Determinants and Effects of Vitamin D Supplementation in Postmenopausal Women: A Systematic Review

Hormonal fluctuations, excessive clothing covering, sunscreen use, changes in body fat composition, a vitamin D-deficient diet, and a sedentary lifestyle can all predispose postmenopausal women to vitamin D deficiency. An effective supplementation plan requires a thorough understanding of underlying factors to achieve the desired therapeutic concentrations. The objective of this study was to conduct a systematic review of the predictors that affect vitamin D status in postmenopausal women. From inception to October 2022, we searched MEDLINE, Embase, Web of Science, Scopus, and clinical trial registries. Randomized clinical trials of postmenopausal women taking supplements of vitamin D with serum 25-hydroxyvitamin D (25(OH)D) measurement as the trial outcome were included. Two independent reviewers screened selected studies for full-text review. The final assessment covered 19 trials within 13 nations with participants aged 51 to 78. Vitamin D supplementation from dietary and pharmaceutical sources significantly increased serum 25(OH)D to optimal levels. Lower baseline serum 25(OH)D, lighter skin color, longer treatment duration, and prolonged skin exposure were all associated with a better response to vitamin D supplementation in postmenopausal women.


Introduction
Menopause marks a significant shift in vitamin D requirements. Postmenopausal women are particularly predisposed to vitamin D deficiency due to changes in body composition, increased age, race, inadequate sun exposure, lack of vitamin D dietary consumption, and adiposity [1][2][3][4][5]. It is becoming increasingly clear from the evidence that vitamin D deficiency is linked to various menopausal health conditions, such as vasomotor symptoms [6], vaginal atrophy or genitourinary syndrome of menopause [7][8][9], sexual dysfunction [10], and postmenopausal osteoporosis [11]. With the recognition of widespread vitamin D deficiency and its impact on the health and well-being of postmenopausal women, the importance of accurate vitamin D status assessment and a thorough understanding of the determinants of vitamin D supplementation in this population is becoming more widely recognized.
The large interlaboratory variations in assay methods used to measure 25(OH)D serum levels, the best measure of vitamin D status [12], have made defining vitamin D sufficiency difficult. Moreover, different patient characteristics and vitamin D inadequacy thresholds partly explain variations in optimal serum concentrations reported by clinical trials. However, the World Health Organization (WHO) [13], the Institute of Medicine (IMO) 2010 [14], the Endocrine Society Clinical Practice Guidelines [15], and the American Association of Clinical Endocrinologists [16] defined vitamin D deficiency as below 20 ng/mL and sufficiency as ≥30 ng/mL. These limits were established in response to evidence that secondary hyperparathyroidism becomes more common when serum 25(OH)D levels fall below 30 ng/mL. Several interventional studies have revealed that a variety of factors influence vitamin D status and the effects of vitamin D supplementation. Body mass, basal serum 25(OH)D levels, and season of the year have all been found to be significant predictors of the vitamin D supplementation response and the impact on its status [17][18][19][20][21][22][23]. Additionally, genetic studies have demonstrated that the genotype affects serum vitamin D levels. However, findings on genetic diversity's effect on the vitamin D supplementation response are limited [24][25][26][27][28].
Disparities in the ideal blood concentration, dose, and duration of vitamin D administration highlight the importance of considering the many factors influencing vitamin D status during menopause when implementing a supplementation plan. This is critical, given the high prevalence of vitamin D-related conditions in postmenopausal women. This review aimed to collect evidence on the factors that influence the response to vitamin D supplementation from dietary and pharmaceutical sources, as well as their impact on vitamin D status in postmenopausal women.

Study Protocol and Guidance
The International Platform of Registered Systematic Review and Meta-Analysis Protocols (INPLASY) assigned this protocol the registration number INPLASY202260116. This systematic review followed the recommended reporting items for systematic reviews and meta-analyses (PRISMA) 2020 [29]. Before conducting the actual search, the protocol and search strategy were peer-reviewed. This systematic review did not require any ethical approval.

Databases Searched and Search Strategy
The review team conducted an initial search for vitamin D-related systematic reviews and identified the literature relevant to the review questions. To validate and peer review the search strategy, the PRESS Checklist was used to evaluate the quality and completeness of the electronic search strategy [30]

Study Selection
The identification of references was completed in two stages. First, all studies published from inception to October 2022 that assessed vitamin D status in postmenopausal women and associated factors were identified. EndNote 20.4 was used to import the identified studies, and duplicates were removed. Then, the remaining records were exported to Covidence Software for further screening and data extraction. Two reviewers independently assessed the eligibility of the title and abstract in an unblended standardized manner . A record was included for full-text review if at least one reviewer coded it as potentially eligible. Irrelevant studies were omitted. Full texts were obtained for further review if a decision could not be reached based on the information provided in the abstract. Differences in data extraction were settled by referring to original articles and discussions in order to reach an agreement. The senior reviewer (H.Z.H.) made the final decision based on the established eligibility criteria. Additional studies were manually retrieved from the references cited in the critical articles chosen for evaluation.

Inclusion/Exclusion of Studies
All randomized, placebo-controlled, double-blind, single-blind trials conducted in humans and published in any language were eligible. Inclusion criteria were as follows: (1) postmenopausal women with physiological or iatrogenic causes, (2) administration of vitamin D, regardless of the source or dose, (3) at least one outcome of interest, including serum 25(OH)D or one of its metabolites, 1,25(OH)D, and (4) comparison of the outcome of interest between any pair of the following: other doses or forms of vitamin D supplementation or placebo. Abstracts were included if they contained enough information to allow data extraction. Cross-sectional, observational, and non-human studies, case reports, and trials with and without end-of-trial outcomes were excluded. We pilot-tested the eligibility criteria on a sample of reports (n = 6) to refine and clarify the criteria and ensure reproducibility in future research.

Strategy for Data Extraction and Synthesis
Three reviewers conducted the extraction process independently to minimize bias and error (M.M.H., H.Z.H., and A.R.A.). A systematized data extraction sheet was used to extract data from each record. All pertinent data were extracted from the records, and no additional information was obtained from the authors. Citations for each article were extracted, as well as the study's first author, full title, publication date, study design, study aim, eligibility criteria, participant characteristics, types of interventions and comparators (including dosage forms, dose, and frequency), total number of participants, adverse outcomes, and study outcomes measures. Potential confounders in randomized clinical trials, if the trial was uncontrolled, the analysis technique or assay of serum 25(OH)D, and the attrition rate were also reported. We attempted to contact the authors of studies that were only published as abstracts in order to obtain more information about the study methodology.

Risk of Bias (RoB) Assessment
Two reviewers (M.M.H. and H.Z.H.) assessed the methodological quality of the included studies, and one reviewer (A.R.A.) arbitrated conflicts that were not due to assessor error using the Cochrane RoB 2.0 quality assessment. All included studies were evaluated for bias following the guidelines outlined in the Cochrane Handbook for Systematic Reviews of Interventions Version 6.3, 2022 [31]. Sequence generation, allocation concealment, the blinding of participants and personnel, the blinding of the outcome assessment, incomplete outcome data, and selective reporting were all investigated as potential areas of bias. Each item was assigned a RoB of low, unclear, or high.

Study Characteristics
All 19 included studies were randomized clinical trials published in English between 2005 and October 2021. The trials included 13 countries (Thailand, Malaysia, Philippines, Indonesia, Spain, Italy, France, Greece, Switzerland, Turkey, Brazil, Argentina, and USA). The 19 trials included a total of 4677 subjects. The sample size ranged from n = 20 to n = 2077. All 19 trials were conducted on postmenopausal women who ranged in age from 51 to 78 years old. Women with osteoporosis were included in four trials [41,44,45,48]. The durations of interventions ranged from 8 weeks to 3 years. Six trials administered vitamin D as a dietary supplement (fortified milk, yogurt, or cheese) [35,[37][38][39][40]46]. Other trials provided vitamin D in the form of oral supplements of vitamin D2, vitamin D3, and

Study Characteristics
All 19 included studies were randomized clinical trials published in English between 2005 and October 2021. The trials included 13 countries (Thailand, Malaysia, Philippines, Indonesia, Spain, Italy, France, Greece, Switzerland, Turkey, Brazil, Argentina, and USA). The 19 trials included a total of 4677 subjects. The sample size ranged from n = 20 to n = 2077. All 19 trials were conducted on postmenopausal women who ranged in age from 51 to 78 years old. Women with osteoporosis were included in four trials [41,44,45,48]. The durations of interventions ranged from 8 weeks to 3 years. Six trials administered vitamin D as a dietary supplement (fortified milk, yogurt, or cheese) [35,[37][38][39][40]46]. Other trials provided vitamin D in the form of oral supplements of vitamin D 2 , vitamin D 3 , and calcidiol. Tables 1 and 2 summarize the key findings and participant characteristics from the studies included.  Did not report The study intervention was administered monthly at the clinic under the direct supervision of the investigator

Risk of Bias
The RoB assessment is detailed in Figure 2. Except for one trial [39], all trials were rated as having a low or unclear risk of bias in all aspects considered. Nine of them were double-blind to both participants and investigators [32][33][34]36,43,44,46,47,50], and one was single-blinded to participants [40]. In six trials, there was an unclear degree of selection, performance, and detection bias due to a lack of information on randomization and the concealment of the intervention allocation [35,37,38,40,45,48]. One trial had a high dropout rate (n = 94) for personal reasons [46]. The lack of assay standardization of serum 25(OH)D serum levels was a source of concern for all studies.

Discussion
We consider our work to be the first systematic review and a key investigation that compiled existing evidence on the effects and predictors of vitamin D supplementation on vitamin D status in postmenopausal women. Postmenopausal women are the most affected by vitamin D deficiency and the most likely to benefit from effective vitamin D supplementation. Additionally, the lack of agreement on optimal and adequate vitamin D supplementation plans in postmenopausal women has pushed this to the forefront of medical research. The findings of the 19 trials revealed that the factors linked to vitamin D status in menopausal women could be classified into two major categories: factors related to vitamin D supplementation dosage regimens, including the dose administered, frequency, formulation, and duration of treatment, and factors related to patients' characteristics and demographics, including serum baseline, lifestyle habits, ethnicity, and genetics.

The Effect of Treatment Duration and Dose on Vitamin D Status
Different trials have investigated the effects of various forms and dosages of vitamin D supplementation on overall vitamin D status. A high single oral dose of 300,000 IU was found to be superior to low doses of 800 IU/day and shown to significantly increase the vitamin serum concentration [33]. Another study concluded the same with a dose of 250,000 IU of vitamin D [51]. On the other hand, Pignotti et al. concluded that low doses of 400 IU/day were ineffective at raising serum concentrations of 25(OH)D to levels considered optimal for bone health [45]. In both trials, with the administration of high doses, serum 25(OH)D returned to baseline after three months. This highlights that a maintenance dose with regular intervals would be reasonable in patients undergoing single-, large-dose vitamin D replacement. Mueangpaisarn and Chaiamnuay et al. found the same with doses of 40,000 IU and 100,000 IU of cholecalciferol after three months of treatment [43].

The Effects of Type of Vitamin D on Vitamin D Status
It has also been proposed that the type of vitamin D affects vitamin D status. Vitamin D 2 was found to be effective at increasing serum 25(OH)D concentrations but required higher than the usual recommended doses of vitamin D 3 [41]. Several studies have found that the response of serum 25(OH)D to vitamin D 2 and vitamin D 3 supplementation differs, with vitamin D 2 being less effective than vitamin D 3 [52][53][54][55]. Research attributed these differences in response between the two calciferol forms to differing affinities for the vitamin D receptor (VDR), which appear to be related to an additional step of 24-hydroxylation that inactivates calcitriol [56,57]. Moreover, it is hypothesized that vitamin D 3 is potentially a more favorable substrate for 25-hydroxylase [58].
Bischoff et al. found that supplementation with the 25(OH)D 3 metabolite itself is more effective and faster than vitamin D 3 in raising 25(OH)D levels in postmenopausal women, compared to typical doses of 800 IU vitamin D 3 [34,44]. This is because 25(OH)D 3 is hydrophilic, has a much shorter half-life, and causes a rapid rise in serum 25(OH)D levels. Furthermore, the fact that 25(OH)D 3 enters the circulation and bypasses first-pass metabolism makes it preferable when the fast replacement of 25(OH)D is required [59,60]. These findings are consistent with many previous studies [61][62][63][64][65][66]. Minisola et al. studied different doses of calcidiol (20,40, and 125 ug/day) in postmenopausal women and found that all dosage regimens significantly increased serum 25(OH)D at the end of the treatment of 12 weeks; however, no difference was noticed between vitamin D-insufficient and vitamin D-deficient patients [42].

The Effects of Baseline Serum 25(OH)D on Vitamin D Status
Venugopal et al. found that large doses of 25,000 IU/4 weeks and 50,000 IU/4 weeks of cholecalciferol can maintain vitamin D sufficiency. However, this effect was only shown after 16 weeks of treatment in women with low baseline serum 25(OH)D receiving 25,000 IU, in contrast to women who received 50,000 IU, who started to show a rise in serum 25(OH)D at eight weeks only [48]. Similarly, after 12 weeks of treatment, patients who received standard doses of 800 IU/day and began with sufficient serum baseline 25(OH)D concentrations of 50 nmol/L were unable to achieve mean serum 25(OH)D concentrations of >75 nmol/L [67].
Additionally, Bonjour et al. and Talwar et al. emphasized the significance of baseline 25(OH)D serum concentrations and their impact on vitamin D status. They found that the lower the baseline levels, the higher the response to vitamin D supplementation, showing an inverse relationship between the two [35,47]. Other studies confirmed similar findings [19,68,69]. Many different mechanisms can explain this inverse relation. First, baseline or initial vitamin D status influences the serum 25(OH)D distribution, or the hepatic hydroxylation rate of the cholecalciferol molecule is increased by its product. Another possible mechanism is that vitamin D status could be affected by the strength of the interaction between the vitamin D molecule and its binding protein. The activity of the catabolic vitamin D 24-hydroxylase enzyme may be diminished in response to a sustained decrease in 25(OH)D serum levels [70][71][72]. These findings indicate that basal serum 25(OH)D is a significant predictor of vitamin D status, independent of the dose.

The Effect of Sun Exposure on Vitamin D Status
Serum 25(OH)D levels were significantly lower in patients exposed to sunlight without additional vitamin D supplementation. Sun exposure combined with vitamin D supplementation, on the other hand, resulted in a significant increase in serum 25(OH)D but only at high doses that ranged between 20,000 IU and 50,000 IU of both vitamin D 2 and D 3 given monthly for at least three months [48,49]. Both trials involved people of Asian descent who lived in tropical areas. For example, Wicherts et al. demonstrated that sunlight exposure had lower efficacy compared to vitamin D supplementation [73]. In addition, a six-month intervention with sunlight exposure reduced serum 25(OH)D concentrations in young women, and it was inferior to oral vitamin D supplementation or vitamin D-fortified milk in a study conducted in Saudi Arabia [74]. A meta-analysis of seven trials by Moradi et al. [75] confirmed that vitamin D 3 significantly increased serum 25(OH)D compared to sun exposure only. Interestingly, Chel et al., on the other hand, found that UV irradiation was as effective as oral vitamin D supplementation in the elderly. However, it is worth mentioning that they used very low doses of 400 IU/day of vitamin D 3 [76]. Furthermore, two Australian studies found that as solar UV exposure increased, serum 25(OH)D levels gradually increased, with limited sun exposure being a risk of developing a deficiency [73].
Most studies evaluating vitamin D supplementation have been conducted in countries at higher latitudes. The effect of the seasons on the response to vitamin D supplementation is well established by different studies [77][78][79]. This emphasizes the importance of supplemental vitamin D consumption during the winter season. Fortified milk with vitamin D has been demonstrated to be beneficial in boosting vitamin D status in postmenopausal women who are at increased risk due to insufficient sun exposure and calcium consumption [22,[37][38][39][80][81][82]. Interestingly, Talwar et al. [47] empirically noted a pronounced peak in the serum concentration of 25(OH)D from June to September.

The Effect of Lifestyle Habits and Dietary Intake on Vitamin D Status
Talwar et al. and Reyes-Garcia et al. [46,47] found that age and weight-related variables were not significantly associated with changes in vitamin D serum levels. Mueangpaisarn and Chaiamnuay et al. concluded that body mass index (BMI) is not an independent factor in attaining optimal 25(OH)D levels [43]. However, one trial found that body mass index was a significant predictor of vitamin status and the dose of vitamin D 3 [36]. Moreover, Mason et al. [83] found that weight loss through dietary restriction and exercise had no significant effect on serum vitamin D status. Lower 25(OH)D serum concentrations are associated with decreased bioavailability and increased adiposity, implying that weight loss can lead to increased 25(OH)D concentrations via decreased peripheral sequestration in a dose-dependent manner and is unrelated to changes in dietary vitamin D intake.
Manios et al. [40] examined the effects of vitamin D 3 -enriched cheese on serum 25(OH)D levels in postmenopausal women in the winter season. They found that in addition to the usual dietary intake of around 2 µg/day, a daily dose of 5.7 µg of vitamin D 3 significantly raised serum 25(OH) D levels. Johnson et al. [84], in their partially double-blind randomized clinical trial, found that, surprisingly, the daily intake of vitamin D-enriched cheese (15 µg/day) resulted in a significant reduction in serum 25(OH)D of 6 nmol/L. In contrast, the groups that received non-enriched cheese or no cheese had either a significant increase or no change in serum 25(OH)D. These differences could be related to differences in the age of participants, the doses administered, the treatment duration, participants' compliance with the intervention, and seasonality. Reyes-Garcia et al. [46] showed that 600 IU/day of vitamin D-enriched milk effectively raised serum levels in a large proportion of women.

Effect of Ethnicity and Genetics
African-American women typically require higher amounts than the recommended upper daily allowance of 2000 IU/day to achieve an optimal serum 25(OH)D concentration of 75 nmol/L, which reflects a dose of 2800 IU/day for those with concentrations >45 nmol/L and 4000 IU/day for those with serum concentrations of <45 nmol/L [32,47]. Although studies on genetic differences with regard to vitamin D supplementations are scarce, one study found that only eleven single-nucleotide polymorphisms (SNPs) were identified to be significantly linked to basal serum 25(OH)D in postmenopausal Caucasian women [50].

Limitations
The variation in the intervention strategy across all studies in terms of the vitamin D supplementation type, dose, duration, frequency of administration, and serum concentration assay methods has contributed to varying levels of heterogeneity, which may limit the ability to generalize the findings toward general practice guidelines or recommendations. However, the consistency in the results that shows the same factors that influence vitamin D status in postmenopausal women is clear from the evidence compiled from clinical trials. Furthermore, while vitamin D supplementation is important for postmenopausal women due to the effects on bone biomarkers, and while bone health is an essential and significant target for vitamin D, future research may focus on the effects of vitamin D deficiency on a broader range of postmenopausal women's conditions, given the potential link between vitamin D deficiency and many other conditions.

Conclusions
The evidence gathered in this review on the factors influencing vitamin D status was consistent across all trials. A minimum dose of 800 IU/day and a minimum treatment duration of 12 weeks was required to reach serum 25(OH)D levels considered optimal for the health of postmenopausal women. Baseline serum 25(OH)D concentrations, below the sufficient serum baseline 25(OH)D concentrations of 50 nmol/L, were associated with a better response to vitamin D supplementation. To achieve adequate serum 25(OH)D levels, high doses of vitamin D supplementation up to 100,000 IU/week were considered safe and effective. Furthermore, a single 300,000 IU bolus dose was superior to low daily doses. Still, their inability to maintain optimal serum concentrations necessitates the regular use of maintenance doses. Vitamin D 3 was superior to vitamin D 2 in improving vitamin D status. Active vitamin D metabolite 25(OH)D 3 supplementation was more effective than vitamin D 3 . Vitamin D-fortified foods can be an excellent source of increasing serum 25(OH)D, especially when sun exposure is limited. Although there is no agreement on the optimal doses or concentrations for different patient groups, clinical practice can benefit from the fact that higher doses were always preferred in specific patient groups with large safety margins.

Data Availability Statement:
The datasets generated during and/or analyzed during the current study will be available upon request from (Mohammed M. Hassanein, email: s2163718@siswa.um.edu.my). Data will be available for 1 year from the date the study has ended by email.

Conflicts of Interest:
The authors have no conflict of interest associated with the material presented in this paper.