Day 3 embryo quality is a predictor for the clinical pregnancy rate and live birth rate in single frozen- thawed poor-quality blastocyst transfer

doi:10.21203/rs.3.rs-4313510/v1

Objective: This study aimed to investigate the utility of day 3 embryo quality in the selection of same-grade blastocysts, particularly focusing on poor-quality blastocysts, to predict clinical pregnancy and live birth rates.

Methods: Seven hundred and thirty-four frozen-thawed single blastocyst transfer cycles carried out between January 2018 to October 2021 were retrospectively analyzed. Cycles were categorized into four groups based on embryo grade: G-G group (good-quality blastocysts derived from day 3 good-quality embryos group, n=194), G-P group (good-quality blastocysts derived from day 3 poor-quality embryos group, n=230), P-Ggroup (poor-quality blastocysts derived from day 3 good-quality embryos group, n=94) and P-P group (poor-quality blastocysts derived from day 3 poor-quality embryos group, n=216). The analysis focused on the role of the day 3 embryo quality in selecting single frozen-thawed good- or poor- quality blastocyst for transfer.

Results: Good-quality blastocysts derived from day 3 poor-quality embryos had comparable pregnancy outcomes to those derived from day 3 good-quality embryos (clinical pregnancy rates: 63.91% vs. 65.98%; live birth rates: 53.04% vs. 55.15%, respectively P≥0.05). However, poor-quality blastocysts derived from day 3 good-quality embryos exhibited significantly higher clinical pregnancy rates (58.51% vs. 37.96%, P＜0.01; OR 2.006,95% CI 1.185-3.395, P＜0.05) and live birth rates (52.13% vs. 28.70%, P＜0.001; OR 2.318, 95% CI 1.355-3.966, P＜0.01) than those derived from day 3 poor-quality embryos.

Conclusion(s): Day 3 embryo quality serves as a predictor for the clinical pregnancy and live birth rate after single frozen-thawed poor-quality blastocyst transfer. When only poor-quality blastocysts are available, selecting those derived from day 3 good-quality embryo for transfer may expedite the process of achieving a live birth.

Single blastocyst transfer

Day 3 embryo quality

Live birth rate

Clinical pregnancy rate

It has been well known that single embryo transfer (SET) is the best method for reducing multiple pregnancy rate, resulting in decreased risks for both maternal and neonatal outcomes in assisted reproductive technology (ART) ^[1-4]. The key of SET is selecting the most viable embryo for transfer to reduce multiple pregnancies while maintaining a high rate of successful pregnancies during in vitro fertilization(IVF) treatment^[5,6]. Now, the most commonly used method for evaluating embryo development is still static morphological evaluation.

There is evidence that single blastocyst transfer has been shown to yield superior pregnancy outcomes compared to single cleavage-stage embryo transfer ^[7,8]. Blastocyst morphological grading is a useful method for blastocyst selection, however, its predictive value for pregnancy outcomes is still limited ^[9-^11]. The study examining chromosomal status of good-quality blastocysts still reports overall aneuploidy rates of 39-57% ^[12]. Moreover, it is difficult that determining which one has better developmental potential when multiple same grade blastocysts are obtained. Therefore, having effective methods for blastocyst selection is crucial.

Day 3 embryo quality is an important evaluation parameter for cleavage stage embryo selection, is it still an important parameter for blastocyst selection? Xia et al ^[13]indicated that day 3 poor-quality embryos may have a negative impact on the pregnancy outcomes, but it is not clear whether these differences still exist when different quality embryo at day 3 are cultured to same morphological grade blastocysts. Several studies showed that there was controversy regarding the effects of day 3 embryo quality on pregnancy outcomes after single good-quality blastocyst transfer^[14-19]. Moreover, as we known, to date, only two studies have analyzed the relationship between day 3 embryo quality and pregnancy outcomes in single poor-quality blastocyst transfer cycles ^[17,18]. And their results showed that there was no relationship between them. Despite these results, it is still unclear whether and how we should focus attention on day 3 embryo quality when transferring single particular grade frozen- warmed blastocysts, especially poor-quality blastocysts .

Therefore, the purpose of our study is to investigate correlation between day 3 embryo quality and the pregnancy outcomes after single good- or poor-quality frozen-thawed blastocyst transfer. It will help us to know the role of day 3 embryo quality in the same grade blastocyst selection.

Patients

A retrospective analysis was performed between January 2018 to October 2021 at the Centre for Reproductive Medicine of Guangzhou Women and Children’s Medical Center. Patients undergoing frozen embryo transfer (FET) cycles with single blastocyst transfer were included. The cycle exclusion criteria are as follows: (1) cycles involving preimplantation genetic testing (PGT); (2) cycles involving female aged over 40 years; (3) cycles involving congenital uterine malformations or endometrium <7mm; (4) cycles involving day 7 blastocyst transfer;(5) cycles involving blastocyst accessed as less than 3BC/3CB.The participant selection process is detailed in Fig. 1. Finally, 734 single frozen-thawed blastocyst transfer cycles were analyzed. These cycles were categorized into four groups according to embryo grade: the good-quality blastocysts derived from day 3 good-quality embryos group (G-G group, n=194), the good-quality blastocysts derived from day 3 poor-quality embryos group (G-P group, n=230), the poor-quality blastocysts derived from day 3 good-quality embryos group (P-G group, n=94) and the poor-quality blastocysts derived from day 3 poor-quality embryos group (P-P group, n=216). The primary outcome of the study was the live birth rate (LBR), while the secondary outcomes included clinical pregnancy rate (CPR), miscarriage rate.

Cleavage stage embryos and blastocyst scoring

Conventional IVF and intracytoplasmic sperm injection (ICSI) procedures were conducted 3-6 hours after oocyte retrieval, followed by a fertilization check at 16-18 hours post-insemination. A zygote with two pronuclei (2PN) and were cultured in G1^TM (Vitrolife, Sweden) under mineral oil. Embryos that underwent extended culture were transferred to G2^TM (Vitrolife, Sweden) on day 3 and cultured until day 5 or 6 under mineral oil. All cultures took place in incubators set at a temperature of 37°C with 6% CO2, 5% O2 and 89% N2. Day 3 embryos were assessed according to the Istanbul consensus ^[20]. Blastocysts were assessed according to Gardner and Schoolcraft's classification. In our laboratory, day 3 good-quality embryos are characterized by having 7-8 cells on day 3, less than 10% fragmentation, symmetrical blastomeres, and an absence of multinucleation. A good-quality blastocyst was defined as blastocysts of grade 3BB or higher.

Blastocyst vitrification and thawing procedures

The criterion for cryopreservation and transfer of blastocysts in our study was a blastocyst grade of at least 3BC/3CB. Vitrification was employed for cryopreservation, and the embryos were thawed using a standardized procedure (Jieying thawing medium, Jieying Laboratory Inc., Canada).

Embryo transfer and pregnancy

Cryopreserved blastocyst transfers were scheduled during either a natural or exogenous steroid replacement cycle, with warmed blastocysts cultured for 2 hours before transfer. Serum HCG levels were assessed 12 days after blastocyst transfer, and clinical pregnancy was defined as the presence of a gestational sac exhibiting fetal heart activity detected via vaginal ultrasound at 7 weeks' gestation.

Statistical analysis

The statistical analysis was conducted using SPSS 22.0 software. For quantitative data with a normal distribution, Student's t-test was applied, while the Mann-Whitney U test was utilized for quantitative data with an abnormal distribution. Qualitative data underwent testing using the chi-squared test or Fisher's exact test when appropriate. Additionally, a multivariate logistic regression analysis was performed to identify confounding factors independently related to the LBR and CPR. The odds ratio (OR) with 95% confidence intervals (CIs) was calculated. A P value < 0.05 was considered statistically significant.

The inclusion process of patients is detailed in Fig. 1. From the initial cohort of 1188 single frozen-thewed blastocyst transfer cycles conducted in our reproductive center during the study period, 454 single frozen-thewed blastocyst transfer cycles were excluded for various reasons: PGT cycles (n=364), female age over 40 years (n=41), and cycles with day 7 blastocysts, uterine malformations, or endometrium less than 7mm (n=49). Consequently, a total of 734 single frozen-thewed blastocyst transfer cycles were included. Within this cohort, patients were divided into four groups: G-G group(n=194), G-P group(n=230), P-G group(n=94) and P-P group (n=216).

Patient and embryo characteristics of frozen-thawed single good- or poor-quality blastocysts with different day 3 embryo quality

As shown in Table 1, no significant differences were observed in female age, female body mass index (BMI), infertility duration, endometrial thickness, infertility types, endometrial preparation, blastocyst frozen day (day 5/6) and degree of expansion (B3/4/5/6) between G-G and G-P groups(P≥0.05). Additional, patient and embryo characteristics were comparable between P-G and P-P groups, except the proportion of vitrified day 5 blastocysts of P-G group was higher than P-P group (74.47% vs. 49.07%, P<0.001).

Pregnancy outcomes of frozen-thawed single good- or poor-quality blastocysts with different day 3 embryo quality

In Table 2, the CPR (65.98% vs. 63.91%, P≥0.05), abortion rate (14.06% vs. 17.01%, P≥0.05) and LBR (55.15% vs. 53.04%, P≥0.05) did no differ significantly between G-G and G-P groups. The abortion rates were similar between P-G and P-P groups (10.91% vs. 20.73%, P≥0.05), however, the CPR (58.51% vs. 37.96%, P<0.01) and LBR (52.13% vs. 28.70%, P<0.001) of the P-G group were significantly higher than those of the P-P group.

The clinical outcomes of the four groups were compared in pairs

In Supplementary Fig1, the CPR and LBR of four groups were compared in pairs. The results revealed that the CPR and LBR of the P-P group were significantly lower than those of G-G, G-P, and P-G groups (P<0.01, respectively). However, there were no significant differences in the CPR and LBR among G-G, G-P and P-G groups (P≥0.05, respectively).

Multivariate logistic regression analysis

Multivariate logistic regression analysis was conducted to examine the variables influencing CPR and LBR of single frozen-thewed poor-quality blastocyst transfer cycles. As presented in Table 3, both day 3 good-quality embryo (OR=2.006 [1.185-3.395], P<0.05) and blastocyst frozen on day 5 (OR=2.128 [1.271-3.562], P<0.01) had a significant positive impact on CPR. Furthermore, age (OR=0.920 [0.855-0.990], P<0.05), endometrial thickness (OR=1.157 [1.005-1.331], P<0.05), day 3 embryo quality (OR=2.318 [1.355-3.966], P<0.01), and blastocyst frozen day (OR=2.472 [1.418-4.307], P<0.01) were found to be significantly associated with LBR. However, the effects of other factors on CPR and LBR did not exhibit significant differences (P≥0.05).

Our finding showed that transferring the single frozen-thewed poor-quality blastocysts derived from day 3 good-quality embryos resulted in higher CPR and LBR compared to those derived from day 3 poor-quality embryos and achieved similar CPR and LBR as the good-quality blastocysts derived from day 3 good- and poor- quality embryos.

Previous studies invested that the development potential of good-quality blastocysts from poor-quality cleavage embryos. Kaartinen et al. ^[21] showed that the CPR of vitrified–warmed good-quality blastocysts derived from poor-quality cleavage embryos was 24.6%, whereas the CPR of vitrified–warmed good-quality cleavage embryos was 29.4%. Sallem et al^[22] reported that CPR was 40.8% after extended culture of embryos with poor developmental potential into good-quality blastocyst. In our study, the transfer of good-quality blastocysts from the extended culture of day 3 poor-quality embryos resulted in CPR of 63.91%. These results indicated that the extended culture of poor-quality cleavage embryos can still develop to the blastocyst stage and result in good pregnancy outcomes. This could be explained by embryo plasticity, the proportion of chromosomally abnormal cells varies during the culture, and the corrupted cells can be eliminated resulting in good-quality blastocysts developing from poor-quality cleavage embryos ^[23]. However, it is not clear whether they have comparable pregnancy outcomes to good-quality cleavage embryos when cultured to the good-quality blastocysts. Therefore, in the present study, we compared the pregnancy outcomes between them. The results showed the CPR, LBR and abortion rate are not influenced by good or poor quality at day 3 when good-quality blastocysts are transferred. The conclusion is in line with previous studies^[14-16]. The study indicated that day 3 embryo quality was not a useful predictor for the pregnancy outcomes in single frozen- thawed good-quality blastocysts.

As we known, to date, only two studies have analyzed the relationship between day 3 embryo quality and pregnancy outcomes in poor-quality blastocyst transfer cycles ^[18,19]. Shen et al ^[18] found that the day 3 embryo grade (I-II, III-IV) did not influence the CPR and LBR in either BC or CB groups after adjusting for confounding factors. Qiu et al^[19]showed that the LBR and miscarriage rate of poor-quality blastocyst from day 3 high-grade embryos were similar with those from day 3 poor-grade embryos(LBR:26.1% VS.28.0%;miscarriage rate:6.6% VS.8.3%).In our study, the CPR and LBR of good-quality blastocysts from day 3 good-quality embryos were significantly higher than those from poor-quality embryos, and achieved similar CPR and LBR as the good-quality blastocysts derived from day 3 good- and poor- quality embryos. These discrepancies between our findings and theirs could be attributed, in part, to a different definition of day 3 good-quality embryos. Moreover, the conventional microscopic assessment of embryo morphology still remains inherently subjective, frequently demonstrating significant variability between different observers and even within the same observer ^[24-26], thus posing a limitation in this study.

Limitations

This study is subject to several limitations. Firstly, its retrospective design introduces the potential for residual confounding, as inherent biases may not have been completely addressed. Further validation through prospective studies is necessary to strengthen the reliability of our findings. Secondly, the subjective nature of morphological evaluation constrains the objectivity of our findings. Therefore, efforts to standardize evaluation protocols or incorporate more objective measures may enhance the robustness of future studies in this area.

In conclusion, our study suggests that day 3 embryo quality is not a useful predictor for the pregnancy outcomes in single frozen-thawed good-quality blastocysts. However, day 3 embryo quality is a strong predictor for the clinical pregnancy rate and live birth rate in single frozen-thawed poor-quality blastocysts. This finding is particularly crucial for patients who only produce poor-quality blastocysts, as it optimizes blastocyst freezing and transfer sequencing, ultimately reducing patients' waiting time to achieve successful live births.

SET: single embryo transfer;

ART: assisted reproductive technology;

IVF: In vitro fertilization;

ICSI: Intracytoplasmic sperm injection;

FET: frozen embryo transfer;

PGT: Preimplantation genetic testing;

CPR: Clinical pregnancy rate;

LBR: Live birth rate;

BMI: body mass index;

2PN: Two pronuclei;

HCG: Human Chorionic Gonadotrophin;

OR: Odds Ratio;

CI: Confidence interval.

Acknowledgements

My colleagues for their invaluable input and for being a great source of support to me during this study.

Author contributions

Shuai Liu, Li Yang contributed to the conception and design of the study. Ling Sun, Minna Yin, Jian Xu and Jun Liu contributed to data interpretation. Wang Hui, Yunhao Liang, Huijiao Wu contributed to data analysis. Yu Jiang, Meiyi Li, and Zhu feng Wu contributed to data acquisition. Li Yang and Minna Yin drafted the first version of the manuscript. Shuai Liu and Zhiheng Chen wrote the final version. The authors read and approved the final manuscript.

Funding

The study was funded by Guangzhou Health and Technology Project (20221A011036).

Data availability

The datasets during the current study are available from the corresponding- g author on reasonable request.

Ethics approval and consent to participate

The study received approval from the Independent Ethics Committee of Guangzhou Women and Children’s Hospital (No.001A01).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Table 1 Patient and embryo characteristics of frozen-thawed single good- or poor-quality blastocysts with different day 3 embryo quality

Blastocyst quality grades	Good-quality blastocyst transfers cycles (n=424)			Poor-quality blastocyst transfers cycles (n=310)
Groups	G-G group (n=194)	G-P group (n=230)	P1	P-G group (n=94)	P-P group (n=216)	P2
Cycles	194	230		94	216
Female Age(year)	31.42±3.88	31.50±3.42	0.826	31.80±3.72	32.11±3.63	0.489
Female BMI(kg/m²)	20.93±3.16	21.46±2.89	0.071	21.24±3.29	20.96±3.23	0.481
Duration of infertility(year)	2.55 (1.00,4.00)	2.00 (1.00,3.00)	0.097	2.00 (1.20,3.25)	2.00 (1.20,3.00)	0.754
Endometrial thickness(mm)	9.73±1.64	9.76±1.81	0.876	9.68±1.72	9.88±1.85	0.377
Infertility type			0.409			0.749
Primary infertility	43.81 (85/194)	47.83 (110/230)		43.62 (41/94)	41.67 (90/216)
Secondary infertility	56.19 (109/194)	52.17 (120/230)		56.38 (53/94)	58.33 (126/216)
Endometrial preparation			0.289			0.165
Natural cycles	10.31 (20/194)	7.39 (17/230)		11.70 (11/94)	6.94 (15/216)
Hormone therapy cycles	89.69 (174/194)	92.61 (213/230)		88.30 (83/94)	93.06 (199/216)
Blastocyst frozen day			0.737			< 0.001
Day5	82.99 (161/194)	81.74 (188/230)		74.47 (70/94)	49.07 (106/216)
Day6	17.01 (33/194)	18.26 (42/230)		25.53 (24/94)	50.93 (110/216)
Expansion degree			0.713		F	0.111
3	15.46 (30/194)	12.61 (29/230)		20.21 (19/94)	21.30 (46/216)
4	51.03 (99/194)	52.61 (121/230)		61.70 (58/94)	50.93 (110/216)
5	32.99 (64/194)	33.48 (77/230)		18.09 (17/94)	24.07 (52/216)
6	0.52 (1/194)	1.30 (3/230)		0.00 (0/94)	3.70 (8/216)

P1:P value between G-G and G-P groups; P2 :P value between P-G and P-P groups

Table 2 Pregnancy outcomes of frozen-thawed single good- or poor-quality blastocysts with different day 3 embryo quality

Blastocyst quality grades	Good-quality blastocyst transfers cycles (n=424)				Poor-quality blastocyst transfers cycles (n=310)
Groups	G-G group (n=194)	G-P group (n=230)		P1	P-Ggroup (n=94)		P-P group (n=216)	P2
Clinical pregnancy rate	65.98 (128/194)	63.91（147/230）	0.657		58.51 (55/94)	37.96 (82/216)			0.001
Abortion rate	14.06 (18/128)	17.01 (25/147)	0.503		10.91 (6/55)	20.73 (17/82)			0.132
Live birth rate	55.15 (107/194)	53.04 (122/230)	0.664		52.13 (49/94)	28.70 (62/216)			< 0.001

P1:P value between G-G and G-P groups; P2 :P value between P-G and P-P groups

Table 3 Multivariate logistic regression analysis of the risk factors affecting the clinical pregnancy rate and live birth rate after frozen-thawed single poor-quality blastocyst transfer

Predictors	Clinical pregnancy rate		Live birth rate
Predictors	OR (95% CI)	P1	OR (95% CI)	P2
Age(year)	0.953 (0.890-1.021)	0.168	0.920 (0.855-0.990)	0.025
BMI(kg/m²)	1.050 (0.974-1.132)	0.207	1.013 (0.939-1.093)	0.736
Duration of infertility(year)	1.049 (0.928-1.186)	0.444	1.093 (0.961-1.242)	0.176
Endometrial thickness(mm)	1.134 (0.991-1.299)	0.068	1.157 (1.005-1.331)	0.042
Infertility type (primary infertility VS. secondary infertility)	0.789 (0.473-1.315)	0.363	0.719 (0.420-1.231)	0.230
Endometrial preparation (natural cycles VS. hormone therapy cycles)	0.781 (0.332-1.834)	0.570	1.013 (0.418-2.458)	0.977
Day 3 embryo quality (good VS. poor)	2.006 (1.185-3.395)	0.010	2.318 (1.355-3.966)	0.002
Blastocyst frozen day (day5 VS. day6)	2.128 (1.271-3.562)	0.004	2.472 (1.418-4.307)	0.001
Expansion degree
4(VS.3)	1.276 (0.681-2.392)	0.446	1.457 (0.756-2.810)	0.261
5(VS.3)	0.952 (0.442-2.050)	0.899	1.125 (0.497-2.547)	0.778
6(VS.3)	2.004 (0.400-10.045)	0.398	2.123 (0.352-12.801)	0.412

P1: P value of the risk factors that affected the clinical pregnancy rate after frozen-thawed single poor-quality blastocyst transfer; P2: P value of the risk factors that affected the live birth rate after frozen-thawed single poor-quality blastocyst transfer

No competing interests reported.

Day 3 embryo quality is a predictor for the clinical pregnancy rate and live birth rate in single frozen- thawed poor-quality blastocyst transfer

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Abstract

Introduction

Materials And Methods

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

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