Andrea Tenley Timing of Multiple Factors in the Development and Treatment of Neural Tube Defects FCS 306, Section

01 Advanced Nutrition Science the Macronutrients Western Illinois University March 18, 2013

I. INTRODUCTION The assigned topic is clinical/experimental research of neural tube defects (NTD) and five articles were reviewed. The following observations were made: Best practice for prevention of NTDs is supplementation of 400g folic acid daily and supplementation should begin at least four weeks prior to conception. A dosage of less than 400g daily may still lower the risk of an NTD. Hyperglycemia in pregnant women and in pregnant mice with diabetes will produce an environment favorable for the development of NTDs. Finally, prenatal surgery for fetuses with NTDs is a superior strategy for preventing future NTDrelated developmental issues compared to postnatal surgery. Whether the topic is prevention, causation, or repair, timing was a key factor.

II. TIMING OF MULTIPLE FACTORS IN THE DEVELOPMENT AND TREATMENT OF NEURAL TUBE DEFECTS The first article was reviewed because the focus of the experiment was on folic acid supplements, which correlates to the prevention of NTDs. The randomized control trial conducted by Norsworthy, Skeaff, Adandk, and Green (2004) examined both the timing of consumption and the amount of folic acid consumed by women from Dunedin, New Zealand, and their resulting folate concentrations were taken via blood samples. The hypothesis was not clearly stated, but the objective of the study was to determine if a weekly high dosage of folic acid supplement would increase a womans red blood cell (RBC) folate concentration to or above 905 nanomole per liter (>905nmol/L), which is the concentration that is associated

with a lowered risk of a NTD-related pregnancy. The participants were selected by convenience sampling; the participants voluntarily joined the study by responding to newspaper ads and posters. The subjects of the trial included 114 women whose initial RBC folate concentrations were between 295 and 905nmol/L. Women with levels below 295nmol/L were deemed folate deficient and referred to their general practitioners. Originally 279 women volunteered, 255 of which were eligible for blood testing and 24 were excluded. Exclusion criteria included the following: Pregnancy within the last year, current consumption of a folic acid supplement over 100 micrograms (g) per day, consumption of a folate metabolism-interfering drug, history of an NTD pregnancy, chronic disease, and/or an RBC folate concentration over 905nmol/L. After the women were screened, 138 were determined eligible and voluntarily joined the trial. Of the 138 participants, 125 women completed the trial. Ultimately, 11 women were excluded due to missing folate level data on week six and/or week 12. Lastly, the data secured for the conclusion of the trial were derived from a total of 114 women. Of the 114 women who participated, 90% were non-smokers, 81% were less than 30 years of age, and 88% had never been pregnant before. The study design consisted of a 12 week randomized control trial conducted July through October 2001. The women were randomly selected for three different groups: Group one received a placebo, group two received a dose of 2,800g per week folic acid, and group three received 400g per day folic acid. The participants RBC folate concentration was assessed on day one of the study, taken again after six weeks, and taken a third and final time at the 12 week conclusion of the study. For accurate analysis, the participants were instructed not to take a dose of their assigned pills within 24 hours of the either of the blood tests

because this may increase the RBC folate concentration significantly and report an inaccurately higher concentration. The participants were also instructed to bring their assigned pills to the screenings at six and at 12 weeks. Compliance was measured by counting and weighing: The women in the placebo and daily consumption groups brought their pills to be weighed and the estimated consumption was recorded. The women in the weekly consumption group brought their pills, and the pills were counted to determine consumption amount. Additionally, at weeks six and 12, the participants were instructed to bring a three-day weighted diet history in order for the researchers to record the average nutrient and energy consumption by the participants. At the 12 week conclusion, results revealed the weekly 2,800g folic acid supplement consumption group had RBC folate concentrations 95% higher than the placebo group. The 400g daily folic acid supplement consumption group had RBC folate concentrations 15% higher than the 2,800g weekly folic acid supplement consumption group. The daily and weekly supplement consumption group participants experienced more rapidly rising RBC folate concentration levels during weeks one through six, compared to weeks six through 12. At the conclusion of the study, 50% of the weekly consumption group participants had an RBC folate concentration level over 905nmol/L by week 12, compared to 74% of the daily consumption participants. Weekly 2,800g supplementation was deemed wise for women who had difficulty maintaining a daily supplementation schedule because it is better than going without. In the discussion section of the article, the researchers measured the outcomes of the study; they noted the United States recommendation of 400g daily folic acid for women in childbearing years to lower the risk of NTD pregnancy occurrences. While the lowest risks of

NTD pregnancies are associated with RBC folate concentration at or above 905nmol/L, this concentration is not known to be the threshold for the absolute lowest risk of NTDs and further studies are warranted. The weekly dose of 2,800g folic acid is successful in increasing RBC folate concentrations, but not as successful as the 400g daily dose. The researchers also recommended that women start folic acid supplementation at least four weeks prior to conception to lower the risk of NTDs; the timing was important because the neural tube is known to close early in the pregnancy. Additionally, most women in the general population do not have the RBC folate concentration of 905nmol/L or higher, therefore any supplementation of folic acid, whether weekly or daily, is better than none at all. A future study topic was suggested by the researchers: Which is more likely for women to consume on a regular basis and maintain compliance, a weekly dose or a daily dose of folic acid? If given the option of a weekly or a daily dose, which would result in better compliance? Ultimately, the goal stated was to increase a womans RBC folate concentration level at least four weeks prior to pregnancy. The researchers determined the concentration levels can be raised with a high level, weekly dose of folic acid, but they concluded the best evidence-based practice remains to be the consumption of a daily folic acid dose of 400g. The second article reviewed was a study of preconception vitamin supplementation and the incidence of NTD pregnancies that followed in women who previously had at least one NTD-related pregnancy. The experiment conducted by Smithells et al. (1981) examined 200 women who were fully supplemented, 50 women who were partially supplemented, and 300 women who were unsupplemented with a folic acid, ascorbic acid, and riboflavin-containing vitamin mixture. The hypothesis questioned if there was a relationship between minor

vitamin deficiencies and NTD causation. The study design included convenience sampling of participants who were recruited through referrals from genetic counselors, obstetricians, general practitioners, and other health related contacts. The women voluntarily participated in the experiment. The researchers explained the vitamin mixture was chosen because it was one of the only supplements available that included folic acid. The vitamin mixture contained: 4000IU vitamin A, 400IU vitamin D, 1.5mg thiamin, 1.5mg riboflavin, 1mg pyridoxine, 15mg nicotinamide, 40mg ascorbic acid, .36mg folic acid, 75.6mg ferrous sulfate, and 480mg calcium phosphate. Inclusion criteria for the participants included women with at least one prior NTD pregnancy, women who were not pregnant at the time of the study, but who were planning another pregnancy in the near future. Women were asked to use a birth control method other than birth control pills for at least 28 days after starting the supplements. The exclusion criteria included women who were not planning on having another pregnancy in the near future, and women who did not have a history of NTD pregnancies. For the conclusion of the study, women who had moved away, who had not yet conceived, and who had inconsistent compliance with the dosage were also excluded. Maternal age was not considered. Originally the researchers proposed a double blind placebo study, but this was rejected by two different ethics committees. Therefore, the new study was designed. All women who were trying to become pregnant were immediately started on the supplement. All of the women were asked to take one supplement tablet three times per day. All women were asked to take the supplement for a minimum of 28 days prior to conceiving and to continue taking them until their second missed period. The women in this group who were able to fully

comply were considered the fully supplemented group. Next, the women who either conceived before allowing supplementation for 28 days, or who started the supplements after they conceived, but prior to neural tube closure were considered partially supplemented. Additionally, women who reportedly missed at least one supplement in the time prior to conceiving were also considered partially supplemented. Later in the study, pregnant women from the same geographical area were recruited in the same manner as the first two groups; if they had a previous NTD pregnancy, and were pregnant and still in their first trimester, and they voluntarily consented to the study, were considered the unsupplemented group. Data analysis was conducted after the women gave birth. The women were not randomly assigned to groups but rather fell into the groups based on the timing of their conceptions and their compliance with taking the supplements. Data collected after the women gave birth were analyzed and determined that the difference between the fully supplemented mothers and the unsupplemented mothers was substantial; the supplemented mothers incidence of an NTD newborn was .5% and the unsupplemented mothers incidence of an NTD newborn was 4%. There was one baby born with an NTD in the fully supplemented group, none in the partially supplemented group, and 13 born to the unsupplemented group. The discussion section of the article examined the possibilities of other nutrients in the supplement having had an impact on NTDs and the need for further studies. The possibility was also considered that pregnant women need to take only one dose of folic acid, or mixture supplement as used in this trial, at one specific time during the first trimester to prevent an NTD. However, this would be a difficult study to conduct with humans due to ethical reasons. Additionally, the researchers noted that other aspects in the mothers environment

may have influenced the participants compliance regarding consumption of the supplements, such as social class, since this was a multi-center study (referrals from many locations). In conclusion, the researchers rationalize that folic acid supplements indeed prevent NTDs, as this is consistent with other published studies, but it still cannot be claimed as proven. The researchers confirmed the need for further studies on multiple types of supplements in addition to folic acid, and also the need to continue to study women for extended preconception periods for a closer examination of supplementation, nutritional, and environmental variables. The third article reviewed was an experimental study conducted by Phelan, Ito, and Loeken (1997) in which there was not a stated hypothesis, but rather a stated objective. The objective was to develop a system, a model, in which the researchers could study the causes of NTD malformations associated with pregnant women who have diabetes. The subjects used in the experiment were mice, female, 9 weeks old. The number of mice and the number of the mices embryos used in the study were not mentioned. As seen before, timing became a crucial factor in the design of this study as the researchers scrutinized over how to understand the connection between diabetes and NTD development based on the timing of hyperglycemia and the expression of a certain gene. The Pax-3 gene is responsible for the closure of specific areas of the neural tube: the hindbrain and the midbrain. Embryos from mice with diabetes have been found to have tremendously lower amounts of Pax-3 genes. Not only was the Pax-3 gene missing or in very low quantity from the areas that suffer from NTDs, but researchers have also found excessive amounts of cells in the process of apoptosis in the areas. Researchers hypothesize that the Pax-3 gene is also responsible for the cells undergoing the high levels of apoptosis.

Researchers believe that the expression of the Pax-3 gene, which is found to be disrupted in embryos of diabetic mice, is essential for the closure of the neural tube. Therefore, the researchers designed a model to test and study the cause of the disruption of the Pax-3 gene. Many other genes have been identified to play a role in closure of neural tubes, just as many other deficiencies have been recorded in children and in mice due to the mother being diabetic during the gestation period. This study was interested in the examination of how Pax-3 was disrupted in the embryos of diabetic mice. Expression of Pax-3 begins in day 8.5 of gestation in mice. When Pax-3 was inhibited in mice embryos, a mutation on an allele called Splotch phenotype occurs. The Splotch phenotype always resulted in exencephaly and/or spina bifida; these are the same NTDs that have been recorded as occurring in the fetuses carried by women with diabetes. In order to understand how the Pax-3 gene is disturbed on a molecular level, the researchers tested embryos of diabetic mice. The mice used in the experiments were 9 week old females that were insulin dependent diabetic mellitus (IDDM) induced with the use of a drug called strptozotocin (STZ). The number of mice in the study was not stated. After the mice were screened and determined to be diabetic, they were then implanted with slow-release insulin pellets subcutaneously to control incidents of hyperglycemia. After controlling the mices glucose levels successfully for three weeks, the females were mated with non-diabetic, control male mice. The regulation of glucose in the females was necessary for proper ovulation. After impregnation, the female mice again became hyperglycemic and remained hyperglycemic until the day they were terminated and their embryos were collected. At the same time, another set of control female mice were not administered the STZ, were not IDDM, and their glucose levels were also recorded. The control female mice were not diabetic; they were also mated on the same day

with non-diabetic, control male mice. The control female mice maintained normal glucose levels during gestation. Some of the diabetic mice were allowed to carry their pups full term and deliver. The diabetic pregnant mice that were killed had a greater amount of embryos versus the number of pups birthed. The researchers suggested that diabetic mice ovulate less, or they lost some embryos throughout different stages of gestation. The pups born to diabetic mice were deformed more often than those born to the non-diabetic mice: 75% deformity was recorded in diabetic mice versus 24% from non-diabetic mice. Embryos collected from diabetic mice on day 11.5 gestation showed NTDs compared to the normally developed embryos collected from non-diabetic mice on the same day. Some of the control mice became diabetic spontaneously during gestation, and at that point they were studied further, and one mouses embryos were collected on day 13.5 of gestation. These embryos displayed massive NTDs that researchers suggested were caused by the hyperglycemia brought on by the gestational diabetes. This specific mouse that was studied after her sudden onset of diabetes was found to be remarkable because her embryos had developed severe NTDs, yet she had previously littered pups that were born without defect prior to her becoming diabetic. The Pax-3 gene was severely under-expressed in the embryos of the diabetic mice. Since Pax-3 was grossly under-expressed, it was not able to code for enough of the DNA-binding protein necessary to close the neural tube. The embryos from the diabetic mice carrying the Splotch allele all developed exencephaly and/or spina bifida 100% of the time. Researchers noted the timing of the mice being induced IDDM and the periods of hyperglycemia that coincided with the period of time that Pax-3 would need to be expressed (to code for binding proteins to close the neural tube). Therefore, in order to correlate the maternal diabetes to the

under-expression of Pax-3, researchers removed the diabetic mices embryos on day 8.5 because this is the day Pax-3 expression should begin, and less Pax-3 was obtained from the embryos from the diabetic mice versus the non-diabetic mice. A control examination was conducted on fibronectin mRNA and unlike the Pax-3 mRNA, the fibronectin mRNA was not disturbed. Thus this supports the suggestion that maternal diabetes affects the Pax-3 gene but not necessarily other genes. In some of the embryos of diabetic mice the Pax-3 gene was entirely undetectable. Embryos were also collected at day 9.5 and the embryos from the diabetic mice continued to display little or no trace of Pax-3 gene expression. After fusion of the neural tube in non-defective embryos, it is normal for apoptosis to then occur because the job of that cell has been completed. The researchers observed and recorded the disruption of the timing of the apoptosis in the embryos from the diabetic mice, thus suggesting the result is an NTD. To further assay the misfiring of apoptosis, the researchers collected embryos from both the diabetic mice and the control mice on day 9.5 and 10.5 gestation. Both embryo groups were in the same development stage, yet the embryos from diabetic mice displayed apoptotic nuclei along the neural tube where no fusion had yet occurred, while the control embryos did not display any apoptotic nuclei. The investigation of whether Pax-3 under expression can cause premature apoptosis in the embryos of diabetic mice has yet to be done. Therefore, the researchers were unable to say if Pax-3 under expression caused the premature apoptosis or if it is a simple correlation. Another experiment was conducted: Homozygous Splotch embryos (missing the Pax-3 genes) and heterozygous Splotch/+ embryos (which had the Pax-3 gene) were collected on day 10.5 gestation. In the homozygous embryos, apoptotic cells were present only in the unfused sections of the neural tube, while in the heterozygous embryos, no apoptosis was

found. The apoptotic cells located only in unfused sections of the neural tube confirmed the researchers suggestion that lack of Pax-3 expression will lead to apoptosis in the defected areas on the neural tube. Therefore, apoptosis observed in the embryos from the diabetic mice likely could be a result of the under expression of the Pax-3 gene. In the discussion section of this article, the researchers concluded that the molecular mechanisms are still not known, but many studies agree that maternal diabetes during gestation can cause severe birth defects. The correlation is officially stated between the under expression of Pax-3 and the development of NTDs. The researchers suggested that the molecular mechanisms and cellular processes that are displayed with Splotch phenotypes are the same as with embryos from diabetic mice. The researchers noted specifically the interesting observation of the apoptotic cells present along the sections of the unfused neural tube only, not the fused sections; this occurred while Pax-3 was missing from the embryo. Also, the NTDs occurring in the embryos (diabetic and Splotch) were primarily in the part of the tube that fuses last. Many questions arise in the conclusion of this study and the researchers recommend more studies should continue. For example, is it possible that Pax-3 only codes for closure of the neural tube at specific sites or is it also specific times? Does the molecular process of the Pax-3 gene work to code for closure of the neural tube by suppressing apoptosis? Is it just the timing of Pax-3 expression that is disrupted by the maternal diabetes? Do excessive free radicals play a part in the NTD development in embryos due to maternal diabetes and the toxic environment? Finally, the researchers reiterate the gene expression that is controlled, regulated, or disrupted by diabetes is not yet understood. Pax-3 is not the only gene in control of the development of NTDs. More studies must be done to further understand what occurs in

this environment, also noted by the researchers, the crucial time frame that these gene expression alterations are made need to be studied further. Timing was essential and researchers aim to determine if there is an effect on Pax-3 gene expression that could possibly be controlled by dosing a medication at a specific time to control the maternal glucose levels and prevent NTDs. The fourth article was reviewed studied glucose levels, specifically hyperglycemia, as a potential causation factor in the development of NTDs and it also stressed the importance of timing in regards to specific development periods when high levels of glucose may inhibit full neural tube closure. This experimental research was conducted by Fine, Horal, Chang, Fortin, and Loeken (1999) and the hypothesis is not clearly stated. Many objectives were scattered throughout the article however, one being the investigation of whether glucose causes reduced expression of the Pax-3 gene? Another objective was does the underexpression of the Pax-3 gene cause NTDs due to less coding for transcription factors that regulate neural tube development? The researchers experimented with placing the embryos in high glucose environments to determine if the same alterations to gene expression and occurrence of apoptosis would be observed as in diabetic pregnancies and if blood glucose levels in mice are correlated to the development of NTDs. The subjects of these experiments were mice, 9 week old females that were IDDM induced, they were mated with control male mice, terminated, and their embryos collected on specific days depending on the data being collected. Embryos collected on day 8.5 were used for preparation of RNA examination, on day 9.5 for glucose environment testing, or on day 10.5 for assay of apoptosis. Just as with the previous study, these researchers induced IDDM with the use of STZ, implanted the insulin extended release tablets subcutaneously to control

hyperglycemia, and the mice were then observed for glucose level controls prior to being mated with control male mice. The first experiment involved the removal of embryos from the diabetic mice on gestation day 9.5; they were placed in a normal glucose solution of five micromoles per liter (5mmol/L) and also an elevated glucose environment of 15mmol/L. The researchers tested the impact the level of glucose had on Pax-3 expression in the embryos. The results indicated that glucose elevated to similar levels of that of a diabetic mouse will inhibit the expression of Pax-3. Next the researchers wanted to reproduce the test in vivo. Therefore, hyperglycemia was induced in a pregnant diabetic mouse on day 7.5 and day 8.5 (the day prior to and the day of neural tube fusing). The result indicated that inducing hyperglycemia the day prior and the day of neural tube fusion resulted in inhibited expression of the Pax-3 gene. Finally, the researchers chose to test the relationship between under expressed Pax-3 genes and apoptosis when the diabetic mouse has had hyperglycemia induced. The result confirmed that exposing the embryo to elevated levels of blood glucose, such as in a diabetic pregnancy, absolutely caused the same increase in apoptosis as seen in a diabetic pregnancy. Since the previous tests demonstrated glucose alone, when elevated, can imitate the effects of diabetes on an embryo and lead to the formation of NTDs then another question had to be asked. Is glucose necessary to cause NTDs? The researchers then tested the pregnant diabetic mice by reducing their glucose levels with the use of Phlorizin, a drug that blocks renal reabsorption of glucose, on days 6.5 through 9.5 gestation; the days before, during, and after neural tube fusion. The drug worked to lower the diabetic mices blood glucose levels. The conclusion of that experiment was that it worked; the mice treated with

Phlorizin had the same rate of NTDs as the non-diabetic pregnant mice. This experiment demonstrated that the more critical the hyperglycemia, the higher the rate of NTDs. In the discussion section researchers state that the evidence in the studies conducted support the hypothesis that the molecular activity that results in NTDs needs hyperglycemia in order to occur. When non-diabetic mice were subjected to induced hyperglycemia, the same inhibition of the Pax-3 gene and signaling for apoptosis was observed just as in a diabetic pregnancy. The timing of the hyperglycemia and the inhibition of the Pax-3 gene, increasing the incidence of NTDs, was significant due to the periods in which the hyperglycemia was induced and the inhibition of the Pax-3 expression that immediately followed. Ultimately, the researchers concluded that severe hyperglycemia is associated with the development of NTDs. The fifth and final article was reviewed because the previous articles were concerned with the prevention and likely causation of NTDs, while this article addressed procedures that are utilized to treat NTDs. The randomized trial conducted by Adzick et al. (2011) explored the consequences of prenatal versus postnatal surgery to repair NTDs. The hypothesis in this article was lacking, however, the researchers discussed their objective: Compare the outcomes of in utero surgery versus postnatal surgery for the most common type of spina bifida, myelomeningocele. Postnatal repair is considered standard, while in utero is not. The design included convenience sampling of 1,087 preliminary subjects; after screening, 605 were excluded, 258 declined to participate for numerous reasons, and 41 refused consent. A total of 183 subjects were randomized into the trial. Inclusion factors were: Singleton pregnancy with myelomeningocele, hindbrain herniation in fetus, 19-25.9 weeks gestational

age, US resident, maternal age of 18 or older, and a normal karyotype. The exclusion factors were: Unrelated fetal anomaly, risk of preterm birth, placental abruption, severe kyphosis, body mass index of 35 or greater, and any contraindication to surgery. The trial ran February 2003 through December 2010. The trial was terminated on December 7, 2010 so the final conclusions were drawn upon 158 subjects who were randomized prior to July 1, 2009. The women in the trial were referred to the study by participating centers, and contacting the trial administrators was voluntary on behalf of the subject. First, the women gave written consent, next they were screened for eligibility, and then they were evaluated further for eligibility. The randomization was web-based and conducted by the coordinating center. This randomization resulted in the subjects being placed in one of two groups: Postnatal surgery for their childs NTD or in utero surgery for treatment of the NTD. According to the committee responsible for monitoring the safety and data of the experimental surgeries, it was recommended that the trial conclude on December 7, 2010. Therefore, of the original 183 subjects in the trial, conclusion data were drawn from the 158 subjects randomized before July 2009. During the trial, no maternal death occurred. The complications from the surgeries occurred most with the prenatal surgery group subjects. Complications related to the prenatal surgery subjects included choriamniotic separation, oligodramnios, spontaneous membrane rupture, and placental abruption. Prenatal fetus subjects experienced averaged gestational age of 34.1 weeks and 13% of the prenatal surgery fetus subjects were born prior to 30 weeks gestation. In comparison, the average post-natal surgery fetus subject was born at 37.3 weeks gestation and none of the post-natal surgery subjects gave birth before 30 weeks gestation. Despite these notable differences, in general the unfavorable neonatal outcomes were comparable between the two trial groups.

The researchers conducted analysis of outcomes at two specific times: Child age 12 months, and child age 30 months. The first analysis considered fetal or neonatal death, and/or whether the child required a cerebrospinal fluid shunt. The incidence of death or need for a shunt occurred in 68% of the prenatal surgery group and in 98% of the post-natal surgery group. Of the children that needed the shunt, 40% of the prenatal surgery group had a shunt placed while 82% of post-natal surgery group had a shunt placed. There was no evidence of hindbrain herniation recorded in 36% of the prenatal surgery group, while only 4% of the post-natal surgery had no evidence of hindbrain herniation. The children who did display hindbrain herniation, 25% were in the prenatal surgery group while 67% were in the postnatal surgery group. The prenatal surgery group also had lower incidences of abnormal fourth-ventricle location, brain stem kinking, and syringomyelia than the post-natal surgery group. The secondary outcome analysis at 30 months utilized the Bayley Mental Development Index and differences in anatomical and functional levels of the lesions. These tools observed that the prenatal surgery group had significantly higher scores than the post-natal surgery group (p=0.007). The children from the prenatal surgery group displayed anatomical function levels two or more levels higher than the post-natal surgery group children (p=0.005). Ability to walk without devices or orthotics was observed in 42% of the prenatal surgery group versus 21% in the post-natal surgery group (p=0.03). Resulting analysis of data collected at child age 30 months: Despite more severe anatomical levels of lesions, the prenatal surgery group displayed markedly better motor function compared to the post-natal surgery group. Between 12 and 30 months of age two deaths occurred, one per surgery group. The death in

the post-natal surgery group was due to complications from chemotherapy and the prenatal surgery group death was due to coxsackie septicemia. The discussion section of the article reiterated the benefits of prenatal surgery for myelomeningocele executed prior to 26 weeks gestation compared to post-natal surgery. Benefits included lower risk of shunting required and lower risk of death by 12 months of age, as well as markedly superior motor function and mental abilities. Further benefits included improved ability to walk without assistance and a lower degree of hindbrain herniation. The researchers suggested the reasons behind the improved outcomes for the prenatal surgery group was due to the timing of the surgery to repair the NTD, which allowed the nervous system to continue to develop prenatally. Researchers also suggested the reason for less shunting requirements in the prenatal surgery group was due to less hindbrain herniation; this allowed for better cerebrospinal fluid flow. The researchers discussed in depth the risks versus the benefits of pre-natal surgery. Risks such as prematurity of childbirth and maternal morbidity must be evaluated. Although long term benefits of prenatal surgery were evidenced, the impediments associated with the surgery included: Uterine scar defects, preterm birth, intraoperative complications, and the need for maternal blood transfusion at the time of childbirth. Of the prenatal surgery subjects, one-fourth displayed chorioamniotic separation via ultrasound following the procedure; chorioamniotic separation increases the likelihood of premature membrane rupturing. Other possible known complications with prenatal surgery include placental abruption and pulmonary edema. Also, mothers who agreed to the prenatal surgery also had to be in agreement that with subsequent pregnancies, it was advised that delivery be via scheduled cesarean prior to natural labor occurring.

Finally, the researchers stressed the importance of this type of prenatal repair only be conducted in experienced centers with the use of a specific stapling device. The stapling device used absorbable staples which does not prevent future fertility like metal staples might and also minimizes blood loss. A multidisciplinary team of experts were involved in every surgery, and it was recommended that similar surgeries must be done in an experienced environment, with experienced surgeons, with subjects who meet the inclusion factors noted in this study; the results of this study are not to be generalized. Additionally, it is imperative that the children in this study be continuously followed to further evaluate the outcome of the prenatal versus the post-natal surgery repair of myelomeningocele, especially concerning the childs mental capacity, sexual function, and bladder and bowel continence. III. CONCLUSIONS The timing of numerous aspects, such as fetus neural tube development, supplementation with folic acid, incidence of hyperglycemia, and repair of an NTD are instrumental in determining factors of prevention, causation, and repair of NTDs. As seen in the first article, the timing of the dose of folic acid was considered. At the end of seven days, the subjects had ingested the same amount of folic acid, and it was determined that while any folic acid supplementation is better than none, and the while the weekly dose was relatively effective, the daily dose worked best in raising RBC concentrations of folate. This lends credence to the conclusion that when taken on a regular basis, the RBC folate concentrations are better replenished. Thus, the timing of the supplementation is important. The second article suggested the possibility of pinpointing the timing of neural tube closure and simultaneously administering a single dose of folic acid and/or other nutrients to prevent an NTD. This is an interesting theory and while it should be examined further, it

would be difficult to experiment on human subjects, but could be done with mice. Additionally, if NTDs occur at specific times and hyperglycemia is a factor in causation (such as with the mice experiments), would folic acid doses during times of hyperglycemia prevent an NTD? It would certainly be worth experimenting with more mice to explore the possibility. It was determined in the third and fourth articles that the expression of the Pax-3 gene is responsible (it is not the only gene responsible, but plays a vital role) for neural tube closure and this gene is inhibited by hyperglycemia. If the hyperglycemia occurs the day before or the day of neural tube closure, Pax-3 inhibition causes the tube not to close. However, when hyperglycemia was controlled the day before and the day of closure, Pax-3 was not inhibited and allowed for neural tube closure. This suggests that women who are diabetic (type one, type two, or gestational) should be monitored as high risk. Pregnant women with diabetes should check their blood glucose levels several times per day especially in the early stages of the pregnancy when the neural tube is under development. Additional studies should be done regarding the amount of cells observed undergoing apoptosis and whether this too was a result of the hyperglycemia or from the inhibition of the Pax-3 gene. Finally, the repair of an NTD once it has occurred is necessary, but when is the right time to repair the neural tube? Researchers worked with mothers who voluntarily decided to be a part of an intense study to find out. The timing of the repair was evidenced to decrease many long term risks associated with NTDs if performed prenatally. If repaired prenatally, timing of the repair allows for further normal development of the nervous system, but it also presents a whole new set of risks for the mother and her child. Ultimately, it comes down to the mother and the risks she is willing to take. It is truly terrible that anyone should have to

make such choices, but the timing of all aspects from prevention, causation, and repair all determine the outcome, so such risks and benefits must be weighed. Further studies should continue to examine all causation factors and discover the key efforts to prevent NTDs.

References Adzick, N. S., Thom, E. A., Spong, C. Y., Brock III, J. W., Burrows, P. K., Johnson, M. P., Howell, L. J., . . . Farmer, D. L. (2011). A randomized trial of prenatal versus postnatal repair of myelomeningocele. The New England Journal of Medicine, 364(11), 993-1004. doi:10.1056/NEJMoa1014379 Fine, E. L., Horal, M., Chang, T. I., Fortin, G., & Loeken, M. R. (1999). Evidence that elevated glucose causes altered gene expression, apoptosis, and neural tube defects in a mouse model of diabetic pregnancy. Diabetes, 48, 2454-2462. doi: 10.2337/diabetes.48.12.2454 Norsworthy, B., Skeaff, C. M., Adank, C., & Green, T. J. (2004). Effects of once-a-week or daily folic acid supplementation on red blood cell folate concentrations in women. European Journal of Clinical Nutrition, 58, 548-554. doi:10.1038/sj.ejcn.1601843 Phelan, S. A., Ito, M., & Loeken, M. R. (1997). Neural tube defects in embryos of diabetic mice: Role of the pax-3 gene and apoptosis. Diabetes, 46, 1189-1197. doi:10.2337/diab.46.7.1189 Smithells, R. W., Sheppard, S., Schorah, C. J., Seller, M. J., Nevin, N. C., Harris, R., . . . Fielding, W. (1981). Apparent prevention of neural tube defects by periconceptional vitamin supplementation. Archives of Disease in Childhood, 56, 911-918. doi:10.1136/adc.56.12.911