Fetoplacental Units Research Articles

Exposure to Extracellular Mitochondrial DNA During Pregnancy Induces a Pro-Inflammatory Response in Rat Placenta

Introduction: Mitochondrial DNA (mtDNA) is released in the cytosol or the extracellular space due to mitochondrial dysfunction or cell death. Extracellular mtDNA is recognized by the innate immune receptor Toll-like receptor 9 (TLR9) and induces pro-inflammatory and immunogenic responses. Circulating cell-free mtDNA (ccf-mtDNA) increases with advancing gestational age and abnormal concentrations have been reported in obstetric complications such as preeclampsia (PE). Placentas from women with PE exhibit signs of extensive inflammation; however, the contribution of ccf-mtDNA to placental inflammation during pregnancy has not been investigated. We hypothesized that in vivo exposure to extracellular mtDNA during pregnancy would induce an inflammatory response in rat placenta via activation of TLR9 signaling. Methods: We isolated mtDNA from liver of Sprague Dawley pregnant rats (gestational day: 14-15, term=22-23 days) and treated gestational age-matched dams with mtDNA (350ng/kgBW) or saline (control) via intravenous injection. Following 4 hours after treatment, maternal organs and fetoplacental units were collected and gene expression of cytokines and TLR9 were measured. Results: Plasma levels of mtDNA 4 hours after treatment were not different between saline and mtDNA-treated rats (2−ΔCT, median (IQR); saline: 2.75 (1.61) vs. mtDNA: 2.64 (1.29), p>0.05). Treatment with mtDNA increased mRNA expression of pro-inflammatory tumor necrosis factor-alpha (TNF-α) (saline: 0.65 (1.1) vs. mtDNA: 2.84 (1.9), p=0.015), interleukin (IL)-6 (saline: 0.32 (0.2) vs. mtDNA: 3.37 (4.3), p=0.008), and IL-1β (saline: 0.2 (0.93) vs. mtDNA: 4.7 (6.0), p=0.01) and decreased expression of anti-inflammatory IL-10 (saline: 1.6 (0.8) vs. mtDNA: 0.6 (0.8), p=0.001) in rat placentas. Expression of macrophage marker F4/80 (saline: 0.4 (1.0) vs. mtDNA: 2.41 (4.7), p=0.04) and monocyte chemoattractant protein-1 (MCP-1) (saline: 0.68 (0.54) vs. mtDNA: 2.0(1.96), p=0.041) also increased in rat placentas in response to mtDNA treatment. To assess whether this inflammatory response was tissue-specific, we evaluated mRNA expression of inflammatory genes in maternal kidneys. Treatment with mtDNA increased the levels of IL-1β (2−ΔCT, median (IQR); saline: 1.05 (1.1) vs. mtDNA: 10.17 (8.0), p=0.008) and IL-6 (saline: 0.98 (1.76) vs. mtDNA: 6.8 (3.5), p=0.002). mtDNA treatment did not affect kidney TNF-α and IL-10 expression (p>0.05). In contrast, expression of F4/80 (saline: 0.87 (2.2) vs. mtDNA: 12.5 (12.4), p=0.003) and MCP-1 (saline: 0.9 (1.7) vs. mtDNA: 7.4 (8.3), p=0.011) was greater in maternal kidneys from mtDNA-treated rats vs. controls. TLR9 expression increased in placentas (saline: 1.29 (0.5) vs. mtDNA: 4.0 (7.8), p<0.0001) and maternal kidneys (saline: 1.2 (1.8) vs. mtDNA: 4.7 (4.6), p=0.045) in response to mtDNA treatment. Conclusion: Exposure to an in vivo challenge with extracellular mtDNA during pregnancy induces a pro-inflammatory response in placentas and kidneys from pregnant rats. These responses may be mediated by TLR9 signaling activation. NIH R01-HL0146562. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Assessment of feto-placental oxygenation and perfusion in a rat model of placental insufficiency using T2* mapping and 3D dynamic contrast-enhanced MRI

IntroductionPlacental insufficiency may lead to preeclampsia and fetal growth restriction. There is no cure for placental insufficiency, emphasizing the need for monitoring fetal and placenta health. Current monitoring methods are limited, underscoring the necessity for imaging techniques to evaluate fetal-placental perfusion and oxygenation. This study aims to use MRI to evaluate placental oxygenation and perfusion in the reduced uterine perfusion pressure (RUPP) model of placental insufficiency. MethodsPregnant rats were randomized to RUPP (n = 11) or sham surgery (n = 8) on gestational day 14. On gestational day 19, rats imaged using a 7T MRI scanner to assess oxygenation and perfusion using T2* mapping and 3D-DCE MRI sequences, respectively. The effect of the RUPP on the feto-placental units were analyzed from the MRI images. ResultsRUPP surgery led to reduced oxygenation in the labyrinth (24.7 ± 1.8 ms vs. 28.0 ± 2.1 ms, P = 0.002) and junctional zone (7.0 ± 0.9 ms vs. 8.1 ± 1.1 ms, P = 0.04) of the placenta, as indicated by decreased T2* values. However, here were no significant differences in fetal organ oxygenation or placental perfusion between RUPP and sham animals. DiscussionThe reduced placental oxygenation without a corresponding decrease in perfusion suggests an adaptive response to placental ischemia. While acute reduction in placental perfusion may cause placental hypoxia, persistence of this condition could indicate chronic placental insufficiency after ischemic reperfusion injury. Thus, placental oxygenation may be a more reliable biomarker for assessing fetal condition than perfusion in hypertensive disorders of pregnancies including preeclampsia and FGR.

The balance between fetal oxytocin and placental leucine aminopeptidase (P-LAP) controls human uterine contraction around labor onset.

A fetal pituitary hormone, oxytocin which causes uterine contractions, increases throughout gestation, and its increase reaches 10-fold from week 32 afterward. Oxytocin is, on the other hand, degraded by placental leucine aminopeptidase (P-LAP) which exists in both terminal villi and maternal blood. Maternal blood P-LAP increases with advancing gestation under the control of non-genomic effects of progesterone, which is also produced from the placenta. Progesterone is converted to estrogen by CYP17A1 localized in the fetal adrenal gland and placenta at term. The higher oxytocin concentrations in the fetus than in the mother demonstrate not only fetal oxytocin production but also its degradation and/or inhibition of leakage from fetus to mother by P-LAP. Until labor onset, the pregnant uterus is quiescent possibly due to the balance between increasing fetal oxytocin and P-LAP under control of progesterone. A close correlation exists between the feto-placental and maternal units in the placental circulation, although the blood in the two circulations does not necessarily mix. Fetal maturation results in progesterone withdrawal via the CYP17A1 activation accompanied with fetal oxytocin increase. Contribution of fetal oxytocin to labor onset has been acknowledged through the recognition that the effect of fetal oxytocin in the maternal blood is strictly regulated by its degradation by P-LAP under the control of non-genomic effects of progesterone. In all senses, the fetus necessarily takes the initiative in labor onset.

Evaluation of fetoplacental oxygenation in a rat model of gestational diabetes mellitus using BOLD-MRI at 7.0-T

Objective: To compare the differences in blood oxygen level-dependent (BOLD) parameters following maternal hyperoxia between normal pregnancy and pregnancy in the rat model of gestational diabetes mellitus (GDM). Methods: GDM was induced by high-fat and sucrose diet (HFS) combined with an intraperitoneal injection of streptozotocin (STZ). On embryonic day 19 (E19), the two groups of pregnant rats were imaged using a 7.0-T animal MRI scanner. TurboRARE was initially used to localize the fetoplacental units (FPUs). Next, multiple gradient echo BOLD was performed during the air and oxygen inhalation periods. T2* map was then generated, and the baseline T2* and absolute changes in T2* value (ΔT2*, difference between T2*oxy and T2*air) were calculated. Following the MRI scan, the placentas and fetuses were aseptically stripped, weighed, and immunostained. Results: Nine rats were used in this study. After maternal oxygen inhalation, T2* increased significantly in all subjects in both groups. The ΔT2* for the placenta (5.97 vs. 7.81 msec; P = 0.007) and fetal brain (2.23 vs. 3.97 msec; P = 0.005) differed significantly between the GDM and control groups. Histochemical detection of placental glycogen content and inflammatory cytokines (IL-6 and TNF-α) showed significantly higher levels in the GDM than in the normal placenta. Conclusions: BOLD-MRI revealed abnormalities in the fetoplacental response to maternal hyperoxygenation in rats with GDM. We believe that this approach can potentially be used to evaluate placental dysfunction and assess the state of the fetus during pregnancy with GDM.

Nanoparticle Contrast-enhanced MRI for Visualization of Retroplacental Clear Space Disruption in a Mouse Model of Placental Accreta Spectrum (PAS)

Prior preclinical studies established the utility of liposomal nanoparticle blood-pool contrast agents in visualizing the retroplacental clear space (RPCS), a marker of normal placentation, while sparing fetuses from exposure because the agent does not cross the placental barrier. In this work, we characterized RPCS disruption in a mouse model of placenta accreta spectrum (PAS) using these agents. Contrast-enhanced MRI (CE-MRI) and computed tomography (CE-CT) using liposomal nanoparticles bearing gadolinium (liposomal-Gd) and iodine were performed in pregnant Gab3-/- and wild type (WT) mice at day 16 of gestation. CE-MRI was performed on a 1T scanner using a 2D T1-weighted sequence (100×100×600 µm3 voxels) and CE-CT was performed at a higher resolution (70×70×70 µm3 voxels). Animals were euthanized post-imaging and feto-placental units (FPUs) were harvested for histological examination. RPCS conspicuity was scored through blinded assessment of images. Pregnant Gab3-/- mice showed elevated rates of complicated pregnancy. Contrast-enhanced imaging demonstrated frank infiltration of the RPCS of Gab3-/- FPUs. RPCS in Gab3-/- FPUs was smaller in volume, demonstrated a heterogeneous signal profile, and received lower conspicuity scores than WT FPUs. Histology confirmed in vivo findings and demonstrated staining consistent with a thinner RPCS in Gab3-/- FPUs. Imaging of the Gab3-/- mouse model at late gestation with liposomal contrast agents enabled in vivo characterization of morphological differences in the RPCS that could cause the observed pregnancy complications. An MRI-based method for visualizing the RPCS would be valuable for early detection of invasive placentation.

Abnormal growth and morphogenesis of placenta at term is linked to adverse fetal development after perigestational alcohol consumption up to early gestation in mouse.

Gestation alcohol consumption produces fetal growth restriction and malformations by affecting the embryo-fetal development. Recently a relationship between abnormal placentation and fetal malformation and intrauterine growth retardation has been suggested. However, the effects of perigestational alcohol ingestion up to early pregnancy on the placenta at term and its association with fetal abnormalities are little known. In female mice, ethanol 10% in water was administered for 15 days previous and up to days 4 (D4), 8 (D8), or 10 (D10) of gestation (TF), and gestation continues without ethanol exposure. Control females (CF) received ethanol-free water. At day 18, feto-placental units and implantation sites were studied. TF had increased resorptions and only fetuses from D8-TF and D10-TF had significantly increased weights versus CF. D4 and D10-TF-placentas had significantly reduced weights. All TF had increased junctional zone (JZ) and reduced labyrinth (Lab) areas (PAS-histology and morphometry) compared with CF. Fetuses with mainly with craniofacial abnormalities and skeletal defects (Alizarin red staining), significantly increase; while the fetal bone density (alizarin color intensity, ImageJ) was reduced in D4, D8 and D10-TF versus CF. Although all TF-placentas were histo-structural affected, TF-abnormal fetuses had the most severe placental anomalies, with junctional abundant glycogenic cells into the labyrinth, disorganized labyrinthine vascularization with signs of leukocyte infiltrates and feto-maternal blood mix. Perigestational alcohol consumption up to early gestation induces at term fetal growth alterations, dysmorphology and defective skeleton, linked to deficient growth and abnormal morphogenesis of placenta, highlighting insight into the prenatal etiology of FASD.

Artificial Synchronization of Sex Steroid Hormones Normalizes Kisspeptin Expression and Placentation in the Preeclamptic‐Like BPH/5 Mouse

Insufficient invasion of the maternal decidua by conceptus‐derived trophoblast cells is a key event in the pathogenesis of early‐onset preeclampsia (ePE), a subclass of PE associated with high maternal morbidity and fetal intrauterine growth restriction (IUGR). Kisspeptins (KP) are a family of small peptides previously shown to inhibit trophoblast cell invasion. Interestingly, KP expression is higher in the placentae of women with ePE than in those with uncomplicated pregnancies. Nonetheless, the mediators of placental KP upregulation remain largely unknown. The Blood Pressure High Subline 5 (BPH/5) is a mouse model that spontaneously develops the main features of PE, including decreased trophoblast cell invasion, lower decidual placental expansion, reduced placental labyrinth endothelial remodelling and IUGR. We have previously shown that Kiss1is higher at the BPH/5 maternal‐fetal interface during early gestation, at embryonic days (e) 4.5 and 7.5, when compared with control C57 mice. Moreover, BPH/5 females have abnormal sex steroid hormone profile during early gestation, including an earlier and depressed surge of estradiol‐17ß (E2) and higher blood progesterone (P4) at e2.5. Upregulation of uterine Kiss1expression has been previously demonstrated by administration of E2 and P4 to non‐pregnant ovariectomized CD‐1 mice. Therefore, we aimed to investigate the effects of artificial synchronization of sex steroid hormones (AS‐SSH) in BPH/5 uteroplacental Kiss1expression and placental morphology. It was hypothesized that AS‐SSH during early gestation would prevent the BPH/5 Kiss1upregulation at the embryonic implantation sites (eIS) and result in higher decidual placental expansion. AS‐SSH was performed via ovariectomy of pregnant BPH/5 and C57 females (n = 8/strain) at e2.5; administration of a single dose of E2 (25 ng/mouse, subcutaneously) to stimulate embryonic implantation and daily subcutaneous injections of P4 (1mg/mouse) until sample collection. The eIS were collected at e5.5 and Kiss1 expression was assessed via RT‐PCR. Additionally, e12.5 fetoplacental units from BPH/5 and C57 natural (Nat) and AS‐SSH pregnancies were stained with Isolectin, and the placental decidual expansion was assessed using Image J® (n = 4‐6/group). The placental expansion into the maternal decidua was calculated as the ratio of the labyrinth and junctional zone in relation to the entire placental disc (placenta + decidua). Comparisons were done using unpaired T‐tests and ANOVA with post‐hoc Newman‐Keuls tests. Contrary to Nat BPH/5 pregnancies, eIS Kiss1 expression was not different between AS‐SSH BPH/5 and C57 (p = 0.77). Nat BPH/5 had lower placental expansion (p < 0.05) than Nat C57, whereas AS‐SSH BPH/5 presented higher placental expansion than Nat BPH/5 (p < 0.05). In conclusion, normalization of the E2 and P4 profile in the PE‐like BPH/5 mouse early gestation not only mitigated the Kiss1upregulation at the maternal‐fetal interface, but also ameliorated the placental defects previously reported in this mouse model.

120 Reproductive Performance, Early Embryo Survival, Fetal Characteristics, and Tissue Fatty Acid Profiles of Sows Fed Linoleic or α-Linolenic Acid

Abstract The objective of this study was to determine the potential reproductive benefits of supplementing sows with linoleic (LA) or α-linolenic acid (α-LA) throughout the preceding lactation and subsequent gestation until 30 d post-rebreeding. Thirty-three second parity sows were allotted to either a control (0.88% LA, 0.10% α-LA), high LA (3.3% LA, 0.10% α-LA,) or high α-LA (0.88% LA, 0.60% α-LA) diet fed throughout lactation and early gestation. At 30 d post-breeding, a subset of sows (n = 18) was harvested to determine embryo survival and fatty acid composition of reproductive and fetal tissues. Number of pigs weaned per litter (P = 0.77) and litter average daily gain (P = 0.34) did not differ among treatments; however, litters from sows supplemented with LA tended to be heavier at weaning (P = 0.09; 55.3, 61.3, and 57.1 kg for control, LA, and α-LA, respectively). Sows supplemented with LA had higher embryo survival (P < 0.01; 57.2, 79.5, and 58.7% for control, LA, and α-LA, respectively), heavier feto-placental units (P < 0.01; 210.3, 279.4 and 235.0 g for control, LA, and α-LA, respectively), and elevated volume of allantoic fluid (P < 0.01; 185.9, 247.9, and 200.8 mL for control, LA, and α-LA, respectively). Concentrations of EFA increased in maternal serum by the dietary EFA supplemented (P < 0.05). Supplementation of LA did not impact linoleic acid or arachidonic acid concentrations in endometrium, chorioallantoic membrane, or fetus; however, α-LA increased (P < 0.05) concentrations of α-linoleic acid in chorioallantoic membrane and eicosapentaenoic acid in serum and endometrium. Supplementation of LA, but not α-LA, throughout lactation and early subsequent gestation was beneficial for embryo survival. The lesser influence of dietary LA and α-LA in fetal tissues may be associated with the intake, transfer, and metabolism of EFA in the chorioallantoic membrane and endometrium.

Importance of Windows of Exposure to Maternal High-Fat Diet and Feto-Placental Effects: Discrimination Between Pre-conception and Gestational Periods in a Rabbit Model.

Context and Aim: Lipid overnutrition in female rabbits, from prepuberty, leads to impaired metabolism (dyslipidemia and increased adiposity) and follicular atresia, and, when continued during gestation, affects offspring phenotype with intrauterine growth retardation (IUGR) and leads to placental and lipid metabolism abnormalities. Growth retardation is already observed in embryo stage, indicating a possible implication of periconceptional exposure. The objective of this study was to discriminate the effects of preconception and gestational exposures on feto-placental development.Materials and Methods: Rabbit 1-day zygotes were collected from female donors under control (CD) or high-fat-high-cholesterol (HD) diet and surgically transferred to the left and right uterus, respectively, of each H (n = 6) or C (n = 7) synchronized recipients. Close to term, four combinations, CC (n = 10), CH (n = 13), HC (n = 13), and HH (n = 6), of feto-placental units were collected, for biometry analyses. Fatty acid (FA) profiles were determined in placental labyrinth, decidua, fetal plasma, and fetal liver by gas chromatography and explored further by principal component analysis (PCA). Candidate gene expression was also analyzed by RT-qPCR in the placenta and fetal liver. Data were analyzed by Kruskal–Wallis followed by Dunn’s pairwise comparison test. Combinations of different data sets were combined and explored by multifactorial analysis (MFA).Results: Compared to controls, HH fetuses were hypotrophic with reduced placental efficiency and altered organogenesis, CH presented heavier placenta but less efficient, whereas HC presented a normal biometry. However, the MFA resulted in a good separation of the four groups, discriminating the effects of each period of exposure. HD during gestation led to reduced gene expression (nutrient transport and metabolism) and big changes in FA profiles in both tissues with increased membrane linoleic acid, lipid storage, and polyunsaturated-to-saturated FA ratios. Pre-conception exposure had a major effect on fetal biometry and organogenesis in HH, with specific changes in FA profiles (increased MUFAs and decreased LCPUFAs).Conclusion: Embryo origin left traces in end-gestation feto-placental unit; however, maternal diet during gestation played a major role, either negative (HD) or positive (control). Thus, an H embryo developed favorably when transferred to a C recipient (HC) with normal biometry at term, despite disturbed and altered FA profiles.

227 Awardee Talk: Advances in Lipid Nutrition of Modern Sows

Abstract The modern lactating sow is a highly productive female capable of producing large quantities of milk to support large, rapidly growing litters. The associated high energy and nutritional demands are challenging to meet, especially during heat stress, when tissue loss is extensive. Supplemental lipids are efficient energy sources with a high energy density and low heat increment associated with digestion and metabolism. Besides serving as concentrated energy sources, unsaturated lipids are important sources of essential fatty acids (EFA; linoleic acid, C18:2n-6; and α-linolenic acid, C18:3n-3). These EFA are precursors of various prostanoids that are important regulators of reproductive processes. Lactating sows fed diets devoid of supplemental lipids exhibited poor reproduction but was significantly improved by inclusion of only 2% unsaturated lipids. It was shown that lactation diets were deficient in both linoleic and α-linolenic acid and tissue reserves were inadequate in mature sows. A multi-trial meta-analysis showed that more sows were successfully bred and maintained pregnancy when they consumed supplemental linoleic acid. Further, increased linoleic acid intake during lactation progressively reduced the number of sows removed from the herd due to reproductive failure (sows not returning to estrus, returning to estrus after breeding, pregnancy loss). Based on dose-response curves, the near maximum response was achieved when sows consumed a minimum of 125 g/d of linoleic acid and 10 g/d of α-linolenic acid during lactation. Recent studies also demonstrated improved subsequent reproduction with supplemental linoleic and α-linolenic acid during lactation, but not during the post-weaning period. Responses appeared to be related to heavier fetoplacental units, elevated allantoic fluid volume, and higher embryo survival measured on day 30 of gestation in sows supplemented with linoleic acid during lactation and early gestation. Research demonstrated the importance of both EFA in lactating sows and their critical role in improving reproduction, thus resolving seasonal anestrus.

Single-cell transcriptome analysis reveals defective decidua stromal niche attributes to recurrent spontaneous abortion.

ObjectivesSuccessful pregnancy involves the homeostasis between maternal decidua and fetoplacental units, whose disruption contributes to compromised pregnancy outcomes, including recurrent spontaneous abortion (RSA). The role of cell heterogeneity of maternal decidua in RSA is yet to be illustrated.Materials and methodsA total of 66,078 single cells from decidua samples isolated from patients with RSA and healthy controls were analysed by unbiased single‐cell RNA sequencing (scRNA‐seq).ResultsOur scRNA‐seq results revealed that stromal cells are the most abundant cell type in decidua during early pregnancy. RSA samples are accompanied by aberrant decidualization and obviously obstructed communication between stromal cells and other cell types, such as abnormal activation of macrophages and NK cells. In addition, the over‐activated TNF superfamily member 12 (TNFSF12, TWEAK) and FASLG in RSA are closely related to stromal cell demise and pregnancy failure.ConclusionsOur research reveals that the cell composition and communications in normal and RSA decidua at early pregnancy and provides insightful information for the pathology of RSA and will pave the way for pregnancy loss prevention.

Genome-Wide fitness analysis of group B Streptococcus in human amniotic fluid reveals a transcription factor that controls multiple virulence traits.

Streptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This analysis showed that one gene, which encodes a GntR-class transcription factor that we named MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic targeted deletion of the mrvR gene, which had a growth defect in amniotic fluid relative to the wild type parent strain. The mrvR deletion strain also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that while the mutant was able to cause persistent murine vaginal colonization, pregnant mice colonized with the mrvR deletion strain did not develop preterm labor despite consistent GBS invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with wild type GBS consistently deliver prematurely. In a sepsis model the mrvR deletion strain showed significantly decreased lethality. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed RNA-seq on wild type and mrvR deletion GBS strains, which revealed that the transcription factor affects expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and salvage pathways were highly represented among the set of differentially expressed genes, suggesting that MrvR may be involved in regulating nucleotide availability.

Identification of appropriate reference genes for qPCR analyses of porcine placentae and endometria, supplying foetuses of different size and sex, at multiple gestational days.

Recent studies suggest associations exist between foetal size and sex, and gene expression at the porcine feto-maternal interface. It is essential to identify reference genes which have stable expression throughout gestation in feto-placental units associated with foetuses of different size and sex. qPCR was performed for 11 genes within porcine placentae and endometria at gestational days (GD) 30, 60 and 90. Several reference genes were found to have stable expression in these samples. The combination of B2m1 and Tbp1, and Hprt1 and Tbp1 had the most stable expression in endometria and placentae, respectively. Reference genes identified as having stable expression were utilized in a larger experiment with placentae and endometria associated with foetuses of different size and sex at four GD. The average expression of B2m1 and Tbp1 mRNAs was suitable for the normalization of temporal changes in endometria, and comparison between endometria supplying foetuses of different size throughout gestation. The average expression of Hprt1 and Tbp1 mRNAs was suitable for the normalization of placental mRNA expression for comparison of temporal changes and sex differences between placentae supplying foetuses of different sex throughout gestation. This combination was suitable for the normalization of mRNA expression in placentas supplying GD30, GD60 and GD90 foetuses of different size. This study has identified reference genes with stable expression in placentae and endometria across multiple gestational days, in tissues associated with foetuses of different size and sex. The results of these experiments highlight the importance of selecting appropriate reference genes for the biological comparison under investigation.

High-resolution diffusion MRI studies of development in pregnant mice visualized by novel spatiotemporal encoding schemes.

This study introduces an MRI approach to map diffusion of water in vivo with high resolution under challenging conditions; the approach's potential is then used in diffusivity characterizations of embryos and fetoplacental units in pregnant mice, as well as of newborn mice in their initial postnatal period. The method relies on performing self-referenced spatiotemporal encoded MRI acquisitions, which can achieve the motional and susceptibility immunities needed to target challenging regions such as a mouse's abdominal cavity in a single shot. When suitably combined with zooming-in and novel interleaving procedures, these scans can overcome the inhomogeneity and sensitivity challenges arising upon targeting ≈100μm in-plane resolutions, and thereby enable longitudinal development studies of abdominal organs that have hitherto eluded in vivo diffusion-weighted imaging. This is employed here to follow processes related to embryonic implantation and placentation, including the final stages of mouse gastrulation, the development of white matter in fetal brains, the maturation of fetal spines, and the evolution of the different layers making up mouse hemochorial placentas. The protocol's ability to extract diffusivity information in challenging regions as a function of embryonic mouse development is thus demonstrated, and its usefulness as a tool for visualizing pregnancy-related developmental changes in rodents is discussed.

Natural embryonic death in the plain viscacha Lagostomus maximus: are there previous anatomo-histological variations that would intervene in the differential survival of the implantations?

Visualization of the retroplacental clear space (RPCS) may provide critical insight into the development of abnormally invasive placenta (AIP). In this pre-clinical study, we characterized the appearance of the RPCS on magnetic resonance imaging (MRI) during the second half of gestation using a liposomal gadolinium contrast agent (liposomal-Gd).Studies were performed in fifteen pregnant C57BL/6 mice at 10, 12, 14, 16, and 18 days of gestation. MRI was performed on a 1T permanent magnet scanner. Pre-contrast and post-contrast images were acquired using T1-weighted gradient-recalled echo (T1w-GRE) and T2-weighted fast spin echo (T2w-FSE) sequences. Animals were euthanized after imaging and feto-placental units harvested for histological examination. Visualization of the RPCS was scored by a maternal-fetal radiologist and quantified by measuring the contrast-to-noise ratio (CNR) on T1w images. Feto-placental features were segmented for analysis of volumetric changes during gestation.Contrast-enhanced T1w images enabled the visualization of structural changes in placental development between days 10–18 of gestation. Although the placental margin on the fetal side was clearly visible at all time points, the RPCS was partially visible at day 10 of gestation, and clearly visible by day 12. Hematoxylin and eosin (H&E) staining of the placental tissue corroborated MRI findings of structural and morphological changes in the placenta.Contrast-enhanced MR imaging using liposomal-Gd enabled adequate visualization of the retroplacental clear space starting at day 12 of gestation. The agent also enabled characterization of placental structure and morphological changes through gestation.

The GRB2-associated binding protein 3 is required for IL-2 and IL-15 induced NK cell expansion and limits trophoblast invasion during pregnancy

Abstract The Grb2-associated binding protein 3 (Gab3) is a scaffolding protein that belongs to the Grb2-assocated binding family of proteins along with Gab1 and Gab2. Gab3 is expressed exclusively in immune cells including NK cells, CD8+T cells, mast cells and macrophages, however the function of Gab3 has remained elusive. Based on two independent mouse models; a hypomorph germline mouse model carrying a missense mutation in the PH domain of Gab3 and complete Gab3-KO mice, we uncovered a key role for Gab3 in peripheral NK cell function. Specifically, loss of Gab3 results in impaired IL-2/IL-15-induced NK cell priming and expansion, due to a selective impairment in MAPK- but not STAT5-signaling. In vivo, we show that Gab3 is required for recognition and elimination of “missing-self” and tumor targets. Gab3-deficient mice exhibit impaired uterine NK cell expansion that is associated with abnormal spiral artery remodeling and increased trophoblast invasion in the decidua basalis. This coincides with stillbirth, retained placenta, maternal hemorrhage and undelivered fetoplacental units at term. Thus, our studies identify Gab3 as a novel component required for IL-2/IL-15-mediated NK cell priming and expansion that is essential for anti-tumor responses and for controlling fetal trophoblast invasion during pregnancy.

The identification of differentially expressed genes between extremes of placental efficiency in maternal line gilts on day 95 of gestation

BackgroundPlacental efficiency (PE) describes the relationship between placental and fetal weights (fetal wt/placental wt). Within litters, PE can vary drastically, resulting in similarly sized pigs associated with differently sized placentas, up to a 25% weight difference. However, the mechanisms enabling the smaller placenta to grow a comparable littermate are unknown. To elucidate potential mechanisms, morphological measurements and gene expression profiles in placental and associated endometrial tissues of high PE and low PE feto-placental units were compared. Tissue samples were obtained from eight maternal line gilts during gestational day 95 ovario-hysterectomies. RNA was extracted from tissues of feto-placental units with the highest and lowest PE in each litter and sequenced.ResultsMorphological measurements, except placental weight, were not different (P > 0.05) between high and low PE. No DEG were identified in the endometrium and 214 DEG were identified in the placenta (FDR < 0.1), of which 48% were upregulated and 52% were downregulated. Gene ontology (GO) analysis revealed that a large percentage of DEG were involved in catalytic activity, binding, transporter activity, metabolism, biological regulation, and localization. Four GO terms were enriched in the upregulated genes and no terms were enriched in the downregulated genes (FDR < 0.05). Eight statistically significant correlations (P < 0.05) were identified between the morphological measurements and DEG.ConclusionMorphological measures between high and low PE verified comparisons were of similarly sized pigs grown on different sized placentas, and indicated that any negative effects of a reduced placental size on fetal growth were not evident by day 95. The identification of DEG in the placenta, but absence of DEG in the endometrium confirmed that the placenta responds to the fetus. The GO analyses provided evidence that extremes of PE are differentially regulated, affecting components of placental transport capacity like nutrient transport and blood flow. However, alternative GO terms were identified, indicating the complexity of the relationship between placental and fetal weights. These findings support the use of PE as a marker of placental function and provide novel insight into the genetic control of PE, but further research is required to make PE production applicable.

Pre-clinical magnetic resonance imaging of retroplacental clear space throughout gestation

IntroductionVisualization of the retroplacental clear space (RPCS) may provide critical insight into the development of abnormally invasive placenta (AIP). In this pre-clinical study, we characterized the appearance of the RPCS on magnetic resonance imaging (MRI) during the second half of gestation using a liposomal gadolinium contrast agent (liposomal-Gd). Materials and methodsStudies were performed in fifteen pregnant C57BL/6 mice at 10, 12, 14, 16, and 18 days of gestation. MRI was performed on a 1T permanent magnet scanner. Pre-contrast and post-contrast images were acquired using T1-weighted gradient-recalled echo (T1w-GRE) and T2-weighted fast spin echo (T2w-FSE) sequences. Animals were euthanized after imaging and feto-placental units harvested for histological examination. Visualization of the RPCS was scored by a maternal-fetal radiologist and quantified by measuring the contrast-to-noise ratio (CNR) on T1w images. Feto-placental features were segmented for analysis of volumetric changes during gestation. ResultsContrast-enhanced T1w images enabled the visualization of structural changes in placental development between days 10–18 of gestation. Although the placental margin on the fetal side was clearly visible at all time points, the RPCS was partially visible at day 10 of gestation, and clearly visible by day 12. Hematoxylin and eosin (H&E) staining of the placental tissue corroborated MRI findings of structural and morphological changes in the placenta. ConclusionsContrast-enhanced MR imaging using liposomal-Gd enabled adequate visualization of the retroplacental clear space starting at day 12 of gestation. The agent also enabled characterization of placental structure and morphological changes through gestation.

Abstract 17312: Hand1 Impairs Angiogenesis in Heart and Placenta

Introduction: Congenital heart defects affect approximately 1% of live births, often requiring complex surgeries at birth. The most significant risk factor for surgery survival is birthweight. Proper placental development and function is vital for normal fetal growth. We have previously demonstrated abnormal placental development and vascularization in human CHD placentas. Hand1 has roles in heart and placental development and has been implicated in multiple types of CHD including double right outlet, hypoplastic left heart syndrome, and septal defects. We utilized the Hand1 A126fs/+ mouse to investigate the role of Hand1 in placentation and vascularization. Methods: Hand1 A126fs/+ female mice were time-mated with Nkx2.5cre or Cdh5cre males. Feto-placental units were harvested at E10.5 and E12.5 for histological analysis, vascular assessment by IHC for CD-31, and RNA expression by qPCR. Results: Nkx2.5cre/Hand1 a126fs/+ fetuses demonstrated embryonic lethality by E10.5 due to lack of placental labyrinth formation and vascularization (Figure 1). In contrast, ablation of Hand1 in vascular endothelium (Cdh5cre) did not disrupt placental labyrinth or heart at E12.5. Expression of VegFb, Ang1, Ang2, Flt1, Flk was reduced in Hand1 A126fs/+ ; Nkx2.5cre placentas compared to control littermates, but VegFa expression was increased. Conclusion: Our data demonstrate that Hand1 expression in placental trophoblast, but not endothelium, is necessary for vascularization of the labyrinth and may disrupt multiple angiogenic factors known to be expressed in trophoblast. Alterations in Hand1 may represent a mechanism for abnormal placentation in cases of CHD. Figure 1. H/E (A-C) and CD31 (D-F) images of Hand1 +/+ (A, D), Hand1 A126fs/+ ; Nkx2.5cre (B, E), and Hand1 A126fs/+ ; Cdh5cre (C, F) placentas at day E12.5. Hand1A 126fs/+ ; Nkx2.5cre placentas fail to form labyrinth and fetal vasculature, while Hand1 A126fs/+ ; Cdh5cre placentas develop normally at this timepoint.

Factors contributing to the variation in placental efficiency on days 70, 90, and 110 of gestation in gilts.

Variations in placental efficiency (PE), a measure of grams of fetus produced per gram of placenta, were initially researched between swine breeds, where increased PE was associated with larger litters. Placental efficiency was also found to vary greatly within production herds and individual litters; however, the use of PE as a selection tool has been debated. Nonetheless, PE is an index of feto-placental adaptation and may help identify compensatory mechanisms that maintain fetal growth when placental size is reduced, potentially providing an opportunity to address production concerns like low birth weights and preweaning survival. Since the nutrient transport capacity of the placenta largely depends on vasculature and nutrient transporter abundance, the objectives of this experiment were to 1) determine the mRNA expression of genes encoding nutrient transporters in the placenta and adjacent endometrium, and 2) evaluate if a relationship existed between PE and vascular density and/or nutrient transporters. Gilts (n = 19) were ovario-hysterectomized on day 70, 90, or 110 of gestation to collect placental and adjacent endometrial samples. The mean litter size was 11.1. Placental efficiency increased (P < 0.0001) throughout the end of gestation, while the range of PE increased from day 70 to 90 and was reduced on day 110 (P < 0.0001). Placental efficiency and placental weight were negatively correlated throughout gestation (70 d, r = -0.83, P < 0.0001; 90 d, r = -0.81, P < 0.0001; 110 d, r = -0.44, P < 0.0007), but the negative correlation between PE and fetal weight was not maintained as gestation progressed (70 d, r = -0.58, P < 0.0001; 90 d, r = -0.36, P < 0.0005; 110 d, r = 0.09, P = 0.51). Based on conditional effects plots, variations in PE were associated with alterations in amino acid transporter expression in the placenta (SLC7A7, SLC3A1) and endometrium (SLC7A1) on day 70. On day 90, PE had a positive relationship with placental expression of a glucose transporter (SLC2A3), and on day 110 PE was positively related to placental vascular density. The results suggest utero-placental adaptations occur as a compensation for reduced placental size to meet the increasing nutrient demands of the growing fetus during late gestation in swine. Furthermore, nutrient requirements differ for individual feto-placental units on a given day; therefore, optimizing nutrient availability during late gestation may improve fetal growth and survival.