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 Prof Dr 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 Prof Dr 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 i ACKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATION .vii PART 1: INTRODUCTION PART 2: LITERATURE REVIEW 2.1 General concept 2.2 Clinical assessment 2.3 Causes 2.4 AhRR and male infertility 2.5 International research 2.6 Vietnam research PART 3: MATERIALS AND METHODS 11 3.1 Study subjects 11 3.2 Materials 11 3.3 Methods 12 3.3.1 Extraction of total DNA from blood samples 12 3.3.2 Primer design 13 3.3.3 Amplification of the DNA region containing AhRR rs2292596 13 3.3.4 PCR-RFLP 13 3.3.5 PCR purification 14 3.3.7 Statistical analysis 16 PART 4: RESULTS 18 4.1 Extraction of total DNA from blood samples 18 4.2 Amplification of the DNA region containing AhRR rs2292596 18 4.3 PCR-RFLP 19 4.4 Sanger sequencing 20 4.4 Statistical analysis 21 PART 5: DISCUSSION 23 iii PART 6: CONCLUSION 26 PART 7: REFERENCES 27 APPENDIX 32 iv LIST OF TABLES Table Four categorizes of underlying causes of male infertility Table Primers information 11 Table Number and size of DNA bands of genotypes of AhRR rs2292596 14 Table Quantity summary of rs2292596 genotype 20 Table Allele frequency of polymorphism AhRR rs2292596 21 Table Correlation between rs2292596 and male infertility 22 v LIST OF FIGURES Figure Structure of Human AhR, ARNT, and AhRR (Sakurai et al., 2017) Figure 2 Feedback regulation model of AhR/ AhRR signaling pathway Figure Discovery of genes related to male infertility (Xavier et al., 2020) Figure Condition of PCR reaction 13 Figure Recognition site of BsuRI (HaeIII) 14 Figure Total DNA of six samples in 0.8% agarose gel 18 Figure PCR products amplifying DNA region containing AhRR rs2292596 19 Figure BsuRI-digested PCR products of six samples on agarose gel 2.5 % 19 Figure 4 Genotyping of AhRR rs2292596 using Sanger sequencing 21 Figure 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 7.7% 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 onethird 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” 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 VSC150 VSC151 VSC152 VSC153 VSC154 VSC155 VSC156 VSC157 VSC158 VSC159 VSC160 VSC161 VSC162 VSC163 VSC164 VSC165 VSC166 VSC167 VSC168 VSC169 VSC170 VSC171 VSC172 VSC173 VSC174 Phạm Xuân C Trịnh Tuấn A Lò Thế H Lưu Quang B Lê Văn L Trần Thái K Đặng Việt S Đỗ Ngọc M Đỗ Xuân H Bùi Duy C Phan Công T Vũ H Lê Văn B Nguyễn Văn C Phạm Văn Đ Nguyễn Thành L Nguyễn Vĩnh B Phan Quốc P Nguyễn Phùng D Phạm Văn N Đoàn Trung L Long Minh H Quách Ngọc H Nguyễn Văn H Trịnh Xuân T 1987 1997 1993 1995 1988 1992 1992 1980 1975 1985 1958 1980 1986 1991 1978 1986 1993 1974 1986 1993 1990 1985 1989 1987 1992 Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE 15.342 23.147 30.799 19.262 36.181 14.091 22.703 18.524 11.454 5.394 6.262 12.309 9.261 13.873 13.485 8.303 11.741 13.101 27.558 15.949 34.026 27.021 14.854 14.442 4.374 1.752 1.788 1.827 1.724 1.755 1.817 1.827 1.756 1.719 1.775 1.745 1.628 1.885 1.702 1.723 1.832 1.835 1.657 1.83 1.765 1.79 1.764 1.71 1.814 1.79 47 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 VSC175 VSC176 VSC177 VSC178 VSC179 VSC180 VSC181 VSC182 VSC183 VSC184 VSC185 VSC186 VSC187 VSC188 VSC189 VSC190 VSC191 VSC192 VSC193 VSC194 VSC195 VSC196 VSC197 VSC198 VSC199 Dương Ngô T Nguyễn Văn H Đậu Khắc T Trần Minh T Đào Văn T Nguyễn Viết A Nguyễn Hoàng H Nguyễn Văn T Lê Văn L Trịnh Viết H Nguyễn Trường D Ngơ Thế T Trần Đình T Đình Đức D Phạm Quốc D Quàng Mạnh H Thân Văn T Lê Quang T Đỗ Mạnh Q Vũ Quý N Nguyễn Hải T Bùi Văn C Nguyễn Văn T Đinh Văn T Trần Nam L 1972 1982 1989 1992 1986 1989 1996 1991 1986 1985 1992 1993 1986 1990 1995 1991 1992 1991 1994 1968 1988 1992 1993 1986 1993 Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE 8.43 35.457 19.401 16.045 13.402 7.24 12.09 16.25 33.537 17.917 29.614 19.126 20.231 14.389 8.175 22.739 13.817 12.297 17.008 9.448 26.436 13.021 33.12 33.285 13.916 1.863 1.817 1.808 1.807 1.718 1.846 1.872 1.82 1.816 1.771 1.756 1.771 1.8 1.723 1.848 1.764 1.761 1.794 1.807 1.81 1.821 1.796 1.828 1.825 1.758 48 418 419 420 421 422 VSC200 VSC201 VSC202 VSC203 VSC204 Vi Quang H Mai Quang L Bùi Văn S Trần Văn D Vũ Văn H 1992 1989 1995 1987 1989 Male Male Male Male Male FALSE FALSE FALSE FALSE FALSE 19.741 10.347 20.523 26.776 18.508 1.796 1.75 1.779 1.718 1.667 49 Appendix 2: DNA extraction result 50 51 Figure Electrophoresis results of total DNA of 422 samples – 43: Patient VSN001 to VSN043; 44 – 150: Patient VSN045 to VSN151; 151 – 218: Patient VSN163 to VSN230; 219 – 422: Control VSC001 to VSC204 52 Appendix 3: PCR result 53 54 Figure PCR products of DNA region containing AhRR rs2292596 of 422 samples M: Marker 100 bp; N: Negative control – 43: Patients VSN001 to VSN043 44 – 150: Patients VSN045 to VSN151 151 – 218: Patients VSN163 to VSN230 219 – 422: Control VSC001 to VSC204 55 Appendix 4: PCR-RFLP result 56 57 58 Figure BsuRI-digested PCR products of 422 samples M: Marker 100 bp; – 43: Patients VSN001 to VSN043 44 – 150: Patients VSN045 to VSN151 151 – 218: Patients VSN163 to VSN230 219 – 422: Control VSC001 to VSC204 59 Appendix 5: Sanger sequencing result (1) (2) VSN001 (3) VSN021 (4) VSN039 (5) VSN040 (6) VSN082 (7) VSN119 (8) VSN148 (9) VSN170 (10) VSN176 (11) VSN186 (12) VSC011 VSC015 60 (13) (14) VSC027 (15) VSC028 (16) VSC033 (17) VSC034 (18) VSC035 (19) VSC036 (20) VSC052 VSC064 Figure Sanger sequencing results of 20 samples – 10: Patients; 11 – 20: Controls; Wildtype form CC: 4, 5; Heterozygous form CG: 1, - 20; Homozygous form GG: 2, 3, 61