Discussion Abstract

ResultsNull Hypothesis

Acknowledgements and References

Biomarker of Vasodilation in Decidual Tissue Associated

with Obesity in Non-Human Primates

The tissue in this experiment was from Japanese macaques that were fed either a control or a high fat diet (36% fat calories), two

Four slides from each animal were taken. The primary antibody was endothelial nitric oxide synthase antibody at a 1:200 dilution. After a night of incubation, the slides were rinsed and then a secondary antibody was applied. The secondary antibody was an anti-rabbit diluted at 1:1000. After an hour of incubation, A/B Solution was applied. Finally DAB solution was added and then the slides were counterstained with hematoxylin.

Tissue

Immunohistochemistry

Analysis

The placenta is a very important part of pregnancy. It acts as the barrier between mother and child that every substance necessary for life and development must pass through. (Fig. 1) Any change in this can cause many problems in the pregnancy.

The obesity epidemic is increasing the occurrence of complications in pregnancy that are related to a high fat diet (HFD). Previous studies have shown that a high fat diet in pregnancy is related to a decrease in the uteroplacental blood flow. (Fig 2) A large amount of blood flowing

through the placenta is necessary for proper development of the fetus and a decrease in this can cause complications such as stillbirth. Several enzymes control the dilation of the blood vessels in the placenta. One of these is endothelial nitric oxide synthase (eNOS). eNOS, a commonly used marker for vasodilation as it produces nitric oxide which has a vasodilatory affect on placental blood vessels. In this experiment, decidua from obese and control Japanese macaques was tested for this enzyme. In the end, eNOS, was found to be less abundant in obese tissue than control tissue.

Materials and Methods

Previous studies have shown that the blood flow in high fat diet pregnancies is decreased compared to control. Thus the alternate hypothesis is; the expression of eNOS will be decreased in the decidua of animals consuming a high fat diet.

Hypothesis

If the presence of eNOS is compared in decidua tissue from high fat diet and control Japanese macaques then there will be no significant difference in average number of positive stained cells.

from each treatment group. Placenta was collected by Caesarean Section at 130 days of gestation. 5µm sections of the decidua (Fig.3) of the placenta were taken using a microtome and mounted on slides. Four slides from each animal were used.

Four images were taken from each of the four animals (two HFD, two Control) from the eNOS slides at 100x magnification. The images were then opened using ImageJ and a grid was applied. At each intersection of lines, if positive staining the intersection was counted (Fig. 4). The researcher was kept blind from the treatment group. The means for each animal were then taken.

Fig.3: Labeled placental tissue stained with hematoxylin and eosin. 40x. Photo courtesy of ONPRC.

Fig. 2: Blood flow is reduced in pregnancy of animals fed a high fat diet.(Frias et. al)

After the positive stained intersections for each animal were counted, the treatment group was revealed to the researcher. Then the mean for each treatment group was taken. The mean number of positive stained intersections in the HFD slides was 12 intersections while the mean for the control slides was 21 intersections (Fig. 6&7).

At the end of the experiment the difference between the two groups was significant (p= 0.0082). Therefore the null hypothesis can be rejected in favor of the alternate hypothesis: that the expression of eNOS will be decreased in the decidua of animals consuming a high fat diet. This finding is in accordance with the findings of Frias et. al. In that experiment it was found that pregnancies complicated by a high fat diet have lower rate of blood flow than control diet pregnancies. In this experiment, tissue from HFD animals had less eNOS which subsequently would mean the blood vessels would be less dilated and therefore have less blood flow (Fig 7&8).There are several sources of possible experimental error that could have affected the results. These being pipetting errors, fluctuation in room temperature, and inaccurate measuring or timing. Finally during the analysis of the slides it was difficult to distinguish between positive staining and nuclei which could cause inaccurate counting.In the future I plan to perform immunohistochemistry on more decidual sections in order to expand my sample. I also intend to stain decidual tissue with Thromboxane A2 as it is known vasoconstrictor. Finally I plan on testing different areas of the placenta to see if other parts have similar patterns of eNOS staining. This will help gain a more complete understanding on how obesity affects pregnancy.

Treatment Mean number of positive stained intersections

HFD 12Control 21

Fig. 4&5: Decidua stained with eNOS. To count the cells on each slide, a program called ImageJ was used to apply a graph with 5000 pixels, then all the line intersections with positive staining were counted. 100x magnification. Photo by researcher.

In conclusion, these findings support the idea that obesity can affect pregnancy. With less eNOS in the tissue, less blood will reach the fetus causing complications. More research must be done to discover more affects of obesity on pregnancy and how to alleviate the consequences. Additionally with more knowledge being gathered, doctors will be able to better advise obese women on becoming pregnant so they will know the implications of their pregnancy.

Conclusion

I would like to thank Dr. Victoria Roberts and Jessica Walker from the ONPRC for providing the tissue and materials and for all their time helping me through the research process. Finally I would like to especially thank Mrs. Newman and Mr. Wright for being my mentors in this project. Frias, A.E., Morgan, T.K., Evans, A.E., Rasanen, J., Oh, K.Y., Thornburg, K.L., Grove, K.L.

2011 Maternal High-Fat Diet Disturbs Uteroplacental Hemodynamics and Increases the Frequency of Stillbirth in a Nonhuman Primate Model of Excess Nutrition. Endocrinology 152(6):2456-2464.

Kulandavelu, S., Whiteley, K.J., Bainbridge, S.A., Qu, D., Adamson, S.L. 2013 Endothelial NO Synthase Augments Fetoplacental Blood Flow, Placental Vascularization, and Fetal Growth in Mice. Hypertension 61:259-266.

Positive stained cells

Blood Vessel

Fig 6&7: The mean number of positive stained intersections in HFD tissue was 12 while the mean for contol was 21.

Fig.8&9: There was significant difference between the presence of eNOS in HFD (left) and Control tissue (right). Photo by researcher, 100x magnification.

Spiral artery Basal plateEndometrial vein

Decidua basalis

Placental septum

Chorionic villus main stem

Intervillous space

Umbilical arteries Umbilical vein

Umbilical cord

Chorionic plate

Amnion

Chorionic vessels

Fig. 1: Ragavendra & Tarantal, Placenta (2001)