Aneuploidy Rates and Morphokinetic Parameters in Sibling Embryos Cultured in Distinct Culture Media
Phase: NA
Conditions: Embryonic Mosaicism, Embryo Culture Media, Embryo Morphokinetics, Infertility
Sponsor: University of California, San Francisco

Aneuploidy rates and morphokinetic parameters in sibling embryos cultured in distinct culture media
NCT03503877
Document Date: June 28, 2017

Title: Aneuploidy rates and morphokinetic parameters in sibling embryos cultured in 
distinct culture media 
Principal Investigator: Mitchell Rosen, MD 
Background:  
Embryonic aneuploidy is the underlying etiology for the majority of failed implantation and 
miscarriage. Preimplantation genetic screening (PGS) with transfer of a euploid embryo has 
been advocated as a strategy for increasing live birth rates with a single embryo transfer. 
Culturing embryos to the blastocyst stage for trophectoderm biopsy is a requirement for PGS. 
Several commercially-available single-step embryonic culture media with varying 
composition have been established for use in the IVF laboratory. Early reports have suggested 
differences in clinical outcomes, such as aneuploidy and miscarriage rates, with distinct 
culture media currently in standard use.1,2 However, there have been no clinical trials 
demonstrating the superiority of any one commercially-available culture media formulation. 
As a result, clinics use media with varying composition based upon familiarity and cost. 
Specific Aims: 
1. To determine if aneuploidy rates differ between sibling embryos cultured in distinct
media.
2. To determine if embryo quality differs between sibling embryos cultured in distinct
media.
3. To determine if morphokinetic parameters, as assessed by time lapse imaging, differ
between sibling embryos cultured in distinct media.
4. To determine if and how morphokinetic parameters and aneuploidy rates differ
between embryos with zona breach performed on day three of embryo development 
versus at the blastocyst stage. 
5. To assess the accuracy of non-invasive genetic testing of embryos from spent culture
media.
Design: 
Prospective sibling embryo study in an academic IVF laboratory comparing clinical 
parameters for embryos cultured in two different single-step culture media with time lapse 
imaging. 
Study Population: 
Couples planning in vitro fertilization (IVF) with intent for blastocyst culture for 
preimplantation genetic screening (PGS) will be offered enrollment prior to initiation of their 
treatment cycle. Intend to enroll 124 subjects (see sample size calculation below). 
Inclusion Criteria: 
All subjects planning blastocyst culture for preimplantation genetic screening (PGS) will be 
offered enrollment. 
AFC >8 
Male and female partner > 18 years of age 
Exclusion Criteria: 
Female partner age >42 years of age 
Cleavage stage biopsy 

 
Outcome Measures: 
Aneuploidy rate for embryos cultured in one of two distinct media from the same patient 
(Primary outcome) 
Embryo quality at cleavage and blastocyst stages 
Morphokinetic parameters from time lapse imaging 
 
Study Implementation: 
 
Couples planning in vitro fertilization (IVF) with intent for blastocyst culture for 
preimplantation genetic screening (PGS) will be offered enrollment prior to initiation of their 
treatment cycle. Ovarian stimulation will be performed with standard treatment chosen by the 
couple's primary physician. The day of ovulation trigger will be determined by the primary 
physician. Oocyte retrieval will be performed according to clinic standard 36 hours following 
ovulation trigger. All oocytes aspirated from the left ovary will be allocated to Global Media 
while all oocytes from the right ovary will be allocated to SAGE media (see supplement with 
media systems). Follicular fluid will be collected from a large and a medium-sized follicle 
from each ovary in order to perform a direct correlation of follicular size to oocyte recovery, 
maturation, and embryo development. The collected follicular fluid will also be assayed for 
estradiol, follicle stimulating hormone, leutinizing hormone, and progesterone. 
 
 
On the day of oocyte retrieval, the male partner will produce a semen sample per usual clinic 
protocol. Following 2-3 hours of incubation, hyaluronidase will be used to denude cumulus 
cells surrounding oocytes per usual clinic protocol. Fertilization will be performed by 
intracytoplasmic sperm injection (ICSI) of all mature oocytes by a trained embryologist. After 
16-18 hours, fertilization will be evaluated by the existence of two pro-nuclei. Fertilized 
embryos from each patient's cohort will remain split for culturing in SAGE or Global media 
systems as previously designated. All embryos will be cultured in the EmbryoScope+ time 
lapse incubator. Once placed in the EmbryoScope, embryos will be cultured at 37 degrees C 
with 6% CO2 and 5.5% O2 or up to 6 days without media exchange. 
 
The EmbryoScope+ incubation chamber contains a built-in microscope and camera allowing 
for continuous monitoring of embryonic development. Image acquisition software will be 
used to obtain high-contrast images every 10 minutes from several focal planes to create time-
lapse videos. Conventional embryonic assessment will be made by observations at pre-
specified time points. Cleavage-stage embryos will be assessed for cell number, symmetry, 
percentage fragmentation, evidence of multinucleation, and progression of compaction. 
Blastocysts will be evaluated to assess for blastocele volume and expansion, inner cell mass 
development, and trophectoderm organization. Additional morphokinetic parameters will be 
assessed with time lapse videos including time to pronuclear fading or syngamy (tPNf), time 
to 2-8 cells, time to morula, start of blastulation, blastocyst, and expanded blastocyst. 
Cleavage anomalies will be recorded. Zona breach will be performed with the use of a laser 
per usual clinical protocol on day 3 of embryo development or at the blastocyst stage. All 
spent media will be collected and sent for quantification of embryonic DNA content.  
 
Embryonic biopsy for preimplantation genetic screening will be performed at blastocyst stage. 
As per usual clinical protocol, on the day of biopsy, 5-10 trophectoderm cells will be gently 
aspirated. All embryos making it to the blastocyst stage will be eligible for biopsy, including 
low quality blastocysts. Arrested embryos may be biopsied to determine a cause for arrest. 

Preimplantation genetic screening will be performed per usual clinical protocol by 
PacGenomics.  All embryos will be vitrified following biopsy according to standard clinic 
protocol awaiting PGS results. Embryos subsequently determined to be euploid will be 
transferred as a controlled or natural cycle frozen embryo transfer according to standard 
clinical protocols. Number of embryos to be transferred will be determined by the patient's 
primary physician. 
 
Additional data will be collected on clinical pregnancy rate (ultrasound demonstrating 
gestational sac with yolk sac per transfer) and live birth rate. These data are routinely 
collected and reported to the Society for Assisted Reproductive Technology Clinical 
Outcomes Reporting System (SART CORS) according to the Fertility Clinic Success Rate 
and Certification Act of 1992. 
 
 
 
 
Statistical Analysis: 
The primary outcome is rate of aneuploidy for embryos cultured in one of two distinct media 
from the same patient. We will perform a paired t-test to compare number of euploid 
blastocysts in embryos split for culture in two different media from the same patient. Our 
secondary outcome is embryo quality for split embryos. Embryo quality will be graded at the 
cleavage and blastocyst stages. High quality cleavage embryos will have at least 6 
blastomeres or greater and scores of "1"or "2" for symmetry and fragmentation. Expanded 
blastocysts will be considered high quality where inner cell mass and trophectoderm score are 
"A" or "B." We will perform paired t-tests to compare number of high quality cleavage and 
blastocyst stage embryos from the cohort of sibling embryos cultured in different media.  
 
 
Sample Size Calculation: 
The primary outcome is aneuploid fraction for embryos cultured in two distinct media from 
the same patient. To detect same difference in euploid fraction as Hickman et al (29% vs 
16%) we would need 176 blastocysts per arm. This calculation does not account for clustering 
of embryos from the same patient; however, we will obtain a substantially higher number of 
blastocysts per arm as we are planning to power for our secondary outcome (see below). 
 
Our secondary outcome relates to differences in embryo quality among split embryos cultured 
in distinct media. Previous review of internal data demonstrates that interventions targeting 
couples with a history of poor embryo quality have been able to demonstrate a four-fold 
increase in high quality day 3 fraction. We need 62 subjects (31/arm) to detect four-fold 
increase in high quality day 3 fraction within this population with history of poor embryo 
quality (< 40% good quality cleavage stage fraction). We estimate that 25% of clinic patients 
have poor quality cleavage stage fraction. Therefore, would need 4 x 62= 248 patients. 
However, since we are splitting embryos from the same patient, each cohort of embryos 
serves as its own control. Therefore, we need to enroll 124 patients to power for our 
secondary outcome of clinically meaningful differences in embryo quality. 
 
 
 
References: 

1Campbell A, Smith R, Montgomery S et al. A prospective multi-center comparison of two 
different single step culture media using sibling embryos. In: Human Reproduction Vol 31, 
Suppl 1; 2016 July 3-6; Helsinki, Finland: ESHRE 2017. Abstract 265. 
 
2Hickman C, Wells D, Gwinnett D et al. Euploid rate sensitivity to laboratory culture 
environment: a blind, prospective, randomized, sibling study. In: Human Reproduction Vol 
31, Suppl 1; 2016 July 3-6; Helsinki, Finland: ESHRE 2017. Abstract 203. 
 
3Goodman LR, Goldberg J, Falcone T, Austin C, Desai N. Does the addition of time-lapse 
morphokinetics in the selection of embryos for transfer improve pregnancy rates? A 
randomized controlled trial. Vol 105 (2); 2016: 275-85. 
 
4Minasi MG, Colasante A, Riccio T, Ruberti A, Casciani V, Scarselli F et al. Correlation 
between aneuploidy standard morphology evaluation and morphokinetic development in 1730 
biopsied blastocysts: a consecutive case series study. Hum Reprod 31(10); 2016:2245-54. 
 
5Morbeck DE, Baumann NA, Oglesbee D. Composition of single-step media used for human 
embryo culture. Fertil Steril 107(4);2017: 1055-60.