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Completed

NCT Number: NCT05898178

Embryo Culture Under Constant 5% vs Gradient 8%, 5%, 2% Oxygen Concentration

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.

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Key information

Conditions

Embryonic Development Embryo Morphokinetics Embryo Morphometry

Age range

18 year–35 year

Sex eligibility

Female

Study type

Interventional

Phase

Not applicable

Primary location

University Medical Centre Maribor

Maribor, 2000, Slovenia

About this study

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.

Who can participate

Healthy volunteers accepted: Yes

Only the study team can determine whether someone qualifies for participation.

Inclusion criteria

  • 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.

Exclusion criteria

  • Presence of endometriosis.
  • Previous clinical intervention on ovaries.
  • Connected endocrine or metabolic diseases.
  • Presence of polycystic ovary syndrome.

Treatment and study plan

Experimental intervention: 8-5-2% oxygen gradient concentration in the incubator

Other

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.

Primary outcomes

  1. 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.

Secondary outcomes

  1. 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.

    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)

  2. Blastocyst and Inner Cell Mass (ICM) Surface Area Measurement on Day 5

    Time frame: Fix time (116 hours) post insemination

    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.

  3. Number of Trophectoderm Cells on Day 5

    Time frame: Fix time (116 hours) post insemination.

    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.

  4. Incidence of Atypical Embryo Cleavages

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

    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.

Sponsors and collaborators

Lead sponsor

University Medical Centre Maribor

Other

Registry information

Official study title

The Effect of Constant 5% Versus Gradient 8%, 5%, 2% Oxygen Concentration on the Development of Sibling Human Embryos in Vitro

Important dates

Study start
2022
Primary completion
2023
Study completion
2023
First posted
Jun 12, 2023
Registry last updated
Jun 8, 2025

OpenTrials presents study information sourced from ClinicalTrials.gov. The official registry record should be consulted for the latest information.

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This listing is for discovery and informational purposes only. It is not medical advice, does not guarantee that a study is recruiting, and does not determine eligibility. Contact the study team and a qualified healthcare professional when considering participation.

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