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, 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 , 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- ( 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 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 ( 5 106 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 as having infertility due to azo- sequence-tagged sites: sY84, sY86 (AZFa); ospermia or severe oligozoosperimia. A total sY127, sY134, sY1161, CDY2, SCMY of 60 male volunteers who had previously (AZFb); sY157, sY254, sY255, sY1191, produced offspring were recruited as con- DAZ, and CDY1 (AZFc). The SRY gene trols. The 57 infertile male patients and 60 (located on the y chromosome) and ZFX/ controls were assessed by cytogenetic and ZFY gene (located on the X and Y chro- molecular analysis for karyotype and AZF mosome) were used as internal controls. microdeletions, respectively (Figure 1). Multiplex PCRs were performed for The mean age was 28 5 years in each sample using the Y chromosome patients with azoospermia (n ¼ 33), 27 4 microdeletion detection kit (Microread) years in patients with severe oligozoosper- with BioRad DNA amplification system mia (n ¼ 24) and 31 5 years in controls (Bio-Rad, CA, USA), according to the (n ¼ 60). Karyotyping revealed that manufacturers’ instructions, with the 22.81% (13/57) of males diagnosed as infer- following amplification conditions: 5 min tile had chromosome abnormalities, includ- initial denaturation at 95C, and 30 cycles ing 30.30% (10/33) of males with of 60 s denaturation at 94C, 60 s extension azoospermia and 12.50% (3/24) of males at 60C and 90 s elongation at 72C, fol- with severe oligozoospermia (Table 1). No lowed by a final annealing process of chromosome abnormalities were found in 60 min at 60C. PCR products were added the control group. to a mix containing ROX 500 probes (ROX The most frequent abnormal karyotype ¼ fluorescence-labelled DNA fragment) and was 47, XXY (n 8) followed by two < formamide, then denatured at 95C for 3 patients with 46, XY (Y G), one patient þ min and placed on ice for 3 min. PCR prod- with 46, XX, one patient with 46, X, Yqh ucts were then sequenced using an Applied and one patient with 46, X, del(Y)(q11.23). Biosystem PRISM 3730xl Analyzer No statistically significant differences in (Applied Biosystems, Foster City, CA, abnormal karyotype were found between USA). No-template and female blood sam- patients with azoospermia compared with severe oligozoospermia (v2 ¼ 2.501, ples were used as negative controls, and a P ¼ 0.200) (Table 1). Representative Y male blood sample was used as a posi- chromosome G-banding patterns found tive control. during karyotype analyses are shown in Figure 2. Statistical analyses Multiplex PCR to assess AZF microdele- Data are presented as n (%) prevalence or tions in 57 Hakka male patients diagnosed as mean SD, and were statistically analysed infertile revealed deletions in the AZFa, using SPSS software, version 20.0 (SPSS AZFb, AZFc, AZFbþc, AZFaþbþc, partial Inc., Chicago, IL, USA). Between-group AZFc, and partial AZFb/c loci, and duplica- differences in frequencies were evaluated tion of AZFc (Table 2). The proportion of using v2-test and a P value <0.05 was rec- infertile males found to have AZF deletions ognized as being statistically significant. was 36.84% (21/57) overall, with a frequency of 33.33% (11/33) in male patients with azo- ospermia, and 41.67% (10/24) in male Results patients with severe oligozoospermia. There A total of 918 male patients who were receiv- was no statistically significance difference in ing clinical counselling for infertility were terms of frequency of AZF microdeletions enrolled into the study, and of these, 57 between the two groups (v2 ¼ 0.415, 1118 Journal of International Medical Research 47(3)

Figure 1. Flow chart showing selection and cytogenetic and molecular analyses for karyotype and AZF microdeletions in male patients diagnosed with infertility associated with azoospermia or oligozoospermia

Table 1. Clinical features regarding sperm count and chromosome abnormalities shown by analysis of karyotype in Hakka male patients diagnosed with infertility

Clinical group

Severe Azoospermia oligozoospermia Cases with AZF Karyotype n ¼ 33 n ¼ 24 microdeletion

47, XXY 7 (21.21) 1 (4.17) 0 46, XX 1 (3.03) 0 (0.00) 1 46, XY(Y

Data presented as n (%) prevalence of cases.

P ¼ 0.585) (Table 2). The most commonly with azoospermia and in 8.33% (2/24) detected microdeletions were within the par- males with oligozoospermia. tial AZFc region in male patients with azoo- spermia (15.15% [5/33]) and AZFc region in male patients with severe oligozoospermia Discussion (25.00% [6/24]). Duplication of the AZFc Chromosomal anomalies have been shown region was found in 3.03% (1/33) males to play an important role in male Zhao et al. 1119

Figure 2. Representative photomicrographs showing karyotype G-banding patterns in abnormal chro- mosomes from Hakka male patients diagnosed with infertility associated with azoospermia or oligozoo- spermia, and in normal male control chromosomes, showing: (a) Yqhþ; (b) Y (Y

Table 2. Azoospermia factor (AZF) microdeletions in Hakka male patients diagnosed with infertility associated with azoospermia or severe oligozoosperimia, and in fertile male controls

Study group

Severe Azoospermia oligozoospermia Controls AZF region Sequence-tagged site deletions n ¼ 33 n ¼ 24 n ¼ 60

AZFa sY84, sY86 1 (3.03) 0 (0.00) 0 (0.00) AZFc sY157, sY254, sY255, sY1191, DAZ, CDY1 2 (6.06) 6 (25.00) 0 (0.00) AZFbþc sY127, sY134, sY1161, SMCY, sY157, sY254, 1 (3.03) 1 (4.00) 0 (0.00) sY255, sY1191, DAZ, CDY1 AZFaþbþc sY84, sY86, sY127, sY134, sY1161, CDY2, 1 (3.03) 0 (0.00) 0 (0.00) SCMY, sY157, sY254, sY255, sY1191, DAZ, CDY1 Partial AZFc CDY2/CDY1>1.5, DAZ/DAZL<1.5 5 (15.15) 3 (12.5) 0 (0.00) Partial AZFb/c sY1161, sY1191, DAZ/DAZL<1.5 1 (3.03) 0 (0.00) 0 (0.00) Total 11 (33.33) 10 (41.67) 0 (0.00)

Data presented as n (%) prevalence. 1120 Journal of International Medical Research 47(3) infertility,16 but the prevalence of these by Naasse et al.22 This difference may be abnormalities has rarely been reported in attributed to population variance and lim- the Hakka male population of China. In ited study subjects in the present study. studies of different populations, the occur- Infertile male patients with 46, XY rence of chromosomal abnormalities in (Y

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