Evaluation of the Impact of Reduced Oxygen Concentration on Embryonic Development
Condition: Infertility
MeSH terms: Infertility
Study type: Interventional

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)

        1. All care including oocyte retrieval, fertilization by ICSI, and cleavage stage culture
           (days 0, 1, and 2) will be completely per routine.

        2. 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

        3. 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.

        4. The number of ongoing day 3 embryos in each condition will then be recorded and given to
           data assessors.

        5. Embryos will not be examined or manipulated at all until day 5 of development as per
           routine protocol.

        6. 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.

        7. 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.

        8. 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.

        9. 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.
    

Intervention name(s): 2% oxygen concentration in the incubator
Intervention type(s): Other
Intervention description(s): 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.
Allocation: Randomized
Intervention model: Single Group Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Treatment
Keywords: Embryonic development; blastocyst stage