International Journal of

Introduction
 
Infertility influences one in seven couples generally and is a growing problem worldwide (1). Assisted reproductive techniques are currently accountable for up to 7% of childbirths in developed countries (2). Intracytoplasmic sperm injection (ICSI) has been developed as one of the best efficient therapeutic approach for male factor infertility (3). Whilst some crucial stages in normal fertilization are bypassed in ICSI, the average fertilization rate remains at 60-70% (4, 5). Fertilization failure after ICSI can be defined by imperfections in oocyte, sperm, as well as, ICSI technique (5, 6). After ICSI, while more than 80% of oocytes have spermatozoa fertilization failure is probably due to lack of activation of the oocyte, or an incapability of the oocyte to decondense the sperm (7-9).
Oocyte activation is one of the first events that occurs at fertilization and characterized by two basic molecular events (5, 10): an elevation of intracellular calcium concentration, due to the initial release from the endoplasmic reticulum which occurs 1-3 minutes after binding of the sperm to the oolemma, and a transient calcium elevation in oocyte as a main trigger of meiotic resumption during fertilization (6). Many investigators have tried different protocols for artificially activating oocytes to overcome fertilization failure after ICSI, including chemical, electrical and mechanical methods (4, 6). Among them, chemical activation is the most commonly used method for artificial oocyte activation (4).
Many chemical substances are known to induce an intracellular calcium surge and subsequently activate the oocytes, such as calcium ionophore (A23187), ethanol, ionomycin, puromycin, strontium chloride, probol ester and thimerosal (5, 6). Several experiments have revealed that the artificial oocyte activation with calcium ionophore led to an increase in intracellular free calcium, mimicking physiologic mechanisms that cause oocyte activation (11, 12). Therefore, this study was designed to investigate the effect of artificial oocyte activation with calcium ionophore (A23187) on fertilization, cleavage and pregnancy rate after ICSI in infertile men suffering from teratoospermia.
 
Materials and methods
 
This study was approved by Ethics Committee of Yazd Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
 
Patient Selection
This prospective, randomized, unblinded, clinical trial was performed on 38 women with teratoospermic partner (normal morphology <14%) undergoing to ICSI cycles in Yazd Research and Clinical Center for Infertility, during the April to December 2012. Teratoospermia in men confirmed based on their semen analysis. The patients were randomly allocated into two groups using a computer based randomization list. Written informed consent was obtained from all of the couples prior to the study. The women with aged more than 40, FSH ≥12 IU/L, immature oocyte, oocyte deformity were eliminated from this study.
 
Ovarian stimulation and ovum pickup
Controlled ovarian stimulation was performed by using the antagonist protocol. All patients received low-dose oral contraceptive pills (Ocp LD) (30 μg Ethinyl Estradiol and 0.3 mg Norgestrel, Aburaihan Pharmaceutical Co., Tehran, Iran) was began on 2^nd day of the cycle then discontinued till menstruation occurred. Once menses began, gonadotropin stimulation by Gonal-F (Gonal-F, Serono, Italy) was started from the 2nd day of the menstrual cycle .The starting dose of gonadotropin was 150-300 mIU/d, according to the patient’s age and body weight.
Monitoring was started on day 7 of stimulation and dose of gonadotropin was adjusted based on the serum E₂ concentrations and ovarian response as observed by ultrasound. Once the leading follicles reached 14 mm in diameter, Cetrorelix (Merck-Serono, Germany) 0.25 mg subcutaneously was added and continued every day till day of hCG administration. In both groups, 10,000 IU of hCG (pregnyl, Daropakhsh, Iran) was administered IM when at least three follicles reached ≥18 mm in diameter. Ovum pickup was done by ultrasound-guided 36 hours after hCG injection.
 
Semen processing before ICSI
The semen samples were gathered by masturbation following 3-4 days of abstinence on the day of ovum pick up. Sperm concentration and total sperm count was assessed using a Makler chamber. Sperm motility and morphology analysis was performed under light microscopy according to the Kruger’s strict criteria and World Health Organization criteria (13, 14). Sperms with normal head in size, shape, and acrosome, without midpiece or tail defects were considered normal. After complete liquefaction, the semen was washed using density gradient sperm-separation techniques, and the final pellet was resuspended in 0.2 mL of medium.
 
Oocyte activation method
After retrieval, oocytes were washed with G-MOPS medium (Vitrolife-Sweden) and incubated in culture medium (GIVF-plus; Vitrolife ,Sweden) then covered with mineral oil (Ovoil; Vitrolife) for 2 hours at 37^oC, 6% CO₂ and 5% O₂. Cumulus cells removed mechanically by a 30-second exposure to Hyase medium containing 80 IU/mL of hyaluronidase (Vitrolife, Sweden). Denuded oocytes were then evaluated for nuclear status.
Oocytes showing the release of the first polar body were considered mature and were selected for ICSI. Oocytes with single, three, or more pronuclei, immature, malformed, and postmature were eliminated from this research. Eppendorf micromanipulator mounted on a Nikon inverted microscope was used to perform ICSI. In control group (n=145 oocyte), routine ICSI was applied and oocytes transferred in the culture media for incubation at 37^oC and 6% CO₂ and 5% O₂.
In the artificial oocyte activation (AOA) group (n=168 oocyte) immediately after ICSI, oocytes were entered and incubated in culture medium containing 5 µM of calcium ionophore (A23187) (Sigma, St. Louis, MO, USA) for 5 minutes. Then injected oocytes washed at least five times with G-MOPS solution and incubated in the same culture medium. The oocytes were checked 16-18 h after injection to determine the presence of pronuclei using a Nikon inverted microscope.
Activated oocytes were defined with observation of at least one pronucleus or cleaved oocytes. The oocytes were cultured in-vitro for 2-3 days to assess their development and cell division.
 
Evaluation of embryo quality and embryo transfer
Fertilization and embryo quality were assessed by a skilled embryologist. Cleavage stage embryos are graded according to the Hill’s criteria (15): Grade A was considered the high-quality embryo, without fragmentation, equal-sized homogenous blastomeres (4-cell embryo on day 2 or 8- cell embryo on day 3) and homogeneous cytoplasm. Grade B: embryos with ≤10% fragmentation, equal sized homogenous blastomeres. Grade C: embryos with ≤50% fragmentation, unequal sized blastomeres, and large granules. Grade D: embryos with >50% fragmentation, unequal sized blastomeres, and large black granules.
Embryo transfer was performed by ultrasound guidance 48-72 hours after oocyte retrieval. For each couple, from one to three embryos were transferred, depending on the embryo quality and the age of the woman using a Labotect catheter (Labotect, Gotting, Germany). Remaining embryos were frozen in liquid nitrogen using verification method. All the patients received progesterone 100 mg/day, IM (Aburaihan Pharmaceutical Co., Tehran, Iran) and estradiol valerate 6 mg/day (Aburaihan Pharmaceutical Co., Tehran, Iran) as luteal support, starting on the day of oocyte retrieval.
 
Outcome measures
The primary outcome, including fertilization rate, chemical and clinical pregnancy rate were analyzed. The secondary outcomes, such as cleavage rate and percentage of high-quality embryos were also measured. The percentage of cleaved embryo was calculated as follow: total of cleaved embryos/number of zygotes Χ 100. Fertilization rate in both groups was calculated by the ratio of fertilized oocytes to the total number of survived injected metaphase II (MII) oocytes multiplied by 100.
Serum βhCG concentration was measured two weeks after embryo transfer. After pregnancy confirmation, progesterone and estradiol valerate were continued until the tenth week of pregnancy. Chemical pregnancy was defined as serum βhCG ≥25 IU/L measured 2 weeks after embryo transfer. Clinical pregnancy was documented by the presence of a gestational sac with heart beat on vaginal or abdominal ultrasound at 4-5 weeks after embryo transfer.
The implantation rate was characterized as the fraction of gestational sac (s) to the number of embryos transferred. Clinical abortion rate was defined as clinically recognized pregnancy losses before 20 weeks of gestation.
 
Statistical analysis
The SPSS 19 software package was used to perform all the statistical analyses. The normality of distribution of variables was tested by using the Kolmogorov-Smirnov test. Independent sample T-test was used for continuous variables which were normally distributed and Mann-Whitney U test for data not normally distributed. Chi-squared test or Fisher exact test were used for qualitative variables as appropriate. p<0.05 was considered statistically significant.

Table I. Basic and demographic characteristics of patients

 
Table II. Semen parameters in two groups


Table III. Cycle characteristics


Table IV. Embryo data of the study and control group



Table V. Pregnancy outcome





Figure 1. Recruitment follow-up and drop outs over the course of the study.


Discussion
 
Despite great advances in clinical and laboratory aspects of intra-cytoplasmic sperm injection (ICSI) procedure, fertilization failure still occurs in 2-3% of ICSI cycles (16). One of the reasons for failed fertilization after ICSI is lack of oocyte activation (4, 7, 16-18). In these cases, various mechanical, electrical and chemical methods are known to use for artificial oocyte activation. Among these methods, chemical oocyte activation was currently used with substances such as calcium ionophore. In ICSI cycles, oocyte activation with calcium ionophore has been effective at increasing  the fertilization rate (11, 19).
In the present study to improve outcomes of ICSI , 168 selected injected  oocytes were chemically activated with calcium ionophore (A23187) immediately after ICSI. Our results showed that there was no significant difference in fertilization and cleavage rate between two groups. Implantation, chemical and clinical pregnancy rate was higher in the AOA group compared with control group; although the difference was not significant. The results of this study are in contrast with the results of Borges and coworkers (11) . They reported an improvement in ICSI outcomes following calcium ionophore oocyte activation in azoospermic patients. The researchers found that calcium ionophore improved ICSI outcomes only the epididymal, but not testicular spermatozoa, were injected.
In another study, Nasr-Esfahani et al evaluated efficiency of ionomycin on ICSI outcomes in teratozoospermic patients . They reported that the fertilization and cleavage rate at 72 hours post-ICSI were significantly higher in study group. Moreover, there was no significant difference in cleavage rate 48 hours after ICSI and the percentage of high-quality embryo 48 and 72 hours after ICSI between the two groups. The overall pregnancy rate was  reported 37% in this study (4).
In a study by Eftekhar et al, the researchers analyzed the effect of artificial oocyte activation (AOA) on unfertilized oocytes 24 hours after ICSI using calcium ionophore (A23187) (5). In their study, fertilization and cleavage rate were72.5% and 62.7%, respectively. However high quality embryos remained low. According to a study in 2006 by Lu et al, exposure of unfertilized oocytes to a combination of calcium ionophore and puromycin could effectively activate them within 20-68  hour after ICSI (20). After 20 hours fertilization rate, cleavage rate and good quality embryos were 91%, 64% and 44% respectively. They detected passing of time after ICSI, resulted low fertilization and cell division rates. So time scale of exposure with calcium ionophore may affect ICSI outcome. Our study results were in agreement with Eftekhar et al and Lu et al studies (5, 20). These studies reported a similar fertilization rate in spite of different timing of oocyte activation after ICSI that demonstrating an optimal time of 24 h to expose the oocyte with calcium ionophpre.
 In our research cleavage rate (89.56%) was comparable with the results reported by Nasr-Esfahani (74%); but was higher than Eftekhar study results (62.7%)(4), (5) . These discrepancies may be due to differences in the different timing of oocyte activation after ICSI. The results of our study indicated a no significant difference in the implantation, chemical and clinical pregnancy rates between two groups; however there was a trend toward increasing these variables in AOA group. Although some inconsistency was observed, but these findings show that artificial oocyte activation using calcium ionophore in ICSI cycles improve the implantation, chemical and clinical pregnancy rates (5, 11).
The percentage of good quality embryos in our research were lower than that reported by Nasr-Esfahani et al (35.3% vs. 70%) (6). It's known that some of the chemical agents can cause intracellular calcium elevation in the oocyte through single or multiple signaling pathways (21). So, there is no doubt that different chemical agent and duration of exposure can result in various ICSI outcomes after oocyte activation. To stimulate oocyte activation in the present work, we applied calcium ionophore (A23187) for 5 minutes; nevertheless, in the study of Nasr-Esfahani et al, the investigators have used Ionomycin for 10 minutes (4).
In addition, in Nasr-Esfahani et al study, Grade “A” and “B” embryos were considered high-quality embryos; but in our study, only Grade “A” embryos were defined as high-quality embryos (4). Our findings in this study showed a higher fertilization rate than those reported in Eftekhar et al study (95.33% vs. 72.5%), which is probably due to difference in duration time after ICSI and oocyte activation. In this study oocyte activation was done 24 hours and in our study immediately after ICSI. Oocyte aging is the most common cause of poor results in IVF/ICSI procedures (22).
Therefore, the time interval between oocyte retrieval and fertilization is critical to obtain good quality embryos. Long term culture of oocyte results in zona hardening, increase the rate of parthenogenesis, disturbed embryo development, and poor embryo quality (23). In our investigation, abortion rate in AOA group was higher than control group; although, this rate was comparable with general population. This finding is in consistent with the prior researches that showed a normal chromosomal status in embryos derived through artificial oocyte activation (20).
 
Conclusion
 
In this study, chemical oocyte activation with calcium ionophore resulted in an insignificant improvement in the implantation, fertilization, cleavage and pregnancy rates after ICSI. On the other, oocyte activation could significantly increase the number of fertilized oocytes and embryos obtained. Finally, calcium ionophore may improve ICSI outcomes in infertile men suffering from teratoospermia. Therefore further study with more patients is suggested to provide greater value.
 
Acknowledgments
 
We would like to express our gratitude to the Yazd Research and Clinical Center for Infertility for their great insights and invaluable assistance to realize the present research project. This study was supported by Shahid Sadoughi University of Medical Sciences.
 
Conflict of interest
 
The authors declare they have no conflict of interest.