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Abstract

Volume 11, Issue 6 (November 2009) 11, 703–709; 10.1038/aja.2009.65

Quick recovery and characterization of cell-free DNA in seminal plasma of normozoospermia and azoospermia: implications for non-invasive genetic utilities

Hong-Gang Li, Shi-Yun Huang, Hui Zhou, Ai-Hua Liao, Cheng-Liang Xiong

Family Planning Research Institute and Centre for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

Correspondence: Prof. Ai-Hua Liao, aihualiao@hotmail.com

Received 12 June 2009; Revised 26 July 2009; Accepted 7 September 2009; Published online 12 October 2009.

Abstract

We established a quick and reliable method for recovering cell-free seminal DNA (cfsDNA), by using the binding-washing-elution procedure on the DNA purification column. Low variations (below 15%) among the triplicate values of cfsDNA quantity verified the reproducibility of our cfsDNA recovery method. Similar cfsDNA yield and size distribution between seminal plasma acquired by filtration and centrifugation confirmed the presence of cfsDNA. To investigate the general characterization of cfsDNA, the quantitation and size distribution of cfsDNA from normozoospermic and azoospermic semen were analyzed by real-time PCR and electrophoresis, respectively. CfsDNA concentration in semen with normozoospermia (n = 11) was 1.34 ±0.65 μg mL-1, whereas a higher cfsDNA concentration was observed in azoospermia (2.56 ± 1.43 μg mL-1, n = 9). The continuous distribution of DNA fragments ranging from ~1 kb to 15 kb and a spectrum of multiples of 180-bp fragments were observed in each normozoospermic and azoospermic sample. Distinct characteristic DNA ladder fragmentations in some azoospermic samples implicated that cfsDNA originate partly from apoptotic cells. CfsDNAs of 36 selected azoospermic patients with known information of Y chromosome microdeletion were subjected to the same microdeletion analysis by multiplex PCR and PCR amplification of sY114 (1 450 bp). All multiplex PCR reactions with cfsDNA amplified successfully and provided the same result as leukocyte DNA. PCR amplification of sY114 gave a 1 450-bp amplicon as expected. Our data suggested the potential use of cfsDNA in search of biomarker or diagnostic procedures.

Keywords: azoospermia, cell-free DNA, normozoospermia, seminal plasma, Y chromosome microdeletion

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