Occurrence Of Neural Tube Defects Research Articles

Epidemiology of neural tube defects

Epidemiologic studies have established clear variations in the occurrence of neural tube defects (NTDs) associated with demographic and other factors. Methods of counting NTD-affected pregnancies and other birth defects rely on multiple sources of information such as hospital records and birth certificates. Widespread prenatal diagnosis of NTDs presents special challenges for birth defects monitoring programs. Even before prenatal diagnosis was possible, epidemiologists noted geographic variation in NTD rates; distinctive recurrence patterns within families; variations in NTD rates by race/ethnicity, socioeconomic strata, and sex of the infant or fetus; and changing rates over time. We review highlights of studies of these factors and some conclusions that can be drawn from NTD epidemiologic data. These conclusions include the importance of maternal diet, a finding that has led to preventive interventions, and the suggestion of multiple causes of NTDs, indicating that no single prevention program may prevent the occurrence of all NTDs. We also discuss the relevance of epidemiologic data to NTD screening and counseling policies and the importance of prenatal diagnosis data. Future epidemiologic studies will need to incorporate prenatal records, particularly to study trends. Other emerging themes in epidemiologic research include collaboration between different birth defects monitoring programs, new information from unique geographic areas, new techniques to study gene-environment interactions, and a multidisciplinary emphasis on measuring the effectiveness of prevention programs. MRDD Research Reviews 1998;4:241–246. Published 1998 Wiley-Liss, Inc.

Epidemiology of neural tube defects

Epidemiologic studies have established clear variations in the occurrence of neural tube defects (NTDs) associated with demographic and other factors. Methods of counting NTD-affected pregnancies and other birth defects rely on multiple sources of information such as hospital records and birth certificates. Widespread prenatal diagnosis of NTDs presents special challenges for birth defects monitoring programs. Even before prenatal diagnosis was possible, epidemiologists noted geographic variation in NTD rates; distinctive recurrence patterns within families; variations in NTD rates by race/ethnicity, socioeconomic strata, and sex of the infant or fetus; and changing rates over time. We review highlights of studies of these factors and some conclusions that can be drawn from NTD epidemiologic data. These conclusions include the importance of maternal diet, a finding that has led to preventive interventions, and the suggestion of multiple causes of NTDs, indicating that no single prevention program may prevent the occurrence of all NTDs. We also discuss the relevance of epidemiologic data to NTD screening and counseling policies and the importance of prenatal diagnosis data. Future epidemiologic studies will need to incorporate prenatal records, particularly to study trends. Other emerging themes in epidemiologic research include collaboration between different birth defects monitoring programs, new information from unique geographic areas, new techniques to study gene-environment interactions, and a multidisciplinary emphasis on measuring the effectiveness of prevention programs. MRDD Research Reviews 1998;4:241–246. Published 1998 Wiley-Liss, Inc.

Maternal illness, including fever and medication use as risk factors for neural tube defects.

We investigated if selected maternal illnesses or medications used during the periconceptional period increased risk of having neural tube defect (NTD)-affected pregnancies. We used a population-based case-control study of fetuses and liveborn infants with NTDs among 1989-1991 California births. In-person interviews were conducted with mothers of 538 (88% of eligible) NTD cases and 539 (88%) nonmalformed controls, usually within 5 months of delivery. A maternal fever or febrile illness episode in the first trimester was associated with an increased risk for having a NTD-affected pregnancy, odds ratio (OR) = 1.91 (95% confidence interval, 1.35-2.72) for fever and OR = 2.02 (1.20-3.43) for febrile illness. Risk estimates were not substantially altered after adjustment for maternal age, race/ethnicity, education, vitamin use, and body mass index. Other reported illnesses were generally not associated with risks of 1.5 or greater, or were too infrequent to adequately estimate risk. An OR of 1.5 or greater was observed for maternal use of guaifenesin, OR = 2.04 (0.79-5.28), and an OR of 0.5 or less was observed for maternal use of calcium-containing medicines, OR = 0.38 (0.14-1.03). Our findings are consistent with previous reports that suggested elevated NTD risks from maternal fever. We could not discriminate, however, whether the increased risks observed for maternal fever were indicative of a causal relation or due to reporting bias. Our findings suggest that many of the illnesses common to reproductive-aged women and the medications commonly used to treat them during pregnancy, except, perhaps, for those illnesses that are febrile-related, do not appear to substantially contribute to the occurrence of NTDs in the population.

Folate intake in Europe: recommended, actual and desired intake.

To evaluate possible inconsistencies between recommended, actual and desired folate intake in European adult populations. Review of dietary recommendations, of food consumption surveys, and of intervention and observational studies relating folate intake to the risk of neural tube defects and plasma homocysteine levels. In Europe, mean dietary folate intake in adults is 291 micrograms/d (range 197-326) for men and 247 micrograms/d (range 168-320) for women. The recommended intakes vary between 200-300 micrograms/d (men) and 170-300 micrograms/d (women). However, women with a previous pregnancy affected by a neural tube defect (NTD), are recommended to take 4000 micrograms/d of supplemental folic acid when planning a subsequent pregnancy. For those without a history of NTD, the use of 400 micrograms/d of supplemental folic acid is the best option to prevent the occurrence of NTDs. A daily dose of 650 micrograms supplemental folic acid normalises elevated plasma homocysteine levels, which is a risk factor for cardiovascular diseases. A dietary folate intake of at least 350 micrograms/d is desired to prevent an increase in plasma homocysteine levels of the adult population in general. Mean dietary folate intake in Europe is in line with recommendations, but the desired dietary intake of > 350 micrograms/d is only reached by a small part of studied European populations. It is considered unethical to investigate whether supplements with a dose lower than 400 micrograms/d of folic acid are also protective against NTDs. However, research to establish the lowest effective dose of dietary folate/supplemental folic acid to optimise homocysteine levels and research on the bioavailability of folate is required. This will enable the choice of a strategy to achieve desired folate intakes in the general population. In the meantime, consumption of plant foods like vegetables, fruits, and cereals should be stimulated to reach the desired level of 350 micrograms of dietary folate per day.

Neural Tube Defects: A Primary Prevention Role for Nurses

Neural tube defects are among the most common and serious birth defects. Most of these defects are caused by multifactorial inheritance. Research over the past decade has led to advances in understanding the etiology of these congenital anomalies. Folic acid has been shown to reduce the risk of first-time occurrence of neural tube defects as well as recurrent risk. Other genetic and environmental factors are under investigation. In this article, the nurse's role in the primary prevention of these birth defects is described.

Surveillance for Anencephaly and Spina Bifida and the Impact of Prenatal Diagnosis—United States, 1985–1994

The reported prevalence of anencephaly and spina bifida in the United States has steadily declined since the late 1960s. During this time, the ability to diagnose these defects prenatally has progressed rapidly. Many U.S. birth defects surveillance systems ascertain defects only among live-born infants or among infants and fetuses beyond a certain gestational age, thus excluding defects among pregnancies prenatally diagnosed as being affected by a neural tube defect (NTD) and electively terminated before the gestational age limit. The impact of prenatal diagnosis and subsequent pregnancy termination on the reported prevalence of anencephaly and spina bifida in the United States has not been well established. However, assessment of this impact is crucial to the use of surveillance data to monitor trends in the occurrence of NTDs and the effectiveness of interventions for these defects (e.g., increased consumption of folic acid).This report presents data from birth defects surveillance systems in six states over different time periods: Arkansas, 1985-1989; California, 1989-1991; Georgia, 1990-1991; Hawaii, 1988-1994; Iowa, 1985-1990; and South Carolina, 1992-1993.Population-based data about a) live-born and stillborn infants with anencephaly and spina bifida and b) pregnancies electively terminated after prenatal diagnosis of these defects were analyzed from the Arkansas Reproductive Health Monitoring System; the California Birth Defects Monitoring Program; CDC's Metropolitan Atlanta Congenital Defects Program; the Iowa Birth Defects Registry, the University of Iowa, and the Iowa Department of Public Health; and the Greenwood Genetic Center in South Carolina. Data also were analyzed from the Hawaii Birth Defects Monitoring Program, which includes data for some women who were not residents of the state. The systems differed in the size and racial/ethnic composition of the populations studied, the surveillance methods used, the completeness of ascertainment, and the availability and utilization of prenatal testing and pregnancy termination.Among all pregnancies ascertained in which the infant or fetus had anencephaly or spina bifida, the percentages that were electively terminated ranged from 9% in Arkansas to 42% in Atlanta and Hawaii, with a corresponding increase in the adjusted prevalence of these defects compared with the prevalence at birth. In each system, pregnancies associated with anencephaly were terminated more frequently than were those associated with spina bifida. These data indicate that the impact of prenatal diagnosis and subsequent pregnancy termination on the prevalence at birth of anencephaly and spina bifida differs among geographic areas and populations. Comprehensive surveillance for these defects requires inclusion of pregnancies that are prenatally diagnosed and then terminated.CDC will use these data to promote the inclusion of prenatally diagnosed and terminated pregnancies in estimates of the prevalence of anencephaly and spina bifida generated by birth defects surveillance programs in the United States. Including such pregnancies is crucial to the ability of these programs to monitor trends accurately and to establish the effectiveness of interventions, including the use of folic acid, for these defects.

Surveillance for Anencephaly and Spina Bifida and the Impact of Prenatal Diagnosis—United States, 1985–1994

Problem/Condition The reported prevalance of anencephaly and spina bifida in the United States has steadily declined since the late 1960s. During this time, the ability to diagnose these defects prenatally has progressed rapidly. Many U.S. birth defects surveillance systems ascertain defects only among live-born infants or among infants and fetuses beyond a certain gestational age, thus excluding defects among pregnancies prenatally diagnosed as being affected by a neural tube defect (NTD) and electively terminated before the gestational age limit. The impact of prenatal diagnosis and subsequent pregnancy termination on the reported prevelance of anencephaly and spina bifida in the United States has not been well established. However, assessment of this impact is crucial to the use of surveillance data to monitor trends in the occurrence of NTDs and the effectiveness of interventions for these defects (e.g., increased consumption of folic acid). Reporting Period This report presents data from birth defects surveillance systems in six states over different time periods: Arkansas, 1985–1989; California, 1989–1991; Georgia, 1990–1991; Hawaii, 1988–1994; Iowa, 1985–1990; and South Carolina, 1992–1993. Description of Systems Population-based data about a) live-born and stillborn infants with anencephaly and spina bifida and b) pregnancies electively terminated after prenatal diagnosis of these defects were analyzed from the Arkansas Reproductive Health Monitoring System; the California Birth Defects Monitoring Program; CDC's Metropolitan Atlanta Congenital Defects Program; the Iowa Borth Defects Registry, the University of Iowa, and the Iowa Department of Public Health; and the Greenwood Genetic Center in South Carolina. Data also were analyzed from the Hawaii Birth Defects Monitoring Program, which includes data for some women who were not residents of the state. The systems differed in the size and racial/ethnic composition of the populations studied, the surveillance methods used, the completeness of ascertainment, and the availability and utilization of prenatal testing and pregnancy termination. Results and Interpretation Among all pregnancies asscertained in which the infant or fetus had anencephaly or spina bifida, the percentages that were electively terminated ranged from 9% in Arkansas to 42% in Atlanta and Hawaii, with a corresponding increase in the adjusted prevalence of these defects compared with the prevelance at birth. In each system, pregnancies associated with anencephaly were terminated more frequently than were those associated with spina bifida. These data indicate that the impact of prenatal diagnosis and subsequent pregnancy termination on the prevelance at birth of anencephaly and spina bifida differs among geographic areas and populations. Comprehensive surveillance for these defects requires inclusion of pregnancies that are prenatally diagnosed and then terminated. Actions Taken CDC will use these data to promote the inclusion of prenatally diagnosed and terminated pregnancies in estimates of the prevalence of anencephaly and spina bifida generated by birth defects surveillance programs in the United States. Including such pregnancies is crucial to the ability of these programs to monitor trends accurately and to establish the effectiveness of interventions, incuding the use of folic acid, for these defects.

Unmetabolized folic acid in serum: acute studies in subjects consuming fortified food and supplements

Periconceptual consumption of folic acid has been shown to decrease the incidence of neural tube defects. The strategy of universal fortification of staple foodstuffs with folic acid presents the possibility of life-long exposure to unmetabolized folic acid. Chief among the risks of exposure to folic acid in the circulation is that of masking the diagnosis of cobalamin deficiency in pernicious anemia and the progression of neurologic disease. Other effects are unknown. For instance, the effect of in vivo chronic exposure of adult and fetal cells to the synthetic form of the vitamin has never been investigated at the population level. This study examined the acute appearance of unmetabolized folic acid in serum in response to the consumption of some fortified foodstuffs by young and elderly volunteers. Subjects on a 5-d regimen of fortified ready-to-eat-cereal and bread in addition to their normal diet had a threshold intake of 266 micrograms folic acid per meal at which unaltered folic acid appeared in the serum. Subjects given folic acid in either isotonic saline, milk, or white bread also had a threshold > 200 micrograms. From patterns of food consumption in the United States, the implementation of flour fortification at 1.4 mg/kg is unlikely to lead to folic acid appearance in serum, assuming that consumption is spread throughout the day. Increasing this level of fortification, however, as has been advocated by some agencies, may result in the repeated appearance of folic acid in serum over many years, particularly in consumers in nontargeted populations of large amounts of fortified foods. The "safe level of intake" of 1 mg folate/d set by the US Food and Drug Administration may cause a serum folic acid effect. Furthermore, a repeated serum folic acid response is likely to be found in many women complying with the advice to take 400 micrograms folic acid/d to prevent the occurrence of neural tube defects.

Periconceptional folic acid and the prevention of neural tube defects: Some food for thought for Canadian paediatricians.

To review the data supporting the periconceptional use of folic acid for the prevention of neural tube defects and to explore how Canadian physicians can help foster preventive strategies. Articles found through a MEDLINE search, 1990 to 1996, and Cochrane Database of Systematic Reviews 1995. Questionnaire survey of selected group of women of childbearing age (November 1994). Chart review of all children with newly diagnosed neural tube defects admitted to the Children's Hospital of Eastern Ontario, Ottawa, Ontario, between 1989 and 1994. Epidemiological studies and randomized trials of supplemental folic acid for prevention of neural tube defects. Epidemio-logical studies and randomized trials of dietary folic acid. An increased periconceptional folic acid intake can prevent the recurrence of and the first occurrence of neural tube defects. However, consumption of adequate folic acid as natural food folate is relatively ineffective. Furthermore, few women are aware of the need for increased folic acid intake, despite Health Canada recommendations, nor do physicians seem to discuss the prevention of neural tube defects with patients. Better physician awareness, public education and supplementation of a staple food with folic acid are necessary to make the preventive strategy against neural tube defects effective.

Are blastogenetic anomalies sporadic?

Opitz [Birth Defects, 1993, 1:3-37] postulated that sporadic defects of blastogenesis generally are highly lethal and have a low recurrence risk. We have observed that mothers of infants with blastogenetic defects have more previous abortions than mothers of children with nonblastogenetic defects or than mothers of control infants. Thus the high lethality of blastogenetic abnormalities may be responsible for the spontaneous abortions, and there may be a potential for an increased recurrence risk in some cases. Our results also show that the increased rate of spontaneous abortions is not similar for all blastogenetic defects, since it is not elevated in mothers of infants with neural tube defects (NTD). Further, our analysis does not confirm the relationship between spontaneous abortions in the preceding pregnancy and the occurrence of NTD previously reported by other authors.

Multigeneration maternal transmission in Italian families with neural tube defects.

Periconceptional vitamin supplementation with folate prevents about three-quarters of expected cases of neural tube defects (NTDs) in clinical trials. However, vitamin action may be regulated at the level of the gene, and individual susceptibility to environmental agents, including dietary components, also may be under genetic control. We investigated the presence of familial factors in a retrospective case control study of neural tube defects in Genoa, Italy. Cases included all patients treated at a single pediatric neurosurgical service. Controls matched on age and sex came from the same hospital. We found strong evidence for the contribution of genetic factors in this study. There was an excess risk of 14 for the occurrence of NTDs in first-degree relatives compared to controls (P < .0005). There was no difference in sex ratio in any group of relatives, but maternal grandparents of children with a high spinal lesion had 14% fewer off-spring than paternal grandparents (P < .005), possibly because of excess miscarriages. Our study is the first to show complex patterns of inheritance in spina bifida families affecting three generation in one clinical subgroup and preferentially on the mother's side. These results support a role for genomic imprinting and highlight the value of multidisciplinary epidemiologic and clinical studies that include multiple generations. New studies incorporating dietary and genetic approaches will help clarify and extend these findings.

Population-based case control study of folic acid supplementation during pregnancy

Previous studies have demonstrated that periconceptional folic acid/multivitamin supplementation reduced the occurrence of neural tube defects. A case control analysis has been conducted in the dataset of the Hungarian Case Control Surveillance of Congenital Abnormalities, 1980-1991. In the study period, 54.9% of 30,663 pregnant women who had healthy babies (negative control group) were supplemented with high doses (in general 2 x 3 mg) of folic acid per day. In those 17,300 pregnant women who had offspring with congenital abnormalities, the rate of folic acid supplementation was 50.4%. Exposure histories: preconceptional, I, II, III, and IV-IX postconceptional months were determined by record reviews and questionnaire assessment. The case control pair analysis showed a significant protection after folic acid supplementation during the critical period of cardiovascular defects, neural tube defects, cleft lip with or without cleft palate and posterior cleft palate.

Effect of supplemental folic acid on valproic acid-induced embryotoxicity and tissue zinc levels in vivo.

Valproic acid (VPA) is an anti-convulsant drug known to cause spina bifida in humans. Administration of the vitamin, folic acid, has been shown to decrease the recurrence and possibly also the occurrence of neural tube defects, primarily spina bifida, in humans. Additionally, treatment with a derivative (folinic acid) of folic acid has been reported to decrease the frequency of VPA-induced exencephaly in mice treated with the drug in vivo. A protective effect by folinic acid has not been observed in vitro. The purpose of this investigation was to reexamine the ability of folinic acid to decrease the incidence of VPA-induced neural tube defects in vivo. We also examined the effect of increased intake of folic acid on zinc levels in various maternal and embryonic tissues. Folinic acid, whether administered by intraperitoneal injection or in osmotic mini-pumps, did not decrease the number of mouse fetuses with VPA-induced exencephaly. Dietary supplementation with 10-20 times the daily required intake of folic acid in rodents also failed to decrease the embryotoxicity of VPA. Such dietary supplementation had no effect on zinc levels in maternal liver, brain, or kidney, nor in embryonic tissues. These results indicate that folic acid is not able to reverse the embryotoxicity induced by the anticonvulsant, that there is no apparent effect of high dietary folate intake on maternal or embryonic zinc levels and suggest that folate is probably not involved in the mechanism of VPA-induced embryotoxicity.

Periconceptional vitamin use, dietary folate, and the occurrence of neural tube defects.

With a case-control study, we investigated whether periconceptional intake of supplemental or dietary folate reduced the risk of having a neural tube defect (NTD)-affected pregnancy. Mothers of 549 (88% of eligible) cases and 540 (88%) controls were interviewed in person about vitamin supplements used in either the 3 months before or the 3 months after conception and also about usual diet in the 3 months before conception. Women with any use of a folic acid-containing vitamin in the 3 months before conception had a lower risk of having an NTD-affected pregnancy [odds ratio (OR) = 0.65; 95% confidence interval (CI) = 0.45-0.94]. ORs were similar for 3 levels (< 0.4, 0.4-0.9, and > 0.9 mg per day) of average daily intake of folic acid. Any level of use in the first 3 months after conception resulted in a lowered risk as well (OR = 0.60; 95% CI = 0.46-0.79). Reduced risks were less marked for Hispanics and were not observed among women who graduated from college. Modest reduced risks were noted among non-vitamin users whose estimated daily dietary intake of folate was more than 0.227 mg. We observed decreasing risk with increasing folate intake from combined dietary sources and vitamin supplements. A reduction in NTD risk associated with folate intake is consistent with other studies; however, the reduced risk may be particular to subsets of the population, primarily non-Hispanic women and women whose education does not exceed high school.

Congenital Malformations in Primary Health Care in Al-Qassim Region

The objective of this predesigned research was to determine the prevalence, sociodemographic correlates, and pattern of congenital malformations (CMs) in a population less than 20 years of age. From January 1991 to December 1991, the extensive examination of family health files coupled with door-to-door survey identified 1736 patients with CMs. The results showed that the prevalence of CMs was 6.9/1000 population. The male to female ratio was 1.3:1. The patient's age, sibling order, co-morbid disorders of CMs, parental age, chronic maternal diseases, and the family history of CMs were significantly associated with groups of arbitrarily classified CMs. The most frequently encountered CMs were of central nervous system (39.2%) followed by cardiovascular (22.3%), alimentary (13.1%), musculoskeletal (6.85%), urogenital (6.16%), communicative and audiovisual (5.5%), and miscellaneous (6.9%). Further one-way ANOVA found significant differences among CM categories and age of patients and parents. Unlike Western trends, our study tentatively concludes that overall the inheritance rather than socioenvironmental factors contributes significantly in the etiology of different congenital malformations. We further suggest that the basic information of this research might be useful foundation data in future analytic studies on congenital malformations.

Use of local neural tube defect registers to interpret national trends.

To conduct a number of studies into the prevalence of neural tube defects (NTD) in the area covered by the Oxford Record Linkage Study (ORLS), multiple sources were used to build a local register of cases occurring in Oxfordshire and West Berkshire between 1968-1990. One source of potential cases--namely, termination and congenital malformation monitoring data available for the locality from the Office of Population Censuses and Surveys (OPCS) data--were kept separate. Comparison of the local cases recorded by OPCS and those known to the register from 1974-1990, using the method of capture-recapture, suggested that national data are only about two thirds complete, but that this underreporting is likely to be reasonably consistent from year to year. OPCS data can therefore be used to study NTD trends if not absolute risks. The local register seemed, by the same yardstick, to be very complete and is being used in a variety of studies of the occurrence of NTD. Survival to one year in this area, over the period 1968-1990, has only improved in the recent past, if at all. Most NTD pregnancies now end in termination rather than birth, and there has been a true decline in the occurrence of NTDs, and likewise the different subtypes.

Review of Neural Tube Defects: Risk Factors in Parental Occupation and the Environment

We conducted a study of published work to evaluate the evidence for the hypothesis that environmental exposure and parental occupation are risk factors for neural tube defects. As other risk factors such as maternal illnesses, medication, and dietary factors have been reviewed before, this review only summarizes this information. In studies concerning environmental pollution, only a few weak associations were found. It appears that specific studies on the topic of parental occupation and neural tube defects are scarce. Therefore, studies on broader malformation categories, such as central nervous system defects, were also taken into account. Both maternal and paternal occupation seem to be associated with the occurrence of neural tube defects. However, results are not always consistent with each other, and relevant recommendations concerning prevention thus cannot be given before more studies with larger populations are conducted to confirm or refute the findings reviewed.

Review of neural tube defects: risk factors in parental occupation and the environment.

We conducted a study of published work to evaluate the evidence for the hypothesis that environmental exposure and parental occupation are risk factors for neural tube defects. As other risk factors such as maternal illnesses, medication, and dietary factors have been reviewed before, this review only summarizes this information. In studies concerning environmental pollution, only a few weak associations were found. It appears that specific studies on the topic of parental occupation and neural tube defects are scarce. Therefore, studies on broader malformation categories, such as central nervous system defects, were also taken into account. Both maternal and paternal occupation seem to be associated with the occurrence of neural tube defects. However, results are not always consistent with each other, and relevant recommendations concerning prevention thus cannot be given before more studies with larger populations are conducted to confirm or refute the findings reviewed.

Preaxial ray reduction defects as part of valproic acid embryofetopathy

It is well known that prenatal exposure of valproic acid (VPA) may be associated with the occurrence of neural tube defects (Robert and Gibaud, 1982). Additional adverse effects related to VPA exposure include craniofacial abnormalities, skeletal defects, brain defects, cardiovascular defects, and urogenital defects (DiLiberti et al., 1984; Winter et al., 1987; Huot et al., 1987; Jager-Roman et al., 1986; Martinez-Frias, 1990). Recently, radial ray reduction has been reported as a severe type of skeletal defect associated with VPA exposure (Jager-Roman et al., 1986; Huot et al., 1987; Verloes et al., 1990). We report two additional cases of this severe limb reduction defect as part of a broader pattern of altered morphogenesis in fetuses exposed to VPA and to confirm an association between such severe specific skeletal defects and VPA exposure. We briefly review teratologic studies in experimental animals which document the biologic plausibility of this association, and suggest that fetuses exposed to VPA undergo detailed prenatal ultrasonographic evaluation of the limbs.

Prevention of the First Occurrence of Neural-Tube Defects by Periconceptional Vitamin Supplementation

Prevention of the First Occurrence of Neural-Tube Defects by Periconceptional Vitamin Supplementation