Bone health in pregnant women

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Introduction

One of the important issues for all women is bone health, particularly during pregnancy. Pregnancy is associated with main changes in calcium metabolism, because the developing baby needs plenty of calcium for developing a skeleton which contains approximately 20-30 grams of calcium and mainly deposited in the latest trimester (1-2). Adaptive mechanisms in pregnant female involved to protect a calcium source to the fetus include:1) increased maternal skeleton resorption of calcium and 2) greater than before intestinal absorption of calcium (3). Enhanced maternal serum free and total 1, 25 hydroxivitamin D (4) sound to be responsible for the better intestinal calcium absorption through pregnancy (5). However, large number of investigation show that increased dietary intake and intestinal absorption are not adequate to provide the calcium required by the fetus (6-8). Therefore the maternal skeleton is potential source of calcium for the fetus (9). The large number of documents suggests that bone turnover in pregnant female increases (4-5) and bone mineral density (BMD) decreases during gestational period (4-11). The OPG/RANKL/RANK system was discovered recently and seems to play a fundamental role in bone homeostasis (12). RANKL is a cytokine belongs to TNF superfamily that regulates the formation and activation of osteoclasts and bone resorption (13). OPG is another cytokine was discovered in 1997 and capable of protecting bone mass by inhibiting osteoclast differentiation and activation (14).The OPG/RANKL ratio is considered to be better expose of bone remodeling environment signs. A high ratio indicates bone formation but a low ratio favors bone resorption (15).Changes in the OPG/RANKL ratio have been concerned in the pathogenesis of bone diseases characterized by bone resorption, such as post-menopausal osteoporosis(16), and glucocorticoid-induced osteoporosis(17). Documents indicate that maternal plasma OPG concentrations increased in the third trimester of pregnancy, a time when the demand for calcium for fetal bone mineralization is at its maximum level and telling that OPG may protect the maternal skeleton from extreme catabolism (14, 18-20).The effect of folic acid on bone turnover and bone metabolism has been evaluated in previous studies (21-22). Furthermore, there is evidence that folic acid supplementation has beneficial effects on bone status (23,24). TNF-α is among the most potent of the osteoclastogenic cytokines that stimulates bone resorption both in vitro and in vivo by increasing the proliferation and differentiation of osteoclast precursors (25).There is no document about TNF- α and pregnancy until now.

Based on the fact that, the maximum rate of bone turnover occurs in the last trimester of the pregnancy, and folic acid supplementation during pregnancy could represent low bone resorption rates, The present study was carried out to compare the effects of high dose(5mg/day) and low dose(0.5mg/day) folic acid on the RANKL/OPG ratio and TNF-α concentration during the pregnancy.

Materials and Methods:

This was a randomized, double-blind study. Ninety Nulliparous women who were visited the AL-Zahra Hospital and one auxiliary clinic, Specialized and Sub-specialized Sheykholrais Clinic of Tabriz University, Tabriz, Iran for prenatal test and planning pregnancy, were enrolled in this follow-up study. Our study’s protocol was approved by Ethical Committee of Tabriz University of Medical Sciences(Ethics code:9149) and registered in the Iranian Registry of Clinical Trials (IRCT) and given the ID, IRCT201206234105N9. All the percipients completed the informed consent form. The inclusion criteria included singleton pregnancy, no history of any disease or medication identified to affect bone metabolism, chronic hypertension, diabetes mellitus, chronic renal disease, cigarette smoking or alcohol Consumption, early miscarriage, assisted conception, and age between 20-30 years old. Sixty women consequently became pregnant. Baseline venous blood samples were collected at 3-month intervals before conception. After collecting baseline sample, percipients receiving different dose of folic acid (5 mg/day and 0.5 mg/day) until 36 week of pregnancy. The participants contacted every week and were asked during each visit to declare whether they took folic acid supplementation or not. Second sampling was done in the 36 week of pregnancy. The percipients that have abortion, multiple pregnancy, and usage of calcium and ferrous sulfate during pregnancy and didn’t complete follow up were excluded from study. Serum was collected after centrifugation (at 3000 RPM) of the sample for 20 minutes, then stored at−70°C till analysis. We used the date of mother’s last menstrual period (LMP) for calculating Gestation age. Serum Osteoprotegerin (OPG) and Receptor Activator of Nuclear Factor-κ B Ligand (sRANKL) concentrations were measured by commercially Enzyme-Linked Immunosorbent Assay (ELISA) kits available( Bioassay Technology Laboratory, Cat No:E1558HU and Cat.No:CK-E90425, with intra- and inter- assay CV of<10% and <12% respectively) .TNF-α was analyzed in serum by ELISA using Immunodiagnostic kit ( DIAsource Immuno Assay S.A, Cat.No:KAP1751 with intra- and inter- assay CV of 6.6% and 4.5%). All of the statistical analyses were performed using SPSS version 18 software (SPSS Ins, Chicago, IL). Median (minimum-maximum values), or mean±SD were used to express results. For unpaired data, The Mann–Whitney U-test and Independent Sample t-test were used, and the Wilcoxon test and a paired Student t-test were used for paired data. Evaluation of Correlation was done by Spearman's test. In all investigated cases ≤ 0.05, was considered statistically significant.

Result:

At the end of study, 45 pregnant women completed follow up: twenty-three treated with 5 mg of folic acid per day in group 1 and 22 treated with 0 .5 mg folic acid per day in group 2(15 patients did not complete the study procedure: 8 cases because of abortion and 2 cases because of multiple pregnancy and 5 cases because of quitting intervention ).The demographic data and baseline characteristics of the two groups are presented in Table1.No significant difference was present between two groups at the beginning of the study (except LDH).

The maternal serum levels of OPG, sRANKL and TNFα at the baseline and at the 36th of pregnancy in high dose and low dose group of folic acid have been shown in Table 2.

Maternal serum evaluation demonstrated that the levels of OPG in both group was increased but there was statically significant difference in group 1 at the 36th week of pregnancy compare to baseline (p=0.008).The concentration of sRANKL (p<0.001) and TNFα (p=0.005) significantly decreased at the 36th week of pregnancy compare to baseline in group 1. As shown in table 2 sRANKL/OPG ratio was decreased in both group but it was statically significant only in group 1.

Variable

Group1(mean±SD)

Group2(mean±SD)

P value

Maternal age(Years)

25.34±3.31

27.00±3.67

.091

Pre pregnancy Weight(Kg)

68.26±7.75

63.77±5.87

.060

Height(cm)

164.39±3.22

162.50±3.67

.071

Pre pregnancy BMI(kg/m2)

25.19±2.09

24.14±1.73

.125

Diastolic BP(mmHg)

76.52±.77

77.72±.92

.499

Systolic BP(mmHg)

105.65±1.82

107.27±1.16

.850

Platelets(x 1,000 mm3)

233.52±48.16

253.18±36.94

.064

LDH(IU/L)

367±98.99

266.54±44.07

.000

Ca(mg/dl)

9.01±0.20

8.95±0.21

.074

P(mg/dl)

3.89±0.15

3.90±.12

.98

ALP (IU/L)

119.17±36.69

112.42±34.7

.364

interval

49.91±20.29

50.54±19.56

.294

Serum creatinine(mg/dl)

.76±.08

.79±.11

.470

Urea(mg/dl)

27.47±1.44

27.50±1.05

.616

Urine protein(mg/24h)

84.62±15.77

72.13±34.47

.095

Urine creatinine(mg/dl)

.75±.10

.71±.07

.159

Education

Under diploma

High education

13(56%)

10(43%)

11(50%)

11(50%)

.317

Variable

Group1(n=23)

Group2(n=22)

P-valuea

Serum OPG (pg/ml)

Baseline

36thweek

340(120-1200)

710(120-1500)

(p=.008)b

375(160-1310)

470(130-1400)

(p=.592)b

.488

Serum sRANKL (pg/ml)

Baseline

36thweek

91.6(33.40-198.60)

40(20.10-100.20)

(p<.001)b

86.10(39.70-198.60)

76.50(43.70-188.5)

(p=.426)b

.302

Serum TNFα (pg/ml)

Baseline

36thweek

5.30(4.10-10.20)

4.8(2.5.-9.00)

(p=.005)b

5.85(4.20-8.9)

6.5(5.00-8.8)

(p=.135)b

.

.251

Serum sRANKL/OPG

Baseline

36thweek

.22 (.09-1.47)

.07(.02-.45)

(p<.001)b

.27(.04-.74)

.24(.04-.67)

(p=.211)b

.291



Bivariate correlation analyses confirmed a positive relationship between the decreased soluble receptor activator of nuclear factor- kappa B ligand after 9 months treating of high dose folic acid (Delta sRANKL) with decreased tumor necrosis factor alpha serum level (Delta TNFα) (r=0.451, p=0.031, Delta sRANKL= (at the end of study of High dose folic acid treatment)—sRANKL (Basal), Delta TNFα=TNFα (at the end of study of High dose folic acid treatment)—TNFα (Basal)). But increased OPG levels did not demonstrate an inverse correlation with decreased sRANKL and TNFα levels (r=0.299, p=0.166 and r=351, p=100 respectively).The mean changes in serum OPG, sRANKL and TNFα at the end of study with high dose and low doses folic acid are shown in Fig. 1

Fig.1. Effect of folic acid supplementation on Percentile variations in serum osteoprotegerin (OPG), soluble receptor activator of nuclear factor-kappa B ligand (sRANKL) and tumor necrosis factor alpha (TNFα) at the end of study between group 1(5mg/day) vs. group 2 (0.5mg/day)(mean).

Discussion: Osteoporosis is a universal problem and affects particularly women (26). Pregnancy-associated osteoporosis (PAO) seemed to be an uncommon problem of pregnancy but it leads to serious difficulties such as fragility fractures and elongated back pain in affected women. Definite prevalence of Pregnancy-associated osteoporosis (PAO) is unknown. (27, 28) but to date about 120 cases have been defined since first report by Nordin and Roper. (29) Most cases develop PAO throughout the first pregnancy and Recurrence of (PAO) with future pregnancies is uncommon, but has been informed to occur (26, 27, 28). No standard treatment protocol has been established for the treatment of pregnancy-associated osteoporosis, due to the absence of a convinced etiology. (27) Document shows that PAO causes significant morbidity in the procedure of pain and disability, the most of patients showing various vertebral fractures in the early puerperium and also show loss of height which can develop at the end of pregnancy, in puerperium, or even later. (30, 31, 32, 33, 34).

Osteoprotegerin, named as “bone protector" and known as a cytokine that increases the density and volume of bone tissue by reducing the amount of active osteoclasts(35). RANKL is a member of TNF superfamily(15) and its Overexpression of soluble RANKL in transgenic mice results in a skeletal phenotype with many similarities to postmenopausal osteoporosis, including reduced BMD, increased bone resorption cortical porosity and skeletal fragility (34). Some investigation showed that OPG reserved bone loss in models of sex-steroid insufficiency and glucocorticoid-induced osteoporosis, rheumatoid arthritis, multiple myeloma, and metastatic bone disease (12). Since OPG directly counter all RANKL mediated activities through RANK, RANKL/OPG ratio is an important determinant of bone mass and skeleton integrity. In malignant diseases, such as myeloma, osteolytic bone metastases of prostate and breast cancer enhance in expression of RANKL by tumor cells and tumor-inducted increase of the RANKL/OPG ratio in bone microenvironment can be observed. (35).

Previous investigations in humans have demonstrated that serum OPG level has progressive increase during human pregnancy, with a decrease in serum RANKL (14, 18, 20). In accordance, in this study, the pregnant women in both group showed higher levels of OPG in comparison with baseline but it was significant only in high dose group and significant decrease of sRANKL was observed only in high dose group .Few studies evaluated the effect of folic acid on bone turnover and bone metabolism (22-23) and only one study evaluated the effect of folic acid on bone metabolism and turnover during pregnancy. Bagher Larijani et al have evaluated the effect of folic acid on OPG, RANKL concentration during pregnancy. They showed that women who took 1 mg of folic acid daily supplement from the beginning of the pregnancy until the delivery time had significantly higher plasma levels OPG concentration in comparison with women who took until the end of the second trimester while sRANKL concentration was significantly lower. For the first time, their results demonstrated evidence that folic acid supplementation during pregnancy could represent low bone resorption rates by higher OPG and lower sRANKL concentrations (24). In our study potential effect of high dose and low dose of folic acid supplementation on prevention of bone resorption and PAO during pregnancy and in future in pregnant women is investigated. Our data for the first time show that high dose (5 mg/day) of folic acid significantly decrease the concentration of the serum sRANKL and increase serum OPG and also for the first time we show that sRANKL/ OPG ratio decrease in high dose group while low dose of folic acid doesn’t have such effects. Also Manizheh sayyah M et al showed that folic acid supplements during pregnancy, regardless dosage could decrease Hcy levels although it was reduced more significant in the high dose group and they show that folic acid supplementation specially high dose of it have beneficial effect on pregnant women and can prevent hypertensive disorder .(36) Furthermore Bagher Larijani et al reported low concentration of Hcy in women who took 1 mg of folic acid daily supplement from the beginning of the pregnancy until the delivery time(24). This two study are in agreement with our study and confirm beneficial effect of folic acid supplementation during pregnancy. Herrmann et al. reported that the proliferation of osteoclasts and the apoptosis of osteoblasts motivated via HCY and B vitamin deficiency interact with bone metabolism, which results in a negative bone turnover (37). There is evidence that the expression of RANKL and RANK genes in peripheral blood mononuclear cells are significantly higher in hyperhomocysteinemic individuals than in controls. Folic acid treatment for 6 weeks significantly reduced gene expression of RANKL/RANK in peripheral blood mononuclear cells from these individuals (38).How ever in our study the variation of serum Hcy level didn’t measure but our result also present significant decrease in sRANK level and conform the capacity of folic acid in decreasing sRANKL level.

It is now clear that inflammatory cytokines such as IL-1, TNF and M-CSF that have long been associated with osteoclastic bone loss, function by stimulating RANKL production by osteoblast precursors and/or developed osteoblasts [39]; and/or by decreasing OPG production and/or by up regulating RANK receptor place on osteoclast precursors ,hence increasing their sensitivity to normal RANKL concentrations.(40) According to subjects were told, in this study for the first time we evaluated the effect of high dose and low dose of folic acid in serum TNFα level. Our data show that in high dose group TNFα level decrease significantly and its decrease has correlation with decrease sRANKL level. Also previous studies have presented that TNFα prompts osteoclastogenesis via the RANKL system, as TNFα up-regulated RANKL mRNA expression (41) and our findings are in agreement with this study.

The complete nature of folic acid’s special effects on bone metabolism has not yet been absolutely understood, therefore a series of researches are necessary to reveal unknown features of this process and it seems that larger sample size and also measuring new markers for bone resorption, such as serum C-telopeptide of type 1 collagen, urine N-telopeptide of type 1 collagen and also bone mineral densitometry will be of great importance to prove our outcomes. In summary, our findings suggest that high dose of folic acid supplementation could decrease bone resorptive bio-markers and prevent PAO in pregnant women by increasing OPG level and decreasing sRANKL and TNFα levels

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