The selection of Y chromosome microdeletion detection methods based on seminal analysis results: a comparison of high-throughput sequencing and fluorescence quantitative polymerase chain reaction (qPCR) applications

Introduction

Male infertility is a common issue affecting couples worldwide, significantly impacting their reproductive health (1). It is estimated that male factors contribute to approximately 40% of infertility cases (2). The Y chromosome, unique to males, carries important genes that determine male sex and reproductive function (3,4). Y chromosome microdeletions are genetic variations associated with male infertility, specifically related to sperm production and reproductive capability (5,6). Therefore, accurate detection of Y chromosome microdeletions is crucial for the diagnosis and personalized treatment of infertility etiology.

Various methods are available for the detection of Y chromosome microdeletions, including high-throughput sequencing [next-generation sequencing (NGS)] and fluorescence quantitative polymerase chain reaction (qPCR). NGS allows the DNA in a sample to accurately reflect an individual’s genomic state, with uniform sequencing coverage and high accuracy in bioinformatics analysis (7). NGS offers high sensitivity and comprehensiveness, enabling the detection of subtle gene deletions and rearrangements. qPCR allows primers to specifically bind to target sequences, amplifying only the target gene with consistent efficiency and maintaining template integrity. Fluorescence qPCR, as a routine molecular biology technique, is known for its simplicity, speed, and cost-effectiveness, making it suitable for rapid screening and preliminary diagnosis (8). However, there is a lack of systematic comparative studies to guide the selection of Y chromosome microdeletion detection methods based on seminal analysis results, leaving uncertainty regarding the most appropriate method for different types of infertility patients.

Seminal analysis is the initial step in infertility diagnosis, providing valuable information about sperm quantity, quality, and motility, which helps to assess male fertility potential and guide further diagnostic directions (9,10). Selecting the appropriate Y chromosome microdeletion detection method based on seminal analysis results can facilitate individualized and accurate diagnosis and treatment for infertility patients (11).

To address the existing challenges in selecting Y chromosome microdeletion detection methods based on seminal analysis results, this study aims to compare the application of high-throughput sequencing and fluorescence qPCR in different types of infertility patients. By dividing infertility patients into azoospermia and oligoasthenoteratozoospermia (OAT) groups and applying high-throughput sequencing and fluorescence qPCR methods, we evaluate the sensitivity, specificity, feasibility, and cost-effectiveness of these two methods for Y chromosome microdeletion detection. The findings aim to provide scientific evidence for clinicians and patients in selecting appropriate detection methods and support personalized infertility treatment.

Methods

Participant selection

Between January and July 2023, six male infertility patients were seen in the outpatient clinic of the Reproductive Medicine Center (the Affiliated Hospital of Inner Mongolia Medical University). According to the World Health Organization (WHO)’s “Laboratory Manual for the Examination and Processing of Human Semen” (5^th edition) criteria, three cases were diagnosed with azoospermia and three cases were diagnosed with OAT (12).

Analysis of semen parameters

Semen parameters were analyzed using the sperm quality analyzer from Sis Medical Technology Company (XXX, XXX), following the diagnostic criteria outlined in the WHO Laboratory Manual for the Examination and Processing of Human Semen (5^th edition) (13,14). According to the WHO semen analysis standards: absence of spermatozoa in three consecutive examinations, even after centrifugation, indicated azoospermia; the presence of spermatozoa observed only in the sediment after fresh semen microscopy was categorized as cryptozoospermia; oligozoospermia was defined as a sperm concentration of less than 15×10⁶/mL and a progressive motility (PR) percentage of less than 32%; asthenozoospermia was defined as a PR percentage of less than 32% and a normal morphology percentage of less than 4%; samples with a sperm concentration of ≥15×10⁶/mL, a PR percentage of ≥32%, and a normal morphology percentage of ≥4% were classified as normozoospermia (15,16).

Genomic DNA extraction and multiplex fluorescent polymerase chain reaction (PCR) detection

According to the instructions provided by Shanghai Bai’Ao Biotechnology Co., Ltd. (Shanghai, China), peripheral blood DNA was extracted from each sample using a DNA extraction kit. The azoospermia factor (AZF) detection kit from Beijing Aipu Yisheng Biotechnology Co., Ltd. (Beijing, China) was employed. The kit targeted eight sequence-tagged sites (STS) in the AZF region of the Y chromosome. PCR and fluorescently labeled probe technology were used, with four triple PCR reactions performed for each sample. Each PCR reaction system contained an internal control, two sets of specific primers and probes for targeted loci, and fluorescence detection in three channels (FAM/VIC/Cy5). The Roche 480II fluorescent qPCR instrument was used for DNA amplification. Ct values >24 or the absence of distinct S-shaped curves for the sY127, sY134, sY152, sY254, and sY255 loci were interpreted as non-amplification (locus deletion). Ct values >28 or the absence of distinct S-shaped curves for the sY86, sY84, and sY145 loci were interpreted as non-amplification (locus deletion).

High-throughput sequencing (NGS) analysis

For each sample, the amplified DNA was prepared into libraries following the manufacturer’s guidelines for VeriSeq PGS (Illumina, XXX, XXX). The reference genome used is hg19/GRCh37 (human_g1k_v37.fasta.gz). Briefly, the diluted DNA underwent fragmentation, tagging, and dual-indexed sequencing. Subsequently, the products were purified using size selection and normalized to ensure equal sample quantity. The pooled and denatured final products were then sequenced on a Miseq System (Illumina) using the Miseq Reagent Kit (v.3; Illumina). The resulting bioinformatics data were analyzed using the BlueFuse Multi Software (Illumina). In this analysis, samples were distinguished based on the deviation of the median chromosomal copy number from the default copy number. A copy number greater than 2 indicated a possible gain (trisomy) of autosomal chromosomes, while a copy number less than 2 indicated a possible loss (monosomy).

Statistical analysis

Statistical analyses were performed using SPSS version 25 (IBM, Armonk, NY, USA). Categorical data were presented as counts and percentages. The significance of the differences was assessed using the Chi-squared test (Chi-square test is a widely used hypothesis testing method based on the Chi-squared distribution. It is a non-parametric test) or Fisher’s exact test. A two-sided P value equal to or less than 0.05 was considered statistically significant.

Ethical statement

The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the ethics committee of the Affiliated Hospital of Inner Mongolia Medical University (No. YKD202101026). Written informed consent was obtained from all study participants.

Results

Seminal analysis results of two groups

In the azoospermia group, no motile sperm were detected in the seminal samples. The semen volume was 3.3, 3.8, and 3.3 mL, with pH values of 7.2, 7.2, and 6.5, respectively. The sperm concentration was 0 million/mL, and both the PR and non-PR (PR + NP) were 0%. Consequently, the total sperm count was 0 million. In the OAT group, a few sperm were present in the seminal samples, but their quantity was limited, and their motility was compromised. The semen volume was 8.6, 3.7, and 6.1 mL, with pH values of 7.2 in all cases. The sperm concentration was 2.36, 4.01, and 0.35 million/mL, respectively. The PR was 26.00%, 7.06%, and 16.67%, and the total motility rate (PR + NP) was 30.00%, 10.59%, and 50.00%, respectively. The total sperm count was 20.30 million, 14.84 million, and 2.14 million, respectively. In summary, no motile sperm were detected in the seminal samples of the azoospermia group, while the OAT group showed a limited quantity of sperm with compromised motility. These seminal analysis results provide important reference information for subsequent selection of Y chromosome microdeletion detection methods (Table 1).

Fluorescence qPCR results of two groups

In the azoospermia group, three cases were included. Fluorescence qPCR analysis confirmed the presence of specific Y chromosome sequences in the AZFa, AZFb, AZFc, and AZFd regions of all patients. Specifically, cases 01, 02, and 03 exhibited specific fluorescent signals at the sY84, sY86, sY127, sY134, sY254, and sY255 loci, indicating no significant deletions in the AZFa, AZFb, AZFc, and AZFd regions of their Y chromosomes. In the OAT group, three cases were also included. Fluorescence qPCR analysis revealed the presence of specific Y chromosome sequences in the AZFa, AZFb, and AZFc regions of all patients. Specifically, cases 01, 02, and 03 exhibited specific fluorescent signals at the sY84, sY86, sY127, sY134, sY254, and sY255 loci, indicating no significant deletions in the AZFa, AZFb, AZFc, and AZFd regions of their Y chromosomes. However, case 01 did not show fluorescence signals at the sY254, sY255, and sY152 loci, suggesting the possibility of deletions in the AZFc and AZFd regions (Table 2).

High-throughput sequencing results of two groups

In the azoospermia group, three cases were included. High-throughput sequencing analysis revealed whole genome amplification (WGA) concentrations ranging from 22 to 46 ng/µL and library concentrations ranging from 8.43 to 9.92 ng/µL. Approximately 2.7–2.8 million unique reads were generated, covering 12.44–13.03% of the genome. Regarding Y chromosome microdeletion detection, all patients showed normal results, with no apparent deletions detected. In the OAT group, three cases were also included. The WGA concentrations of these patients ranged from 20.6 to 44.6 ng/µL, and the library concentrations ranged from 9.73 to 10.5 ng/µL. Approximately 2.5–2.8 million unique reads were generated, covering 11.86–12.81% of the genome. In terms of Y chromosome microdeletion detection, case 1 exhibited a deletion in the AZFc region, with a deletion length of 3.50 Mb, while case 2 exhibited a deletion in the b2/b3 region, with a deletion length of 1.80 Mb. Case 3 showed normal results, with no apparent deletions detected (Figure 1).

Figure 1 High-throughput abnormality analysis results. (A) In the azoospermia group, Y chromosome microdeletion analysis through high-throughput sequencing showed normal results, with no overall abnormalities detected, indicating Klinefelter syndrome patients. (B) In the oligoasthenoteratozoospermia group, case 1 exhibited a deletion in the AZFc region with a length of 3.50 Mb. (C) In the oligoasthenoteratozoospermia group, case 2 exhibited a deletion in the b2/b3 region with a length of 1.80 Mb.

Discussion

The main limitation of this study is the small sample size (n=6), which affects the reliability of statistical conclusions and the general applicability of the results. Future studies with larger sample sizes will be needed to validate these findings. Despite these limitations, the study provides some reference for further exploration of Y chromosome microdeletions and male infertility.

The NGS reference genome used was hg19/GRCh37, which has certain limitations. The GRCh37 reference genome has insufficient coverage in some complex regions, such as highly repetitive sequence regions. For example, certain areas of the Y chromosome, like the pseudoautosomal region (PAR), may have issues with repeated placement or incomplete coverage in GRCh37. This can lead to false-negative results in variant detection and analysis in these regions, especially when using standard analysis pipelines. GRCh37 includes alternative sequences [alternate contigs (ALT)] whose flanking regions are highly similar to the main genome sequence. Most alignment software gives low map quality values (e.g., 0) in these regions, reducing the sensitivity of variant identification.

For Y chromosome testing, fluorescence qPCR is based on STS sequence tag sites, covering discrete sites such as AZFa:SY84/SY86. However, this method can only detect known deletions covered by preset probes. qPCR can identify chimeric deletions at a lower limit of 10% and copy number changes at single sites. High-throughput sequencing (NGS) covers the entire region (AZFa/b/c/d and flanking sequences) and can continuously detect the Yq11.23 region, identifying partial deletions such as gr/gr and b2/b3. NGS can detect chimeric proportions as low as 5%, while also analyzing SNVs/CNVs/structural variations.

In the azoospermia group, both high-throughput sequencing and fluorescence qPCR methods yielded normal results for Y chromosome microdeletion detection. This indicates the consistency and reliability of these two methods in detecting Y chromosome microdeletions in the azoospermia group. In the OAT group, the high-throughput sequencing method detected specific Y chromosome deletions, such as deletions in the AZFc and b2/b3 regions. However, the fluorescence qPCR method did not provide corresponding results for the detection of deletions in the AZFc and AZFd regions, possibly due to the lack of coverage of these loci. In AZFc region testing, qPCR can only determine whether SY254/SY255 is missing, while NGS can clearly distinguish between b2/b3 (partial deletion) and complete AZFc deletion. Therefore, the high-throughput sequencing method demonstrated higher sensitivity and accuracy in the OAT group.

The high-throughput sequencing method offers broader genome coverage and higher resolution, allowing for the detection of smaller Y chromosome deletions. It also provides more detailed information, such as the specific location and length of the deletions, which can better aid in diagnosis and treatment decisions. However, the high-throughput sequencing method is more costly and requires longer analysis time (16). The fluorescence qPCR method, on the other hand, is relatively convenient, rapid, and cost-effective. It can be used for preliminary screening and rapid diagnosis, especially in the OAT group. However, the fluorescence qPCR method has a narrower coverage range and may not detect smaller Y chromosome deletions (17,18). Based on the above analysis, we recommend prioritizing the use of high-throughput sequencing method in the azoospermia group to obtain comprehensive and accurate results for Y chromosome microdeletion detection. For patients in the OAT group, the qPCR method can be used as the preferred initial screening method to obtain rapid results. If the qPCR method shows abnormalities, further confirmation and detailed analysis can be conducted using the high-throughput sequencing method. In actual clinical practice, we need to consider diagnostic needs, resource availability, and cost-effectiveness, and flexibly choose the appropriate testing method. In actual clinical practice, based on the costs of reagents, equipment, manpower, and other factors, the total cost for single-sample qPCR detection of Y chromosome deletions is approximately 350–550 yuan, while the total cost for single-sample NGS detection of Y chromosome deletions ranges from thousands to tens of thousands of yuan. Therefore, adopting a combined detection strategy offers certain cost advantages in clinical practice. It ensures accurate qualitative testing while effectively controlling testing costs and improving testing efficiency.

The qPCR is primarily used to detect microdeletions in specific regions of the Y chromosome, such as AZFa, AZFb, and AZFc. Deletions in AZFa or AZFb typically result in the testes being unable to produce sperm, making it difficult for patients to conceive naturally or through conventional assisted reproductive techniques like intracytoplasmic sperm injection (ICSI). In such cases, patients may need to consider using donor sperm or adoption to fulfill their desire to have children. Patients with deletions in the AZFc region may have varying sperm quality, ranging from none to normal, and sperm count may progressively decline with age. For men with low sperm count, it is advisable to freeze sperm in advance; for those with no sperm, sperm can be retrieved via testicular sperm extraction (TESE) and combined with ICSI to achieve conception. However, since Y chromosome microdeletions are sex-linked, male offspring of affected individuals will inherit the deletion, leading to infertility. Therefore, preimplantation genetic diagnosis can be considered to select female offspring before conception. NGS can identify broader regions of Y chromosome deletions, including combined deletions in multiple areas such as AZFa, AZFb, and AZFc. For patients with complex deletions, fertility may be completely lost, and treatment options may lean more towards using donor sperm or adoption. Accurate diagnosis and personalized treatment plans; NGS can detect small deletions or variations that traditional methods might miss, providing more precise diagnostic information. This aids in formulating tailored treatment strategies.

In summary, this study provides recommendations for the selection of Y chromosome microdeletion detection methods based on seminal analysis results: a comparison between high-throughput sequencing and fluorescence qPCR. These research findings are of great significance in guiding individualized diagnosis and treatment of male infertility patients and provide valuable references for further exploring the association between Y chromosome microdeletions and male infertility.

Conclusions

The selection of Y chromosome microdeletion detection methods based on seminal analysis results is individualized and depends on the specific conditions of the patients. This study recommends that patients with azoospermia prioritize the use of high-throughput sequencing method, while patients with OAT can initially use the fluorescence qPCR method for preliminary screening, and then decide whether to proceed with high-throughput sequencing based on the situation. By considering cost issues and testing needs, the Y chromosome microdeletion detection and individualized treatment of infertile patients can be better guided.

Acknowledgments

We are grateful to the Yikang and Beikang Limited Liability Company for their support with the single cell sequencing experiment.

Footnote

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-593/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-593/prf

Funding: This work was supported by the Natural Science Foundation of Inner Mongolia Autonomous Region, China (No. 2023QN08038); Natural Science Foundation of Inner Mongolia Autonomous Region, China (No. 2024QN08042); Inner Mongolia Medical University Youth Project (No. YKD2024QN004); Inner Mongolia Archives Management Science and Technology Project of Inner Mongolia Autonomous Region, China (No. 2023-10); research project of Inner Mongolia Medical University Affiliated Hospital of the Inner Mongolia Autonomous Region, China (No. 2023NYFYGGO14); Inner Mongolia Grassland Talents Innovation Team Project (2022), Translational medicine project of Inner Mongolia Medical University, Inner Mongolia Medical University Doctoral Program Construction Unit Fund Matching Project (No. RC2300002962); Inner Mongolia Autonomous Region Science and Technology Plan Project (Nos. 2022YFSH0067, 2023KJHZ0038); Science and Technology Program of the Joint Fund of Scientific Research for the Public Hospitals of Inner Mongolia Academy of Medical Sciences (project No. 2024GLLH0264); Natural Science Foundation of the Inner Mongolia Autonomous Region (Project No. 2024QN08017); Youth Project of Inner Mongolia Medical University (project No. YKD2024QN004); and Innovation and Entrepreneurship Project for College Students of Inner Mongolia Medical University (project No. 101322024095).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-24-593/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). This study was approved by the ethics committee of the Affiliated Hospital of Inner Mongolia Medical University (No. YKD202101026).Written informed consent was obtained from all study participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Ascenção C, Sims JR, Dziubek A, et al. A TOPBP1 allele causing male infertility uncouples XY silencing dynamics from sex body formation. Elife 2024;12:RP90887. [Crossref] [PubMed]
2. Coticchio G, Ezoe K, Lagalla C, et al. The destinies of human embryos reaching blastocyst stage between Day 4 and Day 7 diverge as early as fertilization. Hum Reprod 2023;38:1690-9. [Crossref] [PubMed]
3. Huang R, Fu F, Zhou H, et al. Prenatal diagnosis in the fetal hyperechogenic kidneys: assessment using chromosomal microarray analysis and exome sequencing. Hum Genet 2023;142:835-47. [Crossref] [PubMed]
4. Cai M, Guo N, Fu M, et al. Prenatal diagnosis of genetic aberrations in fetuses with pulmonary stenosis in southern China: a retrospective analysis. BMC Med Genomics 2023;16:119. [Crossref] [PubMed]
5. Arasteh H, Gilani MAS, Ramezani-Binabaj M, et al. Microdissection testicular sperm extraction outcomes in azoospermic patients with bilateral orchidopexy. Andrology 2024;12:157-63. [Crossref] [PubMed]
6. De Falco A, Iolascon A, Ascione F, et al. New Insights in 9q21.13 Microdeletion Syndrome: Genotype-Phenotype Correlation of 28 Patients. Genes (Basel) 2023;14:1116. [Crossref] [PubMed]
7. Mottola F, Santonastaso M, Ronga V, et al. Polymorphic Rearrangements of Human Chromosome 9 and Male Infertility: New Evidence and Impact on Spermatogenesis. Biomolecules 2023;13:729. [Crossref] [PubMed]
8. Serpen JY, Presley W, Beil A, et al. A Novel 13q12 Microdeletion Associated with Familial Syndromic Corneal Opacification. Genes (Basel) 2023;14:1034. [Crossref] [PubMed]
9. Chen X, Wu B, Shen X, et al. Relevance of PUFA-derived metabolites in seminal plasma to male infertility. Front Endocrinol (Lausanne) 2023;14:1138984. [Crossref] [PubMed]
10. Leir SH, Paranjapye A, Harris A. Functional genomics of the human epididymis: Further characterization of efferent ducts and model systems by single-cell RNA sequencing analysis. Andrology 2024;12:991-1000. [Crossref] [PubMed]
11. Veneruso I, Cariati F, Alviggi C, et al. Metagenomics Reveals Specific Microbial Features in Males with Semen Alterations. Genes (Basel) 2023;14:1228. [Crossref] [PubMed]
12. Huang J, Yao Y, Jia J, et al. Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis. J Vis Exp 2021; [Crossref] [PubMed]
13. Cao H, Wang C, Cai R, et al. Seminal Plasma ExLncRNA Pairs: Updating Perspectives in the Search for Testicular Spermatozoa Retrieval Biomarkers in Nonobstructive Azoospermia Patients with mTESE by WGCNA. Urol J 2023;20:246-54. [PubMed]
14. Cheng Q, Li L, Jiang M, et al. Extend the Survival of Human Sperm In Vitro in Non-Freezing Conditions: Damage Mechanisms, Preservation Technologies, and Clinical Applications. Cells 2022;11:2845. [Crossref] [PubMed]
15. Habibi M, Abbasi B, Fakhari Zavareh Z, et al. Alpha-Lipoic Acid Ameliorates Sperm DNA Damage and Chromatin Integrity in Men with High DNA Damage: A Triple Blind Randomized Clinical Trial. Cell J 2022;24:603-11. [PubMed]
16. Yousef MS, Megahed GA, Abozed GF, et al. Exogenous gonadotropin-releasing hormone counteracts the adverse effect of scrotal insulation on testicular functions in bucks. Sci Rep 2022;12:7869. [Crossref] [PubMed]
17. Bonsu DNO, Higgins D, Simon C, et al. Quantitative PCR overestimation of DNA in samples contaminated with tin. J Forensic Sci 2023;68:1302-9. [Crossref] [PubMed]
18. Duanmu L, Yu Y, Meng X. Microdroplet PCR in Microfluidic Chip Based on Constant Pressure Regulation. Micromachines (Basel) 2023;14:1257. [Crossref] [PubMed]