Long-term pregestational ethanol exposure induced altered fertilization and preimplantation embryogenesis. We evaluated preimplantational embryo-trophoblast differentiation, growth and invasiveness after perigestational ethanol 10% ingestion for 15 days preceding and up to day 4 (treated females [TF]: TF-D4 group) or 5 (TF-D5) of CD-1 gestation (control females [CF] with water). In TF-D4, expanded and hatched blastocyst numbers were significantly reduced (p < 0.05) versus CF-D4. Abnormal embryos and percentage of pyknotic nuclei were increased, and early blastocyst growth (nuclear number/embryo) and mitotic index was reduced (p < 0.05) versus CF-D4. On day 5 of gestation, TF-D5 presented significantly reduced total embryos and advanced embryo type 3 number versus CF-D5 (p < 0.05). During in vitro development, up to 72-hour culture, TF-D5 had reduced embryo type 1 (the least developed) and 3 percentages (p < 0.05) versus controls, whereas embryo type 2 percentage increased (p < 0.05) versus CF-D5. Embryo-trophoblast growth was studied during culture by morphometry. Embryo size ranges were classified as small, medium and large embryos. At 48-hour culture, small and medium embryos of TF had significantly increased mean area versus CF (p < 0.05), whereas large embryos had reduced mean area at 24-hour culture. Perigestational alcohol exposure up to days 4-5 induced embryo differentiation retardation, abnormal blastocyst growth and alterations of embryo-trophoblast growth and expansion during implantation, suggesting impaired regulation of trophoblast invasion and a relation with early pregnancy loss after mouse perigestational alcohol consumption.

The placenta plays a major role in embryo-fetal defects and intrauterine growth retardation after maternal alcohol consumption. Our aims were to determine the oxidative status and cellular and molecular oxidative stress effects on uterine myometrium and trophoblast-decidual tissue following perigestational alcohol intake at early organogenesis. CF-1 female mice were administered with 10% alcohol in drinking water for 17 days prior to and up to day 10 of gestation. Control females received ethanol-free water. Treated mice had smaller implantation sites compared to controls (p < 0.05), diminished maternal vascular lumen, and irregular/discontinuous endothelium of decidual vessels. The trophoblast giant cell layer was disorganized and presented increased abnormal nuclear frequency. The myometrium of treated females had reduced nitrite content, increased superoxide dismutase activity, and reduced glutathione (GSH) content (p < 0.05). However, the trophoblast-decidual tissue of treated females had increased nitrite content (p < 0.05), increased GSH level (p < 0.001), increased thiobarbituric acid-reactive substance concentration (p < 0.001), higher 3-nitrotyrosine immunoreaction, and increased apoptotic index (p < 0.05) compared to controls. In summary, perigestational alcohol ingestion at organogenesis induced oxidative stress in the myometrium and trophoblast-decidual tissue, mainly affecting cells and macromolecules of trophoblast and decidual tissues around early organogenesis, in CF-1 mouse, and suggests that oxidative-induced abnormal early placental formation probably leads to risk of prematurity and fetal growth impairment at term.

Perigestational alcohol consumption by CF-1 mouse, from before mating up to the period of embryo organogenesis, leads to retarded early embryo development and neural tube defects. Here, we addressed if perigestational alcohol ingestion up to Day 10 of pregnancy induces oxidative stress and changes in macromolecules and organ tissues of early organogenic embryos. Adult CF-1 female mice were administered 10% ethanol in their drinking water for 17 days prior to mating and until Day 10 of gestation, whereas control females were administered ethanol-free water. Our results demonstrated significantly reduced Catalase abundance and activity and increased glutathione content in the embryos of ethanol-treated females. The nitrite level was significantly reduced, but TBARS (thiobarbituric acid reactive substances) content, an index of lipid peroxidation, did not change. Embryos derived from ethanol-treated females also showed higher abundance of 3-nitrotyrosine (3-NT)-containing proteins in all tissues, compared to the control group. Apoptosis was significantly increased in the ectoderm and mesoderm, but not in the heart-although this organ did contain more cleaved Caspase-3-positive cardiomyocytes per area of ventricular myocardium than controls. In sum, moderate perigestational alcohol ingestion up to Day 10 of gestation in mice induces oxidative stress by altering radical nitrogen species and antioxidant enzymatic and non-enzymatic mechanisms in embryos. Further, generalized protein nitration, due to unbalanced nitric oxide levels associated with tissue-specific apoptosis, was detected in embryos, suggesting that oxidative mechanisms may play an important role in the perigestational alcohol-induced malformation of organogenic embryos exposed to ethanol.

Background: Gestation alcohol consumption produces fetal growth restriction and malformations by affecting the embryo-fetal development. Recently a relationship between abnormal placentation and fetal malformation and intrauterine growth retardation has been suggested. However, the effects of perigestational alcohol ingestion up to early pregnancy on the placenta at term and its association with fetal abnormalities are little known. Methods: In female mice, ethanol 10% in water was administered for 15 days previous and up to days 4 (D4), 8 (D8), or 10 (D10) of gestation (TF), and gestation continues without ethanol exposure. Control females (CF) received ethanol-free water. At day 18, feto-placental units and implantation sites were studied.Results: TF had increased resorptions and only fetuses from D8-TF and D10-TF had significantly increased weights versus CF. D4 and D10-TFplacentas had significantly reduced weights. All TF had increased junctional zone (JZ) and reduced labyrinth (Lab) areas (PAS-histology and morphometry) compared with CF. Fetuses with mainly with craniofacial abnormalities and skeletal defects (Alizarin red staining), significantly increase; while the fetal bone density (alizarin color intensity, ImageJ) was reduced in D4, D8 and D10-TF versus CF. Although all TF-placentas were histo-structural affected, TF-abnormal fetuses had the most severe placental anomalies, with junctional abundant glycogenic cells into the labyrinth, disorganized labyrinthine vascularization with signs of leukocyte infiltrates and feto-maternal blood mix. Conclusions: Perigestational alcohol consumption up to early gestation induces at term fetal growth alterations, dysmorphology and defective skeleton, linked to deficient growth and abnormal morphogenesis of placenta, highlighting insight into the prenatal etiology of FASD.