Toward pre-conceptual genetic analysis of human spermatozoa.
Nuclei of mature mammalian spermatozoa are extraordinarily resistant to chemical and thermal injury. Additionally, decondensation of spermatozoa DNA can be accompanied by little or no visual changes of the sperm head. This study tested whether human spermatozoa could be recovered following several cycles of primer extension preamplification (PEP) and used to achieve fertilization and subsequent development of human oocytes. An attempt was also made to amplify PEP buffer after spermatozoon removal. The results demonstrate that the sperm head can be successfully recovered following treatment with KOH or proteinase K followed by one to four cycles of PEP. It is also shown that following this treatment, the spermatozoa can be injected into the oocytes and will transform into a pronucleus if the oocyte is activated by sperm cytosolic fraction. In some cases, it was also possible to obtain polymerase chain reaction signals using a buffer after sperm cells were removed following several cycles of PEP. Although sperm participation in development was confirmed by fluorescence in-situ hybridization, light microscopy revealed some degree of damage to spermatozoal chromosomes. It is concluded that pre-conceptual analysis of sperm cells may be possible, but more research is necessary to determine the optimal conditions that would preserve sperm DNA integrity while allowing accurate diagnoses.
Dozortsev
Dmitri
D
Clínica e Centro de Pesquisa em Reprodução Humana Roger Abdelmassih, São Paulo, Brazil 01433-000. dmitrid@sbcglobal.net
Serafim
Rui
R
Cardoso
J Jakson
JJ
Abdelmassih
Soraya
S
Nagy
Peter
P
Diamond
Michael P
MP
Abdelmassih
Roger
R
D002467:Cell Nucleus / Q000378:metabolism; D002875:Chromosomes / Q000648:ultrastructure; D004247:DNA / Q000378:metabolism; D019286:Endopeptidase K / Q000494:pharmacology; D005260:Female; D005306:Fertilization; D005307:Fertilization in Vitro; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007248:Infertility, Male / Q000628:therapy*; D008297:Male; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D019836:Preimplantation Diagnosis; D013077:Sperm Head; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000378:metabolism / Q000502:physiology*; D013696:Temperature; D013997:Time Factors
D004247:DNA; D019286:Endopeptidase K