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 (Figure 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.
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.
Timelines from the first positive NP swab to sample collection are reported in Figure 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).
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 nasopharyngeal 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 '+' and '−' indicate a positive or a negative nasopharyngeal swab result for SARS-CoV-2 RNA. 'S': azoospermic; 'S*': oligospermic; •: sample collection time; #: median proven healing time: 31 days (range 3–65); §: median SARS-CoV-2-free time: 35 days (range: 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 require oxygen therapy, 10 (23.2%) were treated with low flow O2 therapy, whereas 14 (32.6%) necessitated high flow/invasive O2 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 relationship. 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, P = 0.470; 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)= 37.04); 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 = 34.99 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 infection 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 pharyngeal, vaginal, and rectal swabs (partner): all samples tested negative.
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 (P = 0.152).
Semen analysis demonstrated that eight patients (18.6%) were azoospermic 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 hospitalized in the medicine department and only in one among the 12 nonhospitalized (P < 0.001). No relation was found between occurrence of azoospermia and severity of oxygen therapy (P = 0.417). 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 intensive care unit as compared to non-hospitalized patients or those admitted to the medicine department, but was not related to the intensity of oxygen therapy (36.8% in men without O2 vs. 30.0% with low flow O2 and 42.9% with high flow/invasive O2).
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 (P = 0.142) (Figure 3A). Pathological levels of sIL-8 were related to the severity of oxygen therapy (r = 0.356; P = 0.050). As shown in Figure 3B, high levels of sIL-8 were found in 12 out of 19 (63.2%) men without oxygen therapy, 9 out of 10 (90.0%) with low flow O2-therapy and 12 out of 14 (85.7%) necessitating high flow/invasive O2 therapy.
The 95% CIs for mean interleukin-8 concentration in semen of the enrolled patients (N543). Data were stratified by clinical management (not hospitalized N=12, medicine department N=26, intensive care unit N=5) (A) and need of oxygen support (no oxygen N=19, low flow O2 N=10, high flow O2 N=14) (B) for COVID-19. The time of collection is shown in Figure 2. IL-8: interleukin-8. aCalculated using ANOVA test. Low flow O2 Therapy includes nasal cannula, simple face mask, and partial rebreather mask. High flow oxygen/invasive O2 includes trans tracheal catheters, venturi mask, aerosol mask, tracheostomy collars, non-rebreathing mask with reservoir and one way valve and high humidity face tents.
In univariate analysis, age, hospitalization and sIL-8 were all significant 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 (P = 0.039) (data not shown).
Hum Reprod. 2021;36(6):1520-1529. © 2021 Oxford University Press