Morphometrics and Morphokinetics of Thawed Oocytes and Embryos
Condition: Infertility
MeSH terms: Infertility
Study type: Observational

Summary: 
      The aim of this observational study is to describe for the first time a complete set of
      morphometric and morphokinetic parameters of frozen-thawed human embryos in order to select
      the embryo with the highest implantation potential. These parameters will be correlated with
      (1) data of the fresh embryos and their subsequent survival, the evolution of the contact
      surfaces between surviving blastomeres and the resumption of mitosis; (2) patient
      characteristics and clinical outcome of frozen-thawed embryos and (3) a comparison between
      embryos cryopreserved with vitrification or the slow method regarding these parameters will
      be performed.

      This project aims to introduce new evaluation criteria of the frozen-thawed embryos to
      improve success rate of FET cycles. By establishing new precise, reliable and non-invasive
      measurable parameters, we aim to (1) select which supernumerary embryos are at chance to
      survive the freezing/thawing procedures, (2) establish cut off parameters for the survival
      rate of the embryo, (3) assess implantation, pregnancy and live birth rates depending on the
      developmental characteristics after thawing and after 24h of culture.

      Our strategy for improving the outcome of FET cycles is based on the study of morphometric
      and morphokinetic parameters in frozen-thawed embryos and the correlation with embryological,
      clinical and cycle parameters. Transferring a well-defined thawed embryo with a high
      implantation potential may lead towards an increase in the implantation and life birth rate
      and an increase in the cumulative live birth rate with a reduction of the IVF costs in order
      to optimize the health-economic situation of reproductive medicine.
    

Detailed description: 
      Cryopreservation of supernumerary embryos is an essential part of Assisted Reproduction
      Techniques (ART) and is nowadays a standard routine procedure in the IVF laboratory. Its
      clinical application consists in (1) the possibility of increasing cumulative delivery rates
      per cycle and (2) the reduction of the number of transferable embryos in both fresh and
      successive frozen thawed embryo transfer cycles (FET) decreasing the risk of multiple
      pregnancies.

      Two different techniques to cryopreserve embryos are commonly used: slow freezing and
      vitrification. During slow freezing, the embryo is exposed to subzero temperatures in a
      controlled rate with the use of low concentration of cryoprotectant. Alternatively, using
      vitrification the embryo rapidly enters into a glass-like state with the use of high
      concentrations of cryoprotectant and rapid cooling rates. Several studies comparing
      cryopreservation of human embryos by vitrification or slow freezing are available and
      meta-analyses have been published. Although vitrification has been found to have
      significantly higher survival rates than slow freezing, results on implantation rate,
      clinical pregnancy rate and live birth rate remain unclear, mostly due to a paucity of
      well-designed and powered prospective randomized controlled studies assessing live birth
      rates.

      Embryo evaluation to select the embryo(s) with the highest implantation potential is
      routinely based on embryo developmental and morphological characteristics using light
      microscopy. The embryo parameters usually correlated to the implantation capacity of an
      embryo in a fresh cycle are the number of blastomeres, their symmetry and the percentage
      cytoplasmic fragmentation. For the evaluation of frozen-thawed embryos, the percentage of
      blastomeres that survive the freezing-thawing process and their developmental capacity after
      overnight culture have to be added to these factors.

      Current research in the development of non-invasive methods for scoring embryos and ranking
      them according to their ability to implant and give rise to a healthy birth include both
      morphometrics and morphokinetics.

      Morphometrics Multilevel computerized images enable assessment over an unlimited time period
      and allow a detailed evaluation of the embryo. Several morphometric studies of human fresh
      oocytes and embryos with embryo implantation and development have been published.
      Morphometric studies using multilevel images combined with a computer-assisted scoring system
      (CASS) may be superior compared to standard scoring system (SSS) to predict implantation
      and/or live birth based on the number and size of blastomeres on day 3. Semi-automated
      morphometric analysis also revealed correlations between total embryo volume and clinical
      pregnancy. Another study evaluating morphometric characteristics including the absolute
      volumes, coefficients of form and the coefficient of diversity of human embryos and/or
      oocytes showed a higher coefficient of diversity between sister blastomeres in embryos that
      successfully implanted and progressed to birth. Morphometric characteristics including the
      external and internal diameter of the zona pellucida, the thickness of the zona pellucida and
      the embryo cell mass diameter were described. They stated that image analysis could be used
      to predict the criteria needed for more efficient embryo assessment before transfer. The mean
      blastomere size was correlated with the degree of fragmentation and multinucleation in a
      study using computer-controlled multilevel analyses with 232 embryos. However, until now, no
      morphometric studies in human frozen thawed embryos have been described.

      Morphokinetics Time-lapse imaging allows documentation of early growth without disturbing the
      culture environment. At this moment, no prospective randomized trial data are available
      showing that embryos selected from time-lapse imaging have significantly improved
      implantation rates compared with embryos selected with conventional morphological evaluation.

      However, time lapse imaging of embryos have revealed a correlation between first cleavage
      time, time between cleavages, blastomere size and multinucleation and implantation potential
      of human embryos. A correlation between the times of each embryo cleavage between 2 to 8 cell
      stage, the ability to become a blastocyst and implantation potential. Moreover, morphokinetic
      studies have demonstrated that direct cleavage from 2 cells to 3 cells has been correlated
      with a low implantation and ongoing pregnancy rate. According to time-lapse recording in 38
      oocytes, embryo quality was related to fertilization events and periodicity of the
      cyctoplasmic wave: good quality embryos arose from oocytes that had more uniform timing from
      injection to pronuclear abuttal and longer cytoplasmic wave. Furthermore, cellular fragments
      in human embryos can disappear during in vitro culture.

      Only a few studies on morphokinetics in human cryopreserved embryos have been published.
      Using videocinematography on respectively 50 and 103 frozen-thawed embryos, it was shown that
      the occurrence of smooth membranes and the cell-cell adherence were predictive for the
      implantation capacity of frozen-cleaved embryos. Wong et al. followed the development of 242
      supernumerary frozen d1-old embryos in a study combining time lapse microscopy and gene
      expression profiling: success in progression to the blastocyst stage could be predicted by
      measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization before
      embryonic genome activation: (1) duration of the first cytokinesis, (2) time interval between
      the end of the first mitosis and the initiation of the second and (3) the time interval
      between the second and third mitoses.

      This observational study aims to describe morphometric and morphokinetic parameters of thawed
      human embryos after freezing (slow freezing or vitrification). The study will focus on the
      evolution of the blastomere volume, the evolution of the contact surfaces between surviving
      blastomeres and the development of the embryo using time lapse imaging. Embryo morphometric
      and morphokinetic parameters will be analyzed together with patient characteristics and
      clinical parameters using cluster analysis techniques. The correlation between
      morphometrics/morphokinetics and the implantation potential of frozen-thawed embryos will be
      studied in cycles with single embryo transfer and in cycles with dual implantation after
      double embryo transfer.
    

Observational model: Cohort
Time perspective: Retrospective
Sampling method: Non-Probability Sample
Study population: 
        Patients with frozen thawed embryos for an ART treatment cycle