Maternal periconceptional folic acid supplements use and fetus risk for limb defects.

BACKGROUND: Associations between the periconceptional folic acid only (FAO) or multiple micronutrients containing folic acid (MMFA) supplementation and risk for limb defects are inconsistent. OBJECTIVE: To explore the association between periconceptional folic acid supplements use and risk for limb defects, including clubfoot, polydactyly, syndactyly, and limb deficiencies. METHODS: Data were derived from a cohort based on a pregnancy registry in a district of Beijing, China, from 2013 to 2018. Information on maternal periconceptional FAO and MMFA supplementation was collected via face-to-face interviews at first trimester. Pregnancy outcomes including limb defects were ascertained in livebirths, stillbirths, and elective pregnancy terminations and were recorded into the system. Propensity score methods were used to adjust for potential confounders. RESULTS: A total of 63 969 women with a singleton delivery were included. The overall prevalence of limb defects was 47.5 per 10 000 (n = 63 969) singleton deliveries. Decreased prevalence of limb defects was found among FAO/MMFA users compared with women who did not take supplements (nonusers) (46.1 vs. 61.9 per 10 000 births, adjusted risk ratio [RR] 0.80, 95% confidence interval [CI] 0.56, 1.12). Compared with nonusers (n = 6462, 10.2%), women who took either FAO (n = 26 567, 42.0%) or MMFA (n = 30 259, 47.8%) had a lower risk for total clubfoot (RR 0.40, 95% CI 0.20, 0.84), and for isolated clubfoot (RR 0.41, 95% CI 0.17, 0.97). For other limb defects except clubfoot, FAO supplementation did not appear to be associated with reduced risk, while MMFA supplementation group had 30%-50% reduced risks for other limb defects. A lower risk for limb defects or isolated limb defects was found with MMFA supplementation when FAO supplementation was used as a control. CONCLUSIONS: Maternal periconceptional supplements with either FAO or MMFA had inverse association with clubfoot in offspring, and MMFA was associated with lower risk for isolated limb defects compared with FAO.

Folic acid supplementation and risk for congenital limb reduction defects in China.

BACKGROUND: Folic acid (FA) supplementation prevents neural tube defects, but there are mixed results for its ability to prevent limb reduction defects. We examined whether a preventive effect of FA supplementation exists for congenital limb reduction defects in a large population in China. METHODS: Data from a large population-based cohort study in China were used to evaluate the effects of FA supplementation on birth defects. All births at 20 complete gestational weeks, including live births, stillbirths and pregnancy terminations, and all structural congenital anomalies, regardless of gestational week, were recorded. A total of 247 831 singleton live births delivered at gestational ages of 20-42 weeks to women from northern and southern China with full information on FA intake were included. Limb reduction defects were classified by subtype and maternal FA supplementation. RESULTS: The prevalence of limb reduction defects was 2.7 per 10 000 births among women who took FA compared with 9.7 per 10 000 births among those who did not take FA in northern China; the prevalence was 4.5 and 3.8 per 10 000 births, respectively, in southern China. In both unadjusted and adjusted analyses, the estimated relative risk for upper limb reduction defects [odds ratio (OR) = 0.17, 95% confidence interval (CI): 0.04, 0.63] and total limb reduction defects (OR = 0.24, 95% CI: 0.08, 0.70) in northern China, but not for lower limb reduction defects ,was significantly decreased in association with FA supplementation in northern China. There was no association between FA supplementation and either an increased or decreased risk for limb reduction defects in southern China. CONCLUSIONS: FA supplementation successfully reduces the prevalence of limb reduction defects in northern China, whose population has low folate concentrations.

Fetoscopic Release of Amniotic Bands Causing Limb Constriction: Case Series and Review of the Literature.

The aim of this study was to review the perioperative complication rates and neonatal outcomes after fetoscopic release of amniotic bands that caused bilateral limb constrictions. We present 5 cases of limb constriction by amniotic bands occurring spontaneously or following fetoscopic surgery and also include a review of 21 previously published cases. The cases were analyzed for indication, surgical technique, and postoperative follow-up. In our population and the literature, the majority of the children acquired a functional limb (75%), with few perioperative complications (15%). Fetal morbidity was mainly linked to the consequences of preterm premature rupture of the membranes (38.4%) and preterm birth (34.7 GW). The mortality rate was low (7.7%). This review only describes amniotic bands causing limb constriction, and illustrates that fetoscopic surgery for their release is technically feasible with an acceptable perioperative complication rate. However, the 75% success rate is very likely to be an overestimation of the true success rate. In view of these observations we cannot recommend treatment for cases where the fetus has been extensively affected by the bands. We believe, however, that we could consider this technique for a fraction of amniotic band syndrome cases isolated to the limb constrictions. This kind of surgery should be proposed as a potential treatment for amniotic band syndrome.

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Maternal asthma medication use and the risk of selected birth defects.

OBJECTIVES: Approximately 4% to 12% of pregnant women have asthma; few studies have examined the effects of maternal asthma medication use on birth defects. We examined whether maternal asthma medication use during early pregnancy increased the risk of selected birth defects. METHODS: National Birth Defects Prevention Study data for 2853 infants with 1 or more selected birth defects (diaphragmatic hernia, esophageal atresia, small intestinal atresia, anorectal atresia, neural tube defects, omphalocele, or limb deficiencies) and 6726 unaffected control infants delivered from October 1997 through December 2005 were analyzed. Mothers of cases and controls provided telephone interviews of medication use and additional potential risk factors. Exposure was defined as maternal periconceptional (1 month prior through the third month of pregnancy) asthma medication use (bronchodilator or anti-inflammatory). Associations between maternal periconceptional asthma medication use and individual major birth defects were estimated by using adjusted odds ratios (aOR) and 95% confidence intervals (95%CI). RESULTS: No statistically significant associations were observed for maternal periconceptional asthma medication use and most defects studied; however, positive associations were observed between maternal asthma medication use and isolated esophageal atresia (bronchodilator use: aOR = 2.39, 95%CI = 1.23, 4.66), isolated anorectal atresia (anti-inflammatory use: aOR = 2.12, 95%CI = 1.09, 4.12), and omphalocele (bronchodilator and anti-inflammatory use: aOR = 4.13, 95%CI = 1.43, 11.95). CONCLUSIONS: Positive associations were observed for anorectal atresia, esophageal atresia, and omphalocele and maternal periconceptional asthma medication use, but not for other defects studied. It is possible that observed associations may be chance findings or may be a result of maternal asthma severity and related hypoxia rather than medication use.

Folic acid and birth defects: a case study (Iran).

The aim of this study was to evaluate the impact of folic acid use in pregnancy for the reduction of neural tube defects (NTDs) in the northwest region of Iran. We studied 243 women with pregnancies complicated by some forms of birth defect(s). These patients were identified by medical diagnostic tests as having a fetus with some types of congenital anomalies. The prevalence of NTDs among pregnant women who were referred for therapeutic termination of pregnancy was 24.7 percent. Consumption of folic acid prevented NTDs by 79 percent (Odds Ratio = 0.21, CI 95%: 0.12-0.40) and 94 percent (Odds Ratio = 0.06, CI 95%: 0.03-0.15) compared to pregnancies complicated by other anomalies and normal pregnancies, respectively. Hydrops fetalis, hydrocephaly, Down syndrome, and limb anomalies did not have any significant association with the folic acid use. Along with the advice for the consumption of folic acid for pregnant women, they should be offered prenatal screening or diagnostic tests to identify fetal abnormalities for possible termination of pregnancy.

Systemic human minidystrophin gene transfer improves functions and life span of dystrophin and dystrophin/utrophin-deficient mice.

Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disease, caused by mutations in the dystrophin gene. No efficacious treatment is currently available. Here we report AAV vector systemic delivery and therapeutic benefits of the functional human minidystrophin gene in a severe and more reliable DMD mouse model, the dystrophin/utrophin double deficiency mouse (dys-/-:utrn-/-, dKO). These mice show many pathologic and phenotypic signs typical of DMD in humans including kyphosis and shorter life span, all of which are not seen in the mdx mice due to their utrophin upregulation that partially compensates the loss of dystrophin functions and leads to mild phenotypes. The therapeutic value of this new approach was demonstrated in both mdx and dKO murine models, in which we observed highly efficient minidystrophin gene expression, ameliorated muscle pathologies, improvement in growth and motility, inhibition of spine and limb deformation, and prolongation of life span.

[Folic acid and congenital malformation: scientific evidence and public health strategies]. / Acido folico e difetti congeniti: evidenze scientifiche e strategie di sanità pubblica.

In Italy at least 3% of babies are born with some congenital malformation. The intake of folic acid (FA) prior to conception and during the early stages of pregnancy plays an important role in preventing neural tube defects, severe anomalies of brain embryogenesis, and other malformations such as cardiac and urinary tract anomalies, oro-facial clefts and limb reduction defects. The Italian Network for Folic Acid Promotion, coordinated by the National Center on Rare Diseases of the Italian National Institute of Health, has elaborated and diffused a recommendation for the periconceptional FA supplementation: "Women of child-bearing age, are recommended to consume 0,4 mg/day of FA, to reduce the risk of congenital defects. The intake of folic acid should start at least one month before the conception and should continue for the first quarter of pregnancy". This paper discusses various strategies in order to promote FA intake during periconceptional period. Food fortification, adopted in several countries such as USA, has raised concerns about the risk of an excessive FA intake which may lead to adverse effect such as tumour promotion. Currently, periconceptional supplementation and healthy dietary habits promotion appear to be the most effective strategies.

Folate intake and the primary prevention of non-neural birth defects.

OBJECTIVES: To investigate whether maternal periconceptional folate intake is associated with a reduction in selected non-neural birth defects in Western Australia (WA). METHODS: Case-control study of folate intake in women whose infants had orofacial clefts (62); congenital heart defects (151); urinary tract defects (117); limb reduction defects (26); or other major birth defects (119); and 578 control women. RESULTS: Neither folic acid supplements nor dietary folate intake in women not using supplements was significantly associated with a reduction in risk in any of the case groups. In contrast to neural tube defects, WA population data for orofacial clefts, heart defects, limb reduction defects and urinary tract defects showed no fall in prevalence since the introduction of folate promotion and voluntary food fortification. CONCLUSIONS: This study provides no evidence of folate being an important factor in the prevention of birth defects other than neural tube defects.

Prebreeding maternal immunostimulation with Freund's complete adjuvant reduces placental damage and distal limb defects caused by methylnitrosourea.

PROBLEM: Immunostimulation reduces murine teratogen-induced birth defects. It is unclear if placental improvement contributes to this outcome. The current study examined murine placental ultrastructure and fetal limb development following maternal methylnitrosourea (MNU) exposure, +/-Freund's complete adjuvant (FCA) immunostimulation. METHOD OF STUDY: Two murine strains (CD-1, C57BL/6N) were administered MNU on gestation day 9 (GD9), FCA pre-breeding, or FCA + MNU. Fetal limb and placental development were examined on GD14. RESULTS: MNU decreased placental weight and reduced placental cellular viability; FCA reversed these effects. MNU shortened fetal limbs and increased digital defects in both strains. Placentas were less damaged in C57BL/6N versus CD-1 mice, and distal limb malformations improved only in CD-1 mice. FCA immunostimulation also increased pregnancy rate. CONCLUSION: Improved fetal outcome from immune-stimulated mice may not be dependent on improved placental morphology. However, placental function and morphology in immune-stimulated mice may not directly correlate, thus functional improvements should be examined for possible relationship to reduced birth defects.

Role of maternal dietary antioxidant supplementation in murine placental and fetal limb development.

Methylnitrosourea (MNU) is a multisystem teratogen that damages proliferating cells through macromolecule alkylation and generation of reactive oxygen species (ROS). Murine dams exposed to MNU midgestation produce offspring with distal limb malformations, an outcome reduced by maternal immune stimulation. Immunostimulatory effects of antioxidant therapy may in part explain this improved birth outcome. The present study hypothesizes that placental, rather than fetal, damage from excessive ROS may contribute to MNU-induced embryopathy. Fetal limbs and placentas were examined in immunotolerant CD-1 and immunosensitive C57BL/6N mice exposed to MNU, dietary antioxidant butylated hydroxytoluene (BHT), or both. MNU increased fetal resorptions and incidence of syndactyly, oligodactyly, polydactyly, and interdigital webbing, and decreased fetal size in both mouse strains. BHT reduced syndactyly and oligodactyly in both strains, and reduced polydactyly in C57BL/6N mice. Increased webbing in MNU and MNU+BHT groups likely represented maturational delay. Placentas from CD-1 and C57BL/6N MNU-exposed dams demonstrated decreased trophoblasts and increased necrosis of endothelium. Similar to distal limb defects, placental damage was reduced in mice receiving MNU+BHT. These results suggest that placental damage and fetal defects caused by MNU are in part ROS-mediated, and reduced distal limb defects following MNU+BHT may be related to improved placental integrity and function.

Reduced birth defects caused by maternal immune stimulation in methylnitrosourea-exposed mice: association with placental improvement.

BACKGROUND: Methylnitrosourea (MNU) is a potent carcinogen and teratogen that is associated with central nervous system, craniofacial, skeletal, ocular, and appendicular birth defects following transplacental exposure at critical time points during development, and preliminary studies have suggested that nonspecific maternal immunostimulation may offer protection against development of these birth defects. METHODS: Our study examined morphologic alterations in fetal limb and digital development and placental integrity following maternal exposure to MNU on GD 9 in CD-1 mice, and characterized the improvement in placental integrity and abrogation of fetal defects following maternal immune stimulation with interferon-gamma (IFN-gamma) on GD 7. RESULTS: Fetal limbs were significantly shortened (p < 0.0001) and incidence of limb and digital defects (syndactyly, polydactyly, oligodactyly, clubbing, and webbing) was dramatically increased following mid-gestational maternal MNU exposure. Maternal immune stimulation with IFN-gamma on GD 7 lessened incidence of fetal limb shortening and maldevelopment on GD 12 and 14. Further, disruption of placental spongiotrophoblast integrity, increased cell death in placental trophoblasts with increased intercellular spaces in the spongiotrophoblast layer and minimal inflammation, and increased loss of fetal labyrinthine endothelial cells from MNU-exposed dams suggested that MNU-induced placental breakdown may contribute to fetal limb and digital maldevelopment. MNU + IFN-gamma was associated with diminished cell death within all layers of the placenta, especially in the labyrinthine layer. CONCLUSIONS: These data verify improved distal limb development in MNU-exposed mice as a result of maternal IFN-gamma administration, and suggest a link between placental integrity and proper fetal development.

Teratological consequences of nitric oxide synthesis inhibition.

Nitric oxide (NO) is generated by a family of NO synthase (NOS) enzymes, including endothelial (eNOS), inducible (iNOS) and neuronal (nNOS). NO is an important bioregulator of a wide variety of physiological processes. Recent experimental evidence indicates that inhibition of NO synthesis can lead to teratogenesis. The current review focuses on this aspect of NOS. Exposure of pregnant rodents to non-selective NOS inhibitors, such as N(G)-nitro-L-arginine-methyl ester (L-NAME) and N(G)-nitro-L-arginine (L-NNA), has been linked to limb reduction defects. The teratogenic phenotype, characterized by hemorrhage and transverse terminal tissue destruction, has been regarded to be compatible with a vascular origin. The critical time for teratogenic response was traced to advanced stages of gestation. Similar limb reduction defects have been described in mice deficient in eNOS, but not in other NOS isoforms. Several observations have led to the proposal that hypoxia and possible consequential generation of reactive oxygen species are involved in the causation of NOS inhibitors induced limb defects.

Protection from ethanol-induced limb malformations by the superoxide dismutase/catalase mimetic, EUK-134.

Based on previous in vitro studies that have illustrated prevention of ethanol-induced cell death by antioxidants, using an in vivo model, we have tested the anti-teratogenic potential of a potent synthetic superoxide dismutase plus catalase mimetic, EUK-134. The developing limb of C57BL/6J mice, which is sensitive to ethanol-induced reduction defects, served as the model system. On their ninth day of pregnancy, C57BL/6J mice were administered ethanol (two intraperitoneal doses of 2.9 g/kg given 4 h apart) alone or in combination with EUK-134 (two doses of 10 mg/kg). Pregnant control mice were similarly treated with either vehicle or EUK-134, alone. Within 15 h of the initial ethanol exposure, excessive apoptotic cell death was observed in the apical ectodermal ridge (AER) of the newly forming forelimb buds. Forelimb defects, including postaxial ectrodactyly, metacarpal, and ulnar deficiencies, occurred in 67.3% of the ethanol-exposed fetuses that were examined at 18 days of gestation. The right forelimbs were preferentially affected. No limb malformations were observed in control fetuses. Cell death in the AER of embryos concurrently exposed to ethanol and EUK-134 was notably reduced compared with that in embryos from ethanol-treated dams. Additionally, the antioxidant treatment reduced the incidence of forelimb malformations to 35.9%. This work illustrates that antioxidants can significantly improve the adverse developmental outcome that results from ethanol exposure in utero, diminishing the incidence and severity of major malformations that result from exposure to this important human teratogen.

The nitric oxide synthesis inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) causes limb defects in mouse fetuses: protective effect of acute hyperoxia.

In the present study the relationship between exposure to the nitric oxide synthesis inhibitor Nomega-nitro-l-arginine methyl ester (l-NAME) and the induction of limb defects, with respect to stage specificity and dose dependency, was investigated in the mouse. ICR (CD-1) mice were dosed s.c with l-NAME at 50 or 90 mg/kg on gestation d 12, 13, 14, 15, or 16. A group of animals treated with vehicle on gestation d 14 served as control. Uterine contents were evaluated for teratogenesis on gestation d 18. A treatment-related disruption of limb development was noted. The effect was dose dependent and phase specific. l-NAME became teratogenically operational on gestation d 13 and elicited its maximum effect on gestation d 14, whereas no significant teratogenicity was observed when exposure occurred after gestation d 15. In utero exposure to l-NAME also reduced embryo viability relative to controls. When the higher dose was injected on gestation d 16, a significant number of dams delivered preterm. In a parallel study, the ability of hyperoxia to prevent limb teratogenesis was investigated. To this aim, a group of l-NAME-treated animals (90 mg/kg s.c. on gestation d 14) were exposed to 98 to 100% O(2) for 12 h. l-NAME-treated mice breathing room air served as positive controls. In response to hyperoxia, a significant decrement of l-NAME-induced limb defects was found. This study characterizes for the first time the teratogenic capacity of l-NAME in the mouse. Results obtained with hyperoxia fit the hypothesis that hypoxic tissue damage may play a contributory role in l-NAME-induced limb defects.

Nonspecific stimulation of the maternal immune system. I. Effects On teratogen-induced fetal malformations.

BACKGROUND: Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice. METHODS: Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freund's complete adjuvant (FCA). RESULTS: Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe. CONCLUSIONS: For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation.

The role of physician preferences in the choice of amniocentesis or chorionic villus sampling for prenatal genetic testing.

Our objective was to determine the effect of physician preferences, as well as physician demographic, obstetric, and practice-related factors, on the choice of prenatal test made by their patients. We studied preferences for prenatal outcomes for 372 pregnant women who either chose amniocentesis (AMN) (n = 288) or chorionic villus sampling (CVS) (n = 84) for the indication of maternal age. We also studied preferences for these outcomes for the 92 physicians that referred them for testing. Preferences were assessed using written scenarios and were measured on linear rating scales. According to patients, the choice of prenatal test was made entirely or mostly by the physician in 14% of cases and was shared equally between patient and physician in 37% of cases. After adjustment for patient preferences, physician concern about spontaneous abortion of a normal fetus after CVS (odds ratio 0.71; CI, 0.48-1.05; p = 0.08), and a limb reduction (LRD) birth after CVS (odds ratio 0.85; CI, 0.68-1.05; p = 0.12), tended to decrease their patients' odds of choosing CVS, but the results were not statistically significant. No other physician preference, and no physician demographic, obstetric, or practice-related factor, influenced patient test choice. We conclude that after taking patient preferences into account, physician preferences and practice-related factors did not emerge as significant determinants of the choice of prenatal test made by their patients. It remains possible, however, that physician concern about spontaneous abortion and about LRD increase the likelihood of their patients choosing AMN over CVS.