VIETNAM UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ***************** UNDERGRADUATE THESIS TITLE: “STUDY ON THE ASSOCIATION OF AhRR rs2292596 AND MALE INFERTILITY IN VIETNAMESE POPULATION” Student : Tran Huu Dinh Major : Biotechnology Supervisor : Dr. Nguyen Thuy Duong Assoc. Nguyen Xuan Canh Hà Nội, 2021 DECLARATION I hereby declare that this is my own work and that neither any part of this thesis nor the whole of the thesis has not been previously published in other theses or dissertations. I certify that, to the best of my knowledge, my thesis does not violate anyone’s copyright and that any ideas, methods, citation and other materials are fully acknowledged in accordance with the standard referencing practices.
Hanoi, February 2nd, 2021 Student Tran Huu Dinh i ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere gratitude to my supervisor, Dr. Nguyen Thuy Duong, for her permission to conduct my thesis in her laboratory and also for her patience, motivation, and immense knowledge. Her enthusiasm for biology kept me constantly expanded my knowledge and continued my research. Without her guidance, this thesis would have never been accomplished.
I could not have imagined better supervisors for my undergraduate thesis. Additionally, I would like to thank Assoc. Nguyen Xuan Canh for his encouragement and instruction to study outside the the university campus. His support continuously motivated me to get involve in the scientific research field.
My sincere thanks goes to other lecturer in faculty of Biotechnology in Vietnam University of Agriculture for supporting and sharing their valuable knowledge and experiences during my thesis implementation time. Last but not least, I would like to thank my labmates in human genome laboratory for their kind assistance, and inspiration during my hard time, especially Msc. Duong Thi Thu Ha. Without her presious advices, I could not have overcome difficultities during conducting experiments.
Student Tran Huu Dinh ii CONTENTS DECLARATION. iii LIST OF TABLES. v LIST OF FIGURES. vi LIST OF ABBREVIATION.vii PART 1: INTRODUCTION.
1 PART 2: LITERATURE REVIEW. AhRR and male infertility. 9 PART 3: MATERIALS AND METHODS. Extraction of total DNA from blood samples.
Amplification of the DNA region containing AhRR rs2292596. Extraction of total DNA from blood samples. Amplification of the DNA region containing AhRR rs2292596. 23 iii PART 6: CONCLUSION.
32 iv LIST OF TABLES Table 2. Four categorizes of underlying causes of male infertility. Number and size of DNA bands of 3 genotypes of AhRR rs2292596. Quantity summary of rs2292596 genotype.
Allele frequency of polymorphism AhRR rs2292596. Correlation between rs2292596 and male infertility. 22 v LIST OF FIGURES Figure 2. Structure of Human AhR, ARNT, and AhRR.
(Sakurai et al. 2 Feedback regulation model of AhR/ AhRR signaling pathway. Discovery of genes related to male infertility (Xavier et al. Condition of PCR reaction.
Recognition site of BsuRI (HaeIII). Total DNA of six samples in 0. PCR products amplifying DNA region containing AhRR rs2292596. BsuRI-digested PCR products of six samples on agarose gel 2.
Genotyping of AhRR rs2292596 using Sanger sequencing. Frequency of allele G of Vietnamese and other populations. 25 vi LIST OF ABBREVIATION AhR Aryl hydrocarbon receptor AhRR Aryl hydrocarbon receptor repressor ARNT Aryl hydrocarbon receptor nuclear translocator AIS Androgen insensitivity syndrome aCGH Microarray-based Comparative Genomic Hybridization ART Assisted reproductive technology AZF Azoospermia factor bHLH basic helix–loop–helix bp Base pair CBAVD Congenital bilateral absence of the vas deferens CNV Copy Number Variation CUAVD Congenital unilateral absence of the vas deferens DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid EtOH Ethanol FISH Fluorescence in situ hybridization HWE Hardy – Weinberg equilibrium MMAF Multiple morphological abnormalities of the sperm flagella n Number NOA Non-obstructive azoospermia OA Obstructive azoospermia OD Optical density OR Odd ratio PAIS Partial androgen insensitivity syndrome PAS Per-Arnt-Sim vii PCR Polymerase chain reaction SNP Single nucleotide polymorphism TAE Tris – Acetate - EDTA WHO World Health Organization 95% CI 95% Confident interval viii PART 1: INTRODUCTION Reproductive impairment is an increasingly urgent problem in developing countries, especially Vietnam since the national infertile rate of couples during childbearing age accounts for up to 7. In general, male reproductive concerns such as low sperm production; abnormal sperm morphology and function; obstruction of sperm delivery ducts, etc.
are responsible for approximately one- third of the childless cases. Moreover, male infertility is a multifactorial disease caused by varicoceles, retrograde ejaculation, genetic factors, immunologic infertility, overexposure to certain environmental factors (pesticides, chemicals, alcohol, smoking, etc.), and many other sources. Among them, genetic factors contribute a significant part to the infertility since more than 2000 genes are involving the spermatogenic processes. Worldwide researches have been carried out in different directions from clinical features to triggered elements of infertility to discover the underlying causes of infertile problems, among which studies on the association of genetic polymorphism in specific populations are receiving more and more global attraction.
In Vietnam, most of the studies mainly concentrate on clinical properties along with infertile treatments, whereas genetic aspects of male infertility especially genes related to environmental conditions are extremely limited. As a result, we conducted the study “Study on the association of AhRR rs2292596 and male infertility in the Vietnamese population”. 1 PART 2: LITERATURE REVIEW 2. General concept According to World Health Organization (WHO), infertility is a disease characterized by the incapacity to achieve a clinical pregnancy after at least 12 months of unprotected sexual intercourse or due to the deterioration in the reproductive system of either partner or both (Zegers-Hochschild et al.
Male infertility is further classified as primary and secondary (Asha, 2017; Leaver, 2016; Mélodie and Christine, 2018; Zegers-Hochschild et al. The former type refers to infertile men have never successfully established a clinical pregnancy before. The latter indicates men who are unable to initiate a pregnancy but had previously initiated one or more pregnancies. Infertility globally affects about 7% of male human population (Krausz et al., 2015; Krausz and Riera-Escamilla, 2018; Xavier et al., 2020) and is currently deemed as a global public health issue, resulting in an increase in the number of governmental concerns in population growth, fertility and mortality rate, demographic balance, and national health.
Furthermore, it is an increasing problem in the entire world since the age-standardized prevalence rate of male infertility increased by 8.224% from 1990 to 2017, with an increasing rate of 0.291% per year (Sun et al. Clinical assessment Semen analysis is a credible investigation for measuring clinical andrology, male infertile, reproductive issues, epidemiology, and pregnancy risk assessments with a sensitivity of nearly 89.6%, which means that it can detect problems from 9 out of 10 men (Butt and Akram, 2013). In 2010, World Health Organization (WHO) generates a lower reference limit for human semen features by conducting an analysis of semen samples on a reference male population of over 4500 men whose partner had a time-to-pregnancy (TTP) of equal or less than 12 months. The following criteria are based on the fifth centiles (with 95 th percent confidence 2 intervals) (Cooper et al.5 ml; total sperm number, 39 million per ejaculate; sperm concentration, 15 million per ml; vitality, 58% live; progressive motility, 32%; total (progressive and nonprogressive) motility, 40%; morphologically normal forms, 4.
The term "Azoospermia" refers to the condition of incapable of producing sperms, while "Oligospermia" represents a condition, in which the sperm is at a low concentration (< 15 million sperm/ ml). “Asthenospermia” is a term used to point out motility problems of sperms, in which less than 40% of the sperm are moving, and less than 32% are swimming progressively. Abnormalities in spermatozoa are characterized by the phrase "Teratozoospermia", which means that less than 4% of the sperm are normally shaped. However, some men in reality can not produce sperm at all probably due to the blockage along the vas deferens as well as ejaculatory ducts; thus, completely no semen can be manufactured.
This status is named "Obstructive azoospermia" (OA) to separate it from “Non-obstructive azoospermia” (NOA), where sperms are still produced without any damage to the delivery system but more likely due to dampening in the production process. Causes Generally, male reproductive concerns are assumed to account for at least 50% of infertile cases (Mélodie and Christine, 2018). However, male infertility is a multifactorial disease and can be divided into non-genetic and genetic factors (Asha, 2017). Genetics accounts for more than 15% of prevalent causes of male infertility diseases, ranging from chromosome abnormalities to single nucleotide alterations (Fainberg and Kashanian, 2019; Krausz et al.
The underlying causes of male infertility based on clinical aspects are categorized in 4 groups (Table 2.1): (1) spermatogenic quantitative defects; (2) ductal dysfunction; (3) hypothalamic-pituitary axis disturbances; and (4) spermatogenic qualitative defects (Krausz and Riera-Escamilla, 2018; Tournaye et al. Four categorizes of underlying causes of male infertility. Aetiological groups Diseases Genetic candidates Chromosomal anomalies (numerical or structural) Azoospermia Y chromosome deletions (AZFa, Spermatogenic AZFb, and AZFc) quantitative defects TEX11 Oligozoospermia gr/gr deletion (AZFc region) Hypoandrogenization owing AR to PAIS CBAVD Ductal dysfunction CFTR CUAVD Kallman syndrome Hypothalamic-pituitary FGFR1, ANOS1, TAC3, GNRH1, Normosmic hypogonadotropic axis disturbances GNRHR hypogonadism Globozoospermia DPY19L2 Spermatogenic Sperm macrocephaly AURKC qualitative defects MMAF DNAH1 PCD DNAH1, DNAH11, DNAH5 AZF: Azoospermia factor; CBAVD: congenital bilateral absence of the vas deferens; CUAVD: Congenital unilateral absence of the vas deferens; MMAF, multiple morphological abnormalities of the sperm flagella; PAIS, partial androgen insensitivity; PCD, primary ciliary dyskinesia; Despite the efforts of performing many genetic methods to identify the causes of male infertility, there are still a large number of affected cases remaining as unknown causes or as idiopathic cases. The reason is probably due to the extreme genetic complexity as semen and testis histological phenotypes are heterogeneous, and more than 2,000 genes participate in spermatogenesis (Krausz and Riera-Escamilla, 2018).
Recently, genetic polymorphisms in genes with common cell function, specific spermatogenic function, and endocrine function 4 have been studied for their association with male infertility (Krausz et al. Among them, polymorphisms of AhRR and genes involved in the AhR signaling pathway had received more attention for their correlation with several diseases including male infertility. AhRR and male infertility Aryl Hydrocarbon Receptor (AhR) Repressors (AhRR), located on 5p15.33, spans over 166 kb, and contains 12 exons (Karchner et al. AhRR protein is identified as the members of the basic helix-loop-helix (bHLH) Per-ARNT-Sim (PAS) family (Hollie, 2011).
Family members of bHLH-PAS form a heterodimer with other members of the same family through their N-terminal bHLH-PAS domains (Figure 2. While the bHLH domain is responsible for DNA binding, the PAS domains (PAS-A and PAS-B) are involved in protein-protein interaction and ligand binding, which is in the PAS-B domain of AhR but not in AhRR (Sakurai et al. The protein of AhRR is involved in the regulation of cell growth and differentiation and participates in the AhR ligand-triggered pathway, which includes 3 major molecules: aryl hydrocarbon receptor (AhR), aryl hydrocarbon receptor repressors (AhRR), aryl hydrocarbon receptor nuclear translocator (ARNT) Figure 2. Structure of Human AhR, ARNT, and AhRR.
(Sakurai et al., 2017) In AhR pathway, AhR is a ligand-dependent transcription factor, and diverse exogenous and endogenous ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxins 5 (TCDD) induce it. The AhR then triggers the expressions of genes encoding molecules involved in detoxication, metabolism, and cell differentiation and proliferation. The main specified AhR target genes are CYP1A1 and AhRR. In the absence of ligands, AhR resides in the cytoplasm by forming a complex with heat shock protein 90 (Hsp90), X-associated protein 2, and p23 (Figure 2.
If TCDD presents in the cytoplasm, it binds to AhR, and the ligand-bound AhR is translocated to the nucleus and dimerizes with aryl hydrocarbon receptor nuclear translocator (ARNT). The ligand-AhR-ARNT complex binds to the cognate xenobiotic responsive elements (XREs) in the promoter/enhancer regions of multiple genes, and regulates their transcription. Meanwhile, the expression of AhRR, which is upregulated by activated AhR, negatively represses the activity of AhR (Fujii‐ Kuriyama and Kawajiri, 2011). Thus, polymorphisms in such genes might affect the mediating of dioxins into the body, resulting in male infertility.
2 Feedback regulation model of AhR/ AhRR signaling pathway 2.