Synthetic Estrogen compounds, an ingredient typically found in contraceptives, are invading water systems around the world. The growing use of oral contraceptives, which are subsequently eliminated through human feces, is causing environmental levels of 17α-ethynyl estradiol to rise. 17α-Ethynyl-estradiol (EE2) was initially created to be resistant to metabolic processes in the human liver, which makes it less biodegradable than natural estrogen molecules. EE2 levels in water supplies are impacted by a variety of factors, from agricultural operations and sewage treatment to population and lifestyle. The present amount of EE2 in surface water in the United States is 0.50 ng/L, which is lower than other countries but will still have an effect on the majority of aquatic creatures, particularly during their most sensitive developmental phases. 

Effects on embryogenesis in current levels of EE2 in U.S water supplies have not yet been thoroughly evaluated. Some EE2 research has been done with chicken embryos, but not at environmental concentration. With just a low concentration, this drug will leave subtle and long-lasting effects in the developing embryos’ gene expression. These effects will be related to its growth and developmental abnormalities. EE2 is commonly known for its feminization of aquatic life and its impact on the estrogen receptor. Very little research has been done to test other factors that EE2 can cause during development, and that includes its immune response. At an embryonic stage, the organism starts to create the foundation for their body, and one of the earliest systems they develop, after their vascular system, is their immune response. Around day 10 is when the first sign of an immune response of a chicken embryo is observed and in just 24 hours is when T-cells and B-cells are present.

The study investigated if concentrations of the environmental estrogen EE2 found in US water systems affect chicken embryonic development. While the researchers hypothesized that EE2 would decrease the embryo's size and development, they found no statistical difference in the embryo's weight, length, or developmental stage. They suggest this might be due to the low EE2 concentration used. However, a significant difference was observed in the heterophil to lymphocyte ratio, suggesting that EE2 at these levels may affect blood cell development, which could potentially impact the immune system and organ development. The study's finding of a higher heterophil/lymphocyte ratio, which suggests immunosuppression, aligns with Mo et al. (2019), who found that EE2 exposure reduced lymphocytes in juvenile yellow catfish. However, Mo et al. (2019) also observed increased weight gain from EE2, which contradicts the current study's weight findings. The authors note that the weight difference in the prior study was greater when EE2 was combined with another chemical DEHP, suggesting EE2's impact on weight may depend on other factors. Further research is needed to fully understand these results. 

This study offers novel insights into the early immune and non-reproductive effects of environmentally relevant EE2 exposure on chicken embryogenesis, demonstrating a significant change in the heterophil/lymphocyte ratio, an immune stress marker. This finding suggests sub-lethal, non-reproductive impacts of EE2 that could affect bird populations in contaminated ecosystems. The authors claim to be the first to show this effect on early embryonic immune development. However, the interpretation is subject to limitations from the course-based research approach: the blood parameter analysis used a very small subset of the total sample, the overall sample size for gross morphology should be larger, and only one EE2 concentration was tested. Future research should investigate a dose-dependent relationship, increase sample sizes, and extend the harvest date past E10 to check for later developmental changes. This preliminary work underscores the need for continued investigation into how environmental endocrine disruptors affect early immune and developmental processes.