Table_1_The Association of HLA-G Gene Polymorphism and Its Soluble Form With Male Infertility.docx

Successful reproduction depends on many factors. Male factors contribute to infertility in approximately 50% of couples who fail to conceive. Seminal plasma consists of secretions from different accessory glands containing a mixture of various cytokines, chemokines, and growth factors, which together can induce a local immune response that might impact on a male’s as well as a female’s fertility. Human leukocyte antigen (HLA)-G expression has been suggested as an immunomodulatory molecule that influences pregnancy outcome. The HLA-G gene encodes either membrane-bound or/and soluble proteins. The aim of this study was the evaluation of HLA-G polymorphisms and their impact on soluble HLA-G (sHLA-G) production. We tested the HLA-G polymorphism in three positions: rs1632947: c.-964G>A; rs1233334: c.-725G>C/T in the promoter region; rs371194629: c.∗65_∗66insATTTGTTCATGCCT in the 3′ untranslated region. We tested two cohorts of men: 663 who participated in in vitro fertilization (test material was blood or sperm), and 320 fertile controls who possessed children born after natural conception (test material was blood). Since 50% of men visiting assisted reproductive clinics have abnormal semen parameters, we wondered if men with normal sperm parameters differ from those with abnormal parameters in terms of HLA-G polymorphism and secretion of sHLA-G into semen. We found that certain rs1632947-rs1233334-rs371194629 HLA-G haplotypes and diplotypes were associated with male infertility, while others were protective. Normozoospermic men with the A-C-del haplotype and A-C-del/A-C-del diplotype secreted the most sHLA-G into semen (574.1 IU/mL and 1047.0 IU/mL, respectively), while those with the G-C-ins haplotype and G-C-ins/G-C-ins diplotype – the least (80.8 IU/mL and 75.7 IU/mL, respectively). Men with the remaining haplotypes/diplotypes secreted sHLA-G at an intermediate level. However, only in one haplotype, namely G-C-ins, did we observe strong significant differences in the concentration of sHLA-G in the semen of men with teratozoospermia compared to men with normal sperm parameters (p = 0.009). In conclusion, fertile men differ in the profile of HLA-G polymorphism from men participating in IVF. Among all HLA-G haplotypes, the most unfavorable for male fertility is the G-C-ins haplotype, which determines the secretion of the lowest concentration of the soluble HLA-G molecule. This haplotype may reduce sperm parameters.

成功受孕受诸多因素影响。约50%的不孕夫妇的不育问题与男性因素相关。精浆由多种附属性腺的分泌物组成，内含多种细胞因子、趋化因子与生长因子的混合物，这些成分可共同诱导局部免疫应答，进而对男女双方的生育能力均可能产生影响。人类白细胞抗原G（HLA-G）已被证实为一种可影响妊娠结局的免疫调节分子。HLA-G基因可编码膜结合型或/和可溶性蛋白。本研究旨在评估HLA-G基因多态性及其对可溶性HLA-G（sHLA-G）产生的影响。我们对3个位点的HLA-G基因多态性进行了检测：启动子区域的rs1632947（c.-964G>A）、rs1233334（c.-725G>C/T），以及3'非翻译区的rs371194629（c.∗65_∗66insATTTGTTCATGCCT）。我们纳入了两类男性队列：663名参与体外受精（IVF）的男性（检测样本为血液或精液），以及320名已通过自然受孕诞下子女的生育能力正常的对照男性（检测样本为血液）。鉴于就诊于辅助生殖诊所的男性中有50%存在精液参数异常，我们探究了精子参数正常的男性与异常男性在HLA-G基因多态性以及sHLA-G向精液中的分泌水平方面是否存在差异。研究结果显示，部分rs1632947-rs1233334-rs371194629的HLA-G单体型和双倍型与男性不育相关，而另有部分单体型/双倍型则具有保护作用。携带A-C-del单体型及A-C-del/A-C-del双倍型的正常精子症男性，其精液中sHLA-G的分泌水平最高（分别为574.1 IU/mL和1047.0 IU/mL）；而携带G-C-ins单体型及G-C-ins/G-C-ins双倍型的正常精子症男性，其精液中sHLA-G分泌水平最低（分别为80.8 IU/mL和75.7 IU/mL）。携带其余单体型/双倍型的男性，其精液中sHLA-G的分泌水平处于中间区间。不过，仅在G-C-ins这一单体型中，我们观察到畸形精子症男性与精子参数正常男性的精液sHLA-G浓度存在显著差异（p=0.009）。综上，生育能力正常的男性与参与体外受精的男性在HLA-G基因多态性特征上存在差异。在所有HLA-G单体型中，对男性生育能力最不利的为G-C-ins单体型，该单体型可导致可溶性HLA-G分子的分泌浓度最低，或可降低精子参数。