Impact of a gestational exposure to diesel exhaust on offspring gonadal development: experimental study in the rabbit.

The aim of the present work was to address experimentally the possible impact of exposure to air pollution during gestation on the differentiation and function of the gonads of the offspring using a rabbit model. Rabbits were exposed daily to diluted diesel exhaust gas or filtered air from the 3rd until the 27th day of gestation, during which time germ cells migrate in genital ridges and divide, and fetal sex is determined. Offspring gonads were collected shortly before birth (28th day of gestation) or after puberty (7.5 months after birth). The structure of the gonads was analyzed by histological and immunohistological methods. Serum concentrations of testosterone and anti-Müllerian hormone were determined using ELISA. The morphology and the endocrine function of the gonads collected just at the arrest of the exposure were similar in polluted and control animals in both sexes. No differences were observed as well in gonads collected after puberty. Sperm was collected at the head of the epididymis in adults. Sperm motility and DNA fragmentation were measured. Among all parameters analyzed, only the sperm DNA fragmentation rate was increased three-fold in exposed males. Mechanisms responsible for these modifications and their physiological consequences are to be further clarified.

Placental function and structure at term is altered in broodmares fed with cereals from mid-gestation.

INTRODUCTION: Feeding pregnant broodmares with cereal concentrates has been shown to increase maternal insulin resistance and affect foal metabolism in the short and long-term. These effects are likely to be mediated by the placenta. Here, we investigated feto-placental biometry and placental structure and function at term in mares fed with or without cereals concentrates. MATERIAL AND METHODS: From 7 months of gestation, 22 multiparous mares were fed forage only (group F (n = 12)) or received forage and cracked barley (group B (n = 10)) until foaling. Foals and placentas were weighed and placental samples were collected above the umbilical cord insertion at birth. Placental histological structure was studied by stereology. A RNAseq analysis was performed on 9 placentas of each group. Enrichment of gene sets was analysed using the Gene Set Enrichment Analysis (GSEA) software using the KEGG and GO databases. RESULTS: No difference in feto-placental biometry was observed between groups. The volume of microcotyledonary vessels was decreased in B placentas and the vascular wall of allantoic arterioles was thickened. Gene sets involved in neutral amino acids, folate and anions transport and fatty acids, cholesterol and folate degradation were down-regulated while gene sets involved in RNA expression, inflammation and vascularisation were up-regulated in B placentas. CONCLUSION: Feeding pregnant mares with concentrates from mid-gestation alters the placental function and structure as observed in other species in cases of maternal insulin resistance.

Maternal obesity increases insulin resistance, low-grade inflammation and osteochondrosis lesions in foals and yearlings until 18 months of age.

INTRODUCTION: Obesity is a growing concern in horses. The effects of maternal obesity on maternal metabolism and low-grade inflammation during pregnancy, as well as offspring growth, metabolism, low-grade inflammation, testicular maturation and osteochondrotic lesions until 18 months of age were investigated. MATERIAL AND METHODS: Twenty-four mares were used and separated into two groups at insemination according to body condition score (BCS): Normal (N, n = 10, BCS ≤4) and Obese (O, n = 14, BCS ≥4.25). BCS and plasma glucose, insulin, triglyceride, urea, non-esterified fatty acid, serum amyloid A (SAA), leptin and adiponectin concentrations were monitored throughout gestation. At 300 days of gestation, a Frequently Sampled Intravenous Glucose Tolerance Test (FSIGT) was performed. After parturition, foals' weight and size were monitored until 18 months of age with plasma SAA, leptin, adiponectin, triiodothyronine (T3), thyroxine (T4) and cortisol concentrations measured at regular intervals. At 6, 12 and 18 months of age, FSIGT and osteoarticular examinations were performed. Males were gelded at one year and expression of genes involved in testicular maturation analysed by RT-qPCR. RESULTS: Throughout the experiment, maternal BCS was higher in O versus N mares. During gestation, plasma urea and adiponectin were decreased and SAA and leptin increased in O versus N mares. O mares were also more insulin resistant than N mares with a higher glucose effectiveness. Postnatally, there was no difference in offspring growth between groups. Nevertheless, plasma SAA concentrations were increased in O versus N foals until 6 months, with O foals being consistently more insulin resistant with a higher glucose effectiveness. At 12 months of age, O foals were significantly more affected by osteochondrosis than N foals. All other parameters were not different between groups. CONCLUSION: In conclusion, maternal obesity altered metabolism and increased low-grade inflammation in both dams and foals. The risk of developing osteochondrosis at 12 months of age was also higher in foals born to obese dams.

Placental alterations in structure and function in intra-uterine growth-retarded horses.

BACKGROUND: Following embryo transfer (ET), the size and breed of the recipient mare can affect fetal development and subsequent post natal growth rate and insulin sensitivity in foals. OBJECTIVES: To investigate placental adaptation in pregnancies where increased or restricted fetal growth was induced through ET between Pony, Saddlebred and Draught horses. STUDY DESIGN: In vivo experiment. METHODS: Control Pony (P, n = 21) and Saddlebred (S, n = 28) pregnancies were obtained by artificial insemination. Increased pregnancies were obtained by transferring Pony (P-D, n = 6) and Saddlebred (S-D, n = 8) embryos into Draught mares. Restricted pregnancies were obtained by transferring Saddlebred embryos into Pony mares (S-P, n = 6). Placental weight and surface were recorded and samples collected for stereology and analysis of expression of genes involved in placental growth, vascularisation and nutrient transport. Data were analysed by linear model. RESULTS: S-P foals were growth retarded when compared with controls despite increased gestational length. Placental weight was reduced but placental surface density and volume fraction were increased. Placental expression of genes involved in growth and development and nutrient transfer was strongly reduced. In contrast, placental size and weight were increased in enhanced growth P-D and S-D foals. The trophoblastic surface density and the allantoic vessels surface density were decreased in P-D and S-D, respectively, both with very few modifications in gene expression. MAIN LIMITATIONS: Control embryos were produced by artificial insemination whereas experimental embryos were produced by ET. CONCLUSIONS: Placental structure and gene expression are modified after ET into a smaller or larger breed than that of the embryo. These adaptations contribute to the observed phenotype of foal growth restriction or enhanced growth at birth.

Placental structure and function in different breeds in horses.

Ponies and sometimes draft horses are often used as experimental models for horses although size and metabolic parameters are known to vary between horse breeds. So far, there is little information about differences of placental structure and no information about differences of placental function between breeds. The aim of this study was to investigate differences in placental size, structure and function at birth in relation to foal size and weight in ponies, Saddlebred and draft horses. Pony, Saddlebred and draft horse pregnancies were obtained by artificial insemination over 2 successive breeding seasons. Foals and total fetal membranes (TFM) were weighed and placentas measured for surface area at term. Placentas were sampled above the umbilical cord insertion. Surface density and volume fraction of the different cellular components of the placenta were measured on histological sections using stereology. The expression of genes involved in growth and development, nutrient transfer and vascularization was compared between groups. Foals and TFM were lighter at birth in ponies than Saddlebred horses, and both were lighter compared to draft horses. The surface density and volume fraction of microcotyledonary vessels was increased in pony compared to Saddlebred placentas. The relative expression of genes involved in growth and development was different between breeds and increased with maternal, fetal and placental weight. Primiparous dams produced lighter foals and smaller placentas, associated with a decreased volume fraction of microcotyledonary vessels and genes involved in growth and development and vascularization. Foal sex had little effect on placental structure and function as the expression of only one gene differed according to sex, with EGFR expression being decreased in placentas of females compared to males. In conclusion, foal and placental weight, as well as placental expression of genes involved in growth and development were correlated with maternal size. Placental structure also differed between breeds, with a stronger difference between ponies and both breeds of horses.

Maternal parity affects placental development, growth and metabolism of foals until 1 year and a half.

Primiparous mares are known to produce smaller foals than multiparous mares. This difference seems to be partly explained by the reduced exchange surface and volume of the placental villi in primiparous compared to multiparous placentas. The effect of maternal parity on foals' post-natal growth, metabolism and sexual maturation, however, has been given little consideration. The objectives of this work were to analyse placental biometry and structure at term, growth of foals and yearlings, their metabolism and testicular maturation at one year of age. Twenty multiparous mares (M), aged over 6 years and 12 primiparous mares (P), aged up to 5 years were artificially inseminated with the same stallion and monitored the same way until foaling. At birth, foals and placentas were measured and placentas were sampled above at the umbilical cord insertion, as well as in the pregnant and the non-pregnant horn to perform stereological analyses. Foals were weighed and measured until 540 days of age. At 120 and 360 days of age, an Intravenous Glucose Tolerance Test was performed on foals and yearlings. At 360 days of age, the males were castrated and testicular maturation analysed by RT-qPCR. At birth, P dams produced lighter and smaller foals and placentas. The foal birth weight to placental surface ratio was lower in the P compared to the M group. P Foals remained lighter than M foals until 360 days of age and smaller until at least 540 days of age. At 120 days of age, P foals had a higher glucose tolerance than M foals, and then may be less mature than M foals in terms of the control of their glucose homeostasis. At 360 days of age, the testicles of prepubertal P stallions were less mature in the P vs the M group. In conclusion, primiparous dams produce intrauterine growth restricted, less mature and smaller foals compared to multiparous dams with altered metabolism and growth until at least 540 days of age. These differences could affect the sport career of these foals, especially if it begins at an early age.

Non-invasive evaluation of placental blood flow: lessons from animal models.

In human obstetrics, placental vascularisation impairment is frequent as well as linked to severe pathological events (preeclampsia and intrauterine growth restriction), and there is a need for reliable methods allowing non-invasive evaluation of placental blood flow. Uteroplacental vascularisation is complex, and animal models are essential for the technical development and safety assessment of these imaging tools for human clinical use; however, these techniques can also be applied in the veterinary context. This paper reviews how ultrasound-based imaging methods such as 2D and 3D Doppler can provide valuable insight for the exploration of placental blood flow both in humans and animals and how new approaches such as the use of ultrasound contrast agents or ultrafast Doppler may allow to discriminate between maternal (non-pulsatile) and foetal (pulsatile) blood flow in the placenta. Finally, functional magnetic resonance imaging could also be used to evaluate placental blood flow, as indicated by studies in animal models, but its safety in human pregnancy still requires to be confirmed.

IFPA meeting 2014 workshop report: Animal models to study pregnancy pathologies; new approaches to study human placental exposure to xenobiotics; biomarkers of pregnancy pathologies; placental genetics and epigenetics; the placenta and stillbirth and fetal growth restriction.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2014 there were six themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of animal models, xenobiotics, pathological biomarkers, genetics and epigenetics, and stillbirth and fetal growth restriction.

Impact of maternal hyperlipidic hypercholesterolaemic diet on male reproductive organs and testosterone concentration in rabbits.

The concept of Developmental Origins of Health and Disease initially stemmed from the developmental programming of metabolic diseases. Reproductive functions and fertility in adulthood may also be programmed during foetal development. We studied the impact of dietary-induced maternal hyperlipidaemia and hypercholesterolaemia (HH), administered at 10 weeks of age and throughout the gestation and lactation, on male reproductive functions of rabbit offspring. Male rabbits born to HH dams and fed a control diet had significantly lighter testes and epididymes compared with rabbits born to control dams at adulthood. No significant changes in sperm concentration, sperm DNA integrity and sperm membrane composition were observed, but serum-free testosterone concentrations were decreased in HH males. This study confirms the importance of maternal metabolic status for the development of male reproductive organs.

Analysis of placental vascularization in a pharmacological rabbit model of IUGR induced by L-NAME, a nitric oxide synthase inhibitor.

OBJECTIVES: We have previously validated the use of L-nitro-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, to induce placental hypoperfusion in a rabbit model. Here, the effects of L-NAME on placental vascularization were explored. Transplacental transfer of L-NAME and/or its active metabolite, NG-nitro-L-arginine (L-NOARG), was evaluated. METHODS: 25 pregnant female rabbits were allocated on day 24 to one of 5 groups: L-NAME groups (31.35, 62.5, 125 and 250 mg/kg/day) or Control group (C). On Day 28, the labyrinthine area was analyzed for stereology and gene expression. L-NAME and L-NOARG were quantified in maternal and fetal blood. RESULTS: The volume density of fetal vessels was significantly decreased in L-NAME (including 62.5-250 mg/kg/day which induced an IUGR) compared to C groups. L-NAME induced an increase of the volume and surface density of the maternal blood space. The trophoblast volume density remained unchanged as well as the surface density of fetal vessels. Relative expression of eNOS, VEGFA, VEGFR-1 and VEGFR-2 in placentas was not affected by 125 mg/kg/day L-NAME treatment, whereas IGF-2 expression was significantly increased in this L-NAME group compared to C. L-NAME was not detected in maternal nor fetal plasma. In contrast, fetal to maternal L-NOARG ratio was 100% in all L-NAME groups. CONCLUSION: These data demonstrate that L-NAME induced placental hypovascularization. The active L-NOARG metabolite is found in maternal and fetal plasma at similar concentrations. This could impact the fetal growth and reduces the interest of this model to study fetal outcomes of placental hypoperfusion.

The use of ruminant models in biomedical perinatal research.

Animal models are of critical importance in biomedical research. Although rodents and lagomorphs are the most commonly used species, larger species are required, especially when surgical approaches or new medical devices have to be evaluated. In particular, in the field of perinatal medicine, they are critical for the evaluation of new pharmacologic treatments and the development of new invasive procedures in fetuses. In some areas, such as developmental genetics, reproductive biotechnologies and metabolic programming, the contribution of ruminants is essential. The current report focuses on some of the most outstanding examples of great biomedical advances carried out with ruminant models in the field of perinatal research. Experiments recently carried in our research unit using ruminants are also briefly described.

[Developmental origins of health and disease in adults: role of maternal environment]. / Origines développementales de la santé et des maladies de l'adulte : rôle de l'environnement maternel.

Many epidemiological studies indicate that environmental conditions during embryonic and fetal development can have an impact on health at adulthood. Animal studies clearly demonstrate that maternal, and even paternal undernutrition or nutritional excess durably modify some epigenetic marks in their offspring, affecting gene expression and physiological adaptations to the environment. It is crucial to better define the effects of early environment on adult phenotype and epigenetic marks in humans and to develop, with the help of animal models, new preventive strategies and treatments.

Quantification of utero-placental vascularization in a rabbit model of IUGR with three-dimensional power Doppler angiography.

OBJECTIVES: Our objective was to evaluate the 3D power Doppler angiography (PDA) in terms of feasibility and ability to detect placental hypo-perfusion in an experimental rabbit model of intrauterine growth restriction (IUGR). STUDY DESIGN: 14 pregnant females were treated with NG-nitro-L-arginine methylester (L-NAME), a nitric oxide synthase inhibitor, from day 24 to day 28 of gestation, to induce an IUGR. Concomitantly, 8 pregnant rabbits were used as controls. On day 28, 3D power Doppler indices were quantified in each utero-placental unit. Morphological examination of the placentas for the control group (n = 4) and the L-NAME group (500 mg/day, n = 4) were performed with immunohistochemical staining to discriminate the fetal capillaries in the labyrinthine area. RESULTS: A total of 180 live fetuses were obtained, 108 from the L-NAME group and 72 from the control group. G28 fetal weight was significantly lower in the L-NAME group than in the control group (27.40 ± 0.55 g vs 33.14 ± 0.62 g, p < 0.0001). In the L-NAME group the vascularization index (VI), flow index (FI) and vascularization flow index (VFI) were significantly lower than in the control group (2.6 [1.4; 6.0] vs 7.6 [3.5; 12.6], p < 0.05; 28.7 [26.5; 31.3] vs 32.9 [28.3; 38.1], p < 0.05; 0.8 [0.4; 1.8] vs 2.5 [1.1; 4.1], p < 0.05, for VI, FI and VFI, respectively). Morphological examinations revealed a substantial disorganization of the placental vascular architecture in the L-NAME group. CONCLUSION: This experimental study demonstrates that quantitative 3D PDA indices are sensitive enough to detect placental vascular insufficiency in an experimental rabbit model of IUGR.

OS047. Quantification of placental vascularization in a rabbit model ofIUGR with three-dimensional power doppler angiography.

INTRODUCTION: Placental dysfunction is known to be a major cause of pregnancy complications, such as perinatal loss, preeclampsia, and intrauterine growth restriction (IUGR). Inadequate remodeling of the spiral arteries resulting in decreased blood flowto the placenta has been implicated in the pathophysiology of preeclampsia and IUGR. 3D power Doppler angiography (PDA) is a noninvasive and safe way to study blood flow within an organ or region of interest. The aim of this study was to evaluate PDA as a method to quantify placental perfusion in a pharmacological rabbit model of vascular IUGR induced by inhibition of NO synthesis. OBJECTIVES: Our objective was to evaluate the 3D power Doppler angiography (PDA) in terms of feasibility and ability to detect placental hypo-perfusion in an experimental rabbit model of intrauterine growth restriction (IUGR). METHODS: Fourteen pregnant females were treated with NG-nitro-L-arginine methylester (L-NAME), a nitric oxide synthase inhibitor, from day 24 to day 28 of gestation, to induce an IUGR. Concomitantly, 8 pregnant rabbits were used as controls. On day 28, 3D power Doppler indices were quantified in each uteroplacental unit. RESULTS: A total number of 180 live fetuses were obtained, 180 from the L-NAME group and 72 from the control group. G28 fetal weight was significantly lower in the L-NAME group than in the control group (27.40±0.55g vs 33.14±0.62g,p<0.0001). In the L-Name group the vascularization index (VI) was significantly lower than in the control group (2.6 [1.4;6.0] vs 7.6 [3.5;12.6],p<0.05). Similar results were obtained for the Flow Index (FI) and the Vascularization Flow Index (VFI). The number of fetuses considered as small for gestational age (SGA;weight<10th centile) was significantly higher in the L-NAME group than in the control group (47/108 vs 7/72,p<0.0001). The VI was significantly lower in the SGA group than in the eutrophic group (3.46 [0.46;5.9] vs 7.50 [4.22;10.9] p<0.05). Similar results were obtained for FI and VFI. CONCLUSIONS: This experiment study demonstrates that quantitative 3D PDA indices are sensitive enough to detect placental vascular insufficiency in an experimental model of IUGR.

PPARgamma/RXRalpha heterodimers control human trophoblast invasion.

The ligand-dependent nuclear receptors PPARgamma and RXRalpha/beta were recently determined to be essential for murine placental development and trophoblast differentiation. In the current study we examined the expression and role of the PPARgamma/RXRalpha heterodimers in human invasive trophoblasts. We first report that in human first trimester placenta, PPARgamma and RXRalpha are highly expressed in cytotrophoblasts at the feto-maternal interface, especially in the extravillous cytotrophoblasts involved in uterus invasion. The coexpression of PPARgamma and RXRalpha genes in extravillous cytotrophoblast nuclei were then confirmed by immunocytochemistry, immunoblot, and real-time quantitative PCR using cultured purified primary extravillous cytotrophoblasts. We next examined, using the extravillous cytotrophoblast culture model, the biological role of PPARgamma/RXRalpha heterodimers in vitro, and we showed that both synthetic (rosiglitazone) and natural [15-deoxy-delta-(12,14)PGJ(2)] PPARgamma agonists inhibit extravillous cytotrophoblast invasion in a concentration-dependent manner and synergize with pan-RXR agonists. Conversely, PPARgamma or pan-RXR antagonists promoted extravillous cytotrophoblast invasion. Furthermore, the pan-RXR antagonist abolished the inhibitory effect of the PPARgamma agonists. Together these data underscore an important function of PPARgamma/RXRalpha heterodimers in the modulation of trophoblast invasion.

Characterization of human villous and extravillous trophoblasts isolated from first trimester placenta.

Trophoblasts of the human placenta differentiate along two pathways to give either extravillous cytotrophoblasts (EVCT) with invasive properties and that are implicated in the implantation process, or villous cytotrophoblasts (VCT) that by cell fusion form multinucleated syncytiotrophoblasts. We report the first isolation and purification of these two cell types from the same chorionic villi of first trimester human placenta. We also studied their differentiation in vitro. Electron microscopy showed that in contrast to VCT, EVCT had no microvilli but contained large fibrinoid inclusions. EVCT cultures required a matrix to invade, and as previously established, VCT cultured on plastic dishes aggregated and fused to form syncytiotrophoblasts. These differentiation processes were characterized by a particular pattern of gene expression as assessed by real-time PCR and confirmed by immunocytochemical analysis of the corresponding proteins. EVCT cultured in vitro expressed high levels of HLA-G, c-erbB2, human placental lactogen, and very little human chorionic gonadotropin. Interestingly, TGFbeta2 was a marker of EVCT in vitro and in situ. These data offer a new tool for cell biologists to study the molecular mechanisms involved in human placental development and its pathology.

PPAR gamma/RXR alpha heterodimers are involved in human CG beta synthesis and human trophoblast differentiation.

Recent studies performed with null mice suggested a role of either RXR alpha or PPAR gamma in murine placental development. We report here that both PPAR gamma and RXR alpha are strongly expressed in human villous cytotrophoblasts and syncytiotrophoblasts. Moreover, specific ligands for RXRs or PPAR gamma (but not for PPAR alpha or PPAR delta) increase both human CG beta transcript levels and the secretion of human CG and its free beta-subunit. When combined, these ligands have an additive effect on human CG secretion. Pan-RXR and PPAR gamma ligands also have an additive effect on the synthesis of other syncytiotrophoblast hormones such as human placental lactogen, human placental GH, and leptin. Therefore, in human placenta, PPAR gamma/RXR alpha heterodimers are functional units during cytotrophoblast differentiation into the syncytiotrophoblast in vitro. Elements located in the regulatory region of the human CG beta gene (beta 5) were found to bind RXR alpha and PPAR gamma from human cytotrophoblast nuclear extracts, suggesting that PPAR gamma/RXR alpha heterodimers directly regulate human CG beta transcription. Altogether, these data show that PPAR gamma/RXR alpha heterodimers play an important role in human placental development.

Defect of villous cytotrophoblast differentiation into syncytiotrophoblast in Down's syndrome.

The syncytiotrophoblast (ST) is one of the major components of the human placenta, as it is involved in feto-maternal exchanges and the secretion of pregnancy-specific hormones. The aim of this study was to elucidate the formation and function of the ST in trisomy 21 (Down's syndrome). We first used the in vitro model of cytotrophoblast differentiation into ST. Cytotrophoblasts were isolated from 15 trisomy 21-affected placentas (12-35 weeks gestation) and 10 gestational age-matched control placentas. In vitro cytotrophoblasts isolated from normal placenta fused to form the ST. This was associated with an increase in transcript levels and in the secretion of hCG, human placental lactogen, placental GH, and leptin. In trisomy 21-affected placentas, we observed a defect (or a delay) in ST formation and a dramatic decrease in the synthesis and secretion of these hormones compared to those in cultured cells isolated from control age-matched placentas. These results were confirmed by a significant (P < 0.001) decrease in gene expression in total homogenates of trisomy 21-affected placentas compared to controls. These results will be of help in understanding the maternal hormonal markers of fetal trisomy 21 and the consequences of placental defects for fetal development.

The expression of nuclear retinoid receptors in human implantation.

Vitamin A and retinoids play an important role during development. They affect morphogenesis, cell growth and differentiation by interacting with two types of receptor, the RARs and the RXRs. Despite the well known established teratogenic effects of retinoids during human pregnancy, little is known about the effect of retinoids on human placental development. We studied the possible involvement of retinoids during the implantation process by investigating the spatial distribution of retinoid receptors in the human implantation site by in situ hybridization and immunohistochemistry. For in situ hybridization, we used digoxigenin-labelled antisense riboprobes. Immunochemical staining was performed with specific antibodies against the various retinoid receptors and a streptavidin-alkaline phosphatase immunostaining kit. We found that only two types of receptors were expressed at the implantation site: RARalpha and RXRalpha. Both types of receptors were present in the proliferative intermediate trophoblast, the invasive extravillous trophoblast and decidual cells. Both receptors were also present in the villous cytotrophoblasts. The presence of this retinoid receptor in the cytotrophoblasts suggests a key role for all-trans retinoic acid and/or 9-cis retinoic acid in the development of human placenta.