Abstract
Background
Sperm immobilization is an important step in intracytoplasmic sperm injection (ICSI) as it is necessary for oocyte activation by the spermatozoa. Several techniques are available for sperm immobilization. However, the effects of sperm mobilization techniques on intracytoplasmic sperm injection outcomes have not been established as most of the reports of studies on sperm immobilization techniques are inconsistent. This study aimed to evaluate a mild aggressive mechanical sperm immobilization technique: micro-rubbing, and investigate the effectiveness of micro-rubbing sperm immobilization in ICSI.
Methods
Matched retrospective cohort study of patients who underwent intracytoplasmic sperm injection between April 2018 and June 2023 were enrolled. Non-preimplantation preimplantation genetic testing (non-PGT) and preimplantation genetic testing for aneuploidy (PGT-A) cycles were categorised into two groups based on sperm immobilization method: micro-rubbing and single-touch. Each couple was matched with separate control patients treated by the same operator for ICSI and oocyte denudation, maternal age, duration of infertility, primary or secondary infertility, sperm source, embryo condition, and endometrial thickness on the day of embryo transfer. Main Outcome Measures included: fertilization, oocyte utilization, and blastocyst chromosomal aneuploidy rates.
Results
Overall, 2967 ICSI cycles were evaluated, of which 330 and 318 cycles performed using micro-rubbing sperm immobilization in non-PGT and PGT-A groups, respectively. Compared with the ICSI cycles performed using single-touch sperm immobilization, micro-rubbing sperm immobilization cycles have significantly higher fertilization (non-PGT: 85.28% vs. 76.06%, P < 0.001; PGT-A: 85.26% vs. 75.45%, P < 0.001), day-3 embryo utilization per 2 pronucleus, and embryo utilization rate on day 3 per oocyte injected (non-PGT: 64.13% vs. 50.24%, P < 0.001; PGT-A:63.43% vs. 48.79%, P < 0.001). There were no statistically significant differences in embryo cleavage, top-quality embryos on day 3, blastocyst formation, blastocyst chromosomal aneuploidy, implantation, pregnancy, live birth, or miscarriage rates between both groups.
Conclusions
Micro-rubbing sperm immobilization appears to be effective, with a statistically significant improvement in fertilization and embryo utilization rate on day 3 per oocyte injected. However, it does not seem to affect clinical pregnancy or live birth rates per transfer.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13048-025-01829-6.
Keywords: ICSI, Sperm immobilization, Micro-rubbing, Fertilization, Oocyte utilization
Background
Intracytoplasmic sperm injection (ICSI) is one of the main techniques used in assisted reproductive technology, especially for the treatment of severe male factor infertility [1]. Although the initial indication for ICSI was low sperm count or failed fertilization, the application of ICSI has expanded because it has a higher fertilization rate [2]. The use of ICSI for in vitro fertilization (IVF) cycles has increased to approximately 70% worldwide [3, 4].
Sperm immobilization before ICSI is important for successful fertilization because it plays a key role in oocyte activation by the spermatozoa [5–7]. The disruption of the sperm tail is thought to damage the cell membrane and trigger subsequent physiological and biochemical reactions that may facilitate the decondensation of the sperm nucleus and oocyte activation [8, 9]. Various immobilization techniques have been used to induce sperm membrane permeabilization. Traditional sperm immobilization techniques, primarily a single touch of the midpiece, have been widely used since the early days of ICSI [10, 11]. More aggressive mechanical methods, including triple touching the tail in the midpiece [12], permanently crimping the tail in the midpiece [13], cutting the tail below the midpiece [14], and dissecting the tail at the tip [15], as well as lasers [8] or piezo-pulses [16] can be used to immobilize sperm tails before ICSI.
More aggressive sperm immobilization methods may lead to full sperm membrane rupture and sperm nucleus and centriole damage [12, 13]. The sperm centrosome is critical for fertilization and early human embryo development, as it acts as a microtubule-organizing center, facilitating mitotic spindle assembly, chromosomal segregation, and zygotic division while influencing other aspects of cytoskeletal organization in early-stage embryos [17]. However, reports on whether different sperm immobilization techniques affect ICSI outcomes have been inconsistent. Some studies have found that, compared with the standard single-touch method, higher fertilization rates for ejaculated spermatozoa were achieved via the more aggressive mechanical [13, 14] or piezo-pulse induced [18] sperm immobilization. Other studies have reported no differences when the tail of the spermatozoon was cut at different places [19] or when laser-induced sperm immobilization was performed [8, 20]. Velaers et al. demonstrated that triple-touch sperm immobilization decreased the number of high-quality embryos on day 3 compared with single-touch sperm immobilization [12].
Although several studies have evaluated different techniques for sperm immobilization before ICSI, the location and extent of sperm membrane damage are not fully understood. Additionally, the majority of the aforementioned studies were published more than 10 years ago and were fraught with potential bias and confounders. Factors such as the ICSI operator, oocyte denudation, maternal and paternal age, sperm source, and oocyte maturity were not matched in these studies, which could have led to inaccurate conclusions. Few studies have focused on whether aggressive sperm immobilization causes chromosomal aneuploidy in embryos. This study aimed to investigate the effectiveness of micro-rubbing sperm immobilization in ICSI.
Materials and methods
Patients and study design criteria
We performed a matched retrospective cohort study of all ICSI cycles at the Center for Reproductive Medicine, Guangzhou Women and Children’s Medical Center, between April 2018 and June 2023. The study was approved by the Independent Ethics Committee of Guangzhou Women and Children’s Hospital. We included 1064 ICSI cycles. Only preimplantation genetic testing for aneuploidy (PGT-A) cycles were included in this study. The exclusion criteria were as follows: (1) half-ICSI or rescue ICSI (2), in vitro maturation (3), donor oocytes or sperm (4), frozen sperm or oocytes, and (5) testicular or epididymal sperm. All ICSI procedures were performed by the same trained embryologist with >15 years of experience in performing ICSI. Among the included cycles, the non-preimplantation genetic testing (PGT) and PGT-A cycles were categorized into two groups based on the sperm immobilization method that was used: the micro-rubbing (MRB-ICSI) and single touch (S-ICSI) groups. The study periods for each technique were as follows: S-ICSI was implemented from April 2018 to December 2019, while MRB-ICSI was used from January 2020 to June 2023.
Based on a systematic review of the literature and our clinical experience, the two matched groups were selected according to the following criteria: maternal age; same operator for ICSI and oocyte denudation; history of pregnancy (primary or secondary infertility); duration of infertility; sperm source; paternal age; maternal body mass index (BMI); cause of infertility; the type of protocol used for controlled ovarian hyperstimulation; embryo condition (embryo stage, number, and quality); and endometrial thickness on the day of embryo transfer.
Sperm and oocyte preparation
Ejaculated semen samples were processed using two-layer discontinuous density gradient centrifugation (Vitrolife, Gothenburg, Sweden), with a centrifugal force of 300 ×g for 20 min. The supernatant was removed, and the pellet was collected for further use. Only sperm showing progressive motility are used for ICSI. Oocytes were denuded from cumulus-oocyte complexes by needle dissection and pipetted into a 20 IU/ml hyaluronidase solution (Sage, Trumbull, USA). The oocytes were then transferred to a culture medium. Only oocytes in the MII stage were selected for microinjection.
Sperm immobilization
In the single-touch group, immobilization was achieved by pressing the tail of the spermatozoon once against the bottom of the dish using an injection pipette and then quickly withdrawing this pipette until a clear bend in the middle of the flagellum was observed (Supplementary Video 1). In the micro-rubbing group, the spermatozoon was initially immobilized by gently pressing the end of the principal piece to ensure immobility. Subsequently, the middle of the principal piece was triple touched (withdrawn-push-withdrawn), and the region after the principal piece was cut off after the final state (Fig. 1, Supplementary Video 2). It should be noted that the middle of the principal piece was compressed no more than thrice. If the withdrawal and pushing forces are not perpendicular to the spermatozoon, ensuring that the injection pipette moves in the opposite direction to the head of the spermatozoon during withdrawal and pushing is necessary.
Fig. 1.
MRB-ICSI. a The tip of the injection micropipette is gently pressing the end of the principal piece. b The spermatozoon is withdrawn against the lid of the dish and quickly pushed forward, and then push withdrawn back. c The morphology of spermatozoon post immobilization
Sperm membrane disruption test using eosin
A solution of 0.25% eosin Y (Sigma, Missouri, USA) in EBSS was prepared prior to experiment. A small drop of eosin staining solution and sperm suspension was placed to create two elongated droplets on the ICSI dish, covered with paraffin oil. ICSI immobilization was performed in the sperm suspension droplet. After immobilization, the sperm were transferred into the eosin droplet for staining. Spermatozoa that stained red or pink were considered to have plasma membrane damage [7, 21].
ICSI
The micromanipulation procedure was performed using commercially produced injection micropipettes (inside diameter 5 μm, Sunlight, Florida, USA) and combined with matching holding micropipettes (Origio, Malov, Denmark). Spermatozoa with the best morphology were selected and placed in 2% polyvinylpyrrolidone (Vitrolife, Gothenburg, Sweden) and immobilized for subsequent injection. The injection micropipette was then positioned in the droplet of buffer culture medium containing the oocyte. The fixation micropipette was lowered, and the oocyte was gently manipulated to position the polar body at the 11 o’clock position, then fix the oocyte. Push the sperm to the tip of the injection micropipette, and insert the injection micropipette into the oocyte at the 3 o’clock position through the zona pellucida. As the injection micropipette pierces the oolemma near the center of the oocyte, slightly aspirate some cytoplasm. When the cytoplasm starts to be rapidly aspirated into the injection micropipette (indicating that the oolemma has been penetrated), immediately stop aspirating and then inject the sperm along with the aspirated cytoplasm into the cytoplasm. The sperm is injected into the oocyte head-first. Once the sperm head enters the cytoplasm, slowly withdraw the injection micropipette. During the withdrawal, keep the fixation micropipette in its original position. Throughout the ICSI procedure, the droplet temperature is maintained at 37 °C to mimic physiological conditions and prevent any thermal stress to the oocytes or sperm. Micromanipulation was performed using an inverted microscope with modulated contrast at 200 × magnification.
Fertilization assessment and embryo culture
The fertilization was assessed 16 to 18 h after the ICSI procedure by observing the presence of two pronuclei and two polar bodies. Fertilized oocytes were individually cultured in 35 µL droplets of G-1 PLUS medium (Vitrolife, Gothenburg, Sweden) until Day 3. Subsequently, they were transferred to G-2 PLUS culture medium (Vitrolife, Gothenburg, Sweden), which is designed to support blastocyst development for culture until Day 6. All embryos were cultured under mineral oil (Vitrolife, Gothenburg, Sweden) at 37 °C in 6% CO2, 5% O2, 89% N2 using conventional incubators (Labotect, Labor-Technik-Göttingen, Germany).
Day 3 cleavage stage embryos were graded based on cell number, fragmentation level, evenness of cellular division, and the presence of anomalies such as vacuoles and multinucleation. Top embryos on day 3 were defined as having 7 to 9 cells, ≤ 10% fragmentation, evenly sized blastomeres, and no vacuolization. Usable day-3 embryos were defined as those with 2 PN origins, more than five cells, and fragmentation ≤ 50% on day 3. At the blastocyst stage, embryo quality was evaluated on Day 5 and 6 according to the Gardner classification, which considers expansion grade as well as the inner cell mass (ICM) and trophectoderm (TE) [22]. Usable blastocysts were defined as those reaching achieving a blastocyst stage of ≥ 3, with concurrent development of both the ICM and TE, excluding those graded as C.
Blastocyst biopsy and genetic testing
According to the Gardner grading system, artificial shrinkage of usable blastocysts was performed by applying a laser pulse with a non- contact 1.48- µm diode laser (RI, Falmouth, UK) to the zona pellucida (ZP) at the junction between trophectoderm cells, away from the ICM. Five to ten trophectoderm cells were aspirated using a biopsy pipette with gentle suction. Biopsy cells were detached using a combination of intercellular laser pulses and mechanical flicking.
PGT-A was performed using next-generation sequencing (NGS) protocol. Briefly, A multiple annealing and loop-based amplification cycle (MALBAC)-based single-cell whole-genome amplification (WGA) kit (Yikon Genomics Ltd, Suzhou, China) was used to amplify DNA from trophectoderm (TE) cells, following the manufacturer’s instructions. To analyze the ploidy status of the blastocysts, the amplified TE DNA was sequenced using a NextSeq 550 sequencer (Illumina, Inc., San Diego, USA), with a single-ended read length of 55 bp. Approximately two million raw reads were generated per TE sample. Genome-wide copy number variations (CNVs) were analyzed to determine the ploidy status of each embryo. Embryos with less than 20% aneuploidy in the TE sample were classified as euploid, those exceeding 80% aneuploidy were classified as aneuploid, and those between 20% and 80% were described as mosaic. Mosaic embryos were not transferred.
Embryo transfer
Endometrial preparation was achieved through either a natural cycle (NC) or hormone replacement treatment (HRT). In NC, transvaginal ultrasonography was used to monitor follicular growth and endometrial thickness. If endometrial thickness reached ≥ 8 mm, the cycle was considered suitable for transfer. In the HRT cycle, oral estrogen (4–6 mg/d) was initiated on the third day of the menstrual cycle. When the endometrial thickness reached ≥ 8 mm, progesterone injections (60 mg/d) were administered before transfer. Cleavage-stage embryos or blastocysts were transferred under ultrasound guidance 3 or 5 days after progesterone supplementation in HRT cycle. In frozen embryo transfer (FET) cycles, warming protocols were conducted following traditional methods according to the instructions of the Vit Kit (Kitazato Biopharma, Japan). Embryos were gradually warmed to 37 °C and cultured for 2 h in the blastocyst medium after the warming procedure and before transfer.
Outcome measurements
The primary outcome measure was the post-injection fertilization rate. The rates were calculated according to the Vienna consensus [23]. The fertilization rate was calculated as the number of oocytes with 2PN and 2 PB divided by the number of MII oocytes injected. The embryo utilization rate on day 3 per oocyte injected was calculated as the number of usable day-3 embryos divided by the number of oocytes with 2PN and 2 PB. The top embryos rate on day 3 was calculated as the number of top embryos on day 3 divided by the number of oocytes with 2PN and 2 PB. The blastocyst utilization rate was calculated as the number of usable blastocysts divided by the number of oocytes with 2PN and 2 PB. The implantation rate was calculated as the number of gestational sacs per transferred embryo. Clinical pregnancy was defined as the detection of a gestational sac with fetal heart pulsations on ultrasonography at 4–5 weeks post-transfer. A live birth was defined as the delivery of a viable infant after 24 weeks of gestation. Miscarriage was defined as the spontaneous loss of pregnancy before 20 weeks of gestation.
Statistical analysis
The SPSS Statistics (Version 25.0, IBM Corp., Armonk, NY, USA) and R software packages (Version 3.3 http://www.R-project.org, the R Foundation) were used for the statistical analyses. Continuous variables are presented as the mean ± standard deviation (SD), and groups were compared using the independent samples t-test. Categorical variables were described as the number of cases and percentages (n [%]), and comparisons were made using the Chi-square or Fisher’s exact test, as appropriate. Significance was set at P < 0.05.
Propensity score matching (PSM) was used to reduce potential confounding between the laser S-ICSI and MRB-ICSI groups [24, 25]. All baseline factors were included in the propensity score, including maternal age; same operator for ICSI and oocyte denudation; history of pregnancy (primary or secondary infertility); duration of infertility; sperm source; paternal age; maternal body mass index (BMI); cause of infertility; the type of protocol used for controlled ovarian hyperstimulation; embryo condition (embryo stage, number, and quality); and endometrial thickness on the day of embryo transfer. There were no significant differences in outcome variables. Using the estimated propensity scores as weights, an inverse probability weighting (IPW) model was used to generate a weighted cohort. The probability of exposure for each patient was estimated using logistic regression. The non-PGT groups were matched in a 1:1 ratio and PGT-A groups were matched in a 1:3 ratio with a caliper width of 0.05. Standardized mean differences < 0.1 were considered balanced.
Results
Overall, 1005 ICSI cycles, comprising 648 non-PGT and 357 PGT-A cycles, were performed from April 2018 to June 2023 by a trained embryologist with > 15 years of ICSI experience. The non-PGT cycles included 318 S-ICSI cycles with 264 patients and 330 MRB-ICSI cycles with 279 patients, while the PGT-A cycles included 39 S-ICSI cycles with 34 patients and 318 MRB-ICSI cycles with 245 patients (Fig. 2).
Fig. 2.
Trial flow chart
Embryological and clinical outcomes of S-ICSI and MRB-ICSI groups in non-PGT cycles
The cycle characteristics of the S-ICSI and MRB-ICSI groups are presented in Table 1. Before matching, micro-rubbing sperm immobilization resulted in significantly higher rates of fertilization, day-3 embryo utilization per 2 PN, day-3 embryo utilization per oocyte injected, top-quality embryos on day 3, blastocyst development, and blastocyst utilization compared to single-touch sperm immobilization in ICSI (all P < 0.05). There were no statistically significant differences in the cleavage, implantation, live births, or miscarriage rates between the two groups (Table 2).
Table 1.
Baseline characteristics of non-PGT groups
| Characteristic | Unmatched | Matched | ||||
|---|---|---|---|---|---|---|
| S-ICSI group (n = 264) |
MRB-ICSI group (n = 279) |
P value | S-ICSI group (n = 89) |
MRB-ICSI group (n = 94) |
P value | |
| Maternal age (y) | 34.86 ± 5.26 | 34.52 ± 5.06 | 0.393 | 34.43 ± 5.19 | 34.30 ± 5.21 | 0.858 |
| Maternal BMI (kg/m2) | 21.73 ± 3.15 | 21.84 ± 2.86 | 0.654 | 21.52 ± 3.15 | 21.87 ± 2.77 | 0.417 |
| Duration of infertility (y) | 3.85 ± 3.02 | 3.56 ± 2.71 | 0.193 | 3.24 ± 2.38 | 3.06 ± 2.32 | 0.617 |
| History of pregnancy | 0.341 | 0.774 | ||||
| Primary infertility | 47.17 (150) | 50.91 (168) | 46.39 (45) | 48.45 (47) | ||
| Secondary infertility | 52.83 (168) | 49.09 (162) | 53.61 (52) | 51.55 (50) | ||
| Cause of infertility | 0.365 | 0.584 | ||||
| Male factor | 72.33 (230) | 75.45 (249) | 82.47 (80) | 79.38 (77) | ||
| Other reason | 27.67 (88) | 24.55 (81) | 17.53 (17) | 20.62 (20) | ||
| Ovarian stimulation protocol | 0.283 | 0.468 | ||||
| Antagonist protocol | 50.31 (160) | 57.27 (189) | 42.27 (41) | 49.48 (48) | ||
| Long protocol | 23.27 (74) | 20.61 (68) | 26.80 (26) | 20.62 (20) | ||
| Luteal-phase ovulation | 9.12 (29) | 6.36 (21) | 10.31 (10) | 6.19 (6) | ||
| Other protocols | 17.30 (55) | 15.76 (52) | 20.62 (20) | 23.71 (23) | ||
| Paternal age (y) | 36.89 ± 6.40 | 36.64 ± 5.90 | 0.605 | 36.07 ± 6.20 | 36.08 ± 6.16 | 0.991 |
| Endometrial thickness (mm) | 10.29 ± 3.18 | 9.64 ± 3.28 | 0.009 | 9.21 ± 2.52 | 9.21 ± 2.43 | 0.814 |
| Day of embryos transfer | < 0.001 | 0.834 | ||||
| Day 3 | 75.40 (233) | 55.62 (203) | 54.05 (60) | 55.45 (61) | ||
| Day 5/6 | 24.60 (76) | 44.38 (162) | 45.95 (51) | 44.55 (49) | ||
| Number of embryos transferred (per ET cycles) | ||||||
| Day 3 | 2.00 ± 0.40 | 1.73 ± 0.47 | < 0.001 | 1.63 ± 0.45 | 1.61 ± 0.42 | 0.716 |
| Day 5/6 | 1.70 ± 0.46 | 1.30 ± 0.46 | < 0.001 | 1.41 ± 0.39 | 1.41 ± 0.35 | 0.891 |
Data are presented as mean ± standard deviation and n (%)
BMI body mass index, MII metaphase II, ET embryos transfer
Table 2.
Embryological and clinical outcomes of S-ICSI and MRB-ICSI groups in non-PGT cycles
| Unmatched | Matched | ||||||
|---|---|---|---|---|---|---|---|
| S-ICSI group (n = 318) |
MRB-ICSI group (n = 330) |
P value | S-ICSI group (n = 97) |
MRB-ICSI group (n = 97) |
P value | ||
| Oocytes injected | 2506 | 1979 | - | 848 | 591 | - | |
| Fertilization rate | 74.50 (1867/2506) | 83.27 (1648/1979) | < 0.001 | 76.06 (645/848) | 85.28 (504/591) | < 0.001 | |
| Cleavage rate | 98.82 (1845/1867) | 98.97 (1631/1648) | 0.678 | 98.29 (634/645) | 99.21 (500/504) | 0.201 | |
| Embryo utilization rate on day 3/2 PN | 68.24 (1274/1867) | 75.97 (1252/1648) | < 0.001 | 66.05 (426/645) | 75.20 (379/504) | 0.001 | |
| Embryo utilization rate on day 3/oocyte injected | 50.84 (1274/2506) | 63.26 (1252/1979) | < 0.001 | 50.24 (426/848) | 64.13 (379/591) | < 0.001 | |
| Top embryos rate on day 3 | 30.37 (567/1867) | 34.71 (572/1648) | 0.006 | 29.61 (191/645) | 34.33 (173/504) | 0.088 | |
| Blastocyst cultured | 857 | 1207 | - | 283 | 373 | - | |
| Blastocyst development rate | 52.39 (449/857) | 60.48 (730/1207) | < 0.001 | 58.66 (166/283) | 57.10 (213/373) | 0.690 | |
| Blastocyst utilization rate | 26.95 (231/857) | 39.69 (479/1207) | < 0.001 | 34.28 (97/283) | 37.53 (140/373) | 0.390 | |
| Number of embryos transferred | 594 | 562 | - | 170 | 167 | - | |
| Implantation rate | D3 ET | 34.62 (161/465) | 36.47 (128/351) | 0.586 | 35.71 (35/98) | 37.76 (37/98) | 0.767 |
| D5/6 ET | 43.41 (56/129) | 54.50 (115/211) | 0.047 | 41.67 (30/72) | 44.93 (31/69) | 0.696 | |
| Pregnancy rate | D3 ET | 50.21 (117/233) | 48.28 (98/203) | 0.686 | 45.00 (27/60) | 50.82 (31/61) | 0.522 |
| D5/6 ET | 51.32 (39/76) | 56.79 (92/162) | 0.429 | 50.98 (26/51) | 55.10 (27/49) | 0.680 | |
| Live birth rate | D3 ET | 41.20 (96/233) | 40.89 (83/203) | 0.947 | 36.67 (22/60) | 42.62 (26/61) | 0.503 |
| D5/6 ET | 43.42 (33/76) | 51.85 (84/162) | 0.225 | 43.14 (22/51) | 48.98 (24/49) | 0.558 | |
| Miscarriage rate | D3 ET | 17.09 (20/117) | 15.31 (15/98) | 0.724 | 18.52 (5/27) | 16.13 (5/31) | 1.000 |
| D5/6 ET |
15.38 (6/39) |
10.87 (10/92) | 0.561 | 15.38 (4/26) | 11.11 (3/27) | 0.704 | |
Data are presented as n (%)
PN stands for pronuclei, which are the nuclei of the sperm and egg that form after fertilization, ET embryos transfer
To reduce potential bias, 97 S-ICSI cycles with 89 patients were matched with 97 MRB-ICSI cycles with 94 patients in the non-PGT cycles. There were no significant differences between the two groups in terms of maternal age, maternal BMI, duration of infertility, paternal age, and MII rate. Table 2 presents the main reproductive outcomes in the two groups. The rates of oocyte fertilization (85.28% vs. 76.06%, P < 0.001) was significantly higher in the MRB-ICSI than in the S-ICSI group. Moreover, the embryo utilization rate on day-3 per oocyte injected improved greatly in the MRB-ICSI group compared to that in the S-ICSI group (64.13% vs. 50.24%, P < 0.001). There were no statistically significant differences in the rates of cleavage, top-quality embryos on day 3, blastocyst development, blastocyst utilization, implantation, pregnancy, live birth, or miscarriage between the two groups.
Embryological and clinical outcomes of S-ICSI and MRB-ICSI groups in PGT-A cycles
To further analyze whether micro-rubbing sperm immobilization increased the incidence of chromosomal aneuploidy in embryos, we compared the main embryological and clinical outcomes of the S-ICSI and MRB-ICSI groups in PGT-A cycles. The baseline patient characteristics are shown in Table 3. Before matching, between the MRB-ICSI and S-ICSI groups, there was no significant difference in blastocyst chromosomal aneuploidy rates, cleavage, top-quality embryos on day 3, blastocyst development, blastocyst utilization, and implantations, pregnancies, live births, and miscarriage (all P > 0.05). The rates of fertilization, day-3 embryo utilization per 2 PN, and day-3 embryo utilization per oocyte injected in the MRB-ICSI group were significantly higher than those in the S-ICSI group (Table 4).
Table 3.
Baseline characteristics of PGT-A groups
| Characteristic | Unmatched | Matched | ||||
|---|---|---|---|---|---|---|
| S-ICSI group (n = 34) |
MRB-ICSI group (n = 245) |
P value | S-ICSI group (n = 34) |
MRB-ICSI group (n = 104) |
P value | |
| Maternal age (y) | 37.61 ± 3.57 | 38.12 ± 3.72 | 0.396 | 37.61 ± 3.57 | 37.67 ± 3.90 | 0.834 |
| Maternal BMI (kg/m2) | 21.75 ± 2.73 | 22.18 ± 2.47 | 0.312 | 21.75 ± 2.73 | 21.95 ± 2.48 | 0.392 |
| Duration of infertility, y | 1.93 ± 2.70 | 2.66 ± 2.61 | 0.100 | 1.93 ± 2.70 | 1.96 ± 2.14 | 0.553 |
| History of pregnancy | 0.349 | 0.905 | ||||
| Primary infertility | 17.95 (7) | 12.58 (40) | 17.95 (7) | 18.80 (22) | ||
| Secondary infertility | 82.05 (32) | 87.42 (278) | 82.05 (32) | 81.20 (95) | ||
| Cause of infertility | 0.020 | 0.908 | ||||
| Male factor | 20.51 (8) | 39.62 (126) | 20.51 (8) | 19.66 (23) | ||
| Other reason | 79.49 (31) | 60.38 (192) | 79.49 (31) | 80.34 (94) | ||
| Ovarian stimulation protocol | 0.328 | 0.943 | ||||
| Antagonist protocol | 56.41 (22) | 47.17 (150) | 56.41 (22) | 52.99 (62) | ||
| Long protocol | 23.08 (9) | 35.53 (113) | 23.08 (9) | 27.35 (32) | ||
| Luteal-phase ovulation | 10.26 (4) | 5.66 (18) | 10.26 (4) | 8.55 (10) | ||
| Other protocols | 10.26 (4) | 11.64 (37) | 10.26 (4) | 11.11 (13) | ||
| Paternal age (y) | 38.82 ± 6.89 | 39.37 ± 5.59 | 0.191 | 38.82 ± 6.89 | 38.62 ± 5.97 | 0.792 |
| Endometrial thickness, (mm) | 9.89 ± 3.26 | 9.63 ± 3.23 | 0.311 | 9.25 ± 2.11 | 9.24 ± 2.23 | 0.806 |
| Day of embryos transfer | - | - | ||||
| Day 3 | 0.00 (0) | 0.00 (0) | 0.00 (0) | 0.00 (0) | ||
| Day 5/6 | 100.00 (34) | 100.00 (145) | 100.00 (34) | 100.00 (71) | ||
| Number of embryos transferred (per ET cycles) | ||||||
| Day 5/6 | 1.00 ± 0.00 | 1.00 ± 0.00 | 1.000 | 1.00 ± 0.00 | 1.00 ± 0.00 | 1.000 |
Table 4.
Embryological and clinical outcomes of S-ICSI and MRB-ICSI groups in PGT-A cycles
| Unmatched | Matched | |||||
|---|---|---|---|---|---|---|
| S-ICSI group (n = 39) |
MRB-ICSI group (n = 318) |
P value | S-ICSI group (n = 39) |
MRB-ICSI group (n = 117) |
P value | |
| Oocytes injected | 330 | 1650 | - | 330 | 916 | - |
| Fertilization rate | 75.45 (249/330) | 82.18 (1356/1650) | 0.004 | 75.45 (249/330) | 85.26 (781/916) | < 0.001 |
| Cleavage rate | 99.20 (247/249) | 99.41 (1348/1356) | 0.659 | 99.20 (247/249) | 99.23 (775/781) | 1.000 |
| Embryo utilization rate on day 3/2 PN | 64.66 (161/249) | 73.01 (990/1356) | 0.007 | 64.66 (161/249) | 74.39 (581/781) | 0.003 |
| Embryo utilization rate on day 3/oocyte injected | 48.79 (161/330) | 60.00 (990/1650) | < 0.001 | 48.79 (161/330) | 63.43 (581/916) | < 0.001 |
| Top embryos rate on day 3 | 31.73 (79/249) | 34.51 (468/1356) | 0.394 | 31.73 (79/249) | 36.62 (286/781) | 0.160 |
| Blastocyst development rate | 59.84 (149/249) | 62.54 (848/1356) | 0.420 | 59.84 (149/249) | 64.40 (503/781) | 0.193 |
| Blastocyst utilization rate | 40.56 (101/249) | 39.16 (531/1356) | 0.677 | 40.56 (101/249) | 42.64 (333/781) | 0.564 |
| Biopsied blastocysts sent for DNA detecting | 96 | 512 | - | 96 | 326 | - |
| Blastocyst chromosomal aneuploidy rate | 51.04 (49/96) | 49.61 (254/512) | 0.797 | 51.04 (49/96) | 43.25 (141/326) | 0.178 |
| Number of embryos transferred | 34 | 145 | - | 34 | 71 | - |
| Implantation rate | 58.82 (20/34) | 61.38 (89/145) | 0.783 | 58.82 (20/34) | 63.38 (45/71) | 0.653 |
| Pregnancy rate | 58.82 (20/34) | 61.38 (89/145) | 0.783 | 58.82 (20/34) | 63.38 (45/71) | 0.653 |
| Live birth rate | 50.00 (17/34) | 51.72 (75/145) | 0.856 | 50.00 (17/34) | 56.34 (40/71) | 0.542 |
| Miscarriage rate |
15.00 (3/20) |
15.73 (14/89) | 1.000 | 15.00 (3/20) | 11.11 (5/45) | 0.693 |
Data are presented as n (%)
PN stands for pronuclei, which are the nuclei of the sperm and egg that form after fertilization
We matched 39 S-ICSI cycles with 34 patients to 117 MRB-ICSI cycles with 104 patients. There were no significant differences in maternal age, maternal BMI, duration of infertility, paternal age, or MII rate between the two groups. The main embryological and clinical outcomes are summarized in Table 4. Compared with that after single-touch sperm immobilization, the blastocyst chromosomal aneuploidy rate slightly decreased, following micro-rubbing sperm immobilization; however, the differences were not significant (43.25% vs. 51.04%, P = 0.178). There were significantly higher rates of oocyte fertilization (85.26% vs. 75.45%, P < 0.001), day-3 embryo utilization per 2 PN (74.39% vs. 64.66%, P = 0.003) and day-3 embryo utilization per oocyte injected (63.43% vs. 48.79%, P < 0.001) in the MRB-ICSI group than in the S-ICSI group. The proportion of cleaved embryos, top-quality embryos on day 3, blastocyst development, blastocyst utilization, implantations, pregnancies, live births, and miscarriages were similar between the MRB-ICSI and S-ICSI groups.
Discussion
In this study, we aimed to achieve full sperm membrane rupture without damaging the sperm nucleus and centriole using a mildly aggressive sperm immobilization method known as micro-rubbing. To minimize the impact of embryological and clinical heterogeneity among the patients, we used multiple matching criteria. We attempted to match maternal and paternal age, oocyte denudation, MII rate, history of pregnancy, duration of infertility, sperm source, BMI, cause of infertility, and type of protocol used for controlled ovarian hyperstimulation, embryo condition (embryo stage, number, and quality), and endometrial thickness on the day of embryo transfer which are known to affect ICSI success [26, 27]. A strictly matched case-control study found that compared with single-touch sperm immobilization, micro-rubbing sperm immobilization can significantly improve the rates of fertilization, day-3 embryo utilization, and oocyte utilization and does not have negative effects on embryo chromosomal or clinical outcomes.
During normal fertilization, the spermatozoon undergoes several key processes, including capacitation, calcium influx-mediated hyperactivation, acrosomal reaction, and penetration of the zona pellucida. Once inside the zona pellucida, the spermatozoon becomes immobilized, fuses with the oolemma, and is incorporated into the ooplasm in a demembranated state, it then releases sperm factors that activate the oocyte, and finally, the head is decondensed [28–30]. In ICSI, damage to the plasma membrane can invoke events similar to those occurring during natural fertilization and facilitate the decondensation of the sperm nucleus and activation of the oocyte [31, 32]. Therefore, it is crucial to inject spermatozoa with a fully disrupted plasma membrane into the ooplasm. Based on our experience, a clear bend in the middle of the flagellum can be achieved via single-touch sperm immobilization; occasionally, after some time, the sperm returns to its original state. This is because the microtubule structure at the tail of the spermatozoon is flexible and, in cases of insufficient pressure, reverts to its original state. At this point, the phospholipid bilayer of the cell membrane restores the integrity of the membrane, causing insufficient rupture of the sperm membrane and leading to failed fertilization. In this study, after the principal piece was cut by micro-rubbing, the spermatozoon plasma membrane was fully disrupted, releasing sperm-associated oocyte-activating factors into the ooplasm and inducing oocyte activation with calcium release [5, 33].
Eosin staining showed that 100% (50/50) of sperm heads in the micro-rubbing group were stained red within 30 s, while only 88% (44/50) in the single touch group were stained. Significant differences were observed between the two immobilization methods (P < 0.001, Supplementary Table 1). These results suggest that single deceleration may cause incomplete membrane disruption in some sperm cells. We believe that this is one of the main reasons why the micro-rubbing technique significantly improves embryological outcomes. Compared with the standard method, more aggressive mechanically immobilized spermatozoa are associated with a higher ICSI fertilization rate in patients with suboptimal semen samples [13, 15, 28]. Moreover, our results demonstrated that aggressive mechanical procedures have a positive effect on embryological outcomes in individuals with normal semen parameters.
The entire spermatozoon, encompassing the head, neck, midpiece, principal piece, and tail, is covered with a plasma membrane. Membrane integrity can be disrupted at the midpiece [12, 32], midportion [14] or tail tip [15]. A randomized trial comparing the standard procedure (pressing the tail of the spermatozoon once) and an aggressive procedure (based on the application of the standard procedure twice and compression of the sperm midpiece once) in male patients with infertility found no difference in fertilization rate between the two procedures. Notably, the proportion of good-quality day-3 embryos significantly decreased in the aggressive procedure group [12]. This may be related to the compression of the midpiece of the spermatozoa. The midpiece is adjacent to the centriole and chromosome, making it susceptible to damage from the coarse tip of the injection pipette during movement. The micro-rubbing technique targets the sperm principal piece region for immobilization and considers the direction of the injection pipette movement (opposite to the direction of the sperm head). This technique has benefits in circumventing the potential hazard of damaging the centriole and chromosome, which may occur when compressing the midpiece. To the best of our knowledge, no study has strictly limited the number of compressions applied to the spermatozoa for aggressive sperm immobilization. Some studies have pressed the distal half of the tail of spermatozoa approximately15 times [28]. We believe that each compression by the injection pipette has a severe impact on the sperm, which may potentially damage sperm centrioles and chromosomes. Therefore, we limited the number of compressions to three. This is another reason why the micro-rubbing technique significantly improves embryological outcomes.
The third factor contributing to the significant improvement in embryological outcomes with the micro-rubbing technique is the differential handling of the sperm tail between the micro-rubbing method and the single-touch technique. In the micro-rubbing technique, to ensure complete immobilization and destruction of the sperm, the sperm tail is partially severed. In contrast, the single-touch technique preserves the integrity of the sperm tail. Consequently, the length of the whole sperm subjected to the micro-rubbing technique tend to be shorter compared to those processed with the single-touch method. This reduction in sperm length may result in a lower volume of sperm being introduced into the oocyte culture medium during injection, potentially reducing the risk of detrimental effects on embryo development and improving embryological outcomes.
To assess the reproducibility of the micro-rubbing sperm immobilization technique, we compared the embryological outcomes of three different operators using this method. The results demonstrated a significant higher in both fertilization rates and embryo utilization rates on day 3/2 PN with the micro-rubbing technique compared to the single-touch technique across all three independent operators (all P < 0.05, Supplementary Table 2).
One unusual finding in this study was that although MRB-ICSI improved fertilization rates and increased day-3 embryo utilization, it did not result in improved blastocyst utilization rates. One possible explanation is that while early embryo development is crucial, factors influencing blastocyst formation are likely more complex and may involve additional factors not solely dependent on early-stage development. These factors could include variations in the quality of the culture environment, genetic and epigenetic regulation of embryo development, differences in cellular signaling pathways between day-3 and blastocyst stages, or the intrinsic developmental potential of embryos beyond day 3.
This study had some limitations. First, it was a retrospective study, which cannot exclude selection bias. Secondly, due to the small number of S-ICSI cycles in the PGT-A group, which may lead to a bias in the results. Furthermore, the two groups were performed at different times (S-ICSI: 2018–2019 vs. MRB-ICSI: 2020–2023). Laboratory protocols, stimulation regimens, embryo culture conditions, and PGT analysis methods have evolved during this period, which may have confounded the results. Moreover, the MRB-ICSI group was performed later, which inevitably means that the operators were more experienced, potentially introducing bias in favor of the MRB-ICSI group. Additionally, as data collection was finalized in June 2023, embryos were not fully transferred in the MRB-ICSI group, and clinical outcomes, such as embryo implantation rate, pregnancy, live birth, and miscarriage, could not be compared objectively and unequivocally. Therefore, whether the use of micro-rubbing to immobilize spermatozoa for ICSI can improve clinical outcomes still requires a sibling oocyte design study with a larger sample to confirm its efficacy.
Conclusion
This study suggests that the use of the micro-rubbing method to immobilize spermatozoa was associated with increased fertilization, day-3 embryo utilization, and embryo utilization rate on day 3 per oocyte injected compared with the conventional single-touch sperm immobilization. These findings provide evidence that MRB-ICSI results in superior embryology outcomes compared to S-ICSI. However, it does not appear to influence clinical outcomes.
Supplementary Information
Acknowledgements
The authors would like to gratefully acknowledge the clinical and laboratory staff for their commitment and support to this project.
Abbreviations
- ICSI
Intracytoplasmic sperm injection
- IVF
In vitro fertilization
- OR
Ovarian retrieval
- PN
Pronuclei
- PGT
Preimplantation genetic testing
- PGT-A
Preimplantation genetic testing for aneuploidy
Authors’ contributions
S.L., H.W. and M.-Y.L. contributed to the conception of the study, manuscript drafting and revising. Y.-H.L. and H.-J.W. contributed to the data collection and data analysis. L.Y., Y.J. and Z.-H.C. participated in the interpretation of the data and critical revision. S.L. was responsible for the final approval. All authors were involved in revising the article and approved this final version for publication.
Funding
This study was supported by the Clinical Research Project of Chinese Medical Association (grant number 18010210750) and Health Science and Technology Project of Guangzhou (grant number 20191A011027).
Data availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Guangzhou Women and Children’s Medical Center (2024-135A01). Informed patient consent was not required as the study was retrospective in nature and analyzed patient data anonymously. A statement from the Ethics Committee of Guangzhou Women and Children’s Medical Center waived the need for informed consent.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Shuai Liu, Hui Wang and Meiyi Li contributed equally to this work.
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Supplementary Materials
Data Availability Statement
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.