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.

'Two countries-two labs': the transnational gamete donation (TGD) programme to support egg donation.

PURPOSE: To evaluate the effectiveness, efficiency, and safety of a transnational gamete donation (TGD) programme based on the shipping of vitrified donor oocytes. METHODS: A retro-prospective observational study was conducted in the Assisted Reproductive Technology Center of the University Hospital of Florence, Italy. The study population included 622 consecutive donor oocyte cycles. A mean number of 6 vitrified oocytes per couple were shipped from two Spanish biobanks. In the receiving centre, gametes were warmed and inseminated and the subsequent embryo transfer (ET) was performed. The main outcome measurement was LBR. Secondary outcomes included oocyte survival rate, ICSI damage rate, normal fertilization, cleavage, and implantation rate (IR) in both 'fresh' and cryotransfer cycles. RESULTS: A total number of 3536 mature oocytes were warmed with 81.4% surviving oocytes. 2PN oocytes were 1941 with an ICSI normal fertilization rate of 70.4% and a cleavage rate of 93.4%; 857 day-3 embryos were transferred in 498 women, 63 blastocysts in 44. Couples with at least one vitrified embryo were 181 (32.3%). IR was 25.1% and 33.1% for day-3 ET and blastocyst stage respectively. Crude pregnancy rate and LBR after the first ET were 35.5% and 27% correspondingly with a conservative cumulative LBR of 34% and an optimal LBR of 51.4%. CONCLUSION: Imported vitrified donor oocytes retain their competence and are capable of resulting in ongoing pregnancies and healthy babies in a proportion comparable to other existing systems as egg donation with vitrification/warming in the same laboratory and transnational fresh oocyte donation.

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.

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.

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.

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.

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.

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.

Relevance of testicular histopathology on prediction of sperm retrieval rates in case of non-obstructive and obstructive azoospermia.

INTRODUCTION: The aim of our research was to establish the relevance of testicular histopathology on sperm retrieval after testicular sperm extraction in patients with non-obstructive azoospermia and in patients with obstructive azoospermia, who already underwent a previous failure testicular fine needle aspiration. METHODS: We evaluated a total of 82 azoospermic men, underwent testicular sperm extraction, referring to the Assisted Reproductive Technology Centre of the University of Florence, Italy between January 2008 and March 2017. A general and genital physical examination, scrotal and trans-rectal ultrasound, semen analysis, hormone measurements, including follicle-stimulating hormone, luteinizing hormone and total testosterone, were collected. RESULTS: Successful sperm retrieval was obtained in 36 men of total (43.9%). Successful sperm retrieval was 29.5% in non-obstructive azoospermia patients, while men with obstructive azoospermia, who, underwent a previous failure testicular fine needle aspiration, had sperm retrieval in 86% of cases. Mean luteinizing hormone was 6.55 IU/L, total testosterone 4.70 ng/mL, right testicular volume 13.7 mL and left testicular volume 13.6 mL. Mean Follicle-stimulating hormone was 13.45 IU/L in patients with negative sperm retrieval and 8.18 IU/L in men with successful sperm retrieval. According to histology, 20.7% had normal spermatogenesis, 35.3% hypospermatogenesis, 35.3% maturation arrest and 8.5% Sertoli cell-only syndrome. Successful sperm retrieval was 88.2% in patients with normal spermatogenesis, 24.1% in the maturation arrest group and 48.27% in patients with hypospermatogenesis, while negative sperm retrieval was reported in Sertoli cell-only syndrome patients. Seven cases with maturation arrest showed a successful sperm retrieval. CONCLUSION: Testicular histopathology after testicular sperm extraction offers important information on prediction of sperm retrieval and can guide the surgeon in choosing the more suitable therapeutic practice.

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.

Biological meaning of ubiquitination and DNA fragmentation in human spermatozoa.

The ubiquitin-proteasome is an ubiquitous system mainly devoted to protein degradation. The presence of ubiquitinated proteins in male gametes suggests a role for this system also in reproduction. Available evidence indicate that ubiquitin in spermatozoa may have a role in semen quality control, as ubiquitinated defective spermatozoa in the epididymis are subsequently phagocytosed by epididymal epithelial cells. Moreover, a role both in the regulation of mitochondrial inheritance in mammals (paternal mitochondria are eliminated and their ubiquitination appears to be important for this process) and in sperm-oocyte interaction at fertilization (which is inhibited by an inhibitor of proteasome) have been also suggested. We found that both morphologically normal and abnormal human spermatozoa in semen may be ubiquitinated and that the percentage of ubiquitinated sperm in the ejaculate positively correlates with normal morphology and motility, suggesting that sperm ubiquitination may have a positive role in sperm functions. It remains to be defined if and which patterns of ubiquitination of spermatozoa may distinguish between the different biological functions of this system. In an attempt to answer this question, we set up a method to detect simultaneously ubiquitination and DNA fragmentation by FACScan since the latter parameter is related to a poor quality of semen; in particular, abnormal morphology. We found that DNA fragmented human spermatozoa are also ubiquitinated. Studies are in progress to determine the correlation between the fraction of ubiquitinated-non DNA fragmented spermatozoa and parameters of semen analysis.

Lack of Histocompatibility Leukocyte Antigen-G expression in early embryos is not related to germinal defects or impairment of interleukin-10 production by embryos.

The expression of Histocompatibility Leukocyte Antigen (HLA)-G molecules is a mandatory prerequisite for the development of pregnancy but no hypotheses have yet been advanced regarding the lack of HLA-G modulation expression in a percentage of early embryos obtained by in vitro fertilization (IVF). One possible hypothetical model assumes that the absence of regulation of HLA-G or impaired interleukin (IL)-10 secretion could be related to germinal defects. We investigated the presence of soluble HLA-G antigens in supernatants of single embryo cultures from couples admitted to a second fertilization procedure; these couples showed a complete absence of HLA-G modulation in the first cycle's embryo supernatants (0/31). The results obtained in the second IVF cycle showed embryo supernatants positive for HLA-G (14/40), suggesting that the previous lack of antigen modulation is independent of germinal defects. Furthermore, since it has been reported that oocytes and early embryos can secrete IL-10, an anti-inflammatory cytokine produced by type 2 helper T cells that induces upregulation of HLA-G expression in monocytes and trophoblasts, we investigated the levels of IL-10 and soluble HLA-G in 40 embryo culture supernatants from 21 IVF cycles. No associations were observed between the presence of IL-10 and the production and concentrations of soluble HLA-G, or between IL-10 levels and pregnancy outcome. These results indicate that the lack of HLA-G production in early embryos is not related to germinal defects or to impairment in embryo IL-10 secretion but could be ascribed to possible uncorrected fertilization processes.

HLA-G expression in early embryos is a fundamental prerequisite for the obtainment of pregnancy.

Different mechanisms mediated by the expression of the HLA-class Ib HLA-G products are suggested to account for the induction of immune tolerance against the paternal antigens of the fetus during pregnancy. Soluble HLA-G antigens, mainly produced by cytotrophoblast cells at the materno-fetal interface and circulating in the body fluids, show a capacity analogous to that of membrane-boundstructures to inhibit NK cells. In the present report we have investigated, using specific ELISA, the presence of sHLA-G molecules in culture supernatants of early embryos obtained by in vitro fertilization (IVF) before transfer. The data obtained from the analysis of 285 supernatants corresponding to 101 IVF procedures (43 IVF, 58 intracytoplasmic sperm injection) identify two groups of patients on the basis of sHLA-G antigen presence. No differences in clinical parameters were observed between the groups, but positive embryo implantations occurred only in women showing sHLA-G molecules in culture supernatants (Fisher's exact p value 2.56 x 10(-3)). The results obtained indicate that expression of HLA-G products in embryo cells is a mandatory, but not sufficient, prerequisite for the development of pregnancy.