Defining regions of the Y-chromosome responsible for male infertility and identification of a fourth AZF region (AZFd) by Y-chromosome microdeletion detection

Cytogenetic and molecular deletion analyses of azoospermic and oligozoosper mic males have suggested the existence of AZoospermia Factor(s) (AZF) resid ing in deletion intervals 5 and 6 of the human Y-chromosome and coinciding with three functional regions associated with spermatogenic failure. Nonpol ymorphic microdeletions in AZF are associated with a broad spectrum of test icular phenotypes. Unfortunately, Sequence Tagged Sites (STSs) employed in screening protocols range broadly in number and mapsite and may be polymorp hic. To thoroughly analyze the AZF region(s) and any correlations that may be drawn between genotype and phenotype, we describe the design of nine mul tiplex PCR reactions derived from analysis of 136 loci. Each multiplex cont ains 4-8 STS primer pairs, amplifying a total of 48 Y-linked STSs. Each mul tiplex consists of one positive control: either SMCX or MIC2. We screened f our populations of males with these STSs. Population I consisted of 278 pat ients diagnosed as having significant male factor infertility: either azoos permia, severe oligozoospermia associated with hypogonadism and spermatogen ic arrest or normal sperm counts associated with abnormal sperm morphology. population II consisted of 200 unselected infertile patients. population I II consisted of 36 patients who had previously been shown to have aneuploid y, cytological deletions or translocations involving the Y-chromosome or no rmal karyotypes associated with severe phenotype abnormalities. Population IV consisted of 920 fertile (control) males. The deletion rates in populations I, II and III were 20.5%, 7% and 58.3%, r espectively. A total of 92 patients with deletions were detected. The delet ion rate in population IV was 0.87% involving 8 fertile individuals and 4 S TSs which were avoided in multiplex panel construction. The ability of the nine multiplexes to detect pathology associated microdeletions is equal to or greater than screening protocols used in other studies. Furthermore, the data suggest a fourth AZF region between AZFb and AZFc, which we have term ed AZFd. Patients with microdeletions restricted to AZFd may pre sent with mild oligozoospermia or even normal sperm counts associated with abnormal s perm morphology. Though a definitive genotype/phenotype correlation does no t exist, large deletions spanning multiple AZF regions or microdeletions re stricted to AZFa usually result in patients with Sertoli Cell Only (SCO) or severe oligozoospermia, whereas microdeletions restricted to AZFb or AZFc can result in patients with phenotypes which range from SCO to moderate oli gozoospermia. The panel of nine multiplexed reactions, the Y-deletion Detection System (Y DDS), provides a fast, efficient and accurate method of assessing the integ rity of the Y-chromosome. To date, this study provides the most extensive s creening of a proven fertile male population in tandem with 514 infertile m ales, derived from three different patient selection protocols. (C) 1999 Wi ley-Liss, Inc.