The Alterations of Serum IgG Fucosylation as a Potential Additional New Diagnostic Marker in Advanced Endometriosis

Figures & data

Table 1 Relative Reactivities of Serum Native IgG and Isolated Serum IgG Glycans with Fucose-Specific Lectins

Groups	Relative Reactivity with Lectins (AU)
	AAL (s)	AAL (i)	LCA (s)	LCA (i)	LTA (s)	LTA (i)	UEA (s)	UEA (i)
E n=40	0.029±0.010	0.055±0.033	0.587±0.124	0.540±0.151	0.421±0.149	0.603±0.347	0.048±0.032	0.012±0.015
NE n=36	0.028±0.014	0.093±0.022 p^A<0.000001	0.461 ± 0.109 p^A=0.000020	0.524±0.110	0.352±0.152	0.540±0.280	0.055±0.041	0.016±0.010 p^A=0.004
Control n=19	0.056±0.015 p^A<0.000001 p^B<0.000001	0.140±0.043 p^A<0.000001 p^B= 0.000026	0.467±0.093 p^A=0.000677	0.903±0.105 p^A<0.000001 p^B<0.000001	0.213±0.293 p^A=0.000008 p^B=0.00052	0.185±0.155 p^A<0.000001 p^B<0.000001	0.067±0.045	0.193±0.097 p^A<0.000001 p^B<0.000001

Notes: The results are expressed in absorbance units (AU) as mean values ± standard deviation (SD). Serum IgG glycan reactivities with lectins were examined by lectin-ELISA and expressed in absorbance units (AU). For lectins specificity see Materials and methods section. Significant differences versus groups: ^Awith endometriosis (E), ^Bnon-endometriosis (NE). Control - group of healthy women. Statistically significant differences were accepted for a p-value of less than 0.05.

Abbreviations: s-IgG, serum native IgG; i–IgG, isolated serum IgG; AAL, Aleuria aurantia agglutinin, LCA, Lens culinaris agglutinin; LTA, Lotus tetragonolobus agglutinin; UEA, Ulex europaeus agglutinin.

Figure 1 Relative reactivities of serum native IgG (s-IgG), and isolated serum IgG (i–IgG) glycans with fucose-specific lectins: AAL - Aleuria aurantia lectin, LCA - Lens culinaris agglutinin, LTA - Lotus tetragonolobus agglutinin, UEA - Ulex europaeus agglutinin (A–D). For lectins specificity see Materials and methods section. Significant differences versus groups: ^AE, ^BNE. Median is indicated as a square. A two-tailed p-value of less than 0.05 was considered as significant (*p < 0.05; ***p < 0.001).

Abbreviations: E, endometriosis; NE, non-endometriosis; C, control group of healthy women.

Table 2 The Correlations Between Relative Reactivities of IgG Glycans with Fucose-Specific Lectins

Correlations Between Lectins’ Relative Reactivities	s-IgG	i–IgG
	Spearman Rank Coefficient (r)	Spearman Rank Coefficient (r)
AAL vs LCA		r=0.566 p<0.000001
AAL vs LTA	r=−0.350 p=0.0005	r=−0.292 p=0.004
AAL vs UEA	r=0.437 p=0.000009	r=0.610 p<0.000001
LCA vs LTA		r=−0.352 p=0.0004
LCA vs UEA		r=0.475 p=0.000001
LTA vs UEA		r=−0.270 p=0.008

Notes: For lectins specificity see Materials and methods section. A two-tailed p-value of less than 0.05 was considered as significant.

Abbreviations: s-IgG, serum native IgG; i–IgG, isolated serum IgG; AAL, Aleuria aurantia lectin; LCA, Lens culinaris agglutinin; LTA, Lotus tetragonolobus agglutinin; UEA, Ulex europaeus agglutinin.

Figure 2 The correlations between relative reactivities of s-IgG and i–IgG glycans with fucose-specific lectins (A–D). For lectins’ specificity see Materials and methods section. A two-tailed p-value of less than 0.05 was considered as significant.

Abbreviations: s-IgG, serum native IgG; i–IgG, isolated serum IgG; AAL, Aleuria aurantia lectin; LCA, Lens culinaris agglutinin; LTA, Lotus tetragonolobus agglutinin; UEA, Ulex europaeus agglutinin.

Table 3 Receiver Operating Characteristic Curve Analysis of Serum Native IgG and Isolated Serum IgG Relative Reactivities with Fucose-Specific Lectins

Reactivity with Lectin	AUC	AUC with 95% Confidence Interval	Cut-Off Point	Sensitivity	Specificity	p
s-IgG
AAL	0.926	0.861–0.992	0.044	0.950	0.737	0.0000
LCA	0.775	0.647–0.903	0.470	0.825	0.579	0.0000
LTA	0.862	0.723–1	0.226	0.925	0.842	0.0000
UEA	0.631	0.480–0.782	0.045	0.600	0.632	0.0889
i–IgG
AAL	0.953	0.899–1	0.082	0.875	1	0.0000
LCA	0.978	0.947–1	0.734	0.950	0.947	0.0000
LTA	0.923	0.838–1	0.273	0.825	0.947	0.0000
UEA	0.997	0.99–1	0.045	0.975	1	0.0000

Notes: For lectins’ specificity see Materials and methods section. The analysis was done for advanced endometriosis and control group of healthy women. The values of area under the curve (AUC) ≥0.775 were bolded. AUC value can be defined as: 0–0.5 – zero, 0.5–0.7 – limited, 0.7–0.9 – moderate and > 0.9 high.

Abbreviations: s-IgG, serum native IgG; i–IgG, isolated serum IgG; AAL, Aleuria aurantia lectin; LCA, Lens culinaris agglutinin; LTA, Lotus tetragonolobus agglutinin; UEA, Ulex europaeus agglutinin.

Figure 3 Receiver operating characteristic curve analysis of serum native IgG - s-IgG (s) and isolated serum IgG - i–IgG (i) relative reactivities with AAL (Aleuria aurantia lectin), LCA (Lens culinaris agglutinin), LTA (Lotus tetragonolobus agglutinin) and UEA (Ulex europaeus agglutinin) was done for women with endometriosis and healthy subjects (A–H). For lectins’ specificity see Materials and methods section.

Table 4 Results of Cluster Analysis for Values of Serum Native IgG Relative Reactivities with AAL, LCA and LTA

Group Cluster No	C N=19	E N=40
	The Number of Samples (Percentage Participation in Whole Group)
4 (n=24)	16 (84%)	8 (20%)
3 (n=16)	0 (0%)	16 (40%)
2 (n=18)	2 (10.5%)	16 (40%)
1 (n=1)	1 (5.5%)	0 (0%)

Notes: E – endometriosis, C – control group of healthy women. Cluster analysis was performed only for parameters for which in receiver operating characteristic curve analysis the value of area under the curve was moderate or high (≥ 0.775), and which significantly differentiated endometriosis patients from healthy women. Each serum sample is represented by a vector of three features: AAL (Aleuria aurantia lectin), LCA (Lens culinaris agglutinin) and LTA (Lotus tetragonolobus agglutinin) relative reactivity.

Table 5 Results of Cluster Analysis for Values of Isolated Serum IgG Relative Reactivities with AAL, LCA and LTA

Group Cluster No.	C N=19	E N=40
	The Number of Samples (Percentage Participation in Whole Group)
3 (n=29)	0 (0%)	29 (72.5%)
2 (n=21)	19 (100%)	2 (5%)
1 (n=9)	0 (0%)	9 (22.5%)

Notes: E – endometriosis, C – control group of healthy women. Cluster analysis was performed only for parameters for which in receiver operating characteristic curve analysis the value of area under the curve was moderate (≥ 0.775), and which significantly differentiated endometriosis patients from healthy women. Each isolated serum IgG sample is represented by a vector of three features: AAL (Aleuria aurantia lectin), LCA (Lens culinaris agglutinin) and LTA (Lotus tetragonolobus agglutinin) relative reactivity.

Figure 4 Dendrogram of cluster analysis for values of serum native IgG (s-IgG) relative reactivities with AAL (Aleuria aurantia lectin), LCA (Lens culinaris agglutinin) and LTA (Lotus tetragonolobus agglutinin). The cluster analysis was done only for parameters for which in receiver operating characteristic curve analysis the value of area under the curve was moderate or high (≥ 0.775), which significantly differentiates endometriosis patients from healthy women. Each serum sample is represented by a vector of three features: AAL, LCA and LTA relative reactivities with s-IgG.

Abbreviations: E, endometriosis; C, control group of healthy women.

Figure 5 Dendrogram of cluster analysis for values of isolated serum IgG (i–IgG) relative reactivities with AAL (Aleuria aurantia lectin), LCA (Lens culinaris agglutinin) and LTA (Lotus tetragonolobus agglutinin). Cluster analysis was performed only for parameters for which in receiver operating characteristic curve analysis the value of area under the curve was higher than ≥ 0.775, and which significantly differentiated endometriosis patients from healthy women. Each serum i–IgG sample is represented by a vector of three features: AAL, LCA and LTA relative reactivity.

Abbreviations: E, endometriosis; C, control group of healthy women.