| Objectives: Polycystic Ovary Syndrome(PCOS)is a syndrome caused by endocrine and metabolic disorder,which has the clinical characters of the ovulation or non-ovulation and cause the infertility for around 20% women of child-bearing age.Previous studies have shown that both nuclear genes and environment were the two main factors participated into the process of the pathogenesis of PCOS,but the Pathogenesis of PCOS was still unclear.In this study,the relationships between mitochondria DNA(mt DNA)haplogroup(HG)and PCOS were disclosed by analyzing the dataset of PCOS populations and the function of different mt DNA haplogroup on mitochondrial function.Methods:(1)To understand the impact of PCOS on infertility,we selected347 patients with PCOS factor and 260 patients with tubal factor,from January2016 to December 2019,who received IVF-ET treatment in the Department of the First Affiliated Hospital of Kunming Medical University.and clinical datas were analyze.(2)To further explore the correlation between different mt DNA haplogroups and PCOS,11 families containing two or more PCOS patients were involved,and one representative individual was selected for peripheral blood(PB)samples collection,genomic DNA extraction,amplification,measurement,sequence comparison and haplogroup division of mt DNA genome full sequence.The mt DNA phylogenetic relationship tree of PCOS families was constructed.The proportion of haplogroups of 11 PCOS families was counted,and then compared with the data of our previous research(the proportion of mt DNA haplogroups in 817 control colonies).(3)In order to further verify the influence of different mt DNA haplotypic groups on PCOS at the population level,70 PCOS patients were randomly selected and their PB samples were collected,by performing the D-loop region sequence determination of mt DNA and the division of haplogroups.Based on the proportion of different haplogroups as input factors,the PCOS colony and the control colonies in adjacent areas were clustered.Yuxi Han ethnic groups with similar genetic structure to PCOS colony was selected for statistical analysis of haplogroups and mutation sites of D-loop region in PCOS colony and Yuxi Han ethnic groups.(4)In order to further study the effect of different mt DNA haplogroups on cellular energy supply,considering that the exertion of mitochondrial function is jointly regulated by mitochondria and nuclear genes,15 healthy individuals were randomly selected for PB samples collection.By sequence determination,we determined the haplogroup corresponding to each individual.After that,collected PB samples again,and we extracted the desired individual platelets and fused it with 143 B rho 0 cells to construct a fusion cell line.Then,we carried out relevant mitochondrial function and damage detection experiments.The cells were stained with JC-1 to determine the mitochondrial membrane potential level of cells.2’,7’-Dichlorofluorescein diacetate(DCFH-DA)stained cells to determine cell ROS production,and nonylacridine orange(NAO)stained cells to quantify the quality of cell mitochondria;the above index assays were analyzed using flow cytometry.Results:(1)According to clinical data analysis,the PCOS group had higher BMI,AFC and AMH levels than the control group.The number of oocytes,mature oocytes,fertilized embryos,normal fertilization,cleavage,available embryos,highquality D3 embryos and blastocysts were higher than those in the control group(P<0.05),but the incidence rate of high-quality embryos and blastocyst rate in the PCOS group were significantly lower than those in the control group(P<0.05).(2)According to the comparative analysis of PCOS family data,11 families matched mt DNA haplogroup A(2/11),B(2/11),D(4/11),R9(2/11)and M7(1/11),among which mt DNA haplogroup D accounted for about 36.36%(4/11)of the PCOS family.Moreover,the proportion of this haplogroup in PCOS families was much higher than that of 13.1% from 817 control groups on the Asian continent in the previous study.(3)By analysing the data of PCOS colony and adjacent regional colonies,similar genetic structure was found between PCOS colony and Yuxi Han population in Yunnan.Then,we performed OR analysis on haplogroup from these two groups,the results suggested that there were a total of 8 variations with statistical differences,including 16164(P=0.011,OR=5.933),16182C(P=0.003,OR=2.385),16187(P=0.046,OR=8.687),16360(P=0.019,OR=4.843)in the hypervariable I region and44+C(P=0.016,OR=24.971),150(P<0.001,OR=10.628),154(P=0.016,OR=24.971),193(P=0.012,OR=9.407)in the hypervariable II region.In addition,mt DNA variations 16182 C from HVR-I and mt DNA variations 150 from HVR-II are boundary anchors for haplogroup D and its clade D5.(4)According to the data of flow cytometry,the MMP fluorescence intensity ratio(PE/FITC)of haplogroups D4 a and D4 h were lower than that of the control group(P<0.05),the ROS fluorescence intensity of the experimental group cells was lower than that of the control group(P<0.05),and the MM fluorescence intensity of the cells of the experimental group was higher than that of the control group(P<0.05).Conclusions: Compared with the control group,PCOS patients had a highlevel ovarian reserve function,but a lower proportion of high-quality embryos.mt DNA might affects the occurrence and development of PCOS,and mt DNA haplogroup D has an important impact on the pathogenesis of PCOS.mt DNA haplogroup D is more resistant to oxidative damage and apoptosis than group M9,that means individuals with mt DNA haplogroup D presents a potential benefit role to PCOS patients. |
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