FREQUENCY OF DIFFERENT CONGENITAL ANOMALIES IN PRENATALLY VALPROIC ACID TREATED CHICK EMBRYOS.
Objective: To determine the frequency of different congenital anomalies in surviving chick embryo on hatching after the prenatal administration of valproic acid by comparing with age-matched controls.
Study Design: Experimental study.
Place and Duration of Study: Anatomy Department, College of Physicians and Surgeons Pakistan (CPSP) Regional Centre, Islamabad, from February 2010 to February 2011.
Material and Methods: Thirty fertilized chicken eggs were injected with valproic acid, incubated and then evaluated for different gross congenital anomalies, on hatching or day 22 of incubation whichever was earlier. Chicks of this group were labeled as experimental group-A. Similarly, another group of thirty fertilized chicken eggs labeled as control group-B, underwent sham treatment using normal saline. The weight and length of alive chicks, the total number of chicks with gross anomalies and the number of different types of gross anomalies in both groups were noted and statistically compared.
Results: In control group-B, 28 chicks hatch out on 21 day of hatching with no visible gross deformities. Whereas in experimental group-A, 23 chicks were alive, out of which, 9 chicks were with delayed hatching on 22 days of hatching. The chicks with gross deformities were 8 (p=0.0008) which included: limb abnormalities (i.e. inverted feet) in 6 chicks (p=0.006), eye abnormality (i.e. closed palpebral fissure of both eyes) in 2 chick (p=0.2), 1 chick showed multiple deformities including gastroschisis, closed palpebral fissures and inverted foot (p=0.45). There were behavioral changes in 10 chicks (p=0.0001). There was statistically significant difference in their weights (p=0.03).
Conclusion: Prenatal exposure of chick embryos to valproic acid increased the incidence of different gross deformities.
Keywords: Chick embryo, Gross deformities, Valproic acid.
Epilepsy is a common neurological disorder and has a prevalence of 5.25 per1000. About one third of epileptic patients are women of reproductive age1. Most of the epileptic women need to continue taking medication during pregnancy, since uncontrolled seizures may be harmful to the women as well as to the fetuses. However, medication may still be an issue as some drugs used to treat seizures may contribute to birth defects.
Valproic acid (VPA) is considered to be a drug of first choice for the treatment of generalized and focal epilepsies. It is one of the most frequently-prescribed antiepileptic drugs worldwide due to its broad-spectrum and good tolerability2,3. Both the usage and the therapeutic indications of VPA are increasing. Now in addition to epilepsy, it is also employed in the treatment of different pathologic conditions including schizophrenia, bipolar disorders, different forms of headache, as an anticancer drug, retinitis pigmentosa (RP), autoimmune encephalomyelitis and HIV (human immunodeficiency virus) infection4-10.
Its human teratogenic effects have been reported since 198011. In 1984, DiLiberti suggested the term 'Fetal Valproate Syndrome' (FVS) for major and minor anomalies caused by teratogenic effects of VPA. These include defects in neural tube (NTDs), heart, craniofacial features, urogenital structures and limbs12.
Table-1: Comparison of chicks: with gross abnormalities of experimental group-A and control group-B using Fisher exact test.
Number of chicks
Alive chicks###23###28###51###between A and B
Limb deformities###p= 0.006*
Behavioral changes###pless than 0.0001*
Table-2: Gross comparison of the newly hatched chicks of valproic acid exposed group- A and control group-B using student's t test.
###N###Mean SEM###N###Mean SEM
Length (cm)###23###8.68 0.044###28###8.77 0.038###0.12
Weight (g)###23###38.69 0.098###28###38.92 0.049###0.03*
Previous studies have shown that the VPA readily crosses the placental barrier to the fetus13. Different studies have shown that the risk of major congenital malformations is two to four times as high with the use of valproate as with the use of other antiepileptic drugs, absolute rates of congenital malformations among offspring exposed to valproate in utero have range from 6 to 11%14.
Many animal studies have been carried out, on the different individual organs in developing embryo, in order to mimic the effects of VPA on human embryo. No trials are available regarding the pattern of incidence of different anomalies in single study and it is still remains to be learned. With this background, this study was undertaken to determine the teratogenic effects of valproic acid in terms of different gross congenital anomalies in chick embryo.
MATERIAL AND METHODS
An experimental study was carried out at the Department of Anatomy, Islamabad, Regional Centre of College of Physicians and Surgeons Pakistan (CPSP) between February 2010 and February 2011. Freshly laid fertilized chicken eggs, belonging to "Rhode Island Red" breed of Gallus domesticus were collected from Poultry Research Institute (PRI), Punjab, Murree Road Rawalpindi. The eggs which were damaged, dirty and stored for more than 03 days were excluded from study. The eggs were randomly selected by lottery method. For this purpose the eggs were numbered starting from 1 to the total count of the eggs. Then numbers were put on the pieces of paper, placed in a container and thoroughly mixed. Sixty numbers were selected without looking. Randomly selected eggs were divided into two groups, labelled as experimental group-A and control group-B. Each group comprised of 30 eggs. To limit bacterial contamination, the eggs were swabbed with 70% alcohol rapidly and gently, and then given labels.
Then eggs were placed in the racks with their blunt ends above and pointed ends below, and left in this position for 5 to 15 minutes to let the blastoderm to rotate and come to lie above at the blunt end. This was done to protect it from getting damaged during injecting the drug at the lower end. A sterilized thumb pin was used to drill two holes in the egg shell to inject the drug in the yolk sac. First hole was just one finger breath above the lower pointed end for the injection and second one was at the top of the blunt end to allow escape of air during injecting drug otherwise the drug would not stay inside, it would come out. The eggs were injected with 0.4 mg VPA in 20ul normal saline in the yolk, following the dose adopted by Whitsel et al15. Eggs of control group were injected with the same volume of normal saline in the same way. Holes in the shells were then sealed with melted wax and the eggs were placed in the incubator (Beschickung-loading model 100-800).
The day when eggs were placed in the incubator was taken as day 0. The eggs were then incubated under standard conditions. The temperature was maintained at 38 0.5o C. The relative humidity was kept between 60-70%. The eggs were manually rotated 1/2 turn twice daily. Candling of eggs was done after every 3 or 4 days to keep track of progress of egg incubation. The chicks were allowed to hatch by themselves till day 22 of incubation, afterwards the remaining were manually taken out by breaking the shell.
The day of hatching and the number of alive and dead chicks was recorded. The newly hatched alive chicks were observed for abnormal posture, gait, behavior, gross abnormalities and their length and weight were taken. The data was analyzed by using Statistical Package for Social Sciences (SPSS) computer software program, version 10. Student's t test was applied to detect any significant difference in means SE of gross weight and length of chicks of both groups. Fisher exact test was used to detect any significant difference in total number of chicks with gross anomalies and the number of different types of gross anomalies in both groups. A p-value of [?]0.05 was considered statistically significant. The relative frequency of different malformation within the experimental group-A, was measured by taking their percentages
All the hatched chicks of the control group were normal. Chick embryos exposed to VPA in ovo showed: increased mortality, increased incidence of gross deformities and delayed hatching. There were number of congenital malformations observed in experimental group, which included limb abnormalities (i.e. inverted feet) and eye abnormality (i.e. closed palpebral fissure of both eyes) as indicated in table-1. One chick showed multiple deformities including gastroschisis, closed palpebral fissures and inverted foot (fig-1). This chick was hatched out by manual assistance out of 23 alive chicks. The probable need for assisted hatching appeared to be decreased mobility due to multiple deformities. In the control group, all the 28 alive chicks hatched by themselves.
Regarding the behavior, Out of 23 alive chicks of the experimental group, 10 chicks showed behavioral changes. Some of them were listless, slow and sluggish in behavior, could not stand erect, showed diminished mobility and did not follow the voices of other chicks when taken away from them (fig-2a ). The chicks of control group were active, tried to remain together in group and followed the voices of other chicks when taken away from them (fig-2b).
On gross examination many of the chicks of experimental group A were weak. When means of the weights of newly hatched chicks of two groups were compared, there was significant increase in low birth weights as indicated by p- value of 0.03. There was no statistically significant difference between the means of the lengths of chicks of two groups as indicated by p- value of 0.12 (table-2).
Fig-1 a chick of experimental group (A), which was hatched by manual assistance, showed multiple deformities; inverted foot (1), closed palpebral fissures (2) and gastroschisis (3).
There was significant increase in the number of congenital malformations in the experimental group as indicated by p-value of 0.0008 (table-1). The comparison of percentages of chicks with gross abnormalities, within the experimental group-A, and with the control group- B has been shown in fig-3
Embryonic development is regulated by the hierarchies of signaling and gene regulatory networks. During pregnancy, maternal exposure to exogenous agents induces disruption of such networks resulting in chemically induced birth defects.
VPA is a known teratogen. It has now become evident from systematic review of all cohort studies that use of VPA monotherapy in the first trimester resulted in significantly higher rates of major congenital malformations, as compared with no use of antiepileptic drugs or with use of other antiepileptic drugs16. This is in accordance with our study. In the present study, there were number of congenital malformations observed in the experimental group. Previous studies have shown that exposure to VPA during embryogenesis may result in the multiple birth defects. The most commonly reported anomalies in VPA exposed embryos, in both human and animals are musculoskeletal deformities. In human patients exposed to VPA in utero, 63% had musculoskeletal deformities17. This is in accordance with findings in our study. The most commonly observed deformities were limb deformities.
In the previous studies it has been shown that the mechanism underlying limb deformities was disruption of genes that regulate pattern formation of somities in embryo. Barnes et al have showed that the VPA induced somite teratogenesis by inhibiting the expression of Pax-1 genes18.
The limb abnormalities after VPA exposure may include pre- and postaxial polydactyly, overlapping digits, talipes (clubfoot), clinodactyly, arachnodactyly, hip dislocation, limb deficiencies, preaxial and postaxial polydactyly, reduction malformations of the arms and hands and radial ray defects19,20 . Contractures of the small joints of the hands are also common musculoskeletal manifestation in children with VPA exposure17.
Likewise eye abnormalities have also been observed previously, in chicks exposed to VPA. The underlying mechanism was found to be disruption of genes; Pax-2 and Pax-6 genes15. Abnormal ophthalmic findings are common in children with confirmed FVS syndrome, which include myopia, strabismus, astigmatism, anisometropia, epicanthus, color vision deficiency and bilateral congenital cataract21.
The chicks of experimental group showed behavioral changes and delayed hatching. The result of our study corresponds with the previous observations made by Moore et al who noticed that the anticonvulsants, especially VPA taken during pregnancy were associated with an increased risk of developmental delay and different behavioral problems in the children. The reported behavioral problems included autistic features or autism, Asperger's syndrome, learning difficulties, speech delay, gross and fine motor delay22. Different studies identified valproate as a drug carrying potential risks for developmental delay, growth restrictions and cognitive impairment23.
The experimental work on the pregnant rats has shown that disruption of the early embryonic serotonergic neuronal development might be involved in the etiology of these behavioral changes24. Another possible cause might be altered folate metabolism because it has been implicated in the metabolism of neurotransmitter molecules25. In 2015, Baker et al reported that in utero exposure to valproate as compared with other antiepileptic agents was associated with a lower IQ in children26.
The chicks of experimental group (A) were weak as compared to control group (B). This was in accordance with the previous human study in which 10% of babies are small for gestational age after VPA exposure during pregnancy27. Another study showed that exposure of Sprague-Dawley rats to VPA during pregnancy resulted in intra uterine growth retardation of pups28.
Prenatal exposure of chick embryos to VPA increased the incidence of different gross deformities. The most commonly observed deformity was limb deformity.
CONFLICT OF INTEREST
This study has no conflict of interest to declare by any author.
1. Coban D, Kurtoglu S, Akin MA, Akcakus M, Gunes T. Neonatal episodic hypoglycemia: a finding of valproic Acid withdrawal. J Clin Res Pediatr Endocrinol, 2010; 2: 92-4.
2. Loscher w. Basic pharmacology of valproate: a review after 35 years of clinical use for the treatment of epilepsy. CNS Drugs 2002; 16: 669-94.
3. Gerstner T, Bell N, Konig S. Oral valproic acid for epilepsy-long- term experience in therapy and side effects. Expert Opin Pharmacother. 2008; 9: 285-92.
4. Horowitz E, Bergman LC, Ashkenazy C, Moscona-Hurvitz I, Grinvald-Fogel H, Magnezi R. Off-label use of sodium valproate for schizophrenia. PLoS One. 2014; 9: e92573.
5. Cipriani A1, Reid K, Young AH, Macritchie K, Geddes J. Valproic acid, valproate and divalproex in the maintenance treatment of bipolar disorder. Cochrane Database Syst Rev. 2013; 10: CD003196.
6. Kalita J, Bhoi SK, Misra UK. Amitriptyline vs divalproate in migraine prophylaxis: a randomized controlled trial. Acta Neurol Scand. 2013; 128: 65-72.
7. Gotfryd K, Skladchikova G, Lepekhin EA, Berezin V, Bock E, Walmod PS. Cell type-specific anti-cancer properties of valproic acid: independent effects on HDAC activity and Erk1/2 phosphorylation. BMC Cancer. 2010; 10: 383.
8. Clemson CM, Tzekov R, Krebs M, Checchi JM, Bigelow C, Kaushal S. Therapeutic potential of valproic acid for retinitis pigmentosa. Br J Ophthalmol. 2011; 95: 89-93.
9. Lv J, Du C, Wei W, Wu Z, Zhao G, Li Z et al. The antiepileptic drug valproic acid restores T cell homeostasis and ameliorates pathogenesis of experimental autoimmune encephalomyelitis. J Biol Chem. 2012; 287 (34): 28656-28665.
10. Lehrman G, Hogue IB, Palmer S, Jennings C, Spina CA, Wiegand A, et al. Depletion of latent HIV-1 infection in vivo: a proof-of-concept study. Lancet. 2005; 366: 549-55.
11. Brown NA, Kao J, Fabro S. Teratogenic potential of valproic acid. Lancet 1980; 1: 660-1.
12. DiLiberti JH, Farndon PA, Dennis NR, Curry CJ. The fetal valproate syndrome. Am J Med Genet 1984; 19: 473-81.
13. Brigs GG, Freeman RK, Yaffe SJ. Drugs in Pregnancy and Lactation: A Reference Guide to Fetal and Neonatal Risk. 8th Edition. Philadelphia: Lippincott Williams and Wilkins 2008.
14. Wyszynski DF, Nambisan M, Surve T, Alsdorf RM, Smith CR, Holmes LB. Increased rate of major malformations in offspring exposed to valproate during pregnancy. Neurology. 2005; 64: 961-5.
15. Whitsel AI, Johnson CB, Forehand CJ. An in Ovo Chicken Model to Study the Teratogenic Effects of Valproic acid. Teratology 2002; 66: 153-63.
16. Veiby G, Daltveit AK, Engelsen BA, Gilhus NE. Fetal growth restriction and birth defects with newer and older antiepileptic drugs during pregnancy. J Neurol. 2014; 261(3): 579-88.
17. 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.
18. Barnes GL Jr, Mariani BD, Tuan RS. Valproic acid-induced somite teratogenesis in the chick embryo: relationship with Pax- 1 gene expression. Teratology 1996; 54: 93-102.
19. Rodriguez- Pinilla E, Arroyo I, Fondevilla J, Garcia MJ, Martinez-Frias ML. Prenatal exposure to valproic acid during pregnancy and limb deficiencies: a case-control study. Am J Med Genet, 2000; 90: 376-81.
20. Seidahmed MZ, Miqdad AM, Al-Dohami HS, Shareefi O.A case of fetal valproate syndrome with new features expanding the phenotype. Saudi Med J. 2009; 30: 288-91.
21. Glover SJ, Quinn AG, Barter P, Hart J, Moore SJ, Dean JC et al. Ophthalmic findings in fetal anticonvulsant syndrome(s). Ophthalmology, 2002; 109: 942-7.
22. Moore SJ, Turnpenny P, Quinn A, Glover S, Lloyd DJ, Montgomery T et al. A clinical study of 57 children with fetal anticonvulsant syndromes. J Med Genet, 2000; 37: 489-97.
23. Adab N, Kini U, Vinten J, Ayres J, Baker G, Clayton-Smith J et al. The longer term outcome of children born to mothers with epilepsy. J Neurol Neurosurg Psychiatry, 2004; 75: 1575-83.
24. Miyazaki K, Narita N, Narita M. Maternal administration of thalidomide or valproic acid causes abnormal serotonergic neurons in the offspring: implication for pathogenesis of autism. Int J DevNeurosci. 2005; 23: 287-97.
25. Adams M, Lucock M, Stuart J, Fardell S, Baker K, Ng X. Preliminary evidence for involvement of the folate gene polymorphism 19bp deletion-DHFR in occurrence of autism. NeurosciLett, 2007; 422: 24-9.
26. Baker GA, Bromley RL, Briggs M, Cheyne CP, Cohen MJ, Garcia-Finana M et al. IQ at 6 years after in utero exposure to antiepileptic drugs: a controlled cohort study. J Neurol. 2015 Jan 27; 84(4): 382-90.
27. Clayton-Smith J, Donnai D. Fetal valproate syndrome. J Med Genet. 1995; 32: 724-7.
28. Binkerd PE, Rowland JM, Nau H, Hendrickx AG. Evaluation of valproic acid (VPA) developmental toxicity and pharmacokinetics in Sprague- Dawley rats. Fundam Appl Toxicol, 1988; 11: 485-93.
|Printer friendly Cite/link Email Feedback|
|Publication:||Pakistan Armed Forces Medical Journal|
|Date:||Sep 30, 2016|
|Previous Article:||KNOWLEDGE ABOUT OSTEOPOROSIS IN WOMEN OF CHILD BEARING AGE (15-49 YEARS) ATTENDING FAUJI FOUNDATION HOSPITAL RAWALPINDI.|
|Next Article:||EXTERIORIZATION OR IN-SITU REPAIR, COMPARISON OF OPTIONS FOR UTERINE REPAIR AT CESAREAN DELIVERY.|