Failing maternal–fetal tolerance in SLE (FaMaLE): a prospective cohort study for finding the molecular mechanisms behind pregnancy complications

Abstract

Introduction

Pregnant women with SLE have an increased risk of maternal complications and adverse fetal outcomes. These include pre-eclampsia, preterm birth and fetal growth restriction. Interestingly, this increased risk persists in subsequent pregnancies, whereas it decreases in healthy women due to the development of maternal–fetal tolerance. As maternal–fetal tolerance is crucial for a healthy pregnancy, we hypothesise that its failure contributes to the increased risk of pregnancy complications in women with SLE. Therefore, we initiated the failing maternal–fetal tolerance in SLE (FaMaLE) study to investigate the failure of maternal–fetal tolerance in pregnant women with SLE.

Methods and analysis

In the FaMaLE study, women with SLE and healthy women are included in their first trimester of pregnancy (<14 weeks gestational age) at Amsterdam UMC. Throughout the pregnancy, data on SLE disease activity, pregnancy course and medication use are collected. Peripheral blood is collected once per trimester, within 48 hours before delivery and 5–12 weeks post partum. In addition, the placenta is collected after delivery. Whole blood, peripheral blood mononuclear cells and placenta samples are freshly analysed by flow cytometry to assess immune cell composition. The resulting data are analysed in relation to SLE disease course, pregnancy course and pregnancy outcomes.

Ethics and dissemination

The study has been approved by the Amsterdam UMC Medical Ethics Committee and all participating women will be asked to provide informed consent. The findings will be disseminated through peer-reviewed publications, presentations at scientific meetings and via patient organisations.

Correspondence to Dr Wendy Dankers; [email protected] ; Dr Irene E M Bultink; [email protected]

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Women with SLE have a strongly increased risk of pregnancy complications. Failing maternal–fetal tolerance is believed to contribute to this increased risk, but the underlying mechanisms remain unknown.

WHAT THIS STUDY ADDS

• This study presents a standardised and feasible protocol for the prospective collection and integrative analysis of maternal blood and placental tissue in pregnant women with SLE. It addresses practical and ethical challenges in translational research during pregnancy, offering a reproducible approach for immunological studies in this vulnerable population.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• By providing a clear methodological framework, this protocol can guide researchers aiming to investigate the mechanisms of pregnancy complications in SLE and related conditions. It may serve as a template for future translational studies and help improve understanding and ultimately the management of adverse pregnancy outcomes in both autoimmune and non-autoimmune settings.

Introduction

SLE is a chronic autoimmune disease with the potential to affect all organ systems in the body. It is characterised by a disruption in immunological tolerance, leading to the production of autoantibodies targeting nuclear self-antigens.^{1 2} SLE affects 1 in 1000 people worldwide, with a higher prevalence among individuals of non-Caucasian descent, and predominantly affects women during their childbearing years.¹ Unfortunately, up to 50% of pregnant women with SLE experience complications, such as pre-eclampsia, fetal growth restriction and preterm birth.^{3 4} Importantly, almost 50% of preterm births have a spontaneous onset. The other preterm births are induced mostly because of pre-eclampsia or fetal growth restriction.⁵ Partially due to (the increased risk of) pregnancy complications, 60% of patients with SLE have fewer children than desired.^{6 7}

Despite the significant impact on the health and well-being of pregnant women with SLE and their children, the exact underlying cellular and molecular mechanisms driving these complications are still poorly understood. Understanding these biological processes is crucial to enable prediction, early detection and novel treatments for placenta-related pregnancy complications in SLE.

One of the requirements for a healthy pregnancy is a well-functioning maternal immune system, which facilitates proper placentation, triggers the labour process at the end of pregnancy and ensures that the semiallogeneic fetus is not immunologically rejected during pregnancy.⁸ This latter phenomenon is called maternal–fetal tolerance and is thought to be the reason why generally the risk of pregnancy complications decreases in subsequent pregnancies with the same father. Interestingly, this protective effect is absent in women with SLE,³ suggesting that impaired maternal–fetal tolerance may play a central role in the increased risk for pregnancy complications in women with SLE.

Previous data suggest an altered immune response in pregnant women with SLE who experience pregnancy complications.⁹ Furthermore, there are some studies reporting an increased deposition of neutrophils extracellular traps and higher numbers of natural killer cells, macrophages and extravillous trophoblasts in the placentas of women with SLE.^10–12 However, if and how this dysregulation contributes to pregnancy complications in women with SLE is currently unknown. Therefore, to address these knowledge gaps, we have initiated the failing maternal–fetal tolerance in SLE (FaMaLE) study in which we aim to delineate the differences in cellular composition and function at the maternal–fetal interface in women with SLE, both with and without pregnancy complications, compared with healthy pregnant women.

Methods and analysis

Study overview and design

This single-centre prospective cohort study was initiated in May 2022 and will include 60 pregnant SLE patients and 18 healthy pregnant controls, all aged 18 years or older, who receive their antenatal care from the first trimester onwards at Amsterdam UMC. Women with SLE must be diagnosed according to the American College of Rheumatology (ACR) 1997, Systemic Lupus International Collaborating Clinics (SLICC) and/or ACR/European Alliance of Associations for Rheumatology 2019 criteria and must be under treatment for their disease at Amsterdam UMC. The treating gynaecologist and rheumatologist will screen potentially suitable participants during their first trimester of pregnancy at the Department of Obstetrics and Gynaecology and the Department of Rheumatology and Clinical Immunology of Amsterdam UMC. Eligible subjects will be informed about the aim, procedures and potential risks of the study by members of the research team. Participants can be enrolled before 14 weeks gestational age after providing informed consent. An overview of the study protocol is shown in figure 1.

Figure 1. FaMaLE study flow chart. ACR, American College of Rheumatology; BMI, body mass index; EULAR, European Alliance of Associations for Rheumatology; FaMaLE, failing maternal-fetal tolerance in SLE; HC, healthy control; PBMC, peripheral blood mononuclear cell; SLE-CP, SLE complicated pregnancy; SLE-NCP, SLE non-complicated pregnancy; SLICC, Systemic Lupus International Collaborating Clinics. Created in BioRender. Dankers, W. (2025). https://BioRender.com/t19u876

Data collection includes demographic information (age, ethnicity, highest level of education), medical history (smoking status, alcohol/drug use, general and SLE-specific medical history, current and prior medication use) and obstetric history (number of previous pregnancies, any previous miscarriages, course and outcome of previous pregnancies). SLE disease activity will be assessed using the SLE pregnancy disease activity index (SLEPDAI) during pregnancy,¹³ and SLE disease activity index 2000 (SLEDAI-2K)¹⁴ before pregnancy and after delivery. Preconceptional disease-related damage is assessed by the SLICC/ACR Damage Index.¹⁵ Additionally, gestational age at delivery and gender, weight and delivery outcomes for the newborn will be recorded. Apart from routine laboratory investigations for regular clinical care in pregnant women with SLE, at each visit, an additional 45 mL of blood is collected in four heparin tubes and one coagulation tube. From these serum, plasma and peripheral blood mononuclear cell (PBMC) are isolated. The blood sample from time point 4 is collected within 48 hours before delivery, when the woman is admitted at the delivery room. After delivery, the placenta will be collected for further analysis.

Medical data will be recorded by the treating gynaecologist and rheumatologist. Histopathological examination of all placentas will be performed by one assigned pathologist according to the Amsterdam Placental Workshop Group Consensus Statement.¹⁶ Pregnancies are classified as complicated if they meet any of the following criteria: preterm delivery (<37 weeks gestational age), pregnancy-induced hypertension (de novo or superimposed), pre-eclampsia (ISSHP criteria 2018,¹⁷), small for gestational age child (<p10), placental abruption and/or intrauterine fetal death (>16 weeks gestational age).

Patient and public involvement

Two patient research partners were involved in the design of the study, critically reviewed the patient information folders and contributed to this manuscript.

Inclusion and exclusion criteria

Inclusion criteria for patients with SLE

• Age 18 years or older.

• Gestational age at inclusion <14 weeks.

• Antenatal care from first trimester onwards in the Department of Obstetrics and Gynaecology of Amsterdam UMC.

• Diagnosed with SLE, fulfilling ACR 1997, SLICC and/or ACR/EULAR 2019 criteria.

• Signed informed consent.

Inclusion criteria for healthy pregnant women

• Age 18 years or older.

• Gestational age at inclusion <14 weeks.

• Antenatal care and delivery in the Department of Obstetrics and Gynaecology of Amsterdam UMC.

• Signed informed consent.

Exclusion criteria for patients with SLE

• Diagnosed with a systemic inflammatory disease other than SLE.

• Diagnosed with a malignant disease in the previous 5 years, except for basal cell or squamous epithelial cell skin cancers.

• Multifetal pregnancy.

Exclusion criteria for healthy pregnant women

• Diagnosed with a systemic inflammatory disease.

• Diagnosed with a malignant disease in the previous 5 years, except for basal cell or squamous epithelial cell skin cancers.

• Multifetal pregnancy.

• Treatment with immunosuppressive agents.

• Pre-existing hypertensive disorder.

• Obstetric history of hypertensive disorder of pregnancy, fetal growth restriction (birth weight <p10), preterm birth, placental abruption or intrauterine fetal death.

• Obesity (body mass index >30 kg/m²).

• Any disease for which the woman receives specialist treatment other than obstetric care.

Sample size calculation

The rate of pregnant SLE patients to be included in this study is expected to be 15–20 per year based on our experience over the last >15 years in the multidisciplinary SLE pregnancy care team in Amsterdam UMC. Pregnancy complications due to placental insufficiency as defined above are expected in 30% of patients with SLE as previously published by our research group.³ In the present study, women are categorised into three groups: healthy control (HC), SLE complicated pregnancy (SLE-CP) or SLE not CP (SLE-NCP).

In the analysis, differences in cell composition in whole blood, PBMC and placental tissue between these groups will be compared. We aim to detect differences greater than 10%, so at least 16 patients per group are needed for 80% power to detect differences (with an α of 0.05). Accounting for a 10% dropout rate throughout this study, 18 subjects per group need to be included. Considering an expected 30% incidence of pregnancy complications in patients with SLE, 60 pregnant women with SLE are needed to include at least 18 patients with complications. Additionally, 18 healthy pregnant women will be included.

Study aims

The primary aims of this study are (1) to compare the cellular profile of placental cells from women in the HC, SLE-CP, SLE-NCP groups; (eg, surface markers, transcription factors, gene expression), (2) to analyse the cellular function, cell–cell interactions and the development of tolerance at the in vitro maternal–fetal interface and (3) to identify potential peripheral blood biomarkers associated with pregnancy complications.

As secondary objectives, the study aims to establish an in vitro organoid-based model for the maternal–fetal interface in SLE and to compare the cellular profile and different proteins in the peripheral blood of women in the three groups.

Study procedures

Blood sampling

From the blood drawn at the five time points throughout pregnancy, serum and plasma will be isolated within 4 hours after blood draw from anti-coagulation and heparin tubes, respectively. They are stored at −80°C for later use for the detection of biologic markers (antibodies, cytokines, (disease-related) proteins, cell-free RNA). 100 µL of whole blood is saved for analysis using flow cytometry, and then PBMCs are isolated within 24 hours from the heparin tubes, using Lymphoprep (Serumwerk, Bernburg, Germany) density gradient centrifugation. 2–4 million PBMCs are analysed using flow cytometry, and remaining PBMCs are frozen per 5–10 million cells in freezing medium (10% dimethyl sulfoxide (DMSO) (VWR, Amsterdam, The Netherlands) and 90% Iscove’s Modified Dulbecco’s Medium (Gibco) supplemented with 30% FCS, 0.05 mg/mL gentamicin (Gibco) and 0.00036 (v/v)% β-mercaptoethanol.

Placenta sampling

After delivery, the placenta will be collected by the research team and stored in 0,9% NaCl at 4°C until processing, which starts within 2 hours after delivery. Since most current protocols for processing placenta focus on obtaining either immune cells or trophoblasts, a customised protocol was used to capture both cell types based on previously published protocols.^{18 19} The umbilical cord is removed from the placenta, leaving the insertion intact. A 4 cm of umbilical cord is stored in formalin for histopathological examination and classification by the assigned pathologist, as well as one-third of the chorioamniotic membranes. The remaining decidua parietalis and chorion (PC) are separated from the amnion, cut-off and placed in 1x PBS. Once the membranes and umbilical cord are removed, the placenta weight is determined. Three biopsies from random locations at the placenta are taken using an 8 mm biopsy punch, covered in O.C.T., snap-frozen in liquid nitrogen and stored at −80°C. The placenta is then cut into slices of ~1–1.5 cm thick, to assess the percentage of calcifications throughout the tissue. At least two blocks of 2×1 cm, of which one includes the umbilical cord insertion site, and both blocks covering the entire depth of the placenta, are harvested and stored in formalin for histopathological examination and classification by the assigned pathologist. Subsequently, the basal plate (BP; decidua basalis and ~5 mm of villi) is dissected and put in PBS. Now, both PC and BP are cut into small fragments (~2–3 mm diameter) and washed with 1x PBS until the PBS is clear. After washing with Roswell Park Memorial Institute (RPMI) medium, tissues are transferred to C-tubes (Miltenyi Biotec, Bergisch Gladbach, Germany) and resuspended in digestion mix (200 mg/mL collagenase IV (Sigma-Aldrich, St. Louisa, MO, USA) and 10 mg/mL DNaseI (Roche) in RPMI). Tissue is then dissociated on GentleMACS (BP: 1x m-heart-02.01, PC: 2x h-tumor-02.01) and left to digest for 1 hour at 37°C in a shaking water bath. After 30 min, there is another round of dissociation (all: 1x m-heart-02.01). Digested tissues are filtered through 100 µm cell strainers (Miltenyi Biotec), after which red blood cells (RBCs) are lysed using 1x RBC lysis buffer (BioLegend). Finally, cells are filtered through a 70 µm cell strainer, after which ~5 million cells are analysed using flow cytometry, 1 million cells are plated for subsequent culture and remaining cells are frozen as described for PBMC.

Flow cytometry

2–4 million PBMCs, 5 million cells from BP and PC, and 100 µL whole blood are freshly analysed for cellular composition using five flow cytometry panels: myeloid cells, B cells, T cells, ILC and non-immune cells (tables 1–5). With each sample, a reference control sample is taken along to account for batch effects during analysis.

Table 1

Flow cytometry panel for PBMC and placenta: myeloid cells

Target	Fluorochrome	Clone	Company	Purpose
BDCA2	APC	201A	BioLegend	DC differentiation
CD3	PE-Dazzle594	UCHT1	BioLegend	T cells
CD11b	BUV395	ICRF44	BD Biosciences	DC differentiation, macrophages, neutrophils
CD11c	AlexaFluor700	Bu15	BioLegend	DC differentiation, macrophages
CD14	FITC	61D3	eBioscience	DC- and monocyte differentiation, macrophages
CD15	BV605	W6D3	BioLegend	Neutrophils
CD16	APC-eFluor780	CB16	eBioscience	Monocyte differentiation, Neutrophils, NK cells
CD19	BV650	HIB19	BioLegend	B cells
CD45	PerCP-Cy5.5	HI30	BioLegend	Immune cells
CD56	PE	NCAM 16	BD Biosciences	NK(T) cells
CD103	BV421	Ber-ACT8	BioLegend	CD8+ T cell, DC and NK cell differentiation
CD123	PE-Cy7	6H6	eBioscience	DC differentiation
Viability	eF506	–	Invitrogen	Cell viability

Viability is measured using fixable viability dye.

BDCA2, blood dendritic cell antigen 2; CD, cluster of differentiation; DC, dendritic cell; NK cell, natural killer cell; PBMC, peripheral blood mononuclear cell.

Table 2

Flow cytometry panel for PBMC and placenta: B cells

Target	Fluorochrome	Clone	Company	Purpose
CD19	BV650	HIB19	BioLegend	B cells
CD21	PE-Cy7	B-ly4	BD Biosciences	B cell differentiation
CD24	BV421	ML5	BioLegend	B cell differentiation
CD27	APC-Fire750	O323	BioLegend	B-cell and CD8+ T cell differentiation
CD38	PE	HIT2	BioLegend	B cell differentiation
CD45	PerCP-Cy5.5	HI30	BioLegend	Immune cells
CD138	APC	MI15	BioLegend	B cell differentiation
IgD	BV785	IA6-2	BioLegend	B cell differentiation
IgM	FITC	G20-127	BD Biosciences	B cell differentiation
Viability	eF506	–	Invitrogen	Cell viability

Viability is measured using fixable viability dye.

CD, cluster of differentiation; PBMC, peripheral blood mononuclear cell.

Table 3

Flow cytometry panel for PBMC and placenta: T cells

Target	Fluorochrome	Clone	Company	Purpose
CCR7	PE-Cy7	3D12	BD Biosciences	CD4+-, and CD8+ T cell differentiation
CD3	APC-R700	UCHT1	BD Biosciences	T cells
CD4	BUV563	SK3	BD Biosciences	CD4+ T cells
CD8	BV785	RPA-T8	BioLegend	CD8+ T cells
CD25	APC	M-A251	BioLegend	CD4+ T cell differentiation
CD45	PerCP-Cy5.5	HI30	BioLegend	Immune cells
CD45RA	eFluor450	HI100	eBioscience	γδ- CD4+-, CD8+ T cell differentiation
CD45RO	PE	UCHL1	BioLegend	γδ- CD4+-, CD8+ T cell differentiation
CD56	BV605	HCD56	BioLegend	γδ differentiation, NK(T) cells
CD127	APC-eFluor780	eBioRDR5	eBioscience	Non-NK ILC
TCRγδ	FITC	B1	BD Biosciences	γδ T cells
Viability	eF506	–	Invitrogen	Cell viability

Viability is measured using fixable viability dye.

CD, cluster of differentiation; ILC, innate lymphoid cells; NK cell, natural killer cell; PBMC, peripheral blood mononuclear cell.

Table 4

Flow cytometry panel for PBMC and placenta: innate lymphoid cells (ILC)

Target	Fluorochrome	Clone	Company	Purpose
CD45	PerCP-Cy5.5	HI30	BioLegend	Immune cells
CD56	BV605	HCD56	BioLegend	NK(T) cells
CD103	BV421	Ber-ACT8	BioLegend	CD8+ T cell, DC and NK cell differentiation
CD117	PE	104D2	eBioscience	Non-NK ILC differentiation
CD127	PE-Cy7	A019D5	BioLegend	Non-NK ILC
Lineage	FITC	Multiple*	eBioscience	Identify non-ILC
NKp44	AlexaFluor647	P44-8	BioLegend	Non-NK ILC differentiation
Viability	eF506	–	Invitrogen	Cell viability

Viability is measured using fixable viability dye.

*RPA-2.10, OKT3, 61D3, CB16, HIB19, TULY56, HIR2.

CD, cluster of differentiation; NK cell, natural killer cell; PBMC, peripheral blood mononuclear cell.

Table 5

Flow cytometry panel for placenta: non-immune cells

Target	Fluorochrome	Clone	Company	Purpose
CD31	APC-eF780	WM-59	eBioscience	Endothelial cells
CD45	PerCP-Cy5.5	HI30	BioLegend	Immune cells
CD235a	PE-Cy5	HIR2	BioLegend	Red blood cells
CK7*	AF647	W16155A	BioLegend	Trophoblasts
HLA-DR	BV711	L243	BioLegend	Non-syncytiotrophoblasts
HLA-G	PE	MEM-G/9	Exbio	Cytotrophoblasts, extravillous trophoblast, syncytiotrophoblasts
Viability	eF506	–	Invitrogen	Cell viability
Vimentin*	AF488	O91D3	BioLegend	Decidual stromal cells, trophoblasts

Viability is measured using fixable viability dye.

*Intracellular staining.

CD, cluster of differentiation; CK, cytokeratin; HLA, human leucocyte antigen.

Whole blood is directly stained with the myeloid panel after which RBCs are lysed using 1x RBC lysis buffer (BioLegend). PBMC and placenta cells are stained with the respective panels in PBA (PBS supplemented with 0.5% BSA, 0.01% NaN₃), and then with fixable viability dye eF506 (Invitrogen, Carlsbad, California, USA) in PBS. After staining, cells are fixed using Fix/Perm buffer (eBioscience, ThermoFisher Scientific). For the immune panels, the cells are then measured at the LSR Fortessa (BD Biosciences). For the non-immune panel, cells are stained with the intracellular antibodies in Perm buffer (eBioscience, ThermoFisher) and measured at the Sony SP6800. Data are analysed using FlowJo V.10.9.0.

Statistical analysis

All statistical tests will be performed with a two-sided approach, using a 5% type I error threshold. CIs will be set at 95%, unless otherwise indicated. Missing values will not be imputed. Collected data from patients lost to follow-up will be included in analysis where possible. Variables will be reported as either means with SE of the mean (normal distribution), medians with 95% CI (skewed distribution) or frequencies depending on the type and distribution of the data.

Ethics and dissemination

This study is conducted according to the principles of the Declaration of Helsinki (version October 2013), and in accordance with the Medical Research Involving Human Subjects Act (WMO). This study was approved by the Medical Ethics Committee of Amsterdam UMC under registration number 2021.0774. Eligible women with SLE are given a verbal and written explanation of the study and the procedures involved, by their treating physician. Eligible HCs are asked by their treating physician whether they are interested in participating in the study, and subsequently are given a verbal and written explanation of the study procedures by a member of the research team. Subjects who show interest in participation are contacted by a certified member of the research team. An independent physician is available to answer any question the subject may have. If the subject decides to participate, written informed consent will be obtained before the performance of any study-related activity.

Through identifying mechanisms underlying FaMaLE, this study will identify potential therapeutic targets and biomarkers for the development of pregnancy complications in women with SLE. In the long term, this may improve the development of preventive and therapeutic measures to reduce the incidence of pregnancy complications in SLE. Additionally, these findings may also be relevant to understanding the pathogenesis of pregnancy complications in the general population.

The burden and risks of participation are negligible, considering the placenta is a waste product after childbirth and the blood collection will be performed simultaneously with routine clinical care as much as possible. There is no individual benefit from participation in this study.

Data will be disseminated through peer-reviewed publications in scientific journals, scientific presentations and through articles published in patient magazines and presentations for patient organisations.

Results from additional analyses performed on the stored biomaterials in a later stage, such as single-cell RNA sequencing, will be deposited in the appropriate data repositories (eg, GEO Datasets).

We would like to thank the NVLE and Lupus Europe for their input and support for the FaMaLE study. We also want to thank L. Verleng and Dr. M.L.P van der Hoorn for their advice on placenta dissection. In addition, we acknowledge the European Reference Network on Rare and Complex Connective Tissue Diseases (ERN ReCONNET) for declaring the Department of Rheumatology and Clinical Immunology of our centre as a member.

Data availability statement

Data sharing not applicable as no datasets generated and/or analysed for this study. No datasets were analysed for this study.

Ethics statements

Patient consent for publication

Not applicable.

Ethics approval

This study is conducted according to the principles of the Declaration of Helsinki (version October 2013), and in accordance with the Medical Research Involving Human Subjects Act (WMO). This study was approved by the Medical Ethics Committee of Amsterdam UMC under registration number 2021.0774.

Footnote

Contributors WD, MdB, LGMvB and IEMB designed the study, with input on patient information and study setup from KC and DCR-H. WD and JFvR wrote the manuscript. WD, ARPS and MH designed the study protocols. SAG and MFHMvG collected clinical data. WD is the guarantor of the content of this manuscript. All authors provided input on the manuscript and approved the final version.

Funding This work was supported by research grants from FOREUM Foundation for Research in Rheumatology, Amsterdam UMC and the NVLE Foundation.

Competing interests None declared.

Patient and public involvement Patients and/or the public were involved in the design, conduct, reporting or dissemination plans of this research. Refer to the Methods section for further details.

Provenance and peer review Not commissioned; externally peer reviewed.

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