Evaluation of the Impact of Reduced Oxygen Concentration on Live Birth Rate
| Status: | Recruiting |
|---|---|
| Conditions: | Women's Studies, Infertility |
| Therapuetic Areas: | Reproductive |
| Healthy: | No |
| Age Range: | 18 - 40 |
| Updated: | 4/6/2019 |
| Start Date: | May 15, 2017 |
| End Date: | December 1, 2021 |
| Contact: | Christine Whitehead, BSN |
| Email: | cwhitehead@rmanj.com |
| Phone: | 9736562089 |
Lo2 Phase II: A Comparison of Live Birth Rates Between Embryos Cultured in 2% vs. 5% After Day 3 of Embryo Culture
Clinical in vitro fertilization relies on successful embryo culture. The primary goal of
embryo culture is to attempt to recapitulate the in vivo conditions as much as possible. In
the past decade, the majority embryo culture has been performed at 5% oxygen due to the
discovery that the oxygen tension in the fallopian tube (where the embryo is located for the
first 3 days after fertilization) is 5%. However, relatively recent studies have demonstrated
that the oxygen tension in the uterus (where the embryo is located after day 3) is closer to
2%. This study is a randomized controlled trial that will compare pregnancy rates between
embryos cultured in 2% versus 5% after day 3 of development.
embryo culture is to attempt to recapitulate the in vivo conditions as much as possible. In
the past decade, the majority embryo culture has been performed at 5% oxygen due to the
discovery that the oxygen tension in the fallopian tube (where the embryo is located for the
first 3 days after fertilization) is 5%. However, relatively recent studies have demonstrated
that the oxygen tension in the uterus (where the embryo is located after day 3) is closer to
2%. This study is a randomized controlled trial that will compare pregnancy rates between
embryos cultured in 2% versus 5% after day 3 of 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.
This theory has been corroborated in two recent pilot studies. The first study, recently
awarded the Prize Paper Award at the 2016 American Society for Reproductive Medicine
Scientific Meeting, randomized embryos donated to research to 2% or 5% oxygen concentration
after day 3 of development and found that embryos were twice as likely to blastulate if they
were cultured in 2% oxygen concentration. However, embryos studied in this investigation were
either abnormally fertilized or warmed from cryopreservation on day 3 after being donated to
research. None were intended for clinical use.
With these limitations in mind, our group has recently completed a study utilizing embryos
intended for clinical use (Copernicus IRB: RMA-2016-02). In this study, we enrolled 60
patients and split all ongoing embryos on day 3 of development to culture in either 2% or 5%
oxygen until the blastocyst stage. The results have not yet been published, but culture in 2%
oxygen after day 3 produced more blastocysts than culture in 5% oxygen. In total, 30 patients
had more embryos reach blastocyst in 2% oxygen, while only 17 patients had more embryos reach
blastocyst in 5% oxygen. Ten patients had an equal number in both conditions (3 withdrew from
the study). This difference reached statistical significance.
This initial study was designed to specifically evaluate the developmental performance of
embryos in both conditions. However, there are also some pregnancy data available from those
patients who have proceeded to transfer these embryos. In this initial study, culture
conditions had no impact on the decision of which embryo to transfer. This was blinded to
embryologists and patients and only typical morphology criteria were utilized. Again, of
patients who have proceeded to embryo transfer, embryos that had been cultured in 2% oxygen
have performed better than those exclusively in 5% (88.9% [16/18] vs. 62.5% [15/24]
However, this initial study was not designed to specifically test the question of whether 2%
versus 5% oxygen in extended culture produces better pregnancy outcomes. The patients were
not randomized to either 2% or 5% oxygen. Thus, current pregnancy data is prone to some bias
and a better controlled study is needed evaluate the impact of reduced oxygen concentration
in culture on the most important outcomes: live birth rate.
Purpose of Proposed Study This study seeks to compare clinical outcomes of embryos cultured
at 2% oxygen concentration and 5% oxygen concentration after compaction. The primary outcome
under study will be live birth rate. Secondary outcomes will include miscarriage rate,
gestational age at delivery, birth weight at delivery, embryo blastulation rate, embryo
ploidy status (aneuploid or euploid) and morphologic parameters (expansion, inner cell mass
grade, trophectoderm grade). The cumulative live birth rate of all embryos that originate
from the study stimulation cycle will also be collected and analyzed. This will theoretically
reveal whether any advantage in the number of available blastocysts for transfer translates
to greater reproductive efficiency when extended culture is performed at 2% oxygen.
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.
This theory has been corroborated in two recent pilot studies. The first study, recently
awarded the Prize Paper Award at the 2016 American Society for Reproductive Medicine
Scientific Meeting, randomized embryos donated to research to 2% or 5% oxygen concentration
after day 3 of development and found that embryos were twice as likely to blastulate if they
were cultured in 2% oxygen concentration. However, embryos studied in this investigation were
either abnormally fertilized or warmed from cryopreservation on day 3 after being donated to
research. None were intended for clinical use.
With these limitations in mind, our group has recently completed a study utilizing embryos
intended for clinical use (Copernicus IRB: RMA-2016-02). In this study, we enrolled 60
patients and split all ongoing embryos on day 3 of development to culture in either 2% or 5%
oxygen until the blastocyst stage. The results have not yet been published, but culture in 2%
oxygen after day 3 produced more blastocysts than culture in 5% oxygen. In total, 30 patients
had more embryos reach blastocyst in 2% oxygen, while only 17 patients had more embryos reach
blastocyst in 5% oxygen. Ten patients had an equal number in both conditions (3 withdrew from
the study). This difference reached statistical significance.
This initial study was designed to specifically evaluate the developmental performance of
embryos in both conditions. However, there are also some pregnancy data available from those
patients who have proceeded to transfer these embryos. In this initial study, culture
conditions had no impact on the decision of which embryo to transfer. This was blinded to
embryologists and patients and only typical morphology criteria were utilized. Again, of
patients who have proceeded to embryo transfer, embryos that had been cultured in 2% oxygen
have performed better than those exclusively in 5% (88.9% [16/18] vs. 62.5% [15/24]
However, this initial study was not designed to specifically test the question of whether 2%
versus 5% oxygen in extended culture produces better pregnancy outcomes. The patients were
not randomized to either 2% or 5% oxygen. Thus, current pregnancy data is prone to some bias
and a better controlled study is needed evaluate the impact of reduced oxygen concentration
in culture on the most important outcomes: live birth rate.
Purpose of Proposed Study This study seeks to compare clinical outcomes of embryos cultured
at 2% oxygen concentration and 5% oxygen concentration after compaction. The primary outcome
under study will be live birth rate. Secondary outcomes will include miscarriage rate,
gestational age at delivery, birth weight at delivery, embryo blastulation rate, embryo
ploidy status (aneuploid or euploid) and morphologic parameters (expansion, inner cell mass
grade, trophectoderm grade). The cumulative live birth rate of all embryos that originate
from the study stimulation cycle will also be collected and analyzed. This will theoretically
reveal whether any advantage in the number of available blastocysts for transfer translates
to greater reproductive efficiency when extended culture is performed at 2% oxygen.
Inclusion Criteria:
- Anti-mullerian hormone level (AMH) > 1.0 ng/mL
- Must have at least one surviving embryo on day three 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
- Completion of the protocol requires a single embryo transfer of an embryo as part of
the study. Thus, patients pursuing embryo banking cycles will be excluded from the
study.
- Double embryo transfer
We found this trial at
2
sites
Basking Ridge, New Jersey 07960
Principal Investigator: Scott J Morin, M.D.
Phone: 973-656-2841
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