Downregulation of miR-211 expression in the blood plasma of infertile men compared to the fertile controls.

OBJECTIVES: Infertility is inability to conceive after 12 months of regular unprotected sex. MiRNA expression changes can serve as potential biomarkers for infertility in males due to impaired spermatogenesis. This research was conducted to measure the expression level of miR-211 in plasma samples as a factor identifying infertility in comparison with the control group. METHODS: In this study, blood plasma were taken from the infertile men (n = 103) nonobstructive azoospermia (NOA) or severe oligozoospermia (SO) and the control group (n = 121). The expression of circulating miR-211 in plasma was assessed by qRT-PCR. A relative quantification strategy was adopted using the 2-ΔΔCT method to calculate the target miR-211 expression level in both study groups. RESULTS: Plasma miR-211 levels were significantly lower in infertile men compared to the control group (0.544 ± 0.028 and 1.203 ± 0.035, respectively, p < 0.001). Pearson's correlation analysis showed that miR-211 expression level has a positive and significant correlation with sperm parameters, including sperm concentration, sperm total motility, progressive motility, and normal morphology (p < 0.001). CONCLUSIONS: Decreased expression of miR-211 in blood plasma seems to be associated with male infertility. This experiment showed that miR-211 can be considered as a biomarker for evaluation, diagnosis, and confirmation of the results of semen analysis in male infertility.

Splicing Mutation in DNALI1 Causes Male Infertility with Severe Oligoasthenoteratozoospermia in Humans.

Oligo-astheno-teratozoospermia (OAT), which is a common cause of male infertility, can be caused by genetic factors. This study reports on a case of a male patient suffering from infertility concomitant with OAT. Whole-exome sequencing (WES) confirmed the presence of a homozygous variant (NM_003462: c.464-1G > A) in the DNALI1 gene via Sanger sequencing. Immunofluorescence staining demonstrated that the DNALI1 signal was nearly undetectable in the patient's sperm. Bioinformatics analysis revealed that this mutation could reverse the splicing of the exon 4 acceptor splice site. A minigene experiment was performed to verify the mutation and the results confirmed that the mutation disrupted the splicing. Our findings show that this rare mutation in DNALI1 contributes to male infertility and OAT in humans, thereby expanding our understanding of the causes and pathogenesis of male infertility. This knowledge facilitates genetic counseling, clinical diagnosis, and therapeutic development of male infertility.

EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions: State of the art 2023.

Testing for AZoospermia Factor (AZF) deletions of the Y chromosome is a key component of the diagnostic workup of azoospermic and severely oligozoospermic men. This revision of the 2013 European Academy of Andrology (EAA) and EMQN CIC (previously known as the European Molecular Genetics Quality Network) laboratory guidelines summarizes recent clinically relevant advances and provides an update on the results of the external quality assessment program jointly offered by both organizations. A basic multiplex PCR reaction followed by a deletion extension analysis remains the gold-standard methodology to detect and correctly interpret AZF deletions. Recent data have led to an update of the sY84 reverse primer sequence, as well as to a refinement of what were previously considered as interchangeable border markers for AZFa and AZFb deletion breakpoints. More specifically, sY83 and sY143 are no longer recommended for the deletion extension analysis, leaving sY1064 and sY1192, respectively, as first-choice markers. Despite the transition, currently underway in several countries, toward a diagnosis based on certified kits, it should be noted that many of these commercial products are not recommended due to an unnecessarily high number of tested markers, and none of those currently available are, to the best of our knowledge, in accordance with the new first-choice markers for the deletion extension analysis. The gr/gr partial AZFc deletion remains a population-specific risk factor for impaired sperm production and a predisposing factor for testicular germ cell tumors. Testing for this deletion type is, as before, left at the discretion of the diagnostic labs and referring clinicians. Annual participation in an external quality control program is strongly encouraged, as the 22-year experience of the EMQN/EAA scheme clearly demonstrates a steep decline in diagnostic errors and an improvement in reporting practice.

AZF microdeletion affects semen parameters, sex hormone levels, and chromosome karyotypes in infertile men in Xinjiang.

This study aimed to analyze the correlation between the microdeletion of different regions of the azoospermia factor (AZF) gene and semen parameters, sex hormone levels, and karyotypes in infertile males by retrospective study. This was performed to obtain a comprehensive understanding of the clinical data of AZF microdeletion in infertile males, to guide clinical diagnoses and treatments, and to improve the efficacy and safety of assisted reproductive technology. For this purpose, Fifty-seven patients with AZF microdeletions and complete data were selected from 1916 patients with AZF microdeletions in our hospital from January 2020 to August 2022. The correlation between semen parameters, sex hormone levels, and chromosome karyotypes of these 57 patients was analyzed. Results showed that among the 57 patients with AZF microdeletions, the region with the highest microdeletion rate was AZFc with 57.89%; single or combined deletions in AZFa and AZFb regions resulted in azoospermia. The deletion frequency of AZFc in the oligospermia group was significantly higher than that in the azoospermia group, and the deletion frequencies of AZFb and AZFb + c in the azoospermia group were significantly higher than those in the oligospermia group (P<0.05). There were statistically significant differences in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, and chromosome karyotypes between patients with azoospermia and oligospermia (P<0.05). Statistically significant differences were observed in prolactin (PRL), FSH, testosterone (T), LH levels, and chromosome karyotypes of patients in different AZF microdeletion regions (P<0.05). In conclusion, AZF microdeletions can lead to a decline in semen quality in men, and different types of deletions have different effects on semen parameters, sex hormone levels, and karyotype analysis. Further treatments should be selected based on the AZF microdeletion area.

Fertility outcomes in men with prior history of anabolic steroid use.

OBJECTIVE: To study sperm parameters recovery and fertility outcomes in men with azoospermia or severe oligospermia caused by anabolic steroid use who underwent a standardized treatment regimen for spermatogenesis recovery. DESIGN AND SUBJECTS: A retrospective analysis of a cohort of men with a prior history of anabolic steroid use and infertility complaints (between 2018 and 2022) was conducted. EXPOSURE: The standardized treatment approach involved discontinuing testosterone replacement therapy and administering a combination regimen of clomiphene citrate and human chorionic gonadotropin for a minimum of 3 to 6 months. MAIN OUTCOME MEASURES: The main outcome measures included changes in sperm parameters, predominantly sperm concentration, and subsequent pregnancy outcomes. RESULTS: A total of 45 men (median age 37 years, IQR 32-45) met the inclusion criteria for this analysis. Median duration of prior T use was 4 years (IQR 1.3-10), with the 2 most common modalities consisting of injection therapy (43.5%) and oral therapy (34.8%). The median initial sperm concentration was 0 million/cc (IQR 0-1.15), and 23 (51.1%) men initially presented with azoospermia. The median duration of combination human chorionic gonadotropin/clomid therapy was 5 months (IQR 3-12). In initially azoospermic men (N: 23), 5 were lost to follow-up, 6 (33.3%) progressed to severe oligospermia (<5 million/cc), 6 (33.3%) to oligospermia (<15 million/cc), 1 (5.6%) to normozoospermia (>15 million/cc), and 5 (27.8%) remained azoospermic after medical treatment for 6 months. Among the 24 couples who responded to the follow-up call, a total of 9 (37.5%) achieved a successful subsequent pregnancy. Of these, 33.3% (3 couples) used assisted reproductive technology, whereas 66.7% (6 couples) conceived naturally. On logistic regression analysis, no significant predictors for improved sperm parameters or successful pregnancy were identified. CONCLUSION: Despite appropriate treatment regimens, a significant proportion of men with a prior history of anabolic steroid use continue to exhibit severe oligospermia, with more than half showing limited improvement in semen parameters after 6 months of treatment. Only a fraction of men achieves normozoospermia after treatment. Further research is needed to explore predictors for improved sperm parameters and successful pregnancy outcomes in men with a history of anabolic steroid use.

Whole exome data prioritization unveils the hidden weight of Mendelian causes of male infertility. A report from the first Italian cohort.

Almost 40% of infertile men cases are classified as idiopathic when tested negative to the current diagnostic routine based on the screening of karyotype, Y chromosome microdeletions and CFTR mutations in men with azoospermia or oligozoospermia. Rare monogenic forms of infertility are not routinely evaluated. In this study we aim to investigate the unknown potential genetic causes in couples with pure male idiopathic infertility by applying variant prioritization to whole exome sequencing (WES) in a cohort of 99 idiopathic Italian patients. The ad-hoc manually curated gene library prioritizes genes already known to be associated with more common and rare syndromic and non-syndromic male infertility forms. Twelve monogenic cases (12.1%) were identified in the whole cohort of patients. Of these, three patients had variants related to mild androgen insensitivity syndrome, two in genes related to hypogonadotropic hypogonadism, and six in genes related to spermatogenic failure, while one patient is mutant in PKD1. These results suggest that NGS combined with our manually curated pipeline for variant prioritization and classification can uncover a considerable number of Mendelian causes of infertility even in a small cohort of patients.

Outcome analysis of ICSI assisted pregnancy using testicular sperm versus ejaculated sperm in man with severe oligozoospermia in the same ART cycle: A case report.

RATIONALE: Intracytoplasmic sperm injection (ICSI) has become the most common method for couples with male factor infertility, and source of sperm for the procedure have evolved over time. but few have examined testicular sperm extraction vs. ejaculated sperm use for severe oligozoospermia in the same assisted reproductive technology (ART) cycle. PATIENT CONCERNS: Here, we evaluated the clinical outcomes after ICSI with testicular sperm or ejaculated in man with severe oligozoospermia in the same ART cycle. A couple who had failed the first ART cycle with ejaculated sperm, using the freshly ejaculated sperm and testicular sperm for ICSI during the second ART cycle by lack of enough sperm to fertilize in an ICSI attempt. DIAGNOSES: The patient was diagnosed with severe oligozoospermia, and routine semen analysis revealed sperm concentration is less than 2 million/mL. INTERVENTIONS: The patient using testicular sperm versus ejaculated sperm with ICSI assisted pregnancy in the same ART cycle. OUTCOMES: We found that superior cleavage rate, number of embryos transferred and blastocyst rate with the use of testicular rather than ejaculated sperm-ICSI in the couple. The results described here suggest that use of testicular sperm may improve biologic outcomes, especially for couples with male-partner oligozoospermia who previous ICSI failures. LESSONS: Our case report supported the efficacy of testicular sperm preference over ejaculated sperm for ICSI in men with severe male factor infertility. It is a paradigm shift concerning the use of ejaculated sperm as the preferable source of sperm for ICSI, add to the small amount of literature on testicular sperm extraction vs. ejaculated sperm use for severe oligozoospermia in the same ART cycle.

Compensated Hypospermatogenesis: Elevated Follicle-stimulating Hormone Predicts Decline in Semen Parameters Among Men With Normal Index Semen Analysis.

OBJECTIVE: To determine whether men with elevated follicle-stimulating hormone (FSH) and normal semen analysis (SA) are more likely to experience a decline in semen parameters over time compared to men with normal FSH. METHODS: Men presenting for fertility evaluation between 2002 and 2020 with normal initial SA were dichotomized according to baseline FSH as normal (<7.6 IU/mL) vs elevated (≥7.6 IU/mL). Primary outcomes included the development of abnormal sperm concentration (<15 million/mL) and total motile sperm count <9 million. Secondary outcomes included abnormal sperm motility (<40%), morphology (<4%), and total number of SA abnormalities. RESULTS: The final sample consisted of 858 men; 776 had normal FSH, and 82 had elevated FSH at presentation. Compared to men with normal FSH, men with elevated FSH had lower total motile sperm count (64.1 vs 107.3, P < .001) and higher testosterone levels (339 ng/dL vs 309 ng/dL, P = .03). At each follow-up timepoint, more men with elevated FSH had oligospermia compared to men with normal FSH. Men with elevated FSH were more likely to experience a decline in total motile sperm count below the intrauterine insemination threshold of 9 million and more likely to develop SA abnormalities over time. CONCLUSION: In men presenting for fertility evaluation with normal index SA, elevated FSH was associated with subsequent decline in semen parameters over time. Men with elevated FSH and normal SA, a condition we have termed compensated hypospermatogenesis, represent an at-risk population for whom close follow-up is warranted.

The comparison of Y chromosome microdeletion incidence in blood DNA and sperm cell DNA.

BACKGROUND: During gamete development and spermatogenesis, certain genes on the Y chromosome (Yq) in the Male-Specific Region (MSR) are responsible for human gametes formation. The long arm Yq is composed of both euchromatin and the genetically inactive heterochromatin regions. This region contains the Azoospermia factors AZFa, AZFb and AZFc. In the case of male infertility, microdeletions on the Yq chromosome appear to be structural chromosomal anomalies linked to sperm abnormality. METHODS: The present study aimed to look at the incidence, of Asthenospermia (AS), Teratospermia (TS), Oligospermia (OS) and Oligoasthenoteratospermia (OAT) patterns of Y chromosomal microdeletions in Indian infertile men with an (AZF a, b, c). This study was conducted with 75 infertile men as cases and 75 fertile men as a control for AZF locus microdeletion utilizing sequence-tagged sites. RESULTS: The AZFc region of germ cell DNA (50.6%) was the most deleted section in infertile men when compared to blood DNA (21.3%), followed by deletions in the AZFb region (21.3%) in germ cell DNA whereas blood DNA had no microdeletion in the AZFa region in both germ cell DNA and blood DNA. Infertile men displayed significant Yq microdeletion in both AZFb and also AZFc. Around 33% (25) of 75 infertile men had AZF (a, b, c) region microdeletion in blood DNA, compared to it germ cell DNA had a larger percentage of 72% (54) of Y chromosome microdeletions in the study samples. CONCLUSION: A high-frequency rate of microdeletions seen in germ cell DNA. PCR-based Y chromosome microdeletion screening using germ cell DNA along with Genomic DNA might help in screening for genetic abnormality in infertile men who endure assisted reproductive technology treatments. This study might be attributable to the interplay of lifestyle and genetic factors, both contributing to the risk of developing these germ-line deletions.

Initial Andrological Evaluation of the Infertile Male.

Evaluation of a man presenting for couple infertility starts with obtaining a thorough medical history, and the couple's reproductive and sexual history. Semen analysis, according to World Health Organization recommendations, is the cornerstone of male partner evaluation. A comprehensive physical examination is essential for every male partner, with adequate assessment of the development of secondary sexual characteristics. However, more complex testing than semen analysis (eg, sperm DNA fragmentation index) may be required in selected cases. For men with oligozoospermia or azoospermia, a hormonal evaluation should be performed, including at least total testosterone, follicle stimulating hormone, and luteinising hormone. Genetic testing should be offered to azoospermic men and those with severe oligozoospermia. Ultrasound of the genitourinary tract can provide additional information in select cases. PATIENT SUMMARY: A complete medical history, physical examination, and semen analysis are essential components of the initial evaluation of men being assessed for infertility. The evaluation must proceed in parallel for male and female partners to optimise management for infertile couples.

Metabolomic analysis of seminal fluids in infertile individuals.

OBJECTIVE: Infertility impacts a substantial number of couples worldwide, and about 50% of cases are linked to male factors. The analysis of seminal fluid composition can improve diagnostic accuracy and offer deeper insights into the pathophysiology of male factor infertility. This study seeks to identify novel markers for diagnosing and treating male infertility by comparing organic acid profiles in the seminal fluid of individuals with normospermia, oligospermia, and azoospermia. PATIENTS AND METHODS: Semen samples were collected from men with normospermia, oligospermia, and azoospermia. The organic acid profile in the seminal fluid was analyzed using liquid chromatography-mass spectrometry/mass spectrometry (LC/MS-MS). Data analysis was performed using SPSS and MetaboAnalyst. RESULTS: The study revealed significant differences in metabolite levels among normospermic, oligospermic, and azoospermic individuals. In groups with oligospermia, there were significant decreases in the levels of 2-OH-Isovaleric Acid, 3-Methyl-2-Oxovaleric Acid, Ethyl-Malonic Acid, Citric Acid, Oxoproline, Malic Acid, N-Acetyl-Aspartic Acid, Suberic Acid, Glutaconic Acid, and Succinic Acid. Similarly, individuals with azoospermia exhibited a notable reduction in the levels of Citric Acid, Malic Acid, and Suberic Acid. Furthermore, according to the Variable Importance in the Projection (VIP) score analysis, Ethyl-Malonic Acid, Glycolic Acid, and 3-Methyl-2-Oxovaleric Acid were identified as crucial factors for diagnosis and potential treatment strategies. CONCLUSIONS: The data obtained from the study highlights the significant potential of metabolites in assessing infertility and gaining a more in-depth understanding of the underlying pathological mechanisms.

A Comparative Investigation Applying Testicular Fine Needle Aspiration Cytology and Open Testicular Biopsy Histology for the Diagnosis of Azoospermia and Severe Oligospermia.

Open testicular biopsy histology and fine needle aspiration cytology (FNAC) are the most popular tests used to diagnose male infertility. This study aimed to assess the cytological characteristics of 186 infertile males aged 24-63 with testicular FNAC. Furthermore, the existing relationship between males with severe oligospermia (sperm count: 5 million/ml) and azoospermia was investigated via both cytological and histological diagnosis methods. With a 1.5-inch and 25-gauge needle, the testis was aspirated from three locations (the upper, middle, and lower poles). Papanicolaou stain or Giemsa stain was used to make smears on albumenized slides, which were then dried in the air and stained. A biopsy of the testicles was performed there, preserved in Bouins solution, processed as usual, and stained with hematoxylin and eosin stain. According to our findings, 66.7% of patients had secondary maturation arrest, whereas 18.3% and 15.1% of them had hypospermatogenesis and Sertoli cell only (SCO). Results of the comparison showed that both procedures were very similar. According to biopsy histological examinations, only 3 (1.6%) of the 28 normal FNAC instances had hypospermatogenesis with lymphocyte infiltration. The majority of SCO patients were over 50 years old. These findings revealed that FNAC is more effective than testicular histology for the assessment of male infertility.

Semen parameter variability among users of at-home sperm testing kits.

BACKGROUND: Despite the generally accepted World Health Organization guidelines on semen analysis, an individual's results can display significant variation when performed across time or in different laboratories. Semen parameters are in fact highly variable measures that can differ significantly between various analyses. Numerous researchers have discovered a wide range of semen parameters within each individual male, but only a few studies included the analysis of semen parameters variability in patients with infertility. The aim of this study was to evaluate the inter- and intra-individual variability of semen parameters in men of reproductive age with normozoospermia and those with oligozoospermia. METHODS: Five hundred and thirteen who provided ≥ 2 semen samples (798 samples in total) using an at-home mail-in kit over a period of about 2 years were enrolled in the study. Semen samples collection using Give Legacy at-home mail-in semen collection kit; semen analysis at a CLIA-certified laboratory. RESULTS: The degree of intra-subject variation across all semen parameters was lower in men with normozoospermia compared to men with oligozoospermia. Men with normozoospermia furthermore demonstrated a level of intra-subject variation that was lower than inter-subject variation across all measured parameters. No association was observed between intra-subject coefficients of variation in any of the semen parameters, including sperm concentration, sperm count, motile sperm count, total motility, progressive motility, the percentage of sperm with normal morphology, and the age, duration of abstinence, and BMI of the men. CONCLUSION: The results of this observational study confirm the significant variability in semen parameters in men with normozoospermia and oligozoospermia, as measured from at-home semen collection kit samples. This further underscore the importance of securing multiple samples for analysis to provide a robust assessment of male fertility.

Cost-effectiveness of Early Screening Home Semen Analysis in Couples Attempting to Conceive.

OBJECTIVE: To study the cost-effectiveness of incorporating home semen analysis in screening for oligospermia and expediting time to evaluation. METHODS: A decision analytic model was built using inputs from the medical literature. The index patient is the male partner in a couple seeking fertility, and entry into the model was assumed to be at the inception of the couple's attempts to conceive via natural means. Three main strategies are described and analyzed: (1) baseline strategy of no testing, (2) utilization of a home semen testing kit, (3) universal testing via a clinic visit and gold standard lab semen analysis. The primary outcome was detection of oligospermia (defined as sperm concentration <15 million/mL). Strategies were ranked by months to evaluation by a male infertility specialist saved. Costs were considered from the patient perspective and were incorporated to determine the incremental cost per month saved to evaluation (ICMS) per 100,000 patients. RESULTS: Compared to a baseline strategy of no screening, utilizing a home test would save 89,000 months at the incremental cost of $7,418,000 for an ICMS of $45.51. Shifting to a strategy of universal gold standard clinic and lab testing saves an additional 3000 months but at an ICMS of $17,691 compared to the home testing strategy. CONCLUSION: Widespread adoption and the early usage of home semen analysis may be a cost-effective method of screening for oligospermia and facilitating further evaluation with an andrology specialist.

Genomic testing for copy number and single nucleotide variants in spermatogenic failure.

PURPOSE: To identify clinically significant genomic copy number (CNV) and single nucleotide variants (SNV) in males with unexplained spermatogenic failure (SPGF). MATERIALS AND METHODS: Peripheral blood DNA from 97/102 study participants diagnosed with oligozoospermia, severe oligozoospermia, or non-obstructive azoospermia (NOA) was analyzed for CNVs via array comparative genomic hybridization (aCGH) and SNVs using whole-exome sequencing (WES). RESULTS: Of the 2544 CNVs identified in individuals with SPGF, > 90% were small, ranging from 0.6 to 75 kb. Thirty, clinically relevant genomic aberrations, were detected in 28 patients (~ 29%). These included likely diagnostic CNVs in 3/41 NOA patients (~ 7%): 1 hemizygous, intragenic TEX11 deletion, 1 hemizygous DDX53 full gene deletion, and 1 homozygous, intragenic STK11 deletion. High-level mosaicism for X chromosome disomy (~ 10% 46,XY and ~ 90% 47,XXY) was also identified in 3 of 41 NOA patients who previously tested normal with conventional karyotyping. The remaining 24 CNVs detected were heterozygous, autosomal recessive carrier variants. Follow-up WES analysis confirmed 8 of 27 (30%) CNVs (X chromosome disomy excluded). WES analysis additionally identified 13 significant SNVs and/or indels in 9 patients (~ 9%) including X-linked AR, KAL1, and NR0B1 variants. CONCLUSION: Using a combined genome-wide aCGH/WES approach, we identified pathogenic and likely pathogenic SNVs and CNVs in 15 patients (15%) with unexplained SPGF. This value equals the detection rate of conventional testing for aneuploidies and is considerably higher than the prevalence of Y chromosome microdeletions. Our results underscore the importance of comprehensive genomic analysis in emerging diagnostic testing of complex conditions like male infertility.

Prevalence of Hyperprolactinemia and Clinically Apparent Prolactinomas in Men Undergoing Fertility Evaluation.

OBJECTIVE: To determine prevalence of hyperprolactinemia and prolactinoma among men presenting for initial fertility evaluation. METHODS: We performed a retrospective review of men presenting for initial fertility evaluation at a tertiary care, academic health system between 1999 and 2018. Men with measured prolactin levels were analyzed to determine prevalence of hyperprolactinemia and prolactinoma. We compared clinical characteristics of men with and without hyperprolactinemia. Univariable and multivariable analysis were used to determine factors associated with hyperprolactinemia. We assessed effects of hyperprolactinemia and prolactinoma on testosterone levels, semen parameters and pregnancy outcomes after treatment. RESULTS: A total of 3101 men had serum prolactin level measured. 65 (2.1%) had hyperprolactinemia. Patients with hyperprolactinemia had lower testosterone (median 280 ng/dL vs 313 ng/dL, P = 0.038) and lower total motile sperm count (median 7.0 million vs 34.7 million, P = 0.001) compared to men without hyperprolactinemia. 43.1% of men with hyperprolactinemia had oligospermia vs 21.5% of men without hyperprolactinemia (P<0.001). Univariable analysis demonstrated that men with elevated luteinizing hormone (LH) (OR 1.077, P = 0.001) and follicle-stimulating hormone (FSH) (OR 1.032, P = 0.002) were more likely to have hyperprolactinemia. Men with oligospermia were more likely to have hyperprolactinemia (OR 2.334, P = 0.004). On multivariable analysis, neither hormone parameters nor oligospermia were associated with elevated prolactin (P>0.05). Of the 65 men with hyperprolactinemia, 11 (17%) were diagnosed with a prolactinoma, resulting in an overall prevalence of 11 in 3101 (0.35%). CONCLUSION: The overall prevalence of prolactinoma in our cohort of men undergoing fertility evaluation was 35-fold higher than the prevalence in the general male population.

Comprehensive copy number analysis of Y chromosome-linked loci for detection of structural variations and diagnosis of male infertility.

Infertility affects about 15% of heterosexual couples and male factors account for ~45-50% of clinical cases. Genetic factors play an important role in male infertility and thus we try to develop a cost-effective method for screening the genetic factors in male infertility. In our retrospective proof-of-concept study, we employed the high-throughput ligation-dependent probe amplification (HLPA) to examine the copy number by 115 genomic loci covering the Y chromosome, and 5 loci covering the X chromosome-specific region. We identified 8 sex chromosome aneuploid people from the low sperm concentration (LSC) group, and Y chromosome-specific microdeletion/duplications in 211 samples from the LSC group, and in 212 samples from the control group. 35 samples showed complete loss of AZFc (BPY2 to CDY1B deletion), which was not observed in controls. Nevertheless, a partial loss of AZFc (BPY2 to BPY2B deletion) was detected at comparable frequencies in both groups (68/211 vs. 108/212, respectively). And we further found structural variations in 28.6 and 26.9% samples from infertility and fertility groups. Moreover, we found that there were lower copy numbers for heterochromatic sequences in men with LSC. Especially, we reported that ultra-low relative copy number (RCN) (<0.5) type and low RCN (0.5 to <0.75) type in Yq12 were more often in the LSC group for the first time. Our results not only shed light on the potential role of low RCN in Yq12 in male infertility but also showed that HLPA can be a powerful and cost-effective tool for clinical screening in male infertility.

Abnormal Y chromosome detection in infertile males using multiplex ligation-dependent probe amplification.

Y chromosome abnormalities are the leading cause of male infertility. The clinical detection of abnormalities is necessary for appropriate genetic counselling. This study describes the prevalence, distribution and characteristics of Y chromosome abnormalities, which should be considered in the clinical management of infertile males. A total of 121 patients with oligozoospermia, 120 with azoospermia and 88 normal individuals were recruited between June 2019 and July 2021. Y chromosome microdeletions were assessed using multiplex ligation-dependent probe amplification (MLPA). The abnormal Y chromosome prevalence was 30.70%, and it was most common in patients aged 26-40 years. The frequencies of azoospermia factor (AZF) deletion, duplication and deletions/duplications were 19.76%, 9.42% and 1.52% respectively. The most common abnormalities were AZFc deletion (19.80%), AZFc partial deletion (40.59%) and AZFc partial duplication (17.82%). Oligozoospermia was associated with an increased incidence of AZF deletion. In the subgroup analysis, patients <30 years old with azoospermia exhibited elevated follicle-stimulating hormone levels and oestradiol. Moreover, the incidence of AZF deletion was higher in those with azoospermia (OR: 2.12; 95% CI: 1.05-5.28; p = 0.023) or oligozoospermia (OR: 2.54; 95% CI: 1.13-5.79; p = 0.008) than in normal individuals for ages ≥30 years.