The effect of cult-active medium on pregnancy outcomes after intracytoplasmic sperm injection in azoospermic men: A case-control study

Background: Failed oocyte activation following intracytoplasmic sperm injection (ICSI) as a result of calcium deficiency is a major challenge. Objective: We compared the effect of cult-active medium (CAM) on ICSI outcomes in obstructive azoospermia cases. Materials and Methods: The present study was conducted with 152 ICSI cases, classified into CAM and control groups. The injected oocytes in the control group were cultured in the cleavage medium, while in the artificial oocyte activation group, oocytes were chemically activated through exposure to 200 µL of CAM for 15 min. Fertilization and cleavage rates, quality of embryos, and biochemical pregnancy and live birth rates were assessed in both groups. Results: There were significant differences between the groups in terms of fertilization and cleavage rates after using the CAM in the percutaneous epididymal sperm aspiration (PESA) subgroup (p = 0.05, p ≤ 0.001) and in the testicular sperm extraction subgroup (p = 0.02, p = 0.04), compared to their control groups. Also, the pregnancy rate was significantly higher in the PESA-CAM subgroup (p = 0.03). The PESA-CAM subgroup demonstrated a significant difference in embryo quality after ICSI (p = 0.04). Unsuccessful embryo transfer and abortion were lower in both subgroups compared to the control groups, but this difference was not significant. Surprisingly, live birth rate was higher in the PESA-CAM subgroup (p = 0.03). Conclusion: CAM treatment could improve fertilization and cleavage rates in obstructive azoospermia participants. It had a significant effect on embryo quality, and pregnancy and live birth rates in PESA cases. Key words: Calcium ionophore, Obstructive azoospermia, Fertilization, ICSI.

Combined Use of Whole Exome Sequencing and CRISPR/Cas9 to Study the Etiology of Non-Obstructive Azoospermia: Demonstration of the Dispensable Role of the Testis-Specific Genes C1orf185 and CCT6B

The genetic landscape of male infertility is highly complex. It is estimated that at least 4000 genes are involved in human spermatogenesis, but only few have so far been extensively studied. In this study, we investigated by whole exome sequencing two cases of idiopathic non-obstructive azoospermia (NOA) due to severe hypospermatogenesis. After variant filtering and prioritizing, we retained for each patient a homozygous loss-of-function (LoF) variant in a testis-specific gene, C1orf185 (c.250C>T; p.Gln84Ter) and CCT6B (c.615-2A>G), respectively. Both variants are rare according to the gnomAD database and absent from our local control cohort (n = 445). To verify the implication of these candidate genes in NOA, we used the CRISPR/Cas9 system to invalidate the mouse orthologs 4930522H14Rik and Cct6b and produced two knockout (KO) mouse lines. Sperm and testis parameters of homozygous KO adult male mice were analyzed and compared with those of wild-type animals. We showed that homozygous KO males were fertile and displayed normal sperm parameters and a functional spermatogenesis. Overall, these results demonstrate that not all genes highly and specifically expressed in the testes are essential for spermatogenesis, and in particular, we conclude that bi-allelic variants of C1orf185 and CCT6B are most likely not to be involved in NOA and male fertility.

Lipidomic profile of seminal plasma in non-obstructive azoospermia with sperm maturation arrest

Introduction. The difference between obstructive and non-obstructive azoospermia with sperm maturation arrest is important for the choice of treatment tactics and adequate counseling of a married couple.Purpose of the study. The study aimed to assess the semen lipid profile in patients with sperm maturation arrest. Materials and methods. Samples of seminal plasma for lipid composition of 24 men with normozoospermia and 64 men with azoospermia were studied. Patients with azoospermia underwent microdissection testicular biopsy followed by the detection of testicular tissue pathology. Lipid extracts were analyzed by liquid chromatography with mass spectrometry. Lipid data were compared with the results of pathomorphological studies.Results. Comparison of two groups revealed a statistically significant concentration differences for 22 lipids detected in positive-ion mode and 11 lipids detected in negative-ion mode. Those lipids mainly belong to the classes hexosylceramides, sphingomyelins and phosphatidylcholines — simple ethers and oxidized lipids. In multivariate analysis, the following lipids were found to be statistically significant predictors of sperm maturation arrest: PC 16: 0_22: 6 lipid (β-coefficient: -0.73; 95% confidence interval (95% CI): -1.42 to -0.27; odds ratio (OR): 0.48; OR CI: 0.24 to 0.76; Wald's test: -2.58; p = 0.01), SM d20: 1/22:2 lipid (β-coefficient 4.96; 95% CI 2.29 to 9.13; OR: 142.31; OR CI: 9.90 to 9.22^103; Wald's test: 2.93; p = 0.003); PG 20:3_22: 6 lipid (β-coefficient 2.52; 95% CI 1.13 to 4.49; OR: 12.37; OR CI: 3.10 to 89.27; Wald's test: 3.02; p = 0.002); PC O- 16: 1/16:0 lipid (β-coefficient 1.96; 95% CI -4.12 to 0.27; OR: 0.14; OR CI: 0.02 to 0.76; Wald's test: -2.05; p = 0.04). The prediction model characteristics of sperm maturation arrest, obtained during cross-validation in the positiveion mode composed: sensitivity 91%, specificity 85%; in negative-ion mode: sensitivity 75%; specificity 81%.Conclusions. Even though early stages of spermatogenesis are equally preserved in both fertile men and men with homogeneous sperm maturation arrest, the semen in the studied group of patients differed in its lipid profile. Patients with non-obstructive azoospermia, associated with meiosis arrest, may have unique lipidomic characteristics of seminal plasma, which in the future will make it possible to differentiate various variants of severe male infertility using non-invasive methods.

Update on the Management of Non-Obstructive Azoospermia: Current Evidence and Unmet Needs

Azoospermia, defined as the absence of sperm in the ejaculate after examination of the centrifuged specimens, affects about 1% of the male population and 10–15% of infertile men [...]

Novel Bi-Allelic Variants of FANCM Cause Sertoli Cell-Only Syndrome and Non-Obstructive Azoospermia

Non-obstructive azoospermia (NOA) is the most severe disease in male infertility, but the genetic causes for the majority of NOA remain unknown. FANCM is a member of Fanconi Anemia (FA) core complex, whose defects are associated with cell hypersensitivity to DNA interstrand crosslink (ICL)-inducing agents. It was reported that variants in FANCM (MIM: 609644) might cause azoospermia or oligospermia. However, there is still a lack of evidence to explain the association between different FANCM variants and male infertility phenotypes. Herein, we identified compound heterozygous variants in FANCM in two NOA-affected brothers (c. 1778delG:p. R593Qfs*76 and c. 1663G > T:p. V555F), and a homozygous variant in FANCM (c. 1972C > T:p. R658X) in a sporadic case with NOA, respectively. H&E staining and immunohistochemistry showed Sertoli cell-only Syndrome (SCOS) in the three patients with NOA. Collectively, our study expands the knowledge of variants in FANCM, and provides a new insight to understand the genetic etiology of NOA.

Non-obstructive idiopathic azoospermia vs azoospermia with antecedents of cryptorchidism: ways and probabilities of becoming parents

Background Non-obstructive azoospermia (NOA) with history of cryptorchidism and idiopathic NOA are the most common forms of NOA without genetic aetiology. Of all patients with one of these two types of NOA, only a few will have a positive TEsticular Sperm Extraction (TESE). Of those with positive extraction followed by sperm freezing, not all will have a child after TESE-ICSI. What are the ways and probabilities of taking home a baby for patients with NOA and a history of cryptorchidism compared with patients with idiopathic NOA? Results Patients with idiopathic NOA or NOA and a history of cryptorchidism who underwent their first TESE were included. The patients were divided into two groups: Group 1 was composed of 125 patients with idiopathic NOA and Group 2 of 55 patients with NOA and a history of surgically treated cryptorchidism. Our results showed that more than half of the NOA patients succeeded in becoming parents. The main way to fulfil their plans for parenthood is to use sperm or embryo donation (72%) for men with idiopathic NOA, whereas the majority of men with NOA and a history of cryptorchidism had a child after TESE-ICSI (58.8%). Conclusions In our centre, before considering TESE for a patient with NOA, we explain systematically TESE-ICSI alternatives (sperm donation, embryo donation or adoption). As a result, the couple can consider each solution to become parents.

Analysis of 29 Targeted Genes for Non-obstructive Azoospermia: the Relationship Between Genetic Testing and Testicular Histology

BACKGROUND. Few studies have evaluated the relationship between testicular histology and pathogenic variations of genes regulating spermatogenesis.AIM. To analyze the presence of potentially pathogenic variants of 29 candidate genes known to cause spermatogenic failure (SPGF) in patients with non-obstructive azoospermia (NOA) who underwent testicular histology.PATIENTS AND METHODS. Sixty patients with NOA referred to the Department of Transfusion Medicine and Transplantation Biology, University Hospital Center Zagreb, Croatia, for testicular biopsy were consecutively assessed for eligibility. Twelve patients were excluded from the study because they had Klinefelter syndrome (n=1), Yq microdeletions (n=6), testicular trauma (n=2), or in-situ germ cell neoplasia (n=3). Therefore, 48 patients were considered eligible and included in this study. They were divided into three groups: those who had cryptorchidism (n=9), those with varicocele (n=14), and those with idiopathic NOA (n=25). All included patients underwent blood withdrawal for next-generation sequencing analysis and gene sequencing.RESULTS. We found a possible genetic cause in 4 patients with idiopathic NOA (16%) and in 2 with cryptorchidism (22%). No pathogenic or possibly pathogenic mutations were identified in patients with varicocele. Variants of undetermined significance (VUS) were found in 11 patients with idiopathic NOA (44%), 3 with cryptorchidism (33%), and 8 patients with varicocele (57%). VUSs of the USP9Y gene were the most frequently as they were found in 14 out of 48 patients (29%). In particular, the VUS USP9Y c.7434+14del was found in 11 patients. They showed varied histological pictures, including Sertoli cell-only syndrome, mixed atrophy, and hypospermatogenesis, regardless of cryptorchidism or varicocele. No direct correlation was found between the gene mutation/variant and the testicular histological picture. CONCLUSION. Different mutations of the same gene cause various testicular histological pictures. These results suggest that it is not the gene itself but the type of mutation/variation that determines the testicular histology picture. Based on the data presented above, it remains challenging to design a genetic panel with prognostic value for the outcome of testicular sperm extraction in patients with NOA.

Successful Treatment of Testicular Failure Type IV Without Micro-Testicular Epididymal Sperm Extraction: A Case Report

Objective: Sperm donation and hormonal therapy with micro-Testicular Epididymal Sperm Extraction (TESE) for infertility from testicular failure might not always be available in some contexts. We report a successful embryo transfer from the patient-by ‘cumulative sperm collection’ strategy. Case report: A 42 year-old male presented with non-obstructive azoospermia from testicular failure. Hormonal treatments were given along with the patient-initiated ‘cumulative sperm collection’ strategy, which eventually resulted in 17 sperms retrieved. Twelve mature oocytes were selected for intracytoplasmic sperm injection (ICSI) with the retrieved sperms, of which 8 oocytes were successfully fertilized but only two reached the early blastocyst stage; the first embryo transfer was not successful. Another five eggs were thawed and fertilized with the remaining 5 sperms and 3 oocytes were successfully fertilized: Seven cells were grade 3, 6 cells were grade 3, and 3 cells were grade 3. The second embryo transfer was successful, and the term female infant was successfully delivered by cesarean section. Conclusion: At a center without micro-TESE availability, successful embryo transfer for testicular failure type IV could be achieved by hormonal therapy plus a ‘cumulative sperm collection’ strategy.