MOLECULAR GENETICS OF Y CHROMOSOME IN MALE INFERTILITY LIOW SWEE LIAN DVM (UPM), M Sc (NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 ACKNOWLEDGEMENT I wish to express my deep appreciation to my supervisor, Professor Ng Soon Chye for his wisdom, foresight, invaluable guidance and understanding throughout the full course of this study. I am also grateful to Professor Yong Eu Leong who has introduced and guided me on the basics of molecular genetics. My gratitude also goes to Ms Tan Lay Geok, Staff Nurse Dorothy Tan and Mr John Tan Beng Lee for their support throughout this study. My special thanks to my other colleagues who have encouraged me to complete this study. Last but not least, I am indebted to my wife for her patience, encouragement and unselfish support without which this study would not have been accomplished. i TABLE OF CONTENTS Page i ACKNOWLEDGEMENT SUMMARY vii LIST OF TABLES x LIST OF FIGURES xii LIST OF PUBLICATIONS xiv CHAPTER LITERATURE REVIEW 1.0 Spermatogenesis 1.1 Genetic Control of Spermatogenesis 1.2 Human Male Infertility 1.3 1.3.1. 1.3.1.1 8 1.3.1.2 1.3.1.3 1.3.1.4 1.3.1.5 Genetic Causes of Male Infertility Chromosomal aneuploidies and rearrangements Klinefelter’s syndrome and variants (47,XXY; 46,XY/47, XXY mosaicism) 47,XYY 45,XO/46,XY mosaic or mixed dysgenesis Y chromosomal translocations Autosomal translocations 10 10 10 11 1.3.2 Androgen Insensitivity Syndromes 13 1.3.3 Submicroscopic Deletions or Microdeletions of Spermatogenesis Genes in Yq11 15 1.4 1.4.1 1.4.2 1.4.3 1.4.4 1.4.5 The Human Y Chromosome Evolution of the human Y chromosome Structure of the human Y chromosome Gene content of the human Y chromosome Molecular mapping of the human Y chromosome Non-coding and repetitive DNA 15 15 18 18 21 25 1.5 1.5.1 Azoospermia Factors and Male Infertility AZFa and candidate genes 26 28 ii 1.5.2 1.5.3 1.5.3.1 1.5.3.2 1.5.3.3 1.5.3.4 AZFb and candidate genes AZFc and candidate genes Deleted-in-AZoospermia gene CDY gene Other spermatogenesis genes in AZFc region AZF deletions are caused by homologous recombination of specific repetitive sequence blocks AZF genes as candidates for Azoospermia Factor 29 33 33 36 37 38 40 1.6.1 Assisted Reproduction Techniques and Male Infertiity The concern of assisted reproduction techniques 40 1.7 The Objectives of This Study 43 1.5.3.5 1.6 CHAPTER MATERIALS AND METHODS 39 45 2.1 Patients and controls 46 2.2 Hormone measurements 47 2.3 Cytogenetic analyses 47 2.4 Isolation of DNA from blood samples 47 2.5 Y chromosomal microdeletion analysis using multiplex PCR 48 2.6 Screening for VCY2 deletions in infertile men 49 2.7 Molecular genetic analysis of deletion of the heterochromatic region of Yq12 in an azoospermic man 53 2.8 Y chromosomal microdeletion analysis on single spermatozoon and spermatid Optimization of single cell PCR Isolation of single spermatozoa and spermatids PCR analysis of single sperm and spermatid 53 2.8.1 2.8.2 2.8.3 2.9 2.9.1 Y chromosomal microdeletion analysis on fathers and their sons conceived by intracytoplasmic sperm injection Collection of umbilical cord blood from ICSI male offspring 53 54 54 56 56 iii 2.10 56 Analysis on the effect of Y microdeletions on the outcome of Intracytoplasmic sperm injection (ICSI) Preparation of oocytes for ICSI Preparation of ejaculated sperm for ICSI Preparation of testicular sperm for ICSI Preparation of microinjection dish The technique of ICSI Fertilization, embryo growth and embryo development Pregnancy monitoring 56 57 60 60 61 61 62 RESULTS 65 Prevalence of Y chromosomal microdeletions in infertile men Ethnic composition in the cohort of infertile men Confirmation of men with Y chromosomal microdeletions in this study Y chromosomal microdeletions in men with karyotype 46XX Y microdeletion analysis in men with obstructive azoospermia Y microdeletion analysis in men with non-obstructive azoospermia Y microdeletion analysis in men with Klinefelter’s syndrome Y microdeletion analysis in men with idiopathic infertility Y microdeletions anlaysis in men with varicocele Y microdeletion analysis in men with cryptochidism Y microdeletion analysis in men with mutation in the androgen receptor (AR) gene and in females with complete androgen insensitivity syndrome (CAIS) 67 3.1.11 3.1.12 Summary of findings Discussion 82 87 3.2 3.2.1 3.2.2 3.2.3 VCY2 gene in DAZ-deleted infertile men Introduction Results of VCY deletion analysis on infertile men Discussion 94 94 95 95 2.10.1 2.10.2 2.10.3 2.10.4 2.10.5 2.10.6 2.10.7 CHAPTER 3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.1.7 3.1.8 3.1.9 3.1.10 67 67 71 72 74 76 76 78 78 80 iv 3.3. 3.3.1 3.3.2. 3.3.3 3.3.4 3.4 3.4.1 3.4.2 3.4.3 3.4.4 3.5 AZF Microdeletions of the Y Chromosome and the Outcome of Intracytoplasmic Sperm Injection Introduction Results Discussion Conclusion Y microdeletion analysis of single spermatozoa and sperm cells in infertile men Introduction Results Discussion Conclusion 103 103 104 106 109 112 112 112 117 122 123 Deletion of heterochromatic region of the Y chromosome in an infertile man with azoospermia Introduction Results Conclusion 123 124 125 DISCUSSION 130 4.1 Y chromosomal microdeletion analysis in infertile men 131 4.2 Deletions of DAZ, RBMY1 and VCY2 genes in infertile men DAZ deletions VCY2 deletions RBMY1 deletions AZFc region 131 3.5.1 3.5.2 3.5.3 CHAPTER 4.2.1 4.2.2 4.2.3 4.2.4 132 133 134 134 4.3 Y chromosomal microdeletions and germinal mosaicism in infertile men 135 4.4 Deletion of Yq heterochromatin in an azoospermic man 136 4.5 Detection of double mutations in a severe oligozoospermic man 136 4.6 Intracytoplasmic sperm injection and Y chromosomal microdeletions 136 v 4.7 137 4.7.3 4.7.4 4.7.5 Prognostic value of Y chromosomal microdeletions - how reliable is the outcome of Y deletion analysis in providing a sound prognosis? Correlation between type of Y microdeletions and phenotype Number and selection of Y-specific STS for microdeletion analysis Efficacy of Y chromosomal microdeletion analysis Consequence of deletions of spermatogenesis genes Functions of spermatogenesis genes in the AZF locus 140 141 141 4.8 Conclusion 142 BIBLIOGRAPHY 144 4.7.1 4.7.2 138 139 vi SUMMARY The successful mapping of the entire Y chromosome had resulted in the availability of a method of identifying and locating genes based on PCR analysis of specific sequence-tagged sites (STS). This STS-PCR analysis using 26 Y-specific STS is a simple, highly sensitive and effective method in isolating genes that are found within the AZF locus and the euchromatic region of Yq. This study, in predominantly ethnic Chinese patients in Singapore, defines the subgroup at risk of Y mcirodeletions, and clarifies the relative roles of different Y deletion subregions to male infertility. Although the majority of Y microdeletions were found in men with non-obstructive azoospermia and severe oligozoospermia, these deletions have also been found in a man with obstructive azoospermia. Of the 511 patients with male infertility and 101 fertile controls examined, 4.1% of azoospermic and 2.8% of severely oligozoospermic patients were found to have Y microdeletions. The prevalence of Y deletions in this study was 2.9%. Microdeletions were about twice as frequent in these azoospermic patients when compared with the severely oligozoospermic patients (2:1 ratio). Y microdeletions were not detected in the oligozoospermic patients, infertile men due to varicocele, cryptochidism or Klinefelter’s syndrome, and in fertile men. vii Complete or partial absence of genes particularly in the AZFb and AZFc regions in these infertile men have concurred their important role in spermatogenesis. Two gene families have been identified to have a major influence on male fertility, i.e. DAZ and RBMY1 genes that both encode distinct RNA-binding proteins. While DAZ located in AZFc region is found missing in most of the Y-deleted men, deletion of RBMY1 genes (located in AZFb region) only or together with DAZ are found in two individuals. The prevalence of DAZ deletions among the azoospermic and severe oligozoospermic men was almost equally distributed (Table 14; 4% and 3% respectively). However, in moderately oligozoospermic and normozoospermic men, and fertile men, Y microdeletions were not detected. The loss of the DAZ gene affected spermatogenesis leading to poor sperm production or azoospermia but does not completely impede the production of spermatozoa. Other Y-specific genes found within the AZF locus such as USP9Y, DBY1, DBY2, CDY1, CDY2, remained intact in all patients. This indicates that these genes play a minor role in spermatogenesis. Deletion of VCY2 has been found in DAZ-deleted men because VCY2 is located within the DAZ clusters in the AZFc region. However, partial deletion of VCY2, particularly VCY2-exon could result in spermatogenic failure without the deletions of other Y-specific genes. The combination of a point mutation in the AR gene and DAZ deletion has resulted in severe spermatogenic failure in a man. The loss of the entire heterochromatin of Yq, viii particularly the DYZ1 gene in another man has resulted in complete spermatogenic failure leading to SCOS phenotype. Germinal mosaicisms for Y microdeletions analyzed by PCR of single sperm cell are not common in infertile men and, if they occur the number of Ydeleted sperm cells is negligible. Y-deleted spermatozoa can participate in in vitro fertilization but may have a negative impact on the outcome of ICSI. In conclusion, this study has established that 1) the prevalence of Y microdeletions in infertile men in Singapore is 2.9%; 2) there is the cause and effect relationship between Y microdeletions and male infertility; 3) DAZ gene and the AZFc region are the most commonly deleted; 4) DAZ gene is a strong candidate for the Azoospermia Factor; 5) the occurrence of Y microdeletions is de novo; 6) germinal mosaicisms for Y microdeletions are present but uncommon; 7) there is no defined phenotype for particular pattern of Y microdeletions; 8) there is a risk of transmission of Y microdeletions from father to son by ICSI; 9) Y microdeletion analysis by STS-PCR is a simple, highly sensitive and effective method and 10) Y microdeletion analysis can be a useful diagnostic tool in identifying affected infertile men so that proper counselling can be given before treatment by ICSI. ix Ludena P, Fernandez-Piqueras J, Sentis C (1993). 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Monocentric Y chromosome with breakpoint in long Y arm; (B) Dicentric iso-Yp chromosomes and; (C) ring -Y chromosome are often found together with a 45,XO cell line in the patient’s lymphocyte nuclei pointing to the instability of the Y chromosome after these breakage events; (E) Translocations of the Y chromosome broken in Yq are possible to the X chromosome and to autosomes (F,G), mainly the acrocentric chromosomes... Klinefelter patients with increasing number of X chromosomes in their karyotype shift their sexual phenotype to the female site This suggests an X chromosomal dosage effect on the males’ gonad development (Vogt, 2004) In male infertility, the 47,XXY karyotype is found in 11% of azoospermic and 0.7% of oligozoospermic men (Huynh et al, 2002) Men with Klinefelter mosaicism 46,XY/47,XXY have severely... composition in the cohort of men with male infertility and Y chromosomal microdeletions 68 Table 6 Y microdeletion analysis in men with obstructive azoospermia 73 Table 7 Y microdeletion analysis in men with non-obstructive azoospermia 73 Table 8 Y microdeletion analysis in men with Klinefelter’s syndrome 77 Table 9 Y microdeletion analysis in men with idiopathic infertility 77 Table 10 Y microdeletions anlaysis... oligozoospermic Klinefelter patients (Bhasin et al, 2000; Krausz and Forti, 2000) 9 1.3.1.2 47,XYY Men with 47,XYY karyotype occur frequently (1:750) but their fertility status is variable (Vogt, 2004) If infertile, they are usually oligozoospermic Although the normal formation of the X -Y pairing complex in the germ cells is inhibited resulting in infertility (Vogt, 2004), the majority of spermatozoa... point of view, the process of spermatogenesis can be assumed to be regulated by orchestrated co-ordinated expression of many genes For instance, in mice, there are many genes involved in spermatogenesis (Figure 2; Matzuk and Lambs, 2002) Mutation of the genes involved in male reproduction affects specific testicular cell types and reproductive function, resulting in male infertility or subfertility in. .. Figure 30 Y microdeletion analysis of a single spermatozoon from a severe oligozoospermic man 116 Figure 31 Histological findings of testicular biopsy in a Yq12deleted man 126 Figure 32 Cytogenetics analysis of patient SOE124 127 Figure 33 STS-PCR analysis of Y microdeletions in patient SOE124 128 xiii LIST OF PUBLICATIONS Liow SL, Ghadessy FJ, Ng SC, Yong EL(1998) Y chromosome microdeletions, in azoospermic... anlaysis in men with varicocele 79 Table 11 Y microdeletion analysis in men with cryptochidism 79 Table 12 Y microdeletion analysis in men with mutation in the androgen receptor (AR) gene 83 Table 13 Y microdeletion analysis in females with complete androgen insensitivity syndrome (CAIS) 83 Table 14 Prevalence of Yq11 microdeletions in relation to sperm concentrations of patients with male infertility 92... However, up to 50% of the cause of male infertility is idiopathic (Foresta et al, 2001) and in some individuals are due to genetic defects 5 Figure 2 Genes involved in the regulation of male reproduction in the mouse Source: Matzuk and Lambs (2002) 6 Figure 3 Genetic etiologies of human male infertility Developmental disorders causing human male infertility result from a failure of gonadal development... spermatogenesis The human Y chromosome is mostly devoid of genes, while the X chromosome, its meiotic pairing partner, contains several thousand genes (Graves 1995) The Y chromosome is probably the most bizarre part of the human genome, reflecting its unique status as a huge block of largely non-recombining DNA, maintained in a permanently heterozygous state, and transmitted solely through males (Bull, 1983)... populations, since it is composed mainly of two highly repetitive sequences families, DYZ1 and DYZ2, containing about 5000 and 2000 copies of each respectively Whereas PAR1 and PAR2 represent the 5% of the entire chromosome, the majority of the length of the Y (95%) is made by the non-recombining region,NRY This includes the euchromatic and heterochromatic regions of the chromosome Whereas the heterochromatic . BIBLIOGRAPHY 144 vi SUMMARY The successful mapping of the entire Y chromosome had resulted in the availability of a method of identifying and locating genes based on PCR analysis of. 74 3.1.6 Y microdeletion analysis in men with Klinefelter’s syndrome 76 3.1.7 Y microdeletion analysis in men with idiopathic infertility 76 3.1.8 Y microdeletions anlaysis in men with. Human Male Infertility 4 1.3 Genetic Causes of Male Infertility 8 1.3.1. Chromosomal aneuploidies and rearrangements 8 1.3.1.1 Klinefelter’s syndrome and variants (47,XXY; 46,XY/47, XXY mosaicism)