O-251 LIN28B gene expression is downregulated in spermatozoa of oligozoospermic men and associates with genetic variants previously linked to pubertal onset

Abstract

Study question Is there an association between genetic polymorphisms in LIN28B with LIN28B expression and semen quality in spermatozoa from oligozoospermic men? Summary answer LIN28B is downregulated in spermatozoa of oligozoospermic men and explains 39% of sperm count variability. One SNP (rs314280) was associated with low LIN28B mRNA expression. What is known already Large-scale genomic studies have identified genetic variants in or near LIN28B to be robustly associated with pubertal traits such as age at menarche and age at voice break. The LIN28 family of genes are known to control cell division, growth and differentiation. The LIN28B orthologue is exclusively expressed at high levels in the testis and placenta; however, we have limited knowledge about the role of LIN28B in relationship to testicular function. Interestingly, other studies have showed that later onset of puberty is associated with poor semen quality, suggesting that pubertal development may influence adult testicular function. Study design, size, duration Twenty-nine oligozoospermic (cases) and fifty-four men with normal sperm concentration (controls) were recruited from patients seeking andrological work-up for male/couple infertility at a University Hospital. Subjects with abnormal karyotype, Yq-microdeletions, varicocele GIII-IV, hypogonadotropic hypogonadism, seminal infection, diabetes, BMI>35, excessive consumption of alcohol/drugs, testicular cancer, chemotherapy/radiotherapy were excluded. Semen samples were obtained for analysis (WHO, 2010) and sperm isolation by density gradient (PureSperm40®). Peripheral blood samples were analysed for reproductive hormones and used for DNA extraction. Participants/materials, setting, methods LIN28B and GAPDH-S mRNA abundance were measured by qRT-PCR (2-ΔCT method) in RNA isolated from purified sperm using SYBR® MasterMix (Takyon). Five SNPs in linkage disequilibrium (rs7759938, rs395962, rs314268, rs314277 and rs314280) were genotyped (TaqMan™ SNP Genotyping Assay). Statistical differences between groups were assessed by Mann-Whitney test and genetic association was analysed using “SNPassoc” package in R. Values are showed as median, Q1-Q3. Main results and the role of chance Cases and controls had similar age and BMI but as expected sperm concentration, morphology and vitality were differed between groups (9.9, 4.9-13.1 vs 82.8, 46.0-146.5 mill/ml; 1, 1-2 vs 3, 1-4% normal forms and 83%, 76-89 vs 86%, 82-90, respectively) (p < 0.05). No differences were observed in progressive motility, semen volume and days of abstinence. FSH and LH serum levels were higher in cases compared with controls (4.6, 2.6-7.8 vs 3.2, 2.5-4 mIU/ml and 4.2, 2.9-5.2 vs 3.1, 2.1-4.2 mIU/ml, respectively; p = 0.019 and p = 0.017). Relative expression of LIN28B was significantly diminished in cases compared with controls (0.0034, 0.0015-0.0103 vs 0.0446, 0.0122-0.1026; p < 0.001), while expression of the internal housekeeping control GAPDH-S was similar between groups. Linear regression model showed significant association of sperm concentration and total sperm count with LIN28B relative expression adjusted by age, abstinence time, % progressive motility and % normal morphology (beta=0.408, R2=0.241, p < 0.001 and beta=0.461, R2=0.393, p < 0.001). LIN28B rs314280 was associated with mRNA expression in the dominant model (p = 0.025) and two other SNPs (rs7759938, rs314268) showed the same trend (p = 0.09). Men with at least one minor allele of these 3 SNPs had a lower LIN28B mRNA abundance (p = 0.006; p = 0.07; p = 0.055). Limitations, reasons for caution Genotype frequency comparisons between cases and control were not conducted because of the small sample size. A larger cohort will be studied to address this assumption and association of genotypes with parameters of semen quality. Wider implications of the findings To our knowledge this study represents the first report on LIN28B expression and human testicular function. The significant direct association of LIN28B mRNA expression with sperm concentration suggests that LIN28B may be involved in spermatogonial proliferation. Trial registration number not applicable