Evaluation of the Impact of Reduced Oxygen Concentration on Embryonic Development

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.

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