Elisabetta Trabucco

The pattern of expression of Notch protein members in normal and pathological endometrium.

The objective of this study was to investigate the pattern of expression and the localization of Notch‐1, Notch‐4 and Jagged‐1 in physiological and pathological human endometrium and to evaluate the expression levels of two major regulators of the G1 checkpoint, namely cyclin D1 and p21. Sixty samples of physiological endometrium and 60 samples of pathological endometrium were used for the study. Evaluation of the expression level and the distribution of Notch pathway members and cell‐cycle proteins was performed by immunohistochemistry. In the physiological endometrium we observed an increase of Notch‐1 and Jagged‐1 from proliferative to secretory phase and an opposite trend for Notch‐4. In menopause, the level of expression of all three members of the Notch pathway decreased. We also observed a cyclin D1 increase from proliferative to secretory phase. By contrast, p21 showed a slight increase from proliferative to secretory phase. In the pathological endometrium, we observed an increase of Notch‐1 expression from polyps to carcinoma and decrease for Notch‐4 and Jagged‐1. Moreover, we observed a higher expression of cyclin D1 in all the endometrial pathologies. By contrast, the expression level of p21 slightly increased from polyps to carcinoma. We concluded that in human endometrium Notch‐4 seems to be more involved in controlling proliferation, whereas Notch‐1 seems to be more involved in differentiation programming. Deregulation of these functions may induce the onset of several endometrial pathologies from polyps to cancer.

Effectiveness of the association micronized N-Palmitoylethanolamine (PEA)–transpolydatin in the treatment of chronic pelvic pain related to endometriosis after laparoscopic assessment: a pilot study

OBJECTIVE Aim of our study was to evaluate the effectiveness of the association between N-Palmitoylethanolamine and transpolydatin in the management of chronic pelvic pain related to EMS. STUDY DESIGN This was a randomized, double-blind, parallel-group, placebo-controlled clinical trial involving 61 subjects, submitted to a first line laparoscopic conservative surgery, who were randomized into 3 groups receiving: group A (n=21) the association N-Palmitoylethanolamine-transpolydatin 400 mg + 40 mg twice a day for 3 months; group B (n=20) the placebo for 3 months; group C (n=20) a single course of Celecoxib 200mg twice a day for 7 consecutive days. Assessments of the severity of pelvic endometriosis (pelvic pain, dysmenorrhoea and dyspareunia) were recorded before and after treatment on a questionnaire and a 10-point VAS. Differences between groups were verified with Kruskal-Wallis ANOVA for non-parametric multiple comparisons. RESULTS A marked decrease in dysmenorrhoea, dyspareunia and pelvic pain was observed in all groups, and the association between N-Palmitoylethanolamine and transpolydatin resulted to be more effective than placebo (P<.001). Additionally, the treatment with Celecoxib resulted in a decrease in pelvic pain more effective either than the association N-Palmitoylethanolamine and transpolydatin or placebo. CONCLUSION These preliminary results show that the association between micronized N-Palmitoylethanolamine and transpolydatin is effective in the management of pelvic pain related to endometriosis after laparoscopy. Additionally, this association seems to be safe, shows an optimal control of pain and can be used in patients who are unable to receive other therapies.

CYP17 and CYP19 gene polymorphisms in women affected with endometriosis

OBJECTIVE To investigate whether CYP17 T>C polymorphism and polymorphisms C1558T and Val80 of CYP19 are related to endometriosis. DESIGN Clinical study. PATIENT(S) Women affected with endometriosis (n = 104) and control group (n = 86). The diagnosis of endometriosis was confirmed by the histologic examination of the endometriotic lesions. RESULT(S) In patients affected with endometriosis, we observed that AA and CC genotypes were significantly represented in Val80 and C1558T polymorphisms of CYP19. CONCLUSION(S) The molecular mechanisms that underlie the development of endometriosis are unclear. Both environmental and genetic factors are involved in the pathogenesis of the disease. The inheritable susceptibility to endometriosis justifies the growing interest in identifying genes and/or genetic polymorphisms that predispose women to an increased risk of developing endometriosis. The identification of single-nucleotide polymorphism (SNP), probably linked to endometriosis, could help to explain its pathogenesis.

Minilaparoscopic Myomectomy: A Mini-invasive Technical Variant

OBJECTIVE We propose a mini-invasive technical variant for laparoscopic myomectomy, which is currently less invasive and more feasible. STUDY DESIGN This was a prospective, controlled, randomized trial, involving 170 patients, who underwent laparoscopic myomectomy. Patients were randomized into two groups: Group A (n=98) underwent standard laparoscopic myomectomy, and Group B (n=72) underwent the mini-invasive technique. The current mini-invasive variant is performed with a 10-mm umbilical trocar and only two 5-mm ancillary trocars. Morcellation is transumbilical: a 0° 5-mm optical system is used and is inserted either in the left or in the right iliac trocar according to the surgeons preference. RESULTS The degree of surgical difficulty, evaluated using a visual analog scale (VAS), was similar in the two groups (P=nonsignificant). Postoperative pain measured on a VAS scale showed there was less pain experienced in patients in Group B than in Group A (P<.01). Esthetic results measured on a VAS scale showed a higher compliance for patients in Group B than Group A (P<.01). CONCLUSIONS The mini-invasive laparoscopic myomectomy described is, in our opinion, currently less invasive and more feasible than techniques usually used. Finally, this technique is a valid approach for the surgeon, and it gives women very acceptable aesthetic results.

Luteal phase anovulatory follicles result in the production of competent oocytes: intra-patient paired case-control study comparing follicular versus luteal phase stimulations in the same ovarian cycle

Abstract STUDY QUESTION Are the mean numbers of blastocysts obtained from sibling cohorts of oocytes recruited after follicular phase and luteal phase stimulations (FPS and LPS) in the same ovarian cycle similar? SUMMARY ANSWER The cohorts of oocytes obtained after LPS are larger than their paired-FPS-derived cohorts and show a comparable competence, thus resulting in a larger mean number of blastocysts. WHAT IS KNOWN ALREADY Three theories of follicle recruitment have been postulated to date: (i) the ‘continuous recruitment’ theory, (ii) the ‘single recruitment episode’ theory and (iii) the ‘wave’ theory. Yet, a clear characterization of this crucial biological process for human reproduction is missing. Recent advances implemented in in vitro fertilization (IVF), such as blastocyst culture, aneuploidy testing and vitrification, have encouraged clinicians to maximize the exploitation of the ovarian reserve through tailored stimulation protocols, which is crucial especially for poor prognosis patients aiming to conceive after IVF. LPS has been already successfully adopted to treat poor prognosis or oncological patients through Duostim, LPS-only or random-start ovarian stimulation approaches. Nevertheless, little, and mainly retrospective, evidence has been produced to support the safety of LPS in general. Feasibility of the LPS approach would severely question the classic ‘single recruitment episode’ theory of follicular development. STUDY DESIGN, SIZE, DURATION This case-control study was conducted with paired follicular phase- and luteal phase-derived cohorts of oocytes collected after stimulations in the same ovarian cycle (DuoStim) at two private IVF clinics between October 2015 and December 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS The study included 188 poor prognosis patients undergoing DuoStim with preimplantation genetic testing for aneuploidies (PGT-A). FPS and LPS were performed with the same daily dose of recombinant-gonadotrophins in an antagonist protocol. Blastocyst culture, trophectoderm biopsy, vitrification and frozen-warmed euploid single blastocyst transfers were performed. The primary outcome was the mean number of blastocysts obtained per oocyte retrieval from paired-FPS- and LPS-derived cohorts (required sample size = 165 patients; power = 90%). Mean blastulation and euploidy rates were monitored, along with the number of oocytes, euploid blastocysts and clinical outcomes. MAIN RESULTS AND THE ROLE OF CHANCE Significantly fewer blastocysts were obtained after FPS than LPS (1.2 ± 1.1 vs. 1.6 ± 1.6, P < 0.01), due to fewer oocytes collected (3.6 ± 2.1 vs. 4.3 ± 2.8, P < 0.01) and a similar mean blastocyst rates per retrieval (33.1% ± 30.3% vs. 37.4% ± 30.8%, P = NS). The number of oocytes collected were correlated (R = 0.5, P < 0.01), while the blastocyst rates were uncorrelated among paired-FPS- and LPS-derived cohorts. Overall, a significantly lower chance of producing blastocyst(s) was reported after FPS than after LPS: 67.6% (n = 127/188, 95%CI: 60.3–74.1) vs. 77.1% (n = 145/188, 95%CI: 70.3–82.8; P = 0.05). The mean euploidy rates per retrieval were similar between FPS- and LPS-derived cohorts of oocytes (13.6% ± 22.8% vs. 16.3% ± 23.4%, P = NS). Therefore, on average fewer euploid blastocysts (0.5 ± 0.8 vs. 0.7 ± 1.0, P = 0.02) resulted from FPS. Similar ongoing-pregnancy/delivery rates were reported, to date, after FPS- and LPS-derived euploid single blastocyst transfers: 42.4% (n = 28/66, 95%CI: 30.5–55.2) vs. 53.8% (n = 35/65, 95%CI: 41.1–66.1; P = NS). LIMITATIONS, REASONS FOR CAUTION More studies need to be conducted in the future to confirm the safety of LPS, especially in terms of ovarian and follicular environment, as well as the clinical, peri-natal and post-natal outcomes. Here, we showed preliminary data suggesting a similar ongoing implantation/delivery rate (>22 weeks) between FPS- and LPS-derived euploid blastocysts, that need to be extended in the future, to populations other than poor prognosis patients and using approaches other than DuoStim together with a constant monitoring of the related peri-natal and post-natal outcomes. WIDER IMPLICATIONS OF THE FINDINGS These data, from a paired study design, highlight that LPS-derived oocytes are as competent as FPS-derived oocytes, thereby adding some evidence to support the use of LPS for poor prognosis and oncological patients and to question the ‘single recruitment episode’ theory of follicle recruitment. These findings also encourage additional studies of the basics of folliculogenesis, with direct clinical implications for the management of ovarian stimulation in IVF. TRIAL REGISTRATION None. STUDY FUNDING/COMPETING INTEREST(S) No external funds were used for this study and there are no conflicts of interest.

Double Stimulation in the Same Ovarian Cycle (DuoStim) to Maximize the Number of Oocytes Retrieved From Poor Prognosis Patients: A Multicenter Experience and SWOT Analysis

A panel of experts known as the POSEIDON group has recently redefined the spectrum of poor responder patients and introduced the concept of suboptimal response. Since an ideal management for these patients is still missing, they highlighted the importance of tailoring the ovarian stimulation based on the chance of each woman to obtain an euploid blastocyst. Interestingly, a novel pattern of follicle recruitment has been defined: multiple waves may arise during a single ovarian cycle. This evidence opened important clinical implications for the treatment of poor responders. For instance, double stimulation in the follicular (FPS) and luteal phase (LPS) of the same ovarian cycle (DuoStim) is an intriguing option to perform two oocyte retrievals in the shortest possible time. Here, we reported our 2-year experience of DuoStim application in four private IVF centers. To date, 310 poor prognosis patients completed a DuoStim protocol and underwent IVF with blastocyst-stage preimplantation-genetic-testing. LPS resulted into a higher mean number of oocytes collected than FPS; however, their competence (i.e., fertilization, blastocyst, euploidy rates, and clinical outcomes after euploid single-embryo-transfer) was comparable. Importantly, the rate of patients obtaining at least one euploid blastocyst increased from 42.3% (n = 131/310) after FPS to 65.5% (n = 203/310) with the contribution of LPS. A summary of the putative advantages and disadvantages of DuoStim was reported here through a Strengths–Weaknesses–Opportunities–Threats analysis. The strengths of this approach make it very promising. However, more studies are needed in the future to limit its weaknesses, shed light on its putative threats, and realize its opportunities.

Characterization of the effects of reverse transcriptase and protease inhibitors on ovarian cells

In Italy, every year new cases of ovarian cancer are approximately 5,000, 3,000 are the deaths; this means that 1 woman out of 70 is destined to fall ill and 1 out of 100 to die for this malignancy. Drugs of choice for the treatment of HIV, able to determine a decrease in viral load, have proven to be effective also against neoplasms (Kaposi sarcoma [1] and non-Hodgkin lymphomas [2]) frequent in subjects with acquired immunodeficiency syndrome (AIDS) that thanks to these drugs, are now in evident decline. Our research aims to highlight and characterize, therefore, the effects of anti-HIV drug therapy on Skov3 cells (ovarian adenocarcinoma), hoping for a possible role in the control of ovarian cancer, which is not among the cancers specific of HIV infection. Skov3 cells were cultured and then treated with the chosen drugs: abacavir, tenofovir, efavirenz, etravirine (reverse transcriptase inhibitors) and darunavir (protease inhibitor). At the cytofluorimetric analysis treated cells showed the following results: Abacavir (NRTIs) and Darunavir (PI) do not show any particular modulation of the cell cycle. Tenofovir (NtRTIs) showed an increase amount of cells in the G0/G1 phase of the cell cycle progression. Instead Efavirenz (NNRTIs) and Etravirine (NNRTIs) showed a block in the G0/G1 phase. Between the two drugs of the same class (NNRTIs) the block activity of Etravirine in G0/G1 of cell cycle is higher as compared to Efavirenz; at the same time, Etravirine determines the differentiation of Skov3. DAPI staining has allowed the identification of DNA damage in cells treated with efavirenz and an increased condensation of DNA in cells treated with etravirine. After treatment with tenofovir, efavirenz and etravirine, in Skov3 cells, we observed through immonoblotting: reduction of cyclin D1 concentration, hypophosphorylation of Rb and increase of p21 concentration. Only Skov3cells treated with etravirine showed high levels of E-cadherin expression. Reverse transcriptase, based on these data, can be considered an epigenetic regulator of cell proliferation and differentiation and may represent a new target in cancer therapy. Furthermore, we believe that the drugs used, modulating cell proliferation and differentiation, may represent a new frontier in the development of a therapeutic approach to ovarian cancer.