ANZCTR - Registration

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Trial registered on ANZCTR

Registration number

ACTRN12618000339257

Ethics application status

Approved

Date submitted

2/03/2018

Date registered

6/03/2018

Date last updated

1/06/2020

Date data sharing statement initially provided

3/07/2019

Type of registration

Prospectively registered

Titles & IDs

Public title

ECOBABe - Early Colonisation with Bacteria After Birth

Scientific title

Maternal bacteria to correct abnormal gut microbiota in babies born by C-section

Secondary ID [1]

nil

Universal Trial Number (UTN)

U1111-1206-7066

Trial acronym

ECOBABe

Linked study record

Health condition

Health condition(s) or problem(s) studied:

Abnormal gut microbiota in infants born by C-section

Condition category

Condition code

Metabolic and Endocrine

Metabolic disorders

Oral and Gastrointestinal

Other diseases of the mouth, teeth, oesophagus, digestive system including liver and colon

Intervention/exposure

Study type

Interventional

Description of intervention(s) / exposure

Phase 1. The purposes of this phase are to ensure that the gauze incubated in the maternal vagina will contain vaginal bacteria, and to determine the viability of these bacteria.
We will recruit 3 pregnant women aged 18 years and above, who are carrying a singleton fetus and are scheduled for an elective C-section (CS) at 34 to 41 weeks of gestation. Administration of maternal vaginal bacteria to the offspring is not part of Phase 1.
The woman (or the research midwife) will insert a sterile gauze into her vagina for approximately 30 minutes prior to the CS. After removal the gauze will be vertically divided in two equal sized pieces:
• piece one will be placed in a sterile syringe with 5 ml of sterile water to obtain a vaginal-water supernatant
• piece two will be kept at 4ºC in a sterile container
Both the vaginal-water supernatant (obtained from piece one) and the gauze itself (piece two) will undergo: (i) bacterial extraction using the QIAmp DNA extraction kit; and (ii) bacterial characterization by 16S amplicon sequencing. In addition, the vaginal-saline supernatant (obtained from piece one) will undergo: (iii) viability of bacteria using a culture-independent method that directly counts the numbers of live, dead and damaged cells by fluorescent cell sorting (i.e. FACS).

Phase 2. The aim of this phase of the trial is to determine whether babies receiving the maternal vaginal microbiota will have different gut microbiota profile compared to babies receiving placebo, with treated babies more closely resembling the bacterial profile of vaginal-born babies.
We aim to recruit 45 pregnant women and 45 offspring: 30 women carrying singletons (n=30 babies) and scheduled for an elective CS; and 15 women carrying singletons (n=15 babies) and having a vaginal birth (VB). All included women will give birth at 37 weeks of gestation or later. Briefly, approximately 30 minutes before CS, a sterile gauze will be inserted into the maternal vagina by the woman herself or the research midwife. After removal, the gauze will be placed in a sterile syringe with 5 ml of sterile water to obtain a vaginal-water supernatant, which will be kept at 4ºC in a sterile container. Shortly after birth, babies will be randomised to receive either 3 ml of the vaginal-water supernatant or placebo (3 ml of sterile water) orally by syringe. Parents will be blinded to their infant's exposure to maternal vaginal microbiota. The vaginal birth controls will receive neither vaginal-water supernatant nor placebo.

Intervention code [1]

Treatment: Other

Comparator / control treatment

The case group (15 CS-born babies who will receive the vaginal-water supernatant) will be compared to two control groups:
- control group 1: 15 CS-born babies who will receive placebo (3 ml of sterile water orally by syringe); and
- control group 2: 15 vaginal-born babies who will receive neither the vaginal-water supernatant nor placebo.

Control group

Placebo

Outcomes

Primary outcome [1]

Our primary outcome is a difference in the community structure of the gut microbiota between CS babies who received the active treatment (maternal vaginal microbiota) compared to those who received placebo (sterile water) at 1 month of age. This will be assessed using Bray-Curtis dissimilarity and stastically tested by Permutational Multivariate Analysis of Variance (PERMANOVA).

Timepoint [1]

1 month of age

Secondary outcome [1]

Difference in overall gut community structure between CS groups. This will be assessed using Bray-Curtis dissimilarity and stastically tested by Permutational Multivariate Analysis of Variance (PERMANOVA).

Timepoint [1]

24 hours and 3 months

Secondary outcome [2]

Infant anthropometry (weight SDS and length SDS)

Timepoint [2]

At birth, at 1 month, and at 3 months (secondary timepoint) of age

Secondary outcome [3]

Infant body composition (assessed using whole-body dual-energy X-ray absorptiometry -DXA-)

Timepoint [3]

At 3 months (secondary timepoint) of age.

Secondary outcome [4]

Similarity of CS seeded and placebo gut profiles to vaginally born cohort. This will be assessed using Bray-Curtis dissimilarity and statistically tested by Permutational Multivariate Analysis of Variance (PERMANOVA).

Timepoint [4]

At 24 hours, at 1 month, and at 3 months of age.

Secondary outcome [5]

Degree of maternal vaginal strain transfer in CS-born babies. Strain-level identification will be assessed by SNP haplotype based profiling using StrainPhlAn software. The resulting SNP haplotypes will be used to identify the proportion of shared and unrelated strains between mother-infant pairs, comparing these proportions across treatment groups.

Timepoint [5]

At 24 hours, at 1 month, and at 3 months of age.

Eligibility

Key inclusion criteria

Maternal inclusion criteria:
- greater than or equal to 18 years of age
- delivery at greater than or equal to 37 weeks of gestation

Offspring (CS and vaginal) inclusion criteria:
- APGAR score greater or equal to 7 at 5 minutes after birth

Minimum age

0 Years

Maximum age

No limit

Sex

Both males and females

Can healthy volunteers participate?

Key exclusion criteria

Maternal exclusion criteria:
- emergency c-section
- multiple pregnancy
- carrying a fetus with chromosomal/single gene defects or syndromes
- type 1 diabetes, type 2 diabetes, or gestational diabetes
- use of probiotic supplements or antibiotics in the last two weeks of pregnancy
- a history suggestive of chorioamnionitis
- premature rupture of membranes (PROM) and prolonged PROM
- intrapartum fever >38°C
- history of genital herpes (CS group only)
- a previous group B streptococcus (GBS)-infected baby
- GBS bacteriuria of any count or a re-occurring GBS infection during the current pregnancy
- any transmissible viral, bacterial, or protozoan pathogens

Offspring exclusion criteria:
- birth before 37 weeks of gestation
- congenital abnormalities detected at birth
- perinatal asphyxia (5-minute Apgar score <7)
- respiratory distress requiring support/oxygen therapy with subsequent admission to NICU
- respiratory distress requiring support/oxygen therapy without NICU admission, but without approval of attending clinician for study inclusion

Study design

Purpose of the study

Treatment

Allocation to intervention

Randomised controlled trial

Procedure for enrolling a subject and allocating the treatment (allocation concealment procedures)

Randomisation sequences will be computer generated. Participants will be randomised in a 1:1 ratio to either treatment or placebo group, using block randomisation with variable block sizes of 2 and 4. Parents will be blinded to infants' treatment, To maintain the integrity of the trial evaluation, analysis of the primary outcome (i.e. microbial investigations) will be performed on encoded data, so that the researcher(s) who will perform these analyses will be blinded to group allocation.

Methods used to generate the sequence in which subjects will be randomised (sequence generation)

Computer-generated randomisation sequences

Masking / blinding

Blinded (masking used)

Who is / are masked / blinded?

The people receiving the treatment/s

The people analysing the results/data

Intervention assignment

Parallel

Other design features

Phase

Not Applicable

Type of endpoint/s

Efficacy

Statistical methods / analysis

Metagenomic sequencing data will be processed using bioBakery workflows using docker images available at http://huttenhower.sph.harvard.edu/biobakery_workflows. Briefly, sequence data quality control, including removal of any human reads, will be conducted using KneadData. Taxonomic and functional profiles of the microbiota will be generated using MetaPhlAn v2.6 (Segata et al., 2012) and HUMAnN2 (Fransoza et al., 2018) respectively. Additionally, strain-level taxonomic profiling will be achieved by SNP haplotype based profiling using StrainPhlAn software (Truong et al., 2017). The resulting SNP haplotypes will be used to identify the proportion of shared and unrelated strains between mother-infant pairs, comparing these proportions across treatment groups.
Permutational multivariate analysis of variance (PERMANOVA) will be used to identify any significant differences in the overall microbial community structure between treatment groups. Additionally, Multivariate Association with Linear Models (MaAsLin2) will be used to identify any significant associations between specific microbial taxa and treatment groups. Both taxonomic and functional profiles will be compared. Microbiota stability across time points will be assessed using Bray Curtis dissimilarity. Microbial alpha diversity will be assessed using Shannon’s diversity index and gene counts.
Treatment effects on clinical outcomes will be assessed on the principle of intention to treat, using data collected from all randomised participants. Generalised linear models will be used, adjusting for the baseline outcome value (where appropriate) and sex, and maybe gestational age. Model-adjusted estimates and the differences among the three groups will be calculated and tested. Planned subgroup analyses by sex will be conducted on primary and secondary outcomes to evaluate the consistency of possible treatment effects in males and females. Data analyses will be performed in SAS v.9.4, SPSS v26, and/or Minitab v16. Statistical tests will be two-tailed and significance maintained at 5% level.

References:
Segata N, Waldron L, Ballarini A, Narasimhan V, Jousson O, Huttenhower C. Metagenomic microbial community profiling using unique clade-specific marker genes. Nature methods. 2012;9(8):811-814.
Franzosa EA, McIver LJ, Rahnavard G, et al. Species-level functional profiling of metagenomes and metatranscriptomes. Nature methods. 2018;15(11):962-968.
Truong DT, Tett A, Pasolli E, Huttenhower C, Segata N. Microbial strain-level population structure and genetic diversity from metagenomes. Genome research. 2017;27(4):626-638.

Recruitment

Recruitment status

Stopped early

Data analysis

No data analysis planned

Reason for early stopping/withdrawal

Other reasons/comments

Other reasons

Recruitment has stopped because of the NZ government imposed national lockdown (i.e. mandatory physical isolation) as a result of the Covid-19 outbreak.

Date of first participant enrolment

Anticipated

3/09/2018

Actual

3/12/2018

Date of last participant enrolment

Anticipated

31/03/2020

Actual

16/03/2020

Date of last data collection

Anticipated

30/06/2020

Actual

Sample size

Target

Accrual to date

Final

Recruitment outside Australia

Country [1]

New Zealand

State/province [1]

Auckland

Funding & Sponsors

Funding source category [1]

Government body

Name [1]

Health Research Council (HRC) of New Zealand

Address [1]

PO Box 5541, Wellesley Street, Auckland 1141

Country [1]

New Zealand

Primary sponsor type

University

Name

University of Auckland

Address

Liggins Institute
University of Auckland
85 Park Road
Grafton 1023
Auckland
New Zealand

Country

New Zealand

Secondary sponsor category [1]

None

Name [1]

n/a

Address [1]

n/a

Country [1]

Ethics approval

Ethics application status

Approved

Ethics committee name [1]

Health and Disability Ethics Committees (HDEC)

Ethics committee address [1]

Ministry of Health
Health and Disability Ethics Committees
PO Box 5013
Wellington 6140
New Zealand

Ethics committee country [1]

New Zealand

Date submitted for ethics approval [1]

29/03/2018

Approval date [1]

31/05/2018

Ethics approval number [1]

18/NTA/49

Summary

Brief summary

HYPOTHESIS - Oral administration of maternal vaginal microbiota in babies born by c-section (CS) will alter their gut microbiota so that they more closely resemble that of vaginally-born babies.
AIMS - To explore whether oral administration of maternal vaginal microbiota in babies born by CS will: i) change the overall gut microbiota community structure, to more closely approximate gut microbiota of vaginally-born babies and ii) lead to strain transfer from mother to baby.
BACKGROUND - While CS may cause acute complications, an increased risk of obesity and immune disorders in the offspring has also been identified. This association may be driven by disruption of the gut microbiota, which is essential for many host physiological processes. Contact with maternal vaginal microbes during vaginal delivery is a critical source of symbiotic gut bacteria, which appear to be protective against inflammation and obesity. Thus, we aim to perform a proof-of-concept, pilot RCT to assess the effectiveness of oral administration of maternal vaginal bacteria to restore the gut microbiota in CS-born babies.
METHODS
*Phase 1. To ensure that the gauze incubated in the maternal vagina contains vaginal bacteria, and to determine the viability of these bacteria. We will recruit 3 pregnant women aged 18 to 42 years, carrying singletons and scheduled for elective CS at 34 to 41 weeks of gestation. A sterile gauze will be inserted in the vagina for 30 minutes prior to CS. After removal, the gauze will undergo bacterial extraction using the QIAmp DNA extraction kit, and bacterial characterization by 16S amplicon sequencing.
Phase 2
Aim - To determine whether babies receiving the maternal vaginal microbiota will have different gut microbiota profile compared to babies receiving placebo, with treated babies more closely resembling the bacterial profile of singletons vaginally-delivered.
Participants - 30 women carrying singletons and scheduled for an elective CS; and 15 women carrying singletons born vaginally. Women will be aged greater than or equal to 18 years and will deliver at greater than or equal to 37 weeks of gestation.
Procedures - 30 minutes before CS, a sterile gauze will be inserted into the maternal vagina, and kept in place for 30 minutes. After removal, the gauze will be placed in a sterile syringe with 5 ml of sterile water to obtain a vaginal-water supernatant, Soon after birth, some babies will receive 3ml of the vaginal-water supernatant orally, while others will receive placebo (3 ml of sterile water). The vaginally-born controls will not receive vaginal-water supernatant nor placebo. Infants will be assessed once within 24 hours of life, and subsequently at 1 month and 3 months of age (auxology and stools). Infant stool samples, as well as the remaining 2 ml of vaginal supernatant from CS-mothers will undergo whole metagenomic shotgun sequencing to assess gut microbiota composition and quantify strain transmission from mother to baby.

Trial website

https://www.auckland.ac.nz/en/liggins/in-the-community/clinical-studies/clinical-studies-pregnancy/ecobabe-study.html

Trial related presentations / publications

n/a

Public notes

Our original study protocol was approved by HDEC on 31/5/2018. However, a number of amendments have been made, notably the replacement of twin pregnancies with singleton pregnancies for the c-section groups. All amendments made to our study protocol have since been approved by HDEC, the latest one on 16/5/2019 with approval number 18/NTA/49/AM04.

Contacts

Principal investigator

Name

Prof Wayne Cutfield

Address

Liggins Institute
University of Auckland
85 Park Road
Grafton
Auckland 1023
New Zealand

Country

New Zealand

Phone

+64 9 3737599 Ext 84476

Fax

[email protected]

Contact person for public queries

Name

Prof Wayne Cutfield

Address

Liggins Institute
University of Auckland
85 Park Road
Grafton
Auckland 1023
New Zealand

Country

New Zealand

Phone

+64 9 3737599 Ext 84476

Fax

[email protected]

Contact person for scientific queries

Name

Prof Wayne Cutfield

Address

Liggins Institute
University of Auckland
85 Park Road
Grafton
Auckland 1023
New Zealand

Country

New Zealand

Phone

+64 9 3737599 Ext 84476

Fax

[email protected]

Data sharing statement

Will individual participant data (IPD) for this trial be available (including data dictionaries)?

No/undecided IPD sharing reason/comment

The clinical data cannot be made available in a public repository due to strict conditions of the ethics approval of our study. We plan to make the study protocol publicly available through its publication in an open access journal. Further, anonymized and unidentifiable data, as well as study documents and materials could be made available to other investigators upon request to the principal investigator.

What supporting documents are/will be available?

Doc. No.	Type	Email
2740	Study protocol	[email protected]
2741	Statistical analysis plan	[email protected]
2742	Informed consent form	[email protected]
2743	Ethical approval	[email protected]
2744	Analytic code	[email protected]

Results publications and other study-related documents

Documents added manually

No documents have been uploaded by study researchers.

Documents added automatically

Source	Title	Year of Publication	DOI
Embase	Oral administration of maternal vaginal microbes at birth to restore gut microbiome development in infants born by caesarean section: A pilot randomised placebo-controlled trial.	2021	https://dx.doi.org/10.1016/j.ebiom.2021.103443
Dimensions AI	An update on the current understanding of the infant skin microbiome and research challenges	2023	https://doi.org/10.1016/j.mib.2023.102364

N.B. These documents automatically identified may not have been verified by the study sponsor.

Trial Review

Documents added manually

Documents added automatically