Impact of having surplus blastocysts cryopreserved on the ongoing pregnancy rate following a fresh transfer

Abstract

Purpose

This study aimed to investigate whether a surplus of vitrified blastocysts correlated with ongoing pregnancy by analyzing the clinical outcomes of fresh transfer cycles with/without a surplus of vitrified blastocysts.

Methods

This was a retrospective analysis carried out in the Reproductive Medicine Center of Guizhou Medical University Affiliated Hospital between January 2020 and December 2021. Overall, 2482 fresh embryo transfer cycles were included in this study, including 1731 cycles with a surplus of vitrified blastocysts (group A) and 751 cycles with no surplus of vitrified blastocysts (group B). The clinical outcomes of fresh embryo transfer cycles were analyzed and compared between the two groups.

Results

In total, the clinical pregnancy rate (CPR) and ongoing pregnancy rate (OPR) after fresh transfer in group A were significantly higher than those in group B (59% vs. 34.1%, p < .001; 51.9% vs. 27.8%, p < .001, respectively). Moreover, the miscarriage rate was significantly lower in group A when compared to that in group B (10.8% vs. 16.8%, p = .008). When grouped by either female age or the number of good-quality embryos transferred, the same trends for CPR and OPR were seen in all subgroups. After adjusting for potential confounding factors in multivariate analysis, a surplus of vitrified blastocysts remained significantly associated with a higher OPR (OR: 1.52; 95% CI:1.21–1.92).

Conclusion

Ongoing pregnancy outcome increases significantly in fresh transfer cycle with a surplus of vitrified blastocysts.

Keywords:

• Surplus vitrified blastocysts
• fresh transfer
• clinical pregnancy
• ongoing pregnancy
• miscarriage

Introduction

Over 6 million babies have been born via artificial reproductive technology (ART) treatment since the first in vitro fertilization (IVF) baby was born in 1978 [1]. However, the success rate of ART is still not satisfactory. Many factors are involved in the success of ART, including female age, the number of aspirated oocytes, embryo quality and endometrial receptivity. Clinics and researchers hope to improve or predict the success rate of IVF/intracytoplasmic sperm injection (ICSI) based on the analysis and manipulation of these factors. Recently, a retrospective study was conducted to predict the likelihood of success in IVF/ICSI cycles based upon female age and the number of retrieved oocytes [2]. Theoretically, a younger female with a suitable number of oocytes would produce more high-quality embryos for transfer. A recent study investigating how oocyte number might affect the outcomes of IVF treatment reported that the live birth rate per fresh transfer increased as the oocyte number increased up to 11–15 oocytes but then plateaued [3]. A review by Christos Venetis also concluded that the retrieval of 12–18 oocytes is the optimal range for maximal live birth rate following fresh transfer [4].

Selecting good embryos for transfer is extremely important for successful IVF/ICSI cycles. In IVF lab, embryo quality is evaluated by considering morphological parameters. Over the past decades, many studies have been conducted to predict clinical outcomes according to embryo morphology. For blastocysts, it has been reported that the morphology of the inner cell mass (ICM) and trophectoderm can be used to predict the live birth rate following single blastocyst transfer [5–7]. Moreover, post-warmed ICM grade is considered to represent a good predictive indicator for selecting the best blastocysts for single blastocyst transfer [8]. The dynamics of embryo culture are also associated with embryo quality and clinical pregnancy rate (CPR) in ART; this led to the development of time-lapse imaging, a new system for embryo culture and evaluation. Several studies have reported that selecting embryos for transfer by time-lapse can improve the rate of implantation [9–11].

Although previous studies have reported the potential factors affecting the clinical outcomes of fresh transfer and strategies for improving implantation rate in the IVF lab, the potential value of a surplus of vitrified blastocysts for predicting the outcomes of fresh transfer has yet to be evaluated. Here, we retrospectively compared the CPR and ongoing pregnancy rate (OPR) of fresh transfer cycles with and without surplus vitrified blastocysts to evaluate the potential value of a surplus of vitrified blastocysts in fresh transfer cycles.

Materials and methods

Study subjects

This single-center, retrospective and cohort analysis included infertile patients who received the transfer of fresh embryos in IVF/ICSI cycles from December 2020 to January 2021 according to our electronic medical database. The exclusion criteria were (i) thawed oocyte cycles and (ii) donor oocyte cycles. In total, 2482 fresh transfer cycles from 2281 patients were selected for analysis. This study was approved by the Ethics Committee of Reproductive Medicine, Affiliated Hospital of Guizhou Medical University.

Methods

Patients underwent controlled ovarian stimulation depending on age, ovarian reserve tests and the number of basal follicles. Embryos were cultured in sequential culture media (ART-1026/1029 medium, Quinn’s, USA) in an incubator (5.5% CO₂, 5% O₂ and 89.5% N2, 37 °C). Good-quality embryos on day 3 were defined as 6- or 10-cell embryos with <5% fragmentation or 7–9 cell embryos with <20% fragmentation.

Usually, day 3 embryos were selected for transfer if this practice was permitted medically. Day 2 embryos were transferred when the number of 2PN zygotes was less than 3 on day 1. Embryos showing the best morphology were prioritized for transfer. The number of embryos used for transfer was based on embryo quality and the patient’s individual situation. All surplus embryos were cultured for an additional two or three days to form blastocyst. Blastocyst quality was evaluated on day 5 or 6 by the degree of blastocoel expansion, ICM and trophectoderm cells [12]. Full blastocysts with loosely grouped cells in the ICM or few trophectoderm cells formed in the loose epithelium were cryopreserved.

Clinical outcome assessment

The primary outcome was ongoing pregnancy rate. Ongoing pregnancy was defined as a live-birth pregnancy or pregnancy with visible fetal cardiac activity by ultrasound from 12 weeks of gestation onward. The secondary outcome was clinical pregnancy; this was defined as the presence of at least one intrauterine gestational sac at 7 weeks of gestation. In addition, we also reported the rate of miscarriage; this was defined as a pregnancy loss between clinical pregnancy and ongoing pregnancy. A serum concentration of maternal β-human chorionic gonadotropin (β-hCG) >25 mIU/mL at ∼14 days after transplantation was considered as a positive hCG result.

Statistical analysis

Statistical analysis was performed using IBM SPSS version 20.0. A multivariate logistic regression model was used to identify potential factors that may be associated with ongoing pregnancy. The confounders were as follows: female age at oocyte retrieval (<30, 30–34, 35–39 and ≥40 years-of-age), the number of aspirated oocytes, surplus vitrified blastocysts (Yes, No), OPU cycles (1, >1) and the number of good quality embryos transferred. We calculated odds ratios (OR) with 95% confidence intervals (95% CI). A p value <.05 was considered statistically significant. The Chi-squared test was used for comparisons and the analysis of categorical data, such as hCG-positive rate and CPR. Differences in binary results are presented as relative risk (RR) with 95% CIs. Continuous variables are reported as mean and standard deviation. Differences between groups were evaluated by one-way analysis of variance (ANOVA). Categorical variables are presented as frequencies with percentages.

Results

In total, we included 2281 infertile women undergoing 2482 IVF/ICSI cycles at the Reproductive Medicine Center of Affiliated Hospital of Guizhou Medical University from January 2020 to December 2021. Of these, 1731 cycles transferred fresh embryos and had a surplus of vitrified blastocysts (group A); 751 cycles did not have a surplus of vitrified blastocysts (group B). The baseline characteristics of the two groups are shown in Table 1. The maternal age was 31.6 ± 4.5 years in group A; this was younger than that in group B (34.7 ± 5.6 years). The mean numbers of oocytes collected and embryos transferred in group A were significantly higher than those in group B. The hCG positive rate in group A was 65.2%; this was significantly higher than that in group B (42.1%). The CPR and OPR in group A were also significantly higher than those in group B (59% vs. 34.1%, p < .001; 51.9% vs. 27.8%, p < .001, respectively). Meanwhile, the miscarriage rate in group A was significantly lower than in group B (10.8% vs. 16.8%, p = .008).

Table 1. Patients’ characteristics grouped according to whether they have surplus vitrified blastocysts in fresh embryo transfer cycle.

	Group A (1,731)	Group B (751)	p value
Female age (years)	31.6 (4.5)	34.7 (5.6)	<.001
BMI (kg/m^2)	23.36 (3.34)	23.31(3.32)	.673
AMH (ng/mL)	3.59 (2.30)	2.31 (1.89)	<.001
Infertility duration (years)	3.65 (3.29)	4.01 (3.89)	.025
OPU cycles, n (%)
1	1,426 (82.4)	460 (61.3)
>1	305 (17.6)	291 (38.7)
Diagnosis
Decreased ovarian reserve	22 (31.4)	48 (68.6)
Tubal factor	668 (74.9)	224 (25.1)
Ovulation disorder	15 (62.5)	9 (37.5)
Endometriosis	10 (45.5)	12 (54.5)
Male factor	435 (74.2)	151 (25.8)
Combined factors	204 (55.3)	165 (44.7)
Unexplained	377 (72.6)	142 (27.4)
Stimulation protocol, n (%)
GnRH ultra-long	1,167 (67.4)	306 (40.7)
GnRH antagonist	380 (22)	327 (43.5)
Others	184 (10.6)	118 (15.8)
Number of aspirated oocytes	11.4 (4.5)	6.1 (3.8)	<.001
Method of fertilization, n (%)
IVF	1,470 (84.9)	597 (79.5)
ICSI	261 (15.1)	154 (20.5)
Endometrial thickness (mm)	11.1 (2.9)	10.6 (2.8)
Number of embryos transferred	1.99 (0.18)	1.90 (0.47)	<.001
Transferred embryo stage
Cleavage stage	1,588 (91.7)	714 (95.1)
Blastula stage	143 (8.3)	37 (4.9)
Positive hCG, n (%)	1 128 (65.2)	308 (42.1)	<.001
Clinical pregnancy, n (%)	1,021 (59.0)	256 (34.1)	<.001
Miscarriage, n (%)	110 (10.8)	43 (16.8)	.008
Ongoing pregnancy, n (%)	899 (51.9)	209 (27.8)	<.001

Group A, fresh transfer cycle with a surplus of vitrified blastocysts; Group B, fresh transfer cycle with no surplus of vitrified blastocysts.

AMH, anti-Mullerian hormone; BMI, body mass index; OPU, ovum pick-up; GnRH-a, gonadotropin-releasing hormone analogue.

Continuous data are shown as the median (standard error) and were analyzed using one-way Anova.

Categorial variables are shown as number (percentage) and were analyzed using the chi-square test.

It is known that female age and the number of oocytes retrieved are extremely important for the success of ART. Younger females may potentially exhibit a reduced ovarian reserve. Therefore, we performed sub-group analysis between the two groups when we retrieved 6–15 oocytes according to female age. Similar to the trend for the whole population, subgroup analysis of female age <35 years showed that the OPR was significantly higher in group A when compared to that in group B (56.1% vs. 43.4%, p = .001; Table 2). However, the miscarriage rate did not differ significantly when compared between the two groups (8.3% vs. 12.5%, p = .156; Table 2). Subgroup analysis of female age ≥35 years showed that the OPR was also significantly higher in group A when compared to that in group B (35.9% vs. 25%, p < .027; Table 2). In addition, the miscarriage rate in group A was higher than that in group B (24% vs. 15%, respectively, p = .223) although this was not statistically significant (Table 3).

Table 2. Comparison of clinical outcomes between group A and B when retrieved 6 ∼ 15 oocytes in subgroups by female age.

	Subgroups of female age < 35 years					Subgroups of female age ≥ 35 years
	Group A (n = 950)	Group B (n = 226)	Relative risk	95% CI	p value	Group A (n = 312)	Group B (n = 128)	Relative risk	95% CI	p value
Positive hCG	652 (68.6)	127 (56.2)	1.22	1.08–1.38	<.001	171 (54.8)	48 (37.5)	1.46	1.14–1.87	.001
Clinical pregnancy	589 (62)	112 (49.6)	1.25	1.09–1.44	.001	150 (48.1)	40 (31.2)	1.54	1.16–2.04	.001
Miscarriage	49 (8.3)	14 (12.5)	0.67	0.38–1.16	.156	36 (24)	6 (15)	1.60	0.73–3.53	.223
Ongoing pregnancy	533 (56.1)	98 (43.4)	1.29	1.10–1.52	.001	112 (35.9)	32 (25)	1.44	1.03–2.01	.027

Group A, fresh transfer cycle with a surplus of vitrified blastocysts; Group B, fresh transfer cycle with no surplus of vitrified blastocysts.

Data presented as n (%).CI, confidence interval.

Table 3. Comparison of clinical outcomes between group A and B after transferring two day 3 embryos.

	Subgroup of two good-quality embryos transferred					Subgroup of two embryos transferred ,one of which is of good quality
	Group A (n = 1,368)	Group B (n = 220)	Relative risk	95%CI	p value	Group A (n = 158)	Group B (n = 132)	Relative risk	95%CI	p value
Positive hCG	899 (65.7)	116 (52.7)	1.25	1.09–1.42	<0.001	94 (59.5)	62 (47)	1.27	1.01–1.58	0.033
Clinical pregnancy	810 (59.2)	99 (45)	1.32	1.13–1.53	<0.001	86 (54.4)	55 (41.7)	1.31	1.02–1.67	0.030
Miscarriage	86 (10.6)	12 (12.1)	0.88	0.50–1.54	0.649	8 (9.3)	8 (14.5)	0.64	0.26–1.6	0.338
Ongoing pregnancy	718 (52.5)	87 (39.5)	1.33	1.12–1.58	<0.001	77 (48.7)	46 (34.8)	1.40	1.05–1.86	0.017

Group A, fresh transfer cycle with a surplus of vitrified blastocysts; Group B, fresh transfer cycle with no surplus of vitrified blastocysts.

Data presented as n (%).

Next, we analyzed clinical outcomes between the two groups after transferring two good-quality day 3 embryos or two day 3 embryos, one of which was a good-quality embryo. Results showed that when transferring either two good-quality day 3 embryos, or two day 3 embryos, one of which was a good-quality embryo, the OPR was significantly higher in group A when compared with the OPR in group B (52.5% vs. 39.5%, p < .001; 48.7% vs. 34.8%, p = .017, respectively; Table 3). Miscarriage rate did not differ significantly between the two groups when analyzed by subgroup analysis (10.6% vs. 12.1%, p = .649; 9.3% vs. 14.5%, p = .338; Table 3).

Finally, multivariable logistic regression analysis was used to assess the influence of potential factors affecting ongoing pregnancy. After controlling for female age, OPU cycles, the number of aspirated oocytes, the number of good-quality embryos transferred, surplus vitrified blastocysts (OR: 1.52; 95% CI: 1.21–1.92) remained significantly associated with ongoing pregnancy (Table 4).

Table 4. Multivariable logistic regression analysis of potential factors affecting ongoing pregnancy.

Variables	OR	95% CI	Adjusted p value
OPU cycles
1	Reference
>1	0.79	0.64 ∼ 0.98	0.034
Female age (years)
<30	Reference
30–34	0.96	0.78–1.17	0.672
35–39	0.63	0.49–0.80	<0.001
39	0.11	0.07–0.19	<0.001
Number of aspirated oocytes	1.02	1.00–1.04	0.03
Surplus vitrified blastocyst
No	Reference
Yes	1.52	1.21–1.92	<0.001
Number of good-quality embryos transferred
0	Reference
1	1.96	1.42–2.71	<0.001
2	2.00	1.50–2.68	<0.001

OR, odds ratio; CI, confidence interval.

Discussion

Surplus embryos were cryopreserved after fresh transfer and blastocyst culture is a process used to screen embryos. Only embryos with developmental potential survive and reach the blastocyst stage after extended periods of culture. Previous studies found that the live birth rate after transferring blastocysts was significantly higher than after transferring cleavage stage embryos [13,14]. Although blastocyst transfer may increase the pregnancy rate when single embryos are transferred to reduce multiple gestations, this practice also has some disadvantages [15]. Theoretically, the in vitro environment is inferior to that of the in vivo environment; consequently, cleavage embryos with implantation potential may fail to form blastocysts when cultured in vitro. Moreover, some studies have reported that blastocyst transfer is associated with an increased rate of transfer cancelation. Therefore, we transferred cleavage embryos and performed extended culture for surplus embryos after fresh transfer; then, we cryopreserved the resultant blastocysts. However, whether a surplus of vitrified blastocysts is associated with the success of fresh transfer has not been reported previously. In this study, we found that a surplus of vitrified blastocysts was positively correlated with the clinical outcome of fresh transfer cycles.

It is believed that female age is the main factor affecting the success of IVF/ICSI cycles. Female age is inherently associated with ovarian function and oocyte quality. Older women (≥35 years) have a higher risk of oocytes and embryos with aneuploidy [16,17], thus leading to a lower CPR. Similarly, the CPR for fresh transfer in females in the <35 years group was higher than that in the ≥35 years group, as derived from multivariable regression and subgroup analysis. Considering that the mean age of females in group A was significantly younger than that in group B (31.6 vs. 34.7, p < .001), we hypothesized whether the younger female age contributed to the higher CPR and OPR in group A. In addition, many patients with a low ovarian reserve even in the younger age group would have low oocyte numbers, thus leading to lower pregnancy rates. Therefore, the patients who had 6–15 oocytes retrieved were sub-grouped by female age. Subgroup analysis showed that higher CPRs and OPRs were independent of female age and the number of oocytes retrieved. Miscarriage is thought to have multiple etiologies, including embryos with aneuploidy, immunological dysregulation and embryo quality [18]. Advanced maternal age has also been reported to be associated with oocyte aneuploidy [19]. As expected, the miscarriage rate in females aged ≥ 35 years was significantly higher than that in females aged <35 years. Interestingly, we noticed that the difference in miscarriage rate between the two groups was not statistically significant when analyzed by subgroups. We propose that the difference of miscarriage rate within the entire population was associated with multiple factors.

Embryo quality is a critical factor affecting the success of implantation in fresh transfer cycles [20–22]. In one study, the transfer of two poor quality cleavage embryos resulted in a lower OPR and a higher miscarriage rate, when compared with the transfer of good quality embryos [23]. Recently, a retrospective cohort study discussed the association between embryo quality, the number of transferred embryos and pregnancy outcomes in vitrified blastocysts per transfer cycle; analysis indicated that the live birth rate was significantly higher after transferring two cleavage embryos (one good quality and one poor quality embryo) compared with the transfer of a single good quality embryo [24]. It is possible that the higher CPR and OPR in group A arose because of the transfer of good quality embryos. To clarify this, we compared pregnancy outcomes between group A and B in subgroups relating to the transfer of two good quality embryos and two embryos (one of which was of good quality). According to subgroup analysis, we hypothesize that the better outcomes in group A were not completely caused by the transfer of more embryos and morphologically good quality embryos.

Clinically, the stimulation protocol is extremely important for IVF success. In our center, an ultra-long GnRH-a strategy is commonly used; this leads to a higher implantation rate when compared with other stimulation protocols. In this study, the proportion of cases involving GnRH-a ultra-long protocols differed significantly between group A and B. Therefore, we compared the clinical outcomes between group A and B when using the GnRH-a ultra-long protocol. Analysis showed that the clinical outcomes in group A were better than those in group B (Supplementary Table 1); this finding was similar to that of the entire study cohort.

Although previous studies reported that the success of IVF/ICSI was not affected in women undergoing recurrent IVF failure [25], the results of multivariable logistic regression demonstrated the importance of OPU cycles in terms of ongoing pregnancy in this study. Consistently, several previous studies showed that the clinical pregnancy rate of women with repeated failure IVF cycles or embryo-transfer cycles decreased [26–28]. Since the proportion of first OPU cycles was higher in group A than in group B (82.4% vs. 61.3%), we considered whether the better clinical outcome in group A was associated with OPU cycles. After comparing clinical outcomes between the two groups, we found that the CPR and OPR in group A were significantly higher than those in group B (Supplementary Table 2), thus suggesting that the higher CPR and OPR in group A did not relate to OPU cycles.

It is important that we consider whether the pregnancy rate increases with the number of available embryos. Two previous studies analyzed the first fresh IVF single-blastocyst transfer cycles and found that clinical pregnancy rate and live birth rate increased significantly with every additional blastocyst up to five blastocysts (or each additional fertilized oocyte up to nine oocytes) but then declined thereafter [29,30]. Here, we analyzed cycles that included fresh transfer (mostly cleavage embryos) but not single-blastocyst transfer; we observed an association between having a surplus of vitrified blastocysts and a higher ongoing pregnancy rate. Because having a surplus of vitrified blastocysts is equal to more blastocysts or fertilized oocytes, at least in part, our conclusion is essentially consistent with previous studies.

The higher the morphological score of the embryo, the better its developmental potential. However, this is not always the case for different females. We believe that this rule is more suitable for embryos from the same woman. This is because embryos from different women often show differential developmental potential; this is associated with age, infertility factors, as well as lifestyles. In addition to the morphological score of cleavage embryos, some other factors were also strictly associated with the potential of embryos to develop to the blastocyst stage and clinical pregnancy, such as the morphokinetics of embryos development and genetic factors. Therefore, when considering embryos from the same woman, if embryos with a relatively low score form a blastocyst after extended culture, embryos with a high morphological score, which are selected for transfer, are more capable of reaching the blastocyst stage.

This study had some limitations that need to be considered. By nature, this retrospective study cannot exclude selection bias. Moreover, 338 cycles were included from the same women in the study; in other words, some women had more than two fresh transfer cycles. Of these cycles, 132 cycles belonged to group A (with a surplus of vitrified blastocysts), thus accounting for 39.05%. However, the independence of these data was not taken into account in our analysis.

In conclusion, our research analyzed the outcomes of fresh transfer cycles with or without a surplus of vitrified blastocysts. We demonstrated that embryos transferred in cycles with a surplus of vitrified blastocysts led to a higher ongoing pregnancy rate. Our study could help to evaluate the developmental potential of embryos and select embryos for transfer in an attempt to maintain the OPR and reduce the multiple pregnancy rate.

Authors contribution

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Rui Wu, Zhuo Chen, Wenfang Xu, Chao Yang, Hua Zhou, Wenjie Xu, and Guanyou Huang. The first draft of the manuscript was written by Rui Wu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Statements and declarations

This retrospective cohort study involving human participants was approved by the Ethics Committee of Reproductive Medicine, Affiliated Hospital of Guizhou Medical University. All methods were performed in accordance with the relevant guidelines and regulations (Declaration of Helsinki). The informed consent was obtained from all subjects.

Acknowledgment

The authors express their gratitude to EditSprings (https://www.editsprings.cn) for the expert linguistic services provided.

Disclosure statement

The authors declare that they have no competing interests.

Additional information

Funding

This work was supported by Science and technology Program of Guiyang city (grant number [2019]-9-1), Doctor Startup Fund of Affiliated Hospital of Guizhou Medical University (grant number gyfybsky-2021-2). The Cultivate project 2021 for National Natural Science Foundation of China, the Affiliated Hospital of Guizhou Medical University (grant number gyfynsfc-2021-38).