A Benchtop Incubator Prospective Study

Recruiting

• Conditions: Infertility
• Interventions: Other: Insemination and culture; Other: Media refreshment Group 1; Other: Culture media preparation and refreshment; Other: Embryo grading; Other: Media refreshment Group 2

• Registration Number: NCT04969575
• Lead Sponsor: ART Fertility Clinics LLC

Brief Summary

The use of extended embryo culture to the blastocyst stage and preimplantation genetic testing for aneuploidy (PGT-A) has enabled identification and selection of embryos with the best developmental potential and improved in vitro fertilization (IVF) clinical outcomes. While numerous types of commercially available human embryo culture media exist for culture to the blastocyst stage, the impact of culture conditions on blastocyst development and aneuploidy formation is not well understood.

Culture conditions are very important for the success of the IVF cycle, many of the factors involved in the process have been extensively studied, including the use of sequential culture medium vs single step culture medium, humidified incubators vs dry incubators and refreshing the medium on day 3 vs uninterrupted culture till day 5. However, none of the studies investigated the effect on euploid rate in a sibling oocyte design with PGT-A, which requires culture till day 7.

Detailed Description

It is considered a routine practice to grade the embryos on day 3 of development, which will require disturbing the culture conditions by annotating the embryos using inverted microscopes. Extended culture to day 7 is required in order to perform trophectoderm biopsy required for PGT-A. There are several culture factors that can affect embryo development, such as the pH, which is controlled through the concentration of CO2, and also the osmolality, which is the concentration of solute per liter of solution. Evaporation of the solution due to the exposure to high temperature (37°C) can change the concentrations and hence compromise embryo development.

To reduce the rate of evaporation, culture droplets are covered with a layer of mineral oil. Humidified incubators can also reduce the rate of evaporation. However, in the case of dry incubators, such as benchtop incubators, replenishing the culture medium at least once -by moving the embryos to a new culture dish- during these 7 days of culture might be the solution.

Since single-step medium is designed to sustain embryo development for 5 continuous culture days, while some laboratories culture embryos for 7 days, refreshing the culture medium is inevitable either on day 3 or day 5 of development.

There is no concrete evidence on which of these practices will provide a better environment for embryo development. Moreover, none of the studies investigated the effect of single-step culture medium changeover on day 3 or day 5, on the chromosomal errors (aneuploidy/mosaicism). Investigators aim in this prospective sibling oocyte study, to explore which day of culture medium refreshment (day 3 or day 5) provides a higher rate of euploid blastocysts.

Study Timeline

• Study First Submitted: 2021-07-11
• Study First Submitted QC: 2021-07-11
• Study Start: 2021-07-15
• Study First Posted: 2021-07-20
• Status Verified: 2023-03
• Last Update Submitted: 2023-03-23
• Last Update Posted: 2023-03-24
• Primary Completion: 2023-06-30
• Study Completion: 2023-12-31

Recruitment & Eligibility

• Status: RECRUITING
• Sex: Female
• Target Recruitment: 50

Inclusion Criteria

• Patients with a minimum of 8 - 10 follicles during ovarian stimulation
• Patients with a minimum of 8 mature oocytes
• PGT-A
• ICSI
• BMI<35
• Age 18- 40
• Fresh or frozen ejaculated sperm >10,000 motile sperm
• Any ovarian stimulation
• Embryos cultured in benchtop incubator

Exclusion Criteria

• PGT-M or PGT-SR
• Previous history of embryo arrest
• Endometriosis
• History of previous treatment which may impact the ovarian reserve (adnexal surgery, chemotherapy, radiation...)

Study & Design

• Study Type: OBSERVATIONAL
• Study Design: Not specified

Arm && Interventions

Group	Intervention	Description
day 3 refreshment	Media refreshment Group 1	All MII oocytes will be inseminated by ICSI 39-41 hours post final oocyte maturation trigger. Following ICSI, oocytes will be cultured in 2-4 culture dishes placed in 2 separate chambers. Media refreshment: Zygote scoring on day 1 (18-20hpi). Culture medium refreshment will be done 67-69 hours post insemination (Day3). Embryo grading will not be done day2-5. Culture media preparation and refreshment: 30µL of culture medium (Global total LP is added to each well of the 8 wells of LifeGlobal GPS Dish (EGPS-010) and 50µL to the center 3 drops. Dishes will be equilibrated overnight in a CO2 incubator. Embryo grading should be done prior to media refreshment. Time out of the incubator during media refreshment will be assessed on day 3 and day 5. One operator should perform the ICSI. In case of multiple operators, each operator's injected oocytes will be split between the 2 groups.
day 3 refreshment	Insemination and culture	All MII oocytes will be inseminated by ICSI 39-41 hours post final oocyte maturation trigger. Following ICSI, oocytes will be cultured in 2-4 culture dishes placed in 2 separate chambers. Media refreshment: Zygote scoring on day 1 (18-20hpi). Culture medium refreshment will be done 67-69 hours post insemination (Day3). Embryo grading will not be done day2-5. Culture media preparation and refreshment: 30µL of culture medium (Global total LP is added to each well of the 8 wells of LifeGlobal GPS Dish (EGPS-010) and 50µL to the center 3 drops. Dishes will be equilibrated overnight in a CO2 incubator. Embryo grading should be done prior to media refreshment. Time out of the incubator during media refreshment will be assessed on day 3 and day 5. One operator should perform the ICSI. In case of multiple operators, each operator's injected oocytes will be split between the 2 groups.
day 3 refreshment	Culture media preparation and refreshment	All MII oocytes will be inseminated by ICSI 39-41 hours post final oocyte maturation trigger. Following ICSI, oocytes will be cultured in 2-4 culture dishes placed in 2 separate chambers. Media refreshment: Zygote scoring on day 1 (18-20hpi). Culture medium refreshment will be done 67-69 hours post insemination (Day3). Embryo grading will not be done day2-5. Culture media preparation and refreshment: 30µL of culture medium (Global total LP is added to each well of the 8 wells of LifeGlobal GPS Dish (EGPS-010) and 50µL to the center 3 drops. Dishes will be equilibrated overnight in a CO2 incubator. Embryo grading should be done prior to media refreshment. Time out of the incubator during media refreshment will be assessed on day 3 and day 5. One operator should perform the ICSI. In case of multiple operators, each operator's injected oocytes will be split between the 2 groups.
day 3 refreshment	Embryo grading	All MII oocytes will be inseminated by ICSI 39-41 hours post final oocyte maturation trigger. Following ICSI, oocytes will be cultured in 2-4 culture dishes placed in 2 separate chambers. Media refreshment: Zygote scoring on day 1 (18-20hpi). Culture medium refreshment will be done 67-69 hours post insemination (Day3). Embryo grading will not be done day2-5. Culture media preparation and refreshment: 30µL of culture medium (Global total LP is added to each well of the 8 wells of LifeGlobal GPS Dish (EGPS-010) and 50µL to the center 3 drops. Dishes will be equilibrated overnight in a CO2 incubator. Embryo grading should be done prior to media refreshment. Time out of the incubator during media refreshment will be assessed on day 3 and day 5. One operator should perform the ICSI. In case of multiple operators, each operator's injected oocytes will be split between the 2 groups.
day 5 refreshment	Insemination and culture	All MII oocytes will be inseminated by ICSI 39-41 hours post final oocyte maturation trigger. Following ICSI, oocytes will be cultured in 2-4 culture dishes placed in 2 separate chambers. Media refreshment and embryo grading timings: Zygote scoring on day 1 (18-20hpi). Embryo grading and culture media refreshment will be done 114-118 hours post insemination (Day5). Culture media preparation and refreshment: 30µL of culture medium (Global total LP is added to each well of the 8 wells of LifeGlobal GPS Dish (EGPS-010) and 50µL to the center 3 drops. Dishes will be equilibrated overnight in a CO2 incubator. Embryo grading and media refreshment is done prior to blastocyst biopsy. Time out of the incubator during media refreshment will be assessed on day 3 and day 5. One operator should perform the ICSI. In case of multiple operators, each operator's injected oocytes will be split between the 2 groups.
day 5 refreshment	Culture media preparation and refreshment	All MII oocytes will be inseminated by ICSI 39-41 hours post final oocyte maturation trigger. Following ICSI, oocytes will be cultured in 2-4 culture dishes placed in 2 separate chambers. Media refreshment and embryo grading timings: Zygote scoring on day 1 (18-20hpi). Embryo grading and culture media refreshment will be done 114-118 hours post insemination (Day5). Culture media preparation and refreshment: 30µL of culture medium (Global total LP is added to each well of the 8 wells of LifeGlobal GPS Dish (EGPS-010) and 50µL to the center 3 drops. Dishes will be equilibrated overnight in a CO2 incubator. Embryo grading and media refreshment is done prior to blastocyst biopsy. Time out of the incubator during media refreshment will be assessed on day 3 and day 5. One operator should perform the ICSI. In case of multiple operators, each operator's injected oocytes will be split between the 2 groups.
day 5 refreshment	Embryo grading	All MII oocytes will be inseminated by ICSI 39-41 hours post final oocyte maturation trigger. Following ICSI, oocytes will be cultured in 2-4 culture dishes placed in 2 separate chambers. Media refreshment and embryo grading timings: Zygote scoring on day 1 (18-20hpi). Embryo grading and culture media refreshment will be done 114-118 hours post insemination (Day5). Culture media preparation and refreshment: 30µL of culture medium (Global total LP is added to each well of the 8 wells of LifeGlobal GPS Dish (EGPS-010) and 50µL to the center 3 drops. Dishes will be equilibrated overnight in a CO2 incubator. Embryo grading and media refreshment is done prior to blastocyst biopsy. Time out of the incubator during media refreshment will be assessed on day 3 and day 5. One operator should perform the ICSI. In case of multiple operators, each operator's injected oocytes will be split between the 2 groups.
day 5 refreshment	Media refreshment Group 2	All MII oocytes will be inseminated by ICSI 39-41 hours post final oocyte maturation trigger. Following ICSI, oocytes will be cultured in 2-4 culture dishes placed in 2 separate chambers. Media refreshment and embryo grading timings: Zygote scoring on day 1 (18-20hpi). Embryo grading and culture media refreshment will be done 114-118 hours post insemination (Day5). Culture media preparation and refreshment: 30µL of culture medium (Global total LP is added to each well of the 8 wells of LifeGlobal GPS Dish (EGPS-010) and 50µL to the center 3 drops. Dishes will be equilibrated overnight in a CO2 incubator. Embryo grading and media refreshment is done prior to blastocyst biopsy. Time out of the incubator during media refreshment will be assessed on day 3 and day 5. One operator should perform the ICSI. In case of multiple operators, each operator's injected oocytes will be split between the 2 groups.

Primary Outcome Measures

Name	Time	Method
Blastocyst ploidy	3 days	determined after biopsy of trophectoderm cells, taken from the blastocyst on day 5, 6 or 7 from development

Secondary Outcome Measures

Name	Time	Method
Mosaicism Frequency	1 day	defined as the rate of mosaic errors in aneuploid embryos. This includes full mosaic embryos or aneuploid embryos with 1 or more mosaic chromosome aberration
Blastulation rate	5 days	ability of the embryo to form a cavity (=to blastulate) 114-118 hours post ICSI. Number of blastocysts divided by the number of 2PN
Cycle ploidy rate	1 day	the number of euploid embryos in the group divided by the total number of 2PN (both groups)
Blastocyst quality at time of biopsy	1 day	Four Embryo Quality Scores (EQ1, 2, 3, 4) based on the expansion stage of the blastocyst, the grade of the inner cell mass and the grade of the trophectoderm.

Trial Locations

Locations (1)

ART Fertility Clinics LLC; 🇦🇪 Abu Dhabi, United Arab Emirates