Background: Despite the high success rate of ICSI, total fertilization failure still occurs in 1-3% of all ICSI cycles and can recur in subsequent cycles, even when a sufficient number of oocytes and motile spermatozoa are available. Several reports show that the majority of couples suffering from ICSI failure benefit from the application of ICSI combined with assisted oocyte activation. A variety of artificial activating methods is used in human assisted reproduction treatment, including physical, mechanical or chemical stimuli, which provoke one or more calcium rises in the oocyte cytoplasm. Study Design: Randomized controlled trial. Setting: A university fertility center. Methods: 150 infertile patients who underwent ICSI and all had history of recurrent fertilization failure. The patients were randomly allocated into 2 equal groups. Group1=75 patients who underwent ICSI without oocyte activation. Group2 patients =75 and underwent ICSI Patient underwent ICSI with oocyte activation. Reproductive outcomes were compared between both groups. Results: there were significant differences between groups regarding number of oocytes retrieved, number of mature oocyte, fertilization rate and pregnancy rate. Conclusion: Assisted oocyte activation with calcium ionophore results in significant improvement in the fertilization, cleavage and pregnancy rates after ICSI.

This study is a randomized controlled trial included 150 ICSI patients with history of recurrent fertilization failure. Patients had undergone ICSI trials in ART unit, Al-Azhar University, Cairo, Egypt, from July 2017 to November 2018. The study was approved by the university medical ethical committee (under registry number 202010454) and all couples had signed a written consent before initiation of the study and the treatment cycles. AOA candidate couples were counseled regarding the procedure. The patients were selected according to the following criteria:

Inclusion Criteria: -

1. Age between 20 and 40 years old.
2. Cases with history of total fertilization failure in previous ICSI cycles
3. Oocytes with normal morphology.
4. Male factor infertility. Exclusion Criteria: - Abnormal oocyte morphology degenerated or immature oocytes.

Two groups were randomly designated:

The first Group: The oocytes were treated by calcium ionophore. This group involved 75 ICSI cycles.

The Second Group: Oocytes were not treated by calcium ionophore. This group involved 75 ICSI cycles.

Methods:

1. Ovarian stimulation:

   All women received ovarian stimulating drugs according to the ART protocols (long agonist, flare up or antagonist protocols). Deca 0.1 was given s.c. daily, for down-regulation in short and long protocol while cetrotide 0.25 was given s.c. daily for down-regulation in antagonist protocol. Follicular development was monitored by ultrasound scanning and serum estradiol. Patients received 10,000 IU of Human Chronic Gonadotrophin (HCG) when most of the follicles measured more than 17-20 mm in diameter.

2. Semen preparation:

   • The husband was asked to submit a semen sample in a sterile plastic container after a 2 to 3-day period of abstinence and about 2 hours before the ICSI procedure. The specimen container must be clean, sterile and wide mouthed to minimize collection error.

3. Oocyte retrieval:

   • Under general anesthesia, the oocytes were aspirated by a specialized, ultrasound-guided needle (Labotect aspiration catheter, Germany) at 34-36 h after HCG injection. Warmed HEPES buffered medium (Irvine Scientific, Irvine, CA, USA) was used for handling and washing of oocytes.
   • After ICSI, the whole injected oocytes were washed with Global total Fertilization Medium (Global pharm, Life Global, Brussels, Belgium, Europe) and the patients were randomly allocated into two equal groups (75 patients each) by using computer-based randomization program. Group one in which, the injected oocytes were transferred to a medium that contain 10 µmol of calcium ionophore (GM508 Cult-Active, Gynemed, Germany) and were incubated for 10 minutes, then again oocytes were washed with Global total Fertilization Medium (Global pharm, Life Global, Brussels, Belgium, Europe) and were incubated at 37°C in 6% CO2. In group two, the injected oocytes were not submitted to calcium ionophore activation.
   • 18 hours after injection, the whole injected oocytes in both groups were evaluated for fertilization, cleavage and quality at the day of embryo transfer (ET).

Embryo Transfer

• After ET, luteal phase support was conducted (intramuscularly progesterone injection 100 mg daily) for 14 days until pregnancy test.

After data collection, both groups were compared regarding oocyte number, oocyte fertilization rate, number and quality of embryos, implantation rate and clinical pregnancy rate.

Inclusion Criteria:

• (1) Age between 20 and 40 years old. (2) Cases with history of total fertilization failure in previous ICSI cycles (3) Oocytes with normal morphology.

Exclusion Criteria:

• (1) Abnormal oocyte morphology degenerated or immature oocytes. (2) Husbands with spermatogenic arrest or Sertoli/Leydig cells only.