Our study will include two phases. In Phase I (a retrospective study), archived data will
be collected to train the CNN-enhanced MK with RS method on embryo selection, leading to
the integrated approach (MK-RS). In Phase II (a prospective study), the integrated MK-RS
method established will be used to select embryos, assess the clinical pregnancy rate and
evaluate the efficacy of our approach in a randomized controlled trial.
In Phase I, images of the embryo will be captured every 10 minutes by the in-built
microscope and camera in the time-lapse incubator. Images will then be assessed by the
CNN algorithm for day one human embryo segmentation to identify three distinct features:
the zona pellucida (ZP), cytoplasm and pronucleus (PN). The morphodynamics of these three
features during the fertilisation to first division will be wrapped up as time series
data for the integration. The morphology changes after the first division will be
semi-auto annotated, which will be analysed by the commercial MK scoring system (KID
Score).
After removal of embryos/blastocysts from the culture dish, the corresponding spent
culture medium (SCM) will be collected in sterile polymerase chain reaction (PCR) tubes.
Blank culture mediums will also be collected with the same operating standard. A
specifically designed sampler will be used to pipette 7μL SCM of each sample, passing
through the oil layer of the SCM, and then drop it onto a disposable quartz glass slide
and illuminated by RS system (Basecare Raman 200, China). The RS system will be
calibrated to 520.5 cm-1 by silicon wafer before testing. Laser excitation parameters are
set as follows: 785 nm wavelength, 320 mW power and 100 μm laser spot diameter. Signals
are captured in standard mode with a chargecoupled device (CCD) camera with a 20-seconds
integration time. Three replicates will be done for each aliquot. Re-calibration is
essential when different culture media is tested, considering that G-1 medium is used for
embryos before Day 3 and G-2 medium is used for embryos after Day 3. All obtained spectra
will be pre-processed by subtracting the background signal. Spectroscopy signals within
the near-infrared region (600 cm-1- 1800 cm-1) are analysed for vector normalisation
using Labspec 6 software (Horiba, Japan). Our previous SCM samples with known TE ploidy
results will be used as a training dataset to establish euploid-aneuploid classification
standards. Stacking classification algorithm will be adopted, considering its high
overall accuracy (95.9%, unpublished data).
As the segmented time series of CNN algorithm, KID Score annotations and RS profiling
results have hundreds of subparameters, we will assemble them together with the ensemble
learning, which considers each subparameter as a weak classifier and re-allocated their
weights during the training. The primary index for the training is the clinical pregnancy
outcome and for the ultimation of the information beneath CNN-enhanced MK and RS, the
blastocyst formation results will be used as a secondary index. The ratio of the training
set and test set will be 1:1 and in the training set, a 5-fold cross-validation will be
performed for monitoring the overfitting.
Phase II will comprise a prospective, single-blinded, randomised controlled trial
designed to validate the trained MK-RS method for embryo selection. Metabolomic SCM
profiling using RS with CNN-enhanced MK analysis will be used to assess embryo
developmental potential along with traditional morphological embryo assessments. The
embryo developmental potential results will be used to select the best quality of
embryos. Sensitivity and specificity will be assessed using the patient's TE biopsy or
non-invasive prenatal testing (NIPT) results will further confirm the scoring of MK-RS
method, if available.
Randomisation:
In Phase II, participants who fulfil all the inclusion and exclusion criteria and consent
to join the study will be randomised into experimental group and control group in 1 to 1
ratio using a computer-generated randomisation list. For the experimental group, embryo
selection will be based on the MK-RS method established in Phase I, whereas embryo
selection for the control group will rely on the traditional embryo morphology grading
results alone. All participants will be blinded in this trial.