Effect of early rescue ICSI in multiple cycles of couples with successful fertilization in the initial cycle of conventional IVF.

Our data indicated that couples undergoing their first C-IVF cycle demonstrated a significantly higher early R-ICSI rate compared to those in their second or subsequent treatments (4.00 versus 1.09%; p  < 0.001 and 4.00 versus 0.68%; p  < 0.001). We observed no significant difference in early R-ICSI rates between second and multiple C-IVF attempt cycles (1.09 versus 0.68%; p  = 0.162) (Fig.  1 ). Fig. 1 Incidence of early R-ICSI in different C-IVF treatment cycles Incidence of early R-ICSI in different C-IVF treatment cycles We observed a significant reduction in oocyte yield in previous C-IVF cycles compared to subsequent early R-ICSI cycles among couples undergoing early R-ICSI in their second treatment cycles (8.13 versus 10.56, p = 0.010). No other parameters showed statistically significant differences between the two groups ( p  > 0.05). No significant differences in general characteristics were observed between the two groups among couples undergoing early R-ICSI for multiple treatment cycles ( p  > 0.05) (Table  1 ). Table 1 General characteristics of the enrolled patients Parameter Patients of R-ICSI in the second cycle Patients of R-ICSI in the multiple cycle (≥ 3) Previous C-IVF Early R-ICSI P Previous C-IVF Early R-ICSI P Patients (n) 62 62 / 10 10 / Cycles (oocyte retrievals) 62 62 / 22 10 / Female age (y) 31.85 ± 4.85 32.32 ± 4.72 0.588 30.59 ± 3.36 32.60 ± 3.41 0.129 Protocol / / 0.337 / / 0.687 Long protocol (%, n) 61.29 (38/62) 56.45 (35/62) / 45.45 (10/22) 50.00 (5/10) / Antagonist protocol (%, n) 24.19 (15/62) 35.48 (22/62) / 40.91 (9/22) 30.00 (3/10) / Short protocol (%, n) 14.52 (9/62) 8.06 (5/62) / 13.64 (3/22) 20.00 (2/10) / BMI for women (kg/m 2 ) 23.16 ± 3.29 23.37 ± 3.32 0.965 22.98 ± 3.14 23.29 ± 3.36 0.786 Basal FSH(IU/L) 6.28 ± 2.20 6.18 ± 2.13 0.936 6.36 ± 2.91 6.61 ± 3.15 0.802 Total Gn dosage (IU) 2314.34 ± 926.87 2625.20 ± 1025.49 0.080 2132.96 ± 812.04 2900.00 ± 1724.94 0.094 Stimulation duration (days) 9.93 ± 2.44 10.29 ± 2.21 0.398 9.55 ± 3.39 10.30 ± 3.97 0.584 Number of oocytes retrieved (n) 8.13 ± 4.64 10.56 ± 4.91 0.010 7.09 ± 4.98 9.90 ± 5.88 0.172 Male age (y) 33.84 ± 5.38 34.24 ± 5.41 0.678 31.09 ± 3.65 33.10 ± 3.60 0.158 Sperm concentration (10 6 /mL) 50.54 ± 25.50 50.08 ± 26.75 0.921 56.13 ± 32.53 46.99 ± 30.99 0.455 Progressive motility (a + b) (%) 42.35 ± 15.97 43.31 ± 16.34 0.898 48.53 ± 16.82 38.85 ± 11.98 0.237 Mean number of embryos transferred (n) 1.67 ± 0.47 1.77 ± 0.46 0.255 1.52 ± 0.51 1.56 ± 0.53 0.878 General characteristics of the enrolled patients In second cycles, the 2PN rate following early R-ICSI was comparable to that achieved with previous C-IVF (55.42 versus 55.27%; p  = 0.929). The D3 available embryo rate was significantly higher following early R-ICSI compared to conventional C-IVF (79.34 versus 68.71%; p  = 0.002). No significant differences were observed in D3 good quality embryo (33.45 versus 37.74%; p  = 0.262) and blastocyst formation (45.65 versus 56.40%; p  = 0.096) rates between groups (Table  2 ). Table 2 Comparison of embryo development after previous C-IVF and subsequent early R-ICSI cycles Parameter Patients of R-ICSI in the second cycle Patients of R-ICSI in the multiple cycle (≥ 3) Previous C-IVF Early R-ICSI P Previous C-IVF Early R-ICSI P Patients (n) 62 62 / 10 10 / Cycles (oocyte retrievals) 62 62 / 22 10 / Cumulus-oocyte complexes (n) 504 655 / 156 99 / 2PN (2PN/oocytes) (%, n) 55.27 (278/504) 55.42 (363/655) 0.929 62.18 (97/156) 44.44 (44/99) 0.006 ≥ 3PN (2PN/oocytes) (%, n) 3.77 (19/504) 4.43 (29/655) 0.578 0.06 (1/156) 2.02 (2/99) 0.320 1PN (2PN/oocytes) (%, n) 2.18 (11/504) 1.68 (11/655) 0.534 2.56 (2/156) 2.02 (2/99) 0.644 D3 good quality embryo (%, n) 33.45 (93/278) 37.74 (137/363) 0.262 30.93 (30/97) 29.55 (13/44) 0.869 D3 available embryo (%, n) 68.71 (191/278) 79.34 (288/363) 0.002 70.10 (68/97) 52.27 (23/44) 0.040 Extended culture to blastocyst-stage (n) 92 172 / 37 13 / Blastocyst formation (%, n) 45.65 (42/92) 56.40 (97/172) 0.096 37.84 (14/37) 23.08 (3/13) 0.334 Comparison of embryo development after previous C-IVF and subsequent early R-ICSI cycles In multiple cycles (≥ 3), early R-ICSI showed significantly lower 2PN (44.44 versus 62.18%; p  = 0.006) and D3 available embryo (52.27 versus 70.10%; p  = 0.040) rates compared to previous C-IVF. No significant differences were observed in D3 good quality embryo (30.93 versus 29.55%; p  = 0.869) and blastocyst formation (37.84 versus 23.08%; p  = 0.334) rates between groups (Table  2 ). In second cycles, early R-ICSI showed significantly lower embryo cancellation rate (no embryos available) than previous C-IVF (0 versus 6.45%; p  = 0.042). Early R-ICSI demonstrated significantly superior clinical outcomes compared to previous C-IVF, with higher pregnancy (58.06 versus 24.13%; p  < 0.001), clinical pregnancy (48.39 versus 20.69%; p  = 0.002), ongoing pregnancy (38.71 versus 8.62%; p  < 0.001), and live birth rates (38.71 versus 5.17%; p  < 0.001) (Table  3 ). Table 3 Comparison of clinical outcomes after previous C-IVF and subsequent early R-ICSI cycles Parameter Patients of R-ICSI in the second cycle Patients of R-ICSI in the multiple cycle (≥ 3) Previous C-IVF Early R-ICSI P Previous C-IVF Early R-ICSI P Cycles (oocyte retrievals) 62 62 / 22 10 / Cycles (embryo transfers) 58 62 / 21 9 / Cancellation with no embryos available (%, n) 6.45 (4/62) 0 (0/62) 0.042 4.55 (1/22) 10.00 (1/10) 0.555 Stage of embryos transferred (%, n) 0.056 0.815 Cleavage-stage 87.93 (51/58) 74.19 (46/62) 85.71 (18/21) 88.89 (8/9) Blastocyst-stage 12.07 (7/58) 25.81 (16/62) 14.29 (3/21) 11.11 (1/9) Number of embryos transferred (%, n) 0.108 0.873 1 32.76 (19/58) 16.13 (10/62) 47.62 (10/21) 44.44 (4/9) 2 67.24 (39/58) 83.87 (52/62) 52.38 (11/21) 55.56 (5/9) Pregnancy (%, n) 24.13 (14/58) 58.06 (36/62) < 0.001 28.57 (6/21) 44.44 (4/9) 0.398 Clinical pregnancy (%, n) 20.69 (12/58) 48.39 (30/62) 0.002 14.29 (3/21) 44.44 (4/9) 0.074 Ongoing pregnancy (%, n) 8.62 (5/58) 38.71 (24/62) < 0.001 9.52 (2/21) 33.33 (3/9) 0.109 Live birth rate (%, n) 5.17 (3/58) 38.71 (24/62) < 0.001 9.52 (2/21) 33.33 (3/9) 0.109 Comparison of clinical outcomes after previous C-IVF and subsequent early R-ICSI cycles In multiple cycles (≥ 3), early R-ICSI demonstrated no statistically significant differences in clinical outcomes compared to previous C-IVF, including pregnancy (44.44 versus 28.57%; p  = 0.398), clinical pregnancy (44.44 versus 14.29%; p  = 0.074), ongoing pregnancy (33.33 versus 9.52%; p  = 0.109), and live birth (33.33 versus 9.52%; p  = 0.109) rates (Table  3 ). We compared embryo development and clinical outcomes between all previous C-IVF and early R-ICSI cycles. Early R-ICSI demonstrated comparable rates of 2PN (56.82% versus 53.98%; p  = 0.284) and D3 good quality embryos (36.86% versus 32.80%; p  = 0.235) relative to previous C-IVF cycles. Nevertheless, early R-ICSI was associated with significantly superior clinical outcomes, including higher clinical pregnancy (47.89% versus 18.99%; p < 0.001), ongoing pregnancy (38.03% versus 8.86%; p < 0.001), and live birth (38.03 versus 7.04%; p  < 0.001) rates compared to previous C-IVF (Fig.  2 ). Fig. 2 Comparison of embryo development and clinical outcomes between total previous C-IVF and early R-ICSI cycles Comparison of embryo development and clinical outcomes between total previous C-IVF and early R-ICSI cycles

A retrospective cohort study was performed between January 2014 and December 2023, including 72 couples who had a normal or nearly normal two pronuclei (2PN) rate (≥ 40%) in previous C-IVF cycles but subsequently underwent TFF or near TFF in repeated C-IVF cycles. TFF was defined as the absence of a second polar body in all mature oocytes. Near TFF was defined as fewer than 1/3 of mature oocytes exhibited a second polar body (second polar body rate < 33.33%). Among the study cohort, 62 couples exhibited a normal/near normal 2PN rate in the first cycle but experienced TFF or near TFF in the second cycle. The remaining 10 couples maintained a normal/near normal 2PN rate in at least two cycles but subsequently experienced TFF or near TFF in multiple cycles (≥ 3). For each initiated cycle, only the first embryo transfer was compared. Embryo development and clinical outcomes after early R-ICSI were compared to previous C-IVF. Previous C-IVF cycles were considered as a control group. Most patients received ovarian stimulation using either the GnRH agonist long or GnRH antagonist protocols in this study. The proportion of patients with primary infertility was 47.22%. Most of the couples were diagnosed with tubal factor ( n  = 41), unexplained female infertility ( n  = 12), or male factor infertility ( n  = 9). The remaining patients were diagnosed with ovulation failure ( n  = 5), diminished ovarian reserve ( n  = 3), and endometriosis ( n  = 2). Patients with oocyte retrieved ≤ 2 were excluded because early cumulus cell removal combined with early R-ICSI was not performed for such patients in our center. All patients gave written informed consent and this study was approved by the Ethics Committee of Northwest Women’s and Children’s Hospital (No. 2023003). All participants in our study underwent controlled ovarian hyperstimulation. The ovarian stimulation protocols in our reproductive medicine center include the GnRH agonist long protocol, GnRH agonist short protocol, and GnRH antagonist protocol, as detailed in previous literature [ 13 ]. Notably, recombinant follicle-stimulating hormone (FSH) or urinary FSH and/or human menopausal gonadotropins were used with daily doses between 100 and 450 IU based on patients’ characteristics as calculated previously [ 13 ]. C-IVF was performed 2–2.5 h after oocyte retrieval and each oocyte was incubated with approximately 40 000 sperm. Short-term insemination was adopted and the cumulus granule cells were peeled off 4.5–5 h post-fertilization. Oocytes were analyzed for the release of the second Pb at 5–6 h after the initial insemination. If there was no second Pb (TFF), or less than 1/3 of the oocytes had a second Pb (near TFF), R-ICSI was performed immediately on the oocytes with only one Pb observed. Our skilled ICSI operators injected the oocytes with only one Pb by the direct penetration technique. Oocytes were placed individually into 5 μl droplets of G-MOPS (Vitrolife, Goteborg, Sweden) solution covered under warm mineral oil. Sperm were placed in a central 5 μl droplet of polyvinylpyrrolidone solution, and the details of ICSI protocol were as previously described [ 14 ]. Oocytes were examined for the presence of two pronuclei (2PN) to confirm fertilization 19–20 h after insemination. Normal fertilization was defined by the presence of 2PN and the extrusion of the second polar body. After 64–68 h of culture, the morphologic score was given for day-3 embryo according to the number of blastomeres, homogeneous degree of blastomeres and degree of cytoplasmic fragmentation: grade I (8–10 blastomeres, even homogeneous blastomeres  10 blastomeres with even homogeneous blastomeres of no cytoplasmic fragmentation, 8–10 blastomeres, even homogeneous blastomeres with 10%-20% cytoplasmic fragmentation), grade III (uneven and nonhomogeneous blastomeres with 20–50% cytoplasmic fragmentation), and grade IV (uneven and non-homogeneous blastomeres with > 50% cytoplasmic fragmentation). The D3 good quality embryos were graded I and II. The D3 available embryos were graded I, II, and III. Blastocysts were observed on the fifth and sixth morning after oocyte retrieval, and the scoring system for blastocyst evaluation was a combination of the stage of development from 1 to 6 (early, blastocyst, full blastocyst, expanded, hatching/hatched) and of the grade of the inner cell mass (ICM; A, tightly packed, many cells; B, loosely grouped, several cells; or C, very few cells.) and of the trophectoderm (TE; A, many cells forming a cohesive epithelium; B, few cells forming a loose epithelium; or C, very few large cells.). Three methods of luteal support are implemented in our center. I. Vaginal progesterone gel (90 mg q.d; Crinone, Serono, Hertfordshire, UK); II. Vaginal progesterone soft capsules (0.2 g t.i.d; Utrogestan, Besins, France); III. Intramuscular progesterone (60 mg q.d; Xianju, Zhejiang, China). Patients from both groups could select one of these three luteal support methods and receive oral progesterone (10 mg t.i.d; Dydrogesterone, Abbott Biologicals B.V., Amsterdam, Netherlands) simultaneously. The luteal support was maintained until week 10 of gestation. Pregnancy was defined as β-HCG value more than 50 mIU/ml after 12 days of transfer. Clinical pregnancy was characterized as the presence of an intrauterine gestational sac on ultrasonography during the first trimester. Ongoing pregnancy was defined as a clinical pregnancy that continued for at least 12 weeks. Live birth was defined as a pregnancy that ended with the birth of a live infant. Statistical analysis between groups in the case of continuous variables was performed with Student’s t test for data with normal distribution. Non-parametric Mann–Whitney U-test was performed for data with skewed distribution. Statistical analysis between groups in the case of categorical variables was expressed as number and percentage and Chi-square test or Fisher exact test was performed. The statistical analysis was performed with SPSS version 23 (IBM Corp.; NY, USA). A p -value of less than 0.05 was considered to indicate statistical significance.

In this study, we demonstrated that couples undergoing early R-ICSI in the second cycle had significantly higher D3 available embryo rate and more superior clinical outcomes compared to previous C-IVF. Nevertheless, we observed that couples undergoing early R-ICSI in multiple cycles (≥ 3) showed significantly lower 2PN and D3 available embryo rates compared to previous C-IVF. And early R-ICSI demonstrated no statistically significant differences in clinical outcomes compared to previous C-IVF in multiple cycles. TFF or near TFF after C-IVF is still prevalent and unpredictable. It has been reported that in C-IVF cycles, the proportion of TFF or near TFF ranges from 3.5% to as high as 15–20% [ 15 , 16 ]. Although the causes of TFF have not yet been completely elucidated, various studies have shown that male factors related to sperm abnormalities are the major contributors to this issue [ 8 , 17 ]. Nevertheless, other studies showed that sperm count, motility, and morphology are poor indicators of the likely sperm-oocyte interaction, as 15 to 29% of patients showing normal semen characteristics were reported to have fertilization failure [ 10 ]. In recent year, several studies indicated that patients with fewer than 5 oocytes had a high risk of TFF after C-IVF [ 18 – 20 ]. In this study, semen from male patients has demonstrated the capability of achieving normal fertilization in previous C-IVF. Additionally, most female patients in this cohort exhibited an optimal oocyte yield. Thus, it is difficult to identify the underlying cause of fertilization failure in such cases. In fact, our primary concern lies in understanding the clinical outcomes of these patients following early R-ICSI. The advent of early R-ICSI provides new hope for patients facing fertilization failure. However, it's more challenging to perform early R-ICSI for couples who had normal 2PN rate in previous C-IVF cycles. The main worry is the risk of ≥ 3PN formation following early R-ICSI due to our incorrect assessment. It is very important to balance the time-related risks of oocyte aging and ≥ 3PN incidence for R-ICSI because both of them can result in compromised developmental outcomes. To prevent the aging of oocytes, the early identification of fertilization signs (two or more polar bodies) after 5–6 h of insemination is evaluated in our center. This protocol has been validated to be safe and effective for early observation of fertilization failure [ 21 ]. Notably, the ≥ 3PN rate following early R-ICSI was comparable to previous C-IVF which indicated that it was appropriate to perform early R-ICSI in such cases. Our data also showed no significant difference in the D3 good quality embryo rate following early R-ICSI compared with previous C-IVF. The findings further suggested that the timing of early R-ICSI was optimal, as it did not adversely affect embryo quality due to oocyte aging. Nevertheless, the present study showed controversial results in the D3 available embryo rate following early R-ICSI compared to previous C-IVF for couples undergoing different treatment attempts. We noted that the 2PN rate was only 55.27% in previous C-IVF for couples undergoing R-ICSI during their second treatment cycles, which was significantly lower than the average 2PN rate (62.45%) for total C-IVF cycles in our center. It might be associated with more immature oocytes or less activated oocytes. It has been demonstrated that oocytes derived from a cohort with high incidence of maturation failure may have detrimental embryo development [ 22 ]. Calcium signaling plays a crucial role in initiating and regulating the complex mechanisms required for oocyte activation [ 23 ]. It is closely linked to embryonic development, playing a critical role in regulating early cell divisions and the cell cycle during embryogenesis [ 24 ]. And we also observed that the cancellation rate with no embryos available was significantly reduced following early R-ICSI. By salvaging oocytes that would otherwise remain unfertilized, early R-ICSI could enhance the likelihood of embryo formation. Therefore, it is logical that the D3 available embryo rate has a marked improvement following early R-ICSI. For couples undergoing their multiple cycles (≥ 3), we observed that the D3 available embryo rate was significantly decreased following early R-ICSI compared to previous C-IVF. The possible explanation is that the total quality of oocytes retrieved has declined. For such patients, the average 2PN rate (62.18%) is roughly normal in previous C-IVF cycles. Nevertheless, the 2PN rate (44.44%) was significantly decreased following early R-ICSI. After at least two C-IVF cycles, the semen of male patients has been confirmed to be capable of obtaining normal fertilization rate. Therefore, we suspected that the poor fertilization outcome was mainly due to the oocyte factor. The oocyte is the major determinant of embryo developmental competence, which might lead to the decreased D3 available embryo rate following early R-ICSI [ 12 ]. Fertilization failure may further impose psychological burden for patients with multiple treatment cycles. Multiple researches have demonstrated that early cumulus cell removal combined with early R-ICSI can yield favorable clinical outcomes [ 7 , 25 , 26 ]. Our study also indicated that early R-ICSI demonstrates better clinical results than previous C-IVF for couples undergoing two or more treatment cycles. Early R-ICSI represents an effective solution to address their concerns. The limitations of this study include its retrospective design and the limited sample size, which is attributable to the rare incidence of fertilization failure in couples who had a normal or nearly normal 2PN rate in previous C-IVF cycles. Due to limited blastocyst culture data, the influence of fertilization failure on subsequent embryonic development remains uncertain in this population. Furthermore, the cumulative live birth rate may be a more significant indicator but it is difficult to make a statistical analysis. Nevertheless, few researches have explored the embryo development and clinical outcomes in such cases. Thus, these findings provide clinically actionable insights for optimizing treatment decisions. In summary, fertilization failure is not a predictor of poor treatment outcomes for patients undergoing two or more IVF treatment cycles. Early R-ICSI at 5–6 h post-insemination represents a safe and effective strategy. Given the limited data and methodological constraints, further data accumulation is needed to obtain more reliable conclusions.

Total fertilization failure (TFF) occurs in 3.5–20% of conventional in vitro fertilization (C-IVF) cycles [ 1 ]. This outcome may lead to cycle cancellation, which is a highly frustrating experience for patients and presents a significant challenge for clinicians. In earlier period, intracytoplasmic sperm injection (ICSI) has been routinely employed as a late remediation of unfertilized oocytes 16–18 h after sperm incubation in cases of TFF [ 2 , 3 ]. However, the clinical outcome of ICSI using 1-day-old oocytes is always unsatisfactory which has been confirmed to be associated with oocyte aging [ 4 ]. To salvage fertilization failure and mitigate the effects of oocyte aging, early rescue ICSI (R-ICSI) has been implemented 5–6 h post-insemination which demonstrate a promising clinical result [ 5 – 7 ]. Recently, the cause of TFF is not fully elucidated and remains controversial. A robust body of evidence has identified male factors, particularly sperm abnormalities, as major contributors to this issue [ 8 , 9 ]. Conversely, other studies have demonstrated that sperm count, motility, and morphology serve as poor indicators of the likely sperm-oocyte interaction [ 10 , 11 ]. Aziz et al. showed that nearly a quarter of patients exhibiting normal semen characteristics experienced fertilization failure [ 10 ]. Mahutte et al. demonstrated that nearly half of couples with fertilization failure had a normal pre-IVF semen analysis, highlighting the limitations of standard semen analyses and the criteria used to correlate semen characteristics with fertilization outcomes [ 11 ]. Consequently, the predictive value of sperm abnormalities for fertilization failure is limited. It has been reported that several female-related factors influencing the response to treatment can also impact fertilization outcomes and may be associated with fertilization failure [ 11 ]. Given the complexity of fertilization causes, we designed this study to evaluate the effect of fertilization failure on a special type of patients who had a normal or nearly normal two pronuclei (2PN) rate in previous C-IVF cycles but subsequently underwent TFF or near TFF in repeated C-IVF cycles. It has been confirmed that such male patient’s semen has normal fertilization capacity. These findings strongly implicate oocyte competence deficiencies may be associated with fertilization failure in such cases. It is well known that the oocyte is the major determinant of embryo developmental competence in women [ 12 ]. It suggests that fertilization failure may adversely affect embryo quality in such patients. And fertilization failure may further impose psychological burden for patients with two or more treatment cycles. Thus, it is significant to provide critical evidence regarding the efficacy of early R-ICSI as a potential salvage intervention for fertilization failure in such cases.