Transrectal high-intensity focused ultrasound (HIFU) for management of rectosigmoid deep infiltrating endometriosis: results of Phase-I clinical trial.

OBJECTIVES: Deep infiltrating endometriosis (DIE) of the rectosigmoid is associated with painful symptoms. When medical treatment is ineffective, surgical resection remains the standard treatment, despite significant risk of adverse events. High-intensity focused ultrasound (HIFU) is a minimally invasive ablative procedure. Focal One® is a transrectal HIFU (TR-HIFU) device used in prostate cancer treatment. The primary objective of this study was to confirm the feasibility of treatment with TR-HIFU in patients presenting with posterior DIE with rectosigmoid involvement. We also assessed its safety and clinical efficacy in this context. METHODS: This was a non-controlled, prospective, Phase-I clinical trial in a French University Hospital which is a multidisciplinary center for management of endometriosis. Included were patients older than 25 years, without plans to conceive within 6 months, who presented with a single lesion of posterior DIE, with rectosigmoid invasion, after failure of hormonal therapy. All lesions were assessed preoperatively using transvaginal sonography and magnetic resonance imaging. Patients completed questionnaires on gynecological and intestinal symptoms (similar to a visual analog scale (VAS)), and on quality of life (Medical Outcomes Study 36-item short-form survey (SF-36) and, for the second half of patients recruited, symptom scoring system for constipation (KESS), female sexual function index (FSFI) and endometriosis health profile short-version score (EHP-5)), before, and at 1, 3 and 6 months after, TR-HIFU treatment with a Focal One real-time ultrasound-guided HIFU device. RESULTS: Twenty-three consecutive patients were included in the study between September 2015 and October 2019. All 23 lesions were visualized, giving a detection rate of 100%. Twenty lesions were treated ('feasibility rate', 87.0%): in 13 the whole lesion was treated and in seven the lesion was treated partially. The mean duration of the TR-HIFU procedure was 55.6 min. We observed a significant improvement in VAS score at 6 months, with differences relative to preoperative scores as follows, for: dysmenorrhea (-3.6, P = 0.004), dyspareunia (-2.4, P = 0.006), diarrhea (-3.0, P = 0.006), constipation (-3.0, P = 0.002), dyschezia (-3.2, P = 0.003), false urge to defecate (-3.3, P = 0.007), posterior pelvic pain (-3.8, P = 0.002) and asthenia (-3.8, P = 0.002). There was also a significant improvement in the SF-36 score, with an increase at 6 months relative to the preoperative score in both the physical component summary (+ 9.3%, P = 0.002) and mental component summary (+ 10.9%, P = 0.017). No major complications occurred during or after any procedure. CONCLUSIONS: TR-HIFU therapy for posterior DIE is feasible. If its efficacy and safety are confirmed, it could be a minimally invasive alternative to surgery for the treatment of rectosigmoid endometriosis. © 2019 Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Formation of three-dimensional thyroid follicle-like structures by polarized FRT cells made communication competent by transfection and stable expression of the connexin-32 gene.

Pig thyrocytes, either in the intact gland or cultured under conditions leading to thyroid follicle reconstitution, coexpress two gap junction proteins, connexin-32 (Cx32) and connexin-43 (Cx43). As thyrocytes cultured in the form of a monolayer only express Cx43, we hypothesized that Cx32 could play a role in thyroid folliculogenesis. In the present work, we analyzed the ability of polarized FRT cells (that are gap junction deficient) to form follicle-like structures after stable transfection with either Cx32 or Cx43 genes. Wild-type and transfected FRT cells, while growing, showed the capacity to form three-dimensional structures corresponding to domes that result from the accumulation of fluid underneath limited areas of the cell layer. The number of domes formed by FRT cells expressing Cx32 (FRT-Cx32) was 2- to 3-fold higher than that obtained with either wild-type or Cx43-transfected FRT cells (FRT-Cx43). Domes generated by FRT-Cx32 cells were stable (beyond 3 weeks of culture), whereas those formed from wild-type or FRT-Cx43 cells were transient, disappearing when cells reached confluence. Inspection of the cell organization within domes formed from FRT-Cx32 cells by phase contrast and confocal microscopy revealed a progressive transition from domes toward closed structures with a lumen. The tightness of the lumen was demonstrated by the retention of a fluorescent probe, lucifer yellow, introduced by microinjection. Electron microscope examinations showed that the neoformed follicle-like structures had an inside-out polarity. Analyses of cell motion and division with time, by fluorescence video microscopy, indicated that the transformation of domes into inside-out follicles brings into play the migration of cells and, to a lesser extent, cell multiplication underneath the domes. In conclusion, FRT cells forced to express Cx32 give rise to domes that transform into closed inside-out follicles. This gain of function appears Cx specific, as FRT-Cx43 cells did not form similar structures. Our data suggest that the formation and/or functioning of Cx32 gap junctions might represent a key event in thyroid epithelium morphogenesis, i.e. formation of a lumen from a tight epithelial cell layer.

Susceptibility of differentiated thyrocytes in primary culture to undergo apoptosis after exposure to hydrogen peroxide: relation with the level of expression of apoptosis regulatory proteins, Bcl-2 and Bax.

Thyrocytes, that generate and use hydrogen peroxide (H2O2) to synthesize thyroid hormones, undergo apoptosis, as do most cell types, when exposed in vitro to H2O2. We have studied 1) the kinetics and the amplitude of the apoptotic response to H2O2 and 2) the relationship between the extent of the apoptosis-inducing effect of H2O2, the H2O2 degradation activity, and the level of expression of apoptosis regulatory proteins, Bcl-2 and Bax, in pig thyrocytes in primary culture. Cells were seeded at high density to obtain confluent monolayers and were cultured in the presence of TSH to maintain the expression of differentiation. H2O2 (10-300 microM) induced the appearance of cells with fragmented DNA (terminal transferase deoxy-UTP-fluorescein isothiocyanate nick end labeling-positive cells) at a maximum of 3-4 h after H2O2 addition and then the detachment of apoptotic cells from the cell monolayer. The proportion of detached cells increased with H2O2 concentration and amounted to up to 30% of the initial cell number after 24 h. The transient effect of H2O2 was related to its rapid degradation by cells and culture medium components (rate constant, approximately 0.1 min(-1)). Iterative additions of H2O2 produced cumulative apoptotic waves. The amplitude of the apoptotic response of thyrocytes to H2O2 progressively increased with the time of culture, up to 4-fold from days 1-8. This was not related to a change in the capacity of thyrocytes to degrade H2O2. During the same period of culture, the Bcl-2 cell content progressively decreased, whereas that of Bax concomitantly increased; thus, the Bcl-2/Bax ratio varied from about 6 on day 1 to 0.5 on day 10. These data show that the susceptibility of thyrocytes to undergo apoptosis increases with the time of culture and that the pronounced changes in the apoptotic status ofthyrocytes might be linked to coordinate modifications of the level of expression of pro- and antiapoptotic regulatory proteins.

Restoration of cell-to-cell communication in thyroid cell lines by transfection with and stable expression of the connexin-32 gene. Impact on cell proliferation and tissue-specific gene expression.

Normal thyroid epithelial cells coexpress connexin-32 and connexin-43, which form distinct gap junctions. In primary culture, connexin-43 is expressed by thyrocytes in monolayers or reorganized into follicles, whereas the expression of connexin-32 is dependent upon the reconstitution of follicles. To study the functional impact of connexin-32 gap junctions in thyroid cells, we transfected connexin-32 cDNA in two thyroid-derived communication-deficient cell lines, FRT and FRTL-5. The selected clones, which stably expressed connexin-32 at high levels and exhibited high gap junction-mediated dye-coupling, presented a reduced proliferation rate as compared with that of the corresponding wild-type FRT and FRTL-5 cells; the mean population doubling time was increased by approximately 35%. The proliferation of connexin-32-transfected FRTL-5 cells remained thyrotropin-dependent; the range of thyrotropin concentrations that stimulated growth was the same in transfected and control cells. The expression of connexin-32 led to an increase of thyroglobulin gene expression in FRTL-5 cells. The expression of two other tissue-specific proteins, thyroid transcription factor-1 and Pax-8, was unchanged. These findings provide evidence that connexin-32 gap junction-mediated cell-to-cell communication participates in the control of growth and differentiation of thyroid cells.