Azoospermia Factor Research Articles

Towards a Multi-Omics of Male Infertility.

Infertility is a common problem affecting one in six couples and in 30% of infertile couples, the male factor is a major cause. A large number of genes are involved in spermatogenesis and a significant proportion of male infertility phenotypes are of genetic origin. Studies on infertility have so far primarily focused on chromosomal abnormalities and sequence variants in protein-coding genes and have identified a large number of disease-associated genes. However, it has been shown that a multitude of factors across various omics levels also contribute to infertility phenotypes. The complexity of male infertility has led to the understanding that an integrated, multi-omics analysis may be optimal for unravelling this disease. While there is a vast array of different factors across omics levels associated with infertility, the present review focuses on known factors from the genomics, epigenomics, transcriptomics, proteomics, metabolomics, glycomics, lipidomics, miRNomics, and integrated omics levels. These include: repeat expansions in AR, POLG, ATXN1, DMPK, and SHBG, multiple SNPs, copy number variants in the AZF region, disregulated miRNAs, altered H3K9 methylation, differential MTHFR, MEG3, PEG1, and LIT1 methylation, altered protamine ratios and protein hypo/hyperphosphorylation. This integrative review presents a step towards a multi-omics approach to understanding the complex etiology of male infertility. Currently only a few genetic factors, namely chromosomal abnormalities and Y chromosome microdeletions, are routinely tested in infertile men undergoing intracytoplasmic sperm injection. A multi-omics approach to understanding infertility phenotypes may yield a more holistic view of the disease and contribute to the development of improved screening methods and treatment options. Therefore, beside discovering as of yet unknown genetic causes of infertility, integrating multiple fields of study could yield valuable contributions to the understanding of disease development. Future multi-omics studies will enable to synthesise fragmented information and facilitate biomarker discovery and treatments in male infertility.

Microdeletions and vertical transmission of the Y-chromosome azoospermia factor region.

Spermatogenesis is regulated by several Y chromosome-specific genes located in a specific region of the long arm of the Y chromosome, the azoospermia factor region (AZF). AZF microdeletions are the main structural chromosomal abnormalities that cause male infertility. Assisted reproductive technology (ART) has been used to overcome natural fertilization barriers, allowing infertile couples to have children. However, these techniques increase the risk of vertical transmission of genetic defects. Despite widespread awareness of AZF microdeletions, the occurrence of de novo deletions and overexpression, as well as the expansion of AZF microdeletion vertical transmission, remains unknown. This review summarizes the mechanism of AZF microdeletion and the function of the candidate genes in the AZF region and their corresponding clinical phenotypes. Moreover, vertical transmission cases of AZF microdeletions, the impact of vertical inheritance on male fertility, and the prospective direction of research in this field are also outlined.

Hormonal, clinical, and genetic profile of infertile patients with azoospermia in Morocco.

azoospermia affects more than 10%-15% of infertile male subjects attending the infertility center. In Morocco, there have been no studies on male infertility with azoospermia. Thereby, our objective was to evaluate the clinical, hormonal, and genetic characteristics of infertile men with azoospermia in Morocco. we conducted a retrospective descriptive study performed with a convenience sample of 80 infertile men from 2021 to 2022, in the Assisted Reproductive Technology Unit of the Mohammed VI University Hospital Center in Oujda-Morocco. All patients with azoospermia were subjected to a quantitative hormone assay to evaluate the functionality of the sertolic and leydigial compartments. Human karyotyping and AZF microdeletion analysis are routinely performed in azoospermic patients. the results show that the mean age of patients in the study was 45.7 ± 3.5 years. Primary infertility accounts for the majority, with a rate of 96% (n=77). There were 12 cases of azoospermia of secretory origin, 22 cases of excretory origin, and 3 of undetermined origin. Azoospermia was associated with hydrocele in 29% (n=27) of cases. The average levels of FSH, LH, testosterone, and inhibin B were 15.54 ± 5.5 mIU/mL, 7.71 ± 2.7 mIU/mL, 405.09 ± 6.13 ng/dl and 38.44 ± 5.13 pg/ml, respectively. The prevalence of chromosomal abnormalities was 30.7%. Of these, the sex chromosome aneuploidy with 47, XXY karyotype (Klinefelter syndrome) accounted for 11% (n=9). The incidence of microdeletions of azoospermia factors (AZF) was 9%, and AZFc deletion was the most common at the rate of 3%. our research shows that hydrocele, varicocele, and chromosomal abnormalities are the leading causes of azoospermia. In the Moroccan population, azoospermia is essentially of excretory origin.

Citogenetička analiza odabranih genetski uvjetovanih bolesti u istočnoj Slovačkoj

This work presents the results of cytogenetic analysis performed during the period 1990-2021. The work focuses on cytogenetic analysis of selected diseases that represent a serious medical and social problem in the Prešov region of eastern Slovakia. The analysis also included the determination of cytogenetic and molecular-genetic marker frequency related to selected clinical genetic diseases in a specified population of Roma ethnicity. Chromosome analysis confirmed a wide spectrum of chromosomal aberrations in patients with Down’s syndrome and Turner syndrome, revealing a spectrum of aberrations from monosomy X, isochromosome Xq, and deletions Xp to marker chromosomes. Chromosomal aberrations cause 5.5% of fertility disorders in couples, with numerical and structural chromosomal aberrations found in 2.1 and 3.4% respectively, revealing a risk finding for offspring of carriers of balanced translocations. Microdeletions, combined microdeletions (AZFb,c) and complete deletion of the AZF region of the Y chromosome were found in men diagnosed with azoospermia. In addition, pathological karyotypes were detected in men and women (13 and 10%). Another set of analyses in patients with onco-haematological diseases revealed presence of Philadelphia chromosome (Ph1) in 94.4% of patients with chronic myeloid leukaemia, complex translocation of chromosomes 8, 9, 22; mosaic karyotype of Ph1. Chromosomal aberrations in patients with myelodysplastic syndrome also included also atypical and as yet unpublished cytogenetic markers. Myeloproliferative diseases were detected in 28.3% of patients with heterogenous chromosomal aberrations. Revelations from cytogenetic analysis enable improvement in the efficiency of health care, diagnostics, therapeutic significance and prognosis of affected people in the majority population and Roma minority in this region of Slovakia.

Y-chromosome haplogroups and Azoospermia Factor (AZF) analysis in Tunisian infertile male

The aim of the present study was to clarify the implication of Y chromosome genetic variations and haplogroups in Tunisian infertile men. A total of 27 Y-chromosomal binary markers partial microdeletions (gr/gr, b1/b3 and b2/b3) and copy number variation of DAZ and CDY genes in the AZFc region were analysed in 131 Tunisian infertile men with spermatogenic failure and severe reduced sperm concentrations and in 85 normospermic men as controls. Eleven different haplogroups in the overall population study (E3b2; J1J*, E1, E3b*, F, G, K, P/Q, R*, R1* and R1a1) were found. Interestingly, the J1J* haplogroup was significantly more frequent in azoo/oligospermic patients than in normospermic men (35.1% and 22.3%, respectively (p value = 0.04)). Results showed also that patients without DAZ/CDY1 copies loss and without partial microdeletions belonged to the R1 haplogroup. The relative high frequencies of two haplogroups, E3b2 (35.1%) and J (30%) was confirmed in Tunisia. We reported in the present study and for the first time, that J1J* haplogroup may confer a risk factor for infertility in the Tunisian population and we suggested that R1 haplogroup may ensure certain stability to Y-chromosome in Tunisian men.

The correlation between azoospermia factor microdeletion in the Y chromosome and anti-sperm antibody generation in infertile Iraqi patients

Objective: This study aims to evaluate the occurrence of Y chromosome azoospermia factor (AZF) microdeletions and assess immune infertility incidence and whether there is a correlation between them. Materials and Methods: The sample included 75 infertile men who were referred to private clinics in Ramadi city as a test group and 25 healthy fertile men as a control group. The tests included analysis of seminal fluid and detection of the antibodies against the sperms at both seminal fluid and serum and diagnosed chromosomal deletions via real-time polymerase chain reactions using specific sequence-tagged sites. Results: The results reveal that 46 (61.33%) patients in the group of infertile men have AZF microdeletions, while no deletions are present in the control group. Out of the 75 infertile men, 5 patients (6.66%) from all the study groups have positive results for anti-sperm antibodies (ASA), while the others have negative results for ASA in both serum and seminal plasma. Conclusions: The prevalence of AZF microdeletions in our study population is high in Anbar Governorate of Iraq, while we find a low incidence of ASA in this study. All the patients with positive ASA results are without deletions in the Y chromosome’s AZF region. So there is no correlation between AZF microdeletion and ASA generation in infertile men in the current study.

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

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. 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. 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. 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.

45,X male – rare case of unbalanced translocation of Y chromosome to chromosome 2 presenting with developmental delay, learning difficulty and obesity

A male phenotype accompanied by a 45,X karyotype is rare. It may occur due to Y chromosomal translocation or insertion to X/autosome. Clinical presentation may vary depending on the presence of the Y chromosomal locus and the degree of loss of autosome material. 45,X males can present with short stature and Turner syndrome phenotype due to haploinsufficiency of genes which are normally expressed in both X and Y chromosomes. The presence of the sex-determining region Y (SRY) gene leads to the differentiation of bipotential gonads to testis. Most individuals go through puberty normally, but some may need pubertal induction for delayed puberty. Rarely some can have a pubertal arrest. The risk of gonadoblastoma is minimal in these individuals due to functioning testicular tissue. The azoospermia factor (AZF) region is found on the long arm of the Yq chromosome and is needed for spermatogenesis. In a 45,X male with unbalanced translocation of Y chromosome, spermatogenesis can be affected due to the lack of AZF leading to Sertoli cell-only syndrome. This will have an implication on fertility in adult life. We present a 14-year-old boy with developmental delay, learning difficulties and subtle dysmorphic features who was diagnosed with 45,X,der(2)t(Y:2)(?:p25). Fluorescence in situ hybridisation analysis revealed translocation of SRY (Yp11.3) to the terminal part of the short arm of chromosome 2 resulting in the deletion of most of the Y chromosome (Yp11.2-q12) and part of chromosome 2(2p25.3). This is the first case where SRY translocation to chromosome 2 presents with the above clinical presentation. 45,X karyotype is rare in male. It may occur due to SRY translocation or an insertion to X/autosome. SRY gene translocation to chromosome 2 has been not reported in the literature. Clinical presentation can be varied due to degree of loss of chromosomal material. Due to loss of AZF region found on the long arm of the Yq, spermatogenesis can be affected. Loss of 2p25 leads to learning difficulty and obesity.

Micro-TESE strategy in patients with NOA caused by AZFc deletion: synchronous or asynchronous?

In the treatment of infertile patients with non-obstructive azoospermia (NOA) caused by the deletion of the azoospermia factor c region (AZFc) on the Y chromosome, synchronous and asynchronous surgical strategies are discussed. Clinical data from NOA patients with the AZFc deletion who underwent micro-TESE were analyzed retrospectively. The sperm retrieval rate (SRR) and sperm utilization rate of synchronous and asynchronous operation groups were followed up and compared. The fertilization rate, high-quality embryo rate, clinical pregnancy rate, abortion rate, and cumulative live birth rate of ICSI in patients with successful sperm retrieval were compared between the two groups. The two groups had sperm utilization rates of 98.9% (93/94) and 50.0% (14/28), respectively. The asynchronous group's sperm consumption rates were much lower than those of the synchronous operation group. Fertilization rate, high-quality embryo rate, clinical pregnancy rate of fresh transfer cycle, abortion rate, and cumulative live birth rate of patients in the synchronous operation group with fresh sperm, and the asynchronous operation group with thawed sperm, respectively, were 30.6% vs 33.8%, 33.8% vs 40.7%, 40.0% vs 12.5%, 30.4% vs 7.1%. Between the two groups, there was no significant difference. This suggests that individuals with NOA caused by the AZFc deletion have a high possibility of successfully acquiring sperm using micro-TESE and ICSI to conceive their own offspring. Synchronous micro-TESE is recommended to improve sperm utilization rate and the cumulative live birth rate.

European Academy of Andrology: Collaboration with other societies and organisations

European Academy of Andrology: Collaboration with other societies and organisations

The significance of karyotyping and azoospermia factor analysis in patients with nonobstructive azoospermia or oligozoospermia

ObjectiveWe present our study about the significance of karyotyping and azoospermia factor(AZF) analysis in patients with azoospermia or oligozoospermia. Materials and methodsWe retrospectively reviewed 141 Taiwanese patients with nonobstructive azoospermia and 45 Taiwanese patients with oligozoospermia at MacKay Memorial Hospital, Taiwan, from 2010 to 2021 to determine the significance of karyotyping and azoospermia factor analysis. The karyotyping was analyzed using the Giemsa banding method. The AZF microdeletions were determined using multiplex polymerase chain reaction using primers specifically flanking the AZF subregions. ResultsWe found that 7.80% of patients with nonobstructive azoospermia had AZF microdeletions and 19.86% of patients with nonobstructive azoospermia had chromosomal anomalies or polymorphic variations. Furthermore, 4.44% of patients with oligozoospermia had AZF microdeletions, and 4.44% of patients with oligozoospermia had chromosomal anomalies or polymorphic variations. ConclusionIn this study, 25.53% of patients with nonobstructive azoospermia and 8.88% of patients with oligozoospermia had abnormal findings. The significance of karyotyping and azoospermia factor analysis is more critical in patients with nonobstructive azoospermia than patients with oligozoospermia. Both karyotyping and AZF analysis could prevent delayed treatment for male infertility through accurate diagnosis and appropriate treatment. The number of our patients with AZFc microdeletion was also higher than that of patients with AZFa or AZFb. The spermatogenic potential may gradually decline in patients with AZFc microdeletion. The earlier is the diagnosis, the earlier will be the retrieval of testicular spermatozoa.

Microdeleciones del gen AZF en varones azoospérmicos-oligozoospérmicos

Background and objectiveThe presence of microdeletions in the Y-chromosome azoospermia factor (AZF) region (YCMs) is considered the most frequent genetic cause of male infertility along with Klinefelter syndrome. The objective of this study was to investigate the frequencies and type of YCMs in infertile men in Aragon and to analyze the relationship between sex hormones, sperm count and microdeletions in them. Patients and methodsRetrospective descriptive study of 644 men who during 2006–2019 were screened for YCMs using YChromStrip (Operón, Spain) by PCR+reverse hybridization, spermiogram, karyotype and quantification of sex hormones. ResultsThe frequency of YCMs was 3.88% (25/644), not being detected in any patient with mild or normospermic oligozoospermia, that is, in sperm counts higher than 5×106/mL. The group of azoospermic patients was the one that presented a higher frequency of YCMs (14.58%, 14/96). Deletions in the AZFc region were the most frequent (68%). 20% (5/25) of patients with YCMs also presented some type of karyotype abnormality that included aneuploidies, deletions, duplications and/or translocations. Sperm count was significantly lower and FSH and LH concentrations significantly higher in the group of patients with YCMs. ConclusionsYCMs screening is a key test in the diagnostic approach to male infertility. Obtaining an adequate result allows choosing suitable assisted reproduction techniques, preventing unnecessary treatments and the transmission of genetic defects to offspring.

Clinical and molecular cytogenetic findings and pregnancy outcomes of fetuses with isochromosome Y

BackgroundThe mosaic forms and clinical phenotypes of fetuses with isochromosome Y are difficult to predict. Therefore, we summarized the cases of nine fetuses with isochromosome Y identified in prenatal diagnosis with a combination of molecular cytogenetic techniques, providing clinical evidence for prenatal genetic counseling.MethodsThe prenatal diagnosis and pregnancy outcomes of nine fetuses with isochromosome Y were obtained by a retrospective analysis. Isochromosome Y was identified prenatally by different approaches, such as conventional karyotyping, chromosomal microarray analysis (CMA), quantitative fluorescent polymerase chain reaction (QF-PCR) and fluorescence in situ hybridization (FISH).ResultsSeven idic(Y) fetuses and two i(Y) fetuses were identified. One fetus was complete for i(Y)(p10), and the rest with 45,X had mosaic forms. A break and fusion locus was identified in Yp11.3 in one fetus, in Yq11.22 in six fetuses and in Yp10 in two fetuses. The CMA results suggested that different deletions and duplications were found on the Y chromosome. The deletion fragments ranged from 4.7 Mb to the entire Y chromosome, and the duplication fragments ranged from 10.4 to 18.0 Mb. QF-PCR analysis suggested that the AZF region was intact in one fetus, four fetuses had AZFb+c+d deletion, one fetus had AZFa+b+c+d deletion, and one fetus had AZFc+d deletion. Finally, four healthy male neonates were delivered successfully, but the parents of the remaining five fetuses, including three healthy and two unhealthy fetuses, chose to terminate their pregnancies.ConclusionThe fetus and neonate phenotype of prenatally detected isochromosome Y usually is that of a normally developed male, ascertained in the absence of other indicators of a fetal structural anomaly. Our study provides clinical reference materials for risk assessment and permits better prenatally counseling and preparation of parents facing the birth of isochromosome Y fetuses.

O-207 Incidence of Y chromosome microdeletions and microdissection testicular sperm extraction (micro TESE) in patients with Japanese azoospermic patients

Abstract Study question What is the frequency of azoospermia factor (AZF) microdeletions and sperm retrieval rate (SRR) by micro TESE in patients with these deletions? Summary answer AZFc is most frequent of Y chromosome microdeletions and a predictor of micro TESE outcome in Japanese azoospermic men. What is known already After Klinefelter syndrome, Y chromosome microdeletions are the second most frequent genetic cause of male infertility, with a prevalence of 2%-10% in non-obstructive azoospermia (NOA) and three spermatogenesis loci in the Y chromosome long arm (Yq11) have been classified as AZFa, AZFb, and AZFc. The classical correlation of histopathology phenotypes with these three microdeletions comprises of complete absence of germ cells (Sertoli cell-only syndrome) in patients with AZFa microdeletions, maturation arrest of meiosis in patients with AZFb microdeletions, and hypospermatogenesis in patients with AZFc microdeletions, however, individual variation in the extent of deletions has led to various spermatogenic phenotypes. Study design, size, duration We performed a retrospective study based on two reproduction centers in Japan and evaluated 1373 azoospermic patients in our clinics between September 2013 and December 2021. We investigated the frequency of AZF microdeletions and SRR by micro TESE in patients with these microdeletions and therefore aimed to evaluate the correlation between AZF microdeletions and micro TESE results. Participants/materials, setting, methods A total of 1373 azoospermic were enrolled. After the diagnosis of azoospermia, karyotype analysis and detection of Y chromosome microdeletions were performed on peripheral blood lymphocytes of these patients. Y chromosome microdeletions in AZFa, AZFb, and AZFc regions were detected using Promega Y Chromosome AZF Analysis System version 2.0 (Promega Co.). Twenty sequence-tagged sites within the AZF region of Yq11 and the sex-determining region Y gene were targeted for polymerase chain reaction (PCR) amplification. Main results and the role of chance One hundred and fifty-two AZF microdeletions (11.1%) were detected in the azoospermic patients. The most common deleted region was AZFc (60 cases, 4.4%). Among the patients, 17 (1.2%), 1 (0.1%), 42 (3.1%), 13 (1.0%), and 6 (0.5%) had AZFa, AZFa+b, AZFb+c, AZFb, and AZFa+b+c microdeletions, respectively. When the cases were grouped according to causes of infertility that could be detected, no Y chromosome microdeletions were detected in some groups (cases with Klinefelter Syndrome, hypogonadotropic hypogonadism, congenital absence of vas deferens, and 47, XYY karyotype). Fifty-three azoospermic men with AZFc microdeletions underwent micro TESE, and spermatozoa were detected in 88.7% (47/53) of these men. In contrast, we detected spermatozoa in only 20.4% (109/534) of the azoospermic men without AZF microdeletions. The SRR was much higher in patients with AZFc microdeletions than that of patients without AZF deletions. Although three azoospermic men with AZFb+c microdeletions had also undergone micro TESE following patient request, we did not retrieve spermatozoa. Limitations, reasons for caution We excluded post chemotherapy NOA showing 46, XX and AZFa+b+c deletions post bone marrow transplantation from female donor. Additionally, we did not detect AZFc partial deletion including gr/gr deletion. The cohort size of this study is not small, however, our screened population of infertile men may be biased. Wider implications of the findings NOA patients with AZFc microdeletions had a high percentage of successful sperm retrieval by micro TESE. Our study emphasizes that diagnosis of Y chromosome microdeletions is critical for preconception genetic counseling and provides clinically valuable prognostic information to couples considering surgical sperm retrieval. Trial registration number None

O-154 Genetic screening in male infertility: time for new guidelines?

Abstract Approximately 1 in 7 couples wishing to conceive fail to do so after one year of trying. About half of the cases can be assigned to male factors. Currently, genetic causes explain approximately 4-9% of all cases of male infertility, with strong variation among the different subtypes of infertility. Presently a majority of all infertile males (60-70%) remain without a clear diagnosis, despite the expectation that genetic causes explain a substantial fraction of these patients. Without a genetic diagnosis, it is impossible for a clinician to accurately provide counseling to couples with questions about the causes of their infertility, possible co-morbidities, the potential success of ART treatment and the reproductive health of their offspring. Hence, knowing and understanding the genetic causes of infertility is of enormous value to patients and clinicians. Already for more than 25 years, genetic testing is recommended for patients with severe oligozoospermia or azoospermia. These tests typically include karyotyping, azoospermia factor (AZF) deletion screening and/or CFTR mutation testing (in case of CBAVD). For a long time, further genetic testing was time-consuming and expensive, resulting in slow progress to unveil novel causes of male infertility in both research and clinic. The uptake of next-generation sequencing (NGS) methods has revolutionized genetic testing for a plethora of disorders, allowing for cost-effective screening of variants in limited gene panels, all coding parts of genes (exome), or the entire genome. NGS is now routinely used by many laboratories for genetic testing in diagnostics and research. Using these new technologies, we and others have identified numerous novel causal variants in genes indispensable for human fertility. Although the use of NGS in male infertility research is skyrocketing in recent years, it has not yet broadly found its way into diagnostics. This means that 1) numerous patients with over a hundred newly discovered monogenic causes of male infertility are presently left undiagnosed and 2) large groups of patients with phenotypes such as globozoospermia or multiple morphological abnormalities of the sperm flagellum do not routinely receive genetic testing, even though diagnostic yields of up to 60% can be expected. The field of genetics of male infertility is rapidly evolving and it is time for patients to benefit from these advances. This talk will cover the steps we and others are taking towards bringing NGS methods such as whole exome sequencing to the clinic for male infertility and will show the first results on the validation of our whole exome sequencing-based test for male infertility.

P-086 AZF Microdeletions: A New Look at Past Paradigms

Abstract Study question To elucidate whether AZF screening is indicated in men with proven non-obstructive azoospermia (NOA) or severe oligozoospermia (<5x106 sperm/mL) who concomitantly harbor chromosomal abnormalities. Summary answer Some chromosomal aberrations are concomitant with AZF microdeletions, and hence Y chromosome microdeletion (YCM) screening is necessary for these patients. What is known already In the era of assisted reproduction, finding cost-minimization strategies in infertility clinics without affecting the quality of diagnosis and treatment is becoming one of the top-priority topics for future research. Formulating definitive guidelines concerning the indications for paraclinical testing could be one of the strategies. Accordingly, definitive guidelines for AZF screening do not exist especially in men who also have chromosomal abnormalities. The current clinical practice is to decide per case whether to pursue further molecular testing, with the accompanying added psychological and socio-economic burden. Study design, size, duration An observational retrospective cohort study of 10,388 consecutive cases from a tertiary infertility referral center (Royan institute, Tehran, Iran) over the last 12 years. A comprehensive literature review was also performed to summarize scientific evidence. Participants/materials, setting, methods The study recruited the largest cohort of men with primary infertility (NOA or severe oligozoospermia) ever presented who underwent screening for cytogenetic abnormalities and YCMs using sequence-tagged sites-polymerase chain reaction (STS-PCR) with a primer set covering the AZF regions according to the EAA/EMQN guidelines, as part of the infertility workup prior to ART. Main results and the role of chance In total, one-third of men with YCMs concomitantly had an abnormal karyotype (excluding those with heteromorphisms) (169/565; 29.9%, 95% CI: 26.3-33.8). In respect to the frequency of YCMs, our findings suggest that the cases diagnosed with 46,X,add(X) with incidence of 1.8% (3/169; 95% CI: 0.6-5.1); 45,X and mosaic forms: 45,X/46,X,inv(Y)(p11.2q12); 45,X/46,X,del(Y); 45,X,der(Y;Autosome); 45,X/46,X,idic(Y)(p11.2); 45,X/46,XY,r(Y); and 45,X/46,X,idic(Y)(q11.2) (19/169; 11.2%, 95% CI: 7.3-16.9); and inv(Y)(p11.2q12) (2/169; 1.2%, 95% CI: 0.3-4.2) should also be referred for AZF deletion screening, as data suggests they are accompanied with YCMs. Limitations, reasons for caution The extension of the outcomes beyond the described population could introduce concerns on appropriate medical management. Confirmatory studies in other geographic/ethnic groups are still necessary to reach a consensus regarding the outcomes. Wider implications of the findings It has been recommended that all men with NOA who have chromosomal abnormalities, except those with 46,XY/45,X karyotype, do not need AZF testing. The results reflect a crucial need for reconsidering whether YCM testing is indicated in the population of men with certain karyotypic abnormalities. Trial registration number Not Applicable

P-105 Genetic variants in the spermatogenic transcription regulators RFX2 and TAF7 enhance the incidence of azoospermia

Abstract Study question What are the genetic variants of meiotic regulator genes that are implicated in male infertility among men from West Bengal, India? Summary answer TAF7 C16T (MW827584 G > A) and RFX2 562delT (MZ560629delA) new variations may have a major impact on optimal fertility in men from West Bengal. What is known already According to published research, roughly 50% of reported infertility cases in India are due to male reproductive abnormalities. Because of the involvement of several gene regulatory networks in the spermatogenesis process, the genetic etiology of idiopathic male infertility remains a mystery. The AZF region of the Y chromosome microdeletion has been discovered as a prevalent cause of male infertility across all ethnicities. Infertility in men has also been linked to sex chromosomal copy number variation and autosomal gene polymorphisms. Study design, size, duration This is a case-control study to infer whether the genetic variants are significantly associated in favor of cases or in favor of controls. The study included 160 healthy male controls and 150 azoospermic cases submitted to the Institute of Reproductive Medicine in Kolkata (IRM). All of the subjects agreed to participate in the study and provided blood and sperm samples. The duration of the study is three years. Seminograms were made to filter azoospermic individuals. Participants/materials, setting, methods 2 ml of venous blood samples were taken from azoospermic men using the venipuncture procedure and stored in EDTA coated vacutainer tubes at -20°C for DNA isolation.QIAamp Blood Mini Kit was used to isolate genomic DNA. For PCR analysis, unique primers for TAF7 and RFX2 functional domains were created. Taq Dye Deoxy Terminator sequencing kit (Applied Biosystems, Foster City, USA) and ABI Prism 377 DNA sequencer were used to analyze PCR results. Main results and the role of chance We have found two significant genetic alterations in the coding region of TAF7 and RFX2 which are MW827584 G > A(C16T) and MZ560629delA(562delT) respectively. Both were found to have a strong association to the occurrence of azoospermia. For MW827584 G > A, minor allele ‘A’ has shown elevated risk of azoospermic against the odd 4.684 (95% C.I = 2.900-7.564, Relative risk =2.110, p value = <0.001). Fisher’s exact test was performed to calculate Odds ratios with respective 95% confidence interval (CI). After Bonferroni's correction, a two-tailed P-value of less than 0.02 was considered statistically significant throughout. Only the case sample cohort had the RFX2 deletion MZ560629 delA. Only 9 cases with this unique deletion in heterozygous form were found out of 150 azoospermic patients. This frameshift mutation results in the production of an early termination signal at exon 6, resulting in the creation of a truncated protein. The wild-type RFX2 protein has 729 amino acids but this frameshift mutation results in a 212-amino-acid RFX2 protein that lacks several secondary motifs and domains, rendering the wild-type function useless. In silico analysis of SIFT, PROVEAN, MUTATION TASTER and REGULATION SPOTTER predicted that these genetic variants are disease-causing. Limitations, reasons for caution There are some potential limitations to our research. A larger sample size investigation is needed to reconfirm the genetic association study. Moreover, confounding effects of allelic variation of those genetic modifiers may cause differences in the manifestation of fertility state along with the allelic variants of TAF7 and RFX2 genes. Wider implications of the findings This diagnostic technique will not only reduce the hazards connected with assisted reproductive technologies (ART), but it will also provide insight into previously unknown areas of infertility and enable clinicians in conducting a more thorough investigation of the patients. Trial registration number not applicable

Genetic Architecture of Azoospermia—Time to Advance the Standard of Care

BackgroundCrypto- and azoospermia (very few/no sperm in the semen) are main contributors to male factor infertility. Genetic causes for spermatogenic failure (SPGF) include Klinefelter syndrome and Y-chromosomal azoospermia factor microdeletions, and CFTR mutations for obstructive azoospermia (OA). However, the majority of cases remain unexplained because monogenic causes are not analysed. ObjectiveTo elucidate the monogenic contribution to azoospermia by prospective exome sequencing and strict application of recent clinical guidelines. Design, setting, and participantsSince January 2017, we studied crypto- and azoospermic men without chromosomal aberrations and Y-chromosomal microdeletions attending the Centre of Reproductive Medicine and Andrology, Münster. Outcome measurements and statistical analysisWe performed exome sequencing in 647 men, analysed 60 genes having at least previous limited clinical validity, and strictly assessed variants according to clinical guidelines. Results and limitationsOverall, 55 patients (8.5%) with diagnostic genetic variants were identified. Of these patients, 20 (3.1%) carried mutations in CFTR or ADGRG2, and were diagnosed with OA. In 35 patients (5.4%) with SPGF, mutations in 20 different genes were identified. According to ClinGen criteria, 19 of the SPGF genes now reach at least moderate clinical validity. As limitations, only one transcript per gene was considered, and the list of genes is increasing rapidly so cannot be exhaustive. ConclusionsThe number of diagnostic genes in crypto-/azoospermia was almost doubled to 21 using exome-based analyses and clinical guidelines. Application of this procedure in routine diagnostics will significantly improve the diagnostic yield and clinical workup as the results indicate the success rate of testicular sperm extraction. Patient summaryWhen no sperm are found in the semen, a man cannot conceive naturally. The causes are often unknown, but genetics play a major role. We searched for genetic variants in a large group of patients and found causal mutations for one in 12 men; these predict the chances for fatherhood.

AZFa Y gene, DDX3Y, evolved novel testis transcript variants in primates with proximal 3´UTR polyadenylation for germ cell specific translation

Translational control is a major level of gene expression regulation in the male germ line. DDX3Y located in the AZFa region of the human Y chromosome encodes a conserved RNA helicase important for translational control at the G1-S phase of the cell cycle. In human, DDX3Y protein is expressed only in premeiotic male germ cells. In primates, DDX3Y evolved a second promoter producing novel testis-specific transcripts. Here, we show primate species-specific use of alternative polyadenylation (APA) sites for these testis-specific DDX3Y transcript variants. They have evolved subsequently in the 3´UTRs of the primates´ DDX3Y transcripts. Whereas a distal APA site (PAS4) is still used for polyadenylation of most DDX3Y testis transcripts in Callithrix jacchus; two proximal APAs (PAS1; PAS2) are used predominantly in Macaca mulatta, in Pan trogloydates and in human. This shift corresponds with a significant increase of DDX3Y protein expression in the macaque testis tissue. In chimpanzee and human, shift to predominant use of the most proximal APA site (PAS1) is associated with translation of these DDX3Y transcripts in only premeiotic male germ cells. We therefore assume evolution of a positive selection process for functional DDX3Y testis transcripts in these primates which increase their stability and translation efficiency to promote its cell cycle balancing function in the human male germ line.

Birth of mice from meiotically arrested spermatocytes following biparental meiosis in halved oocytes.

Microinjection of spermatozoa or spermatids into oocytes is a major choice for infertility treatment. However, the use of premeiotic spermatocytes has never been considered because of its technical problems. Here, we show that the efficiency of spermatocyte injection in mice can be improved greatly by reducing the size of the recipient oocytes. Live imaging showed that the underlying mechanism involves reduced premature separation of the spermatocyte's meiotic chromosomes, which produced much greater (19% vs. 1%) birth rates in smaller oocytes. Application of this technique to spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans, resulted in the production of live offspring. Thus, the microinjection of primary spermatocytes into oocytes may be a potential treatment for overcoming a form of nonobstructive azoospermia caused by meiotic failure.