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).

BACKGROUND AND SCIENTIFIC RATIONALE Embryo fragmentation is one of the most frequently observed morphological abnormalities during in vitro culture in assisted reproductive technology (ART). Anucleate cytoplasmic fragments arise from asymmetric cell division or apoptotic processes and can affect a variable proportion of the embryo volume. Their presence has been consistently associated with reduced implantation potential, lower blastocyst formation rates, and poorer clinical outcomes in IVF/ICSI cycles.

Current embryo grading systems, including the Istanbul Consensus and the Gardner scoring system for blastocysts, incorporate fragmentation as a key morphological parameter. Embryos with fragmentation ≥10% at cleavage stages are generally considered to have a reduced but not absent developmental potential, and their management - particularly whether active intervention to remove fragments is beneficial - remains a subject of ongoing debate.

Mechanical and laser-assisted embryo defragmentation at early cleavage stages (Day 2-Day 3) has been practiced in selected ART laboratories for several decades. The rationale is that physical removal of anucleate fragments may relieve developmental constraints on blastomeres, reduce cytotoxic or apoptotic signaling, and improve compaction and blastocyst formation. However, published evidence on early defragmentation is heterogeneous: some studies report improvements in blastocyst development and pregnancy rates, while others show no significant benefit or even potential harm due to mechanical stress during manipulation.

A critical gap in the literature concerns defragmentation performed at Day 4 (D4) of culture, corresponding to the morula and early compaction stage. By D4, embryos that have successfully maintained developmental competence are undergoing compaction - a key morphogenetic event involving cell polarization, tight junction formation, and the establishment of the inner cell mass and trophectoderm lineages. The biological rationale for D4 defragmentation differs from earlier interventions: at this stage, the relationship between fragments and compacting blastomeres may be more clearly defined, and removal of fragments may less likely interfere with ongoing cell division while potentially supporting the compaction process.

Preliminary observational data from our center suggest that D4 defragmentation may be associated with higher rates of usable blastocysts compared to historical controls, particularly in embryos with moderate fragmentation (10-50%). These preliminary findings, while encouraging, were obtained from non-randomized data and are subject to selection bias. A prospective randomized controlled trial (RCT) is therefore necessary to determine whether D4 defragmentation genuinely improves developmental and clinical outcomes.

STUDY DESIGN This is a prospective, randomized, controlled, single-blind trial (DEBRIS-D4 / DEFRAG-D4 trial). The study is designed according to CONSORT guidelines for reporting of randomized trials and follows the SPIRIT checklist for clinical trial protocols.

Single-blind design: The embryologist performing blastocyst quality assessment at D5/D6 and the sonographer evaluating clinical pregnancy will be blinded to the treatment arm assignment. Blinding of the embryologist performing the D4 procedure is not feasible due to the nature of the intervention.

Unit of randomization: Randomization occurs at the patient level (not the embryo level), to avoid contamination between groups and to reflect the clinical reality of embryo cohort management.

Stratification factors:

Patient age: <35 years vs. ≥35 years Number of embryos with ≥10% fragmentation at D4: 1-2 vs. ≥3 Use of preimplantation genetic testing for aneuploidy (PGT-A): yes vs. no

Randomization method: Pre-generated block randomization lists with variable block sizes (4 and 6), managed through sequentially numbered sealed opaque envelopes. Envelopes are opened on the morning of Day 4, after morphological evaluation confirms the presence of at least one embryo meeting inclusion criteria.

STUDY POPULATION

Inclusion Criteria:

Female patients undergoing IVF/ICSI cycles at the participating center Age 18-43 years Basal FSH < 20 mIU/mL Presence of ≥1 embryo with ≥10% cytoplasmic fragmentation at D4 morphological evaluation Embryos intended for extended culture to blastocyst stage (D5/D6) Written informed consent obtained prior to any study-related procedure

Exclusion Criteria:

Donor gamete cycles (oocyte or sperm donation) Cycles with preimplantation genetic testing for monogenic disease (PGT-M) as primary indication Embryos with diffuse fragmentation >50% or severe morphological compromise at Day 3 Concurrent participation in other interventional studies that could interfere with study outcomes Inability to provide informed consent

INTERVENTIONS

Group A - D4 Defragmentation (Intervention):

Mechanical or laser-assisted defragmentation will be performed on Day 4 (96 ± 2 hours post-fertilization), immediately following the morning morphological assessment. All embryos belonging to patients randomized to Group A with ≥10% fragmentation will undergo the procedure.

The defragmentation procedure will be carried out under standard micromanipulation conditions:

Low light exposure environment Controlled temperature at 37°C (heated stage) Maximum time outside the incubator per patient: ≤10 minutes Technique: mechanical defragmentation using a biopsy pipette or laser-assisted removal (as per center-specific standard operating procedure) Following defragmentation, embryos will be returned to culture under the same standard conditions (time-lapse incubator or conventional incubator, as applicable)

Group B - Standard Culture (Control):

Embryos in the control arm will not receive any additional manipulation beyond routine morphological assessment at D4. To ensure comparability of culture conditions between groups, the D4 assessment in control patients will be performed by the same operator with the same duration of incubator opening as in the intervention group (i.e., embryos will be briefly removed for evaluation but immediately returned without intervention).

Laboratory Standardization:

For both arms, culture conditions will be identical throughout the study:

Culture medium: standardized product (to be specified in final protocol) Incubator type: identical for both arms (time-lapse preferred) Gas atmosphere: standardized CO₂/O₂/N₂ composition Morphological assessments at: Day 1 (fertilization check), Day 3, Day 4 (randomization), Day 5, Day 6 Blastocyst scoring: Gardner grading system (expansion, ICM grade, TE grade)

OUTCOME MEASURES

Primary Outcome:

Rate of usable blastocysts (Gardner score ≥3BB) per embryo included in the study, assessed at Day 5 and Day 6 of in vitro culture (time frame: 6 days from fertilization).

Secondary Outcomes (with time frames):

Overall blastocyst development rate (D5 + D6 combined) - time frame: Day 6 Distribution of Gardner scores among obtained blastocysts - time frame: Day 6 Blastocyst cryopreservation rate - time frame: Day 6-7 Implantation rate (gestational sac per blastocyst transferred) - time frame: 5 weeks post-transfer Clinical pregnancy rate (fetal heartbeat detected at ultrasound at 7 weeks) - time frame: 7 weeks post-transfer Ongoing pregnancy rate (viable pregnancy beyond 12 weeks of gestation) - time frame: 12 weeks post-transfer Live birth rate per embryo transfer - time frame: approximately 9 months post-transfer Morphokinetic analysis (if time-lapse incubator available for both arms) - time frame: Day 6 Aneuploidy rate in embryos undergoing PGT-A (exploratory endpoint) - time frame: Day 5-6 (biopsy) plus laboratory turnaround

STATISTICAL ANALYSIS PLAN

Sample Size Calculation:

The sample size is based on the primary endpoint (rate of usable blastocysts ≥3BB per embryo). Assuming a blastocyst rate of 42% in the control group and 57% in the intervention group (absolute difference of 15%), with 80% power, two-tailed α = 0.05, using the chi-square test with Fleiss continuity correction:

N per arm ≈ 100-110 patients With 15% dropout correction: N per arm ≈ 115-130 patients Total estimated sample: N ≈ 230-260 patients

The sample size calculation will be updated with definitive preliminary data from the center prior to final registration. Statistical consultation with a biostatistician will be obtained before the start of enrollment.

Primary Analysis:

The rate of usable blastocysts will be compared between the two arms using the chi-square test (or Fisher's exact test for small samples). The primary analysis will follow the Intention-To-Treat (ITT) principle, including all randomized patients regardless of protocol deviations.

Secondary Analyses:

Multivariable logistic regression adjusting for pre-specified covariates (patient age, degree of fragmentation, operator, incubator type) Pre-specified subgroup analyses: age <35 vs. ≥35 years; fragmentation grade (10-25% vs. 26-50%); incubator type (time-lapse vs. conventional) Per-protocol (PP) analysis as a sensitivity analysis Kaplan-Meier survival analysis for ongoing pregnancy and live birth outcomes

Interim Analysis:

A pre-planned interim analysis will be conducted at 50% enrollment (approximately N=120 patients), reviewed by an independent Data Safety Monitoring Board (DSMB). Stopping rules will follow the O'Brien-Fleming boundary for efficacy and pre-specified criteria for safety.

Missing Data:

Missing data for the primary endpoint will be handled using worst-case scenario imputation. For clinical outcome data, multiple imputation will be applied for data missing at random.

DATA COLLECTION AND MANAGEMENT

Data will be collected prospectively using a standardized Case Report Form (CRF). Key data domains include:

Baseline patient data: age, BMI, FSH, AMH, antral follicle count (AFC), ART indication, number of prior cycles, stimulation protocol, number of retrieved and fertilized oocytes.

D4 laboratory data: number of embryos assessed, number with ≥10% fragmentation, degree of fragmentation per embryo (%), morphodynamic stage at D4 (compacting morula, full morula, etc.), randomization arm, defragmentation technique (Group A only), procedure duration, operative notes.

D5/D6 laboratory data: number of blastocysts per day, Gardner score per blastocyst (assessed by blinded embryologist), number of blastocysts cryopreserved.

Clinical outcome data: embryo transfer performed (yes/no), number of blastocysts transferred, biochemical pregnancy (beta-hCG), clinical pregnancy (heartbeat at 7 weeks), ongoing pregnancy (12 weeks), live birth.

All data will be pseudonymized. Data management will comply with GDPR (EU Regulation 679/2016) and applicable Italian data protection legislation (D.Lgs. 196/2003). Data will be stored in a secure, access-controlled database.

ETHICS AND REGULATORY COMPLIANCE The study has obtained a favorable opinion from the local Ethics Committee (Comitato Etico). All patients will receive a detailed patient information sheet and will provide written informed consent prior to any study-related procedure. The study consent is separate from the standard IVF/ICSI cycle consent.

The study will be registered on ClinicalTrials.gov prior to enrollment of the first participant, in accordance with ICMJE requirements for clinical trial registration. Parallel registration on ISRCTN (Europe) is also planned.

The study is conducted in accordance with the Declaration of Helsinki, ICH Good Clinical Practice (GCP) guidelines, and applicable Italian and European regulatory requirements.

STUDY TIMELINE (ESTIMATED)

Protocol finalization and team training: 1months ClinicalTrials.gov and ISRCTN registration: 2 weeks Run-in phase (procedure calibration and internal audit): 1 month Active enrollment: 6 months Interim DSMB analysis: Month 6 Clinical follow-up (ongoing pregnancy and live birth): 6 months post-enrollment Final statistical analysis: 1 months Manuscript preparation and submission: 1 months Total estimated duration: ~20 months

Inclusion Criteria:

1. Female patients undergoing ICSI cycles at the participating center
2. Age 18-42 years
3. 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%)
4. Embryos intended for extended culture to blastocyst stage (Day 5/Day 6)
5. All embryo cultures performed in time-lapse incubator (Geri, Genea Biomedex)
6. Written informed consent obtained prior to any study-related procedure -

Exclusion Criteria:

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

Individual participant data will not be shared due to patient privacy protection requirements under the European General Data Protection Regulation (GDPR, EU Regulation 679/2016) and applicable Italian data protection legislation (D.Lgs. 196/2003). All data are pseudonymized and stored in a secure, access-controlled database. Aggregate results will be made available through peer-reviewed publication upon study completion.