The impact of yoga in pregnancy on placental growth factor levels and mean arterial pressure in pre-eclampsia: A randomized controlled trial

Context : Pre-eclampsia is a pregnancy-related condition characterized by hypertension, proteinuria, and edema occurring after 20 weeks of pregnancy or during the postpartum period. Several markers are often assessed to determine the suitable option for confirming pre-eclampsia diagnosis, one of which is the examination of placental growth factor (PlGF) as a placental ischemia indicator. Aims : To evaluate the impact of prenatal yoga on PlGF and mean arterial pressure (MAP) levels in pre-eclampsia within the Madura region. Methods : A randomized controlled trial was conducted on fifty subjects that were randomly allocated to the experimental group with pre-post control groups consisting of 50 participants, namely 25 pregnant women with pre-eclampsia and 25 healthy individuals. The selection was conducted based on the following inclusion criteria: belonging to Madura ethnicity spanning three generations, exhibiting pregnancy duration >20 weeks, maternal ages ranging from 20 – 35 years, pre-pregnancy body mass index (BMI) >25, a history of hypertension, diabetes mellitus (DM), kidney disorders, pre-eclampsia, chronic hypertension, or family history of chronic high blood pressure and pre-eclampsia among those at risk. PlGF levels were estimated using an ELISA kit, while blood pressure was measured with an Omron brand sphygmomanometer. Results : Statistical analysis performed with the Wilcoxon sign test and T-test indicated significant differences (p<0.005) in systolic blood pressure, diastolic blood pressure, and MAP between the groups. Furthermore, a significant difference was observed in PlGF levels before and after engaging in prenatal yoga practice (p<0.05). Conclusions : The results showed that prenatal yoga contributed to the elevation of PlGF levels in women with pre-eclampsia, compared to their healthy counterparts.


INTRODUCTION
Pre-eclampsia is one of the most common serious complications characterized by hypertension, proteinuria, and edema, occurring in 3-5% of all pregnancies (Brosens et al., 2019;Chaiworapongsa et al., 2018;Poon et al., 2020;Redman and Sargent, 2012;Tessema et al., 2015).This is considered a significant contributor to maternal mortality in Indonesia, specifically in East Java, where it ranks high among the leading causes, including bleeding and infections (Dinkes Jatim, 2021).Pre-eclampsia manifests after 20 weeks of pregnancy or during postpartum and can escalate into eclampsia, marked by the onset of seizures (Douglas and Redman, 1994;Poon et al., 2020).
In the absence of eclampsia, those suffering from pre-eclampsia are at a higher risk of developing strokes (McDermott et al., 2018;Miller, 2020), acute cardiovascular complications (Orabona et al., 2018;Vaught et al., 2021), and a condition known as HELLP syndrome (hemolysis, increased liver enzymes, and low thrombocyte count) (Sibai, 1990).Pre-eclampsia directly affects the fetus, leading to dysfunction, growth obstruction, and death (Basso et al., 2006).Additionally, it has long-term health implications, such as an increased risk of cardiovascular disease (Lu and Hu, 2019;Mongraw-Chaffin et al., 2011;Staff, 2019).Consequently, pre-eclampsia is one of the leading causes of morbidity and mortality among mothers and babies worldwide.
The diagnosis of pre-eclampsia relies on various markers, including placental ischemia, which is evaluated through the examination of the placental growth factor (PlGF).PlGF serves as a laboratory marker for hypoxia, playing a crucial role in the development of endothelial damage and clinical manifestations of pre-eclampsia.Research showed that, in patients with pre-eclampsia, PlGF levels exhibit a significant decline before reaching 37 weeks of pregnancy (Grill et al., 2009;Palei et al., 2013).In uncomplicated pregnancy, serum concentrations of PlGF increase during weeks 8-12, peak between weeks 29-32, and decrease at weeks 33-40.Meanwhile, PlGF levels in women at lower risks begin to decrease around weeks 13-16 until delivery time and remain lower than the value found in normal pregnancy (Miller et al., 2021;Carmeliet et al., 2001;Weed et al., 2012).
PlGF research focused on the Madura ethnic group is limited, and the impact of prenatal yoga has not been investigated among women with pre-eclampsia.The incidence of pre-eclampsia without known causes is very high in Madura, specifically in Syarifah General District Hospital, Bangkalan City, where 2-3 cases are reported daily and 40-50 cases monthly.Therefore, preventive measures are needed to reduce elevated blood pressure and hinder pre-eclampsia from progressing into eclampsia.Prenatal yoga tends to help in achieving this goal by regulating blood pressure and improving blood circulation in expectant mothers.
The potential impact of prenatal yoga on autonomic nervous system activity is thought to be mediated through the limbic system and higher central nervous system regions (Ankad et al., 1995).Regular yoga exercises have been shown to increase baroreflex sensitivity and reduce sympathetic tone, leading to the normalization of blood pressure in individuals with essential hypertension, but their specific influence on PGIF levels remains unclear.Therefore, this research aims to evaluate the impact of yoga in pregnancy on placental growth factor levels and mean arterial pressure in pre-eclampsia.During the execution process, all participants engaged in the same amount of exercise, namely 12 sessions.The results are expected to provide insights into the application of prenatal yoga in managing pre-eclampsia, using MAP and PlGF levels as monitoring parameters, while serving as a means of preventing elevated blood pressure.

Research design
This research used a randomized pre-post-test control group design.The participants consisted of 50 pregnant women.Samples were taken at two independent midwife practice places, one treatment place and one place as a control with the same characteristics of pregnant women, pregnant women belonging to Madura ethnicity spanning three generations, categorized into two groups, namely 25 healthy individuals with pregnancy period >20 weeks, age 20-40 years, and without pregnancy complications.Meanwhile, the other 25 were pregnant women with preeclampsia meeting additional criteria, including maternal age <20 to >35 years, pre-pregnancy Body Mass Index (BMI) >25, a history of hypertension, diabetes mellitus (DM), kidney disorders, chronic hypertension, and a family history of chronic hypertension.Samples were selected from those presenting one or two risk factors and provided with comprehensive information and an opportunity to sign an informed consent form.All participants were screened according to the stated criteria before being included in this research.Pregnant women who were selected as respondents according to the criteria were contacted by the local midwife by telephone and put into a special group, risk of pre-eclampsia and non-pre-eclampsia, because the local midwife had the area so that the patient followed the midwife's instructions, then the midwife coordinated with the researcher, and pregnant women came to the midwife's practice to do prenatal yoga exercises according to the schedule determined by the researcher.Sample size calculation was performed using the Lameshow 1997 formula (Lwanga and Lemeshow, 1991) with reference values derived from previous similar investigations, leading to the selection of a total of 50 pregnant women (Rakhsani et al., 2012).Treatment was carried out twice a week for 12 weeks, from August-October 2023.Evaluate the implementation of prenatal yoga exercises at each exercise, while blood tests were only carried out before and after yoga exercises, once before yoga exercises, and at week 12, and the evaluations were performed as described in Fig. 1.
The study was conducted under the principles of ethical standards set out in the Declaration of Helsinki of the World Medical Association, and the Ethical Committee approved the adequacy of Nonprofit Organization TACTICS.The institution's IRB approval number was 2018-124, Ethical Principles for Medical Research Involving Human Subjects.This research has been declared ethically sound by the Ngudia Husada Madura College of Health Sciences ethics commission with No. ethical clearance number: 1762/KEPK/STIKES-NHM/EC/VI/2023.

Prenatal yoga twice a week for 12 weeks
Prenatal yoga sessions were conducted and supervised by a certified yoga trainer, in collaboration with local research experts and midwives, ensuring the correct execution of practices to minimize the risk of injury.The program consisted of light-intensity movements beginning with meditation, breathing exercises, core exercises, and a cooling phase.Prenatal yoga movements were carefully implemented to guarantee the safety of pregnant women with preeclampsia.
This research comprised a control group (healthy pregnant women, n = 25) and an intervention group (pregnant women with pre-eclampsia, n = 25).The control group did not participate in any training programs and were instructed not to make significant changes to their physical activity or dietary habits.Participants in this group met specific criteria, including three generations of Madura ethnicity, pregnancy duration >20 weeks, maternal age 20-40 years, absence of pregnant complications, pre-pregnancy BMI <25, and having good health indicated by normal blood pressure and no urine protein.
Prenatal yoga sessions were conducted twice a week over a period of 12 weeks, with a duration of 30 minutes.These featured 10 minutes of meditation and breathing exercises, 10 minutes of core exercises incorporating a combination of movements, and 10 minutes of cooling, during which participants lay on their left side with closed eyes while listening to soothing music for relaxation.The meditation exercises included padmasana, vajrasana, left and right head turns, hand movements sideways and upward, cow, butterfly, child, and side angle poses, as well as body rotation, warrior poses (I, II, and III), leg relaxing, and gentle body shaking (right and left), meditation, and cooling.
Blood pressure and fetal heart rate assessments were conducted before and after prenatal yoga sessions.Participants were advised to wear comfortable, moisture-absorbing clothing, and yoga mattresses were provided by the research experts.The sessions were performed outdoors to ensure adequate air circulation.Data collection, pre-eclampsia screening, prenatal yoga practice and blood sampling can be seen in Fig. 2.

Blood sample collection and analysis of MAP and PlGF
In the morning, 3 mL of blood was collected twice from the cubital vein before initiating prenatal yoga and after reaching week 12 of the exercise.The blood samples were centrifuged for 15 minutes at 3000 rpm (Daskalopoulou et al., 2014).Subsequently, the serum was separated, and PGIF levels were analyzed immediately using the ELISA kit (Human Phosphatidylinositol-glycan Biosynthesis Class F Protein, PlGF ELISA kit, 96 test Brand BT Lab Cat No. E3793Hu).The detection range was 0.05-30 ng/mL, and the sample used consisted of serum, plasma, and cell culture supernatants sourced from Shanghai Korain Biotech.PlGF assessments were conducted at the Institute of Tropical Disease (ITD) of Unair.Additionally, measurements of blood pressure comprising systolic and diastolic readings and mean arterial pressure (MAP) were performed for both the intervention and control groups.The analysis included the evaluation of prepregnancy BMI and demographic characteristics of participants.

Statistical analysis
Data analysis executed in this research featured several stages, including descriptive tests, normality tests, and homogeneity tests employing the Shapiro-Wilk and Levene tests.The data exhibiting normal distribution and homogeneous variance were subjected to paired parametric tests, such as t-test samples, Mann-Whitney, Wilcoxon sign test, and advanced tests using post-hoc HSD Tukey tests.Non-normally distributed data without homogeneous variance, non-parametric tests, such as the Wilcoxon Signed Ranks Test, were performed.All data were presented as mean ± standard deviation (SD), and statistical significance was established at a 5% level.

RESULTS
The analysis of the characteristics of pregnant women with pre-eclampsia and their healthy counterparts showed both insignificant and significant abnormalities, as presented in Table 1.
The age of participants ranged from 20−44 years, with an average of 30.0 ± 4.54 years for those with pre-eclampsia and 29.3 ± 4.85 years for healthy individuals without significant pregnancy complications.The statistical analysis showed a p>0.05, indicating no significant difference between both groups (Table 2).
The average parity for pregnant women with preeclampsia was 2.12 ± 5.24 (∑), while the healthy participants had 2.21 ± 1.05 (∑).The statistical analysis showed no significant difference (p>0.05) between the two groups.Similarly, the average BMI was 26.9 ± 5.24 and 26.40 ± 5.34 (kg/m 2 ) for the intervention and control groups, respectively, with a p=0.46.Moreover, all evaluated risk factors, including a history of DM (p<0.05),chronic hypertension (HT) (p<0.05), and pre-eclampsia, as well as a family history of hypertension (p<0.05),showed significant differences (Table 2).
The normality test indicated non-normally nondistributed data for systolic blood pressure, and the Wilcoxon statistical test was performed.Meanwhile, PlGF, systolic blood pressure, diastolic blood pressure, and MAP parameters followed a normal distribution and were subjected to t-test statistical analysis (Table 3).For healthy pregnant women without intervention, heavy activity, or dietary changes before and after the yoga sessions, no significant differences were observed in their PlGF (p>0.05),systolic blood pressure (p>0.05),diastolic blood pressure (p>0.05), and MAP (p>0.05).
Non-normal distribution was identified through normality tests for systolic blood pressure data, leading to the application of Wilcoxon statistical tests.Meanwhile, diastolic blood pressure, PlGF, and MAP data parameters exhibited normal distribution and were analyzed using t-test statistics.The results of pregnant women with pre-eclampsia examined before and after 12 weeks of prenatal yoga practice (twice a week, 30 minutes per session) showed significant differences in PlGF (p<0.05),systolic blood pressure (p<0.05),diastolic blood pressure (p<0.05), and MAP (p<0.01).

DISCUSSION
This pre-experimental research aimed to evaluate the impact of prenatal yoga on PlGF and MAP in pregnant women with pre-eclampsia and healthy individuals.Furthermore, it was performed in Burneh Bangkalan and comprised 50 participants.Pregnant women with pre-eclampsia were engaged in prenatal yoga twice a week for 12 weeks, each with a duration of 30 minutes, supervised by local experts and certified instructors.
Initially, blood pressure readings indicated no significant changes, but after several treatments, blood pressure levels remained stable, and some decreased.However, PGIF assessments showed that most pregnant women with pre-eclampsia had lower PlGF levels compared to their healthy counterparts.Blood tests were conducted only twice due to certain considerations related to the comfort and willingness of patients.
Pre-eclampsia is characterized by elevated blood pressure reaching 140/90 mmHg during pregnancy, accompanied by proteinuria (Matsubara et al., 2021;Zeisler et al., 2016).Women with chronic hypertension face an increased risk of several pregnancyrelated complications, such as overlapping preeclampsia (PE), fetal growth obstruction, placenta abruption, preterm birth, and the need for cesarean delivery (Seely and Jeffrey, 2014).Chronic hypertension is a significant risk factor for pre-eclampsia, with a risk ratio of 5.1 at a 95% confidence interval (CI) of 4.0-6.5 (Bartsch et al., 2016).In this research, pregnant women with pre-eclampsia were selected based on previously identified risk factors, including maternal age <20->35 years, pre-pregnancy BMI >25, a history of hypertension, DM, kidney disorder, pre-eclampsia, and chronic high blood pressure, as well as a family history of chronic hypertension and pre-eclampsia.Individuals with one or two risk factors were selected as samples (Wheeler et al., 2022).
The results of this study were that pregnant women who were not preeclamptic before and after being given prenatal yoga exercises had no changes in systolic, diastolic, and MAP blood pressure (p<0.05)(Table 2).Meanwhile, pregnant women with preeclampsia showed significant changes in systolic, diastolic, and MAP blood pressure parameters after prenatal yoga practice (p<0.05), as indicated in (Table 3).Small alterations in blood pressure can serve as risk markers for the development of pre-eclampsia.Women with pre-eclampsia tend to have higher systolic blood pressure and MAP before the onset of clinical symptoms.Moreover, MAP is easily calculated by dividing the summation of both systolic blood pressure and twice the diastolic blood pressure value by three.This parameter predicts pre-eclampsia in the first or second trimester more than systolic and diastolic readings (Cnossen et al., 2008). Spencer et al. (2007) reported that blood pressure and MAP measured between 11 and 19 weeks of pregnancy had a 10% false-positive rate with detection rates of 74.3, 62.9, and 49.3% for pre-eclampsia at the onset stage, preterm pre-eclampsia, and total pre-eclampsia, respectively.At 20-24 weeks, the false-positive rate remained at 10%, with 84.3, 65.7, and 52.5% detection rates, respectively (Gallo et al., 2014).
Research has reported significant reductions in mean heart rate and systolic and diastolic blood pressure after six months of yoga (Ankad et al., 1995).Similarly, Bharshankar et al. (2003) observed a substantial decrease in resting heart rate, systolic heart rate, and blood pressure following yoga practice.The results were consistent with other investigations performed on the physiological effects of yoga exercises (Ankad et al., 1995;Bharshankar et al., 2003).
Prenatal yoga modifications are designed to accommodate the physical changes associated with pregnancy.Although direct research into the effects of prenatal yoga on maternal and fetal physiological parameters remains limited, no significant adverse effects have been recorded.An evaluation was previously conducted on these parameters in pregnant women exposed to a series of 26 yoga postures during the third trimester.The results showed that the essential signs of the mother, pulmonary oximetry, and uterine activity monitoring remained normal during the examination period.Furthermore, no adverse obstetric clinical outcomes, such as contractions or vaginal bleeding, were discovered within 24 hours of a yoga session, as indicated by the follow-up survey of participants, and no injuries or falls occurred through the evaluation duration (Babbar and Shyken, 2016;Babbar et al., 2016;Dangel et al., 2021).Another research supported the safety of yoga for mothers and fetuses during the third trimester.Additionally, it found that the fetal heart rate and activity, maternal blood pressure, heart rate, and uterine artery Doppler index persisted within normal limits after a yoga session (Polis et al., 2015).This preliminary evidence suggested prenatal yoga as a safe form of modified physical activity appropriate for execution during pregnancy.
Angiogenesis, the process of new blood vessel formation, plays a crucial role in diagnosing placental conditions, as it reflects fetal development (Robinson and Johnson, 2007).The levels of free PlGF in the blood have been found to decrease in pre-eclampsia patients, while they increased during the first 30 weeks.The results of this research showed that there was a significant difference in PlGF levels.Previously, pregnant women with pre-eclampsia had lower PlGF than healthy individuals, while after prenatal yoga practice, PlGF increased.
A current theory suggests that pre-eclampsia occurs from placental hypoxia and ischemia due to inadequate placental perfusion, leading to endothelial dysfunction.Several markers, such as the measurement of serum PlGF levels, have shown specificity and sensitivity in predicting pregnancy related to placenta hypoxia and endothelial dysfunction.PlGF levels are significantly lower in patients with preeclampsia before attaining the pregnancy period of 37 weeks compared to those with normal pregnancy (Gilbert et al., 2008;Granger et al., 2001).
PlGF and vascular endothelial growth factor (VEGF) influence endothelial cells, a crucial aspect in stimulating uteroplacental vasculogenesis.The most recent evidence indicates that the presence of receptor blockers for these two substances in the maternal circulation and the placenta is more common in pregnancy with pre-eclampsia than in normal pregnancy (Carty et al., 2008).The perceived imbalance between proangiogenic and antiangiogenic factors is closely associated with the development of pre-eclampsia.Several investigations reported that the ratio of proangiogenic to antiangiogenic factors in circulation was altered before the onset of pre-eclampsia (Grill et al., 2009).PlGF has been proposed as a marker and mediator of endothelial dysfunction in pre-eclampsia, with various sources confirming significantly lower PGIF concentrations of around 3 to 10 times below the levels in normal pregnancy.
The limitation of this research was that blood tests were conducted twice due to considerations related to the comfort and willingness of participants since they were selected from the general population and not in a hospital setting.Many pregnant women were scared of multiple blood collections, leading to sampling only before initiating the prenatal yoga practice and at the end of the 12 weeks.

CONCLUSION
The results showed that 12 weeks of prenatal yoga, performed twice a week for 30 minutes, significantly increased PlGF levels in pregnant women with preeclampsia while reducing systolic and diastolic blood pressure and MAP. research provided valuable insights into the potential of prenatal yoga to stabilize blood pressure in pregnant women, including those with hypertension.The documented observations could serve as guidelines for preventing and managing pre-eclampsia, considering crucial factors such as the type, intensity, and frequency of exercise and monitoring blood pressure and fetal heart rate before and after prenatal yoga practice.Additionally, the results suggested the benefits of combined training, incorporating pregnancy exercise and walking, for pre-eclampsia prevention.

Table 1 .
General characteristics of pregnant women in two groups.

Table 2 .
Analysis of parameters in pregnant women.

Table 3 .
Analysis of parameters in pregnant women with pre-eclampsia.