Abstract
Abstract
Objective
To investigate the effects of in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) on the clinical outcomes of non-male factor patients aged >35 with three or fewer oocytes retrieved.
Design
Retrospective cohort study.
Setting
Reproductive centre of a university-affiliated hospital in China.
Participants
547 women with non-male factor infertility who underwent assisted reproductive technology (ART) treatment with three or fewer oocytes retrieved were identified from June 2019 to May 2022. Of these 547 patients, 334 were treated with IVF and 213 with ICSI.
Outcome measures
The primary outcomes were normal fertilisation rate, live birth rate per transfer and cumulative live birth rate per retrieval.
Results
The baseline characteristics were comparable between the two groups, except for the proportion of primary infertility, which was higher in the ICSI group (17.66% vs 32.86%, p=0.000). Compared with the IVF group, the ICSI group showed higher normal fertilisation rate and lower cycle cancellation rate (65.99% vs 76.56%, p=0.002; 33.53% vs 24.41%, p=0.023). However, no significant differences were found in clinical pregnancy rate per transfer (23.86% vs 18.92%, p=0.545), miscarriage rate per fresh embryo transfer (19.05% vs 28.57%, p=0.595), live birth rate per transfer (17.05% vs 13.51%, p=0.623), cumulative clinical pregnancy rate per retrieval (12.87% vs 11.27%, p=0.576) and cumulative live birth rate per retrieval (9.28% vs 6.57%, p=0.261) between the two groups (p>0.05).
Conclusions
In non-male factor ART cycles, ICSI was not associated with improved pregnancy outcomes in older women with a low number of oocytes retrieved. Routine use of ICSI is not recommended in older women who are infertile due to non-male factors.
Keywords: GYNAECOLOGY, Reproductive medicine, Male infertility, Subfertility
Strengths and limitations of this study.
The study includes over 500 participants, and due to the strict inclusion and exclusion criteria the statistical results demonstrate strong reliability and stability.
This study provides reference data for clinicians to make decisions on the fertilisation method for patients suffering from infertility due to non-male factors.
The single-institution setting limits the generalisability of the results.
This is a retrospective study and residual confounding could be a relevant consideration.
Introduction
Intracytoplasmic sperm injection (ICSI) has been used for treatment of couples with severe male factor infertility who want to achieve normal fertilisation and pregnancy. Although ICSI was originally used for moderate or serious male factor infertility,1 its indications have expanded widely over the past decade. The latest data from the European Society of Human Reproduction and Embryology involved more than 500 000 fresh assisted reproductive technology (ART) treatments, showing that ICSI was performed in 71.3% of cycles, including non-male factor infertility cycles, and showed a significant annual increase in fertilisation and pregnancy.2 These data are consistent with recent studies in some countries: ICSI is thought to prevent about 30% of fertilisation failures3 and it is performed even in all in vitro fertilisation (IVF) cycles.4
With the increasing incidence of age-related infertility, ICSI is being recommended over IVF.5 Advanced age in women is often associated with decreased ovarian function, with a general decline in oocytes quality and quantity. Decreased oocyte quality may lead to fertilisation failure or abnormal fertilisation. It is thought that IVF in these patients may lead to lower fertilisation rates, and ICSI can overcome the negative interference in sperm–oocyte interaction and the penetration problems associated with advanced age rather than sperm abnormalities.6 7 Therefore, ICSI is preferred to increase the available embryos and maximise implantation rates, especially in women of advanced age in whom fewer oocytes are retrieved. However, despite its increasing use, the clear advantages of ICSI over IVF have not yet been confirmed. Some studies have even shown a negative impact of ICSI on pregnancy outcomes after ART treatment.8 9 A study involving 745 women over 40 years of age with non-male factor infertility did not demonstrate ICSI being superior to conventional IVF.10 For older women treated with ART, the quality and quantity of oocytes obtained are important indicators of success and are independent predictors of fresh and cumulative live birth rates.11 12
Currently, data on the comparison between ICSI and IVF in those with non-male factors with fewer oocytes retrieved are limited. Whether ICSI could improve the reproductive outcome of couples with non-male factor infertility undergoing ART remains controversial. Additionally, there are few reports on the role of ICSI in women with non-male factor infertility and with no more than three oocytes retrieved. There is an urgent need for an international consensus on the method of fertilisation appropriate for this group. Therefore, this study seeks to assess the role of ICSI and IVF in the reproductive outcomes of older women with fewer oocytes retrieved and with non-male factor infertility.
Methods
Study population
The research was performed at the ART Center of the Affiliated Reproductive and Women-Children Hospital, Chengdu University of Traditional Chinese Medicine. A total of 547 women with non-male factor infertility with three or fewer oocytes retrieved and who underwent ART were identified from June 2019 to May 2022. Of these 547 patients, 334 were treated with IVF and 213 with ICSI. Inclusion criteria included age ≥35 years (the patients belonged to the POSEIDON groups 2 and 4), first ART cycle, presence of bilateral ovaries and male partner with normal semen variables based on the reference values recommended by the 2010 WHO Fifth Edition Guideline. Patients with abnormal uterine cavity that may reduce endometrial receptivity were excluded. In terms of selecting the fertilisation method, embryologists tended to choose more mature oocytes for ICSI. If a couple had a history of primary infertility for more than 5 years, we were more inclined to choose ICSI for older women with less than three oocytes retrieved. When choosing the fertilisation method, patients’ rights to know the efficacy and risk of each of IVF and ICSI must be fully ensured.
Each participant has provided written informed consent.
Controlled ovarian hyperstimulation protocol
All recognised protocols for ovarian stimulation were included. The choice of controlled ovarian hyperstimulation protocol was based on the patient’s condition. Drugs for ovarian stimulation include recombinant follicle-stimulating hormone (rFSH), rFSH plus recombinant luteinising hormone or human menopausal gonadotropin. Body mass index (BMI), age, anti-Mullerian hormone (AMH) and antral follicular count were considered when choosing the dose of gonadotropin.
Human chorionic gonadotropin (hCG) trigger was used for final oocyte maturation when two or more follicles reached 17 mm. Oocytes aspiration by transvaginal B-ultrasound was performed approximately 34–36 hours after the hCG trigger.
Laboratory procedures
The sperm prepared for insemination underwent gradient centrifugation. IVF insemination was performed 40 hours after the hCG trigger, with about 20 000 motile spermatozoa for each oocyte. Cumulus cells were removed 2 hours after retrieval for ICSI. At 16–18 hours after ICSI or IVF insemination, fertilisation was determined when two pronuclei (2PN) were observed. Non-fertilised oocytes, including Metaphase of meiosis II (MII), Metaphase of meiosis I (MI) or Germinal vesicle, were also recorded.
Good-quality cleavage embryos were defined as four cells on day 2 and/or seven to eight cells on day 3, with no apparent morphological abnormalities and with <20% fragmentation. Based on the size of the inner cell mass (ICM) on assessment and trophectoderm development (≥3 BB), blastocysts were graded as basic and high. Blastocysts of good quality should meet the following standards: the blastocele completely filled the embryo, the ICM loosely grouped with several cells and the trophectoderm formed a loose epithelium. All embryos were cryopreserved by vitrification.
Outcome measurement
Laboratory indexes and clinical pregnancy outcomes were analysed, including the number of oocytes retrieved, MII oocytes, rate of normal fertilisation (2PN fertilised eggs/number of mature eggs), total fertilisation failure (all oocytes failed to fertilise with a given fertilisation method), rate of good-quality embryo (good-quality embryos/normal fertilised eggs), cycle cancellation rate, clinical pregnancy rate per transfer, live birth rate per transfer, miscarriage rate, cumulative clinical pregnancy per retrieval and cumulative live birth rate (CLBR) per retrieval. In this study, the cancellation rate referred to cancelled ART cycles because there were no available embryos. Clinical pregnancy was confirmed when cardiac activity was monitored 4 weeks after embryo transfer, whereas abortion was defined as pregnancy loss before 28 weeks of gestation.
The primary endpoint was CLBR, including fresh and frozen embryo transfers. The secondary endpoints were normal fertilisation rate, embryo implantation rate and abortion rate.
Statistical analysis
SPSS V.16.0 was used for data analysis. Kolmogorov-Smirnov (K-S) test and Shapiro-Wilk (S-W) test were conducted to assess the normality of the data. When p>0.05, the data conformed to a normal distribution. When it did not conform to a normal distribution, non-parametric tests were used. Comparison between groups was performed by Student’s t-test, χ2 test, Fisher’s exact test and Mann-Whitney U test. Statistical significance was defined at p<0.05. The confounding factors for clinical pregnancy rate were evaluated using multivariate logistic regression analysis, and the results were additionally adjusted for female age, BMI, years of infertility, type of infertility, basal serum follicle-stimulating hormone (FSH), AMH, E2 on hCG trigger day and endometrial thickness.
Patient and public involvement
None.
Results
Baseline characteristics
During the study period, the ART cycles of 547 women with non-male factors and with three or fewer oocytes retrieved were analysed, including 334 IVF cycles and 213 ICSI cycles. The two groups had comparable baseline characteristics, with no significant differences in female age, BMI, years of infertility, basal serum FSH, AMH, E2 on hCG trigger day and total gonadotropin dose (p>0.05). The ICSI group showed a higher proportion of primary infertility (17.66% vs 32.86%, p=0.000). The baseline characteristics are shown in table 1.
Table 1. Baseline characteristics of included women.
| IVF (n=334) | ICSI (n=213) | P value | |
| Female age (years) | 40.04±3.49 | 40.19±3.64 | 0.708 |
| Ethnicity (Yi nationality/Han nationality) | 45/289 | 29/184 | 0.962 |
| BMI (kg/m2) | 22.60±2.76 | 22.53±2.49 | 0.819 |
| Years of infertility | 4 (2, 8) | 3 (2, 9) | 0.796 |
| Primary infertility, n (%） | 59/334 (17.66) | 70/213 (32.86) | 0.000 |
| Basal serum FSH (IU/L) | 10.12±4.55 | 10.32±4.75 | 0.771 |
| AMH (ng/mL) | 0.77±0.51 | 0.72±0.61 | 0.417 |
| Data missing, n (%) | 2 (0.60) | 1 (0.47) | – |
| E2 on hCG trigger day (pg/mL) | 644.60±328.96 | 665.51±357.58 | 0.597 |
| Data missing, n (%) | 2 (0.60) | 2 (0.60) | – |
| Total gonadotropin dose (IU) | 1906.33±825.35 | 1829.72±820.19 | 0.432 |
| Data missing, n (%) | 2 (0.60) | 2 (0.60) | – |
AMH, anti-Mullerian hormoneBMI, body mass index; FSH, follicle-stimulating hormonehCGhuman chorionic gonadotropinICSIintracytoplasmic sperm injectionIVFin vitro fertilisation
Our data had missing values for AMH, oestrogen levels and total gonadotropin dose, with only one or two missing values for each indicator. Additionally, we applied a missing data method to handle these gaps in our data set. These missing values are described in table 1.
Cycle outcomes of the IVF and ICSI groups
The cycle outcome variables are shown in table 2. There was no significant difference in the number of oocytes retrieved between the two groups (1.68±0.77 vs 1.65±0.74, p=0.672). However, the number of MII oocytes and the cycle cancellation rate in the IVF group were significantly higher than in the ICSI group (1.62±0.83 vs 1.28±0.76, p=0.000; 33.53% vs 24.41%, p=0.023). There were also no significant differences in the rate of good-quality embryo and the number of fresh embryos transferred between the two groups (49.58% vs 51.20%, p=0.711; 1.50±0.55 vs 1.36±0.49, p=0.188). Compared with the IVF group, the normal fertilisation rate was higher in the ICSI group (65.99% vs 76.56%, p=0.002).
Table 2. Cycle outcomes of the IVF and ICSI groups.
| IVF (n=334) | ICSI (n=213) | P value | |
| Number of oocytes retrieved | 1.68±0.77 | 1.65±0.74 | 0.672 |
| Number of MII oocytes | 1.62±0.83 | 1.28±0.76 | 0.000 |
| Normal fertilisation rate, n (%) | 357/541 (65.99） | 209/273 (76.56) | 0.002 |
| Total fertilisation failure, n (%) | 34/334 (10.18) | 8/213 (3.76) | 0.006 |
| Good-quality embryo rate, n (%) | 177/357 (49.58) | 107/209 (51.20) | 0.711 |
| Cycle cancellation rate, n (%) | 112/334 (33.53) | 52/213 (24.41) | 0.023 |
| Number of fresh embryos transferred | 1.50±0.55 | 1.36±0.49 | 0.188 |
| Number of cryopreserved embryos | 1 (1, 1) | 1 (1, 1） | 0.013 |
| Number of transfer cycles for fresh/frozen embryos | 88/71 | 37/59 | – |
| Endometrial thickness of fresh embryo transfer | 8 (7, 9.75) | 9 (8, 10) | 0.217 |
| Endometrial thickness of frozen embryo transfer | 8 (8, 9） | 8 (7, 10) | 0.734 |
ICSIintracytoplasmic sperm injectionIVFin vitro fertilisationMIIMetaphase of meiosis II
Reproductive outcomes
Detailed information on the comparison of clinical outcomes between the two groups is shown in table 3. No significant differences were found in the clinical pregnancy rate per transfer (23.86% vs 18.97%, p=0.545), miscarriage rate (19.05% vs 28.57%, p=0.595) and live birth rate per transfer (17.05% vs 13.51%, p=0.623) between the two groups.
Table 3. Reproductive outcomes.
| IVF (n=334) | ICSI (n=213) | P value | |
| Clinical pregnancy rate per transfer | 21/88 (23.86) | 7/37 (18.92) | 0.545 |
| Miscarriage rate per fresh embryo transfer | 4/21 (19.05) | 2/7 (28.57) | 0.595 |
| Live birth rate per transfer | 15/88 (17.05) | 5/37 (13.51) | 0.632 |
| Cumulative clinical pregnancy per retrieval | 43/334 (12.87) | 24/213 (11.27) | 0.576 |
| Cumulative live birth rate per retrieval | 31/334 (9.28) | 14/213 (6.57) | 0.261 |
Values are presented as numbern (%).
ICSIintracytoplasmic sperm injectionIVFin vitro fertilisation
Adjusting for confounding factors (table 4), such as female age (OR 0.943; 95% CI 0.782, 1.136), BMI (OR 0.998; 95% CI 0.990, 1.007), years of infertility (OR 0.998; 95% CI 0.882, 1.128), type of infertility (OR 2.508; 95% CI 0.603, 10.431), basal serum FSH (OR 1.030; 95% CI 0.926, 1.145), AMH (OR 0.994; 95% CI 0.331, 2.989), E2 on hCG trigger day (OR 1.000; 95% CI 0.998, 1.002) and endometrial thickness (OR 1.137; 95% CI 0.879, 1.471), the cumulative clinical pregnancy per retrieval and CLBR per retrieval were similar in both groups (12.87% vs 11.27%, p=0.576; 9.28% vs 6.57%, p=0.261).
Table 4. Logistic regression analysis of the association between potential influence factors and clinical pregnancy.
| Confounding factors | Adjusted OR (95% CI) | P value |
| Female age | 0.943 (0.782, 1.136) | 0.534 |
| BMI (kg/m2) | 0.998 (0.990, 1.007) | 0.703 |
| Years of infertility | 0.998 (0.882, 1.128) | 0.973 |
| Primary infertility (%） | 2.508 (0.603, 10.431) | 0.206 |
| Basal serum FSH (IU/L) | 1.030 (0.926, 1.145) | 0.590 |
| AMH (ng/mL) | 0.994 (0.331, 2.989) | 0.992 |
| E2 on hCG trigger day (pg/mL) | 1.000 (0.998, 1.002) | 0.825 |
| Endometrial thickness | 1.137 (0.879, 1.471) | 0.327 |
AMH, anti-Mullerian hormoneBMI, body mass index; FSH, follicle-stimulating hormonehCGhuman chorionic gonadotropin
Discussion
Patients of advanced age with diminished ovarian reserve usually have a low number of oocytes. Additionally, oocyte quality decreases significantly with age, making the patient more prone to fertilisation failure or abnormal fertilisation, leading to poor clinical outcomes and even cycle cancellation.10 Therefore, choosing the optimal fertilisation strategy to harvest more fertilised embryos with good quality and quantity is key to improving the outcomes of these patients. Since the application of ICSI technology, its indications have expanded from severe male infertility to low number of oocytes retrieved in the absence of male factor infertility.13 14
The present study demonstrated that ICSI achieved a higher normal fertilisation rate and a lower total fertilisation failure rate compared with IVF in patients of advanced age with non-male factors and whose retrieved oocytes were three or fewer. However, adjusting for potential confounding factors, including female sex, age, BMI, years of infertility, type of infertility, basal serum FSH, AMH, E2 on hCG trigger day and endometrial thickness, did not lead to an increase in clinical pregnancy rate and CLBR. There was also no significant difference in pregnancy outcomes between ICSI and IVF. All patients were treated individually, involving various ovulation stimulating drugs, and there was no difference in drug composition between the two groups.
The molecular mechanism of fertilisation is complex, mainly related to oocyte quality, sperm–oocyte interactions, culture environment and in vitro manipulation techniques. Nearly 41% of couples with infertility with normal semen parameters had problems such as sperm not being able to bind and penetrate the zona pellucida, or the zona pellucida having abnormal function. In such patients, switching to ICSI can significantly improve fertilisation outcomes in the following cycle. Liu et al15 showed that ICSI can overcome fertilisation failure or polyfertilisation caused by age-related abnormal sperm–oocyte binding and the abnormal function of the zona pellucida of oocytes and is therefore effective for such patients. In the present study, the 2PN fertilisation rate of ICSI was higher than that of IVF, which is consistent with Liu et al’s15 study. Additionally, we found that the cycle cancellation rate in the ICSI group was lower than in the IVF group, which may be related to the lower total fertilisation failure rate. However, the present study failed to detect any difference in cumulative clinical pregnancy rate and CLBR between the two groups. Although ICSI may be beneficial in reducing the risk of cycle cancellation among those with non-male factors with a low number of oocytes retrieved, it has not shown benefits in improving embryo rate and CLBR. The possible reasons for this include the following: the puncture of ICSI would cause physical damage to the oocyte membrane, especially oocytes from older women with reduced self-repair function. A negative pressure would be generated after puncture into the cytoplasm, causing part of the cytoplasm to be sucked into the needle tube, which may lead to reduced embryo quality and fertilisation failure. ICSI is an invasive manipulation, and its safety is still not well demonstrated.16 It may damage the cytoskeleton or the spindle during meiosis, resulting in loss of genetic material.17 18 At the same time, the puncturing process has the risk of carrying exogenous genes, such as acrosomal enzymes, into the oocytes. In addition, during the ICSI procedure, sperm selection is based only on microscopic morphological characteristics and motility, and sperm quality cannot be judged from chromosomes and DNA levels. Therefore, it is possible to artificially select sperm with abnormal chromosomes or DNA for insemination. The presence of sperm DNA fragments may also affect the rate of fertilisation; however, there is no reliable method to completely filter out spermatozoa with fragmented DNA manually.19 Live births may occur, however with increased risk of birth defects. ICSI insemination bypasses the natural selection process of the sperm–oocyte combination, whereas IVF is considered a natural selection without too much manual intervention and with an impact that is much smaller than ICSI.
According to the present study, although the ICSI group showed a lower rate of MII oocytes, it had higher 2PN fertilisation rate than the IVF group. Some possible reasons were that oocyte denudation was performed 40 hours after the hCG trigger in the ICSI group, while in the IVF group it was about 56 hours. Longer in vitro culture time in the IVF group increased the chance of immature oocytes developing into mature oocytes. Oocytes selected for ICSI tend to be of higher quality and may have higher cleavage potential. Before the ICSI operation, it is necessary to judge the quality of oocytes. Only if the quality is good, which will be conducive to ovum cleavage, can the ICSI be continued; the quality of the oocyte cannot be assessed in IVF and therefore the cleavage rate may be lower than with ICSI. In ICSI, MII oocytes are generally selected to let sperm pass through the zona pellucida by artificial means, thereby saving some ova and reducing the occurrence of complete fertilisation failure and cycle cancellation rate. Additionally, in patients of advanced age, the ICSI procedure could avoid polyspermic fertilisation due to zona pellucida dysfunction, increasing normal fertilisation rate. However, despite the higher 2PN fertilisation rate, ICSI failed to show its advantage in terms of CLBR, which suggests that ICSI cannot improve age-related decline in embryonic development potential. Advanced age alone or a low number of eggs retrieved should not be an indication for ICSI.
Although in our country insurance does not cover the cost of infertility treatment, this will have a certain financial impact. However, in our study, the main reasons for cancelling transplants included women with only one cleavage stage embryo available for transfer and the progestin-primed ovarian stimulation (PPOS) protocol. The PPOS protocol does not allow transfer of fresh embryos. In older women, the pregnancy rate following transfer of one cleavage embryo is low. With full informed consent, many patients choose to give up fresh single embryo transplantation and demand accumulating embryos. In this situation, patients are more willing to choose the more expensive ICSI fertilisation method in order to achieve a higher likelihood of success.
The choice of the primary endpoint is very important. Many previously published similar trials chose clinical pregnancy rate or live birth rate per transplant cycle as the primary outcome. In this study, we chose CLBR per retrieved cycle as the endpoint. With a longer follow-up period, the CLBR is more convincing than the clinical pregnancy rate. The cumulative live rate can fully reflect the final treatment outcome of a treatment cycle, and therefore it is the most important indicator of a successful ART treatment. It is also a more clinically valuable indicator has been widely recognised internationally in recent years.
The data from this study may provide a useful reference for clinical decision-making. However, this study also has several limitations. First, as a retrospective analysis with limited level of evidence, the study results will be affected by some confounding variables, such as differences in smoking habits, the man’s semen condition, ovum quality and patient ethnicity. Prospective randomised controlled studies are needed. Second, we did not consider sperm morphology as part of the eligibility criteria for study participation. The reason is that, aside from some specific sperm defects associated with genetic diseases, there is little evidence on the prognostic power of sperm morphology in the outcomes after IVF or ICSI. Third, based on semen analysis at the time of the initial assessment, most of the male partners in this study only had asthenospermia. In clinical practice, fluctuations in semen parameters often occur. Therefore, our results should be interpreted with caution in non-study populations.
Conclusions
ICSI was not associated with improvements in pregnancy outcome in couples with a low number of oocytes retrieved. The fertilisation method was selected according to semen variables. ICSI is an invasive procedure and its routine use is not advised for older women with non-male factor infertility.
Footnotes
Funding: This study was supported by the Key Research and Development Projects of Sichuan Science and Technology Program of Sichuan Province, China (grant no: 2022YFS0251).
Prepublication history for this paper is available online. To view these files, please visit the journal online (https://doi.org/10.1136/bmjopen-2023-080688).
Data availability free text: The datasets used in the current study are available from the corresponding author on reasonable request.
Patient consent for publication: Not required.
Ethics approval: This study involves human participants and was approved by the Ethics Review Board of the Reproductive and Women-Children Hospital, Chengdu University of Traditional Chinese Medicine (ethics approval number: SP23-09). It has been undertaken according to the Helsinki Declaration. Participants gave informed consent to participate in the study before taking part.
Provenance and peer review: Not commissioned; externally peer reviewed.
Patient and public involvement: Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Contributor Information
Shaomi Zhu, Email: Zhumi1982@163.com.
Hengli Li, Email: lihengli2014@163.com.
Zili Lv, Email: 758672669@qq.com.
Xin Liang, Email: liangxin@cdutcm.edu.cn.
Liang Dong, Email: dongliang513@sina.com.
Dongmei Tian, Email: caixue1984@163.com.
Data availability statement
Data are available upon reasonable request.
References
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