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Molecular and Cytogenetic Analysis of Infertile Hakka Men With

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Molecular and Cytogenetic Analysis of Infertile Hakka Men With Clinical Research Report Journal of International Medical Research 2019, Vol. 47(3) 1114–1123 Molecular and cytogenetic ! The Author(s) 2019 Article reuse guidelines: analysis of infertile Hakka sagepub.com/journals-permissions DOI: 10.1177/0300060518816253 men with azoospermia and journals.sagepub.com/home/imr severe oligozoospermia in southern China Pingsen Zhao1,2,3,4,5,6,* , Xiaodong Gu1,2,3,4,5,6,*, Heming Wu1,2,3,4,5,6 and Xunwei Deng1,2,3,4,5,6 Abstract Objective: To determine the prevalence of chromosome abnormalities and azoospermia factor (AZF) microdeletions in Hakka men with infertility in southern China. Methods: Hakka male patients, who received clinical counselling for infertility between August 2016 and October 2017, and fertile male controls, were enrolled into this retrospective study. Patients diagnosed with infertility and controls underwent cytogenetic analysis by standard G-banding; AZF microdeletions were examined by multiplex polymerase chain reaction and capillary electrophoresis. Results: Out of 918 male patients who received fertility counselling, 57 were diagnosed with infertility due to azoospermia or severe oligozoospermia. Of these infertile patients, 22.81% (13/ 57) carried chromosome abnormalities, with 47, XXY being the most common abnormal kar- yotype. In addition, 36.84% (21/57) presented with Y chromosome microdeletions, most 5Meizhou Municipal Engineering and Technology Research Centre for Molecular Diagnostics of Major Genetic 1Clinical Core Laboratory, Meizhou People’s Hospital Disorders, Meizhou, China (Huangtang Hospital), Meizhou Academy of Medical 6Guangdong Provincial Key Laboratory of Precision Sciences, Meizhou Hospital Affiliated to Sun Yat-sen Medicine and Clinical Translational Research of Hakka University, Meizhou, China Population, Meizhou, China 2Centre for Precision Medicine, Meizhou People’s Hospital (Huangtang Hospital), Meizhou Academy of *These authors contributed equally to this work. Medical Sciences, Meizhou Hospital Affiliated to Sun Yat- Corresponding author: sen University, Meizhou, China Pingsen Zhao, Clinical Core Laboratory, Centre for 3Guangdong Provincial Engineering and Technology Precision Medicine, Meizhou People’s Hospital (Huangtang Research Centre for Molecular Diagnostics of Hospital), Meizhou Hospital Affiliated to Sun Yat-sen Cardiovascular Diseases, Meizhou, China University, 63 Huangtang Road, Meijiang District, Meizhou 4Meizhou Municipal Engineering and Technology Research 514031, China. Centre for Molecular Diagnostics of Cardiovascular Emails: [email protected]; Diseases, Meizhou, China [email protected] Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). Zhao et al. 1115 frequently in the complete AZFc and partial AZFc region. Duplication of the AZFc region was found in three patients. No AZF microdeletions were found in 60 fertile male controls. Conclusion: The high AZF microdeletion frequency in the current Hakka population suggests that AZF microdeletion analysis is essential in fertility screening, and combined with cytogenetic analysis, may influence the choice of assisted reproductive techniques and reduce the risk of inherited genetic disease. Keywords Male infertility, Y chromosome, karyotype, AZF microdeletions, azoospermia, oligospermia, Hakka population Date received: 1 August 2018; accepted: 5 November 2018 Introduction chromosome and are recognized as being important for spermatogenesis.8,9 Infertility can be defined as a failure of fer- Microdeletions in these regions have been tilization in a couple after a year of regular 1 shown to have adverse effects on spermato- unprotected sex, and about half of infertil- 3 ity in couples is caused by male factors, genesis, thus, AZF microdeletions are most commonly seen to occur in patients with either acting alone or in combination with 10 female factors.2 Major genetic causes of azoospermia or severe oligospermia. male infertility are chromosomal abnormal- With the development of assisted repro- ities and Y chromosome microdeletions.3 ductive technology, infertile males with Y Cytogenetic analysis can be used to detect chromosome microdeletions are able to numerical and structural chromosomal produce offspring through intracytoplasmic 11 abnormalities,4 and is recommended by the sperm injection. However, the potential American Urological Association and risks of passing genetic abnormalities to 12 European Academy of Andrology guideline their offspring are increased. in all men with a motile sperm cell count The primary purpose of the current study <5 million and who are considered to have was to assess the prevalence and major types non-obstructive azoospermia.5 Y chromo- of chromosomal abnormalities among infer- some microdeletions in the azoospermia tile Hakka male patients with azoospermia factor (AZF) region cannot be observed or severe oligospermia, and to emphasize the cytogenetically, however, some studies sug- need for genetic consultation and examina- gest that a Y chromosome microdeletion test tion prior to providing fertility therapy using should be offered to men with non- assisted reproductive technology in Hakka obstructive azoospermia or severe oligosper- male patients in southern China. mia (total motile sperm count <5 million),6 while other studies recommended this test should be carried out in men with total Patients and methods < 7 motile sperm count 10 million. The AZF Study population locus includes three subregions, namely, AZFa, AZFb and AZFc, which are com- This observational study included Hakka monly found in the q11.23 band of the Y male patients receiving clinical counselling 1116 Journal of International Medical Research 47(3) for infertility who were referred to the duct obstruction, gonad anomalies, varico- Centre for Reproductive Medicine of cele, cryptorchidism, orchiditis, ionizing Meizhou People’s Hospital (Huangtang radiation exposure, testicular trauma or Hospital), Meizhou Academy of Medical other possible causes of male infertility Sciences, Meizhou Hospital Affiliated to were excluded from the study. Sun Yat-sen University, China between August 2016 and October 2017, including Cytogenetic analysis patients diagnosed with azoospermia or severe oligozoospermia. A control group Peripheral blood (2.0 ml) was collected from of male volunteers, who had previously all patients into aseptic 30 U/ml heparin produced offspring, were also recruited vacutainer tubes and 0.5 ml blood of each from the Centre for Reproductive sample were transferred into two sterile Medicine of Meizhou People’s Hospital tubes with lymphocyte culture medium (Huangtang Hospital). The study was per- (Guangzhou Baidi Biotech, Guangzhou, formed in accordance with the Declaration China) and cultured for 72 h at 37 C of Helsinki, and was approved by the Ethics (shaken twice daily). Three hours before Committee of the Meizhou People’s the end of the culture, two drops of Hospital (Huangtang Hospital), Meizhou 20 ug/ml colcemid (Baidi Biotech, Academy of Medical Sciences, Meizhou Guangzhou, China) was added to each Hospital Affiliated to Sun Yat-sen tube. Microscopy images of karyotype anal- University. All study participants provided ysis were captured using a ZEISS Axio written informed consent. Imager.Z2 system (ZEISS, Hensoldt Semen specimens, acquired after 3–7 Wetzlar, Germany). Karyotypes were ana- days of abstinence from sexual activity, lysed using standard G-banding and were sent at room temperature to the reported in line with the International andrology laboratory of the Centre for System for Human Cytogentic Reproductive Medicine of Meizhou Nomenclature 2009.14 Atotalof20GTG- People’s Hospital and analysed for several banded metaphases were routinely counted clinical parameters, measured according to with five metaphases analysed on each slide. the 2010 criteria of the World Health The analysis was performed by two labora- 13 Organization. Additionally, if no sperm tory technicians (HMW and XWD) with were found following examination of the qualifications in prenatal diagnosis. samples under the microscope, the samples were centrifuged at 3000 Â g for 15 min at Molecular analysis room temperature, and the precipitate was m re-examined. Genomic DNA was extracted from 200 l Patients were diagnosed with infertility if venous blood in EDTA-coated tubes using they were found to have severe oligozoo- QIAamp DNA blood Mini kit (Qiagen, spermia ( 5 Â 106 sperm cells/ml) or azoo- Duesseldorf, Germany) according to the spermia (no sperm observed following manufacturer’s instructions. Multiplex centrifugation of semen samples obtained polymerase chain reaction (PCR) analysis more than twice at intervals of 1–3 of AZF microdeletions was performed as weeks).13 Cytogenetic analysis and AZF recommended by the European Academy microdeletion tests were conducted for all of Andrology and European Molecular infertile male patients and controls. Genetics Quality Network,15 using a Y Patients with normal sperm concentra- chromosome deletion kit (Microread, tion, abnormal endocrinology, seminal Beijing, China) according to the Zhao et al. 1117 manufacturer’s instructions, including were defined
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