Microfluidics: The future of sperm selection in assisted reproduction.

BACKGROUND: Obtaining functional sperm cells is the first step to treat infertility. With the ever-increasing trend in male infertility, clinicians require access to effective solutions that are able to single out the most viable spermatozoa, which would max out the chance for a successful pregnancy. The new generation techniques for sperm selection involve microfluidics, which offers laminar flow and low Reynolds number within the platforms can provide unprecedented opportunities for sperm selection. Previous studies showed that microfluidic platforms can provide a novel approach to this challenge and since then researchers across the globe have attacked this problem from multiple angles. OBJECTIVE: In this review, we seek to provide a much-needed bridge between the technical and medical aspects of microfluidic sperm selection. Here, we provide an up-to-date list on microfluidic sperm selection procedures and its application in assisted reproductive technology laboratories. SEARCH METHOD: A literature search was performed in Web of Science, PubMed, and Scopus to select papers reporting microfluidic sperm selection using the keywords: microfluidic sperm selection, self-motility, non-motile sperm selection, boundary following, rheotaxis, chemotaxis, and thermotaxis. Papers published before March 31, 2023 were selected. OUTCOMES: Our results show that most studies have used motility-based properties for sperm selection. However, microfluidic platforms are ripe for making use of other properties such as chemotaxis and especially rheotaxis. We have identified that low throughput is one of the major hurdles to current microfluidic sperm selection chips, which can be solved via parallelization. CONCLUSION: Future work needs to be performed on numerical simulation of the microfluidics chip prior to fabrication as well as relevant clinical assessment after the selection procedure. This would require a close collaboration and understanding among engineers, biologists, and medical professionals. It is interesting that in spite of two decades of microfluidics sperm selection, numerical simulation and clinical studies are lagging behind. It is expected that microfluidic sperm selection platforms will play a major role in the development of fully integrated start-to-finish assisted reproductive technology systems.

Evaluation of Predicting Factors Affecting Sperm Retrieval in Patients with Klinefelter Syndrome: A Prospective Study.

BACKGROUND: This study aimed to evaluate the predicting factors affecting sperm retrieval. We prospectively assessed the relationship between sonographic and microdissection testicular sperm extraction (mTESE) findings in Klinefelter syndrome (KS). MATERIALS AND METHODS: In this prospective study, 44 azoospermic men with 47, XXY karyotypes participated in this study. In order to evaluate the amount of blood supply in different parts of testicular tissue, a doppler ultra-sonographic was performed. Also, for the detection of sperm in this group mTESE technique was performed. RESULTS: The age average of positive mTESE and negative mTESE groups was 29.4 and 33.6 years, respectively. By comparing the testicle volume (based on the data obtained from the clinical examinations conducted by the urologist) it was determined that there is no significant difference between mTESE positive and negative groups. Folliclestimulating hormone (FSH) levels in men with negative mTESE (P=0.03) and testosterone levels in men with positive mTESE significantly increased (P=0.017). The overall rate of testis vascularity was significantly higher in the positive mTESE group than in the negative mTESE group. The clinical pregnancy rate in positive mTESE men was 9% per cycle, 16.6% per embryos were transferred (ET), and 12.5% per cycle. CONCLUSION: Totally, our observation indicated that there is not a significant relationship between sonographic and mTESE results in KS patients. However, more investigations with bigger sample Size can be useful to validate our results.

Nuclear Factor Kappa-B Protein Levels in Sperm of Obese Men with and without Diabetes; Cellular Approach in Male Infertility.

OBJECTIVE: Although the role of obesity and diabetes mellitus (DM) in male infertility is well established, little information about the underlying cellular mechanisms in infertility is available. In this sense, nuclear factor kappa-B (NF-kB) has been recognized as an important regulator in obesity and DM; However, its function in the pathogenesis of male infertility has never been studied in obese or men who suffer from diabetes. Therefore, the main goal of current research is assessing NF-kB existence and activity in ejaculated human spermatozoa considering the obesity and diabetics condition of males. MATERIALS AND METHODS: In an experimental study, the ELISA technique was applied to analyze NF-kB levels in sperm of four experimental groups: non-obese none-diabetic men (body mass index (BMI) <25 kg/m2; control group; n=30), obese non-diabetic men (BMI >30 kg/m2; OB group; n=30), non-obese diabetic men (BMI <25 kg/m2; DM group; n=30), and obese diabetic men (BMI >30 kg/m2; OB-DM group; n=30) who were presented to Royan Institute Infertility Center. In addition, protein localization was shown by Immunocytofluorescent assay. Sperm features were also evaluated using CASA. RESULTS: The diabetic men were older than non-diabetic men regardless of obesity status (P=0.0002). Sperm progressive motility was affected by obesity (P=0.035) and type A sperm progressive motility was affected by DM (P=0.034). The concentration of sperm (P=0.013), motility (P=0.025) and morphology (P<0.0001) were altered by obesity × diabetes interaction effects. The NF-kB activity was negatively influenced by the main impact of diabetics (P=0.019). Obesity did not affect (P=0.248) NF-kB activity. Uniquely, NF-kB localized to the midpiece of sperm and post-acrosomal areas. CONCLUSION: The current study indicated a lower concentration of NF-kB in diabetic men, no effect of obesity on NF-kB was observed yet. Additionally, we revealed the main obesity and diabetes effects, and their interaction effect adversely influenced sperm characteristics.

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

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Alpha-Lipoic Acid Supplementation for Male Partner of Couples with Recurrent Pregnancy Loss: A Post hoc analysis in Clinical Trial.

BACKGROUND: Increased sperm DNA damage is known as one of the causes of recurrent pregnancy loss (RPL) which can be due to increased levels of oxidative stress. Therefore, the aim of this study was to assess the effect of alpha-lipoic acid (ALA) on sperm parameters and sperm functions in couples with a history of RPL. MATERIALS AND METHODS: In this post hoc analysis in clinical trial study, a total of 37 couples with RPL (n=12 and n=25 for placebo and ALA groups, respectively) were considered. Men were treated with ALA (600 mg/day) or placebo for 80 days. Semen samples were acquired from the participants before initiation and after completion of the medication course and assessed regarding conventional sperm parameters, chromatin damage/integrity, intracellular oxidative stress, lipid peroxidation, and seminal antioxidant characteristics. Individuals were further followed up for twelve months for pregnancy occurrence and outcomes. Finally, after excluding patients with no history of RPL, the data was analyzed. RESULTS: No significant differences were observed between the baseline measures of the aforementioned parameters except for seminal volume. After the intervention, the mean sperm DNA damage, protamine deficiency, and persisted histones were significantly lower in the ALA group than in placebo receivers (P<0.05). A decrease in the mean of seminal total antioxidant capacity (P=0.03), malondialdehyde (P=0.02), and sperm DNA damage (P=0.004) as well as an increase in sperm total motility (P=0.04) after treatment with ALA was noticed. In addition, the mean of protamine deficiency and persisted histones were declined post-ALA therapy (P=0.003 and 0.002, respectively). The percentage of spontaneous pregnancy in the ALA group (4 of 25 cases; 16%) was higher than in the placebo group (1 of 12, 8.3%). CONCLUSION: ALA-therapy attenuates sperm DNA damage and lipid peroxidation while enhancing sperm total motility and chromatin compaction in the male partner of couples with PRL (registration number: IRCT20190406043177N1).

Evaluation of Nrf2/ARE Signaling Pathway in the Presence of Pentoxifylline as a Cryoprotectant in Mouse Spermatogonial Stem Cells.

The process of spermatogonial stem cell cryopreservation (SSCs) in young male cancer survivors is associated with increased reactive oxygen species (ROS), DNA fragmentation, apoptosis, decreased cell activity, and finally reduced fertility of SSCs. Therefore, it is necessary to add cryoprotectants to the freezing medium to minimize the injuries associated with cryopreservation. In addition, the Nrf2/ARE pathway is a main cellular pathway that regulates the antioxidant defense system. The purpose of this study was to evaluate the cryoprotective effect of pentoxifylline (PTX) on SSCs after freezing-thawing through the Nrf2/ARE pathway. SSCs extracted from neonatal mice testes were isolated and their purity was measured by flow cytometry with GDNF family receptor alpha-1 (GFRα1) and inhibitor of differentiation 4 (ID4). After culturing, the cells were frozen in different groups for 1 month. After freezing-thawing, cell viability, colonization rate, and intracellular ROS, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were evaluated. Quantitative real-time polymerase chain reaction and western blotting were done to assess the expression levels of Nrf2, Keap-1, PI3K, and AKT genes and proteins. The survival and colonization rates of SSCs, SOD, and CAT levels, and Nrf2, PI3K, and AKT expression levels were significantly higher in the PTX group compared with the other cryopreservation groups. The Keap-1 expression level and the ROS and MDA production levels also decreased significantly in the PTX group (p-value <0.05). According to our findings, PTX can activate the antioxidant defense through the Nrf2/ARE signaling pathway; therefore, it could be a suitable cryoprotectant candidate for freezing and long-term storage of SSCs in the clinical setting.

Sperm Retrieval in Non-azoospermic Patients with Persistent Ejaculation Dysfunction.

Infertility is a common disease that affects 15 to 20% of couples at some point in their lives. Among infertile couples, male factor accounts for 50% of infertile cases. Assisted reproductive techniques are the gold standard approach in case of failure in medical or surgical treatments. Moreover, the role of the urologist in these approaches is to provide appropriate sperm on the day of oocyte pick-up. However, sperm retrieval procedure is quite different in azoospermic and non-azoospermic men. Although most cases of infertile patients are not azoospermic, their ejaculation disorder prevents obtaining sperm for assisted reproductive techniques. This review article explains common problems of sperm retrieval in non-azoospermic patients with persistent ejaculatory dysfunction and introduces some management strategies. In fact, it is possible to design a classic approach for managing such patients, which definitely reduces the problems faced by clinicians as well.

Alpha-Lipoic Acid Ameliorates Sperm DNA Damage and Chromatin Integrity in Men with High DNA Damage: A Triple Blind Randomized Clinical Trial.

OBJECTIVE: Evidence suggests the contributory role of oxidative stress (OS) to sperm DNA damage and eventually, male infertility. Antioxidant supplementation has exhibited favorable results regarding seminal OS, sperm DNA damage, and chromatin integrity. We aimed to evaluate the effect of alpha-lipoic acid (ALA) supplementation on semen analysis, sperm DNA damage, chromatin integrity, and seminal/intracellular OS in infertile men with high sperm DNA damage. MATERIALS AND METHODS: In this randomized triple-blind placebo-controlled clinical trial study, we opted for a triple-blind controlled clinical trial design. Considering the study's inclusion criteria for the level of sperm DNA fragmentation (higher than the threshold of 30 and 15%), 70% of participants were selected for this clinical research study. Subjects were divided into case and control groups receiving oral ALA (600 mg/day) and placebo for eighty days, respectively. Sperm parameters and functional tests were examined and compared before and after treatment. The final sample size was 34 and 29 for ALA and placebo receivers, respectively. RESULTS: No significant differences were observed about anthropometrics and baseline measures of semen analysis, DNA damage, OS, and chromatin integrity between the two groups. Conventional semen parameters were enhanced insignificantly in both groups (P>0.05). DNA damage decreased significantly in the ALA group, as per sperm chromatin structure assay (SCSA, P<0.001). Moreover, chromomycin A3 (CMA3) staining results indicated a decrease in nuclear protamine deficiency post-ALA therapy (P=0.004). Lipid peroxidation decreased significantly after treatment with ALA (P=0.003). Further, seminal antioxidant capacity/activity did not differ significantly in either of the groups (registration number: IRCT20190406043177N1). CONCLUSION: An 80-day course of oral ALA supplementation (600 mg/day) alleviates sperm OS, DNA damage, and chromatin integrity in men with high sperm DNA damage.

Evaluation of PGK2 and ACR proteins in seminal plasma: suggestion of potential new biomarkers for prediction of sperm retrieval in non-obstructive azoospermia patients.

This study aimed to assess the role of testis-specific proteins, PGK2 and ACR, in the prediction of sperm retrieval results by microdissection testicular sperm extraction (micro-TESE) in men with non-obstructive azoospermia (NOA). This was a case-control study including 48 semen samples of NOA patients undergoing the micro-TESE procedure, 15 semen samples from normozoospermic men as the positive control, and 12 semen samples from obstructive azoospermia/post-vasectomy (OA/PV) as negative controls. We investigated the levels of PGK2 and ACR proteins by ELISA tests in seminal plasma samples. The ELISA results revealed a significantly higher concentration of PGK2 and ACR in the NOA patients with successful sperm retrieval (NOA+) in comparison to NOA patients with failed sperm retrieval (NOA-) group (p = 0.0001 in both cases). For the first time, the data from this study suggests that a seminal PGK2 concentration of 136.3 pg/ml and ACR concentration of 21.75 mIU/ml can be used as cut-off values for the prediction of micro-TESE outcomes in NOA patients. These findings may be useful to avoid unnecessary micro-TESE operations. Overall, the seminal levels of the PGK2 and ACR proteins may be useful in predicting sperm retrieval success by micro-TESE in NOA patients.

Epigenetic modifications at DMRs of imprinting genes in sperm of type 2 diabetic men.

High rates of infertility in type 2 diabetic (T2DM) men have led to attempts to understand the mechanisms involved in this process. This condition can be investigated from at least two aspects, namely sperm quality indices and epigenetic alterations. Epigenetics science encompasses the phenomena that can lead to inherited changes independently of the genetics. This study has been performed to test the hypothesis of the relationship between T2DM and the epigenetic profile of the sperm, as well as sperm quality indices. This research included 42 individuals referred to the infertility clinic of Royan Institute, Iran in 2019-2021. The study subjects were assigned to three groups: normozoospermic non-diabetic (control), normozoospermic diabetic (DN) and non-normozoospermic diabetic (D.Non-N). Sperm DNA fragmentation was evaluated using the sperm chromatin structure assay technique. The global methylation level was examined using 5-methyl cytosine antibody and the methylation status in differentially methylated regions of H19, MEST, and SNRPN was assessed using the methylation-sensitive high-resolution melting technique. The results showed that the sperm global methylation in spermatozoa of D.Non-N group was significantly reduced compared with the other two groups (P < 0.05). The MEST and H19 genes were hypomethylated in the spermatozoa of D.Non-N individuals, but the difference level was not significant for MEST. The SNRPN gene was significantly hypermethylated in these individuals (P < 0.05). The results of this study suggest that T2DM alters the methylation profile and epigenetic programming in spermatozoa of humans and that these methylation changes may ultimately influence the fertility status of men with diabetes.

Epigenetic Dysregulation of BRDT Gene in Testis Tissues of Infertile Men: Case-Control Study.

Objective: Bromodomain testis associated (BRDT), a testis-specific member of the Bromo- and Extra-Terrminal domain (BET) protein family, is involved in spermatogenesis and, more specifically, chromatin remodeling. In the post-meiotic spermatogenic cells, BRDT protein binds to the hyperacetylated histones and facilitates their replacement with transition proteins (TPs), particularly protamines, which are essential for chromatin condensation. The current research was conducted to assess the expression and epigenetic profile of BRDT in the testis tissues of infertile men. Materials and Methods: In this case-control study, three groups were included: positive control group: obstructive azoospermia (OA, n=10), round spermatid maturation arrest group (SMA, n=10) and negative control group: sertoli cellonly syndrome (SCOS, n=10). Using quantitative real-time polymerase chain reaction (PCR), the expression profile of BRDT was generated. Also, ChIP-real time PCR was used to measure the following histone marks: H3K9ac, H3K9me3, H3K4me3, H3K27me3 on the promoter region of BRDT. Results: Our data indicated that BRDT expression decreased in the SMA group in comparison with the positive control group and this finding is in line with the ChIP results obtained in this group. Conclusion: Based on these data, we postulate that BRDT gene has a vital role in the spermatogenesis and its decreased expression due to an aberrant epigenetic signaling might be associated with male infertility.

Direct visualization of pre-protamine 2 detects protamine assembly failures and predicts ICSI success.

Histone-to-protamine transition is an essential step in the generation of fully functional spermatozoa in various mammalian species. In human and mouse, one of the two protamine-encoding genes produces a precursor pre-protamine 2 (pre-PRM2) protein, which is then processed and assembled. Here, we design an original approach based on the generation of pre-PRM2-specific antibodies to visualize the unprocessed pre-PRM2 by microscopy, flow cytometry and immunoblotting. Using mouse models with characterized failures in histone-to-protamine replacement, we show that pre-PRM2 retention is tightly linked to impaired nucleosome disassembly. Additionally, in elongating/condensing spermatids, we observe that pre-PRM2 and transition protein are co-expressed spatiotemporally, and their physical interaction suggests that these proteins act simultaneously rather than successively during histone replacement. By using our anti-human pre-PRM2 antibody, we also measured pre-PRM2 retention rates in the spermatozoa from 49 men of a series of infertile couples undergoing ICSI, which shed new light on the debated relation between pre-PRM2 retention and sperm parameters. Finally, by monitoring 2-pronuclei embryo formation following ICSI, we evaluated the fertilization ability of the sperm in these 49 patients. Our results suggest that the extent of pre-PRM2 retention in sperm, rather than pre-PRM2 accumulation per se, is associated with fertilization failure. Hence, anti-pre-PRM2 antibodies are valuable tools that could be used in routine monitoring of sperm parameters in fertility clinics, as well as in experimental research programmes to better understand the obscure process of histone-to-protamine transition.

A Comprehensive Guide to Sperm Recovery in Infertile Men with Retrograde Ejaculation.

Retrograde ejaculation (RE) is a condition defined as the backward flow of the semen during ejaculation, and when present can result in male infertility. RE may be partial or complete, resulting in either low seminal volume or complete absence of the ejaculate (dry ejaculate). RE can result from anatomic, neurological or pharmacological conditions. The treatment approaches outlined are determined by the cause. Alkalinizing urinary pH with oral medications or by adding sperm wash media into the bladder prior to ejaculation may preserve the viability of the sperm. This article provides a step-by-step guide to diagnose RE and the optimal techniques to retrieve sperm.

Role of peroxisome proliferator-activated receptor gamma (PPARγ) in the regulation of fatty acid metabolism related gene expressions in testis of men with impaired spermatogenesis.

Although male infertility is a multifactorial syndrome in which genetic factors are responsible for up to 15 % of cases, there are few studies of genes involved in lipid metabolism and male infertility. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor in testis tissue. PPARγ binds to DNA and regulates the genes for fatty acid (FA) metabolism. Thus, it has a key role in male reproduction. The current study assessed the expressions of fatty acid desaturase 2 (FADS2), elongation of very-long-chain fatty acids-like 2 (ELOVL2), stearoyl-CoA desaturase-1 (SCD), and lipoprotein lipase (LPL) and incorporation of PPARγ in the promoter regions of these genes in testicular tissue biopsies from 30 infertile males who underwent testicular sperm extraction. The samples were classified into three groups: obstructive azoospermia (OA), which was the positive control (n = 10); round spermatid maturation arrest (SMA, n = 10); and Sertoli cell-only syndrome (SCOS, n = 10). There were significantly lower relative mRNA expression levels of the FADS2, ELOVL2, SCD, and LPL genes in the SCOS (P < 0.01) and SMA (P < 0.01) groups compared to the OA control group. We observed a significant decrease in chromatin incorporation of PPARγ on the promoter regions of the candidate FA metabolism genes (P < 0.05). For the first time, the present study results show that PPARγ is a strong mediator for regulation of FA metabolism in human testis tissue and we confirmed its critical role in normal spermatogenesis.

Vasectomy reversal: Unilateral versus bilateral vasovasostomy.

The aim of this study was to evaluate and compare the efficiencies of unilateral and bilateral vasovasostomies as the vasectomy reversal procedures. A total of 95 patients with a history of bilateral vasectomy were evaluated. 42 of them had undergone unilateral surgery, and bilateral surgery had been done for the other 53 patients. Their information including the age, the time interval between the initial vasectomy to the reversal surgery and other underlying illnesses or medications was gathered. Patency rates in the unilateral and bilateral groups were 88.1% (38 patients) and 88.7% (48 patients), respectively, the difference of which was not statistically significant (p = .907). Successful pregnancies occurred in 22 (52.4%) and 29 (54.7%) patients, respectively, which did not show any statistically significant difference too (p = .713). Based on the multivariate logistic regression model, only the time interval between vasectomy and the reversal (duration of obstruction) was predictive of patency (OR = 1.112, p = .037). The outcomes of the unilateral and bilateral vasovasostomies in terms of patency and pregnancy rates were not significantly different. We suggest that performing unilateral, instead of bilateral, vasovasostomy can reduce the time of anaesthesia and surgery and save costs and consumables without having a significant negative impact on the surgical outcomes.

A Global Survey of Reproductive Specialists to Determine the Clinical Utility of Oxidative Stress Testing and Antioxidant Use in Male Infertility.

PURPOSE: The use of antioxidants is common practice in the management of infertile patients. However, there are no established guidelines by professional societies on antioxidant use for male infertility. MATERIALS AND METHODS: Using an online survey, this study aimed to evaluate the practice pattern of reproductive specialists to determine the clinical utility of oxidative stress (OS) testing and antioxidant prescriptions to treat male infertility. RESULTS: Responses from 1,327 participants representing 6 continents, showed the largest participant representation being from Asia (46.8%). The majority of participants were attending physicians (59.6%), with 61.3% having more than 10 years of experience in the field of male infertility. Approximately two-thirds of clinicians (65.7%) participated in this survey did not order any diagnostic tests for OS. Sperm DNA fragmentation was the most common infertility test beyond a semen analysis that was prescribed to study oxidative stress-related dysfunctions (53.4%). OS was mainly tested in the presence of lifestyle risk factors (24.6%) or sperm abnormalities (16.3%). Interestingly, antioxidants were prescribed by 85.6% of clinicians, for a duration of 3 (43.7%) or 3-6 months (38.6%). A large variety of antioxidants and dietary supplements were prescribed, and scientific evidence were mostly considered to be modest to support their clinical use. Results were not influenced by the physician's age, geographic origin, experience or training in male infertility. CONCLUSIONS: This study is the largest online survey performed to date on this topic and demonstrates 1) a worldwide understanding of the importance of this therapeutic option, and 2) a widely prevalent use of antioxidants to treat male infertility. Finally, the necessity of evidence-based clinical practice guidelines from professional societies is highlighted.

Observational retrospective study of Intracytoplasmic sperm injection outcomes of 117 infertile men with severe short tail sperm defect.

Intracytoplasmic sperm injection (ICSI) is increasingly used to treat male-factor infertility when sperm parameters are not proper for intrauterine insemination (IUI) or in vitro fertilization (IVF). Among sperm abnormalities, short tail sperm defect is a rare kind of teratozoospermia, which is a severe cause of male infertility. In this study, we evaluated the ICSI outcomes of infertile men with severely short tail sperm defect. 117 infertile men with primary infertility were included in this study. We evaluated the impact of short tail sperm defect on large ICSI series (228 cycles) outcomes. The fertilisation rate (FR) was 49.0%, the clinical pregnancy rate (PR) was 21.7%, and the delivery rate (DR) was 17.5%. The results of statistical analysis show that there is no relationship between short tail sperm defect and clinical pregnancy. According to the present study, there were patients with successful ICSI outcomes despite the severe defect in their spermatozoa flagella. Our results can be considered in two main aspects: (a) it seems that ICSI could be a proper therapy for infertile men with short-tailed sperm defect and (b) the abnormal sperm morphology (especially in sperm flagellum) is not a reliable predictor for the ICSI outcomes. In conclusion, our study suggests that ICSI should be considered as a proper treatment way for infertile men with severe short tail sperm defect and probably other sperm flagella abnormalities.

The Germ Cell-Specific Markers ZPBP2 and PGK2 in Testicular Biopsies Can Predict the Presence as well as the Quality of Sperm in Non-obstructive Azoospermia Patients.

To assess the role of three testis-specific genes including ZPBP2, PGK2, and ACRV1 in the prediction of sperm retrieval result and quality of retrieved sperm by microdissection testicular sperm extraction (micro-TESE) in non-obstructive azoospermia (NOA) patients. This was a case-control study including 57 testicular samples of NOA patients including 32 patients with successful sperm retrieval (NOA+) and 25 patients with failed sperm retrieval (NOA-), and 9 samples of men with normal spermatogenesis in the testes as the positive control (OA). We investigated the expression of candidate genes by RT-qPCR and germ cell population patterns by DNA flow cytometry in testicular biopsy samples. The association between PGK2 expressions with the quality of retrieved spermatozoa was also evaluated. The RT-qPCR data revealed a significantly higher expression of ZPBP2 and PGK2 in the NOA+ in comparison to NOA- group (P = 0.002, and P = 0.002, respectively). Flow cytometry results revealed that the haploid cell percentage was significantly higher in NOA+ vs. NOA- group (P = 0.0001). In samples with a higher percentage of haploid cells, expression levels of ZPBP2 and PGK2 were higher (P = 0.001). The PGK2 expression was significantly associated with retrieved sperm quality (P = 0.01). Our results contribute to the search for the biomarkers for predicting the presence of testicular sperm and would be useful to avoid unnecessary multiple micro-TESE. Overall, the expression pattern of the ZPBP2 and PGK2 may be useful in predicting sperm recovery success and quality of retrieved sperm in NOA patients.

Sertoli cell-only syndrome: etiology and clinical management.

Almost 50% of infertility cases are due to male factors, and spermatogenesis failure is one of the most severe forms of male infertility. Sertoli cell-only syndrome (SCOS) also known as germ cell aplasia is characterized by azoospermia in which the seminiferous tubules of testicular biopsy are lined only with Sertoli cells. The definitive diagnosis of SCOS is by diagnostic testicular biopsy. Although SCOS may be a result of Klinefelter syndrome, most of the SCOS men have a normal karyotype. Along with genetic aberrations, signaling pathways and endocrine processes might be major factors in the development of SCOS. Sperm retrieval and intracytoplasmic sperm injection (ICSI) are available treatments for SCOS. However, some SCOS patients do not have therapeutic options to help them having a biological child. This review aims to summarize our present knowledge about SCOS and to highlight the importance of future researches in the diagnosis and treatment of this disorder.

Rare missense variant in MSH4 associated with primary gonadal failure in both 46, XX and 46, XY individuals.

STUDY QUESTION: Can whole-exome sequencing (WES) reveal a shared pathogenic variant responsible for primary gonadal failure in both male and female patients from a consanguineous family? SUMMARY ANSWER: Patients with primary ovarian insufficiency (POI) and non-obstructive azoospermia (NOA) were homozygous for the rare missense variant p. S754L located in the highly conserved MSH4 MutS signature motif of the ATPase domain. An oligozoospermic patient was heterozygous for the variant. WHAT IS KNOWN ALREADY: MSH4 is a meiosis-specific protein expressed at a certain level in the testes and ovaries. Along with its heterodimer partner MSH5, it is responsible for double-strand Holliday junction recognition and stabilization, to ensure accurate chromosome segregation during meiosis. Knockout male and female mice for Msh4 and Msh5 are reportedly infertile due to meiotic arrest. In humans, MSH4 is associated with male and female gonadal failure, with distinct variations in the MutS domain V. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of a consanguineous family with multiple cases of gonadal failure in both genders. The subject family was recruited in Iran, in 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: The proband who is affected by POI, an NOA brother, a fertile sister and their parents were subjected to WES. The discovered variant was validated in these individuals, and the rest of the family was also genotyped by Sanger sequencing. The variant was not detected in 800 healthy Iranian individuals from the Iranome database nor in 30 sporadic NOA and 30 sporadic POI patients. Suggested effect in aberrant splicing was studied by RT-PCR. Moreover, protein homology modeling was used to further investigate the amino acid substitution in silico. MAIN RESULTS AND THE ROLE OF CHANCE: The discovered variant is very rare and has never been reported in the homozygous state. It occurs in the ATPase domain at Serine 754, the first residue within the highly conserved MutS signature motif, substituting it with a Leucine. All variant effect prediction tools indicated this variant as deleterious. Since the substitution occurs immediately before the Walker B motif at position 755, further investigations based on protein homology were conducted. Considering the modeling results, the nature of the substituted amino acid residue and the distances between p. S754L variation and the residues of the Walker B motif suggested the possibility of conformational changes affecting the ATPase activity of the protein. LARGE SCALE DATA: We have submitted dbSNP entry rs377712900 to ClinVar under SCV001169709, SCV001169708 and SCV001142647 for oligozoospermia, NOA and POI, respectively. LIMITATIONS, REASONS FOR CAUTION: Studies in model organisms can shed more light on the role of this variant as our results were obtained by variant effect prediction tools and protein homology modeling. WIDER IMPLICATIONS OF THE FINDINGS: Identification of variants in meiotic genes should improve genetic counseling for both male and female infertility. Also, as two of our NOA patients underwent testicular sperm extraction (TESE) with no success, ruling out the existence of pathogenic variants in meiotic genes in such patients prior to TESE could prove useful. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by Royan Institute in Tehran, Iran, and Institut Pasteur in Paris, France. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.