BackgroundEndometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1). Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and the involvement of nuclear estrogen receptors (ER) and G-coupled protein estrogen receptor (GPER)-1.ResultsUpstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) secretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of USF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERα) or G1 (specific agonist for GPER1); (b) no increase in USF2 binding to SF-1 E-Box/DNA consensus sequence in E2-treated cells; (c) USF2 variants protein contents were not modified by PGE2; (d) SF-1 nuclear protein content was significantly higher than basal when treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) cytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was additive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF-1 and P450Arom protein contents in basal condition were unmodified.ConclusionThese data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERα and GPER1, whereas SF-1 through GPER1, visualized by the response of the cells obtained from control endometria, being unaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF-1 E-Box/DNA-sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction that they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 variants protein expression during the secretory phase in eutopic endometria from women with endometriosis may participate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial invasion to ectopic sites in the pelvic cavity.

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&gt;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 &lt; 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 &lt; 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 &lt; 0.001 and beta=0.461, R2=0.393, p &lt; 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