Role of calcium inophore in assisted oocyte activation and accelerated capacitation in sperm: A literature review

INTRODUCTION

In in-vitro fertilization (IVF) or ICSI resulting in few or no embryos for transfer continues to be a significant clinical challenge during treatment.^[1,2,3] The introduction of ICSI significantly improved the clinical outcome for patients with male factor infertility, particularly those with low sperm counts, who could not achieve normal levels of fertilization with conventional IVF. Typical fertilization rates with ICSI average 70% for most patients,^[4,5] including those with poor semen parameters or surgically retrieved sperm. A significant proportion of ICSI cycles still results in fertilization rates below 50%, with 3% resulting in total failed fertilization.^[16] Although ICSI is invasive, and operator-dependent factors may contribute to the proportion of oocytes not fertilizing normally, most commonly the failure of an oocyte to fertilize after sperm injection is failure of oocyte activation or a failure of sperm activation. In most mammals, the mature ovulated oocyte is arrested in metaphase of the second meiotic division (meiosis II) until fertilization by a sperm. Sperm binding with the oolemma activates the oocyte, triggering a series of pulsatile increases in intracellular calcium concentration, which in turn results in the resumption and completion of meiosis II, extrusion of the second polar body (PB2), and the initiation of preimplantation development. Phospholipase C zeta 1 (PLCz), a sperm-specific phospholipase, is considered the trigger for the molecular pathway within the oocyte, resulting in the release of calcium stores from the endoplasmic reticulum. Recent studies have shown that fertilization failure after ICSI can be linked to sperm devoid of PLCz or sperm with abnormal PLCz function.^[6,7,8] It has also been demonstrated that oocyte factors as well as sperm factors are involved in failed fertilization after ICSI. This is even with normal sperm parameters and a good ovarian response. The persistent fertilization failure raises the question of an underlying cause and whether other factors, apart from calcium ionophore being located within the oocyte cytoplasm, may play a role in fertilization.

Sperm and oocyte activation are induced by a variety of chemical agents, such as 6-dimethylaminopurine, strontium chloride, or calcium ionophores.^[13] A deficiency in intracellular calcium can be compensated by approaches that aim for an artificial calcium entrance or release.^[9,10] A number of studies have been carried out to assess the value of calcium ionophore as a method of sperm and oocyte activation. This study aimed to evaluate the use of calcium ionophore during assisted oocyte activation (AOA) and achieving hyperactivation in sperms.

MATERIALS AND METHODS

An online search was conducted on articles related to use of calcium ionophore in assisted oocyte activation (AOA) and achieving hyperactivation in sperm on PubMed, Google Scholar, ScienceDirect, and the Wiley online library. Search words like calcium ionophore, sperm capacitation, and assisted oocyte activation were used. The studies, which met the following criteria were recruited for analysis:

Inclusion criteria: Studies involving calcium ionophore for oocyte activation and sperm capacitation.

Exclusion criteria: Older studies (>10 years). The current review recruited studies conducted over the past decade to ensure a uniform criterion for calcium ionophore use.

Outcome measures: The studies were evaluated for the number of embryos fertilized, clinical pregnancy rate, miscarriage rates, and congenital birth defects.

RESULTS

Our study initially yielded 1521 articles via the database search. Nineteen articles, including reviews and meta-analysis. Eventually, nine full-text articles were included in our review, consisting of two meta-analyses (Shan et al.,^[9]; Murugesu et al.^[12]), two prospective studies (Miller et al.^[18]; Bonte et al.^[16]), four randomized clinical trials (Nikiforaki et al.^[13]; Navarrete et al.^[17]; Tateno et al.^[8]; Karabulut et al.^[15]), and one retrospective study (Miller et al.^[18]; Table 1). The flowchart of literature identification and selection is summarized in Figure 1.

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Figure 1:

Selection of studies.

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In most of the studies, the oocyte was exposed to 10 μM calcium ionophore dissolved in medium for 10 minutes before ICSI. The oocyte was washed extensively and then exposed 30 minutes later to 10 μM calcium ionophore for 10 minutes again. Finally, it was washed medium and continued for 2 to 3 hours in a medium drop under oil at 37 °C under 6% CO₂. Sperms were activated by 10 minutes of exposure to 20 μM calcium-ionophore before doing ICSI.

DISCUSSION

Calcium ionophore treatment for AOA is a common chemical activation method and is always used in cases of low fertilization rates or failed fertilization. Similarly, use of calcium ionophore for achieving hyperactivation in sperms is also used in human sperms. Several studies showed that the use of AOA with calcium ionophore treatment improved pregnancy and the live birth rate to a certain degree. As the number of studies increases, it is necessary to find more reliable evidence of the role of calcium ionophore in improving pregnancy outcomes using both oocytes and sperms. This study was done to explore the efficacy and safety of calcium ionophore treatment in improving pregnancy outcomes and decreasing the incidence of congenital birth defects.

Murugesu et al.^[12] studied 14 papers on AOA, where a total 5771 oocytes were taken for the AOA. Where calcium ionophore increased the overall clinical pregnancy rate (per ET; 3.48; 95% confidence interval [CI], 1.65–7.37) and the live birth rate (3.33; 95% CI, 1.50–7.39). This effect of adding calcium ionophore was further demonstrated with fertilization, cleavage, blastocyst, and implantation rates. Subgroup analysis further supported our findings (studies where n > 10 in both arms; random and fixed effects models). The study increases that the effect of calcium ionophore is significantly more pronounced with regards to the overall pregnancy rate.

Antonio et al.,^[11] did a sub group analysis on 49 oocytes and the results revealed that most oocytes activated normally. Twenty-seven of these were analyzed, and 16 (59.3%) were euploid, showing no effect of AOA on meiotic segregation. No difference in the proportion of meiosis II^[14] type errors was observed between artificially activated oocytes (28.6%; 95% confidence interval 3.7%–71.0%) compared with embryos obtained from normally fertilized oocytes (44.4%; 95% confidence interval 13.7%–78.8%). The abnormally activated oocytes, with >2PN (4 of 39, 10.3%) were diploid, indicating a failure to coordinate the telophase of meiosis II with polar body extrusion.

Miller et al.^[18] studied on the basis of a cohort of 595 ICSI pregnancies and 83 ICSI-Ca pregnancies (total number of oocytes taken for AOA not mentioned) theyfound no difference in birth defects rate for singletons or for twins. Additionally, no significant difference was found between defect type (chromosomal aberrations or structural malformations) and malformation type (heart, urogenital, and limb) between the ICSI and ICSI-Ca groups.

Karabulut et al.^[15] selected 78 patients (total number of oocytes taken for AOA not mentioned) and Nikiforaki et al.^[13] did AOA on 37 oocytes. Shan et al.^[9] found fertilization rates and quality of embryos observed in the AOA groups were higher than those of the controls, groups suggesting that not only total fertilization failure patients but also in globozoospermia.

Bonte et al.^[16] studied AOA (191 cycles, 1743 oocytes) significantly improved fertilization, pregnancy, and live birth rates in all mouse oocyte activation test (MOAT) groups compared with previous ICSI attempts (243 cycles). Fertilization rates after AOA were significantly different among groups 1 (70.1%), 2 (63.0%), and 3 (57.3%). Depending on the activation rate, patients were classified into MOAT group 1 (20% activation), MOAT group 2 (21–84% activation), or MOAT group 3 (85% activation). Between group 1 and 3, significant differences in pregnancy (49.0% vs. 29.4%) and live birth (41.2% vs. 22.1%) rates were observed. In total, 225 embryo transfers resulted in 60 healthy live births following AOA.

Navarrete et al.^[17] used a concentration of 1 million sperm cells/mL on 20 to 30 oocytes and demonstrated that transient incubation of mouse sperm with Ca²⁺ ionophore accelerated capacitation and rescued fertilizing capacity in sperm with inactivated PKA function. We now show that a pulse of Ca²⁺ ionophore induces fertilizing capacity in sperm from infertile CatSper1 (Ca²⁺ channel), Adcy10 (soluble adenylyl cyclase), and Slo3 (K+ channel) KO mice. In contrast, sperm from infertile mice lacking the Ca²⁺ efflux pump PMACA4 were not rescued. These results indicate that a transient increase in intracellular Ca²⁺ can overcome genetic infertility in mice and suggest this approach may prove adaptable to rescue sperm function in certain cases of human male infertility. Roles of cAMP were confirmed using KO genetic mouse models lacking either the PKA sperm-specific catalytic splicing variant Cα219, or sAC18. These cycles work the same as humans.

Tateno et al.^[8] used a concentration of 1 to 2 million sperm cells/mL on 70 to 140 oocytes. They found that, when spermatozoa were treated with A23187, more than 95% of oocytes were fertilized in the constant presence of the protein kinase A inhibitor, H89. Ionophore-treated spermatozoa also fertilized 80% of oocytes, even in the absence of HCO³⁻, a component essential for cAMP synthesis under normal in vitro conditions. Under these conditions, fertilized oocytes developed into normal offspring. These data indicate that mouse spermatozoa treated with ionophores are able to fertilize without activation of the cAMP/PKA signaling pathway. Furthermore, they suggest that the cAMP/PKA pathway is upstream of an intracellular Ca²⁺ increase required for the acrosome reaction and hyperactivation of spermatozoa under normal in vitro conditions.

CONCLUSION AND RECOMMENDATIONS

Calcium ionophore treatment on oocytes during ICSI and on sperms for achieving capacitation results in an improvement in fertilization rate. Though overall blastulation, implantation, pregnancy rate, and live-birth rates have yet to be proven statistically significant. It can be recommended for couples where ICSI alone yields poor fertilization rates. This analysis may have clinical application in patients with repeated fertilization failures in ICSI. However, larger randomized clinical trials are needed to recommend its standard use.