POST-PARTUM SUB-CLINICAL ENDOMETRITIS- ALTERATION IN UTERINE HAEMODYNAMICS, INFLAMMATORY MARKERS ON TREATMENT WITH MODIFIED ESTRUS SYNCHRONIZATION PROTOCOLS IN DAIRY COWS

Forty-one dairy cows (n=41) were enrolled to envisage the changes in uterine haemodynamics during sub-clinical endometritis (SCE) and its treatment with modified estrus synchronization protocols at 8 weeks post-partum. Trans-rectal Doppler sonography of both the middle uterine arteries (MUAs) was carried out for assessment of uterine perfusion whereas serum inflammatory markers i.e. IL-6 and C-RP were measured at 8 weeks post-partum and estrus (induced or spontaneous). Modified estrus synchronization protocols (MG6G and MG6GP) were used to adjudge their efficacy in post-partum dairy cows diagnosed with SCE and reproductive parameters were recorded. As a part of result, Doppler indices of both the MUAs at estrus i.e. TAMEAN, TAMAX, Blood flow volume-TAMEAN and TAMAX and diameter of MUA, were significantly lower (P<0.05) after application of MG6G and MG6GP protocols in SCEP as compared to SCEP control cows. Similarly, the IL-6, C-RP concentrations and PMNCs proportion (%) were significantly higher (P<0.05) in SCEP control as compared to cows treated with MG6G and MG6GP protocols. Moreover, on the day of estrus, the uterine haemodynamics, concentration of serum inflammatory markers and PMNCs proportion (%) in treated cows was at par with SCE negative control (SCEN) cows. In terms of reproductive performance, days open were recorded to be significantly lower (P<0.01) in treated and SCEN group as compared to SCEP control cows. In conclusion, sub-clinical endometritis led higher uterine perfusion, release of proinflammatory cytokines and PMNCs proportion which happened to plummet the post-partum reproductive performance was successfully managed with modified estrus synchronization protocols.

Uterine perfusion in patients with MMP2 gene polymorphisms

Aim. To investigate the effect of allelic polymorphisms of the MMP2 gene on uterine perfusion in patients planning pregnancy. Materials and methods. 95 women planning pregnancy were examined. The patients underwent clinical and laboratory examination, analysis of single nucleotide polymorphisms rs2285052 and rs243865 of the MMP2 gene, ultrasound examination on days 1825 of the menstrual cycle with an assessment of the pulsatility index of blood flow of the uterine vessels and a qualitative assessment of endometrial and subendometrial perfusion. Depending on the genotype, the patients were divided into 3 groups: first with haplotype rs2285052(A)rs243865(T), second with haplotype rs2285052(A)rs243865(С), and a third control group with rs2285052(С/С)rs243865(С/С) genotype. Results. Decreased perfusion in the subendometrial zone was found in 40.6, 44.4 and 19.4% of patients in the 1, 2 and 3 groups, respectively; decreased perfusion of endometrium in 68.8, 55.6 and 36.1% of patients in the 1, 2 and 3 groups, respectively. Spiral arteries were not visualized in 28.1, 14.8 and 11.1% of patients in the 1, 2 and 3 groups, respectively. No statistical differences were found in the pulsatility index of uterine blood flow depending on the genotype. Conclusion. In patients with the A rs2285052 and T rs243865 alleles of the MMP2 gene poor vascular patterns of the endometrium and subendometrial zone of the uterus were statistically significantly more frequent, which can lead to infertility. These associations are more pronounced for the rs2285052 polymorphism. The simultaneous determination of the rs2285052 and rs243865 polymorphisms does not provide additional information compared to the definition of rs2285052 alone.

Angiotensin II type 1 receptor agonistic autoantibody blockade improves postpartum hypertension and cardiac mitochondrial function in rat model of preeclampsia

Women with preeclampsia (PE) have a greater risk of developing hypertension, cardiovascular disease (CVD), and renal disease later in life. Angiotensin II type I receptor agonistic autoantibodies (AT1-AAs) are elevated in women with PE during pregnancy and up to 2-year postpartum (PP), and in the reduced uterine perfusion pressure (RUPP) rat model of PE. Blockade of AT1-AA with a specific 7 amino acid peptide binding sequence (‘n7AAc’) improves pathophysiology observed in RUPP rats; however, the long-term effects of AT1-AA inhibition in PP is unknown. Pregnant Sprague Dawley rats were divided into three groups: normal pregnant (NP) (n = 16), RUPP (n = 15), and RUPP + ‘n7AAc’ (n = 16). Gestational day 14, RUPP surgery was performed and ‘n7AAc’ (144 μg/day) administered via osmotic minipump. At 10-week PP, mean arterial pressure (MAP), renal glomerular filtration rate (GFR) and cardiac functions, and cardiac mitochondria function were assessed. MAP was elevated PP in RUPP vs. NP (126 ± 4 vs. 116 ± 3 mmHg, p < 0.05), but was normalized in in RUPP + ‘n7AAc’ (109 ± 3 mmHg) vs. RUPP (p < 0.05). PP heart size was reduced by RUPP + ’n7AAc’ vs. RUPP rats (p < 0.05). Complex IV protein abundance and enzymatic activity, along with glutamate/malate-driven respiration (complexes I, III, and IV), were reduced in the heart of RUPP vs. NP rats which was prevented with ‘n7AAc’. AT1-AA inhibition during pregnancy not only improves blood pressure and pathophysiology of PE in rats during pregnancy, but also long-term changes in blood pressure, cardiac hypertrophy, and cardiac mitochondrial function PP.

Elastin-Like Polypeptide: VEGF-B Fusion Protein for Treatment of Preeclampsia

Preeclampsia is characterized by the development of elevated blood pressure during the second and third trimesters of pregnancy that is accompanied by end organ dysfunction. The pathogenesis of preeclampsia is multifactorial but is commonly characterized by endothelial dysfunction and the overproduction of antiangiogenic factors, including the soluble VEGF (vascular endothelial growth factor) receptor sFlt-1 (soluble Fms-like tyrosine kinase receptor 1). Previously, administration of exogenous VEGF-A, bound to a carrier protein called ELP (elastin-like polypeptide), significantly reduced free sFlt-1 levels and attenuated the hypertensive response in a rodent model of preeclampsia. However, VEGF-A administration induces multifactorial effects mediated through its direct activation of the Flk-1 receptor. In response to this, we developed a therapeutic chimera using ELP bound to VEGF-B, a VEGF isoform that binds to sFlt-1 but not to Flk-1. The purpose of this study was to evaluate the in vitro activity and pharmacological properties of ELP-VEGF-B and to test its efficacy in the reduced uterine perfusion pressure rat model of placental ischemia. ELP-VEGF-B was less potent than ELP-VEGF-A in stimulation of endothelial cell proliferation and matrix invasion, indicating that it is a weaker angiogenic driver. However, after repeated subcutaneous administration in pregnant rats, ELP-VEGF-B was maternally sequestered and reduced blood pressure when compared with saline treated animals following induction of placental ischemia (123.38±11.4 versus 139.98±10.56 mm Hg, P =0.0129). Blood pressure reduction was associated with a restoration of the angiogenic capacity of plasma from rats treated with ELP-VEGF-B. ELP-VEGF-B is a nonangiogenic, maternally sequestered protein with potential efficacy for treatment of preeclampsia.

Low Dose of IL-2 Normalizes Hypertension and Mitochondrial Function in the RUPP Rat Model of Placental Ischemia

IL-2 is a cytokine released from CD4+T cells with dual actions and can either potentiate the inflammatory response or quell a chronic inflammatory response depending on its circulating concentration. IL-2 is elevated in many chronic inflammatory conditions and is increased during preeclampsia (PE). PE is characterized by new-onset hypertension during pregnancy and organ dysfunction and increasing evidence indicates that proinflammatory cytokines cause hypertension and mitochondrial (mt) dysfunction during pregnancy. The reduced uterine perfusion pressure (RUPP) model of placental ischemia is a rat model of PE that we commonly use in our laboratory and we have previously shown that low doses of recombinant IL-2 can decrease blood pressure in RUPP rats. The objective of this study was to determine the effects of a low dose of recombinant IL-2 on multi-organ mt dysfunction in the RUPP rat model of PE. We tested our hypothesis by infusing recombinant IL-2 (0.05 ng/mL) into RUPP rats on GD14 and examined mean arterial pressure (MAP), renal, placental and endothelial cell mt function compared to control RUPP. MAP was elevated in RUPP rats (n = 6) compared to controls (n = 5) (122 ± 5 vs. 102 ± 3 mmHg, p < 0.05), but was reduced by administration of LD recombinant IL-2 (107 ± 1 vs. 122 ± 5 mmHg, n = 9, p < 0.05). Renal, placental and endothelial mt ROS were significantly increased in RUPP rats compared to RUPP+ IL-2 and controls. Placental and renal respiration rates were reduced in RUPP rats compared to control rats but were normalized with IL-2 administration to RUPPs. These data indicate that low-dose IL-2 normalized multi-organ mt function and hypertension in response to placental ischemia.

Abstract 29: Acute Systemic Treatment With Sphingosine -1- Phosphate Receptor 1 Agonist Diminishes Sex-difference In Renal Function Seen In Intrauterine Growth Restricted Mice.

IUGR is a risk factor for the early development of cardiorenal diseases in life. We previously showed that our IUGR mouse model exhibits sex differences in blood pressure (BP) and kidney functions as males have elevated BP and impaired kidney function while females are not. Sphingosine-1-Phosphate Receptor 1 (S1PR1) is reported to be involved in developing and progressing several cardiorenal diseases. We found that acute activation of S1PR1 transiently reduced BP in male IUGR; however, the effects of SIPR1 on renal function in IUGR are still unknown. We hypothesize that S1PR1 plays a role in the sex differences of impaired kidney function in IUGR. Here we investigated the acute effects of a specific S1PR1 agonist (SEW2178) on kidney function in IUGR offspring generated through placental insufficiency. Methods: C57BL IUGR or control offspring were obtained from the Reduced Uterine Perfusion Pressure (RUPP) or sham surgeries on the 13 th day of gestation. At 6 months, nephron number was measured by analyzing kidney histology while Glomerular Filtration Rate (GFR) was measured using FITC-Anulin decay after retroorbital injection. Mice were treated with 3mg/kg SEW2871 i.p before GFR measurement. Results: Both male and female IUGR had a decrease in nephron number compared to same-sex control (371.8± 28.8 vs 284.0± 11.3, P<0.01, male control vs IUGR) and (333.6±13.3 vs 256± 13.0, P<0.05, female control vs IUGR). Furthermore, IUGR impaired kidney function in male but not female mice as indicated by reduced GFR (8.9 ±0.6 vs 13.9±1.1 μL/min/kg BW, P<0.05, IUGR vs control males) and (12.3±0.9 vs 15.9 ±1.9 L/min/kg BW, control females vs IUGR). Administration of SEW2871 diminished the difference of GFR between male control and IUGR (9.5±0.6 vs 9.7±1.1 μL/min/kg BW, P> 0.05 control vs IUGR). While in females, both IUGR and control had a similar response to SEW2178 (7.4±1.0 vs 10.6 ±1.9 μL/min/kg BW, control vs. IUGR). Conclusion: IUGR impairs renal function in male offspring but not in females, indicating sex differences in fetal programming of kidney disease. Male and female IUGR offspring respond differently to acute systemic activation of S1PR1. Further studies are required to investigate the kidney-specific and cell-specific of S1PR1 activation/inhibition.

Abstract P235: Reduced Uterine Perfusion Pressure Impairs Seizure-induced Downregulation Of Neurotransmitter Transporters In Mice

Preeclampsia, a hypertensive disorder of pregnancy, can advance to eclampsia, if new onsetseizures occur. Previous work showed increased susceptibility to chemically-induced seizures inthe reduced uterine perfusion pressure (RUPP) rat model of preeclampsia; however, theunderlying mechanisms are unknown. Because seizures occur due to neurotransmitter activityimbalance, we hypothesized that RUPP mice have elevated excitatory and reduced inhibitoryactivity and that seizures exacerbate this imbalance.Timed-pregnant SMA-GFP mice (n=5-6 per group/treatment) were subjected to sham or RUPPsurgery on gestational day (GD) 13.5 and seizures were induced using 40mg/kg pentylenetetrazolon GD18.5. Tissues were harvested 30 minutes post-seizure induction. Maximum seizure scoreswere similar in sham (4.7±0.3) and RUPP (4.5±0.3) mice; p=0.37. Fluorometric assay showsseizures increased [F (1, 16) = 5.99, p=0.03], while RUPP had no effect [F (1, 16) = 1.15, p=0.3]on hippocampal glutamate concentration. No pairwise differences were observed within the shamand RUPP groups exposed to seizures (p>0.05). Seizures increased [F (1, 16) = 6.96, p=0.02],while RUPP had no effect [F (1, 16) = 0.61, p=0.45] on GABA concentration, with no significantpairwise difference in GABA concentration (p>0.05).Western blot analysis shows seizures significantly reduced hippocampal NMDAR1 (1.0±0.5 vs0.6±0.96, p=0.02; 1.0±0.1 vs 0.6±0.0, p=0.04) and GABAAR receptor expression (1.0±0.4 vs0.35±0.1, p<0.5; 1.05±0.3 vs 0.3±0.1, p<0.05) in sham and RUPP mice. Following seizureexposure, vesicular glutamate transporter (VGLUT1: sham: 1.0±0.3 vs 0.5±0.1; p<0.01, RUPP:0.8±0.2 vs 0.6±0.1; p=0.11), excitatory amino acid transporter 1 (EAAT1: sham: 1.0±0.4 vs0.6±0.2; p=0.02, RUPP: 0.9±0.1 vs 0.6±0.2; p=0.17) and GABA transporter (GAT1: sham: 1.0±0.5vs 0.4±0.1, p=0.01; RUPP: 0.8±0.2 vs 0.5±0.1, p=0.12) was reduced in sham mice, but not RUPPmice.Although RUPP does not change baseline GABA or glutamate related receptor or transporterexpression, our findings suggest seizure-induced reductions in vesicular and astrocyticneurotransmitter transporters is impaired by the RUPP procedure. Ongoing studies assesswhether other receptors and transporters are affected.

Abstract 31: Esomeprazole Improves Blood Pressure, Intrauterine Growth, Inflammation, And Vascular Function During Placental Ischemia Through Inhibition Of NLRP3

Preeclampsia (PE), a multisystem hypertensive disorder of pregnancy is characterized by intrauterine growth restriction (IUGR), inflammation, and vascular dysfunction. NLRP3 inflammasome is a cytoplasmic complex that mediates inflammation and is implicated in CVD. Clinical studies show an association between PE and increased placental NLRP3 expression. We hypothesized that inhibition of NLRP3 using (1) a specific NLRP3 small molecule inhibitor, MCC950 (M9, 20 mg/kg/d, i.p.) or (2) esomeprazole (ESO, 3.5 mg/kg/d, oral), a therapeutic that is safe in pregnancy, would improve MAP, inflammation, IUGR, and vascular dysfunction in the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia. Sham (S) or RUPP surgery was performed in pregnant Sprague Dawley rats on gestation day (GD) 14. A subset of rats from both groups received either vehicle, M9, or ESO on GD14-19 (n=9/group). On GD18, Uterine Artery Resistance Index (UARI) was measured via Doppler Ultrasound. MAP, fetal, and placental weight were measured, and blood and tissues were processed for additional analyses on GD19. MAP (mmHg) was elevated in RUPP (133±1) vs S (108±2; p<0.05). Treatment with M9 or ESO in RUPP decreased MAP (111±1 and 115±3, respectively; p<0.05 vs RUPP). Fetal weight (g) was reduced in RUPP (2.1±0.04) vs S (2.4±0.05), and ESO normalized fetal weight (2.4±0.01; p<0.05 vs RUPP). Placental NLRP3 mRNA expression increased 5-fold in RUPP vs S (p<0.05); and was less than 2 fold in M9 and ESO treated RUPP rats (p<0.05 vs RUPP). Inflammatory T-helper 17 and cytolytic NK cells, evaluated by flow cytometry, were increased in the circulation, placenta, and kidney of RUPP vs S controls. Treatment with M9 or ESO normalized both cell populations in all tissues (p<0.05 vs RUPP). UARI was increased in RUPP (0.71±0.03) versus S (0.56±0.01; p<0.05) and was decreased to 0.48±0.01 in M9 and 0.61±0.03 in ESO-treated RUPPs (p<0.05 vs RUPP). Renal vascular resistance (mmHg/mL/min/g) was increased in RUPP (42±8) vs S (23±4, p<0.05) and was normalized to 26±3 after treatment with M9 (p<0.05 vs RUPP). These data implicate NLRP3 in mediating inflammation and vascular dysfunction to cause maternal HTN and IUGR in RUPP; and identify NLRP3 as a potential target and ESO as a potential therapeutic for PE.

Uterine artery hemodynamics in female dogs with open- and closed-cervix pyometra

Although pyometra is a common disease, the mechanisms that determine cervical opening remain unknown. Knowing that the vascular structures are crucial in pathophysiology, it was observed need for hemodynamic studies assessing uterine artery of female dogs with pyometra and its relation to the neck opening. Thirty-five female dogs were selected and separate into three groups: control group (CG) (n = 12), open-cervix pyometra group (OCG) (n = 11) and closed-cervix pyometra group (CCG) (n = 12), with the objective of evaluating and comparing the hemodynamic changes of the uterine artery [peak systolic velocity (PSV), end diastolic velocity (EDV), and resistance index (RI)] in female dogs with open- and closed-cervix pyometra and correlate them with measurements of uterine diameter (UD) and endometrial thickness (ET). The correlation analysis showed that, with the exception of PSV, the hemodynamics indices were associated with UD and ET, presenting a moderate and positive correlation between UD and EDV (r = 0.62; P<0.01), a moderate and negative correlation between UD and RI (r =-0.68; P<0.01) and also moderate and negative correlation between ET and RI (r = -0.62; P<0.01). These results suggest that alterations of uterine artery hemodynamics are similar in dog females with open- or closed-cervix pyometra, although the UD and the ET can influence in the uterine perfusion.

POST-PARTUM SUB-CLINICAL ENDOMETRITIS IN DAIRY COWS: INCIDENCE AND DIAGNOSIS

Post-partum uterine infections such as metritis, clinical endometritis and sub-clinical endometritis, are the most common cause for decreased productivity and fertility in dairy cows. Being the least severe form of endometritis, sub-clinical endometritis (SCE) is defined as the superficial inflammation of endometrium with no signs of systemic illness and characterized by an increase in number of polymorphonuclear cells (PMNCs) inside the uterine lumen. The impact of sub-clinical endometritis on fertility of dairy cows is well known probably due to absence of any clinical signs and thus, difficulty in diagnosis and treatment. Different techniques such as endometrial cytology, uterine biopsy, biochemical analysis of uterine fluid, and measurement of acute phase proteins and inflammatory markers have been employed for the diagnosis of SCE. Doppler and B-mode ultrasonography of middle uterine arteries and uterus have also been used to diagnose the inflammation via assessment of uterine perfusion, respectively. Among all methods, endometrial cytology via cytotape is one of the most advanced and frequently employed methods for diagnosis of cytological endometritis based on the fact that proportion of PMNCs increase during uterine inflammation. The review focuses mainly on current status of incidence and diagnosis of post-partum sub-clinical endometritis in dairy cows.