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A B S TRACT The early events of fertilization that precede and cause activation of an egg have not been fully elucidated. The earliest electrophysiological change in the sea urchin egg is a sperm-evoked increase of the egg's membrane conductance. The resulting depolarization facilitates entry of the fertilizing sperm and precludes the entry of supernumerary sperm. The sequence of the increase in the egg's membrane conductance, gamete membrane fusion, egg activation, and sperm entry, including causal relationships between these events, are not known. This study reports the use of whole egg voltage clamp and loose patch clamp to monitor simultaneously changes of membrane conductance and capacitance at the site of sperm-egg contact. Measurements were made during sperm-egg interactions where sperm entry readily proceeded or was precluded by maintaining the egg's membrane potential either at large, negative values or at positive values. Whenever the sperm evoked an increase of the egg's membrane conductance, that increase initiated abruptly, was localized to the site of sperm attachment, and was accompanied by a simultaneous abrupt increase of the membrane capacitance. This increase of capacitance indicated the establishment of electrical continuity between gametes (possibly fusion of the gametes' plasma membranes). If sperm entry was blocked by large negative membrane potentials, the capacitance cut off rapidly and simultaneously with a decrease of the membrane conductance, indicating that electrical continuity between gametes was disrupted. When sperm entry was precluded by positive membrane potentials, neither conductance nor capacitance increased, indicating that sperm entry was halted before the fusion of membranes. A second, smooth increase of capacitance was associated with the exocytosis of cortical granules near the sperm in eggs that were activated. Electrical continuity between the gametes always preceded activation of the egg, but transient electrical continuity between the gametes alone was not always sufficient to induce activation. Early during fertilization of sea urchin eggs, the fertilizing sperm attaches to the egg and then, after a brief (10-30 s) latent period (Allen and Griffin, 1958), the egg begins to activate. The early events that lead up to and cause activation have not been fully elucidated.In the sea urchin egg, an increase of membrane conductance that occurs within seconds after sperm attachment is the earliest physiological event indicating intimate interaction between the sperm and the egg. The increase of conductance induced by the sperm depolarizes the egg from a resting value near -70 mV (Chambers and de Armendi, 1979;Lynn and Chambers, 1984;Lynn, McCulloh, and Chambers, 1988). Depolarization initiates an activation potential consisting of three distinct phases (Chambers and de Armendi, 1979). Phases 1 and 2 maintain the membrane potential more positive than + 10 mV as the fertilization envelope elevates over the surface of the egg. Phase 3 returns the membrane potential to a value ...

Background Progesterone (P4) is essential for support of the endometrium and implantation of an embryo in the normal menstrual cycle. In programed frozen embryo transfer cycles using exogenous P4 is necessary, as the endogenous production of P4 requires a functioning corpus luteum that is not present in programed cycles. To date, there is continuing debate about ideal serum estradiol and P4 values in frozen embryo transfer cycles. Methods Patients underwent single euploid embryo frozen transfer cycles from 2010 to 2013 at a single large academic center. Patients using donor oocytes and patients with changes in progesterone dose during the cycles in question were excluded. All cycles were programed and intramuscular P4 was used exclusively. Only patients administering the same daily dose of P4 throughout the cycle were included (N=213 patients). Main outcomes were ongoing pregnancy/live birth rates (OPR/LBR), clinical pregnancy rates (CPR), and spontaneous abortions/biochemical pregnancies. CPR was defined by the presence of a sac on 1st trimester ultrasound. Missed abortions were calculated per pregnancy with a sac. Receiver operator characteristic curves (ROC curves) and chi-squared tests were performed for statistical analysis. Results Two groups based on day 19 P4 levels were compared (group A, P4<20 ng/ml; group B, P4>20 ng/ml). OPR/LBRs were 65 vs. 49 %, group A vs. B, p value=0.02, RR=1.33 (1.1-1.7). Missed abortion and biochemical rates were higher in group B as opposed to group A, 27 vs. 12 %, p=0.01, RR=0.45(0.24-0.86). When P4 was stratified into five groups based on nanogram per milliliter of progesterone on day 19 (10-15, 15-20, 20-30, 30-40, and >40), there was a trend downward in OPR/LBR (70, 62, 52, 50, and 33 %, respectively). There was also an increase in missed abortion/biochemical rates (7, 15, 27, 32, and 20 %, respectively). Multiple logistic regression showed an increase in OPR/LBR when accounting for age, day 2 FSH, weight, number of embryos biopsied, and number of euploid embryos. Conclusion P4 levels >20 ng/ml on the day of transfer (during frozen single euploid embryo transfer cycles) were associated with decreased OPR/LBR.

Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism.

Preimplantation genetic screening using NGS significantly improves pregnancy outcomes versus PGS using aCGH in STEET cycles. Next-generation sequencing has the ability to identify and screen for embryos with reduced viability such as mosaic embryos and those with partial aneuploidies or triploidy. Pregnancy outcomes with NGS may be improved due to the exclusion of these abnormal embryos.