The combination of intracytoplasmic sperm injection (ICSI) with artificial oocyte activation has overcome repeated fertilization failure after ICSI due to sperm defects in activating oocyte, resulting in pregnancies and births for many couples.1-3 However, in comparison to the growing data illustrating the effectiveness of artificial oocyte activation in reproductive medicine, the safety of this approach has not been well studied. Previously, we demonstrated that the activated embryos derived from unfertilized oocytes after ICSI could develop to blastocyst stage, similar to normal embryos, as evidenced by dual color fluorescence in situ hybridization (FISH) on sex chromosomes.3 However, before artificial oocyte activation is accepted as a clinical strategy, further safety studies of the offspring need to be carefully evaluated.
Array comparative genomic hybridization (aCGH) analysis has emerged as a primary diagnostic tool for prenatal diagnosis and the evaluation of developmental delay and structural malformations in children because it is more accurate and efficient than conventional cytogenetic analysis and FISH.4,5 Although aCGH analysis possesses obvious advantages for prenatal and genetic diagnoses, it also has certain shortcomings with regard to the detection on triploidies and balanced rearrangements. Therefore, the present study was designed to employ a conventional cytogenetic analysis in combination with aCGH on three infants compared to their parents to explore any possible occurrence of genetic alterations associated with artificial oocyte activation. To the best of our knowledge, this is the first study to completely evaluate whether the artificial oocyte activation technique may result in adverse impacts on a human offspring.
After obtaining approval of our institutional review from the ethics committee of the People's Hospital, Peking University, the blood of three infants derived from artificial oocyte activation and their parents were obtained following signing informed consents. A total of nine samples were recruited in this study.
All three couples had experienced repeated total or near total fertilization failure after ICSI because of severe oligoasthenozoospermia. The semen analysis, fertilization and embryo development in the three families are shown in Table 1. The baby in family one was derived from an activated, one-day-old unfertilized oocyte.2 For the other two couples, oocyte activation via 5 μmol/L calcium ionophore A23187 (A23187) was performed on thirty minutes after ICSI in the next cycle, according to our previously study method.3 Umbilical cord blood of the infants and the parents' blood were collected. Each sample was split into two, with one aliquot used for a standard karyotyping analysis and the other for DNA extraction.
The cytogenetic analysis was performed using the GTGbanding technique on metaphase chromosomes obtained by standard procedures from peripheral blood lymphocytes. Briefly, the blood was inoculated in 5 ml of 1640 culture medium (Yishishengda Ltd., Beijing, China) and cultured for 72 hours at 37°C. The cells were then harvested, and chromosomal samples were prepared according to the ISCN (1985) standard for karyotype analysis.
An aCGH analysis was utilized to examine all of the interrogated samples. Total DNA was extracted from the peripheral blood using Genomic DNA Extraction Kit (BioChain Institute, Inc., CA, USA) according to the manufacturer's protocol. For each aCGH experiment, 400 ng each of purified DNA and normal sex-matched DNA (BioChain Institute) was digested with 10 U AluI and 10 U RsaI (Promega, USA) and differentially labeled with cyanine-5 (cy5) and cyanine-3 (Cy3) fluorescent dyes using Agilent's Genomic DNA Enzymatic Labeling Kit. The aCGH analysis was performed using Agilent's 8×60 K commercial arrays; this platform contains 60-mer oligonucleotide probes spanning the entire human genome, with an overall mean probe spacing of 50 kb. After hybridization, the arrays were scanned using Agilent's duallaser scanner, and the images were extracted and analyzed with Agilent Feature Extraction software and Workbench genomics software, respectively.
The three infants derived from ICSI and oocyte activation with A23187 revealed no physical and mental developmental disorders during follow-up for at least one year. They all showed a normal karyotype, which included 46, XX for the two girls and 46, XY for the boy.
Nine aCGH analyses were performed for the three families; Table 2 shows an overview of the results. Similar to the cytogenetic analysis, all of the karyotype analyses showed normal chromosome numbers. However, small genetic deviations could be detected. Chromosomes 10 and 15 were frequently subjected to deletions or duplication. For the first family, even though the father and mother had small genetic deviation, the daughter exhibited no duplication or deletion. In the second family, the son's deviation was also detected in his mother. The variation in the daughter of the last family was not completely the same as her parents, and the genetic abnormalities were found to be polymorphisms.4
Repeatedly total or near-total fertilization failure after ICSI is a rare event yet is one of the most distressful experiences for an infertile couple and the fertility clinic because of the significant emotional and financial involvement in assisted reproductive techniques. The combination of ICSI with artificial oocyte activation has been gradually implemented for couples with a previous history of total or near-total fertilization failure after ICSI because such artificial interventions can overcome sperm defects in activating oocytes. At present, calcium ionophore A23187, the most common chemical compounds used for inducing calcium increase and initiating oocyte activation, has resulted in pregnancies and live births.1-3 Although studies have not shown the cytotoxic activity of such chemical compounds on gametes and embryos, the potential risks, such as teratogenetic and mutagenetic activity of A23187, still need to be explored before the extensive clinical application of this technique. Therefore, it is urgently necessary to conduct a series of studies concerning the safety of this technique. Using a cytogenetic examination and aCGH, we here first demonstrate that artificial oocyte activation with a chemical stimulus, e.g., calcium ionophore A23187, did not increase chromosomal alterations in the offspring.
We also reported that the three infants derived from ICSI and oocyte activation with A23187 showed normal physical and mental development after at least one year of observation. The cytogenetic analysis and aCGH showed a normal chromosome number and no appearance of smaller genetic deviations in the three infants compared to their parents. As aCGH can detect an increased incidence of genomic abnormalities at a high resolution, it has been the first-tier test for prenatal diagnosis and in children with unexplained developmental delay or intellectual disability.4 Furthermore, the shortcoming of aCGH in identifying structural chromosome aberrations, such as balanced reciprocal translocations or inversions, was overcome when combined with a cytogenetic analysis. Thus, we believe that cytogenetic analysis in combination with aCGH definitely decreased the probability of missing mutations in the infants. All of these findings indicated that artificial oocyte activation with A23187 might not introduce any genetic abnormalities. Therefore, A23187 at an optimum concentration did not exert teratogenetic and mutagenetic activity on gametes and embryos. This finding will be important and reassuring for the further clinical application of ICSI combined with artificial oocyte activation in women who have experienced total or nearly total fertilization failure after ICSI.
It is well known that human gametes and the earliest developmental stages are critically sensitive to external perturbations. Indeed, any external intervention during these stages may cause a variety of alterations of early gene expression and methylation status that may lead to long-term alterations, such as the arrest of embryonic development and dysplasia. Therefore, further studies on the methylation status are required, and the safety of different activation stimuli should also be evaluated.
Acknowledgements: The authors would like to thank Zhang Liang, PhD, from BioChainScience & Technology Inc. (Beijing, China), for providing scientific advice.
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