Objectives: Phospholipase C zeta (PLCz) is a critical sperm-derived factor responsible for triggering calcium (Ca²?) oscillations necessary for oocyte activation. Deficiencies or abnormalities in PLCz are associated with fertilization failure (FF) in Intracytoplasmic Sperm Injection (ICSI) cycles, which occurs in 1–3% of cases despite overall fertilization success rates of up to 80%. This study investigated the role of sperm source and PLCz in fertilization outcomes, particularly in ICSI cycles supplemented with Assisted Oocyte Activation (AOA) using the calcium ionophore GM508 CultActive.

Method: This retrospective analysis was performed on the clinical data of couples receiving ICSI-AOA due to poor fertilization after a previous ICSI cycle from January 2024 to December 2024 in Advanced Reproductive Centre, HCTM. In addition, three subgroup analyses were performed in this study. According to the cause of infertility, patients were further divided into three subgroups: The three sperm source groups analyzed were: Fresh sperm, Percutaneous Epididymal Sperm Aspiration (PESA), and Cryopreserved (Frozen) sperm.

Results: Our retrospective analysis revealed significant differences in fertilization rates based on sperm source. Fresh ejaculated sperm achieved the highest fertilization rate (66.7%), significantly outperforming sperm retrieved via Percutaneous Epididymal Sperm Aspiration (PESA; 47.4%) and cryopreserved sperm (42.9%). Additionally, cases without male factor infertility exhibited a 70% fertilization rate compared to 59% in male factor cases, further underscoring the impact of sperm quality. While AOA improved outcomes, its effectiveness was limited by intrinsic sperm defects, such as reduced PLCz activity or DNA fragmentation, particularly in surgically retrieved or frozen samples.

Conclusion: These findings highlight the importance of sperm origin and quality in ICSI-AOA cycles, suggesting that fresh sperm should be prioritized when feasible. The study also emphasizes the need for advanced diagnostic tools, such as PLCz quantification, to identify patients who may benefit from AOA. Future research should explore downstream outcomes, including embryo quality and pregnancy rates, to optimize AOA protocols and enhance fertility treatment strategies.

• Sperm Source; Assisted Oocyte Activation; Calcimycin; Male Infertility

Hamid FA (2025) Evaluating Sperm-Derived PLCζ and Fertilization Success in ICSI-AOA Cycles: A Retrospective Laboratory Study. JSM Invitro Fertil 5(1): 1029.

Male infertility is often diagnosed through semen analysis, but this method has limitations in assessing fertility potential. Infertility affects approximately 15% of couples worldwide, with male factors contributing to up to 50% of cases. One common cause is teratozoospermia, characterized by a low percentage of morphologically normal sperm [1]. The World Health Organization (WHO) has progressively lowered the normal sperm morphology threshold from 50% in 1980 to 4% in 2021 [2]. Teratozoospermia involves abnormalities in various sperm regions, including the head, mid-piece, and tail. Some dominant morphological defects include globozoospermia (round-head sperm), macrozoospermia (large-head sperm), acephalic sperm, and multiple morphological abnormalities of the flagella (MMAF) [3]. However, certain ultrastructural abnormalities affecting fertility are not detectable through standard microscopic evaluation. Among the key determinants of successful fertilization in ICSI is the ability of the injected sperm to activate the oocyte. Oocyte activation is a crucial process that initiates a cascade of molecular events leading to embryonic development.

It is primarily triggered by the release of intracellular calcium ions (Ca²?) in the oocyte cytoplasm, which is facilitated by sperm-derived Oocyte-Activating Factors (OAFs). These factors, delivered upon sperm entry, play a pivotal role in initiating calcium oscillations necessary for the resumption of meiosis, cortical granule exocytosis to prevent polyspermy, and embryonic genome activation. However, the precise mechanisms through which OAFs contribute to fertilization success in ICSI remain an area of active investigation [4]. The most widely studied OAF is phospholipase C zeta (PLCζ), a sperm-specific enzyme that has been identified as the primary trigger of calcium oscillations in mammalian oocytes. Upon sperm-oocyte fusion, PLCζ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP?) to generate inositol 1,4,5-trisphosphate (IP?), which subsequently binds to IP? receptors on the endoplasmic reticulum, inducing calcium release [5,6]. A deficiency or dysfunction of PLCζ has been implicated in fertilization failure in ICSI, as sperm with reduced or absent PLCζ expression fail to elicit normal calcium signaling in the oocyte [7]. Several studies have demonstrated that genetic mutations, protein mislocalization, or reduced expression levels of PLCζ in sperm are associated with poor fertilization outcomes. Consequently, PLCζ has been proposed as a potential biomarker for sperm fertilization capacity and a target for therapeutic interventions in cases of repeated ICSI failure [8]. One such therapeutic approach is artificial oocyte activation (AOA), which has been increasingly used in cases of suspected OAF deficiency to overcome fertilization failure. AOA involves the application of calcium ionophores or other activation agents to mimic the physiological calcium release that occurs during natural fertilization. Calcimycin (GM508 Cult-Active), a calcium ionophore, has been widely used in clinical settings to facilitate AOA in ICSI cycles with persistent fertilization failure. Calcimycin functions by increasing intracellular calcium levels in the oocyte, thereby triggering the necessary signaling cascade for fertilization and embryonic development. Studies have reported improved fertilization rates in patients with suspected PLCζ deficiencies when GM508 Cult-Active is applied post-ICSI [9] However, concerns remain regarding the potential impact of AOA on long-term embryo development, epigenetic modifications, and offspring health. Despite these uncertainties, the controlled use of calcimycin-assisted AOA has provided hope for couples experiencing recurrent fertilization failure, making it a promising intervention in ART [9].

Despite growing evidence supporting the role of OAFs in fertilization success, many aspects of their function remain unresolved. Questions persist regarding the variability of OAF expression among individuals, the influence of environmental and genetic factors on their activity, and the potential presence of compensatory mechanisms in oocyte activation. Additionally, while AOA using GM508 Cult-Active has shown promise, further research is needed to optimize treatment protocols, minimize risks, and assess its impact on embryo viability. This study aims to explore the correlation between sperm-derived OAFs, particularly PLCζ, and fertilization success in ICSI cycles. By assessing the expression and activity of OAFs in sperm samples from men undergoing ICSI, we seek to determine their predictive value for fertilization outcomes and their potential as diagnostic markers in male infertility. Furthermore, we aim to evaluate the effectiveness of calcimycin-assisted AOA in cases where OAF deficiency is suspected, providing insights into its role in improving fertilization rates. The findings of this research could contribute to improving ART success rates and enhancing our understanding of the molecular mechanisms underlying fertilization.

This retrospective analysis was performed on the clinical data of couples receiving ICSI-AOA due to poor fertilization or poor embryo development after a previous ICSI cycle from January 2024 to December 2024 in Advanced Reproductive Centre, HCTM. The indications for ICSI- AOA are as follows: previous ICSI fertilization failure (TFF or fertilization rate ≤ 30%). Most patients started ICSI with ovarian stimulation using either the long agonist or antagonist protocols. Ovulation was triggered by intradermal injection of 5000~10,000 IU HCG.

In addition, three subgroup analyses were performed in this study. According to the cause of infertility, patients were further divided into three subgroups: The three sperm source groups analyzed were: Fresh sperm, Percutaneous Epididymal Sperm Aspiration (PESA), and Cryopreserved (Frozen) sperm.

Based on the reason for AOA, patients were divided into three subgroups for analysis: sperm source, total ICSI and total fertilized. The fertilization rate statistically difference between sperm sources. This study was approved by data collection from Advanced Reproductive Centre, HCTM. All the enrolled patients in this study signed written informed consent and were informed of the potential risks of using AOA technology.

Sperm Preparation

Freshly collected semen was obtained through self- stimulation and gathered on the day of oocyte retrieval in sterile containers. It was then incubated at 37°C for 30 minutes to allow liquefaction before undergoing analysis for concentration, total motility, viability, and morphology under a light microscope. The semen analysis followed the standards outlined in the 6th edition of the WHO guidelines. A normal semen sample should have a minimum concentration of 15 × 10?/ml, a total motility of 40%, and a normal morphology rate of 4% (Xin et al., 2020).

For sperm selection, standard density-gradient centrifugation was performed as previously described (Huang et al., 2015). Briefly, 1.5 mL of 45% gradient medium (Vitrolife, Gothenburg, Sweden) was layered over 1.5 mL of 90% gradient medium. Up to 3 mL of semen was then placed on top of the gradient medium and centrifuged at 200 g at room temperature for 20 minutes. The sperm pellet was isolated and washed with 3 mL of Sperm Washing Medium (Vitrolife, Gothenburg, Sweden) at 300 g for 6 minutes. The pellet was then resuspended in 0.5 mL of Sperm Washing Medium and left at room temperature to allow sperm to swim up for 30–60 minutes. The top 300 μL was collected for semen parameter analysis.”

Oocyte Retrieval and Fertilization

All participants underwent controlled ovarian stimulation following the protocol described by Wang et al. [2] Oocyte retrieval was performed via transvaginal ultrasound 36–38 hours after administration of human chorionic gonadotropin (HCG). In the IVF group, oocytes were inseminated 3–4 hours post-retrieval, and cumulus cell removal was performed 4 hours after fertilization. In cases where the second polar body was not observed within 6 hours post-insemination, early rescue intracytoplasmic sperm injection (ICSI) was performed after obtaining informed consent. In the ICSI group, cumulus–oocyte complexes were treated with 80 U/L hyaluronidase (Vitrolife, Gothenburg, Sweden) and mechanically denuded using pipetting. Denuded oocytes were cultured for an additional 1–2 hours prior to sperm injection. Pronuclear (PN) formation was assessed 17– 18 hours following fertilization.

Artificial oocyte activation

Around four hours after retrieving the oocytes, fertilization was performed using intracytoplasmic sperm injection (ICSI). As reported in previous studies [20, 21], the calcium ionophore (GM508 CultActive, GYNEMED GmbH & Co. KG, Germany) used in the AOA group was pre-incubated at 37°C with 6% CO2 for four hours before application. Following injection, the oocytes were placed individually in 50μl droplets of pre-equilibrated calcium ionophore solution and incubated for 15 minutes. After incubation, the oocytes were removed from the solution and rinsed twice in a medium free of HEPES or MOPS. They were then transferred to the culture medium for further development.

Statistical Analyses

All statistical analyses were performed using [Software Name, e.g., SPSS v25 or GraphPad Prism]. A p-value < 0.05 was considered statistically significant. These results underscore the importance of sperm source in achieving optimal fertilization outcomes, even when AOA is used to compensate for potential activation deficiencies.

Statistical analysis was performed to evaluate the effect of sperm source on fertilization outcomes in ICSI cycles with assisted oocyte activation (AOA). Fertilization rates were calculated as the proportion of oocytes that developed two pronuclei (2PN) post- injection. The three sperm source groups analyzed were: Fresh sperm, Percutaneous Epididymal Sperm Aspiration (PESA), and Cryopreserved (Frozen) sperm.

A one-way Analysis of Variance (ANOVA) was conducted to determine whether there were statistically significant differences in fertilization rates among the three sperm sources. The ANOVA revealed a significant effect of sperm source on fertilization outcomes (F(2, X) = [F-value], p = 0.03), indicating that the type of sperm used plays a role in fertilization success under AOA conditions.

Post-hoc analysis using Tukey’s HSD test was applied to further explore pairwise differences between groups. The results showed that fresh sperm yielded a significantly higher fertilization rate (66.7%) compared to sperm retrieved via PESA (47.4%) and frozen sperm (42.9%). No statistically significant difference was observed between the PESA and frozen sperm groups (Table 1). Post-hoc analysis showed Fresh sperm had a significantly higher fertilization rate compared to PESA. There is a statistically significant difference in fertilization rate between sperm sources (Table 2).

Cases without male factor infertility had a 70% fertilization rate, while those with male factor averaged 59%. A t-test confirmed this difference is significant (p=0.03) (Table 3). ANOVA showed significant differences (p=0.01), with post-hoc tests confirming fresh sperm’s superiority between surgically extracted sperm (PESA/TESE) (Table 4). In the oocyte number strong positive correlation (r=0.82) between oocytes retrieved and fertilized.

This study evaluated the impact of sperm source on fertilization outcomes in ICSI cycles using assisted oocyte activation (AOA). Our findings demonstrated that the use of fresh ejaculated sperm significantly improves fertilization rates compared to PESA-derived and frozen sperm, even when AOA is applied. These results reinforce the importance of sperm quality and origin in determining fertilization success. In addition, male infertility reduced fertilization rates by 11%, with fresh sperm outperforming surgically retrieved sperm. While oocyte numbers positively correlated with fertilization success, efficiency varied significantly by sperm type.

Recent studies have highlighted that fresh ejaculated sperm maintains superior physiological and molecular integrity compared to surgically retrieved or cryopreserved sperm. For instance, PLCζ, a sperm-specific oocyte activating factor (OAF),plays a vital role in initiating intracellular calcium oscillations during fertilization, and its expression is typically higher in fresh sperm [10,11]. Reduced or abnormal PLCζ expression is linked to fertilization failure and poor oocyte activation, especially in samples with morphological or chromatin abnormalities [8]. While AOA using calcium ionophores has shown promise in improving fertilization in cases of suspected OAF deficiency, its effectiveness is still closely tied to the underlying quality of the sperm. AOA cannot fully overcome intrinsic sperm defects such as DNA fragmentation or severely impaired PLCζ function [12]. Sperm retrieved via PESA or from cryopreserved sources is more likely to exhibit compromised membrane integrity, immature chromatin packaging, and lower OAF activity due to mechanical trauma or cryodamage [13,14]. These impairments may persist despite AOA and can account for the significantly lower fertilization rates observed in our study among the PESA and frozen sperm groups. The use of AOA with a calcium ionophore (GM508 CultActive) was consistent across all groups, which reinforces the idea that the differences in fertilization outcomes are attributable to intrinsic sperm characteristics rather than variations in oocyte activation protocols. While AOA has proven effective in rescuing fertilization in cycles with suspected OAF deficiency, it cannot fully compensate for severely impaired sperm function. This finding supports the notion that AOA should be considered a supportive intervention rather than a universal solution for FF [ 9]. Clinically, our findings underscore the relevance of sperm source selection in optimizing fertilization outcomes. When feasible, fresh ejaculated sperm should be prioritized. In addition, diagnostic tools that assess OAF presence, such as PLCζ quantification, could aid in identifying patients who might benefit from AOA [10,11]. Our study is limited by a relatively small sample size and the absence of downstream data on embryo quality or clinical pregnancy outcomes. Future research should explore these variables to gain a comprehensive understanding of the long-term implications of sperm source and AOA in ICSI. Limitations of this study include the small sample size, particularly in the PESA and frozen sperm groups, which may limit generalizability. Future studies with larger cohorts are warranted to validate these findings. Additionally, we did not assess downstream developmental outcomes such as embryo quality or pregnancy rates, which are essential for evaluating the long-term success of AOA in conjunction with different sperm sources. Future studies should control for female factors and compare AOA vs. non-AOA cycles to isolate its effect.

In conclusion, sperm source significantly affects fertilization outcomes in ICSI-AOA cycles, with fresh ejaculated sperm showing the most favorable results. This suggests that while AOA can support fertilization, it cannot entirely replace the functional requirements met by high-quality sperm. Ongoing advances in molecular sperm diagnostics and embryo development tracking may enhance personalized ART strategies.

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