Trial Outcomes & Findings for Embryo Culture Under Constant 5% vs Gradient 8%, 5%, 2% Oxygen Concentration (NCT NCT05898178)

Last Updated: 2025-06-08

Results Overview

The primary outcome measure will be the proportion of oocytes that will develop to the morphologically optimal day-5 blastocysts, scored 4-5AA according to Gardner criteria. According to the scoring system of Gardner, blastocyst morphology parameters such as the degree of blastocoel expansion (1-5), the morphological appearance of the inner cell mass (ICM) (A, B, C) and the cohesiveness of trophectoderm (TE) (A, B, C) will be measured.

Recruitment status

COMPLETED

Study phase

Target enrollment

44 participants

Primary outcome timeframe

Embryos will be annotated on day 5 post insemination (at 8:00 am).

Results posted on

2025-06-08

Participant Flow

Participants were recruited between January 2022 and January 2023 at the University Medical Centre Maribor. Women under 35 undergoing ICSI for male infertility were included. All patients provided informed consent. A total of 44 participants were enrolled, with a total of 658 cumulus-oocyte complexes (COCs) collected.

Following oocyte pickup, 625 cumulus-oocyte complexes from 44 ICSI cycles were allocated to two groups by quasi randomisation. After excluding immature (GV/MI) or degenerated oocytes, 521 metaphase II (MII) oocytes remained, with 258 assigned to the control group (5% O₂) and 263 to the intervention group (8-5-2% O₂).

Unit of analysis: Injected sibling MII oocytes

Participant milestones

Participant milestones
Measure	8-5-2% Oxygen Gradient Concentration in the Incubator The intervention group simulated a dynamic oxygen gradient designed to mimic reported conditions found in vivo, with gas concentrations adjusted according to embryonic developmental stages. From Day 0 to Day 3, the culture environment was set to 6% CO₂, 8% O₂, and 86% N₂. On Day 3, the oxygen concentration was reduced to 5% (6% CO₂, 5% O₂, 89% N₂), and from the end of Day 3 through Day 5, it was further lowered to 2% (6% CO₂, 2% O₂, 92% N₂).	Control (no Intervention) The control group was maintained under fixed atmospheric conditions with 5% O₂ (6% CO₂, 5% O₂, 89% N₂) throughout the entire culture period, reflecting an arbitrarily used static oxygen culture system.
Overall Study STARTED	44 263	44 258
Overall Study COMPLETED	44 263	44 258
Overall Study NOT COMPLETED	0 0	0 0

Reasons for withdrawal

Withdrawal data not reported

Baseline Characteristics

Race and Ethnicity were not collected from any participant.

Baseline characteristics by cohort

Baseline characteristics by cohort
Measure	All Study Participants n=44 Participants As a sibling oocyte study, individual patients were not assigned to groups; instead, all 44 enrolled female patients had their sibling oocytes allocated between the intervention and control conditions. Thus, a single cohort of patients served as the baseline population.
Age, Continuous	31.4 Years STANDARD_DEVIATION 2.36 • n=44 Participants
Sex: Female, Male Female	44 Participants n=44 Participants
Sex: Female, Male Male	0 Participants n=44 Participants

PRIMARY outcome

Timeframe: Embryos will be annotated on day 5 post insemination (at 8:00 am).

Population: The primary outcome measure is the proportion of inseminated MII oocytes that develop into morphologically optimal day-5 blastocysts, defined as grade 4-5AA according to the Gardner criteria and not the enrolled patient coupled (n=44)

Outcome measures

Outcome measures
Measure	8-5-2% Oxygen Gradient Concentration in the Incubator n=263 Injected metaphase II [MII] oocytes The intervention group simulated a dynamic oxygen gradient designed to mimic reported conditions found in vivo, with gas concentrations adjusted according to embryonic developmental stages. From Day 0 to Day 3, the culture environment was set to 6% CO₂, 8% O₂, and 86% N₂. On Day 3, the oxygen concentration was reduced to 5% (6% CO₂, 5% O₂, 89% N₂), and from the end of Day 3 through Day 5, it was further lowered to 2% (6% CO₂, 2% O₂, 92% N₂).	Control (no Intervention) n=258 Injected metaphase II [MII] oocytes The control group was maintained under fixed atmospheric conditions with 5% O₂ (6% CO₂, 5% O₂, 89% N₂) throughout the entire culture period, reflecting an arbitrarily used static oxygen culture system.
Proportion of Inseminated Oocytes Developed to the Morphologically Optimal Blastocysts on Day 5	27 Oocytes	38 Oocytes

SECONDARY outcome

Timeframe: Morphokinetic timings were recorded continuously (every 5 minutes) throughout embryo culture, up to 124 hours (Day 5) post-insemination.

Population: Embryos included in the analysis were those that progressed to the blastocyst stage and were deemed suitable for clinical use, either through transfer or cryopreservation.

Using time-lapse software, embryo development videos were reviewed by manually advancing the images frame by frame. Morphokinetic timings were recorded continuously (every 5 minutes) throughout embryo culture, up to 124 hours (Day 5) post-insemination. The following developmental milestones were recorded for each clinically used embryo that reached the blastocyst stage (cryopreserved or transferred): tPNa - Appearance of individual pronuclei t2 - Time to 2-cell stage t3 - Time to 3-cell stage t4 - Time to 4-cell stage t5 - Time to 5-cell stage t6 - Time to 6-cell stage t7 - Time to 7-cell stage t8 - Time to 8-cell stage tSC - First evidence of compaction tM - Completion of the compaction process (morula stage) tSB - Initiation of blastulation tB - Full blastocyst (last frame before zona pellucida starts to thin) tEB - Initiation of blastocyst expansion (first frame showing zona thinning)

Outcome measures

Outcome measures
Measure	8-5-2% Oxygen Gradient Concentration in the Incubator n=97 Clinically utilized embryos The intervention group simulated a dynamic oxygen gradient designed to mimic reported conditions found in vivo, with gas concentrations adjusted according to embryonic developmental stages. From Day 0 to Day 3, the culture environment was set to 6% CO₂, 8% O₂, and 86% N₂. On Day 3, the oxygen concentration was reduced to 5% (6% CO₂, 5% O₂, 89% N₂), and from the end of Day 3 through Day 5, it was further lowered to 2% (6% CO₂, 2% O₂, 92% N₂).	Control (no Intervention) n=122 Clinically utilized embryos The control group was maintained under fixed atmospheric conditions with 5% O₂ (6% CO₂, 5% O₂, 89% N₂) throughout the entire culture period, reflecting an arbitrarily used static oxygen culture system.
Measured Times From Insemination to Different Embryonic Stages Reached tPNa	11.4 Hours post-insemination (HPI) Interval 11.0 to 12.8	11.7 Hours post-insemination (HPI) Interval 11.1 to 12.6
Measured Times From Insemination to Different Embryonic Stages Reached t2	25.7 Hours post-insemination (HPI) Interval 23.6 to 27.7	25.7 Hours post-insemination (HPI) Interval 24.9 to 28.5
Measured Times From Insemination to Different Embryonic Stages Reached t3	36.6 Hours post-insemination (HPI) Interval 31.8 to 39.0	35.1 Hours post-insemination (HPI) Interval 31.7 to 39.2
Measured Times From Insemination to Different Embryonic Stages Reached t4	38.8 Hours post-insemination (HPI) Interval 35.4 to 41.3	37.6 Hours post-insemination (HPI) Interval 36.0 to 41.0
Measured Times From Insemination to Different Embryonic Stages Reached t5	49.5 Hours post-insemination (HPI) Interval 46.1 to 53.0	48.1 Hours post-insemination (HPI) Interval 45.0 to 51.3
Measured Times From Insemination to Different Embryonic Stages Reached t6	53.2 Hours post-insemination (HPI) Interval 50.5 to 57.7	53.1 Hours post-insemination (HPI) Interval 50.3 to 57.7
Measured Times From Insemination to Different Embryonic Stages Reached t7	61.5 Hours post-insemination (HPI) Interval 55.7 to 67.6	63.4 Hours post-insemination (HPI) Interval 56.8 to 67.8
Measured Times From Insemination to Different Embryonic Stages Reached t8	69.5 Hours post-insemination (HPI) Interval 65.8 to 74.6	71.9 Hours post-insemination (HPI) Interval 66.4 to 75.1
Measured Times From Insemination to Different Embryonic Stages Reached tSC	86.0 Hours post-insemination (HPI) Interval 78.5 to 89.5	83.8 Hours post-insemination (HPI) Interval 79.0 to 87.5
Measured Times From Insemination to Different Embryonic Stages Reached tM	91.2 Hours post-insemination (HPI) Interval 86.8 to 96.5	88.3 Hours post-insemination (HPI) Interval 84.4 to 92.8
Measured Times From Insemination to Different Embryonic Stages Reached tSB	106.1 Hours post-insemination (HPI) Interval 101.4 to 111.1	100.8 Hours post-insemination (HPI) Interval 96.0 to 106.6
Measured Times From Insemination to Different Embryonic Stages Reached tB	112.4 Hours post-insemination (HPI) Interval 108.6 to 117.0	106.9 Hours post-insemination (HPI) Interval 103.4 to 110.3
Measured Times From Insemination to Different Embryonic Stages Reached tEB	117.0 Hours post-insemination (HPI) Interval 115.0 to 122.4	111.4 Hours post-insemination (HPI) Interval 108.4 to 114.1

SECONDARY outcome

Timeframe: Fix time (116 hours) post insemination

Population: Blastocysts graded Gardner ≥3 were included, as they exhibited sufficient morphological development for accurate measurement of blastocyst and inner cell mass (ICM) surface areas.

Surface measurements of blastocysts and inner cell mass (ICM) will be recorded on time-lapse photos at 116 hours after insemination. The surfaces will be measured in square micrometres using the Primo vision software's measuring tool. An ellipse will be generated around the trophectoderm's outer edge or the inner cell mass. These measurements will exclude the zona pellucida. The measurements will be taken at the focus plane with the largest surface area.

Outcome measures

Outcome measures
Measure	8-5-2% Oxygen Gradient Concentration in the Incubator n=72 Blastocysts graded Gardner ≥ 3 The intervention group simulated a dynamic oxygen gradient designed to mimic reported conditions found in vivo, with gas concentrations adjusted according to embryonic developmental stages. From Day 0 to Day 3, the culture environment was set to 6% CO₂, 8% O₂, and 86% N₂. On Day 3, the oxygen concentration was reduced to 5% (6% CO₂, 5% O₂, 89% N₂), and from the end of Day 3 through Day 5, it was further lowered to 2% (6% CO₂, 2% O₂, 92% N₂).	Control (no Intervention) n=110 Blastocysts graded Gardner ≥ 3 The control group was maintained under fixed atmospheric conditions with 5% O₂ (6% CO₂, 5% O₂, 89% N₂) throughout the entire culture period, reflecting an arbitrarily used static oxygen culture system.
Blastocyst and Inner Cell Mass (ICM) Surface Area Measurement on Day 5 Blastocyst surface area (P, µm²)	25994 µm² Standard Deviation 4657	28198 µm² Standard Deviation 4139
Blastocyst and Inner Cell Mass (ICM) Surface Area Measurement on Day 5 Inner cell mass surface area (ICM, µm²)	3695 µm² Standard Deviation 732.5	3628 µm² Standard Deviation 831.9

SECONDARY outcome

Timeframe: Fix time (116 hours) post insemination.

Population: Blastocysts graded Gardner ≥3 were included, as these embryos exhibited sufficient morphological development to enable accurate count of trophectoderm (TE) cell number.

At 116 hours following insemination, the number of trophectoderm cells will be counted using time-lapse photos. Images will be focused on the trophectoderm's outermost edge to better determine the boundaries of each individual cell.

Outcome measures

Outcome measures
Measure	8-5-2% Oxygen Gradient Concentration in the Incubator n=72 Blastocysts graded Gardner ≥3 The intervention group simulated a dynamic oxygen gradient designed to mimic reported conditions found in vivo, with gas concentrations adjusted according to embryonic developmental stages. From Day 0 to Day 3, the culture environment was set to 6% CO₂, 8% O₂, and 86% N₂. On Day 3, the oxygen concentration was reduced to 5% (6% CO₂, 5% O₂, 89% N₂), and from the end of Day 3 through Day 5, it was further lowered to 2% (6% CO₂, 2% O₂, 92% N₂).	Control (no Intervention) n=110 Blastocysts graded Gardner ≥3 The control group was maintained under fixed atmospheric conditions with 5% O₂ (6% CO₂, 5% O₂, 89% N₂) throughout the entire culture period, reflecting an arbitrarily used static oxygen culture system.
Number of Trophectoderm Cells on Day 5	14.4 Number of cells Standard Deviation 2.3	15.3 Number of cells Standard Deviation 2.8

SECONDARY outcome

Timeframe: Continuously (a picture will be taken every 5 minutes) during 5 days of embryo culture.

Population: The analysis included all embryos that progressed to the blastocyst stage.

Time-lapse videos for each embryo will be reviewed for atypical cleavage features, such as; pseudofurrows, direct cleavage, reverse cleavage, multinucleation, irregular chaotic division, cell exclusion and blastocyst collapse, using Primo vision software by manually forwarding the images frame by frame. The frequency of abnormal cleavage patterns will be recorded.

Outcome measures

Outcome measures
Measure	8-5-2% Oxygen Gradient Concentration in the Incubator n=130 Embryos reaching blastocyst stage The intervention group simulated a dynamic oxygen gradient designed to mimic reported conditions found in vivo, with gas concentrations adjusted according to embryonic developmental stages. From Day 0 to Day 3, the culture environment was set to 6% CO₂, 8% O₂, and 86% N₂. On Day 3, the oxygen concentration was reduced to 5% (6% CO₂, 5% O₂, 89% N₂), and from the end of Day 3 through Day 5, it was further lowered to 2% (6% CO₂, 2% O₂, 92% N₂).	Control (no Intervention) n=141 Embryos reaching blastocyst stage The control group was maintained under fixed atmospheric conditions with 5% O₂ (6% CO₂, 5% O₂, 89% N₂) throughout the entire culture period, reflecting an arbitrarily used static oxygen culture system.
Incidence of Atypical Embryo Cleavages Pseudofurrows	36 Number of events	49 Number of events
Incidence of Atypical Embryo Cleavages Direct cleavage	25 Number of events	30 Number of events
Incidence of Atypical Embryo Cleavages Reverse cleavage	14 Number of events	9 Number of events
Incidence of Atypical Embryo Cleavages Irregular chaotic division	21 Number of events	15 Number of events
Incidence of Atypical Embryo Cleavages Blastomere exclusion	45 Number of events	34 Number of events
Incidence of Atypical Embryo Cleavages Blastocyst collapse	55 Number of events	67 Number of events

Adverse Events

8-5-2% Oxygen Gradient Concentration in the Incubator

Serious events: 0 serious events

Other events: 0 other events

Deaths: 0 deaths

Control (no Intervention)

Serious events: 0 serious events

Other events: 0 other events

Deaths: 0 deaths

Serious adverse events

Adverse event data not reported

Other adverse events

Adverse event data not reported

Additional Information

Borut Kovačič

Maribor University Medical Centr

Email: [email protected]

Results disclosure agreements

Principal investigator is a sponsor employee
Publication restrictions are in place