Abstract
Purpose
To evaluate the euploidy rate and obtain the biochemical pregnancy, implantation, clinical pregnancy, miscarriage and live birth rates for embryos biopsied on days 5, 6, 7 and 8 of development.
Methods
Observational, retrospective and multicenter study. In total, 1845 IVF‒ICSI cycles with preimplantation genetic testing for aneuploidy (PGT-A) were included. The euploidy rate was determined according to the biopsy day (5, 6, 7 or 8), as were the reproductive outcomes of 710 single embryo transfers (SETs).
Results
Seven thousand three hundred thirty-eight biopsied embryos were analyzed by means of PGT-A. Of these, 414 procedures resulted in amplification failure (5.6%), leaving 6924 embryos. In total, 5042 (72.8%) were biopsied on day 5, 1779 (25.7%) on day 6, 101 (1.5%) on day 7, and 2 (0.03%) on day 8. The euploidy rates on days 5, 6, 7 and 8 were 41.6%, 32.7%, 27.7%, and 50%, respectively. The euploid embryo biopsied on day 8 was not transferred. For blastocysts biopsied on days 5, 6 or 7, the biochemical pregnancy rates were 62.3%, 53.2% and 33.3%; the implantation rates were 59.8%, 50.3% and 22.2%; the clinical pregnancy rates were 59.7%, 42.2% and 22.2%; the live birth rates were 52.5%, 43.2% and 22.2%; and the miscarriage rates were 12.2%, 14.0% and 0%, respectively.
Conclusions
The euploidy rate decreased with increasing time required to reach the blastocyst stage. Compared with day 6 and day 7 euploid embryos, day 5 euploid embryos had better clinical outcomes. However, the findings in this study demonstrate that embryos with slow development can result in live births.
Clinical trial registration
Due to being a retrospective study, the clinical trial was not registered.
Keywords: Blastocyst, Preimplantation genetic testing for aneuploidy, Implantation rate, Trophectoderm biopsy
Introduction
In vitro fertilization (IVF) is one of the most effective assisted reproductive treatments, with significant advances in recent decades. Improvements in ovarian stimulation protocols, embryo vitrification procedures, delayed transfer and advances in embryo culture have led to higher implantation, clinical pregnancy and live birth rates.
The optimization of culture conditions has allowed embryos to reach to the blastocyst stage in a prolonged manner. Blastocyst transfer improves reproductive outcomes, so its application has been increasing [1]. However, there are cases in which blastocyst formation is delayed, and this stage is reached within 140 ± 2 h after insemination (day 6), and even up to day 7 or 8, these represent 10 to 15% of blastocysts [2].
Preimplantation genetic testing for aneuploidy (PGT-A) is a modern assisted reproductive technology (ART) tool whose use enables the selection of euploid embryos to improve the implantation rate, reduce the miscarriage rate and avoid live births with chromosomal abnormalities [2].
The results of multiple studies have demonstrated that slower developing blastocysts have higher aneuploidy rates, which could be attributed to meiotic spindle abnormalities, mitochondrial deficiencies or gene expression [3–8]. These deficiencies could affect the ability of blastocysts to implant and develop in utero [3]. However, in more recent studies, it has been observed that embryos with slow development can also be viable, euploid and lead to a healthy live birth [9].
In IVF‒ICSI cycles without PGT-A, when embryos exhibit comparable morphological qualities, the transfer of day-5 embryos is preferred over that of day-6 and day-7 embryos [10]. However, due to PGT-A, we can identify slow-developing euploid embryos that may have a good reproductive prognosis.
Hernández-Nieto et al. 2019, Tiegs et al. 2019 and, Liu et al.2022 [4–6] have reported lower euploidy rates in embryos biopsied on day 7 compared to those biopsied on day 5 or 6. Euploidy rates ranged from 48.8% to 67.7% on day 5, 35.1% to 52.9% on day 6, and 30.3% to 43.1% on day 7, showing a progressive decrease as the time required to reach the blastocyst stage.
In a sub-analysis of reproductive outcomes, Hernández-Nieto et al. 2019 [4] found that implantation rates declined as the day of biopsy advanced: 77.9% on day 5, 69.6% on day 6 and 44.8% on day 7. A similar pattern was also observed in clinical pregnancy and live birth rates. These findings are consistent with those reported by other authors (Tiegs et al.2019, Liu et al. 2022, and, Abdala et al. 2023) [5, 6, 11] who likewise documented a decline in these outcomes.
Objective
The main objective of this study was to evaluate euploidy rates in blastocysts biopsied on days 5, 6, 7 and 8 of development. The secondary objectives included comparing reproductive outcomes, specifically biochemical pregnancy, implantation, clinical pregnancy, miscarriage and live birth rates, following single embryo transfers of blastocysts biopsied on these respective days.
Materials and methods
An observational, retrospective, multicenter study was performed. In total, 1845 IVF‒ICSI cycles with PGT-A from January 2018 to December 2023 were included.
A total of 7338 embryos were biopsied on days 5, 6, 7 and 8 of development and analyzed for preimplantation genetic testing for aneuploidy. The biopsy was performed according to the degree of expansion and morphological quality [12]. Five to ten cells were taken from the trophectoderm and deposited in PCR tubes with 2.5 µl of 1 X Buffered PBS (Gibco™, USA). Biopsies were frozen at − 20 °C until shipment, and blastocysts were vitrified following the protocols of each IVF laboratory pending study results. The method for detecting aneuploidies through next-generation sequencing (NGS) of trophectoderm cells has been extensively described in previous publications [13, 14]. The embryos were classified as euploid, aneuploid or mosaic (also known as intermediate chromosome copy number embryos) following the standard criteria for interpretation of PGT-A by NGS [14].
The embryos were divided into four groups according to the day on which trophectoderm biopsy was performed: day 5, 6, 7 and 8. Then, euploidy, aneuploidy and mosaicism rates were calculated in each group. The euploidy rate was also analyzed based on the day of biopsy and maternal age. For the analysis, embryos were classified into one of the following age groups as defined by the Society for Assisted Reproductive Technology (SART): < 35 years, 35–37 years, 38–40 years, 41–42 years and > 42 years.
Controlled ovarian stimulation protocols were applied according to the individual characteristics and needs of each patient using gonadotropins, and follicular monitoring was performed with ultrasound. Oocyte maturation was triggered when at least one follicle reached 18 mm, with hCG (5000–10,000 UI), recombinant hCG (250–500 µl) or 0.2 mg of triptorelin acetate with or without 1500 UI of hCG, according to each physician’s assessment and the risk of developing ovarian hyperstimulation syndrome. Oocyte retrieval was performed vaginally under ultrasonographic guidance 36–37 h later.
Only transfers with SET were included in natural cycle and hormone replacement therapy (HRT) protocol endometrial preparations, based on the criteria of each treating physician. The embryo transfer was performed on day 5 + 1 of progesterone exposure, regardless of the day of embryo development at the time of biopsy and vitrification.
Couples with indications for preimplantation genetic testing for structural rearrangements (PGT-SR) or monogenic diseases (PGT-M) were excluded from the study, as were those with cycles initiated for PGT-A, in which no blastocysts were obtained for biopsy.
A subanalysis was performed to determine the following reproductive outcomes: the biochemical pregnancy rate, defined by the International Glossary in Infertility and Fertility 2017 [15] as a pregnancy diagnosed only by the detection of beta hCG in serum or urine per number of embryo transfers; the implantation rate, defined as the number of gestational sacs observed per number of embryos transferred; the rate of clinical pregnancy with a fetal heartbeat, defined as the number of clinical pregnancies with a fetal heartbeat (ultrasound-diagnosed pregnancy of one or more gestational sacs, with at least one fetus with a discernible heartbeat) per number of embryo transfers; the live birth rate, defined as the number of newborns after 22 weeks of gestation showing signs of life per the number of embryo transfers; and the miscarriage rate, defined as the loss of a clinical pregnancy before 22 completed gestational weeks per number of clinical pregnancies.
Statistical analysis was performed with SPSS Statistics software, version 21. All p values are bilateral, and p < 0.05 was the threshold for significance in all statistical tests.
Results
In total, 1845 ICSI cycles with 7338 embryos with PGT-A data were evaluated. The mean age of the patients was 37.3 ± 4.5 years. Among the total number of embryos biopsied, 414 (5.6%) were amplification failures due to the limited amount of initial DNA, resulting in a total of 6924 embryos. Out of the embryos analyzed, 5042 (72.8%) were biopsied on day 5, 1779 (25.7%) were biopsied on day 6, 101 (1.5%) were biopsied on day 7, and 2 (0.03%) were biopsied on day 8 (Fig. 1).
Fig. 1.
Percentage of embryos analyzed on the day of biopsy
The overall rates of euploidy, aneuploidy and mosaicism were 39.2% (n = 2711), 53.8% (n = 3724) and 7.0% (n = 487), respectively. An analysis of the euploidy rate based on biopsy day revealed a decreasing trend as the time required for blastulation increased from 41.6% (n = 2100) on day 5 to 32.7% (n = 583) on day 6 to 27.7% (n = 28) on day 7. Significant differences were observed between day 5 and day 6 (p < 0.0001); however, differences in the euploidy rate were not significant between day 6 and day 7. Among the 2 embryos biopsied on day 8, one was euploid, and the other was aneuploid, resulting in a euploidy rate of 50%; the available euploid embryo has not been transferred to date. These embryos were not included in the statistical analysis because of the small number of blastocysts in this group (Fig. 2). The aneuploidy rate on day 5 was 51% (n = 2576), that on day 6 was 60.5% (n = 1078), and that on day 7 was 69.3% (n = 70). As expected, this increased with a prolonged blastulation time. Like the euploidy rate, significant differences were observed between day 5 and day 6 (p < 0.0001); however, no significant differences were observed between day 6 and day 7. The rates of mosaicism, also known as intermediate chromosome copy number embryos, were very similar, with no significant differences among the three groups: 7.5% (n = 366) on day 5, 6.6% (n = 118) on day 6 and 2.9% (n = 3) on day 7.
Fig. 2.
Euploidy, aneuploidy and mosaicism rates per day of biopsy
An analysis of the proportion of euploid embryos was performed according to the day of trophectoderm biopsy, and the embryos were subsequent stratified by age group (Table 1). On days 5 and 6, the biopsied embryos had a parallel behavior, in which the euploidy rate was lower in the day 6 group than in the day 5 group, in both groups euploidy rate decreased progressively with increasing maternal age. Embryos biopsied on day 7 showed unexpected behavior, no progressive decline in the euploidy rate was observed as patients age increased. The rates in some age groups were higher than those in embryos biopsied on days 5 and 6 (Fig. 3). We believe that these results may be due to the limited number of days with 7 euploid embryos (n = 28).
Table 1.
Euploidy rate by maternal age and biopsy day
| Maternal age | Day 5, n/N (%) | Day 6, n/N (%) | Day 7, n/N (%) |
|---|---|---|---|
| < 35 | 1190/2140 (55.6) | 316/667 (47.4) | 14/22 (63.6) |
| 35–37 | 380/869 (43.7) | 106/295 (35.9) | 9/21 (42.9) |
| 38–40 | 388/1223 (31.7) | 118/450 (26.2) | 2/30 (6.7) |
| 41–42 | 112/546 (20.5) | 33/236 (14) | 1/12 (8.3) |
| > 42 | 30/264 (11.4) | 10/131 (7.6) | 2/16 (12.5) |
Data are presented as proportions according to the total number of embryos biopsied per day of biopsy and maternal age
Fig. 3.
Euploidy rate by maternal age and day of biopsy
To date, 1317 (48.6%) of 2711 euploid embryos have been transferred. Among the total number of transfers performed, 710 were SETs. These transfers were the only ones analyzed in the present study to accurately identify the embryo that was implanted. Among the 710 SETs, 560 were day 5 embryos (78.9%), 141 were day 6 embryos (19.9%), and 9 were day 7 embryos (1.3%). A single day 8 euploid embryo has not been transferred to date.
The biochemical pregnancy rate of embryos transferred on day 5 was 62.3% (n = 349/560), that on day 6 was 53.2% (n = 75/141), and that day 7 was 33.3% (n = 3/9), with significant differences only between day 5 and day 6 (p = 0.04). The implantation and clinical pregnancy rates were very similar because only SETs were included, and we did not have twin pregnancies; the implantation rates were 59.8% (n = 335/560) on day 5, 50.3% (n = 71/141) on day 6 and 22.2% (n = 2/9) on day 7. The clinical pregnancy rates were 59.6% (n = 334/560) on day 5, 49.6% (n = 70/141) on day 6, and 22.2% (n = 2/9) on day 7. The results revealed that the implantation and clinical pregnancy rates were higher on day 5 than on day 6 (p = 0.04) or day 7 (p = 0.02), with no significant difference between day 6 and day 7 (Fig. 4).
Fig. 4.
Biochemical pregnancy, implantation, live birth and miscarriage rates on the day of trophectoderm biopsy. Only the statistically significant rates are shown
The live birth rate for embryos biopsied on day 5 was 52.5% (n = 294/560), that on day 6 was 43.2% (n = 61/141), and that on day 7 was 22.2% (n = 2/9). The analysis revealed a significantly higher live birth rate on day 5 than on day 6 (p = 0.04) but not between day 5 and day 7 or between day 6 and day 7. This could be explained by the smaller number of day 7 embryo transfers. The miscarriage rate on day 5 was 12.2% (n = 41/335), and that on day 6 was 14.0% (n = 10/71) (p = 0.6). No miscarriages were reported in the day 7 cohort (Fig. 4). Additionally, two pregnancies were ectopic.
Discussion
In our study, a total of 7338 embryos underwent biopsy for PGT-A, amplification failure occurred in 414 embryos (5.6%). Yielded results for 6924 embryos. Among these, 27.3% (1882) of the blastocysts had slower development (1779 on day 6, 101 on day 7 and 2 on day 8). Traditionally, embryos that require more time to reach the blastocyst stage are considered to have a poorer prognosis [1, 9, 16–18]. Previously, a consensus established that delayed blastocyst development (day 7 or 8) is a poor prognostic indicator for implantation [19]. However, the publication of the first pregnancy achieved with a day 7 embryo [20] prompted a change in recent practices, allowing extended embryo culture and embryo transfer [21]. The amplification failure rate observed in our study was above average, however, it is worth mentioning that include non-concurrent outcomes [22] and biopsies from multiple centers starting their PGT-A program. Actually, our amplification failure has decreased to 3.1% [13, 23–25].
Our study provides insight into the success rates of late developing embryos, offering relevant information for cycle management in infertile patients with day 6, 7 and 8 blastocysts. Hernández-Nieto et al. 2019 [4] found that the euploidy rate progressively decreased, with rates of 54.7%, 52.9%, and 40.8% on biopsy days 5, 6, and 7, respectively. This same trend has been replicated in more recent studies, reporting euploidy rates of 48.8% on day 5, 35.1% on day 6, and 30.3% on day 7 [6]. In our cohort, we observed that as the time required to reach the blastocyst stage increased, the euploidy rate decreased, with significant differences in the euploidy rate between day 5 and day 6 embryos (41.6% vs. 32.7%, p < 0.0001) but not between day 6 and day 7 embryos (32.7% vs. 27.7%, p = 0.292). Additionally, we evaluated two embryos biopsied on day 8; one was euploid and remains untransferred. While the sample size is limited, this finding suggests that extended embryo culture may be a viable option in select clinical scenarios.
In addition to the analysis of PGT-A results according to the biopsy day, we evaluated these results in relation to maternal age, observing, as expected, an increase aneuploidy rate with advanced maternal age, as in previous studies [26]. In our study, the euploidy rate gradually decreased with increasing maternal age and time required to reach the blastocyst stage. This pattern was evident in embryos biopsied on days 5 and 6 but not in embryos biopsied on day 7, where euploidy was reduced in the 38 to 40 and 41 to 42 age groups than in the other age groups in the cohort. We attribute this observation to the number of embryos biopsied on day 7 and consider that, as this number increases, the results could resemble those observed on days 5 and 6.
One of the objectives of this work was to evaluate the reproductive potential of embryos with delayed blastulation. Although initial studies with a small number of transfers showed no differences between day 5 and day 6 euploid embryos [27], the opposite was reported in other studies with a larger number of transfers. In two meta-analyses that included cycles without PGT-A, the clinical pregnancy and live newborn rates were reported to be higher following the transfer of day 5 blastocysts than after the transfer of day 6 blastocysts (included fresh transfers frozen-thawed embryo transfers) [1, 9]. These results were also replicated in other studies with a high number of euploid embryo transfers, which revealed that the implantation rate, clinical pregnancy rate and live birth rate were significantly higher among day 5 transfers than among day 6 and day 7 transfers [4, 5, 28, 29]. Recently, Abdala et al. 2023 [11] reported pregnancy rates of 70.4%, 59.3%, and 16.7%, and live birth rates of 48.7%, 35.5%, and 11.1% on biopsy days 5, 6, and 7, respectively. These findings are similar to ours, which showed pregnancy rates of 62.3%, 53.2%, and 33.3%, and live birth rates of 52.5%, 43.2%, and 22.2%, respectively. The miscarriage rates reported by other authors did not differ significantly among pregnancies derived from embryos biopsied on days 5, 6, and 7 [4, 5, 11], which is consistent with our findings.
These data demonstrate that despite confirmed embryonic euploidy, the longer the time required to reach the blastocyst stage is, the lower the reproductive success rate. However, it is important to consider that extended culture until day 6, 7 or 8 may increase the number of embryos available for transfer, particularly in the older patient group, increasing the chances of achieving a live birth.
Finally, it is important to recognize other variables can influence the reproductive success rates of euploid embryos, regardless of the day of biopsy. These variables include embryo morphological quality, endometrial preparation protocol, endometrial receptivity and particularly errors in transcription of oocyte mRNA or genome activation may disrupt gene expression patterns linked to implantation [4, 27, 30–33].
Chen et al. 2022 [34] demonstrated that embryo transfers with higher morphological grade PGT-A embryos had better implantation rates (71.4%) than did those with lower-morphological-grade PGT-A embryos (59% for regular-quality embryos and 42% for poor-quality embryos). Similarly, the live birth rates were higher among good-quality embryos (59.3%) than among poor-quality embryos (39.3%), with no significant difference in the miscarriage rate. These findings are supported by those of other studies. In particular, Zhang et al. 2022 [35] evaluated the impact of morphology on euploid embryo transfer and reported that embryos with better morphological quality had higher clinical pregnancy and live birth rates. Similarly, in a paper published by our group, we reported concordant reproductive results and a positive association between better morphological quality and a higher euploidy rate [25]. It would be important to assess the euploidy rate related to morphological quality per biopsy day.
The evidence regarding endometrial preparation protocols for euploid embryo transfer remains inconclusive. While Greco et al. 2016 and Jin et al. 2021 [36, 37] found no differences, Li et al. 2022 [38] reported higher live birth rates in natural versus artificial endometrial preparation protocol (68.8% vs 58.35%; p = 0.034, respectively). These findings are supported by other authors, who also reported improved reproductive outcomes and fewer perinatal complications with natural cycles [39–42].
The results of our study demonstrated that euploid embryos with slow development can result in healthy newborns. This provides valuable information for daily clinical practice, especially when day 7 or even day 8 embryos are involved, and opens the door for further studies on the reproductive potential of slowly developing euploid embryos. A limitation of our study is the lack of analysis of relevant confounding factors such as body mass index, sperm quality, ovarian stimulation protocol, embryo quality, and endometrial patterns. Because of the retrospective and multicenter design of our study we were unable to perform the analysis. Future studies should consider performing such analysis to strengthen and validate our findings.
Conclusions
Our results reveal gradual decrease in the euploidy rate as the required blastulation time is prolonged. The rates of biochemical pregnancy, implantation and live birth also tend to decrease as the extent of embryonic development increases, and they are significantly higher among embryos biopsied on day 5 than between those biopsied on day 6 or 7. However, the results of this study demonstrate that embryos with slow development can result in live births, suggesting that extended culture of these embryos may increase the chances of achieving a live birth.
Author contribution
− Rosa Alhelí Abarca-Rodríguez: conceptualization, resources, investigation, writing—review & editing.
− Anna Calull-Bagó: conceptualization, data curation, investigation, formal analysis, methodology, writing—original draft.
− Claudia González-Ortega: conceptualization, methodology, investigation, resources, writing—review & editing, supervision.
– Ariana A. Ramírez-Macías: conceptualization, supervision, writing—review & editing.
– Andrea Luna-Gutiérrez: conceptualization, resources, writing—review & editing.
– Antonio M. Gutiérrez-Gutiérrez: conceptualization, methodology, project administration, supervision, writing—review & editing.
Data availability
Data presented are available upon request from the corresponding authors.
Code availability
The code used in this study is the property of Institute of Sciences in Human Reproduction Vida and cannot be publicly shared due to confidentiality restrictions.
Declarations
Ethics approval
Ethical approval for retrospective review was obtained from the institutional review board.
Competing interests
The authors have no competing interests to declare that are relevant to the content of this article.
Consent to publish
All the authors are affiliated with the Institute of Sciences in Human Reproduction “Vida” to be considered for publication as an original article in the Journal of Assisted Reproduction and Genetics.
Footnotes
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data presented are available upon request from the corresponding authors.
The code used in this study is the property of Institute of Sciences in Human Reproduction Vida and cannot be publicly shared due to confidentiality restrictions.