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. 2022 Oct 17;11:e79720. doi: 10.7554/eLife.79720

Genetically predicted high IGF-1 levels showed protective effects on COVID-19 susceptibility and hospitalization: a Mendelian randomisation study with data from 60 studies across 25 countries

Xinxuan Li 1, Yajing Zhou 1, Shuai Yuan 1,2, Xuan Zhou 1, Lijuan Wang 1, Jing Sun 1, Lili Yu 1, Jinghan Zhu 3, Han Zhang 1, Nan Yang 1, Shuhui Dai 1, Peige Song 4, Susanna C Larsson 2,5, Evropi Theodoratou 6,7,†, Yimin Zhu 1,†, Xue Li 1,†,✉
Editors: Evangelos J Giamarellos-Bourboulis8, Jos W van der Meer9
PMCID: PMC9576268  PMID: 36250974

Abstract

Background:

Epidemiological studies observed gender differences in COVID-19 outcomes, however, whether sex hormone plays a causal in COVID-19 risk remains unclear. This study aimed to examine associations of sex hormone, sex hormones-binding globulin (SHBG), insulin-like growth factor-1 (IGF-1), and COVID-19 risk.

Methods:

Two-sample Mendelian randomization (TSMR) study was performed to explore the causal associations between testosterone, estrogen, SHBG, IGF-1, and the risk of COVID-19 (susceptibility, hospitalization, and severity) using genome-wide association study (GWAS) summary level data from the COVID-19 Host Genetics Initiative (N=1,348,701). Random-effects inverse variance weighted (IVW) MR approach was used as the primary MR method and the weighted median, MR-Egger, and MR Pleiotropy RESidual Sum and Outlier (MR-PRESSO) test were conducted as sensitivity analyses.

Results:

Higher genetically predicted IGF-1 levels have nominally significant association with reduced risk of COVID-19 susceptibility and hospitalization. For one standard deviation increase in genetically predicted IGF-1 levels, the odds ratio was 0.77 (95% confidence interval [CI], 0.61–0.97, p=0.027) for COVID-19 susceptibility, 0.62 (95% CI: 0.25–0.51, p=0.018) for COVID-19 hospitalization, and 0.85 (95% CI: 0.52–1.38, p=0.513) for COVID-19 severity. There was no evidence that testosterone, estrogen, and SHBG are associated with the risk of COVID-19 susceptibility, hospitalization, and severity in either overall or sex-stratified TSMR analysis.

Conclusions:

Our study indicated that genetically predicted high IGF-1 levels were associated with decrease the risk of COVID-19 susceptibility and hospitalization, but these associations did not survive the Bonferroni correction of multiple testing. Further studies are needed to validate the findings and explore whether IGF-1 could be a potential intervention target to reduce COVID-19 risk.

Funding:

We acknowledge support from NSFC (LR22H260001), CRUK (C31250/A22804), SHLF (Hjärt-Lungfonden, 20210351), VR (Vetenskapsrådet, 2019-00977), and SCI (Cancerfonden).

Research organism: Viruses

Introduction

The COVID-19 pandemic has emerged as the most important health concern across the globe since December 2019. A notable finding that has been noted in many affected countries is a male predominance of COVID-19-related hospitalization and death (Grasselli et al., 2020; Peckham et al., 2020). Globally, more than 60% of deaths from COVID-19 are reported in males (Richardson et al., 2020). This epidemiological pattern indicates the need for urgent public health actions, as well as for further investigations on the contributing factors of sex differences in COVID-19 risk and its underlying biological mechanisms.

Sex hormones play important roles in the immune response in which estrogen was thought to be immune boosting and testosterone to be immunosuppressing (Strope et al., 2020). Due to the higher levels of testosterone in male than female, it has been hypothesized that testosterone might be a promoter of SARS‐CoV‐2 infection and progression in males, considering the regulatory effect of androgen receptor (AR) and testosterone on the transcription of a transmembrane protease serine 2, which is a critical factor enabling cellular infection by coronaviruses, including SARS‐CoV‐2 (Peckham et al., 2020; Pozzilli and Lenzi, 2020; Cattrini et al., 2020). Estrogen has been shown not only to enhance immunological markers and response, but also to be linked to T-cell proliferation, which might be involved in the immune response to the infection of SARS-CoV-2 (Taneja, 2018). Most hormone (about 60%) is tightly bound to sex hormone-binding globulin (SHBG), which is an important regulator of the bioactivities of estrogens and testosterone (Raverot et al., 2010; Dimou et al., 2021). In addition, sex hormone signaling could also regulate the insulin-like growth factor (IGF-1) concentrations, which were also reported to be associated with acute respiratory distress syndrome (Ahasic et al., 2012). It is therefore hypothesized that sex hormone and its related biomarkers might contribute to the sex difference of COVID-19 outcomes. A number of observational studies examined the associations between sex hormones and COVID-19 risk, however, the causality of these associations remains unestablished due to potential limitations of observational studies (e.g., residual confounding and reverse causality) and lack of high-quality data from randomized trials (Tsang et al., 2016).

Mendelian randomization (MR) analysis is an epidemiological approach that can strengthen the casual inference by utilizing genetic variants as instrumental variables to mimic biological effects of related biomarkers (Burgess and Thompson, 2015). Here, we conducted a two-sample MR (TSMR) study to explore the causal associations testosterone, estrogen, SHBG, and IGF-1 with the risk of COVID-19 (susceptibility, hospitalization, and severity) using genome-wide association study (GWAS) summary level data from the COVID-19 Host Genetics Initiative (COVID-19 HGI). Sex-stratified MR analyses for testosterone and estradiol were further performed to explore the associations in males and females separately.

Materials and methods

Study design

We firstly conducted a TSMR analysis to explore the causal links between testosterone, estrogen, SHBG, IGF-1, and the risk of COVID-19 (susceptibility, hospitalization, and severity), based on GWAS summary level data from COVID-19 HGI. We then performed sex-stratified MR analysis to further examine the associations between genetically determined circulating levels of testosterone and estrogen and COVID-19 outcomes in males and females separately. The design of this study is explained in Figure 1.

Figure 1. Overall study design.

Figure 1.

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Abbreviation: IGF-1, insulin-like growth factor-1; GWAS, genome-wide association study; SNP, single-nucleotide polymorphism; LD, linkage disequilibrium; IVW, inverse variance weighting; MR, Mendelian randomization.

Genetic instruments of testosterone, estradiol, SHBG, and IGF-1

Single-nucleotide polymorphisms (SNPs) associated with testosterone, estradiol, SHBG, and IGF-1 levels were identified from genome-wide association analyses in up to 425,097 participants of European ancestry (Ruth et al., 2020; Sinnott-Armstrong et al., 2021). Sex-stratified SNPs related to estradiol were obtained from a GWAS including 147,690 males and 163,985 females in UK Biobank (Schmitz et al., 2021). We restricted the analysis to SNPs in linkage equilibrium which were identified in the relevant GWAS at p<5 × 10−8 clumped on r2=0.01 within 10,000 kb using the 1000 genomes reference panel (Hemani et al., 2018) to ensure sufficient statistical effectiveness. Among those pairs of SNPs that had LD r2 above the specified threshold (r2 = 0.01), only the SNP with the lower p value would be retained. SNPs absent from the LD reference panel were also removed. To test whether there was a weak instrumental variable bias, namely genetic variants selected as instrumental variables had a weak association with exposure, we calculated the F statistic if it is much greater than 10 for the instrument-exposure association, the possibility of weak instrumental variable bias is small. These analyses were conducted using the R package ‘TwoSampleMR’ (Yavorska and Burgess, 2017). Consequently, a total of 320, 316, 7, and 18 SNPs were used as instrumental variables for SHBG, testosterone, estradiol, and IGF-1, respectively. Given that genetic variants predicting testosterone and estradiol levels differ for men and women, we selected sex-specific SNPs for testosterone (130 SNPs in males, 151 SNPs in females) and estradiol (10 SNPs in males and females) separately for MR sensitivity analyses. Detailed information on the genetic instruments were provided in Supplementary file 1a-d. We used the STROBE case-control checklist when writing our report (von Elm et al., 2014).

Data source from COVID-19 HGI

We obtained the summary level data of COVID-19 susceptibility, hospitalization, and severity from the COVID-19-HGI GWAS meta-analyses of data across 60 studies from 25 countries (Round 5, European population) where UK Biobank data were excluded (COVID-19 Host Genetics Initiative, 2020). The HGI dataset included 1,348,701 participants (32,494 laboratory-confirmed cases of SARS-CoV-2 infection and 1,316,207 population controls) for COVID-19 susceptibility, 1,557,411 participants (8316 hospitalized COVID-19 patients and 1,549,095 population controls) for COVID-19 hospitalization, and 1,059,456 participants (4792 very severe respiratory-confirmed COVID-19 cases and 1,054,664 controls) for COVID-19 severity. COVID-19-HGI defined very severe respiratory-confirmed COVID-19 cases as patients hospitalized for laboratory-confirmed SARS-CoV-2 infection who died or were given respiratory support. The characteristics of the participants are shown in Table 1.

Table 1. Sources of data for Mendelian randomization analysis in COVID-19 HGI.

Phenotype Participants
Susceptibility Meta-analysis of 35 GWAS performed in individuals of European ancestry
 Cases: 32,494 individuals with COVID-19 by laboratory confirmation, chart review, or self-report
 Controls: 1,316,207 individuals without confirmation or history of COVID-19
Hospitalization Meta-analysis of 23 GWAS performed in individuals of European ancestry
 Cases: 8316 hospitalized individuals with COVID-19
 Controls: 1,549,095 individuals without confirmation or history of COVID-19
Severity Meta-analysis of 14 GWAS performed in individuals of European ancestry
 Cases: 4792 SARS-CoV-2 infected hospitalized individuals who died or required respiratory support (intubation, CPAP, BiPAP, continuous external negative pressure, high flow nasal cannula).
 Controls:1,054,664 individuals without confirmation or history of COVID-19
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Notes: COVID-19 outcomes are taken from the COVID-19 HGI.

HGI, Host Genetics Initiative; GWAS, genome-wide association study; UKB, UK Biobank; CPAP, continuous positive airway pressure ventilation; BiPAP, bilevel positive airway pressure ventilation.

TSMR analyses

We applied the inverse variance weighted (IVW) method under the random-effects model as the primary MR analysis. We performed sensitivity analyses, including the weighted median, MR-Egger regression, leave-one-out analysis, and MR Pleiotropy RESidual Sum and Outlier (MR-PRESSO) methods, to examine the consistency of associations and to detect and correct for potential pleiotropy. The weighted median method was performed to provide unbiased causal estimates if at least 50% instrumental variables were valid (Bowden et al., 2016). MR-Egger regression was used to observe and correct potential directional pleiotropy, which was assessed by its intercept test (Bowden et al., 2015). MR-PRESSO method can detect SNP outliers and estimate the association after removal of these outliers. The differences in estimates between before and after outlier removal were examined by the embedded distortion test (Wu et al., 2020). Cochrane’s Q value was used to assess the heterogeneity among estimates of genetic instruments and the p value for intercept in MR-Egger was used to detect horizontal pleiotropy (Bowden et al., 2015). All statistical analyses were two-sided and performed in R 4.0.4 software using the R package TwoSampleMR and MR-PRESSO (Yavorska and Burgess, 2017).

Sensitivity analyses

We additionally used the SNP rs7173595 in CYP19A1 gene, which encodes aromatase, an enzyme that converts androgens to estrogens. Rs7173595 has previously been shown to be strongly associated with serum E2 levels in GWAS of men (Ruth et al., 2020; Eriksson et al., 2018) and postmenopausal women (Thompson et al., 2016). This SNP was also associated with serum E2 in 25,502 premenopausal European women (<50 years of age and not reporting a hysterectomy or that menopause has occurred) in UK Biobank. The associations of serum E2 instrumented by rs7173595 in the CYP19A1 gene region with COVID-19 outcomes were estimated using the Wald ratio method. We further performed a sensitivity analysis using a list of genetic instruments consisting of 10 correlated SNPs (r2 < 0.4) located in the IGF-1 gene region (genomic position on build GRCh37/hg19: chromosome 12:102789652–102874341) and associated with IGF-1 levels at the genome-wide significance level. A matrix of linkage disequilibrium among these SNPs was introduced in the MR analysis model. To control potential data confounder, we selected SNPs associated with testosterone, estrogen, SHBG, and IGF-1 only, excluding SNPs associated with BMI which is thought to be a causal risk factor for COVID-19 (Freuer et al., 2021) at the threshold of 5×10–8 in European ancestry samples by querying PhenoScanner (Yavorska and Burgess, 2017). SNPs in estrogen were not excluded because their irrelevance to BMI.

Results

Table 2 presents the TSMR estimates for the associations between sex hormones, SHBG, IGF-1, and the risk of COVID-19 susceptibility, hospitalization, and severity based on the data from HGI. Higher genetically predicted IGF-1 levels have nominally significant association with reduced risk of COVID-19 susceptibility and hospitalization. For one standard deviation increase in genetically predicted IGF-1 levels, the odds ratio was 0.77 (95% confidence interval [CI], 0.61–0.97, p=0.027) for COVID-19 susceptibility, 0.62 (95% CI: 0.25–0.51, p=0.018) for COVID-19 hospitalization, and 0.85 (95% CI: 0.52–1.38, p=0.513) for COVID-19 severity. Associations of IGF-1 levels with COVID-19 susceptibility and hospitalization were not statistically significant after Bonferroni correction, albeit showing a nominal significance at p<0.05. No outlying SNPs were identified by MR-PRESSO analyses. Estimates from the MR-Egger and weighted mode analyses were in the same direction as those from the IVW analysis (Figure 2, Figure 2—figure supplement 1, Figure 2—figure supplement 2). The MR-Egger intercept p was 0.614 and 0.595 for susceptibility and hospitalization, respectively, indicating the absence of directional pleiotropy. The associations remained directionally consistent in the sensitivity analysis based on SNPs located in the IGF-1 gene region as instrumental variables with risk of COVID-19 susceptibility (OR = 0.99, 95% CI: 0.91–1.07, p=0.777), hospitalization (OR = 0.90; 95% CI: 0.74–1.10, p=0.645), and severity (OR = 1.01; 95% CI: 0.82–1.24, p=0.415) (Table 3).

Table 2. Sex hormones, SHBG, IGF-1, and COVID-19 outcomes in Mendelian randomization (MR) analyses.

Exposure Method Susceptibility Hospitalization Severity
SNPs OR (95% CI) p Effect p Heterogeneity p Intercept SNPs OR (95% CI) p Effect p Heterogeneity p Intercept SNPs OR (95% CI) p Effect p Heterogeneity p Intercept
Testosterone IVW 315 0.94 (0.83, 1.06) 0.309 0.006 – 303 0.82 (0.64, 1.04) 0.103 0.055 – 316 0.83 (0.60, 1.15) 0.256 0.041 –
MR-Egger 0.93 (0.76, 1.12) 0.430 0.005 0.860 0.79 (0.55, 1.15) 0.217 0.051 0.819 0.78 (0.48, 1.27) 0.313 0.038 0.732
Weighted median 0.89 (0.71, 1.12) 0.329 – – 0.81 (0.52, 1.28) 0.370 – – 0.71 (0.40, 1.26) 0.246 – –
Simple mode 1.13 (0.73, 1.77) 0.584 – – 0.77 (0.27, 2.20) 0.623 – – 0.44 (0.09, 2.18) 0.316 – –
Weighted mode 0.91 (0.77, 1.08) 0.300 – – 0.77 (0.52, 1.13) 0.180 – – 0.65 (0.40, 1.05) 0.081 – –
MR-PRESSO 0.94 (1.06, 0.84) – – – 0.82 (1.04, 0.65) – – – 0.83 (1.15, 0.59) – – –
SHBG IVW 319 0.91 (0.80, 1.04) 0.182 0.002 – 309 0.86 (0.66, 1.11) 0.255 0.087 – 320 0.92 (0.65, 1.29) 0.618 0.096 –
MR-Egger 0.96 (0.78, 1.18) 0.708 0.002 0.494 0.83 (0.57, 1.22) 0.352 0.081 0.818 0.92 (0.56, 1.51) 0.730 0.090 0.994
Weighted median 0.90 (0.72, 1.13) 0.360 – – 0.82 (0.52, 1.29) 0.391 – – 0.72 (0.41, 1.27) 0.255 – –
Simple mode 1.09 (0.66, 1.81) 0.735 – – 1.18 (0.40, 3.44) 0.767 – – 1.16 (0.25, 5.41) 0.850 – –
Weighted mode 0.94 (0.78, 1.14) 0.547 – – 0.81 (0.56, 1.18) 0.279 – – 0.79 (0.47, 1.33) 0.376 – –
MR-PRESSO 0.91 (1.05, 0.80) – – – 0.86 (1.11, 0.67) – – – 0.91 (1.28, 0.65) – – –
Estradiol IVW 7 0.54 (0.15, 1.94) 0.346 0.188 – 7 0.87 (0.11, 6.70) 0.895 0.769 – 7 0.50 (0.03, 7.64) 0.620 0.987 –
MR-Egger 0.73 (0.04, 14.11) 0.845 0.123 0.830 0.34 (0.00, 29.54) 0.657 0.685 0.662 0.04 (0.00, 17.04) 0.345 1.000 0.401
Weighted median 0.36 (0.10, 1.35) 0.130 – – 0.35 (0.03, 4.21) 0.407 – – 0.30 (0.01, 7.26) 0.458 – –
Simple mode 0.29 (0.03, 2.60) 0.313 – – 0.71 (0.01, 44.94) 0.875 – – 0.33 (0.00, 43.56) 0.673 - -
Weighted mode 0.34 (0.07, 1.73) 0.241 – – 0.38 (0.03, 4.81) 0.482 – – 0.29 (0.01, 9.43) 0.511 - -
MR-PRESSO 0.54 (1.94, 0.15) – – – 0.87 (3.93, 0.19) – – – 0.51 (1.52, 0.17) - - -
IGF-1 IVW 16 0.77 (0.61, 0.97) 0.027 0.175 – 16 0.62 (0.25, 0.51) 0.018 0.715 – 18 0.85 (0.52, 1.38) 0.513 0.601 -
MR-Egger 0.84 (0.56, 1.26) 0.408 0.145 0.614 0.72 (0.37, 1.38) 0.336 0.668 0.595 1.45 (0.67, 3.10) 0.358 0.758 0.096
Weighted median 0.76 (0.57, 1.02) 0.071 – – 0.75 (0.44, 1.28) 0.294 – – 0.76 (0.38, 1.53) 0.446 - -
Simple mode 0.64 (0.39, 1.05) 0.097 – – 0.66 (0.30, 1.45) 0.318 – – 0.82 (0.27, 2.47) 0.730 - -
Weighted mode 0.77 (0.58, 1.02) 0.084 – – 0.71 (0.44, 1.17) 0.199 – – 0.70 (0.35, 1.38) 0.319 - -
MR-PRESSO 0.77 (0.98, 0.61) – – – 0.62 (0.88, 0.43) – – – 0.85 (1.34, 0.54) - - -
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SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; IVW, inverse variance weighting; SHBG, sex hormones-binding globulin; IGF-1, insulin-like growth factor-1.

Figure 2. IGF-1 and COVID-19 outcomes in Mendelian randomization (MR) analyses.

Abbreviation: IGF-1, insulin-like growth factor-1; SNP, single-nucleotide polymorphism; IVW, inverse variance weighting; OR, odds ratio; CI, confidence interval.

Figure 2.

Figure 2—figure supplement 1. Leave-one-out plot for IGF-1 and COVID-19 susceptibility, hospitalization and severity in Mendelian randomization analysis.

Figure 2—figure supplement 1.

Figure 2—figure supplement 2. Funnel plot for IGF-1 and COVID-19 susceptibility, hospitalization and severity in Mendelian randomization analysis.

Figure 2—figure supplement 2.

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Table 3. Sensitive analysis between serum IGF-1 levels instrumented by 10 SNPs in the IGF-1 gene region and COVID-19 outcomes.

Method Susceptibility Hospitalization Severity
OR (95% CI) p Effect p Heterogeneity p Intercept OR (95% CI) p Effect p Heterogeneity p Intercept OR (95% CI) p Effect p Heterogeneity p Intercept
IVW 0.99 (0.91, 1.07) 0.777 0.596 – 0.90 (0.74, 1.10) 0.645 0.104 – 1.01 (0.82, 1.24) 0.415 0.437 –
MR-Egger 0.99 (0.93, 1.05) 0.732 0.541 0.527 0.97 (0.84, 1.11) 0.338 0.108 0.375 1.09 (0.92, 1.30) 0.953 0.372 0.590
Weighted median 1.01 (0.96, 1.06) 0.739 – – 0.97 (0.86, 1.10) 0.620 – – 1.05 (0.93, 1.20) 0.310 – –
Simple mode 0.98 (0.89, 1.08) 0.685 – – 1.12 (0.88, 1.43) 0.395 – – 1.16 (0.88, 1.51) 0.316 – –
Weighted mode 0.98 (0.92, 1.05) 0.596 – – 0.94 (0.82, 1.09) 0.439 – – 1.12 (0.92, 1.37) 0.279 – –
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IGF-1, insulin-like growth factor-1; SNP, single-nucleotide polymorphism; IVW, inverse variance weighting; OR, odds ratio; CI, confidence interval.

In the analyses based on data from the genetic consortia, we found no causal associations of genetically predicted testosterone with the risk of COVID-19 susceptibility (OR = 0.94; 95% CI: 0.83–1.06, p=0.309), hospitalization (OR = 0.82; 95% CI: 0.64–1.04, p=0.103), risk of severity (OR = 0.83; 95% CI: 0.60–1.15, p=0.256). Null association was also noticed between SHBG and COVID-19 susceptibility (OR = 0.91; 95% CI: 0.80–1.04, p=0.182), hospitalization (OR = 0.86; 95% CI: 0.66–1.11, p=0.255), risk of severity (OR = 0.92; 95% CI: 0.65–1.29, p=0.618). Overall, no significant associations between testosterone, estrogen, SHBG, and COVID-19 outcomes were observed from TSMR analyses. Sex-specific associations of genetically testosterone and estradiol levels with COVID-19 risk (Table 4) were still nonsignificant. We noticed that the p for intercept in MR-Egger regression analysis was more than 0.05 for both genders, and no outlier was detected. Genetic predisposition to higher serum E2 levels proxied by rs7173595 in the CYP19A1 gene was not associated with the risk of COVID-19 susceptibility (OR = 0.32; 95% CI, 0.06–1.80, p = 0.195), hospitalization (OR = 0.28; 95% CI: 0.01–6.46, p=0.426), and severity (OR = 0.22; 95% CI: 0.00–12.73, p=0.469) in females; similarly, the associations remained directionally consistent in males with susceptibility (OR = 0.37; 95% CI, 0.08–1.67, p = 0.195), hospitalization (OR = 0.33; 95% CI: 0.02–5.11, p=0.426), and severity (OR = 0.27; 95% CI: 0.01–9.26, p=0.469) (Table 5). As shown in Table 6, after removing SNPs associated with BMI, we found similar associations of genetically predicted IGF-1 levels with the risk of COVID-19 susceptibility (OR = 0.76; 95% CI: 0.60–0.96, p=0.021), hospitalization (OR = 0.61; 95% CI: 0.41–0.90, p=0.014), risk of severity (OR = 0.84; 95% CI: 0.52–1.38, p=0.497) in which we detected no moderate heterogeneity, and no indication of horizontal pleiotropy in MR-Egger, and no outlier in MR-PRESSO analyses. No causal associations of genetically predicted testosterone and SHBG with COVID-19 were found, but the directions were consistent with results in Table 2.

Table 4. Sex-specific associations of genetically testosterone and estradiol levels with COVID-19 risk.

Exposure Method Susceptibility Hospitalization Severity
Male Female Male Female Male Female
OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p
Testosterone IVW 0.96 (0.90, 1.05) 0.463 1.06 (0.97, 1.15) 0.214 0.96 (0.83, 1.10) 0.547 1.03 (0.87, 1.22) 0.731 1.07 (0.89, 1.27) 0.479 0.88 (0.69, 1.11) 0.269
MR-Egger 0.97 (0.86, 1.09) 0.644 1.04 (0.85, 1.26) 0.713 0.88 (0.71, 1.10) 0.270 1.13 (0.76, 1.69) 0.549 0.81 (0.62, 1.08) 0.152 0.68 (0.39, 1.18) 0.169
Weighted median 0.93 (0.83, 1.04) 0.184 1.06 (0.94, 1.19) 0.370 0.89 (0.72, 1.10) 0.277 1.08 (0.84, 1.39) 0.523 0.89 (0.67, 1.19) 0.438 0.81 (0.57, 1.14) 0.227
p for intercept 1.00 (1.00, 1.00) 0.998 1.00 (0.99, 1.01) 0.854 1.00 (1.00, 1.01) 0.348 1.00 (0.99, 1.01) 0.615 1.01 (1.00, 1.02) 0.017 1.01 (0.99, 1.03) 0.314
MR-PRESSO 0.97 (0.90, 1.05) 0.464 1.06 (0.97, 1.15) 0.216 0.96 (0.83, 1.10) 0.549 1.03 (0.87, 1.22) 0.732 1.07 (0.89, 1.27) 0.478 0.88 (0.69, 1.11) 0.270
Estradiol IVW 0.99 (0.89, 1.11) 0.923 0.95 (0.71, 1.26) 0.724 0.98 (0.81, 1.18) 0.826 1.04 (0.63, 1.73) 0.873 0.90 (0.71, 1.15) 0.403 1.39 (0.74, 7.15) 0.310
MR-Egger 1.00 (0.73, 1.36) 0.993 0.89 (0.59, 1.34) 0.598 0.93 (0.52, 1.67) 0.812 1.15 (0.56, 2.34) 0.719 0.61 (0.29, 6.15) 0.233 1.76 (0.74, 3.15) 0.234
Weighted median 1.05 (0.92, 1.20) 0.432 0.95 (0.68, 1.32) 0.745 0.93 (0.74, 1.16) 0.508 1.32 (0.67, 2.57) 0.422 0.88 (0.65, 1.15) 0.411 1.96 (0.81, 5.15) 0.135
p for intercept 1.00 (0.96, 1.04) 0.980 1.00 (0.99, 1.02) 0.669 1.01 (0.94, 1.08) 0.856 0.99 (0.96, 1.02) 0.707 1.05 (0.96, 0.15) 0.312 0.99 (0.95, 0.15) 0.441
MR-PRESSO 0.99 (0.89, 1.11) 0.925 0.95 (0.71, 1.26) 0.732 0.98 (0.81, 1.18) 0.831 1.04 (0.63, 1.73) 0.877 0.90 (0.71, 1.15) 0.425 1.39 (0.74, 2.63) 0.335
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OR, odds ratio; CI, confidence interval; IVW, inverse variance weighting.

Table 5. Associations of serum E2 levels instrumented by rs7173595 in the CYP19A1 gene region with COVID-19 outcomes.

Sex Phenotype beta SE OR (95% CI) p Effect
Female Susceptibility –1.14 0.88 0.32 (0.06, 1.80) 0.195
Hospitalization –1.27 1.60 0.28 (0.01, 6.46) 0.426
Severity –1.49 2.06 0.22 (0.00, 12.73) 0.469
Male Susceptibility –1.00 0.77 0.37 (0.08, 1.67) 0.195
Hospitalization –1.11 1.40 0.33 (0.02, 5.11) 0.426
Severity –1.31 1.80 0.27 (0.01, 9.26) 0.469
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E2, estradiol; OR, odds ratio; CI, confidence interval.

Table 6. Testosterone, SHBG, IGF-1, and COVID-19 outcomes in Mendelian randomization (MR) analyses adjusting BMI.

Exposure Method Susceptibility Hospitalization Severity
SNPs OR (95% CI) p Effect p Heterogeneity p Intercept SNPs OR (95% CI) p Effect p Heterogeneity p Intercept SNPs OR (95% CI) p Effect p Heterogeneity p Intercept
Testosterone IVW 306 0.95 (0.83,1.07) 0.386 0.006 – 294 0.83 (0.64,1.06) 0.134 0.041 – 307 0.84 (0.60,1.17) 0.304 0.030 –
MR-Egger 0.93 (0.77,1.13) 0.484 0.006 0.855 0.83 (0.56,1.21) 0.324 0.038 0.991 0.83 (0.50,1.37) 0.466 0.027 0.949
Weighted median 0.90 (0.72,1.12) 0.331 – – 0.82 (0.52,1.28) 0.375 – – 0.71 (0.42,1.21) 0.214 – –
Simple mode 1.13 (0.70,1.82) 0.610 – – 0.68 (0.24,1.91) 0.465 – – 0.37 (0.07,1.88) 0.229 – –
Weighted mode 0.95 (0.79,1.13) 0.540 – – 0.81 (0.56,1.17) 0.273 – – 0.65 (0.40,1.06) 0.085 – –
MR-PRESSO 0.94 (0.83,1.07) – – – 0.83 (0.64,1.06) – – – 0.83 (0.64,1.06) – – –
SHBG IVW 308 0.90 (0.79,1.04) 0.160 0.002 – 198 0.84 (0.64,1.10) 0.209 0.047 – 309 0.89 (0.62,1.26) 0.511 0.058 –
MR-Egger 0.94 (0.76,1.15) 0.538 0.001 0.663 0.81 (0.54,1.21) 0.299 0.043 0.794 0.89 (0.53,1.49) 0.666 0.054 0.978
Weighted median 0.90 (0.71,1.13) 0.356 – – 0.81 (0.52,1.28) 0.377 – – 0.72 (0.42,1.23) 0.230 – –
Simple mode 1.05 (0.60,1.84) 0.860 – – 1.25 (0.42,3.78) 0.689 – – 0.97 (0.22,4.22) 0.967 – –
Weighted mode 0.94 (0.77,1.15) 0.570 – – 0.81 (0.55,1.20) 0.295 – – 0.72 (0.43,1.22) 0.224 – –
MR-PRESSO 0.90 (0.79,1.04) – – – 0.84 (0.64,1.10) – – – 0.89 (0.62,1.26) – – –
IGF-1 IVW 15 0.76 (0.60,0.96) 0.021 0.172 – 15 0.61 (0.41,0.90) 0.014 0.688 – 17 0.84 (0.52,1.38) 0.497 0.534 –
MR-
Egger		 0.88 (0.58,1.33) 0.554 0.168 0.390 0.77 (0.39,1.50) 0.458 0.676 0.403 1.55 (0.71,3.39) 0.284 0.757
Weighted median 0.75 (0.57,0.99) 0.046 – – 0.75 (0.45,1.24) 0.260 – – 0.75 (0.38,1.48) 0.410 – –
Simple mode 0.65 (0.38,1.11) 0.135 – – 0.64 (0.30,1.37) 0.265 – – 0.75 (0.25,2.31) 0.629 – –
Weighted mode 0.76 (0.56,1.03) 0.096 – – 0.71 (0.44,1.15) 0.185 – – 0.72 (0.36,1.47) 0.383 – –
MR-PRESSO 0.76 (0.60,0.96) – – – 0.61 (0.43,0.86) – – – 0.84 (0.53,1.35) – – –
Open in a new tab

SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; IVW, inverse variance weighting; SHBG, sex hormones-binding globulin; IGF-1, insulin-like growth factor-1.

Discussion

In this study, we assessed whether there were any causal associations between sex hormone-related biomarkers and the risk of COVID-19 outcomes. We found suggestive evidence for associations between genetic liability to high IGF-1 levels and decreased risk of COVID-19 susceptibility and hospitalization. Our findings suggest a potential role of IGF-1 in COVID-19 risk and have implications for tailored treatment of COVID-19 patients.

Our MR findings were consistent with the multiple epidemiological studies that reported a nominal association between measured IGF-1 levels and COVID-19 illness. There is one observational study that demonstrated an inverse association between pre-diagnostic circulating levels of IGF-1 and COVID-19 mortality risk among COVID-19 patients in UK Biobank (Fan et al., 2021). Another observational study in Greece reported lower IGF-1 levels in critically ill COVID-19 patients compared to their counterparts with less severe disease or without COVID-19 (Ilias et al., 2021). A single-cell analysis revealed that the exhaustion of CD8+ T cells together with several cytokines including IGF-1 was associated with the pathogenesis of severe SARS-CoV-2 infection (He et al., 2021). Our MR analyses found a negative association between genetically determined high circulating IGF-1 levels and decreased risk of COVID-19 susceptibility and hospitalization, indicating IGF-1 may be a protective factor of COVID-19 risk.

IGF-1 has been found to be pro-survival/anti-aging, anti-inflammatory, and antioxidant with neuro- and hepatoprotective properties. A study by the Narasaraju group demonstrated that IGF-1 plays an important role in the repair of lung tissue by regulating the proliferation and differentiation of alveolar epithelial cells (AECs) (Narasaraju et al., 2006). Airway inflammation can be mitigated when apoptotic cells are engulfed by pulmonary epithelial cells (Juncadella et al., 2013). IGF-1 has also been shown to upregulate engulfment by professional phagocytes such as dendritic cells (Xuan et al., 2017), and inhibit IL-6 production from lipopolysaccharide-induced AECs (Wang et al., 2019). Both of these mechanisms are beneficial to the regression of local inflammation. Jakn et al. showed that IGF-1 binds to IGF-1 receptor (IGF-1R) on airway epithelial cells of non-professional phagocytic cells, which can promote the phagocytosis of microparticles by airway epithelial cells (Han et al., 2016). Transforming growth factor β1 derived from AECs activated alveolar macrophages (AMs) to secrete IGF-1 into the alveolar fluid in response to stimulation of the airway by inflammatory signals. This AM-derived IGF-1 attenuated the p38 mitogen-activated protein kinase inflammatory signal in AECs and promoted the phagocytosis of apoptotic cells by AECs. This two-way communication between AECs and AMs represents a well-tuned system for the regulation of the inflammatory response in alveoli (Mu et al., 2020). Taken together, these studies provide biological evidence supporting that IGF-1 might be an important anti-inflammatory factor in the alveolar microenvironment and thus may contribute to improve COVID-19 outcomes. More studies are required to determine whether novel therapeutic strategy targeting on IGF-1 pathway might improve COVID-19 prognosis.

IGF-1 level is regulated by estrogen and the functional interactions between estradiol and IGF-1 signaling system involve several transcriptional and posttranscriptional mechanisms. Specifically, IGF-1 can affect estrogen receptor α action by enhancing its expression and potentiating its transcriptional activity in a ligand-independent manner (Lange, 2004; Edwards et al., 1993; Shupnik, 2004). On the other hand, E2 can enhance IGF-1 signaling by upregulating the expression of IGF-1 (Umayahara et al., 1994), IGF-1R (Bartucci et al., 2001), and some IGF-1-binding proteins (Qin et al., 1999). This may explain the same direction from the IVW analysis of IGF-1, estradiol, and COVID-19 outcomes. Estrogen is found to have immune enhancing effect (Taneja, 2018) to trigger the local immune response by activating a plethora of cells such as phagocytes, dendritic cells, natural killers, and CD8+ T cells. Once these immune cells are activated, they could fight against the infection by destroying the virus and thus preventing its diffusion to the lower respiratory tract or by decreasing the viral load. Experimental tests have also reported that estradiol can affect angiotensin-converting enzyme 2 and FURIN expression, with the potential of mitigating SARS-CoV-2 infection (Glinsky, 2020). However, our study did not find any supportive evidence for the associations between estradiol and COVID-19, which might be due to the small variance of estradiol explained by genetic instruments.

Our studies showed that SHBG or testosterone may not be associated with COVID-19 outcomes, which is consistent with the research findings of Liu et al., 2022. They also observed a null causal relationship for testosterone or SHBG levels with COVID-19 outcomes in females and males. Meanwhile, epidemiologic data (Peckham et al., 2020) indicate that while men are not more predisposed to contracting COVID-19, they are more likely to develop severe illness following the infection compared with women. However, our study observed null causal relationship for testosterone levels with COVID-19 outcomes in both females and males. According to the available evidence on the role of testosterone in COVID-19, it appears that both high and low testosterone levels can be associated with poor COVID-19 outcomes (Ho et al., 2022). A study demonstrated androgen deprivation therapy (ADT) exposure was associated with a reduction in COVID-19 severity (Lee et al., 2022). By contrast, the Ohio study did not identify any protective effect of ADT on the severity of COVID-19 outcomes (Klein et al., 2021). Androgen-related treatments showed that transmembrane serine protease 2 (TMPRSS2) expression and SARS-CoV-2 entry in human lung cells have been reduced by antiandrogens (Leach et al., 2021; Deng et al., 2021; Qiao et al., 2020). Additionally, androgens have numerous immunosuppressive effects such as decreasing proinflammatory cytokine release (e.g., IFNγ and TNF) or increasing anti-inflammatory cytokine release (e.g., IL-4 and IL-10), reducing T helper 1 (Th1) and T helper 17 (Th17) cell differentiation, inducing Treg differentiation and regulating B-cell development (Olsen and Kovacs, 2011; Henze et al., 2020; Trigunaite et al., 2015). Paradoxically, these immunosuppressive effects of testosterone might be beneficial to overcome the heightened inflammatory environment that predisposes to severe COVID-19. Recent research has revealed that males with COVID-19 have lower testosterone levels (Ma et al., 2021). Another study found a negative association between total testosterone levels and biochemical markers of COVID-19 severity (Rastrelli et al., 2021). Lower testosterone concentrations were associated with higher concentrations of IL-6, CRP, IL-1 receptor antagonist, hepatocyte growth factor, and IFNγ-inducible protein 10 (Dhindsa et al., 2021). Therefore, additional research efforts need to be made to investigate the complex relationships furtherly.

The major advantage of our study is the design taking the advantages of MR approach and used several sensitivity analyses to test the robustness of the MR findings. The application of MR analysis reduces the influence of confounding factors and reverse causality so that reliable causal estimations were obtained to complement the observational findings. The potential limitations of this study also need to be acknowledged. Our study may suffer from weak instrument bias, especially within sensitivity analyses that restricted to smaller sets of genetic instruments. In TSMR, this bias would tend to make estimates closer to the null. Since there is no available data on recovery status for COVID-19 patients in UK Biobank, the current study did not take recovery as a potential competing risk into account. We could not assess the sex-specific associations in IGF-1 and COVID-19 due to no data by sex in HGI. Moreover, the MR was merely based on individuals of European ancestry. Our findings might not be generalized to other populations. It should also be noted that the study findings are based on evidence from genetic data, additional large and prospective cohort studies with available IGF-1 data and information on COVID-19 susceptibility and clinical outcomes are needed to validate the findings.

In conclusion, our study indicated that genetically predicted high IGF-1 levels were associated with decrease the risk of COVID-19 susceptibility and hospitalization, but these associations did not survive the Bonferroni correction of multiple testing. Further studies are needed to validate the findings and explore whether IGF-1 could be a potential intervention target to reduce COVID-19 risk.

Data availability statement

Data analyzed in the present study are GWAS summary statistics, which have been made publicly available. GWAS summary level data of COVID-19 HGI could be downloaded from https://www.covid19hg.org/results/. GWAS summary level data of sex hormones and IGF-1 in UK Biobank could be downloaded from GWAS catalog. All genome-wide significant SNPs have been provided in Supplementary file 1a–d. All analyses were performed using R statistical package freely available at https://cran.r-project.org/mirrors.html. The TSMR package is available at https://mrcieu.github.io/TwoSampleMR/(Hemani et al., 2020).

Acknowledgements

The authors are thankful for all the participants that contributed to the UK Biobank study. Funding Statement: The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication Funding information: This paper was supported by the following grants: Natural Science Fund for Distinguished Young Scholars of Zhejiang Province (LR22H260001) to Xue Li. CRUK Career Development Fellowship(C31250/A22804) to Evropi Theodoratou, the Swedish Heart Lung Foundation (Hjärt-Lungfonden, 20210351), the Swedish Research Council (Vetenskapsrådet, 2019-00977), and the Swedish Cancer Society (Cancerfonden) to Susanna C Larsson.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Xue Li, Email: xue.li@ed.ac.uk.

Evangelos J Giamarellos-Bourboulis, National and Kapodistrian University of Athens, Medical School, Greece.

Jos W van der Meer, Radboud University Medical Centre, Netherlands.

Funding Information

This paper was supported by the following grants:

  • Natural Science Foundation of Zhejiang Province Distinguished Young Scholars to Xue Li.

  • Cancer Research UK CRUK Career Development Fellowship to Evropi Theodoratou.

  • Swedish Cancer Foundation to Susanna C Larsson.

  • Swedish Research Council to Susanna C Larsson.

  • Swedish Heart Lung Foundation to Susanna C Larsson.

  • Science Fund for Distinguished Young Scholars of Zhejiang Province to Susanna C Larsson.

Additional information

Competing interests

No competing interests declared.

No competing interests declared.

Author contributions

Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft.

Formal analysis, Writing - original draft.

Writing - original draft, Writing – review and editing.

Resources, Formal analysis.

Formal analysis.

Validation, Methodology.

Resources, Validation.

Visualization.

Validation.

Resources, Methodology.

Resources.

Conceptualization, Writing – review and editing.

Funding acquisition, Writing – review and editing.

Supervision, Funding acquisition, Writing – review and editing.

Conceptualization, Methodology, Writing – review and editing.

Conceptualization, Data curation, Supervision, Funding acquisition, Project administration, Writing – review and editing.

Ethics

UK Biobank received ethical approval from the North West Multi-centre Research Ethics Committee, the National Information Governance Board for Health and Social Care in England and Wales, and the Community Health Index Advisory Group in Scotland. All participants provided written informed consent. All institutions contributing cohorts to the COVID-19 HGI received ethics approval from their respective research ethics review boards. The ethical permit for MR analyses based on summary-level data was unnecessary.

Additional files

MDAR checklist
Supplementary file 1. Genetic instruments for testosterone, estrogen, SHBG, and IGF-1.
elife-79720-supp1.docx (279.1KB, docx)
Reporting standard 1. Reporting checklist for case-control study.

Data availability

Data analysed in the present study are GWAS summary statistics, which have been made publicly available. GWAS summary level data of COVID-19-HGI could be downloaded from https://www.covid19hg.org/results/. GWAS summary level data of sex hormones and IGF-1 in UK biobank could be downloaded from GWAS catalog (http://ftp.ebi.ac.uk/pub/databases/gwas/summary_statistics/GCST90019001-GCST90020000/). All genome-wide significant SNPs have been provided in Supplementary Tables 1 to 4 in Supplementary file 1. All analyses were performed using R statistical package freely available at https://cran.r-project.org/mirrors.html. The Two-sample MR package is available at https://mrcieu.github.io/TwoSampleMR/.

The following previously published dataset was used:

COVID-19 Host Genetics Initiative. 2021. COVID-19 Host Genetics Initiative round 5. COVID-19 HGI. covid19hg

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Editor's evaluation

Evangelos J Giamarellos-Bourboulis 1

Using publicly available genetic data, Li and colleagues tested the association and inferred the causality of genetic variants predicted to alter the levels of testosterone, estrogen, SHBG, or IGF-1, against susceptibility, severity and outcome of SARS-Cov2 infection. The main strength of the study is the large cohort which adds to the robustness of the data.

Decision letter

Editor: Evangelos J Giamarellos-Bourboulis1
Reviewed by: Evangelos J Giamarellos-Bourboulis2

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for submitting your article "Genetically predicted high IGF-1 levels showed protective effects on COVID-19 susceptibility and hospitalization: A Mendelian Randomisation study with data from 60 studies across 25 countries" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Evangelos J Giamarellos-Bourboulis as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jos van der Meer as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Recommendations for the authors:

– The main limitation of the study is the lack of any real-world validation cohort where IGF-1 is measured.

– Another limitation is the lack of any transcriptomic data to support the findings. Can the authors describe any eQTLs to recompensate for this?

– The authors leveraged previous reports of genetic variants in association with testosterone, estrogen, SHBG, and IGF-1 levels, outside of COVID-19 cohorts. Testosterone, estrogen, SHBG, and IGF-1 levels were not measured specifically in the context of the COVID-19 Host Genetics Initiative. Secondly, no attempts were made to further validate, either experimentally or in other external cohorts, their findings related to IGF-1 levels. Thirdly, the statistics is seemingly lacking appropriate control of the false discovery rate. Although the study is a targeted meta-analysis, by examining 657 different markers one is bound to identify an association by chance. In fact, applying a Bonferroni correction to the reported p-values for IGF-1 related SNPs yields non-significant probabilities. Fourth, the methods section lacks important details, for example handling of the known "winner's curse" bias in inverse variance weighting mendelian randomization, thereby decreasing the potential for reproducibility by other groups. Lastly, the methods employed did not adequately address potential data confounders, for example treatment of patients with dexamethasone or the influence of BMI (inferred in other studies as a causal risk factor) on model estimates. Against this backdrop, the interpretation of the findings by Li and colleagues is complicated and conclusions related to genetic proxies of IGF-1 levels are unreliable.

– Based on the cited references (13 and 14), selection of SNPs in the meta-analysis is subject to a bias towards non-infectious conditions. It is important to consider that levels of testosterone, estradiol, SHBG, and IGF-1 could be substantially altered during the course of the infection, either in the acute phase or later stages. What are the levels of testosterone, estradiol, SHBG and IGF-1 in the context of viral infections, as well as crucially in COVID-19? How do those levels influence the association of the selected SNPs?

– Throughout the results, particularly in Figure 2, confidence intervals are large and indicates a potential complication due to unaddressed confounders or subgroups within the cohort. How many of the COVID-19 patients were administered corticosteroid therapy? How many were administered tocilizumab? How does treatment effect the model estimates? The authors should provide more characteristics of the COVID-19 patients with a more extensive investigation on the potential confounders and treatment effects on MR estimates. How does BMI influence model estimates and results?

– Inverse-variance weighted two-sample Mendelian randomization is indeed the most extensively used method utilizing GWAS summary stats to infer on causality of the exposure (ie. SNP variant) and outcome variables. The approach suffers from various biases that were seemingly not handled in the study herein described, for example, the winner's curse and potential pleiotropy. The authors should provide a more objective assessment of potential biases and address them accordingly.

– Notwithstanding the large sample size of the discovery cohort obtained from the host genetics in COVID-19 initiative, it is imperative to expand on the findings by ascertaining the robustness of the IFG-1 related SNPs. Do the IGF-1-related SNPs influence IGF-1 levels in the context of a viral infection? Preferably of course in COVID-19. Also, do the SNPs constitute eQTLs for IGF-1 expression in primary organs, particularly lungs, liver, and kidneys?

– The methods section needs to be expanded substantially by the addition of important details. For example, how was LD clumping performed? Using PLINK? Why was an r2 cutoff of < 0.01 used? Why not r2 < 0.001?

– The sentence on page 5 line 38 reading "postmenopausal women (22) and men(13, 23)." needs correction as it implies men were also postmenopausal.

– Page 7, line 196, the statement that reads "The cytokine storm related respiratory distress syndrome…" should be revised, avoiding the sensationalistic "cytokine storm" description of what has been reported elsewhere as merely a cytokine breeze at best.

– The Discussion on cytokine storm is arbitrary and should be omitted. The authors do not provide any evidence on the association of IGF-1 with the cytokine storm.

– This is another trial finding no association of sex hormones to COVID-19. This has to be discussed more extensively in the discussion; now discussion is more focused on IGF (the positive finding) but negative results should be put into context. Please refer also to PMID: 35577073, PMID: 35470422, PMID: 35602518

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Genetically predicted high IGF-1 levels showed protective effects on COVID-19 susceptibility and hospitalization: A Mendelian Randomisation study with data from 60 studies across 25 countries" for further consideration by eLife. Your revised article has been evaluated by Jos van der Meer (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

– The study hinges solely on MR analysis of selected SNPs used as proxies of testosterone, estradiol, SHBG, and IGF-1 levels. None of the markers were directly measured, and no information on their levels in the context of infections or COVID-19 was given. The authors argue at length that measuring the markers in the specific cohort is of limited value because their genetic causality modelling approach supersedes the need for such measurements. While MR models have indeed emerged as interesting tools to infer causality, it is also important to provide a potential mechanism linking genetic variants to the phenotype. Without direct measurements of the said markers minimize the potential to provide readers with a possible mechanism bridging genetic variant and phenotype.

– The number of tests performed in the study is equivalent to the number of SNPs that were investigated, that is, 657. Hence, the multiple-testing correction must take into account 657 tests. The authors are wrong in adjusting their probabilities just for the 4 markers, which is more attuned to p-value hacking. Therefore, my initial comments on the lack of significant findings after considering multiple-test corrections remain a concern. Hence, the conclusions pertaining to IGF-1 variants do not reflect on the author's findings. Based on this result, I suggest being more objective by stating that none of the genetic variants can be deemed as a causal variant.

eLife. 2022 Oct 17;11:e79720. doi: 10.7554/eLife.79720.sa2

Author response

Recommendations for the authors:

– The main limitation of the study is the lack of any real-world validation cohort where IGF-1 is measured.

Thanks for this insightful suggestion. We agree with the reviewer that cohort study is a straightforward and effective way to validate whether the IGF-1 levels are associated with the risk of COVID-19 outcomes. There is one observational study that demonstrated an inverse association between measured circulating levels of IGF-1 and COVID-19 mortality risk among COVID-19 patients in the UK biobank (1), however, the causality of the association remains unestablished due to the potential limitations of this study (e.g., residual confounding and reverse causality) and the shortcoming that the blood samples were taken over a decade ago in UK biobank and are unlikely to be representative of participants’ IGF-1 levels at the time of the pandemic. We therefore applied the MR design as an alternative approach to explore this association, which has several advantages. By employing genetic variants as instrumental variables to proxy the life-long time exposure, the MR approach can strengthen causal inference by minimizing unobserved confounding and diminishing reverse causality, since genetic variants are randomly assorted at conception, and thus unrelated to common confounding factors and unaffected by the onset and progression of the disease. We now highlight this proposed point as future research efforts in the Discussion section.

Discussion, page 11, line 289-291: “It should also be noted that our study findings are based on evidence from genetic data, additional large and prospective cohort studies with available IGF-1 data and information on COVID-19 susceptibility and clinical outcomes are needed to validate the findings.”

– Another limitation is the lack of any transcriptomic data to support the findings. Can the authors describe any eQTLs to recompensate for this?

Many thanks for your constructive suggestion. We agree with the reviewer that the lack of any transcriptomic data is a limitation. As suggested, we searched 157 significant single-tissue eQTLs of IGF-1 (ENSG00000017427.15) in all tissues by GTEs and identified 2 eQTLs of IGF-1 in small intestine-terminal ileum, 9 in spleen, 15 in muscle skeletal and 131 in testis, whilst we didn’t find any eQTLs of IGF-1 in blood cells or lung tissue. Considering the transcriptomic effect is different across tissues, we are therefore on the side of caution to use the eQTLs in other tissues to perform analysis.

– The authors leveraged previous reports of genetic variants in association with testosterone, estrogen, SHBG, and IGF-1 levels, outside of COVID-19 cohorts. Testosterone, estrogen, SHBG, and IGF-1 levels were not measured specifically in the context of the COVID-19 Host Genetics Initiative. Secondly, no attempts were made to further validate, either experimentally or in other external cohorts, their findings related to IGF-1 levels. Thirdly, the statistics is seemingly lacking appropriate control of the false discovery rate. Although the study is a targeted meta-analysis, by examining 657 different markers one is bound to identify an association by chance. In fact, applying a Bonferroni correction to the reported p-values for IGF-1 related SNPs yields non-significant probabilities. Fourth, the methods section lacks important details, for example handling of the known "winner's curse" bias in inverse variance weighting mendelian randomization, thereby decreasing the potential for reproducibility by other groups. Lastly, the methods employed did not adequately address potential data confounders, for example treatment of patients with dexamethasone or the influence of BMI (inferred in other studies as a causal risk factor) on model estimates. Against this backdrop, the interpretation of the findings by Li and colleagues is complicated and conclusions related to genetic proxies of IGF-1 levels are unreliable.

Many thanks for these helpful comments. Firstly, we agree with reviewer that testosterone, estrogen, SHBG, and IGF-1 levels were not measured specifically in the context of the COVID-19 Host Genetics Initiative. But it does not affect our MR analysis of causality since genetic alleles associated with the exposure are randomly assorted at conception and thus unrelated to self-selected lifestyle and environmental factors, and are not modified by disease either. Mendelian randomization (MR) study, which employs genetic variants as unbiased proxies for the risk factor, is a genetic method that can strengthen the inference on the causal nature of exposure-outcome associations by diminishing the likelihood of confounding and eliminating reverse causality in conventional observational studies (2, 3).

Secondly, we approve of the reviewer that it’s better to further validate our findings. MR study use genetic variants as instruments, which are not associated with a wide range of behavioral, social and physiological factors that confound associations with outcomes. On the other hand, they can serve as unconfounded indicators of particular trait values. Genetic variants and their effects are subject to relatively little measurement error or bias.(4) We agree it should be acknowledged as a limitation or future research effort that additional large and prospective cohort studies with available IGF-1 data and information on COVID-19 susceptibility and clinical outcomes are needed to validate the findings.

Thirdly, we totally used 657 SNPs, including 315 for testosterone, 319 for SHBG, 7 for estradiol and 16 for IGF-1. The 16 SNPs were analyzed as a whole for IGF-1. We apologize for not considering correction. When applying the Bonferroni correction for multiple testing, the adjusted P value < 0.05/4 = 0.013 (four biomarkers) should be considered as statistically significant. As suggested, we have now added Bonferroni correction in the Materials and methods section as below. When adopting the adjusted P-value threshold, we observed marginally significant association between IGF-1 and COVID-19 hospitalization with a p-value of 0.018. Any association with P-value<0.05 is now described as nominally significant, throughout the main text.

Lastly, we agree with the reviewer that addressing potential data confounders is inadequate in our study. We therefore used PhenoScanner to find out all reported associations for each SNP and exclude several SNPs to be associated with body mass index in the subsequent analysis additionally. Besides, we did not find any SNP associated with dexamethasone therapy at the threshold of 5×10-8 in European ancestry samples, as examined by the PhenoScanner. As suggested by the reviewer, we conduct the sensitive analysis and add the results in Table 6. Revisions in the manuscript are made as below.

Materials and methods section, page 5, line 155-159: “To control potential data confounder, we selected SNPs associated with testosterone, estrogen, SHBG, and IGF-1 only, excluding SNPs associated with BMI which is thought to be a causal risk factor for COVID-19(5) at the threshold of 5×10-8 in European ancestry samples by querying PhenoScanner. SNPs in estrogen were not exclude for their irrelevance to BMI.”

Results section, page 7, line 167-169: “Associations of IGF-1 levels with COVID-19 susceptibility and hospitalization were not statistically significant after Bonferroni correction, albeit showing a nominal significance at P<0.05.”

Results section, page 7, line 194-200: “As shown in Table 6, after removing SNPs associated with BMI, we found similar associations of genetically predicted IGF-1 levels with the risk of COVID-19 susceptibility (OR=0.76; 95%CI: 0.60-0.96, P=0.021), hospitalization (OR=0.61; 95%CI: 0.41-0.90, P=0.014), risk of severity (OR=0.84; 95%CI: 0.52-1.38, P=0.497) in which we detected no moderate heterogeneity, and no indication of horizontal pleiotropy in MR-Egger, and no outlier in MR-PRESSO analyses. No causal associations of genetically predicted testosterone and SHBG with COVID-19 were found, but the directions were consistent with results in Table 2.”

– Based on the cited references (13 and 14), selection of SNPs in the meta-analysis is subject to a bias towards non-infectious conditions. It is important to consider that levels of testosterone, estradiol, SHBG, and IGF-1 could be substantially altered during the course of the infection, either in the acute phase or later stages. What are the levels of testosterone, estradiol, SHBG and IGF-1 in the context of viral infections, as well as crucially in COVID-19? How do those levels influence the association of the selected SNPs?

Many thanks for this comment. Agreeing with the reviewer that these SNPs were selected from non-infectious conditions, in which case, the genetic alleles associated with the exposure are randomly assorted at conception and thus unrelated to self-selected lifestyle and environmental factors and are not modified by disease either; in other word, disease conditions would not influence the selection of SNPs. We acknowledge that the levels of testosterone, estradiol, SHBG, and IGF-1 might be changed during the course of infection, which means that the association of interest would be easier to suffer from reverse causality. Given the fact that we were unbale to obtain the measured levels of biomarkers during the course of infection, we therefore alternatively used the genetically predicted levels to proxy the lifelong-time exposure, which was believed to be more persistent than a single measurement of these biomarkers and to minimize the potential of reverse causation.

– Throughout the results, particularly in Figure 2, confidence intervals are large and indicates a potential complication due to unaddressed confounders or subgroups within the cohort. How many of the COVID-19 patients were administered corticosteroid therapy? How many were administered tocilizumab? How does treatment effect the model estimates? The authors should provide more characteristics of the COVID-19 patients with a more extensive investigation on the potential confounders and treatment effects on MR estimates. How does BMI influence model estimates and results?

Thank you for this comment. This study was designed as Two-sample MR based on the GWAS summary data, with the advantage to resist the common confounders. As explained above, MR study use genetic variants as instruments, which are not associated with a wide range of behavioral, social and therapeutic factors that might confound associations with the outcome. Therefore, whether the patient has received medication does not affect our analysis of the relationship between IGF-1 and COVID-19. However, as proposed by the reviewer as an example, if the used genetic IVs have any pleiotropic effect on BMI, this will violate the MR assumptions. To address this point, we have added more sensitivity analyses in our study. The revisions have been made as below.

Materials and methods section, page 5, line 155-159: “To control potential data confounder, we selected SNPs associated with testosterone, estrogen, SHBG, and IGF-1 only, excluding SNPs associated with BMI which is thought to be a causal risk factor for COVID-19(5) at the threshold of 5×10-8 in European ancestry samples by querying PhenoScanner.(6) SNPs in estrogen were not exclude for their irrelevance to BMI.”

Results section, page 7, line194-200: “As shown in Table 6, after removing SNPs associated with BMI, we found similar associations of genetically predicted IGF-1 levels with the risk of COVID-19 susceptibility (OR=0.76; 95%CI: 0.60-0.96, P=0.021), hospitalization (OR=0.61; 95%CI: 0.41-0.90, P=0.014), risk of severity (OR=0.84; 95%CI: 0.52-1.38, P=0.497) in which we detected no moderate heterogeneity, and no indication of horizontal pleiotropy in MR-Egger, and no outlier in MR-PRESSO analyses. No causal associations of genetically predicted testosterone and SHBG with COVID-19 were found, but the directions were consistent with results in Table 2.”

– Inverse-variance weighted two-sample Mendelian randomization is indeed the most extensively used method utilizing GWAS summary stats to infer on causality of the exposure (ie. SNP variant) and outcome variables. The approach suffers from various biases that were seemingly not handled in the study herein described, for example, the winner's curse and potential pleiotropy. The authors should provide a more objective assessment of potential biases and address them accordingly.

Many thanks for this helpful comment. We agree with the reviewer that the MR approach might suffer from various biases, we therefore performed a series of sensitivity analysis to examine the presence of pleiotropy and test the robustness of the MR results. In our study, MR-Egger regression was used to observe and correct potential directional pleiotropy, which was assessed by its intercept test. The weighted median method was performed to provide unbiased causal estimates if at least 50% instrumental variables were valid. MR-PRESSO method can detect SNP outliers and estimate the association after removal of these outliers. The differences in estimates between before and after outlier removal were examined by the embedded distortion test. Cochrane’s Q value was used to assess the heterogeneity among estimates of genetic instruments. As a result, we detected no moderate heterogeneity, and no indication of horizontal pleiotropy in MR-Egger, and no outlier in MR-PRESSO analyses. The influence of winner's curse was considered when selecting data for two-sample MR analysis, for which we used the modified version of Host Genetics Initiative (COVID-19-HGI) (Round 5, European population) where UKB data were excluded to avoid any overlapped samples between the exposure and the outcome GWAS datasets.

– Notwithstanding the large sample size of the discovery cohort obtained from the host genetics in COVID-19 initiative, it is imperative to expand on the findings by ascertaining the robustness of the IFG-1 related SNPs. Do the IGF-1-related SNPs influence IGF-1 levels in the context of a viral infection? Preferably of course in COVID-19. Also, do the SNPs constitute eQTLs for IGF-1 expression in primary organs, particularly lungs, liver, and kidneys?

Thanks for your insightful comment. Based on the Mendelian genetic law, MR refers to the use of genetic variants to develop causal inferences from observational data, if the variant genotype is associated with the phenotype and the variant genotype associated with the risk of disease of interest through the phenotype. Thus, whether IGF-1-related SNPs influence IGF-1 levels in the context of a viral infection, it would not affect the application of MR approach for the inference of causality. As suggested, we searched 157 significant single-tissue eQTLs of IGF-1 (ENSG00000017427.15) in all tissues by GTEs and identified 2 eQTLs of IGF-1 in small intestine-terminal ileum, 9 in spleen, 15 in muscle skeletal and 131 in testis, whilst we didn’t find any eQTLs of IGF-1 in blood cells or lung tissue. Considering the transcriptomic effect of eQTLs is differential across tissues, we are therefore on the side of caution to use the eQTLs in other tissues to perform analysis.

– The methods section needs to be expanded substantially by the addition of important details. For example, how was LD clumping performed? Using PLINK? Why was an r2 cutoff of < 0.01 used? Why not r2 < 0.001?

Many thanks for the useful comment. We agree with the reviewer that the Methods section could be improved by providing more methodological details. In this study, all statistical analyses were two-sided and performed in R 4.0.4 software using the R package Two Sample MR and MR-PRESSO.(6) Therefore we performed LD clumping using not PLINK but R package “TwoSampleMR”. It should be acknowledged that with the decrease of r2 cutoff and the increase of kb cutoff, the less the numbers of IVs left which result in the less confounding and pleiotropy, but the corresponding statistical effect is lower; While the increase of IV numbers can improve the statistical effectiveness, it will also bring more bias. We therefore chose the r2 cutoff of < 0.01 which we thought is moderate. Moreover, given the confounding and pleiotropy, we used the Cochrane’s Q value to assess the heterogeneity among estimates of genetic instruments and the p value for intercept in MR-Egger to detect horizontal pleiotropy. Revisions in the manuscript are made as below.

Materials and methods section, page 4, line 101-110: “We restricted the analysis to SNPs in linkage equilibrium which were identified in the relevant GWAS at P<5 × 10−8 clumped on r2 = 0.01 within 10,000 kb using the 1000 genomes reference panel(7) to ensure sufficient statistical effectiveness. Among those pairs of SNPs that had LD r2 above the specified threshold (r2 = 0.01) only the SNP with the lower P value would be retained. SNPs absent from the LD reference panel were also removed. To test whether there was a weak instrumental variable bias, namely genetic variants selected as instrumental variables had a weak association with exposure, we calculated the F statistic if it is much greater than 10 for the instrument-exposure association, the possibility of weak instrumental variable bias is small. These analyses were conducted using the R package “TwoSampleMR”.(6)”

– The sentence on page 5 line 38 reading "postmenopausal women (22) and men(13, 23)." needs correction as it implies men were also postmenopausal.

Thank you for this careful comment. We have now revised it as “men (13,22) and postmenopausal women (23)” to be clear.

– Page 7, line 196, the statement that reads "The cytokine storm related respiratory distress syndrome…" should be revised, avoiding the sensationalistic "cytokine storm" description of what has been reported elsewhere as merely a cytokine breeze at best.

Many thanks for this insightful comment. Combined with comment 10 and comment 11, we agree with the reviewer that description and discussion on cytokine storm are inappropriate. As suggested, we have now removed the part of cytokine storm from manuscript.

– The Discussion on cytokine storm is arbitrary and should be omitted. The authors do not provide any evidence on the association of IGF-1 with the cytokine storm.

Many thanks for this helpful comment. We agree with the reviewer that discussion on the association of IGF-1 with the cytokine storm is not well-grounded. As suggested, we have now removed the discussion on cytokine storm.

– This is another trial finding no association of sex hormones to COVID-19. This has to be discussed more extensively in the discussion; now discussion is more focused on IGF (the positive finding) but negative results should be put into context. Please refer also to PMID: 35577073, PMID: 35470422, PMID: 35602518

Thank you very much for this comment. We agree with the reviewer that negative results should also be included in the discussion. We thank the reviewer for providing these important references. After carefully reading through the referred papers and other relevant articles, we have now discussed the negative results in the Discussion section. Revisions are made as below to extend our Discussion section.

Discussion section, page 10, line 253-277: “Our study showed that SHBG or testosterone may not be associated with COVID-19 outcomes, which is consistent with the research findings of Liu et al.(8) They also observed a null causal relationship for testosterone or SHBG levels with COVID-19 outcomes in females and males. Meanwhile, epidemiologic data (9) indicate that while men are not more predisposed to contracting COVID-19, they are more likely to develop severe illness following the infection compared with women. However, our study observed null causal relationship for testosterone levels with COVID-19 outcomes in both females and males. According to the available evidence on the role of testosterone in COVID-19, it appears that both high and low testosterone levels can be associated with poor COVID-19 outcomes.(10) A study demonstrated androgen deprivation therapy (ADT) exposure was associated with a reduction in COVID-19 severity.(11) By contrast, the Ohio study did not identify any protective effect of ADT on the severity of COVID-19 outcomes.(12) Androgen-related treatments showed that transmembrane serine protease 2 (TMPRSS2) expression and SARS-CoV-2 entry in human lung cells have been reduced by antiandrogens.(13-15) Additionally, androgens have numerous immunosuppressive effects such as decreasing proinflammatory cytokine release (e.g., IFNγ and TNF) or increasing anti-inflammatory cytokine release (e.g., IL-4 and IL-10), reducing T helper 1 (Th1) and T helper 17 (Th17) cell differentiation, inducing Treg differentiation and regulating B-cell development.(16-18) Paradoxically, these immunosuppressive effects of testosterone might be beneficial to overcome the heightened inflammatory environment that predisposes to severe COVID-19. Recent research has revealed that males with COVID-19 have lower testosterone levels.(19) Another study found a negative association between total testosterone levels and biochemical markers of COVID-19 severity.(20) Lower testosterone concentrations were associated with higher concentrations of IL-6, CRP, IL-1 receptor antagonist, hepatocyte growth factor, and IFNγ-inducible protein 10.(21) Therefore additional research efforts need to be made to investigate the complex relationships furtherly.”

Reference:

1. Fan X, Yin C, Wang J, Yang M, Ma H, Jin G, et al. Pre-diagnostic circulating concentrations of insulin-like growth factor-1 and risk of COVID-19 mortality: results from UK Biobank. Eur J Epidemiol. 2021;36(3):311-8.

2. Davey Smith G, Hemani G. Mendelian randomization: genetic anchors for causal inference in epidemiological studies. Hum Mol Genet. 2014;23(R1):R89-98.

3. Smith GD, Ebrahim S. 'Mendelian randomization': can genetic epidemiology contribute to understanding environmental determinants of disease? Int J Epidemiol. 2003;32(1):1-22.

4. Smith GD, Lawlor DA, Harbord R, Timpson N, Day I, Ebrahim S. Clustered environments and randomized genes: a fundamental distinction between conventional and genetic epidemiology. PLoS Med. 2007;4(12):e352.

5. Freuer D, Linseisen J, Meisinger C. Impact of body composition on COVID-19 susceptibility and severity: A two-sample multivariable Mendelian randomization study. Metabolism. 2021;118:154732.

6. Yavorska OO, Burgess S. MendelianRandomization: an R package for performing Mendelian randomization analyses using summarized data. Int J Epidemiol. 2017;46(6):1734-9.

7. Hemani G, Zheng J, Elsworth B, Wade KH, Haberland V, Baird D, et al. The MR-Base platform supports systematic causal inference across the human phenome. eLife. 2018;7.

8. Liu L, Fan X, Guan Q, Yu C. Bioavailable testosterone level is associated with COVID-19 severity in female: A sex-stratified Mendelian randomization study. J Infect. 2022;85(2):174-211.

9. Peckham H, de Gruijter NM, Raine C, Radziszewska A, Ciurtin C, Wedderburn LR, et al. Male sex identified by global COVID-19 meta-analysis as a risk factor for death and ITU admission. Nat Commun. 2020;11(1):6317.

10. Ho JQ, Sepand MR, Bigdelou B, Shekarian T, Esfandyarpour R, Chauhan P, et al. The immune response to COVID-19: Does sex matter? Immunology. 2022;166(4):429-43.

11. Lee KM, Heberer K, Gao A, Becker DJ, Loeb S, Makarov DV, et al. A Population-Level Analysis of the Protective Effects of Androgen Deprivation Therapy Against COVID-19 Disease Incidence and Severity. Front Med (Lausanne). 2022;9:774773.

12. Klein EA, Li J, Milinovich A, Schold JD, Sharifi N, Kattan MW, et al. Androgen Deprivation Therapy in Men with Prostate Cancer Does Not Affect Risk of Infection with SARS-CoV-2. J Urol. 2021;205(2):441-3.

13. Leach DA, Mohr A, Giotis ES, Cil E, Isac AM, Yates LL, et al. The antiandrogen enzalutamide downregulates TMPRSS2 and reduces cellular entry of SARS-CoV-2 in human lung cells. Nat Commun. 2021;12(1):4068.

14. Deng Q, Rasool RU, Russell RM, Natesan R, Asangani IA. Targeting androgen regulation of TMPRSS2 and ACE2 as a therapeutic strategy to combat COVID-19. iScience. 2021;24(3):102254.

15. Qiao Y, Wang XM, Mannan R, Pitchiaya S, Zhang Y, Wotring JW, et al. Targeting transcriptional regulation of SARS-CoV-2 entry factors ACE2 and TMPRSS2. Proc Natl Acad Sci U S A. 2020;118(1).

16. Olsen NJ, Kovacs WJ. Evidence that androgens modulate human thymic T cell output. J Investig Med. 2011;59(1):32-5.

17. Henze L, Schwinge D, Schramm C. The Effects of Androgens on T Cells: Clues to Female Predominance in Autoimmune Liver Diseases? Front Immunol. 2020;11:1567.

18. Trigunaite A, Dimo J, Jørgensen TN. Suppressive effects of androgens on the immune system. Cell Immunol. 2015;294(2):87-94.

19. Ma L, Xie W, Li D, Shi L, Ye G, Mao Y, et al. Evaluation of sex-related hormones and semen characteristics in reproductive-aged male COVID-19 patients. J Med Virol. 2021;93(1):456-62.

20. Rastrelli G, Di Stasi V, Inglese F, Beccaria M, Garuti M, Di Costanzo D, et al. Low testosterone levels predict clinical adverse outcomes in SARS-CoV-2 pneumonia patients. Andrology. 2021;9(1):88-98.

21. Dhindsa S, Zhang N, McPhaul MJ, Wu Z, Ghoshal AK, Erlich EC, et al. Association of Circulating Sex Hormones With Inflammation and Disease Severity in Patients With COVID-19. JAMA Netw Open. 2021;4(5):e2111398.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

– The study hinges solely on MR analysis of selected SNPs used as proxies of testosterone, estradiol, SHBG, and IGF-1 levels. None of the markers were directly measured, and no information on their levels in the context of infections or COVID-19 was given. The authors argue at length that measuring the markers in the specific cohort is of limited value because their genetic causality modelling approach supersedes the need for such measurements. While MR models have indeed emerged as interesting tools to infer causality, it is also important to provide a potential mechanism linking genetic variants to the phenotype. Without direct measurements of the said markers minimize the potential to provide readers with a possible mechanism bridging genetic variant and phenotype.

Many thanks for your constructive suggestion. We agree with the reviewer that measurements of these biomarkers in study populations to verify the association are important. There are several epidemiological studies in which they measured IGF-1 levels in their study populations and followed up the clinical outcomes of COVID-19, and observed a significant association between low IGF-1 levels and severe COVID-19 outcomes. For instance, in the paper by Xikang Fan et al. (1), they demonstrated an inverse association between pre-diagnostic circulating levels of IGF-1 and COVID-19 mortality risk. Another observational study in Greece reported lower IGF-1 levels in critically ill COVID-19 patients compared to their counterparts with less severe disease or without COVID-19.(2)

Findings from these observational studies are the basis for the generation of hypothesis to be tested in this MR study. The adoption of MR design in this study aimed to provide different tiers of evidence to validate the observed association from previous studies. Considering the creativity and novelty of study findings, we highlighted the relevant findings from previous observational studies in the Discussion section, instead of repeating their work to explore the association between direct measurement of the marker levels and COVID-19 outcomes. We acknowledge that future studies, in particular laboratorial experiments, are warranted to investigate the possible mechanisms for the involvement of IGF-1 in the recovery of COVID-19. In order to provide some clues on the underlying mechanisms, we reviewed the relevant literature and tried to proposed some hypotheses to bridging up these findings.

Discussion section, page 9, line 209-214: “Our MR findings were consistent with the multiple epidemiological studies that reported an association between measured IGF-1 levels and COVID-19 illness. There is one observational study that demonstrated an inverse association between pre-diagnostic circulating levels of IGF-1 and COVID-19 mortality risk among COVID-19 patients in UK biobank. (26) Another observational study in Greece reported lower IGF-1 levels in critically ill COVID-19 patients compared to their counterparts with less severe disease or without COVID-19.(27) ”

Discussion section, page 9, line 220-239: “IGF-1 has been found to be pro-survival/anti-aging, anti-inflammatory, and antioxidant with neuro- and hepatoprotective properties. A study by the Narasaraju group demonstrated that IGF-1 plays an important role in the repair of lung tissue by regulating the proliferation and differentiation of alveolar epithelial cells (AECs).(29) Airway inflammation can be mitigated when apoptotic cells are engulfed by pulmonary epithelial cells.(30) IGF-1 has also been shown to up-regulate engulfment by professional phagocytes such as dendritic cells,(31) and inhibit IL-6 production from lipopolysaccharide (LPS)-induced AECs. (32) Both of these mechanisms are beneficial to the regression of local inflammation. Jakn et al. showed that IGF-1 binds to insulin-like growth factor-1 receptor (IGF-1R) on airway epithelial cells of non-professional phagocytic cells, which can promote the phagocytosis of microparticles by airway epithelial cells.(33) Transforming growth factor β1 (TGF-β1) derived from AECs activated alveolar macrophages (AMs) to secrete IGF-1 into the alveolar fluid in response to stimulation of the airway by inflammatory signals. This AM-derived IGF-1 attenuated the p38 mitogen-activated protein kinase (MAPK) inflammatory signal in AECs and promoted the phagocytosis of apoptotic cells by AECs. This two-way communication between AECs and AMs represents a well-tuned system for the regulation of the inflammatory response in alveoli.(34) Taken together, these studies provide biological evidence supporting that IGF-1 might be an important anti-inflammatory factor in the alveolar microenvironment and thus may contribute to improve COVID-19 outcomes. More studies are required to determine whether novel therapeutic strategy targeting on IGF-1 pathway might improve COVID-19 prognosis.”

Discussion section, page 11, line 291-293: “It should also be noted that the study findings are based on evidence from genetic data, additional large and prospective cohort studies with available IGF-1 data and information on COVID-19 susceptibility and clinical outcomes are needed to validate the findings.”

– The number of tests performed in the study is equivalent to the number of SNPs that were investigated, that is, 657. Hence, the multiple-testing correction must take into account 657 tests. The authors are wrong in adjusting their probabilities just for the 4 markers, which is more attuned to p-value hacking. Therefore, my initial comments on the lack of significant findings after considering multiple-test corrections remain a concern. Hence, the conclusions pertaining to IGF-1 variants do not reflect on the author's findings. Based on this result, I suggest being more objective by stating that none of the genetic variants can be deemed as a causal variant.

Many thanks for this comment and apologies for not addressing the previous comment properly. This Mendelian randomization study was designed to use genetic variants as instrumental variables to proxy the circulating level of 4 biomarkers, but not intends to examine the association between individual genetic variant and COVID-19 outcome. Thus, when applying the Bonferroni correction for multiple testing, the number of tests should be the number of biomarkers examined in the study, instead of the number of genetic variants used as IVs, therefore the adjusted P value should be < 0.05/4 = 0.013 (four biomarkers). The p-value for the association between IGF-1 and COVID-19 hospitalization was 0.018, which didn’t reach the significance threshold when adopting the Bonferroni correction. Given that there is also prior evidence for IGF-1 and COVID-19, this could be regarded as possibly a stand-alone prior hypothesis, in which case, P-value < 0.05 is acceptable for the validation of a prior hypothesis. However, to address this comment carefully, we have now revised the manuscript to describe the association as nominally significant and state this association did not survive the Bonferroni correction, and the strength of the conclusion has also been toned down. Revisions are made as below.

Abstract, page 2, line 49-53: “Conclusions: Our study indicated that genetically predicted high IGF-1 levels were associated with decrease the risk of COVID-19 susceptibility and hospitalization, but these associations did not survive the Bonferroni correction of multiple testing. Further studies are needed to validate the findings and explore whether IGF-1 could be a potential intervention target to reduce COVID-19 risk.”

Results, page 7, line 169-171: “Associations of IGF-1 levels with COVID-19 susceptibility and hospitalization were not statistically significant after Bonferroni correction, albeit showing a nominal significance at P<0.05.”

Discussion section, page 11, line 294-298: “In conclusion, our study indicated that genetically predicted high IGF-1 levels were associated with decrease the risk of COVID-19 susceptibility and hospitalization, but these associations did not survive the Bonferroni correction of multiple testing. Further studies are needed to validate the findings and explore whether IGF-1 could be a potential intervention target to reduce COVID-19 risk.”

References:

1. Fan X, Yin C, Wang J, Yang M, Ma H, Jin G, et al. Pre-diagnostic circulating concentrations of insulin-like growth factor-1 and risk of COVID-19 mortality: results from UK Biobank. Eur J Epidemiol. 2021;36(3):311-8.

2. Ilias I, Diamantopoulos A, Botoula E, Athanasiou N, Zacharis A, Tsipilis S, et al. Covid-19 and Growth Hormone/Insulin-Like Growth Factor 1: Study in Critically and Non-Critically Ill Patients. Front Endocrinol (Lausanne). 2021;12:644055.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Citations

    1. COVID-19 Host Genetics Initiative. 2021. COVID-19 Host Genetics Initiative round 5. COVID-19 HGI. covid19hg

    Supplementary Materials

    MDAR checklist
    elife-79720-mdarchecklist1.docx (99.9KB, docx)
    Supplementary file 1. Genetic instruments for testosterone, estrogen, SHBG, and IGF-1.
    elife-79720-supp1.docx (279.1KB, docx)
    Reporting standard 1. Reporting checklist for case-control study.
    elife-79720-repstand1.docx (18.7KB, docx)

    Data Availability Statement

    Data analyzed in the present study are GWAS summary statistics, which have been made publicly available. GWAS summary level data of COVID-19 HGI could be downloaded from https://www.covid19hg.org/results/. GWAS summary level data of sex hormones and IGF-1 in UK Biobank could be downloaded from GWAS catalog. All genome-wide significant SNPs have been provided in Supplementary file 1a–d. All analyses were performed using R statistical package freely available at https://cran.r-project.org/mirrors.html. The TSMR package is available at https://mrcieu.github.io/TwoSampleMR/(Hemani et al., 2020).

    Data analysed in the present study are GWAS summary statistics, which have been made publicly available. GWAS summary level data of COVID-19-HGI could be downloaded from https://www.covid19hg.org/results/. GWAS summary level data of sex hormones and IGF-1 in UK biobank could be downloaded from GWAS catalog (http://ftp.ebi.ac.uk/pub/databases/gwas/summary_statistics/GCST90019001-GCST90020000/). All genome-wide significant SNPs have been provided in Supplementary Tables 1 to 4 in Supplementary file 1. All analyses were performed using R statistical package freely available at https://cran.r-project.org/mirrors.html. The Two-sample MR package is available at https://mrcieu.github.io/TwoSampleMR/.

    The following previously published dataset was used:

    COVID-19 Host Genetics Initiative. 2021. COVID-19 Host Genetics Initiative round 5. COVID-19 HGI. covid19hg

    Articles from eLife are provided here courtesy of eLife Sciences Publications, Ltd

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