Abstract
Purpose
This study aims to know what proportion of culture day 5 pre-blastocyst-stage embryos develop into blastocysts by culture day 6 and what patient and cycle characteristics are associated with delayed blastocyst formation.
Methods
A retrospective observational cohort analysis was performed including a total of 9886 embryos from 1008 IVF cycles in 835 patients, who underwent treatment between January 1, 2016, and December 31, 2018. Autologous fresh in vitro fertilization (IVF) cycles at a single academic center were included in the analysis. Embryos were group-cultured using single-step culture media. Blastulation was defined as the presence of a new blastocyst. Usable blastulation was defined as the presence of a new good or excellent quality, expanded, hatching, or hatched blastocysts.
Results
The mean blastulation rate between days 5 and 6 of extended embryo culture was 30.9%. The mean percentage of embryos developing into usable blastocyst-stage embryos was 19.8%. The factors associated with blastulation on day 6 included the total number of embryos and the number of pre-blastocysts on day 5, as well as the use of ICSI. Age, the number of total embryos, those remained in culture and pre-blastocysts, as well as the blastulation rate on day 5 were associated with usable blastulation.
Conclusion
It is important to know the usable blastocyst development rate between culture days 5 and 6 in order to adequately counsel patients debating whether to proceed with fresh ET on day 5 or forego ET with the expectation that embryos will be biopsied for PGT and/or cryopreserved on culture day 6. Our findings provide evidence to help guide patients in this difficult decision.
Keywords: Blastocyst, Blastulation rate, Embryo culture
Introduction
Human blastocysts typically form on day 5 of the culture following fertilization; however, some embryos may take longer to get to this stage of development. It is a common practice to allow embryos one additional day in culture before determining their eligibility for cryopreservation or preimplantation genetic testing (PGT), both of which benefit from using a blastocyst for the best outcome [1, 2]. Embryo development and quality criteria for cryopreservation or biopsy for PGT vary among embryology laboratories. Some centers may have a more permissive policy in embryo biopsy and/or cryopreservation, as well as allowing the extended culture to the seventh day. In our center, day 6 embryos must reach the expanded blastocyst stage and be a good or excellent quality to be eligible for cryopreservation; otherwise they will be discarded [3]. The availability and number of such “usable” blastocysts are difficult to predict, especially in older patients and those with diminished ovarian reserve. This creates a difficult situation for patients who are contemplating PGT for aneuploidy due to advanced maternal age or delaying transfer for other reasons but may prefer undergoing a fresh embryo transfer on culture day 5 to avoid the possibility of not having any usable blastocysts on day 6. We do not extend the embryo culture to the seventh day in our center. There is a paucity of data regarding the blastulation rate between culture days 5 and 6 in group-cultured embryos in a single-step culture medium. Despite the difficulties in designing such a study, information is needed by couples who are faced with difficult decisions as indicated above. The purpose of this study was to determine the blastulation rate of pre-blastocyst-stage embryos specifically from culture days 5 to 6 in group culture in a single-step culture medium and to calculate the proportion of embryos that would be “usable” or eligible for cryopreservation. We also sought to identify which patient and cycle variables were associated with the day 6 blastulation and usable blastulation rates during a fresh IVF cycle.
Materials and methods
Fresh in vitro fertilization (IVF) cycles occurring between January 1, 2016, and December 31, 2018, at a single academic center were retrospectively queried from the database. Patients, who, after embryo transfer (ET) or cryopreservation on culture day 5, had additional embryos cultured to day 6, were included in the study. Similarly, patients, who did not have an ET on day 5 but had viable embryo(s) in culture on day 5, were also included. Cycles involving donor oocytes or embryos were excluded. Any embryo which did not become a blastocyst on day 5 (such as cleavage stage or morula) was defined as a pre-blastocyst-stage embryo. Embryo cohorts were group-cultured; therefore, documenting the development of individual embryos was not possible. To account for this, and for data validation, only the fresh IVF cycles, which had an equal number of embryos reported cultured on day 5 and evaluated on day 6, were included in this study (Fig. 1).
Fig. 1.
Study sample selection process
Controlled ovarian stimulation was performed based on GnRH agonist or antagonist protocols using recombinant follicle-stimulating hormone and human menopausal gonadotropins in the usual fashion. Ovulation was triggered by human chorionic gonadotropin, GnRH agonist, or a combination of the two after 2 or more follicles reached a diameter of ≥ 18 mm, and oocyte retrieval was performed 36 h later under transvaginal ultrasound guidance. Oocyte maturity was assessed at the time of retrieval by quickly spreading the cumulus oocyte complex and observing the oocyte for the presence of a polar body at 320× magnification. Oocytes were inseminated 5 to 6 h post-retrieval by conventional insemination or intracytoplasmic sperm injection (ICSI) with the insemination decision being based on previous semen analyses. Fertilization was confirmed 16–18 h after insemination. Embryos were cultured in groups of 4 in 50 μl drops of Global culture media supplemented with 10% Quinn’s Advantage Serum Protein Substitute (CooperSurgical, Inc., Trumball, CT) under oil in 6% CO2 in air at 37 °C. The single-step culture media was refreshed on culture day 3.
Blastocyst-stage embryo development and grading were performed at 110–114 h post-insemination on culture day 5 and 140 h post-insemination on culture day 6. Embryos were scored according to the criteria described by Gardner and Schoolcraft [3]. In order to control for the variation of embryo grading among embryologists, a quarterly quality control exercise that consisted of 20 embryos being graded simultaneously by all embryologists was conducted (n =5). Corrective action was required if an embryologist incorrectly graded ≥ 10% of the blastocysts as usable/unusable compared to the group consensus. Blastulation was defined on a per cycle basis as the presence of any new blastocyst. Hence, the day 5 blastulation rate was calculated by dividing the number of blasts on day 5 by the number of 2PN embryos, while the day 6 blastulation rate was calculated by subtracting the number of blastocysts cultured on day 5 from the number of blastocysts on day 6 (i.e., new blastocysts) and then dividing by the number of pre-blastocysts cultured between days 5 and 6. A usable blastocyst was defined as a new good or excellent quality expanded, hatching, or hatched blastocyst (expansion stage ≥ 4, BB grade or better). The day 5 usable blastulation rate was calculated by dividing the number of day 5 usable blastocysts by the number of 2PN embryos, and the day 6 usable blastulation rate was calculated by subtracting the number of usable blastocysts cultured on day 5 from the number of usable blastocysts on day 6 (i.e., new usable blastocysts) and then dividing by the number of embryos not classified as usable blasts cultured between day 5 and 6 again on a per cycle basis.
Patient and cycle characteristics were extracted from our institution’s IVF database and were summarized as mean with standard deviation (SD) or frequency with percentage. Data obtained included age; BMI; race; gravidity; parity; antral follicle count (AFC); male factor diagnosis; history of or current smoking status; cycle duration; size and proportion of mature oocyte cohort (number and ratio of metaphase 2 oocytes) at retrieval; intracytoplasmic sperm injection (ICSI) use; the number of embryos of each stage and grade on day 5, cultured from days 5 to 6, and in culture on day 6; and whether a fresh ET occurred on day 5. Statistical analysis was performed with the use of SPSS version 25.0.
Generalized estimating equations were used to control for inclusion of multiple cycles per patient. Variables were selected for model inclusion in accordance with Bursac et al.’s purposeful selection method [4]. Briefly, odds ratios (OR) were calculated along with 95% confidence intervals (CI) for each candidate for model inclusion. Candidates for model inclusion included age, BMI, gravidity, parity, antral follicle count (AFC), male factor diagnosis, size of mature oocyte cohort, ICSI use, number of pre-blastocyst embryos on day 5, number of usable blastocysts on day 5, day 5 blastulation rate, fresh ET on day 5, cycle duration, and number of embryos cultured. Factors which met the prescribed criteria of p < 0.25 in bivariate analysis were entered into the multivariate logistic regression model. A p value cut point of 0.25 was used to limit the risk of failing to identify important candidates for model inclusion. Through an iterative process of variable selection, covariates were removed from the model if they did not meet a significance of 0.1 alpha level or were not found to change any remaining parameter estimate by greater than 15%. After the iterative process of deleting, refitting, and verifying, each variable not selected for inclusion in the original multivariate model was added back one at a time, with any significant at an alpha level of 0.1 retained. Model construction was also performed separately for two subgroups of cycles, cycles without usable blastocysts on day 5, and cycles without blastocysts on day 5. ANOVA with Hochberg post hoc tests was used to compare blastulation rates between age categories.
Results
A total of 1008 cycles from 835 patients were included in the study. The patient and cycle baseline characteristics are summarized in Table 1. Overall, the most common infertility diagnosis was male factor (34%). Approximately 75% (754/1008) of cycles had a fresh embryo transfer performed on day 5, and the mean number of usable blastocysts on day 5 was 1.7 ± 2.2 including those transferred or cryopreserved. The mean blastulation rate between days 5 and 6 per cycle was 30.9% (standard deviation [SD] 31.5%). The mean usable blastulation rate per cycle was 19.8% (SD 15.4%).
Table 1.
Demographic and some laboratory parameters of the study group
| Variable | Mean ± SD or n (%) |
|---|---|
| Total embryo cohort (2PN) | 9.81 ± 6.20 |
| Embryo cohort cultured on day 5 | 7.02 ± 5.15 |
| Age | 33.58 ± 4.60 |
| BMI | 28.03 ± 6.83 |
| White race* | 889 (89) |
| Gravidity | 1.04 ± 1.42 |
| Parity | 0.40 ± 0.68 |
| Antral follicle count | 24.23 ± 12.86 |
| Male factor diagnosis | 343 (34) |
| History of smoking | 175 (17) |
| Current smoker | 27 (3) |
| Total number of metaphase 1 and 2 oocytes | 13.91 ± 8.27 |
| Total oocytes retrieved | 16.64 ± 10.21 |
| Intracytoplasmic sperm injection performed | 580 (58) |
| Preimplantation genetic testing done | 157 (16) |
| Number of pre-blastocyst embryos on day 5 | 4.88 ± 4.06 |
| Blastulation rate on day 5 | 48.9 ± 25.8 |
| Usable blastulation rate on day 5 | 16.5 ± 17.7 |
| Number of usable blastocysts on day 5 | 1.7 ± 2.2 |
| Fresh ET on day 5 | 754 (75) |
*Solely intended to indicate the relative lack of diversity in our patient population
Table 2 illustrates logistic regression models for day 6 blastulation and usable blastulation. The model for blastulation included ICSI (OR, 0.73; 95% confidence intervals [CI], 0.54–0.98), total number of embryos (OR, 1.10; 95% CI, 1.05–1.15), and number of pre-blastocysts on day 5 (OR, 1.16; 95% CI, 1.06–1.26), whereas the model for usable blastulation consisted of age (OR, 0.95; 95% CI, 0.91–0.98), total number of embryos (OR, 0.92; 95% CI, 0.83–1.01), number of embryos in culture on day 5 (OR, 2.07; 95% CI, 1.74–2.46), number of pre-blastocysts on day 5 (OR, 0.61; 95% CI, 0.50–0.76), and blastulation rate on day 5 (OR, 4.06; 95% CI, 1.12–14.76). Both blastulation and usable blastulation rates were then compared between patients who had no usable blastocysts on day 5 and those who had at least one such embryo. As seen in Table 3, the differences were significant, and the usable blastulation rate was almost doubled in patients who had at least one usable blastocyst on day 5 as compared to others. Similarly, patients who had at least one blastocyst had superior blastulation and usable blastulation rates as compared to those without any blastocyst on day 5 (data not shown). Lastly, we observed that usable blastulation decreased significantly in women older than 42 years of age. Figure 2 displays the blastulation and usable blastulation rates per age category. Subgroup analyses by only including cycles that did not have any blastocysts (or usable blastocysts) on day 5 did not contain enough cycles to build reliable models (n = 69).
Table 2.
Patient and cycle parameters associated with blastulation and usable blastulation on day 6
| Blastulation | Adjusted OR (95% CI)* |
|---|---|
| Proportion of mature oocytes at retrieval | 1.86 (0.93–3.73) |
| Number of pre-blastocysts on day 5 | 1.71 (1.52–1.93) |
| Blastulation rate on day 5 | 0.43 (0.16–1.17) |
| Occurrence of fresh ET on day 5 | 1.53 (1.08–2.17) |
| Number of usable blastocysts on day 5 | 1.32 (1.17–1.51) |
| Usable blastulation | |
| Proportion of mature oocytes at retrieval | 1.99 (0.97–4.11) |
| Blastulation rate on day 5 | 17.82 (6.45–49.22) |
| Number of embryos in culture | 1.45 (1.30–1.63) |
| Number of usable blastocysts on day 5 | 0.63 (0.53–0.75) |
| Number of pre-blastocysts on day 5 | 0.84 (0.75–0.95) |
*OR, odds ratio; CI, confidence intervals
Table 3.
Comparison of day 6 blastulation rates between patients with or without usable blastocysts in day 5
| No usable blastocyst on day 5 (%, n = 370) | One or more usable blastocyst on day 5 (%, n = 638) | p | |
|---|---|---|---|
| Blastulation | 61.3 ± 48.8 | 71.1 ± 45.4 | 0.002 |
| Usable blastulation | 13.8 ± 20.6 | 22.6 ± 22.8 | < 0.001 |
*data presented as mean ± standard deviation
Fig. 2.
Blastulation and usable blastulation rate per age category
Discussion
The goal of this study was to identify the blastocyst and usable blastocyst development rate, i.e., blastulation between days 5 and 6 of extended embryo culture, in order to guide patients who are conflicted between waiting to see if they will have embryos which qualify for PGT for aneuploidy testing or embryo banking on day 6 versus undergoing embryo transfer on day 5 with the best available embryos, which may not qualify for cryopreservation or biopsy within the next 24 h. Although there have been several reports on blastulation rate of human embryos under various culture conditions, ours is the only study addressing blastulation between days 5 and 6 of group culture in single-step media to the best of our knowledge. Based on our findings, the chances of having a new blastocyst developing during this time are about 30.9%, and having a new, usable blastocyst is about 19.8%. Oocyte maturity at retrieval, the size, and quality of the existing embryo cohort in the culture had a positive impact on culture day 6 blastulation based on the associated factors identified by logistic regression, whereas day 5 blastulation rate had a negative impact. However, it is more difficult to interpret the factors associated with the development of usable blastocysts. Although the number of embryos remaining in culture was positively associated with usable blastocysts developing on day 6, both the number of existing usable blastocysts and pre-blastocysts on day 5 seem to have a negative impact on the occurrence of new, usable blastocysts on day 6. A possible explanation of this finding may be that a higher number of pre-blastocysts on day 5 may be an indicator of slower embryo development rate, which may be the underlying cause of decreased odds for usable blastulation on day 6. Both oocyte maturity at retrieval and day 5 blastulation rate had a positive impact on the odds of blastulation on day 6. The latter of these factors has an interesting impact; although it decreases the odds of blastulation on day 6, if blastulation occurs, it increases the odds of usable blastulation most robustly as compared to other parameters included in the analysis. Additionally, usable blastulation is significantly reduced in women older than 42 years of age.
Blastulation rate on day 5 shows significant variation between cycles and based on patient age. Laboratory conditions are likely to have an impact on this parameter as well. Therefore, a wide range is reported in the literature from 31 to 55% [5–7], and our day 5 blastulation rate (48.9±25.8) seems comparable with those.
Kort et al. found that about 54–61% of day 5 morulae would progress to the blastocyst stage by day 6 in women 30–35 years old, while blastulation rates for women 36–40 years and 41 and older were 12 and 11%, respectively [8]. The major limitation of their study was the size of their embryo cohort of 296 embryos. Despite having lower blastulation and usable blastulation rates, we believe our numbers may be more realistic based on our cohort size of 9986 embryos. Haas et al. reported 20.4% of morulae progressing to blastocyst on day 6 and 39.2% of cavitating morulae developing to blastocysts on day 6 [9]. They also calculated the blastulation rate of embryos progressing to top-quality embryos, defined as an embryo graded ≥ 3BB on the Gardner grading scale, similar to our definition of usable blastulation. These were 17.7% for morulae and 35.9% for cavitating morulae in their cohort of 10,304 embryos [9]. One advantage they had was being able to separately keep track of morulae and cavitating morulae in order to calculate their respective blastulation rates based on individual embryo culture, an analysis we were unable to perform due to our practice of group culturing embryos. Our blastulation rate of 30.9% and usable blastulation rate of 19.8% are similar to their findings within the constraints of comparing group embryo culture to individual one.
Blastulation has been reported to be negatively impacted by women’s age by some studies [10], whereas others reported no such association [11, 12]. We did find a significantly reduced usable blastulation rate in our patients older than 42 years of age, although the sample size was relatively small in this category (n = 19). In combination with the reduced euploidy rate of blastocysts in this age group, the true impact of reproductive senescence becomes quite evident by this data in our opinion.
The main purpose of our study was to be able to guide a subset of patients, who struggle between two difficult choices on day 5: whether to take the risk of waiting one more day for the development of a new, usable blastocyst for PGT and/or embryo banking purposes or proceeding with a fresh untested ET sometimes with a pre-blastocyst instead. In order to specifically target this group, we performed a subgroup logistic regression analysis by only including cycles that did not develop a usable blastocyst by day 5. The statistical strength of this analysis was inferior due to the smaller sample size as expected, and the predictive variables for usable blastulation that were included in the model were essentially the same ones that were identified in the main analysis.
The strengths of our study include presenting data from an ART lab with strict quality assurance criteria in embryo culture and monitoring as well as our large embryo cohort size. During this study period, our center’s live birth rate ranged from 69.3 to 11.1% based on patient age group with the number of embryos transferred ranging from 1.1 to 1.8 as a reference for the reader (https://www.sartcorsonline.com/rptCSR_PublicMultYear.aspx? ClinicPKID=1931). We believe that based on these strengths, our results should be helpful in guiding patients who are struggling to make a difficult decision especially with a small cohort of cultured embryos. Further studies with larger number of such cycles are needed to confirm our findings.
Our study is a retrospective cohort with the inherent weaknesses of this design, which were minimized by including all cycles that matched our selection criteria during the study period. Our center’s embryo group culture policy, while based on data from quality assurance and control studies that suggest improved embryo outcomes from group vs single embryo culture [13, 14], limited our study to analysis at the embryo cohort level. Individual embryo culture would be necessary to more accurately monitor each embryo and its outcome allowing for analysis of factors at the embryo level that are associated with blastulation. However, we believe that our findings may be helpful for those centers that would prefer to continue providing group embryo culture to their patients.
In conclusion, we found that 30.9% of pre-blastocysts cultured become blastocysts on day 6 and 19.8% of pre-blastocysts and poor quality blastocysts develop into good or excellent, advanced stage, i.e., usable blastocysts that would perform well when biopsied and/or cryopreserved. Usable blastulation rate was significantly lower for patients older than 42 years of age. Patients with a higher proportion of mature oocytes at retrieval and larger embryo cohorts that contained good-quality embryos were more likely to develop new blastocysts on day 6 of the embryo culture. We believe that our findings provide valuable information for counseling patients about their options during this critical time period.
Author contribution
NEB and JEA equivalently contributed to the drafting of the article; KVE contributed to the conception of the study question and provided intellectual input; SAE contributed to data acquisition, drafting, and critical review of the manuscript; SKM performed statistical analyses and contributed to drafting and critical review of the manuscript; VVBJ provided intellectual input and critical review of the manuscript; and DEH participated in the conception of the study question, statistical analysis, interpretation of the results, drafting, and critical review of the manuscript.
Declaration
Conflict of interest
The authors declare no competing interests.
Footnotes
E. B. Nguyen and E. A. Jacobs should be regarded as joint First Authors
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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