Folic acid intake during pregnancy reduced the incidence of pregnancy-induced hypertension in Tibetan Nationality of Tibet Plateau: a population based cohort study

Objective The objective of this study was to investigate the effect of maternal supplementation with folic acid on pregnancy induced hypertension (PIH) and fetal outcomes in Tibetan Nationality of Tibet Plateau . Design A retrospective cohort study. Primary and secondary outcomes Negative maternal and fetal outcomes between mothers who received folic acid supplement and mothers did not. Frequency of pregnancy induced hypertension were main outcome. Results 6700 pregnant women were included in the study, 18.37% (1231/6700) mothers (group A)received folic acid supplement during pregnancy and 24.37%(300/1231) of them(group A1) having supplement of folic acid longer than 3months. 1230 mothers did not receive folic acid supplement were selected as control (group B) through propensity score matching. In comparison, mothers of group A have a lower frequency of PIH than group B ( 5.8 % vs.19.3 %, P<0.001), including gestational hypertension (1.87%vs.4.47 % ), mild Pre-eclampsia (2.11 %vs.5.69 % ) ,severe Pre-eclampsia(1.87% vs.8.54 %) and Eclampsia(0% vs.0.57% ). However, Longer duration of folic acid supplement did not reduced the incidence of PIH. Before delivery, mothers of group A had less anemia ( 31.7 %vs.20.1%, P <0.001) and much higher hemoglobin (122.3±16.02g vs.115.5±16.76g, P <0.001) than group B. After delivery, more preterm infants, more neonatal asphyxia , lighter weight, shorter height , smaller head circumference and pediatric admission were observed in infants of group B than group A. Conclusion Among pregnant women of Tibetan Nationality on Tibet Plateau , folic acid supplement could reduce the incidence of pregnancy induced hypertension, anemia and related negative outcomes of infants.


Background
Pregnancy induced Hypertension(PIH) is a common hypertensive disorders during pregnancy, which include gestational hypertension, preeclampsia and eclampsia 1 .It is estimated that PIH complicates 6-10% of pregnancies and preeclampsia complicates 2-8% pregnancies globally [2][3] . PIH are one of the leading causes of maternal and perinatal mortality worldwide,which are responsible for 9% of deaths hypertensive disorders in Africa and Asia, even almost 26% of maternal deaths in Latin America and the Caribbean [4][5] . Also, pregnancy induced hypertension is a risk factor for diabetes and cardiovascular disease in later life [6][7] . Preeclampsia is thought to be caused by super cial placenta invasion, however, other risk factors for preeclampsia remain unclear 8 .
Folic acid(FA) are required for methionine-Hcy metabolism and intake de ciency can result in increased Homocysteine(Hcy) concentrations 9 . Hyperhomocysteinemia affects the walls of blood vessels, causing endothelial changes and smooth muscle proliferation which futther lead to cardiovascular disease and vasculopathy 10 . It has been assumed that hyperhomocysteinemia may contribute to the development of placental microvascular diseases and affecting the endothelium adversely,then elevated levels of blood homocysteine is a risk of gestational hypertension and preeclampsia 11 .A small but growing body of evidence has suggested that folic acid supplementation could decrease the incidence of preeclampsia and gestational hypertension [12][13][14] .However, other studies thought that daily consumption of 400 μg folic acid during early pregnancy cannot prevent the occurrence of gestational hypertension and preeclampsia 15 . Now, the 0.4mg folic acid were free distributed in many part of China. Whether folic acid supplementation alone in pregnancy can prevent the occurrence of gestational hypertension/ preeclampsia remains uncertain in several meta-analysis 16-17 . Tibetan people live at a high altitude of average 3,650 metres above the sea level, where people have to adapt to the unavoidable environmental stress of high altitude hypoxia for the reduced atmospheric pressure. People lived in high altitude have large quantitativehave differences in numerous physiological and molecular traits involved in oxygen delivery. The genetic variance avail themselves of essentially the full range of oxygen-using metabolism as populations at sea level 18 . An average increase of 1 lnHcy (log transformation of total Hcy level) was associated with an increase of 3.78mmHg of systolic blood pressure (BP) and 3.02mmHg of diastolic BP among Tibetans in China 19 .
Because the aforementioned studies showed different results between the occurrence of preeclampsia or gestational hypertension and the consumption of folic acid alone during early pregnancy. Besides, little is known about the role of folic acid in Tibetan wemen, then more comparative study is needed to enhance the body of knowledge and determine whether there is the need to explore folic acid supplement on pregnancy induced hypertensions in Tibetan Nationality of Tibet Plateau.

Study Design :
This was a retrospective cohort study performed from January 2012 to January 2016 at the Lasha People's Hospital, which provided health care service for 50 percent pregnant wemen of Tibet Plateau. The trial was approved by the ethics review committee of Lasha People's Hospital (approval number: SYLL2217001 ).Subjects were screened for the following eligibility criteria: age between 20 and 45 years; all participants had singleton pregnancies and progressed into the third trimester. Patients who had a history of chronic diseases before pregnancy, such as primary hypertension, diabetes mellitus, kidney and liver diseases were excluded.
Mothers received folic acid supplement during early pregnancy were assigned into group A and did not received folic acid supplement were assigned into group B, respectively.

Data Collections:
Using an electronic medical record system and paper charts, the following data from the clinic and inpatient services at Lasha People's Hospital were collected for analysis: baseline information before delivery including age, ways of conception, history of pregnancy or delivery; pregnancy and obstetric complications; pertinent physical ndings; and laboratory information including sonography results.The study only reported on maternal and fetus and infant complications without details on patient symptoms and laboratory abnormalities.Data were assessed at the following time points: 4-week intervals from baseline information to delivery; postpartum weeks 6-8. Data were allocated to the more recent time point if the tests were performed between two time points.
Maternal habitation at recruitment was categorized into two groups: countryside and town/city. Educational level was dichotomized into illiteracy, elementary education(grade school/junior high school), Secondary education(high school/technical secondary school),Higher education( college/university). Habitation altitude was grouped into low altitude(< 1500), middle altitude(1500 3500), and high altitude (≥3500). Occupation was rough classi ed into light physcial labor (sitting work and no need overtime), moderate physical labor (continous work of arms and/or legs) and Heavy physical labor(long time work with load). 5 min Apgar score was dichotomized using the cut-off value 7.
Propensity score matching for each group was computed for each case determined by multivariable logistic regression analysis. Patient demographics, tumor characteristics, and treatment patterns were entered in the propensity score model(PSM). One-to-one propensity score matching between the postoperative radiation group and the ovarian conservation group was performed through an automated algorithm with the propensity score difference cutoff being 1%.

Outcome Measurements
The primary outcomes were the frequency of pregnancy induced hypertension ( including gestational hypertension, preeclampsia, eclampsia, HELLP syndrome) between two groups. The secondary assessment was other negative maternal and fetal outcomes between two groups, including birth defects, fetal growth retardation, and intrapartum complications (e.g. fetal distress, neonatal death, premature delivery and postpartum hemorrhage). Safety reports in newborns or infants were tabulated using data acquired from infant during the prenatal period or the postnatal period up to 72 hours (e.g., ultrasonography examination, reports of birth defects and APGAR scores, measurements from growth charts, and development milestones).
The incidence rates of GH and PE were described according to the characteristics of participants, including maternal age, gravid, parity, education, habitation altitude and folic acid supplementation.

Statistical Analysis
Based on the studies, we estimated 5.9 % of patients with GH or PE in tibetan wemen 20 and OR of preeclampsia with folic acid supplementation in comparison to no folic acid supplementation were 0.78 (95% CI 0.63, 0.98) 21 , the number of patients needed was calculated to be 309, with a signi cance level of 0.05 (one-tailed) and detection power of 0.90.Considering a 10 % drop-outs rate, a sample size of patient enrollment more than 340 was a reasonable estimation for each group.
The intention to treatment analysis was de ned as analysis included all enrolled patients, including those with protocol deviations. We included all enrolled infants to perform ITT analysis of thePIH rates. Data of mothers who were lost to followup were still included in the analysis and counted as having no PIH. Baseline characteristics and laboratory results were summarized for two groups by means of descriptive statistics, including percentage, means±standard deviation (SD), and 95% CI. For the quantitative variable, the t-test was used to compare group differences. For categorical variables, the chisquare test was used for group comparisons. Signi cance level was set at P < 0.05; all data were analyzed by SPSS 23.0 (SPSS, IBM., NewYork).

Study Population and Baseline values
A total of 6902 pregnant patients were retrospectively screened and 202 patients were excluded due to other reasons of multiple gestation and other ethinity. Among 6700 patients were included in the nal study, 18.37% (1231/6700) patients received folic acid supplement during pregnancy were assigned into group A and 81.63% (5469/6700) patients did not receive folic acid supplement. 24.37%(300/1231) patients having supplement of folic acid longer than 3months were further assigned into group A1 and 75.63%(931/1231) having supplement of folic acid shorter than 3months were assigned into group A2. Compared to the mothers without folic acid supplement , the mothers with folic acid supplement were older (28.59±3.83vs. 28.23±4.90, P=0.015) , having fewer multipara(49.6%vs.70.2%,p=2.53*10 -43 ) and living in lower altitude (3706.95±449.42vs.3800.39±475.14, P=3.26*10 -10 ). More mothers in folic acid supplement group had higher level of Education, engaged in jobs with less physical labor and lived in town or city(see table 1). 1230 mothers did not received folic acid supplement were selected through PSM (based on age, educational levels, living altitude, multipara or not , occupation and residence) and and enrolled group B. The patients who were screened and enrolled into the different study groups are shown in Fig. 1. After propensity score matching, there was no statistical signi cance of the baseline between group A and group B. The baseline characteristics of maternal showed in the table 1.
The mean (±SD) duration of folic acid supplement was 2.07±1.41 months in group A, 3.87±1.74 months in group A1 and 1.48±0.51 months in group A2.From baseline visit to the last visit at postpartum week 6, there was no patients drop-out from our study. Thus, the on-protocol analyses also represented the outcomes of the intention-to-treat analyses in the our study.
In the term of infants complications, more preterm delivery were observed in the infants of group B than group A( To further evaluate the role of folic acid supplement on reducing the incidence of PIH, subset analysis was performed based on the dosing course of folic acid. Patients with dosing course longer than 3 months were assigned into group A1,and those without into group A2. Compared to patients in groupA2, longer dosing course of folic acid seems to further reducing the incidence of PIH but without signi cant difference (6.0% vs.5.4%, p =0.662)(see suppl table 1) .

Discussion
In this study, we report on retropective data on folic acid supplement for reducing pregnancy induced hypertension in selected Tibetan Nationality of Tibet Plateau.To our knowledge, this is the rst and largest study involving 2461 subjects in this special population. Our results indicate that folic acid supplement during early stage of pregnancy could reduce the incidence of pregnancy induced hypertension and antepartum anemia.In combination with regularlly antepartum examination,a signi cant reduction of preterm delivery and asphyxia of newborn was observed in mothers received folic acid supplement, compared to those did not receive.
Whether folic acid supplementation in pregnancy can reduce the incidence PIH remains uncertain for the inconsistency of previous studies [16][17] . The prevalence of gestational hypertension/preeclampsia are associated with maternal age, parity, ethics, education, socioeconomic status, history of preeclampsia and chronic hypertension, antenatal care among other factors [22][23] .The inconsistency of ndings may be attributable to different heterogeneity of population, sample size, education, socioeconomic status, and different antenatal care. Among 6700 tibetan mothers screened in our study, only 18.37% patients received folic acid supplement during pregnancy. The mothers received folic acid supplement were older (28.59±3.83 vs. 28.23±4.90) and lived in lower altitude (3706.95±449.42mvs. 3800.39±475.14m). Less of them were illiteracy(3.6%vs. 29.1%), multipara(49.6%vs.70.2%), heavy physical labor (29.1%vs.61.5%) and lived in countryside(34.4%vs.63.4%). All the above features might re ect a more easily acquiring of prenatal education or sanitary condition. After adjusted the age, parity,living altitude,education, occupation and residence through propensity score matching, our results indicated that folic acid supplement from 3 months before conception to rst trimester could reduce the incidence of PIH from 19.3 % to 5.8 % in this special population. The folic acid supplement could reduce PIH from Gestational hypertension (4.47 % vs.1.87%), mild Pre-eclampsia(5.69 % vs.2.11 %) ,severe Pre-eclampsia(8.54 % vs.1.87%) to Eclampsia( 0.57% vs.0%) comprehensively .However, longer duration of folic acid supplement did not reduced the incidence of PIH.
Previous studies only reported on the incidence of pIH without details on maternal and fetus and infant complications.Our study also showed that less anemia ( 20.1%vs.31.7 %) and much lower hemoglobin (122.3±16.02 vs.115.5±16.76) were found in group B than in group A before delivery. There is evidence that natural selection is ongoing in the Tibetan population, where women estimated to have genotypes for high oxygen saturation of hemoglobin (and less physiological stress) have higher offspring survival 18 .Due to the higher incidence of PIH, more preterm infants were observed in mothers did not receive folic acid supplement than mothers received ( 8.0%vs.1.0%) and resulted in a shorter gestational ages(38.74±1.93 vs. 39.10±1.12). More preterm and mother complications further led to more full-term low birth weight infants ( 8.5%vs.3.0%), neonatal asphyxia (9.0 % vs.2.1%) and Pediatric admission ( 17.1% vs. 4.1%). In antepartum examinations, folic acid was mainly used to prevent the Neural tube defects 24 . The incidence of fetal/infant congenital deformities( 1.1% vs.0.7 % )and newborn death( 0.4% vs.0.1%) seems more in mothers did not receive folic acid supplement than mothers received, but no signi cant differences were found.
In ammation and oxidative stress is considered to play a role in the pathophysiology of preeclampsia 25 .Hyperhomocysteinemia might damage the vascular endothelium of the developing placenta by promoting oxidative stress, thereby increasing contractile response and the production of procoagulants and vasoconstrictors [26][27] .Folic acid supplementation might reduce the risk of gestational hypertension or preeclampsia through reduce elevated homocysteine [28][29] .However, other studies thought hyperhomocysteinemia may be a consequence rather than a cause of hypertensive disorders of pregnancy 30 .The concentration of blood folic acid level were affected by many factors except folic acid supplement, like dietary habit. In their pastoral pattern, 85% did not consume packaged vegetables or consumed this food group less than once per month; whereas onions and garlic were consumed as condiments in minimal amounts as parts of culinary dishes 31 .Besides, maternal and fetal MTHFR C677T polymorphism may be associated with a moderately increased risk of gestational hypertension, and there is a suggestion that this association may be diminished among women receiving folate supplementation during Pregnancy 32 .
At sea level, maternal uterine artery blood at a pO2 of 100 mmHg enters the intervillous spacewhere it is stirred by oscillations of fetal villi and equilibrates with uterine vein blood at a pO2 of 45 mmHg. While in High altitude at 3600 m, maternal uterine artery pO2 is only 55 mmHg, the same gradient, need a lower uterine vein blood or an adaptive uterine artery pressure 33 .Then lead to an altitude-related increase in the incidence of PE and GH 34 .However, different to previous study in european, Tibetan women had higher rates of pre-eclampsia/gestational hypertension than han chinese women (5.9% vs 10.3%, P = 0.04) 20 . Also, altitude was found to be negatively associated with the risk of DM in tibetan people. Compared to individuals living at < 3500 meters, the risk of DM decreased by 65% for those living at 3500-3999 meters (P = 0.034) and by 89% for those living at ≥ 4000 meters (P = 0.015) 35 . The screening of Gestational Diabetes Mellitus were not commonly perfomed among Tibetanswithout symptoms or abnormal laboratory results.Our study did not include the patients of Diabetes Mellitus and other chronic diseases which is also the risk factor of PIH.Further prospective studies are need to include the data of blood Hcy levels, hcy polymorphism and chronic diseases in this population.
In conclusion,Folic acid supplement in the rst trimester could reduce the incidence of pregnancy induced hypertension in Tibetan Nationality of Tibet Plateau.Continuing free distribution of 0.4mg folic acid and closely follow-up were highly recommended in this popultaion.