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A retrospective analysis of morphokinetic parameters according to the implantation outcome of IVF treatment
European Journal of Obstetrics & Gynecology and Reproductive Biology 197 (2016) 186–190
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
A retrospective analysis of morphokinetic parameters according to the implantation outcome of IVF treatment Lihong Wu, Wei Han, Xiaodong Zhang, Jiang Wang, Weiwei Liu, Shun Xiong, Guoning Huang * Chongqing Reproductive and Genetics Institute, 64 Jing Tang ST, Yu Zhong District, Chongqing 400013, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 July 2015 Received in revised form 22 November 2015 Accepted 9 December 2015
Objective: The aim of this study was to identify candidates for a morphokinetic embryo selection model for day-3 transfer through the analysis of known implantation data. Study design: A diagnostic trial study was conducted. Two hundred twelve embryos from 109 patients participating in in-vitro fertilization (IVF) protocols. Results: In this diagnostic trial study, we analyzed cleavage times of transferred embryos with either failed (n = 102) or full implantations (n = 110). The logistic regression (LR) model of the relationship between the morphokinetic parameters and the implantation rate had a sensitivity of 79.61% and specificity of 47.56%. Results also showed that morphokinetic parameters such as tPNf (the time when pronuclei disappearanced), t2 (t2 for two cells), t4 (t4 for four cells) and t4-t3 were significantly different between embryos in the two groups (p < 0.037). Implantation rates of embryos within the time interval of 21.27 h < t2 < 26.725 h were higher than those of embryos outside this period (60.83% vs. 40.21%, p = 0.003). Fifty-seven patients with fully implanted embryos included five patients with pregnancy loss and 52 patients with live births. Embryos that met the time interval criteria for t2 and were also within the range of 11 h < t5-t4 < 13 h had significantly higher implantation rates than those out of this range for t5-t4 (78.05% vs. 51.9%, p = 0.005). Conclusions: Selection and transfer of embryos that reach specific developmental stages within critical time intervals during IVF may improve the outcome of transfers and result in higher implantation rates and live birth rates. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Time-lapse monitoring (TLM) Embryonic morphokinetic parameters Implantation Live births
Introduction Selective single embryo transfer (eSET) is becoming increasingly applied to clinical practice, as it can effectively reduce the risk for multiple pregnancies that can cause maternal and infant complications. However, eSET may reduce pregnancy rates after IVF [1]. Therefore, selecting the optimal embryo to transfer constitutes a major challenge in assisted reproductive technology. Presently, morphology assessment of embryos at a few discrete time points during development remains the preferred method of evaluating developmental potential [2]. However, this classical approach can only reveal static information, omitting the dynamics and exact timing of early mitotic divisions [3,4].
* Corresponding author. E-mail address: [email protected] (G. Huang). http://dx.doi.org/10.1016/j.ejogrb.2015.12.002 0301-2115/ß 2015 Elsevier Ireland Ltd. All rights reserved.
In recent years, the time-lapse monitoring (TLM) system has provided a novel non-invasive method to continuously monitor and assess the dynamic processes of early embryonic development [5]. Selection of embryos for transfer can be improved by TLM [6], as those which would be deemed viable using a static evaluation but display aberrant cleavage patterns could be excluded. Wong et al. [7] demonstrated that two morphologically similar embryos, which would have been classified at the same developmental stage in a time-point analysis, were actually products of completely different developmental processes. Another advantage of the TLM system is the ability to maintain embryos in an optimal culture environment that facilitates embryonic growth during the assessments [8,9]. Frequent evaluations outside the incubator allow assessments regarding timing of events, but at the same time, embryos are also exposed to undesirable changes in temperature, humidity and gas composition [10]. The application of the TLM system avoids the need to remove embryos from the optimal culture conditions andminimizes their
L. Wu et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 197 (2016) 186–190
transfer between incubator and microscopic platforms during daily observations, thus decreasing the risk for deleterious effects. Stable culture conditions and embryo selection by TLM are also of great clinical importance, because using the TLM system compared with a standard incubator, the relative probability of clinical pregnancies could be significantly improved [11]. The potential benefit of evaluating morphokinetic variables measured by TLM for improved embryo selection was reported by several retrospective observational studies, which focused primarily on the duration and synchrony of the cell cycles as predictors of blastocyst formation [7,12–16], implantation [3,14] and pregnancy [5,16,17,11]. The aim of this study was to identify candidates for a morphokinetic embryo selection model for day-3 transfers through retrospective analysis of known implantation data, and to develop a simple model for in-house use based upon our findings.
Materials and Methods Study Design and Patient Selection A total of 272 cycles in women (one cycle/person) who underwent IVF treatment in the Chongqing Reproductive and Genetics Institute, Chongqing, China, between April 2013 and June 2014 were included in the present study. Participants also had to fulfil the following criteria: age, 37 years; BMI, 18–25 Kg/m2; first IVF treatment; simple tubal factor infertility. Subsequently, 89 cycles were excluded from the study because of various reasons (aspirated oocytes were 20 or 5, n = 32 cases; rescued ICSI, n = 5 cases; canceled cycles due to oocytes or embryos being of poor quality, n = 8 cases; and whole-embryo freezing, n = 44 cases). Finally, embryos at Day 3 (n = 358) from 183 cycles were transferred. The 74 cycles were excluded from further analysis, because the number of gestational sacs did not match the number of transferred embryos. Fully implanted embryos (n = 110) from 57 cases, and embryos that failed to implant (n = 102) from 52 cases were included in the analysis (Fig. 1). This study was approved by the ethics committee of Chongqing Obstetrics and Gynecology Hospital, Chongqing, China. All included patients had previously provided informed consent.
IVF cycles 272 cases
Excluding 89 cases: 32 cases where aspirated oocytes were ≥ 20 or ≤ 5; 5 cases of rescued ICSI cycles; 8 cases of canceled cycles due to oocytes or embryos being of poor quality; 44 cases of whole-embryo freezing
Embryo transfer 183 cases
Fully implanted embryos (n=110)
Ovarian Stimulation and Oocyte Retrieval Before the ovaries were stimulated with recombinant FSH (Gonal-F, Merck Serono, Switzerland), down-regulation was carried out using a GnRHagonist (Decapeptyl, Ferring, Switzerland). When at least three leading follicles reached a mean diameter >18 mm, hCG (Ovidrel, Merck Serono, Italy) was administered. Transvaginal oocyte retrieval was performed 36–38 h after hCG injection. IVF Insemination, Culture of Embryos and TLM System Oocytes were fertilized using routine IVF methods [18]. Briefly, each oocyte was inseminated at 39–40 h post-hCG treatment. Before 9 AM on Day 1, the 2PN zygotes were chosen and placed into the microwells of a custom-made well-of-the-well dish (Primo Vision, Vitrolife Kft., Sweden), containing a 50 ul equilibrated G-1 (Vitrolife Sweden AB, Sweden) microdroplet over the microwells and covered with 2.5 ml Ovoil (Vitrolife Sweden AB, Sweden). Subsequently, the dish was maintained in the TLM system and cultured at 37 8C with 5% O2, 6% CO2 in compressed air and 95% relative humidity in a standard incubator (SANYO, MCO-5 M, Japan) until embryo transfer on Day 3. The main features of the TLM system included an inert, compact, sealed digital inverted microscope unit (2560 1920 pixels) installed inside a conventional incubator. The microscope used a homogenous green LED light (550 nm) as light source, and seven planar focal images were generated every 5 min (Primo Vision, Vitrolife Kft., Sweden). Embryo Score and Transfer Early cleavage was evaluated at 27–29 h post IVF insemination, and embryo morphology was scored on the morning of Day 2 (43– 45 h post IVF) based on the acquired digital images of the TLM system. On the morning of Day 3 (67–69 h post IVF), continuous monitoring and recording were finished. The embryos were moved from the incubator and scored by an embryologist using a conventional inverted microscope (Olympus IX70, Japan). Embryo quality was evaluated using a morphologic scoring system, taking into account regularity of the blastomeres, degree of fragmentation and microscopic appearance of the embryos, as described previously by our group [19]. Accordingly, all embryos were scored for cell number and embryo quality on a scale from 1 to 3. On day 3, those embryos having more than four blastomeres and a quality score of 1–3 were considered to be transferrable embryos. Clinical Follow-Up To monitor IVF outcome, we measured serum hCG concentrations 14 days after embryo transfer. Clinical pregnancy was confirmed by the presence of gestational sacs using ultrasonographic examination at week 4. The live birth rate was also used as a clinical outcome. Evaluation of Morphokinetic Parameters
Excluding partial implantation in 74 cases (146 embryos)
Fully implanted 57 cases
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Non-implanted 52 cases
Non-implanted embryos (n= 102)
Fig. 1. Flow chart showing the diagnostic trial study selection process.
Embryonic development using precise morphokinetic parameters was analyzed by the image analyzer software. The start time (t0) was defined as the time of IVF insemination. The tPNf was the time when pronuclei disappearanced [20]. The t2, t3, t4, t5, t6, t7 and t8 were defined as the times when the corresponding numbers of blastomeres as discrete cells first appeared (t2 for two cells, t3 for three cells, etc.). The t3-t2 was the time from division into a two-blastomere embryo until division to a three-blastomere embryo. Other morphokinetic parameters (t4-t3, t5-t3, t8-t5, t5-t4, t6-t5, t7-t6 and t8-t7) were calculated in the same manner. The time
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L. Wu et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 197 (2016) 186–190
for all events was expressed as hours and fractions of an hour post IVF insemination.
Table 2 Morphokinetic parameters of non-implanted and fully implanted embryos.
Statistical Analyses As the end-point of the study was the implantation rate, logistic regression (LR) was used to test the association between implantation rate and time-lapse parameters of the embryo. The cut-off values of significant TLM parameters were determined by ROC curve analysis or stratification analysis. All morphokinetic parameters for fully implanted and nonimplanted embryos were expressed as mean SD. A pvalue < 0.05 was considered to be significant. All results were obtained using the statistical software Stata11 (StataCorp LP, Stanford, USA). Results Timing of Embryonic Developmental Events and Implantation The characteristics of the patients between viably implanted and non-implanted embryos did not differ (Table 1). In viably implanted embryos, tPNf, t2 and t4 occurred at an earlier time than in those embryos that failed to implant (p = 0.010, p = 0.009 and p = 0.009, respectively) (Table 2). The calculated timings assessed indirectly from the cleavage times are also presented in Table 2. The period of t4-t3 was shorter (p = 0.037) in viably implanted compared with non-implanted embryos (0.94 1.44 and 1.5 2.9 h, respectively), while the other parameters did not differ significantly. The LR model of the relationship between the morphokinetic parameters and the implantation rate showed a sensitivity of 79.61% and specificity of 47.56%. The morphokinetic parameters’ 95% CI are given in Table 2. Indeterminate results and missing data were excluded from the study. Evaluation of Potential Selection Parameters The cut-off value for t2 (t2 = 26.725 h) was used in order to determine the first time interval (21.27 h < t2 < 26.725 h; t2 = 21.27 h, which was derived from this study and included 212 embryos) for selection. Implantation rates of embryos that reached 21.27 h < t2 < 26.725 h within this interval were higher than in those outside the range (60.83% vs. 40.21%; p = 0.003).
tPNf (h) (95% CI) t2 (h) (95% CI) t3 (h) (95% CI) t4 (h) (95% CI) t5 (h) (95% CI) t6 (h) (95% CI) t7 (h) (95% CI) t8 (h) (95% CI) t3-t2 (h) (95% CI) t4-t3 (h) (95% CI) t5-t3 (h) (95% CI) t8-t5 (h) (95% CI) t5-t4 (h) (95% CI) t6-t5 (h) (95% CI) t7-t6 (h) (95% CI) t8-t7 (h) (95% CI)
Non-implanted embryo (N)
Fully implanted embryo (N)
p-value
25.25 2.90 (102) (24.64–25.85) 27.58 3.46 (102) (26.70–28.05) 38.28 5.36 (102) (37.23–39.34) 39.79 5.16 (102) (38.77–40.80) 51.8 5.25 (97) (50.74–52.86) 53.74 5.15 (95) (52.69–54.79) 55.42 5.4 (94) (54.31–56.53) 57.32 5.49 (84) (56.13–58.51) 10.7 3.46 (102) (10.02–11.38) 1.5 2.9 (102) (0.93–2.07) 14.07 2.68 (97) (13.53–14.62) 5.66 5.15 (84) (4.55–6.78) 12.53 4.12 (97) (11.70–13.36) 1.91 3.27 (95) (1.24–2.58) 1.79 2.92 (94) (1.20–2.39) 2.32 2.97 (84) (1.70–3.00)
24.38 2.47 (110) (23.89–24.88) 26.58 2.59 (110) (25.80–26.78) 37.45 3.42 (110) (36.80–38.09) 38.38 3.37 (110) (37.75–39.02) 50.92 5.36 (110) (49.91–51.94) 52.89 5.09 (110) (51.93–53.86) 54.33 5 (105) (53.37–55.30) 56.25 5.47 (104) (55.19–57.32) 10.86 1.98 (110) (10.49–11.23) 0.94 1.44 (110) (0.66–1.21) 13.47 3.40 (110) (12.83–14.12) 5.66 5.47 (104) (4.59–6.72) 12.53 3.64 (110) (11.85–13.22) 1.97 3.44 (110) (1.32–2.63) 1.81 2.74 (105) (1.28–2.34) 2.02 2.24 (104) (1.58–2.45)
0.010 0.009 NS 0.009 NS NS NS NS NS 0.037 NS NS NS NS NS NS
Data are presented as mean SD. h: hours after IVF insemination; N: number of embryos; CI: confidence interval.
Embryo blastomere numbers and embryo quality scores between the two groups were also significantly different (Table 3). Embryos that met the time interval criteria for t2 and were also within the range of 11 h < t5-t4 < 13 h had significantly higher implantation rates than those out of this range for t5-t4 (78.05% vs. 51.9%, p = 0.005); Table 4). Clinical Follow-Up There were 57 cases with fully implanted embryos, including five cases of pregnancy loss, and 52 cases of live births.
Table 1 Characteristics of patients with non-implanted and fully implanted embryos.
Age (years) Infertility (years) BMI (kg/m2) Total gonadotropin dose (IU) Endometrium thickness (mm) Number of oocytes retrieved (n) Number of mature oocytes (n) Percentage of mature oocytes (%) Number of 2PN oocyte s(n)
Patients with non-implanted embryos (N = 52)
Patients with fully implanted and live birth embryos (N = 57)
p-value
30.5 3.54 4.98 2.83 21.04 3.03 2376.44 106.07
29.16 6.36 4.57 2.12 21.50 2.98 2080.26 1343.50
NS NS NS NS
10.29 0.71
10.09 2.12
NS
11.13 0
11.40 2.12
NS
10.10 0.71
10.21 1.41
NS
90.67 (525/579)
89.54 (582/650)
NS
7.77 2.12
8.05 0
NS
Data are presented as mean SD. N: number of patients, NS = not statistically significant.
Comments In this diagnostic trial study, we compared the morphokinetic parameters between fully implanted embryos and those that failed to implant after IVF and transfer on Day 3 (the IVF cycles were derived from the ‘good prognosis’ patients). The LR model of the
Table 3 Characteristics and implantation rate of embryos that developed to 2-blastomere stage within or outside the time interval 21.27 h < t2 < 26.725 h. Embryos developed within time interval (N = 120) Blastomere numbers (n) Embryo score Implantation rate (%)
Embryos developed outside of time interval (N = 92)
p-value
9.13 2.29
7.96 1.51
0.000
2.39 0.45 60.83
2.52 0.56 40.21
0.031 0.003
Data are presented as mean SD or %. N: number of embryos.
L. Wu et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 197 (2016) 186–190 Table 4 Characteristics and implantation rate of embryos that all developed to 2-blastomere stage within the time interval 21.27 h < t2 < 26.725 h but were within or outside the second time range 11 h < t5-t4 < 13 h. Embryos developed within second time interval (n = 41) Blastomere numbers (n) Embryo score Implantation rate (%)
Embryos developed outside of second time interval (n = 79)
9.54 2.68
8.92 2.29
2.43 0.54 78.05
2.37 0.48 51.9
p-value
NS NS 0.005
Data are presented as mean SD or %. N: number of embryos.
relationship between the morphokinetic parameters and the implantation rate showed a sensitivity of 79.61% and specificity of 47.56%. Conaghan et al. [15] found that the Eeva (Early Embryo Viability Assessment, another time-lapse system) prediction and cell-tracking software plus Day 3 morphology correctly predicted which embryos would become usable blastocysts, with a sensitivity of 58.8% and specificity of 84.2%. The prediction models for blastocysts or implantation potential, to some extent, improve the ability of selecting embryos of potentially high developmental capability. However, it is worth noting that the sensitivity and specificity of the model could not simultaneously efficiently predict embryonic potential. Therefore, this area requires further research in order to improve these modes of embryonic analysis. Some of these parameters were significantly different between the two groups suggesting that timing of particular embryonic developmental events may be crucial for successful transfer and pregnancy. In our study, PN disappearance (tPNf) occurred earlier in the fully implanted group than in the non-implanted one. Aguilar et al. [21] also found that timing of pronuclei fading between 22.2 and 25.9 h was linked to successful embryonic implantation, and early PN disappearance was correlated with a higher number of blastomeres on Day 2 after IVF or ICSI [5]. However, Azzarello et al. [22] showed that too early disappearance of PNB (before 20 h 45 min) may be detrimental and results in failure of live births. Early cleavage embryos have higher implantation and pregnancy rates [23,24] and higher number of blastomeres on Day 2 [5]. Van Montfoort and colleagues reported increased pregnancy rates in the early-cleavage transfer group compared with the latecleavage transfer group [25]. We also confirmed that cleavage, detected by morphokinetic parameters during time-lapse monitoring, occurs earlier in the fully implanted embryos than in the embryos that fail to implant. Along with the extensive application of TLM, several recent studies have investigated the importance of early morphokinetic parameters during embryonic development. In the retrospective analysis of 100 embryos during 48 h of dynamic developmental processes, Wong et al. showed that duration of first cytokinesis, cc2 (t3-t2), and s2 (t4-t3) can predict blastocyst formation. However, none of the embryos in this particular study were transferred [7]. In the present study, 212 embryos were transferred, and we reliably predicted the relation between morphokinetic parameters (tPNf, t2, t4, t4-t3) and implantation potential. Another study where the authors predicted implantation potential [3] found that t5, cc2 and s2 were positively associated with implantation potential; however, morphokinetic parameters were recorded only until the 5-cell stage rather than the 8-cell stage in our study. Another similar finding between our and other two studies above was that the synchronized embryonic development from three to four cells was associated with a higher chance of implantation. Thus, 3-cell and 4-cell synchronization was essential for blastocyst
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formation and potential for implantation. The morphokinetic parameters t2 were also significantly different between fully implanted and non-implanted embryos in our study. This supports the findings of Desai et al. [26] and Dal Canto et al. [27] who detected different timings of t2, t3, t5 and t8 between implanted and non-implanted blastocysts, and shorter development of implanting embryos to the 8-cell stage, respectively. t4 was only significantly different between fully implanted and non-implanted embryos in our study, suggesting that t4 may be specific to our IVF laboratory, and this could provide us with more information regarding embryo selection. The results from several IVF centers were not always consistent, which may be due to the different sample populations. Additionally, the length of the cell cycle may also be affected by the culture conditions and fertilization methods used. Wale and Gardner [27] found that the developmental stages of mouse embryos cultured in 20% oxygen were prolonged compared with embryos in 5% oxygen. It has already been reported that culture media also affects human embryonic cleavage rates [28–31]. Moreover, some studies have shown that the method of fertilization has an effect on cleavage time with ICSI inducing earlier cleavage than conventional IVF [32,33]. Therefore, it is important to note that some data and selection models cannot be completely extrapolated from one laboratory to another. Our second goal was to establish an embryo selection model at our IVF center. Therefore, we determined the best embryonic cleavage time intervals by using the LR model and ROC curves. In the range of 21.27 h < t2 < 26.725 h, embryos showed increased blastomere numbers, higher embryo score and higher implantation rate. Previously, early cleavage was observed to occur at 27– 29 h after IVF fertilization and 25–27 h after ICSI fertilization using a traditional inverted microscope [2]. When the combined ranges of 21.27 h < t2 < 26.725 h and 11 h < t5-t4 < 13 h were selected, the implantation rate, was still significantly increased. In a previous retrospective TLM analysis comparing the morphokinetic parameters of 72 fully implanted embryos with 106 nonimplanted embryos, Day-5 embryos with a cc3 (from 3 cells to 5 cells) between 9.7 and 21 h had the highest probability to implant [14]. In this study, we obtained optimal time intervals for t2 and t5-t4 using a time-lapse monitoring system for early embryonic development, which was associated with a higher embryonic implantation rate. Based on these results, an embryo selection model in our IVF center can be established. However, it should be confirmed in a prospective, randomized, controlled study so as to ascertain whether use of the model can consistently improve clinical pregnancy rates. One limitation of this study was however that the times for second polar body extrusion and PN appearance were not observed due to placing the normally fertilized zygotes into the time-lapse culture system on the next morning after insemination. The second limitation was that we confirmed clinical pregnancy by the presence of gestational sacs at week 4 and not heart beat. In conclusion, embryos that reach specific developmental stages during early cleavage at critical times have higher potential for implantation and live births. Importantly, an embryo selection model can be established by using specific time intervals for t2 and t5-t4, which may facilitate the selection of optimal embryos for eSET, thereby avoiding subjectivity and limitations of a singlemorphology scoring system.
Funding This article and study was funded by ‘‘Chongqing municipal health and family planning commission’’, and the project number is ‘‘2015MSXM087’’.
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
A retrospective analysis of morphokinetic parameters according to the implantation outcome of IVF treatment Lihong Wu, Wei Han, Xiaodong Zhang, Jiang Wang, Weiwei Liu, Shun Xiong, Guoning Huang * Chongqing Reproductive and Genetics Institute, 64 Jing Tang ST, Yu Zhong District, Chongqing 400013, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 July 2015 Received in revised form 22 November 2015 Accepted 9 December 2015
Objective: The aim of this study was to identify candidates for a morphokinetic embryo selection model for day-3 transfer through the analysis of known implantation data. Study design: A diagnostic trial study was conducted. Two hundred twelve embryos from 109 patients participating in in-vitro fertilization (IVF) protocols. Results: In this diagnostic trial study, we analyzed cleavage times of transferred embryos with either failed (n = 102) or full implantations (n = 110). The logistic regression (LR) model of the relationship between the morphokinetic parameters and the implantation rate had a sensitivity of 79.61% and specificity of 47.56%. Results also showed that morphokinetic parameters such as tPNf (the time when pronuclei disappearanced), t2 (t2 for two cells), t4 (t4 for four cells) and t4-t3 were significantly different between embryos in the two groups (p < 0.037). Implantation rates of embryos within the time interval of 21.27 h < t2 < 26.725 h were higher than those of embryos outside this period (60.83% vs. 40.21%, p = 0.003). Fifty-seven patients with fully implanted embryos included five patients with pregnancy loss and 52 patients with live births. Embryos that met the time interval criteria for t2 and were also within the range of 11 h < t5-t4 < 13 h had significantly higher implantation rates than those out of this range for t5-t4 (78.05% vs. 51.9%, p = 0.005). Conclusions: Selection and transfer of embryos that reach specific developmental stages within critical time intervals during IVF may improve the outcome of transfers and result in higher implantation rates and live birth rates. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Time-lapse monitoring (TLM) Embryonic morphokinetic parameters Implantation Live births
Introduction Selective single embryo transfer (eSET) is becoming increasingly applied to clinical practice, as it can effectively reduce the risk for multiple pregnancies that can cause maternal and infant complications. However, eSET may reduce pregnancy rates after IVF [1]. Therefore, selecting the optimal embryo to transfer constitutes a major challenge in assisted reproductive technology. Presently, morphology assessment of embryos at a few discrete time points during development remains the preferred method of evaluating developmental potential [2]. However, this classical approach can only reveal static information, omitting the dynamics and exact timing of early mitotic divisions [3,4].
* Corresponding author. E-mail address: [email protected] (G. Huang). http://dx.doi.org/10.1016/j.ejogrb.2015.12.002 0301-2115/ß 2015 Elsevier Ireland Ltd. All rights reserved.
In recent years, the time-lapse monitoring (TLM) system has provided a novel non-invasive method to continuously monitor and assess the dynamic processes of early embryonic development [5]. Selection of embryos for transfer can be improved by TLM [6], as those which would be deemed viable using a static evaluation but display aberrant cleavage patterns could be excluded. Wong et al. [7] demonstrated that two morphologically similar embryos, which would have been classified at the same developmental stage in a time-point analysis, were actually products of completely different developmental processes. Another advantage of the TLM system is the ability to maintain embryos in an optimal culture environment that facilitates embryonic growth during the assessments [8,9]. Frequent evaluations outside the incubator allow assessments regarding timing of events, but at the same time, embryos are also exposed to undesirable changes in temperature, humidity and gas composition [10]. The application of the TLM system avoids the need to remove embryos from the optimal culture conditions andminimizes their
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transfer between incubator and microscopic platforms during daily observations, thus decreasing the risk for deleterious effects. Stable culture conditions and embryo selection by TLM are also of great clinical importance, because using the TLM system compared with a standard incubator, the relative probability of clinical pregnancies could be significantly improved [11]. The potential benefit of evaluating morphokinetic variables measured by TLM for improved embryo selection was reported by several retrospective observational studies, which focused primarily on the duration and synchrony of the cell cycles as predictors of blastocyst formation [7,12–16], implantation [3,14] and pregnancy [5,16,17,11]. The aim of this study was to identify candidates for a morphokinetic embryo selection model for day-3 transfers through retrospective analysis of known implantation data, and to develop a simple model for in-house use based upon our findings.
Materials and Methods Study Design and Patient Selection A total of 272 cycles in women (one cycle/person) who underwent IVF treatment in the Chongqing Reproductive and Genetics Institute, Chongqing, China, between April 2013 and June 2014 were included in the present study. Participants also had to fulfil the following criteria: age, 37 years; BMI, 18–25 Kg/m2; first IVF treatment; simple tubal factor infertility. Subsequently, 89 cycles were excluded from the study because of various reasons (aspirated oocytes were 20 or 5, n = 32 cases; rescued ICSI, n = 5 cases; canceled cycles due to oocytes or embryos being of poor quality, n = 8 cases; and whole-embryo freezing, n = 44 cases). Finally, embryos at Day 3 (n = 358) from 183 cycles were transferred. The 74 cycles were excluded from further analysis, because the number of gestational sacs did not match the number of transferred embryos. Fully implanted embryos (n = 110) from 57 cases, and embryos that failed to implant (n = 102) from 52 cases were included in the analysis (Fig. 1). This study was approved by the ethics committee of Chongqing Obstetrics and Gynecology Hospital, Chongqing, China. All included patients had previously provided informed consent.
IVF cycles 272 cases
Excluding 89 cases: 32 cases where aspirated oocytes were ≥ 20 or ≤ 5; 5 cases of rescued ICSI cycles; 8 cases of canceled cycles due to oocytes or embryos being of poor quality; 44 cases of whole-embryo freezing
Embryo transfer 183 cases
Fully implanted embryos (n=110)
Ovarian Stimulation and Oocyte Retrieval Before the ovaries were stimulated with recombinant FSH (Gonal-F, Merck Serono, Switzerland), down-regulation was carried out using a GnRHagonist (Decapeptyl, Ferring, Switzerland). When at least three leading follicles reached a mean diameter >18 mm, hCG (Ovidrel, Merck Serono, Italy) was administered. Transvaginal oocyte retrieval was performed 36–38 h after hCG injection. IVF Insemination, Culture of Embryos and TLM System Oocytes were fertilized using routine IVF methods [18]. Briefly, each oocyte was inseminated at 39–40 h post-hCG treatment. Before 9 AM on Day 1, the 2PN zygotes were chosen and placed into the microwells of a custom-made well-of-the-well dish (Primo Vision, Vitrolife Kft., Sweden), containing a 50 ul equilibrated G-1 (Vitrolife Sweden AB, Sweden) microdroplet over the microwells and covered with 2.5 ml Ovoil (Vitrolife Sweden AB, Sweden). Subsequently, the dish was maintained in the TLM system and cultured at 37 8C with 5% O2, 6% CO2 in compressed air and 95% relative humidity in a standard incubator (SANYO, MCO-5 M, Japan) until embryo transfer on Day 3. The main features of the TLM system included an inert, compact, sealed digital inverted microscope unit (2560 1920 pixels) installed inside a conventional incubator. The microscope used a homogenous green LED light (550 nm) as light source, and seven planar focal images were generated every 5 min (Primo Vision, Vitrolife Kft., Sweden). Embryo Score and Transfer Early cleavage was evaluated at 27–29 h post IVF insemination, and embryo morphology was scored on the morning of Day 2 (43– 45 h post IVF) based on the acquired digital images of the TLM system. On the morning of Day 3 (67–69 h post IVF), continuous monitoring and recording were finished. The embryos were moved from the incubator and scored by an embryologist using a conventional inverted microscope (Olympus IX70, Japan). Embryo quality was evaluated using a morphologic scoring system, taking into account regularity of the blastomeres, degree of fragmentation and microscopic appearance of the embryos, as described previously by our group [19]. Accordingly, all embryos were scored for cell number and embryo quality on a scale from 1 to 3. On day 3, those embryos having more than four blastomeres and a quality score of 1–3 were considered to be transferrable embryos. Clinical Follow-Up To monitor IVF outcome, we measured serum hCG concentrations 14 days after embryo transfer. Clinical pregnancy was confirmed by the presence of gestational sacs using ultrasonographic examination at week 4. The live birth rate was also used as a clinical outcome. Evaluation of Morphokinetic Parameters
Excluding partial implantation in 74 cases (146 embryos)
Fully implanted 57 cases
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Non-implanted 52 cases
Non-implanted embryos (n= 102)
Fig. 1. Flow chart showing the diagnostic trial study selection process.
Embryonic development using precise morphokinetic parameters was analyzed by the image analyzer software. The start time (t0) was defined as the time of IVF insemination. The tPNf was the time when pronuclei disappearanced [20]. The t2, t3, t4, t5, t6, t7 and t8 were defined as the times when the corresponding numbers of blastomeres as discrete cells first appeared (t2 for two cells, t3 for three cells, etc.). The t3-t2 was the time from division into a two-blastomere embryo until division to a three-blastomere embryo. Other morphokinetic parameters (t4-t3, t5-t3, t8-t5, t5-t4, t6-t5, t7-t6 and t8-t7) were calculated in the same manner. The time
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for all events was expressed as hours and fractions of an hour post IVF insemination.
Table 2 Morphokinetic parameters of non-implanted and fully implanted embryos.
Statistical Analyses As the end-point of the study was the implantation rate, logistic regression (LR) was used to test the association between implantation rate and time-lapse parameters of the embryo. The cut-off values of significant TLM parameters were determined by ROC curve analysis or stratification analysis. All morphokinetic parameters for fully implanted and nonimplanted embryos were expressed as mean SD. A pvalue < 0.05 was considered to be significant. All results were obtained using the statistical software Stata11 (StataCorp LP, Stanford, USA). Results Timing of Embryonic Developmental Events and Implantation The characteristics of the patients between viably implanted and non-implanted embryos did not differ (Table 1). In viably implanted embryos, tPNf, t2 and t4 occurred at an earlier time than in those embryos that failed to implant (p = 0.010, p = 0.009 and p = 0.009, respectively) (Table 2). The calculated timings assessed indirectly from the cleavage times are also presented in Table 2. The period of t4-t3 was shorter (p = 0.037) in viably implanted compared with non-implanted embryos (0.94 1.44 and 1.5 2.9 h, respectively), while the other parameters did not differ significantly. The LR model of the relationship between the morphokinetic parameters and the implantation rate showed a sensitivity of 79.61% and specificity of 47.56%. The morphokinetic parameters’ 95% CI are given in Table 2. Indeterminate results and missing data were excluded from the study. Evaluation of Potential Selection Parameters The cut-off value for t2 (t2 = 26.725 h) was used in order to determine the first time interval (21.27 h < t2 < 26.725 h; t2 = 21.27 h, which was derived from this study and included 212 embryos) for selection. Implantation rates of embryos that reached 21.27 h < t2 < 26.725 h within this interval were higher than in those outside the range (60.83% vs. 40.21%; p = 0.003).
tPNf (h) (95% CI) t2 (h) (95% CI) t3 (h) (95% CI) t4 (h) (95% CI) t5 (h) (95% CI) t6 (h) (95% CI) t7 (h) (95% CI) t8 (h) (95% CI) t3-t2 (h) (95% CI) t4-t3 (h) (95% CI) t5-t3 (h) (95% CI) t8-t5 (h) (95% CI) t5-t4 (h) (95% CI) t6-t5 (h) (95% CI) t7-t6 (h) (95% CI) t8-t7 (h) (95% CI)
Non-implanted embryo (N)
Fully implanted embryo (N)
p-value
25.25 2.90 (102) (24.64–25.85) 27.58 3.46 (102) (26.70–28.05) 38.28 5.36 (102) (37.23–39.34) 39.79 5.16 (102) (38.77–40.80) 51.8 5.25 (97) (50.74–52.86) 53.74 5.15 (95) (52.69–54.79) 55.42 5.4 (94) (54.31–56.53) 57.32 5.49 (84) (56.13–58.51) 10.7 3.46 (102) (10.02–11.38) 1.5 2.9 (102) (0.93–2.07) 14.07 2.68 (97) (13.53–14.62) 5.66 5.15 (84) (4.55–6.78) 12.53 4.12 (97) (11.70–13.36) 1.91 3.27 (95) (1.24–2.58) 1.79 2.92 (94) (1.20–2.39) 2.32 2.97 (84) (1.70–3.00)
24.38 2.47 (110) (23.89–24.88) 26.58 2.59 (110) (25.80–26.78) 37.45 3.42 (110) (36.80–38.09) 38.38 3.37 (110) (37.75–39.02) 50.92 5.36 (110) (49.91–51.94) 52.89 5.09 (110) (51.93–53.86) 54.33 5 (105) (53.37–55.30) 56.25 5.47 (104) (55.19–57.32) 10.86 1.98 (110) (10.49–11.23) 0.94 1.44 (110) (0.66–1.21) 13.47 3.40 (110) (12.83–14.12) 5.66 5.47 (104) (4.59–6.72) 12.53 3.64 (110) (11.85–13.22) 1.97 3.44 (110) (1.32–2.63) 1.81 2.74 (105) (1.28–2.34) 2.02 2.24 (104) (1.58–2.45)
0.010 0.009 NS 0.009 NS NS NS NS NS 0.037 NS NS NS NS NS NS
Data are presented as mean SD. h: hours after IVF insemination; N: number of embryos; CI: confidence interval.
Embryo blastomere numbers and embryo quality scores between the two groups were also significantly different (Table 3). Embryos that met the time interval criteria for t2 and were also within the range of 11 h < t5-t4 < 13 h had significantly higher implantation rates than those out of this range for t5-t4 (78.05% vs. 51.9%, p = 0.005); Table 4). Clinical Follow-Up There were 57 cases with fully implanted embryos, including five cases of pregnancy loss, and 52 cases of live births.
Table 1 Characteristics of patients with non-implanted and fully implanted embryos.
Age (years) Infertility (years) BMI (kg/m2) Total gonadotropin dose (IU) Endometrium thickness (mm) Number of oocytes retrieved (n) Number of mature oocytes (n) Percentage of mature oocytes (%) Number of 2PN oocyte s(n)
Patients with non-implanted embryos (N = 52)
Patients with fully implanted and live birth embryos (N = 57)
p-value
30.5 3.54 4.98 2.83 21.04 3.03 2376.44 106.07
29.16 6.36 4.57 2.12 21.50 2.98 2080.26 1343.50
NS NS NS NS
10.29 0.71
10.09 2.12
NS
11.13 0
11.40 2.12
NS
10.10 0.71
10.21 1.41
NS
90.67 (525/579)
89.54 (582/650)
NS
7.77 2.12
8.05 0
NS
Data are presented as mean SD. N: number of patients, NS = not statistically significant.
Comments In this diagnostic trial study, we compared the morphokinetic parameters between fully implanted embryos and those that failed to implant after IVF and transfer on Day 3 (the IVF cycles were derived from the ‘good prognosis’ patients). The LR model of the
Table 3 Characteristics and implantation rate of embryos that developed to 2-blastomere stage within or outside the time interval 21.27 h < t2 < 26.725 h. Embryos developed within time interval (N = 120) Blastomere numbers (n) Embryo score Implantation rate (%)
Embryos developed outside of time interval (N = 92)
p-value
9.13 2.29
7.96 1.51
0.000
2.39 0.45 60.83
2.52 0.56 40.21
0.031 0.003
Data are presented as mean SD or %. N: number of embryos.
L. Wu et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 197 (2016) 186–190 Table 4 Characteristics and implantation rate of embryos that all developed to 2-blastomere stage within the time interval 21.27 h < t2 < 26.725 h but were within or outside the second time range 11 h < t5-t4 < 13 h. Embryos developed within second time interval (n = 41) Blastomere numbers (n) Embryo score Implantation rate (%)
Embryos developed outside of second time interval (n = 79)
9.54 2.68
8.92 2.29
2.43 0.54 78.05
2.37 0.48 51.9
p-value
NS NS 0.005
Data are presented as mean SD or %. N: number of embryos.
relationship between the morphokinetic parameters and the implantation rate showed a sensitivity of 79.61% and specificity of 47.56%. Conaghan et al. [15] found that the Eeva (Early Embryo Viability Assessment, another time-lapse system) prediction and cell-tracking software plus Day 3 morphology correctly predicted which embryos would become usable blastocysts, with a sensitivity of 58.8% and specificity of 84.2%. The prediction models for blastocysts or implantation potential, to some extent, improve the ability of selecting embryos of potentially high developmental capability. However, it is worth noting that the sensitivity and specificity of the model could not simultaneously efficiently predict embryonic potential. Therefore, this area requires further research in order to improve these modes of embryonic analysis. Some of these parameters were significantly different between the two groups suggesting that timing of particular embryonic developmental events may be crucial for successful transfer and pregnancy. In our study, PN disappearance (tPNf) occurred earlier in the fully implanted group than in the non-implanted one. Aguilar et al. [21] also found that timing of pronuclei fading between 22.2 and 25.9 h was linked to successful embryonic implantation, and early PN disappearance was correlated with a higher number of blastomeres on Day 2 after IVF or ICSI [5]. However, Azzarello et al. [22] showed that too early disappearance of PNB (before 20 h 45 min) may be detrimental and results in failure of live births. Early cleavage embryos have higher implantation and pregnancy rates [23,24] and higher number of blastomeres on Day 2 [5]. Van Montfoort and colleagues reported increased pregnancy rates in the early-cleavage transfer group compared with the latecleavage transfer group [25]. We also confirmed that cleavage, detected by morphokinetic parameters during time-lapse monitoring, occurs earlier in the fully implanted embryos than in the embryos that fail to implant. Along with the extensive application of TLM, several recent studies have investigated the importance of early morphokinetic parameters during embryonic development. In the retrospective analysis of 100 embryos during 48 h of dynamic developmental processes, Wong et al. showed that duration of first cytokinesis, cc2 (t3-t2), and s2 (t4-t3) can predict blastocyst formation. However, none of the embryos in this particular study were transferred [7]. In the present study, 212 embryos were transferred, and we reliably predicted the relation between morphokinetic parameters (tPNf, t2, t4, t4-t3) and implantation potential. Another study where the authors predicted implantation potential [3] found that t5, cc2 and s2 were positively associated with implantation potential; however, morphokinetic parameters were recorded only until the 5-cell stage rather than the 8-cell stage in our study. Another similar finding between our and other two studies above was that the synchronized embryonic development from three to four cells was associated with a higher chance of implantation. Thus, 3-cell and 4-cell synchronization was essential for blastocyst
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formation and potential for implantation. The morphokinetic parameters t2 were also significantly different between fully implanted and non-implanted embryos in our study. This supports the findings of Desai et al. [26] and Dal Canto et al. [27] who detected different timings of t2, t3, t5 and t8 between implanted and non-implanted blastocysts, and shorter development of implanting embryos to the 8-cell stage, respectively. t4 was only significantly different between fully implanted and non-implanted embryos in our study, suggesting that t4 may be specific to our IVF laboratory, and this could provide us with more information regarding embryo selection. The results from several IVF centers were not always consistent, which may be due to the different sample populations. Additionally, the length of the cell cycle may also be affected by the culture conditions and fertilization methods used. Wale and Gardner [27] found that the developmental stages of mouse embryos cultured in 20% oxygen were prolonged compared with embryos in 5% oxygen. It has already been reported that culture media also affects human embryonic cleavage rates [28–31]. Moreover, some studies have shown that the method of fertilization has an effect on cleavage time with ICSI inducing earlier cleavage than conventional IVF [32,33]. Therefore, it is important to note that some data and selection models cannot be completely extrapolated from one laboratory to another. Our second goal was to establish an embryo selection model at our IVF center. Therefore, we determined the best embryonic cleavage time intervals by using the LR model and ROC curves. In the range of 21.27 h < t2 < 26.725 h, embryos showed increased blastomere numbers, higher embryo score and higher implantation rate. Previously, early cleavage was observed to occur at 27– 29 h after IVF fertilization and 25–27 h after ICSI fertilization using a traditional inverted microscope [2]. When the combined ranges of 21.27 h < t2 < 26.725 h and 11 h < t5-t4 < 13 h were selected, the implantation rate, was still significantly increased. In a previous retrospective TLM analysis comparing the morphokinetic parameters of 72 fully implanted embryos with 106 nonimplanted embryos, Day-5 embryos with a cc3 (from 3 cells to 5 cells) between 9.7 and 21 h had the highest probability to implant [14]. In this study, we obtained optimal time intervals for t2 and t5-t4 using a time-lapse monitoring system for early embryonic development, which was associated with a higher embryonic implantation rate. Based on these results, an embryo selection model in our IVF center can be established. However, it should be confirmed in a prospective, randomized, controlled study so as to ascertain whether use of the model can consistently improve clinical pregnancy rates. One limitation of this study was however that the times for second polar body extrusion and PN appearance were not observed due to placing the normally fertilized zygotes into the time-lapse culture system on the next morning after insemination. The second limitation was that we confirmed clinical pregnancy by the presence of gestational sacs at week 4 and not heart beat. In conclusion, embryos that reach specific developmental stages during early cleavage at critical times have higher potential for implantation and live births. Importantly, an embryo selection model can be established by using specific time intervals for t2 and t5-t4, which may facilitate the selection of optimal embryos for eSET, thereby avoiding subjectivity and limitations of a singlemorphology scoring system.
Funding This article and study was funded by ‘‘Chongqing municipal health and family planning commission’’, and the project number is ‘‘2015MSXM087’’.
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