Abstract - 26th Annual Meeting of ESHRE - Rome 2010

Permanent embryo monitoring and exact timing of early cleavages allow reliable prediction of human embryo

D. Hlinka1, M. Dudas2, J. Rutarova3, J. Rezacova3, S. Lazarovska1.

1 Prague Fertility Centre, Embryology, Prague, Czech Republic.
2 P. J. Safarik University, Institute of Biology and Ecology, Kosice, Slovakia.
3 Institute for the Care of Mother and Child, embryology, Prague, Czech Republic.

Keywords:
permanent embryo monitoring;cell cycle analysis;prediction of human embryo viability;

Introduction
Multiple different embryo selection strategies have been proposed up to date. The most usual evaluation of morphological parameters is based on daily microscopic observations. However, this classical approach can only reveal limited and static information, omitting the dynamics and timing of early mitotic divisions. Here we analyze the chronology of early mitotic events by continuous human embryo monitoring, demonstrating a strong correlation of cell cycle timing and cleavage synchrony with the outcomes of pregnancy tests.

Materials and methods:
A total of 210 human pronuclear embryos were subjected to time-lapse monitoring (PrimoVision, 1 picture/10 min; with the institutional ethical approval) under standard culture conditions. The exact timing of the three interphases (IP) occurring after the two-cell stage (IP2 defined as the period between 2 and 3 cells stages, IP3 between 3 and 5, and IP4 between 5 and 9 cells embryo), and the synchrony of daughter cell cleavages in these three cycles (i.e. timing of the transition from 3 to 4 cells, from 5 to 8 and from 9 to16 cells) were analyzed. After 5 days of culture, the morphologically best blastocysts with distinct embryoblasts were transferred, and the above data were correlated with the outcomes of pregnancy tests (fetal heart beat).

Results
In 28 embryos giving viable pregnancies (12 singletons and 8 twins), the durations of IP2 and IP3 were similar, taking 12–14 hrs. The IP4 between 5 and 9 cells stage was taking 22–24 hrs. In these embryos, the sister blastomeres cleaved in a very synchronous manner: from 3 to 4 cell stage in 10–20 min; from 5 to 8 cells in 30–50 min, and from 9 to 16 cells within 40–70 min. The timing of early events correlated strongly with the abilities of the embryos to develop to fully expanded blastocysts. 78 out of 92 (85 %) analyzed embryos that cleaved in the above described ranges reached the blastocyst stage within 5 days of culture. On the other hand, when the cleavage of some cells occurred during the IP period (frequently occurring at the beginning of IP) it always led to asynchronous and/or uneven cleavage of the resulting daughter cells. They were frequently arrested within the next few cleavages and partly/fully abnormal embryo development occurred. The sooner the untimely cleavage occurred – with the respect to the order of IP’s – the more abnormal development was detected. None of 18 embryos exhibiting abnormal premature cleavage during the IP2 period (mostly in three cells stages) developed to blastocyst stage. 8 (19 %) of 42 embryos detected in 5 cell stage before ending IP3 reached blastocyst, and finally, 19 (32 %) of 58 embryos with some cleaved cells in the IP4 interval formed blastocysts. http://www.youtube.com/watch?…

Discussion
Taking in account the biological importance of S-phase during interphase, there is not a surprise that timing of the early postfertilization processes correlates with viability of the resulting embryos. As we show in this study, any untimely premature cleavage was associated with the time-dependent impairment of embryonic development. In these cases, the insufficient duration of the IP period may not be apparent when counting blastomeres once a day, and can only be revealed by a precise time-lapse monitoring. Taken together, permanent embryo monitoring opens a new opportunity for exact measurement of the early phases of human embryo development and provides a novel powerful strategy for objective early embryo pre-selection.

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