Non-obstructive Azoospermia Cases Research Articles

Non-invasive biomarkers for sperm retrieval in non-obstructive patients: a comprehensive review.

Recent advancements in reproductive medicine have guided novel strategies for addressing male infertility, particularly in cases of non-obstructive azoospermia (NOA). Two prominent invasive interventions, namely testicular sperm extraction (TESE) and microdissection TESE (micro-TESE), have emerged as key techniques to retrieve gametes for assisted reproduction technologies (ART). Both heterogeneity and complexity of NOA pose a multifaceted challenge to clinicians, as the invasiveness of these procedures and their unpredictable success underscore the need for more precise guidance. Seminal plasma can be aptly regarded as a liquid biopsy of the male reproductive tract, encompassing secretions from the testes, epididymides, seminal vesicles, bulbourethral glands, and prostate. This fluid harbors a variety of cell-free nucleic acids, microvesicles, proteins, and metabolites intricately linked to gonadal activity. However, despite numerous investigations exploring potential biomarkers from seminal fluid, their widespread inclusion into the clinical practice remains limited. This could be partially due to the complex interplay of diverse clinical and genetic factors inherent to NOA that likely contributes to the absence of definitive biomarkers for residual spermatogenesis. It is conceivable that the integration of clinical data with biomarkers could increase the potential in predicting surgical procedure outcomes and their choice in NOA cases. This comprehensive review addresses the challenge of sperm retrieval in NOA through non-invasive biomarkers. Moreover, we delve into promising perspectives, elucidating innovative approaches grounded in multi-omics methodologies, including genomics, transcriptomics and proteomics. These cutting-edge techniques, combined with the clinical and genetics features of patients, could improve the use of biomarkers in personalized medical approaches, patient counseling, and the decision-making continuum. Finally, Artificial intelligence (AI) holds significant potential in the realm of combining biomarkers and clinical data, also in the context of identifying non-invasive biomarkers for sperm retrieval.

Evaluation of human bone marrow mesenchymal stem cells in the treatment of non obstructive azoospermia.

Non-obstructive azoospermia (NOA) represents an infertility problem that is usually difficult to treat. Such patients usually have testicular biopsy of germ cell aplasia or spermatogenic arrest. In recent decades, Mesenchymal Stem Cells (MSCs) had been studied thoroughly and proved safe and effective regarding their capability for trans-differentiation into different cell types. The aim of this study was to evaluate the effect of MSCs local intratesticular injection in induction of spermatogenesis. The current study included 87 infertile non-obstructive azoospermic patients. Clinical assessment and repeated semen analysis with centrifugation were done to confirm azoospermia. Karyotyping and AZF study were done. Some of the patients had previous testicular biopsy proving a lack of sperm in the testes. Single intratesticular injection of purified MSCs suspension was done. 20.7% of patients showed sperm in their semen after variable period of time. Hormonal profile among treated patients showed significant improvement regardless success of treatment. Also most of the treated patients appreciated the improvement of their sexual function and libido. Bone marrow derived MSCs could be a new hope and therapeutic modality for treatment of refractory cases of NOA.

Microsurgical Testicular Sperm Extraction: Predictive Factors and Outcomes for Men with Nonobstructive Azoospermia

Background. Nonobstructive azoospermia (NOA) is a frequent cause of male infertility, and microsurgical testicular sperm extraction (micro-TESE) is an effective technique for sperm retrieval in NOA cases. However, the predictive factors for successful sperm retrieval and the outcomes of micro-TESE in NOA patients are not fully understood. Methods. In this retrospective study, we evaluated the predictive value of preoperative data and outcomes of micro-TESE in 172 men with NOA who underwent the procedure between April 2012 and February 2021. Patients’ age, medical history, testicular size, hormonal profile, testicular histology, and sperm retrieval were recorded. Results. 172 cases of micro-TESE were performed (99 unilateral and 73 bilateral) and testicular sperm retrieval rate (SRR) was 72.7%. There was no statistical difference in age, testicular size, and hormonal profiles between positive SRR and negative SRR groups. Clinical varicocele and previous orchitis did not significantly affect the result of micro-TESE. Y chromosome microdeletions were detected in seven patients. On histological examination, hypospermatogenesis was found in 39% of patients who underwent testicular biopsy. SRR was higher in patients with hypospermatogenesis than in other groups. Conclusions. Testicular microdissection is an effective and safe procedure for nonobstructive azoospermia patients, regardless of the different etiologies and preoperative data. Age, testicular size, and hormonal levels did not predict the success of micro-TESE. The presence of hypospermatogenesis was associated with a higher SRR. Clinical varicocele and previous orchitis were not significantly associated with the outcome of micro-TESE.

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%.

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

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

A successful outcome in a case of nonobstructive azoospermia with repeat surgical sperm retrieval (SSR)-testicular sperm aspiration followed by microsurgical testicular sperm extraction

Azoospermia is a major cause of male infertility, either obstructive or nonobstructive azoospermia (NOA). However, such couples should be offered surgical sperm retrieval to enable them to become parents with their own gametes. Cases of NOA will have higher retrieval rate with microsurgical testicular sperm extraction (micro-TESE), even though, in some cases, it may be a repeat procedure. Presenting here a case of successful twin gestation after intracytoplasmic sperm injection with sperms retrieved successfully by micro-TESE after a previous failed frozen embryo transfer with a very poor retrieval by a testicular sperm aspiration in a case of NOA.

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.

Varicocele Surgery: Three Years Experience

Despite available treatments like varicocelectomy, a standard therapy remains controversial. Varicocele repair has shown promise in improving sperm parameters, but further research is needed, especially in nonobstructive azoospermia cases. Techniques such as microscopic testicular sperm extraction and intracytoplasmic sperm injection have shown some success in achieving pregnancies. Objective: To articulate our hands-on experience and understanding pertaining to varicocele. Methods: This study included patients who presented to our clinic due to varicocele and underwent surgical procedures between November 2020 and January 2023. Results: This research involved 26 varicocele patients, primarily age rage was 27.5 years old. The majority (85%) had varicocele on the left side, and 15% had bilateral varicocele. The mean vein diameter was 3.58mm and 2.6mm for the left side and bilateral cases respectively, with a general average of 3.45mm. Analysis of sperm count, motility, and morphology reflected an average of 32.6 million/ml, 23.2%, and 5.23%, respectively. An average of 4.45 veins was ligated during surgery, with a slight variance based on the side. One patient lost approximately 85% of the testicular blood supply due to a complication during surgery, leading to testicle hardening without atrophy. Two patients experienced recurrence (7.6% recurrence rate). Negative and positive correlations were found between the number of ligated veins and preoperative sperm count (p=0.043, r=-0.408), and vein diameter and preoperative sperm morphology (p=0.004, r=0.544), respectively. Conclusions: Despite the limitations, our study provides valuable insights into the relationship between varicocele severity and sperm parameters.

O-137 Mapping microRNA profiling and dysregulated pathways in non-obstructive azoospermia cases with maturation arrest: A systematic review and in-silico analysis of the affected pathways

Abstract Study question Could microRNA profiling be a useful tool towards better understanding the pathophysiological mechanisms leading to non-obstructive azoospermia (NOA) due to maturation arrest? Summary answer MicroRNA profiling may be an efficient method towards unveiling maturation arrest pathophysiology as microRNAs constitute master posttranscriptional regulators of cell-cycle control during spermatogenesis. What is known already Non-obstructive azoospermia constitutes the most severe form of male factor infertility, affecting approximately 10% of infertile men. According to the histological subtype, NOA is classified into three main categories namely hypospermatogenesis, maturation arrest, and Sertoli-cell only syndrome. Maturation arrest is one of the most complex forms of NOA characterized by failure of spermatogenesis. Despite advances, the pathophysiology of maturation arrest remains a conundrum rendering management complex. Recent studies indicate that alterations on microRNA expression patterns could lead to maturation arrest. Mapping microRNA profiling and highlighting dysregulated pathways of maturation arrest cases may lead the way forward. Study design, size, duration A systematic review was performed in PubMed/Medline and Embase up to April 2022. Only full-length original studies in humans were included. Strict inclusion-exclusion criteria were applied aiming to isolate studies comparing microRNA profiling between maturation arrest cases versus normal fertile men, and/or men with obstructive azoospermia (OA). Following study selection, data on altered microRNA expression patterns were analyzed to underline differences between the abovementioned groups. Subsequently, in-silico analysis was performed to compare affected gene pathways. Participants/materials, setting, methods The studied population consisted of maturation arrest cases. Control groups consisted of men with normal spermatogenesis and/or men with OA. Predicted microRNA–target pairs were retrieved from microT-CDS, while a 0.8 cutoff threshold was applied. The GTEx repository was used to identify microRNA-targeted genes in the testis. Annotations derived from Ensembl and miRbase. Gene-set enrichment analysis was performed employing the KEGG-database. Fisher’s exact test was performed in R package limma, setting a 0.01 p-value threshold. Main results and the role of chance Ten studies reported altered microRNA expression patterns in maturation arrest versus normal spermatogenesis cases and six studies in maturation arrest versus OA cases. Considering the maturation arrest-normal spermatogenesis arm, analysis revealed that eight microRNAs, which were upregulated in maturation arrest cases, namely hsa-miR-449a, hsa-miR-370-3p, hsa-miR-10b-3p, hsa-miR-539-5p, hsa-miR-22-5p, hsa-miR-605-5p, hsa-miR-491-3p and hsa-miR-302d-3p, affected 106 statistically significant gene-targets in the testis. The three most significantly affected pathways included the “microRNAs in cancer” pathway (55 affected genes, p-value <0.001), the “EGFR tyrosine kinase inhibitor resistance pathway” (29 affected genes, p-value = 0.007), and the “Hedgehog signaling pathway” (22 affected genes, p-value=0.008). Regarding the second arm of maturation arrest-OA, functional analysis indicated that four microRNAs, presenting to be downregulated in maturation arrest cases, namely hsa-miR-181a-5p, hsa-miR-449a, hsa-miR-182-5p and hsa-miR-138-5p, affected 421 statistically significant gene-targets in testis. The three most significantly affected pathways included the “pathways in cancer” pathway (86 affected genes, p-value =0.009), the “microRNAs in cancer” pathway (48 affected genes, p-value < 0.001), and the “ cGMP-PKG signaling” pathway (36 affected genes, p-value=0.008). In summary, microRNAs affect the expression of master genes regulating cell-cycle and maturation processes during spermatogenesis, leading to maturation arrest as well as to other associated pathologies, including cancer. Limitations, reasons for caution The limited number of the included studies, as well as the small size population characterizing the great majority of them, constitute the main limitations of this study. Another reason for caution is the great heterogeneity observed among the studies regarding the molecular methods employed for microRNA profiling. Wider implications of the findings Data presented herein indicate that microRNA functional analysis may be a significant tool towards better understanding the pathophysiological basis of spermatogenesis maturation arrest, indicating future personalized diagnostic and therapeutic targets. Moreover, microRNA analysis may also constitute an efficient method of evaluating predisposition of NOA patients to other pathologies including cancer. Trial registration number Not applicable

P-030 Clinical outcomes of microdissection testicular sperm extraction (micro TESE) and intracytoplasmic sperm injection (ICSI) in non-obstructive azoospermia (NOA) with the history of cryptorchidism

Abstract Study question What is sperm retrieval rate (SRR) by micro TESE and the clinical outcomes using testicular sperm in couples with the history of cryptorchidism? Summary answer NOA couples with the history of cryptorchidism had a higher SRR by micro TESE but lower clinical outcomes by ICSI compared of unexplained NOA. What is known already Undescended testis (UT) which is exposed to a higher temperature compared with the scrotal temperature is associated with impairment of germ cell maturation, and progressive Leydig and Sertoli cell atrophy, and subsequent infertility in adulthood. There have been very few studies of ICSI with a focus on, or large enough numbers to examine, the specific outcomes associated with male factor infertility, however improvement in sperm retrieval techniques including micro TESE and micromanipulation techniques, such as ICSI, has led to excellent fertilization and pregnancy outcomes of treatment cycles. Study design, size, duration We performed a retrospective study based on two reproduction centers in Japan and evaluated 1521 azoospermic patients in our clinics between September 2013 and December 2022. We investigated SRR by micro TESE in these patients and therefore aimed to evaluate the prevalence and the significance of ICSI outcomes with embryonic development in NOA couples with the history of cryptorchidism. Participants/materials, setting, methods We evaluated SRR of micro TESE, two pronuclei (2PN) oocyte rates, blastocyst development, good-quality blastocyst (Grade 3BB and above on day 5 by the Gardner scoring), and clinical pregnancy rates per embryo transfer (ET) in 72 NOA cases with the history of UT, 953 cases of unexplained NOA, not including after orchidopexy, Klinefelter syndrome, cryptozoospermia, mumps orchitis, and 284 cases of obstructive azoospermia (OA). Statistical analysis was performed using unpaired t-tests and chi-squared tests. Main results and the role of chance SRR of first attempt micro TESE in UT (44/56=78.6%) was higher than unexplained NOA (160/748=21.4%) (p < 0.001). Spermatozoa were successfully retrieved in 13 of 22 (59.1%) UT group and 36 of 226 (15.9%) unexplained NOA who had previously undergone micro TESE with no sperm found. No correlation was found between serum FSH, LH, and T level with the success of sperm retrieval. Testicular volume and patient age at orchidopexy also did not affect the SRR for micro TESE. 2PN oocytes, blastocysts development, and good-quality blastocysts rates were 45.5%, 47.4%, and 18.9% in UT, 51.2%, 45.0%, and 20.9% in unexplained NOA, and 62.6%, 52.1%, and 23.7% in OA, respectively. Clinical pregnancy rates per ET were 29.3% in UT, 32.3% in unexplained NOA, and 41.6% in OA. Significant differences were only observed in 2PN oocytes between unexplained NOA and OA (p < 0.05) and clinical pregnancy between UT and unexplained NOA (p < 0.05), and OA and unexplained NOA (p < 0.01). Several UT patients showed a very small number of spermatozoa and even only immotile sperm of retrieved by micro TESE. Limitations, reasons for caution We included the patients only after a surgery of cryptorchidism, but not a delayed testicular descent without surgery. The cohort size of this study is not small, however, our screened population of azoospermic patients may be biased. Wider implications of the findings This study shows a high impact in micro TESE and ICSI outcomes with embryonic development for the NOA couples with the history of cryptorchidism. Our study emphasizes that history of cryptorchidism provides clinically valuable prognostic information to couples considering surgical sperm retrieval. Trial registration number not applicable

P-089 effect of testis tissue retrieval on “Micro Mapping Testicular Extraction” (MMTE): comparison with microscopic testicular sperm extraction (Micro TESE) in non-obstructive azoospermia (NOA)

Abstract Study question Is a new sperm retrieval method, Micro Mapping Testicular Extraction (MMTE) comparable to that of Microscopic testicular sperm extraction (Micro TESE)? Summary answer MMTE is comparable to that of Micro TESE regarding sperm retrieval rate (SRR). It can reduce tissue damage and improve intracytoplasmic sperm injection (ICSI) results. What is known already Micro TESE is currently the standard technique for sperm retrieval in NOA. However, the large incision severely damages testicles, complicating repeated Micro TESE, and there is a high risk of testicular atrophy and hypogonadism. New technologies, such as FNA mapping, have also been reported, but these require multiple surgeries, long operation times, and high examination costs. A more accurate and minimally invasive method of sperm retrieval is needed, using a shorter and simpler procedure. Study design, size, duration MMTE requires only a small amount of testicular tissue, obtained through multiple needle holes in the tunica albuginea by micro-squeezing under a microscope. Tissue is divided into four testicular areas: ventral-upper, ventral-lower, dorsal-upper, and dorsal-lower, and samples are immediately searched for sperm. If sperm are found, a small incision is made in the area with good sperm and additional tissue is taken under the microscope. If no sperm are found, routine Micro TESE is performed. Participants/materials, setting, methods From January to November 2022, 25 NOA cases (35.9+/-4.85 years) with indications for Micro TESE were eligible: 15 idiopathic, 6 gr/gr deletion, 2 Klinefelter’s syndrome, 1 b2b4 deletion and 1 other. MMTE was performed on 45 testes: 5 unilateral (Group A) cases and 20 bilateral (Group B) cases. If sperm were found, they were cryopreserved and later used or will be used for ICSI. Main results and the role of chance After MMTE, sperm were found in all 5 cases in Group A. In group B, sperm were found in 6 cases after MMTE. For those sperm positive 11 cases, additional sampling was performed to gain enough sperm for ICSI. Micro TESE was performed for the rest of group B (14 cases, 28 testes), in which no sperm was found by MMTE. Of 2 cases out of 14 cases, only a few sperm were found by Micro TESE. No sperm was found in the rest of 12 cases. As a result, the SRR per patient was 52% (13/25). The weight of tissue sampled was 56.2 mg (15.6-96.2 mg) by MMTE and 224.6mg (206.4-433.2 mg) by Micro TESE, a significant difference. (p < 0.05). To date, ICSI has been performed in the MMTE-positive 11 cases with a partner (33.8+/-3.4 years). In those cases, the fertilization rate was 62.7% (79/126). The blastocyst rate was 31.7% (40/126), and the pregnancy rate per transfer was 75% (6/8). The total testosterone levels before and 1 month after surgery were significantly reduced only in the cases in which no sperm were found by MMTE. Limitations, reasons for caution Two cases that were sperm-positive only by Micro TESE had severe tissue degeneration and tissue was difficult to retrieve from needle holes, so countermeasures were necessary. It is necessary to consider the number of needle holes depending on the size of the testis. The number of cases is still small. Wider implications of the findings MMTE can correct tissue just under the albuginea and can cover areas that are difficult to reach with Micro TESE. The sample can be taken through a small incision, so tissue damage is limited to that of Simple TESE, which improves ICSI results by taking sperm from the best areas. Trial registration number non applicable

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.

A Novel Frameshift Microdeletion of the TEX12 Gene Caused Infertility in Two Brothers with Nonobstructive Azoospermia.

Male infertility is a growing health problem, which affects approximately 7% of the global male population. Nonobstructive azoospermia (NOA) is one of the most severe forms of male infertility caused by genetic defects, including chromosome structural abnormalities, Y chromosome microdeletions, or single-gene alterations. However, the etiology of up to 40% of NOA cases is unidentified. By whole-exome sequencing, we detected a homozygous 5-bp-deletion variant in exon 4 of the TEX12 gene (c.196-200del, p.L66fs, NM_031275.4) in two brothers with NOA of a nonconsanguineous Vietnamese family. This deletion variant of 5 nucleotides (ATTAG) results in a premature stop codon in exon 4 and truncation of the C-terminal. Segregation analysis by Sanger sequencing confirmed that the deletion variant was inherited in an autosomal recessive pattern. The 1st and 3rd infertile sons were homozygous for the deletion, whereas the 2nd fertile son and both parents were heterozygous. The new deletion mutation identified in TEX12 gene caused loss of function of TEX12 gene. The loss of TEX12 function has already caused infertility in male mice. Therefore, we concluded that the loss of TEX12 function may cause infertility in men. To our knowledge, this is the first case reported so far indicating disruption of human TEX12, which leads to infertility in men.

Co-Culture of Cryopreserved Healthy Sertoli Cells with Testicular Tissue of Non-Obstructive Azoospermia (NOA) Patients in Culture Media Containing Follicle-Stimulating Hormone (FSH)/Testosterone Has No Advantage in Germ Cell Maturation.

Different cell culture conditions and techniques have been used to mature spermatogenic cells to increase the success of in vitro fertilization. Sertoli cells (SCs) are essential in maintaining spermatogenesis and FSH stimulation exerts its effect through direct or indirect actions on SCs. The effectiveness of FSH and testosterone added to the co-culture has been demonstrated in other studies to provide microenvironment conditions of the testicular niche and to contribute to the maturation and meiotic progression of spermatogonial stem cells (SSCs). In the present study, we investigated whether co-culture of healthy SCs with the patient's testicular tissue in the medium supplemented with FSH/testosterone provides an advantage in the differentiation and maturation of germ cells in NOA cases (N = 34). In men with obstructive azoospermia (N = 12), healthy SCs from testicular biopsies were identified and purified, then cryopreserved. The characterization of healthy SCs was done by flow cytometry (FC) and immunohistochemistry using antibodies specific for GATA4 and vimentin. FITC-conjugated annexin V/PI staining and the MTT assay were performed to compare the viability and proliferation of SCs before and after freezing. In annexin V staining, no difference was found in percentages of live and apoptotic SCs, and MTT showed that cryopreservation did not inhibit SC proliferation compared to the pre-freezing state. Then, tissue samples from NOA patients were processed in two separate environments containing FSH/testosterone and FSH/testosterone plus co-culture with thawed healthy SCs for 7 days. FC was used to measure 7th-day levels of specific markers expressed in spermatogonia (VASA), meiotic cells (CREM), and post-meiotic cells (protamine-2 and acrosin). VASA and acrosin basal levels were found to be lower in infertile patients compared to the OA group (8.2% vs. 30.6% and 12.8% vs. 30.5%, respectively; p < 0.05). Compared to pre-treatment measurements, on the 7th day in the FSH/testosterone environment, CREM levels increased by 58.8% and acrosin levels increased by 195.5% (p < 0.05). Similarly, in medium co-culture with healthy SCs, by day 7, CREM and acrosin levels increased to 92.2% and 204.8%, respectively (p < 0.05). Although VASA and protamine levels increased in both groups, they did not reach a significant level. No significant difference was found between the day 7 increase rates of CREM, VASA, acrosin and protamine-2 in either FSH/testosterone-containing medium or in medium additionally co-cultured with healthy SCs (58.8% vs. 92.2%, 120.6% vs. 79.4%, 195.5% vs. 204.8%, and 232.3% vs. 198.4%, respectively; p > 0.05). Our results suggest that the presence of the patient's own SCs for maturation of germ cells in the culture medium supplemented with FSH and testosterone is sufficient, and co-culture with healthy SCs does not have an additional advantage. In addition, the freezing-thawing process would not impair the viability and proliferation of SCs.

Characterization of Non-Obstructive Azoospermia in Men Using Gut Microbial Profiling.

(1) Background: Non-obstructive azoospermia (NOA) is a complex multifactorial disease and the causes of most NOA cases remain unknown. (2) Methods: Here, we performed comprehensive clinical analyses and gut microbial profiling using shotgun metagenomic sequencing in patients with NOA and control individuals. (3) Results: The gut microbial alpha and beta diversity significantly differed between patients with NOA and controls. Several microbial strains, including Bacteroides vulgatus and Streptococcus thermophilus, were significantly more abundant in the NOA group, whereas Bacteroides thetaiotaomicron and Parabacteroides sp. CT06 were enriched in the control group. Moreover, functional pathway analysis suggested that the altered microbiota in NOA suppressed the carbohydrate metabolism pathway, while amino acid metabolism and methane metabolism pathways were enhanced. We observed that the differential microbial species, such as Acinetobacter johnsonii, had a strong correlation with clinical parameters, including age, body mass index, testosterone, and follicle-stimulating hormone. Communication and interplay among microbial genera were significantly increased in NOA than in the control group. (4) Conclusions: Altered microbial composition and functional pathways in the NOA group were revealed, which highlight the utility of gut microbiota in understanding microbiota-related pathogenesis of NOA and might be helpful to the clinical management of NOA.

Bridging the Gap between AZF Microdeletions and Karyotype: Twelve Years' Experience of an Infertility Center.

Despite all past efforts, the current guidelines are not explicit enough regarding the indications for performing azoospermia factor (AZF) screening and karyotype, burdening clinicians with the decision to assess whether such tests are meaningful for the infertile male patient. These assessments can be costly and it is up to the healthcare practitioner to decide which are necessary and to weigh the benefits against economic/psychological harm. The aim of this study is to address such gaps and provide update on current management options for this group of patients. To address such gaps in male infertility management and to elucidate whether AZF screening is indicated in individuals who concomitantly harbor chromosomal abnormalities we conducted a retrospective cohort analysis of 10,388 consecutive patients with non-obstructive azoospermia (NOA) and severe oligozoospermia. Previously, it has been suggested that all NOA cases with chromosomal defects, except males with 46,XY/45,X karyotype, have no indication for AZF screening. Our findings revealed that cases carrying the following chromosomal abnormalities inv(Y)(p11.2q12); idic(Y)(q11.2); 46,XY,r(Y); idic(Y)(p11.2) and der(Y;Autosome) (76/169; 44.9%; 95% CI, 37.7-52.5) should also be referred for AZF deletion screening. Here, we also report the correlation between sperm count and AZF deletions as a secondary outcome. In accordance with previously reported data from North America and Europe, our data revealed that only 1% of cases with >1×106 sperm/mL had Y chromosome microdeletions (YCMs). In the era of assisted reproduction, finding cost-minimization strategies in infertility clinics without affecting the quality of diagnosis is becoming one of the top prioritized topics for future research. From a diagnostic viewpoint, the results reflect a need to reconsider the different karyotype presentations and the sperm count thresholds in male infertility guidelines as indicators for YCM screening during an infertility evaluation.

ZMYM3 : a new candidate gene in nonobstructive azoospermia?

ZMYM3 : a new candidate gene in nonobstructive azoospermia?

Diverse monogenic subforms of human spermatogenic failure

Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven challenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome-sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human “knockouts”, and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification.

Estimation of Serum and Seminal Plasma Levels of Glactin-1 in Non-Obstructive Azoospermia Cases and Their Correlations with the Rate of Sperm Retrieval: A Comparative Prospective Study

Remarkably, the current study is one of the first to deploy galectin-1 (Gal-1) in determining the degree of impairment of spermatogenesis among cases with non-obstructive azoospermia (NOA) as well as utilizing it as a biomarker to predict the rate of sperm retrieval in these patients. The purpose of the study was to evaluate the seminal plasma and serum levels of Gal-1 in NOA patients as well as their correlations with Johnsen's tubular biopsy scoring (JTBS). The current case control study included totally 48 patients with NOA whose ages ranged from 24 to 46 years old and 50 age matched healthy controls. Gal-1 levels were measured in both seminal plasma and serum of all subjects by the enzyme-linked immunosorbent assay (ELISA). A significant negative correlation between seminal plasma levels of Gal-1 and JTBS was detected (r= -0.281, p=0.048) in the NOA cases. Interestingly, the receiver operating characteristic (ROC) curve had demonstrated that the cutoff value of seminal plasma levels of Gal-1 in determining azoospermia was >0.735 ng/ml and the area under the curve (AUC) was 0.858. The sensitivity, specificity, positive predictive, and negative predictive values for seminal plasma levels of Gal-1 were 76, 92, 90.5, and 79.3, respectively. In addition, sensitivity, specificity, positive predictive, and negative predictive values for serum levels of Gal-1 were 38, 66, 52.8, and 51.6, respectively. Seminal plasma levels of Gal-1 are higher in NOA men versus healthy controls. Interestingly, negative correlation of seminal plasma levels of Gal-1 with JTBS was determined. Thus, it can be used as a good predictor for NOA cases.

A homozygous missense variant in DND1 causes non-obstructive azoospermia in humans.

Non-obstructive azoospermia (NOA) is a severe factor of male infertility; it affects approximately 1% of the global male population and accounts for 40% of male infertility cases. However, the majority of NOA cases remain idiopathic. This is the first study using whole-exome sequencing (WES) to identify a novel missense mutation in the DND1 gene (c.212A>C, p. E71A) from a Pakistani family, that includes three males with NOA. This mutation is predicted to cause DND1 protein misfolding and weaken the DND1 interaction with NANOS2, a significant regulator in primordial germ cell development. Our study identified a DND1 pathogenic mutation in NOA patients and highlighted its critical role in male fertility in humans.