Quantitative Fluorescent-PCR Detection of Sex Chromosome Aneuploidies and AZF Deletions/Duplications

Abstract

The most common genetic causes of spermatogenic failure are sex chromosomal abnormalities (most frequently Klinefelter's syndrome) and deletions of the azoospermia factor (AZF) regions (AZFa, AZFb, and AZFc) of the Y chromosome. Several studies have proposed that partial AZFc deletions/duplications may be a risk factor for spermatogenic impairment. We describe a multiplex quantitative fluorescent-polymerase chain reaction (QF-PCR) method that allows simultaneous detection of these genetic causes and risk factors of male infertility. The 11-plex QF-PCR permitted the amplification of the amelogenin gene, four polymorphic X-specific short tandem repeat (STR) markers (XHPRT, DXS6803, DXS981, and exon 1 of the androgen receptor gene), nonpolymorphic Y-specific marker (SRY gene), polymorphic Y-specific STR marker (DYS448), and coamplification of DAZ/DAZL, MYPT2Y/MYPT2, and two CDY2/CDY1 fragments that allow for determination of the DAZ, MYPT2Y, and CDY gene copy number. A total of 357 DNA samples from infertile/subfertile men (n = 205) and fertile controls (n = 152) was studied. We detected 14 infertile males with sex chromosome aneuploidy (10 with Klinefelter's syndrome, 2 XX, and 2 XYY males). All previously detected AZF deletions, that is, AZFc (n8), AZFb (n1), AZFb + c (n1), gr/gr (n11), gr/gr with b2/b4 duplication (n3), and b2/b3 (n5), gave a specific pattern with the 11-plex QF-PCR. In addition, 32 DNA samples showed a pattern consistent with presence of gr/gr or b2/b4 and 4 with b2/b3 duplication. We conclude that multiplex QF-PCR is a rapid, simple, reliable, and inexpensive method that can be used as a first-step genetic analysis in infertile/subfertile patients.