Cases Of Male Factor Infertility Research Articles

Evaluation of serum inhibin B and inhibin B/FSH ratio in the diagnosis of non-obstructive azoospermia and oligozoospermia.

Infertility affects approximately 15 % of couples globally, with 50 % cases of male factor infertility. Precise assessment of spermatogenesis is essential for evaluating male infertility. Recent studies suggest serum inhibin B as a promising biomarker for testicular function. This study aims to evaluate the diagnostic utility of serum inhibin B in predicting male infertility, particularly focusing on its relationship with sperm count. A cross-sectional study was conducted on 80 adult men (mean age 31.4±6.89 years) presenting with infertility at gynecology and urology outpatient departments. Semen analysis was performed following WHO (2010) guidelines, and serum inhibin B levels were quantified. The correlation between serum inhibin B levels and sperm parameters was assessed using Pearson's correlation test. Receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic accuracy of serum inhibin B and the inhibin B/FSH ratio for non-obstructive azoospermia (NOA) and oligozoospermia. A significant positive correlation was observed between serum inhibin B and sperm count (r=0.94, p<0.001). ROC analysis demonstrated that the inhibin B/FSH ratio had the highest diagnostic accuracy for NOA and oligozoospermia (AUC=0.986), with sensitivity of 100 % and specificity of 91.67 %. Serum inhibin B alone also showed high diagnostic value (AUC=0.965 for NOA and 0.969 for oligozoospermia). Serum inhibin B is a reliable biomarker for assessing male infertility, particularly in evaluating spermatogenic function. The inhibin B/FSH ratio provides superior diagnostic accuracy for NOA and oligozoospermia, offering valuable clinical utility in male infertility diagnosis.

Development of Recombinant PLC-Zeta Protein as a Therapeutic Intervention for the Clinical Treatment of Oocyte Activation Failure.

The sperm-specific phospholipase C zeta (PLCζ) protein is widely considered as the predominant physiological stimulus for initiating the Ca2+ release responsible for oocyte activation during mammalian fertilization. The increasing number of genetic and clinical reports that directly link PLCζ defects and/or deficiencies with oocyte activation failure (OAF) necessitates the use of a powerful therapeutic intervention to overcome such cases of male factor infertility. Currently, in vitro fertilization (IVF) clinics treat OAF cases after intracytoplasmic sperm injection (ICSI) with Ca2+ ionophores. Despite their successful use, such chemical agents are unable to trigger the physiological pattern of Ca2+ oscillations. Moreover, the safety of these ionophores is not yet fully established. We have previously demonstrated that recombinant PLCζ protein can be successfully used to rescue failed oocyte activation, resulting in efficient blastocyst formation. Herein, we produced a maltose binding protein (MBP)-tagged recombinant human PLCζ protein capable of inducing Ca2+ oscillations in mouse oocytes similar to those observed at fertilization. Circular dichroism (CD) experiments revealed a stable, well-folded protein with a high helical content. Moreover, the recombinant protein could retain its enzymatic properties for at least up to 90 days after storage at -80 °C. Finally, a chick embryo model was employed and revealed that exposure of fertilized chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control, giving a first indication of its safety. Our data support the potential use of the MBP-PLCζ recombinant protein as an effective therapeutic tool but further studies are required prior to its use in a clinical setting.

Pregnancy Rate In Non-Azoospermia With Normal Or Suboptimal Semen Parameter Versus Azoospermic Male Treated By IVF-ICSI Cycle

Background: Intracytoplasmic sperm injection (ICSI) opens the gate for many cases of male factor infertility to be the biological fathers of their sibling since 1992. Most of cases were non-obstructive azoospermia and different levels of oligoastheno-teratozoospermia. Nowadays many cases of reduced semen parameter or female factor infertility are treated by IVF-ICSI Cycle for better pregnancy rate, biochemical and clinical, and live birth rate. Aim of the study: The study aims to compare biochemical, clinical pregnancy rate and outcome of pregnancies in the group with ejaculated sperm with normal or suboptimal semen parameter and group with non-obstructive azoospermia in whom sperm retrieved by TESE, using ICSI. Method: A retrospective cohort study was conducted between January, 2016 and February, 2023 in the fertility center of Al-Sader Medical City, a total of 372 couple, 90 of the males gave semen sample by masturbation and 282 of the males were non-obstructive azoospermia and their sperms were retrieved by TESE; all are treated by ICSI, all of their female partner were under age of 37 year, the maternal medical condition and obstetric history were not included in this study. Simple random sampling was depended, SPSS version 26 was used to perform the statistical analysis processes. Results: There was a highly significant difference in pregnancy rate by β.HCG between ejaculate group (43.3 %) and azoospermia (26.6 %) with p. value =0.003. A significant difference in clinical pregnancy rate by ultrasound between the ejaculate group (31.1%) and azoospermia (20.9%) with p. value =0.047. There was no statistically significant difference in live birth rate between ejaculate group as (24.4 %) and azoospermia as (17.4 %) with p.value=0.137. Conclusion: Freshly ejaculated sperm with normal or suboptimal semen parameter gave a better biochemical and clinical pregnancy rate than obtained from NOA by TESE, while live birth rate was not largely different in both groups

O-298 Poor semen parameters alter embryo morphokinetic patterns during preimplantation development but do not compromise clinical outcomes

Abstract Study question Does semen quality affect preimplantation development and clinical outcomes in ICSI cycles using donor oocytes? Summary answer Poor semen parameters led to altered morphokinetic parameters during preimplantation development but did not compromise blastocyst quality nor clinical outcomes in oocyte donation cycles. What is known already The quality of semen used for in vitro fertilization (IVF) may compromise the kinetics and morphology of developing embryos, ultimately impairing implantation and reducing live birth rates. Such effects are particularly pronounced when testicular or epididymal spermatozoa are used for IVF treatment. However, current studies assessing the impact of poor semen parameters on embryo development and clinical outcomes are often confounded by the use of oocytes from infertile patients. Performing such evaluations in oocyte donation cycles will provide more definitive insights into the role of semen during preimplantation development. Study design, size, duration This retrospective study included 616 embryos from 99 oocyte donation ICSI cycles, performed between February 2018 and July 2019. We compared embryo morphokinetics and clinical outcomes (cumulative pregnancy and live birth rates) across three groups with variable semen parameters. These included donor semen (control group, DD, n = 294), partner sperm obtained by testicular sperm extraction (TESE group, n = 72) and partner semen with altered parameters (including poor concentration, motility and morphology, ALT group, n = 250). Participants/materials, setting, methods We assessed several morphokinetic parameters, including second polar body extrusion (tPB2), pronuclei appearance (PN1a, PN2a), 2-3-4-5-8-cell stages (t2 to t8), start of blastulation (tSB) and blastocyst formation (tB). Kaplan-Meier survival curves were built for each parameter per study group. Survival curves were analysed by Cox regression. Qualitative parameters (even size PN and blastomeres, percent fragmentation, abnormal cleavages, blastocyst grade) were assessed using a Chi-Squared test, while Fisher’s exact test was used to evaluate clinical outcomes. Main results and the role of chance Interestingly, TESE embryos reached several developmental stages faster than the DD group: tPB2 (3.03 vs. 3.08, p &amp;lt; 0.001), PN1a (5.55 vs. 6.19, p &amp;lt; 0.001), PN2a (6.78 vs. 7.36, p &amp;lt; 0.001), tPNf (21.30 vs. 22.20, p = 0.001), t3 (34.02 vs. 35.79, p = 0.016), t4 (35.90 vs. 36.92, p = 0.015), t5 (46.70 vs. 48.07, p = 0.036), t8 (52.48 vs. 55.28, p = 0.049), tSB (85.53 vs. 94.62, p &amp;lt; 0.001) and tB (104.09 vs. 106.60, p = 0.002). ALT embryos were faster in early stages: PN1a (6.63 vs. 6.19, p = 0.005), PN2a (7.84 vs. 7.36, p &amp;lt; 0.001), t5 (48.12 vs. 48.07, p = 0.028), t8 (55.57 vs. 55.28, p = 0.032), but slower in the late stages: tSB (93.71 vs. 94.62, p = 0.001), tB (105.60 vs. 106.60, p = 0.002). Compared to DD, ALT and TESE embryos showed a lower rate of even pronuclei (ALT p = 4.6x10-06; TESE p = 8x10-05) and even blastomeres at the 2-cell (ALT p &amp;lt; 0.001; TESE p = 0.006) and 4-cell stage (ALT p = 0.004; TESE p = 0.005). Moreover, embryos derived from testicular sperm showed significantly higher fragmentation rates at the 8-cell stage (p = 0.005), while no significant differences in the frequency of irregular divisions nor in the number of top-quality blastocysts were detected in ALT and TESE. Importantly, cumulative live birth rates per cycle were similar across all study groups (DD 31/53, ALT 31/51, TESE 8/11; p &amp;gt; 0.05). Limitations, reasons for caution The main limitation resides in the retrospective nature of this study and the limited number of embryos in the TESE group. We also note variability in the testicular biopsy (obstructive and non-obstructive azoospermia) and non-normozoospermic diagnoses (oligozoospermia, teratozoospermia or asthenozoospermia), which may confound the analysis. Wider implications of the findings Although poor semen parameters significantly altered embryo morphokinetics, they did not compromise embryo quality, pregnancy or cumulative live birth rates, resulting in similar clinical outcomes as with double donation. Overall, our findings support the use of autologous sperm with donor oocytes, even in severe cases of male factor infertility. Trial registration number not applicable

P-782 The influence of microfluidic preparation of spermatozoa for ICSI (intracytoplasmic sperm injection) in an unselected IVF population on fertilization rate, embryo-quality and pregnancy rate

Abstract Study question Does the microfluidic technique as a new preparation option for sperm in ICSI during IVF have a better outcome in fertilization, blastocysts and pregnancy rate? Summary answer The patients that had the sperm preparation for ICSI with the microfluidic sperm selection had similar results as with the gradient centrifugation selection. What is known already Semen analysis is a poor predictor of the fertilization potential of spermatozoa and a male factor may contribute to poor outcomes of IVF procedure despite a normal semen analysis. Traditionally, sperm processing is accomplished by density gradient centrifugation or swim-up methods but this have been shown in some studies to increase reactive oxygen species and induce sperm DNA fragmentation. The microfluidic sperm selection (ZyMot- ICSI) is based on the selection of the spermatozoa with the lowest DNA fragmentation rate but studies do not prove better clinical outcomes after this method. Study design, size, duration This is a prospective randomized study carried out in a university level fertility clinic in Oradea, Romania from January 2022 to December 2022, including 239 patients in total. The patients that underwent IVF-ICSI procedures for various indications were randomized in two groups: the control group swim-up technique was used for sperm selection for ICSI during IVF and the study group with the ZyMot-ICSI (microfluidic sperm selection technique) was used. Participants/materials, setting, methods We conducted a prospective trial comparing 119 couples that were allocated to the classic swim-up technique (control group) and 120 couples that were allocated with the microfluidic technique was used (study group) in our university level clinic to go through IVF. Inclusion criteria were couples with known infertility requiring IVF and willing to participate in the study. Exclusion criteria were: severe oligoasthenospermia (concentration &amp;lt; 5x10^6 spermatozoa/ml or motility &amp;lt;10%), oocyte or sperm donation. Main results and the role of chance The statistical analysis showed that there is no significant difference between the fertilization rate (study vs control p = 0.37 with absolute difference -1.182, 95% CI (-7.2, 5.4)) or blastocyst rate (study vs control p = 0.88 with absolute difference -0.5 95% (-6.5, 6)) nor is there a difference in clinical pregnancy. Even if microfluidic preparation of spermatozoa did not seem to improve the results it may be utilized more broadly for standard IVF, intrauterine insemination and could also improve workflow, decrease intervention by laboratory personal and provide more consistent incubation conditions. Limitations, reasons for caution The population studied was inclusive with unselected IVF indications and did not attempt to isolate male factor infertility cases or patients with a history of elevated sperm DNA fragmentation. Wider implications of the findings Microfluidic sperm preparation performs similarly to density gradient centrifugation in sperm preparation for IVF in an unselected population. Trial registration number Not applicable

P-024 Identification of spermatogenic infertility phenotypes using next generation sequencing

Abstract Study question Can next generation sequencing (NGS) contribute to diagnoses male idiopathic infertility? Summary answer A male factor gene panel identifies pathogenic variants associated to spermatogenic failure in oligozoospermia and cryptozoospermia patients. What is known already In 50% of cases, infertility is due to a male factor problem. Although the causes of male infertility are heterogeneous, genetic causes account for approximately 30% of cases. Some phenotypes have been associated with specific genetic disorders such as chromosomal abnormalities and chromosome Y microdeletions. However, current genetic studies explain only 4% of cases, whilst most cases of male factor infertility remain without a clear diagnosis. Therefore, new techniques that explain the cause of male infertility are needed. Advances in NGS allowed us to study a large number of genes involved in spermatogenesis process in patients with idiopathic infertility. Study design, size, duration A retrospective study was performed from July 2020 until May 2021. A total of 30 patients with abnormal seminal count parameters (oligozoospermic and cryptozoospermic) were included in the male factor gene panel study. Patients carrying Y-chromosome microdeletions or abnormal karyotype were excluded. The control group included 20 normozoospermic healthy donors selected on the basis of normal semen parameters according to the WHO criteria (2010). Participants/materials, setting, methods Genomic DNA extraction from blood-EDTA of the patients was performed using the commercial MagMax DNA MultiSample Ultra kit and the King-Fisher automated extractor (ThermoFisher®). Next Generation Sequencing (NGS) was done using a panel with 426 genes involved in the spermatogenesis process. Panel sequencing for identification of genetic variants was performed using Nextera Enrichment technology (Illumina®). FASTAQ data were processed using BWA and GATK algorithms. VCF files were analyzed using Variant Interpreter software. Main results and the role of chance After data analysis, we observed that eight of the thirty patients studied were carriers of mutations in least one of the genes included in the panel (8/30, 26.7%). We identified the following pathogenic variants: a missense mutation (Phe1052Val) and a deletion (Phe508del) of CFTR gene (2/30, 6.6%), two frameshifts (Asp128GlufsTer34 and Lys1299Ter) of CEP290 (2/30, 6.6%), a missense mutation (Tyr284Cys) of GNRHR gene (1/30, 3.3%), a missense mutation (Tyr416Cys) of SCN5A gene (1/30, 3.3%), a deletion (Ser83del) of NANOS1 gene (1/30, 3.3%), a stop gained in splice region Arg341Ter of TEX14 gene (1/30, 3.3%), a splicing donor c.362 + 2T&amp;gt;C of ESR2 gene (1/30, 3.3%) and a missense mutation (Ser321Leu) of DNAH5 gene (1/30, 3.3%), which are related to spermatogenesis failure. Additionally, some variants classified as benign have been identified, which are not associated with pathogenicity. All the variants identified are related with male infertility, affecting spermatogenesis process, such as congenital bilateral absence of the vas deferens (CFTR), reproductive system syndrome (CEP190), endocrine disorder (GNRHR, hypogonadotropic hypogonadism), testis expressed (SCN5A), spermatogenic failure (NANOS1, TEX14 and ESR2) and syndromic infertility (DNAH5). Nevertheless, no pathogenic mutations associated to spermatogenic failure were observed in the control group. Limitations, reasons for caution The main limitation of this study is the small number of patients included. Further studies including a higher number of males with idiopathic infertility are warranted to confidently link the genetic variants included in our gene panel to spermatogenic failure. Wider implications of the findings The gene list included in our panel represents a step-forward in the diagnosis screening of males with altered sperm parameters. Our results may add in the knowledge of male factor infertility in order to provide etiologic factors towards a personalized treatment and adequate genetic counselling. Trial registration number Not applicable

O-046 Reproductive prospects for ICSI children

Abstract Intracytoplasmic sperm injection (ICSI), developed 30 years ago in our Center, allowed men with low to extremely low sperm quality to become the genetic father of their child. Overall, ICSI is now applied in up to 70% of all ART cycles and has resulted in the birth of millions of children worldwide. ICSI circumvents natural sperm selection mechanisms and concerns related to transgenerational inheritance of male infertility following ICSI could not be fully answered till today, given the young age of the offspring born after ICSI. Since genetic abnormalities account for one third of all cases of male factor infertility, it is essential to identify these genetic abnormalities because of the potential risk of their transmission to the offspring through the use of ART. As ICSI is nowadays performed even in couples with normal semen parameters, the disentanglement of procedure-related and parental background-related factors on the health of the offspring is important. Due to the safety concerns regarding the health of children born after ART, a longitudinal follow-up program was set up in our Center in the 80’s, resulting in data of more than 50 000 pregnancies. To date, more than 25 000 children are conceived after ICSI in our Center. We have not only the worldwide eldest cohort of ICSI offspring but also a uniform cohort of children and young adults conceived by ICSI because of (severe) male factor infertility. We present the results of these prospective studies with focus on the reproductive health in ICSI children between 8 and 22 years and compare these with available data from others. First of all, we will show data on the gonadal function assessed both clinically and by means of biomarkers at pre-pubertal and pubertal ages in ICSI boys. Secondly, we will present findings on the reproductive status of young adult men and women conceived after ICSI between 1992 and 1996: results of hormonal work-up, physical examination and semen parameters for ICSI men and results of hormone levels and antral follicle counts for ICSI women. Finally, we will discuss the findings in these cohorts of children and young adults conceived because of male infertility in view of the available literature regarding reproductive function in offspring conceived following ICSI.

O-311 Evaluating the Reproductive Potential of Azoospermic Men by Profiling the Genome of Surgically Retrieved Spermatozoa

Abstract Study question Can whole exome sequencing of surgically retrieved spermatozoa from azoospermic men pinpoint mutations related to the etiology of their infertility and ability to support pregnancy? Summary answer Identifying key germline mutations involved in spermatogenesis helps explain reproductive failure, regardless of the etiology of the azoospermia. What is known already Azoospermia accounts for approximately 15% of male factor infertility cases. Although it can be caused by pre-testicular factors, the most recognized forms are testicular and post-testicular. Post-testicular azoospermia is mainly attributed to a mechanical obstruction, whereas testicular azoospermia, the most challenging form, is characterized by scattered functional germinal epithelia that may fail to support the meiotic process during sperm development. To elucidate the etiology of this condition, genetic studies on somatic cells have been carried out. Here, we perform whole exome sequencing (WES) on surgically retrieved spermatozoa to preferentially detect germline mutations that may be passed on to offspring. Study design, size, duration Over 3 years, we recruited patients undergoing epididymal sperm aspiration for acquired obstructive azoospermia (OA; n = 19) or testicular biopsy for nonobstructive azoospermia (NOA; n = 11). Eight men were included as fertile controls. Copy number variants (CNVs) and gene mutation profiles were obtained through WES and compared between the OA and NOA cohorts, followed by sub-analyses within those two categories according to whether they generated a clinical pregnancy (fertile) or not (infertile), while controlling for maternal age. Participants/materials, setting, methods Spermatozoal DNA was extracted and amplified from the surgically retrieved specimens (concentration, 742±520 ng/ul; quality, 1.7±0.1 nm). CNVs and gene mutations were detected using CLC Genomic Server 9.0. Genes were considered duplicated or deleted when the read depth was &amp;gt;1.5 or &amp;lt; 0.5 times the median read depth in the control. Common mutations were compared between the OA and NOA cohorts, as well as according to the couples’ clinical outcomes. Female partners had negative infertility workups. Main results and the role of chance Of 30 men (paternal age, 42.3±7yrs), 19 OA men underwent epididymal sperm retrievals (concentration, 1.1±4x106/ml, 9±12% motility), while 11 NOA men underwent testicular biopsies (concentration, 0.03±0.4x106/ml, 0.5±1% motility). WES did not indicate a significant difference in sperm aneuploidy between the two etiologies (OA, 1.7%; NOA, 1.8%) compared to the control (1.1%). In OA patients, only 3 housekeeping-related genes were deleted, while in the NOA cohort, 5 genes involved in RNA transcription (POLR2L), apoptosis (AP5M1), and basic spermiogenic functions (AP1S2, AP1G2, APOE) were deleted. OA patients and their partners (maternal age, 36.8±4yrs) underwent 19 ICSI cycles resulting in a delivery rate of 47.4% (9/19). Those able to reproduce (n = 9) shared a mutation in ZNF749, specifically affecting sperm production. The infertile men (n = 10) all shared a PRB1 deletion, controlling essential DNA replication. NOA men and their partners (maternal age, 38.2±2yrs) underwent 11 ICSI cycles, yielding a delivery rate of 72.7% (8/11). The fertile men (n = 8) all shared a MPIG6B deletion, involved in stem cell lineage differentiation. All of their infertile counterparts (n = 3) presented gene deletions not only involved in spermato/spermio-genesis (n = 4) but, most importantly, also superimposed with those encoding early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). Limitations, reasons for caution Although maternal age was controlled for, confounding factors related to the female partner cannot entirely be excluded. While men unable to reproduce shared common gene mutations providing information about their condition, these findings still need to be confirmed in larger observations. Wider implications of the findings By performing WES, we were able to identify specific mutations associated with compromised embryo developmental competence of surgically retrieved spermatozoa. DNA sequencing technologies help identify gametes capable of sustaining a pregnancy even in the most severe form of male infertility, laying the groundwork for precision medicine in this field. Trial registration number N/A

Microfluidic preparation of spermatozoa for ICSI produces similar embryo quality to density-gradient centrifugation: a pragmatic, randomized controlled trial.

Does processing of spermatozoa for IVF with ICSI by a microfluidic sperm separation device improve embryo quality compared with density-gradient centrifugation? Patients randomized to microfluidic sperm preparation had similar cleavage- and blastocyst-stage embryo quality and clinical and ongoing pregnancy rates to those who underwent standard sperm processing for IVF with ICSI. Microfluidic sperm preparation can isolate spermatozoa for clinical use with minimal DNA fragmentation but with unclear impact on clinical outcomes. A prospective randomized controlled trial of 386 patients planning IVF from June 2017 through September 2021 was carried out. One hundred and ninety-two patients were allocated to sperm processing with a microfluidic sperm separation device for ICSI, while 194 patients were allocated to clinical standard density-gradient centrifugation (control) at an academic medical centre. In an intention to treat analysis, there were no differences in high-quality cleavage-stage embryo fraction [66.0 (25.8)% control versus 68.0 (30.3) microfluidic sperm preparation, P = 0.541, absolute difference -2.0, 95% CI (-8.5, 4.5)], or high-quality blastocyst fraction [37.4 (25.4) control versus 37.4 (26.2) microfluidic sperm preparation, P = 0.985, absolute difference -0.6 95% CI (-6, 5.9)] between groups. There were no differences in the clinical pregnancy or ongoing pregnancy rates between groups. The population studied was inclusive and did not attempt to isolate male factor infertility cases or patients with a history of elevated sperm DNA fragmentation. Microfluidic sperm separation performs similarly to density-gradient centrifugation in sperm preparation for IVF in an unselected population. No external funding to declare. M.P.R. is a member of the Clinical Advisory Board for ZyMōt® Fertility, Inc. NCT03085433. 21 March 2017. 16 June 2017.

O-119 Evaluating the reproductive potential of azoospermic men by germline mutation profiling

Abstract Study question Can whole exome sequencing (WES) of spermatozoa from azoospermic men identify mutations related to the etiology of their infertility and ability to support a pregnancy? Summary answer Key de novo germline mutations that affect sperm production and/or embryo developmental competence may explain reproductive failure in azoospermic men, regardless of the etiology. What is known already Azoospermia accounts for approximately 15% of male factor infertility cases. Although it can be caused by pre-testicular factors, the most recognized forms are testicular and post-testicular. While post-testicular azoospermia is mainly due to a mechanical obstruction, testicular azoospermia, the most severe form, is characterized by scattered functional germinal epithelia that strive to support the meiotic process during gamete development. To shed light on the etiology of this condition, genetic studies have been performed, albeit exclusively on peripheral blood. We chose to perform a genomic assessment of spermatozoa to preferentially detect germline mutations that may be passed to the progeny. Study design, size, duration In a 2-year period, we recruited infertile men undergoing epididymal aspiration for acquired obstructive azoospermia (OA; n = 19) or testicular retrieval for nonobstructive azoospermia (NOA; n = 10). Four additional men were included as fertile controls. Following WES, copy number variants (CNVs) and gene mutation profiles were compared between the OA and NOA patients, and within those two categories, in relation to whether they generated a clinical pregnancy (fertile) or not (infertile). Participants/materials, setting, methods Spermatozoal DNA was extracted and amplified from the surgically retrieved specimens of consenting men (DNA concentration, 762±492 ng/ul; quality, 1.7±0.1 nm). CNVs, gene mutations, duplications, and deletions were detected using the CLC Genomic Server 9.0. Genes were considered duplicated or deleted when the read depth was &amp;gt;1.5 or &amp;lt; 0.5 times the median read depth in the control. Common mutations in the OA and NOA cohorts were assessed according to the couples’ clinical outcome. Main results and the role of chance Of 29 couples (maternal age, 41.9±7yrs; paternal age, 42.5±7yrs), 19 OA men underwent epididymal sperm retrieval (1.1±4x106/ml concentration, 9±12% motility) while 10 NOA men underwent testicular biopsy (0.03±0.2x106/ml concentration, 0.5±1% motility). WES did not reveal a significant difference in sperm aneuploidy between the two etiologies (OA, 1.8%; NOA, 1.9%). In OA patients, only 3 genes were deleted, mainly housekeeping-related, while in the NOA cohort, 5 genes were deleted, involved in RNA transcription (POLR2L) and apoptosis (AP5M1), in addition to spermiogenic functions (AP1S2, AP1G2, APOE). OA patients and their partners (maternal age, 36.8±4yrs) underwent 19 ICSI cycles that resulted in a pregnancy and delivery rate of 47.4% (9/19). Those able to reproduce (n = 9) shared a mutation in ZNF749, a gene affecting only sperm production. The infertile individuals (n = 10) all had a deletion on PRB1, controlling essential DNA replication. NOA men and their partners (maternal age, 38.2±2yrs) underwent 10 ICSI cycles, yielding a clinical pregnancy rate of 70% (7/10). The fertile men (n = 7) had a concurrent gene deletion involved in stem cell lineage differentiation (MPIG6B). Their infertile counterparts (n = 3) had deleted genes involved in spermato/spermio-genesis (n = 6) and, most importantly, in early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC9, REPIN1, MAPRE3, and ARL4C). Limitations, reasons for caution This is a novel study with limited observations. The presence of housekeeping-related mutations in fertile OA men as well as the DNA replication mutation in infertile OA patients, considering the acquired condition, remains puzzling. Although maternal age was controlled for, confounding factors related to the female partner cannot be excluded. Wider implications of the findings Screening men for germline mutations provides valuable information on their ability to reproduce, regardless of the etiology of azoospermia. Genome profiling was able to identify reasons for failed reproductive performance in azoospermic men, particularly those individuals with secretory azoospermia (NOA). Genomic profiling may identify gametes with retained embryo developmental competence. Trial registration number n/a

Telomere Distribution in Human Sperm Heads and Its Relation to Sperm Nuclear Morphology: A New Marker for Male Factor Infertility?

Infertility is a problem affecting an increasing number of couples worldwide. Currently, marker tests for male factor infertility are complex, highly technical and relatively subjective. Up to 40% of cases of male factor infertility are currently diagnosed as idiopathic therefore, there is a clear need for further research into better ways of diagnosing it. Changes in sperm telomere length have been associated with infertility and closely linked to DNA damage and fragmentation, which are also known to be related to infertility. However, telomere distribution is a parameter thus far underexplored as an infertility marker. Here, we assessed morphological parameters of sperm nuclei in fertile control and male factor infertile cohorts. In addition, we used 2D and 3D fluorescence in situ hybridization (FISH) to compare telomere distribution between these two groups. Our findings indicate that the infertile cohort sperm nuclei were, on average, 2.9% larger in area and showed subtle differences in sperm head height and width. Telomeres were mainly distributed towards the periphery of the nuclei in the control cohort, with diminishing telomere signals towards the center of the nuclei. Sperm nuclei of infertile males, however, had more telomere signals towards the center of the nuclei, a finding supported by 3D imaging. We conclude that, with further development, both morphology and telomere distribution may prove useful investigative tools in the fertility clinic.

Efficacy of L-Carnitine Therapy in Selected Cases of Male Factor Infertility

Efficacy of L-Carnitine Therapy in Selected Cases of Male Factor Infertility

Shedding light into the relevance of telomeres in human reproduction and male factor infertility†.

Sperm telomere length (STL) is a promising new parameter for sperm quality analysis that may elucidate the molecular mechanisms underlying the idiopathic cases of male factor infertility, which represent almost half of all the male factor infertility cases worldwide. Telomeres consist of nucleoprotein structures present at the ends of eukaryotic chromosomes, whose protective functions maintain the genomic stability. Their role in reproduction includes an active intervention during gametogenesis, fertilization, and preimplantation embryo development. In consonance, studies have shown that compromised telomere homeostasis is associated with male infertility. Since critically short telomeres have their function affected, assessing STL may be a fast and economic method for sperm quality analysis and expectantly contribute to improve the success of fertility treatments. This hypothesis is supported by several reports associating STL with seminal parameters, sperm genome integrity, and clinical outcomes. However, there are other studies in the literature that do not demonstrate these associations. Additionally, it is still not clear whether the lengthening mechanisms of telomeres occurring during early embryo development resume the inherited telomere length. Further research is essential to clarify the suitability of STL as a biomarker for male infertility, before it could be routinely implemented in medically assisted reproduction centers. Understanding the molecular mechanisms underlying STL function and dynamics will provide us new insights into the origins of male infertility and a possible new useful tool as an outcome predictor for assisted reproduction.

Female obesity is associated with complete failure to fertilize with conventional insemination

In a woman’s first ART procedure our center restricts intracytoplasmic sperm injection (ICSI) to cases of male factor infertility. In cases of unexplained infertility, we use “partial ICSI” where some oocytes are inseminated conventionally, and some are inseminated via ICSI. Our objective was to better define factors outside of male factor infertility in which ICSI is indicated.

Identification of IL-7R as a candidate proteomic marker for asthenozoospermia by high throughput screening (HTS) of human spermatozoa

Despite advances in the field of human reproduction, the underlying etiology of most male factor infertility cases is unknown. Some de novo maturation, genetic abnormalities, and cell intrinsic factors have recently been suggested to impair sperm function. In the current work we hypothesized that dysfunction related to interactions between spermatozoa and its surrounding microenvironment might play a role in some cases of male factor infertility. The aim was to identifying novel cell surface receptors on mature human spermatozoa and compare their expression in sperm from patients with asthenozoospermia with normal controls.

Genetic susceptibility to male infertility: news from genome‐wide association studies

A thorough understanding of the genetic basis of male infertility has eluded researchers in spite of significant efforts to identify novel genetic causes of the disease, particularly over the past decade. Approximately half of male factor infertility cases have no known cause; however, it is likely that the majority of idiopathic male factor infertility cases have some unidentified genetic basis. Well-established genetic causes of male infertility are limited to Y chromosome microdeletions and Klinefelter's syndrome, together accounting for 10-20% of cases of severe spermatogenic failure. In addition to these, several genetic polymorphisms have been demonstrated to be significantly associated with male infertility. The discovery of new genetic associations with male infertility has been hampered by two primary factors. First, most studies are underpowered because of insufficient sample size and ethnic and phenotypic heterogeneity. Second, most studies evaluate a single gene, an approach that is very inefficient in the context of male infertility, considering that many hundreds of genes are involved in the process of testicular development and spermatogenesis. Significant recent advances in microarray and next-generation sequencing technologies have enabled the application of whole-genome approaches to the study of male infertility. We recently performed a pilot genome-wide association study (GWAS) for severe spermatogenic failure, and several additional male infertility GWAS have since been published. More recently, genomic microarray tools have been applied to the association of copy number variants with male infertility. These studies are beginning to shed additional light on the genetic architecture of male infertility, and whole-genome studies have proven effective in identifying novel genetic causes of the disease. This review will discuss some of the recent findings of these whole-genome studies as well as future directions for this research that will likely be the most productive moving forward.

Sperm retrieval rates and ICSI outcomes for men with nonobstructive azoospermia and the health of resulting offspring.

report on the results of a reasonably large (365) retrospective series of patients with nonobstructive azoospermia (NOA) who had sperm collected by microscopically assisted microdissection (microTESE) for intracytoplasmic sperm injection (ICSI). The results from NOA patients are compared with previously published data from a group of men with obstructive azoospermia collected during the same time frame. In addition, as is standard of care, the couples were offered ICSI with donor sperm if the microTESE procedure failed to yield sperm, providing a group from the same cohort who had ejaculated sperm used for ICSI. NOA describes the condition in which there are no sperm in the semen due to lack of sperm production by the testes, rather than due to posttesticular obstruction of the excurrent ducts. NOA accounts for about 7% of male factor infertility cases and includes patients with Y‑chromosome deletions, Klinefelter syndrome and prior cancer treatment. Until the advent of surgical collection of testicular sperm and ICSI, men with NOA were unable to father biological offspring. Now, using TESE, sperm can be collected for injection into oocytes. 2

Focus on intracytoplasmic morphologically selected sperm injection (IMSI): a mini-review

Intracytoplasmic sperm injection (ICSI) is the recommended treatment in many cases of male-factor infertility. Several studies have demonstrated a positive correlation between optimal sperm morphology and positive ICSI outcomes. In fact, spermatozoa with severe abnormalities of the head are well documented to be associated with low fertilisation, implantation and pregnancy rates. However, a spermatozoon which is classified as 'normal' by microscopic observation at low magnification could contain ultrastructural defects that impair both the fertilisation process and embryonic development. The intracytoplasmic morphologically selected sperm injection (IMSI) procedure changed the perception of how a spermatozoon suitable for injection should appear. Sperm selection is carried out at ×6000 magnification, allowing improved assessment of the sperm nucleus. Currently, standardized clinical indications for IMSI are lacking and the candidates are selected on the grounds of their medical history or of a careful analysis of the sperm suspension. Further prospective randomized studies are needed to confirm the advantages of IMSI in specific groups of patients. In addition to providing a brief overview of the IMSI procedure, this study aims to review the literature, which explains the theoretical basis and the clinical outcomes of this technique. Several reports show that IMSI is associated with improved implantation and clinical pregnancy rates as well as lower abortion rates when compared to ICSI. Although a possible correlation between the sperm's abnormal nucleus shape, increased DNA fragmentation and negative laboratory and clinical outcomes has been long investigated, the results are conflicting.

Comprehensive Genomic Study in Patients with Idiopathic Azoospermia and Oligoasthenoteratozoospermia

ABSTRACTInfertility affects 10% to 15% of all couples. In about 40% of the cases the male factor is reported to be the main reason for unsuccessful fertilization. Although in 70% of the male factor infertility cases the etiology is recognizable, in the remaining 30% the reason is unknown and the male infertility is named idiopathic. In the current study, we selected 10 patients with idiopathic infertility, affected by azoospermia and oligoasthenoteratozoospermia. All patients were subjected to DNA analysis for deletions of the Y chromosome and cytogenetic analysis for chromosomal aberrations. After these analyses, the patients without such kind of abnormalities were further analyzed by microarray-based comparative genomic hybridization (array CGH). The cytogenetic analysis revealed two patients with chromosomal mutations—inv (9)(p11;q13) and t(Y;9)(q12.3;q21.1). Two other patients were detected to have deletions of the AZFc region of the Y chromosome. Thus, we applied array CGH analysis for the six patients without chromosomal or Y-chromosome aberrations. Apart from the presence of known polymorphisms, we established copy number alteration in 17q12-17q21.2—a region containing the gene for zona pellucida binding protein ZPBP2. This protein plays a crucial role for the proper spermatogenesis. Our results prompted for investigation of this gene and protein as a potential candidate for spermatogenic failure.

Celebrating ICSI's twentieth anniversary and the birth of more than 2.5 million children--the 'how, why, when and where'

The first major breakthrough for the alleviation of tubal infertility occurred in the last quarter of the twentieth century. Louise Brown was born on the 25 July, 1978 inOldham, England as a result of the pioneering work of Bob Edwards and the late Patrick Steptoe with the help of the late Jean Purdy. Bob Edwards received the ‘Nobel Prize for Physiology or Medicine’ for this work in 2010. In the early eighties IVF practice was gradually introduced in many countries and became a successful treatment for the alleviation of longstanding female-factor and idiopathic infertility. However, the hypothesis that bringing the male gametes closer to the oocytes would be an effective treatment in couples with reduced numbers of spermatozoa was not supported. As soon as sperm parameters were below a certain threshold, fertilization of oocytes was much reduced or failed completely. It became obvious that conventional IVF could not solve many cases of male-factor infertility. For couples with a very low sperm count or azoospermia it was necessary to resort to the use of artificial insemination with donor sperm (AID). As a consequence, several groups embarked on research aimed at assisting the fertilization process. Micromanipulation procedures were introduced. The two initial assisted fertilization procedures: zona drilling and partial zona dissectionwere not successful. Zona drilling involvedmaking a hole in the zona pellucida of an oocyte, whichwas then incubated in a sperm suspension. Zona drilling worked well in mice but not in humans. Partial zona dissection was introduced afterwards. A mechanical slit was made in the oocytes, which were then incubated in a sperm suspension. Fertilization was obtained by this method but when it occurred there was a similar percentage of monospermic and polyspermic fertilization. Although some pregnancies and births occurred, inconsistent results meant that partial zona dissection was not widely applied in the clinic. Around the same time a few case reports were published on the next assisted fertilization procedure: subzonal insemination (SUZI), a micromanipulation technique involving the insertion of a few spermatozoa between the zona pellucida and the membrane of the oocyte. We decided to embark on this approach for assisted fertilization. What did we do?