Daily versus stat vitamin D supplementation during pregnancy; A prospective cohort study.

BACKGROUND: Despite favorable climatic conditions, vitamin D deficiency (VDD) is widespread in Pakistan. Current study was aimed to evaluate the prevalence of VDD in Pakistani pregnant women and effectiveness of various regimen of Vitamin D supplementation.
METHODOLOGY: This hospital-based prospective cohort study included pregnant women at 12th to 24th weeks of gestation attending Gynae clinic from October 2018 to April 2019. Patients were classified into control and treatment groups (Groups: G1, G2 and G3) according to the dose of vitamin D supplementation. Patients received various regimens of vitamin D including 2000 IU/day (G1), 5000 IU/day (G2) and stat 200000 IU (G3). The levels of vitamin D were measured before and after supplementation. The effectiveness of dosages were compared between and within the groups. Moreover, factors associated with vitamin D sufficiency and insufficiency were ascertained using appropriate statistical methods.
RESULTS: Among 281 pregnant women (mean age: 28.22 ± 4.61 years), VDD was prevalent in 47.3% cases. Vitamin D supplementation caused significant rise in the levels 25(OH)D in treatment groups, while there was no significant difference in control group. The highest mean increment in vitamin D (23.14 ± 11.18 ng/ml) was observed with dose 5000 IU/day followed by doses 200000 IU stat (21.06 ± 13.73 ng/ml) and 2000 IU/day (10.24 ± 5.65 ng/ml). Vitamin D toxicity was observed in one patient who received 200000 IU stat of vitamin D. The frequency of VDD following the supplementation was 5.7%. Education status, duration of sun exposure and use of sunblock was substantially associated with vitamin D sufficiency in the current study.
CONCLUSION: Our findings underscore the high proportion of VDD among pregnant women in Pakistan. Maternal vitamin D supplementation substantially improved the levels of 25(OH)D. Of three used regimens, the dose of 5000 IU/day is considered safe and equally effective as of 200000 IU stat. Since pregnancy is a time of tremendous growth and physiological changes for mother and her developing fetus with lifelong implications for the child, gestational vitamin D supplementation should be considered to ensure the optimal vitamin D accrual in pregnant women. This study generates the hypothesis that vitamin D supplementation at a dose of 5000 IU/day during pregnancy is superior to the other regimens. However, well-controlled randomized trials are needed to confirm these findings.

Introduction

Vitamin D deficiency (VDD) is widespread around the globe and associates with negative maternal and neonatal health outcomes. Despite the geographical region with a warm climate, VDD is widely prevalent in Pakistan. The National survey conducted in 2011, concluded that 68.9% pregnant women were vitamin D deficient [1]. Majority of the women in Pakistan practice Hijab (veil) due to religious or cultural reasons, and spend most of their time indoor and thus have predilection to suffer from VDD [2]. Though the definition of VDD is quite debated but plasma levels of 25-hydroxyvitamin D (25(OH)D) < 20 ng/ml is widely accepted in the literature [2-4].

Vitamin D can be naturally obtained from direct sun-exposure to skin (Ultra Violet-B radiations). Sunlight converts 7-dehydrocholesterol to pre-vitamin D3 in the skin, which is further metabolized to vitamin D3 and then to 25(OH)D in the liver. Renal conversion of 25(OH)D to 1, 25-dihydroxyvitamin D3 (1, 25-OH2D3) maintains calcium hemostasis [2, 5]. Sun exposure of face and forearms at mid-day for about 20–30 minutes produces around 2000 IU equivalent of vitamin D in light-fair skinned population. However, duration of sun exposure is 2 to 10 times for dark skinned population to produce the equivalent amount of vitamin D [6]. Dietary sources of Vitamin D include oily fish, egg yolk, milk, juices, yogurts, cereals, soy, mushrooms, margarine and cod liver oil. VDD can lead to musculoskeletal manifestations such as osteomalacia in adults, rickets in children, metabolic disorders (secondary hyperparathyroidism), obstetric complications such as pre-eclampsia (PET), gestational diabetes mellitus (GDM) and gestational hypertension (GHT), increased probability of cesarean section, pre-term delivery, and decreased bone mineral density (BMD) [7].

Vitamin D supplementation is not routinely recommended during antenatal care, as there is not enough evidence to support its benefits during pregnancy [2, 8–11]. The Recommended Dietary Allowance (RDA) in pregnancy is 600 IU (15 mcg) and 400 to 600 IU according to Institute of Medicine (IOM) [12]. However, revision of guidelines during gestation and lactation were suggested by several investigators that supplementation must be evidence-based and in accordance with the clinical relevance [13]. For modestly dressed pregnant female, 1000 IU (25mg) per day is recommended due to inadequate sun-exposure [14]. The dose of 2000 IU/day is also considered safe but inadequate in most of the studies and therefore the dose of 4000 IU was preferred [5, 15–20]. Daily tolerable upper intake limit according Institute of medicine (IOM) is 4000 IU and according to The Endocrine Society Clinical Practice Guidelines by Holick et al. (2011) is 10,000 IU and no evidence of toxicity was associated at these doses [2, 4, 21, 22].

Increased prevalence of VDD globally and its associated health related intricacies have raised a major concern and hence needs to be addressed, especially in developing countries such as Pakistan. However, there is unavailability of regional data on high dose supplementation during gestation. Current study was aimed to ascertain the prevalence of VDD during pregnancy, effectiveness of various regimens of Vitamin D supplementation (200000 IU single dose, daily high dose of 2000 IU and 5000 IU) and proportion of the study population attaining sufficient vitamin D levels following treatment.

Methodology

Ethical approval

This study was approved by the Mid City Hospital`s Ethical Review Board (Reference: MCH/EXC/CEO-01). Informed written consent was obtained and purpose of the study was explained to the participants. All the patient`s identities were anonymised before analysis.

Study design and location

This hospital-based prospective cohort study was conducted in the Outpatient Department (OPD) of the Mid City Hospital (MCH), a multi-disciplinary hospital known due its specialty in Gynaecology which serves hundreds of patients daily.

Study population

All the pregnant women at 12-24th weeks of gestation attending OPD of MCH during October 2018 to April 2019 were consecutively recruited into the study. Gestational age was calculated in weeks on the basis of Last Menstrual Period (LMP). Fetal ultrasound was also done to ensure the gestational age or any other anomalies. Pregnant women with renal disease, chronic Liver disease (CLD), or those using anti-tubercular or anti-epileptic drugs during last three months were excluded as they can affect the study outcomes.

Treatment groups

Since vitamin D screening is routinely performed for patients registered in the hospital, the baseline levels of vitamin D were available for all patients. Pregnant women were classified by the researcher into four different groups according to the dose of Vitamin D prescribed. The choice of vitamin D supplementation and dose was at the discretion of the individual treating physician. Patients in which vitamin D supplementation was initiated were classified into three groups (Supplementation Groups (G) i.e. G1, G2 and G3) according to the dose they received. Patients in G1 received 2000 IU/day, G2 received 5000 IU/day and G3 received 200,000 IU single stat dose. Patients who were not prescribed any vitamin D dose were classified as control group (CG). Patients in CG received conventional antenatal management. In our hospital, patients are encouraged to report any adverse event to the antenatal clinic or directly to the pharmacist and side effects for overdose were monitored in all patients receiving supplementation during the study period. A follow up was scheduled two months after initiation of dose, in compliance with the current antenatal care follow up visit. The process of study flow is described in Fig 1.

Fig 1

Study flow diagram.

Outcome measure

Serum 25(OH)D level was used as the measuring outcome to assess the vitamin D status at baseline and follow-up to compare the effectiveness of prescribed supplementation.

Safety measures

Vitamin D toxicity was defined as circulating level of 25(OH)D >100 ng/ml. Vitamin D3 supplementation was stopped in case of toxicity. Hypercalcemia was measured using serum calcium level and routinely Ultrasonography (USG) was conducted for the high-risk patients to observe the kidney stones formation. Patients were monitored followed by the supplementation for the rest of the study period (2 months).

Biochemical analysis

Vitamin D status was evaluated by measuring serum 25(OH)D level. Maternal blood sample was collected, centrifuged and stored at -80°C followed by Chemiluminescence or CLIA (Chemiluminescence Immunoassay Analyzer) using state of the art Maglumi® 600 fully automated system. Maternal blood (5 milliliters) was collected at baseline and again at the follow-up. CLIA is a quantitative method which measures total 25(OH)D and other hydroxylated vitamin D metabolites in serum sample. CLIA is two-incubation assay in which antibody-antigen complex is formed; 25(OH)D is dissociated from the binding protein followed by its binding to 25(OH)D antibody. The chemiluminescent reaction was as relative light units which are inversely proportional to the 25(OH)D in the sample. Serum Vitamin D concentration was measured in Nano grams per milliliter (ng/ml). The cut-off reference points used to define vitamin D status in this study were < 20 ng/ml as deficiency, 20 to < 30 ng/ml as insufficiency, 30–100 ng/ml as sufficiency and > 100 ng/ml as toxicity [2–4, 23–31]. Maternal serum calcium levels were assessed using spectrometry method at the follow-up to rule out any manifestation of vitamin D intoxication.

Data collection

Data Collection was devised to gather the information regarding demographics, gestation, parity of recruits, medical history, medication history, Clinical features indicating osteomalacia (muscle weakness, bone pain, tenderness, or fractures). Patient’s demographics were recorded directly from patients and their medical records. Patient compliance to the regimen was assessed by self-reporting.

Statistical analysis

An IBM SPSS version 25 was used to perform all statistical analysis. The data was recorded as the Mean ± standard deviation (SD) for the continuous variables and as frequencies with percentages (proportion) for the categorical variables. The comparisons between more than two treatment groups for normally distributed data was done using one way ANOVA or Kruskal Wallis test, where appropriate. The comparison (univariate) between two categorical variables and dichotomous data was carried out using χ2 (chi square) or Fisher Exact test, where appropriate. Comparison of vitamin D levels between baseline and follow-up within each treatment groups was made using paired t-test. Comparison of patients with vitamin D sufficiency and insufficiency was conducted by chi-square test for categorical variables. Chi-square was used to check the association between the educational status and self-medication. A logistic regression model was performed to determine the factors independently associated with vitamin D insufficiency. Odds ratio and 95% confidence interval were also calculated. One-way ANCOVA was performed to compare different interventions and to control the effect of confounders. A two-tailed p value of 0.05 was considered significant.

Results

Characteristics of study participants

Out of 305 patients, 296 were recruited into the study. Of these, 9 patients were excluded from the analysis due to their refusal to participate in the study (n = 3), miscarriage (n = 2) and gestational age > 24 weeks (n = 4) (Fig 1). A total of 281 patients completed the study and were available for the analysis. Of these, 61 patients were in control group, 64 in G1, 76 in G2 and 80 patients were in G3 group.

The mean maternal and gestational age of study participants was 28.2 ± 4.6 years and 18.2 ± 4.2 week, respectively. The demographics were equally distributed between the control and treatment groups. Approximately half of the study participants (49.5%) were over-weight, 172 (61.2%) were graduates, 201 (71.5%) were housewives and 280 (99.6%) were Asian. Thirty two (11.4%) pregnant women reported self-medication of analgesics (n = 25), folic acid (n = 4) and multivitamins (n = 3). Demographics and clinical features were equally distributed between the study groups. The vitamin D rich food consumption was also shown to be equally distributed between the treatment groups. The patients were recruited in three different season with decreased UV index (UVI) and were equally distributed across the treatment groups; (Autumn = 22nd September– 21st December, Winter = 22nd December– 20th March, Spring = 21st March - 21st June) (Table 1). The mean 25(OH)D level was lowest in winters (20.46 ± 10.53 ng/ml), with concentration recovering in spring (20.60 ± 9.70 ng/ml) and highest in the current study in autumn (23 ± 8.31 ng/ml). Moreover, there was no statistically significant difference between the mean 25(OH)D level between the groups (p = 0.149).

Table 1

Demographic characteristics of the treatment groups recorded at baseline.

Characteristics	Total (N = 281)	Control (n = 61)	G1 (n = 64)	G2 (n = 76)	G3 (n = 80)	P value
Maternal age (years ± SD)	28.22 ± 4.61	27.61 ± 4.18	28.41 ± 5.09	28.71 ± 4.80	28.09 ± 4.36	0.552
Maternal age range, n (%)						0.320
18–27 years	128 (45.6%)	33 (54.1%)	27 (42.4%)	33 (43.4%)	37 (46.3%)
28–37 years	138 (49.1%)	28 (45.9%)	34 (53.1%)	36 (47.4%)	37 (46.3%)
38–47 years	15 (5.3%)	0	3 (4.7%)	6 (7.9%)	6 (7.5%)
Education; n (%)						0.428
Un-educated	4 (1.4%)	0	2 (3.1%)	2 (2.6%)	0
Primary	2 (0.7%)	0	1 (1.6%)	1 (1.3%)	0
Matric	7 (2.5%)	3 (4.9%)	2 (3.1%)	1 (1.3%)	1 (1.3%)
Intermediate	38 (13.5%)	10 (16.4%)	10 (15.6%)	12 (15.8%)	6 (7.5%)
Graduate	172 (61.2%)	34 (55.7%)	39 (60.9%)	51 (67.1%)	48 (60.0%)
Post-Graduate	58 (20.6%)	14 (23.0%)	10 (15.6%)	9 (11.8%)	25 (31.3%)
Occupation; n (%)						0.052
Un-employed	201 (71.5%)	44 (72.1%)	40 (62.5%)	64 (84.2%)	53 (66.3%)
Student	14 (5.0%)	5 (8.2%)	3 (4.7%)	4 (5.3%)	2 (2.5%)
Professional	64 (22.8%)	12 (19.7%)	19 (29.7%)	8 (10.5%)	25 (31.3%)
Self-employed	2 (0.7%)	0	2 (3.1%)	0	0
Gestational age (weeks)	18.21 ± 4.17	17.77 ± 4.05	18.30 ± 4.20	17.47 ± 4.23	19.16 ± 4.04	0.064
BMI	24.82 ± 4.40	25.12 ± 3.78	24.53 ± 4.75	24.63 ± 4.31	24.99 ± 4.69	0.844
BMI categories						0.655
Underweight	14 (5.0%)	1 (1.6%)	3 (4.7%)	3 (3.9%)	7 (8.8%)
Normal	77 (27.4%)	21 (34.4%)	19 (29.7%)	20 (26.3%)	17 (21.3%)
Over-weight	139 (49.5%)	29 (47.5%)	32 (50.0%)	38 (50.0%)	40 (50.0%)
Obese	51 (18.1%)	10 (16.4%)	10 (15.6%)	15 (19.7%)	16 (20.0%)
Gravidity (Median)	2	2	2	2	2
Total Number of pregnancies						0.676
PG/None	130 (46.3%)	25 (41%)	30 (46.9%)	39 (51.3%)	36 (45%)
MG/More	151 (53.7%)	36 (59%)	34 (53.1%)	37 (48.7%)	44 (55%)
Parity (Range)	0–4	0–4	0–4	0–3	0–4
Mode of last delivery; n (%)						0.206
Normal	72 (25.6%)	16 (26.2%)	20 (31.3%)	19 (25.0%)	17 (21.3%)
C-section	62 (22.1%)	13 (21.3%)	18 (28.1%)	12 (15.8%)	19 (23.8%)
Milk Consumption (per day)						0.078
None	111 (39.5%)	28 (45.9%)	26 (40.6%)	36 (47.4%)	21 (26.3%)
Once	128 (45.6%)	27 (44.3%)	28 (43.8%)	32 (42.1%)	41 (51.2%)
Twice	42 (14.9%)	6 (9.8%)	10 (15.6%)	8 (10.5%)	18 (22.5%)
Fish Consumption (per day)						0.530
None	273 (97.2%)	58 (95.1%)	63 (98.4%)	75 (98.7%)	77 (96.3%)
Once	8 (2.8%)	3 (4.9%)	1 (1.6%)	1 (1.3%)	3 (3.8%)
Egg Consumption (per day)						0.053
None	122 (43.4%)	25 (41.0%)	30 (46.9%)	43 (56.6%)	24 (30%)
Once	137 (48.8%)	30 (49.2%)	29 (45.3%)	30 (39.5%)	48 (60%)
Twice	22 (7.8%)	6 (9.8%)	5 (7.8%)	3 (3.9%)	8 (10%)
Recruitment Season						0.072
Autumn	112 (39.9%)	24 (39.3%)	24 (37.5%)	25 (32.9%)	39 (48.8%)
Winter	104 (37.0%)	19 (31.1%)	30 (46.9%)	27 (35.5%)	28 (35.0%)
Spring	65 (23.1%)	18 (29.5%)	10 (15.6%)	24 (31.6%)	13 (16.3%)

P values are calculated between control and treatment groups using χ2 or Fisher Exact Test (categorical variables) and One-way ANOVA or Kruskal-Wallis Test (continuous variable), where appropriate

Gravidity: Total pregnancies, regardless of outcome; Parity: Number of births after 24 weeks, live or still birth

Abbreviations: G: Treatment Group; PG (Primigravida)–First time pregnancy; MG (Multigravida)—Multiple pregnancies, regardless of outcome; BMI (Body Mass Index): < 18.5 kg/ m2 as under-weight, 18.5–22.9 kg/m2 as Normal weight, 23.0–24.9 kg/m2 as over-weight and ≥ 25.0 kg/m2 as obese (Asian cut-off values)

Impact of supplementation on 25(OH)D levels

Table 2 demonstrates that the baseline levels of 25(OH)D were equally distributed between control and treatment groups (p = 0.245). During follow-up, the levels of 25(OH)D were significantly improved in treatment groups, with the highest mean serum 25(OH)D achieved in G2 group. Sub-group analysis showed that there was no statistically significant (P = 0.686) difference of vitamin D levels between 5000 IU/day (43.92 ± 16.95 ng/ml) and 200,000 IU stat (41.50 ± 15.33 ng/ml) regimens. The highest proportion of patients (78.8%) achieved sufficient levels of 25(OH)D were in G3 group. Our results showed that supplementation improved the proportion of patients with vitamin D sufficiency from 18.1% to 65.8%. Only one patient attained serum 25(OH)D > 100 ng/ml. However, serum biochemical indices were within the normal range and USG showed no stones in the kidneys. Levene’s test and normality checks were carried out and assumptions met.

Table 2

Vitamin D status between the treatment groups.

Measure	Total (N = 281)	Control (n = 61)	G1 2000 IU/day; (n = 64)	G2 5000 IU/day; (n = 76)	G3 200000 IU stat; (n = 80)	P Value
Baseline
25(OH)D (mean ± SD); ng/ml	21.51 ± 10.49	23.82 ± 9.41	21.51 ± 8.87	20.77 ± 2.46	20.43 ± 10.34	0.245
Vitamin D status						0.005
Deficiency < 20	133 (47.3%)	16 (26.2%)	30 (46.9%)	44 (57.9%)	43 (53.8%)
Insufficiency 20 to < 29.9	97 (34.5%)	29 (47.5%)	24 (37.5%)	17 (22.4%)	27 (33.8%)
Sufficiency 30–100	51 (18.1%)	16 (26.2%)	10 (15.6%)	15 (19.7%)	10 (12.5%)
Toxicity > 100	0	0	0	0	0
Follow-up
25(OH)D (mean ± SD); ng/ml	36.85 ± 15.16	27.29 ± 10.82	31.75 ± 8.41	43.92 ± 16.95	41.50 ± 15.33	<0.001
Vitamin D status						0.016
Deficiency < 20	16 (5.7%)	12 (19.7%)	2 (3.1%)	2 (2.6%)	0
Insufficiency 20 to < 30	79 (28.1%)	24 (39.3%)	24 (37.5%)	15 (19.7%)	16 (20.0%)
Sufficiency 30–100	185 (65.8%)	25 (41.0%)	38 (59.4%)	59 (77.6. %)	63 (78.8%)
Toxicity > 100	1 (0.4%)	0	0	0	1 (1.3%)

Sufficient vitamin D concentration = 30–100 ng/ml

P values are calculated between the groups.

G1 group with 2000 IU/day dose

G2 group with 5000 IU/day dose

G3 group with 200000 IU stat dose

Control group with no treatment

P values are calculated between control and treatment groups using one-way ANOVA

All the confounding variables i.e. sun-exposure, seasonal variation, use of sunblock, body area covered and baseline VD level were adjusted using General linear model to assess the effect of intervention. One-way ANCOVA was conducted to compare the effectiveness of different interventions (CG, G1, G2, G3) on patient’s VD level. There is no significant relationship between the covariate and the dependent variable, after controlling for the independent variable (treatment group) and adjusting covariates i.e. baseline VD level, seasonal variation, sun-exposure, body area covered and use of sunblock. There was a strong relationship between the baseline and follow-up VD level, as indicated by a partial eta squared value of 0.375 (Table 3).

Table 3

Relationship of confounding factors with respect to treatment groups.

COVARIATES	Estimated Marginal mean	p-value	Partial eta squared
Seasonal variation		0.288	0.012
Autumn	37.32 ± 1.45
Winter	37.00 ± 1.49
Spring	34.00 ± 1.76
Average sun-exposure		0.240	0.006
< 15 min	37.21 ± 1.26
> 15 min	34.95 ± 1.37
Use of sunblock	36.88 ± 1.93	0.607	0.001
Body area covered		0.758	0.000
Fully covered	36.56 ± 1.74
Partially covered	35.94 ± 1.00
Baseline VD level		0.245	0.375
Control	24.73 ± 1.87
G1	31.40 ± 2.16
G2	43.07 ± 1.77
G3	42.23 ± 1.48

P values are calculated using, General Linear Model (one-way ANCOVA) to adjust the confounding variables

Supplementation with different vitamin D doses had a variable effect on circulating vitamin D. Current study demonstrated significant increment in serum vitamin D level in treatment groups following supplementation (Table 4). However, the increase in the levels of vitamin D in control group was insignificant (p = 0.061). The highest mean increment (23.14 ± 11.18 ng/ml) was observed with dose 5000 IU/d followed by dose 200000 IU stat (21.06 ± 13.73 ng/ml) (p < 0.001). The Vitamin D increment was statistically different between the control group and treatment [p-value: CG and G1: 0.001; CG and G2: < 0.001; CG and G3: 0.001]. Moreover, the VD increment in G1—G2 and G1—G3 were also statistically different (p-value < 0.001). However, VD increment was statistically insignificant between G2 and G3 (p = 0.579).

Table 4

Mean increment in the vitamin D level after supplementation (within the group analysis).

Group	Mean 25(OH)D at baseline (ng/ml) a	Mean 25(OH)D at follow-up (ng/ml) a	Mean increment in 25(OH)D level a (ng/ml)*	t value	p value
CG	23.82 ± 9.41	27.29 ± 10.82	3.47 ± 6.41	4.23	0.061
G1	21.51 ± 8.87	31.75 ± 8.41	10.24 ± 5.65	14.49	< 0.001 b
G2	20.77 ± 12.46	43.92 ± 16.95	23.14 ± 11.18	18.05	< 0.001 b
G3	20.43 ± 10.34	41.50 ± 15.33	21.06 ± 13.73	13.72	< 0.001 b

* Mean increment (from baseline to follow-up) in vitamin D serum concentration is measured as ng/ml

a Data is tabulated as Mean ± SD

b Paired t-test significant value of < 0.05

Risk factors of vitamin D insufficiency among study participants

Table 5 indicates the factors associated with vitamin D sufficiency and insufficiency. The patients who had vitamin D sufficiency (30–100 ng/ml) (n = 51) and those with insufficiency (n = 230) were compared with each other. There was a significant difference between the two groups for their educational status. Patients with lower education level, sun exposure for less than 30 minutes or no sun-exposure were associated with vitamin D deficiency. In the sufficiency group 74.5% were graduate and 27.5% had a daily sun-exposure for more than 1 hour.

Table 5

Comparison between patients having sufficient and insufficient status of vitamin D at baseline.

Characteristics	Sufficient (n = 51)	Insufficient (n = 230)	p value
Maternal age range; n (%)			0.308
18–27 years	21 (41.2%)	107 (46.5%)
28–37 years	29 (56.8%)	109 (47.4%)
38–47 years	1 (2.0%)	14 (6.1%)
BMI			0.153
Under-weight	0	14 (6.1%)
Normal	12 (23.5%)	65 (28.2%)
Over-weight	31 (60.8%)	108 (47.0%)
Obese	8 (15.7%)	43 (18.7%)
Mode of last delivery; n (%)			0.671
Normal	13 (25.5%)	59 (25.7%)
C-section	9 (17.6%)	53 (23.0%)
Total Number of pregnancies			0.421
PG/None	21 (41.2%)	109 (47.4%)
MG/More	30 (58.8%)	121 (52.6%)
Education; n (%)			0.004
Un-educated	0	4 (1.7%)
Primary	0	2 (0.9%)
Matric	4 (7.8%)	3 (1.3%)
Intermediate	6 (11.8%)	32 (13.9%)
Graduate	38 (74.5%)	134 (58.3%)
Post-Graduate	3 (5.9%)	55 (23.9%)
Occupation; n (%)			0.506
Un-employed	40 (78.4%)	161 (70.0%)
Student	3 (5.9%)	11 (4.8%)
Professional	8 (15.7%)	56 (24.3%)
Self-employed	0	2 (0.9%)
Average daily sun exposure			< 0.001
None	5 (9.8%)	81 (35.2%)
< 15 min	1 (2.0%)	53 (23.0%)
15–30 min	9 (17.6%)	79 (34.3%)
31–60 min	22 (43.1%)	15 (6.52%)
> 1 hour	14 (27.5%)	2 (0.9%)
Body area covered			0.440
Partially covered	44 (86.3%)	188 (81.7%)
Fully covered	7 (13.7%)	42 (18.3%)
Sunblock use			0.027
Yes	2 (3.9%)	36 (15.7%)
No	49 (96.1%)	194 (84.3%)
Use of Fish	0	8 (3.5%)	0.358
Use of Egg	27 (52.9%)	132 (57.4%)	0.562*
Use of Milk	28 (54.9%)	142 (61.7%)	0.366
Recruitment season			0.198
Autumn	26 (51.0%)	86 (37.4%)
Winter	15 (29.4%)	89 (38.7%)
Spring	10 (19.6%)	55 (23.9%)

: PG (Primigravida)–First time pregnancy; MG (Multigravida)—Multiple pregnancies, regardless of outcome

Chi-square test or Fisher Exact test was used to assess the association of variables

*Fisher exact test, while all other p values are from Chi-square test

To identify possible risk factors of vitamin D insufficiency among pregnant women, a series of logistic regression analysis was performed for clinically relevant and statistically tested variables (Table 6). Out of five tested variables, average daily sun-exposure (OR: 14.8, p = 0.009) and use of sunblock (OR: 4.4, p = 0.045) were two factors with a higher likelihood of vitamin D insufficiency. Patients with average daily sun exposure less than 15 minutes and those using sun block while going outside presented a higher risk of vitamin D insufficiency in this study. Seasonal variation was adjusted as covariate, there was no significant result of season on the baseline vitamin D levels.

Table 6

Risk factors for vitamin D insufficiency by regression model.

	Univariate analysis			Multivariate analysis
Variables	P value	OR	95% CI for OR	P value	OR	95% CI for OR
Education Level	0.765	0.9	0.41–1.9	-	-	-
Average daily Sun-exposure	0.008	14.9	2.1–110.9	0.009	14.8	2.0–109.7
Milk Consumption	0.367	1.3	0.7–2.5	-	-	-
Egg Use	0.562	1.1	0.7–2.2	-	-	-
Use of sunblock	0.042	4.6	1.1–19.5	0.047	4.4	1.1–19.3

p-values with > 0.250 were excluded from the Multivariate analysis

Odds Ratio (OR) and Confidence Interval (CI) have been rounded off

Codes for logistic regression

Education level—0: intermediate level or less, 1: graduation level or above

Average daily sun exposure—1: 0: More than 15 minutes per day, Less than 15 minutes per day

Milk consumption—0: No, 1: Yes

Egg use—0: No, 1: Yes

Use of sunblock while going outside—0: No, 1: Yes

Discussion

To the best of our knowledge, this is the first study to explore the impact of various vitamin D antenatal supplementation regimens among pregnant women in Pakistan. The key findings of the present study demonstrated the high prevalence of VDD during gestation. Women classified to G1, G2 and G3 groups when compared to those receiving no treatment experienced improved vitamin D status during the follow-up of 2 months. The high proportion of VDD in study population can be attributed to the various cultural, social, demographic and socioeconomic factors.

Despite adequate sunlight, high prevalence of VDD is reported in Pakistan. The reported prevalence of VDD as of 47.3%, insufficiency of 34.5% and sufficiency of only 18.1% is similar to the results of previously conducted studies in Pakistan [32-34]. Speculating from the results of the current study, VDD is probably much higher at national level than that reported in this study. The antenatal vitamin D supplementation is mainstay of therapy and widely recommended. On the other hand, comparatively higher prevalence (89% to 99.5%) of VDD was reported in some studies [33, 35]. Methodological variations among available studies led to the great disparity in the incidence as well as the epidemiology of VDD, making it difficult or even impossible to compare findings across the studies. Such varying prevalence might be attributable to several factors including different inclusion criteria with variable gestational age and BMI, variation in population with respect to financial status (our study site receives financially stable patients), different laboratory techniques for the estimation of serum 25(OH)D, different cut-off references for VDD and sufficiency, and inconsistent VDD definitions. There is an on-going debate on utility of criteria for vitamin D status [36, 37]. However, majority of the studies suggest the level of < 20 ng/ml for 25(OH)D as a cut-off value for VDD [23-26].

A recent systematic review concluded that VDD is highly prevalent and supplementation proved to be an effective intervention during gestation in Pakistan [38]. Sun-exposure and vitamin D rich diet alone cannot maintain adequate levels in pregnant women. Food fortification and creating awareness through public health programs will be of paramount importance to curb the growing burden of VDD in Pakistan. However, vitamin D supplementation is required in addition to diet and sun-exposure to achieve optimal concentration. It is evident from the previous investigations that vitamin D supplementation during pregnancy can improve both maternal and neonatal status for vitamin D [38]. The findings of these studies corroborate with our results. Socio-religious restrictions or limited outdoor activity results in decreased sun-exposure and in majority of cases even low dose of 600 IU is not prescribed. Present study used high doses of vitamin D including 2000 IU/day, 5000 IU/day and 200000 IU stat. It is evident from the previous studies that high dose of 200,000 IU is effective and considered safe [39-43].

Our findings indicate that vitamin D supplementation significantly increases the levels of serum 25(OH)D during pregnancy, particularly if the supplementation regimen was daily versus stat. However, this response was highly heterogeneous in different studies [15, 44–46]. Increment in 25(OH)D in G2 group receiving daily supplement was higher as compared to group receiving single dose (G3). It is important to note that in daily supplemented groups (G1 and G2), only high dose of 5000 IU (G2) showed higher serum 25(OH)D level in the current study.

Similar to the earlier studies, the beneficial effects of the dose 2000 IU/day are evident from the present study [15, 17, 18, 20, 47, 48]. However, the results and conclusions are heterogeneous, several studies concluded that 2000 IU/day dose do not achieve sufficiency in majority of the patients [17]. Despite the rise in serum 25(OH)D levels with the use of 2000 IU group, majority of the patients remained insufficient in their vitamin D status and hence higher dose should be preferred and recommended. Moreover, where low dose of vitamin D is recommended, patients should be encouraged and counselled to increase their daily sun-exposure up to 1 hour.

In the present study, the dose of 5000 IU/day was used to achieve the optimal vitamin D status. Yap et al., (2014) conducted a study on high and low daily doses of vitamin D and showed significantly higher plasma 25(OH)D levels achieved with 5000 IU/day dose. Authors concluded that supplementation with 5,000 IU/day vitamin D3 during pregnancy can safely and effectively elevate the serum 25(OH)D concentrations into the desired target range in 90% of the women [49]. In another similar study, 97% of women attained vitamin D concertation as of 80 nmol/l (32 ng/ml) at the time of delivery with dose 5000 IU/day [50]. These results are consistent with the findings of the present study and dose of 5000 IU/day was concluded as safe and effective to achieve the optimal concentration of 25(OH)D.

Existing data indicate that the high dose of 200,000 IU stat is effective and safe to achieve desired vitamin D status [44, 51]. These results are in line with our findings where sufficient serum 25(OH)D status was achieved in maximum number of patients. Our results in corroboration with other studies suggest that dose of 200,000 IU is effective and carries the advantage of compliance. Moreover, patients prescribed with high dose of vitamin D should be monitored for their serum 25(OH)D levels. It is pertinent to mention that one safety measure was taken and further supplementation was stopped as per safety protocol in the G3 group. Serum vitamin D concentration was 108 ng/ml in this patient. Further investigations revealed that patients was taking drug at multiple times along with other multivitamins. Fortunately, the serum biochemical indices were within the normal range and USG findings indicates no stones in either kidneys. These findings suggest the periodic monitoring of patients receiving high dose. These patients must be educated to avoid concurrent use of other multivitamins, excessive sun-exposure, use of sunblock and to monitor any unwanted effects. The baseline 25(OH)D levels should be estimated before administering 200000 IU dose. The use of HD should be avoided if patients have sufficient vitamin D status at baseline. In such cases, lower doses of vitamin D would be effective and preferred. Our findings manifested that supplemented patients showed substantial improvements in the vitamin D status. The doses of 5000 IU/day and 200000 IU stat are comparable but the high dose necessitate monitoring. Moreover, the dose of 200000 IU stat carries an advantage of compliance and can effectively be used with vigorous monitoring of any toxicity.

Prevalence of VDD was evidently associated with various factors including practice of veil, limited sun-exposure and ethnicity in South Asian countries [36, 52–57]. Similar to the other studies [32, 53, 58], factors such as exposure to sunlight and use of sunblock are found to be independent predictor of vitamin D insufficiency the current study (Table 5). We analysis revealed that patients having average daily sun exposure of less than 15 minutes portend high propensity of vitamin D insufficiency. Similarly, the use of sunblock before going outside increase the risks of vitamin D insufficiency by four times. These findings underscore that in addition to supplementation, pregnant female must be encouraged to have adequate sun-exposure which could be beneficial for attaining the optimal serum 25(OH)D [59]. Findings of the previous investigations with significant association of sun-exposure with insufficient vitamin D level were consistent with this study [32]. However, dressing habits and impact of vitamin D rich diet did not show any significant association with VDD in the present study. In contrast to the previous studies, there was no association between BMI and vitamin D status during our analysis. It is pertinent to mention that women in Pakistan spend most of their time indoor due to household activities and cultural norms. This indoor time further increases among pregnant women due to common beliefs of rest and restricted movement for baby care. These cultural or societal norms must be considered during the interpretation of results.

It is important to note that VDD has been found to be positively associated with low socioeconomic status [60]. However, these findings are contrary to the results of the present study. The majority of the patients in our study were from good socioeconomic status but still had a high prevalence of VDD similar to another study conducted in Pakistan in which nursing mothers belonged to upper socioeconomic class [61]. These findings urge the need of education and awareness as a pivotal key to reduce the growing encumbrance of VDD in Pakistan. Patients should be educated about the significance of supplementation, factors associated with VDD and sources of vitamin D. Educational campaigns and patient counselling at the antenatal visit regarding VDD could be of paramount importance for pregnant women. A clinical pharmacist can play a crucial role in this regard.

Screening for VDD at gestation and implementation of vitamin D supplementation could be considered during antenatal care. Policy makers, nutritionists and other healthcare professionals can establish their roles to increase the awareness regarding VDD consequences. Moreover, food fortification of staple food should be initiated at National level. Health programs creating awareness regarding sun-exposure, dietary modification and supplementation should be initiated at both public and private healthcare facilities.

Study limitations and strengths

This study was a single-centered study, results of which cannot be extrapolated to larger population. Current study was only conducted in Lahore city and hence requirements of women living in rural areas, other provinces, and different latitudes could be different. Inter-laboratory variation may have affected the serum 25(OH)D value of an individual as different cut-off reference values and varying techniques may have been used in different laboratories. No follow-up of neonates was done to determine the effects of adequate maternal 25(OH)D levels on neonatal health. Follow-up with neonate and measuring cord 25(OH)D levels would have further enlighten the significance of maternal supplementation. It must be noted that most of the study participants were recruited during the winter and autumn months of the year in which UV index is comparatively low and most of the women reside in their homes due to the cold waves. In this context, caution must be carried out to interpret the results for summer recruits. Last but not least, we tried to rigorously adjust the co-variates during the analysis. However, data on many important confounders which may affect vitamin D levels were missing for many patients due to observational nature of the study. This confounding affect can be explicitly adjusted in randomized controlled trials. Nonetheless, equal distribution of co-variates among treatment groups in the current study minimizes the risks of bias. We suggest careful consideration of this limitation during the interpretation of results and validation of findings. Moreover, there was no validated form used to assess the patient compliance with the regimen but compliance was reassured through self-reporting. Future studies incorporating these limitations are direly suggested.

Despite aforementioned shortcomings, this study provides data on prevalence of vitamin D specifically in Pakistani pregnant population and contains important information which can be used to address appropriate supplementation regimens in the country which could be translated into improved vitamin D status during gestation. Findings of the current study may provide the basis to formulate guidelines and recommendations for vitamin D supplementation among pregnant women. Considering the dearth of regional investigations in Pakistan, results of the present study will serve to strengthen the field of research in the country. This study generates the hypothesis that vitamin D supplementation at a dose of 5000 IU/day during pregnancy is superior to the other regimens. However, well-controlled randomized trials are needed to confirm these findings.

Conclusions

Current study suggests high proportion of VDD among pregnant women in Pakistan. Antenatal vitamin D supplementation proved to be an effective intervention and may benefit all the VD insufficient pregnant female. The stat dose of 200000 IU is equally effective as 5000 IU/day dose and also carries additional advantage of compliance but the propensity of drug toxicity cannot be disregarded. Future research should evaluate neonatal consequences of VDD and determine any association between the vitamin D status and BMI. There is a dire need to have randomized control trials (RCTs) to ascertain the effectiveness of various dosing regimens of vitamin D in mothers and neonates.

13 Dec 2019

PONE-D-19-29625

Daily versus Stat Vitamin D Supplementation During Pregnancy; A prospective Cohort Study

PLOS ONE

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Reviewer #1: This study, conducted in Pakistan, evaluate the effectiveness of three different regimes of vitamin D (VD) supplementations among pregnant women. Some details should be clarified before further evaluation.

1. Pregnant women were stratified into four groups (one control group, and three VD intervention groups). Were they randomly allocated to each group? If not, how was the dose of VD determined for each woman?

2. How long was the interval of side effects of VD supplementation monitored?

3. There were two methods employed to measure VD levels. The reason for using two different methods should be described. Besides, did the author assess the difference between the two methods. Moreover, the author said vitamin D3 (likely to be 25(OH)D3) were measured to assess the 25(OH)D concentrations. Why 25(OH)D2 was not measured.

4. Fisher exact test should be noted in the Tables if such a test was used. The Chi square test (or Fisher exact test) provided only one P-value, why there were multiple P-values provided for each category of a variable in Table 1 (also Table 4). Gravidity is defined as the number of times that a woman has been pregnant, which is equal to the “Total Number of pregnancies” in the table. Parity in Table 1 should better be presented as a categorical variable.

5. The difference in mean VD level at baseline and follow-up between all groups were tested using one-way ANOVA. Were the data satisfied the assumptions of one-way ANOVA?

6. The differences in VD levels between baseline and follow-up within each treatment group were tested, while the differences among groups were not tested. Thus, it is not rigorous to conclude that the group with 5,000 IU/d VD supplementation had the highest level of VD increment. Additionally, season is a crucial influencing factor of VD levels, and the authors should take it into account when analyzing and interpreting the result.

7. Multivariate analysis, other than Chi square test, should be performed to test the risk factors between women with sufficient and insufficient VD.

8. The sum of each category of the variable in Table 4 was not equal to the total number of each arm. The authors should recheck the data carefully.

9. English writing should be improved. There are numerous typos and grammar errors (a lot of missing articles).

Reviewer #2: First study to explore the impact of various vitamin D antenatal supplementation regimens among pregnant women in Pakistan.

1) Information about Food source of vitamin D should be added on data collection. Have the authors asked about the participants’ diet? Food frequency questionnaire could be applied.

2) Maternal vitamin D status should be followed through pregnancy. Comparison between early and late gestation might be important.

3) Follow up with neonates should be addressed and/or umbilical cord blood could be used to measure levels of 25(OH)D.

4) Explain how the maternal blood was collected and processed.

5) Deficiency of Vitamin D has been associated with risks for preeclampsia. Have the authors collected information about blood pressure?

6) The follow reference should be added on the current paper. PMID: 31669079; DOI: 10.1016/j.jand.2019.07.002

Reviewer #3: This study is important because it indicates the amount of vitamin D needed to achieve normal levels. Vit D could plan an important role to control inflammation and hypertension during pregnancy.

What does the G stand for “Patients received various regimens of vitamin D including 2000 IU/day (G1), 5000 IU/day (G2) and stat 200000 IU (G3). The g should define group I thought it was a typo

Can the authors retrieve data on blood pressure or any inflammatory markers. Could the prevalence of hypertension be determined. Could extrapolations to fetal weight or health be made?this would make the paper much more exciting.

The clinical methods description is very vague, there is now detail about how blood was collected.

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Reviewer #1: Yes: Yunxian Yu, Bule Shao

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Reviewer #3: No

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8 Jan 2020

Point-by-Point response to reviewers

Respected Editor,

We have received revisions/suggestions for our submitted manuscript. All the concerns and suggestions of the reviewers have been addressed and we hope that the revised version of the manuscript will satisfy the concerns of all reviewers. We have attached point-by-point response to each reviewer and revised version of manuscript is highlighted to track the changes. Please let us know if any other changes are required in this regard. Last but not least, we are very thankful to editor and reviewers for their time and efforts to put their valuable suggestions. Indeed their recommendations made this manuscript more scientifically elegant and sound.

Reviewer 1

This study, conducted in Pakistan, evaluate the effectiveness of three different regimes of vitamin D (VD) supplementations among pregnant women. Some details should be clarified before further evaluation.

Query – 1: Pregnant women were stratified into four groups (one control group, and three VD intervention groups). Were they randomly allocated to each group? If not, how was the dose of VD determined for each woman?

Response – 1: Respected Reviewer, Thank you very much for pointing out the ambiguity in the manuscript. All the pregnant women attending OPD of the Hospital and fulfilling the inclusion criteria during the study period were consecutively included into the analysis. Patients were classified into four groups according to the VD dose they received during their visit. The choice of vitamin D dose was beyond our control and was at the complete discretion of the treating physician. Due to the observational nature of the study, it was not possible for us to randomly allocate the patients into the different treatment groups. This information on patients’ stratification has been elaborated and updated in the manuscript. We hope that the correct version would be satisfactory. There is another study (Wenisch et al.) comparing three different dosage regimens of antibiotic which has followed the similar methodology.

• Wenisch JM, Schmid D, Kuo H-W, Allerberger F, Michl V, Tesik P, et al. Prospective observational study comparing three different treatment regimes in patients with Clostridium difficile infection. Antimicrobial agents and chemotherapy. 2012;56(4):1974-8.

Query – 2: How long was the interval of side effects of VD supplementation monitored?

Response – 2: Respected Reviewer, side-effects of VD supplementation was monitored throughout the follow-up period of the study (2 months). This information has been updated in the manuscript as well.

Query – 3: There were two methods employed to measure VD levels. The reason for using two different methods should be described. Besides, did the author assess the difference between the two methods. Moreover, the author said vitamin D3 (likely to be 25(OH)D3) were measured to assess the 25(OH)D concentrations. Why 25(OH)D2 was not measured.

Response – 3: Respected Reviewer, Thank you very much for highlighting the ambiguity. There was only one method used to measure VD status, using serum total 25(OH)D level. Use of 1,25(OH)2D and 25(OH)D tests to assess the vitamin D status is still debatable. However, in order to address this concern, studies conducted by Kennel et al (2010) and Lips et al (2007) can be used as reference. In these studies, authors concluded that although 1,25(OH)2D is the active form of vitamin D, serum 25(OH)D is the barometer for the vitamin D status of an individual due to its longer half-life. Heaney (2004) also considered 25(OH)D to be the best indicator of Vitamin D status.

The technique used to ascertain the vitamin D status in the current manuscript was chemiluminescent immunoassay (CLIA), a quantitative method which measures total 25(OH)D and did not quantify D2 and D3 separately. This method is widely used by clinicians to assess Vitamin D status during their practice (Moon et al., 2016, Yu et al., 2009). CLIA has been used in various studies and a recent systematic review by Gallo et al., provides the utility of this method in wide range of studies. We hope that our response is satisfactory to address the concern of reviewer. Following is the list of references discussed in this response.

• Kennel KA, Drake MT, Hurley DL. Vitamin D deficiency in adults: when to test and how to treat. Mayo Clinic proceedings. 2010;85(8):752-8.

• Lips P. Relative value of 25 (OH) D and 1, 25 (OH) 2D measurements. Journal of Bone and mineral Research. 2007;22(11):1668-71.

• Moon RJ, Harvey NC, Cooper C, D'Angelo S, Crozier SR, Inskip HM, et al. Determinants of the Maternal 25-Hydroxyvitamin D Response to Vitamin D Supplementation During Pregnancy. The Journal of Clinical Endocrinology & Metabolism. 2016;101(12):5012-20.

• Yu CKH, Sykes L, Sethi M, Teoh TG, Robinson S. Vitamin D deficiency and supplementation during pregnancy. Clinical Endocrinology. 2009;70(5):685-90.

• Gallo S, McDermid JM, Al-Nimr RI, Hakeem R, Moreschi JM, Pari-Keener M, et al. Vitamin D Supplementation during Pregnancy: An Evidence Analysis Center Systematic Review and Meta-Analysis. J Acad Nutr Diet. 2019.

Query – 4: Fisher exact test should be noted in the Tables if such a test was used. The Chi square test (or Fisher exact test) provided only one P-value, why there were multiple P-values provided for each category of a variable in Table 1 (also Table 4). Gravidity is defined as the number of times that a woman has been pregnant, which is equal to the “Total Number of pregnancies” in the table. Parity in Table 1 should better be presented as a categorical variable.

Response – 4: Respected Reviewer, Thank you for correcting the mistake regarding the p-values. Based on your suggestion, all the p values are corrected and only one value is presented for more than 2 by 2 variables. Chi square test or Fisher exact test are described in the methodology and have been indicated in the tables, where applicable.

Query – 5: The difference in mean VD level at baseline and follow-up between all groups were tested using one-way ANOVA. Were the data satisfied the assumptions of one-way ANOVA?

Response – 5: Respected Reviewer, the groups were compared using One-Way ANOVA or Kruskal-Wallis Test depending on the data distribution. There were only four continuous variables (Mean VD level at baseline, Mean VD level at follow-up, age and BMI) and all of them were normally distributed.

Query – 6: The differences in VD levels between baseline and follow-up within each treatment group were tested, while the differences among groups were not tested. Thus, it is not rigorous to conclude that the group with 5,000 IU/d VD supplementation had the highest level of VD increment. Additionally, season is a crucial influencing factor of VD levels, and the authors should take it into account when analyzing and interpreting the result.

Response – 6: Respected Reviewer, the difference among the groups was also tested using paired t-test. Table 3 shows the difference at baseline and follow-up within each treatment group. Please refer to the Table 2 in the manuscript which shows the difference among the groups in the baseline as well as during follow-up. However, seasonal variation was not considered during the study, which should have been taken into account. However the data was collected in the relatively colder months of year in the country. Moreover, the data was collected in Peak gynae season in Pakistan which occurs from September to Feb 2019. To increase the applicability of the study and to get sufficient number of patients this study was conducted in these months. However, we have taken into account the potential impact of this season on VD. Since November to February is coldest period in the country in which sun exposure increases, we suggested considering such impact during the interpretation of results. Such impact of influencing factors has been discussed in the discussion section of the manuscript.

Query – 7: Multivariate analysis, other than Chi square test, should be performed to test the risk factors between women with sufficient and insufficient VD.

Response – 7: Respected Reviewer, Thank you very much for the recommendation. Based on your suggestion, we performed logistic regression analysis to ascertain the independent predictors associated with vitamin D insufficiency. The manuscript has been updated accordingly. Please refer to the Result where a new table (Table No. 5) is added. All the variables included for the analysis were either statistically tested or those having clinical plausibility with the outcome of interest.

Query – 8: The sum of each category of the variable in Table 4 was not equal to the total number of each arm. The authors should recheck the data carefully.

Response – 8: Respected Reviewer, Thank you very much for pointing out the mistakes in Table 4, the information has been updated in the manuscript.

Query – 9: English writing should be improved. There are numerous typos and grammar errors (a lot of missing articles).

Response – 9: Respected Reviewer, The manuscript has been proofread by native English speaker. Numerous typos, grammar and syntax errors have been addressed. We hope that the current version will satisfy the Reviewer`s concerns regarding the readability of the manuscript.

REVIEWER # 2

First study to explore the impact of various vitamin D antenatal supplementation regimens among pregnant women in Pakistan

Query - 1: Information about Food source of vitamin D should be added on data collection. Have the authors asked about the participants’ diet? Food frequency questionnaire could be applied.

Response – 1: Respected Reviewer, the data regarding the intake of vitamin D rich food source were collected as the part of data collection. Food frequency questionnaire was used to obtain the patient’s data on the food intake prior to the supplementation. However the data was not used previously. Relevant data has been used and the manuscript has been updated accordingly. As per you suggestion, this data is added in Table 1 of the manuscript.

Query – 2: Maternal vitamin D status should be followed through pregnancy. Comparison between early and late gestation might be important.

Response – 2: Respected reviewer, thank you so much for highlighting this important issue. Undoubtedly, data on pregnancy outcomes and gestational timing is of great importance. Unfortunately, we were unable to extract this information due to time constraints. Since this study was a postgraduate project to secure Master degree, investigators were only able to collect limited data required to test pre-defined objectives due to restricted time to complete the project. All the procedures or tasks related to the current project including synopsis writing, proposal defense, ethical approval, data collection, data analysis, drafting of results and submission of the report were supposed to be completed in one year of student candidature. In this context, only limited data was retrieved to achieve the predefined study objectives. However, to strengthen the impact of the data, vitamin D status was assessed twice (at baseline and during follow-up). Te objective of current studies were determined by keeping in view the current problem faced by Pakistan i.e. which dose of vitamin D is effective to maintain optimal vitamin D status in pregnant women. A similar study is conducted by Kaloczi et al (2014) where vitamin D levels were measured twice (at baseline and 28th week of gestation), as we did in our study. In another study by Moon et al (2016) assessed the vitamin D after 34th weeks of gestation. We were only able to include follow-up data of two months due to the limitations as described above. However, we have mentioned this limitation in the manuscript. We will definitely incorporate this suggestion in our future clinical trials we have planned in order to assess the effectiveness of vitamin D dosages in varying population of pregnant women. The trial will be initiated in this year. Following are the list of references discussed above in this response.

• Kaloczi LD, Deneris A. Rate of Low Vitamin D Levels in a Low-Risk Obstetric Population. Journal of Midwifery & Women's Health. 2014;59(4):405-10.

• Moon RJ, Harvey NC, Cooper C, D’Angelo S, Crozier SR, Inskip HM, et al. Determinants of the maternal 25-hydroxyvitamin D response to vitamin D supplementation during pregnancy. The Journal of Clinical Endocrinology & Metabolism. 2016;101(12):5012-20.

Query – 3: Follow up with neonates should be addressed and/or umbilical cord blood could be used to measure levels of 25(OH)D.

Response – 3: Respected reviewer, thank you so much for highlighting this important issue. Undoubtedly, data on neonatal outcomes and vitamin D status is of paramount importance. Unfortunately, we were unable to extract this information due to time constraints, as described in the previous query. However, we have addressed these limitations in the limitation section of the study. Furthermore, we would like to underscore that previous studies also did not record the cord blood 25(OH)D level as the study objectives were not extended to the measure the neonatal outcome of vitamin D supplementation. Kaloczi et al (2014) conducted a study in which 25(OH)D level was measured initially at first visit and at 28th week of gestation to evaluate effectiveness of vitamin D supplementation and no cord blood was tested. Moreover, the hospital setup was a non-charity structure institution and co-investigators could not bear the cost of the test as it was much expensive. However, these recommendations will be incorporated in future studies that we are planning. We are designing a clinical trial to be conducted in 2020 based on the current findings and we are in the phase of getting funding for this project. Your recommendations of measuring the levels of 25(OH)D throughout the gestation and at the time of delivery (using cord blood) will definitely be incorporated to improve the study and its impact in clinical practice. Following are the references used to support the query.

• Kaloczi LD, Deneris A. Rate of Low Vitamin D Levels in a Low-Risk Obstetric Population. Journal of Midwifery & Women's Health. 2014;59(4):405-10.

Query – 4: Explain how the maternal blood was collected and processed.

Response – 4: Respected Reviewer, Thank you for highlighting the missing information. Informed patient consent was obtained before the commencement of the study. Purpose of the study was briefed to the patients. 5 milliliters of maternal blood sample was obtained for the assessment purposes. Vitamin D status was assessed using Immunoassay technique i.e. Chemiluminescence Immunoassay Analyzer (CLIA). The detailed process of maternal blood collection has been added in the manuscript (under the heading methodology, sub-heading of Biochemical Analysis.

“Biochemical Analysis:

Vitamin D3 estimation was performed by chemiluminescence or CLIA (Chemiluminescence Immunoassay Analyzer) technique using state of the art Maglumi® 600 fully automated system. Maternal blood (5 milliliters) was collected at baseline and again at the follow-up. Blood samples were centrifuged and stored at -80° C followed by Chemiluminescence to assess the 25(OH)D level (ng/ml). Serum Vitamin D3 concentration was measured in Nano grams per milliliter (ng/ml). The cut-off reference points used to define vitamin D status in this study were < 20 ng/ml as deficiency, 20 to < 30 ng/ml as insufficiency, 30-100 ng/ml as sufficiency and > 100 ng/ml as toxicity (3-14). Maternal serum calcium levels were assessed using spectrometry method at the follow-up to rule out any manifestation of vitamin D intoxication.”

Query – 5: Deficiency of Vitamin D has been associated with risks for preeclampsia. Have the authors collected information about blood pressure?

Response – 5: Respected Reviewer, Blood pressure was measured by attending physician in the clinic but was not recorded in the file. However, BP measurements were recorded for those diagnosed with pre-eclampsia or eclampsia. The diagnosis of pre-eclampsia was mentioned on the file. We re-checked the patients` record and were unable to find any case of pre-eclampsia for the present study.

Since the primary purpose of the current study was to compare the supplementation regimen, obstetric complications such as pre-eclampsia was not considered for the current study. Blood pressure was measured by the attending physician but not documented unless the patient was diagnosed with pre-eclampsia or eclampsia. The diagnosis was mentioned on the files of the patients. However in this study, no case of pre-eclampsia or eclampsia was present. We have again checked the patient record and found no such data with the indication of pre-eclampsia. However, we would like to underscore that various studies conducted earlier with a primary objective of supplementation comparison during gestation also did not record the Blood pressure as the parameter to be studied. Hollis et al (2011) conducted a study and primary objective was to evaluate safety and effectiveness of Vitamin D during pregnancy; however less attention was paid to the epidemiological features and other parameters associated with VDD. Similarly, other studies conducted by Rodda et al (2015), Cooper et al (2016) and Kaloczi et al (2014) did not record these parameters throughout the study as their primary objective was comparing of dosing regimen. Since your comments are of much value and helpful for us, any further suggestion/guidance in the analysis will be warmly welcomed. Following are the list of references provided to support the current query.

• Kaloczi LD, Deneris A. Rate of Low Vitamin D Levels in a Low-Risk Obstetric Population. Journal of Midwifery & Women's Health. 2014;59(4):405-10.

• Hollis BW, Johnson D, Hulsey TC, Ebeling M, Wagner CL. Vitamin D supplementation during pregnancy: double-blind, randomized clinical trial of safety and effectiveness. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2011;26(10):2341-57.

• Rodda CP, Benson JE, Vincent AJ, Whitehead CL, Polykov A, Vollenhoven B. Maternal vitamin D supplementation during pregnancy prevents vitamin D deficiency in the newborn: an open-label randomized controlled trial. Clinical Endocrinology. 2015;83(3):363-8.

• Cooper C, Harvey NC, Bishop NJ, Kennedy S, Papageorghiou AT, Schoenmakers I, et al. Maternal gestational vitamin D supplementation and offspring bone health (MAVIDOS): a multicentre, double-blind, randomised placebo-controlled trial. The Lancet Diabetes & Endocrinology. 2016;4(5):393-402.

Query – 6: The follow reference should be added on the current paper. PMID: 31669079; DOI: 10.1016/j.jand.2019.07.002

Response – 6: Respected Review, Thank you very much for the recommendation. The recommended reference has been added in the discussion part of the manuscript.

REVIEWER # 3

Query-1: This study is important because it indicates the amount of vitamin D needed to achieve normal levels. Vit D could plan an important role to control inflammation and hypertension during pregnancy.

Response-1: Respected reviewer, thank you for acknowledging the significance of the current study. Though the impact of vitamin D on inflammation control and hypertension is of significant value, but we were unable to analyze such factors in the current study due to several reasons. First, data on blood pressure was not available on patient`s file and record. In our hospital, attending physician check blood pressure in the clinic and most of the time does not record the readings in the patient`s file. Such readings are recorded for patients with pre-eclampsia and there was no diagnosis of pre-eclampsia in the current study. Secondly, due to financial constraints inflammatory markers were not determined for the current study. Since the primary objective of the current study was to compare the dosing regimens of supplementation, the least attention was paid to other confounders. However, in align to your recommendations we will consider these parameters in our future study which we will start in 2020. We are planning to conduct a clinical trial based on the current findings and your recommendations will definitely be incorporated in the objectives of the study.

Query-2: What does the G stand for “Patients received various regimens of vitamin D including 2000 IU/day (G1), 5000 IU/day (G2) and stat 200000 IU (G3). The g should define group I thought it was a typo

Response-2: Respected reviewer, Thank you so much for pointing out the mistake. We have addressed this typo throughout the manuscript. The G represents the Group:

G = Group; G1 = Group 1 with 2000 IU dose, G2 = Group 2 with 5000 IU dose and G3 = Group 3 with 200,000 IU dose

Query-3: Can the authors retrieve data on blood pressure or any inflammatory markers. Could the prevalence of hypertension be determined. Could extrapolations to fetal weight or health be made?this would make the paper much more exciting.

Response-3: Respected reviewer, these variables were not considered before the commencement of the study. Blood pressure was measured by attending physician in the clinic but was not recorded in the file on every visit. However, BP measurements were recorded for those diagnosed with pre-eclampsia or eclampsia. The diagnosis of pre-eclampsia was mentioned on the file. We re-checked the patient record and were unable to find any case of pre-eclampsia for the present study. Moreover, as previously described, the study the study was purely observational in nature and ordering new tests was beyond the protocol. Moreover, the hospital where current study is conducted in no-charity institution and ordering of such tests was quite costly for patients as well as researchers.

Moreover, we would like to underscore that various studies conducted earlier with a primary objective of supplementation comparison during gestation also did not record the Blood pressure during the study period. Hollis et al (2011) conducted a study of similar nature and their primary objective was to evaluate safety and effectiveness of Vitamin D during pregnancy; however less attention was paid to the other obstetric complications and their association with VDD. Some other studies conducted by Rodda et al (2015), Cooper et al (2016) and Kaloczi et al (2011) also did not record these parameters throughout the study as their primary objective was regimen comparison. Moreover, the data regarding the inflammatory markers is not a part of routine tests in our hospital, so we were unable to retrieve the data. Last but not least, current project was part of postgraduate study for which we have only one year to plan a study, conducting a study, preparing results and submission of research report. Therefore, neonatal and gestational outcomes were not evaluated in the current study. However, we have incorporated your suggestions in our upcoming project which will start in 2020 as a doctorate degree project. Following is the list of references used to support the current query.

• Kaloczi LD, Deneris A. Rate of Low Vitamin D Levels in a Low-Risk Obstetric Population. Journal of Midwifery & Women's Health. 2014;59(4):405-10.

• Rodda CP, Benson JE, Vincent AJ, Whitehead CL, Polykov A, Vollenhoven B. Maternal vitamin D supplementation during pregnancy prevents vitamin D deficiency in the newborn: an open-label randomized controlled trial. Clinical Endocrinology. 2015;83(3):363-8.

• Cooper C, Harvey NC, Bishop NJ, Kennedy S, Papageorghiou AT, Schoenmakers I, et al. Maternal gestational vitamin D supplementation and offspring bone health (MAVIDOS): a multicentre, double-blind, randomised placebo-controlled trial. The Lancet Diabetes & Endocrinology. 2016;4(5):393-402.

Query-4: The clinical methods description is very vague, there is now detail about how blood was collected.

Response-4: Respected reviewer, the clinical methods description is revised in the manuscript. The detail of blood sample collection has been added to the manuscript; under sub-heading of “outcome measures”, in the Methodology section. We hope the current version will satisfy the concern of the Reviewers.

At the end, we are greatly thankful to all three reviewers for their valuable time and suggestions. Indeed their recommendations helped us a lot to make the current manuscript more scientifically sound and correct. Any further suggestions related to the current manuscript is warmly welcomed.

Dr Yusra Habib Khan

Corresponding author

Submitted filename: Response to reviewers.docx

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3 Feb 2020

PONE-D-19-29625R1

Daily versus Stat Vitamin D Supplementation During Pregnancy; A prospective Cohort Study

PLOS ONE

Dear Dr Khan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Kind regards,

Frank T. Spradley

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:

The authors have addressed most of the questions. However, some were yet to be solved.

Season or month of the year at the baseline should be taken into account throughout all analyses, given it is a crucial influencing factor of vitamin D (VD) level.

As is shown in Table 2, VD levels at the baseline and follow-up were compared among the four groups, respectively. Besides, paired t-tests were performed to examine the VD increment after VD supplementation (Table 3) within each group. However, we would like to know whether the difference of VD increment among the four groups is statistically different (especially between G2 and G3). Thus, the VD increment of each group should be compared, and a multivariate model should be employed to control the confounding of baseline VD level, and more importantly, the season (or month of the year) at the baseline, as well as other potential confounders.

Did the authors assess the compliance of each intervention group regarding VD supplementation?

In the Statistical Analysis section, "Fischer exact test" is supposed to be “Fisher exact test.”

**********

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Reviewer #1: Yes: Yunxian Yu, Ph.D. Bule Shao

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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Submitted filename: Comments on PONE-D-19-29625_R1.docx

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9 Mar 2020

RESPONSE TO REVIEWERS

Respected Editor,

We have received second round of revisions/suggestions for our submitted manuscript. All the concerns and suggestions of the reviewer have been addressed and we hope that the revised version of the manuscript will satisfy the concerns of all reviewer. We have attached point-by-point response to the reviewer and revised version of manuscript is also highlighted to track the changes. Please let us know if any other changes are required in this regard. Last but not least, we are very thankful to editor and reviewers for their time and efforts to put their valuable suggestions. Indeed their recommendations made this manuscript more scientifically elegant and sound.

REVIEWER # 1

Query – 1: Season or month of the year at the baseline should be taken into account throughout all analyses, given it is a crucial influencing factor of vitamin D (VD) level.

Response – 1: Respected reviewer, thank you for the recommendation. The study was carried out in the colder months of the country i.e. October 2018 to April 2019 [Autumn = 22nd September – 21st December, Winter = 22nd December – 20th March, Spring = 21st March - 21st June]. A total of 112 patients were recruited in Autumn season, 104 in Winter and 65 in Spring. The UV index (UVI) is comparatively low in these months. The optimal UVI for the production of Vitamin D (D-UV) is greater than 3.1,2 However, the UVI decreases from October to December and then raises from January to April. Moreover, Chi-square test was preformed to check association of season with the VD level at the baseline; the test was insignificant (p = 0.072). With respect to your conerns, we have also highlighted the same issue in the limitation section of the manuscript.

1. Holick MF. Vitamin D Deficiency. N Engl J Med 2007; 357: 266-81.

2. Hollick MF, Chen TC. Vitamin D deficiency: a worldwide problem with health consequences. Am J Clin Nutr 2008; 87: 1080S-6S.

Query – 2: As is shown in Table 2, VD levels at the baseline and follow-up were compared among the four groups, respectively. Besides, paired t-tests were performed to examine the VD increment after VD supplementation (Table 3) within each group. However, we would like to know whether the difference of VD increment among the four groups is statistically different (especially between G2 and G3). Thus, the VD increment of each group should be compared, and a multivariate model should be employed to control the confounding of baseline VD level, and more importantly, the season (or month of the year) at the baseline, as well as other potential confounders.

Response – 2: Respected Reviewer, Thank you for the suggestion. VD increment between all the groups has been checked as per your recommendation. All groups shows that there is a significant difference between the control group and treatment cohorts (G1, G2 and G3); (p < 0.001). The treatment groups were also compared with each other and there was statistically significant difference [G1 and G2; (p < 0.001); G1 and G3; (p < 0.001)]. However, there was no statistically significant difference between G2 and G3 (p = 0.579). This information has been updated in the manuscript in the text before Table 3. To the best of our knowledge, if covariates are equally distributed between the treatment groups then confounder adjustments are not necessary during the analysis. However, we respect your suggestion and carried out regression analysis in which we adjusted the season to determine the potential contributors of vitamin D insufficiency among study participants.

Query – 3: Did the authors assess the compliance of each intervention group regarding VD supplementation?

Response – 3: Respected Reviewer, patient compliance was self-reported by patients and was assessed during the follow-up visit. All the patients in the treatment groups were compliant with their medication regimen.

Query – 4: In the Statistical Analysis section, “Fischer exact test” is supposed to be “Fisher exact test.”

Response – 4: Respected Reviewer, Thank you for pointing out the typo. Manuscript has been updated accordingly.

Corresponding author: Yusra Habib Khan

Submitted filename: Point by Point Response.docx

Click here for additional data file.

13 Mar 2020

PONE-D-19-29625R2

Daily versus Stat Vitamin D Supplementation During Pregnancy; A prospective Cohort Study

PLOS ONE

Dear Dr Khan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

SPECIFIC ACADEMIC EDITOR COMMENT: There are few minor comments that still need addressing by the authors.

We would appreciate receiving your revised manuscript by Apr 27 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Frank T. Spradley

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. In the abstract, please provide the VDD frequency after intervention.

2. Conclusion has better be simplified.

3. While the effect of VD supplement was compared between groups, the VD influence factors such as sun-exposure time, Use of sunblock and baseline VD level must be adjusted. This part is the main result for evaluating the effect of intervention. Then add this part to manuscript.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

25 Mar 2020

RESPONSE TO REVIEWERS

Respected Editor,

We have received third round of minor revisions/suggestions for our submitted manuscript. All the concerns and suggestions of the reviewer have been addressed and we hope that the revised version of the manuscript will satisfy the concerns of the reviewer. We have attached point-by-point response to the reviewer and revised version of manuscript is also highlighted to track the changes. Please let us know if any other changes are required in this regard. Last but not least, we are very thankful to editor and reviewers for their time and efforts to put their valuable suggestions. Indeed their recommendations made this manuscript more scientifically elegant and sound.

REVIEWER # 1

Query – 1: 1. In the abstract, please provide the VDD frequency after intervention.

Response – 1: Respected Reviewer, VDD frequency has been added in the abstract section.

Query – 2: 2. Conclusion has better be simplified.

Response – 2: Respected Reviewer, as per your suggestion we have simplied the conclusion section of the manuscript. I hope that current version of conclusion will satisfy the concern.

Query – 3: While the effect of VD supplement was compared between groups, the VD influence factors such as sun-exposure time, Use of sunblock and baseline VD level must be adjusted. This part is the main result for evaluating the effect of intervention. Then add this part to manuscript.

Response – 3: Respected Reviewer, the confounding factors has been adjusted. This resulted in addition of another table controlling the impact of covariates on outcome variable. We have also modified the results according to the new table. We really hope that the current version of manuscript will satisfy your concern in this regard.

At the end, are are greatly thankful for your time and interest in our research . Indeed, your suggestions were quite valuable for us. Our believe on peer review is more strenghthened amid healthy discussion pertaining to current research.

Corresponding author: Yusra Habib Khan

Submitted filename: Point by Point Response.docx

Click here for additional data file.

27 Mar 2020

Daily versus Stat Vitamin D Supplementation During Pregnancy; A prospective Cohort Study

PONE-D-19-29625R3

Dear Dr. Khan,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Frank T. Spradley

Academic Editor

PLOS ONE

3 Apr 2020

PONE-D-19-29625R3

Daily versus Stat Vitamin D Supplementation During Pregnancy; A prospective Cohort Study

Dear Dr. Khan:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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