Embryo Development Clinical Trial
Official title:
Blastocyst Developmental Rate in Two Different Single Step Culture Media: a Sibling Oocyte Prospective Non-interventional Study
The aim of this non-interventional study is to compare the blastulation rate per fertilized oocyte in two different single step culture media commercially available (GEMS and Irvine culture media).
SUMMARY:
In ART procedures, blastocyst transfer can be considered the most physiological situation, as
the embryo is placed in the uterine cavity at a stage most similar to that which occurs in
nature. These potential benefits have induced more and more centres to move from cleavage
stage to blastocyst stage embryo transfer all over the world (Maheshwari et al., 2016).
Despite this, there are also some potential disadvantages. This approach decreases the total
number of usable embryos (defined as those available for transfer or cryopreservation)
(Glujovsky et al., 2016). Some concerns regarding the safety of this approach have also been
raised. In particular, the extended duration of the in vitro culture may have long-term
effect in the embryo development. In a recent meta-analysis, blastocyst stage transfer was in
fact associated with increased risks of preterm birth (<37 weeks), very preterm birth (<32
weeks), large for gestational age and perinatal mortality, although the latter was only
identified from one study. Conversely, blastocyst transfer was associated with a decrease in
the risk of small for gestational age and vanishing twins, although this latter was reported
by only one study (Martins et al., 2017).
Culture media plays a crucial role in this context. Two distinct approaches aimed at
supporting embryo development to the blastocyst stage have been proposed: the "back to
nature" sequential approach and the "let the embryo choose" single medium approach (Summers
et al., 2003; Machtinger et al., 2012, Quinn 2012).
Sequential culture media system is designed to meet identified changing metabolic and
nutritional requirements of the developing embryo from day 1 to day 5 (Gardner et al. 1996,
1997, 1998, 1999). With this approach, embryos are grown up to day 3 in a first medium and
then, at the cleavage stage, are moved to a second one. The goal is to expose the embryos to
stage-specific media, designed to reflect observed changes in concentrations of pyruvate,
lactate, and glucose in the Fallopian tube versus the uterus (Gardner et al., 1996).
On the other hand, single culture media aim at allowing developing embryos to choose the
nutrients they require, while at the same time minimizing stress from exposure to an abrupt
change in their culture environment on day 3. Single media can be used with a medium change
on day 3, or in a single-step uninterrupted culture, in which there is no medium refreshing
on day 3 [Biggers et al., 2008]. Available data comparing the performance of single vs.
sequential culture media suggest that both types of media seem to provide similar support to
the developing embryo (Sfontouris et al., 2017).
Aim of the study and design
The aim of this non-interventional study is to compare the blastulation rate per fertilized
oocyte in two different single step culture media commercially available (GEMS and Irvine
culture media).
Oocyte collection
Consecutive egg collection will be performed (with inclusion of oocytes coming from woman not
older than 42 years of age, presenting between 4 and 8 normal appearing MII oocytes and
undergoing ICSI treatment with ejaculated sperm in the Centre for Reproductive Medicine
GENERA in Rome). To exclude potential negative paternal effect on blastulation rate, all
oocytes coming from couples with surgically extracted spermatozoa and very severe
oligoastenoteratozoospermia (motile sperm count <500.000/ml after preparation) will be
excluded. Oocytes coming from patients enrolled in PGD program for monogenic diseases or
structural chromosomal abnormalities will be excluded too.
It is estimated, based centre experience, the study will last for 18 months. The informed
consent is the GENERA standard format in ordinary use. The study will be approved by the
Institutional Review Board of the Clinic.
Oocyte denudation, evaluation and injection All the procedures will be performed according to
standard practice without any kind of intervention.
Just after the pick-up procedure, oocytes will be denuded from the cumulus oophorus by a
brief exposure to 40 IU/ml hyaluronidase solution followed by mechanical removal of the
corona radiata with the use of plastic pipettes of defined diameters in a controlled
temperature environment. This procedure will be performed between 37 and 40 hours post hCG
administration. Metaphase II (MII) oocytes will be then separated from the immature oocytes
and evaluated at the stereomicroscope. Those with severe morphological abnormalities will be
considered of lower quality (according to Rienzi et al., 2008) and will thus be excluded from
comparison. All the MII oocyte will be placed in the same Petri Dish.
Using randomization tables, oocytes from each patient will be randomly split in two groups
that will be independently inseminated and subsequently kept in 2 single step culture media,
GEMS (GERI Medium) and Irvine (Continuous Single Culture Complete with HSA). Oocytes will be
subjected to ICSI using previously described techniques and instrumentations (Rienzi et al.,
1998). To be able to follow the developmental progression of individual oocyte, inseminated
oocytes from each group will be cultured in separate dishes and each individual oocyte will
be identified according to its position within the dish microwells. Embryo culture will last
up to day 5 and will be performed in a time-lapse incubator (GERI, Genea) in hypoxic
atmosphere containing 6%CO2 and 5%O2.
Embryo assessment
As per GENERA procedure, detailed analysis of various events during embryo development
(syngamy, cleavages, compaction and blastulation) will be assessed using morphokinetic
analysis. Various parameters will be assessed: time between the end of the ICSI procedure and
syngamy, completion of cleavage to 2, 3, 4, 5 and 8 cells (T2, T3, T4, T5, T8, respectively);
length of the first, second and third cell cycle (CC1, CC2, and CC3 respectively) and
synchrony in the division from three to four and five to eight cells (S2 and S3,
respectively). Standard blastocyst morphological assessment will be performed according to
the criteria reported by Gardner and Schoolcraft (1999). In brief, the blastocysts will be
evaluated according to the degree of expansion, quality of the inner cell mass (ICM) and of
the trophectoderm cells (TE). The ICM is evaluated according to the number of cells, and the
degree of compaction, while the TE is evaluated according to the number, dimension of the
cells and the appearance of the epithelium (cohesive or loose). Morphokinetic parameters
related to blastocyst formation will also be recorded and in particular: initiation of
compaction, initiation of blastulation, and completion of blastulation (TSC, TSB, TB,
respectively).
Statistical Considerations The primary study endpoint is the blastulation rate, computed as
the percentage of fertilized eggs, cultured in either medium, that develop to the blastocyst
stage in each half of the cohort. Since only women with between 4 and 8 retrieved oocytes
will be eligible, and oocytes from each woman will be randomly split in 2 groups, assuming a
fertilization rate of 80% the study population to be analysed will be composed by X*2 (where
X is the number of enrolled women) groups of 2+ oocytes, half cultured in one medium and the
other cultured in the second medium. The most obvious approach to the analysis of the
resulting data is to consider it as a set of X (= number of women) 2x2 tables, where the
quantity to be estimated is the odds ratio of blastulation of one medium relative to the
other, to be analysed by means of the Mantel Haenszel procedure, with woman as the
stratification factor (primary analysis). Assuming an overall blastulation rate of 34% a
minimal relevant difference of 7% corresponds to an odds ratio of blastulation of 1.4. In
order to detect with alfa=5% (2-sided) and power = 80% an odds ratio of 1.4, approximately
1200 (1182) oocytes need to be included (Hulley et al., 2013) split in 2 groups of equal
sizes, that correspond approximately to 160 - 200 women (aprox. 6 oocytes/woman retrieved and
considering a fertilization rate 80%).
In secondary exploratory analysis, a logistic model will be fitted to the data with
blastulation as the dependent binary variable and woman as stratum, to assess the predictive
value of various prognostic factors and the possible interactions between each of them and
the culture medium.
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