Abstract
Purpose
To explore the effects of traditional vs. intracytoplasmic sperm injection (ICSI) insemination method on the outcome of high-quality blastocyst development in a split sibling oocyte cohort.
Methods
In this retrospective cohort study, we analyzed 62 ICSI/IVF split cycles. Sibling oocytes were randomly assigned to ICSI or IVF insemination. Two hundred thirty-four ICSI-only cycles and 152 IVF-only cycles were also analyzed for comparison. Blastocysts were graded by Gardner’s embryo grading and were considered a high-quality blastocyst if 3BB or better (Gardner 1999).
Results
In the ICSI/IVF split group, (1) ICSI oocytes had a higher fertilization rate per oocyte allocated (73% vs 62%, p < 0.001), (2) more high-quality day 2 embryos (69% vs 55%, p < 0.005), (3) ICSI oocytes had a lower blastulation rate per 2PN (46% vs 54%, p < 0.05), but a higher blastulation rate when calculated per oocyte allocated (40% vs 32%, p < 0.05). The ICSI-only group had a lower fertilization rate (65% vs 70%, p < 0.001) but more high-quality day 2 embryos in comparison to the IVF-only group (68% vs 64%, p < .05). The total high-quality blastulation rate was higher for the IVF-only group per 2PN (49% vs 43%, p < 0.05) and per oocyte retrieved (34% vs 28%, p < 0.05).
Conclusions
This distinctive IVF/ICSI sibling oocyte split design demonstrated a higher-quality blastulation rate in the IVF group compared to the ICSI group when calculated per 2PN, but not per oocyte allocated to each insemination procedure.
Keywords: Intracytoplasmic sperm injection (ICSI), In vitro fertilization, Blastulation rate, Assisted reproductive technology (ART)
Introduction
Intracytoplasmic sperm injection (ICSI) is indicated for male factor infertility and after fertilization failure. However, the use of ICSI for other infertility indications despite evidence indicating there is no benefit over traditional IVF is increasing [1]. In patients with unexplained infertility, one reason for ICSI use without clear evidence is to avoid total fertilization failure (TFF). A recent cross-sectional analysis of the SART database revealed that ICSI use for non–male-factor infertility cycles was 34.6% in 2000 and increased to 73.9% in 2015 [2]. Despite this growing trend in un-indicated ICSI use, there remains various health concerns with ICSI, including imprinting disorders or higher rates of minor/major malformations [3, 4]. In addition, data to support that ICSI-derived embryo development is equivalent to conventional IVF is lacking.
In general, many assisted reproductive cycles utilizing ICSI are in a population with severe male factor and abnormal sperm parameters, making it difficult to compare ICSI-derived embryo quality to those from traditional IVF insemination. Severe male factor has been shown to impair embryo development [5]. Therefore, it is difficult to disentangle the potential male factor detrimental impact on embryo development when using ICSI, leading to a tremendous source of confounding. In this study, we used a split sibling oocyte design to compare embryo development in an ICSI versus IVF cohort. Using a split design significantly reduces multiple confounders in patient characteristics, which can affect embryo development. Previous sibling oocyte studies are controversial with some reporting reduced blastulation rates of ICSI embryos [6, 7], no difference in ICSI versus IVF embryos [8], and others showing improved embryo development with ICSI [9]. While the data is mixed, it is difficult to compare previous studies due to differences in how blastulation rate is defined and how embryo quality is reported. For example, some studies report on all blastocysts instead of high-quality blastocysts, while others evaluate cleavage stage embryo quality.
Therefore, our goal was to evaluate the impact of the insemination method on the development of high-quality embryos from sibling oocytes in split IVF/ICSI cycles and to determine if using two pronuclei (2PN) or oocytes allocated to each group would affect our primary outcome of high-quality blastulation rate. For comparison, we also analyzed the high-quality blastulation rate in our IVF- vs. ICSI-only cohorts.
Material and methods
Human subjects and clinical data collection
This retrospective cohort study included data extracted from the medical record of a single reproductive medicine practice at Women & Infants Hospital in Providence, RI. Subjects who underwent assisted reproductive technology utilizing IVF, ICSI, or both and had at least one fresh blastocyst transferred at the Women and Infants Fertility Center from August 2014 to July 2017 were included in the present study. Donor egg cycles, freeze-only cycles for high risk of ovarian hyperstimulation syndrome (OHSS) or planned pre-implantation genetic testing for aneuploidy (PGT-A), day 3 transfers, and patients with no transfer were excluded from this study.
Three study populations were evaluated based on insemination method: (1) IVF/ICSI split, (2) ICSI-only, and (3) IVF-only. IVF/ICSI split insemination was offered to couples with the diagnosis of unexplained infertility, normal semen parameters based on WHO 4 criteria, and at least 10 oocytes obtained at retrieval. Oocytes were randomly allocated 1:1 to insemination with either ICSI or traditional IVF. Couples intended for the split protocol who had < 10 oocytes underwent ICSI-only and were not included in the analysis of this group. Oocyte yields with odd numbers had the additional oocyte allocated to ICSI. The oocytes were allocated under low magnification without consideration for perceived oocyte quality or maturity.
In the separate ICSI-only and IVF-only cohorts, the decision to inseminate with ICSI vs IVF was based on provider discretion; however, in general ICSI cycles were reserved for patients with abnormal semen parameters or prior fertilization failure. More specifically the following cutoffs were used to recommend ICSI: density < 10 million/mL, motility < 25% forward (3+ and 4+), total motile count < 2 million post-processing, sperm morphology < 2%, Epididymal/testicular sperm, previous failed or poor (< 40%) fertilization of mature eggs.
Baseline demographic characteristics such as age, semen analysis parameters, ovarian reserve, and BMI were collected and compared by insemination methods, where appropriate. This study was approved by the Institutional Review Board at Women & Infants Hospital (Providence, RI).
Ovarian hyperstimulation and oocyte retrieval
Patients underwent ovarian hyperstimulation cycles utilizing daily injection of recombinant FSH (rFSH) with either GNRH agonist or antagonist protocols for pituitary suppression and prevention of premature ovulation. Serum estradiol levels were monitored and oocyte growth was evaluated with transvaginal ultrasound. rFSH dosing was were adjusted accordingly. In the GNRH antagonist cycles, Menopur (Ferring Pharmaceutical) was added at time of antagonist initiation. When 3 or more follicles were 18 mm in average diameter or greater, 10,000 units of human chorionic gonadotropin (hCG) was injected to trigger oocyte maturation. Oocyte retrieval was carried out 36 h after hCG injection by transvaginal ultrasound-guided puncture of the follicles. Luteal-phase supplementation with either daily intravaginal progesterone or by single intramuscular progesterone injection was started the morning after retrieval.
Semen preparation and insemination techniques
Semen processing was the same in all groups. After abstinence for 48 to 72 h an ejaculated sample was produced the day of the retrieval. The sample was allowed to liquefy for up to 1 h and then washed to remove the seminal fluid and processed by centrifugation through a density gradient. The sample was then washed again, concentrated and analyzed on a Makler chamber to obtain a final count and motility. The final parameters were used to calculate the volume needed for conventional IVF insemination
Conventional IVF was performed using a Microdrop insemination technique. Oocytes were trimmed of the majority of their cumulus cells after the retrieval. Insemination was performed approximately 4–4.5 h post-retrieval. Each drop of media contained two to three oocytes to which was added 1.0 × 105 motile sperm. Oocytes were then co-incubated with sperm in a small drop of culture medium (100 μl) for 16–18.5 h to facilitate fertilization.
For oocytes inseminated with ICSI, cumulus cells were first completely denuded from cumulus-oocyte complexes by hyaluronidase treatment using a Pasteur pipette 30–45 min prior to the ICSI procedure. A 60 mm Hyaluronidase dish with 300 μL drop of hyaluronidase solution in center surrounded by 8–10: 50 μL drops of HEPES medium/10%SPS was used. Oocytes (maximum 10) were placed into the hyaluronidase drop for a 15–30-s exposure. Oocytes were then transferred through 8-10 HEPES drops and pipetted up and down decreasing pipet tip size (600–170 μm) as needed to remove the cumulus cells. Oocytes were then placed in a Rinse dish (Global media/10% PS). Sperm were then immobilized, aspirated into a glass pipet, and a single sperm was injected into each oocyte. Sperm were injected at either 6 or 12 o’clock in relationship to the polar body. Only M2 oocytes were injected; however immature appearing oocytes were cultured for 1 h after cumulus cell removal and subsequently injected if they had matured in vitro. Resulting zygotes were cultured for subsequent embryo development.
Embryo evaluation and transfer
Oocytes were examined for successful fertilization 12–18 h after insemination. On the morning of day 1, remaining cumulus cells were mechanically stripped from IVF-inseminated oocytes. Fertilization rate was defined as the number of oocytes with 2PN divided by the number of retrieved cumulus–oocyte complexes. All embryos were evaluated on day 2 and developing embryos remained in continuous single culture media until day 5 or day 6. Blastocyst transfer was performed utilizing either a Wallace (Cooper Surgical) or a Soft-Pass (Cook Medical) embryo transfer catheter. Supernumerary high-quality blastocysts were vitrified. High-quality cleavage stage embryos contained the following characteristics: (1) 2PN on day 1, (2) between 4 and 6 cells on day 2, and (3) a fragmentation score of < 10%. Gardner’s embryo grading scale was used to determine high-quality blastocyst formation, which was defined as 3BB or better [10]. Embryos were graded on both days 5 and 6 of development. Both sibling oocyte cohorts were graded at the same time. If the embryos were evaluated on day 5 and met the cut off of a 3BB or greater they were frozen or transferred and not reevaluated on day 6. These embryos were considered high-quality blastocysts and that grade was used for the analysis. If they did not meet the cut-off on day 5, they were re-evaluated on day 6 and the day 6 grade was the one used for the analysis High-quality blastulation rates for the ICSI/IVF split group and the ICSI- or IVF-only groups were then calculated based on number of 2PN’s as well as number of oocytes allocated/retrieved.
Statistics
This analysis was restricted to patients who had a fresh blastocyst transfer 5 days following the oocyte retrieval. Univariate distributions of all variables were examined. Baseline characteristic data were analyzed by Wilcoxon rank-sum comparing ICSI and IVF insemination methods. Data for split subjects who had undergone both ICSI and IVF insemination methods were analyzed using the Wilcoxon signed-rank test for paired comparisons. Data were not normally distributed; therefore, non-parametric methods were used to compare outcomes by insemination method. p values < 0.05 were considered statistically significant and all tests were two-sided. We conducted a sensitivity analysis restricting to first cycles only to adjust for multiple cycles per patient. Statistical analyses were conducted using Stata/SE 15.1 (College Station, TX).
Results
Table 1 demonstrates the baseline characteristics of the cohort. A total of 62 ICSI/IVF sibling oocyte split cycles (53 subjects), 234 ICSI-only cycles (124 subjects), and 152 IVF-only cycles (107 patients) were included for analysis. The mean age for the study population was 33.9 ± 3.9 years. There were no significant differences in age, BMI, and ovarian reserve markers between the ICSI and IVF groups. The ICSI-only cycles had a higher percentage of abnormal semen parameters, including lower total motile count and motility. Patients in the ICSI-only group had a higher number of previous cycle attempts in comparison to the IVF-only group (0.79 vs 0.45 p < .001).
Table 1.
Baseline characteristics
| Entire Cohort | ICSI | IVF | ICSI vs IVF | IVF/ICSI splits | |
|---|---|---|---|---|---|
| N=284 patients | (N=284) | (n=124) | (n=107) | p-Value | (n=53) |
| Age | |||||
| Mean (SD) | 33.9 (3.9) | 33.7 (3.8) | 34.2 (4.2) | 0.2711 | 33.6 (3.7) |
| BMI (kg/m2) | |||||
| Mean (SD) | 27.1 (6.5) | 26.7 (5.9) | 28.2 (6.8) | 0.1211 | 25.9 (6.9) |
| FSH-Day 3 (mIU/ml) | |||||
| Mean (SD) | 7.3 (2.7) | 7.5 (2.8) | 7.1 (2.4) | 0.2811 | 7.2 (2.4) |
| Total motile Count (106) | |||||
| Mean (SD) | 111.3 (115.7) | 48.0 (75.7) | 151.9 (117.2) | <0.0011 | 172.4 (119.3) |
| SA Motility (%) | |||||
| Mean (SD) | 58.2 (20.9) | 47.0 (22.6) | 66.3 (16.6) | <0.0011 | 65.9 (12.9) |
1Wilcoxon rank-sum
In the ICSI/IVF split group there were 53 patients with 62 IVF/ICSI split cycles. Two cycles had failed fertilization with IVF (Table 2). More oocytes were inseminated by ICSI compared to IVF (9.6 vs 9.1, p < 0.001). ICSI oocytes in the split group had a higher fertilization rate (73% vs 62%, p < 0.001) and more high-quality day 2 embryos (69% vs 55%, p < 0.005). High-quality blastulation rate was superior with IVF in the ICSI/IVF split cohort when calculated per 2PN as the denominator (54% vs 46%, p < 0.05). Conversely, when calculated per oocyte allocated the ICSI group resulted in a higher-quality blastulation rate (40% vs 32%, p < 0.05). These findings were similar when we restricted our analysis to first cycles only. Pregnancy and implantation rate calculations were limited in the sibling oocyte groups due to the low number of embryo transfer cycles of only ICSI or I VF embryos. There were 30 ICSI-only transfer cycles with a 46% CPR rate and 20 IVF-only transfer cycles with a 60% CPR rate which was not statistically different.
Table 2.
Embryo quality. Sibling oocyte splits with fresh day 5 transfers
| Total patients = 60 | |||
| Total cycles = 62 | |||
| Oocytes retrieved | |||
| Mean (SD) | 18.7 (7.0) | ||
| Oocytes by ICSI | Oocytes by IVF | p-Value | |
| Oocytes allocated to each group | |||
| Mean (SD) | 9.6 (3.6) | 9.1 (3.5) | <0.0012 |
| Fertilization rate (#2pn/oocyte) | |||
| Mean (SD) | 0.73 (0.16) | 0.62 (0.25) | <0.0012 |
| High quality day 2 rate (day 2/2pn) | |||
| Mean (SD) | 0.69 (0.25) | 0.55 (0.29) | 0.00522 |
| High quality blastulation rate/2pn† | |||
| Mean (SD) | 0.46 (0.25) | 0.54 (0.26) | 0.0422 |
| High quality blastulation rate/oocyte* | |||
| Mean (SD) | 0.40 (0.23) | 0.32 | 0.0222 |
2Wilcoxon signed-rank
† (# day 5 high quality blast + # day 6 high quality blast)/ (# 2pn)
* (# day 5 high quality blast + # day 6 high quality blast)/ (# oocytes)
When the ICSI-only and IVF-only cycles were compared oocytes retrieved were similar between the two groups, reflecting similar ovarian reserve levels (Table 3). The ICSI group had a higher percentage of high-quality embryos on day 2 in comparison to the IVF group (68% vs 64% p < 0.05), similar to the findings in the sibling oocyte group. However, the total high-quality blastulation rate per 2PN was higher in the IVF group than in the ICSI group (49% vs 43%, p < 0.05). When high-quality blastocyst development was calculated per oocyte retrieved, IVF remained superior to ICSI for high-quality blastulation (34% vs 28% p < 0.05). Implantation and clinical pregnancy rates were not statistically different between IVF and ICSI groups. First cycle blastulation rates were also evaluated, and there was a trend for more high-quality blastocyst development in the IVF group.
Table 3.
Embryo quality and Pregnancy outcomes. ICSI vs IVF cohort with day 5 fresh embryo transfers
| N=386 cycles | ICSI | IVF | p-Value |
|---|---|---|---|
| (n=234) | (n=152) | ||
| Oocytes retrieved | |||
| Mean (SD) | 15.4 (7.6) | 16.0 (7.9) | 0.521 |
| Fertilization rate (#2pn/oocytes retrieved) | |||
| Mean (SD) | 0.65 (.18) | 0.70 (0.15) | <0.0011 |
| High quality day 2 rate (day2/2pn) | |||
| Mean (SD) | 0.68 (0.22) | 0.64 (0.19) | 0.0211 |
| High quality blastulation rate/2pn† | |||
| Mean (SD) | 0.43 (0.27) | 0.49 (0.27) | 0.0311 |
| High Quality blastulation rate/oocyte* | |||
| Mean (SD) | 0.28 (0.19) | 0.34 (0.19) | 0.00111 |
| Implantation rate (heart beat/number transferred) | |||
| Mean (SD) | 0.32 (0.43) | 0.38 (0.45) | 0.331 |
| Clinical pregnancy rate (CPR) | |||
| Mean (SD) | 0.30 (0.41) | 0.39 (0.46) | 0.081 |
1Wilcoxon rank-sum
† (# day 5 good blast + # day 6 good blast)/ (# 2pn)
* (# day 5 high quality blast + # day 6 high quality blast)/ (# oocytes)
Discussion
Many couples with non-male factor infertility prefer ICSI due to the higher fertilization rates in comparison to IVF and their desire to avoid TFF. Although our study found a higher fertilization rate for ICSI in our split group, ICSI embryos may not have the same development potential when compared to embryos fertilized with traditional insemination techniques. One explanation for hindered embryo progression is that an M2 oocyte fertilized by ICSI may possess chromosomal maturity, this may not equate to cytoplasmic maturity. Additionally, with ICSI, there is potential for selection and injection of a sperm with low natural fertilization potential leading to “forced fertilization”, which may impact subsequent blastulation. There are also concerns about damaging the oocyte during the sperm injection process. Hence, the goal of this study was to compare the impact of ICSI versus traditional IVF insemination on high-quality blastocyst formation in sibling oocytes.
Blastulation rate is commonly reported as the number of blastocysts by number of 2PN. However, some reports have used oocytes retrieved as the denominator [7, 9, 11]. In the ICSI/IVF split oocyte group, ICSI lead to a higher fertilization rate, more high-quality day 2 embryos, but high-quality blastulation rate was higher in the IVF fertilized oocytes when analyzed per 2PN. Conversely, when oocytes were used as the denominator, ICSI inseminated oocytes produced more high-quality embryos in comparison to IVF. In the IVF- and ICSI-only cohorts, this discrepancy in denominator-dependent blastulation rates was nonexistent. One potential explanation for the difference in blastulation rates based on 2PN is that M1 oocytes allocated to the ICSI group were incubated in culture media post cumulus removal for an extra hour, which could allow for in-vitro maturation of M1s to M2s. These in vitro-matured oocytes may fertilize, but likely have a lower blastulation probability [12]; leading to a falsely elevated denominator in the ICSI group when calculating blastulation rates per 2PN. Additionally, ICSI fertilized embryos may lack the necessary cellular machinery required for high-quality blastocyst formation due to cytoplasmic immaturity, again falsely increasing the 2PN number [12]. When comparing the IVF- and ICSI-only groups, the detrimental impact of male factor infertility and subsequent poor sperm quality reduces embryo development so significantly in the ICSI group that high-quality blastulation rate was lower regardless of the denominator used.
Our findings are in agreement with previous studies by Griffiths et al. and Yoeli et al., who also found a significantly reduced blastulation rate when calculated per 2PN in their ICSI vs IVF population utilizing a sibling oocyte design [6, 7]. Unfortunately, these studies did not report rates per oocyte allocated. However, in contrast to our findings Landuyt et al. did not find a difference in embryo development rates per fertilized oocyte in their split cohort [9, 13]. These differences could be explained by Landuyt et al. consisting of a more heterogeneous population with multiple infertility diagnoses and the inclusion of patients who underwent day 3 transfers [13]. Another study by Khamsi et al. found higher good quality embryos per oocyte with ICSI compared to IVF, however they evaluated day 2 embryo quality and not blastulation rate [9]. In an effort to simplify our primary outcome of blastulation rates in a split ICSI/IVF cohort, we only included patients who underwent blastocyst transfers. In our center, we offer day 3 transfers to patients with less than 3 dividing embryos or if the majority of embryos have increased fragmentation and abnormal cell number. Patients with day 3 transfers were excluded because the “best” embryo(s), based on morphology was transferred, hence were not available for evaluation of progression to blastocyst.
There is concern that ICSI may be harmful; however this may depend on the technique used. Although data is controversial, some believe that the ICSI procedure itself may be detrimental to the development of an embryo. This damage to the oocyte varies by technique and could occur if there is trauma to organelles such as the mitochondria or meiotic spindles. Motoishi et al. report their findings of a sham ICSI procedure on bovine oocytes and showed no differences in blastulation rates in comparison to oocytes that had not been injected [14]. Conversely, Hewitson et al. evaluated the impact of ICSI in rhesus monkey oocytes and showed a higher degree of spindle variation than IVF inseminated oocytes [15]. Two studies utilizing human oocytes revealed that location of injection impacted embryo quality, with injection near the meiotic spindle causing lower day 3 embryo quality [16, 17].
Sperm quality is a major source of confounding when comparing ICSI and IVF outcomes. One large retrospective study comparing ICSI vs IVF cycles did not control for sperm quality [11]. Another study comparing IVF and ICSI outcomes consisted of patients with abnormal semen parameters in the ICSI group versus IVF patients with normal semen parameters [18]. Previous studies have demonstrated that sperm from patients with severe male factor infertility have decreased blastulation rates compared to patients with normal sperm parameters [5, 18]. While these abnormal sperm allow for fertilization with ICSI and formation of 2PN, the detrimental impact of the sperm is not recognized until embryonic genome activation at the 4–8 cell stage. This may explain why our high-quality day 2 rate was improved with ICSI, but subsequent blastulation was reduced in our ICSI-only cohort. Other causes for our improved high-quality day 2 embryos include quicker formation of 2PN seen with ICSI embryos and the time lag seen in cleavage of IVF embryos [19–21]. The higher day 2 quality with the ICSI-only cohort in comparison to the IVF cohort also supports the theory that ICSI does not damage the embryo. If the procedure was detrimental, it would cause a decrease in embryo quality within first day or two of ICSI. Instead, we found that the ICSI-only group had a higher day 2 quality embryos than the IVF group but a reduced high-quality blastulation rate per 2PN and oocyte allocated likely a result of the detrimental impact of sperm that is not recognized until later in embryo development.
Our study has several strengths including the use of a sibling oocyte design in which each patient also serves as a control. This design is a highly effective way to reduce confounding factors that impact embryo blastulation rates. The IVF/ICSI split controls for the impact of sperm quality, as the sperm source is the same for both groups. In addition, we compared both high-quality day 2 and high-quality blastocysts between ICSI and IVF whereas other studies only evaluated one or the other. Furthermore our embryologists blindly allocated the oocytes to either ICSI or IVF prior to evaluating their appearance, reducing bias in oocyte distribution. This study also has a few limitations. While the split design reduces confounding bias, the retrospective nature of the study allows for the potential for other forms of bias. Another limitation is the generalizability of this study to freeze-only patients or patients with a poor prognosis that underwent day 3 embryo transfers, as we only included blastocyst transfer patients in our sibling oocyte cohort as well as our IVF- and ICSI-only cohorts. Therefore, our evaluation is limited to good prognosis patients and may not be valid in other populations. Lastly, we only evaluated patients who underwent a day 5 transfer, so we cannot provide the percentage of patients with total fertilization failure in the ICSI versus IVF group. Results of our study should be confirmed in a larger trial where day 3 transfers are not performed.
In conclusion, our findings from this ICSI/IVF sibling cohort study suggest that the conventional method of calculating blastocyst formation per 2PN as the denominator may not provide an accurate assessment of blastocyst potential in vitro. In our sibling cohort, ICSI resulted in higher fertilization rates, more high-quality day 2 embryos and more high-quality blastocysts per oocyte in comparison to IVF. While in this sibling cohort IVF resulted in a higher blastulation rate per 2PN, the increased fertilization rate with ICSI is able to overcome this lower blastulation rate per 2PN and actually resulted in more high-quality blastocyst per oocyte allocated. Therefore, in our institution ICSI can achieve more high-quality blastocysts than IVF per retrieval cycle.
Funding information
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Compliance with ethical standards
Declarations of interest
None
Footnotes
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