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Diagnostics for Preterm Birth

Clinicians have no effective diagnostic tools to predict preterm birth (PTB), and women are often asymptomatic until contractions begin. With current therapeutics, clinicians can delay labour by only days. Our goal is to develop a simple, inexpensive diagnostic that will allow clinicians to test for and predict preterm birth early enough so that therapeutics can work (see Therapeutics).

 

Before labour symptoms, parturition is characterized by a sterile inflammatory process. Our team is developing diagnostics for preterm labour by harnessing two key physiological steps within this process. First, as labour approaches, the human fetal membranes produce and release a chemotactic factor that attracts a massive influx of inflammatory cells to the intrauterine tissues. Second, the mother’s peripheral leukocytes are activated to mobilize and infiltrate the uterus. 

 

Leukocyte Migration Assay:

This assay measures the stage of maternal peripheral leukocyte activation by assessing their response to chemoattractants. Using a Boyden chamber (Figure 1), a 3μm membrane filter separates the upper from the lower compartments. Chemoattractant is placed in the bottom, whereas the maternal peripheral leukocytes are placed in the upper chamber. After an incubation time, the leukocytes that crossed the membrane into the bottom compartment are counted. The number of leukocytes that respond to the chemoattractant directly correlates to how close the mother is to labour. The closer a woman is to labour, the more her leukocytes will react to the chemoattractant. Our LMA is patented in the US and Canada and has a predictive capacity of over 91% within seven days of delivery.

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Figure 1: Flow Diagram of the Leukocyte Migration Assay (Created using BioRender)

The source of chemoattractant we now use for the LMA is tissue homogenate from fetal membranes isolated from the placentas of mothers who have given birth. We need to develop a standardized synthetic chemoattractant cocktail for this assay to become a mass-produced diagnostic for clinical labs. We are now determining the composition of chemoattractants from women in term vs. preterm labour. For a summary of our plans, check Commercialization.

 

Leukocyte Activation Markers:

Our lab is characterizing the four steps necessary for maternal peripheral leukocyte activation before labour. These are changes in leukocyte chemokine receptor mRNA expression, leukocyte cytokine mRNA expression, F-actin protein polarization in cells, and changes in intracellular F-actin pathway mRNA expression. If successful, detecting and quantifying one or a combination of these four stages may be a faster and less expensive way of diagnosing women at risk for preterm labour. 

For example, activated leukocytes change their shape as they prepare to cross the endothelium of the blood and lymphatic vessels to reach the uterus. This process is called diapedesis, or transendothelial migration. After activation, leukocytes roll, adhere to the vessel wall, and squeeze between the junctions of endothelial cells of the vessel to enter the interstitial space. We can visualize the morphological changes by staining for F-actin (Figure 2). We can also use qRT-PCR to quantify changes in the mRNA expression of genes that control this process.

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Figure 2: F-actin polarization in neutrophils increases at term labour (TL) compared to preterm, not in labour (PNL), and term not in labour (TNL). *p < 0.05 (Rodezno et al. unpublished data)

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