AZF Deletions Research Articles

Prevalence of AZFс Y chromosome microdeletions and association with spermatogenesis in Russian men from the general population

The Y chromosome contains a set of genes with testis-specific expression that are responsible for the development of testes and spermatogenesis, and it is the most important target in the search for genetic causes of male infertility. Most of these genes are located in the “azoospermia factor” AZF locus (regions AZFa, AZFb, and AZFc) on the long arm of the Y chromosome. Microdeletions of the Y chromosome, leading to the removal of the entire AZF locus as well as one or more regions (complete deletions), are one of the leading causes of spermatogenesis impairment and infertility. However, the role of partial AZFc deletions (gr/gr, b2/b3, b1/b3) in spermatogenesis failure is unclear, and their impact on spermatogenesis varies between populations. The aim of the present study was to assess the frequency of various types of AZFc microdeletions and to search for associations with spermatogenesis parameters in men of Slavic ethnicity from the general Russian population (n = 700, average age 25.8 years). To identify AZF microdeletions, the presence/ absence of 15 STS markers was analyzed using multiplex real-time polymerase chain reaction. Age, weight, height, and the volume, concentration, total count, proportion of motile and morphologically normal spermatozoa in the ejaculate were recorded for all participants. In the studied sample, 19.9 % (139/700) of men were found to have AZFc microdeletions, of which 16.7 % (117/700) were carriers of a partial b2/b3 deletion, 3.0 % (21/700) had a partial gr/gr deletion, and 0.14 % (1/700) had a complete b2/b4 deletion. Neither AZFa nor AZFb microdeletions nor other types of AZF deletions were detected. The overall frequency of all types of AZFc deletions, as well as each type of partial microdeletion, b2/b3 and gr/gr, did not differ in the groups of azoospermia, severe oligozoospermia (≤5.0 mill/ml), oligozoospermia (5.0 < SC < 16.0 mill/ml), and normal sperm concentration (≥16.0 mill/ml). Comparison of semen parameters in groups with different types of partial AZFc deletions and the control group (without deletions) also did not reveal significant differences. Thus, partial AZFc microdeletions b2/b3 and gr/gr do not have a significant impact on spermatogenesis in Slavic men. It is suggested that in Slavs, partial AZFc microdeletions b2/b3 and gr/gr are fixed in Y haplogroups N3 and R1a, respectively, and their negative impact on spermatogenesis is balanced by other genetic factors. The higher frequency of partial AZFc deletions (19.7 %) in Slavs compared to European populations (7.3 %) established in our study may be explained by the widespread distribution of these Y haplogroups in the Slavic population of Russia.

New Insights beyond Established Norms: A Scoping Review of Genetic Testing for Infertile Men.

From a diagnostic standpoint, certain approaches to genetic screening in clinical practice remain ambiguous in the era of assisted reproduction. Even the most current guidelines do not provide definite guidance on testing protocols, leaving clinicians to carefully determine which tests best serve patients struggling with infertility. The lack of uniformity in the current practice of male fertility evaluation can prove to be quite costly, thus necessitating healthcare practitioners to carefully appraise the necessity and weigh the advantages against potential economic and psychological detriments. The objective of this review is to map the existing literature on the general topic of the clinical indications of routine karyotyping and/or AZF screening in infertile men, identify key concepts, determine where the gaps are, and lastly, provide an overview of the conclusions drawn from a body of knowledge that varies widely in terms of methodologies or disciplines. A thorough search was conducted for the published findings up until July 2023, utilizing PubMed (MEDLINE). This comprehensive search involved the use of specific search keywords, either individually or in combination. The search terms employed were as follows: "Karyotype", "Klinefelter" or "KS" or "47,XXY", "AZF" or "Azoospermi*" and/or "microdeletion*" in the title or abstract. Once the titles and abstracts of selected articles were obtained, the complete texts of linked papers were meticulously scrutinized. A total of 191 records were identified from PubMed. During screening, 161 records (84.3%) were eliminated. Finally, 30 papers were included in this scoping review, which was conducted in 18 countries. The number of sequence tag sites (STSs) used in the studies varied from 5 to 59. The rate of AZF deletions among patients with NOA ranged from 1.3% to 53%. The mean frequency was estimated to be 5.6%. The rate of YCM among patients with XXY karyotype was nil in 19 out of 30 studies (63%), whilst, in the remaining studies, the rate varied from 0.8% to 67%. This review provides insights into managing male infertility. The presence of spermatozoa in ejaculation and successful surgical retrieval cannot be excluded for individuals with AZFb/AZFbc microdeletions. Screening for Y chromosome microdeletions is not needed for mosaic or classic KS. Only 1% of individuals with sperm concentration exceeding 1×106 sperm/mL and less than 5×106 sperm/mL exhibit AZF microdeletions; therefore, testing referral for such populations may need reassessment. Individuals with mosaic monosomy X karyotype and certain chromosomal anomalies should be referred for AZF deletion screening. These findings have implications for male infertility management and future research.

Optimization of Multiplex-PCR Technique To Determine Azf Deletions in infertility Male Patients.

To optimize the multiplex polymerase chain reaction (M-PCR) technique to diagnose microdeletions of azoospermia factors (AZF) on the Y chromosome and initially apply the technique to diagnose male patients with sperm density less than 5×106 million sperm/mL was assigned to do a test to check for AZF microdeletions on the Y chromosome. Based on the positive control samples which belong to male subjects who have had 2 healthy children without any assisted reproductive technologies, the M-PCR method was developed to detect simultaneously and accurately AZF microdeletions on 32 male patients with sperm densities below 5×106 million sperm/mL of semen at the Department of Biology and Medical Genetics - Vietnam Military Medical University. Successful optimization of the M-PCR technique including 7 reactions arranged according to each AZFabc region using 24 STS/gene on the Y chromosome. Initial application to diagnose AZF deletion on 32 azoospermic and oligospermic men reveals that AZFa deletion accounts for 6.25% (2/32); deletion of all 3 regions AZFa,b,c with 18.75% (6/32 cases); The combined deletion rate of AZFb,c is highest, accounting for 56.24% (18/32 patients). Successfully optimized the M-PCR technique in identifying AZF microdeletions using 24 sequence tagged sites (STS)/gene for azoospermic and oligozoospermic men. The M-PCR technique has great potential in the application of AZF deletion diagnosis.

Clinical features and Y chromosome abnormalities in children with 45, X/46, XY mosaicism

Objective: To investigate the clinical and genetic characteristics of children with 45, X/46, XY mosaicism. Methods: The retrospective study included 20 children diagnosed with 45, X/46, XY and 45, X/46, X,+mar mosaicism in the First Affiliated Hospital of Zhengzhou University from 2018 to 2022. The clinical features, gonadal pathology, treatment and follow-up were summarized. Genetic tests were performed by SRY gene test, azoospermia factor region (AZF) deletion test, copy number variation-sequencing (CNV-seq). Age at first diagnosis was compared between boys and girls using independent sample t-test. Results: The 20 patients included 3 boys and 17 girls, and the age at first diagnosis were (7.6±5.5) years, it is (2.1±1.9) years in boys, (8.7±5.4) years in girls, significantly younger for boys (t=-3.86, P=0.004). The chief complaint was external genitalia malformation for boys, and short stature (13 cases) and dysplastic external genital for girls (4 cases). Five girls presented with features of Turner syndrome. The gonadal phenotypes included mixed gonadal dysplasia (MGD, 6 cases), complete gonadal dysplasia (CGD, 10 cases), unilateral ovotestis (2 cases), possible ovaries (1 case) and undetermined gonad (1 case). One female with dysplastic genital was reassigned to male, and the gender of the remaining cases remained unchanged. Seven females were treated with recombinant human growth hormone. The height increased by (17±7) cm during the (2.9±1.2) years follow-up. No gonadal malignancy was observed. The karyotype was 45, X/46, XY in 16 cases, and 45, X/46, X,+mar in 4 cases. All of the 4 marker chromosomes were derived from Y chromosome confirmed by CNV-seq. SRY gene was detected in all 20 patients genome, and AZF deletion was found in 7 girls. Conclusions: 45, X/46, XY mosaicism presented with dysplastic external genital or female with remarkable short stature. Gonadal phenotypes included MGD, CGD and ovotestis. AZF microdeletions were found in the majority of female cases.

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.

Primary azoospermia factor C duplication associated with spermatogenic impariment: a case-control study based on Y-chromosome haplogrouping in a Han Chinese population.

Azoospermia factor C (AZFc) in the male-specific region of Y-chromosome (MSY) presents wide structure variation mainly due to frequent non-allele homologous recombination, leading to significant copy number variation of the AZFc-linked coding sequences involving in spermatogenesis. A large number of studies had been conducted to investigate the association between AZFc deletions and male infertility in certain Y chromosome genetic backgrounds, however, the influence ofprimary AZFc duplication on spermatogenesis remained controversial and the cause of the discrepant outcomes is unknown. In the present study, a total of 1,102 unrelated Han Chinese males without any detectable AZF deletions were recruited from 2014 to 2019, including 411 controls with normozoospermia and 691 patients with idiopathic spermatogenic failure. Using multiple paralog ratio tests (PRTs), the structure duplications were classified by the copy number of the AZFc-linked amplicons and genes. The Y-chromosome haplogroup (Y-hg) was categorized by genetyping of MSY-linked polymorphism loci. The association of primary AZFc duplication with spermatogenic phenotype was investigated in males with the same Y-hg. Within Y-hg O3* group, the frequency of the gr/gr duplication in patients is significantly higher than that of controls (P=1.29×10-3 , odds ratio (OR) 7.64, 95% confidence interval (CI) 1.79-32.57). Moreover, Y-hg O3* males with the gr/gr duplication presented a significantly lower sperm production compared with non-AZFc duplicated ones (sperm concentration: P=1.46×10-3 ; total sperm count: P=1.82 ×10-3 ). The b2/b3 duplication were identified clustered in Y-hg Cα2*, and the significant difference in the distribution was not observed between patients with spermatogenic failure and controls. The results suggest that, in the Han Chinese population, the gr/gr duplication is a predisposinggenetic factor for spermatogenic impairment in males harboring Y-hg O3* . Meanwhile, the b2/b3 duplication may be fixed ona yet-unidentified subbranch of Y-hg Cα2* without significantly deleterious effect on spermatogenesis. Our findings provide evidence that the difference in the Y-hg composition may cause the discrepancy on the association of AZFc duplication with spermatogenic failure among the studied populations.

High incidence of AZF duplications in clan-structured Iranian populations detected through Y chromosome sequencing read depth analysis

The ampliconic region of the human Y chromosome consists of large duplicated sequences that can undergo non-allelic homologous recombination (NAHR), resulting in structural rearrangements that may cause infertility, especially when they occur in the azoospermia factor b/c (AZFb/c) region. Although AZF duplications have long been neglected due to the technical limitations of STS-based studies that focused mainly on deletions, recent next generation sequencing (NGS) technologies provided evidence for their importance in fertility. In this study, a NGS read depth approach was used to detect AZFb/c rearrangements in 87 Iranians from different ethnic groups. The duplication frequency in Iran proved to be twice as high as in the "1000 Genomes" dataset. Interestingly, most duplications were found in patrilineal ethnic groups, possibly as a consequence of their lower male effective population size which can counteract negative selection. Moreover, we found a large 8.0 Mb duplication, resulting in a fourfold increase in the copy number of AZFc genes, which to our knowledge is the largest duplication ever reported in this region. Overall, our results suggest that it is important to consider not only AZF deletions but also duplications to investigate the causes of male infertility, especially in patrilineal clan-based populations.

O-015 Spontaneous alterations in semen parameters are associated with age, accessory gland function and the FSHB c.-211G>T variant

Abstract Study question Which parameters are associated with spontaneous alterations in semen analysis in infertile men? Summary answer Changes in semen parameters are modulated by abstinence time, accessory gland function and fluctuations in FSH serum concentration, which is bound to FSHB c.-211G>T variant. What is known already There is a strong within-subject alteration of semen parameters in men with infertility. The WHO therefore recommends two ejaculate analyses. However, it remains unknown in which subgroup variations are likely to occur and which semen parameters are affected. We assume that in some men with idiopathic infertility, ejaculate parameters may spontaneously change without interventional medication apart from regression to the mean effects. The study aims to identify factors in conventional semen analysis based on the patients’ clinical data, including reproductive hormones like follicle stimulating hormone (FSH), and factors impacting endocrine activity, like the FSHB-211 promoter gene. Study design, size, duration In this retrospective longitudinal study we selected 3456 deeply phenotyped men with idiopathic infertility who had visited our centre for fertility workup from 2010-2020. The patients were selected from our well curated database Androbase© for analysis of repeated ejaculate samples. Participants/materials, setting, methods Men with idiopathic infertility and at least two ejaculate analyses (70-90 days distance) were included. Exclusion criteria comprised: sperm concentration <1 million/ml, abnormal FSH or low testosterone, bitesticular volume (bTV) <10ml, orchiectomy, cryptorchidism, varicocele testis, oncological disease, genetic findings (i.e. abnormal karyotype, AZF deletions), medication affecting spermatogenesis. Grouped linear 2-level nested mixed-effect models were applied. Analyzed parameters included: somatic, hormonal (including FSH and FSHB c.-211 variants), and semen parameters (including abstinence time, accessory gland markers). Main results and the role of chance Groups were built according to sperm concentration: A (n = 397): ≥1.0 to < 5.0 mill/ml, B (n = 708): ≥5.0 to < 15.0 mill/ml, C (n = 2351): ≥15.0 mill/ml. A, B and C: changes in ejaculate volume were associated with alterations in total sperm count and motility (p < 0.003). Changes were, controlled for abstinence time (p < 0.001), related to alpha-glucosidase, fructose or zinc (p = 0.005-0.02). A+B: fluctuations in FSH concentration influenced sperm concentration/count (p = 0.004-0.02), albeit only in men with FSHB c.-211 GG (p = 0.007-0.02). T-allele carriers did not show changes in FSH concentration (p > 0.1). B: age <50 years (p = 0.007-0.01) and normal bTV (p = 0.008-0.02) were associated with spontaneous increases of sperm concentration, count and motility. Semen parameters exhibit intra-individual alterations associated with organic, hormonal and genetic variables. Changes are pronounced in younger men with normal bTV and oligo- to almost normozoospermia. The effect is modulated by abstinence time, accessory gland function and fluctuations in FSH concentrations which, in turn, are bound to the FSHB-211 promotor gene polymorphism. Limitations, reasons for caution A limitation of our approach is the retrospective nature of the study, which cannot exclude the possibility of bias within the data set. Wider implications of the findings Judgement of semen analysis should be based on two semen samples, with abstinence time between 4-5 days. In future clinical trials, abstinence time should be kept constant. It might be investigated if younger men with normal bTV and FSHB c.-211 GT/TT benefit from improving accessory gland function and increasing FSH. Trial registration number not applicable

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 ...

Testicular Architecture of Men with 46,XX Testicular Disorders of Sex Development

Background: A subtype of disorders of sex development (DSD) in individuals with a 46,XX karyotype who are phenotypically male is classified as testicular DSD (46,XX TDSD). These individuals develop testes but are infertile due to germ cell loss. However, little is known about their testicular architecture. Methods: We analyzed biopsies of four SRY positive 46,XX TDSD men for testicular architecture, Sertoli (SCs) and Leydig cells (LCs). These were compared with biopsies of men with normal spermatogenesis (NS, n = 4), men with Klinefelter syndrome, 47 XXY (KS, n = 4), and men with AZF deletions (AZF, n = 5). Testicular architecture was evaluated and SCs and LCs were analyzed for specific markers (SC: SOX9, DMRT1; LC: INSL3). Results: A smaller number of tubules, more SOX9-negative but similar proportions of DMRT1-negative SCs were found in 46,XX TDSD compared to NS. The lower number of tubules and severe LC hyperplasia observed in 46,XX TDSD were similar to KS. Conclusion: Testicular architecture and marker expression of SCs and LCs in 46,XX TDSD men display unique patterns, which are discernable from chromosomal aneuploidies. Given the reduced Y-chromosomal gene content in 46,XX TDSD, the supernumerary X chromosome effects may be decisive regarding the damage on testicular composition and endocrine function.

Делеции в AZF локусе и нарушения сперматогенеза у мужчин c мозаицизмом по хромосоме Y

Введение. Аномалии кариотипа и микроделеции в локусе AZF (Yq11.2) хромосомы Y являются частыми генетическими причинами мужского бесплодия, вызванного нарушением сперматогенеза, однако эффект их сочетанного влияния недостаточно исследован. Цель: сравнительный анализ сперматологических показателей у мозаиков по хромосоме Y, имеющих и не имеющих делеции в локусе AZF. Методы. Обследованы 16 мужчин с мозаицизмом по хромосоме Y. Анализ кариотипа выполнен с использованием стандартного цитогенетического исследования на культивированных лимфоцитах периферической крови. Молекулярно-цитогенетическое (FISH) исследование проводили для выявления/верификации мозаицизма и структурных аномалий хромосомы Y. Микроделеции хромосомы Y детектировали методом мультиплексной ПЦР. Спермиологическое исследование выполняли в соответствии с рекомендациями Руководства ВОЗ (2010). Результаты. Цитогенетически идентифицируемые несбалансированные аномалии хромосомы Y обнаружены у 13 (81,25%) из 16 мужчин. Делеции в локусе AZF детектированы у 8 (50%) из 16 пациентов. Выборка разделена на две группы: группа I - без AZF делеций (n=8); и II - с AZF делециями (AZFb+c, n=7 и AZFc, n=1). Выявлены различия в структуре сперматологических диагнозов между группами: в группе I отмечены различные формы патозооспермии (азооспермия - 3; олигоастенотератозооспермия - 3; астенотератозооспермия - 2); в группе II - только тяжелые формы патозооспермии (азооспермия - 7; олигозооспермия тяжелой степени - 1), а также по частоте олигоспермии - 12,5% и 37,5%, соответственно. Более высокая концентрация сперматозоидов обнаружена у пациентов без AZF делеций (группа I - 15,9±31,0 млн/мл, группа II - 0,003±0,009 млн/мл; р=0,026). Не выявлено статистически значимых различий между группами по среднему возрасту пациентов, объёму, pH и вязкости эякулята. Выводы. Структурные аномалии и микроделеции хромосомы Y в локусе AZF (Yq11.2) встречаются с высокой частотой у пациентов с мозаицизмом по хромосоме Y. Несбалансированные перестройки хромосомы Y и патогенные микроделеции в локусе AZF у мужчин-мозаиков по хромосоме Y характеризуются тяжелой степенью нарушения сперматогенеза. Сохранение фертильностного потенциала мужчин с мозаицизмом по хромосоме Y возможно при отсутствии несбалансированных перестроек и тяжелых типов AZF делеций. Introduction. Chromosome abnormalities and microdeletions in the AZF (Yq11.2) locus of the Y chromosome are common genetic causes of male infertility caused by impaired spermatogenesis, however, the effect of their combined influence has not been sufficiently investigated. Aim: a comparative analysis of semen parameters in the Y-chromosome mosaics with and without AZF deletions. Methods. 16 male patients with Y chromosome mosaicism were examined. Chromosome analysis was done using by standard cytogenetic examination. Fluorescence in situ hybridization (FISH) was performed to identify/verify mosaicism and determine the structural Y chromosome abnormalities. The Y chromosome microdeletions were detected by multiplex PCR. The semen examination was carried out with the recommendations of the WHO Guidelines (2010). Results. Cytogenetically identifiable unbalanced the Y chromosome abnormalities were found in 13 patients (81.2%) of 16 patients. AZF deletion were detected in 8 patients (50%) of 16 patients. The patients were divided into two groups: group I - patients without AZF deletions (n=8), group II - patients with AZF deletions: AZFb+c, n=7 and AZFc (b2/b4), n=1 (n=8). Prominent difference in sperm diagnosis was revealed between the groups: in group I, there was a various degree of pathozoospermia (azoospermia - 3; oligoastenoteratozoospermia - 3; asthenoteratozoospermia - 2); in group II, only severe forms of pathozoospermia (azoospermia - 7; oligozoospermia severe - 1), as well as the frequency of oligospermia - 12.5% and 37.5%, respectively. A higher concentration of spermatozoa was found in patients without AZF deletions (group I - 15.9±31.0 million/ml, group II - 0.003±0.009 million/ml; p=0.026). There was no statistically significant difference between the average age of patients in the groups, ejaculate volume, pH and viscosity of ejaculate. Conclusion. There is high frequency of structural Y chromosome abnormalities and microdeletions in the AZF (Yq11.2) locus in the Y chromosome mosaics. Unbalanced cytogenetic Y chromosome rearrangements and pathogenic AZF microdeletions are characterized by a severe degree of spermatogenesis disorder in male patients with Y chromosome mosaicism. The preservation of fertility potential in men with Y chromosome mosaicism is possible in the absence of unbalanced rearrangements and severe types of AZF deletions.

Tỷ lệ vô tinh, thiểu tinh nhẹ thiểu tinh nặng và nồng độ hormone sinh sản ở bệnh nhân vô sinh có vi mất đoạn AZF tại Bệnh viện Nam học Hiếm muộn Hà Nội

Mục tiêu: Khảo sát một số đặc điểm tinh dịch đồ và một số chỉ số nội tiết sinh sản ở nam giới vô sinh có mất đoạn AZF. Đối tượng và phương pháp: Nghiên cứu hồi cứu, mô tả, cắt ngang trên 166 nam giới Việt Nam có vi mất đoạn AZF trên các bệnh nhân (BN) thiểu tinh hoặc vô tinh tại Bệnh viện Nam học và Hiếm muộn Hà Nội từ 12/2016 – 6/2021. Kết quả: Mất đoạn AZFc chiếm tỷ lệ cao nhất trong mất đoạn AZF (51,81%). Mất đoạn AZF phối hợp chiếm 42,77%. Không có BN nào mất đoạn AZFb đơn độc trong nghiên cứu này. Tỷ lệ mất đoạn AZFa là 1,81%. 3/3 BN vi mất đoạn AZFa đơn độc đều vô tinh. Mất đoạn AZFc có 27% vô tinh, 58% thiểu tinh nặng và 13% thiểu tinh. 100% vi mất đoạn phối hợp nếu chứa AZFa vô tinh. Tỷ lệ vô tinh ở mất đoạn AZFbc là 71,42%, ở mất đoạn AZFbcd là 52%. Vi mất đoạn AZFa có nồng độ FSH cao (36,67 ± 12,70 IU/l). Kết luận: Tỷ lệ mất đoạn AZFc cao nhất (51,81%). Mất đoạn AZFa trầm trọng hơn so với các loại mất đoạn khác. Các loại mất đoạn còn lại (AZFb, c, d và phối hợp của chúng) ở mức độ có từ vô tinh tới thiểu tinh nhẹ.

Гены системы HLA класса II у супружеских пар с неэффективными попытками вспомогательных репродуктивных технологий

Aim: to study immunogenetic patterns of couples who have appealed to assisted reproductive technologies (ARTs). Patients and Methods: HLA class II typing was performed in 345 couples with different outcomes of ARTs, i.e., ineffective in vitro fertilization (IVF) or intrauterine insemination (IUI), and missed miscarriage after IVF. Inclusion criteria were primary or secondary ART implantation failures. HLA class II typing was performed by real-time PCR. In addition, HLA genotypes were investigated in women examined for female infertility (ICD-10 cone N97) and men examined for urogenital infections plus spermogram, MAR test, and assessment of AZF deletions. All participants were divided into five groups of pairs based on obstetric and gynecologic history, i.e., one failed IVF attempt (n=100), two failed IVF attempts (n=64), three or more failed IVF attempts (n=87), ineffective IUI (n=48), and missed miscarriage after IVF (n=10). The comparision group included 36 couples with one or more childbirths after ARTs. Results: ART efficacy depends on the HLA genotypes of couples. In women, DQA1*01:02 is associated with IVF failure. In men, DQA1*01:01 is associated with failed ART, and DQA1*05:01 is associated with childbirth after ARTs. In men, DRB1*11 favors effective IUI. DQB1*03:03 in women and DRB1*01, DQA1*01:01, and DQB1*05:01 in men predispose to missed miscarriages after IVF and IUI. In men, DQB1*02:01 contributes to childbirths after ARTs. The outcomes of IVF and IUI are not related to husband-wife matching for HLA class II alleles. Conclusion: HLA class II typing is crucial for diagnosing infertility when using assisted reproductive technologies. KEYWORDS: infertility, in vitro fertilization, alleles, genes, HLA. FOR CITATION: Zagarskikh A.N., Butina E.V., Zaytseva G.A. HLA class II alleles in couples with failed ART attempts. Russian Journal of Woman and Child Health. 2022;5(1):5–10 (in Russ.). DOI: 10.32364/2618-8430-2022-5-1-5-10.

An NGS-based approach to identify Y-chromosome variation in non-obstructive azoospermia.

Copy number variations (CNVs), including deletions and duplications on the Y chromosome, are known genetic factors in azoospermia. Therefore, it is important to identify novel pathogenic CNVs related to azoospermia. In this study, we compared CNVs detected by STS-PCR and NGS in 107 individuals with nonobstructive azoospermia (NOA). STS-PCR analysis revealed that 8.14% (9/107) of patients had AZF deletions. The highest percentage of deletions was located in the AZFc region, followed by AZFa and AZFb+c. Positive CNVs, including four duplications, six deletions and three complex CNVs, were detected using NGS methods in 12.15% (13/107) of NOA patients. Both the duplications and deletions detected in q11.223 were confirmed to increase the genetic risk for NOA. A comparison between the STS-PCR results and NGS methods revealed concordant CNV-positive results in 4 of 107 cases (3.74%). The discrepancies included 6 cases with CNVs identified by NGS but not detected by STS-PCR, and two cases were detected by STS-PCR but not by NGS. Notably, four duplications were not identified and three complex CNVs were detected as simple deletions using STS-PCR analysis. The NGS method provides comprehensive results in detecting Y chromosome-linked CNVs, including deletions and duplications, which might broaden our understanding of NOA.

Investigation of genotype-phenotype correlation in patients with AZF microdeletion in a single-reference centre.

In this study, we aimed to elucidate the relationship between AZF deletion type and clinical information of azoospermic patients with AZF microdeletion in the Turkish population. Azoospermic patients with normal karyotype and AZF microdeletion were analysed retrospectively by collecting clinical data including hormone profile, demographic characteristics and micro-TESE results. As a result of the AZF microdeletion tests of 42 cases with 46 XY karyotype, AZFa deletion was detected in 3 cases, AZFb deletion in 2 cases, AZFc deletion in 31 cases, AZFb+AZFc deletion in 4 cases and AZFa+AZFb+AZFc deletion in 2 cases respectively. Spermatozoon was obtained in 16 cases with AZFc microdeletion with micro-TESE. Pregnancy was achieved in 2 cases. There was no statistically significant difference between the type of deletion and age, height, weight, body mass index, hormone profile and testicular volume. When AZF is evaluated according to the type of microdeletion, it will be appropriate to plan the medical and surgical options more carefully in a multidisciplinary manner in cases with deletions including AZFa, AZFb or their combinations. Also, genotype-phenotype correlation was found to be consistent with the literature; particularly patients having AZFc deletions were found to have a chance for pregnancy.

Study on clinical and genetic characteristics of male patients with non-obstructive azoospermia

We examined 501 patients with non - obstructive azoospermia to evaluate clinical, subclinical, and genetic characteristics. The results show that the average age of patients in the study was 29.8 ± 5.5 years. Primary infertility accounts for the majority, with a rate of 90.3%. There was 38.6% of patients had a history of mumps orchitis. The average levels of FSH, LH, testosterone were 31.6 ± 16.5 mIU/mL, 15.5 ± 10 mIU/mL and 12.8 ± 7.13 nmol/L, respectively. The prevalence of chromosomal abnormalities was 30.7%. Of these, the sex chromosome aneuploidy with 47,XXY karyotype (Klinefelter syndrome) accounted for 27.3%. The incidence of AZF microdeletion was 13.8%. Of these, AZFc deletion was the most common at the rate of 42.1%, AZFa deletion, which accounted for 2.6%, were the least prevalent, and the frequency of AZFd deletion was 5.3%. However, there was no solitary AZFb deletion, which combined with other AZF deletions with 34.2%. Our research shows that mumps orchitis and chromosomal abnormalities are the leading causes of azoospermia. Screening for genetic abnormalities plays an important role in infertile patients with non - obstructive azoospermia.

Methylation of the SNRPN gene in infertile men without mutations in common candidate genes for reduced fertility

Introduction. Male infertility is a common problem in andrology and occurs in 45 % of infertile couples. Some cases of male infertility caused by genetic reasons: point mutations at some monogenic diseases, AZF deletions or a CFTR mutation compounds; reduced fertility is also associated with polymorphic variants of the genes AR and GSTT1/GSTM1. At the same time, increasing amount of data are being published about the role of epigenetic mechanisms (aberrant methylation and imprinting alterations) in defective spermatogenesis.Materials and methods. We have studied 49 sperm samples obtained from unrelated infertile men using polymerase chain reaction, fragment analysis, and sequencing.Results. Five patients were excluded from the initial cohort: one with a repeat length of 29 (CAG) in the first exon of the AR, three with null genotypes in the GSTT1 and GSTM1, and one with the delF508/5T heterozygous compound in the CFTR. Thereafter, methylation of the imprinted gene SNRPN was determined using methyl-specific polymerase chain reaction and bisulfite sequencing. Aberrant SNRPN methylation was detected in 11.4 % of male infertility samples. In total, molecular genetic and epigenetic alterations were determined in 20 % of patients.Conclusions. Obtained data demonstrate a significant proportion of (epi)genetic disorders in a heterogeneous cohort of men with reduced fertility.

Y chromosome copy number variation and its effects on fertility and other health factors: a review.

The Y chromosome is essential for testis development and spermatogenesis. It is a chromosome with the lowest gene density owing to its medium size but paucity of coding genes. The Y chromosome is unique in that the majority of its structure is highly repetitive sequences, with the majority of these limited genes occurring in 9 amplionic sequences throughout the chromosome. The repetitive nature has its benefits as it can be protective against gene loss over many generations, but it can also predispose the Y chromosome to having wide variations of the number of gene copies present in these repeated sequences. This is known as copy number variation. Copy number variation is not unique to the Y chromosome but copy number variation is a well-known cause of male infertility and having effects on spermatogenesis. This is most commonly seen as deletions of the AZF sequences on the Y chromosome. However, there are other implications for copy number variation beyond just the AZF deletions that can affect spermatogenesis and potentially have other health implications. Copy number variations of TSPY1, DAZ, CDY1, RBMY1, the DYZ1 array, along with minor deletions of gr/gr, b1/b3, and b2/b3 have all be implicated in affecting spermatogenesis. UTY copy number variations have been implicated in risk for cardiovascular disease, and other deletions within gr/gr and the AZF sequences have been implicated in cancer and neuropsychiatric diseases. This review sets out to describe the Y chromosome and unique susceptibility to copy number variation and then to examine how this growing body of research impacts spermatogenesis and other health factors.

The effects of Y chromosome microdeletions on in vitro fertilization outcomes, health abnormalities in offspring and recurrent pregnancy loss.

Male factor infertility accounts for approximately 50% of all infertility evaluations. A common cause of severe oligozoospermia and azoospermia is Y chromosome microdeletions (YCMs). Men with these genetic microdeletions must typically undergo assisted reproductive technology (ART) procedures to obtain paternity. In this review, we performed a thorough and extensive search of the literature to summarize the effects of YCMs on in vitro fertilization (IVF) outcomes, health abnormalities in offspring and recurrent pregnancy loss (RPL). The PubMed database was searched using specific search terms and papers were identified using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Sperm retrieval amongst men with complete AZFa and/or AZFb deletions is extremely rare and thus data on ARTs is largely unavailable. In AZFc-deleted men undergoing assisted reproduction, the collective fertilization rate (FR) is 59.8%, the clinical pregnancy rate is 28.6% and the live birth rate is 23.4%. When successful, the YCM is always transmitted to the male offspring and the deletion size either remains unchanged or widens. YCMs generally result in decreased fertilization, clinical pregnancy and live birth rates compared to men with intact Y chromosomes during ART interventions. There is a minimal or absent association of YCMs with abnormalities in the offspring or RPL.

Association of MSY haplotype background with nonobstructive azoospermia is AZF-dependent: A case-control study.

Identifying causal genes of spermatogenic failure on the male-specific region of Y chromosome (MSY) has been a challenging process. Due to the nonrecombining nature of MSY, haplotype-based approaches have recently been shown to be promising in identifying associated MSY haplogroups. We conducted an MSY analysis of nonobstructive azoospermia (NOA) patients in a case-control setting (N=278 and 105 respectively) to identify modal haplogroups strongly associated with NOA. Patients with AZF deletions (AZF+) and no AZF deletions (AZF-) were compared with the control group. Given the larger sample set of AZF- NOA patients, we further investigated the association based on histopathological severity, namely Sertoli cell-only syndrome and maturation arrest subtypes. We observed no significant enrichment of MSY haplogroups in AZF- azoospermic patients (or its subtypes). However, we observed a strongly significant association between haplogroup J2a* and AZF+patients (FDR-corrected p=.0056; OR=7.02, 95%CI 1.89 to 39.20), a haplogroup which also showed significant enrichment for AZFa/b deletions (p=4x10-4 ). We conclude that unlike AZF+patients, AZF- NOA are less likely to have an MSY causative factor with large effect size, thus indicating that the aetiology of AZF- NOA, and to some extent AZFc NOA, is more likely to be based on non-MSY factors.