VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY --------🙡 🕮 🙣-------- UNDERGRADUATE THESIS “STUDY ON THE ASSOCIATION BETWEEN TDRD1 rs541192490 AND MALE INFERTILITY IN A VIETNAMESE POPULATION” HANOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY --------🙡 🕮 🙣-------- UNDERGRADUATE THESIS “STUDY ON THE ASSOCIATION BETWEEN TDRD1 rs541192490 AND MALE INFERTILITY IN A VIETNAMESE POPULATION” STUDENT : NGUYEN MINH NGUYET CLASS : K62CNSHE MAJOR : BIOTECHNOLOGY SUPERVISORS : NGUYEN THUY DUONG, PhD : TRAN THI HONG HANH, MSc HANOI – 2022 COMMITMENT I declare that this thesis has been composed solely by myself and has not been submitted, in whole or in part, for any other degree or professional qualification. I confirmed that all methods, data, and results mentioned in this thesis are true and all references are fully cited according to standard reference practice. Hanoi, February 22nd, 2022 Student Nguyen Minh Nguyet i ACKNOWLEDGEMENT I would like to express my deepest gratitude to my supervisor, Dr. Nguyen Thuy Duong, who permitted me to work in Human Genomics Laboratory as a research student to conduct my undergraduate thesis.
While doing my thesis, she guided, supported and provided favorable conditions that helped me learn more knowledge in molecular biology, especially in research methods, then achieve targeted results. I also want to thank MSc. Tran Thi Hong Hanh for her monitoring and instruction to my study progression. I want to appreciate my sincere to all staff at Human Genomic Laboratory for their assistance and mentoring.
Especially, I want to express my thanks to MSc. Duong Thi Thu Ha for her careful and precious guidance, which was extremely valuable for my theoretical and practical study. I would like to thank the Vietnam National University of Agriculture and Faculty of Biotechnology for providing a helpful chance to do an undergraduate thesis at the Institute of Genome Research, which is a good opportunity to learn and experience after studying in the lecture hall. Last but not least, I am strongly grateful for my family’s support and encouragement throughout my study journey.
Without them, my thesis can not be completed. Student Nguyen Minh Nguyet ii CONTENTS COMMITMENT .iii LIST OF ABBREVIATIONS. v LIST OF TABLES. vi LIST OF FIGURES.
vii Part 1: INTRODUCTION. 1 Part 2: LITERATURE REVIEW. Physical and sexual factors. Environmental and lifestyle factors.
TDRD1 and male infertility. Principle of PCR-RFLP method for SNP detection. International and Vietnam research. 17 Part 3: MATERIALS AND METHODS.
Total DNA extraction from whole blood samples. Amplification of the target DNA region. Total DNA extraction from whole blood samples. Amplification of the target DNA region.
Association between TDRD1 rs541192490 and male infertility. 47 iv LIST OF ABBREVIATIONS AZF Azoospermia factor CBAVD Congenital bilateral absence of the vas deferens CF Cystic fibrosis CFTR Cystic Fibrosis Transmembrane Conductance Regulator CI Confidence interval DNA Deoxyribonucleic acid FSH Follicle-stimulating hormone GWAS Genome-wide association study HWE Hardy-Weinberg Equilibrium LINE Long interspersed nuclear elements NOA Non-obstructive azoospermia OR Odds ratio PCR Polymerase chain reaction PiRNA Piwi-interacting RNA Piwi P-element-induced wimpy testis RE Restriction enzyme RFLP Restriction fragment length polymorphism SHBG Sex hormone-binding globulin SNPs Single nucleotide polymorphisms TDRD1 Tudor domain-containing 1 WHO World Health Organization v LIST OF TABLES Table 2. The normal values for semen parameters over the years. The primer pair for amplification of TDRD rs541192490.
Genotypes and allele frequency of TDRD1 rs541192490. Association between polymorphism TDRD1 rs541192490 and male infertility. 29 vi LIST OF FIGURES Figure 2. piRNA biogenesis (Pek et al.
Structure of the typical eTudor domain (PDB: 3OMC). Restriction enzyme cleavage for SNP genotyping (Kim & Misra, 2007). PCR reaction condition. Recognition site of Psp1406I (AclI).
Total DNA of six samples in 1% agarose gel. PCR products amplified DNA region containing SNP TDRD1 rs541192490 on agarose gel 1%. Psp1406I (AclI) – digested PCR products of six samples on agarose gel 1. Genotyping result of TDRD1 rs541192490 using Sanger sequencing.
29 vii Part 1: INTRODUCTION In the recent decades, along with the population explosion, infertility has become a major health concern resulting in substantial psychological and social distresses (Bak et al. In Vietnam, a nationwide study of the National Hospital of Obstetrics and Gynecology and Hanoi Medical University also showed an ominous 7.7% of infertility rate in the childbearing age group. Infertility affects 7% of the global male partner and about 40-50% of all infertility cases are due to the “male factor” (Sharlip et al., 2002; Dada et al. The spermatogenic defect is the most common form of male infertility and idiopathic primary testicular dysfunction.
Many genetic factors are considered to contribute to this complex disease (Anawalt, 2013; Krausz et al. Spermatogenesis is a complex differentiation process of cells transformation from spermatogonia to mature spermatozoa, which is regulated by up to 2000 genes. About 20% of these genes are found to be expressed only in male germlines (Schultz et al. Thus mutations or genetic alterations in these genes may lead to abnormalities in spermatogenesis resulting in male infertility (Stouffs et al.
Piwi-interacting RNAs (piRNAs) are novel non-coding RNAs that play an essential role in spermatogenesis via piRNA biogenesis to protect the genomic DNA from transposon elements (Klattenhoff & Theurkauf, 2008; Yan, 2009). Numerous piRNA pathway-associated genes are found to be vital for the generation of piRNA (Olivieri et al., 2010; Saito et al. In particular, the Tudor domain-containing 1 (TDRD1) gene is original from the Tudor gene encoding a protein that function in the suppression of transposable elements during spermatogenesis (Chen et al. It is located on the long arm of chromosome 10 at position 10q25.
The knockout model in mouse revealed the mutation in TDRD1 leading to complete male infertility due to spermatogenic defects (Chuma 1 et al. In addition, a previous study suggested an association between single nucleotide polymorphism (SNP) rs77559927 in the TDRD1 gene and spermatogenic impairment (Zhu et al. Therefore, we decided to conduct the study "Study on the association between TDRD1 rs541192490 and male infertility in a Vietnamese population" to achieve two main purposes of determination: 1. The distribution of TDRD1 rs541192490 in the Vietnamese population.
The association between TDRD1 rs541192490 and male infertility in the Vietnamese population. 2 Part 2: LITERATURE REVIEW 2. General concepts The International Committee for Monitoring Assisted Reproductive Technology, World Health Organization (WHO) defines infertility as a disease of the reproductive system that fails to conceive the clinical pregnancy after at least 1 year of regular, unprotected sexual intercourse (Zegers-Hochschild et al. There are two types of infertility.
Primary infertility refers to couples who have not conceived a child after at least 12 months of having sex without any birth control method. Secondary infertility refers to couples who have been able to get one or more pregnancies, but now are unable. Infertility is a psychological, economic, and physiological disease-causing pain and stress, especially in a society like ours that heavily emphasizes childbearing (Kumar & Singh, 2015). An estimate of a survey reported during the time of 27-year, the age-standardized prevalence of infertility rose annually in women and men by 0.291%, respectively (Sun et al.
In half of all infertility cases, the cause of a solely male factor accounts for 20-30% and 20-30% due to a combination of both genders (Sharlip et al. Male infertility or infertility due to the "male factor" can be completed or partially defined as subfertility referring to a male's inability to conceive a child with a fertile female partner. It could be caused by a decrease in the number of spermatozoa (oligozoospermia), a decrease in sperm motility (asthenozoospermia) (Curi et al., 2003), a decrease in sperm vitality (necrozoospermia), an aberrant sperm morphology (teratozoospermia), or a combination of these factors (Sharma, 2017). Spermatogenic failure is the most severe form of male infertility affecting up to 10% of all male infertility patients leading to non-obstructive azoospermia (NOA), i.
the absence of sperm in the ejaculate (Kumar, 2013; Chiba et al. Spermatogenesis is one of the most crucial processes in the seminiferous tubules wherein the haploid mature spermatozoa are produced from diploid cells (Neto et al. A complex network of stages requiring approximately 74 days in humans comprises four main phases: (1) mitotic proliferation and differentiation into spermatocytes (spermatogoniogenesis), (2) meiotic division of spermatocytes to form spermatids (meiosis) and maturation of spermatids (changing of shape and nuclear content, spermatogenesis), finally releasing of highly specialized mature spermatozoa are released into testicular tubule lumen (spermatogenesis) (Potter & Defalco, 2017). Thousands of genes are involved in regulating and processing of spermatogenesis (Sha et al., 2002; Schultz et al.
, 2003; Schlecht et al., 2004; Ellis et al., 2007; Zamudio et al. In recent years, the association between polymorphisms in the specific genes relating to spermatogenesis and male infertility was mainly focused on most published studies (Massart et al. Clinical evaluation According to a recommendation of the American Society for Reproductive Medicine and the European Association of Urology, an initial examination includes a reproductive history and at least one semen analysis, whereas the American Urological Association requires two (Jarow et al. If the initial results are abnormal, a reproductive specialist will indicate a more thorough examination (Agarwal et al.
A crucial step in infertility workup is taking a good medical history and physical examination. These should include investigating frequency and duration of sexual intercourse, previous fertility issues or successful pregnancies; any medical history about sexually transmitted diseases, history of occupational or therapeutic exposures to certain toxins and chemicals, childhood conditions (e., cryptorchidism, postpubertal mumps orchitis, and testicular torsion or trauma) or problems with childhood development; any serious illnesses such as diabetes, previous medication, allergies, lifestyle factors (alcohol consumption, weight gain and smoking); and relevant family history of fertility problems should be taken. A thorough of body 4 habitus, secondary sexual characteristics, and genitalia (testicles, penis, and perineum) should be included in a physical examination which is a key part of male infertility evaluation. (Leaver, 2016; Agarwal et al.
Semen analysis remains the single most useful and fundamental investigation with a sensitivity of 89.6% that can detect 9 out of 10 men with a genuine male infertility problem (Butt & Akram, 2013). To detect infertility in men, specialists considered the most significant of sperm parameters below the WHO standard features consisting of low sperm concentration (oligozoospermia), poor sperm motility (asthenozoospermia), and abnormal sperm morphology (teratozoospermia) (Plachot et al. Oligozoospermia is the most significant cause of infertility accounting for 90% of male infertility problems and there is a positive association between the abnormal semen parameters and sperm count (Sabra & Al-Harbi, 2014). The problems with sperm count, motility, and morphology stem from control mechanism disorder, including pre-testicular, testicular, and post-testicular factors (Iwamoto et al.
Semen analysis reveals a piece of useful information for the initial evaluation of the infertile male. It is not a test of fertility (Jequier, 2010). The normal values for semen parameters over the years. WHO WHO WHO WHO WHO Parameters manual 1st manual 2nd manual 3rd manual 4th manual 5th edn (1980) edn (1987) edn (1992) edn (1999) edn (2010) Volume ND ≥2.5 mL Sperm 20– ≥20x10⁶/mL ≥20x10⁶/mL ≥20x10⁶/mL ≥15x10⁶/mL concentration 200x10⁶/mL Total sperm ≥40x10⁶/mL ≥40x10⁶/mL ≥40x10⁶/mL ≥40x10⁶/mL ≥39x10⁶/mL count 5 Sperm mobility (% ≥60% ≥50% ≥50% ≥50% ≥32% progressive) Sperm ND ≥50% ≥75% ≥75% ≥58% vitality (%) Sperm morphology ≥80.5% * ≥50% ≥30% ** ≥15% *** ≥4% (% normal) Note: Data extracted from the WHO Laboratory Manual for the Examination and Processing of Human Semen and Sperm–Cervical Mucus Interaction.
ND = not defined. *Mean of fertile population. ***Value not defined but strict criteria and in-vitro fertilization data suggest a 14% cutoff value. According to large-scale molecular genetics research, about 3000 genes are involved in male reproduction (Li & Zhou, 2012).
About 15% of infertility cases in men were affected by genetic defects (Krausz & Riera-Escamilla, 2018). A recent review study showed a linkage between 78 genes and 92 phenotypes of male infertility (Oud et al.