The Effect of Piezoelectric Stimulation in Patients with Low Fertilization Potential

Objective: To assess the value of the electrical activation of oocytes in ICSI patients with previous limited fertilization outcomes. Design: Prospective randomized study. Settings: Clinical IVF laboratory. Patient(s): A hundred and seven couples undergoing ICSI with possible low fertilization outcomes. Intervention(s): TESE, TESA, ICSI with Piezoelectric Activation Main Outcome Measure(s): Fertilization, clinical pregnancy rates, embryo grades. Result(s): Patients were subdivided into six study groups. In Group I, testicular elongated spermatids were used and 27.9% fertilization, 2.3% clinical pregnancy rates were evaluated. In testicular immotile spermatozoa injected group (Group II), 56.5% fertilization and 50% clinical pregnancy rates were obtained. 66.7% fertilization and 30%clinical pregnancy rates were achieved in Group III with testicular motile spermatozoa. In Group IV, patients with severe oligozoospermia, 64% fertilization and 28.6% clinical pregnancy rates were achieved. Group V included patients with total immotile spermatozoa and fertilization and clinical pregnancy rates were 50% and 57.1% respectively. In patients with history of low fertilization rate (Group VI), 38.7% fertilization and 19% clinical pregnancy rates were obtained. Conclusion(s): Piezoelectric stimulation can be used for patients with low fertilization rates and total immotile spermatozoa; as we detected an improvement in the fertilization and clinical pregnancy rates of these patients.


Introduction
Intra Cytoplasmic Sperm Injection (ICSI) has been widely used since 1992 in the era of assisted reproduction [1]. Although this method has become the preferred technique for male infertility, total fertilization failure or low fertilization rates are still the problem that has to be solved. Many studies have focused on this problem and it was revealed that more than 80% of unfertilized oocytes were arrested at the metaphase II stage, possibly due to failed oocyte activation [2].
The exact mechanism of oocyte activation is still unclear. Once the spermatozoon binds to receptors on oocyte plasma membrane, either a signal transduction pathway initiates [3,4] or Sperm-borne Oocyteactivating Factors (SOAF) are introduced into the oocyte cytoplasm [5,6]. Intracellular Ca 2+ increase causesa series of metabolic reactions in oocyte activation process. Higher Ca 2+ concentration inactivates Metaphase-Promoting Factor (MPF) that blocks the oocyte cell cycle at the metaphase stage of second meiotic division [7]. Following the inactivation of MPF; oocyte activation occurs and meiosis continues with the anaphase/ telophase transition followed with the exclusion of the second polar body and the exocytosis of cortical granules [8][9][10].
Although oocyte activation can be performed by several methods, the potential toxic effects of chemical activation on different developmental stages of embryos have not been adequately estimated yet. A nonchemical activation method such as electrical stimulation might be used as an alternative against insufficiently tested drugs for activation [24,25].
The aim of this study was to estimate the value of the electrical activation of oocytes in ICSI patients with previously failed or limited fertilization outcomes.

Experimental design
This study included 107 patients undergoing ICSI treatment at Gen-Art Woman Health and Reproductive Biotechnology Center between January 2006 and May 2009. All the patients were informed about the piezoelectric stimulation procedure and signed an appropriate written consent form. The patients were distributed into six groups. The first group consisted of 43 cycles in which testicular elongated spermatids were used (Group I). In Group II, 6 patients with testicular immotile spermatozoa, in Group III, 10 patients with testicular motile spermatozoa were included. Group IV comprised of 7 patients with severe oligozoospermia, Group V of 7 patients with total immotile spermatozoa and Group VI of 21 patients who had low fertilization rates (≤ 25%) in their previous IVF cycles.

Ovarian stimulation and embryo development
The protocols and procedures for ovarian stimulation and oocyte handling have been published previously [11]. Fertilization was assessed 12-16 hours post microinjection. Normally fertilized oocytes were left for further culture. Embryos were classified according to Veeck's morphological criteria: Grade I embryos had equal-sized blastomeres and no cytoplasmic fragmentation, grade II embryos had blastomeres of equal size and minor cytoplasmic fragmentation covering 10% of the preembryo surface, grade III embryos had blastomeres of distinctly unequal size and variable fragmentation, grade IV embryos had blastomeres of equal or unequal size and moderate-to-significant cytoplasmic fragmentation covering >10% of the pre-embryo surface, and grade V embryos had few blastomeres of any size and severe fragmentation covering 50% of the preembryo surface. None of the embryos were classified as grade V in this study.
All embryos were transferred on the third day of culture, using transabdominal sonographic guidance. Biochemical pregnancy was established when serum β-HCG was found >20 IU/L on the 12th day after embryo transfer, and clinical pregnancy was defined as the presence of a gestational sac on ultrasound examination at 6 week's gestation.

Semen preparation
For the preparation of ejaculated semen, gradient or swim-up, techniques were performed regarding to sperm parameters [26-28]. Testicular Sperm Extraction (TESE) procedure was performed under local anesthesia by widely opening the testis in an equatorial plane. The collected tissue was mechanically dispersed in a petri dish (Falcon Plastics, Becton-Dickinson) with an insulin syringe. All the elongated spermatids, immotile and motile spermatozoa were collected directly from the pool of testicular tissue samples or testicular sperm aspirates

Piezoelectric stimulation of injected oocytes
Piezoelectric stimulation was applied 20 minutes following microinjection with a BTX Electro-cell manipulator (BTX, San Diego, CA) at room temperature with a chamber with two stainless steel electrodes 0.5 mm apart, filled with activation buffer (pH ¼ 7.0) including Mannitol (0.3 M), MgSO 4 (0.1 mM), CaCl 2 (0.1 mM), HSA (0.05 mg/mL), and HEPES (0.5 mM). Injected oocytes were activated with a single pulse of 1.5 kV/cm DC for 100 msec. Stimulated oocytes were immediately transferred back to culture medium G1 (Vitro life; IVF Science) droplets in a humidified atmosphere of 6% CO 2 at 37 º C.

Statistical analysis
Data analysis was performed using Statistical Package for Social Sciences (SPSS) version 11.5 software (SPSS Inc., , , ).

Results
A total of 498 embryos were obtained after piezoelectric stimulation from 107 patients. The effect of electro activation on fertilization and clinical pregnancy rates are described in Table 1. In Group I, 27.9% fertilization and 2.3% clinical pregnancy rates were achieved. In Group II, the fertilization and clinical pregnancy rates were 56.5% and 50% respectively whereas the rates were 66.7% and 30% in Group III. Piezoelectric stimulation was also applied to patients with severe oligozoospermia in order to increase the fertilization rate and 64% fertilization and 28.6% clinical pregnancy rates were achieved (Group IV). In Group V, fertilization rate was 50% and clinical pregnancy rate was 57.1%. In patients with a history of low fertilization rate (Group VI), 38.7% fertilization and 19% clinical pregnancy rates were obtained.  The grades of embryos were also evaluated in each group to see whether electro stimulation had apparent effects on embryo morphology or not. The grades of embryos were also classified among 6 groups and individually figured out in Table 2.  Oocyte activation method has been used as an efficient treatment option in cases of complete fertilization failure and low fertilization outcomes [19,24,[34][35][36][37][38][39][40].

Groups
Cytoplasmic aspiration in conventional ICSI procedure may not always be sufficient for oocyte activation [16,23]. There are several studies focusing on different artificial oocyte activation (AOA) techniques. According to some of the previously published data, artificially activated oocytes (either calcium ionophore or electrically) develop similarly to fertilized oocytes [41][42][43][44].
Tejera et al. [19], showed that activation of oocytes with calcium ionophore increased the fertilization rates from 35.7% to 55.6% and they achieved a successful pregnancy and childbirth [19]. On the contrary Borges et al. [18] reported that AOA with Calcium ionophore did not improve ICSI outcomes with epidydimal or testicular spermatozoa. Also Kahraman previously reported that oocyte activation prior to ICSI did not lead up to better fertilization rates [45].
The knowledge about the potential cytotoxic, teratogenic and mutagenic effects of assisted activation techniques on oocytes and embryos in IVF is inadequate [26,46]. While designing our studies, we preferred the nonchemical activation method such as electrical stimulation [12,24,26] as chemical activation was shown to induce parthenogenetic development of oocytes [47]. On the other hand about 70%-80% of unfertilized oocytes after ICSI responded to electro activation and formed two pronuclei [24,36].
In the recent study we aimed to evaluate the effect of piezoelectric stimulation in patients with possible low fertilization outcomes. In the beginning we decided to evaluate elongated spermatid injected oocytes and observed a fertilization rate of 27.9% whereas various studies have reported 24% [48,49] and 71% [50]. Although we had piezoelectrically stimulated the oocytes of patients with elongated spermatids; our data is still similar to non-activated studies. Although we achieved one successful clinical and one biochemical pregnancy, the results were still unsatisfactory. Furthermore, in the study of Kumagai et al., 15% of pregnancy rate was reported with electro activation of spermatid injection [51].
Testicular immotile spermatozoa injection combined with piezoelectric stimulation, the fertilization rates were observed as 56.5% while the clinical pregnancy rates were 50%. In case of using testicular motile spermatozoa; fertilization rates were 66.7% and clinical pregnancy rates were 30%. Konc et al. [52] have evaluated the nonactivated TESE-ICSI-ET cycles and found a fertilization rate of 68% and 60% and a pregnancy rate of 33% and 29% with TESE motile spermatozoon and TESE immotile spermatozoon respectively. When we compare our results with this study, we observed no significant difference in fertilization and pregnancy rates in testicular motile spermatozoon injected patients. However the results of TESE immotile group indicated an impressive effect of piezoelectric activation on pregnancy rates. Implantation period, postimplantational development, fetal morphology [53] and weight variations in offspring could be affected via the differences in the calcium oscillation [54]. The exact mechanism by which intracellular calcium influences embryonic development remain subject to debate, [18] Borges et al. [18] have demonstrated higher pregnancy rates in artificial oocyte activation by calcium ionophore A23187 and also indicated that more patients are needed to be evaluated [18]. In our study, it was an unexpected result for us to find that electro activation had affected only pregnancy rate but not fertilization rate in TESE immotile group. So we concluded that it would be appropriate to increase the number of patients in order to obtain statistically significant data.
In a previous study of Mansour et [12]. We also intended to evaluate the effects of piezoelectric stimulation on severe oligozoospermia patients' IVF cycles and couldn't observe any significant difference as our fertilization rate was 64%.
On the other hand we obtained sufficient results in total immotile group (Group V); a pregnancy rate of 57.1% and good quality embryo rate of 71.4%. A fertilization rate of 44% was reported in Westlander et al. [55] study and 2 of 6 embryos were found to be good quality. As Nijset et al. [56], obtained low quality embryos with total immotile spermatozoa, our results referred the positive effect of piezoelectric stimulation on embryo grades and clinical pregnancy rates of total immotile patients. It has always been questioned whether to use testicular or seminal sperm for ICSI in total immotile patients. Westlander et al. [55], preferred to use testicular immotile spermatozoa instead of seminal immotile as DNA damage increases in ejaculated semen [55]. In contrary to this, our results demonstrated that higher pregnancy rates and better quality of embryos were obtained by using seminal immotile sperm for microinjection.
Hee-Jun Chi et al. demonstrated that the fertilization rate was increased from 25% to 80% after calcium ionophore activation in a normozoospermic patient with a history of low fertilization rate and obtained a successful pregnancy and a twin born following the activation [43]. Our results have not suggested such a significant increase in fertilization rates but still, piezoelectric could be referred as a sufficient activation method in patients with low fertilization histories.
In our previous study; we obtained very encouraging results of piezoelectric combined ICSI in Total Fertilization Failure (TFF) patients and reported apregnancy rate of 12.8% [11]. Yanagida et al.
[24] reported that ICSI followed by electrical oocyte activation resulted in the delivery of healthy twins for a couple with previously failed fertilization [24]. Mansour et al. [12], also recommended that the patients should be electro activated if there was a failure/limitation in previous cycles or the patient was severe OAT, azoospermic or has total immotile spermatozoa [12].
In studies related with total or limited failure of fertilization, all the activation procedures are performed on oocytes. On the other hand, it has been estimated that sperm soluble factors also plays a role in oocyte activation. Yanagida et al. [57] compared the fertilization, cleavage and pregnancy rates among three types of sperm immobilization (pipetting, squeezing and piezo application) techniques and concluded that the piezo activation method was the most efficient one. In order to use artificial activation techniques as a part of the conventional IVF procedures; the effects of sperm soluble factors could also be taken into consideration in multifactorial oocyte activation studies.
As a conclusion; our results agree with the usage of piezoelectric stimulation for patients with low fertilization rates and total immotile spermatozoa; as we detected an improvement in the fertilization and clinical pregnancy rates of these patients. But further research is still needed with an increased number of patients in order to evaluate the clinical effectiveness and safety of this method.