DNA Amplification in Blastocoel Fluid

Embryo Implantation Clinical Trial

Official title:

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

Clinical Trial Details — Status: Terminated

Administrative data

• NCT number: NCT04744844
• Other study ID #: BF 13.1.2021:51
• Status: Terminated
• Phase: N/A
• Start date: January 1, 2022
• Est. completion date: January 31, 2024

Study information

• Verified date: February 2024
• Source: Antalya IVF
• Contact: n/a
• Is FDA regulated: No
• Study type: Interventional

Clinical Trial Summary

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.

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.

Recruitment information / eligibility

• Status: Terminated
• Enrollment: 15
• Est. completion date: January 31, 2024
• Est. primary completion date: January 31, 2024
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: Female
• Age group: 18 Years to 35 Years

Eligibility

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.

Related Conditions & MeSH terms

• Clinical Pregnancy
• Embryo Implantation

Intervention

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.

Locations

Country	Name	City	State
Turkey	Antalya IVF	Antalya

Sponsors (1)

Lead Sponsor	Collaborator
Antalya IVF

Country where clinical trial is conducted

Turkey, 

References & Publications (9)

Capalbo A, Rienzi L, Cimadomo D, Maggiulli R, Elliott T, Wright G, Nagy ZP, Ubaldi FM. Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts. Hum Reprod. 201 — View Citation

Gleicher N, Metzger J, Croft G, Kushnir VA, Albertini DF, Barad DH. A single trophectoderm biopsy at blastocyst stage is mathematically unable to determine embryo ploidy accurately enough for clinical use. Reprod Biol Endocrinol. 2017 Apr 27;15(1):33. doi — View Citation

Iwayama H, Hochi S, Yamashita M. In vitro and in vivo viability of human blastocysts collapsed by laser pulse or osmotic shock prior to vitrification. J Assist Reprod Genet. 2011 Apr;28(4):355-61. doi: 10.1007/s10815-010-9522-4. Epub 2010 Dec 9. — View Citation

Magli MC, Albanese C, Crippa A, Tabanelli C, Ferraretti AP, Gianaroli L. Deoxyribonucleic acid detection in blastocoelic fluid: a new predictor of embryo ploidy and viable pregnancy. Fertil Steril. 2019 Jan;111(1):77-85. doi: 10.1016/j.fertnstert.2018.09. — View Citation

Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod. 2 — View Citation

Ozgur K, Berkkanoglu M, Bulut H, Yoruk GDA, Candurmaz NN, Coetzee K. Single best euploid versus single best unknown-ploidy blastocyst frozen embryo transfers: a randomized controlled trial. J Assist Reprod Genet. 2019 Apr;36(4):629-636. doi: 10.1007/s1081 — View Citation

Palini S, Galluzzi L, De Stefani S, Bianchi M, Wells D, Magnani M, Bulletti C. Genomic DNA in human blastocoele fluid. Reprod Biomed Online. 2013 Jun;26(6):603-10. doi: 10.1016/j.rbmo.2013.02.012. Epub 2013 Mar 13. — View Citation

Paulson RJ, Reichman DE, Zaninovic N, Goodman LR, Racowsky C. Time-lapse imaging: clearly useful to both laboratory personnel and patient outcomes versus just because we can doesn't mean we should. Fertil Steril. 2018 Apr;109(4):584-591. doi: 10.1016/j.fe — View Citation

Veeck LL: Atlas of the Human Oocytes and Early Conceptus. 1991, Vol. 2. Baltimore, Williams & Willkins Co.

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	clinical implantation	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	pregnancy	Pregnancy will be defined as a cycle with an arbitrary serum ßHCG level of >30 mIU/mL	Blood serum pregnancy tests will be performed 9 days after blastocyst transfer
Secondary	ongoing pregnancy	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