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MicroRNAs (miRNAs) are class of small RNA molecules with major impact on gene regulation. We analyzed the potential of miRNAs secreted from pre-implantation embryos into the embryonic culture media as biomarkers to predict successful pregnancy. Using microarray analysis, we profiled the miRNome of the 56 spent culture media (SCM) after embryos transfer and found a total of 621 miRNAs in the SCM. On average, we detected 163 miRNAs in SCM of samples with failed pregnancies, but only 149 SCM miRNAs of embryos leading to pregnancies. MiR-634 predicted an embryo transfer leading to a positive pregnancy with an accuracy of 71% and a sensitivity of 85%. Among the 621 miRNAs, 102 (16.4%) showed a differential expression between positive and negative outcome of pregnancy with miR-29c-3p as the most significantly differentially expressed miRNA. The number of extracellular vehicles was lower in SCM with positive outcomes (3.8 × 109/mL EVs), as compared to a negative outcome (7.35 × 109/mL EVs) possibly explaining the reduced number of miRNAs in the SCM associated with failed pregnancies. The analysis of the miRNome in the SCM of couples undergoing fertility treatment lays the ground towards development of biomarkers to predict successful pregnancy and towards understanding the role of embryonic miRNAs found in the SCM.

X-linked protoporphyria (XLP) (MIM 300752) is a recently recognized erythropoietic porphyria due to gain-of-function mutations in the erythroid-specific aminolevulinate synthase gene (ALAS2). Previously, two exon 11 small deletions, c.1699_1670ΔAT (ΔAT) and c.1706_1709ΔAGTG (ΔAGTG), that prematurely truncated or elongated the ALAS2 polypeptide, were reported to increase enzymatic activity 20- to 40-fold, causing the erythroid accumulation of protoporphyrins, cutaneous photosensitivity and liver disease. The mutant ΔAT and ΔAGTG ALAS2 enzymes, two novel mutations, c.1734ΔG (ΔG) and c.1642C>T (p.Q548X), and an engineered deletion c.1670-1671TC>GA p.F557X were expressed, and their purified enzymes were characterized. Wild-type and ΔAGTG enzymes exhibited similar amounts of 54- and 52-kDa polypeptides on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), whereas the ΔAT and p.F557X had only 52-kDa polypeptides. Compared to the purified wild-type enzyme, ΔAT, ΔAGTG and Q548X enzymes had increased specific activities that were only 1.8-, 3.1- and 1.6-fold, respectively. Interestingly, binding studies demonstrated that the increased activity Q548X enzyme did not bind to succinyl-CoA synthetase. The elongated ΔG enzyme had wild-type specific activity, kinetics and thermostability; twice the wild-type purification yield (56 versus 25%); and was primarily a 54-kDa form, suggesting greater stability in vivo. On the basis of studies of mutant enzymes, the maximal gain-of function region spanned 57 amino acids between 533 and 580. Thus, these ALAS2 gain-of-function mutations increased the specific activity (ΔAT, ΔAGTG and p.Q548X) or stability (ΔG) of the enzyme, thereby leading to the increased erythroid protoporphyrin accumulation causing XLP.

H-rev107-1 is a growth inhibitory RAS target gene capable of suppressing anchorage independent growth in vitro and in vivo. Using a tumour tissue array with 241 matched tumour and normal tissue cDNA pools, we found down-regulation of H-REV107-1 in 7 out of 14 ovary-derived cDNAs. RT-PCR analysis and immunohistochemical investigation confirmed expression of H-REV107-1 in normal ovarian epithelial cells but down-regulation in high grade ovarian carcinomas. H-REV107-1 is also strongly expressed in immortalized rat and human ovarian epithelial cells in vitro, but suppressed in transformed cells by two different mechanisms. KRAS-transformed rat ovarian cells and PA1 teratocarcinoma cells, reversibly repress H-REV107-1 via MAP/ERK signaling. In contrast, treatment of A27/80 and OVCAR-3 epithelial ovarian cancer cells with IFNgamma stimulated H-REV107-1 expression. In NIH3T3 cells harbouring an estrogen-inducible IRF-1, H-rev107-1 is directly induced after activation of IRF-1, indicating that H-rev107-1 is a target of IRF-1. Stimulation of H-REV107-1 expression was also observed in ovarian epithelial cells suggesting that IRF-1 is involved in H-REV107-1 regulation in human ovarian epithelium. In the IFNgamma-sensitive cell line A27/80, H-REV107-1 suppresses colony formation. A27/80 and OVCAR-3 cells overexpressing H-REV107-1 protein underwent apoptosis. These results demonstrate down-regulation of the class II tumour suppressor H-REV107-1 in human ovarian carcinomas and suggest an involvement of H-REV107-1 in interferon-dependent cell death.