Optimization of Adipose Tissue Cryopreservation Techniques in a Murine Model.

BACKGROUND: The aim of this study was to develop an adipose tissue (AT) cryopreservation protocol that is effective, simple, and maintains the functionality and viability of AT after thawing and transplantation. METHODS: Two cryopreservation temperatures (T°), -20°C and -80°C, and two cryoprotective agents (CPAs), trehalose and hydroxyethyl starch (HES), were compared first in an experimental study, using a slowfreezing protocol. The five experimental groups were the following: (a) Fresh AT (control group), (b) T = -20°C, 10%HES, (c) T = -80°C, 10%HES, (d) T = -20°C, 0.35M trehalose, (e) T = -80°C, 0.35M trehalose. We evaluated the morphology (histological studies) and tissue viability by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genic expression. Based on the results of the preliminary study, an in vivo study was performed, choosing as cryopreservation T° -20°C. HES and trehalose were compared as cryoprotective agents and with a control group (fresh AT). AT grafts were transplanted into immunodeficient mice. After 1 month of inoculation, animals were euthanized and samples were recovered. Samples were weighted and processed for histological study, viability study (GAPDH genic expression), and vascularization study (VEGF genic expression). RESULTS: The initial histological study demonstrated that all AT cryopreserved group samples showed typical histological features of AT, similar to that of the control group. Statistically significant differences were not observed (P > 0.05) in GAPDH expression between different groups related to temperature or CPA. Referring to the in vivo studies, cryopreserved groups showed good take of the graft and normal AT architectural preservation, as well as a clear vascular network. Statistically significant differences were not found (P > 0.05) with regard to graft take (%), GAPDH, or VEGF expression. CONCLUSION: Slow freezing at -20°C using trehalose, and -20°C using HES as cryoprotective agents are both straightforward and easy AT cryopreservation procedures, with results similar to those of fresh AT in terms of tissue viability and morphohistological characteristics.

Effect of GnRH agonist before IVF on outcomes in infertile endometriosis patients: a randomized controlled trial.

RESEARCH QUESTION: Does 3-months of gonadotrophin releasing hormone agonist (GnRHa) treatment before IVF improve clinical pregnancy rate in infertile patients with endometriosis? DESIGN: Single-blind, placebo-controlled clinical trial of 200 infertile women with endometriosis assigned to use GnRHa (study group) or placebo (control group) for 3 months before IVF. Clinical, embryological outcomes and stimulation parameters were analysed. Clinical pregnancy rate was the primary endpoint. In a subgroup of 40 patients, follicular fluid levels of oestradiol, testosterone and androstendione were measured. Gene expression profile of CYP19A1 was analysed in cumulus and mural granulosa cells. RESULTS: Implantation or clinical pregnancy rate were not significantly different between the two groups. Clinical pregnancy rates were 25.3% and 33.7% in the study and control groups, respectively (P = 0.212). Cumulative live birth rate was not significantly different: 22.0% (95% CI 13.0 to 31.0) in the study group and 33.7% (95% CI 24.0 to 44.0) in the control group (P = 0.077). Ovarian stimulation was significantly longer and total dose of gonadotrophins significantly higher in the study group (both P < 0.001). Serum oestradiol levels on the day of HCG were significantly lower in the study group (P = 0.001). Cancellation rate was significantly higher in the study group (P = 0.042), whereas cleavage embryos were significantly more numerous in the control group (P = 0.023). No significant differences in the expression of CYP19A1 gene in mural or cumulus granulosa cells or steroid levels in follicular fluid between the two groups were observed, but testosterone was significantly lower in the study group (P < 0.001). CONCLUSION: Three-months of GnRHa treatment before IVF does not improve clinical pregnancy rate in women with endometriosis.

Aplicación del diagnóstico genético preimplantacional en la distrofia miotónica tipo 1 o enfermedad de Steinert / Application of pre-implantation genetic diagnosis in Myotonic dystrophy type 1 - Steinert's disease

La distrofia miotónica tipo 1 (DM1) es la forma más común de distrofia muscular en adultos causada por la expansión de una repetición de trinucleótidos CTG en el gen DMPK (Distrofia miotónica proteína quinasa) y su herencia es autosómica dominante. Hasta el momento, la técnica de Diagnóstico Genético Preimplantacional parece ser la aproximación reproductiva más eficaz para aquellas parejas en las que alguno de sus progenitores es afecto de DM1. En este estudio se evaluó la técnica de Diagnóstico Genético Preimplantacional en función del sexo del progenitor afecto y el número de repeticiones CTG y su influencia sobre el resultado reproductivo de la misma. Un total de 13 parejas, del programa de Diagnóstico Genético Preimplantacional de la Unidad de Reproducción Asistida del Hospital Universitari i Politècnic La Fe, fueron incluidas en el estudio. En 8 de éstas la afecta es la mujer, mientras que en las 5 restantes es el hombre el portador de la enfermedad. En todas ellas se llevaron a cabo todos los pasos del programa. Los embriones fueron analizados mediante mTP-PCR y PCR multiplex. El efecto de ambas variables se analizó mediante análisis bivariado (prueba T, Chi-cuadrado, regresión lineal y test Anova) y análisis multivariante utilizando análisis de correlación (Rho de Spearman). Los resultados obtenidos revelaron una transmisión preferencial de los alelos expandidos en DM1, independiente del sexo del progenitor afecto. A pesar de ello, no mostraron ninguna relación estadísticamente significativa entre el sexo del progenitor afecto ni el número de repeticiones CTG y el resultado reproductivo de la técnica. De modo que, atendiendo a nuestros resultados, el Diagnóstico Genético Preimplantacional sería una buena aproximación reproductiva en casos de DM1 con una tasa de gestación de 53,8 % y de nacidos sanos de 38,5 %, independientemente del sexo del progenitor afecto y del número de repeticiones CTG

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults caused by the expansion of a CTG trinucleotide repeat in the DMPK gene (dystrophia myotonica-protein kinase gene) and its inheritance is autosomal dominant. So far, the technique of Preimplantation Genetic Diagnosis (PGD) seems to be the most effective reproductive approach for those couples in which one of their parents is affected by DM1. In this study, the Preimplantation Genetic Diagnosis technique was evaluated according to the sex of the affected progenitor and the number of CTG repetitions and their influence on the reproductive result of itself. A total of 13 couples from the Preimplantation Genetic Diagnosis program of the Assisted Reproduction Unit of the Hospital Universitario i Politècnic La Fe were included in the study were included in the study. In 8 of them the affected parent is the woman, whereas the 5 remaining is the man who is the disease carrier. In all of them, all the steps of the program were followed. The embryos were analyzed by mTP-PCR and multiplex PCR. The variables effect was analyzed by bivariate analysis (T-test, Chi-square, linear regression and Anova test) and multivariate analysis using correlation analysis (Spearman's rho). The results obtained revealed a preferential transmission of the expanded alleles in DM1, regardless of the sex of the affected parent. In spite of, the results obtained do not show any statistically significant relation between the sex of the progenitor and the number of repetitions and the reproductive result of the technique. Thus, according to our results, Preimplantation Genetic Diagnosis would be a good reproductive approach in cases of DM1 with a gestation rate of 53.8% and healthy births of 38.5%, regardless of the sex of the affected parent and the number of CTG repetitions

Influence of temperature, serum, and gonadotropin supplementation in short- and long-term organotypic culture of human immature testicular tissue.

OBJECTIVE: To study how temperature, serum, and gonadotropin supplementation affect the organotypic culture of human immature testicular tissue (ITT) in vitro. DESIGN: Experimental basic science study. SETTING: Reproductive biology laboratory. PATIENT(S): ITT from 4 boys with cancer that had testicular tissue cryopreserved as part of their fertility preservation treatment. INTERVENTION(S): In vitro organotypic culture of ITT, exposed to different temperatures (37°C vs. 34°C), serum (fetal bovine serum [FBS] vs. Knockout Serum Replacement [KOS]), and gonadotropin supplementation (with and without FSH and LH). MAIN OUTCOME MEASURE(S): Characterization of the tissue was performed at days 0, 14, and 70 with the use of reverse-transcription quantitative polymerase chain reaction, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, histologic analysis by means of hematoxylin-eosin staining, and immunohistochemical staining. Hormonal secretion was determined at days 3, 14, 28, and 70 by means of immunofluorescent assay. RESULT(S): The 37°C conditions showed an accelerated loss of tubular morphology and higher intratubular apoptosis. KOS supplementation triggered the up-regulation of STAR, SOX9, DAZL, DDX4, PLZF, and UTF1, the percentage of SOX9+/androgen receptor (AR)-positive mature Sertoli cells at day 14, and testosterone secretion. Gonadotropin supplementation increased the numbers of both undifferentiated UTF1+ spermatogonia and premeiotic VASA+/SYCP3+ spermatogonia at day 14, and the number of SOX9+ Sertoli cells at day 70. The low SOX9+/AR+ colocalization, the disorganized pattern of ZO-1, and the progressive decrease of antimüllerian hormone secretion indicated inefficient Sertoli cell maturation in vitro. CONCLUSION(S): The 34°C condition in KOS showed the best results for the survival of both spermatogonia and Sertoli cells. FSH/LH supplementation also improved long-term survival of Sertoli cells and the maturation of spermatogonia up to meiotic initiation in short-term culture.

Effect of Human Fat Graft on Breast Cancer Metastasis in a Murine Model.

BACKGROUND: Isolated adipose stem cells have been reported to encourage migration and early metastasis of breast cancer. Mimicking a surgical situation, the authors developed a human breast cancer model to evaluate in vivo whether human adipose tissue promotes tumor growth and invasion. METHODS: Human adipose tissue was obtained from four patients. The MDA-MB-468 cell line was cultured with a lentiviral vector encoding a puromycin resistance gene and mCherry fluorescent protein. Virus-infected cells were selected. Animals were injected in the left renal capsule and divided into three experimental groups: group A, MDA-MB-468 cells (n = 4); group B, MDA-MB-468 cells/human adipose tissue (n = 4); and group C, Dulbecco's Modified Eagle Medium/F-12 medium (negative control, n = 4). Metastatic development was monitored using an in vivo imaging system. Small breast epithelial mucin (SBEM), human hypoxanthine-guanine phosphoribosyltransferase (HPRTh), and murine hypoxanthine-guanine phosphoribosyltransferase (HPRTm) expression were analyzed by real-time polymerase chain reaction to detect multifocal metastases in right/left renal capsule, liver, spleen, and pancreas. RESULTS: Metastasis was observed between postinjection days 37 and 44. No significant differences were found in survival rates between groups (group A, 157 ± 42.60 days; group B, 169 ± 40.17 days). All samples expressed HPRTm. HPRTh and SBEM were expressed in left renal capsules from all group A and B mice, whereas in spleen, liver, pancreas, and right renal capsule the HPRTm and SBEM expression was not constant in all samples of group A and B mice. Differences were found between groups in HPRTh and SBEM expression but were not statistically significant. CONCLUSION: Human adipose tissue used to restore breast defects after oncologic resection did not increase metastasis development risk when there were residual breast cancer cells in proximity.

Osmotic-shock produced by vitrification solutions improves immature human oocytes in vitro maturation.

BACKGROUND: During cytoplasmic oocyte maturation, Ca(2+) currents are vital for regulating a broad range of physiological processes. Recent studies have demonstrated that DMSO and EG cause large transient increases in intracellular Ca(2+) in mouse oocytes. The CP used in vitrifying protocols also increases the intracellular calcium transient. The aim of this study is to evaluate the effects of vitrifying time (before and after IVM) and exposure to the vitrification solutions and ionomycin on oocyte quality and embryonic development. METHODS: 221 GV-oocytes unsuitable for IVF-ICSI cycles were randomly distributed into one of the following three groups. G1 (control group): 41 GV-oocytes IVM until MII; G2: 43 oocytes vitrified at GV stage and IVM until MII stage; and G3: 53 GV-oocytes IVM until MII and then vitrified. In order to clarify the effect of vitrification solutions (VS) on human oocyte IVM through the intracellular Ca(2+) oscillation, the following two groups were also included. G4: 43 GV-oocytes exposed to VS and IVM until MII; and G5: 41 GV-oocytes exposed to ionomycin and IVM until MII. All GV-oocytes that reached MII-stage were parthenogenetically activated to assess oocyte viability. IVM was performed in IVF-medium (24-48 h). Chemical treatment (ionomycin) and osmotic treatment (vitrification solutions) were performed without liquid-N2 immersion. The following rates were evaluated: survival (SR), in-vitro maturation (IVMR), activation (AR), development to 2-cell (DRC), development to morula (DRCM) and development to blastocyst (DRB). Ratios between the different treatment groups were compared using contingency tables analysis (chi-square test). RESULTS: A high survival rate was obtained in G2 (95.5 %) and G4 (96.6 %). In-vitro maturation rate was significantly higher for G4 (86 %) and G2 (83.7 %) compared to G1 (63.4 %), G3 (56.6 %) and G5 (48.8 %). DRCM was significantly higher for G1 and G2 compared to G3 (G1: 15.8 %, G2: 20.7 % and G3: 0 %). DRB was only obtained for the oocytes vitrified before IVM (G2: 3.4 %). AR was also significantly higher for G2 and G4 compared to G5 (G2: 80.5 %, G4: 86.5 % and G5: 55 %). DRCM and DRB were only obtained in G2 and G4. DRCM was significantly higher for oocytes vitrified at GV stage (G2) and for oocytes exposed to the VS in G4 compared to the oocytes exposed to the ionomycin in G5 (G2: 20.7 %; G4: 37.5 % and G5: 0 %). CONCLUSIONS: Vitrifying GV-oocytes improves their IVM. Further investigation could look to increase the oocyte pool and improve fertility preservation options.

Bacterial and fungal contamination risks in human oocyte and embryo cryopreservation: open versus closed vitrification systems.

OBJECTIVE: To study the contamination risk in open and closed vitrification devices for oocyte/embryo cryopreservation by evaluating the contaminants present (bacteria and fungi) in the thaw medium and in liquid nitrogen (LN) storage containers. DESIGN: Retrospective study. SETTING: Human reproduction unit. PATIENT(S): None. INTERVENTION(S): Retrospective study of vitrification device safety and LN sterility performed from July to October 2014. MAIN OUTCOME MEASURE(S): From each bank container, both open and closed vitrification devices, devitrification media and LN in the containers and as supplied by the company were evaluated for contaminants. An automated system and the corresponding susceptibility to antibiotics were used for bacteria identification. Fungus detection was performed by evaluating the colony morphology and their microscopic characteristics. RESULT(S): No bacteria or fungi were observed in any of the devitrification media regardless of the type of device used, nor in the LN supplied by the company. No fungi were observed in any of the LN samples tested. Stenotrophomonas maltophilia and Bacillus spp. were found in all oocyte/embryo bank LN containers. There was no relationship between the number of samples or the time that each container had been used and the presence of microbiologic contaminants in the LN. At the container's bottom, Acinetobacter lwoffii, Alcaligenes faecalis ssp. faecalis, and Sphingomonas paucimobilis were found. CONCLUSION(S): Bacteria cross-contamination may not occur in oocyte/embryo banking in either open or closed storage devices. However, microorganisms can survive in LN. The bacteria cross-contamination risk is no greater for open than for closed containers. Storage containers should be cleaned periodically owing to the risk of lost straws or small particles of contaminated material.

New methods to improve the safety assessment of cryopreserved ovarian tissue for fertility preservation in breast cancer patients.

OBJECTIVE: To develop a novel molecular panel of markers to detect breast cancer (BC) disseminated malignant cells in ovarian tissue, and to improve the safety of ovarian tissue transplantation. DESIGN: Experimental study. SETTING: University hospital. PATIENT(S): Ten ovarian biopsies from healthy patients, 13 biopsies with diagnosed BC metastasis, and 4 biopsies from primary BC tumor for designing a diagnostic panel of BC cell contamination; 60 ovarian biopsies from BC patients undergoing fertility preservation for validating the panel. ANIMAL(S): Female nude mice. INTERVENTION(S): A novel panel for BC malignant cell detection by reverse-transcription polymerase chain reaction (RT-PCR), inmmunohistochemical analysis, in vitro invasion assay and xenotransplantation assayed in ovarian tissue from BC patients. MAIN OUTCOME MEASURE(S): Expression of GCDFP15, MGB1, SBEM, MUC1, WT-1, and NY-BR-01, selected as markers, assessed by quantitative RT-PCR in samples with confirmed BC metastasis. The most sensitive markers were confirmed by immunohistochemistry, and tested in vitro and in vivo. RESULT(S): GCDFP15, MGB1, and SBEM were the most sensitive and specific markers to detect BC metastatic cells when at least one was expressed by quantitative RT-PCR. The panel was validated in 60 patients and confirmed in an in vitro invasion assay, where no invasive cells were observed. Samples negative for BC cells cannot develop disease when xenografted. CONCLUSION(S): GCDFP15, MGB1, and SBEM were the most sensitive molecules to create a diagnostic panel for BC malignant cell contamination, which may make ovarian tissue cryopreservation and transplantation a safe technique for fertility preservation in BC patients.

Short-Term PTEN Inhibition Improves In Vitro Activation of Primordial Follicles, Preserves Follicular Viability, and Restores AMH Levels in Cryopreserved Ovarian Tissue From Cancer Patients.

INTRODUCTION: In vitro activation and growth of primordial dormant follicles to produce fertilizable oocytes would provide a useful instrument for fertility preservation. The employment of Phosphatase and TENsin homolog (PTEN) inhibitors, in combination with Protein kinase B (Akt) stimulating molecules, has been previously employed to increase follicular activation through the stimulation of the PTEN-Akt pathway. METHODS: We aim to establish improved in vitro activation also for cancer patients whose ovarian tissue has already been cryopreserved. Fresh and previously cryopreserved human ovarian cortex were exposed to short-term, low-concentration and ovary-specific treatment with only a PTEN inhibitor. RESULTS: Our in vitro activation protocol enhances the activation mechanisms of primordial follicles in both fresh and cryopreserved samples, and enlarges growing populations without inducing apoptosis in either follicles or the surrounding stroma. Treatment augments estradiol secretion and restores the expression levels of the previously diminished Anti-Müllerian hormone by means of cryopreservation procedures. Genomic modulation of the relative expression of PTEN pathway genes was found in treated samples. CONCLUSION: The in vitro activation protocol offers new alternatives for patients with cryopreserved tissue as it increases the pool of viable activated follicles available for in vitro growth procedures. The combination of ovarian tissue cryopreservation and in vitro activation of primordial follicles, the main ovarian reserve component, will be a major advancement in fertility preservation.

Time evolution of in vivo articular cartilage repair induced by bone marrow stimulation and scaffold implantation in rabbits.

PURPOSE: Tissue engineering techniques were used to study cartilage repair over a 12-month period in a rabbit model. METHODS: A full-depth chondral defect along with subchondral bone injury were originated in the knee joint, where a biostable porous scaffold was implanted, synthesized of poly(ethyl acrylate-co-hydroxyethyl acrylate) copolymer. Morphological evolution of cartilage repair was studied 1 and 2 weeks, and 1, 3, and 12 months after implantation by histological techniques. The 3-month group was chosen to compare cartilage repair to an additional group where scaffolds were preseeded with allogeneic chondrocytes before implantation, and also to controls, who underwent the same surgery procedure, with no scaffold implantation. RESULTS: Neotissue growth was first observed in the deepest scaffold pores 1 week after implantation, which spread thereafter; 3 months later scaffold pores were filled mostly with cartilaginous tissue in superficial and middle zones, and with bone tissue adjacent to subchondral bone. Simultaneously, native chondrocytes at the edges of the defect started to proliferate 1 week after implantation; within a month those edges had grown centripetally and seemed to embed the scaffold, and after 3 months, hyaline-like cartilage was observed on the condylar surface. Preseeded scaffolds slightly improved tissue growth, although the quality of repair tissue was similar to non-preseeded scaffolds. Controls showed that fibrous cartilage was mainly filling the repair area 3 months after surgery. In the 12-month group, articular cartilage resembled the untreated surface. CONCLUSIONS: Scaffolds guided cartilaginous tissue growth in vivo, suggesting their importance in stress transmission to the cells for cartilage repair.

Improving ovarian tissue cryopreservation for oncologic patients: slow freezing versus vitrification, effect of different procedures and devices.

OBJECTIVE: To compare slow freezing (SF) with four vitrification techniques (VT) for cryopreservation of ovarian tissue (OT) and to evaluate the best protocol for human OT in a xenograft model. DESIGN: Experimental study. SETTING: University hospital. PATIENT(S): Patients undergoing fertility preservation. ANIMAL(S): Ovariectomized nude mice. INTERVENTION(S): Cryopreservation of bovine OT after SF and four VTs (VT1, VT2, VT3, and VT4) by combining two cryoprotectant vitrification solutions (VS1 and VS2) and two devices (metallic grid and ethyl vinyl acetate bag), after which the cryopreservation of human OT by SF and VT1 and xenograft into nude mice. MAIN OUTCOME MEASURE(S): Follicular densities, proliferation, vascularization, fibrosis, apoptosis, tissue viability. RESULT(S): The in vitro study in bovine OT showed a lower percentage of quiescent follicles in the SF group but not in the vitrification groups (VT1-VT4). Apoptosis increased and cell proliferation decreased in all the experimental groups except VT1 (20% ethylene glycol, 20% dimethyl sulfoxide, 0.5 M sucrose, and 20% synthetic serum substitute in HEPES-buffered M199 culture media with Cryotissue metallic grids). Tissue viability was diminished in VT3, and the SF-xenografted human samples showed reduced primordial and secondary densities and unbalanced follicular populations when compared with fresh and VT1 tissue. CONCLUSION(S): VT1 offers similar conditions to fresh tissue for follicular density, proliferation, viability, and cell death and preserves a larger population of quiescent follicles than SF after transplantation, thus ensuring the maintenance of graft potential fertility.

Spontaneous in vitro maturation of oocytes prior to ovarian tissue cryopreservation in natural cycles of oncologic patients.

PURPOSE: To evaluate the recovery rate and spontaneous in vitro maturation (IVM) of immature oocytes enclosed within or released from follicles during the processing of ovarian tissue prior to its cryopreservation. METHODS: Thirty-three oncologic patients who had not previously undergone chemo or radiotherapy underwent ovarian tissue cryopreservation (OTC) during natural menstrual cycles. Immature oocytes, enclosed within follicles or released during ovarian cortex processing, were collected and matured spontaneously in vitro for 48 h. Nuclear maturation was assessed every 24 h and the ability of the IVM oocytes to display a normal activation response following parthenogenetic activation was evaluated. The following outcome measures were also evaluated: disease, age, FSH, LH, E2, P4 and AMH serum levels, menstrual cycle day, recovery and spontaneous IVM and parthenogenetic activation rates. RESULTS: Oocytes recovered per patient were 3.3 ± 0.7 (1.8-4.7 oocytes, 95CI), regardless of the menstrual phase. The mean number of IVM oocytes per patient was 1.3 ± 0.2 oocytes (95CI: 0.8-1.8), regardless of menstrual phase (p = 0.86) and oocyte origin (p = 0.61). Forty-one percent of oocytes extruded the second polar body and formed one pronucleus after parthenogenetic activation. CONCLUSION: Twenty-one of the 33 women (63.6 %) requesting OTC produced at least one mature oocyte.

Effect of antiangiogenic treatment on peritoneal endometriosis-associated nerve fibers.

OBJECTIVE: To investigate the effect of antiangiogenic treatment on experimental endometriotic lesion nerve fibers. DESIGN: Heterologous mouse model of endometriosis. SETTING: University Institute IVI, University Hospital La Fe. ANIMAL(S): Ovariectomized nude mice (n = 16) receiving human endometrial fragments from oocyte donors (n = 4). INTERVENTION(S): Endometrium fragments stuck in the peritoneum of 5-week-old female nude mice treated with vehicle (n = 8) and antiangiogenic agent cabergoline (n = 8; Cb(2,) 0.05 mg/kg/day) for 14 days. MAIN OUTCOME MEASURE(S): Immunofluorescence analysis of von-Willebrand factor (vWF) and vascular smooth muscle cells (αSMA) for evaluating the number of immature blood vessels (IBV) and microvascular density (MVD); immunochemical analysis of protein-gene product 9.5 (PGP 9.5) to assess nerve fibers density (NFD), and blue toluidine staining to confirm presence of mast cells and macrophages in endometriotic lesions. RESULT(S): All the results were quantified by morphometric techniques. The IBV, NFD, and number of macrophages and mast cells were statistically significantly decreased in the Cb2-treated group when compared with controls. CONCLUSION(S): Antiangiogenic treatment statistically significantly diminishes new blood vessel formation after macrophage, mast cell, and nerve fiber reduction, providing a rationale to test antiangiogenic agents as a novel therapeutic approach to severe pelvic pain associated with human peritoneal endometriosis.

Identification and quantification of dopamine receptor 2 in human eutopic and ectopic endometrium: a novel molecular target for endometriosis therapy.

Previous studies in an experimental mouse model of endometriosis have shown that the dopamine agonist (DA) cabergoline (Cb2) reduces angiogenesis and endometriotic lesions, hypothetically binding to the dopamine receptor type-2 (DRD2). To date, this has not been described in human endometrium and/or endometriotic lesions. Thus, we aimed to investigate the presence of DRD2 in said tissues. Endometrium fragments were implanted in nude mice treated with different doses of Cb2. Polymerase chain reaction assays and immunohistochemistry were performed to analyze the gene and protein expressions (respectively) of DRD2, VEGF, and VEGF receptor-2 (KDR). In addition, lesions and endometrium from women with mild and severe endometriosis and endometrium from healthy women were collected to analyze their gene expression profile. In experimental endometriosis, DRD2 was expressed at gene and protein levels in all three groups. VEGF gene and protein expressions were significantly lower in lesions treated with Cb2 than in controls. KDR protein expression was significantly lower in experimental lesions treated with Cb2 than in controls. In eutopic endometria, there was a significant decrease in DRD2 expression and an increase in VEGF in women with mild and severe endometriosis with respect to healthy patients. In endometriosis, KDR expression was significantly higher in red than in white and black lesions. VEGF expression was significantly lower in black than in red lesions. DRD2 is present in the human eutopic and ectopic endometrium and is regulated by DA, which provides the rationale for pilot studies to explore its use in the treatment of endometriosis.

Mimicking natural dentin using bioactive nanohybrid scaffolds for dentinal tissue engineering.

Synthetic materials mimicking the internal porous structure of natural dentin were prepared as nanohybrid matrix scaffolds made of poly(ethyl methacrylate-co-hydroxyethyl acrylate), pure and with a sol-gel-derived interpenetrated silica nanophase, with aligned tubular pores in the micrometer range typical of dentinal tissue. Some of them were internally coated with a layer of hydroxyapatite by immersion in simulated body fluid. Their physicochemical and mechanical properties were investigated. The different types of scaffolds were implanted subcutaneously into immunocompromised nude mice for 4, 6, and 8 weeks and their biological response were analyzed. Optical microscopy was employed to study the scaffold structure and neovascularization. Cells origin, inflammation, and macrophagic responses were evaluated by optical microscopy, immunohistochemistry, and transmission electron microscopy. The scaffold ultrastructural pattern imitates dentinal histological structure. The materials allowed cell colonization and neoangiogenesis. These biomaterials were colonized by murine cells fenotypically different to those of dermal connective tissue, showing structural differentiations. Colonization and viability were improved by the use of mineralized interphases, which showed a cellular distribution resembling a neodentinal pattern. Invasion of the scaffold tubules by single odontoblast-like processes was ascertained both in the noncoated and coated scaffolds. Such materials thus seem promising in tissue engineering strategies for dentin regeneration.

Malignant cells are not found in ovarian cortex from breast cancer patients undergoing ovarian cortex cryopreservation.

BACKGROUND: Breast cancer is a frequent indication for ovarian cortex cryopreservation due to its high incidence. The main concern of this procedure is the possibility of reintroducing metastatic cells within the implant, an issue that has not been addressed systematically. Thus, a study was designed to analyse the presence of ovarian metastases in breast cancer patients undergoing ovarian tissue cryopreservation. METHODS: Morphological and immunohistochemical studies following the concept of the sentinel lymph node (SLN) were performed on 100 cortical ovarian biopsies obtained from 63 patients and on six frozen-thawed entire cortex from patients with the diagnosis of infiltrating ductal breast carcinoma undergoing ovarian cortex extraction and cryopreservation. The antibody panel included Cytokeratin CAM 5.2, Gross Cystic Disease Fluid Protein-15 (GCDFP15), Wilms' tumour antigen-1 (WT1) and Mammaglobin 1. RESULTS: Employing only morphologic criteria, suspicious neoplastic cells were detected in five biopsies, but in none of the six entire cortex analysed. These five cases were reclassified as hyperplasic surface epithelium-inclusion cysts (CAM 5.2+, WT1+) or apoptotic granulosa cells (CAM 5.2-, GCDFP15+, WT1-). CONCLUSIONS: Using the methodology of the SLN our data suggest the absence of tumour cells in biopsies obtained from patients undergoing ovarian cortex cryopreservation to preserve their fertility potential, although future methods of cancer screening may change our perception of this procedure.

Dopamine agonist administration causes a reduction in endometrial implants through modulation of angiogenesis in experimentally induced endometriosis.

BACKGROUND: Implantation of a retrogradely-shed endometrium during menstruation requires an adequate blood supply. The endometrium has angiogenic potential, and endometriotic lesions grow in areas with a rich vascularization, suggesting that angiogenesis is a prerequisite for endometriosis development. Targeting vascular endothelial growth factor (VEGF) leads to an inhibition of endometriosis. Dopamine and its agonists, such as cabergoline (Cb2), promote VEGF receptor-2 (VEGFR-2) endocytosis in endothelial cells, preventing VEGF-VEGFR-2 binding and reducing neoangiogenesis. The aim of this study was to evaluate the anti-angiogenic properties of Cb2 on growth of established endometriosis lesions and investigate the molecular mechanisms by which Cb2 exerts the anti-angiogenic effect. METHODS: Human endometrium fragments were implanted in female nude mice peritoneum, and mice were treated with vehicle, 0.05 or 0.1 mg/kg/day oral Cb2 for 14 days. After treatment, the implants were processed to assess proliferative activity, neoangiogenesis, VEGFR-2 phosphorylation and angiogenic gene expression. RESULTS: A significant decrease in the percentage of active endometriotic lesions (P < 0.05) and cellular proliferation index (P < 0.001) was found with Cb2 treatment. Neoangiogenesis was reduced by Cb2 treatment, as observed at gross morphological level and by significant changes in gene expression. The degree of VEGFR-2 phosphorylation was significantly lower in Cb2-treated animals than controls. CONCLUSIONS: Cb2 treatment in experimental endometriosis has an anti-angiogenic effect acting through VEGFR-2 activation. These findings support the testing of dopamine agonists as a novel therapeutic approach to peritoneal endometriosis in humans.

Dopamine agonist cabergoline reduces hemoconcentration and ascites in hyperstimulated women undergoing assisted reproduction.

CONTEXT: Ovarian hyperstimulation syndrome (OHSS) results from increased vascular permeability (VP) caused by ovarian hypersecretion of vascular endothelial growth factor (VEGF), which activates its receptor-2. In animals, the dopamine receptor 2 agonist cabergoline (Cb2) inactivates VEGF receptor-2 and prevents increased VP. OBJECTIVE: Our objective was to test whether Cb2 reduces VP and prevents OHSS in humans. DESIGN: We conducted a prospective, randomized, double-blind study on oocyte donors at risk of developing OHSS (>20 follicles, >12 mm developed, and >20 oocytes retrieved). INTERVENTIONS: Cb2 0.5 mg/d (n = 37) or a placebo (n = 32) was administered from the day of human chorionic gonadotropin (d 0) until d 8. Ascites (a pocket of peritoneal fluid > 9 cm(2) in lithotomy position), hemoconcentration, and serum prolactin were recorded. Pharmacokinetic studies with magnetic resonance employing the transfer constant rate (K(trans), measure of permeability) and the extravascular extracellular space (upsilon(e), marker of cellular leakage) were performed to measure VP objectively. RESULTS: Hematocrit (P < 0.01), hemoglobin (P = 0.003), and ascites (P = 0.005) were significantly lower on d 4 and 6 after treatment with Cb2 as compared with placebo. The incidence of moderate OHSS was 20.0 and 43.8%, respectively (P = 0.04). Magnetic resonance studies showed an increase in VP and extravascular leakage of fluid 5 d after human chorionic gonadotropin injection that was significantly prevented with Cb2 (K(trans) P = 0.04 and upsilon(e) P = 0.001, respectively). CONCLUSIONS: Given that Cb2 is a well-established and safe medication, this study provides proof of concept for the use of dopamine agonists in the prevention of OHSS in women undergoing assisted reproduction.

Low-dose dopamine agonist administration blocks vascular endothelial growth factor (VEGF)-mediated vascular hyperpermeability without altering VEGF receptor 2-dependent luteal angiogenesis in a rat ovarian hyperstimulation model.

No specific treatment is available for ovarian hyperstimulation syndrome (OHSS), the most important complication in infertile women treated with gonadotropins. OHSS is caused by increased vascular permeability (VP) through ovarian hypersecretion of vascular endothelial growth factor (VEGF)-activating VEGF receptor 2 (VEGFR-2). We previously demonstrated in an OHSS rodent model that increased VP was prevented by inactivating VEGFR-2 with a receptor antagonist (SU5416). However, due to its toxicity (thromboembolism) and disruption of VEGFR-2-dependent angiogenic processes critical for pregnancy, this kind of compound cannot be used clinically to prevent OHSS. Dopamine receptor 2 (Dp-r2) agonists, used in the treatment of human hyperprolactinemia including pregnancy, inhibit VEGFR-2-dependent VP and angiogenesis when administered at high doses in animal cancer models. To test whether VEGFR-2-dependent VP and angiogenesis could be segregated in a dose-dependent fashion with the Dp-r2 agonist cabergoline, a well-established OHSS rat model supplemented with prolactin was used. A 100 microg/kg low-dose Dp-r2 agonist cabergoline reversed VEGFR-2-dependent VP without affecting luteal angiogenesis through partial inhibition of ovarian VEGFR-2 phosphorylation levels. No luteolytic effects (serum progesterone levels and luteal apoptosis unaffected) were observed. Cabergoline administration also did not affect VEGF/VEGFR-2 ovarian mRNA levels. Results in the animal model and the safe clinical profile of Dp-r2 agonists encouraged us to administer cabergoline to oocyte donors at high risk for developing the syndrome. Prophylactic administration of cabergoline (5-10 microg/kg x d) decreased the occurrence of OHSS from 65% (controls) to 25% (treatment). Therefore, a specific, safe treatment for OHSS is now available.