Y Chromosome Microdeletion Detected By Using Multi-analyte Suspension Array And Its Applications In Clinical Diagnosis

Objective Infertility is defined as an inability to conceive or produce an offspring. It is affecting about 15% of all the couples attempting to generate pregnancy. In the case of infertility approximately 50% of the cases can be attributed to male factors. Because of the increasing interest and popularization of Y-chromosome STS scanning as a routine analysis in the work-up of the infertile male, the need for technologies that allow rapid, cost-effective, high-throughput detection of specific nucleic acid sequences becomes apparent. Currently, the most common method is based on PCR multiplex and conventional agarose electrophoresis. But it is possible to cause the laboratory contaminant and the presence of false positive and false negative. Here we present a novel and rapid method to scan AZF region microdeletion of Y chromosome using multi-analyte suspension array technology. By using multi-analyte suspension array technology to compare the patients idiopathic azoospermia and oligozoospermia and normal fertility to validate the feasibility of the methods, and also to confirm men the microdeletion rate of these patients. It is studied the relations between azoospermia factor (AZF) with the patients and the distribution rule of microdeletions of AZF in these patients.Methods Primers covering only hot spot regions were chosen according to previous reports. A series of five STSs from AZF region on the long arm of Y chromosome were used to detect microdeletions. These included sY84 for AZFa, sY127/ sY134 for AZFb, s254/sY255 for AZFc. In addition, testing for the Yp was performed using sY14, an STS located within the gene SRY and testing for the 12 chromosome was GAPDH(a house keeper gene) (internal positive control). According to the sequence data from GeneBank Database, the special PCR primers and probes were designed and synthesized. PCR system and hybridization have been optimized. Fertile male and female samples were used as positive and negative controls. Seven pairs of primers were amplified in a single reaction. Screening for AZF microdeletion by multiplex PCR- multianalyte suspension array technology was performed in a total of 312 patients including 178 infertile patients with azoospermia and 134 infertile patients with oligozoospermia who had normal karyotype as well as 40 fertile man controls.Results 1. Established seven multiplex PCR system with sY14 (SRY), sY84, sY127, sY134, sY254, sY255, GAPDH.2. Established seven multiplex PCR - multianalyte suspension array system. The repeatability of the system had been studied. The range of CV% was between 2.2%～7.8%.3. 40(12.8%) of 312 patients were found to have deletions in AZF region. The microdeletion frequency was 14%(25/178) in the azoospermic group and 11.2%(15/134) in the oligospermic group. Among 40 patients with microdeletions, 19 patients had deletions in AZFc region(Figurel), 13 had deletions in AZFa and 4 had deletions in AZFb. In addition, 4 patients had both AZFb and AZFc deletions.4. No microdeletion in AZF region was found in fertile controls. There were significient difference after statistics compare(X~2 = 5.788, P<0.05).Conclusion 1. We developed a high-throughput multiplex, fast and simple assay to scan AZF region microdeletions of Y chromosome. 2. The distributions of Y chromosome microdeletions have relations with idiopathic azoospermia and oligozoospermia patients. 40(12.8%) patients were found to have deletions in AZF region. The microdeletion frequency was 14% in the azoospermic group, 11.2% in the oligospermic group.