DNA Amplification in Blastocoel Fluid

February 17, 2024 updated by: Kevin Coetzee, Antalya IVF

Does Absence of DNA Amplification in Blastocoel Fluid Enhance Conventional Blastocyst Selection and IVF Outcome?

Introduction: Although innovative procedural changes in frozen embryo transfer (FET) cycles have increased the implantation rate of blastocysts transferred significantly, blastocyst selection remains a significant limiting factor in implantation outcomes. To improve implantation rates requires conventional microscopic blastocyst morphology scoring/selection technique to be replaced by an enhanced blastocyst selection technique or for the conventional morphology selection technique to be strengthened by novel supplementary selection techniques. Blastocoel fluid biopsy with DNA amplification is a minimally invasive (mi) technique that may supplement a blastocyst morphology score variables with a genetic variable.

Objective: In the present randomized controlled trial (RCT), DNA amplification in blastocoel fluid biopsies (BF-biopsy) will be investigated as a supplementary measure to select blastocysts for transfer in conjunction with blastocyst morphology scores. The objective will be to develop a minimally invasive blastocyst selection technique, which will improve selection and increase clinical implantations, while not increasing costs.

Materials and Methods: A single IVF centre double-blind randomised controlled trial, with patients recruited having female age 18 to 35 years from infertile patients presenting for freeze-all-IVF treatment. Enrolled patients (N = 500) with ≥five 2PN zygotes after ICSI will be randomised (1:1) to the two arms of the trial (i.e., test and control arm). In the test arm, 3 blastocysts will undergo blastocoel fluid biopsy (BF-biopsy) and whole-genomic amplification. Single blastocysts with no DNA amplification will be transferred in FETs of the test arm and single top-scoring blastocysts will be transferred in FETs of the control arm. The primary outcome measure of the trial will be clinical implantation (i.e., gestational sac with fetal heartbeat).

Results: The clinical implantation outcomes of FETs in which score-selected single blastocyst with no DNA amplification and score-selected single blastocysts were transferred will be compared.

Study Overview

Status

Terminated

Conditions

Intervention / Treatment

Detailed Description

Introduction The goal of assisted reproductive technology (ART) is the delivery of a healthy singleton, with the treatment strategy in IVF having the best chance being the transfer of the single most viable embryo from a patient's embryo cohort. Embryo selection, therefore, is paramount to the success of this goal. The microscopic assessment and scoring of embryo morphology was developed as an embryo selection technique in the early years of IVF (Veeck, 1991) and has ever since remained the most widely practised embryo selection technique. However, the technique's limited ability to maximise embryo implantation rates has for long been lamented and, therefore, more enhanced embryo selection techniques have for long been sought. Time-lapse monitoring and scoring (Paulson et al., 2018) and euploid embryo transfer using PGT-A (preimplantation genetic testing for aneuploidy; Gleicher et al., 2017) are recent innovations that have become contenders to replace conventional microscopic assessment and scoring of embryo morphology. An advantage of PGT-A is its independence of embryo development and morphological feature assessments, as euploid blastocysts implant at the same rate irrespective of blastocyst morphology score (Capalbo et al., 2014). Moreover, besides PGT-A's technical and biological limitations, the costs of implementation PGT-A as an embryo selection technique may be regarded prohibitive by many.

In the evolution of PGT in human IVF, PGT has progressed from PGT-v1 to PGT-v2 based on the adverse outcomes of biopsying 1-2 blastomeres from cleavage-stage embryos (PGT-v1) and the improvement in blastocyst development conditions. The evidence suggested that PGT-v.1 posed a significant risk to embryo viability, with no benefit resulting from the transfer of euploid embryos (Gleicher et al., 2017). The majority of studies have reported that PGT-v2, which requires the biopsying of 5-10 trophectoderm (TE) cells, poses no significant risk to the developmental potential (implantation viability) of biopsied blastocysts. However, recently there have been reports, suggesting that TE biopsy may not be completely free of risk (Gleicher et al., 2017, Ozgur et al., 2019). Currently, PGT-A faces four challenges preventing its routine use as a blastocyst selection technique; (1) the need for high technical expertise, (2) the invasiveness of TE biopsy, (3) the cost of the full technique, and (4) that TE biopsy is subject to sampling bias - a single TE biopsy of 5-10 cells may not accurately represent the ploidy of the whole blastocyst.

A spin-off of conventional PGT is the analysis of DNA in blastocoel fluid (BF). This phenomenon was first reported by Palini et al. (2013). The origin of the DNA in BF has been suggested to be the result of embryo euploidisation, i.e., the elimination of aneuploidy DNA or aneuploidic cells through regulatory processes such as apoptosis and or necrosis (Leaver and Wells, 2019). The practice of blastocyst collapse before vitrification (Mukaida et al., 2006, Iwayama et al., 2011) provides the opportunity to perform minimally invasive PGT-A (miPGT-A), i.e., the biopsying BF. In a study to investigate ploidy concordance between PGT-A with TE biopsy and PGT-A with BF biopsy, the authors found that the clinical pregnancy rate was 77% for blastocysts with no DNA amplification were transferred and 37% for blastocysts with DNA amplification (Magli et al., 2018). The mere accumulation of DNA in the BF, therefore, could be used as a supplementary measure to prioritise blastocysts for transfer. Implementing BF-biopsy and whole-genomic amplification (WGA) as a supplementary selection measure limits/reduces three of PGT-A's main challenges; (1) technical expertise, (2) invasiveness, and (3) costs.

In the present randomized controlled trial (RCT), the investigators will investigate DNA amplification in blastocyst fluid biopsies (BF-biopsy) as a supplementary selection technique to select blastocysts for transfer in conjunction with blastocyst morphology scores. The clinical implantation outcomes of FETs in which score-selected single blastocyst with no DNA amplification and score-selected single blastocysts were transferred will be compared.

Study Type

Interventional

Enrollment (Actual)

15

Phase

  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

  • Turkey
      • Antalya, Turkey, 07080
        • Antalya IVF

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

18 years to 35 years (Adult)

Accepts Healthy Volunteers

Yes

Description

Inclusion Criteria:

  • Patients with female age 18≤35 years on the day of consultation (with the clinician projecting female age to be ≤35 on the day of oocyte retrieval).
  • Patients who provide informed consent to participate in the trial and for the use of their anonymized data in research.
  • Patients with ≤2 previous IVF treatments.
  • Patients predicted to have single blastocyst transfers.

Exclusion Criteria:

  • Patients with female age >35 years on the day of consultation.
  • Female patients with insulin-dependent diabetes or non-insulin-dependent diabetes mellitus and female patients with gastrointestinal, cardiovascular, pulmonary, liver or kidney disease.
  • Female patients with any contraindications or allergies to the drugs used in routine freeze-all-IVF.
  • Patients undergoing conventional PGT-A (aneuploidy) or PGT-M (monogenic disorders)
  • Patients with less than 5 2-PN zygotes on day 1 of embryo development will be excluded from randomization.

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Primary Purpose: Treatment
  • Allocation: Randomized
  • Interventional Model: Parallel Assignment
  • Masking: Double

Arms and Interventions

Participant Group / Arm
Intervention / Treatment
Experimental: DNA-amplification selection
In the experimental arm, all blastocysts will undergo routine morphological assessment, with the 3 top-scoring blastocysts undergoing blastocoel fluid biopsy (BF-biopsy) and whole-genomic amplification. A single blastocyst with no DNA amplification will be selected for transfer in a frozen embryo transfer cycle.
Procedure: blastocoel fluid biopsy
In the present study, blastocoel fluid biopsy (BF-biopsy) and collapse will be performed using a microinjection pipette similar to that used to perform ICSI. The pipette will be pushed gently through the zona pellucida and TE, and up to 90% of the BF will be aspirated.
Active Comparator: Morfological-score selection
In the active comparator arm, all blastocysts will undergo routine morphological assessment. The (single) top-scoring blastocyst will be selected for transfer in a frozen embryo transfer cycle.
Procedure: blastocoel fluid biopsy
In the present study, blastocoel fluid biopsy (BF-biopsy) and collapse will be performed using a microinjection pipette similar to that used to perform ICSI. The pipette will be pushed gently through the zona pellucida and TE, and up to 90% of the BF will be aspirated.

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
clinical implantation
Time Frame: Transvaginal ultrasound examinations will be performed after 5 weeks of gestation
Clinical implantation will be defined as a cycle with an ultrasound confirmed normal gestational sac and heartbeat.
Transvaginal ultrasound examinations will be performed after 5 weeks of gestation

Secondary Outcome Measures

Outcome Measure
Measure Description
Time Frame
pregnancy
Time Frame: Blood serum pregnancy tests will be performed 9 days after blastocyst transfer
Pregnancy will be defined as a cycle with an arbitrary serum βHCG level of >30 mIU/mL
Blood serum pregnancy tests will be performed 9 days after blastocyst transfer
ongoing pregnancy
Time Frame: Transvaginal ultrasound examinations will be performed after 12 weeks of gestation
Ongoing pregnancy will be defined as a cycle with an ultrasound confirmed normal gestational sac and heartbeat.
Transvaginal ultrasound examinations will be performed after 12 weeks of gestation

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Antalya IVF

Investigators

  • Study Director: Kemal Ozgur, MD, Antalya IVF
  • Principal Investigator: Kevin Coetzee, PhD, Antalya IVF

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

January 1, 2022

Primary Completion (Actual)

January 31, 2024

Study Completion (Actual)

January 31, 2024

Study Registration Dates

First Submitted

February 4, 2021

First Submitted That Met QC Criteria

February 4, 2021

First Posted (Actual)

February 9, 2021

Study Record Updates

Last Update Posted (Actual)

February 20, 2024

Last Update Submitted That Met QC Criteria

February 17, 2024

Last Verified

February 1, 2024

More Information

Terms related to this study

Keywords

Other Study ID Numbers

  • BF 13.1.2021:51

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

YES

IPD Plan Description

The IPD plan sharing plan:

The protocol will be published (word document) on the completion of registration on ClinicalTrials.gov The relevant clinical data will be published on journal acceptance of the study (excel spreadsheet)

IPD Sharing Time Frame

To be published immediately after publication acceptance.

IPD Sharing Access Criteria

Open access at the Mendeley Data at the URLs provided

IPD Sharing Supporting Information Type

  • STUDY_PROTOCOL
  • CSR

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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