Embryonic DevelopmentNCT05898178
Embryo Culture Under Constant 5% vs Gradient 8%, 5%, 2% Oxygen Concentration
A study testing Experimental intervention: 8-5-2% oxygen gradient concentration in the incubator in people with Embryonic Development and Embryo Morphokinetics, run by University Medical Centre Maribor at 1 site in Slovenia.
Trial summary
The purpose of this study is to investigate the development of human embryos in vitro under two different oxygen concentrations; a static 5% during all five days of culture or under an oxygen gradient, starting with 8% from day-0 to day-3, continuing with 5% on day-3 and following with 2% of oxygen from the end of… Read more about this study
- Who is it for?
- Ages 18 to 35 years, women onlyHealthy volunteers can take part
- Conditions the study is for
- Embryonic Development, Embryo Morphokinetics, Embryo Morphometry
- What is being tested?
- Experimental intervention: 8-5-2% oxygen gradient concentration in the incubator
- Could I receive a placebo?
- No, everyone receives an active treatment
- How many people
- 44 participants
- Where
- 1 siteSlovenia
- Study dates
- 1 Jan 2022to 1 Jan 2023
- Phase
- Interventional
- Are there results?
- Yes, posted in the registrySummary in the doctors' view
Key requirements
Who can take part, as listed by the sponsor. Only the study team can confirm whether you are eligible, so take this list to your doctor.
- Ages 18 to 35 years
- Women only
- Healthy volunteers accepted
- Adult
Who may be able to join
- Age of women between 18 and 35 years.
- Body mass index (BMI) between 18 and 30 kg/m².
- Only patients with ICSI procedure (male factor of infertility, excluding azoospermia) and blastocyst culture.
- Patients from first and second ICSI cycle attempt.
- Gonadotropin hormone-releasing hormone (GnRH) antagonist cycles.
Who cannot join
- Presence of endometriosis.
- Previous clinical intervention on ovaries.
- Connected endocrine or metabolic diseases.
- Presence of polycystic ovary syndrome.
Trial locations and contacts
1 site in 1 country.
About this study
Official title: The Effect of Constant 5% Versus Gradient 8%, 5%, 2% Oxygen Concentration on the Development of Sibling Human Embryos in Vitro
The purpose of this study is to investigate the development of human embryos in vitro under two different oxygen concentrations; a static 5% during all five days of culture or under an oxygen gradient, starting with 8% from day-0 to day-3, continuing with 5% on day-3 and following with 2% of oxygen from the end of day-3 to day-5.
Read the detailed description
Several studies have shown that embryonic morphological parameters improved when the oxygen concentration in embryo culture was reduced from 20% to 5%. Early mammalian embryos developed faster, had shorter cell cycles, a higher blastocyst formation rate and a better integrity of the inner cell mass (ICM) compared to embryos cultured at 20% oxygen concentration.
Recent studies have shown that the oxygen concentration in the female reproductive tract is not static. In the fallopian tubes of higher mammals it is at around 8%, while in the uterus, at the time of embryo implantation, the oxygen concentration is almost anoxic (2%).
Mimicking such physiological conditions that better reflect the in vivo environment in the human reproductive tract is the goal of assisted reproductive technology (ART).
The aim of this study is to assess whether changing the static 5% oxygen during five days of in vitro embryo culture to the gradient of oxygen, starting with 8% from day-0 to day-3, continuing with 5% on day-3 and following with 2% of oxygen from the end of day-3 to day-5, better reflects conditions found within the human reproductive tract and improves embryo developmental characteristics.
- Being tested
- Experimental intervention: 8-5-2% oxygen gradient concentration in the incubator
- Keywords
- Embryo culture, reduced oxygen, time-lapse, blastocyst
What participants receive
What participants receive. In a randomised study each person is assigned to one group by chance.
Experimental intervention: 8-5-2% oxygen gradient concentration in the incubatorOther
After oocyte retrieval and insemination, half of a given patient's embryos will be randomly allocated in an incubator set at a gradient of oxygen tension (8-5-2%). The embryos will remain in the incubator until their developmental assessments on day 5.
| Study group | Role | What they receive |
|---|---|---|
| 8-5-2% oxygen gradient concentration in the incubator | Experimental | A physiological oxygen gradient concentration in the incubator (8-5-2% O2). Receives: Other: Experimental intervention: 8-5-2% oxygen gradient concentration in the incubator |
| Control (no intervention) | No intervention | Arbitrary cultivation conditions (static 5% 02). |
What is being measured
The primary outcome is the main question the study is designed to answer.
Proportion of Inseminated Oocytes Developed to the Morphologically Optimal Blastocysts on Day 5
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.
Measured: Embryos will be annotated on day 5 post insemination (at 8:00 am).
Secondary outcomes (4)
Measured Times From Insemination to Different Embryonic Stages Reached
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)
Measured: Morphokinetic timings were recorded continuously (every 5 minutes) throughout embryo culture, up to 124 hours (Day 5) post-insemination.
Blastocyst and Inner Cell Mass (ICM) Surface Area Measurement on Day 5
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.
Measured: Fix time (116 hours) post insemination
Number of Trophectoderm Cells on Day 5
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.
Measured: Fix time (116 hours) post insemination.
Incidence of Atypical Embryo Cleavages
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.
Measured: Continuously (a picture will be taken every 5 minutes) during 5 days of embryo culture.
Timeline
From the start of the study to its completion; the mark is today. Estimated dates are the sponsor’s plan.
- First posted12 Jun 2023
- Study start1 Jan 2022
- Primary completion1 Jan 2023
- Study completion1 Jan 2023
Resources
- Official record on ClinicalTrials.govThe registry entry this page is built from, with its full change history.
- Posted resultsParticipant flow, outcome measures and adverse events reported by the sponsor.
- Other trials for Embryonic DevelopmentStudies of the same condition, open and under way.
- What the trial phases meanHow a treatment moves from first tests in people to approval.
More details
For doctors and researchers: how the study is designed, who runs it, and its registry IDs.
Study design
- Study type
- Interventional
- Phase
- Phase N/A
- Primary purpose
- Other
- Allocation
- Randomised
- Intervention model
- Parallel groups
- Model description
- 496 sibling oocytes will be randomly allocated for culture either at low oxygen tension (5% O2) (n = 248 oocytes) or a more physiological oxygen gradient (8-5-2% O2) (n = 248 oocytes) for the next 5 days. Immediately after allocation, the oocytes will be inseminated through Intracytoplasmic Sperm Injection (ICSI). The sibling oocytes will always be injected by the same embryologist. Under standard (5%) oxygen concentration, which is currently being used in the cultivating incubators, approximately 15% of all embryos manage to reach a morphological optimal blastocyst, in a patient group younger than 35 years (see Inclusion Criteria). According to the hypothesis that cultivating embryos under a more physiological atmosphere (gradient from 8% to 2%) improves the blastulation rate of embryos to 25%, a sample size of 496 Injected oocytes (248 in each arm), will be needed. Whereby a statistical strength of 80% and α = 0.05 is considered (calculated using power analysis).
- Masking
- Single blind
- Who is masked
- Investigator
- Enrolment
- 44 participants (actual)
Sponsor and investigators
- Lead sponsor
- University Medical Centre Maribor
- Sponsor type
- University, hospital or other
- Responsible party
- Principal investigator, Borut Kovačič, University Medical Centre Maribor
IDs, dates and publications
- ClinicalTrials.gov ID
- NCT05898178
- Sponsor's protocol ID
- 0120-405/2021/4
- Other IDs
- P3-0327
- Status last verified
- Jun 2025
- First submitted
- 26 Apr 2023
- Last updated
- 8 Jun 2025
- Results posted
- Yes, on ClinicalTrials.gov
- Data monitoring committee
- No
- FDA-regulated drug
- No
- FDA-regulated device
- No
Study at a glance
From the registry record, last updated by the sponsor on 8 Jun 2025. Recruitment status is as the sponsor reported it: confirm with the study team.
- Official title
- The Effect of Constant 5% Versus Gradient 8%, 5%, 2% Oxygen Concentration on the Development of Sibling Human Embryos in Vitro
- ClinicalTrials.gov ID
- NCT05898178
- Acronym / sponsor ID
- 0120-405/2021/4
- Keywords
- Embryo culture, reduced oxygen, time-lapse, blastocyst
- Study type
- Interventional
- Recruitment status
- Completed
- Enrolment
- 44 participants (actual)
- Lead sponsor
- University Medical Centre MariborUniversity, hospital or other
- Study start
- 1 Jan 2022 (actual)
- Primary completion
- 1 Jan 2023 (actual)
- Study completion
- 1 Jan 2023 (actual)
- First posted
- 12 Jun 2023 (actual)
- Results
- Posted 8 Jun 2025
Clinical summary
The research question as the sponsor registered it. This describes what the study plans to test, not what it found.
The purpose of this study is to investigate the development of human embryos in vitro under two different oxygen concentrations; a static 5% during all five days of culture or under an oxygen gradient, starting with 8% from day-0 to day-3, continuing with 5% on day-3 and following with 2% of oxygen from the end of day-3 to day-5.
Detailed description
Several studies have shown that embryonic morphological parameters improved when the oxygen concentration in embryo culture was reduced from 20% to 5%. Early mammalian embryos developed faster, had shorter cell cycles, a higher blastocyst formation rate and a better integrity of the inner cell mass (ICM) compared to embryos cultured at 20% oxygen concentration.
Recent studies have shown that the oxygen concentration in the female reproductive tract is not static. In the fallopian tubes of higher mammals it is at around 8%, while in the uterus, at the time of embryo implantation, the oxygen concentration is almost anoxic (2%).
Mimicking such physiological conditions that better reflect the in vivo environment in the human reproductive tract is the goal of assisted reproductive technology (ART).
The aim of this study is to assess whether changing the static 5% oxygen during five days of in vitro embryo culture to the gradient of oxygen, starting with 8% from day-0 to day-3, continuing with 5% on day-3 and following with 2% of oxygen from the end of day-3 to day-5, better reflects conditions found within the human reproductive tract and improves embryo developmental characteristics.
Study design
- Primary purpose
- Other
- Allocation
- Randomised
- Intervention model
- Parallel groups
- Masking
- Single blind (investigator)
- Number of arms
- 2
- Model description
- 496 sibling oocytes will be randomly allocated for culture either at low oxygen tension (5% O2) (n = 248 oocytes) or a more physiological oxygen gradient (8-5-2% O2) (n = 248 oocytes) for the next 5 days. Immediately after allocation, the oocytes will be inseminated through Intracytoplasmic Sperm Injection (ICSI). The sibling oocytes will always be injected by the same embryologist. Under standard (5%) oxygen concentration, which is currently being used in the cultivating incubators, approximately 15% of all embryos manage to reach a morphological optimal blastocyst, in a patient group younger than 35 years (see Inclusion Criteria). According to the hypothesis that cultivating embryos under a more physiological atmosphere (gradient from 8% to 2%) improves the blastulation rate of embryos to 25%, a sample size of 496 Injected oocytes (248 in each arm), will be needed. Whereby a statistical strength of 80% and α = 0.05 is considered (calculated using power analysis).
| Arm | Type | Receives |
|---|---|---|
| 8-5-2% oxygen gradient concentration in the incubator | Experimental | Other: Experimental intervention: 8-5-2% oxygen gradient concentration in the incubatorA physiological oxygen gradient concentration in the incubator (8-5-2% O2). |
| Control (no intervention) | No intervention | –Arbitrary cultivation conditions (static 5% 02). |
Who may qualify
- Ages
- 18 to 35 years
- Sex
- Female only
- Healthy volunteers
- Accepted
- Age groups
- Adult
Key inclusion criteria (5 listed)
- Age of women between 18 and 35 years.
- Body mass index (BMI) between 18 and 30 kg/m².
- Only patients with ICSI procedure (male factor of infertility, excluding azoospermia) and blastocyst culture.
- Patients from first and second ICSI cycle attempt.
- Gonadotropin hormone-releasing hormone (GnRH) antagonist cycles.
Key exclusion criteria (4 listed)
- Presence of endometriosis.
- Previous clinical intervention on ovaries.
- Connected endocrine or metabolic diseases.
- Presence of polycystic ovary syndrome.
Eligibility is decided by the study team after screening; this overview does not establish that a patient qualifies. Full inclusion and exclusion criteria.
Interventions
| Intervention | Type | Arms |
|---|---|---|
| Experimental intervention: 8-5-2% oxygen gradient concentration in the incubatorAfter oocyte retrieval and insemination, half of a given patient's embryos will be randomly allocated in an incubator set at a gradient of oxygen tension (8-5-2%). The embryos will remain in the incubator until their developmental assessments on day 5. | Other | 8-5-2% oxygen gradient concentration in the incubator |
Primary outcome
Proportion of Inseminated Oocytes Developed to the Morphologically Optimal Blastocysts on Day 5
Time frame: Embryos will be annotated on day 5 post insemination (at 8:00 am).
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.
4 secondary outcomes
Measured Times From Insemination to Different Embryonic Stages Reached
Time frame: Morphokinetic timings were recorded continuously (every 5 minutes) throughout embryo culture, up to 124 hours (Day 5) post-insemination.
Blastocyst and Inner Cell Mass (ICM) Surface Area Measurement on Day 5
Time frame: Fix time (116 hours) post insemination
Number of Trophectoderm Cells on Day 5
Time frame: Fix time (116 hours) post insemination.
Incidence of Atypical Embryo Cleavages
Time frame: Continuously (a picture will be taken every 5 minutes) during 5 days of embryo culture.
Results and safety
Loading the posted results from ClinicalTrials.gov…
Research links
- Official record on ClinicalTrials.govLast updated by the sponsor 8 Jun 2025.
- Posted resultsParticipant flow, baseline, all outcome measures and adverse events.
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Referral and contact
AzTrials does not enrol participants or take referrals. Contact the study team or a site listed below; they decide who can take part. This study is not recruiting at the moment.
The record lists no contact. Use the official record to reach the sponsor.
1 site in 1 country: see every site and its status.
Questions about this trial
What is this clinical trial studying?
A study testing Experimental intervention: 8-5-2% oxygen gradient concentration in the incubator in people with Embryonic Development and Embryo Morphokinetics, run by University Medical Centre Maribor at 1 site in Slovenia.
The full registered description is in the About this study section and the official record.
Who may be eligible to take part?
The sponsor lists 5 inclusion and 4 exclusion criteria. Eligibility is decided by the study team after screening, so read the full criteria and contact a listed site.
Is this clinical trial currently recruiting?
The record says: finished, as of the sponsor's last update on 8 Jun 2025. Status can change between updates, so confirm with the study contact before making plans.
Where is the study taking place?
The record lists 1 site in Slovenia. Whether a site is taking part right now should be confirmed with that site. See every site.
What are the possible benefits and risks?
They depend on the study and the treatment, and no study can promise a benefit. The study team explains the known risks during informed consent, before anyone joins; the record itself does not list them.
Are results available for this study?
Yes. Results are posted in the registry (first posted 8 Jun 2025). A summary is in the For doctors view, and the full results are on ClinicalTrials.gov.
How can a doctor refer a patient or contact the research team?
The record lists no contact. The official record names the sponsor, who can be asked directly. AzTrials does not enrol participants or take referrals.
What does the primary outcome measure?
Proportion of Inseminated Oocytes Developed to the Morphologically Optimal Blastocysts on Day 5 (time frame: Embryos will be annotated on day 5 post insemination (at 8:00 am).). It is what the study was designed to measure, not a finding.
Where can I read the original study information?
Does a listing mean the treatment is approved or proven to work?
No. A listing means the study is registered. It does not show that the treatment is safe, works, or is approved by a regulator.
How often is this information updated?
AzTrials copies changed records from ClinicalTrials.gov every night and reloads the whole registry every week. The sponsor last updated this record on 8 Jun 2025.