MINISTRY OF EDUCATION MINISTRY OF HEALTH HANOI MEDICAL UNIVERSITY    VU THI HUYEN EVALUATE THE VARIABILITY OF SOME GENES ENCODING XENOBIOTIC METABOLISM IN INFERTILE MEN Specialization : Biomedical - Genetic Code : 62720111 SUMMARY OF DOCTORAL DISSERTATION HANOI - 2019 THIS STUDY WAS COMPLETED IN: HANOI MEDICAL UNIVERSITY Spervisor: Assoc Prof MD Tran Duc Phan PhD Nguyen Thi Trang Reviewer 1: Assoc Prof MD Phan Thi Hoan Reviewer 2: Assoc Prof MD Tran Van Khoa Reviewer 3: Assoc Prof MD Nguyen Nam Thang The thesis will be defended before the Examining Board at university level in Hanoi Medical University At …… o’clock……… Date …… Month …… Year… This thesis could be found at - National Library - Centre Medical Information Library INTRODUCTION Infertility is a pathological condition that tends to increase There are many causes of male infertility The important basis of infertility treatment involves diagnostic procedures that allow discovering its reason and introducing effective treatment A xenobiotics are non-biotic substances that are usually naturally produced Xenobiotics metabolism disorders can lead to increased production of free radicals and the risk of male infertility In humans, CYP1A1, NAT2, and GSTP1 are genes that regulate antioxidant enzymes The gene mutation can lead to dysfunction of detoxifying enzymes leading to chronic diseases, including male infertility, cancer In Vietnam, there are no studies evaluating the effects of these three genes in male infertility patients, so we conducted this study with two objectives: Determine the nucleotide mutations of CYP1A1, NAT2, and GSTP1 genes in primary male infertile Analysis of the relationship between the common nucleotid variants of CYP1A1, NAT2, GSTP1 genes with male infertility New finding of the thesis: This is the first study in Vietnam using ARMS-PCR technique to determine polymorphisms of CYP1A1, NAT2 and GSTP1 genes: CYP1A1 2455A>G, NAT2 481C>T, NAT2 590G>A, GSTP1 313G>A, GSTP1 341C>T were higher in the infertile group than in the control group The study found that CYP1A1 2455A>G genotype AG increased the risk of male infertility 4.09 times, polymorphism NAT2 481C>T (rs1799929), CT genotype increased the risk of male infertility by 4.1 times, polymorphism NAT2 590G>A (rs1799930) genotype GA increased the risk of male infertility by 3.3 times, GSTP1 313G>A increased the risk of male infertility by 3.25 times, GSTP1 polymorphism 341C>T (rs1138272), CT genotype increased the risk of male infertility 6.9 times Combining polymorphisms GSTP1 and NAT2 or CYP1A1 and NAT2 or GSTP1 and CYP1A1 increased the risk of infertility than only one polymorphism In this research, we found that the gene polymorphic complexes of male infertility in Vietnam include: Gene polymorphisms combination [GSTP1 (341C> T); NAT2 (590G> A); CYP1A1 (2455A> G)] or [GSTP1 (341C> T); CYP1A1 (2455A> G); NAT2 (481C> T)] expression of additive interaction, but [NAT2 (590G> A); CYP1A1 (2455A> G)] or [CYP1A1 (2455A> G)]; NAT2 (481C> T)] is a complementary interaction Using oxysperm kit, we found that infertile men with mutations in the xenobiotics metabolism are 29.87 times increased risk of oxidative stress than those who not The structure of the thesis: The thesis included 122 pages with 27 tables, 21 figures and graphs, 193 references, of which 178 are in English and 15 Vietnamese Thesis structure including pages introduction, 39 page overview, 20 page object and methodology, 26 page results, 32 page discussion, page conclusions and recommendations Chapter OVERVIEW 1.1 Infertility and male infertility According to the WHO, infertility is found in 12% -15% of reproductive age couples, equivalent to 50-80 million people in the world There are still cases unknown reason infertility Today, environmental problems, toxic chemicals and stress have also increased the rate of infertility and become a matter of concern 1.2 The causes of male infertility 1.2.1 Genetic causes 1.2.1.1 Cyto- genetic causes Numeral and structural chromosomal abnormalities including autosme and X/Y chromosome, especially, Klinerfelter syndrome These abnormalities reduce the reproductive process resulting in malformation of male reproductive function 1.2.1.2 Molecular genetic causes Base on the development of molecular biology, the causes of male infertility at the molecular level have also been discovered They are micro-deletion of Y chromosome (deletation of AZF locus), DNA sperm fragmentation, polymorphisms such as CFTR, AR, Kallmann's syndrome, folate metabolism, metabolism of xenobiotics 1.2.2 Biochemical causes Fructose, zinc, phosphatase, and citric acid have been shown to play an important role in sperm motility, morphology and density 1.2.3 Endocrine causes GnRH, GH, Testosterone, Inhibin participate in the process of sperm establist as well as the differentiation and development of sperm 1.2.4 Pathology affects male fertility The common diseases that affect the number and quality of sperm are: varicocele, testicular inflammation, testicular cancer, systemic diseases, infections 1.2.5 Age of reproduction Studies have shown that the higher the age, the less number sperm decreases 1.2.6 Environment The most common environmental factors causing male infertility are heavy metals, cigarette smoke, ethylene dibromide, chromium, ethylene dibromide, pesticides, dioxin These agents inhibit the growth of sperm reducing, density and mobility of sperm reducing 1.3 Xenobiotics and the metabolism of xenobiotics in the body Xenobiotics are chemicals that are not produced by the organism, which, if not metabolized and excreted by the body, will cause increasing free radicals, which oxidize the molecule, and causes DNA to mutate And causing diseases including infertility in men 1.4 Genes coding for the enzyme metabolize xenobiotics primarily 1.4.1 CYP1A1 gene In the cytochrome P450 system, CYP1A1 is the major enzyme responsible for the metabolism of aromatic hydrocarbons, heterocyclic amino acids If this metabolism is not good leads to increased free radicals causing DNA changes leading to infertility in men CYP1A1 is a gene on the 15th chromosome (15q24.2-4) consist of 6,069 base pairs, exons and introns CYP1A1 Protein has 512 amino acids with 58165 Dalton, it’s plays a major role in phase I metabolism of xenobiotics 1.4.2 Gen GSTP1 The GSTP1 gene is located on the longitudinal branch of chromosome 11 consist of 3066 base pairs, located at 11q13.3 This enzyme catalyzes the detoxification reaction by binding glutathione, neutralizing toxic substances, xenobiotics and products of oxidative stress The polymorphisms of the GSTP1 gene can cause non-normal metabolism, causing free radical increasing and causing chronic diseases including infertility 1.4.3 Gene NAT2 Located on chromosome 8, there are exons, intron Protein encoded by NAT2 has 290 amino acids, molecular weight is 33,542 Dalton This enzyme participates in phase metabolism of xenobiotics, primarily active in the acetylation of xenobiotics, which makes products easily excreted The common polymorphism of NAT2 disrupts the detoxification process of the enzyme metabolizing xenobiotics which leads to male infertility Chapter OBJECTIVES AND METHODOLOGY 2.1 Research subjects 2.1.1 Sample size 4C Sample size is determined by the formula: n  (ln OR)2 pq Where: p = 0.29 is the frequency of CYP1A1 gene polymorphism, according to a study by Sena Erdogan Aydes (this study has the smallest p, so the sample size is large) C: Constants are related to type I and type II errors Take the value α= 0.05; β = 0.2 then C = 7.85 Replace the values into n = 82.5 In fact, we studied the 170 infertile group and the 170 control group 2.1.2 Criteria for selecting research subjects Infertility group: unknown reason infertility male: with azoospermia or severe oligospermia (A of the GSTP1 gene, all other genes with a different distribution to the Hardy-Weinberg distribution have been shown to have the effect of polymorphisms that alter the frequency of infertility 4.2.2 Genotype distribution and relationship between polymorphism of CYP1A1 2455A> G with male infertility Studies by Luo H (2014, Nejati M (2016), Ramgir S.S (2017) have confirmed that CYP1A1 polymorphism is closely related to male infertility increased 4.09 times inferiority to the control group, GG and AG conjugated genes increased the likelihood of infertility 4.39, and G-induced infertility increased to 3.27 4.2.3 Relationship between polymorphic NAT2 481C>T (rs1799929) and NAT2 590 G>A (rs1799930) with male infertility For the polymorphic NAT2 590G> A gene (rs1799930), the 18 heterologous GA gene was 53.5% larger than the control group at 25.9% Allele A in the group of infertility 31.5%, control group 12.9% GA heterogeneity increased the infertility rate by 3.3 times; People with GA and AA combinations, the possibility of infertility increased by 3.99: GA + AA increased 3.99 times; Bring alleles A, the possibility of infertility increased 3.09 times In the NAT2 gene, position 481, transformed genotype (CT) increased the likelihood of infertility by 4.1 Alenes metabolism (T) was greater in the control group than in the control group Our research is consistent with studies by Dinh Doan Long (2010) and Yarosh (2014) 4.2.4 Relationship between polymorphism of GSTP1 313G> A (rs1695) and GSTP1 341C> T (rs1138272) with male infertility On polymorphism of GSTP1 313G>A (rs1695), the results showed that the rate of mutant genotypes in the infertile group was higher than the control group; Incorporating GA + AA, infertility increased 5.16 times; AG GSTP1 gene localization at position 313 increased the risk of infertility 3.25 times, heterologous and mutant genotypes (GA and AA) increased the likelihood of infertility 5.16 The mutant carriers (A) increased the risk of infertility by 5.18 times With the GSTP1 341C>T gene polymorphism (rs1138272): the rate of mutant genotypes in the infertile group was higher than that of the control group The mutant and mutant genotypes of GSTP1 at position 341 increased the risk of infertility by 6.9 The mutant (T) gene of GSTP1 at 341 increased the risk of infertility by 5.5 times that of normal allelic carriers (C) 4.3 Correlation between common nucleotid variants of CYP1A1, GSTP1 and NAT2 genes for male infertility 19 4.3.1 Correlation between polymorphisms of CYP1A1, GSTP1 and NAT2 with male infertility According to our study, people with genotype (590GA or 590AA) and (2455AG or 2455GG), the risk of infertility increased 4.86 times; Except for genotypes that carry only one of the 590GG polymorphisms and 2455AG or 2455GG and 2455AA and 590GA or 590AA People with both polymorphism (481CT or 481TT) and (2455AG or 2455GG) increased the risk of infertility by 6.37 times We have not found any authors to study the association of CYP1A1 2455A>G and NAT2 (590G>A) polymorphisms; (481C>T) with male infertility Our study found that the greater the polymorphism of CYP1A1, NAT2, the higher the risk of infertility Persons with simultaneous CYP1A1 polymorphism (2455A>G) and GSTP1 (313G>A); GSTP1 (341C>T) increased 4.02 times, with genotypes (313GA or 313AA) and (2455AG or 2455GG) and increased 10.51 with genotypes (341CT or 341TT) and 2455AG or 2455GG) About the additive effect of the NAT2 polymorphic polymorphism (590G>A); (481C>T) with GSTP1 (313G>A); (341C>T), we found that genotypes with both polymorphisms (481CT or 481TT) and (313GA or 313AA) increased the risk of infertility by 9.18 times Both polymorphisms (481CT or 481TT) and (341CT or 341TT) increased the risk of infertility by 4.28 Both polymorphisms (590GA or 590AA) and (313GA or 313AA) increased the risk of infertility by 7.64 Both polymorphisms (590GA or 590AA) and (341CT or 341TT) increased the risk of infertility by 8.56 Thus, the cumulative effect of CYP1A1, NAT2, and GSTP1 polymorphisms on the infertility of male infertility can be observed Although only one polymorph is present in one of these genes, there is a risk of infertility in men 20 To assess the gene-wide interaction with large numbers of loci, we used the MDR method All SNP interactions were tested using 10fold crossover analysis of 2, 3, and genotypes including both high-risk genotypes and low-risk genotypes This is the first study in Vietnam to analyze gene combination From all genetic interactions, the most valuable combination was the GSTP1 (341C>T) and CYP1A1 (2455A>G) and NAT2 (590G>A) combinations; with a reproducibility of 30.8% (Monte-Carlo, p G) and NAT2 (590G>A) and NAT2 (481C>T) with a 100% repeatability and a predicted error of 22.3% (predicted error T) and CYP1A1 (2455A>G) and NAT2 (590G>A) polymorphisms; or GSTP1 (341C>T) and CYP1A1 (2455A>G) and NAT2 (481C>T) show cumulative interaction, in which CYP1A1 (2455A>G) and NAT2 (590G>A) or CYP1A1 (2455A>G) and NAT2 (481C>T) are complementary interactions Therefore, individuals with mixed polymorphic genes have a significantly increased risk of male infertility Our study indicates that combinations of GSTP1, NAT2 and CYP1A1 polymorphisms may increase the risk of infertility This may be due to oxidative stress due to excessive activity of ROS leading to alterations in GSTP and CYP1A1 genes and non-recovery of sperm DNA by abnormal metabolism when transgenic NAT2 4.3.2 Relationship between oxidative stress levels in semen in male patients with polymorphisms in xenobiotics metabolism OS has four levels from to and is divided into two groups of HOS (including Levels and 4) and LOS (Level and OS) Comparison between the two groups: Patients with spermatozoa in 21 semen and at least one polymorphisms that metabolize xenobiotics; Controls are those without any gene polymorphism We collected 71 patients in the disease group and 72 patients in the control group In the group of patients, high OS (HOS) was 78.9%, low OS (LOS) was 21.1% In contrast, in the control group, high OS (HOS) was only 11.1%, low OS was 88.9% This suggests that infertile men with polymorphisms that metabolize xenobiotics have a 29.87-fold higher risk of oxidative stress than normal people The difference was statistically significant with p G polymorphism in the infertile group was 52.4% and 1.8%, higer in the control group Frequency of CT genotype of NAT2: polymorphism 481C>T was 50.6% in the infertile group and 20% in the control group; Frequency of GA and AA genotypes of NAT2 590 polymorphism in the infertile group was 53.5% and 4.7%; in the control group was 25.9% and 0% - Gene GSTP1: Frequency of GA and AA genotypes 313G>A in the infertile group was 35.9% and 11.2%; in the control group was 14.7% and 0%; Frequency of GA and AA genotypes 341C>T in the inferior group was 38.2%; in the control group was 8.2% Relationship between polymorphism of GSTP1, NAT2, and CYP1A1 with infertility - Polymorph CYP1A1 2455A> G, A allele replaced by G allele increases the risk of male infertility compared to A allele 3.27 times - Polymorphic NAT2 481C> T (rs1799929), C allele replaced by T allele increased the risk of infertility compared to C allele up 3.05 times - Polymorph NAT2 590 G> A (rs1799930), G allele replaced by A allele increases the risk of male infertility compared to G allele 3.09 times - GSTP1 313G polymorphism> A: G allele was replaced by A allele to increase the risk of male infertility compared to G allele 5.18 times - GSTP1 341C> T polymorphism (rs1138272): C allele replaced by T allele increased the risk of male infertility compared to C allele 5.5 times 23 Combining polymorphs: - Combining two GSTP1 and NAT2 polymorphisms, the risk of infertility is higher when there is only one type of polymorphism - Combining two polymorphisms CYP1A1 and NAT2 the risk of infertility is higher when there is only one type of polymorphism - Combining polymorphisms GSTP1 and CYP1A1 are at higher risk of infertility when there is only polymorphic type Combining the polymorphisms of NAT2, GSTP1 and CYP1A1: Complex GSTP1(341C>T); NAT2(590G>A); CYP1A1(2455A>G) with 100% reproducibility and prediction error of 30.8% Type GSTP1 (341C>T); NAT2 (590G>A); CYP1A1 (2455A>G); NAT2 (481C>T) with a 100% repeatability and a predicted error of 22.3% Complex of polymorphisms GSTP1 (341C>T); NAT2 (590G>A); CYP1A1 (2455A>G) or GSTP1 (341C>T); CYP1A1 (2455A>G); NAT2 (481C>T) exhibits additive interaction; NAT2 (590G>A); CYP1A1 (2455A>G) or CYP1A1 (2455A>G); NAT2 (481C>T) is a complementary interaction - On oxidative stress and polymorphism, xenobiotics metabolize There is a correlation between oxidative stress in semen in infertile men with polymorphisms that metabolize xenobiotics Infertile men with mutations in the xenobiotics metabolism, were 29.87 times more likely to develop oxidative stress than those who did not 24 RECOMMENDATION It is necessary to widely apply tests to identify polymorphisms of xenobiotics to determine the risk of infertility for male infertility cases of unknown causes, thereby taking preventive measures to limit infertility More research is needed on gene-environment interactions related to the expression of CYP1A1, NAT2 and GSTP gene polymorphisms with male infertility, especially the assessment of cigarette smoke and alcohol sensitivity with infertility in males LIST OF PUBLICATION RELATED TO THE DISSERTATION Nguyen Thi Trang, Vu Thi Huyen (2018) Polymorphism of xenobiotic detoxification genes and male infertility In: Male infertility Intech Open, United Kingdom, pp.1-19 Nguyen Thi Trang, Vu Thi Huyen, Nguyen Thanh Tuan, Tran Duc Phan (2018) Association of N-acetyltransferase-2 and glutathione Stransferase polymorphisms with idiopathic male infertility in Vietnam male subjects Chemico-Biological interactions 286(2018)11-16 Vu Thi Huyen, Nguyen Thi Trang, Uong Ngoc Nguyen, Tran Duc Phan (2017) Indentification of genetic polymorphism of xenobiotic metabolic enzyms NAT2 and GSTP1 in infertile men by ARMS-PC method Journal of Military Pharmaco-medicine 42(9):297-302 Vu Thi Huyen, Nguyen Thi Trang, Tran Duc Phan (2018) Indentification of polymorphism of NAT2, GSTP1 genes and frimary infertille men, oxidative stress Vietnam Medical jounal, 463: 70-73 Vu Thi Huyen, Tran Duc Phan, Nguyen Thi Trang (2018) Relationship between oxidative stress and sperm DNA fragmention in male infertility Journal of Medical Reseach.Volume 110, (1), 25-31 Bui Thi Nga, Vu Thi Huyen, Nguyen Thi Trang, Luong Thi Lan Anh, Tran Duc Phan (2018) Cytochrome P450 1A1 gene polymorphism and the risk of idiopathic male infertility in Vietnam Vietnam Medical jounal 8(469):391-396 Vu Thi Huyen, Nguyen Thi Trang, Tran Duc Phan (2018) Improved technical protocol to determine the leve of oxidative stress in seminal plasma n male infertility patients with sperm DNA fragmentation Vietnam Medical jounal 8(469):391-396 ... GSTP1 are genes that regulate antioxidant enzymes The gene mutation can lead to dysfunction of detoxifying enzymes leading to chronic diseases, including male infertility, cancer In Vietnam, there... Thi Lan Anh, Tran Duc Phan (2018) Cytochrome P450 1A1 gene polymorphism and the risk of idiopathic male infertility in Vietnam Vietnam Medical jounal 8(469):391-396 Vu Thi Huyen, Nguyen Thi Trang,... mutant genotypes in the infertile group was higher than that of the control group The mutant and mutant genotypes of GSTP1 at position 341 increased the risk of infertility by 6.9 The mutant