Human embryonic stem cells derived by somatic cell nuclear transfer.

Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

Insulin and insulin-like growth factor stimulation of vascular endothelial growth factor production by luteinized granulosa cells: comparison between polycystic ovarian syndrome (PCOS) and non-PCOS women.

CONTEXT: Vascular endothelial growth factor A (VEGF-A) is a potent cytokine that promotes angiogenesis and vascular permeability. After controlled ovarian stimulation (COS) for in vitro fertilization (IVF), excessive VEGF-A production can occur, particularly in women with polycystic ovarian syndrome (PCOS); however, it is unclear whether the regulation of VEGF-A production is different between PCOS and non-PCOS women. OBJECTIVE: The aim of this study was to determine whether there were differences in the dose- and time-dependent effects of insulin and IGFs on VEGF-A production by luteinized granulosa cells (LGCs) from women with and without PCOS. DESIGN AND SETTING: A prospective comparative experimental study was conducted at an institutional practice. PATIENTS: Patients included six PCOS and six non-PCOS women undergoing COS and IVF. INTERVENTIONS: Interventions included COS for IVF. MAIN OUTCOME MEASURES: VEGF-A levels in culture media were collected daily for 3 d from LGCs after incubation with variable doses of insulin, IGF-I, and IGF-II in the presence and absence of LH. RESULTS: In both study groups, exposure to LH alone did not alter VEGF-A levels. However, insulin or IGF increased VEGF-A levels within 1 d and appeared to synergize with LH at 3 d. VEGF-A production by non-PCOS LGCs was more sensitive to IGF exposure, whereas PCOS cells were more sensitive to insulin. Although an increase in DNA content (P < 0.05) was noted in cultures of PCOS cells, progesterone levels were lower compared with non-PCOS LGCs. CONCLUSION: Insulin and IGFs promote VEGF-A production in LGCs, but the response patterns are different when cells from PCOS and non-PCOS women are compared.

Precision of progesterone measurements with the use of automated immunoassay analyzers and the impact on clinical decisions for in vitro fertilization.

OBJECTIVE: To compare the precision of progesterone measurements obtained with the use of immunoassays and of liquid chromatography-tandem mass spectrometry (LC-MS/MS). DESIGN: Comparative study. SETTING: Academic, private practice, and in vitro fertilization (IVF) research centers. PATIENT(S): A total of 189 human serum samples were collected during controlled ovarian hyperstimulation and early pregnancy in women undergoing IVF. INTERVENTION(S): Serum progesterone pools (n = 10; 0.2-4 ng/mL) were sent to four laboratory centers that used four different automated immunoassay analyzers. Progesterone was measured by immunoassay in triplicate at three separate time points (n = 9 per pool) and by LC-MS/MS in triplicate once (n = 3 per pool). MAIN OUTCOME MEASURE(S): Inter- and intraassay coefficients of variation (CVs) of progesterone measurements were compared for each analyzer and LC-MS/MS. RESULT(S): Progesterone measurements by immunoassay were highly correlated with those by LC-MS/MS. Only two analyzers had intraassay CVs <10% at all three experimental time points, and only two analyzers had an interassay CV <10%. Mean progesterone levels by the analyzers were different across multiple progesterone pools. CONCLUSION(S): Our results indicate that progesterone threshold measurements used for IVF clinical decisions should be interpreted cautiously and based on laboratory- and method-specific data. A validated progesterone standard incorporated into daily immunoassays could improve medical decision accuracy.