First‐trimester plasma extracellular heat shock proteins levels and risk of preeclampsia

Abstract Preeclampsia (PE) occurs annually in 8% of pregnancies. Patients without risk factors represent 10% of these. There are currently no first‐trimester biochemical markers that accurately predict PE. An increase in serum 60‐ and 70‐KDa extracellular heat shock proteins (eHsp) has been shown in patients who developed PE at 34 weeks. We sought to determine whether there is a relationship between first‐trimester eHsp and the development of PE. This was a prospective cohort study performed at a third level hospital in Mexico City from 2019 to 2020. eHsp levels were measured during the first‐trimester ultrasound in singleton pregnancies with no comorbidities. First‐trimester eHsp levels and biochemical parameters of organ dysfunction were compared between patients who developed preeclampsia and those who did not. All statistical analyses and model of correlation (r) between eHsp and clinical parameter were performed using bootstrapping R‐software. p‐values <0.05 were considered significant. The final analysis included 41 patients. PE occurred in 11 cases. eHsp‐60 and eHsp−70 were significantly higher at 12 weeks in patients who developed PE (p = 0.001), while eHsp‐27 was significantly lower (p = 0.004). Significant differences in first‐trimester eHsp concentration suggest that these are possible early biomarkers useful for the prediction of PE.


| INTRODUC TI ON
Preeclampsia (PE) is a heterogeneous, multisystemic hypertensive disorder of pregnancy of variable severity that is associated with 60,000 deaths per year worldwide. 1 Preeclampsia complicates from 2 to 8% of pregnancies and continues to be one of the leading causes of maternal 2,3 and foetal death. 4 It is associated with complications such as foetal growth restriction (FGR) [5][6][7] and placental abruption. 5,8,9 Preeclampsia development is divided in two stages. 10,11 During the first stage, inefficient cytotrophoblast invasion early in the first trimester causes inadequate and incomplete remodelling of the spiral arteries, with consequent high vessel resistance and low blood flow which cause placental ischemia. 12,13 The measurement of blood flow through uterine arteries reflect these changes and, coupled with maternal blood pressure, are the only validated first-trimester prediction models. 14 During the second stage of this abnormal placentation, there is a proangiogenic and antiangiogenic imbalance, 15,16 including soluble Fms-like tyrosine kinase-1 (sFlt-1), vascular endothelial growth factor (VEGF) and placental growth factor (PIGF). [17][18][19][20] The decrease in these proteins in plasma is correlated with the severity of PE and with the development of adverse pregnancy outcomes [21][22][23] attributed to an increase in pro-inflammatory cytokines. 24,25 These serum markers and their indices (sFlt-1/PIGF ratio) are only used in prediction models later on in pregnancy. 26 Extracellular heat shock proteins (eHsp), first described by Ferruccio Ritossa in 1962, are molecules stable at high temperature present in both prokaryotic and eukaryotic cells. 27 These molecules are expressed during cellular stress and regulate cellular homeostasis, 28 proliferation and differentiation of the professional immune system cells. [29][30][31] Extracellular heat shock proteins have been classified as being of high (60,70,90, or low (20, 27-kDa) molecular weight. 32,33 When released into the extracellular space, eHsp function as cellto-cell mediators. 27,34 eHsp-60 (HSPD1; heat shock protein family D member 1A) and eHsp-70 (HSPA1A; heat shock protein family A member 1A) stimulate pro-inflammatory cytokines production, 27,34 while eHsp-27 (HSPB1; heat shock protein family B [small] 1) has an important anti-inflammatory function. [35][36][37] Their concentration has also been shown to increase in the serum of patients with severe trauma, 37,38 inflammatory processes 39,40 and PE. [41][42][43] Therefore, eHsp have been used as sensible indicators of the physiological status during the onset and resolution of different pathological conditions. 44,45 Recently, our research group has confirmed significantly higher levels of eHsp-60 and eHsp-70 in patients with PE compared with same gestational age controls, as well as a linear relationship between their concentration and levels of inflammatory cytokines (IL-1β and TNFα), uric acid, creatinine, lactate dehydrogenase and liver transaminases (AST and ALT) 41 ; eHsp concentrations in the first trimester of pregnancy were not assessed. The aim of this study was to determine whether first-trimester serum levels of eHsp are associated with the development of PE and if there is a correlation between eHsp and serum biomarkers of end-organ dysfunction present in PE.

| Ethics statements
This study was reviewed and approved by the National Institute of Perinatology Ethics and Research Committees (registration number 212250-3210091). All patients were explained the purpose of the study, and informed consent was obtained.

| Study design and patients
We performed a prospective cohort study. The data were derived from screening in women attending their first-trimester routine ultrasound at the National Institute of Perinatology in Mexico City, México, between February 2019 and January 2020.

| Clinical definitions and inclusion criteria
Inclusion criteria were singleton pregnancies without a history of PE who were able to attend a first-trimester ultrasound and have their blood drawn for eHsp measurement. Patients were excluded from the study when the amount of blood collected for the quantification of eHsp was insufficient (plasma <1200 μl), as well as patients di-

| Obtaining blood samples and biochemical assays
During the first ultrasound visit, which is held at 11-13 weeks of gestation, peripheral blood samples (5 ml) were collected in K 2 -EDTA vacutainer tubes (Becton-Dickinson; NJ, USA) and centri-

| Data analysis
All assays were independently replicated at least three times.
Laboratory parameters, clinical data and eHsp concentrations between healthy and PE patients were compared using Student's t-test.
The Spearman rank correlation (r) between eHsp and clinical parameters was compared by bootstrapping analysis for small samples. 47 The data are presented as means with SD. Statistical analysis was carried out using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA; USA). A p-value <0.05 was considered significant.
All statistical analyses were performed using the R software statistical computing version 4.1.2 (R Foundation, Vienna, Austria; https:// www.R-proje ct.org/).

| RE SULTS
A total of 48 women were followed up after eHsp measurement during the first-trimester ultrasound. Of these, seven were eventually excluded for the following reasons: premature rupture of membranes (n = 4), placental abruption (n = 2) and foetal growth restriction (FGR) without PE (n = 1). In total, 41 women with singleton pregnancies were included in this study, of which 11 developed PE. Of these, three developed early PE and eight late PE. One patient part of late PE group developed HELLP syndrome and two patients FGR + PE.  Table 2 shows the values of serum biochemical parameters at the time of PE diagnosis. No significant differences were found in haemoglobin  Note: Data are shown as the mean ± SD and were analysed using the bootstrapping with 10,000times repeated. Statistically significant difference in <0.05 was considered.

TA B L E 1
Comparison of maternal characteristics at the time of admission, at the development of preeclampsia and neonatal at the delivery between the two groups of pregnant patients  Table 3).

| DISCUSS ION
Inefficient cytotrophoblast invasion and abnormal placentation promotes an imbalance between angiogenic and antiangiogenic factors. 48 Note: Data are shown as the mean ± SD and were analysed using the bootstrapping with 10,000times repeated. Statistically significant difference in <0.05 was considered.
Abbreviation: CI, confidence intervals.  41 In the same study, there was a significant correlation between both eHsp-60 and eHsp-70 and biochemical markers of organ dysfunction. Nevertheless, the association between these markers and the development of PE has not been studied in the first trimester.
In this study, we noted that pregnant women who subsequently In the next phase of the study, we intend to follow-up with the patients and take blood samples from the first, second and third trimesters of pregnancy. At the time of delivery, we will take a sample of the placental tissue and assess how it relates with eHsp levels at systemic level. Three months after the baby is born, a final blood sample will be drawn from patients. These results will allow us to establish: Some limitations in the present study must be addressed.
First, the small sample size may limit statistical power. Second,

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interests with the research or the authorship publication. All authors carefully read the final version of the manuscript and gave their consent for journal submission.

DATA AVA I L A B I L I T Y S TAT E M E N T
All of the relevant information from the study is described in the manuscript.