Day-4 Embryo Defragmentation: Blastocyst Rate and clInical Outcomes

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

Interventions

PROCEDURE

Mechanical embryo defragmentation at Day 4 (morula stage)

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.

OTHER

Standard embryo culture without intervention

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.

OTHER

Standard embryo culture without intervention

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.

Sponsors & Collaborators

• Momo Fertilife

  lead OTHER

Principal Investigators

• Giorgio Maria baldini, Medicine · Department of Interdisciplinary Medicine, University of Bari "Aldo Moro"

• domenico baldini, medicine · IVF Center MOMO' FERTILIFE

• Daniele Ferri, Biology · IVF Center MOMO' FERTILIFE

Study Design

Allocation

RANDOMIZED

Purpose

TREATMENT

Masking

SINGLE

Model

PARALLEL

Eligibility

Min Age

20 Years

Max Age

42 Years

Sex

FEMALE

Healthy Volunteers

No

Timeline & Regulatory

Start

2026-03-24

Primary Completion

2026-03-24

Completion

2026-12-31

Countries

• Italy

Study Locations