Y Chromosome Microdeletion Research Articles

A New Sperm Concentration Threshold for Y Chromosome Microdeletion Analysis in Infertile Men: Could It Be Azoopermia?

We aimed to assess the frequency of Y-chromosome microdeletions (YCMs) in a non-multiethnic urban population in our region, define predictive factors, and determine a new clinical threshold for YCMs in infertile men. A total of 281 patients with a sperm concentration ≤5 million/mL were retrospectively evaluated. Oligozoospermic and/or azoospermic patients with a sperm concentration of ≤5 million/mL were screened for the YCM analysis. Y-chromosome microdeletion was detected in 9 (3.2%) of the 281 patients. All patients with YCM were azoospermic. The presence of azoospermia, a high folliclestimulating hormone level, and a high luteinizing hormone level were found to be important determinants for the identification of a microdeletion (P = .002, P = .002, and P=.021, respectively). If the presence of azoospermia and a sperm concentration threshold of <1 million/mL had been applied for the YCM test, the number of tests performed would have been reduced by 54.4% (153 tests) and 42.7% (120 tests), respectively, resulting in cost saving of approximately $11 474 and $9000, respectively. We recommend that the threshold for sperm concentration for YCM analysis be set at <1 million in individuals in developed countries and only in patients with azoospermia in developing countries, in order to reduce costs and save labor by excluding unnecessary tests. These proposed thresholds (azoospermia and sperm counts less than <1 million/mL) provide cost-effectiveness by significantly reducing the number of genetic tests ordered without affecting the diagnosis rate.

A nomogram predicting testicular sperm extraction success in men with non-obstructive azoospermia: A multi-center study

ObjectivesTo develop an efficient and easy-to-use nomogram which can predict testicular sperm extraction(TESE) success in men with non‐obstructive azoospermia(NOA) by using pre-operative parameters. Materials and MetodsData of 3093 men who underwent TESE for NOA were included in this study. Demographic data, testis volumes, presence of varicocele, length of infertility, history of previous surgeries, history of genitourinary infections, smoking status, chromosome abnormalities, presence of Y-chromosome microdeletion, family history of infertility, testis biopsy, surgical data, sperm retrieval rate, final pathology obtained at testicular sperm extraction, follicle stimulating hormone, luteinizing hormone, testosterone levels were recorded. The primary outcome was to develop an efficient nomogram which can predict the TESE success in men with NOA. The secondary outcomes were identifying the significant pre-operative parameters which are associated with success in TESE. ResultsSperm retrieval rate was 50.2%(1553/3093). Testis volume, history of varicocelectomy, chromosome abnormalities, presence of Y-chromosome microdeletion were shown to affect sperm retrieval rate significantly(p<0.05). Sperm retrieval success was higher in men with older age, higher testis volume(>10mL), lower follicle stimulating hormone level(≤ 12.92mIU/mL), lower luteinizing hormone level, and higher testosterone level(p<0.05). Only testis volume and patient’s age were associated with succesful sperm retrieval in multivariate logistic regression analysis. ConclusionIn men with NOA, high testis volume, old age, low follicle stimulating hormone level, low luteinizing hormone level, and high testosterone level are advantageous for successful sperm retrieval in TESE. Herein we present a nomogram which can predict the outcome of TESE in men with NOA with adequate success.

Innovative all-in-one exome sequencing strategy for diagnostic genetic testing in male infertility: Validation and 10-month experience.

Current guidelines indicate that patients with extreme oligozoospermia or azoospermia should be tested for chromosomal imbalances, azoospermia factor (AZF) deletions and/or CFTR variants. For other sperm abnormalities, no genetic diagnostics are recommended. To determine whether exome sequencing (ES) with combined copy number variant (CNV) and single nucleotide variant (SNV) analysis is a reliable first-tier method to replace current methods (validation study), and to evaluate the diagnostic yield after 10 months of implementation (evaluation study). In the validation study, ES was performed on DNA of patients already diagnosed with AZF deletions (n=17), (non-)mosaic sex chromosomal aneuploidies or structural chromosomal anomalies (n=37), CFTR variants (n=26), or variants in known infertility genes (n=4), and 90 controls. The data were analyzed using our standard diagnostic pipeline, with a bioinformatic filter for 130 male infertility genes. In the evaluation study, results of 292 clinical exomes were included. All previously reported variants in the validation cohort, including clinically relevant Y-chromosomal microdeletions, were correctly identified and reliably detected. In the evaluation study, we identified one or more clinically relevant genetic anomalies in 67 of 292 of all cases (22.9%): these included aberrations that could have been detected with current methods in 30 of 67 patients (10.2% of total), (possible) (mono)genetic causes in the male infertility gene panel in 28 of 67 patients (9.6%), and carriership of cystic fibrosis in nine of 67 patients (3.1%). ES is a reliable first-tier method to detect the most common genetic causes of male infertility and, as additional genetic causes can be detected, in our evaluation cohort the diagnostic yield almost doubled (10.2%-19.8%, excluding CF carriers). A genetic diagnosis provides answers on the cause of infertility and helps the professionals in the counseling for treatment, possible co-morbidities and risk for offspring and/or family members. Karyotyping will still remain necessary for detecting balanced translocations or low-grade chromosomal mosaicism.

Updates to Male Infertility: AUA/ASRM Guideline (2024).

In 2023 the American Urological Association (AUA) requested an Update Literature Review (ULR) to incorporate new evidence generated since the 2020 publication of this Guideline. The resulting 2024 Guideline Amendment addresses updated recommendations to provide guidance on the appropriate evaluation and management of the male partner in an infertile couple. In 2023, the Male Infertility Guideline was updated through the AUA amendment process in which newly published literature is reviewed and integrated into previously published guidelines. An updated literature search identified 4093 new abstracts. Following initial abstract screening, 125 eligible study abstracts met inclusion criteria. On data extraction, 22 studies of interest were included in the final evidence base to inform the Guideline amendment. The Panel developed evidence- and consensus-based statements based on an updated review to provide guidance on evaluation and management of male infertility. These updates are detailed herein. This update provides several new insights, including revised thresholds for Y-chromosome microdeletion testing, indications for pelvic MRI imaging in infertile males, and guidance regarding the use of testicular sperm in nonazoospermic males. This Guideline will require further review as the diagnostic and treatment options in this space continue to evolve.

Clinical Analysis of Y Chromosome Microdeletions and Chromosomal Aberrations in 1596 Male Infertility Patients of the Zhuang Ethnic Group in Guangxi.

The long arm of the Y chromosome (Yq) contains many amplified and palindromic sequences that are prone to self-reorganization during spermatogenesis, and tiny submicroscopic segmental deletions in the proximal Yq are called Y chromosome microdeletions (YCM). A retrospective study was conducted on male infertility patients of Zhuang ethnicity who presented at Reproductive Medical Center of Nanningbetween January 2015 and May 2023. Seminal fluid was collected for standard examination. YCM were detected by using a combination of multiplex PCR and agarose gel electrophoresis. Preparation of peripheral blood chromosomes and karyotyping of chromosomes was performed. 147 cases (9.22%) of YCM were detected in 1596 maleinfertility patients of Zhuang ethnicity. Significant difference was found in the detection rate of YCM between the azoospermia group and the oligospermia group (P < 0.001). Of all types of YCM, the highest detection rate was AZFc (n = 83), followed by AZFb + c (n = 28). 264 cases (16.54%) of sex chromosomal aberrations were detected. The most prevalent karyotype was 47, XXY (n = 202). The detection rate of sex chromosomal aberrations in azoospermia group was higher than that in severe oligospermia group and oligospermia group, and the differences were significant (P < 0.001). 28 cases (1.57%) of autosomal aberrations and 105 cases (6.58%) of chromosomal polymorphism were identified. The current research has some limitations due to the lack of normal men as the control group but suggests that YCM and chromosomal aberrations represent key genetic factors influencing spermatogenesis in infertile males of Zhuang ethnicity in Guangxi.

Testicular sperm aspiration (TESA) outcome in Middle Eastern patients with non-obstructive azoospermia: a retrospective cohort study

BackgroundInfertility is an important health problem, affecting couples worldwide. Non-obstructive azoospermia is the most severe form of azoospermia, which is mostly idiopathic or caused by different causes such as chemotherapy and genetic disorders. Testicular fine needle aspiration (or testicular sperm aspiration (TESA)) is simple, cost-effective and less invasive than testicular sperm extraction.Materials and methodsThree hundred twenty Middle Eastern patients with NOA were recruited in this study. The patients underwent routine infertility assessment including medical, surgical, and reproductive history, physical examination, semen analysis, and hormonal profile including FSH, Testosterone, and inhibin B in addition to Genetic assessment including karyotype and Y-chromosome micro-deletion.ResultsTesticular sperm aspiration was positive in 70 patients (22.18%). Serum FSH levels were clearly elevated in the patients with negative sperm retrieval (mean = 21.39 U/L), while they were reduced in the patients with positive sperm retrieval (mean = 14.61 U/L). Testosterone value did not clearly correlate with the results of testicular sperm aspiration in the two groups of patients, and testicular volume was normal for most of the patients in the two groups. Patients with Y-chromosome micro-deletion were 11.22% of the total patients studied and they had negative TESA results, while 13.12% of patients had Klinefelter Syndrome and their TESA results were negative.ConclusionWe confirmed that there are many factors that negatively affect Testicular sperm aspiration results: high FSH and low inhibin B levels, smoking, and genetic disorders. Despite the absence of sperm in the semen, some NOA patients have a chance to have children by using this technique.

A Comprehensive Analysis of Y-Chromosome Microdeletions and Their Relationship to Male Infertility and Lifestyle Variables.

Male infertility is the inability of a male to conceive a fertile female during at least a year of unprotected sexual activity. A variety of medical conditions and treatments cause male infertility. Y chromosome microdeletion is an important cause of infertility among males. Various epidemiological factors also play a role in the occurrence of infertility. Our study aims to determine the association between Y-chromosome microdeletion and age, sperm count, body mass index (BMI), alcohol, and tobacco consumption. This study was conducted in 70 male infertility cases. Data was collected from 2018 to 2023 at the Genetic Lab, Department of Anatomy, All India Institute of Medical Sciences, Raipur, Chhattisgarh, India. Demographic profiles, including age, sperm count, weight and height, and history of smoking and drinking, were collected from individuals. BMI was calculated, and chromosome analysis was done for Y chromosome microdeletion. Both multiplex and singleplex methods were used to determine the microdeletion using a thermocycler (Applied Biosystems, VeritiTM96-well Fast Thermal Cycler, 0.2 ml USA) in AZF, and the association between age, sperm count, BMI, alcohol, and tobacco was determined. The number of regions deleted among individuals varies from one to seven. Regions Sy746, Sy143, and Sy145 were found to be commonly deleted. We found a positive, but not statistically significant, correlation between age and microdeletion (point biserial correlation coefficient (r) = 0.2, p-value = 0.097). When comparing age with sperm count, the results showed a negative correlation, highlighting the influence of age on sperm count (r (68) = 0.284, p = 0.017). In comparing BMI and microdeletion, no significant relationship (χ² = 3.7, p = 0.296) indicated independence between them. According to our observations, microdeletion affects all smokers and 45% of non-smokers. We found a significant association between smoking and microdeletion (χ2 = 4.49, P = 0.034). There was no statistically significant relationship between microdeletion and drinking (χ²(3) = 5.65, p = 0.13). We discovered a significant positive association between smoking and a positive, but not statistically significant, correlation between age, BMI, and drinking, as well as a microdeletion. There are probably a lot of unidentified variables that affect successful fertilization and implantation. These could include variables that affect fertility and the success of reproduction on an environmental, genetic, and epigenetic level. The study reveals that Y chromosome microdeletion and other epidemiological factors coexist concurrently in cases of infertility. Assessing these variables is crucial for infertile patients. A community-based, comprehensive survey is required to assess the overall consequences of various epidemiological factors on infertility.

Molecular Study on Y Chromosome Microdeletion in Male Infertility: A Cross-Sectional Design in Indonesian Men

Y chromosome microdeletions (YCMs) are one kind of genetic disorder that contributes to male infertility. This study aims to determine the profile of YCMs in the infertile male population in Indonesia. This cross-sectional study was conducted by identifying YCMs testing data on 49 infertile male patients identified with azoospermia and oligoasthenoteratozoospermia (OAT) based on their sperm analysis, who visited andrology polyclinics in several hospitals in Yogyakarta Province between March 2021 to August 2022. Study participants underwent YCMs testing at the Molecular Medicine and Therapy Research Laboratory, Muhammadiyah University of Yogyakarta, Indonesia, using the Polymerase Chain Reaction (PCR) method according to the procedures established by the laboratory. Four out of 49 (8.2%) participants were identified to have YCMs with deletions in the Azoospermia Factor C (AZFc) subregion. Two participants identified with YCMs had cryptozoospermia in their sperm analysis. Hormonal examination showed variable results in 4 participants, including hypergonadotropic, hypogonadism, and normogonad. All participants in the study identified with YCMs showed a deletion in the AZFc subregion. This type of deletion is different from previous studies in Indonesia, so broad examinations of infertile male patients are required to figure out the deletion profile in a larger population of Indonesian sterile males.

An analysis of Y-chromosome microdeletion in infertile Korean men with severe oligozoospermia or azoospermia.

Infertility affects 10% to 15% of couples, and male factor accounts for 50% of the cases. The relevant male genetic factors, which account for at least 15% of male infertility, include Y-chromosome microdeletions. We investigated clinical data and patterns of Y-chromosome microdeletions in Korean infertile men. A total of 919 infertile men whose sperm concentration was ≤5 million/mL in two consecutive analyses were investigated for Y-chromosome microdeletion. Among them, 130 infertile men (14.1%) demonstrated Y-chromosome microdeletions. Medical records were retrospectively reviewed. In 130 men with Y-chromosome microdeletions, 90 (69.2%) had azoospermia and 40 (30.8%) had severe oligozoospermia. The most frequent microdeletions were in the azoospermia factor (AZF) c region (77/130, 59.2%), followed by the AZFb+c (30/130, 23.1%), AZFa (8/130, 6.2%), AZFb (7/130, 5.4%), AZFa+b+c (7/130, 5.4%), and AZFa+c (1/130, 0.7%) regions. In men with oligozoospermia, 37 (92.5%) had AZFc microdeletion. Chromosomal abnormalities were detected in 30 patients (23.1%). Higher follicle-stimulating hormone level (23.2±13.5 IU/L vs. 15.1±9.0 IU/L, p<0.001), higher luteinizing hormone level (9.7±4.6 IU/L vs. 6.0±2.2 IU/L, p<0.001), and lower testis volume (10.6±4.8 mL vs. 13.3±3.8 mL, p<0.001) were observed in azoospermia patients compared to severe oligozoospermia patients. Y-chromosome microdeletion is a common genetic cause of male infertility. Therefore, Y-chromosome microdeletion test is recommended for the accurate diagnosis of men with azoospermia or severe oligozoospermia. Appropriate genetic counseling is mandatory before the use of assisted reproduction technique in men with Y-chromosome microdeletion.

AZFa, AZFb, AZFc and gr/gr Y-chromosome microdeletions in azoospermic and severe oligozoospermic patients, analyzed from a neural network perspective

AZFa, AZFb, AZFc and gr/gr Y-chromosome microdeletions in azoospermic and severe oligozoospermic patients, analyzed from a neural network perspective

Y chromosome microdeletions in Chinese men with infertility: prevalence, phenotypes, and intracytoplasmic sperm injection outcomes

BackgroundThe incidence of Y chromosome microdeletions varies among men with infertility across regions and ethnicities worldwide. However, comprehensive epidemiological studies on Y chromosome microdeletions in Chinese men with infertility are lacking. We aimed to investigate Y chromosome microdeletions prevalence among Chinese men with infertility and its correlation with intracytoplasmic sperm injection (ICSI) outcomes.MethodsThis single-center retrospective study included 4,714 men with infertility who were evaluated at the Reproductive Center of the First Affiliated Hospital of Sun Yat-sen University between May 2017 and January 2021. Semen analysis and Y-chromosome microdeletion via multiplex polymerase chain reaction were conducted on the men. The study compared outcomes of 36 ICSI cycles from couples with male azoospermia factor (AZF)cd deletions with those of a control group, which included 72 ICSI cycles from couples without male Y chromosome microdeletions, during the same period. Both groups underwent ICSI treatment using ejaculated sperm.ResultsAmong 4,714 Chinese men with infertility, 3.31% had Y chromosome microdeletions. The combined deletion of sY254 and sY255 in the AZFc region and sY152 in the AZFd region was the prevalent pattern of Y chromosome microdeletion, with 3.05% detection rate. The detection rates of AZF deletions in patients with normal total sperm count, mild oligozoospermia, severe oligozoospermia, cryptozoospermia, and azoospermia were 0.17%, 1.13%, 5.53%, 71.43%, and 7.54%, respectively. Compared with the control group, the AZFcd deletion group exhibited no significant difference in the laboratory results or pregnancy outcomes of ICSI cycles using ejaculated sperm.ConclusionsThis is the largest epidemiological study on Y chromosome microdeletions in Chinese men with infertility. The study results underline the necessity for detecting Y chromosome microdeletion in men with infertility and severe sperm count abnormalities, especially those with cryptozoospermia. The combined deletion of sY254 and sY255 in the AZFc region and sY152 in the AZFd region was the most prevalent Y chromosome microdeletion pattern. Among patients with AZFcd deletion and ejaculated sperm, ICSI treatment can result in pregnancy outcomes, similar to those without AZFcd deletion.

#110 : Diagnostic Approach and Clinical Recommendations for Non-obstructive Azoospermia: Global Andrology Forum Survey Results

Background and Aims: Non-obstructive azoospermia (NOA) is the most common form of male infertility, characterized by an absence of sperm in ejaculate and is caused by testicular failure. While NOA afflicts 10-38.3% of infertile males, there is no specific recommendations in EAA/EAU guidelines on its clinical diagnosis or WHO guidelines on semen analysis. Therefore, we conducted a global survey to assess the standard approach of practitioners in the diagnosis and management of NOA. Method: The Global Andrology Forum (GAF) conducted a survey on 436 independent practicing entities from 55 countries. We compiled data from 336 respondents (andrologists, urologists, fertility specialists). Information collected included frequency of NOA, varicocele, frequency of obtaining semen, interval between obtaining semen samples, hormone evaluation (FSH, LH, prolactin, total/free testosterone, SHBG, estradiol, inhibin), genetic testing (karyotype, Y-chromosome deletion, CFTR, Kal1) and referral to genetic counsellors. Results: The majority of participants (80%) either reported &lt;10% or 10-25% NOA frequency in their population (Fig. 1A). 40.6% of participants stated that 10-25% of men with NOA cases have a clinically significant varicocele (Fig. 1B). For NOA diagnosis, majority of participants cited the requirement of at least two semen samples, with a period of 14-29 days as the interval between two semen analyses. To achieve high-quality semen, consideration must be given to an abstinence period (2-7days) and the complete collection of ejaculates. The survey reported FSH (97%), LH (86.9%) and total testosterone (92.9%) tests as most frequently used for NOA patients (Fig. 2A). The initial hormonal evaluation of a patient with suspected NOA should include total testosterone levels and LH, as these hormones are the primary regulators of spermatogenesis. Karyotype analysis (86.2%), Y-microdeletion (88.3%), CFTR (21.9%) and Kal1 (2.7%) were most routinely performed genetic tests (Fig. 2B). Referrals to genetic counsellors is recommended as these defects have the potential to be passed to future generations. Conclusion: This global study evaluates NOA frequency, clinical diagnosis and management by specialists, in an effort to fill the gap in NOA research. Concomitantly, we provide GAF expert recommendations to enhance our collective understanding of NOA, optimize patient care and improve the management of NOA.

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 nolonger 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.

P-022 The ICSI Outcome of Frozen Thawed Testicular Sperm Utilization in Azoospermic Men with AZFc Microdeletion

Abstract Study question What is the efficacy of frozen thawed surgically retrieved spermatozoa in azoospermic men with AZFc microdeletion undergoing Introcytoplasmic sperm injection (ICSI)? Summary answer The comparison between frozen thaw to fresh testicular sperm did not show statistically significant difference in terms of fertilization, blastocyst development and pregnancy rates. What is known already In male infertility, Y chromosome microdeletion (YCM) may be the cause up to 10% of men with oligozoospermia or azoospermia. AZFc region deletion was mostly detected in YCM. In men with azoospermia and AZFc deletion spermatozoa can be retrieved via testicular sperm extraction (TESE). Today, couples suffering azoospermia due to AZFc deletion may have genetically own child by using testicular sperm with ICSI. Because of the rare incidence of AZFc deletion, the outcome of ICSI cycles in which frozen thawed spermatozoa utilized for assisted conception has not been widely known. Study design, size, duration The data of 611 men with non-obstructive azoospermia (NOA) who underwent microTESE surgery between 2014 and 2022 was retrospectively analyzed. Assisted reproduction treatment (ART) outcome of couples whose men with AZFc deletion was further analyzed according to the fresh or frozen thawed spermatozoa usage for ICSI. Participants/materials, setting, methods All of the patients were genetically screened with karyotype and Y chromosome microdeletion analysis and microTESE operation was performed for testicular sperm recovery. The patients who diagnosed AZFc deletion and had successful sperm recovery divided to two groups according to cryopreserved or fresh sperm utilization for ICSI. The spouse and men age, fertilization rate, blastocyst development, pregnancy and live birth rate was compared between the groups. Main results and the role of chance Twenty-seven (4.4%) men were diagnosed YMC deletion in total of 611 men with NOA who underwent microTESE. Spermatozoa was successfully retrieved in 14 men (51.8%) men who were diagnosed AZFc deletion. The mean age of women and men were 30.5 (3.5) and 32.8 (4.1) years, respectively. Oocyte retrieval and ICSI were accomplished in 18 cycles. In 7 (38.8%) cycles fresh testicular spermatozoa and in 11(61.2%) cycles frozen thawed testicular spermatozoa were used. In one cycle, embryo transfer was cancelled due to total fertilization failure. In total, fertilization rate as 2PN/M2 oocytes was 57% (119/206). In 5 cycles cleavage stage embryos were transferred whereas in remaining 13 cycles blastocysts were transferred. Blastocyst development rate as day 5 blastocyst/2PN was 45.2% (42/95). Comparison of fertilization and blastocyst development rates according to fresh TESE spermatozoa and frozen thawed TESE spermatozoa groups were 63.2% vs 54.4% and 50.0% vs 38.7% respectively (p &amp;gt; 0.5 for both). Clinical and live birth rates per embryo transfer cycle were 57.1% vs 40.0% and 42.8% vs 30.0% respectively (p &amp;gt; 0.5 for both). Limitations, reasons for caution Fresh and frozen thawed groups demonstrated no statistical difference in terms of ICSI outcome. However, relatively reduced fertilization and blastocyst development rates should be interpreted cautiously because beta type error. Further data is needed to clarify this difference. Wider implications of the findings The best of our knowledge our study is the largest in literature comparing frozen thawed and fresh sperm utilization for ICSI in men with AZFc deletion. This data may help for physicians and couples to give decision diagnostic microTESE surgery and cryopreservation of sperm further ICSI treatment. Trial registration number Not applicable

P-069 A male factor panel of NGS can identify the genetic cause of more than half patients with seminal alterations

Abstract Study question Is Next Generation Sequencing (NGS) an effective diagnostic tool for sperm abnormalities? Summary answer NGS male factor gene panel can be a potent diagnostic tool for discovering pathogenic variants linked to sperm abnormalities. What is known already Male infertility, responsible for half of infertility cases, often shows as absence or decreased sperm count (azoospermia, criptozoospermia or oligozoospermia), poor motility (asthenozoospermia) or a higher proportion of morphologically abnormal sperm (teratozoospermia). While the spermiogram remains the gold standard for evaluating male fertility, the potential to identify the molecular basis of sperm abnormalities may make NGS a valuable approach for such patients. The aim of our study was to assess NGS panel's effectiveness in diagnosing patients with abnormal sperm count, motility and morphology. Study design, size, duration A prospective study was conducted from May 2021 to May 2022 in 82 patients including 20 patients with oligozoospermia, 19 with asthenozoospermia and 23 with teratozoospermia. No other sperm alterations were taken into account in each group. The control group consisted of 20 normozoospermic healthy donors selected on the basis of normal sperm parameters according to the WHO criteria (2010). Patients carrying Y-chromosome microdeletions or abnormal karyotype were excluded. Participants/materials, setting, methods Genomic DNA extraction from blood-EDTA of the patients was performed using the commercial MagMax DNA MultiSample Ultra kit and the King-Fisher automated extractor (ThermoFisher®). Next Generation Sequencing (NGS) was done using a panel with 426 genes involved in the spermatogenesis process. Panel sequencing for identification of genetic variants was performed using Nextera Enrichment technology (Illumina®). FASTAQ data were processed using BWA and GATK algorithms. VCF files were analysed using Variant Interpreter software and in silico predictors. Main results and the role of chance Data analysis showed that thirty-seven of the sixty-two patients were carriers of pathogenic mutations in at least one of the genes included in the panel (37/62,59.6%). In the oligozoospermic group, eleven patients (11/20,55%) were carriers of pathogenic mutations in:CFTR, CEP290, WDR66, ESR1, DNAI2, POLG, PIWIL3, GNRHR, MSH5 and CTNS. In the asthenozoospermic group, ten out of nineteen patients (10/19,52.6%) were carriers of pathogenic mutations in:HS6ST1, PMS2, CYP19A1, DNAI2, POLG, LRRC6, G6PD, CCDC39 and PIWIL3. In the teratozoospermic group, six of the twenty-three patients (6/23,26%) were carriers of pathogenic mutations in:CFTR, PIWIL3, CYP21A2, SRD5A. No pathogenic mutations were detected in the control group. The analysis showed comparable results among the various spermiogram alterations with no significant difference (p &amp;gt; 0.05). Each variant was found in a single patient per group, except POLG and CCDC39, which were found in two cases in the oligozoospermic and asthenozoospermic group, respectively, also CFTR and PIWIL3 in two teratozoospermic patients. Besides, it is remarkable that some of the pathogenic variants were present in genes in more than one group:CFTR (3/62), DNAI2 (2/62), POLG (3/62) and PIWIL3 (4/62). All identified variants were linked with processes of spermatogenesis such as dynein assembly and DNA integrity. Limitations, reasons for caution The main limitation of this study is the limited number of patients included. Functional studies with a larger cohort of males with seminal disorders is warranted to confidently correlate the genetic variants identified in this analysis with spermatogenic failure. Wider implications of the findings The gene list included in our panel represents a step-forward in the screening of males with altered sperm parameters. Our results may add in the knowledge of male factor infertility to provide etiologic factors towards a personalized treatment and adequate genetic counselling. Trial registration number Not applicable

(255) Operating Characteristics of Sperm Concentration and FSH for Predicting Chromosomal Abnormalities

Abstract Introduction Microdeletions of the Y-chromosome (Yq) and karyotype abnormalities are frequent causes of male factor infertility. Current AUA/EUA guidelines recommend obtaining Yq microdeletion analysis, karyotype, and genetic counseling in men with non-obstructive azoospermia or severe oligospermia (&amp;lt;5 mil/mL). A recent retrospective cohort study suggests that Yq microdeletions are primarily found at a sperm concentration &amp;lt; 0.5 mil/mL and that by lowering the concentration threshold for genetic testing, specificity could be increased and relative financial cost decreased without adversely affecting the sensitivity. Studies have shown that FSH is the best predictor of NOA in combination with testicular volume as well as a significant predictor of microdeletion presence. Identification of Yq microdeletions and karyotype abnormalities have implications when counseling patients (and their partners) about the likelihood of retrieving sperm and need for further genetic counseling. Objective We hypothesized that the combination of sperm concentration and FSH would better predict the presence of chromosome abnormalities in infertile men. Methods A retrospective cohort study was conducted with subjects who were referred to a military tertiary Urology clinic between 2010 to 2020 and who underwent an infertility evaluation to include genetic testing (karyotype, Y-chromosome analysis), hormone profile (FSH), and at least one semen analysis. Sperm density and FSH were analyzed as independent predictors of chromosomal abnormalities using logistic regression. Receiver operating characteristics were generated for each independent variable that achieved significance in regression. Results A total of 356 patients met inclusion criteria. Over the ten-year period, the prevalence of Y-chromosome microdeletions and karyotype abnormalities was 3.3% (n=12) and 8.4% (n=30), respectively. The ability to predict chromosomal abnormality based on logistic regression was statistically significant for each independent variable. The area under the curve for sperm concentration and FSH was found to be statistically better in combination than concentration alone (0.71 vs 0.66, p=0.033). The best balance of sensitivity and specificity (i.e. for a diagnostic test) for concentration and FSH were found at cut-points of 8.345 mIU/mL, respectively. To optimize performance as a screening test and maintain sensitivity &amp;gt;90%, the cut points for concentration and FSH were 2.74 mIU/mL, respectively. There was no difference in sensitivity for concentration between 2-7 mil/mL and no abnormalities were found if concentration exceeded 7 mil/mL; thus a cutoff of 7 mil/mL would capture 100% of all chromosome abnormalities. Conclusions Based on receiver operating characteristics, lowering the threshold for chromosomal analysis to a sperm concentration of &amp;lt;1 mil/mL maintains acceptable performance as a screening test for genetic abnormality, with resulting cost savings expected. Incorporating FSH into this decision appears to improve both sensitivity and specificity in prediction of chromosomal abnormalities. Our military single-institution ten-year cohort, exemplifies the potential improvement in patient selection and results that can be achieved if the sperm concentration is lowered from the current cutoff Disclosure No

Gene panel analysis of literature-based infertility genes in Sertoli cell-only syndrome patients

Sertoli cell-only syndrome (SCOS) is a condition of male infertility characterized by a total absence of spermatogenic cells in nearly all seminiferous tubules. Apart from well-established genetic changes such as Klinefelter syndrome, CFTR variants, and Y-chromosome microdeletions, several hundred candidate genes were reported as associated with male infertility. We selected 92 evidence-based genes associated with infertility and investigated data from whole-exome sequencing in 6 individuals with clinically diagnosed SCOS. Eight heterozygous variants passed our filtering criteria, including population frequency ≤ 0.1% and high functional impact indicated by Sift, Polyphen, and CADD scores. Out of them, we considered only variants with putative autosomal dominant effects on infertility that were subsequently validated by Sanger sequencing. This filtering pipeline has led to the final likely causative variants detected in CHD7 and SCYP3 genes that potentially explain SCOS in two of our patients. Our discoveries suggest that gene panel testing of patients with SCOS could improve the diagnostic outcome; however, assembling a gene panel consisting of only genuine causative genes is crucial.

Deciphering the Molecular Characteristics of Human Idiopathic Nonobstructive Azoospermia from the Perspective of Germ Cells.

Nonobstructive azoospermia (NOA) is one of the most important causes of male infertility, accounting for 10-15% of infertile men worldwide. Among these, more than 70% of cases are idiopathic NOA (iNOA), whose pathogenesis and molecular basis remain unknown. This work profiles 3696 human testicular single-cell transcriptomes from 17 iNOA patients, which are classified into four classes with different arrest periods and variable cell proportions based on the gene expression patterns and pathological features. Genes related to the cell cycle, energy production, and gamete generation show obvious abnormalities in iNOA germ cells. This work identifies several candidate causal genes for iNOA, including CD164, LELP1, and TEX38, which are significantly downregulated in iNOA germ cells. Notably, CD164 knockdown promotes apoptosis in spermatogonia. Cellular communications between spermatogonial stem cells and Sertoli cells are disturbed in iNOA patients. Moreover, BOD1L2, C1orf194, and KRTCAP2 are found to indicate testicular spermatogenic capacity in a variety of testicular diseases, such as Y-chromosome microdeletions and Klinefelter syndrome. In general, this study analyzes the pathogenesis of iNOA from the perspective of germ cell development, transcription factor (TF) regulatory networks, as well as germ cell and somatic cell interactions, which provides new ideas for clinical diagnosis.

MP30-07 EVALUATION OF MTESE OUTCOMES IN PATIENTS WITH PARTIAL AZFb Y CHROMOSOME MICRODELETIONS

You have accessJournal of UrologyCME1 Apr 2023MP30-07 EVALUATION OF MTESE OUTCOMES IN PATIENTS WITH PARTIAL AZFb Y CHROMOSOME MICRODELETIONS Jonathan Gal, Jessica Marinaro, Anna Mielnik, and Peter Schlegel Jonathan GalJonathan Gal More articles by this author , Jessica MarinaroJessica Marinaro More articles by this author , Anna MielnikAnna Mielnik More articles by this author , and Peter SchlegelPeter Schlegel More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003258.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Y chromosome microdeletions (YCMD) are a common cause of non-obstructive azoospermia. While men with deletions in the azoospermia factor C (AZFc) region have a high likelihood of having sperm in the ejaculate, or sperm retrievable with effective microdissection testicular sperm extraction (mTESE) procedure, those with complete deletions in the AZFa or AZFb regions have a poor prognosis for identification of sperm. Men with AZFa deletions typically do not have any germ cells, and no men, to-date, with complete AZFb deletions have had usable sperm identified. Partial deletions of AZFb appear to portend a different prognosis for fertility potential. Our objective was to evaluate semen parameters, sperm retrieval rates and fertility outcomes in men with partial deletions of the AZFb region. METHODS: We retrospectively reviewed 468 men with YCMDs who underwent testing at our institution from 4/1996 to 8/2022. Of these, we identified 20 men with partial AZFb deletions, defined as a deletion of some but not all tested segments from sY1227 to sY1291. Of these, 8 had documented semen analysis and underwent mTESE by a single, high-volume surgeon (PS). Sperm retrieval rate (SRR) was defined as the presence of mature sperm identified after processing by a trained andrologist. RESULTS: Of the 8 men with a partial AZFb deletion, 7 (87.5%) had a sY117-sY1291 deletion, and one (12.5%) had an sY1227-sY1192 deletion. All 8 men (100%) were azoospermic. Mean testosterone levels were 450.5 ± 350.5 (ng/dL), Mean FSH 6.5±2.8 (mIU/mL). After mTESE, 1 of 7 men (14%) with an sY117-sY1291 deletion was found to have sperm, and the 1 man with a sY1227-sY1192 deletion was found to have round spermatids on histology. Overall, the SRR was 12%. All pathology specimens (n=8, 100%) demonstrated maturation arrest. CONCLUSIONS: In this large series, we demonstrate that while patients with partial AZFb deletions are azoospermic, it is possible to find sperm at least for some men with mTESE. Distinguishing partial from complete AZFb deletions may have prognostic value. Further work is needed to determine which men with partial AZFb deletions are more likely to have a successful mTESE procedure. Source of Funding: Author JG is supported in part by the Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e393 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jonathan Gal More articles by this author Jessica Marinaro More articles by this author Anna Mielnik More articles by this author Peter Schlegel More articles by this author Expand All Advertisement PDF downloadLoading ...

MP30-10 OPERATING CHARACTERISTICS OF SPERM CONCENTRATION AND FSH FOR PREDICTING CHROMOSOMAL ABNORMALITIES

You have accessJournal of UrologyCME1 Apr 2023MP30-10 OPERATING CHARACTERISTICS OF SPERM CONCENTRATION AND FSH FOR PREDICTING CHROMOSOMAL ABNORMALITIES Colin McLain, Matthew Christman, Eric Biewenga, and Donald Crain Colin McLainColin McLain More articles by this author , Matthew ChristmanMatthew Christman More articles by this author , Eric BiewengaEric Biewenga More articles by this author , and Donald CrainDonald Crain More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003258.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Current AUA/EUA guidelines recommend obtaining Yq microdeletion analysis, karyotype, and genetic counseling in men with non-obstructive azoospermia or severe oligospermia (<5 mil/mL). A recent retrospective cohort study suggests that Yq microdeletions are primarily found at a sperm concentration < 0.5 mil/mL and that by lowering the concentration threshold for genetic testing, specificity could be increased and relative financial cost decreased without adversely affecting the sensitivity. Studies have shown that FSH is the best predictor of non-obstructive azoospermia (NOA) in combination with testicular volume as well as a significant predictor of microdeletion presence. We hypothesized that the combination of sperm concentration and FSH would better predict the presence of chromosome abnormalities in infertile men. METHODS: A retrospective cohort study was conducted with subjects who were referred to a military tertiary Urology clinic between 2010 to 2020 and who underwent an infertility evaluation to include genetic testing, hormone profile, and at least one semen analysis. Sperm density and FSH were analyzed as independent predictors of chromosomal abnormalities using logistic regression. Receiver operating characteristics were generated for each independent variable that achieved significance in regression. RESULTS: 356 patients met inclusion criteria. Over the ten-year period, the prevalence of Y-chromosome microdeletions and karyotype abnormalities was 3.3% (n=12) and 8.4% (n=30), respectively. The ability to predict chromosomal abnormality based on logistic regression was statistically significant for each independent variable. The area under the curve for sperm concentration and FSH was found to be statistically better in combination than concentration alone (0.71 vs 0.66, p=0.033). The best balance of sensitivity and specificity (i.e. for a diagnostic test) for concentration and FSH were found at cut-points of <0.215 mil/mL and >8.345 mIU/mL, respectively. To optimize performance as a screening test and maintain sensitivity >90%, the cut points for concentration and FSH were <1 mil/mL and >2.74 mIU/mL, respectively. There was no difference in sensitivity for concentration between 2-7 mil/mL and no abnormalities were found if concentration exceeded 7 mil/mL. CONCLUSIONS: Our military single-institution ten-year cohort, exemplifies the potential improvement in patient selection and results that can be achieved if the sperm concentration is lowered from the current cutoff as well as possible addition of FSH to algorithm. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e394 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Colin McLain More articles by this author Matthew Christman More articles by this author Eric Biewenga More articles by this author Donald Crain More articles by this author Expand All Advertisement PDF downloadLoading ...