STZ (Cat#: S0130) was purchased from Sigma (Oakville, Ontario, Canada). Anti-$\alpha$SMA rabbit antibody (Cat#: ab7817), anti-Cytokeratin18 mouse antibody (Cat#: ab668), anti-Tpbp$\alpha$ rabbit antibody (Cat#: ab104401) and anti-Histone H3 (phospho S10) (p-H3S10) antibody (Cat#: ab5176) were purchased from Abcam (Cambridge, UK). Anti-CD31 rabbit antibody (Cat#: 77699) was purchased from Cell Signaling Technology (Danvers, MA). DAPI mounting medium (Cat#: F6057) was purchased from Sigma (Oakville, Ontario, Canada).

Wildtype (WT), C57BL/6J female mice, between the ages of 8–11 weeks, were maintained with a chow diet (9% fat) and water ad libitum under a 12 h light/dark cycle throughout the experiment. Prior to treatment, mice body weights, basal blood glucose levels and age were recorded. The mice were then randomly selected and administered STZ, prepared in citrate buffer (pH 7.4), at a dose of 100 mg/kg body weight (HG), or citrate buffer only (NG) on day 1 and day 4, intraperitoneally (Fig. 1A). After the second injection, the female mice were mated with male mice (C57BL/6J) overnight, and the following day at noon was considered as embryonic day 0.5 (E0.5). Random blood glucoses were measured daily after the first injection to identify the initial onset of diabetic conditions. Maternal blood glucose levels higher than 200 mg/dL were defined as a hyperglycemic pregnancy. Embryos and placentas were collected at E10.5, E12.5, E14.5 and E16.5.

Fig. 1.

STZ induced GDM results in larger placental and embryonic weight in later stages of pregnancy. (A) STZ treatment timeline. (B) Maternal age at start of treatment (NG n = 5; HG n = 10). (C) Maternal body weight at start of treatment (NG n = 5; HG n = 10). (D) Basal blood glucose at start of STZ treatment (NG n = 5; HG n = 10). (E) Maternal glucose level changes over time (NG n = 5; HG n = 10). (F) Litter size of samples collected at E14.5 (NG n = 5; HG n = 10). (G) Placenta weight changes over time (NG n = 5; HG n = 10). (H) Embryo weight changes over time (NG n = 5; HG n = 10). Data are presented as Mean $\pm$ SD.

All mouse experiments were completed according to a protocol reviewed and approved by the Institutional Animal Care and Use Committee of Texas A&M University, in compliance with the USA Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Placentas were sagittally bisected. Half of the placenta was stored at –80 ${}^{\circ }$C, while the embryos and the remaining halves were fixed in 10% formalin at room temperature. The formalin fixed embryos and placentas were processed through an ethanol-xylene series and embedded in paraffin. 5 $\mu$m thick sections were collected. Routine procedures were used for H&E staining, periodic acid–Schiff (PAS) staining, immunohistochemical (IHC) staining and immunofluorescent (IF) staining. Isotype IgG was used for immunostaining negative control (Supplementary Fig. 1).

SpA remodeling during murine pregnancy involves the replacement of VSMCs by SpA-TGCs to form high circulation, low resistance blood vessels. A well-remodeled SpA is surrounded by SpA-TGCs, with a dilated lumen to support an increased blood volume during pregnancy. The remodeling process was assessed by the level of TGCs associated with the SpAs at E14.5. Based on the percentage of the arterial wall surrounded by TGCs (Cytokeratin 18 (Cytok) positive), all observed SpAs were categorized as either unremodeled (0–25%), partially remodeled (25–75%), or remodeled (75–100%). Based on the ratio of the remodeled SpAs of each placenta, placentas were further classified as “TGC-associated” if more than 50% of the spiral arteries were remodeled or “TGC-unassociated” if less than 50% of the spiral arteries were remodeled. In addition, the lumen diameter (LD) to outer diameter (OD) ratio of the SpA was measured to determine the lumen size of the SpA.

RT-PCR was performed to measure the mRNA expression of TGC migration markers.

The LZ angiogenesis was assessed by measuring the area of the maternal lacunae space, traced by Cytok expression, and the fetal blood space, labeled by CD31 positive staining, at both E14.5 and E16.5. Images of the LZ from CD31 and Cytok co-IF staining were captured with a Leica confocal microscope. In each sample, outlines of fetal capillary and maternal lacune areas in 10,000 $\mu$m${}^2$ of the LZ layer were drawn based on CD31 staining (green) and Cytok staining (red), respectively, and the total areas were measured. The barrier between the fetal and maternal circulations, composed of endothelial cells, syncytiotrophoblasts and sinusoidal trophoblast giant cells, is defined as the IHM, and is an important structure for efficient nutrient and gas exchange [16]. To investigate placental nutrient transportation capacity, the IHM thickness was measured. Ten IHMs were randomly selected in each picture and the thinnest portion of the membrane between the fetal capillary and maternal lacunae was measured. All analyses were performed with ImageJ.

The total protein from the placentas was extracted using Cell Lysis buffer (Cell Signaling Technology, Cat#: 9803). The concentrations were measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Cat#: 23225). 15–30 $\mu$g proteins were loaded on 7–12% SDS-PAGE electrophoresis gels and transferred onto PVDF membranes (Fisher Scientific, Hampton, NH, Cat#: 45-004-110). The membranes were then blocked with 5% fat-free milk, and incubated in the primary antibodies overnight at 4 ${}^{\circ }$C. After incubation with the anti-rabbit secondary antibody (Cell Signaling Technology, Cat#: 7040) for 1 h, the signal was detected with the HRP substrate, and analyzed with the ImageJ.

The total RNA of mouse placentas was extracted using Trizol and RNA spin columns. cDNA was then synthesized using the SuperScript III Reverse Transcriptase kit (Thermo Fisher, Waltham, MA, Cat#: 18080051). RT-PCR was performed using a SYBR Green PCR master mix (Bio-Rad). Results were analyzed using the comparative C (T) method. The primers are listed in Table 1.

Embryos and placentas from at least 5 female mice per group were analyzed in this study. Data was analyzed by the student’s t-test, Chi-square test, or one-way ANOVA. Results were considered significant when the p values were less than 0.05. All statistical analyses were carried out using GraphPad Prism.

Before pregnancy, female mice demonstrated synonymous age and body weight (Fig. 1B,C). The blood glucose of the HG and NG groups were normal (Fig. 1D). Daily random blood glucose checks identified a significant increase of blood glucose levels in the HG group beginning at E2.5. At E3.5, 80% (8 out of 10) of the HG mice reached a hyperglycemic condition ($>$200 mg/dL), displaying blood glucose levels of 289 $\pm$ 123.9 mg/dL. By E4.5, all of the HG mice had obtained this hyperglycemic condition, which persisted throughout the pregnancy. At E14.5, the blood glucose levels of the HG group were as high as 555.7 $\pm$ 32.2 mg/dL, while the NG group remained at 160.3 $\pm$ 15.6 mg/dL (Fig. 1E). To be noted, the hyperglycemic status did not affect the litter size in this study (Fig. 1F).

Increased placental weights were observed in the HG group beginning at E12.5 and reached significance at E16.5 (Fig. 1G). The HG group also showed a significantly larger embryonic weight at E14.5 and E16.5 (Fig. 1H). The placental efficiency of the HG placentas was lower at E12.5, but this observation was reversed at E16.5 (Supplementary Fig. 1).

The effect of GDM on placenta morphology was assessed at E14.5. An increased thickness in the junctional zone (JZ) was observed in the HG placentas (Fig. 2A, white arrow). Additionally, the HG placenta displayed a larger portion of the whole placenta to be occupied by the JZ and the LZ to take up a smaller portion, when compared to the NG placenta (Fig. 2B). This resulted in a significantly smaller LZ to JZ area ratio in HG placentas (Fig. 2C). PAS staining was performed to specifically label the glycogen trophoblast cells (GlyTs), which are enriched with glycogen (Fig. 2D). Thus, the PAS negative cell population was majorly composed of spongiotrophoblast cells (SpTs). The PAS staining showed no difference in the SpT area ratios in the JZ area of the HG and NG groups. However, a larger ratio of GlyT area was noticed in the HG group (Fig. 2F). In addition, the total area of the venous sinus was significantly decreased in the JZ of the HG group (Fig. 2G). Tpbpɑ staining (Fig. 2H) showed that the number of SpTs is not changed, while the GlyT cell count increased significantly (Fig. 2J,K). Western blots performed on the whole placenta showed that hyperglycemia enhanced the level of Tpbpɑ (Fig. 2I), a protein highly expressed in GlyT, which is consistent with the increased total area of GlyTs.

Fig. 2.

E14.5 GDM placentas have larger JZ areas with increased GlyTs. (A) JZ visibly occupies a larger area of the placenta. Slide image from PAS staining. (B) JZ and LZ area proportion within the placenta (NG n = 8; HG n = 9). (C) LZ to JZ ratio (NG n = 8; HG n = 9). (D) PAS staining of GlyTs within the JZ and IF staining of Tpbp$\alpha$. (E) Area proportion of JZ occupied by SpTs (NG n = 9; HG n = 10). (F) Area proportion of JZ occupied by GlyTs (NG n = 9; HG n = 10). (G) Area proportion of JZ occupied by venous sinuses (NG n = 9; HG n = 9). (H) Tpbpɑ protein expression localization in placenta. (I) Tpbpɑ protein expression level in whole placenta lysate. (J) Numbers of SpT cell in the entire JZ (NG n = 5; HG n = 5). (K) Numbers of GlyT cell in the entire JZ (NG n = 5; HG n = 5). Data are presented as Mean $\pm$ SD.

The decidua (DZ) layer was found to have fewer TGCs, marked by Cytok expression (Cytok+), surrounding the SpAs (CD31+) in the HG placentas (Fig. 3A). When all TGC associations to SpAs were evaluated together, the HG placenta presented more unremodeled SpAs, and less partially and fully remodeled SpAs (Fig. 3B). As shown in Table 2, 9 out of the 10 NG placentas were graded as “TGC-associated”, while the HG group only had 3 out of 9 TGC-associated placentas. The placentas from the HG group also had a significantly smaller LD/OD ratio (Fig. 3C,D). The SpA remodeling level was further evaluated by the expression of ɑ-SMA, a marker of VSMCs that surround the SpA. The HG placenta showed a higher ɑ-SMA expression on the SpA (Fig. 3E), and less apoptotic cells surrounding the SpAs (Fig. 3F), suggesting hindered remodeling. Consistently, the mRNA expression level of Acta2, which is an alias for ɑ-SMA [17], was found to be significantly higher in the HG placentas at E12.5 (Fig. 3G), suggesting an increased presence of VSMCs, hence decreased remodeling by TGCs.

Fig. 3.

GDM hinders SpA remodeling evidenced by decreased TGC association, VSMC breakdown, and mRNA expression of migratory genes. (A) Co-IF of CD31, Cytok, and DAPI. SpAs and TGCs are visualized by CD31 and Cytok, respectively. (B) Ratio of classification of TGC association by sample (NG n = 9; HG n = 10). (C) Vascular wall structure visualized by H&E staining. (D) LD to OD ratio indicating degree of vascular remodeling (NG n = 10; HG n = 10). (E) SpA VSMC visualized by ɑ-SMA IHC. (F) TUNEL staining on SpA. SpA indicated by the black arrow. #TUNEL+ cell around the SpA (NG n = 5; HG n = 5). (G) mRNA level of genes involved in TGC migration and SpA vascular wall composition at E12.5 (NG n = 4; HG n = 4). Data are presented as Mean $\pm$ SD.

The reduced number of SpA-TGCs and discrepancies observed in the SpA wall suggested a migration problem of the TGCs. Thus, the influence of GDM on the expression of genes involved in TGC migration was then examined. Mmp2, Mmp9 and Mmp14 are pro-migratory proteins, while Tgf$\beta$ is anti-migratory [10, 18]. Wnt5a is known to induce cell migration [19, 20]. A significant decrease in Mmp2, Mmp9, Mmp14, and Wnt5a mRNA transcripts, and an increase in the Tgf$\beta$ levels were observed in the HG placentas (Fig. 3G).

The HG group had a decreased trend of fetal blood space and the maternal lacunae area at both E14.5 and E16.5, however neither showed statistical significance (Fig. 4A–C). The mean of the maternal lacunae and fetal capillary space also did not show any differences between the HG and NG groups (Fig. 4D). The HG placentas were found to display a thicker IHM compared to the NG placentas at E16.5 (Fig. 4E).

Fig. 4.

GDM increases IHM thickness. (A) Co-IF of CD31, Cytok, and DAPI. Zoomed in example shows the IHM (white bar), MLA (yellow line) and FCA (cyan line) that was measured for (B) (C) (E). (B) FCA defined by area enclosed by CD31 (NG n = 7; HG n = 7). (C) MLA defined by area encircled by Cytok and not occupied by fetal capillary or DAPI (NG n = 7; HG n = 7). (D) Mean of FCA and MLA (NG n = 7; HG n = 7). (E) IHM thickness (NG n = 7; HG n = 7). Data are presented as Mean $\pm$ SD.

To gain an insight of how hyperglycemia impacts embryonic development, embryos were harvested at E14.5. The number of congenital defects, including the neural tube defects (NTD), ocular defects, craniofacial defects, and heart defects, were counted (Fig. 5A,B). 11 out of 73 of the HG embryos displayed at least one type of the evaluated birth defects, while no birth defects were observed in the NG group (Table 3, p 0.001 by Chi-square test). These congenital defects included 6 cases of NTDs, 3 cases of ocular defects and 1 case of a craniofacial defect (Fig. 5A,B). Statistically, the HG group had significantly more incidences of NTDs than the NG group (Table 3, p 0.01 by Chi-square test). The incidence of heart defects, including atrial septum defects, ventricular septum defects and outflow tract defects, was not different between the HG and NG groups, according to the examination of 7 NG and 17 HG hearts randomly selected (Table 4, 1/17 vs 0/7, p $>$ 0.05). Edema was also observed in 2 out of 73 HG embryos, showing no statistical difference from the NG group (Table 3). Embryonic hearts were further examined for the interventricular septum (IVS) thickness and the presence of cardiac hypertrophy or hyperplasia. Overall, dense ventricles and increased ventricular weight of the embryonic heart were observed in the HG group compared to the NG group (Fig. 5C,D). Specifically, a thicker IVS was observed in the HG embryo (Fig. 5C,E). The HG embryonic heart also had an increased myocardial area of both the left and right ventricle (Fig. 5C,F). IF staining of p-H3S10 was performed on heart sections at E12.5 to determine if proliferation of ventricular cardiomyocytes was affected by hyperglycemia. A significant increase in the percentage of proliferating cardiomyocytes, labeled by p-H3S10 expression, was found in the HG group, when compared to the NG group (Fig. 5G,H). A significant increase in Cdk6, Cyclin A, Cyclin B2 and Cyclin D2 mRNA transcripts was observed in the HG heart ventricle (Fig. 5I). These results suggested cardiac hyperplasia in the HG embryos.

Fig. 5.

GDM increases the incidence of hyperplastic ventricular disease and congenital malformations. IHM thickness is increased in placentas from malformed embryos. (A) Normal embryo at E14.5. (B) Observed congenital malformations include NTD, ocular defect, craniofacial defect, and edema. (C) H&E staining of E14.5 embryonic hearts labeled with LV, RV, and IVS. (D) Heart weight (NG n = 7; HG n = 7). (E) IVS thickness (NG n = 4; HG n = 4). (F) Myocardium tissue area that occupies the left and right ventricle individually (NG n = 10; HG n = 10). (G) p-H3S10 IF staining of heart sections at E12.5. (H) p-H3S10 positive cell percentage at E12.5 (NG n = 4; HG n = 4). (I) mRNA level of genes involved in cell cycle and proliferation at E12.5 (NG n = 4; HG n = 4). (J) IHM thickness in placenta from normal embryos and embryos with congenital defects (NG n = 5; HG n = 3). (K) GlyT/SpT ratio in placenta from normal embryos and embryos with congenital defects (NG n = 5; HG n = 3). (L) SpA LD/OD ratio in placenta from normal and congenital defect embryos (NG n = 5; HG n = 3). Data are presented as Mean $\pm$ SD.

To understand if the congenital birth defects were associated with any abnormal feature of the placenta, placental samples with congenital defects, regardless of them being in the NG or HG group, were pooled in a “Congenital Defect” group, while the rest were grouped as “Normal”. One-way ANOVA was performed on the data of GlyT/SpT ratio, the LD/OD ratio and the IHM thickness. A Chi-square test was performed on the TGC-SpA association data in terms of congenital defect group versus the normal group. The results showed a marginal significance of IHM thickness and the congenital defects (p = 0.085), but not for the GlyT/SpT ratio (p = 0.200), the LD/OD ratio (p = 0.207) and the TGC-SpA association (p = 0.222) (Fig. 5J–L).