Day-4 Embryo Defragmentation: Blastocyst Rate and clInical Outcomes
Effect of Embryo Defragmentation on Day 4 (D4) on Blastocyst Development Rate and Clinical Outcomes in ART Cycles: a Prospective RCT
Momo Fertilife
320 participants
Mar 24, 2026
INTERVENTIONAL
Conditions
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).
Eligibility
Inclusion Criteria6
- 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 -
Exclusion Criteria11
- 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
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Interventions
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.
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.
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.
Locations(1)
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NCT07500337