Analysis of copy number variations of the autosomal genome in 156 patients with azoospermia
Introduction
Infertility affects about 10–15% of couples of gestational age. It has attracted attention as a common and frequently occurring condition, half of which is caused by male factors (1). Male factors mainly manifest as sexual dysfunction, oligoasthenospermia, azoospermia, abnormal seminal plasma, and immune infertility, among others. Azoospermia has a serious impact due to its complex etiology and was one of the most important factors of infertility. At present, the causes of spermatogenesis disorder are mainly involved the endocrine system, reproductive system ultrasound, chromosome abnormalities, and gene mutation (2). However, the etiology of most azoospermia cases is unknown, and the exact mechanism of spermatogenesis block has not yet been determined. Therefore, in the present study, we screened 156 azoospermia patients with normal chromosomes and measured their genomic copy number variations (CNVs) by next-generation sequencing (NGS) technology, in order to determine the relationship between CNVs and azoospermia and lay a foundation for clarifying the possible mechanism of spermatogenic block caused by CNVs. We present the following article in accordance with the MDAR reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-22-301/rc).
Methods
General information
A total of 156 azoospermia patients treated in our hospital from October 2018 to May 2021 were selected. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of Yinchuan Maternal and Child Health Hospital (No. 2018-079) and the patients provided informed consent.
Inclusion criteria
B-ultrasound was performed to indicate that there was no abnormality in the bilateral testis, epididymis, and spermatic cord. Other inclusion criteria were as follows: genetic testing shown 46, XY, and no Y chromosome microdeletion was detected; no history of systemic diseases such as hepatitis, tuberculosis, and mumps; no history of exposure to radiation and chemicals; no urogenital infection, malformation, and varicocele. The ages ranged from 24 to 43 years, with an average of 30.79±6.43 years.
Semen analysis
According to the fifth edition of the WHO standard (1) and the Guidelines on Male Infertility of the European Society of Urology, patients were required to abstain for 3 to 5 days then masturbate and collect semen into a sterile cup. The sample was placed in a 36 °C incubator for 30 to 60 minutes to liquefy, and any patient who had no sperm after microscopic examination and centrifugal precipitation was determined as infertile.
Testicular biopsy
After routine disinfection and laid the drapes, 2% lidocaine spermatic cord block was performed. The left ring finger and middle finger on the opposite side of the biopsy were fixed to make the scrotal skin on the biopsy side in a tense state. The left thumb and index finger were twisted tightly in the middle of the testis to counter the biopsy pressure, then the vas deferens separation forceps were held in the right hand to slowly separate the skin and subcutaneous tissue at the needle eye during spermatic cord block to reach the testicular white membrane. The white membrane continued to be blunt friction to make it thinner. When there was a sense of breakthrough, the vas deferens separation forceps entered the testis. Some testicular tissue was clamped and sent to the laboratory for microscopic examination. After the procedure, gentle pressure was applied for a moment to stop bleeding.
Chromosome detection
The fasting venous blood of the patient’s upper limb was collected for chromosome detection which was performed by G-banding karyotype analysis technology, and at least 20 cells were analyzed.
Detection of CNVs by NGS
The venous blood of the upper limbs was collected to extract genomic DNA, and 10 ng DNA was used to construct the library using “rapid PCR free library construction technology”, cut the DNA, repaired the enzyme digestion gap and double stranded ends, phosphorylate and modify them and connect them with primers, measure the sequence with the NextSeqCN500 gene sequencer, and analyze the data with the data analysis software from Hangzhou Berry Genomics Diagnosis Technology Company. The results were determined by comprehensively referring to the latest published data of public databases such as GRCh37 (hg19): ENSEMBL release_59/61/64/68/69/75, Database of Genomic Variants (DGV), human chromosome imbalance and phenotype database (DECIPHER), Online Mendelian Inheritance in Man (OMIM) database, University of California Santa Cruz (UCSC) database, and PubMed. Analyses were completed by Beijing Berry Genomics Biotechnology Co., Beijing, China.
Statistical analysis
The statistical analyses were performed using the Statistical Package for the Social Sciences version 20.0 (SPSS Inc., Chicago, IL, USA). Non-normally distributed metric variables were analyzed by the Kruskal-Wallis test and Mann-Whitney U-test. The mean surface areas of the endometriotic implants between the same group (before and after the medical treatment) were analyzed by Wilcoxon’s signed-rank test. P≤0.05 was considered statistically significant. Values were expressed as mean ± standard deviation, unless stated otherwise.
Results
Comparison between testicular puncture results and CNVs
Among the 156 patients, 81 had sperm in the testicular puncture and 75 had no sperm in the testicular puncture. There was a significant difference in CNV detection between the 2 groups (P<0.05), as shown in Table 1.
Table 1
| Operation | Result | CNV test results, n (%) | χ2 | P | |
|---|---|---|---|---|---|
| Normal | Abnormal | ||||
| Testicular puncture | Sperm | 49 (60.49) | 32 (39.51) | 4.958 | 0.037 |
| Azoospermia | 32 (42.67) | 43 (57.33) | |||
CNV, copy number variation.
Analysis of CNVs on each chromosome
Among the 156 patients, 75 patients had CNVs, with an incidence of 48.08%, including 59 patients with 1 CNV, 9 patients with 2 CNVs, 5 patients with 3 CNVs, 1 patient with 4 CNVs, and 1 patient with 5 CNVs. The chromosomes with CNVs which accounted for more than 5% were 2, 3, 5, 10, 11, and Y, as shown in Table 2.
Table 2
| Chromosome | Defect | Repeat | Total | |||||
|---|---|---|---|---|---|---|---|---|
| Amount | Proportion (%) | Amount | Proportion (%) | Amount | Proportion (%) | |||
| 1 | 0 | 0.00 | 3 | 4.23 | 3 | 2.97 | ||
| 2 | 3 | 10.00 | 8 | 11.27 | 11 | 10.89 | ||
| 3 | 2 | 6.67 | 5 | 7.04 | 7 | 6.93 | ||
| 4 | 0 | 0.00 | 2 | 2.82 | 2 | 1.98 | ||
| 5 | 4 | 13.33 | 6 | 8.45 | 10 | 9.90 | ||
| 6 | 3 | 10.00 | 2 | 2.82 | 5 | 4.95 | ||
| 7 | 2 | 6.67 | 2 | 2.82 | 4 | 3.96 | ||
| 8 | 0 | 0.00 | 4 | 5.63 | 4 | 3.96 | ||
| 9 | 2 | 6.67 | 0 | 0.00 | 2 | 1.98 | ||
| 10 | 0 | 0.00 | 7 | 9.86 | 7 | 6.93 | ||
| 11 | 1 | 3.33 | 7 | 9.86 | 8 | 7.92 | ||
| 12 | 1 | 3.33 | 2 | 2.82 | 3 | 2.97 | ||
| 13 | 1 | 3.33 | 0 | 0.00 | 1 | 0.99 | ||
| 14 | 3 | 10.00 | 2 | 2.82 | 5 | 4.95 | ||
| 15 | 0 | 0.00 | 3 | 4.23 | 3 | 2.97 | ||
| 16 | 2 | 6.67 | 2 | 2.82 | 4 | 3.96 | ||
| 17 | 0 | 0.00 | 2 | 2.82 | 2 | 1.98 | ||
| 18 | 0 | 0.00 | 1 | 1.41 | 1 | 0.99 | ||
| 19 | 0 | 0.00 | 1 | 1.41 | 1 | 0.99 | ||
| 20 | 0 | 0.00 | 2 | 2.82 | 2 | 1.98 | ||
| 21 | 1 | 3.33 | 0 | 0.00 | 1 | 0.99 | ||
| 22 | 0 | 0.00 | 0 | 0.00 | 0 | 0.00 | ||
| X | 1 | 3.33 | 4 | 5.63 | 5 | 4.95 | ||
| Y | 4 | 13.33 | 6 | 8.45 | 10 | 9.90 | ||
| Total | 30 | 100 | 71 | 100 | 101 | 100 | ||
CNV, copy number variation.
Detailed analysis of CNVs on chromosomes 2, 3, 5, 10, and 11
A total of 132 genes were found on autosomes with CNVs and the percentage of CNVs was more than 5%, including 8 deletions and 124 repetitions. CNVs on chromosome 2 found 11 genes, of which 3 were deleted and 8 were duplicated; 17 genes were found in CNVs on chromosome 3, including 3 deletions and 14 duplications; 12 genes were found in CNVs on chromosome 5, of which 2 were deleted and 10 were repeated; 72 genes were found in CNVs on chromosome 10, all of which were duplicates; CNVs on chromosome 11 found 20 genes, all of which were duplicates. The specific location, size, and genes of CNVs on chromosomes 2, 3, 5, 10, and 11 are shown in Tables 3-7, respectively.
Table 3
| Zone | Type | Specific location | Gene | Size (Mb) |
|---|---|---|---|---|
| q24.2 | del | 161320001_161420000 | RBMS1 | 0.1 |
| p15 | dup | 61660000_61820000 | USP34, XPO1 | 0.16 |
| p24.3 | dup | 13560001_14340000 | There is no gene in this region | 0.78 |
| p23.3 | dup | 25620001_25980000 | ASXL2, DTNB | 0.36 |
| p16.3p16.2 | del | 52860001_53040000 | MIR4431 | 0.18 |
| q12.3 | dup | 109160001_109400000 | RANBP2, LIMS1 | 0.24 |
| q22.2 | dup | 142900001_143500000 | There is no gene in this region | 0.60 |
| p24.3 | dup | 13560000_14340000 | There is no gene in this region | 0.78 |
| p16.3 | del | 50940000_51060000 | NRXN1 | 0.12 |
| q37.3 | dup | 237380001_237720000 | IQCA1, ACKR3 | 0.34 |
| q14.1 | dup | 116920001_117340000 | There is no gene in this region | 0.42 |
CNV, copy number variation; del, deletion; dup, duplication.
Table 4
| Zone | Type | Specific location | Gene | Size (Mb) |
|---|---|---|---|---|
| q11.2 | del | 94700001_94980000 | LINC00879 | 0.28 |
| q29 | dup | 197020001_197340000 | DLG1-AS1, BDH1, MIR4797, DLG1 | 0.32 |
| q26.1 | del | 165180001_165940000 | BCHE, LINC01322 | 0.76 |
| q25.32 | dup | 158060001_158600000 | MFSD1, GFM1, RARRES1, LXN, RSRC1, LOC100996447, MLF1 | 0.54 |
| q26.31q26.32 | dup | 175540001_175880000 | There is no gene in this region | 0.34 |
| p26.3 | dup | 820000_1460000 | LINC01266, CNTN6 | 0.64 |
| p24.2 | dup | 25160000_25380000 | RARB | 0.22 |
CNV, copy number variation; del, deletion; dup, duplication.
Table 5
| Zone | Type | Specific location | Gene | Size (Mb) |
|---|---|---|---|---|
| q23.1 | del | 116740001_116920000 | LINC00992 | 0.18 |
| p15.31 | del | 6960000_7200000 | There is no gene in this region | 0.24 |
| p14.1 | del | 25080001_25360000 | There is no gene in this region | 0.28 |
| p15.32p15.31 | dup | 5720000_6500000 | UBE2QL1, MED10, LINC02145 | 0.78 |
| p12p11 | dup | 45980000_46400000 | There is no gene in this region | 0.42 |
| p13.3 | dup | 32040000_32200000 | PDZD2, GOLPH3 | 0.16 |
| q23.1 | del | 117100001_117320000 | LOC102467224 | 0.22 |
| q15 | dup | 94020001_94180000 | SLF1, MCTP1 | 0.16 |
| q13.2 | dup | 72620001_72800000 | FOXD1, BTF3, LINC01386 | 0.18 |
| q34 | dup | 161640000_162360000 | There is no gene in this region | 0.72 |
CNV, copy number variation; del, deletion; dup, duplication.
Table 6
| Zone | Type | Specific location | Gene | Size (Mb) |
|---|---|---|---|---|
| p14p13 | dup | 11940001_12300000dup | MIR548AK, UPF2, NUDT5, DHTKD1, SEC61A2, CDC123 | 0.36 |
| p11.21 | dup | 35700001_36000000dup | CCNY, MIR4683, GJD4, FZD8 | 0.3 |
| q26.3 | dup | 135260000_135440000dup | SPRNP1, FRG2B, SCART1, SYCE1, CYP2E1 | 0.18 |
| q11.22q11.23 | dup | 46960000_51600000dup | ANTXRL, GDF10, BMS1P5, LRRC18, OGDHL, SYT15, GPRIN2, AGAP9, ZNF488, FRMPD2, C10orf128, C10 or f71, DRGX, SLC18A3, MSMB, TIMM23, HNRNPA1P33, ANXA8, LOC107001062, LOC105378292, RBP3, FAM25C, FAM170B, PGBD3, PARG, LINC00842, MAPK8, ARHGAP22, WDFY4, FAM170B-S1, ERCC6, AGAP7P, ANXA8L1, ANTXRLP1, CTSLP2, PTPN20, CHAT, GDF2, VSTM4, C10 or f53 | 4.64 |
| p12.1 | dup | 28000001_28760000dup | ARMC4, MKX, MIR8086, MKX-AS1, MPP7 | 0.76 |
| p12.33 | dup | 17540001_17640000dup | PTPLA | 0.10 |
| q11.22 | dup | 46960000_47700000dup | ANTXRL, SYT15, GPRIN2, FAM25BP, FAM35DP, LOC102724593, NPY4R, HNRNPA1P33, LINC00842, ANXA8L1, ANTXRLP1 | 0.74 |
CNV, copy number variation; dup, duplication.
Table 7
| Zone | Type | Specific location | Gene | Size (Mb) |
|---|---|---|---|---|
| p15.1 | dup | 16820001_17060000 | PLEKHA7 | 0.24 |
| q22.3 | dup | 105860001_106620000 | AASDHPPT, MSANTD4, KBTBD3, LOC101928535, GUCY1A2 | 0.76 |
| p14.3 | dup | 22840001_23280000 | CCDC179, SVIP | 0.44 |
| q22.1 | del | 97440001_97640000 | There is no gene in this region | 0.20 |
| q22.3 | dup | 105860001_106620000 | AASDHPPT, MSANTD4, KBTBD3, LOC101928535, GUCY1A2 | 0.76 |
| q22.3 | dup | 106660000_107020000 | GUCY1A2 | 0.36 |
| p15.4 | dup | 3780001_4200000 | MIR4687, PGAP2, RRM1, RHOG, NUP98, STIM1 | 0.42 |
| q24.3 | dup | 129540001_129660000 | There is no gene in this region | 0.12 |
CNV, copy number variation; del, deletion; dup, duplication.
Discussion
Spermatogenesis is a programmed, multigene, and complex process (3-7). Any abnormality in any link may lead to the stagnation of spermatogenesis, which may then lead to severe oligozoospermia or azoospermia (8-10). The deletion of azoospermia factor (AZF) confirmed by a large number of studies is a typical example. In fact, there are thousands of genes involved in spermatogenesis, and most of the mechanisms are not clear (3,11,12). It is particularly important to find and clarify the mechanisms of genes related to spermatogenesis, which can aid in many aspects, such as species evolution, spermatogenic arrest, in vitro culture, and clinical intervention (13-15).
In this study, we examined the CNVs of 156 azoospermia patients and found that each chromosome had more or less CNVs. We further screened for chromosomes with CNVs which accounted for more than 5% which yielded chromosomes 2, 3, 5, 10, 11, and Y, indicating that there may be important gene fragments affecting spermatogenesis. A large number of genes on the Y chromosome have been confirmed to cause spermatogenesis arrest, while autosomal gene abnormalities have rarely been reported (16-18). In this experiment, 132 genomic CNVs were found in the 5 autosomal chromosomes 2, 3, 5, 10, and 11, including 8 deletions and 124 repeats. A large number of studies have been carried out on gene deletions and mutations, which could be verified by animal experiments through gene knockout, but gene duplication has always been a research blind spot until the emergence of NGS (19-23). This technique provides a strong technical guarantee for the detection of gene duplication, but how to confirm the impact of gene duplication on spermatogenesis in the next step will be challenging.
The size of CNV fragments detected in this study was 0.1–4.64 Mb, and gene free fragments accounted for 25.58% (11/43), suggested that even if there were no known genes in CNVs, the changes in chromosome structure caused by CNVs are of great importance and may also affect spermatogenesis, which is worthy of attention and in-depth research.
NGS technology analyzes Y chromosome aberrations by detecting genomic CNVs over 100 kb. Zhu et al. (24) showed that the size of CNVs correlated with male infertility. This study found that duplication (DUP) of Y chromosome CNVs detected by NGS is currently undetectable by sequence tagged site (STS)-polymerase chain reaction (PCR). Therefore, we speculated that gene duplication also has the potential to cause azoospermia. In terms of detection of gene deletions, there are no reports on the concordance between Y-chromosome AZF microdeletions detected by STS-PCR and Y-chromosome CNVs deletions (del) detected by NGS. It has been documented that CNVs in the AZFc region are associated with an increased risk of male spermatogenic failure, but the significance with respect to the detection of deletions in both genes is not clear. Since the current tests are for CNVs over 100kb, there may be some cases of missed STS loci, especially novel STS such as sY1192 or small deletions. If future technologies allow detection of smaller segments of CNVs, it will be of great value to explore Y chromosome gene level contributions to male infertility (2).
The use of STS-PCR to detect Y-chromosomal AZF microdeletions is an important laboratory genetic test for azoospermia, but exactly how many STS loci are more appropriate to detect remains to be explored. The use of NGS to detect Y-chromosome CNVs has not been widely adopted, and we believe that the 2 assays complement each other and that, typically, NGS may be considered for screening in patients with azoospermia, whereas STS-PCR may be used for validation.
According to the overall analysis, among the 156 patients, 75 had CNVs, with an incidence of 48.08%. Especially in those without sperm in the testicular puncture, the incidence of CNVs was as high as 57.33% (43/75). This phenomenon itself deserves attention. Therefore, it could be inferred that the chromosomal changes caused by CNVs in structure or function may affect the component of spermatogenesis. In this way, it interferes with mitosis and/or meiosis in the process of spermatogenesis, making spermatogenesis stagnate in a certain spermatogenesis stage, resulting in partial or complete spermatogenesis block, clinically manifesting as severe oligozoospermia or azoospermia. Based on this, the next step is to interfere with the related CNVs in vitro or in vivo, open the obstacle of spermatogenic block, and then restore spermatogenesis, which will be the subject of our future studies.
Acknowledgments
Funding: This research was supported by the Ningxia Natural Science Foundation (Item No. 2020AAC03495), and the Key Research and Development Program of Ningxia Hui Autonomous Region (2021BEG02029).
Footnote
Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-22-301/rc
Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-22-301/dss
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-22-301/coif). The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of Yinchuan Maternal and Child Health Hospital (No. 2018-079) and written informed consent was obtained from all patients.
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(English Language Editor: C. Betlazar-Maseh)
