Abstract
Purpose
In vitro developing embryos may apparently show no developmental progress during 24 h and resume their development up to the blastocyst stage. The present study was conducted to assess their ability to implant and to give rise to a live birth when replaced at day 5 (fresh or vitrified/warmed) as compared to continuously developing embryos.
Methods
Embryo development follow-up and grade were prospectively recorded in a photo database. The studied period was from April 2011 to July 2017. The studied embryos included transient arrested embryos (TAE) that showed the same developmental stage at two subsequent observations, i.e. between day 2 and day 3 (d2 and d3), between day 3 and day 4 (d3 and d4) and between day 4 and day 5 (d4 and d5). TAE were compared to continuously developing embryos (CDE). Elective day 5 embryo transfers were performed.
Results
Woman age was higher in TAE (34.3±3.9) than in CDE (32.9±4.8) (p<0.01). TAE were more frequently (63.1%) observed after ICSI than after conventional IVF (55.9%) (p<0.01). Implantation rate was reduced in TAE as compared to CDE, after both fresh (10.0% vs 23.8% [p<0.01]) and vitrified/warmed (12.9% vs 19.0% [p<0.01]) embryo transfers. Delivery rate was also lower after the transfer of fresh (8.3% vs 19.4% [p<0.01]) and vitrified/warmed (8.5% vs 14.1% [p<0.01]) TAE as compared to CDE. Implantation and delivery rates were not statistically different whether embryo arrested between day 2 and day 3 (d2 and d3), between day 3 and day 4 (d3 and d4) or between day 4 and day 5 (d4 and d5).
Conclusion
TAE may be considered for transfer at a lower priority than CDE and associated with inferior prognosis than CDE.
Keywords: Transient arrested embryo, Continuously developing embryos, Implantation rate, Delivery rate, ICSI, IVF
Introduction
The human embryo undergoes a series of crucial changes during in vitro pre-implantation development. These events and their timing are generally used as indicators of embryo ability to give rise to a pregnancy and live birth. The timing of crucial events that should undergo a so-called good prognosis embryo was discussed in a consensus document [1]. This recommendation document also tackles the fate that should be given to embryos that do not meet good embryo prognosis criteria [1]. Embryo development disruption is possibly caused by various factors. On the one hand, suboptimal in vitro culture conditions were proved to negatively impact embryo development, which in turn impairs embryo viability [2]. Oxygen tension, embryo density and culture media composition were identified as physical and chemical stressors that may cause apoptosis and cleavage anomalies in in vitro developing embryos [2]. Although culture systems have improved over the past decades, in practice, in vitro developing embryos show variable morphokinetics and may even show developmental arrest. On the other hand, intrinsic factors may also be responsible for embryo developmental arrest. Indeed, it is well known that the first steps of pre-implantation embryo development are under the control of oocyte intracellular stores until the activation of embryonic genome that occurs at the 8-cell stage [3]. Although oocyte was shown to be capable of repairing sperm-borne DNA damage and to support the first embryo cleavages before the embryo activates its own genome, defective embryonic genome activation ultimately leads to embryo developmental arrest [4, 5]. Other causes for aberrant embryo development were identified. They encompass chromosomal status, genome integrity [6–11], environmental factors [12–14] and patient-related factors, including woman age, endometriosis and smoking habits [15–17]. Complete developmental embryo arrest was estimated to happen in 10–32% of embryos obtained after conventional IVF or ICSI [18, 19]. Nevertheless, it was recommended to consider these arrested embryos as “non-viable” and that they “should not be replaced if morphologically better embryos are available” [1].
A metabolic activity was detected in arrested embryos and they are considered to be in a non-apoptotic “senescence-like” state [18]. This atypical phenomenon is likely not irreversible. Extended embryo culture unmasks the fact that developmental embryo arrest can be transitory; embryos may show no developmental progress during 24 h and resume their development to the blastocyst stage.
As embryos displaying such atypical features are often not utilised, little is known about their competence and their ability to give rise to pregnancy and to live birth. The present study was conducted to assess the competence of embryos showing transient development arrest (TAE). Their ability to implant and to give a live birth when replaced at day 5 (either fresh or after vitrification/warming cycle) was compared to continuously developing embryos (CDE).
Materials and methods
Studied embryos
This is a retrospective study conducted between April 2011 and July 2017. Amongst the 7548 extended cultured embryos, there were 883 TAE that showed the same developmental stage at two subsequent observations, i.e. between day 2 and day 3 (d2 and d3), between day 3 and day 4 (d3 and d4) or between day 4 and day 5 (d4 and d5) (Fig. 1). When the embryo developmental transient arrest happened between day 4 and day 5, the evolution was checked a second time before transfer or cryopreservation, 4–5 h after daily embryo observation (e.g. Fig. 1, emb03). These TAE were compared to 6665 CDE. All studied embryos (TAE and CDE) were transferred or cryopreserved at day 5.
Fig. 1.
Illustration of the 3 studied scenarios in TAE. Transient developmental arrest between day 2 and day 3 (d2 and d3) (emb01), between day 3 and day 4 (d3 and d4) (emb02) and between day 4 and day 5 (d4 and d5) (emb03). Photos of oocytes (before and after injection) and injected spermatozoon were taken at day 0, after oocyte collection (d0) up to day 5 (d1, d2, d3, d4, d5). When the embryo developmental transient arrest happened between day 4 and day 5, the evolution was checked a second time before transfer or cryopreservation, 4–5 h after daily embryo observation (e.g. emb03). ovo, oocyte; spz, spermatozoon; emb, embryo; T or C, transferred or cryopreserved
Embryo culture and data collection
Embryos obtained after conventional IVF and ICSI were cultured in single drops under the same conditions, in individual microdrops of global medium (LifeGlobal®, USA) covered with mineral oil (Ferticult™) under standard culture conditions (37 °C, CO2 6%). As recommended for zygote and embryo scoring, fertilisation check was performed on day 1 (17 h±1 post insemination). IVF and ICSI embryos were scored on day 2 (44 h±1 post insemination), day 3 (68 h±1 post insemination), day 4 (92 h±2 post insemination) and day 5 (116 h±2 post insemination) (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011).
Embryo morphology was assessed at cleavage stages using the BLEFCO grading system characterising cell number, percentage of fragmentation and synchrony of the blastomere division [20]. Blastocyst stage embryos were scored using a simplified Gardner grading system [21]. Day 5 embryos were classified into 3 categories based on their expansion degree: Grade 1 embryos included compacting morulas and blastocysts with early forming blastocoele, grade 2 embryos encompassed early blastocysts with clear blastocoele that occupied up to half of the embryonic volume (B1–B2) and grade 3 embryos were full blastocysts displaying a blastocoele occupying more than half the volume of the embryo and/or increased size (>B3).
Elective single embryo transfers (fresh or vitrified/warmed) were performed on day 5.
A prospective image collection was started in 1998 in which a photo of each embryo was manually taken by an embryologist and recorded daily (from fertilisation to day 5) in a photo database (PEGASE software, 2iSYSTEM, France) [22]. Embryo grade and fate were given every day by an embryologist. The studied period started in 2011 which corresponds to the introduction of vitrification technique in our laboratory. Data regarding embryo development from April 2011 to July 2017, post-warming survival rate and clinical outcome were analysed retrospectively using the photo database and clinical registry.
Embryo vitrification and warming cycles
Vitrification and warming procedures were performed following the protocol published by Kuwayama and colleagues using Kitazato media® and Cryotop Device® [23].
During vitrification, embryos were incubated 10 to 15 min at room temperature in the equilibration solution then exposed during 1 min to the vitrification solution, placed on the Cryotop Device® strip and were directly immersed into liquid nitrogen. Embryos were individually vitrified on the device.
Embryo warming cycles were performed using the following protocol: The strip of the Cryotop Device® was immersed into the thawing solution at 37 °C during 1 min. Embryos were transferred at room temperature into the diluent solution (for 3 min) and washed twice in drops of wash solution for 5 min and then 1 min. Embryos were incubated at least 1 h in global protein supplemented medium 37 °C, CO2-6% prior to transfer. A photo was also taken on the day of warming to assess survival rate by comparing with the picture taken prior cryopreservation. Post-warming survival rate was calculated as the ratio between the number of embryo with more than 80% of intact cells and the total number of warmed embryos.
Outcomes definition
Implantation rate was the ratio between the number of transferred embryo and the number of gestational sac with foetal heartbeat. Delivery was considered if pregnancy was achieved after 22 gestational weeks. Delivery rate was calculated as the ratio between the number of deliveries and the number of embryo transfers.
Statistics
Statistical analyses were performed using Statistical Package for Social Sciences, version 17 (SPSS, IBM Corporation, USA). Qualitative variables appeared as percentages and quantitative variables as means with standard deviations. Student’s t test was used to compare quantitative variables and Pearson chi-square or Fisher’s exact test was used for qualitative variables. Implantation and delivery rates were compared with univariate analyses. Multivariate analyses were performed to compare implantation and delivery rates according to woman age, fertilisation technique and embryo grade. A two-sided p value of <0.05 was considered statistically significant.
Results
Embryo follow-up using PEGASE software is depicted in Fig. 1. The 3 studied scenarios in TAE (d2–d3, d3–d4 and d4–d5) are illustrated.
A total of 883 TAE and 6665 CDE were studied, either transferred fresh or vitrified for later treatment. The overall description of the embryos studied is provided in Table 1. The mean woman age was significantly higher in TAE (34.3±3.9) than in CDE (32.9±4.8) (p<0.01). We have also found that TAE were more frequently observed (63.1%) after ICSI than after conventional IVF (55.9%) (p<0.01). TAE displayed significantly more compacting morulas and early blastocysts (grade 1 embryos) (71.6%) than CDE (36.5%) (p<0.01) and fewer expanded blastocysts (8.5%) than CDE (22.5%) (p<0.01). TAE were less frequently selected for fresh embryo transfer than CDE (38.0% vs 42.0%, p<0.01) but more likely vitrified for use in later treatment (62%). TAE had a reduced ability to survive vitrification/warming cycle than CDE (69.2% vs 85.2%, p<0.01) (Table 1).
Table 1.
Description of analysed embryos
| TAE | CDE | OR | 95% CI | p value | ||
|---|---|---|---|---|---|---|
| Mean woman age (SD) | 34.3 (3.9) | 32.9 (4.8) | - | - | <0.01 | |
| Number of studied embryos | 883 | 6665 | - | - | - | |
| Embryos obtained after conventional IVF (%) | 326 (36.9) | 2939 (44.1) | 0.7 | [0.7–0.8] | <0.01 | |
| Embryos obtained after ICSI (%) | 557 (63.1) | 3726 (55.9) | 1.4 | [1.2–1.5] | <0.01 | |
| Embryo grade at day 5 (%) | 1 | 632 (71.6) | 2432 (36.5) | 4.4 | [3.8–5.1] | <0.01 |
| 2 | 176 (19.9) | 2731 (41.0) | 0.4 | [0.3–0.4] | <0.01 | |
| 3 | 75 (8.5) | 1502 (22.5) | 0.3 | [0.3–0.4] | <0.01 | |
| Number of fresh transferred embryos (%) | 335 (38.0) | 2801 (42.0) | 0.8 | [0.7–1.0] | <0.05 | |
| Number of Vitrified embryos (%) | 548 (62.0) | 3864 (58.0) | 1.2 | [1.0–1.4] | <0.05 | |
| Number of warmed embryos | 292 | 1930 | - | - | - | |
| Post-warming survival rate (%) | 69.2 | 85.2 | 0.4 | [0.3–0.5] | <0.01 | |
After controlling for woman age, fertilisation technique and embryo grade, overall implantation rate turned out to be significantly reduced in TAE as compared to CDE, after either fresh (10.0% vs 23.8% [β=−0.06; p<0.01]) or vitrified/warmed embryo transfer (12.9% vs 19.0% [β=−0.08; p<0.01]). Delivery rate was also lower after the transfer of fresh (8.3% vs 19.4% [β=−0.05; p<0.01]) and vitrified/warmed (8.5% vs 14.1% [β=−0.06; p<0.01]) TAE as compared to CDE (Fig. 2).
Fig. 2.
Implantation and delivery rate of fresh and vitrified/warmed TAE as compared to CDE. Embryos were transferred or cryopreserved on day 5. *p value<0.01
After adjusting for woman age, fertilisation technique and embryo grade, implantation rate was not significantly different whether developmental arrest happened between day 2 and day 3 (d2 and d3) (14.5%), between day 3 and day 4 (d3 and d4) (12.5%) or between day 4 and day 5 (d4 and d5) (5.7%) (Fig. 3). No difference was observed after the transfer of vitrified/warmed embryos (9.5%, 13.8% and 13.3% in d2 and d3, d3 and d4, d4 and d5, respectively). Besides, the differences observed in delivery rates did not reach statistical significance after fresh (12.9%, 11.9% and 4.4% in d2–d3, d3–d4 and d4–d5 TAE, respectively) and after vitrified/warmed embryo transfer (6.1%, 11.4% and 8.2% in d2–d3, d3–d4 and d4–d5 TAE, respectively) (Fig. 3).
Fig. 3.
Implantation rate and delivery rate of TAE depending on the time frame of transient embryo developmental arrest; between day 2 and day 3 (d2 and d3), between day 3 and day 4 (d3 and d4) and between day 4 and day 5 (d4 and d5). Embryos were transferred or cryopreserved at day 5
Discussion
As far as we know, this is the first report on the competence of in vitro cultured human embryo displaying transient developmental arrest. We show that although they display lower capacity to develop into expanded blastocysts, to recover vitrification/warming cycle, to implant and to lead to live birth, they ensure a non-negligible chance of success.
Data reported here were not obtained using time-lapse imaging for the technology was implemented in our laboratory only in 2017. We are completely aware that embryo grading may vary between embryologists. However, our high-quality image database has proved its worth during the past 21 years since it provides an objective retrospective overview of embryo development. Besides, it enables to verify and correct embryo scores in a retrospective manner. Like time-lapse technology, daily photos of embryos are substantial objective records that provide a superior approach to traditional embryo development scoring, with no image record. Besides, we are not limited to the number of photos taken and we can focus on specific embryonic structures. Interestingly, it was reported that only a minority of IVF laboratories worldwide use time-lapse technology (17% in the USA, 39% in France) and therefore, daily embryo development monitoring is still being performed in a traditional manner (at static time points) in a large proportion of laboratories [24, 25]. Therefore, we believe that at least nonusers of time-lapse technology will show some interest in the present work. Nevertheless, we expect to conduct a similar study using time-lapse imaging and possibly to compare the two approaches. A time-lapse study will help to refine the description of TAE and to discriminate real developmental arrest and discrete slow continuous evolution.
The arrest of in vitro cultured embryos is likely caused by multiple factors. Developmental embryo arrest is under the influence of oxygen tension, embryo density, telomere dysfunction, oxidative stress and irregular cleavage [2, 18, 26]. A modification of culture conditions like decreasing oxygen tension may potentially change the occurrence of TAE. Our data show that TAE are related to woman age and method of fertilisation (IVF or ICSI) (Table 1). As already known, early embryo cleavage is under oocyte control [3]. Therefore, low developmental competent oocytes as reported in advanced age women may compromise embryo development, ultimately resulting in cleavage arrest [27, 28]. This is in line with our result showing higher woman age observed in TAE [28]. The relationship between fertilisation technique and embryo arrest rate was questioned in few studies with controversial outcomes; some data tend to show a higher frequency of this anomaly after conventional IVF [29], while others found no impact of fertilisation technique on the arrest of embryo development [30]. Nevertheless, our data indicate that transient arrest is more frequently observed after ICSI than after conventional IVF. This is consistent with the finding that morphokinetics defects are perceived at a higher occurrence in ICSI embryos than after conventional IVF [12, 31]. This observation is a reminder that ICSI is an invasive technique that was suggested to be performed in definite cases since no benefit was found to its extensive non-male factor utilisation [32, 33]. Another interpretation of this observation is that male factor infertility is more frequent in ICSI group and aetiologies of male infertility were reported to impact embryo development [34]. Overall, embryo developmental arrest is expected to be a multifactorial feature whose cause is complex to identify with a high degree of confidence.
TAE show general compromised competence when compared to CDE. Indeed, TAE demonstrated a reduced ability to develop into expanded blastocysts and to survive vitrification/warming cycle. The reported post-warming survival rate in CDE is slightly lower than published values for blastocyst vitrification in autologous cycles [35]. In the present study, CDE include early blastocyst but also compacted morulas. We are aware that our embryo selection policy for cryopreservation is likely less strict than in other laboratories which may explain the post-warming survival rate observed in CDE [36]. Not surprisingly, we described impaired implantation and delivery rates in TAE when compared to CDE after adjusting for confounding variables. This observation is possibly a result of higher chance of TAE to carry abnormalities such as chromosomal imbalance [6–11]. For legal reasons, we do not perform pre-implantation genetic testing. Therefore, we were unable to evaluate ploidy status of TAE. Our figures also show that the time frame at which the transient arrest occurs does affect neither implantation rate nor delivery rate in a significant manner (Fig. 3).
Previous reports on arrested embryos involved complete developmental arrest and data about embryos resuming their development is scarce. Developmental arrest observed in TAE might correspond to a “self-correction” time frame where the embryo restores chromosomal balance or prolonged cell cycles that are likely associated with DNA repair or incorrect chromosome attachment to the spindle [26, 37–39]. One can suppose that TAE that do not implant are those that failed to “correct” the genetic errors. Deeper analyses of TAE possibly using PGT-A are needed to tackle this hypothesis.
In the present study, we have shown that although TAE have reduced competence, based on our figures, TAE should not be considered as “non-viable”. Therefore, such embryos may be used at a lower priority than CDE. The present report on TAE may benefit patient for counselling purposes in cases where no better embryos are available for transfer associated with obviously a follow-up of clinical outcomes.
Acknowledgements
We warmly thank the clinical staff for contributing in patient recruitment. We also acknowledge the laboratory staff for technical support.
Footnotes
Publisher’s note
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Contributor Information
Debbie Montjean, Email: debbie_montjean@hotmail.com.
Cendrine Geoffroy-Siraudin, Email: csiraudin@hopital-saint-joseph.fr.
Marie-José Gervoise-Boyer, Email: mboyer@hopital-saint-joseph.fr.
Pierre Boyer, Email: pboyer@hopital-saint-joseph.fr.
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