CD163 positive alternatively activated monocytes as a marker of early miscarriage

Threatened spontaneous miscarriage is the most common pregnancy complication. According to the literature, 10–20% of pregnancies end in a miscarriage and up to 80% of losses of pregnancy occur in the first trimester [1]. The etiology of this pathology is multifactorial. According to current research evidence, one of the most significant pathogenetic factors of this pathology is impaired immune mechanisms, including congenital immunity factors [2]. Macrophage cells have high plasticity and a wide spectrum of action, which implies their important role in the pathogenesis of pregnancy failure [3]. Monocytes are subdivided into three subpopulations, including classically, intermediately, and alternatively activated monocytes; all of them have different functions [4]. There is evidence that alternatively activated monocytes are involved in the formation of immunological tolerance to the semi-allogeneic fetus [5].

The study aimed to develop a new method for predicting spontaneous miscarriage in women with threatened miscarriage in early pregnancy by detecting alternatively activated monocytes that express the scavenger receptor CD163 in peripheral venous blood.

Materials and methods

This retrospective study included 71 women who had threatened spontaneous miscarriage at 5–12 weeks of gestation. Patients were treated at the gynecological department of the clinic according to the protocol "Miscarriage in early pregnancy: diagnosis and management strategy" dated 16.05.2016. The women were monitored throughout pregnancy and outcomes were evaluated. Fifty-four women (Group 1) gave birth at full term (Group 1) and 17 women had early and late spontaneous miscarriages (Group 2). The study material was peripheral venous blood, which was collected upon admission to the hospital, before the start of treatment to prevent miscarriage. An enriched population of peripheral blood mononuclear cells was isolated by standard high-speed centrifugation on a double density gradient of Ficoll -urographine (d=1.06 and d=1.078) at 1500 rpm for 30 minutes. The surface phenotype of monocytes was determined by standard multicolor flow cytofluorometry on a FACScanto II flow cytometer (Becton Dickinson, USA) using FACSDiva software. We used anti-CD14, anti-CD16, and anti-CD163 (Invitrogen, USA) labeled peridinine chlorophyll (PerCP) conjugated with either FITC or PE. The relative abundance of classically activated (CD14+CD16-), intermediately activated (CD14+CD16+) and alternatively activated (CD14+CD16+) monocytes was determined in the monocyte gait according to the standard methodology [6] (Fig. 1, 2). The proportion of cells expressing the CD163 receptor was assessed in the gates of classically, intermediately, and alternatively activated monocytes (Fig. 3).

Statistical analysis

Statistical analysis was performed using Statistica for Windows 13.0, Microsoft Excel 2010, and MedCalc software. Categorical variables were presented as counts and percentages. Pearson's χ2 test with Yates's continuity correction was used to compare categorical variables. The significance threshold was defined as p<0.05. For non-normally distributed parameters, the Mann–Whitney U-test was utilized for comparisons; quantitative variables were presented as the median (Me) and quartiles (Me (Q25%–Q75%)). An ROC analysis was performed to determine the cut-off value of the proposed diagnostic method. Data are presented as area under the curve (AUC) with 95% confidence interval (CI).

Results

Women in Group 2 were older than in Group 1, but the difference was statistically insignificant [32 (28; 35) vs. 30 (28; 34) years, respectively; p˃0.05] (Table 1). Group 2 patients were more likely to have working specialty and unhealthy behaviors such as smoking compared to Group 1. Women whose pregnancies ended in spontaneous miscarriage more often had a history of acute respiratory viral infections (ARVI), chronic recurrent nasolabial herpes, infertility, early and late spontaneous miscarriages.

As shown in Table 2, the proportion of classically activated monocytes expressing the scavenger receptor CD163 did not differ between the two groups (p˃0.05). The expression of CD163 scavenger receptor by intermediate monocytes was significantly lower than on classically activated cells. At the same time, there were no differences between the parameters between the two groups (p<0.05). A comparison of the content of alternatively activated monocytes expressing the CD163 scavenger receptor between the groups revealed that women whose pregnancies ended in spontaneous miscarriage had 3-fold higher parameters than those whose pregnancies ended in full-term birth (p=0.0007).

Based on the results of ROC analysis, a method was developed to predict spontaneous miscarriage in women with threatened miscarriage in early pregnancy by determining the content of alternatively activated monocytes expressing the scavenger receptor CD163 in the peripheral venous blood of a pregnant woman at 5–12 weeks' gestation before the start of preventive therapy. The cutoff value for monocytes expressing the CD163 scavenger receptor, calculated from ROC analysis, was 15.6%. A value of 15.6% or higher predicted spontaneous miscarriage with a high accuracy of 87.50%, sensitivity of 90.0% (95% CI 55.5–98.3) and specificity of 81.8% (95% CI 59.7–94.7) (Fig. 4), confirming the advantages of this method. The area under the ROC curve is 90%.

Discussion

The leading risk factors for spontaneous miscarriage in women with threatened miscarriage in early pregnancy included older reproductive age, occupation, unhealthy behavior in the form of tobacco smoking, as well as a history of acute respiratory infections, recurrent nasolabial herpes, infertility, and reproductive loss in the form of early and late spontaneous miscarriage, which is consistent with the literature [7, 8].

CD163 is a hemoglobin scavenger receptor that is specifically expressed in activated monocyte subpopulations and plays an important role in immune homeostasis, performing protective, anti-inflammatory and immunomodulatory functions [9]. CD163 serves as a diagnostic parameter for monocyte activation in inflammatory diseases [10]. All monocyte populations carry the scavenger receptor CD163 [11]. In our study, we found no significant differences in the content of classically activated monocytes expressing the scavenger receptor CD163 between the study groups. However, literature data suggest the possibility of using this parameter as a predictor of the outcome of autoimmune, cancer, infectious, and inflammatory diseases [12–14]. There are only a few studies that evaluate these cells in pregnancy and focus on preeclampsia [15]. The authors suggest a relationship between elevated levels of pro-inflammatory cytokines and plasma levels of the soluble form sCD163 in women with severe preeclampsia, which implies the role of impaired modulation of the systemic inflammatory response in these patients.

To date, no studies have investigated changes in the content of alternatively activated monocytes expressing the scavenger receptor CD163 in pregnancy. We hypothesize that alternatively activated monocytes expressing the CD163 scavenger receptor are normally required to maintain immune balance and prolong pregnancy by regulating the immunocompetent cell activity and inflammation process seen in threatened miscarriage in early pregnancy.

Conclusion

In women with threatened miscarriage in early pregnancy, the expression of the CD163 scavenger receptor in monocytes was highest in the population of classically activated cell population and lowest in the population of alternatively activated cells. The expression of CD163 scavenger receptor in the population of alternatively activated monocytes in patients with this pathology was associated with pregnancy outcome. The content of alternatively activated monocytes expressing the CD163 scavenger receptor in peripheral blood can be used as a predictor of spontaneous miscarriage in women with a threatened miscarriage in early pregnancy. Early prediction of miscarriage will optimize the treatment of pregnant women with threatened miscarriage in early pregnancy.