Forms Of Male Factor Infertility Research Articles

Epigenetics of nonobstructive azoospermia.

Nonobstructive azoospermia (NOA) is a severe and heterogeneous form of male factor infertility caused by dysfunction of spermatogenesis. Although various factors are well defined in the disruption of spermatogenesis, not all aspects due to the heterogeneity of the disorder have been determined yet. In this review, we focus on the recent findings and summarize the current data on epigenetic mechanisms such as DNA methylation and different metabolites produced during methylation and demethylation and various types of small noncoding RNAs involved in the pathogenesis of different groups of NOA.

Non-obstrüktif azospermiye güncel yaklaşım

Infertility affects 1 in 6 couples and male factor infertility is the cause in 50% of cases. Azoospermia is defined as the absence of spermatozoa in the ejaculate and is considered the most extreme form of male factor infertility. In the past, these men were considered infertile, but with the advent of testicular sperm extraction and assisted reproductive technologies, men with azoospermia can biologically father their own children. Non-obstructive azoospermia (NOA) occurs when there is a defect in spermatogenesis. This review describes the contemporary management of NOA and discusses the role of hormone stimulation therapy, surgical and embryological factors, and new technologies such as proteomics, genomics and artificial intelligence systems in the diagnosis and treatment of men with NOA. We also emphasise that men with NOA represent a vulnerable population at high risk of developing cancer and cardiovascular comorbidities. Keywords: azoospermia, non-obstructive azoospermia, infertility, hypogonadism

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

Are Clinical Outcomes of Microdissection Testicular Sperm Extraction in Non-Obstructive Azoospermic Men Affected by the Use of Fresh or Frozen Gametes?

Background: Non-obstructive azoospermia (NOA) is a severe form of male factor infertility, evident in approximately 5% of infertile couples seeking fertility treatment. The combination of microsurgical testicular sperm extraction (mTESE) and ICSI has become one of the treatment options, with sperm motility positively associated with outcome success rates. Few studies have investigated whether use of fresh or thawed gamete combinations affect clinical outcomes. Aim: To determine the clinical outcomes of ICSI cycles using mTESE recovered testicular sperm of NOA patients and whether these are affected by the use of fresh or thawed gametes. Method: A retrospective study of NOA patients who underwent mTESE between March 2017 and September 2020. Clinical outcomes investigated included successful surgical sperm retrieval (SSR), sperm motility, fertilisation, embryo utilisation and live-birth/ongoing pregnancy rates. Results: A total of 105 NOA patients underwent mTESE, with a 65.7% successful SSR. In total 58 patients contributed to 72 ICSI cycles, with a 37.5% 2PN fertilisation rate and 29.4% live-birth/ongoing pregnancy rate. Cycles using only motile sperm had a greater fertilisation rate than those using a motile/non-motile sperm combination (51.7% vs 31.1%, p[Formula: see text]0.05). Compared with fresh, thawed testicular spermatozoa didn’t affect clinical outcomes, including live-birth/ongoing pregnancy rates (Fresh 18.2%; Thawed 17.9%; Odds ratio (OR) 1.02 (0.34-2.86) p=0.98). In contrast, use of thawed oocytes decreased fertilisation rate [Fresh-oocytes: 45.0%; Vitrified-oocytes: 35.5%; OR 1.47(1.03-2.10), p=0.03] but had no differences for other clinical outcomes, including live-birth/ongoing pregnancy rate (p=0.74). Importantly, injection of exclusively non-motile-spermatozoa (n=26 cycles) resulted in a very low fertilisation rate (2.7%) and no live-birth/ongoing pregnancies were recorded. Conclusion: Microdissection TESE is an effective treatment for NOA patients, with no clear negative effects of using thawed spermatozoa, however, the use of vitrified oocytes requires further investigation. The use of non-motile spermatozoa in mTESE ICSI cycles is discouraged.

O-045 ICSI: From ‘this cannot work' to ‘remarkably safe'

Abstract ICSI From “this cannot work” to “remarkably safe” It is now 30 years since the first child was born after ICSI treatment. Since then there has been an ongoing debate on the use and safety of the technique. Initially, ICSI was used to treat severe forms of male factor infertility but today it is also used to treat mild male factor infertility, mixed male/female infertility, unexplained infertility and fertilization failures. Both the latest European ESHRE report and the global ICMART report reveal an increasing global use of ICSI, with more than 70% of cycles using ICSI. Due to the invasiveness of the ICSI procedure as well as the arbitrary selection of the spermatozoon and genetic and epigenetic parental factors, concerns have been expressed about the health of ICSI children. While most births after ART are uncomplicated, ART is associated with potential adverse obstetric outcomes for both mothers and infants, including hypertensive disorders of pregnancy, preterm delivery, and low birth weight ART has also been associated with an increased risk of birth defects .Many of these adverse outcomes can be attributed to a higher rate of multiple pregnancies after ART. With the increasing use of single embryo transfer, the multiple pregnancy rate has been significantly reduced but is still unacceptably high in many countries. When comparing ICSI and conventional IVF, most large studies have found similar or lower risks of very preterm and preterm birth, very low birth weight, low birth weight and peri/neonatal mortality in ICSI children. A possible explanation for the better outcome in ICSI singletons may be that in ICSI the majority of the women are reproductively healthy, which could give a more favorable intrauterine environment. Most studies have found an increased rate of birth defects in ART children, ranging between 30% and 70% higher, but with no difference between conventional IVF and ICSI and the different sperm sources used in ICSI do not seem to influence the rate of birth defects negatively. However, the incidence of de novo and inherited sex chromosomal abnormalities in ICSI offspring is slightly higher, which probably relates to the genetics of the infertile couples. Although children born after IVF and ICSI have a higher risk to be born preterm and with low birth weight they usually catch up during the first years. A large systematic review from Denmark showed no differences in weigh and length up to 22 years of age, between children born after IVF and ICSI and children born after spontaneous conception. Looking at general physical health similar results are observed for ICSI and IVF-conceived children. Most large cohort studies do not show any increase in childhood cancer in general in ART children and provide no evidence of an increased risk in the ICSI group. Most studies comparing children up to eight years of age born after ICSI, conventional IVF and spontaneous conception, suggest their neurocognitive development is comparable. Sperm source or individual semen parameters do not affect neurodevelopment. Two recent large studies reporting associations between ICSI and autism and autistic disorders should be interpreted with caution, since the absolute risks of autism and autistic disorders are small. School performances in 15-16 years old adolescences have recently been studied in several large Nordic register studies. Children born after ART performed better in unadjusted analyses but after adjustment, mainly for parental education, these differences disappeared. No differences were observed between ICSI and IVF. Continuous supervision after ART is needed to ensure safety and quality, especially when new techniques are introduced. National ART registries such as those existing in the Nordic countries enable follow-up studies of ART children and should be encouraged.

O-094 Utilization of ultrastructural analysis and genomics of spermatozoa to better characterize subtle forms of male factor infertility

Abstract Study question Can sequencing the sperm genome provide insight into the various forms of male factor infertility caused by sperm organelle ultrastructural defects? Summary answer A comprehensive genomic assessment of spermatozoal DNA is able to identify genetic causes of ultrastructural defects visualized by transmission electron microscopy (TEM). What is known already To evaluate a man’s reproductive potential, a conventional semen analysis through the assessment of concentration, motility, and morphology can indicate the proficiency of male gametes. Among those, conventional morphology assay can only provide indirect information on the different components of the sperm cell. The assessment of nanoscopic details such as chromatin, centriolar, mitochondrial, and axonemal components can only be observed by TEM. Indeed, TEM has been used to identify defects in the acrosome, chromatin compaction, and axonemal/periaxonemal structures. Furthermore, exome sequencing of spermatozoal DNA may identify novel causes and candidate genes for these ultrastructural defects. Study design, size, duration In the past 2 years, 20 men with history of fertilization failure or severe astheno-/terato-zoospermia were selected for TEM analysis of their spermatozoa, while 3 fertile men served as controls. Sperm head characteristics, intactness of fibrous sheath, and axonemal/periaxonemal structure were examined by diagnostic TEM. For consenting patients, NGS assessment was concurrently performed to identify mutations responsible for the structural abnormalities identified by TEM. Participants/materials, setting, methods TEM was performed on the ejaculates of 20 infertile patients and 3 fertile controls. Post-centrifugation cell pellets were resuspended, fixed, and dehydrated to be infiltrated and embedded onto the resin. Fixed specimens were sliced by ultramicrotome to 100-nm sections, then viewed by JEOL-1400 electron microscope at 300,000X magnification. At least 100 spermatozoa were evaluated by TEM. For consenting patients, DNA was extracted and amplified from at least 500 spermatozoa for concurrent NGS analysis. Main results and the role of chance Four types of sperm ultrastructural defects were observed, including globozoospermia, dysplasia of fibrous sheath (DFS), proximal centriole defect, and primary ciliary dyskinesia (PCD). One combined case of globozoospermia and DFS was identified. In globozoospermic patients (n = 13), 97-100% of the spermatozoa displayed characteristic spherical heads with absence of acrosomes, dispersed chromatin, and perinuclear theca deformities. Centrosomal and axonemal structures were conserved. NGS identified gene deletions (DPY19L, PICK1, SPATA16) directly related to the globozoospermic phenotype. In patients with DFS (n = 4), complete absence of flagellum was observed in 90-100% of spermatozoa. These defective gametes also displayed mitochondria disorganization, microtubular deformities, and cytoplasmic residues containing coiled flagellum with deformed capitulum within the plasma membrane. Contrary to the globozoospermia, acrosomes and nuclei appeared normal, indicating incomplete late spermiogenesis. Indeed, NGS confirmed gene deletions involved in flagellar development/function (AKAP4, SPAG16, CATSPER1). For the patient with proximal centriole defect (n = 1), sperm nucleus, fibrous sheath, and flagellar structure were conserved. However, 90% of proximal centrioles assessed exhibited microtubular disorganization, confirmed by ODF2 mutation per NGS. In the PCD patient (n = 1), chaotic flagellar microtubule arrangement and absence of outer dynein arms were prevalent in 90% of axonemal cross-sections examined, which was explained by a DNAH5 gene deletion. Limitations, reasons for caution While TEM can overcome the limitations of conventional semen analysis by providing direct visualization of the inner organelle arrangement of spermatozoa to accurately diagnose rare sperm pathologies, it is not routinely applied in clinics due to its high cost and technical specifications. Therefore, confirmatory NGS can provide additional diagnostic value. Wider implications of the findings Ultrastructural analysis with a concurrent genomic assessment characterized phenotypes and genotypes of rare sperm pathologies in infertile men. The utilization of TEM, corroborated by genomic assay, is therefore crucial for clinical and translational reproductive medicine to better characterize male factor infertility. Trial registration number N/A

Omics in Seminal Plasma: An Effective Strategy for Predicting Sperm Retrieval Outcome in Non-obstructive Azoospermia.

Non-obstructive azoospermia (NOA) is a severe form of male factor infertility resulting from the impairment of sperm production. Surgical sperm retrieval followed by intracytoplasmic sperm injection (ICSI) is the only alternative for NOA patients to have their own genetic children. Nevertheless, due to an approximately 50% chance of success, harvesting sperm from these patients remains challenging. Thus, discovering noninvasive biomarkers, which are able to reliably predict the probability of sperm acquisition, not only can eliminate the risk of surgery but also can lower the costs of NOA diagnosis and treatment. Seminal plasma is the non-cellular and liquid portion of the ejaculate that consists of the secretions originating from testes and male accessory glands. In past years, a wide range of biomolecules including DNAs, RNAs, proteins, and metabolic intermediates have been identified by omics techniques in human seminal plasma. The current review aimed to briefly describe genomic, transcriptomic, proteomic, and metabolomic profiles of human seminal plasma in an attempt to introduce potential candidate noninvasive biomarkers for sperm-retrieval success in men with NOA.

Non-obstructive azoospermia: current and future perspectives.

Infertility affects 1 in 6 couples, and male factor infertility has been implicated as a cause in 50% of cases. Azoospermia is defined as the absence of spermatozoa in the ejaculate and is considered the most extreme form of male factor infertility. Historically, these men were considered sterile but, with the advent of testicular sperm extraction and assisted reproductive technologies, men with azoospermia are able to biologically father their own children. Non-obstructive azoospermia (NOA) occurs when there is an impairment to spermatogenesis. This review describes the contemporary management of NOA and discusses the role of hormone stimulation therapy, surgical and embryological factors, and novel technologies such as proteomics, genomics, and artificial intelligence systems in the diagnosis and treatment of men with NOA. Moreover, we highlight that men with NOA represent a vulnerable population with an increased risk of developing cancer and cardiovascular comorbodities.

Towards a better testicular sperm extraction: novel sperm sorting technologies for non-motile sperm extracted by microdissection TESE.

Non-obstructive azoospermia (NOA) is the most severe form of male factor infertility. It is characterized by a lack of spermatogenesis in the seminiferous tubules. Microdissection testicular sperm extraction (microTESE) has significantly improved testicular sperm retrieval rates compared to conventional techniques for NOA. Following testicular biopsy, the sperm is usually non-motile and contained within seminiferous tubules requiring extensive laboratory processing to find individual sperm sufficient for artificial reproductive technologies (ART). Current techniques include mechanical and enzymatic processing which is time-consuming and often damaging to sperm. We review novel techniques that may help improve sperm retrieval rates after microTESE including microfluidics (dielectrophoretic cell sorting, spiral channel sorting, and pinched flow fractionation), fluorescence-activated cell sorting (FACS), and magnetic-activated cell sorting (MACS).

Essential Role of Sperm-Specific PLC-Zeta in Egg Activation and Male Factor Infertility: An Update.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus responsible for generating calcium (Ca2+) oscillations that induce egg activation and early embryonic development during mammalian fertilization. In the mammalian testis, PLCζ expression is detected at spermiogenesis following elongated spermatid differentiation. Sperm-delivered PLCζ induces Ca2+ release via the inositol 1,4,5-trisphosphate (InsP3) signaling pathway. PLCζ is the smallest known mammalian PLC isoform identified to date, with the simplest domain organization. However, the distinctive biochemical properties of PLCζ compared with other PLC isoforms contribute to its unique potency in stimulating cytosolic Ca2+ oscillations within mammalian eggs. Moreover, studies describing PLCζ “knockout” mouse phenotypes confirm the supreme importance of PLCζ at egg activation and monospermic fertilization in mice. Importantly, a number of clinical reports have highlighted the crucial importance of PLCζ in human fertilization by associating PLCζ deficiencies with certain forms of male factor infertility. Herein, we give an update on recent advances that have refined our understanding of how sperm PLCζ triggers Ca2 + oscillations and egg activation in mammals, while also discussing the nature of a potential “alternative” sperm factor. We summarise PLCζ localization in mammalian sperm, and the direct links observed between defective PLCζ protein in sperm and documented cases of male infertility. Finally, we postulate how this sperm protein can be used as a potential diagnostic marker, and also as a powerful therapeutic agent for treatment of certain types of male infertility due to egg activation failure or even in more general cases of male subfertility.

Shorter leukocyte telomere length is associated with risk of nonobstructive azoospermia

To determine the association between leukocyte telomere length and the risk of nonobstructive azoospermia (NOA). The mean leukocyte telomere length (LTL) among men with NOA, obstructive azoospermia (OA), and normospermic subjects was determined by quantitative polymerase chain reaction (PCR). We used logistic regression to investigate the association between LTL and the risk of NOA after adjustment for age and body mass index (BMI). Partial correlation analysis was also used to evaluate the relationship of clinical parameters with the mean LTL among men with OA and NOA. Reproductive medicine center. A total of 866 men, including 270 normospermic controls, 247 OA and 349 NOA patients. None. Leukocyte telomere length. The mean relative LTL of men with NOA was significantly shorter than that of those with OA and in normospermic controls (odds ratio [OR] 0.81, 95% confidence interval [CI] 0.64-0.98 vs. OR 0.92, 95% CI 0.70-1.24 vs. OR 0.99, 95% CI 0.83-1.22), respectively). Subjects with shorter telomeres (lowest tertile) had a significantly higher risk of NOA than those with longer telomeres (highest tertile). Interestingly, we also found that a low relative LTL was associated with poor efficiency of spermatogenesis using the Johnsen score after testis biopsy and histopathology in azoospermic patients, after adjusting for patient age and BMI. This is the first report that short LTL is associated with NOA, shedding light on an important biological pathway involved in the etiology of this form of male factor infertility.

Methods of surgical sperm extraction and implications for assisted reproductive technology success.

Intracytoplasmic sperm injection offers a chance to surpass severe forms of male factor infertility, including azoospermia. Retrieval of the male gamete from the epididymis or testis provides the chance for biological parenthood for the affected men. In this review, we scrutinize the recent evidence about the surgical sperm retrieval methods for use in association with ICSI. We provide a historical overview of the surgical sperm retrieval methods development, its indication in both azoospermic and non-azoospermic men, and the technical aspects of each method. We also present and critically discuss the evidence concerning the success of ICSI using non-ejaculated sperm and the consequences of this approach to the health of resulting offspring.

MP60-15 TRANSCRIPTOME ANALYSIS TO IDENTIFY HUMAN SPERMATOGONIAL CELLS FROM SERTOLI CELL-ONLY TESTES

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology1 Apr 2018MP60-15 TRANSCRIPTOME ANALYSIS TO IDENTIFY HUMAN SPERMATOGONIAL CELLS FROM SERTOLI CELL-ONLY TESTES Koji Shiraishi and Hideyasu Matsuyama Koji ShiraishiKoji Shiraishi More articles by this author and Hideyasu MatsuyamaHideyasu Matsuyama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1907AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Non-obstructive azoospermia (NOA), particularly Sertoli cell-only (SCO), is the most severe form of male factor infertility and is classically thought to lack germ cells and spermatogenic stem cells (SSCs). However, evidence from microsurgical testicular sperm extraction (micro-TESE) on men with SCO has revealed the presence of focal areas of full spermatogenesis, suggesting the possible presence of SSCs in some patients with SCO lacking mature sperm. To determine whether gene products from SSCs could be detected in SCO, transcriptome analysis using next-generation sequencing (NGS) was performed using human biopsied testicular samples. METHODS Under IRB approval, testicular samples were obtained from 20 men (mean age: 35.7 years) with SCO in which no germ cells were found during laboratory examination and from five men with obstructive azoospermia (mean age: 38.5 years) with normal spermatogenesis. Men with sex chromosome or AZF abnormality were excluded. Transcriptomes on the Illumina platform were expressed as FPKM, and Cuffdiff analysis was performed to compare individual differences among testicular tissues from obstructive azoospermia (OA). Expression of mRNA and localization of targeted genes were confirmed by RT-PCR and immunohistochemical analyses, respectively. RESULTS From the 23,003 genes screened with using NGS with a significant signal after background correction and normalization, a total of 5,666 genes were analyzed. As representative markers of immature germ cells, UTF1, OCT4, DDX4, MMP1, GFRα, GPX1, SCP3, CD14, KLF9, GFRA1 and KIT mRNA were detected in SCO samples by transcriptome analysis. RT-PCR showed significant mRNA expression of UTF1, OCT4, DDX4, SCP3 and KIT from SCO samples. Immunohistochemistry showed the localization of DDX4 and/or KIT-positive cells in the seminiferous tubules and basement membrane in eight (40%) of the SCO samples. All the gene products evaluated in SCO samples were present in testes with obstructive azoospermia. There was no significant difference in the patients' background and hormonal data between the men with and without DDX4 and/or KIT-positive cells. CONCLUSIONS Germ cells are present in 40% of SCO samples as immature forms, namely SSCs. These observations help our understanding of the pathophysiology of spermatogenic failure where germ cell development stops during early stages of spermatogenesis in some men with SCO. The azoospermia seen in men with SCO appears to be, at least in part, secondary to a defect in proliferation and differentiation of SSCs. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e795 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Koji Shiraishi More articles by this author Hideyasu Matsuyama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The pioneering of intracytoplasmic sperm injection: historical perspectives.

Intracytoplasmic sperm injection (ICSI) has often been heralded as a ground-breaking technique that has transformed the treatment of couples with infertility. By injecting a single spermatozoon into the cytoplasm of the oocyte, ICSI bypasses the zona pellucida and increases the chances of fertilization and subsequent embryo development, independent of semen parameters. Ever since the first live births using ICSI were reported in 1992, ICSI has become the mainstay of treating male factor infertility as well as overcoming fertilization failure associated with conventional in vitro insemination. Today, ICSI is utilized in nearly 66% of all assisted reproductive treatments worldwide and has resulted in the birth of millions of babies. The primary goal of this review is to provide historical perspectives about the pioneering of ICSI. We begin by highlighting the scientific work of early investigators who elucidated the mechanisms central to mammalian fertilization. Furthermore, we briefly discuss how these findings contributed to the development of IVF for the treatment of infertility. We then emphasize the shortcomings of IVF in treating severe forms of male factor infertility and enumerate the micromanipulation techniques that were developed to circumvent these shortcomings. Finally, we indicate how the inadequacies of these micromanipulation techniques lead to the inception, application and popularity of ICSI.

MP70-11 EVIDENCE OF SPERMATOGONIAL STEM CELL PRESENCE IN MEN WITH SERTOLI CELL-ONLY SYNDROME

INTRODUCTION AND OBJECTIVES: Sertoli cell-only syndrome (SCOS) is one of the most severe forms of male factor infertility and is classically thought to be devoid of germ cells. However, evidence from microsurgical testicular sperm extractions (TESE) on patients with SCOS has revealed the presence of rare focal areas of spermatogenesis, thereby suggesting the presence of spermatogonial stem cells (SSC) in at least some patients with SCOS. We hypothesize that SSCs are present in most patients with SCOS and that it is abnormal differentiation of the SSC into spermatogonia (SPG) A and/or B that leads to SCOS. METHODS: Testicular samples were obtained from 5 men with pure SCOS on biopsy during TESE and from 5 cadaveric men with normal spermatogenesis. Specimens were assessed for mRNA markers of SSC, SPG A, and SPG B by RNA sequencing. Immunofluorescence was then used to visualize SSCs and SPG A in the testicular samples. Finally, flow cytometry using known markers for SSC (SSEA4) and SPG A (GFRa1) was used to provide quantitative evidence of cellular presence in testicular samples. RESULTS: On RNA sequencing, mRNA markers for SSC and SPG A are expressed in similar numbers in men with normal spermatogenesis and in those with SCOS. On the other hand, there was a dramatic overexpression of mRNA markers for differentiated cells, such as spermatocytes, in men with normal spermatogenesis (Table 1). Immunofluorescence showed increased expression of GFRa1 in the testes of men with SCOS when compared to the testes of men with normal spermatogenesis. Flow cytometry analysis of four testicular samples from men with SCOS and four samples from those with normal spermatogenesis showed a similar number of SSEA4+ cells (0.11%), but a nearly four-fold increase in the number of GFRa1+ cells in men with SCOS compared to those with normal spermatogenesis (0.7% versus 0.18%, respectively). CONCLUSIONS: These findings suggest that SSCs are present in patients with SCOS in similar numbers as in those with normal spermatogenesis. However, there is a surprising accumulation of SPG in SCOS patients. Rather than differentiating and proceeding down the spermatogenesis pathway, the SSCs proliferate, but remain undifferentiated. The azoospermia seen in men with SCOS appears to be secondary to a defect in SSC differentiation and entry into meiosis. Source of Funding: The project was supported by The Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust. This project was also supported by grant number T32HS00066 from the Agency for Healthcare Research and Quality. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Agency for Healthcare Research and Quality.

Azoospermia and testicular biopsy before intra-cytoplasmic sperm injection: Does the type of anesthesia make a difference?

Aim:Azoospermia is the most common form of male factor infertility, due to which sperms are retrieved for intra-cytoplasmic sperm injection (ICSI) under general or local anesthesia. The aim of the present study was to compare the efficacy of general and local anesthesia in an attempt to extract a sufficient quality of sperm for men with azoospermia, who intend to undergo ICSI.Materials and Methods:A total number of 50 patients with azoospermia, who were within the age range of 20-40, were randomly scheduled for testicular sperm extraction with either general or local anesthesia before ICSI. The hormonal abnormalities and severe varicocele were evaluated and treated before the testicular sperm extraction. The inclusion criteria obliged the researchers to focus on men with azoospermic, those who were diagnosed by two semen analyses after a 3-day abstinence from coitus according to the modified WHO criteria, and 19< body mass index ≤28. The concentration, motility, and morphology of the spermatozoa of the two groups were also compared.Results:A total number of 76 men, with a mean age of 35.1 ± 6.0, were selected, 26 were excluded from the study, and the remaining participants were randomly divided into two groups such as general anesthesia and lidocaine group. According to the results, the differences between the values of sperm parameters within various hours after the testicle biopsy were not statistically significant. Also, there was no significant difference between the two groups in terms of sperm motility and sperm morphology during various timing after the processing.Conclusion:The present study demonstrated that there was no evidence found for values of sperm parameters between the two groups, undergoing local and general anesthesia, within various hours after the testicular biopsy. Further investigations with more focus on concentration-dependent lidocaine on human sperm parameters need to be carried out.

Mutational screening of NR5A1 gene encoding steroidogenic factor 1 in cryptorchidism and male factor infertility and functional analysis of seven undescribed mutations

To study the role of NR5A1 in cryptorchidism and male factor infertility. Mutations in NR5A1 have been initially associated with primary adrenal insufficiency and 46,XY gonadal dysgenesis and more recently with less severe phenotypes, including preliminary descriptions in severe forms of male factor infertility. Far less clear is the possible involvement of NR5A1 mutations in cryptorchidism. Retrospective cross-sectional cohort study and functional analysis of mutant proteins. University department. Nine hundred fifty-nine subjects, including children with cryptorchidism and adults with different semen phenotypes associated or not associated with a history of cryptorchidism. None. Mutation screening of NR5A1 by sequencing all exons. Functional analysis of mutant proteins by transactivation assays of CYP11A1 and CYP17A1 promoters. We identified seven undescribed and one previously described missense mutation in subjects with severe spermatogenic impairment, without (4/236, 1.7%) and with (3/85, 3.5%) a history of cryptorchidism. Newborns with cryptorchidism carry NR5A1 mutations at low frequency (0.7%), whereas no mutations were found in milder forms of infertility and normozoospermia, irrespective of the presence of cryptorchidism. The mutant proteins showed impaired transactivation of gonadal promoters. A single nucleotide polymorphism (rs1110061; c.437 G→C; p.Gly146Ala) was also associated with more severe forms of spermatogenic impairment with cryptorchidism. This study, combined with what is already known about NR5A1-associated phenotypes, suggests considering mutations in this gene as a novel genetic cause of more severe forms of male factor infertility, especially when associated with a history of cryptorchidism.

Detection and Management of Obstructive Azoospermia

IntroductionObstructive azoospermia represents a treatable form of male factor infertility. With greater demand for assisted reproductive technologies the general urologist may be tasked with initiating the infertility evaluation and providing counsel for treatment options. In this review we discuss appropriate laboratory, radiographic and genetic testing for the patient with obstructive azoospermia. We also outline surgical treatment options. MethodsThe Medline® database was searched for relevant studies of the evaluation and treatment of men with obstructive azoospermia. Key words included obstructive azoospermia, vasovasostomy, vasoepididymostomy, testicular sperm extraction, sperm aspiration and sperm retrieval. ResultsMost published reports were based on small cohorts followed at single institutions. There were sufficient data to characterize the current state of the art and review the standard of practice. ConclusionsThe initial evaluation of azoospermia is primarily based on differentiating obstructive from nonobstructive etiologies with a substantial reliance on history, physical examination and screening laboratory studies. Various treatment options exist for obstructive azoospermia, including reproductive tract reconstruction (vasovasostomy or vasoepididymostomy) or numerous surgical sperm extraction approaches.

Early Versus Late Maturation Arrest: Reproductive Outcomes of Testicular Failure

There is a paucity of data characterizing infertile men with maturation arrest. We hypothesized that men with early stage maturation arrest could be clinically distinguished from men with late maturation arrest and would have worse reproductive outcomes. We retrospectively reviewed the records of all patients with nonobstructive azoospermia and cryptozoospermia who underwent testis mapping and sperm extraction from 2002 to 2009 and for whom histopathological findings were available. Patients had uniform maturation arrest if multiple biopsies revealed maturation arrest at the spermatogonia/spermatocyte (early maturation arrest) or the spermatid (late maturation arrest) stage. Clinical parameters and pregnancy outcomes of in vitro fertilization/intracytoplasmic sperm injection were examined. Statistical analysis consisted of univariate and multivariate analysis. Uniform maturation arrest was identified in 49 of 219 men (22.3%) undergoing testicular sperm extraction. On multivariate analysis men with maturation arrest had significantly larger testes (p=0.01), decreased follicle-stimulating hormone (p=0.05) and more detectable genetic abnormalities (p=0.01) than men with other histopathological conditions. Men with late maturation arrest had decreased follicle-stimulating hormone (p=0.02), increased testosterone (p=0.03) and a higher sperm retrieval rate at testicular sperm extraction (p=0.01) than men with early maturation arrest. Predictors of successful sperm retrieval were larger testes, cryptozoospermia, late maturation arrest and hypospermatogenesis (each p≤0.05). Pregnancy outcomes for men with maturation arrest were not significantly different from those for men with other histopathological conditions. Maturation arrest is a common, diverse histopathological subtype of severe male infertility. Compared to men with late maturation arrest those with early maturation arrest have increased follicle-stimulating hormone, decreased testosterone and a decreased probability of mature spermatozoa. In vitro fertilization/intracytoplasmic sperm injection outcomes were similar when spermatozoa were discovered during testicular sperm extraction.

Varicocele Repair in Patients With Nonobstructive Azoospermia: A Meta-Analysis

Multiple small case series have reported sperm in the ejaculate and spontaneous pregnancies in patients with nonobstructive azoospermia after varicocele repair. We hypothesized that men with favorable testicular histopathology on testis biopsy such as maturation arrest or hypospermatogenesis would have a higher probability of success than those with more ablative pathology, eg Sertoli-cell-only. A review of the literature on varicocele repair in patients with nonobstructive azoospermia was performed and 11 publications from the previous 20 years were evaluated. Histopathological data were presented in 8 publications, and were categorized as Sertoli-cell-only, maturation arrest and hypospermatogenesis. Maturation arrest was further differentiated by 4 publications. Early maturation arrest was defined as maturation ending at the secondary spermatocyte and late maturation arrest was defined as maturation ending at the spermatid without spermatozoa present. Success after repair was defined as having sperm in the ejaculate or spontaneous pregnancy. A total of 233 patients were analyzed. After varicocele repair 91 (39.1%) patients had motile sperm in the ejaculate and 14 spontaneous pregnancies were reported. Success rates in patients with maturation arrest (42.1%) or hypospermatogenesis (54.5%) were significantly higher than in those with Sertoli-cell-only (11.3%, p <0.001 in both groups). Patients with late maturation arrest had a higher probability of success (45.8%) than those with early maturation arrest (0%, p = 0.007). Infertile men with nonobstructive azoospermia can have improvement in semen analysis and achieve spontaneous pregnancy after repair of clinical varicoceles. This meta-analysis demonstrates that men with late maturation arrest and hypospermatogenesis have a higher probability of success and, therefore, histopathology should be considered before varicocele repair in men with nonobstructive azoospermia.