Metformin potentiates immunosuppressant activity and adipogenic differentiation of human umbilical cord-mesenchymal stem cells.

Metformin, a first-line drug for type-2 diabetes, displays pleiotropic effects on inflammation, aging, and cancer. Obesity triggers a low-grade chronic inflammation leading to insulin resistance, characterized by increased pro-inflammatory cytokines produced by adipocytes and infiltrated immune cells, which contributes to metabolic syndrome. We investigated metformin's differentiation and immunoregulatory properties of human umbilical cord-mesenchymal stem cells (UC-MSC), as cellular basis of its beneficial role in metabolic dysfunctions. Isolation, characterization and multilineage differentiation of UC-MSC were performed using standard protocols and flow-cytometry. Metformin effects on UC-MSC growth was assessed by colony formation and MTT assay, gene and protein expression by qRT-PCR, and western blot analysis. Proliferation of peripheral blood mononuclear cells (PBMCs) co-cultured with metformin-treated UC-MSC-conditioned media was evaluated by dye dilution assay. We show that metformin decreases proliferation and colony formation of UC-MSCs and enhances their adipogenic lineage commitment. Metformin (3 mM) increases PPARγ and downregulates FABP4 mRNA both in basal and in adipogenic culture conditions; however, the modulation of PPARγ expression is unrelated to the antiproliferative effects. Moreover, metformin inhibits UC-MSC inflammatory activity reducing the expression of IL-6, MCP-1, and COX-2. Conditioned media, collected from metformin-treated UC-MSCs, down-regulate CD3+ T lymphocyte growth in stimulated PBMCs and, in particular, reduce the CD8+ T cell population. These results indicate that metformin may favor new adipocyte formation and potentiate immune suppressive properties of UC-MSCs. Thus, adipose tissue regeneration and anti-inflammatory activity may represent possible mechanisms by which metformin exerts its positive effect on lipid metabolism.

Uterine Myoma Position-based Radiofrequency Ablation (UMP-b RFA): 36 months follow-up clinical outcomes.

OBJECTIVE: To assess the efficacy of Uterine Myoma Position-based Radiofrequency Ablation (UMP-b RFA) at 36 months. An analysis of a new uterine fibroid ablation technique that allows personalized access of delivering radiofrequency energy (transvaginal, hysteroscopic or laparoscopic) based on myoma localization. STUDY DESIGN: Prospective observational cohort study in a community-based secondary care medical center enrolled 61 premenopausal women with 112 symptomatic uterine myomas. 112 fibroids were ablated in 5 ways with single or combined accesses: 82 Vaginal Ultrasound (VU)-guided RFA, 19 Laparoscopic (L)-RFA, 5 Hysteroscopic (H)-RFA, 5 VU+H-RFA, and 1 VU+L-RFA. The primary endpoint of this study was to evaluate the 3-year clinical outcome of UMP-b RFA. The secondary endpoint was the possible identification of predictors of its success. The outcomes evaluated at 12, 24, and 36 months after UMP-b RFA were myoma size, type of symptomatology suffered, quality of life based on the "Uterine Fibroid Symptom and Quality of Life" questionnaire, and interviews on the degree of satisfaction with this surgery. The reintervention and complication rates were also recorded and analyzed. RESULTS: Fibroids volume and diameter were significantly reduced by -90.2 % / -55.7 % at 36 months post-intervention (p < 0.001) and the reduction of Symptom Severity scores was -71.8 % three years after UMP-b RA (p < 0.001). The overall improvement in the quality of life was demonstrated by an increase in the Quality-of-Life score of + 26.0 % at the third follow-up (p < 0.001). 88.5 % of the patients interviewed would have the surgery done again if they went back in time. The reintervention rate was 10/61 (16.4 %): 3 hysterectomies, 3 myomectomies, 3 operative hysteroscopies and 1 VU-RFA reoperation. In this group of unsuccessful surgeries, the mean diameter of the dominant myomas was found to be greater than that of the successes (5.3 vs 4.4 cm.). Out of the 61 cases, no major complications occurred, and the 2 minor complications observed were self-limiting. CONCLUSION: Uterine Myoma Position-based Radiofrequency Ablation is a safe, effective, and minimally invasive solution for the treatment of symptomatic fibroids. Indeed, these clinical outcome data at 36 months shows how UMP-b RFA can treat the symptomatology of uterine fibromatosis. Hysterectomies or myomectomies were successfully avoided in more than 80 % of women bearing myomas with an average diameter of less than 5 cm.

Impact of the COVID-19 pandemic on voluntary terminations of pregnancy in an Italian metropolitan area.

PURPOSE: The COVID-19 pandemic and national lockdown from 9 March to 4 May 2020 changed social, familial, and sexual relationships, as well as how citizens interact with the health services. How these profound changes have affected sexuality, contraception and voluntary terminations of pregnancy is still largely undescribed. The main objective of this study was therefore to find out whether the COVID-19 pandemic and ensuing lockdown affected the demand for legal abortion. MATERIAL AND METHODS: The study period was divided into three phases: the pre-pandemic (January and February 2020); lockdown (March and April); and post-lockdown (May and June). The number and characteristics of women requesting pregnancy termination each month during that time were compared with the stats for the same months in the preceding three years (2017-2019). RESULTS: Immediately after national lockdown, the number of voluntary abortions markedly declined (-40.45%). The effect was more evident in women below 20 years of age (-66.67%), employed versus unemployed women (-42.71% vs. -21.05), and non-Italian versus Italian citizens (-53.01 vs. -32.85). No difference was found in the mean time from request to execution of the procedure, or in the type of the procedure used. CONCLUSION(S): National lockdown reduced the number of unwanted pregnancies, especially in younger women. The Italian health service's response to the demand appears to have been unaffected by the pandemic. However, as the demand for abortion is still high, probably due to unplanned pregnancies among cohabitants within a stable relationship, contraception guidance should be improved among women traditionally deemed low-risk in terms of sexual behaviour.

A Prospective Intervention Trial on Tailored Radiofrequency Ablation of Uterine Myomas.

Background and Objective: Investigating the use of radiofrequency myolysis (RFM) for the treatment of fibroids through less invasive access by combining transvaginal ultrasound, hysteroscopy and laparoscopy. Materials and Methods: Fifty-four premenopausal women with 106 symptomatic uterine myomas. Patients underwent RFM in three ways: Vaginal Ultrasound-guided RFM (VU-RFM), Laparoscopic RFM (L-RFM) and Hysteroscopic-RFM (H-RFM). The mean patient age was 43 years; 52 symptomatic uterine myomas were subserosal, 44 intramural and 10 submucosal. The outcomes evaluated at 1 and 12 months after RFM were myoma size (volume-diameter), "Uterine Fibroid Symptom and Quality of Life (UFS-QOL)" questionnaire and a 10-point Visual Analogue Scale (VAS). The therapy was completed with a single ablation in all patients, no complication was registered. The average number of fibroids treated per intervention was two with the use of different accesses: 64/106 VU-RFMs (60.4%), 32/106 L-RFMs (30.2%) and 10/106 H-RFMs (9.4%). Results: Volume and diameter of fibroids were significantly reduced by, respectively, 51.3% and 20.1% in the first 30 days post-intervention (p < 0.001) up to a maximum of 73.5% and 37.1% after the second follow-up visit at 12 months (p < 0.001). A similar trend was shown in terms of disability with a progressive and significant reduction of symptoms (menorrhagia, dysmenorrhea, dyspareunia and pollakiuria) demonstrated by percentage variation of UFS-QOL Symptom Severity and VAS scores to -74.3% and -45.3% as well as -84.9% and -74.3%, respectively, at 1 and 12 months after RFM (p < 0.001). An overall improvement in the quality of life was also demonstrated by a significant increase in the UFS-QOL total score of +38.2% in the first 30 days post-intervention up to +44.9% after the second follow-up visit at 12 months (p < 0.001). The overall average surgery time of the RFM for each patient was 48 minutes, and the time to treat each fibroid by Vaginal Ultrasound-guided RFM (23 min) was found to be significantly less than those of laparoscopy or hysteroscopy (respectively 35 and 34 min) (p < 0.05). An electromagnetic virtual needle tracking system (VNTS) was successfully tested during the RFM procedures, and real-time contrast-enhanced ultrasound (CEUS) has proven to be effective in determining the duration of myolysis through the identification of eventual residual areas of enhancement within the fibroids. Conclusion: Radiofrequency can be considered a minimally invasive and safe procedure for the treatment of uterine myomas through the customization and possible combination of transvaginal, laparoscopic or hysteroscopic accesses. The standardization of the ablation technique with pre-intervention biopsy and new technologies such as VNTS and CEUS spares healthy uterine tissue and may change the future management of symptomatic uterine fibroids.

Ultrasound-guided transvaginal radiofrequency ablation of uterine fibroids assisted by virtual needle tracking system: a preliminary study.

PURPOSE: The purpose of this study was to assess the feasibility and outcome of transvaginal ultrasound (US)-guided radiofrequency ablation of uterine fibroids assisted by a real-time virtual needle tracking (VT) system. METHODS: Between January 2017 and February 2018, 19 patients (age 45 ± 8 y, range 36-53 y) with 25 symptomatic uterine fibroids underwent transvaginal radiofrequency ablation (RFA) at a single center. Mean number of fibroids for patient was 1.7 (min, max: 1-3). Patients with more than one fibroid were 10 (52.6%). Uterine fibroids (mean volume: 13.6 mL; range: 5.3-41.9 mL) were treated with a dedicated internally cooled 17 G 35 cm RF needle with 1 cm or variable active tip and the moving shot technique. An electromagnetic system was used for showing a virtual needle during the procedure. Contrast-enhanced ultrasound evaluation was performed before and immediately at the end of procedure. Feasibility of the procedure, technical success rate, volume percentage reduction at 1, 3 and 6 months, clinical outcome (QOL score) and complications were analyzed. RESULTS: Procedure was feasible in 19/19 patients (100%). Technical success was achieved in 100% of 25 treated fibroids. Mean fibroids volume decreased from 13.6 ml at baseline to 5.9 ml at 6 month (reduction rate 62.7%, range 48.5-76.9; p < .05). No major immediate or late complications occurred. Minor complications occurred in two patients. QOL score significantly improved from 68 ± 36 at baseline to 97 ± 16 at six-months follow-up (p < .05). CONCLUSION: Transvaginal US-guided RFA assisted by a real-time VT system is a feasible, safe and effective technique for the treatment of uterine fibroids.

Different Effects of Human Umbilical Cord Mesenchymal Stem Cells on Glioblastoma Stem Cells by Direct Cell Interaction or Via Released Soluble Factors.

Glioblastoma (GBM), the most common primary brain tumor in adults, is an aggressive, fast-growing and highly vascularized tumor, characterized by extensive invasiveness and local recurrence. In GBM and other malignancies, cancer stem cells (CSCs) are believed to drive invasive tumor growth and recurrence, being responsible for radio- and chemo-therapy resistance. Mesenchymal stem cells (MSCs) are multipotent progenitors that exhibit tropism for tumor microenvironment mediated by cytokines, chemokines and growth factors. Initial studies proposed that MSCs might exert inhibitory effects on tumor development, although, to date, contrasting evidence has been provided. Different studies reported either MSC anti-tumor activity or their support to tumor growth. Here, we examined the effects of umbilical cord (UC)-MSCs on in vitro GBM-derived CSC growth, by direct cell-to-cell interaction or indirect modulation, via the release of soluble factors. We demonstrate that UC-MSCs and CSCs exhibit reciprocal tropism when co-cultured as 3D spheroids and their direct cell interaction reduces the proliferation of both cell types. Contrasting effects were obtained by UC-MSC released factors: CSCs, cultured in the presence of conditioned medium (CM) collected from UC-MSCs, increased proliferation rate through transient ERK1/2 and Akt phosphorylation/activation. Analysis of the profile of the cytokines released by UC-MSCs in the CM revealed a strong production of molecules involved in inflammation, angiogenesis, cell migration and proliferation, such as IL-8, GRO, ENA-78 and IL-6. Since CXC chemokine receptor 2 (CXCR2), a receptor shared by several of these ligands, is expressed in GBM CSCs, we evaluated its involvement in CSC proliferation induced by UC-MSC-CM. Using the CXCR2 antagonist SB225002, we observed a partial but statistically significant inhibition of CSC proliferation and migration induced by the UC-MSC-released cytokines. Conversely, CXCR2 blockade did not reduce the reciprocal tropism between CSCs and UC-MSCs grown as spheroids. In conclusion, we show that direct (cell-to-cell contact) or indirect (via the release of soluble factors) interactions between GBM CSCs and UC-MSCs in co-culture produce divergent effects on cell growth, invasion and migration, with the former mainly causing an inhibitory response and the latter a stimulatory one, involving a paracrine activation of CXCR2.

Metformin repositioning as antitumoral agent: selective antiproliferative effects in human glioblastoma stem cells, via inhibition of CLIC1-mediated ion current.

Epidemiological and preclinical studies propose that metformin, a first-line drug for type-2 diabetes, exerts direct antitumor activity. Although several clinical trials are ongoing, the molecular mechanisms of this effect are unknown. Here we show that chloride intracellular channel-1 (CLIC1) is a direct target of metformin in human glioblastoma cells. Metformin exposure induces antiproliferative effects in cancer stem cell-enriched cultures, isolated from three individual WHO grade IV human glioblastomas. These effects phenocopy metformin-mediated inhibition of a chloride current specifically dependent on CLIC1 functional activity. CLIC1 ion channel is preferentially active during the G1-S transition via transient membrane insertion. Metformin inhibition of CLIC1 activity induces G1 arrest of glioblastoma stem cells. This effect was time-dependent, and prolonged treatments caused antiproliferative effects also for low, clinically significant, metformin concentrations. Furthermore, substitution of Arg29 in the putative CLIC1 pore region impairs metformin modulation of channel activity. The lack of drugs affecting cancer stem cell viability is the main cause of therapy failure and tumor relapse. We identified CLIC1 not only as a modulator of cell cycle progression in human glioblastoma stem cells but also as the main target of metformin's antiproliferative activity, paving the way for novel and needed pharmacological approaches to glioblastoma treatment.

Metformin selectively affects human glioblastoma tumor-initiating cell viability: A role for metformin-induced inhibition of Akt.

Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect.

Incisional hernia on the 5-mm trocar port site and subsequent wall endometriosis on the same site: a case report.

A 26-year-old nulliparous woman underwent a laparoscopy to remove a 10-cm endometrial cyst on the left ovary (type II Nezhat). The cyst was extracted from the 10-mm umbilical incision; the other 2 trocars were inserted through 5-mm incisions. One year later, in correspondence to the previous 5-mm incision site, a hernia occurred that contained omentum and was reduced easily with a local anesthetic. After 2 years of good health, an aching nodule occurred on the same trocar site; vaginal ultrasound examination showed another left ovarian cyst. A second laparoscopy was performed; the cyst was very adherent and was removed in fragments. The wall nodule was removed, and the histologic examination classified it as endometriosis.

Prevalence of human papillomavirus cervical infection in an Italian asymptomatic population.

BACKGROUND: In the last decade many studies have definitely shown that human papillomaviruses (HPVs) are the major cause of cervical carcinogenesis and, in the last few years, HPV testing has been proposed as a new and more powerful tool for cervical cancer screening. This issue is now receiving considerable attention in scientific and non scientific press and HPV testing could be considered the most important change in this field since the introduction of cervical cytology. This paper reports our prevalence data of HPV infection collected in the '90s, while a follow up of these patients is ongoing. METHODS: For this study we used polymerase chain reaction (PCR) to search HPV DNA sequences in cervical cell scrapings obtained from 503 asymptomatic women attending regular cervical cancer screening program in the city of Genova, Italy. All patients were also submitted to a self-administered, standardized, questionnaire regarding their life style and sexual activity. On the basis of the presence of HPV DNA sequences women were separated into two groups: "infected" and "non infected" and a statistical analysis of the factors potentially associated with the infection group membership was carried out. RESULTS: The infection rate was 15.9% and the most frequent viral type was HPV 16. CONCLUSION: Our HPV positivity rate (15.9%) was consistent to that reported by other studies on European populations.