Soraya Labied

Isoform 111 of vascular endothelial growth factor (VEGF111) improves angiogenesis of ovarian tissue xenotransplantation

Background Cryopreservation of cortex ovarian tissue before anticancer therapy is a promising technique for fertility preservation mainly in children and young women. Ischemia in the early stage after ovarian graft causes massive follicle loss by apoptosis. VEGF111 is a recently described vascular endothelial growth factor (VEGF) isoform that does not bind to the extracellular matrix, diffuses extensively, and is resistant to proteolysis. These properties confer a significantly higher angiogenic potential to VEGF111 in comparison with the other VEGF isoforms. Methods We evaluated the morphology of cryopreserved sheep ovarian cortex grafted in the presence or absence of VEGF111. Ovarian cortex biopsies were embedded in type I collagen with or without VEGF111 addition before transplantation to severe combined immunodeficient mice ovaries. Transplants were retrieved 3 days or 3 weeks later. Follicular density, vasculature network, hemoglobin content, and cell proliferation were analyzed. Results Addition of VEGF111 increased density of functional capillaries (P=0.01) 3 days after grafting. By double immunostaining of Ki-67 and von Willebrand factor, we demonstrated that proliferating endothelial cells were found in 83% of the VEGF111 group compared with 33% in the control group (P=0.001). This angiostimulation was associated with a significant enhancement of hemoglobin content (P=0.03). Three weeks after transplantation, the number of primary follicles was significantly higher in VEGF111 grafts (P=0.02). Conclusion VEGF111 accelerates blood vessel recruitment and functional angiogenesis and improves the viability of ovarian cortex by limiting ischemia and ovarian cortex damage.

Strategies for using the sheep ovarian cortex as a model in reproductive medicine.

Objective To evaluate and compare the distribution and density of primordial follicles within a whole sheep ovary and to gain insight into how to overcome the impact of natural follicular heterogeneity on the experimental results. Design Histological study. Setting Academic research center. Animals Five- to nine-month-old ewes. Interventions Freshly sampled whole sheep ovaries were collected and prepared for histological analysis. Main Outcome Measure(s) The follicular densities and distributions were determined for hematoxylin and eosin sections. A mathematical model was derived based on the follicle counts and Monte-Carlo simulations. Results Heterogeneous distributions and densities of primordial follicles were identified 1) for distinct areas of the same ovarian cortex, 2) between the ovaries of the same animal and 3) across different ewes. A mathematical model based on the analysis of 37,153 primordial follicles from 8 different ovaries facilitated the estimation of the number of cortical biopsies and sections that had to be analyzed to overcome such heterogeneity. Conclusion The influence of physiological follicular heterogeneity on experimental and clinical results can be overcome when a definite number of cortical pieces and sections are taken into consideration.

Persistence of an Intact Endometrial Matrix and Vessels Structure in Women Exposed to VA-2914, a Selective Progesterone Receptor Modulator

BACKGROUND VA-2914 is a selective progesterone receptor modulator with potential contraceptive activity that induces amenorrhea, whereas progestins cause endometrial spotting and bleeding. This abnormal bleeding due to progestins is a consequence of focal stromal proteolysis by an increase in naked vessel size and density. OBJECTIVE Our objective was to quantify the effects of VA-2914 on endometrial vascularization, fibrillar matrix, and vascular endothelial growth factor (VEGF)-A expression in endometrial biopsies from 41 women before and after 12 wk daily treatment with a placebo, or 2.5, 5, or 10 mg VA-2914. METHODS Collagen fibrillar network was stained by silver impregnation. Vessel area, density, and structure were quantified with a computer-assisted image analysis system after double immunostaining using an anti-von Willebrand factor (endothelial cells) and an anti-alpha smooth muscle actin (vascular smooth muscle cells) marker antibody. VEGF-A mRNAs were quantified by RT-PCR and localized by immunohistochemistry. RESULTS The endometrial vessels, collagen network, and mRNA levels of VEGF-A were identical during the luteal phase at baseline and in VA-2914 treated women. VEGF-A distribution was unchanged. CONCLUSIONS VA-2914 does not alter the endometrial matrix and cells, and does not modify the endometrial vessel morphology as compared with baseline biopsies.

Transient reduction of placental angiogenesis in PAI-1-deficient mice.

Murine placentation is associated with the invasion of maternal endometrium by trophoblasts and an extensive maternal and fetal angiogenesis. Plasminogen activator inhibitor-1 (PAI-1) is transiently produced by spongiotrophoblasts and trophoblast giant cells at 10.5-11.5 days postcoitum (dpc). Knowing the key contribution of PAI-1 in the regulation of angiogenesis, we have now analyzed the consequence of PAI-1 deficiency on murine placentation. Morphological and quantitative computer-assisted image analysis revealed abnormal placental morphology in PAI-1-/- mice at 10.5 and 12.5 dpc. At 10.5 dpc, the genetic ablation of PAI-1 resulted in a transient reduction of both maternal and fetal vascularizations in the placenta and increased trophoblast cell density. This was associated with a poorer development of the labyrinth and an extension of the decidua. A larger spongiotrophoblast layer appeared at 12.5 dpc in PAI-1-deficient mice. Placental morphology was normalized at 14.5 dpc. Microarray analyses performed on laser capture microdissected labyrinths revealed that 46 genes were differentially expressed between the two genotypes at 10.5 dpc. However, only 11 genes were still differently modulated at 14.5 dpc, when normalization of placental morphology had taken place. This transcriptomic profiling highlighted a dysregulation in the expression of placenta-related cathepsin family members. Altogether our data provide evidence for a transient impaired placental morphology in PAI-1-deficient mice that is then normalized, leading to normal embryonic development.

Development of an animal experimental model to study the effects of levonorgestrel on the human endometrium

BACKGROUND This study was designed to develop an animal model to test the response of endometrium to local progestin delivery. METHODS Proliferative human endometrium was subcutaneously grafted in two groups of SCID mice that received, 2 days before, a subcutaneous estradiol (E2) pellet and, for half of them, an additional implant of levonorgestrel (LNG). Mice were sacrificed 1, 2, 3 or 4 weeks after endometrial implantation and grafts were histologically analysed. Proliferation, steroid hormone receptors, blood vessels and stromal decidualization in both groups (E2 and LNG) were immunohistologically evaluated and compared with proliferative endometrium and endometrium from women with an LNG intrauterine device. RESULTS Grafts presented normal morphological endometrial characteristics. The expression of progesterone receptors was significantly decreased in glands and stroma of the LNG group as compared with the E2 group at all times. A significant decrease was also observed in the stromal expression of estrogen receptor-α in the LNG group. At 4 weeks, the mean cross-sectional area of vessels was significantly higher after LNG treatment. CONCLUSIONS These morphological and immunohistochemical characteristics are similar to those observed in women treated with local LNG. This mouse model might facilitate further investigations needed to understand the mechanisms responsible for the breakthrough bleeding frequently observed in progestin users.

Differential elevation of matrix metalloproteinase expression in women exposed to levonorgestrel-releasing intrauterine system for a short or prolonged period of time

BACKGROUND The levonorgestrel-releasing intrauterine system (LNG-IUS) is an effective contraceptive and has many non-contraceptive health benefits. However, it is commonly associated with irregular endometrial bleeding. Metalloproteinases contribute to extracellular matrix (ECM) remodelling and regulate bleeding during the menstrual cycle. Enhanced metalloproteinase expression participates in the pathogenesis of breakthrough bleeding. Thus the objective of this study was to compare matrix metalloproteinase (MMP) expression in endometrium during luteal phase and in short-term (1 month) and long-term (> or =6 months) LNG-IUS users. METHODS MMP expression was analysed by semi-quantitative RT-PCR and immunohistochemistry. Gelatinase activity was determined by gelatin zymography. RESULTS MMP-1, -2, -3, -7, -9 and -12 mRNAs levels were increased, whereas that of MMP-26 was decreased in the endometrium of LNG-IUS users. MMP-1, -2, -3, -7 and -9 were localized by immunohistochemistry in all biopsies in the short-term group but in only 0-27% in the control group. The incidence of positive immunostaining for MMP-2 and -3 decreased significantly in the long-term compared with short-term LNG-IUS users. MMP-26 was localized in all biopsies from the control group but in only 14 and 25% from the short- and long-term LNG-IUS groups, respectively. In both LNG groups, the numbers of macrophages (the major source of MMP-12) was increased. CONCLUSIONS MMP-1, active MMP-2, MMP-3, MMP-7, MMP-9 and MMP-12 are more prevalent in the short-term LNG-IUS group, suggesting their important contribution to ECM breakdown and transient bleeding. The decrease in the percentage of women expressing MMP-2 and -3 might contribute to the decreased occurrence of unwanted spotting and bleeding in long-term LNG-IUS users.

Comparison between paraffin and mineral oil covering on early human embryo culture: a prospective randomized study

ABSTRACT The oil overlay in microdrop culture systems prevents medium evaporation, helps to maintain appropriate pH and osmotic conditions and protects from microbial contamination. In the present study, we prospectively compared covering by Ovoil™, a paraffin oil, and LiteOil®, a mineral oil, on the in vitro development of human embryos and their suitability for transfer/freezing at day 3 and live birth rate. One hundred and one patients undergoing in vitro fertilization (IVF) treatment by intracytoplasmic sperm injection (ICSI) were enrolled in our study. After ICSI, 1237 oocytes were 1:1 randomly allocated into 2 groups according to the type of overlaying oil: Ovoil™ (616 oocytes) or LiteOil® (621 oocytes). Fertilization rate was assessed around 18 hours post-insemination (hpi) and embryos were checked for early cleavage at 25 hpi. Embryo morphology was recorded on days 2 and 3. A total of 437 (Ovoil™) and 438 day 3 embryos (LiteOil®) were analyzed. There were no differences between the two groups in terms of fertilization rate and occurrence of early cleavage. The proportion of top quality embryos (41.7% vs. 41.2%) and the final utilization rates (92.2% vs. 92.0%) were similar in Ovoil and LiteOil groups, respectively, at day 3. Live birth rate per transfer was essentially the same with Ovoil™ overlay (26.9%) when compared to LiteOil® (26.2%). Live birth rate in patients who simultaneously received embryos from both overlay types was 17.2%. Despite the different characteristics of these two oils regarding hydrocarbon saturation, packing and temperature storage, Ovoil™ and LiteOil® can be used in parallel in the same IVF protocol. Abbreviations: ART: assisted reproductive technologies; hpi: hours post-insemination; hSA: human serum albumin; HTF: human tubal fluid; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; MII: metaphase II; MEA: mouse embryo assay; RT: room temperature.