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Published in final edited form as: Lab Invest. 2023 Jan;103(1):100017. doi: 10.1016/j.labinv.2022.100017

FoxO1 Deficiency Enhances Cell Proliferation and Survival Under Normoglycemia and Promotes Angiogenesis Under Hyperglycemia in the Placenta

Zehuan Ding a, Naomi McCauley a, Yushu Qin a, Lauren Lawless a, Shaodong Guo a, Lanjing Zhang b,c, Ke K Zhang a,d, Linglin Xie a,*
PMCID: PMC11890199  NIHMSID: NIHMS2058037  PMID: 36748194

Abstract

FoxO1 is an important transcriptional factor that regulates cell survival and metabolism in many tissues. Deleting FoxO1 results in embryonic death due to failure of chorioallantoic fusion at E8.5; however, its role in placental development during mid-late gestation is unclear. In both human patients with gestational diabetes and pregnant mice with hyperglycemia, placental FoxO1 expression was significantly increased. Using FoxO1+/− mice, the effects of FoxO1 haploinsufficiency on placental development under normoglycemia and hyperglycemia were investigated. With FoxO1 haploinsufficiency, the term placental weight increased under both normal and hyperglycemic conditions. Under normoglycemia, this weight change was associated with a general enlargement of the labyrinth, along with increased cell proliferation, decreased cell apoptosis, and decreased expression of p21, p27, Casp3, Casp8, and Rip3. However, under hyperglycemia, the placental weight change was associated with increased fetal blood space, VEGFA overexpression, and expression changes of the angiogenic markers, Eng and Tsp1. In conclusion, FoxO1 plays a role in regulating cell proliferation, cell survival, or angiogenesis, depending on blood glucose levels, during placenta development.

Keywords: angiogenesis, apoptosis, FoxO1, hyperglycemia, placental development, proliferation

Introduction

Diabetes during pregnancy is known to affect the health of both the mother and their infant. Gestational diabetes mellitus (GDM) occurs in approximately 7.6% of pregnancies in the United States.1 In 2017, there were an estimated 21.3 million births affected by gestational hyperglycemia, globally.2 Because nearly half of the population with diabetes remains undiagnosed,2 the actual number of mothers and newborns affected by diabetes or hyperglycemia is estimated to be even greater.

The placenta, an interactive barrier between the mother and fetus, is responsible for the exchange of nutrients and gas and the generation of important hormones that support pregnancy and fetal development. To satisfy the increasing nutritional requirements of the fetus, maturation of the placental villi is accomplished by well-controlled proliferation and angiogenesis. Maternal diabetes can obstruct the development of the placenta by impacting many different aspects, such as placental weight, angiogenesis, vasculature maturity, and inflammation levels.3–5 Placentas in GDM are characterized by villous immaturity and edema, syncytial nodes, fibrin thrombi, and fibrinoid necrosis.6 Immature vascularization leads to fetal growth restriction and even death.7,8 The physiological progression of pregnancy can be complicated by various factors, including the incidence and severity of hyperglycemia, the presence of insulin resistance, and the time of onset. Therefore, more studies are warranted to unveil the specific mechanisms in which diabetes affects placental and fetal development.

FoxO1 is an essential transcription factor involved in many developmental and metabolic processes. Its activity is tightly controlled by posttranslational modifications, such as phosphorylation and acetylation. FoxO1 phosphorylation occurs at 3 different conserved amino acid sites, Thr24, Ser256, and Ser319, and is mediated by AKT or serum/glucocorticoid-regulated kinase signaling, which further induces its nuclear exportation and protein degradation.9,10 One major role of FoxO1 is to promote gluconeogenesis through the IRS-PI3K-AKT pathway.11,12 FoxO1 has also been found to regulate angiogenesis via downstream factors, such as VEGFA and AKT.13,14 Notably, FoxO1 has been reported to play an important role in placental development.15–17 It is expressed in both endothelial and syncytiotrophoblast cells in the human placenta during late gestation16 and in the endothelial cells of the mouse placenta.17 Deleting FoxO1 results in embryonic death due to failure of chorioallantoic fusion at E8.5.15

In this study, FoxO1-deficient pregnant mice with streptozotocin (STZ)–induced hyperglycemia were used to examine the placenta for morphologic changes and its underlying mechanisms. In this study, we examined how FoxO1 deficiency impacts placental development under normoglycemia and hyperglycemia.

Materials and Methods

Antibodies

The antibodies against Histone H3 (phospho-S10) (ab5176), Tpbpα (ab104401), VEGFR2 (ab2349), VEGFA (46154), and Cytokeratin (ab668) were purchased from Abcam. The antibodies against FoxO1 (2880), FoxO1 (phospho-S256) (9461), and CD31 (77699) were purchased from Cell Signaling Technology.

Mice

Generation of FoxO1fl/+ mice has been described previously.18 They were crossed with EIIa-Cre mice to generate a germline knockdown of FoxO1. FoxO1+/−; EIIaCre/+ mice were then crossed with wild-type (WT) mice to exclude the EIIa-Cre allele. As shown in Supplementary Figure S1, the knockdown of the FoxO1 allele was confirmed by genotyping the tissues from both the placenta and the embryo.

Study Design

WT or FoxO1+/− female mice aged 9 to 10 weeks were maintained on a chow diet and water ad libitum, under a 12-hour light/dark cycle, throughout the experiment. Hyperglycemia was induced using an STZ injection. The time and the dose of STZ were determined previously, in which hyperglycemic conditions were achieved after pregnancy establishment to avoid ovulation and implantation issues.19 Before treatment, the body weights and basal blood glucose levels of the mice were recorded (Supplementary Fig. 2A–C). Mice were randomly selected and intraperitoneally injected with either citrate buffer (pH 7.4) only (normoglycemia group) or STZ (Sigma-Aldrich) dissolved in citrate buffer at a dose of 100 mg/kg of body weight (hyperglycemia group) on day 1 and day 4. After the second injection, the female mice were mated with WT males overnight, and the next day at noon was considered embryonic day 0.5 (E0.5). Because the placental tissue is composed of cells from both maternal and fetal origin, a combination of different genotypes is possible. To address this, the placentas collected were divided into 3 groups: WT-WT, F-WT, and F-F. The “-” separates the placental and fetal genotypes (WT: wild-type; F: FoxO1+/−). Random blood glucose levels were measured daily to track the blood glucose changes. Embryos and placentas were collected and weighed at E10.5, E12.5, E14.5, E16.5, and term. Placentas were bisected, and half of each placenta was then microdissected to separate the labyrinth from the decidua and stored at −80 °C, whereas the remaining half was fixed in 10% formalin. Amniotic fluid glucose levels were measured during sample collection.

Human Placenta Samples

Decoded human paraffin-embedded placenta slides were obtained (from L.Z.; institutional review board number, BN2313, approved at Princeton Medical Center). Immunohistochemistry (IHC) was performed on these slides. The samples were then photographed and evaluated in a single-blinded manner for the staining intensity of the villi and basal plate. The staining intensity was scored as 1, 2, or 3, with 1 being the lowest.

Histology of Mouse Placenta

The histology of the mouse placenta was evaluated using hematoxylin and eosin staining. IHC staining was performed as previously described.19 Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining, using the ApopTag Peroxidase or ApopTag Red In Situ Apoptosis Detection Kit (MilliporeSigma) and performed according to the manufacturer’s instructions.

Western Blot

Total protein from the labyrinth and decidual tissue was extracted using Cell Lysis Buffer (Cell Signaling Technology). Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). An amount of 15 to 30 μg of protein was loaded on a 7% to 12% SDS-PAGE gel for electrophoresis and transferred onto Immun-Blot polyvinylidene fluoride membranes (Bio-Rad). The membranes were then blocked with 5% fat-free milk and incubated in primary antibodies overnight at 4 °C. After incubation with anti-rabbit secondary antibodies (Cell Signaling Technology) for 1 hour, the signal was detected with the HRP substrate and analyzed with the ImageJ.

Real-Time PCR

Total RNA was extracted from the labyrinth and decidua using Trizol reagent (Thermo Fisher Scientific) and purified using RNA spin columns. Total RNA (500 ng) was reverse transcribed using a SuperScript III Reverse Transcriptase Kit (Invitrogen). qPCR was performed using a SYBR Green PCR Master Mix (Bio-Rad). Primer sequences are listed in Supplementary Table S1. Results were analyzed using the comparative threshold cycle (CT) method, with Cyclophilin A as a normalization control.20

Results

FoxO1 Was Expressed in the Syncytiotrophoblasts and Endothelial Cells of the Placenta

To identify the localization of FoxO1, IHC staining on human term placentas and mouse placentas, from E10.5 to term, was performed. In the human placenta, positive staining was observed in the placental villi and the maternal basal plate. Within the villi, FoxO1 was localized in the fetal vascular endothelial cells (Fig. 1A, red arrow) and the cytotrophoblast (Fig. 1A, black arrow). In the decidua, FoxO1 was strongly expressed in the spiral artery endothelial cells (Fig. 1A, green arrow). Endometrial stromal cells also displayed a weaker expression of FoxO1 (Fig. 1A, yellow arrow). In the mouse placenta, FoxO1 was consecutively expressed in both endothelial cells and syncytiotrophoblasts in the labyrinth, as well as the spiral artery endothelial cells in the decidua (Fig. 1B). There was no expression of FoxO1 in the junctional zone (Fig. 1B). The IHC staining also showed a temporal expression pattern of FoxO1 between E10.5 and term.

Figure 1.

Figure 1.

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FoxO1 expression in placentas of human and mouse. (A) FoxO1 immunohistochemistry staining in human term placenta. Red arrow: endothelial cells in villi; black arrow: cytotrophoblasts in villi; green arrow, spiral artery endothelial cells; yellow arrow, endometrial stromal cells. (B) Immunohistochemistry staining of FoxO1 in mice placentas at E10.5, E12.5, E14.5, E16.5, and term. Red lines indicate the borderline of different layers in the placenta; green arrows indicate FoxO1-positive signals. D, maternal decidua; JZ, junctional zone; LZ, labyrinth.

FoxO1 Expression Was Increased in the Human and Mouse Placenta Under Gestational Diabetes Mellitus

To investigate whether the placental FoxO1 expression is affected by GDM, FoxO1 IHC staining was performed on the term placentas from women who were either healthy or diagnosed with GDM (Fig. 2A, B). In both the placental villi and basal plate, the intensity scores were significantly higher in the GDM group than in the normal group (Fig. 2C, D).

Figure 2.

Figure 2.

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FoxO1 expression in human and mouse placentas under normal and hyperglycemic conditions. (A and B) FoxO1 immunohistochemistry staining performed in healthy women and those with GDM. (C) Staining intensity score in the placental villi. (D) Staining intensity score in the basal plate. (E) Western blot analysis of FoxO1 expression in E14.5 WT placentas under normal and hyperglycemic conditions. (F) Quantifications of the FoxO1:GAPDH ratio. Data are presented as the mean ± SE, N = 4 to 5, *P < .05, **P < .01. GDM, gestational diabetes mellitus; HG, hyperglycemia; NG, normoglycemia.

To test whether FoxO1 expression is affected by hyperglycemia consistently in the mouse placenta, western blot was performed using proteins from the WT placenta under normoglycemia (normoglycemia group) or hyperglycemia (hyperglycemia group). Hyperglycemia was induced using STZ injection, as previously reported.19 The glucose level after the injection was monitored (Supplementary Fig. S2D–F). Consistent with the observations in human placentas, the FoxO1 expression level was significantly upregulated under hyperglycemia (Fig. 2E, F).

FoxO1 Heterozygous Mice Expressed Lower Levels of FoxO1 in the Placenta

FoxO1 expression was subsequently evaluated in E14.5 FoxO1+/− placentas. Compared with the WT-WT group, the F-WT group showed a decreasing trend in FoxO1 expression (P =.0735), whereas the F-F mice demonstrated significant downregulation of FoxO1 (Fig. 3A, B). Consistent with the western blot results, FoxO1 IHC staining also exhibited a decreased staining intensity in the F-F placentas compared with the WT-WT (Fig. 3C).

Figure 3.

Figure 3.

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FoxO1 expression in E14.5 WT and FoxO1+/− placentas. (A) Western blot analysis of FoxO1 expression in placentas. (B) Quantifications of the FoxO1:GAPDH ratio. (C) Immunohistochemistry staining of FoxO1 in decidua and labyrinth of mouse placentas. Red arrows indicate representative FoxO1-positive signals. Data are presented as the mean ± SE, N = 4 to 5, **P < .01. WT, wild-type.

FoxO1 Deficiency and Hyperglycemia Independently Increased Embryo and Placenta Weight Toward the End of Gestation

Consistent with previous reports, the FoxO1−/− embryos displayed severe developmental delays or defects and did not survive through E10.5 (Supplementary Fig. S3A). Thus, the outcome of FoxO1 haploinsufficiency and maternal hyperglycemia was evaluated by measuring the embryonic and placental weights. At E16.5, the embryonic and placental weights of the WT-WT offspring were increased under a maternal hyperglycemic condition, which was maintained until term (Fig. 4A–D). Interestingly, FoxO1 deficiency (F-F group) led to a further increase in both the embryonic and placental weight at term but not at E16.5 under both normoglycemia and hyperglycemia (Fig. 4A–D). Overall, the F-WT group did not show any changes in either the embryonic or placental weights at E16.5 or term, regardless of the glucose level. From a gross perspective, no birth defects or placental abnormalities were observed (Supplementary Fig. S4). The number of offspring per treatment group was evaluated to understand whether the changes in embryonic or placental weights were due to litter size. However, litter size differences were not observed among all groups (Fig. 4E). These results suggested that FoxO1 deficiency can lead to placental and embryonic overgrowth, independent of the glucose level.

Figure 4.

Figure 4.

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Embryonic and placental weight in WT and FoxO1+/− mice under normal or hyperglycemic conditions at different gestational stages. (A) Embryo weight at E16.5. (B) Placenta weight at E16.5. (C) Embryo weight at term. (D) Placenta weight at term. (E) Litter size of the pregnancy. Data are presented as the mean ± SE, N = 6 to 7, *P < .05. HG, hyperglycemia; NG, normoglycemia; WT, wild-type.

FoxO1 Deficiency Increased Labyrinth Size Under Normal Conditions

Tpbpα IHC staining was performed on the E14.5 placentas to evaluate morphologic alterations in the placental layers. Tpbpα is a specific marker for the spongiotrophoblasts and glycogen trophoblasts in the junctional zone, allowing the 3 layers of the placenta to be distinguished. The results showed that the labyrinth layer was significantly enlarged in the F-F group compared with the WT-WT group under normoglycemia but not under hyperglycemia (Fig. 5A, B). The relative junctional zone size was not different between any 2 groups (Fig. 5A, C).

Figure 5.

Figure 5.

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FoxO1 deficiency increased relative labyrinth volume under normal conditions but not hyperglycemic conditions. (A) Immunohistochemistry staining of Tpbpα in placentas at E14.5. (B) Relative labyrinth volumes were evaluated using the ratio of the labyrinth area to the placenta area. (C) Relative junctional zone volumes were evaluated using the ratio of the junctional zone area to the placenta area. Data are presented as the mean ± SE, N = 6 to 7, *P < .05. HG, hyperglycemia; NG, normoglycemia.

Decreased Apoptosis and Increased Proliferation Were Observed in the Labyrinth of the FoxO1-Deficient Placentas Under Normal Conditions

To understand the mechanisms underlying the enlargement of the labyrinth layer in the FoxO1-deficient placentas, terminal deoxynucleotidyl transferase dUTP nick end labeling and pH3S10 staining were performed to examine the apoptosis and proliferation levels, respectively. The placenta experiences accelerated growth after E10.5, reaches its growth peak around E12.5, and continues to grow in size and complexity from E14.5 to term.21,22 Thus, placental samples at E12.5 and E14.5 were evaluated. At E14.5, a limited number of apoptotic cells were observed, and there was no significant differences between any 2 groups (Supplementary Fig. S5). At E12.5, the number of apoptotic cells per given labyrinth area was significantly lesser in the F-F group than in the WT-WT group only under normoglycemia (Fig. 6A, B). The expression of a panel of genes involved in apoptosis was further detected in the E12.5 placenta by qPCR. The results showed decreased levels of Casp3 and Casp8 in the F-F placenta versus the WT-WT placenta under normoglycemia but not under hyperglycemia (Fig. 6C). In addition, the expression of Rip3, a marker for necrosis, was also lower in the F-F placenta than in the WT-WT placenta under normoglycemic conditions only.

Figure 6.

Figure 6.

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FoxO1 disruption promoted proliferation and inhibited apoptosis under euglycemic conditions but not hyperglycemic conditions. (A) TUNEL staining in the labyrinth for placenta at E12.5. Red arrows indicate the cell with TUNEL-positive staining. (B) The number of TUNEL+ cells per square micrometer in the labyrinth layer. (C) Relative expression levels of Casp2, Casp3, Casp8, Parp1, and Rip3 in the placenta at E12.5. (D) pH3S10 staining in the labyrinth of the placenta at E12.5. (E) The number of pH3S10+ cells per square micrometer in the labyrinth layer. (F) Relative expression levels of p21, p27, and Pten. Data are presented as the mean ± SE, N = 4, *P < .05. HG, hyperglycemia; NG, normoglycemia; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WT, wild-type.

The pH3S10 IHC staining, which marks proliferating cells at the G2/M phase, was used to evaluate cell proliferation at E12.5 and E14.5. At E14.5, the percentage of proliferating cells was similar among all groups (Supplementary Fig. S6). However, at E12.5, the number of pH3S10+ cells per given area was significantly increased in the F-F group compared with the WT-WT group, only under normoglycemic conditions (Fig. 6D, E). It has previously been reported that expressions of p21, p27, and Pten are dependent on FoxO1.23–25 Consistently, the expression of p21 and p27 was decreased in the F-F placenta at E12.5 under normal glucose level conditions, compared with the WT-WT placenta (Fig. 6F). However, expression levels of these genes were not different among the 3 groups under hyperglycemia (Fig. 6F).

FoxO1 Deficiency Increased Labyrinth Angiogenesis Under Hyperglycemia

The proper formation of both the fetal vascular network and maternal lacunae in the labyrinth is essential to ensure the normal growth of the embryos. Therefore, the fetal vascular area and maternal lacunae area were further evaluated by PECAM-Cytokeratin co-IF staining (Fig. 7A). The area of the fetal vasculature and maternal lacunae were defined by PECAM and cytokeratin, respectively.19 The hyperglycemic condition induced significantly larger fetal vascular spaces in both the F-WT and F-F groups compared with their normal glucose level counterparts (Fig. 7B), suggesting that maternal hyperglycemia can promote fetal angiogenesis. In addition, the F-F group showed a significant increase in fetal vascular spaces compared with the WT-WT group, only under hyperglycemic conditions (Fig. 7B). However, no significant differences were observed in the maternal lacunae area between the groups (Fig. 7C).

Figure 7.

Figure 7.

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FoxO1 disruption promoted labyrinth angiogenesis. (A) Co-IF staining of PECAM (green) and cytokeratin (red) in the labyrinth, counterstained with DAPI (blue). (B) Relative fetal blood space (normalized to the WT-WT NG group) observed in a randomly selected 0.025-mm2 area. (C) Relative maternal lacunae space (normalized to the WT-WT NG group) observed in a randomly selected 0.025-mm2 area. (D) Western blot analysis of FoxO1 and p-FoxO1 expression in WT-WT, F-WT, and F-F placentas under normal or hyperglycemic conditions. (E) Western blot analysis of VEGFA and VEGFR2 expression in WT-WT and F-F placentas under normal or hyperglycemic conditions. (F) Relative expression levels of Eng and Tsp1 in the placenta at E14.5. Data are presented as the mean ± SE, N = 6 to 7, *P < .05, **P < .01. HG, hyperglycemia; NG, normoglycemia; WT, wild-type.

A significant reduction in FoxO1 expression in the F-F, but not the F-WT, placenta compared with the WT-WT placenta was confirmed using western blot (Fig. 7D). Interestingly, the F-F placenta also had an increased level of p-FoxoO1/FoxO1, suggesting more active gene degradation in the mutant placenta (Fig. 7D). This is possibly due to the removal of exon 2, which may produce unstable FoxO1 and increase its degradation in the FoxO1+/− cells. Moreover, hyperglycemia did not result in further changes in the p-FoxO1:FoxO1 ratio.

Expression levels of VEGFA and VEGFR2 were measured in the dissected labyrinth layer of the placenta at E12.5, which is when angiogenesis dramatically occurs in the placenta. There was a trend of increase in the expression of VEGFA in the F-F group versus the WT-WT group, and this difference was enhanced under hyperglycemia (Fig. 7E). Unlike VEGFA, VEGFR2 expression remained unchanged between the groups (Fig. 7E). The expressions of additional angiogenic markers in the labyrinth layer, including Eng and Tsp1, were measured by qPCR. Consistently, a significant increase in Eng and decrease in Tsp1 expression was detected in the F-WT and the F-F groups under hyperglycemia, compared with the respective groups under normoglycemia (Fig. 7F).

Discussion

FoxO1 is an important transcriptional factor that regulates cell survival and metabolism in many tissues. Our findings showed similarity with previous studies that reported placental FoxO1 expression to be significantly increased in both human GDM and hyperglycemic pregnant mice. This study further investigated the effects of FoxO1 haploinsufficiency on placental development under normoglycemia and hyperglycemia. Using FoxO1+/− mice, we showed that FoxO1 deficiency promotes cell proliferation and inhibits cell apoptosis, which ultimately enlarged the labyrinth under normoglycemia. However, under hyperglycemia, FoxO1 deficiency promoted angiogenesis without changing the relative size of the labyrinth.

Under normal glucose level conditions, FoxO1 deficiency increased both placental and embryonic weights at term, suggesting that FoxO1 plays an important role during the late stages of pregnancy when rapid growth occurs. Consistent with this finding, the labyrinth was significantly larger in FoxO1-deficient placentas under normal glucose level conditions, possibly owing to decreased apoptosis and enhanced proliferation. These findings are consistent with previous reports that showed FoxO1 upregulation to inhibit cell proliferation and induce apoptosis in other tissues, such as the skin and pancreas.26,27 It has been reported that inactivating FoxO1 decreases the expression of negative cell cycle regulators, such as p27.23–25,28 This study confirmed that FoxO1-deficient placentas have lower messenger RNA levels of the negative cell cycle genes (p21 and p27), proapoptotic genes (Casp3 and Casp8), and the necrotic gene (Rip3). Together, these results support the idea that FoxO1 plays a role in placental development by regulating cell survival and proliferation in the labyrinth layer.

Pathologic disorders such as obesity, insulin resistance, and diabetes interfere with FoxO1 function in various tissues. It has been reported that the loss of FoxO1 leads to severe vasculature malformation during early placental development.15 The vascular endothelial growth factor family is one of the major angiogenic factors that function throughout placental development.29,30 FoxO1 deficiency, specifically under hyperglycemia, enhanced VEGFA and Eng expressions while suppressing Tsp1 expression, which suggests increased labyrinth angiogenesis under hyperglycemia. This finding is consistent with the observation that FoxO1 deficiency enlarged the labyrinth area under hyperglycemic conditions. These data suggested that FoxO1 regulates placenta angiogenesis through VEGFA and other angiogenic factors under hyperglycemia. The explanation as to why this is only observed under hyperglycemia remains unclear. Interestingly, ischemia caused by diabetes is known to disrupt the function of endothelial cells, leading to defective angiogenesis, which could justify this effect.31 In the WT-WT placentas, hyperglycemia increased VEGFA levels without changing the fetal blood space area. It is possible that FoxO1 deficiency augmented this effect owing to some unknown mechanisms.

Another intriguing point is that the F-F labyrinth, under hyperglycemia, displayed significantly enlarged fetal capillaries without any changes in cell survival or cell proliferation at E12.5. This could be due to FoxO1 acting as an essential endothelial growth checkpoint by inducing endothelial quiescence and synchronously decreasing the proliferation and metabolism of endothelial cells.13,32 Furthermore, it must be noted that cell proliferation and angiogenesis are dynamic processes. Cell proliferation and VEGFA expression were only examined at E12.5. Therefore, further investigation should focus on detecting dynamic changes in cell survival, proliferation, and angiogenesis at multiple time points during placental development to clarify this phenomenon.

It has been suggested that there is a strong association between diabetic pregnancies and increased birth weights.33 In addition, placental weight has also been shown to be increased during pregestational diabetes or gestational diabetes.33,34 In this study, STZ-induced diabetes in mice significantly increased the weight of both the placenta and embryo from E16.5 and beyond, which is consistent with clinical observations in humans.35 Additionally, FoxO1 deficiency increased both placental and embryonic weights at term, under both hyperglycemic and normoglycemic conditions. However, hyperglycemia and FoxO1 deficiency did not have synergic effects on increasing the placental and fetal weight. Under normoglycemia, this could be due to increased cell proliferation and decreased cell death in the labyrinth layer. Under hyperglycemia, it is possible that the heavier term placenta was at least partially due to increased fetal blood in the enlarged fetal capillaries. However, the data found in this study could not explain why FoxO1 deficiency led to a larger fetus at term. Thus, we hypothesize that additional mechanisms regarding placenta-fetal adaptation must be involved. Nonetheless, these data suggest that an enlarged placenta leads to enlarged embryos under FoxO1 insufficiency.

In summary, the role of FoxO1 during placental development was investigated using FoxO1-deficient mice under different glucose level conditions. Results from this study demonstrate that FoxO1 regulates placental development by altering cell proliferation, cell survival, or angiogenesis, depending on the glucose level. This knowledge broadens our understanding of placental development in normal and GDM pregnancies.

Supplementary Material

Supplementary material

Acknowledgementss

FoxO1fl/+ mice were obtained as a gift from the Department of Nutrition, Texas A&M University, College Station, Texas (S.G.’s laboratory).

Funding

This work was funded, in part, by a grant from the National Institute of Environmental Health Sciences (P30 ES0 29067).

Footnotes

Declaration of Competing Interest

The authors declare no conflict of interest.

Ethics Approval and Consent to Participate

The retrospective human study component of this article used deidentified human tissues that were obtained during routine health care. The study has been reviewed and approved as an exempt human study (category 4) by the institutional review board at Princeton Medical Center, Plainsboro, New Jersey.

Supplementary Material

The online version contains supplementary material available at https://doi.org/10.1016/j.labinv.2022.100017

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Supplementary material
NIHMS2058037-supplement-Supplementary_material.pdf (3.6MB, pdf)

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