Comparison of the ability of three radioimmunoassay to detect pregnancy-associated glycoproteins in bovine plasma

Pregnancy-associated glycoproteins (PAGs) constitute a large family of glycoproteins that are synthesized in the superficial layer of the ruminant placenta according to a spatial and temporal expression pattern. When PAGs are released in the maternal blood they can be used for pregnancy diagnosis, pregnancy follow-up and for the monitoring of the trophoblastic function. Three different radioimmunoassay systems (RIA 1, RIA 2 and RIA 3) using antisera produced against PAG I67 (RIA 1), PAG55+62 (RIA 2) and PAG55+59 (RIA 3) were used in this investigation in order to measure the PAG concentration in plasma samples withdrawn from pregnant cows and heifers during different periods following artificial insemination (AI). These systems were able to detect PAG molecules in the maternal blood as early as 21 days after AI in different concentrations (RIA 1: 0.43 +/- 0.24 ng/ml, mean +/- SD; RIA 2: 0.48 +/- 0.24 ng/ml; RIA 3: 0.64 +/- 0.37 ng/ml). On days 32 and 42 RIA 2 (4.30 +/- 1.32 ng/ml and 5.56 +/- 1.95 ng/ml) and RIA 3 (4.17 +/- 1.15 ng/ml and 5.60 +/- 1.89 ng/ml) presented significantly (p /= 0.929) were determined between the three systems. Additionally the three individual PAG profiles presented in this study showed that PAG molecules secreted in the maternal blood between 21 and 50 days after AI were better recognized by the RIA 2 and RIA 3 systems. This study clearly indicated that the ability of a RIA test to recognize PAG molecules in the maternal blood can be improved by carefully selecting the antiserum

Aspartic proteinase members secreted by the ruminant placenta: Specificity of three radioimmunoassay systems for the measurement of pregnancy-associated glycoproteins

Pregnancy-associated glycoproteins (PAGs) isolated from the placenta of various ruminant species are enzymatically inactive members of the aspartic proteinase family. The measurement of these proteins in the maternal blood can be a good indicator of the presence of a live embryo. As certain aspartic proteinases are present in biological fluids in physiological and pathological conditions at various concentrations, it was necessary to determine the specificity of three radioimmunoassay (RIA) systems currently used for the detection of PAG molecules. Commercially available members of the aspartic proteinase family like pepsinogen, pepsin, chymosin, rennet, cathepsin D and renin were tested in a wide concentration range (10 ng/ml - 1 mg/ml). Pepsinogen cross-reacted in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 50 microg/ml and 500 microg/ml concentrations, respectively. In the presence of pepsin, cross-reaction was observed in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 500 microg/ml and 1 mg/ml concentrations, respectively. Chymosin and rennet could cross-react in RIA 2 and RIA 3, while renin and cathepsin D did not decrease the binding of the tracer to antisera more, than that of the minimal detection limit. As the plasma/serum concentrations of the examined aspartic proteinases reported in the literature were outside the concentration range where cross-reaction was observed, it can be concluded that these RIA systems were specific for the detection of PAGs in biological fluids

The monitoring of bovine pregnancies derived from transplantation of in vitro produced embryos

International audienceBoth an increased rate of embryonic, foetal and perinatal losses, and the occurrence of deviations in foetal and placental development are associated with bovine pregnancies obtained from in vitro produced embryos. This thus requires for a more accurate and frequent monitoring of foetal and maternal functions during pregnancies. Such approaches will enable to establish the period during which these losses and deviations in development occur and to plan possible clinical interventions. This paper reviews some recent data on return rates, late embryonic and foetal losses in recipients after the transfer of either MOET, IVF or nuclear transfer embryos. Special attention is paid to the diagnostic value of measurements of pregnancy specific/associated proteins and progesterone in maternal plasma. Possibilities to measure foetal body sizes, size of placentomes and foetal heart rate by means of transrectal or transabdominal ultrasonography are illustrated with data from the literature and with recent results from our own large field study with MOET, IVP-co-culture and IVP-SOF embryos

Fetometry and fetal heart rates between Day 35 and 108 in bovine pregnancies resulting from transfer of either MOET, IVP-co-culture or IVP-SOF embryos

The Large Offspring Syndrome has frequently been reported for in vitro produced calves. The objective of this study was to determine whether any differences in body dimensions (biparietal diameter of the cranium (BPD), cross-section of the abdomen at the insertion of the umbilical cord (CAU)) and heart rate (FHR) can be detected during the first 108 days of gestation between bovine foetuses derived from different methods of embryo production. Three groups of pregnancies with calvings at term resulted from non-surgical transfers of three types of embryos: recipients carrying an embryo obtained by standard MOET procedures (n = 25); recipients carrying an embryo produced in vitro from OPU-derived oocytes, using co-culture-medium (n = 14) or SOF-medium (n = 22). Transrectal ultrasonographic examinations were performed weekly. Ultrasound images were recorded and during off-line analysis FHR, BPD and CAU were determined. For each foetus a curve was fitted and the estimates on fixed time intervals were used as dependent variables in an analysis of variance to detect differences between the three pregnancy groups. Neither gestation length nor birth weight differed significantly between the three pregnancy groups, nor could any differences with respect to BPD, CAU or FHR be detected between Days 35 and 108 of gestation. It is concluded that no differences exist between the early development of bovine foetuses, derived from MOET, IVP-co-culture or IVP-SOF embryos, and resulting in calves with normal birth weights

Plasma concentrations of bovine pregnancy-associated glycoprotein (bPAG) do not differ during the first 119 days between ongoing pregnancies derived by transfer of in vivo and in vitro produced embryos

Calves derived from IVP embryos may suffer from the large offspring syndrome that has been related to effects of in vitro culture on the intrinsic quality of the embryo. Limited information is available on the role of the placenta in such cases. In this study, bovine pregnancy-associated glycoprotein (bPAG) was used as a marker to test whether placental function is influenced by the route of embryo production. Therefore, from day 7 until day 119 of ongoing gestations, resulting from transfer of MOET (n = 53), IVP-co-culture (n = 21) and IVP-SOF (n = 38) embryos, bPAG levels were compared in peripheral plasma of recipients. Plasma progesterone levels were compared as well. From day 25 of gestation onwards, bPAG could be detected in all recipients and the levels were significantly influenced by the day of gestation. Although IVP calves were significantly heavier than the in vivo produced calves, this difference was not reflected in the bPAG profiles of the embryo production groups. Yet, the mean bPAG level of the three last sampling moments (days 105-119) tended to be positively related to the birth weight of the calves, irrespective of the embryo production technique. Progesterone concentrations were not influenced by route of embryo production, but were significantly affected by parity of the recipient and day of gestatio

Development of nanoelectrospray high resolution isotope dilution mass spectrometry for targeted quantitative analysis of urinary metabolites: application to population profiling and clinical studies

An automated chip-based electrospray platform was used to develop a high-throughput nanoelectrospray high resolution mass spectrometry (nESI-HRMS) method for multiplexed parallel untargeted and targeted quantitative metabolic analysis of urine samples. The method was demonstrated to be suitable for metabolic analysis of large sample numbers and can be applied to large-scale epidemiological and stratified medicine studies. The method requires a small amount of sample (5 μL of injectable volume containing 250 nL of original sample), and the analysis time for each sample is three minutes per sample to acquire data in both negative and positive ion modes. Identification of metabolites was based on the high resolution accurate mass and tandem mass spectrometry using authentic standards. The method was validated for 8 targeted metabolites and was shown to be precise and accurate. The mean accuracy of individual measurements being 106% and the intra- and inter-day precision (expressed as relative standard deviations) were 9% and 14%, respectively. Selected metabolites were quantified by standard addition calibration using the stable isotope labelled internal standards in a pooled urine sample, to account for any matrix effect. The multiple point standard addition calibration curves yielded correlation coefficients greater than 0.99, and the linear dynamic range was more than three orders of magnitude. As a proof-of-concept the developed method was applied for targeted quantitative analysis of a set of 101 urine samples obtained from female participants with different pregnancy outcomes. In addition to the specifically targeted metabolites, several other metabolites were quantified relative to the internal standards. Based on the calculated concentrations, some metabolites showed significant differences according to different pregnancy outcomes. The acquired high resolution full-scan data were used for further untargeted fingerprinting and improved the differentiation of urine samples based on pregnancy outcome

The incidence and significance of anti-natalizumab antibodies - Results from AFFIRM and SENTINEL

Objective: To determine the incidence and clinical effects of antibodies that develop during treatment with natalizumab