RNA interference (RNAi) of the placental glucose transporter SLC2A3 resulted in smaller hypoglycemic fetuses with reduced umbilical artery insulin and glucagon concentrations at mid-gestation (75 d of gestation [dGA]) in sheep. Our current objective was to determine the ramifications of SLC2A3-RNAi throughout gestation, and to assess fetal insulin secretion in response to glucose and arginine challenges. We successfully generated SLC2A3-RNAi (n = 6) and NTS-RNAi (non-targeting sequence) control sheep pregnancies (n = 6). Near-term they underwent surgical catheterization followed by in vivo metabolic studies at 133 ± 2 dGA. A baseline metabolic study, which included assessment of uterine and umbilical blood flow rates, was followed by a square-wave hyperglycemic clamp of the fetus (GSIS), followed by infusion of an arginine bolus to assess maximal fetal insulin secretion. The baseline metabolic study determined that uterine glucose uptake (μmol/min) was reduced 29% (P = 0.03), as was placental glucose utilization by 40% (P = 0.05), but umbilical glucose uptake was not impacted in SLC2A3-RNAi pregnancies. By contrast, amino acid carbon uptake/kg of uterus increased 76%, as did placental utilization of amino acid carbon (P = 0.09), indicating placental compensation supported by increased mRNA concentrations of NOS3, IGF2, IGF1R, and IGF2R (P ≤ 0.05 to ≤ 0.10). Unlike the findings at 75 dGA, near-term fetal body and pancreas weights were no longer significantly (P ≥ 0.10) impacted by SLC2A3-RNAi. While baseline umbilical artery concentrations of glucose and insulin were not different (P ≥ 0.10), two-way ANOVA revealed a significant (P ≤ 0.01) SLC2A3-RNAi treatment effect during GSIS and arginine stimulated insulin secretion (ASIS), demonstrating significant enhancement of fetal insulin secretion capacity. In summary, placental compensatory mechanisms appeared to rescue fetal growth and umbilical glucose concentrations between mid-gestation and near-term. Decreasing placental glucose utilization while increasing amino acid utilization may be a mechanism aiding recovery of glucose transfer to the fetus. While microvillous glucose uptake to the placenta appears to be rate-limiting to fetal growth and development early in gestation, placental glucose transfer and fetal growth are rescued later in gestation. However, increased nutrient stimulated fetal insulin secretion persists near-term, as a possible sequela of the earlier impacts.

Dietary supplementation with long-chain omega-3 fatty acids such as docosahexaenoic acid (DHA) is recommended to women during pregnancy to prevent preterm birth and support optimal fetal development. DHA supplementation also decreases circulating triglyceride levels through diverse effects on systemic lipid metabolism, but its impacts on fetal metabolism are largely unknown due to inherent limitations of such investigations during pregnancy. The aim of the present study was to validate a large animal model suitable for investigating impacts of prenatal DHA supplementation on fetal development and metabolism. White-faced ewes were fed either a control diet (Show-rite NewCo Lamb Feed) or a DHA-supplemented diet (control diet + 3% algae-derived DHA) from 2-3 weeks before pregnancy until mid-gestation (75 days), after which serum, placenta and fetal tissues were collected for biochemical analysis of phospholipid fatty acid composition and fatty acid transporter expression, and a broader characterization of nutrient metabolism of fetal heart, skeletal muscle, and liver. Prenatal DHA supplementation reduced maternal serum triglycerides and significantly enriched all six fetal tissues examined. These results were paralleled by tissue-specific impacts on fetal fatty acid transporters and metabolism, a greater capacity of fetal muscle and heart to oxidize lipids over carbohydrate substrates, and reduced expression of insulin receptor substrate-1 in fetal muscle and liver. In conclusion, this study demonstrates the utility of an ovine model for investigating the biological effects of prenatal DHA supplementation on fetal development and metabolism, and highlights the need for a better understanding of its impacts on fetal and offspring metabolic health trajectory.

Abstract Pregnancy in any mammal requires the integration of three distinct compartments; the maternal, placental and fetal compartments. Compromised function of any of the three compartments can result in suboptimal pregnancy outcomes, including increased morbidity and mortality at delivery, reduced livestock production efficiency and adult-onset of metabolically-related disease in humans. To accurately understand the progression of both normal and compromised pregnancies, therefore, requires the simultaneous assessment of all three compartments, which for most species is not technically feasible. Consequently, over the past 60 yr the pregnant sheep has been used extensively for in vivo investigation of pregnancy physiology. The ability to place indwelling catheters, within both maternal and fetal vessels, allows the simultaneous steady-state assessment of uterine and umbilical blood flows, hormone secretion, and nutrient uptake and utilization of nutrients by each of the three compartments, under non-stressed and non-anesthetized conditions. This has provided unrivalled insight into the physiology of pregnancy, and the realization that the placenta is a highly metabolic tissue, utilizing the majority of oxygen and glucose taken up by the uterus. However, the deficit in studying pregnant sheep is the lack of genetic models, readily available in rodents, that could help define specific gene function during pregnancy. Consequently, we developed in vivo lentiviral-mediated RNA interference (RNAi) methods to specifically alter the abundance of specific gene transcripts/proteins within the placenta, beginning at 9 d of gestation (dGA). Our lentiviral-mediated approach limits the RNAi to the trophectoderm derivatives of the placenta. Initially we used this approach to determine the importance of PRR15 and LIN28 during the establishment of pregnancies, but turned our efforts towards combining in vivo RNAi with steady-state physiological assessments during late gestation to examine the function of genes involved in placental nutrient transport (SLC2A3) and placenta derived hormones (CSH). By creating a placental deficiency in SLC2A3, not only was the fetus smaller and hypoglycemic at mid-gestation, but by interfering with placental glucose uptake, maternal hormone concentrations were also impacted, yet near-term, a distinctly different phenotype was present. For the placental hormone CSH (a.k.a. placental lactogen), CSH RNAi not only impacts placental and fetal growth, but also reductions in uterine blood flow, nutrient fluxes to the fetus, and produces an altered hormonal environment. Combining these methodological approaches provides new and often unexpected insights into the physiological integration of the maternal, placental and fetal compartments. Supported by NIH-NICHD grants HD093701 and HD094952.

Glucose, the primary energy substrate for fetal oxidative processes and growth, is transferred from maternal to fetal circulation down a concentration gradient by placental facilitative glucose transporters. In sheep, SLC2A1 and SLC2A3 are the primary transporters available in the placental epithelium, with SLC2A3 located on the maternal-facing apical trophoblast membrane and SLC2A1 located on the fetal-facing basolateral trophoblast membrane. We have previously reported that impaired placental SLC2A3 glucose transport resulted in smaller, hypoglycemic fetuses with reduced umbilical artery insulin and glucagon concentrations, in addition to diminished pancreas weights. These findings led us to subject RNA derived from SLC2A3-RNAi (RNA interference) and NTS-RNAi (non-targeting sequence) fetal pancreases to qPCR followed by transcriptomic analysis. We identified a total of 771 differentially expressed genes (DEGs). Upregulated pathways were associated with fat digestion and absorption, particularly fatty acid transport, lipid metabolism, and cholesterol biosynthesis, suggesting a potential switch in energetic substrates due to hypoglycemia. Pathways related to molecular transport and cell signaling in addition to pathways influencing growth and metabolism of the developing pancreas were also impacted. A few genes directly related to gluconeogenesis were also differentially expressed. Our results suggest that fetal hypoglycemia during the first half of gestation impacts fetal pancreas development and function that is not limited to β cell activity.

We previously demonstrated impaired placental nutrient transfer in chorionic somatomammotropin (CSH) RNA interference (RNAi) pregnancies, with glucose transfer being the most impacted. Thus, we hypothesized that despite experimentally elevating maternal glucose, diminished umbilical glucose uptake would persist in CSH RNAi pregnancies, demonstrating the necessity of CSH for adequate placental glucose transfer. Trophectoderm of sheep blastocysts (9 days of gestational age; dGA) were infected with a lentivirus expressing either nontargeting control (CON RNAi; n = 5) or CSH-specific shRNA (CSH RNAi; n = 7) before transfer into recipient sheep. At 126 dGA, pregnancies were fitted with vascular catheters and underwent steady-state metabolic studies (3H2O transplacental diffusion) at 137 ± 0 dGA, before and during a maternal hyperglycemic clamp. Umbilical glucose and oxygen uptakes, as well as insulin and IGF1 concentrations, were impaired (P ≤ 0.01) in CSH RNAi fetuses and were not rescued by elevated maternal glucose. This is partially due to impaired uterine and umbilical blood flow (P ≤ 0.01). However, uteroplacental oxygen utilization was greater (P ≤ 0.05) during the maternal hyperglycemic clamp, consistent with greater placental oxidation of substrates. The relationship between umbilical glucose uptake and the maternal-fetal glucose gradient was analyzed, and while the slope (CON RNAi, Y = 29.54X +74.15; CSH RNAi, Y = 19.05X + 52.40) was not different, the y-intercepts and elevation were (P = 0.003), indicating reduced maximal glucose transport during maternal hyperglycemia. Together, these data suggested that CSH plays a key role in modulating placental metabolism that ultimately promotes maximal placental glucose transfer.NEW & NOTEWORTHY The current study demonstrated a novel, critical autocrine role for chorionic somatomammotropin in augmenting placental glucose transfer and maintaining placental oxidative metabolism. In pregnancies with CSH deficiency, excess glucose in maternal circulation is insufficient to overcome fetal hypoglycemia due to impaired placental glucose transfer and elevated placental metabolic demands. This suggests that perturbations in glucose transfer in CSH RNAi pregnancies are due to compromised metabolic efficiency along with reduced placental mass.

Supplemental figures accompanying manuscript titled "Increasing maternal glucose concentrations is insufficient to restore placental glucose transfer in chorionic somatomammotropin RNA interference pregnancies."

Maternal diet during pregnancy is associated with offspring metabolic risk trajectory in humans and animal models, but the prenatal origins of these effects are less clear. We examined the effects of a high-fat diet (HFD) during pregnancy on fetal skeletal muscle metabolism and metabolic risk parameters using an ovine model. White-faced ewes were fed a standardized diet containing 5% fat wt/wt (CON), or the same diet supplemented with 6% rumen-protected fats (11% total fat wt/wt; HFD) beginning 2 wk before mating until midgestation (GD75). Maternal HFD increased maternal weight gain, fetal body weight, and low-density lipoprotein levels in the uterine and umbilical circulation but had no significant effects on circulating glucose, triglycerides, or placental fatty acid transporters. Fatty acid (palmitoylcarnitine) oxidation capacity of permeabilized hindlimb muscle fibers was >50% higher in fetuses from HFD pregnancies, whereas pyruvate and maximal (mixed substrate) oxidation capacities were similar to CON. This corresponded to greater triacylglycerol content and protein expression of fatty acid transport and oxidation enzymes in fetal muscle but no significant effect on respiratory chain complexes or pyruvate dehydrogenase expression. However, serine-308 phosphorylation of insulin receptor substrate-1 was greater in fetal muscle from HFD pregnancies along with c-jun-NH2 terminal kinase activation, consistent with prenatal inhibition of skeletal muscle insulin signaling. These results indicate that maternal high-fat feeding shifts fetal skeletal muscle metabolism toward a greater capacity for fatty acid over glucose utilization and favors prenatal development of insulin resistance, which may predispose offspring to metabolic syndrome later in life.NEW & NOTEWORTHY Maternal diet during pregnancy is associated with offspring metabolic risk trajectory in humans and animal models, but the prenatal origins of these effects are less clear. This study examined the effects of a high-fat diet during pregnancy on metabolic risk parameters using a new sheep model. Results align with findings previously reported in nonhuman primates, demonstrating changes in fetal skeletal muscle metabolism that may predispose offspring to metabolic syndrome later in life.

While fetal growth is dependent on many factors, optimal placental function is a prerequisite for a normal pregnancy outcome. The majority of fetal growth-restricted (FGR) pregnancies result from placental insufficiency (PI). The insulin-like growth factors (IGF1 and IGF2) stimulate fetal growth and placental development and function. Previously, we demonstrated that in vivo RNA interference (RNAi) of the placental hormone, chorionic somatomammotropin (CSH), resulted in two phenotypes. One phenotype exhibits significant placental and fetal growth restriction (PI-FGR), impaired placental nutrient transport, and significant reductions in umbilical insulin and IGF1. The other phenotype does not exhibit statistically significant changes in placental or fetal growth (non-FGR). It was our objective to further characterize these two phenotypes by determining the impact of CSH RNAi on the placental (maternal caruncle and fetal cotyledon) expression of the IGF axis. The trophectoderm of hatched blastocysts (9 days of gestation, dGA) were infected with a lentivirus expressing either a non-targeting sequence (NTS RNAi) control or CSH-specific shRNA (CSH RNAi) prior to embryo transfer into synchronized recipient ewes. At ≈125 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies. Nutrient uptakes were determined, and tissues were harvested at necropsy. In both CSH RNAi non-FGR and PI-FGR pregnancies, uterine blood flow was significantly reduced (p ≤ 0.05), while umbilical blood flow (p ≤ 0.01), both uterine and umbilical glucose and oxygen uptakes (p ≤ 0.05), and umbilical concentrations of insulin and IGF1 (p ≤ 0.05) were reduced in CSH RNAi PI-FGR pregnancies. Fetal cotyledon IGF1 mRNA concentration was reduced (p ≤ 0.05) in CSH RNAi PI-FGR pregnancies, whereas neither IGF1 nor IGF2 mRNA concentrations were impacted in the maternal caruncles, and either placental tissue in the non-FGR pregnancies. Fetal cotyledon IGF1R and IGF2R mRNA concentrations were not impacted for either phenotype, yet IGF2R was increased (p ≤ 0.01) in the maternal caruncles of CSH RNAi PI-FGR pregnancies. For the IGF binding proteins (IGFBP1, IGFBP2, IGFBP3), only IGFBP2 mRNA concentrations were impacted, with elevated IGFBP2 mRNA in both the fetal cotyledon (p ≤ 0.01) and maternal caruncle (p = 0.08) of CSH RNAi non-FGR pregnancies. These data support the importance of IGF1 in placental growth and function but may also implicate IGFBP2 in salvaging placental growth in non-FGR pregnancies.

In the ruminant placenta, glucose uptake and transfer are mediated by facilitative glucose transporters SLC2A1 (GLUT1) and SLC2A3 (GLUT3). SLC2A1 is located on the basolateral trophoblast membrane, whereas SLC2A3 is located solely on the maternal-facing, apical trophoblast membrane. While SLC2A3 is less abundant than SLC2A1, SLC2A3 has a five-fold greater affinity and transport capacity. Based on its location, SLC2A3 likely plays a significant role in the uptake of glucose into the trophoblast. Fetal hypoglycemia is a hallmark of fetal growth restriction (FGR), and as such, any deficiency in SLC2A3 could impact trophoblast glucose uptake and transfer to the fetus, thus potentially setting the stage for FGR. By utilizing in vivo placenta-specific lentiviral-mediated RNA interference (RNAi) in sheep, we were able to significantly diminish (p ≤ 0.05) placental SLC2A3 concentration, and determine the impact at mid-gestation (75 dGA). In response to SLC2A3 RNAi (n = 6), the fetuses were hypoglycemic (p ≤ 0.05), exhibited reduced fetal growth, including reduced fetal pancreas weight (p ≤ 0.05), which was associated with reduced umbilical artery insulin and glucagon concentrations, when compared to the non-targeting sequence (NTS) RNAi controls (n = 6). By contrast, fetal liver weights were not impacted, nor were umbilical artery concentrations of IGF1, possibly resulting from a 70% increase (p ≤ 0.05) in umbilical vein chorionic somatomammotropin (CSH) concentrations. Thus, during the first half of gestation, a deficiency in SLC2A3 results in fetal hypoglycemia, reduced fetal development, and altered metabolic hormone concentrations. These results suggest that SLC2A3 may be the rate-limiting placental glucose transporter during the first-half of gestation in sheep.

Dietary supplementation of omega-3 fatty acids such as docosahexaenoic acid (DHA) during pregnancy is often recommended to support optimal fetal brain development and cognitive function of the offspring. DHA supplementation also influences cardiometabolic risk parameters in adults, but its effect on fetal metabolism and subsequent risk is poorly understood. The aim of this study was to determine the effects of maternal DHA supplementation (MDS) on placental and fetal nutrient handling during pregnancy. White-faced ewes were fed either a control diet (Show-rite NewCo Lamb Feed) or a DHA-supplemented diet (control diet + 3% w/w algae-derived DHA) from 2–3 weeks before pregnancy until mid-gestation (75 days), after which a C-section was performed to collect the placenta and fetal tissues for metabolic analyses. MDS significantly increased serum DHA levels and decreased serum triglycerides in the uterine (maternal) circulation, but not umbilical (fetal) circulation. Nevertheless, MDS resulted in significant DHA enrichment of the placenta and all fetal tissues examined, and differentially affected the protein expression of the four major fatty acid transport proteins FATP1, FATP4, CD36 and FABP in placenta, muscle, liver and heart, but had no effect on kidney or brain. Consistent with these findings, MDS tended to increase the capacity for fat over pyruvate oxidation in fetal muscle and heart, but favored a greater capacity for glucose uptake and oxidation in fetal liver. This study is the first to validate use of an ovine model for investigating the impact of maternal DHA supplementation on fetal metabolism and development. Results demonstrate a complex tissue-specific effect of MDS on fetal tissue carbohydrate and fatty acid metabolism that favors a greater capacity for serum glucose disposal and fatty acid oxidation. Whether these changes ultimately impact nutrient metabolism and cardiometabolic risk in the offspring later in life merits further investigation. Colorado Agricultural Experiment Station.

The placenta facilitates the transport of nutrients to the fetus, removal of waste products from the fetus, immune protection of the fetus and functions as an endocrine organ, thereby determining the environment for fetal growth and development. Additionally, the placenta is a highly metabolic organ in itself, utilizing a majority of the oxygen and glucose derived from maternal circulation. Consequently, optimal placental function is required for the offspring to reach its genetic potential in utero. Among ruminants, pregnant sheep have been used extensively for investigating pregnancy physiology, in part due to the ability to place indwelling catheters within both maternal and fetal vessels, allowing for steady-state investigation of blood flow, nutrient uptakes and utilization, and hormone secretion, under non-stressed and non-anesthetized conditions. This methodology has been applied to both normal and compromised pregnancies. As such, our understanding of the in vivo physiology of pregnancy in sheep is unrivalled by any other species. However, until recently, a significant deficit existed in determining the specific function or significance of individual genes expressed by the placenta in ruminants. To that end, we developed and have been using in vivo RNA interference (RNAi) within the sheep placenta to examine the function and relative importance of genes involved in conceptus development (PRR15 and LIN28), placental nutrient transport (SLC2A1 and SLC2A3), and placenta-derived hormones (CSH). A lentiviral vector is used to generate virus that is stably integrated into the infected cell's genome, thereby expressing a short-hairpin RNA (shRNA), that when processed within the cell, combines with the RNA Induced Silencing Complex (RISC) resulting in specific mRNA degradation or translational blockage. To accomplish in vivo RNAi, day 9 hatched and fully expanded blastocysts are infected with the lentivirus for 4 to 5 h, and then surgically transferred to synchronized recipient uteri. Only the trophectoderm cells are infected by the replication deficient virus, leaving the inner cell mass unaltered, and we often obtain ~70% pregnancy rates following transfer of a single blastocyst. In vivo RNAi coupled with steady-state study of blood flow and nutrient uptake, transfer and utilization can now provide new insight into the physiological consequences of modifying the translation of specific genes expressed within the ruminant placenta.

The proper conceptus elongation in ruminants is critical for the successful placentation and establishment of pregnancy. We have previously shown that the trophectoderm-specific knockdown of LIN28A/B in day 9 ovine blastocysts resulted in increased let-7 miRNAs and reduced conceptus elongation at day 16 of gestation. In this current study, by transcriptome analysis of LIN28A knockdown (AKD) or LIN28B knockdown (BKD) trophectoderm (TE), we explored the downstream target genes of the LIN28-let-7 axis and their roles in the placental and fetal development. We identified 449 differentially expressed genes (DEGs) in AKD TE and 1214 DEGs in BKD TE compared to non-targeting control (NTC). Our analysis further revealed that 210 downregulated genes in AKD TE and 562 downregulated genes in BKD TE were the potential targets of let-7 miRNAs. Moreover, 16 downregulated genes in AKD TE and 57 downregulated and 7 upregulated genes in BKD TE were transcription factors. The DEGs in AKD and BKD TE showed enrichment in the biological processes and pathways critical for placental development and function, and fetal development and growth. The results of this study suggest the potential roles of the LIN28-let-7 axis in placental and fetal development beyond its involvement in trophoblast proliferation and conceptus elongation.

Pregnant sheep have been used extensively for investigating pregnancy physiology, providing valuable information about the progression of ruminant pregnancy. The ability to place indwelling catheters, within both maternal and fetal vessels, allows for steady-state investigation of blood flow, nutrient uptakes and utilization, and hormone secretion, under non-stressed and non-anesthetized conditions. As such, our understanding of the in vivo physiology of pregnancy in sheep is unrivalled by any other species. However, until recently, a significant deficit existed in determining the specific function or significance of individual genes expressed by the placenta in livestock. To that end, we developed and have been using in vivo RNA interference (RNAi) within the sheep placenta to examine the function and relative importance of genes involved in conceptus development (PRR15 and LIN28), placental nutrient transport (SLC2A1 and SLC2A3), and placenta derived hormones (CSH). The lentiviral vector LL3.7 is used to generate virus that is stably integrated into the infected cell’s genome, thereby expressing a short-hairpin RNA (shRNA), that when processed within the cell, combines with the RNA Induced Silencing Complex (RISC) resulting in specific mRNA degradation or translational blockage. To accomplish in vivo RNAi, day 9 hatched and fully expanded blastocysts are infected with the lentivirus for 4–5 hours, and then surgically transferred to synchronized recipient uteri. Only the trophectoderm cells are infected by the replication deficient virus, leaving the inner cell mass unaltered, and we typically obtain 70–80% pregnancy rates following transfer of a single blastocyst. Data will be presented from two projects. One is focused on generating a deficiency in placental glucose transporters at mid-gestation, and the other on the impact of CSH RNAi during late gestation, demonstrating the utility of this experimental approach for examining gene function within the placenta of livestock. Supported by NIH-NICHD grants HD093701 and HD094952.

Deficiency of the placental hormone chorionic somatomammotropin (CSH) can lead to the development of intrauterine growth restriction (IUGR). To gain insight into the physiological consequences of CSH RNA interference (RNAi), the trophectoderm of hatched blastocysts (nine days of gestational age; dGA) was infected with a lentivirus expressing either a scrambled control or CSH-specific shRNA, prior to transfer into synchronized recipient sheep. At 90 dGA, umbilical hemodynamics and fetal measurements were assessed by Doppler ultrasonography. At 120 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies with the 3H2O transplacental diffusion technique at 130 dGA. Nutrient uptake rates were determined and tissues were subsequently harvested at necropsy. CSH RNAi reduced (p ≤ 0.05) both fetal and uterine weights as well as umbilical blood flow (mL/min). This ultimately resulted in reduced (p ≤ 0.01) umbilical IGF1 concentrations, as well as reduced umbilical nutrient uptakes (p ≤ 0.05) in CSH RNAi pregnancies. CSH RNAi also reduced (p ≤ 0.05) uterine nutrient uptakes as well as uteroplacental glucose utilization. These data suggest that CSH is necessary to facilitate adequate blood flow for the uptake of oxygen, oxidative substrates, and hormones essential to support fetal and uterine growth.

Abstract Histone lysine demethylase 1A is a master regulator of genes necessary for trophoblast cell proliferation. A proper functioning placenta is critical for pregnancy, fetal growth and development and postnatal health. Trophoblast cell proliferation and differentiation is critical for placental development and function. Recently we demonstrated that the histone lysine demethylase KDM1A binds to androgen receptor (AR) in human and sheep trophoblast cells, and targets the same promoter region of vascular endothelial growth factor A (VEGFA), suggesting a role for KDM1A and AR in early placental angiogenesis. The goal of this study was to determine the function of KDM1A during early placental development. We hypothesized that KDM1A regulates genes that are necessary for trophoblast cell proliferation, and early placental development. To this end, both in vitro and in vivo approaches were used in this study. ACH-3P cells (human first trimester trophoblast cells (CT and EVT) fused with the choriocarcinoma cell line AC1-1) were used, and a KDM1A knock out (KO) cell line was generated using CRISPR-Cas 9 based genome editing. KDM1A KO in ACH-3P cells led to significant (P<0.05) reduction in AR and VEGFA. Furthermore, factors important for cell proliferation and trophoblast cell development high mobility group AT-hook 1 (HMGA1), LIN28, and MYC protooncogene (cMYC) were significantly (P<0.05) lower in KDM1A KO ACH-3P cells. Cell proliferation assays revealed a significant (P<0.05) reduction in KDM1A KO ACH-3P cells compared to scramble controls. An in vivo experiment was conducted to demonstrate a role for KDM1A in placental development, using the sheep as a model. Day 9 hatched blastocysts were flushed and infected with a Lenti-CRISPRv2 KDM1A target construct (n=4) to knockout KDM1A specifically in the trophectoderm, or with SC (n=5). Infected embryos were transferred to recipient ewes and embryos were collected at gestational day 16. Data suggests that KDM1A KO in trophoblast cells is necessary for conceptus elongation. Current experiments are ongoing to determine the effects of KDM1A and AR knockdown using shRNA lentiviral target vectors on conceptus elongation and pregnancy. Collectively these results indicate that KDM1A plays a central role in regulating genes necessary for trophoblast cell proliferation. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2019-67015-29000 from the USDA National Institute of Food and Agriculture.