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

Study Overview
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Brief Summary
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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
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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.

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

Conditions
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Embryonic Development, Embryo Morphokinetics, Embryo Morphometry

Intervention / Treatment
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* Other: Experimental intervention: 8-5-2% oxygen gradient concentration in the incubator


Participation Criteria
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Eligibility Criteria
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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.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 35 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Other
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional 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


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: 8-5-2% oxygen gradient concentration in the incubator<br>A physiological oxygen gradient concentration in the incubator (8-5-2% O2). | Other: Experimental intervention: 8-5-2% oxygen gradient concentration in the incubator<br>* 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.<br>|
| No Intervention: Control (no intervention)<br>Arbitrary cultivation conditions (static 5% 02). | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| 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. | Embryos will be annotated on day 5 post insemination (at 8:00 am). | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Measured times from insemination to different embryonic stages reached | Using the time-lapse software, videos will be reviewed directly by manually advancing the images frame by frame. Thus morphokinetic timings such as: 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 - time to start of compaction, tM - time to completion of morula, tSB - time to initiation of blastulation, tEB - time to initiation of expansion, tB - time to full blastocyst, will be recorded for each embryo. | : Morphokinetic timings will be recorded continuously (a picture will be taken every 5 minutes) during 5 days of embryo culture. | 
| 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. | 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. | 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. | Continuously (a picture will be taken every 5 minutes) during 5 days of embryo culture. | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Embryo culture, reduced oxygen, time-lapse, blastocyst