Dose-Dependent Teratogenic Effects of Retinoic Acid on Cardiac Morphogenesis: A Folic Acid Rescue

Video Transcript

Every year, millions of children are born with congenital heart defects, yet in many cases, the exact cause remains unknown.

Hi, my name is Farheen, and my project explores how retinoic acid, a vitamin A derivative, disrupts heart development—and whether folic acid can help prevent that damage.

Using a chicken embryo model, I found that retinoic acid severely impaired cardiogenesis, preventing normal heart looping and chamber formation. This resulted in elevated heart rates, a sign of physiological stress.

However, when folic acid was introduced, embryos showed partial structural recovery and improved cardiac efficiency, despite reduced survival rates.

These findings suggest that folic acid may play a protective role during early heart development, with potential applications in preventing congenital defects.

Ultimately, this research contributes to a deeper understanding of how simple nutritional compounds could influence complex developmental outcomes and potentially save lives.

Background Research

Congenital heart defects (CHDs) are structural abnormalities present at birth, affecting cardiac function and circulation, with many causes still unknown (Cardiovasc Pathol., 2010).

Chicken embryos are widely used in research due to their accessibility, rapid development, and ability to model cardiac growth in a controlled environment.

Retinoic acid (RA), a metabolite of vitamin A, regulates gene expression during heart development, including cardiac patterning and cell differentiation (Development, 2012). However, excess RA disrupts normal development, causing defects such as ventricular septal defects (VSD) and double outlet right ventricle (DORV).

Certain nutrients may reduce these effects. Folic acid (vitamin B9) supports normal cardiac development, while vitamin C and taurine may help reduce oxidative stress and cellular damage.

Project Objectives

Developing a novel treatment strategy to pivot towards using highly-accessible pharmaceuticals. Traditional methods focus heavily on pre-marital screening and genetic counseling, often considered financially burdensome.

Research Question

Does folic acid preserve normal heart morphology and rhythmic function during early-stage chick embryo development under retinoic acid-induced stress conditions?

Hypotheses

H0: Folic acid is predicted to counteract abnormal levels of apoptosis in neural crest cells, preventing the disruption of cardiac morphogenesis, ultimately mitigating heart defects from excessive RA signaling.

H1: Folic Acid may not be able to repair some aspects of the heart defects caused by retinoic acid, as this agent targets other forms of stressors, such as ethanol and caffeine.

Impact

This could protect millions by reducing CHD mortality rates and save billions of dollars lost to traditional methods (Pickles & Keller, 2024).

Stage 1: Incubated Egg Development Analysis

Eight fertilized chicken eggs were incubated at ~99°F and 60% humidity to initiate development. After incubation, selected eggs were cracked into sterile Petri dishes for direct observation of embryonic structures. Key indicators, including blastoderm formation, vascularization, and early morphology, were documented to assess developmental progression.

Stage 2: Chick Embryo Treatment

Remaining eggs were labeled and randomly assigned to four groups: (1) untouched control, (2) vehicle control (20 µL DMSO), (3) TTNPB-treated (20 µL of 10 mM TTNPB in DMSO), and (4) TTNPB + folate. Approximately three eggs per group were used.

On day 0, eggs were incubated under standard conditions. On day 2, embryos in treatment groups were candled to confirm viability, windowed, and injected with assigned solutions. Eggs were resealed and returned to the incubator. Observations, including leakage or bleeding, were recorded. Control eggs remained unmanipulated. On day 3, all eggs were checked for contamination or damage.

Stage 3: Morphology and Analysis

On day 4, embryos were harvested and assessed. Survival and developmental stage were recorded. Trypan blue staining enhanced visualization, and imaging was standardized. Primary endpoints included survival, cardiac looping, and rhythmic functions. Secondary endpoints included edema, hemorrhage, and overall cardiac morphology. Scoring followed consistent criteria (e.g., normal/abnormal, severity levels).

Stage 4: Safety and Waste Disposal

All procedures followed standard lab safety protocols. Gloves were worn when handling TTNPB and DMSO. Workspaces were disinfected, and all biological and chemical waste was disposed of properly. For proper ethics, embryos are observed and properly disposed of after 7 days of incubation (6-9 Days before nociception).

Rationale

Treatment at day 2 (HH12–14) was selected to avoid disrupting early embryonic patterning while targeting cardiac development. Analysis at day 4 (HH22–24) allowed a clear assessment of heart morphology, improving the reliability of results.

1) Retinoic Acid (RA) Acts as a Potent Teratogen to Cardiogenesis

The injected RA concentrations arrested chick heart development at the primitive tubular stage, effectively blocking the critical process of cardiac looping and subsequent chamber formation.

This structural failure resulted in a physiologically compromised system; the RA group exhibited a high heart rate of 47.7 BPM, which can be interpreted as compensatory tachycardia. Additionally, cardiac efficiency remained low due to lack of chamber formation.

Because the heart remained a simple, inefficient tube, the embryo likely increased its heart rate to move blood through the body, though it still failed to reach the "overly healthy" baseline of the DMSO control (52 BPM), possibly due to temperature gradients.

2) The Introduction of Folic Acid as a Rescue Agent Successfully Countered the RA signal

Folic acid allowed for the retention of key cardiac structures and evident chamber formation. While these rescue embryos experienced a significant developmental delay and a lower survival rate (1/5) in the Day 10 eggs, their physiological profile was superior; the lower heart rate of 34 BPM suggests a transition toward stroke-volume efficiency.

By forming chambers, the heart became a more powerful, functional pump that could move a higher volume of blood with fewer beats, reducing the metabolic stress seen in the RA-only group.

Comparative analysis (Group 4) indicates a shift toward normal physiological function.

3) Statistical Analysis Confirms Significant Differences Between Groups

One-way ANOVA revealed statistically significant differences in cardiac outcomes across treatment groups (p < 0.05) (Graph 1).

Post-hoc comparisons (Welch’s T-test) showed significant differences between RA and control, as well as RA and RA + folate groups (p < 0.05), confirming that both RA exposure and folate intervention produce measurable effects.

These results validate that observed differences are not due to random variation.

4) New Discovery: DMSO Could Potentially Advance Embryonic Development

Although not the primary variable, this suggests DMSO may influence embryonic physiology. Statistical comparisons showed variation relative to other groups, though interpretation remains cautious due to possible environmental factors.

Unexpected results could be due to...

DMSO known as a highly polar aprotic solvent known to cross biological membranes easily, often acting as a carrier that increases the permeability of cells to other substances.

DMSO can act as a free radical scavenger. This antioxidant role might help protect the delicate cells of the early embryo from endogenous stress.

5) Morphological Outcomes Correlate with Functional Data

RA-treated embryos displayed abnormal looping and poorly defined structures, while folate-treated embryos showed partial morphological recovery (Group 3).

Secondary observations, including edema and developmental delay, were more prominent in RA groups.

These structural differences aligned with heart rate data (Graph 1), demonstrating a direct relationship between morphology and cardiac function.

6) Folic Acid Demonstrates Cardioprotective Potential

Overall, results indicate that folic acid mitigates RA-induced cardiac defects by improving both structural development and physiological efficiency.

Although not fully restorative, statistical significance (p < 0.05) and graphical trends (Graph 1) support its role as a potential cardioprotective agent in developmental models.

Conclusions

1) Retinoic acid significantly disrupts normal cardiac development in embryos, particularly by impairing the process of cardiac looping.

2) When folic acid was added alongside retinoic acid, a small reduction in chamber structure impairment was observed, suggesting that folic acid may provide limited protective support during heart development.

3) The DMSO vehicle control developed normally, confirming that the solvent did not negatively affect embryonic development and only emphasized retinoic acid's impacts.

Errors & Challenges

1) Temperature Gradient

Cold temperatures slow chick embryo heart rates primarily by acting on pacemaker cells, reducing their intrinsic firing rate due to lowered metabolic demand (hypometabolism) and decreased action-potential kinetics. As a result of the lab environment, these eggs were situated once taken out of the incubator, which potentially impacted the rhythmic analysis, explaining the abnormally low heart rates for the chicken embryos

2) Fertilization Rate of Eggs

As Specific Pathogen Free Eggs were purchased from the Canadian Food Industry, the majority of the eggs either developed significantly or did not develop at all, highly resembling grocery-bought eggs. This posed a major hardship for sampling sizes, as it introduced uncertainty about obtaining adequate results to ultimately support our findings, while also demonstrating the effects of retinoic acid and folic acid.

Future Application & Impact

These findings can lead to exploring pharmaceuticals available from mainstream pharmacies and grocery stores to treat these significant cardiovascular diseases. This can increase awareness of transitioning towards simpler chemicals to treat forecasted Congenital Heart Defects (CHDs) instead of expensive and time-consuming alternatives.

Immediate Next Steps

Ensuring Higher Fertility Rate of Eggs

The majority of the eggs purchased either developed significantly or did not develop at all, posing a major hardship for sampling sizes and correlating results.

Alternative Quantitative Variable

This can include a severity scale for pericardial edema or the number of embryos with impaired chamber formation.

Calculating Chick Embryo Internal Heart Rate

Mathematical calculations can allow more accurate rhythmic functions and heart rate without temperature gradient influence.

Alternative Applications

Due to promising results, further experiments could explore modifying genes through CRISPR-Cas9 techniques or using alternative animal models to evaluate cardioprotective mechanisms.

Acknowledgements

Dr. Alexandru Moise

Despite knowing him personally, Dr. Moise served as the foundational backbone for the contributions to this project. From supplying fertilized Specific-Pathogen-Free (SPF) eggs for research to providing valuable insights into chick cardiogenesis, he strengthened my passion for biomedical sciences. All success in this project was sparked by his creativity, wit, and friendly persona.

Mathiew Dykstra

My science teacher, Mr. Dykstra, also aided with various tasks, such as managing chicken embryos, providing useful advice on presentation, and being supportive throughout the many challenges we faced along the way.

Pedro Orcon Romero

While being a talented photographer and a great friend, Pedro played an important behind-the-scenes role, capturing the highest quality of my experimental process. He is renowned as one of the official photographers for Lo Ellen Park Secondary School, which ensured his proficiency, skill, and ability to capture images that come to life. (Instagram Commisions: @photography.pedro)

Works Cited

[1] Bartman, T., & Hove, J. (2005). Mechanics and function in heart morphogenesis. Developmental Dynamics, 233(2), 373–381. https://doi.org/10.1002/dvdy.20367

[2] Cleveland Clinic. (2021, August 13). Congenital heart disease (CHD): Causes, symptoms and treatment. https://my.clevelandclinic.org/health/diseases/21674-congenital-heart-disease

[3] Ford, S. M., et al. (2021). Folic acid prevents functional and structural heart defects induced by prenatal ethanol exposure. AJP Heart and Circulatory Physiology, 320(4), H1313–H1320. https://doi.org/10.1152/ajpheart.00817.2020

[4] Lai, W.-T., et al. (2022). Right ventricular pump efficiency in secundum-type atrial septal defect. Acta Cardiologica Sinica, 38(1), 47. https://doi.org/10.6515/ACS.202201_38(1).20210721A

[5] Linask, K. K. (2003). Regulation of heart morphology: Current molecular and cellular perspectives on the coordinated emergence of cardiac form and function. Birth Defects Research Part C: Embryo Today: Reviews, 69(1), 14–24. https://doi.org/10.1002/bdrc.10004

[6] Manasek, F. J. (1968). Embryonic development of the heart. I. A light and electron microscopic study of myocardial development in the early chick embryo. Journal of Morphology, 125(3), 329–365. https://doi.org/10.1002/jmor.1051250306

[7] Oullin, J. (2005). The development of cardiac rhythm. Heart, 91(7), 874–875. https://doi.org/10.1136/hrt.2004.047415

[8] Rhinn, M., & Dolle, P. (2012). Retinoic acid signalling during development. Development, 139(5), 843–858. https://doi.org/10.1242/dev.065938

[9] Samarut, E., & Rochette-Egly, C. (2012). Nuclear retinoic acid receptors: Conductors of the retinoic acid symphony during development. Molecular and Cellular Endocrinology, 348(2), 348–360. https://doi.org/10.1016/j.mce.2011.03.025

[10] Wang, G., et al. (2022). Epigenetics in congenital heart disease. Journal of the American Heart Association, 11(7). https://doi.org/10.1161/JAHA.121.025163

[11] Xu, J., et al. (2025). Global, regional, and national epidemiology of congenital heart disease in children from 1990 to 2021. Frontiers in Cardiovascular Medicine, 12. https://doi.org/10.3389/fcvm.2025.1522644

[12] Zimmerman, M. S., et al. (2020). Global, regional, and national burden of congenital heart disease, 1990–2017: A systematic analysis for the Global Burden of Disease Study 2017. The Lancet Child & Adolescent Health, 4(3), 185–200. https://doi.org/10.1016/S2352-4642(19)30402-X

Book References

Brand, T. (2003). Heart development: Molecular insights into cardiac specification and early morphogenesis. Developmental Biology, 258(1), 1–19

Buja, L. M., et al. (1993). Apoptosis and necrosis: Basic types and mechanisms of cell death. Archives of Pathology & Laboratory Medicine, 117(12), 1208–1214.

Feinstein, A., & Prevention Magazine Health Books. (1996). Prevention’s healing with vitamins: The most effective vitamin and mineral treatments for everyday health problems and serious disease—from allergies and arthritis to water retention and wrinkles. Rodale Press.

Lynch, S. R., & Cook, J. D. (1980). Interaction of vitamin C and iron. Annals of the New York Academy of Sciences, 355(1), 32–44.

Additional References

Abdullah, M., et al. (2023, May 1). Vitamin C (ascorbic acid). StatPearls Publishing; National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/books/NBK499877/

Asthma + Lung UK. (2022, November 30). Oxygen level tests. https://www.asthmaandlung.org.uk/symptoms-tests-treatments/tests/oxygen-level-tests

BC Children’s Hospital Research Institute. (2025). Using chicken embryos, BC researchers develop new approach for difficult-to-treat cancers. https://www.bcchr.ca/brave-initiative/using-chicken-embryos-bc-researchers-develop-new-approach-difficult-treat-cancers

Boullin, J. (2005). The development of cardiac rhythm. Heart, 91(7), 874–875. https://doi.org/10.1136/hrt.2004.047415

Brade, T., et al. (2013). Embryonic heart progenitors and cardiogenesis. Cold Spring Harbor Perspectives in Medicine, 3(10), a013847. https://doi.org/10.1101/cshperspect.a013847

Bruneau, B. G. (2013). Signaling and transcriptional networks in heart development and regeneration. Cold Spring Harbor Perspectives in Biology, 5(3), a008292. https://doi.org/10.1101/cshperspect.a008292

Chippewa Valley Technical College. (2025). Congenital heart defects. StatPearls Publishing; National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/books/NBK615354/

Cleveland Clinic. (2022). Collagen: What it is, types, function & benefits. https://my.clevelandclinic.org/health/articles/23089-collagen

Fang, X., et al. (2016). In utero caffeine exposure induces transgenerational effects on the adult heart. Scientific Reports, 6, 34106. https://doi.org/10.1038/srep34106

Hawkins, J. A., et al. (1984). Effect of caffeine on cardiovascular function in the stage 24 chick embryo. Developmental Pharmacology and Therapeutics, 7(5), 334–343. https://doi.org/10.1159/000457182

Heart and Stroke Foundation of Canada. (2024). Valvular heart disease. https://www.heartandstroke.ca/heart-disease/conditions/valvular-heart-disease

Jelínek, R., & Kistler, A. (1981). Effect of retinoic acid upon the chick embryonic morphogenetic systems: I. The embryotoxicity dose range. Teratology, 23(2), 191–195. https://doi.org/10.1002/tera.1420230204

Johns Hopkins Medicine. (2022). Heart valve diseases. https://www.hopkinsmedicine.org/health/conditions-and-diseases/heart-valve-diseases

Kalra, A., et al. (2024). Bicuspid aortic valve disease: Classifications, treatments, and emerging transcatheter paradigms. Structural Heart, 8(1), 100227. https://doi.org/10.1016/j.shj.2023.100227

Kamino, K. (1991). Optical approaches to ontogeny of electrical activity and related functional organization during early heart development. Physiological Reviews, 71(1), 53–91. https://doi.org/10.1152/physrev.1991.71.1.53

Kaplan-Arabaci, O., et al. (2025). The chicken embryo: An alternative animal model in development, disease and pharmacological treatment. Pharmacology Research & Perspectives, 13(2). https://doi.org/10.1002/prp2.70086

Kelly, R. G., et al. (2014). Heart fields and cardiac morphogenesis. Cold Spring Harbor Perspectives in Medicine, 4(10), a015750. https://doi.org/10.1101/cshperspect.a015750

Klimek-Piotrowska, W., et al. (2016). Geometry of Koch’s triangle. Europace. https://doi.org/10.1093/europace/euw022

Mayo Clinic. (2022, October 21). Ventricular septal defect (VSD): Symptoms and causes. https://www.mayoclinic.org/diseases-conditions/ventricular-septal-defect/symptoms-causes/syc-20353495

MedlinePlus. (2023, January 19). Vitamin C: MedlinePlus medical encyclopedia. https://medlineplus.gov/ency/article/002404.htm

MedlinePlus. (2023, October 28). Subcutaneous (SQ) injections: MedlinePlus medical encyclopedia. https://medlineplus.gov/ency/patientinstructions/000430.htm

Nakano, H., et al. (2021). The role of glucose in physiological and pathological heart formation. Developmental Biology, 475, 222–233. https://doi.org/10.1016/j.ydbio.2021.01.020

National Heart, Lung, and Blood Institute. (2022, March 24). Congenital heart defects: What are congenital heart defects? https://www.nhlbi.nih.gov/health/congenital-heart-defects

National Institutes of Health. (2021, March 22). Vitamin C. https://ods.od.nih.gov/factsheets/VitaminC-Consumer/

Peterson, J. K., et al. (2024). Trisomy 21 and congenital heart disease: Impact on health and functional outcomes from birth through adolescence. Journal of the American Heart Association, 13(19), e036214. https://doi.org/10.1161/JAHA.124.036214

Ripps, H., & Shen, W. (2012). Taurine: A “very essential” amino acid. Molecular Vision, 18, 2673.

Santulli, G., et al. (2023). Functional role of taurine in aging and cardiovascular health. Nutrients, 15(19), 4236. https://doi.org/10.3390/nu15194236

ScienceDirect. (n.d.). Renal clearance. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/renal-clearance

Smolich, J. J. (1995). Ultrastructural and functional features of the developing mammalian heart. Reproduction, Fertility and Development, 7(3), 451. https://doi.org/10.1071/rd9950451

Starship Children's Health. (2026, January 22). Pulse oximetry screening in the newborn. https://www.starship.org.nz/guidelines/pulse-oximetry-screening-in-the-newborn/

Wang, X. L., et al. (2019). Differential proteomic analysis revealed crucial egg white proteins for hatchability of chickens. Poultry Science, 98(12), 7076–7089. https://doi.org/10.3382/ps/pez459

Wiesinger, A., et al. (2021). Retinoic acid signaling in heart development. Stem Cell Reports, 16(11), 2589–2606. https://doi.org/10.1016/j.stemcr.2021.09.010

Willems, E., et al. (2014). Importance of albumen during embryonic development in avian species. World’s Poultry Science Journal, 70(3), 503–518. https://doi.org/10.1017/S0043933914000567