The present study is a secondary analysis of a previous randomized trial in which control (n = 15) and GDM patients with a singleton fetus (n = 30) were enrolled between weeks 24 and 28 of pregnancy and either treated (n = 15) or not (n = 15) with a diet enriched in three tablespoons of crude EVOO daily (36 g/day) from enrollment and until term (22). The present work evaluated in the same cohort circulating insulin resistance markers and MMP activity as well as placental morphology, storage metabolites and proteins, and regulators of the placental endocytic pathway.

The patients were enrolled at Hospital Ignacio Pirovano, Buenos Aires, Argentina. GDM was diagnosed according to the diagnostic criteria of Latin American Diabetes Association (ALAD)/Argentine Society of Diabetes (SAD) (glycemia values >99 mg/dl in two measurements or glycemia values >140 mg/dl at 2 h after a universal p75g oral glucose tolerance test) (38). The study was approved by the Ethics and Research Committee of the Pirovano Hospital (Review Board Project: DI-2016-29-HGAIP) and registered at the Ministry of Health of the City of Buenos Aires, IF-2016-22533767. All participants provided written informed consent. Exclusion criteria included body mass index (BMI) over 30 kg/m² and concurrent pathologies. Detailed exclusion criteria, design, adherence to the dietary treatment, and outcomes of the randomized trial have been previously reported (22).

Maternal blood samples were collected at enrollment and at term, whereas both blood samples and placentas were collected at term. Immediately after delivery, the umbilical cord was rapidly clamped, and the placenta carefully cleaned up from excess blood (39). Placental diameter and perimeter were measured with adequate measuring tools. Placental thickness at the center of the chorionic plate was measured by piercing the disc with a knitting needle in which millimeter marks were inscribed (40). Then, placental tissues from central cotyledons were obtained and appropriately stored as previously described for further analysis (22).

To study the ratio between circulating triglycerides (TG) and HDL cholesterol as a marker of insulin resistance (41), TG and HDL cholesterol levels were evaluated by colorimetric methods (Wiener lab, Rosario, Argentina) in 8-h fasting plasma at enrollment (gestational weeks 24–28) and at term (gestational week 37). Plasma was conserved at −80°C for further evaluation of fatty acid binding protein 4 (FABP4) [an insulin resistance marker (24)] and MMP2 and MMP9 activities [remodeling enzymes and markers of the proinflammatory state (25)]. BMI, defined as weight in kilograms divided by the square of the height in meters, was evaluated at enrollment and at term. At delivery, placental diameter, thickness, and perimeter were measured, and the placentas were prepared for histological studies and preserved at −80°C for either Western blot, lipid content evaluation, or RT-qPCR studies.

The gelatinase activities of MMP9 and MMP2 were studied in frozen preserved maternal plasma from control and GDM patients treated or not with the EVOO-enriched diet, and evaluated by zymography as previously described (26). Briefly, from maternal plasma, an amount equivalent to 30 µg of protein was mixed with loading buffer (2% SDS, 10% glycerol, 0.1% bromophenol blue, and 50 mM Tris-HCl, pH 6.8) and subjected to a 7.5% SDS-PAGE containing 1 mg/ml gelatin (type A from porcine skin). Following electrophoresis, gels were rinsed in 30% Triton X-100 for 60 min to remove SDS. Gels were incubated on 50 mM Tris Buffer, pH 7.4, containing 0.15 mM NaCl and 30 mM CaCl₂ for 18 h at 37°C. Gels were stained with Coomassie blue and destained with 10% acetic acid–30% methanol in water. The areas of proteolytic activity appeared as negatively stained bands in a dark background. The identities of MMPs were based on their molecular weights and a positive internal control (conditioned medium of human fibrosarcoma HT-1080 cells). The enzymatic activity was quantified using ImageJ software and expressed as arbitrary densitometric units, which were normalized to the internal control. Data are shown as relative to a value of 1 assigned to the mean value for MMP activity in the control group.

FABP4 levels were evaluated in frozen preserved maternal plasma samples from the three experimental groups by Western blot. Proteins from maternal plasma (60 µg of protein) were separated by SDS-PAGE and transferred to nitrocellulose membranes (35V constant, overnight at 4°C), as previously described (42). The membranes were stained with Ponceau Red staining solution for total proteins (Sigma-Aldrich) to confirm proper loading and transfer. Blocking was carried out in 1% BSA in TBS-Tween solution for 1 h at room temperature. Next, the membranes were incubated with the primary antibody, diluted in 2% BSA in TBS-Tween, overnight at 4°C. A primary antibody from Genetex (CA, USA), diluted 1/500, was used to determine the protein expression of FABP4. A primary antibody from Sigma-Aldrich, diluted 1/500, was used to determine the protein expression of actin. After washing, the membranes were incubated with the appropriate peroxidase-conjugated anti-rabbit secondary antibody. The bands were visualized using ECL detection solution (Thermo Scientific, MA, USA) and captured in a Chemiluminescence imaging system (GeneGnomeXRQ, Syngene). Densitometry analysis was performed with ImageJ software. The relative intensity of protein signals was quantified by densitometric analysis using the ImageJ Software (NIH, MD, USA). Results are expressed as protein of interest/actin protein ratio.

Placental slides were paraffinized and serially sectioned (5 μm). The slides obtained were deparaffinized with xylol and rehydrated through a graded series of ethanol. To perform periodic acid–Schiff staining (PAS), a histological technique that allows the evaluation of glycogen depots (32), the slides were incubated with 0.5% periodic acid solution for 5 min, washed with distilled water, and stained with Schiff’s reagent, protected from light, for 15 min.

To perform Masson’s trichrome staining, a histological technique in which light blue color indicates collagen deposition (31), the slides were incubated with Weigert’s hematoxylin for 7 min, and then washed for 5 min. Subsequently, the slides were stained in scarlet-acid fuchsin for 2 min, rinsed in distilled water, incubated in phosphomolybdic acid for 15 min, stained with Light Green 2% for 10 min, and incubated in 1% acetic acid for 1 min.

To perform immunohistochemical studies, the slides were incubated for 20 min at 95°C to unmask the antigens. Next, slides were incubated with H₂O₂ 0.3% for 20 min to block the activity of the endogenous peroxidase. The sections were then incubated overnight with the primary antibody (anti-megalin mouse antibody, Santa Cruz Biotechnology, diluted 1/40; anti-Cubilin rabbit antibody, Millipore, diluted 1/20) at room temperature. Then, slides were incubated with the corresponding secondary antibody (Vector Laboratories, CA, USA, diluted 1/200) for 1 h and, after rinsing, incubated with the avidin–biotin complex (Vectastain, Vector Laboratories) for 1 h. The stain was developed with 3,3′-diaminobenzidine, as previously described (22). Negative controls were performed in the absence of primary antibody and by replacing the primary antibody by a pooled serum of the same species that contains a spectrum of the IgG subclasses (Vector Laboratories). In all cases, the tissues were dehydrated and mounted, and examined by two skilled blinded observers using light microscopy (Nikon Eclipse E200) and photographed (Nikon DS-Fi1). Immunoreactivity intensity was quantified using the ImageProPlus software. Data are shown as relative to a value of 1, assigned to the mean value of the control group.

Placentas were each homogenized in 1,000 μl of PBS and protein content in the homogenates measured by the Bradford assay. As previously carried out (43), tissue lipids were extracted from 500 μl of each homogenate by three rounds of organic extraction in methanol:chloroform (2:1), following the method of Bligh and Dyer. The lipids extracted (equivalent to 400 μg of protein) were developed by thin-layer chromatography in 0.2- mm silica gel plates (Merck, Darmstadt, Germany). The developing solvent mixture was hexane:ether:acetic acid (80:20:2, v:v:v). Lipid species were stained with iodine vapors, identified and quantified by comparison with known amounts of standards on the same plate, and analyzed densitometrically with the ImageJ software.

Fatty acid methyl esters (FAMEs) extracted from placental lipids were prepared by reaction with 5% HCl in methanol at 70°C for 2 h. After cooling, water was added, and FAMEs were extracted with chloroform. FAMEs were analyzed by gas chromatography, as previously mentioned (43) on a Focus gas chromatograph (Thermo Finnigan Corporation), equipped with an Innowax capillary column (Agilent, 100% polyethylene glycol, 30 m length, 0.25 mm i.d., and 0.5 µm film thickness). Nitrogen was the carrier gas (0.8 ml/min continuous flow rate). The injector and detector temperatures were set at 240°C and 300°C, respectively. Column temperature was programmed from 100°C (1 min), then at a rate of 15°C/min up to 200°C, maintained at this temperature for 1 min, then at a rate of 2°C/min up to 240°C and maintained at 240°C for 17 min. Individual FAMEs were identified by comparing retention time data with those obtained from authentic laboratory standards (Sigma-Aldrich Co.). Quantitation was done by comparing the percent of area of each FAME peak on the chromatogram with that of the internal standard of known weight (nonadecanoate methyl ester, Sigma-Aldrich Co.). Individual FAMEs were expressed as percentage of total fatty acids.

Total RNA and microRNA were isolated from 100 mg of placental explants using RNAzol^® (MCR Inc., OH, USA) according to the manufacturer’s recommendations. The concentrations of total RNA and microRNA were determined using the NanoDrop spectrophotometer. For mRNA analysis, cDNA was synthesized incubating 2 μl (1 μg/μl) of extracted RNA and random primer hexamers (Promega, WI, USA) at 72°C for 5 min. Then, we added a reaction mixture containing MMLV reverse transcriptase (Promega) and each of all four dNTPs (Invitrogen) and performed an incubation at 37°C for 60 min and then at 72°C for 15 min, as previously mentioned (44). cDNA was used to perform the amplification in reaction buffer containing dNTPs mix (Solis BioDyne), GoTaq Polymerase (Promega), Eva Green 20× (Biotium, CA, USA), and the gene-specific primers described in Table 1 . The qPCR started with a denaturation step at 95°C for 5 min and followed by up to 40 cycles of denaturation, annealing, and primer extension. mRNA levels were normalized to the geometric mean of the mRNA levels of the 60s ribosomal protein L30 and β2-microglobulin.

The evaluation of miR-199 was performed as previously mentioned (44). Briefly, cDNA was obtained using the TaqMan MicroRNA reverse transcription kit (Applied Biosystems, CA, USA). The relative expression of miR-199 was determined using the TaqMan detection system (assay ID 000498, Applied Biosystems). U6 spliceosomal RNA (assay ID 001973, Applied Biosystems) was used as endogenous control.

For both mRNA and microRNA, the course of PCR amplification was followed in each cycle by the fluorescence measurement on Corbett Rotor-Gene 6000 (QIAGEN, MD, USA). The expression of the mRNAs evaluated and miR-199 was quantified using the 2^−ΔΔCt method. Relative mRNA levels are shown as fold values of the control.

Data are presented as means ± SEM. Homogeneity of variance was verified with Levene’s test and normality of the variable distribution was checked with the Shapiro–Wilk test using the SPSS 19 software. Once the ANOVA assumptions were verified, the experimental groups were compared by one-way ANOVA followed by Bonferroni’s post-hoc test, using the GraphPad Prism Version 8 software (GraphPad, Inc., San Diego, CA, USA). A p-value lower than 0.05 was considered statistically significant.

To examine the putative beneficial effects of an EVOO-enriched diet supplementation on insulin resistance markers in GDM women, TG/HDL cholesterol ratio and FABP4 levels were evaluated in plasma samples in a cohort of control (n = 15), GDM (n = 15), and GDM patients who received the dietary EVOO supplementation from enrollment to term (n = 15).

At enrollment, the TG/HDL cholesterol ratio was unchanged in the evaluated experimental groups ( Figure 1A ). Differently, at term, the TG/HDL cholesterol ratio was increased in the GDM group compared to controls (p < 0.05), an alteration prevented in the GDM women who received the EVOO-enriched diet (p < 0.01, GDM-EVOO vs. GDM) ( Figure 1B ). At enrollment, FABP4 levels were unchanged in the experimental groups evaluated ( Figure 1C ). Differently, at term, FABP4 levels were increased in the GDM group compared to controls (p < 0.05), an alteration prevented in the GDM women who received the EVOO-enriched diet (p < 0.05 GDM-EVOO vs. GDM) ( Figure 1D ). BMI was also unchanged at enrollment in the three experimental groups ( Figure 1E ), whereas at term, it was increased in the GDM group compared to controls (p < 0.05), an alteration prevented by the EVOO-enriched diet (p < 0.05 GDM-EVOO vs. GDM) ( Figure 1F ).

As previous studies have shown increases in MMP9 in term placentas and in MMP2 and MMP9 in cord blood from GDM patients (22), the activities of MMP2 and MMP9 were evaluated in maternal plasma in the same cohort of control and GDM patients treated or not with the dietary EVOO supplementation.

Maternal plasma MMP2 activity was unchanged in the experimental groups both at enrollment and at term ( Figures 2A, B ). Differently, MMP9 activity was unchanged in the maternal plasma of the experimental groups at enrollment but increased in the GDM group at term compared to controls (p < 0.05, Figure 2C ). The increase in maternal plasma MMP9 activity in the GDM group was prevented by the maternal diet enriched in EVOO (p < 0.05 vs. GDM) ( Figure 2D ).

The placenta is a crucial organ for the mother, the fetus, and the offspring’s health (6, 45). Previously, we found increased proinflammatory markers in the placenta from GDM patients, an alteration prevented by the maternal diet enriched in EVOO (22). Here, we focused on placental morphometric parameters and the evaluation of profibrotic markers and metabolic substrates that are stored in the placenta. As shown in Table 2 , the diameter and perimeter of the placenta showed no differences in the groups evaluated. Differently, the thickness of the placenta was increased in the GDM group compared to controls (p < 0.05), an alteration prevented in the placenta of GDM women who received the EVOO-enriched diet (p < 0.01 GDM-EVOO vs. GDM) ( Table 2 ).

Aiming to address the placental content of collagen, an abundant extracellular matrix protein, relevant for placental structure, development, and function and marker of fibrosis when increased (31, 46), we performed a trichrome Masson staining in term placentas from control women and from GDM patients who received or did not receive the EVOO dietary supplementation. We found increased collagen depots in the placental villi of GDM patients compared to controls (p < 0.01), an alteration prevented by the maternal diet supplemented with EVOO (p < 0.01 GDM-EVOO vs. GDM) ( Figures 3A, B ). In addition, chorionic vessels also showed increased collagen depots in the placenta from GDM patients compared to controls (p < 0.05), an alteration prevented by the maternal diet supplemented with EVOO (p < 0.05 GDM-EVOO vs. GDM) ( Figures 3C, D ).

Next, we addressed the placental levels of glycogen, the main storage form of carbohydrates in different tissues, which can be evaluated by PAS histochemical staining (47). Increased glycogen depots, as indicated by increased PAS staining, was observed in the placenta from GDM patients compared to controls (p < 0.001), an alteration prevented by the maternal diet supplemented with EVOO (p < 0.001 GDM-EVOO vs. GDM) ( Figures 4A, B ). On the other hand, the evaluation of term placenta lipid content showed no differences in triglycerides, cholesterol, cholesteryl esters, phospholipids, and free fatty acid levels when the three experimental groups were compared ( Figure 5 ). Similarly, no changes in the fatty acid percentual composition were observed when the three experimental groups were compared ( Table 3 ).

Although we found no changes in lipid content in the placenta, we previously found increased circulating triglycerides in the GDM patients, an alteration prevented by the EVOO-enriched diet (22). As lipoproteins rich in triglycerides may be transferred from maternal circulation to the placenta through the endocytic pathway (33), we evaluated the levels of cubilin and megalin, two proteins involved in the endocytosis of lipoproteins (48). The cubilin levels in term placentas showed no differences when the three experimental groups were compared ( Figures 6A, B ). Differently, megalin levels were reduced in the placenta from GDM patients compared to controls (p < 0.05), an alteration prevented by the maternal diet supplemented with EVOO (p < 0.05 GDM-EVOO vs. GDM) ( Figures 6C, D ).

The mRNA expression of LRP2 (which codifies for megalin), as well as the mRNA of other genes involved in the endocytic pathway—LRP1, LDLR, and CLTC, was further evaluated. We found reduced expression of LRP2, LRP1, LDLR, and CLTC in the placenta from GDM patients compared to controls (p < 0.05) ( Figures 7A–D ). The reduced mRNA levels of LRP2, LDLR, and CLTC were prevented by the EVOO-enriched diet (p < 0.05) ( Figures 7A, C, D ). As these genes are targets of the miR-199 (37), we evaluated its expression. Interestingly, miR-199 showed a 30-fold increase in its expression in the placentas of GDM patients compared to controls (p < 0.01), and alteration prevented in the placentas of the GDM patients treated with the EVOO-enriched diet (p < 0.01 GDM-EVOO vs. GDM) ( Figure 7E ).