Title page Title: Non-isolated neural tube defects with comorbid malformations are responsive to population-level folic acid supplementation in Northern China Authors

Title page Title: Non-isolated neural tube defects with comorbid malformations are responsive to population-level folic acid supplementation in Northern China Authors Xiaoyu Che, BD 1,2, Jufen Liu, Ph.D. 1,2, *, Gabriel L. Galea,, Ph.D 3, Yali Zhang, Ph.D. 1,2, Nicholas D. E. Greene, Ph.D.3, Le Zhang, Ph.D. 1,2, Lei Jin, Ph.D. 1,2, Linlin Wang, Ph.D. 1,2, Aiguo Ren, Ph.D.1,2, Zhiwen Li, Ph.D. 1,2* Authors affiliation 1 Institute of Reproductive and Child Health / Key Laboratory of Reproductive Health, National Health Commission of the People’s Republic of China 2 Department of Epidemiology and Biostatistics, School of Public Health, Peking University, Beijing 100191, P. R. China 3 Developmental Biology and Cancer Department, UCL Great Ormond Street Institute of Child Health, University College London, London WC1N 1E, United Kingdom *Equal contribution. Corresponding author Zhiwen Li, or Jufen Liu, 38 College Rd, Haidian District, Institute of Reproductive and Child Health, Peking University Health Science Center, Beijing 100191, China. Tel.: +86-10-82801140; Fax: +86-10-82801141. Email: lizw@bjmu.edu.cn; liujufen@bjmu.edu.cn Running head: Comorbid malformations with neural tube defects Funding: This work was supported by the National Key Research and Development Program, Ministry of Science and Technology from P.R. China (Grant No. 2021YFC2701001), and the Natural Science Foundation of China (No.81973056). GLG acknowledges funding from the Wellcome Trust (211112/Z/18/Z) and the NIHR Great Ormond Street Institute of Child Health. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.12.22277037 doi: medRxiv preprint


Introduction
Neural tube defects (NTDs) are severe congenital malformations of the central nervous system (CNS) which affect approximately 1 in every 1,000 birthds globally 1  Some malformations occur secondary to the NTD, such as Chiari II malformation, hydrocephalus, congenital hip dislocation and talipes in individuals who have spina bifida [3][4][5] . Hower, animal studies strongly corroborate the potential for pleiomorphic effects of gene mutations and teratogen exposure on multiple organ systems. For example, mutations in the planar cell polarity pathway can cause NTDs, abdominal wall closure defects and limb reduction defects in mice 6,7 In humans, genetic predispositions contribute to NTD risk, but their penetrance is complex and involves gene/environment interactions. The best known environmental factor is maternal folate status. This is clearly shown by clinical trials and by the dramatic reduction in NTD prevalence in northern China from 12 % in 2004 before folic acid supplementation, to 3.2 % in 2014 after supplementation 8 .
Although most epidemiological studies focused on isolated NTDs, those with comorbidities are also clinically important. In countries where fetal surgery for spina bifida is available, independent comorbid malformations are typically a surgical exclusion criterion 9 When counselling parents whose pregnancy has been affected by a non-isolated NTD 10 , the evidence for a beneficial effect of folic acid is less robust than for isolated NTDs. A previous study in the USA found a progressive reduction in NTD rates between 1992 and 2009, but no significant change in non-isolated NTD All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.12.22277037 doi: medRxiv preprint rates over the same period 11 . In mice, folic acid does not rescue NTDs caused by mutations in some genes 12 potentially changing the spectrum of associated malformations in supplemented populations.
Population differences might begin to explain variability in previous estimates of non-isolated NTD prevalence. The proportion of NTDs described as having comorbid malformations in community-or hospital-based studies ranges from 9.1% to 66% 13-19 .
Our previous pathological anatomy study showed that 75.8% of NTD cases had additional malformations, of which the majority were musculoskeletal or visceral defects 20 . However, the definition of comorbid malformations is not standardised across studies, with those using sensitive methodologies such as fetal autopsy potentially being biased by analysing more severe terminated or non-viable cases.
Additionally, spina bifida in particular is known to cause secondary malformations due to CSF leakage during gestation and musculoskeletal malformations due to deinnervation.
To our knowledge, few studies have described the population-level epidemiological status of non-isolated NTDs, excluding potentially secondary malformations. Therefore, we aimed to assess the epidemiological characteristics of comorbid malformations associated with NTDs in Northern China over a time period spanning introduction of folic acid supplementation.

Birth Defects Surveillance
Five counties in Shanxi province, namely Pingding, Xiyang, Taigu, Zezhou, and Shouyang were included in the population-based birth defects surveillance system in the current study. The system was established in the early 2000s, and more than 20,000 births were recorded each year. All pregnant women who resided in the study area for more than 1 year were monitored. All live births (births of 28 or more complete gestational weeks), all stillbirths of at least 20 weeks' gestational age, and pregnancy terminations at any gestational age following the prenatal diagnosis of NTDs were recorded 21 . NTD diagnosis was conducted by local specialists in All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.12.22277037 doi: medRxiv preprint maternal-fetal medicine and confirmed by pediatricians in Peking University. The study protocol was reviewed and approved by the Institutional Review Board of Peking University.

Case Classification
The present study includes NTDs cases observed between 2002 and 2021. In addition to NTDs, there were 20 types of birth defects monitored, including congenital hydrocephalus, cleft palate and/or cleft lip, congenital ear defects, oesophageal atresia/stenosis, anorectal atresia/stenosis, hypospadias, hydronephrosis, club foot, polydactyly, syndactyly, limb reduction defects, omphalocele, gastroschisis, conjoined twins, Down syndrome, and congenital heart disease, among others. We also included other defects types recorded in the database, such as exposed viscera and cystic hygroma.
All NTD cases were classified as isolated or non-isolated. Congenital hydrocephalus, club foot and congenital dislocation of hip are considered to be secondary to spina bifida and were therefore included in the isolated NTDs cohort, but were considered non-isolated when they occurred with cranial NTDs. Additional defects were classified according to the organ system primarily affected: central nervous system (CNS) defects, craniofacial defects, gastrointestinal system defects, musculoskeletal system defects, abdominal wall defects, urogenital system defects, and others. Due to the possibility of a case having multiple major defects, subgroup categories are not mutually exclusive.

Statistical Analysis
For NTDs, three types of prevalence were calculated: prevalence of total NTDs, isolated NTDs, and non-isolated NTDs. In calculating these indicators, the denominator remains the same: the total number of all live births. For other associated defects, two types of prevalence were calculated: prevalence of associated defects in the NTD group (the numerator is the number of cases with NTDs and associated defects; the denominator is the number of NTD cases) and prevalence of associated All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.12.22277037 doi: medRxiv preprint defects in the non-NTD group (the numerator is the number of cases with associated defects but no NTDs; the denominator is the number of live births without NTDs).
Cochran-Armitage Trend Tests were used to analyze the trend of prevalence of total, isolated and non-isolated NTDs. Chi-square tests were used to compare the effects of the residence of the mother, infant sex, gestational weeks and delivery time on isolated and non-isolated NTDs. Chi-square tests were also used to compare the prevalence of isolated and non-isolated NTDs before and after national folic acid supplementation. Poisson test was used to compare the prevalence of associated defects in NTDs group and non-NTDs group. The types and prevalence of defects associted with NTDs and the prevalence of three subtypes of NTDs combined with other defects were described. Two-tailed p<0.05 was considered statistically significant. All statistical analyses were performed using the R 4.0.2 software.
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The Prevalence, Types and Proportion of Defects Co-occurring with NTDs
From 2002 to 2021, a total of 296,306 births and 2,031 cases of NTDs were recorded in the Shanxi surveillance system. 28.4% (334/1175) of spina bifida cases suffered from defects believed to arise secondarily to their NTD, of which 293 had congenital hydrocephalus, 26 cases had clubfoot, and 15 cases had both congenital hydrocephalus and horseshoe foot. These "secondary" co-morbid malformations were included in the isolated NTD grouping.
Additionally These 97 infants had 105 associated defects (some infants had defects in more than one site). There were six cases of spina bifida with two additional defects (limb reduction defect and gastroschisis; limb reduction defect and cleft palate; limb reduction defect and reproductive system defect; syndactyly and gastroschisis; cleft lip and palate and polydactyly; anorectal atresia or stenosis and reproductive system defect). There was also a case of multiple NTDs (anencephaly and spina bifida), with limb reduction defect and diaphragmatic hernia. Another case of multiple NTDs (anencephaly and encephalocele) had cleft lip and palate and clubfoot. In summary, co-morbid malformations in individuals who have NTDs are uncommon, but can affect multiple organ systems and have the potential to diminish quality of life of individuals who survive.

Comorbid malformations over-represented in individuals with NTDs
We next assessed whether malformations of specific structures are over-represented in individuals who have NTDs, suggesting common genetic or environmental causation.
The prevalence of cleft lip or/and palate (P<0.01), limb reduction defects (P<0.01), hip dislocation (P<0.05, excluding cases which also have spina bifida), omphalocele All rights reserved. No reuse allowed without permission.
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Associations with the three NTD sub-types were also tested individually (Table 1).
Cleft lip and/or palate was more commonly observed in those with spina bifida (P < 0.01) or encephalocele (P < 0.05) than non-NTD controls. Limb reduction defects were more common in those with spina bifida (P < 0.01) or anencephaly (P < 0.01), whereas polydactyly was more common in those with encephalocele (P < 0.01).
Diaphragmatic hernia was more common in those with anencephaly (P < 0.05). Hip dislocation was excluded from analysis in those with spina bifida but was over-represented in those with encephalocele (P < 0.01). Similarly, hydrocephalus was excluded from the analysis of those with spina bifida but was over-represented in those with sub-total anencephaly (P < 0.05) and encephalocele (P < 0.01).
Omphalocele and gastroschisis were over-represented in those with either spina bifida (P < 0.01) or anencephaly (P < 0.01). Urogenital defects were specifically over-represented in those with spina bifida (P < 0.01).

Epidemiological Characteristics of Isolated and Non-isolated NTD Cases
No differences in the distribution of maternal residence, infant sex and gestational weeks were observed between the isolated NTD group and those with NTDs and co-morbid malformations ( Table 2). Individuals with encephalocele tended to be more likely to have additional malformations.

Temporal Trends in the Prevalence of NTDs
We next asked whether non-isolated NTDs follow the same temporal change in prevalence as isolated NTDs which are the primary target of prevention programmes.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.12.22277037 doi: medRxiv preprint The 3-year rolling average curves indicate progressively diminishing prevalence of both isolated and non-isolated NTDs ( Figure 3B and 3C). Cochran-Armitage Trend Test results showed that the prevalence of total NTDs, isolated, non-isolated and multiple NTDs decreased significantly over the past two decades (P<0.01). Both isolated and non-isolated NTD subtypes all showed a significant downward trend, except for non-isolated spina bifida for which this did not reach significance (P>0.05)

Acid Supplementation
The population survailance period inculdes various changes in public policy. The After national folic acid supplementation, the prevalence of all NTD groupings, including both isolated cases and those with co-morbid malformations, decreased significantly (P<0.05) ( Table 3). Non-isolated cases accounted for 4.7% of NTDs before folic acid supplementation and 4.9% of NTDs after supplementation.

Discussion
NTDs remain prevalent and clinically important globally. Surgical advances have tremendously improved outcomes for individuals who have spina bifida, but the priority remains population-wide primary prevention. Cases which occur despite adequate maternal folate status are often assumed to reflect genetic/teratogenic causes not responsive to fortification or supplements. Using population-based surveillance data, this study reveals that 4.8% of NTDs have gross comorbid malformations, but that the epidemiology of non-isolated NTDs is comparable to isolated cases including a reduction in prevalence following population-wide folic acid supplementation. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Opitz et al. 23 suggested that midline structures share a particular common developmental vulnerability. Changes in the central nervous system may affect the lip and palate, diaphragm, heart, abdominal wall and genitalia that share the same midline. Although previous studies 15,16 provide no evidence to support the above two hypotheses, many of the conditions proposed are associated with NTDs in our study population. Compared with non-NTD controls, infants with NTDs were more likely to have cleft lip and/or palate, limb reduction defects, hip dislocation, gastroschisis, omphalocele, hydrocephalus and urogenital system defects.
These associations could be either genetic or teratogenic, if teratogen exposure spans the relevant susceptibility periods. For example, fetal valproate syndrome involves limb reduction defects as well as spina bifida. Genetic causes can also be shared between NTD subtypes and comorbid malformations: Grhl3 Cre deletion of Rac1 can cause abdominal call defects, spina bifida, exencephaly and/or encephalocele in mice 24 . Encephalocele etiology is poorly understood and future work will be needed to explain our observation that they account for a larger proportion of non-isolated NTD cases.
Our results showed that there are no significant differences between isolated and non-isolated NTDs in key demographic characteristics, including residence of the mother, infant sex, subtype of NTDs, gestational weeks and delivery time, which suggested that non-isolated NTDs are more likely to be caused by multiple genetic factors. Due to the serious consequences of non-isolated NTDs, we need to further clarify the cause and identify other ways to reduce the risk of non-isolated NTDs.
Further prospective cohort or case-control studies are needed to detect influences by more factors such as maternal diseases, childbirth history, family history and so on.
An unanswered question is whether folic acid prevents both NTDs and their comorbid malformations, or only the NTDs. Our study showed that the prevalence of all types All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.12.22277037 doi: medRxiv preprint of NTDs decreased significantly after national folic acid supplementation. A recent study in China also showed that periconceptional folic acid use prevents both rare and common NTDs 25 . In our previous study, we found that periconceptional folic acid supplementation can prevent congenital limb reduction defects in people with extremely low folate concentrations in northern China 26 . Some previous studies showed that perinatal use of multivitamin supplements effectively reduced the risk of multiple birth defects, even after excluding NTDs 11 . However, several studies showed that folic acid supplementation can reduce the risk of NTDs, but cannot reduce the risk of other birth defects 27 .
The prevalence of total NTDs, isolated NTDs and non-isolated NTDs in China showed a downward trend, which also reflected the remarkable results of preventing and controlling NTDs in the five counties of Shanxi Province in recent 20 years.
However, when we conducted trend test according to different subtypes, the prevalence of non-isolated spina bifida did not show a downward trend. A previous study in South Carolina showed that the prevalence of total NTDs and isolated NTDs decreased significantly from 1992 to 2009, but the prevalence of non-isolated NTDs did not change significantly 28 . It is worth noting that since the three different subtypes of NTDs may be caused by different genetic factors, it is necessary to analyze the epidemiological trends according to different subtypes. Therefore, our results suggested that there may be etiological heterogeneity between non-isolated spina bifida and isolated spina bifida. A previous epidemiological study of NTDs also showed that isolated NTDs were more sensitive to environmental factors, whereas non-isolated NTDs were sensitive to both environmental and genetic factors 29 . In summary, the etiology of non-isolated NTDs needs to be confirmed by more population studies and laboratory studies. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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an increased risk of NTDs 30,31 . Other studies have found the opposite association, with high birth prevalence of spina bifida among younger mothers 32 . Our study showed that after the implementation of the universal two-child policy, the prevalence of non-isolated NTDs continued to decline. We speculate that there are two possible reasons. First, population-level folic acid supplementation can effectively prevent non-isolated NTDs, so the universal two-child policy had little impact on the prevalence of NTDs. Secondly, the impact of two-child policy on the overall population is not yet clear. We did not observe a rising trend of birth population after 2016 in the five counties of Shanxi Province. It is worth noting that the universal three-child policy was implemented in China in 2021, which means that further research on birth defects is needed to evaluate any impact on health outcomes in pregnancy.
Our study has several strengths. First, the surveillance is population-based and used quality control to ensure data quality. Our previous retrospective study showed that the surveillance covered 95.6% of births 33 . Second, previous to the present study, only a few studies have reported the epidemiological characteristics of isolated and non-isolated NTDs in China. We provided more detailed information about associated defects.
Despite the clear strengths of our study, some limitations should be acknowledged.
First, the study included data from only five counties, and the results were not representative of other provinces or countries. Second, this study did not collect information on demographic and socio-economic factors, which prevented us from exploring the risk factors associated with isolated and non-isolated NTDs. Third, the monitoring system mainly reported external defects, so we may have missed some internal defects.

Conclusion
Epidemiologically, non-isolated NTDs follow similar trends as isolated cases and are responsive to primary prevention by folic acid supplementation. Various clinically-important congenital malformations are over-represented in individuals All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 12, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.12.22277037 doi: medRxiv preprint in China in the era of the two-child policy. BMJ global health. 2020;5:e002157. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.12.22277037 doi: medRxiv preprint  (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.12.22277037 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.12.22277037 doi: medRxiv preprint  Table 2 Characteristics of isolated and non-isolated NTD cases in five counties in Shanxi province of northern China, 2002-2021, N (%) Table 3 Prevalence of isolated and non-isolated NTDs before and after national folic acid supplementation in five counties in Shanxi province of northern China, 2002-2021, N (/10,000) All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.12.22277037 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.