Journal Reading Fetomaternal Division

Identification of Potential Early Biomarkers of Preeclampsia

Presenter: Dr. Gerry Irawan

Moderator: Dr. H. Wim T. Pangemanan, SpOG(K)

DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY

FACULTY OF MEDICINE SRIWIJAYA UNIVERSITY
Dr. MOH. HOESIN GENERAL HOSPITAL
PALEMBANG
Presented on Wednesday, March 28th, 2018, at 07.00 AM
 OUTLINE
2
1 INTRODUCTION 4 DISCUSSION

2 MATERIALS & METHODS 5 CONFLICT OF INTEREST

3 RESULTS 6 ACKNOWLEDGEMENTS
I. Introduction
3
Preeclampsia (PE), occurs in 3-5% of pregnant women, the leading causes
of maternal and perinatal mortality. PE is considered to occur following a
two-step process.

Stage 1: placentation defect, and abnormal differentiation of cytotrophoblast

cells → ↓placental size and restricted utero-placental blood flow →
ischaemia/hypoxia → ↑ syncytiotrophoblast apoptosis and necrosis and
release of damaging factors of placental origin

stage 2: systemic endothelial dysfunction in many organs and systems

including the kidneys, cardiovascular system, liver, brain, and others

Identification of Potential Early Biomarkers of

Preeclampsia
I. Introduction
The application of different
molecular biology
approaches has enabled the
identification of the entire
gamut of differentially
expressed genes.
4
miRNAs that epigenetically
Some of the miRNA usable
control of the expression
in early terms of pregnancy
target genes in the
and are therefore
regulation of placental
interesting potential
development and function
biomarkers.
has been analyzed.
Presentation Title Here
II. Materials and Methods
1st Pantient Control 5
Inclusion Exclusion

1. fullterm physiological 1. assisted reproductive

pregnancy techniques to increase
2. pregnant women with fertility
an indication for an 2. multifoetal pregnancies
54 pregnant women (27- 40 years with emergency Caesarean 3. severe somatic
Caesarean section indications (Table 1). section pathology
3. moderate and severe 4. foetal aneuploidy
early-onset PE (24-34 5. vaginal delivery
weeks)
4. moderate and severe
late-onset PE (>34 GW)

Identification of Potential Early Biomarkers of

Preeclampsia
II. Materials and Methods
Clinical Characteristics of the 1st pregnant women cohort 6

Identification of Potential Early Biomarkers of

Preeclampsia
 II. Materials and Methods
2nd Patient Control 7

16 pregnant women (26-46 years) underwent a women with

three-stage screening at 11-13, 24-26 and 30- physiological women with severe
32 GW and comprised the 2 groups.
pregnancy earlyonset PE
manifesting the
disease at 28-33 GW.

Identification of Potential Early Biomarkers of

Preeclampsia
II. Materials and Metods
Clinical charactheristic of the 2nd pregnant women cohort 8

Identification of Potential Early Biomarkers of

Preeclampsia
II. Materials and Metods
9
RNA isolation from the placental tissue RNA isolation from peripheral blood plasma

Venous blood samples collected into

Sampled placental tissue was washed in 0.9%
VACUETTE® tubes containing EDTA, centrifuged
NaCl and immediately frozen in liquid nitrogen for
for 20 min at 300 g (4 C) followed by plasma
subsequent storage at 80 C
collection and re-centrifugation for 10 min

The RNA was extracted from 200 ml

The RNA concentration was measured
blood plasma using the Serum Plasma
using a Qubit fluorimeter 3.0
kit

The sample quality of the total RNA was

into the mixture containing plasma and
examined on the Agilent Bioanalyzer
the phenol reagent Qiazol.
2100 using the RNA 6000 Nano Kit

Identification of Potential Early Biomarkers of

Preeclampsia
II. Materials and Methods
10
Reverse transcription and quantitative
miRNA deep sequencing real-time PCR
Five hundred nanograms of total RNA
from the placenta and 7 of 14 ml total
RNA column eluate extracted from 200
cDNA libraries were the ml blood plasma → cDNA in a reaction
NEBNext® Multiplex mixture
Small RNA Library Prep
Set for Illumina®,
amplified for 14 and 18
PCR cycles the sample volume was adjusted
with deionized water to 200 ml

sequenced on
The synthesized cDNA (3 ml) was
the NextSeq used as a template for realtime
500 platform. PCR using a forward primer
specific for the studied RNA

Identification of Potential Early Biomarkers of

Preeclampsia
II. Materials and Methods
Statistical data processing 11
Two-sided WilcoxonMann-Whitney test
To analysis of the significant differences in the delta Ct of
the studied miRNAs

A logistic regression
analyse the relationship between the miRNA levels and the
probability of PE development.

Identification of Potential Early Biomarkers of

Preeclampsia
III. Results
miRNA sequencing 12

Identification of Potential Early Biomarkers of

Preeclampsia
III. Results
miRNA sequencing
placenta and blood plasma
samples from 12 women in
the 1st cohort of patients ->
analyzed by miRNA deep
sequencing
miRNAs (P < 0.05) were
plotted as heat maps (Fig.
1A) separated the PE
groups from the control
groups based on the miRNA
in the placenta.
13
Comparison of the severe
early-onset PE and
moderate late-onset PE
groups was performed with
the control group for the
corresponding gestational
age
Fig. 2A, miRNA expression
patterns in blood plasma;
cluster 1 differed from
cluster 2 without considering
samples 163p and 188p.
Identification of Potential Early Biomarkers of
Preeclampsia
III. Results
Comparison of miRNA expression patterns in early- and lateonset PE 14
291 miRNAs was observed in both forms of PE → miRNAs specific to
trophoblasts, 42 miRNAs(C19MC, miR371-3), 33 miRNAs (C14MC).

no crossing on the lists of up-regulated miRNAs in early- and late-onset

PE (Fig. 2B)

in late-onset PE, none of the up-regulated miRNAs was encoded by the

C19MC/miR-371-3 and C14MC clusters, contrast to the early-onset PE.
(Fig. 2C)

early- and late-onset PE showed no overall changes, except for 1 miRNA

(Fig. 2D). In late-onset PE, 10 of 192 miRNAs had elevated expression levels
(Fig. 2E), and 182 of 192 miRNAs had decreased expression levels (Fig. 2F),

Identification of Potential Early Biomarkers of

Preeclampsia
 15

Identification of Potential Early Biomarkers of

Preeclampsia
III. Results
Pairwise comparison between plasma and placenta samples 16
using the DESeq2 package →
multiple-comparison
Bonferroni-Holm correction test

83 up-regulated miRNAs in the plasma

vs placenta, none of them encoded by
the C19MC/miR-371-3 or C14MC

Fewer differentially expressed

miRNAs were found in the plasma
in the late-onset PE
Identification of Potential Early Biomarkers of
Preeclampsia
III. Results
Detection of genes with no expression changes in the plasma samples 17

miRNAs were hsa-miR532-5p

plasma groups compatible: hsa- was chosen as
were compared miR-532-5p, hsa- an optimal
all vs all miR-106b-3p, endogenous
and hsalet-7a-5p control

Identification of Potential Early Biomarkers of

Preeclampsia
III. Results
Validation of the miRNA sequencing data by RT-qPCR 18

Identification of Potential Early Biomarkers of

Preeclampsia
III. Results
Validation of the miRNA sequencing data by RT-qPCR 20

Presentation Title Here

III. Results
Validation of the miRNA sequencing data by RT-qPCR 21

women who developed

clinical signs of early-
onset severe PE after excluded the use of
this miRNA as an
28 → ↑imiR532-5p endogenous control
expression at 11-13
GW

Presentation Title Here

 III. Results
Logistic regression model 22

ROC curve (AUC) = 0.844, 87.5%

sensitivity, 80% specificity, and 87.5% , p-
value= 0.01008 → “very good” models →
miR-423-5p as potentially early diagnosis of
PE before the clinical manifestation of the
disease.

Identification of Potential Early Biomarkers of

Preeclampsia
III. Results
Maternal plasma miR-423-5p as an early predictor of preeclampsia at 11-13 GW 23

miR-423-5p could clearly differentiate the early-onset PE group from the control group at 11-13 GW although the
manifestation of PE clinical signs only occurred at 28-33 GW.
Identification of Potential Early Biomarkers of
Preeclampsia
III. Results
Identification of signalling pathways potentially regulated by the associated PE miRNAs 24
processing
and
presentation
of antigen
cell apoptosis
graft-versus-
and DNA
host disease
repair

Potential
regulators
miR-532-5p, -423-
5p, -127-3p, -539-
5p, -519a-3p, and - complement
controlling 629-5p and let-7c- and
the cell cycle 5p coagulation
cascades

reninangioten
focal
sin system,
adhesion and
progesterone
p53-mediated
-mediated
signalling
oocyte
pathway
maturation

Identification of Potential Early Biomarkers of

Preeclampsia
Identification of Potential Early Biomarkers of
Preeclampsia
IV. Discussion
26
miRNA expression pattern
in early- and late-onset PE
were different.

↑ hypoxia/ischaemia
in the placenta ↑ expressed C14MC
miRNAs in trophoblast
→changes in the cells during the 1st
expression of miRNAs
trimester, ↑ C19MC
due to ↑ trophoblast miRNAs in the 3rd
cell shedding and trimester
necrosis.

Identification of Potential Early Biomarkers of

Preeclampsia
IV. Discussion
27
Changes in miR-532-5p, miR-423-5p, miR-127-3p, miR-539-5p,
miR-629-5p, and let-7c-5p expression →revealed in this study

high diagnostic value of miR-423-5p for the development of

early-onset PE

miR-532-5p in early stages of gestation →regulating genes

in decidualization and maintaining pregnancy

expression of miRNAs in the blood plasma should be corrected

for gestational age and normalized to exogenous cel-miR

Identification of Potential Early Biomarkers of

Preeclampsia
IV. Discussion
Factors associated with PE 28
excessive or restricted
failed endovascular villous cytotrophoblast
and interstitial fusion with
cytotrophoblast syncytiotrophoblasts
migration and
invasion

this study revealed

the differential
expression of
miRNAs in PE for the
first time

Preeclampsia
Identification of Potential Early Biomarkers of
Preeclampsia
IV. Discussion
Factors associated with PE 29
miR-127-3p is the miR-539-5p in
main mitochondrial let-7 in cytotrophoblast
posttranscriptional fragmentation and proliferation
regulator cell apoptosis

miR-423-5p in
inhibiting miR-629-5p in cell
miR532 in thrombosis
progesterone motility and invasion
receptor transcription

Identification of Potential Early Biomarkers of

Preeclampsia
 30

Conflict of interest Acknowledgements

• Sample collection, total RNA isolation,
• no conflicts between NGS and RT-PCR were performed as part
of the State assignment. Bioinformatics
any of the authors processing of the NGS and RT-PCR data
was supported by the Russian Science
Foundation project

Identification of Potential Early Biomarkers of

Preeclampsia
Thank You!
Any Questions?

Identification of Potential Early Biomarkers of

Preeclampsia