Introduction
Obesity remains a public health issue worldwide. The prevalence of obesity tripled since 1975. Nearly 50% of women of childbearing age and 20–25% of pregnant women in Europe are overweight or obese (WHO 2022 Reference Stevens, Singh and Lu1). Overweight and obesity are associated with reproductive difficulties.Reference Poston, Caleyachetty and Cnattingius2 Obesity in pregnant women is also associated with an increased risk of morbidity and mortality during pregnancy or labour including gestational diabetes mellitus, arterial hypertension or preeclampsia, and in the neonate with preterm birth, macrosomia, shoulder dystocia, or stillbirth.Reference Lawlor, Relton, Sattar and Nelson3–Reference Declercq, MacDorman, Cabral and Stotland5 Maternal obesity can also have consequences on children in later life, with a higher prevalence of metabolic syndrome which include arterial hypertension, insulin resistance, dyslipidemia or obesity.Reference Catalano and Shankar6–Reference Bellver and Mariani9
In addition with lifestyle modifications, bariatric surgery can be recommended for patients with a morbid obesity (i.e a BMI ≥ 40 kg/m2) or for those with a BMI ≥ 35 kg/m2 and some comorbidities, with superior outcomes than nutritional care alone, on weight loss and obesity-related comorbidities resolution.Reference Colquitt, Pickett, Loveman and Frampton10,Reference Kwak, Mehaffey and Hawkins11 Preconceptional bariatric surgery may improve maternal outcomes with a significant decrease of pregnancy comorbidities,Reference Kwong, Tomlinson and Feig12 and may be associated with long term benefits in the offspring, with a reduced risk of obesity, insulin resistance, or arterial hypertension.Reference Guénard, Deshaies and Cianflone13 Two types of interventions are commonly proposed: the Sleeve Gastrectomy, a restrictive procedure, or the Roux-en-Y Gastric Bypass, a restrictive and malabsorptive procedure, considered as a gold standard for bariatric surgery. Nevertheless, complications can occur after bariatric surgery despite appropriate dietary management and micronutrient supplementation particularly in the form of nutritional deficiencies (protein, vitamins, iron, trace elements).Reference O’Kane14,Reference Jans, Matthys and Bogaerts15 Recommendations have therefore been established to ensure the best possible monitoring of patients and the nutritional supplements they should receive (BARIA-MAT group).Reference Ciangura, Coupaye and Deruelle16
Recent clinical studies moderated the beneficial effects of those interventions in neonates with an increased risk of fetal growth restriction, moderate prematurity or possible nutritionalReference Kwong, Tomlinson and Feig12,Reference Bel Lassen, Tropeano and Arnoux17,Reference Rives-Lange, Poghosyan and Phan18,Reference Spann and Grayson19,Reference Johansson, Cnattingius and Näslund20 and micronutrient deficiencies.Reference Gascoin, Gerard and Sallé21,Reference Mead, Sakkatos and Sakellaropoulos22
The placenta is the interface between the mother and the fetus and it regulates nutrient and oxygen transfer to the fetus during pregnancy and consequently contributes to fetal homeostasis. In animal models, reactive placental adaptations to an obesogenic environment can result in alterations of implantation, placental development, or function accompanied by gene and epigenetic modifications.Reference Saben, Lindsey and Zhong23 However, there is no animal study in the literature about placental adaptations after bariatric surgery. Human studies only focus on fetal consequences. Thus, difficulties in the placenta adapting to maternal bariatric surgery linked to maternal insufficient energy intake could be responsible for clinical consequences observed in the fetus and child.
However, women who have undergone bariatric surgery may be considered to be in a state of relative malnutrition or undernutrition du to nutritional deficiencies, especially if their weight has not yet stabilised. Some studies on women in undernutrition situation have shown a deleterious effect on the placenta, with defects in development and implantation,Reference Belkacemi, Nelson, Desai and Ross24 as well as altered placental transport of carbohydrates, lipids and amino acids,Reference Satterfield, Edwards and Bazer25 leading to an increased risk of growth restriction in the child.Reference Belkacemi, Nelson, Desai and Ross24 Fetal programming phenomena may occur. To cope with this environment of undernutrition, the fetus will develop a ‘thrifty’ phenotype in order to ensure its survival and the growth of key tissues (brain, heart) at the expense of others (muscles, intestines, etc.). The result is fetal growth restriction (Barker’s theory).Reference Barker26
Maternal bariatric surgery is an effective method for improving the maternal phenotype and reducing the risk of certain comorbidities in the child linked to maternal obesity. However, our hypothesis is that maternal nutritional deficiencies linked to maternal undernutrition after bariatric surgery can also cause growth restriction in the foetus and even increase mortality, all mediated by changes in the placenta which is the interface between the mother and the foetus. These effects may also be more detrimental after a gastric by-pass due to the restrictive and malabsorptive nature of this procedure. In order to understand the pathophysiological mechanisms involved in this context of maternal obesity and pre-conceptional bariatric surgery, we established an animal model of bariatric surgery in obese female rats, before reproduction.
The aim of this preliminary study was to describe feto-placental units in terms of phenotype and molecular/immunohistochemical modifications, induced by obesity and then by bariatric surgery performed before gestation on an obese rat model.
Methods
Animals
This first study is exploratory and without sample size calculation. All animal procedures were approved by the local Ethic committee in animal experimentation (CEEAPdL-006) and approved by the Education, Research and Innovation Minister (APAFIS#10697-2017091422557044v1, 14/11/2017). These procedures were in accordance with the institutional guidelines (3R principles). All methods are reported in accordance with ARRIVE guidelines.
The animals were Sprague Dawley rats coming from Janvier LABS. They were housed on 12/12h light/dark cycle at 21°C with ad libitum access to food and water.
Thirty-three female Sprague Dawley rats, aged 7 weeks were randomly assigned to one of two diets fed: a standard diet (3.1% lipid, 59.9% carbohydrate, 16.1% protein, 2.791kCal/g = 11.685 kJ/g) or a validated obesogenic diet (High Fat High Sugar diet (HFHS))Reference Fernandez-Twinn, Gascoin and Musial27,Reference Schoonejans and Ozanne28 composed of High Fat Diet (research Diet D12451, SDS France)(24% lipid, 41% carbohydrate, 24% protein, 4.54 kCal/g = 19.0 kJ/g) supplemented with High Sugar Diet (sweetened condensed milk, Nestle UK) (8.7% lipid, 54.4% carbohydrate, 7.91% protein, 3.24 kCal/g = 13.565 kJ/g) (Fig. 1). Animals receiving the standard diet constituted the ‘Control Group’. Animals receiving the obesogenic diet were then randomly divided into 3 other groups: the ‘Obese group’, the ‘Sleeve Gastrectomy group’ or the ‘Gastric Bypass group’. After 9 weeks on the diet, all animals underwent surgery.
Flow chart and experimental design of the study.
Pre-operative tests
All animals underwent the same pre-operative tests.
Glucose tolerance test – One week before surgery, animals were fasted for 12h. Baseline tail vein blood glucose was quantified using a glucometer (StatStrip® glucose, Nova). Subsequently, animals received an intraperitoneal injection of 1 g/kg D-glucose at time (t) = 0 min after which blood glucose was measured at 15, 30, 60 and 120 min post-injection.
Food intake – One week before surgery, animals were housed 24 h in individual metabolic cages (Techniplast France, Lyon, France). They were provided with access to water and their respective diet. Food and water were quantified before and after the 24 h to determine the consumption and then calculate the caloric intake.
Surgery
Pre-operative care – Animals were all fasted 4 h before surgery. After a general anaesthesia by Isoflurane 2% v/v O2, they all received subcutaneous injections of analgesics (Buprenorphine: 0.05 mg/kg), antiemetic (Metoclopramide: 0.5 mg/kg), iron (Fercobsang®: 0.175 mg/kg), antibiotics (amoxicillin-clavulanic acid: 30 mg/kg) and NaCl 0.9% (1 mL).
Sleeve gastrectomy – Some obese rats underwent a sleeve gastrectomy by midline laparotomy.
After exteriorisation of the stomach, the right gastro-epiploic artery was ligated and the lateral 70% of the stomach was excised. A gastric tube in continuity with the oesophagus and duodenum was reconstructed using linear 6/0 sutures. This gastric sleeve was then reintegrated into the abdominal cavity and the abdominal wall was closed in layers using 3/0 coated vicryl suture (18/15L, OPTIME® Peters Surgical).
Gastric bypass – Some obese rats underwent a Roux-en-Y gastric bypass surgery via midline laparotomy. The jejunum was cut at 12 cm from the Treitz angle to create the duodenal loop and end-to-side anastomosed to the distal jejunum with 8/0 semi-linear sutures, creating a 18 cm long digestive loop. A sleeve gastrectomy as described previously was created after closure of the pylorus. Finally, the end of the digestive loop was anastomosed to the anterior part of the stomach, before reintegration into the abdominal cavity and closure of the abdominal wall in layers using 3/0 coated vicryl suture (18/15L, OPTIME® Peters Surgical).Reference Couchot, Schmitt, Mermet, Fassot and Mabilleau29
Sham surgery – Rats from the obese group and the control group underwent a ‘sham surgery’ consisting only in a midline laparotomy (opening and then closure of the abdominal wall without any organ resection).
Post-operative care – Following surgery, all rats were fed with Osmolite liquid diet (Gel Diet Energy®, Safe Diet) during 3 days and then rats from the control group received their standard food while rats of the obese and bariatric surgery groups received their HFHS diet. No dietary changes were made after bariatric surgery in order to rule out weight and metabolic improvements attributable to dietary changes rather than surgery. Rats were hydrated by water ad libitum, to which were added fructose (37g for 200 mL of water) the first week, and vitaminic supplementations (Vita rongeur®, Vibrac: 2 spoons for 100 mL of water) until the end of the protocol. They also received care for 3 days: twice a day subcutaneous injections of Buprenorphine (0.05mg/kg), Amoxicillin-Clavulanic Acid (30mg/kg) and NaCl 0,9% (1 ml), and once a day a subcutaneous injection of iron (0.175mg/kg) until day 5.
In women, it is recommended to wait at least 12 months before becoming pregnant in order to achieve weight stability. In this exploratory study and given the limited data available in the literature, we determined a period of one month after surgery before allowing our females to reproduce. They were therefore mated with a non-obese Sprague-Dawley male for one night. Vaginal smear, searching for sperm, was performed the next day to confirm the mating and then the weekly body weight gain and abdominal palpation were used as supplemental indicators of a successful gestation. Placentae and foetuses were surgically removed at D19 of gestation to analyse placentae before the delivery and their degradation.
Sample collection
At D19 of gestation, female rats were anaesthetized by Isoflurane 2% v/v O2, and received a subcutaneous injection of Buprenorphine (0.05 mg/kg). Following midline laparotomy, the uterus was excised and feto-placental units exteriorised. Placement and number of the feto-placental units were noticed. Placentae and fetuses were extracted and weighted individually (for a descriptive analyse) before being preserved at −80°C.
Females were then euthanized by exsanguination under Isoflurane anaesthesia.
Blood collection – Blood was collected in the inferior vena cava and centrifuged (4°C, 5000 rpm, 15 min) to isolate plasma which was stored at -20°C.
Leptin and insulin were quantified by ELISA (leptin kit: A05176.96 wells, Bertin technologies; insulin kit: 80-INSRT-E01, ALPCO).
Fetus sexing – Fetuses were sexed by visual inspection, and yolk sacs DNA were subjected to PCR for the SRY gene. An amplification of the SRY gene confirmed the fetus and associated placenta to be male.
Placental analyses
Placental analyses were designed to detect lipotoxicity (lipid accumulations), inflammation and oxidative stress that may alter the transport of nutrients (proteins, carbohydrates) through the placenta. Placentae of around median weight of the whole litter were selected for immunohistochemistry, and molecular analyses. When it was possible, one placenta of a male pup and one of a female pup were selected from each gestating rat.
Oil Red O – Frozen placentae sections of 8 μm thickness were stained with Oil Red O (Sigma-Aldrich, UK) and counterstained with Haematoxylin solution to detect lipidic accumulations within the various structures. Images were digitalised by an APERIO ScanScope Console (0.5 µm/pixel). Single blinded analyses were conducted using ImageJ software on four fields of view per sample (n = 5–6 per group). Lipid droplets (red) were detected and quantified as a percentage of positively stained pixels in the field of view, using Image J software.
RTqPCR – For gene expression analyses, total RNA was isolated from placentae using QIAzol lysis reagent (Qiagen, France) and Qiagen RNeasy mini kit (Qiagen, France). cDNA was generated with 1000 ng of RNA using a High-Capacity cDNA Reverse Transcription Kit (QuantiTect reverse transcription, Qiagen, France). Quantitative polymerase chain reaction was performed on a LightCycler480 PCR System (Roche, France), using 300 nM primer concentrations, 1×SYBR Green master mix (Applied Biosystems, UK) and a 1:10 dilution of sample cDNA. The thermal cycling conditions consisted of a denaturation 3 min at 95°C and 40 cycles at 95°C for 15 sec and 60°C for 1 min. The Cq method was used for calculation of genes relative expression levels using comparative method. mRNA expression was normalised to the geometric mean of selected reference genes (POLR2A, GUSB and ACTB). Primer sequences was performed using primer blast and they were validated by testing PCR efficiency using standard curve as MIQE guidelinesReference Bustin, Benes and Garson30. Primer sequences used in the PCR analyses are listed in the Supplementary Table S1. They explored lipotoxicity, oxidative stress, inflammation and insulin signalling pathways.
Protein extraction and Western blot – Proteins were extracted from half of a placenta using the RIPA lysis buffer system (Santa Cruz Biotechnology, Dallas, TX). Concentrations were determined using a Pierce BCA protein assay kit (23235, Thermo Fisher Scientific, Rockford, IL), and spectrometry was performed with a Tecan Infinite 200 PRO. Proteins were combined at a 1:1 ratio with Laemmli sample buffer (BioRad Laboratories, Hercules, CA) and denatured at 95° C for 5 min. Proteins (40 μg) were loaded onto BioRad 4-20% polyacrylamide Mini Protean TGX gels, and electrophoresis were performed in a BioRad Tetra-Cell 2 gel system (100V during 1h30). Subsequently, proteins were transferred to PVDF membranes using a BioRad Trans-Turbo transfer system. Membranes were incubated for 1h30 at room temperature with Pierce Protein-Free (TBS) Blocking Buffer (Thermo Scientific, Rockford, IL). Primary antibodies used in Western Blot are detailed in Supplementary Table S2. They explored lipotoxicity, oxidative stress, inflammation and insulin signalling pathways. In between incubations, membranes were washed with TBS containing 0.05% Tween. Anti-rabbit HRP conjugate (1:5000, #sc-2004, Santa Cruz Biotechnology, Dallas, TX) and anti-mouse HRP conjugate (1:5000, #sc-2005, Santa Cruz Biotechnology, Dallas, TX) were used to incubate membranes for 1h at room temperature before applying Clarity TM Western ECL (BioRad) or Clarity Max TM ECL (BioRad). Images were captured using a BioRad ChemiDoc XRS system and analysed using BioRad Image Lab software.
Lipid quantification – Phospholipids, triglycerides, free fatty acids and total cholesterol were quantified in placentae using fluorometric kits (Ab65336 Triglyceride Assay Kit, Ab234050 Phospholipid Assay Kit, Ab65341 Free Fatty Acid Assay Kit – Quantification, Ab65390 Cholesterol Assay Kit – HDL and LDL/VLDL; Abcam) to study placental lipid distribution.
Statistical analyses
Statistical analyses were performed using GraphPad Prism v10 (GraphPad Software, San Diego, California, USA). Differences in quantitative variables were assessed by using non-parametric Mann–Whitney tests when comparing 2 groups (HFHS vs standard diet groups), and Kruskall-Wallis tests when comparing more than 2 groups. To observe time-wise differences, two-way ANOVA methods (variables: surgical group and time) were used. Due to a small sample size, all results are given as median, extremes.
Results were considered statistically significant when p < 0.05.
Results
Eight rats were fed with a standard diet and 25 with HFHS (High Fat High Sugar) diet.
After mating of all the female rats, we finally obtained 17/33 successful mating and establishment of pregnancy. We constituted a ‘control group’ of 5 rats, an ‘obese group’ of 5 rats, a ‘sleeve group’ of 4 rats and a ‘bypass group’ of 3 rats (Fig. 1).
Maternal phenotype before surgery
Rats fed with obesogenic diet had an increased caloric intake (0.198 kcal/g [0.137–0.253]) in comparison to rats under standard diet (0.157 Kcal/g [0.134–0.218], p = 0.04) (Fig. 2A). It was associated with a significant increase of the HFHS rats’ body weight, from the 5th week of diet.
Mother’s phenotype during obesogenic diet and after bariatric surgery. A Animals caloric intake measurement after 24 h in metabolic cages. B Animals weight gain after HFHS or standard diet. C Glucose Tolerance Test one week before surgery. D Weight monitoring after surgery. E Leptin levels at sacrifice. Data are expressed as median ± extremes and analysed by a two-way ANOVA method.
At week 10 (before surgery), HFHS rats weighed 372 g [315–406] versus 327 g [310–355] for rats under standard diet (p < 0.0001), resulting in an additional weight gain of 11.7% in the HFHS group (Fig. 2B).
The glucose tolerance test, one week before surgery, showed a significantly elevated glycaemic response at 15, 30 and 60 minutes after glucose injection in the HFHS group (Fig. 2C).
At surgery, rats in the obese, gastric bypass and sleeve gastrectomy groups had similar weights but were always significantly heavier than those of the control group (Fig. 2D).
Maternal phenotype after surgery
Rats after sleeve gastrectomy or gastric bypass had similar phenotypes (weight, glycaemia, leptin levels) with no significant difference so these rats were pooled into a larger group called ‘bariatric surgery’ for all the analyses to improve their power (Fig. S1).
From the first postoperative week, weight loss was observed in the bariatric group, reaching a weight similar to the control group. Four weeks after surgery, we observed that rats after bariatric surgery and controls had a significantly lower weight than the obese group (control: 357 g [335–402]; obese: 402 g [350–425]; bariatric surgery: 348 g [300–385], p ≤ 0.05) (Fig. 2D).
At sacrifice, leptin levels were significantly lower in the control group as compared to the obese group (2997 pg/mL [2472–5884] vs 19,103 pg/mL [8513–30041]), p = 0.002). In the bariatric surgery group, leptin levels were comparable to the control group (Fig. 2E).
Placental and fetal phenotype
There was no significant difference in pups’ number per litter between the different groups (control: 11 [5–16]; obese: 7 [1–13]; bariatric surgery: 9 [1–13]) (Fig. 3A). However, obese rats tend to have a lower number of fetuses than controls and this number seems to increase after bariatric surgery. The number of dead in utero fetuses (residual placentae and/or fetuses in maternal uterine horns) was significantly higher in the bariatric surgery group (4 [1–8]) as compared to the control group (0 [0–2], p = 0.02) (Fig. 3B). This number was not significantly higher in the bariatric surgery group as compared to the obese group.
Description of the feto-placental units at D19 of gestation. A Comparison of number of pups per litter. B Comparison of number of dead in utero fetuses per litter. C Comparison of placentae weight. D Comparison of fetus weight. E Comparison of placenta/fetus weight ratios. Data are expressed as median ± extremes and analysed by a Kruskall–Wallis test.
Placentae weights were not significantly different between the groups but there was a trend towards higher placental weights in the bariatric surgery group (Fig. 3C) (control: 0.54 g [0.40–0.82]; obese: 0.54 g [0.37–0.86]; bariatric surgery: 0.58 g [0.40–0.91]). Fetuses were significantly smaller in the bariatric surgery group as compared to the control (p = 0.0005) and obese groups (p = 0.01) (control: 1.54 g [0.82–3.14]; obese: 1.53 g [1.08–1.89]; bariatric surgery: 1.30 g [0.7–1.63]) (Fig. 3D).
Placenta/fetus weight ratio was significantly higher in the bariatric surgery as compared to the two other groups, in accordance with a lower fetal weight for a similar placental weight, p = 0.002 (Fig 3E).
There was no sexual dimorphism in terms of fetal weight, placental weight, or placenta/fetus weight ratio within each group (Fig S2). Details by bariatric subgroups are shown in Figs. S3 and S4.
Placental levels of phospholipids, triglycerides, free fatty acids and total cholesterol were not significantly different between groups (Fig. S5).
Placental immunohistochemistry
After Oil Red O staining, placental sections of obese rats showed a higher lipid accumulation (1.6 % [1.01–2.04]) than in control rats (0.82% [0.37–1.25], p = 0.02) and operated rats (0.79% [0.47–1.69], p = 0.02) (Fig. 4A and B).
Lipid accumulations in the placenta. A Total placental lipidic accumulations after Oil-Red-O staining. Data are expressed as median ± extremes and analysed by a Kruskall–Wallis test. B Oil-Red-O staining on placental sections. Scale bar = 1 mm.
Placental metabolic pathways analyses
Inflammation pathway (Fig. 5A)
The expression of Interleukin-6 gene (IL-6) was significantly increased in the obese mothers’ placentae as compared to the control group and then was significantly decreased in the bariatric surgery group placentae (median fold change (MFC): 0.80 [0.51–0.82]) as compared to obese rats’ placentae (MFC: 1.28 [0.95–2.36]) (p = 0.003).
Placental molecular analyses. A Molecular analyses of placental inflammation pathway. B Molecular analyses of placental oxydative stress pathway. C Molecular biology and biochemical analyses of placental lipotoxicity pathway. D Molecular analyses of placental nutritional exchange pathway. Data are expressed as median ± extremes and analysed by a Kruskall–Wallis test.
Oxidative stress pathway (Fig. 5B)
The expression of Matrix MetalloPeptidase 9 gene (MMP9) tend to be higher in the obese group without significance as compared to the control group. This expression was significantly decreased in bariatric surgery group placentae as compared to the obese group (MFC in control: 1.21 [0.71–1.59]), obese: 1.67 [0.72–2.9], bariatric surgery: 0.31 [0.007–0.96], p < 0.02).
Lipotoxicity pathway (Fig. 5C)
The expression of Fatty Acid Synthase gene (FASN) was significantly decreased in the bariatric surgery placentae as compared to the obese group placentae (MFC in bariatric surgery: 0.87 [0.70–1.07] vs obese: 1.18 [1.01–1.39], p = 0.007) and non-significantly lower than in controls.
The protein expression of Gamma Receptor Activated by Peroxisome Proliferators (PPARγ) was significantly higher in bariatric surgery group subjects as compared to obese subjects and near the controls (MFC in bariatric surgery: 1.0 [0.51–2.13] and control: 1.0 [0.42–1.53] vs obese: 0.35 [0.22–0.84], p = 0.02).
Nutritional exchange pathway (Fig. 5D)
By RTqPCR, the expression of the Insulin Receptor gene (INSR) was significantly decreased in the placentae of bariatric surgery group (bariatric surgery: 0.71 [0.52-0.94]) as compared to controls (MFC in the control group: 1.05 [0.78–1.30], p = 0.008).
The Insulin Receptor Substrate 1 (IRS1) expression was significantly reduced in the obese group (MFC: 0.38 [0.07–0.89]) and the bariatric surgery group (MFC: 0.43 [0.23–0.59]) as compared to controls (MFC: 0.88 [0.53–1.8], p < 0.03) in Western Blot.
The expression of RAC-beta serine/threonine-protein kinase (AKT2) in the placentae of bariatric surgery group was significantly decreased as compared to the control group (MFC: 0.8 [0.67–1.08] vs control: 0.93 [0.85–1.13], p = 0.04).
The expression of RAC-alpha serine/threonine-protein kinase (AKT1) was significantly decreased in the placentae of bariatric surgery group (MFC: 0.86 [0.69–0.97]) as compared to the control one (MFC: 0.99 [0.82–1.24]), p = 0.02.
The gene expression of Mitogen-Activated Protein kinase 3 (MAPK3) was significantly decreased in the placentae of bariatric surgery group (MFC: 0.83 [0.70–1.03]) as compared to controls (MFC: 1.02 [0.85–1.3]), p = 0.01.
The expression of mammalian Target Of Rapamycin gene (mTOR) was significantly decreased in the placentae of bariatric surgery group (MFC: 0.81 [0.70–1.1]) as compared to controls (MFC: 0.98 [0.82–1.24]), p = 0.02.
The expression of Slc38a1 was significantly decreased in the bariatric surgery group as compared to controls (MFC: 0.68 [0.58–1.0] vs 0.90 [0.77–1.44], p = 0.02).
Discussion
In our preliminary study on induced-obesity on a pregnant rat model, we showed that preconceptional bariatric surgery is associated with the reversal of placental lipid accumulations and consequently lipotoxicity, induced by maternal obesity. It is also associated with the reversal of placental inflammation and oxidative stress induced by maternal obesity. Maternal preconceptional bariatric surgery is correlated to fetal intrauterine growth restriction potentially induced by a reduction of placental glucose and protein exchanges.
Rats fed with the obesogenic HFHS diet had a significant higher weight gain and a glucose intolerance before surgery, in accordance with the literature and with a previous study performed by our team.Reference De Moura E Dias, Dos Reis and Da Conceição31,Reference Payen, Guillot and Chaigneau32 Sleeve gastrectomy and gastric bypass resulted in a significant weight loss. At sacrifice, maternal blood leptin levels were significantly higher in the obese group as compared to the control group. After sleeve gastrectomy and gastric bypass, these rates reached thus of the control group as described with a previous study performed by our team.Reference Payen, Guillot and Chaigneau32 As well as in the literature, it was associated with a decrease of adipose tissue and a normalisation of the weight and body composition of female rats after bariatric surgery as well as a normalisation of leptin and insulin one month after surgery, stabilised over time.Reference De Moura E Dias, Dos Reis and Da Conceição31
Leptin is more secreted by adipocytes in case of obesity. Some studies in human literature showed a normalisation of leptin level after bariatric surgery because of a reduction in adipose mass.Reference Stefater, Pacheco and Bullock33,Reference Spann, Lawson and Bidwell34
Rats that have undergone sleeve gastrectomy or gastric bypass were thus pooled as ‘bariatric surgery group’ to analyse the effects of obesity and bariatric surgery on the feto-placental unit.
The small number of rats per group precluded the ability to demonstrate a statistically significant difference in the number of pups per litter. However, there was a trend towards a reduction in the number of fetuses in obese rats with a partial restoration after surgery. This may suggest that obese rats have more reproductive difficulties than normal-weight rats, with improvement after bariatric surgery, in accordance with human literature.Reference Al Qurashi, Qadri and Lund35 The number of fetal deaths occurring in utero was significantly higher in the bariatric surgery group as compared to the control group. Two studies by Spann et al.Reference Spann, Lawson and Bidwell34 and Grayson et al.Reference Grayson, Schneider, Woods and Seeley36 on sleeve gastrectomy yielded comparable results. Our study showed that fetuses from rats that have undergone bariatric surgery were smaller than in the other groups, which may indicate intrauterine growth restriction. We found the same results using a placenta/fetus weight ratio to take into account the influence of the number of pups per litter. Indeed, this ratio must be constant, regardless of the number of pups. This is concordant with the only study, written by Spann, on the effect of sleeve gastrectomy on rats.Reference Al Qurashi, Qadri and Lund35 Moreover, some human studies demonstrated that offspring of mothers who underwent preconceptional bariatric surgery had a higher intrauterine growth restriction incidence.Reference Bel Lassen, Tropeano and Arnoux17,Reference Rives-Lange, Poghosyan and Phan18,Reference Gascoin, Gerard and Sallé21,Reference Maric, Kanu and Muller37,Reference Chevrot, Kayem and Coupaye38 Bariatric surgery may induce a fetal nutritional stress that remains poorly described, due to maternal nutritional deficiencies and possibly to secondary placental insufficiency. In other studies conducted in our laboratory, almost all of the pups born from bypass mothers died before weaning.Reference Payen, Guillot and Chaigneau32 We supposed that maternal caloric restriction can cause fetal caloric restriction. Intrauterine growth restriction can lead to fetal death or metabolic and cardiovascular diseases.Reference Gaudineau39,Reference Bukowski, Hansen and Willinger40 In contrast to the study by Spann et al.,Reference Spann, Lawson and Bidwell34 fetuses of operated mothers had a smaller weight but a placenta of equivalent weight to the others. This observation suggests that the placenta might compensate, at least in part, the caloric restriction by hypertrophying, without success on the fetal growth conservation.
To be as representative as possible in the placental analyses, placentae of median litter weight were selected for immune-histochemistry and molecular analyses.
FASN is a gene encoding for a group of enzymes involved in fatty acids’ synthesis. The greater expression of FASN in obese rats’ placentae as compared to rats that have undergone bariatric surgery, suggests a greater transformation of circulating fatty acids in obese rats and consequently an excessive accumulation of lipids. The accumulation of lipids in non-adipose tissues such as the placenta can lead to lipotoxicity, responsible for cell dysfunction and apoptosis.Reference Belkacemi, Nelson, Desai and Ross24
Histological analyses of placental sections revealed a significant increase in placental lipid accumulations in obese mothers’ placentae as compared to controls, consistent with the results of previous studies and with our molecular analyses.Reference Malti, Merzouk and Merzouk41 Reversibility of these placental lipid accumulations was observed after bariatric surgery. There is no study in literature that explored these modifications after bariatric surgery but the same results are described after physical exercise in obese rat mothers, another way to improve mother’s weight and metabolism.Reference Malti, Merzouk and Merzouk41
Placental lipotoxicity described in maternal obesityReference Belkacemi, Nelson, Desai and Ross24 is involved in increased inflammationReference Gaudineau39 and oxidative stress.Reference Bukowski, Hansen and Willinger40 In our study, IL-6 expression, a proinflammatory cytokine, was increased in the placentae of obese mothers. This inflammation was then significantly decreased in the placentae of operated rats as compared to obese rats, suggesting a reversibility of the placental inflammatory effects of obesity after bariatric surgery. As well, the decreased expression of MMP9 in placentae of operated rats suggests normalisation of the placental inflammation/oxidative stress profile by bariatric surgery. These results were also found after maternal weight loss through physical exercise.Reference Malti, Merzouk and Merzouk41
Indeed, the MMP9 gene encodes the MMP9, a metalloproteinase expressed in macrophages, particularly in atherosclerotic plaques, promoting the degradation of elastin in the arterial wall and activated by the release of oxygen free radicals. MMP9 is more expressed in tissues with high oxidative stress. On the contrary, Spann et al.Reference Spann, Lawson and Bidwell34 found an increase in MMP9 expression in rats’ placentae after maternal sleeve gastrectomy, associated with inflammation and cellular apoptosis. The reduction in this inflammation and oxidative stress may be partly linked to the decline in placental lipid accumulation and lipotoxicity associated with maternal obesity. Thus, maternal bariatric surgery could reduce the fetal and neonatal risks associated with maternal obesity, and even reduce the cardiovascular risk in children’s later life.
Molecular biology and biochemistry analyses revealed numerous modifications in the cascade of insulin function. Western blot analyses demonstrated a decreasing trend in the protein expression of IRS1 in placentae of obese rats and a decrease in placentae of rats that have undergone bariatric surgery.
RTqPCR showed a declining trend in placental gene expression associated with the insulin cascade (AKT1, AKT2, MAPK3, INSR, and mTOR) in placentae of obese rats and a significant reduction in placentae from rats following bariatric surgery. These disparate modifications suggest that placental obesity-induced glucose transport and metabolism troubles may not be corrected by bariatric surgery, despite mothers’ phenotype improvement and normal mothers’ weight. Glucose is the primary stimulus for insulin secretion. It reaches islet β-cells via the GLUT4 transporter. An increase in intracellular glucose concentration leads to a cascade resulting in insulin secretion via an exocytosis mechanism. Once secreted, insulin binds to its receptor (INSR) with tyrosine kinase activity located in target cells (hepatocytes, skeletal muscle cells and adipocytes), phosphorylates IRS1 and activates a PI3K/Akt pathway involved in the control of energy metabolism. Thus, activation of the insulin receptor leads to translocation of the GLUT4 transporter to the cell membrane allowing glucose to enter the cell and reducing blood glucose levels. Glucose is then stored in the form of glycogen and fatty acids.Reference Schultze, Hemmings, Niessen and Tschopp42 In addition, there is another important insulin signalling pathway, the MAPK pathway, which is common to many growth factors and ultimately activates gene expression and proliferation. The PI3 kinase/PKB and MAPK pathways are interconnected, both participating in each other’s activation. IRS1 is the first mediator activated in response to insulin release.Reference Schultze, Hemmings, Niessen and Tschopp42,Reference Ruiz-Palacios, Ruiz-Alcaraz, Sanchez-Campillo and Larqué43
The decreased expression of AKT1, AKT2, MAPK3, mTOR and SCL38A1 in rats’ placentae after bariatric surgery may indicate a restriction in the transport of carbohydrates to the fetus. Furthermore, the insulin pathway influences the amino acid transport through the placenta via AKT1.Reference Barnes and Ozanne44 A disruption to this pathway can induce a protein deficiency in the fetus. This, results in a global fetal caloric restriction based on both carbohydrates and proteins, which are the main components allowing fetuses to grow.
Consequently, maternal preconceptional bariatric surgery not only reduces placental inflammation and lipotoxicity, but may also reduce fetal caloric intake, inducing a fetal ‘thrifty phenotype’, characterised by an attempt to maintain vital functions.Reference Hales and Barker45,Reference Ford and Long46 This thrifty phenotype is responsible for a drop in fetal weight as in humans, as well as an increased of fetal death incidence. This notion of maternal energy deficit after bariatric surgery despite normal maternal weight has been reported in human studies.Reference Jans, Matthys and Bogaerts15,Reference Gascoin, Gerard and Sallé21,Reference Maric, Kanu and Muller37
In this study, no sexual dimorphism was found possibly due to a lack of power. In literature, mother obesity is associated with a greater risk of poor outcomes in male pups and placental modifications are more pronounced in males (inflammation, oxidative stress). No data are available after bariatric surgery on placenta.Reference Santos, Hernández and Sérazin47
Only one animal model study in literature analysed the effect of bariatric surgery, sleeve gastrectomy, on placenta in an obese rat model. Our study is the first to focus on inflammation and nutrient exchanges in the placenta. Human literature is also non-existent.
The main limitation of this preliminary study is the small number of pregnant rats in each group and the high number of miscarriages after bariatric surgery. Nevertheless, it remains the only study reported on the effects of maternal obesity and bariatric surgery on the feto-placental unit using a rat model.
In conclusion, this study is the first to analyse the feto-placental unit using an animal model of preconceptional bariatric surgery in pregnant obese rats. In our model, we observed obesity-induced placental modifications including lipid accumulations, inflammation and increased oxidative stress that all seemed to be at least partially corrected by maternal preconceptional bariatric surgery. We also observed a reduction in placental glucose and protein transfers to the fetus after bariatric surgery that can lead to fetal caloric restriction, the adoption of a ‘thrifty phenotype’ and subsequently fetal growth restriction. Further studies remain necessary to confirm these results and to evaluate the impact on cardiovascular and metabolic health of children in later life.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S2040174425100408.
Acknowledgements
We acknowledge all members of Mitovasc Laboratory, Angers University Hospital Biochemistry Laboratory and the pet store caregivers for their help to conduct this study.
Author contribution
Marion Plourde: Conceptualisation, Methodology, Validation, Formal Analyses, Investigation, Writing-Original Draft, Writing – Review & Editing. Inès Batellier: Conceptualisation, Methodology, Validation, Formal Analyses, Investigation, Writing-Original Draft, Writing – Review & Editing. Mathilde Rémy: Conceptualisation, Methodology. Daniel Henrion: Validation, Resources, Writing – Review & Editing, Project Administration. Céline Fassot: Conceptualisation, Methodology, Project Administration. Anne-Laure Guihot: Investigation, Linda Grimaud: Investigation, Manuela Garcia: Investigation, Jennifer Deschamps: Investigation, Clément Tétaud: Investigation, Agnès Barbelivien: Investigation, Françoise Joubaud: Investigation, Florence Boux de Casson: Investigation, Agnès Sallé: Conceptualisation. Régis Coutant: Writing – Review & Editing. Françoise Schmitt: Conceptualisation, Methodology, Validation, Writing – Review & Editing, Supervision, Project Administration, Funding Acquisition. Géraldine Gascoin: Conceptualisation, Methodology, Validation, Writing – Review & Editing, Supervision, Project Administration, Funding Acquisition.
Financial support
This project was supported by the Angers University Hospital.
Competing interests
None.
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national guides on the care and use of laboratory animals (ARRIVE Guidelines and approved by the Education, Research and Innovation Minister (APAFIS#10697-2017091422557044v1, 14/11/2017)) and has been approved by the institutional committee (CEEAPdL- 006).
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