Genetic Variants Of Mitochondria DNA(mtDNA) On Spermatogenic Impairment

Infertility remains a major clinical problem in field of reproductive health. The World Health Organization(WHO) estimates that approximately 10-15% of couples experience some form of infertility problem, and male factor infertility accounts for about 50% of all infertility cases. A significant proportion of male infertility is accompanied by spermatogenesis failure.It has been suggested that spermatogenesis failure may result mainly from genetic factors, environment risk factors and epigenetic modification. Oxidative phosphorylation(OXPHOS) is located on the inner membrane of mitochondrion and supplies ~90% of the energy of the human body, which is crucial to spermatogenesis, differentiation and optimal functioning of germ cells. Mitochondria also play an important role in reactive oxygen species(ROS) production, apoptosis and cell signal regulation. Since mitochondrial DNA(mt DNA), which is the only genetic material outside nuclear, codes the necessary units of OXPHOS and their RNA translation elements. mt DNA mutation would ultimately affect the production of ATP, consequently may lead to abnormal spermatogenesis, impaired differentiation and hypospermatogenesis.Many mt DNA variations were reported to be associated with spermatogenesis failure in previous studies. However, the contributions of whole mt DNA genome to male infertility have not yet been systematically and comprehensively evaluated. Therefore, we hypothesized that:(1) Genetic variants of mitochondria DNA are associated with spermatogenesis failure and male infertility;(2) Specific genetic background such as mt DNA haplogroup may confer risks of abnormal male reproduction;(3) mt DNA content may be effected through a feedback mechanism to compensate for the respiratory function decline of the mitochondria harboring mt DNA with oxidative damage or mutation in men with idiopathic oligoasthenspermia.Part I Genetics Variants of mt DNA genome in NOAObjective Non-obstructive azoospermia(NOA) is one of the most severe forms of idiopathic male infertility, the pathophysiology of which is largely unknown. Mitochondrial DNA(mt DNA) is believed to be both the source and target of reactive oxygen species(ROS), and its variants could induce meiotic arrest. To explore the role of mt DNA in NOA, a two-stage study was performed in a Han Chinese Population.Methods In the screening stage, the mt DNA genome was sequenced in 96 NOA cases and 96 healthy controls using NGS. In the validation stage, fourteen population polymorphisms were used to define 13 haplogroups for further study using Multiplex SNa Pshot Kit. Selected genetic variants were genotyped by a custom-by-design SNPscan TM Kit. Assay kits for superoxide dismutase(SOD) and total antioxidant capability(T-AOC) in serums were all determined spectrophotometrically using commercially available assay kits(Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer’s protocols.Results In the screening stage, thirteen mt DNA haplogroups were ascertained, and ten susceptible variants were observed, including six potential amino acid substitution variants(m.3394T>C, m.6881A>G, m.8684C>T, m.11696G>A, m.12358A>G, m.13135G>A), one variant in the t RNA gene(m.15968T>C) and three variants in the hypervariable region I(HVRI)(m.16224 T >C, m.16319G>A, m.16497A>G).In the validation stage, We found that individuals with haplogroup M8* were associated with increased risk of NOA(odds ratio [OR] 2.61, 95% confidence interval [CI] 1.47-4.61)(P=6.76×10-4). Unexpectedly, the frequency of m.8684C>T, the defining marker for haplogroup M8 a, was also higher in the case group(OR 4.14, 95% CI 1.56-11.03)(P=2.09×10-3). Subsequently, we compared the frequency distributions of the sub-haplogroups of haplogroup M8*(including haplogroups M8 a and Z) and, intriguingly, no significance was found in haplogroup Z. Additionally, antioxidant capacity, including total antioxidant capability(T-AOC) and superoxide dismutase(SOD) were evaluated. The level of T-AOC was significantly decreased(P<0.05) compared to the control group.Conclusion These results demonstrated that haplogroup M8 a background in general enhances the penetrance of m.8684C>T in NOA. And mt DNA genetic variants, causing low antioxidant levels, might increase mitochondrial damage and impair normal spermatogenesis. Further investigation in larger populations and fuctional characterizations are needed to validate our findings.Part II Genetics Variants of mt DNA genome in oligoasthenospermiaObjective Oligoasthenospermia is an obvious and general cause of male idiopathic infertility. Mitochondrial DNA(mt DNA) is believed to be both the source and target of reactive oxygen species(ROS) and mt DNA genetic alterations responsible for human disease have been associated with molecular defects in the OXPHOS system. To investigate the potentially susceptible mt DNA genetic variants in oligoasthenospermia, we conducted a two-stage study in 921 idiopathic infertile men of oligoasthenospermia and 766 healthy controls using comprehensive molecular analysis.Methods In the screen stage, we used next generation sequencing(NGS) in 233 cases and 233 controls to screen oligoasthenospermia susceptible mitochondrial genetic variants.In the validation stage, fourteen population polymorphisms were used to define 13 haplogroups for further study using Multiplex SNa Pshot Kit. Selected genetic variants were replicated in 688 cases and 533 healthy controls by a custom-by-design SNPscan TM Kit.Results In the screen stage, a total of 13 mt DNA haplogroups were defined, no significant difference was found in the mt DNA haplogroups distribution between these two groups. Totally, the frequency distributions of seven genetic variants were screened to be different among the case group and the control group. Among which, four coding variants were accompanied with amino acid substitution(m.12338 T >C, m.12361 A >G, m.13928G>C in ND5 and m.A15235 A >G in Cyt B).In the validation stage, we observed a significant increased risk of idiopathic infertility in association with m.16179 C>T(odds ratio(OR) 3.10, 95% CI 1.41-6.79)(P =3.10×10-3). To elucidate the exact role of the genetic variants in spermatogenesis, two main sperm parameters(sperm count and motility) were taken into account. We found that m.16179 C>T was associated with both low sperm count and motility, with ORs of 4.18(95% CI 1.86-9.40)(P =1.90×10-4) and 3.17(95% CI 1.40-7.16)(P =3.50×10-3), respectively. Additionally, m.12361 A >G was found to be assoctated with low sperm count, with an OR of 3.30(95% CI 1.36-8.04)(P =5.50×10-3).Conclusion These results indicated that m.16179 C>T influenced both the process of spermatogenesis and sperm motility, while m. 12361 A >G may just only participated in the process of spermatogenesis. Further investigation in larger populations and functional characterizations are needed to validate our findings.Part III mt DNA copy number variations in sperm from oligoasthenospermiaObjective Mitochondrial DNA copy number is also an important factor in mitochondrial genetic variability and pathology. Increased mt DNA content was also observed in spermatozoa collected from abnormal semen samples.To figure out whether the mt DNA copy number variations is associated with oligoasthenospermia, we compared mt DNA copy number among men with normal and abnormal semen parameters.Methods We used the real-time PCR to test the relative mt DNA copy numbers of 100 oligoasthenospermia cases and 80 controls.Results A significant increase in mt DNA copy number was observed in the spermatozoa samples from patients with abnormal semen parameters(79.02±10.07) compared to the patients with normal semen parameters(21.40±3.69)(P<0.001). The copy number of mt DNA was significantly correlated with sperm quality.Conclusion Mitochondrial DNA quantity may serve as useful diagnostic markers of sperm quality in infertile men.