Medicine, Faculty of
Obstetrics and Gynaecology, Department of
DSpace
UBCV
Tang, Steven Siu Yan
Master of Science - MSc
University of British Columbia
Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients.
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ANEUPLOIDY AND DNA FRAGMENTATION IN MORPHOLOGICALLY ABNORMAL SPERM by STEVEN SIU YAN TANG B.Sc., The University of British Columbia, 1997 A THESIS SUMBITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS IN SCIENCE in THE FACULTY OF GRADUATE STUDIES THE UNIVERSTIY OF BRITISH COLUMBIA (Vancouver) June 2008 (Reproductive and Developmental Sciences) © Steven Siu Yan Tang, 2008 ii ABSTRACT Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed- supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients. iii TABLE OF CONTENTS Abstract .................................................................................................................................... ii Table of Contents.................................................................................................................... iii List of Tables .......................................................................................................................... vi List of Figures ........................................................................................................................ vii List of Abbreviations ............................................................................................................ viii Glossary .................................................................................................................................. xi Acknowledgements................................................................................................................ xii CHAPTER I General Introduction.................................................................................... 1 1.1 Development of Male Germ Cells......................................................... 1 1.1.1 Development of Primordial Germ Cells ...................................... 1 1.1.2 Spermatogenesis and Meiosis ...................................................... 1 1.1.3 Spermiogenesis ............................................................................ 3 1.1.4 Hormonal Control of Spermatogenesis........................................ 4 1.1.5 Germ Cell Death and Apoptosis .................................................. 5 1.2 Abnormalities of Spermatogenesis........................................................ 6 1.2.1 Errors in Meiosis and Aneuploidy ............................................... 6 1.2.2 Abnormal Sperm Maturation ....................................................... 8 1.3 Male-factor Infertility ............................................................................ 9 1.3.1 Causes of Male-factor Infertilty................................................... 9 1.3.2 Genetic Origin of Male-factor Infertility ................................... 10 1.3.2.1 Chromosomal Abnormalities ....................................... 10 1.3.2.1.1 Constitutional Aneuploidy ............................. 10 1.3.2.1.2 Structural Chromosomal Aberrations ............ 11 1.3.2.2 Specific Gene Disorders ............................................... 12 1.3.2.3 Other Genetic Factors................................................... 14 1.4 Treatment of Male-factor Infertility .................................................... 15 1.4.1 Standard Assessments................................................................ 16 1.4.2 Application of ICSI in Male-factor Infertility ........................... 18 1.4.2.1 Safety of ICSI............................................................... 19 iv 1.4.2.1.1 Chromosomal Abnormalities in ICSI Conceptions .................................................. 19 1.4.2.1.2 Genetic Abnormalities in ICSI....................... 20 1.5 Sperm Selection Criteria for ICSI ....................................................... 21 1.5.1 Genetic Determinants in Sperm from Infertile Men.................. 21 1.5.1.1 Analysis of Numerical Chromosomal Abnormalities in Sperm ........................................................................ 21 1.5.1.1.1 Sperm Karyotyping ........................................ 21 1.5.1.1.2 FISH Analysis of Sperm ................................ 22 1.5.1.2 DNA Fragmentation in Sperm...................................... 26 CHAPTER II Statement of Problem and Research Methodology ................................... 29 2.1 Aim of the study .................................................................................. 29 2.2 Significance of the Study..................................................................... 30 2.3 Subject Inclusion Criteria .................................................................... 31 2.4 Additional Sperm Morphological Assessment .................................... 31 2.4.1 Preparation for Morphological Assessment .............................. 32 2.4.2 Microscopic Analysis of Sperm Morphology............................ 32 2.5 Fluorescence in situ Hybridization (FISH).......................................... 34 2.5.1 Sample Preparation for FISH of Whole Semen......................... 34 2.5.2 Sample Preparation for FISH of Morphologically Separated Spermatozoa .............................................................................. 34 2.5.3 FISH Preparation and Analysis.................................................. 35 2.6 DNA Fragmentation Methodology...................................................... 37 2.7 Statistical Analysis .............................................................................. 37 CHAPTER III Results ....................................................................................................... 40 3.1 Sperm Morphology Assessment.......................................................... 40 3.1.1 Results of Morphologic Assessment.......................................... 40 3.1.1.1 Comparison of Morphology in Raw, Unselected Sperm of Control and Teratozoospermic Subjects ..... 41 3.1.1.2 Effects of Swim-up Sperm Selection ........................... 43 3.1.1.3 Correlation of Sperm Morphology ............................... 44 v3.2 Chromosomal Analysis in Sperm........................................................ 48 3.2.1 Chromosomal Analysis of Raw, Unselected Spermatozoa ....... 48 3.2.2 Chromosomal Analysis of Morphologically Separated Spermatozoa .............................................................................. 50 3.3 DNA Fragmentation ............................................................................ 51 3.4 Correlation Sperm Morphology to Genetic Integrity .......................... 52 3.4.1 Correlation Between Sperm Morphology and Chromosomal Abnormalities ............................................................................ 52 3.4.2 Correlation Between Sperm Morphology and DNA Fragmentation............................................................................ 54 CHAPTER IV General Discussion .................................................................................... 55 4.1 Sperm Morphology in Control and Teratozoospermic Subjects ......... 55 4.1.1 Sperm Morphology After Swim-up Fractionation..................... 61 4.2 Chromosomal Analysis in Control and Teratozoospermic Subjects ... 63 4.2.1 Correlation of Sperm Morphology to Chromosomal Abnormalities ............................................................................ 64 4.2.2 Chromosomal Analysis of Morphologically Separated Spermatozoa .............................................................................. 67 4.3 DNA Fragmentation in Control and Teratozoospermic Subjects........ 68 4.4 Conclusions ......................................................................................... 69 TABLES ................................................................................................................................ 72 FIGURES............................................................................................................................... 90 BIBLIOGRAPHY.................................................................................................................. 96 vi LIST OF TABLES Table 1.1 Reference values for semen assessment and nomenclature .................................. 72 Table 3.1 Percentage of morphologic characteristics in sperm from control subjects .......... 73 Table 3.2 Percentage of morphologic characteristics in sperm from teratozoospermic subjects .................................................................................................................. 74 Table 3.3 Group means of morphologic characteristics: comparison between control and teratozoospermic groups in raw semen and swim-up sperm.......................... 75 Table 3.4 Cross correlation of specific abnormal morphology in raw, unselected sperm of fertile control subjects....................................................................................... 76 Table 3.5 Cross correlation of specific abnormal morphology in raw, unselected sperm of teratozoospermic subjects ................................................................................. 77 Table 3.6 Cross correlation of specific abnormal morphology in swim-up sperm of fertile control subjects ........................................................................................... 78 Table 3.7 Cross correlation of specific abnormal morphology in swim-up sperm of teratozoospermic subjects...................................................................................... 79 Table 3.8 Chromosome 18, X and Y (Triple-colour FISH) Results ..................................... 80 Table 3.9 Chromosome 13 and 21 (Dual-colour FISH) Results ........................................... 81 Table 3.10 Summary of Chromosomal Abnormalities in Control and Teratozoospermic Groups ................................................................................................................... 82 Table 3.11 Chromosomal Abnormalities in Morphologically Separated Sperm .................... 83 Table 3.12 DNA Fragmentation in Control and Teratozoospermic Subjects ......................... 84 Table 3.13 Linear Correlation Analysis of Sperm Morphology and Aneuploidy in Control Subjects (n=5) .......................................................................................... 85 Table 3.14 Linear Correlation Analysis of Sperm Morphology and Multiple Chromosomal Abnormalities in Control Subjects (n=5)....................................... 86 Table 3.15 Linear Correlation Analysis of Sperm Morphology and Aneuploidy in Teratozoospermic Subjects (n=10)........................................................................ 87 Table 3.16 Linear Correlation Analysis of Sperm Morphology and Multiple Chromosomal Abnormalities in Teratozoospermic Subjects (n=10) .................... 88 Table 3.17 Linear Correlation Analysis of Sperm Morphology and DNA Fragmentation..... 89 vii LIST OF FIGURES Figure 1.1 Spermatogenesis.................................................................................................. 90 Figure 1.2 Meiosis in Male Germ Cells ............................................................................... 90 Figure 1.3 Male Germ Cell Maturation Across Seminiferous Epithelium .......................... 91 Figure 2.1 Morphologically Normal Sperm ......................................................................... 92 Figure 2.2 Sperm with Abnormal Head Morphology ......................................................... 92 Figure 2.3 Sperm with Abnormal Neck, Midpiece, or Tail Abnormalities ......................... 93 Figure 2.4 FISH of Chromosomes 18, X, and Y Probe Set on Sperm ................................ 94 Figure 2.5 FISH of Chromosomes 13 and 21 Probe Set on Sperm ..................................... 94 Figure 2.6 DNA Fragmentation by TUNEL Assay ............................................................. 95 viii LIST OF ABBREVIATIONS ABP androgen binding protein AR androgen receptor ART assisted reproductive technique AT asthenoteratozoospermia ATP adenosine triphosphate AZF azoospermic factor BEDO bilateral ejaculatory duct obstruction CBAVD congenital bilateral absence of vas deferens CDY1 chromodomain on Y chromosome (in AZFc) CF Cystic Fibrosis CFTR CF transmembrane conductance regulator gene D-JUN broadly expressed proto-oncogene of JUN family (the name JUN comes from the Japanese 'ju-nana,' meaning the number 17 named after the transforming gene of avian sarcoma virus 17) DAZ deleted in azoospermia DAPI 4’,6-diamidino-2-phenylindole: fluorescent stained that binds to DNA DBY DEAD box Y (in AZFa) DFRY Drosophila fat facets related on Y (in AZFa) DHT dihydrotestosterone DM Myotonic dystrophy DNA deoxyribonucleic acid ET embryo transfer FAS cell surface receptor that mediates apoptotic signalling FASL FAS ligand: cell surface ligand, when complexed with FAS receptor, mediates apoptotic signalling FADD Fas-associated death domain FISH fluorescence in situ hybridization FITC Fluorescein isothiocyanate FGFR1 fibroblast growth factor receptor 1 FSH follicle stimulating hormone ix GNRH gonadotrophin releasing hormone: synthesized by hypothalamic neurons, and carried to the anterior pituitary to stimulate secretion of gonadotropic hormones GNRHR GNRH receptor: expressed on the surface of gonotropes in the anterior pituitary GPR54 G protein-coupled receptor 54: mutation has an autosomal dominant role in IHH development hCG human chorionic gonadotrophin HspA2 heat shock protein A2: testis-specific chaperone protein expressed in spermatocytes and elongating spermatids during cytoplasmic extrusion HSP70-2 mouse homologue of HspA2 HTF human tubal fluid ICSI intracytoplasmic sperm injection IHH idiopathic hypogonadotrophin hypogonadism IUI intrauterine insemination IVF in vitro fertilization KAL-1 gene responsible for X-linked form of Kallmann syndrome; encodes anosmin protein, that plays a key role in the migration of GnRH neurons and olfactory nerves to the hypothalamus LH luteinizing hormone MI meiosis I MII meiosis II MESA microsurgical epididymal sperm aspiration NELF nasal embryonic LHRH factor: expressed in peripheral and central nervous system tissues during embryonic development with a role in the neurophilic migration of LHRH cells, mutation has an autosomal dominant role in IHH development NOR nucleolar organizer region OAT oligoasthenoteratozoospermia PAR pseudo-autosomal region (found in sex chromosomes) PBS phosphate buffered saline PESA percutaneous sperm aspiration PGC primordial germ cell Prbp mouse gene encoding Prm-1 binding protein involved in protamines mRNA translation delay PWS Prader-Will syndrome xPZD partial zona dissection RBM RNA binding motif RNA ribonucleic acid SCF stem cell factor (anti-apoptotic factor produced by Sertoli cells) SDI sperm deformity index (total number of morphologic defects / number of sperm counted) SHBG sex hormone-binding globulin SRY sex-determining region of Y chromosome SUZI subzonal insemination T testosterone TDF testis-determining factor TESE testicular sperm extraction TRBP TAR RNA binding protein: human homologue of Prbp TUNEL terminal dUTP nick-end labelling TZI teratozoospermic index (total number of morphologic defects / number of sperm with defects) UTY ubiquitous transcribed tetrapeptide repeat gene on Y (in AZFa) WHO World Health Organization xi GLOSSARY aneuploidy a single chromosome change (gain or loss of one chromosome) from the normal complement; aneuploidy of specific chromosomes in results section is the combination of disomy and nullisomy of the particular chromosome complete globozoospermia close to 100% of sperm with round heads and no or very little acrosome constitutional chromosomal abnormalities chromosomal abnormalities involving entire chromosomes disomy two copies of a particular chromosome (abnormal in the case of haploid complement) gonocyte post-migratory primordial germ cell hypogonadism inadequate function of the sex organs hypogonadotrophism abnormally reduced secretion of gonadotrophins microphallus abnormally small penis nullisomy zero copies of a particular chromosome (abnormal in the case of haploid complement) proliferation growth by mitotic division (as opposed to meiotic division) supernumerary chromosomal abnormalities gains or losses (relative to the normal complement) involving multiple chromosomes xii ACKNOWLEDGEMENTS There are several people I must acknowledge for their assistance and support throughout the process of my completing this research project in fulfillment of my graduate studies requirements. I am extremely grateful for the guidance and support of my research supervisor, Dr. Sai Ma, who has been an invaluable mentor in my research endeavours. I am thankful for the opportunities she has provided me, and most importantly, for what I have learned under her tutelage. I would also like to acknowledge the support of Haijun Gao, Agata Minor, Chiho Hatakeyama, Edgar Chan Wong, Kyle Ferguson - past and present members of Dr. Sai Ma’s research laboratory. I am very appreciative to the members of my thesis committee, Drs. Dan Rurak, Helène Bruyere, Anthony Perks, for their guidance throughout the process of my graduate studies. Additionally, I would like to thank Dr. Yulian Zhao, Director of ART Laboratories in the John’s Hopkins Outpatient Center, for her support in the provision of a portion of the subject samples needed for me to complete this research project. The research conducted in this project was financially supported by a research grant to Dr. Sai Ma from the Canadian Institutes of Health (CIHR). Last but not least, I would like to thank my family (my mother, father and younger brother) for their support and encouragement during my studies. 1Chapter I. General Introduction 1.1 Development of Male Germ Cells 1.1.1 Development of Primordial Germ Cells Between the fourth and sixth weeks of embryonic development, primordial germ cells (PGCs) proliferate and migrate from the yolk sac to the genital ridge and arrive in undifferentiated gonads (Eddy et al., 1981). Wolffian (mesonephric) and Mullerian (paramesonephric) ducts develop and the epithelium of the genital ridge proliferates and penetrates the underlying mesenchyme forming irregularly shaped cords (primitive sex cords) of an indifferent, bipotential gonad. The expression of the testis-determining factor (TDF) gene in the sex-determining region of a Y chromosome (SRY) (Koopman et al., 1991), initiates a cascade of events that result in the development of internal and external genitalia of the human male, including the differentiation of the undifferentiated gonad into the testis (Polanco and Koopman, 2007; Sinisi et al., 2003). Under the influence of SRY, primitive sex cords continue to proliferate and penetrate into the medulla and epithelial cells begin to differentiate into Sertoli cells. The Sertoli cells polarize and, along with peritubular myoid cells, aggregate around the PGCs to organize the testis cords at around the 8th week of development. Sertoli cells produce antimullerian hormone (AMH) which causes regression of mullerian ducts. Interstitial cells between the testis cords also begin to differentiate into Leydig cells (mesenchymal origin). Leydig cells produce steroid hormones such as testosterone, which promotes virilization of Wolffian (mesonephric) ducts to become vas deferens and epididymis. As well, testosterone is converted by 5α-reductase to dihydrotestosterone to control the development of the prostate (from the urogenital sinus) and penis (from the genital tubercle). The post- migratory PGCs, or gonocytes, enclosed by Sertoli cells continue to proliferate until about a month after birth, when the germ cells enter a quiescent period of growth in which proliferation tapers off and the level of apoptosis increases (Muller and Skakkebaek, 1984; Berensztein et al., 2002). 1.1.2 Spermatogenesis and Meiosis Spermatogenesis is the process by which a diploid male germ cell divides meiotically to produce four progenitor haploid germ cells. This process includes the 2production of spermatogonial stem cells and the reduction/division (meiosis) that produces haploid cells. In the early stages of puberty, bursts of gonadotrophin releasing hormone (GNRH) from the hypothalamus stimulate the production and release of gonadotrophins, LH and FSH, in the pituitary, which in turn leads to further development of the testis along with other secondary sex characteristics; the combinatory effects of both LH and FSH are required for normal spermatogenic function (Grumbach, 2002; Hiort, 2002). As Sertoli cells differentiate, they develop tight junctions (the eventual blood-testis barrier). Subsequent secretion of fluid by the Sertoli cells generates a tubule lumen in the development of the seminiferous tubule. Gonocytes, attached to the basal lamina of the seminiferous epithelium on the other side of the Sertoli cell tight junctions, differentiate into spermatogonial stem cells and continue to proliferate throughout adult life. At regular intervals the spermatogonal stem cells give rise to Type A spermatogonia, which marks the start of spermatogenesis. Type A spermatogonia undergo a limited number of mitotic divisions, producing a population of cells that can replenish itself (stem cells) as well as cells that are more differentiated/committed toward the path of spermatozoa development, the intermediate type A spermatogonia. Intermediate type A spermatogonia divide mitotically once more to form type B spermatogonia, which are the precursors of primary spermatocytes. Spermatogonial cell divisions are incomplete, producing clones of cells still bound together by thin bridges of cytoplasm. These cytoplasmic bridges are maintained through spermatocyte development and disperse only in advanced phases of spermatid development; this facilitates a synchronous development of successive germ cell spermatocytes with double the genetic complement. Primary spermatocytes enter into an extended prophase, lasting about 22 days. During this extended prophase, replicated chromatin condenses to chromosomes (leptotene), and homologous chromosomes approach each other (zygotene) and begin to pair in structures called synaptonemal complexes (completed at pachytene). Within the synaptonemal complexes, chiasmata are formed between sister chromatids and genetic recombination occurs between chromatids of different parental origin. When the synaptonemal complexes disperse (in diplotene), the chiasmata are maintained between homologous chromosomes. Microtubules from the generations (Figure 1.1 and 1.3). Type B spermatogonia replicate to produce primary 3meiotic spindle attach to the centromeres of homologous chromosomes but the chiasmata balance the opposing forces from each pole and help facilitate proper alignment at the metaphase plate I. This is followed by a rapid completion of meiosis I (MI) and the formation of secondary spermatocytes (Page and Hawley, 2003; Baarends and Grootegoed, 1999). Each primary spermatocyte divides, separating the homologous chromosome pairs and producing two secondary spermatocytes. The secondary spermatocytes undergo the second meiotic division (MII) where the chromatids of each chromosome will migrate to each pole of the cell and division occurs to produce haploid cell interactions, spermatogenesis begins in the basal compartment of the epithelium, differentiating preleptotene/leptotene spermatocytes traverse the blood-testis barrier from the basal to the adluminal compartments, and spermatogenesis is completed in the ). 1.1.3 Spermiogenesis Spermiogenesis is the morphological development of round spermatids to highly specialized, elongated, mature spermatozoa well adapted for traversing the male and female reproductive tracts and achieving fertilization of an oocyte. No further division occurs. During spermiogenesis, the acrosome forms, nuclear reorganization occurs, the flagellar apparatus forms, and excess cytoplasm (the residual body) is shed (de Krester et al., 1988). The Golgi apparatus in early spermatids forms the acrosome vesicle, attaches to one pole (anterior) of the nucleus and spreads as a cap over the nucleus, progressively elongating as the chromatin condenses. The acrosome of mature spermatozoa is a flattened sac filled with hydrolytic enzymes that covers two-thirds of the nucleus. As the acrosome cap is formed, the nucleus rotates so that the cap faces the basal membrane of the seminiferous epithelium. The centriole organises the formation of microtubules to form a flagellum and approaches the nucleus at the opposite (posterior) pole ensuring a linear alignment of the tail with the longitudinal axis of the head. The assembly of the axoneme central element begins in the cytoplasm and subsequently protrudes from the cell (initial flagellum). The progeny – spermatids (Figure 1.1 and 1.2). Through the coordination of Sertoli cell-germ adluminal compartment (Cheng and Mruk, 2002) (Figure 1.3 4axoneme is composed of a circumferential array of nine peripheral microtubular doublets surrounding a central pair of microtubules (9 + 2 configuration). Dynein arms connect the peripheral doublets capable of utilizing ATP as energy to generate movement. The flagellum is anchored onto the nuclear membrane by the cytoskeletal connecting piece formed around the centrioles. The sperm flagellum is composed of: (a) the neck with two centrioles (proximal and distal); (b) the mid-piece consisting of a sheath of ring-shaped mitochondria surrounding the axoneme to provide the energy for the flagellar movement; (c) and the principle piece with a sheath of ring fibres (fibrous sheath) around the axoneme (Baccetti et al., 1981). Histones, that are characteristically associated to DNA in somatic cells and germ cells up to round spermatids, are replaced by protamines (Brewer et al., 2002; Dadoune, 2003), which associate side-to-side with the groove of the DNA helix and results in linear, parallel packaging of nucleoprotein fibres stabilized by disulphide bonds – a high degree of DNA compaction (Ward and Coffey, 1991). The cytoplasm of the spermatids that is no longer needed is extruded and phagocytosed by Sertoli cells or is disposed of in the lumen of the seminiferous tubules. A clump of cytoplasm, though, can remain hanging on the neck and mid piece of the sperm cell for a little while. Finally, the mature spermatozoa are released into the lumen of the tubule - spermiation. 1.1.4 Hormonal Control of Spermatogenesis Endocrine control is not only critical in normal testicular development, but also for ongoing normal function after the onset of spermatogenesis. The pulsatile secretion of GNRH by the hypothalamic neurons into the hypothalamo-hypophyseal portal system directly stimulates the anterior pituitary to release the gonadotrophins, LH and FSH, into the peripheral circulation. Regulation of pituitary gonadotrophins release is control by higher central nervous system inputs as well as testicular negative feedback mechanisms. LH binds to specific membrane receptors on Leydig cells in the testis and stimulates testicular steroidogenesis and ultimately, the secretion of testosterone (T). The majority of T is secreted into blood circulation bound to plasma proteins, such as sex hormone- binding globulin (SHBG) and albumin, as well as in non-protein-bound, physiologically 5active form. Deficiency of T in circulation results in characteristics such as reduced libido, infertility, behavioural changes, weakness and fatigue. The level of T in adult circulation acts as negative feedback control of gonadotrophin secretion at both the hypothalamic and pituitary levels. The local secretion of T within the testis is important to the maintenance of spermatogenesis. In the testis, T is taken up by Sertoli cells and converted to a more potent form, dihydrotestosterone (DHT) by 5α-reductase. DHT or unconverted T bind to intracellular androgen receptors (ARs), and the androgen-androgen receptor complexes interact with the genome to stimulate DNA transcription, mRNA translation and protein synthesis related to the functions of Sertoli cells, including germ cell support. High intratesticular T concentration, which is important in the maintenance of normal spermatogenesis, is maintained in part by T binding to androgen binding protein (ABP), which is produced by Sertoli cells. The other pituitary gonadotrophin, FSH, binds to receptors on the surface of Sertoli cells and spermatogonia. The effect of FSH binding to spermatogonia is not known, but FSH binding to Sertoli cells upregulates the production of a variety of proteins that include AR, ABP, transferring, and inhibin-B. Inhibin-B in turn, has a negative feedback effect that directly inhibits FSH release in the pituitary gland (Burger, 1987; Hiort, 2002). 1.1.5 Germ Cell Death and Apoptosis Equally important as the proliferation and differentiation that occurs in germ cell development is the level of germ cell death (Wang et al., 1998; Blanco-Rodriguez and Martinez-Garcia, 1998). Germ cell death in human spermatogenesis occurs mainly via apoptosis, or programmed coordinated cell death (Blanco-Rodriguez and Martinez-Garcia, 1998; Rodriguez et al., 1997). Apoptosis is structurally characterized by cell shrinkage, chromatin condensation, cytoplasmic vacuolization and apoptotic bodies and biochemically characterized by activation of caspase cascades and DNA cleavage and fragmentation (King and Cidlowski, 1995; Matsui, 1998). In the normal development and function of the testis in mice, germ cell apoptosis is critical during the migration of PGCs to the eventual gonads and in spermatocytes during the first round of spermatogenesis (Wang et al., 1998). In the adult testis, apoptosis of germ cells is intricately associated to and controlled by the Sertoli cells. Sertoli cells 6apoptosis acts to limit the number of germ cells that are be supported by the Sertoli cells. Sertoli cells also contribute in the modulation of germ cell apoptosis. Sertoli cells produce and secrete stem cell factor (SCF), also known as Steel factor, which binds to c-kit receptors, which are expressed in Type A spermatogonia, and initiates multiple downstream signalling pathways that promotes cell survival (Yan et al., 2000). The tight association that germ cells have with Sertoli cells also allow for the classical cell death signalling pathway (i.e., caspase activation followed by targeting for phagocytosis by phosphatidylserine externalization) (Tesarik et al., 2004). The FASL ligand, produced by Sertoli cells, when bound to its FAS receptor on germ cells (Lee et al., 1997; Pentikainen et al., 1999), induces trimerization of FAS receptors and recruits FADD (Fas-associated death domain). The FAS/FADD complex binds to initiator caspase 8 or 10 and subsequently activates caspases 3, 6 and 7 (Nagata and Golstein, 1995), which actively cleave proteins and cause morphological changes in the cell and nucleus characteristic of apoptosis. Altered levels of expression of pro- and anti-apoptotic proteins can lead to disruption of normal spermatogenesis and infertility (Rodriguez et al., 1997). In addition, physiological and environmental factors have been found to bring about germ cell apoptosis, including: gonadotrophin withdrawal (Hikim et al., 1995) cryptorchidism (Shikone et al., 1994), irradiation (Henriksen et al., 1996), heat exposure (Yin et al., 1997) and vasectomy (Lue et al., 1997). 1.2 Abnormalities of Spermatogenesis 1.2.1 Errors in Meiosis and Aneuploidy Meiosis in spermatogenesis progresses through precise control, and stalls or terminates at specific checkpoints when errors in chromosomal segregation occur (Gazvani et al., 2000). When abnormal segregation escapes the meiotic checkpoints (Hassold et al., 1992), the result is an abnormal chromosomal complement, aneuploidy, in mature spermatozoa. A common mechanism leading to aneuploidy is meiotic non-disjunction between the homologous chromosomes at anaphase of MI and between sister chromatids at support and nourish developing germ cells (Mruk and Cheng, 2004) (Figure 1.3), and 7anaphase of MII. Disomy and nullisomy are complementary results of meiotic non- disjunction in gametes. Anaphase lag, or a delay in the movement of one or more chromosomes from the metaphase plate during anaphase, is another mechanism that can potentially lead to nullisomy in mature gametes (Chandley, 1987). Other mechanisms leading to aneuploidy that have been observed in animal models include: non-conjunction (chromosomes fail to maintain paired state and univalent chromosomes segregate randomly at anaphase I); premature centromere division (sister chromatids separate); and extra replication of chromosome (additional chromosome copy without the concomitant production of hypohaploids) (reviewed in Bond and Chandley, 1983). These modes of aneuploidy production have been seen in human gametes (Cupista et al., 2003) but knowledge about non-disjunction in man is still limited (Nicolaidis and Petersen, 1998). Altered recombination is considered one of the major factors leading to non- disjunction. It has been shown that reduced levels of recombination are associated with increased incidences of non-disjunction (Sherman et al., 1994; Hassold et al., 1995; Robinson et al., 1998; Savage et al., 1998; Shi et al., 2001; Thomas et al., 2001; Ferguson et al., 2007). The relationship between recombination and non-disjunction is chromosome specific (Warburton and Kinney, 1996). Smaller chromosomes appear to have fewer chiasmata (recombination foci), and bivalents of these chromosomes are more prone to premature separation and non-disjunction (Cupista et al., 2003). In addition, bivalents without chiasmata are unable to orient properly on the metaphase plate (Bascom-Slack et al., 1997); this makes them more prone to abnormally segregate to daughter cells (Sun et al., 2004). During normal male meiosis, pairing of the X and Y sex chromosomes is limited to pseudo-autosomal regions (PAR) 1 and 2 (2.5Mb and 0.33Mb, respectively) (Freije et al., 1992; Rappold, 1993), with one obligate recombination loci occurring within PAR to ensure proper disjunction (Burgoyne, 1982; Hassold et al., 1991; Shi et al., 2001). Consequently, XY disomy occurs more frequently than with any other chromosomes (Spriggs et al., 1996). Similarly, the short chromosome 21 permits single chiasmata and is prone to non-disjunction (Laurie and Hulten, 1985). 81.2.2 Abnormal Sperm Maturation Abnormalities in chromatin maturation and compaction have been described as “lacunar” defects (2-3um in diameter) with granulo-fibrillar ‘empty’ areas occupy as much as 20-50% of the nucleus (Zamboni 1987). It has been suggested that these defects represent apoptotic changes (Baccetti et al., 1996), however the association was not confirmed (Muratori et al., 2000). Sperm with chromatin abnormalities frequently display abnormal head shapes, have diminished fertility potential, or associate with abortions of first trimester (Chemes, 2000). Abnormalities in acrosome formation have also been described. The lack or insufficient formation of acrosome leads to conditions known as acrosomal aplasia, which includes globozoospermia (round headed, acrosomeless spermatozoa) and acrosomal hypoplasia (small and detached acrosomes). Acrosomeless sperm occurs at around 0.5% in semen of fertile individuals, while it is up to 2-3% in infertile patients (Kalahanis et al., 2002). Acrosomal aplasia, more specifically defines the cases when it is the predominant anomaly (up to 100% of ejaculated spermatozoa). These sperm pathologies are the result of failure of the Golgi apparatus to attach normally to the nucleus and coincident faulty development of the acrosome granule. As a result, the acrosome doesn’t spread over the nucleus, exists as a cytoplasmic lobule, and is frequently phagocytosed by Sertoli cells. When the sperm’s centriole is unable to attach normally to the spermatid’s nucleus at the opposite pole, heads and tails develop independently, and the heads are usually phagocytosed by Sertoli cells or lost in the epididymis. The result is acephalic spermatozoa. This occurs in very small number in semen of fertile individuals and increase up to 10-20% in subfertile men (Chemes et al., 1987b; Panidis et al., 2001). In some teratozoospermic (sperm with abnormal morphologic features) patients, 90-100% of sperm are acephalic. A variation of abnormal centriole attachment to the nucleus would include failure to localize to the caudal pole of the spermatid nucleus resulting in the implantation to the middle piece, with heads attaching either to the tip or the sides of the mid-piece without linear alignment with the sperm axis (Chemes et al., 1999). This abnormality incidentally has been associated with a failure to nucleate a functional sperm aster in the developing zygote, and impaired normal syngamy and cleavage (Chemes et al., 1999; Saias Magnan et al., 1999; Rawe et al., 2002). 9Abnormalities in the flagella inherently affect the motility of spermatozoa and the ability to traverse the male and female reproductive tracts to fertilize an oocyte. They can include axonemal defects like lack of both dynein arms, absence of central microtubules or radial spokes, transposed microtubules, lack of axoneme, and fibrous sheath aberrations. 1.3 Male-factor Infertility Infertility is defined as the inability to conceive after one year of unprotected intercourse, and affects one in six couples (WHO, 2000). A male-factor is identified in about half the couples, with 20% due solely to a male-factor. To simply put, male-factor infertility is the inability of a man to produce sufficient functional sperm capable of fertilizing an oocyte, ultimately resulting in a viable conception. Several factors can be involved that would effect the number and / or functional capacity of the sperm produced by a given man. 1.3.1 Causes of Male-factor Infertility Male-factor infertility involves many factors that include: anatomical and physiological abnormalities, the presence of infection, testicular trauma, heat, and the presence of drugs or radiation that may be toxic to sperm production (Lipshultz and Howard, 1997). The most commonly diagnosed condition related to male infertility is varicocele (dilation of spermatic veins), occurring in about 40% of the cases. Although varicocele has been associated with infertility, the exact effect has not been isolated; the effect may be related to an increase in testicular temperature or substances introduced by the back flow of blood that may be detrimental to sperm production. A ductal obstruction, caused by vasectomy, repeated infection, inflammation, or developmental defect, is also quite common, accounting for 14% of the cases. Cryptorchidism, which is the developmental failure of the testes to descend into the scrotal sack, occurs in 3-6% of infertile men; increased testicular temperature and hormonal insufficiencies are possible effects leading to infertility. Other physiologic factors that are associated to male infertility include: hormone dysfunction (e.g., hypogonadism), metabolic disorders (e.g., hemochromatosis), immunologic conditions (e.g., antisperm antibodies, infectious diseases) and systemic illness (e.g., cancer). Of all the cases of male infertility, only a 10 small proportion (0.1%) has been associated to an identifiable genetic defect (discussion to follow), although many of the above factors are likely to have a genetic component. It has been estimated that about 30% of the cases of male infertility is genetically determined, taking account of chromosomal abnormalities and spermatogenic disorders (Engel et al., 1996). Nonetheless, roughly 23% of the male infertility cases cannot be explained. 1.3.2 Genetic Origin of Male-factor Infertility 1.3.2.1 Chromosomal Abnormalities An association between male infertility and chromosomal abnormalities first became evident in a large karyotype survey, in which the incidences of chromosomal abnormalities were higher in comparison to the general population (5.3% vs. 0.6% respectively; Chandley, 1979). Moreover, the incidences of sex chromosomal and autosomal abnormalities have been shown to be 15-fold and 6-fold higher than the respective rates in the general population. It has been estimated that about 5% of infertile males have a chromosomal abnormality, with 4% involving the sex chromosomes (Bhasin et al., 2000). 1.3.2.1.1 Constitutional Aneuploidy Constitutional chromosomal abnormalities occur in 2-14% of infertile men is (Shi and Martin, 2001), the most common being sex chromosomes abnormalities and trisomy 21. Some of the more common constitutional chromosomal abnormalities involving male infertility will be discussed. Klinefelter syndrome is the most frequent chromosomal disorder associated with male infertility and occurs in one in 500 to one in 1000 males and (Bielanska et al., 2000; Bojesen et al., 2003). A 47,XXY karyotype is found in ~93% of the cases, while variants such as 47,XXY/46,XY mosaicism; 48,XXXY; 48XXYY; and 49,XXXXY can also manifest Klinefelter symptoms. The 47,XXY karyotype derives from a paternal meiosis I non-disjunction of an XY bivalent in >50% of the cases, a maternal meiotic error in 40% of the cases, and post-zygotic mitotic errors in the remainder of the cases (Hassold et al., 1996). Men with Klinefelter syndrome typically are diagnosed with testicular atrophy and 11 non-obstructive azoospermia (Rives et al., 2000). However, cases with extremely reduced levels of sperm (oligozoospermia) have been found in men with mosaicism. Sperm that are found have an increased frequency of sex chromosome disomy (2-25%, reviewed by Shi and Martin, 2001), which are the result of absent recombination between the X and Y chromosomes and their subsequent non-disjunction in meiosis I (Hassold et al., 1991). 47,XYY occurs in one in 1000 males and arises from paternal meiotic II non- disjunction of the Y chromosome. It has also been reported that abnormal sex chromosome configuration can occur as early as the pachytene stage of meiotic prophase I (Wong et al., 2008). The chromosomal imbalance leads to hormonal imbalances in the gonads (Attanasio et al., 1982) and some spermatogenic impairment (Skakkebaek et al., 1973) with significantly increased aneuploidy (0.3 – 15%) in sperm resulting from meiotic errors. Trisomy 21, or Down syndrome, occurs in 1 in 650 to 1,000 live births. When trisomy 21 occurs in males, it is associated with spermatogenic arrest and reduction in the number of germ cells that is thought to result from an accelerated rate of apoptosis of chromosomally abnormal primordial germ cells (Patrizio and Broomfield, 1999). 1.3.2.1.2 Structural Chromosomal Aberrations Reciprocal translocations can lead to reduced fertility. In order to proceed through meiosis, autosome-autosome translocations synapse and form quadrivalent pairings. The mechanics and time constraints of a resulting quadrivalent can impede meiosis; as well, the disjunction of this pairing cross is prone to genetically unbalanced gametes (Forejt et al., 1982). Unpaired regions may also result, leading to failure of meiosis and elimination of germ cells. In addition, translocated segments of chromosomes attempt non- homologous pairing with X and Y chromosomes during meiosis I. The early condensation of the sex chromosome bivalent in meiotic prophase I relative to the other chromosomes seen in primary spermatocytes and the invariable sterility accompanied with X-autosome translocations in mice suggests that X inactivation may have a role in the progression of spermatogenesis from primary spermatocytes (Lifshytz and Lindsley, 1972). Inactivation of asynapsed regions of a reciprocal translocation in humans is evidenced by both the localization of asynapsed quadrivalents to the sex chromosomes and the localization of 12 BRCA1 and gammaH2AX, two proteins implicated in meiotic sex chromosome incactivation, along asynapsed regions (Ferguson et al., 2008). Additionally, reciprocal X- autosome translocations can occur, and men with such translocations present with spermatogenic arrest and frequently are azoospermic (no sperm in ejaculate) (Madan, 1983), as well as severe oligozoospermia in rare cases (Ma et al., 2003). Robertsonian translocations, which occur in about one in 625 births (Van Dyke et al., 1983), have been observed in infertile males and this association may be related to the loss of nucleolar organizer regions (NOR) of heterochromatic short arms of acrocentric chromosomes. These NORs are required to associate with the sex vesicle and their loss can increase the likelihood of cell disruption and germ-cell death (Gabriel-Robez, 1996). Chromosomal inversions, 13 times more likely in infertile men than the general population, can also cause infertility by: the mechanistic and time constraints the resulting pairing loop has on meiosis (Forejt et al, 1982), and the reduced recombination within the pairing loop increases the likelihood of aneuploid gametes (Chandley et al., 1987). 1.3.2.2 Specific Gene Disorders In addition to the constitutional chromosomal abnormalities, mutations of specific genes have been identified in etiology of male infertility; some of the most prevalent will be described. Congenital bilateral absence of vas deferens (CBAVD) is a form of obstructive azoospermia. CBAVD is the most common extra-testicular ductal obstruction, affecting 1- 2% of infertile men (Quinzii et al., 2000). CBAVD is commonly associated to mutations in the cystic fibrosis (CF) transmembrane conductance regulator gene (CFTR). The CFTR protein product functions as a chloride ion channel and has a role in the formation of the ejaculatory ducts, seminal vesicle, vas deferens and distal two-thirds of the epididymis (Shah et al., 2003). In 50-83% of CBAVD cases, individuals have at least one known CFTR mutation; while in 10% of the cases, individuals have two known CFTR mutations (Donat et al., 1997). The most common cause of CBAVD is a combination of the 5T allele, which lacks exon 9, in one copy of the CFTR gene, resulting in low expression of functional CFTR protein (Chu et al., 1991), and a mutation in the other copy, the most common being ∆F508 (Chillon et al.,1995). 13 CFTR mutations have also been found to be associated to bilateral ejaculatory duct obstruction (BEDO) and Young syndrome, which can cause complete bilateral plugging of the epididymal lumen (Meschede et al., 1997, 1998). Myotonic dystrophy (DM), with a prevalence of one in 10,000, results from erroneous meiotic expansion of a CTG repeat in a gene (on chromosome 19) that encodes for a regulatory protein kinase found in skeletal muscle. Male infertility may be the result of deficient capacitation and acrosome reaction in spermatozoa (Hortas et al., 2000) or (obstructive) azoospermia from the sclerosis of the tubuli seminiferi contorti (Hauser et al., 1991). Genetic disorders that affect the secretion and action of gonadotrophins can also lead to male infertility. Reduced secretion of gonadotrophins (hypogonadotrophism) results in inadequate function of the sex organs (hypogonadism) exhibited by either androgen deficiency or infertility due to disrupted germ cell development (Bhasin et al., 2000). The effect of androgen deficiency depends on time of onset and magnitude of the deficiency. For instance, general androgen deficiency during gestation may result in a range of mal-developed Wolffian structures; whereas in isolated hypogonadotrophism of the fetus, normal development of the fetal testis can be stimulated by placental hCG in early development, but absence of critical fetal pituitary LH and FSH during later developmental stages often results in undescended testes and microphallus. Pre-pubertal androgen deficiency results in delayed or arrested sexual development; post-pubertal androgen deficiency results in deterioration of secondary sex characteristics, sexual dysfunction, and infertility. The varying degrees of defective hypothalamic GNRH secretion in idiopathic hypogonadotrophin hypogonadism (IHH), result in corresponding degrees of sexual developmental impairment, which can include: complete absence of pubertal development, sexual infantilism and undescended testes (Crowley et al., 1985; Spratt et al., 1987; Waldstreicher et al., 1996). About 20% of IHH have an X-linked pattern of inheritance (mapped to the KAL-1 gene loci of Xp22.3), one-third an autosomal recessive mode of inheritance (mapped to the FGFR1 gene at 8p11.2-p11.1), and the remainder an autosomal dominant mode of inheritance (mapped to GPR54 at 19p13.3, 9q34.3, GNRHR at 4q21.2, and NELF at 9q34.3). 14 Prader-Willi syndrome (PWS) is another hypothalamic disorder associated with impaired GNRH activity, occurring in one in 25,000 births (Butler, 1990). With respect to the male infertility, PWS commonly presents with hypogonadism, cryptorchidism, micropenis, and testes with Sertoli cells only (Cassidy and Schwartz, 1998). Like other hypothalamic disorders, PWS includes varying degrees of gonadotrophin deficiency and phenotypes. PWS is a disorder of genomic imprinting, and arises from a loss of the paternal copy of 15q11-q13 by deletions in 70-80% of the cases and maternal uniparental disomy, unbalanced translocation and mutiation of the imprinting centre in the remainder of the cases. 1.3.2.3 Other Genetic Factors The Y chromosome contains several genes critical in testis development (Lahn and Page, 1997; Hargreave, 1999). As mentioned earlier, the testis-determining factor (SRY) is located on the Y chromosome at Yp11.3 (Koopman et al., 1991); mutations and deletions that inactivate SRY would affect sexual differentiation and testes formation. In addition to SRY, many other genes have been found to be deleted from the Y chromosome in infertile men. These genes, located in the Yq11 region, have been designated azoospermia factors (AZF) critical in normal spermatogenesis (Vogt et al., 1996). Y chromosome microdeletions occur in 4% of oligozoospermia (low sperm count in semen) cases, 14% in idiopathic severe oligozoospermia, 11% in acquired azoospermia, and 18% in idiopathic azoospermia (Foresta et al., 2001). Critical deletions can be localized to three distinct regions: AZFa, AZFb, and AZFc. Deletions in the AZFa region are associated with lack of germ cells or Sertoli cell only syndrome. Genes found in AZFa include: DFFRY (Drosophila fat facets related on Y) involved in ubiquitin-dependent degradation of proteins of D-JUN signal transduction, DBY (DEAD box Y) possibly involved in RNA metabolism, and UTY (ubiquitous transcribed tetrapeptide repeat gene on Y) with unknown function. Deletions in AZFb region are associated with spermatogenesis arrest. Genes that have been isolated to the AZFb region include RNA binding motif genes, RBM 1 and 2, and RBMY; absence of these genes leads to defects in RNA metabolism and processing (Ma et al., 1993a, and b). Incidentally, RBMY protein is localized to the nucleus of germ cells at all stages of 15 spermatogenesis except elongating spermatids (Elliott et al., 1997). Deletions in AZFc region are associated with abnormal maturation (Vogt et al., 1996). The DAZ (deleted in azoospermia) gene can be found in AZFc, and produces an RNA binding protein with a possible role in RNA metabolism (Reijo et al., 1995). Of the three AZF regions, AZFc deletions are the most frequent (60% of all AZF deletions) followed by AZFb deletions, alone or with other AZF regions (AZFb + AZFc or AZFa + AZFb + AZFc) (35% of deletions); and finally, AZFa deletions (5% of deletions) (Krausz et al., 2003). Deletions on the Y chromosome are predominantly large deletions, which are thought to be the due to the large numbers of repetitive sequences causing unequal sister chromatid exchanges (Blanco et al., 2000). The variable rearrangements involving different genes, coupled by the presence of genes in multiple copy number and possible dosage effects have made the correlation between genotype and phenotype difficult (Affara and Mitchell, 2000). 1.4 Treatment of Male-factor Infertility Many of causes of male infertility can be treated medically, surgically, or both. Some varicocele diagnoses can be treated by surgical ligation of dilated vein (Dubin and Amelar, 1970). Ductal obstruction (e.g., epididymal, vasal, or ejaculatory) to sperm delivery can also be surgically treated to bypass or relieve the obstruction (Lipshultz and Howards, 1997). Hormonal replacement or stimulation may be used in cases where endocrine disorders cause insufficient release of specific hormones. If an identifiable infection is the sole cause of infertility, antibiotics can be used to treat the infection. As well, if infertility involves the effect of antisperm antibodies, immunosuppression agents can be employed. When infertility cannot be amenable to medical or surgical therapies, assisted reproductive techniques (ARTs) can be employed. If sufficient motile sperm are available, intrauterine insemination (IUI) may be used to place the sperm directly in the uterus, bypassing the cervical barrier. However, if the availability of motile sperm is insufficient, in vitro fertilization (IVF) can be used, in which ova aspirated from the female are incubated with a concentrated sperm sample to produce an embryo, which is then transferred (ET) back into the females uterus (Edwards et al., 1980; Mahadevan et al., 1983). Conventional IVF-ET is less effective, however, for patients with severely 16 abnormal semen parameters (Tournaye et al., 1992). With the discovery that membrane fusion events can be bypassed without compromising oocyte activation and initiation of embryonic development, micromanipulation techniques were developed. The initial micro-insemination techniques that included partial zona dissection (PZD) and subzonal insemination (SUZI) failed to increase success in severe male infertility cases. The most significant improvement has been the introduction of intracytoplasmic sperm injection (ICSI), in which a single spermatozoon is injected directly into cytoplasm of mature oocyte to facilitate fertilization (Palermo et al., 1992), allowing for the use of sperm extracted from the testis or epididymis (Schoor et al., 2002). 1.4.1 Standard Assessments Since male-factor infertility can be caused by a variety of defects, detailed assessment of sperm are important for accurate diagnosis and choice of appropriate treatments. Standard semen assessments include initial macroscopic examinations such as liquefaction, appearance, volume, viscosity, and pH of semen. This is then followed by microscopic investigations which include the estimation of sperm concentration, assessment of sperm motility, assessment of level of cellular elements other than spermatozoa, sperm vitality tests and sperm morphology (WHO, 1999). Although each laboratory should determine its own reference ranges, the large numbers needed and the complexity of the relationships between semen characteristics make prospective studies difficult. However, the World Health Organization (WHO, 1999) has provided reference values for each variable based on the clinical experience of many investigators of in vitro or in vivo fertility in a sub-fertile population. Sperm concentration is measured as the number of sperm per millilitre of seminal fluid. The reference value that is used to define a normal semen sample is to have at least 20 x 106 spermatozoa / ml; individuals with sperm concentrations less than this value are clinically recognized as having oligozoospermia (Table 1.1). Sperm motility is assessed by degree of progressive motility. Grade A sperm have rapid progressive motility (≥25µm/s at 37ºC and ≥20µm/s at 20ºC); grade B sperm have slower or sluggish progressive motility; grade C sperm have non-progressive motility 17 (<5µm/s); grade D sperm are immotile. Semen with less than 50% progressively motile sperm (Grade A and B) is abnormal and recognized as asthenozoospermia (Table 1.1). Sperm morphology is assessed on stained semen smears, identifying specific characteristics of sperm head, neck, mid-piece, and flagellum. Because of the subjective nature of this type of assessment, strict criteria have been created to identify normal sperm morphology. The length of the head should be 4-5µm and the width 2.5-3.5µm, the length-to-width ratio of 1.5-1.75. There should be a well defined acrosome region, comprising 40-70% of the head area. The midpiece should be less than 1µm wide and about one and half times the length of the head. Cytoplasmic droplets should be no more than one-third the size of the head. There should be no neck, midpiece or tail defects and all borderline forms are considered abnormal (WHO, 1999). Using these strict criteria, abnormal sperm morphology has been correlated to lack IVF success, with significantly more success using semen with greater than 14% morphologically normal sperm (Kruger et al., 1986, 1988; Mortimer et al., 1986, Jouannet et al., 1988; Liu and Baker 1992; Grow et al., 1994; Toner et al., 1995; Garret et al., 1997). Consequently, this reference value is used to identify individuals with clinically relevant levels of abnormal sperm, termed teratozoospermia (Table 1.1). Incidentally, a normal fertile semen sample can have a very high proportion of morphologically abnormal forms. Although the critical anomalies have yet to be identified, sperm with abnormal morphology have reduced fertilizing potential, and abnormal morphology has been correlated with specific deficiencies such as poor zona pellucida binding and penetration, poor response to agonist that modulate intracellular calcium concentrations, and with biochemical markers such as reactive oxygen species production and enhanced creatine phosphokinase activity. Also, sperm of poor morphology may possess loosely packaged chromatin and may contribute to a failure in sperm decondensation during fertilization (Elder and Dale, 2000). Different combinations of abnormal sperm concentration, motility and morphology are also recognized clinically (Table 1.1) with the most severe having less than normal reference values for all three concentration, motility and morphology parameters, oligoasthenoteratozoospermia (OAT). 18 1.4.2 Application of ICSI in Male-factor Infertility Since the first pregnancy achieved by ICSI in 1992 (Palermo et al., 1992), it has become a routine treatment in most IVF centers. Most importantly, ICSI can be used to treat the most severe cases of male-factor infertility, even those previously deemed untreatable. OAT patients, with severely abnormal semen parameters have been successfully treated with ICSI (Ng et al., 1991; Ma and Ho Yuen, 2001). Semen with high concentration of antisperm antibodies and sperm retrieved from patients who underwent cancer therapy have also been successfully applied to ICSI (Lahteen Mati et al., 1995; Raziel et al., 2003; Meseguer et al., 2003). ICSI has also been successful in treating azoospermic patients who have no sperm in their ejaculate: CBAVD, Young syndrome, failed vaso-epididymostomy, failed vasovasostomy, bilateral inguinal obstruction of ejaculatory duct, non-obstructive azoospermia due to maturation arrest (Silber et al., 1996) or spermatogenesis impairment (Schoysman et al., 1993). The requirement of only a single sperm has allowed for expansion of sources from which sperm can be used. Conventional IVF is effective when semen samples consist of at least 80-90% progressively motile sperm, >14% morphologically normal sperm (by Kruger’s strict criteria), and at concentrations where 50-100 x106 sperm per oocyte can be achieved in a Petri dish. However, ICSI is applied when: 1) semen samples consist of <1x106 sperm / ml and <50% progressively motile sperm or <5% morphologically normal sperm (Kruger’s criteria); 2) at least one failed IVF attempt with an adequate number of inseminated oocytes; 3) the fertilization rate was <20% in a previous IVF attempt (Van Steirteghem et al., 1994). Sperm can be retrieved from epididymis, via microsurgical epididymal sperm aspiration (MESA) (Tournaye et al., 1994) or percutaneous sperm aspiration (PESA) (Tsirigotis et al., 1996). When no sperm can be retrieved from even the epididymis, sperm can be retrieved from testes via testicular biopsy with testicular sperm extraction (TESE) (Silber et al., 1995). Normal fertilization, cleavage, and pregnancy have even been achieved with fresh or frozen-thawed epididymal and testicular spermatozoa (Nagy et al., 1995; Ma and Ho Yuen, 2001; Raziel et al., 2003). However, with the requirement of the availability of a single sperm comes the assumption that meiosis is occurring at least to some degree; no therapeutic approach (ICSI included) is available for men with spermatogenesis halted prior to the completion of meiosis. 19 1.4.2.1 Safety of ICSI Although the selection of sperm for injection is done on the basis of visual inspection of physical characteristics of sperm, the technical limitations of light microscopy do not allow for identification of micronuclear abnormalities that may influence functional competence. By circumventing physiological / physical barriers encountered in the natural fertilization process, ICSI has the potential to introduce sub- cellular abnormalities into a conception. The prospect of utilizing inherently abnormal spermatozoa for fertilization not only has implications on the viability of the conceptions, but also may risk transmitting the genetic abnormalities underlying the infertility of the men pursuing the use of ICSI to their offspring. Thus, concerns about the safety of ICSI have been raised. 1.4.2.1.1 Chromosomal Abnormalities in ICSI Conceptions Prenatal and postnatal diagnoses of fetuses conceived through ICSI found a significantly elevated incidence of chromosomal abnormalities compared to natural conceptions (Lam et al., 2001; Bonduelle et al., 2002; Van Steirteghem et al., 2002). Specifically, a significantly higher rate of inherited chromosomal abnormalities in the ICSI conceptions was found compared to the natural conceptions (1.4% vs. 0.3-0.4%; P<0.001), which suggests that ICSI can transmit abnormalities from parent to offspring. In addition, the rate of de novo chromosomal abnormalities involving sex chromosomes and autosomes was also significantly higher in conceptions from ICSI compared to natural conceptions (1.6% vs. 0.5%; P<0.007) (Bonduelle et al., 2002). This is noteworthy because chromosomal abnormalities that appear de novo are not constitutionally transmitted from the parent and can only arise as a result of abnormalities in the gametes, due to the conception specifically, or the result of post-zygotic errors. Inherited abnormalities found in ICSI conceptions demonstrate the capability of ICSI to transmit abnormalities from parent to offspring (Bonduelle et al., 2002). When chromosomal abnormalities found in ICSI conceptions originate from karyotypically normal fathers, the use of chromosomally abnormal sperm is likely the cause. A fetus conceived via ICSI with a 47,XXY karyotype was found to have an additional X chromosome of paternal origin (Bonduelle et al., 2002). Another fetus conceived by ICSI 20 was found to have a 45,X karyotype that was missing a paternal sex chromosome. Incidentally, further inspection of the sperm from the father of this child revealed an extremely high rate of sperm with a missing sex chromosome (19%) balanced by the rate of sperm with an extra sex chromosome (19%; Tang et al., 2004). Other studies have also suggested increased incidence of paternally derived chromosomal abnormalities in ICSI conceptions (7 paternal abnormalities of 9 total byVan Opstal et al., 1997; one case by Bartels et al., 1998). Unfortunately, this is the extent to which parental origin of chromosomal abnormalities in ICSI conceptions have been investigated, and it is clear that more work needs to be done to identify the true risk of ICSI in generating abnormal conceptions. 1.4.2.1.2 Genetic Abnormalities in ICSI Potential risks of transmission of paternal genetic defects to the embryo / offspring also exist when ICSI is applied (Georgiou et al., 2006). Epididymal or testicular spermatozoa can be recovered from men with obstructive azoospermia due to various CF mutations; when ICSI is employed, there is a risk of producing an affected offspring. In infertile males with hypogonadotrophic hypogonadism with sufficient testosterone levels to support sexual differentiation and development, spermatogenesis can be stimulated by gonadotrophins; sperm carrying the mutation responsible for hypogonadotrophic hypogonadism can subsequently be recovered and used in ICSI procedures. Myotonic dystrophy, characterized by dynamic trinucleotide repeat expansion at the DM gene, commonly presents with decreased sperm function or obstructive azoospermia. Again, sperm can be recovered from patients with myotonic dystrophy, used in ICSI, and propagate the genetic defect to the offspring. Transmission of Y-chromosome deletions from father to son occurs through ICSI: Page et al. (1999) reported father-to-son transmission of AZFc deletions in three families; Cram et al. (2000) reported 2 father-to- son transmissions of AZFc/d; Kamischke et al. (1999) reported a father-to-son transmission of an AZFc deletion containing the DAZ and CDY1 genes; Minor et al. (2007) reported an AZFc gr/gr deletion over three generations. Therefore, the application of ICSI procedures in cases of male-factor infertility associated to known genetic defects does have a real risk in vertical transmission of the defect. In these situations, genetic 21 profiles of the patients should be assessed, proper genetic counselling provided to assist in making informed decisions, and the use of preimplantation genetic diagnosis (PGD) (although not currently offered in Canada) be considered to minimize transmission of a possible genetic defect to the offspring. 1.5 Sperm Selection Criteria for ICSI Ideally, the sperm that is destined for use in ICSI is normal in function and viability. This is routinely identified as mature, morphologically normal, and motile spermatozoa. However this is not always possible in the most severely infertile cases, in which case, ICSI is attempted with the most “normal” sperm available. Although immature sperm have been used successfully with ICSI, including round spermatids (Tesarik et al., 1995) and elongated spermatids (Kahraman et al., 1998; Sofikitis et al., 1998) from patients with severely impaired spermatogenesis, the success rates are much lower. 1.5.1 Genetic Determinants in Sperm from Infertile Men With the ability of ICSI to by-pass natural barriers to abnormal conceptions coupled with the increased application of ICSI in the clinical setting, it would be important to assess the genetic integrity of the sperm potentially being used, namely that from severe male-factor infertility. In addition, the relatively high rates of fertilization from ICSI have not been matched with improved implantation and pregnancy rates. The cause of this may also lie in the genetic integrity of the sperm. 1.5.1.1 Analysis of Numerical Chromosomal Abnormalities in Sperm Methods that have been used extensively in the investigation of chromosomal constitution in spermatozoa include sperm karyotyping (Martin et al., 1988) and fluorescence in situ hybridization (FISH) (Martin et al., 1993). Each has its advantages and disadvantages, of which will be discussed. 1.5.1.1.1 Sperm Karyotyping Human sperm karyotyping, or the cytogenetic analysis of condensed chromosomes in sperm, is achieved with the fusion of a human sperm to a zona-free hamster oocyte. As 22 a result, both chromosome number and structure can be assessed. However, this method is very labor intensive, time consuming and is influenced by the fertilization potential of sperm. As result, very low numbers of sperm / patient have been analyzed. To date only about 20,000 sperm from 200 normal men have been analyzed from 8 centers worldwide (Guttenbach et al., 1997). By this method, normal men have structural aberrations occurring in 6.6% of sperm and numerical aberrations in 2.4%, with a mean of 0.03% for disomy of each autosome and 0.11% for sex chromosome disomy. The difficulties encountered by this technique have limited its ability to assess sperm of infertile men. In five infertile men with oligozoospermia, teratozoospermia and asthenozoospermia, the total aneuploidy rate was 19.7%, which was significantly higher than that of fertile men (14.5%) (Moosani et al., 1995) A variation of the human sperm / hamster oocyte fusion system that have been used is the microinjection of human sperm into mouse oocytes. With this technique, abnormal sperm could be assessed and the mouse oocytes more efficiently cleave compared to hamster oocytes (Lee et al., 1996). One study utilized this human sperm-mouse oocyte fusion test to assess the chromosomal constitution of sperm with different morphologic categories: normal, small, large, amorphous, round and elongated heads (Lee et al., 1996). They found no significant increase in chromosomal abnormalities in small and large heads compared to normal heads, but the number of sperm studied was quite small (nine sperm in the each of the small and large categories). The only statistical increase that was found was structural anomalies in the amorphous, round and elongated sperm compared to the normal sperm (26.1% vs. 6.9%, respectively; P<0.01). Again, because of the limitations of this technique, the numbers of sperm studied will be appreciably small. 1.5.1.1.2 FISH Analysis of Sperm FISH is a molecular technique that can be used to analyze the chromosomal complement in sperm. It consists of using sequence specific DNA probes labeled with fluorescent tags to hybridize with the complementary DNA sequence in the target cells of interest. The resulting fluorescent signals can then be counted, allowing for assessment of the copy number of the sequence of interest (Egozcue et al., 1997). When sequences unique to specific chromosomes are used, assessment of numerical chromosomal 23 abnormalities can be made. Also, FISH can be applied to non-dividing interphase chromatin, thus allowing for the analysis of thousands of sperm. In this context however, FISH cannot be used to assess structural chromosomal abnormalities. In addition, the number of chromosomes that can be examined per experiment is also limited by the number of spectrally distinct fluorescent labels. Despite this, disomy rates have been reported for all chromosomes (Downie et al., 1997; Egozcue et al., 1997; Martin et al., 1999). The mean frequency of disomy from FISH analyses on sperm of normal healthy men has been estimated to be 0.13% for autosomes and 0.37% for sex chromosome; this, in addition to the rate of diploid sperm (0.06-0.24%), has lead to a conservatively calculated rate of around 6.7% of chromosomal abnormality in the sperm of normal men (Vidal et al., 2001). Slight differences in rates of aneuploidy have been reported from study to study, and experimental differences and inter-individual variability are likely causes. Variability decreases significantly when analyses were done on 1x104 spermatozoa per set of FISH probes (Egozcue et al., 1997). It has also been suggested that some normal men are more prone to specific forms of sperm aneuploidy, who were termed stable variants, accounting for some of the variability in the results across different groups (Rubes et al., 2005). However, men with severely impaired semen parameters have increased rates of aneuploidy in sperm compared to normal men. Significant increases in aneuploidy have been found in infertile men with low sperm concentration (Rives et al., 1999; Vegetti et al., 2000; Calogero et al., 2001; Martin et al., 2003; Gianaroli et al., 2005; Pang et al., 2005; Rives, 2005) and significant correlation between the incidence of aneuploidy and decrease in sperm concentration has also been found (Ohashi et al., 2001; Martin et al.¸2003). Percentages of aneuploid sperm have also been found to be elevated in cases of: asthenozoospermia (Bernardini et al., 2005; Templado et al., 2005) and teratozoospermia (Machev et al., 2005; Templado et al., 2005; Kirkpatrick et al., 2008), including structural flagellar anomalies (Carrell et al., 2004; Rives et al., 2005). As mentioned earlier, ICSI conceptions have been found to have increased incidences of de novo chromosomal abnormalities compared the natural conceptions (Bonduelle et al., 2002; Lam et al., 2008). Patients undergoing ICSI have extremely poor semen parameters; the above studies suggest that these patients may have sperm with 24 significantly higher rates of aneuploidy compared to fertile men. Studies have attempted to find a correlation between abnormal semen characteristics, the most relevant to ICSI being morphology, and aneuploidy (reviewed by Shi and Martin, 2006). The basis of a relationship between sperm morphology and chromosomal aneuploidy stems from the finding that sperm immaturity was correlated to sex chromosome and chromosome 17 disomy (Kovanci et al., 2001). Sperm immaturity has been associated to cytoplasmic retention, increased size and roundness of sperm heads, amorphous sperm, and shorten tails (Huszar and Vigue, 1993; Gergely et al., 1999, Cayli et al., 2004). A connection between sperm maturation has also been suggested by the expression patterns of HspA2, a testis-specific chaperone protein that is expressed in spermatocytes and later in elongating spermatids during cytoplasmic extrusion and plasma membrane remodelling (Huszar et al., 1997, 2000). Furthermore, the mouse homologue of HspA2, HSP70-2, has been shown to be a component of synaptonemal complex during meiosis (Dix et al., 1996; Eddy, 1999). Individuals with complete globozoospermia, characterized by close to 100% of their sperm with round heads and no or very little acrosome do appear to have moderate increases in chromosomally abnormal sperm, incidences ranging from 0-5% involving different chromosomes (Carrell et al., 1999, 2001; Viville et al., 2000; Vicari et al., 2002; Martin et al., 2003; Morel et al., 2004; Ditzel et al., 2005). One study (Carrell et al., 1999) of 2 siblings affected with globozoospermia, which might suggest the existence of a heritable component, however found rates of aneuploidy to be quite elevated in only one of the siblings (13.5% for sex chromosome aneuploidy, 35.5% for chromosome 13 aneuploidy, 38.5% for chromosome 21 aneuploidy). But, only 7 studies involving 10 globozoospermic patients exists, limiting the ability to draw conclusive correlations between the phenotype and specific aneuploidy. Studies that investigated the chromosomal constitution in the sperm of teratozoospermia patients with close to 100% of the sperm with enlarged heads not only found that most of the sperm are chromosomally abnormal, particular with polyploidy, but that the incidence of macronuclear spermatozoa are also correlated to the incidence of polyploidy (Yurov et al., 1996; Benzacken et al., 2001; Devillard et al., 2002; Lewis-Jones et al., 2003; Vicari et al., 2003; Mateu et al., 2006). 25 Some studies looked at the incidence of chromosomal abnormalities in the sperm of individuals with a heterogeneous collection of morphologic abnormalities of sperm, polymorphic teratozoospermia (Calogero et al., 2001; Gole et al., 2001; Härkönen et al., 2001); however, the variability in clinical presentations is also matched by variability in the level and type of chromosomal abnormality. Calogero et al, (2001) found an increased rate of disomy for chromosomes 8 and 18 (but not chromosome 12 and XY disomy) in a group of eight teratozoospermic patients compared to 13 normozoospermic individuals. No difference was found in aneuploidy of teratozoospermic patients when compared to 19 OAT patients. The authors did concede that 3 of the teratozoospermic patients had less than normal levels of motile sperm, but closer inspection reveals that 3 others had borderline normal levels of motile sperm (close to but less than 50% progressively motile sperm), leaving only 2 individuals to be strictly isolated teratozoospermic. Therefore, the majority of their teratozoospermic group would actually be asthenoteratozoospermic (AT), and this may explain the similarity in aneuploidy rates with their OAT group; AT possibly being a more moderate form of OAT. Gole et al. (2001) also compared the aneuploidy rate in the sperm of teratozoospermic men to that of OAT and normozoospermic men, and found that sex chromosomal disomy was significantly more frequent in the OAT and teratozoospermic patients compared to their controls, but only XY disomy (not XX or YY disomy) was higher in the OAT than teratozoospermic patients (0.5% vs. 0.26%, respectively). Again, five of the eight patients classified as teratozoospermic had borderline normal levels of motile sperm. Härkönen et al. (2001) examined the rate of aneuploidy for chromosomes 1, 7, 18, X and Y in teratozoospermic patients that were segregated by severity in abnormal sperm morphology, by WHO criteria (group A: < 10% normal forms, n=7; group B: 10-19%, n=6; group C: 20-29%, n=7). The mean sperm concentration for all groups were normal; the mean percent of progressively motile sperm were above 50% for groups A and C (53.3±15.9 and 52.3±10.7, respectively; group B was 44.2±7.4), but the individual values were not published. So the actual number of strictly teratozoospermic patients is speculative. Nonetheless, they found significantly higher incidences of disomy 7, 18, XY and YY and diploidy, as well as inverse correlations between semen parameters and various aneuploidy (including the percent of morphologically normal sperm and disomy 1 and 7 and diploidy). However, other than 26 the association between enlarged headed sperm and polyploidy, the connection between other morphologic features and specific chromosomal abnormalities has yet to be identified, let alone any functional relationship. 1.5.1.2 DNA Fragmentation in Sperm DNA fragmentation is typically characterized by strand breaks. DNA strand breaks can be indicative of apoptosis. Since it is unlikely that spermatocytes with extensive DNA breaks could undergo the profound structural and functional chromatin changes that take place during the process of meiosis, DNA fragmentation likely occurs during spermiogenesis (Rodriguez et al., 2005). DNA strand breaks can be signs of incomplete sperm maturation; endogenous nicks occur during replacement of histones by protamines in chromatin packaging (McPherson and Longo, 1992; Marcon and Boissonneault, 2004). There is also evidence that indicates DNA damage in sperm is often oxidatively induced, rather than the result of other processes such as defective apoptosis (Kodoma et al., 1997; Barroso et al., 2000; Kemal Duru et al., 2000). To test for the level of DNA strand breaks, several methods can be employed. The sperm chromatin structure assay (SCSA) is a fluorescence-activated cell sorter test that measures the susceptibility of sperm nuclear DNA to heat- or acid-induced DNA denaturation followed by staining with acridine orange. Acridine orange is a metachromatic dye that fluoresces red when associated with denatured (fragmented) DNA and green when associated with double-stranded (normal) DNA (Evenson and Jost, 2000). However, SCSA measures the level of breaks in a given cell population and not in a single cell. The Comet assay involves the electrophoresis of cells, digested of plasma and nuclear membranes, in agarose gel (Collins et al, 1997). Fragmented and unfragmented DNA differentially migrate away from the nucleus. The migration of fragmented DNA is visualized with dyes and appears as a comet, where the tail length and signal intensity related to the degree of DNA fragmentation. Computer software subsequently measures the comet parameters (head density, comet tail length, comet moment) and calculates the magnitude of DNA fragmentation in each cell. The terminal deoxynucleotidyl transferase- mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) technique labels single and double stranded DNA breaks (Gorczyca et al, 1993). In TUNEL, terminal 27 deoxynucleotidyl transferase (TdT) catalyzes the polymerization of nucleotides (added in the assay reaction mixture) on the 3’-OH ends of double and single stranded DNA. With the use of nucleotides incorporated with fluorescent labels or antibodies (such a digoxigenin), DNA strand breaks can be identified. Several studies have linked male infertility to DNA damage in sperm (Aitken, 1999; Larson et al., 2000; Henkel et al., 2004; Tesarik et al., 2004). Loosely packed chromatin and endogenous DNA strand breaks have been found more often in both testicular germ cells and ejaculated spermatozoa in men with abnormal semen parameters (Irvine et al., 2000). DNA fragmentation has also been correlated negatively to sperm concentration and motility (Zini et al., 2001) and additionally to sperm morphology (Varum et al., 2007) in infertile men of mixed clinical classifications. Spermatozoa possess endonuclease activity that may be responsible for some DNA repair (Maione et al.,, 1997). Oocyte cytoplasm also has the capacity to repair chromosomal DNA lesions in sperm after fertilization (Genescà et al., 1992; Ashwood-Smith et al., 1996). Despite this, DNA damage in sperm has been associated with reduced rates of fertilization in vitro, impaired preimplantation development of the embryo, increased rates of pregnancy loss (Sakkas et al., 1998; Aitken and Krausz, 2001; Zini et al., 2001; Duran et al., 2002; Morris et al., 2002; Benchaib et al., 2003; Loft et al., 2003; Saleh et al., 2003; Bungum et al., 2004; Virro et al., 2004; Tesarik et al., 2006; Zini and Libman, 2006). So, there appears to be a threshold of DNA fragmentation that determines a viable conception. In addition, incomplete or incorrect repair of DNA damage may have genetic and epigenetic implications on future disease. A higher incidence of disease have been recorded in the offspring of men exhibiting significant DNA damage in their spermatozoa, including infertility and cancer (Aiken and Krausz, 2001; Aitken et al., 2004). Since DNA damaged sperm can achieve fertilization with the use of ICSI (Twigg et al., 1998; Gandini et al., 2004), transmission of DNA damage is a concern. So it seems that recognition of abnormalities that would be indicative of high levels of DNA fragmentation is important. Yet, limited investigation exists in associating excessive DNA fragmentation to specific sperm morphology. Conflicting reports involving globozoospermia exist: increase in DNA fragmentation has been found in sperm of globozoospermic men compared to fertile controls (10 vs. 0.1%, Baccetti et al., 1996; 37 vs. 22.5%, Vicari et al., 2002), while no difference was found in 28 DNA fragmentation in sperm of one globozoospermic man compared to controls (Larson et al. 2001). 29 Chapter II. Statement of Problem and Research Methodology 2.1 Aim of the study The ability of ICSI to circumvent some of the biological barriers associated with natural and in vitro fertilization is of particular concern when the sperm of infertile men could contain sub-cellular abnormalities detrimental to conception or health of potential offspring. Detection of known genetic defects associated with male-infertility is possible for the most part; however, not only is the cause of infertility unknown for a large proportion of infertile population, but so is the underlying genetic defect. Additionally, the integrity of the genetic material has been known to be sub-optimal in the sperm of infertile men. While procedures are available to maximize the selection of functionally viable sperm, the selection of the sperm during ICSI is still reliant on the recognition of morphologic characteristics of the sperm. In some infertile patients, the selection of morphologically normal sperm may not be possible. So it would be valuable to determine if sperm morphology can be a reliable indicator for the level of genetic integrity, particularly in application of ICSI. There have been studies that have attempted to correlate the level of chromosomal abnormalities to abnormal morphology in the sperm of infertile patients. Earlier studies tried to make such correlations in individuals with heterogeneous clinical presentations of abnormal sperm concentration, motility, and morphology; no correlation could be found between the level of normal morphology and the incidence of chromosomal abnormalities. However, the use of heterogeneous study populations to study the specific association of sperm morphology and chromosomal abnormality introduces possible confounding effects of the underlying causes of other seminal abnormalities; for instance, the etiology of reduced sperm concentration would be different from that of abnormal sperm morphology. Rare cases have been identified in which a single type of abnormal morphology is predominant in virtually all of the individual’s sperm (e.g., round headed sperm and enlarged headed sperm). Chromosomal analysis of the sperm in these cases have found significant correlation of enlarged headed sperm and polyploidy. So, the possibility of identifying specific types of sperm morphology at risk of containing chromosomal abnormalities exists. By focussing on individuals with infertility isolated to only abnormal sperm morphology, it may be possible to identify a subset of individuals whose abnormal 30 sperm morphology shares a common genetic etiology. It is at least conceivable that by focussing the investigation to individuals with abnormalities in only sperm morphology, certain factors responsible for reduced sperm number and low motility are eliminated. Although attempts of investigation with this strategy have been made for chromosomal abnormalities, the numbers of subjects that meet this criteria have been too limited to make any significant conclusions. Investigaton of another aspect of genetic integrity, DNA fragmentation, in relation to specific sperm morphology is even more limited, with only cases studies reported with respect to globozoospermia. To aid in the selection of viable sperm, particularly in the application of ICSI, the overall objective of this project is to determine if sperm morphology can be used to identify the level of genetic integrity. The specific objectives of this study are: 1. to determine if sperm from men of with infertility isolated to abnormal morphology have in increased rates of chromosomal abnormalities and DNA fragmentation compared to sperm from men of proven fertility; 2. to determine if specific forms of sperm morphology can be associated to different levels of chromosomal abnormalities and DNA fragmentation. 2.2 Significance of the Study This study will expand upon the knowledge gained from investigations on chromosomal abnormalities in the sperm of men with isolated teratozoospermia. The underlying hypotheses of this study is that sperm of men with isolated teratozoospermia have higher rates of chromosomal abnormalities and DNA fragmentation compared to the sperm from normal fertile men and that specific forms of sperm morphology can be identified as more likely to have chromosomal abnormalities and fragmented DNA. The strict inclusion of isolated teratozoospermic men in the examination of chromosomal abnormalities in sperm would provide a more convincing argument for or against the existence of a relationship between sperm morphology and chromosomal abnormalities. Any specific chromosomal abnormality that emerges to be significantly higher in the isolated teratozoospermic patients compared to the fertile men would also incite future investigation of its pathogenicity of abnormal sperm morphology and infertility. Since investigation of DNA fragmentation in the sperm of isolated teratozoospermic men thus 31 far have been quite limited, the information gained from this portion of the study would certainly be valuable in the management of these patients. The identification of specific types of sperm morphology that are associated to chromosomal abnormality and fragmented DNA would have a direct impact in the selection of sperm employed in ICSI procedures; avoiding the selection of such sperm may improve ICSI pregnancy outcomes. 2.3 Subject Inclusion Criteria Semen samples are collected from each subject and initially processed by standard procedures for semen evaluation by experienced technicians in the local fertility clinic. For the study group, this semen evaluation is part of their standard fertility treatment; for the control group semen evaluation will solely be for the purposes of participation in this research study. Standard assessments that pertain to this study include the values of sperm concentration, percent of motile sperm, and percent of normal morphology (by Kruger’s strict criteria) (WHO, 1999). The control group consists of healthy men who have proven fertility (conceived a healthy child born within a year of semen collection). They must also have normal values in terms of sperm concentration, motility, and morphology (i.e., >20x106 sperm / ml of semen, >50% of sperm are progressively motile, and >14% of sperm being morphologically normal by Kruger’s strict criteria). The study group consists of men whose sub-fertility is determined to be primarily the result of less than normal levels of morphologically normal sperm (<5%) that would have made them eligible for ICSI, but have normal values of sperm concentration (>20x106 sperm / ml of semen) and motility (>50% of sperm are progressively motile). This fertility status is also known as isolated teratozoospermia. 2.4 Additional Sperm Morphological Assessment In addition to the overall sperm morphology count included in the standard evaluations of semen assessed by the respective fertility clinics, a more detailed assessment of sperm morphology is repeated in our lab on all available raw semen samples. This detailed assessment provides a breakdown of the specific types of morphologic abnormalities that can be found in sperm and includes: specific head defects, mid-piece defects and tail defects. Furthermore, the same detailed morphologic 32 assessment is done on semen samples processed by swim-up technique to evaluate the types of morphologic abnormalities in potentially viable, motile sperm that would normally be selected for ICSI in a clinical setting. 2.4.1 Preparation for Morphological Assessment Preparation of semen for sperm morphology assessment is done according to protocols indicated by the WHO laboratory manual (WHO, 1999) with some modification. A small drop of raw semen from each subject is applied to a thoroughly cleaned glass slide and the semen is spread across the slide with the side of a clean, sterile glass pipette. The sample on the slide is air dried. In the swim-up procedure, an additional aliquot (1-3 drops) of each available whole semen sample is transferred to a 1.5ml conical centrifuge tube, and 1ml of modified human tubal fluid (HTF) is gently layered over the semen. The tube is then tilted to a 45º angle and incubated for 1 hour at 37ºC. The tube is then gently returned to upright position and the uppermost 200µl of the suspension is collected. Drops of the collected suspension are applied to a clean glass slide. When adequate cell density is achieved (sufficiently spread that will allow for efficient counting of sperm at 1000x magnification), the slide is allowed to air dry. Both air-dried samples are stained by a differentially staining set (Hemacolor Stain Set, EM Science, Gibbstown, NJ) similar to the Diff-Quik staining set indicated in the WHO laboratory manual (WHO, 1999). Each specimen slide is immersed five times for one second each in the supplied Solution 1 Fixative (99.8% methyl alcohol). Excess solution is allowed to drain off. Each slide is then immersed in Solution 2 (1.25g/L phosphate buffered eosin solution; 0.1g/L sodium azide) four times for one second each and allowing excess to drain in air for one second between each immersion. Each slide is then immersed in Solution 3 (1.2g/L phosphate buffered thiazine solution) four times for one second each and allowing excess to drain in air for one second between each immersion. Finally, each slide is rinsed in distilled water for 30 seconds and allowed to air dry. 2.4.2 Microscopic Analysis of Sperm Morphology The classification of sperm morphology is done according to the classification scheme detailed in the WHO laboratory manual for the examination of human semen and 33 sperm-cervical mucus interaction (WHO, 1999). For a spermatozoan to be normal, the sperm head, neck, midpiece, and tail must strictly conform to the “normal” criteria (Figure 2.1). The head should be oval, 4.0-5.0µm in length and 2.5-3.5µm in width. The head should have a well-defined acrosomal region, about 40-70% of the head area, and have less than 20% of head with unstained vacuolar areas. Any presence of a cytoplasmic droplet should be less than half the size of the normal head. The midpiece should be axially attached, less than 1µm thick and one and a half times the length of the head. The tail should be straight, uniform, uncoiled and roughly 45µm long. In the detailed morphological assessment, all details that deviate from the normal criteria are recorded and include: head defects, neck defects, abnormally large cytoplasmic droplets, midpiece defects, and tail defects (Figures 2.2 and 2.3). Head defects can include: small heads <4µm length and <2.5µm width; large heads >5.5µm length and >3.5 width; tapered, pyriform, round, thin and amorphous heads; vacuolated heads with >20% of head with vacuolar areas; and absence or small acrosomal areas (<40% of head). Neck defects can include bent necks (insertion angle >90º to longitudinal head axis) and asymmetrical insertion (non-axial attachment). Midpiece defects can include thick and thin midpieces. Tail defects can include short, coiled, bent, and multiple tails. Analysis of sperm morphology is done on a phase-contrast microscope (Nikon Eclipse E600) equipped with x10 and x40 phase objectives, a x100 oil-immersion objective, and x10 eyepieces equipped with a micrometer. The eyepiece micrometer (10mm/100 divisions) is calibrated with a stage micrometer (0-1mm/100 divisions) to allow differentiation of 1 micrometer at x1000 combined magnification, so head dimensions can be measured accurately. At least 200 consecutive spermatozoa are counted by systematically moving from field to field. Overlapping spermatozoa and those with the head lying on edge are not counted. All defects are recorded and represented as a percent of the cells counted per sample. In addition, two customary measures of sperm function, the teratozoopsermic index (TZI; or multiple abnormalities index) and sperm deformity index (SDI) is also calculated. TZI can be interpreted as the number of different morphologic abnormalities in each abnormal sperm and is determined as a ratio of the total number of defects / number of sperm with defects. A TZI of 1.00 represents one type of defect in each abnormal spermatozoon, while a TZI of 3.00 represents three 34 categorically different (e.g. head, midpiece, or tail) defects in every abnormal sperm. A TZI of more than 1.6 has been associated with lower pregnancy rates in untreated infertile couples (Jouannet et al., 1988). SDI also accounts for multiple morphologic abnormalities in each sperm, but considers both normal and abnormal sperm; SDI is determined as a ratio of total number of defects / number of spermatozoa counted. An SDI of more than 1.6 has been associated with IVF failures (Aziz et al., 1996). 2.5 Fluorescence in situ Hybridization (FISH) Fluorescence in situ hybridization is the molecular cytogenetic technique that employs sequence specific single stranded DNA probes incorporated with fluorescently labeled nucleotides to anneal to the denatured DNA targeted for study. This study will utilize probes unique to specific chromosomes of interest and will enable the enumeration of the number of chromosomes in each sperm of the study subjects. 2.5.1 Sample Preparation for FISH of Whole Semen For each subject, an aliquot (1-3 drops) of whole semen sample (either fresh or thawed from cryogenic storage in liquid nitrogen) is added to a centrifuge tube and washed 3 times in 1X phosphate buffered saline (PBS) by repeated centrifugation (500g), removal of supernatant, and re-suspension of pellet. The final sperm suspension of each sample is aspirated by a glass pasteur pipette and dropped onto clean glass slides. When the desired cell density (enough to efficiently count 10,000 sperm within a 22mm x 22mm area) is achieved on the slide, the cells are allowed to air dry. The dried sample is then fixed onto the glass slides with 3:1 methanol: glacial acetic acid. This preparation can then be processed by FISH to assess chromosome constitution. 2.5.2 Sample Preparation for FISH of Morphologically Separated Spermatozoa An additional aliquot of the whole semen sample from each subject will be processed by swim-up fractionation (as mentioned in section 2.4.1 on page 31) above to isolate normal, motile spermatozoa. A small drop of the selected sperm is then placed on a Petri dish prepared with addition of droplets of modified HTF. The spermatozoa in each 35 sample are separated by specific morphologic criteria with the use of a micromanipulation system (Narishige) fitted to an inverted microscope (Nikon Eclipse TE200) equipped with Hoffman modulation contrast condensers, x4, x10, x20, x40 objectives, and x10 eyepieces equipped with a micrometer. The eyepiece micrometer (10mm/100 divisions) is calibrated with a stage micrometer (0-1mm/100 divisions) to allow differentiation of sperm head dimensions. The technical limitations of the optics feasibly allows for the recognition of only the exterior head characteristics. Consequently, sperm are separated by: size of uniformly oval heads (small heads <4µm in length, medium heads between 4 and 6µm in length, large heads >6µm in length), and amorphous heads of any size. Spermatozoa of particular morphologic criteria will be aspirated by a micropipette controlled by the micro-manipulator and transferred to separate droplets of modified HTF prepared on the Petri dish. Entire droplets of particular morphologic criteria are then transferred to a clean glass slide and allowed to air dry. Prior to the droplets drying, the boundaries of the droplets are etched with a carbide pen to aid in the location of the sperm after subsequent processing of the sperm. After the sperm are dry, they are fixed onto glass slides and processed by FISH for assessment of chromosomal constitution. 2.5.3 FISH Preparation and Analysis The methods of sperm preparation, probe hybridization, and FISH analysis were described previously (Tang et al., 2004). Sperm are fixed onto glass slides by treating the slides in with 3:1 methanol:acetic acid for five minutes and subsequent air drying. The fixed sperm are treated in 2 x SSC (standard saline citrate solution) for 5 minutes at room termperature. The sperm nuclei are then decondensed by incubated in dithiothreitol (10mM, 100mmM Tris-HCl) at room temperature initially for 30 minutes, and for additional 10 minute treatments, until the sperm heads have sufficiently increased in size while still preserving sperm tails. Preservation of sperm tails allows for differentiation between mature spermatozoa and other cell types that may be present in the sample. After sperm are decondensed, the slides are treated with 2 x SSC for 5 minutes at room temperature. Triple-colour FISH, with α-satellite DNA probes for chromosomes 18 (SpectrumAqua), X (SpectrumGreen) and Y (SpectrumOrange), and dual-colour FISH, with probes for chromosomes 13 (SpectrumGreen) and 21 (SpectrumOrange) (Vysis Inc., 36 Downers Grove, IL) are used for both study and control samples. Hybridization procedures follow protocols supplied for probes. The slides are de-hydrated in an ethanol series (70, 80, 90, and 100%) for 2 minutes each at room temperature and allowed to air dry. The slides are then treated in denaturation solution (70% formamide, 2 x SSC) for 5 minutes at 73ºC. The denatured slides are then de-hydrated in an ethanol series (70, 80, 90, and 100%) for 1 minute each at room temperature. The slides are allowed to dry on a slide warmer at 45ºC. Because the DNA probes being used are pre-denatured, the only preparation for the probes is thawing to room temperature. Once the slides are completely dried, 10µl of a probe mixture is applied directly to the slide and a 22 x 22 mm glass coverslip is laid on top of the probe mixture. When the probe has fully spread underneath the coverslip, the coverslip is sealed with rubber cement, and the slide is incubated overnight at 37ºC in a humidified container. After the hybridization period, the coverslip is removed, and the slide is washed in 0.4xSSC / 0.3% NP-40 for 1 minute at 73ºC and then in 2xSSC / 0.1% NP-40 for 30 seconds at room temperature. The slides are then rinsed in PBS for 1 minute, treated with 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich Canada Ltd., Oakville, ON) counterstain for 1 minute, rinsed again in PBS for 1 minute, and allowed to air dry all at room temperature. Once dry, anti-fade solution in applied to the hybridization area and a coverslip is overlain. The slides are then stored in the dark and at -20ºC until they are ready to be analyzed. Analysis of the FISH assays are done on research microscopes (Nikon Eclipse E600 and Zeiss Axioplan 2) equipped with 100W halogen bulb and triple band-pass filters for DAPI/FITC/Texas Red, and single band-pass filters for FITC (specific for SpectrumGreen), Cyan 3 (specific for SpectrumOrange), and Aqua. Only sperm with intact head and tail morphology, within an area of the slide where consistent hybridization is evident, are scored (Figures 2.4 and 2.5). Two signals of the same colour are scored as two copies of the corresponding chromosome when they are comparable in brightness and size and are separated from each other by a distance longer than the diameter of each signal. Nullisomy of any individual chromosome is considered when the sperm clearly contain at least one of the other chromosomal signals (internal control). Simultaneous hybridization of three chromosomal probes (i.e., chromosome 18, X, and Y) are used to differentiate between disomy (result of non-disjunction) and diploidy (result of non- 37 reduction). A minimum of 10,000 sperm is scored for each probe combination for each individual. Sperm with a complete lack of signals (complete nullisomy) are compared against the total cells counted to calculate the hybridization efficiency. The incidences of chromosomal abnormalities are reported as percentages of the cells counted less the cells that were completely nullisomic (those with no signals) as true nullisomic sperm can not be distinguished from cells that could not be adequately hybridized. Aneuploidy, for the purposes of this project, is strictly defined as a departure from normal euploidy (in the case of haploid sperm: a single copy of any autosome and a single copy of any gonosome) by a single chromosome change (gain or loss of one whole chromosome). Any departures from euploidy involving multiple chromosomes are classified as a supernumerary chromosomal abnormality (e.g., Figure 2.4c). Ploidy changes (changes of whole chromosomal complement) are identified by matched changes in the number of autosomes and the number of gonosomes (eg. a sperm is diploid if it has two chromosomes 18 and two sex chromosomes). For this project, ploidy changes are only considered in the hybridization of the 18,X,Y chromosome probe set (Figure 2.4d). The hybridization of the 13,21 chromosome probe set is not used to evaluate ploidy changes because it is difficult to distinguish single chromosomal gain from a haploid complement to that of a single chromosomal loss from a diploid complement (eg. 24, X, +13 or 24, Y, +13 will look the same as 45, XY, -21 when hybridized with probes for just chromosomes 13 and 21: both will have 2 signals for chromosome 13 and one signal for chromosome 21). 2.6 DNA Fragmentation Methodology Sperm DNA fragmentation is evaluated with the TUNEL assay (In Situ Cell Death Detection Kit, Roche, Mannheim, Germany). To evaluate the level of DNA fragmentation in viable sperm, motile sperm is selected for by the swim-up technique described early (page 31). Swim-up selected sperm are dropped onto slides, and allowed to air dry. The sperm are fixed onto the slides with freshly prepared 4% paraformaldehyde in PBS (pH 7.4) for 1 hour at room temperature and then rinsed in PBS. The slides are then incubated in a solution of 0.1% Triton X-100 (Sigma, St. Louis, MO) and 0.1% sodium citrate (Sigma) for 2 minutes on ice. After incubation, the slides are washed twice in PBS and 38 air-dried. A 50μL TUNEL reaction mixture, containing terminal deoxynucleotidyl transferase (TdT) and fluorescein-deoxyuridine triphosphate, is added to each slide, covered with 22 × 22mm coverslips and then incubated for 60 minutes at 37°C in a moist dark chamber. After incubation, the slides are washed three times with PBS and air-dried. Anti-fade is added to the treated slides and a coverslip is applied. Each TUNEL assay is performed with accompanying positive and negative control slides. Positive control sperm samples are fixed and treated with DNase I (100U in 50mM Tris-HCl, pH 7.5; Sigma) for 10 minutes at room temperature to induce DNA fragmentation prior to the procedures described above. The negative controls are prepared as above, but without TdT. Analysis is done with a research microscope (Zeiss Axioplan 2) equipped with a 100W halogen bulb, a single band-pass FITC filter, and a triple band-pass filter for DAPI/FITC/Texas Red, which allow for the differentiation of sperm labelled with fluorescein (positive for DNA strand breaks) and those without (negative for DNA strand breaks) (Figure 2.6). The level of DNA fragmentation in each sample is represented by a comparison of cells with or without the fluorescein label and is recorded as a percentage of fluorescein- labelled cells per total cells counted (at least 500 cells per sample). 2.7 Statistical Analysis Statistical analysis of the collected data is done with the aid of statistics software (GraphPad Prism Version 5.0). Although the data collected in this study are on a ratio scale, all are proportional data (percentage of total counted). Percentages form a binomial distribution and deviate from a normal distribution particularly at small and large percentages. Since the frequencies of chromosomal abnormalities found in sperm are historically low, raw frequencies (rather than percentages) from the chromosomal analysis is statistically analysed with parametric tests such as Student’s t test and analysis of variance (ANOVA). The Student’s t test is also used to compare the level of DNA fragmentation between the control and teratozoospermic subjects as the incidences are not expected to be extremely low or high. When the use of raw frequencies are invalid (i.e., when data are recorded as percentages of different sample sizes or the variable, by definition is a proportion), non-parametric statistical tests are used to avoid the effects of departures from normality. As a result, comparison of the frequencies of specific sperm 39 morphologic types is accomplished through non-parametric statistical tests such as Mann- Whitney test (for bivariate comparisons) and Kruskal-Wallis test (for multi-variate comparisons). Simple linear correlation analysis are also used to determine if specific sperm morphologic types occur simultaneously with other morphologic types, as well with levels of chromosomal abnormality and DNA fragmentation. Because data included may again deviate from normal, the non-parametric Spearman’s rank correlation procedure is employed. More detailed information about the specific statistical tests used and the reasons for their application will be provided in the relevant results sections to follow. Statistical significance is determined with at least 95% confidence (P<0.05). 40 Chapter III. Results 3.1 Sperm Morphology Assessment A detailed morphologic assessment on washed, unselected sperm further resolves the specific morphologic variations than can be provided by the routine clinical assessment. As well, a detailed morphologic assessment of swim-up selected sperm allows for a better appreciation of the morphology of sperm that would typically be used in cases of severe male-factor infertility. Comparison of detailed sperm morphology was made between the fertile control and isolated teratozoospermic subjects to determine what the morphologic differences are, if any, between the two groups. Doing a detailed morphologic assessment on raw, unselected and swim-up selected sperm for each semen sample allowed for the evaluation of the effect of the swim-up selection on sperm morphology from both fertile control and isolated teratozoospermic subjects. 3.1.1 Results of Morphologic Assessment Semen samples from seven control subjects and ten isolated teratozoospermic subjects were available at the time the detailed assessment of sperm morphology was done. The results of the detailed morphologic assessment for each subject are detailed in Table 3.1 for the control subjects and Table 3.2 for the teratozoospermic subjects. The group means of the morphologic assessments for the control and teratozoospermic subjects are presented in Table 3.3 for easier comparison of the raw, unselected and the swim-up selected sperm. The incidences of each morphologic feature represent purely their presence in any particular sperm; the presence of each morphologic feature is not mutually exclusive and multiple abnormal features can be present in a given sperm. Correspondingly, linear correlation analysis was applied to determine if the incidence of any specific abnormal sperm morphology can be correlated to each other in the control and teratozoospermic subjects (Tables 3.4 and 3.5 for raw, unselected sperm; Tables 3.6 and 3.7 for swim-up sperm, respectively). The data collected in the morphologic assessments are represented as percentages of the specific morphologic types out of the total cells counted (at least 200), with some morphologic types being quite rare. The data will also include index values such as TZI and SDI. Consequently, normal distribution of 41 the data cannot be assumed, and comparisons of the data between the control and teratozoospermic subjects as well as between the raw, unselected and swim-up samples were done with non-parametric statistical tests such as Mann Whitney, for bivariate, and Kruskal-Wallis (and Dunn multiple comparison post-test), for multi-variate comparisons. The non-parametric Spearman’s rank correlation coefficient (rs) was calculated for the linear correlation analysis between the specific types of abnormal sperm morphology. Statistical significance was determined with 95% confidence (P<0.05). 3.1.1.1 Comparison of Morphology in Raw, Unselected Sperm of Control and Teratozoospermic Subjects Although all control subjects that participated in the study satisfied the study inclusion criteria, information linking the clinical information (eg. clinical semen parameters) of the samples to their coded identification (to maintain anonymity) was unfortunately unavailable for 6 (of the 7 available) control samples (see “Kruger’s Measurement” in Table 3.1). This prevented a valid statistical comparison of the clinical sperm morphology value (Table 3.3) between the control and teratozoospermic subjects. However, the detailed morphological assessment done on the seven available semen samples allowed for the comparison of the level of normal sperm (without any morphologic abnormality) in the control and teratozoospermic subjects. The proportion of normal sperm in the raw, unselected sperm sample was significantly higher in the control subjects than in the teratozoospermic subjects (21.53±4.92% vs. 10.36±2.27, respectively; P=0.0001) (Table 3.3). Comparison of the specific types of abnormal morphology found in the raw, unselected sperm of control and teratozoospermic subjects presented in order of descending prevalence are as follows. The most prevalent category of morphologic abnormality encountered in the raw, unselected sperm in both the control and teratozoospermic subjects was internal head abnormalities (53.72±9.27% and 58.44±14.73%, respectively). The high incidence of internal sperm head abnormalities in control and teratozoospermic subjects largely consisted of abnormally vacuolated areas (48.33±10.57% and 49.45±19.01%, respectively) (e.g., Figure 2.2h), followed by the incidence of acrosomal abnormalities (that includes small or absent of acrosomal areas) (5.46±2.01% and 9.68±11.74%, respectively) (e.g., 42 Figure 2.2c, e; Figure 2.3a). Although the mean incidences of the internal head abnormalities (individually and combined) in the teratozoospermic subjects were all higher than the respective values from the control subjects, none were statistically significant. The second most prevalent category of morphologic abnormality in the raw, unselected sperm was head shape abnormalities, which was significantly less frequent in the control subjects compared to the teratozoospermic subjects (23.53±2.92% vs. 37.83±10.91%, respectively; P=0.0046). The incidence of the amorphous heads was highest of the head shape category (e.g., Figure 2.2e, g, h; Figure 2.3d), and was significantly lower in the control subjects than in the teratozoospermic subjects (10.22±4.38% vs. 18.97±5.94%, respectively; P=0.0097). Although the incidences of the other recorded head shape abnormalities were not significantly different between the two groups, the incidences of round, thin, and pyriformed heads were lower in the control group compared to the teratozoospermic group (4.01±1.84% vs. 5.53±5.49%, 0% vs. 0.59±0.79%, 4.26±3.25% vs. 10.06±8.53%, respectively). Contrarily, the incidence of tapered sperm heads was higher in the control subjects than in the teratozoospermic subjects (5.04±4.43% vs. 2.68±3.83%, respectively). The category of morphologic abnormality with the next highest incidence in both the control and teratozoospermic groups is that of tail abnormalities (e.g., Figure 2.3f, g, h). While the incidence of each of the recorded tail abnormalities (coiled, bent, irregular, 2-tailed, and combined) was lower in control subjects compared to that of the teratozoopsermic subjects (10.61±7.21% vs. 14.21±7.62%, 0.27±0.38% vs. 1.99±1.29%, 0% vs. 0.25±0.35%, 0.27±0.26% vs. 0.55±0.92%, 11.15±7.39% vs. 16.99±7.30%), no difference was statistically significant. The incidences of abnormally small and large head sperm were not significantly different between the control and teratozoospermic groups (3.05±1.70% vs. 8.27±11.62%; 2.65±1.87% vs. 1.00±0.90%, respectively). The incidences of neck abnormalities (bent or asymmetric insertion; Figure 2.3a, b) were also not significantly different between the control and teratozoospermic groups (5.34±1.39% vs. 4.68±2.67%, 0.33±0.35% vs. 1.09±1.09%, respectively). As well, the incidences of midpiece abnormalities (thin or 43 thick; Figure 2.3c, e) were not significantly different between the control and teratozoospermic groups (0% vs. 0.20±0.41%, 4.85±2.45% vs. 9.39±7.24%, respectively). The incidence of sperm with abnormally large cytoplasmic droplets (larger than half the head) was significantly lower in the control group than that in the teratozoospermic group (7.22±6.24% vs. 2.39±3.15%, respectively; P=0.0431). In addition to the individual sperm categories of sperm morphology, the teratozoopsermic index (TZI; or multiple abnormalities index) and the sperm deformity index (SDI) were also calculated for the raw, unselected sperm. The TZI, interpreted as the number of different morphologic abnormalities per abnormal sperm, of the control subjects were significantly different than the teratozoopsermic subjects (means of 1.41±0.16 vs. 1.56±0.07, respectively; P=0.0431). The SDI similarly takes into account multiple abnormalities in each sperm, but considers both normal and abnormal sperm. The SDI of the control subjects was significantly different than that of the teratozoopermic subjects (1.09±0.13 vs. 1.40±0.08, P=0.0001). 3.1.1.2 Effects of Swim-up Sperm Selection Selection of motile sperm by the swim-up technique to do the TUNEL assay provided the opportunity to assess the effect of swim-up selection on the distribution of abnormal morphology in sperm of our control and teratozoopsermic subjects. The swim-up selection of sperm did not significantly alter the incidence of any of the recorded morphologic characteristics in the control subjects (detailed in Table 3.1). The incidence of the normal sperm was similar between the raw and swim-up samples (21.53±4.92% vs. 21.31±7.16%). None of the incidences of recorded morphologic abnormalities in the raw samples significantly differed from the swim-up samples: internal head abnormalities (vacuolated and acrosomal abnormalities combined) were 53.72±9.27% and 49.81±16.88%, abnormally large cytoplasmic droplets were 7.22±6.24% and 2.86±2.45%, combined head shape abnormalities were 23.53±2.92% and 22.11±6.10%, combined neck and midpiece abnormalities were 10.03±1.50% and 14.68±4.69%, and combined tail abnormalities were 11.15±7.39% and 19.51±12.17%; respectively. There was also no significant difference in the TZI and SDI between the raw 44 and swim-up sperm in the control subjects (1.42±0.14 vs. 1.45±0.15, 1.11±0.11 vs. 1.15±0.20; respectively). With respect to the teratozoospermic subjects, the swim-up technique did increase the proportion of morphologically normal sperm (10.36±2.27% in raw vs. 26.10±11.48% in swim-up, P<0.0001). The incidences of each of the specific morphologic abnormalities in the swim-up selected sperm were lower than the respective values in the raw, unselected samples; however, no differences were statistically different (Table 3.2). In spite of the lack of statistical difference in the individual morphologic abnormalities, the TZI and SDI did improve significantly with the use of the swim-up technique (1.56±0.07 in raw vs. 1.42±0.15 in swim-up, P=0.0185; 1.40±0.08 in raw vs. 1.06±0.25 in swim-up, P=0.0002; respectively). In addition, the significant differences seen in sperm morphology in raw, unselected selected sperm between the control and teratozoospermic subjects were not seen in the swim-up selected sperm (Table 3.3). The occurrence of all recorded morphologic characteristics (normal, abnormal, and derived indices) in the swim-up selected sperm of the teratozoospermic subjects were not significantly different from that of the control subjects. 3.1.1.3 Correlation of Sperm Morphology Since the TZI values of the control and teratozoopsermic subjects in both the raw, unselected and swim-up sperm samples (1.42±0.14 and 1.56±0.07 in raw; 1.45±0.15 and 1.42±0.15 in swim-up, respectively; Table 3.3) indicate that there is slightly more than one morphologic abnormality per abnormal sperm, it would be valuable to investigate whether any morphologic features tend to occur in concert with others. This may shed light into possible shared mechanisms of development and have implications when considering genetic origins. To determine the possibility of concurrence of specific morphologic features in the sperm, statistical linear correlation analysis was applied to the incidences of recorded features from the detailed morphologic assessment. Again, because the prevalence of specific morphologic features cannot be assumed to be normally distributed, the non-parametric Spearman’s rank correlation coefficient (rs) was calculated. The control and teratozoospermic samples were compared independently since they are 45 considered clinically distinct and the mechanisms of abnormal sperm development may also be correlatively distinct. The raw, unselected samples and swim up samples were also considered separately to allow for the results to be evaluated appropriately with the chromosomal analyses (done on raw, unselected sperm) and DNA fragmentation analysis (done on swim-up sperm). Based on the seven available raw, unselected sperm of the control subjects, a few morphologic features of sperm correlated to each other with statistical significance (with 95% confidence) (Table 3.4). There was a positive correlation between the incidence of morphologically normal sperm (from detailed morphologic assessment) and the incidence of asymmetric neck insertion (rs = 0.8233, P=0.0341). The incidence of coiled tails positively correlated to the incidence of bent tails (rs = 0.7684, P=0.048). There was actually a negative correlation between the incidence of large headed sperm and abnormally vacuolated sperm (rs = -0.8929, P=0.0123). In other words, the larger the head of the sperm, the smaller the likelihood that the sperm will have an abnormal amount of vacuoles, and that the presence of vacuoles in the sperm nuclei do not contribute an increased size of sperm head. With respect to the morphologic indices, the incidence of amorphous shaped sperm heads positively correlated to the TZI (rs = 0.9286, P=0.0067), and thus, a higher TZI more often involves the presence of amorphous shaped sperm heads in fertile men. Finally, there was significant correlation between the TZI and SDI (rs = 0.7857, P=0.048), which illustrates that the two indices similarly identify the number of morphologic abnormalities per sperm. In the ten raw, unselected semen samples from teratozoospermic subjects, there were quite a few more morphologic features of sperm correlated, both positively and negatively, to each other with statistical significance (Table 3.5). The clinically measured level of morphologically normal sperm (“Kruger’s measurement in Table 3.5) negatively correlated to the incidence of amorphous shaped sperm heads (rs = -0.7377, P=0.0174). The incidence of morphologically normal sperm (from detailed morphologic assessment) positively correlated to the incidence of asymmetric neck insertion (rs = 0.7212, P=0.0234), as was the case of the raw, unselected sperm in the control subjects. With respect to sperm head characteristics, the incidence of small headed sperm positively correlated to the incidences of acrosomal abnormalities (rs = 0.8667, P=0.0022) and round 46 headed sperm (rs = 0.6727, P=0.039), but negatively to the incidences of abnormally vacuolated sperm (rs = -0.6485, P=0.049) and pyriform shaped sperm heads (rs = -0.7939, P=0.0088). The incidence of abnormally vacuolated sperm negatively correlated to the incidences of acrosomal abnormalities (rs = -0.7091, P=0.0268) and bent sperm tail (rs = - 0.6848, P=0.0347). The incidence of acrosomal abnormalities negatively correlated to the incidence of pyriform shaped sperm heads (rs = -0.697, P=0.0306). The incidence of round headed sperm also negatively correlated to the incidence of pyriform shaped sperm heads (rs = -0.8182, P=0.0058) but positively correlated to the incidence of tapered shaped sperm heads (rs = 0.7295, P=0.0202). The incidence of thin shaped sperm heads correlated positively to the incidence of amorphous shaped sperm heads (rs = 0.7943, P=0.0088). Moving along the longitudinal axis of the sperm, the incidence of abnormally large cytoplasmic droplets negatively correlated with the incidence of irregular tail structure (rs = -0.6575, P=0.0438). The incidence of bent necks positively correlated with the incidence of 2-tailed sperm (rs = 0.6486, P=0.049). The incidence of thin midpieces negatively correlated to the incidence of coiled tails (rs = -0.6833, P=0.0347). The incidence of bent tails coincided significantly with the incidences of irregular tails (rs =0.6691, P=0.039) and 2-tails (rs = 0.7579, P=0.0149). With respect to the morphologic indices, the incidence of small heads positively correlated to the TZI (rs = 0.6727, P=0.039), and thus, a higher TZI more often involves the presence small sperm heads in teratozoospermic men. As in the raw unselected sperm from the control subjects, TZI significantly correlated to SDI (rs = 0.8182, P=0.0058), again illustrating the two indices similarly identify the number of morphologic abnormalities per sperm. There were similar number of statistically significant correlations between the different sperm morphology in the swim-up selected sperm (Table 3.6) as in the raw, unselected sperm of the six control subjects; however, the correlations involved different pairings of morphologic features. The clinical value of normal sperm morphology was not considered in the swim-up sperm as the clinical evaluation in usually done on raw, unselected sperm. The incidence of small headed sperm positively correlated to the incidence of sperm with irregular tails (rs = 0.8018, P=0.0341). The incidence of large headed sperm positively correlated to the incidence of acrosomal abnormalities (rs = 0.7928, P=0.0480). The incidence of amorphous shaped sperm heads positively correlated 47 to the incidence of acrosomal abnormalities (rs = 0.8571, P=0.0238), but negatively to the incidence of vacuolated sperm (rs = -0.8214, P=0.0341). The incidence of tapered shaped sperm head negatively correlated to the incidence of bent tails (rs = -0.8829, P=0.0123). Not only did TZI and SDI positively correlate to each other (rs = 0.8929, P=0.0123), TZI and SDI both positively correlated to acrosomal abnormalities (rs = 0.8929, P=0.0123; rs = 0.7857, P=0.0480, respectively) and coiled tails (rs = 0.8571, P=0.0238; rs =0.8214, P=0.0341, respectively). Additionally, SDI negatively correlated to the level of normal sperm (determined from the detailed morphologic assessment) (rs = -0.7857, P=0.0480). As was the case in the raw, unselected sperm, there were more morphologic features that correlated with one another in the swim-up selected sperm of the ten teratozoospermic subjects (Table 3.7) compared to the control subjects. The incidence of normal sperm in swim-up samples significantly correlated to quite a few more morphologic characteristics than what was seen for the raw, unselected samples. The incidence of normal sperm positively correlated to the incidence of thin shaped sperm heads (rs = 0.8057, P=0.0072), but negatively correlated to the incidences of tapered shaped sperm heads (rs = -0.8254, P=0.0047), abnormally large cytoplasmic droplets (rs = -0.6659, P=0.0390), asymmetric neck insertions (rs = -0.6833, P=0.0347), thick midpiece (rs = -0.9879, P<0.0001). The incidence of small headed sperm positively correlated to the incidence of round headed sperm (rs = 0.9362, P=0.0002) but negatively correlated to the incidences of large headed sperm (rs = -0.7134, P=0.0234) and pyriform shaped heads (rs = -0.7538, P=0.0149). Also, the incidence of round headed sperm negatively correlated to the incidence of thin shaped sperm heads (rs = -0.7033, P=0.0268), while the incidence of pyriform shaped sperm heads negatively correlated to the incidence of acrosomal abnormalities (rs = -0.6606, P=0.0438). The incidence of abnormally large cytoplasmic droplets positively correlated to the incidences of tapered shaped sperm heads (rs = 0.6870, P=0.0347) and thick midpieces (rs = 0.7435, P=0.0174); incidence of tapered shaped sperm heads positively correlated to incidence thick midpieces (rs = 0.8567, P=0.0029). The incidence of asymmetric neck insertion positively correlated to the incidences of bent necks (rs = 0.7028, P=0.0268), thin midpieces (rs = 0.7454, P=0.0174), and thick midpieces (rs = 0.6833, P=0.0347). The morphologic indices, TZI and SDI, appeared to have a more defined pattern of correlation to specific morphologic features in the swim- 48 up selected sperm, compared to the raw, unselected sperm, of the teratozoopsermic subjects. Both the TZI and SDI negatively correlated to the incidence of morphologically normal sperm (rs = -0.7333, P=0.0202 and rs = -0.8788, P=0.0016; respectively). Both the TZI and SDI positively correlated to the incidences of abnormally large cytoplasmic droplets (rs = 0.8728, P=0.0016 and rs = 0.8469, P=0.0029, respectively) and thick midpieces (rs = 0.7818, P=0.0105 and rs = 0.9030, P=0.0008; respectively), while the SDI also had an additional positive correlation to the incidence of tapered shaped sperm heads (rs = 0.6753, P=0.0390). Again, there was good correlation between the TZI and SDI (rs = 0.9515, P=0.0001). 3.2 Chromosomal Analysis in Sperm 3.2.1 Chromosomal Analysis of Raw, Unselected Spermatozoa Chromosomal analysis was done by FISH on raw, unselected samples of nine control and ten teratozoospermic subjects with a chromosome 18,X,Y probe set (Table 3.8) and a chromosome 13,21 probe set (Table 3.9). The control and teratozoospermic group means of the pertinent chromosomal abnormalities are also summarized in Table 3.10. Although the results are summarized and presented as percentages of the cells counted, the statistical analyses were done on the raw frequencies to allow for the use of parametric tests without violating underlying assumptions of normality. Statistical significance was determined with 95% confidence (P<0.05). One-way ANOVA for repeated measures (done for counts from the same probe set) were used to determine if there are differences in disomy and nullisomy within each of the control and teratozoospermic groups. Relatively, the aneuploidy (combined disomy and nullisomy) of chromosome 13 was compared to that of chromosome 21 (same probe set) and aneuploidy of chromosome 18 was compared to that of the sex chromosomes (the other probe set) by paired-sample t tests in each of the control and teratozoospermic groups. To compare the different types of aneuploidy among all the chromosomes studied in each of the control and teratozoospermic groups, all aneuploidy types were analysed by one-way ANOVA followed by the Tukey test (if a significant difference is found in ANOVA) for multiple comparisons to elucidate which pairs of aneuploidy are significantly different. Student t test was also used to compare the different types of chromosomal abnormalities in the 49 control subjects to the respective chromosomal abnormalities in the teratozoospermic subjects. In the control subjects (n = 9), incidence of disomy for each of the chromosomes studied was not significantly different from the incidence of nullisomy for the corresponding chromosome (Table 3.8 and 3.9). However, there were significant differences between the disomy of chromosome 18 and sex chromosomes (means of 0.06±0.05% vs. 0.45±0.19% respectively; P<0.001) and the nullisomy of chromosome 18 and sex chromosomes (means of 0.07±0.08% vs. 0.41±0.25, respectively; P<0.001) (Table 3.8). When the aneuploidy (combined disomy and nullisomy) of each of the chromosomes were compared against each other (Table 3.10), the aneuploidy rate was lowest for chromosome 18 (0.13±0.09%), followed by the increasing incidences of aneuploidy for chromosomes 21, 13 and sex chromosomes (0.50±0.37%, P<0.05; 0.52±0.23%, P<0.05; 0.86±0.36%, P<0.001; respectively). The incidence of aneuploidy for chromosomes 21 was also significantly lower than that for the sex chromosomes (0.50 ± 0.37 vs. 0.86 ± 0.36, P<0.05). In the teratozoozpermic patient group (n = 10), no significant difference was also found between the incidences of disomy and nullisomy for each of the chromosomes studied (Tables 3.8 and 3.9). However, the disomy rate of the chromosome 18 was significantly different from the disomy of sex chromosomes (0.23±0.53% vs. 0.76±0.54%, P<0.05) (Table 3.8). Unlike in the control subjects, no significant difference could be found when the aneuploidy of each of chromosomes studied in the teratozoospermic patients were compared against each other (Table 3.10). In comparison of the aneuploidy rates between the control and teratozoospermic subjects, the order of prevalence differed. In decreasing order of most to least prevalent, the aneuploidy rates in the control subjects were: 0.86±0.36% for sex chromosomes, 0.52±0.23% for chromosome 13, 0.50±0.37% for chromosome 21, and 0.13±0.09% for chromosome 18 (statistical difference indicated above). In decreasing order of most to least prevalent, the aneuploidy rates in the teratozoospermic subjects were: 1.51±1.52% for chromosome 21, 1.30±0.83% for chromosome 13, 0.94±0.54% for sex chromosomes, and 0.43±0.59% for chromosome 18 (none were statistically different from one another, as indicated above). When the disomy, nullisomy, and aneuploidy (combined disomy and 50 nullisomy) for all the chromosomes investigated of the control subjects were compared against that of the teratozoospermic patients, the incidences of disomy and aneuploidy for only chromosome 13 was significantly different between the control and teratozoopsermic groups (0.20±0.14% vs. 0.54±0.52%, P=0.0046; 0.52±0.23% vs. 1.30±0.83%, P=0.0148, respectively). The total aneuploidy (combined disomy and nullisomy of all chromosomes analysed) of the control subjects was significantly different from that of the teratozoospermic subjects (1.99±0.65% vs. 4.90±2.82%, P=0.0087). Similarly, the total of all chromosomal abnormalities was significantly different between the control and teratozoospermic subjects (2.35±0.87% vs. 5.90±3.74%, P=0.0128). 3.2.2 Chromosomal Analysis of Morphologically Separated Spermatozoa In an effort to assess chromosomal content directly in sperm with morphological differences, sperm were separated with a micromanipulator into four morphologic categories (small, medium, large, and amorphous heads) and analyzed by FISH. To assure the FISH analysis was made on viable sperm, the selection of sperm was taken from swim-up samples. Due to the scarcity of most of the head categories (namely the small, large, and amorphous heads), the selection of sperm by micromanipulation was quite time consuming and made the completion of this part of the project unfeasible. However, analyses with a chromosome 18,X,Y FISH probe set were done on samples from five teratozoopsermic subjects and one fertile control subject (for comparison) (Table 3.11). Fisher’s exact test was used to compare the incidences of chromosomal abnormalities between the different cell types (i.e., small, medium, large, and amorphous) in the control subject (zero frequencies were not considered). Only four large headed sperm produced analysable FISH signals, none of which were chromosomally abnormal, and thus, could not be included in the statistical comparison of chromosomal abnormalities between different cell types. Nevertheless, the amorphous shaped heads had the highest proportion of cells with [total] chromosomal abnormalities (40.74% of 27 cells), which was significantly higher than that of the small heads (14.71% of 68 cells, P=0.0118) and medium sized heads (8.97% of 78 cells, P=0.0005). Of the chromosomes abnormalities, the incidence of disomy 18 in amorphous headed sperm (18.52%) was significantly higher than that of the medium headed sperm (6.41%, P=0.0078) and small 51 headed sperm (2.94%, P=0.0018). Significant difference between the different cell types in the control subjects was not found for any other chromosomal abnormality. The number of cells analyzable by FISH from some of the cell types (particularly the small, large, and amorphous) from some of the teratozoospermic subjects was small. Consequently, the pooled incidences of chromosomal abnormalities found in the specific cell types of teratozoopsermic subject were considered in statistical comparisons. Analysis of variance in the frequencies of chromosomal abnormalities was done to evaluate if the individuals belonged to a homogeneous population sample. Because the number of cells counted was variable for each individual and for each cell type, the non- parametric Kruskal-Wallis test was used to compare the incidences of chromosomal abnormalities expressed in percentages. No significant difference was found between each individual for each chromosomal abnormality across all cell types. Subsequently, the pooled incidences of chromosomal abnormalities between the different cell types in the teratozoospermic subjects were compared (by Fisher’s exact test), and only the incidences of sex chromosomal disomy were significantly different between the medium and large headed sperm (1.81% vs. 11.11%, respectively; P=0.0079). When the pooled incidences of chromosomal abnormalities between the different cell types in the teratozoospermic subjects were compared to those of the control subject, the incidence of total chromosomal abnormalities in the medium sperm were significantly different (8.97% vs. 6.86%, respectively; P= 0.03). 3.3 DNA Fragmentation As mentioned in the methodology chapter, motile sperm were selected by the swim-up technique from each subject. Because of the time needed to do the assessment of aneuploidy, all samples that were assayed by TUNEL were thawed from cryogenic storage in liquid Nitrogen. Consequently, samples for seven control subjects and ten isolated teratozoospermic subjects were available for testing. The level of DNA fragmentation was assessed on at least 500 spermatozoa (when possible) and up to 1000 spermatozoa for some subjects. As a result of the fixation method (4% paraformaldehyde overlay) and the possible length of cryogenic storage, less than 500 spermatozoa were available for analysis for one of the control subjects (CONTROL14) and three of the 52 teraozoospermic subjects (TERATO1, 2, and 5). Because the number of cells counted per sample was not uniform, the data was recorded as a percentage of spermatozoa that indicated signs of DNA fragmentation (labelling of green) out of the total number of spermatozoa counted. Percentages typically fit a binomial distribution, rather than a normal distribution, and to avoid any effects of departures from normality, the level of DNA fragmentation in the control subjects were compare to that in the teratozoospermic subjects with the non-parametric Mann-Whitney test. The results of the assessment of DNA fragmentation by TUNEL assay are summarized in Table 3.12. There was large amount of variability in both control and teratozoospermic subject groups. Despite the variability seen in the two groups, there was a significant difference in the mean level DNA fragmentation between the control and teratozoospermic subjects (32.40±17.20% vs. 60.28±21.40%, respectively; P=0.0136). 3.4 Correlation of Sperm Morphology to Genetic Integrity 3.4.1 Correlation Between Sperm Morphology and Chromosomal Abnormalities Correlation analyses were done between the levels of morphologic abnormalities and the incidences of chromosomal abnormality found in the sperm of five control and ten teratozoospermic subjects (Tables 3.13, 3.14, 3.15 and 3.16). Since the FISH analysis was done on raw, unselected sperm samples, the correlation analyses of the chromosomal abnormalities were compared against the morphologic abnormalities of the raw, unselected sperm. The non-parametric Spearman’s test of correlation was used to compare the data, as the sperm morphology data cannot be assumed to be normally distributed. Statistical comparisons involving the clinical value of percentage of normal sperm (“Kruger’s measurements” in Tables 3.13 and 3.14) could not be done for the control subjects, because only one of the semen samples of the control subjects could be matched to his clinical morphological assessment (problem of matching clinical identity to the coded identity for the project mentioned earlier). The incidence of normal sperm in the five control subjects, available from the detailed morphologic assessment, was correlatively evaluated against the chromosomal abnormalities, but no significant correlation was found. With respect to the morphologic abnormalities in the sperm of the five control subjects, none correlated significantly to the aneuploidy of the individual 53 chromosomes in the sperm (Table 3.13). However, when the chromosomal abnormalities were assessed together (“Total Chromosomal Abnormality”, Table 3.14), there was a significant negative correlation with the incidence of pyriform shaped sperm heads (rs = - 0.9747, P=0.0167). There was also a significant positive correlation between the incidence of tapered shaped sperm heads and the incidence of supernumerary chromosomal abnormalities (rs = 0.9747, P=0.0167). The clinical value of morphologically normal sperm was available for the ten teratozoospermic subjects (“Kruger’s measurements” in Tables 3.15 and 3.16). Kruger’s measurements of the teratozoospermic subjects negatively correlated significantly to the incidence of sex chromosome disomy (rs = -0.7078, P=0.0220; Table 3.15). Alternatively, the incidence of normal sperm, as recorded from detailed morphologic assessment, positively correlated to the incidences of disomy 18 (rs = 0.6646, P=0.0360; Table 3.15) and diploidy (rs = 0.6647, P=0.0360; Table 3.16). The comparison of chromosomal abnormalities and morphologic abnormalities in the sperm of the ten teratozoospermic subjects yielded a few more statistically significant correlations (Tables 3.15 and 3.16). In terms of sperm head size, the incidence of large headed sperm negatively correlated to the incidence of sex chromosomal nullisomy (rs = -0.8971, P=0.0004) and total sex chromosomal aneuploidy (rs = -0.7394, P=0.0145). Of the internal head abnormalities, the incidence of abnormally vacuolated sperm correlated negatively to the incidences of sex nullisomy (rs = -0.6485, P=0.0425) and diploidy (rs = -0.6893, P=0.0274). The incidence of amorphous shaped sperm positively correlated to the incidences of chromosome 18 disomy (rs = 0.6391, P=0.0466) and sex chromosome total aneuploidy (rs = 0.8049, P=0.0050). The incidence of coiled sperm tails negatively correlated to the incidences of chromosomal 13 nullisomy (rs = -0.7333, P=0.0202) and total aneuploidy (rs = -0.6364, P=0.0479) and supernumerary chromosomal abnormalities (rs = -0.7356, P=0.0153). But, significant positive correlations were found between the incidences of bent tails and the incidences of chromosomal 13 disomy (rs = 0.7939, P=0.0061) and total aneuploidy (rs = 0.6606, P=0.0376), as well as between the incidences of two-tailed sperm and the incidences of chromosomal 13 disomy (rs = 0.8193, P=0.0037) and total aneuploidy (rs = 0.8330, P=0.0028) and supernumerary chromosomal abnormalities (rs = 0.7534, 54 P=0.0119). Finally, the teratozoospermic index negatively correlated the incidence of chromosomal 13 nullisomy (rs = -0.7212, P=0.0234). 3.4.2 Correlation Between Sperm Morphology and DNA Fragmentation Linear correlation analyses were done between the different sperm morphology and the level of DNA fragmentation in the sperm of seven fertile control and ten teratozoospermic subjects (Table 3.17). The TUNEL assay was applied to swim-up selected sperm, so the DNA fragmentation data was correlated to the detailed morphologic assessment of corresponding swim-up samples. Because clinical assessment of morphology is routinely done on unselected semen samples, the clinical value of morphologic normal sperm (“Kruger’s measurements”) was not considered in the correlation analyses. Since there is a risk that the data collected from the morphologic assessment and DNA fragmentation analysis may not be normally distributed, the non- parametric Spearman’s test of correlation was used to compare the data. In the fertile control subjects, the linear correlation analysis yielded two significant correlations with the level of DNA fragmentation: a positive correlation with the incidence of bent necks (rs = 0.8571, P=0.0238) and negative correlation with the incidence of thin shaped sperm heads (rs = -0.8018, P=0.0341). No other significant correlation could be found between any other sperm morphology and the level of DNA fragmentation in the control subjects. There was no occurrence of thin midpiece in the swim-up selected sperm of the control subjects and therefore, its correlation to DNA fragmentation could not be evaluated. In the teratozoospermic subjects, however, a significant positive correlation was only found between in the level of DNA fragmentation and the incidence of round headed sperm (rs = 0.6727, P=0.0390). There was no occurrence of irregular tails in the swim-up selected sperm of the teratozoospermic subjects; so the correlation between irregular tails and DNA fragmentation could not be evaluated. 55 Chapter IV. General Discussion Because ICSI bypasses many barriers encountered during natural conception, many severely infertile men are now able to father biological offspring. Fertilization is achieved so long as the appropriate components of the sperm are introduced into the oocyte cytoplasm. This includes the male proximal centriole responsible for building the first zygotic spindle and creation of the 2-cell stage. The proximal centriole is detectable in the middle spermatid stages, indicating that these immature germ cells are viable for use in ICSI (Sathananthan et al., 1991; Palermo et al., 1994; Simerly et al, 1995). Fertilization also requires an oocyte-activating factor to trigger the cascade of ooplasmic events that result in the resumption of meiosis of the female gamete (Kimura et al., 1995a and b; Tesarik et al., 1994). However, successful fertilization by ICSI does not ensure that the zygote in genetically viable. Aytoz et al. (1998) have shown that the rate of intrauterine death after ICSI techniques was higher in a severely defective sperm subgroup than in better quality sperm subgroups. Even when an ICSI conception develops to term, there is in increased risk of chromosomal abnormality (Bonduelle et al., 2002, Van Steirteghem et al., 2002), childhood disease and infertility (Aiken and Krausz, 2001; Aitken et al., 2004). Consequently, all efforts should be made to minimize the selection of abnormal sperm to use in procedures such as ICSI. This project endeavors to provide meaning tools to aid in this selection. 4.1 Sperm Morphology in Control and Teratozoospermic Subjects By definition, teratozoospermic patients have abnormally low levels of morphologically normal sperm. The results of the detailed morphological assessment of raw, unselected samples were consistent with this definition: significant difference in morphologically normal sperm between the fertile control and teratozoospermic subjects (21.53±4.92% vs. 10.36±2.27, respectively; P=0.0001) (Table 3.3). However, the levels of morphologically normal sperm that were detected in the detailed morphologic assessment were higher than what was clinically recorded (Table 3.2), and this is likely indicative of the difference in recognition of morphologic abnormalities between the clinical technician and the observer in this project [myself]. Nonetheless, detailed assessment of both the control and teratozoospermic samples were done by the same observer and a significant 56 difference was still found between the two groups. Also, the TZI and SDI of the teratozoospermic subjects were significantly higher than that of the control subjects (1.56±0.07 vs. 1.41±0.16, P=0.0431; 1.40±0.08 vs. 1.09±0.13, P=0.0001, respectively), indicating that the number of abnormalities per sperm is higher in the teratozoospermic subjects than in the control subjects, in addition to the overall number of morphologically abnormal sperm. The most prevalent morphologic abnormality seen in sperm of both the control and teratozoospermic men is the presence of abnormally large amounts of vacuoles (48.33±10.57% and 49.45±19.01%, respectively); suggesting that this characteristic is a large determinant of morphologic abnormality in sperm in general. However, the incidence of the abnormal vacuolation did not correlate to the TZI or SDI values, two classical measures of sperm function (Tables 3.4-3.7). This, along with the similarity in the frequency of abnormal vacuolation between the fertile and teratozoospermic subjects, suggests that the presence of large amounts of vacuoles does not functionally affect the fertilization ability of sperm. This may be true to some extent with the most critical requirement of fertilization being a functional paternal centrosome and oocyte activating factor. However, abnormal chromatin packaging and DNA damage that have been associated to vacuolated nuclei (Berkovitz et al., 2005) could have later developmental implications. The incidence of abnormal vacuolation in sperm did not correlate positively to any other morphologic abnormality. Conversely, significant negative correlations were found with the incidences of small heads and acrosomal abnormalities in the raw sperm samples of the teratozoospermic subjects (rs=-0.6485 and rs=-0.7091, respectively, P<0.05; Table 3.5), but not from that of the control subjects (Table 3.4). Vacuoles were most often observed in and around the acrosome cap. Since acrosomal abnormality was defined in the morphologic assessment as a reduced or absence of acrosome, small or absent acrosomes rarely would be accompanied by large amounts of vacuoles in the same sperm. Also, sperm with smaller or no acrosomal areas would tend to also have smaller head dimensions. It so happens that the incidence of small heads correlated positively to that of acrosomal abnormalities (rs=0.8667, P=0.0022), as well as round shaped heads (rs=0.6727, P=0.039). All three morphologic features incidentally are characteristics of the rounded 57 headed sperm found in globozoospermia. Acrosome formation and nucleus elongation appear to be involved in the formation of round-headed sperm. The possible mechanisms include: 1) the acrosome develops separately from the nucleus to be lost with residual body and taken up by Sertoli cells; 2) the acrosome vesicle fails to fuse with the nuclear membrane and is subsequently lost with the residual body; 3) malfunctioning Golgi apparatus resulting in degeneration of acrosome vesicle attached to nuclear membrane in late spermatid stage; 4) absent or malfunctioning nuclear manchette (cytoskeletal component involved in determining the shape of nucleus) (reviewed in Dam et al., 2007). The incidences of small heads, acrosomal abnormalities and round shaped heads were elevated in the teratozoospermic samples relative to the control samples, but not significantly so (8.27±11.62% vs. 3.05±1.70%, 9.68±11.74% vs. 5.46±2.01%, 5.53±5.49% vs. 4.01±1.84%; respectively) and they were not present in a particularly large proportion of the sperm in the teratozoospermic group. Also, no individual teratozoospermic patient carried extremely high levels of round headed sperm that would qualify him as having even a mild form of globozoospermia (Table 3.2), but round- headed sperm can be found at lower levels in semen in subfertile men (Kalahanis et al., 2002). The positively correlated incidences of small, round and reduced acrosome in our morphologically heterogeneous patient samples do suggest that at least some sperm possess this distinct morphological form. The incidence of small headed sperm also positively correlated to the TZI, which further illustrates the role this morphologic abnormality has in determining overall morphology in isolated teratozoospermic patients. Elongated sperm heads, with increased head length and slightly reduced width, are caused by abnormally elongated nuclei. The development of elongated nuclei is not well understood, but could involve the formation of an abnormally narrow microtubular manchette in elongating spermatids (Rouy and Sentein, 1977 [sited in Prisant et al., 2007]). Analyses using light microscopy, however, often record sperm with elongated nuclei as sperm with pyriformed heads (Kubo-Irie et al., 2005; Rousso et al., 2002) and tapered heads (Auger et al., 1990; Urry et al., 1990). Likewise in this study, accurate identification of nuclear length was not possible with magnification used to assess the sperm morphology, but pyriform, tapered, and thin shaped sperm were identified. The incidence of pyriform shaped sperm, like that of abnormal vacuolation, negatively 58 correlated to the incidences of small, round, and acrosomally abnormal sperm (rs=-0.7939, P=0.0088; rs=-0.8182, P=0.0058; rs=-0.697, P=0.0306; respectively) in the raw, unselected teratozoospermic samples. Pyriform shaped sperm occurred more frequently, but not significantly, in the teratozoospermic samples than in the control samples (10.06±8.53% vs. 4.26±3.25%). Contrarily, Rousso et al., (2002) found that subfertile men (with unspecified semen parameters) had significantly higher levels of pyriformed sperm than fertile men (22±14.9% vs. 13±7.8%, respectively; P<0.001), with a positive correlation between pyriformed sperm and acrosomeless sperm (P<0.05), along with acephalus sperm (P<0.01) and amorphous headed sperm (P<0.05).The negative correlation of pyriformed heads and reduced acrosome found in the current results may be indicative of a common mechanism (possibly involving the Golgi apparatus) that, when malfunctioning, leads to the mutually exclusive events such as reduced acrosome area (anterior head reduction) and pyriform shape (anterior head expansion). However, pyriformed shaped sperm have been scarcely studied and its pathogenesis is unknown. The incidence of tapered sperm heads positively correlated to the incidence of round heads in the raw, unselected sperm of the teratozoospermic subjects (rs=0.7295, P=0.0202; Table 3.5). Since tapered heads appeared to be unrelated to the acrosomal abnormalities (no significant correlation; P=0.4069), the tapered shape of the sperm heads is not likely the result of reduced acrosomal area. More likely, the process that actively determines the shape of sperm (e.g., nuclear manchette) is differentially malfunctioning, leading to an increased production of tapered sperm heads and round sperm heads. The incidence of tapered sperm heads, though, was relatively low and not statistically different in the teratozoospermic and control subjects; the incidence was actually slightly lower in the teratozoospermic subjects (2.68±3.83% vs. 5.04±4.43%, respectively; P= 0.1560, Table 3.3). This suggests that tapered shape is not a large contributor to morphologic abnormality in sperm of teratozoospermic men and that is has little effect on fertility. Like tapered sperm heads, thin shaped sperm heads occurred infrequently in the raw, unselected sperm of the teratozoospermic subjects (0.59±0.79%), suggesting a small, if any, significance on fertility. Nonetheless, a low fertilization rate has been found in men with severely elongated sperm heads (Osawa et al., 1999). 59 The incidence of thin shaped heads positively correlated to that of amorphous sperm heads (rs=0.7943, P=0.0088). Next to abnormal vacuolation, amorphous shaped sperm heads was the most prevalent morphologic abnormality in sperm of the teratozoospermic subjects, and it was the only morphologic abnormality that was significantly more frequent than in the control subjects (18.97±5.94% vs. 10.22±4.38%, respectively; P=0.0097). Both the magnitude and statistical significant elevation in frequency of amorphous sperm heads in teratozoospermic subjects compared to control subjects may suggest a biological significance. This significance would likely involve a malfunction in nuclear shape determination and its possible influence to chromatin packaging. Abnormally large cytoplasmic droplets, on the other hand, occured more frequently in the sperm of control subjects than in teratozoospermic subjects (7.22±6.24% vs. 2.39±3.15%, respectively; P=0.0431). This abnormality is probably the result of a transient disruption in testicular environment that reduces the efficiency of residual body extrusion during spermiation and is not likely to have an effect on fertilization potential. The incidence of abnormally large cytoplasmic droplets, however, showed a negative correlation to the incidence of irregular tails (rs=-0.6575, P=0.0438). This association is curious since cytoplasmic retention is not known to involve microtubular organization that determines flagellar (tail) development. Sperm are able to shed cytoplasm even when membrane remodelling is absent at the midpiece, indicating the residual body formation is independent from flagellum morphogenesis (Escalier, 2006). The significant correlation found may have been the result of assessment bias: recognition of large cytoplasmic droplets superseded that of irregular tails, given the relative difficulty to detect tail irregularities with light microscopy. The recorded incidence of irregular tails (0.25±0.35% in raw unselected teratozoospermic samples) likely represented the most severe alterations. Other tail abnormalities occurred in the raw, unselected sperm (Table 3.3). Athough the incidences were all higher in the teratozoospermic subjects compared to the control subjects, no difference was statistically significant. The most prevalent of the tail abnormalities in the teratozoospermic and control subjects were coiled tails (14.21±7.62% and 10.61±7.21%, respectively), followed by thick midpieces (9.39±7.24% and 60 4.85±2.45%, respectively). Both abnormalities likely relate to abnormal fribrous sheath organization of the principle and mid-pieces, respectively, of the flagellum. The remaining recorded tail abnormalities were found in the ranges of 0-5% (Table 3.3) and thus, are small contributors to the overall morphologic abnormality in isolated teratozoospermic and fertile men. It is notable that a positive correlations existed between the incidence of 2-tail and that of bent tails (rs=0.7579, P=0.0149), and irregular tails (rs=0.6691, P=0.039) were seen in the teratozoospermic samples and between incidences of coiled tails and bent tails (rs=0.7684, P=0.048) in the control samples. The exact mechanism from which these abnormalities are derived cannot be known without ultrastructural examination of the individual abnormalities. However, a possible explanation could relate to irregularities in the fibrous sheath organization: coiled tails, irregular tails and 2-tails (as seen by light microscopy) could derive from deteriorated or absent fibrous sheath of the principle piece allowing the internal microtubules of the axoneme to uncouple; bent tail could derive from localized abnormality in the flagellar organization involving sheath or axoneme components. Most tail abnormalities naturally affect sperm motility and would influence natural fertilization potential, but would not affect assisted fertilization procedures such as ICSI. However, abnormalities of head-neck attachment that leads to bent necks, asymmetric neck insertions, and acephalic sperm can involve a dysfunctional proximal centriole that in turn would fail to nucleate a functional sperm aster in the developing zygote and prevent normal syngamy and cleavage (Chemes et al., 1999, Rawe et al., 2002). However, the incidences of bent necks and asymmetric neck insertions (4.68±2.67% and 1.09±1.09%, respectively) were low in the sperm of our isolated teratozoospermic samples and not significantly different from their incidences in the control samples (5.34±1.39% and 0.33±0.35%, respectively). There was also a positive correlation between the incidence of bent necks and 2-tails in the teratozoospermic samples (rs=0.6486, P=0.049). Bent necks arise from the inability of the proximal centriole to migrate completely to the caudal pole of the spermatid nucleus causing a misaligned positioning of the nucleus (Chemes et al., 1999; Rawe et al., 2002). Although there was a general lack of specific morphologic abnormalities in the sperm with significant difference between of the fertile control and teratozoospermic subjects (aside from abnormal cytoplasmic droplets), the teratozoospermic subjects did 61 have a lower frequency of normal sperm and higher TZI and SDI values. Also, the number of significant correlations between the specific morphologic abnormalities in the teratozoospermic samples, relative to the control samples, were not only greater, but also different (Table 3.4 and Table 3.5). This may suggest that there are distinct mechanisms at work in generating morphologically abnormal sperm in isolated teratozoospermic men. While most abnormal morphologic forms would likely have an impact on the natural fertilization potential, only those that are associated with nuclear organization and oocyte activation will ultimately influence ICSI outcome. This study has provided a glimpse into the prevalence of abnormal forms in polymorphic teratozoospermic men and the potential sperm that could be encountered during selection in ICSI. 4.1.1 Sperm Morphology After Swim-up Fractionation The intent of swim-up fractionation of the semen samples is to select for motile, viable sperm, with the assumption that motile sperm are also more functionally viable. The results of the detailed morphologic assessment of the teratotozoospermic samples in this study show that swim-up selection does effectively increase the proportion of morphologically normal sperm (10.36±2.27% to 26.10±11.48%; P<0.0001) and decrease the number of abnormalities in each sperm (TZI: 1.56±0.07 to 1.42±0.15, P=0.0185; SDI: 1.40±0.08 to 1.06±0.25, P=0.0002) (Table 3.2). Although swim-up selection appeared to decrease a number of specific morphologic abnormalities (most notably abnormal vacuolation, pyriform and amorphous shaped heads, large cytoplasmic droplets), no change was significantly different from what was seen in the raw, unselected samples. The lack of statistical significance may be a result of individual variability, but swim-up selection can result in large decreases in abnormalities in a given individual (e.g. incidence of abnormal vacuolation was 60.98% and 27.80% before and after swim-up). The swim-up fractionation did not have much effect on reducing morphologic abnormalities in fertile control subjects: the proportion of morphologically normal sperm, and the TZI and SDI (along with all of the individual morphologic abnormalities) in the swim-up fractions were similar to respective values in the raw, unselected samples (Table 3.1). This suggests that the swim-up fractionation is an effective technique for reducing but not excluding morphologic abnormalities in individuals with a large proportion of 62 sperm with morphologic abnormalities (i.e. teratozoospermic patients), but is not as effective for fertile individuals with significantly less morphologically abnormal sperm. Also, swim-up fractionation appears to generate sperm samples from teratozoospermic subjects that aproximated that of fertile controls: the frequencies of all morphologic characteristics were similar (not statistically different) between the control and teratozoospermic subjects (Tables 3.3). However, persistently elevated presence of specific abnormalities, even after swim-up fractionation, may be indicative of a predominant functional abnormality (e.g., prevalence of acrosomal abnormalities in Terato1 was 38.5% and 34.60% before and after swim-up; prevalence of abnormal vacuolation in Terato8 was 75.60% and 60% before and after swim-up; Table 3.2). Although a significant change in the incidences of specific morphologic abnormalities was not detected after swim-up selection, the different significant correlations found can be informative. A positive correlation between the incidences of coiled tails and bent tails was seen in swim-up control samples, suggesting that deformities in flagellar organization can exist even in motile sperm. The absence of the relationship between amorphous shaped sperm heads and bent necks, seen in the raw, unselected control samples, may indicate this combination is not conducive for sperm motility. It is interesting that the swim-up selection produce consistent correlation of both the TZI and SDI to the incidences of acrosomal abnormalities (rs=0.8929, P=0.0123; rs=0.7857, P=0.0480; respectively) and coiled tails (rs=0.8571, P=0.0238; rs=0.8214, P=0.0341; respectively) (Table 3.6). However, it is difficult to explain how these two abnormalities become more significant in the identification of abnormal morphology or how amorphous shaped heads (positively correlated to TZI in raw, unselected control samples) lose its significance after motile sperm are selected, keeping in mind that the incidences of these morphologic abnormalities did not change significantly after the swim-up selection. In the teratozoospermic samples, many significant correlations seen in the raw, unselected samples were maintained after swim-up selection (Table 3.7). The significant correlation between the incidences of round heads and small heads (rs=0.9362, P=0.0002) was present after swim-up selection, indicating that “round headed sperm” are motile and can persist after swim-up selection. Of note, the correlation of abnormal vacuolation with 63 “round headed sperm” seen in the raw, unselected sperm was absent in swim-up samples and may demonstrate that swim-up fractionation is able to select for viable sperm, vacuolation possibly indicative of deteriorating sperm. The significant negative correlation between the incidences of pyriform heads and acrosome abnormalities was also maintained in swim-up samples (rs=-0.6606, P=0.0438). Again, there appears to be some developmental mechanism that maintains their mutually exclusive occurrence in motile sperm – this is not too surprising since neither abnormalities are related to the function of the flagella. What is a little suprising is the persistence of correlations in tail defects in the swim-up selected samples. The incidence of asymmetric neck insertion was positively correlated to the incidences of bent necks (rs=0.7454, P=0.0174), thin midpieces (rs=0.6833, P=0.0347), and thick midpieces (rs=0.7028, P=0.0268). Swim-up selection did reduce the group incidences of these tail abnormalities, but the differences were not statistically different. This may indicate that such abnormalities do not significantly impact motility of sperm. Finally, both the TZI and SDI of the teratozoospermic subjects positively correlated to the incidences of large cytoplasmic droplets (rs=0.8728, P=0.0016; rs=0.8469, P=0.0029; respectively) and thick midpieces (rs=0.7818, P=0.0105; rs=0.9030, P=0.0008; respectively) in the swim-up samples. In spite of this, both abnormalities occurred in relatively low frequencies and their significance to fertility is corresponding low. 4.2 Chromosomal Analysis in Control and Teratozoospermic Subjects The results of the chromosomal analysis in sperm of our control subjects (summarized in Table 3.10) indicate that aneuploidy of sex chromosomes is greater than that of the 3 autosomes studied, significantly so for chromosomes 18 (P<0.001) and 21 (P<0.05) and not for 13. This is consistent with the notion that the sex chromosomes are prone to meiotic I non-disjunction in males (Hassold et al., 1991). The same could not be said in the isolated teratozoospermic subjects. The rates of aneuploidy among the different chromosomes were similar, with only a slightly significant difference between the aneuploidy of the sex chromosomes and that of chromosome 18 (P<0.05) when the incidences were compared in a paired analysis (observation of values in the same hybridization). 64 The frequencies of chromosomal abnormalities in the sperm of teratozoospermic subjects were significantly higher than that of the fertile control subjects, albeit only moderately with a little more than a 2-fold increase. The total chromosomal abnormalities in the teratozoospermic vs. control subjects were: 5.90 ± 3.74% vs. 2.35 ± 0.87%, P<0.05; total aneuploidy 4.90 ± 2.82% vs. 1.99 ± 0.65%; P<0.001, respectively. Furthermore, there appears to be a general increase in aneuploidy of the individual chromosomes, with the mean incidences of disomy, nullisomy, and aneuploidy for all chromosomes of the teratozoospermic subjects higher than their respective values of the control subjects (Table 3.10); but only the disomy and aneuploidy of chromosome 13 were statistically different from that of the controls. This is contrary to the degree of increase and predominant chromosomal involvement (i.e. sex chromosomes) of chromosomal abnormality found in the sperm of severely infertile men (Aran et al., 1999; Pang et al., 1999; Vegetti et al., 2000; Calogero et al., 2001; Rubio et al., 2001). With respect to polymorphic teratozoospermic men (Calogero et al., 2001; Gole et al., 2001; Härkönen et al., 2001) the total aneuploidy rates were similar to ours. However, contrary to our results, all maintained a preponderance of sex chromomosome aneuploidy over the other chromosomes studied, and this was due to lower autosomal disomy rates compared to ours (sex disomy rates were similar to ours). The sex chromosome aneuploidy rate was significantly higher than the respective rates in the controls, which was not found in the results of this study. Gole et al. (2001) also found the MI derived XY disomy was significantly higher than the MII derived XX/YY forms, which was not the case in our results. None of the studies included chromosome 13 in their chromosomal analyses, so the significance of increase in chromosome 13 aneuploidy in the sperm of our teratozoospermic men could not be measured against the incidence in other teratozoospermic men. 4.2.1 Correlation of Sperm Morphology to Chromosomal Abnormalities While the establishment of incidence of chromosomal abnormality in the sperm of isolated teratozoospermic men is important in assessing the risks in this sub-group of infertility, much more important would be the ability to identify specific morphologic characteristics that are associated to genetic defects. In assessing the sperm of men whose 65 subfertility is isolated to abnormal sperm morphology, the likelihood may be improved for detecting morphologically abnormal sperm that also carry genetic insufficiencies, such as chromosomal abnormalities. Since the concentration and motility in these patients are normal, it is hoped that any associations made with specific morphologic abnormalities and chromosomal abnormalities can be transferred to sperm from other clinical presentations of infertility. In the case of sperm from fertile control subjects, very few significant correlations between the morphology of the sperm and chromosomal abnormalities were found (Table 3.14 and Table 3.15). This would indicate that morphology of sperm is a relatively poor indicator of chromosomal content in fertile men. Nonetheless, a positive correlation between tapered heads and supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167) and a negative correlation between pyriform heads and total chromosomal abnormalities (rs=-0.9747, P=0.0167) were found. Both tapered heads and pyriform heads have been classified as variations of elongated head malformation and possibly result from alternate malfunction of a common mechanism that determines nuclear (and head) shape. It may not be just coincidence that the apparent contrary manifestations of elongated heads (i.e., tapered vs. pyriformed heads) also have opposite associations with the incidence of chromosomal abnormalities. The increased level of morphologically abnormal sperm in the teratozoospermic subjects was also accompanied by an increased number of significant correlations with chromosomal abnormalities. Thus, the morphology of sperm in teratozoospermic subjects appears to be a much better indicator for chromosomal abnormality. Even in the clinical assessment of normal morphology, there was a significant correlation: the level of morphologically normal sperm (Kruger’s Measurement, Table 3.14) negatively correlated with sex chromosomal disomy (rs=-0.6391, P=0.0343). This was the case in spite of the lack of significant increase in the rate of sex chromosomal aneuploidy in the teratozoospermic patients relative to the control values. The incidence of amorphous shaped heads in the sperm was the only sperm abnormal head morphology in teratozoospermic subjects that correlated positively to the incidence of sperm with chromosome 18 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050). Increases in chromosomal abnormalities 66 have also been found in amorphous sperm, investigated by human sperm injection into mouse oocytes (Lee et al., 1996), and by two different imaging software-assisted strategies of simultaneously investigating morphology and aneuploidy in the same sperm (Celik-Ozenci et al., 2004; Strassburger et al., 2007). This suggests a genuine risk exists in amorphous sperm of having chromosomal abnormalities. While sperm from globozoospermic men have been found to have increased rates of aneuploidy (Carrell et al., 1999, 2001; Morel et al., 2004), the incidence of round headed sperm in this investigation did not correlate significantly with any chromosomal abnormalities. Strassburger et al. (2007) also found an increased rate of aneuploidy in the globozoospermic patients they studied (6.7% vs. 2.0 % in the controls); but their ability to assess the morphology and chromosomal content in the same cell allowed them to determine that the rate of aneuploidy specifically in round-headed sperm (4.7%) was small in comparison to other abnormal forms in the globozoospermic patients (e.g. 28.4% in amorphous heads; 50% in two headed/two tails). Moreover, the seven teratozoospermic patients included in their study had a 3.7 ± 2.60% incidence of round headed sperm (incidence in this study was 5.53±5.49%) and an aneuploidy rate of 0.2 ± 0.55% in round headed sperm; this indicates that the significance of aneuploidy in this abnormal form is relatively low in teratozoospermic patients. Although macronuclear spermatozoa have been found to have high levels of hyperploidy (Yurov et al., 1996; Vicari et al., 2003) and tetraploidy (Benzacken et al., 2001; Devillard et al., 2002; Lewis-Jones et al., 2003; Mateu et al., 2006), the incidence of large headed sperm in this study did not correlate to diploidy. The lack of positive correlation is likely the result of the low incidences of large-headed sperm (1.00±0.90%) and diploidy (0.17 ± 0.16%) in our teratozoospermic subjects. Although the risk of chromosomal abnormality is high when large-headed sperm is the predominant morphologic abnormality, it may not be as relevant in more heterogeneous forms of teratozoospermia. The incidence of 2-tails correlated to the incidences of chromosome 13 disomy and supernumerary chromosomal abnormalities in the teratozoospermic subjects (rs=0.8193, P=0.0037; rs=0.7534, P=0.0119; respectively). A genuine association of this morphologic abnormality to chromosomal abnormality seems apparent (Strassburger et al., 67 2007); the likely connection being dysfunctional centriole organization. The incidence of bent tails was also positively correlated to disomy 13 (rs=0.7939, P=0.0061). However, the connection between this morphologic abnormality and chromosomal aneuploidy is more difficult to explain and may be related to the incidences of 2-tail and bent tails being positive correlations to each other. 4.2.2 Chromosomal Analysis of Morphologically Separated Spermatozoa In spite of the low number of morphologically separated sperm that were assessed in the fertile patient (Table 3.11), an elevation in total chromosomal abnormalities was detected in the amorphous shaped sperm heads (40.74%) compared to small sperm heads (14.71%, P=0.0118) and medium sized sperm heads (8.97%, P=0.0005). Lee et al. (1996) used a similar strategy of looking at small, normal, large and amorphous sperm of a fertile man, but injected the sperm into mouse oocytes to facilate the examination of chromosomal content. They found no significant increase in the rate of chromosomal aberrations in small- or large-headed spermatozoa (1.3% aneuploidy rate in normal head; 4.3% in amorphous heads, 0% in small and large). The overall elevation in chromosomal abnormality seen in the results of our fertile control subject may be related to: the low number of sperm assessed; the influence of other morphologic defects (not assessed) such as internal head defects, neck, midpiece and tail defects. When comparing the pooled results of the teratozoospermic subjects, no significant difference was seen in total chromosomal abnormalities between the different cell types. Only sex chromosome disomy between the medium and large headed sperm were significantly different (1.81% vs. 11.11%; respectively; P=0.0079; Table 3.11). However, due to the disproportionate contribution of cells from each teratozoospermic subject to the sperm categories and the possible inter-individual variability that may exist, drawing conclusions from this result should be cautioned against. Comparison of the pooled terazoospermic results to the control results revealed only a significant difference in total chromosomal abnormalities in medium sperm (8.97% in control, 6.86% in teratozoospermic group; P<0.05). Again, the significance may be overstated due the low number of cells analysed in the control patient. Although analysis of more cells may produce more convincing results, the labour intensive nature of the 68 micro-manipulated sperm selection, may suggest this approach in finding correlation of specific sperm morphology to genetic abnormality is less promising than originally thought, particularly when more efficient approaches are available, such as the software aided identification of abnormal morphology and chromosomal abnormalities in the same sperm (Strassburger et al., 2007). 4.3 DNA Fragmentation in Control and Teratozoospermic Subjects The potential of ICSI to facilitate the fertilization of sperm containing fragmented DNA warrants the investigation of possible relationships between sperm morphology and high levels of DNA fragmention. The significant increase in the level of DNA fragmentation found in the sperm of our isolated teratozoospermic subjects as compared to our fertile control subjects (60.28±21.40% vs. 32.40±17.20%, P= 0.0121; Table 3.12) suggests that a relationship between abnormal sperm morphology and DNA fragmentation may exist. The rate of DNA fragmentation in both our groups is somewhat elevated relative to other studies (Sun et al, 1997; Lopes et al, 1998; Irvine et al, 2000; Muratori et al, 2000), and this could be related to the use of HTF media to do the the swim-up selection. Swim-up human sperm incubated in HTF medium results in progressive increase in percentage of DNA fragmented sperm (Estop et al., 1993). The effects of cryogenic storage of the semen samples that was needed until this analysis could be done may also induce unintended levels of DNA fragmentation (Gandini et al., 2006). In any case, any deficiencies in the procedures were encountered equivalently in both the teratozoospermic and control samples, and a significant difference was still found. In addition to the increase in DNA fragmentation in the morphologically abnormal sperm samples, significant correlations were found between the level of DNA fragmentation and specific morphologic abnormalities in sperm. In control samples, incidence of bent necks correlated to DNA fragmention in sperm (rs=0.8571, P=0.0238). This is consistent with the positive correlation to DNA fragmentation of other tail defects such midpiece, broken necks, abnormal necks, and curled tails (Cohen-Bacrie et al., 2008). Mice with disrupted dsRNA binding protein Prbp (TRBP is human homolog) have altered translational control in nuclear remodelling and chromatin compaction and this can lead to a range of morphologic abnormalities that include abnormal acrosomal organization, 69 inappropriate flagellum attachment, and gaps in midpiece (Zhong et al., 1999). Sperm immaturity may also be a critical component to high levels of DNA fragmentation in human sperm, and the abnormal chromatin compaction increases the susceptibility of DNA strand breaks. Sperm with diminished maturity have also been reported to have higher ROS production and DNA fragmentation, and this was associated with abnormal head morphology (Ollero et al, 2001), in addition to mid-piece and tail abnormalities (Gergely et al, 1999). In the sperm of our teratozoospermic subjects, a positive correlation was seen between round-headed sperm and DNA fragmentation (rs=0.6727, P=0.0390); this is consistent with the finding of increased percentage of DNA fragmentation in globozoospermic sperm cells as compared to fertile controls (Baccetti et al., 1996; Vicari et al., 2002). While Prisant et al. (2007) found poor chromatin compaction in a high percentage of elongated sperm heads in polymorphic teratozoospermic patients with >30% of sperm with the elongated form, the incidence of thin-headed sperm in our control subjects negatively correlated to DNA fragmention (rs=-0.8018, P=0.0341). The incidence of thin-headed sperm in our teratozoospermic samples, however, was 0.59±0.79% in the raw unselected samples and 0.64±1.11% in the swim-up sample (swim-up incidences applied to correlation analysis). Thus, the relationship between elongated sperm and DNA fragmentation is less than conclusive, and the relevance of elongated sperm in teratozoospermic men may be proportional to the incidence of this abnormal form. 4.4 Conclusions In summary, the sperm of isolated teratozoospermic patients do have higher rates of chromosomal abnormality compared to the sperm of proven fertile men. This increase in chromosomal abnormality appears to be non-specific in terms of the chromosomes involved, because aneuploidy of chromosome 13, chromosome 21, and sex chromosomes were not significantly different from one another (chromosome 18 had a significantly lower incidence of aneuploidy compared to the sex chromosomes). As a result, only the disomy of chromosme 13 was higher in the teratozoospermic subjects as compared to the control men. The sperm of teratozoospermic men also have higher levels of DNA fragmentation relative to fertile men. 70 Consistent with other studies that were unable to find correlation between sperm morphology and chromosomal abnormality, a relative lack of significant correlation between morphology to chromosomal abnormalities was seen in the sperm of the fertile controls in this study. This may be the result of fertile individuals having fewer morphologically abnormal sperm and that fertile individuals are just not suitable for such comparisons. On the other hand, morphologic abnormalities in sperm are more critical to the sub-fertility of isolated teratozoospermic men. The analysis of sperm from teratozoospermic individuals with extremely low levels of morphologically normal sperm (3.25±1.50%) has proven to be informative with respect to the clinically relevant morphologic forms. The selection of morphologically normal sperm is preferable for ICSI (Bartoov et al., 2003); when this is not possible, specific morphologic abnormalities should be avoided. Selection of amorphous-headed sperm from teratozoospermic patients should be avoided because of their association to chromosome 18 disomy and sex chromosome aneuploidy. Also, the high incidence of amorphous heads relative to other abnormal forms and relative to the incidence in fertile men, suggests that the structural dysfunction responsible for the amorphous shape is significant in teratozoospermic men; these structural abnormalities likely affect the nuclear organization and genetic integrity. Abnormal flagellar defects (2-tailed, bent tails, irregular tails) were associated to chromosome 13 disomy and supernumary chromosomal abnormalities. In addition, bent necks were associated to DNA fragmentation in control samples. The cytoskeletal abnormalities (including centriolar defects) central to these morphologic forms not only induce the genetic abnormalities in question, but also may hinder successful fertilization. Incidentally these flagellar defects persist after swim-up selection; so careful inspection for these defects is still required after swim-up fractionation. The risks of round-headed sperm and taper-headed sperm would be considered moderate to low, depending on their prevalence in a given individual (their incidences in this study were low). While an association between round headed sperm and chromosomal abnormalities was not indicated, the incidence of round heads was correlated to DNA fragmentation. Tapered heads, though, was associated to supernumerary chromosomal abnormalities. The incidences of both morphologic forms 71 were correlated to each other in the teratozoospermic samples and both are related to acrosome reduction. Because acrosomeless sperm have alterations in the perinuclear theca and associated proteins that are probably responsible for oocyte activation after fertilization (Longo et al., 1987; Escalier, 1990; Sutovsky et al., 1997), the use of such sperm may result in reduced fertilization potential in addition to the effects of the genetic insufficiencies they carry. Although no significant correlation was found between the incidence of large-headed sperm and chromosomal abnormality, selection of this abnormality should still be avoided due to the clear association found with polyploidy when enlarged heads is the predominant abnormal form. No other morphologic abnormality in sperm is associated to chromosomal abnormalities and high levels of DNA fragmentation. Apart from the above morphologic abnormalities, risk to fertilization and conception by ICSI with the use of other abnormal morphologic forms is not indicated, and could be used in the absence of normal sperm. 72 TABLES Table 1.1. Reference values for semen assessment and nomenclature Semen ParametersNomenclature Concentration (106/ml) Motility (% motile) Morphology (Kruger’s Criteria) (% normal) Normozoospermia ≥ 20 ≥ 50 ≥ 14 Oligozoospermia < 20 ≥ 50 ≥ 14 Asthenozoospermia ≥ 20 < 50 ≥ 14 Teratozoospermia ≥ 20 ≥ 50 < 14 Oligoasthenozoospermia (OA) < 20 < 50 ≥ 14 Oligoteratozoospermia (OT) < 20 ≥ 50 < 14 Asthenoteratozoospermia (AT) ≥ 20 < 50 < 14 Oligoasthenoteratozoospermia (OAT) < 20 < 50 < 14 Azoospermia No sperm in ejaculate Aspermia No ejaculate 73 CONTROL5 CONTROL8 CONTROL9 CONTROL10 CONTROL11 CONTROL13 CONTROL14 CONTROL MEAN Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Kruger’s Measurement 75 - no data no data - no data - no data - no data - no data - no data - normal 22.12 19.70 23.94 10.09 30.59 33.99 16.25 18.75 20.00 19.71 21.47 23.64 16.35 23.27 21.53 21.31 small head 1.84 1.48 2.82 6.42 3.20 1.97 0.83 4.33 6.09 8.17 4.19 3.10 2.40 5.45 3.05 4.42 large head 2.30 1.97 1.88 2.29 6.39 1.97 1.25 0.96 1.30 1.44 1.57 0.00 3.85 1.49 2.65 1.45 vacuolated 47.00 44.83 44.60 62.84 34.25 16.26 63.75 51.44 61.30 54.81 45.55 46.51 41.83 27.23 48.33 43.42 acrosomal abnormalities 4.61 7.39 8.45 10.55 6.39 4.43 2.08 4.33 5.22 5.29 4.71 3.88 6.73 9.90 5.46 6.54 round head 4.61 2.46 3.76 1.83 5.48 7.88 7.08 1.44 2.61 5.29 2.62 1.94 1.92 0.00 4.01 2.98 thin head 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.48 0.00 0.78 0.00 0.00 0.00 0.18 pyriform head 8.29 3.94 1.88 6.88 2.28 1.48 3.75 1.92 1.30 2.40 9.42 7.75 2.88 4.95 4.26 4.19 tapered head 2.76 0.49 1.88 0.46 8.22 7.39 3.33 1.44 6.09 8.17 0.00 7.36 12.98 2.97 5.04 4.04 amorphous head 5.53 10.34 16.90 9.63 9.59 16.26 4.58 10.10 12.17 7.21 13.61 8.14 9.13 13.37 10.22 10.72 cytoplasmic droplet 2.76 0.49 1.88 3.67 3.20 2.96 3.33 7.69 18.70 1.44 11.52 0.78 9.13 2.97 7.22 2.86 bent neck 5.07 2.46 5.16 4.13 6.39 5.42 5.42 2.88 3.48 1.44 4.19 1.94 7.69 4.46 5.34 3.25 asymmetric neck insertion 0.46 2.46 0.94 0.46 0.46 0.49 0.00 0.96 0.43 0.48 0.00 0.00 0.00 1.98 0.33 0.98 thin midpiece 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 thick midpiece 3.69 9.85 8.45 7.34 2.74 12.32 3.75 9.13 8.26 10.10 4.19 8.53 2.88 17.33 4.85 10.66 coiled tail 9.22 34.48 23.47 26.61 7.31 4.43 5.42 10.58 1.30 12.50 13.61 7.36 13.94 27.23 10.61 17.60 bent tail 0.46 2.46 0.47 1.38 0.00 0.00 0.00 2.40 0.00 0.00 0.00 0.39 0.96 0.50 0.27 1.02 irregular tail 0.00 0.00 0.00 0.46 0.00 0.00 0.00 0.00 0.00 1.44 0.00 0.00 0.00 0.00 0.00 0.27 2-tailed 0.46 0.00 0.00 0.46 0.00 0.49 0.00 0.96 0.43 0.96 0.52 0.00 0.48 1.49 0.27 0.62 teratoxoopermic index 1.27 1.55 1.59 1.61 1.39 1.26 1.24 1.36 1.57 1.51 1.48 1.29 1.40 1.58 1.42 1.45 sperm deformity index 0.99 1.24 1.21 1.45 0.96 0.83 1.04 1.10 1.26 1.22 1.16 0.98 1.17 1.21 1.11 1.15 Table 3.1. Percentage of morphologic characteristics in sperm from control subjects. Raw: washed, unselected sperm sample Swim-up: swim-up selected sperm sample 74 TERATO1 TERATO2 TERATO3 TERATO4 TERATO5 TERATO6 TERATO8 TERATO9 TERATO10 TERATO11 TERATO MEAN Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Raw Swim- up Kruger’s Measurement 5 - 2 - 1 - 4 - 5 - 5 - 3.5 - 3 - 1.5 - 2.5 - 3.25 - normal 10.00 18.48 8.13 19.50 9.13 23.15 8.00 14.29 11.17 20.29 10.36 16.98 7.32 45.85 12.56 44.39 12.87 22.77 14.00 35.29 10.36 26.10a small head 40.00 38.86 2.87 1.50 4.81 0.49 7.00 9.09 2.13 0.48 4.15 5.66 0.00 1.46 4.83 1.46 12.38 20.30 4.50 2.45 8.27 8.18 large head 0.00 0.47 2.39 0.50 1.44 8.37 1.50 1.73 1.60 2.90 1.55 0.47 0.00 1.46 0.00 0.49 0.00 0.00 1.50 0.98 1.00 1.74 vacuolated 29.00 14.69 75.60 60.00 71.15 37.44 46.50 29.44 63.83 47.83 57.51 55.19 60.98 27.80 20.77 27.80 34.65 42.08 34.50 50.00 49.45 39.23 acrosomal abnormalities 38.50 34.60 4.31 2.00 3.37 9.36 6.50 11.69 3.19 5.31 1.04 0.94 0.98 1.95 9.66 13.17 21.29 13.86 8.00 3.43 9.68 9.63 round head 2.50 9.00 1.91 7.00 2.40 0.49 13.50 8.23 0.00 0.97 7.25 4.72 1.46 1.46 2.90 2.44 16.34 7.92 7.00 3.43 5.53 4.57 thin head 0.00 0.00 0.48 0.00 0.48 3.45 0.00 0.00 0.00 0.00 0.00 0.00 2.44 1.46 0.48 0.98 0.50 0.00 1.50 0.49 0.59 0.64 pyriform head 1.00 1.90 10.53 22.50 18.27 7.88 2.00 0.43 18.62 13.53 5.70 3.77 25.85 7.80 10.63 1.95 0.50 0.00 7.50 1.96 10.06 6.17 tapered head 0.50 0.95 0.00 2.00 1.44 0.49 12.50 10.39 0.53 1.45 1.55 0.94 0.00 0.00 5.31 0.00 1.49 0.00 3.50 0.00 2.68 1.62 amorphous head 14.50 6.64 16.27 5.50 20.67 12.81 14.50 14.29 9.04 7.25 16.06 17.92 25.37 15.61 26.57 7.32 26.73 15.35 20.00 11.27 18.97 11.39 cytoplasmic droplet 0.50 0.95 0.48 0.50 3.37 1.48 2.50 3.03 4.26 0.00 10.36 0.47 0.98 0.00 0.00 0.00 1.49 0.00 0.00 0.00 2.39 0.64 bent neck 4.50 0.95 2.87 0.50 1.92 3.45 7.50 5.19 1.06 2.90 7.77 11.32 3.90 0.98 5.31 2.93 2.97 2.48 9.00 0.98 4.68 3.17 asymmetric neck insertion 0.00 0.00 0.48 0.00 0.96 0.00 0.50 1.30 1.60 0.00 0.52 1.42 0.49 0.00 3.86 0.00 0.99 0.00 1.50 0.00 1.09 0.27 thin midpiece 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.42 0.98 0.00 0.00 0.00 0.99 0.00 0.00 0.00 0.20 0.14 thick midpiece 3.50 9.95 6.22 6.50 3.37 4.43 6.00 13.42 6.91 5.80 4.15 11.32 7.32 1.46 25.60 1.95 13.86 3.96 17.00 2.45 9.39 6.12 coiled tail 13.50 18.48 16.27 8.50 18.27 26.11 25.50 17.32 17.02 22.22 12.95 6.13 1.95 0.00 23.19 10.24 3.47 1.98 10.00 2.45 14.21 11.34 bent tail 2.00 0.00 1.44 0.00 0.96 2.46 1.50 1.73 0.53 0.00 2.59 0.94 1.95 0.98 2.90 1.95 0.99 1.98 5.00 0.00 1.99 1.00 irregular tail 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.49 0.00 0.48 0.00 0.00 0.00 0.50 0.00 0.25 0.00 2-tailed 0.00 0.00 0.00 0.00 0.00 0.99 0.00 0.43 0.00 0.48 0.52 0.00 1.95 0.49 0.48 0.00 0.00 1.98 2.50 0.00 0.55 0.44 TZI 1.66 1.69 1.54 1.45 1.68 1.56 1.61 1.48 1.48 1.39 1.49 1.48 1.47 1.16 1.62 1.31 1.53 1.44 1.54 1.23 1.56 1.42b SDI 1.49 1.37 1.42 1.17 1.52 1.20 1.48 1.27 1.31 1.11 1.34 1.23 1.36 0.63 1.42 0.73 1.34 1.11 1.33 0.79 1.40 1.06c Table 3.2. Percentage of morphologic characteristics in sperm from teratozoospermic subjects. Raw: washed, unselected sperm sample; Swim-up: swim-up selected sperm sample TZI: (Teratozoospermic index) = total number of defects / number of sperm with defects SDI: (Sperm deformity index) = total number of defects / number of spermatozoa counted a statistically significantly difference between Raw and Swim-up, P<0.0001 b statistically significantly difference between Raw and Swim-up, P= 0.0185 c statistically significantly difference between Raw and Swim-up, P= 0.0002 75 Table 3.3. Group means of morphologic characteristics: comparison between control and teratozoospermic groups in raw semen and swim-up sperm * Statistically significant difference between Control and Terato groups, P<0.05. RAW SWIM-UP CONTROL (n=7) TERATO (n=10) CONTROL (n=7) TERATO (n=10) Kruger’s Measurement no data 3.25±1.50% not applicable normal 21.53±4.92% 10.36±2.27%* 21.31±7.16% 26.10±11.48% small head 3.05±1.70% 8.27±11.62% 4.42±2.43% 8.18±12.39% medium head 94.30±2.41% 90.74±11.25% 94.14±2.61% 90.09±11.78% large head 2.65±1.87% 1.00±0.90% 1.45±0.77% 1.74±2.48% vacuolated 48.33±10.57% 49.45±19.01% 43.42±16.24% 39.23±14.32% acrosomal abnormalities 5.46±2.01% 9.68±11.74% 6.54±2.77% 9.63±10.04% TOTAL internal head abnormalities 53.72±9.27% 58.44±14.73% 49.81±16.88% 48.81±9.43% round head 4.01±1.84% 5.53±5.49% 2.98±2.69% 4.57±3.25% thin head 0.00 0.59±0.79% 0.18±0.32% 0.64±1.11% pyriform head 4.26±3.25% 10.06±8.53% 4.19±2.46% 6.17±7.13% tapered head 5.04±4.43% 2.68±3.83% 4.04±3.48% 1.62±3.16% amorphous head 10.22±4.38% 18.97±5.94%* 10.72±3.12% 11.39±4.44% TOTAL head shape abnormalites 23.53±2.92% 37.83±10.91%* 22.11±6.10% 24.39±7.30% cytoplasmic droplet 7.22±6.24% 2.39±3.15%* 2.86±2.45% 0.64±0.98% bent neck 5.34±1.39% 4.68±2.67% 3.25±1.45% 3.17±3.21% asymmetric neck insertion 0.33±0.35% 1.09±1.09% 0.98±0.91% 0.27±0.57% thin midpiece 0.00 0.20±0.41% 0.00 0.14±0.45% thick midpiece 4.85±2.45% 9.39±7.24% 10.66±3.32% 6.12±4.15% TOTAL neck/midpiece abnormalities 10.03±1.50% 15.12±8.85% 14.68±4.69% 9.57±7.31% coiled tail 10.61±7.21% 14.21±7.62% 17.60±11.64% 11.34±9.16% bent tail 0.27±0.38% 1.99±1.29% 1.02±1.07% 1.00±0.97% irregular tail 0.00 0.25±0.35% 0.27±0.54% 0.00 2-tailed 0.27±0.26% 0.55±0.92% 0.62±0.55% 0.44±0.64% TOTAL tail abnormalities 11.15±7.39% 16.99±7.30% 19.51±12.17% 12.79±9.37% TZI 1.42±0.14 1.56±0.07* 1.45±0.15 1.42±0.15 SDI 1.11±0.11 1.40±0.08* 1.15±0.20 1.06±0.25 76 Spearman’s Rank Correlation Coefficient “rs” (P-value) Kr ug er ’s m ea su re m e nt no rm al sm al l h ea d la rg e h ea d va co ul at e d ac ro so m al ab n or m al iti es cy to pl as m ic d ro p ro un d h ea d py ri fo rm h ea d ta p er ed h e ad am or ph ou s he ad th ic k m id pi ec e be n t n ec k as ym m et ri c ne ck in se rt io n co ile d ta il be n t t ai l 2 ta il te ra to z oo s pe rm ic in de x sp er m d e fo rm ity in de x Kruger’s measurement -0.7 (0.2333) -0.6 (0.35) -0.4 (0.5167) 0.7 (0.2333) -0.8 (0.1333) 0.1 (0.95) 0.1 (0.95) 0.6 (0.35) -0.4 (0.5167) -0.6 (0.35) 0 (1.05) -0.5 (0.45) -0.4104 (0.5167) -0.1 (0.95) 0.1118 (0.95) 0.6708 (0.2333) -0.6 (0.35) 0.1 (0.95) normal 0.3214 (0.4976) 0.6071 (0.1667) -0.6071 (0.1667) 0.4286 (0.3536) -0.5714 (0.2) 0.2143 (0.6615) -0.1786 (0.7131) -0.2143 (0.6615) 0.5 (0.2667) -0.07143 (0.9063) -0.03571 (0.9635) 0.8233* (0.0341) 0.2857 (0.556) 0.0197 (0.9635) -0.2594 (0.556) 0.2857 (0.556) -0.3214 (0.4976) small head 0 (1.0365) -0.2143 (0.6615) 0.3571 (0.4444) 0.5357 (0.2357) -0.5 (0.2667) -0.3929 (0.3956) -0.07143 (0.9063) 0.75 (0.0663) 0.3214 (0.4976) -0.5357 (0.2357) 0.09356 (0.8397) -0.1429 (0.7825) -0.3744 (0.3956) 0.2224 (0.6615) 0.7143 (0.0881) 0.4286 (0.3536) large head -0.8929* (0.0123) 0.5357 (0.2357) -0.3929 (0.3956) -0.1071 (0.8397) -0.03571 (0.9635) 0.4286 (0.3536) 0.03571 (0.9635) -0.6786 (0.1095) 0.5714 (0.2) 0.3742 (0.3956) 0.4286 (0.3536) 0.4532 (0.3024) 0.07412 (0.9063) -0.03571 (0.9635) -0.4286 (0.3536) vacuolated -0.75 (0.0663) 0.2857 (0.556) 0.2143 (0.6615) 0.1429 (0.7825) -0.3214 (0.4976) -0.3571 (0.4444) 0.4643 (0.3024) -0.5714 (0.2) -0.2994 (0.4976) -0.5714 (0.20) -0.3941 (0.3956) -0.03706 (0.9635) -0.25 (0.5948) 0.2143 (0.6615) acrosomal abnormalities -0.2143 (0.6615) -0.5 (0.2667) -0.6071 (0.1667) 0.25 (0.5948) 0.6429 (0.1389) 0.1071 (0.8397) 0.3214 (0.4976) 0.4304 (0.3536) 0.6071 (0.1667) 0.5714 (0.2) -0.1112 (0.8397) 0.7143 (0.0881) 0.4286 (0.3536) cytoplasmic drop -0.5357 (0.2357) 0 (1.0365) 0.2143 (0.6615) 0.03571 (0.9635) 0.07143 (0.9063) -0.3571 (0.4444) -0.7298 (0.0663) -0.4643 (0.3024) -0.4335 (0.3536) 0.5189 (0.2357) 0.1429 (0.7825) 0.3929 (0.3956) round head 0.2143 (0.6615) -0.1786 (0.7131) -0.4286 (0.3536) -0.2143 (0.6615) 0.1786 (0.7131) 0.2433 (0.5948) -0.3214 (0.4976) -0.4335 (0.3536) -0.6671 (0.1095) -0.6429 (0.1389) -0.7143 (0.0881) pyriform head -0.4286 (0.3536) -0.3571 (0.4444) -0.2857 (0.556) 0 (1.0365) -0.4678 (0.3024) 0.1786 (0.7131) -0.07881 (0.9063) 0.5559 (0.2) -0.5357 (0.2357) -0.5357 (0.2357) tapered head -0.4286 (0.3536) -0.6429 (0.1389) 0.6071 (0.1667) -0.1684 (0.7131) -0.2857 (0.556) 0.1576 (0.7131) -0.1482 (0.7825) -0.25 (0.5948) -0.07143 (0.9063) amorphous head 0.6071 (0.1667) -0.3929 (0.3956) 0.3742 (0.3956) 0.4286 (0.3536) 0.03941 (0.9635) 0.07412 (0.9063) 0.9286** (0.0067) 0.5357 (0.2357) thick midpiece -0.6786 (0.1095) 0.1871 (0.7131) 0.1071 (0.8397) -0.03941 (0.9635) -0.07412 (0.9063) 0.6786 (0.1095) 0.75 (0.0663) bent neck -0.09356 (0.8397) 0.3214 (0.4976) 0.4335 (0.3536) -0.2594 (0.556) -0.3929 (0.3956) -0.3929 (0.3956) asymmetric neck insertion 0.2058 (0.6615) 0.2168 (0.6615) -0.5243 (0.2357) 0.3181 (0.4976) -0.05614 (0.9063) coiled tail 0.7684* (0.048) 0.2594 (0.556) 0.3929 (0.3956) 0.1429 (0.7825) bent tail 0.184 (0.7131) 0.2167 (0.6615) 0.2561 (0.5948) 2 tail 0.1112 (0.8397) 0.1482 (0.7825) teratozoospermic index 0.7857* (0.048) Table 3.4. Cross correlation of specific abnormal morphology in raw, unselected sperm of fertile control subjects * statistically significant correlation, P<0.05 ** statistically significant correlation, P<0.01 rs values highlighted green indicate statistically significant positive correlation; rs values highlighted red indicate statistically significant negative correlation 77 Spearman’s Rank Correlation Coefficient “rs” (P-value) Kr ug er ’s m ea su re m e nt no rm al sm al l h ea d la rg e h ea d va co ul at e d ac ro so m al ab n or m al iti es cy to pl as m ic d ro p ro un d h ea d th in h ea d py ri fo rm h ea d ta p er ed h e ad am or ph ou s he ad th in m id pi ec e th ic k m id pi ec e be n t n ec k as ym m et ri c ne ck in se rt io n co ile d ta il be n t t ai l ir re gu la r ta il 2 ta il te ra to z oo s pe rm ic in de x sp er m d e fo rm ity in de x Kruger’s measurement 0.09232 (0.8113) -0.04924 (0.8916) 0.2548 (0.4697) -0.1785 (0.6321) -0.08616 (0.8113) 0.355 (0.3129) 0.03077 (0.946) -0.5436 (0.1049) -0.2646 (0.4483) -.006173 (1) -0.7377* (0.0174) -0.1845 (0.6073) -0.1969 (0.5837) 0.3016 (0.3869) -0.1785 (0.6321) -0.2093 (0.5603) 0.16 (0.6567) 0.1907 (0.6073) 0.1005 (0.785) -0.4124 (0.2325) -0.4 (0.2475) normal 0.2727 (0.4483) -0.07528 (0.8382) -0.5273 (0.1231) 0.4788 (0.1663) -0.2249 (0.5367) 0.3697 (0.2957) 0.1204 (0.733) -0.297 (0.4069) 0.4195 (0.2325) 0.231 (0.5135) -0.0173 (0.973) 0.5273 (0.1231) 0.2242 (0.5367) 0.7212* (0.0234) -0.1636 (0.6567) 0.2727 (0.4483) 0.1366 (0.7072) 0.2117 (0.5603) -0.0303 (0.946) -0.5394 (0.1139) small head -0.5081 (0.1334) -0.6485* (0.049) 0.8667* (0.0022) -0.1763 (0.6321) 0.6727* (0.039) -0.2471 (0.4918) -.7939** (0.0088) 0.4438 (0.2044) 0.1033 (0.785) -0.0173 (0.973) -0.1394 (0.7072) 0.2485 (0.4918) -0.05455 (0.8916) 0.2485 (0.4918) 0.1394 (0.7072) 0.157 (0.6567) -0.4028 (0.2475) 0.6727* (0.039) 0.4667 (0.1786) large head 0.6211 (0.0603) -0.4391 (0.2044) 0.3146 (0.3679) -0.2258 (0.5367) -0.3803 (0.2788) 0.1568 (0.6567) -0.05978 (0.8651) -0.5978 (0.0734) -0.5372 (0.1139) -0.2196 (0.5367) -0.1192 (0.733) -0.01255 (0.973) 0.2447 (0.4918) -0.2447 (0.4918) -0.5371 (0.1139) -0.1307 (0.7072) -0.2196 (0.5367) -0.3325 (0.3487) vacuolated -0.7091* (0.0268) 0.4802 (0.1663) -0.5394 (0.1139) -0.01267 (0.973) 0.4909 (0.1548) -0.5471 (0.1049) -0.2067 (0.5603) -0.02595 (0.946) -0.4303 (0.2182) -0.6364 (0.0544) -0.2606 (0.4697) 0.0303 (0.946) -0.6848* (0.0347) -0.6213 (0.0603) -0.2936 (0.4069) -0.2364 (0.5135) -0.0303 (0.946) acrosomal abnormalities -0.5471 (0.1049) 0.4909 (0.1548) -0.03801 (0.9184) -0.697* (0.0306) 0.2979 (0.4069) 0.1641 (0.6567) -0.0173 (0.973) 0.2 (0.5837) 0.2 (0.5837) 0.05455 (0.8916) 0.1273 (0.733) 0.2364 (0.5135) 0.3551 (0.3129) -0.3004 (0.3869) 0.5758 (0.0883) 0.2242 (0.5367) cytoplasmic drop 0 (1) -0.5211 (0.1231) 0.04255 (0.9184) -0.0122 (0.973) -0.3963 (0.2632) 0.00867 6 (1) -0.5836 (0.0806) -0.2918 (0.4069) -0.01824 (0.973) 0.09119 (0.8113) -0.5593 (0.0963) -0.6575* (0.0438) -0.3082 (0.3869) -0.2796 (0.4271) -0.08511 (0.8113) round head -0.05068 (0.8916) -.8182** (0.0058) 0.7295* (0.0202) 0.2492 (0.4918) 0.1557 (0.6567) 0.1152 (0.7589) 0.6242 (0.0603) 0.1152 (0.7589) -0.01818 (0.973) 0.3455 (0.3304) -0.1229 (0.733) 0.0478 (0.8916) 0.1758 (0.6321) 0.01818 (0.973) thin head 0.2724 (0.4483) -0.1843 (0.6073) 0.7943** (0.0088) 0.6149 (0.0667) 0.5829 (0.0806) 0.01901 (0.973) 0.1394 (0.7072) -0.5892 (0.0806) 0.1774 (0.6321) 0.3105 (0.3869) 0.5103 (0.1334) -0.2407 (0.4918) -0.2217 (0.5367) pyriform head -0.3587 (0.3129) 0.04863 (0.8916) -0.07785 (0.8382) 0.1273 (0.733) -0.4182 (0.2325) 0.2364 (0.5135) 0.07879 (0.8382) -0.2 (0.5837) 0 (1) 0.2731 (0.4483) -0.2727 (0.4483) -0.09091 (0.8113) tapered head 0.06707 (0.8651) -0.2646 (0.4483) 0.2614 (0.4697) 0.6261 (0.0603) 0.5471 (0.1049) 0.4377 (0.2044) 0.3951 (0.2632) -0.113 (0.7589) 0.1815 (0.6073) 0.2432 (0.4918) 0 (1) amorphous head 0.6247 (0.0603) 0.5167 (0.1334) 0.01824 (0.973) 0.2796 (0.4271) -0.3526 (0.3129) 0.1641 (0.6567) 0.09246 (0.8113) 0.3082 (0.3869) 0.01824 (0.973) 0.02432 (0.946) thin midpiece 0.3546 (0.3129) -0.1816 (0.6073) -0.0519 (0.8916) -0.6833* (0.0347) -0.1989 (0.5837) 0.00487 3 (1) 0.09258 (0.8113) -0.493 (0.1548) -0.2768 (0.4271) thick midpiece 0.2242 (0.53670 0.6 (0.0734) -0.2485 (0.4918) 0.3576 (0.3129) 0.2595 (0.4697) 0.4848 (0.1548) -0.3818 (0.2788) -0.5758 (0.0883) bent neck 0.00606 1 (1) -0.1152 (0.7589) 0.8909 (0.0011) 0.4233 (0.2182) 0.6486* (0.049) 0.01818 (0.973) -0.04242 (0.9184) asymmetric neck insertion 0.2 (0.5837) 0.0303 (0.946) -0.1502 (0.6821) 0.2185 (0.5367) -0.1152 (0.7589) -0.4667 (0.1786) coiled tail -0.1879 (0.6073) -0.3414 (0.3304) -0.4848 (0.1548) 0.6121 (0.0667) 0.503 (0.144) bent tail 0.6691* (0.039) 0.7579* (0.0149) 0.06667 (0.8651) -.006061 (1) irregular tail 0.5038 (0.144) 0.1161 (0.7589) 0.08876 (0.8113) 2 tail -.4165 (.2325) -.3687 (.2957) teratozoospermic index .8182** (.0058) Table 3.5. Cross correlation of specific abnormal morphology in raw, unselected sperm of teratozoospermic subjects rs values highlighted green indicate statistically significant positive correlation; rs values highlighted red indicate statistically significant negative correlation * statistically significant correlation, P<0.05 ** statistically significant correlation, P<0.01 78 Spearman’s Rank Correlation Coefficient “rs” (P-value) n o rm al s m al l h e ad la rg e h e a d v ac o ul a te d a c ro s o m a l a b n o rm a lit ie s c yt o pl as m ic d ro p ro u n d h e a d th in h e ad p y rif o rm h e a d ta p e re d h e ad a m o rp ho us h ea d th ic k m id p ie ce b e n t ne ck a sy m m e tr ic n e ck in se rt io n c oi le d t ai l b e n t ta il ir re g u la r ta il 2 t a il te ra to z o os p e rm ic in d ex s pe rm d e fo rm ity in d ex normal -0.3571 (0.4444) -0.2883 (0.5560) -0.7500 (0.0663) -0.4643 (0.3024) -0.4286 (0.3536) 0.4286 (0.3536) 0.3563 (0.4444) -0.1429 (0.7825) 0.7500 (0.0663) 0.3214 (0.4976) 0.5714 (0.2000) 0.1786 (0.7131) -0.1071 (0.8397) -0.5357 (0.2357) -0.7027 (0.0881) -0.4009 (0.3956) 0.01818 (0.9635) -0.6786 (0.1095) -0.7857* (0.0480) small head -0.05406 (0.9063) 0.6786 (0.1095) 0.3571 (0.4444) 0.4643 (0.3024) -0.2857 (0.5560) 0.2227 (0.6615) 0.1786 (0.7131) 0.1429 (0.7825) -0.5714 (0.2000) -0.07143 (0.9063) -0.2143 (0.6615) -0.3929 (0.3956) 0.1071 (0.8397) -0.3063 (0.4976) 0.8018* (0.0341) 0.5637 (0.2000) 0.4643 (0.3024) 0.3929 (0.3956) large head -0.03604 (0.9635) 0.7928* (0.0480) 0.09009 (0.8397) 0.1802 (0.7131) -0.6742 (0.1095) -0.09009 (0.8397) -0.4685 (0.3024) 0.4325 (0.3536) 0.03604 (0.9635) 0.5406 (0.2357) 0.2342 (0.5948) 0.3964 (0.3956) 0.1909 (0.6615) 0.2247 (0.5948) -0.1376 (0.7825) 0.5045 (0.2667) 0.5225 (0.2357) vacuolated 0.1786 (0.7131) 0.2857 (0.5560) -0.1786 (0.7131) 0.2673 (0.5560) 0.3214 (0.4976) -0.2857 (0.5560) -0.8214* (0.0341) -0.7143 (0.0881) -0.5357 (0.2357) -0.5000 (0.2667) 0.1429 (0.7825) 0.1622 (0.7131) 0.7572 (0.0663) -0.07274 (0.9063) 0.4286 (0.3536) 0.6071 (0.1667) acrosomal abnormalities 0.1786 (0.7131) -0.2500 (0.5948) -0.5345 (0.2357) 0.2143 (0.6615) -0.5000 (0.2667) 0.1786 (0.7131) 0.1071 (0.8397) 0.3214 (0.4976) 0.2857 (0.5560) 0.7500 (0.0663) 0.2703 (0.5560) 0.4009 (0.3956) 0.1818 (0.7131) 0.8929* (0.0123) 0.7857* (0.0480) cytoplasmic drop -0.5714 (0.2000) -0.4900 (0.2667) -0.2143 (0.6615) -0.2857 (0.5560) 0.1429 (0.7825) -0.1071 (0.8397) 0.5000 (0.2667) -0.03571 (0.9635) -0.1429 (0.7825) 0.1261 (0.7825) 0.08909 (0.8397) 0.6001 (0.1667) 0.2143 (0.6615) 0.0000 (1.0365) round head 0.2673 (0.5560) -0.4286 (0.3536) 0.5357 (0.2357) -0.03571 (0.9635) 0.1429 (0.7825) -0.1786 (0.7131) -0.1786 (0.7131) -0.3929 (0.3956) -0.5225 (0.2357) 0.2227 (0.6615) -0.3819 (0.3956) -0.5357 (0.2357) -0.2143 (0.6615) thin head 0.4009 (0.3956) 0.5791 (0.2000) -0.7572 (0.0663) -0.2227 (0.6615) -0.7572 (0.0663) -0.6682 (0.1095) -0.3563 (0.4444) -0.5169 (0.2357) 0.2778 (0.5560) -0.2495 (0.5948) -0.3563 (0.4444) -0.2227 (0.6615) pyriform head -0.3571 (0.4444) -0.4286 (0.4444) -0.5000 (0.2667) -0.2857 (0.5560) -0.4286 (0.3536) 0.3214 (0.4976) 0.1802 (0.7131) 0.08909 (0.8397) -0.4001 (0.3956) 0.4643 (0.3024) 0.3571 (0.4444) tapered head -0.1429 (0.7825) 0.5357 (0.2357) -0.2143 (0.6615) -0.2500 (0.5948) -0.5714 (0.2000) -0.8829* (0.0123) 0.1336 (0.7825) 0.3091 (0.4976) -0.6429 (0.1389) -0.6071 (0.1667) amorphous head 0.5714 (0.2000) 0.8571* (0.0238) 0.6429 (0.1389) 0.03571 (0.9635) 0.1442 (0.7825) -0.6682 (0.1095) 0.1818 (0.7131) -0.1071 (0.8397) -0.3214 (0.4976) thick midpiece 0.3571 (0.4444) 0.5714 (0.2000) 0.07143 (0.9063) -0.3784 (0.3956) -0.2227 (0.6615) 0.6183 (0.1389) -0.1429 (0.7825) -0.3214 (0.4976) bent neck 0.2857 (0.5560) -0.1071 (0.8397) 0.0000 (1.0365) -0.4009 (0.3956) 0.2910 (0.5560) 0.03571 (0.9635) -0.2500 (0.5948) asymmetric neck insertion 0.5714 (0.2000) 0.5225 (0.2357) -0.4454 (0.3024) 0.2910 (0.5560) 0.2143 (0.6615) 0.1429 (0.7825) coiled tail 0.6307 (0.1389) 0.1336 (0.7825) 0.03637 (0.9635) 0.8571* (0.0238) 0.8214* (0.0341) bent tail -0.3146 (0.4976) -0.2477 (0.5948) 0.4865 (0.2667) 0.5045 (0.2667) irregular tail 0.1361 (0.7825) 0.4009 (0.3956) 0.5791 (0.2000) 2 tail 0.1455 (0.7825) -0.1091 (0.8397) teratozoospermic index 0.8929* (0.0123) Table 3.6. Cross correlation of specific abnormal morphology in swim-up sperm of fertile control subjects * statistically significant correlation, P<0.05 rs values highlighted green indicate statistically significant positive correlation; rs values highlighted red indicate statistically significant negative correlation 79Spearman’s Rank Correlation Coefficient “rs” (P-value) no rm al sm al l h ea d la rg e h ea d va co ul at e d ac ro so m al ab n or m al iti es cy to pl as m ic d ro p ro un d h ea d th in h ea d py ri fo rm h ea d ta p er ed h e ad am or ph ou s he ad th in m id pi ec e th ic k m id pi ec e be n t n ec k as ym m et ri c ne ck in se rt io n co ile d ta il be n t t ai l 2 ta il te ra to z oo s pe rm ic in de x sp er m d e fo rm ity in de x normal -0.5228 (0.1231) 0.1581 (0.6567) -0.2432 (0.4918) -0.07879 (0.8382) -0.6659* (0.0390) -0.6364 (0.0544) 0.8057** (0.0072) 0.1636 (0.6567) -0.8254** (0.0047) 0.06667 (0.8651) -0.4062 (0.2475) -0.9879*** (P<0.0001) -0.2614 (0.4697) -0.6833* (0.0347) -0.3455 (0.3304) 0.2564 (0.4697) 0.2521 (0.4697) -0.7333* (0.0202) -0.8788** (0.0016) small head -0.7134* (0.0234) -0.1341 (0.7072) 0.3951 (0.2632) 0.2788 (0.4271) 0.9362*** (0.0002) -0.5616 (0.0963) -0.7538* (0.0149) 0.09408 (0.7850) 0.1337 (0.7072) 0.1746 (0.6321) 0.4377 (0.2044) -0.1311 (0.7072) 0.3384 (0.3304) -0.2918 (0.4069) -0.08154 (0.8382) -0.2205 (0.5367) 0.4073 (0.2475) 0.5714 (0.0883) large head -0.01220 (0.9730) -0.2432 (0.4918) 0.2399 (0.4918) -0.6383 (0.0544) 0.4452 (0.2044) 0.5228 (0.1231) 0.2760 (0.4271) -0.09119 (0.8113) -0.3492 (0.3129) -0.04863 (0.8916) 0.2073 (0.5603) -0.09110 (0.8113) 0.4924 (0.1548) 0.06586 (0.8651) 0.3178 (0.3679) -0.04863 (0.8916) -0.1398 (0.7072) vacuolated -0.6018 (0.0734) -0.09727 (0.7850) -0.09726 (0.7850) -0.3356 (0.3487) 0.4742 (0.1663) 0.2133 (0.5603) -0.01216 (0.9730) 0.4074 (0.2475) 0.2310 (0.5135) 0.02134 (0.9460) 0.2169 (0.5367) -0.1702 (0.6321) -0.3199 (0.3679) -0.1686 (0.6321) -0.1033 (0.7850) 0.0000 (1.0000) acrosomal abnormalities 0.1616 (0.6567) 0.4061 (0.2475) -0.1366 (0.7072) -0.6606* (0.0438) -0.02501 (0.9460) -0.3333 (0.3487) -0.5222 (0.1231) 0.04242 (0.9184) -0.07903 (0.8382) -0.3114 (0.3869) 0.3697 (0.2957) 0.2939 (0.4069) 0.1616 (0.6567) 0.3818 (0.2788) 0.2848 (0.4271) cytoplasmic drop 0.3556 (0.3129) -0.1602 (0.6567) -0.03233 (0.9460) 0.6870* (0.0347) -0.1099 (0.7589) 0.06190 (0.8651) 0.7435* (0.0174) 0.2270 (0.5367) 0.4245 (0.2182) 0.5625 (0.0963) 0.1201 (0.7330) -0.07586 (0.8382) 0.8728** (0.0016) 0.8469** (0.0029) round head -0.7033* (0.0268) -0.6364 (0.0544) 0.3439 (0.3304) -0.1030 (0.7850) 0.05803 (0.8651) 0.5515 (0.1049) -0.2249 (0.5367) 0.3200 (0.3679) --0.1758 (0.6321) -0.2189 (0.5367) -0.3297 (0.3487) 0.4303 (0.2182) 0.6000 (0.0734) thin head 0.2185 (0.5367) -0.6164 (0.0603) 0.1912 (0.5837) -0.2615 (0.4697) -0.7169* (0.0234) 0.06849 (0.8651) -0.3898 (0.2632) -0.02731 (0.9460) 0.4614 (0.1786) 0.2404 (0.4918) -0.3482 (0.3304) -0.5394 (0.1139) pyriform head 0.2376 (0.5135) -0.3091 (0.3869) 0.05803 (0.8651) -0.07879 (0.8382) -0.1885 (0.6073) -0.2249 (0.5367) 0.2242 (0.5367) -0.3502 (0.3129) -0.07112 (0.8382) -0.1515 (0.6821) -0.2364 (0.5135) tapered head -0.3814 (0.2788) 0.05987 (0.8651) 0.8567** (0.0029) 0.07527 (0.8382) 0.4105 (0.2325) 0.5565 (0.0963) -0.3710 (0.2957) -0.2068 (0.5603) 0.6003 (0.0734) 0.6753* (0.0390) amorphous head 0.5222 (0.1231) -0.09091 (0.8113) 0.5897 (0.0806) 0.5449 (0.1049) -0.5030 (0.1440) 0.5253 (0.1231) 0.4202 (0.2325) -0.1636 (0.6567) -0.09091 (0.8113) thin midpiece 0.4062 (0.2475) 0.5238 (0.1231) 0.7454* (0.0174) -0.1741 (0.6321) -0.05987 (0.8651) -0.3095 (0.3869) 0.1741 (0.6321) 0.2901 (0.4069) thick midpiece 0.2979 (0.4069) 0.6833* (0.0347) 0.4424 (0.2044) -0.2439 (0.4918) -0.2651 (0.4483) 0.7818* (0.0105) 0.9030*** (0.0008) bent neck 0.7028* (0.0268) 0.2614 (0.4697) 0.5488 (0.1049) 0.1816 (0.6073) 0.2188 (0.5367) 0.2128 (0.5603) asymmetric neck insertion -0.02595 (0.9460) 0.07140 (0.8382) -0.2122 (0.5603) 0.3373 (0.3304) 0.5103 (0.1334) coiled tail 0.01251 (0.9730) -0.01940 (0.9730) 0.6364 (0.0544) 0.4909 (0.1548) bent tail 0.6203 (0.0603) 0.1063 (0.7589) -0.06253 (0.8651) 2 tail -0.04526 (0.9184) -0.1616 (0.6567) teratozoospermic index 0.9515*** (0.0001) Table 3.7. Cross correlation of specific abnormal morphology in swim-up sperm of teratozoospermic subjects rs values highlighted green indicate statistically significant positive correlation; rs values highlighted red indicate statistically significant negative correlation * statistically significant correlation, P<0.05 ** statistically significant correlation, P<0.01 *** statistically significant correlation, P<0.001 Table 3.8. Chromosome 18, X and Y (Triple-colour FISH) Results Chromosome 18 Sex chromosomes Total Sex Disomy %Patients Cells counted Disomy % Nullisomy % XX YY XY Nullisomy % Total aneuploidy % Diploidy % CONTROL SUBJECTS 0.15 CONTROL1 10311 0.08 0.01 0.00 0.12 0.03 0.19 0.43 0.01 0.52 CONTROL4 10047 0.06 0.08 0.12 0.16 0.24 0.56 1.21 0.04 0.56 CONTROL5 10019 0.03 0.01 0.21 0.05 0.30 0.34 0.94 0.02 0.34 CONTROL6 10098 0.02 0.27 0.06 0.12 0.16 0.36 0.99 0.06 0.38 CONTROL7 10265 0.18 0.11 0.03 0.10 0.25 0.22 0.89 0.06 0.48 CONTROL8 10226 0.00 0.08 0.11 0.11 0.26 0.80 1.36 0.00 0.54 CONTROL9 10015 0.08 0.02 0.05 0.05 0.44 0.79 1.46 0.25 0.77 CONTROL10 10178 0.08 0.03 0.23 0.49 0.05 0.23 1.08 0.04 0.19 CONTROL11 10223 0.05 0.00 0.06 0.05 0.08 0.18 0.41 0.52 0.45aCONTROL (pooled) 91382 0.06a 0.07b 0.10 0.14 0.22 0.41b 0.97 0.11 TERATOZOOSPERMIC SUBJECTS 0.45 TERATO1 10127 0.02 0.24 0.06 0.05 0.35 0.95 1.66 0.18 0.88 TERATO2 10010 0.00 0.00 0.16 0.22 0.50 0.10 0.98 0.00 1.96 TERATO3 11022 0.01 0.75 0.56 0.45 0.94 0.73 3.47 0.00 0.41 TERATO4 10086 0.00 0.00 0.07 0.15 0.19 0.11 0.52 0.00 0.12 TERATO5 10239 0.01 0.18 0.02 0.06 0.04 0.27 0.58 0.17 0.81 TERATO6 10009 0.16 0.09 0.11 0.40 0.30 0.65 1.73 0.33 0.68 TERATO8 10177 0.08 0.06 0.13 0.32 0.24 0.88 1.71 0.09 0.87 TERATO9 10083 0.23 0.23 0.25 0.24 0.38 1.81 3.13 0.49 0.54 TERATO10 10057 1.72 0.27 0.07 0.28 0.19 0.92 3.46 0.23 0.79 TERATO11 10083 0.09 0.14 0.13 0.23 0.43 0.62 1.68 0.17 0.75cTERATO (pooled) 101893 0.23c 0.20 0.16 0.24 0.36 0.70 1.90 0.17 a significant difference (P<0.001) in disomy incidence for chromosome 18 and sex chromosomes in control subjects b significant difference (P<0.001) in nullisomy incidence for chromosome 18 and sex chromosomes in control subjects c significant difference (P<0.05) in disomy incidence for chromosome 18 and sex chromosomes in teratozoospermic subjects Table 3.9. Chromosome 13 and 21 (Dual-colour FISH) Results Chromosome 13 Chromosome 21 Patient Cells counted Disomy % Nullisomy% Disomy % Nullisomy % Total aneuploidy % CONTROL SUBJECTS CONTROL1 10029 0.06 0.31 0.22 0.15 0.72 CONTROL4 10118 0.12 0.26 0.09 0.17 0.63 CONTROL5 10254 0.10 0.20 0.05 0.07 0.42 CONTROL6 10222 0.12 0.25 0.21 0.07 0.68 CONTROL7 10129 0.10 0.20 0.19 0.11 0.59 CONTROL8 10086 0.26 0.43 0.24 0.47 1.40 CONTROL9 10030 0.47 0.11 0.17 0.12 0.91 CONTROL10 10135 0.23 0.72 0.35 0.65 1.92 CONTROL11 10009 0.38 0.36 0.80 0.36 1.88 CONTROL (pooled) 91012 0.20 0.32 0.26 0.24 1.01 TERATOZOOSPERMIC SUBJECTS TERATO1 10051 0.82 0.44 0.45 0.55 2.33 TERATO2 10033 0.25 0.26 0.15 0.18 0.84 TERATO3 10024 0.45 0.33 0.48 1.79 3.04 TERATO4 10013 0.16 0.02 0.14 0.07 0.39 TERATO5 10156 0.18 0.80 0.16 0.79 1.90 TERATO6 10158 1.59 1.57 2.50 0.85 7.44 TERATO8 10098 1.10 0.81 4.59 0.29 7.37 TERATO9 10024 1.14 0.25 0.22 0.33 2.01 TERATO10 10001 0.75 0.47 0.39 0.43 2.20 TERATO11 10011 1.22 0.42 0.43 0.35 2.60 TERATO (pooled) 100579 0.77 0.54 0.95 0.56 3.02 82 Table 3.10. Summary of Chromosomal Abnormalities in Control and Teratozoospermic Groups Control Group (% mean ± SD) Teratozoospermic Group (% mean ± SD) Chromosome 13 Disomy 0.20 ± 0.14** 0.77 ± 0.50** Nullisomy 0.32 ± 0.18 0.54 ± 0.44 Aneuploidy 0.52 ± 0.23 a* 1.30 ± 0.83* Chromosome 18 Disomy 0.06 ± 0.05 0.23 ± 0.53 Nullisomy 0.07 ± 0.08 0.20 ± 0.22 Aneuploidy 0.13 ± 0.09ab 0.43 ± 0.59d Chromosome 21 Disomy 0.26 ± 0.22 0.95 ± 1.46 Nullisomy 0.24 ± 0.21 0.56 ± 0.50 Aneuploidy 0.50 ± 0.37 ac 1.51 ± 1.52 Sex Chromosomes Disomy 0.45 ± 0.19 0.75 ± 0.49 Nullisomy 0.41 ± 0.25 0.70 ± 0.50 Aneuploidy 0.86 ± 0.36bc 0.94 ± 0.54d TOTAL Aneuploidy (for all chromosomes) 1.99 ± 0.65** 4.90 ± 2.82** Diploidy 0.11 ± 0.17 0.17 ± 0.16 TOTAL Chromosomal Abnormalities 2.35 ± 0.87* 5.90 ± 3.74* a significant difference (P<0.05) between aneuploidy of chromosome 18 and aneuploidy of chromosomes 13 and 21 in control subjects b significant difference (P<0.001) between aneuploidy of chromosome 18 and aneuploidy of sex chromosomes in control subjects c significant difference (P<0.05) between aneuploidy of chromosome 21 and aneuploidy of sex chromosomes in control subjects d significant difference (P<0.05) between aneuploidy of chromosome 18 and aneuploidy of sex chromosomes in teratozoospermic subjects * significant difference (P<0.05) between control vs. teratozoospermic subjects ** significant difference (P<0.001) between control vs. teratozoospermic subjects 83 small heads medium heads large heads amorphous heads Control 8 # of sperm 68 78 4 27 disomy 18 2.94%b 6.41%b 0 % 18.52%b nullisomy 18 0 % 0 % 0 % 7.41% sex disomy 4.41% 0 % 0 % 3.70% sex nullisomy 4.41% 3.85% 0 % 7.41% diploidy 2.94% 0 % 0 % 3.70% total chr. abnormality 14.71%a 8.97%c* 0 % 40.74%ac Terato8 # of sperm 17 121 4 10 disomy 18 0 % 0 % 0 % 0 % nullisomy 18 0 % 0 % 0 % 0 % sex disomy 0 % 2.48% 50.00% 0 % sex nullisomy 5.88% 2.48% 0 % 0 % diploidy 0 % 0 % 0 % 0 % total chr. abnormality 5.88% 4.96% 50.00% 0 % Terato9 # of sperm 23 78 23 12 disomy 18 0 % 0 % 0 % 0 % nullisomy 18 0 % 0 % 0 % 0 % sex disomy 4.55% 1.30% 8.70% 9.09% sex nullisomy 0 % 5.19% 0 % 0 % diploidy 9.09% 1.30% 4.35% 0 % total chr. abnormality 13.64% 7.79% 13.04% 9.09% Terato10 # of sperm 30 96 7 4 disomy 18 0 % 1.08% 0 % 0 % nullisomy 18 0 % 0 % 14.29% 0 % sex disomy 0 % 5.38% 0 % 0 % sex nullisomy 0 % 4.30% 0 % 0 % diploidy 0 % 0 % 0 % 0 % total chr. abnormality 0 %* 10.75% 14.29% 0 % Terato11 # of sperm 18 190 0 9 disomy 18 0 % 1.59% n/a 0 % nullisomy 18 0 % 0 % n/a 0 % sex disomy 0 % 0.53% n/a 0 % sex nullisomy 5.56% 3.17% n/a 0 % diploidy 0 % 0.53% n/a 0 % total chr. abnormality 5.56% 5.82%* n/a 0 % Terato12 # of sperm 14 74 2 10 disomy 18 0 % 1.35% 0 % 0 % nullisomy 18 0 % 0 % 0 % 0 % sex disomy 0 % 0 % 0 % 0 % sex nullisomy 7.14% 4.05% 0 % 0 % diploidy 0 % 1.35% 0 % 0 % total chr. abnormality 7.14% 6.76% 0 % 10.00% # of sperm 102 559 36 45Terato (pooled) disomy 18 0 % 0.90% 0 % 0 % nullisomy 18 0 % 0 % 2.78% 0 % sex disomy 1.01% 1.81%d 11.11%d 2.27% sex nullisomy 3.03% 3.61% 0 % 0 % diploidy 2.02% 0.54% 2.78% 0 % total chr. abnormality 6.06% 6.86%* 16.67% 4.55% a significant difference (P<0.05) in respective cells of the control subject b significant difference (P<0.01) in respective cells of the control subject c significant difference (P<0.0001) in respective cells of the control subject d significant difference (P<0.001) in respective cells of the teratozoospermic subjects pooled * significant difference (P<0.05) between control and (pooled) teratozoospermic subjects Table 3.11. Chromosomal Abnormalities in Morphologically Separated Sperm 84 Table 3.12. DNA Fragmentation in Control and Teratozoospermic Subjects % of fragmented sperm / total sperm CONTROL SUBJECTS TERATOZOOSPERMIC SUBJECTS CONTROL5 (1151 cells) 25.54 TERATO1 (442 cells) 66.11 CONTROL8 (1019 cells) 49.26 TERATO2 (322 cells) 80.10 CONTROL9 (1009 cells) 61.04 TERATO3 (808 cells) 19.93 CONTROL10 (1032 cells) 33.14 TERATO4 (1002 cells) 92.71 CONTROL11 (954 cells) 15.09 TERATO5 (127 cells) 49.58 CONTROL13 (717 cells) 15.06 TERATO6 (591 cells) 45.35 CONTROL14 (329 cells) 27.66 TERATO8 (1032 cells) 44.09 TERATO9 (500 cells) 62.60 TERATO10 (637 cells) 61.85 TERATO11 (1023 cells) 80.25 CONTROL MEAN 32.40 ± 17.20 TERATO MEAN 60.28 ± 21.40* * Significantly different (P= 0.0136) from control group 85 Chromosome 13 Chromosome 18 Chromosome 21 Sex Chromosomesrs (P-value) D N T D N T D N T D N T Kruger’s Measurement n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a normal 0.5000 (0.4500) -0.7000 (0.2333) -0.7000 (0.2333) -0.2052 (0.7833) 0.2000 (0.7833) -0.1000 (0.9500) -0.6000 (0.3500) -0.5000 (0.4500) -0.6000 (0.3500) -0.3000 (0.6833) 0.8000 (0.1333) 0.7000 (0.2333) small head 0.8000 (0.1333) -0.5000 (0.4500) -0.1000 (0.9500) -.05130 (0.9500) -0.5000 (0.4500) -0.3000 (0.6833) 0.3000 (0.6833) -0.3000 (0.6833) 0.3000 (0.6833) -0.9000 (0.0833) -0.1000 (0.9500) -0.1000 (0.9500) large head 0.3000 (0.6833) -0.9000 (0.0833) -0.9000 (0.0833) -0.1026 (0.9500) -0.1000 (0.9500) -0.3000 (0.6833) -0.8000 (0.1333) -0.8000 (0.1333) -0.8000 (0.1333) -0.1000 (0.9500) 0.6000 (0.3500) 0.5000 (0.4500) vacuolated -0.5000 (0.4500) 0.7000 (0.2333) 0.7000 (0.2333) 0.2052 (0.7833) -0.2000 (0.7833) 0.1000 (0.9500) 0.6000 (0.3500) 0.5000 (0.4500) 0.6000 (0.3500) 0.3000 (0.6833) -0.8000 (0.1333) -0.7000 (0.2333) acrosomal abnormalities 0.6000 (0.3500) -0.3000 (0.6833) -0.3000 (0.6833) -0.4617 (0.4500) 0.3000 (0.6833) -0.1000 (0.9500) -0.1000 (0.9500) -0.1000 (0.9500) -0.1000 (0.9500) -0.7000 (0.2333) 0.7000 (0.2333) 0.5000 (0.4500) cytoplasmic droplet 0.3000 (0.6833) 0.1000 (0.9500) 0.6000 (0.3500) 0.6669 (0.2333) -0.6000 (0.3500) 0.2000 (0.7833) 0.7000 (0.2333) 0.2000 (0.7833) 0.7000 (0.2333) -0.1000 (0.9500) -0.9000 (0.0833) -0.5000 (0.4500) round head -0.2000 (0.7833) 0.1000 (0.9500) 0.1000 (0.9500) 0.6669 (0.2333) 0.4000 (0.5167) 0.7000 (0.2333) -0.3000 (0.6833) 0.2000 (0.7833) -0.3000 (0.6833) 0.9000 (0.0833) 0.1000 (0.9500) 0.5000 (0.4500) thin head no data no data no data no data no data no data no data no data no data no data no data no data pyriform head -0.7000 (0.2333) -0.1000 (0.9500) -0.4000 (0.5167) 0.1539 (0.7833) 0.1000 (0.9500) 0.0000 (1.0500) -0.7000 (0.2333) -0.3000 (0.6833) -0.7000 (0.2333) 0.9000 (0.0833) 0.1000 (0.9500) 0.1000 (0.9500) tapered head 0.7000 (0.2333) -0.5000 (0.4500) 0.1000 (0.9500) 0.8208 (0.1333) -0.5000 (0.4500) 0.3000 (0.6833) 0.2000 (0.7833) -0.2000 (0.7833) 0.2000 (0.7833) -0.1000 (0.9500) -0.4000 (0.5167) 0.1000 (0.9500) amorphous head 0.5000 (0.4500) -0.1000 (0.9500) -0.1000 (0.9500) -0.6156 (0.3500) 0.1000 (0.9500) -0.3000 (0.6833) 0.2000 (0.7833) 0.0000 (1.0500) 0.2000 (0.7833) -0.9000 (0.1315) 0.4000 (0.5167) 0.1000 (0.9500) bent neck 0.3000 (0.6833) -0.1000 (0.9500) 0.0000 (1.0500) 0.5643 (0.3500) 0.6000 (0.3500) 0.8000 (0.1333) -0.3000 (0.6833) 0.2000 (0.7833) -0.3000 (0.6833) 0.5000 (0.4500) 0.5000 (0.4500) 0.9000 (0.0833) asymmetric neck insertion 0.1026 (0.9500) -0.3591 (0.5167) -0.6669 (0.2333) -0.7105 (0.2333) 0.3591 (0.5167) -0.3591 (0.5167) -0.5643 (0.3500) -0.3591 (0.5167) -0.5643 (0.3500) -0.3591 (0.5167) 0.8721 (0.0833) 0.4617 (0.4500) thin midpiece no data no data no data no data no data no data no data no data no data no data no data no data thick midpiece -0.1000 (0.9500) 0.7000 (0.2333) 0.5000 (0.4500) -0.6156 (0.3500) 0.3000 (0.6833) -0.1000 (0.9500) 0.6000 (0.3500) 0.6000 (0.3500) 0.6000 (0.3500) -0.5000 (0.4500) 0.0000 (1.0500) -0.3000 (0.6833) coiled tail -0.3000 (0.6833) -0.1000 (0.9500) -0.6000 (0.3500) -0.6669 (0.2333) 0.6000 (0.3500) -0.2000 (0.7833) -0.7000 (0.2333) -0.2000 (0.7833) -0.7000 (0.2333) 0.1000 (0.9500) 0.9000 (0.0833) 0.5000 (0.4500) bent tail -0.4472 (0.4500) 0.1118 (0.9500) -0.4472 (0.4500) -0.9177 (0.0833) 0.4472 (0.4500) -0.4472 (0.4500) -0.4472 (0.4500) -0.1118 (0.9500) -0.4472 (0.4500) -0.1118 (0.9500) 0.6708 (0.2333) 0.1118 (0.9500) irregular tail no data no data no data no data no data no data no data no data no data no data no data no data 2-tailed -0.4472 (0.4500) -0.3354 (0.5167) -0.4472 (0.4500) -0.3441 (0.5167) -0.7826 (0.1333) -0.8944 (0.0833) -0.2236 (0.7833) -0.6708 (0.2333) -0.2236 (0.7833) -0.1118 (0.9500) -0.4472 (0.4500) -0.7826 (0.1333) TZI 0.5000 (0.4500) -0.1000 (0.9500) -0.1000 (0.9500) -0.6156 (0.3500) 0.1000 (0.9500) -0.3000 (0.6833) 0.2000 (0.7833) 0.0000 (1.0500) 0.2000 (0.7833) -0.9000 (0.0833) 0.4000 (0.5167) 0.1000 (0.9500) SDI 0.0000 (1.0500) 0.6000 (0.3500) 0.6000 (0.3500) -0.4104 (0.5167) -0.1000 (0.9500) -0.2000 (0.7833) 0.8000 (0.1333) 0.5000 (0.4500) 0.8000 (0.1333) -0.6000 (0.3500) -0.4000 (0.5167) -0.6000 (0.3500) Table 3.13. Linear Correlation Analysis of Sperm Morphology and Aneuploidy in Control Subjects (n=5) D = disomy; N = nullisomy; T = total aneuploidy (disomy and nullisomy combined) TZI = teratozoospermic index; SDI = sperm deformity index n/a = not applicable (data not complete for all subjects) 86 rs (P-value) Total Chromosomal Abnormality Total Aneuploidy Supernumerary Chromosomal Abnormality Diploidy Kruger’s Measurement n/a n/a n/a n/a normal 0.2052 (P=0.7833) -0.2000 (P=0.7833) 0.1539 (P=0.7833) -0.2000 (P=0.7833) small head 0.7182 (P=0.2333) -0.4000 (P=0.5167) 0.5643 (P=0.3500) 0.6000 (P=0.3500) large head -0.1539 (P=0.7833) -0.5000 (P=0.4500) 0.1539 (P=0.7833) -0.1000 (P=0.9500) vacuolated -0.2052 (P=0.7833) 0.2000 (P=0.7833) -0.1539 (P=0.7833) 0.2000 (P=0.7833) acrosomal abnormalities 0.7182 (P=0.2333) 0.0000 (P=1.0500) 0.05130 (P=0.9500) -0.2000 (P=0.7833) cytoplasmic droplet 0.1026 (P=0.9500) 0.0000 (P=1.0500) 0.6669 (P=0.2333) 0.9000 (P=0.0833) round head -0.6669 (P=0.2333) 0.5000 (P=0.4500) 0.1539 (P=0.7833) -0.1000 (P=0.9500) thin head no data no data no data no data pyriform head -.9747* (P=0.0167) -0.1000 (P=0.9500) -0.3591 (P=0.5167) -0.4000 (P=0.5167) tapered head 0.0513 (P= 0.9500) -0.1000 (P=0.9500) 0.9747* (P=0.0167) 0.9000 (P=0.0833) amorphous head 0.8721 (P= 0.0833) -0.1000 (P=0.9500) -0.05130 (P=0.9500) -0.1000 (P=0.9500) bent neck -0.2052 (P=0.7833) 0.6000 (P=0.3500) 0.3591 (P=0.5167) -0.1000 (P=0.9500) asymmetric neck insertion 0.2632 (P=0.6833) -0.2052 (P=0.7833) -0.3947 (P=0.5167) -0.6156 (P=0.3500) thin midpiece no data no data no data no data thick midpiece 0.6669 (P=0.2333) 0.3000 (P=0.6833) -0.4617 (P=0.4500) -0.3000 (P=0.6833) coiled tail -0.1026 (P=0.9500) 0.0000 (P=1.0500) -0.6669 (P=0.2333) -0.9000 (P=0.0833) bent tail 0.05735(P=0.9500) -0.1118 (P=0.9500) -0.8603 (P=0.0833) -0.8944 (P=0.0833) irregular tail no data no data no data no data 2-tailed -0.3441 (P=0.5167) -0.8944 (P=0.0833) -0.2868 (P=0.6833) 0.1118 (P=0.9500) TZI 0.8721 (P=0.0833) -0.1000 (P=0.9500) -0.05130 (P=0.9500) -0.1000 (P=0.9500) SDI 0.6669 (P=0.2333) 0.1000 (P=0.9500) -0.2052 (P=0.7833) 0.1000 (P=0.9500) Table 3.14. Linear Correlation Analysis of Sperm Morphology and Multiple Chromosomal Abnormalities in Control Subjects (n=5) TZI = teratozoospermic index; SDI = sperm deformity index * = statistically significant correlation, P<0.05 rs values highlighted green indicate statistically significant positive correlation rs values highlighted red indicate statistically significant negative correlation n/a = not applicable (data not complete for all subjects) 87 Chromosome 13 Chromosome 18 Chromosome 21 Sex Chromosomesrs (P-value) D N T D N T D N T D N T Kruger’s Measurement 0.1046 (0.7736) 0.5231 (0.1231) 0.2954 (0.4073) -.03406 (0.9256) -0.2315 (0.5199) -0.2809 (0.4318) 0.07385 (0.8393) 0.1908 (0.5975) 0.2216 (0.5384) -.7078* (0.0220) -0.1785 (0.6218) -0.4568 (0.1844) normal 0.4182 (0.2291) 0.1030 (0.7850) 0.3091 (0.3848) 0.6646* (0.0360) 0.4985 (0.1425) 0.5714 (0.0844) -.06667 (0.8548) 0.3576 (0.3104) -0.1515 (0.6761) -.03030 (0.9338) 0.2848 (0.4250) 0.3161 (0.3736) small head -.05455 (0.8810) -0.4182 (0.2325) -0.2364 (0.5109) 0.2195 (0.5423) 0.4863 (0.1541) 0.5350 (0.1111) -0.1879 (0.6032) 0.04242 (0.9074) -0.3212 (0.3655) -0.1515 (0.6761) 0.4545 (0.1869) 0.1824 (0.6141) large head -0.3074 (0.3876) -.01882 (0.9730) -0.3199 (0.3675) -0.5586 (0.0933) -0.5254 (0.1188) -0.5820 (0.0775) -0.4140 (0.2342) 0.00627 (0.9863) -0.2698 (0.4510) 0.05646 (0.8769) -.8971*** (0.0004) -0.7394* (0.0145) vacuolated -0.4788 (0.1615) 0.1515 (0.6821) -0.3939 (0.2600) -0.6159 (0.0580) -0.2796 (0.4339) -0.4012 (0.2505) -.01818 (0.9602) 0.1030 (0.7770) 0.1879 (0.6032) 0.2485 (0.4888) -0.6485* (0.0425) -0.3587 (0.3088) acrosomal abnormalities -.00606 (0.9867) -0.4667 (0.1786) -0.2000 (0.5796) 0.2622 (0.4643) 0.4255 (0.2202) 0.4377 (0.2058) -0.3455 (0.3282) -0.1273 (0.7261) -0.5030 (0.1383) -0.1394 (0.7009) 0.4182 (0.2291) 0.1337 (0.7126) cytoplasmic droplet -0.2675 (0.4550) 0.4742 (0.1663) -.09726 (0.7892) -0.1713 (0.6362) 0.06707 (0.8539) 0.03659 (0.9201) 0.1763 (0.6261) 0.5471 (0.1017) 0.4499 (0.1921) -0.2492 (0.4874) -0.2492 (0.4874) -0.1220 (0.7372) round head 0.2242 (0.5334) -0.1879 (0.6073) 0.05455 (0.8810) 0.4512 (0.1905) 0.07903 (0.8282) 0.3343 (0.3450) -0.1152 (0.7514) -.09091 (0.8028) -0.3212 (0.3655) -.06667 (0.8548) 0.1515 (0.6761) 0.1398 (0.7001) thin head 0.3294 (0.3526) 0.06335 (0.8651) 0.3041 (0.3930) 0.3761 (0.2842) 0.05401 (0.8822) 0.1239 (0.7331) 0.3231 (0.3625) -0.2661 (0.4574) 0.1140 (0.7538) 0.3548 (0.3144) 0.2154 (0.5501) 0.4448 (0.1977) pyriform head - 0.04242 (0.9074) 0.1273 (0.7330) 0.1030 (0.7770) -0.2378 (0.5082) -0.1216 (0.7379) -0.2675 (0.4550) 0.2000 (0.5796) 0.04242 (0.9074) 0.3212 (0.3655) 0.2485 (0.4888) -0.1152 (0.7514) 0.09119 (0.8022) tapered head 0.1520 (0.6751) -0.4195 (0.2325) 0.0000 (1.0000) 0.2844 (0.4258) 0.02744 (0.9400) 0.1860 (0.6070) -0.2979 (0.4032) -.08511 (0.8152) -0.4559 (0.1854) -.06687 (0.8544) 0.04255 (0.9071) 0.05183 (0.8869) amorphous head 0.3891 (0.2665) -.04863 (0.8916) 0.2796 (0.4339) 0.6391* (0.0466) 0.3384 (0.3388) 0.5274 (0.1172) 0.2979 (0.4032) -0.1033 (0.7763) 0.07903 (0.8282) 0.5106 (0.1315) 0.5471 (0.1017) 0.8049** (0.0050) bent neck 0.6242 (0.0537) -.07879 (0.8382) 0.5030 (0.1383) 0.3598 (0.3072) -0.3161 (0.3736) -.08511 (0.8152) 0.1394 (0.7009) -0.2485 (0.4888) -0.1515 (0.6761) -.00606 (0.9867) 0.09091 (0.8028) -.03647 (0.9203) asymmetric neck insertion 0.1636 (0.6515) -.05455 (0.8916) 0.1394 (0.7009) 0.4634 (0.1774) 0.3708 (0.2915) 0.3951 (0.2584) -0.1758 (0.6272) 0.2485 (0.4888) -0.1636 (0.6515) 0.01818 (0.9602) 0.1636 (0.6515) 0.3647 (0.3001) thin midpiece 0.06920 (0.8493) 0.4152 (0.2325) 0.2249 (0.5322) 0.4700 (0.1705) 0.1388 (0.7021) 0.2343 (0.5148) 0.3027 (0.3952) -0.1470 (0.6852) 0.3027 (0.3952) -0.1816 (0.6155) 0.3546 (0.3146) 0.4902 (0.1503) thick midpiece 0.2970 (0.4047) -.06667 (0.8651) 0.3091 (0.3848) 0.5427 (0.1050) -.08511 (0.8152) 0.02432 (0.9468) -0.2121 (0.5563) -0.4303 (0.2145) -0.3333 (0.3466) -.06667 (0.8548) 0.2000 (0.5796) 0.3100 (0.3833) coiled tail -0.5152 (0.1276) -.7333* (0.0202) -.6364* (0.0479) -0.5000 (0.1411) -.01824 (0.9601) -0.1155 (0.7507) -0.6121 (0.0600) -.09091 (0.8028) -0.5394 (0.1076) 0.05455 (0.8810) -0.2121 (0.5563) -0.2796 (0.4339) bent tail .7939**(0.0061) -.05455 (0.8916) 0.6606* (0.0376) 0.4573 (0.1839) -0.2067 (0.5667) 0.0000 (1.0000) 0.2606 (0.4671) -0.2242 (0.5334) -.05455 (0.8810) 0.1879 (0.6032) 0.3091 (0.3848) 0.1641 (0.6505) irregular tail 0.5189 (0.1243) 0.05462 (0.8916) 0.5189 (0.1243) 0.2679 (0.4542) 0.1027 (0.7776) 0.06164 (0.8657) 0.3755 (0.2849) -0.1297 (0.7209) 0.1775 (0.6237) -.09559 (0.7928) 0.5053 (0.1363) 0.1815 (0.6158) 2-tailed .8193**(0.0037) 0.3277 (0.3487) .8330** (0.0028) 0.4946 (0.1461) -0.2534 (0.4799) -.08218 (0.8214) 0.5257 (0.1186) -.08876 (0.8074) 0.3004 (0.3990) 0.2185 (0.5442) 0.1639 (0.6510) 0.2637 (0.4617) TZI -0.1636 (0.6515) -.7212* (0.0234) -0.4424 (0.2004) -0.2317 (0.5195) 0.4012 (0.2505) 0.3404 (0.3358) -0.1879 (0.6032) 0.07879 (0.8287) -0.3333 (0.3466) 0.3697 (0.2931) 0.2242 (0.5334) 0.04863 (0.8939) SDI -0.2000 (0.5796) -0.5394 (0.1139) -0.3576 (0.3104) -0.3537 (0.3161) 0.1641 (0.6505) 0.1277 (0.7253) 0.05455 (0.8810) -.05455 (0.8810) -.04242 (0.9074) 0.3455 (0.3282) 0.2485 (0.4888) 0.08511 (0.8152) Table 3.15. Linear Correlation Analysis of Sperm Morphology and Aneuploidy in Teratozoospermic Subjects (n=10) D = disomy; N = nullisomy; T = total aneuploidy (disomy and nullisomy combined) TZI = teratozoospermic index; SDI = sperm deformity index * statistically significant correlation, P<0.05 ** statistically significant correlation, P<0.01 *** statistically significant correlation, P<0.001 rs values highlighted green indicate statistically significant positive correlation rs values highlighted red indicate statistically significant negative correlation 88 rs (P-value) Total Chromosomal Abnormality Total Aneuploidy Supernumerary Chromosomal Abnormality Diploidy Kruger’s Measurement -0.1354 (P=0.7092) -0.1416 (P=0.6965) 0.02161 (P=0.9528) 0.2469 (P=0.4917) normal 0.03030 (P=0.9338) 0.1030 (P=0.7770) 0.2067 (P=0.5667) 0.6647* (P=0.0360) small head -0.1515 (P=0.6761) -0.1879 (P=0.6032) -0.2432 (P=0.4984) 0.2708 (P=0.4492) large head -0.4454 (P=0.1970) -0.3952 (P=0.2583) -0.3996 (P=0.2526) -0.3791 (P=0.2800) vacuolated -0.06667 (P=0.8548) -0.01818 (P=0.9602) -0.1581 (P=0.6628) -0.6893* (P=0.0274) acrosomal abnormalities -0.3455 (P=0.3282) -0.3697 (P=0.2931) -0.3161 (P=0.3736) 0.3262 (P=0.3577) cytoplasmic droplet 0.2310 (P=0.5208) 0.2553 (P=0.4765) 0.06098 (P=0.8671) -0.09569 (P=0.7926) round head 0.006061(P=0.9867) 0.07879 (P=0.8287) 0.1277 (P=0.7253) 0.3508 (P=0.3203) thin head 0.2661 (P=0.4574) 0.3738 (P=0.2873) 0.5624 (P=0.0906) -0.08042 (P=0.8252) pyriform head 0.2242 (P=0.5334) 0.1879 (P=0.6032) 0.1459 (P=0.6876) -0.2893 (P=0.4176) tapered head -0.09726 (P=0.7892) -0.07295 (P=0.8413) -0.02744 (P=0.9400) 0.3025 (P=0.3956) amorphous head 0.4924 (P=0.1482) 0.5471 (P=0.1017) 0.5213 (P=0.1223) 0.2531 (P=0.4805) bent neck 0.1030 (P=0.7770) 0.1152 (P=0.7514) 0.3283 (P=0.3544) 0.3570 (P=0.3113) asymmetric neck insertion 0.07879 (P=0.8287) 0.1273 (P=0.7261) 0.1398 (P=0.7001) 0.4308 (P=0.2139) thin midpiece 0.3200 (P=0.3673) 0.4152 (P=0.2328) 0.4946 (P=0.1462) 0.1230 (P=0.7350) thick midpiece -0.06667 (P=0.8548) -0.006061(P=0.9867) 0.2188 (P=0.5436) 0.4062 (P=0.2441) coiled tail -0.4182 (P=0.2291) -0.5273 (P=0.1173) -0.7356* (P=0.0153) -0.2277 (P=0.5269) bent tail 0.2364 (P=0.5109) 0.2000 (P=0.5796) 0.3891 (P=0.2665) 0.4801 (P=0.1603) irregular tail 0.1297 (P=0.7209) 0.06828 (P=0.8513) 0.3082 (P=0.3863) 0.2600 (P=0.4681) 2-tailed 0.4780 (P=0.1624) 0.5189 (P=0.1243) 0.7534* (P=0.0119) 0.3224 (P=0.3636) TZI -0.1636 (P=0.6515) -0.2606 (P=0.4671) -0.4377 (P=0.2058) -0.1477 (P=0.6838) SDI 0.06667 (P=0.8548) -0.06667 (P=0.8548) -0.2979 (P=0.4032) -0.3077 (P=0.3870) Table 3.16. Linear Correlation Analysis of Sperm Morphology and Multiple Chromosomal Abnormalities in Teratozoospermic Subjects (n=10) TZI = teratozoospermic index; SDI = sperm deformity index * statistically significant correlation, P<0.05 rs values highlighted green indicate statistically significant positive correlation rs values highlighted red indicate statistically significant negative correlation 89 DNA Fragmentation rs (P-value) Control Subjects (n=7) Teratozoospeermic Subjects (n=10) normal -0.1429 (P=0.7825) -0.3697 (P=0.2957) small head -0.07143 (P=0.9063) 0.5046 (P=0.1440) medium head -0.2143 (P=0.6615) -0.07879 (P=0.8382) large head 0.6487 (P=0.1389) -0.1763 (P=0.6321) total internal head abnormalities -0.1071 (P=0.8397) 0.1758 (P=0.6321) vacuolated -0.1786 (P=0.7131) 0.07903 (P=0.8382) acrosomal abnormalities 0.3214 (P=0.4976) 0.2848 (P=0.4271) total head shape abnormalities -0.07143 (P=0.9063) -0.03030 (P=0.9460) round head 0.0000 (P=1.0365) 0.6727* (P=0.0390) thin head -0.8018* (P=0.0341) -0.4643 (P=0.1786) pyriform head -0.5000 (P=0.2667) -0.3818 (P=0.2788) tapered head -0.2857 (P=0.5560) 0.3564 (P=0.3129) amorphous head 0.6429 (P=0.1389) -0.4303 (P=0.2182) cytoplasmic droplet 0.6429 (P=0.1389) 0.1875 (P=0.6073) total neck/midpiece abnormalities 0.4286 (P=0.3536) 0.06667 (P=0.8651) asymmetric neck insertion 0.1786 (P=0.7131) 0.1124 (P=0.7589) bent neck 0.8571* (P=0.0238) -0.2796 (P=0.4271) thin midpiece no data -0.2901 (P=0.4069) thick midpiece 0.1071 (P=0.8397) 0.3212 (P=0.3679) total tail abnormalities -0.1786 (P=0.7131) -0.05455 (P=0.8916) coiled tail -0.1786 (P=0.7131) -0.04242 (P=0.9184) bent tail 0.05406 (P=0.9063) -0.4064 (P=0.2475) irregular tail -0.08909 (P=0.8397) no data 2-tailed 0.2364 (P=0.5948) -0.5237 (P=0.1231) teratozoospermic index 0.03571 (P=0.9635) 0.05455 (P=0.8916) sperm deformity index -0.07143 (P=0.9063) 0.2242 (P=0.5367) * statistically significant correlation, P<0.05 rs values highlighted green indicate statistically significant positive correlation rs values highlighted red indicate statistically significant negative correlation Table 3.17. Linear Correlation Analysis of Sperm Morphology and DNA Fragmentation 90 FIGURES Figure 1.1 Spermatogenesis Figure 1.2 Meiosis in Male Germ Cells Meiosis I Meiosis II Mitosis Type A Spermatogonia (2n) Intermediate Type A Spermatogonia (2n) Type B Spermatogonia (2n) Primary Spermatocytes (2n) Secondary Spermatocytes (n) Spermatids (n) Meiosis I Meiosis II Type B Spermatogonia (2n) Primary Spermatocyte (2n) Secondary Spermatocytes (n) Spermatids (n) Prophase I Metaphase I leptotene zygotene pachytene diplotene Anaphase I Metaphase II Anaphase II 91 Figure 1.3 Male Germ Cell Maturation Across Seminiferous Epithelium Sertoli cell Sertoli cell Tight junctions Type A Spermatogonia Intermediate Type A Spermatogonia Type B Spermatogonia Primary Spermatocytes Secondary Spermatocytes Spermatids Mature Spermatozoa basal lamina tubule lumen 92 Figure 2.1 Morphologically Normal Sperm Figure 2.2 Sperm with Abnormal Head Morphology 5µm a b c d e f g h Hemacolor stained sperm with characteristically normal morphology Hemacolor stained sperm. a) slightly tapered sperm head with bent neck and small cytoplasmic droplet; b) pyriform sperm head; c) small, round sperm head with no acrosome and thick midpiece; d) round sperm head with normal acrosome area; e) amorphous sperm head with no acrosome; f) amorphous sperm head (small posterior indentation); g) amorphous sperm head with coiled tail; h) amorphous sperm head with abnormally large amount of vacuoles. 93 Figure 2.3 Sperm with Abnormal Neck, Midpiece, or Tail Abnormalities a gf ed c b h Hemacolor stained sperm. a) asymmetric neck insertion with small acrosome area; b) bent neck; c) thick midpiece; d) cytoplasmic droplet with amorphous sperm head; e) thin midpiece; f) bent tail; g) 2-tailed sperm; h) coiled sperm tail. 94 Figure 2.4 FISH of Chromosomes 18, X, and Y Probe Set on Sperm Figure 2.5 FISH of Chromosomes 13 and 21 Probe Set on Sperm 18,18,XXY 18,18,XY 18,X 18,Y a c d b 18,XY 13,21 13,0 0,21 13,13,21 a b DAPI counterstained sperm with FISH probes labelling centromeres of chromosomes 18 (aqua colored arrows), X (green colored arrows), and Y (red colored arrows): a) euploid (normal haploid) sperm: one sperm with one chromosome 18 and one Y chromosome, one sperm with one chromosome 18 and one X chromosome; b) aneuploid sperm with one chromosome 18 and two sex chromosomes (X and Y); c) sperm with supernumerary chromosomal abnormality (numerical chromosomal abnormalities involving multiple chromosomes); d) diploid sperm (two autosomes with two sex chromosomes) DAPI counterstained sperm with FISH probes labelling centromeres of chromosomes 13 (green colored arrows) and 21 (red colored arrows): a) one euploid sperm (one chromosome 13 and one chromosome 21), one sperm missing chromosome 13, one sperm missing chromosome 21; b) aneuploid sperm with one chromosome 21 and two chromosome 13’s 95 Figure 2.6 DNA Fragmentation by TUNEL Assay TUNEL labeling of fragmented DNA with fluorescein dUTP in one sperm (green arrow) and not in the other sperm. 96 BIBLIOGRAPHY Affara NA, Mitchell MJ (2000) The role of human and mouse Y chromosome genes in male infertility J of Endocrin Invest 23, 630–645 Aitken RJ (1999) The Amoroso Lecture. 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Thesis/Dissertation
eng
Reproductive and Developmental Sciences
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Aneuploidy and DNA fragmentation in morphologically abnormal sperm
Text