Maternal 25-hydroxyvitamin D correlates with weight gain and bone metabolism during pregnancy

Background: Vitamin D is involved in the regulation of 3% of the human genome, which encode proteins affecting bone health, immune system and other systems. The status of maternal vitamin D may program neonatal skeletal development. Besides, vitamin D is associated with obesity during pregnancy. Obesity has a detrimental effect on bone metabolism and the level of several hormones related to bone metabolism Methods: This cross-sectional study recruited 103 families who visited Anzhen Hospital from January 2017 to June 2017. The age of included pregnant women ranged from 21 to 42 years old. Patients were assigned to different groups according to their body mass index (BMI) and weight gain during pregnancy. The level of 25-hydroxyvitamin D (25-(OH)D) in serum, parathyroid hormone (PTH), total procollagen type 1 N-terminal propeptide (P1NP), osteocalcin (OC), and C-terminal telopeptides type I collagen (CTX) from mothers and neonates were compared. Results: The results showed that the average level of 25-(OH) D in serum from mothers was 15.09ng/ml during pregnancy and 16.23ng/ml in the umbilical cord. The maternal P1NP increased significantly when the weight gain increased during pregnancy (P<0.01), as well as the fetal weight (P<0.05). Conclusions: Vitamin D deficiency is prevalent during pregnancy and led to more weight gain during pregnancy, also influence the fetal bone health. Obesity negatively correlates with 25-(OH)D level in serum and has a detrimental effect on bone metabolism.

population in high-risk groups from obesity and its related symptoms. A lower level of vitamin D in obese people is consistently found across age, ethnicity, and geography, though it may not always reflect a clinical symptom. 25-hydroxyvitamin D 25-(OH)D is a secosteroid hormone. The serum level of 25-(OH)D is used as an index of vitamin D in the body. The obese population may need to take higher-level vitamin D to achieve the same level of serum 25-(OH)D in people with normal weight.
Obesity is a notable risk factor for vitamin D deficiency, which may be caused by vitamin D3 sequestration in adipose tissue (9). Although many researchers have demonstrated a dramatic change of 25-(OH) D concentration along with the overweight severity (3,(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24), we are unaware of any study investigating the relationship between 25-(OH)D level and obesity in the context of pregnancy. Pregnancy is a particular period of time susceptible to vitamin D deficiency, in that mothers must sustain their vitamin D level as well as those of their fetuses (25).
Furthermore, hormonally active form of vitamin D play important roles in placental development and function, inflammation, angiogenesis, immunomodulation, and insulin sensitivity (25)(26)(27)(28)(29). All of these findings indicate the potential roles of vitamin D deficiency in several well-known adverse pregnancy outcomes related to the overweight during pregnancy, including miscarriage, preeclampsia, and gestational diabetes (30). The level of serum 25-(OH)D in pregnant women not only affect the health of the mother's, but also the health of their children's.

Subjects
This study was approved by the Anzhen Hospital Ethics Committee (Anzhen18029X), the signed consent form from each patient/parents/legal guardians was obtained. And written informed consent was obtained from the patient/parents/legal guardians for publication of any accompanying images and videos. A copy of the written consent is available for review by the editor of this journal. This study was a cross-sectional study analyzing data from 103 pregnant women who visited Anzhen Hospital to have a delivery from January 2017 to June 2017. The mean (±SD) values of age, BMI, and pregnancy weight gain of mothers were 31.09 (±4.1) years old, 25.8 (±3.8) kg/m 2 , and 13.1 (±4.4) kg respectively. Ninety-six cases were full-term delivery, seven cases were premature delivery. Blood samples were collected from mothers during pregnancy and from umbilical cords at birth for analysis of serum 25-(OH)D, PTH, and bone remodeling markers.

Measurements
BMI values ranged in 24.0 to 27.9 kg/m 2 were defined as "overweight," while values equal to or above 28kg/m 2 were described as "obese" according to the reference values for an adult population (31).
The overweight gain was defined as a weight gain of 16kg during the pregnancy for the normalweight women before pregnancy, for the overweight was 11.5kg and for the obese was 9kg, respectively.
A level of total serum 25-(OH)D level less than 20 ng/ml was considered as vitamin D deficiency, and vitamin D insufficiency was defined when its level fell into the range from 20 to 30 ng/ml (32,33). The method of blood glucose determination was hexokinase method, which was determined by glucose assay kit. Calcium Arsenazo (Beckman Coulter AU Chemistry System, United States) was used to quantitatively determine the calcium concentration, this step was done by the laboratoryChemiLuminescence (DiaSorin Inc.) was the methodology used to measure 25-(OH)D3, PTH and the markers of bone metabolism (P1NP, OC, CTX).

Statistical analyses
Patients were divided into three groups (normal weight, overweight, and obese), according to their BMI before pregnancy. Statistical analyses were performed with the software SPSS, version 22.0 for Windows. T-test for independent samples was performed when comparing two groups; if the variances were not equal, the Wilcoxon test was performed. One-Way ANOVA was used to compare the means among the three groups. Correlation coefficients were calculated by Pearson correlation.
Results were presented as mean (±SD), unless otherwise indicated. The differences were considered significant when P<0.05; P values between 0.05 and 0.10 were considered different in trends.

Results
The characteristics of the 103 mothers and their newborns were presented separately for vitamin D deficiency or insufficiency ( Table 1). The mean serum 25-(OH)D level of the whole group was 14.9 ng/ml. Briefly, no difference was found between the vitamin D deficient group and the vitamin D insufficient group in terms of mothers' age, height and BMI before delivery (P=0.359, 0.587, 0.723, 0.676 and, respectively). Meanwhile, the level of PTH in the vitamin D insufficient group was significantly lower than that in the vitamin D deficient group (18.  Data represents as mean (±SD). *: P<0.05; "cord" means "umbilical cords". Table 2 showed the relationships between BMI and maternal 25-(OH)D, PTH, OC, and CTX, respectively. A significant difference of maternal P1NP was found among the three groups. The concentration of P1NP in overweight and obesity were both higher than normal group (P=0.0008,  "nw" stands for normal weight; "ov" means overweight; "ob" means obesity. Data represents as mean (±SD). * P<0.05.
All patients were divided into three groups, normal weight group, overweight group and obesity group, according to their BMI (  Similarly, the authors also analyzed the relationship between the weight gain of the mother during pregnancy and the level of maternal P1NP, OC, and cord 25-(OH)D occurred. The results (  *, The overweight gain was defined as a weight gain of 16kg during the pregnancy for the normal-weight women before pregnancy, for the overweight was 11.5kg and for the obese was 9kg, respectively.

Discussion
Vitamin D is a fat-soluble nutrient which is mainly obtained from consuming dairy supplements and fish oils. Also, it can be produced endogenously in the skin with exposure to sunlight. To be active, vitamin D must undergo hydroxylation in the liver, being converted to 25-(OH)D, then hydroxylized to the physiologically active 1,25-dihydroxyvitamin D primarily in kidney. This active form is nesessary to promote absorption of calcium in the gut, and enables normal bone mineralization and growth. The newborn disordered skeletal homeostasis, congenital rickets, and fractures is highly considered with severe maternal vitamin D deficiency (34,35).
The level of vitamin D is affected by many different factors, such as the geographical region, race, Consequently, infants of those mothers with or at high risk of vitamin D deficiency are also at risk of vitamin D deficiency (38,39). Lower maternal vitamin D level is associated with lower bone mineral concentration in newborn infants (40).

Vitamin D and obesity
Obesity is a significant health problem worldwide, particularly in developed nations. Recently, the relationship between obesity and vitamin D drew more and more attention around the world. Some researchers have found that obese people tend to suffer from vitamin D decline or deficiency; consistently, the decrease in the level of vitamin D increases the risk of obesity. Parikh and his co-

Vitamin D and bone metabolism
Taking optimum dose of vitamin D can not only control the weight gain during pregnancy and lower the risk of obesity complication (48),but also benefit fetal bone growth. Our data showed that newborn's 25-(OH)D level in the vitamin D insufficient group was higher than that in the vitamin D deficient group, which was also the case in terms of mothers' 25-(OH)D.
Maternal vitamin D level during pregnancy can program offspring's skeletal development (49)and body composition in the offspring (50)by influencing the interaction between osteoblasts and adipocytes. Low maternal serum 25-(OH)D level is associated with shorter duration of gestation and, consequently, reduced growth of long bones in newborns (51). In vitamin D-deficient mothers, maternal PTH is elevated, probably due to the increased mineral demands of larger babies (51).
Children of mothers with low vitamin D status during late pregnancy had reduced whole-body bone mineral content, bone area, and areal bone mineral density (BMD) at the age of 9 (52). This suggests that vitamin D has an influence on skeletal programming, and the tracking of bone mass lasts throughout childhood. Furthermore, birth weight and growth during the first year of life may contribute to skeletal fragility later in life (53)(54)(55).
Previous studies showed that maternal vitamin D status, defined by serum 25-OH-D concentration, is tightly associated with cord blood vitamin D concentration (56)(57)(58).

Obesity and bone metabolism
Although obesity has been considered as a condition of low risk for osteoporosis traditionally, this classic view has recently been questioned (59). Bodyweight is directly associated with BMD. A low BMI has been identified as an essential risk factor for lower BMD and predicts greater bone loss in older age (60,61) and in younger persons in the absence of menses and/or an eating disorder (62) . On the other hand, a high body weight can be due to the increased physical activity or obesity, both of which will increase BMD. However, increasing evidence has suggested that excess weight due to adiposity is detrimental to bone and increases fracture risk. In an animal test, short-term and extended high-fat diet-induced obesity caused significant bone loss. Recently, more and more index of bone metabolism has been shown correlated with obesity, such as OC, PTH, P1NP, CTX, and serum calcium.

Osteocalcin
Reinehr and Roth found that OC levels were lower in obese children and increased when weight loss was achieved (63). OC, also known as bone gamma-carboxyglutamic acid-containing protein, is a noncollagenous protein hormone found in bone and dentin, giving bones strength and flexibility. It is secreted solely by osteoblasts and thought to play a role in metabolic regulation. In addition, it is proosteoblastic, or bone-building, by nature (64). It is also implicated in bone mineralization and calcium

Parathyroid hormone
PTH, response to the low level of serum calcium, however, the over-responses of PTH will destroy the balance between the bone remodeling and resorbing. Previous publication indicated that the obese people have higher PTH level (66), and Pitroda thought the reason might be the low 25-(OH)D level in the obese people (67). The PTH in the vitamin D insufficient group is clearly lower than that in the vitamin D deficient group. Meanwhile, the PTH in the obese pregnant women were higher than that in the non-obesity pregnant women, and it is also the case in terms of calcium level in serum.

Total procollagen type 1 N-terminal propeptide and C-terminal telopeptides type I collagen
The organic composition of the bone includes mainly typeⅠcollagen (90%), bone binding protein (10%) and trace albumin. Collagen is the crucial component for maintaining the bone integrity. P1NP reflects the change of the newly synthesized typeⅠcollagen, which can be regarded as a symbol of bone remodeling. CTX is the degradation product of the typeⅠcollagen and it is an index of bone resorbing.
Some researchers think that the uncontrollable bone resorbing is the main reason leading to the sharply decline of bone mass in obesity (68). Table 3 and 4 showed that the maternal P1NP increased clearly when the weight gain increased during pregnancy and the CTX of the obese pregnant women was higher than that in the normal weight group. The P1NP and CTX were increased to meet the bigger demand of the baby's bone growth which was indicated by the finding that obese pregnant women have higher probability to deliver a baby whose weight is over 4000 g.

Conclusions
Vitamin D deficiency is prevalent during pregnancy. Obesity negatively correlates with the level of serum 25-(OH)D and has a detrimental effect on bone metabolism and fetal weight. The decrease of vitamin D not only increased the weight of fetal, but also affected their bone health. The levels of PTH, P1NP, CTX and serum calcium of patients in the obesity group were higher than that of patients in the normal weight group. This phenomenon can be explained by a feedback mechanism trying to meet the demand of their own and fetal bone growth. Thus it is significant for pregnant women to maintain a reasonable 25-OHD level.

Ethics approval and consent to participate
This study was approved by the Anzhen Hospital Ethics Committee (Anzhen18029X), the signed consent form from each patient/parents/legal guardians was obtained. And written informed consent was obtained from the patient/parents/legal guardians for publication of any accompanying images and videos. A copy of the written consent is available for review by the editor of this journal.
Consent to publish available from the corresponding author upon reasonable request

Competing interests
The authors declare that there is no conflict of interest regarding the publication of this article,

Funding Support
The work was supported by The Capital Medical University sustentation fund of the undergraduate scientific research innovation.

Authors' Contributions
We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.