Presentation on theme: "Dr. Attila Vereczkey, M.D., M.A."— Presentation transcript:
1 Dr. Attila Vereczkey, M.D., M.A. Primary Prevention of Birth Defects by Periconceptional Folic-Acid Containing Multivitamin SupplementationProf. Andrew E. Czeizel, MD., C.Sc., D.Sc. (Scientific director of the Foundation for Community Control of Hereditary Diseases,Budapest, Hungary)Dr. Attila Vereczkey, M.D., M.A.(Medical Director of the Versys Clinics, Human Reproduction Institute, Budapest, Hungary)
2 The deficiency or overdosage of certain nutrients may have a role in the origin of birth defects. First in 1932 Fred Hale demonstrated that a vitamin A-free diet during early pregnancy of sows resulted in offspring without eyeballs, oral clefts, accessory ears, malposition of kidney and defects of hind legs.The deficiency or overdosage of certain nutrients may have a role in the origin of birth defects. First in 1932 Hale (1) demonstrated that a vitamin A-free diet during early pregnancy of sows resulted in offspring without eyeballs, oral clefts, accessory ears, malposition of kidney and defects of hind legs. Hale's conclusion was "the condition is illustrative of the marked effect that a deficiency may have in the disturbance of the internal factors that control the mechanism of development".
3 Joseph Warkany ( ), known as „ father of teratology”, recognized the importance of purified diets and used these to test various vitamin deficiencies for their teratogenic effects.Warkany found that maternal dietary deficiency can induce structural birth defects, i.e., congenital abnormalities (CAs).Joseph Warkany ( ), one of the founders of teratology, recognized the importance of purified diets and used these to test various vitamin deficiencies for their teratogenic effects. Warkany (2, 3) found that maternal dietary deficiency can induce structural birth defects, i.e., congenital abnormalities (CAs).
4 Richard W Smithells (1924 - 2002) MD, FRCPCH, FRCP, FRCPE, FRCOG, DCH Professor of Paediatrics and Child Health,In 1964 Hibbard reported a higher rate of CAs (3%) in the infants of folate-deficient mothers than in controls (1.6%)Hibbard and Smithells showed a relationship between human embryopathy and a deficiency of folate metabolismSmithells et al demonstrated the role of vitamin deficiencies in the origin of neural-tube defects (NTD).He was the first who hypothesized that among triggering environmental factors in the origin of NTDs, undernutrition could be the common and major denominator.In 1964 Hibbard (13) reported a higher rate of CAs (3%) in the infants of folate-deficient mothers than in controls (1.6%) and later Hibbard and Smithells (14) showed a relationship between human embryopathy and a deficiency of folate metabolism. Smithells et al (15) demonstrated the role of vitamin deficiencies in the origin of neural-tube defects (NTD).
5 NTD : AnencephalusAnencephaly and spina bifida (aperta or cystica) are the major classes of NTD followed by occipital encephalocele and craniorachischisis due to the multi-site closure defects of the neural-tube in humans (16). However, it is necessary to differentiate the so-called isolated or non-syndromic and multiple-syndromic NTD (17). In multiple NTD cases, NTD associates with other CAs and are caused by chromosomal aberrations (e.g. trisomy 18), gene mutations (e.g. Meckel-Gruber syndrome) or teratogenic factors (such as valproic acid or diabetes mellitus).Anencephaly is a serious birth defect in which a baby is born without parts of the brain and skull. It is a type of neural tube defect (NTD). These are birth defects that happen during the first month of pregnancy, usually before a woman knows she is pregnant. Anencephaly happens if the upper part of the neural tube does not close all the way. This often results in a baby being born without the front part of the brain (forebrain) and the thinking and coordinating part of the brain (cerebrum). The remaining parts of the brain are often not covered by bone or skin.Unfortunately, almost all babies born with anencephaly will die shortly after birth. CDC estimates that each year, about 1 in every 4,859 babies in the United States will be born with anencephaly.1Scientists believe that many factors such as genes, behaviors, and things in the environment are involved. CDC researchers have reported important findings about some factors that affect the risk for anencephaly:Low intake of folic acid before getting pregnant and in early pregnancy increases the risk of having a pregnancy affected by neural tube defects, including anencephaly.2There has been a 27% decline in pregnancies affected by neural tube defects (spina bifida and anencephaly) since the United States began fortifying enriched grains with folic acid.2Babies born to Hispanic mothers are at an increased risk for anencephaly.3 Reasons for the increased risk among Hispanic mothers are not well understood. DiagnosisAnencephaly can be diagnosed during pregnancy or after the baby is born.During PregnancyDuring pregnancy, there are screening tests (prenatal tests) to check for birth defects and other conditions. Anencephaly would result in an abnormal result on a blood or serum screening test or it might be seen during an ultrasound (which creates pictures of the body).After the Baby is BornIn some cases, anencephaly might not be diagnosed until after the baby is born. Anencephaly is immediately seen at birth. TreatmentsThere is no known cure or standard treatment for anencephaly. Unfortunately, almost all babies born with anencephaly will die shortly after birth.
6 NTD: Encephalocele, occipital Encephaloceles are rare neural tube defects characterized by sac-like protrusions of the brain and the membranes that cover it through openings in the skull. These defects are caused by failure of the neural tube to close completely during fetal development. The result is a groove down the midline of the upper part of the skull, or the area between the forehead and nose, or the back of the skull. When located in the back of the skull, encephaloceles are often associated with neurological problems. Usually encephaloceles are dramatic deformities diagnosed immediately after birth, but occasionally a small encephalocele in the nasal and forehead region can go undetected. Encephaloceles are often accompanied by craniofacial abnormalities or other brain malformations. Symptoms and associated abnormalities of encephaloceles may include hydrocephalus (excessive accumulation of cerebrospinal fluid in the brain), spastic quadriplegia (paralysis of the arms and legs), microcephaly (abnormally small head), ataxia (uncoordinated movement of the voluntary muscles, such as those involved in walking and reaching), developmental delay, vision problems, mental and growth retardation, and seizures. Some affected children may have normal intelligence. There is a genetic component to the condition; it often occurs in families with a history of spina bifida and anencephaly in family members.Is there any treatment?Generally, surgery is performed during infancy to place the protruding tissues back into the skull, remove the sac, and correct the associated craniofacial abnormalities. Even large protrusions can often be removed without causing major functional disability. Hydrocephalus associated with encephaloceles may require surgical treatment with a shunt. Other treatment is symptomatic and supportive.What is the prognosis?The prognosis for individuals with encephaloceles varies depending on the type of brain tissue involved, the location of the sacs, and the accompanying brain malformations.
7 3/a 3/b NTD: spina bifida aperta NTD: spina bifida cystica Spina bifida (SB) is a neural tube defect (a disorder involving incomplete development of the brain, spinal cord, and/or their protective coverings) caused by the failure of the fetus's spine to close properly during the first month of pregnancy. Infants born with SB sometimes have an open lesion on their spine where significant damage to the nerves and spinal cord has occurred. Although the spinal opening can be surgically repaired shortly after birth, the nerve damage is permanent, resulting in varying degrees of paralysis of the lower limbs. Even when there is no lesion present there may be improperly formed or missing vertebrae and accompanying nerve damage. In addition to physical and mobility difficulties, most individuals have some form of learning disability. The types of SB are: myelomeningocele, the severest form, in which the spinal cord and its protective covering (the meninges) protrude from an opening in the spine; meningocele in which the spinal cord develops normally but the meninges and spinal fluid) protrude from a spinal opening; closed neural tube defects, which consist of a group of defects in which development of the spinal cord is affected by malformations of the fat, bone, or meninges; and and occulta, the mildest form, in which one or more vertebrae are malformed and covered by a layer of skin. SB may also cause bowel and bladder complications, and many children with SB have hydrocephalus (excessive accumulation of cerebrospinal fluid in the brain).Is there any treatment?There is no cure for SB because the nerve tissue cannot be replaced or repaired. Treatment for the variety of effects of SB may include surgery, medication, and physiotherapy. Many individuals with SB will need assistive devices such as braces, crutches, or wheelchairs. Ongoing therapy, medical care, and/or surgical treatments may be necessary to prevent and manage complications throughout the individual's life. Surgery to close the newborn's spinal opening is generally performed within 24 hours after birth to minimize the risk of infection and to preserve existing function in the spinal cord.What is the prognosis?The prognosis for individuals with SB depends on the number and severity of abnormalities. Prognosis is poorest for those with complete paralysis, hydrocephalus, and other congenital defects. With proper care, most children with SB live well into adulthood.3/a3/b
9 NTD: spinal dysraphism Spinal Dysraphism is a type of neural tube defect referred to as NTD. It is the term used to describe a group of irregularities in the spine that develops before birth. These anomalies are the result of a closing of the neural tube early in their fetal life or an abnormal development of the caudal cell mass. Spinal Dysraphism comes in two forms, open and closed. The open form is called Spina Bifida Cystica while the closed form is called Spina Bifida Occulta. The effects of a Spinal Dysraphism disfect range from mild to severe. Some of the common indicators of this condition include an irregularity in the structure of the midline of the back, neural arch deficiences, skin defects, and failure in the nerves, and spinal cord. The most severe effects can result in a total failure of the neural tube and an exposure of the spinal cord at the surface. When dealing with the closed form of Spinal Dysraphism, "occult" means "hidden" and means that most signs are not clearly visible in a newborn. The effects of the Dysraphism do not usually become apparent until the baby grows and unfortunately by this time, cord tethering has already taken place, causing permanent and irreversible neurological damage. Beginning signs of Dysraphism include deep dimples in the lumbo-scral area, an atypical hairy patch of skin located on the back, a mass such as a lipoma on the midline of the back, a "tail" that projects from the lower back, a skin marking that resembles a cigarette burn, and anorectal abnormalitities. Open form of Spinal Dysraphism is often the most immediately recognizable and produces a great deal of abnormalities. These abnormalities that are associated with Spinal Dysraphism include Myelocele, Meningocele, and Myelomeningcocele. Myelocele is a sac that projects through the faulty neural arches and contains only the spinal cord. Meningocele is the term for an opening in the spine. Through this opening, a fluid-filled sac sticks out. This usually has a very slight adverse affect and is simple to deal with. The most complicated abnormality associated with Spinal Dysraphism is a condition called Myelomeningocele. This is a condition where the neural arches are not successful in closing during the embryonic stage. This exposes the nervous and the meningeal tissue of the spinal cord and spinal canal causing a great deal of problems and complications. These complications include motor damage as well as weakness, incontinence, and disability. Disabilities that can result include paralysis, bladder and bowel dysfunction, weakness, kidney and liver damage, scoliosis, hip dislocations, tethered spinal cord, and other orthopaedic problems. Most couples who give birth to a child with Spinal Dysraphism do not have a family history of the disease. The cause of Dysraphism is unknown but is considered to be caused by a multitude of factors including genetics factors and environmental factors.
10 Characteristics of NTD Origin of isolated NTDs (92% of all cases) can be explained by gene-environmental interaction.Polygenic predisposition: fact that recurrence in first degree relatives is 10 times higher than their occurrenceEnvironmental factors: very wide range ( per 1000) of NTD incidences in different populations, rapid secular changes and seasonal variation of births with NTD’s were observed. Socio-economic status dependence (a low risk in the highest class to an above-average risk in the lowest class) which was found in several populationsEarly critical period: between 15th and 28th postconceptional days, this explains the use of "periconceptional supplementation".Estimated annual number of cases affected with NTD throughout the world is about 400,000The origin of isolated NTDs (92% of all cases) can be explained by gene-environmental interaction. The most obvious proof for the genetic (polygenic) background of isolated NTD is the fact that recurrence in first degree relatives is 10 times higher than their occurrence (17). However, the occurrence of NTD is modified by environmental factors as well, e.g. there is a very wide range ( per 1000) of NTD incidences in different populations, rapid secular changes and seasonal variation of births with NTD were observed (18). An important indicator for the great sensitive of NTD regarding environmental factors is its socio-economic status dependence (a low risk in the highest class to an above-average risk in the lowest class) which was found in several populations (18).The estimated annual number of cases affected with NTD throughout the world is about 400,000 (18).Our topic includes the prevention of isolated NTD cases.
11 Periconceptional vitamin supplementation: Commence 28 days prior to conceptionContinue until the second missed MP21-26 days). In general, the closure of neural-tube occurs between postconceptional days 15 and 28 (i.e. between gestational days 29 and 42 calculated from the first day of the last menstrual period) in humans, thus the so-called critical (the most sensitive/vulnerable) period of anencephaly and spina bifida is between postconceptional days and 23-28, respectively. The correction of undernutrition needs some time, therefore dietary supplementation should commence at least 28 days prior to conception and continue to the date of the second missed menstrual period explaining the new term “periconceptional vitamin supplementation”. The major practical problem is that the start of the closure of neural-tube overlaps with the first missed menstrual period, thus women, who do not plan and prepare their conception, are unaware of their pregnancy at the “critical period” of NTD. Periconceptional vitamin supplementation therefore needs the deliberate preparation of conception, i.e. family planning.menstruationconceptionClosure of neural tubes
12 Hungarian Periconceptional Service (HPS) 1984. The Hungarian Periconceptional Service (HPS) was launched in 1984 by A.E.Czeizel.It embraces all the ethods for the prevention of structural birth defects (i.e. congenital abnormalities) and pre-term birth known at that time.prefer to use the term “periconceptional” rather than “preconceptional”The most sensitive and vulnerable early period of fetal development, is not covered by the standard medical health service, leaving embryos uncared for and in general unprotectedProf. A.E. CzeizelThe Hungarian Periconceptional Service (HPS) was launched in 1984 (Czeizel, 1999). It embraces all the methods for the prevention of structural birth defects (i.e. congenital abnormalities) and pre-term birth known at that time. We prefer to use the term “periconceptional” rather than “preconceptional” because prenatal care usually begins between the 8th and 12th weeks of pregnancy. Thus the most sensitive and vulnerable early period of fetal development, from the 5th to the 10th gestational week calculated from the first day of the last menstrual period (i.e. from the 3rd week post-conception until the 8th week) is not covered by the standard medical health service, leaving embryos uncared for and in general unprotected
13 The three stages of the Hungarian Periconceptional Service, and activities undertaken at each stage 1) Reproductive Health check-upa) Family history of prospective mother and father, and obstetric history of females.b) Case history and available medical records of females, e.g., epilepsy, diabetes,c) Vaginal and cervical smear screening for sexually transmitted infections/disorders.d) Sperm analysis to detect subfertility and pyosperm (i.e. pus cells in the semen as indicators of sexually transmitted infections)e) Psychosexual assessment.f) Blood screening of women to detect rubella seronegativity, or lack of previous exposure to varicella (vaccination will be offered), or HIV positivity. In addition, carrier screening for cystic fibrosis, and, more recently, predictive genetic diagnostic tests are carried out at this stage.The HPS begins 3 months before a pregnancy is planned, and continues for the first three months after conception. It comprises the provision of information and counselling, examinations and interventions in three stages by qualified nurses (Table 1).I) The Reproductive Health Check-Up is carried out prior to conception. This involves screening for risk identification and assessment, and couples or persons judged to be at high risk being referred for appropriate secondary care.
14 The three stages of the Hungarian Periconceptional Service, and activities undertaken at each stage 2) The 3-month preparation for conception perioda.) Protection of germ cells: avoidance of tobacco, alcohol or narcotic consumption, and taking of unnecessary drugs.b) Discontinuation of oral contraception, and removal of IUDs (condoms are provided).c) Occupational history of femalesd) Menstrual history; measurement of basal body temperature for detection of hormonal dysfunction (and commencement of further investigation and treatment, if necessary).e) Start of pre-conceptional multivitamin supplementation.f) Recommendation that dental status be checked.h) Guidelines for physical exercise.i) Guidelines for healthy dietPreparation for conception. This three-month period allows sufficient time for the cessation of smoking and alcohol consumption, and the taking of narcotic or recreational drugs that are hazardous to germ cells, and later, the fetus. This is also the optimal time to commence periconceptional folic acid or multivitamin supplementation
15 The three stages of the Hungarian Periconceptional Service, and activities undertaken at each stage 3) Better protection of early pregnancya) Undertaking of all additional investigation/treatment necessitated by conditions and disorders detected at the pre-conception check-up.b) Appropriate investigation and treatment of women shown to suffer from hormonal dysfunctionc) Optimal timing of conception in relation to ovulation.d) Early pregnancy confirmation using pregnancy tests and ultrasound scanning.e) Post-conceptional multivitamin supplementation.f) Avoidance of teratogenic and other risks.g) Referral of pregnant women to prenatal care clinics.The better protection of early pregnancy. Women are asked to consult the HPS immediately after the first missed menstrual period, for confirmation of conception by a sensitive pregnancy test and ultrasound scanning. They are asked to continue taking folic acid or multivitamin supplements until the 14th gestational week, when they are referred for prenatal care, taking with them a discharge summary from the HPS.
16 Data and Results of Previous Intervention Studies for the Reduction of Recurrent NTD Based upon the results of the MRC Vitamin Study, the Centers for Disease Control (CDC) in 1991 recommended daily supplementation of diet with 0,4 mg of folic acid under medical supervision in the periconception period for women at high risk (i.e. who had one or more previous offspring with NTD) for the reduction of NTD recurrence.R. W. Smithells ( ) was the first who hypothesized that among triggering environmental factors in the origin of NTDs, undernutrition could be the common and major denominator. His group tested therefore the effect of diet supplemented with folic acid and some other vitamins in the first intervention study .The final results of the Smithells’ study were published separately for the Yorkshire region of the UK (20) and Northern Ireland (21), and they found 91% and 83% reduction in NTD recurrence (Table 1), respectively. However, their results were not accepted by some experts due to possible selection bias because in general more educated people are willing to take part in intervention trials and these women have a lower risk for NTD. Two ethical committees refused to give permission for the original protocol of a randomized controlled trial of the Smithells’ study, thus the control group was made up of women who had had one or more previous infants with NTDs and were already pregnant when referred to the study centers or women who declined to take part in the intervention portion of the trial.Thus in the early 1980s, the Medical Research Council (MRC) in the UK (22) decided to organize a multicenter (43% of participants came from Hungary) randomized double-blind controlled trial (RCT). There were four supplementation groups (Table 1); one of them was 4 mg folic acid because previously Laurence et al (23) used this dose in their study. The MRC Vitamin Study found that a pharmacological dose (4 mg) of folic acid supplementation alone can reduce NTD recurrence significantly by 71%, (0.8% vs. 4.3%; RR with 95% CI was 0.29, ). There was 40% reduction in the recurrent NTD after the use of other vitamins (2.6% vs. 4.3%) but it did not exceed the level of significance.Based upon the results of the MRC Vitamin Study, the Centers for Disease Control (CDC) (24) in 1991 recommended daily supplementation of diet with 4 mg of folic acid under medical supervision in the periconception period for women at high risk (i.e. who had one or more previous offspring with NTD) for the reduction of NTD recurrence.19. Smithells RW, Sheppard S, Schorah CJ, et al. Possible prevention of neural tube defects by periconceptional vitamin supplementation. Lancet 1980; 1:20. Smithells RW, Sheppard S, Wild J, Schorah CJ. Prevention of neural tube defect recurrences in Yorkshire: final report. Lancet 1989; 2:21. Nevin NC, Seller MJ. Prevention of neural tube defect recurrences. Lancet 1990; 1:
17 Goals of the Hungarian randomized double-blind controlled trial (RCT) About 95% of women with NTD offspring have no previous NTD pregnancies.- Thus the question is whether the periconceptional folic acid- containing multivitamin supplementation can reduce the first occurrence of NTD?The pharmacological dose (> 1 mg, e.g., 4 mg) of folic acid cannot be recommended for the population at large or without medical supervision.- Thus, the question is whether a physiological dose (< 1 mg) is effective or not?Possible other beneficial or adverse effects of periconceptional multivitamin supplementation.There were two major questions following the publications of the above two 'recurrence' studies. The first question was: "Does folic acid or folic acid-containing multivitamin supplementation also reduce the risk of first occurrence of NTD?” About 95% of women who deliver infants with isolated NTD have no previous NTD pregnancies, thus the prevention of first occurrence of this CA group would be a real public health success. The second question was connected with the dose. The pharmacological dose (e.g. 4 mg) of folic acid may have some adverse effects, thus it cannot be recommended for the healthy pregnant women without previous NTD offspring and/or without medical supervision. The Hungarian RCT attempted to provide data to answer these questions.
18 Composition of Supplements The Hungarian RCT was carried out at the HPS co-ordinating centre in Budapest, using a multivitamin supplement (Elevit prenatal) containing 12 vitamins including folic acid (0.8 mg), vitamin B12 (4.0 μg), B6 (2.6 mg), B2 (1.8 mg), C (100.0 mg), four minerals and three trace elements (copper, manganese and zinc). The Ethical Committee would not sanction use of a true placebo, and accordingly a placebo-like combination of trace elements (including the three trace element components included in the formulation of Elevit prenatal) was used. The Hungarian RCT was launched on 1st February 1984, and recruitment ceased on 30th April Pregnancy outcomes, and in particular, data on informative offspring (livebirths, stillbirths and malformed fetuses prenatally diagnosed and terminated) were evaluated until the end of April  The total data set of the Hungarian RCT is available: Czeizel AE: Randomized Controlled Trial of Multivitamin Supplementation on Birth Defects and Pregnancy Outcomes, Complementary and Alternative Medicine Data Archive, Data Set 16. October Sociometric Corporation, National Institute of Health
19 Result of the Hungarian RCT: Reduction of the First Occurrence of NTD Centers for Disease Control and Prevention, Atlanta, USA, were interested in our trial, and they organized a Scientific Advisory Committee, of which Dr. Smithells was a member, to assist in the evaluation of our data. We found no NTD cases amongst the informative offspring in the ‘multivitamin’ group, but 6 NTD cases among the informative offspring of the ‘trace-element’ group (p = 0.01) (Table 2). Thus, the Hungarian RCT demonstrated that a multivitamin containing 0.8 mg of folic acid prevented around 90% of primary NTDs
20 Based upon the Hungarian RCT and some observational studies, the CDC in September 1992 recommended that "all women of childbearing age who are capable of becoming pregnant should consume 0.4 mg of folic acid per day for the purpose of reducing their risk of having a pregnancy affected with spina bifida or other NTD” and this recommendation was subsequently followed by several countries.In 1992, on the basis of these results, the US Public Health authorities recommended periconceptional folic acid (0.4 mg) supplementation for all women seeking to become pregnant .However, at that time there was no scientific evidence to support the recommended dose (in the Smithells study, 0.36 mg folic acid was a component of the multivitamin preparation used, and had not been individually tested). Subsequently the efficacy of this amount of folic acid in preventing primary NTDs was evaluated in a Chinese intervention study (Berry et al., 1999). These workers found that folic acid (0.4 mg) daily was sufficient to reduce the risk of NTD in areas with a high rate of NTD (6.5 per 1000) by about 79%, while in areas with low rates of NTD (0.8 per 1000) NTDs were reduced by 41%.CDC. Recommendations for the use of folic acid to reduce the number of cases of spina bifida and other neural tube defects. MMWR 1992; 41:
21 Categories of CAs Group of CAs Number and rate (per 1000) of different CA-groups in multivitamin and no multivitamin supplemented groupCategories of CAs Group of CAsMultivitamin (N=2,471)No multivitamin (N=2,391)RR (with 95% CI)No.RateIsolated CAs NTD Orofacial clefts Cardiovascular CAs CAs of urinary tract Limb deficiencies Cong. pyloric stenosis Others0.07 (0.04, 0.13) 0.77 (0.22, 2.69) 0.42 (0.19, 0.98) 0.21 (0.05, 0.95) 0.19 (0.03, 1.18) 0.24 (0.05, 1.14) 0.68 (0.37, 1.10)Multiple CAs104.05125.020.81 (0.36, 1,26)Total5120.649740.570.53 (0.35, 0.70)Furthermore the Hungarian RCT also generated an unexpected finding. Periconceptional multivitamin supplementation was also associated with a significant reduction in the rate of informative offspring with congenital abnormalities (CA). The rate of major CAs was 20.6 per 1000 in the ‘multivitamin’ group, and 40.6 per 1000 in the ‘trace-element’ group (RR = 0.53, 95% CI: ). When 6 NTD cases were excluded, this difference in the rates of major CA between the two study-groups remained very highly significant (p < ). Thus, periconceptional multivitamin supplementation reduced not only the occurrence of NTD, but also the rate of other major CA. Dr. Smithells encouraged me to publish these results as soon as possible (Czeizel, 1993).The detailed analysis of the final data set from the Hungarian RCT, which was based on our personal medical examination of all children born to participants in the RCT, indicated a significant reduction in two further groups of CA: those of the urinary tract and the cardiovascular system (Czeizel, 1996) (Table 3). The reduction was most marked in the case of obstructive CAs of the urinary tract and conotruncal cardiovascular malformations, including ventricular septal defects (3 vs. 10, RR: 0.29, 95% CI: ). There was also some reduction in the prevalence at birth of congenital limb deficiencies, congenital pyloric stenosis and Down’s syndrome in the ‘multivitamin’ group, but this difference between the two groups in the RCT was not significant. There was no difference in the rate of cases with unclassified multiple CAs in the ‘multivitamin’ and ‘trace element’ groups.
22 OTHER EXPERIENCES OF THE HUNGARIAN RANDOMIZED CONTROLLED TRIAL Female cycle become more regularNo difference between sexual activity7% higher rate of conceptionsTime to become pregnant was slightly but significantly shorterSignificantly lower rate of severe morning sickness, neusea vomiting in pregnancy (3,0 vs 6,6%)No difference in maternal weight gainConstipation (1,8 vs 0,8%) diarrhoea (1,4 vs 0,4%) more oftenMultiple birth was 40% higher in multivitamin groupNo significant difference in fetal deaths ( biochemical PR, ectopic PR, miscarriages, stillbirths), somewhat higher in multivitamin group ( no terathanasia- multiple PR)Sex ratio showed slightly girl excess vs 51% boy predominanceNo difference in gestational age at birth, and birth weightNo difference in postnatal somatic and mental development until 6 yrsThe great majority of the women in the Hungarian RCT were healthy and not malnourished (49), therefore this RCT was appropriate to study several other effects of periconceptional folic acid-containing multivitamin supplementation.During the preconceptional multivitamin supplementation the female cycle became more regular, i.e. the variance was lower (50). Thus, multivitamin supplementation may have a beneficial effect for women with irregular menstrual cycles.There was no difference in the sexual activity (measured by the rate of weekly sexual intercourse) of couples between the multivitamin and the no multivitamin groups in the preconceptional period (51). However, only women were supplemented and sexual activity is often determined by males.A 7% higher rate of conceptions occurred in women within one year who were treated with the multivitamin preconceptionally compared with those who were not supplemented. The time taken to become pregnant was slightly but significantly shorter in the multivitamin group (52).A significantly lower rate of severe (treated) morning sickness, i.e. nausea and vomiting in early pregnancy occurred after periconceptional multivitamin supplementation (3.0% versus 6.6% in the no multivitamin group) (53).There was no difference in maternal weight gain between the multivitamin and no multivitamin groups before and during early pregnancy (54). In Hungary this possible side effect caused the major concern among females.All other possible side effects were monitored continuously. The number of female participants in the Coordinating Center of the Hungarian Periconception Service was 14,540 until January, 1999 and patients with pernicious anaemia have not been recorded among these reproductive aged women. Of 14,540 female participants, 66 (0.46%) were epileptic and 60 wanted to use multivitamin supplementation. There was no case with multivitamin-related side effects of epilepsy during the periconception period (55). However, a 22-yr-old epileptic woman was treated continuously with carbamazepine, who stopped with our multivitamin intake in the 12th gestational week but used another folic acid (1 mg) containing multivitamin from the 20th week, and she had repeated status epilepticus after this new supplementation parallel with the manifestation of symptoms of systemic lupus erythematosus. Her pregnancy ended in the delivery of stillborn fetus on the 39th gestational week (55). Autoimmune disease of epileptic pregnant women could damage the blood-brain barrier and the pharmacological dose (>1 mg) of folic acid may trigger a cluster of seizures. Of 14,540 female participants, four (0.03%) had severe allergic exanthema, and of these 4, three discontinued the use of multivitamin (all of them had a history of drug induced allergic diseases). Among all other possible side effects, constipation (1.8 vs. 0.8%) and diarrhoea (1.4 vs. 0.4%) were reported somewhat more often after multivitamin supplementation in the preconceptional period (54).The rate of multiple births, namely twins was about 40% higher after periconceptional multivitamin supplementation (56). The higher rate of twin conceptions could not be explained by maternal factors (age, parity) or by a higher rate of infertility drug use (57), and that time in vitro fertilization was not used in Hungary. This finding was confirmed later in the USA (58) and Sweden (59), but not in China (60). However, the prevalence of twins is lowest in the Oriental race (including Chinese population) and Chinese people have an extremely high occurrence of TT genotype of MTHFR-gene (55%) (61), and this genotype associates with a very low dizygotic twin rate (62). These conflicting data stimulated us to check the large material of the Hungarian Case-Control Surveillance of Congenital Abnormalities (HCCSCA), , including 38,151 controls without CA (63). There was a somewhat higher prevalence of multiple pregnancies both after periconceptional high doses (mainly 6 mg) of folic acid and folic acid ( mg) containing multivitamin supplementations (64). A systematic review of the recent literature, July 1994 to July 2006 resulted in the conclusion: “Overall…there is possible evidence for a relationship between periconceptional folic acid intake and increased twinning” (65).There was no significant difference in pregnancy outcomes of singletons including four types of fetal deaths: (i) chemical pregnancies (positive pregnancy test without clinical symptoms of pregnancy later), (ii) ectopic pregnancies, (iii) miscarriages (including the so-called missed abortions or blighted ova), (iv) late fetal death (stillbirths) and livebirth between the multivitamin and no multivitamin groups (66). The rate of all kinds of fetal deaths together was somewhat higher in the multivitamin group than in no multivitamin group, in addition, the sex ratio showed a slight girl excess in the multivitamin group while the well-known 51 % boy predominance was seen in the no multivitamin group. Thus, a small change in the pattern of prenatal selection cannot be excluded. This finding was confirmed in the USA (67) but not in China (68). In my opinion the slightly higher rate of fetal death cannot be explained by terathanasia (69) but – if it is not caused by chance – by the higher proportion of multiple conceptions in the multivitamin group (70) because twin conceptions associate with some excess of the fetal death.There was no difference in gestational age at birth and birth weight, in addition the rate of preterm birth and low birthweight newborns of live-born babies of mothers with or without supplementation of the multivitamin in the periconception period (66).Postnatal somatic (body weight, body length, head circumference) and mental (measured by three tests) development until one (71) and six years (72) of age did not show any significant difference between the multivitamin and no multivitamin groups. Thus, the previously found higher rate of worrying, fussiness and fearfulness in girls born to mothers who previously had NTD infants and were supplemented in the next pregnancy by a multivitamin during the periconceptional period (73) was not confirmed. However, it is necessary to stress that our pregnant women used a folic acid-containing multivitamin until the 12th gestational week, and the major part of brain development is in the second, but mainly in the third trimester of pregnancy. The measurement of mental development of children born to mothers with folic acid or multivitamin supplementation until the end of pregnancy would worth studying.Last but no least there was a significant reduction in the total rate of informative offspring with CA after periconceptional multivitamin supplementation. The rate of major CAs was 20.6 per 1000 in the multivitamin and 40.6 per 1000 in the no-multivitamin group (RR = 0.53, 95% CI: ) (74). After the exclusion 6 NTD cases, the difference in the rate of major CAs between the two study groups remained very highly significant (p0.0001). In conclusion, periconceptional multivitamin supplementation reduced not only the occurrence of NTD but also the rate of other major CAs (75).The final data set of the Hungarian RCT indicated a significant reduction beyond NTD in two other CA groups: CAs of the urinary tract and cardiovascular CAs (76) (Table 3). The difference was most obvious in the obstructive CAs of the urinary tract and conotruncal CAs (3 vs. 10, RR: 0.29, 95% CI: ) including ventricular sepal defect of cardiovascular CAs. There was also some reduction in the prevalence at birth of congenital limb deficiencies, congenital pyloric stenosis and Down’s syndrome, but it did not reach the level of significance between the multivitamin and no-multivitamin groups (Table 3).
23 Hungarian Cohort-Controlled Trial ( CCT ) of Periconceptional Multivitamin Supplementation Supplemented cohort:Participants of the Hungarian Periconceptional Service (HPS) with the same multivitamin use(0.8mg folic acid), until 14th week of gestationNo. of participants:3056Unsupplemented cohort:Participants of regional Antenatal care, matched to age socioeconomic status and region, without folic acid /multivitamin supplementation use after 14th week of gestation3056For ethical reasons, were unable to continue the Hungarian RCT. Therefore a cohort controlled trial (CCT) was designed to collect further data, in order to confirm or reject the efficacy of periconceptional folic acid-containing multivitamin supplementation in preventing CAs other than NTD.Supplemented women were recruited via the HPS co-ordinating centre in Budapest, and the 31 HPS subsidiary centres between 1st May 1993 and 30th April The examination of newborn infants with CAs was carried out until 30th April 1999, to allow for a twelve-month infant follow-up period. All participants were supplemented with the same folic acid (0.8 mg)-containing multivitamin (Elevit prenatal) during the periconceptional period. Women in the supplemented cohort were followed until the 14th week of gestation, while a cohort of unsupplemented women was recruited from the Regional Prenatal Care Clinics at the 14th week of pregnancy. None of these women had received supplementation with folic acid, folic acid-containing multivitamins, or any multivitamins either before conception or in the first trimester of pregnancy. Women in the unsupplemented cohort were matched to each pregnant woman in the supplemented cohort on the basis of age, socio-economic status and place of residence. As in the Hungarian RCT, the informative offspring of women from both cohorts were evaluated for CAs at three stages (during pregnancy, at birth, and during the first year of life).
24 Reduction of NTD by periconceptional folic acid-containing multivitamin supplementation in two Hungarian intervention studiesIntervention studiesSupplementedUnsupplementedRandomized controlled trial No. of informative offspring No. of NTD offspring RR (95% CI)2,471 02,391 6Cohort controlled trial No. of informative offspring No. of NTD offspring OR (95% CI)3,056 13,056 9Together No. of informative offspring No. of NTD offspring OR (95% CI)5,527 15,447 150.06 (0.04,0.13)0.11 (0.01,0.91)The CCT confirmed the protective effect of folic acid-containing multivitamin supplementation, and its role in reducing the incidence of NTDs: we found one NTD among the informative offspring in the supplemented group, and nine NTDs among informative offspring in the unsupplemented cohort0.08 (0.01,0.47)
25 Number of informative offspring with cardiovascular CAs in multivitamin (MV) and no multivitamin (No-MV) groupsCardiovascular CAsRCTCCTPooled dataMV (N=2,471) No.No-MV (N=2,391) No.MV (N=3,056) No.No-MV (N=3,056) No.MV (N=5,527) No.No-MV (N=5,447) No.Conotruncal Ventricular septal defect Others Subtotal2 1 38 2 105 3 87 4 11Others710233040Total2031504170OR (with 95% CI)0.42 (0.19, 0.98)0.60 (0.38, 0.96)0.57 (0.39, 0.85)the incidence of other CAs in the supplemented and unsupplemented groups. Cardiovascular CAs are heterogeneous in their origin and manifestation, and have different critical periods, but their total occurrence (31 vs. 50) was again significantly reduced in the supplemented cohort. This can be explained mainly by the reduction in the number of ventricular septal defects (5 vs. 19) (OR: 0.26; 95% CI: ) in the supplemented cohort.
26 Number of informative offspring with urinary tract’s CAs in multivitamin (MV) and no multivitamin (No-MV) groupsCAs of urinary tractRCTCCTPooled dataMV (N=2,471) No.No-MV (N=2,391) No.MV (N=3,056) No.No-MV (N=3,056) No.MV (N=5,527) No.No-MV (N=5,447) No.Renal a/dysgenesisCystic kidneyObstructive CAs Pelvicureteric Others Subtotal10 1 134 1 522 8 102 9 11Total914191628OR (with 95% CI)0.21 (0.05, 0.95)0.71 (0.33, 1.50)0.50 (0.30, 1.04)Urinary tract CAs are also very heterogeneous in their origin and manifestation, but there was no significant difference in their occurrence between the supplemented and unsupplemented cohorts (14 vs. 19 cases). However, within the subgroup of obstructive CAs (10 vs 19), stenosis of the pelvicoureteric junction (2 vs. 13) showed a significant reduction in the supplemented cohort (OR: 0.19; 95% CI: ).
27 Number of informative offspring with other „candidate” CAs in multivitamin (MV) and no multivitamin (No-MV) groupsOther „candidate” CAsRCTCCTPooled dataMV (N=2,471) No.No-MV (N=2,391) No.MV (N=3,056) No.No-MV (N=3,056) No.MV (N=5,527) No.No-MV (N=5,447) No.Orofacial cleftsCleft lip ± palate43275Posterior cleft palate1Total8OR (with 95% CI)0.77 (0.22, 2.69)1.63 (0.31, 28.8)0.99 (0.37, 2.63)Limb deficiencies0.19 (0.03, 1.18)0.33 (0.01, 3.71)0.25 (0.05, 1.16)Cong. pyloric stenosis100.24 (0.05, 1.14)0.00 (0.00, 26.8)0.20 (0.04, 0.90)The causes and manifestation of limb reduction defects are many and heterogeneous, and there was again a trend towards reduction in incidence in the supplemented cohort (1 vs. 3): it is perhaps worth mentioning that all children in the unsupplemented cohort had unimelic terminal transverse type defects. Congenital pyloric stenosis was diagnosed in two infants of the unsupplemented cohort but was not found in the supplemented cohort. There was no reduction in the incidence of cleft lip + palate or cleft palate in the supplemented group.
28 Congenital limb deficiencies 3 0 Congenital pyloric stenosis 0 1 Other observational studies regarding periconceptional (folic acid containing) multivitamin supplementation“Other” CAs Association confirmed refusedCardiovascular CAsCAs of urinary tractCongenital limb deficiencies 3 0Congenital pyloric stenosis 0 1The efficacy of folic acid-containing multivitamin supplementation in preventing cardiovascular CAs (mainly conotruncal defects eg common truncus, transposition of the great vessels and tetralogy of Fallot, and certain types of ventricular septal defects) was also demonstrated in two US studies (Botto et al., 1996; Botto et al., 2000). Their results, together with our results from the RCT and CCT trials, suggest that periconceptional multivitamin supplementation was associated with an approximately 40% reduction in risk for cardiovascular CAs. The effect of early postconceptional supplementation with a pharmacological dose (6 mg) of folic acid alone in protecting against cardiovascular CAs was also discernible in the data set of the Hungarian Case-Control Surveillance of Congenital Abnormalities (HCCSCA) (Czeizel et al., 1996). Recently, two groups reported raised plasma homocysteine levels and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms in association with cardiovascular CA (Kapusta et al., 1999, van Beynum et al., 2006).
29 Metabolism of Homocysteine and the Effect of Folate-Folic Acid (Vitamin B11), Vitamin B2, Vitamin B6 and Vitamin B12MTHFR-geneVitamin BFolate(polyglutamate)Folic acid(monoglutamate)11Reductase5-methyl-THF5,10-methylene-THFVitamin CTetrahydrofolate =THFDihydrofolateMonoglutamateZincConjugaseMethylene-THF-reductase=MTHFRB2ProteinsMethionineS-adenosylmethionineHomocysteineHomocystinuriaCystathioneCysteineSulphate6Cystathione-betasynthaseSerinCystathionase12Methionine-CH+3The available epidemiological and biochemical findings suggest that the origin of NTD is not primarily the lack of sufficient folate in the diet, but arises from genetically determined changes in the uptake, metabolism, or both in maternal and, particularly, fetal cells (30). Thus, a gene-environmental interaction between vitamin dependency (e.g. an inborn error of folate metabolism) and nutrition (such as dietary deficiency) may have a causal role in the origin of NTD. Supplementation with folic acid alone or folic acid-containing multivitamins may cause an increase in folate metabolite concentrations of tissue fluids and it may overcome the failure of the local folate metabolite supply.Obviously some genes and several environmental factors contribute to the origin of NTD. Here only one – the most established – hyperhomocysteinemia (31, 32) is discussed, because the primary prevention of NTD by folic acid or folic acid-containing multivitamin is partly connected with the reduction of hyperhomocysteinemia.The causes of hyperhomocysteinemia need a longer explanantion. When meat, fish or plant proteins are digested, amino acids, among others, methionine, are released. (See Figure 1 in Chapter 8.) There is a multistep conversion of methionine to homocysteine. Homocysteine is a toxic metabolite; therefore humans neutralize it normally as soon as possible. On the one hand, homocysteine can be metabolized via the transsulfuration pathway to form cystathionine caused by the condensation with serine and catalyzed by cystathionine -synthase. This enzyme requires pyridoxal 5'‑phosphate, the biologically active form of vitamin B6 as a cofactor. The final product of this metabolic pathway is cysteine. On the other hand, remethylation of homocysteine to methionine is catalyzed by methionine-synthase. This enzyme requires vitamin B12 as a cofactor and 5-methyl-tetrahydrofolate as the methyl donor. The latter explains the importance of folate-folic acid deficiency in the origin of NTD.This vitamin was discovered by Lucy Wills (33, 34) in 1931, and she stressed the similarity of this "curative agent" with vitamin B12, as a “twin” vitamin. The generic term folate describes the many different naturally occurring polyglutamate forms of the vitamin, whereas folic acid is the synthesized monoglutamate form, chemically designated as pteroylglutamic acid. The different name of two forms of the same vitamin is disturbing and are confused frequently, thus their joint name as vitamin B11 has been used in some countries, e.g. the Netherlands, while their name is vitamin B9 in France. Folate is required for cell division and cell maintenance, because it acts as a co-enzyme in the transfer and processing of one-carbon units and plays an important role in nucleotide (thymidine) synthesis which is essential for the de novo construction or repair of DNA. In addition folate is key factor in the “site-specific” methylation of the cytosine base in DNA, which regulates gene expression. Here only the third major function of folate, the remethylation of plasma homocysteine to methionine will be summarized.Humans cannot produce folate. The major dietary sources of folates are fresh and frozen green leafy vegetables, citrus fruits and juices, liver, wheat bread and legumes. Food folates are converted to monoglutamates by conjugase enzymes in the upper part of the small intestine. Monoglutamate folic acid, however, can be absorbed directly. After the active and passive absorption of monoglutamates, they are converted to dihydrofolate and then to tetrahydrofolate (THF) by reductase enzymes. THF is the parent compound of all biological active folates. The most important cause of hyperhomocysteinemia and/or lack of methionine are the polymorphism of methylene-THF-reductase (MTHFR) gene (35). A 677CT mutation has been identified in the MTHFR gene and it results in thermolabile variant (Ala225Val) of MTHFR enzyme with 30-50% activity of the CT (heterozygote) and with very low (less than 20%) activity of TT (homozygote) forms (36). These enzyme variants cannot effectively catalyze the pathway of 5,10,methylene-THF to 5,methyl-THF, i.e., the methyl donor for methionine-synthase. The MTHFR gene polymorphism is common in different populations (37). There are several studies that provide evidence for the confirmation of hyperhomocysteinemia-related NTDs.
30 MTHFR gene Gene location: Chromosome 1, short arm 36.3 Mutation: 677 C TFrequency ofmutant homozygosity (TT): % (11%)heterozygosity (CT): % (45%)A 677CT mutation has been identified in the MTHFR gene and it results in thermolabile variant (Ala225Val) of MTHFR enzyme with 30-50% activity of the CT (heterozygote) and with very low (less than 20%) activity of TT (homozygote) formsa) The thermolabile variants of MTHFR enzyme, thus the polymorphism of MTHFR gene is an obvious risk factor for NTD (38, 39). The Hungarian population has a moderate total (birth + fetal) prevalence of NTD (3/1000) (40) while the frequency of TT and CT genotypes of MTHFR genes occur in 11.1% and 45.2% of people, respectively (37). These very high frequencies need some explanation. The highly significant excess of CT heterozygosity in male first-degree relatives of patients with NTD may demonstrate CT heterozygote advantage in the context of their genetic background (41). However, other common mutations of MTHFR gene were identified (e.g. A1298C) and these genes polymorphisms may also have a role in folate metabolism, and have an interaction in the origin of NTD (42). The point is that there is an inverse relation between plasma homocysteine and folate levels in the humans. However, the plasma/serum folate level reflects the recent intake of folate/folic acid, the concentration of folates in red blood cells represents the body storage over the last 7 weeksb) Hyperhomocysteinemia and/or lack of methionine can induce NTD in animal experiments (43, 44).c) Mothers who gave birth to a child with NTD have higher blood (45) and amniotic fluid (46) levels of homocysteine.d) Low maternal folate status, particularly in the first trimester was a strong risk factor for NTD (r = 0.99) in the MRC Vitamin Study (29).e) Folic acid-containing multivitamins and folic acid alone are able to reduce the hyperhomocysteinemia, and consequently the hyperhomocysteinemia-related NTD as it was shown in the previous subchapter.However, the polygenic system in the origin of NTD includes other folate-independent factors as well. For example inositol deficiency leads to NTD in mice, and prevent NTD in curly tail mice, in addition the mothers of NTD offspring had lower inositol concentration than controls (47). The latter may explain the lack of NTD prevention by folic acid or folic acid-multivitamins in some cases.
31 Optimal Dosage ? Good 400 microgram of folic acid Better BestMultivitamin containing 800 microgram folic acidThe second question concerns the optimal dose of folic acid. At present there is no unequivocal evidence for, or universal consensus regarding, the optimal dose of folic acid. Folic acid has two forms (known as vitamin B11 in the Netherlands, and vitamin B9 in France): dietary polyglutamate folate and synthetic monoglutamate folic acid. These two forms have different (active and passive) modes of absorption from the gastrointestinal tract, and their levels have to be combined when calculating the dose of folic acid. The Institute of Medicine, the US National Academy (1998) and the European Commission Scientific Committee on Food (1998) all recommend 1 mg folate/folic acid as the highest amount suitable for ingestion by healthy individuals, including pregnant women. It is important, therefore, to make a distinction between a physiological dose of folic acid (less than 1 mg daily), being taken by healthy individuals for preventive purposes, and a pharmacological dose (more than 1 mg daily), used to treat patients
32 Comparison of different preventive approaches of NTD There are two pubic health tasks to help prospective pregnant women use ofpericonceptional folic acid or multivitamins.The first is a strong and widespread educational campaign, to suggest the start of the use of folic acid or multivitamins immediately after the discontinuation of oral contraceptive pills or other contraceptive methods when couples decide to have a baby. However, unfortunately these campaigns have had only a limited success in all countries (155).The second important task is to establish a network of pre-or periconceptional care within the primary heath care (* incl. termination of pregnancy
33 Conclusion I:. Periconceptional multivitamin (containing 0 Conclusion I: Periconceptional multivitamin (containing 0.8 mg folic acid) supplementation1. Very effective (about 90%) for the prevention of NTD2. Effective for the reduction of cardiovascular CAs (ventricular septal defect), CAs of urinary tract (stenosis/atresia of pelvicureteric junction) and congenital limb deficiencies (terminal transverse type)3. No effective for the prevention of orofacial cleft (dose-dependent effect of folic acid alone?)4. Effective for the reduction of total (birth+fetal) prevalence of major CAs at least by one-third
34 Conclusion II:Neural-tube defects are preventable by periconceptional folic acid or multivitamin supplementation.The incidence of some other structural birth defects can also be reduced by folic acid-containing multivitamin use during the periconception period.All women of childbearing age who are capable of becoming pregnant should consume folic and/or folic acid containing multivitamin during the periconception period.The primary prevention of birth defects by periconceptional folic acid/multivitamin supplementation is much better than the so-called secondary prevention, i.e. the termination of pregnancy due to severe fetal defects.Periconceptional care – beyond other benefits – is optimal for the introduction of periconceptional folic acid/multivitamin supplementation.Proper preparation for conception is the earliest and most effective method for the prevention of birth defects.
35 Thus G. P. Oakley is right: “Inertia on folic acid fortification equals public health malpractice”.Oakley GP. Inertia on folic acid fortification: Public health malpractice. Teratology 2002; 66:
36 International Society of Periconceptional Medicine The founding of the first World Scientific Society of Periconeptional Medicine is under process!International Society of Periconceptional MedicineISPMEnvironmental SciencesLifestyle SciencesNutritionDieteticsToxicologyTeratologyPharmacologyBiotechnologyGenetics-EpigeneticsAssisted Reproductive TechnologiesEmbryologyPsychologyRegister online:
37 Thank you for your attention! 1st world congress of theISPM in Budapest, Hungary,2012 !!ispm2012
39 Preventive efficacy of folic acid alone70%?Key factorLowmultivitamin90%At least three other CA-groupsVitamin B12, B2 and B6 are independent factorsModerate (reimbursement)NTDOther CAsOther argumentsin hyperhomocysteinemia related NTDCost
40 Cardiovacular defects: Government of Western Australia, WA Register of Developmental Anomalies source: Department of Health, AU
41 Chromosomal defects: Government of Western Australia, WA Register of Developmental Anomalies source: Department of Health, AU
42 NTD trends: Government of Western Australia, WA Register of Developmental Anomalies source: Department of Health, AU
43 NTD trends: Government of Western Australia, WA Register of Developmental Anomalies source: Department of Health, AU
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