Objective The angiotensin II type 1 receptor (AT1R) can be activated under conditions of mechanical stretch in some cellular systems. Whether this activity influences signaling within the abdominal aorta to promote to abdominal aortic aneurysm (AAA) development remains unknown. We evaluated the hypothesis that mechanical AT1R activation can occur under conditions of hypertension (HTN) and contribute to AAA formation. Methods BPH/2 mice, which demonstrate spontaneous neurogenic, low-renin HTN, and normotensive BPN/3 mice underwent AAA induction via the calcium chloride model, with or without an osmotic minipump delivering 30 mg/kg/d of the AT1R blocker Losartan. Systolic blood pressure (SBP) was measured at baseline and weekly via a tail cuff. The aortic diameter (AoD) was measured at baseline and terminal surgery at 21 days by digital microscopy. Aortic tissue was harvested for immunoblotting (phosphorylated extracellular signal-regulated kinase-1 and -2 [pERK1/2] to ERK1/2 ratio) and expressed as the fold-change from the BPN/3 control mice. Aortic vascular smooth muscle cells (VSMCs) underwent stretch with or without Losartan (1 μM) treatment to assess the mechanical stimulation of ERK1/2 activity. Statistical analysis of the blood pressure, AoD, and VSMC ERK1/2 activity was performed using analysis of variance. However, the data distribution was determined to be log-normal (Shapiro-Wilk test) for ERK1/2 activity. Therefore, it was logarithmically transformed before analysis of variance. Results At baseline, the SBP was elevated in the BPH/2 mice relative to the BPN/3 mice ( P < .05). Losartan treatment significantly reduced the SBP in both mouse strains ( P < .05). AAA induction did not affect the SBP. At 21 days after induction, the percentage of increase in the AoD from baseline was significantly greater in the BPH/2 mice than in the BPN/3 mice (101.28% ± 4.19% vs 75.59% ± 1.67% above baseline; P < .05). Losartan treatment significantly attenuated AAA growth in both BPH/2 and BPN/3 mice (33.88% ± 2.97% and 43.96% ± 3.05% above baseline, respectively; P < .05). ERK1/2 activity was increased approximately fivefold in the BPH/2 control mice relative to the BPN/3 control mice ( P < .05). In the BPH/2 and BPN/3 mice with AAA, ERK1/2 activity was significantly increased relative to the respective baseline control ( P < .05) and effectively reduced by concomitant Losartan therapy ( P < .05). Biaxial stretch of the VSMCs in the absence of angiotensin II demonstrated increased ERK1/2 activation ( P < .05 vs static control), which was significantly inhibited by Losartan. Conclusions In BPH/2 mice with spontaneous neurogenic, low-renin HTN, AAA growth was amplified compared with the normotensive control a...

Introduction Elevated interleukin-6 (IL-6) plasma levels have been associated with abdominal aortic aneurysm (AAA), but whether this cytokine plays a causative role in the degenerative remodeling or represents an effect from the inflammatory cascades initiated by infiltrating leukocytes remained unclear. This project aims to demonstrate that within the aortic wall, signaling from IL-6 through the STAT3 transcription factor is necessary for infiltration of proteolytically-active macrophages and development of small AAA. Methods Following measurement of baseline infrarenal aortic diameter (AoD, digital microscopy), C57Bl/6 and IL-6 knockout (IL-6KO) mice underwent AAA induction by application of peri-adventitial CaCl2 (0.5 M) +/− implantation of an osmotic mini-pump delivering IL-6 (4.36 µg/kg/day over 21 days). At the terminal procedure, AoDs were measured by digital microscopy and aortas harvested for immunoblot (pSTAT3/STAT3), matrix metalloproteinase (MMP) quantification, or flow cytometric analysis of macrophage content. Plasma was collected for cytokine analysis. Results IL-6 infusion significantly increased the plasma IL-6 levels in C57Bl/6 and IL-6KO animals. The C57Bl/6 + CaCl2 group developed AAA (AoD >50% above baseline) but IL-6KO + CaCl2 did not. In the IL-6KO + IL-6+CaCl2 group, AAA developed to match that of C57Bl/6 + CaCl2 mice. STAT3 activity was significantly increased in animals with advanced stages of dilation (>40% from baseline), compared to those with ectasia (≤25%). Although cytokine profiles did not support T-cells or neutrophils as being active contributors in this stage of aortic remodeling, changes in the profile of elaborated MMPs suggested macrophage activity with a trend toward alternatively activated pathways. Flow cytometry confirmed significantly increased macrophage abundance specifically in animals with upregulated STAT3 activity and advanced aortic dilation. Conclusion In this murine model of AAA, progressive dilation to development of true AAA was only accomplished when IL-6 signaling upregulated STAT3 activity to effect accumulation of proteolytically-active macrophages. This pathway warrants further investigation to identify potential therapeutic avenues to abrogate growth of small AAA.

Purpose The aim of this study was to determine if pregnancy-associated plasma protein-A (PAPP-A), typically measured in maternal serum and a potential predictor of adverse maternal and fetal outcomes such as spontaneous miscarriage, pre-eclampsia, and stillbirth, is expressed in blastocoel fluid–conditioned media (BFCM) at the embryonic blastocyst stage. Design This is an in vitro study. Methods BFCM samples from trophectoderm-tested euploid blastocysts (n = 80) from in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) patients were analyzed for PAPP-A mRNA. BFCM was obtained from blastocyst stage embryos in 20 uL drops. Blastocysts underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy prior to blastocyst vitrification and BFCM collection for snap freezing. cfDNA was synthesized using BFCM collected from 80 individual euploid blastocysts. Next, real-time qPCR was performed to detect expression of PAPP-A with GAPDH for normalization of expression in each sample. Results PAPP-A mRNA was detected in 45 of 80 BFCM samples (56.3%), with varying levels of expression across samples. Conclusion Our study demonstrates the expression of PAPP-A in BFCM. To our knowledge, this is the first study to report detection of PAPP-A mRNA in BFCM. Further studies are required and underway to investigate a greater number of BFCM samples as well as the possible correlation of PAPP-A expression with pregnancy outcomes of transferred euploid blastocysts. If found to predict IVF and obstetric outcomes, PAPP-A may provide additional information along with embryonic euploidy for the selection of the optimal blastocyst for embryo transfer.

Preimplantation embryo development refers to the maturation of a fertilized ovum to a blastocyst. This process is highly regulated and required for proper implantation of the blastocyst into the endometrium. During this phase, several tasks must be accomplished. The differentiated zygotic genome must undergo reprogramming back to totipotency in order to generate all of the different types of tissue making up a human. Next, certain cells begin to differentiate to prepare for implantation which occurs at approximately day 7 post-fertilization. This progression is a result of a careful interplay between maternally persistent RNA transcripts and activation of the zygotic genome. After the embryonic genome activation, blastomere differentiation begins to occur. Cellular polarity has been shown to be the signal transduction that initiates this differentiation. Understanding the molecular and cellular mechanisms regulating preimplantation embryo development is of fundamental importance for reproductive science and has numerous applications in fields such as assisted reproductive technology and stem cell therapy.

POV procedure. These samples consisted of 12 conditions with 6 samples each. The 12 conditions were constituted of three separate manufactured lots, each with two package sizes, and each of these was evaluated at the end of shelf life and also several weeks after opening. The standard samples represented one test per bottle. 9 separate samples were tested using an optimized preparation and testing procedure. The optimized procedure was employed to first address the turbidity reported in the oil-preparation reagent mixture. This included increased heat during mixing and adding a stand time after mixing in order to allow the turbidity to resolve. The method was also modified to test the oil after spiking it with one of the calibrators to bring the value to the middle of the calibration curve, where there tends to be lower error. The spiked sample was run alongside a spike control of the calibrator in an equal volume of preparation reagent. The difference between the spiked oil and spike control was calculated and multiplied by the dilutions. The optimized samples represented nine tests per one bottle. The experiments ran over three months from November 2020 to January 2021. The means and standard deviations were calculated for both the standard and optimized measurements.RESULTS: The testing of the first 72 samples revealed a high relative standard deviation (RSD) of 244%. This error was further compounded by an additional dilution intended to offset the impact of the turbidity on the measurement. For the 9 samples, the optimized testing achieved more precise results with an RSD of 3.2%.CONCLUSIONS: Commercially available methods of POV determination in white mineral oil can be optimized for precise quantitation of low peroxide with a RSD of less than 5%.IMPACT STATEMENT: This shows that low POV in embryo culture oil may be a challenge for commercial methods to precisely quantify but can be optimized to a particular matrix. A cost-effective option, commercial testing allows for increased access to this assay for future studies. Results should not be generalized to other mineral, paraffin, or silicone oils, which may differ in chemical or physical properties, such as viscosity, or other POV test methods which do not employ optical density measurements and calculations against a standard curve; Further testing is needed to assess whether the standard POV procedures are appropriate or if optimization is necessary for other matrices.