Nonpregnant Research Articles

Calcium signaling cascades differentially regulate PGF2α-induced myometrial contractions in water buffaloes (Bubalus bubalis).

This study unravels the differential involvement of calcium signaling pathway(s) in PGF2α-induced contractions in myometrium of nonpregnant (NP) and pregnant buffaloes. Compared to the myometrium of pregnant animals, myometrium of NP buffaloes was more sensitive to PGF2α as manifested by changes in mean integral tension (MIT) and tonicity. In the presence of nifedipine, myometrial contraction to PGF2α was significantly attenuated in both NP and pregnant uteri; however, mibefradil and NNC 55-0396 produced inhibitory effects only in uterus of pregnant animals, thus suggesting the role of extracellular Ca2+ influx through nifedipine-sensitive L-type Ca2+-channels both in NP and pregnant, but T-type Ca2+ channels seem to play a role only during pregnancy. Entry of extracellular Ca2+ is triggered by enhanced functional involvement of Pyr3-sensitive TRPC3 channels and Rho-kinase pathways as evidenced by a significant rightward shift of the concentration-response curve of PGF2α in the presence of Pyr3 and Y-27632 in NP myometrium. But significant down-expressions of TRPC3 and Rho-A proteins during pregnancy apparently facilitate uterine quiescence. In the presence of Ca2+-free solution and cyclopiazonic acid (SERCA blocker), feeble contraction to PGF2α was observed in both NP and pregnant myometrium which suggests minor role of intracellular source of Ca2+ in mediating PGF2α-induced contractions in these tissues.

Anisotropic Mechanical Properties of the Human Uterus Measured by Spherical Indentation.

The mechanical function of the uterus is critical for a successful pregnancy. During gestation, uterine tissue grows and stretches to many times its size to accommodate the growing fetus, and it is hypothesized the magnitude of uterine tissue stretch triggers the onset of contractions. To establish rigorous mechanical testing protocols for the human uterus in hopes of predicting tissue stretch during pregnancy, this study measures the anisotropic mechanical properties of the human uterus using optical coherence tomography (OCT), instrumented spherical indentation, and video extensometry. In this work, we perform spherical indentation and digital image correlation to obtain the tissue's force and deformation response to a ramp-hold loading regimen. We translate previously reported fiber architecture, measured via optical coherence tomography, into a constitutive fiber composite material model to describe the equilibrium material behavior during indentation. We use an inverse finite element method integrated with a genetic algorithm (GA) to fit the material model to our experimental data. We report the mechanical properties of human uterine specimens taken across different anatomical locations and layers from one non-pregnant (NP) and one pregnant (PG) patient; both patients had pathological uterine tissue. Compared to NP uterine tissue, PG tissue has a more dispersed fiber distribution and equivalent stiffness material parameters. In both PG and NP uterine tissue, the mechanical properties differ significantly between anatomical locations.

Differential sperm proteomic profiles according to pregnancy achievement in intracytoplasmic sperm injection cycles: a pilot study.

To describe the proteomic profiles in semen samples and define the differences in sperm proteomic profiles among samples that ultimately achieved pregnancy (P) via intracytoplasmic sperm injection (ICSI) in an oocyte donation program and those that were unsuccessful (NP). Prospective, analytical, observational nested case and control study evaluating the proteomic profile of spermatozoa from patients' ejaculates where pregnancies were (group pregnant (P), n= 4) or were not (group non-pregnant (NP), n=4) achieved after ICSI in an oocyte donation program aiming to standardize female factor. Proteins were separated and analyzed by means of SWATH-MS) and compared between P/NP groups to identify sperm biomarkers of fertility/infertility. Proteins are available via ProteomeXchange. We identified and quantified 2228 proteins, with 37 significantly higher in the P group and 16 higher in NP. Enrichment analysis revealed that the increased proteins in P group sperm were related to motility, anaerobic metabolism, and protein biosynthesis functions, while the increased proteins in the NP group were involved in protein biosynthesis, protein folding, aerobic metabolism, and signal transduction, all of which are functions not previously described as influencing sperm success. Some proteins identified (e.g., SLC2A3, or CD81) are located in the cell membrane and thus may be employed to select spermatozoa by magnetic-activated cell sorting (MACS). This work revealed differences in the proteomic profiles of sperm samples successful in achieving pregnancy and those that were not, expanding our understanding of sperm function and infertility-related molecular markers, and enabling the future development of male fertility diagnostic tools and therapies.

Identification of differentially expressed miRNAs in serum extracellular vesicles (EVs) of Kazakh sheep at early pregnancy.

MiRNAs-containing extracellular vesicles (EVs) possess the unique function of mediating intercellular communication and participating in many biological processes such as post-transcriptional gene regulation of embryo implantation and placental development. In the present study, Illumina small-RNA sequencing was used to identify differentially expressed (DE) miRNAs in serum EVs of pregnant (P) and non-pregnant (NP) Kazakh sheep at Day 17 from mating. The specifically and differentially expressed miRNAs at early pregnancy in sheep were verified by using RT-PCR. The target genes of DE miRNAs were predicted by bioinformatics software, and the functional and pathway enrichment analysis was performed on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) terms. A total of 562 miRNAs (210 novel miRNAs) were identified by sequencing, of which 57 miRNAs were differentially expressed, 49 were up-regulated, 8 were down-regulated and 22 novel miRNAs were specifically expressed in the pregnant sheep. Eight highly expressed known miRNA (miR-378-3p, miR-320-3p, miR-22-3p, let-7b, miR-423-3p, miR-221, miR-296-3p, miR-147-3p) in pregnant group were down-regulated in the control group. miRNAs-containing pregnancy-related terms and regulatory pathways regulation were enriched using both GO and KEGG analyses. Moreover, we also envisioned a miRNA-mRNA interaction network to understand the function of miRNAs involved in the early pregnancy serum regulatory network. The results of RT-PCR verification confirmed the reliability of small-RNA sequencing. Among them, miR-22-3p and miR-378-3p were significantly differentially expressed (DE) between pregnant sheep and non-pregnant group (p< 0.01). The site at which oar-miR-22-3p binds MAPK3 was determined with a dual-luciferase system. This is the first integrated analysis of the expression profiles of EV-miRNAs and their targets during early pregnancy in ewes. These data identify key miRNAs that influence the implantation of sheep in the early stage of pregnancy, and provide theoretical basis for further molecular regulatory mechanisms research.

Sodium acetate ameliorated systemic and renal oxidative stress in high-fructose insulin-resistant pregnant Wistar rats.

Pregnancy is an insulin-resistant condition especially at near term predisposing maternal kidneys to hyperinsulinemia-induced oxidative stress. The impact of fructose on renal metabolic dysregulation and oxidative stress in pregnancy requires elucidation. Short-chain fatty acids (SCFAs) are known for protective roles in oxidative stress conditions. Therefore, the study aimed at investigating fructose-induced glucose dysregulation and renal oxidative stress in pregnant and non-pregnant rats and the possible preventive role of SCFA, acetate. Thirty female Wistar rats were grouped (n = 5/group). Three groups were made pregnant (P); the other three remained non-pregnant (NP). Both pregnant and non-pregnant rats received drinking water (control), 10% fructose (w/v) (NP+F or P+F), and 10% (w/v) fructose plus sodium acetate (200 mg/kg) (NP+F+A or P+F+A) for 3 weeks. Renal and plasma glutathione antioxidant index (GSH/GSSG), G6PDH, and adenosine were significantly lower in NP+F and P+F groups compared with control while renal and plasma adenosine deaminase (ADA), xanthine oxidase (XO), uric acid (UA), lactate dehydrogenase (LDH), and malonaldehyde (MDA) were significantly elevated in NP+F and P+F groups compared with controls. HOMA-IR showed marked impairment in both NP+F and P+F groups. The P+F group revealed greater suppression in plasma and renal G6PDH-dependent antioxidant index, adenosine, and aggravation of LDH, MDA compared with the NP+F group (p < 0.05). Sodium acetate reduces plasma and renal surrogate oxidative stress markers, improved G6PD-dependent antioxidant index, and HOMA-IR in NP+F and P+F groups. Pregnancy exacerbates fructose-induced insulin resistance and renal oxidative stress whereas acetate ameliorated fructose-induced redox and glucose dysregulation in pregnant and non-pregnant rats.

Fecal Progestins Differentiate Pregnant From Nonpregnant Early Luteal Bitches

Considering the high perimplantation progesterone (P4) secretion that occurs in female domestic dogs, we hypothesize that fecal P4 concentrations distinguishes pregnant from nonpregnant early luteal bitches. The objective of this study was to compare fecal P4 metabolites in early pregnant vs. nonpregnant early luteal bitches. Eighteen 2-6 years of age, 5-15 kg, pure-bred bitches, that were followed up for breeding management, and 2 ovariectomized (OVX) bitches were included in this study. On pregnancy day 18-28, a single fecal sample was collected from each animal. Fecal samples were frozen at −20°C until analysis. Pregnancy diagnosis was carried out by ultrasonography on day 21. Fecal samples were also collected from the 2 OVX bitches. Fecal P4 metabolites were extracted as based on methods described by Brown et al. (2008) and, then, measured by an electrochemiluminescence immunoassay. Eleven bitches were pregnant (PREG) while 7 were non-pregnant (N-PR). Fecal P4 metabolites concentrations were 283.9 ± 60.4 and 102.7 ± 19.1 (ng/g; P < .05) for PREG and N-PR, respectively. Both values were higher than those of the OVX bitches (9.9±1.1; P < .05). This noninvasive technique to measure P4 metabolites in feces appears as a simple and noncostly alternative for early pregnancy diagnosis in the domestic dog. Further large scale studies are necessary before this test could be widely recommended.

34 Long term effects of pregnancy on cardiovascular function: serial echocardiograms in non-hypertensive and hypertensive mice

The specific impact of pregnancy on long term cardiovascular function (CVF) is challenging to study due to multiple confounders. A mouse model allows us to study the effect of pregnancy while controlling other factors. Our aim was to evaluate CVF in pregnancy and up to the equivalent of 1 year postpartum (PP) in non-hypertensive and hypertensive murine models compared to non-pregnant (NP) controls. Wild type (WT) and endothelial nitric oxide synthase (eNOS) heterozygous females were used to generate the non-hypertensive and moderately hypertensive models, respectively. Mice were divided between pregnant/PP and NP groups. Mice underwent echocardiography (echo) at age-matched time points: 0 = baseline (10 weeks of age), 1 = gestational day 18.5, 2 = 1 week PP or 4 months of life, 3 = 2 months PP or 6 months of life. After assessing mouse weights, echos were performed under anesthesia using the Vevo system. Left ventricle (LV) parameters were measured three times and averaged: ejection fraction (EF), fractional shortening (FS), cardiac output (CO), stroke volume (SV), LV mass index (LVMI), and ascending aortic (AA) diameter and velocity. Two-way and RM ANOVA were used for statistical analysis, followed by Holm-Sidak post hoc test. 121 murine echos were performed. In WT mice, pregnancy significantly increased LVMI, a measure of LV hypertrophy (Fig. 1A). Pregnancy had no effect on LVMI in the hypertensive eNOS mice (Fig. 1B). EF and FS was not different between the pregnant and NP groups in both WT and eNOS animals. In WT mice, CO, SV, and AA diameter and velocity were persistently increased PP compared to the NP; while in the eNOS mice, they were increased only in the initial PP period (Table 1). Mouse weights were not different among groups. In these animal models, pregnancy persistently adversely impacts CVF in non-hypertensive mice. However, only vascular function is altered after pregnancy in the setting of preexisting hypertension (eNOS). A longer study period is needed to determine if these findings contribute to early-onset of maternal cardiovascular disease.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

456 The involvement of placental and extracellular vesicles micro-RNA in the pathophysiology of gestational vascular complications

Gestational vascular complications (GVC) include gestational hypertension and preeclampsia are leading cause of maternal morbidity and mortality. Extracellular vesicles (EV) are small, membrane-bound vesicles, budding from different cells and released to body fluids, containing various particles as microRNA (figure 1). Elevated levels of EV in GVC have been linked with vascular injury. MicroRNAs (miRNAs), are involved in various disorders, including GVC. We studied the role of placental and circulating EVs-miRNA in GVC and the effect of HP EVs versus GVC EVs on endothelial and trophoblast cells. Blood samples obtained from healthy non-pregnant (NP), healthy pregnant (HP) and GVC women. Plasma EVs' size and concentration were measured by Nanoparticles Tracking Analysis (NTA) and miRNA-EVs were screened. The effects of study cohort EVs on miRNA expression of early-stage trophoblasts and endothelial cells culture were evaluated. From 800 miRNAs screened, 104 significantly expressed in pregnant women, with higher concentrations contained in EVs. Alterations in expression of three miRNAs (miR-16-5p, miR-210 and miR-29b-3p), involved in gene regulations related to PI3K-Akt signaling pathway were detected. Expression of miR-16, miR-185 and miR-210 were low in EVs obtained from NP, elevated in HP-EVs and significantly decreased in GVC. Except for miR-29, which was upregulated in GVC, no changes were found in the levels of the other miRNAs in placenta sections of HP or GVC (figure 2). Additionally, exposure of villous trophoblasts to GVC-EVs significantly increased miR-29 in the treated cells, compared to HP-EVs but did not affects endothelial cells. miRNAs in EVs, play significant role in pathogenesis of GVC, with miR-16 and miR-210 are maternal factors, whereas miR-29 placental. This may distinguish between placental versus maternal pathology related to preeclampsia.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Postpartum perineal muscle sonogram in Madura beef cow.

Background and Aim:Ultrasonography (USG) is useful for non-invasively identifying changes that occur in soft tissue architecture. The objective of this research was to demonstrate postpartum (PP) uterine involution through the changes of perineal muscle intensity and thickness in Madura beef cow by ultrasonography.Materials and Methods:Madura’s breed cows used in the research consist of eight non-pregnant (NP) cows and three PP cow. The transrectal and transperineal USG imaging of NP cows was performed on days 1, 33, and 65. USG imaging of PP cows was performed every day starting from day 1 (24 h after parturition) until day 21 PP. Transrectal USG of the reproductive tract was performed for the cervix, corpus uteri, and cornua uteri. USG was performed transcutaneously over the perineal area for coccygeus and levator ani muscles at the longitudinal and transverse angles. Reproductive tract diameter and perineal muscle intensity and thickness were measured with ultrasound imaging.Results:The analysis of the sonogram of PP cows showed that the diameter of the cervix, corpus uteri, and cornua uteri decreased within 21 days PP. The transverse view of the coccygeus muscle of PP cows showed decreased muscle intensity and thickness. On the other hand, the longitudinal view showed increased coccygeus muscle intensity and thickness. The transverse view of the coccygeus muscle of NP cows showed increased muscle intensity, while muscle thickness was reduced. Sonogram analysis of the levator ani muscle of PP cows showed decreased muscle intensity with increasing muscle thickness. However, imaging of the levator ani muscle of NP cows showed a decrease in both intensity and muscle thickness. There was a significant difference in the mean value intensity of the scanning view analysis results of the levator ani muscle of the PP cow (523.6 AU increased to 672.1 AU) and the NP cow (515.9 AU decreased to 465.4 AU). Furthermore, there was a significant difference (p<0.05) in the mean value thickness of both scanning view analyses of the coccygeus and levator ani muscles of PP cows (5.8 mm increased to 6.5 mm and 3.8 mm increased to 4.8 mm, respectively) and NP cows (8.8 mm increased to 9.1 mm and 5.9 mm decreased to 4.9 mm, respectively).Conclusion:We found that the perineal muscles, namely, the levator ani muscle and coccygeus muscle, change in size, and intensity during uterine involution as demonstrated on Madura beef cow.

New Insights Into the Role of β-NGF/TrKA System in the Endometrium of Alpacas During Early Pregnancy.

One striking reproductive feature in South American camelids is that more than 90% of gestations are established in the left uterine horn (LUH). This phenomenon could be related to a differential vascular irrigation of the LUH. An increase of vascularization in llama endometrium was observed after systemic administration of Beta Nerve Growth Factor (β-NGF), a neurotrophin present in the uterus and placenta of various mammals that is involved in pregnancy development. We hypothesized that the β-NGF signaling pathway is related to embryo implantation in the LUH in camelids. The aim of this study was to characterize the spatial expression of β-NGF and its high-affinity receptor, TrKA, between LUH and right uterine horn (RUH) of non-pregnant (NP) and early pregnant alpacas (15 and 30 days of gestation, 15 and 30P, respectively). In addition, β-NGF, TrKA, and Vascular Endothelium Growth Factor A (VEGFA) temporal gene expression patterns and counting of blood vessels were evaluated among groups. The β-NGF and TrKA were localized in the luminal, glandular, and vascular epithelium of the alpaca uterus and in the embryonic membranes of the 30-days-old conceptus. β-NGF and TrKA immunosignal were stronger in 15P females than that of NP and 30P. In addition, TrKA signal was higher in the LUH luminal epithelium of NP and 15P alpacas than that of NP-RUH and 15P-RUH. β-NGF mRNA relative abundance was higher in the 30P-RUH than that of NP-RUH; whereas TrKA mRNA abundance only differed between 15P-RUH and NP-LUH. VEGFA mRNA relative abundance was higher in NP females compared to the LUH of 15P and 30P alpacas, and lower to their right counterparts. The number of vessels per field was higher in 15P than that of 30P. A positive correlation was observed between the number of vessels per field and β-NGF immunosignal in 15P-LUH. In contrast, the area occupied by vessels was higher in 30P alpacas than of NP and 15P females. The changes of β-NGF/TrKA expression pattern in the peri-implantation endometria between LUH and RUH and their localization in the extraembryonic membranes support the implication of the neurotrophin during implantation and pregnancy development in South American Camelids.

Follicular dynamics during the pre-ovulatory and subsequent first follicular wave stages affect the pregnancy outcome in Japanese Black cows.

The diameters of the pre-ovulatory follicles (PF) and the largest follicle during the subsequent first follicular wave (W1LF), and plasma estradiol-17β (E2) concentrations were monitored on Days 0, 1, 3, 5, and 7 (ovulation = Day 1). Pregnancy was diagnosed on Day 30. Cows were classified into two groups according to the location of the dominant follicle ipsilateral (IG) or contralateral (CG) to the corpus luteum on Day 7. From Days 3 to 7, some follicles that had been determined as the subordinate in the previous examination exceeded the W1LF located in the opposite ovary in terms of the diameter. These were defined as switching (SW), whereas others were defined as non-switching (NSW). The diameter of PF was significantly smaller in pregnant (P) animals than in non-pregnant (NP) animals. The plasma E2 concentration on Day 0 was significantly higher in P animals than in NP animals and tended to be higher in NSW than in SW. In addition, plasma E2 concentrations around Days 3 to 7 tended to be higher in P animals of NSW than in NP animals of SW. The conception rates did not differ between IG and CG but were significantly higher in NSW than in SW. In the IG group, the conception rate tended to be higher in NSW than in SW.

Pregnancy decreases O-GlcNAc-modified proteins in systemic arteries of normotensive and spontaneously hypertensive rats

AimWe determined the role played by O-linked N-acetylglucosamine (O-GlcNAc) of proteins in systemic arteries during late pregnancy in normotensive and hypertensive rats. Main methodsO-GlcNAc levels and O-GlcNAc modification of endothelial nitric oxide synthase (eNOS) were determined in aorta (conductance vessel) and mesenteric arteries (resistance vessels) of non-pregnant (NP) and pregnant (P) Wistar rats and spontaneously hypertensive rats (SHR). Vascular O-GlcNAc-modified proteins, O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT) expression, and OGA activity were analyzed. Concentration–response to phenylephrine (PE) curves were constructed for arteries with and without endothelium. Arteries were treated with vehicle or PugNAc (OGA inhibitor, 100 μmol/L) in the presence of L-NAME (NOS inhibitor, 100 μmol/L). Key findingsThe content of vascular O-GlcNAc-modified proteins was lower, OGT and OGA expression did not change, and OGA activity was higher in arteries of P-Wistar rats and P-SHR compared to arteries of NP-groups. Reactivity to PE increased in arteries of P-Wistar rats treated with PugNAc compared to vehicle. O-GlcNAcylation of eNOS decreased in P-SHR compared to NP-SHR. PugNAc partially inhibited the effects of endothelium removal and L-NAME on reactivity to PE in arteries of P-Wistar rats. However, PugNAc did not alter reactivity to PE in arteries of P-SHR. Our data showed that pregnancy decreased the content of vascular O-GlcNAc-modified proteins. SignificanceIncreased OGA activity and decreased O-GlcNAc modification of eNOS boosts eNOS activity in arteries of P-Wistar rats. In P-SHR, altered OGA activity may lower the content of O-GlcNAc-modified proteins, but decreased OGT activity seems a potential mechanism to reduce glycosylation.

46 Endometrial transcriptome profiles associated with bovine preimplantation pregnancy outcome and parity

Abstract Pregnancy failure in dairy cattle appears to be attributable to losses during embryogenesis, mainly within the first month. Models have estimated that pregnancies lost after day 30 of fertilization cost producers US $550/cow. The reasons for these losses are not well understood, and the transcriptome profile of the reproductive tract during the establishment of pregnancy may point to mechanisms. Thirty-eight cows in lactations 1 to 3 were artificially inseminated, and endometrial biopsies were collected on day 15 after artificial insemination to investigate the effect of parity on the preimplantation pregnancy outcome. Pregnancy status was determined by the presence of interferon-tau in the uterine flushing, revealing 19 pregnant (P) and 19 non-pregnant (NP) cows. Nineteen biopsy samples [9 cows in lactation 1 (6 P and 3 NP), 4 in lactation 2 (2 P and 2 NP), and 6 in lactation 3 (3P and 3NP)] with an average RNA integrity number of 7 were selected for RNA sequencing with an Illumina HiSeq analyzer. Sequence reads were assembled to the ARS_USD1.2.99 bovine reference genome using the CLC genomics workbench software. On average, the samples generated ~56 million reads and 94.99% were mapped to the reference genome. Differential gene expression analysis between P and NP cows identified 187, 60, and 136 differentially expressed genes (DEG) in lactation 1, 2 and 3, respectively (P &amp;lt; 0.01, FDR &amp;lt; 0.05, FC &amp;gt; ±2). Metabolic pathway enrichment analysis identified several DEG upregulated in lactations 2 and 3 associated with IL-17 signaling pathway, although expression of IL-17 itself was not different. This pathway is part of the protective response against extracellular bacteria and likely participates in pregnancy maintenance through local regulation of immune function. Further functional genomic analyses will be performed to determine additional metabolic pathways, key regulator genes, and functional SNPs associated with the establishment of pregnancy.

Enhanced Stromal Cell CBS-H2S Production Promotes Estrogen-Stimulated Human Endometrial Angiogenesis.

Angiogenesis is a physiological process for endometrial regeneration in the menstrual cycle and remodeling during pregnancy. Endogenous hydrogen sulfide (H2S), produced by cystathionine-β synthase (CBS) and cystathionine-γ lyase (CSE), is a potent proangiogenic factor; yet, whether the H2S system is expressed in the endometrium and whether H2S plays a role in endometrial angiogenesis are unknown. This study was to test whether estrogens stimulate endometrial H2S biosynthesis to promote endometrial microvascular endothelial cell (EMEC) angiogenesis. CBS messenger RNA/protein and H2S production significantly differed among endometria from postmenopausal (POM), premenopausal secretory (sPRM), and proliferative (pPRM) nonpregnant (NP) and pregnant (Preg) women (P < .05) in a rank order of POM approximately equal to sPRM is less than pPRM is less than Preg, positively correlating with angiogenesis indices and endogenous estrogens and with no difference in CSE expression. CBS and CSE proteins were localized to stroma, glands, and vessels in endometrium, and greater stromal CBS protein was observed in the pPRM and Preg states. Estradiol-17β (E2) (but not progesterone) stimulated CBS (but not CSE) expression and H2S production in pPRM endometrial stromal cells (ESCs) in vitro, which were attenuated by ICI 182 780. The H2S donor sodium hydrosulfide promoted in vitro EMEC angiogenesis. Co-culture with sPRM, pPRM, and Preg ESCs all stimulated EMEC migration with a rank order of sPRM less than pPRM approximately equal to Preg. CBS (but not CSE) inhibition attenuated ESC-stimulated EMEC migration. E2 did not affect EMEC migration but potentiated ESC-stimulated EMEC migration. Altogether, estrogens stimulate specific receptor-dependent stromal CBS-H2S production to promote endometrial EMEC angiogenesis in women.

Abstract P220: Differential Cardiac Geometry During Pregnancy In Lean Versus Obese Mice

Objectives: Women with a history of pregnancy complications are at increased risk for subsequent cardiovascular disease (CVD), but mechanisms linking these conditions are unclear. Obesity is associated with pregnancy complications, and is an independent risk factor for CVD. We hypothesized that obesity during pregnancy in mice would promote impaired cardiac function and/or adverse cardiac remodeling. Methods/Results: Female C57BL/6J mice (8 weeks old) were fed a high fat (HF; 60% kcal from fat) or a control low fat (LF; 10% kcal from fat) diet for 8 weeks, then were either crossed with male mice to become pregnant (P) or remained non-pregnant (NP) controls. At gestational day 18, cardiac function was quantified by echocardiography in LF- and HF-fed P and NP females. On gestational 19 day, mice were euthanized for tissue collection. HF-fed females had significantly increased body weight compared to LF-fed controls (P&lt;0.001), and body weight was increased in P compared to NP mice (P&lt;0.001; LF NP: 22.3 + 0.8 [n=20], LF P: 34.8 + 1.3 [n=10], HF NP: 32.7 + 0.9 [n=18], HF P: 39.2 + 1.1 [n=12] g). In response to pregnancy, LF-, but not HF-fed, mice had significantly increased LV mass (LF NP: 60.0 + 2.4, LF P: 75.5 + 3.1, HF NP: 70.3 + 2.0, HF P: 75.1 + 3.8 mg; P&lt;0.01) and LV end-diastolic diameter (LVED; LF NP: 3.60 + 0.05, LF P: 3.95 + 0.07, HF NP: 3.67 + 0.05, HF P: 3.73 + 0.01 mm; P&lt;0.01) compared to NP controls. In contrast, HF-fed pregnant mice had smaller LVED (P&lt;0.01) and increased relative wall thickness (RWT; LF NP: 0.36 + 0.01, LF P: 0.35 + 0.01, HF NP: 0.39 + 0.01, HF P: 0.40 + 0.02 [2* LV posterior wall thickness/LV end-diastolic diameter], P&lt;0.05) compared to LF-fed pregnant mice. Preliminary gene expression analysis indicate mRNA abundance of TGFb1 is elevated by 50% (P&lt;0.05) hearts of HF-fed compared to LF-fed pregnant mice. Conclusions: These data suggest obesity during pregnancy promotes concentric remodeling, versus eccentric remodeling in lean mice. These data are clinically meaningful, as they suggest that obesity during pregnancy may promote pathological cardiac remodeling, which may contribute to subsequent maternal risk for cardiovascular disease.

Early pregnancy-induced transcripts in peripheral blood immune cells in Bos indicus heifers

Immune cells play a central role in early pregnancy establishment in cattle. We aimed to: (1) discover novel early-pregnancy-induced genes in peripheral blood mononuclear cells (PBMC); and (2) characterize the temporal pattern of early-pregnancy-induced transcription of select genes in PBMC and peripheral blood polymorphonuclear cells (PMN). Beef heifers were artificially inseminated on D0 and pregnancies were diagnosed on D28. On D10, 14, 16, 18, and 20, blood was collected for isolation of PBMC and PMN from heifers that were retrospectively classified as pregnant (P) or non-pregnant (NP). PBMC samples from D18 were submitted to RNAseq and 220 genes were differentially expressed between pregnant (P) and non-pregnant (NP) heifers. The temporal abundance of 20 transcripts was compared between P and NP, both in PBMC and PMN. In PBMC, pregnancy stimulated transcription of IFI6, RSAD2, IFI44, IFITM2, CLEC3B, OAS2, TNFSF13B, DMKN and LGALS3BP as early as D18. Expression of IFI44, RSAD2, OAS2, LGALS3BP, IFI6 and C1R in PMN was stimulated in the P group from D18. The novel early-pregnancy induced genes discovered in beef heifers will allow both the understanding of the role of immune cells during the pre-attachment period and the development of technologies to detect early pregnancies in beef cattle.

Dexamethasone increases renal free fatty acids and xanthine oxidase activity in female rats: could there be any gestational impact?

Dexamethasone (DEX) is used for various conditions in female and even during pregnancy. We tested the hypothesis that DEX exposure in female rats would lead to renal free fatty acid (FFA) accumulation with elevated xanthine oxidase (XO) activity that would be aggravated by pregnancy. Twenty-four female rats (n = 6/group) were randomly assigned to non-pregnant (NPR), DEX-exposed non-pregnant (NPR + DEX), pregnant (PRE) and DEX-exposed pregnant (PRE + DEX), respectively. NPR and PRE rats received vehicle (po) while NPR + DEX and PRE + DEX groups received DEX (0.2 mg/kg; po), between gestational days 14 and 19. Data showed that DEX exposure caused increased plasma creatinine, urea, renal FFA accumulation, lipid peroxidation, aminotranferases, depressed glutathione, increased activity of XO, and elevated uric acid in both pregnant and non-pregnant rats. The findings of this study indicate that DEX exposure would cause renal FFA accumulation and glutathione depletion that are accompanied by increased activity of XO/uric acid independently of gestation. The study also implies that DEX-induced renal damage could be worsened by gestation.

ST2-expressing B1 B cells acquire an anti-inflammatory capacity during pregnancy in mice

Abstract In the context of pregnancy, it is known that IL-33 induces the production of anti-inflammatory molecules by decidual B1 cells, protecting against preterm birth (PTB) in human and mouse. We have previously showed that expression of IL-33 receptor, Il1rl1 (ST2) is significantly upregulated in splenic B cells during mid pregnancy, predominantly in B1 cell subset. Furthermore, using an LPS induced PTB mouse model, we observed increased numbers of splenic and decidual ST2-expressing B1 cells in the acute phase of PTB as compared to term pregnant females. We aimed to investigate here the anti-inflammatory properties of ST2-expressing B1 cells during pregnancy. Total splenocytes from pregnant (P) and non-pregnant (NP) mice were isolated and cultured for 24h with/without LPS (10 μgr/ml), ST2 expression and cytokine production by ST2+ B1 cells was evaluated by flow cytometry. B1 cells from P mice stimulated with LPS showed significantly higher levels of ST2 expression. ST2+ B1 cells produced significantly higher levels of IL-10 and significantly lower levels of TNF-α and IL-17 as compared to ST2+ B1 cells from NP mice. In a separate project in our laboratory we demonstrated that prophylactic treatment with probiotic Lactobacillus kefiri prevented LPS-induced PTB in mice. Interestingly, we observed that numbers of splenic and decidual ST2-expressing B1 cells were increased in L. kefiri treated mice that were protected against LPS-induced PTB. Our data strongly suggest an anti-inflammatory capacity of ST2-expressing B1 cells during gestation, presumably to ensure pregnancy wellbeing and prevent inflammation induced PTB.

O-GlcNAc impairs endothelial function in uterine arteries from virgin but not pregnant rats: The role of GSK3β

Increased O-Linked β-N-acetylglucosamine (O-GlcNAc) is observed in several pathologies, and unbalanced O-GlcNAcylation levels favor endothelial dysfunction. Whether augmented O-GlcNAc impacts the uterine artery (UA) function and how it affects the UA during pregnancy remains to be elucidated. We hypothesized that glucosamine treatment increases O-GlcNAc, leading to uterine artery dysfunction and this effect is prevented by pregnancy. Pregnant (P) and non-pregnant (NP) Wistar rats were treated with glucosamine (300 mg/kg; i.p.) for 21 days. Concentration response-curves (CRC) to acetylcholine (in the presence or absence of L-NAME) and sodium nitroprusside were performed in UAs. In NP rats, glucosamine treatment increased O-GlcNAc expression in UAs accompanied by decreased endothelium-dependent relaxation, which was abolished by L-NAME. Endothelial nitric oxide synthase (eNOS) activity and total Akt expression were decreased by glucosamine-treatment in NP rats.Further, NP rats treated with glucosamine displayed increased glycogen synthase kinase 3 beta (GSK3β) activation and O-GlcNAc-transferase (OGT) expression in the UA. P rats treated with glucosamine displayed decreased O-GlcNAc in UAs and it was accompanied by improved relaxation to acetylcholine, whereas eNOS and GSK3β activity and total Akt and OGT expression were unchanged. Sodium nitroprusside-induced relaxation was not changed in all groups, indicating that glucosamine treatment led to endothelial dysfunction in NP rats. The underlying mechanism is, at least in part, dependent on Akt/GSK3β/OGT modulation. We speculate that during pregnancy, hormonal alterations play a protective role in preventing O-GlcNAcylation-induced endothelial dysfunction in the UAs.

Development of a novel 21-day reinsemination program, ReBreed21, in Bos indicus heifers

The aim was to develop a program for resynchronization of ovulation (ReBreed21) that allowed reinsemination of non-pregnant Bos indicus heifers every 21 d using timed AI (TAI) without the need for detection of estrus. The Rebreed21 program begins 12 d after previous TAI (Day 0) by inserting an intravaginal P4 implant (Day 12) that is removed 7 d later (Day 19) combined with treatment with 300 IU of eCG. On Day 21, early pregnancy diagnosis (Doppler PD) is performed based on CL vascularity. Non-pregnant (NP) heifers immediately received AI combined with 100 μg of GnRH. The program is replicated 12 d after second TAI to produce a breeding season (BS) of 42 d with 3 potential TAIs. Two experiments were conducted as a proof of concept for this rapid rebreeding program. In Experiment 1, 76 heifers were enrolled in ReBreed21, as explained above. In Experiment 2, 300 Nellore heifers were synchronized for 1st TAI and randomly assigned to one of two groups: ReBreed21 (n = 147) or another early resynchronization procedure, Resynch14 (n = 153) with P4 implant inserted 14 d after previous TAI plus 50 mg of long-acting injectable P4; 8 d later P4 implant removed (Day 22) and early Doppler PD performed; NP heifers received 150 μg of cloprostenol, 0.5 mg of ECP, and 300 IU of eCG with TAI on Day 24. In both experiments, the largest follicle (LF) was measured at each Resynch TAI. Ultrasound was later used to confirm the early Doppler PD and to determine ovulation (OV) to Resynch at 12 d after TAI in ReBreed21 (Day 33 of pregnancy) and 14 d after TAI in Resynch14 (Day 38 of pregnancy). Final PD was performed 40 d after 3rd TAI. Results for Experiment 1 were: diameter of LF 11.8 ± 0.23 mm; 88.9% OV; 20.5% false positives; 38.1% P/AI at 1st TAI; 44.4% overall P/AI for ReBreed21 TAIs; 72.3% total pregnant at end of BS. In experiment 2, Rebreed21 vs. Resynch14 were different for: diameter of LF (10.9 ± 0.17 vs. 10.0 ± 0.17 mm, P = 0.0003), heifers with LF < 8.5 mm (10.2 vs. 26.4%, P = 0.04), or LF ≥ 11 mm (50.0 vs. 37.2%, P = 0.001), and P/AI at first TAI (29.3% [43/147] vs. 20.3% [31/153], P = 0.074) but similar for OV (overall 86.8% [239/275], P = 0.82), false positives (P = 0.52) overall P/AI for Resynch TAIs (33.6 vs. 28.8%, P = 0.4), and total pregnant at end of BS (58.5% [86/147] vs. 55.6% [85/153], P = 0.64). In addition, median time to pregnancy was 9 d earlier (P = 0.0007) for ReBreed21 than Resynch14. Thus, ReBreed21 is a novel protocol that allows earlier reinseminations than Resynch14 but with similar fertility.