Non-obstructive Azoospermic Men Research Articles

P-570 A cell-free DNA test in semen of nonobstructive azoospermic men to predict spermatogenesis

Abstract Study question Can a genetic assessment of cell-free DNA, isolated from seminal plasma of nonobstructive azoospermic (NOA) men, predict successful sperm extraction at testicular biopsy? Summary answer Seminal cell-free DNA (cfDNA) isolated from the ejaculates of NOA men with failed testicular sperm retrieval outcomes concurrently and consistently displayed mutations on three genes. What is known already The most puzzling form of azoospermia is the testicular type, where the chances of successful microdissection testicular sperm extraction (micro-TESE) are unpredictable. Therefore, developing a method that could identify or predict individuals with absent spermatogenesis would be most useful. While histopathology is considered the most reliable method for predicting successful micro-TESE, it is invasive and often inconclusive. Recently, cfDNA testing has been gaining momentum as a noninvasive method for prenatal screening and even with the potential for eventually replacing PGT-A. However, the prognostic value of using cfDNA to predict the presence/absence of spermatozoa in men undergoing micro-TESE is relatively unknown. Study design, size, duration Over a period of 8 months, we recruited azoospermic men with normal peripheral karyotypes and for whom an extensive semen analysis did not identify spermatozoa. For consenting patients (n = 12), we performed genetic assessments on cfDNA from their ejaculates (IRB 1006101085). These patients subsequently underwent micro-TESE, and their cfDNA mutation profiles were compared according to whether spermatozoa were successfully retrieved ((+)Sperm, n = 5), or not ((-)Sperm, n = 7). Participants/materials, setting, methods Using a commercially available kit, circulating cfDNA was eluted from the ejaculates of consenting NOA men, as well as a normozoospermic control (n = 2). The isolated cfDNA specimens subsequently underwent whole exome sequencing (WES) on an Illumina HiSeq at 2x150bp. Gene mutations were detected by CLC Genomic Server 9.0 and compared within, as well as between, the +Sperm and -Sperm cohorts in comparison to the control group. Main results and the role of chance All 12 men (35.5±4yrs) had normal peripheral karyotypes and tested negative for Y-microdeletions. They had no history of genetic diseases and were off any supplements, medications, and/or hormones before or during the study. Genetic assessment on cfDNA from the +Sperm cohort (n = 5; 38.3±2yrs) identified 8 genes (ADAMTS7, ATP9B, CBFA2T3, FAM20C, FCGBP, PEBP4, PHF2, RPS9) that were concurrently mutated among all NOA men from this group. Although ADAMTS7 plays a potential role in sperm-egg fusion, the remaining genes are unrelated to sperm production. Whole exome sequencing of cfDNA from the -Sperm cohort (n = 7; 33.5±4yrs) consistently detected mutations on 3 genes, including one involved in spermatogenesis (CFAP54), one essential for oxidative regulation of spermatozoa (GSTT4), and one that encodes testis antigens that are normally expressed during spermatogenesis (BAGE2). Moreover, we concurrently and consistently identified pathogenic mutations on an additional gene in the -Sperm cohort. This gene (MUC2) is involved in the epithelial coating of mucus membrane-containing organs, and its expression has been correlated with spermatogenesis. Interestingly, these genes were all unaffected in men with successful micro-TESE outcomes. Limitations, reasons for caution Although we detected candidate gene mutations that correlate with micro-TESE outcome, these are preliminary findings that need further validation in a larger study cohort. Furthermore, while cfDNA analysis has shown great promise, it is not specific enough on its own and should be corroborated by epigenetic testing. Wider implications of the findings Genetic screening of seminal cfDNA provides a noninvasive alternative to histopathology and can be used to predict the absence of spermatogenesis, sparing these men from unnecessary surgery and cost. This novel biomarker approach lays the groundwork for precision medicine in male infertility testing. Trial registration number n/a

P-069 The expression patterns of ALOX15 and GPX4 in the seminiferous epithelium of men with non-obstructive azoospermia (NOA) due to primary testicular failure

Abstract Study question Does the expression of ALOX15 and GPX4 differ in NOA men with a history of cryptozoospermia from NOA who never had sperm in their ejaculate? Summary answer There was no significant difference between these 2 groups of patients. What is known already Oxidative stress (OS) has been investigated as a prime suspect for many unexplained pathologies, including primary testicular failure (PTF). The fact that male germ cells are rich in polyunsaturated fatty acids (PUFAs) but have a limited amount of cytoplasmic scavenging enzymes, is the reason behind their extreme vulnerability to OS. The accumulation of lipid peroxidation markers leads to the inactivation of glutathione peroxidase 4 (GPX4), the lipid repair enzyme, and the exacerbation of arachidonate 15-lipoxygenase (ALOX15), a lipoxygenase enzyme that promotes lipid degradation. This study investigates the activity of these antagonizing enzymes in the testicular tissue of NOA patients. Study design, size, duration This observational study was conducted in the period from May 2020, following the Ethics Committee of the University of Split School of Medicine approval, to October 2023. The testicular tissue from 57 patients diagnosed with NOA (according to WHO criteria) and referred to their first microTESE as a part of fertility treatment was collected and processed. Participants/materials, setting, methods Samples were prepared for histological evaluation and immunodetection (fixation; embedding in paraffin; H&E staining; and staining with anti-ALOX15 and anti-GPX4 antibodies). Two groups of NOA patients were observed: Crypto-NOA and NOA group. The main outcome measure was the number of ALOX15 positive germ cells per 1 mm² of the seminiferous tubule surface (N/ mm²) and the percentage (%) of GPX4 positive surface per surface of the tubule measured by the ImageJ program. Main results and the role of chance Spermatozoa were retrieved in 37 of 57 (64,91 %) men; samples of 4 patients had to be excluded due to poor fixation. There were 18 patients in the Crypto-NOA group, and 10 in the NOA group, while 5 patients were diagnosed with obstructive azoospermia (OA) and were used as controls. For comparison of the baseline data of Crypto-NOA and NOA patients, we used the Mann-Whitney test, and with statistical significance set at the level of P < 0.05, we found that there were significantly more patients with cryptorchism in the NOA group. As a tool for histological evaluation, we used the diagnostic code designed by McLachlan et al. and found similar spermatogenic phenotypes between Crypto NOA and NOA patients. The most represented phenotype was hypospermatogenesis with a moderate reduction in late spermatid number mixed with normal tubules (38,8 % in Crypto-NOA; 40% in NOA), following the phenotype where late spermatids are present in some tubules but mixed with tubules showing no progression past the spermatocyte stage. Using the Mann-Whitney test, we did not find a significant difference in the expression of ALOX15 and GPX4 in the seminiferous epithelium of Crypto-NOA and NOA patients. Limitations, reasons for caution The testicular tissue of NOA patients is characterized by focal or patchy spermatogenesis which led to the exclusion of several samples due to the fixation of tissue with a homogenous pattern of Sertoli cell-only or germ-cell arrest. Wider implications of the findings This study will provide additional information regarding the cause of impaired spermatogenesis and help to determine if the patient is a potential candidate for antioxidant therapy. Our next step is to associate the expression patterns of ALOX15 and GPX4 with the success rates of in vitro fertilization procedure outcomes. Trial registration number not applicable

O-263 The risk of hypogonadism after microdissection testicular sperm extraction (micro TESE) in non-obstructive azoospermia (NOA) patients with various etiologies

Abstract Study question What is the risk factor of hypogonadism after micro TESE in NOA patients? Summary answer Klinefelter syndrome (KS), past history of cryptorchidism, preoperative testicular volume <8 ml or testosterone <287 ng/dL are significant risk factors of hypogonadism after micro TESE. What is known already Micro TESE combined with intracytoplasmic sperm injection (ICSI) is currently standard procedure to treat infertility in cases of NOA. Although many studies have reported the effectiveness of the treatment concerning about sperm retrieval rates (SRR) and the live birth rates, data on safety, especially on the risk of hypogonadism, are limited. Most previous studies, including our past study, regarding the risk of hypogonadism after TESE have been limited to relatively few in number of patients and the investigations only of the changes of mean serum testosterone levels. Study design, size, duration This study retrospectively investigated 535 NOA patients including 336 cases of unexplained NOA, 91 KS, 16 other chromosomal abnormalities, 31 azoospermia factor (AZF)c deletions, 31 past history of cryptorchidism, and 30 post anticancer chemotherapy, who had undergone micro TESE at our institution from September 2013 to August 2018. All of them had been examined serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone levels before and at least once in 1-3 months after micro TESE. Participants/materials, setting, methods In this study, hypogonadism was defined as total testosterone <250 ng/dL, which is established by the Japanese Society of Men’s Health as diagnostic criteria for late onset hypogonadism syndrome, and/or starting testosterone replacement therapy (TRT). We assessed risk factors of hypogonadism after micro TESE using indicators such as age, preoperative testicular volume and testosterone levels, past medical history, unilateral or bilateral, first time or not micro TESE. Main results and the role of chance In total of 535 men, FSH and LH after micro TESE significantly increased vs baseline (26.2 vs 29.9 IU/L, 9.6 vs 12.8 IU/L, respectively). On the other hand, testosterone levels significantly decreased after surgery (351.2 vs 297.5 ng/dL). Although postoperative hypogonadism was found in 42% (227/535), many of whom were cases of KS or preoperative testosterone levels <250 ng/dL. Sixteen men with KS, 3 men with unexplained NOA, and 2 men with past history of cryptorchidism started TRT because they complained clinical symptoms after micro TESE. Of the 21 men who started TRT, only 5 had new-onset low testosterone levels after micro TESE. According to the assessment of any association of various indicators and low postoperative total testosterone levels using multiple logistic regression analysis, significant risk factors of hypogonadism early after micro TESE were cases of KS (OR 11.4, 95% CI 4.4-29.9), preoperative testosterone levels <287 ng/dL (OR 7.5, 95% CI 4.7-12.0), preoperative testicular volume <8 ml (OR 3.9, 95% CI 2.3-6.4), past history of cryptorchidism (OR 2.9, 95% CI 1.1-7.8). Limitations, reasons for caution Most examinations of postoperative hormone levels were performed in short-term period after micro TESE, so we could only assess the risk of postoperative hypogonadism in a short time course in this cohort study. Wider implications of the findings The strength of the current study is that the risk factors of hypogonadism after micro TESE was assessed in a large population of NOA men with various etiologies. The results of this study provide useful clinical information about the risk of postoperative hypogonadism for NOA patients considering micro TESE. Trial registration number not applicable

Parameters affecting the success rate of microscopic testicular sperm extraction in male patients with a solitary testis and non-obstructive azoospermia.

We aimed to compare the success rate of spermatozoa retrieval through microscopic testicular sperm extraction (mTESE) in non-obstructive azoospermic (NOA) men with a solitary testis with that of mTESE in NOA men with bilateral testes and the parameters affecting these rates. A retrospective cross-sectional study of factors contributing to infertility in NOA patients with a solitary testis and men with bilateral testes was carried out. In this multicenter study, 74 patients with NOA with a solitary testis were matched with 74 patients with bilateral testes in terms of age, duration of infertility, and volume of the solitary testis from 2770 patients with NOA with bilateral testes. Hormonal parameters, presence of varicocele, history of varicocelectomy, history of undescended testis and karyotype analysis results were compared. Spermatozoa were obtained from 40 (54.1%) patients with a solitary testis and 42 (56.76%) patients with bilateral testes. No differences were found regarding age, duration of infertility, or mean testicular volume between patients with a solitary testis and patients with bilateral testes. When serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were compared regardless of sperm retrieval status, it was observed that both levels were greater in the group of patients with a solitary testis (p < 0.01). Patients with solitary and bilateral testes from whom spermatozoa were obtained had larger testes than those from whom spermatozoa could not be obtained (p < 0.05). Similarly, the serum levels of FSH and LH were significantly greater in patients with a solitary testis than in those with bilateral testes (p < 0.05). To the best of our knowledge, this is the first study in the literature to evaluate the parameters that influence mTESE outcome in NOA patients with a solitary testis and NOA patients with bilateral testes. Greater testicular volume was found to positively affect spermatozoa retrieval for patients with a solitary testis. The higher levels of FSH and LH in patients with a solitary testis than in patients with bilateral testes of similar testicular volume may be due to a compensatory mechanism developed by the hypothalamic-pituitary-gonadal axis. The fact that these hormones are higher in patients with a solitary testis does not mean that the number of spermatozoa obtained through mTESE will be decreased.

Genetic profiling of azoospermic men to identify the etiology and predict reproductive potential

PurposeTo identify germline mutations related to azoospermia etiology and reproductive potential of surgically retrieved spermatozoa, and to investigate the feasibility of predicting seminiferous tubule function of nonobstructive azoospermic men by transcriptomic profiling of ejaculates.Materials and methodsSperm specimens were obtained from 30 men (38.4 ± 6 years) undergoing epididymal sperm aspiration for obstructive azoospermia (OA, n = 19) acquired by vasectomy, or testicular biopsy for nonobstructive azoospermia (NOA, n = 11). To evaluate for a correlation with azoospermia etiology, DNAseq was performed on surgically retrieved spermatozoa, and cell-free RNAseq on seminal fluid (n = 23) was performed to predict spermatogenesis in the seminiferous tubule.ResultsOverall, surgically retrieved sperm aneuploidy rates were 1.7% and 1.8% among OA and NOA cohorts, respectively. OA men carried housekeeping-related gene mutations, while NOA men displayed mutations on genes involved in crucial spermiogenic functions (AP1S2, AP1G2, APOE). We categorized couples within each cohort according to ICSI clinical outcomes to investigate genetic causes that may affect reproductive potential. All OA-fertile men (n = 9) carried mutations in ZNF749 (sperm production), whereas OA-infertile men (n = 10) harbored mutations in PRB1, which is essential for DNA replication. NOA-fertile men (n = 8) carried mutations in MPIG6B (stem cell lineage differentiation), whereas NOA-infertile individuals (n = 3) harbored mutations in genes involved in spermato/spermio-genesis (ADAM29, SPATA31E1, MAK, POLG, IFT43, ATG9B) and early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). Transcriptomic assessment of cell-free RNAs in seminal fluid from NOA men allowed the prediction of residual spermatogenic foci.ConclusionsSperm genome profiling provides invaluable information on azoospermia etiology and identifies gene-related mechanistic links to reproductive performance. Moreover, RNAseq assessment of seminal fluid from NOA men can help predict sperm retrieval during testicular biopsies.

A step closer to parenthood with non-obstructive azoospermia: Unveiling the impact of microdissection testicular sperm extraction in Australia's largest single-centre study.

Non-obstructive azoospermia (NOA) diagnosis poses challenges for couples seeking parenthood. Microdissection testicular sperm extraction (MD-TESE) excels in retrieving testicular sperm cells for NOA cases. However, limited live birth data in Australian NOA patients hinders accurate counselling. This study aimed to determine the likelihood of infertile couples with a male partner diagnosed with NOA conceiving biological children using MD-TESE / intracytoplasmic sperm injection (ICSI). A retrospective cohort study included 108 NOA men treated at a public fertility unit and a private fertility centre (May 2009-May 2022). live birth rate (LBR); secondary outcomes: sperm retrieval rate, pregnancy rate, and neonatal outcomes. Among 108 patients undergoing MD-TESE, the positive sperm retrieval rate (PSRR) was 64.8% (70/108). Histology best predicted sperm retrieval success, with hypo-spermatogenesis yielding a 94.1% PSRR. Age, testicular volume, and hormonal parameters had no significant impact. Mean male age: 35.4 years; mean partner age: 32.7 years. Fertilisation rate: 50.7%. LBR per initiated cycle: 58.7% (37/63); per embryo transfer: 63.8% (37/58); per initially diagnosed NOA man: 34.3% (37/108). Cumulative LBR: 74.1% (43/58); twin rate: 10.8% (4/37). No neonatal deaths or defects were observed among 47 live offspring. This study provides valuable data for counselling NOA couples on the probability of conceiving biological offspring. MD-TESE and ICSI yielded favourable PSRR (64.8%) and LBR (63.8%). However, couples should be aware that once NOA is confirmed, the chance of taking home a baby is 34%.

Intratesticular Testosterone and Its Precursors among Azoospermic Men: A Pilot Study.

The study aimed to comprehensively analyze testosterone and precursor concentrations in the testicular interstitial fluid (TIF) of men with azoospermia, exploring their significance in the testicular microenvironment and their correlation with testicular sperm retrieval outcomes. We analyzed 37 TIF samples, including 5 from men with obstructive azoospermia (OA) and 32 from men with non-obstructive azoospermia (NOA). Liquid chromatography with tandem mass spectrometry quantified testosterone and precursor levels. Comparative assessments of the outcomes of testicular sperm retrieval were performed between the OA and NOA groups as well as among men with NOA. Men with NOA who had not undergone hormone treatment exhibited significantly higher intratesticular concentrations of testosterone (median 1,528.1 vs. 207.5 ng/mL), androstenedione (median 10.6 vs. 1.9 ng/mL), and 17-OH progesterone (median 13.0 vs. 1.8 ng/mL) than men diagnosed with OA. Notably, in the subgroup of patients with NOA subjected to medical treatment, men with successful sperm retrieval had significantly reduced levels of androstenedione (median androstenedione 5.7 vs. 18.5 ng/mL, p=0.004). Upon a more detailed analysis of these men who underwent hormone manipulation treatment, the testosterone/androstenedione ratio (indicative of HSD17B3 enzyme activity) was markedly increased in men with successful sperm retrieval (median: 365.8 vs. 165.0, p=0.008) compared with individuals with NOA who had unsuccessful sperm recovery. Furthermore, within the subset of men with NOA who did not undergo medical treatment before microdissection testicular sperm extraction but achieved successful sperm retrieval, the ratio of 17-OH progesterone/progesterone (indicative of CYP17A1 activity) was substantially higher. The study suggests distinct testosterone biosynthesis pathways in men with compromised spermatogenesis and those with normal spermatogenesis. Among NOA men with successful retrieval after hormone optimization therapy, there was decreased androstenedione and increased HSD17B3 enzyme activity. These findings have diagnostic and therapeutic implications for the future.

Тестикулярный микробиом у инфертильных мужчин.

Introduction. The concept of microbiome is a collective term for many microorganisms, such as bacteria, archaea, fungi, protists, and viruses, inhabiting various anatomical areas of the human body. The relevance of studying their quantitative and qualitative relationships is associated with the size of microbial populations, as well as the lack of awareness of the modern scientific community about the true role of most of them, including the connection between the microbiome diversity of male testicles and sperm with the presence of azoospermia and the development of idiopathic variants of infertility. The purpose: identification of the composition of the microbiome of testicular tissue in infertile men and determination of its clinical significance. Studying the influence of microbial populations of the urogenital tract of men on the development and course of diseases associated with infertility, such as azoospermia. Materials and methods. A literature review was performed based on data published in the PubMed (https://www.ncbi.nlm.nih.gov/pubmed/) and Scientific Electronic Library eLibrary.ru (https://elibrary.ru/) databases. The search was carried out using the following keywords: microbiome; microbiota; testicular tissue; azoospermia; sequencing; genome; male infertility. A total of 167 sources were analyzed. For this publication, 48 publications were selected. Results. Lactobacillus iners, Gardnerella vaginalis, Escherichia faecalis, Escherichia coli and Staphylococcus aureus are associated with the development of non-obstructive azoospermia. In men with different forms of azoospermia, a greater number of Bacteroidetes and Firmicutes phyla were observed, while Proteobacteria and Actinobacteria were significantly less common compared to healthy controls. Also, the microbial diversity between obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) had a higher degree of similarity than the control group. Interestingly, the microbiome modifications found in men with NOA are similar to those previously identified in the gut of older adults, which can be considered as further evidence of microbiologically early aging of NOA men at the testicular level. Conclusions. Microbiome analysis demonstrated for the first time that human testicular tissue is not a microbiologically sterile environment, and also provided new data on the bacteriological composition of the male urogenital tract and allowed us to consider excluding any potential contribution of microbial communities present in neighboring anatomical regions. Therefore, larger studies are needed to confirm our preliminary findings that the testes of azoospermic men contain a more dysbiotic bacterial community.

Seminal TEX101 May Predict Seminal Sperm Recovery after Varicocelectomy in Nonobstructive Azoospermic Patients with Varicocele

Background. Infertile men with nonobstructive azoospermia (NOA) associated with varicocele may benefit from varicocele repair with recovered sperms in semen. Currently, there is no clinically applied method to predict the success of seminal sperm recovery in this subset of azoospermic men. Objective. We aimed to evaluate the predictive role of the seminal testis expressed protein (TEX101) in sperm recovery in the semen of NOA men with palpable bilateral varicocele after microsurgical varicocelectomy. Patients and Methods. This prospective cross-sectional cohort study included 40 patients with NOA and bilateral varicocele. Seminal TEX101 levels were measured followed by subinguinal microsurgical varicocele repair. Two seminal analyses were carried out at 3- and 6-month follow-up periods to evaluate seminal sperm recovery. Results. After varicocele repair, sperms were recovered in the semen of 10/40 patients (25%) along the follow-up periods (seven patients after 3 months and additional three patients after 6 months). The preoperative median seminal TEX101 level was significantly higher in NOA men with seminal sperm recovery compared with NOA patients without seminal sperm recovery (13.5 vs. 9.8 ng/ml, p = 0.014 ). Besides, the serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) median levels were significantly higher in NOA men without seminal sperm recovery compared with NOA men with seminal sperm recovery ( p = 0.001 , p = 0.01 ). There were nonsignificant differences comparing the preoperative testicular volume or serum testosterone levels between the two investigated groups ( p = 0.072 , p = 0.272 ). A cutoff value of 9.9 ng/ml was demonstrated to have preoperative TEX101 prediction of seminal sperm recovery (sensitivity of 90% and specificity of 57%). Conclusion. Preoperative seminal TEX101 could be considered as a biomarker for sperm recovery for seminal sperm recovery in NOA cases associated with varicocele after its surgical repair. Further work at this point with larger number of cases should be carried out to verify its potential importance.

Tip-microVapour Fast Freezing: A novel easy method for cryopreserving severe oligozoospermic samples.

Sperm cryopreservation is an important procedure for oligozoospermic subjects at risk of azoospermia and after surgical recovery of spermatozoa in non-obstructive azoospermic men. Conventional procedures for sperm cryopreservation might be, however, not suitable for samples with a very low sperm number. In this pilot study, we investigated the recoveries of sperm motility and viability in severe oligozoospermic subjects (n=39) after cryopreservation with a tip-microVapour Fast Freezing, a procedure previously developed by our group for men with good semen quality. Sperm DNA fragmentation was also evaluated in a second group of oligozoospermic samples (n=16). We used a Vapour Fast Freezing procedure using 10μL tips as carrier, and Test Yolk Buffer as freezing medium (tip-microVapour Fast Freezing). In a subset of samples (n=22), we compared recovery of motility and viability as obtained with tip-microVapour Fast Freezing and with a Vapour Fast Freezing procedure using 500μL straws. Sperm DNA fragmentation was evaluated by the sperm chromatin dispersion test. We found a recovery rate (median [interquartile range]) of 0.29 (0.13-0.41) for progressive motility, 0.30 (0.21-0.52) for total motility and 0.48 (0.29-0.60) for viability. Interestingly, we observed that samples with the poorest motility were apparently less damaged by freezing/thawing. In a subset of samples (n=22), we directly compared values of viability, progressive motility and total motility by freezing/thawing with tip-microVapour Fast Freezing and Vapour Fast Freezing conducted with 500μL straws. We found much better values of all sperm parameters in samples after freezing/thawing with tip-microVapour Fast Freezing than with Vapour Fast Freezing in 500μL straws: that is, progressive motility: 7.00 (3.00-8.50)% versus 2.00 (0.00-4.25)%, p<0.001; total motility: 12.00 (8.00-16.25)% versus 6.50 (1.00-9.25)%, p<0.001; viability: 29.75 (23.75-45.25) versus 22.50 (13.75-28.13), p<0.001, respectively. In the second group of oligozoospermic samples, we found that tip-microVapour Fast Freezing produced lower levels of sperm DNA fragmentation than straws (33.00 [19.75-36.00]% vs. 36.00 [22.75-41.87]%, p<0.001). Tip-microVapour Fast Freezing appears to be a very promising method to cryopreserve semen samples from severe oligozoospermic patients.

Stepwise mini-incision microdissection testicular sperm extraction in NOA patients with a history of cryptorchidism: a case–control study

BackgroundAlthough the orchiopexy is recommended for cryptorchidism to preserve male fertility, non-obstructive azoospermia (NOA) may occur in adulthood. Fortunately, a great many of azoospermic men may obtain sperm by microdissection testicular sperm extraction (mTESE). Due to the potential injuries caused by testicular diagnostic biopsy and vascular damage at the time of orchidopexy, minimal invasiveness is particularly important during mTESE, aims to reduce the surgical damage and avoids secondary testicular failure. This comparative study aims to investigate the efficacy of stepwise mini-incision mTESE technique by comparison with standard mTESE in the treatment of NOA patients with a history of cryptorchidism.ResultsA total of 73 mTESE procedures were divided into two groups: Group 1 included 37 cases performed by stepwise mini-incision mTESE, while Group 2 included 36 cases with standard mTESE. The overall sperm retrieval rate (SRR) in the two groups was 68.5% (50/73), with no significant difference in SRR between Group 1 (78.4%, 29/37) and Group 2 (58.3%, 21/36) (P = 0.1). In addition, 46.0% of the patients (17/37) obtained sperm in the first mini-incision step in Group 1, which was also equal to an overall SRR in Group 2 (58.3%, 21/36) (P = 0.3). The operation time in Group 1 (72.6 ± 33.9 min) was significantly shorter than that in Group 2 (90.4 ± 36.4 min) (P = 0.04). Patients with an orchidopexy age no more than 10 years old had a higher SRR (79.5%, 31/39) than others (55.9%, 19/34) (P = 0.03). There were no postoperative complications including wound infection, scrotal hematoma, persistent pain, and testicular atrophy during a follow-up period of at least 6 months.ConclusionsIn conclusion, our study suggests that the stepwise mini-incision mTESE could be a promising approach for sperm retrieval in NOA men with a history of cryptorchidism. While the technique may potentially reduce operation time and surgical invasiveness, further research is needed to validate these findings on a larger scale. The results also suggest that age at orchidopexy may affect SRR and have important implications for the management of cryptorchidism.

Factors associated with undergoing microdissection testicular sperm extraction among men with non-obstructive azoospermia following evaluation by a reproductive urologist.

Microdissection testicular sperm extraction (mTESE) is the gold standard treatment for men with non-obstructive azoospermia (NOA). However, many men do not elect to pursue this surgical intervention. We aimed to identify factors associated with NOA patients undergoing mTESE after initial evaluation by a reproductive urologist (RU) through a retrospective cohort study. We retrospectively reviewed NOA patient who underwent evaluation by a RU between 2002-2018. Demographic and clinical data were collected. Our primary outcome was electing to undergo mTESE. 44.4% (75/169) of NOA men underwent mTESE. These patients earned significantly higher median neighborhood income ($133,000 vs. $97,000, P<0.001), spent fewer years trying to conceive before seeking care {1.3 [interquartile range (IQR): 1-3] vs. 2.3 (IQR: 1-5), P=0.012}, and were more likely to be married (79.7% vs. 53.9%, P=0.001). On univariate analysis, married men [odds ratio (OR) 3.37, 95% confidence interval (CI): 1.67-6.79, P=0.001] and men with higher neighborhood income (OR 1.14, 95% CI: 1.06-1.21, P<0.001) were more likely to undergo mTESE, while couples attempting to conceive for a longer period of time prior to initial evaluation were less likely to undergo mTESE (OR 0.79, 95% CI: 0.68-0.92, P=0.003). On multivariable regression analysis, marital status and years attempting to conceive remained significantly associated with NOA patients undergoing mTESE (OR 4.61, 95% CI: 1.16-18.25, P=0.03; OR 0.67, 95% CI: 0.52-0.88, P=0.003, respectively). Higher neighborhood income and marital status were positively associated with patients undergoing mTESE, while couples who attempted to conceive for a longer period of time before seeking infertility care were less likely to undergo mTESE.

P-086 Prevalence and clinical factors associated with biochemical hypogonadism in infertile men with non-obstructive azoospermia: a single-center cohort study including 761 patients

Abstract Study question What is the prevalence, and which factors are associated with hypogonadism in infertile men with non-obstructive azoospermia (NOA)? Summary answer The prevalence of biochemical hypogonadism among NOA males is significant. The condition is associated with testicular volume, estradiol levels, paternal age, and testicular histopathology results. What is known already NOA is a severe and irreversible condition that accounts for approximately 60% of azoospermia cases, primarily associated with intrinsic testicular deficiencies of various causes. NOA men can present signs of hypogonadism, associated or not with elevated circulating FSH levels; however, the exact prevalence is ill-reported. There is also a lack of data on the clinical factors associated with hypogonadism. We, therefore, estimated the prevalence of biochemical hypogonadism using real-world data and investigated its relationship with clinical factors. Study design, size, duration An observational cohort study was conducted, including 767 consecutive NOA patients with primary spermatogenic failure seeking fertility treatment at a University-affiliated tertiary center for male reproductive health between January 2014 and September 2021. Biochemical hypogonadism was defined as total testosterone (T) levels &amp;lt;350 ng/dL, measured by chemiluminescence immunoassay from a venous sample, collected in the morning, from 8:00 to 10:00 am, and confirmed on a second analysis at least one week apart. Participants/materials, setting, methods All included patients (age range: 23-55 years) had complete clinical, hormonal, genetic, seminal, and histopathology data and were naïve concerning previous sperm retrieval attempts. Patients with NOA due to hypogonadotropic hypogonadism and those using hormonal therapy were excluded. Prevalence rates were computed by Goodman’s method. The relationship between clinical factors and hypogonadism was assessed by logistic regression analysis. We also assessed the frequency of patients with high and within-range FSH levels in the cohort. Main results and the role of chance NOA patients with biochemical hypogonadism represented 80.3% (95% confidence interval [CI] 77.3–43.7) of the studied population. The median (interquartile range [IQR]) of T and FSH levels in hypogonadal men were 276 ng/dL (233.0-303 ng/dL) and 12.3 IU/L (8.9-18.2). Among them, hypergonadotropic (FSH&amp;gt;12 IU/L) and normogonadotropic (within-range FSH levels 1.5-12.0 IU/L) patients were 52.1% (95% CI 48.1-56.0) and 47.8% (43.9-51.8) of studied individuals, respectively. Logistic regression analyses revealed that testicular volume (p &amp;lt; 0.0001) and estradiol levels (p = 0.0005) had a significant inverse relationship with hypogonadism, whereas paternal age (p = 0.02) showed a positive relationship. Moreover, testicular histopathology was a relevant predictor (p = 0.003), with an inverse relationship between hypogonadism and germ cell maturation arrest and a positive relationship between hypogonadism and hypospermatogenesis or Sertoli cell-only. Using these variables, a model constructed to predict hypogonadism had an area under the ROC curve of 0.72. Limitations, reasons for caution Limitations include possible unmeasured confounding factors and the intra- and inter-assay electrochemiluminescence method variability. Another limitation is that the study center is a referral facility for azoospermic patients; thus, hypogonadism prevalence rates reported herein might not represent that of the general population of NOA males attending general fertility centers. Wider implications of the findings Efforts are needed to provide adequate care for this population due to its association with poor quality of life. Moreover, as recent evidence has suggested that hormonal therapy to boost T production might increase sperm retrieval success rates, studies investigating hormonal therapy in such patients may be warranted. Trial registration number Not applicable

Next-generation sequencing analysis of semen microbiome taxonomy in men with nonobstructive azoospermia vs. fertile controls: a pilot study

To study how the semen microbiome profile in men with nonobstructive azoospermia (NOA) differs from that of fertile controls (FCs). Using quantitative polymerase chain reaction and 16S ribosomal RNA, we sequenced semen samples from men with NOA (follicle-stimulating hormone >10 IU/mL, testis volume <10 mL) and FCs and performed a comprehensive taxonomic microbiome analysis. All patients were identified during evaluation at the outpatient male andrology clinic at the University of Miami. In total, 33 adult men, including 14 diagnosed with NOA and 19 with proven paternity undergoing vasectomy, were enrolled. Bacterial species in the semen microbiome were identified. Alpha-diversity was similar between the groups, suggesting similar diversity within samples, whereas beta-diversity was different, suggesting differences in taxa between samples. In the NOA men, the phyla Proteobacteria and Firmicutes were underrepresented, and Actinobacteriota were overrepresented compared with FC men. At the genus level, Enterococcus was the most common amplicon sequence variant in both groups, whereas 5 genera differed significantly between the groups, including Escherichia and Shigella, Sneathia, and Raoutella. Our study showed significant differences in the seminal microbiome between men with NOA and fertile men. These results suggest a loss of functional symbiosis may be associated with NOA. Further research into the characterization and clinical utility of the semen microbiome and its causal role in male infertility is necessary.

Evaluation of the Utility of Seminal Plasma Resistin and Leptin in Predicting Successful Surgical Sperm Retrieval in Men with Non-Obstructive Azoospermia.

The purpose of the current study was evaluation of the utility of seminal plasma (SP) resistin and leptin in predicting successful surgical sperm retrieval (SSR) in men with non-obstructive azoospermia (NOA). This prospective comparative study was conducted in the andrology clinic of a specialized fertility center. In total, 53 NOA men as candidates for either first time micro-testicular sperm extraction (micro-TESE) or repeat micro-TESE and 28 normozoospermic controls were included. ELISA was used for measurement of SP resistin and leptin levels in all participants. Significance level was defined as p<0.05. The current study demonstrated a significant positive correlation between estradiol (E2) level in serum and SP resistin (r=0.342, p=0.025). Also, there was a highly significant positive correlation between SP leptin and SP resistin (r=0.568, p= 0.001). Interestingly, SP leptin was the only variable that demonstrated a significant correlation with eventful micro-TESE outcome in men who underwent micro-TESE for the first time. Finally, ROC curve showed that SP leptin level of 4.05 ng/ml predicted successful SSR in men who underwent micro-TESE for the first time with a sensitivity of 73.3% and a specificity of 75% as 11 out of 27 (41%) cases showed eventful micro-TESE at or above this cut-off level [AUC of 0.747, 95% CI, lower bound of 0.555, and upper bound of 0.939, p=0.030]. SP leptin can be used as a non-invasive biomarker to predict successful SSR in NOA cases undergoing first time micro-TESE, while SP resistin failed to play the same role.

Testicular Apoptosis and Proliferation in Relation to Body Mass Index and m-TESE Success in Nonobstructive Azoospermic Men

Male infertility occurs due to the disruption of the balance of proliferation and apoptosis in the testicular tissue. It is important to show the effect of increased body mass index, which is one of the factors that disrupts this balance of apoptosis and proliferation, not only at the blood hormone level but also at the tissue level. For this reason, the present study is aimed at demonstrating the relation between body mass index and cell turnover in the testis using immunohistochemical methods. In the present study, patients were grouped as normal, overweight and obese, and as m-TESE positive and negative. The sperm retrieval rate with microsurgical testicular sperm extraction (m-TESE) was 41.67%. Histological diagnosis of testicular tissues was made with hematoxylin and eosin and Masson’s trichrome staining. Apoptosis and proliferation in the testicular tissue were demonstrated by TUNEL and PCNA immunohistochemical methods, respectively. It was concluded that BMI had no significant effect on reproductive hormone profile (FSH, LH, and testosterone), m-TESE success, apoptosis, and proliferation in testicular tissue in nonobstructive azoospermic men. In addition TUNEL positivity and proliferative index was found to be significantly correlated with testicular histology and m-TESE outcome.

MP01-07 A NEXT-GENERATION SEQUENCING ANALYSIS OF THE SEMEN MICROBIOME IN MEN WITH NON-OBSTRUCTIVE AZOOSPERMIA

You have accessJournal of UrologyCME1 Apr 2023MP01-07 A NEXT-GENERATION SEQUENCING ANALYSIS OF THE SEMEN MICROBIOME IN MEN WITH NON-OBSTRUCTIVE AZOOSPERMIA Katherine Campbell, Farah Rahman, Camila Suarez, Aaron Miller, Emad Ibrahim, Scott D. Lundy, and Ranjith Ramasamy Katherine CampbellKatherine Campbell More articles by this author , Farah RahmanFarah Rahman More articles by this author , Camila SuarezCamila Suarez More articles by this author , Aaron MillerAaron Miller More articles by this author , Emad IbrahimEmad Ibrahim More articles by this author , Scott D. LundyScott D. Lundy More articles by this author , and Ranjith RamasamyRanjith Ramasamy More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Detection of a microbiome profile coupled with advanced genetic sequencing has opened a new research frontier. We hypothesized that the semen microbiome profile in men with non-obstructive azoospermia (NOA) would differ from that of fertile controls (FC). METHODS: NOA was diagnosed using semen analysis and clinical phenotype (small testis volume, FSH >10 IU/mL). FC were identified as men undergoing vasectomy after fathering at least one child. Semen samples were collected and underwent Next Generation Sequencing (NGS) including quantitative PCR and 16s rRNA V1/V2 region comprehensive sequencing using Illumina MiSeq technology. Data was processes and analyzed in dada2. Alpha diversity was analyzed via Margalef. Beta diversity was analyzed via Bray-Curtis difference. Differential abundance was evaluated with ANCOMBC procedure. RESULTS: After rarefaction analysis, 33 samples were included (18 NOA and 15 FC) determined samples had sufficient sequencing depth to capture microbiome diversity. Alpha diversity was lower in the NOA men but did not reach statistical significance. Beta diversity showed a difference between the NOA and fertile groups primarily driven by differences in ethnicity. At the phylum level, NOA patients demonstrated lower levels of Proteobacteria and Firmicutes and higher levels of Actinobacteriota compared to the FC group consistent with prior published work. At the genus level, the known uropathogens Ureaplasma (1.3% vs 0.16%) and Eschrichia/Shigella (2.26% vs 0.42%) were higher in the NOA group compared to fertile controls. Other notable differences were Raoultella (39-fold higher in Fertile – 2% vs 0.09%) and Sneathia (9.5-fold higher in Fertile – 1% vs. 0.1%). Differential abundance analysis largely confirmed these findings. CONCLUSIONS: Compared to fertile controls, men with NOA have a less diverse seminal microbiome enriched for known pathogens and several understudied organisms. This work supports the presence of a testicular microbiome, which may become dysbiotic with infertility and could be leveraged as a biomarker to guide treatment decision making in the future. Source of Funding: MicrogenDX © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e4 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Katherine Campbell More articles by this author Farah Rahman More articles by this author Camila Suarez More articles by this author Aaron Miller More articles by this author Emad Ibrahim More articles by this author Scott D. Lundy More articles by this author Ranjith Ramasamy More articles by this author Expand All Advertisement PDF downloadLoading ...

Assessment of Serum Anti-Müllerian Hormone (AMH) as an Independent Marker for Oligozoospermia and Non-Obstructive Azoospermia in Infertile Nigerian Men

Background: Anti-Müllerian hormone (AMH) is a Sertoli cell-derived glycoprotein that mediates regression of Müllerian duct in male embryos. The present study aims to evaluate the diagnostic efficacy of serum AMH in the detection of oligozoospermia and non-obstructive azoospermia (NOA) in a homogenous population of Nigerian men. Methods: This case-controlled prospective study was conducted on eighty male subjects (aged 18-45 years), at the Jos University Teaching Hospital, Nigeria. Subjects were classified as control (n=30), oligozoospermic (n=27) and non-obstructive azoospermia (NOA; n=23) (World Health Organization, 2010). Serum concentrations of various hormones were measured. Statistical analyses were performed using MedCalc. (v.19.5.1, Ostend, Belgium). Results: Serum AMH levels did not differ significantly among the study groups (P&gt;0.05). Serum levels of testosterone were significantly lower, while serum FSH levels were significantly higher in the infertile groups than the control (P&lt;0.000001). Serum LH levels were significantly higher in the NOA men (P&lt;0.000001), while oligozoospermic men showed no significant difference, compared to control. Receiver operating characteristics (ROC) curve analysis depicted the same cut-off value (≤1.7 ng/ml) of serum AMH for oligozoospermia and NOA with low sensitivity and moderate specificity. Conclusion: The findings suggest that serum AMH is not a potent stand-alone marker of NOA or oligozoospermia among Nigerian men.

Does Clomiphene citrate administration increase success rate of micro TESE in non-obstructive azoospermic men? A randomized controlled trial

Does Clomiphene citrate administration increase success rate of micro TESE in non-obstructive azoospermic men? A randomized controlled trial

How to improve the clinical outcome of round spermatid injection (ROSI) into the oocyte: Correction of epigenetic abnormalities.

First successful human round spermatid injection (ROSI) was conducted by Tesarik et al. in 1996 for the sole treatment of nonobstructive azoospermic men whose most advanced spermatogenic cells were elongating round spermatids. Nine offsprings from ROSI were reported between 1996 and 2000. No successful deliveries were reported for 15 years after that. Tanaka et al. reported 90 babies born after ROSI and their follow-up studies in 2015 and 2018 showed no significant differences in comparison with those born after natural conception in terms of physical and cognitive abilities. However, clinical outcomes remain low. Clinical and laboratory data of successful cases in the precursor ROSI groups and those of Tanaka et al. were reviewed. Differences were found between the two groups in terms of identification of characteristics of round spermatid and oocyte activation. Additionally, epigenetic abnormalities were identified as underlying causes for poor ROSI results, besides correct identification of round spermatid and adequate oocyte activation. Correction of epigenetic errors could lead to optimal embryonic development. Correction of epigenetic abnormalities has a probability to improve the clinical outcome of ROSI.