Introduction

Intracytoplasmic sperm injection (ICSI) is the insemination method that entails the injection of a single spermatozoon into a mature oocyte. Its application has seen a significant surge globally since 1992, establishing it as the most effective solution for fertilization failure resulting from male factor infertility1. The increased utilization of ICSI is primarily attributable to Europe and the USA2,3. However, in China, the application of ICSI remains relatively low, accounting for only 19.2% of all in vitro fertilization (IVF) cycles4. The effectiveness of ICSI in couples with non-male factor infertility, such as unexplained infertility, low oocyte yield, advanced female age, previous fertilization failure, and cryopreserved or in vitro maturation (IVM) oocytes, has been a contentious topic in recent years1. Notably, ICSI is a highly sophisticated and invasive technique that could potentially compromise the integrity of the oocyte membrane and organelles, potentially leading to oocyte degeneration, reduced embryonic development potential, and increased risk of birth defects5.

Frozen-thawed embryo transfer (FET) presents a viable option for those patients considering additional children, those who have experienced failed embryo transfer, and crucially, for those cycles unsuitable for fresh embryo transfer. The advancements in cryopreservation technology, which effectively preserves the developmental potential of the cryopreserved embryo, has led to a significant increase in the number of women undergoing FET treatment6,7. Several factors, including embryo quality, endometrial preparation protocol, the number of embryos transferred, and body mass index (BMI), have been identified as factors independently influencing the live birth rate in women undergoing FET treatment8,9,10,11,12,13,14,15,16,17,18.

Numerous studies have investigated the effectiveness of ICSI in couples with non-male factor infertility undergoing fresh embryo transfer in ovarian stimulation cycles19,20,21,22,23. However, there is a dearth of evidence demonstrating whether ICSI, compared to conventional IVF (cIVF), could influence reproductive outcomes in couples with non-male factor infertility undergoing FET treatment24,25,26,27. To gain a more comprehensive understanding of the impact of ICSI insemination in ovarian stimulation cycles on the reproductive outcomes in subsequent FET cycles, we conducted this retrospective cohort study.

Methods

Study design and populations

This study was a retrospective analysis that included patients who underwent FET treatment at the Third Affiliated Hospital of Zhengzhou University between January 2016 and September 2022. All embryos were derived from oocytes inseminated via either cIVF or ICSI. Initial inclusion criteria encompassed all cycles associated with non-male factor infertility. However, patients were excluded if they met any of the following conditions: (1) Embryos that were biopsied and tested for genetic anomalies; (2) Oocytes that had previously undergone vitrification; (3) Embryos that were derived from donor oocytes, assisted oocyte activation (AOA) treatment, or rescue ICSI; (4) Embryos that were not transferred due to various reasons in the FET cycles; and (5) Women who had previously undergone embryo transfer in fresh treatment cycles. This study was performed in accordance with with the institutional review board on human experimentation and with the Helsinki Declaration of 1964 and its later amendments, and was approved by the the Institutional Review Board of the Third Affiliated Hospital of Zhengzhou University (2024-145-01). Written informed consent were obtained from the patients before embryo transfer.

Following the application of these criteria, a total of 10,143 cycles were included in the analysis. Patients were categorized into two groups based on the insemination methods used for the transferred embryos. A summary of the study population can be found in the flowchart presented in Fig. 1.

Fig. 1
figure 1

The flowchart of the included couples with non-male factor infertility.

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Ovarian stimulation, insemination, and embryo culture

Individualized standard ovarian stimulation protocols were offered to patients, tailored to their ovarian responses. Specifically, patients under the age of 43 years old with a basal follicle-stimulating hormone (FSH) level below 15 IU/L, an antral follicle count (AFC) greater than 5, and anti-Müllerian hormone (AMH) levels above 7.14 pmol/L were offered pituitary down-regulation induced by gonadotropin-releasing hormone agonist (GnRH-a). For patients with low ovarian reserve and those not suitable for GnRH-a protocols, GnRH-antagonist (GnRH-ant) protocols and mild ovarian stimulation protocols were provided. Oocyte maturation in all patients was triggered by either GnRH-a or human chorionic gonadotropin (hCG), or a combination of both.

Oocyte retrieval was performed approximately 36–38 h post-trigger. On the same day, semen samples were collected from the husbands through masturbation. The specific details of insemination methods were described as follows. For cIVF insemination which was performed 38–40 h post-trigger, a total of 40–60 μl sperm which were prepared to the concentration of (8–10)*106/ml were introduced into the central well dish containing no more than 12 female partner's oocytes. Two or more central well dishes were used if more than 12 oocytes were picked up. No more than 6 oocytes were stripped of granulosa cells and removed to the cleavage-stage culture medium 4–6 h later for the observation of second polar body. The left oocytes were kept in the central well dished till 7:00 on the next morning. For ICSI insemination, granulosa cells were enzymatically digested by hyaluronidase for less than 1 min, and then mechanically and softly stripped from the oocytes prior to injection. The residual hyaluronidase and granulosa cells were washed away by repeated inhalation and vomiting in the gametic buffer. The oocytes were then visually accessed for egg maturity. Only mature oocytes were used for sperm injection, which was performed 38–40 h post-trigger by experienced embryologists who were trained with conventional ICSI method.

Embryos were cultured in sequential culture medium within incubators maintained at a temperature of 37 °C and CO2 concentration of 6%. Fertilization was assessed by experienced embryologists on the first day (defined as the day following insemination). Cleavage-stage embryo quality was evaluated on the third day. For the patients who underwent freeze-all strategies, viable cleavage-stage embryos were either cryopreserved during fresh cycles or cultured to the blastocyst stage for subsequent vitrification.

FET protocols

Protocols were tailored based on the patient's menstrual cycle and previous ovulation monitoring. Natural cycles were recommended for women who exhibited regular menstrual cycles and normal follicular development in the past. Hormone replacement therapy cycles were suggested for women with irregular menstruation and ovulation disorders, or for those who were unable to undergo ovulation monitoring due to personal reasons. Stimulated cycles were provided to those with extended menstrual cycles, ovulation disorders, thickened endometrium, and contraindications for estrogen use. The number of transferred embryos were chosen based on the women’s specific conditions. For the young women with first transfer cycle, or with women scarred uterus, single embryos transferred with the best quality was chosen for transfer. For the women with (recurrent) failed FET treatment cycles, no more than two embryos were chosen for transfer. Blasocyst stage embryos were generally chosen for transfer with priority.

Outcome measures

The primary outcome measures included clinical pregnancy rate, miscarriage rate, and live birth rate. Clinical pregnancy was characterized by the presence of a gestational sac as confirmed by ultrasound following a positive β-hCG test. Miscarriage was defined as the spontaneous loss of a clinical pregnancy before 28 weeks of gestation. Live birth was identified as the delivery of any live-born fetus exhibiting cardiac activity at > 24 weeks of gestation. Secondary outcome measures encompassed obstetric outcomes such as gestational age, neonatal length and weight at birth, preterm birth rate, and pregnancy-related complications.

Statistical analysis

Continuous data were presented as mean (standard deviation, SD) or median (interquartile range, IQR), and comparisons between two groups were conducted using the Student's t-test or Mann–Whitney U test. Categorical data were expressed as number (percentage), and comparisons were made using chi-square analysis or Fisher exact test. Given the significant differences in general characteristics between the two groups, propensity score matching (PSM) which could effectively reduce confounding bias and achieve similar effects to randomized controlled studies throughout the study design phase was utilized to ensure comparability of reproductive outcomes at a ratio of 1:1 with the caliper value being 0.2. Additionally, subgroup analyses of primary outcomes were conducted based on factors such as female age, type of infertility, and stage of transferred embryos. A generalized estimating equation (GEE) was employed to explore the impact of insemination methods on clinical outcomes. Variables that showed significant differences between the two groups were employed to adjust the GEE model, otherwise variables such as female age, endometrial thickness, cause and type of infertility, quantity and developmental stage of embryos, and FET protocols which were proved to impact the reproductive outcomes were used as confounding factors. All statistical analyses were conducted using the IBM Statistical Package for the Social Sciences (IBM SPSS v.25; IBM Corporation Inc.). A P-value < 0.05 (two-tailed) was considered statistically significant.

Results

As depicted in Fig. 1 and Table 1, this retrospective study initially incorporated 10,143 cycles from 6206 couples for analysis. The study included 9316 cycles in the cIVF group and 827 in the ICSI group. Upon employing PSM by a ratio of 1:1 with a caliper value of 0.2, each group comprised of 822 cycles (Fig. 1). Prior to PSM, notable disparities were observed in the general characteristics between the cIVF and ICSI groups. The mean age of females in the ICSI group was significantly higher compared to the cIVF group [33.5(5.7) years vs. 35.9(5.8) years, P < 0.001]. Correspondingly, the AFC in the ICSI group was significantly reduced [15.8(9.3) vs. 10.7(7.6), P < 0.001]. Moreover, the ovarian stimulation protocols of the oocyte retrieval cycles, the etiologies of infertility, and the transferred embryos exhibited significant differences (all P < 0.001). Post-PSM, the general characteristics were all comparable across both groups (all P > 0.05) (Table 1).

Table 1 General characteristics of included patients with non-male factor infertility after PSM.
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The clinical pregnancy rates (37.6 vs. 40.0%, P = 0.311), miscarriage rates (21.4 vs. 23.1%, P = 0.597), and live birth rates (28.2 vs. 30.4%, P = 0.329) exhibited no significant disparity post-PSM between the two groups. Detailed comparisons of clinical outcomes, considering variables such as female age, stage of transferred embryos, type of infertility, and number of oocytes retrieved in fresh cycles, were also conducted. Notably, a marginally elevated live birth rate was observed in the ICSI group when blastocysts were transferred (39.8 vs. 48.3%, P = 0.050) (Table 2). Furthermore, ICSI was significantly associated with higher live birth when more than 15 oocytes were retrieved in the fresh cycles (40.8 vs. 53.2%, P = 0.023). However, the GEE models indicated that ICSI did not improve the likelihood of clinical pregnancy [Odds Ratio (OR): 1.11 (0.90–1.36), P = 0.326], miscarriage [OR: 1.11 (0.76–1.61), P = 0.601], or live birth [OR: 1.11 (0.90–1.38), P = 0.339] compared to cIVF. Following adjustments for variables such as female age, BMI, types and causes of infertility, ovarian stimulation protocols of fresh cycles, endometrial preparation regimens, and transferred embryos, the insemination method of ICSI yielded consistent OR values [1.14(0.92–1.42), P = 0.241; 1.08 (0.72–1.61), P = 0.703; and 1.16 (0.92–1.46), P = 0.220, respectively] (Table 3).

Table 2 Clinical outcomes of included patients with non-male factor infertility after PSM.
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Table 3 The impact of ICSI compared to cIVF on the reproductive outcome by GEE.
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Lastly, obstetric outcomes were compared between the two groups. The gestational weeks, proportion of singleton births, and cesarean section rate in the ICSI group were comparable to those in the cIVF group. A significantly higher incidence of preterm birth was observed in the ICSI group (20.0 vs. 12.5%, P = 0.026). However, statistical significance vanished when comparisons in the subgroups based on the number of live births were made (8.5 vs. 14.0%, P = 0.088; 38.7 vs. 55.6%, P = 0.222). While pregnancy complications were comparable between the two groups, neonates born as twins in the ICSI group exhibited significantly reduced live birth-weight [2520.8(572.9)g vs. 2691.9(423.7) g, P = 0.043] and birth length [47.5(2.8) cm vs. 48.5(2.3) cm, P = 0.031]. Correspondingly, the rate of low birthweight was significantly higher in the ICSI group (38.9 vs. 21.0%, P = 0.025) (Table 4).

Table 4 Comparisons of obstetric outcomes between cIVF and ICSI groups in women with non-male factor infertility.
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Discussion

In this retrospective cohort study, we incorporated a sample of infertile women experiencing non-male factor infertility who underwent a freeze-all strategy during ovarian stimulation cycles. The results indicated that ICSI did not enhance the reproductive outcomes. Conversely, ICSI was found to be correlated with an increased risk of low birthweight rate when twins were born.

According to data from the US National Assisted Reproductive Technology Surveillance System spanning from 1996 to 2012, the utilization of ICSI among couples experiencing non-male factor infertility escalated from 15.4% in 1996 to 66.9% in 2012. This surge occurred despite the fact that only 35.8% of these couples were diagnosed with male factor infertility2. More strikingly, data from the Latin American Registry of Assisted Reproductive Technology revealed that for couples not dealing with male factor infertility, ICSI was employed in four out of five cycles20.While both studies exhibited minimal enhancement of reproductive outcomes in the ICSI group as compared to the cIVF group among couples with non-male factor infertility, it had to be noted that there were significant disparities in the general characteristics between the ICSI and cIVF groups. A separate population-based cohort from Australia corroborated these findings, reached the same conclusion after adjusting for the general characteristics28. Correspondingly, in this retrospective analysis, we discerned significant disparities in terms of female age, AFC, etiologies of infertility, and notably, the transferred embryos, which indisputably affect reproductive outcomes. Consequently, the PSM analysis was implemented to render the general characteristics between the two equivalent groups, following which, comparisons of reproductive outcomes were undertaken. However, ICSI did not demonstrate a significant association with the improvement of clinical pregnancy rate, miscarriage rate, or live birth rate.

The application of ICSI for women of advanced age or women with poor ovarian response and non-male factor infertility continues to be a subject of debate. Farhi et al. incorporated 52 eligible patients with advanced age (≥ 35 years old) who underwent their first IVF cycle, from which 50% of the retrieved oocytes were inseminated via cIVF and the remaining 50% through ICSI. Drawing from data of sibling oocytes with comparable quality, they inferred that ICSI was correlated with an elevated fertilization rate and an increased number of top quality embryos. However, the positive correlation were no longer significant in those aged 40–45 years old19. Tannus et al. illustrated comparable fertilization and live birth rates between the cIVF and ICSI groups in a retrospective study which encompassed women aged over 40 years21. In contrast, both McPherson et al. and Butts et al. provided evidence that ICSI was associated with a reduced live birth rate when compared to cIVF16,29. The study conducted by Farhi et al. was well designed to compare the efficacy between cIVF and ICSI. However, it was undermined by a limited sample size and a lack of clinical outcomes. Meanwhile, the other studies, while bolstered by a sufficient sample size, were weakened by the selection bias among the studied groups30. In the current retrospective study, a subgroup analysis based on female age was also conducted. The findings indicated that ICSI was not associated with improved clinical pregnancy rate, miscarriage rate, and live birth rate in both younger and advanced age subgroups. Studies that encompassed women with poor ovarian response and non-male factor infertility demonstrated that ICSI did not surpass cIVF in enhancing reproductive outcomes14,15. Similarly the subgroup analysis from this study revealed comparable clinical pregnancy rate, miscarriage rate, and live birth rate between the cIVF and ICSI groups in women who had fewer than five oocytes. However, it was interesting to find that for those who had more than 15 oocytes in the fresh cycles, significantly higher live birth rate were observed in the ICSI group. More interestingly, although the clinical pregnancy rate and miscarriage rate were similar between the two groups no matter what stage of the transferred embryos were, the live birth rate were slightly higher in the ICSI group with the statistical P value being 0.05 when blastocysts were transferred. The rational behind this result was yet unknown. One explanation of the marginally significance may be that the transfer of blastocyst inferred that the couples were with good ovarian reserve, which was associated with high yield of oocytes, and were offered with changed ovarian stimulation protocols compared to the previous cycles. Anyhow, the results remained to be confirmed by studies with larger sample size.

Supramaniam et al. conducted a comparison of clinical outcomes between the cIVF and ICSI groups based on the developmental stage of the transferred embryos. Their results indicated that the clinical pregnancy rate and live birth rate remained consistent, regardless of the stage of embryo development22. The subgroup analysis from the current study also yielded similar results. Moreover, a study that encompassed couples with unexplained and non-male factor infertility concluded that the embryo development potential and clinical outcomes in the cIVF group did not exceed those in the ICSI group. On the contrary, the clinical outcomes in the cIVF group were markedly improved when the couples presented with secondary infertility. This suggests that the specific type of infertility should be considered when determining the appropriate insemination method for couples with unexplained and non-male factor infertility31. However, the reproductive outcomes appeared to be consistent between the cIVF and ICSI groups, regardless of the type of infertility presented by the couple. Lastly, several studies have explored the role of ICSI in preventing total fertilization failure, a factor not analyzed in the current study. Nevertheless, it appears that ICSI does not perform effectively in preventing total fertilization failure17,32.

The new published multi-centre, open-label, randomised controlled trial conducted by Wang et al. included couple with infertility with non-severe male factor, and found that ICSI neither improve live birth rate, nor impact on the neonatal outcomes compared to cIVF. The well-designed RCT made it possible to include the cycles with both fresh and frozen-thawed embryo transfer. However, the comparisons of cycles with FET cycles in the RCT were lacking33. Both Dang et al. and Bhattacharya et al., through well-designed randomized controlled trials, demonstrated that ICSI did not enhance reproductive outcomes compared to cIVF in women with non-male factor infertility18,23. They also suggested that ICSI should not be routinely employed as the insemination method for couples without male factor infertility. After adjusting for confounding factors, our studies also indicated that ICSI did not offer significant advantages in terms of clinical pregnancy, live birth, and miscarriage rates compared to cIVF. In the RCT conducted by Dang et al. and Wang et al., obstetric and perinatal outcomes between ICSI and cIVF were also compared, which were often overlooked in previous studies that included couples with non-male factor infertility. In the current study, obstetric and perinatal outcomes were comparable between the ICSI and cIVF groups. When single live births were considered, the neonatal outcomes were consistent with the RCT conducted by Dang et al. When twin live births were considered, ICSI was significantly associated with lower birth weight, shorter birth length, and correspondingly a higher low-birth-weight rate. The differences between the studies are difficult to explain. However, we tend to believe that it was the women in our study undergoing FET treatment, instead of fresh embryo transfer, that may have partially contributed to the different results between the studies. On one hand, the transferred embryos in our study were mostly subjected to assisted hatching, which was lacking in the study conducted by Dang et al. On the other hand, it was thought that the endometrium was significantly thinner in the FET cycles compared to the fresh cycles, and endometrium showed better synchronism with the transferred embryos34,35. The study performed by Drakopoulos et al. also investigated the impact of ICSI compared to cIVF on the clinical outcomes in couples with non-male factor infertility across different ovarian response categories36. One strength of their study was the inclusion of FET treatment as well. However, their excessive use of ICSI, which was apparently different from our study and the exclusion of fresh embryo transfer in our study made the comparison between the two studies rather difficult.

This retrospective study has several limitations. Firstly, the retrospective design may lead to selection bias among the included patients. Specifically, in our center in addition to the presence of male factor infertility, we mainly offer ICSI to those with previous IVF failure, which meant that a second or more treatment cycles were subjected to the ICSI group. As shown in Table 1, significant differences in general characteristics were observed between the two groups. However, propensity score matching at a ratio of 1:1 was performed to ensure comparability of general characteristics. Secondly, the use of ICSI in ART has been strictly regulated in China, limiting the sample size and potentially reducing the statistical power. However, the large population base in China may partially remedy the limited sample size. Thirdly, although the cause of infertility was categorized as non-male factor, various infertility causes attributable to the female partner were not specified. However, without restriction of female factor infertility, the results could be applied to a broader population. With an increasing data pool of couples with non-male factor infertility and specific female factor infertility, such as advanced female age, decreased ovarian response, and polycystic ovary syndrome, the impact of ICSI on reproductive and neonatal outcomes could be better understood. Finally, this study only compared the primary outcomes of clinical pregnancy rate, miscarriage rate, and live birth rate. It did not compare early reproductive outcomes such as embryo quality and fertilization rate. There was also no follow-up tracking conducted, making it difficult to observe long-term outcomes such as children's growth and development status.

The strength of this study was the inclusion of patients with non-male factor infertility undergoing FET treatment. To our knowledge, evidence of whether ICSI impacts reproductive outcomes in this study population is scarce. FET treatment offers infertile women who are planning for additional children, who are not suitable for fresh embryo transfer due to high risk of ovarian hyperstimulation syndrome, elevated progesterone levels on the trigger day, or embryo biopsy, a viable option. With these data, we were able to further understand that the value of ICSI should be thoroughly discussed with patients with non-male factor infertility before ovarian stimulation. However, it should be noted that the sample size was still too small to make the comparisons of neonatal outcomes convincing.

Conclusion

Although subgroup analysis showed that ICSI was associated with slightly improved live birth rate for infertile couples with non-male factor infertility compared to cIVF, the logistical regression analysis proved its negative impact on reproductive outcomes. The infertile women with twin pregnancies should be further informed of the lower birthweight and lower birth length when their oocytes were inseminated with ICSI. The findings of this study provide valuable insights for clinicians when discussing the benefits and risks of ICSI with patients with non-male factor infertility.