https://orcid.org/0000-0001-5518-2209
ABSTRACT
The ICSI procedure was invented to treat severe male infertility but is often used even if the sperm quality parameters are normal. This practice has recently been called into question, but there is still no clear answer especially in terms of sperm morphology, regarding when it is necessary to perform ICSI and when conventional IVF is indeed more beneficial. In borderline cases it seems logical to fertilize oocytes using ICSI and conventional IVF at the same time. Since we also use this approach we performed a retrospective analysis of such cycles to elucidate, which procedure results in a better clinical outcome in terms of fertilization rate, the quality of day 3 and day 5 embryos, and the pregnancy rate. The data from fifty-one couples who were treated with ART and whose male factor of infertility was defined as teratozoospermia were included. The fertilization rates were similar between ICSI and conventional IVF groups (per COCs: 54.5% vs. 58.2%, P = 0.322; per MII oocytes: 63.9% vs. 67.2%; P = 0.399), but more oocytes degenerated after ICSI (11.7% vs. 4.3%; P = 0.0003). The quality of cleaved embryos was similar between the groups, but more embryos reached the blastocyst stage after conventional IVF (43.7% vs. 55.0%; P = 0.032) and furthermore, more of them were of good quality (19.8% vs. 29.2%; P = 0.037). The pregnancy rate did not significantly differ between the groups (21.4% vs. 45.5%; P = 0.175), although there was a trend in favor of conventional IVF. This retrospective analysis suggests that when sperm morphology is not severely impaired and sperm concentration and motility are normal, it is better to use conventional IVF to fertilize oocytes and not ICSI. The main advantage of conventional IVF is reflected in improved blastocyst rate and quality.
Abbreviations: ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; COC: cumulus-oocyte complex; COH: controlled ovarian hyperstimulation
Introduction
The ICSI procedure was originally developed to treat severe male infertility, although it was later frequently used in the treatment of a subfertile population of patients and even in cases where there is no diagnosis requiring this invasive procedure. The latest report from The European IVF-monitoring Consortium showed that in the year 2014, ICSI was used in 71.3% of fresh treatments, which is slightly more than the year before (De Geyter et al. Citation2018). The reason for this overuse is due to the fact that ICSI has become an acceptable method for treating female infertility in cases where oocytes are impaired in either quality or number as well as in cases of polyspermy or low fertilization (Practice Committee of American Society for Reproductive Medicine Citation2008). ICSI is also recommended in cases when PGD is planned (Practice Committee of American Society for Reproductive Medicine Citation2008), although this has recently been challenged and it is generally recommended that in PGD cycles, ICSI should only be used in cases of male factor infertility (Feldman et al. Citation2017). In clinical practice, the decision of when to use ICSI or conventional IVF should depend mostly on semen quality, which means that ICSI should be used only when the quality parameters of semen are impaired. The issue is that interpretations of these parameters can vary and there can be different understandings of which threshold should be considered to be normal, especially in terms of sperm morphology. Fundamental studies in the eighties at the Tygerberg Hospital determined that there is a threshold among normal sperm morphologic features at 14% and that this threshold was strongly correlated with the fertilization of oocytes (Kruger et al. Citation1986, Citation1987). Furthermore, by proposing strict criteria it was shown that only patients with less than 4% of normal forms had significantly lower fertilization rate of oocytes after conventional IVF compared to a normal morphology between 4% and 14% and only in cases when more than 14% of normal forms were observed were oocytes fertilized within the normal laboratory rate (Kruger et al. Citation1988). Kruger’s morphology assessment was later implemented in the World Health Organization manual for the examination and processing of human semen (WHO Citation1999, Citation2010). Although the same approach of assessing sperm morphology was described in both manuals, the reference values for normal morphology has changed from 15% (WHO Citation1999) to 4% (WHO Citation2010). The reason for this is that the fourth edition’s (WHO Citation1999) interpretation relied on data from assisted reproductive technology showing a decreased fertilization rate when sperm morphology falls below 15%, and the fifth edition’s (WHO Citation2010) interpretation implemented data obtained by Cooper et al. (Citation2010), who studied semen samples from a large number of various populations of men. In this study, the reference distributions for semen parameters were calculated from men whose partners achieved pregnancy within a year or earlier. With regard to morphology, the reference value for morphologically normal forms was determined to be at 4%. The reference values for normal sperm morphology from both the latest WHO manuals are frequently used as a criterion when deciding to use ICSI or conventional IVF for oocyte fertilization. The issue is that the percentage of normal sperm morphology has to be taken with caution, because the number of assessed spermatozoa in diagnostics is usually around 200 and this does not ensure a reliable result due to the possibility of high standard deviations in assessment (WHO Citation2010). Therefore, it is not always clear which sperm morphology threshold should be used to decide for ICSI in patients whose sperm concentration and motility are normal. In such borderline cases it seems reasonable, at least in the first cycle, to fertilize a proportion of the obtained oocytes using ICSI and the rest with conventional IVF. Since our IVF center also uses this approach, we decided to retrospectively analyze such cycles and to elucidate, which procedure results in a better clinical outcome in terms of fertilization rate, quality of day 3 and day 5 embryos, and the pregnancy rate.
Results
Fifty-one couples were included in this retrospective study, all of whom were treated at our center with ICSI and with conventional IVF at the same time (sibling oocyte), whereas the male partner had a diagnosis of slight to moderate teratozoospermia. The baseline sperm characteristics of patients are presented in . Other results are presented in and . Briefly, the number of divided COCs (oocytes) were similar between the groups (i.e., the ICSI vs. conventional IVF groups; 6.4 ± 3.1 vs. 7.3 ± 5.0, P = 0.088) as was the fertilization rate (ICSI vs. IVF, 54.5% vs. 58.2%; P = 0.322 (if obtained COCs were used for calculation), and 63.9% vs. 67.2%; P = 0.399 (if MII oocytes were used for calculation)). We observed a significantly higher proportion of degenerated oocytes after ICSI (11.7% vs. 4.3%; P = 0.0003), but this is not surprising due to the nature of the procedure used to fertiliz oocytes. The number of cleaved embryos obtained and their quality was similar among groups, but in the case of blastocysts it was different. We observed that the proportion of blastocysts with regard to embryos that were cultured until day 5/6 was significantly higher in a conventional IVF group (43.7% vs. 55.0%; P = 0.032) and additionally, even the proportion of good quality blastocysts was also higher (19.8% vs. 29.2%; P = 0.037). Interestingly, if the quality of the blastocyst was compared with regard to the number of embryos that reached the blastocyst stage, then no difference was observed.
Table 1. The baseline sperm characteristics of patients included in this retrospective study.
Table 2. The outcome of the ICSI/IVF cycles in terms of oocytes and embryos according to the procedure used to fertilize oocytes.
Table 3. The outcome of the ICSI/IVF cycles in terms of pregnancies according to the procedure used to fertilize oocytes (ICSI vs. conventional IVF).
The outcome of ETs in terms of pregnancies (for day 3 and day 5 ETs together) did not differ significantly between the treatments (21.4% vs. 45.5%; P = 0.175), although there was a trend in favor of the conventional IVF, but the number of all the included retrospectively analyzed cycles was probably too low to enable one to observe statistically significant differences from this aspect (statistical power of this analysis was 0.298). Since the number of cycles, ETs and statistical power were low, we calculated the number of ETs in each treatment group that would need to be performed to achieve statistically significant difference in pregnancy rate. The analysis showed (with power 0.8) that altogether at least 126 ETs would need to be performed, 49 ETs in ICSI group and 77 in conventional IVF group, if we assume that the same proportion of patients would get pregnant in each group as in this retrospective analysis.
Discussion
Deciding how to fertilize oocytes should depend on the quality of the semen, but it is still not completely clear at what level of quality one should draw the line to determine whether to use ICSI or conventional IVF. When the number of spermatozoa is low, or their motility is impaired, the use of ICSI seems to be justified. However, in terms of sperm morphology it is more complicated to pin down, especially when concentration and motility are normal. The problem is which criteria to use to determine a threshold for normal morphology. Should it be taken into account that 15% means normal morphology or 4% (when Kruger strict criteria are used to evaluate sperm morphology)? The reference values set at 4% were generated in 2010 by Cooper et al. who studied semen samples from a large group of men from 14 countries. The reference values were determined by analyzing men whose partners got pregnant within one year, but these values have since been debated since they can cause some confusion (Niederberger Citation2010). It was suggested that in ART it would be more appropriate to set reference values generated from an actual population treated with ART instead of with patients with proven fertility (Tocci and Lucchini Citation2010). Similarly, as Cooper et al. (Citation2010) reference values Kruger’s strict criteria, and their prognostic value in the ICSI cycles have already been questioned (French et al. Citation2010). Therefore, in such borderline cases (morphology from 4% to 14%) it is the most practical and safe for patients, at least in their first cycle, to perform ICSI and conventional IVF at the same time, meaning that one part of the oocytes is fertilized using ICSI and the rest using conventional IVF. We followed this practice in our clinic and this retrospective analysis suggests that when sperm morphology is not severely impaired, it is better to use conventional IVF to fertilize oocytes and not ICSI. The benefit of conventional IVF was revealed in the better development of embryos up to the blastocyst stage in terms of both quantity and quality. The difference in the pregnancy rate between procedures was not significant, although the data suggest a trend towards better results after conventional IVF. However, the number of cycles included in this study was probably too low to achieve statistical significance and consequently the statistical power of this analysis is not sufficient. To show with higher statistical power (0.8) that the difference between the treatments is significant, more than 3-times more of ETs should be performed in each group if we would consider the same approach for fertilization of oocytes (half with ICSI and half with conventional IVF).
Similar results for the usual IVF/ICSI cycles were also reported several times in recent years. Check et al. (Citation2010a, Citation2010b) compared cycles where sperm morphology was subnormal (2-5%) and ICSI or conventional IVF was used for fertilization. They observed that there was a difference in the pregnancy rate after a fresh embryo transfer and that the live birth rate approached statistical significance in favor of conventional IVF; this difference was also found after a frozen embryo transfer, but the beneficial effect was probably not due to the formation of embryos with more rapid cleavage in a conventional IVF group. Grimstad et al. (Citation2016) showed that in couples with a history of tubal ligation as their infertility diagnosis, and without male factor ICSI, did not show any significant improvement in their fertilization rate. More importantly, the odds of clinical intrauterine gestation and of a live birth rate were even lower when ICSI was used. Tannus et al. (Citation2017), who studied only women aged 40 years and older, included in IVF treatments for non-male factor infertility, did not find any difference in live birth rates between ICSI and the conventional IVF group. They also did not find any difference in fertilization rate and fertilization failure, but observed a higher proportion of cycles with the transfer of blastocyst stage embryos and of cycles with embryos available for cryopreservation in a conventional IVF group. A similar result was observed in a sibling oocytes study, where half of the oocytes from a patient were fertilized by ICSI and the rest with conventional IVF (Speyer et al. Citation2019). The authors of this study noticed that the blatocysts from the conventional IVF group were significantly more often chosen to be transferred and they explained this with the assumption that IVF-derived embryos develop faster to the blastocyst stage than ICSI-derived embryos. Otherwise, they did not observe any other important differences in terms of fertilization, pregnancy or live birth rate. Similarly, the number of embryos transferred or cryopreserved was significantly higher after conventional IVF in patients with increased oocyte maturity, where the fertilization rate was also improved by conventional IVF, although the quality of embryos did not differ (Taylor et al. Citation2008). The cryopreserved embryos were taken into consideration by Li et al. (Citation2018) who evaluated the cumulative live birth rate in cycles without male factor infertility. They discovered that 36.8% of ICSI cycles were performed in non-male factor infertility cases, which led to a similar cumulative live birth rate compared to conventional IVF. Additionally, no differences in embryo development or in blastocyst formations or in pregnancy rates were reported several times before (Staessen et al. Citation1999; Van Landuyt et al. Citation2005; Foong et al. Citation2006; Tobler et al. Citation2014), but similarly to other topics in ART, also on this topic the opposite results were published. It was shown that ICSI can increase fertilization rates and good-quality embryo rates in patients with non-male factor indications (Khamsi et al. Citation2001), can increase fertilization rates and lower complete fertilization failure in patients with polycystic ovarian syndrome and normozoospermic semen and in cases of unexplained inferility (Hershlag et al. Citation2002; Hwang et al. Citation2005), can increase normal and lower triploid fertilization rates leading to a higher mean number of embryos (but no improvement in pregnancy and implantation rate) in patients with endometriosis and normozoospermic semen (Komsky-Elbaz et al. Citation2013) and can improve the fertilization rate, while at the same time lowering the pregnancy and delivery rate in patients with unexplained infertility (Check et al. Citation2009).
To conclude, this retrospective analysis suggests that when sperm morphology is not severely impaired and sperm concentration and motility are normal, it is better to use conventional IVF to fertilize oocytes and not ICSI. The advantage of conventional IVF was reflected in improved blastocyst rates and quality, but there was no statistically significant difference in pregnancy rates between procedures, although the data suggest a trend towards better results after conventional IVF. Since this is a retrospective study suggesting interesting results, but with limitation in number of cycles included, an appropriately powered study should be designed and performed to determine, which procedure of oocyte fertilization is more appropriate to use in patients with mild or moderate teratozoospermia.
Materials and methods
In this retrospective study, conducted at the IVF Unit of the University Medical Centre Ljubljana, an analysis was done on the outcome of conventional IVF/ICSI cycles performed between October 2017 and July 2018. We included 51 couples in this study, who were treated with ART and where the male factor of infertility was defined as teratozoospermia, by which teratozoospermia means that less than 15% of spermatozoa were assessed with normal morphology (as defined by strict Kruger criteria). Therefore, most (48) of the included patients had morphology in the range of 4% to 14%, but there were also three patients with lower morphology (one with a morphology of 2% and two with a morphology of 3%). The women in the couples were aged from 26 to 43 years old. We included data from all cycles performed in the mentioned time frame. In all cycles there were at least two cumulus-oocyte complexes (COCs) intended for ICSI or for conventional IVF in the same patients (sibling oocyte), meaning that each patient had at least 4 COCs.
Controlled ovarian hyperstimulation (COH) protocols
For controlled ovarian hyperstimulation (COH) two different protocols were used, namely, GnRH antagonist (in 39 women) and GnRH agonist protocols (in 12 women), as previously described (Stimpfel et al. Citation2018). Briefly, in both approaches a daily dose of 200–225 IU recombinant FSH (follitropin alfa, Gonal F, Merck Serono or follitropin beta, Puregon, MSD) was used to stimulate ovaries. In the the GnRH antagonist protocol GnRH antagonist cetrorelix acetate (Cetrotide; Asta Medica AG) was used and in the GnRH agonist protocol GnRH analogue Suprefact (Hoechst AG) was used. For oocyte maturation triggering Ovitrelle (Merck Serono) was administered at a dose of 6500 IU. In both approaches of COH oocyte retrieval was performed 36 h after HCG administration.
Semen quality evaluation (concentration, motility, and morphology) and criteria
The semen quality was evaluated as previously described by Golob et al. (Citation2014). To determine the proportion of normal and abnormal morphology spermatozoa were examined using strict Kruger criteria (Kruger et al. Citation1986, Citation1987). The men were considered teratozoospermic if the sperm concentration was ≥15 million spermatozoa/ml, the total motility was ≥40%, and the percentage of spermatozoa with normal morphology was ≤14%.
Preparation of semen sample for conventional IVF and ICSI
The semen sample was prepared as previously described (Stimpfel et al. Citation2018). Briefly, on the day of oocyte retrieval, the sample was assessed for volume, concentration and motility and then prepared using density gradient centrifugation, followed by swim-up. After applying the swim-up technique, one part of the sperm fraction was used to make droplets under paraffin oil, which were used to perform ICSI while another part of the sperm fraction was used to perform conventional insemination of the cumulus-oocyte complexes. The oocytes after ICSI and conventionally inseminated cumulus-oocyte complexes were cultured until the next day in Fertilization Medium (Cook) or Universal IVF Medium (Origio).
Embryo culture and embryo transfer (ET)
To check if the fertilization was successful, the oocytes were examined for the presence of pronuclei 16–18 hours after fertilization. Normally fertilized oocytes (with 2PN) were further cultured in a continuous culture medium SAGE 1-Step and transferred into the uterus on day 3 or on day 5. The quality of embryos on day 3 was assessed as previously described by Stimpfel et al. (Citation2018) and on day 5/6, the blastocysts were graded as described by Gardner et al. (Citation2000). When ET was performed on day 3, the supernumerary embryos were cultured further until the fifth/sixth day and were cryopreserved if they developed to the blastocyst stage. When ET was performed on day 5, the supernumerary blastocysts were cryopreserved. The rest of the embryos were cultured until day 6 and then cryopreserved if they developed into a blastocyst. In each patient, at most two embryos/blastocysts were transferred. When calculating the pregnancy rate, the embryo transfers on day 3 and day 5 were not separately analyzed but were put together.
Statistical analysis
To determine the differences between the groups Pearson’s chi-squared, a Fisher’s exact test and t-test were used to analyze the data as appropriate. The P-values under 0.05 were recognized as statistically significant. G*Power program, version 3.1.9.4, (Faul et al. Citation2007) was used to calculate statistical power of analysis and sample size of couples (only couples in which ET would be performed) needed to be included in to the study to achieve statistically significant difference in pregnancy rate between the treatments.
Ethical approval
Due to a retrospective nature of data analysis, the study does not need ethical approval.
Author contributions
Study design: MS, IVK; analysis, interpretation of data and manuscript writing: MS; revision of manuscript: NJ, EVB, IVK.
Acknowledgments
The authors would like to thank all gynecologists, clinical embryologists, medical nurses, and other staff at the Department of Human Reproduction, Division of Gynaecology, University Medical Centre Ljubljana, for their support.
Disclosure statement
No potential conflict of interest was reported by the authors.
Additional information
Funding
References
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