes in semen is unclear and the diagnostic
significance is still debated (Grande et al., 2018). New seminal bio-
markers, including some cytokines, have been reported as the most
promising putative markers of infection (Grande et al.,2018). IL-8 is a
chemokine involved in several inflammatory diseases, comprising in-
flammation of the prostate, seminal vesicles, and epididymis (Penna
et al.,2007;Lotti and et al.,2012;Lotti and Maggi, 2013). Therefore,
IL-8 has been proposed as a specific marker for silent MAGI (Lotti
et al.,2011). The high levels of sIL-8 found in a high percentage of our
patients support the persistence of an inflammatory condition within
the male genitourinary tract (Penna et al., 2007) after healing. In addi-
tion, reanimation treatments (e.g., invasive ventilation, administration
of sedative drugs, specific organ support therapy) and body distress
can affect testicular function (Vishvkarma and Rajender, 2020). Clearly,
in order to understand whether these men can recover from poor
testicular function, they should be reassessed at least 3 months after
complete healing from COVID-19.
Our study has some limitations. First, the number of enrolled
patients (43) may limit the statistical power of our study regarding the
cause-effect relationship between COVID-19 infection, semen quality,
and sexual transmission. However, ours is the largest among studies
published to date reporting on semen quality and occurrence of virus
genome in the semen of sexually active men who were previously
infected and who recovered from COVID-19 (12 patients in Ma et al.,
2020;23inGuo et al.,2020). Another limitation of our study is that
semen quality before infection of the men that demonstrated severe
oligo-crypto-azoospermia after COVID-19 was not known. However,
all patients who were crypto-azoospermic had previously had children.
The strength of our study is that that the SARS-CoV-2 genome
has been analyzed in four different biological samples, from different
anatomical sites, collected simultaneously. Finally, the study was
performed over a short time frame, and in a single center: this allows
us to minimize biases related to virus modifications or analytic
procedures.
Conclusion
The SARS-CoV-2 genome can be detected in biological fluids such as
saliva, urine, and semen in a small percentage of sexually active men
who have recovered from COVID-19. Even if our evidence suggests
that the risk of SARS-CoV-2 sexual transmission after recovery in sta-
ble partners seems negligible, caution should be taken when managing
the semen of healed COVID-19 patients during assisted reproduction
and cryopreservation.
Most importantly, one-quarter of men who recovered from
COVID-19 demonstrated oligo-crypto-azoospermia and signs of male
genital tract inflammation, strictly related to disease severity.
Therefore, our study suggests that COVID-19 patients of reproductive
age should undergo a careful follow up for reproductive function and
semen parameters.
Figure 3. The 95%CIs for mean interleukin-8 concentration in semen of the enrolled patients (N543). Data were stratified by clini-
cal management (not hospitalized N ¼12, medicine department N ¼26, intensive care unit N¼5) (A) and need of oxygen support (no oxygen
N¼19, low flow O
2
N¼10, high flow O
2
N¼14) (B) for COVID-19. The time of collection is shown in Fig. 2. IL-8: interleukin-8.
a
Calculated using
ANOVA test. Low flow O
2
Therapy includes nasal cannula, simple face mask, and partial rebreather mask. High flow oxygen/invasive O
2
includes trans
tracheal catheters, venturi mask, aerosol mask, tracheostomy collars, non-rebreathing mask with reservoir and one way valve and high humidity face
tents.
8Gacci et al.
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Supplementary data
Supplementary data are available at Human Reproduction online.
Data availability
The data underlying this article will be shared on reasonable request
to the corresponding author.
Authors’ roles
Conception and Design: M.G., S.S., E.B., F.A., M.M., L.V., A.B.
and G.M.R. Acquisition of data: A.M., A.P., R.N., C.B., A.L. and
L.G. Analysis and interpretation of data: A.S., M.G., E.B., S.M.,
S.M., S.D. and S.D. Drafting the manuscript: M.G. and A.S. Critical
revision of the manuscript: all the Authors. Statistical analysis: S.M.,
M.G., E.B. and S.P. Administrative, technical or material support: A.A.,
S.P., F.L. and M.C. Supervision: S.G. Ethical approval: S.M. and C.Z.
Funding
None.
Conflict of interest
None.
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