To evaluate the effects of lavender oil aromatherapy on state anxiety, semen collection-related anxiety and difficulty, satisfaction with the inhaled scent, and semen parameters during semen sample collection. Semen sample collection may be associated with anxiety, perceived stress, and discomfort in men undergoing infertility evaluation. Supportive approaches that improve men’s experience during this process remain limited. This parallel-group randomised controlled trial included 102 men undergoing infertility evaluation. Participants were randomised to either the aromatherapy group (n = 51), which inhaled lavender oil aromatherapy during semen sample collection, or the placebo group (n = 51), which inhaled coconut oil using the same diffuser procedure. State anxiety was assessed using the State-Trait Anxiety Inventory-State form (STAI-S). Semen collection-related anxiety, difficulty, and satisfaction with the inhaled scent were evaluated using the Visual Analog Scale (VAS). Semen parameters were assessed through semen analysis. The groups were comparable at baseline. Repeated-measures analysis showed a borderline significant time × group interaction for STAI-S scores, suggesting a greater reduction in state anxiety in the aromatherapy group (p = 0.048). The VAS anxiety interaction was not statistically significant, and the post-procedure VAS anxiety difference did not remain significant after Bonferroni correction. Satisfaction with the inhaled scent was significantly higher in the aromatherapy group (p < 0.001). No significant differences were found in VAS difficulty or semen parameters. Lavender oil aromatherapy may support short-term state anxiety reduction and improve satisfaction with the semen collection environment, but it did not affect procedural difficulty or semen parameters. ClinicalTrials.gov, NCT06241495. Registered on January 29, 2024. Available at: https://clinicaltrials.gov/study/NCT06241495 .

PURPOSE:Antioxidant (AOX) therapy has long been investigated for the management of male infertility. It has potential benefits, but persistent controversy affects its broad acceptance and clinical utility. This study aimed to develop standardized, evidence-based guidelines for AOX use by synthesizing the best available evidence and achieving global expert consensus. MATERIALS AND METHODS:A comprehensive literature review was conducted by senior experts of the Global Andrology Forum (GAF). Additionally, data on the real-life use of oxidative stress (OS) testing and AOX use obtained from the largest global survey of practicing physicians conducted by GAF, which served as a reference point for this study. In phase one, 151 international specialists (>5 years' experience) participated in a modified Delphi process to evaluate AOX therapy in male infertility. Experts reviewed draft statements and rated agreement on a 10-point Likert scale. Statements achieving >80% consensus (with score >7/10) were accepted. The original eight statements were expanded to 19 and finalized into 18. In phase two, 84 senior physicians (>10 years' experience) graded the Delphi-approved statements using the GRADE approach. RESULTS:Four out of the 18 statements failed to reach consensus and were excluded. Of the final 14 statements, seven (50%) were graded as "Strong" and seven (50%) as "Weak." Current evidence indicates that AOX therapy can reduce OS, improve sperm quality, and potentially enhance reproductive outcomes. However, benefits vary by agent, regimen, and patient population. The guidelines emphasize the need to document OS and to consider underlying factors before initiating AOX therapy, and caution against indiscriminate or prolonged use. CONCLUSIONS:Developed through a dual-validation process and endorsed by a globally diverse expert panel, these GAF guidelines represent the first standardized, evidence-based guidelines for AOX use in male infertility. By addressing heterogeneity in research and practice, they provide clinicians with practical, safe, and patient-centered recommendations for rational AOX therapy worldwide.

Background: Male infertility is increasingly being conceived as a biomarker of general male health status other than being a significant reproductive health issue. Nonobstructive azoospermia (NOA) patients have the worst health status impairment as well as highest risk of developing cancer. It’s well known that cancer susceptibility gene, MLH1 have roles in homologous recombination during meiosis. Mlh1 mutant mice have azoospermia with meiotic arrest. Aim: We aimed to identify risk alleles in MLH1 gene in males with nonobstructive azoospermia related maturation arrest (MA) to investigate further whether this cancer susceptibility gene is also related to male infertility. Patients and Methods: MLH1 variants were genotyped in 42 patients with MA. The variants of interest then compared to GnomAD v.3.1.2 (non-cancer) male population database and to the repository exome data of a national genetic disease evaluation center (NGDEC). Results: rs1800734 allele frequencies were significantly higher in the patient group compared to GnomAD (p

Sperm DNA fragmentation is associated with poor sperm quality and reproductive outcomes. Free radicals are a significant cause of DNA fragmentation, with mitochondria being the primary intrinsic source. To investigate the relationship between midpiece measurements containing mitochondria and sperm DNA fragmentation, and to determine the ideal midpiece area. Demographic data, semen analysis results, and DNA fragmentation values were prospectively collected from 239 men with infertility complaints. Detailed analyses and morphometric measurements were performed on 50 spermatozoa from each patient, totaling 11,950 spermatozoa. The ideal DNA fragmentation index (DFI) cutoff value was calculated. Patients were classified into three subgroups based on midpiece length, midpiece width, and midpiece area measurements. The correlation between mid-piece measurements and DFI was investigated. The cutoff value for DFI was determined as 19.50. The odds ratio evaluating the relationship between morphology status and high DFI was 0.159 (95% CI: 0.089-0.282). It was observed that midpiece length and width have statistically significant but low correlations with DFI, whereas midpiece area shows a higher correlation. Finally, based on DFI values, the ideal midpiece area was between 2.31 and 3.13 µm2. There is a significant correlation between sperm midpiece area and DFI value, surpassing that of length and width. Future studies may yield important insights by exploring the impact of midpiece area measurements on reproductive outcomes.

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.

PURPOSE:The purpose of this meta-analysis is to study the impact of varicocele repair in the largest cohort of infertile males with clinical varicocele by including all available studies, with no language restrictions, comparing intra-person conventional semen parameters before and after the repair of varicoceles.MATERIALS AND METHODS:The meta-analysis was performed according to PRISMA-P and MOOSE guidelines. A systematic search was performed in Scopus, PubMed, Cochrane, and Embase databases. Eligible studies were selected according to the PICOS model (Population: infertile male patients with clinical varicocele; Intervention: varicocele repair; Comparison: intra-person before-after varicocele repair; Outcome: conventional semen parameters; Study type: randomized controlled trials [RCTs], observational and case-control studies).RESULTS:Out of 1,632 screened abstracts, 351 articles (23 RCTs, 292 observational, and 36 case-control studies) were included in the quantitative analysis. The before-and-after analysis showed significant improvements in all semen parameters after varicocele repair (except sperm vitality); semen volume: standardized mean difference (SMD) 0.203, 95% CI: 0.129-0.278; p<0.001; I²=83.62%, Egger's p=0.3329; sperm concentration: SMD 1.590, 95% CI: 1.474-1.706; p<0.001; I²=97.86%, Egger's p<0.0001; total sperm count: SMD 1.824, 95% CI: 1.526-2.121; p<0.001; I²=97.88%, Egger's p=0.0063; total motile sperm count: SMD 1.643, 95% CI: 1.318-1.968; p<0.001; I²=98.65%, Egger's p=0.0003; progressive sperm motility: SMD 1.845, 95% CI: 1.537%-2.153%; p<0.001; I²=98.97%, Egger's p<0.0001; total sperm motility: SMD 1.613, 95% CI 1.467%-1.759%; p<0.001; l2=97.98%, Egger's p<0.001; sperm morphology: SMD 1.066, 95% CI 0.992%-1.211%; p<0.001; I²=97.87%, Egger's p=0.1864.CONCLUSIONS:The current meta-analysis is the largest to date using paired analysis on varicocele patients. In the current meta-analysis, almost all conventional semen parameters improved significantly following varicocele repair in infertile patients with clinical varicocele.

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.

BACKGROUND:The question of whether patients are more likely to succeed with testicular sperm intracytoplasmic sperm injection (T-ICSI) after unsuccessful ICSI with ejaculated sperm (Ej-ICSI) remains unknown. OBJECTIVE:The study aimed to identify potential predictors of successful T-ICSI in men with idiopathic infertility and oligozoospermia (sperm concentration < 15 × 106/mL, non-azoospermic) who had previously experienced unsuccessful Ej-ICSI. MATERIALS AND METHODS:In total, 154 couples with male partners who had oligozoospermic conditions after two unsuccessful cycles of Ej-ICSI switched to T-ICSI. Before initiating T-ICSI, the sperm DNA fragmentation index (DFI) was assessed in ejaculated specimens. Participants were divided into two groups: group A (live birth (+), n = 60) and group B (live birth (-), n = 94). RESULTS:Fertilization, clinical pregnancy, live births, and miscarriages had rates of 72.7%, 44.2%, 39%, and 5.2%, respectively. The total motile sperm (TMS) count in group A was significantly higher (3.8 ± 1.5 million) than in group B (3 ± 1.6 million; p = 0.002). DFI was significantly higher in group A (24.2 ± 12.3) than in group B (18.1 ± 11; p = 0.001). Hormone levels and oocyte counts showed no statistically significant differences between groups. Multivariate regression analysis revealed that TMS (odds ratio [OR]: 1.46; 95% CI, 1.14-1.87, p = 0.003) and DFI (OR: 1.04; 95% CI, 1.01-1.08, p = 0.009) were found to be significant predictors of live birth outcomes. At a cutoff point of 2.55 (area under the curve [AUC] = 0.65), the optimal sensitivity and specificity values for TMS were 78% and 48%, respectively. At a cutoff point of 25.8 (AUC = 0.65), DFI had a maximum sensitivity of 51.7% and a specificity of 78.7%. CONCLUSIONS:TMS and DFI were found to be significant predictors of live birth outcomes in couples with oligozoospermic male partners undergoing T-ICSI. These findings may help clinicians tailor treatment strategies for this specific patient population.

PURPOSE:Sperm DNA fragmentation (SDF) is a functional sperm abnormality that can impact reproductive potential, for which four assays have been described in the recently published sixth edition of the WHO laboratory manual for the examination and processing of human semen. The purpose of this study was to examine the global practices related to the use of SDF assays and investigate the barriers and limitations that clinicians face in incorporating these tests into their practice.MATERIALS AND METHODS:Clinicians managing male infertility were invited to complete an online survey on practices related to SDF diagnostic and treatment approaches. Their responses related to the technical aspects of SDF testing, current professional society guidelines, and the literature were used to generate expert recommendations via the Delphi method. Finally, challenges related to SDF that the clinicians encounter in their daily practice were captured.RESULTS:The survey was completed by 436 reproductive clinicians. Overall, terminal deoxynucleotidyl transferase deoxyuridine triphosphate Nick-End Labeling (TUNEL) is the most commonly used assay chosen by 28.6%, followed by the sperm chromatin structure assay (24.1%), and the sperm chromatin dispersion (19.1%). The choice of the assay was largely influenced by availability (70% of respondents). A threshold of 30% was the most selected cut-off value for elevated SDF by 33.7% of clinicians. Of respondents, 53.6% recommend SDF testing after 3 to 5 days of abstinence. Although 75.3% believe SDF testing can provide an explanation for many unknown causes of infertility, the main limiting factors selected by respondents are a lack of professional society guideline recommendations (62.7%) and an absence of globally accepted references for SDF interpretation (50.3%).CONCLUSIONS:This study represents the largest global survey on the technical aspects of SDF testing as well as the barriers encountered by clinicians. Unified global recommendations regarding clinician implementation and standard laboratory interpretation of SDF testing are crucial.

Abstract Introduction LIM15, also known as DNA meiotic recombinase 1 (DMC1), is a meiosis-specific recombinase. LIM15 is specifically expressed as a meiotic recombination protein in spermatogenesis. Objective Does the LIM15 gene expression value measured in the ejaculate predict the success of the second micro–testicular sperm extraction (mTESE) after the failed first mTESE? Methods Data from 111 men who failed the first mTESE and underwent the second mTESE between August 2018 and March 2022 were prospectively collected. LIM15 gene expression level was determined by real-time PCR in the materials from the ejaculate. Results Sperm was recovered successfully in 23 out of 111 (20.7%) men with second mTESE. The mean age of men for whom sperm could not be retrieved and men with successful sperm recovery were 34.6 ± 3.5 and 33.7 ± 5.2, respectively. The mean patient age, serum total testosterone and FSH levels, and testicular volume did not show any statistical difference between men with successful sperm recovery and men with failed sperm recovery (Table 1). According to the testicular histology, the sertoli cell-only, maturation arrest, and hypospermatogenesis in men with successful sperm recovery were 26.1%, 65.2%, and 8.7%, and in men for whom sperm could not be retrieved were 0%, 27.3%, and 72.7%. Comparing the groups in terms of testicular histology demonstrated meaningful correlation between sperm retrieval rate (SRR) and testis histology (p<0.001). The LIM15 gene expression level measured in ejaculate significantly correlated with the SRR in mTESE (p = 0.039). As shown in Figure 1, the maximal sensitivity (50%) and specificity (87%) were obtained for the LIM15 gene with a cutoff point of 4.61 and AUC of 0.65. Conclusions According to our results, LIM15 measured in the ejaculate before the second mTESE can provide information on sperm retrieval and reduce the potential complications and unnecessary costs associated with assisted reproductive techniques. Disclosure No

Different cell culture conditions and techniques have been used to mature spermatogenic cells to increase the success of in vitro fertilization. Sertoli cells (SCs) are essential in maintaining spermatogenesis and FSH stimulation exerts its effect through direct or indirect actions on SCs. The effectiveness of FSH and testosterone added to the co-culture has been demonstrated in other studies to provide microenvironment conditions of the testicular niche and to contribute to the maturation and meiotic progression of spermatogonial stem cells (SSCs). In the present study, we investigated whether co-culture of healthy SCs with the patient’s testicular tissue in the medium supplemented with FSH/testosterone provides an advantage in the differentiation and maturation of germ cells in NOA cases (N = 34). In men with obstructive azoospermia (N = 12), healthy SCs from testicular biopsies were identified and purified, then cryopreserved. The characterization of healthy SCs was done by flow cytometry (FC) and immunohistochemistry using antibodies specific for GATA4 and vimentin. FITC-conjugated annexin V/PI staining and the MTT assay were performed to compare the viability and proliferation of SCs before and after freezing. In annexin V staining, no difference was found in percentages of live and apoptotic SCs, and MTT showed that cryopreservation did not inhibit SC proliferation compared to the pre-freezing state. Then, tissue samples from NOA patients were processed in two separate environments containing FSH/testosterone and FSH/testosterone plus co-culture with thawed healthy SCs for 7 days. FC was used to measure 7th-day levels of specific markers expressed in spermatogonia (VASA), meiotic cells (CREM), and post-meiotic cells (protamine-2 and acrosin). VASA and acrosin basal levels were found to be lower in infertile patients compared to the OA group (8.2% vs. 30.6% and 12.8% vs. 30.5%, respectively; p < 0.05). Compared to pre-treatment measurements, on the 7th day in the FSH/testosterone environment, CREM levels increased by 58.8% and acrosin levels increased by 195.5% (p < 0.05). Similarly, in medium co-culture with healthy SCs, by day 7, CREM and acrosin levels increased to 92.2% and 204.8%, respectively (p < 0.05). Although VASA and protamine levels increased in both groups, they did not reach a significant level. No significant difference was found between the day 7 increase rates of CREM, VASA, acrosin and protamine-2 in either FSH/testosterone-containing medium or in medium additionally co-cultured with healthy SCs (58.8% vs. 92.2%, 120.6% vs. 79.4%, 195.5% vs. 204.8%, and 232.3% vs. 198.4%, respectively; p > 0.05). Our results suggest that the presence of the patient’s own SCs for maturation of germ cells in the culture medium supplemented with FSH and testosterone is sufficient, and co-culture with healthy SCs does not have an additional advantage. In addition, the freezing–thawing process would not impair the viability and proliferation of SCs.

Purpose: Sperm DNA fragmentation (SDF) testing was recently added to the sixth edition of the World Health Organization laboratory manual for the examination and processing of human semen. Many conditions and risk factors have been associated with elevated SDF; therefore, it is important to identify the population of infertile men who might benefit from this test. The purpose of this study was to investigate global practices related to indications for SDF testing, compare the relevant professional society guideline recommendations, and provide expert recommendations. Materials and Methods: Clinicians managing male infertility were invited to take part in a global online survey on SDF clinical practices. This was conducted following the CHERRIES checklist criteria. The responses were compared to professional society guideline recommendations related to SDF and the appropriate available evidence. Expert recommendations on indications for SDF testing were then formulated, and the Delphi method was used to reach consensus. Results: The survey was completed by 436 experts from 55 countries. Almost 75% of respondents test for SDF in all or some men with unexplained or idiopathic infertility, 39% order it routinely in the work-up of recurrent pregnancy loss (RPL), and 62.2% investigate SDF in smokers. While 47% of reproductive urologists test SDF to support the decision for varicocele repair surgery when conventional semen parameters are normal, significantly fewer general urologists (23%; p=0.008) do the same. Nearly 70% would assess SDF before assisted reproductive technologies (ART), either always or for certain conditions. Recurrent ART failure is a common indication for SDF testing. Very few society recommendations were found regarding SDF testing. Conclusions: This article presents the largest global survey on the indications for SDF testing in infertile men, and demonstrates diverse practices. Furthermore, it highlights the paucity of professional society guideline recommendations. Expert recommendations are proposed to help guide clinicians.

INTRODUCTION AND OBJECTIVES:It is necessary to be able to predict sperm retrieval before microdissection testicular sperm extraction (mTESE) in azoospermic men. This study established the importance of proliferating cell nuclear antigen (PCNA) and LIM15 gene expression levels in predicting the success of sperm retrieval by mTESE.MATERIALS AND METHODS:One hundred and forty-three men who were diagnosed with non-obstructive azoospermia (NOA) were included in the study. Patients' age, total testosterone and follicle stimulating hormone values, testicular volume and testicular histology were recorded by prospectively. PCNA and LIM15 gene expression levels were determined by real-time PCR in the materials from both ejaculate and testicular specimens.RESULTS:Testis volume and histology were the most important factors in predicting the sperm retrieval rate (SRR). The PCNA and LIM15 gene expression levels measured in testicular tissues and the LIM15 gene expression levels measured in ejaculate significantly correlated with the SRR in mTESE (p=0.038, p=0.022, and p=0.004, respectively). Although the PCNA gene expression level measured in ejaculate was higher in men with successful sperm retrieval, the difference was not statistically significant (p=0.061). According to the multivariate logistic regression analysis, testicular volume and LIM15 gene expression level in ejaculate were independent predictive parameters for sperm retrieval.CONCLUSION:The data showed that LIM15 gene expression level in ejaculate is a useful molecular marker to predict the SRR before mTESE.

This study examines the association of the -617 C > A polymorphism in the Nrf2 gene (rs6721961) with male infertility in a Turkish population and determines its functional role in spermatogenesis in correlation with the impact of different levels of DNA damage on the genotypes. A total of 100 infertile men and 100 healthy fertile men were included in the study. Nrf2 genotyping was performed with the PCR-based restriction fragment length gene polymorphism (RFLP-PCR) analysis. According to our results, the Nrf2 CC, CA, and AA genotype distribution frequencies were 58.6%, 38.4%, and 3% in the control group, respectively, and 38%, 48%, and 14% in the infertile men, respectively. The AA genotype was significantly higher in the patient group. In smokers, a significant difference was found in progressive motility values between the genotypes (p = 0.001). Also, sperm progressive motility and concentration decreased significantly in those smokers with the AA genotype; smokers carrying this genotype were also 5.75 times more likely to have oligoasthenozoospermia than those with CC (p < 0.05). There was a significant relationship between the number of cases with high sperm-DNA damage when comparing the frequency of Nrf2 AA genotype carriers with the CC genotype 16.3% vs. 6.9%, respectively (p < 0.001). These results suggest the importance of the Nrf2 gene C > A (rs 6,721,961) polymorphism in the etiology of sperm DNA damage as a risk factor for male infertility. Smokers carrying the AA genotype are more likely to impair seminal parameters through antioxidant mechanisms.Abbreviations: Polymerase chain reaction (PCR)-based restriction fragment length gene polymorphism (RFLP-PCR); reactive oxygen species (ROS); deoxyribonucleic acid (DNA); catalases (CATs); superoxide dismutase (SOD); glutathione peroxidase (GPX); glutathione-S-transferase (GST); Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2); basic leucine zipper (bZIP); antioxidant response element (ARE); World Health Organization (WHO);normospermia(NS);asthenozoospermia(AS);oligozoospermia(OS);oligoasthenozoospermia (OAS); follicle stimulating hormone (FSH); ultraviolet (UV); low-melting-point agarose (LMA); normal-melting-point agarose (NMA); arbitrary units (AU); total comet score (TCS); A one-way analysis of variance (ANOVA); standard deviation (SD); N-acetyltransferase (NAT2); small non-coding RNAs (ncRNAs); microRNAs (miRNA).