Trans-Uterine Tracer Injections in Mammalian Foetuses

Intra-uterine manipulation of mammalian foetuses for experimental purposes was first described at the beginning of this century (Wolff, 1919). Though numerous publications have appeared since which bear witness to the feasibility of intra-uterine manipulation, its application has remained rather restricted. In this paper we describe a technique for the trans-uterine injection of neuronal tracers into rat foetuses. Uterine wall and foetal membranes are pierced only with a micro pipette, and are thus left virtually intact, preventing loss of amniotic fluid. Surgical mortality is 24% overall, but the experimental success rate is much lower (23%). Even so these results are comparable to more complicated procedures, because the technique is simple (i.e. requires no micro surgical skill), and because up to 6 foetuses can be injected per dam. Technical problems, such as foetal anaesthesia and the detection of false-negative results due to imponderable factors are discussed.     

Critical periods for foetal mortality in gilts identified by analysing the length distribution of mummified foetuses and frequency of non-fresh stillborn piglets

The objective of this study was to investigate the timing of foetal mortality in gilts of a segregating F2 cross of Large White and Meishan pigs on the basis of the length distribution of mummified foetuses and the frequency of non-fresh stillborn piglets in order to establish whether critical periods for foetal mortality exist. All expelled conceptuses and placentae of 192 farrowing gilts with a normal health status were meticulously investigated to recover all mummified foetuses. The length of each mummified foetus was measured. The predicted number of foetuses present per gilt at the early foetal stage of gestation was calculated as the sum of numbers of mummified foetuses and non-fresh stillborn, fresh stillborn and liveborn piglets. Foetal loss was calculated as the sum of mummified foetuses and non-fresh stillborn piglets. The average foetal mortality rate per gilt was 8.7%. In total 162 mummified foetuses were found (average 0.84 per litter), ranging in length from 0.4 to 33.0 cm. This indicates a range in foetal age at death of approximately 35-100 days. Although mummified foetuses of all lengths within the above mentioned range were found, relatively many had a length of less than 4 cm or of 10-21 cm. The total number of non-fresh stillborn piglets (i.e. late foetal deaths) was 58 (average 0.30 per litter). It can be concluded that foetal mortality occurred in these gilts throughout the period from day 35 to term, with relatively high incidences at the early foetal stage (days 35-40), shortly after mid-pregnancy (days 55-75) and after approximately day 100 of gestation. These three periods coincide with reported periods of change in porcine placental growth.     

Comparison of the IFAT and Iscom-ELISA response in bovine foetuses with Neospora caninum infection

The study was carried out to evaluate the efficacy of foetal serology in the diagnosis of Neospora-associated bovine abortions. Fluids from 14 foetuses of cows with confirmed neosporosis (Group A), seven foetuses with confirmed bovine viral diarrhoea virus (BVD infection) (Group B) and 11 aborted foetuses without demonstrable infection (Group C) were examined. The age of the foetuses ranged from 4.5 months to 9 months. Albumin concentration (measured by Rocket Immunoelectrophoresis) was not significantly different in Group A compared with that in both Groups B and C, while that in Group B was significantly lower than in Group C. Levels of total IgG ranged from 0.01 to 1.78 (mg IgG) ml(-1) measured by single radial immunodiffusion technique. A measurable level of total IgG was found in all foetuses from Groups A and B, with no significant difference between levels in the two groups. Only one foetus in Group C had a detectable level of IgG. All foetuses in Group A had a specific IgG response (titre> or = 20) against Neospora caninum using the IFAT, while no positive responses in IFAT were found in Groups B and C. Measurement of specific IgG1 and IgG2 by Iscom-ELISA showed one and three false-negative results, respectively, in Group A. The IgG1 and IgG2 response in Group A was correlated according to the Spearman test (r = 0.66). Increasing age of the foetuses correlated significantly with the foetal IgG concentration, the specific IgG and IgG1 + IgG2. On the basis of the results obtained, it was concluded that the IFAT with a cut-off titre of 1:20, was a specific method for diagnosis of neosporosis in foetuses older than 4.5 months. The Iscom-ELISA also showed promising results as a method for screening specific antibodies against N. caninum in foetal fluid.     

The use of foetal ovarian stromal cell culture for cytogenetic diagnosis. Stromal ovarian culture cytogenetic diagnosis

Some studies have been carried out to analyze human female first meiotic prophase. Most of them use samples from foetuses collected after legal interruption of pregnancy. In some cases, a control population is needed and foetuses aborted for non-chromosomal reasons are used. The assumption of these samples as being euploids could perhaps represent an error. In this article, we describe an easy methodology to certify the euploidy of foetal ovarian tissue using an one-week somatic culture. Using this protocol, we have obtained a primary culture in 88.2% of the studied cases, material usable for being karyotyped in 93.3% of the cases, and a cytogenetic diagnosis was performed in 100% of these cases. Finding the same karyotype in cultured cells in cases in which we had a prenatal cytogenetic diagnosis has validated the technique, and in applying this protocol we have been able to check our prophase meiotic-study control population.     

Synthesis and secretion of albumin and transferrin by foetal rat hepatocyte cultures

Hepatocytes derived from foetal rat liver synthesize and secrete albumin and transferrin when maintained in primary culture. These proteins are produced for at least seven days under the conditions of culture. Studies on hepatocyte cultures derived from 12, 13, 14, 15 and 19-day foetal rats show that the maximal cellular rate of secretion of both proteins increases about 50-fold over this period. The maximal rate of albumin secretion in all cultures is achieved after one day in culture and decreases in hepatocytes from early foetuses after the fourth to sixth day in culture. Transferrin secretion by hepatocytes from 12 to 15 day foetuses increases markedly during the second day of culture and is relatively constant thereafter. In contrast, secretion of transferrin by hepatocytes from 19-day foetuses is constant from the first day of culture. The results show that both albumin and transferrin are synthesized and secreted by the foetal liver as early as the twelfth day of gestation. The increase in the rate of transferrin secretion that occurs during culture of hepatocytes from 12 to 15 day foetuses may reflect the development of a secretory mechanism that is different from that for albumin.     

Materno-foetal exchanges and utilisation of nutrients by the foetus: comparison between species

Several general features of nutrient uptake and utilisation by foetuses are similar among mammalian species. Nevertheless, there are also differences linked mainly to differences in placental permeability. Glucose and lactate are the main energetic substrates of the foetus. In normal conditions, the oxidation of carbohydrates accounts for about 75, 60 and 50% of oxygen uptake in the foetal pig, foal and lamb, respectively, and acetate accounts for about 10% in ruminants. Acidic amino acids are synthesised by the foetus, whereas neutral and basic amino acids are transported from the placenta. As shown by the high urea level in foetal blood, amino acids are partly involved in the oxidative metabolism of foetuses; their contribution is higher in ruminants than in humans, horses and pigs. Fatty acids cross the haemochorial placenta of rodents, rabbits and primates, and are incorporated into the foetal lipids, whereas their uptake by ruminant, pig and horse foetuses is very low.     

Influence of the stage of pregnancy on Neospora caninum distribution, parasite loads and lesions in aborted bovine foetuses

In the present work we have studied in Neospra caninum aborted bovine foetuses the influence of foetal age (first, second and third gestational periods) on parasite distribution by nested PCR, parasite loads by real-time PCR and N. caninum associated lesions. For this purpose, a total of 220 aborted foetuses were analysed and detection of N. caninum infection was accomplished by nested-PCR in brain, heart and liver, detecting the presence of the parasite in 72 (32.7%) bovine foetuses. When the different age classes were compared, parasite DNA-detectability in heart and liver was reduced over time of gestation (P < 0.05, Fisher F-test). N. caninum distribution, parasite loads and lesions were studied on 34 out of 72 N. caninum-infected foetuses selected according to the stage of pregnancy and organs recovered. A higher number of positive-PCR tissue samples were observed in the foetuses corresponding to the first and second pregnancy periods. In the last trimester, the parasite could only be detected in the brain and, sporadically, in the diaphragm, heart and lymph nodes. The parasite loads decreased during pregnancy and the foetuses from the first period had higher parasite burdens in brain, heart, kidney and lung (P < 0.05, Kruskal-Wallis H-test) than in those corresponding to the other two trimesters of pregnancy. In addition, the observed lesions were more severe in foetuses from the first and second pregnancy periods than those from the third period (P > 0.05, Kruskal-Wallis H-test). Our results confirm the influence of N. caninum foetal age on pathogenesis in natural N. caninum infections.     

Turkey liver xanthine dehydrogenase. Reactivation of the cyanide-inactivated enzyme by sulphide and by selenide

1. The glycogen present in the liver of rat foetuses was labelled by injecting a trace amount of [6-(3)H]glucose into the mother at 19.5 days of gestation. The radioactivity incorporated in the glycogen 4h after the administration of the label was still present 38h later. A large proportion of this radioactivity was on the outer chains of the polysaccharide. These results indicate that there is normally almost no glycogen degradation in the foetal liver. In contrast, glycogen breakdown occurs very rapidly in the livers of foetuses whose mother is anaesthetized. 2. Glycogen synthetase is present in the liver at day 16 of gestation at a concentration as high as 30% of that in the adult, but essentially as an inactive (b) enzyme. The appearance of synthetase phosphatase between days 18 and 19 corresponds to that of synthetase a and to the beginning of glycogen synthesis. From day 19 to 21.5 the amount of synthetase a present in the foetal liver is just sufficient to account for the actual rate of glycogen deposition. 3. The content of total phosphorylase in the foetal liver increases continuously from day 16 to birth. However, a precise measurement of the a and b forms of the enzyme in the liver of non-anaesthetized foetuses is not possible. Taking the rate of glycogenolysis as an appropriate index of phosphorylase activity, we conclude that this enzyme is almost entirely in the inactive form in the foetal liver under normal conditions. 4. The accumulation of glycogen in the liver during late pregnancy may therefore be explained by a relatively slow rate of synthesis and a nearly total absence of degradation.     

Chromosomal mosaicism detected by karyotyping and chromosomal microarray analysis in prenatal diagnosis

To investigate the incidence and clinical significance of chromosomal mosaicism (CM) in prenatal diagnosis by G-banding karyotyping and chromosomal microarray analysis (CMA). This is a single-centre retrospective study of invasive prenatal diagnosis for CM. From 5758 karyotyping results and 6066 CMA results, 104 foetal cases with CM were selected and analysed further. In total, 50% (52/104) of foetal cases with CM were affected by ultrasound-detectable phenotypes. Regardless of whether they were singleton or twin pregnancies, isolated structural defects in one system (51.35%, 19/37 in singletons; 86.67%, 13/15 in twins) and a single soft marker (18.92%, 7/37 in singletons; 13.33%, 2/15 in twins) were the most common ultrasound anomalies. Mosaic autosomal trisomy (19.23%, 20/104) was the most frequent type, and its rate was higher in phenotypic foetuses (28.85%, 15/52) than in non-phenotypic foetuses (9.62%, 5/52). There was no difference in mosaic fractions between phenotypic and non-phenotypic foetuses based on specimen sources or overall classification. Discordant mosaic results were observed in 16 cases (15.38%, 16/104) from different specimens or different testing methods. Genetic counselling and clinical management regarding CM in prenatal diagnosis remain challenging due to the variable phenotypes and unclear significance. Greater caution should be used in prenatal counselling, and more comprehensive assays involving serial ultrasound examinations, different specimens or testing methods verifications and follow-up should be applied.     

The effect of hypertransfusion on the growth of mouse foetal liver cells transplanted into irradiated, adult recipients: failure to detect a differential effect between cells from early and late foetuses

Erythropoiesis can be reinstated in lethally irradiated mice by transplantation of adult bone marrow or liver cells from foetuses at any stage of gestation beyond the eleventh day. Hypertransfusion of the recipients abolishes erythropoiesis resulting from the transplants, even when the transplants are derived from foetuses at a very early stage of development. This suggests that erythropoietin is necessary for the successful growth of these early foetal transplants, at least in the environment of the irradiated recipient. These results are at variance with an earlier report on the susceptibility of early foetal liver transplants to suppression by hypertransfusion.     

Plasma amino acid concentrations in pregnant rats and in 21-day foetuses.

Plasma amino acid concentrations were determined in virgin female rats, in pregnant rats (12 and 21 days after impregnation) and in 21-day foetuses. The total amino acid concentration in plasma decreases significantly with pregnancy, being lower at 12 than at 21 days. Alanine, glutamine+glutamate and other 'gluconeogenic' amino acids decrease dramatically by mid-term, but regain their original concentrations at the end of the pregnancy. With most other amino acids, mainly the essential ones, the trend is towards lower concentrations which are maintained throughout pregnancy. These data agree with known nitrogen-conservation schemes in pregnancy and with the important demands on amino acids provoked by foetal growth. In the 21-day foetuses, concentrations of individual amino acids are considerably higher than in their mothers, with high plasma foetal/maternal concentration ratios, especially for lysine, phenylalanine and hydroxy-proline, suggesting active protein biosynthesis and turnover. All other amino acids also have high concentration ratios, presumably owing to their requirement by the foetuses for growth. Alanine, glutamine+glutamate, asparagine+aspartate, glycine, serine and threonine form a lower proportion of the total amino acids in foetuses than in the virgin controls or pregnant rats, probably owing to their role primarily in energy metabolism in the adults. The results indicate that at this phase of foetal growth, the placental amino acid uptake is considerable and seems to be higher than immediately before birth.     

Transplacental Transmission of Bluetongue Virus Serotype 1 and Serotype 8 in Sheep: Virological and Pathological Findings

The Bluetongue virus serotype 8 (BTV-8) strain, which emerged in Europe in 2006, had an unusually high ability to cause foetal infection in pregnant ruminants. Other serotypes of BTV had already been present in Europe for more than a decade, but transplacental transmission of these strains had never been demonstrated. To determine whether transplacental transmission is a unique feature of BTV-8 we compared the incidence and pathological consequences of transplacental transmission of BTV-8 to that of BTV-1. Nine pregnant ewes were infected with either BTV-8 or BTV-1. The BTV strains used for the infection were field strains isolated on embryonated chicken eggs and passaged twice on mammalian cells. Blood samples were taken to monitor the viraemia in the ewes. Four weeks after the infection, the foetuses were examined for pathological changes and for the presence of BTV. BTV-8 could be demonstrated in 12 foetuses (43%) from 5 ewes (56%). %). BTV-1 was detected in 14 foetuses (82%) from 6 ewes (67%). Pathological changes were mainly found in the central nervous system. In the BTV-8 group, lympho-histiocytic infiltrates, gliosis and slight vacuolation of the neuropil were found. BTV-1infection induced a severe necrotizing encephalopathy and severe meningitis, with macroscopic hydranencephaly or porencephaly in 8 foetuses. In our experimental setting, using low passaged virus strains, BTV-1 was able to induce transplacental transmission to a higher incidence compared to BTV-8, causing more severe pathology.     

Pathogenic copy number variations are associated with foetal short femur length in a tertiary referral centre study

Shortened foetal femur length (FL) is a common abnormal phenotype that often causes anxiety in pregnant women, and standard clinical treatments remain unavailable. We investigated the clinical characteristics, genetic aetiology and obstetric pregnancy outcomes of foetuses with short FL and provided a reference for perinatal management of such cases. Chromosomal microarray analysis was used to analyse the copy number variations (CNV) in short FL foetuses. Of the 218 foetuses with short FL, 33 foetuses exhibited abnormal CNVs, including 19 with pathogenic CNVs and 14 with variations of uncertain clinical significance. Of the 19 foetuses with pathogenic CNVs, four had aneuploidy, 14 had deletions/duplications, and one had pathogenic uniparental diploidy. The 7q11.23 microdeletion was detected in three foetuses. The severity of short FL was not associated with the rate of pathogenic CNVs. The duration of short FL for the intrauterine ultrasound phenotype in foetuses carrying a pathogenic CNV was independent of the gestational age. Further, maternal age was not associated with the incidence of foetal pathogenic CNVs. Adverse pregnancy outcomes occurred in 77 cases, including termination of pregnancy in 63 cases, postnatal dwarfed foetuses with intellectual disability in 11 cases, and three deaths within 3 months of birth. Pathogenic CNVs closely related to foetal short FL were identified, among which the 7q11.23 microdeletion was highly associated with short FL development. This study provides a reference for the perinatal management of foetuses with short FL.     

Effects of ethanol infusion on foetal EEG and breathing movements

Ethanol (0.3 g/kg and 0.5 g/kg administered over 2 hours) was infused intravenously into 15 chronically instrumented pregnant ewes between 128 to 135 days of gestation (0.85 to 0.92 gestation time, term 147 days). Brainstem dissection above the pons was made in 7 foetuses. Foetal breathing movements were suppressed for 7 hours following a 30 ml ethanol infusion. Low voltage foetal electrocortical activity was suppressed or replaced by an intermediate voltage electrocortical activity for 5 and 3 hours following the 60 ml and 30 ml ethanol infusions, respectively. In brainstem dissected foetuses the effects of ethanol infusion on the foetal EEG were similar. Foetal blood gases and pH were not altered. These data suggest that ethanol moves across the foetal blood-brain barrier and suppresses foetal breathing movements by a direct central mechanism.     

Evaluation of prenatal diagnosis of cleft lip/palate by foetal ultrasonographic examination

Ultrasound scans in the midtrimester of pregnancy are now a routine part of antenatal care in many countries. This type of screening procedure can detect serious foetal anomalies. Thanks to our registry of congenital anomalies a study was undertaken. The objective of the study was to evaluate prenatal detection of cleft lip (palate)(CL/P) by routine ultrasonographic examination of the foetus in 265679 consecutive pregnancies from 1979 to 1998. The percentage of prenatal detection of CL/P was low. For isolated malformation (foetuses with only CL/P) the detection rate was low: 17.8%; however, this detection rate increased from 5.3% during the period 1979-1988 to 26.5% during the period 1989-1998, for foetuses with associated malformations (foetuses with CL/P and one or more additional major malformations) these detection rates were 34.6, 13. 3 and 50.0%, respectively. In foetuses with associated malformations with CL/P this detection rate was higher for chromosomal abnormalities with CL/P and for non-syndromic, non-chromosomal multiply malformed children with CL/P than for non-chromosomal recognized syndromes with CL/P.     

FOETAL ERYTHROPOIESIS IN GENETICALLY ANAEMIC, FLEXED-TAILED (f/f) MICE

This paper presents measurements of foetal weights, haemoglobin concentrations, red cell counts, blood volumes, erythroblast thymidine labelling indices and cell cycle parameters in genetically anaemic, flexed-tailed ( f/f ) and haematologically normal ( f/+ ) foetuses.
The production of red blood cells from the livers of ( f/f ) foetal mice lags behind that of heterozygote ( f/+ ) foetuses, and the cells that are produced are small and poorly haemoglobinized. The anaemia is not due to an abnormal cell cycle in f/f erythroblasts, which appear to be capable of responding to the anaemia by a small increase in their rates of proliferation. The number of red cells in the foetal circulation can be calculated from the cell cycle time and number of hepatic erythroblasts; the calculated number agrees quite well with the measured number in both f/f and f/+ foetuses. Many characteristics of the anaemia of the f/f foetus can be accounted for if it is assumed that there is a deficiency in the production or activity of an enzyme involved early in the haem synthetic pathway.     

The effect of foetal decapitation on the antenatal development of the enzymes monoamine oxidase and catechol O-methyl transferase in rabbit foetuses

The development of the enzymes monoamine oxidase and catechol O-methyl transferase in rabbit foetuses was studied. The adrenocortical system of the rabbit foetuses was inactivated by hypophysectomy on the 18th day of foetal age. The hypophysectomy of the foetuses by decapitation in utero resulted in marked increases in the activities of the enzymes monoamine oxidase and catechol O-methyl transferase in most of the body organs determined 12 days after the operation. A single administration of adrenocorticotropic hormone or hydrocortisone to the decapitated foetuses reduced the activities of both the enzymes studied in most of the body organs. The results suggest that glucocorticoids present in the circulation of foetal animals act as inhibitory factor for enzymes of catecholamine degradation. The inactivation of glucocorticoidogenesis by removal of the hypophysis takes away this rate-limiting control and highly significant increases in the activities of the enyzmes monoamine oxidase and catechol O-methyl transferase occur.     

DNA methylation patterns in tissues from mid-gestation bovine foetuses produced by somatic cell nuclear transfer show subtle abnormalities in nuclear reprogramming

Background  

Cloning of cattle by somatic cell nuclear transfer (SCNT) is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. These abnormalities are thought to be due, in part, to incomplete re-setting of the epigenetic state of DNA in the donor somatic cell nucleus to a state that is capable of driving embryonic and foetal development to completion. Here, we tested the hypothesis that DNA methylation patterns were not appropriately established during nuclear reprogramming following SCNT. A panel of imprinted, non-imprinted genes and satellite repeat sequences was examined in tissues collected from viable and failing mid-gestation SCNT foetuses and compared with similar tissues from gestation-matched normal foetuses generated by artificial insemination (AI).