Gender selection: separation techniques for X- and Y-chromosome bearing human spermatozoa

Van Der Linde, Michelle (2013-12)

Thesis (MScMedSc)-- Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Preconceptual sex selection is an ethically justifiable process whereby X- and Y-chromosome bearing spermatozoa are isolated prior to fertilization of the oocyte in order to generate either a male or a female offspring. Although various separation techniques are available, none can guarantee 100% accuracy. There are various physiological differences between X- and Y-chromosome bearing spermatozoa which can be used to separate these two populations of sperm. For the purpose of this study, X- and Y-chromosome bearing spermatozoa were separated based on (1) their respective abilities to remain viable when subjected to adverse environments, including extreme pH values, increased temperatures and various hydrogen peroxide (H2O2) concentrations; (2) the ability of Y-chromosome bearing spermatozoa to swim faster and/or more progressively than X-chromosome bearing spermatozoa; and (3) the X-chromosome bearing spermatozoa’s increased size and weight when compared to the Y-chromosome bearing spermatozoa. The efficacy of live and dead cell separation through (i) Magnetic Antibody Cell Separation (MACS) and (ii) a modified swim-up technique was also assessed and compared. Changes in the sex-chromosome ratio of samples were established by double-label fluorescent in situ hybridization (FISH) before and after processing. Sperm motility (CASA) and viability (eosin/nigrosin) was assessed before and after each intervention. Ethical clearance for this study was granted by the Health Research Ethics Committee 1 (Ethics #: S13/04/068). The results indicated successful enrichment of X-chromosome bearing spermatozoa upon incubation in acidic media, increased temperatures, and H2O2. In contrast, Y-chromosome bearing spermatozoa were successfully enriched through a direct swim-up method as well as discontinuous gradient centrifugation. In conclusion, this study demonstrated the potential role for physiological differences between X- and Y-chromosome bearing spermatozoa in the development of preconceptual gender selection through sperm sorting.

AFRIKAANSE OPSOMMING: Prekonsepsie geslagselektering is 'n eties regverdigbare proses waardeur X- en Y- chromosoom draende spermatosoë geïsoleer word voordat bevrugting van die oösiet plaasvind, om óf 'n manlike óf 'n vroulike nageslag te genereer. Alhoewel verskeie skeidingstegnieke beskikbaar is, kan geeneen 100% akkuraatheid waarborg nie. Daar bestaan verskeie fisiologiese verskille tussen X- en Y- chromosoom draende spermatosoë wat skeiding van hierdie twee groepe spermatosoë moontlik kan maak. Vir die doel van hierdie studie is skeidingsmetodes vir die X- en Y- chromosoom draede spermatosoë gebaseer op (1) hul onderskeie vermoëns om lewensvatbaar te bly tydens blootstelling aan ‘n ongunstige milieu, insluitend ekstreme pH waardes, verhoogde temperature en verskeie waterstofperoksied (H2O2) konsentrasies; (2) die vermoë van die Y-chromosoom draende spermatosoon om vinniger en/of meer progressief as X-chromosoom draende spermatosoë te swem; en (3 ) die X-chromosoom draende spermatosoon se verhoogde grootte en gewig in vergelyking met die Y- chromosoom draende spermatosoon. Die effektiwiteit van die (i) Magnetiese Anti-liggaam Sel Skeidingstegniek (MACS) en (ii) 'n aangepaste weergawe van die op-swem tegniek om lewendige en dooie selle te skei is ook bepaal en vergelyk. Veranderinge in die geslagschromosoom verhouding van die monsters is bepaal deur dubbel-etiket fluoresensie in situ hibridisering (FISH) voor en na verwerking. Spermmotiliteit (CASA) en lewensvatbaarheid (eosien/nigrosin) is bepaal voor en na elke intervensie. Etiese goedkeuring vir hierdie studie is verleen deur die Gesondheids-Navorsingsetiekkomitee 1 (Etiese # : S13/04/068). Die resultate dui suksesvolle verryking van X-chromosoom draende spermatosoë deur inkubasie in suur media, verhoogde temperature, en H2O2. Y-chromosoom draende spermatosoë is verryk deur middel van 'n direkte op-swem metode sowel as diskontinue gradiënt sentrifugering . Ten slotte, hierdie studie toon die potensiële rol vir fisiologiese verskille tussen X- en Y- chromosoom draende spermatosoë in die ontwikkeling van prekonsepsie geslagselektering metodes deur skeiding van X- en Y-chromosoom draende sperme.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/85629
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