ESHRE PGT Consortium good practice recommendations for the detection of structural and numerical chromosomal aberrations.

The field of preimplantation genetic testing (PGT) is evolving fast, and best practice advice is essential for regulation and standardisation of diagnostic testing. The previous ESHRE guidelines on best practice for PGD, published in 2005 and 2011, are considered outdated, and the development of new papers outlining recommendations for good practice in PGT was necessary. The current paper provides recommendations on the technical aspects of PGT for chromosomal structural rearrangements (PGT-SR) and PGT for aneuploidies (PGT-A) and covers recommendations on array-based comparative genomic hybridisation (aCGH) and next-generation sequencing (NGS) for PGT-SR and PGT-A and on fluorescence in situ hybridisation (FISH) and single nucleotide polymorphism (SNP) array for PGT-SR, including laboratory issues, work practice controls, pre-examination validation, preclinical work-up, risk assessment and limitations. Furthermore, some general recommendations on PGT-SR/PGT-A are formulated around training and general risk assessment, and the examination and post-examination process. This paper is one of a series of four papers on good practice recommendations on PGT. The other papers cover the organisation of a PGT centre, embryo biopsy and tubing and the technical aspects of PGT for monogenic/single-gene defects (PGT-M). Together, these papers should assist everyone interested in PGT in developing the best laboratory and clinical practice possible.

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.

Nuclear organisation of sperm remains remarkably unaffected in the presence of defective spermatogenesis.

Organisation of chromosome territories in interphase nuclei has been studied in many systems and positional alterations have been associated with disease phenotypes (e.g. laminopathies, cancer) in somatic cells. Altered nuclear organisation is also reported in developmental processes such as mammalian spermatogenesis where a "chromocentre" model is proposed with the centromeres and sex chromosomes repositioning to the nuclear centre. The purpose of this study was to test the hypothesis that alterations in nuclear organisation of human spermatozoa are associated with defects upstream in spermatogenesis (as manifest in certain infertility phenotypes). The nuclear address of (peri-) centromeric loci for 18 chromosomes (1-4, 6-12, 15-18, 20, X and Y) was assayed in 20 males using established algorithms for 3D extrapolations of 2D data. The control group comprised 10 fertile sperm donors while the test group was 10 patients with severely compromised semen parameters including high sperm aneuploidy. All loci examined in the control group adopted defined, interior positions thus providing supporting evidence for the presence of a chromocentre and interior sex chromosome territories. In the test group however there were subtle alterations in the nuclear address for certain centromeres in individual patients and, when all patient results were pooled, some different nuclear addresses were observed for chromosomes 3, 6, 12 and 18. Considering the extensive impairment of spermatogenesis in the test group (evidenced by compromised semen parameters and increased chromosome abnormalities), the observed differences in nuclear organisation for centromeric loci compared to the controls were modest. A defined pattern of nuclear reorganisation of centromeric loci in sperm heads therefore appears to be a remarkably robust process, even if spermatogenesis is severely compromised.

The association between male infertility and sperm disomy: evidence for variation in disomy levels among individuals and a correlation between particular semen parameters and disomy of specific chromosome pairs.

BACKGROUND: The association between infertility and sperm disomy is well documented. Results vary but most report that men with severely compromised semen parameters have a significantly elevated proportion of disomic sperm. The relationship between individual semen parameters and segregation of specific chromosome pairs is however less well reported as is the variation of disomy levels in individual men. METHODS: In order to address these questions the technique of fluorescent in-situ hybridisation (FISH) was utilised to determine the disomy levels of chromosomes X, Y and 21 in 43 sperm samples from 19 infertile males. The results generated from this study were analysed using logistic regression. RESULTS: In this study we compared levels of sperm concentration, motility and morphology with levels of sperm disomy for chromosome 21 and the sex chromosomes. Our results suggest that there is considerable variation in disomy levels for certain men. They also suggest that oligozoospermic males have significantly elevated levels of sex chromosome disomy but not disomy 21; they suggest that severe asthenozoospermic males have significantly elevated levels of disomy 21 but not sex chromosome disomy. Surprisingly, severe teratozoopsermic males appeared to have significantly lower levels of sperm disomy for both the sex chromosomes and chromosome 21. CONCLUSION: We suggest that the association between sex chromosome disomy and oligozoospermia may be due to reduced recombination in the XY pairing region and discuss the relevance of our findings for the correlations between sperm disomy and sperm motility and morphology.