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

Xuehua Yan The Assisted Reproduction Center, Northwest women’s and Children’s Hospital Xitong Liu The Assisted Reproduction Center, Northwest women’s and Children’s Hospital Wennan Chen The Assisted Reproduction Center, Northwest women’s and Children’s Hospital Xueli Yan The First Afliated Hospital of Xi'an Jiaotong University Yating Zhu The Assisted Reproduction Center, Northwest women’s and Children’s Hospital Xia Xue The Assisted Reproduction Center, Northwest women’s and Children’s Hospital Juanzi Shi (  [email protected] ) Northwest Women and Children's Hospital https://orcid.org/0000-0002-9040-7829

Research Article

Keywords: artifcial oocyte activation (AOA), ICSI, multivariate logistic regression, clinical pregnancy rate.

Posted Date: July 21st, 2021

DOI: https://doi.org/10.21203/rs.3.rs-625260/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Page 1/13 Abstract

Research question: Is the pregnancy rate affected by artifcial 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 frst 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 prespecifed 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.

Introduction

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 defciencies (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].

Artifcial 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 signifcantly improve the efciency of IVF with an improved live birth rate per embryo transfer [2].

Page 2/13 In the feld 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 artifcial oocyte activation.

Materials And Methods

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 fve 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, defned as one or more observed gestational sac or defnitive 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 frst 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.

Page 3/13 The outcome terms were defned 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 frst 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% confdence 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 signifcance was set at p< 0.05.

Results

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 signifcant

Page 4/13 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 signifcantly 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 stratifcation 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 fnding was persisted in most subgroups after controlled other confounding except stratifed 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 confdence interval, 1.09 to 3.27, p = 0.03).

Discussion

To the best of our knowledge, this is the frst 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 defciency. At the same time, research found that AOA can signifcantly 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. Page 5/13 We excluded all patients who were the frst 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 frst cycle.

Declarations

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）

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

Page 6/13 References

1. Aydinuraz B, Dirican EK, Olgan S, Aksunger O, Erturk OK (2016) Artifcial oocyte activation after intracytoplasmic morphologically selected sperm injection: A prospective randomized sibling oocyte study. Hum Fertil (Camb) 19(4):282-288. 10.1080/14647273.2016.1240374 2. Bonte D, Ferrer-Buitrago M, Dhaenens L, Popovic M, Thys V, De Croo I, De Gheselle S, Steyaert N, Boel A, Vanden MF, De Sutter P, Heindryckx B (2019) Assisted oocyte activation signifcantly increases fertilization and pregnancy outcome in patients with low and total failed fertilization after intracytoplasmic sperm injection: a 17-year retrospective study. Fertil Steril 112(2):266-274. 10.1016/j.fertnstert.2019.04.006 3. Caglar AP, Kilicdag EB, Haydardedeoglu B, Simsek E, Cok T, Parlakgumus HA (2015) Can calcium ionophore "use" in patients with diminished ovarian reserve increase fertilization and pregnancy rates? A randomized, controlled study. Fertil Steril 104(5):1168-1174. 10.1016/j.fertnstert.2015.07.1163 4. Cheung S, Xie P, Parrella A, Keating D, Rosenwaks Z, Palermo GD (2020) Identifcation and treatment of men with phospholipase Cζ–defective spermatozoa. Fertil Steril 114(3):535-544. 10.1016/j.fertnstert.2020.04.044 5. Chi HJ, Koo JJ, Song SJ, Lee JY, Chang SS (2004) Successful fertilization and pregnancy after intracytoplasmic sperm injection and oocyte activation with calcium ionophore in a normozoospermic patient with extremely low fertilization rates in intracytoplasmic sperm injection cycles. Fertil Steril 82(2):475-477. 10.1016/j.fertnstert.2004.01.038 6. Fawzy M, Emad M, Mahran A, Sabry M, Fetih AN, Abdelghafar H, Rasheed S (2018) Artifcial oocyte activation with SrCl2 or calcimycin after ICSI improves clinical and embryological outcomes compared with ICSI alone: results of a randomized clinical trial. Hum Reprod 33(9):1636-1644. 10.1093/humrep/dey258 7. Ferrer-Buitrago M, Dhaenens L, Lu Y, Bonte D, Vanden Meerschaut F, De Sutter P, Leybaert L, Heindryckx B (2018) Human oocyte calcium analysis predicts the response to assisted oocyte activation in patients experiencing fertilization failure after ICSI. Hum Reprod 33(3):416-425. 10.1093/humrep/dex376 8. Karabulut S, Aksunger O, Ata C, Sagiroglu Y, Keskin I (2018) Artifcial oocyte activation with calcium ionophore for frozen sperm cycles. Syst Biol Reprod Med 64(5):381-388. 10.1080/19396368.2018.1452311 9. Kashir J, Heindryckx B, Jones C, De Sutter P, Parrington J, Coward K (2010) Oocyte activation, phospholipase C zeta and human infertility. Hum Reprod Update 16(6):690-703. 10.1093/humupd/dmq018 10. Li B, Zhou Y, Yan Z, Li M, Xue S, Cai R, Fu Y, Hong Q, Long H, Yin M, Du T, Wang Y, Kuang Y, Yan Z, Lyu Q (2019) Pregnancy and neonatal outcomes of artifcial oocyte activation in patients undergoing frozen–thawed embryo transfer: a 6-year population-based retrospective study. Arch Gynecol Obstet 300(4):1083-1092. 10.1007/s00404-019-05298-3

Page 7/13 11. Li J, Zheng X, Lian Y, Li M, Lin S, Zhuang X, Chen L, Liu P, Qiao J (2019) Artifcial oocyte activation improves cycles with prospects of ICSI fertilization failure: a sibling oocyte control study. Reprod Biomed Online 39(2):199-204. 10.1016/j.rbmo.2019.03.216 12. Liu X, Qu P, Bai H, Shi W, Shi J (2019) Endometrial thickness as a predictor of ectopic pregnancy in 1125 in vitro fertilization-embryo transfer cycles: a matched case–control study. Arch Gynecol Obstet 300(6):1797-1803. 10.1007/s00404-019-05353-z 13. Mingrong Lv DZXH (2020) Artifcial oocyte activation to improve reproductive outcomes in couples with various reproductive outcomes in couples with various causes of infertility: a retrospective cohort study. RBMO 14. Murugesu S, Saso S, Jones BP, Bracewell-Milnes T, Athanasiou T, Mania A, Serhal P, Ben-Nagi J (2017) Does the use of calcium ionophore during artifcial oocyte activation demonstrate an effect on pregnancy rate? A meta-analysis. Fertil Steril 108(3):468-482. 10.1016/j.fertnstert.2017.06.029 15. Nazarian H, Azad N, Nazari L, Piryaei A, Heidari MH, Masteri-Farahani R, Karimi M, Ghaffari-Novin M (2019) Effect of Artifcial Oocyte Activation on Intra-Cytoplasmic Sperm Injection Outcomes in Patients with Lower Percentage of Sperm Containing Phospholipase Czeta: A Randomized Clinical Trial. J Reprod Infertil 20(1):3-9 16. O'Neill CL, Chow S, Rosenwaks Z, Palermo GD (2018) Development of ICSI. Reproduction 156(1):F51- F58. 10.1530/REP-18-0011 17. Sfontouris IA, Nastri CO, Lima MLS, Tahmasbpourmarzouni E, Raine-Fenning N, Martins WP (2015) Artifcial oocyte activation to improve reproductive outcomes in women with previous fertilization failure: a systematic review and meta-analysis of RCTs. Hum Reprod 30(8):1831-1841. 10.1093/humrep/dev136 18. Vanden Meerschaut F, Nikiforaki D, Heindryckx B, De Sutter P (2014) Assisted oocyte activation following ICSI fertilization failure. Reprod Biomed Online 28(5):560-571. 10.1016/j.rbmo.2014.01.008 19. Zegers-Hochschild F, Adamson GD, Dyer S, Racowsky C, de Mouzon J, Sokol R, Rienzi L, Sunde A, Schmidt L, Cooke ID, Simpson JL, van der Poel S (2017) The International Glossary on Infertility and Fertility Care, 2017. Fertil Steril 108(3):393-406. 10.1016/j.fertnstert.2017.06.005

Tables

Page 8/13 Table 1 The baseline characteristics of the patients in the two groups. Patient characteristics Routine ICSI group ICSI-AOA group p-value

(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%)

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).

Page 9/13 Patient characteristics Routine ICSI group ICSI-AOA group p-value

(n = 308) (n = 82)

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 627/800 1.09(0.91–1.32) 0.37

(76.82) (78.38)

Top-quality embryo rate 1575/2422 415/627 1.05(0.87–1.27) 0.61

(65.03) (66.19)

Available embryo rate 2187/2422 570/627 1.07(0.80–1.46) 0.70

(90.30) (90.91)

Blastocyst formation rate 1104/2151 308/552 1.19(0.99–1.44) 0.06

(51.32) (55.80)

Clinical pregnancy rate 177/308 59/82 1.90(1.12–3.23) 0.02 (57.47) (71.95)

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.

Page 10/13 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

Page 11/13 Data are presented as n (%) or mean ±SD, we controlled other confounding except stratifed variables. OR odds ratio, CI confdence 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) OR (95% CI) OR (95% CI)

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.

Figures

Page 12/13 Figure 1

Flow chart of present study. ICSI intracytoplasmic sperm injection, AOA artifcial oocyte activation, other protocol patients in other protocol were people who undergoing protocol except antagonist and agonist protocol.

Page 13/13