"In House" assays for the quantification of Annexin V and its autoantibodies in patients with recurrent pregnancy loss and in vitro fertilisation failures

Abstract

Several studies have been shown that Annexin V (ANXV) autoantibodies concentrations are associated with both early recurrent pregnancy losses (RPLs) or in vitro fertilization failure (IVFf). We investigated the association between ANXV autoantibodies and ANVX levels in RPL, IVFf and normal group women. The study was conducted on 22 female patients with RPLs, 66 patients with IVFf, and 16 normal samples from women who had given birth. ANXV autoantibodies were measured using an ELISA test developed by fixing a homemade recombinant ANXV protein and examined with labeled human antibodies, while ANXV concentrations were measured by a competitive ELISA using a homemade anti ANXV polyclonal antibody. The results showed a clear relationship between the high levels of ANXV autoantibodies and the recurrent abortion. On the other hand, ANXV measurement in those patients showed decreased concentrations compared to normal samples. Negative correlation between ANXV and its autoantibodies levels was reported in almost all patients' samples. Our data supports the possibility that ANXV autoantibodies are a risk factor for reproductive failures associated with both RPLs and/or IVFf and the significant role for ANXV in the maintenance of pregnancy.

Figure 1

Preparation of rhANXV and its polyclonal antibody. (A) Purified proteins (0.5–2 µg/lane) were separated by SDS-PAGE then detected either by blue staining or by immunoblotting using rabbit anti-ANXV polyclonal antibody (R-a-ANXV/1:2000). Protein molecular weight ladder (M) (lane 1), rhANXV (lane 3), green fluorescent protein (GFP) was used as the negative control (lane 5), (line 2, 4 empty). (B) ELISA testing the reactivity of several dilutions (v:v) of R-a-ANXV antibody against serial concentrations (ng/mL) of immobilised rhANXV. Ab/Ag complexes were detected using goat anti-rabbit HRP-conjugated antibody (1:3000).

Figure 2

Testing the sensitivity of ANXV detection systems. Two ELISA systems were developed for quantitating ANXV and its autoantibodies levels. (A) Standard ELISA in pre-coated wells with serial concentrations (ng/mL) of immobilised rhANXV. (B) Competitive ELISA were performed after incubating R-a-ANXV (1:2000) with serial concentrations (ng/mL) of free ANXV then the mixture was added to the immobilised rhANXV (300 ng/mL). Ab/Ag complexes were detected using goat anti-rabbit HRP-conjugated antibodies (1:3000). Goodness of fit (R²) was shown for each curve.

Figure 3

Detection of ANXV autoantibodies in RPL, IVFf patients. ANXV autoantibodies in the plasma of RPL, IVFf and normal samples were detected using standard ELISA. (A) Detection values were expressed as a fold increase in ELISA signal between rhANXV-coated wells (300 ng/mL) and uncoated blank wells. Dotted line defines the cutoff of positive samples (> 1.5 folds). (B) The means of fold signals for all samples (Total Fold Signal) in each group or for those in the group having detection value above the cutoff (Limited Fold Signal) are shown (Bars, left Y-axes). Percentage of samples having detection value above the cutoff for each group is shown (•, right Y- axes). (C) Relative frequencies (%) of patients according to their fold detection values in the different groups.

Figure 4

Detection of ANXV in RPL, IVFf patients. (A) The graph shows the average concentrations (ng/mL) of ANXV (Bars, left Y-axes), determined by competitive ELISA and ANXV autoantibodies absorbance detection signals (Points, right Y-axes), obtained by standard ELISA, for RPL, IVFf and normal samples. Percentage of total, normal and patient samples categorized in several ranges according to their ANXV concentration (ng/mL) (B) or ANXV autoantibodies absorbance (C).

Figure 5

Relationship between ANXV and its autoantibodies in same patients. A scatter plot of ANXV concentration (ng/ml) and its autoantibodies levels (absorbance) of Normal (Red), RPL (Blue) and IVF (Green) samples. Analyses were performed using GraphPad Prism 9.