Enhancing Embryo Quality through Meiotic Spindle- Aligned Microinjection in POSEIDON Group 4 Patients Undergoing ICSI Cycles: A Retrospective Cohort Study

Enhancing Embryo Quality through Meiotic Spindle- Aligned Microinjection in POSEIDON Group 4 Patients Undergoing ICSI Cycles: A Retrospective Cohort Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancing Embryo Quality through Meiotic Spindle- Aligned Microinjection in POSEIDON Group 4 Patients Undergoing ICSI Cycles: A Retrospective Cohort Study Chyi-Uei Chern, Pei-Fen Liao, Chia-Jung Li, Pei-Hsuan Lin, Wan-Ping Su, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3828724/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Embryologists have traditionally depended on the polar body (PB) as a reference point for guiding the process of intracytoplasmic sperm injection (ICSI). However, its accuracy in predicting the precise location of the meiotic spindle (MS) is constrained. To overcome this limitation, polarized light microscopy has emerged as a noninvasive technique for visualizing and precisely positioning the MS during ICSI procedures. The primary aim of our study was to assess whether implementing MS alignment in ICSI results in improved embryo quality compared to conventional PB alignment, specifically in the POSEIDON group 4 population. Methods This retrospective cohort study included 315 women who met the POSEIDON Group 4 criteria and underwent ICSI cycles. The study group comprised 168 women who underwent MS-aligned microinjection, while 147 women in the control group underwent the PB-aligned method. We conducted a comparative analysis of baseline and cycle characteristics as well as embryo quality between these two groups. Results At baseline, the MS-aligned group had older age and more previous IVF attempts than the PB-aligned group. The spindle-aligned group demonstrated a higher fertilization rate, a greater cleavage rate, an increased rate of top-quality day 3 embryos (TQE), and a larger number of TQE when compared to the PB-aligned group. Multivariate analysis revealed that the MS-aligned group had a positive impact on the likelihood of achieving ≥ 1 TQE (odds ratio [OR] 4.22, 95% confidence interval [CI] 2.24–7.96, P < 0.001) and reaching a TQE rate of ≥ 50 (OR 3.63, 95% CI 2.06–6.39, P < 0.001). Conclusions Our findings suggest that MS-aligned microinjection may enhance embryo quality in older patients with diminished ovarian reserve undergoing ICSI cycles. embryo quality intracytoplasmic sperm injection (ICSI) meiotic spindle polar body diminished ovarian reserve Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Meiotic spindles (MS) represent dynamic microtubular structures that play a pivotal role in facilitating the separation of homologous chromosomes and sister chromatids during meiosis, thus serving as a critical determinant of oocyte developmental potential. Disruptions to MS integrity can result in improper chromosomal segregation, subsequently leading to maturation arrest, diminished fertilization rates, and aneuploidy (1–3). Traditionally, embryologists have relied on the polar body (PB) as a spatial reference point for orienting the injection site during intracytoplasmic sperm injection (ICSI). This practice aimed to protect MS from potential damage caused by the ICSI procedures, assuming that the MS closely neighbored the PB. However, studies have revealed disparities between the actual MS location and that of the PB. In essence, the PB's location cannot reliably predict the precise location of the MS (4–6). Consequently, the utilization of PB-aligned ICSI may inadvertently lead to MS damage in cases where the MS location does not align with that of the PB. Fortunately, recent advances in technology have enabled the noninvasive visualization of MS through the application of polarized light. The utilization of a polarized light microscopy system (Polscope) allows for the visualization of structures within living oocytes based on their birefringence properties (7, 8). Given that MS exhibit pronounced birefringence due to microtubule orientation, they can be readily identified using the Polscope (9, 10). Therefore, MS-aligned ICSI, when performed using the Polscope, has the capacity to protect the MS from harm during microinjection procedures (11, 12). It is imperative to acknowledge that MS are highly susceptible to physical and chemical perturbations that may occur during oocyte retrieval and handling (13, 14). Additionally, advanced maternal age and oocyte in vitro aging have been correlated with aberrations in MS architecture (9, 15, 16). A prior investigation that compared MS in oocytes from two distinct groups of women—those aged 20–25 and those aged 40–45—revealed that older women exhibited a significantly higher incidence of abnormalities related to chromosome placement and structure within their MS (15). With this backdrop, we hypothesized that PB-aligned ICSI may inflict more substantial damage to MS in older women when the MS location does not align with that of the PB. Conversely, MS-aligned ICSI may yield more favorable outcomes in older women. Nevertheless, there is a paucity of published studies that have explored this specific issue. In 2016, the Patient-Oriented Strategies Encompassing Individualized Oocyte Number (POSEIDON) group introduced a novel classification system aimed at tailoring approaches for patients with a less favorable prognosis (17). Specifically, POSEIDON Group 4 comprises women characterized by advanced age and diminished ovarian reserve. Accordingly, we designed a retrospective cohort study to compare embryo quality between MS-aligned and PB-aligned ICSI procedures in patients meeting the criteria of POSEIDON Group 4, who subsequently underwent ICSI treatments. Materials and methods Study Design and Participants This retrospective cohort study was conducted at the Reproductive Medical Center of Kaohsiung Veterans General Hospital during the period from January 2019 to March 2023. The study received ethical approval from the Institutional Review Board of Kaohsiung Veterans General Hospital (Institutional Review Board reference number: KSVGH23-CT8-02). Given its retrospective nature, the requirement for obtaining informed consent was waived by the Institutional Review Board of Kaohsiung Veterans General Hospital. Data for this study were meticulously extracted from electronic medical records and in vitro fertilization (IVF) treatment documentation. The study cohort consisted of women who met the criteria of POSEIDON Group 4 and had undergone their initial ICSI cycle at our center. POSEIDON Group 4 criteria referred to women aged ≥ 35 years, accompanied by an anti-Müllerian hormone (AMH) level < 1.2 ng/ml and/or antral follicle counts (AFCs) < 5. In addition, the study included women aged ≤ 45 years with a body mass index (BMI) falling within the range of 18–30 kg/m². Exclusion criteria included women who had undergone preimplantation genetic testing for aneuploidy (PGT-A), oocyte recipients, cases involving testicular sperm extraction (TESE) in their partners, and individuals lacking metaphase II (MII) oocytes. Finally, a total of 315 patients were identified and subsequently categorized into two groups: the MS-aligned group (n = 168) and the PB-aligned group (n = 147) for microinjection procedures. In the PB-aligned group, oocyte alignment was achieved with reference to the first polar body, a cellular structure generated through the process of oocyte meiosis, encapsulating a nucleus formed during meiotic division and a minimal cytoplasmic volume. In this group, the first polar body was positioned at the 12 o'clock position, while the exact location of the MS remained undetermined, and sperm microinjection was carried out at the 3 o'clock position. However, in the MS-aligned group, oocytes were manipulated to align their MS at the 12 o'clock position, a process confirmed using a polarized light microscope. A schematic representation of the study's workflow is depicted in Fig. 1. Figure 1 Study flow chart. IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; BMI, body mass index; PGT-A, preimplantation genetic testing for aneuploidy; TESE, testicular sperm extraction; MII, metaphase II; MS, meiotic spindle; PB, polar body; D3, day 3. Preparation of sperm Ejaculated spermatozoa were collected through masturbation following a period of 3 to 5 days of ejaculatory abstinence. After the semen had undergone liquefaction at room temperature, the sperm samples were meticulously processed via a one-layer density-gradient centrifugation method. In this process, the lowermost fraction was gently aspirated, underwent two sequential washes utilizing SepaSperm wash medium (KITAZATO, Japan), each at a speed of 2,500 rpm for 5 minutes, and was subsequently incubated at 37°C. Oocyte denudation Following retrieval, the collected oocytes were incubated in fertilization medium (LGTF, LifeGlobal, CooperSurgical, Denmark) and covered with paraffin oil (Liquid Paraffin, Origio, CooperSurgical, Denmark). This incubation was carried out at 37°C in an environment with 6% CO2 for a duration of 2 hours. Cumulus cells were subsequently removed by exposing the oocytes to ICSI Cumulase (Origio, CooperSurgical, Denmark) for 30 seconds, followed by a wash using HEPES-buffered HTF containing 5 mg/ml human serum albumin (LGTH, LifeGlobal, CooperSurgical, Denmark). The corona cells were meticulously removed using a series of finely drawn glass Pasteur pipettes. Following denudation, the oocytes were assessed for both structural integrity and meiotic maturity. Thereafter, they were placed in culture dishes (Oosafe, Astec Bio, USA) within a fertilization medium (LGTF, LifeGlobal, CooperSurgical, Denmark) and covered with a paraffin oil overlay (Liquid Paraffin, Origio, CooperSurgical, Denmark) in preparation for microinjection. Intracytoplasmic sperm injection PB-aligned oocytes were gently transferred into 4 µl of prewarmed HEPES-buffered HTF containing 5 mg/ml human serum albumin (LGTH, LifeGlobal, CooperSurgical, Denmark) during the microinjection procedures. Sperm samples were prepared in a polyvinylpyrrolidone solution (SAGE, CooperSurgical, Denmark) in the microinjection dish immediately before sperm injection. The oocytes were positioned under an inverted microscope (Olympus IX73, Japan) equipped with a heated stage maintained at 37.0 ± 0.5°C and were observed at ×400 magnification. Subsequently, sperm injection into the mature oocytes was performed, with the polar body positioned at 12:00. After injection, the oocytes were transferred to a culture medium (LGGT, LifeGlobal, Coopersurgical, Denmark) and placed in an environment maintained at 37°C with 6% CO2. For the MS-aligned oocytes, a volume of 5 µl of HEPES-buffered HTF containing 5 mg/ml human serum albumin (LGTH, LifeGlobal, CooperSurgical, Denmark) was added, and the oocytes were then covered with paraffin oil (Liquid Paraffin, Origio, CooperSurgical, Denmark) within a glass-bottomed dish (MATSUNAMI, Japan). The temperature was maintained at 37.0 ± 0.5°C using a heated stage (Integra3, RI, UK). MS visualization was achieved using a Polscope system integrated with an inverted microscope (Olympus IX73, Japan). The oocyte was immobilized with a holding pipette and gently rotated using the injection pipette until both the MS and PB were clearly visible within the oocyte. ICSI was promptly performed following this imaging. For oocytes with evident spindles, oocytes were positioned such that the MS was at the 12 o'clock position, and sperm injection was carried out at the 3 o'clock position to minimize disruption of the MS. For oocytes without visible spindles, the oocytes were injected with their PB at the 12 o’clock position. However, oocytes lacking a visible spindle were excluded from the analysis of the MS-aligned group, as no spindle was present for alignment. Following microinjection and imaging procedures, the oocytes were transferred to individual 50 µl culture medium droplets (LGGT, LifeGlobal, Coopersurgical, Denmark) within sterile plastic dishes. These dishes were subsequently placed in an incubator with a controlled atmosphere of 6% CO2 at 37°C. Fertilization and embryo development Fertilization status was assessed 16–18 hours following sperm injection. Oocytes displaying two distinct pronuclei with extrusion of the second PB were categorized as having undergone normal fertilization. On day 3, embryonic cleavage was examined, and embryo quality was assessed in accordance with the criteria established in the Istanbul consensus workshop (18). Embryos were stratified into Grade 1 to Grade 3 categories based on several parameters, including the percentage of fragmentation, uniformity of blastomeres, and the presence of multinucleation. Grade 1 embryos exhibited less than 10% fragmentation, uniform-sized blastomeres, and an absence of multinucleation. Grade 2 embryos displayed 10–25% fragmentation, predominantly uniform-sized blastomeres, and no multinucleation. Grade 3 embryos featured more than 25% fragmentation, uneven blastomere sizes, and evidence of multinucleation. Top-quality Day 3 embryos (TQE) were specifically defined as embryos with 6–10 cells and a Grade 1 classification, signifying that they had less than 10% fragmentation, uniform-sized blastomeres, and no multinucleation. Our reproductive medical center implemented a freeze-all policy. Given the diminished quality of embryos within our study cohort, the majority of embryos were cryopreserved at the cleavage stage. Only a limited number of embryos were subjected to extended culture to the blastocyst stage. Outcome measures The primary outcome measure in this study was the rate of TQE. Secondary outcome measures encompassed the assessment of the fertilization rate, the incidence of total fertilization failure, and the cleavage rate. The fertilization rate was calculated as the number of observed two pronuclei zygotes divided by the total number of oocytes subjected to microinjection. Total fertilization failure was defined as the absence of fertilization for all available oocytes within a single ICSI cycle. The cleavage rate was defined as the ratio of the number of day 3 embryos to the number of fertilized oocytes. The TQE rate was determined by dividing the number of top-quality day 3 embryos by the total number of day 3 embryos. Statistical analysis The normal distribution of continuous variables was assessed using the Kolmogorov–Smirnov test. Student’s t test was employed for quantitative variables that exhibited a normal distribution, had sufficiently large sample sizes, or both; conversely, the Mann–Whitney U test was utilized for nonnormally distributed data. For categorical data, a comparison was made using either the chi-squared test or Fisher's exact test, as appropriate. To identify the independent effects of MS-aligned microinjection on the attainment of a rate of at least one TQE and a TQE rate ≥ 50, multivariable logistic regression was performed. This analysis was adjusted for various covariates, including age, body mass index, previous IVF attempts, types of infertility, basal follicle-stimulating hormone (FSH) levels, AMH levels, and the number of MII oocytes. The results of this analysis are expressed as odds ratios (ORs) with corresponding 95% confidence intervals (CIs). Statistical significance was determined using a two-tailed P value of < 0.05. Data processing and statistical analyses were conducted using IBM SPSS Statistics version 20.0 (IBM Corp., Armonk, NY, USA). Results Table 1 summarizes the baseline characteristics of the study cohort. The MS-aligned group displayed a higher mean age and a greater number of previous IVF attempts than the PB-aligned group. However, no statistically significant differences were observed between the two groups in terms of parameters such as body mass index, duration of infertility, and infertility types. Additionally, the basal levels of FSH and AMH were comparable between the two groups. Table 1 Baseline characteristics of women undergoing MS-aligned or PB-aligned ICSI cycles Parameters MS-aligned group (n=168) PB-aligned group (n=147) p value Age (years) 41.1±2.9 40.0±2.5 <0.001 Body mass index (kg/m 2 ) 22.7±3.0 22.7±2.9 0.972 Infertility duration (years) 4.7±3.4 4.5±3.5 0.609 Previous IVF attempts (%) 0.017 0-1 28.6% (48/168) 36.1% (53/147) 2 30.4% (51/168) 38.1% (56/147) ≧3 41.1% (69/168) 25.9% (38/147) Types of infertility (%) 0.964 Primary infertility 49.4% (83/168) 49.7% (73/147) Secondary infertility 50.6% (85/168) 50.3% (74/147) Basal FSH (IU/l) 6.8±5.3 6.1±4.8 0.203 Anti-Müllerian hormone (ng/mL) 0.77±0.42 0.73±0.32 0.412 Data are presented as the mean ± standard deviation or %. MS, meiotic spindle; PB, polar body; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; FSH, follicle-stimulating hormone Table 2 presents a detailed comparison of various treatment-related parameters between the two groups. No statistically significant differences were observed in the stimulation duration, gonadotropin dosage, number of retrieved oocytes, number of MII oocytes, number of fertilized oocytes, or number of day 3 embryos between the two groups. However, the MS-aligned group achieved significantly superior outcomes in several key parameters, including a higher fertilization rate (87.8±19.8% vs. 79.5±30.5%, P = 0.006), a greater cleavage rate (88.7±22.6% vs. 72.3±41.0%, P < 0.001), an elevated TQE rate (54.2±37.2% vs. 32.2±35.8%, P < 0.001), a higher rate of achieving ≥ 1 TQE (78.6% vs. 55.1%, P = 0.003) and a greater TQE rate of ≥ 50 (61.9% vs. 38.1%, P < 0.001) when compared to the PB-aligned group (Figure 2). Furthermore, the MS-aligned group displayed a notably reduced incidence of total fertilization failure (0.6% vs. 8.8%, P < 0.001) and a significantly increased number of TQE (1.7±1.6 vs. 1.3±1.8, P = 0.040) in comparison to the PB-aligned group. Table 2 Cycle characteristics of women undergoing MS-aligned or PB-aligned ICSI cycles Parameters MS-aligned group (n=168) PB-aligned group (n=147) p value Stimulation duration (days) 10.7±2.3 10.3±2.4 0.139 Gonadotropin dosage (IU) with corifollitropin alfa 1727.6±515.9 1440.0±997.5 0.051 without corifollitropin alfa 2839.0±834.6 2929.3±834.6 0.456 No. of retrieved oocytes (n) 5.3±3.4 5.1±3.5 0.624 No. of metaphase II oocytes (n) 4.0±2.7 4.1±3.0 0.786 Maturation rate (%) 77.0±20.5 78.9±24.9 0.440 No. of fertilized oocytes (n) 3.4±2.1 3.5±2.7 0.703 Fertilization rate (%) 87.8±19.8 79.5±30.5 0.006 Total fertilization failure (%) 0.6% (1/168) 8.8% (13/147) <0.001 No. of Day 3 embryos (n) 3.0±2.0 2.9±2.8 0.618 Cleavage rate (%) 88.7±22.6 72.3±41.0 <0.001 No. of top-quality Day 3 embryos (n) 1.7±1.6 1.3±1.8 0.040 Rate of top-quality Day 3 embryos (%) 54.2±37.2 32.2±35.8 <0.001 ≧1 top-quality Day 3 embryos rate (%) 78.6% (132/168) 55.1% (81/147) <0.001 Top-quality Day 3 embryos rate≧50 (%) 61.9% (104/168) 38.1% (56/147) <0.001 Data are presented as the mean ± standard deviation or % (n). MS, meiotic spindle; PB, polar body; ICSI, intracytoplasmic sperm injection Figure 2 Comparison of fertilization rate, cleavage rate, TQE rate, rate of ≥ 1 TQE, and TQE rate ≥ 50 between the MS-aligned group (blue) and PB-aligned group (red). ** P<0.01, *** P<0.001 TQE, top-quality day 3 embryo; MS, meiotic spindle; PB, polar body Table 3 presents the results of a binary logistic regression analysis conducted to evaluate the impact of MS-aligned microinjection on the likelihood of achieving ≥ 1 TQE and a TQE rate of ≥ 50 in women belonging to POSEIDON Group 4. The analysis accounted for potential confounding variables, including age, body mass index, previous IVF attempts, types of infertility, basal FSH levels, AMH levels, and the number of MII oocytes. The multivariate analysis demonstrated that the MS-aligned group exerted favorable effects on the probability of achieving ≥ 1 TQE (OR 4.22, 95% CI 2.24–7.96, P < 0.001) and attaining a TQE rate of ≥ 50 (OR 3.63, 95% CI 2.06–6.39, P < 0.001). Additionally, it was noted that the number of MII oocytes represented an independent factor capable of influencing the likelihood of achieving ≥ 1 TQE. Table 3 Logistic regression analyses of factors influencing top-quality day 3 embryos in POSEIDON group 4 patients ≧ 1 TQE TQE rate ≧ 50 Adjusted OR* (95% CI) p value Adjusted OR* (95% CI) p value MS-aligned vs. PB-aligned 4.22(2.24–7.96) <0.001 3.63(2.06–6.39) <0.001 Age (years) 0.99(0.87–1.11) 0.806 0.91(0.82–1.02) 0.105 BMI (kg/m 2 ) 1.02(0.92–1.13) 0.701 1.09(1.00–1.20) 0.061 Previous IVF attempts 0.75(0.51–1.11) 0.152 0.81(0.58–1.15) 0.236 Types of infertility 0.61(0.33–1.16) 0.269 0.65(0.37–1.14) 0.132 Basal FSH (IU/l) 0.98(0.93–1.04) 0.378 0.98(0.94–1.04) 0.530 AMH (ng/mL) 1.69(0.69–4.15) 0.251 1.02(0.47–2.22) 0.966 No. of MII oocytes (n) 1.45(1.23–1.69) <0.001 1.07(0.95–1.20) 0.289 TQE, top-quality day 3 embryos; OR, odds ratio; CI, confidence interval; MS, meiotic spindle; PB, polar body; BMI, body mass index; IVF, in vitro fertilization; FSH, follicle-stimulating hormone; AMH, anti-Müllerian hormone; MII, metaphase II. *Adjustment for age, BMI, previous IVF attempts, types of infertility, basal FSH, AMH and number of MII oocytes In the MS-aligned group, a total of 441 oocytes were assessed. However, 8.4% (n=37) of these oocytes exhibited no visible MS and were subsequently inseminated using the PB-aligned method. When comparing oocytes with a visible MS to those without, the latter group displayed significantly lower fertilization rates (73.0% vs. 85.4%, P = 0.046), cleavage rates (59.5% vs. 80.2%, P = 0.003), and TQE rates (18.9% vs. 35.9%, P = 0.038), as illustrated in Figure 3A. Among the 404 oocytes with a visible MS, 42.6% (n=172) exhibited no MS deviation (MS at 0° in relation to the PB), while 57.4% (n=232) displayed MS deviation, which was corrected using MS-aligned microinjection. Consequently, the fertilization rate, cleavage rate, and TQE rate were comparable between these two groups, as depicted in Figure 3B. However, among the oocytes with MS deviation, 59.5% exhibited a deviation angle of less than 30° (MS < 30° in relation to the PB). Figure 4 illustrates the oocytes without visible MS and those with visible MS, whether or not they displayed deviation. Figure 3 (A) Comparison of fertilization rate, cleavage rate, and TQE rate between oocytes with a visible spindle (blue) and those without a visible spindle (red). (B) Comparison of fertilization rate, cleavage rate, and TQE rate between oocytes with no MS deviation (blue) and oocytes with corrected MS deviation using MS-aligned microinjection (red). * P<0.05, ** P<0.01 TQE, Top-quality Day 3 embryo; MS, meiotic spindle Figure 4 Meiotic spindle visualization under Polscope. (A) Oocytes displaying an absence of a visible spindle. (B) The oocyte spindle is positioned beneath the first polar body. (C) The oocyte spindle exhibits a deviation angle of less than 30° relative to the polar body. (D) The oocyte spindle exhibits a deviation angle of more than 30° relative to the polar body. Blue arrow: meiotic spindle, red arrow: polar body. Discussion This retrospective study represents the first investigation into the impact of MS-aligned microinjection on embryo quality in patients meeting the criteria of POSIDEN Group 4. Our findings indicate that the MS-aligned group exhibited significantly higher fertilization rates, cleavage rates, and TQE rates than the PB-aligned group. Additionally, the multivariate analysis demonstrated a 4.22-fold increase in the likelihood of achieving more than 1 TQE (95% CI 2.24–7.96, P < 0.001) and a 3.63-fold increase in the likelihood of attaining a TQE rate ≥ 50 (95% CI 2.06–6.39, P < 0.001) in POSEIDON Group 4 patients undergoing MS-aligned microinjection compared to those subjected to the PB-aligned method. During the ICSI procedure, oocytes are arrested at the metaphase II stage of the meiotic cell cycle when chromosomes are aligned at the equatorial region of the MS. Microtubules within the MS are responsible for the precise separation of chromosomes and are highly susceptible to oocyte manipulations (1, 3, 14). However, the position of the MS cannot be accurately predicted by the first PB’s location (4–6). Our study provides support for this notion. We observed that among oocytes with a visible MS, 57.4% exhibited a deviation in the MS position relative to the PB. Within this subset of oocytes with MS deviation, 59.5% displayed a deviation angle of less than 30° relative to the PB, while 40.5% exhibited a deviation angle of more than 30°. Heindryckx et al. demonstrated a significantly lower prevalence of MS located adjacent to the first PB in in vivo matured oocytes compared to in vitro matured oocytes (64% vs. 94%, P < 0.0001) (19). Furthermore, findings from Hardarson et al. indicated that in vivo matured oocytes exhibited significantly greater degrees of MS deviation from the PB position than in vitro matured oocytes (41.7° vs. 26.6°, P = 0.005) (5). These findings suggest that the increased MS deviation in in vivo matured oocytes may result from lateral displacement of the first PB during manipulations for cumulus and corona removal since in vitro matured oocytes were denuded before the extrusion of the first PB. Consequently, the use of PB-aligned ICSI could result in MS injury when the MS is not located directly adjacent to the PB. However, the needle used for ICSI is relatively small in size in comparison to the oocyte, making it unlikely to cause MS damage during needle insertion. Instead, MS injury is more likely to occur during the aspiration of the oocyte cytoplasm just prior to sperm injection. Therefore, the utilization of MS-aligned ICSI with the Polscope has the potential to effectively protect the MS from harm during microinjection procedures. A study conducted by Asa et al. involved a randomized controlled trial with the enrollment of 264 oocytes from 24 patients. These oocytes were randomly assigned to either the spindle-aligned group (n = 138) or the PB-aligned group (n = 126). The results of this study revealed a significantly higher fertilization rate in the spindle-aligned group than in the PB-aligned group (78.5% vs. 65.0%, P < 0.05) (11). Furthermore, in a separate randomized controlled trial led by Cooke et al., sibling oocytes within patients were allocated to either the spindle-aligned group (n = 124) or the PB-aligned group (n = 122) for microinjection. This investigation demonstrated that embryos originating from the spindle-aligned group exhibited significantly higher embryo quality scores than those from the PB-aligned group (12). It is important to note that the design of previous studies was based on a per-oocyte analysis. However, our research specifically targeted a distinct patient subgroup—patients meeting the criteria of POSEIDON Group 4. POSEIDON Group 4 comprises women characterized by advanced age and diminished ovarian reserve. In women of advanced age, MS may be more susceptible to damage during oocyte manipulations due to the increased prevalence of MS abnormalities with age (15, 16). Additionally, for women with diminished ovarian reserve, obtaining a greater number of high-quality embryos may reduce the necessity for repeated IVF cycles. Therefore, we hypothesized that MS-aligned microinjection would yield more substantial benefits within this particular patient group. Our findings indeed support this hypothesis. The MS-aligned group exhibited significantly higher fertilization rates, cleavage rates, and TQE rates than the PB-aligned group, validating our initial speculation. Furthermore, within the spindle-aligned group, women with MS deviation corrected using MS-aligned microinjection exhibited fertilization rates, cleavage rates, and TQE rates similar to those of women without MS deviation. This suggests that MS-aligned microinjection can effectively prevent MS damage during ICSI. However, it is imperative to emphasize that additional large-scale prospective studies are necessary to further corroborate and validate these results. The incidence of oocytes lacking a visible MS under Polscope examination ranged from 7–34.1% across different studies (11, 12, 20–23). In our current investigation, within the spindle-aligned group, 8.4% of oocytes exhibited a nonvisible MS. Oocytes with a visible MS demonstrated significantly higher rates of fertilization, cleavage, and TQE than those without a visible MS. These findings are similar to the outcomes reported in previous studies (6, 10, 21, 23–25). In a meta-analysis encompassing data from 10 studies, a substantial increase in fertilization success was observed in 4,684 oocytes with visualized MS compared to 1,264 oocytes where no MS was observed (OR 1.79, 95% CI 1.57–2.05, P < 0.0001). Furthermore, embryos derived from oocytes with visible MS exhibited higher cleavage rates (OR 1.85, 95% CI 1.47–2.32, P < 0.0001), TQE rates (OR 1.70, 95% CI 1.20–2.42, P = 0.003), and blastocyst formation rates (OR 2.61, 95% CI 1.74–3.91, P < 0.0001) (24). Additionally, a retrospective cohort investigation demonstrated that embryos resulting from oocytes with MS angles ranging from 0° to 29° were associated with higher pregnancy rates and live birth rates when compared to oocytes lacking a visible MS (25). Thus, the presence or absence of a visible MS within oocytes can serve as a valuable predictor for outcomes in ICSI procedures. This study is subject to several limitations that warrant consideration. First, its retrospective design and the relatively limited sample size represent the primary limitations. To validate our findings, large-scale prospective studies are imperative. Second, the decision to perform either MS-aligned or PB-aligned microinjection relied on the clinical judgment and preferences of physicians and embryologists, potentially introducing bias. Third, the exclusion of oocytes lacking a visible spindle from the MS-aligned group's analysis might introduce additional bias. In conclusion, our study findings suggest that MS-aligned microinjection could lead to improved embryo quality compared to PB-aligned microinjection, particularly among older patients with diminished ovarian reserve undergoing ICSI cycles. Nonetheless, it is crucial to acknowledge and address these limitations, emphasizing the need for further extensive prospective investigations to validate and reinforce our conclusions. Abbreviations AFCs, antral follicle counts; AMH, anti-Müllerian hormone; BMI, body mass index; CI, confidence interval; FSH, follicle-stimulating hormone; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; MII, metaphase II; MS, meiotic spindle; OR, odds ratio; PB, polar body; PGT-A, preimplantation genetic testing for aneuploidy; TESE, testicular sperm extraction; TQE, top-quality day 3 embryos Declarations Ethics approval and consent to participate This study adhered to the principles set forth in the Declaration of Helsinki regarding medical research involving human subjects. Furthermore, it received ethical approval from the Institutional Review Board of Kaohsiung Veterans General Hospital, identified as KSVGH23-CT8-02 . The study was conducted in strict accordance with the approved guidelines. Notably, the need for informed consent was waived by the Institutional Review Board of Kaohsiung Veterans General Hospital due to the retrospective nature of the study. Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding Not applicable Authors’ contributions Pei-Hsuan Lin, Yu-Chen Chen and Kuan-Hao Tsui contributed to the study's conception and design; Pei Fen Liao and Wan Ping Su were responsible for organizing the database; Chia-Jung Li and Li-Te Lin conducted the statistical analysis; Chyi-Uei Chern drafted the initial manuscript, with Li-Te Lin contributing to specific sections. All authors participated in the manuscript's review and approval. Acknowledgments Not applicable References Howe K, FitzHarris G. Recent insights into spindle function in mammalian oocytes and early embryos. Biology of reproduction. 2013;89(3):71. Wang WH, Keefe DL. Prediction of chromosome misalignment among in vitro matured human oocytes by spindle imaging with the PolScope. Fertil Steril. 2002;78(5):1077-81. Severson AF, von Dassow G, Bowerman B. Oocyte Meiotic Spindle Assembly and Function. Curr Top Dev Biol. 2016;116:65-98. Silva CP, Kommineni K, Oldenbourg R, Keefe DL. The first polar body does not predict accurately the location of the metaphase II meiotic spindle in mammalian oocytes. Fertil Steril. 1999;71(4):719-21. Hardarson T, Lundin K, Hamberger L. The position of the metaphase II spindle cannot be predicted by the location of the first polar body in the human oocyte. Hum Reprod. 2000;15(6):1372-6. Konc J, Kanyó K, Cseh S. Visualization and examination of the meiotic spindle in human oocytes with polscope. J Assist Reprod Genet. 2004;21(10):349-53. Keefe D, Liu L, Wang W, Silva C. Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF. Reprod Biomed Online. 2003;7(1):24-9. Wang WH, Keefe DL. Spindle observation in living mammalian oocytes with the polarization microscope and its practical use. Cloning and stem cells. 2002;4(3):269-76. Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil Steril. 2001;75(2):348-53. Wang WH, Meng L, Hackett RJ, Keefe DL. Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination. Hum Reprod. 2001;16(7):1464-8. Asa E, Tabatabaee R, Farrokhi A, Nejatbakhsh R. Relationship between meiotic spindles visualization and intracytoplasmic sperm injection outcomes in human oocytes. Anat Cell Biol. 2017;50(1):26-32. Cooke S, Tyler JP, Driscoll GL. Meiotic spindle location and identification and its effect on embryonic cleavage plane and early development. Hum Reprod. 2003;18(11):2397-405. Can A, Semiz O, Cinar O. Bisphenol-An induces cell cycle delay and alters centrosome and spindle microtubular organization in oocytes during meiosis. Molecular human reproduction. 2005;11(6):389-96. Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy. Hum Reprod. 2001;16(11):2374-8. Battaglia DE, Goodwin P, Klein NA, Soules MR. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod. 1996;11(10):2217-22. Wang ZB, Schatten H, Sun QY. Why is chromosome segregation error in oocytes increased with maternal aging? Physiology (Bethesda, Md). 2011;26(5):314-25. Alviggi C, Andersen CY, Buehler K, Conforti A, De Placido G, Esteves SC, et al. A new more detailed stratification of low responders to ovarian stimulation: from a poor ovarian response to a low prognosis concept. Fertil Steril. 2016;105(6):1452-3. Alpha Scientists in Reproductive M, Embryology ESIGo. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26(6):1270-83. Heindryckx B, De Gheselle S, Lierman S, Gerris J, De Sutter P. Efficiency of polarized microscopy as a predictive tool for human oocyte quality. Hum Reprod. 2011;26(3):535-44. Rienzi L, Ubaldi F, Martinez F, Iacobelli M, Minasi MG, Ferrero S, et al. Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI. Hum Reprod. 2003;18(6):1289-93. Moon JH, Hyun CS, Lee SW, Son WY, Yoon SH, Lim JH. Visualization of the metaphase II meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI. Hum Reprod. 2003;18(4):817-20. Kilani S, Cooke S, Tilia L, Chapman M. Does meiotic spindle normality predict improved blastocyst development, implantation and live birth rates? Fertil Steril. 2011;96(2):389-93. Madaschi C, de Souza Bonetti TC, de Almeida Ferreira Braga DP, Pasqualotto FF, Iaconelli A, Jr., Borges E, Jr. Spindle imaging: a marker for embryo development and implantation. Fertil Steril. 2008;90(1):194-8. Petersen CG, Oliveira JB, Mauri AL, Massaro FC, Baruffi RL, Pontes A, et al. Relationship between visualization of meiotic spindle in human oocytes and ICSI outcomes: a meta-analysis. Reprod Biomed Online. 2009;18(2):235-43. Mahfoudh AM, Moon JH, Henderson S, Garcia-Cerrudo E, Son WY, Dahan MH. Relationship between pre-ICSI meiotic spindle angle, ovarian reserve, gonadotropin stimulation, and pregnancy outcomes. J Assist Reprod Genet. 2017;34(5):609-15. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3828724","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":264871351,"identity":"529b19c6-3b11-4695-9dd8-d8d50c9d09e3","order_by":0,"name":"Chyi-Uei Chern","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chyi-Uei","middleName":"","lastName":"Chern","suffix":""},{"id":264871352,"identity":"052b5175-82ba-4cee-8d1e-fc7b357740bf","order_by":1,"name":"Pei-Fen Liao","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pei-Fen","middleName":"","lastName":"Liao","suffix":""},{"id":264871353,"identity":"898ef88f-af6d-424a-b652-8ea9ac76bbbc","order_by":2,"name":"Chia-Jung Li","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chia-Jung","middleName":"","lastName":"Li","suffix":""},{"id":264871354,"identity":"1ca8ad3f-d623-4530-b7e8-e849ecd4aef3","order_by":3,"name":"Pei-Hsuan Lin","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pei-Hsuan","middleName":"","lastName":"Lin","suffix":""},{"id":264871355,"identity":"6688dca1-e31f-418a-bfac-64cbb0d5fcd6","order_by":4,"name":"Wan-Ping Su","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wan-Ping","middleName":"","lastName":"Su","suffix":""},{"id":264871356,"identity":"9100b56c-aa96-4459-b5d0-39cb7faf1ab4","order_by":5,"name":"Yu-Chen Chen","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yu-Chen","middleName":"","lastName":"Chen","suffix":""},{"id":264871357,"identity":"53f5f7ab-ae3b-47b3-b44a-1ba5525cbcb0","order_by":6,"name":"Kuan-Hao Tsui","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kuan-Hao","middleName":"","lastName":"Tsui","suffix":""},{"id":264871358,"identity":"ae34bf44-ecaf-4119-a290-3d45a32b43a5","order_by":7,"name":"Li-Te Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYBACPmYg8cCAgYefvfngAyCbh4+QFjZmoJ4EAwYZyZ5jyQYgLWwEtTCAtDAw2BjcyDGTgIgQ0sLOf/BBQsEdHoMzB8wqv+bYyQANefjoBn6HMRskGDzjkTzekHZbdlsy0GFsxsY5+LWwSSQYHObhO3Pg2G3JbcxALTxs0gS0sP8AaWG4kdhWLLmtnigtbAwgLQI3ktkYP247TJQWY7DDgIHMLM247TgPGzMBv/DzH3z44cOfw/b87P0fP/7cVg1kND98jE8LCmDmAZPEKgcBxh+kqB4Fo2AUjIIRAwCWuUAyQ7DZWQAAAABJRU5ErkJggg==","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":true,"prefix":"","firstName":"Li-Te","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2024-01-02 06:29:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3828724/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3828724/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49238472,"identity":"66b0ab53-bb2e-4619-8231-6f5045cda51e","added_by":"auto","created_at":"2024-01-05 18:12:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58900,"visible":true,"origin":"","legend":"\u003cp\u003eStudy flow chart.\u003c/p\u003e\n\u003cp\u003eIVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; BMI, body mass index; PGT-A, preimplantation genetic testing for aneuploidy; TESE, testicular sperm extraction; MII, metaphase II; MS, meiotic spindle; PB, polar body; D3, day 3.\u003c/p\u003e","description":"","filename":"Figure1studyflowchart.png","url":"https://assets-eu.researchsquare.com/files/rs-3828724/v1/8776cfcdbc61b4d4fc2a637e.png"},{"id":49238473,"identity":"bdb79b56-a94d-44ca-ad96-2859ae62b19f","added_by":"auto","created_at":"2024-01-05 18:12:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33148,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of fertilization rate, cleavage rate, TQE rate, rate of ≥ 1 TQE, and TQE rate ≥ 50 between the MS-aligned group (blue) and PB-aligned group (red). ** P\u0026lt;0.01, *** P\u0026lt;0.001\u003c/p\u003e\n\u003cp\u003eTQE, top-quality day 3 embryo; MS, meiotic spindle; PB, polar body\u003c/p\u003e","description":"","filename":"Figure2MSalignedvsPBaligned.png","url":"https://assets-eu.researchsquare.com/files/rs-3828724/v1/ae197026a83dc48d49b1860d.png"},{"id":49238470,"identity":"0afed349-9fa3-4588-a457-9fc76362eb2c","added_by":"auto","created_at":"2024-01-05 18:12:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":57851,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Comparison of fertilization rate, cleavage rate, and TQE rate between oocytes with a visible spindle (blue) and those without a visible spindle (red). (B) Comparison of fertilization rate, cleavage rate, and TQE rate between oocytes with no MS deviation (blue) and oocytes with corrected MS deviation using MS-aligned microinjection (red). * P\u0026lt;0.05, ** P\u0026lt;0.01\u003c/p\u003e\n\u003cp\u003eTQE, Top-quality Day 3 embryo; MS, meiotic spindle\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3828724/v1/ce519627c554ca364cafdd55.png"},{"id":49238471,"identity":"9e4914fa-328d-446a-b4e2-b161648601f3","added_by":"auto","created_at":"2024-01-05 18:12:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":225847,"visible":true,"origin":"","legend":"\u003cp\u003eMeiotic spindle visualization under Polscope. (A) Oocytes displaying an absence of a visible spindle. (B) The oocyte spindle is positioned beneath the first polar body. (C) The oocyte spindle exhibits a deviation angle of less than 30° relative to the polar body. (D) The oocyte spindle exhibits a deviation angle of more than 30° relative to the polar body. Blue arrow: meiotic spindle, red arrow: polar body.\u003c/p\u003e","description":"","filename":"Figure4MSviewunderpolscope.png","url":"https://assets-eu.researchsquare.com/files/rs-3828724/v1/7dbf957b6d7a6711ac844e64.png"},{"id":49402121,"identity":"9ff4ca8c-baee-4eb4-a80f-873cf224eacc","added_by":"auto","created_at":"2024-01-10 06:52:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":816439,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3828724/v1/9d40acc9-a306-4dcc-871a-0ba603dcbaf5.pdf"},{"id":49238474,"identity":"b69faa87-e418-4f9e-bf1d-23f2e673ab75","added_by":"auto","created_at":"2024-01-05 18:12:03","extension":"xls","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":123392,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryinformation.xls","url":"https://assets-eu.researchsquare.com/files/rs-3828724/v1/04dd1ee9c017d2254e247cc7.xls"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing Embryo Quality through Meiotic Spindle- Aligned Microinjection in POSEIDON Group 4 Patients Undergoing ICSI Cycles: A Retrospective Cohort Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMeiotic spindles (MS) represent dynamic microtubular structures that play a pivotal role in facilitating the separation of homologous chromosomes and sister chromatids during meiosis, thus serving as a critical determinant of oocyte developmental potential. Disruptions to MS integrity can result in improper chromosomal segregation, subsequently leading to maturation arrest, diminished fertilization rates, and aneuploidy (1\u0026ndash;3).\u003c/p\u003e \u003cp\u003eTraditionally, embryologists have relied on the polar body (PB) as a spatial reference point for orienting the injection site during intracytoplasmic sperm injection (ICSI). This practice aimed to protect MS from potential damage caused by the ICSI procedures, assuming that the MS closely neighbored the PB. However, studies have revealed disparities between the actual MS location and that of the PB. In essence, the PB's location cannot reliably predict the precise location of the MS (4\u0026ndash;6). Consequently, the utilization of PB-aligned ICSI may inadvertently lead to MS damage in cases where the MS location does not align with that of the PB. Fortunately, recent advances in technology have enabled the noninvasive visualization of MS through the application of polarized light. The utilization of a polarized light microscopy system (Polscope) allows for the visualization of structures within living oocytes based on their birefringence properties (7, 8). Given that MS exhibit pronounced birefringence due to microtubule orientation, they can be readily identified using the Polscope (9, 10). Therefore, MS-aligned ICSI, when performed using the Polscope, has the capacity to protect the MS from harm during microinjection procedures (11, 12).\u003c/p\u003e \u003cp\u003eIt is imperative to acknowledge that MS are highly susceptible to physical and chemical perturbations that may occur during oocyte retrieval and handling (13, 14). Additionally, advanced maternal age and oocyte in vitro aging have been correlated with aberrations in MS architecture (9, 15, 16). A prior investigation that compared MS in oocytes from two distinct groups of women\u0026mdash;those aged 20\u0026ndash;25 and those aged 40\u0026ndash;45\u0026mdash;revealed that older women exhibited a significantly higher incidence of abnormalities related to chromosome placement and structure within their MS (15).\u003c/p\u003e \u003cp\u003eWith this backdrop, we hypothesized that PB-aligned ICSI may inflict more substantial damage to MS in older women when the MS location does not align with that of the PB. Conversely, MS-aligned ICSI may yield more favorable outcomes in older women. Nevertheless, there is a paucity of published studies that have explored this specific issue. In 2016, the Patient-Oriented Strategies Encompassing Individualized Oocyte Number (POSEIDON) group introduced a novel classification system aimed at tailoring approaches for patients with a less favorable prognosis (17). Specifically, POSEIDON Group 4 comprises women characterized by advanced age and diminished ovarian reserve. Accordingly, we designed a retrospective cohort study to compare embryo quality between MS-aligned and PB-aligned ICSI procedures in patients meeting the criteria of POSEIDON Group 4, who subsequently underwent ICSI treatments.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design and Participants\u003c/h2\u003e \u003cp\u003eThis retrospective cohort study was conducted at the Reproductive Medical Center of Kaohsiung Veterans General Hospital during the period from January 2019 to March 2023. The study received ethical approval from the Institutional Review Board of Kaohsiung Veterans General Hospital (Institutional Review Board reference number: KSVGH23-CT8-02). Given its retrospective nature, the requirement for obtaining informed consent was waived by the Institutional Review Board of Kaohsiung Veterans General Hospital. Data for this study were meticulously extracted from electronic medical records and in vitro fertilization (IVF) treatment documentation. The study cohort consisted of women who met the criteria of POSEIDON Group 4 and had undergone their initial ICSI cycle at our center. POSEIDON Group 4 criteria referred to women aged\u0026thinsp;\u0026ge;\u0026thinsp;35 years, accompanied by an anti-M\u0026uuml;llerian hormone (AMH) level\u0026thinsp;\u0026lt;\u0026thinsp;1.2 ng/ml and/or antral follicle counts (AFCs)\u0026thinsp;\u0026lt;\u0026thinsp;5. In addition, the study included women aged\u0026thinsp;\u0026le;\u0026thinsp;45 years with a body mass index (BMI) falling within the range of 18\u0026ndash;30 kg/m\u0026sup2;. Exclusion criteria included women who had undergone preimplantation genetic testing for aneuploidy (PGT-A), oocyte recipients, cases involving testicular sperm extraction (TESE) in their partners, and individuals lacking metaphase II (MII) oocytes.\u003c/p\u003e \u003cp\u003eFinally, a total of 315 patients were identified and subsequently categorized into two groups: the MS-aligned group (n\u0026thinsp;=\u0026thinsp;168) and the PB-aligned group (n\u0026thinsp;=\u0026thinsp;147) for microinjection procedures. In the PB-aligned group, oocyte alignment was achieved with reference to the first polar body, a cellular structure generated through the process of oocyte meiosis, encapsulating a nucleus formed during meiotic division and a minimal cytoplasmic volume. In this group, the first polar body was positioned at the 12 o'clock position, while the exact location of the MS remained undetermined, and sperm microinjection was carried out at the 3 o'clock position. However, in the MS-aligned group, oocytes were manipulated to align their MS at the 12 o'clock position, a process confirmed using a polarized light microscope. A schematic representation of the study's workflow is depicted in Fig.\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eFigure 1 Study flow chart.\u003c/p\u003e \u003cp\u003eIVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; BMI, body mass index; PGT-A, preimplantation genetic testing for aneuploidy; TESE, testicular sperm extraction; MII, metaphase II; MS, meiotic spindle; PB, polar body; D3, day 3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of sperm\u003c/h2\u003e \u003cp\u003eEjaculated spermatozoa were collected through masturbation following a period of 3 to 5 days of ejaculatory abstinence. After the semen had undergone liquefaction at room temperature, the sperm samples were meticulously processed via a one-layer density-gradient centrifugation method. In this process, the lowermost fraction was gently aspirated, underwent two sequential washes utilizing SepaSperm wash medium (KITAZATO, Japan), each at a speed of 2,500 rpm for 5 minutes, and was subsequently incubated at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eOocyte denudation\u003c/h2\u003e \u003cp\u003eFollowing retrieval, the collected oocytes were incubated in fertilization medium (LGTF, LifeGlobal, CooperSurgical, Denmark) and covered with paraffin oil (Liquid Paraffin, Origio, CooperSurgical, Denmark). This incubation was carried out at 37\u0026deg;C in an environment with 6% CO2 for a duration of 2 hours. Cumulus cells were subsequently removed by exposing the oocytes to ICSI Cumulase (Origio, CooperSurgical, Denmark) for 30 seconds, followed by a wash using HEPES-buffered HTF containing 5 mg/ml human serum albumin (LGTH, LifeGlobal, CooperSurgical, Denmark). The corona cells were meticulously removed using a series of finely drawn glass Pasteur pipettes. Following denudation, the oocytes were assessed for both structural integrity and meiotic maturity. Thereafter, they were placed in culture dishes (Oosafe, Astec Bio, USA) within a fertilization medium (LGTF, LifeGlobal, CooperSurgical, Denmark) and covered with a paraffin oil overlay (Liquid Paraffin, Origio, CooperSurgical, Denmark) in preparation for microinjection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIntracytoplasmic sperm injection\u003c/h2\u003e \u003cp\u003ePB-aligned oocytes were gently transferred into 4 \u0026micro;l of prewarmed HEPES-buffered HTF containing 5 mg/ml human serum albumin (LGTH, LifeGlobal, CooperSurgical, Denmark) during the microinjection procedures. Sperm samples were prepared in a polyvinylpyrrolidone solution (SAGE, CooperSurgical, Denmark) in the microinjection dish immediately before sperm injection. The oocytes were positioned under an inverted microscope (Olympus IX73, Japan) equipped with a heated stage maintained at 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C and were observed at \u0026times;400 magnification. Subsequently, sperm injection into the mature oocytes was performed, with the polar body positioned at 12:00. After injection, the oocytes were transferred to a culture medium (LGGT, LifeGlobal, Coopersurgical, Denmark) and placed in an environment maintained at 37\u0026deg;C with 6% CO2. For the MS-aligned oocytes, a volume of 5 \u0026micro;l of HEPES-buffered HTF containing 5 mg/ml human serum albumin (LGTH, LifeGlobal, CooperSurgical, Denmark) was added, and the oocytes were then covered with paraffin oil (Liquid Paraffin, Origio, CooperSurgical, Denmark) within a glass-bottomed dish (MATSUNAMI, Japan). The temperature was maintained at 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C using a heated stage (Integra3, RI, UK). MS visualization was achieved using a Polscope system integrated with an inverted microscope (Olympus IX73, Japan). The oocyte was immobilized with a holding pipette and gently rotated using the injection pipette until both the MS and PB were clearly visible within the oocyte. ICSI was promptly performed following this imaging. For oocytes with evident spindles, oocytes were positioned such that the MS was at the 12 o'clock position, and sperm injection was carried out at the 3 o'clock position to minimize disruption of the MS. For oocytes without visible spindles, the oocytes were injected with their PB at the 12 o\u0026rsquo;clock position. However, oocytes lacking a visible spindle were excluded from the analysis of the MS-aligned group, as no spindle was present for alignment. Following microinjection and imaging procedures, the oocytes were transferred to individual 50 \u0026micro;l culture medium droplets (LGGT, LifeGlobal, Coopersurgical, Denmark) within sterile plastic dishes. These dishes were subsequently placed in an incubator with a controlled atmosphere of 6% CO2 at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFertilization and embryo development\u003c/h2\u003e \u003cp\u003eFertilization status was assessed 16\u0026ndash;18 hours following sperm injection. Oocytes displaying two distinct pronuclei with extrusion of the second PB were categorized as having undergone normal fertilization. On day 3, embryonic cleavage was examined, and embryo quality was assessed in accordance with the criteria established in the Istanbul consensus workshop (18). Embryos were stratified into Grade 1 to Grade 3 categories based on several parameters, including the percentage of fragmentation, uniformity of blastomeres, and the presence of multinucleation. Grade 1 embryos exhibited less than 10% fragmentation, uniform-sized blastomeres, and an absence of multinucleation. Grade 2 embryos displayed 10\u0026ndash;25% fragmentation, predominantly uniform-sized blastomeres, and no multinucleation. Grade 3 embryos featured more than 25% fragmentation, uneven blastomere sizes, and evidence of multinucleation. Top-quality Day 3 embryos (TQE) were specifically defined as embryos with 6\u0026ndash;10 cells and a Grade 1 classification, signifying that they had less than 10% fragmentation, uniform-sized blastomeres, and no multinucleation. Our reproductive medical center implemented a freeze-all policy. Given the diminished quality of embryos within our study cohort, the majority of embryos were cryopreserved at the cleavage stage. Only a limited number of embryos were subjected to extended culture to the blastocyst stage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOutcome measures\u003c/h2\u003e \u003cp\u003eThe primary outcome measure in this study was the rate of TQE. Secondary outcome measures encompassed the assessment of the fertilization rate, the incidence of total fertilization failure, and the cleavage rate. The fertilization rate was calculated as the number of observed two pronuclei zygotes divided by the total number of oocytes subjected to microinjection. Total fertilization failure was defined as the absence of fertilization for all available oocytes within a single ICSI cycle. The cleavage rate was defined as the ratio of the number of day 3 embryos to the number of fertilized oocytes. The TQE rate was determined by dividing the number of top-quality day 3 embryos by the total number of day 3 embryos.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe normal distribution of continuous variables was assessed using the Kolmogorov\u0026ndash;Smirnov test. Student\u0026rsquo;s t test was employed for quantitative variables that exhibited a normal distribution, had sufficiently large sample sizes, or both; conversely, the Mann\u0026ndash;Whitney U test was utilized for nonnormally distributed data. For categorical data, a comparison was made using either the chi-squared test or Fisher's exact test, as appropriate. To identify the independent effects of MS-aligned microinjection on the attainment of a rate of at least one TQE and a TQE rate\u0026thinsp;\u0026ge;\u0026thinsp;50, multivariable logistic regression was performed. This analysis was adjusted for various covariates, including age, body mass index, previous IVF attempts, types of infertility, basal follicle-stimulating hormone (FSH) levels, AMH levels, and the number of MII oocytes. The results of this analysis are expressed as odds ratios (ORs) with corresponding 95% confidence intervals (CIs). Statistical significance was determined using a two-tailed P value of \u0026lt;\u0026thinsp;0.05. Data processing and statistical analyses were conducted using IBM SPSS Statistics version 20.0 (IBM Corp., Armonk, NY, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eTable 1 summarizes the baseline characteristics of the study cohort. The MS-aligned group displayed a higher mean age and a greater number of previous IVF attempts than the PB-aligned group. However, no statistically significant differences were observed between the two groups in terms of parameters such as body mass index, duration of infertility, and infertility types. Additionally, the basal levels of FSH and AMH were comparable between the two groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1 Baseline characteristics of women undergoing MS-aligned or PB-aligned ICSI cycles\u003c/strong\u003e\u003c/p\u003e\n\u003ctable width=\"645\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003eParameters\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003eMS-aligned group (n=168)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003ePB-aligned group\u003c/p\u003e\n\u003cp\u003e(n=147)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003eAge (years)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e41.1\u0026plusmn;2.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e40.0\u0026plusmn;2.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003eBody mass index (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e22.7\u0026plusmn;3.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e22.7\u0026plusmn;2.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e0.972\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003eInfertility duration (years)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e4.7\u0026plusmn;3.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e4.5\u0026plusmn;3.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e0.609\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003ePrevious IVF attempts (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e0.017\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003e0-1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e28.6% (48/168)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e36.1% (53/147)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e30.4% (51/168)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e38.1% (56/147)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003e≧3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e41.1% (69/168)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e25.9% (38/147)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003eTypes of infertility (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e0.964\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003ePrimary infertility\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e49.4% (83/168)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e49.7% (73/147)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003eSecondary infertility\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e50.6% (85/168)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e50.3% (74/147)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003eBasal FSH (IU/l)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e6.8\u0026plusmn;5.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e6.1\u0026plusmn;4.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e0.203\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"257\"\u003e\n\u003cp\u003eAnti-M\u0026uuml;llerian hormone (ng/mL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"151\"\u003e\n\u003cp\u003e0.77\u0026plusmn;0.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"149\"\u003e\n\u003cp\u003e0.73\u0026plusmn;0.32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e0.412\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eData are presented as the mean \u0026plusmn; standard deviation or %.\u003c/p\u003e\n\u003cp\u003eMS, meiotic spindle; PB, polar body; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; FSH, follicle-stimulating hormone\u003c/p\u003e\n\u003cp\u003eTable 2 presents a detailed comparison of various treatment-related parameters between the two groups. No statistically significant differences were observed in the stimulation duration, gonadotropin dosage, number of retrieved oocytes, number of MII oocytes, number of fertilized oocytes, or number of day 3 embryos between the two groups. However, the MS-aligned group achieved significantly superior outcomes in several key parameters, including a higher fertilization rate (87.8\u0026plusmn;19.8% vs. 79.5\u0026plusmn;30.5%, P = 0.006), a greater cleavage rate (88.7\u0026plusmn;22.6% vs. 72.3\u0026plusmn;41.0%, P \u0026lt; 0.001), an elevated TQE rate (54.2\u0026plusmn;37.2% vs. 32.2\u0026plusmn;35.8%, P \u0026lt; 0.001), a higher rate of achieving \u0026ge; 1 TQE (78.6% vs. 55.1%, P = 0.003) and a greater TQE rate of \u0026ge; 50 (61.9% vs. 38.1%, P \u0026lt; 0.001) when compared to the PB-aligned group (Figure 2). Furthermore, the MS-aligned group displayed a notably reduced incidence of total fertilization failure (0.6% vs. 8.8%, P \u0026lt; 0.001) and a significantly increased number of TQE (1.7\u0026plusmn;1.6 vs. 1.3\u0026plusmn;1.8, P = 0.040) in comparison to the PB-aligned group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2 Cycle characteristics of women undergoing MS-aligned or PB-aligned ICSI cycles\u003c/strong\u003e\u003c/p\u003e\n\u003ctable width=\"662\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eParameters\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003eMS-aligned group\u003c/p\u003e\n\u003cp\u003e(n=168)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003ePB-aligned group\u003c/p\u003e\n\u003cp\u003e(n=147)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eStimulation duration (days)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e10.7\u0026plusmn;2.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e10.3\u0026plusmn;2.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.139\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eGonadotropin dosage (IU)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003e\u0026nbsp; with corifollitropin alfa\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e1727.6\u0026plusmn;515.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e1440.0\u0026plusmn;997.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.051\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003e\u0026nbsp; without corifollitropin alfa\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e2839.0\u0026plusmn;834.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e2929.3\u0026plusmn;834.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.456\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eNo. of retrieved oocytes (n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e5.3\u0026plusmn;3.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e5.1\u0026plusmn;3.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.624\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eNo. of metaphase II oocytes (n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e4.0\u0026plusmn;2.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e4.1\u0026plusmn;3.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.786\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eMaturation rate (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e77.0\u0026plusmn;20.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e78.9\u0026plusmn;24.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.440\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eNo. of fertilized oocytes (n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e3.4\u0026plusmn;2.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e3.5\u0026plusmn;2.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.703\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eFertilization rate (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e87.8\u0026plusmn;19.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e79.5\u0026plusmn;30.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.006\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eTotal fertilization failure (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e0.6% (1/168)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e8.8% (13/147)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eNo. of Day 3 embryos (n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e3.0\u0026plusmn;2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e2.9\u0026plusmn;2.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.618\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eCleavage rate (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e88.7\u0026plusmn;22.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e72.3\u0026plusmn;41.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eNo. of top-quality Day 3 embryos (n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e1.7\u0026plusmn;1.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e1.3\u0026plusmn;1.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.040\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eRate of top-quality Day 3 embryos (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e54.2\u0026plusmn;37.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e32.2\u0026plusmn;35.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003e≧1 top-quality Day 3 embryos rate (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e78.6% (132/168)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e55.1% (81/147)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"284\"\u003e\n\u003cp\u003eTop-quality Day 3 embryos rate≧50 (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e61.9% (104/168)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e38.1% (56/147)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eData are presented as the mean \u0026plusmn; standard deviation or % (n).\u003c/p\u003e\n\u003cp\u003eMS, meiotic spindle; PB, polar body; ICSI, intracytoplasmic sperm injection\u003c/p\u003e\n\u003cp\u003eFigure 2 Comparison of fertilization rate, cleavage rate, TQE rate, rate of \u0026ge; 1 TQE, and TQE rate \u0026ge; 50 between the MS-aligned group (blue) and PB-aligned group (red). ** P\u0026lt;0.01, *** P\u0026lt;0.001\u003c/p\u003e\n\u003cp\u003eTQE, top-quality day 3 embryo; MS, meiotic spindle; PB, polar body\u003c/p\u003e\n\u003cp\u003eTable 3 presents the results of a binary logistic regression analysis conducted to evaluate the impact of MS-aligned microinjection on the likelihood of achieving \u0026ge; 1 TQE and a TQE rate of \u0026ge; 50 in women belonging to POSEIDON Group 4. The analysis accounted for potential confounding variables, including age, body mass index, previous IVF attempts, types of infertility, basal FSH levels, AMH levels, and the number of MII oocytes. The multivariate analysis demonstrated that the MS-aligned group exerted favorable effects on the probability of achieving \u0026ge; 1 TQE (OR 4.22, 95% CI 2.24\u0026ndash;7.96, P \u0026lt; 0.001) and attaining a TQE rate of \u0026ge; 50 (OR 3.63, 95% CI 2.06\u0026ndash;6.39, P \u0026lt; 0.001). Additionally, it was noted that the number of MII oocytes represented an independent factor capable of influencing the likelihood of achieving \u0026ge; 1 TQE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3 Logistic regression analyses of factors influencing top-quality \u003c/strong\u003e\u003cstrong\u003eday\u003c/strong\u003e\u003cstrong\u003e 3 embryos in POSEIDON \u003c/strong\u003e\u003cstrong\u003egroup 4 patients\u003c/strong\u003e\u003c/p\u003e\n\u003ctable width=\"105%\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"30%\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"32%\"\u003e\n\u003cp\u003e\u003cstrong\u003e≧ \u003c/strong\u003e\u003cstrong\u003e1 TQE\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"1%\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"3\" width=\"35%\"\u003e\n\u003cp\u003e\u003cstrong\u003eTQE rate \u003c/strong\u003e\u003cstrong\u003e≧ \u003c/strong\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"19%\"\u003e\n\u003cp\u003eAdjusted OR*\u003c/p\u003e\n\u003cp\u003e(95% CI)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"13%\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"1%\"\u003e\n\u003cp\u003e \u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"18%\"\u003e\n\u003cp\u003eAdjusted OR*\u003c/p\u003e\n\u003cp\u003e(95% CI)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"16%\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003eMS-aligned vs. PB-aligned\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"19%\"\u003e\n\u003cp\u003e4.22(2.24\u0026ndash;7.96)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"13%\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"1%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"19%\"\u003e\n\u003cp\u003e3.63(2.06\u0026ndash;6.39)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003eAge (years)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"19%\"\u003e\n\u003cp\u003e0.99(0.87\u0026ndash;1.11)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"13%\"\u003e\n\u003cp\u003e0.806\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"1%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"19%\"\u003e\n\u003cp\u003e0.91(0.82\u0026ndash;1.02)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003e0.105\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"19%\"\u003e\n\u003cp\u003e1.02(0.92\u0026ndash;1.13)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"13%\"\u003e\n\u003cp\u003e0.701\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"1%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"19%\"\u003e\n\u003cp\u003e1.09(1.00\u0026ndash;1.20)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003e0.061\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003ePrevious IVF attempts\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"19%\"\u003e\n\u003cp\u003e0.75(0.51\u0026ndash;1.11)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"13%\"\u003e\n\u003cp\u003e0.152\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"1%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"19%\"\u003e\n\u003cp\u003e0.81(0.58\u0026ndash;1.15)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003e0.236\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003eTypes of infertility\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"19%\"\u003e\n\u003cp\u003e0.61(0.33\u0026ndash;1.16)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"13%\"\u003e\n\u003cp\u003e0.269\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"1%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"19%\"\u003e\n\u003cp\u003e0.65(0.37\u0026ndash;1.14)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003e0.132\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003eBasal FSH (IU/l)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"19%\"\u003e\n\u003cp\u003e0.98(0.93\u0026ndash;1.04)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"13%\"\u003e\n\u003cp\u003e0.378\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"1%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"19%\"\u003e\n\u003cp\u003e0.98(0.94\u0026ndash;1.04)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003e0.530\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003eAMH (ng/mL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"19%\"\u003e\n\u003cp\u003e1.69(0.69\u0026ndash;4.15)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"13%\"\u003e\n\u003cp\u003e0.251\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"1%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"19%\"\u003e\n\u003cp\u003e1.02(0.47\u0026ndash;2.22)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003e0.966\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003eNo. of MII oocytes (n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"19%\"\u003e\n\u003cp\u003e1.45(1.23\u0026ndash;1.69)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"13%\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"1%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"19%\"\u003e\n\u003cp\u003e1.07(0.95\u0026ndash;1.20)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003e0.289\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTQE, top-quality day 3 embryos; OR, odds ratio; CI, confidence interval; MS, meiotic spindle; PB, polar body; BMI, body mass index; IVF, in vitro fertilization; FSH, follicle-stimulating hormone; AMH, anti-M\u0026uuml;llerian hormone; MII, metaphase II.\u003c/p\u003e\n\u003cp\u003e*Adjustment for age, BMI, previous IVF attempts, types of infertility, basal FSH, AMH and number of MII oocytes\u003c/p\u003e\n\u003cp\u003eIn the MS-aligned group, a total of 441 oocytes were assessed. However, 8.4% (n=37) of these oocytes exhibited no visible MS and were subsequently inseminated using the PB-aligned method. When comparing oocytes with a visible MS to those without, the latter group displayed significantly lower fertilization rates (73.0% vs. 85.4%, P = 0.046), cleavage rates (59.5% vs. 80.2%, P = 0.003), and TQE rates (18.9% vs. 35.9%, P = 0.038), as illustrated in Figure 3A. Among the 404 oocytes with a visible MS, 42.6% (n=172) exhibited no MS deviation (MS at 0\u0026deg; in relation to the PB), while 57.4% (n=232) displayed MS deviation, which was corrected using MS-aligned microinjection. Consequently, the fertilization rate, cleavage rate, and TQE rate were comparable between these two groups, as depicted in Figure 3B. However, among the oocytes with MS deviation, 59.5% exhibited a deviation angle of less than 30\u0026deg; (MS \u0026lt; 30\u0026deg; in relation to the PB). Figure 4 illustrates the oocytes without visible MS and those with visible MS, whether or not they displayed deviation.\u003c/p\u003e\n\u003cp\u003eFigure 3 (A) Comparison of fertilization rate, cleavage rate, and TQE rate between oocytes with a visible spindle (blue) and those without a visible spindle (red). (B) Comparison of fertilization rate, cleavage rate, and TQE rate between oocytes with no MS deviation (blue) and oocytes with corrected MS deviation using MS-aligned microinjection (red). * P\u0026lt;0.05, ** P\u0026lt;0.01\u003c/p\u003e\n\u003cp\u003e\u003cbr /\u003e TQE, Top-quality Day 3 embryo; MS, meiotic spindle\u003c/p\u003e\n\u003cp\u003eFigure 4 Meiotic spindle visualization under Polscope. (A) Oocytes displaying an absence of a visible spindle. (B) The oocyte spindle is positioned beneath the first polar body. (C) The oocyte spindle exhibits a deviation angle of less than 30\u0026deg; relative to the polar body. (D) The oocyte spindle exhibits a deviation angle of more than 30\u0026deg; relative to the polar body. Blue arrow: meiotic spindle, red arrow: polar body.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis retrospective study represents the first investigation into the impact of MS-aligned microinjection on embryo quality in patients meeting the criteria of POSIDEN Group 4. Our findings indicate that the MS-aligned group exhibited significantly higher fertilization rates, cleavage rates, and TQE rates than the PB-aligned group. Additionally, the multivariate analysis demonstrated a 4.22-fold increase in the likelihood of achieving more than 1 TQE (95% CI 2.24\u0026ndash;7.96, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and a 3.63-fold increase in the likelihood of attaining a TQE rate\u0026thinsp;\u0026ge;\u0026thinsp;50 (95% CI 2.06\u0026ndash;6.39, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in POSEIDON Group 4 patients undergoing MS-aligned microinjection compared to those subjected to the PB-aligned method.\u003c/p\u003e \u003cp\u003eDuring the ICSI procedure, oocytes are arrested at the metaphase II stage of the meiotic cell cycle when chromosomes are aligned at the equatorial region of the MS. Microtubules within the MS are responsible for the precise separation of chromosomes and are highly susceptible to oocyte manipulations (1, 3, 14). However, the position of the MS cannot be accurately predicted by the first PB\u0026rsquo;s location (4\u0026ndash;6). Our study provides support for this notion. We observed that among oocytes with a visible MS, 57.4% exhibited a deviation in the MS position relative to the PB. Within this subset of oocytes with MS deviation, 59.5% displayed a deviation angle of less than 30\u0026deg; relative to the PB, while 40.5% exhibited a deviation angle of more than 30\u0026deg;. Heindryckx et al. demonstrated a significantly lower prevalence of MS located adjacent to the first PB in in vivo matured oocytes compared to in vitro matured oocytes (64% vs. 94%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (19). Furthermore, findings from Hardarson et al. indicated that in vivo matured oocytes exhibited significantly greater degrees of MS deviation from the PB position than in vitro matured oocytes (41.7\u0026deg; vs. 26.6\u0026deg;, P\u0026thinsp;=\u0026thinsp;0.005) (5). These findings suggest that the increased MS deviation in in vivo matured oocytes may result from lateral displacement of the first PB during manipulations for cumulus and corona removal since in vitro matured oocytes were denuded before the extrusion of the first PB. Consequently, the use of PB-aligned ICSI could result in MS injury when the MS is not located directly adjacent to the PB. However, the needle used for ICSI is relatively small in size in comparison to the oocyte, making it unlikely to cause MS damage during needle insertion. Instead, MS injury is more likely to occur during the aspiration of the oocyte cytoplasm just prior to sperm injection.\u003c/p\u003e \u003cp\u003eTherefore, the utilization of MS-aligned ICSI with the Polscope has the potential to effectively protect the MS from harm during microinjection procedures. A study conducted by Asa et al. involved a randomized controlled trial with the enrollment of 264 oocytes from 24 patients. These oocytes were randomly assigned to either the spindle-aligned group (n\u0026thinsp;=\u0026thinsp;138) or the PB-aligned group (n\u0026thinsp;=\u0026thinsp;126). The results of this study revealed a significantly higher fertilization rate in the spindle-aligned group than in the PB-aligned group (78.5% vs. 65.0%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (11). Furthermore, in a separate randomized controlled trial led by Cooke et al., sibling oocytes within patients were allocated to either the spindle-aligned group (n\u0026thinsp;=\u0026thinsp;124) or the PB-aligned group (n\u0026thinsp;=\u0026thinsp;122) for microinjection. This investigation demonstrated that embryos originating from the spindle-aligned group exhibited significantly higher embryo quality scores than those from the PB-aligned group (12). It is important to note that the design of previous studies was based on a per-oocyte analysis. However, our research specifically targeted a distinct patient subgroup\u0026mdash;patients meeting the criteria of POSEIDON Group 4. POSEIDON Group 4 comprises women characterized by advanced age and diminished ovarian reserve. In women of advanced age, MS may be more susceptible to damage during oocyte manipulations due to the increased prevalence of MS abnormalities with age (15, 16). Additionally, for women with diminished ovarian reserve, obtaining a greater number of high-quality embryos may reduce the necessity for repeated IVF cycles. Therefore, we hypothesized that MS-aligned microinjection would yield more substantial benefits within this particular patient group. Our findings indeed support this hypothesis. The MS-aligned group exhibited significantly higher fertilization rates, cleavage rates, and TQE rates than the PB-aligned group, validating our initial speculation. Furthermore, within the spindle-aligned group, women with MS deviation corrected using MS-aligned microinjection exhibited fertilization rates, cleavage rates, and TQE rates similar to those of women without MS deviation. This suggests that MS-aligned microinjection can effectively prevent MS damage during ICSI. However, it is imperative to emphasize that additional large-scale prospective studies are necessary to further corroborate and validate these results.\u003c/p\u003e \u003cp\u003eThe incidence of oocytes lacking a visible MS under Polscope examination ranged from 7\u0026ndash;34.1% across different studies (11, 12, 20\u0026ndash;23). In our current investigation, within the spindle-aligned group, 8.4% of oocytes exhibited a nonvisible MS. Oocytes with a visible MS demonstrated significantly higher rates of fertilization, cleavage, and TQE than those without a visible MS. These findings are similar to the outcomes reported in previous studies (6, 10, 21, 23\u0026ndash;25). In a meta-analysis encompassing data from 10 studies, a substantial increase in fertilization success was observed in 4,684 oocytes with visualized MS compared to 1,264 oocytes where no MS was observed (OR 1.79, 95% CI 1.57\u0026ndash;2.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Furthermore, embryos derived from oocytes with visible MS exhibited higher cleavage rates (OR 1.85, 95% CI 1.47\u0026ndash;2.32, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), TQE rates (OR 1.70, 95% CI 1.20\u0026ndash;2.42, P\u0026thinsp;=\u0026thinsp;0.003), and blastocyst formation rates (OR 2.61, 95% CI 1.74\u0026ndash;3.91, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (24). Additionally, a retrospective cohort investigation demonstrated that embryos resulting from oocytes with MS angles ranging from 0\u0026deg; to 29\u0026deg; were associated with higher pregnancy rates and live birth rates when compared to oocytes lacking a visible MS (25). Thus, the presence or absence of a visible MS within oocytes can serve as a valuable predictor for outcomes in ICSI procedures.\u003c/p\u003e \u003cp\u003eThis study is subject to several limitations that warrant consideration. First, its retrospective design and the relatively limited sample size represent the primary limitations. To validate our findings, large-scale prospective studies are imperative. Second, the decision to perform either MS-aligned or PB-aligned microinjection relied on the clinical judgment and preferences of physicians and embryologists, potentially introducing bias. Third, the exclusion of oocytes lacking a visible spindle from the MS-aligned group's analysis might introduce additional bias.\u003c/p\u003e \u003cp\u003eIn conclusion, our study findings suggest that MS-aligned microinjection could lead to improved embryo quality compared to PB-aligned microinjection, particularly among older patients with diminished ovarian reserve undergoing ICSI cycles. Nonetheless, it is crucial to acknowledge and address these limitations, emphasizing the need for further extensive prospective investigations to validate and reinforce our conclusions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAFCs, antral follicle counts; AMH, anti-M\u0026uuml;llerian hormone; BMI, body mass index; CI, confidence interval; FSH, follicle-stimulating hormone; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; MII, metaphase II; MS, meiotic spindle; OR, odds ratio; PB, polar body; PGT-A, preimplantation genetic testing for aneuploidy; TESE, testicular sperm extraction; TQE, top-quality\u0026nbsp;day\u0026nbsp;3 embryos\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study adhered to the principles set forth in the Declaration of Helsinki regarding medical research involving human subjects. Furthermore, it received ethical approval from the Institutional Review Board of Kaohsiung Veterans General Hospital, identified as KSVGH23-CT8-02\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eThe study was conducted in strict accordance with the approved guidelines. Notably, the need for informed consent was waived by the Institutional Review Board of Kaohsiung Veterans General Hospital due to the retrospective nature of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePei-Hsuan Lin, Yu-Chen Chen and Kuan-Hao Tsui contributed to the study\u0026apos;s conception and design; Pei Fen Liao and Wan Ping Su were responsible for organizing the database; Chia-Jung Li and Li-Te Lin conducted the statistical analysis; Chyi-Uei Chern drafted the initial manuscript, with Li-Te Lin contributing to specific sections. All authors participated in the manuscript\u0026apos;s review and approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHowe K, FitzHarris G. Recent insights into spindle function in mammalian oocytes and early embryos. Biology of reproduction. 2013;89(3):71.\u003c/li\u003e\n\u003cli\u003eWang WH, Keefe DL. Prediction of chromosome misalignment among in vitro matured human oocytes by spindle imaging with the PolScope. Fertil Steril. 2002;78(5):1077-81.\u003c/li\u003e\n\u003cli\u003eSeverson AF, von Dassow G, Bowerman B. Oocyte Meiotic Spindle Assembly and Function. Curr Top Dev Biol. 2016;116:65-98.\u003c/li\u003e\n\u003cli\u003eSilva CP, Kommineni K, Oldenbourg R, Keefe DL. The first polar body does not predict accurately the location of the metaphase II meiotic spindle in mammalian oocytes. Fertil Steril. 1999;71(4):719-21.\u003c/li\u003e\n\u003cli\u003eHardarson T, Lundin K, Hamberger L. The position of the metaphase II spindle cannot be predicted by the location of the first polar body in the human oocyte. Hum Reprod. 2000;15(6):1372-6.\u003c/li\u003e\n\u003cli\u003eKonc J, Kany\u0026oacute; K, Cseh S. Visualization and examination of the meiotic spindle in human oocytes with polscope. J Assist Reprod Genet. 2004;21(10):349-53.\u003c/li\u003e\n\u003cli\u003eKeefe D, Liu L, Wang W, Silva C. Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF. Reprod Biomed Online. 2003;7(1):24-9.\u003c/li\u003e\n\u003cli\u003eWang WH, Keefe DL. Spindle observation in living mammalian oocytes with the polarization microscope and its practical use. Cloning and stem cells. 2002;4(3):269-76.\u003c/li\u003e\n\u003cli\u003eWang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil Steril. 2001;75(2):348-53.\u003c/li\u003e\n\u003cli\u003eWang WH, Meng L, Hackett RJ, Keefe DL. Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination. Hum Reprod. 2001;16(7):1464-8.\u003c/li\u003e\n\u003cli\u003eAsa E, Tabatabaee R, Farrokhi A, Nejatbakhsh R. Relationship between meiotic spindles visualization and intracytoplasmic sperm injection outcomes in human oocytes. Anat Cell Biol. 2017;50(1):26-32.\u003c/li\u003e\n\u003cli\u003eCooke S, Tyler JP, Driscoll GL. Meiotic spindle location and identification and its effect on embryonic cleavage plane and early development. Hum Reprod. 2003;18(11):2397-405.\u003c/li\u003e\n\u003cli\u003eCan A, Semiz O, Cinar O. Bisphenol-An induces cell cycle delay and alters centrosome and spindle microtubular organization in oocytes during meiosis. Molecular human reproduction. 2005;11(6):389-96.\u003c/li\u003e\n\u003cli\u003eWang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy. Hum Reprod. 2001;16(11):2374-8.\u003c/li\u003e\n\u003cli\u003eBattaglia DE, Goodwin P, Klein NA, Soules MR. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod. 1996;11(10):2217-22.\u003c/li\u003e\n\u003cli\u003eWang ZB, Schatten H, Sun QY. Why is chromosome segregation error in oocytes increased with maternal aging? Physiology (Bethesda, Md). 2011;26(5):314-25.\u003c/li\u003e\n\u003cli\u003eAlviggi C, Andersen CY, Buehler K, Conforti A, De Placido G, Esteves SC, et al. A new more detailed stratification of low responders to ovarian stimulation: from a poor ovarian response to a low prognosis concept. Fertil Steril. 2016;105(6):1452-3.\u003c/li\u003e\n\u003cli\u003eAlpha Scientists in Reproductive M, Embryology ESIGo. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26(6):1270-83.\u003c/li\u003e\n\u003cli\u003eHeindryckx B, De Gheselle S, Lierman S, Gerris J, De Sutter P. Efficiency of polarized microscopy as a predictive tool for human oocyte quality. Hum Reprod. 2011;26(3):535-44.\u003c/li\u003e\n\u003cli\u003eRienzi L, Ubaldi F, Martinez F, Iacobelli M, Minasi MG, Ferrero S, et al. Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI. Hum Reprod. 2003;18(6):1289-93.\u003c/li\u003e\n\u003cli\u003eMoon JH, Hyun CS, Lee SW, Son WY, Yoon SH, Lim JH. Visualization of the metaphase II meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI. Hum Reprod. 2003;18(4):817-20.\u003c/li\u003e\n\u003cli\u003eKilani S, Cooke S, Tilia L, Chapman M. Does meiotic spindle normality predict improved blastocyst development, implantation and live birth rates? Fertil Steril. 2011;96(2):389-93.\u003c/li\u003e\n\u003cli\u003eMadaschi C, de Souza Bonetti TC, de Almeida Ferreira Braga DP, Pasqualotto FF, Iaconelli A, Jr., Borges E, Jr. Spindle imaging: a marker for embryo development and implantation. Fertil Steril. 2008;90(1):194-8.\u003c/li\u003e\n\u003cli\u003ePetersen CG, Oliveira JB, Mauri AL, Massaro FC, Baruffi RL, Pontes A, et al. Relationship between visualization of meiotic spindle in human oocytes and ICSI outcomes: a meta-analysis. Reprod Biomed Online. 2009;18(2):235-43.\u003c/li\u003e\n\u003cli\u003eMahfoudh AM, Moon JH, Henderson S, Garcia-Cerrudo E, Son WY, Dahan MH. Relationship between pre-ICSI meiotic spindle angle, ovarian reserve, gonadotropin stimulation, and pregnancy outcomes. J Assist Reprod Genet. 2017;34(5):609-15.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"embryo quality, intracytoplasmic sperm injection (ICSI), meiotic spindle, polar body, diminished ovarian reserve","lastPublishedDoi":"10.21203/rs.3.rs-3828724/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3828724/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEmbryologists have traditionally depended on the polar body (PB) as a reference point for guiding the process of intracytoplasmic sperm injection (ICSI). However, its accuracy in predicting the precise location of the meiotic spindle (MS) is constrained. To overcome this limitation, polarized light microscopy has emerged as a noninvasive technique for visualizing and precisely positioning the MS during ICSI procedures. The primary aim of our study was to assess whether implementing MS alignment in ICSI results in improved embryo quality compared to conventional PB alignment, specifically in the POSEIDON group 4 population.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis retrospective cohort study included 315 women who met the POSEIDON Group 4 criteria and underwent ICSI cycles. The study group comprised 168 women who underwent MS-aligned microinjection, while 147 women in the control group underwent the PB-aligned method. We conducted a comparative analysis of baseline and cycle characteristics as well as embryo quality between these two groups.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAt baseline, the MS-aligned group had older age and more previous IVF attempts than the PB-aligned group. The spindle-aligned group demonstrated a higher fertilization rate, a greater cleavage rate, an increased rate of top-quality day 3 embryos (TQE), and a larger number of TQE when compared to the PB-aligned group. Multivariate analysis revealed that the MS-aligned group had a positive impact on the likelihood of achieving\u0026thinsp;\u0026ge;\u0026thinsp;1 TQE (odds ratio [OR] 4.22, 95% confidence interval [CI] 2.24\u0026ndash;7.96, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and reaching a TQE rate of \u0026ge;\u0026thinsp;50 (OR 3.63, 95% CI 2.06\u0026ndash;6.39, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings suggest that MS-aligned microinjection may enhance embryo quality in older patients with diminished ovarian reserve undergoing ICSI cycles.\u003c/p\u003e","manuscriptTitle":"Enhancing Embryo Quality through Meiotic Spindle- Aligned Microinjection in POSEIDON Group 4 Patients Undergoing ICSI Cycles: A Retrospective Cohort Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-05 18:11:58","doi":"10.21203/rs.3.rs-3828724/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e04796ef-5718-4241-bc5c-c1e87e1b7050","owner":[],"postedDate":"January 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-10T06:44:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-05 18:11:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3828724","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3828724","identity":"rs-3828724","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}