JBRA Assist. Reprod. 2020;24(4):411-415
ORIGINAL ARTICLE
doi: 10.5935/1518-0557.20200035
Morphokinetic parameters as auxiliary criteria for selection of blastocysts cultivated in a time-lapse monitoring system
1Clínica Reproferty, São José dos Campos, SP, Brazil
2UNIFUNVIC- Centro Universitário, Pindamonhangaba, SP, Brazil
The authors declare no conflict of interest.
ABSTRACT
Objective: To describe embryonic profile up to blastocyst stage in a time-lapse
system.
Methods: A retrospective, longitudinal, analytical study of patients submitted to in vitro fertilization. The embryos were grouped
according to the degree of expansion, internal cell mass and trophectoderm
classification, the morphokinetic parameters were associated with the time
periods stated in each evolution phase.
Results: The appearance of a second polar corpuscle (CPap) occurred earlier in the
embryos classified as excellent (2.99h; p<0.05), in
relation to the embryos classified as good (3.40h), average (3.48h) and poor
(3.55h). The embryos classified as excellent took less time for the
pronuclei to disappear (PNbd) (21.80h; p<0.05), when
compared to the good embryos (22.96h), the average (23.21h) and the poor
(23.47h). As for the morphokinetic parameter, the end of the two-cell
division (T2) occurred first in the excellent blastocysts (24.38h; p<0.05), when compared to the other groups: good
(25.57h), average (25.53h) and poor (25.78h). With respect to
synchronization with the division of three to four cells (S2), the poor
embryos presented longer times for such division to occur (3.67h; p<0.05). When compared to the embryos from the
groups excellent (1.97h), good (2.70h) and average (2.09h). At the time
point of the blastocoel formation (TB), the excellent embryos (104.04h) did
not differ from the good embryos (104.10h). However, when compared to
average (107.27h) and poor (106.86h) embryos, there was statistical
significance (p<0.05).
Conclusions: Embryos of better quality had a shorter time in some morphokinetic parameters
when compared to the other groups, thus increasing the possibilities to
establish new parameters for the classification and selection of
embryos.
Keywords: blastocyst, time-lapse monitoring system, blastocyst quality
INTRODUCTION
Despite efforts to improve outcomes in assisted reproduction processes, pregnancy
rates are relatively low, and in order to increase these percentages the transfer of
multiple embryos per cycle has been performed, which can lead to an increase in
multiple pregnancies, which is associated with neonatal complications and maternal
health problems (Svendsen et al.,
1996; Alasmari et al.,
2016; Klitzman, 2016; Wintner et al., 2017).
In recent years, efforts to improve embryo selection in assisted reproduction cycles
have been directed to laboratory practice. There is a documented correlation between
the morphological characteristics and the stage of embryo development at certain
times and its parameters. In order to reduce the number of embryos in the transfer
without reducing the chances of pregnancy, we have been seeking the best way to
identify high implantation capacity embryos (Meseguer et al., 2011; Rubio et al., 2014; Macer et al., 2017). One of the non-invasive and
objective evaluation methods to distinguish embryos has been the Time-Lapse (MTL)
monitoring system with the use of EmbryoScope (Vitrolife A/S, Denmark?).
This tool enables the assessment of morphokinetic parameters in order to select
embryos with greater implantation potential, and minimize human handling (Marcos et al., 2015; Peavey et al., 2015; Bodri et al., 2016).
Recent studies have suggested that this monitoring can introduce dynamic markers of
embryonic competence as well as offer the possibility of monitoring for 24 hours
during embryo development. In this way, there is an increase in the quantity and
quality of information, without disturbing the conditions of the culture medium,
since the embryos remain in a stable and controlled environment (Kirkegaard et al., 2013a; Tejera et al., 2013; Drejza et al., 2017).
The main objective of this study was to provide information regarding parameters that
could be relevant, and thus guide on the choice of the best embryo for transfer
during the in vitro fertilization process. The specific objectives
were: - to classify the blastocysts as to excellent, good, average and poor, so as
to achieve the best embryo transfer profile to use in IVF. Trace the embryo
development profile during 24 hours until expanded blastocyst, in groups of embryos
classified as Excellent, Good, Average and Poor; Compare the results obtained among
the studied groups by analyzing how these factors interfere in the final quality of
these embryos according to Gardner and Lane’s classification (Schoolcraft et al., 1999).
Thus, we expect to shed light on the most suitable profile for a possible clinical
indication of the best embryos to be transferred or frozen for in
vitro fertilization processes.
MATERIALS AND METHODS
This is a retrospective, longitudinal, analytical study; involving patients who
underwent an IVF procedure in the Assisted Reproduction program of a clinic in the
Metropolitan Region of Vale do Paraiba/SP, from September 2018 to March 2019, which
was submitted for approval from the Research Ethics Committee.
Inclusion criteria
We evaluated all the cases in which there was an evolution from embryo to
blastocyst.
Exclusion Criteria
Those patients who were not grouped according to the study variables.
Stimulation protocol
All women in the study were treated with GnRH antagonist, and ovarian
hyperstimulation was considered with the administration of gonadotropins at
doses adjusted according to clinical response. Human chorionic gonadotropin
(hCG) was given to trigger ovulation when the follicles reached values greater
than 18mm in diameter. Embryonic culture removal of the oocytes was performed 36
hours after hCG administration, and they were inseminated by intracytoplasmic
sperm injection (ICSI) and placed in culture medium. The embryos were grown in a
special incubator, EmbryoScope (Vitrolife A/S, Denmark).
Sample
The embryos were classified according to their degree of expansion, MCI, and
trophectoderm. The total number of blastocysts studied was 337, from which 136
were classified as A (Excellent); 32 were classified as B (Good); 89 were C
(Average), and 80 were D (Poor).
The embryos used for the study were grouped according to quality, and divided
into A (Excellent): 3AA, 4AA, 5AA and 6AA; B (Good): 3AB, 4AB and 4BA; C
(Average): 3BB, 4BB, 5BB, 6BB, 3AC, 4AC and 3CA, and D (Bad): 3BC, 4BC, 5BC,
3CB, 3CC, 4CC and 5CC; according to the degree of trophectoderm cohesion; we
compared the internal cell mass and degree of expansion of the blastocoel to the
morphokinetic parameters (times of cell division) (Zhao et al., 2018).
Morphokinetic parameters
Mesegueret al. (2011) parameters were defined from the time ICSI was performed, and the second polar
corpuscle (CPap) was seen; pronuclei appearance (PNap); pronuclei disappearance
(PNbd); end of the division into two cells (T2); end of the division into three
cells (T3); end of the division into four cells (T4); end of the division into
five cells (T5); duration of the second cell cycle (Cc2 / T3-T2); time between
the division of three cells and five cells (Cc3 / T5-T3); synchronization of the
three to four cells division (S2 / T4-T3); when the blastocyst begin to expand,
the embryo grows in size, and the zona thins out (TB)
Blastocyst Classification
The quality of blastocysts was classified according to Gardner and Lane’s (Schoolcraft et al., 1999)
Morphological Assessment System, as well as the degree of trophectoderm
cohesion, the internal cell mass, and degree of blastocoel expansion.
Data collection
We collected the data from the GS-Doctor software (Golden Skill-it solution) and
passed it to spreadsheets coded by the researcher responsible for the other
members of the team, guaranteeing patient confidentiality under study, according
to the ethical norms of CONEP/MS.
Statistical evaluation
We plotted the data by medium and standard deviation. We analyzed the results
using the ANOVA test to compare the groups studied (analysis of variance test).
We used the Tukey's test to compare mean values using the 5 AS Program-South
Western Sydney PHN software. A p value <0.05 was considered
significant.
RESULTS
We noticed that there were 337 embryos in the blastocyst stage; the number of
excellent embryos (136) was higher than in other groups.
Table 1 shows the comparison between the
groups mentioned above vis-a-vis the morphokinetic parameters obtained in a
time-lapse monitoring system.
Table 1. Morphokinetic parameters (mean ± standard deviation) obtained from the
time-lapse monitoring system of blastocyst culture, according to embryo
classification groups
Second polar corpuscle (CPap) could be seen, based on the results obtained, occurring earlier in embryos classified as excellent (2.99h; p<0.05), when compared to embryos classified as good (3.40h), average (3.48h), and poor (3.55h).
Embryos classified as excellent took less time to have their pronuclei disappear (PNbd) (21.80h; p<0.05), when compared to good (22.96h), average (23.21h), and poor (23.47h) embryos.
As for the morphokinetic parameter, the end of the two-cell division (T2) occurred first among the excellent blastocysts (24.38h; p<0.05) when compared to the other groups: good (25.57h), average (25.53h), and poor (25.78h). Regarding the synchronization of the three-to-four-cell (S2) division, poor embryos took longer to divide (3.67h; p<0.05) when compared to the excellent (1.97h), the good (2.70h), and the average ones (2.09h).
Upon assessing the onset of blastulation (TB), the excellent embryos (104.04h) did not differ in relation to the good embryos (104.10h). However, when compared to the average (107.27h) and the poor (106.86h) ones, there was statistical significance (p<0.05).
Regarding the parameters: appearance of the pronuclei (PNap), the end of the division to 3 cells (T3); duration of the second cell cycle (Cc2); the end of the division to 4 cells (T4); the end of the division to 5 cells (T5) and time between the division to 3 and 5 cells (Cc3), there was no significant statistical difference among the blastocysts in the studied groups.
Regarding the results, it is possible to see that better-quality embryos had lower times in some morphokinetic parameters when compared to the other groups, thus increasing the possibilities of establishing new embryo classification and selection parameters.
DISCUSSION
The Time Lapse Embryo Monitoring System (MTL) is the most recent technology developed
for evaluation and selection of embryos with high implantation capacity. This
technique enables the collection of more information about the in
vitro development of the embryos through monitoring the embryo for 24
hours. In addition, embryos are not removed from the culture environment, and the
morphokinetic information and noninvasive conditions during culture are useful in
selecting the most appropriate embryo (Kirkegaard et al., 2015; Kovacs, 2016).
In this study, we used the morphological characteristics according to Gardner and
Lane’s (Schoolcraft et al.,
1999) classification to classify the blastocysts as excellent, good,
average or poor. Table 2 shows statistically
significant values for the morphokinetic parameters defined as: appearance of the
second polar corpuscle (CPap); disappearance of the protons (PNbd); termination of
the division into two cells (T2); synchronization of the 3 to 4 cell division (S2)
and time at which the blastocyst began to expand (TB).
In this sense, Meseguer et al. (2011) defined a hierarchical prediction model combining statistical
evaluation with the dynamic parameters and the most predictive parameters of that
study, according to the authors, were the times of T5, S2 and Cc2 occurrence. The S2
parameters were also defined and used in this study; and there was agreement of
results for the S2 time. Already at the end of division to 5 cells (T5), and
duration of the second cell cycle (Cc2), did not show statistically significant
differences between the groups studied.
In relation to the PNbd, there was statistical significance for the embryos
classified as excellent with the time, when compared to the good, average and poor
embryos. Although the significant statistical difference between the excellent and
the embryos of the other groups was revealed, all the embryos of this study,
regardless of the group, showed less time than Azzarello et al. (2012), who proved it (24.9h) to be
associated with the prediction of live births.
Coticchio et al. (2018), using
MTL, reported that male and female pronuclei (PN) appeared simultaneously 6.2 hours
after ICSI. However, the initial position of the male CP can be cortical,
intermediate or central in the following proportions, respectively: 15%, 31%, 2% and
53.8%. They also revealed that PN juxtapositions involve rapid movements of the
female PN towards the male PN. Thus, PN disappearance times and the first cleavage
showed a consistent relationship, occurring progressively later, depending on
whether the position of the initial male PN was central, intermediate or cortical.
In this way the time intervals between fertilization events were strongly associated
with embryo quality on day three.
In this study we found that in relation to the T2 parameter, the same occurred, where
the excellent embryos showed less time when compared to the good, average and poor
groups. Lee et al. (2012) reinforced, in consonance with other authors (Pfeffer et al., 2005; Fu et al., 2009), that early cleavage in the first
embryonic division resulting in two cells, about 25 to 27 hours post ICSI is a
relevant data, since there are several reports describing the transfer of embryos
with cleavage. Precocious cells have high implantation rates and some studies still
state that higher rates occur in the formation of blastocysts in early cleaved
embryos.
In our results, the embryos classified as poor showed longer time for second
generation cell division when compared to the embryos of the excellent, good, and
average groups. Data by Chen et al.
(2013) show that better quality blastocysts developed under significantly
lower times for second-generation cell divisions. Our data substantially support
these findings.
In addition, they further state that ICSI time for the five-cell stage and for the
morula stage were indicative of good blastocyst quality. In the study by Ciray et al. (2014), the
researchers state that the time that the embryo passes from three to five cells was
confirmed as a key parameter, associating it with higher implantation rates in
comparison to other evaluated criteria. However, when we evaluated the time between
the division to 3 and 5 cells (Cc3), these findings were not significant.
Hashimoto et al. (2012) and Dal Canto et al. (2012) highlighted the third generation of cell division, the stages of four to eight, or
five to eight cells, as indicative of expanded blastocysts. This study has not
focused on evaluating the time of division to eight cells.
Regarding blastulation onset (TB), the excellent embryos did not differ in relation
to the good embryos; but when compared to the average and poor embryos, there was a
significant difference. Herrero et
al. (2013) found time differences related to embryo
morphology in the cleavage and blastocyst stages, highlighting the time of 104.1
hours for blasting into embryos with a high probability of implantation.
These results demonstrate clear and precise conclusions, showing the existence of a
relationship between each stage of development with its predictive power for the
same phase, so that the earliest parameters are those that allow predicting the
initial development potential.
In addition to the focus on the definition of parameters to select the best embryos,
other studies have described new criteria to be analyzed, such as Kirkegaard et al. (2013b) in
relation to the influence of oxygen on embryo stability, corroborating the early
cleavage and obtaining higher rates of term pregnancy in the development of embryos
with the use of time lapse.
Other morphokinetic criteria for blastocyst development and rates of implantation and
live births (Kovacs, 2016) were used in
heterogeneous populations under non-standardized culture conditions, thus increasing
the range of possibilities to establish new parameters for the best use of MTL.
Limitations
More comprehensive and well-designed studies must be performed to evaluate the
effectiveness of time lapse in clinical use, considering the recent clinical
technology and its potential new variables, which will be introduced for better
use, but future revised guidelines are required.
CONCLUSIONS
Embrioscope is the latest technology proposed for the evaluation and selection of
embryos; and through this study, data can be obtained to report on the morphokinetic
profile of the population of a human reproduction clinic in the Metropolitan Region
of the Paraiba Valley, in order to obtain tools to develop new selection criteria or
to fine-tune existing ones for a more widespread use of Time-lapse monitoring.
We inferred the importance of establishing blastocyst parameters with greater
implantation potential, a subject already extensively discussed in the
literature.
The morphokinetic parameters appearance of the second polar corpuscle (CPap);
disappearance of the protons (PNbd); termination of the division into two cells
(T2); synchronization of the 3 to 4 cell division (S2) and the time when the
blastocyst began to expand (TB), had reduced times in the group of excellent
embryos. Thus, the selection of better embryos through this technique will reduce
the number of embryos in the transfer, without reducing the chances of pregnancy,
and in the future it will minimize the problem caused by excess frozen embryos, in
order to guarantee greater possibilities of obtaining a term pregnancy.
On the other hand, MTL has been only recently introduced IVF laboratories across the
world, and because it is a high-cost device, more studies are needed to prove the
real cost-effectiveness in obtaining better live birth rates.
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