Single-Cell Sequencing Analysis Reveals the Correlation between Obesity and Circadian Rhythm in Spermatogenesis and Its Microenvironment

Background: Male infertility has gradually increased morbidity during these years. Due to the lack in understanding of specific mechanism of male infertility, the patients could not receive efficient treatment for a long period. Obesity was universally thought to correlate with male infertility. However, the underlying pathway of obesity inducing male infertility is still uncertain. Previous studies indicated that obesity could impact circadian rhythm in multiple tissues. Therefore, it's worth exploring the correlation between obesity and circadian rhythm and their effect to male infertility. Methods Based on a single-cell RNA sequencing dataset containing 12 human testicular tissues, we divided the testicular cells into different clusters. According to Body Mass Index (BMI), the cells were divided into normal (18.5 ≤ BMI ≤ 24.9), little fat (25.0 ≤ BMI ≤ 29.9) and very fat (30.0 ≤ BMI ≤ 34.9) groups. hdWGCNA was used to construct co-expression modules of circadian rhythm-related genes. CRD_scores were constructed to describe circadian rhythm conditions based on the modules. Differentially expressed genes (DEGs) between two groups were identified using the R package "seurat". Enrichment analysis were performed to explore the function of DEGs using relevant R packages. Statistical analysis including comparison and correlation analysis were also conducted using R. Results Co-expression modules were found to all correlate with BMI. Furthermore, circadian rhythm showed a significant correlation with obesity in every cell clusters. In spermatogenesis, obesity could induce circadian rhythm disruption (CRD) in related cells. In Leydig cells, obesity could disturb androgen production by inducing CRD, which will lead to androgen level disruption and further impact reproduction function. Two hub genes (STAT3 and CEBPD) in the red co-expression module were found to play an important part in the process of obesity inducing CRD in Leydig cells. In macrophages, relationship between obesity and circadian rhythm was found to correlate with inflammation response and spermatogenesis. Conclusions We determined the correlation between obesity and circadian rhythm in testicular cells. It's found that obesity-related CRD in testis could lead to disrupted spermatogenesis and androgen level, inflammation, which will cause male reproductive dysfunction and infertility.