Choosing the right sperm for ART: gender and viability selection of spermatozoas by flow cytometry

Abstract

Objective: Currently the method to choose the spermatozooa to use for ICSI is still a blind manoeuvre, and it can explain failures of fertilization or to allow the transmision of a genetic disease to the offspring due to the selection of a wrong sperm. The aim of this study was to determine the spermatozoa gender and its viability by MicroSort of them with flow cytometry, to enrich the sample by sex and normal spermatozoas, and test the utility of this method to choose the right sperm to use in ART.Design: Prospective and experimental study.Materials/Methods: We analyse by FISH 10 ejaculates samples from sperm donnors. The probes used were: X, Y, 18, and 21, to determine the genetic status of spermatozoas. After that we sorted the remaining sample in a flow cytometry (FACS Vantage, Becton Dickinson, CA, USA). The spermatozoas were stained with the vital fluorochrome bisbenzimide (Hoeschst, Behring, CA, USA), then the sample was passed through the cytometer and sorted for X-bearing spermatozoa and Y-bearing spermatozoa, and chromosomically abnormal, separed and enriched electronically to the diferents fractions. An alliquote of each fraction was again reanalyze by FISH, to check the precision of cytometer procedure. After that the X and Y fractions were stained with fluoresceine diacetate and MicroSorted and enriched by viability.Results: The enrichment by flow cytometry for X, Y and abnormal spermatozoa was almost 90% per group. The viability of spermatozoas in groups X and Y was 95% and 63% in the abnormal group (p <0.05). This sorting and enrichment was confirmed by the second round of FISH.Conclusions: The possibility to diagnose these alterations by FISH of spermatozoa, and further MicroSort by flow cytometry and enrich normal spermatozoas, could avoid the posibility of transmition of some genetic pathology to the offspring and maybe improve the results of some Assisted Reproductive Techniques and sex selection. The flow cytometry is an interesting new technique in human biology of reproduction, that probably will change the current way to prepare the seminal fluid for ART, and probably how to choose the rigth spermatozoa for ICSI procedure. However the safety of these procedures needs to be tested before to use in human clinical practice.Supported by: Reproduccion y Genetica, Hospital Angeles del Pedregal, Universidad La Salle, Biotechnology & Bioengineering Department of Center for Research and Advanced Studies - National Polytechnic Institute (CINVESTAV-IPN), Mexico City. Objective: Currently the method to choose the spermatozooa to use for ICSI is still a blind manoeuvre, and it can explain failures of fertilization or to allow the transmision of a genetic disease to the offspring due to the selection of a wrong sperm. The aim of this study was to determine the spermatozoa gender and its viability by MicroSort of them with flow cytometry, to enrich the sample by sex and normal spermatozoas, and test the utility of this method to choose the right sperm to use in ART. Design: Prospective and experimental study. Materials/Methods: We analyse by FISH 10 ejaculates samples from sperm donnors. The probes used were: X, Y, 18, and 21, to determine the genetic status of spermatozoas. After that we sorted the remaining sample in a flow cytometry (FACS Vantage, Becton Dickinson, CA, USA). The spermatozoas were stained with the vital fluorochrome bisbenzimide (Hoeschst, Behring, CA, USA), then the sample was passed through the cytometer and sorted for X-bearing spermatozoa and Y-bearing spermatozoa, and chromosomically abnormal, separed and enriched electronically to the diferents fractions. An alliquote of each fraction was again reanalyze by FISH, to check the precision of cytometer procedure. After that the X and Y fractions were stained with fluoresceine diacetate and MicroSorted and enriched by viability. Results: The enrichment by flow cytometry for X, Y and abnormal spermatozoa was almost 90% per group. The viability of spermatozoas in groups X and Y was 95% and 63% in the abnormal group (p <0.05). This sorting and enrichment was confirmed by the second round of FISH. Conclusions: The possibility to diagnose these alterations by FISH of spermatozoa, and further MicroSort by flow cytometry and enrich normal spermatozoas, could avoid the posibility of transmition of some genetic pathology to the offspring and maybe improve the results of some Assisted Reproductive Techniques and sex selection. The flow cytometry is an interesting new technique in human biology of reproduction, that probably will change the current way to prepare the seminal fluid for ART, and probably how to choose the rigth spermatozoa for ICSI procedure. However the safety of these procedures needs to be tested before to use in human clinical practice. Supported by: Reproduccion y Genetica, Hospital Angeles del Pedregal, Universidad La Salle, Biotechnology & Bioengineering Department of Center for Research and Advanced Studies - National Polytechnic Institute (CINVESTAV-IPN), Mexico City.