Correlation between maternal and neonatal blood Vitamin D level: Study from Pakistan

Abstract In Pakistan, there is limited evidence for the levels and relationship of 25 (OH) Vitamin D (25(OH)D) status in pregnant women and their newborns, while the association between maternal 25(OH)D and newborn anthropometric measurements remains unexplored. Sociodemographic data were collected from 213 pregnant mothers during their visit to a tertiary care hospital at the time of childbirth. Anthropometric measurements were performed on all mothers and their newborns and blood samples collected from both for 25(OH)D levels. Participants were classified into two groups according to their 25(OH)D status: sufficient (25(OH)D ≥50 nmol L−1) and deficient (25(OH)D <50 nmol L−1). Simple and multiple regression models were used for analysis. Among 213 pregnant women, prevalence of 25(OH)D deficiency was 61.5%, and their newborn was 99.5% (mean 25(OH)D levels: 46.3 [11.3] and 24.9 [5.4] nmol L−1, respectively). Maternal sociodemographic characteristics were similar between 25(OH)D deficient and sufficient mothers, whereas newborn 25(OH)D levels were significantly lower in the former (22.60 [4.53] vs. 27.67 [3.82] nmol L−1, respectively, P < 0.001). There was a strong positive association between maternal and newborn 25(OH)D levels (r, 0.66; r 2, 43%, B [SE], 0.3 [0.02]; P < 0.001). Association of maternal 25(OH)D levels with newborn weight, length and head circumference was not significant (all P > 0.05). Our study shows a high prevalence of 25(OH)D deficiency in pregnant women and their newborns and a strong positive association between maternal and newborn 25(OH)D levels. Findings of this study indicate the importance of maintaining sufficient 25(OH)D levels during pregnancy.


| Data collection
All participants completed a pro forma, at the time of admission to the hospital, including questions on demographics, monthly income, education, typical daily duration of sun exposure, type of clothing worn when outdoors, colour of the skin and use of 25(OH)D supplements.
Anthropometric measurements were performed on both the mother (at the time of admission) and the newborn (immediately after birth) using standardized scales. Maternal anthropometry included weight and height measurements. Weight was measured to the nearest 0.1 kg using digital scales mounted on a hard and flat surface.
Height was measured to the nearest 0.1 cm, using a stadiometer, mounted on a hard and flat surface. Newborn anthropometry included weight, length and head circumference. Newborn weight was

Key messages
• Vitamin D deficiency is very high in pregnant mothers and their newborns.
• Our findings support the evidence that newborns are dependent on their mothers for Vitamin D supply.
• Antenatal visits should include education on the safety and importance of Vitamin D supplementation and sufficient sunlight exposure during pregnancy.
• Pregnancy specific Vitamin D cut-offs are required for optimal health of the mothers and the newborns and to enable comparisons.
• Prospective and interventional studies are required to investigate the optimum Vitamin D requirement for pregnant mothers and their newborns and determine maternal Vitamin D associations with newborn outcomes. measured to the nearest 5 g using baby scales. Length and height were measured to the nearest 0.1 cm using a nonstretchable measuring tape.
Blood samples from the mothers and cord blood of their newborns were collected immediately after the delivery. All maternal and newborn blood samples were sent to the biochemistry laboratory of the Armed Forces Institute of Pathology (AFIP) Rawalpindi, for estimation of 25(OH)D levels, where they strictly follow internal and external quality control checks (https://www.afip.gov.pk/index.php?page= accreditation). Blood samples were collected by trained nurses and sent immediately to the lab for analysis according to the available SOPs of the hospital. Blood samples were analysed by the liquid chromatography-tandem mass spectroscopy assays. The test principle is based on an electro-chemiluminescence immuno assay (ECLIA). The assay uses a 25(OH)D-binding protein (VDBP) as capture protein that binds to Vitamin D3 (25-hydroxyvitamin).
Monthly income was categorized into low-, middle-and highincome groups according to the World Bank's classification of income groups based on gross national income (GNI) per capita (The World Bank, 2017). Educational status was classified into two categories: illiterate, defined as having no formal schooling and unable to read and write, and literate, defined as any number of years of formal schooling and able to read and write.
Due to the sociocultural reasons, sun exposure means the exposure of hands and/or arms, feet, face and neck (bare skin) to the sunlight in this setting. Through literature search, it was identified that the minimum duration of sun exposure, for any beneficial effects, is usually 10 min. Hence, this cut-off of 10 min day −1 was chosen to classify the women as adequately or inadequately sun exposed for this study.

| Statistical analysis
Continuous data were expressed as mean (standard deviation), and categorical data as percentage (%). Distribution of the study variables was assessed using histograms. Univariate tests of the differences were carried out between 25(OH)D sufficient and deficient mothers using the independent samples t test and chi-squared test for continuous and categorical variables, respectively. Pearson's correlation coefficient was used for initial univariate assessment of the relationship between the 25(OH)D status of mother and newborn. Effect of possible confounders on the relationship between maternal and newborn 25(OH)D levels was assessed using multiple linear regression.
Relationship between maternal and newborn anthropometric measurements (weight, length and head circumference) was determined using multiple linear regression. P value of less than 0.05 was considered statistically significant. Mothers taking Vitamin D supplements were excluded from the final analysis. Statistical analyses were performed using STATA 14.

| Characteristics of participants
We studied a total of 213 healthy pregnant mothers (attending the hospital at the time of delivery) and their 213 newborns (N = 416). Table 1 presents the characteristics of our study participants. Among these participants, 67% were aged less than 30 years, and 33% were aged 30 or above. Mean maternal age was 27.8 (4.1) years. The women were, on average, multiparous, and the mean gestational age was 38 (1.7) weeks. Mean maternal body mass index was 27.8 and 99.5% of the newborns were 25(OH)D deficient. The average birthweight was 2.9 (0.5) kg with 11.4% prevalence of low birthweight. Mean length and head circumference of the newborns were 47.4 (2.8) and 33.9 (2.2) cm, respectively (Table 1).
Maternal and newborn clinical characteristics were compared between the 25(OH)D sufficient and deficient mothers ( Table 2).
Sociodemographic characteristics were similar between the two groups (all P > 0.05). Compared with the 25(OH)D sufficient mothers, the newborn 25(OH)D levels were significantly lower among the 25(OH)D deficient mothers (P < 0.001). There was no significant difference in other newborn characteristics between the two groups (Table 2).  Table 3).

| Association between maternal and newborn anthropometric measurements
The relationship between maternal and newborn anthropometric measurements (weight, length and head circumference) was determined using multiple linear regression. Results were presented before and after adjustment for age, gestational age, BMI, education, monthly income, skin colour, parity and sun exposure (Table 4)  confounding variables. Blood samples from the mothers were taken in the months of September to February, which corresponds to autumnwinter season in Rawalpindi (Pakistan) where the average length of day and the sun exposure is less than compared with the summer season. It may therefore be likely that the high prevalence of 25(OH)D deficiency is associated with seasonality, as reported in previous studies too (Goswami et al., 2000;Sheikh, Saeed, Jafri, Yazdani, & Hussain, 2012). Maternal demographic and physical characteristics did pregnant women found a significant relationship between maternal 25(OH)D levels and newborn birthweight only after adjusting for several confounders, including ethnicity, pre-pregnancy BMI, trimester at maternal blood withdraw and study site (Gernand et al., 2013). In contrast, a prospective study conducted in Spain reported no association between maternal and newborn birthweight in either adjusted or unadjusted analysis (Rodriguez et al., 2015). Furthermore, research studies observed there was no relationship between maternal 25(OH) D inadequacy with the infant's height and head circumference (Ong et al., 2016;Wierzejska et al., 2018).
Nevertheless, some authors have suggested the presence of an association between maternal 25(OH)D levels and newborn T A B L E 2 Characteristics of the study participants by mother Vitamin D status   (Nobles et al., 2015) . Moreover, another study reported that newborns whose mothers received Vitamin D had greater head circumference compared with babies of mothers who did not get Vitamin D (Abbasian et al., 2016)  however, there was no significant effect on birth length and head circumference (Aghajafari et al., 2013). A systematic review suggested that evidence from the available literature is not sufficient to confirm an association between maternal 25(OH)D levels and newborn outcomes (Harvey et al., 2014). The evidence, therefore, regarding the which may account for the high prevalence of obesity in our study.
The current study was conducted in a hospital setting where mostly families of army employee visit, so caution is advised before generalizing the results.

ACKNOWLEDEGEMENT
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest.

CONTRIBUTIONS
SR and ZH conceived and designed the study. SR, MK, YY and SHH were involved in data collection. SA and SF analysed the data. SA, NL and ZH interpreted the data, revised the manuscript and approved the final draft.