key: cord-0313431-tbqd99ab authors: Toscano-Guerra, E.; Martinez-Gallo, M.; Arrese-Munoz, I.; Gine, A.; Diaz, N.; Gabriel-Medina, P.; Riveiro-Barciela, M.; Labrador-Horrillo, M.; Martinez-Valle, F.; Hernandez-Gonzalez, M.; Rodriguez-Frias, F.; Pujol Borrell, R.; Ferrer, R.; Thomson, T. M.; Paciucci, R. title: Recovery of serum testosterone levels is an accurate predictor of survival from COVID-19 in male patients date: 2021-07-01 journal: nan DOI: 10.1101/2021.06.29.21259693 sha: 6ecf1848db496b9eef3ff937bb790cb64c4f5f16 doc_id: 313431 cord_uid: tbqd99ab Infection with SARS-CoV-2 portends a broad range of outcomes, from a majority of asymptomatic cases or mild clinical courses to a lethal disease. Robust correlates of severe COVID-19 include old age, male sex, poverty and co-morbidities such as obesity, diabetes or cardiovascular disease. A precise knowledge is still lacking of the molecular and biological mechanisms that may explain the association of severe disease with male sex. Here, we show that testosterone trajectories are highly accurate individual predictors (AUC of ROC = 0.928, p < 0.0001) of survival in male COVID-19 patients. Longitudinal determinations of blood levels of luteinizing hormone (LH) and androstenedione suggest an early modest inhibition of the central LH-androgen biosynthesis axis in a majority of patients, followed by either full recovery in survivors or a peripheral failure in lethal cases. Moreover, failure to reinstate physiological testosterone levels was associated with evidence of impaired T helper differentiation and decrease of non-classical monocytes. The strong association of recovery or failure to reinstate testosterone levels with survival or death from COVID-19 in male patients is suggestive of a significant role of testosterone status in the immune responses to COVID-19. The COVID-19 pandemic caused by the new SARS-CoV-2 virus is characterized by a diversity of clinical manifestations, including exacerbated inflammatory states accompanied with tissue and organ destruction beyond direct cytopathic effects of SARS-CoV-2. From the outset of the pandemic, it became clear that, while men and women present a similar prevalence of infection 1,2 , a higher risk of severe disease and death is significantly associated with male sex 2,3 . Similar observations come from studies of outbreaks of pathogenic coronaviruses with SARS-CoV and the Middle East respiratory syndrome (MERS-CoV), or other viral respiratory infections 4, 5 . Multiple factors have been considered to explain the sex disparity observed in the development of severe COVID-19, including differential biological and pathophysiological impacts of age and comorbidities such as cardiovascular disease, high blood pressure, chronic obstructive pulmonary disease (COPD), diabetes, obesity or active cancer 6 . Sexual differences in disease severity are also observed in racial and ethnic minority groups, disproportionately affected by COVID-19 6 . The underlying mechanisms that may account for these differences are not completely understood. As the main cellular receptor for SARS-CoV-2, ACE2, and the major viral fusogenic membrane-associated protease, TMPRSS2, are under transcriptional regulation by androgens 7 , it had been predicted that men would present a higher propensity of infection by SARS-CoV-2 and to develop more severe disease than women 7, 8 . However, men and women show comparable risks of infection 1, 2 and observational studies of male COVID-19 patients under androgendeprivation therapy have yielded contrasting results with regards to risk of developing severe COVID-19 (ref. 9, 10) . Contrariwise, there is growing evidence that severe COVID-19 in male patients is accompanied with diminished levels of circulating testosterone [11] [12] [13] , suggesting a critical role for androgens 11, 14 in preventing the innate and/or adaptive immune dysfunctions that lead to the development of severe forms of the disease [15] [16] [17] . The sexual dimorphism of immune responses to pathogens has been long recognised 18 , pointing to women having stronger antiviral mechanisms, stronger Tregulatory cells, higher numbers of group 2 innate lymphoid cells (ILC2), and superior immune-mediated tissue repair capacities as compared to men 19, 20 . In addition, sex hormones may differentially impact the frequency and severity of many autoimmune and inflammatory diseases, generally more prevalent in women than men 21, 22 . It should be noted that sex hormones may exert apparently contrasting effects. For example, an immunosuppressive role for testosterone was observed in response to influenza vaccination 23 , while testosterone supplementation following influenza infection in aged male mice, which caused decreased serum testosterone levels, reduced mortality 24 . In order to better understand the relationship between testosterone status and disease severity, we have studied a population of male and female COVID-19 patients for serum and blood biomarkers in association with disease outcome, and have performed a longitudinal analysis in a sub-cohort of male patients. We have found that male patients who survive the disease eventually reinstate physiological levels of testosterone, while those who die from COVID-19 fail to do so. As such, we show that the trajectories of serum testosterone levels are highly accurate predictors of survival [area under the curve (AUC) of receiver operating characteristic (ROC) curve = 92.8%, p < 0.0001] in male COVID-19 patients, independent of associated co-morbidities or clinical management, and superior in predictive power to blood All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint lymphocyte or neutrophil counts. Furthermore, we have found that male COVID-19 patients with a fatal outcome display a late coordinated depletion of circulating subsets of differentiated CD4+ T lymphocytes and monocytes, mirrored by a relative enrichment of undifferentiated CD4+ T cells and monocytes. With the aim of exploring factors that may underlie the worse progression of COVID- Tables 1 and 2 and Fig. SF1 and Tables ST1 and ST2 . Patients were All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint grouped according to their eventual outcomes at discharge/death into mild, moderate, severe-survivor and severe-deceased (Table ST3 ). In spite of the comparable overall age distributions in male and female patients (Fig. SF1) , a higher proportion of female patients fell into the mild and moderate outcome groups (56%) as compared to males (44%). Conversely, a higher proportion of male patients fell into the severe-survivor and severe-deceased outcome groups (56% and 58.46%, respectively) as compared to female patients (44% and 41.5%). In both male and female patients, the median age severe-deceased outcome groups were significantly older than those in mild or moderate outcome groups, as expected 25 . However, women in the severe-deceased group were significantly older than males in the same outcome group (Fig. SF1 ). As an approach to capture global patterns of association between biochemical parameters and outcomes, we applied principal component analysis (PCA), followed by Spearman multivariate correlation analysis (Fig. 1a, b) . In male patients (Fig. 1a) , we observed a clear correlation between mild or moderate outcomes with known predictors of good outcome, such as higher lymphocyte counts or hemoglobin levels, while severe outcome groups correlated with low neutrophil counts, and high IL-6, CRP, D-dimer, ferritin or LDH levels, confirming abundant prior evidence 1, 25, 26 . Patients with moderate outcomes had significantly lower serum testosterone levels compared to those with mild outcomes (Fig. 1c ), in agreement with other studies 12, 13 . Additionally, severe outcome patients had significantly lower serum testosterone levels compared to those with mild or moderate outcomes (Fig. 1c) . Interestingly, the low testosterone levels found in the admission time-point determinations for both severe outcome groups were not significantly different between survivor and deceased patients (Fig. 1c) . Furthermore, older age presented a stronger correlation All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint with a severe-deceased outcome than biochemical parameters predictive of poor outcome, such as D-dimer, ferritin, LDH or IL-6 ( Fig. 1a) , whereas the reverse was the case for correlations with a severe-survivor outcome, in which biochemical parameters were more strongly correlated with this group than age (Fig. 1a) . In female patients, PCA and multivariate analysis also highlighted significant differences between mild-moderate and severe outcome groups ( Fig. 1b and d) . Similar to male patients, the mild outcome group of female patients showed strong correlations to lymphocyte counts and hemoglobin levels, while the severe outcome groups were correlated with high IL-6, CRP, D-dimer, ferritin and LDH, (Fig. 1b and d) . Mirroring male patients, older age showed the strongest correlation to a severedeceased group in female patients (Fig. 1b) . Age, platelet counts and fibrinogen levels significantly discriminated severe-survivor from severe-deceased female patients ( Fig. 1 b and d) . The risk of ICU admission for patients with mild-moderate outcomes was assessed by odds ratio (OR) estimates and logistic regression analysis. In male patients, the most significant OR of ICU admission were found for IL-6 (OR 12 25, 27 . A significant OR was also found for testosterone (0.16, 95% CI 0.10-0.26) also in line with other studies 12, 13 . In female patients, the most significant OR of ICU admission were for IL-6 (OR 6.41, 95% CI 3.86-11.60), LDH (OR 4.86, 95% CI 2.82-9.15), aspartate aminotransferase (OR 2.58, 95% CI 1.60-4.28) and lymphocytes percentage of WBC (OR 0.34, 95% CI 0.21-0.54) (Fig. 2a) . Interestingly, testosterone levels at admission were not significantly associated with the occurrence of comorbidities in older men (Fig. SF2 ). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint The power of these parameters to predict severe disease was corroborated by multiple logistic regression analysis. The resulting receiver operating characteristic (ROC) curves yielded areas under the curve (AUC), which, in male patients, were > 0.7 (p < 0.0001) for IL-6, LDH and neutrophilia, and < 0.23 for lymphopenia and testosterone. In females, AUC of ROC curves were > 0.7 (p < 0.0001) for IL-6 and LDH (Fig. 2b) . The same parameters showed a weaker power to predict risk of death from COVID-19, with the exception of serum IL-6 levels, in both male and female patients (OR 4.45, 95% CI 2.14 -10.71 and AUC of ROC 0.7189, p = 0.0002 for males; and females had OR 4.21 and AUC of ROC 0.7123, p = 0.0014 for females) (Fig. 2c, d) . In sum, this baseline analysis reveals that critically low serum testosterone levels in male patients are a risk factor for severe COVID-19. Although other factors predictive of severity are in line with prior evidence 25, 27 , our observations also suggest that, compared to age-matched female patients, male COVID-19 patients present a higher risk of progression to a severe-critical disease, with higher levels of inflammatory markers (serum IL-6, blood neutrophil counts) and tissue damage (LDH), and more marked lymphopenia 2 . Importantly, biochemical and hematological parameters determined at or near hospital admission do not reveal strong predictive markers of death from COVID-19, with the exception of serum IL-6 levels. A significant correlation of serum testosterone levels with lymphocyte (absolute counts, r = 0.3122; percentages of WBC, r = 0.4187) and neutrophil (r = -0.3586) counts suggests that these three parameters may be mutually coupled (Fig. 2e) . Recovery of serum testosterone levels accurately predicts survival in male COVID- All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint 0.9205, 95% CI 0.8664 to 0.9747, p < 0.0001) comparisons (Fig. 3b) . Lymphocyte counts (absolute numbers per dL or percentage of WBCs) were also highly accurate predictors of outcome, as were neutrophil counts (Fig. 3b) . Interestingly, the trajectories of IL-6 or LDH, which values on admission were predictive of severity and death from COVID-19 in male patients, were not significantly different in these longitudinal comparisons (Fig. SF3 ). Age is a factor predictor of COVID-19 severity 25 . In our cohort of male patients, the testosterone trajectory slopes significantly and inversely correlated with age (r = -0.3801, p < 0.0001) (Fig. 3c) . Consistently, a majority of patients with severe deceased outcomes had low or negative testosterone trajectory slopes (Fig. 3c) . However, and interestingly, the median age of patients with severe deceased outcomes was not significantly different from the median age of patients with mildmoderate outcomes (Fig. 3c) , and a substantial proportion of patients with severe survivor outcomes were aged older than 60 (Fig. 3c) . Likewise, while the frequency of comorbidities was higher among patients with fatal outcomes as compared with those who survived severe disease, it was not significantly different from the frequency of the moderate outcome group (Fig. SF1 ). This suggests that old age, with or without accompanying comorbidities, may impact the ability of a subset of COVID-19 patients to reinstate testosterone production, coupled to a failure to recover from the disease. These results suggest a role for testosterone in deregulation of the immune response in deceased patients, in particular for the observed lymphopenia and neutrophilia. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. following the onset of COVID-19, for which several causal mechanisms may be proposed. One possible mechanism is an inhibition of the LH-androstenedione axis, previously associated with non-specific critical illness 29 and the deleterious action on the hypophysis of inflammatory cytokines 30 . A second possible mechanism may involve infection and damage by SARS-CoV-2 of testicular cells expressing ACE2, mainly Leydig cells 31, 32 . In the first scenario, acute declines in LH and adrostenedione levels would be expected, while in the latter scenario they would be either unaffected or increased for LH due to a negative feedback loop with testosterone 29 . We determined circulating LH and androstenedione levels in a longitudinal series of samples in a patient subcohort for which testosterone trajectories had been concomitantly determined, as described above. The median levels of LH were within normal ranges, independent of patient outcome (Fig. 4a) . Similarly, longitudinal LH trajectories were not significantly different between patients in the survivor vs. deceased outcomes, in stark contrast with the strongly divergent testosterone trajectories (Fig. 4b) . Nevertheless, LH levels determined in the last of the longitudinal samples showed a decline in the deceased outcome group as compared to the severe survivor group, although without reaching statistical significance. On All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint the other hand, although androstenedione levels fell within normal ranges in the majority of patients in all outcome groups and throughout the longitudinal analysis ( Fig. 4a, b) , deceased patients showed an increase as compared to the survivor outcome groups, without a concomitant increase in testosterone levels (Fig. 4a) . It should be noted that circulating androstenedione is produced mostly by adrenal glands 28 and its synthesis might be affected by the corticosteroids used to treat these patients that may block the endogenous production of cortisol, corticosterone and aldosterone, deviating the synthesis from cholesterol to androstenedione 28 . Therefore, the failure of patients with fatal outcomes to recover their physiological levels of testosterone in spite of LH and androstenedione levels within normal ranges suggests the development of an irreversible peripheral failure in the biosynthesis of testosterone in these patients. As such, an irreversible damage of Leydig cells 33 in patients with fatal outcomes could explain these observations, while a resolution of viral infection would explain the recovery of a normal production of testosterone in survivors. A number of studies have found substantial differences in immune responses to SARS-CoV-2 between male and female patients 15, 17 , although the mechanisms underlying these differences are still unclear. In order to address the relationship between testosterone trajectories, outcome and immune status in male patients, we analyzed circulating immune subpopulation repertoires in a subset of our patient cohort, in at least two independent determinations, separated by 5-20 days. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. 5a ). The subpopulations with the most significant changes in relative abundance as a function of outcome tended to correlate with serum testosterone levels sampled in the same period (1-3 days from sampling for immune repertoire analyses) (Fig. 5a) . These correlations were more evident in multivariate correlation analyses, particularly for Sample 2 (Fig. 5b) . These analyses show a generalized loss of representation of circulating CD4 + (CD4 + central memory, CD4 + effector memory, CD4 + TEMRA) and T helper (Th) T lymphocyte subpopulations (Th1, Th17, Th1-Th17, Th2) in deceased patients compared to surviving patients, accompanied with a reciprocal increased representation of naïve CD4 + T cell compartments (recent thymus emigrant (RTE) CD4 + , naïve CD4 + CD45RA + CCR7 + ) (Fig. 5b-d) . They also point to an association of monocyte differentiation with outcome, with a predominant correlation with non-classical monocytes in moderate and severe survivor patients and, conversely, with more pro-inflammatory 34 classical monocytes in deceased patients ( Fig. 5b-d) . There are additional associations of cytotoxic T cell (particularly diminished CD8 + effector memory) or B cell subpopulations (diminished transitional All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. Failure to recover from such imbalances is paralleled by a failure to reinstate physiological levels of testosterone and is associated with a lethal outcome in male patients. Numerous studies have identified prognostic markers able to discern severe COVID-19 patients, including older age, male sex, co-morbidities such as obesity, diabetes or cardiovascular disease, elevated circulating markers of inflammation, lymphopenia, neutrophilila 2, 12, 16, [25] [26] [27] 41 , or the presence of autoantibodies to class-I interferons 42 . Nomograms or scores that combine several independent parameters have been proposed as predictors of COVID-19 outcome 2,12,25 . However, relatively All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint few studies have addressed sex differences in predictive markers of disease outcome 2, 12, 25, [43] [44] [45] . Interestingly, inflammation markers, but not co-morbidities, BMI or age have been found to be associated with outcome differences between male and female COVID-19 patients 45 Our comparative analysis of biochemical and hematological parameters has revealed that both sexes share markers with significant predictive power of disease outcome, including IL-6, LDH, D-dimer, lymphopenia and neutrophilia, although the levels of these markers, and the strength of their predictive power, are consistently higher in male patients as compared to female patients. This becomes more evident when evaluating predictive markers of lethal COVID-19, which yields IL-6 and lymphocyte (percentage of total WBC) as the only two significantly predictive markers shared in both male and female patients. Other significant markers predictive of lethal COVID-19 in male, but not female, patients are LDH levels, neutrophilia and absolute lymphocyte counts, in addition to testosterone levels, which are exclusively masculine in our patient cohorts. These observations suggest that male COVID-19 patients with severe and lethal disease suffer from more deleterious underlying pathogenic and inflammatory processes than female patients with comparable clinical severity 2 , a situation also observed in other respiratory viral infections 4,5 . Interestingly, markers of inflammation (IL-6, CRP) or tissue damage (LDH), with good outcome predictive power in first sample determinations, lost their predictive power in longitudinal analyses of samples in male patients, collected up to the time of discharge or fatality. In contrast, testosterone levels, whose determinations on admission provided relatively modest outcome predictive power, corroborating other studies 11, 13 , gained remarkable levels of significance when analyzed longitudinally. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Other biochemical parameters indicative of pathological inflammatory or procoagulant states, such as elevated IL-6, CRP or D-dimer levels, eventually return to near-physiological levels in both survivors and patients with fatal outcomes. This has been observed in other studies 45, 46 , and suggests that the normalization of these factors is insufficient, per se, to abate the pathological hyperinflammation and hypercoagulation accompanying severe COVID-19 with a fatal outcome, which would require the concomitant alleviation of lymphopenia and neutrophilia. On the other hand, although different stimuli and conditions such as mechanical ventilation, muscle immobilization, severe sepsis, and multiple organ dysfunction as well as neuro/myotoxic agents may contribute to a critical status amongst patients admitted to ICU 47 , all severe patients in our study, with either survivor or deceased outcomes, were under comparable pharmacological and physical management (Tables ST1 and ST2) . A relevant factor associated with late-onset hypogonadism 48 , as well as with an irreversible failure to reinstate testosterone production after critical situations that may compromise the LH-androstenedione axis is old age 49 , which has been linked to senescent dysfunction of Leydig cells 50 . The fact that a majority of non-survivor All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint patients in our study who failed to reinstate testosterone levels are older than 60 years of age would be consistent with the senescence hypothesis. However, our cohort has more patients older than 60 who reinstated their testosterone levels and survived severe COVID-19. Therefore, either Leydig cell senescence only affects a small subset of older patients, or other mechanisms may be invoked to explain failure to restore testosterone production. Conversely, these observations also suggest that, while the reinstatement of physiological testosterone levels may be mechanistically linked to a return to lymphocyte and neutrophil homeostasis, it may not be required for the relative normalization of other inflammatory pathways, arguably driven by an unmitigated production of IL-6 and other pro-inflammatory cytokines triggered by acute viral infection 51 . In this regard, IL-6 can be produced by a broad range of cells, beyond immune cells 51 , in response, or not, to production of IL-1b or IL-18 by epithelial or endothelial cells through viral activation of the inflammasome 52 , aided by viral blunting of class I and III interferon pathways 53 . Pro-inflammatory actions of cytokines such as IL-6 are the main inducers of the production of CRP in the liver during infectious and inflammatory processes 54 , and can independently trigger the activation of coagulation pathways, explaining the rise in blood D-dimer levels 51 , as well as complement pathways, which, coupled to direct viral cytopathic effects, explains the elevated circulating levels of tissue damage markers such as LDH and a further exacerbation of a pro-coagulant state 55 . As such, an eventual control of viral replication is expected to lead to a return of these pathways to homeostasis. However, our observations suggest that sufficient and timely resolution of pathogenic hyperinflammation to prevent a lethal outcome may require the additional return to homeostasis of innate and/or adaptive immune cell dynamics and function, possibly All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint assisted in male patients by reinstatement of testosterone production. There is now a wealth of studies describing the dynamics of immune responses to acute and subacute infection with SARS-CoV-2, including multiparameter and functional analyses of circulating and tissue-associated innate and adaptive immune subpopulations 56, 57 . Some of these studies have addressed sex differences in such responses 11, 14 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint equivalent critical illness and management, makes it appealing to hypothesize a mechanistic relationship bonding these coincident phenotypes. Sex hormones have a profound influence on innate and adaptive immune system development, differentiation and response to challenge 14, 17 More specifically, androgens have a global anti-inflammatory effect 23, 59 , reflected in higher frequencies of autoimmune diseases in women or in acquired or genetically determined hypogonadism 18 , as compared to men with a normal XY chromosome complement. On the other hand, testosterone replacement therapy in hypogonadal men attenuates inflammation 60 and androgens suppress thymic precursor development 61 and promote the terminal differentiation of T cell subpopulations 62 and monocyte precursors 63 . Conversely, androgen deprivation through surgical or pharmacological castration in animal models prompts the regeneration of the thymus in aged mice, leading to a relative accumulation of naïve T cell populations (RTE and naïve T cells) 64 and classical monocytes 65 . A similar effect of androgen deprivation on T cell development and differentiation has been observed in prostate cancer patients, with an expansion of RTE and naive T cells, particularly among CD4 + cells 64, 66 . To sum up, the tight association between reinstatement of testosterone and survival from COVID-19 in male patients, along with a reversal of signs of excessive inflammation and immune dysfunction, suggests a functional role for testosterone, beyond being a mere biomarker of outcome, in such recovery. The limitations of our study include its observational nature on retrospective patients and samples, which has precluded the collection of samples at precisely equivalent time points after symptom onset for all patients. This has most notably impacted longitudinal studies of immune populations which, in the present study, was limited to two temporally separate determinations per male patient studied. A follow-up study is All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. pre-clinical animal models would be required for a robust experimental demonstration of mechanistic relationships between testosterone status (e. g., deprivation and replacement) and SARS-CoV-2 infection outcomes, which should also contemplate factors such as age. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Immunophenotyping studies of peripheral blood cells underwent a separate review and approval process (protocol number PR(AG)242/2020). Patients were classified as mild, moderate, severe-survivor and severe-deceased as per a 4-point scale (Table ST3) . Clinical data, demographics, co-morbidities, hospital admission, discharge, death dates, time from symptoms onset to hospitalization, length of hospital stay, treatments, requirement for oxygen support, and ICU requirement, were collected from the Hospital database. For reference analyses (497 patients, males and females), data were obtained at (near) hospital admission date. For longitudinal analysis, a sub-cohort (114) of male patients were analyzed throughout the hospitalization. Serum biochemical variables were measured by automated analyzers at the Core Laboratory Facility at the Biochemistry Service of the Vall d'Hebron Hospital. Serum All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. (Table ST4) : All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. DCs were studied selecting the population negative for the following markers: CD3, CD14, CD16, CD19, CD20, and CD56. High expression of HLA-DR and CD11c and CD123 was used to identify plasmacytoid DCs (HLA-DR + CD123 + ) and myeloid DCs (HLA-DR + CD11c + ). Panel 6: Recent thymic emigrant cells (RTEs) were studied using CD3, CD4, CD27, CD31, CD45RA, and CD62L expression. Peripheral whole blood (50 μL) was All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Longitudinal determinations (≥ 3 samples) of serum LH, androstenedione and testosterone levels, analysed as in Fig. 3 . Comparisons of trajectories (linear regression slopes) were performed by two-way ANOVA. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Serum testosterone (ng/dL) Serum testosterone (ng/dL) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.29.21259693 doi: medRxiv preprint Gender Differences in Patients With COVID-19: Focus on Severity and Mortality. Front Public Health 8 Male sex identified by global COVID-19 meta-analysis as a risk factor for death and ITU admission Baseline Characteristics and Outcomes of 1591 Patients Infected With SARS-CoV-2 Admitted to ICUs of the Lombardy Region Do men have a higher case fatality rate of severe acute respiratory syndrome than women do? 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in middle-aged and elderly men Pulsatile iv infusion of recombinant human LH in leuprolide-suppressed men unmasks impoverished Leydig-cell secretory responsiveness to midphysiological LH drive in the aging male FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice The Role of Interleukin 6 During Viral Infections Inflammasomes and Pyroptosis as Therapeutic Targets for COVID-19 COVID-19 and the human innate immune system IL-6 in inflammation, immunity, and disease Complement Activation in the Disease Course of Coronavirus Disease 2019 and Its Effects on Clinical Outcomes Different Innate and Adaptive Immune Responses to SARS-CoV-2 Infection of Asymptomatic, Mild, and Severe Cases Single-cell landscape of immunological responses in patients with COVID-19 Human Monocyte Subsets and Phenotypes in Major Chronic Inflammatory Diseases Enhanced immunological response by dendritic cells in male hypogonadism Androgen receptor influences on body defense system via modulation of innate and adaptive immune systems: lessons from conditional AR knockout mice An Inconvenient Variable: Sex Hormones and Their Impact on T Cell Responses Androgen Receptor Signaling Positively Regulates Monocytic Development Activation of Thymic Regeneration in Mice and Humans following Androgen Blockade Regeneration of dendritic cells in aged mice Prostate cancer patients on androgen deprivation therapy develop persistent changes in adaptive immune responses The authors wish to thank A. Sánchez Montalvá for advice and the Vall d'HebronBiochemical Service staff for technical support. This study was funded by grants from the Ministerio de Ciencia e Innovación