Can cytomegalovirus infection affect male reproductive function? Results of a retrospective single-centre analysis.

Our objective was to investigate whether the chronic cytomegalovirus (CMV) infection can affect semen parameters in men with couple infertility and to assess the impact of male CMV infection on the reproductive outcomes of CMV-seronegative women suffering from tubal factor. Group 1 included CMV IgG-seropositive men, Group 2 CMV IgG-seronegative patients. Seminal parameters, two-pronuclear (2PN) fertilization rate (FR), 1-2-3PN FR, cleavage rate (CR), miscarriage rate (MR), pregnancy rate (PR) and live birth rate (LBR) were collected. Two hundred and twenty-two men were included: 115 (51.8%) in Group 1 and 107 (48.2%) in Group 2. There was reported a low trend towards higher sperm concentration/ml, total sperm count and viability in CMV IgG-seronegative males, compared to CMV IgG-seropositive (p > .05). Semen volume, pH, motility and normal sperm morphology were similar among groups. Considering the subgroup of men, partners of CMV IgG-seronegative females, 65 couples (29.2%) were selected. Median 2PN FR was 67%, total FR 83%, CR 100%, PR/cycle 26.2%, MR 10.8%, LBR/cycle 15.4%. No significant differences were found regarding the reproductive outcomes between CMV IgG-seropositive men and those seronegative. CMV did not seem to play a key role in male reproductive function, as well as in influencing sperm fertility potential in the assisted reproductive outcomes.

A Biochemical Approach to Detect Oxidative Stress in Infertile Women Undergoing Assisted Reproductive Technology Procedures.

Oxidative stress plays a major role in critical biological processes in human reproduction. However, a reliable and biologically accurate indicator of this condition does not yet exist. On these bases, the aim of this study was to assess and compare the blood and follicular fluid (FF) redox status of 45 infertile subjects (and 45 age-matched controls) undergoing in vitro fertilization (IVF), and explore possible relationships between the assessed redox parameters and IVF outcomes. Reactive Oxygen Species (ROS) production, assessed by flow cytometry analysis in blood leukocytes and granulosa cells, significantly increased (p < 0.05) in infertile patients. Also, oxidative stress markers-ThioBarbituric Acid-Reactive Substances (TBARS) as an index of lipid peroxidation, and Oxygen Radical Absorbance Capacity (ORAC) to account for total antioxidant capacity, both assayed by fluorometric procedures-in blood and FF were significantly (p < 0.001) modified in infertile patients compared to the control group. Moreover, a significant correlation between blood redox markers and FF redox markers was evident. An ORAC/TBARS ratio, defined as the redox index (RI), was obtained in the plasma and FF of the patients and controls. In the patients, the plasma RI was about 3.4-fold (p < 0.0001) lower than the control, and the FF RI was about six-fold (p < 0.0001) lower than the control. Interestingly, both the plasma RI and FF RI results were significantly correlated (p < 0.05) to the considered outcome parameters (metaphase II, fertilization rate, and ongoing pregnancies). Given the reported findings, a strict monitoring of redox parameters in assisted reproductive techniques and infertility management is recommended.

Blood Leukocyte ROS Production Reflects Seminal Fluid Oxidative Stress and Spermatozoa Dysfunction in Idiopathic Infertile Men.

A large proportion of infertile men do not receive a clear diagnosis, being considered as idiopathic or unexplained cases due to infertility diagnosis based on standard semen parameters. Particularly in unexplained cases, the search for new indicators seems mandatory to provide specific information. In the etiopathogenesis of male infertility oxidative stress displays important roles by negatively affecting sperm quality and function. In this study, performed in a population of 34 idiopathic infertile men and in 52 age-matched controls, redox parameters were assessed in blood, leukocytes, spermatozoa, and seminal fluid and related to semen parameters. The main findings indicate that blood oxidative stress markers reflect seminal oxidative stress. Interestingly, blood leukocyte ROS production was significantly correlated to sperm ROS production and to semen parameters. Overall, these results suggest the potential employ of blood redox markers as a relevant and adjunctive tool for sperm quality evaluation aimed to preconception care.

5-Methyltetrahydrofolate and Vitamin B12 Supplementation Is Associated with Clinical Pregnancy and Live Birth in Women Undergoing Assisted Reproductive Technology.

The homocysteine pathway in the preconception period should be evaluated to highlight micronutrient deficiencies and warrants optimal multivitamin supplementation, before Assisted Reproduction, as preconception care. We conducted a retrospective study aimed at investigating the role of vitamin B complex (5-methyltetrahydrofolate, vitamin B12, vitamin B6) supplement use compared with the role of only folic acid supplement use, in relation to clinical pregnancy and live birth in infertile women undergoing homologous ART. We investigated 269 Caucasian women referred to the Centre for Assisted Reproductive Technology for homologous ART. In these women, 111 (Group A) were daily supplemented with vitamin B complex and 158 (Group B) with only folic acid. In group A the mean number of Metaphase II oocytes and the 2PN Fertilization Rate were higher in comparison to group A (p = 0.04; p = 0.05, respectively). A higher percentage of women in group A had a clinical pregnancy and live birth in comparison to group B (p = 0.01; p = 0.02, respectively). Vitamin B complex supplementation remained independently associated, after multivariable adjustment, with clinical pregnancy (OR 2.03, p = 0.008) and live birth (OR 1.83, p = 0.03). Women supplemented with 5-MTHF and vitamin B12, have a higher chance of clinical pregnancy and live birth in comparison to those supplemented with only folic acid.

Cardiometabolic and Thrombotic Risk Profile in Women Undergoing Oocyte Donation for Assisted Reproduction.

Background: The last two decades have seen a growing number of pregnancies in women who needed the donation of oocytes. With oocyte donation pregnancies, studies on obstetric outcomes among these women revealed an increased incidence of pre-eclampsia and pregnancy-induced hypertension. Furthermore, several studies have found a higher incidence of low birth weight, preterm birth, and delivery by cesarean section in oocyte donation rather than in women subjected to assisted reproduction techniques (ART) with autologous oocytes. Numerous studies have also shown a deep connection between cardiovascular and thrombotic risk factors and adverse pregnancy outcomes. In this setting, to strictly assess the preconceptional risk for women who undergo egg donation to achieve pregnancy, the aim of our study is to draw a detailed assessment of the vascular risk profile of patients with gamete donation ART indications through the evaluation of comorbidities and cardiometabolic and thrombophilic markers Materials and Methods: Patients undergoing ART with oocyte or sperm donation or double donation of gametes underwent a careful clinical assessment through a detailed personal and family anamnesis and they were evaluated for cardiometabolic and thrombophilic profile. Clinical and demographic characteristics, comorbidities, and biohumoral parameters were collected. The study was approved by the Regional Ethical Committee(Em 2018-017 CINECA 10189). Results: We evaluated 525 women. Around 73.1% were >40 years and 35% of them were older than 45 years. There was a high prevalence of dyslipidemias (58.1%), smoking habit (24.6%), a body mass index >25 in 28.6% of patients, a high abdominal circumference in 58.1% of cases, a prevalence of acquired thrombophilia in about 7% and hereditary of 19.2%. Around 39.2% of patients had total cholesterol >200 mg/dL, 19.5% had high-density lipoprotein <48 mg/dL and 43.6% had low-density lipoprotein >115 mg/dL, and 6.9% had triglyceride values >150 mg/dL. Conclusions: A careful assessment of the preconceptional status of patients undergoing ART programs with oocyte donation can be highly recommended.

Sperm retrieval by conventional testicular sperm extraction for assisted reproduction in patients with Zinner syndrome.

We present data from three Caucasian men with Zinner syndrome who attended our center for the treatment of primary couple's infertility. Each patient was scheduled for conventional testicular sperm extraction (cTESE) and cryopreservation. Sperm analysis confirmed absolute azoospermia. Patient 1 had right and left testis volumes of 24 mL and 23 mL, respectively; left seminal vesicle (SV) agenesis, severe right SV hypotrophy with right renal agenesis. Follicle-stimulating hormone (FSH) was 3.2 IU/L. Patient 2 exhibited right and left testis volumes of 18 mL and 16 mL, respectively; a left SV cyst of 32 × 28 mm, ipsilateral kidney absence, and right SV agenesis. FSH was 2.8 IU/L. Patient 3 showed a testicular volume of 10 mL bilaterally, a 65 × 46 mm left SV cyst, right SV enlargement, and left kidney agenesis. FSH was 32.0 IU/L. Sperm retrieval was successful in all patients. Nevertheless, cTESE should be performed on the day of oocyte retrieval.

Hepatitis B Surface Antigen Seropositive Men in Serodiscordant Couples: Effects on the Assisted Reproductive Outcomes.

PURPOSE: To evaluate the influence of hepatitis B virus (HBV) infection in men of serodiscordant couples on the reproductive outcomes. MATERIALS AND METHODS: A total of 134 infertile couples were included in this retrospective single-center cohort study. Sixty-six couples had hepatitis B surface antigen (HBsAg)-seropositive men and seronegative partners, while 68 couples were controls with both seronegative men and women. Overall, 134 fresh in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatments were performed. As the main outcome measures, on the day of the fresh IVF/ICSI cycle, we assessed seminal parameters Before and after sperm preparation techniques. Two-pronuclear (2PN) fertilization, 1-2-3PN fertilization, cleavage, miscarriage, pregnancy and live birth rates were collected. RESULTS: No significant differences were found between groups in terms of oocytes retrieved, oocytes injected and embryos obtained (p=0.64, p=0.97, and p=0.40, respectively). The 2PN fertilization rate (FR) was comparable among groups (p=0.51). The 1-2-3PN FR was significantly lower in the HBsAg group than in the control group (66.6% vs. 69.7%, respectively). The clinical pregnancy per cycle, implantation, miscarriage and live birth rate were comparable between the HBsAg group and the control group. The median sperm concentration/ml and total sperm count, measured at baseline and after sperm preparation, was comparable between groups (p>0.05). There was a trend toward significant lower progressive motility (35.0% vs. 55.0%; p<0.05) in the HBsAg group at baseline and after sperm preparation (p<0.05). CONCLUSIONS: HBV infected men have the same chance to became father, compared to seronegative patients.

Sperm morphology: What implications on the assisted reproductive outcomes?

OBJECTIVE: To evaluate the impact of sperm morphology (SM) on laboratory and pregnancy outcomes in conventional intracytoplasmic sperm injection (c-ICSI) cycles, using the egg donation model to minimize female confounding variables. MATERIALS AND METHODS: We retrospectively collected data of oocyte donation cycles from October 2016 to February 2020. Median seminal parameters, total (1-2-3PN) fertilization rate (FR), 2PN FR, cleavage rate (CR), implantation rate (IR), pregnancy rate (PR), miscarriage rate (MR), and live birth rate (LBR) were collected. The study population was divided into three groups: Group 1 with SM < 4%, Group 2 with SM between 4% and 6%, and Group 3 with SM > 6%. RESULTS: Of 741 fresh ICSI cycles and 4507 warmed oocytes were included. Male age was 46.0 (31.0-72.0) years, and recipients' age was 44.0 (29.0-54.0) years. Normal SM was 5.0% (1.0%-15.0%). Male age was negatively correlated with normal SM (P = .002; Rho -0.113). Oocyte survival rate was 83.3% (16.7%-100.0%). Total FR was 75.0% (11.1%-100.0%), 2PN FR was 66.7% (11.1%-100.0%) %, and CR was 100% (0.0%-100%). Comparing samples with SM > 6% and those with SM < 4%, 2PN FR was significantly higher in the first group (P = .04). No significant associations were found among groups in terms of CR. IR was 27.7%, resulting significantly higher when normal SM was > 6% (P < .01). Clinical PR was 36.0%, MR was 23.9%, and LBR was 25.9%. PR and LBR were significantly higher in samples with normal SM > 6%, compared to other groups (P = .02 and P < .01, respectively). CONCLUSIONS: Although c-ICSI technique allows the embryologist to select the best quality spermatozoa, male factor plays a key role in achieving successful assisted reproductive outcomes. Normal SM has been shown to have implications not only for laboratory outcomes, in terms of fertilization, but also for clinical findings, as regards implantation, pregnancy, and live birth.

Blood plasma miR-20a-5p expression as a potential non-invasive diagnostic biomarker of male infertility: A pilot study.

BACKGROUND: Recently, alterations in miRNAs expression profile in semen have been linked to damaged spermatogenesis, suggesting miRNAs could be used as potential infertility biomarkers. In previous animal studies, miR-20a-5p was found to be down-expressed in low motile spermatozoa, implying its potential target of genes associated with cell apoptosis. OBJECTIVE: To investigate miR-20a-5p expression in blood plasma of patients suffering from non-obstructive azoospermia (NOA), compared to normozoospermic controls. MATERIALS AND METHODS: Between January 2018 and December 2019, from 52 infertile couples eligible for the study, 24 couples were finally enrolled in this monocentric observational prospective pilot study. Patients were included into two groups: Group 1 comprised men with NOA (n = 14) and Group 2 fertile men partners of women with female tubal factor infertility (n = 10). All NOA patients underwent testicular sperm extraction. The expression of circulating miR-20a-5p in plasma samples was assessed by RT-qPCR. A relative quantification strategy was adopted using the 2-ΔCq method to calculate the target miR-20a-5p expression with respect to miR-16-5p as endogenous control. RESULTS: Median blood plasma miR-20a-5p was significantly higher in patients affected by NOA (0.16 2-ΔCt , range: 0.05-0.79 2-ΔCt ) than in fertile controls (0.06 2-ΔCt , range: 0.04-0.10 2-ΔCt ), P < .001. MiR-20a-5p was positively correlated with follicle-stimulating hormone (FSH) (rrho  = -0.490, P = .015) and luteinizing hormone (LH) (rrho  = -0.462, P = .023), and negatively correlated with serum total testosterone (TT) (rrho  = -0.534, P = .007) and right and left testicular size (rrho  = -0.473, P = .020 and rrho  = -0.471, P = .020, respectively). Successful sperm retrieval (SR) rate was 50.0%. Median value of miR-20a-5p did not differ significantly among patients with successful SR and those with negative SR. Testicular histological examination showed: hypospermatogenesis in 6/14 (42.8%), maturation arrest in 4/14 (28.6%), sertoli cell-only syndrome in 4/14 (28.6%). No significant differences in miR-20a-5p were found between histopathological patterns (P > .05). CONCLUSIONS: MiR-20a-5p could represent a novel non-invasive diagnostic biomarker of male infertility.

Reproductive Outcomes in Infertile Men With Spinal Cord Injury (SCI): A Retrospective Case-Control Analysis.

OBJECTIVE: To evaluate semen characteristics and reproductive outcomes after assisted ejaculation methods with fresh in vitro fertilization/intracytoplasmic sperm injection cycles in patients suffering from spinal cord injury (SCI), compared to controls, affected by idiopathic male infertility (non-SCI group). METHODS: SCI patients first underwent penile vibratory stimulation. Men "non-responders" to penile vibratory stimulation underwent electroejaculation. Third, testicular sperm aspiration was proposed. RESULTS: This retrospective monocenter case-control study included 193 couples: 53 couples in SCI group and 140 couples in non-SCI group. Overall, 210 fresh in vitro fertilization/intracytoplasmic sperm injection cycles were performed. Median semen volume of SCI was significantly lower, compared to non-SCI (1.5 mL vs 3.1 mL; P < .01). Median sperm concentration/mL and total sperm count was considerably higher in SCI. Mean sperm progressive motility was significantly lower in SCI (5.0% vs 35.0%; P < .01). Normal fertilization rate was significantly lower in SCI (46.0% vs 71.0%; P < .01). Total fertilization rate was 50.0% and 75%, respectively, in SCI and non-SCI groups. A trend toward higher pregnancy rates per cycle was found in non-SCI (31.4% vs 21.4%), or in the live birth rate, which was 27.1% and 20.0%, respectively. No significant differences were found in pregnancy, miscarriage, and live birth rates per cycle, between the 2 groups. CONCLUSION: The assisted ejaculation methods in SCI proved to be efficacious and safe to obtain viable sperm for assisted reproductive technologies. Overall, pregnancy and live birth rates were similar to non-SCI patients. Thus, SCI men have the same opportunity to father biological children, compared to men without SCI.

Redox status assessment in infertile patients with non-obstructive azoospermia undergoing testicular sperm extraction: A prospective study.

BACKGROUND: Oxidative stress (OS) is one of the most prevalent causes of sperm damage, through the toxic effects of endogenously generated hydrogen peroxide, superoxide anion, and hydroxyl radicals. Peripheral leukocytes represent a feasible model for studying the pathophysiology of OS-mediated homeostasis, which can be responsible for cell dysfunction and cell injury. OBJECTIVE: To evaluate the redox status in patients with non-obstructive azoospermia (NOA), establishing the potential role exerted by reactive oxygen species (ROS) in the genesis of testicular secretory injury. MATERIAL AND METHODS: From May 2018 to March 2019, 39 patients were enrolled in this prospective single-center cohort study and divided into two groups. Group 1 included 19 patients with NOA, and Group 2 included 20 normozoospermic men, partners of women with infertility tubal factor. All patients underwent serum blood tests. NOA underwent testicular sperm extraction (TeSE). ROS production (in lymphocytes, monocytes, and granulocytes) was assessed by fluorescence-activated cell sorting (FACS) analysis. Plasma oxidative stress was evaluated by lipid peroxidation markers (MDA) and total antioxidant capacity (TAC) both assessed by fluorometric techniques. RESULTS: Mean lymphocyte ROS production resulted 967.0 ± 224.5 vs 728.0 ± 98.0 (NOA vs Controls, P < .001), monocyte ROS resulted 2102.5 ± 517.5 vs 1253 ± 171 (P < .001), and granulocyte ROS were 2366.5 ± 595.4 vs 1751.0 ± 213.0 (P < .001). Significant increases plasma lipid peroxidation markers were found in NOA patients compared with controls (2.7 ± 0.8 vs 0.37 ± 0.2 nmol/mL, P < .001). Significant decreased TAC was evident in NOA compared with controls (13.4 ± 3.9 vs 3.0 ± 0.2 µmol/mL Trolox equivalents, P < .001). No significant differences were found in blood leukocyte subpopulations ROS production, plasma lipid peroxidation, and TAC comparing groups (positive vs negative sperm retrieval, P > .05). CONCLUSION: ROS production can be directly related to disorders of spermatogenesis, leading to severe conditions of male infertility, including azoospermia.

Successful pregnancy in double factor infertility: Sperm in vitro culture by modified testicular fine-needle aspiration.

INTRODUCTION: Infertility may depend up to 27% of couples on both partners. In patients with obstructive azoospermia, testicular fine-needle aspiration represents a good option to retrieve spermatozoa, in order to perform an assisted reproductive treatment. In vitro maturation of testicular spermatozoa could be the better choice of treatment in view of the increased motility, improving fertilization and pregnancy rates. CASE DESCRIPTION: A 34-year-old azoospermic man and his 33-year-old partner referred for treatment of simultaneous male and female infertility factor. The woman presented a diminished ovarian reserve, with serum follicle stimulating hormone value of 27.15 IU/L. The man underwent trans-rectal and testicular ultrasounds that detected the congenital absence of proximal vas deferens on the right side and the absence of seminal vesicle and distal vas deferens on the left side. We proposed a chance to have their own biological child. The man underwent modified testicular fine-needle aspiration using a 18-gauge butterfly needle. Sperm retrieval was successful with 0.001 × 106 spermatozoa/mL and absence of motility. Testicular sperm suspension was cultured for 24 h to identify sperm viability, achieving 10% of sperm motility. Two metaphase II oocytes were retrieved and processed with intracytoplasmic sperm injection. Clinical pregnancy with live birth was obtained. CONCLUSION: Performing modified testicular fine-needle aspiration increases successful sperm retrieval. Testicular sperm in vitro culture for 24 h proved to be a real and practical technique to increase sperm motility, in order to select mature and viable spermatozoa and improve successful intracytoplasmic sperm injection outcomes.

Testicular Fine-Needle Aspiration for Sperm Retrieval in Azoospermia: A Small Step toward the Technical Standardization.

PURPOSE: The aim was to describe our preliminary experience performing testicular fine-needle aspiration (TEFNA) with a larger needle in infertile patients with obstructive azoospermia, and to provide a systematic literature review of the different testicular sperm aspiration techniques, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement recommendations. MATERIALS AND METHODS: We prospectively collected data between March 2017 and June 2018. All men underwent bilateral TEFNA under analgo-sedation, using a larger disposable 18-gauge butterfly needle with 60 mL Luer-Lock syringe attached to it. RESULTS: Thirty consecutive patients were enrolled. Median operative time was 16 minutes (interquartile range [IQR]: 12-30 minutes). No intraoperative complications occurred. Two/thirty patients (6.7%) reported postoperative adverse events: 1 patient had prolonged orchialgia, 1 patient presented scrotal hematoma. Successful sperm retrieval was found in 28/30 cases (93.3%). Median sperm concentration was 0.05 ×106/mL (IQR: 0.001-0.1 ×106/mL). Median total sperm motility was 10% (IQR: 0%-15%). In 20/30 men (66.7%) sperm retrieved was used for fresh intracytoplasmic sperm injection cycle, in 8/30 (26.7%) sperm cryopreservation was necessary, because on the day of sperm retrieval the female resulted not responder to ovarian stimulation. In this cases mean number of 3 (IQR: 1-4) bio system straws was cryopreserved. CONCLUSIONS: TEFNA with 18-gauge needle proved to be a feasible, safe and effective treatment, even if future prospective studies will be addressed to clarify what type of azoospermia benefits from this procedure, and if a larger needle permits to improve Assisted Reproductive Technologies (ART) outcomes.

Male Inflammatory Parameters Are not Useful to Predict the Outcomes of Intracytoplasmic Sperm Injection: Results from a Cross-Sectional Study.

PURPOSE: The relationship between male systemic inflammation and fertility seems intriguing, but no data about its impact on the assisted reproductive technology outcomes has been reported. Here, we aimed to evaluate the prognostic role of male systemic inflammatory parameters in intracytoplasmic sperm injection (ICSI) outcomes prediction, in couples undergoing an ovum donation program. MATERIALS AND METHODS: From January 2016 to December 2017, one hundred-ten couples were considered for this cross-sectional study. Neutrophil-to-lymphocyte ratio (NLR), monocyte-to-eosinophil ratio (MER), platelet-to-lymphocyte ratio (PLR), seminal parameters, fertilization rate (FR), cleavage rate (CR), pregnancy rate (PR) were evaluated. Male patients were divided into Group A with FR ≤70%, Group B with FR >70%. RESULTS: Overall, FR was 74.5%, CR 90.9%, PR 41.8%. Group A included 43 patients, Group B 67 men. Group A showed a median NLR of 1.55, PLR of 106.09, MER of 2.33. Group B reported a median NLR of 1.64, PLR 109.0, MER 2.76. We found no statistically differences between two groups with respect to NLR, PLR, MER (p=0.90, p=0.70, p=0.96, respectively). The age-adjusted linear regression analysis demonstrated only a relationship between NLR and sperm motility count (r=-0.02; p<0.05). Using the univariate logistic regression analysis, we found no significant associations. CONCLUSIONS: We did not find any relationship between ICSI outcomes and male inflammation parameters.

Impact of advanced paternal age on the intracytoplasmic sperm injection (ICSI) outcomes in donor egg cycles.

BACKGROUND: The purpose of this study is to assess the impact of paternal age on the oocyte-donation outcomes in intracytoplasmic sperm injection (ICSI) cycles. METHODS: Two hundred and seventy-eight infertile couples were retrospectively involved. Inclusion criteria were: infertility from almost 1 year, normal or sub-fertile seminal parameters, overall oocyte survival rate greater than 85%. Baseline characteristics included male age, recipient age, male body mass index (BMI), smoking, drinking status. Main outcome measures: fertilization rate (FR), cleavage rate (CR), pregnancy rate (PR). RESULTS: Patients were categorized in group 1 ≤45, group 2 >45 years. A total of 1,724 frozen oocytes were included. After warming, 1,642 oocytes survived. Median overall oocyte survival rate was 100% [interquartile range (IQR), 85-100%]. Median male age was 44±5.60 years (IQR, 31-70 years). Median recipients age was 42±3.62 years (IQR, 29-50 years). Group 1 included 166 men, group 2 112 men. Two hundred and seventy-eight fresh ICSI cycle were performed. "Two-pronuclear" (2PN) FR was 72.6%±0.20%, CR 93.0%±0.16%, PR 39.6%. Miscarriage rate was 25.5%. Live birth rate per cycles was 29.5%. Comparison between group 1, group 2 and ICSI outcomes confirmed an association with FR, resulting 80.0% (IQR, 67.0-83.0%) and 67.0% (IQR, 50.0-80.0%), respectively (P<0.01). There were no significant differences between the two groups with respect to seminal parameters. CONCLUSIONS: It would be recommended more emphasis on the advancing male age when counselling older couples who undergo egg donation program.

Novel method of histopathological analysis after testicular sperm extraction in patients with nonobstructive and obstructive azoospermia.

OBJECTIVE: To assess whether the "testicular pool" could be used for histological analysis and whether it gave more accurate information than the standard testicular biopsy. METHODS: Between January 2017 and March 2018, this single-center prospective study included 60 azoospermic men undergoing conventional bilateral testicular sperm extraction. Six samples were excised from each testicle and transferred to an embryologist. One additional biopsy was randomly taken from each testis for a histological analysis. After processing, the testicular pool was also sent for a histological analysis, which showed normal spermatogenesis (NS), hypospermatogenesis (HYPO), maturation arrest (MA), Sertoli cell-only syndrome (SCOS), and tubular atrophy (TA). RESULTS: Twenty of the 60 patients (33.3%) had obstructive azoospermia (OA), while the remaining 40 (66.6%) had nonobstructive azoospermia. Their mean age was 40.5 years. All patients with OA had previously undergone unsuccessful testicular fine-needle aspiration. Successful sperm retrieval (SSR) occurred in 93.3% of patients. Histological analysis of the testicular biopsy revealed NS in 12 patients (20%), HYPO and TA in 28 patients (46.6%), MA in eight patients (13.3%), and SCOS in 12 patients (20%). The testicular pool analysis showed NS in 12 patients (20%), HYPO and TA in 44 patients (73.3%), MA in four patients (6.6%), and SCOS in no patients. In four patients with MA (6.6% of the total sample) and 12 patients with SCOS (20% of the total sample) according to the standard testicular biopsy, the embryologist found SSR with cryopreservation. Overall, in 44 patients (73.3%), the testicular pool analysis confirmed the histological findings of the standard testicular biopsy. In the 16 cases (26.6%) with a discrepancy between the single-biopsy histological findings and SSR, the testicular pool analysis confirmed the embryological data on SSR. CONCLUSION: The testicular pool proved to be easily analyzable, practical, manageable, and more accurate for predicting sperm retrieval than standard testicular biopsy.

Potential Pitfalls of SDH Immunohistochemical Detection in Paragangliomas and Phaeochromocytomas Harbouring Germline SDHx Gene Mutation.

BACKGROUND/AIM: Germline mutations in any of the succinate dehydrogenase (SDH) genes result in destabilization of the SDH protein complex and loss of SDHB expression at immunohistochemistry. SDHA is lost together with SDHB in SDHA-mutated tumours, but its expression is retained in tumours with other SDH mutations. We investigated whether SDHA/SDHB immunohistochemistry is able to identify SDH-related tumours in a retrospective case series of phaeochromocytomas (PCCs) and paragangliomas (PGLs). MATERIALS AND METHODS: SDHA and SDHB immunostaining was performed in 13 SDH gene-mutated tumours (SDHB: n=3; SDHC: n=1; SDHD: n=9) and 16 wild-type tumours. Protein expression by western blot analysis and enzymatic activity were also assessed. RESULTS: Tumours harbouring SDH gene mutations demonstrated a significant reduction in enzymatic activity and protein expression when compared to wild-type tumours. SDHB immunostaining detected 76.9% of SDH mutated PCCs/PGLs (3/3 SDHB-mutated samples; 1/1 SDHC-mutated sample; 6/9 SDHD-mutated samples). In three SDHD-related tumours with the same mutation (p.Pro81Leu), positive (n=2) or weakly diffuse (n=1) SDHB staining was observed. All wild-type PCCs/PGLs exhibited SDHB immunoreactivity, while immunostaining for SDHA was positive in 93.8% cases and weakly diffuse in one (6.2%). SDHA protein expression was preserved in all tumours with mutations. CONCLUSION: SDHA and SDHB immunohistochemistry should be interpreted with caution, due to possible false-positive or false-negative results, and ideally in the setting of quality assurance provided by molecular testing. In SDHD mutation, weak non-specific cytoplasmic staining occurs commonly, and this pattern of staining can be difficult to interpret with certainty.

Sunitinib in the therapy of malignant paragangliomas: report on the efficacy in a SDHB mutation carrier and review of the literature.

Metastatic pheochromocytomas (PHEOs) and paragangliomas (sPGLs) are rare neural crest-derived tumors with a poor prognosis. About 50% of them are due to germ-line mutations of the SDHB gene. At present, there is no cure for these tumors. Their therapy is palliative and represented by different options among which antiangiogenic drugs, like sunitinib, have been hypothesized to be effective especially in malignant SDHB mutated tumors. We report the effects of sunitinib therapy in a SDHB mutation carrier affected by a malignant sPGL. During 101 weeks of therapy at different doses, sunitinib was able to cause a partial response and then a stable disease for a total of 78 weeks. This favorable response is the longest, out of the 35 so far reported in the literature, registered in a patient treated exclusively with sunitinib but, similarly to the other responses, the effect was limited in time. From our analysis of the scanty data present in the literature, the effect of sunitinib does not seem to be different among wild-type patients and those carrying a cluster 1 germ-line mutation. Sunitinib seems able to slow the disease progression in some patients with malignant PHEO/PGL and therefore may represent a therapeutic option, although randomized controlled studies are needed to assess its efficacy definitively in the treatment of these aggressive tumors.

Metformin as a new anti-cancer drug in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare heterogeneous malignancy with poor prognosis. Since radical surgery is the only available treatment, more specific and effective drugs are urgently required. The anti-diabetic drug metformin has been associated with a decreased cancer prevalence and mortality in several solid tumors, prompting its possible use for ACC treatment.This paper evaluates the in vitro and in vivo anti-cancer effects of metformin using the ACC cell model H295R.Metformin treatment significantly reduces cell viability and proliferation in a dose- and time-dependent manner and associates with a significant inhibition of ERK1/2 and mTOR phosphorylation/activation, as well as with stimulation of AMPK activity. Metformin also triggers the apoptotic pathway, shown by the decreased expression of Bcl-2 and HSP27, HSP60 and HSP70, and enhanced membrane exposure of annexin V, resulting in activation of caspase-3 apoptotic effector. Metformin interferes with the proliferative autocrine loop of IGF2/IGF-1R, which supports adrenal cancer growth. Finally, in the ACC xenograft mouse model, obtained by subcutaneous injection of H295R cells, metformin intraperitoneal administration inhibits tumor growth, confirmed by the significant reduction of Ki67%.Our data suggest that metformin inhibits H295R cell growth both in vitro and in vivo. Further preclinical studies are necessary to validate the potential anti-cancer effect of metformin in patients affected by ACC.

15 YEARS OF PARAGANGLIOMA: Metabolism and pheochromocytoma/paraganglioma.

The discovery of SDHD as a pheochromocytoma/paraganglioma susceptibility gene was the prismatic event that led to all of the subsequent work highlighting the key roles played by mitochondria in the pathogenesis of these tumors and other solid cancers. Alterations in the function of tricarboxylic acid cycle enzymes can cause accumulation of intermediate substrates and subsequent changes in cell metabolism, activation of the angiogenic pathway, increased reactive oxygen species production, DNA hypermethylation, and modification of the tumor microenvironment favoring tumor growth and aggressiveness. The elucidation of these tumorigenic mechanisms should lead to novel therapeutic targets for the treatment of the most aggressive forms of pheochromocytoma/paraganglioma.