Reproductive Outcomes of Artificial Oocyte Activation after ICSI Undergoing Blastocyst Transfer: A 6-year Retrospective Cohort Study

doi:10.21203/rs.3.rs-625260/v1

Research question: Is the pregnancy rate affected by artificial oocyte activation (AOA) with A23187 after intracytoplasmic sperm injection (ICSI) in infertile patients?

Design: Our retrospective study included 308 patients who transferred blastocyst from routine intracytoplasmic sperm injection (ICSI) and 82 patients who transferred blastocyst from routine ICSI combined with AOA (ICSI-AOA) from January 2014 to April 2020. Pregnancy outcomes of couples who received routine ICSI or ICSI-AOA were analyzed after the first blastocyst transfer, which covered frozen-thawed blastocyst transfer and fresh blastocyst transfer. AOA was performed with A237817. We used multivariable logistic regression analysis to determine which variables could be independently associated with the pregnancy rate. Effect sizes were summarized as odds ratios (ORs), with precision evaluated by 95% CIs.

Results: The clinical pregnancy rate was 71.95% in the AOA group and 57.47% in the routine ICSI group. The effect size of the AOA on clinical rate was evaluated in prespecified and exploratory subgroups in each subgroup. And multivariable logistic regression analysis was performed to identify factors associated with the clinical rate. The AOA group had a higher chance of clinical pregnancy in all subgroups: female age at oocyte retrieval, female BMI, protocol in the fresh cycle, female infertility type, MII oocyte numbers, fresh or frozen blastocyst transfer, No. of blastocyst transfer and blastocyst quality. Multivariable analysis showed AOA to be associated with an increased likelihood of clinical pregnancy compared with routine ICSI (p=0.03; adjusted OR 1.89, 95% CI 1.09–3.27).

Conclusions: This study suggested that AOA can increase the rate of clinical pregnancy obviously, which helps clinicians to advise patients on AOA risks.

Obstetrics & Gynecology

artificial oocyte activation (AOA)

ICSI

multivariate logistic regression

clinical pregnancy rate.

Intracytoplasmic sperm injection (ICSI) has become a more and more popular assisted reproductive technique applied for oligozoospermia, asthenozoospermia, and teratoszoopermia[16]. Although average fertilization rates are estimated at ~ 70%, total fertilization failure (TFF) still occurs in 1–5% of all ICSI cycles [9]. The leading cause for TFF after ICSI has been attributed to oocyte activation deficiencies (OADs), which can be related to sperm or oocyte factors. Studies have shown that AOA can be helpful to couples who have clear sperm-related OAD [2, 4, 6].

Artificial oocyte activation is a critical step, which is a prerequisite for fertilization and embryo development [10]. This process inclusive calcium oscillations which increase the intracellular calcium levels in the oocyte, This is essential for a series of nuclear and cytoplasmic changes before the formation of pronucleus. AOA has been used to effectively improve the clinical outcomes of infertile couples with ineffective or low fertilization rates [1, 2, 7, 11, 16, 17], poor embryo development [11] and also can significantly improve the efficiency of IVF with an improved live birth rate per embryo transfer [2].

In the field of assisted reproduction technology, the application of calcium (Ca²⁺)-dependent oocyte activation has been reseached in depth [9]. In the past few decades, AOA induced by several stimuli or combining calcium ionophore treatment after ICSI, which has been successfully used in cases of oglobozoospermia or other severe forms of teratozoospermia. For instance, chemical, electrical, mechanical, or thermal stimuli are commonly used in clinical practice, but chemical stimuli is the primary choice for AOA treatment. A23187 and ionomycin are the leading method of AOA in clinical application [18]. A meta-analysis has shown that AOA can improve the pregnancy rate [14]. However, these studies did not take confounding factors into account. And they transferred the embryo, concluding both cleavage embryo and blastocyst.

Here, the present study aimed to evaluate the effect of AOA on pregnancy outcomes of infertile patients undergoing blastocyst transfer, and pregnancy outcomes were compared between patients with routine intracytoplasmic sperm injection (ICSI) and ICSI-AOA. These results would be helpful for clinicians counseling patients about the risk of artificial oocyte activation.

Study design and patients

This retrospective cohort study included parents who received ICSI or ICSI-AOA treatment from January 2014 to March 2020 in the Center for Assisted Reproductive Technology of Northwest Women’s and Children’s Hospital in the People’s Republic of China. In our center, the couples with male factor infertility, female factor infertility, or both, were offered ICSI–AOA according to the following criteria: (1) patients with severe teratozoospermia, such as acephalic spermatozoa syndrome, globozoospermia or tiny acrosome spermatozoa; (2) patients who had failed fertilization (fertilization rate<20%) after one ICSI procedure when at least five oocytes were injected without oocyte abnormality; (3) patients with embryo developmental problems (total embryo developmental arrest), complete developmental delay (no morula/blastocyst on day 5), reduced blastocyst formation on day 5 (≤15%). The follow-up time was clinical pregnancy, defined as one or more observed gestational sac or definitive clinical signs of pregnancy under ultrasonography at seven weeks of gestation (including clinically documented ectopic pregnancy). After The selection process for ICSI cycles is illustrated in Fig.1, The exclusion criteria were: (1) patients in the first cycle of ICSI; (2) patients who were undergoing other protocol (except antagonist and agonist protocol).

AOA and outcome

Oocyte retrieval, preparation, and insemination were carried out according to previously published guidelines [12]. Thirty minutes after ICSI injection, the surviving oocytes were moved into the AOA dish with A23187 for 15 minutes. Following oocyte activation, embryos were cultured in Sydney IVF Cleavage Medium (vitro-life) until day 3 and in Sydney IVF Blastocyst Medium (vitro-life) from day 3 to day 5.

The outcome terms were defined based on The International Glossary on Infertility and Fertility Care,2017 [19]. Fresh blastocyst transfer on day 5 of embryo culture or frozen-thawed blastocyst transfer (F-TBT) cycles were performed. Pregnancy outcomes after the first fresh or frozen embryo transfer will be used as endpoint for this study.

Blastocyst grading

Blastocysts were divided into three groups based on their morphologic grading assessed: excellent (≥3BB); poor (2BB, 2CC, 3CC, 4CC, 5CC, 6CC, early blastocyst); good:one excellent blastocyst and one poor blastocyst, or 2CB, 3CB, 4CB, 5CB, 6CB, 2BC, 3BC, 4BC, 5BC, 6BC).

Confounding variables

Based on clinical experience and previously published articles[10], We determined the following covariates: female age; No. of embryos transferred; Duration of infertility; MII numbers; female BMI; protocol; embryo quality; fresh or frozen transfer; infertility type.

Ethical approval

The Human Research Ethics Committee of the Northwest Women’s 676 RBMO VOLUME 39 ISSUE 4 2020 and Children’s Hospital approved this study in January 2020 (approval number:2019013).

Statistical analysis

The descriptive data were presented as mean ± standard deviation (SD). Mean values were compared by Student’s t-test and the categorical variables were compared by the Chi-squared test. Use chi-square test (for categorical variables) or analysis of covariance (normally distributed continuous variables) or Kruskal-Wallis test (abnormally distributed continuous variables) to compare groups. In our study, we used logistic regression analysis to assess the association between AOA and clinical pregnancy rate. We calculated the original odds ratio (OR) and adjusted the OR with a 95% confidence interval (CI).

Univariate and multivariable conditional logistic regression was used to analyze the relationship between AOA and clinical pregnancy rate in different models. In the crude model, we did not adjust any covariates, In model I, we only adjusted the female age, the number of embryos transferred, embryo quality, MII numbers, female BMI, protocol, embryo quality, fresh or frozen blastocyst transfer, infertility type. All statistical analyses were performed with SPSS Version 13.0 and the level of significance was set at p< 0.05.

After exclusions, a total of 390 patients undergoing ICSI were analyzed:(1) 308 routine ICSI patients, and (2) 82 ICSI-AOA patients. The demographic characteristics are presented in Table 1. Compared with the routine ICSI group, the ICSI-AOA group women were more with primary infertility type. No significant differences in the female age at oocyte retrieval, body mass index, duration of infertility, collected oocyte numbers, MII oocyte numbers, clinical protocol, fresh or frozen blastocyst transfer, the number of blastocyst transfer and blastocyst quality of transfer.

Table 2 shows the embryological outcome of the two groups. In the ICSI-AOA group, the pregnancy rate was significantly higher than the routine ICSI group (71.95% VS. 57.47%). The fertilization rate (1.09 (0.91–1.23), p = 0.37), the top-quality embryo rate (1.05 (0.87–1.27), p = 0.61), and the available rate (1.07 (0.80–1.46) p = 0.70) on day 3 and the rate of blastocyst formation (1.19 (0.99–1.44), p = 0.06) on day 5 was increase slightly.

The results of the stratification analysis are shown in Table 3, a higher chance of clinical pregnancy rate was observed in the AOA-ICSI group in all subgroups. It shows that our finding was persisted in most subgroups after controlled other confounding except stratified variables. As such, we considered the results in this study reliable and stable.

The relationship between AOA and clinical pregnancy rate in the different models is shown in Table 4. After adjusting for female age, female BMI, protocol, metaphase II (MII) oocyte number, the number of blastocyst transfer, the blastocyst quality, fresh or frozen blastocyst transfer, infertility type. We found that AOA was associated with an increased likelihood of clinical pregnancy compared with the routine ICSI group (OR,1.89 confidence interval, 1.09 to 3.27, p = 0.03).

To the best of our knowledge, this is the first study to describe the pregnancy outcomes after AOA, undergoing blastocyst transfer. Our data demonstrate that the clinical pregnancy rate in the routine ICSI group and the AOA-ICSI group was 71.95% vs. 57.47%. We have considered the potential confounding factors of female age, female BMI, protocol, the metaphase II (MII) oocyte numbers, number of blastocyst transfer, the blastocyst quality, fresh or frozen blastocyst transfer, infertility type. We found that compared with the routine ICSI group, the AOA group has a higher chance of clinical pregnancy (OR,1.89; 95% CI,1.09 to 3.27, p = 0.03).

Several studies have been published on the association between clinical outcome and AOA, but the results have been contradictory. Previous studies with the sample size from 21 to 194, suggested that AOA has no effects on the clinical pregnancy rate [1, 3, 5, 10, 11, 13]. By contrast, some studies [2, 6], with sample sizes ranging from 122 to 343, have indicated that the AOA may be helpful to couples who have clear sperm-related oocyte activation deficiency. At the same time, research found that AOA can significantly improve the clinical pregnancy of certain types of infertility. Our results are consistent with those of Fawzy et al. We transferred a single type of embryo and found that the AOA has a higher chance of clinical pregnancy compared with the routine ICSI group. It enabled us to assess the clinical outcomes independently from the patient’s characteristics, which varied considerably between the two groups. This kind of study is great value to the current three-child policy in the People's Republic of China Opening is being opening up.

We excluded all patients who were the first cycle and only treated patients with multiple cycles as controls. This is because the treatment principle of AOA in clinical practice, patients in the AOA group are all multiple cycles, it was more rigorous to design like this. At the same time, we use the number of the MII oocytes as a covariate rather than the number of collected oocytes, because in the ICSI cycle, the mature oocyte number is the key factor for success of ICSI rather than the number of oocytes obtained.

Our study shows that the fertilization rate, the rate of top-quality embryos and the blastocyst formation rate was increased slightly after using AOA than the routing ICSI group, showing that AOA has no effect on the developmental potential of the embryo from oocyte to blastocyst, similar results were published in [7, 8, 10, 15],but it can increase the probability of every blastocyst becoming pregnant.

There are some strengths of our current work. First, we adjusted more variables to make the results more dependable. Second, appropriate statistical methods and sensitivity analysis can ensure the stability of the research. Third, our research comes from actual clinical data rather than clinical trials, so strict inclusion and exclusion criteria are avoided, which may cause certain limitations on the representativeness and authenticity of randomized controlled trials. Moreover, the nature of retrospective research can prevent observation bias.

The present study also has some limitations. First, because this was a retrospective study from a single center, we cannot investigate other confounding factors, including emotional state, nutritional supplements, and endometrial conditions. Second, as patients or oocytes were not randomized, the patients were divided into groups based on clinical practice, so there may be result in selection bias. Third, the embryos we transferred are all blastocysts. Therefore, the results might not be suitable for people who was transferred cleavage stage embryo. Forth, the patients are all multiple cycles, as such, our conclusion may not be applicable to patients who was the first cycle.

Acknowledgements: The author thanks the staff of Northwest Women and Children's Hospital for their assistance in data collection, and we also thank all participants in this study.

Author contributions

XH Yan: manuscript writing; XT Liu: manuscript editing; WN Chen: data collection; XL Yan: data analysis; YT Zhu: data collection; X Xue: manuscript editing; JZ Shi: protocol development.

Funding

This work was supported General Projects of Social Development in Shaanxi Province (No. 2020SF-305）

Conflict of interest: The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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Table 1

The baseline characteristics of the patients in the two groups.
Patient characteristics	Routine ICSI group	ICSI-AOA group	p-value
Patient characteristics	(n = 308)	(n = 82)
Female age at oocyte retrieval	31.69 ± 4.19	31.17 ± 3.78	0.311
Female BMI	23.00 ± 3.44	22.69 ± 3.32	0.460
Cycles			0.668
2	261 (84.74%)	68 (82.93%)
≥ 3	47 (15.26%)	14 (17.07%)
Duration of infertility	3.80 ± 3.05	3.91 ± 2.36	0.763
MII oocyte numbers	10.24 ± 4.85	9.76 ± 4.13	0.412
Collected oocyte numbers	11.81 ± 5.45	11.55 ± 4.82	0.695
Protocol			0.566
Antagonist	117 (37.99%)	34 (41.46%)
Agonist	191 (62.01%)	48 (58.54%)
Infertility type			0.018
Primary	135 (43.83%)	48 (58.54%)
Secondary	173 (56.17%)	34 (41.46%)
Fresh or frozen blastocyst transferred			0.499
Fresh cycles	198 (64.29%)	56 (68.29%)
Frozen cycles	110 (35.71%)	26 (31.71%)
No. of blastocyst transferred			0.396
1	185 (60.06%)	45 (54.88%)
2	123 (39.94%)	37 (45.12%)
Blastocyst quality of transfer			0.567
Excellent	128 (41.56%)	37 (45.12%)
Good	39 (12.66%)	7 (8.54%)
Poor	141 (45.78%)	38 (46.34%)
MII metaphase II, BMI body mass index, Embryo quality (excellent:3BB and above; poor: 2BB,2CC, 3CC, 4CC, 5CC, 6CC, early blastocyst; good: one excellent blastocyst and one poor blastocyst, or 2CB, 3CB, 4CB, 5CB, 6CB, 2BC, 3BC, 4BC, 5BC, 6BC).

Table 2

**Embryological outcome of the two groups.**
	Routine ICSI		ICSI-AOA	OR	p-value
	(n = 308)		(n = 82)
Fertilization rate(2PN/MII)		2422/3153 (76.82)	627/800 (78.38)	1.09(0.91–1.32)	0.37
Top-quality embryo rate	1575/2422 (65.03)		415/627 (66.19)	1.05(0.87–1.27)	0.61
Available embryo rate	2187/2422 (90.30)		570/627 (90.91)	1.07(0.80–1.46)	0.70
Blastocyst formation rate	1104/2151 (51.32)		308/552 (55.80)	1.19(0.99–1.44)	0.06
Clinical pregnancy rate	177/308 (57.47)		59/82 (71.95)	1.90(1.12–3.23)	0.02
Note: the reference group is routine ICSI group. Fertilization rate = No. of two pronuclei/metaphase II numbers; top-quality embryo rate = No. of top-quality Day 3 embryo /No. of two pronuclei; available embryo rate = No. of available-quality embryo/ No. of two pronuclei; blastocyst formation rate = blastocysts numbers/No. of Day 3 embryo culture.

Table 3 Subgroup analysis of AOA associated with clinical pregnancy

rate according to covariates by logistic regression.

Subgroup	N	OR (95% CI)	p-value
Female age
<30	131	2.35 (0.81, 6.78)	0.12
≥30, <35	164	1.71 (0.66, 4.44)	0.27
≥35	95	1.65 (0.36, 7.56)	0.52
Female BMI
<24	249	1.96 (0.92, 4.14)	0.08
≥24, <28	110	2.37 (0.73, 7.72)	0.15
≥28	30	/
Protocol
Antagonist	151	2.66 (0.94, 7.55)	0.07
Agonist	239	1.77 (0.87, 3.59)	0.12
Infertility type
Primary	183	1.74 (0.79, 3.85)	0.17
Secondary	207	1.91 (0.82, 4.47)	0.13
MII oocyte number
<8	119	1.61 (0.54, 4.80))	0.39
≥8, <15	214	2.68 (1.20, 5.99)	0.02
≥15	57	1.83 (0.24, 14.14)	0.56
Fresh or frozen transfer
Fresh	254	2.08 (1.05, 4.13)	0.04
Frozen	136	11.89 (0.64, 5.63)	0.25
No. of embryos transferred
1	230	1.86 (0.87, 3.99)	0.11
2	160	1.56 (0.60, 4.06)	0.36
Blastocyst quality
Excellent	165	2.44 (0.87, 6.89)	0.09
Good	46	/
Poor	179	1.32 (0.58, 2.99)	0.50
Data are presented as n (%) or mean ±SD, we controlled other confounding except stratified variables. OR odds ratio, CI confidence interval. /, means that the sample sizes of the two groups are too different to be comparable.

Table 4 Relationship between the AOA and the clinical pregnancy rate

in different models.

Exposure	Crude model	p value	Model I	p value	Model II	p-value
	OR (95% CI)	p value	OR (95% CI)	p value	OR (95% CI)	p-value
ICSI	reference		reference		reference
ICSI+AOA	1.90 (1.12, 3.23)	0.02	1.81 (1.06, 3.10)	0.04	1.89 (1.09, 3.27)	0.03
Non-adjusted model adjust for: None; Adjust I model adjust for: female age; No. of embryos transferred; embryo quality. Adjust II model adjust for: female age; No. of embryos transferred; Duration of infertility; MII numbers; female BMI; protocol; embryo quality; fresh or frozen transfer; infertility type.

Reproductive Outcomes of Artificial Oocyte Activation after ICSI Undergoing Blastocyst Transfer: A 6-year Retrospective Cohort Study

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Declarations

References

Tables

Status:

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