Hormonal regulation of pigment epithelium-derived factor (PEDF) in granulosa cells.

Angiogenesis is critical for the development of ovarian follicles. Blood vessels are abrogated from the follicle until ovulation, when they invade it to support the developing corpus luteum. Granulosa cells are known to secrete anti-angiogenic factors that shield against premature vascularization; however, their molecular identity is yet to be defined. In this study we address the physiological role of pigment epithelium-derived factor (PEDF), a well-known angiogenic inhibitor, in granulosa cells. We have shown that human and mouse primary granulosa cells express and secrete PEDF, and characterized its hormonal regulation. Stimulation of granulosa cells with increasing doses of estrogen caused a gradual decrease in the PEDF secretion, while stimulation with progesterone caused an abrupt decrease in its secretion. Moreover, We have shown, by time- and dose-response experiments, that the secreted PEDF and vascular endothelial growth factor (VEGF) were inversely regulated by hCG; namely, PEDF level was nearly undetectable under high doses of hCG, while VEGF level was significantly elevated. The anti-angiogenic nature of the PEDF secreted from granulosa cells was examined by migration, proliferation and tube formation assays in cultures of human umbilical vein endothelial cells. Depleting PEDF from primary granulosa cells conditioned media accelerated endothelial cells proliferation, migration and tube formation. Collectively, the dynamic expression of PEDF that inversely portrays VEGF expression may imply its putative role as a physiological negative regulator of follicular angiogenesis.

The effect of two distinct levels of oxygen concentration on embryo development in a sibling oocyte study.

PURPOSE: This prospective randomized study used sibling oocytes of 258 women with ≥8 oocytes to compare the effect of 5 % O(2) versus 20 % O(2) concentrations on embryo development and clinical outcome. METHODS: Oocytes of each case were divided between incubators with either 5 % or 20 % O(2) concentration. Outcome measures were fertilization, cleavage, embryo quality, blastocyst formation, and implantation, pregnancy and live birth rates. RESULTS: Fertilization and cleavage rates were similar in both groups. The 5 % O(2) group had significantly more blastomeres (P < 0.05) and more top-quality embryos on day 3 (P < 0.02), as well as significantly more available embryos for transfer (31.6 % vs. 23.1 % for the 20 % O(2) group; P < 0.0001). There were significantly more cycles with good embryos in the 5 % group (76/258) than in the 20 % group (38/258) (P < 0.0001). Implantation and pregnancy rates were significantly higher for 5 % O(2) embryos (P < 0.03 and P < 0.05, respectively). Live birth rates per embryo transfer were 34.2 % and 15.8 %, respectively, P < 0.05. CONCLUSIONS: Implantation, pregnancy and live birth rates are higher, and more good quality embryos are available for transfer and freezing with reduced rather than with atmospheric oxygen concentrations during embryo incubation.

Preimplantation genetic diagnosis (PGD) for SHOX-related haploinsufficiency in conjunction with trisomy 21 detection by molecular analysis.

PURPOSE: Development of a molecular PGD protocol for a male with an X-linked deletion in the SHOX gene region, located in the pseudoautosomal region of the X/Y chromosomes. Due to excessive recombination in this region, the deletion can be found in male offspring. METHODS: We developed a 13 marker multiplex fluorescent PCR protocol: 3 markers within the deleted SHOX region, 5 flanking markers, 3 informative markers on chromosome 21 (advanced maternal age) and 2 markers for sex determination. RESULTS: Of four embryos, two wild type males, diploid for chromosome 21 were transferred resulting in twin boys. One embryo was an affected female and another embryo was Turner. Amniocentesis confirmed the implanted embryos were males (46XY), with no recombinations. CONCLUSIONS: While many X-linked disorders can be analyzed by sexing, genes located in the pseudoautosomal regions have high XY recombination rates, requiring multiple markers to enable an accurate diagnosis.