Abstract
BackgoundY-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers.Principal FindingsThe widely established “EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)” were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size.SignificanceWith these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection.
Highlights
In Western countries, infertility is a major health problem that affects about 15% of couples trying to conceive [1]
An obvious target are patients subjected to intracytoplasmic sperm injection (ICSI) treatment which present with severe oligozoospermia (,16106/mL) [10]
With the popularization of the polymerase chain reaction (PCR) methodology, rapid testing for the presence or absence of Y-chromosomal microdeletions (YCMD) by amplification of the respective AZF loci is achievable in matter of a few hours
Summary
In Western countries, infertility is a major health problem that affects about 15% of couples trying to conceive [1]. Y-chromosomal microdeletions (YCMD), along with Klinefelter syndrome, are the major genetic cause for primary spermatogenic failure on the male side, accounting for 5–10% of all non-obstructive azoospermic men [2]. The testicular phenotype in men bearing YCMD depends on the type of deletion, ranging from Sertoli cell-only syndrome (SCO) in AZFa [6,7], SCO to meiotic arrest in cases of complete AZFb/AZFb+c deletions [2], up to the very heterogeneous pattern from hypospermatogenesis to SCO in AZFc deletions [8,9]. There are no defined criteria as to when patients should undergo YCMD genetic testing. There are strong indications that an increased frequency of nullisomic sperm in the ejaculate of men with AZFc deletions might raise the potential risk of 45,X Turner’s syndrome [13]
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