Dopamine receptor 2 activation inhibits ovarian vascular endothelial growth factor secretion in vitro: implications for treatment of ovarian hyperstimulation syndrome with dopamine receptor 2 agonists.

OBJECTIVE: To ascertain whether vascular endothelial growth factor (VEGF) secretion by luteinized granulosa cells (GCs) is modulated by the dopaminergic system in a dose-dependent fashion and how this is related to the differential efficacy of dopamine receptor 2 (D2)-agonists (D2-ag) in preventing ovarian hyperstimulation syndrome (OHSS). DESIGN: The relationship between the dopaminergic system and VEGF secretion in luteinized GCs was evaluated. Archived human ovaries were immunostained to characterize D2 expression. SETTING: University affiliated infertility center. PATIENT(S): Premenopausal women and egg donors. INTERVENTION(S): Luteinized GCs were cultured with the D2-ag cabergoline. Human ovarian sections were immunostained for D2. MAIN OUTCOME MEASURE(S): The VEGF was measured by ELISA and D2 expression was evaluated by In-Cell ELISA. The D2 expression throughout the luteal phase was characterized by immunohistochemistry. RESULT(S): The VEGF secretion was decreased by the D2-ag in a dose-dependent fashion. The efficiency of this process was correlated with the amount of D2 expressed by luteinized GCs. A decrease in D2 expression in ovarian sections was observed during the late luteal phase. CONCLUSION(S): The efficacy of D2-ags in preventing OHSS might rely on their capacity to inhibit VEGF secretion by luteinized GCs. Because this capacity is dose-dependent, increasing the intraovarian concentration of D2-ags should be explored as a means of increasing the efficacy of these drugs in preventing OHSS.

Efficiency and purity provided by the existing methods for the isolation of luteinized granulosa cells: a comparative study.

BACKGROUND: Several protocols for the isolation of luteinized granulosa cells (LGCs) contained in follicular fluid have been described but no previously published study has compared the relative efficiency of these protocols. Our objective is to obtain conclusive scientific evidence for the superiority of one method over another. METHODS: Different purification methods for LGCs based on the recognition of specific cell markers, aggregates, differential adhesion and LGC size were evaluated. We compared the levels of CD45 cell contamination and the percentage of total cell viability in paired aliquots of cells (before and after purification) derived from the follicular fluid obtained from women who were donating oocytes (n = 72). Each of the six purification methods was performed six times using pooled follicular fluids from two women. RESULTS: Samples processed by means of recognition of specific cell markers were characterized by their greater purity (0.1-1.33% CD45+) but low rate of LGC recovery (17.13-25.4%) when compared with the other methods (3.29-12% CD45+, P < 0.05 and 51.67-73.20% LGC, P < 0.05). It is noteworthy that the filter method, which is based on the LGC size, combined one of the highest rates of LGC recovery (∼70%) with acceptable low levels of contamination (<5%). CONCLUSIONS: There is currently no gold standard method for the isolation of LGCs, and protocols should be chosen depending on the purpose in question. We conclude that fluorescence-activated cell sorting is the best protocol for isolating LGCs when purity is the principal criterion, and magnetic separation when both purity and viability are essential. However, cell straining (filter) is probably the least laborious and, overall, the most efficient method to isolate LGCs.

The effects of ergot and non-ergot-derived dopamine agonists in an experimental mouse model of endometriosis.

Implantation of a retrogradely shed endometrium during menstruation requires an adequate blood supply, which allows the growth of endometriotic lesions. This suggests that the development of endometriosis can be impaired by inhibiting angiogenesis. The growth of endometriotic foci is impaired by commercial oncological antiangiogenic drugs used to block vascular endothelial growth factor (VEGF) signaling. The dopamine agonist cabergoline (Cb2) inhibits the growth of established endometriosis lesions by exerting antiangiogenic effects through VEGFR2 inactivation. However, the use of ergot-derived Cb2 is associated with an increased incidence of cardiac valve regurgitation. To evaluate the potential usage of non-ergot-derived dopamine agonists for the treatment of human endometriosis, we compared the efficacy of quinagolide with that of Cb2 in preventing angiogenesis and vascularization in a heterologous mouse model of endometriosis. Nude mice whose peritoneum had been implanted with eutopic human endometrial fragments were treated with vehicle, 50  µg/kg per day oral Cb2, or 50 or 200  µg/kg per day quinagolide during a 14-day period. At the end of the treatment period, the implants were excised in order to assess lesion size, cell proliferation, degree of vascularization, and angiogenic gene expression. Neoangiogenesis was inhibited and the size of active endometriotic lesions, cellular proliferation index, and angiogenic gene expression were significantly reduced by both dopamine agonists when compared with the placebo. Given that Cb2 and quinagolide were equally effective in inhibiting angiogenesis and reducing lesion size, these experiments provide the rationale for pilot studies to explore the use of non-ergot-derived dopamine agonists for the treatment of endometriosis in humans.

Evidences for the existence of a low dopaminergic tone in polycystic ovarian syndrome: implications for OHSS development and treatment.

CONTEXT: The dopamine/dopamine receptor 2 (D2/Drd2) pathway modulates vascular endothelial growth factor (VEGF)-dependent vascular permeability and angiogenesis in the ovary. Deregulation of the VEGF/VEGF receptor (VEGFR)-2 pathway leading to increased risk of ovarian hyperstimulation syndrome has been described in the ovary of patients suffering from polycystic ovarian syndrome (PCOS). OBJECTIVE: The objective of the study was to ascertain whether deregulation of the VEGF/VEGFR-2 might a least be partially due to abnormalities of the D2/Drd2 pathway in PCOS women. DESIGN: Dated, archived ovaries from PCOs and control group patients as well as human chorionic gonadotropin-stimulated luteinized granulosa cells form PCOS and non-PCOS oocyte patients were used. SETTING: The study was conducted at a private research center. PATIENTS OR OTHER PARTICIPANTS: PCOS and nonpolycystic ovarian patients and oocyte patients participated in the study. INTERVENTION(S): Human ovarian sections were stained against the Drd2 antibody. Human chorionic gonadotropin-stimulated luteinized granulosa cells (LGC) were cultured in the presence/absence and the Drd2 agonist cabergoline. MAIN OUTCOME MEASURE(S): Drd2 and vascularized stained area in the theca layer of antral (< 8 mm) and luteinized follicles was quantified. VEGF, D2, and its related metabolites were measured in the supernatant of cultured LGC by ELISA and HPLC, respectively. VEGFR-2 and Drd2 expressed by LGC was quantified through an In-Cell ELISA. RESULTS: Decreased Drd2 expression and increased vascularization in the theca layer of antral and luteinized follicles of PCOS ovaries was observed. A lower dopamine production and reduced efficacy of cabergoline in inhibiting VEGF secretion was uncovered in LGC from PCOS. CONCLUSIONS: Decreased dopaminergic tone as well as deregulated Drd2 signaling might explain higher VEGF and vascularization leading to increased ovarian hyperstimulation syndrome risk in PCOS.

Human endometrial side population cells exhibit genotypic, phenotypic and functional features of somatic stem cells.

During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis for women's cycling and its dysfunction may be involved in the etiology of pathological disorders. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cells (SSC) population. Here we explore the hypothesis that human endometrial side population (SP) cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium after subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC.

A new automatic device for routine cord blood banking: critical analysis of different volume reduction methodologies.

BACKGROUND AIMS: Volume reduction is the usual process in cord blood banking that has some advantages regarding reducing the storage space and dimethyl sulfoxide (DMSO) quantity in the final product. The volume reduction methodology must guarantee high cell recovery and red blood cell (RBC) depletion by reducing all the umbilical cord blood (UCB) units to a standard volume. METHODS: We analyzed and compared critically three different volume reduction methods [hydroxyethylstarch (HES), top and bottom with Optipress II and Compomat G4, and AXP] used at the Valencia Cord Blood Bank over 10 years. RESULTS: The highest significant RBC depletion was achieved with the AXP system (P<0.001), while the top and bottom system with Compomat G4 and an adjusted buffy coat (BC) volume to 41 mL enabled the best total nucleated cell (TNC) recovery (P<0.001). TNC recovery and RBC depletion were similar for AXP and HES with an adjusted volume to 21 mL. In the multivariate analysis, when analyzing all cases, the BC volume set significantly influenced TNC, CD34+ and lymphocyte recoveries and RBC depletion (P<0.001). RBC depletion was significantly influenced by the initial volume and initial RBC content of UCB units (P<0.001). CONCLUSIONS: AXP is a highly efficient method for RBC depletion, providing the same TNC recovery as HES method with a final volume of 41 mL. AXP has the advantages of being an automatic and functionally closed system that shortens and better standardizes the proceedings. Top and bottom is a closed system that allows better TNC recoveries when the BC volume set is 41 mL.