key: cord-299940-nvlcwcz8
authors: Rastrelli, Giulia; Di Stasi, Vincenza; Inglese, Francesco; Beccaria, Massimiliano; Garuti, Martina; Di Costanzo, Domenica; Spreafico, Fabio; Greco, Graziana Francesca; Cervi, Giulia; Pecoriello, Antonietta; Magini, Angela; Todisco, Tommaso; Cipriani, Sarah; Maseroli, Elisa; Corona, Giovanni; Salonia, Andrea; Lenzi, Andrea; Maggi, Mario; De Donno, Giuseppe; Vignozzi, Linda
title: Low testosterone levels predict clinical adverse outcomes in SARS‐CoV‐2 pneumonia patients
date: 2020-06-03
journal: Andrology
DOI: 10.1111/andr.12821
sha: 
doc_id: 299940
cord_uid: nvlcwcz8

BACKGROUND: The pandemic of new severe acute respiratory syndrome (SARS) due to coronavirus (CoV) 2 (SARS‐CoV‐2) has stressed the importance of effective diagnostic and prognostic biomarkers of clinical worsening and mortality. Epidemiological data showing a differential impact of SARS‐CoV‐2 infection on women and men have suggested a potential role for testosterone (T) in determining gender disparity in the SARS‐CoV‐2 clinical outcomes. OBJECTIVES: To estimate the association between T level and SARS‐CoV‐2 clinical outcomes (defined as conditions requiring transfer to higher or lower intensity of care or death) in a cohort of patients admitted in the respiratory intensive care unit (RICU). MATERIALS AND METHODS: A consecutive series of 31 male patients affected by SARS‐CoV‐2 pneumonia and recovered in the respiratory intensive care unit (RICU) of the “Carlo Poma” Hospital in Mantua were analyzed. Several biochemical risk factors (ie, blood count and leukocyte formula, C‐reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), ferritin, D‐dimer, fibrinogen, interleukin 6 (IL‐6)) as well as total testosterone (TT), calculated free T (cFT), sex hormone–binding globulin (SHBG), and luteinizing hormone (LH) were determined. RESULTS: Lower TT and cFT were found in the transferred to ICU/deceased in RICU group vs groups of patients transferred to IM or maintained in the RICU in stable condition. Both TT and cFT showed a negative significant correlation with biochemical risk factors (ie, the neutrophil count, LDH, and PCT) but a positive association with the lymphocyte count. Likewise, TT was also negatively associated with CRP and ferritin levels. A steep increase in both ICU transfer and mortality risk was observed in men with TT < 5 nmol/L or cFT < 100 pmol/L. DISCUSSION AND CONCLUSION: Our study demonstrates for the first time that lower baseline levels of TT and cFT levels predict poor prognosis and mortality in SARS‐CoV‐2‐infected men admitted to RICU.

The new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has spread rapidly around the world in the last few months, thus resulting in more than 3. disease pathogenesis and to explore factors, which could have an impact toward its prognosis. Earlier prediction models suggest that elderly patients or patients with comorbidities (eg, hypertension, diabetes mellitus, chronic obstructive pulmonary disease, cardiovascular disease) run a greater risk for disease progression and even fatal outcomes. [1] [2] [3] However, delineating efficient prognostic factors for both worse clinical course and mortality is claimed as a major unmet need in order to tackle clinical management of the severe cases. 4 SARS-CoV-2 infection has been shown to have a differential impact on women and men. Sex disaggregated data indeed showed greater mortality rates in men as compared with women. In particular, men are up at threefold higher odds of lethality than women, 5 thus indicating a firm gender inequality in SARS-CoV-2-associated sequelae. Several social factors, genetic, immunological, and hormonal differences, as well as lifestyle habits (ie, smoking and alcohol consumption), have been considered to play a role in this gender disparity. 6 Among sex hormones, a potential role for testosterone (T) in defining sex-related differences in terms of SARS-CoV-2 infection clinical outcomes has been hypothesized. 7, 8 Obviously, due to the known higher level of T in men than in women, the prevailing theory is that T might be a promoter of SARS-CoV-2 infection and progression. 7, 8 This theory stems from the positive regulatory effect of androgen receptor (AR) and T on the transcription of a transmembrane protease, serine 2 (TMPRSS2), a critical factor enabling cellular infection by coronaviruses, including SARS-CoV-2. Conversely, others have hypothesized that low T levels could theoretically be detrimental, increasing the risk of severe disease among patients with SARS-CoV-2 infection, 8 as for other comorbidities. 9, 10 Indeed, T has been also described to induce the angiotensin-converting enzyme 2 (ACE2) expression, 11 an important lung protective enzyme. 12 This view would agree with the evidence reporting that in some countries (at least Italy and Spain), SARS-CoV-2 infection among female health workers is twice that of their male counterpart (https://data.unwom en.org/resou rces/Covid -19-emerg ing-gende r-data-and-why-it-matters). Of relevance, it is well established that T level declines according to aging and in the presence of comorbidities like obesity, diabetes mellitus, and cardiovascular diseases, which have been found to be highly prevalent in SARS-CoV-2 patients. 13 Owing to the high global prevalence of hypogonadism (estimated to be 15%-20% among middle-aged/elderly men), 14, 15 the relation between hypogonadism and SARS-CoV-2 infection outcomes deserves deep a thorough investigation. More importantly, the prevalence of hypogonadism in elderly male patients admitted to the hospital for acute illness raises up to 53%. 16, 17 Thereof, we sought to estimate the association between the T levels and the SARS-CoV-2 infection clinical outcomes (defined as conditions requiring to transfer to a higher or lower intensity of care units or even death) as well as the biochemical prognostic predictors of severe and fatal SARS-CoV-2 infection in a cohort of patients admitted in the respiratory intensive care unit (RICU) of a single Hospital in Mantua, one of the epicenter of the global SARS-CoV-2 pandemic in Italy. This study reports findings from 31 patients with SARS-CoV-2 infection and follows 25 of them through to discharge or death. The follow-up until discharge or death is a point of difference from other case series to date.

Data from a consecutive series of 31 male patients with SARS-CoV-2 pneumonia and recovered in the respiratory intensive care unit (RICU) of the "Carlo Poma" Hospital in Mantua, Italy, were analyzed.

A laboratory (pharyngeal-nose swab positivity) of SARS-CoV-2 infection was confirmed by chest X-ray. Comorbidity burden was assessed by the Charlson comorbidity index (CCI).

Acute respiratory distress syndrome (ARDS) was defined by "Berlin definition," and patients were segregated into mild ARDS (PaO2/FiO2 ≤ 300 mm Hg and > 200 mm Hg), moderate ARDS (PaO2/FIO2 ≤ 200 mm Hg and > 100 mm Hg), and severe ARDS (PaO2/FiO2 ≤ 100 mm Hg) 18 ; therefore, for the specific purpose of this analysis, patients have been further subdivided into either men with severe ARDS or with mild-moderate ARDS at the RICU admission.

Discussion and conclusion: Our study demonstrates for the first time that lower baseline levels of TT and cFT levels predict poor prognosis and mortality in SARS-CoV-2-infected men admitted to RICU.

COVID-19, inflammatory markers, mortality, prognosis, sex hormones According to the protocol (approved by Local Ethics Committee Val Padana, Mantua, Italy), each patient underwent a standardized diagnostic workup. Specifically, blood samples were drawn the first morning after the admission at the RICU within 8.00 AM, after an overnight fast, for determination of blood count and leukocyte formula, creatinine, uric acid, electrolytes, transaminases, creatine phosphokinase (CPK), C-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), ferritin, D-dimer, fibrinogen, interleukin 6 (IL-6), total testosterone (TT), sex hormone-binding globulin (SHBG), and luteinizing hormone (LH). Blood sample analyses were performed in the central laboratory of the "Carlo Poma" Hospital (Mantua, Italy) with commercially available kits normally used for clinical practice of the hospital. TT was measured once by immunoassay (Electrochemiluminescence Immunoassay, ECLIA), and free T was calculated by the Vermeulen formula (http://www.issam.ch/ freet esto.htm). 19 Hence, patients were divided according to the outcome through- 

Differences among groups with different prognoses were evaluated eventually merging the two groups with the worst outcomes (the aforementioned third and fourth groups). Thereafter, a sensitivity analysis was performed to compare the groups of which post-RICU outcome was known at the time of the analysis (thus excluding the aforementioned second group). Either Mann-Whitney U test or one-way ANOVA on ranks (Kruskal-Wallis test) was used to test the differences concerning continuous variables between two or more than two groups, respectively. Differences between continuous cat- 

Of 31 patients, 21 (67.7%) were transferred from RICU to IM after overall improvement of the respiratory conditions, six (19.4%) were stable at time of the present analysis and maintained in RICU, and four (12.9%) worsened their conditions and were either transferred to ICU (n = 2) or eventually died (n = 2). Table 1 details descriptive statistics of the whole cohort of patients as segregated according to the different outcomes. Overall, significant difference among groups was found in terms of previous history of psychiatric diseases and of prevalence of severe ARDS at RICU admission. Moreover, lymphocyte count was the lowest in both RICU and ICU/deceased patients, whereas PCT and LDH had the highest values in the ICU/deceased group. Of note, no differences in D-dimer serum levels were observed. LH, TT, and cFT were significantly different among groups, with higher LH and lower TT and cFT in the ICU/deceased group. These differences were confirmed when comparing the groups transferred from RICU because of either improved respiratory conditions or adverse outcomes, respectively (Table 1 ). In addition, the comparison of these two groups revealed that men in the adverse outcome group (ie, transferred to ICU or deceased) more frequently had a history of arrhythmia and were less frequently obese. Besides the aforementioned biochemical and hormonal parameters, men in the ICU/deceased group had significantly higher neutrophil count, potassium, and CRP levels, as compared with men transferred to IM.

In order to further explore the trends of hormonal levels according to the severity of the outcome, hormones were analyzed in each category after splitting the adverse outcome group into men transferred to ICU and men eventually deceased. Figure As both TT and cFT showed a significant and progressive decline according to worsening outcomes, we evaluated the relationship between T and markers of disease severity, as emerged by Table 1 . Table 2 shows the correlation for TT and cFT with markers of disease severity. Both TT and cFT showed a negative significant correlation with the neutrophil count, LDH, and PCT levels and a positive one with the lymphocyte count. TT was also negatively correlated with CRP and ferritin; cFT showed a similar but not statistically significant trend. Neither TT nor cFT was correlated with potassium. In order to further explore these relationships, we conducted LOWESS analyses to check for their non-linearity. The estimated probability of being transferred to IM and ICU, or to die based on T levels is reported in Figure 4 . A non-linear change in the probability of different outcomes is well recognizable. When applying the aforementioned thresholds, the probability of being We also showed that low baseline TT and cFT levels are related to a more severe ARDS at RICU admission. Noteworthy, these data highlight a novel potential mechanism of frailty and mortality by identifying low T as a risk factor for the severe respiratory failure and inflammatory storm in SARS-CoV-2 infections. Our observations are further substantiated by several epidemiological evidence demonstrating that male hypogonadism represents a risk factor for a higher morbidity and mortality. 10, 21 In particular, it has been well demonstrated that the so-called functional hypogonadism is associated with conditions like obesity and inflammation in males. 22 Therefore, it could be also speculated that obesity and low T could even foster the cytokine storm aggravating further the clinical condition.

Consistently, over the last decades it has become clear that T is involved in a multitude of biological processes in males, other than reproduction and sexuality. In particular, a novel aspect of the physiology of T is its anti-inflammatory role. 23, 24 Several preclinical and clinical evidence showed that low T boosted pro-inflammatory cytokines and that T treatment blunted their levels. 23, 25, 26 There is evidence for an immunomodulatory and protective effect of T by regulating differentiation of T lymphocytes. 23, 27, 28 Interestingly, we observed that lymphocyte count increased, while neutrophil levels decreased, as a function of increasing TT and cFT.

Accumulating evidence suggests that SARS-CoV-2 infection severity is influenced by a dysregulation of the immune response.

A drastic lymphopenia with reduction in numbers of CD4 + T cells, CD8 + T cells, B cells, and natural killer (NK) cells is a common feature in patients with severe SARS-CoV-2 infection, but not in patients with mild disease, 29 with neutrophil-to-lymphocyte ratio reflecting an enhanced inflammatory process and a poor prognosis. 30 AR is expressed on CD4 + T lymphocytes, CD8 + T lymphocytes, and macrophages supporting the possibility of direct action of T on these cells. 31 A worsening of clinical status was coupled not only with reduced T level, but also with increased LH levels, even though the latter association did not maintain significance when the four groups were compared, thus supporting the presence of a primary hypogonadism. Accordingly, an orchitis-like syndrome has been hypothesized in SARS-CoV-2 men. 32 Several pathogenic mechanisms occurring during the SARS-CoV-2 infection might be responsible for the impairment of testicular function. First, ACE2 is highly expressed within the human testis, being a constitutive product of adult-type Leydig cells. 33, 34 Because angiotensin II reduced both basal and LHstimulated testosterone synthesis by Leydig cells, ACE2 has been hypothesized to modulate the steroidogenic activity of these cells and to shield testis by limiting angiotensin II detrimental effects. 35, 36 ACE2 on Leydig cells could also alter local microvascular flow and permeability 33 and favoring inflammation. 37 Moreover, increased LH levels could contribute further to testicular alterations by modulating testicular blood flow, endothelial cell permeability, and fluid accumulation within the testis. 38 Finally, in our study, SARS-CoV-2 men are affected by a severe form of hypogonadism associated with a primary testicular impairment. A number of well-designed longitudinal studies have shown that late-onset hypogonadism (LOH) represents a common clinical entity among aging males. In most men, there is a slow decline in serum TT levels with aging, even in the absence of disease. 39 However, in our cohort of men T level was sharply reduced without showing any significant age-dependent modulation, further supporting the view of a potential direct effect of SARS-CoV-2 on testicular function.

A study on 6 men (mean age 39 years; ranged from 20 to 58 years old) has previously reported LH and T levels in SARS men. 35 The authors compared SARS men with age-matched healthy men, showing a rise of LH similar to our study, but associated with normal T level. 37 Our study is the first to report novel evidence of a clear-cut reduction in T level along with an LH increase in SARS-CoV-2 men; however, while TT and cFT were associated with a higher risk of clinical deterioration, increased LH levels tended to have a similar relationship, without reaching a statistical significance. We would like to recognize that low serum T levels might also be a consequence and not a reason for the patients' condition. However, the causal effect relationship could only be tested by randomized trials with testosterone treatment vs placebo in hypogonadal men upon submission to the ICU. Also, the presence of either a psychiatric disorder or cardiac arrhythmias was associated with a higher risk of a poor prognosis, whereas obese patients were paradoxically less prone to worse outcomes.

Some limitations should be recognized. Firstly, the sample size is relatively small with a limited number of adverse outcomes. However, a small sample size represents a concern for lack of significant associations, more than for significant ones. Secondly, six patients were still in charge of the RICU at the time of the present analysis;

hence, we do not know their definitive outcome. However, the days passed in RICU are comparable among the groups. Unfortunately, information on the onset of infection before RICU was not collected.

Moreover, this study lacks of a control group of patients not affected by SARS-CoV-2 but hospitalized and evaluated in the same way. This comparison could have strengthen our study as the prevalence of hypogonadism in elderly male patients admitted to the hospital for acute illness is reported to be close to 50%. 16, 17 Finally, T was assessed only one time and not by the gold standard method (such as mass spectrometry, which was not available in this clinical setting) but it was measured by a commercially available immunoassay used in a high-volume hospital and undergoing quality control programs.

Free T was calculated rather than measured.

Our study demonstrated that, besides a clear pattern of inflammatory, hematologic, biochemical, and immune biomarker abnormalities, lower TT and cFT levels enable significant discrimination between SARS-CoV-2 patients with or without poor clinical outcomes. Therefore, both TT and cFT level assessment may potentially aid in terms of risk stratification modeling to predict severe and even fatal SARS-CoV-2 infection. To the best of our knowledge, this is the first study unraveling the prognostic role of T levels toward either severity or mortality associated with SARS-CoV-2 pneumonia.

Whether testosterone therapy could theoretically be beneficial, mitigating the steep increase in clinical deterioration among severely hypogonadal SARS-CoV-2 men may be worth further studies.

Risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in Wuhan

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Diabetes is a risk factor for the progression and prognosis of COVID-19

Prediction models for diagnosis and prognosis of covid-19 infection: systematic review and critical appraisal

Similarity in case fatality rates (CFR) of COVID-19/SARS-COV-2 in Italy and China

COVID-19: the gendered impacts of the outbreak

SARS-CoV-2, Testosterone and frailty in males (PROTEGGIMI): A multidimensional research project

Testosterone, a key hormone in the context of COVID-19 pandemic

The complex association between metabolic syndrome and male hypogonadism

Endogenous testosterone levels and cardiovascular risk: meta-analysis of observational studies

Individual Variation of the SARS-CoV2 Receptor ACE2 Gene Expression and Regulation

Assessing ACE2 expression patterns in lung tissues in the pathogenesis of COVID-19

Cardiovascular disease, drug therapy, and mortality in Covid-19

Characteristics of secondary, primary, and compensated hypogonadism in aging men: evidence from the European Male Ageing Study

Prevalence of symptomatic androgen deficiency in men

Hypogonadism in aged hospitalized male patients: prevalence and clinical outcome

Associations between low serum testosterone and all-cause mortality and infection-related hospitalization in male hemodialysis patients: a prospective cohort study

Acute respiratory distress syndrome: the Berlin Definition

A critical evaluation of simple methods for the estimation of free testosterone in serum

Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan. China

Clinical review: Endogenous testosterone and mortality in men: a systematic review and meta-analysis

European Academy of Andrology (EAA) guidelines* on investigation, treatment and monitoring of functional hypogonadism in males

Antiinflammatory effect of androgen receptor activation in human benign prostatic hyperplasia cells

Testosterone protects from metabolic syndrome-associated prostate inflammation: an experimental study in rabbit

Testosterone: a metabolic hormone in health and disease

The relationship between circulating testosterone and inflammatory cytokines in men

Testosterone replacement effectively inhibits the development of experimental autoimmune orchitis in rats: evidence for a direct role of testosterone on regulatory T cell expansion

Fat boosts, while androgen receptor activation counteracts, BPH-associated prostate inflammation

COVID-19: immunopathology and its implications for therapy

Neutrophil-to-lymphocyte ratio and lymphocyte-to-C-reactive protein ratio in patients with severe coronavirus disease 2019 (COVID-19): A meta-analysis

Testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production

The SIAMS-ED trial: A national, independent, multicentre study on cardiometabolic and hormonal impairment of men with erectile dysfunction treated with vardenafil

The novel angiotensin-converting enzyme (ACE) homolog, ACE2, is selectively expressed by adult Leydig cells of the testis

scRNA-seq profiling of human testes reveals the presence of the ACE2 receptor, a target for SARS-CoV-2 infection in spermatogonia, leydig and sertoli cells

Multistep regulation of Leydig cell function

Angiotensin II receptors and inhibitory actions in Leydig cells

Orchitis: a complication of severe acute respiratory syndrome (SARS)

Relationship between human chorionic gonadotrophin-induced changes in testicular microcirculation and the formation of testicular interstitial fluid

Hypothalamic-pituitary-testicular axis disruptions in older men are differentially linked to age and modifiable risk factors: the European Male Aging Study

Low testosterone levels predict clinical adverse outcomes in SARS-CoV-2 pneumonia patients

The authors declare that there are no conflicts of interest in connection with this article.