Current Diagnosis of von Willebrand Disease in Italy: 3 Years Following the Release of the International Guidelines.

The American Society of Hematology-International Society on Thrombosis and Haemostasis-National Hemophilia Foundation-World Federation of Hemophilia 2021 International Guidelines (IGL) on von Willebrand disease (VWD) have pointed out many challenges, mainly in the diagnostic approach of VWD patients. To determine the impact of these IGL on the current clinical and laboratory diagnosis of Italian VWD patients, we have recently conducted a survey among 43 centers affiliated with the Italian Association of Hemophilia Centers (AICE). Directors and colleagues responsible for the management of VWD patients were invited to report in a detailed questionnaire how IGL recommendations about the assessment of the specific activities of von Willebrand Factor (VWF) could be applied at their local sites. Results from such a survey showed that bleeding assessment tools, VWF antigen, and factor VIII procoagulant are currently in use in all centers. The automated assays for platelet-dependent VWF activity with or without ristocetin described in IGL have been used since 2021 in 37/43 (86%) centers. Among other laboratory tests, VWF collagen binding, ristocetin-induced platelet agglutination, multimeric analysis, VWF propeptide, VWF:FVIII binding assay were available in 49, 63, 26, 7, and 28% of AICE, respectively. Analyses of VWF gene defects are available only at 3/43 (7%) centers. Desmopressin (DDAVP) infusion trials at diagnosis, with measurements of VWF activities at 1 and 4 hours post-DDAVP, is currently performed at 38/43 (88%) centers. Based on this information, a simplified clinical diagnosis using a few automated tests before and after DDAVP has been proposed. Such a diagnostic approach will be validated prospectively in a large cohort of Italian VWD patients.

Real-World Efficacy and Safety of Plasma-Derived Von Willebrand Factor-Containing Factor VIII Concentrates in Patients With Von Willebrand Disease in Italy.

Plasma-derived von Willebrand factor-containing factor VIII concentrates (pd-VWF/FVIII-C) are the mainstay of treatment in von Willebrand disease (VWD). Real-world data on efficacy and safety of these pd-VWF/FVIII-C are required. To retrospectively evaluate the efficacy and safety of pd-VWF/FVIII-C (Fanhdi® and Alphanate®, Grifols) in clinical practice in Italy. A multicentric, observational, retrospective study at 10 Italian centers was conducted. Eligible patients diagnosed with inherited VWD (ISTH criteria) were treated with either Fanhdi® or Alphanate® for bleeding episodes, prevention of surgical bleeding and secondary long-term prophylaxis (SLTP) according to clinical practice with medical records collected from January 2007 to December 2019. Efficacy/safety of pd-VWF/FVIII-C was assessed according to FDA-agreed objective criteria following regulatory procedures. Fifty-seven patients (M/F: 21/36) were enrolled in the study with the following VWD types: VWD1 (n = 29, 52%), VWD2A (n = 10, 18%), VWD2B (n = 7, 12%), VWD2M (n = 2, 4%), VWD2N (n = 1, 2%), VWD2 unclassified (n = 1, 2%), and VWD3 (n = 7, 12%). These pd-VWF/FVIII-C were used to manage 58 bleeding episodes (n = 24 patients), 100 surgeries (n = 47 patients), and 7 SLTP (n = 6 patients). Global clinical efficacy with these pd-VWF/FVIII-C was reported to be excellent/good in 85% of bleeding episodes, 98% of surgeries, and 100% of SLTP. As far as safety, no adverse-drug-related episodes, immunogenic or thrombotic events were reported. This study confirmed that Fanhdi® and Alphanate® were effective and safe in the management of bleeding episodes, the prevention of bleeding during surgeries and for SLTP in Italian patients with inherited VWD.

Tetrapod biodiversity in sambaquis from southern Brazil.

Fishermen-hunter-gatherers of sambaquis (Brazilian shell mounds) had an intimate affinity with marine-coastal environments, where they exploited a great variety of fish and mollusks that comprise the best documented fauna from sambaquis. However, other groups of animals as mammals, birds, reptiles, and amphibians are also present in these sites, but are relatively less studied. This paper is the first one focused exclusively on the Tetrapoda biodiversity of sambaquis and aimed to identify tetrapods of ten sites from southern Brazil. We present a faunal inventory and data regarding animal capture and environmental exploitation. We identified the specimens anatomically and taxonomically, analyzed them concerning fragmentation, and quantified the data for the number of identified specimens (NISP) and minimum number of individuals (MNI). Despite the high degree of fragmentation of remains, we identified 46 taxa. As expected, most were from marine animals: cetaceans (total NISP = 2,568 and MNI = 27), otariids (total NISP = 248 and MNI = 32), and seabirds (total NISP = 65 and MNI = 23), indicating great relevance of marine tetrapod fauna as a resource for sambaqui builders (79.39% of NISP). We thus document the close bond between fishermen-hunter-gatherers of sambaquis and the marine tetrapods in southern Brazil.

Follicular fluid concentration of soluble Human-G Leukocytic Antigen (sHLA-G) in in vitro fertilization cycles of women with and without peritoneal endometriosis.

OBJECTIVE: The objective of this research is to investigate the association between the concentrations of soluble human leukocyte G antigen (sHLA-G) in the follicular fluid (FF) in infertile patients with peritoneal endometriosis submitted to in vitro fertilization. METHODS: We performed a cross-sectional study, including ninety-six women undergoing in vitro fertilization (IVF) ageing ≤ 40 years. Infertile patients were classified into two groups: with endometriosis diagnosed by laparoscopy and without endometriosis due to tubal factor. ELISA measured soluble HLA-G in the FF of a pool of punctured (more than 17mm) follicles from women with endometriosis and without endometriosis who were subjected to ovulation induction for IVF. Embryos obtained after fertilization were classified according to the graduated embryo score (GES). RESULTS: Groups were comparables in terms of age, the number of follicles, AMH, FSH and all included reproductive outcomes. There was no association between sHLA-G concentrations and the average score of the generated embryos (p>0.05). Measurement of sHLA-G in the follicle fluid in women with endometriosis and without endometriosis (tubal factor) showed no significant difference (p>0.05). We also compared sHLA-G per follicle and per embryo, which were not different between both groups (p>0.05). CONCLUSIONS: Patients with peritoneal endometriosis submitted to IVF did not demonstrate an altered sHLA-G in the follicular fluid compared to the follicular fluid sHLA-G concentration in tubal factor patients. Also, this molecule was not linked to any other reproductive outcome.

Hereditary breast cancer next-generation sequencing (NGS) panel evaluation in the south region of Brazil: a novel BRCA2 candidate pathogenic variant is reported.

In this article, we delineate a loosely selected cohort comprising patients with a history of early-onset breast cancer and/or a familial occurrence of cancer. The aim of this study was to gain insights into the presence of breast cancer-related gene variants in a population from a micro-region in southern Brazil, specifically the Metropolitan Region of Curitiba. This area exhibits a highly genetically mixed population, mirroring the general characteristics of the Brazilian people. Comprehensive next-generation sequencing (NGS) multigene panel testing was conducted, involving the evaluation of twelve patients. Two pathogenic variants and one candidate pathogenic variant were identified: BRCA2:c.8878C>T, p.Gln2960Ter; CHEK2:c.1100delAG>A, p.Thr367Metfs*15 and BRCA2:c.3482dupG>GA, p.Asp1161Glufs*3, a novel variant, previously unpublished, is reported.

Seminal glucose levels: a prognostic factor of sperm survival to cryopreservation?

OBJECTIVE: Considering that glucose is an important component of seminal plasma and is a cryoprotectant at high concentrations, the aim of this study was to investigate the possible association of glucose levels in fresh semen with the sperm survival and motility rates following cryopreservation. METHODS: This was a prospective study including 149 men undergoing semen analysis due to male and/or female infertility. The seminal samples were analyzed according to the World Health Organization standards and glucose concentrations were measured using a dipstick glucometer. Samples were cryopreserved with Test Yolk Buffer-Gentamicine freezing medium under liquid nitrogen for an average of 120 days. The frozen aliquots were thawed at 37°C for 10 minutes and analyzed using the same methods and protocols used pre-freezing. RESULTS: Glucose levels ranged from 14 to 99 mg/dL and were similar in individuals with normal (n=100) vs. abnormal (n=49) semen analysis. The rates of sperm recovery (total, alive or motile sperm) in the cryopreserved samples did not change among samples with different glucose levels (p>0.05, Kruskal-Wallis ANOVA and Spearman's correlation coefficient). CONCLUSIONS: There appears to be no association between glucose levels in human semen samples and their resistance to cryopreservation.

Von Willebrand Disease: Gaining a global perspective.

INTRODUCTION: Recent guidelines for von Willebrand Disease (VWD) highlighted the challenges in diagnosis and management. Identifying the number of persons with VWD (PwVWD) internationally will help target support to aid diagnosis of PwVWD. AIM: To examine international registration rates of PwVWD, the influence of income status, geographical region and the age and sex profile. Cumulatively, these data will be used to inform future strategy from the World Federation of Haemophilia (WFH) to address unmet clinical and research needs. METHODS: Data from the 2018/2019 WFH Annual Global Survey (AGS) were analysed, providing a global perspective on VWD registration. RESULTS: Registration rates are lowest in South Asia (0.6/million population) and highest in Europe/Central Asia (50.9/million population, 0.005%), but below the expected prevalence rate (0.1%). National economic status impacted VWD registration rates, reflecting variation in access to optimal healthcare infrastructure. Females represented the majority of PwVWD globally, however, in low-income countries (LIC) males predominated. Age profile varied, with markedly higher rates of paediatric registrations in North America, Middle East and North Africa and South Asia. Rates of type 3 VWD registrations were significantly influenced by economic status (81% of VWD diagnoses in LIC), suggesting only the most severe VWD types are diagnosed in resource limited settings. CONCLUSION: Significant variation in registration rates of PwVWD exist internationally and is influenced by income status and the presence of HTC networks. Improved understanding of registration rates will enable targeting of advocacy to improve awareness, diagnosis and support for PwVWD internationally. KEY POINTS: Registration rates of People with Von Willebrand Disease (PwVWD) vary internationally and are influenced by national income status Although females represent the majority of PwVWD globally, in low income countries (LIC) males predominated, possibly related to stigma surrounding gynaecological bleeding. Rates of type 3 VWD registration were significantly influenced by economic status (81% of VWD diagnoses in LIC), suggesting only the most severe VWD types are diagnosed in resource limited settings.

von Willebrand factor neutralizing and non-neutralizing alloantibodies in 213 subjects with type 3 von Willebrand disease enrolled in 3WINTERS-IPS.

BACKGROUND: Type 3 von Willebrand disease (VWD) is the most severe form of this disease owing to the almost complete deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived products containing VWF or recombinant VWF rarely cause the development of alloantibodies against VWF that may be accompanied by anaphylactic reactions. OBJECTIVE: The objective of this study was to assess the prevalence of anti-VWF alloantibodies in subjects with type 3 VWD enrolled in the 3WINTERS-IPS. METHODS: An indirect in-house enzyme-linked immunosorbent assay has been used to test all the alloantibodies against VWF. Neutralizing antibodies (inhibitors) have been tested with a Bethesda-based method by using a VWF collagen binding (VWF:CB) assay. Samples positive for anti-VWF antibodies were further tested with Bethesda-based methods by using the semiautomated gain-of-function glycoprotein-Ib binding (VWF:GPIbM) and a VWF antigen (VWF:Ag) enzyme-linked immunosorbent assay. RESULTS: In total, 18 of the 213 (8.4%) subjects tested positive for anti-VWF antibodies and 13 of 213 (6%) had VWF:CB inhibitors. These 13 were among the 18 with anti-VWF antibodies. Of the 5 without VWF:CB inhibitors, 3 had non-neutralizing antibodies, 1 only inhibitor against VWF:GPIbM, and one could not be tested further. Ten of the 13 subjects with VWF:CB inhibitors also had VWF:GPIbM inhibitors, 6 of whom also had VWF:Ag inhibitors. Subjects with inhibitors were homozygous for VWF null alleles (11/14), homozygous for a missense variant (1/14), or partially characterized (2/14). CONCLUSIONS: Anti-VWF antibodies were found in 8.4% of subjects with type 3 VWD, whereas neutralizing VWF inhibitors were found in 6%, mainly in subjects homozygous for VWF null alleles. Because inhibitors may be directed toward different VWF epitopes, their detection is dependent on the assay used.

A need to increase von Willebrand disease awareness: vwdtest.com - A global initiative to help address this gap.

Von Willebrand disease (VWD) is an inherited bleeding disorder caused by quantitative or qualitative deficiencies in von Willebrand factor (VWF). People with VWD may experience excessive, recurrent or prolonged bleeding, particularly during menstruation, childbirth, surgery or following trauma. However, many VWD patients are undiagnosed, and therefore inadequately treated. Reasons for the underdiagnosis of VWD include its relatively mild symptoms, complex diagnosis, lack of awareness among non-specialist healthcare providers and the general population, and a lack of prioritisation of disorders disproportionately affecting females. The vwdtest.com platform was launched as part of a global initiative to raise awareness and improve diagnosis of VWD. Besides providing VWD-specific educational resources, the website includes an online bleeding self-assessment tool and offers diagnostic support for individuals, and their providers, who have a score suggestive of a bleeding disorder. vwdtest.com helps to address these unmet needs, especially in regions with limited access to educational and diagnostic resources.

Sugarcane multitrophic interactions: Integrating belowground and aboveground organisms.

Sugarcane is a crop of major importance used mainly for sugar and biofuel production, and many additional applications of its byproducts are being developed. Sugarcane cultivation is plagued by many insect pests and pathogens that reduce sugarcane yields overall. Recently emerging studies have shown complex multitrophic interactions in cultivated areas, such as the induction of sugarcane defense-related proteins by insect herbivory that function against fungal pathogens that commonly appear after mechanical damage. Fungi and viruses infecting sugarcane also modulate insect behavior, for example, by causing changes in volatile compounds responsible for insect attraction or repelling natural vector enemies via a mechanism that increases pathogen dissemination from infected plants to healthy ones. Interestingly, the fungus Fusarium verticillioides is capable of being vertically transmitted to insect offspring, ensuring its persistence in the field. Understanding multitrophic complexes is important to develop better strategies for controlling pathosystems affecting sugarcane and other important crops and highlights the importance of not only studying binary interactions but also adding as many variables as possible to effectively translate laboratory research to real-life conditions.

Sixth Åland Island Conference on von Willebrand disease.

INTRODUCTION: The sixth Åland Islands Conference on von Willebrand disease (VWD) on the Åland Islands, Finland, was held from 20 to 22 September 2018. AIM: The meeting brought together experts in the field of VWD from around the world to share the latest advances and knowledge in VWD. RESULTS AND DISCUSSION: The topics covered both clinical aspects of disease management, and biochemical and laboratory insights into the disease. The clinical topics discussed included epidemiology, diagnosis and treatment of VWD in different countries, management of children with VWD, bleeding control during surgery, specific considerations for the management of type 3 VWD and bleeding control in women with VWD. Current approaches to the management of acquired von Willebrand syndrome were also discussed. Despite significant advances in the understanding and therapeutic options for VWD, there remain many challenges to be overcome in order to optimise patient care. In comparison with haemophilia A, there are very few registries of VWD patients, which would be a valuable source of data on the condition and its management. VWD is still underdiagnosed, and many patients suffer recurrent or severe bleeding that could be prevented. Awareness of VWD among healthcare practitioners, including non-haematologists, should be improved to allow timely diagnosis and intervention. Diagnosis remains challenging, and the development of fast, simple assays may help to facilitate accurate and rapid diagnosis of VWD.

Use of the von Willebrand factor concentrate with low factor VIII content to manage patients with inherited von Willebrand disease requiring surgical or secondary long-term prophylaxis: An expert opinion paper from an Italian panel.

OBJECTIVES: The present review aims to summarize the state-of-the-art von Willebrand disease (VWD) treatment focusing on specific clinical settings (obstetrics, surgery, long-term prophylaxis and comorbidities) as well as on the use of a Von Willebrand factor (VWF) concentrate with low FVIII content. METHODS: Literature research and case reports. RESULTS AND CONCLUSIONS: Considering that patients affected by VWD have an intact ability to synthesize FVIII, in order to avoid excessive levels of FVIII, a highly purified plasma VWF concentrate with low FVIII content could be particularly useful in those patients and clinical circumstances at high thrombotic risk as well as for long-term prophylaxis. When deciding the optimal therapeutic strategy, physicians should take into account both the patient's history and the differences among available concentrates according to the clinical situations requiring treatment.

Von Willebrand factor propeptide and pathophysiological mechanisms in European and Iranian patients with type 3 von Willebrand disease enrolled in the 3WINTERS-IPS study.

BACKGROUND: Type 3 von Willebrand disease (VWD) is a severe bleeding disorder caused by the virtually complete absence of von Willebrand factor (VWF). Pathophysiological mechanisms of VWD like defective synthesis, secretion, and clearance of VWF have previously been evaluated using ratios of VWF propeptide (VWFpp) over VWF antigen (VWF:Ag) and factor (F)VIII coagulant activity (FVIII:C) over VWF:Ag. OBJECTIVE: To investigate whether the VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios may also be applied to understand the pathophysiological mechanism underlying type 3 VWD and whether VWFpp is associated with bleeding severity. METHODS: European and Iranian type 3 patients were enrolled in the 3WINTERS-IPS study. Plasma samples and buffy coats were collected and a bleeding assessment tool was administered at enrolment. VWF:Ag, VWFpp, FVIII:C, and genetic analyses were performed centrally, to confirm patients' diagnoses. VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios were compared among different variant classes using the Mann-Whitney test. Median differences with 95% confidence intervals (CI) were estimated using the Hodges-Lehmann method. VWFpp association with bleeding symptoms was assessed using Spearman's rank correlation. RESULTS: Homozygosity/compound heterozygosity for missense variants showed higher VWFpp level and VWFpp/VWF:Ag ratio than homozygosity/compound heterozygosity for null variants ([VWFpp median difference, 1.4 IU/dl; 95% CI, 0.2-2.7; P = .016]; [VWFpp/VWF:Ag median difference, 1.4; 95% CI, 0-4.2; P = .054]). FVIII: C/VWF:Ag ratio was similarly increased in both. VWFpp level did not correlate with the bleeding symptoms (r = .024; P = .778). CONCLUSIONS: An increased VWFpp/VWF:Ag ratio is indicative of missense variants, whereas FVIII:C/VWF:Ag ratio does not discriminate missense from null alleles. The VWFpp level was not associated with the severity of bleeding phenotype.

The effect of association of aspirin and omega 3 in rat temporomandibular joint with induced arthritis.

This study aimed to evaluate the effects of omega-3 (ω3) polyunsaturated fatty acids, in association with aspirin (AA), on the morphology of cytokine release in the temporomandibular joint (TMJ) of rats induced with rheumatoid arthritis (IR) by injecting 100 µL of complete Freund's adjuvant with bovine type II collagen at the tail base. Thirty-two adult male rats were divided into treatment groups: Sham, treated with 0.9% sodium chloride (NaCl) p.o.; IR-control, treated with 0.9% NaCl p.o.; IR-ω3 treated with ω3 PUFAS (85 mg/kg/day p.o.); and IR-ω3 + AA treated with ω3 (85 mg/kg/day p.o.) + AA (20 mg/kg/day i.p.). After maintained treatment for seven days, the animals were euthanized. Bilateral TMJs from each rat were removed and one was subjected to histological immunoassays and enzyme-linked immunosorbent assays to assess interleukin (IL)-1ß, tumor necrosis factor-α, and IL-10 levels. Data analysis was performed using the Kruskal-Wallis and Dunn tests. In the IR-ω3 and IR-ω3 + AA groups, the TMJ was greater than in the IR-control group (P < 0.0001). The addition of AA did not improve the effects of ω3 (P = 0.0698). Similarly, the addition of AA conferred no additional effects on the cytokine levels (P > 0.05); however, it increased the proteoglycan density, compared with ω3 alone. We found that ω3 exhibited anti-inflammatory activity in arthritic rats, and the addition of AA increased proteoglycan density, but did not affect cytokine expression.

Are green roofs the path to clean air and low carbon cities?

Green roofs, as part of urban green structures, have been pointed out as the solution to pursuit the goal of healthy cities. This study aims to investigate the direct, focused on meteorological changes, and indirect, related to both meteorological and emissions changes, impacts of green roofs on air quality (PM10, NO2 and O3). For that, the numerical modelling system composed by the WRF-SLUCM-CHIMERE models was applied to a 1-year period (2017), having as case study the Porto urban area. The EnergyPlus model was also applied to estimate the green roofs impacts on the building's energy needs and related impacts on air quality and atmospheric emissions. The analysis of the direct impacts showed that green roofs promote a temperature increase during the autumn and winter seasons and a temperature decrease during the spring and summer seasons. Both negative - concentrations increase - and positive - concentrations decrease - impacts were obtained for the primary, PM10 and NO2, and secondary, O3, air pollutants, respectively, due to changes in the dynamical structure of the urban boundary layer. The indirect effects of green roofs showed their potential to enhance the buildings energy efficiency, reducing the cooling and heating needs. These changes in energy consumption promoted an overall decrease of the environmental and economic indicators. Regarding air quality, the impact was negligible. The obtained results highlight the need for a multipurpose evaluation of the impacts of green roofs, with the different effects having to be traded off against each other to better support the decision-making process.

Genotypes of European and Iranian patients with type 3 von Willebrand disease enrolled in 3WINTERS-IPS.

Type 3 von Willebrand disease (VWD3) is a rare and severe bleeding disorder characterized by often undetectable von Willebrand factor (VWF) plasma levels, a recessive inheritance pattern, and heterogeneous genotype. The objective of this study was to identify the VWF defects in 265 European and Iranian patients with VWD3 enrolled in 3WINTERS-IPS (Type 3 Von Willebrand International Registries Inhibitor Prospective Study). All analyses were performed in centralized laboratories. The VWF genotype was studied in 231 patients with available DNA (121 [115 families] from Europe [EU], and 110 [91 families] from Iran [IR]). Among 206 unrelated patients, 134 were homozygous (EU/IR = 57/77) and 50 were compound heterozygous (EU/IR = 43/7) for VWF variants. In 22 patients, no or only one variant was found. A total of 154 different VWF variants (EU/IR = 101/58 [5 shared]) were identified among the 379 affected alleles (EU/IR = 210/169), of which 48 (EU/IR = 18/30) were novel. The variants p.Arg1659*, p.Arg1853*, p.Arg2535*, p.Cys275Ser, and delEx1_Ex5 were found in both European and Iranian VWD3 patients. Sixty variants were identified only in a single allele (EU/IR = 50/10), whereas 18 were recurrent (≥3 patients) within 144 affected alleles. Nine large deletions and one large insertion were found. Although most variants predicted null alleles, 21% of patients carried at least 1 missense variant. VWD3 genotype was more heterogeneous in the European population than in the Iranian population, with nearly twice as many different variants. A higher number of novel variants were found in the Iranian VWD3 patients.

Combined OX40 Agonist and PD-1 Inhibitor Immunotherapy Improves the Efficacy of Vascular Targeted Photodynamic Therapy in a Urothelial Tumor Model.

PURPOSE: Vascular targeted photodynamic therapy (VTP) is a nonsurgical tumor ablation approach used to treat early-stage prostate cancer and may also be effective for upper tract urothelial cancer (UTUC) based on preclinical data. Toward increasing response rates to VTP, we evaluated its efficacy in combination with concurrent PD-1 inhibitor/OX40 agonist immunotherapy in a urothelial tumor-bearing model. EXPERIMENTAL DESIGN: In mice allografted with MB-49 UTUC cells, we compared the effects of combined VTP with PD-1 inhibitor/OX40 agonist with those of the component treatments on tumor growth, survival, lung metastasis, and antitumor immune responses. RESULTS: The combination of VTP with both PD-1 inhibitor and OX40 agonist inhibited tumor growth and prolonged survival to a greater degree than VTP with either immunotherapeutic individually. These effects result from increased tumor infiltration and intratumoral proliferation of cytotoxic and helper T cells, depletion of Treg cells, and suppression of myeloid-derived suppressor cells. CONCLUSIONS: Our findings suggest that VTP synergizes with PD-1 blockade and OX40 agonist to promote strong antitumor immune responses, yielding therapeutic efficacy in an animal model of urothelial cancer.

A fertility-oriented method for histological processing of testicular biopsies in men with azoospermia.

The present study was designed to evaluate whether tissue preparation by glutaraldehyde and glycol methacrylate (G/GMA) improves the diagnostic assessment of testicular biopsies from azoospermic men when compared to the standard tissue preparation using Bouin's solution and paraffin. We prospectively included a total of 21 testicular biopsies of sexually mature men aged 29-50 years with infertility and azoospermia. One testicular biopsy fragment from each patient was processed by the G/GMA method, whereas another tissue fragment was contemporarily processed by the conventional Bouin/paraffin (B/P) method. The G/GMA method provided better resolution of cytological details of the seminiferous epithelium, changing the final diagnosis in four cases. The medians of Bergmann's spermatogenesis scores were 0.25 (interquartile range 0.04-0.88) for B/P preparations and 0.79 (interquartile range 0.17-0.96) for G/GMA preparations. Both techniques allowed accurate prediction of sperm recovery from the biopsies (B/P, area under the receiver operating characteristics [ROC] curve 0.88, 95% confidence interval [CI] 0.75-1.00; G/GMA, area under the ROC curve 0.94, 95% CI 0.86-1.00). We conclude that human testicular biopsy preparation with G/GMA improved image resolution under light microscopy and produced more reliable results for the evaluation of spermatogenesis in comparison with B/P, allowing a more precise fertility-oriented diagnosis in azoospermic men.Abbreviations: B/P: Bouin/paraffin; GMA: glycol methacrylate; G/GMA: glutaraldehyde and glycol methacrylate; ICSI: intracytoplasmic sperm injection; OA: obstructive azoospermia; NOA: nonobstructive azoospermia; TESE: testicular sperm extraction.