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ORIGINAL ARTICLE
Year : 2020  |  Volume : 69  |  Issue : 1  |  Page : 48-52

An experimental study on the effect of maternal folate diet on microstructure of some vital organs of offspring


1 Department of Anatomy, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, Karnataka, India
2 Department of Anatomy, Government Medical College, Palakkad, Kerala, India

Date of Submission08-Jun-2019
Date of Acceptance04-Feb-2020
Date of Web Publication11-Apr-2020

Correspondence Address:
Dr. Lokadolalu Chandracharya Prasanna
Kasturba Medical College, Manipal Academy of Higher Education, Manipal - 576 104, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JASI.JASI_73_19

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  Abstract 


Introduction: Folic acid (FA) deficiency or its supplementation during pregnancy affects fetal development at critical periods and can cause more potentially impaired organ development through epigenetic gene regulations. The present study aimed to analyze the effect of maternal FA deficiency and/or its supplementation in diet during gestation on offspring's few major fetal organs, including brain, liver, pancreas, and kidney. Material and Methods: This experimental study was done on 18 female Wistar rats to study the effects of dietary FA intake (absence/supplementation,/normal amount) during pregnancy on the development of offspring's liver, kidney, and pancreas development. Results: The present study revealed that with maternal FA supplementation, there was increase in the number of islets in pancreas, number of hepatic lobules, and renal glomeruli in offspring, which, in turn, correlates with increased risk of cardiovascular, renal, and metabolic diseases in their later life. Discussion and Conclusion: The findings in this study support the possible negative effects of higher FA supplementation during peri-conceptional and pregnancy phase on offspring and indicates long-term health hazards in their later life.

Keywords: Development, folic acid, offspring, organ, pregnancy, supplementation


How to cite this article:
Prasanna LC, Vinaykumar N, Ramesh A. An experimental study on the effect of maternal folate diet on microstructure of some vital organs of offspring. J Anat Soc India 2020;69:48-52

How to cite this URL:
Prasanna LC, Vinaykumar N, Ramesh A. An experimental study on the effect of maternal folate diet on microstructure of some vital organs of offspring. J Anat Soc India [serial online] 2020 [cited 2020 May 28];69:48-52. Available from: http://www.jasi.org.in/text.asp?2020/69/1/48/282305




  Introduction Top


Folic acid (FA) has an important role in reducing the incidence of birth defects in offspring, and this fact has gained considerable attention in recent generation.[1] Prenatal supplementation of FA and its usage or consequence of long-term health hazards in offspring has remained debatable and unsettled.[2]

Due to a lack of epidemiological findings, the reference range for serum folate in women, especially during reproductive age, has not been mentioned.[3] In general, pregnancy demands high FA availability due to a greater need for the growth of the fetus and uteroplacental organs as well as the maternal excess FA urinary excretion.[4] Various studies have revealed that there is a positive correlation between the maternal plasma FA levels and the incidence of cardiovascular diseases,[5] structural brain changes/neurodegenerative conditions,[6],[7],[8] insulin resistance,[9] and childhood asthma[10] in offspring in the later period of life. Few clinical studies have reported on the incidence of decreased cognitive behavior,[11] glomerular sclerosis, systemic blood pressure,[12] and conotruncal defects[13] in the postnatal life.

The purpose of the present study was to evaluate the effect of maternal folate diet during gestation (either its absence or its supplementation) on the microstructure of their offspring's few vital organs (brain, liver, pancreas, and kidney). This would help us to correlate the risk of development of neurological, metabolic, and renal diseases in the offspring in their adult life.


  Material and Methods Top


This experimental study was conducted from June 2017 to February 2018, after the approval letter was obtained by our institutional animal ethical committee. Eighteen female Wistar rats were obtained from the central animal research facility of our institution and their body weight was approximately 120 g.

Selection of groups

The female Wistar rats were divided randomly into three groups with six rats in each group. The first (control) group of animals was administered a diet containing a normal recommended dose of FA as provided by the central animal lab. The second group of animals was administered a diet with no FA content (purchased from Vinod Ramakrishna (VRK) Biotech Pvt Ltd, Miraj, Maharashtra, India. To ensure complete FA absence, we added 1% succinyl sulfathiazole to the diet with no FA. This reduces or inhibits the gut flora which is responsible for the synthesis of FA and also avoids coprophagy during gestation. The third group of animals was administered a diet with an excess amount of FA (4 mg/kg body weight, daily).

All rats were caged in separate polypropylene cages in the central animal research facility with (12 × 12 h light and dark cycle, 25°C, and 35% humidity) with free access to water. All rats were given diets specific to each group, 5 weeks before gestation, throughout the gestation, and 3 weeks after their delivery. The litter size, number, and weight of the pups were recorded after delivery. All the pups from each group were weaned on the 21st day, and then the male and female pups were separated and maintained in separate cages till adult age.

Collection of specimens

On the 75th day of birth, rats from each group were sacrificed. Upon dissecting the abdominal cavities, few organs (liver, pancreas, and kidneys) were carefully dissected, fixed in 10% formalin, and processed further for H and E staining. On microscopic examination, we studied three different fields of the same tissue, and the average was considered as the final value to avoid intraobserver variations.

Statistical analysis

Data were analyzed using the Statistical Package for the Social Sciences statistical software (version 6.0). The number of classical hepatic lobules and its hepatocytes, number of  Islets of Langerhans More Details along with their cells, and the number of glomeruli were compared with each group. P < 0.05 was considered statistically significant.


  Results Top


Microscopic details

Liver

Microscopic sections of the liver [Figure 1]a of the control group showed hepatocytes with normal microstructure and prominent nucleoli. The number of classical hepatic lobules per field in low magnification (×10) was 12–13 and in each lobule [Graph 1]a, the number of hepatocytes from central to peripheral zone as counted in higher magnification (×40) was found to be 8–9 [Graph 1]b.
Figure 1: Photomicrograph of liver (a) at × 40, pancreas (b) at × 40, and kidney (c) at x10 magnification (C – Control group, F Ab: Folic acid-absent group, F Inc: Folic acid-supplemented group)

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In FA-absent diet group sections, hepatocytes showed partial nucleomegaly with dispersed nucleoli, whereas in FA-supplemented group, hepatocytes showed heterogeneous nuclear changes with small and large few nuclei with prominent nucleoli. The classical hepatic lobule per field in FA-absent diet was 10–11, and the number of hepatocytes in each row increased to 9–10, whereas in the FA-supplemented group, the lobules increased to 16/field and the number of hepatocytes increased to 11–12 in each row.

Pancreas

Sections of the control group and the FA-supplemented group did not show any histological changes [Figure 1]b, ×40]. In the control group, we found 3–4 islets per field in low magnification, i.e., ×10 [Graph 2]a and an average of 81–82 cells in each islet of Langerhans [Graph 2]b.



Few exocrine acini of adult born to FA-absent diet group showed nucleomegaly with immature chromatin and single prominent nucleolus suggestive of megaloblastic changes. The islets were reduced to an average of 3, and the number of cells increased to 105/islet, whereas in the FA-supplemented group, we found the number of the islets to be between 6 and 7/field and 84–85 cells per islet.

Kidney

Histological sections of the kidney [Figure 1]c, ×4) in the control group showed no significant changes and the number of glomeruli ranged between 20 and 21/field [Graph 3]. Adults born to mothers treated with absent FA diet showed large nuclei with prominent nucleoli (with average 25 glomeruli per field in × 10) and those born with FA-supplemented mothers showed heterogeneous nuclear changes, with small and large nuclei and prominent nucleoli. In addition, the number of glomeruli was found to be 30–31.



All the three groups maintained near-normal cytoarchitecture with minimal nuclear changes, and no inflammation and necrosis were identified.


  Discussion Top


Alterations in the maternal micronutrients, especially FA in diet during gestation and/or lactation, are associated with an increased risk of cardiovascular, renal, and metabolic diseases in later life.[12],[13] A research study recommended that higher dose of FA (5 mg) should be provided only in special cases such as history of neural tube defects in epileptic and diabetic mothers and patients with inconsistent birth control and recreational substance abuse in the form of alcohol, tobacco, and drugs.[14] Although FA supplementation to pregnant women demonstrates many benefits to their fetuses, concerns have been documented about the detrimental effects of the unmetabolized FA in their offspring.[15]

Accumulating evidence suggests that there is an association between the maternal plasma FA levels and the incidence of vascular diseases[12], structural brain changes and neurodegenerative conditions[15],[16], insulin resistance[16], and childhood asthma[17] in their offspring in the later period of life. Few clinical studies implicate the incidence of decreased behaviour and cognitive behaviour[18], glomerular sclerosis, and systemic blood pressure, and conotruncal defects[19],[20],[21] in the postnatal life.

Lucock and Yates[22] claimed that excess FA use in pregnancy causes T allele of MTHFR in children, and such allele infants have a greater tendency of spontaneous abortion because of increased homocysteine levels in their mother. In addition, carriers of T allele are at greater risk of neurological disorders such as depression,[7] schizophrenia,[8] bipolar disorders,[7] neural tube defect,[23] and rarely Down's syndrome.[24],[25] A cohort study by Valencia, as quoted by Valera-Gran et. al, (2014)[26] demonstrated that the excess maternal FA was associated with reduced fetal growth, which, in turn, reduces the infant's psychomotor development.

An experimental study of Wistar rats born to FA-supplemented mothers during their pregnancy showed no significant changes in liver microarchitecture.[27] Sections of the liver of rats born to mother given with excess/supplemented amount of FA showed enlarged hepatocytes with cytoplasmic vacuolations, consistent with hydropic degeneration or hepatic ballooning.[28] Roncalés et al.'s[29] study revealed a 17% increase in the number of hepatocytes as well as the size of the hepatocytes with FA supplementation. In another study, altered methylation promoters were found in livers of male fetuses born to mothers fed with FA-deficient diet.[11] Contrary to the earlier studies, we found that the number of classical hepatic lobules and the number of hepatocytes in each row increased in rats born to FA-supplemented mothers. In addition, hepatocytes showed heterogeneous nuclear changes with small and large few nuclei with prominent nucleoli.

Maternal FA deficiency can affect the offspring's pancreas and can cause a reduction in the β-cell area as well as a decrease in the number of cells per islet by 50% along with the increased risk of insulin resistance. This could be due to hormonal modification in both mother and fetus, alteration in epigenetic gene regulation, and limited fetal growth and development.[30] Chittiboyina et al.[30] found that there was a remarkable increase in the percentage of islets of Langerhans and decrease in the percentage of the number of acini in mouse embryo after supplementation of folate during gestation, concluding that administration of FA diet influences the development of the pancreas and its deficiency leads to histopathological alterations. In the present study, the number of islets decreased and numerous small and large islet cells were demonstrable in rats born to mothers fed with FA-absent diet and the number of islets increased in rats born to mothers fed with FA-excess diet with normal to a high number of cells per islet.

Maternal FA restriction results in a decrease in the number of nephrons, lower filtration surface area per glomerulus, electrolyte imbalance, an increase in glomerular pressure, glomerular sclerosis, and finally end up in systemic blood pressure and renal disease.[12],[31] Black et al.[32] found that the reduced folate and Vitamin B12 concentrations with increased homocysteine levels in pregnancy can alter the nephrogenesis, leading to smaller kidney and reduced renal function in their offspring. However, few works of literature have stated that both kidney function and risk of metabolic syndrome were greatly benefited by supplementation of FA during pregnancy.[9] We found that there was an increase in the number of glomeruli as compared to other experimental groups.


  Conclusion Top


The present study supports the possible negative effect of higher FA supplementation than recommended during periconceptional and throughout the pregnancy. However, it is hard to conclude conclusively due to the limitation of the small sample size in the present study. Further research is needed to evaluate the most folate-sensitive period of fetal organ development as well as the dose-dependent effects of folate during different periods of gestation (periconceptional, early gestation, mid-gestation, and during the last phase) on the development of vital organs and its association with the development of diseases in the later life.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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