Study design: This is an exploratory, pilot and feasibility study to determine quantitative and functional differences among DC subsets of early onset pre-eclampsia patients based on well established experimental protocols.
Sample size: We aim to recruit 30 early onset pre- eclampsia patients and 30 normal pregnant women based on the feasibility of budget and time. Enrolment of these women started in January 2019 and will be completed by the end of 2021. Both blood and placenta samples are collected from each subject.
Participant recruitment and study sites: Pregnant women already being enrolled/ admitted at Civil hospital, Gandhinagar are first assessed for the eligibility criteria. Informed consent is obtained from eligible study participants. This is followed by sample collection and follow up with a case report form to collect information about the participant’s medical condition, their family medical history and other clinical information. The collected samples (blood and placenta) are brought to the research laboratory at the Indian Institute of Public Health Gandhinagar (IIPHG) where further sample processing procedures are carried out. As the final step, the processed samples are analysed by flow cytometry (Thermofisher Attune Nxt) at the Institute of Science, Nirma University, Ahmedabad. An overview of the study sites is shown in figure 4. SOPs are developed for collection, transfer and processing of samples.
Recruitment of study subjects based on eligibility criteria: An eligibility criteria checklist is being utilized for the recruitment of participants. This form is completed and signed by the obstetrician at Civil hospital, Gandhinagar. There are two sections (A & B) for eligibility assessment. Patients answering ‘NO’ to ‘ANY’ of the section A criteria are further assessed by section B criteria in order to be eligible for the study. In section B, patients answering ‘YES’ are classified as early-onset pre-eclampsia (EOPE) group and those answering ‘NO’ are classified as normal pregnant women.
Section A criteria: Patients answering ‘NO’ for the following conditions will be ELIGIBLE for the study.
- Multiple pregnancy (pregnancy with more than one fetus).
- Women pregnant for the second or more time.
- Late & new onset hypertension and proteinuria developing at or after 34+0 weeks of gestation (Late-onset pre-eclampsia).
- Chronic hypertension (≥140/90mm Hg) diagnosed before pregnancy or in the first half of pregnancy (<20 weeks) and continued for >12 weeks after delivery.
- Atypical pre-eclampsia (pre-eclampsia symptoms <20 weeks of gestation or > 48hrs after delivery.
- Positive for SARS-CoV-2 infection (currently or in the past).
- Medical complications: Urinary tract infections, HIV+, Hepatitis B+, Hepatitis C+, Infectious diseases, Diabetes mellitus, Collagen disorders, Autoimmune disorders (SLE), Thrombocytopenic purpura, antiphospholipid antibody syndrome, Hemolytic uremic syndrome, Acute fatty liver of pregnancy, Fetal malformations, Premature rupture of membranes, Chorioamnionitis/ Chronic villitis, Inflammatory diseases, Renal diseases, Severe extragenital pathology, Post transplantation state, Cancer history, Heart failure/Ischemic heart disease.
- Smoking.
- Any “other” obstetric complications. “Other” term for pre-eclampsia patient group includes obstetric complications except for the early-onset pre-eclampsia condition. Examples include hemorrhage, obstructed labor, amniotic fluid embolism.
- Maternal age >35 years.
- BMI > 27Kg/m2.
- Pregnant via assisted reproductive technology (ART).
Factors such as multiple pregnancy, chronic hypertension diagnosed before pregnancy, atypical pre-eclampsia, stated medical complications and infections (including COVID-19), smoking, higher maternal age, increased BMI and other obstetric complications can introduce biological mechanisms unrelated to the true representation of pre-eclampsia pathogenesis. Therefore, subjects with these scenarios are excluded from the study. Additionally, women who got pregnant via ART procedure are excluded from this study as these women demonstrated increased risk of PE, compared to women with spontaneous pregnancy [24]. It is not clear if the technique of assisted reproductive technology itself influences the placental biology [25]. Among spontaneous pregnancy, the risk of pre-eclampsia is much lower in women who are getting pregnant for the second or more time [26]. Therefore, these women are excluded from the study. As the study focus is on early-onset pre-eclampsia patients, pregnant women with late-onset pre-eclampsia are excluded from the study.
Section B criteria: Patients answering ‘YES’ are eligible for enrolment as early-onset pre-eclampsia participants. Patients who answer ‘NO’ are eligible for enrolment as normal pregnant women participants.
- Early & new onset hypertension (≥140/90mmHg) developing before 34+0 weeks of gestation.
- Early & new onset proteinuria (≥0.3g/24hr) developing before 34+0 weeks of gestation.
Obtaining informed consent: Based on the eligibility criteria, 2 groups of pregnant women are created: a) Early-onset pre-eclampsia (EOPE) patients and b) Normal pregnant women. These pregnant women are given the patient information sheet and sample collection details are clearly explained to them by the obstetrician. After explanation of the study, informed consent from the subjects are obtained.
Sample collection:
Blood Collection: Around 2 ml of blood is collected from each pregnant woman at the time of parturition. Experienced hospital staff/phlebotomy team are performing the blood collection by venipuncture. The sample is collected in sterile blood collection tubes coated with an anticoagulant such as EDTA, stored at room temperature and brought to research lab at IIPHG for further processing.
Placenta collection: Placenta is collected during parturition, placed in a sterile tissue collection bag enclosed in an ice box and brought to the research lab at IIPHG for further processing. Proper and approved biosafety practices for handling and disposal of biological materials is followed.
Case report form (CRF): Clinical data is collected from the pregnant women recruited into the study. These data are entered into the CRF during sample collection (blood and placenta) and post-partum. Briefly, general details of the subject are entered into CRF, including name, date of birth and BMI. In addition, participant and their family history are collected. Clinical parameters are recorded in the CRF; including gestation age, diastolic & systolic blood pressure, mean arterial pressure, proteinuria, any ongoing medical treatments, presence of HELLP/IUGR/atypical pre-eclampsia, recently taken hemoglobin levels, platelet levels and complete blood counts, doppler examination of uterine arteries and any other obstetric complication. Other parameters such as delivery date and gestation, placenta weight, type of delivery, systolic and diastolic blood pressure, any medical treatments done, and any other obstetric complications are also recorded.
Additionally, post-partum clinical parameters are entered into the CRF: Infant birth weight, systolic and diastolic blood pressure, presence of atypical pre-eclampsia, APGAR score, neonatal outcomes (example: Perinatal/fetal death, delivery<34 weeks, fetal distress syndrome, necrotizing enterocolitis, intraventricular haemorrhage) and maternal outcomes (example: Death, Pulmonary edema, acute renal failure, cerebral thrombosis, disseminated intravascular coagulation).
The collected clinical parameters are compared between pre-eclampsia patients and normal pregnant women. Data is presented as mean +/- standard deviation (SD) and range. Differences are considered significant when the p value will be equal to or less than 0.05. All statistical analyses assume a 2- sided significance level. Mann-Whitney U non-parametric test is used for comparisons between groups.
Experimental work plan: An overview of the complete experimental plan with blood and placenta samples is shown in figure 5.
A. Blood sample: A portion of blood sample is processed for direct immunofluorescence surface staining procedure and another portion for DC subset specific TLR stimulation.
Direct immunofluorescence staining of whole blood: This is a well-established methodology for directly detecting dendritic cell subsets in blood samples from normal pregnant women and healthy non-pregnant individuals [27-32]. This method is more efficient compared to other methods as it is shown to improve assay reproducibility and is less likely to show loss of lymphocyte subsets [33-36]. All monoclonal antibodies are titrated for determining optimum antibody concentration for usage.
Around 0.2ml of blood sample is stained with monoclonal antibodies against surface markers (Table 1), followed by RBC lysis. These samples are run on the flow cytometer (Thermo Fisher Attune Nxt) to characterize DC subsets. The 9-color multiparametric flow panel has been designed using FluoroFinder2.0 software (Table 1), with careful consideration given for minimal spectral spill-over values between fluorochromes so that automatic compensation can be easily performed using FlowJo software. AbcTM Anti-Mouse Bead Kit (Thermo Fisher Scientific) is used to set up flow cytometry compensation. The 9-color multiparametric flow panel is designed to identify the 3 DC subsets, simultaneously determining other phenotypic changes, such as activation (CD80), maturation (CD83) and tolerogenic properties (ILT-3). ILT3 (immunoglobulin- like transcript 3), also known as CD85K is highly expressed on myeloid DCs in the decidua of normal pregnant women [13]. ILT3 is involved in the induction of immune tolerance in DCs via interaction with HLA-G on extra villous trophoblasts (EVTs) [37]. Therefore, ILT-3, along with activation and maturation markers are included in the panel to monitor their expression changes in the pro-inflammatory environment of EOPE.
TLR stimulation of whole blood: This procedure has been well established for directly analyzing DC subsets functional responses in whole blood post stimulation/activation with TLR ligands [32]. Briefly, 0.5ml of blood sample is subjected to TLR stimulation, by using LPS 100ng/ml (for TLR-4 stimulation on CD1c+ mDCs) and CpG 2216 30ug/ml (for TLR-9 stimulation on plasmacytoid DCs) and poly I:C 30ug/ml (for TLR-3 stimulation on CD141+ mDCs) along with brefeldin A 10 ug/ml (protein transport inhibitor) for 5hrs at 37c, 5% CO2. Selection of TLRs for each DC subset is based on differential expression of TLRs on these cells [38]. Thereafter, post surface staining, intracellular cytokine staining procedure (permeabilization and fixation) is performed- specifically for IL-12, TNF-A (for myeloid DCs) and IFN-A, TNF-A (for plasmacytoid DCs) (Tables 2-4). These samples are run on the flow cytometer (Thermofisher Attune Nxt). Production of these cytokines by DC subsets is drastically altered in several pro-inflammatory disorders [7,8]. Therefore, these cytokines are being included in the flow-panel to assess their production in EOPE patients and to determine feasibility of including them as biomarkers for diagnosis and/or immunotherapeutic intervention.
B. Placenta (decidua) samples: Procedures for the isolation of decidua, decidual cells and leukocytes are being adapted from well-established studies [39, 40]. Decidua basalis (part of decidua in contact with placenta) and decidua parietalis (rest of the decidua on the maternal myometrium end) are isolated from the placenta. Collected decidua are subjected to mechanical processing to obtain decidual cells. Decidual leukocytes are isolated by Ficoll-Paque density gradient centrifugation method [41]. The leukocytes settled at the interface are carefully collected and washed for immunophenotyping DC subsets. The total yield of leukocytes from this protocol is up to 30x106 cells per decidual tissue per study participant.
Immunophenotyping and functional analysis of decidual DC subsets: A portion of freshly isolated decidual leukocytes is used for cell-surface antigen staining using monoclonal antibodies and another portion of cells will be subjected to specific TLR stimulation. Appropriate mouse anti-human isotype controls are included. In addition, appropriate fluorescence minus one (FMO) controls are used to eliminate any spill-over- induced background.
Around 2 x106 live cells are used per sample (dead cells are excluded by trypan blue counting with a hemocytometer). All monoclonal antibodies are titrated for determining optimum antibody concentration for usage. For surface staining, the cells are stained with appropriate monoclonal antibodies forming the 9-color panel (Table 2). TLR stimulation of decidual cells are performed similar to blood samples and optimal concentration of TLR ligands to stimulate decidual DC subsets are being optimized. Similar to blood samples, surface staining (including activation markers) and intracellular cytokine staining (permeabilization & fixation) are performed for detecting IL-12, TNF-A (myeloid DCs) and IFN-A, TNF-A (plasmacytoid DCs) (Tables 2-4).
Data analyses: At least 200 000 events within the combined lymphocyte-monocyte gate, based on the FSC and SSC parameters per sample is collected in the flow cytometer and data is analyzed using FlowJo software. Appropriate mouse anti-human isotype controls are included to rule out any non-specific background signal caused by primary antibodies. In addition, appropriate fluorescence minus one (FMO) controls is used to eliminate any spill-over- induced background.
Data is analyzed using FlowJo software as follows (Fig-6). As the first step, based on FSC Vs SSC dot plots, cell debris and dead cells are excluded. This is followed by selection of lineage (Lin)- and major histocompatibility complex (MHC)- class II (HLA-DR) hi /+ populations. Dendritic cells do not express lineage-specific markers (CD3+ T cells, CD14+ Monocytes, CD16+ NK cells and granulocytes, CD19+/CD20+ B cells and CD56+ NK cells). Therefore, the DCs in blood is identified as Lin- HLA-DR+/hi [42].
This is followed by determining frequencies and absolute numbers of a) Plasmacytoid DCs: CD11c- CD123+, b) CD1c+ Myeloid DCs: CD11c+ CD1c+ and c) CD141+ Myeloid DCs: CD11c+ CD141+. In addition, the frequencies and mean fluorescence intensity (MFI) of the cytokines and activation, tolerogenic markers expressed by each of the 3 DC subsets is calculated. All the data are compared between normal pregnant women(n=30) and pre-eclampsia patients (n=30).
Statistical analyses: A standard non-parametric test (Mann-Whitney U- test) is used to determine statistical differences of blood and decidua derived DC subsets between the two groups of pregnant women. Differences at P<0.05 is considered statistically significant. IBM SPSS 20 software is used to perform statistical analyses.