Sex hormone and metabolic dysregulations are associated with critical illness in male Covid-19 patients

Males develop more severe SARS-CoV-2 infection related disease outcome than females. Herein, sex hormones were repeatedly proposed to play an important role in Covid-19 pathophysiology and immunity. However, it is yet unclear whether sex hormones are associated with Covid-19 outcome in males and females. In this study, we analyzed sex hormones, cytokine and chemokine responses as well as performed a large profile analysis of 600 metabolites in critically-ill male and female Covid-19 patients in comparison to healthy controls and patients with coronary heart diseases as a prime Covid-19 comorbidity. We here show that dysregulated sex hormones, IFN-γ levels and unique metabolic signatures are associated with critical illness in Covid-19 patients. Both, male and female Covid-19 patients, present elevated estradiol levels which positively correlates with IFN-γ levels. Male Covid-19 patients additionally display severe testosterone and triglyceride deficiencies as compared to female patients and healthy controls. Our results suggest that male Covid-19 patients suffer from multiple metabolic disorders, which may lead to higher risk for fatal outcome. These findings will help to understand molecular pathways involved in Covid-19 pathophysiology.

The current SARS-CoV-2 pandemic continues taking its toll on human health with currently 1.61 4 6 million lives lost worldwide (as of 14 th December 2020). SARS-CoV-2 was first reported in humans 4 7 in December 2019 in Wuhan, China 1 . On February 11 th 2020, the World Health Organization To shed light on the origin of severe testosterone deficiency in male COVID-19 patients observed in 1 4 6 this study, we further analyzed related hormones ( testosterone-to-estradiol aromatization in Covid-19 patients are required. We furthermore observed 2 8 0 that some female Covid-19 patients also presented elevated testosterone levels albeit statistically not 2 8 1 significant. In our female Covid-19 cohort, all except one patient, were postmenopausal. However, 2 8 2 the low female Covid-19 cohort size in our study is a potential limitation with respect to conclusions 2 8 3 on female Covid-19 outcome. This was due to the fact that more men than women were admitted to 2 8 4 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.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 this version posted December 20, 2020.  Second, we found that similar to males, female Covid-19 patients also present elevated estradiol 2 9 0 levels. This is in contrast to the findings in the female SARS-CoV-2 golden hamster model using 2 9 1 young, lean and comorbidity-free animals 4 . Thus, future in depth investigations are required to 2 9 2 understand the role of estradiol in postmenopausal Covid-19 patients. One possible investigation line 2 9 3 might be to analyze whether elevated female testosterone levels might provide a substrate for the 2 9 4 CYP19A1 aromatase resulting in elevated estradiol levels in at-risk postmenopausal women upon 2 9 5 SARS-CoV-2 infection. However, elevated estradiol levels seem to be a risk factor for severe Covid- suffer from severely depleted testosterone and dihydrotestosterone levels, which might present an 2 9 8 additional hit putting them at increased risk compared to females. Third, by analyzing 27 different cytokines/chemokines and correlating their levels to testosterone or 3 0 0 estradiol levels using regression analysis, we identified interferon-γ (IFN-γ) as a key cytokine that 3 0 1 positively correlated with estradiol levels. This finding is of highest importance given that IFN-γ is 3 0 2 the key cytokine responsible for macrophage activation 14,15 . IFN-γ primes macrophages that are Macrophages contain membrane-bound as well as nuclear androgen-and estrogen-receptors. Thus, it 3 0 7 will be of high interest to dissect the role of sex hormones and IFN-γ in orchestrating e.g.  Fourth, analyzing over 600 metabolites representing various biochemical pathways, we identified 3 1 1 lower triglycerides as a unique feature in critically-ill Covid-19 patients. Triglycerides are usually 3 1 2 elevated in patients with sepsis. Inflammation-related inhibition of lipoprotein lipase due to 3 1 3 hyperglycaemia and hyperinsulinaemia produces upregulated hepatic triglyceride production. In 3 1 4 addition, disturbances of the mitochondrial fatty acid β -oxidation also lead to higher levels of 3 1 5 triglycerides 28 . The underlying cause of reduced triglycerides and altered bile acid profiles in male 3 1 6 All rights reserved. No reuse allowed without permission. 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 this version posted December 20, 2020. ;https://doi.org/10.1101https://doi.org/10. /2020 Covid-19 males is not clear but indicate an involvement of the liver, which is the responsible organ 3 1 7 for the secretion of triglyceride-rich lipoproteins and bile acids 29 . In future, the disturbed lipid 3 1 8 metabolism need particular attention given that comorbidities, such as obesity are usually associated 3 1 9 with elevated triglyceride levels 24 . Interestingly, some of these species have a key role in regulating 3 2 0 macrophage functions 30 , as lipids are not only a source of energy for macrophages but they are Collectively, our findings herein highlight that metabolic dysregulations pose a hallmark of severe 3 2 7 Covid-19 outcome. In particular, monitoring sex hormone and triglyceride levels might offer new 3 2 8 diagnostic opportunities for patient management and mitigation of early intervention strategies.

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All rights reserved. No reuse allowed without permission. 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 this version posted December 20, 2020. ;https://doi.org/10.1101https://doi.org/10. /2020  All rights reserved. No reuse allowed without permission. 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 this version posted December 20, 2020. ;https://doi.org/10.1101https://doi.org/10. /2020  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 this version posted December 20, 2020. ; https://doi.org/10. 1101/2020  All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Distress Syndrome (ARDS) using the Berlin definition 9 . Additionally, we recorded antiviral 3 8 2 treatment, supportive and experimental COVID-19 therapies, the need for mechanical ventilation and 3 8 3 extracorporeal membrane oxygenation 10 . Furthermore, we followed the course of the patients and 3 8 4 recorded discharge or death. globulin, thyroid-stimulating hormone, free triiodothyronine (T3), free thyroxine (T4), luteinizing 3 9 0 hormone, follicle-stimulating hormone and cortisol), as well as testosterone, dihydrotestosterone, LH, FSH, TT, E2 and SHBG were analyzed by electro-chemiluminescence immunoassay (ECLIA).

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Cytokine and chemokine levels were measured using a Bio-Plex Pro TM multiplex assay  acid in water (solvent A) and 0.2 % formic acid in acetonitrile (solvent B) as eluent system. The

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The copyright holder for this this version posted December 20, 2020. ; https://doi.org/10.1101/2020.05.07.20073817 doi: medRxiv preprint gradient profile was in accordance with the protocol of the manufacturer. FIA solvent was methanol 4 4 6 with a modifier, which was provided by the kit manufacturer. Data analysis of the UHPLC results 4 4 7 was based on a seven-point curve or one-point calibration and internal standard normalization. The bioinformatics analysis of the raw metabolite quantifications was done mostly in concordance to eliminate any gender-specific variation of baseline metabolite levels between male and female, we are referred to as "scaled" throughout this article. Differential analysis between male and female 4 5 8 Covid-19 samples was done using the aforementioned scaled values, applying an unpaired two-sided 4 5 9 t-test followed by multiple testing correction (false-discovery rate or FDR). Pathway analysis was 4 6 0 done using the KEGG database as provided by the Metaboanalyst implementation, using a 4 6 1 hypergeometric test. Statistical evaluation for quantitative data was performed with two-way-ANOVA including the 4 6 5 cohort and sex as independent variable as well as its interaction. For non-normal data unpaired 4 6 6 Mann-Whitney or Kruskal-Wallis test ignoring any multiple comparisons were used. Statistical 4 6 7 significance was defined as P < 0.05 (* P < 0.05, ** P < 0.01 and *** P < 0.001.  All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. design. GG wrote the manuscript. All authors revised the manuscript. The study funder had no role in study design, data collection, data analysis, data interpretation, or 4 8 7 writing of the report. The corresponding author had full access to all the data in the study and had  All authors declare no competing interests. All rights reserved. No reuse allowed without permission. 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 this version posted December 20, 2020. Medical Center Hamburg-Eppendorf within the period 9 th March-9 th Decemebr 2020. Patients were 5 0 0 further subdivided in surving (males, n=63; females, n=32) and deceased (males, n=25; females, 5 0 1 n=16) patients.   Testosterone (a-c), dihydrotestosterone (d-f), estradiol (g-i) and estrone (j-l) levels were measured in   parametric Student's t-test (Mann-Whitney test). Statistical significance was defined as P < 0.05 5 2 4 (* P < 0.05, ** P < 0.01). Values are shown as median and interquartile range. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  Correlation of IFN-γ levels in male and female COVID-19 patients to sex hormone levels.

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Testosterone and estrogen levels were measured in plasma of COVID-19 male (n=39) and female    unpaired two-sided t-test followed by multiple testing correction was performed. Significant outliers or delta male -female > 0.1699 (reflecting a 12.5% difference). Pathway analysis was performed for male versus female (f); hypergeometric test was performed against the KEGG metabolite database 32 5 6 0 All rights reserved. No reuse allowed without permission. preprint (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. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. ( 2 0 0 1 ) . 6 5 9 6 6 0 All rights reserved. No reuse allowed without permission. preprint (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. 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 this version posted December 20, 2020.   All rights reserved. No reuse allowed without permission. 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 this version posted December 20, 2020. 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 this version posted December 20, 2020. ; https://doi.org/10.1101/2020.05.07.20073817 doi: medRxiv preprint  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 this version posted December 20, 2020.   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 this version posted December 20, 2020. ; https://doi.org/10.1101/2020.05.07.20073817 doi: medRxiv preprint  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 this version posted December 20, 2020. ; https://doi.org/10.1101/2020.05.07.20073817 doi: medRxiv preprint Figure S1 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.