Semaphorin 3C exacerbates liver fibrosis.

BACKGROUND AND AIMS: Chronic liver disease is a growing epidemic, leading to fibrosis and cirrhosis. TGF-ß is the pivotal profibrogenic cytokine that activates HSC, yet other molecules can modulate TGF-ß signaling during liver fibrosis. Expression of the axon guidance molecules semaphorins (SEMAs), which signal through plexins and neuropilins (NRPs), have been associated with liver fibrosis in HBV-induced chronic hepatitis. This study aims at determining their function in the regulation of HSCs. APPROACH AND RESULTS: We analyzed publicly available patient databases and liver biopsies. We used transgenic mice, in which genes are deleted only in activated HSCs to perform ex vivo analysis and animal models. SEMA3C is the most enriched member of the semaphorin family in liver samples from patients with cirrhosis. Higher expression of SEMA3C in patients with NASH, alcoholic hepatitis, or HBV-induced hepatitis discriminates those with a more profibrotic transcriptomic profile. SEMA3C expression is also elevated in different mouse models of liver fibrosis and in isolated HSCs on activation. In keeping with this, deletion of SEMA3C in activated HSCs reduces myofibroblast marker expression. Conversely, SEMA3C overexpression exacerbates TGF-ß-mediated myofibroblast activation, as shown by increased SMAD2 phosphorylation and target gene expression. Among SEMA3C receptors, only NRP2 expression is maintained on activation of isolated HSCs. Interestingly, lack of NRP2 in those cells reduces myofibroblast marker expression. Finally, deletion of either SEMA3C or NRP2, specifically in activated HSCs, reduces liver fibrosis in mice. CONCLUSION: SEMA3C is a novel marker for activated HSCs that plays a fundamental role in the acquisition of the myofibroblastic phenotype and liver fibrosis.

HAPLN1 potentiates peritoneal metastasis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) frequently metastasizes into the peritoneum, which contributes to poor prognosis. Metastatic spreading is promoted by cancer cell plasticity, yet its regulation by the microenvironment is incompletely understood. Here, we show that the presence of hyaluronan and proteoglycan link protein-1 (HAPLN1) in the extracellular matrix enhances tumor cell plasticity and PDAC metastasis. Bioinformatic analysis showed that HAPLN1 expression is enriched in the basal PDAC subtype and associated with worse overall patient survival. In a mouse model for peritoneal carcinomatosis, HAPLN1-induced immunomodulation favors a more permissive microenvironment, which accelerates the peritoneal spread of tumor cells. Mechanistically, HAPLN1, via upregulation of tumor necrosis factor receptor 2 (TNFR2), promotes TNF-mediated upregulation of Hyaluronan (HA) production, facilitating EMT, stemness, invasion and immunomodulation. Extracellular HAPLN1 modifies cancer cells and fibroblasts, rendering them more immunomodulatory. As such, we identify HAPLN1 as a prognostic marker and as a driver for peritoneal metastasis in PDAC.

In male Hodgkin lymphoma patients, impaired fertility may be improved by non-gonadotoxic therapy.

The outcome of patients with Hodgkin lymphoma (HL) has improved significantly in recent years, and now attention is increasingly being focused on the well-being of these young patients. This study aimed to analyse the influence of HL and its treatment on the spermatogenic status of 46 male HL patients with available spermiograms, treated between 2008 and 2016. Analysing prognostic factors at diagnosis, we found that the number of spermatozoa was reduced in stage III-IV; motility and vitality were reduced in stage III-IV and in the presence of B symptoms; and abnormal forms were increased in patients with elevated erythrocyte sedimentation rate (ESR) and low albumin. Furthermore, we found that haematopoietic stem cell transplantation (HSCT) was associated with a severe impairment of fertility in terms of sperm motility. In HL-treated patients who did not undergo HSCT we found a statistically significantly improved fertility in terms of motility. In this study, we found that HSCT induced infertility in the majority of male patients with HL, but that first-line treatment could improve the impaired fertility status caused by disease. Further studies are needed in larger case series to investigate risk factors for impaired fertility at HL diagnosis and after treatment.

Gene Expression Profiles Controlled by the Alternative Splicing Factor Nova2 in Endothelial Cells.

Alternative splicing (AS) plays an important role in expanding the complexity of the human genome through the production of specialized proteins regulating organ development and physiological functions, as well as contributing to several pathological conditions. How AS programs impact on the signaling pathways controlling endothelial cell (EC) functions and vascular development is largely unknown. Here we identified, through RNA-seq, changes in mRNA steady-state levels in ECs caused by the neuro-oncological ventral antigen 2 (Nova2), a key AS regulator of the vascular morphogenesis. Bioinformatics analyses identified significant enrichment for genes regulated by peroxisome proliferator-activated receptor-gamma (Ppar-γ) and E2F1 transcription factors. We also showed that Nova2 in ECs controlled the AS profiles of Ppar-γ and E2F dimerization partner 2 (Tfdp2), thus generating different protein isoforms with distinct function (Ppar-γ) or subcellular localization (Tfdp2). Collectively, our results supported a mechanism whereby Nova2 integrated splicing decisions in order to regulate Ppar-γ and E2F1 activities. Our data added a layer to the sequential series of events controlled by Nova2 in ECs to orchestrate vascular biology.

Endogenous luteinizing hormone concentration and IVF outcome during ovarian stimulation in fixed versus flexible GnRH antagonist protocols: An RCT.

BACKGROUND: Luteinizing hormone (LH) is essential for normal follicular development and oocyte maturation. In particular, fluctuations of LH during the follicular phase have a significant impact on morphological and functional changes of the oocyte and determine its meiotic status and ability to be fertilized. OBJECTIVE: This prospective randomized controlled trial examined effects of endogenous follicular phase LH levels on oocyte maturity and IVF outcomes in fixed vs. flexible in vitro fertilization. MATERIALS AND METHODS: Normo-ovulatory women age <39 yr (n=213) were randomized to fixed or flexible gonadotrophin-releasing hormone (GnRH) antagonist protocols. Follicular phase LH, estradiol, and progesterone profiles were measured. Oocytes retrieved, implantation rate, and pregnancy rate were compared between the two groups. RESULTS: LH profiles were similar in both protocols. A lower trend of LH values at the end of ovarian stimulation correlated significantly with a higher pregnancy rate, regardless of protocol (p=0.02). Estradiol levels were statistically different with respect to time points within treatment groups (p<0.0001), but not between groups (p=0.43), or pregnancy outcomes (p=0.2595). Progesterone profiles were similar between groups. No differences were found in retrieved oocytes numbers, fertilization rate or embryos obtained. Significantly, younger age and a higher number of antral follicles were correlated with positive results. CONCLUSION: Fixed and flexible GnRH antagonist protocols did not produce an oscillation of endogenous LH values correlated to the outcome of ovarian stimulation.

Dynamics of the development of multiple follicles by early versus late hCG administration in ART program.

OBJECTIVE: To evaluate, in patients stimulated with recombinant FSH and GnRH antagonists, whether triggering the final maturation of oocytes affects IVF outcomes. STUDY DESIGN: Five hundred and six IVF procedures were divided into three groups according to the timing of hCG administration: when at least 2 follicles reached the diameter of 17 mm, at least 2 follicles reached 18 mm and at least 2 follicles reached 20 mm. The main outcome was the number of mature oocyte that was the dependent variable of a multivariate model whose independents were, age, AFC, hCG timing, E2 levels at hCG day, number of follicles in different categories of dimension. Secondary endpoints were to compare fertilization, implantation and pregnancy rates in a multilevel multivariate model whose covariates were age, BMI, AFC, embryo quality and cause of infertility. RESULTS: Timing did not result a statistically significant factor influencing the number of oocytes collected, which was influenced by age, AFC, number of follicles between 12.1 and 15.9 mm and E2 levels. Implantation rate and pregnancy rate appear to be affected only by embryo quality. CONCLUSION: The number of oocytes collected and the probability of pregnancy are not associated with the time of hCG administration.

Fertility preservation in males with cancer: 16-year monocentric experience of sperm banking and post-thaw reproductive outcomes.

BACKGROUND: Anticancer treatments can impair male fertility. Cryopreservation of semen is an efficient procedure for fertility preservation. The aim of this study was to evaluate pre-freeze semen parameters among the various types of cancer, post-thaw sperm viability and reproductive outcome of samples used for assisted reproductive treatment (ART). METHODS: This study included 721 men with cancer that had their semen cryopreserved in our bank in 1999-2015. Semen analysis and cryopreservation were performed before the start of antineoplastic treatment, according to the World Health Organization recommendations, European Commission and Italian law. RESULTS: Among the 721 patient, 196 had seminoma of the testis, 173 Hodgkin's lymphoma, 108 mixed testicular tumors, 89 germ cell tumors, 67 other tumors, 46 hematological tumors, and 42 non-Hodgkin's lymphoma. The mean age of patients was significantly lower in Hodgkin's lymphoma compared to other tumors. Statistically significant lower volume, sperm count and number of straws stored were observed respectively in Hodgkin's lymphoma, mixed testicular tumor and hematological tumors. Nineteen patients used their frozen semen for 20 ART cycles. After thawing a significant reduction of motility and vitality was recorded. A lower fertilization rate was observed in patients affected by testicular tumor and lymphoma (35.42% and 50%) compared with other cancers (71.43%). No significant differences were observed in terms of cleavage and implantation rates. A total of five pregnancies and seven healthy newborns were achieved. CONCLUSIONS: Fertility preservation before gonadotoxic therapy is of great importance to patients with cancer and must be indicate before the start of treatment.

The Effects of Chronic Lifelong Activation of the AHR Pathway by Industrial Chemical Pollutants on Female Human Reproduction.

Environmental chemicals, such as heavy metals, affect female reproductive function. A biological sensor of the signals of many toxic chemical compounds seems to be the aryl hydrocarbon receptor (AHR). Previous studies demonstrated the environmental of heavy metals in Taranto city (Italy), an area that has been influenced by anthropogenic factors such as industrial activities and waste treatments since 1986. However, the impact of these elements on female fertility in this geographic area has never been analyzed. Thus, in the present study, we evaluated the AHR pathway, sex steroid receptor pattern and apoptotic process in granulosa cells (GCs) retrieved from 30 women, born and living in Taranto, and 30 women who are living in non-contaminated areas (control group), who were undergoing in vitro fertilization (IVF) protocol. In follicular fluids (FFs) of both groups the toxic and essential heavy metals, such as chromiun (Cr), Manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb), were also analyzed. Higher levels of Cr, Fe, Zn and Pb were found in the FFs of the women from Taranto as compared to the control group, as were the levels of AHR and AHR-dependent cytochrome P450 1A1 and 1B1; while CYP19A1 expression was decreased. The anti-apoptotic process found in the GCs of women fromTaranto was associated with the highest levels of progesterone receptor membrane component 1 (PGRMC1), a novel progesterone receptor, the expression of which is subjected to AHR activated by its highest affinity ligands (e.g., dioxins) or indirectly by other environmental pollutants, such as heavy metals. In conclusion, decreased production of estradiol and decreased number of retrieved mature oocytes found in women from Taranto could be due to chronic exposure to heavy metals, in particular to Cr and Pb.

Anti-laminin-1 antibodies in serum and follicular fluid of women with Hashimoto's thyroiditis undergoing in vitro fertilization.

The aim of this study is to evaluate the presence of anti-laminin-1 antibodies (aLN-1) in sera and follicular fluid (FF) of infertile women affected by Hashimoto's thyroiditis (HT) undergoing in vitro fertilization (IVF) and its impact on oocyte maturation and IVF outcome. aLN-1 were measured by a home-made enzyme linked immunosorbent assay (ELISA) in: (1) sera and FF from 44 infertile women affected by HT (HTIW) with tubal factor or male factor as primary cause of infertility; (2) in sera and FF from 28 infertile women without HT, with tubal factor or male factor as cause of infertility (infertile controls-ICTR); and (3) in sera from 50 fertile women (FW). aLN-1 serum levels were significantly higher in HTIW when compared with both fertile women and ICTR (P <0.001and P <0.01, respectively). Assuming as cutoff the 99th percentile of values obtained in sera of FW, 43.2% of HTIW and 3.6% of ICTR were aLN-1 positive (P = 0.0001). Also aLN-1 detected in FF from HTIW were significantly higher in comparison with those found in FF of ICTR (P = 0.006). In HTIW, metaphase II oocyte count showed inverse correlation with both serum and FF aLN-1 levels (r = 0.34, P = 0.02 and r = 0.33, P = 0.03, respectively). Implantation and pregnancy rates were significantly lower in HTIW (7.9% and 9.1%, respectively) when compared with ICTR (23% and 31.1%, respectively) (P = 0.015 and P = 0.03, respectively). Our results demonstrated for the first time the presence of aLN-1 in a relevant percentage of HTIW and suggest that these auto-antibodies may impair IVF outcome.

Oocyte morphological abnormalities in overweight women undergoing in vitro fertilization cycles.

The effect of elevated body mass index (BMI) on the oocyte quality was investigated in women undergoing in vitro fertilization (IVF) cycles. A total of 268 patients classified on the basis of BMI subject to the first reproductive treatment were included in this study: the normal weight (NW) group consisted of 160 patients with BMI 19-24.9 kg/m(2) and the overweight (OW) group consisted of 108 patients with BMI ≥ 25 kg/m(2). All women were treated with a standard long luteal protocol. The oocyte features were classified as extracytoplasmic or cytoplasmic abnormalities. Outcomes were oocyte morphology, embryo quality, fertilization and implantation rates, and the ovarian response to stimulation. A higher percentage of oocytes with granular cytoplasm was found in women with BMI ≥ 25 (p = 0.04). However, percentages of mature, immature oocytes and germinal vesicle were similar in both groups. No differences were found in fertilization and cleavage rates and percentages of embryo quality. The implantation rate (p < 0.001) was significantly lower in the OW group than in the NW group. The amount of gonadotrophins was significantly higher in OW group (p = 0.003). These findings suggest that the poor reproductive outcome of obese women is influenced by the release of ova with reduced fertilization potential.

Semen quality and hormonal levels in infertile patients with varicocele.

OBJECTIVE: Aim of this study was to evaluate the semen quality and the serum concentration of follicle-stimulating hormone (FSH) and Testosterone (T) in infertile patients with and without varicocele. MATERIAL AND METHODS: 365 infertile patients undergoing Assisted Reproduction Tecnique (ART) were retrospectively included in the study. All subject were evaluated by history, physical examination, semen analysis, semen culture, mixed anti-immunoglobulin reaction test (MAR) for demonstration of sperm agglutination antibodies IgG and IgA, serum FSH and T determination. RESULTS: We observed 97 (26.6%) patients affected by varicocele compared to 268 (73.4%) without varicocele. A significant reduced percentage of motile spermatozoa (24.58 +/- 21.68 vs 21.01 +/- 12.62, p < 0.001) and lower sperm concentration (15.50 +/- 23.30 vs 16.50 +/- 15.22, p < 0.001) were observed in patients with varicocele compared to patients without varicocele. No significant differences were observed in sperm vitality between the two population of men with and without varicocele. Serum FSH (10.42 +/- 10.84 vs 9.11 +/- 18.81, p < 0.001) and Testosterone (5.73 +/- 5.97 vs 5.21 +/- 2.43, p < 0.001) levels were significantly higher in patients with varicocele compared to patients without varicocele. Detection of IgG and IgA sperm antibodies were negative in both man with and without varicocele. CONCLUSION: The direct connection between varicocele and infertility is not clear. The data of the present study suggest that the presence of a clinical varicocele rule out fertility in men affecting the hypothalamic pituitary-gonadal axis.

Follicular growth and oocyte maturation in GnRH agonist and antagonist protocols for in vitro fertilisation and embryo transfer.

BACKGROUND: The aim of this study was to evaluate the response to treatment in a group of patients undergoing IVF and randomised to receive GnRH-antagonist or the GnRH-agonist. The endpoints were the pattern of follicular growth, the maturity of the oocytes collected, the embryo quality and the pregnancy outcome. METHODS: A total of 136 patients undergoing IVF were included. Sixty-seven patients were allocated to the GnRH antagonist and 69 patients to the GnRH agonist. GnRH antagonist was administered when the leading follicle reached a diameter of 12-14 mm. GnRH agonist was administered in a long luteal protocol. RESULTS: The mean numbers of oocytes retrieved and mature oocytes were significantly higher in the agonist than in the antagonist group (p < 0.02 and p < 0.01, respectively). Embryo quality, implantation rate, clinical pregnancy rates, ongoing pregnancy rate and miscarriage rate were similar in both groups. CONCLUSIONS: Better follicular growth and oocyte maturation are achieved with GnRH agonist treatment. However, both regimens seem to have similar efficacy in terms of implantation and pregnancy rates. Further studies clarifying the effect of the GnRH antagonist on ovarian function are needed, as well as a clear definition of the best period of the follicular phase for the GnRH antagonist administration.

Intrauterine insemination with recombinant or urinary human chorionic gonadotropin: A prospective randomized trial.

AIM: Our aim was to compare the efficacy and safety of recombinant and urinary human chorionic gonadotropin (rhCG and uhCG, respectively) for the induction of follicle maturation in women undergoing intrauterine insemination (IUI). METHODS: Patients were randomized to receive rhCG or uhCG. IUI was carried out 24 h (day 1) and 48 h (day 2) after hCG administration, except for all cases in which ovulation occurred after 24 h. RESULTS: The two treatments were comparable in terms of progesterone levels on day 7 and day 12. Pregnancy rates were comparable between the treatment groups. Of the 64 women who received rhCG, 29.7% became pregnant; there were 16.7% clinical pregnancies and 3.1% biochemical pregnancies per started cycle, and an ongoing pregnancy rate of 93.7% was reported. Of the 61 patients who received uhCG, 24.6% became pregnant; there were 15.9% clinical pregnancies and 1.1% biochemical pregnancies per started cycle, and ongoing pregnancy rate was 92.9%. No adverse effects were noted in either group. CONCLUSION: The recombinant products can be effectively used instead of urinary products; moreover, apart from the equivalent efficacy in ovulation induction and safety described in this study, it is necessary to consider the advantages provided by the recombinant form.

Outcome of in vitro fertilization after transabdominal ultrasound-assisted embryo transfer with a full or empty bladder.

The use of ultrasound guidance has proven to be a key factor in performing embryo transfer in a gentle and atraumatic manner. However, despite the lower incidence of brusque maneuvers, bladder distension has not shown any positive impact on the IVF success rate.

The efficiency of sperm washing in removing human immunodeficiency virus type 1 varies according to the seminal viral load.

We evaluated the relationship between the seminal HIV-1 viral load and the efficiency of a standardized sperm-washing procedure in removing HIV-1 RNA from semen samples. The results obtained indicate that the amount of virus present in the original sample affects the efficiency of the procedure and suggest that the seminal viral load should be preevaluated before enrolling an HIV-serodiscordant couple in an assisted reproduction protocol.

Is the occurrence of the empty follicle syndrome a predictor that a subsequent stimulated cycle will be an unfavourable one?

Several hypotheses have been advanced to explain empty follicle syndrome (EFS) but it remains a controversial topic. This paper reports experience with three IVF cycles in which no oocytes were collected. In all cases, an additional IVF cycle was performed. The ovarian stimulation protocol, ultrasound and hormonal surveillance methods, human chorionic gonadotrophin timing and oocyte retrieval technique were similar in all patients. The assessment of additional cycles demonstrated a poor response in terms of oocyte quality, since the number of mature oocytes was low despite the high number of oocytes collected. Thus, the data suggest that in these patients, EFS should be considered as a borderline form of poor response to ovarian stimulation. If this is confirmed, EFS should be a recurrent event and an empty cycle could be a good predictor that a subsequent stimulated cycle will be an unfavourable.