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Evaluation of the Impact of Reduced Oxygen Concentration on Embryonic Development (LO2)

Primary Purpose

Infertility

Status
Completed
Phase
Not Applicable
Locations
United States
Study Type
Interventional
Intervention
2% oxygen concentration in the incubator
Sponsored by
Reproductive Medicine Associates of New Jersey
About
Eligibility
Locations
Arms
Outcomes
Full info

About this trial

This is an interventional treatment trial for Infertility focused on measuring Embryonic development, blastocyst stage

Eligibility Criteria

18 Years - 40 Years (Adult)FemaleAccepts Healthy Volunteers

Inclusion Criteria:

  1. Age 18-40 years and seeking IVF with aneuploidy screening, which is our current recommendation regardless of study participation
  2. Anti-mullerian hormone level (AMH) > 1.0 pmol/L (an assessment of ovarian reserve)
  3. Must have at least two surviving embryos on day 3 of development
  4. Male partner with >100,000 total motile spermatozoa per ejaculate (donor sperm acceptable)
  5. Body Mass Index < 35

Exclusion Criteria:

  1. Diagnosis of endometrial insufficiency, as defined by prior cycle with maximal endometrial thickness <6mm, abnormal endometrial pattern (failure to attain a trilaminar appearance), or persistent endometrial fluid
  2. Use of oocyte donation
  3. Use of gestational carrier
  4. Use of sperm obtained via surgical procedure
  5. Presence of hydrosalpinges that communicate with endometrial cavity
  6. Single gene disorders, chromosomal translocations or any other disorders requiring more detailed embryo genetic analysis
  7. Couples seeking gender selection for family balancing

Sites / Locations

  • Reproductive Medicine Associates of New Jersey

Arms of the Study

Arm 1

Arm 2

Arm Type

Experimental

No Intervention

Arm Label

2% oxygen concentration in the incubator

Control

Arm Description

At the time that embryos are changed from "cleavage stage media" to "blastocyst stage media" on day 3 of development, half of a given patient's embryos will randomly be placed in an incubator set at 2% oxygen concentration. The splitting of the embryos will be done under low magnification such that the embryologist will have no ability to bias allocation of embryos to 2% or 5% oxygen based on embryo morphology on day 3. The embryos will remain in this incubator until their developmental assessments on day 5 and 6.

Embryos in this arm will be cultured at 5% oxygen (current standard of care) from day 3 until blastocyst developmental assessment.

Outcomes

Primary Outcome Measures

Blastulation Rate (number of embryos meeting criteria for biopsy and/cryopreservation divided by number of embryos randomized on day 3 to either the experimental or control arm)
On day 5 and day 6, all embryos are examined under the microscope to see if they 1) meet developmental criteria for embryo biopsy (for chromosomal evaluation) and cryopreservation (since all embryos in this program are cryopreserved while awaiting results from chromosome assessment), or 2) arrest development in the laboratory.

Secondary Outcome Measures

Clinical Pregnancy rate
After embryos are cryopreserved, embryos with the proper number of chromosomes (so called euploid embryos) will be available for transfer to the uterus (as is standard protocol in our center). Clinical pregnancy rate will be defined by the presence of a gestational sac on ultrasound in the embryo transfer cycle.

Full Information

First Posted
September 27, 2016
Last Updated
June 25, 2018
Sponsor
Reproductive Medicine Associates of New Jersey
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1. Study Identification

Unique Protocol Identification Number
NCT02919384
Brief Title
Evaluation of the Impact of Reduced Oxygen Concentration on Embryonic Development
Acronym
LO2
Official Title
Evaluation of the Impact of Reduced Oxygen Concentration on Embryonic Development
Study Type
Interventional

2. Study Status

Record Verification Date
June 2018
Overall Recruitment Status
Completed
Study Start Date
October 6, 2016 (Actual)
Primary Completion Date
February 10, 2017 (Actual)
Study Completion Date
May 2018 (Actual)

3. Sponsor/Collaborators

Responsible Party, by Official Title
Sponsor
Name of the Sponsor
Reproductive Medicine Associates of New Jersey

4. Oversight

Data Monitoring Committee
No

5. Study Description

Brief Summary
During this study, patients will undergo a routine in vitro fertilization cycle as they would otherwise if not participating in the study. After eggs have been fertilized they will be cultured as usual until day 3 of embryo development. On day 3 of development, the embryologist will randomize half of the embryos to be cultured in 2% oxygen concentration and the other half at 5%, which is currently the standard of care. All other embryological care procedures will remain the same. On day 5 or 6 of embryo development, the embryos will be evaluated and each blastocyst stage embryo will be recorded. The primary outcome will be the blastulation rate (or percentage of embryos that reach the blastocyst stage).
Detailed Description
Significant progress has been made in characterizing the optimal environment for a developing embryo in culture. These efforts have been based on the premise that clinical embryo culture should mimic the in vivo environment. To this end, investigators have gone to great lengths to recreate every aspect of the natural setting to which the early embryo is exposed. This focused approach has led to significant modifications of the embryo culture system in the modern in vitro fertilization (IVF) lab and ultimately to improvements in pregnancy rates. One area that has been subject to significant scrutiny is the relationship between incubator oxygen concentration and early embryonic development. Oxygen plays a central role in embryonic metabolism. The mechanism governing its utilization is dependent on the stage of embryonic development. During the first 3 days of development, oxygen reaches the embryo via passive diffusion and its concentration gradient is regulated by oxygen consumption during oxidative phosphorylation. Inefficiencies in this process - due to compromised integrity of the inner mitochondrial membrane or alterations in substrate availability - can result in excessive production of harmful reactive oxygen species which can cause significant damage to cellular machinery and ultimately lead to embryonic arrest. The concentration of oxygen that the embryo in culture is exposed to can also impact this delicately balanced system and alter the metabolic health of an embryo. Historically, atmospheric oxygen concentration (approximately 20%) was exclusively used in human IVF laboratories for embryo culture. However, multiple investigations subsequently found that the physiologic concentration of oxygen within the female reproductive tract is well below atmospheric levels, being consistently measured at <10%. These observations led to multiple trials comparing atmospheric oxygen concentrations to 5% oxygen in embryo culture. These studies demonstrated significant perturbations in gene expression, protein secretion, and suboptimal utilization of amino acids and carbohydrates in embryos cultured in atmospheric oxygen. The same comparisons were made in clinical IVF studies and demonstrated that embryos cultured in 5% oxygen consistently resulted in an increase in clinical pregnancy rate and live birth rate. A meta-analysis of this topic suggested that a clinic with a baseline live birth rate of 30% could expect an improvement as great as 13% when culturing embryos at 5% O2. As a result of these compelling data, most modern IVF programs now exclusively culture embryos at 5% oxygen concentration. However, some have proposed that the oxygen concentration to which the embryo is exposed after day 3 of development is actually lower than 5%. These data originate from the idea that the embryo crosses the utero-tubal junction on day 3 of development in vivo. Multiple studies have demonstrated that the oxygen concentration in the uterus is actually lower than that in the fallopian tube at approximately 2%. Thus, the most physiologic embryo culture system would culture embryos in 5% oxygen until day 3 and then decrease the oxygen concentration to 2% until transfer or cryopreservation on day 5 or 6. A change in the optimal oxygen concentration for an embryo on day 3 would fit with a general shift in metabolic requirements of embryos seen at this stage of development. Activation of the embryonic genome occurs on day 3 which prompts a significant increase in biosynthetic activity. The metabolic behavior of embryos also shifts substantially during this time. The embryo changes its metabolic strategy from oxidative phosphorylation to glucose based metabolism in the form of the aerobic glycolysis and the citric acid cycle. During this process, termed compaction, embryos exhibit greatly increased oxygen consumption. The physiologic environment of the female reproductive tract tends to mirror the metabolic needs of the developing preimplantation embryo. As the embryo shifts its metabolic strategy after compaction and upon entering the uterus, it is certainly possible that a reduced oxygen concentration in the uterus may best support the energy producing mechanisms of this stage in embryonic development. Recapitulating this environment in culture may enhance embryonic development and long term health of pregnancies resulting from IVF. In fact a recent pilot study, using embryos donated to research demonstrated improved embryo development when embryos were cultured in 2% oxygen after day 3 of development compared to 5% (Kaser et al.) Purpose of Proposed Study This study seeks to compare laboratory outcomes of embryos cultured at 2% oxygen concentration and 5% oxygen concentration after compaction. The primary outcome under study will be the proportion of day 3 embryos that are ongoing at the blastocyst stage (known throughout the rest of this document as blastulation rate). A. Location of Study: Patients will be recruited at both participating clinical centers. Oocyte retrieval, embryology, embryo biopsy, and embryo transfer will occur at the clinical facilities of Reproductive Medicine Associates of New Jersey. All genetic diagnostics will occur at the Foundation for Embryonic Competence (FEC). B. Study Population: Infertile couples attempting conception through in vitro fertilization. 60 couples will be enrolled in the study (see sample size calculation below). STUDY PROCEDURES Experimental Design The purpose of the study is to evaluate the embryological impact of 2% oxygen versus 5% concentrations in embryo culture after day 3 of development. As such, the study related procedures begin only after a patient's embryos reach day 3 of development. The experimental design for this study is as follows: (Figure 1) All care including oocyte retrieval, fertilization by ICSI, and cleavage stage culture (days 0, 1, and 2) will be completely per routine. It is standard protocol in our laboratory to remove embryos from the incubator on day 3 to 1) perform assisted hatching and 2) change embryos from cleavage stage media to blastocyst media At time of assisted hatching, two 5-well extended culture dishes will be brought to the isolette where changeover into extended culture will take place. The configuration of this 5-well dish is as follows. One mL of blastocyst media is placed in the center well of the 5-well dish. This well is only used for washing of cleavage stage embryos prior to placement into blastocyst media drops. The outer four wells contain small drops for ongoing blastocyst culture. As part of the study, once assisted hatching is complete, all embryos will be placed into the center well and mixed in the 1 mL of blastocyst media. At low power magnification and with no ability to perform day 3 grading, half of the embryos will be placed into the outer drops in one 5-well dish and half will be placed in the outer drops of the other 5-well dish. These two dishes will then be separated to the left and right side of the isolette. An embryologist, will then open a sequentially numbered, opaque sealed envelope from the box marked "oxygen randomization." The piece of paper in this envelope will direct the embryologist to place the 5-well dish on the left side of the isolette to either the 2 or 5% oxygen concentration incubator. The 5-well dish on the right side of the incubator will go to the opposite condition. The number of ongoing day 3 embryos in each condition will then be recorded and given to data assessors. Embryos will not be examined or manipulated at all until day 5 of development as per routine protocol. On day 5 or 6 of embryo development, the embryos will be evaluated and the number of ongoing blastocysts will be recorded for embryos cultured in each condition. All ongoing embryos will then undergo a trophectoderm biopsy using the standardized technique according to standard laboratory protocol without regard to study. Embryos will then be cryopreserved to allow for results from comprehensive chromosomal screening to be available and to optimize embryonic-endometrial synchrony, as per standard protocol. Further decisions regarding the number of euploid embryos to transfer will be up to the patient. Data will be collected regarding pregnancy outcomes according to the culture conditions that a transferred embryo was exposed. However, all of this information will be analyzed as secondary outcomes. Pregnancy testing and follow up will not be altered. RANDOMIZATION SCHEMA Simple randomization will be performed independently at the study site (RMANJ). A random number sequence will be generated by random.org. Odd numbers will be assigned to A intervention (2%) oxygen and even numbers will be assigned to B intervention (5%). Pieces of paper containing the letter dictated by random number sequence will be placed into a box of sequentially numbered, opaque sealed envelopes labelled "oxygen randomization." As noted above, on day 3, after placing half of the embryos into one 5-well dish and half of the embryos into another 5-well dish, the embryologist will separate those dishes to the left and right hand side of the incubator. The embryologist, will then open a sequentially numbered, opaque sealed envelope from this box marked "oxygen randomization." The piece of paper in this envelope will direct the embryologist to place the 5-well dish on the left side of the isolette to either the 2 or 5% oxygen concentration incubator. The 5-well dish on the right side of the incubator will go to the opposite condition. In the case of an odd number of embryos, the extra embryo will always be placed in the left handed 5-well dish. The numbers are anticipated to even out as the left sided dish has an equal chance of randomization to 2 or 5% oxygen concentration.

6. Conditions and Keywords

Primary Disease or Condition Being Studied in the Trial, or the Focus of the Study
Infertility
Keywords
Embryonic development, blastocyst stage

7. Study Design

Primary Purpose
Treatment
Study Phase
Not Applicable
Interventional Study Model
Single Group Assignment
Masking
ParticipantCare ProviderInvestigatorOutcomes Assessor
Allocation
Randomized
Enrollment
60 (Actual)

8. Arms, Groups, and Interventions

Arm Title
2% oxygen concentration in the incubator
Arm Type
Experimental
Arm Description
At the time that embryos are changed from "cleavage stage media" to "blastocyst stage media" on day 3 of development, half of a given patient's embryos will randomly be placed in an incubator set at 2% oxygen concentration. The splitting of the embryos will be done under low magnification such that the embryologist will have no ability to bias allocation of embryos to 2% or 5% oxygen based on embryo morphology on day 3. The embryos will remain in this incubator until their developmental assessments on day 5 and 6.
Arm Title
Control
Arm Type
No Intervention
Arm Description
Embryos in this arm will be cultured at 5% oxygen (current standard of care) from day 3 until blastocyst developmental assessment.
Intervention Type
Other
Intervention Name(s)
2% oxygen concentration in the incubator
Intervention Description
At the time that embryos are changed from "cleavage stage media" to "blastocyst stage media" on day 3 of development, half of a given patient's embryos will randomly be placed in an incubator set at 2% oxygen concentration. The splitting of the embryos will be done under low magnification such that the embryologist will have no ability to bias allocation of embryos to 2% or 5% oxygen based on embryo morphology on day 3. The embryos will remain in this incubator until their developmental assessments on day 5 and 6.
Primary Outcome Measure Information:
Title
Blastulation Rate (number of embryos meeting criteria for biopsy and/cryopreservation divided by number of embryos randomized on day 3 to either the experimental or control arm)
Description
On day 5 and day 6, all embryos are examined under the microscope to see if they 1) meet developmental criteria for embryo biopsy (for chromosomal evaluation) and cryopreservation (since all embryos in this program are cryopreserved while awaiting results from chromosome assessment), or 2) arrest development in the laboratory.
Time Frame
6 days of embryonic development in the laboratory
Secondary Outcome Measure Information:
Title
Clinical Pregnancy rate
Description
After embryos are cryopreserved, embryos with the proper number of chromosomes (so called euploid embryos) will be available for transfer to the uterus (as is standard protocol in our center). Clinical pregnancy rate will be defined by the presence of a gestational sac on ultrasound in the embryo transfer cycle.
Time Frame
2 months
Other Pre-specified Outcome Measures:
Title
Live birth rate
Description
Delivery of an alive baby will be the criteria for live birth rate assessment
Time Frame
10 months

10. Eligibility

Sex
Female
Minimum Age & Unit of Time
18 Years
Maximum Age & Unit of Time
40 Years
Accepts Healthy Volunteers
Accepts Healthy Volunteers
Eligibility Criteria
Inclusion Criteria: Age 18-40 years and seeking IVF with aneuploidy screening, which is our current recommendation regardless of study participation Anti-mullerian hormone level (AMH) > 1.0 pmol/L (an assessment of ovarian reserve) Must have at least two surviving embryos on day 3 of development Male partner with >100,000 total motile spermatozoa per ejaculate (donor sperm acceptable) Body Mass Index < 35 Exclusion Criteria: Diagnosis of endometrial insufficiency, as defined by prior cycle with maximal endometrial thickness <6mm, abnormal endometrial pattern (failure to attain a trilaminar appearance), or persistent endometrial fluid Use of oocyte donation Use of gestational carrier Use of sperm obtained via surgical procedure Presence of hydrosalpinges that communicate with endometrial cavity Single gene disorders, chromosomal translocations or any other disorders requiring more detailed embryo genetic analysis Couples seeking gender selection for family balancing
Overall Study Officials:
First Name & Middle Initial & Last Name & Degree
Scott J Morin, MD
Organizational Affiliation
Reproductive Medicine Associates of New Jersey
Official's Role
Principal Investigator
Facility Information:
Facility Name
Reproductive Medicine Associates of New Jersey
City
Basking Ridge
State/Province
New Jersey
ZIP/Postal Code
07920
Country
United States

12. IPD Sharing Statement

Plan to Share IPD
No
Citations:
PubMed Identifier
8107053
Citation
Fischer B, Bavister BD. Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits. J Reprod Fertil. 1993 Nov;99(2):673-9. doi: 10.1530/jrf.0.0990673.
Results Reference
background
PubMed Identifier
22786519
Citation
Bontekoe S, Mantikou E, van Wely M, Seshadri S, Repping S, Mastenbroek S. Low oxygen concentrations for embryo culture in assisted reproductive technologies. Cochrane Database Syst Rev. 2012 Jul 11;(7):CD008950. doi: 10.1002/14651858.CD008950.pub2.
Results Reference
background
PubMed Identifier
23312219
Citation
Gardner DK, Wale PL. Analysis of metabolism to select viable human embryos for transfer. Fertil Steril. 2013 Mar 15;99(4):1062-72. doi: 10.1016/j.fertnstert.2012.12.004. Epub 2013 Jan 8.
Results Reference
background
PubMed Identifier
18927130
Citation
Meintjes M, Chantilis SJ, Douglas JD, Rodriguez AJ, Guerami AR, Bookout DM, Barnett BD, Madden JD. A controlled randomized trial evaluating the effect of lowered incubator oxygen tension on live births in a predominantly blastocyst transfer program. Hum Reprod. 2009 Feb;24(2):300-7. doi: 10.1093/humrep/den368. Epub 2008 Oct 16.
Results Reference
background

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Evaluation of the Impact of Reduced Oxygen Concentration on Embryonic Development

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