Precise capture of circulating endometrial cells in endometriosis

Abstract

Background: Endometriosis (EM) is a complex benign gynecological disease, but it has malignant biological behavior and can invade any part of the body. Clinical manifestations include pelvic pain, dysmenorrhea, infertility, pelvic nodules, and masses. Our previous study successfully detected circulating endometrial cells (CECs) in the peripheral blood of patients with EM. The purpose of this study is to overcome the limitation of cell size in the previous microfluidic chip method, to further accurately capture CECs, understand the characteristics of these cells, and explore the relationship between CECs and the clinical course characteristics of patients with EM.

Methods: Human peripheral venous blood used to detect CECs and circulating vascular endothelial cells (CVECs) was taken from EM patients ( n = 34) hospitalized in the Peking University People's Hospital. We used the subtraction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) method to exclude the interference of red blood cells, white blood cells, and CVECs, so as to accurately capture the CECs in the peripheral blood of patients with EM. Then we clarified the size and ploidy number of chromosome 8 of CECs, and a second grouping of patients was performed based on clinical characteristics to determine the relationship between CECs and clinical course characteristics.

Results: The peripheral blood of 34 EM patients and 12 non-EM patients was evaluated by SE-iFISH. Overall, 34 eligible EM patients were enrolled. The results showed that the detection rates of CECs were 58.8% in EM patients and 16.7% in the control group. However, after classification according to clinical characteristics, more CECs could be detected in the peripheral blood of patients with rapidly progressive EM, with a detection rate of 94.4% (17/18). In total, 63.5% (40/63) of these cells were small cells with diameters below 5 μm, and 44.4% (28/63) were aneuploid cells. No significant association was found between the number of CECs and EM stage.

Conclusion: The number and characteristics of CECs are related to the clinical course characteristics of patients with EM, such as pain and changes in lesion size, and may be used as biomarkers for personalized treatment and management of EM in the future.

Figure 1

General process of detecting CECs by the SE-iFISH method. Six milliliters of venous blood is collected from EM patients, and a non-hypotonic method is used to remove RBCs from the blood of EM patients. A combination of various anti-leukocyte antibodies, including CD45, is coupled to specially coated magnetic beads to remove leukocytes to the greatest extent. Then, endometrium-specific antibodies against ER and PR are used for identification, and CECs and CVECs are distinguished according to the characteristics of CD31 immunofluorescence staining. Finally, the ploidy of chromosome 8 is identified by chromosome in situ hybridization. CECs: Circulating endometrial cells; CEP8: Centromere of chromosome 8;CVECs: Circulating vascular endothelial cells; EM: Endometriosis; ER: Estrogen receptor; PR: Progesterone receptor; RBCs: Red blood cells; SE-iFISH: Subtraction enrichment and immunostaining fluorescence in situ hybridization.

Figure 2

Representative microscopic images and sizes of CECs and CVECs. (A) Representative microscopic fluorescence images of CECs and CVECs. Positivity for the target cell was defined as follows: DAPI+/CD45–/CD31–/ER+ or PR+ for CECs and DAPI+/CD45–/CD31+/ER+ or –/PR+ or – for CVECs. Scale bars, 5 μm. (B) Representative microscopic fluorescence images of small and large CECs. The proportions of small cells (diameter <5 μm) and large cells (diameter ≥5 μm) among CECs. Scale bars, 5 μm. CECs: Circulating endometrial cells; CVECs: Circulating vascular endothelial cells; ER: Estrogen receptor; PR: Progesterone receptor.

Figure 3

Comparison of the positive rates and numbers of CECs and CVECs in the EM and control groups. (A) The positive rates of CECs and CVECs and the sum of the two in the EM and control groups. (B) The numbers of CECs and CVECs in the EM and control groups and the sum of the two. ^*P <0.05 vs. non-EM, ^†P <0.01 vs. non-EM, ns P >0.05 vs. non-EM. (C) The correlation coefficient for CECs and CVECs is 0.6704 (P <0.05). CECs: Circulating endometrial cells; CVECs: Circulating vascular endothelial cells; EM: Endometriosis.

Figure 4

Comparison of the positive rates and numbers of CECs and CVECs in different groups. (A) The positive rate of CECs, that of CVECs, and the sum of the two in the active EM, dormant EM, and non-EM control groups; (B) CECs, CVECs, and the sum of the two in the active EM, dormant EM, and non-EM groups. ^†P <0.05 vs. dormant EM; ^*P <0.05 vs. non-EM, ^†P <0.01 vs. dormant EM, ^‡P <0.001 vs. non-EM, ^§P <0.0001 vs. non-EM. CECs: Circulating endometrial cells; CVECs: Circulating vascular endothelial cells; EM: Endometriosis.

Figure 5

The relationships of CECs and CVECs with menstrual cycle and EM stage. (A–C) The relationship of the number of CECs, number of CVECs, or the sum of the two with menstrual cycle phase in the active EM group (^*P <0.05, ^‡P <0.001), as well as the dormant EM and non-EM control groups. (D, E) Number of CECs, number of CVECs, and the sum of the two in active EM and dormant EM and their relationships with the stages of EM. CECs: Circulating endometrial cells; CVECs: Circulating vascular endothelial cells; EM: Endometriosis; ns: Not significant.

Figure 6

Chromosome 8 aneuploidy and subtype analysis of CECs. (A) Representative fluorescence images of aneuploid CECs. (B) Proportions of different chromosome 8 ploidy subtypes; most CECs were normal diploid, 4.1% were haploid, 4.8% were triploid, 3.1% were tetraploid, and 32.4% were polyploid. CECs: Circulating endometrial cells.