Day-4 Embryo Defragmentation: Blastocyst Rate and clInical Outcomes

NCT07500337 | Recruiting

• 1 other identifier: 205/CE/2025
• Study Type: interventional
• Target: 320
• Locations: 1 country
• Sites: 1

Brief Summary

Background: Embryo fragmentation is one of the main morphological parameters assessed during in vitro culture in assisted reproductive technology (ART). The presence of anucleate cytoplasmic fragments is commonly observed in human embryos and may negatively affect developmental potential and clinical outcomes. Embryo defragmentation at early stages (Day 2-3) is an established technique in some centers, but evidence remains heterogeneous. Defragmentation at Day 4 (morula/compaction stage) represents a significantly less explored area, with promising but insufficient data to guide clinical practice. Study Objective: This study aims to evaluate whether mechanical or laser-assisted embryo defragmentation performed on Day 4 (D4) of in vitro culture improves blastocyst development rates and clinical outcomes in ART cycles, compared to standard culture without intervention. Study Design: This is a prospective, randomized controlled trial (RCT) with single-blind assessment. Patients undergoing IVF/ICSI with embryos showing ≥10% fragmentation at D4 morphological evaluation will be randomly assigned (1:1 ratio) to one of two groups: Group A (Intervention): Mechanical/laser defragmentation at D4, followed by standard blastocyst culture Group B (Control): Standard blastocyst culture without any additional manipulation Randomization will be performed at the patient level using pre-generated block randomization lists, stratified by patient age (\<35 vs. ≥35 years), number of fragmented embryos at D4, and use of preimplantation genetic testing (PGT-A). Participants: Women aged 18-43 years undergoing IVF/ICSI cycles, with at least one embryo showing ≥10% fragmentation at D4 and destined for blastocyst culture. Key exclusion criteria include: donor gamete cycles, PGT-M as primary indication, embryos with \>50% fragmentation, or severe morphological compromise at Day 3. Primary Outcome: Rate of usable blastocysts (Gardner score ≥3BB) per embryo included in the study, assessed at Day 5 and Day 6 of culture. Secondary Outcomes: Overall blastocyst development rate (D5/D6), Gardner score distribution, blastocyst cryopreservation rate, implantation rate, clinical pregnancy rate (heartbeat at 7 weeks), ongoing pregnancy rate (beyond 12 weeks), live birth rate per transfer, and morphokinetic analysis (if time-lapse incubator available). Sample Size: Approximately 240 patients total (120 per arm), based on an expected blastocyst rate of \~42% in the control group vs. \~57% in the intervention group (15% absolute difference), with 80% power and α=0.05. A 15% dropout correction is applied. Duration: 6 months of enrollment plus 6 months of clinical follow-up (total \~12 months).

Conditions

Female Infertility; Embryo Development; In Vitro Fertilization

Keywords

Embryo defragmentation; Blastocyst development; Embryo fragmentation; ICSI; Embryo culture; Randomized controlled trial

Outcome Measures

Primary Outcomes (1)

• Clinical pregnancy rate

  Rate of positive beta-hCG tests 12-14 days after single blastocyst transfer, confirmed by transvaginal ultrasound showing gestational sac and fetal viability at 4 weeks post-transfer.

  4 weeks post-embryo transfer

Secondary Outcomes (1)

• Blastocyst formation rate

  Day 5 and Day 6 post-fertilization

Study Arms (3)

D4 Non-Defragmented Group

SHAM COMPARATOR

Embryos with cytoplasmic fragmentation ≥10% at Day 4 morphological assessment, randomized to standard blastocyst culture without any additional manipulation. Day 4 assessment is performed under the same conditions as the intervention group to ensure comparability of culture conditions.

Other: Standard embryo culture without intervention

D4 Defragmented Group

EXPERIMENTAL

Embryos with cytoplasmic fragmentation ≥10% at Day 4 morphological assessment, randomized to undergo mechanical or laser-assisted defragmentation on Day 4 (96 ± 2 hours post-fertilization). Following defragmentation, embryos are returned to standard blastocyst culture conditions until Day 5/Day 6 assessment.

Procedure: Mechanical embryo defragmentation at Day 4 (morula stage)

Control Group (Low Fragmentation)

ACTIVE COMPARATOR

Embryos with cytoplasmic fragmentation \<10% at Day 4 morphological assessment, cultured under standard conditions without intervention. This arm serves as a biological reference benchmark to evaluate whether defragmentation in Group A restores developmental competence to levels observed in low-fragmentation embryos.

Other: Standard embryo culture without intervention

Interventions

Mechanical embryo defragmentation at Day 4 (morula stage) PROCEDURE

Microsurgical aspiration of cytoplasmic fragments from morulae with ≥10% fragmentation, performed on Day 4 of embryo development (96 ± 2 hours post-ICSI), when blastomeres are already compacting and fragments are clearly distinguishable from the compact cell mass. A diode laser (1.48 µm wavelength) is used to breach the zona pellucida, followed by fragment aspiration using a 35 µm biopsy needle mounted on a hydraulic oil microinjection system. Fragments of softer consistency are aspirated directly; harder fragments are anchored with the needle spike and mechanically extracted. The procedure is performed on a heated stage at 37°C using HEPES-buffered medium (G-MOPS Plus, Vitrolife), with a maximum manipulation time of 10 minutes per patient. All embryos are cultured in a time-lapse incubator (Geri, Genea Biomedex) under controlled atmosphere (6% CO2, 5% O2) throughout the study.

D4 Defragmented Group

Standard embryo culture without intervention OTHER

Standard blastocyst culture without any additional manipulation. Day 4 morphological assessment is performed under the same conditions as the intervention group to ensure comparability of culture conditions.

D4 Non-Defragmented Group

Eligibility Criteria

• Age: 20 Years - 42 Years
• Sex: female
• Healthy Volunteers: No
• Age Groups: Adult (18-64)

You may qualify if:

• Female patients undergoing ICSI cycles at the participating center
• Age 18-42 years
• Presence of at least one morula with cytoplasmic fragmentation ≥10% at Day 4 morphological assessment (Grade B: 10-25%, Grade C: 26-50%, Grade D: ≥50%)
• Embryos intended for extended culture to blastocyst stage (Day 5/Day 6)
• All embryo cultures performed in time-lapse incubator (Geri, Genea Biomedex)
• Written informed consent obtained prior to any study-related procedure -

You may not qualify if:

• Severely reduced ovarian reserve (AMH ≤ 0.5 ng/mL)
• Body mass index (BMI) ≥ 32 kg/m²
• Diagnosis of endometriosis
• Polycystic ovary syndrome (PCOS)
• Maternal age ≥ 42 years
• History of repeated implantation failure
• Donor gamete cycles (oocyte or sperm donation)
• Cycles with preimplantation genetic testing for monogenic disease (PGT-M) as primary indication
• Embryos with severe morphological compromise at Day 3
• Concurrent participation in other interventional studies that could interfere with study outcomes
• Inability to provide written informed consent

Sponsors & Collaborators

• Momo Fertilife: lead

Study Sites (1)

MOMO' Fertilife

Bisceglie, BT, 76011, Italy

RECRUITING

Related Publications (5)

• Petrushko M, Yurchuk T, Piniaiev V, Buderatska N. Cryopreservation of incomplete compacted morulae and preliminary biopsy of excluded fragments. Zygote. 2019 Dec;27(6):386-391. doi: 10.1017/S0967199419000455. Epub 2019 Aug 15.

  PMID: 31412967 RESULT

• Eftekhari-Yazdi P, Valojerdi MR, Ashtiani SK, Eslaminejad MB, Karimian L. Effect of fragment removal on blastocyst formation and quality of human embryos. Reprod Biomed Online. 2006 Dec;13(6):823-32. doi: 10.1016/s1472-6483(10)61031-0.

  PMID: 17169204 RESULT

• Kim SG, Kim YY, Park JY, Kwak SJ, Yoo CS, Park IH, Sun HG, Kim JW, Lee KH, Park HD, Chi HJ. Early fragment removal on in vitro fertilization day 2 significantly improves the subsequent development and clinical outcomes of fragmented human embryos. Clin Exp Reprod Med. 2018 Sep;45(3):122-128. doi: 10.5653/cerm.2018.45.3.122. Epub 2018 Sep 3.

  PMID: 30202742 RESULT

• Sordia-Hernandez LH, Morales-Martinez FA, Frazer-Moreira LM, Villarreal-Pineda L, Sordia-Pineyro MO, Valdez-Martinez OH. Clinical Pregnancy After Elimination of Embryo Fragments Before Fresh Cleavage-stage Embryo Transfer. J Family Reprod Health. 2020 Sep;14(3):198-204. doi: 10.18502/jfrh.v14i3.4674.

  PMID: 33603813 RESULT

• Alikani M, Calderon G, Tomkin G, Garrisi J, Kokot M, Cohen J. Cleavage anomalies in early human embryos and survival after prolonged culture in-vitro. Hum Reprod. 2000 Dec;15(12):2634-43. doi: 10.1093/humrep/15.12.2634.

  PMID: 11098037 RESULT

Related Links

• Related Info

MeSH Terms

Conditions

Infertility, Female

Interventions

Methods

Condition Hierarchy (Ancestors)

Genital Diseases, Female; Female Urogenital Diseases; Female Urogenital Diseases and Pregnancy Complications; Urogenital Diseases; Genital Diseases; Infertility

Intervention Hierarchy (Ancestors)

Investigative Techniques

Study Officials

• Giorgio Maria baldini, Medicine

  Department of Interdisciplinary Medicine, University of Bari "Aldo Moro"

  PRINCIPAL INVESTIGATOR

• domenico baldini, medicine

  IVF Center MOMO' FERTILIFE

  STUDY DIRECTOR

• Daniele Ferri, Biology

  IVF Center MOMO' FERTILIFE

  STUDY CHAIR

Central Study Contacts

Giorgio Maria Baldini, Medical Doctor

CONTACT

+39 0803962313; gbaldini97@gmail.com

Domenico Baldini, Medicine

CONTACT

+39 3337845481; dbaldini@libero.it

Study Design

• Study Type: interventional
• Phase: not applicable
• Allocation: RANDOMIZED
• Masking: SINGLE
• Who Masked: OUTCOMES ASSESSOR
• Purpose: TREATMENT
• Intervention Model: PARALLEL
• Sponsor Type: OTHER
• Responsible Party: SPONSOR

Study Record Dates

• First Submitted: March 24, 2026
• First Posted: March 30, 2026
• Study Start: March 24, 2026
• Primary Completion: March 24, 2026
• Study Completion (Estimated): December 31, 2026
• Last Updated: March 30, 2026

Record last verified: 2026-03

Data Sharing

• IPD Sharing: Will not share

Locations

IT (1)