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

• Sponsor: University Medical Centre Maribor (Other)
• Overall Status: Completed
• CT.gov ID: NCT05898178
• Collaborator: (none)
• Enrollment: 44
• Location: 1
• Arms: 2
• Actual Duration (Months): 12
• Patients Per Site Per Month: 3.7

Study Details

Study Description

Brief 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 day-3 to day-5.

Condition or Disease	Intervention/Treatment	Phase
Embryonic Development; Embryo Morphokinetics; Embryo Morphometry	Other: Experimental intervention: 8-5-2% oxygen gradient concentration in the incubator	N/A

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

• Study Type: Interventional
• Actual Enrollment: 44 participants
• Allocation: Randomized
• Intervention Model: Parallel Assignment
• Intervention 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).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 (Investigator)
• Masking Description: The cohort of oocyte-cumulus complexes will be divided into either the study or control group by simple randomization. All embryologists involved in the study will be blinded during embryo assessment, measurement, and selection for embryo transfer or vitrification. A code system will be used on the Petri dishes to ensure the embryologists are blinded.
• Primary Purpose: Other
• Official Title: The Effect of Constant 5% Versus Gradient 8%, 5%, 2% Oxygen Concentration on the Development of Sibling Human Embryos in Vitro
• Actual Study Start Date: Jan 1, 2022
• Actual Primary Completion Date: Jan 1, 2023
• Actual Study Completion Date: Jan 1, 2023

Arms and Interventions

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

Outcome Measures

Primary Outcome Measures

1. Proportion of inseminated oocytes developed to the morphologically optimal blastocysts on day 5 [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 Outcome Measures

1. Measured times from insemination to different embryonic stages reached [: Morphokinetic timings will be recorded continuously (a picture will be taken every 5 minutes) during 5 days of embryo culture.]

   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.

2. Blastocyst and inner cell mass (ICM) surface area measurement on day 5 [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 [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 [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.

Eligibility Criteria

Criteria

• Ages Eligible for Study: 18 Years to 35 Years
• Sexes Eligible for Study: Female
• Accepts Healthy Volunteers: Yes

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.

Contacts and Locations

Locations

	Site	City	State	Country	Postal Code
1	University Medical Centre Maribor	Maribor		Slovenia	2000

Sponsors and Collaborators

• University Medical Centre Maribor

Investigators

None specified.

Study Documents (Full-Text)

None provided.

More Information

Publications

None provided.

• Responsible Party: Borut Kovačič, Prof. Borut Kovačič, PhD, University Medical Centre Maribor
• ClinicalTrials.gov Identifier: NCT05898178
• Other Study ID Numbers: 0120-405/2021/4; P3-0327
• First Posted: Jun 12, 2023
• Last Update Posted: Jun 12, 2023
• Last Verified: May 1, 2023

• Studies a U.S. FDA-regulated Drug Product: No
• Studies a U.S. FDA-regulated Device Product: No
• Keywords provided by Borut Kovačič, Prof. Borut Kovačič, PhD, University Medical Centre Maribor: Embryo culture, reduced oxygen, time-lapse, blastocyst

Study Results

No Results Posted as of Jun 12, 2023