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PreImplantation factor (PIF) detection in maternal circulation in early pregnancy correlates with live birth (bovine model).

Ramu S, Stamatkin C, Timms L, Ruble M, Roussev RG, Barnea ER - Reprod. Biol. Endocrinol. (2013)

Bottom Line: PIF level in pregnant samples was a stringent 3 + SD higher as compared to heifers and steer sera.Anti-PIF mAb is specific and does not interact with circulating proteins.Data herein documents that PIF is a specific, reliable embryo-derived biomarker conveniently detectable in early maternal circulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Society for the Investigation of Early Pregnancy (SIEP), 1697 Lark Lane, Cherry Hill, NJ 08003, USA. barnea@earlypregnancy.org.

ABSTRACT

Background: Early identification of viable pregnancy is paramount for successful reproduction. Detection of specific signals from pre-implantation viable embryos in normal pregnancy circulation would indicate initiation of embryo-maternal interaction and create a continuum to accurately reflect embryo/fetal well-being post-implantation. Viable mammalian embryos secrete PreImplantation Factor (PIF), a biomarker which plays key, multi-targeted roles to promote implantation, trophoblast invasion and modulate maternal innate and adaptive immunity toward acceptance. Anti-PIF monoclonal antibody (mAb-based chemiluminescent ELISA) accurately detects PIF in singly cultured embryos media and its increased levels correlate with embryo development up to the blastocyst stage. Herein reported that PIF levels (ELISA) in early maternal serum correlate with pregnancy outcome.

Methods: Artificially inseminated (AI) blind-coded Angus cattle (N = 21-23) serum samples (day 10,15 & 20 post-AI) with known calf birth were blindly tested, using both non-pregnant heifers (N = 30) and steer serum as negative controls. Assay properties and anti-PIF monoclonal antibody specificity were determined by examining linearity, spike and recovery experiments and testing the antibody against 234 different circulating proteins by microarray. Endogenous PIF was detected using <3 kDa filter separation followed by anti-PIF mAb-based affinity chromatography and confirmed by ELISA and HPLC. PIF expression was established in placenta using anti-PIF mAb-based IHC.

Results: PIF detects viable pregnancy at day 10 post-AI with 91.3% sensitivity, reaching 100% by day 20 and correlating with live calf birth. All non-pregnant samples were PIF negative. PIF level in pregnant samples was a stringent 3 + SD higher as compared to heifers and steer sera. Assay is linear and spike and recovery data demonstrates lack of serum interference. Anti-PIF mAb is specific and does not interact with circulating proteins. Anti-PIF based affinity purification demonstrates that endogenous PIF is what ELISA detects. The early bovine placenta expresses PIF in the trophoblast layer.

Conclusion: Data herein documents that PIF is a specific, reliable embryo-derived biomarker conveniently detectable in early maternal circulation. PIF ELISA emerges as practical tool to detect viable early pregnancy from day 20 post-AI.

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Related in: MedlinePlus

PIF levels, day 10- post AI vs. control and day-20 post AI vs. control. PIF levels were determined by ELISA at different days after AI in cows with documented calf delivery. Levels were compared with non-pregnant never inseminated animal. A stringent +3SD threshold (99.74%) was used vs controls. As expected, non-pregnant control PIF levels overlap with a small number of day-10 post-AI subjects, whereas by day-20, there is clear stratification.
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Figure 2: PIF levels, day 10- post AI vs. control and day-20 post AI vs. control. PIF levels were determined by ELISA at different days after AI in cows with documented calf delivery. Levels were compared with non-pregnant never inseminated animal. A stringent +3SD threshold (99.74%) was used vs controls. As expected, non-pregnant control PIF levels overlap with a small number of day-10 post-AI subjects, whereas by day-20, there is clear stratification.

Mentions: At day-10, 22/23 (95.6%) serum cow samples were blindly diagnosed as pregnant using PIF ELISA (mean + 2SD) and subsequently calved. When using a more stringent criteria, (mean +3SD) as a threshold cut-off for non-pregnant cows only 21/23 (91.3%) were correctly identified as pregnant. One cow tested by PIF as non-pregnant at day 10; however by day 15 and 20 post-AI she was detected to be pregnant by ELISA. At day 15 18/21 (85%) serum samples were correctly diagnosed as pregnant. Remarkably, at day 20, 23/23 (100%) were detected correctly. As negative controls, 30 heifers (never inseminated or mated) and two steer samples were used. All control samples tested negative as compared with PIF positive samples (Figure 2). PIF concentrations in pregnant samples serum range was (175–227) ng/ml at day 10–20 post-AI and no significant changes in the mean concentration were found at the tested days. Background of PIF assay in non-pregnant animals is presented mean+/−SD (14+/−22) (Additional file 1: Figure S1). The data illustrates the quartiles of the individual values at background, day 10 and day 20. The median concentrations increased, and the lowest levels at day 20 were much higher than that observed at day 10. Thus PIF detection in early pregnancy circulation correlates with live calf birth.


PreImplantation factor (PIF) detection in maternal circulation in early pregnancy correlates with live birth (bovine model).

Ramu S, Stamatkin C, Timms L, Ruble M, Roussev RG, Barnea ER - Reprod. Biol. Endocrinol. (2013)

PIF levels, day 10- post AI vs. control and day-20 post AI vs. control. PIF levels were determined by ELISA at different days after AI in cows with documented calf delivery. Levels were compared with non-pregnant never inseminated animal. A stringent +3SD threshold (99.74%) was used vs controls. As expected, non-pregnant control PIF levels overlap with a small number of day-10 post-AI subjects, whereas by day-20, there is clear stratification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3842769&req=5

Figure 2: PIF levels, day 10- post AI vs. control and day-20 post AI vs. control. PIF levels were determined by ELISA at different days after AI in cows with documented calf delivery. Levels were compared with non-pregnant never inseminated animal. A stringent +3SD threshold (99.74%) was used vs controls. As expected, non-pregnant control PIF levels overlap with a small number of day-10 post-AI subjects, whereas by day-20, there is clear stratification.
Mentions: At day-10, 22/23 (95.6%) serum cow samples were blindly diagnosed as pregnant using PIF ELISA (mean + 2SD) and subsequently calved. When using a more stringent criteria, (mean +3SD) as a threshold cut-off for non-pregnant cows only 21/23 (91.3%) were correctly identified as pregnant. One cow tested by PIF as non-pregnant at day 10; however by day 15 and 20 post-AI she was detected to be pregnant by ELISA. At day 15 18/21 (85%) serum samples were correctly diagnosed as pregnant. Remarkably, at day 20, 23/23 (100%) were detected correctly. As negative controls, 30 heifers (never inseminated or mated) and two steer samples were used. All control samples tested negative as compared with PIF positive samples (Figure 2). PIF concentrations in pregnant samples serum range was (175–227) ng/ml at day 10–20 post-AI and no significant changes in the mean concentration were found at the tested days. Background of PIF assay in non-pregnant animals is presented mean+/−SD (14+/−22) (Additional file 1: Figure S1). The data illustrates the quartiles of the individual values at background, day 10 and day 20. The median concentrations increased, and the lowest levels at day 20 were much higher than that observed at day 10. Thus PIF detection in early pregnancy circulation correlates with live calf birth.

Bottom Line: PIF level in pregnant samples was a stringent 3 + SD higher as compared to heifers and steer sera.Anti-PIF mAb is specific and does not interact with circulating proteins.Data herein documents that PIF is a specific, reliable embryo-derived biomarker conveniently detectable in early maternal circulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Society for the Investigation of Early Pregnancy (SIEP), 1697 Lark Lane, Cherry Hill, NJ 08003, USA. barnea@earlypregnancy.org.

ABSTRACT

Background: Early identification of viable pregnancy is paramount for successful reproduction. Detection of specific signals from pre-implantation viable embryos in normal pregnancy circulation would indicate initiation of embryo-maternal interaction and create a continuum to accurately reflect embryo/fetal well-being post-implantation. Viable mammalian embryos secrete PreImplantation Factor (PIF), a biomarker which plays key, multi-targeted roles to promote implantation, trophoblast invasion and modulate maternal innate and adaptive immunity toward acceptance. Anti-PIF monoclonal antibody (mAb-based chemiluminescent ELISA) accurately detects PIF in singly cultured embryos media and its increased levels correlate with embryo development up to the blastocyst stage. Herein reported that PIF levels (ELISA) in early maternal serum correlate with pregnancy outcome.

Methods: Artificially inseminated (AI) blind-coded Angus cattle (N = 21-23) serum samples (day 10,15 & 20 post-AI) with known calf birth were blindly tested, using both non-pregnant heifers (N = 30) and steer serum as negative controls. Assay properties and anti-PIF monoclonal antibody specificity were determined by examining linearity, spike and recovery experiments and testing the antibody against 234 different circulating proteins by microarray. Endogenous PIF was detected using <3 kDa filter separation followed by anti-PIF mAb-based affinity chromatography and confirmed by ELISA and HPLC. PIF expression was established in placenta using anti-PIF mAb-based IHC.

Results: PIF detects viable pregnancy at day 10 post-AI with 91.3% sensitivity, reaching 100% by day 20 and correlating with live calf birth. All non-pregnant samples were PIF negative. PIF level in pregnant samples was a stringent 3 + SD higher as compared to heifers and steer sera. Assay is linear and spike and recovery data demonstrates lack of serum interference. Anti-PIF mAb is specific and does not interact with circulating proteins. Anti-PIF based affinity purification demonstrates that endogenous PIF is what ELISA detects. The early bovine placenta expresses PIF in the trophoblast layer.

Conclusion: Data herein documents that PIF is a specific, reliable embryo-derived biomarker conveniently detectable in early maternal circulation. PIF ELISA emerges as practical tool to detect viable early pregnancy from day 20 post-AI.

Show MeSH
Related in: MedlinePlus