key: cord-0924198-px2nimc7 authors: Guo, Tong-Hang; Sang, Mei-Ying; Bai, Shun; Ma, Hui; Wan, Yang-Yang; Jiang, Xiao-Hua; Zhang, Yuan-Wei; Xu, Bo; Chen, Hong; Zheng, Xue-Ying; Luo, Si-Hui; Xie, Xue-Feng; Gong, Chen-Jia; Weng, Jian-Ping; Shi, Qing-Hua title: Semen parameters in men recovered from COVID-19 date: 2021-05-11 journal: Asian J Androl DOI: 10.4103/aja.aja_31_21 sha: 3cf64ddca0c643cd25fe49d38f0fc439fd2c8daf doc_id: 924198 cord_uid: px2nimc7 The novel coronavirus disease (COVID-19) pandemic is emerging as a global health threat and shows a higher risk for men than women. Thus far, the studies on andrological consequences of COVID-19 are limited. To ascertain the consequences of COVID-19 on sperm parameters after recovery, we recruited 41 reproductive-aged male patients who had recovered from COVID-19, and analyzed their semen parameters and serum sex hormones at a median time of 56 days after hospital discharge. For longitudinal analysis, a second sampling was obtained from 22 of the 41 patients after a median time interval of 29 days from first sampling. Compared with controls who had not suffered from COVID-19, the total sperm count, sperm concentration, and percentages of motile and progressively motile spermatozoa in the patients were significantly lower at first sampling, while sperm vitality and morphology were not affected. The total sperm count, sperm concentration, and number of motile spermatozoa per ejaculate were significantly increased and the percentage of morphologically abnormal sperm was reduced at the second sampling compared with those at first in the 22 patients examined. Though there were higher prolactin and lower progesterone levels in patients at first sampling than those in controls, no significant alterations were detected for any sex hormones examined over time following COVID-19 recovery in the 22 patients. Although it should be interpreted carefully, these findings indicate an adverse but potentially reversible consequence of COVID-19 on sperm quality. China, from January to March 2020, and those who were willing to participate were all recruited in this study. A total of 41 men were recruited, among whom 22 were willing to have a second sampling approximately 1 month later. All these participants had been tested positive for SARS-CoV-2 viral RNAs in throat swabs or respiratory specimens by RT-PCR assays through a two-step confirmation strategy. 15 According to the New Coronavirus Pneumonia Prevention and Control Program published by the National Health Commission of China, the patients were classified into mild, moderate, severe, and critical subtypes on the basis of severity of the illness. 16 All the patients recruited for this study filled in a questionnaire regarding their marriage status, occupations, history of diseases and familial diseases, and any exposure to factors that are potentially harmful to the male reproductive system. The control group was composed of 50 Chinese men of matching ages, who had not suffered from COVID-19. Informed consent forms were obtained from all participants. All the participants underwent comprehensive clinical examination of the genitals and secondary sexual characteristics, all had normal secondary sexual characteristics and normal testicular sizes, and varicoceles were found in four patients and eight control men. Semen samples were obtained by masturbation. After the semen reached complete liquefaction at 37°C, semen parameters were assessed. Briefly, semen volumes were calculated from the sample weights according to the instructions of the WHO laboratory manual. 17 Sperm concentration and sperm motility were assessed by computer-assisted sperm analysis (CASA) under phase contrast microscopy (CX43, Olympus corporation, Tokyo, Japan) equipped with SAS-II system (Beijing Precise Instrument Co., Ltd., Beijing, China) at 10× magnification. The morphology of at least 200 spermatozoa was examined after Diff-Quick staining (Ankebio, Hefei, China) with a light microscope (UB100i, Aopuguangdian, Chongqing, China) at 100× magnification in accordance with the WHO laboratory manual. 16 Sperm vitality was assessed by eosin Y staining method in semen, following the WHO guidelines. 17 Evaluation of sex hormone levels Sera were obtained from participants' peripheral blood samples. The levels of estradiol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone, testosterone (T), prolactin, anti-Müllerian hormone (AMH) and inhibin B were assessed by chemiluminescent immunoassays, using commercial kits (Shenzhen Yahuilong Biotechnology, Shenzhen, China) measured by YHLO iFlash 3000-H immunoassay analyzer for inhibin B and using commercial kits (Beckman Coulter, Brea, CA, USA) measured by Beckman Coulter Unicell DXI 800 immunoassay analyzer for the other hormones. Owing to the small numbers of participants, data are presented as median (interquartile range [IQR] ). Data were compared between controls and men recovered from COVID-19 using the non-parametric Mann-Whitney-Wilcoxon test for semen characteristics, the Student's t-test for quantitative variables (ages, days after the appearance of symptoms, days after hospital discharge, and sex hormone levels), and the Chi-squared test or Fisher's exact test for categorical variables (percentages of patients with semen volume <1.5 ml, oligozoospermia, asthenozoospermia, and teratozoospermia). Data were compared between the first and second samplings using the Wilcoxon signed-rank sum test for semen characteristics and Student's t-test for sex hormone levels. All statistical tests were two-tailed and a P < 0.05 was considered statistically significant for all analyses. A total of 41 men with a median age of 26.0 (IQR: 22.0-34.0) years old who had been admitted to hospital with confirmed COVID-19 were enrolled in the study. The physical information and clinical characteristics related to COVID-19 for these patients are shown in Supplementary Table 1 and 2. Among the 41 patients, 22 (53.7%) were unmarried and had no child, 18 (43.9%) were married and had at least one child, and one (2.4%) who had been married for six months had no child yet. Eight (19.5%) patients had light to moderate alcohol drinking and tobacco smoking habits. None of these patients reported having any exposure to toxic chemicals, radiations, or high temperature over the past 6 months, or having any genetic diseases in their families. Noticeably, the median body mass index (BMI) for these patients was 25.1 (IQR: 21.9-28.0) kg m −2 , above the upper limit of normal range (18.5-23.9 kg m −2 ) for adult men according to the prevention and control guide for overweight and obesity in Chinese adult. All the patients were symptomatic, exhibiting at least one of the typical COVID-19 symptoms 18 during the SARS-CoV-2 infection period. One patient had blood in his urine and one felt burning around the eyes during infection. Twenty-nine patients were categorized as mild, ten as moderate and two as severe cases. They had been hospitalized for a median of 17.0 (IQR: 13.0-26.0) days and discharged after viral clearance. Of the 41 patients, one exhibited only very mild symptoms and did not take any medicine, six patients received corticosteroid therapy during hospitalization, seven patients received only conventional antiviral or anti-inflammatory medication, and the others took a variety of medicines, such as conventional antiviral or anti-inflammatory drugs, lopinavir-ritonavir, diamine glycyrrhizinate, bicyclol tablets, polyene phosphatidylcholine capsules, interferons, and traditional Chinese medicine. All the patients were physically normal with respect to external genitalia, testicular volume and texture, and male secondary sex characteristics. No epididymal or testicular tenderness was found during physical examination of the genitourinary system. None of the patients reported having any scrotum-related symptoms since the appearance of COVID-19 symptoms. All the patients self-reported suffering from psychological stress, such as fear, tension, and anxiety, since appearance of COVID-19 symptoms. Semen parameter values were compared between patients and controls who had not suffered from COVID-19 (Table 1) . Of the 41 patients, 27 men (65.9%) showed an abnormal value for at least one semen parameter as judged from the reference defined by WHO, 17 in contrast to 40.0% (20 out of 50, data not shown) in controls (P = 0.0248; Chi-squared test). The median total sperm count per ejaculate and the median value of sperm concentration for the patients were significantly lower than those for the controls (P = 0.0322). Compared with controls, patients had significantly lower percentages of motile spermatozoa (P = 0.0233) and progressively motile spermatozoa (P = 0.0280) and a higher ratio of asthenozoospermia (P = 0.0450). Among the 41 patients, 18 patients had percentages of progressively motile or motile spermatozoa below the WHO reference values, while in controls, 11 men had low percentages of progressively motile or motile spermatozoa (P = 0.0450; Chi-squared test). Semen volume, sperm vitality, percentage of abnormal sperm morphology, and percentage of men with teratozoospermia were all comparable between patients and controls with no significant between-group difference. Since sex-related steroids can also be used to evaluate the status of the male gonad, and in order to understand better the after-effects of SARS-COV-2 infection on male reproductive function, we next assessed sex hormone levels in the sera of these 41 patients ( Table 1 and Supplementary Table 5 ). The levels of T, LH, FSH, and AMH were largely normal for all the patients. Statistically significantly higher prolactin (P = 0.0088) and lower progesterone levels (P = 0.0050) were observed in patients than those in controls. Though estradiol levels were below the reference range in nine patients, the median value in patients was not significantly different from that in controls. Inhibin B is predominantly secreted by Sertoli cells, correlates positively with sperm concentration, sperm count and testicular volume, and its level is used as a valuable index for spermatogenic function. [19] [20] [21] Inhibin B levels in 13 of the 38 patients examined fell below the reference range and the median value was 103.0 (IQR: 74.8-145.8) pg ml −1 in patients, in comparison with 116.0 (IQR: 85.3-128.5) pg ml −1 in controls, but the difference between patient and control groups did not reach statistical significance. Table 6 ). The values of sperm parameters and sex hormones for these two samplings were compared ( Table 2) . There were significant increases in total sperm count (P = 0.0029), sperm concentration (P = 0.0066), and motile sperm count (P = 0.0391) at the second sampling, compared with those at the first sampling. The percentage of spermatozoa with abnormal sperm morphology was significantly reduced at the second sampling than that at the first sampling (P = 0.0333). No significant alterations in sex hormones were observed. We have described the semen characteristics and sex hormones in a cohort of 41 patients after recovery from COVID-19 and for the first time, to our knowledge, to undertake a longitudinal assessment of spermatogenesis following recovery from COVID-19. Though several pieces of evidence have suggested that the testes could be a target organ of COVID-19, 2,4,5,11,14,22 so far there is only limited information available regarding the association between COVID-19 and spermatogenesis. Recently, Holtmann et al. 8 reported a negative influence of COVID-19 on sperm quality observed in four recovered patients in need of hospital care during COVID-19 course, but not in 18 recovered patients with mild symptoms when home care was possible, but the cohort size was small and the consequences of COVID-19 on spermatogenesis remain to be ascertained. Here, our findings present direct evidence of temporary reductions in total sperm count, sperm concentration and numbers of motile and progressively motile sperm in patients who had recovered from COVID-19 after hospitalization, suggesting an adverse but potentially reversible consequence of COVID-19 on testicular function. When compared with controls, the sperm concentration, motility, and progressive motility were all significantly reduced in the patients at the first sampling that was conducted at a median of 56 days after hospital discharge, while when comparing sperm characteristics over time following COVID-19 recovery, total sperm count, sperm concentration, and numbers of motile and progressively motile spermatozoa in the patients were all significantly increased at the second sampling that was conducted at a median of 84 days after hospital discharge than those at the first sampling for the 22 patients examined, suggesting a potential recovery of sperm numbers. There were also some increases in percentages of motile and progressively motile spermatozoa observed at the second sampling, but the increasements were not statistically significant, which suggest that a longer time may be needed for the recovery of the sperm motility or that these patients had pre-existing low sperm motility before COVID-19. Since the spermatogenic cycle in human is estimated as taking approximately 74 days, 23, 24 these findings suggest that the after-effects of COVID-19 may last for one spermatogenic cycle. Consistent with a preliminary study (preprint) on sex hormones in patients with SARS-CoV-2 infection, 22 higher prolactin levels were observed in our patients who had recovered from COVID-19, which could result from multiple physiological or pathological conditions, or medication. 25 On the other hand, the higher prolactin levels could be preexisting in these patients, as we did not observe any evidence of recovery in patients at the second sampling. In this previous study, the authors also found increased LH levels, along with decreased T/LH and FSH/LH ratios in patients with SARS-CoV-2 infection, 22 while in our study, all the patients have normal levels of LH, and the LH levels, and the ratios of T/LH and FSH/LH in the patients did not differ significantly from those in controls. These differences may be due to the timing of blood sampling -their samples were collected from patients with SARS-CoV-2 infection, while our samples were collected at least 36 days after hospital discharge; this, however, needs to be confirmed by studies on the same patients sampled at different time points after SARS-CoV-2 infection. We also noticed reduced inhibin B levels in patients at the first sampling compared with controls, and increased inhibin B levels at the second sampling than that at first sampling for the 22 patients examined, though the differences did not meet statistical significance. Inhibin B is predominantly produced by Sertoli cells, which support and nurse germ cells and are essential for spermatogenesis, its level positively correlates with Sertoli cell function and sperm number. 19, 26 Hence, the low inhibin B levels at the first sampling may also support the observation of worse semen quality in our patients. Though we found that the progesterone levels in the patients were lower than those in controls, we did not observe any significant change between the two samplings conducted for the 22 patients. Thus, we think that the finding of lower progesterone levels in patients than those in controls may result from variations in men (for example, compared with controls, our patients may have pre-existing lower progesterone levels) given our small cohort size, but other than a consequence of COVID-19. In particular, in our opinion, the temporary sperm parameter alterations (at the first sampling) in the patients may not be solely attributable to a specific pathogenic mechanism of SARS-CoV-2. There are other plausible explanations of the COVID-19 after-effects on male reproduction. First, all our patients self-reported that they suffered from emotional and mental stresses since SARS-CoV-2 infection and a large retrospective cohort study also showed that COVID-19 was linked to higher rates of mental health problems, 27 while existing clinical evidence suggests a negative association between psychological stress and spermatogenesis. 28, 29 Second, in our patients, 85% had fever and several reports have observed temporarily decreased semen parameter values in patients who had experienced febrile illness. 30, 31 In a recent study, sperm concentration, sperm number and motile sperm number in recovered patients with fever symptoms during COVID-19 course were lower than those without fever, but only 18 patients were included for this comparison. 8 Hence, the impact of fever needs to be ascertained by future studies on a larger scale. Third, though we did not observe any obvious correlation between the medications, therapies or the severity of the COVID-19 and the sperm quality in our patients, Holtmann et al. 8 reported that the negative influence of COVID-19 on sperm count and sperm motility was not detected in subjects recovered from mild symptoms when home care was possible, but was detected in patients in need of hospital care. Thus, the possibility that these medications or therapies may be harmful to testicular functions cannot be definitely ruled out. However, our findings of the consequences of COVID-19 on male reproduction should be interpreted cautiously, as the lack of pre-COVID-19 sperm and hormone parameter values are a huge limitation and sperm concentration and sperm motility were determined mainly using CASA, which is not the standard procedure for semen analyses but an optional choice as suggested by the WHO. 17 The data were obtained from a small cohort size of 41 men who had recovered from COVID-19 for a median time of 56 days at the first sampling and controls lack a second sampling at a similarly spaced time point, while sperm parameters could be physiologically subject to intra-patient variability and fluctuations. Subsequent long-term follow-ups on more patients and elucidation of the underlying pathological mechanism and pathways would help a better understanding of the after-effects of COVID-19 on spermatogenesis and to find ways to diminish the consequences during coronavirus infections. QHS and JPW conceived and designed the study. THG, MYS, SB, HM, YYW, BX, HC, XHJ, YWZ, XFX, and CJG recruited the participants, carried out examinations, and analyzed the data. JPW, XYZ, and SHL provided clinical information of the patients. HM, SB, and QHS wrote the manuscript. QHS and JPW edited the manuscript and provided comments and feedback. QHS supervised the study. All authors read and approved the final manuscript. This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. ©The Author(s)(2021) A novel coronavirus from patients with pneumonia in China A pneumonia outbreak associated with a new coronavirus of probable bat origin Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding scRNA-seq profiling of human testes reveals the presence of the ACE2 receptor, a target for SARS-CoV-2 infection in spermatogonia No evidence of SARS-CoV-2 in semen of males recovering from COVID-19 Expression of ACE2, the SARS-CoV-2 receptor, and TMPRSS2 in prostate epithelial cells Absence of 2019 novel coronavirus in semen and testes of COVID-19 patients Assessment of SARS-CoV-2 in human semen -a cohort study Absence of SARS-CoV-2 in semen of a COVID-19 patient cohort Study of SARS-CoV-2 in semen and urine samples of a volunteer with positive naso-pharyngeal swab Clinical characteristics and results of semen tests among men with coronavirus disease 2019 SARS-CoV-2 infection, male fertility and sperm cryopreservation: a position statement of the Italian Society of Andrology and Sexual Medicine (SIAMS) (Società Italiana di Andrologia e Medicina della Sessualità) SARS-CoV-2 presence in seminal fluid: myth or reality Pathological findings in the testes of COVID-19 patients: clinical implications Asymptomatic patients and asymptomatic phases of Coronavirus Disease 2019 (COVID-19): a population-based surveillance study Interpretations of "Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 7 World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. 5 th ed. Geneva: World Health Organization Clinical characteristics of coronavirus disease 2019 in China Serum inhibin B as a marker of spermatogenesis Semen quality and spermatozoal DNA integrity in survivors of childhood cancer: a case-control study The serum inhibin B concentration and reference ranges in normozoospermia Effect of SARS-CoV-2 infection upon male gonadal function: a single centerbased study Spermatogenesis in man: an estimate of its duration Kinetics of the germinal epithelium in man Serum inhibin B levels reflect Sertoli cell function in normal men and men with testicular dysfunction Bidirectional associations between COVID-19 and psychiatric disorder: retrospective cohort studies of 62,354 COVID-19 cases in the USA Psychological stress and testicular function: a cross-sectional study of 1,215 Danish men Effects of psychological stress on male fertility High risk of temporary alteration of semen parameters after recent acute febrile illness History of febrile illness and variation in semen quality We thank all the participants for their cooperation. We thank Dr. Jing Yang, Dr. Wei-Min Yu, Dr. Tai Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.