Global Practice Patterns in the Evaluation of Non-Obstructive Azoospermia: Results of a World-Wide Survey and Expert Recommendations.

PURPOSE: Non-obstructive azoospermia (NOA) represents the persistent absence of sperm in ejaculate without obstruction, stemming from diverse disease processes. This survey explores global practices in NOA diagnosis, comparing them with guidelines and offering expert recommendations. MATERIALS AND METHODS: A 56-item questionnaire survey on NOA diagnosis and management was conducted globally from July to September 2022. This paper focuses on part 1, evaluating NOA diagnosis. Data from 367 participants across 49 countries were analyzed descriptively, with a Delphi process used for expert recommendations. RESULTS: Of 336 eligible responses, most participants were experienced attending physicians (70.93%). To diagnose azoospermia definitively, 81.7% requested two semen samples. Commonly ordered hormone tests included serum follicle-stimulating hormone (FSH) (97.0%), total testosterone (92.9%), and luteinizing hormone (86.9%). Genetic testing was requested by 66.6%, with karyotype analysis (86.2%) and Y chromosome microdeletions (88.3%) prevalent. Diagnostic testicular biopsy, distinguishing obstructive azoospermia (OA) from NOA, was not performed by 45.1%, while 34.6% did it selectively. Differentiation relied on physical examination (76.1%), serum hormone profiles (69.6%), and semen tests (68.1%). Expectations of finding sperm surgically were higher in men with normal FSH, larger testes, and a history of sperm in ejaculate. CONCLUSIONS: This expert survey, encompassing 367 participants from 49 countries, unveils congruence with recommended guidelines in NOA diagnosis. However, noteworthy disparities in practices suggest a need for evidence-based, international consensus guidelines to standardize NOA evaluation, addressing existing gaps in professional recommendations.

Global Practice Patterns and Variations in the Medical and Surgical Management of Non-Obstructive Azoospermia: Results of a World-Wide Survey, Guidelines and Expert Recommendations.

PURPOSE: Non-obstructive azoospermia (NOA) is a common, but complex problem, with multiple therapeutic options and a lack of clear guidelines. Hence, there is considerable controversy and marked variation in the management of NOA. This survey evaluates contemporary global practices related to medical and surgical management for patients with NOA. MATERIALS AND METHODS: A 56-question online survey covering various aspects of the evaluation and management of NOA was sent to specialists around the globe. This paper analyzes the results of the second half of the survey dealing with the management of NOA. Results have been compared to current guidelines, and expert recommendations have been provided using a Delphi process. RESULTS: Participants from 49 countries submitted 336 valid responses. Hormonal therapy for 3 to 6 months was suggested before surgical sperm retrieval (SSR) by 29.6% and 23.6% of participants for normogonadotropic hypogonadism and hypergonadotropic hypogonadism respectively. The SSR rate was reported as 50.0% by 26.0% to 50.0% of participants. Interestingly, 46.0% reported successful SSR in <10% of men with Klinefelter syndrome and 41.3% routinely recommended preimplantation genetic testing. Varicocele repair prior to SSR is recommended by 57.7%. Half of the respondents (57.4%) reported using ultrasound to identify the most vascularized areas in the testis for SSR. One-third proceed directly to microdissection testicular sperm extraction (mTESE) in every case of NOA while others use a staged approach. After a failed conventional TESE, 23.8% wait for 3 months, while 33.1% wait for 6 months before proceeding to mTESE. The cut-off of follicle-stimulating hormone for positive SSR was reported to be 12-19 IU/mL by 22.5% of participants and 20-40 IU/mL by 27.8%, while 31.8% reported no upper limit. CONCLUSIONS: This is the largest survey to date on the real-world medical and surgical management of NOA by reproductive experts. It demonstrates a diverse practice pattern and highlights the need for evidence-based international consensus guidelines.

Consensus and Diversity in the Management of Varicocele for Male Infertility: Results of a Global Practice Survey and Comparison with Guidelines and Recommendations.

PURPOSE: Varicocele is a common problem among infertile men. Varicocele repair (VR) is frequently performed to improve semen parameters and the chances of pregnancy. However, there is a lack of consensus about the diagnosis, indications for VR and its outcomes. The aim of this study was to explore global practice patterns on the management of varicocele in the context of male infertility. MATERIALS AND METHODS: Sixty practicing urologists/andrologists from 23 countries contributed 382 multiple-choice-questions pertaining to varicocele management. These were condensed into an online questionnaire that was forwarded to clinicians involved in male infertility management through direct invitation. The results were analyzed for disagreement and agreement in practice patterns and, compared with the latest guidelines of international professional societies (American Urological Association [AUA], American Society for Reproductive Medicine [ASRM], and European Association of Urology [EAU]), and with evidence emerging from recent systematic reviews and meta-analyses. Additionally, an expert opinion on each topic was provided based on the consensus of 16 experts in the field. RESULTS: The questionnaire was answered by 574 clinicians from 59 countries. The majority of respondents were urologists/uro-andrologists. A wide diversity of opinion was seen in every aspect of varicocele diagnosis, indications for repair, choice of technique, management of sub-clinical varicocele and the role of VR in azoospermia. A significant proportion of the responses were at odds with the recommendations of AUA, ASRM, and EAU. A large number of clinical situations were identified where no guidelines are available. CONCLUSIONS: This study is the largest global survey performed to date on the clinical management of varicocele for male infertility. It demonstrates: 1) a wide disagreement in the approach to varicocele management, 2) large gaps in the clinical practice guidelines from professional societies, and 3) the need for further studies on several aspects of varicocele management in infertile men.

Antisperm Antibody Testing: A Comprehensive Review of Its Role in the Management of Immunological Male Infertility and Results of a Global Survey of Clinical Practices.

Antisperm antibodies (ASA), as a cause of male infertility, have been detected in infertile males as early as 1954. Multiple causes of ASA production have been identified, and they are due to an abnormal exposure of mature germ cells to the immune system. ASA testing (with mixed anti-globulin reaction, and immunobead binding test) was described in the WHO manual 5th edition and is most recently listed among the extended semen tests in the WHO manual 6th edition. The relationship between ASA and infertility is somewhat complex. The presence of sperm agglutination, while insufficient to diagnose immunological infertility, may indicate the presence of ASA. However, ASA can also be present in the absence of any sperm agglutination. The andrological management of ASA depends on the etiology and individual practices of clinicians. In this article, we provide a comprehensive review of the causes of ASA production, its role in immunological male infertility, clinical indications of ASA testing, and the available therapeutic options. We also provide the details of laboratory procedures for assessment of ASA together with important measures for quality control. Additionally, laboratory and clinical scenarios are presented to guide the reader in the management of ASA and immunological male infertility. Furthermore, we report the results of a recent worldwide survey, conducted to gather information about clinical practices in the management of immunological male infertility.

Post-Vasectomy Semen Analysis: Optimizing Laboratory Procedures and Test Interpretation through a Clinical Audit and Global Survey of Practices.

PURPOSE: The success of vasectomy is determined by the outcome of a post-vasectomy semen analysis (PVSA). This article describes a step-by-step procedure to perform PVSA accurately, report data from patients who underwent post vasectomy semen analysis between 2015 and 2021 experience, along with results from an international online survey on clinical practice. MATERIALS AND METHODS: We present a detailed step-by-step protocol for performing and interpretating PVSA testing, along with recommendations for proficiency testing, competency assessment for performing PVSA, and clinical and laboratory scenarios. Moreover, we conducted an analysis of 1,114 PVSA performed at the Cleveland Clinic's Andrology Laboratory and an online survey to understand clinician responses to the PVSA results in various countries. RESULTS: Results from our clinical experience showed that 92.1% of patients passed PVSA, with 7.9% being further tested. A total of 78 experts from 19 countries participated in the survey, and the majority reported to use time from vasectomy rather than the number of ejaculations as criterion to request PVSA. A high percentage of responders reported permitting unprotected intercourse only if PVSA samples show azoospermia while, in the presence of few non-motile sperm, the majority of responders suggested using alternative contraception, followed by another PVSA. In the presence of motile sperm, the majority of participants asked for further PVSA testing. Repeat vasectomy was mainly recommended if motile sperm were observed after multiple PVSA's. A large percentage reported to recommend a second PVSA due to the possibility of legal actions. CONCLUSIONS: Our results highlighted varying clinical practices around the globe, with controversy over the significance of non-motile sperm in the PVSA sample. Our data suggest that less stringent AUA guidelines would help improve test compliance. A large longitudinal multi-center study would clarify various doubts related to timing and interpretation of PVSA and would also help us to understand, and perhaps predict, recanalization and the potential for future failure of a vasectomy.

Willingness of Infertile Couples to Pay for In Vitro Fertilization Treatment in the Integrated Human Reproduction Section of the Escola Paulista de Medicina, São Paulo Federal University.

OBJECTIVE: To describe the willingness to pay (WTP) of infertile couples for in vitro fertilization (IVF) treatment. METHOD: This was a prospective study with an anonymous questionnaire for infertile couples in an academic setting. Clinical characteristics were analyzed by a Student's t test or Mann-Whitney test, categorical variables were compared by a chi-square or Fisher exact test, and correlations were assessed using a Spearman's test. An alpha of 5% was adopted. RESULTS: Mean female and male ages were 31.5 and 35.9 years, respectively; 80.2% were married; 19.8% were in consensual union; 48.1% of women had college degrees; and 49.4% of men had a high school education. Most women (77.8%) and men (75.3%) were white, with a household income of class C. Average duration of union was 8.5 years, and average infertility was 4.7 years. Using a willingness-to-pay (WTP) evaluation and the technique of "direct questioning," the average value was determined to be R$18 720.18 (by payment scale R$22 831.17). WTP positively correlated with household income and each woman's education level. Previous parenthood or use of public health system negatively correlated with WTP. CONCLUSION: We conclude that the higher the couple's monthly income and the woman's educational level, the higher the WTP for an IVF treatment; previous parenthood determined a lower WTP for an IVF treatment, and previous use of the Brazilian Unified Health System, determined a lower WTP for an IVF treatment.

Follicular fluid lipid peroxidation levels in women with endometriosis during controlled ovarian hyperstimulation.

This observational study aimed to establishing a relationship between lipid peroxidation and endometriosis in women undergoing controlled ovarian hyperstimulation. A total of 79 women were divided into two groups: (i) controls (tubal or male factor); and (ii) endometriosis (stages III/IV). The endometriosis diagnosis was confirmed by videolaparoscopy and the controlled ovarian stimulation protocol was similar to all patients. Follicular fluid (FF) lipid peroxidation levels were determined through the quantification of malondialdehyde. Statistical analysis was performed using parametric and non-parametric tests, logistic regression was performed to estimate the chance of achieving a pregnancy in each group and a moving average was calculated for the endometriosis group. Peroxidation levels in the endometriosis group were significantly higher when compared to controls. The moving average showed a decrease of MDA levels in the endometriosis group with increasing female age. Moreover, women with endometriosis who were under 33 years of age were 4.3 times more likely to achieve a pregnancy than women above that age. In conclusion, endometriosis is associated with increased FF oxidative stress (OS) in patients undergoing in vitro fertilization (IVF). Also, increasing age is associated with a decrease in severity of the oxidative status, but a decreased chance of pregnancy.

Analysis of the functional aspects and seminal plasma proteomic profile of sperm from smokers.

OBJECTIVE: To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profile. PATIENTS AND METHODS: Sperm functional tests were performed in 20 non-smoking men with normal semen quality, according to the World Health Organization (2010) and in 20 smoking patients. These included: evaluation of DNA fragmentation by alkaline Comet assay; analysis of mitochondrial activity using DAB staining; and acrosomal integrity evaluation by PNA binding. The remaining semen was centrifuged and seminal plasma was used for proteomic analysis (liquid chromatography-tandem mass spectrometry). The quantified proteins were used for Venn diagram construction in Cytoscape 3.2.1 software, using the PINA4MS plug-in. Then, differentially expressed proteins were used for functional enrichment analysis of Gene Ontology categories, Kyoto Encyclopedia of Genes and Genomes and Reactome, using Cytoscape software and the ClueGO 2.2.0 plug-in. RESULTS: Smokers had a higher percentage of sperm DNA damage (Comet classes III and IV; P < 0.01), partially and fully inactive mitochondria (DAB classes III and IV; P = 0.001 and P = 0.006, respectively) and non-intact acrosomes (P < 0.01) when compared with the control group. With respect to proteomic analysis, 422 proteins were identified and quantified, of which one protein was absent, 27 proteins were under-represented and six proteins were over-represented in smokers. Functional enrichment analysis showed the enrichment of antigen processing and presentation, positive regulation of prostaglandin secretion involved in immune response, protein kinase A signalling and arachidonic acid secretion, complement activation, regulation of the cytokine-mediated signalling pathway and regulation of acute inflammatory response in the study group (smokers). CONCLUSION: In conclusion, cigarette smoking was associated with an inflammatory state in the accessory glands and in the testis, as shown by enriched proteomic pathways. This state causes an alteration in sperm functional quality, which is characterized by decreased acrosome integrity and mitochondrial activity, as well as by increased nuclear DNA fragmentation.

Association between the seminal plasma proteome and sperm functional traits.

OBJECTIVE: To analyze the seminal plasma proteome and biological functions associated with sperm functional alterations. DESIGN: Cross-sectional study. SETTING: University andrology and research laboratories. PATIENT(S): A total of 156 normozoospermic men. INTERVENTION(S): Sperm mitochondrial activity, acrosome integrity, and DNA fragmentation were evaluated in a semen aliquot. Remaining semen was centrifuged, and seminal plasma was utilized for proteomic analysis (liquid chromatography-tandem mass spectrometry). Patients were divided into percentiles (15%) to form the following groups: substudy 1, high (control, n = 26) and low (study, n = 23) sperm mitochondrial activity; substudy 2, high (control, n = 23) and low (study, n = 22) sperm acrosome integrity; and substudy 3, low (control, n = 22) and high (study, n = 22) sperm DNA fragmentation. Groups were compared using univariate and multivariate analyses. Differentially expressed proteins were used for functional enrichment analysis. MAIN OUTCOME MEASURE(S): Seminal plasma proteome and postgenomic pathways are associated with several sperm functional traits. RESULT(S): In total, 506, 493, and 464 proteins were observed in substudies 1, 2, and 3, respectively. Enriched functions in substudy 1 were intramolecular oxidoreductase activity, aminoglycans catabolism, endopeptidases inhibition, lysosomes, and acute-phase response (study group). In substudy 2, main enriched functions were phospholipase inhibition, arachidonic acid metabolism, exocytosis, regulation of acute inflammation, response to hydrogen peroxide, and lysosomal transport (study group). In substudy 3, enriched functions were prostaglandin biosynthesis and fatty acid binding (study group). We proposed eight, six, and eight seminal biomarkers for substudies 1, 2, and 3, respectively. CONCLUSION(S): Seminal plasma proteome reflects sperm mitochondrial activity reduction, acrosome damage, and DNA fragmentation, with several postgenomic functions related to these alterations.

Lipidomics analysis of follicular fluid by ESI-MS reveals potential biomarkers for ovarian endometriosis.

PURPOSE: The aim of the present study was to analyze the lipid profile of follicular fluid from patients with endometriosis and endometrioma who underwent in vitro fertilization treatment (IVF). METHODS: The control group (n = 10) was composed of women with tubal factor or minimal male factor infertility who had positive pregnancy outcomes after IVF. The endometriosis group consisted of women with endometriosis diagnosed by videolaparoscopy (n = 10), and from the same patients, the endometriomas fluids were collected, which composed the endometrioma group (n = 10). From the follicular fluid and endometriomas, lipids were extracted by the Bligh and Dyer method, and the samples were analyzed by tandem mass spectrometry. RESULTS: We observed phosphatidylglycerol phosphate, phosphatidylcholine, phosphatidylserine, and phosphatidylnositol bisphosphate in the control group. In the endometriosis group, sphingolipids and phosphatidylcholines were more abundant, while in the endometrioma group, sphingolipids and phosphatidylcholines with different m/z from the endometriosis group were found in high abundance. CONCLUSION: This analysis demonstrated that there is a differential representation of these lipids according to their respective groups. In addition, the lipids found are involved in important mechanisms related to endometriosis progress in the ovary. Thus, the metabolomic approach for the study of lipids may be helpful in potential biomarker discovery.

Differences in the seminal plasma proteome are associated with oxidative stress levels in men with normal semen parameters.

OBJECTIVE: To study the seminal plasma proteome in association with semen lipid peroxidation levels in men with normal semen parameters. DESIGN: Cross-sectional study. SETTING: University andrology and research laboratories. PATIENT(S): A total of 156 normozoospermic men. INTERVENTION(S): Seminal lipid peroxidation levels were assessed in individual samples through thiobarbituric acid reactive substances quantification. Subsequently, lipid peroxidation data were used to divide the samples into the experimental groups: low lipid peroxidation levels (control group, bottom 15%, n = 23) and high lipid peroxidation levels (study group, top 15%, n = 23). Seminal plasma proteins from these groups were pooled (four pools per group, with biological variation between the pools) and used for a shotgun proteomic analysis using a liquid chromatography-tandem mass spectrometry approach. Quantitative data were used for univariate (unpaired Student's t test) and multivariate (partial least-squares discriminant analysis, logistic regression, and discriminant analyses) statistical analyses. Significant proteins were also used for functional enrichment analysis. MAIN OUTCOME MEASURE(S): Seminal plasma protein profile and postgenomic pathways of seminal plasma are associated with seminal lipid peroxidation levels. RESULT(S): In total, 629 proteins were quantified in seminal plasma. Of these, 23 proteins were absent or underexpressed and 71 were exclusive or overexpressed in the study group. The main enriched functions in association with seminal lipid peroxidation were unsaturated fatty acids biosynthesis, oxidants and antioxidants activity, cellular response to heat stress, and immune response. Moreover, we suggested mucin-5B as a potential biomarker of semen oxidative stress. CONCLUSION(S): The seminal plasma proteome does reflect semen lipid peroxidation status and, thus, oxidative stress.

Follicular fluid alterations in endometriosis: label-free proteomics by MS(E) as a functional tool for endometriosis.

Endometriosis is a chronic gynecological condition that affects 10-32% of women of reproductive age and may lead to infertility. The study of protein profiles in follicular fluid may assist in elucidating possible biomarkers related to this disease. For this, follicular fluid samples were obtained from women with tubal factor or minimal male factor infertility who had pregnancy outcomes after in vitro fertilization (IVF) treatment (control group, n = 10), women with endometriosis (endometriosis group, n = 10), along with the endometrioma from these same patients were included (endometrioma group, n = 10). For proteomic analysis, samples were pooled according to their respective groups and normalized to protein content. Proteins were analyzed by in tandem mass spectrometry (MS(E)) Spectra processing and the ProteinLynx Global Server v.2.5. was used for database searching. Data was submitted to the biological network analysis using Cytoscape 2.8.2 with ClueGO plugin. As a result, 535 proteins were identified among all groups. The control group differentially or uniquely expressed 33 (6%) proteins and equal expression of 98 (18%) proteins was observed in the control and endometriosis groups of which 41 (7%) proteins were further identified and/or quantified. Six (1%) proteins were observed in both the endometriosis and endometrioma groups, but 212 (39%) proteins were exclusively identified and/or quantified in the endometrioma group. There were 9 (1%) proteins observed in both the control and endometrioma groups and there were 139 (25%) proteins common among all three groups. Distinct differences among the protein profiles in the follicular fluid of patients included in this study were found, identifying proteins related to the disease progression and IVF success. Thus, some pathways related to endometriosis are associated with the presence of specific proteins, as well as the absence of others. This study provides a first step to the development of more sensitive diagnostic tests and treatment.

Follicular fluid lipid fingerprinting from women with PCOS and hyper response during IVF treatment.

PURPOSE: Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder that leads to lower natural reproductive potential and presents a challenge for assisted reproductive medicine because patients may exhibit immature oocyte retrieval and a higher risk of ovarian hyper stimulation syndrome during in vitro fertilization (IVF) treatment. This study aimed to identify potential lipid biomarkers for women with PCOS and a hyper response to controlled ovarian stimulation. METHODS: Follicular fluid samples were collected from patients who underwent IVF, including normal responder women who became pregnant (control group, n = 11), women with PCOS and a hyper response to gonadotropins (PCOS group, n = 7) and women with only hyper response to gonadotropins (HR group, n = 7). A lipidomic analysis was performed by electrospray ionization mass spectrometry, and candidate biomarkers were analyzed by tandem mass spectrometry experiment. RESULTS: The lipid profiles indicated particularities related to differences in phosphatidylcholine (PCOS and HR), phosphatidylserine, phosphatydilinositol and phosphatidylglycerol (control), sphingolipids (PCOS) and phosphatidylethanolamine (control and HR). CONCLUSIONS: These findings contribute to the understanding of the molecular mechanisms associated with lipid metabolism in the PCOS-related hyper response, and strongly suggest that these lipids may be useful as biomarkers, leading to the development of more individualized treatment for pregnancy outcome.

Hyaluronidase alters the lipid profile of cumulus cells as detected by MALDI-TOF MS and multivariate analysis.

This research aimed to study the changes in lipid composition in cumulus cells using hyaluronidase according to the intracytoplasmic sperm injection protocol commonly used in human reproduction clinics. The lipid extraction was performed by the Blight-Dyer protocol and the lipid profiles were obtained by MALDI-TOF MS in positive and negative modes. The mass spectra data were processed with MassLynx and the statistical analysis was performed using MetaboAnalyst 2.0. Fifteen ions were selected for each mode as potential markers for differences between the groups. These ions were identified in the human metabolome database as phosphatidylserine with and without treatment, phosphatidylethanolamine in the after treatment group and phosphatidylinositol in the before treatment group, which are lipids that may be involved in cell apoptosis and signaling. We concluded that MALDI-TOF MS coupled with multivariate analysis can be utilized as a strategy to obtain and study the lipid profiles of cumulus cells and as a tool to study the metabolic state of cumulus cells.

Proteomic analysis of seminal plasma in adolescents with and without varicocele.

OBJECTIVE: To compare proteomic profiles of seminal plasma from adolescents with varicocele and changes in semen quality with the plasma from adolescents with varicocele without seminal changes and from adolescents without varicocele. DESIGN: Observational study. SETTING: Patients in an academic research environment. PATIENT(S): Adolescents without varicocele (control group), adolescents with varicocele and normal semen quality (VNS group), adolescents with varicocele and abnormal semen quality (VAS group). INTERVENTION(S): Two semen collections at 1-week interval. Protein separation by two-dimensional protein electrophoresis, analysis by gel densitometry, and identification by mass spectrometry. MAIN OUTCOME MEASURE(S): Overexpressed proteins in each group, observed by increased densitometric signal in gels, and exclusively identified proteins in each group. RESULT(S): No differences were observed among the three groups regarding clinical parameters. In semen analysis, the VAS group presented lower sperm concentration, motility, and morphology compared with the VNS and control groups. Forty-seven protein spots of interest were submitted to mass spectrometry identification. Apoptosis regulation proteins were overexpressed in the VAS group, whereas spermatogenesis proteins were overexpressed in the VNS group. Controls presented proteins related to homeostasis. CONCLUSION(S): Changes in the proteomic profile of adolescents with varicocele and normal semen parameters (VNS group) indicate that normal semen analysis may not reflect alterations in proteins in seminal plasma. Implementation of proteomics will help characterize proteins identified in seminal plasma and will facilitate detection of new proteins associated with spermatogenesis and sperm function.