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.
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 Type
INTERVENTIONAL
Allocation
RANDOMIZED
Purpose
OTHER
Masking
SINGLE
Enrollment
44
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.
University Medical Centre Maribor
Maribor, Slovenia
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.
Time frame: Embryos will be annotated on day 5 post insemination (at 8:00 am).
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)
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
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.
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Time frame: 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.
Time frame: 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.
Time frame: Continuously (a picture will be taken every 5 minutes) during 5 days of embryo culture.