A 27-year-old Chinese male accepted infertility consultation in The Center for Reproductive Medicine and Center for Prenatal Diagnosis at The First Hospital of Jilin University after 1 year of regular unprotected coitus and no pregnancy in June, 2015. The patient's height and weight were 176 cm and 80 kg, respectively. His penis development and growth were normal, and testicular volume was ~4 and ~6 ml for the left and right testes, respectively. No other physical abnormalities were observed. A series of routine examinations were conducted. Semen analysis and levels of sex hormones are presented in Table I. The male was eventually diagnosed with azoospermia according to routine semen examination (17). The Ethics Committee of the First Hospital of Jilin University approved the study protocol (permit no. 2016-416), and the patient provided written informed consent and agreed to participate in this study.

For the patient, a karyotype analysis of peripheral blood lymphocytes was conducted: Peripheral blood (0.5 ml) was cultured in lymphocyte culture medium containing 30 U/ml heparin for 72 h at 37°C (Yishengjun; Baidi Biotechnology) and subsequently treated with 50 µg/ml colchicine (Yishengjun; Baidi Biotechnology) 1 h before culture termination to arrest mitoses. The lymphocytes were hypotonically treated in 0.075 M KCl for 20 min at 37°C and fixed in methanol:acetic acid (3:1) for 10 min at room temperature, G-banding of metaphase chromosomes and chromosomal karyotype analysis were performed according to a previous study (18). The karyotype was described according to the International System for Human Cytogenetic Nomenclature 2016 (19).

Following written consent from the patient, 5 ml peripheral blood was collected from the patient using a standard vacuum extraction blood-collecting system containing EDTA and heparin. Genomic DNA was isolated from whole blood using a QIAamp DNA Mini kit (Qiagen GmbH) according to the manufacturer's protocol. CMA was performed using a CytoScan 750K array (Affymetrix; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The procedure included genomic DNA extraction, digestion and ligation, PCR amplification, PCR product purification, quantification and fragmentation, labeling, array hybridization, washing and scanning. In the PCR amplification process, 250 ng DNA samples was digested with Nsp1, amplified with TITANIUM Taq DNA polymerase (cat. no. 190118EA; Clontech Laboratories, Inc.), fragmented with Affymetrix fragmentation reagent, and labeled with biotin end-labeled nucleotides. PCR was performed using a Veriti Thermal Cycler 96-well, Alpha-SE (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions consisted of an initial denaturation step of 3 min at 94°C, followed by 30 cycles of 30 sec at 94°C, 45 sec at 60°C, and 15 sec at 68°C, with a final extension step at 68°C for 7 min. The platform used included 550,000 nonpolymorphic probes and 200,436 single nucleotide polymorphic probes. Thresholds for genome-wide screening were set at ≥200 kb for gains and ≥100 kb for losses. The detected copy number variations were comprehensively estimated by comparing them with published literature and public databases, including Database of Genomic Variants (DGV; http://dgv.tcag.ca/dgv/app/home), DECIPHR v9.28 (http://decipher.sanger.ac.uk/), ISCA (https://www.iscaconsortium.org/), ECARUCA (http://www.ecaruca.net), and OMIM (http://www.ncbi.nlm.nih.gov/omim).

Based upon the karyotype analysis and CMA results, FISH analysis was carried out to further confirm the characterization of chromosomal Y anomalies. Two sets of probes were used according to the manufacturers' instructions in the present study: Centromere probes specific for chromosomes X, Y (cat. no. F01001, CSPX, spectrum green; CSPY, spectrum red; Beijing GP Medical Technologies, Ltd.) and SRY probe (cat. no. RU-LPU026; Cytocell). The SRY probes, labelled in red, consists of two non-overlapping probes, 30 and 50 kb. The probes cover the entire SRY gene and flanking DNA, including the RPS4Y1 gene. The probe mix also contains control probes for the X centromere (DXZ1), labelled in blue, and for chromosome Y (DYZ1, the heterochromaticblock at Yq12), labelled in green. Lymphocytes cell suspensions fixed with methanol:acetic acid (3:1) for the karyotype analysis were further used for FISH analysis. The cell suspensions were centrifuged (931 × g for 5 min) at 4°C, then were dropped onto dry glass slides (Shitai; Citotest Labware Manufacturing Co., Ltd.) using a dropper. The procedure of slide preparation (including probe application, co-denaturation, hybridization and post-hybridization washes) was performed according to the manufacturer's instructions. The probes were denatured for 2 min at 75°C. The hybridization mixture (1 µl each probe and 7 µl hybridization solution) was applied to each slide and covered with a coverslip 20×20 mm. Then, the slides were sealed with rubber cement before hybridization was carried out overnight in a moist chamber at 37°C. After hybridization, the slides were washed for 2 min in a solution of 0.4X SSC at 72°C and a second time for 30 sec in a solution of 2X SSC, 0.05% Tween-20 at room temperature. Following the final wash, slides were air dried and counterstained with a solution of 4′,6′-DAPI in the dark at 4°C overnight. Appropriate viewing, analysis and imaging of FISH results was accomplished using a fluorescent microscope (Leica DM4000B; Leica Microsystems GmbH; magnification, ×1,000).

Microdeletions in the AZF region were detected using PCR, as previously described in accordance with the recommendations of The European Academy of Andrology and the European Molecular Genetics Quality Network. Specific sequence-tagged sites were mapped in the AZF region, including SY84 and SY86 for AZFa; SY27, SY134, and SY143 for AZFb; SY157, SY254, and SY255 for AZFc; and SY152 for AZFd. Human zinc-finger protein-encoding genes (ZFX/ZFY) located on the X and Y chromosomes were selected as internal control primers (20). Genomic DNA was isolated with the Tiangen Blood DNA Extraction Mini kit (Tiangen Biotech Co., Ltd.) using peripheral blood lymphocytes according to the manufacturer's protocol. The PCR process was performed according to a previous study, and primer sequences are presented in Table II (21). Each 20 ml reaction mix contained 50 ng genomic DNA, 5–10 pmol of each primer (Shanghai Sangon Pharmaceutical Co., Ltd.), 100 mM potassium chloride, 200 mM Tris-HCl (pH 8.8), 15 mM magnesium chloride, 1% Triton X-100, 500 mM of each dNTP and 2 U Taq polymerase (Beijing Dingguo Changsheng Biotechnology Co., Ltd.). Multiplex PCR was carried out using a Veriti 96-well PCR thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling program involved preliminary denaturation at 94°C for 6 min, followed by 35 cycles of denaturation at 95°C for 40 sec, annealing at 58°C for 45 sec, and elongation at 72°C for 60 sec, followed by a final elongation step at 72°C for 10 min. Classical AZF reaction products were analyzed by electrophoresis in 1.5% agarose gels at 80 V for 30 min (Biowest). All gels contained 0.5 mg/ml ethidium bromide and PCR products were visualized under ultraviolet light.

The present study focused on cases presenting neocentric chromosome Y associated with different clinical manifestations. Based upon this selection criterion, a systematic literature search was conducted by means of a PubMed literature search (https://www.ncbi.nlm.nih.gov/pubmed/) using relevant terms and their combinations [including neo(Y), neocentric chromosome Y, chromosomal Y neocentromere, neocentric marker chromosome Y and marker chromosome Y neocentromere], and by searching the sSMC database (http://ssmc-tl.com/sSMC.html) (22).

Cytogenetic G-banding analysis initially described the karyotype as 47,X,i(Y)(q10),+mar (Fig. 1), consisting of an isochromosome Yq [i(Yq)] and an sSMC.

In order to delineate the origin of the marker chromosome, CMA analysis was performed, describing the sSMC as arr[hg19]Yp11.31q11.23(2,650,-424-28,799,654)×2 (Fig. 2), indicating that the patient had an extra Y chromosome but that the karyotype was not 47,XYY. Based on this, the karyotype was characterized as 47,X,i(Y)(p10),i(Y)(q10). The existence of two forms of the Y chromosome was further investigated with SRY and alphoid probes for the Y centromere. FISH with centromere probes showed two close but clearly distinct chromsomal Y centromere signals (red) in the middle region of i(Yq) (Fig. 3A), but no Y centromere signal was detected on the sSMC. Furthermore, two DYZ1 signals (green) and no SRY signal indicated that i(Yq) was actually an idic(Yq) (Fig. 3B). These results imply that two identical Y chromosomes, with partially missing short arms, had integrated together symmetrically (Fig. 4). The sSMC(Y) with two SRY signals (red) revealed the presence of neo(Y)(pter→Yp11.2:: Yp11.2→pter) derived from Ypter to Yp11.2 without centromeric material (Fig. 3B), which might indicate the existing breakpoints in Yp11.2. The existence of the neocentromere requires further verification. In addition, no microdeletions in the AZF region were detected.

A literature review on the cases with neocentric chromosome Y was conducted, which are presented in Table III (13,14,22–28). Of all cases, 4/9 were female, 3/9 were male, and the sex in 2/9 cases was not available. Among their chromosomal karyotypes, 4/9 were mosaic, 4/9 were non-mosaic and 1/9 was not reported. The clinic manifestations of these patients were varied, ranging from normal fetal ultrasound findings to abnormal phenotypes in teenagers and adults.