Hyperlipidic hypercholesterolemic diet in prepubertal rabbits affects gene expression in the embryo, restricts fetal growth and increases offspring susceptibility to obesity.

Maternal hypercholesterolemia has been shown to lead to fetal intra-uterine growth retardation (IUGR) in rabbits. The effects of a long term maternal hyperlipidemic and hypercholesterolemic diet on embryo, fetal and post-natal development, have not been addressed so far. Rabbit does were fed either a hypercholesterolemic (0.2%) hyperlipidic (8%) (HH) or a control (C) diet from 10 weeks of age. Sixteen does (N = 8 HH and N = 8 C) were euthanized at 18 weeks to assess the effect of the diet on dams before mating. Embryos from 18 females (N = 9 HH and N = 9 C) were collected from the oviducts at the 16-20 cell stage (embryonic genome activation stage) for gene expression analysis (micro array and quantitative RT-PCR). Thirty females (N = 16 HH and N = 14 C) were mated naturally and fetal growth was monitored by ultrasound. Six of them (N = 4 HH and N = 2 C) were euthanized at D28 of gestation to collect fetuses and placentas. Finally, the remaining 24 does delivered at term and litters were cross fostered and equilibrated in number to create 4 groups according to the biological dam and the foster dam (C-C, C-HH, HH-C, HH-HH). Growth was monitored until weaning. A subset of 26 offspring from the 4 groups was fed the control diet until 25 weeks of age and then fed the HH diet for three weeks. All does had similar growth rates and bodyweight. Transcriptomic analyses evidenced an overexpression of Adipophilin in HH embryos at the stage of embryonic genome activation. This was confirmed by quantitative RT-PCR. During pregnancy, IUGR was observed from D9 by ultrasound and subsequently, fetal weight at 28 days, birthweight and fat deposition in newborn offspring were significantly decreased in HH (P < 0.05). After weaning, there was no significant difference for weight between HH-HH and HH-C offspring and both groups became significantly heavier (P < 0.0001) than C-C and C-HH offspring. During the 3 weeks when offspring were fed the HH diet, the differences in feed intake were no longer significant between groups but the differences in body weight remained. At post-mortem, offspring from HH does had significantly more abdominal and inter-scapular fat than offspring from C does (P < 0.05). These data illustrate the importance of maternal nutrition before and during gestation in the establishment and control of the growth trajectory of the conceptus and in the onset of disease in adult life.

Correlation between uteroplacental three-dimensional power Doppler indices and true uterine blood flow: evaluation in a pregnant sheep model.

OBJECTIVES: Three-dimensional (3D) Doppler quantification within the uteroplacental unit could be of great help in understanding and screening for pre-eclampsia and intrauterine growth restriction. Yet the correlation between 3D Doppler indices and true blood flow has not been confirmed in vivo. The aim of this study was to evaluate this correlation in a pregnant sheep model. METHODS: A blood flow quantitative sensor and a controllable vascular occlusion system were placed around the common uterine artery in seven sheep in late pregnancy, while all the other arterial supplies were ligated. Several occlusion levels were applied, from 0 to 100%, simultaneously with 3D Doppler acquisitions of several placentomes, using standardized settings. Each placentome was analyzed using VOCAL™ (Virtual Organ Computer-aided AnaLysis) software. The correlation between true blood flow and Doppler indices (vascularization index (VI), flow index (FI) and vascularization flow index (VFI)) was evaluated, together with measurement reproducibility. RESULTS: Forty-eight acquisitions were analyzed. All 3D Doppler indices were significantly correlated with true blood flow. Higher correlations were observed for VI and VFI (r = 0.81 (0.74-0.87), P < 0.0001 and r = 0.75 (0.67-0.82), P < 0.0001) compared with FI (r = 0.53 (0.38-0.64) P < 0.0001). Both intra- and interobserver reproducibility were high, with intraclass correlation coefficients of at least 0.799. CONCLUSION: This is the first in-vivo experimental study confirming a significant correlation between true blood perfusion and quantitative 3D Doppler indices measured within the uteroplacental unit. These results confirm the potential usefulness of 3D Doppler ultrasound for the assessment of placental vascular insufficiency both in clinical cases and in a research setting.

In utero characterisation of fetal growth by ultrasound scanning in the rabbit.

Fetal development is an important factor influencing the susceptibility of adults to metabolic diseases. In order to study the influence of fetal growth on further development in animal models like the rabbit, methods of measurement of fetal and placental size and viability must be established and validated. In this study, 42 New Zealand does bred naturally (N=12) or transferred with in vivo produced embryos (2, 4 or 6 embryos/doe) have been scanned every 2-3 days with a 7.5 MHz transabdominal probe from Day 7 post-coitum until term to measure fetal and placental growth. Vesicle, placental, fetal length and head size have thus been determined according to number of fetuses and time. In late gestation, the fetuses that were transferred in limited numbers to the uterus of does were significantly larger than their natural breeding counterparts probably due to reduced litter size.

Placental expression of major histocompatibility complex class I in bovine somatic clones.

Abnormally increased placental expression of major histocompatibility complex class I (MHC-I) molecules at the trophoblastic surface has been suggested previously to be the cause of early fetal loss in nuclear transfer (NT) bovine pregnancies. Here, we report the lack of expression of MHC-I at the trophoblastic surface at D30 and D60 and in placentomes from D60 to term in placentas obtained by NT from three different genotypes and by artificial insemination, whatever the outcome of the pregnancy. MHC-I expression was assessed by immunohistochemistry using four different antibodies, including a novel beta2-microglobulin antibody. The MHC-I type of the clones was established using reference strand-mediated conformation analysis (RSCA); however, since it proved problematic to type the recipient animals in the same way, outcome of pregnancy could not be related to MHC compatibility. In conclusion, the present study provides no evidence to support abnormal expression of MHC-I on the trophoblastic surface in clones as a major cause of fetal loss during pregnancy after NT.

Large offspring or large placenta syndrome? Morphometric analysis of late gestation bovine placentomes from somatic nuclear transfer pregnancies complicated by hydrallantois.

Somatic nuclear transfer (NT) in cattle is often complicated by fetal oversize (i.e., large offspring syndrome), hydrallantois, and placentomegaly in late gestation. The aims of this work were to obtain data on the placentome structure in NT-recipient cows with hydrallantois (NTH) and to relate these with fetal and placental weights to better understand the abnormalities observed in NTH pregnancies during the third trimester. Pregnant cows were slaughtered between Gestation Days 180 and 280. The fetuses were weighed, and the placentomes were numbered and weighed. Placentomes were examined by histologic and stereological techniques. Macroscopic data showed that placental overgrowth preceded fetal overgrowth, and the ratio of the fetal to the total placentome weight in the NTH group was lower than that in controls after Gestation Day 220. This suggests that placental overgrowth is due to placental default rather than due to fetal overgrowth, as shown also by stereological analysis showing primary deregulation of the growth of cotyledonary tissues. Observed alterations, such as thinning of the maternal epithelium within placentomes and increased trophoblastic surface, could be secondary adaptations. Thus, placental growth deregulations would be due to modifications of the expression of placental factors. Various examples of placental deficiency were observed, suggesting that some fetal abnormalities observed in NTH calves, such as enlarged heart, enlarged umbilical cord, and abdominal ascites, are consequences of placental dysfunction. Therefore, the condition described by the term "large offspring syndrome" might better be described by "large placenta syndrome," because this syndrome affects an average of 50% of late-gestation NT pregnancies. No conclusion can be drawn from this work on apparently normal pregnancies.

Ultrasound fetal measurements and pregnancy associated glycoprotein secretion in early pregnancy in cattle recipients carrying somatic clones.

Somatic cloning in the bovine species leads to high levels of fetal losses which occur throughout pregnancy. These losses are most often associated with fetal overgrowth, a syndrome known as large offspring syndrome (LOS), and excessive maternal plasma pregnancy serum protein 60 (PSP60), a protein similar to a pregnancy-associated glycoprotein of 67 kDa (PAG I67) produced by the bovine placenta. Predicting the outcome of pregnancies initiated from cloned embryos has become an important issue both to prevent potential harm to the mother because of excessive fetal size at birth and also to get a better understanding of the relationships between growth, differentiation and placental functions in developing cloned fetuses. Here, we report on a systematic analysis of fetal and placental development in the first trimester of pregnancy performed by ultrasonographic imaging and by measurement of the maternal concentrations of pregnancy associated glycoproteins (PAGS), using four different radioimmunoassays (RIA) (two homologous RIA systems with PSP60 and PAG I67; two heterologous RIA systems with PAG I67 as standard and tracer, and antisera anti-caprine PAGs). We showed that crown-rump length (CRL) in clones appeared smaller than controls at 35, 50 and 62 days (P<0.05). At 62 days of pregnancy, CRL in cloned fetuses that died before 90 days was smaller compared to the other cloned fetuses (P<0.05) whereas the width of the fetal sack and the biparietal diameter (BPD) was larger in fetuses that developed LOS in late gestation (P<0.05). Maternal PAGs concentrations were statistically different between controls and all clone recipients as early as Day 34, suggesting early abnormal placental glycoprotein synthesis for clone pregnancies regardless of pregnancy outcome. This work provides a practical, non-invasive tool to follow up clone pregnancies and suggests that primary growth retardation and abnormal placental function precedes excessive fetal and placental growth at later stages of pregnancy.

Health status of cloned cattle at different ages.

The procedure of somatic cloning is associated with important losses during pregnancy and in the perinatal period, reducing the overall efficacy to less than 5% in most cases. A mean of 30% of the cloned calves die before reaching 6 months of age with a wide range of pathologies, including, for the most common, respiratory failure, abnormal kidney development, liver steatosis. Heart and liver weight in relation to body weight are also increased. Surviving animals, although mostly clinically normal, differ from controls obtained by artificial insemination (AI) within the first 1-2 months, to become undistinguishable from them thereafter. Hemoglobin concentrations, for instance, are lower, and leptin concentrations are elevated. In response to the lack of prospective studies addressing the health of adult clones, a long-term, 3-4-year study is currently being conducted to assess the health of mature bovine clones at INRA. Preliminary results over 1 year of study do not show any statistical difference between groups for hematological parameters.