Precise Capture of Circulating Endometrial Cells in Endometriosis
Endometriosis (EM) is a complex benign gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity. Despite its benign nature, EM exhibits malignant biological behaviors, including invasive growth, distant metastasis, and high recurrence rates. The disease affects approximately 10–15% of women of reproductive age, with an estimated 190 million women worldwide suffering from its symptoms, which include pelvic pain, dysmenorrhea, infertility, and the formation of pelvic nodules and masses. The diagnosis of EM is often delayed due to the lack of specific biomarkers, and the gold standard for diagnosis remains laparoscopy. However, laparoscopy is invasive and not always accessible, highlighting the need for non-invasive diagnostic methods.
In recent years, circulating endometrial cells (CECs) have emerged as a potential biomarker for EM. These cells, detected in the peripheral blood of EM patients, may provide insights into the disease’s progression and offer a non-invasive diagnostic tool. Previous studies have successfully detected CECs using microfluidic chip methods, but these techniques were limited by cell size, potentially missing smaller cells. Additionally, circulating vascular endothelial cells (CVECs) in peripheral blood, which share similar characteristics with CECs, posed a challenge in accurately identifying CECs. This study aimed to overcome these limitations by employing a more advanced method, subtraction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH), to precisely capture CECs and explore their relationship with the clinical course of EM.
Methods
The study enrolled 34 EM patients and 12 non-EM controls. Peripheral venous blood samples were collected from EM patients hospitalized at Peking University People’s Hospital. The inclusion criteria for EM patients were a diagnosis confirmed by laparoscopy, absence of pregnancy, breastfeeding, or menopause, no history of other tumors or hematological diseases, and no use of steroidal hormone drugs within three months prior to blood collection. The control group included patients with benign gynecological conditions and healthy volunteers.
The SE-iFISH method was used to detect CECs and CVECs in the peripheral blood. This method involves several steps: subtraction enrichment to remove red blood cells, white blood cells, and CVECs, followed by immunostaining to identify CECs based on their expression of estrogen receptor (ER) and progesterone receptor (PR). Fluorescence in situ hybridization (FISH) was then performed to determine the ploidy of chromosome 8 in the captured cells. The SE-iFISH method was chosen for its ability to capture cells of all sizes, overcoming the limitations of previous methods that relied on cell size filtration.
Results
The study successfully detected CECs in the peripheral blood of EM patients using the SE-iFISH method. The detection rate of CECs in EM patients was 58.8%, significantly higher than the 16.7% detection rate in the control group. When patients were stratified based on clinical characteristics, the detection rate of CECs increased to 94.4% in patients with rapidly progressive EM, defined by worsening pelvic pain or dysmenorrhea and an increase in ovarian mass size over the past six months. In contrast, the detection rate was only 18.8% in patients with dormant EM, characterized by stable or mild symptoms.
Analysis of the size of CECs revealed that 63.5% of the cells had diameters below 5 micrometers, while 36.5% were larger than 5 micrometers. This finding highlights the importance of capturing small cells, which were missed by previous methods. Additionally, 44.4% of the CECs exhibited aneuploidy in chromosome 8, with tetraploidy and polyploidy being the most common subtypes.
The study also found a significant correlation between the number of CECs and CVECs in EM patients, with a correlation coefficient of 0.6704. This suggests that the presence of CECs may be associated with increased vascular activity in EM. However, no significant association was found between the number of CECs and the stage of EM, indicating that CECs may be more related to disease activity than severity.
Discussion
The precise capture of CECs using the SE-iFISH method provides new insights into the pathophysiology of EM. The detection of CECs in the peripheral blood of EM patients, particularly those with rapidly progressive disease, suggests that these cells may play a role in the disease’s progression. The presence of CECs in active EM patients, especially during the proliferative phase of the menstrual cycle, aligns with the theory that endometrial fragments shed during menstruation may enter the bloodstream and implant in distant sites, leading to the formation of EM lesions.
The finding that a significant proportion of CECs are small cells underscores the importance of capturing cells of all sizes in EM research. Previous methods, which relied on size filtration, may have missed these smaller cells, leading to an incomplete understanding of the disease. The SE-iFISH method, which does not depend on cell size, offers a more comprehensive approach to detecting CECs.
The detection of aneuploidy in CECs, particularly tetraploidy and polyploidy, provides further evidence of the malignant biological behavior of EM. While EM is a benign disease, the presence of aneuploid cells suggests that it shares some characteristics with cancer, such as genomic instability. This finding may have implications for the early detection of malignant transformation in EM, a rare but serious complication of the disease.
The study also highlights the potential clinical utility of CECs as a biomarker for EM. The detection of CECs in patients with active EM suggests that these cells may be useful in identifying patients who require early intervention. Additionally, the dynamic tracking of CECs in patients with dormant EM may help predict disease progression and guide personalized treatment strategies.
Limitations and Future Directions
The study has several limitations. The sample size was relatively small, and the participants were primarily patients with ovarian EM. Future studies should include a larger and more diverse patient population to validate the findings. Additionally, the study did not explore the long-term outcomes of patients with CECs, particularly those with dormant EM. Further research is needed to determine whether the presence of CECs in these patients predicts the development of new lesions or progression to active EM.
Another area for future research is the role of CECs in adolescent dysmenorrhea. Early-onset dysmenorrhea is a risk factor for EM, and the detection of CECs in adolescents may provide a non-invasive method for early diagnosis and intervention. This could significantly reduce the diagnostic delay associated with EM and improve patient outcomes.
Conclusion
The precise capture of CECs using the SE-iFISH method represents a significant advancement in EM research. The detection of CECs in the peripheral blood of EM patients, particularly those with active disease, provides new insights into the disease’s pathophysiology and offers a potential biomarker for early diagnosis and personalized treatment. The finding that a significant proportion of CECs are small cells and exhibit aneuploidy underscores the importance of capturing cells of all sizes and understanding the genomic instability associated with EM. Future research should focus on expanding the sample size, exploring the long-term outcomes of patients with CECs, and investigating the role of CECs in adolescent dysmenorrhea. The detection of CECs may ultimately lead to improved diagnostic and therapeutic strategies for EM, reducing the burden of this complex and debilitating disease.
doi.org/10.1097/CM9.0000000000002910
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