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Introduction

Preeclampsia is a condition characterized by high blood pressure and proteinuria that develops during pregnancy (1–10). Preeclampsia affects 2.5–10% of all pregnancies (1–8) and causes serious complications for both the mother and the fetus. According to the World Health Organization (WHO) report, 14% of maternal deaths occur due to hypertensive disorders, ranking second overall (11); moreover, preeclampsia can cause serious complications, such as placental abruption, hemolysis, elevated liver enzymes, low platelet count (HELLP) syndrome, and disseminated intravascular coagulopathy (DIC) (2–7,9,10). In addition, infants of mothers with preeclampsia are at twice the risk of neonatal death as those without preeclampsia and have a higher probability of admission to the neonatal intensive care unit due to preterm birth or low birth weight (12–14). Accordingly, prevention and treatment strategies for patients with preeclampsia have been one of the most important topics in obstetrics.

The pathogenesis of preeclampsia has long been shrouded in mystery but has slowly become clearer. Preeclampsia occurs only in the presence of the placenta (2,10,15), and delivery is the ultimate treatment (2–4,15), which strongly suggests a relationship between the placenta and preeclampsia. Although inadequate remodeling of the spiral artery could be one of the mechanisms for the development of preeclampsia, this condition cannot be explained by this single mechanism, and other mechanisms could be present in the development of preeclampsia (2,4,10,15). For example, an increased level of hypoxia-inducible family (HIF) 1-α in the placenta of preeclamptic women has been reported, suggesting an association between preeclampsia and hypoxia in the placenta (16).

Preeclampsia is classified into two types: early-onset (disease onset before 34 weeks) and late-onset diseases (disease onset after 34 weeks) (1). Early-onset preeclampsia is reported to show a poorer prognosis in the neonate period and more frequent intrauterine fetal growth retardation compared with late-onset (12). Sezer et al reported no significant association between angiogenic factors, such as placental growth factor, vascular endothelial growth factor, and HIF-1α, in the placenta, and the number of weeks at preeclampsia onset (17). However, it has been speculated that early- and late-onset preeclampsia have different developmental mechanisms (7,12). Although several research studies have addressed the pathogenesis of preeclampsia, especially the differences between early- and late-onset preeclampsia (7,12,17), these differences have not yet been fully elucidated. CD200 belongs to the immunoglobulin superfamily, and its signaling leads to immune tolerance (18–20). Immune tolerance plays important roles in pregnancy, and the association between CD200 and preeclampsia has been addressed in a study (21). However, difference of CD200 expression between early- and late-onset preeclampsia has not yet been analyzed.

The purpose of this study was to clarify the differences between early- and late-onset preeclampsia using comprehensive gene expression and immunohistochemical analyses.

Materials and methods

Patient selection

This study enrolled patients with preeclampsia, from January 2014 to December 2020, at Kansai Medical University Hospital. Preeclampsia was defined as hypertension (systolic blood pressure of ≥140 mmHg and/or diastolic ≥90 mmHg) and proteinuria (1+ in randomly collected urine or >0.3 g in a 24-h interval) (3,5,22) We excluded patients aged <20 years at delivery, and those with chorioamnionitis, fetal anomaly, twin pregnancy, and placental abruption. Patients with preeclampsia were classified into the following two groups according to the gestational age of onset of preeclampsia: the early-onset group (gestational age <34 weeks) and the late-onset groups (gestational age ≥34 weeks) (1).

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Kansai Medical University Hospital (approval no. 2018138). Before December 2018, informed consent was obtained from patients by implementing the opt-out method, owing to the retrospective design of the study, with assurance of no risk to the participants. Information regarding this study, such as the inclusion criteria and the opportunity to opt out, was provided through the institutional website (https://www.kmu.ac.jp/hirakata/hospital/2671t800000124re-att/a1625627306468.pdf). Patients who went into delivery after January 2019 provided written informed consent for sample collection and its subsequent analysis.

RNA extraction

RNA was isolated from the archived samples. Briefly, for mRNA extraction, 5 µm-thick sections were examined from the archived formalin-fixed and paraffin-embedded (FFPE) blocks of the placenta from five patients each in the early- and late-onset groups, and two blocks from normal controls. For the placental block, the part with the attached umbilical cord was selected for each patient. RNA was extracted using a NucleoSpin total RNA FFPE kit (Macherey-Nagel GmBH & Co. KG), including an on-column DNase treatment. The quantitative evaluation of RNA was performed using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc.). The quality of RNA was evaluated by measuring the ratio of 260/280 nm. We excluded samples in which the total amount of RNA was <50 ng/µl or the 260/280 ratio was <1.6 (23).

Analysis of comprehensive mRNA expression profiles

We studied the expression profiles of 770 immune-related genes using the nCounter PanCancer Immune Profiling Panel (NanoString Technologies, Inc.). The nCounter assay was performed according to the manufacturer's instructions. The RNA was hybridized with the probe sets for 16 h at 67°C, and the samples were processed using an automated nCounter Sample Prep Station (NanoString Technologies, Inc.). Cartridges containing immobilized and aligned reporter complexes were subsequently imaged on an nCounter Digital Analyzer (NanoString Technologies, Inc.) that had been set at a data resolution of 555 fields of view. Reporter counts were collected and normalized using the nSolver analysis software (version 3.0; NanoString Technologies, Inc.). The analysis between the two groups was performed using a volcano plot after adjusting for the control group (23). Sequencing data were deposited into the DNA Data Bank of Japan Sequence Read Archive (accession nos. SAMD00549508-SAMD00549517).

Immunohistochemistry

The protein expression of CD200 was analyzed by immunohistochemistry. FFPE blocks of the placenta of the part with the attached umbilical cord were cut into 4-µm-thick sections. Subsequently, the samples were deparaffinized and rehydrated. Immunohistochemical analyses were performed using an autostainer (Discovery XT System; Roche Diagnostics), according to the manufacturer's instructions. The primary antibody used was a rabbit polyclonal antibody against CD200/OX2 (ab203887; Abcam; dilution 1:100). The staining intensity was classified into four categories as follows: 0=no staining, 1=weak staining, 2=moderate staining, and 3=strong staining. The scoring was independently done by at least two researchers blind to the clinical information.

Statistical Analysis

In the analysis of nCounter, statistical software R version 3.5.2 (R Project, Vienna, Austria) was used to perform significance analysis of the identification of differentially expressed genes between normal and preeclampsia placental tissue samples. The counted raw RNA-seq data were normalized and analyzed between groups using the R software extension package ‘DESeq’ 2 available in Bioconductor (www.bioconductor.org/packages/release/bioc/html/DESeq2.html). Genes with an absolute value of log2 fold change (log2FC) >2 and the adjusted P-value <0.00001 were defined as DEGs. The volcano plot was visualized using the ggplot2 package in R (24).

Other data were presented as mean ± standard error and analyzed by unpaired and paired t-tests using Prism 8 computer software (GraphPad Software). The analysis between the two groups was performed using the Mann-Whitney test, and the analysis between the three groups was performed using the Kruskal-Wallis test (followed by the Dunn or Dunn-Bonferroni posterior test method). A probability level of <0.05 was considered statistically significant.

Results

Comprehensive mRNA expression profiles

Table I summarizes the clinicopathological features of the 10 patients with preeclampsia (5 patients each in the early- and late-onset groups) used for the comprehensive mRNA expression analysis. Gestational age at onset of preeclampsia was significantly different between the early-onset (mean 33.2 weeks) and late-onset (mean 35.6 weeks) groups (P<0.05). Age at delivery, systolic and diastolic blood pressures, birth weight, and placental weight were not significantly different between the two groups.

Table I. Clinicopathological characteristics of ten patients with preeclampsia who were studied the mRNA expression profiling.

Table I.

Clinicopathological characteristics of ten patients with preeclampsia who were studied the mRNA expression profiling.

Characteristics	Early-onset (n=5)	Late-onset (n=5)	P-value
Age, years	28 (26–38)	33 (29–37)	0.22
Primipara, cases (%)	3 (60%)	5 (100%)	0.44
Gestational age at onset, weeks	33.2 (24.0-33.5)	35.6 (34.2-38.1)	<0.01
Gestational age at delivery, weeks	33.5 (27.5-37.3)	35.6 (35.3-38.1)	0.13
Systolic blood pressure, mmHg	160 (143–163)	162 (141–165)	0.60
Diastolic blood pressure, mmHg	96 (89–100)	109 (70–130)	0.39
Vaginal delivery, cases (%)	3 (60%)	5 (100%)	0.44
Birth weight, g	1,512 (529–2,394)	1,806 (1,698–2,715)	0.15
Placenta weight, g	365 (220–480)	380 (310–510)	0.69

[i] The median value is shown, and the maximum and minimum values are shown in brackets.

The volcano plot of different gene expression levels between the early- and late-onset groups is shown in Fig. 1. CD200, AICDA, EBI3, and lactoferrin were significantly upregulated in the early-onset group, as compared to the late-onset group (adjusted P<0.05).

Figure 1. Comprehensive mRNA expression profiles between early- and late-onset preeclampsia patients.

Immunohistochemistry analysis

Table II summarizes the clinicopathological features of the 53 patients with preeclampsia (26 and 27 patients in the early- and late-onset groups, respectively) and 9 normotensive pregnant women (the control group) included in this analysis. The patients who performed the comprehensive mRNA expression analysis were also included in the immunohistochemical analysis. Gestational age at onset of preeclampsia was significantly earlier in the early-onset group than in the late-onset group (P<0.05). Gestational age at delivery was significantly earlier and birth weight was significantly lower in early-onset group than in the late-onset and control groups (P<0.05). Systolic and diastolic blood pressures were significantly higher in the early- and late-onset groups than in the control group (P<0.05). Other factors were not significantly different among the three groups.

Table II. Clinicopathological characteristics of 62 patients with preeclampsia, including the ones performed the immunohistochemistry analysis.

Table II.

Clinicopathological characteristics of 62 patients with preeclampsia, including the ones performed the immunohistochemistry analysis.

Characteristic	Control group (n=9)	Early-onset group (n=26)	Late-onset group (n=27)	P-value
Age, years	35 (24–43)	34 (24–46)	33 (23–40)	0.59
Primipara, cases (%)	9 (100%)	17 (65.3%)	17 (63.0%)	0.08
Gestational age at onset, weeks	ND	31.3 (24.0–33.6)	35.6 (34.0–40.5)	<0.01a
Gestational age at delivery, weeks	40.2 (37.0–41.2)	34.0 (27.0–38.0)	37.1 (34.5–40.5)	<0.01b
Systolic blood pressure, mmHg	116 (79–147)	163 (138–220)	156 (129–192)	<0.01c
Diastolic blood pressure, mmHg	64 (47–84)	102 (82–125)	98 (70–124)	<0.01c
Vaginal delivery, cases (%)	0 (0%)	5 (19.2%)	7 (25.9%)	0.24
Birth weight, g	2,750 (2,450–3,585)	1,514 (529–2,584)	2,355 (1,554–3,315)	<0.01b
Placenta weight, g	510 (350–655)	350 (220–630)	405 (260–690)	<0.01b

a Significant difference between the early onset and late-onset groups;

b significant difference between the early-onset group vs. the late-onset and control groups;

c significant difference between the control group vs. the early-onset and the late-onset groups. The median value is shown, and the maximum and minimum values are shown in brackets.

Table III summarizes the results of the immunohistochemical analysis. CD200 was expressed in the syncytial trophoblasts (Fig. 2). The staining intensity of CD200 was significantly stronger in the early-onset group than in the late-onset and control groups (P<0.05). There was no significant difference in the staining intensity of CD200 between the late-onset and control groups. Moreover, in the early-onset group, the staining intensity of CD200 showed a stronger tendency in patients with an earlier gestational age compared to those with a later gestational age, although the difference was not significant (P=0.09). However, this trend was not observed in the late-onset group (P=0.33) (Fig. 3).

Figure 2. Representative images of the immunohistochemical expression of CD200 in the normal, early- and late-onset preeclampsia placentas. (A) Early-onset group with staining intensity is 1. (B) Early-onset group with staining intensity is 2. (C) Early-onset group with staining intensity is 3. (D) Late-onset group with staining intensity is 1. (E) Late-onset group with staining intensity is 2. (F) Late-onset group with staining intensity is 3. (G) Control group with staining intensity is 1. (H) Control group with staining intensity is 2. (I) Negative control. Scale bars, 20 µm.

Figure 3. Plotting of the staining intensity of CD200 in early- and late-onset preeclampsia. (A) Correlation between the intensity of CD200 expression in the early-onset group placenta and the gestational age of delivery is shown. There was a correlation between expression intensity of CD200 and the gestational age of delivery, but the difference was not significant (P=0.09). (B) Correlation between the intensity of CD200 expression in the late-onset group placenta and the gestational age of delivery is shown. There was no correlation between expression intensity and the gestational age of delivery (P=0.33).

Table III. Results of the immunohistochemical analysis for CD200.

Table III.

Results of the immunohistochemical analysis for CD200.

Staining intensity	Control group (n=9)	Early-onset group (n=26)	Late-onset group (n=27)	P-value
0	0 (0%)	0 (0%)	0 (0%)	<0.01a
1	8 (88.9%)	8 (30.8%)	11 (40.7%)
2	1 (11.1%)	7 (26.9%)	14 (51.9%)
3	0	11 (42.3%)	2 (7.4%)

a Significantly higher expression of CD200 in the early-onset group compared with in the late-onset and the control groups. Staining intensity: 0, no staining; 1, low staining; 2, medium staining; 3, intense staining.

Discussion

Preeclampsia is one of the most important conditions requiring treatment for pregnant women. Especially, early-onset preeclampsia is reported to have a poor prognosis in the neonatal period (12). In particular, early-onset preeclampsia is more likely to cause intrauterine fetal growth retardation than late-onset preeclampsia (25). In fact, significant morphological differences, including villous and vasculature features, have been reported between the placentas of normotensive pregnant women and those of pregnant women with early-onset preeclampsia, while the placentas of pregnant women with late-onset preeclampsia are not significantly different from those of normotensive pregnant women with corresponded gestational age (25,26). These facts indicate that early- and late-onset preeclampsia are not caused by the same mechanism. Although it has been recognized that inadequate remodeling of the spiral artery plays an important role in the development of preeclampsia, a systemic inflammatory response due to placental stress, such as hypoxia, is also considered one of the pathogenic mechanisms (27–29). Microarray studies of placental RNA have shown that gene expressions of immune and inflammatory systems in the placenta of preeclampsia patients are different from those in the normotensive placenta (30). In particular, changes in the levels of angiogenic factors, such as vascular endothelial growth factor and placental growth factor, have been strongly associated with the development of preeclampsia (31). However, the association of these factors between early- and late-onset preeclampsia has not yet been analyzed. Thus, we aimed to clarify the differences of the two subtypes of preeclampsia.

We first used the nCounter immune profiling panel to analyze differences in RNA expression of 770 genes in the placenta between early- and late-onset preeclampsia, and the results showed that four genes, including CD200, were significantly upregulated in early-onset preeclampsia. Among them, we focused on CD200, because this protein was found to be expressed in the placenta in a previous study (21). Following the results of the RNA expression, we performed the immunohistochemical validation study in a larger number of patients with preeclampsia. Immunohistochemical analysis also showed that staining intensity for CD200 in the trophoblasts was significantly stronger in the early-onset preeclampsia group than in the late-onset group. Accordingly, CD200 expression is considered to be significantly upregulated in patients with early-onset preeclampsia by both RNA and protein expression levels.

CD200 belongs to the immunoglobulin superfamily and is expressed on diverse cell types and tissues, from B lymphocytes in the spleen to neurons in the central nervous system, and directly and continuously regulates macrophages and granulocytes through interaction with CD200R (18,19). CD200 signaling has been reported to inhibit classical macrophage activation (M1 polarization) and support an immunosuppressive M2 polarized state, resulting in immune tolerance (19,20). Immune tolerance is an important intrinsic mechanism in implantation. It has been reported that CD200 expression was significantly decreased in the villi of patients with early spontaneous abortion (32), suggesting that CD200 plays important roles in implantation. In addition, the polarization to M2 macrophages, which occurs physiologically during pregnancy, is thought to suppress the development of preeclampsia, and it has been reported that M1 macrophages were increased in the decidual tissue in patients with acute atherosis (19). Accordingly, macrophage polarization plays important roles in pregnancy and its disorders. Xu et al reported that CD200 expression was significantly downregulated in preeclampsia compared with normal placenta (21); however, they did not stratify the cases into early- and late-onset groups and might have included more patients with late-onset preeclampsia because the median gestational week was 36 weeks in their series. In addition, the period between the onset of preeclampsia and delivery might influence CD200 expression and macrophage polarization in the placenta (the data regarding this period was not available in the study by Xu et al (21). They also found that Th1 cytokines were upregulated and Th2 cytokines were downregulated in trophoblasts (21). In contrast, the results of the present study showed that CD200 was significantly upregulated in the early-onset group. The significance of CD200 expression in the trophoblasts might differ between early- and late-onset preeclampsia, although it remains unclear whether CD200 expression is the mainstream of preeclampsia development. CD200 expression might be the outcome or main cause of preeclampsia development. It is possible that excessive macrophage polarization influences the development of early-onset preeclampsia. In addition, significantly lower CD200 expression was noted in oocyte donation pregnancies with preeclampsia compared to those without preeclampsia, and this was not observed in naturally conceived pregnancies (33). Similarly, in preeclampsia pregnancies, significantly lower CD200 expression was noted in oocyte donation pregnancies compared to naturally conceived pregnancies (33). Thus, CD200 might have a role in the gestation processes of oocyte donation pregnancies. Moreover, additional mechanisms other than macrophage polarization might be involved in the development of preeclampsia. Additional studies are needed to clarify this issue.

There are some limitations in this study. First, we analyzed the immunohistochemical expression for CD200 in a relatively large number of patients (53 patients) with preeclampsia; however, RNA expression profiles analysis was performed in only 10 patients. Thus, further studies on both RNA expression and immunohistochemical analysis with a larger sample size of patients with early- and late-onset preeclampsia are needed. Second, the present study demonstrated that CD200 was significantly upregulated in early-onset preeclampsia. Although CD200 is considered to act in immune tolerance via macrophage polarization, distribution of M1 and M2 macrophages using CD68 and CD163 immunostaining in the placenta of patients with early- and late-onset preeclampsia was not analyzed in the present study. Thus, the relationship between CD200 expression and distribution of macrophages in the development of preeclampsia needs to be clarified.

In summary, the present study demonstrated that CD200 expression was significantly upregulated in patients with early-onset preeclampsia, although it remains unknown whether upregulation of CD200 is a cause or effect of development of early-onset preeclampsia. Thus, further studies are needed to clarify the mechanism of these conditions for adequate treatment.

Acknowledgements

The authors would like to thank Mr Ryosuke Yamaka who is a research assistant of Kansai Medical University (Osaka, Japan) who provided assistance in this study.

Funding

This research was partially supported by the research grant D2 from Kansai Medical University.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Sequencing data were deposited into the DNA Data Bank of Japan Sequence Read Archive (accession nos. SAMD00549508 and SAMD00549517; http://ddbj.nig.ac.jp/resource/bioproject/PRJDB14567).

Authors' contributions

HT and HO conceived and designed the study, and performed gene expression analyses. HT and MI performed immunohistochemical analyses. HT, MI, AN, TY, SK, YB, AY, YHi, YHa, TTN, HM, KT and HO acquired and analyzed data. HT, MI, and HO confirmed the authenticity of all the raw data, and drafted the manuscript, tables, and figures. All authors read and approved the final manuscript.

Ethics approval and consent to participate

This retrospective single-institution study was conducted in accordance with the principles of the Declaration of Helsinki, and the study protocol was approved by the Institutional Review Board of the Kansai Medical University Hospital (approval no. 2018138; Hirakata, Japan). All data are completely anonymized. The Institutional Review Board waived the requirement for informed consent due to the retrospective design of the study, using medical records and archival samples with no risk of identity exposure of the patients. Moreover, the present study did not include any minors.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References