INTRODUCTION:

Pregnancy are correlated with the alteration of physic and physiological condition in women where psychosocial, cultural, and environmental stressors experienced during pregnancy exert the influence of maternal and fetal health^1,2. Evidence showed that stress during pregnancy impact the women’s health and labour outcomes ie preterm delivery, prolonged labour, caesarean birth, and low birth weight ^3,4. Previous studies estimated the maternal stress during pregnancy is high and occur in 15,9% pregnancy worldwide ^5-8.

Although mechanism of stress development during pregnancy are unknown, but maternal stress is correlated with the activation of hypothalamus-pituitary-adrenal axis resulting the excessive glucocorticoid secretion from adrenal⁹. In cell levels, maternal stress exert oxygen and hydrogen level and upregulate the rate of reactive oxygen species (ROS) leads to oxidative stress that are able to damage the cell structure ¹⁰.

In order to maintain the pregnancy, the balance of immune system is paramount importance to obtain since the beginning of pregnancy¹¹. Interleukin-6 (IL-6) is a byproduct of abundant level of ROS inside the cells as well as the pro-inflammatory cytokines in maternal and placental circulation¹². Previous study suggested that overexpression of IL-6 in pregnant rats induced the depressive-like behavior while the inhibition of IL-6 promotes the reduction of stress¹³. Other study showed that cytokine profile including IL6 were at higher concentration in the mother with depression rather than the pregnant women without depression¹⁴. Interestingly, abundant level of IL6 during pregnancy also affect the disease in fetal adulthood, injection of IL-6 in pregnant rats directly induce fetal injury including the release of fetal stress hormone which might lead to disease in adult age ¹⁵.

Folic acid (FA) is a synthetic form of vitamin B dissolved in water, and are essential for DNA replication, enzymatic reaction, and involved in amino acid synthesis^16,17. During the last decade, it has been found that folic acid is important in modulating diverse clinical condition during embryogenesis and pregnancy such as birth defects and abnormal development of neural tube¹⁸. Interestingly, FA is also has a potential benefit as the supplementation in pregnancy for prevention of pregnancy-related complication other than neural tube defects¹⁹.

Although several studies have been showed the protective effect of FA in pregnancy and embryogenesis, the direct effect of FA to stress protection in placenta and embryo has not been characterized. To that end, stress mice model was created by chronic restrain stress and 379 lux bright light exposure, this model then treated with FA administration. In this report, we described the effect of FA on fetal birth weight by direct assessment on the fetus. Further, the concentration of IL-6 was also detected with immunohistochemistry (IHC) displayed the similar concentration between the FA treated group and non-treated group. Folic acid has the protective effect to the fetus during pregnancy in stress induced mice model.

MATERIAL AND METHODS:

Animal

All of the procedure in this study was approved by University of Airlangga animal care and use committee (with approval number 459-KE). Twenty-one female Mus musculus aged 12-14 weeks weighed 25-30 gram was divided into 3 groups: normal pregnant mice (n=7), stress pregnant mice (n=7), and stress pregnant mice treated with FA (n=3). Mice were acclimatized for 1 week and maintained with ad libitum supply for standard feed and drinking water in 20-25°C on 13/11 photo period. The mice then injected by Pregnant mare’s Serum Gonadotropin (PMSG) 5 IU intraperitoneal followed by 5 IU hCG intraperitoneal injection. The 48 hours after injection, male and female mice were put together in 1 cage (monomating, 1 female and 1 male) for 17 hours. Vaginal plug was taken as an evidence of pregnancy and stated as gestational day (gd)-0.

Normal pregnant mice received aquabidest via lavage. Stress exposure were given at gd-10 to gd-15, mice were left their home cages and stress induced by giving environment change (individual was restrained in transparent cylinder plastic fix to the mice body and the light was exposed to the mice body in 397 lux for 30 minutes twice a day (morning and evening). Three mg/kg BW folic acid was dissolved in aquadest and emulated in Carboxymethyl cellulose (CMC) 0.5% for with the maximum volume of 500 ml, the solution was administered via lavage once a day before morning stress experiments given. All mice were euthanized at gd 16. The placenta and fetus then collected from the animal.

Histology:

post mortem sample of placenta were fixed in 10% buffered formalin. The 3 μm placenta thickness were obtained and stained for immunochemistry observation. Immunohistochemistry was performed by deparaffinization of the samples in the absolute ethanol, degraded to 70% ethanol followed by washing with aquabidest. Subsequently, the samples were incubated with hydrogen peroxide for 5 minutes followed by washing step with phosphate buffer saline (PBS). Anti-IL6 antibody (Abcam) was used to detect the presence of IL-6 in the placenta followed by IgG secondary antibody. DAB substrate then added for a maximum of 20 minutes to induce the color change. All slides were counterstained using Streptovidin peroksidase to differentiate the background. In the negative control group, primary antibody was replaced by PBS. The presence of IL-6 was assessed by the brownish color under the microscope.

The images were collected using brighfield microscope Nikon H600L with 40x magnification. Semiquantitative assessment was obtained using immunoreactive score (IRS). The section was examined with 40x magnification in 5 random locations in mice placenta. The intensity of the staining was obtained using ImageJ software. Statistical analysis was performed using SPSS program (Version 20.0, SPSS Inc, IBM) with One-way Analysis of Variance (ANOVA) and followed by post hoc test and finally correlation test with Pearson test. Value were expressed as mean±standard deviation (SD), The interval confidence was 95% with significant value of <0.05.

Fetus weight:

The fetus was collected from the mice, weighted directly after collection using electric weighted Camry EHA401.

RESULT:

Fetus weight observation

The fetus weigh was assessed in the 16^th day of pregnancy. The FA treated mice displayed higher fetus weight (0,61±0,15 g) compared to the stress mice without FA, implying the effectiveness of FA treatment during stress. This increase was significantly different in each group.

Figure 1. Fetus weight in mice from control, stress pregnant mice, and stress pregnant mice treated with formic acid 3 mg/kg bw/day

Immunohistochemistry of IL6 in pregnancy-induced stress placenta:

The protective effect of folic acid was observed using placental IL-6 expression with immunohistochemistry. This changing however, were not significantly different (p>0.005). The IL-6 expression with the stress exposure group were increase. In addition, by consuming FA, the mice showed lower placental IL-6 expression but however, this expression is higher than the control group.

Figure 2. IL-6 expression in placenta A. Image from IL-6 counterstained with methyl green B. Immunoreactive score for IL-6 from control, stress mice, and stress mice treated with formic acid 3 mg/kg bw/day. Immunohistochemistry. Yellow arrow showed the IL-6 expression

DISCUSSION:

Prenatal stress is correlated with the adverse effect on maternal health, fetus development and embryogenesis. This effect could be either directly affect the fetal development or indirectly by influencing maternal health which in turn affect the infant prosperities¹. It has been suggested that maternal stress affecting pregnancy by disrupting maternal endocrine, nervous, and immune response²⁰.

A total of 7 stress pregnant mice model, all of them displayed the adverse effect on maternal and fetal response including inflammation and low birth weight of the fetus compared to control. Maternal stress during pregnancy is associated with exaggerate maternal immune response. This condition, however cause number of disorders in maternal health, birth outcomes, and behavioral outcomes of the offspring^9.Placenta is a specific-organ which connect maternal and fetal interface in order to supply all of organs in the fetus’ body by nutrients and oxygen transfer¹⁰. Abnormal placental function that induced by stress were able to influence the fetal health. We hypnotized that the folic acid administration during pregnancy has protective effect to maternal prenatal stress via action in reducing pro-inflammatory cytokines such as IL-6, IL-8, TNF-α, consequently fetus weight gain will improve.

In keeping with previous study, we found that fetus birth weight was significantly increased in FA administration. From systematic review and meta-analysis, Fekete and colleagues found that for every two-fold increase in folate intake, the birth weight was also increase ²¹. Prenatal stress is related to the impairment of many physiological responses, including the alteration of placental vascularization due to the elevated of inflammation process induced by maternal stress^22,23. Folic acid supplementation is important to increase the placental folate transport capacity which is most likely to reduce in IUGR and abnormal environment such as tress ^24,25.

This study examining the direct relationship between the role of FA in reducing IL-6 concentration during stress in pregnancy. We found that there was no relationship between these variables. Regarding the effect of FA on inflammatory cytokine, previous studies demonstrated that FA, when combined with vitamin B12 can reduce nicotine-induced impairment by modulating levels of pro-inflammatory cytokines ie TNF-α and IL6²⁶. However, the supplementation was added with B12 than in our study and the pregnant mice condition, making any direct comparison inappropriate. Given that IL-6 concentration was not statistically altered in pregnant mice, either the higher dose of FA or the combination with B12 might compromise in such condition. In addition, since FA has the potential role in determining the effect of inflammation by reducing pro-inflammatory cytokines, observing other pathway might also be necessary to expand the research.

In this study, we reported that FA has a potential effect to increase the fetus weight in maternal stress mice model without the effect in IL-6 expression. In addition, the observation of other inflammation marker such as cortisol, homocysteine, IL-2, IL-8, TNF-α, apoptosis, VEGF and placental vascularization might be needed.

ACKNOWLEDGEMENT:

This work was supported and funded, by Faculty of medicine, University of Airlangga.

CONFLICT OF INTEREST:

The authors declare they have no conflict of interest.

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Received on 29.06.2020 Modified on 27.07.2020

Research J. Pharm. and Tech. 2021; 14(8):4371-4374.

DOI: 10.52711/0974-360X.2021.00759