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

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).

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)

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.

Time frame: 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.

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.

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

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.

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.

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.

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