Maternal Vitamin D Levels during Pregnancy and Offspring Psychiatric Outcomes: A Systematic Review

Prenatal exposure to vitamin D may play a significant role in human brain development and function. Previous epidemiological studies investigating the associations between maternal vitamin D status and offspring developmental and psychiatric outcomes in humans have been inconclusive. We aimed to systematically assess the results of previously published studies that examined the associations between maternal vitamin D levels, measured as circulating 25(OH)D levels in pregnancy or at birth, and offspring neuropsychiatric and psychiatric outcomes. Systematic searches were conducted using MEDLINE, Embase, PsychINFO and Web of Science for studies published by 10 August 2022. We included human observational studies that examined associations between prenatal or perinatal vitamin D levels and offspring neuropsychiatric and psychiatric outcomes and were published in English in peer-reviewed journals. Of the 3729 studies identified, 66 studies were screened for full texts and 29 studies published between 2003 and 2022 were included in the final review. There was a small amount of evidence for the association between prenatal vitamin D deficiency and autism spectrum disorder. When studies with larger sample sizes and stricter definitions of vitamin D deficiency were considered, positive associations were also found for attention-deficit/hyperactivity disorder and schizophrenia. Future studies with larger sample sizes, longer follow-up periods and prenatal vitamin D assessed at multiple time points are needed.


Introduction
Vitamin D is essential for bone mineralization and bone mass acquisition [1]. It may also be important for the development of other organ systems including the central nervous system [2]. In the past few decades, the role that vitamin D plays in brain development and function has been explored. Abundant vitamin D receptors have been found in brain regions such as the prefrontal cortex and hippocampus. Vitamin D is important for neuronal differentiation and reducing apoptosis in the hippocampus, the area involved in language and memory [3]. It serves as a transcriptional regulator by expressing genes vital to brain development [4,5].
Vitamin D is crucial especially when the central nervous system of the fetus develops and the brain is sensitive to maternal nutritional deficiencies [6,7]. Previous literature has emphasized the role of early prenatal insults in relation to later mental illnesses. The Dutch famine study was one of the earliest epidemiological studies of this kind and it suggested associations between maternal nutritional deficiencies during pregnancy and the subsequent risk of offspring mental illness. Offspring who were exposed to prenatal nutritional deficiency had a twofold risk of schizophrenia later in life, compared to the unexposed cohort [8].
Furthermore, studies from rodents have suggested morphological changes in the brain due to prenatal exposure to vitamin D deprivation in utero [9]. Animal studies suggest that vitamin D plays an important role in fetal brain development. However, human studies that have investigated the associations between maternal vitamin D status and offspring developmental and psychiatric outcomes in humans have been inconclusive. Previous systematic reviews and meta-analyses have explored associations between prenatal vitamin D and some neurodevelopmental or cognitive outcomes including autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD) [4,10]. The reviews reported contradictory results. The systematic review found inconclusive evidence for offspring neurocognitive and psychological outcomes [4] whereas the meta-analysis reported potentially decreased risks of ADHD and autism-related traits with increased exposure to prenatal vitamin D [10]. The first study only included studies published up to 2014, while the other study did not include any other psychiatric outcomes than ASD and ADHD.
Research interest in the possible prenatal effects of vitamin D has been growing and many studies published in the last five years have emphasized the need to summarize and review the rapidly gathered new information. That is why the main aim of this review was to systematically assess the results of previously published studies that examined the relationship between maternal vitamin D levels, measured as circulating 25(OH)D levels in pregnancy or at birth, and offspring psychiatric outcomes. Previous studies focused on neurodevelopmental disorders and that is why we wanted to broaden the study area to encompass other psychiatric outcomes, including both disorders and symptoms.

Materials and Methods
This systematic review was conducted in accordance with the Preferred Reporting Items of Systematic Reviews and Meta-analyses (PRISMA) [11] and the Synthesis Without Meta-analysis reporting guideline [12]. The review protocol was prospectively registered with The Open Science Framework (osf.io/jt4wa) [13].

Search Strategy
Relevant studies were identified by comprehensive searches of electronic databases from inception until 10 August 2022. We conducted systematic searches in MEDLINE, Embase, PsychINFO and Web of Science by combining terms that covered maternal vitamin D during pregnancy or at birth, the use of serum samples, and offspring psychiatric outcomes. Potentially relevant papers were also searched using the backward snowballing technique, which involves looking at the reference lists of the included papers and screening them to find other possible papers to include. The search strategy was developed and refined after consultation with a library information specialist (Supplementary Materials S1).

Inclusion and Exclusion Criteria
The review included human observational studies that used longitudinal study designs and were published in peer-review journals. The included papers were written in English and measured associations between circulating 25(OH)D levels during pregnancy or from newborns and psychiatric outcomes in offspring. As maternal and neonatal vitamin D levels have been shown to be correlated, we also included studies that measured vitamin D levels in cord blood or neonatal dried blood samples [14]. The outcomes in the included studies reported offspring neuropsychiatric or psychiatric outcomes based on registry diagnoses, diagnostic interviews or symptoms.
We excluded any cross-sectional studies, as well as case reports, conference abstracts, editorials, comments, letters and reviews with no relevant primary data.

Study Selection Procedures
The studies were retrieved from search databases and checked for duplicates. Two reviewers (B.A./T.S., S.W./S.U.) screened the titles and abstracts independently, followed by full-text assessments of the papers based on the inclusion and exclusion criteria. Two reviewers (B.A./T.S., S.W./S.U.) cross-checked the papers and any disagreements were resolved after discussion.

Quality Assessment
The quality assessment of the studies was conducted by two independent researchers (B.A./T.S., S.W./S.U.) using the Joanna Briggs Institute (JBI) critical appraisal tools specific to study designs [15]. The reviewers used the JBI tool with 10 questions for case-control studies and the tool with 11 questions for cohort studies to assess the methodological quality of the studies. The tools enable the studies to be categorized as yes, no, unclear or not applicable. Any discrepancies with the JBI scores were resolved through discussion and studies were only included if both reviewers agreed.

Data Extraction and Synthesis
After the quality assessment, the relevant data were extracted and placed in an Excel spreadsheet. The data included the author, year, country, study design, sample size, offspring age at assessment, vitamin D assessment time, vitamin D categorization, outcome assessment, limitations and associations between vitamin D and outcome. We summarized the most relevant information in two tables: the first covered psychiatric disorders or diagnoses and the other was for the outcomes of psychiatric symptoms.

Study Selection
The electronic search using the MEDLINE, Embase, PsychINFO and Web of Science databases yielded 3729 titles. After we removed any duplicates, the number of publications included in this review was 2444. Further screening of the titles and abstracts yielded 66 papers for the full-text review and 28 were found to be relevant. One additional article was found in the references of the included papers. The flow diagram for the screening processes and study selection are shown in Figure 1.
The outcomes for this review were limited to neuropsychiatric or psychiatric disorders and symptoms and therefore neurocognitive outcomes were not reported. The outcomes in the studies varied, but a clear majority of the studies had examined ASD and ADHD ( Figure 2). Some studies had examined diagnoses, which were retrieved from clinical registers or were determined by a clinician during the study. Other studies examined symptoms measured by questionnaires. Some of the questionnaire scores were analyzed by using dichotomous yes/no categorizations for certain cut-off points and others were studied as continuous variables. Most of the studies examined outcomes in children and adolescents ( Figure 2). Few studies had followed the subjects into young adulthood [16,24,27,31] and only one study examined adult outcomes [40]. The age of the offspring when the outcomes were assessed in each study are presented in detail in Figure S1. The vitamin D values were measured at different time points, varying from early pregnancy to neonatal samples ( Figure 3). Only four studies had measured vitamin D concentrations at more than one time point pre-and perinatally [34,35,38,44]. Vitamin D was studied as a continuous variable (22 studies), by clinical categories (18), deciles (2), tertiles (1), quartiles (6) and quintiles (4) (Figure 4). Vitamin D levels were presented as nmol/L in 22 studies or as ng/mL in 7 studies. The definitions for deficient levels varied greatly. In the studies that used nmol/L, the deficiency was defined as between <20 nmol/L and <50 nmol/L. The study characteristics have been described in detail in Table 1a,b. The findings of the reviewed studies have been summarized in detail by each outcome, in Table 2.

Quality Assessment
The included studies had adequate quality, as assessed by the JBI quality appraisal tools. The biggest concerns in the reviewed studies were attrition rates, insufficient strategies used to address attrition, scarce confounders, small sample sizes and insufficient length of follow up. Vitamin D levels were often measured from a subsample and it was unclear if this was because of attrition or based on a planned decision. The sample sizes for the studied outcomes varied greatly and the number of cases with diagnosed outcomes was between 24 and 1558. In addition, the number of cases whose mothers were vitamin D deficient was relatively low in some studies. The follow-up time was considered insufficient in some of the studies, because the offspring's age at assessment was less than five years. Diagnoses based on self-report questionnaires were excluded from the review but symptom-level outcomes of those studies were included [16,28,33]. Furthermore, one study assessed both ASD symptoms and diagnoses, but there were only three diagnosed cases and the ASD diagnoses outcomes were not included [18]. The quality appraisal is presented in detail in Table S1.

ADHD and ADHD Symptoms
ADHD was studied as a diagnostic outcome in four studies [26,31,32,44] and ADHD symptoms in five studies [19,22,28,29,37]. There were 24 [31] to 1067 [32] cases with diagnosed ADHD. Diagnoses of ADHD were either made by a clinician [26], register-based data [31,32] or parental reports [44]. The ADHD symptoms were reported by parents in all but one study, where teacher reports were used [28]. We did not include the diagnostic outcomes from that study, as we did not consider that teacher reports on their own were a valid way of diagnosing ADHD.
Two studies reported significant links between maternal vitamin D levels and the increased odds of offspring being diagnosed with ADHD [32,44]. The largest study that examined ADHD outcomes reported significant odds ratios for both continuous and categorized vitamin D levels and ADHD [32]. The other study with significant associations reported an association between low third trimester values and increased odds for offspring ADHD but no association for values measured in early pregnancy [44].
The other two studies of diagnosed ADHD did not find significant associations between ADHD and continuous or categorized vitamin D levels from umbilical cord samples [26,31] or categorized levels from maternal sera from the third trimester [31]. It is noteworthy that the second study defined vitamin D deficiency below <50 nmol/L, which could be considered a relatively high value compared to some other studies [31].
Three out of five studies on symptom-level outcomes [22,28,29] presented associations between low prenatal or perinatal vitamin D and an increased risk for ADHD symptoms, while the other two studies did not find clear associations [19,37]. Three of the studies measured vitamin D from maternal sera in early pregnancy [22,28,37] and two from cord blood [19,29]. Four studies only assessed toddlers or preschool-aged children [19,22,28,29], which might have had an impact on the validity of the results. One study found no difference in ADHD symptoms by vitamin D category but found an interaction related to vitamin D deficiency in the association between maternal depression and offspring ADHD symptoms [19].
Three studies showed significant associations between maternal vitamin D levels during pregnancy and ASD diagnoses in offspring [20,30,34]. They comprised two studies that examined maternal vitamin D in early pregnancy [20,30] and one study that examined mid-gestation samples [34]. The largest study, with 1558 ASD cases, showed significant odds ratios for the continuous, lowest quintiles compared to the highest quintiles and categorical vitamin D measures [30]. One study reported significant associations between maternal first trimester vitamin D levels in the lower three quartiles (quartiles 1, 2, 3), compared to the highest quartile (quartile 4) [20]. Another study reported an association between deficient vitamin D levels < 25 nmol/L in mid-gestation and offspring ASD, but no association for vitamin D measured from cord blood [34].
In contrast, five studies did not find any associations between maternal vitamin D levels during pregnancy and offspring ASD [23,38,[41][42][43]. Although one study did not find any association in the overall sample, there was a significant association between maternal vitamin D insufficiency (25-<50 nmol/L) and ASD in Nordic-born mothers [38]. Most studies did not include race/ethnicity as a covariate. Two studies reported significant associations between neonatal vitamin D levels and ASD diagnoses in offspring [21,38] and one found similar associations but only in females [41].
ASD symptoms or traits in children were reported by parents in three studies [18,35,37]. Two studies showed significant associations between vitamin D levels and ASD symptoms or traits. One study reported that sufficient vitamin D levels during pregnancy (≥30 ng/mL) were associated with a lower number of ASD symptoms than deficient levels [37]. Another study reported significant associations between lower vitamin D concentrations <25 nmol/L in mid-gestation, or at the time of birth, and ASD traits [35]. However, one study did not find any correlation between low vitamin D during pregnancy and ASD traits [18].

Depressive Disorders and Depressive Symptoms
No associations were found in the two studies that examined associations between vitamin D levels during pregnancy and diagnosed depression [31] or depressive symptoms [36]. The first study measured maternal vitamin D levels at 30 weeks of gestation and the depression diagnoses were obtained from registers. The subjects were either diagnosed or they had been given at least one prescription for antidepressant medication [31]. In the second study, vitamin D levels were measured at any time during pregnancy and depression symptoms were measured using the Short Mood and Feelings Questionnaire [36].

Eating Disorder Symptoms
Eating disorder symptoms were only included in one study [16]. This was a cohort study and the offspring were assessed at 14, 17 or 20 years of age. Eating disorder symptoms or diagnoses were based on the Child Eating Disorder Examination or an Eating Disorder Examination Questionnaire. In addition, the subjects' body mass indexes were measured and the females were asked about their menstruation. The study used both eating disorder and eating disorder symptoms as definitions for the outcomes and this meant that the diagnostic validity remained somewhat unclear. A total of 98 adolescents filled in the diagnostic criteria and only 13 were males. There were no associations in the total sample, but low maternal vitamin D levels were associated with eating disorders in female offspring. Parental psychopathology was not considered as a covariate.

Schizophrenia, Schizoaffective Disorder and Psychotic Symptoms
Psychotic disorders were included in three studies [24,27,40] and one study reported psychotic symptoms and disorders based on a self-report questionnaire [33]. Only the symptoms from that study were included in this review. All three studies examined schizophrenia [24,27,40], and one study also included schizoaffective disorder [40]. The number of cases varied between 26 [40] and 1301 [24] and the offspring were followed until at least young adulthood in all three studies. The two register-based studies found an increased risk for schizophrenia [24,27] in subjects with neonatal vitamin D deficiency. Both studied neonatal dried blood samples and vitamin D levels as quintiles and continuous variables. Interestingly, one study found that the highest quintile had an increased risk for schizophrenia when the fourth quintile was used as a reference [27], while another study did not find any associations between maternal vitamin D levels in the third trimester and schizophrenia or schizoaffective disorder [40].
One study measured vitamin D from maternal sera at any stage of pregnancy and examined the association with psychotic experiences among 18-year-olds. The number of individuals with psychotic experiences was 117 out of more than 2000 subjects. No associations were established [33].

Behavioral or Emotional Symptoms
Four studies assessed behavioral or emotional symptoms [17,22,37,39]. All four studies measured vitamin D levels from maternal sera in the first or second trimester. Two studies assessed very young children [22,39], whereas the other two had multiple assessment points in childhood and adolescence [17,37]. The Infant Toddler Social Emotional Assessment Scale [39], Child Behaviour Checklist (CBCL) [17,37] and Strengths and Difficulties Questionnaire (SDQ) [22,37] were used to assess behavioral and emotional symptoms.
One study reported fewer behavioral and externalizing symptoms in children whose mothers had higher vitamin D levels [22]. One study found an association between low maternal vitamin D levels and increased internalizing scores, but among children of White ethnicity [39]. Two studies did not find associations between prenatal vitamin D levels and offspring's behavioral or emotional symptoms [17,37].
Since the studies were heterogeneous, we also examined these studies in several subgroups based on three factors: first, large sample size and stricter definitions of vitamin D deficiency and with at least 100 mothers in the deficient group (Table 3); second, time of measurement of vitamin D levels (Table S2); and third, geographical locations. There was a tendency to report positive associations between prenatal vitamin D levels and offspring ASD, ADHD or schizophrenia for subgroups with large sample sizes and stricter definitions of vitamin D deficiency. However, there were no clear tendencies to report associations based on categorization by time of measurement of vitamin D levels. Moreover, we observed more tendencies to report positive associations from countries from Nordic regions. Table 3. Large studies with stricter definitions for vitamin D deficiency. Only studies that had defined vitamin D deficiency as ≤40 nmol/L and had at least 100 mothers in the deficiency group were included. An additional limit for the diagnostic outcomes was to have at least 100 cases for the studied outcome.

Outcome
Positive

Discussion
The present systematic review found inconclusive findings with regards to the associations between maternal vitamin D deficiency and neuropsychiatric and psychiatric outcomes. Compared to other outcomes, the most evidence was accumulated for the association between maternal vitamin D deficiency and ASD. When studies with larger sample sizes, stricter definitions for vitamin D deficiency and at least 100 mothers in the deficient group were considered, positive associations were also found for ADHD and schizophrenia. Moreover, studies from Nordic regions with low sunlight exposure during the winter season were more likely to report positive associations between vitamin D levels during pregnancy and offspring psychiatric outcomes. Only one study examined depressive disorder, depressive symptoms, eating disorder symptoms and psychotic experiences and no consensus could be reached for these outcomes. None of the studies examined anxiety or bipolar disorders.

Neuropsychiatric and Psychiatric Outcomes
The findings of this review suggest a small amount of evidence for any association between maternal vitamin D deficiency and offspring ASD diagnoses. This finding was in line with a previous systematic review that suggested the potential role of vitamin D during pregnancy and autism in children [45]. Possible mechanisms for this association are related to the role of vitamin D in brain development. For example, vitamin D seems to participate in the regulation of certain neurotransmitters, including serotonin [46] and dopamine [47]. Previous studies have shown that these neurotransmitters may function abnormally in subjects with ASD [48,49].
Interestingly, when we examined a subsample of studies with larger sample sizes, stricter definitions of vitamin D deficiency and at least 100 mothers in the deficient group, there was a tendency towards reporting more positive findings for ASD, ADHD and schizophrenia ( Table 3). The evidence was inconclusive for symptom level outcomes, including ASD symptoms, ADHD symptoms, and behavioral and emotional symptoms. This was in line with previous reviews that examined neurocognitive outcomes and found mixed results [4,50,51]. The lack of associations in these studies may be attributable to factors such as small sample sizes, low prevalence of vitamin D deficiency, age at the time of the assessment and assessment tools. Many studies were based on small sample sizes that, for example, weakened the statistical power of possible subgroup analyses. While most of the studies controlled for maternal and neonatal factors, only eight studies adjusted for maternal psychiatric illness [19,21,24,27,29,30,32,40], and six for race or ethnicity [18,30,32,38,41,44]. These are both important confounding factors for the associations between maternal vitamin D levels and offspring psychiatric outcomes. One study only reported the association between maternal vitamin D levels and offspring behavioral and emotional symptoms by race, leaving out relevant information about the total sample [39]. In addition, when we examined studies by geographical locations, Nordic countries had a greater tendency to report positive associations. Moreover, the majority of these studies adjusted for the seasons when subjects were born or had blood drawn.
Several of the included studies had some important limitations. For example, the assessments were made at an early age in many studies, including studies on ADHD symptoms [19,22,28,29] and behavioral and emotional symptoms [39]. Furthermore, most of the questionnaire-based studies just relied on information from parental reports. In addition, attrition rates were high in most of the cohort studies and they were not always clearly reported. The included confounders varied: most studies had considered sociodemographic and pre-or perinatal risk factors, but parental psychopathology was included only in ten studies. Parental psychopathology is an important potential confounder because genetic effects are well established in the etiology of most neuropsychiatric and psychiatric disorders.

Timing of Exposure
The time when vitamin D was measured in the included studies varied from early pregnancy to neonatal samples. While most of the studies measured vitamin D levels in early pregnancy or dried blood spots from newborns, few studies had measured vitamin D levels during mid-gestation, late pregnancy or from umbilical cord blood at birth. The studies that measured vitamin D deficiency during early pregnancy or used newborn samples had a greater tendency to report positive associations. Only four studies had measured vitamin D status during multiple stages of pregnancy [34,35,38,44], which limited the ability to compare results across pregnancy. One study examined the association with ADHD using maternal vitamin D during the first and third trimesters and only found a significant association for vitamin D deficiency in the third trimester [44]. However, another measured vitamin D levels in mid-gestation and from a neonatal blood sample and observed significant associations with autistic traits at both time points [35]. Similarly, one study found significant associations between ASD and vitamin D levels in early pregnancy and the neonatal period, but the findings were restricted to the children of Nordic mothers [38]. In contrast, one study only found significant associations between ASD and maternal vitamin D levels in mid-gestation, but not for ASD and newborn vitamin D concentrations [34].
Previous literature suggests that maternal vitamin D concentrations can be seen as the determinant or the proxy of the closely correlated fetal vitamin D status. It also suggests that neonatal vitamin D concentrations measured from cord blood at birth can be used as markers of fetal vitamin D status at the end of gestation [14]. It is difficult to extract evidence from just the results of on these few studies measuring vitamin D levels at two time points in pregnancy. Moreover, these particular studies were based on small sample sizes. Future studies with large sample sizes and vitamin D measurements at multiple time points are warranted.

Categorization of Exposure
There was high heterogeneity in how vitamin D levels were classified in different studies. While 22 of the studies reported vitamin D levels using continuous variables, 18 reported varying clinical categories. The definitions of vitamin D deficiency varied considerably across studies, from <20 nmol/L to <50 nmol/, and this made it difficult to compare the findings. Moreover, there were few cases with deficient vitamin D levels in some studies, which could have limited the power to detect associations.

Age at Assessment
Most studies were conducted among young children, with a few among young adults or adults. Most associations were observed in young children, especially for ASD and ADHD. Only one study followed up on offspring behavioral symptoms until young adulthood and found no associations in the adolescent samples [17]. Based on two studies on schizophrenia, there were some indications that possible adverse effects of vitamin D deficiency might persist into young adulthood [27,40]. However, more studies are needed to confirm these findings. In some studies, the age range of the children was provided, and the studies were categorized by mean age at assessment for comparison; thus, some misclassification may have occurred.

Strengths and Limitations
A key strength of this systematic review is that we focused on a broad range of neuropsychiatric and psychiatric outcomes. This scope has not been explored by previous reviews that have focused on vitamin D in relation to neurodevelopmental or neurocognitive outcomes [4,10]. Furthermore, we investigated the studies rigorously by looking at the timing of vitamin D measurements, the categorization of the vitamin D levels and the age when psychiatric outcomes were assessed. However, there are several limitations that should be considered. First, several previous reviews focused on cognitive, motor and language development outcomes and prenatal vitamin D and we did not include those outcomes in our review. Second, the studies included in the review were heterogeneous. The study populations varied, especially by the timing of the exposure measurements and the categorization of vitamin D, which made it difficult to harmonize the results across studies. Third, only a few studies investigated outcomes other than ASD or ADHD, which prevented us from drawing firm conclusions on most of the other psychiatric outcomes. For example, eating disorders, psychotic experiences, depressive disorders and depressive symptoms were only examined in one study each. None of the studies examined other common psychiatric outcomes, such as anxiety and bipolar disorders. Fourth, only four studies measured vitamin D levels at two time points and the results were not uniform across the different time points. Finally, there were few studies from low-to-middle-income countries and this means that the most of the findings were based on high-income countries.

Conclusions
Our systematic review provides a small amount of evidence for the association between maternal vitamin D levels during pregnancy and offspring ASD. When studies with larger sample sizes and stricter definitions of vitamin D deficiency were considered, positive associations were also found for ADHD and schizophrenia. The findings could have important implications for public health, as vitamin D deficiency can be readily prevented with vitamin supplements. However, as the included studies were all observational, no conclusions can be made regarding the causality of the observed associations. Several other factors including genetic factors also play a role in the multifactorial etiology of neuropsy-chiatric and psychiatric disorders. No consensus was established for other psychiatric outcomes due to inconclusive findings or the low number of studies. Further studies with larger sample sizes and longitudinal designs with long follow-up periods are needed to draw firm conclusions, especially for adulthood-onset disorders. Moreover, future studies should preferably measure vitamin D at multiple time points during pregnancy, and at birth, to establish a window that might be sensitive to vitamin D deficiency. In particular, more studies are needed to explore the associations between prenatal vitamin D and some currently understudied common psychiatric outcomes such as anxiety, depression and bipolar disorders.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement:
As this is a systematic review of previous studies, direct human participation is not involved.