Intracytoplasmic sperm injection (ICSI) is an effective method for human-assisted reproductive technology, especially for male-factor infertility. However, the average fertilization rate of ICSI is 70%, and total fertilization failure occurs in 3% of cycles. Oocyte activation deficiency is the primary cause of fertilization failure.
Typically, sperm penetration evokes intracellular calcium (Ca2+) oscillations and causes oocyte activation. Oocyte activation deficiency can be overcome by artificial oocyte activation (AOA), involving various mechanical, electric, or chemical stimuli. Chemical activation is commonly applied in in vitro fertilization (IVF) clinics using Ca2+ stimulators, such as ionomycin, calcimycin, and strontium chloride (SrCl2). The process induces either a single spike or pulsating waves of intracytoplasmic Ca2+ concentration increases. The conventional AOA protocol, based on a single Ca2+ stimulator, has increased fertilization rates of couples who had previously experienced total fertilization failure after undergoing ICSI. However, conventional AOA is not adequately effective in overcoming fertilization failure in all patients.
In the present study, we performed a before–after study on a couple suffering from repeated fertilization failure despite previously undergoing ICSI with conventional AOA. To explore more effective AOA protocols, various methods of applying Ca2+ stimulators were compared in the patient. Shengjing Hospital Ethics Committee approval was obtained for the study. In addition, the couple provided written informed consent for the procedure and the use and publication of their data (this trial was registered at www.chictr.org.cn as ChiCTR2200055839).
The couple, a 32-year-old woman and her 33-year-old husband, had a 10-year history of primary infertility and repeated fertilization failure. The semen analysis of the husband revealed a semen volume of 3 mL, a sperm count of 21.25 × 106/mL with 20.69% motility (progressive sperm motility), 1% normal morphology, low sperm chromatin maturity, and abnormal polymorphonuclear elastase level. All female diagnostic parameters were normal.
Before visiting our clinic, they failed to achieve pregnancy after one cycle of artificial insemination with the husband's semen and three cycles of IVF at other clinics from 2012 to 2015. For the three cycles of IVF, 40 oocytes were achieved and successively subjected to conventional IVF, ICSI, and ICSI combined with conventional AOA with SrCl2. No oocytes were fertilized normally.
We recommended ICSI combined with AOA when the couple underwent the first cycle of IVF in our clinic. Ovarian hyperstimulation was performed using a gonadotropin-releasing hormone antagonist protocol. AOA medium of 10 μmol/L SrCl2 (Sigma, St. Louis, USA) and 10 μmol/L ionomycin (Sigma) were prepared 1 day before oocyte collection. AOA medium drops were overlaid with tissue culture oil (CooperSurgical, Trumbull, Connecticut, USA) and equilibrated at 37°C, 6% CO2, and 5% O2. Sixteen oocytes were obtained, including 13 oocytes in metaphase of meiosis II (MII), which were inseminated via ICSI. Subsequently, sibling-injected oocytes were activated with SrCl2 (four oocytes) for 1 h immediately after ICSI, ionomycin (five oocytes) for 10 min following 1 h of ICSI manipulation, and SrCl2 combined with ionomycin (four oocytes), which consisted of exposure to SrCl2 immediately after ICSI for 1 h followed by ionomycin for 10 min, at 37°C, 6% CO2, and 5% O2. On day 1 post insemination, no oocytes treated with SrCl2 were fertilized. However, one of five oocytes activated with ionomycin was fertilized but subsequently developed into a poor-grade embryo which was discarded on day 3. Notably, two of the four oocytes treated with ionomycin combined with SrCl2 were fertilized, and both were developed into fair-grade embryos and cryopreserved. The two fair-grade, cleaved embryos were thawed and transferred in the following menstrual cycle. Hormone replacement therapy was used for endometrial preparation for the frozen embryo transfer (FET) cycle. The luteal phase was supported by vaginal progesterone gel and estradiol. However, pregnancy was not achieved.
Three months later, the couple started a second cycle of IVF and underwent a similar procedure. As an unsatisfactory result was achieved via AOA with SrCl2 in the former cycle, 14 injected sibling MII oocytes were divided equally into two AOA treatment groups: seven oocytes were treated with ionomycin and the other seven with SrCl2 and ionomycin. On day 1, only one of the seven oocytes activated with ionomycin was fertilized and developed into a fair-grade embryo on day 3. On the other hand, five of the seven oocytes treated with SrCl2 and ionomycin were fertilized, resulting in five embryos on day 3, of which two were optimal and three were fair-grade embryos. A time-lapse was used to monitor fertilization and embryonic development [supplemental videos 1, https://links.lww.com/CM9/B230 and 2, https://links.lww.com/CM9/B231]. All cleaved embryos on day 3 were cryopreserved for future use. In the subsequent menstrual cycle, two optimal embryos derived from AOA with SrCl2 and ionomycin were thawed and transferred after endometrial preparation with hormone replacement therapy. Luteal support was performed as previously described. The serum human chorionic gonadotropin level was 922.62 mIU/mL 13 days after embryo transfer. A gestational sac with a regular fetal heartbeat was identified via ultrasonography 32 days after FET. Both the pregnancy and delivery process were completed without complications, and a healthy baby was born with no apparent abnormalities.
SrCl2 is widely used to overcome fertilization failure. However, in this study, SrCl2 was repeatedly ineffective. Even though SrCl2 is the most efficient method for AOA in rodents, its efficacy in humans is controversial. SrCl2 induces prolonged Ca2+ oscillations in mouse oocytes and supports embryo development. However, SrCl2 elicits a slight and gradual rise in Ca2+ levels in humans. The discrepancy between human and mouse oocyte adenosine triphosphate (ATP) levels could be the cause of their different responses to SrCl2. Therefore, as the total amount of Ca2+ signals elicited by SrCl2 in humans did not reach a minimum threshold required for oocyte activation, the failure of AOA using SrCl2 in this couple might be related to insufficient Ca2+ signal induction.
To compensate for the induction of Ca2+, we introduced another kind of Ca2+ inducer for AOA, namely the Ca2+ ionophore, which binds extracellular Ca2+ to facilitate its influx and elicits a single, prolonged intracellular Ca2+ transient. Both ionomycin and calcimycin belong to Ca2+ ionophores. Ionomycin rather than calcimycin was selected because it is more potent than calcimycin in provoking a Ca2+ increase with significantly higher amplitude and area. Results showed that the effect of the combined application of SrCl2 and ionomycin was indeed superior to that of SrCl2 alone. Further, the efficacy of ionomycin treatment alone was also assessed, revealing that the average fertilization and cleavage rates of AOA with ionomycin were superior to those of ICSI alone yet inferior to those of AOA with the combined application of SrCl2 and ionomycin. Furthermore, embryo quality obtained from AOA using ionomycin was unsatisfactory, suggesting that the evoking of Ca2+ signal in a transient manner using ionomycin was insufficient for supporting oocyte activation and subsequent embryonic development. It has been proposed that oocyte activation and subsequent embryonic development depend on the cumulative effect of Ca2+ input over downstream effectors. The combined use of SrCl2 and ionomycin likely provides oocytes with a sufficient level of Ca2+ signaling for accomplishing cellular events associated with oocyte activation. Previous studies in mice also showed that combined use of Sr2+ and ionomycin results in higher rates of oocyte activation and improved development vs. Sr2+ or ionomycin alone. Collectively, the results indicate that this innovative AOA protocol involving the combined use of two Ca2+ stimulators compensates for Ca2+ signal insufficiency observed during conventional, single Ca2+ stimulator AOA.
The health of children conceived via ICSI-AOA remains a major concern. Accumulating evidence suggests the utility and safety of ionomycin and SrCl2 for AOA. No significant differences in pregnancy status, baby health, and congenital birth defects (chromosomal aberration and structural malformations) were observed when ICSI-AOA and conventional ICSI were compared. This study provides direct evidence concerning the safety of the combined use of SrCl2 and ionomycin in humans, and a healthy baby was born after using the innovative AOA protocol. Nevertheless, evidence of the safety of the protocol is far from sufficient. Further follow-up exams of the baby are needed to clarify the long-term effects of the protocol, and we look forward to completing additional safety studies on a larger number of patients.
In conclusion, we report the first live birth achieved using an innovative AOA protocol with two types of Ca2+ stimulators, SrCl2 and ionomycin. The efficacy of the innovative AOA protocol was superior to that of conventional AOA based on the separate use of either ionomycin or SrCl2, as it resulted in a markedly improved fertilization rate and embryo outcomes. The compensatory mechanism of Ca2+ signals might be responsible for the superior efficacy of the innovative AOA protocol, which has the potential as a more effective protocol in couples for whom conventional AOA is ineffective [Figure 1].
This work was supported by the National Natural Science Foundation of China (No. 82071607); LiaoNing Revitalization Talents Program (No. XLYC1907071); Fok Ying Tung Education Foundation (No. 151039); Outstanding Scientific Fund of Shengjing Hospital (No. 202003).
Conflicts of interest
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