Nonpregnant Research Articles

ARRHYTHMOGENIC ATRIAL REMODELING DURING PREGNANCY IN MICE

Pregnancy is associated with greater vulnerability to supraventricular tachyarrhythmias (SVT). As the underlying mechanisms remain to be elucidated, we investigated whether pregnancy induces atrial remodeling that might contribute to this. Atrial electrophysiological and contractile properties were examined in non-pregnant (NP) and pregnant (P) mice. Cell shortening and Ca2+ imaging were measured on atrial myocytes. Atrial action potential and ionic currents were recorded using patch-clamp technique. Atrial mRNA and protein expression were analyzed using qPCR and Western blot. The P-wave area on the ECG increased by 50% during pregnancy, suggesting atrial enlargement, confirmed by echocardiography. The atrial myocytes were longer in P mice, adding further evidence to the physiological hypertrophy associated with pregnancy. Echocardiography showed a 50% increase in atrial fractional area change during pregnancy, indicating much stronger contraction. A similar increase in cell shortening was observed in P mice and was associated with a decrease in sarcomere length and changes in myofilament protein phosphorylation. However, pregnancy did not affect L-type Ca2+ current, Ca2+ transients, and SR Ca2+ load. Myocytes from P mice showed twice as many spontaneous contractions and spontaneous diastolic Ca2+ releases. Moreover, pregnancy was associated with a 50% increase in action potential duration, linked to a reduction in the density of the transient outward K+ current Ito and the underlying KV4.3 channel. During pregnancy, atrial tissues undergo substantial remodeling, potentially contributing to the development of SVT.

PSIII-2 Exploring differential gene expression and pathways associated with pregnancy outcomes in beef heifers

Abstract Heifer fertility is a key factor in the success of beef production systems, as the reproductive performance of a heifer correlates with her stayability in the breeding herd. However, despite being reproductively mature and physically healthy, 5 to 15% of these heifers fail to conceive within a designated breeding season. The underlying physiological mechanisms preventing these heifers from achieving pregnancy remain elusive. Therefore, this study aimed to determine differentially expressed genes (DEGs) and biological pathways associated with pregnancy outcomes in beef heifers. Angus-Simmental crossbred heifers were subjected to an estrus synchronization and fixed-time artificial insemination (AI) program (7-d CO-Synch + CIDR) and blood samples were collected at the time of AI for isolation of peripheral white blood cells (PWBC). After pregnancy check, heifers were classified as pregnant (P) and non-pregnant (NP). Based on that, 6 animals per group were randomly selected for total RNA isolation from the PWBC samples, and genome-wide expression was measured through RNA sequencing. The RNA-Seq data were analyzed using an in-house bioinformatics pipeline, with filtered reads aligned to the Bos taurus reference genome using STAR. DEGs were identified using DESeq2, followed by functional analysis using ShinyGO. Our approach unveiled 230 DEGs, comprising 102 up and 128 downregulated genes in the NP group (P-value ≤ 0.05 and |log2FC| > 0.5). Upregulated genes included those from the cytochrome P450 superfamily (CYP27B1 and CYP2U1). The genes SCML2, USIG2, and FBP1 were upregulated and have been associated with immune response and increased PWBC counts. This may be related to the over-representation of biological processes involved in cell division based on the upregulated genes. KEGG pathway over-representation analysis of DEGs retrieved allograft rejection, graft-versus-host disease, type I Diabetes Mellitus, insulin signaling pathway, and transcriptional misregulation of cancer. These pathways included genes such as BOLA-DQB, PRKAR1B, CALML4 and JUP, which were reported as downregulated during times of inflammatory response. Another downregulated gene was GZMB, which is needed for proper cell targeting during immune response. This finding may provide a link between immune function and early pregnancy failure. However, the relationship between the immune system and pregnancy warrants further investigation. Our results shed light on the molecular mechanisms underlying reproductive performance in beef heifers and support previous studies associating immune function with pregnancy outcomes. These differentially expressed genes may be further explored as potential avenues for enhancing fertility in beef production systems.

391 The impact of trace mineral source on systemic immunological status associated with early embryonic loss in lactating dairy cattle

Abstract Milk production has doubled from 1970 to 2009 but has been associated with a decline in fertility. Around 25% of failed pregnancies in dairy cattle are due to embryonic loss. Mineral bioavailability may affect embryonic retention, so we hypothesized that Zn, Cu, and Mn sources will influence systemic immunological status, which affects early embryonic retention. Holstein cows (n = 58) were randomly assigned to one of two daily oral drench trace mineral (Zn, Cu, and Mn) treatments 30 d before breeding (d -30): 1) Inorganic (ITM) or 2) Metal Methionine Hydroxy Analog Chelate (MMHAC). Before treatments began, cows were assigned to a semen type, beef vs. Holstein, equally, across treatments. All cows underwent a double Ovsynch protocol and were artificially inseminated on d 0. Humoral immunological status was assessed in serum samples taken on d 0 and d 7. Immune activity was quantified via a D2Dx systemic immunity score, which assesses samples for the presence of IgG, IgM, cytokines, and complement proteins. Ultrasonography was utilized for pregnancy confirmation on d 35, classifying cows as pregnant (P) or open. Progesterone analysis from d 21 plasma was used to categorize open cows as either non-pregnant (NP) or early embryonic loss (EEL). Statistical analyses in R Studio were completed, in two models based on pregnancy (P vs EEL and NP vs EEL), to determine the effects of mineral treatment, pregnancy status, semen type, and all interactions on humoral immune status. In the P vs EEL model, there were no effects (P > 0.30) of semen type, mineral treatment, pregnancy status, or any interactions on systemic immunity score on the day of breeding (d 0) or d 7. On d 0, cows provided MMHAC had a systemic immunity score of 0.073 ± 0.008 but were not different (P > 0.34) from ITM cows (0.063 ± 0.006). This trend continued on d 7 with systemic immunity scores of MMHAC cows not differing (P > 0.32) from ITM cows (0.055 ± 0.004 and 0.061 ± 0.003; respectively). There was a tendency (P = 0.10) for the three-way interaction of semen type x pregnancy x treatment to influence the immunity score change from d 0 to d 7. Pregnant cows on ITM treatment bred with beef semen had a positive change in immunity score (0.020 ± 0.0085). Whereas ITM cows bred with Holstein semen that underwent EEL had a negative change (-0.015 ± 0.0085); all other means were intermediate. In the NP vs EEL model, there were no effects (P > 0.14) of semen type, treatment, pregnancy status, or any interactions on systemic immunity score on d 0, d 7, or the change between d 0 and d 7. In summary, trace mineral bioavailability tended to affect systemic immunological status in P vs EEL cows but not in NP vs EEL cows, which may allow for the elucidation of immunological status differences during pregnancy establishment.

Pregnancy ameliorates neuropathic pain through suppression of microglia and upregulation of the δ-opioid receptor in the anterior cingulate cortex in late-pregnant mice.

Pregnancy-induced analgesia develops in late pregnancy, but its mechanisms are unclear. The anterior cingulate cortex (ACC) plays a key role in the pathogenesis of neuropathic pain. The authors hypothesized that pregnancy-induced analgesia ameliorates neuropathic pain by suppressing activation of microglia and the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and by upregulating opioid receptors in the ACC in late-pregnant mice. Neuropathic pain was induced in non-pregnant (NP) or pregnant (P) C57BL/6JJmsSlc female mice by partial sciatic nerve ligation (PSNL). The nociceptive response was evaluated by mechanical allodynia and activation of microglia in the ACC was evaluated by immunohistochemistry. The expressions of phosphorylated AMPA receptors and opioid receptors in the ACC were evaluated by immunoblotting. In von Frey reflex tests, NP-PSNL-treated mice showed a lower 50% paw-withdrawal threshold than NP-Naïve mice on experimental day 9. No difference in 50% paw-withdrawal threshold was found among the NP-Naïve, NP-Sham, P-Sham, and P-PSNL-treated mice. The number of microglia in the ACC was significantly increased in NP-PSNL-treated mice compared to NP-Sham mice. Immunoblotting showed significantly increased expression of phosphorylated AMPA receptor subunit GluR1 at Ser831 in NP-PSNL-treated mice compared to NP-Sham mice. Immunoblotting also showed significantly increased δ-opioid receptor in the ACC in P-Sham and P-PSNL-treated mice compared to NP-Sham mice. Pregnancy-induced analgesia ameliorated neuropathic pain by suppressing activation of microglia and the expression of phosphorylated AMPA receptor subunit GluR1 at Ser831, and by upregulation of the δ-opioid receptor in the ACC in late-pregnant mice.

Circulating vascular endothelial growth factor receptor-3, a pro-lymphangiogenic and pro-angiogenic mediator, is decreased in pre-eclampsia.

To compare circulating levels of vascular endothelial growth factor receptor 3 (VEGFR-3) in women with pregnancy-induced hypertension (PIH) and in non-pregnant (NP) and healthy pregnant (HP) women. We conducted a case-control study including PIH (n = 135), HP (n = 68), and NP (n = 49) women from southeastern Brazil. PIH were diagnosed according to international guidelines, and defined as gestational hypertension (GH, n = 61) or pre-eclampsia (n = 74). VEGFR-3 was measured in plasma using ELISA. Plasma VEGFR-3 was increased in HP (1207 pg/mL) compared with NP (133 pg/mL) women; however, PIH (729 pg/mL) patients exhibited lower levels than HP women (both p < 0.05). In addition, plasma VEGFR-3 was decreased in pre-eclampsia compared with GH (537 versus 980 pg/mL; p < 0.05). When pre-eclampsia was classified according to different clinical presentations, plasma VEGFR-3 was further decreased in the cases identified as pre-eclampsia with severe features, preterm pre-eclampsia, and pre-eclampsia accompanied by small for gestational age (all p < 0.05). Our data indicate reduced circulating VEGFR-3 levels in patients with PIH, specifically in those diagnosed with pre-eclampsia. Moreover, decreased VEGFR-3 was associated with adverse clinical outcomes in pre-eclampsia. These findings expand previous evidence of reduced VEGFR-3 expression in pre-eclampsia. Future studies should investigate whether it can be used as a predictive biomarker and/or therapeutic target for pre-eclampsia.

Abstract Tu064: Abnormal Calcium Regulation Leads to Pathological Cardiac Hypertrophy During Pregnancy in the GSNOR-Deficient Mouse Model of Preeclampsia

Introduction: Preeclampsia (PE), a leading cause of maternal mortality, is linked to cardiac hypertrophy and impaired relaxation, predisposing the mother to an increased risk of cardiovascular (CV) injury during pregnancy. Mice lacking S-nitrosoglutathione reductase (GSNOR –/– mice) exhibit all the clinical features of PE. Calcium regulation plays an important role in cardiac relaxation and hypertrophy, but whether alterations in calcium handling contribute to the impairment in these PE mice is unknown. Hypothesis: Cardiac hypertrophy and impaired relaxation in pregnant GSNOR –/– mice may be due in part to altered Ca 2+ handling mechanisms. Methods: Pregnant control (WT [C57B6]) and GSNOR –/– mice were examined at non-pregnant (NP) and late pregnancy (17.5 dpc). Cardiomyocytes (CMs) were isolated (N=3-4 mice/group; n=8-24 CMs/heart) and assessed for intracellular Ca 2+ and sarcomere shortening using IonOptix. Ca 2+ decay and sarcomere relaxation rates were recorded. Ventricular tissue homogenates were assessed for Ca 2+ handling and sarcomere-associated protein expression and post-translational modifications by Western Blot. Results: At late pregnancy, the CMs of GSNOR –/– mothers were significantly wider and systolic and diastolic Ca 2+ levels were elevated as compared to WT. At 17.5 dpc, expression of Ca 2+ handling proteins, RyR2, L-TCC and NCX1 were increased, and phosphorylation of RyR2 and L-TCC were increased in the GSNOR –/– mothers as compared to WT. The Ca 2+ decay rate improved in CMs from pregnant versus NP WT CMs but not in GSNOR –/– CMs. PLB phosphorylation at Thr-17 was augmented in pregnant WT hearts, while phosphorylation at Ser-16 was reduced in pregnant GSNOR –/– . Sarcomere relaxation parameters and sarcomere resting length were improved in CMs from pregnant WT mice but were impaired in pregnant versus NP GSNOR –/– mice in association with increased expression of MyBPC in pregnant GSNOR –/– . Conclusion: The PE-related cardiac hypertrophy and impaired relaxation at late pregnancy in GSNOR –/– mice were associated with elevated cytosolic Ca 2+ levels, altered expression and post-translational modifications of Ca 2+ handling and sarcomeric proteins, which may predispose these mice to higher CV injury during pregnancy.

Thrombin in Pregnancy and Preeclampsia: Expression, Localization, and Vasoactivity in Brain and Microvessels From Rats.

Thrombin is a coagulation factor increased in pregnancy and further increased in preeclampsia (PE), a hypertensive disorder. Thrombin is also expressed in the brain and may have a nonhemostatic role. We characterized thrombin expression and vasoactivity in brain cerebral parenchymal arterioles (PAs) in rat models of pregnancy and PE. PAs were isolated and pressurized from nonpregnant (NP) and late-pregnant (LP) rats and rats with experimental preeclampsia (ePE). Reactivity to thrombin (1-50 U/mL) was measured in the absence and presence of inhibition of cyclooxygenase and nitric oxide synthase. Plasma levels of prothrombin, thrombin-antithrombin (TAT), tissue plasminogen activator, and plasminogen activator inhibitor-1 (PAI-1) and cerebrospinal fluid levels of TAT were compared using enzyme-linked immunosorbent assay. Expression of protease-activated receptor types 1 and 2 in PAs were measured by Western blot and immunohistochemistry. Neuronal thrombin expression was quantified in brains from all groups by immunohistochemistry. Prothrombin and TAT were elevated in ePE plasma compared with NP and LP. TAT was detected in cerebrospinal fluid from all groups and significantly elevated in LP (NP: 0.137 ± 0.014 ng/mL, LP: 0.241 ± 0.015 ng/mL, ePE: 0.192 ± 0.028 ng/mL; P < 0.05). Thrombin caused modest vasoconstriction in PAs from all groups regardless of cyclooxygenase or nitric oxide synthase inhibition. PAR1 and PAR2 were found in PAs from all groups colocalized to smooth muscle. Thrombin expression in central neurons was decreased in both LP and ePE groups compared with NP. These findings suggest a role for thrombin and other hemostatic changes during pregnancy and PE beyond coagulation.

Cardiac mitochondrial metabolism during pregnancy and the postpartum period.

The goal of the present study was to characterize changes in mitochondrial respiration in the maternal heart during pregnancy and after birth. Timed pregnancy studies were performed in 12-wk-old female FVB/NJ mice, and cardiac mitochondria were isolated from the following groups of mice: nonpregnant (NP), midpregnancy (MP), late pregnancy (LP), and 1-wk postbirth (PB). Similar to our previous studies, we observed increased heart size during all stages of pregnancy (e.g., MP and LP) and postbirth (e.g., PB) compared with NP mice. Differential cardiac gene and protein expression analyses revealed changes in several mitochondrial transcripts at LP and PB, including several mitochondrial complex subunits and members of the Slc family, important for mitochondrial substrate transport. Respirometry revealed that pyruvate- and glutamate-supported state 3 respiration was significantly higher in PB vs. LP mitochondria, with respiratory control ratio (RCR) values higher in PB mitochondria. In addition, we found that PB mitochondria respired more avidly when given 3-hydroxybutyrate (3-OHB) than mitochondria from NP, MP, and LP hearts, with no differences in RCR. These increases in respiration in PB hearts occurred independent of changes in mitochondrial yield but were associated with higher abundance of 3-hydroxybutyrate dehydrogenase 1. Collectively, these findings suggest that, after birth, maternal cardiac mitochondria have an increased capacity to use 3-OHB, pyruvate, and glutamate as energy sources; however, increases in mitochondrial efficiency in the postpartum heart appear limited to carbohydrate and amino acid metabolism.NEW & NOTEWORTHY Few studies have detailed the physiological adaptations that occur in the maternal heart. We and others have shown that pregnancy-induced cardiac growth is associated with significant changes in cardiac metabolism. Here, we examined mitochondrial respiration and substrate preference in isolated mitochondria from the maternal heart. We show that following birth, cardiac mitochondria are "primed" to respire on carbohydrate, amino acid, and ketone bodies. However, heightened respiratory efficiency is observed only with carbohydrate and amino acid sources. These results suggest that significant changes in mitochondrial respiration occur in the maternal heart in the postpartum period.

Impact of vaginal microecological differences on pregnancy outcomes and endometrial microbiota in frozen embryo transfer cycles.

This prospective study investigates the correlation between vaginal microecology and pregnancy outcomes and explores their impact on endometrial microbiota composition during frozen embryo transfer (FET) cycles. Additionally, the impact of transvaginal Lactobacillus supplementation on reproductive outcomes in patients with previous failed cycles was assessed. A total of 379 patients undergoing FET at a reproductive medicine center were categorized into clinical pregnancy (CP), miscarriage (MISC), and non-pregnant (NP) groups. Vaginal specimens were collected for microecological evaluation prior to embryo transfer. Endometrial microbiota samples were obtained during embryo transfer for 16S rRNA gene sequencing analysis to assess endometrial microbiota composition. Vaginal microecological indicators, including pH, Lactobacillus dominance, and leukocyte esterase activity, were measured. Transvaginal Lactobacillus supplementation was investigated in 60 patients with previous failed cycles. Vaginal microecology significantly correlated with pregnancy outcomes, with normal microecology associated with a higher clinical pregnancy rate. Vaginal pH and leukocyte esterase activity were significantly associated with clinical pregnancy. Furthermore, vaginal microecological differences significantly impacted endometrial microbiota composition. However, no significant differences were observed in endometrial microbiota composition among the CP, MISC, and NP groups. Notably, transvaginal Lactobacillus supplementation increased the clinical pregnancy rate without affecting the miscarriage rate. This study highlights that normal vaginal microecology, characterized by lower pH and leukocyte esterase negativity, is associated with a higher likelihood of clinical pregnancy following FET. Importantly, vaginal microecological differences influence endometrial microbiota composition. Moreover, transvaginal Lactobacillus supplementation appears promising in improving clinical pregnancy rates in patients with previous failed cycles. These findings contribute to a better understanding of the interplay between vaginal and endometrial microbiota and offer potential interventions to enhance reproductive success in assisted reproductive technologies.

Follicular Fluid Proteomic Analysis of Women Undergoing Assisted Reproduction Suggests That Apolipoprotein A1 Is a Potential Fertility Marker.

Infertility affects millions worldwide, posing a significant global health challenge. The proteomic analysis of follicular fluid provides a comprehensive view of the complex molecular landscape within ovarian follicles, offering valuable information on the factors influencing oocyte development and on the overall reproductive health. The follicular fluid is derived from the plasma and contains various proteins that can have different roles in oocyte health and infertility, and this fluid is a critical microenvironment for the developing oocytes as well. Using the high-performance liquid chromatography-mass spectrometry method, we investigated the protein composition of the follicular fluid, and after classification, we carried out relative quantification of the identified proteins in the pregnant (P) and non-pregnant (NP) groups. Based on the protein-protein interaction analysis, albumin and apolipoprotein A1 (ApoA1) were found to be hub proteins, and the quantitative comparison of the P and NP groups resulted in a significantly lower concentration of ApoA1 and high-density lipoprotein cholesterol in the P group. As both molecules are involved in the cholesterol transport, we also investigated their role in the development of oocytes and in the prediction of fertility.

Blood Metabolites and Faecal Microbial Communities in Nonpregnant and Early Gestation Ewes in Highly Cold Areas.

Ewes undergo complex metabolic changes during pregnancy. Understanding the specific process of these changes is a necessary prerequisite in ewes for regulating and intervening in order to maintain pregnancies. However, there have been relatively few studies on the specific changes that occur in nutritional metabolism in pregnant ewes during early gestation, especially for some landrace ewes in highly cold areas. Therefore, this study aimed to (1) elucidate the changes in metabolites and microbial communities in pregnant ewes during early gestation using metabolomics and 16S ribosomal RNA gene (rDNA) amplicon sequencing approaches, and to (2) discover novel early pregnancy-induced biomarkers in the blood and faeces. Rams were placed together with ewes on D0 and removed on D45. During early gestation, blood and faecal samples were collected from ewes in a highly cold area for analysing the metabolites and microbial communities; these were retrospectively classified as the early gestation pregnant (EP) ewe group or the nonpregnant (NP) ewe group based on the lambing status recorded during the expected delivery period. The differences in the plasma biochemical parameters, plasma metabolites, and faecal microbial communities of pregnant and nonpregnant ewes were characterised. The GC, IL-6, O-acetyl-l-serine, L-glutamine, and 6-acetamido-2-oxohexanoic acid were screened out as potential biomarkers for evaluating the occurrence of early pregnancy. These novel early pregnancy-induced metabolites discovered in ewes might allow for the development of technologies to detect early pregnancies in sheep in highly cold areas.

Exploring myometrial microenvironment changes at the single-cell level from nonpregnant to term pregnant states.

The microenvironment and cell populations within the myometrium play crucial roles in maintaining uterine structural integrity and protecting the fetus during pregnancy. However, the specific changes occurring at the single-cell level in the human myometrium between nonpregnant (NP) and term pregnant (TP) states remain unexplored. In this study, we used single-cell RNA sequencing (scRNA-Seq) and spatial transcriptomics (ST) to construct a transcriptomic atlas of individual cells in the myometrium of NP and TP women. Integrated analysis of scRNA-Seq and ST data revealed spatially distinct transcriptional characteristics and examined cell-to-cell communication patterns based on ligand-receptor interactions. We identified and categorized 87,845 high-quality individual cells into 12 populations from scRNA-Seq data of 12 human myometrium tissues. Our findings demonstrated alterations in the proportions of five subpopulations of smooth muscle cells in TP. Moreover, an increase in monocytic cells, particularly M2 macrophages, was observed in TP myometrium samples, suggesting their involvement in the anti-inflammatory response. This study provides unprecedented single-cell resolution of the NP and TP myometrium, offering new insights into myometrial remodeling during pregnancy.NEW & NOTEWORTHY Using single-cell RNA sequencing and spatial transcriptomics, the myometrium was examined at the single-cell level during pregnancy. We identified spatially distinct cell populations and observed alterations in smooth muscle cells and increased M2 macrophages in term pregnant women. These findings offer unprecedented insights into myometrial remodeling and the anti-inflammatory response during pregnancy. The study advances our understanding of pregnancy-related myometrial changes.

Abstract 18622: The Role of MicroRNA-98 in the Context of Myocardial Ischemia-Reperfusion Injury During Late Pregnancy

BACKGROUND: We have shown that the late-pregnant (LP) rodent exhibits a higher susceptibility to myocardial ischemia-reperfusion injury (IRI) compared to non-pregnant (NP). The molecular mechanisms remain unclear. The dysregulation of multiple microRNAs (miRs) in IRI suggests their potential as promising targets for therapeutic interventions. Methods: Female Sprague-Dawley rats in both NP and LP states underwent occlusion of the left anterior descending coronary artery (LAD) for a duration of 45 minutes, followed by reperfusion for 3 hours or 24 hours. Expression of miR was assessed via fluorescent in situ hybridization. MicroRNA-microarray profiling was conducted on hearts of NP and LP rats subjected to IRI. Female H9c2 rat cardiomyoblast cells were transfected with miR mimics and inhibitors, and subjected to hypoxia/reoxygenation. Blood samples were obtained from NP, LP, and ischemic heart disease (IHD) patients. MiR98 inhibitor was administered at the initiation of reperfusion. Results: MicroRNA-microarray analysis revealed upregulation of microRNA-98-5p (miR-98) in LV of LP at baseline, and a further upregulation after IRI. The expression of Stat3 and Pgc-1α were decreased in LV of LP rats compared to NP rats upon IRI. Fluorescent in situ hybridization revealed expression of miR-98 in cardiomyocytes in NP rats, which was increased in LP rats. Overexpression of miR-98 in vitro in female H9c2 cells decreased Stat3 and Pgc-1α levels, and promoted apoptosis, oxidative stress, and inflammatory markers. MiR-98 inhibitor in LP rats at the onset of reperfusion reduced infarct size, apoptosis, oxidative stress, and inflammatory markers and was associated with upregulation of Stat3 and Pgc-1α. In humans, plasma miR-98 levels were significantly higher in healthy LP compared to healthy NP individuals and even higher in LP patients with IHD patients. CONCLUSIONS: We show the detrimental effects of miR-98 by promoting cardiomyocyte oxidative stress, inflammation, and apoptosis via its targets Stat3 and Pgc-1α in the context of late pregnancy. MiR-98 could be a novel cardio-protective strategy or biomarker in late pregnancy.

293 Impact of Late Embryonic and Early Fetal Mortality on Overall Productivity of Suckled Beef Cows

Abstract The objective of this study was to evaluate the effect of late embryonic and early fetal mortality (LEEFM) on the performance of beef cows exposed to fixed-timed artificial insemination (TAI). Postpartum cows (n = 207) from three locations were exposed to a 7-d CO-Synch+CIDR protocol followed by TAI (day 0) and natural service breeding starting 14 days after TAI. Two pregnancy diagnoses were performed on days 29 and 100 and cows were classified as: 1) Maintained (MAINT): cows were pregnant on day 29 and the same pregnancy was confirmed on day 100; 2) LEEFM: cows were diagnosed as pregnant on day 29 but non-pregnant or pregnant by natural service on day 100; and 3) Non-pregnant (NP): cows were diagnosed as non-pregnant to TAI on both days 29 and 100. Cows were evaluated until calves that were a product of the breeding season described herein were weaned. Final pregnancy rates were decreased (P &amp;lt; 0.01) for LEEFM compared with MAINT and NP. Calving (P &amp;lt; 0.01) and weaning rates (P &amp;lt; 0.01) were also decreased for LEEFM compared with MAINT and NP. Average calving day was decreased (P &amp;lt; 0.01) in MAINT cows compared with both NP and LEEFM; however, no differences were observed between NP and LEEFM (P = 0.46). Weaning weight was increased (P ≤ 0.05) for MAINT compared with both NP and LEEFM; however, there were no differences between NP and LEEFM ( P = 0.52). Cows that experienced LEEFM had decreased (P &amp;lt; 0.01) weaning weights per cow exposed compared with both MAINT and NP. In summary, that conceive to TAI and experience LEEFM have decreased performance compared not only to MAINT cows, but also cows that failed to conceive to TAI (NP).

Plasma eNOS Concentration in Healthy Pregnancy and in Hypertensive Disorders of Pregnancy: Evidence of Reduced Concentrations in Pre-Eclampsia from Two Independent Studies.

Hypertensive disorders of pregnancy (HDP), comprising gestational hypertension (GH) and pre-eclampsia (PE), are leading causes of maternal and perinatal morbidity and mortality. Both GH and PE are characterized by new-onset hypertension, but PE additionally includes proteinuria and/or end-organ damage. Impaired nitric oxide (NO) bioavailability may lead to endothelial dysfunction in GH and PE, and the primary source of vascular NO is endothelial NO synthase (eNOS). However, no previous study has investigated plasma eNOS concentrations in patients with GH and PE. In this study, we compared plasma eNOS concentrations in healthy pregnancies and HDP in two independent cohorts. The primary study included 417 subjects, with 43 non-pregnant (NP) and 156 healthy pregnant (HP) women and 122 patients with GH and 96 with PE. The replication study included 85 pregnant women (41 healthy and 44 pre-eclamptic). Plasma concentrations of eNOS were measured using a commercial ELISA kit provided by R&D Systems, and plasma nitrite concentrations were assessed using two ozone-based chemiluminescence assays. Correlations between plasma eNOS concentrations and plasma nitrite concentrations, as well as clinical and biochemical parameters, were evaluated by either Spearman's or Pearson's tests. In the primary study, NP women and HDP had significantly lower plasma eNOS concentrations compared to HP; concentrations were even lower in PE compared to GH. Plasma eNOS concentrations were reduced but not significant in early-onset PE, PE with severe features, preterm birth, and intrauterine growth restriction. No correlation was observed between plasma eNOS and nitrite levels. In HDP, there was a significant positive correlation between levels of eNOS and hemoglobin (r = 0.1496, p = 0.0336) as well as newborn weight (r = 0.1487, p = 0.0316). Conversely, a negative correlation between eNOS levels and proteinuria was observed (r = -0.2167, p = 0.0179). The replication study confirmed significantly reduced plasma concentrations of eNOS in PE compared to HP. Our findings provide evidence of reduced plasma eNOS concentrations in HDP; they were particularly lower in PE compared to GH and HP in two independent studies.

Abstract P2126: Role Of Microrna-98 In Myocardial Ischemia-reperfusion Injury In Late Pregnancy

Background: We have shown that the late-pregnant (LP) rodent is more vulnerable to myocardial ischemia-reperfusion injury (IRI) compared to non-pregnant (NP). The molecular mechanisms are unknown. Several microRNAs (miRs) are dysregulated in IRI, making them promising therapeutic targets. Methods: The left anterior descending coronary artery (LAD) of female Sprague-Dawley rats (2-3 months old NP and LP, 20-21 days pregnant) was occluded for 45 minutes followed by 3 hours reperfusion. Myocardial necrosis was assessed using TTC staining. MicroRNA-microarray profiling was performed on hearts of NP and LP rats subjected to IRI. In vitro, female cardiomyocytes were transfected either miR98 mimic or inhibitor, followed by 3 hours hypoxia and 6 hours reoxygenation. Blood samples were collected from NP, LP, and coronary heart disease (CHD) patients. To assess therapeutic potential of miR, miR98 inhibitor was administered at the onset of reperfusion. Results: MicroRNA-microarray profiling revealed miR-98-5p (miR-98) is upregulated in the hearts of LP IRI rats compared to NP IRI by 2.3 folds, which we confirmed with qPCR. miR98 is significantly increased in the plasma of healthy LP compared to healthy NP women, and significantly increased in LP with CHD compared to healthy LP. TargetScan revealed STAT3 and PGC-1α to be targets of miR98. We confirmed significantly decreased expression of STAT3 and PGC-1α in LP IRI rat myocardium compared to NP IRI. MiR98 overexpression in female cardiomyocytes subjected to hypoxia/reoxygenation (H/R) decreased STAT3 and PGC-1α, and increased apoptosis, inflammation, and oxidative stress, while miR-98 knockdown resulted in the opposite effect. Therapeutic targeting of microRNA-98-5p in LP rats subjected to IRI reduced infarct size, apoptosis, oxidative stress, and inflammatory markers via upregulating Stat3 and Pgc-1α. Conclusions: MiR98 is upregulated in LP rats subjected to IRI compared to NP IRI and in LP human plasma samples with CHD compared to healthy NP and LP. miR98 promotes oxidative stress, inflammation, and apoptosis in the setting of H/R in vitro by targeting STAT3 and PGC-1α. Inhibition of miR-98 in LP rats reduces myocardial infarct size, apoptosis, oxidative stress, and inflammation via Stat3 and Pgc-1α.

Abstract P1176: What Is The Nature Of The Hypertrophic Response Contributing To Pregnancy-induced Cardiac Growth And Its Reversal?

During pregnancy, the maternal heart undergoes physiological cardiac growth, reversing following parturition; however, the contributing underlying processes are largely unknown. Changes in cardiac metabolism appear to be associated with cardiac growth through a reduction in glucose catabolism. We have also shown that several cardiac metabolic transcripts, proteins, and liver-derived metabolites that are elevated during pregnancy are associated with cardiac growth. However, the nature of the hypertrophic response observed in the maternal heart is unclear. Therefore, this study aimed to examine the metabolic and hypertrophic changes associated with pregnancy-induced cardiac growth and reversal. Studies revealed that both heart weight (HW: TL; p &lt; 0.0001) and liver weight (LIV: TL; p &lt;0.0001) were highest in lactating 1-week post-birth (PB) mice versus mid-pregnant (8 d), late-pregnant (LP; 16 d), and non-pregnant (NP) 12-week-old FVB/NJ mice. Of note, we also found that HW: TL ( p &lt; 0.01) and LIV: TL ( p &lt; 0.0001) were significantly reduced in non-lactating 1-week post-birth mice (PBNL) compared with PB mice. However, the changes in cardiac size observed during pregnancy and post-birth were not associated with changes in either cardiomyocyte cross-sectional area or cardiac fluid content. Hearts and livers remained significantly larger, irrespective of lactation status, following parturition through 6 weeks post birth (PB) versus NP. Consistent with these changes, we found significant reductions in Pdk4 protein between 1- and 3-week PB and PBNL groups that were reversed by six weeks postpartum and a substantial increase in Pdk4 expression at six weeks postpartum, respectively. Interestingly, we see a sustained increase in cardiac Bdh1 expression that persisted at six weeks postpartum, which could contribute to the sustained increases in HW: TL versus NP. Changes in lactation status and metabolism are likely contributing to similar patterns of growth seen in the heart and liver during and following pregnancy. The maternal hypertrophic response is considered eccentric due to the absence of changes in myocyte cross-sectional area and cardiac fluid content.

Abstract P2009: Mice Lacking S-nitrosoglutathione Reductase (gsnor -/- ), A Mouse Model Of Preeclampsia, Exhibit Abnormal Calcium Regulation And Cardiac Hypertrophy During Pregnancy.

Introduction: We recently showed that mice lacking S-nitrosoglutathione reductase (GSNOR -/- mice), a denitrosylase that regulates protein S-nitrosylation, exhibit the clinical features of preeclampsia (PE) a leading cause of maternal mortality. PE predisposes the mother to increased risk of cardiovascular (CV) injury later in life and is linked to cardiac hypertrophy and impaired relaxation. Cardiac hypertrophy and relaxation are regulated by calcium cycling. We hypothesized that GSNOR deficiency in pregnant mice alters calcium cycling, leading to hypertrophy and impaired cardiac relaxation. Methods: Pregnant control (WT [C57B6]) and GSNOR -/- mice were examined at non-pregnant (NP) and late pregnancy (17.5 dpc). Cardiomyocytes (CMs) (N=3-4 mice/group; n=8-24 CMs/heart) were isolated and assessed for intracellular calcium and sarcomere shortening using IonOptix system. Dynamic parameters associated with calcium decay and sarcomere relaxation were recorded (time to peak (ttp), time to 50% of baseline (tt50%); d SL/ d t min , time to d [Ca]/ d t min , etc.). Experiments were conducted at 37°C. Results: At late pregnancy, systolic calcium levels were significantly elevated in CMs isolated from GSNOR -/- as compared to WT mothers. Despite the increased cytosolic calcium, CMs from pregnant GSNOR -/- mice exhibited no change in contractility. The improved calcium decay rate observed in CMs from pregnant versus NP WT mice, was absent in GSNOR -/- mothers. Similarly, sarcomere relaxation parameters and sarcomere resting length, which were also improved by pregnancy in WT CMs, were impaired in CMs from pregnant versus NP GSNOR -/- mice. In addition, hearts were significantly enlarged in GSNOR -/- mothers as compared to WT, even when normalized to tibia length. Conclusion: The elevated systolic calcium levels, impaired cardiac relaxation, and cardiac hypertrophy seen in pregnant GSNOR -/- mice may predisposes them to higher risk for CV injury during pregnancy and later in life.

Characterization of extracellular vesicles in COVID-19 infection during pregnancy.

Introduction: SARS-CoV-2 infection may cause a severe inflammatory response, inflicting severe morbidity and mortality. This risk is modestly increased in pregnant patients. Despite the hypercoagulability and immunosuppression associated with pregnancy, most pregnant women experience a mild COVID-19 infection. Maternal extracellular vesicles (EVs) may interact with endothelial and immune components to facilitate a favorable disease course. This pilot study aimed to explore the characteristics of EVs released during COVID-19 infection occurring during the third trimester of pregnancy. Methods: In this prospective study, blood samples were obtained from 16 healthy non-pregnant (NP), 18 healthy-pregnant (HP), and 22 COVID-19 positive pregnant subjects (CoV-P). Disease course and pregnancy outcomes were assessed and EVs were characterized. Of note, limited volumes of sample acquired from the subjects made it necessary to use smaller and different subsets of samples for each analysis. Results: The majority (91%) of the COVID-19-pregnant subjects (18 mild and 2 moderate disease) experienced good pregnancy-related outcomes. EV concentrations were higher in healthy-pregnant subjects compared to non-pregnant subjects (p = 0.0041) and lower in COVID-19-pregnant subjects compared to healthy-pregnant subjects (p = 0.0150). CD63 exosome marker expression was higher in EVs of healthy-pregnant subjects and COVID-19-pregnant subjects compared to EVs of non-pregnant subjects (p = 0.0149, p = 0.0028, respectively). Similar levels of SARS-CoV-2 entry proteins (ACE-2 and TMPRSS2) were found in all three groups. Cytokine content increased in healthy-pregnant subject-EVs compared to non-pregnant EVs, while IL-2 and IL-6 levels were decreased in COVID-19-pregnant subject-EVs compared to healthy-pregnant subject-EVs (p = 0.043, p = 0.0390, respectively). CD8+, cytotoxic T-cell marker, was lower in non-pregnant EVs compared to healthy-pregnant subject-EVs and to COVID-19-pregnant subjects (p = 0.0108, p < 0.0001, respectively). COVID-19- pregnant subject-EVs demonstrated higher levels of platelet activation marker (CD62P) than non-pregnant (p = 0.0327) and healthy-pregnant subjects (p = 0.0365). Endothelial marker EV-CD144+ was lower in healthy-pregnant subjects versus non-pregnant subjects (p = 0.0093), but similar in COVID-19-pregnant and non-pregnant subjects. Other EVs' coagulation markers/activity, D-Dimer and fibrinogen levels were similar in healthy-pregnant subjects and COVID-19 positive pregnant subjects. Conclusion: COVID-19 positive pregnant subjects' EVs demonstrated an attenuated inflammatory response, with no additional activation of the coagulation system.

Mapping Pregnancy-dependent Sulfhydrome Unfolds Diverse Functions of Protein Sulfhydration in Human Uterine Artery.

Uterine artery (UA) hydrogen sulfide (H2S) production is augmented in pregnancy and, on stimulation by systemic/local vasodilators, contributes to pregnancy-dependent uterine vasodilation; however, how H2S exploits this role is largely unknown. S-sulfhydration converts free thiols to persulfides at reactive cysteine(s) on targeted proteins to affect the entire proteome posttranslationally, representing the main route for H2S to elicit its function. Here, we used Tag-Switch to quantify changes in sulfhydrated (SSH-) proteins (ie, sulfhydrome) in H2S-treated nonpregnant and pregnant human UA. We further used the low-pH quantitative thiol reactivity profiling platform by which paired sulfhydromes were subjected to liquid chromatography tandem mass spectrometry-based peptide sequencing to generate site (cysteine)-specific pregnancy-dependent H2S-responsive human UA sulfhydrome. Total levels of sulfhydrated proteins were significantly greater in pregnant vs nonpregnant human UA and further stimulated by treatment with sodium hydrosulfide. We identified a total of 360 and 1671 SSH-peptides from 480 and 1186 SSH-proteins in untreated and sodium hydrosulfide-treated human UA, respectively. Bioinformatics analyses identified pregnancy-dependent H2S-responsive human UA SSH peptides/proteins, which were categorized to various molecular functions, pathways, and biological processes, especially vascular smooth muscle contraction/relaxation. Pregnancy-dependent changes in these proteins were rectified by immunoblotting of the Tag-Switch labeled SSH proteins. Low-pH quantitative thiol reactivity profiling failed to identify low abundance SSH proteins such as KATP channels in human UA; however, immunoblotting of Tag-Switch-labeled SSH proteins identified pregnancy-dependent upregulation of SSH-KATP channels without altering their total proteins. Thus, comprehensive analyses of human UA sulfhydromes influenced by endogenous and exogenous H2S inform novel roles of protein sulfhydration in uterine hemodynamics regulation.