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New strategies were proposed to improve the developmental competence of calf oocytes through in vitro technologies. Cumulus-oocyte complexes were first prematured for 24 h in the presence of meiosis inhibitors. Both Roscovitine alone (50 microM) or in combination with Butyrolactone-I (12.5 microM Rosco+6.25 microM BL-I) prevented the progression of meiosis. Their effect on nuclear maturation was reversible after a further 17 or 24 h maturation step. However, a dramatic decrease in embryo development was observed after fertilization (abattoir oocytes: 4-9% blastocyst rate versus 14-17% for control embryos). Similar results were obtained with oocytes collected by Ovum Pick Up from living donors. No pregnancy was obtained after single transfer of two blastocysts obtained from prematured oocytes (0/2 versus 4/12 for control embryos). Adding low concentrations (1, 3 or 10 microM) of follicular fluid-meiosis activating sterol (FF-MAS) during the maturation step had a beneficial effect on nuclear maturation (73-86% metaphase II versus 58% for control oocytes). However, subsequent embryo development was not improved. Enriching the maturation medium, namely with hormones, growth factors and precursors of glutathione, induced a sixfold increase in glutathione in the oocyte and had a beneficial effect on embryo development (38% increase in blastocyst rate). In conclusion, in opposition to the results reported with adult oocytes, prematuring calf oocytes had a negative impact on their developmental potential. Although FF-MAS improved nuclear maturation, its addition in the maturation medium did not increase embryo development. However, enriching the maturation medium had a positive effect on embryo development, indicating that cytoplasmic maturation was improved.

Objective: Because oral contraceptives are so widely used, any health consequences may have substantial public health implications. Whether pregravid oral contraceptives could affect subsequent pregnancies has not been adequately studied. The study objectives were to examine whether pregravid oral contraceptive use affects fetal growth and pregnancy hormone levels., Design: A prospective study of pregnant women followed through pregnancy., Setting: A major teaching hospital in Boston, USA., Population: Two hundred and sixty Caucasian pregnant women, with a mean age of 31, and a parity of no more than two. Seventy-nine percent of the women were pregravid oral contraceptive users., Methods: Exposure and covariate data were collected through structured questionnaires. Blood was drawn for hormonal analysis during the 16th and 27th gestational week. Information on pregravid oral contraceptive use included duration and recency of use, and oral contraceptive formulation. Multivariate regression models were used to examine the effect of pregravid oral contraceptive use on birth outcomes and the studied pregnancy hormones., Main Outcome Measures: Birthweight, placental weight, gestational age, pregnancy hormone levels of oestriol and progesterone at 16th and 27th gestational week., Results: Adjusting for confounders, pregravid oral contraceptive use increased birthweight (mean difference =+207.3 g, 95% CI =+77.6 to +337.1) and placental weight (mean difference =+64.9 g, 95% CI =+13.0 to +116.9) compared with never use. Women with prior oral contraceptive use had higher levels of serum progesterone (P= 0.002) and oestriol (P= 0.12) at the 27th gestational week measurement. The effect on birthweight, placental weight and hormones was stronger among those using oral contraceptives in the previous year and those using a high progestin/high oestrogen potency preparation., Conclusions: Pregravid oral contraceptive use is positively associated with fetal growth, and this effect may be mediated through oestriol and progesterone.

This study was conducted to elucidate the role of amino acids added singly or in groups to a chemically defined culture medium in blastocyst formation and blastomere proliferation of bovine embryos. Embryos were generated by in vitro fertilization, and blastocyst formation and hatching, and blastomere number of blastocysts were subsequently monitored after the culture of embryos in synthetic oviduct fluid medium (SOFM). First, one of four non-essential amino acids (asparagine, aspartate, glutamate or serine) was added to SOFM and, compared with no addition, a significant (P <0.05) increase in blastocyst formation was found after the addition of asparagine, aspartate, or glutamate (35-42% versus 22%). Second, one of four essential amino acids (arginine, cystine, isoleucine or leucine) was added and arginine or isoleucine greatly improved blastocyst formation (30-36% versus 16%). Third, the addition of five stimulatory amino acids (aspartate, asparagine, glutamate, arginine and isoleucine) to SOFM significantly improved blastocyst formation compared with no addition (12% versus 21%) and such value was similar to that obtained after the addition of 19 amino acids consisting of MEM amino acid solutions (21-27%). However, five amino acids yielded fewer hatched blastocysts than 19 amino acids. Finally, although five amino acids yielded more cell number of blastocysts than no addition (93 versus 74 cells per blastocyst), it was lower than that from 19 amino acids (131 cells per blastocyst). In conclusion, either single or combined addition of asparagine, aspartate, glutamate, arginine and isoleucine stimulated blastocyst formation, while other amino acids might be necessary for further stimulating blastomere proliferation and blastocyst hatching.

L-leucine, an essential amino acid, is one of the most popular ingredients in dietary supplements. To investigate a possibility of its embryo-fetal toxicity in rats, 11- to 12-week old dams were orally administered an aqueous solution of L-leucine at doses of 300 or 1000 mg/kg body weight on gestational days 7-17. Body weight and feed intake was evaluated throughout the whole course of pregnancy (days 0-20). L-Leucine did not influence body weight, but at a dose of 1000 mg/kg, slightly enhanced feed intake on days 14 and 18 of pregnancy. Caesarean section (day 20) revealed no influences on the litter size and weight of live-born fetuses, the number of corpora lutea, implantation index or the quality of placenta, and the minor increase in feed intake was considered irrelevant to the pregnancy outcomes. Fetuses were evaluated in a battery of external, visceral and skeletal examinations. No effects of L-leucine on gender ratio and external abnormalities, and no significant treatment-related variations in visceral and skeletal pathologies were observed. These results suggested that L-leucine, administered orally during organogenesis at doses up to 1000 mg/kg body weight, did not affect the outcome of pregnancy and did not cause fetotoxicity in rats.

Numerous studies have shown that di-2-ethylhexyl phthalate (DEHP) is teratogenic in animals but the mechanism of developmental toxicity is not well understood. One hypothesis is altered zinc homeostasis. The present study has investigated the effect of DEHP exposure on several key genes in zinc metabolism (MT-I, MT-II, ZnT-1) for early mouse embryos exposed in utero. Time- and dose-dependent effects were examined using expression polymerase chain reaction (PCR) (relative to ACTB) and Western blot analysis of the maternal liver, embryonic brain, and visceral yolk sac at 9 days post-coitus (d.p.c.). Maternal exposure to 800 mg/kg DEHP increased the abundance of MT-I and MT-II transcripts in maternal liver at 3.0, 4.5 and 6.0 h after administration. MT-I and MT-II protein induction was confirmed by Western blot analysis. On the other hand, this exposure down-regulated both transcripts (MT-I, MT-II), as well as transcripts for a zinc transporter (ZnT-1), in the embryonic brain, but not the visceral yolk sac. To examine dose-response relationships, the experiment was repeated for DEHP exposures of 50, 200 and 800 mg/kg. The effect to MT-I and MT-II expression in the maternal liver became significant at the 200 mg/kg dose level. The contrasting effect to MT-I, MT-II and ZnT-1 expression in the embryo was also dose-dependent, and a benchmark computation for the dose resulting in a 5% change in the mean (BMD5) was estimated as 11.6 mg/kg for MT-I, 8.9 mg/kg for MT-II, and 6.6 mg/kg for ZnT-1. We conclude that DEHP exposure to pregnant dams at reasonably low levels during organogenesis stages can alter the expression of several key genes in embryonic zinc homeostasis.

Tributyltin is ubiquitous in the environment and an endocrine disruptor for many wildlife species. However, minimal information is available regarding the effect of this chemical on bone formation. When tributyltin chloride (TBT) (1mg/kg body weight) was administered subcutaneously to pregnant mice at 10, 12, and 14 days post coitus (dpc), fetuses at 17.5 days post coitus revealed the inhibition of calcification of supraoccipital bone. In contrast, 1mg/kg body weight monobutyltin trichloride (MBT) did not affect the fetal skeleton. Therefore, we examined the effects of TBT and its metabolites (dibutyltin dichloride, DBT, and MBT) on bone metabolism using rat calvarial osteoblast-like cells (ROB cells). The viability of ROB cells was not affected by the exposure of the cells to 10(-10) to 10(-7)M TBT. However, TBT reduced the activity of alkaline phosphatase (ALPase) and the rate of deposition of calcium of ROB cells. In addition, the expression levels of mRNA for ALPase and osteocalcin, which are markers of osteoblastic differentiation, were depressed by the treatment with TBT. TBT inhibited ALPase activity and the deposition of calcium to a greater extent than did DBT. MBT had no effect on the osteoblast differentiation of ROB cells. Tributyltin is known to inhibit the activity of aromatase. However, the aromatase inhibitor aminoglutethimide did not reproduce the inhibitory effects of TBT on osteoblast differentiation. Our findings indicate that TBT might have critical effects on the formation of bone both in vivo and in vitro although its action mechanism is not clarified.

Effects of oxygen (O2) tension in the gas atmosphere during in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) on the efficiency of in vitro production of mouse embryos were examined. Mouse oocytes recovered from large antral follicles were subjected to IVM in Waymouth medium for 15, 16 and 17 hr under 5 or 20% O2 and then subjected to IVF and IVC under 5 or 20% O2 tension. Lowering the O2 tension in the gas atmosphere for IVM from 20 to 5% improved the cleavage rate after IVF when the oocytes were subjected to IVM for 15 hr; however, no improvement in the cleavage rate was observed when the culture period for IVM was extended to 16 and 17 hr. Lowering the O2 tension to 5% for IVM and IVC improved the development of the cleaved oocytes to the blastocyst stage, regardless of the culture period for IVM. However, the O2 tension for IVF had no remarkable effect on the subsequent embryonic development. These results demonstrate that 5% O2 is superior to 20% O2 for IVM and IVC, and suggest that 20% O2 for IVM may delay oocyte maturation and/or the acquisition of fertilizability and impair the developmental competence of oocytes.

Embryonic development is a highly coordinated set of processes that depend on hierarchies of signaling and gene regulatory networks, and the disruption of such networks may underlie many cases of chemically induced birth defects. The antiepileptic drug valproic acid (VPA) is a potent inducer of neural tube defects (NTDs) in human and mouse embryos. As with many other developmental toxicants however, the mechanism of VPA teratogenicity is unknown. Using microarray analysis, we compared the global gene expression responses to VPA in mouse embryos during the critical stages of teratogen action in vivo with those in cultured P19 embryocarcinoma cells in vitro. Among the identified VPA-responsive genes, some have been associated previously with NTDs or VPA effects [vinculin, metallothioneins 1 and 2 (Mt1, Mt2), keratin 1-18 (Krt1-18)], whereas others provide novel putative VPA targets, some of which are associated with processes relevant to neural tube formation and closure [transgelin 2 (Tagln2), thyroid hormone receptor interacting protein 6, galectin-1 (Lgals1), inhibitor of DNA binding 1 (Idb1), fatty acid synthase (Fasn), annexins A5 and A11 (Anxa5, Anxa11)], or with VPA effects or known molecular actions of VPA (Lgals1, Mt1, Mt2, Id1, Fasn, Anxa5, Anxa11, Krt1-18). A subset of genes with a transcriptional response to VPA that is similar in embryos and the cell model can be evaluated as potential biomarkers for VPA-induced teratogenicity that could be exploited directly in P19 cell-based in vitro assays. As several of the identified genes may be activated or repressed through a pathway of histone deacetylase (HDAC) inhibition and specificity protein 1 activation, our data support a role of HDAC as an important molecular target of VPA action in vivo.

Purpose: To observe pregnancies exposed to latanoprost, a prostaglandin analog administered in the treatment of glaucoma. Its prescription is limited in pregnancy, because reproduction studies in animals report a high incidence of abortion and human investigations are not adequate. As a consequence it is classified as category C drug according to the United States Food and Drug Administration's use-in-pregnancy ratings., Design: Observational study., Methods: We collected data, referred to our Teratology Information Service, relative to latanoprost exposure in pregnancy. We followed by phone interviews women treated with latanoprost during the first trimester, and we evaluated whether there had been any adverse effects on the fetus., Results: Eleven cases of latanoprost exposure in pregnancy were referred to our Teratology Information Service. One case was lost to follow-up, and one case was complicated by miscarriage. Nine cases had a complete follow-up without congenital anomalies., Conclusions: Our series is too small to perform statistical significance; however, we found no evidence of adverse effects of latanoprost on pregnancy or neonatal outcomes.

Although pesticide use is widespread, little is known about potential adverse health effects of in utero exposure. We investigated the effects of organophosphate pesticide exposure during pregnancy on fetal growth and gestational duration in a cohort of low-income, Latina women living in an agricultural community in the Salinas Valley, California. We measured nonspecific metabolites of organophosphate pesticides (dimethyl and diethyl phosphates) and metabolites specific to malathion (malathion dicarboxylic acid), chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl) phosphoro-thioate], and parathion (4-nitrophenol) in maternal urine collected twice during pregnancy. We also measured levels of cholinesterase in whole blood and butyryl cholinesterase in plasma in maternal and umbilical cord blood. We failed to demonstrate an adverse relationship between fetal growth and any measure of in utero organophosphate pesticide exposure. In fact, we found increases in body length and head circumference associated with some exposure measures. However, we did find decreases in gestational duration associated with two measures of in utero pesticide exposure: urinary dimethyl phosphate metabolites [beta(adjusted) = -0.41 weeks per log10 unit increase; 95% confidence interval (CI), -0.75 -- -0.02; p = 0.02], which reflect exposure to dimethyl organophosphate compounds such as malathion, and umbilical cord cholinesterase (beta(adjusted) = 0.34 weeks per unit increase; 95% CI, 0.13-0.55; p = 0.001). Shortened gestational duration was most clearly related to increasing exposure levels in the latter part of pregnancy. These associations with gestational age may be biologically plausible given that organophosphate pesticides depress cholinesterase and acetylcholine stimulates contraction of the uterus. However, despite these observed associations, the rate of preterm delivery in this population (6.4%) was lower than in a U.S. reference population.

We reported previously that insecticide exposures were widespread among minority women in New York City during pregnancy and that levels of the organophosphate chlorpyrifos in umbilical cord plasma were inversely associated with birth weight and length. Here we expand analyses to include additional insecticides (the organophosphate diazinon and the carbamate propoxur), a larger sample size (n = 314 mother-newborn pairs), and insecticide measurements in maternal personal air during pregnancy as well as in umbilical cord plasma at delivery. Controlling for potential confounders, we found no association between maternal personal air insecticide levels and birth weight, length, or head circumference. For each log unit increase in cord plasma chlorpyrifos levels, birth weight decreased by 42.6 g [95% confidence interval (CI), -81.8 to -3.8, p = 0.03] and birth length decreased by 0.24 cm (95% CI, -0.47 to -0.01, p = 0.04). Combined measures of (ln)cord plasma chlorpyrifos and diazinon (adjusted for relative potency) were also inversely associated with birth weight and length (p < 0.05). Birth weight averaged 186.3 g less (95% CI, -375.2 to -45.5) among newborns with the highest compared with lowest 26% of exposure levels (p = 0.01). Further, the associations between birth weight and length and cord plasma chlorpyrifos and diazinon were highly significant (p < or = 0.007) among newborns born before the 2000-2001 U.S. Environmental Protection Agency's regulatory actions to phase out residential use of these insecticides. Among newborns born after January 2001, exposure levels were substantially lower, and no association with fetal growth was apparent (p > 0.8). The propoxur metabolite 2-isopropoxyphenol in cord plasma was inversely associated with birth length, a finding of borderline significance (p = 0.05) after controlling for chlorpyrifos and diazinon. Results indicate that prenatal chlorpyrifos exposures have impaired fetal growth among this minority cohort and that diazinon exposures may have contributed to the effects. Findings support recent regulatory action to phase out residential uses of the insecticides.

1-beta-D-Arabinofuranosylcytosine (Ara-C), a cytidine analogue cytotoxic to proliferating cells, has a teratogenic effect in the brain of experimental animals and causes neural cell apoptosis in vitro and in vivo. In the present study, pregnant rats were injected with Ara-C on Day 13 of gestation and the fetal brain was collected from 1 to 48 h after treatment. Histopathological examinations revealed marked induction of apoptotic cell death and decrease of mitosis in neuroepithelial cells in the brain of Ara-C-treated fetus, and these changes were most prominent from 9 to 12 h. Expression of p53 protein, which mediates apoptosis and cell cycle arrest after DNA damage, was elevated remarkably and peaked at 3 h. p21, a cyclin-dependent kinase inhibitor responsible for p53-mediated cell cycle arrest, showed intense overexpression in protein and mRNA levels following the increase of p53 protein. The mRNA expressions of other p53 transcriptional target genes, bax, cyclinG1, and fas, also significantly increased and peaked at around 9 h. In conclusion, prenatal treatment of Ara-C is thought to induce apoptosis and inhibition of cell proliferation mediated by p53 and its target genes in the fetal brain., (Copyright 2004 Elsevier Inc.)

Organotin compounds have been implicated as reproductive toxicants and endocrine disruptors primarily through studies in aquatic organisms, with little information available in mammals. Among the organotins, aryltins have been less studied than alkyltins. Extensive data is available on mammalian developmental and reproductive toxicity of one aryltin compound, triphenyltin (TPT), from toxicity studies conducted in connection with the registration of triphenyltin hydroxide (TPTH) as a pesticide and supporting publications from the open literature. Indications of adverse functional and morphological effects on the reproductive tract of rats were reported in a dose range of 1.4-20 mg/kg/d. Gonadal histopathology (both ovaries and testes) and infertility were affected at the higher doses, while reproductive-tract cancer, smaller litter sizes, and reproductive organ weights were affected at the lower end of the dose range. In vitro studies indicate that TPT can directly activate androgen receptor-mediated transcription and inhibit enzymes that are involved in steroid hormone metabolism. These data suggest that the aryltin TPT can be active as a reproductive toxicant in mammals and may be a human endocrine disruptor.

This review covers normal expression of the NMDA receptor in the fetus and newborn, and then the response of the NMDA receptors within the central nervous system (CNS) during early development, to noxious stimuli. In the research setting, hypoxia is a commonly studied noxious stimulus that has been studied in a variety of contexts, including isolated hypoxia, or hypoxia combined with ischemia or hypercapnia, and delivered in single or repeated doses (intermittent stimuli). We review differences and commonalities between these experimental paradigms, and the sequelae of a common outcome, which is cell death, possibly through excitotoxic mechanisms. Finally, based on current literature, we will examine potential directions for clinical therapeutic interventions. By highlighting knowledge gaps in this field, we hope to encourage future research focusing on clinically relevant problems and outcomes in this area., (Copyright 2004 S. Karger AG, Basel)

Vitamin A is required throughout the life cycle, including crucial stages of embryonic and fetal development. With the identification of retinoic acid-specific nuclear transcription factors, the retinoid receptors, considerable advances have been made in understanding the molecular function of vitamin A. The requirement for vitamin A during early embryogenesis has successfully been examined in the vitamin A-deficient avian embryo during neurulation, when in the vertebrates crucial developmental decisions take place. These studies revealed that retinoic acid is essential during these early stages of embryogenesis for the initiation of organogenesis (i.e., formation of the heart). If retinoic acid is not present at this time, abnormal development ensues, leading to early embryonic death. Though the initial insult of the absence of vitamin A appears to be on the specification of cardiovascular tissues, subsequently all development is adversely affected and the embryo dies. Molecular and functional studies revealed that retinoic acid regulates the expression of the cardiogenic transcription factor GATA-4 and several heart asymmetry genes, which explains why the heart position is random in vitamin A-deficient quail embryos. During the crucial retinoic acid-requiring developmental window, retinoic acid transduces its signals to genes for heart morphogenesis via the receptors RARalpha2, RARgamma, and RXRalpha. Elucidation of the function of vitamin A during early embryonic development may lead to a better understanding of the cardiovascular birth defects prevalent in the Western world.

Adverse developmental effects of two haloacetic acids, bromochloroacetic acid (BCA) and dibromoacetic acid (DBA), were determined by using the Frog Embryo Teratogenesis Assay--Xenopus (FETAX). Xenopus embryos (150-400/concentration group) were exposed to 0, 8000, 10,000, 12,000, or 14,000 ppm BCA or 0, 10,000, 12,000, 14,000, or 16,000 ppm DBA for 96 h beginning from stage 8 (mid-blastula) to stage 46 (when primary organogenesis is complete). BCA produced 29, 83, and 100% mortality at 10,000, 12,000 and 14,000 ppm, respectively. Incidence of malformations among surviving embryos at 96 h for 10,000 and 12,000 ppm BCA were 8.4 and 68%. Thus LC50 and EC50 for BCA were between 10,000 and 12,000 ppm. DBA did not produce any significant mortality or malformation at any of the concentrations tested. In summary, BCA affected development of Xenopus embryos only at high concentrations, while DBA did not affect Xenopus development at the concentrations tested.

Among the many deleterious effects of maternal smoking during pregnancy on foetal development, is a higher incidence of persistent pulmonary hypertension. The recent identification of nicotinic acetylcholine receptors (nAChR) on cells of the pulmonary vessel walls suggests that maternal smoking during pregnancy may produce morphological alterations in foetal pulmonary vasculature. Timed-pregnant rhesus monkeys were treated with nicotine (1 mg x kg(-1) x day(-1)) delivered by subcutaneous osmotic mini-pumps from days 26-134 of gestation (term: 165 days). Lung sections from 134-day foetal monkeys were used for morphometric analysis, in situ hybridisation and immunohistochemical staining. Following nicotine treatment, total wall and tunica adventitia thickness of airway associated vessels (AAV) increased significantly. Nicotine exposure significantly increased collagen I and III mRNA and protein in tunica adventitia in all AAV but not in tunica media. By contrast, levels of elastin protein were significantly decreased. alpha7 nAChR were detected in AAV fibroblasts that expressed collagen mRNA. Choline acetyltransferase, the enzyme which synthesises acetylcholine, the ligand for alpha7 nAChR was also detected in endothelium and fibroblasts. These findings suggest that with smoking during pregnancy, nicotine is transported across the placenta and directly interacts with nicotinic acetylcholine receptors in pulmonary vessels to alter connective tissue expression and therefore produce vascular structural alterations.

This study was conducted to characterize the effect of androstenedione on estrous cyclicity, mating behavior and fetal development. Thirty-day old rats received corn oil alone or androstenedione (in corn oil) at one of four concentrations (0, 1.0, 5.0, 10.0 or 30.0 mg/kg body weight) by gavage for two weeks prior to mating, during the mating period and throughout gestation. Dose related increases in serum androstenedione, estradiol and estrone were observed in all androstenedione treated animals at gestation day 20. A statistically significant increase in serum testosterone concentration was observed in the 30 mg/kg dose group. Feed and fluid consumption were not affected by androstenedione treatment during the pre-mating or gestational periods, however a statistically significant decrease in the number of females with regular estrous cycles was observed in the 10.0 and 30.0 mg/kg dose groups. Exposure to androstenedione did not affect mean body weight gain during pre-mating or gestation. Slight not statistically significant reductions in the number of implants, number of viable fetuses and number of viable male fetuses were observed in the 30.0 mg/kg androstenedione group. Reductions were not observed in the number of corpora lutea. Fetal growth in terms of fetal weight, crown-rump length, anogenital distance and the number of external abnormalities was not affected by androstenedione exposure. At the doses given, androstenedione had no specific effect on the development of individual bones, including sternebrae. Dose related effects of androstenedione were not observed on the development of soft tissues. A statistically significant increase in moderately enlarged ureter at the kidney was observed in both the 1.0 and 5.0 mg/kg dose groups. Organ weights (expressed per gram of body weight or per gram of brain weight) were not affected by androstenedione treatment.

In a standard embryotoxicity/teratogenicity study, alpha-cyclodextrin (alpha-CD) was administered to groups of sixteen, artificially inseminated New Zealand White rabbits at dietary concentrations of 0, 5, 10, or 20%. An additional group received a diet containing 20% lactose. Treatment started on day 0 of gestation and ended on day 29 when the animals were killed. Except for the occurrence of transient diarrhoea in one rabbit of the 20% alpha-CD group for a few days, the treatment was well tolerated. A reduced food intake in the 20% alpha-CD group during the first week of treatment resulted in a reduced weight gain from day 0 to 12 of the study. However, the difference to the controls was not significant and at termination of the study body weights were similar in all groups. Even at the highest dose level, which corresponds to an intake of 5.9-7.5 g/kg bw/day, no signs of maternal toxicity were observed. Maternal reproductive performance was not affected by the treatment. Uterine weight, placental weight, fetal weight, number of fetuses, sex ratio, number of implanation sites, resorptions, and corpora lutea did not differ among the groups. Visceral and skeletal examinations of the fetuses did not reveal any malformations, anomalies or variations that could be attributed to treatment. It was concluded that dietary alpha-CD is generally well tolerated by pregnant rabbits, has no adverse effect on maternal reproductive performance and is not embryotoxic, fetotoxic, or teratogenic at dietary concentrations of up to 20%, the highest dose level tested.

The embryotoxicity/teratogenicity of alpha-cyclodextrin (alpha-CD) was examined in Wistar Crl:(WI)WU BR rats. alpha-CD was fed at dietary concentrations of 0, 1.5, 5, 10, or 20% to groups of 25 pregnant female rats from day 0 to 21 of gestation. An additional group received a diet with 20% lactose. The additions to the diet of alpha-CD and lactose were made at the expense of pregelatinized potato starch. Body weight as well as food and water intake were recorded during the treatment period. The rats were killed on day 21 and examined for standard parameters of maternal reproductive performance. The fetuses were examined for external abnormalities, body weight and crown rump length. Fetuses were examined for skeletal and visceral abnormalities. Generally, alpha-CD was well tolerated and no deaths occurred in any group. Weight gain and food consumption were similar in all groups during gestation, except for a slightly yet significantly increased food intake in the 20% alpha-CD group from day 6 to 21. Water intake was similar in all alpha-CD groups; in the lactose group, it was significantly higher than in the controls. Maternal reproductive performance was not affected by the alpha-CD treatment. Examination of the fetuses for external, visceral and skeletal changes did not reveal any fetotoxic, embryotoxic, or teratogenic effects of alpha-CD. In conclusion, no adverse effects were observed at alpha-CD intakes of up to 20% of the diet, the highest dose level tested at which the rats consumed about 13 g/kg bw/day.

Objectives: The purpose of this research was to study the independent effect of the amino acid glutamine on preimplantation mouse embryo development in vitro., Study Design: Two-cell stage mouse embryos were cultured in human tubal fluid medium in the presence and absence of 1 mmol/L of glutamine. Outcomes for morphology and cleavage rates were compared with Fisher's and Mann-Whitney's tests, respectively., Results: Glutamine increased the proportion of embryos that developed to the blastocyst stage (86.4%) compared with those cultured without glutamine (59.1%) (P =.052). The percentages of embryos developing to the morula or hatching blastocyst stages were comparable in the 2 groups. Blastocyst total cell numbers were significantly higher in the glutamine group (34+/-1.7 vs 18.5+/-3.5, respectively; values are mean+/-SEM, P =.044)., Conclusion: The amino acid glutamine independently improves preimplantation mouse embryo development in vitro. Further studies are needed to examine the applicability of these results to humans.

Problem: The immunological success of pregnancy is thought to depend upon the establishment of a balance between favorable and deleterious cytokines, the current paradigm viewing pregnancy as a T helper (Th)2 cytokine-dependent phenomenon. In this context, a particular attention should be directed to the potential role of interleukin (IL)-12, which promotes the development of Th1 responses, in the induction of adverse pregnancy-related phenomena. Indeed, very few data linked the Th1-inducer IL-12 to the event of abortion., Methods: In this study, we have investigated the maternal and fetal effects of exogenous administration of IL-12 to CD1 (BR) ICR mice during the pre- and peri-implantation period (day 2-6 of pregnancy). Animals have been evaluated for parameters of reproductive performance, embryo and fetal developmental toxicity and maternal toxicity., Results: Intraperitoneal administration of IL-12 at concentrations from 2.5 to 10 microg/kg daily did not result in an increase in the murine abortion rate. A statistically significant, although minimal, decrease in the number of somites were found in the embryos of animals treated with IL-12 at a dose of 10 microg/kg/day. However, developmental parameters at birth were similar between the two groups of animals suggesting that alteration of somites might be a transitory state during treatment. An increased body weight gains and reduced feed and water consumption were observed in the mothers treated with the cytokine., Conclusion: In the present experimental conditions and in this specific strain of mice, IL-12 does not exert adverse effects on reproductive performance and induces an only modest harmful action on mothers and embryos.

Sequential culture and coculture are two methods of improving the development of preimplantation embryos in vitro. Direct comparison of the efficiency of these methods is limited. Proliferation and apoptosis determine the total number of blastomere in preimplantation embryo, which is a sensitive parameter for evaluation of the development of embryo in vitro. In this study, we compared the proliferation and apoptosis of mouse embryo in different culture media, including CZB, KSOM, MTF, G1.2/G2.2 sequential culture media, and in human oviductal cell coculture. Sequential culture using G1.2/G2.2 was superior to KSOM, MTF, and CZB/CZB + G with respect to the formation of 3-4 cell embryos, morula, and blastocyst. G1.2/G2.2 cultured blastocyst had significantly more proliferating blastomeres and higher total cell number per blastocyst than those cultured in KSOM or CZB/CZB + G. Compared to embryos cultured in G1.2/G2.2, embryos cocultured in G1.2/G2.2 hatched more frequently. Cocultured blastocysts also had significantly higher percentage of proliferating cell and lower percentage of apoptotic cell per blastocyst than those cultured in G1.2/G2.2. It was concluded that G1.2/G2.2 facilitated the proliferation of blastomere whilst human oviductal cell coculture suppressed apoptosis in addition to stimulating proliferation of blastomere., (Copyright 2004 Wiley-Liss, Inc.)

Occupational exposures to pesticides may increase parental risk of infertility and adverse pregnancy outcomes such as spontaneous abortion, preterm delivery, and congenital anomalies. Less is known about residential use of pesticides and the risks they pose to reproduction and development. In the present study we evaluate environmentally relevant, low-dose exposures to agrochemicals and lawn-care pesticides for their direct effects on mouse preimplantation embryo development, a period corresponding to the first 5-7 days after human conception. Agents tested were those commonly used in the upper midwestern United States, including six herbicides [atrazine, dicamba, metolachlor, 2,4-dichlorophenoxyacetic acid (2,4-D)], pendimethalin, and mecoprop), three insecticides (chlorpyrifos, terbufos, and permethrin), two fungicides (chlorothalonil and mancozeb), a desiccant (diquat), and a fertilizer (ammonium nitrate). Groups of 20-25 embryos were incubated 96 hr in vitro with either individual chemicals or mixtures of chemicals simulating exposures encountered by handling pesticides, inhaling drift, or ingesting contaminated groundwater. Incubating embryos with individual pesticides increased the percentage of apoptosis (cell death) for 11 of 13 chemicals (p

Hydroxyprogesterone caproate is one of the most effective and widely used drugs for the treatment of uterine bleeding and threatened miscarriage in women. Hydroxyprogesterone caproate was administered to pregnant rats in order to assess the effect of intraperitoneal exposure to supranormal levels of hydroxyprogesterone caproate on the male reproductive potential in the first generation. The cauda epididymal sperm count and motility decreased significantly in rats exposed to hydroxyprogesterone caproate during embryonic development, when compared with control rats. The levels of serum testosterone decreased with an increase in follicle stimulating hormone and luteinizing hormone in adult rats exposed to hydroxyprogesterone caproate during the embryonic stage. It was suggested that the impairment of male reproductive performance could be mediated through the inhibition of testosterone production.

Fish embryos exposed to complex mixtures of polycyclic aromatic hydrocarbons (PAHs) from petrogenic sources show a characteristic suite of abnormalities, including cardiac dysfunction, edema, spinal curvature, and reduction in the size of the jaw and other craniofacial structures. To elucidate the toxic mechanisms underlying these different defects, we exposed zebrafish (Danio rerio) embryos to seven non-alkylated PAHs, including five two- to four-ring compounds that are abundant in crude oil and two compounds less abundant in oil but informative for structure-activity relationships. We also analyzed two PAH mixtures that approximate the composition of crude oil at different stages of weathering. Exposure to the three-ring PAHs dibenzothiophene and phenanthrene alone was sufficient to induce the characteristic suite of defects, as was genetic ablation of cardiac function using a cardiac troponin T antisense morpholino oligonucleotide. The primary etiology of defects induced by dibenzothiophene or phenanthrene appears to be direct effects on cardiac conduction, which have secondary consequences for late stages of cardiac morphogenesis, kidney development, neural tube structure, and formation of the craniofacial skeleton. The relative toxicity of the different mixtures was directly proportional to the amount of phenanthrene, or the dibenzothiophene-phenanthrene total in the mixture. Pyrene, a four-ring PAH, induced a different syndrome of anemia, peripheral vascular defects, and neuronal cell death, similar to the effects previously described for potent aryl hydrocarbon receptor ligands. Therefore, different PAH compounds have distinct and specific effects on fish at early life history stages.

Although heparin plays an important role in bovine sperm capacitation, there is no direct evidence for a role in embryonic development. The present study was designed to examine the effect of heparin on early development of bovine zygotes obtained by intracytoplasmic sperm injection (ICSI). Spermatozoa were treated with or without heparin, and the ICSI ova were cultured in a chemically defined medium + BSA, with or without heparin. Treatment of spermatozoa with heparin before ICSI or heparin in post-ICSI culture medium for 18 or 24h had a beneficial effect on pronuclear formation, cleavage rate (63% versus 76-83%), and number of cells in blastocysts (68 cells versus 82-109 cells; P < 0.05).

Objective: Bipolar disorder affects 0.5%-1.5% of individuals in the United States. The typical age at onset is late adolescence or early adulthood, placing women at risk for episodes throughout their reproductive years. General guidelines for the treatment of bipolar disorder are available from the American Psychiatric Association, but additional issues arise when these guidelines are applied in the treatment of peripartum women. The authors summarize knowledge regarding the management of bipolar disorder during pregnancy and the postpartum period, with a focus on managing mania, hypomania, and the psychotic components of the illness., Method: An expert panel reviewed articles that address the management of bipolar disorder and the consequences of the use of mood stabilizers during pregnancy, and a consensus document was generated., Results: The treatment of bipolar disorder in pregnant women involves significant challenges. Some mood stabilizers, e.g., sodium valproate and carbamazepine, are human teratogens. On the other hand, the teratogenicity associated with lithium may have been overestimated in the past., Conclusions: Since treatment can be managed most effectively if pregnancy is planned, clinicians should discuss the issue of pregnancy and its management with every bipolar disorder patient who has childbearing potential, regardless of future reproductive plans. Additional research should address the risks of disturbed sleep to pregnant and postpartum women with bipolar disorder, as well as structural and behavioral consequences to offspring when mood stabilizers are used during pregnancy. Longitudinal and cohort studies can promote these efforts. Given the rate of bipolar disorder in the general population, research efforts will need to be broad based and include multiple collaborating centers.

Ovigerous females of the estuarine crab Chasmagnathus granulatus were exposed to copper (0.01 and 1 mg/L), zinc (0.05, 1, and 10 mg/L), or lead (0.01 and 1 mg/L) during early, late, or whole embryonic development. None of the assayed heavy metals produced a significant mortality of females, neither a decrease in the number of hatched larvae nor a decrease in the egg incubation time, but several morphological abnormalities were detected in hatched larvae. The abnormalities were classified in three categories: eye, body pigmentary, and body morphological abnormalities. Those larvae with eye and body pigmentary abnormalities, particularly those involving retinal pigments and chromatophores, showed the highest incidence by exposure to the assayed metals. In addition, embryos were more susceptible to copper and zinc during the late period of development, whereas the effect of lead was greater during the early period of embryogenesis. Some teratogenic effects observed in C. granulatus embryos exposed to heavy metals, particularly the hypertrophy and hypopigmentation of eyes observed in the laboratory at a lead concentration as low as that reported for the natural environment, could be considered as sensitive biomarkers for this kind of pollutant.

In utero exposure of male C57BL/6 mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents prostatic epithelial buds from forming in the ventral region of the urogenital sinus (UGS) and reduces the number of buds that form in the dorsolateral region. This inhibition of budding is aryl hydrocarbon receptor (AHR) dependent and appears to be the primary cause of lobe-specific prostate abnormalities in TCDD-exposed mice. TCDD can inhibit prostatic epithelial bud formation by acting directly on the UGS in vitro, but whether it does so via AHR in UGS mesenchyme, epithelium, or both was unknown. To address this issue, UGS mesenchyme and epithelium from gestation day (GD) 15 wild-type C57BL/6J male mice were isolated, recombined, and cultured in vitro for 5 days with 10(-8) M 5alpha-dihydrotestosterone (DHT) and either 10(-9) M TCDD or vehicle. Prostatic epithelial buds were viewed by light microscopy after removal of mesenchyme. Effects depended greatly on which portions of the mesenchyme were used: TCDD had little if any effect when whole UGS epithelium (UGE) was recombined with ventral plus dorsolateral mesenchyme, tended to reduce bud numbers in recombinants made with UGE and dorsolateral mesenchyme, and severely reduced bud numbers in recombinants made with UGE and ventral mesenchyme (VM). [VM + UGE] recombinants prepared from wild-type and AHR knockout (Ahr(-/-)) mice were then cultured with DHT to determine the site of action of TCDD. AHR null mutation alone had no effect on budding. TCDD severely inhibited prostatic epithelial bud formation in recombinants that contained mesenchymal AHR, whereas bud formation was not inhibited by TCDD in recombinants lacking mesenchymal AHR, regardless of epithelial AHR status. These results demonstrate that UGS mesenchyme and not UGS epithelium is the site of action of TCDD. Therefore, the initial events responsible for abnormal UGS (and ultimately prostate) development occur within the UGS mesenchyme, and changes in gene expression in the UGS epithelium responsible for inhibited prostatic budding are secondary to the direct effects of TCDD on UGS mesenchyme.

Isocupressic acid (ICA) [15-hydroxylabda-8 (17), 13E-dien-19-oic acid], a labdane diterpene acid, isolated from ponderosa pine (Pinus ponderosa), Lodgepole pine (Pinus contorta), common juniper (Juniperus communis) and Monterey cypress (Cupressus macrocarpa), induces abortion in pregnant cows when ingested primarily during the last trimester. The objective of this study was to investigate the effects of isocupressic acid on bovine oocyte maturation (in vitro maturation (IVM)-Experiment I) and preimplantation embryo development (in vitro culture (IVC)-Experiment II) using in vitro embryo production techniques and to subsequently evaluate viability and developmental competence of ICA-cultured embryos via embryo transfer to recipient heifers (Experiment III). A complete randomized block experimental design was used. In Experiment I and II, isocupressic acid was added to IVM or IVC media at 0 (TRT1, control), 1.3 (TRT2), and 2.6 microg/ml (TRT3) Results from Experiment I and II indicated that ICA did not inhibit oocyte maturation and did not adversely affect preinpiantation embryo development. Furthermore, results from Experiment II demonstrated that isocupressic acid enhanced bovine preimplantation embryo development in vitro in a dose dependent manner. Subsequently, Day 8 (Day 0 = IVF) blastocysts cultured in vitro in the medium containing 2.6 microg/ml ICA were transferred to recipient heifers and resulted in normal pregnancies as determined by ultrasound imaging. Subsequently, all but two births were normal as evaluated by post natal veterinary examination. In conclusion, ICA showed no adverse effects on oocyte maturation and preimplantation embryo development in vitro or subsequent viability in vivo using the ICA concentrations and in vitro culture parameters of this study.

Inner-city, minority populations are high-risk groups for adverse birth outcomes and also are more likely to be exposed to environmental contaminants, including environmental tobacco smoke (ETS), benzo[a]pyrene (BaP), and other polycyclic aromatic hydrocarbons (PAHs) found in urban air. In a sample of nonsmoking African-American and Dominican women, we evaluated the effects on birth outcomes of prenatal exposure to ETS, using questionnaire data and plasma cotinine as a biomarker of exposure, and environmental PAHs using BaP-DNA adducts as a molecular dosimeter. We previously reported that among African Americans, high prenatal exposure to PAHs estimated by prenatal personal air monitoring was associated with lower birth weight (p = 0.003) and smaller head circumference (p = 0.01) after adjusting for potential confounders. In the present analysis, self-reported ETS was associated with decreased head circumference (p = 0.04). BaP-DNA adducts were not correlated with ETS or dietary PAHs. There was no main effect of BaP-DNA adducts on birth outcomes. However, there was a significant interaction between the two pollutants such that the combined exposure to high ETS and high adducts had a significant multiplicative effect on birth weight (p = 0.04) and head circumference (p = 0.01) after adjusting for ethnicity, sex of newborns, maternal body mass index, dietary PAHs, and gestational age. This study provides evidence that combined exposure to environmental pollutants at levels currently encountered in New York City adversely affects fetal development.

Pregnant Wistar rats were exposed to 1500 ppm toluene 6 hr/day from gestational day 7-20 or to chronical mild stress from gestational day 9-20 as single exposure or in combination. Behavioural, immunohistopathological, molecular biological, and neurochemical methods were applied to investigate the offspring for developmental neurotoxicity and level of apoptosis in the brain. The number of apoptotic cells in cerebellum postnatal day 22, 24, and 27 and in hippocampus (postnatal day 22, 24, and 27) were counted after visualization by the TUNEL staining or measured by DNA-laddering technique. Caspase-3 activity was determined in cerebellum (postnatal day 6, 22, 24, and 27) and in hippocampus (postnatal day 6 and 22). TUNEL staining and DNA-laddering technique showed a marked decrease in number of apoptotic cells from postnatal day 22 to 27 in both cerebellum and hippocampus. Apparently, a peak in the number of TUNEL positive cells was identified in cerebellum at postnatal day 22. There was no statistically significant influence of exposure except that DNA-laddering in cerebellum at postnatal day 27 was increased by toluene exposure. Caspase-3 activity decreased in cerebellum and hippocampus with age. At postnatal day 6 stress and toluene, when singly exposed, increased activity in cerebellum whereas co-exposure to stress and toluene did not. Stress increased caspase-3 activity in hippocampus postnatal day 22. There was overall consistency between the results obtained by the three supplementary methods regarding the influence of exposure and age on apoptotic activity in cerebellum and hippocampus. New methods to quantitate the relative level of apoptosis measured as DNA-laddering and the caspase-3 activity in tissue are presented.

Children's exposure to tobacco constituents during fetal development and via environmental tobacco smoke (ETS) exposure is perhaps the most ubiquitous and hazardous of children's environmental exposures. A large literature links both prenatal maternal smoking and children's ETS exposure to decreased lung growth and increased rates of respiratory tract infections, otitis media, and childhood asthma, with the severity of these problems increasing with increased exposure. Sudden infant death syndrome, behavioral problems, neurocognitive decrements, and increased rates of adolescent smoking also are associated with such exposures. Studies of each of these problems suggest independent effects of both pre- and postnatal exposure for each, with the respiratory risk associated with parental smoking seeming to be greatest during fetal development and the first several years of life.

Inorganic arsenic, an environmental contaminant, produces a variety of stress responses in mammalian cells, including metabolic abnormalities accompanied by growth inhibition and carcinogenesis. Much of the toxicity of arsenic is known to stem from its uncoupling effects on mitochondria. Because previously we had shown that mitochondrial dysfunction can disrupt oocyte and embryo development, we investigated effects of arsenite on meiotic progression and early embryo development in mice. Six-week-old CD-1 mice were treated with 0 (solvent as control), 8 mg/kg (a dose previously established in mice as the maternal no-observed-adverse-effect level), and 16 mg/kg doses of sodium arsenite every 2 days for a total of seven i.p. injections ver a period of 14 days. The incidence of meiotic anomalies, characterized by spindle disruption and/or chromosomal misalignment, was significantly increased in arsenite-treated groups (25% after 8 mg/kg and 62.5% after 16 mg/kg), compared to normal metaphase II in control oocytes. Further, arsenite treatment significantly decreased cleavage rates of zygotes at 24 h, morula formation at 72 h, and development to blastocysts at 96 h in a dose-dependent manner. The total cell number in developed blastocysts did not differ significantly between the 8 mg/kg arsenite treatment and control groups, but was significantly reduced in the 16 mg/kg arsenite treatment group. Moreover, the percentage of apoptotic nuclei was significantly increased in blastocysts following 16 mg/kg arsenite treatment. These data suggest that arsenite causes meiotic aberrations, which may contribute to decreased cleavage and preimplantation development, as well as increased apoptosis.

Short interfering RNAs (siRNAs) have proved to be a useful tool in studying gene function in plants, invertebrates and mammalian systems. Here we report the use of siRNAs for targeting the zebrafish dystrophin gene. This study demonstrates the efficacy of siRNA-based gene silencing in this vertebrate model species, and illustrates the potential of this approach for determining the roles of multiple protein products expressed by a single gene during the early stages of development. In addition this study illustrates the usefulness of zebrafish as a model for muscle disease, and highlights the potential of siRNA-based gene targeting for disease analysis in this model organism.

The objective of this work was to study the effect of a preparation of human recombinant gonadotrophins (r-FSH and r-LH) on the in vitro maturation (IVM) and development of sheep oocytes. In addition, the viability of fresh and vitrified blastocysts obtained after transfer was tested. Oocytes collected from slaughtered animals were divided into five different maturation groups. All groups were matured in a medium containing TCM199 with 4 mg/ml BSA, 100 microM cysteamine and 1 microg/ml estradiol-17beta. Each group was also treated with one of the following: 0.1 UI/ml r-FSH (r-FSH group), 0.1UI/ml r-LH (r-LH group), 0.1 UI/ml r-FSH and 0.1 UI/ml r-LH (r-FSH/r-LH group), 5 microg/ml FSH and 5 microg/ml LH hypophysial gonadotrophins (h-G group) as a control, or no gonadotrophins (no-G group). After in vitro fertilization with fresh ram semen, presumptive zygotes were cultured in vitro for 6-7 days and a total of 109 blastocysts were then transferred in pairs into synchronized ewes. To determine the viability of embryos after vitrification, 36 blastocysts from the r-FSH/r-LH group and 30 from the h-G group were vitrified in 10% ethylene glycol (EG) and 10% dimethylsulphoxide (DMSO) for 5min, followed by 20% EG, 20% DMSO and 0.5M Sucrose (S) for <45 s. They were loaded into open pulled straws (OPS) and plunged into LN(2). After warming, the blastocysts were transferred in pairs into synchronized ewes. The highest maturation rate was reached in the r-FSH/r-LH group (91.9%). However, no statistical difference was found when this group was compared with the h-G group (84.0%). Likewise, the cleavage rate of the r-FSH/r-LH group (81.4%) was not significantly different from that of the h-G group (82.3%). The cleavage rates of all other groups, however, were significantly lower than the r-FSH/r-LH and h-G groups. The blastocyst rate was highest in the h-G group (53.6%), and it was statistically higher than in the r-FSH/r-LH group (41.5%). The blastocyst rate was very similar between groups r-FSH and r-FSH/r-LH (42.0 and 41.5%, respectively). The lowest lambing rate (31.8%) was in the no-G group. The highest lambing rate was achieved in the r-FSH/r-LH group (66.6%). The vitrified embryos of h-G and r-FSH/r-LH groups had a very similar lambing rate (16.6% and 19.4%). In conclusion, these data provide support for the hypothesis that sheep oocytes respond to human recombinant gonadotrophins used for in vitro embryo production.

The amount of research into endocrine disruption has exploded over the past decade and a re-evaluation of the state of research in this area is timely. There are debates about whether human male reproductive health is really declining and whether endocrine disrupting chemicals play any role in the perceived decline. Most data currently conclude that there are wide geographical variations in semen quality and in the incidence of testicular cancer, cryptorchidism and hypospadias. This review aims to give a brief overview of the issues surrounding the perceived decline in human male reproductive health and the importance of the hormonal environment for the development of the testis and reproductive tract. The consequences for the male reproductive tract of abnormal androgen levels or action are discussed with reference to environmental anti-androgenic compounds. The in vivo data on several anti-androgenic compounds that have been administered to pregnant rodents during the period of male reproductive tract development are assessed with attention to the effects on the male offspring. Finally, the data on in utero phthalate administration are discussed in detail to illustrate the similarities between the effects of some phthalate esters and the human male reproductive tract disorders which comprise testicular dysgenesis syndrome (TDS).

Objective: To compare the effects of four methods of analysis on the results of randomised controlled trials that recruit women with multiple pregnancies and measure outcomes on their babies., Design: Analysis of one real and two simulated data sets., Setting: Secondary analysis of perinatal randomised controlled trials., Population: Randomised controlled trials including women with multiple pregnancies., Methods: The analytical methods compared were (a) assuming independence among babies, (b) analysing outcomes per women, counting a woman as having an outcome if any of her babies had it (equivalent to selecting the worst outcome among any of a woman's babies), (c) randomly selecting one baby from each set of multiples for inclusion in the analysis, (d) adjustment of the analysis to take account of non-independence of babies from multiple pregnancies, using methods developed for analysis of cluster randomised trials., Main Outcome Measures: Odds ratios for trials' main outcomes., Results: Results from application of cluster trial methods were similar to those from assuming independence among babies, but with slightly wider confidence intervals, reflecting the reduced effective sample size caused by non-independence between babies from the same pregnancy. Results were more variable using the other two methods, and in some cases, departed markedly from the results of the cluster trial methods., Conclusions: Cluster trial methods provide a simple way of adjusting the analysis to take account of non-independence between babies from the same pregnancy. Random selection and analysis by pregnancy (methods (b) and (c)) have disadvantages and do not report outcomes for all of the babies in the trial. This may cause problems with incorporating trials analysed using these methods into systematic reviews.

The present study was designed to compare the effect of two activation regimens for rat oocytes on full-term development after round spermatid injection (ROSI). Oocytes were treated either with 1.25 mM SrCl2 for 20 min or with two direct-current (DC) pulses at 100 V/mm for 99 msec followed by exposure to 2 mM 6-dimethylaminopurine (6-DMAP) for 20 min. Single spermatids then were injected into the oocytes that were ready for polar body extrusion. The proportions of oocytes surviving injection and exhibiting pronuclear formation were comparable between the SrCl2 group (76.8% and 45.6%, respectively) and the DC+6-DMAP group (78.3% and 34.9%, respectively). More SrCl2-activated ROSI oocytes (44.2%) cleaved into the two-cell stage than did those activated with DC+6-DMAP (34.7%). However, the proportions of the two-cell embryos developing to morulae/blastocysts and live offspring were significantly (P = 0.0005 and 0.042) lower in the SrCl2 group (12.3 and 6.1%, respectively) than the DC+6-DMAP group (36.1 and 14.6%, respectively). Our results indicate DC+6-DMAP is better than SrCl2 for oocyte activation prior to successful ROSI of rats.

2-Bromopropane (2-BP), a halogenated propane analogue, is a substitute for chlorofluorocarbones. The present study was carried out to investigate the potential adverse effects of 2-BP on pregnant dams and embryo-fetal development after maternal exposure on gestational days (GD) 6 through 19 in Sprague-Dawley rats. The test chemical was administered subcutaneously to pregnant rats at dose levels of 0, 250, 500, and 1000 mg/kg per day. All dams were subjected to caesarean section on GD 20 and their fetuses were examined for external, visceral and skeletal abnormalities. In the 1000 mg/kg group, maternal toxicity included an increase in the incidence of abnormal clinical signs, a suppression in the body weight and body weight gain, and a decrease in the food intake. Developmental toxicity included an increase in the fetal deaths, a decrease in the litter size, and a reduction in the fetal body weight. In addition, an increase in the incidence of fetal external, visceral, and skeletal abnormalities was seen. In the 500 mg/kg group, minimal developmental toxicity including decreased fetal body weight and increased fetal ossification delay was observed. There were no adverse effects on either pregnant dams or embryo-fetal development in the 250 mg/kg group. These findings suggest that a 14-day subcutaneous dose of 2-BP is embryotoxic and teratogenic at a maternally toxic dose (i.e., 1000 mg/kg per day) and is minimally embryotoxic at a nonmaternally toxic dose (i.e., 500 mg/kg per day) in Sprague-Dawley rats. In the present experimental conditions, the no-observed-adverse-effect level (NOAEL) of 2-BP is considered to be 500 mg/kg per day for dams and 250 mg/kg per day for embryo-fetal development.

The objective of this study was to evaluate the developmental toxicity of beta-thujaplicin (TP) in rats. Pregnant rats were given TP by gastric intubation at 15, 45, or 135 mg/kg on days 6-15 of pregnancy. The maternal body weight gain during administration at 45 and 135 mg/kg and after administration at 136 mg/kg and adjusted weight gain at 45 and 135 mg/kg were significantly reduced. A significant decrease in food consumption during and after administration was found at 45 and 135 mg/kg. A significant increase in the incidence of postimplantation loss was found in pregnant rats given TP at 135 mg/kg. A significantly lower weight was found in female fetuses at 45 and 135 mg/kg and in male fetuses at 135 mg/kg. Although a significantly increased incidence of fetuses with skeletal variations and decreased degree of ossification were found at 135 mg/kg, no significant increase in external, skeletal and internal malformations was detected after administration of TP. The data demonstrated that TP had adverse effects on embryonic/fetal survival and growth only at maternal toxic doses. No adverse effects on morphological development were found in rats fetuses. Based on the significant decreases in maternal body weight gain and weight of female fetuses at 45 mg/kg and higher, it is concluded that the no-observed-adverse-effect levels (NOAELs) of TP for both dams and fetuses are considered to be 15 mg/kg in rats.

Some anticholinesterase organophosphorus compounds introduced into the organism in the course of pregnancy lead to the development of behavioral and morphological disturbances in both human and animal kids. In this context, the potential teratogenic effect of dimephosphon (1,1-dimethyl-3-ketobutylphosphonic acid dimethyl ester), representing nonanticholinesterase derivatives of organophosphoric acids, was evaluated in rats. The drug was introduced in a dose of 200 mg/kg over the period from 1st to 6th day of pregnancy and the results were assessed on the 20th day. It was found that dimephosphon did not lead to the loss of embryos. The results of morphological investigation showed neither anomalies in the development of embryo nor evidence of changes in the sex differentiation. Thus, dimephosphon exhibited no teratogenicity upon introduction in the early stage of pregnancy in rats.

Background/purpose: This study was aimed at comparing albumin and dextran as intrapulmonary hyperoncotic enhancers of fetal lung growth after tracheal occlusion., Methods: Fetal lambs (n = 27) were divided proportionally into 5 groups: group I consisted of sham-operated controls; group II underwent tracheal occlusion (TO); groups III, IV, and V underwent TO and intratracheal infusion of 60 mL of either saline, 6% dextran-70, or 25% albumin, respectively. Multiple fetal lung analyses were performed near term. Statistical analysis was by 1-way analysis of variance (ANOVA) and post-hoc analyses by the Bonferroni correction for multiple comparisons (P <.05)., Results: The lung volume-to-body weight ratio was significantly higher in groups IV and V than in all other groups with no differences between groups II and III, nor between groups IV and V. Airspace fraction was not significantly different among the groups, nor was there any evidence of alveolar cellular damage. Type-II pneumocyte density was higher in group I than in groups II, IV, and V, with no differences among the latter 3 groups. Lung liquid biochemical profile was normal in all groups., Conclusions: Albumin is as effective as dextran as an intrapulmonary hyperoncotic booster of lung growth acceleration after fetal tracheal occlusion, with no lasting effects on its fetal lung liquid levels. As a naturally occurring oncotic agent, albumin may be a safer option in the clinical application of this therapeutic concept.

Background: Placenta immunomodulatory ferritin (PLIF) cDNA was recently cloned from the human placenta, where it is expressed in syncytiotrophoblast and decidual mononuclear cells. PLIF and its subcloned bioactive domain (C48), expressed in Escherichia coli, are immunosuppressive proteins and induce pronounced IL-10 production in vitro and in vivo., Methods and Results: PLIF serum level, measured by enzyme-linked immunosorbent assay, was elevated in pregnant mice throughout gestation and declined towards delivery. Blocking of PLIF activity by vaccination of mice with C48 prior to mating inhibited pregnancy development. Passive transfer of anti-C48 immunoglobulin (Ig) starting at 3.5-12.5 days post coitum (dpc) resulted in high rate of embryo resorption. Furthermore, treatment with anti-C48 Ig resulted in placental and embryonal growth restriction. At gestation day 13.5, growth retardation was especially notable in the placentae, while at 16.5 dpc it was pronounced in the embryos. Histopathological examination revealed that experimental placentae were globally hypoplastic and the labyrinth was strikingly pale and contained less maternal blood compared with control. Immune-activated spleen cells harvested at 13.5 dpc from anti-C48 Ig-treated pregnant mice secreted in vitro increased level of Th1 cytokines (IL-2, TNF-alpha, IL-12) and decreased level of Th2 cytokines (IL-10, IL-4, IL-5, IL-6) as compared with the level of the respective cytokines secreted by spleen cells from control pregnant mice., Conclusion: This study provides the first in vivo evidence that PLIF plays a major role in placentation and embryonic growth.

Background: Inhaled steroids are recommended for the treatment of persistent asthma during pregnancy, but their potential effects on intrauterine growth have been inadequately evaluated., Objective: The purpose of this study was to evaluate the association between maternal use of specific inhaled steroids and inhaled steroid dose during pregnancy and the incidence of infants who are small for gestational age (SGA) and mean birth weight., Methods: Pregnant asthmatic women being treated with inhaled steroids were enrolled in the study before delivery by their managing allergists. Information regarding the specific inhaled steroid and daily dose used, requirement for oral steroids, occurrence of acute asthmatic episodes, maternal race, birth weight, gestational age, and congenital malformations was obtained for each patient. SGA was defined through use of a published normative sample of American births., Results: A total of 474 women were enrolled in the study; of the 451 enrolled participants whose pregnancy ended in a singleton live birth, 396 (88%) completed the study. The incidence of infants with low birth weight, preterm births, and congenital malformations in this cohort was not greater than expected in the general population. The incidence of SGA was 7.1% (95% CI, 5.0% to 10.1%). No significant relationships between specific inhaled steroid or dose of inhaled steroid used and either SGA or mean birth weight were observed., Conclusion: These data suggest that the use of inhaled steroids by pregnant asthmatic women does not reduce intrauterine growth and supports the recommendation that inhaled steroids should be used in the management of persistent asthma during pregnancy.