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ABSTRACT#1 of 2 - Selected for an Oral Presentation Title: 'Leukocyte f-actin polarization for extravasation is upregulated in term and preterm labor' Introduction: Maternal leukocytes are activated by chemotactic ligands derived from the fetal membranes (hFMs) for capillary extravasation and migration into uterine tissues to release pro-inflammatory mediators to initiate term and preterm parturition. Activation of receptors CXCR1, CXCR2, CX3CR1, and CCR3 trigger integrin-dependent adhesion, rolling, and extravasation of migrating leukocytes, which undergo polarization of cytoskeletal component f-actin for mobilization through the vascular endothelium. We hypothesized that term laboring women (TL) have an increased population of polarized peripheral leukocytes compared to term non-laboring women (TNL) and women in their second trimester (CNL, control NIL) and that increased expression ofCXCR1, CXCR2, CX3CR1 and CCR3 in leukocytes from peripheral circulation and hFMs is associated with a decrease in length of gestation for TL and preterm laboring women (PTL). Furthermore, to better understand the hFM-mediated f-actin polarization pathway, we measured changes in gene abundance of ARP3 and VAV1, two proteins directly involved in cytoskeletal rearrangement and actin filament formation in peripheral leukocytes and leukocytes embedded in hFMs. Methods: Peripheral blood was drawn at CNL (n=9), PTL (n=7), TNL (n=21), and TL (n=17). Leukocytes were isolated. F-actin polarization was characterized by phallotoxin staining and fluorescence microscopy. Whole placentas were collected at PTL (n=6), TNL (n=11), and TL (n=15) <1h after delivery. hFMs were isolated using the simple-random collection method. qRT-PCR was used to measure mRNA abundance levels in leukocytes and hFMs. Gestational length was determined from medical records. Data were analyzed using a One-Way ANOVA, Tukey post hoc testing, and Pearson correlations (p<0.05). Results: TL women had a 63.7% increase in polarized leukocytes vs. TNL and CNL (p<0.03). qRT-PCR analysis revealed that CXCR1, CX3CR1, and CCR3 mRNA abundance was respectively 2.7-, 2.0-, and 5.1-fold higher in PTL leukocytes compared to TNL leukocytes (p<0.05). Compared to TNL and TL women, PTL hFMs had significantly elevated levels of CXCR1 (TNL:6.1x; TL:2.8x), CXCR2, (TNL: 7.4x; TL: 5.5x), CX3CR1 (TNL: 5.3x; TL:4.5x), Arp3 (TNL:2.9x; TL:4.5x), and VAV1 (TNL: 3.1x; TL:3.6x) (p<0.05). Increased expression of CCR3 in leukocytes but not in hFMs was associated with shorter gestational length (r= -0.69, p=0.006). Increased expression of CXCR1, CXCR2, CX3CR1, VAV1, and Arp3 in hFMs only was associated with shorter gestational length (r=-0.70, r=-0.52, r=-0.56,r= -0.69, r=-0.70, respectively; p<0.05). Conclusion: These data support that leukocytes are primed prior to migration. F-actin polarization is a quantifiable predictor of the onset of labor, and receptors and mediators associated with activation are upregulated at PTL and TL compared to TNL. ABSTRACT #2 of 2 - Selected for Poster Presentation Title: 'Leukocyte migration at term delivery is evolutionarily conserved' Introduction: Since leukocyte chemotaxis into the uterus is key to parturition initiation in all mammals, could its mechanics be shared by various species? Shortly before labor onset, circulating peripheral leukocytes are recruited by uterine chemotactic factors (CF) to infiltrate the uterus, placenta, fetal membranes, and cervix. We used our Leukocyte Migration Assay (LMA) to examine the migration of leukocytes from several species at term pregnancy to migrate in response to CF from other species. We hypothesized that the CFs from various species were chemically similar and that leukocytes from various species would respond similarly to them suggesting the CFs and leukocyte characteristics are conserved among mammals. Methods: Whole placentas were collected from term laboring sheep (n=4), cows (n=4), pigs (n=3) and humans(n=15) 9 different locations into1x1 cm^2 pieces. For cotyledonary placentas (cow, sheep), cotyledons were collected in the same manner. Lower uterus sections were harvested from term laboring CD-1 mice (n=10) on gestational day (GD) 18.5. CF was extracted by homogenizing the tissue sections in DPBS (100mg/mL) and standardizing them to a total protein content of 1mg/mL. Whole blood was drawn from the arm of term laboring women (n=15) via venipuncture and from pregnant CD-1 mice (n=5) on GD 18.5 via cardiac puncture; leukocytes were isolated using Hetasep. Leukocyte migration was assessed using the LMA, where human or mouse leukocytes (2 x 10^5/100uL) were plated in the top wells of a Boyden Chamber and separated from CFs present in the bottom wells by a membrane with 3um pores. Leukocytes that migrated through the membrane in response to the CF were quantified. A One-Way ANOVA and Tukey post hoc testing were used to determine significance (p<0.05). Results: All CFs tested elicited a significant migratory response in human peripheral leukocytes compared to the control. Human peripheral leukocytes migrated in response to cow, pig and sheep fetal membrane and cotyledon CF (p<0.05) as well as mouse lower uterus CF (p<0.01). Reversing the components, human fetal membrane (hFM) CF stimulated the migration of pregnant CD-1 mouse (GD18.5) leukocytes (p<0.01). hFM CF and mouse lower uterus CF elicited migration of pregnant mouse (GD18.5) leukocytes to a similar degree. Conclusion: These data demonstrate that leukocyte migration mechanisms at term delivery are evolutionarily conserved across several mammalian species. It indicates the CFs from these species have a similar molecular composition and that the cells have similar responsiveness to different species' CFs. Increased CF production is a universal mechanism involved in signaling the end of pregnancy and initiation of labor. The data, therefore, suggest that the fetus is involved in initiating its delivery.