Prevalence of Vitamin D and Calcium Deficiency and Insufficiency in Women of Childbearing Age and Associated Risk Factors: A Systematic Review and Meta-Analysis

Vitamin D deficiency and insufficiency as well as low serum calcium levels can trigger negative health outcomes in women of childbearing age. Therefore, we aimed to estimate the prevalence of serum vitamin D and calcium deficiencies and insufficiencies and associated risk factors in Brazilian women of childbearing age and to assess whether there are differences in prevalence according to regions of the country and the presence or absence of pregnancy. The systematic literature review was performed using the following databases: PubMed, LILACS, Embase, Scopus, and Web of Science. Cross-sectional, cohort, and intervention studies were included. Among pregnant women, the prevalence of vitamin D deficiency ranged from 0% to 27% and of vitamin D insufficiency from 33.9% to 70.4%. Among non-pregnant women, the prevalence of vitamin D deficiency ranged from 0% to 41.7% and of vitamin D insufficiency from 38.5% to 69.3%. We found a high prevalence of vitamin D deficiency and insufficiency in women of childbearing age, with insufficiency affecting more than half of these women. The highest prevalence of vitamin D deficiency and insufficiency was observed in the South region. It was not possible to assess the prevalence and factors associated with calcium deficiency.


Introduction
Nutritional deficiencies affect individuals worldwide [1], especially women of childbearing age during pregnancy [2]. During this period, a woman's body faces major physical and physiological changes due to the needs of the growing baby [3], making them vulnerable to vitamin and mineral deficiencies [4].
In an observational study conducted in Southern Korea, 89% of women of childbearing age had low concentrations of vitamin D [5], and another observational study of pregnant Iranian women showed a 58% prevalence of vitamin D deficiency during pregnancy [6]. Although there is no agreement on the optimal range of vitamin D deficiency, it is predominantly characterized by serum 25(OH)D concentrations below 25-30 nmol /L (10-12 ng/mL) [7]. Vitamin D deficiency is highly prevalent even in countries with high

Search Strategy and Databases
The systematic literature review was performed using the following databases: PubMed (National Library of Medicine), LILACS (Latin American and Caribbean Health Sciences Literature), Embase, Scopus, and Web of Science. The search was conducted in December 2021 to identify articles published until that date for inclusion in this review. The review was updated in March 2022. The search strategy included relevant keywords related to vitamin D deficiency and specific terms related to calcium deficiency to ensure that all articles of interest were identified. Details describing the entire methodology and search strategy with the terms are published in the protocol article [31].

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Studies that provide data on the prevalence of serum calcium and/or vitamin D deficiency in women of childbearing age (15-49 years or menarche and menopause). • Studies with representative population-based samples in hospitals, health centers, or outpatient clinics. • Prevalence data in women of different age groups, such as adolescents, pregnant women, lactating women, and premenopausal adult women. • Studies with a cross-sectional design and data from longitudinal studies (cohort studies) or intervention studies, such as clinical trials or community trials, provided they had prevalence information for a specific time. Articles in English, Portuguese, and Spanish were included.

Exclusion Criteria
Opinion articles, comments, or editorials. Duplicate articles, i.e., the same study found in different databases. Articles with the same database/population/sample, in which case the study with the largest sample size was considered. Articles with primary data not accessible even after request to the authors. Case-control articles, narrative reviews, and case series. Studies conducted among female athletes of any sport. Studies conducted among women with the following specific diseases: autoimmune diseases such as lupus, psoriasis, thyroiditis, rheumatoid arthritis, and multiple sclerosis; eating disorders such as anorexia and bulimia; hematological diseases such as thalassemia and sickle cell disease; respiratory diseases such as chronic obstructive pulmonary disease, asthma, pneumonia, respiratory infections, and tuberculosis; chronic diseases such as heart failure, kidney failure, liver disease, chronic kidney disease, heart disease, nephrotic syndrome, AIDS, inflammatory bowel disease, hypoor hyperthyroidism, sepsis, and cancer; genetic diseases and syndromes such as vitamin D receptor mutation, cystic fibrosis, and Prader-Willi syndrome; neurological or psychiatric disorders such as epilepsy (or antiepileptic medication use), attention deficit hyperactivity disorder, and schizophrenia. Studies conducted among post-surgical patients, patients with trauma or burns, or patients undergoing recent treatment for fractures or orthopedic/osteoarticular diseases. Studies conducted among patients undergoing intensive, urgency or emergency, or palliative care. Studies with fewer than 50 participants. Studies conducted among indigenous women.

Reviewer Training
The authors responsible for assessing the eligibility criteria of the articles were trained. An eligibility test was performed with 50 titles and abstracts before coding the articles. The reviewers also received training in the instruments used to assess the risk of bias through five articles that were not included in the review. Rayyan and Mendeley software were used for the selection steps, the first being the selection of studies and the second being the exclusion of duplicate articles.

Review Process
After completing the search strategy, the identified articles were gathered and imported into the Mendeley software. Duplicate articles were excluded. The articles were selected by two independent reviewers (L.A.N.M. and M.C.R.C.). The titles were read first and then the abstracts. Finally, the entire article was read. Disagreements between the two reviewers were resolved by a third reviewer (E.A.S.). Eligibility was determined according to inclusion and exclusion criteria.

Data Extraction and Risk of Bias Assessment
Data were extracted using a table considering the following aspects: author/year, type of study, study location, age group, sample type/sample size, place of residence (urban/rural), gestational status (yes/no), lactating (yes/no), micronutrients analyzed, technique used, cut-off points and results-that is, prevalence/impact of calcium or vitamin D deficiency. The values of vitamin D are expressed as nanogram per milliliter (ng/mL) or nanol per liter (nmol/L). For conversion purposes, it is enough to multiply ng/mL by 2.5 to obtain the value in nmol/L [32].
The Downs and Black scale used to assess the risk of bias is an instrument with 27 items, but with only 16 items being applicable to observational studies (items 1-3, 5-7, 9-12, 17, 18, 20, 21, 25, and 26). The score was applied to each article according to the number of items, considering the total percentage (0 to 17 points). Low risk of bias was defined as a total score > 70%.
The Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) approach was used to assess the quality of evidence of selected studies. For each study, quality was assigned one of the following four grades: high quality (four filled circles), moderate quality (three filled circles), low quality (two filled circles), or very low quality (one filled circle).
Data were extracted and evaluated by two independent reviewers (L.A.N.M. and M.C.R.C.). Disagreements were resolved by a third reviewer (E.A.S.). A researcher (M.C.R.C.) contacted the authors of the articles to obtain relevant data not reported in their article. Potential conflicts of interest and ethical information from the studies included in the review were also reported.

Statistical Analysis
A meta-analysis was performed to assess the prevalence of vitamin D deficiency and insufficiency in Brazilian women. The analyses were stratified by region of the country and pregnancy status (pregnant or non-pregnant). Given the high heterogeneity between studies, random-effects models were used to reduce differences. We calculated the mean of the vitamin D deficiency and/or prevalence values stratified by gestational trimester in the studies and pooled them for meta-analysis. The analyses were performed using the R language (version 4.1.0) and the Meta package (version 6.0-0) and metaprop command. When the analyses included more than ten publications, a funnel plot was used to assess the asymmetry between studies.

Results
Of the 149 articles with data on serum calcium and vitamin D deficiency and insufficiency in Brazilian women of childbearing age initially identified in the databases, 143 articles remained after the exclusion of duplicates. After applying the eligibility criteria, 73 articles were selected for full reading, of which 16 were included in the systematic review. Seven articles provided information on pregnant women of which five provided information on serum vitamin D and two on calcium ( Figure 1).

Results
Of the 149 articles with data on serum calcium and vitamin D deficiency and insufficiency in Brazilian women of childbearing age initially identified in the databases, 143 articles remained after the exclusion of duplicates. After applying the eligibility criteria, 73 articles were selected for full reading, of which 16 were included in the systematic review. Seven articles provided information on pregnant women of which five provided information on serum vitamin D and two on calcium ( Figure 1).

Prevalence of Vitamin D Deficiency and Insufficiency
The number of women in studies that evaluated vitamin D in pregnant women ranged from 174 to 487, and they were aged between 20 and 40 years, with only two studies including adolescents (Table S1).
The number of non-pregnant women ranged from 15 to 369, with eight studies having a sample of fewer than 100. Five studies included only adolescents, and seven included adult women or adults and adolescents. The number of women in the two studies that evaluated calcium ranged from 99 to 226, and the age range varied between 16 and 44 years of age (Table S2).
Most studies were conducted in the Southeast (six articles) and South (four articles) regions. Only two studies were conducted in the Midwest region, and none were conducted in the North region. The cut-off points for both pregnant and non-pregnant women varied widely in the assessment of vitamin D deficiency and insufficiency, with the lowest value for deficiency being ≤10 ng/mL and the highest being <20 ng/mL. Among pregnant women, the prevalence of vitamin D deficiency ranged from 0% to 27% and the prevalence of insufficiency ranged from 33.9% to 70.4%. One study considered the prevalence of both deficiency and insufficiency in pregnant women, which

Prevalence of Vitamin D Deficiency and Insufficiency
The number of women in studies that evaluated vitamin D in pregnant women ranged from 174 to 487, and they were aged between 20 and 40 years, with only two studies including adolescents (Table S1).
The number of non-pregnant women ranged from 15 to 369, with eight studies having a sample of fewer than 100. Five studies included only adolescents, and seven included adult women or adults and adolescents. The number of women in the two studies that evaluated calcium ranged from 99 to 226, and the age range varied between 16 and 44 years of age (Table S2).
Most studies were conducted in the Southeast (six articles) and South (four articles) regions. Only two studies were conducted in the Midwest region, and none were conducted in the North region. The cut-off points for both pregnant and non-pregnant women varied widely in the assessment of vitamin D deficiency and insufficiency, with the lowest value for deficiency being ≤10 ng/mL and the highest being <20 ng/mL. Among pregnant women, the prevalence of vitamin D deficiency ranged from 0% to 27% and the prevalence of insufficiency ranged from 33.9% to 70.4%. One study considered the prevalence of both deficiency and insufficiency in pregnant women, which reached 82.9%. Among non-pregnant women, the prevalence of deficiency ranged from 0% to 41.7% and the prevalence of insufficiency ranged from 38.52% to 69.3%. Two articles reported the prevalence of both insufficiency and deficiency in non-pregnant women, which reached 74.1%.

Factors Associated with Vitamin D Deficiency and Insufficiency
The factors associated with the risk of vitamin D deficiency in pregnant women were being married, the use of vehicles as a means of transportation, blood collection in winter, only face and hands being exposed to the sun, preeclampsia, first pregnancy, adolescence, and low income [33,34]. However, a study with 226 women found no association between vitamin D deficiency and obstetric, biological, and socioeconomic variables [35]. Three studies with non-pregnant women did not investigate associated risk factors [36][37][38]. Five studies [38][39][40][41][42] did not find an association, whereas those that found an association identified the following risk factors: non-white skin, diabetes, serum glucose, homeostatic model assessment for insulin resistance, obesity, and serum calcium. Only two studies evaluated serum calcium, but they did not investigate the associated risk factors [35,39] ( Table 1). Table 1. Summarizing the association factors as risk or protective in pregnant and non-pregnant women.

Author/Year/Location Investigated Variables Summary of the Association of Vitamin D Deficiency or Insufficiency Pregnant women
Pereira-Santos/2017 [35] San Antonio de Jesus-Bahia Northeast Age, MFI, YS, skin color, MS, GA, number of weekly SEs, region of body exposed to sun, SY, means of transport

Prevalence of Calcium Deficiency and Associated Risk Factors
The hypocalcemia cut-off values ranged from <8.6 to <8.8 mg/dL and only one article with pregnant women showed a 15% prevalence of deficiency (Table S3).

Quality Analysis of the Evidence
The Downs and Black scale scores ranged from 54% to 100% (Table 2). Fourteen studies scored above 70%, indicating a low risk of bias. The GRADE score, which evaluated the methodological quality, revealed 12 studies with moderate quality, three studies with low quality, and one study with very low quality. All studies declared no conflict of interest and 14 (88%) declared an ethical approval.  . Items G and P were applied only to cross-sectional studies. Items K and N were applied only to case-control and cross-sectional studies. # The score reaches 100% with 13, 15, and 17 points for cross-sectional, case-control, and cross-sectional studies, respectively. *, not reported, -, not applicable. GRADE, Grading of Recommendations, Assessment, Development, and Evaluations; a filled circle, very low quality; two filled circles, poor quality; three filled circles, moderate quality; four filled circles, high quality.
A slight asymmetry to the left is observed in the funnel plot for vitamin D deficiency in women of childbearing age, indicating a heterogeneity between studies ( Figure 6). The funnel plot for vitamin D insufficiency in women of childbearing age did not show asymmetry, indicating less heterogeneity than the vitamin D deficiency plot (Figure 7).

Discussion
To the best of our knowledge, this is the first systematic review and meta-analysis to estimate the prevalence of serum concentration of vitamin D and calcium deficiencies and insufficiencies in Brazilian women of childbearing age. The study included 1276 pregnant women and 1436 non-pregnant women and was stratified by Brazilian geographic regions, excluding the Northeast since no study was conducted in this region. We observed a high prevalence of vitamin D deficiency in both pregnant (29.5%) and non-pregnant (29.4%) women without significant differences. The prevalence of insufficiency was 59% and it was similar between pregnant and non-pregnant women. It was not possible to perform a meta-analysis of the studies that estimated the prevalence of calcium deficiency due to the small number of studies, i.e., only two articles. Similarly, we could not conduct a meta-analysis to identify the risk factors associated with vitamin D and calcium deficiency and insufficiency due to the heterogeneity of the investigated factors and the reduced number of studies.
The overall prevalence of vitamin D deficiency (29%) and insufficiency (59%) identified in this meta-analysis were slightly higher than those found in an observational study conducted in Colombia that found a 24% prevalence of vitamin D deficiency and a 47% prevalence of vitamin D insufficiency [50]. An observational study conducted in Pakistan reported that an inadequate vitamin D status is common among women of childbearing age [51]. Another observational study found that 56% of Egyptian women of childbearing age had a vitamin D deficiency or insufficiency [52]. Looking at results from countries other than those with an income level similar to Brazil, we found an observational study conducted in Sweden reporting that more than a third of pregnant women had low levels of 25-hydroxyvitamin D [53]. The high prevalence of inadequate vitamin D status in women of childbearing age can be explained by the fact that a large part of this population lives in urban areas, which is considered a risk factor for vitamin D deficiency. Vitamin D is obtained and synthesized in the skin during exposure to ultraviolet B (UV-B) sunlight (270-300 nm) [54]. Spending time predominantly indoors owing to economic or occupational factors and wearing tight clothing reduces the exposure to sunlight [55]. Urban women generally commute to work, schools, and children's activities using forms of transport that avoid sun exposure, and the following factors influence the vitamin D levels: day-to-day activities, physical locations where they are performed, location, and geoclimatic conditions [56].
Another influencing factor is the dietary intake of vitamin D, whose levels show a statistically positive association with the Mediterranean diet, regardless of body mass index [57]. The average consumption of this nutrient in adult Brazilian individuals ranged from 2.4 to 4.67 µg [34,45,48], which is below the recommendation of estimated average requirement of 10 µg/day for women of childbearing age (15-49 years), and also according to dietary reference intakes (DRIs) of 15 µg/day for women [58]. An article that evaluated Latin American women pointed to an average vitamin D intake of 1.9 µg by Brazilian women [59], and another article that evaluated dietary intake in Brazilian adolescents (15-19 years old) observed a median vitamin D intake of 1.48 µg/day [44]. Even so, dietary intake was not associated with vitamin D deficiency or insufficiency [34].
The meta-analysis by Brazilian geographic regions showed that the prevalence of vitamin D deficiency and insufficiency was higher in the South region. A possible explanation for this is that the South region has a subtropical climate and, due to its proximity to the Tropic of Capricorn, it is characterized by the lowest temperatures in the country. The main source of vitamin D in humans is UVB radiation (290-315 nm) from the sun on the skin [60], and exposure to UV rays appears to be determinant in the epidermal synthesis of vitamin D [61]. The height of the sun determines the path of rays through the ozone layer, and the intensity of the rays depends mainly on latitude (geographical location), season, and time of day [62]. An observational study conducted in nine European countries showed that the availability of UVB radiation decreased with increasing latitude, and the availability of UVB in the winter months was too low to allow for the cutaneous synthesis of vitamin D [63]. The mean dietary calcium intake is 625.1 to 738.72 mg/day [37,45,48]. According to Herrera-Cuenca (2021), 95.16% of Brazilian women had an inadequate calcium intake, which was also below the recommendation (1000-1200 mg/day).
It is important to highlight that we did not find studies conducted in the North region of Brazil, which is characterized by pockets of poverty and the worst indicators of infant mortality and health in the country. Only one study in the Midwest region and three in the North region described regional inequalities, not only from the socioeconomic point of view but also from the perspective of promotion of scientific research.
Vitamin D deficiency cut-offs varied widely in the studies included in this review. This is in line with a systematic review including 33 observational studies that showed wide variations in the cut-off values used by studies to determine vitamin D deficiency [30]. The public health vitamin D food fortification or supplementation programs in Brazil are not mandatory [34,46], which is unusual among women receiving prenatal care from public service [34] and might be challenging due to the differing recommendations of vitamin D status, based on studies conducted in high latitude countries with older Caucasian populations [48]. Therefore, further studies are needed to deepen the discussion of optimal cut-offs for specific vitamin D levels for the Brazilian population [48].
The risk factors most associated with vitamin D deficiency in pregnant women in the studies included were being married, the use of vehicles, sun exposure of only the face and hands, preeclampsia, and low income. Being married was identified as a risk factor for vitamin D deficiency in an observational study in Saudi Arabia [64], whereas low socioeconomic status was associated with vitamin D deficiency in an observational study in China [65]. Furthermore, a systematic review of 13 observational studies demonstrated that vitamin D deficiency was associated with an increased risk of preeclampsia in pregnant women [66]. Vitamin D deficiency has also been associated with obesity since people with obesity may be more sedentary, perform less outdoor activities, and therefore be less exposed to sunlight [67]. In addition, adipose tissue is responsible for vitamin D sequestration, resulting in volumetric dilution of ingested or cutaneous synthesized vitamin D3 [68]. A prospective cohort study conducted in Italy showed that men have higher vitamin D concentrations than women across all body mass index classes [69]. Furthermore, women with a vitamin D deficiency had a higher percentage of fat mass when compared to men with a vitamin D deficiency, which can be explained by the fact that women have more localized fat than men [70]. Although several studies have addressed some factors associated with vitamin D deficiency, there is no consensus about these associations. A cross-sectional study conducted in Brazil demonstrated the low prevalence of vitamin D deficiency in severe obesity (10%) and also identified that serum and dietary vitamin D were not associated with metabolic syndrome [71]. Therefore, more research is needed on these factors to guide public health policies in the development of action plans to reduce the rate of vitamin D deficiency and consequently avoid its negative outcomes in women of childbearing age.
A possible limitation of this study was that most of the articles showing vitamin D deficiency and insufficiency were conducted in the Southeast and South regions of the country (63%), which can be considered a bias with respect to obtaining an overview of the country. In most of the articles included in this systematic review, women who supplemented vitamin D were excluded from the study samples [34,37,38,40,41,43,[45][46][47][48]. As well as not evaluating the dietary intake of vitamin D, not taking into consideration the use of supplements may be a bias in the study, as it may interfere with vitamin D metabolism [72,73]. Furthermore, it was not possible to perform a meta-analysis of calcium deficiency and the factors associated with vitamin D and calcium deficiency in women of childbearing age due to the low number of studies found. Therefore, the findings should be interpreted with caution. The same limitation occurred for the identification of associated risk factors. However, the strengths of this systematic review include the use of scales that assessed the methodological quality of the studies included as well as the absence of restriction on the year of publication. Another positive aspect relates to the fact that the selection and inclusion of articles were conducted separately by two researchers and the disagreements were resolved by a third reviewer to ensure consistency and rigor in the application of the eligibility criteria. In addition, although another systematic review estimated the prevalence and insufficiency of vitamin D, our review was the only one able to identify the associated factors, as well as to assess the risk of bias of the included studies.
This study highlights the high prevalence of vitamin D insufficiency in both pregnant and non-pregnant women. Thus, we highlight the need for the development of public policies, with a focus on preventing and minimizing this problem. From the point of view of clinical implication, national health education campaigns, focusing on behavioral measures such as 10 minutes of daily sun exposure and a balanced diet [74,75] as well as outdoor physical activity practices [23], may be sufficient to expand women's knowledge, favoring the achievement of adequate serum levels of vitamin D and calcium.
The education on and promotion of health, including dietary intake that meets nutrient and micronutrient needs, with the use of supplementation and food fortification if necessary, may constitute policies to improve nutritional deficiencies in general [26,58,76]. However, the gaps in knowledge about calcium and vitamin D deficiency and associated factors in Brazilian women are diverse, making it impossible to establish public policies due to insufficient evidence, particularly for different ethnic/racial groups [34,48]. In this sense, the results of this study may contribute to fostering policies to be increased, aiming at the development of research on calcium and vitamin D deficiency in Brazilian women of childbearing age, as well as at the identification of potential risk and protection factors. We emphasize that no research was conducted in the North region and only two studies were conducted in the Center-West region. Therefore, the development of research on serum calcium deficiency in all Brazilian regions and vitamin D deficiency in the North, Northeast, and Midwest regions is recommended to enrich the discussion related to actions and clinical implications. We also suggest prioritizing the regions with the most gaps in the country's research funding notices.
According to the Brazilian Society of Endocrinology and Metabology, the diagnosis of serum vitamin D deficiency should be performed in pregnant and lactating women [24]. Considering that the prevalence of vitamin D deficiency, in this systematic review, did not differ between pregnant and non-pregnant women, it is necessary that women of reproductive age are also tested to monitor their nutritional status of vitamin D.

Conclusions
This systematic review and meta-analysis showed a high prevalence of vitamin D deficiency and a higher prevalence of vitamin D insufficiency in Brazilian women of childbearing age. There was no significant difference between pregnant and non-pregnant women. We found a higher prevalence of vitamin D deficiency and insufficiency in the South region. It was not possible to carry out a meta-analysis of calcium deficiency, as only two articles addressed the topic, with one presenting only its prevalence and the other just the mean serum calcium levels.
This systematic review revealed a wide variability in the cut-off points used to classify vitamin D deficiency and insufficiency. Therefore, the results demonstrate the importance of standardizing cut-off points to facilitate comparisons between studies and to obtain accurate and reproducible information about the prevalence of vitamin D deficiency and insufficiency. Considering that vitamin D deficiency and insufficiency was common in women of childbearing age in Brazil, public health policies aimed at primary healthcare should be conducted with a focus on preventing vitamin D insufficiency in this population.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nu14204351/s1, Table S1: Prevalence and factors associated with vitamin D deficiency in pregnant women in studies conducted in Brazil; Table S2: Prevalence and factors associated with vitamin D deficiency in non-pregnant Brazilian women of childbearing age; Table S3. Prevalence and factors associated with Ca deficiency in Brazilian pregnant or non-pregnant women of childbearing age.

Conflicts of Interest:
The authors declare no conflict of interest.