Maternal L-proline supplementation during gestation alters amino acid and polyamine metabolism in the first generation female offspring of C57BL/6J mice.

We recently reported that dietary supplementation with L-proline (proline) during gestation improved embryonic survival in C57BL/6J mice. The objective of the present study was to test the hypothesis that the effect of maternal proline supplementation on embryonic survival can be carried forward to the first generation female offspring. In the F0 generation, pregnant dams were fed a purified diet supplemented with 0 (control) or 5 g proline/kg diet. The F1 female adult offsprings were bred to fertile males. Fetal survival at embryonic day (E)12.5 and reproductive outcomes at term birth were recorded. The concentrations of amino acids, ammonia, and urea in plasma and amniotic fluid, as well as concentrations of polyamines in placental tissues and amniotic fluid at E12.5 were determined. Results showed that the F1 generation female offspring from proline-supplemented dams had higher (P < 0.05) concentrations of glutamate and taurine in plasma; of putrescine and spermidine in placental tissues; and of glycine, taurine, and spermidine in amniotic fluid at E12.5, as compared with F1 generation female offsprings from dams without proline supplementation. Concentration of proline in the plasma of offspring mice from proline-supplemented dams were lower (P < 0.05), as compared with the control group. No differences in fetal survival, reproductive outcomes, or concentrations of ammonia and urea in plasma and amniotic fluid were observed between the two groups of F1 female offspring. Collectively, our results indicate that the benefits of maternal proline supplementation during gestation on improving embryonic survival and fetal growth in F0 females are not transmitted to their F1 generation females.

Dynamics of inflammatory reaction and oxidative stress across maternal serum, placenta and amniotic fluid in laboratory rats and the role played by genistein aglycone.

Background Genistein was reported to adversely influence fetal development although this is yet to be fully understood as a mechanism. Methods In this study, pregnant rats were divided into control (Cont.) and genistein force-fed (2-mg/kg and 4-mg/kg) groups. Each group was divided further into five subgroups: GD-0, GD-6, GD-13, GD-18, and GD-20 based on the terminal gestational day (GD). On the respective terminal GD, the rats were sacrificed and blood samples and amniotic fluid were carefully collected and separated and placenta homogenates were prepared. These samples were evaluated for oxidative stress and inflammatory reaction. The weights of embryonic implant and placenta tissue were also recorded. Heat shock protein (Hsp) (60 and 90), corticosterone, and oxidative stress biomarkers were determined in all the samples. Results Fetal and placental weights in all genistein-exposed groups were significantly decreased. A fluctuation in the level of the Hsp was recorded with a significant decrease recorded in Hsp90 level in the placenta and amniotic fluid towards GD-20 along with a concomitant increase in the corticosterone level in the amniotic fluid in all genistein groups compared to control. Maternal serum at GD-18 and GD -20 recorded a significant increase in antioxidant level (SOD, GSH, CAT) in all genistein-exposed groups. However, these antioxidants were significantly reduced in the placenta and the amniotic fluid compared to control. Conclusions Genistein enhances the placenta function in attenuating the risk of oxidative stress in the amniotic fluid and deferentially suppressed inflammatory activities in the placenta during early gestation and towards late gestation period.

Linking expression of aquaporin 3 to activation of JNK pathway.

Aquaporin 3 (AQP3) has been shown to be low in the amnion and chorion tissues of patients with oligohydramnios and that S. miltiorrhiza, a Chinese herbal medicine, results in increased AQP3 in human amniotic epithelial cells (hAECs). Here, we provide evidence for the involvement of the JNK pathway in AQP3 regulation in isolated oligohydramnios tissues in vitro, in hAECs derived from normal amniotic fluid and fluid from patients with isolated oligohydramnios. Phosphorylation of JNK was suppressed by pretreatment of cells with JNK-specific inhibitor (SP600125) and was up-regulated by S. miltiorrhiza; S. miltiorrhiza combined with SP600125 prevented SP600125-induced down-regulation of phospho-JNK both in normal amniotic fluid volume and in isolated oligohydramnios. In isolated oligohydramnios, AQP3 expression was significantly suppressed by SP600125 in a concentration- and time-dependent mannner, while its expression was up-regulated by S. miltiorrhiza. S. miltiorrhiza combined with SP600125 inhibited the increased expression of AQP3 relative to the S. miltiorrhiza treated group. Together, the data suggest that c-jun N-terminal kinase (JNK) pathway unerlies the regulation of AQP3 by S. miltiorrhiza amnion and chorion tissues.

Effect of high-dose fluoride on antioxidant enzyme activities of amniotic Fluid in rats.

OBJECTIVE: To investigate the effect of high-dose fluoride on antioxidant enzyme activities of amniotic fluid and fluoride of serum in rats. METHODS: The experimental study was conducted from January 8, 2008, to December 14, 2010, at the Suleyman Demirel University Experimental Animals Laboratory and the Medical Biochemistry Department Research Laboratory, Isparta, Turkey. Impregnated Wistar albino rats were divided into two equal groups. Group I had controls, while Group II rats were exposed to high-dose fluoride. Group I was given drinking water mixed with 0.1 mg/kg/b.w./day of natrium fluoride, while group II was given drinking water mixed with 10 mg/kg/b.w./day of natrium fluoride for 18 days. At the end of 18 days, amniotic fluid and blood samples were collected from control and experimental groups of pregnancy. Superoxide dismutase, glutathione peroxidase, catalase activities and thiobarbituric acid reactive substances as antioxidant enzymes in amniotic fluid and levels of fluoride in serum samples were investigated. RESULTS: There were 14 rats, with 7(50%) in each group. Foetal weight in group II significantly decreased compared to the control group (p< 0.05). Antioxidant enzyme activities in amniotic fluid were significantly higher in group II than group I (p< 0.05) although thiobarbituric acid reactive substances in amniotic fluid and serum fluoride levels were significantly lower in group II than group I (p< 0.05). CONCLUSIONS: Fluoride that created oxidative stress inhibited lipid peroxidation and apparently increased the antioxidant defence system.

Effects of Pinellia ternata (Thunb.) Berit. on the metabolomic profiles of placenta and amniotic fluid in pregnant rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Banxia (BX) is the root of Pinellia ternata (Thunb.) Berit. Its processed products, such as Jiang Banxia (JBX), have been clinically used in traditional Chinese medicine to treat vomiting, coughing, and inflammation. However, data for their safety for pregnant women are contradictory and confusing. AIM OF THE STUDY: To further explore the safety of BX, an ultra-performance liquid chromatography coupled with liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) metabolomics approach was used to evaluate the metabolic perturbation in pregnant rats caused by BX and JBX. MATERIALS AND METHODS: Placenta and amniotic fluid samples were collected from control Sprague-Dawley pregnant rats and exposed to BX suspension and JBX decoction (1.434g/kg/day). Samples were analyzed using LC-MS and GC-MS. The acquired MS data of above samples were further subjected to multivariate data analysis, and the significantly altered metabolites were identified. The associated pathways were constructed using MetaboAnalyst 3.0. RESULTS: The weight and histopathology of the placenta from each group of rats had no definite difference. However, we found 20 differential endogenous metabolites that changed significantly in the placenta and amniotic fluid samples. The alterations of identified metabolites indicated a perturbation in glycerophospholipid metabolism, amino acid metabolism, and carbohydrate metabolism in pregnant rats exposed to BX and JBX. CONCLUSION: In summary, this work suggested that oral administration of BX and JBX may induce disturbances in the intermediary metabolism in pregnant rats. This work contributes to further understanding the safety of BX and its processed products.

Distribution of grape seed flavanols and their metabolites in pregnant rats and their fetuses.

SCOPE: Polyphenols have been demonstrated to provide health benefits affecting cellular and physiological processes. This study aims to evaluate the bioavailability and distribution of grape seed flavanol compounds during pregnancy and whether fetuses could be exposed to these compounds. METHODS AND RESULTS: The distribution of flavanols and their metabolites in rat plasma, liver, white adipose tissue, brain, amniotic fluid, placenta, and fetuses after 1 and 2 h of an acute intake of a grape seed proanthocyanidin extract was examined by LC-ESI-TOF/MS. Flavanols and their metabolites were widely distributed in both pregnant and nonpregnant rat plasma and tissues. In liver, the conjugated forms of flavanols were less available in pregnant than nonpregnant rats. Flavanol metabolites were abundant in maternal placenta but detected at low levels in fetuses and amniotic fluid. CONCLUSION: Flavanol metabolization appears to be less active in the liver during pregnancy. Moreover, data indicated that transport across the placenta is not efficient and for flavanols and their metabolites, the placenta seems to act as a barrier. However, these compounds target the fetus and are excreted in the amniotic fluid.

Valproic acid confers functional pluripotency to human amniotic fluid stem cells in a transgene-free approach.

Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However, due to risks of random integration of the reprogramming transgenes into the host genome, the low efficiency of the process, and the potential risk of virally induced tumorigenicity, alternative methods have been developed to generate pluripotent cells using nonintegrating systems, albeit with limited success. Here, we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and capacity to differentiate into lineages of the three germ layers, such as definitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling.

Embryo protective effect of pomegranate (Punica granatum L.) fruit extract in adriamycin-induced oxidative stress.

The possible protective role of pomegranate (Punica granatum L.) fruit extract which has shown antioxidant capacity higher than that of red wine and green tea was evaluated against adriamycin-induced oxidative stress in chick embryos. Adriamycin (ADR), an anthracycline broad spectrum of chemotherapeutic drug is used for the treatment of variety of cancers; however, its prolonged use is limited by an irreversible, dose-dependant and progressive cardiomyopathy, hepatotoxicity and general toxicity to other organs in human beings, due to oxidative stress. The morphological changes (malformation of different organs), changes in body weight, volume of amniotic fluid (AF) and biochemical parameters of AF were studied after 24 and 48 h of incubation by comparing ADR alone and pomegranate fruit extract pretreated groups with their respective controls of 12 days old chick embryos. ADR alone at a dose of 70 microg/egg showed a significant dose versus time- dependent reduction in body weight, volume of AF. A dose-related increase in embryo gross morphological deformities and significant changes in the levels of biochemical parameters in AF were observed in ADR-treated group. These changes were significantly ameliorated to normal by pre-administration of pomegranate fruit extract at a dose of 200 microg/egg. Thus, the present study demonstrated the embryo protective nature of pomegranate fruit extract against ADR-induced oxidative stress.

Induction of fetal diuresis with intraamniotic furosemide increases the clearance of intraamniotic substances: An alternative therapy aimed at reducing intraamniotic meconium concentration.

BACKGROUND/PURPOSE: Contact with amniotic fluid (AF) causes intestinal damage in gastroschisis. Intraamniotic meconium has been shown to be responsible for intestinal damage, and occurrence of this damage has been shown to depend on the concentration of intraamniotic meconium. When intraamniotic meconium concentration is lowered below threshold level by exchanging AF with saline in gastroschisis, intestinal damage can be prevented. Theoretically, induction of fetal diuresis with intraamniotic furosemide may increase AF volume and fetal swallowing rate, thus, increase absorption of AF by intestines; therefore, the clearance of meconium from the AF may increase. An experimental study was planned to investigate the effects of intraamniotic diuretic injection on the clearance of intraamniotic substances. METHODS: Pregnant rabbits on the 23rd to 25th gestational day were divided into 2 groups as furosemide and control. Technetium tc99m labeled "tin colloid" was injected into the amniotic cavity, and AF sample was taken 10 minutes later. Furosemide was injected into the amniotic cavity afterwards. Two and 6 hours later, AF samples were obtained. Intestines were harvested at the end of the study. Control group received intraamniotic saline instead of furosemide. Radioactivities of the AF samples and intestines were determined by gamma counter. Clearance of the radioisotope from AF and intestinal accumulation were calculated. RESULTS: The clearance of the radioisotope from AF was increased significantly in the furosemide group (n = 10) compared with the control group (n = 8; P <.01). Gastrointestinal accumulation of the radioisotope in the furosemide group was 4-fold higher than that the control group (P <.01). CONCLUSIONS: Induction of fetal diuresis with intraamniotic furosemide accelerates the clearance of intraamniotic substances. This is probably caused by increased urinary output rate, which increases AF volume and consequently results in increased fetal swallowing of AF. In the diseases like gastroschisis and myelomeningocele, in which the contact with AF causes tissue damage, the elimination of meconium from AF in a somewhat natural manner like this method, should be studied further because it may be an alternative minimal invasive in utero treatment modality.

Correction of hemorrhage-induced anemia with intra-amniotic iron in the ovine fetus.

OBJECTIVE: This study tested the hypothesis that intra-amniotic iron treatment would enhance fetal red blood cell production after an acute, severe fetal hemorrhage of 40% of estimated blood volume over 2 hours. STUDY DESIGN: Three groups of late-gestation ovine fetuses were studied for 10 days: (1) control fetuses (n = 8), (2) fetuses hemorrhaged on day 3 (n = 11), and (3) similarly hemorrhaged fetuses supplemented with a single bolus of 60 mg of iron injected intra-amniotically immediately after the hemorrhage (n = 7). Statistical analysis was by 3-factor analysis of variance. RESULTS: At 24 hours after hemorrhage, red blood cell mass increased 5% in the control group and was reduced equally in both hemorrhage groups by 32% below day 3 prehemorrhage values. At 7 days after hemorrhage, red blood cell mass increased 27.8% +/- 2.6% (SE) above day 3 baseline values in the control fetuses. In the nonsupplemented hemorrhaged fetuses, red blood cell mass was not different from prehemorrhage values after 7 days (+3.7% +/- 4.1%), whereas red blood cell mass increased by 29.9% +/- 6.1% above prehemorrhage values in the iron-supplemented hemorrhage group (P <.001). CONCLUSION: Intra-amniotic iron supplementation resulted in full restoration of red blood cell mass within 7 days after a large loss of blood in fetal sheep, whereas restoration failed without iron supplementation. Intra-amniotic iron treatment may be of therapeutic value in restoring red blood cell mass in human fetuses with certain types of anemia such as that resulting from fetal or fetomaternal hemorrhage.

Garlic ingestion by pregnant women alters the odor of amniotic fluid.

Amniotic fluid samples were obtained from 10 pregnant women undergoing routine amniocentesis procedure. Approximately 45 min prior to the procedure, five of the women ingested placebo capsules, whereas the remaining five ingested capsules containing the essential oil of garlic. Randomly selected pairs of samples, one from a woman who ingested garlic and the other from a woman who ingested placebo capsules, were then evaluated by a sensory panel of adults. The odor of the amniotic fluid obtained from four of the five women who had ingested the garlic capsules was judged to be stronger or more like garlic than the paired samples collected from the women consuming placebo capsules. Thus, garlic ingestion by pregnant women significantly alters the odor of their amniotic fluid.

Effect of Viscum album L. on rapidly proliferating amniotic fluid cells. Sister chromatid exchange frequency and proliferation index.

Amniotic fluid cells (AFC) from 10 women undergoing amniocentesis were investigated. The 5-bromo-2'-deoxyuridine-induced sister chromatid exchange (SCE) frequency of AFC remained stable after the addition of a therapeutical concentration of Viscum album L. preparation Iscador P but decreased significantly after administration of high drug doses. As the proliferation index remained stable, even at extremely high drug concentrations, this effect could not be ascribed to a reduction of proliferation. No indications of cytogenetic damage or effects of mutagenicity were seen after the addition of Viscum album L. preparation P.

Protective effect of Liv.52 on alcohol-induced fetotoxicity.

The adverse effects of maternal alcohol consumption on the development of the fetus are well known. The adverse effects of ethanol on the liver are now believed to be due to acetaldehyde formed as an intermediate metabolite of ethanol. Liv.52 has been shown to bring about faster elimination of acetaldehyde from the body and thus prevent alcoholic liver damage. Other toxic effects of alcohol may also be due to acetaldehyde and may be prevented by Liv.52. In this study, rats were given 20% (v/v) ethanol in drinking water, during the gestation period, and the effect on maternal body weight and fetal outcome was noted. The protective effect of Liv.52 administration during the gestation period was studied. The results show that ethanol ingestion caused a decrease in gestational weight gain, total fetal weight, and number of live fetuses. There were increases in resorptions. Liv.52 administration reduced the deleterious effects of ethanol. The concentration of acetaldehyde in the amniotic fluid of ethanol-consuming animals was 0.727 microgram/ml. Liv.52 administration lowered it to 0.244 microgram/ml. The protective effect of Liv.52 could be due to the rapid elimination of acetaldehyde.

Prenatal dexamethasone exposure in rats: effects of dose, age at exposure, and drug-induced hypophagia on malformations and fetal organ weights.

Glucocorticoids cause stunting and cleft palate in rodents. The aim of this study is to identify fetal organs and developmental periods sensitive to stunting induced by maternal exposure to dexamethasone (DEX). DEX (0.2 or 0.4 mg/kg) or saline was given sc to pregnant CD albino rats on Gestation Days (GD) 9-14 or 14-19. On GD 20 dams were euthanized. Fetuses were weighed and examined for cleft palate. Eight fetuses/litter were randomly selected, and weights were obtained. Fetal skeletons were examined for abnormalities, and long bone measurements were taken. A dose-related decrease in maternal and fetal body weights occurred at both exposure periods. Developmental stage-specific malformations were noted in the high-dose group on GD 9-14 (cleft palate) and on GD 14-19 (wavy ribs). A dose-response in stunting occurred in all organs except cerebellum in at least one exposure period. Across both exposure periods the brain, heart, testes, and long bones were relatively resistant to DEX. Sensitive organs included thymus, spleen, adrenals, lungs, liver, and kidneys. DEX substantially reduced maternal food intake and increased water intake in some dams. Pair-feeding experiments suggested that the hypophagic effect of DEX was not responsible for the noted malformations and had little impact on growth stunting. The present findings have identified fetal organs, skeletal regions, and developmental periods sensitive to DEX exposure.

[In vitro transformation of human amniotic fluid cells in the presence of fecalase. Method for detecting environmental chemicals with oncogenic potential]. / Transformation in vitro de cellules amniotiques humaines en présence de fécalase. Mise au point d'un test de détection du pouvoir oncogène potentiel des substances chimiques de l'environnement.

A short-term method for detecting potential environmental carcinogens is described using in vitro transformation assay of human epithelial-like amniotic fluid cells. According to Styles, after a short exposition to the carcinogen, only transformed cells grow and form large colonies when cultured in semi-solid agar. In our assay addition of liver homogenate was not necessary to activate the carcinogens. Nevertheless adjunction to the exposure medium of human fecalase (after Ames) is advised for studying carcinogenicity of food glycosides. Fecalase efficiency in metabolic activation of glycosidic compounds has been demonstrated in the use of 8-hydroxyquinoline-beta-D-glycopyranoside.

Bacterial growth inhibition by amniotic fluid. VII. The effect of zinc supplementation on bacterial inhibitory activity of amniotic fluids from gestation of 20 weeks.

Twenty human amniotic fluids obtained from gestations of 20 weeks' duration supported bacterial growth. Nine of the 20 fluids could be made inhibitory by adjusting the phosphate to zinc ratios of the fluids to less than 200 mug per milliliter. These fluids contained the phosphate-sensitive bacterial inhibitor previously, but the fluids contained sufficient phosphate to inactivate the antibacterial system. The remaining 11 amniotic fluids did not contain the peptide component of the phosphate-sensitive bacterial inhibitor and could not be made inhibitory by adjusting the phosphate to zinc ratio to less than 200 mug per milliliter. The data obtained suggested synthesis of the peptide component may occur at a gestational age of approximately 20 weeks. The peptide may indirectly be detected in fluids by determining whether antibacterial activity is obtained when the phosphate to zinc ratio of the fluids is adjusted to less than 200 mug per milliliter.