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Trial registered on ANZCTR


Registration number
ACTRN12623000951651
Ethics application status
Approved
Date submitted
11/08/2023
Date registered
4/09/2023
Date last updated
4/09/2023
Date data sharing statement initially provided
4/09/2023
Type of registration
Prospectively registered

Titles & IDs
Public title
Pregnancy Immunobiology Study
Scientific title
The immunobiology of pregnancy, and impacts in women with Multiple Sclerosis (MS) or Neuromyelitis Optica Spectrum Disorder (NMOSD)
Secondary ID [1] 310294 0
None
Universal Trial Number (UTN)
Trial acronym
PREGMOL
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Multiple Sclerosis 330996 0
Neuromyelitis Optica Spectrum Disorder 330997 0
Condition category
Condition code
Neurological 327810 327810 0 0
Multiple sclerosis
Neurological 327811 327811 0 0
Other neurological disorders

Intervention/exposure
Study type
Observational
Patient registry
False
Target follow-up duration
Target follow-up type
Description of intervention(s) / exposure
The study aims to characterise the hormonal, cellular and epigenetic profiles of healthy women and women with MS or NMOSD before, during and after pregnancy. Our study seeks to elucidate the molecular mechanisms behind the potential long-term benefit of pregnancy in women with MS and the potential drawbacks in the context of NMOSD. This exploration may contribute to a deeper understanding of the pathogenesis and pathophysiology of MS or NMOSD, and offer novel therapeutic targets within the epigenome.
During the screening process, participants will be requested to sign the Patient Informed Consent Form and complete questionnaires concerning significant environmental and clinical factors that could impact the epigenome. Subsequently, they will be asked to provide blood samples at various timepoints and to recomplete the questionnaire. These assessments are scheduled for the 1st trimester (10-14 weeks) of pregnancy, the 3rd trimester (32-38 weeks), 3months after giving birth, 1 year, and 3 years. Study nurses will collect blood samples, which will then be transferred to Monash facilities for processing. Plasma, serum, immune cell subpopulations, DNA and RNA will be extracted from whole blood samples.
For women affected by MS or NMOSD, we will request access to routine MRIs that are conducted as a part of regular care at different timepoints. This will allow us to investigate the impact of pregnancy on MRI outcomes.
Participants are enrolled prospectively into four distinct study arms. Arms A and B comprise non-MS controls: a cohort of non-MS nulligravida women (Arm A) and non-MS primigravida women (Arm B). MS-affected primigravida women constitute Arm C, while MS-affected nulligravida women form Arm D.
Intervention code [1] 326753 0
Diagnosis / Prognosis
Comparator / control treatment
The comparator/control treatment strategy for this study involves the establishment of distinct study arms for comparative analysis. Given that women in the general population do not typically attend obstetric clinics prior to falling pregnant, unless presenting complex cases, a precautionary approach is taken to mitigate confounding variables stemming from intricate pregnancy scenarios. To achieve this, we will recruit a dedicated cohort of healthy nulligravida women (Arm A) to serve as a foundational baseline for comparison with our primigravida healthy control group Arm B. Importantly, these women will not be subject to regular monthly follow-ups or SMS reminders when they are not pregnant or before they conceive. Instead, it will be at their discretion to inform us if they become pregnant along the study. In such a case, they will transition into the primigravida healthy control (Arm B). These carefully designed arms facilitate a meaningful comparison against both the MS-affected primigravida women (Arm C) and the MS-affected nulligravida women (Arm D), enabling us to isolate and comprehend the specific effects of MS or NMOSD on pregnancy-associated molecular changes.
Control group
Active

Outcomes
Primary outcome [1] 335725 0
Characterisation of cellular milieu through immunophenotyping of blood samples at each designated timepoint. This will be done through comprehensive high dimentional spectral flow cytometry analysis pipeline.
Timepoint [1] 335725 0
The timing of these tests would be as follows:
- pre-pregnancy baseline (<=12m prior to conception)
- 1st trimester (10-14 weeks) of pregnancy
- 3rd trimester (32-38 weeks) of pregnancy
- 3 months after giving birth
- 1 year after giving birth
- 3 years after giving birth
Primary outcome [2] 335727 0
Assess changes in epigenetic signatures from a nulligravida baseline through pregnancy and postpartum. Epigenetic alterations will include DNA methylation and other mechanisms. This analysis will be conducted through the collection of blood samples at various timepoints and the administration of relevant questionnaires.
Timepoint [2] 335727 0
The timing of these tests would be as follows:
- pre-pregnancy baseline (<=12m prior to conception)
- 1st trimester (10-14 weeks) of pregnancy
- 3rd trimester (32-38 weeks) of pregnancy
- 3 months after giving birth
- 1 year after giving birth
- 3 years after giving birth
Primary outcome [3] 335728 0
Investigate hormone concentration changes during pregnancy in women with MS using blood samples and compare these between who do and do not relapse during pregnancy and postpartum. Hormones assessment to include: estrogen (E2, E3), progesterone (P4), testosterone, anti-mullerian hormone (AMH), luteinizing hormone (LH), and follicle stimulating hormone (FSH).
Timepoint [3] 335728 0
The timing of these tests would be as follows:
- pre-pregnancy baseline (<=12m prior to conception)
- 1st trimester (10-14 weeks) of pregnancy
- 3rd trimester (32-38 weeks) of pregnancy
- 3 months after giving birth
- 1 year after giving birth
- 3 years after giving birth
Secondary outcome [1] 425349 0
Determine how epigenetic changes impact clinical assessments in women with MS and NMOSD. This analysis will be conducted using medical history reviews.
Timepoint [1] 425349 0
The timing of these tests would be as follows:
- pre-pregnancy baseline (<=12m prior to conception)
- 1st trimester (10-14 weeks) of pregnancy
- 3rd trimester (32-38 weeks) of pregnancy
- 3 months after giving birth
- 1 year after giving birth
- 3 years after giving birth
Secondary outcome [2] 425353 0
Determine how epigenetic changes impact radiological assessments in women with MS and NMOSD. This analysis will be conducted using routine and research MRI scans.
Timepoint [2] 425353 0
The timing of these tests would be as follows:
- pre-pregnancy baseline (<=12m prior to conception)
- 1st trimester (10-14 weeks) of pregnancy
- 3 months after giving birth
- 1 year after giving birth
- 3 years after giving birth
Secondary outcome [3] 425407 0
Characterise the gene expression profiles associated with epigenetic signatures in healthy women, as well as those with MS and NMOSD. This analysis will be achieved through the examination of transcriptomic data and methylation arrays derived from peripheral blood samples, utilising advanced sequencing techniques.
Timepoint [3] 425407 0
The timing of these tests would be as follows:
- pre-pregnancy baseline (<=12m prior to conception)
- 1st trimester (10-14 weeks) of pregnancy
- 3rd trimester (32-38 weeks) of pregnancy
- 3 months after giving birth
- 1 year after giving birth
- 3 years after giving birth
Secondary outcome [4] 425408 0
Quantify and compare cytokine levels in healthy individuals, women with MS, and women with NMOSD. This will involve measuring concentrations in blood samples using enzyme-linked immunosorbent assay (ELISA) or similar quantitative methods.
Timepoint [4] 425408 0
The timing of these tests would be as follows:
- pre-pregnancy baseline (<=12m prior to conception)
- 1st trimester (10-14 weeks) of pregnancy
- 3rd trimester (32-38 weeks) of pregnancy
- 3 months after giving birth
- 1 year after giving birth
- 3 years after giving birth
Secondary outcome [5] 426213 0
Quantify and compare hormone levels in healthy individuals, women with MS, and women with NMOSD. This will involve measuring concentrations in blood samples using enzyme-linked immunosorbent assay (ELISA) or similar quantitative methods.
Timepoint [5] 426213 0
The timing of these tests would be as follows:
- pre-pregnancy baseline (<=12m prior to conception)
- 1st trimester (10-14 weeks) of pregnancy
- 3rd trimester (32-38 weeks) of pregnancy
- 3 months after giving birth
- 1 year after giving birth
- 3 years after giving birth
Secondary outcome [6] 426214 0
Quantify and compare neurofilament levels in healthy individuals, women with MS, and women with NMOSD. This will involve measuring concentrations in blood samples using enzyme-linked immunosorbent assay (ELISA) or similar quantitative methods.
Timepoint [6] 426214 0
The timing of these tests would be as follows:
- pre-pregnancy baseline (<=12m prior to conception)
- 1st trimester (10-14 weeks) of pregnancy
- 3rd trimester (32-38 weeks) of pregnancy
- 3 months after giving birth
- 1 year after giving birth
- 3 years after giving birth
Secondary outcome [7] 426215 0
Measure intrapartum serum neurofilament light chain (sNfL) concentrations and postpartum radiological data (brain volume, T2 lesion volume, T1 hyper intense lesion volume, slowly expanding lesion number) in women with MS and NMOSD.
Timepoint [7] 426215 0
The timing of these tests would be as follows:
- pre-pregnancy baseline (<=12m prior to conception)
- 1st trimester (10-14 weeks) of pregnancy
- 3rd trimester (32-38 weeks) of pregnancy
- 3 months after giving birth

Eligibility
Key inclusion criteria
- females
- women with clinically definite MS or NMOSD
- nulligravida healthy women
- primigravida healthy women
- eligible for Medicare
Minimum age
18 Years
Maximum age
45 Years
Sex
Females
Can healthy volunteers participate?
Yes
Key exclusion criteria
- unable or unwilling to give informed consent
- has another neuroimmunological condition
- is not nulligravida/primigravida

Study design
Purpose
Natural history
Duration
Longitudinal
Selection
Defined population
Timing
Prospective
Statistical methods / analysis
The study requires a minimum of 120 participants (30 women per arm), with a risk mitigation strategy involving the recruitment of 249 women (169 with MS, 80 healthy controls).

Methylation:
Power calculations aim to capture at least 30 successful pregnancies per category, ensuring 80% power to detect differences between groups. Using EPIC array recommendations, the study cohort has 80% power to detect methylation changes of 5% and 10% for 125k and 400k CpGs at a genome-wide significance level. Methylation analysis will employ Illumina EPIC beadchips, with preprocessing using ChAMP for quality control, normalisation, and batch effect correction. Methylation data will undergo statistical modeling using a general linear mixed model (GLMM) for within and between-group comparisons. confounding factors will be managed by matching participants based on age, BMI, disease-modifying therapy, and MS disability. Hormonal changes and pregnancy outcomes will also be considered, adjusting for impacts on DNA methylation.

Radiological outcomes:
Predictors of pregnancy-related radiological changes at 1-year postpartum will be analysed using mixed effects linear or logistic regression (depending on data distribution) with a random effect for the individual, and time modelled as a fixed effect. Models will be adjusted for age, pre-pregnancy annualised relapse rate, baseline and third trimester sex steroid concentrations, therapy type, therapy exposure duration in pregnancy, EDSS disability at pregnancy baseline, relapse during pregnancy, breastfeeding, and perceived stress score.

sNfL analyses: sNfL levels will be adjusted for age and BMI and Z scores derived as described by Benkert (Lancet Neurol 21:246-257). Multivariable linear mixed-effects models with a random intercept for the patient, and adjusted for MS immunotherapy use, pregnancy status, relevant comorbid conditions, and sex steroid concentrations will be used to assess associations between MRI metrics of interest (T2 lesion number and volume, whole brain and regional atrophy) and sNfL concentrations.

Association between biological variables and clinical outcomes:
To identify biomarker signatures associated with quiescence, relapse, disability progression (measured using the EDSS score) together with MRI metrics including slowly expanding lesions, whole brain and regional atrophy, we will build per-outcome models using an Elastic Net machine learning approach. Briefly, elastic net regression is useful for uncovering multiple conjoint effects in datasets with correlated features (e.g. hormone concentrations, immune phenotypes, methylation or protein expression changes), and a greater number of features than samples (p>>>n). This method can be useful for identifying important biological signatures with greater sensitivity than conventional proteomic analyses. We will split our data into training (70%) and testing sets (30%) to reduce model overfitting. Training will involve “leave one out cross-validation (LOOCV)” resampling to allow the use of data from all timepoints, without violating a key assumption of elastic net regression, that all features are independent. Our ML models will be optimized, and final elastic net regression models applied to our testing sets.


Recruitment
Recruitment status
Not yet recruiting
Date of first participant enrolment
Anticipated
3/10/2023
Actual
Date of last participant enrolment
Anticipated
3/10/2024
Actual
Date of last data collection
Anticipated
31/10/2028
Actual
Sample size
Target
249
Accrual to date
Final
Recruitment in Australia
Recruitment state(s)
NSW,QLD,TAS,WA,VIC
Recruitment hospital [1] 25359 0
Monash Medical Centre - Clayton campus - Clayton
Recruitment hospital [2] 25360 0
Box Hill Hospital - Box Hill
Recruitment hospital [3] 25361 0
The Alfred - Melbourne
Recruitment hospital [4] 25362 0
John Hunter Hospital - New Lambton
Recruitment hospital [5] 25363 0
Perron Institute for Neurological and Translational Science - Nedlands
Recruitment hospital [6] 25364 0
Royal Brisbane & Womens Hospital - Herston
Recruitment hospital [7] 25369 0
Royal Hobart Hospital - Hobart
Recruitment postcode(s) [1] 41091 0
2305 - New Lambton
Recruitment postcode(s) [2] 41090 0
3004 - Melbourne
Recruitment postcode(s) [3] 41089 0
3128 - Box Hill
Recruitment postcode(s) [4] 41088 0
3168 - Clayton
Recruitment postcode(s) [5] 41093 0
4029 - Herston
Recruitment postcode(s) [6] 41092 0
6009 - Nedlands
Recruitment postcode(s) [7] 41098 0
7000 - Hobart

Funding & Sponsors
Funding source category [1] 314567 0
University
Name [1] 314567 0
Monash University
Country [1] 314567 0
Australia
Funding source category [2] 314568 0
Commercial sector/Industry
Name [2] 314568 0
F. Hoffmann-La Roche Ltd
Country [2] 314568 0
Switzerland
Primary sponsor type
University
Name
Monash University
Address
The Alfred Centre
Level 6
99 Commercial Road
Melbourne VIC 3004
Country
Australia
Secondary sponsor category [1] 316533 0
None
Name [1] 316533 0
Address [1] 316533 0
Country [1] 316533 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 313549 0
Alfred Hospital Ethics Committee
Ethics committee address [1] 313549 0
55 Commercial Road
Melbourne VIC 3004
Australia
Ethics committee country [1] 313549 0
Australia
Date submitted for ethics approval [1] 313549 0
29/03/2023
Approval date [1] 313549 0
12/05/2023
Ethics approval number [1] 313549 0
HREC/72291/Alfred-2021-264207 Local reference: 197/21

Summary
Brief summary
The study aims to understand how pregnancy affects women with Multiple Sclerosis (MS) or Neuromyelitis Optica Spectrum Disorder (NMOSD). In the past, pregnancy was thought to worsen MS, but a 1990s study contradicted this, revealing reduced relapses during pregnancy followed by increased postpartum relapses, especially in severe cases. Recent research, including CI Jokubaitis’ work, suggests that pregnancy might delay MS onset and diminish long-term disability, as symptoms often diminish or entirely disappear during pregnancy.
This study delves into the biological changes during pregnancy in women with MS and NMOSD. A central hypothesis is that epigenetic factors, like altered DNA methylation, underlie enhanced disability outcomes in MS. Additionally, the study explores the role of natural killer (NK) cells in mediating these improvements.
The study has several key goals: revealing cellular and molecular changes, which involves comparing immune cells and genomic and proteomic markers in pregnant women with MS and a non-MS population; identifying relapse biomarkers by investigating whether ratios of immune cells and hormone levels (estrogen, progesterone) correlate with clinical and MRI outcomes during pregnancy and postpartum. Understanding improved disability outcomes by analysing molecular shifts during pregnancy in women with MS and NMOSD with improved disability outcomes versus those without.
The study enrols participants into four groups: non-MS controls, healthy pregnant women, nulligravida MS (or NMOSD) patients, and pregnant MS (or NMOSD) patients. By comparing these groups, researchers aim to unravel the complex connections between pregnancy, immune changes, and the progression of MS/NMOSD. MS and NMOSD women will be analysed separately.
This study holds the promise of shedding light on the potential benefits of pregnancy for women with MS/NMOSD and providing fresh insights into new therapeutic approaches.
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 128510 0
A/Prof Vilija Jokubaitis
Address 128510 0
Department of Neuroscience - Central Clinical SchoolMonash University - The Alfred CentreLevel 699 Commercial RoadMelbourne VIC 3004Australia
Country 128510 0
Australia
Phone 128510 0
+61 3 9903 0880
Fax 128510 0
Email 128510 0
vilija.jokubaitis@monash.edu
Contact person for public queries
Name 128511 0
Dr Gerard Loquet
Address 128511 0
Department of Neuroscience - Central Clinical SchoolMonash University - The Alfred CentreLevel 699 Commercial RoadMelbourne VIC 3004Australia
Country 128511 0
Australia
Phone 128511 0
+61 3 9903 8271
Fax 128511 0
Email 128511 0
gerard.loquet@monash.edu
Contact person for scientific queries
Name 128512 0
A/Prof Vilija Jokubaitis
Address 128512 0
Department of Neuroscience - Central Clinical SchoolMonash University - The Alfred CentreLevel 699 Commercial RoadMelbourne VIC 3004Australia
Country 128512 0
Australia
Phone 128512 0
+61 3 9903 0880
Fax 128512 0
Email 128512 0
vilija.jokubaitis@monash.edu

Data sharing statement
Will individual participant data (IPD) for this trial be available (including data dictionaries)?
No
No/undecided IPD sharing reason/comment
Due to privacy, confidentiality and ethical considerations, no line-by-line individual participant data will be accessible upon request. This is essential to uphold sensitive information and ethical standards.


What supporting documents are/will be available?

No Supporting Document Provided



Results publications and other study-related documents

Documents added manually
No documents have been uploaded by study researchers.

Documents added automatically
No additional documents have been identified.