| Background and objective In recent years,infertility has become a global public health problem as life paces up,work pressures increase,environmental pollution worsens,and diet structures change.In addition to the two major infertility factors due to female ovulation disorders and fallopian tube dysfunction,male factors have become increasingly common,and many studies have shown that male sperm count and fertility are declining.There are many reasons for the decline in sperm quality,exposure to toxic environment,varicocele,physical factors and mental factors.With the deepening of molecular biology and genetic research,some scholars have further turned their attention to sperm mitochondria.Some foreign scholars have found that there is a close relationship between mitochondrial gene fragment deletion and male infertility.We have previously studied the correlation between sperm mitochondrial genes MT-ATP6,MT-ND3,MT-ND6 and MT-ND2 variations and total fertilization failure.But we do not know the variations of MT-CO1 gene,and so a small sample was used to understand the MT-CO1 gene fristly.In this study,we found that human sperm mitochondrial CO1 gene has undergone significant vatiants in the past 5 years,and the genetic variation has a certain regularity.Therefore,we tried to explore whether the sperm mitochondrial CO1 gene mutation in Han people affects male sperm quality and its correlation with complete in vitro fertilization failure.Method1.Sample collection1.1: A retrospective analysis of semen from 343 male patients with IVF cycles at the Reproductive Center of the Second Affiliated Hospital of Zhengzhou University from 2010 to 2018.1.2: 19 puerperal female volunteers who were admitted to the Second Affiliated Hospital of Zhengzhou University from September to November 2018,each taking 2ml of peripheral venous blood,and collecting 0.5ml of peripheral venous blood from8 infants within two years of age.1.3: Six male volunteers were recruited to take their semen,peripheral venous blood and peripheral blood of their own mother.1.4: Five individuals of different years(2012 and 2018)were collected for individual before and after the control study.1.5: In September to November 2018,more than 40 immature eggs discarded during the injection of cytoplasmic spermatozoa were collected in our reproductive center.2.Method2.1 This study used retrospective comparative analysis to analyze the mitochondrial CO1 gene variation by nested PCR and DNA sequencing.The results were compared by human Cambridge sequence(r CRS)recorded in the mimatop database(www.mitomap.org).2.2 Protein function prediction of variant sites was performed using Uniport datebase and Polyphen-2 software;A swiss-model software homology modeling was used to predict protein tertiary structure and TM-Align software was used to calculate wild type and mutant type and protein high-level structure in database.The RMSD difference was used to assess the effect of SNP on the stability of the protein's spatial structure.2.3 343 sperm samples were grouped according to the year,and the mitochondrial CO1 gene variation and characteristics were observed.According to whether the genes were mutated,the relationship between the mutation and the sperm quality was observed.According to whether the complete in vitro fertilization failure group was observed,the gene mutation was observed.Statistical method The continuous count data was used to apply the mean ± standard deviation,and the classification count data was expressed by the number and percentage.The measurement data were analyzed by independent sample t-test,and the count data were analyzed by chi-square test or Fisher's exact test.All analyses were performed on both sides,and P < 0.05 was considered statistically significant.All sample data were calculated by SPSS 21.0 statistical software.Result1.A total of 75 homozygous variants and 66 heterozygous variants were found in the mitochondrial sequencing of sperm in the Han population.Among them,1homozygous locus was newly discovered,and 11 new heterozygous loci were found.2.Observing all the variation sites of the sperm mitochondrial CO1 gene,we found that since 2014,the sperm mitochondrial CO1 gene began to appear in a group of G6023 A,T6221C,C6242 T,A6266C,A6299 G,G6366A,G6383 A,C6410T,C6452 T,C6483T,T6512 C,C6542T,C6569 A,T6641C,C6935 T,C6938T,A7146 G,C7232T,C7256 T and G7316 A.Specific gene variants consisting of 20 fixed sites and the reversion variation of the C7028 T locus.In this paper,the mitochondrial CO1 gene containing all 20 fixed variant sites was defined as a variant gene,and the mutated mitochondrial CO1 gene was defined as a wild-type gene.The variant in mitochondrial CO1 gene was not detected in the blood.3.The prediction of protein function suggested that the two missense variants of G6366 A and A7146 G had no obvious effect on protein function.The swiss-model software homology model predicted the protein tertiary structure,and found that wild type RMSD=0.1;variant RMSD=1.24.This result suggests that the stability of higher structure of the variant protein is lower than that of the wild type protein.4.There was no significant difference in sperm density between wild-type genome and variant genome(P>0.05);sperm motility(a+b grade)was different between the two groups(P<0.05),and the average viability of wild-type genome was significantly higher than that of variant genome.5.There was no significant correlation between sperm mitochondrial CO1 variant gene and complete fertilization failure in vitro(P>0.05).6.To analyze each of the mitochondrial CO1 gene variants,we found that between 2010 and 2013,homozygous variants were T5999C(0% vs 5.26%),G6179A(0% vs 5.26%),and C7028T(87.64% vs 71.05).%)and heterozygous variant site A7241G(0% vs 5.26%)differed between normal fertilization group and complete fertilization failure group(P<0.05).From 2014 to 2018,homozygous variant sites G6383A(59.34% vs 38.89%),C7028T(17.58% vs 47.22%),C7232T(53.85% vs33.33%)and heterozygous variant site G6023A(38.46% vs 11.11%),T6221C(38.46% vs 13.89),C6242T(39.56% vs 13.89%),A6299G(46.15% vs 13.89%),G6366A(50.55% vs 16.67%),C6410T(39.56% vs 13.89%),C6452T(37.36% vs13.89%),C6483T(38.46% vs 13.89%),T6512C(54.95% vs 16.67%),C6542T(45.05% vs 13.89%),C6569A(40.66% vs 13.89%),T6641C(31.87% vs 13.89%),C6935T(43.96% vs 16.67%),C6938T(42.86% vs 13.89%),C7028T(52.75% vs22.22%),A7146G(34.07% vs 13.89%),C7232T(31.87% vs 13.89%),C7256T(58.24)%vs16.67%)and G7316A(48.35% vs 25%)were different between the normal fertilization group and the complete fertilization failure group,P<0.05.Conclusion In the past 8 years,specific gene variants consisting of 20 fixed sites such as G6023 A and the reversion variation of the C7028 T locus appeared in the sperm MT-CO1 gene of the Han population.The mitochondrial CO1 gene containing all 20 fixed variant sites was defined as a variant gene.The variant protein still has certain biological functions,but its spatial structure stability is reduced.The variation has no adverse effect on sperm density.Significantly affected,but this variation may be one of the reasons for the decline of the sperm motility.The effect of sperm mitochondrial CO1 gene variation in Han Chinese population on the in vitro fertilization failure is not static,but rather as a dynamic process. |
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