Application of modern molecular techniques to evaluate sperm sex selection methods.

The aim of this paper is to review modern approaches which have been used to evaluate sex pre-selection procedures. Two approaches can be used, polymerase chain reaction (PCR) and fluorescence in-situ hybridization (FISH). FISH is currently the method of choice for evaluating sex selection procedures because: (i) FISH accurately identifies the sex chromosome of individual spermatozoa using specific probes for the X and Y chromosomes and a two-colour detection system; and (ii) large numbers of spermatozoa can be screened in a short period of time. Of the published sex pre-selection methods tested using FISH, only flow cytometry has been shown to produce a clinically significant enrichment of X- and/or Y-bearing human spermatozoa. Studies have shown that 12-step Percoll gradients produce a slight but clinically insignificant enrichment of X-bearing spermatozoa, swim-up techniques do not appear to enrich either X- or Y-bearing spermatozoa, and discontinuous albumin gradients do not enrich Y-bearing spermatozoa. Despite this evidence, some of these methods continue to be used clinically, so it is vital that sex selection methods are properly evaluated using reliable methods such as double-label FISH before they are introduced for clinical use.