Conventional Intracytoplasmic Sperm Injection Research Articles

High sperm deoxyribonucleic acid fragmentation index is associated with an increased risk of preeclampsia following assisted reproduction treatment

To study the association between sperm DNA fragmentation index (DFI) and the odds of preeclampsia and other adverse perinatal outcomes after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment. A prospective cohort study including infertile couples undergoing conventional IVF or ICSI treatment and their children. Data regarding preeclampsia and perinatal outcomes were derived from the Swedish National Birth Register. 1594 infertile couples undergoing IVF or ICSI treatment and their 1660 children conceived by assisted reproduction. Sperm DNA fragmentation index measured by Sperm Chromatin Structure Assay. The primary outcome was preeclampsia. Secondary outcomes were preterm birth, low birth weight, low Apgar score, and small for gestational age. With DFI < 20% as a reference, the OR for preeclampsia was statistically significantly increased in the group with DFI ≥ 20% when IVF was used as fertilization method (OR 2.2; 95% CI 1.1 to 4.4; p = 0.02). Already at DFI levels ≥ 10%, in IVF pregnancies, preeclampsia odds were increased in a dose-response manner, from a prevalence of 3.1% in the reference group to more than 10% among those with DFI of 30% or higher. The DFI was not associated with preeclampsia odds in the ICSI group. In the entire cohort, DFI ≥ 20% was associated with an increased OR of preterm birth (OR 1.4; 95% CI 1.0 to 2.0; p = 0.03). High DNA fragmentation index was associated with increased odds of preterm birth and, in IVF pregnancies, also increased odds of preeclampsia.

O-186 A novel micro-insemination method, assisted sperm fusion insemination, increases the embryo utilization rate compared to conventional intracytoplasmic sperm injection

Abstract Study question Does a novel micro-insemination method, assisted sperm fusion insemination (ASFI), improve fertilization and embryonic development compared to conventional intracytoplasmic sperm injection (C-ICSI)? Summary answer ASFI showed comparable fertilization and blastocyst formation rates to C-ICSI, while achieving significantly higher embryo utilization rates. Furthermore, there were no degenerated oocytes after ASFI. What is known already We have reported that ASFI yielded fertilization and blastocyst rates similar to those obtained with rescue ICSI performed at 6 hours after in vitro fertilization (IVF). In ASFI, fertilization is achieved by pressing a zona pellucida (ZP)-bound sperm onto the oocyte membrane without breaking the oocyte membrane. However, there was a risk of artificial polyspermy because the sperm were pressed against the unfertilized oocytes after IVF. Moreover, it is uncertain whether fertilization occurred with sperm that penetrated into the oocyte via IVF or that were pressed against the oocyte membrane. Clarifying this requires performing ASFI of the oocyte without IVF. Study design, size, duration We prospectively evaluated the effectiveness of ASFI in a sibling oocyte study including 14 subjects from which at least two metaphase II (MII) oocytes and one immature or degenerated oocyte were retrieved. Between December 2022 and October 2023, 119 MII oocytes were obtained in 22 oocyte retrieval cycles and used in the study. Sibling MII oocytes were randomly assigned to a C-ICSI or ASFI group. The present study was approved by the local ethics committee. Participants/materials, setting, methods In the ASFI group, 10,000 motile sperm were incubated with at least one immature or degenerated oocyte for 3 hours in 100 μL of medium per patient. After incubation, the motile sperm bound to the ZP were aspirated directly using an injection pipette. The heads of these ZP-bound sperm were pressed onto the membrane of MII oocytes for 10 seconds using the injection pipette. C-ICSI was conducted using the conventional method. Main results and the role of chance The mean and standard deviation of age of the women and number of MII oocytes per cycle were 39.9 ± 2.2 years and 5.5 ± 1.8, respectively. All sperm collected from the ZP adhered to the MII oocyte membrane after ASFI. Furthermore, time-lapse imaging revealed that all sperm adhering to the MII oocyte membrane were subsequently incorporated into the oocyte. Comparison of the ASFI and C-ICSI groups did not reveal any significant differences in 2 pronuclear embryo rate (ASFI group: 85.4% vs. C-ICSI group: 71.8%), degeneration rate (0% vs. 5.6%), good-quality embryo rate at day 3 (63.4% vs. 52.9%), blastocyst formation rate (67.6% vs. 51.9%), or good-quality blastocyst (Grade 3BB and above by the Gardner criteria) formation rate (29.4% vs. 15.4%). On the contrary, the embryo utilization rate (defined as the total number of embryos transferred and cryopreserved divided by the number of MII oocytes) was significantly higher in the ASFI group compared to the C-ICSI group (47.9% vs. 25.4%, P &amp;lt; 0.05). Limitations, reasons for caution This study was limited by using the ZP for sperm selection. To expand its applicability, a method to identify sperm capable of fusing with an oocyte without using the ZP must be established. Additionally, since our sample size was small, larger studies are required to confirm our findings. Wider implications of the findings ASFI is expected to enhance the survival rate of oocytes and to increase the number of embryos available for implantation, not only compared to rescue ICSI but also C-ICSI. ASFI could aid in understanding fertilization mechanisms by enabling direct observation of oocyte-sperm fusion during the process. Trial registration number UMIN000050184

O-184 Piezo-ICSI reduces oocyte degeneration, improves ICSI success rates and has shown less dependence on the experience level of embryologists: cumulative data with 65,033 MII oocytes

Abstract Study question Dose piezo-ICSI improve the overall success rates of in ICSI operations compared with the conventional ICSI by the operators with different level of experience? Summary answer Piezo-ICSI showed lower oocyte degeneration rate during or after ICSI operations compared to the conventional ICSI for embryologists with different level of experience. What is known already Intracytoplasmic sperm injection (ICSI) with a glass micropipette can sometimes result in degeneration of the injected oocyte. Piezo-assisted ICSI has been developed as an alternative method to prevent such degeneration occurring during the procedure. It employs gentler and controlled impacts generated from the piezo electric elements attached to the injection micropipette holder to penetrate oolemma, and it does not require reciprocal aspiration of ooplasm required in the conventional ICSI. While there have been numerous study reports demonstrating the improvements in fertilization rates with piezo-ICSI, most of these studies have the limitation in the size study of population. Study design, size, duration We conducted a retrospective analysis of the ICSI results with 65,033 MII oocytes in 15,687 treatment cycles carried out between January 2018 and February 2023. ICSI were performed using the piezo-assisted equipment with flat-tip micropipettes (PIEZO PMM4G, Prime Tech Ltd., [n = 51,484, Piezo-group]) or in the conventional ICSI method with spiked-tip micropipettes (n = 13,549, C-group). Participants/materials, setting, methods Following the insemination with ICSI, the oocyte degeneration rate, fertilization rate per MII oocytes and blastocyst formation rate per 2PN embryos were evaluated in both the Piezo- and C-groups (n = 51,484 and n = 13,549, respectively). We also evaluated the degeneration rates and fertilization rates of ICSI by the operators: i.e., well-experienced embryologists (at least 10 years of experience) and less experienced embryologists (5 years or less of experience). Main results and the role of chance The fertilization and blastocyst formation rates in the Piezo-group were 83.8% (43,140/51,484) and 54.0% (23,300/43,140), respectively, which were significantly higher than the C-group (74.8% [10,132/13,549] and 36.6% [3,674/10,132], respectively, p &amp;lt; 0.01). The oocyte degeneration rate in the Piezo-group was 2.9% (1,493/51,484), which was significantly lower than the C-group (6.9% [933/13,549], p &amp;lt; 0.05). For the patients over 40 years old, the degeneration rate was also significantly lower in the Piezo-group (3.0% [632/20,770]) than the C-group (7.1% [351/4927], p &amp;lt; 0.01), while the fertilization and blastocyst formation rates were higher in the Piezo-group (82.6% [17,164/20,770] and 46.6% [7,997/17,164], respectively) than the C-group (72.8% [3,588/4,927] and 25.8% [926/3,588], respectively, p &amp;lt; 0.01). We found a marked drop in the oocytes degeneration rates in the Piezo group recorded from both well-experienced and less experienced embryologists. The degeneration rates recorded by less experienced embryologists were 2.8% (119/4,242) in Piezo-group and 8.6% (8/93) in the C group (p &amp;lt; 0.01), while the records by well-experienced embryologists were 2.7% (815/29,681) in the Piezo-group and 6.9% (555/8,037) in the C-group, respectively (p &amp;lt; 0.01). The fertilization rates of the Piezo-group by less experienced and well-experienced embryologists were 84.1%(3,569/4,242) and 83.4% (24,757/29,681), respectively, which were significantly higher the C-group (71.0％ [66/93] and 76.7％ [6,167/8,037], respectively, p &amp;lt; 0.01). Limitations, reasons for caution It is a retrospective study, in which the ICSI operations with piezo and the conventional methods were not equally assigned to the embryologists, especially, for those less experienced, thereby the number of conventional ICSI operations was more skewed to well-experienced embryologists than the less experienced colleagues. Wider implications of the findings Our results with piezo-ICSI showed a marked drop in the oocyte degeneration while achieving comparable fertilization rates amongst operators with different levels of experience. We found these as major advantages over the conventional method in standardizing the ICSI operation by filling the gap between embryologists with different length of carrier. Trial registration number Not applicable

P-703 Is the time interval between triggering final oocyte maturation and oocyte retrieval associated with ART outcomes? A systematic review and meta-analysis

Abstract Study question Is the time interval between triggering final oocyte maturation and oocyte retrieval associated with assisted reproductive technology (ART) outcomes? Summary answer Limited evidence suggests that extending the time interval between triggering final oocyte maturation and oocyte retrieval from 33-36h to 37-41h does not affect ART outcomes. What is known already The time interval between final oocyte maturation and oocyte retrieval varies among IVF centres, although an interval of 33–36 h is the most widely adopted. The use of a prolonged time interval of 37-41h has been suggested to increase the number and competence of oocytes retrieved, their maturation, their ability to be fertilized, their capacity to produce usable embryos and the probability of pregnancy. Considering recently published data, a systematic review is warranted to provide clinicians with updated, evidence-based information regarding the optimal time interval between triggering final oocyte maturation and oocyte retrieval. Study design, size, duration A systematic review and meta-analysis was performed aiming to evaluate the effect of the time interval between triggering final oocyte maturation and oocyte retrieval on ART outcomes. For this purpose, a literature search was carried out until January 2024 in PubMed, MEDLINE and CENTRAL. The primary outcome measure was clinical pregnancy rate, while ovulation before oocyte retrieval, oocytes retrieved, maturation rate, fertilization rate, blastulation rate, implantation rate and miscarriage rate were secondary outcomes. Participants/materials, setting, methods Citation, demographic, methodological and clinical data were extracted from the studies by two independent reviewers. Quality was assessed using the RoB2 Tool by Cochrane. Statistical heterogeneity was assessed by I2. In dichotomous data, estimates were expressed as relative risk (RR) with 95% confidence intervals (CIs), using the fixed or random effects method. In continuous data, differences were pooled across resulting in a weighted mean difference (WMD) with 95% CI. Main results and the role of chance Six eligible RCTs (n = 1081) were identified, published between 1991-2023. In three trials, conventional insemination was used for fertilization, in two trials this was performed by intracytoplasmic sperm injection (ICSI), whereas in one trial either conventional insemination or ICSI were used. The time intervals between triggering final oocyte maturation and oocyte retrieval compared, varied between studies (34.5-36 vs 38.5-40h, 33 vs 41h, 34 vs 38h, 35 vs 37h, 34 vs 39h and 36 vs 38h). Patients were grouped in advance based on these time intervals (short interval group: 33-36h versus long interval group: 37-41h). Ovarian stimulation was performed with GnRH analogues (GnRH antagonists:1 GnRH agonists:5) and gonadotrophins (HMG:4 recombinant FSH:2, while triggering of final oocyte maturation was performed with hCG( recombinant hCG:1 urinary hCG:5). None of the studies was deemed as of low risk of bias. No statistically significant differences were observed between the short nd long interval groups regarding clinical pregnancy rate per randomized patient (RR: 0.83; 95%CI: 0.62-1.11; fixed effects model; heterogeneity: I2:36.4%; five studies, 827 patients). Furthermore, it was not feasible to pool any of the secondary outcome data. This was either because they were not reported, or due to the fact that they were reported inappropriately. Limitations, reasons for caution This meta-analysis included a limited number of RCTs, which were generally of low quality due to methodological limitations and of high risk of bias. Moreover, the total number of patients is still not large enough to draw solid conclusions. Wider implications of the findings Taking into account the limitations of the current meta-analysis, it appears that clinicians may elect a shorter (33–36 h) or a longer (37-41h) interval regarding the timing of oocyte retrieval following triggering final oocyte maturation, without affecting clinical pregnancy rate. Trial registration number PROSPERO: CRD42024501986

Does Physiological Intracytoplasmic Sperm Injection Improve Outcome in Men with Abnormal Semen Parameters: A Retrospective Cohort Study.

As the male factor in infertility is on rising trend, we need a promising tool for sperm selection to improve the embryo development. The present study compares the embryological and clinical parameters between physiological intracytoplasmic sperm injection (PICSI) and conventional intracytoplasmic sperm injection (ICSI) in abnormal semen parameters. This was a retrospective cohort study. This study was conducted over the duration of 9 months from April to December 2023. One hundred and four patients, who matched the inclusion and exclusion criteria, were enrolled in the study. The adjusted blastulation rate was evaluated as the primary outcome, and the secondary outcomes studied were fertilisation rate, good-quality cleavage stage embryo rate, clinical pregnancy and miscarriage rate. The outcome measures were calculated using independent Student's t-test, and P < 0.5 was considered statistically significant. The 104 enrolled patients were divided into two groups. In the study population (n = 51), PICSI was used for sperm selection and the controls (n = 53) used conventional ICSI. The adjusted blastulation rate was statistically significant higher in the PICSI group (43.7%) as compared to the controls (34.2%) (P = 0.022). The fertilisation and cleavage rates were not statistically different amongst the groups. The pregnancy rate (37.2% vs. 32%, P = 0.027) and miscarriage rate (5.3% vs. 11.7%, P = 0.005) were statistically better in the PICSI group. PICSI may improve treatment outcomes in couples undergoing assisted reproduction for male factor infertility. However, larger randomised controlled trials are needed for validating the current study findings.

Effect of sperm treatment with lysolecithin on in vitro outcomes of equine intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) in horses is currently employed for clinical and commercial uses, but the protocol could be optimized to improve its efficiency. We have hypothesized that destabilization of plasma and acrosomal membranes prior to injection would positively impact the developmental potential of equine zygotes generated by ICSI. This study evaluated effects of the sperm treatment with lysolecithin on plasma and acrosomal membranes and on oocyte activation ability, initially following heterologous ICSI on bovine oocytes and subsequently employing equine oocytes. The effects of the lysolecithin -treatment on the efficiency of conventional and piezo-assisted equine ICSI were evaluated. To do this, the equine sperm were treated with different concentrations of lysolecithin and the sperm plasma membrane, acrosome and DNA integrity were evaluated by flow cytometry. The results showed that a lysolecithin concentration of 0.08 % destabilized the membranes of all sperm and affected DNA integrity within the range described for the species (8–30 %). In addition, the heterologous ICSI assay showed that lysolecithin treatment was detrimental to the sperm's ability to activate the oocyte, therefore, chemical oocyte activation was used after equine ICSI after injection with lysolecithin -treated sperm. This group showed similar developmental rate to the control group with and without exogenous activation. In conclusion, lysolecithin pre-treatment is not necessary when using ICSI to produce equine embryos in vitro. The results from the current study provide additional insight regarding the factors impacting ICSI in horses.

Comparative analysis of conventional in vitro fertilization and intracytoplasmic sperm injection in patients with polycystic ovarian syndrome, tubal factor infertility, and unexplained infertility whose partners exhibit normal semen parameters: a retrospective study of sibling oocytes.

This study compared the outcomes of conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in patients with polycystic ovarian syndrome (PCOS), tubal factor (TF) infertility, and unexplained infertility whose partners had normal semen parameters. This retrospective study included 360 couples diagnosed with infertility involving PCOS (n=157), unexplained infertility (n=140), and TF infertility (n=63). Sibling oocytes were randomly assigned to undergo ICSI or conventional IVF insemination. The fertilization rate and embryo morphology were evaluated as outcomes. Retrieved cumulus-oocyte complexes from patients with PCOS (2,974), unexplained infertility (1,843), and TF infertility (844) were split and inseminated by conventional IVF and ICSI respectively. In comparison to the ICSI method, the conventional IVF approach was linked to a significantly higher fertilization rate in groups with PCOS (68.81% vs. 77.49%), unexplained infertility (67.62% vs. 78.84%), and TF issues (69.23% vs. 78.63%) (p<0.05). The proportion of embryos with grade A produced by the conventional IVF method was significantly higher than that produced using the ICSI method in the PCOS and unexplained infertility groups (p<0.05). Additionally, the percentage of grade B embryos produced with the ICSI method was significantly higher than that produced with the conventional IVF method in PCOS patients (p=0.002). Our results indicated that the conventional IVF method was associated with higher zygote production and a higher proportion of grade A embryos when all infertile groups were evaluated together. Thus, ICSI is not suggested for patients with these causes of infertility if their partner has normal semen parameters.

Clinical effectiveness of spindle-view intracytoplasmic sperm injection compared to conventional intracytoplasmic sperm injection in patients with poor ovarian response and previous implantation failure.

This study aimed to determine the clinical advantage of spindle-view intracytoplasmic sperm injection (SVICSI; a novel technology) over conventional intracytoplasmic sperm injection (cICSI) in patients with poor ovarian response (POR) and previous implantation failure. The study included 37 patients who underwent SVICSI followed by fresh embryo transfer (FET) at a single fertility clinic from January to December 2022, 58 patients who underwent cICSI followed by FET at the same fertility clinic from January to December 2021 as a control group. All study participants met the Bologna criteria for POR and had at least three or more previous failed embryo transfers. The number of blastocyst transfers was significantly higher in the SVICSI group than in the cICSI group. A good-quality cleavage embryo rate, blastocyst rate, and good-quality blastocyst rate were also significantly higher in the SVICSI group than in the cICSI group. There were no significant differences in the rates of fertilization, implantation, clinical pregnancy, or clinical abortion between the two groups. In patients with POR, those who underwent SVICSI appeared to have better embryos than those who underwent cICSI. However, whether SVICSI improved clinical outcomes such as implantation and pregnancy rates cannot be proven.

Fertilization, pregnancy, and neonatal outcomes after IVF, rescue ICSI, and ICSI in unexplained infertility: A retrospective study

AbstractConventional intracytoplasmic sperm injection (ICSI) is not recommended for nonmale infertile patients to avoid fertilization failure (FF) in view of controversies regarding safety issues. Among the strategies that may help to promote the use of in vitro fertilization (IVF) for women with a high risk of FF, rescue ICSI (R‐ICSI) has attracted attention. This study aimed to evaluate the efficacy of short‐term insemination combined with early R‐ICSI in the treatment of unexplained infertility. A total of 952 controlled ovarian stimulation (COS) cycles for unexplained infertility were divided into short‐term IVF (short‐term insemination without R‐ICSI, n = 500), R‐ICSI (short‐term insemination with R‐ICSI, n = 141), and ICSI (conventional ICSI, n = 311) groups. Patients underwent consecutive transfer cycles until live birth, or until all embryos from the first COS cycle were used. Laboratory data and clinical outcomes from short‐term IVF, R‐ICSI, and ICSI groups were compared. Short‐term IVF and ICSI groups were superior to R‐ICSI group in polyspermy rate, available embryo rate, and top‐quality embryos rate. Short‐term IVF, R‐ICSI, and ICSI groups underwent 705 (500 fresh and 205 frozen‐thawed cycles), 190 (141 fresh and 49 frozen‐thawed cycles), and 445 (311 fresh and 134 frozen‐thawed cycles) transfer cycles, resulting in 294, 76, and 190 live birth cycles, respectively. Comparison of pregnancy outcomes among these three groups demonstrated similar clinical pregnancy rates and live birth rates in fresh and frozen‐thawed embryo transfer cycles. There was no significant difference in the cumulative live birth rate among these three groups. R‐ICSI group showed similar neonatal outcomes compared with short‐term IVF and ICSI groups, including the rates of low birth weight, fetal macrosomia, small for gestational age and large for gestational age. Short‐term insemination combined with early R‐ICSI achieved satisfactory pregnancy and neonatal outcomes, albeit with a high polyspermy rate, which was an effective alternative to avoid excessive use of ICSI in unexplained infertility.

A micro-fabricated device (microICSI) improves porcine blastocyst development and procedural efficiency for both porcine intracytoplasmic sperm injection and human microinjection

PurposeIntracytoplasmic sperm injection (ICSI) imparts physical stress on the oolemma of the oocyte and remains among the most technically demanding skills to master, with success rates related to experience and expertise. ICSI is also time-consuming and requires workflow management in the laboratory. This study presents a device designed to reduce the pressure on the oocyte during injection and investigates if this improves embryo development in a porcine model. The impact of this device on laboratory workflow was also assessed.MethodsPorcine oocytes were matured in vitro and injected with porcine sperm by conventional ICSI (C-ICSI) or with microICSI, an ICSI dish that supports up to 20 oocytes housed individually in microwells created through microfabrication. Data collected included set-up time, time to align the polar body, time to perform the injection, the number of hand adjustments between controllers, and degree of invagination at injection. Developmental parameters measured included cleavage and day 6 blastocyst rates. Blastocysts were differentially stained to assess cell numbers of the inner cell mass and trophectoderm. A pilot study with human donated MII oocytes injected with beads was also performed.ResultsA significant increase in porcine blastocyst rate for microICSI compared to C-ICSI was observed, while cleavage rates and blastocyst cell numbers were comparable between treatments. Procedural efficiency of microinjection was significantly improved with microICSI compared to C-ICSI in both species.ConclusionThe microICSI device demonstrated significant developmental and procedural benefits for porcine ICSI. A pilot study suggests human ICSI should benefit equally.

Optimizing non-invasive preimplantation genetic testing: investigating culture conditions, sample collection, and IVF treatment for improved non-invasive PGT-A results

PurposeThis study aimed to optimize the non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) in the laboratory by comparing two collection timing of the spent culture medium (SCM), two embryo rinsing protocols, and the use of conventional insemination instead of intracytoplasmic sperm injection (ICSI).MethodsResults of two embryo rinsing methods (one-step vs sequential) and SCM collected on day 5 vs day 6 after retrieval were compared against trophectoderm (TE) biopsies as reference. Results from day 6 SCM in cycles fertilized by conventional insemination were compared with PGT-A using ICSI.ResultsThe rate of concordance was higher in day 6 samples than in day 5 samples when the sequential method was used, in terms of total concordance (TC; day 6 vs day 5: 85.0% vs 60.0%, p = 0.0228), total concordance with same sex (TCS, 82.5% vs 28,0%, p < 0.0001), and full concordance with same sex (FCS, 62.5% vs 24.0%, p = 0.0025). The sequential method significantly out-performed the one-step method when SCM were collected on day 6 (sequential vs one-step, TC: 85.0% vs 64.5%, p = 0.0449; TCS: 82.5% vs 54.8%, p = 0.0113; FCS: 62.5% vs 25.8%, p = 0.0021). There was no significant difference in niPGT-A results between cycles fertilized by the conventional insemination and ICSI.ConclusionWe have shown a higher concordance rate when SCM was collected on day 6 and the embryos were rinsed in a sequential manner. Comparable results of niPGT-A when oocytes were fertilized by conventional insemination or ICSI. These optimization steps are important prior to commencement of a randomized trial in niPGT-A.

Bovine oocyte activation with bull or human sperm by conventional ICSI and Piezo-ICSI: Its relationship with PLCɀ activity.

The intracytoplasmic sperm injection (ICSI) technique has low efficiency in cattle. This has mainly been attributed to the oocyte activation failure due to oocyte and/or sperm factors. Our aim was to evaluate the effect of conventional ICSI and Piezo-ICSI with bull or human sperm on bovine oocyte activation and embryo development and to assess its relationship with the phospholipase C zeta (PLCɀ) activity of both species. In vitro matured bovine oocytes were randomly divided into five groups and were fertilized as follows: conventional ICSI using bovine sperm with chemical activation (control), conventional ICSI using bovine sperm, Piezo-ICSI using bovine sperm, conventional ICSI using human sperm, and Piezo-ICSI using human sperm. PLCɀ activity was determined in bull and human sperm samples. Within the groups using bull sperm, the oocytes fertilized by conventional ICSI had the lowest values of 2 pronuclei (PN) formation and cleavage, Piezo-ICSI increased both percentages and ICSI + chemical activation presented the highest 2 PN, cleavage, and blastocyst rates (p < 0.05). Within the groups using human sperm, the oocytes fertilized by Piezo-ICSI presented higher 2 PN and cleavage rates than those activated by conventional ICSI (p < 0.05). Piezo-ICSI with human sperm increased bovine oocyte activation as much as conventional ICSI + chemical activation with bovine sperm (p < 0.05). Higher values of PLCɀ activity were found in human sperm compared with bovine sperm (p < 0.05). Our results suggest that the higher stability of the bovine sperm in combination with its relatively low content of PLCɀ impairs bovine oocyte activation after ICSI.

Improved fertilization, degeneration, and embryo quality rates with PIEZO–intracytoplasmic sperm injection compared with conventional intracytoplasmic sperm injection: a sibling oocyte split multicenter trial

To investigate whether PIEZO-intracytoplasmic sperm injection (PIEZO-ICSI) increases the fertilization rate, decreases the degeneration rate, and increases the utilization rate per oocyte injected compared with conventional intracytoplasmic sperm injection (ICSI). Sibling oocyte split multicenter trial. Fertility clinics. Women with a diagnosis of infertility who used ICSI as their method of insemination and had ≥6 mature oocytes for injection. Participants had their mature oocyte cohort divided, where half were injected using conventional ICSI and the other half were injected using PIEZO-ICSI. For patients with an uneven oocyte number, the extra oocyte was injected using conventional ICSI. The injection technique used first was also randomized to ensure that there was no bias due to order of injection. The primary outcome measure was the fertilization rate after injection. A total of 108 patients underwent a sibling split use of conventional ICSI and PIEZO-ICSI. The fertilization rate was 71.6% in PIEZO-ICSI, which significantly increased compared with that in conventional ICSI 65.6%. In addition, the oocyte degeneration rate decreased in PIEZO-ICSI compared with that in conventional ICSI (6.3% vs. 12.1% respectively), and the blastocyst quality increased, as measured by the number of grade A and B quality blastocysts present on day 5 of development (33.3% vs. 27.5%). No significant differences in the aneuploidy or utilization rate, clinical pregnancy, or live birth outcome after single embryo transfer were noted between the two injection techniques. This trial supports the possibility that PIEZO-ICSI increases the fertilization rates, decreases the oocyte degeneration rates, and increases the blastocyst quality compared with conventional ICSI; however, it does not appear to influence the clinical pregnancy or live birth rate per transfer. Australian and New Zealand Clinical Trial Registry ACTRN12620000407998.

Y chromosome AZFc microdeletion may have negative effect on embryo euploidy: a retrospective cohort study

BackgroundEmbryo aneuploidy is a main of principal reason of pregnancy loss, in vitro fertilization (IVF) failure and birth defects in offspring. Previous researchs have demonstrated that Y chromosome AZFc microdeletion was associated with reproduction outcomes, however, the relationship between Y chromosome AZFc microdeletion and embryo aneuploidy remains unexplored.MethodsThis retrospective cohort study enrolled 513 patients with 603 cycles in the reproductive center of Nanjing Maternity and Child Health Care Hospital from January 1, 2016 to June 30, 2022. The study cohort was divided into two groups: the AZFc microdeletion group, comprising 53 patients and 58 cycles, and the control group, comprising 460 patients and 545 cycles. Statistical methods including restricted cubic spline and generalized estimating equation (GEE) were employed to evaluate the relationship between Y chromosome AZFc microdeletion and embryo euploidy.Results294 and 2833 blastocysts were selected as AZFc microdeletion group and control group, respectively. Patients with Y chromosome AZFc microdeletion had significantly higher embryo aneuploid rate (33.0% vs. 27.3%, P < 0.05), lower rate of normal fertilization rate (81.5% vs. 90.3%, P < 0.05) and lower blastocysts formation rate (47.0% vs. 57.8%, P < 0.05) compared with the control group. However, no significant differences in pregnancy outcomes after euploid embryos transfer were observed between these two groups.ConclusionsOur study underscored the association between Y chromosome AZFc microdeletion and an elevated risk of embryo aneuploidy. Before the conventional intracytoplasmic sperm injection (ICSI) treatment, couples with Y chromosome AZFc microdeletion should be apprised of the heightened susceptibility to embryo aneuploidy. Preimplantation genetic testing for aneuploidy (PGT-A) should be introduced for selection.

Intracytoplasmic sperm injection does not improve the outcome of IVF treatments in patients with advanced maternal age or low oocyte number: A randomized controlled trial.

Intracytoplasmic sperm injection (ICSI) was introduced to achieve fertilization in cases of severe male factor infertility. However, ICSI is often used in cases of non-male factor infertility, such as advanced maternal age or low oocyte number, but the clinical benefit of the method in these indications has not been proven. A prospective randomized study was conducted in a university clinic between 2018 and 2020. Patients with ≥40 years of age and/or ≤4 oocytes with non-sever male factor infertility were randomized into conventional IVF or ICSI groups. Fertilization rate, embryo quality, implantation, clinical pregnancy and live birth rates were compared. A total of 336 IVF cycles (169 conventional IVF and 167 ICSI) were involved in the study. The fertilization rate was higher in the conventional IVF group compared to the ICSI group (IVF: 61.7%, ICSI: 53.4%, P=0.001). Embryo development and morphology did not show considerable difference between groups. Implantation, clinical pregnancy and live birth rate were 13.1%, 24.3% and 11.4% in the conventional IVF and 10.4%, 19.0%, 12.0% in the ICSI group. The differences were not significant. Subgroup analysis showed a significantly better clinical outcome following conventional IVF when advanced maternal age was accompanied by low oocyte number (Implantation: 11.7% vs 2.6%, P=0.027; Clinical pregnancy: 18.5% vs 4%, P=0.020). A significantly higher fertilization rate, a tendency for higher clinical pregnancy rate was found in conventional IVF treatments compared to ICSI. When advanced maternal age was associated with low oocyte number, ICSI resulted in a substantially lower chance of fertilization and clinical pregnancy. These data suggest that ICSI offers no advantage over conventional IVF in terms of fertilization, embryo quality, implantation and pregnancy rates for couples with advanced maternal age or with low oocyte number.

P-259 Intracytoplasmic sperm injection conducted within a micro 3D printed device that requires no holding pipette or vacuum reduces porcine oocyte invagination during microinjection

Abstract Study question Can the use of a custom-designed and 3D printed microICSI™ device reduce the degree of oocyte invagination during intracytoplasmic sperm injection (ICSI)? Summary answer Invagination was significantly less in porcine oocytes when ICSI was performed in microICSI™ device compared to conventional ICSI. What is known already ICSI is a difficult procedure for embryologists to master, yet until recently, technically little had changed. In particular, the holding pipette (HP) with vacuum to hold the oocyte has not been surpassed. However, a source of shear-stress during ICSI of oocytes is the invagination of the zona pellucida and cytoplasm that occurs during microinjection, due to the pressure of the injection pipette against the small surface area of the HP. A recent study demonstrated microinjection within a two-piece 3D printed device. Here we evaluated a design refinement that displaced injection pressure more evenly across the oocyte to improve embryo quality. Study design, size, duration A control (conventional ICSI) and study group (microICSI™) were compared over three replicates investigating the level of invagination (3-point score, 1 is lowest and 3 is greatest) during ICSI. Incidence of cytoplasmic lysis was also recorded 24 h following ICSI. Participants/materials, setting, methods Porcine oocytes collected by follicle aspiration of abattoir-sourced ovaries were matured for 39-44 h in NCSU-17 medium supplemented with eCG, hCG, porcine follicular fluid, EGF and insulin. Mature oocytes, stripped of cumulus, were randomly allocated to either conventional ICSI or microICSI™. ICSI was performed, with treatment order randomised, and the injected oocytes were cultured in NCSU-17 culture medium at 39 C. Main results and the role of chance The microICSI™ device was modelled as a one-piece structure with contoured surfaces within a microwell array to support the oocyte during injection without the requirement for a holding pipette. Two-photon polymerisation 3D micro printing was used to print and assess multiple prototype geometries to optimise the support of the oocyte during ICSI. Over the three replicates (n = 10-20/treatment/replicate), the degree of invagination was consistently lower in the microICSI™ group compared with conventional ICSI (Mean ± SEM): Replicate 1: 1.40 ± 0.16 vs. 2.00 ± 0.19; Replicate 2: 1.20 ± 0.11 vs. 2.00 ± 0.13; Replicate 3: 1.42 ± 0.12 vs. 1.78 ± 0.15, with a paired t-test showing a significant difference between the two groups (p = 0.035). The incidence of lysis was not significantly different between the two treatments (ranging from 15-20% across both groups and varied between replicates). Limitations, reasons for caution The use of a porcine ICSI model may not sufficiently replicate what occurs with human ICSI as human oocytes experience greater levels of invagination (typically scored out of 4 rather than 3). Wider implications of the findings Reducing the invagination due to the injection process may reflect a less stressful procedure, with improvement to fertilization rates and embryo quality. Trial registration number not applicable

O-179 Fertilization topologies that occur seldom or not at all in nature give rise to distinct functional classes of mouse embryos

Abstract Study question Would new embryo properties emerge if fertilization was imposed on oocytes in regions, such as the animal pole, where it does not take place naturally? Summary answer Oocyte fertilization at the animal pole, vegetal pole or equator results in 2-cell embryos with distinguishable transcriptomes and functional peculiarities. What is known already There is fertilization bias in embryos used in basic and clinical research, because: 1) sperm–oocyte fusion hardly occurs at the surface above the meiotic spindle – the animal pole; 2) the region opposite the spindle - vegetal pole - is poorly accessible to sperm due to tiny perivitelline space. These regions are also avoided during intracytoplasmic sperm injection (ICSI) for fear of damaging the spindle or losing the sperm nucleus into the 2nd polar body. Pole materials are held non-essential for mouse development, but this conclusion relies on the 2nd polar body, which is an unreliable topological marker. Study design, size, duration Oocytes were rotated using a micromanipulator fitted with Nomarski optics, using the meiotic spindle as a landmark. Between 9:30 and 10:30 am single sperm heads were microinjected (ICSI) at the animal or vegetal pole (treatments) vs. the naturally prevalent equatorial region (control). This way two fertilization topologies and possibly also two classes of zygotes were created that are otherwise seldom or not at all represented in natural fertilization, in vitro insemination or conventional ICSI. Participants/materials, setting, methods Metaphase II oocytes were collected from 8-week-old B6C3F1 mice stimulated with 10 I.U. eCG+hCG. Sperm heads from a single batch of cryopreserved CD1 semen were deposited via ICSI in the cortex at the animal pole, vegetal pole or half-way between poles i.e. equatorially. Zygotes were cultured in KSOM(aa) and analyzed (triplicate or more) for: cleavage rates, transcriptomes at the 2-cell stage (RNAseq), blastocyst germ layers (immunostaining for trophectoderm, primitive endoderm, epiblast), and postimplantation development. Main results and the role of chance Although full development was supported irrespective of ICSI site, embryos clustered by site, as revealed by single-cell RNAseq of 21, 21 and 13 two-cell embryos whose oocytes were fertilized at the animal pole, vegetal pole or equator, respectively. When examining the sister blastomeres together, 462 genes of the shared transcriptome were differently expressed between ICSI sites, with the equatorial class contributing most to the difference (adj.p&amp;lt;0.05, Wilcoxon test). This was true also when examining the sister blastomeres separately: interblastomere differences of the equatorial class exceeded those of the pole classes (72% vs. 14% of differently expressed genes). Ontology analysis of the differently expressed genes using Enrichr pointed at the endomembrane system – an acquaintance of oocyte polarity studies (PMID 10545249; PMID 29746690). Follow-up of 2-cell embryos to blastocysts revealed that sister blastomere contribution to each germ layer was less balanced in the equatorial class, as measured by linear correlation of cell numbers (e.g. equatorial R2=0.00 vs. polar R2&amp;gt;0.23 for the epiblast). Summing up, it is difficult to reconcile these data with a mainstream view that fertilization at the animal pole is harmful. Rather they support that the topology of fertilization defines functional classes of 2-cell embryos with distinguishable transcriptomes. Limitations, reasons for caution This is an animal study. Mouse ICSI uses mercury-loaded piezo-driven needles, human ICSI does not. The higher consistency of using the one and same batch of cryopreserved spermatozoa was traded off against lower developmental rates. Single-cell resolution posed a limit on RNAseq depth. Results need confirmation in other mouse strains. Wider implications of the findings The higher blastomere similarity observed after ICSI at the vegetal pole compared to ICSI at the equator is not consistent with the prevalent model of first zygotic cleavage that is driven by the topology of the two apposing pronuclei. Trial registration number not applicable

O-136 Artificial intelligence to assist in surgical sperm detection and isolation

Abstract Study question Can an artificial intelligence (AI) improve the speed and accuracy of identifying sperm in complex testicular tissue samples? Summary answer Trained AI can identify sperm in real-time instantly with higher accuracy, not only reducing strain on embryologists but increasing sample coverage in a shorter time. What is known already Non-obstructive azoospermia (NOA) is a form of severe male-factor infertility, affecting nearly 5% of infertile couples seeking treatment. Isolating sperm from macerated testicular tissue for intracytoplasmic sperm injection (ICSI) has changed marginally in the last two decades, and still requires embryologists to search arduously through a background of collateral cells including red blood cells (RBC’s), white blood cells (WBC’s), leydig, sertoli and epithelial cells, causing fatigue and reducing sample coverage. Image analysis using trained AI can instantly identify shapes and forms and thus presents itself as a candidate to dramatically reduce processing times in surgical sperm cases. Study design, size, duration This proof-of-concept consists of two phases over 5 months. A training phase using 7 azoospermic patients to provide samples to train a convolutional neural network with manual annotation. Secondly, a side-by-side live test with an embryologist versus the AI model comparing time taken and accuracy of sperm identification, and precision of identifying sperm in macerated tissue samples (false positives and false negatives). Results were analysed using Mann-Whitney U-test. Differences were considered significant when p-value&amp;lt;0.05. Participants/materials, setting, methods Consenting patients at IVF Australia, Sydney attending for surgical sperm collection donated discarded macerated testicular tissue samples whereby excess tissue was used post-processing in conjunction with donor sperm, to train the machine learning software. Thereafter, testing using conventional ICSI micromanipulator microscope was performed side-by-side on test samples and time taken, accuracy and precision were recorded between a control count, computer vision and trained embryologist. Main results and the role of chance The AI model showed dramatic improvement in time taken to identify sperm per field, improved accuracy in identifying sperm as well as a high level of precision. Precision for the trained embryologist was considered to be 100%. Time taken per field of view to identify all sperm was significantly lower for the AI when compared to the trained embryologist (0.019 ± 1.4 x 10-4s vs 22.87 ± 0.98s, P &amp;lt; 0.0001). There was also a significant difference in accuracy (89.88 ± 1.56% vs 83.22 ± 2.02%, P = 0.017) for the AI model versus the trained embryologist respectively. The precision of the model is 91.27 ± 1.27% which considered false positives and the correct identification of sperm versus the control count. From a total of 688 sperm to be found, 560 were found by the embryologist and 611 were found by the AI model in less than 1000th of the time it took the embryologist. Limitations, reasons for caution This is a proof-of-concept test of a convolutional neural network AI. Following this study, side-by-side on donated clinical samples are needed, and a clinical trial comparing embryologists versus AI is required to prove efficacy and usefulness. A larger number of samples across multiple indications and surgical approaches is required. Wider implications of the findings AI-powered image analysis has the potential for seamless integration into laboratory workflows to reduce time to identify and isolate sperm from surgical sperm samples from hours to minutes, and do this with improved accuracy, thus increasing success rates from these treatments. Trial registration number N/A

Safety of early cumulus cell removal combined with early rescue ICSI in the prevention of fertilization failure

Research questionWhat are the clinical outcomes and safety implications of early cumulus cell removal after short-term insemination combined with early rescue intracytoplasmic sperm injection (ICSI) in preventing fertilization failure? DesignIn this retrospective study, a total of 14,360 cycles were divided into four groups based on insemination method and fertilization ability: conventional IVF group (n = 5519); early cumulus cell removal group (n = 4107); conventional ICSI group (n = 4215); and early rescue ICSI group (where failed or low fertilization was predicted, n = 519). Fertilization outcomes, pregnancy outcomes, neonatal outcomes and birth defects were analysed by comparing the early cumulus cell removal group with the conventional IVF group, and the early rescue ICSI group with the conventional ICSI group. ResultsThere were no significant differences in the outcomes of fertilization, pregnancy, neonates or birth defects between the conventional IVF group and the early cumulus cell removal group (P > 0.05). When compared with the conventional ICSI group, the early rescue ICSI group had similar rates of two pronuclei (2PN) at fertilization, clinical pregnancy, miscarriage, ectopic pregnancy, live birth, sex, mean gestational age, very low birthweight, macrosomia and birth defects (P > 0.05) but a higher polyploidy rate, lower high-quality embryo rate (both P < 0.001), lower twin pregnancy rate (P < 0.01), lower rate of low birthweight, and a higher rate of normal birthweight (both P = 0.024). ConclusionsEarly cumulus cell removal combined with early rescue ICSI led to good pregnancy and neonatal outcomes without an increase in birth defects. This approach could therefore be an effective and safe method for patients with fertilization failure in conventional IVF.

Opportunities and Limits of Conventional IVF versus ICSI: It Is Time to Come off the Fence.

Conventional IVF (c-IVF) is one of the most practiced assisted reproductive technology (ART) approaches used worldwide. However, in the last years, the number of c-IVF procedures has dropped dramatically in favor of intracytoplasmic sperm injection (ICSI) in cases of non-male-related infertility. In this review, we have outlined advantages and disadvantages associated with c-IVF, highlighting the essential steps governing its success, its limitations, the methodology differences among laboratories and the technical progress. In addition, we have debated recent insights into fundamental questions, including indications regarding maternal age, decreased ovarian reserve, endometriosis, autoimmunity, single oocyte retrieval-cases as well as preimplantation genetic testing cycles. The “overuse” of ICSI procedures in several clinical situations of ART has been critically discussed. These insights will provide a framework for a better understanding of opportunities associated with human c-IVF and for best practice guidelines applicability in the reproductive medicine field.