Katerina Vesela

ESHRE PGD Consortium data collection VI: cycles from January to December 2003 with pregnancy follow-up to October 2004

The sixth report of the ESHRE PGD Consortium is presented, relating to cycles collected for the calendar year 2003 and follow-up of the pregnancies and babies born up to October 2004. Since the beginning of the data collections, there has been a steady rise in the number of cycles, pregnancies and babies reported. For this report, 50 centres participated, reporting on 2984 cycles, 501 pregnancies and 373 babies born. Five hundred and twenty-nine cycles were reported for chromosomal abnormalities, 516 cycles were reported for monogenic diseases, 137 cycles were reported for sexing for X-linked diseases, 1722 cycles were reported for preimplantation genetic screening (PGS) and 80 cycles were reported for social sexing. Data VI is compared to the cumulative data for data collections I-V.

Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results

BACKGROUND Several randomized controlled trials have not shown a benefit from preimplantation genetic screening (PGS) biopsy of cleavage-stage embryos and assessment of up to 10 chromosomes for aneuploidy. Therefore, a proof-of-principle study was planned to determine the reliability of alternative form of PGS, i.e. PGS by polar body (PB) biopsy, with whole genome amplification and microarray-based comparative genomic hybridization (array CGH) analysis. METHODS In two centres, all mature metaphase II oocytes from patients who consented to the study were fertilized by ICSI. The first and second PBs (PB1and PB2) were biopsied and analysed separately for chromosome copy number by array CGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by array CGH for concordance analysis. RESULTS Both PBs were biopsied from a total of 226 zygotes from 42 cycles (average 5.5 per cycle; range 1–15) in 41 couples with an average maternal age of 40.0 years. Of these, the ploidy status of the zygote could be predicted in 195 (86%): 55 were euploid (28%) and 140 were aneuploid (72%). With only one exception, there was at least one predicted aneuploid zygote in each cycle and in 19 out of 42 cycles (45%), all zygotes were predicted to be aneuploid. Fresh embryos were transferred in the remaining 23 cycles (55%), and one frozen transfer was done. Eight patients had a clinical pregnancy of which seven were evolutive (ongoing pregnancy rates: 17% per cycle and 30% per transfer). The ploidy status of 156 zygotes was successfully analysed by array CGH: 38 (24%) were euploid and 118 (76%) were aneuploid. In 138 cases complete information was available on both PBs and the corresponding zygotes. In 130 (94%), the ploidy status of the zygote was concordant with the ploidy status of the PBs and in 8 (6%), the results were discordant. CONCLUSIONS This proof-of-principle study indicates that the ploidy of the zygote can be predicted with acceptable accuracy by array CGH analysis of both PBs.

What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD Consortium steering committee

Since 2004, there have been 11 randomized controlled trials (RCTs) mainly for advanced maternal age (AMA), which have shown no benefit of performing preimplantation genetic screening (PGS). Ten of the RCTs have been performed at the cleavage stage and one at the blastocyst stage. It is probable that the high levels of chromosomal mosaicism at cleavage stages, which may result in the tested cell not being representative of the embryo, and the inability to examine all of the chromosomes using fluorescence in situ hybridization, have contributed to the lack of positive outcome from the RCTs. We suggest that future RCTs should examine alternative biopsy timing (polar body and/or trophectoderm biopsy), and should apply technologies that allow more comprehensive testing to include all chromosomes (microarray-based testing) to determine if PGS shows an improvement in delivery rate. Currently there is no evidence that routine PGS is beneficial for patients with AMA and conclusive data (RCTs) on repeated miscarriage, implantation failure and severe male factor are missing. To evaluate benefits of PGS, an ESHRE trial has recently been started on patients with AMA using polar body biopsy and array-comparative genomic hybridization, which should bring more information on this patient group in the near future.

Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation.

Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age in the decade preceding the menopause. Molecular genetic analysis following natural conception and spontaneous miscarriage demonstrates that trisomies arise mainly in female meiosis and particularly in the first meiotic division. Here, we studied copy number gains and losses for all chromosomes in the two by-products of female meiosis, the first and second polar bodies, and the corresponding zygotes in women of advanced maternal age undergoing IVF, using microarray comparative genomic hybridisation (array CGH). Analysis of the segregation patterns underlying the copy number changes reveals that premature predivision of chromatids rather than non-disjunction of whole chromosomes causes almost all errors in the first meiotic division and unlike natural conception, over half of aneuploidies result from errors in the second meiotic division. Furthermore, most abnormal zygotes had multiple aneuploidies. These differences in the aetiology of aneuploidy in IVF compared with natural conception may indicate a role for ovarian stimulation in perturbing meiosis in ageing oocytes.

What next for preimplantation genetic screening

Preimplantation genetic diagnosis for aneuploidy screening (preimplantation genetic screening-PGS) has been used to detect chromosomally normal embryos from subfertile patients. The main indications are advanced maternal age (AMA), repeated implantation failure, repeated miscarriages and severe male factor infertility. Many non-randomized PGS studies have been published and report an increase in implantation rate, and/or a decrease in miscarriage rate. Recently, two randomized controlled trials have been conducted on patients with AMA as the only indication. Neither study showed a benefit in performing PGS using live birth rate as the measure of success. The debate on the usefulness of PGS is ongoing; the only effective way to resolve the debate is to perform more well-designed and well-executed randomized clinical trials.

ESHRE PGD consortium best practice guidelines for fluorescence in situ hybridization-based PGD

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium published a set of Guidelines for Best Practice PGD to give information, support and guidance to potential, existing and fledgling PGD programmes. The subsequent years have seen the introduction of new technologies as well as evolution of current techniques. Additionally, in light of recent advice from ESHRE on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD, the new guidelines are separated into four new documents that apply to different aspects of a PGD programme, i.e. organization of a PGD centre, fluorescence in situ hybridization (FISH)-based testing, amplification-based testing and polar body and embryo biopsy for PGD/preimplantation genetic screening (PGS). Here, we have updated the sections that pertain to FISH-based PGD. PGS has become a highly controversial technique. Opinions of laboratory specialists and clinicians interested in PGD and PGS have been taken into account here. Whereas some believe that PGS does not have a place in clinical medicine, others disagree; therefore, PGS has been included. This document should assist everyone interested in PGD/PGS in developing the best laboratory and clinical practice possible. Topics covered in this guideline include inclusion/exclusion criteria for FISH-based PGD testing, referrals and genetic counselling, preclinical validation of tests, FISH-based testing methods, spreading of cells for analysis, set-up of local IVF centre and transport PGD centres, quality control/ quality assurance and diagnostic confirmation of untransferred embryos.

Sperm and embryo analysis in a carrier of supernumerary inv dup(15) marker chromosome.

We identified a small, paternally inherited, supernumerary marker chromosome, inv dup(15), in a phenotypically normal and normozoospermic male from a couple with reproductive problems. Sperm analysis by fluorescence in situ hybridization (FISH) showed that the marker was present in 26% of sperm nuclei. The disomy 15 was 10 times higher than in normal control donors. FISH analysis for aneuploidies of the other chromosomes showed an increase in nondisjunction of chromosome 21. We also examined 24 embryos by preimplantation genetic diagnosis, and 10 embryos (41.7%) contained the marker. This report provides information about inheritance of inv dup(15) from a male carrier.

Comprehensive meiotic segregation analysis of a 4-breakpoint t(1;3;6) complex chromosome rearrangement using single sperm array comparative genomic hybridization and FISH

Complex chromosomal rearrangements (CCR) represent rare structural chromosome abnormalities frequently associated with infertility. In this study, meiotic segregation in spermatozoa of an infertile normospermic carrier of a 4-breakpoint t(1;3;6) CCR was analysed. A newly developed array comparative genomic hybridization protocol was used, and all chromosomes in 50 single sperm cells were simultaneously examined. Three-colour FISH was used to analyse chromosome segregation in 1557 other single sperm cells. It was also used to measure an interchromosomal effect; sperm chromatin structure assay was used to measure chromatin integrity. A high-frequency of unbalanced spermatozoa (84%) was observed, mostly arising from the 3:3 symmetrical segregation mode. Array comparative genomic hybridization was used to detect additional aneuploidies in two out of 50 spermatozoa (4%) in chromosomes not involved in the complex chromosome rearrangement. Significantly increased rates of diploidy and XY disomy were found in the CCR carrier compared with the control group (P < 0.001). Defective condensation of sperm chromatin was also found in 22.7% of spermatozoa by sperm chromatin structure assay. The results indicate that the infertility in the man with CCR and normal spermatozoa was caused by a production of chromosomally unbalanced, XY disomic and diploid spermatozoa and spermatozoa with defective chromatin condensation.

Hybridization of the 18 alpha–satellite probe to chromosome 1 revealed in PGD

Although the chromosome 18 alpha-satellite probe is considered to have a very low polymorphism rate, the routine use of this probe in prenatal diagnosis revealed rare variants in size and copy number of these sequences. A polymorphic signal was detected in preimplantation genetic diagnosis (PGD) for aneuploidy, in a patient with repeated early miscarriages. A third small signal of chromosome 18 alpha-satellite probe was observed in two of four evaluated embryos. Hybridization to the womans metaphasic lymphocytes revealed that the small signal was localized in the pericentromeric region of chromosome 1. Reanalysis of blastomeres with telomeric probes for chromosome 18q confirmed the presence of only two copies of chromosome 18. Options for verifying PGD analysis results, to prevent misdiagnosis in cases of suspected polymorphism, are discussed. Although some authors speculate about a possible role of heterochromatin polymorphism in infertility, this rare polymorphism of 18 alpha-satellite sequences is in itself probably a normal variant. This is the third report of a cross-hybridization of the chromosome 18 alpha-satellite probe and the first report of the localization of the polymorphic 18 alpha-satellite signal to chromosome 1.