Association Between Sperm Mitochondrial ND2 Gene Variants And Total Fertilisation Failure In Chinese Han Population

Background and ObjectiveAt present,infertility has become an important reproductive health problem involving couples of reproductive age in various countries and regions around the world.In China,about 50% of infertile couples are caused by male factors,and most of them are related to sperm quality decline and sexual dysfunction.Although with the development of medical technology,infertility can use assisted reproductive technology to solve some problems,but still 5-15% of infertile couples have total fertilisation failure.Total fertilisation failure(TFF)refers to the fact that after a period of micro-insemination of conventional in vitro fertilisation(IVF)and intracytoplasmic sperm injection(ICSI),sperm and eggs failed to combine into zygote,and there was no phenomenon of sperm nucleus and egg pronucleus formation.The process of total fertilisation failure is complex and unpredictable,and it will exert great pressure on infertility patients both in life and economically.In recent years,studies have shown that sperm motility and oligozoospermia are related to sperm mitochondria,but there are few studies on total fertilisation failure.Therefore,if we understand the mechanism and causes of the occurrence of TFF,it is of great significance to prevent in vitro fertilisation failure.However,at present,little is known about domestic and foreign research on the specific mechanism for total fertilisation failure.Our previous study found that MT-ATP6 variation may be the cause of IVF failure,but 10397 homologous variation of MT-ND3 may help reduce the risk of in vitro fertilisation failure.In this study,we investigated the association between sperm mitochondrial ND2(MT-ND2)gene mutation and total fertilisation failure(TFF)which provided a useful exploration for predicting total fertilisation failure.Method(1)Clinical data: The subjects were Chinese Han sperm from the fresh in vitro fertilisation(IVF)cycle or part of the intracytoplasmic sperm injection(Half-ICSI)cycle,and A total of 246 cases were collected at the Second Affiliated Hospital of Zhengzhou University from July 2011 to May 2017.(2)In the present study,246 cases fresh sperms of Chinese Han(in the IVF cycle or half-ICSI)were divided into two groups according to the definition of total fertilisation failure.A retrospective comparative study of a total of 59 patients in the total fertilisation failure group and 187 patients in the control group(fertility rate >50%).(3)In this experiment,semen samples were separated by renewal gradient centrifugation,sperm mitochondrial DNA was extracted,and sperm mitochondrial gene mutations were analyzed by nested PCR and direct DNA sequencing.The results were compared with the mitochondrial human Cambridge sequence(r CRS)from the Mitomap database(www.mitomap.org)and analyzed for variation in amino acids encoded by the mutation sites.(4)Statistical analysis: Continuous data was expressed by number and percentages(%),and categorical data was expressed by mean ± standard deviation.We used the independent-samples T test to analyze measurement data and used FDR corrections to perform chi-square test or Fisher’s exact test to analyze continuous data.Multivariate logistic regression analysis was also used to analyze the association between mitochondrial variants and TFF,and 53 pairs of propensity scores were matched.P values less than 0.05 were considered statistically significant.Results(1)In this study,32 homozygous variation sites and 47 heterozygous variation sites were found in the MT-ND2 gene.Among the 32 homozygous variations,there were 7 missense variations and 25 synonymous variations.There was no significant statistical difference of 32 homozygous variations between the two groups.(2)The m.4914C>T,m.5320C>T and m.5426T>C loci in the 47 heterozygous variation sites were all less than 0.05 before FDR correction,but they were not statistically significant after FDR correction.A 53-pair pairwise comparison was performed,and the general characteristics of the subjects in the TFF group and the control group were adjusted in logistic regression analysis.The data showed the frequencies of the m.4914C>T,m.5320C>T,and m.5426T>C locus variants of the MT-ND2 gene between the TFF group and the control group.We found no statistical significance and there was no synergistic cumulative effect among the three heterozygous variant loci.(3)In addition,there was no statistically significant difference between mt DNA haplogroup D and haplogroup G in the TFF group and the control group.Conclusion(1)The ND2 gene variation may not be related to the total fertilisation failure,and there is no cumulative synergy between the m.4914C>T,m.5320C>T and m.5426T>C variation sites.(2)Haplogroup D and haplogroup G are not associated with total fertilisation failure in Chinese Han population.