DNA methylation biomarkers to identify epigenetically abnormal spermatozoa in male partners from couples experiencing recurrent pregnancy loss

Figures & data

Table 1. Diagnostic characteristics of combinations of genes tested.

Sr. No	No of genes	Imprinted genes included in the combination	AUC	Standard error	Diagnostic potential	Threshold value	% Specificity	% Sensitivity
1	7	IGF2-H19, IG-DMR, ZAC, KvDMR, PEG3, MEST, PEG10	0.8906	0.02798	80.14	>0.6547	92.65	69.12
2	6	IGF2-H19, IG-DMR, ZAC, KvDMR, PEG3, MEST	0.8815	0.02796	81.2	>0.5847	90.14	73.08
3	5	IGF2-H19, IG-DMR, ZAC, KvDMR, PEG3	0.8841	0.0271	80.205	>0.6179	90.41	70
4	4	IGF2-H19, IG-DMR, ZAC, KvDMR	0.781	0.03615	69.325	>0.6561	91.03	47.62
5	4	IGF2-H19, IG-DMR, ZAC, PEG3	0.8592	0.0275	78.035	>0.5895	90.11	65.96
6	4	IGF2-H19, IG-DMR, KvDMR, PEG3	0.8657	0.02943	78.74	>0.6227	90.41	67.07
7	4	IGF2-H19, ZAC, KvDMR, PEG3	0.8822	0.02729	79.58	>0.6360	90.41	68.75
8	3	IGF2-H19, IG-DMR, ZAC	0.7073	0.03664	65.36	>0.5912	90.91	39.81
9	3	IGF2-H19, ZAC, KvDMR	0.7651	0.03682	68.19	>0.6654	91.03	45.35
10	3	IGF2-H19, ZAC, PEG3	0.8585	0.02758	78.565	>0.5902	90.11	67.02

Figure 1. Multiple regression and ROC analysis: (a) ROC curve for the probability score of average methylation levels of differentially methylated CpGs in IGF2-H19 DMR, IG-DMR, ZAC, KvDMR, and PEG3 for the samples analysed in the previous study. Arrow indicates the threshold value of 0.61 probability score. Scatter plot with median for the probability score for the control (N = 73) and RPL samples (N = 80) in the previous study (b) and for the probability score for the control (N = 38) and RPL (N = 45) samples in the current validation study (c). The horizontal line indicates the threshold value of 0.61.

Table 2. Unsupervised k-means clustering of predicted probability values of combination of five imprinted genes into two clusters in validation cohort. Cluster 1 comprised of most of the control samples and some RPL samples with predicted probability values comparable to control; while Cluster 2 comprised of a sub-set of RPL samples with a predicted probability value different than the controls.

	Cluster 1	Cluster 2
Number of objects in cluster	54	29
No of controls (%) N = 38	33 (86.84%)	5 (13.15%)
No of RPL (%) N = 45	21 (46.66%)	24 (53.33%)
Cluster centroids	0.164	0.696
Distance between centroids	0.532
Within Cluster Variance	0.015	0.035
Minimum distance to centroid	0.002	0.012
Average distance to centroid	0.101	0.155
Maximum distance to centroid	0.245	0.303
Mean Silhouette scores	0.704	0.542

Data availability statement

All the data contained in the manuscript can be available on request.