Abstract
Purpose
Limited research has been published comparing PIEZO-ICSI with conventional ICSI. While positive effects have been documented in improving fertilization and degeneration, the outcomes in patients with previous poor results from conventional ICSI remain unclear. It is hypothesized that these patients may benefit the most from this form of insemination.
Methods
This retrospective paired within-patient cohort study investigated patients (n=72) undertaking PIEZO-ICSI after a previous conventional ICSI cycle resulted in poor outcomes (including low fertilization (<50%), high degeneration (>15%), and/or poor embryo development and utilization). Patients required at least five oocytes collected in both cycles and a period of less than 2 years between the cycles. The outcomes of both cycles were compared in respect to fertilization, degeneration, embryo utilization, and pregnancy rates. Further analyses were applied to patients <38 and ≥38 years of age, with <50% or ≥50% fertilization with conventional ICSI and with <20% or ≥20% utilization with conventional ICSI.
Results
PIEZO-ICSI resulted in significantly higher fertilization (61.9% vs 45.3%, P<0.0001) and lower degeneration (7.7% vs 18.2%, P=0.0001) when compared to the conventional ICSI cycles. The greatest benefit was seen in patients who had less than 50% fertilization or <20% utilization in their conventional ICSI cycle, with improvements in fertilization and degeneration rates resulting in a significantly higher number of embryos utilized (frozen or transferred) per cycle.
Conclusions
PIEZO-ICSI improved fertilization, degeneration, and utilization rates in patients with previous poor outcomes from conventional ICSI. The number of embryos available for use per cycle was also increased. Further significant improvements were achieved in patients who exhibited poor fertilization (<50%) or low utilization (<20%) from conventional ICSI.
Keywords: ICSI, PIEZO, Fertilization, Degeneration, Advanced maternal age
Introduction
Intracytoplasmic sperm injection (ICSI) has been widely used in assisted reproduction, particularly in cases of severe male factor infertility [1, 2]. While this technique has been widely adopted and accounts for up to 70–80% of the IVF cycles performed worldwide [3, 4], it does come with disadvantages, including increased rates of degeneration and abnormal fertilization post-injection [5]. The process of ICSI involves the mechanical penetration of the zona pellucida and the aspiration of the internal oocyte membrane using a spiked and beveled injection pipette, which causes deformation of the oocyte and increased internal pressure [6, 7]. This change in pressure may have a negative impact on the oocyte and affect fertilization and blastocyst development, especially in older women who are more likely to have oocytes with fragile plasma membranes [1, 8].
PIEZO-ICSI was initially undertaken in animal species, including mice, rabbits, horses, and cattle, where it was used successfully to produce high-quality embryos and live births [9–11]. PIEZO-ICSI is a modified form of insemination that uses a piezoelectric actuator to create high-speed movement of the injection pipette. These movements allow precise microdrilling of the zona and smooth penetration of a blunt-ended injection pipette into the cytoplasm which reduces the need for physical pressure on the oocyte membrane. In addition, as suction of the ooplasm is not necessary to break the plasma membrane and the sperm is simply deposited into the cytoplasm without cytoplasmic agitation, it is considered a gentler form of microinjection [12].
Early studies on the use of PIEZO-ICSI in human oocytes reported improved survival and fertilization when compared to conventional ICSI [6, 12]. Since these initial studies, notable improvements to the PIEZO-ICSI technique and equipment have included the use of thin-walled microinjection pipettes [13, 14] and the use of biocompatible operating fluid [15]. Historically, the operating fluid used for PIEZO-ICSI had been mercury or fluorinert, neither of which was considered safe to be used in a clinical setting. A recent study reported the use of a new operating fluid, perfluoro-n-octane, which is a biocompatible operation liquid that is safe for use in the human body [15]. These improvements result in PIEZO-ICSI being a suitable and safe procedure in human-assisted reproduction, overcoming the safety concerns that have been raised previously [7].
While there are only a small number of studies of PIEZO-ICSI in human oocytes, most have reported improvements in fertilization rates and reduced degeneration rates in comparison to conventional ICSI [8, 15, 16]. These studies however were paired cohort studies comparing outcomes between patients that were case-matched according to their demographics, and direct comparisons in specific patient subgroups were not performed. More recently, studies have identified improved fertilization and blastocyst development rates when compared to conventional ICSI in women >35 years of age [8], and the most beneficial improvements for women >38 years of old [15] highlighting that the effectiveness of PIEZO-ICSI may be greater in certain patient cohorts or infertility etiologies, such as older patients and those considered to be poor prognosis, based on outcomes in their previous treatment cycles.
The benefit of PIEZO-ICSI on pregnancy rates is less documented with most studies focused on the effects on fertilization and oocyte degeneration rates. Early studies reported no significant differences in ongoing pregnancy rate beyond 8 weeks [12], while more recent studies have shown no significant improvements in pregnancy rate and live birth rate per embryo transfer when conventional ICSI was compared to PIEZO-ICSI, despite improvements in fertilization [7]. Reports of increased numbers of good quality embryos available for cryopreservation or transfer per cycle with the use of PIEZO-ICSI suggest that the cumulative pregnancy rates may be increased; however, these studies are ongoing [15].
Consequently, the purpose of this study was to assess the clinical efficacy of PIEZO-ICSI in patients who had previously exhibited poor results (low fertilization and/or high degeneration) from the use of conventional ICSI while also examining outcomes based on advanced maternal age (≥38 years of age). This initial study uses a retrospective paired within-patient experimental design to compare outcomes between a conventional ICSI cycle and the subsequent PIEZO-ICSI cycle with respect to fertilization, degeneration, embryo utilization, and pregnancy rates. Comparison of the two cycles from the same patient allows for within-patient control to reduce the confounders seen in paired cohort studies. It was hypothesized that the use of PIEZO-ICSI would improve fertilization and decrease degeneration in this poor prognosis patient group, with the greatest effects in older patients.
Methods
Patients and study design criteria
Patients received treatment at two large Monash IVF clinics in Melbourne, Australia. At the request of their consulting doctor, patients were selected for PIEZO-ICSI insemination based on poor outcomes in their previous conventional ICSI cycle. These outcomes include low fertilization (<50%), high degeneration (>15%), and/or poor embryo development and utilization. As PIEZO-ICSI and the associated consumables are not yet registered by the Australian Therapeutic Goods Administration (TGA), all patients were granted approval to utilize this technology through the TGA Special Access Scheme. Both the ICSI cycle and the subsequent PIEZO-ICSI cycle were autologous fresh cycles, where the sperm source was the same for both cycles assessed (this included fresh or frozen sperm, including surgical sperm retrieval). The ICSI cycle preceding their request for PIEZO-ICSI was used for analysis with PIEZO-ICSI cycles being completed between March 2020 and October 2021. Patient demographics including maternal age at the commencement of PIEZO-ICSI, body mass index (BMI), infertility diagnosis as per the Australia and New Zealand Assisted Reproduction Database (ANZARD) definitions [17], and the number of previous cycles were obtained from an internal patient treatment database (Table 1). Exclusion criteria included patients who had less than five oocytes collected from either of their cycles or had more than 2 years between the ICSI and the subsequent PIEZO-ICSI cycle.
Table 1.
Demographics of patient cohort
| Parameter | |
|---|---|
| Number of patients (n) | 72 |
| Maternal BMI | 27.0 ± 6.9 |
| Paternal BMI | 27.2 ± 3.8 |
| Infertility diagnosis | |
|
Male factor Endometriosis Tubal factor Other female factor Unexplained |
16 (22%) 14 (19%) 9 (13%) 18 (25%) 15 (21%) |
| Number of previous cycles a | 2.8 ± 2.1 |
| Interval between conventional ICSI and PIEZO-ICSI cycles (days) | 154.1 ± 154.0 |
Values are shown as mean with ± SD or % frequency; BMI, body mass index
aNumber of previous cycles including the conventional ICSI cycle used for analysis
For further analysis, the patients were then divided into three smaller cohorts: the low fertilization group where patients had less than 50% fertilization from their conventional ICSI cycle, the low utilization group where patients had utilization of <20%, and the high maternal age group (≥38 years at the time of PIEZO-ICSI treatment). The age cut of ≥38 years was chosen as this is often the point on success rate graphs where fertility is seen to rapidly decline [18]. Ethics approval for this study was obtained under Monash Health Human Ethics (HREC) Number 15172M as well as the Group Scientific Advisory Committee Internal Review Board.
Ovarian stimulation and oocyte retrieval
Ovarian stimulation was conducted as previously described [19]. Briefly, patients underwent either gonadotrophin-releasing (GnRH) agonist or GnRH antagonist protocols with recombinant FSH. Oocyte retrieval was performed transvaginally under ultrasound guidance 35 to 36 h post-human chorionic gonadotrophin (hCG) trigger. Oocyte cumulus complexes (COCs) were incubated in G-IVF PLUS (Vitrolife, Goteburg, Sweden) after oocyte collection. At 39 h post-trigger, COCs were denuded in 75 IU/mL hyaluronidase (Hyalase, Sanofi-aventis, NSW, Australia) in G-IVF-PLUS (Vitrolife), and stripped oocytes were incubated in G-1 PLUS (Vitrolife) prior to insemination. Metaphase II oocytes were inseminated 40 to 42 h post-trigger using either conventional ICSI or PIEZO-ICSI. All oocytes and embryos were cultured at 37°C in 6% CO2, 5% O2, and 89% N2 in benchtop incubators (MINC, Cook Medical, Brisbane, Australia or PLANER, Origio, Malov, Denmark).
Semen preparation
Semen preparation was performed using either a swim-up technique or a gradient centrifugation (45%/90% SpermGrad, Vitrolife) as described previously [20].
Conventional ICSI protocols
The conventional ICSI procedure was performed using an Olympus inverted microscope (IX-71 or IX-73) equipped with Eppendorf TransferMan NK2 or 4R microinjectors (Eppendorf, Hamburg, Germany). Commercially produced micropipettes with a beveled spike and tip (tip 5μm 5 (outside diameter), bevel 24°; 30° bend, The Pipette Company (TPC), Thebarton, South Australia, LICR-ID4) were combined with matching holding pipettes (tip 120μm (outside diameter), 25μm (inside diameter), 30° bend, TPC). Morphologically normal spermatozoa were selected in polyvinylpyrrolidone (Origio, CooperSurgical, Denmark) and immobilized by twice striking the tail with the micropipette before being aspirated tail first into the injection pipette. The oocyte was held with the polar body orientated at 12 o’clock, and the injection pipette was inserted at 3 o’clock. After penetrating through the zona pellucida, the needle was moved 70% of the way across the oocyte diameter, and suction was gently applied to the injector to break the oocyte membrane. After breakage of the membrane and the resulting sudden flow of cytoplasm into the pipette, positive pressure was applied to stop the flow and return the cytoplasm, along with single spermatozoa into the oocyte.
PIEZO-ICSI protocols
As previously described [15], PIEZO-ICSI was performed using commercially available thin-walled injection pipettes with a flat tip (ultra-thin, 6μm (outside diameter); 30° bend, Vitrolife AB, Sweden/Primetech Ltd, Tokyo, Japan, PINUS06-30FT) and holding pipettes as used for conventional ICSI. Approximately, 15 μl of operating fluid (Arcotane C8F18, Device Technologies, Eagle Farm, QLD, Australia) was placed in the middle of the micropipette and the pipette inserted into the holder and attached to the hydraulic injector (PNJ-T2, Primetech Ltd). The PIEZO micromanipulator drive unit (MB-D, Primetech Ltd) was attached to the micropipette holder, and the drive unit was driven by the PIEZO controller (PMAS-CT4G, Primetech Ltd). Under high-power magnification, the operating fluid was pushed to the end of the pipette, and the tip was primed with PVP (Origio, CooperSurgical, Denmark). The strength of the PIEZO pulse was then tested. Mechanical immobilization of the sperm occurred, and sperm was aspirated tail first into the pipette ready for injection. With the oocyte positioned with the polar body at either 6 or 12 o’clock, the micropipette was placed against the surface of the zona pellucida, and continuous PIEZO pulses were applied to allow gentle drilling of the zona (intensity setting 1.5, speed setting 3). After penetration of the zona, the remnant zona pieces were expelled, and the sperm moved to the tip of the pipette. The pipette was pushed slowly into the cytoplasm until it was positioned 80% of the way across the diameter of the cytoplasm, and a single PIEZO pulse was applied to break the membrane (intensity setting 2.0, speed setting 1). The cytoplasm was not aspirated into the injection pipette as the sperm was deposited into the cytoplasm and the pipette withdrawn slowly.
Embryo culture, transfer, and freezing
Post-insemination, oocytes were cultured in G-1 PLUS medium (Vitrolife) under mineral oil (Ovoil, Vitrolife). Fertilization was assessed 16–18-h post-insemination, and oocytes were classified as normally fertilized if the presence of two pronuclei and two polar bodies was observed. Those showing either one pronucleus or three or more pronuclei were classified as abnormally fertilized. Oocytes were considered degenerate if they had lost oocyte membrane integrity or had lysed following injection. On day 3, culture embryos were transferred to G-2 PLUS media (Vitrolife). Embryo transfer was performed on day 3 or day 5 according to each patient’s treatment plan, and the remaining embryos were assessed for vitrification on days 5 and 6. Patients undergoing a frozen embryo transfer (FET) cycle had their embryo transfer either in a natural cycle or a hormone replacement cycle (with or without oral contraceptive pill and GnRH agonist downregulation). Embryos were thawed on the morning of transfer. Post-embryo transfer, luteal support was provided to patients by daily progesterone administration intravaginally (8% Crinone, Merck). Serum β-HCG levels were measured 16 days post-oocyte collection and clinical pregnancy defined as the presence of at least one gestational sac via ultrasound at 6 weeks post-transfer. Birth outcome data was collected post-delivery from the treating hospital/obstetrician or directly from the patient as per mandatory reporting requirements. All live births were singleton deliveries.
Statistical analyses
Normality of the datasets was tested with the Shapiro–Wilk test. Developmental data (fertilization, degeneration, and abnormal fertilization) underwent Arcsin transformation before statistical analysis. Fisher’s exact test was used for categorical data (embryo utilization and pregnancy data). All continuous data were non-parametric, and Wilcoxon signed-rank test for paired samples was used for statistical analyses. All statistical analyses were undertaken using R (R Foundation for Statistical Computing, Vienna, Austria). P values <0.05 were considered to be statistically significant, and all data are presented as mean ± SD unless otherwise stated.
Results
Patient demographics
A total of 72 patients were included in this retrospective within-patient paired cohort study. Patients were selected who had a period of less than 2 years between their conventional ICSI and PIEZO-ICSI cycles and had more than five oocytes collected in both cycles. The number of previous conventional ICSI cycles prior to PIEZO-ICSI was 2.8 ± 2.1 (Table 1), and the average number of days between ICSI and PIEZO-ICSI cycles was 154.1 ± 154 days. Patients were referred by their consulting doctor for PIEZO-ICSI due to poor outcomes in their previous conventional ICSI cycle. There were no differences between the stimulation protocols used for the conventional ICSI and PIEZO-ICSI cycles, with a similar proportion of agonist and antagonist regimens used. The sperm source used for insemination was consistent for both cycles with fresh (74%), frozen (18%), or surgically retrieved sperm (8%) used. There were no significant differences in the number of oocytes collected, number of oocytes injected, sperm parameters (concentration and motility), the number of fresh transfer cycles, and the number of freeze all cycles (Table 2). No significant difference was seen in the average number of embryos transferred per cycle (1.2 conventional ICSI vs 1.1 PIEZO-ICSI). Single embryo transfer (SET) was used in 84% of conventional ICSI cycles and 91% of PIEZO-ICSI cycles and in 100% of frozen embryo transfer (FETs).
Table 2.
Treatment cycle demographics of the patient cohort for their paired conventional ICSI and subsequent PIEZO-ICSI cycles (n=72 patients)
| Parameter | Conventional ICSI | PIEZO-ICSI | P value |
|---|---|---|---|
| Maternal age (years) | 37.6 ± 4.0 | 38.0 ± 3.9 | <0.0001 |
| Paternal age (years) | 38.7 ± 5.5 | 39.1 ± 5.5 | <0.0001 |
| Sperm concentration (million/mL) | 37.1 ± 36.0 | 37.0 ± 36.8 | 0.944 |
| Sperm motility (%) | 39.7 (0–90) | 44.0 (0-99) | 0.301 |
| Number oocytes collected | 11.0 ± 5.5 | 11.0 ± 5.2 | 0.971 |
| Intended fresh transfer | 43 (59.7%) | 43 (59.7%) | 1.000 |
| Freeze all cycle | 29 (40.3%) | 29 (40.3%) | 1.000 |
Values are mean ± SD for continuous data or % (n/total) for categorical data. P < 0.05 was considered to be statistically significant
Fertilization and oocyte degeneration
Fertilization was significantly increased with PIEZO-ICSI when compared to the patient’s conventional ICSI cycle (61.9% vs 45.3%, P < 0.0001) (Table 3). PIEZO-ICSI compared with conventional ICSI also significantly decreased the proportion of degenerate oocytes (7.7% vs 18.2%, P = 0.0001). The proportion of abnormally fertilized oocytes was not significantly different when PIEZO-ICSI was compared to conventional ICSI (5.6% vs 5.5%, P = 0.169).
Table 3.
Comparison of conventional ICSI to PIEZO-ICSI for fertilization, utilization, and clinical pregnancy rates in the paired cohort (n=72 patients)
| Parameter | Conventional ICSI | PIEZO-ICSI | P value |
|---|---|---|---|
| Total number of oocytes injected | 581 | 557 | |
| Average number of oocytes injected | 8.1 ± 3.9 | 7.7 ± 4.5 | 0.473 |
| Total oocytes fertilized | 45.3% | 61.9% | <0.0001 |
| Total oocytes abnormally fertilized | 5.5% | 5.6% | 0.169 |
| Total oocytes degenerate | 18.2% | 7.7% | <0.0001 |
| Number of embryos utilized per cycle | 1.1 ± 1.3 | 1.6 ± 1.7 | 0.008 |
| Total embryos utilized as % of fertilization | 77/263 (29.3%) | 116/348 (33.3%) | 0.293 |
| Average number of embryos transferred | 1.2 ± 0.37 | 1.1 ± 0.30 | 0.490 |
| Number of fresh transfers (SET, DET) | 25 (21, 4) | 31 (28, 3) | |
| Implantation per embryo transferred | 1/29 (3.4%) | 10/34 (29.4%) | 0.008 |
| Clinical pregnancy per transfer (fresh) | 1/25 (4.0%) | 8/31 (25.8%) | 0.034 |
| Clinical pregnancy per cleavage stage transfer | 1/7 (14.2%) | 2/5 (40.0%) | 0.523 |
| Clinical pregnancy per blastocyst stage transfer | 0/18 (0%) | 6/26 (23.0%) | 0.067 |
| Live birth/transfer | 0/25 (0%) | 6/31 (19.4%) | 0.028 |
| Number of frozen SET blastocyst transfers | 12 | 26 | |
| Clinical pregnancy per transfer (first FET) | 3/12 (25.0%) | 11/26 (42.3%) | 0.472 |
| Live birth/transfer | 1/12 (8.3%) | 10/26 (38.4%) * | 0.121 |
| Merged clinical pregnancy rate (fresh and first FET) | 4/37 (10.8%) | 19/57 (33.3%) | 0.015 |
| Live birth/transfer | 1/37 (2.7%) | 16/57 (28.0%) | 0.002 |
Values are mean ± SD for continuous data or % (n/total) for categorical data. P < 0.05 was considered to be statistically significant
FET, frozen embryo transfer; SET, single embryo transfer; DET, double embryo transfer. *the final clinical pregnancy for PIEZO-ICSI is still ongoing at >20 weeks at time of manuscript submission
Embryo utilization
When embryo utilization rate was compared, as a percentage of oocytes fertilized, there was no significant difference between the two groups (33.3% PIEZO-ICSI vs 29.3% conventional ICSI, P = 0.293); however, the total number of embryos utilized per cycle (transferred and/or frozen) was significantly increased in the PIEZO-ICSI cycles when compared to the conventional ICSI cycles (1.6 ± 1.7 vs 1.1 ± 1.3, P = 0.008). Therefore, the increased rates of fertilization seen with PIEZO-ICSI led to an increased number of embryos available for transfer and cryopreservation in each cycle.
Pregnancy rates
PIEZO-ICSI significantly increased the clinical pregnancy rate per fresh transfer when compared to conventional ICSI (25.8% (8/31) vs 4.0% (1/25), P = 0.034). The one pregnancy in the conventional ICSI group resulted in miscarriage before 20 weeks gestation, whereas there were six live births (all singleton) and two miscarriages from the PIEZO-ICSI group with no recorded abnormalities in the babies born. Analysis of the first frozen embryo transfer (FET) post-cycle for these patients identified a small but not significant improvement in clinical pregnancy rates between conventional ICSI (3/12 (25.0%), resulting in one live birth and two miscarriages) and PIEZO-ICSI (11/26 (42.3%) with ten live births and one ongoing pregnancy at > 20 weeks of gestation) with no recorded abnormalities in the babies born. Analysis of the merged clinical pregnancy rate incorporating all pregnancies from the fresh cycles and the first frozen embryo transfer showed a significant improvement with PIEZO-ICSI when compared to conventional ICSI (19/57 (33.3%) vs 4/37 (10.8%), P = 0.015).
Effects of maternal age
When the data were split into low maternal age (< 38 years at time of PIEZO-ICSI treatment) and high maternal age (≥ 38 years at the time of PIEZO-ICSI treatment), the significant improvements in both fertilization and degeneration were still evident across both age groups when PIEZO-ICSI was used (P < 0.05) (Table 4). The high maternal age group also showed significant improvement in the total number of embryos utilized per cycle (1.7 ± 1.9 PIEZO-ICSI vs 1.1 ± 1.2 conventional ICSI, P = 0.050); however, this was not significant in women aged < 38 years. Clinical pregnancy data was not analyzed due to insufficient power in the subgroup analysis.
Table 4.
Comparison of conventional ICSI and PIEZO-ICSI for fertilization, oocyte degeneration, and embryo utilization in patients with respect to % of fertilization in their previous conventional ICSI cycle based on maternal age (<38 years compared with ≥38 years)
| Parameter | Maternal age <38 years at time of PIEZO-ICSI cycle (n=33) | Maternal age ≥38 years at time of PIEZO-ICSI cycle (n=39) | ||||
|---|---|---|---|---|---|---|
| ICSI | PIEZO-ICSI | P | ICSI | PIEZO-ICSI | P | |
| Total number oocytes injected | 290 | 276 | 291 | 281 | ||
| Average number of oocytes injected | 8.8 ± 3.6 | 8.4 ± 4.2 | 0.557 | 7.5 ± 4.1 | 7.2 ± 4.7 | 0.652 |
| Total oocytes fertilized | 43.4% | 62.0% | 0.004 | 47.1% | 61.9% | 0.009 |
| Total oocytes abnormally fertilized | 5.5% | 6.9% | 0.717 | 5.5% | 4.2% | 0.133 |
| Total oocytes degenerate | 17.2% | 8.0% | 0.004 | 19.2% | 7.4% | 0.008 |
| Number of embryos utilized per cycle | 1.0 ± 1.4 | 1.6 ± 1.5 | 0.094 | 1.1 ± 1.2 | 1.7 ± 1.9 | 0.050 |
| Total embryos utilized as % of fertilization | 34/126 (27.0%) | 51/171 (29.8%) | 0.606 | 43/137 (31.4%) | 65/177 (37.3%) | 0.340 |
Values are mean ± SD for continuous data or % (n/total) for categorical data. P < 0.05 was considered to be statistically significant
Patients with low fertilization in their previous conventional ICSI cycle
To further investigate patients that would benefit the most from the use of PIEZO-ICSI, the cohort was split to investigate those who had exhibited low fertilization in their previous conventional ICSI cycle. For the purpose of this study, low fertilization was set at < 50% fertilization in their conventional ICSI cycle (n = 43). Patients with low fertilization in their conventional ICSI cycle had significantly increased fertilization rates in their subsequent PIEZO-ICSI cycle (58.6% vs 30.1%, P < 0.0001) and decreased degeneration (8.4% vs 21.6%, P = 0.005) (Table 5). Abnormal fertilization (1PN, ≥ 3PN) was determined to decrease with PIEZO-ICSI; however, this failed to meet significance (5.7% vs 6.5%, P = 0.070). The number of embryos utilized per cycle was significantly increased in the poor fertilization group, with around one extra embryo available for use per cycle when PIEZO-ICSI was used (1.6 ± 1.4 PIEZO-ICSI vs 0.7 ± 0.8 conventional ICSI, P = 0.0002). PIEZO-ICSI was not associated with significant improvements in any of the laboratory parameters investigated in patients who had previously exhibited greater than 50% fertilization with conventional ICSI (Table 5). Clinical pregnancy data was not analyzed due to insufficient power in the subgroup analysis.
Table 5.
Comparison of conventional ICSI and PIEZO-ICSI for fertilization, oocyte degeneration, and embryo utilization in patients with respect to % of fertilization in their previous conventional ICSI cycle
| Parameter | Patients with ≥50% fertilization in conventional ICSI cycle (n=29) | Patients with <50% fertilization in conventional ICSI cycle (n=43) | ||||
|---|---|---|---|---|---|---|
| ICSI | PIEZO-ICSI | P | ICSI | PIEZO-ICSI | P | |
| Total number oocytes injected | 245 | 224 | 336 | 333 | ||
| Number of oocytes injected | 8.5 ± 3.9 | 7.7 ± 5.6 | 0.390 | 7.8 ± 3.9 | 7.7 ± 3.6 | 0.890 |
| Total oocytes fertilized | 66.1% | 67.0% | 0.280 | 30.1% | 58.6% | <0.0001 |
| Total oocytes abnormally fertilized | 4.1% | 5.4% | 0.756 | 6.5% | 5.7% | 0.070 |
| Total oocytes degenerate | 13.8% | 6.7% | 0.119 | 21.4% | 8.4% | 0.005 |
| Number of embryos utilized per cycle | 1.7 ± 1.7 | 1.6 ± 2.1 | 0.772 | 0.7 ± 0.8 | 1.6 ± 1.4 | 0.0002 |
| Total embryos utilized as % of fertilization | 48/162 (29.6%) | 46/150 (30.7%) | 0.902 | 29/101 (28.7%) | 70/198 (35.9%) | 0.300 |
Values are mean ± SD for continuous data or % (n/total) for categorical data. P < 0.05 was considered to be statistically significant
Patients with low utilization in their previous conventional ICSI cycle
Subgroup analysis was also performed on patients who had experienced poor utilization in their previous conventional ICSI cycle. In this study, poor utilization was set at < 20% of embryos being transferred or frozen from the cycle (n = 27). Patients who had experienced total failed fertilization in the conventional ICSI cycle were excluded from the analysis (n = 5). Patients with < 20% utilization in their conventional ICSI cycle had significantly increased fertilization rates in their subsequent PIEZO-ICSI cycle (63.3% vs 46.3%, P = 0.012) and decreased degeneration (2.9% vs 14.8%, P = 0.002) (Table 6). Similar significant improvements were observed for fertilization and degeneration in the PIEZO-ICSI cycles of the ≥ 20% utilization group (Table 6). Interestingly patients with < 20% utilization from conventional ICSI showed highly significant increases in the number of embryos utilized per cycle, with one extra embryo available for use per cycle when PIEZO-ICSI was used (1.3 ± 1.6 PIEZO-ICSI vs 0.2 ± 0.4 conventional ICSI, P = 0.0005). This was also reflected in improvements in the total number of embryos utilized as a percentage of the number fertilized when PIEZO-ICSI was used (25.2% PIEZO-ICSI vs 4.9% conventional ICSI, P < 0.0001). No significant improvements in utilization were observed in patients who had previously exhibited greater than 20% utilization with conventional ICSI.
Table 6.
Comparison of conventional ICSI and PIEZO-ICSI for fertilization, oocyte degeneration, and embryo utilization in patients with respect to % utilization in the previous conventional ICSI cycle
| Parameter | Patients with ≥20% utilization in conventional ICSI cycle (n=40) | Patients with <20% utilization in conventional ICSI cycle (n= 27) | ||||
|---|---|---|---|---|---|---|
| ICSI | PIEZO-ICSI | P | ICSI | PIEZO-ICSI | P | |
| Total number oocytes injected | 340 | 310 | 221 | 218 | ||
| Number of oocytes injected | 8.5 ± 4.3 | 7.8 ± 4.8 | 0.879 | 8.2 ± 3.1 | 8.1 ± 4.3 | 0.237 |
| Total oocytes fertilized | 47.2% | 59.5% | 0.006 | 46.3% | 63.3% | 0.012 |
| Total oocytes abnormally fertilized | 4.1% | 2.6% | 0.239 | 7.8% | 8.0% | 0.609 |
| Total oocytes degenerate | 18.4% | 10.6% | 0.024 | 14.8% | 2.9% | 0.003 |
| Number of embryos utilized per cycle | 1.8 ± 1.3 | 1.9 ± 1.8 | 0.837 | 0.2 ± 0.4 | 1.3 ± 1.6 | 0.0005 |
| Total embryos utilized as % of fertilization |
72/160 (45.0%) |
75/197 (38.0%) |
0.196 |
5/103 (4.9%) |
36/143 (25.2%) |
<0.0001 |
Values are mean ± SD for continuous data or % (n/total) for categorical data. P < 0.05 was considered to be statistically significant. Patients with failed fertilization were not included in the analysis (n=5)
Discussion
This retrospective paired within-patient cohort study suggests that PIEZO-ICSI can significantly increase fertilization rates and decrease oocyte degeneration in patients who had previous poor outcomes with conventional ICSI. The number of embryos available for use per cycle was also increased with the use of PIEZO-ICSI. Further significant improvements in fertilization, degeneration, and the number of embryos available per cycle can be achieved with PIEZO-ICSI in patients with advanced maternal age (≥38 years at time of PIEZO-ICSI treatment) and in patients who exhibited poor fertilization (<50%) coupled with increased rates of oocyte degeneration from conventional ICSI.
The results from this study are consistent with other PIEZO-ICSI studies and support the hypothesis that the PIEZO-assisted penetration of the oolemma may lead to superior oocyte activation [6, 21]. Although the actual fertilization rates seen in this study are lower than those reported elsewhere, this reflects the poor prognosis patient cohort selected for analysis in this study. The levels of improvement in fertilization rates achieved in the current study are consistent with previous reports [6, 7, 15] even though these previous studies did not focus on patients with previous poor outcomes with conventional ICSI. The gentle mechanism by which the blunt injection pipette penetrates through the zona pellucida in the PIEZO-ICSI procedure may lead to the decreased degeneration rates observed in this study, due to decreased pressure on the oocyte plasma membrane [7, 8]. This is likely an important factor for poor prognosis patients, who have high degeneration rates in conventional ICSI cycles, as defined by the benchmark of ≤ 5% published by the Vienna consensus [22]. Oocyte degeneration following ICSI is likely a function of the inherent oocyte quality [23], and the high degeneration rate following conventional ICSI suggests that these patients may have oocytes with more fragile plasma membranes that may likely benefit from the gentle nature of PIEZO-ICSI insemination. With average oocyte degeneration rates ranging from 5 to 19% of all oocytes injected with conventional ICSI [3], PIEZO-ICSI has the potential to help increase oocyte survival in thousands of patients worldwide. Since the PIEZO-ICSI cycles were performed up to 2 years later than the conventional ICSI cycles, these improvements in fertilization and degeneration are even more notable, as it would generally be expected that poorer outcomes are associated with the increasing maternal age [24–26].
The increased fertilization rate evident with PIEZO-ICSI usage directly resulted in more embryos being available for transfer or cryopreservation per cycle, in agreement with a previous study [15]. Similarly, significant increases in the number of embryos vitrified per oocyte fertilized have been reported [7]. The current results indicate a potential clinical benefit in the use of this technology, with an even more pronounced effect in patients who had experienced either low fertilization (< 50%) or poor embryo utilization (< 20%) from their conventional ICSI cycle, that resulted in these patients having on average one extra embryo available for transfer or cryopreservation per PIEZO-ICSI cycle. These groups with a history of low fertilization or poor embryo utilization were also found to have the greatest overall improvements in fertilization rate and oocyte degeneration rate. There was also a decrease, although not significant, in the rates of oocytes that were abnormally fertilized in the low fertilization patient group, similar to those previously reported [8, 15].
When patients were split into < 38 years and ≥ 38 years of age at the time of PIEZO-ICSI, there were significant improvements in fertilization and degeneration rates with PIEZO-ICSI in both maternal age groups. The improvements seen in the older patients were similar to the younger patient group which does differ to previous reports where significant improvements were observed only in patients of advanced age (< 35 years of age or ≥ 38 years of age) [8, 15]. Results from the current study suggest that poor prognosis patients, irrespective of age, may benefit from the use of PIEZO-ICSI. Notably, in the current study in women ≥ 38 years of age, there was also an increased embryo utilization rate with PIEZO-ICSI, with an average of one extra embryo being available for transfer or cryopreservation per cycle. This result is clinically beneficial outcome for women of advanced maternal age, due to their likelihood of reduced ovarian reserve and higher rates of chromosomal abnormalities and aneuploidy [27, 28].
The benefits of PIEZO-ICSI on pregnancy rates are not well documented in the literature with increases in fertilization rate being the focus of most previous publications [7, 14]. The present study did show a significant improvement in clinical pregnancy rate between conventional ICSI and PIEZO-ICSI in fresh transfer cycles and an increase (although not statistically significant) from the subsequent frozen embryo transfer (FET). Importantly, significant improvement was seen in the merged clinical pregnancy rates (from both the fresh transfers plus the first FET) and the number of live births per transfer performed from the PIEZO-ICSI cycles. Although the sample size in this study is small which limits the statistical significance in some parameters, these results do suggest a trend towards an improvement in clinical pregnancy rates following PIEZO-ICSI. It however must be noted that these patients were selected for PIEZO-ICSI based on poor outcomes in their previous conventional ICSI cycle, which is reflected in the very low pregnancy rates using conventional ICSI, and further investigation into the direct impact of PIEZO-ICSI on pregnancy rates is warranted which should include larger prospective studies. In addition, it must be noted however that in both the conventional ICSI and PIEZO-ICSI cycles, there were a small number of day 3 embryo transfers. These transfers may have led to an overestimation of the embryo utilization rates in both treatment types and cannot be discounted. Despite these factors, the promising pregnancy rates for PIEZO-ICSI compared with conventional ICSI in this patient cohort suggest a positive effect from the use of PIEZO-ICSI in patients with poor outcomes from conventional ICSI. Ongoing analysis of live births and further frozen embryo transfers is again required as it is anticipated that the cumulative pregnancy rate would be increased in the PIEZO-ICSI cycles, due to more embryos being available for use per cycle. To date, this study has resulted in 16 live births from the use of PIEZO-ICSI, with no reported fetal abnormalities or complications, providing supporting evidence that this technology, in combination with the alternative operating fluid (perfluoro-n-octane), is a safe and effective alternative to conventional ICSI.
While a key strength of the current study was the ability to analyze conventional ICSI and PIEZO-ICSI cycle outcomes from the same patients, there was up to a 2-year time difference between these cycles, and as patient age, BMI, lifestyle, and treatment cycle change over this timeframe, we cannot discount that some of these may have contributed to the differences in results that were observed. To counter this however, patients were always older in their subsequent PIEZO-ICSI cycle, so if anything, their fertility status would likely have decreased, yet despite this, better outcomes were identified in their PIEZO-ICSI cycles. In summary, the findings of the current study demonstrate that PIEZO-ICSI improved fertilization, degeneration, and utilization rates in this patient cohort, with previous poor outcomes from conventional ICSI. This data is supported by previous reports who have assessed that PIEZO-ICSI can improve the survival and fertilization results compared to conventional ICSI [6–8, 14–16, 29]. This study also highlights the significant improvements in utilization and pregnancy rate per patient, notably in a poor prognosis patient cohort. Future prospective studies are needed in the form of sibling split or randomized control trial (RCT) to determine the efficacy of PIEZO-ICSI and to identify specific patient subgroups in which PIEZO-ICSI would be of greatest benefit.
Conclusion
Patients with previous poor outcomes from conventional ICSI demonstrated improvements in clinical embryology parameters when PIEZO-ICSI was used. Fertilization and degeneration rates, as well as embryo utilization number, were all significantly improved with the use of PIEZO-ICSI. Notably, patients who had low (< 50%) fertilization or low utilization (< 20%) from conventional ICSI showed the greatest improvements in their outcome measures and hence may benefit most from the use of this technology. These benefits lie predominantly in increasing the fertilization rate and decreasing the degeneration rate in the oocytes of these patients, rather than overcoming any kind of quality deficit in their resulting embryos. Assessment of clinical pregnancy and live birth outcomes is ongoing. In addition, a regression to the mean effect cannot be excluded, and further RCT studies are warranted to confirm findings of this initial study into the efficacy of PIEZO-ICSI.
Declarations
Conflict of interest
The authors declare no competing interests.
Footnotes
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