Enrichment of X- and Y- bearing chromosome spermatozoa using simple centrifugation techniques and investigation of a potential prevalence of either of the two types of sex chromosome spermatozoa in certain human male populations
Creators: name: Koundouros, Savvas
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: The broad aim of this thesis was to exemplify simple centrifugation techniques being a useful laboratory tool towards enriching either of the two human spermatozoa populations. Further to these laboratory interventions the thesis endeavoured to investigate whether the existing prevalence observed in certain human male populations to produce progenies of only male or female offspring is a coincidental event or the result of potentially deranged mitotic mechanics during spermatogenesis. In the first part of the introduction section of the thesis the two categories of sex determination (environmental and genetic) are examined. Most of the emphasis is allocated to the various mechanisms of genetic sex determination since the environmental mode is less complex and species limited. A detailed reference of the genetic sex determination in invertebrates (hymenoptera, dipterans and nematodes) is described whereas a more elaborate interpretation of genetic sex determination in vertebrates is exemplified. An integral part of the latter is the mechanisms of gonadal development incorporating a complex interplay of genes and the architectural proteins, which they encode. The complex process of spermatogenesis in the human and its dependence on intratesticular and extratesticular hormonal regulatory processes is also reported. Finally a retrospective review of sex selection approaches and a compilation of already documented laboratory techniques towards enriching human male spermatozoa are outlined along with the aims of this thesis. Chapter 2 provides an elaborate description of the materials and methods used during the research studies. The selection of the donor candidates, the screening tests undertaken and the processing of the semen specimens from production to sex chromosome enumeration are depicted. Chapters 3 and 4 demonstrate the effective enrichment of Y- and X- bearing chromosome populations in human spermatozoa respectively using simple yet rigorous centrifugation techniques. During these studies a number of young and proven fertility donor candidates were recruited following sample size estimation. Furthermore, analysis of the prevalence of either of the two types of spermatozoa was made using triple layer fluorescent in situ hybridization in an attempt to reduce potential bias against the results of each of the techniques. The magnitude of the reactive centrifugal force, duration of centrifugal separation and the number of centrifugal rounds are considered critical in achieving optimal isolation. Reference is made to previously published reports and their efficiency and safety is reported. Chapter 5 examines whether the high incidence of same gender children in large families achieved by certain human male populations is a result of chance or due to potentially deranged meiotic mechanics leading to one of the two heteromorphic sex chromosomes being transmitted in progeny disproportionately to normal Mendelian inheritance. The prevalence of X- and Y-bearing chromosome spermatozoa in the semen of eighteen donors between 28 and 45 years of age with large families of either all female or all male offspring naturally conceived was compared with that of an equal number of subjects who had achieved large families of 4 or more children with an equal proportion of male and female progeny also naturally conceived. Fifty four fresh specimens (three from each donor) were examined for the experimental design. The first part of the final chapter of the thesis presents a backdate in the interest of preconception sex selection and a description of the methodologies published in the literature reporting successful X- or Y- bearing human sperm enrichment. Emphasis on the efficacy and safety of the methods in both animals and humans is noted. A detailed numeric analysis of the data demonstrating successful isolation and the importance of the rigorous centrifugation parameters employed are depicted. Furthermore reference is made to the value of the enrichment techniques with respect to reducing X-linked disease risk or to balance the sex distribution of children in families where such interventions are permitted. The findings of the final research chapter are also discussed rejecting the possibility of genetic factors favouring the unequal transmission of sex chromosomes in human males with large families of similar gender children. In essence, it was concluded that biased sex ratios among human male progenies cannot be attributed to genetic distortion. Rather the disproportionate progeny observed can only relate to coincidental events.