key: cord-0738341-tdz73813
authors: Kadihasanoglu, Mustafa; Aktas, Semih; Yardimci, Emre; Aral, Hale; Kadioglu, Ates
title: SARS-CoV-2 Pneumonia Affects Male Reproductive Hormone Levels: A Prospective, Cohort Study
date: 2020-11-27
journal: J Sex Med
DOI: 10.1016/j.jsxm.2020.11.007
sha: 3cf92ab88fc0c8f86eb851abc0671fb9a163cb34
doc_id: 738341
cord_uid: tdz73813

BACKGROUND SARS-CoV-2 which causes coronavirus disease 2019 (COVID-19) binds to angiotensin-converting enyzme 2 (ACE2) and enters the host cell. ACE2 protein is expressed highly in the testis. AIM The aim of this study was to compare male reproductive hormones such as total testosterone (TT), luteinizing hormone (LH), follicular stimulant hormone (FSH), and prolactin between patients with COVID-19, age-matched cases with non-COVID-19 respiratory tract infection, and age-matched controls. METHODS This was a prospective cohort study and included 262 men aged between 20 and 65 years. The study comprised 3 groups including patients with COVID-19 (n = 89), cases with non-COVID-19 respiratory tract infection (n = 30), and age-matched controls (n = 143). All cases were evaluated using TT, LH, FSH, and prolactin. Correlations between TT and clinical parameters of patient groups were investigated using Pearson's correlation test. OUTCOMES The primary outcome of the study was detection of the difference of TT, FSH, LH, and prolactin levels between the groups. Secondary outcome was to correlate TT and hospitalization time and oxygen saturation on hospital admission (SpO2) of patients. RESULTS The mean age of study groups was 49.9 ± 12.5 years, 52.7 ± 9.6 years, and 50 ± 7.8 years, respectively (P = .06). Serum TT levels was median 185.52 ng/dL in patients with COVID-19, median 288.67 ng/dL in patients with non-COVID-19 respiratory tract infection and median 332 ng/dL in control cases, (P < .0001). The proportion of patients with testosterone deficiency in group 1, group 2, and group 3 was 74.2%, 53.3%, and 37.8%, respectively (P < .0001). Serum LH levels (P = 0.0003) and serum prolactin levels (P = .0007) were higher in patients with COVID-19 and patients with non-COVID-19 respiratory tract infection than control cases. Correlation analysis revealed significant negative correlation between serum TT levels and hospitalization time of patients with COVID-19 (r = -0.45, P < .0001). In addition, a significant positive correlation was observed between SpO2 and serum TT levels in patients with COVID-19 ( r = 0.32, P = .0028). CLINICAL IMPLICATIONS Physicians may consider to evaluate male patients with COVID-19 for concomitant androgen deficiency. STRENGTHS & LIMITATIONS Strengths include the evidence about the alteration of male reproductive hormones under COVID-19. Limitations include the analysis limited to one general hospital, only a single measurement of TT was available, free and bioavailable testosterone levels were not evaluated. CONCLUSION This study demonstrates COVID-19 is associated with decreased level of TT and increased level of LH and prolactin. More serious COVID-19 causes more reduction in TT levels and prolongs hospitalization period. M Kadihasanoglu, S Aktas, E Yardimci, et al. SARS-CoV-2 Pneumonia Affects Male Reproductive Hormone Levels: A Prospective, Cohort Study. J Sex Med 2020;XX:XXX-XXX.

In early December 2019, a new contagious disease were identified in China (1) and caused by as a novel beta coronavirus (2) that has currently been named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (3). It causes respiratory illness named by World Health Organization as coronavirus disease 2019 and has become a global viral pandemic and a public health problem of international concern (4). Expression Omnibus and Sequence Read Archive, and showed that ACE2 is highly expressed in spermatogonia, Leydig and Sertoli cells (11) . As a consequence, testicular tissue may be a target tissue of SARS-CoV-2. After the binding of SARS-CoV-2 to ACE2 and virion membrane fusion, downregulation of ACE2 expression leads to excessive production of angiotensin by the related enzyme ACE (12) . A previous study showed that increased replication of coronaviruses downregulate the expression of ACE2 (13). Overproduction of angiotensin enhances oxidative stress mechanisms and leads to tissue injury. In sum, these findings may explain the possibility of harmful effects of SARS-CoV-2 on testis, potentially J o u r n a l P r e -p r o o f affecting TT secretion. In analogy to this relationship, it has been shown that the testis may be the target organ for other viruses, including human immunodeficiency virus, mumps, Zika and Ebola causing orchitis, and occasionally decreased TT production and oligospermia (14) .

Acute respiratory infection is another cause of decreased serum total testosterone (TT) levels. Muehlenbein 

This study was reviewed and approved by institutional ethics committee, on 27 April 2020 and complied with the Declaration of Helsinki. The information for all patients including demographic data, clinical characteristics, laboratory parameters and outcomes, were collected prospectively. Two authors independently reviewed the data collection forms to ensure that there was no duplicated information. Data were analyzed and interpreted by the authors.

The study population comprised three groups including patients hospitalized for COVID-19, hospitalized cases with non-COVID-19 respiratory tract infection between 22

March and 22 May 2020 and age-matched controls admitted to the urology outpatient department before January 2020. The first group was patients with COVID-19, the second groups included patients with non-COVID-19 respiratory tract infection, and the third consisted of control cases admitted to urologic outpatient clinic for reproductive function evaluation.

The first inclusion criteria for whole groups was age between 18 and 65 years.

Additional inclusion criteria for group-1 were (1) positive reverse-transcription polymerase chain reaction (RT-PCR) test of nasal and pharyngeal swab specimens (2) typical chest CT findings including ground-glass opacities, particularly on peripheral and lower lobes, crazy paving and bilateral multiple lobular and subsegmental areas of consolidation. Patients with non-COVID-19 respiratory tract infection had final diagnosis of at least two negative RT-PCR tests and negative chest CT findings. The exclusion criteria for all study subjects were J o u r n a l P r e -p r o o f

(1) previous exposure to exogenous testosterone, 5-alpha reductase inhibitors, luteinizing hormone-releasing hormone agonists, dehydroepiandesterone, clomiphene citrate, or other selective estrogen receptor modulators (2) use of opioid drugs within 3 months prior to study (3) diagnosis of prolactinoma and any cancer (4) history of testicular diseases and surgical interventions known to affect sex hormone levels.

Nasal and pharyngeal swab specimens were collected for the COVID-19 test on the day of admission. RT-PCR assay was used according to the manufacturer's instructions. The degree of severity (mild, moderate, and severe) were determined according to the COVID-19

Treatment Guidelines published by the National Institutes of Health (17) .

The recent medical history, clinical symptoms or signs on admission were extracted from electronic medical records. Radiologic assessments included CT, and all laboratory testing was performed according to the clinical care needs of the patient. Laboratory assessments were drawn before any treatment the first morning after admission between 8-11 am, after an overnight fast, and consisted of a blood count, C-reactive protein (CRP), procalcitonin (PCT), D-dimer and fibrinogen. After analyzing of blood samples for routine tests required for group-1 and group-2 patients' needs, the residual serum samples were Testosterone deficiency was defined as < 300 ng/dL according to American Urological Association testosterone deficiency guideline (18) . Compensated or subclinical hypogonadism was defined as low-normal TT levels (> 300 ng/dL) and elevated LH (> 9.4 IU/L) according to European Male Aging Study criteria (19) .

The primary outcome of the study was detection of the difference of TT, FSH, LH and prolactin levels between the groups. Secondary outcome was to correlate TT and hospitalization time and oxygen saturation on hospital admission (SpO2) of patients.

Sample size calculation was based on changes in serum TT levels. Type I error α was 0.05, type II error β was 0.10, two-tailed P-value was <0.05, study and control group ratio was 1:1, loss to follow-up rate was 10%, and at least 90 patients in each group were studied in the final analysis. Data were checked for suitability for a normal distribution with the Shapiro Wilk test and expressed as mean (SD) 

Eighty nine cases infected with COVID-19 were studied (Table-1 ). Fever (54%) and cough (49.4%) were the most common symptoms. Thirty cases (33.7%) had shortness of breath. Moreover, 29 (32.6%) patients had myalgia, 12 patients (13.5%) had sore throat, and 8 patients (18.56%) had chest pain symptoms. During the diagnostic procedure by RT-PCR, we found that 77 patients (86.5%) got a positive result in the first test, 12 patients (13.5%) got a positive result in the second test.

Among 89 patients with COVID-19, 52.8% (47/89) were diagnosed as "mild type", 33 .7% (30/89) as "moderate type", and 13.5% (12/89) as "severe type".

J o u r n a l P r e -p r o o f Thirty patients had non-COVID-19 respiratory tract infection were studied (Table-1) .

Fever (46.7%) and dyspnea (40%) were the most common symptoms. Seven cases (23.3%) had chest pain and 7 cases (23.3%) had cough. Also, 5 (16.6%) patients had myalgia, 3 patients (10%) had sore throat symptoms, and 2 (6.6%) had diarrhea. Twenty-one patients had two negative RT-PCR, 8 patients had 3 negative RT-PCR tests, and one patient had 5 negative RT-PCR test. All patients with non-COVID-19 respiratory tract infection had nonspecific CT findings.

The clinical presentation of patients with COVID-19 and patients with non-COVID-19 respiratory tract infection was similar (p=0.14) ( Table-1 ). Only the COVID-19 patients had more cough symptom than other group (p=0.018). The comparison of inflammation parameters are presented in Table- 2. The white blood cell count of the COVID-19 group (median 6.08 10 9 /L, IQR 3.36 10 9 /L) was significantly lower than that of the non-COVID-19 respiratory tract infection (median 8.71 10 9 /L, IQR 4.98 10 9 /L) and control group (median 7.7 10 9 /L, IQR 2.52 10 9 /L) (p=0.0001). It was not different between non-COVID-19 respiratory tract infection and control group (p=0.07). The lymphocyte count in the COVID-19 group (median 1.4 10 9 /L, IQR 0.83 10 9 /L) was also significantly lower than that in the non-COVID-19 respiratory tract infection (median 2.13 10 9 /L, IQR 1.46 10 9 /L)and control group (median 2.3 g/L, IQR 0.96 g/L) (p=0.0001). The lymphocyte count was not different between non-COVID-19 respiratory tract infection and control group (p=0.07).

Hemoglobin levels of patient and control groups were not statistically different (p=0.0574) ( Table-2 ). CRP levels were significantly higher in patients with COVID-19 and non-COVID-19 respiratory tract infection than control group (p=0.0007 and p=0.03).

However, COVID-19 and non-COVID-19 respiratory tract infection groups were similar in terms of CRP (p=1). In addition, the D-dimer, procalcitonin and fibrinogen levels were not significantly different between group-1 and group 2 (p=0.03, p=0.51 and p=0.32, respectively).

The comparison of TT levels showed a significant difference between the groups serum PRL (p=0.0007) levels compared to control cases. However, there was not a difference between group-1 and group-2 (p=1 and p=1). When serum FSH levels were compared, the groups had similar levels of FSH levels (p=0.91). The ratio of LH/TT is lowest in patients with COVID-19 (Table-3 ).

The comparison of hormone levels of COVID-19 patients classified into 3 different groups according to disease severity presented in Table- The testicular function may be effected by multiple pathogenic mechanisms occurring during SARS-CoV-2 infection. ACE2 is highly expressed by Leydig cells and its downexpression by SARS-CoV-2 lead to increase of angiotensin II levels (12) . It has been shown that angiotensin II reduced both basal and LH stimulated testosterone synthesis by Leydig J o u r n a l P r e -p r o o f cells, ACE2 may modulate the production of testosterone of these cells and protect testis by limiting angiotensin II detrimental effects (24, 25) . In this context, changes in LH and TT levels of patients were evaluated in present study and it has been determined that patients with COVID-19 had increased levels of LH and decreased levels of TT compared to patients with non-COVID-19 respiratory tract infection and control cases. Moreover, the lowest LH/TT ratio was calculated in patients with COVID-19. In the literature, several studies suggested that increased LH/TT ratio indicates Leydig cell defects and it is an important marker of primary hypogonadism (26) . Primary hypogonadism is a result of factors having negative influence on testicular tissue such as injury, tumor and infections. This finding supports the impact of harmful effect of SARS-CoV-2 on ACE2 expression and correspondingly increase of angiotensin II and reduction of testosterone production.

Compensated hypogonadism is defined TT in the normal range and inappropriately high LH (19) . In European Male Aging Study including 3,369 community-dwelling middleaged and older men, they reported that 9. However, this elevation reached not to levels of hyperprolactinemia. Therefore this is not a cause of pituitary suppression and decreased TT levels. Serum PRL levels may be influenced by stress and infection (28) . In addition, cytokines IL-1, IL-2, and IL-6 stimulate prolactin secretion (29) . The increased levels of IL-1, IL-2 and IL-6 in patients with COVI-19 has been demonstrated in several studies (30) . This relationship between increased levels of interleukins and suppression of prolactin secretion explained the elevated levels of prolactin in patients with COVID-19.

In present study, the TT levels correlated negatively with hospitalization time (r=-0.395, p<0.0001) and positively with SpO2 levels at admission of patients with COVID-19.

This finding demonstrates that serum TT levels is an important factor and predictor for the clinical process of patients. Rastrelli et al. evaluated the association between TT levels and clinical outcomes in a cohort of patients with COVID-19 (31) . They demonstrated that lower TT levels predict poor prognosis in patients with COVID-19. They correlated TT levels with neutrophil and lymphocyte counts and CRP levels and they found a negative correlation between serum TT levels and neutrophil count and CRP levels and a positive correlation between TT levels and lymphocyte count. However, in the present study, serum TT levels did not correlated with neutrophil and lymphocyte counts and CRP levels. g/dL to means of 13.9 at 1 month (34) . With these findings, the indifference of hemoglobin levels between patient and control groups is logical.

This study has several strengths. The study provides the evidence about the alteration of male reproductive hormones under COVID-19. In this study, serum LH and prolactin levels significantly elevated and serum TT levels decreased in COVID-19 patients, which infer to the potential hypogonadism. Because greater portion of patients with COVID-19 were J o u r n a l P r e -p r o o f reproductive-aged, cases, who recovered from this disease, should be evaluated with hormonal tests to detect harmful effects of SARS-CoV-2 on reproductive system.

There are several limitations in our study. First, the analysis in our study was limited to one general hospital, but all cases had fulfilled the laboratory diagnosis of COVID-19.

Control group included patients who admitted to urology outpatient clinic and evaluated for reproductive function had hormonal abnormalities. As another limitation, only a single measurement of TT was available. Moreover, free and bioavailable testosterone levels were not evaluated. Another limitation was neither semen analysis nor determination of SARS-CoV-2 in semen was performed, which are more clear evidence for SARS-CoV-2 induced testis injury. Psychological stress is another factor effecting serum TT levels. A current study evaluated psychological status of patients admitted with COVID-19 and presented that the predominant emotional state of patients for most days during the stay or for almost all the days during the hospital stay was that of anxiety (92 %), remaining worried (96 %), and feeling isolated (90 %) (35) . However, the authors could not compare their results with that of existing literature because of limited data on experiences of people with COVID-19. Several studies evaluating the impact of stress on hormonal function in both human and animals confirmed that psychological stress, especially chronic stress, inhibiting role in testosterone production (36) . Contrary to these studies presenting the decreasing effect of stress in serum testosterone levels, in the literature, it has been shown psychological stress does not constantly inhibit testosterone secretion (37) . In addition to this contradictory studies, a few studies exist which presented that testosterone concentration may be increased at initial stages of acute stress (38, 39) . Based on the aforementioned findings, it is not possible to associate the reduction of serum TT levels in patients with COVID-19 with their psychological stress in J o u r n a l P r e -p r o o f acute phase of the disease. However, even if not the lack of the evaluation of the emotional status of patients and controls is a limitation of the study.

This study demonstrates COVID-19 is associated with decreased level of TT and increased level of LH and prolactin. More serious COVID-19 causes more reduction in TT levels and prolongs hospitalization period. In light of these findings, physicians may consider to evaluate male patients with COVID-19 for concomitant androgen deficiency. 

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