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

Abstract

Cytogenetic and molecular deletion analyses of azoospermic and oligozoospermic males have suggested the existence of AZoospermia Factor(s) (AZF) residing in deletion intervals 5 and 6 of the human Y-chromosome and coinciding with three functional regions associated with spermatogenic failure. Nonpolymorphic microdeletions in AZF are associated with a broad spectrum of testicular phenotypes. Unfortunately, Sequence Tagged Sites (STSs) employed in screening protocols range broadly in number and mapsite and may be polymorphic. To thoroughly analyze the AZF region(s) and any correlations that may be drawn between genotype and phenotype, we describe the design of nine multiplex PCR reactions derived from analysis of 136 loci. Each multiplex contains 4-8 STS primer pairs, amplifying a total of 48 Y-linked STSs. Each multiplex consists of one positive control: either SMCX or MIC2. We screened four populations of males with these STSs. Population I consisted of 278 patients diagnosed as having significant male factor infertility: either azoospermia, severe oligozoospermia associated with hypogonadism and spermatogenic arrest or normal sperm counts associated with abnormal sperm morphology. Population II consisted of 200 unselected infertile patients. Population III consisted of 36 patients who had previously been shown to have aneuploidy, cytological deletions or translocations involving the Y-chromosome or normal 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%, respectively. A total of 92 patients with deletions were detected. The deletion rate in population IV was 0.87% involving 8 fertile individuals and 4 STSs 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 termed AZFd. Patients with microdeletions restricted to AZFd may present with mild oligozoospermia or even normal sperm counts associated with abnormal sperm morphology. Though a definitive genotype/phenotype correlation does not exist, large deletions spanning multiple AZF regions or microdeletions restricted 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 oligozoospermia. The panel of nine multiplexed reactions, the Y-deletion Detection System (YDDS), provides a fast, efficient and accurate method of assessing the integrity of the Y-chromosome. To date, this study provides the most extensive screening of a proven fertile male population in tandem with 514 infertile males, derived from three different patient selection protocols.

Screening for Y microdeletions in men with testicular cancer and undescended testis

“…The prevalence of polymorphisms may vary between the different populations. This assumption has been supported by a very recent study, which found a single STS missing for sY84 in Asian fertile men (1/117) (12), even though this STS previously was suggested not to be polymorphic (5). Thus, the pathogenetic significance of these single missing STSs remains to be determined.…”

“…A high frequency of Y microdeletions has been reported (up to 55%) in men having oligozoospermia or azoospermia (5). An association between testicular cancer and male infertility has been studied and reported in many studies.…”

“…It is believed that microdeletions on the Y chromosome are the cause of 10-15% of cases with idiopathic azoospermia or severe oligozoospermia. However, higher frequencies were found when evaluating the most severe idiopathic primary testiculopathies (up to 55%) (5).…”

Multiplex Sequence-Tagged Site PCR for Efficient Screening of Microdeletions in Y Chromosome in Infertile Males with Azoospermia or Severe Oligozoospermia

“…The compositions of the STSs and the size of their base pairs for each of the multiplex primers are shown in Table 1. These STSs were derived from the map by Vollrath et al [22], Henegariu et al [4], Reijo et al [16], and Kent-First et al [6]. The primer pairs in a multiplex di ered at least 22 base pairs in length from each other to make the screening easier and more e cient.…”

Non-chimerism and chimerism pseudo dicentric Y chromosome: two case reports about azoospermia and cytogenetic/molecular genetic analysis in the Chinese population

“…In 1999, Kent-First et al divided AZF into three regions: AZFa, AZFb and AZFc. Among them, AZFb and AZFc possess an overlapping area of about 1.5 Mb, only the AZFa region is relatively independent [24][25][26]. Microdeletion in the AZF region is closely related to the male infertility, as it is one of the most common molecular genetic causes of azoospermia or severe oligozoospermia.…”