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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 2  |  Page : 39-42

Teratogenic effects (resorptions and reduction in weight and crown rump length) of valproate on fetal mice


1 Assistant Professor, Department of Anatomy, Integral Institute of Medical Sciences and Research, Integral University, Lucknow, Uttar Pradesh, India
2 Assistant Professor, Department of Forensic Medicine and Toxicology, Integral Institute of Medical Sciences and Research, Integral University, Lucknow, Uttar Pradesh, India
3 Professor and Head, Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Date of Submission26-Jun-2020
Date of Decision29-Jul-2020
Date of Acceptance14-Aug-2020
Date of Web Publication10-Sep-2020

Correspondence Address:
Neelesh Kumar Shakya
Department of Forensic Medicine and Toxicology, Integral Institute of Medical Sciences and Research, Integral University, Dasauli, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/NJCA.NJCA_9_20

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  Abstract 


Background and Aims: Teratology is the study of abnormal development in fetus. Teratology first came into existence in 1930 when a number of experiments were conducted. There are various causes of congenital anomalies such as genetic factors, environmental factors such as radiation, changes in temperature, hypoxia, chemical substances, drugs, and maternal infections. At present, valproic acid is the most widely used antiepileptic drug. Valproic acid is salt of dipropyl acetic acid. Most of the studies on the valproate were done at higher doses starting from 400 mg/kg. Hence, the present study was done to assess teratogenic effects at lower dose of valproate i.e., 200 mg/kg. Only few studies are conducted on fetal mice at Valproate dose of 200 mg/kg. The aim of the present study is to report the teratogenic effects of valproate at dose of 200 mg/kg on single gestational day (8th) and multiple gestational days (7th, 8th, and 9th) in fetal mice. Materials and Methods: Forty pregnant mice were taken in the present study, and these are distributed in three groups. Group A which was control group received normal saline. In Group B, Valproate was administered on single gestational day (8th). In Group C, Valproate was administered on multiple gestational days (7th, 8th, and 9th). Dose of valproate given in each group was 200 mg/kg intraperitoneally. On the 18th gestational day, fetuses were collected after uterotomy. The fetuses of all three groups were weighed by digital laboratory weighing scale. Crown rump length (CRL) of fetuses of all groups was documented by means of graph paper. Mean, standard deviation, and P value were calculated. Statistical analysis was done by the ANOVA one-way test. Results: Valproate administered groups showed resorptions. CRL and fetal weight reduction were found in treated groups. These findings were more found in Group C in comparison to Group B. Conclusion: Valproate is teratogenic drug at 200 mg/kg dose, so valproate should be prescribed at lowest efficacious dosage to minimize the teratogenic risk.

Keywords: Fetal mice, gestation, teratogenic, valproate


How to cite this article:
Shakya P, Shakya NK, Mohanty C. Teratogenic effects (resorptions and reduction in weight and crown rump length) of valproate on fetal mice. Natl J Clin Anat 2020;9:39-42

How to cite this URL:
Shakya P, Shakya NK, Mohanty C. Teratogenic effects (resorptions and reduction in weight and crown rump length) of valproate on fetal mice. Natl J Clin Anat [serial online] 2020 [cited 2020 Oct 27];9:39-42. Available from: http://www.njca.info/text.asp?2020/9/2/39/294753




  Introduction Top


Study of abnormal fetal development due to any teratogenic factor is known as teratology. A number of factors can cause the valproate-induced teratogenicity. These factors are drug dosage, the number of co-administered drugs, metabolism of mother, fetal gestational age at the time of exposure.

Valproic acid is used mainly as a mood-stabilizing and an anticonvulsant while now a days there is also guidelines for using in the treatment of bipolar disorders, schizophrenic disorder, and migraine headache. It is the salt of dipropyl acetic acid.

Valproate was licensed to use in the year 1978. First teratogenic effect due to valproate was reported in year 1980. Since that time teratogenicity of valproate has been emphasized.[1]

A number of mechanisms cause valproate teratogenicity. It is thought that valproate acts by inhibition of gamma-aminobutyric acid metabolism and impairment of metabolism of cell.[2] Valproate teratogenic dose significantly change folate metabolism in the fetus by alteration in the level of tetrahydrofolate, 5-formyl and10-formyltetrahydrofolate. These changes could be due to the effect of valproic acid on interruption of the formyl group transfer by the enzyme glutamate formyltransferase.[3] Valproic acid passes through the placental barrier, and its concentration is found more in fetal blood than maternal blood.

Maximum number of malformations due to valproate administration is congenital heart defects, neural tube disorders, oral clefts, limb disorders, craniofacial abnormalities, and genital developmental defect. Valproate also causes bifid ribs, hernia in umbilical and inguinal region, additional digit on the ulnar margin of hand, additional great toe in feet, supernumerary nipple behavioral problems, and learning difficulties.

Unfortunately, most of the publications related to teratogenic effects of antiepileptic drugs are lacking in information to draw any significant inference.

Most of the studies on the valproate were done at higher doses starting from 400 mg/kg. Hence, the present study was done to assess teratogenic effects at lower dose of valproate, i.e., 200 mg/kg.

Reports on valproate teratogenicity at 200 mg/kg dose are very limited in the literature so the present study was planned to evaluate the teratogenic effects of valproate in fetuses of mice.

The aim of this research article is to analyze the abnormal developmental defects of valproate on mice fetuses in terms of resorptions, weight and crown rump length (CRL) reductions.


  Materials and Methods Top


The present research was conducted in Anatomy department, Institute of Medical Sciences, Banaras Hindu University, Varanasi after obtaining ethical clearance from the institute.

Forty female adult Swiss albino mice having weight of 20–25 g were used in the present research work after approval of Institutional Ethical Committee. These animals were obtained from the animal house of Anatomy department of Institute of Medical Sciences, Banaras Hindu University, Varanasi.

The animal house was maintained at an ambient temperature of 25°C ± 2°C and 50%–60% relative humidity with 12 h: 12 h light dark cycle. The animals were housed in polypropylene cages with husk bedding. They were fed on pelleted diet obtained from local Pashu Ahaar Kendra and tap water, ad libitum.

The pregnant mice were separated out and housed individually in different cages. The pregnant mice were weighed every day.

Forty pregnant mice were taken in the present study and distributed into three different groups.

  1. Group A: Equal amount of normal saline (10 mice)-control group
  2. Group B: 200 mg/kg single dose on 8th gestational day (15 mice)
  3. Group C: 200 mg/kg dose on the 7th, 8th, and 9th gestational day (15 mice)


Valproate in Group B and Group C was administered via intraperitoneal route. Normal saline was also administered to Group A through same route.

On the 18th gestational day, deep ether anesthesia was given to mice of all the groups, and then, mice of all groups were sacrificed. Collection of fetuses was done after uterotomy.

The fetuses of all three groups were weighed after blotting by digital laboratory weighing scale. Documentation of CRL of all group's fetuses were done by means of graph paper. CRL was measured from topmost point of head to tip of tail of fetus.

The collected fetuses were also photographed.

Resorptions and intrauterine growth retardation (IUGR) was noted in all the groups during the collection of fetuses.

Resorption is the disintegration of fetuses in uterine horns of mice.

For documentation of fetal resorptions, total number of resorption sites was counted in both uterine horns of the pregnant mice in all the groups after careful observation.

IUGR is percentage of fetuses having weight <10th percentile of fetal weight of control group.

Statistical analysis was performed by the ANOVA one-way test.


  Results Top


Resorptions [Table 1] and [Figure 1] and IUGR [Table 2] and [Figure 2] were seen in valproate treated fetuses when compared to the control group. For the evaluation of IUGR, 10th percentile of fetal weight of control group is calculated, i.e., 1.216 g.
Table 1: Comparison of resorptions of fetuses in different groups

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Figure 1: Resorptions (black arrow) in sodium valproate administered fetuses

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Table 2: Comparison of intrauterine growth retardation of fetuses in different groups

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Figure 2: Intrauterine growth retardation (black arrow) in sodium valproate administered fetuses

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Mean fetal weight [Table 3] and mean CRL [Table 4] were also found less in treated group when compared to the control group.
Table 3: Comparison of mean weight (g) of fetuses in different groups

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Table 4: Comparison of mean crown rump length (mm) of fetuses in different groups

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These effects were found more in multiple dose administered fetuses in comparison to single dose administered fetuses [Table 1], [Table 2], [Table 3], [Table 4].


  Discussion Top


In the present study, there was significantly higher resorption in fetuses exposed to sodium valproate. Rate of resorption was found in 11.54% and 45.54% in single and multiple dose administered fetuses, respectively [Table 1] and [Figure 1].

Various authors[4],[5],[6],[7],[8],[9] found significantly higher resorptions in fetuses exposed to sodium valproate. Faiella et al.[10] reported the lethal effects on embryo having range from 34% to 100% in different strains of mice. It shows that the variation in embryo lethality depends upon the different strains of mice.

In our study, we reported that in valproic acid treated group on the 8th gestational day (Group B) and Valproic acid treated group on the 7th, 8th, and 9th gestational day (Group C), there was significant reduction in CRL, fetal weight in comparison to the control group (Group A). IUGR was found in 70.65% and 100% in single and multiple dose administered fetuses, respectively [Table 2] and [Figure 2]. There was no IUGR in the control group.

IUGR and weight reduction may be due to developmental toxic effect of valproate.[11],[12]

Ong et al.,[4] Binkerd et al.,[5] Elmazar et al.,[6] Khera,[7] Padmanabhan et al.,[8] Al Deeb et al.,[13] Akbari et al.,[14] Hrubec et al.,[15] Tung et al.,[16] Mahabady et al.[9] also observed IUGR, decrease in fetal weight, and reduction in CRL in valproate exposed fetuses.

Vorhees et al.[17] depicted that the exposure of valproic acid to mice at 400 mg/kg on 7th–18th day of gestation produced reduction in fetal weight in 43% fetuses. Faiella et al.[10] reported decrease in fetal weight ranging from 62% to 89% fetuses in different strains of mice. This discrepancy in different strains indicates genetic control on development of lethal effects in embryo.

In a human study by Kozma,[18] 15% of children had growth retardation after exposure to pregnant women with Valproic acid.

From pregnancy registry data, Diav-Citrin et al.[19] study showed that valproic acid produced a significant increased dose dependent teratogenic effects in utero when administered to pregnant women at the dose of 600–1000 mg/day.

Kulaga et al.[20] conducted a study which was based on pregnancy registry data. They reported abortion, still birth, IUGR when epileptic women had taken valproate during their pregnancy.


  Conclusion Top


Resorption rate was higher in groups receiving sodium valproate and it is dose related as the resorption rate was significantly higher in group receiving multiple doses as compared to those receiving single dose.

There was significant decrease in weight and crown lump length of fetuses in valproate exposed groups.

The present study concludes that valproate is teratogenic. Hence, there must be few principles for the use of valproate during pregnancy.

The first principle is polytherapy (multiple anticonvulsant drug combinations) should be avoided in the treatment of women in reproductive age if possible.

The second principle is that if valproate use in pregnancy is unavoidable, then it should be prescribed at lowest efficacious dosage to minimize the teratogenic risk.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Dalens B, Raynaud EJ, Gaulme J. Teratogenicity of valproic acid. J Pediatr 1980;97:332-3.  Back to cited text no. 1
    
2.
Brown JK. Valproate toxicity. Dev Med Child Neurol 1988;30:121-5.  Back to cited text no. 2
    
3.
Wegner C, Nau H. Alteration of embryonic folate metabolism by valproic acid during organogenesis: Implications for mechanism of teratogenesis. Neurology 1992;42:17-24.  Back to cited text no. 3
    
4.
Ong LL, Schardein JL, Petrere JA, Sakowski R, Jordan H, Humphrey RR, et al. Teratogenesis of calcium valproate in rats. Fundam Appl Toxicol 1983;3:121-6.  Back to cited text no. 4
    
5.
Binkerd PE, Rowland JM, Nau H, Hendrickx AG. Evaluation of valproic acid (VPA) developmental toxicity and pharmacokinetics in Sprague-Dawley rats. Toxicol Sci 1988;11:485-93.  Back to cited text no. 5
    
6.
Elmazar MM, Thiel R, Nau H. Effect of supplementation with folinic acid, Vitamin B6, and Vitamin B12 on valproic acid-induced teratogenesis in mice. Fundam Appl Toxicol 1992;18:389-94.  Back to cited text no. 6
    
7.
Khera KS. Valproic acid-induced placental and teratogenic effects in rats. Teratology 1992;45:603-10.  Back to cited text no. 7
    
8.
Padmanabhan R, Hameed MS. Exencephaly and axial skeletal malformations induced by maternal administration of sodium valproate in the MF1 mouse. J Craniofac Genet Dev Biol 1994;14:192-205.  Back to cited text no. 8
    
9.
Mahabady MK, Najafzadeh Varzi H, Ranjbar R, Rahgazar M. Prophylactic effects of melatonin on sodium valproate induced neural tube defects and skeletal malformations in rat embryos. Am J Appl Sci 2011;8:413-9.  Back to cited text no. 9
    
10.
Faiella A, Wernig M, Consalez GG, Hostick U, Hofmann C, Hustert E, et al. A mouse model for valproate teratogenicity: Parental effects, homeotic transformations, and altered HOX expression. Hum Mol Genet 2000;9:227-36.  Back to cited text no. 10
    
11.
Bailey CJ, Pool RW, Poskitt EM, Harris F. Valproic acid and fetal abnormality. Br Med J (Clin Res Ed) 1983;286:190.  Back to cited text no. 11
    
12.
Emmanouil-Nikoloussi EN, Foroglou NG, Kerameos-Foroglou CH, Thliveris JA. Effect of valproic acid on fetal and maternal organs in the mouse: A morphological study. Morphologie 2004;88:41-5.  Back to cited text no. 12
    
13.
Al Deeb S, Al Moutaery K, Arshaduddin M, Tariq M. Vitamin E decreases valproic acid induced neural tube defects in mice. Neurosci Lett 2000;292:179-82.  Back to cited text no. 13
    
14.
Akbari M, Abolhassani F, Azizi M, Dehpour AR, Ansari M, Ahmadi Faghih MA, et al. Altered plasma zinc level contributes to the developmental toxicity of valproic acid in skeletal system of rat. Acta Med Iran 2004;42:10-5.  Back to cited text no. 14
    
15.
Hrubec TC, Yan M, Keying Y, Salafia CM, Holladay SD. Valproic acid induced fetal malformations are reduced by maternal immune stimulationc with granulocyte macrophage colony stimulating factor or interferon Y. Anat Rec A Discov Mol Cell Evol Biol 2006;288:1303-9.  Back to cited text no. 15
    
16.
Tung EW, Winn LM. Valproic acid increases formation of reactive oxygen species and induces apoptosis in postimplantation embryos: A role for oxidative stress in valproic acid-induced neural tube defects. Mol Pharmacol 2011;80:979-87.  Back to cited text no. 16
    
17.
Vorhees CV. Teratogenicity and developmental toxicity of valproic acid in rats. Teratology 1987;35:195-202.  Back to cited text no. 17
    
18.
Kozma C. Valproic acid embryopathy: Report of two siblings with further expansion of the phenotypic abnormalities and a review of the literature. Am J Med Genet 2001;98:168-75.  Back to cited text no. 18
    
19.
Diav-Citrin O, Shechtman S, Bar-Oz B, Cantrell D, Arnon J, Ornoy A. Pregnancy outcome after in utero exposure to valproate: Evidence of dose relationship in teratogenic effect. CNS Drugs 2008;22:325-34.  Back to cited text no. 19
    
20.
Kulaga S, Sheehy O, Zargarzadeh AH, Moussally K, Bérard A. Antiepileptic drug use during pregnancy: Perinatal outcomes. Seizure 2011;20:667-72.  Back to cited text no. 20
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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