The combination of calcium ionophore A23187 and GM-CSF can safely salvage aged human unfertilized oocytes after ICSI.

PURPOSE: Artificial oocyte activation using calcium ionophores and enhancement of embryonic developmental potential by the granulocyte-macrophage colony-stimulating factor (GM-CSF) have already been reported. In this study, we evaluated the synergistic effect of these two methods on aged human unfertilized oocytes after intracytoplasmic sperm injection (ICSI). Then, we cultured the resulting embryos to the blastocyst stage and screened them for chromosomal abnormalities, to assess the safety of this protocol. METHODS: Aged human oocytes deemed unfertilized after ICSI were activated, either by briefly applying the calcium ionophore A23187 alone (group A) or by briefly applying the ionophore and then supplementing the culture medium with recombinant human GM-CSF (rhGM-CSF) (group B). Next, the development was monitored in a time-lapse incubator system, and ploidy was analyzed by array comparative genomic hybridization (aCGH), after whole embryo biopsy and whole genome amplification. Differences between oocytes and resulting embryos in both groups were evaluated statistically. RESULTS: Oocytes unfertilized after ICSI can be activated with the calcium ionophore A23187 to show two pronuclei and two polar bodies. Addition of rhGM-CSF in the culture medium of A23187-activated oocytes enhances their cleaving and blastulation potential and results in more euploid blastocysts compared to the culture medium alone. CONCLUSIONS: This study shows that activating post-ICSI aged human unfertilized oocytes with a combination of a calcium ionophore and a cytokine can produce good-morphology euploid blastocysts.

Polar body analysis by array comparative genomic hybridization accurately predicts aneuploidies of maternal meiotic origin in cleavage stage embryos of women of advanced maternal age.

STUDY QUESTION: How accurate is array comparative genomic hybridization (array CGH) analysis of the first polar body (PB1) and second polar body (PB2) in predicting aneuploidies of maternal meiotic origin in the cleavage stage embryos of women of advanced maternal age? SUMMARY ANSWER: Almost all of the aneuploidies detected in cleavage stage embryos were associated with copy number changes in the polar bodies (93%) and all but one (98.5%) were predicted to be aneuploid. A minority of copy number changes (17%), mainly in PB1, did not result in the predicted changes in the embryo, but many of these were small copy number changes, which are likely to be artefacts. WHAT IS KNOWN ALREADY: Chromosome aneuploidy is a major cause of pregnancy failure and loss, abnormal pregnancy and live births. Most aneuploidy is of maternal meiotic origin and increases exponentially in the decade preceding the menopause. A pilot study demonstrated a high rate of concordance between the chromosomal status predicted by polar body analysis and the corresponding zygotes in women of advanced maternal age. STUDY DESIGN, SIZE AND DURATION: Polar body biopsy and array CGH analysis of mature oocytes, which fertilized normally, to identify segregation errors in meiosis, followed by the analysis of the corresponding cleavage stage embryos (n = 34), in a consecutive series of stimulated and natural IVF cycles in women of advanced maternal age. MATERIALS, SETTING AND METHODS: Twenty couples requesting aneuploidy screening (mean ± SD of maternal age 39 ± 3 years) had 16 controlled ovarian hyperstimulation and 7 natural IVF cycles. PB1 and PB2 were biopsied from mature oocytes, prior to intracytoplasmic sperm injection (ICSI) and following confirmation of normal fertilization, respectively. Array CGH was used to detect chromosome copy number changes and to predict aneuploidy in the corresponding embryos. Embryos with normal copy number in both polar bodies were transferred but, 34 cleavage stage embryos, most of which were predicted to have one or more aneuploidies of maternal meiotic origin, were analysed in whole after removal of the zona by array CGH, on Day 3 post-ICSI. MAIN RESULTS AND THE ROLE OF CHANCE: Thirty cleavage stage embryos, predicted to have one or more aneuploidies, were all confirmed to be aneuploid (100% concordant). Seventy four aneuploidies were detected in these embryos. Sixty-nine (93%) aneuploidies were associated with copy number changes in the polar bodies and 68 (98.5%) of these had been predicted to be aneuploid. Also, 19 of 20 (95%) balanced combinations of chromatid gain/loss in PB1/PB2 accurately predicted normal copy number in the corresponding embryos. However, 17 (12%) copy number changes in the polar bodies did not result in the expected outcome, including 12 false positive predictions of aneuploidy. Most of these involved copy number changes that were smaller than would be expected for whole chromosome or chromatid imbalance and occurred significantly more often in PB1 than PB2 (P < 0.0005). Three other embryos with only small copy number changes and one embryo with a partial chromosome loss in PB2, were all confirmed to be euploid. LIMITATIONS, REASONS FOR CAUTION: Accurate false positive and negative rates will require follow-up of both euploid and aneuploid embryos, ideally using molecular genetic markers to detect aneuploidy independently and to identify their origin. WIDER IMPLICATIONS OF THE FINDINGS: Polar body biopsy and array CGH analysis is efficient and accurately predicts most aneuploidies in cleavage stage embryos. However, the size of the ratio shifts, particularly in PB1, should always be compared with the X chromosome shift before it can be concluded that there is a real copy number change. STUDY FUNDING/COMPETING INTEREST(S): Study funded by Embryogenesis, Athens. P.S. and A.H.H. are employed full time and part time, respectively, by BlueGnome Ltd, Cambridge, UK.