Searching HPV genome for methylation sites involved in molecular progression to cervical precancer.

Background: Human Papilloma Virus has been considered as the main cause for cervical cancer. In this study we investigated epigenetic changes and especially methylation of specific sites of HPV genome. The main goal was to correlate methylation status with histological grade as well as to determine its accuracy in predicting the disease severity by establishing optimum methylation cutoffs. Methods: In total, sections from 145 cases genotyped as HPV16 were obtained from formalin- fixed, paraffin-embedded tissue of cervical biopsies, conization or hysterectomy specimens. Highly accurate pyrosequencing of bisulfite converted DNA, was used to quantify the methylation percentages of UTR promoter, enhancer and 5' UTR, E6 CpGs 494, 502, 506 and E7 CpGs 765, 780, 790. The samples were separated in different groupings based on the histological outcome. Statistical analysis was performed by SAS 9.4 for Windows and methylation cutoffs were identified by MATLAB programming language. Results: The most important methylation sites were at the enhancer and especially UTR 7535 and 7553 sites. Specifically for CIN3+ (i.e. HSIL or SCC) discrimination, a balanced sensitivity vs. specificity (68.1%, 66.2% respectively) with positive predictive value (PPV) and negative predictive value (NPV) (66.2%, 68.2% respectively) was achieved for UTR 7535 methylation of 6.1% cutoff with overall accuracy 67.1%, while for UTR 7553 a sensitivity 60.9%, specificity 69.0%, PPV=65.6%, NPV=64.5% and overall accuracy=65.0% at threshold 10.1% was observed. Conclusion: Viral HPV16 genome was found methylated in NF-1 binding sites of UTR in cases with high grade disease. Methylation percentages of E6 and E7 CpG sites were elevated at the cancer group.

Healing after Resection of Colonic Endometriosis and Growth Factor-enriched Agents: An Experimental Rat Model.

STUDY OBJECTIVE: To examine the potential beneficial effect of platelet-rich plasma (PRP) and fibrin sealant (TISSEEL; Baxter Healthcare Corporation, Deerfield, IL) on bowel wound healing after shaving of an experimentally induced endometriotic lesion. DESIGN: A single-blind, randomized study (Canadian Task Force classification I). SETTING: A certified animal research facility. ANIMALS: Thirty female Sprague-Dawley rats. INTERVENTIONS: Experimental colonic endometriosis was induced by transplanting endometrial tissue to all animals (first surgery). Thirty rats were then randomized to 1 of 3 groups according to treatment; PRP (group 1, n = 10), fibrin sealant (group 2, n = 10), or no agent (group 3, n = 10) was applied after shaving of the endometriotic nodule (second surgery). MEASUREMENTS AND MAIN RESULTS: Colonic endometriosis was successfully induced in all subjects. Four days after the second surgery, the animals were euthanized, and microscopic evaluation was performed. The pathologist was blinded to the treatment method. Histopathologic analysis revealed that compared with the control group, collagen disposition was found in a significantly higher expression in both the PRP and fibrin sealant groups (p = .011 and p = .011, respectively). Distortion of the integrity of the colon layers was statistically more pronounced in the control group compared with the fibrin sealant group (p = .033), whereas greater new blood vessel formation was observed in the fibrin sealant group compared with the control (p = .023). No histologic evidence of residual or recurrent disease was detected. CONCLUSION: Both PRP and fibrin sealant appear to be safe and associated with improved tissue healing during shaving for the excision of colonic endometriosis, attributed to the enhanced collagen disposition, neovascularization, and protection of the integrity of colon layers. Clinical trials are warranted to confirm the feasibility of PRP and fibrin sealant in the clinical setting.

Corifollitropin alfa compared with follitropin beta in GnRH-antagonist ovarian stimulation protocols in an unselected population undergoing IVF/ICSI.

Recombinant DNA technologies have produced Corifollitropin alfa (CFa) used during IVF/ICSI in order to keep the circulating FSH levels above the threshold necessary to support multi-follicular growth for a week. In this prospective case-control study, we compared 70 participants treated with 150 µg CFa combined with 150 IU of follitropin beta (study group) with 70 subfertile participants with matching baseline characteristics, conforming with the same inclusion criteria and treated with an antagonist protocol using follitropin beta (control group). Live birth was the primary outcome, while secondary outcome measures were IVF/ICSI cycles characteristics, including adverse events and complications. Live birth was determined in reduced rates in the study compared to the control group, reaching statistical significance [6/70 versus 20/70, p = 0.002], as also in the respective number of clinical pregnancies [9/70 versus 23/70, p = 0.005], although the incidence of miscarriage was similar for both groups [6/70 versus 5/70, p > 0.99]. Most of the secondary parameters examined were similar between groups. Logistic regression revealed that protocol and AFC had a direct impact on live birth. Ovarian stimulation with CFa does not seem to constitute an equally effective method as compared with follitropin beta to be offered in a general subfertile population seeking IVF/ICSI treatments.

Endometrial injury for RIF patients undergoing IVF/ICSI: a prospective nonrandomized controlled trial.

To evaluate the effect of endometrial injury on clinical outcomes in subfertile women with repeated implantation failures (RIF) undergoing assisted reproduction. In this prospective nonrandomized controlled trial, 103 subfertile women with RIF were included. Fifty-one underwent endometrial injury through hysteroscopy in the early follicular phase of the previous cycle and 52 underwent the standard protocol without any intervention. Live birth and miscarriage were the primary outcomes. Clinical and in vitro fertilization (IVF) cycle characteristics, were also compared between groups. Both groups were comparable in terms of baseline and cycle characteristics. Live birth rates were significantly higher in the study, compared with the control group (18/51 vs. 8/52, odds ratio (OR) = 0.25; 95% confidence interval (CI) = 0.10-0.64; p = 0.020), although miscarriage rates were similar (7/51 vs. 10/52, OR= 0.25; 95%CI= 0.12-0.66; p = 0.452). The rest of the outcomes parameters were comparable between groups. Logistic regression analysis revealed that endometrial injury and duration of subfertility were independent predictors of live birth after control of other variables (OR = 2.818; 95%CI = 1.044-7.605; p = 0.041 and OR = 0.674; 95%CI = 0.461-0.985, p = 0.042, respectively). Endometrial injury induced through office hysteroscopy in the preceding cycle in subfertile women with RIF improves live birth rates.

Estradiol Pretreatment in an Ultrashort GnRH Combined with a GnRH Antagonist Protocol in A Cohort of Poor Responders Undergoing IVF/ICSI: A Case-control Study.

AIM: To provide results of the use of estradiol pretreatment in a combination of an ultrashort gonadotropin-releasing hormone (GnRH) agonist and antagonist protocol, in an attempt to improve the clinical outcomes in "poor responders", according to the Bologna criteria, undergoing in vitro fertilization (IVF). PATIENTS AND METHODS: We applied estradiol pretreatment to 20 participants before the initiation of a combination of an ultrashort GnRH agonist plus an GnRH antagonist protocol followed by high doses of gonadotropins; the control group consisted of 20 subfertile participants with matching age, body mass index (BMI), basal follicle-stimulating hormone (FSH) and anti-Müllerian hormone (AMH), antral follicle count (AFC) and cause of subfertility, conforming with the same inclusion criteria and treated with a fixed GnRH antagonist protocol. The primary outcome measure was live birth, while the secondary outcomes included embryological and cycle parameters. RESULTS: Live birth was determined in reduced rates in the study compared to the control group (0/20 vs. 3/20, p=0.231) as also in the respective number of clinical pregnancies (2/20 vs. 5/20, p=0.407) and cancellations (10/20 vs. 6/20, p=0.197), but none of these differences reached statistical significance. Also, most of the secondary parameters studied were similar for both groups. CONCLUSION: The study protocol does not seem to constitute an equally effective method compared to the GnRH antagonist protocol in the selected study population. The presented dissimilar results with those reported so far in the literature are possibly attributed to the small sample size and the strict criteria applied when labeling participants as "poor responders".

OCT-4 and DAZL expression in precancerous lesions of the human uterine cervix.

AIM: To determine whether octamer-binding transcription factor 4 (OCT-4) and deleted in azoospermia like (DAZL) are expressed among cells with human papilloma virus (HPV) infection and cervical intraepithelial neoplasia (CIN) lesions and quantify their relative expression when compared with normal cervical cultures. METHODS: Cervical cells derived from normal cell cultures, HPV lesions and CIN lesions were cultured in Dulbecco's modified Eagle's medium supplemented with 20% amniotic fluid and 5 ng/mL basic fibroblast growth factor at 37°C and humidified 10% CO2 in air. Real-time polymerase chain reaction (PCR) was carried out using G6PD as a reference. We used REST for statistical analysis of real-time PCR. RESULTS: Whereas DAZL was not expressed either in normal cultures or HPV and CIN lesions, OCT-4 was expressed in all examined cell lines. Moreover its relative expression was significantly upregulated among cultures of HPV-infected cells (RE, 11.003; 95%CI: 0.054-36 704.527, P = 0.042), an observation that was also close to statistical significance among cultures of CIN lesions (P = 0.066). CONCLUSION: The relative expression of OCT-4 is upregulated during the early, preinvasive stages of cervical carcinogenesis. Future studies should investigate its potential as a screening marker and as a possible target of therapy.

The effect of vaginal lubrication on unsatisfactory results of cervical smears.

OBJECTIVE: Speculum lubrication with water-based gels before cervical smear sampling has been investigated and commented on thoroughly during the last decade. The present meta-analysis was based on randomized and quasi-randomized control trials and evaluated its impact on unsatisfactory cytologic results. We also assessed its efficacy in producing lower pain scores during speculum insertion. MATERIALS AND METHODS: We searched MEDLINE (1966- 2013), Scopus (2004-2013), Clinicaltrials.org (1997-2013), Popline (1973-2013), Cochrane Central Register of Controlled Trials (CENTRAL) (1999-2013), and Google Scholar (2004-2013) engines for published randomized control trials, as well as the reference lists from all the included studies. RESULTS: Five randomized trials and 2 quasi-randomized trials were included in the present review involving 8,717 women. Unsatisfactory results based on conventional cytology did not statistically differ among procedures performed with lubricated speculums and those without (odds ratio = 0.94, 95% CI = 0.64-1.37). Consequently, pain scores also did not differ significantly among the 2 groups (odds ratio = -0.37, 95% CI = -1.10 to 0.36). Only 2 studies evaluated unsatisfactory results on liquid-based cytology, precluding firm results. CONCLUSIONS: It seems that speculum lubrication does not interfere with the rates of unsatisfactory results when examination is based on conventional cytology. Conversely, however, the limited number of studies evaluating speculum lubrication on liquid-based cytology precludes firm conclusions.