Clinical Trial Details
— Status: Not yet recruiting
Administrative data
| NCT number |
NCT05612620 |
| Other study ID # |
0140-21-AAA |
| Secondary ID |
|
| Status |
Not yet recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
November 14, 2022 |
| Est. completion date |
September 2023 |
Study information
| Verified date |
September 2022 |
| Source |
Assuta Ashdod Hospital |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational [Patient Registry]
|
Clinical Trial Summary
Recurrent pregnancy loss (RPL) is defined as 2 or more consecutive miscarriages1 This
condition affects about 1-3% of couples during their reproductive years. The role of vaginal
infections in RPL is controversial and microbiological screening is not recommended as per
the international guidelines. Current theories suggest that altered vaginal and uterine
microbiota may trigger an inflammatory response in the endometrium even without the presence
of clinical infection which could affect the success of embryo implantation and future
development of pregnancy2 .Changes in the uterine microbiota can lead to chronic endometritis
(CE). This condition is caused by continuing inflammation of the endometrium, involving a
variety of common bacterial and yeast species and has been associated with RPL3 . Notably, CE
can be found in up to 45% of infertile patients4. Current diagnosis of CE is based on
histopathological examination, immunohistochemistry assay for CD138 cells and morphological
appearance on hysteroscopy. While antibiotic treatment can improve ongoing pregnancy rates in
patients with RPL treatment success is still partial and unpredictable. A mechanistic link is
yet to be established between vaginal and uterine microbiota and RPL and it is unknown
whether restoration of the microbiome in patients with RPL can improve pregnancy outcomes.
Description:
Aims The aim of the current study is to evaluate vaginal and intrauterine microbiota in women
with recurrent pregnancy loss (RPL) as compared to normal controls. Ultimately we would like
to assess whether women with RPL have a characteristic microbiome which may be significantly
different microbiota of healthy patients in our study population. This could later lead to
treatment with a tailored microbiota transplantation approach involving healthy vaginal
microbiota for the treatment of patients with RPL and implement it in a prospective
randomized controlled trial in order to modulate the reproductive tract microbiota and
achieve and improved pregnancy outcome.
Background
A rapidly growing field in biologic research of human health and disease is that of
microbiome research. The microbiome inhabiting the female reproductive tract has long been
suspected to play an important role in health and disease. Current theories suggest that
altered microbiota even without clinical infection may trigger an inflammatory response in
the endometrium. It affects the success of embryo implantation and future development of
pregnancy.5 Most studies deal with genital tract microbiome in patients with repeated
implantation failure in IVF, however, some studies have also found altered microbiome in
patients with recurrent pregnancy loss (RPL)6 Vaginal and endometrial microbiotas might be
independent. Endometrial microbiota has a richer alpha diversity than vaginal microbiota, and
dysbiosis of vaginal or endometrial microbiota can cause implantation failure. Unlike
previous reports, Ichiyama et al reports that the Lactobacillus rate in the vagina, but not
the endometrium, may be a biomarker for repeated implantation failure7 Small studies showed
association between spontaneous abortion and abnormal vaginal microbiome( 60% intermediate
flora, 20% BV condition), with higher levels of selected metabolites in the vaginal
environment (e.g., inosine, fumarate, xanthine, benzoate, ascorbate)8 Recurrent pregnancy
loss (RPL) is defined American Society for Reproductive medicine as two or more spontaneous
miscarriages before gestational week 22 (not necessarily consecutive) The role of vaginal
infections in RPL is controversial and screening for infections is not routinely recommended
in patients with RPL9
Major immune cells in the human endometrium include uterine Natural Killer (uNK) cells,
macrophages, dendritic cells (DCs) and T cells. Each of these cell populations demonstrated a
specific role. uNK are involved in the success of implantation and maintenance of pregnancy
through their ability both to interact through inhibitor receptors with HLA-G, HLA-E and
HLA-C expressed on trophoblast cells and to produce angiogenic factors Kuon et al in their
small study described that RPL patients with elevated pNK suffer more often from colonization
by Gardnerella vaginalis and gram-negative anaerobes . This might indicate an association
between the vaginal microbiota, local inflammation, changes in immune parameters and
miscarriage.10 Like the vaginal microbiome, the endometrial microbiome is usually dominated
by Lactobacilli. 11 A recent studies illustrated that the presence of a
non-Lactobacillus-dominating microbiome in endometrial fluid of IVF patients was associated
with significantly lower implantation, pregnancy and live birth rates 12 13 According to
researches microbiota composition in endometrial fluid does not fully reflect that in
endometrial tissue.14 However, only few studies have directly probed the microbes within the
uterine environment and how these microbes could influence in implantation , the majority of
them in IVF cycles.
First trimester miscarriage can be associated with reduced prevalence of Lactobacillus spp.
and with changes in the relative abundance of several bacterial genera, including
Fam_Finegoldia, Lac_Coprococcus_3, and Lac_Roseburia 15 16
Chen W et al showed the relative abundances of Actinobacteria and Fusobacteria in the so
called Chronic endometritis group at the phylum level.At the genus level, high relative
abundances of Gardnerella were observed in the Chronic endometriosis and non-pregnancy
groups, which significantly referred to the negative IVF outcome17
Settings and participants All patients referred to Assuta Ashdod university hospital due to
RPL, meeting the inclusion criteria, will be offered recruitment.
Inclusion criteria include:
• women aged 18-40 years old, with two or more consecutive miscarriages.
Exclusion criteria will include:
- documented past PID,
- previous history of uterine malformations,
- patients with hyperplasia and endometrial cancer,
- patients with IUD and antimicrobial treatment in the preceding 4 weeks.
Study methodology and techniques
Study design
After obtaining informed consent we will collect samples from 4 anatomical locations: lower
third of vagina (before speculum placement), posterior fornix and cervical canal ( after
sterile speculum placement), and a sample of gut microbiome.
Sample size - 20 women for each pathology in the research group and 20 women control group,
this is similar to other studies on the subject 4.
The following data will be collected from medical records:
- age,
- ethnicity,
- BMI,
- Lifestyle: tobacco use, Number of partners in the past 6 months, diet, hygienic
procedures,
- Gynecological and obstetrical history: gravidity, parity, menarche time, regularity of
menstrual cycle,
- Type and duration of contraception,
- Symptoms: pain, menometrorrhagia,
- Sonographic findings,
Samples from the vagina, fornix and cervical canal will be taken by introducing a flocked
nylon swab to the vagina via a speculum and circular swabbing for 5-10 seconds before using
any antiseptics. Thus a total of 160 samples (3 patients groups (n=20*2) * 4 samples) will be
taken.
Samples will be snap frozen at -80c and kept until further analysis. Batched swab specimens
will undergo microbial DNA extraction using standard protocols of the Human Microbiome
Project (HMP). Following extraction, a PCR reaction for 16S RNA gene amplification will be
performed using HMP primers sets. 16S amplicons will be sequenced using an Illumina
next-generation sequencing platforms (PE 250bpX2).
Sequencing data will be analyzed at the Moran-Gilad lab, Ben-Gurion University. The
bioinformatics analysis will be based on the QIIME software package pipeline18 . Briefly,
microbiome data will be analyzed using standard methods for rarefaction, alpha and beta
diversity and abundance of taxa at different taxonomical levels. The ANOVA test will be used
to determine if OTU relative abundance is different between samples, the G test of
independence will be used to determine association of OTU presence/absence with a specific
category, and significant variation between sample groups will be analyzed using DESeq2 R
package 19on the raw non-normalized OTU biom table.
Recruitment strategy
Patients will be recruited from outpatients clinic at the Samson Assuta University Hospital
in accordance with a protocol that is under approval process of the local Institutional
Review Board (IRB).
The investigator will inform the patient about the study, its purpose, and method of
randomization of study groups. Patients will not be cajoled into participating in this study.
The Investigator will discuss foreseeable risks involved, as well as potential benefits, for
each study group. Patients who have consented to having their information obtained during the
study for analysis of the results will have their confidentiality maintained at all times
using a study code as an identifier in the research database. The patients will be informed
by the Investigator that their medical treatment will not be affected in any mean by their
decision whether to participate or not in the study. Patients will be informed that they may
choose to withdraw from the study at any stage without jeopardizing any further medical care.
A signed and dated Informed Consent must be obtained by the Investigator from the patient
prior to enrolment into this study. The original signed and dated information sheet and
patient consent will be kept by the Investigator. A signed copy will be provided to the
patient.
Statistical analysis
Statistical analysis will be performed using R software environment for statistical computing
and graphics. Categorical variable data will be presented using percentile and statistical
significance will be tested using the X² or Fisher's exact test, as appropriate. Ordinal
variable data will be presented using median and inter quartile range and statistical
significance will be analyzed using Mann Whitney test. Continuous variable data will be
presented using mean and standard deviation, Student t test will be used for statistical
analysis
Ethics
This study protocol will be submitted to Assuta Ashdod University Hospital Ethics committee
for approval. The study will be conducted in accordance with the ethical principles
originating from the Declaration of Helsinki and GCP (ICH E6) and in compliance with local
regulatory requirements. Participants' identity will be kept confidential, and to the extent
permitted by the applicable laws and/or regulations, will not be made publicly available. If
the study results are published, the participants' identity will remain confidential.
Informed consent will be obtained in accordance with ICH/GCP Guidelines and the Declaration
of Helsinki and will be implemented before protocol specific procedures are carried out. The
risks and benefits of participating in the study will be verbally explained to each potential
participant prior to signing the consent form in accordance with local regulatory legal
requirements. The signed consent form will be retained by the investigator and a copy
provided to each participant.
Source Documentation
The investigator will keep accurate separate records of all participants' study data
collected, including all pertinent study related information. The investigators are committed
to thorough documentation of all side effects and Adverse Events during the study in a
participant study binder including any diagnostic tests conducted during the study.
All study documentation will be kept for 15 years after all participants have completed and
all data has been collected. The IRB/IEC will be notified in writing of the study completion
and archive site.
