Intraovarian regulation of gonadotropin-dependent folliculogenesis depends on notch receptor signaling pathways not involving Delta-like ligand 4 (Dll4).

BACKGROUND: In-situ hybridisation studies demonstrate that Notch receptors and ligands are expressed in granulosa cells (GCs) and in the theca layer vasculature of growing follicles. Notch signaling involves cell-to-cell interaction mediated by transmembrane receptors and ligands. This signaling pathway may represent a novel intraovarian regulator of gonadotropin-dependent follicular development to the preovulatory stage. We hypothesized that blocking Notch pathways would disrupt follicular maturation in the mouse ovary. METHODS: Hypophysectomized CD21 female mice were administered pregnant mare serum gonadotropin (PMSG) for 3 days to stimulate follicular development. In one experiment, a pan-notch inhibitor, compound E, was initiated 2 days prior to and throughout stimulation (n = 10), while in a second experiment, a humanized phage Dll4 blocking antibody, YW152F, was used (n = 5). After sacrifice, ovarian histology, serum estradiol levels and uterine weights were compared to controls. The ovarian morphology was evaluated with hematoxylin/eosin staining and immunohistochemistry was performed for Notch1, Notch2, Notch3, Notch4, Jagged1, Dll4, platelet endothelial cell adhesion molecule (PECAM) and alpha-smooth muscle actin (α-SMA) detection. RESULTS: We localized specific Notch ligands and receptors in the following structures: Dll4 is specific to theca layer endothelial cells (ECs); Notch1/Notch4 and Jagged1 are expressed in theca layer ECs and vascular smooth muscle cells (VSMCs), whereas Notch3 is restricted to VSMCs; Notch2 is expressed mostly on GCs of small follicles. Administration of a pan-Notch inhibitor, compound E, inhibits follicular development to the preovulatory stage (8.5 preovulatory follicles in treatment vs. 3.4 preovulatory follicles in control, p < 0.01; average number per ovary) with significant secondary effects on ovarian and uterine weight and estradiol secretion in a setting of uninhibited vascular proliferation, but disorganized appearance of ECs and VSMCs. Inhibition of endothelial Notch1 function through the inactivation of its ligand Dll4 with the blocking antibody YW152F induces mild disorganisation of follicular vasculature, but has no significant effect on gonadotropin-dependent folliculogenesis. CONCLUSIONS: Our experiments suggest that the complete blockage of the Notch signaling pathway with compound E impairs folliculogenesis and induces disruption of gonadotropin stimulated angiogenesis. It seems the mechanism involves Notch1 and Notch3, specifically, causing the improper assembly of ECs and VSMCs in the theca layer, although the potential role of non-angiogenic Notch signaling, such as Jagged2 to Notch2 in GCs, remains to be elucidated.

Does the addition of clomiphene citrate or letrazole to gonadotropin treatment enhance the oocyte yield in poor responders undergoing IVF?

PURPOSE: To evaluate the effect of adjunctive letrazole or clomiphene in IVF stimulation protocols. METHODS: A retrospective analysis of high dose GnRH antagonist IVF cycles (450-600 IU of gonadotropins) that have met poor responder criteria. Selected consecutive cycles in same patients differed solely in presence or absence of adjunctive clomiphene or letrazole. RESULTS: Supplementation with clomiphene citrate in poor responders showed significant improvements (p < 0.05) in estradiol levels (1506 vs. 1044 pg/ml), number of dominant follicles (5.6 vs. 3.9), oocytes retrieved (5.2 vs. 3.4) and number of transferred embryos (1.7 vs. 1.1). It significantly improved biochemical pregnancy rates (18.1% vs. 5.9%) while reducing cycle cancellations (11.7% vs. 32.6%). Letrozole supplementation showed similar effects. CONCLUSION: Both Clomiphene and Letrazole performed similarly and showed significant effects. However, despite increasing oocyte yield and embryo transfer rates, the overall clinical and live birth rate in this population remained low and showed no measurable increase.

Not all women diagnosed with PCOS share the same cardiovascular risk profiles.

Although definitive and confirmatory data are lacking, women with polycystic ovary syndrome (PCOS) are considered to be at increased risk for cardiovascular and metabolic disease. In recent years, the diagnosis of PCOS has broadened considerably to result in several phenotypes. Here we review the evidence for cardiovascular and metabolic risks in PCOS in the classic disorder and the various phenotypes. We conclude that not all women with PCOS should be considered as being similar in terms of cardiovascular risk profiles.

Ectopic pregnancy in a preexisting hydrosalpinx during a spontaneous pregnancy.

OBJECTIVE: To discuss the possible role of abnormal embryo migration as a cause of ectopic pregnancy during IVF with hydrosalpinges. DESIGN: Case report. SETTING: University-based reproductive endocrinology and fertility clinic. PATIENT(S): A patient presenting with a tubal ectopic pregnancy after spontaneous conception in a preexisting hydrosalpinx. INTERVENTION(S): Laparoscopic salpingectomy. MAIN OUTCOME MEASURE(S): Ultrasound and operative findings. RESULT(S): Case demonstration of abnormal embryo migration into a surgically documented preexisting hydrosalpinx during a spontaneous conception. CONCLUSION(S): The mechanism of increased tubal ectopic pregnancy rates during IVF with hydrosalpinges remains unexplained. This case supports abnormal embryo migration due to the hydrosalpinx as a contributing factor.

Adenovirus-mediated gene transfer of enhanced Herpes simplex virus thymidine kinase mutants improves prodrug-mediated tumor cell killing.

The Herpes simplex virus 1 (HSV) thymidine kinase (tk) suicide gene together with ganciclovir (GCV) have been successfully used for the in vivo treatment of various solid tumors and for the ablation of unwanted transfused stem cells in recent clinical trials. With the aim of improving this therapeutic system, we compared the potential efficacy of adenoviral (Ad) vectors expressing enhanced tk mutants in vitro and in vivo. The previously created HSV-tk mutants dm30 and sr39, created by random sequence mutagenesis, were inserted into a standard Ad.RSV E1(-)E3(-) backbone using homologous recombination. GCV killing of Ad.HSV-tk, Ad.dm30-tk and Ad.sr39-tk was assessed in various tumor cell lines with a cell proliferation assay. Cells expressing the two TK mutants were two-to-five-fold more sensitive to GCV when compared with Ad.HSV-tk transduced cells in all cell lines tested (five human mesotheliomas, one human lung cancer, a human cervical carcinoma, a mouse fibrosarcoma, and a rat glioma line) at equal TK expression levels. Flank tumor models, including cell-mixing studies, assessed the in vivo efficacy of the engineered viruses in BALB/C and SCID mice. In all animal studies, Ad.dm30-tk and Ad.sr39-tk showed more tumor growth inhibition than Ad.HSV-tk when GCV was administered. The use of adenovirus-mediated gene transfer of both tk mutants dm30-tk and sr39-tk for cancer suicide gene therapy should provide a more effective and safer alternative to wild-type HSV-tk.

An E2F-responsive replication-selective adenovirus targeted to the defective cell cycle in cancer cells: potent antitumoral efficacy but no toxicity to normal cell.

To improve the transduction and distribution of adenovirus in a tumor mass, we generated an adenovirus to selectively replicate in tumors. We hypothesized that after infection the replicating adenovirus would spread throughout the tumor mass and cause direct oncolysis of tumor cells. E2F transcription factors are critical regulators of cell growth and are often overexpressed in cancer cells because of the frequent aberrations in the pRb/E2F/p16(INK4a) pathway. As a result, a majority of tumor cells exist in a high proliferative state. E2F-1 is a transcription factor that activates its own transcription and that of other genes involved in the G(1) to S transition phase of the cell cycle. We constructed an adenovirus (Ad(E2F-1(RC)) so that E1A expression and viral replication were under the control of the human E2F-1 promoter element. AdE2F-1(RC) virus replicated as efficiently as the wild-type adenovirus and caused extensive cell killing in a panel of tumor cells in vitro. In contrast, nonproliferating normal epithelial, fibroblast, and endothelial cells, which express no E2F-1, were not able to support AdE2F-1(RC) replication. In animal studies, different dosing regimens of AdE2F-1(RC) administered to flank xenografts of ovarian and lung cancers led to a significant therapeutic advantage often surpassing that seen in animals treated with the wild-type adenovirus. This novel selectively replicating adenovirus offers a promising treatment platform for a variety of cancers of which the hallmark is uncontrolled cell growth.