S Alfarawati

The relationship between blastocyst morphology, chromosomal abnormality, and embryo gender.

OBJECTIVE To assess correlation between blastocyst morphology and chromosomal status. DESIGN Observational research study. SETTING An IVF clinic and a specialist preimplanation genetic diagnosis (PGD) laboratory. PATIENT(S) Ninety-three couples undergoing IVF treatment in combination with chromosome screening of embryos. INTERVENTION(S) Five hundred blastocysts underwent trophectoderm biopsy and comprehensive chromosome screening using comparative genomic hybridization (CGH). The morphology of the embryos was evaluated using standard methods. MAIN OUTCOME MEASURE(S) Association of aneuploidy and morphologic score. RESULT(S) A total of 56.7% of blastocysts were aneuploid. One-half of the grade 5/6 blastocysts were euploid, compared with only 37.5% of embryos graded 1/2, suggesting an effect of aneuploidy on blastocyst development. Aneuploidy also had a negative effect on inner cell mass and trophectoderm grades. Morphologically poor blastocysts had a higher incidence of monosomy and abnormalities affecting several chromosomes. The gender ratio was significantly skewed in relation to morphology. A total of 72% of blastocysts attaining the highest morphologic scores (5AA and 6AA) were found to be male, compared with only 40% of grade 3 embryos. CONCLUSION(S) Morphology and aneuploidy are linked at the blastocyst stage. However, the association is weak, and consequently, morphologic analysis cannot be relied on to ensure transfer of chromosomally normal embryos. A significant proportion of aneuploid embryos are capable of achieving the highest morphologic scores, and some euploid embryos are of poor morphology. Gender was associated with blastocyst grading, male embryos developing at a significantly faster rate than females.

Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH

One of the most important factors influencing embryo viability is chromosome imbalance (aneuploidy). Embryos derived from aneuploid gametes have little potential for forming a viable pregnancy, but cannot be distinguished from normal embryos using standard morphological evaluation. For more than a decade, preimplantation genetic screening (PGS) has been used to assist in the identification of aneuploid embryos. However, current strategies, based upon cell biopsy followed by fluorescent in situhybridization, allow less than half of the chromosomes to be screened. In this review, we discuss methods that overcome the limitations of earlier PGS strategies and provide screening of the entire chromosome complement in oocytes and embryos. In recent months, there has been a rapid growth in the number of PGS cycles utilizing one such method, comparative genomic hybridization (CGH). Data from IVF cycles utilizing CGH must be considered preliminary, but appear to indicate a dramatic increase in embryo implantation following comprehensive chromosomal screening. It is expected that methods based upon microarrays will yield similar clinical results and may be sufficiently rapid to permit comprehensive screening without the need for embryo cryopreservation. Some microarray platforms also offer the advantage of embryo fingerprinting and the potential for combined aneuploidy and single gene disorder diagnosis. However, more data concerning accuracy and further reductions in the price of tests will be necessary before microarrays can be widely applied.

First births after preimplantation genetic diagnosis of structural chromosome abnormalities using comparative genomic hybridization and microarray analysis

BACKGROUND Balanced chromosomal rearrangements represent one of the most frequent indications for preimplantation genetic diagnosis (PGD). Although fluorescence in situ hybridization (FISH) has been successfully employed for diagnosis in such cases, this approach usually restricts assessment of the chromosomes involved in the rearrangement. Furthermore, with FISH-based strategies, it is sometimes necessary to create patient-specific protocols, increasing the waiting time and costs. In the current study, we explored the use of two comprehensive chromosome screening methods, conventional metaphase comparative genomic hybridization (CGH) and microarray-CGH (aCGH), as alternatives for PGD of chromosome rearrangements. METHODS The study included 16 patients who underwent 20 cycles of PGD for a variety of chromosome rearrangements (reciprocal or Robertsonian translocations or inversions). Testing was performed at various embryonic stages using CGH (9 cases) or aCGH (11 cases). RESULTS Results were obtained for 121 out of 132 samples (91.7%). Of the diagnosed samples, 48.8% were found to carry abnormalities associated with the rearrangement, either alone or in combination with other chromosomal abnormalities. A further 28.9% of samples were normal/balanced for the rearranged chromosomes, but affected by aneuploidy for other chromosomes. Only 22.3% of samples were chromosomally normal. Of the 15 patients who completed their treatment cycles, 5 became pregnant after one or two cycles resulting in four healthy births. The delivery rate per cycle was 21% (27% per embryo transfer). CONCLUSIONS This is the first study to describe the clinical application of comprehensive chromosome screening applied to polar bodies, blastomeres or trophectoderm cells from patients carrying inversions and translocations. Using these techniques, most patients requesting PGD for a chromosome rearrangement can be treated using a single protocol. Additionally, the detection of abnormalities affecting chromosomes unrelated to the rearrangement may assist in the selection of viable embryos for transfer.

Morphological and cytogenetic assessment of cleavage and blastocyst stage embryos

Morphological assessments are the main way in which fertility clinics select in vitro generated embryo(s) for transfer to the uterus. However, it is widely acknowledged that the microscopic appearance of an embryo is only weakly correlated with its viability. Furthermore, the extent to which morphology is affected by aneuploidy, a genetic defect common in human preimplantation embryos, remains unclear. Aneuploidy is of great relevance to embryo selection as it represents one of the most important causes of implantation failure and miscarriage. The current study aimed to examine whether morphological appearance can assist in identifying embryos at risk of aneuploidy. Additionally, the data produced sheds light on how chromosomal anomalies impact development from the cleavage to the blastocyst stage. A total of 1213 embryos were examined. Comprehensive chromosome analysis was combined with well-established criteria for the assessment of embryo morphology. At the cleavage stage, chromosome abnormalities were common even amongst embryos assigned the best morphological scores, indicating that aneuploidy has little effect on microscopic appearance at fixed time points up until Day 3 of development. However, at the blastocyst stage aneuploidies were found to be significantly less common among embryos of optimal morphological quality, while such abnormalities were overrepresented amongst embryos considered to be of poor morphology. Despite the link between aneuploidy and blastocyst appearance, many chromosomally abnormal embryos were able to achieve the highest morphological scores. In particular, blastocysts affected by forms of aneuploidy with the greatest capacity to produce clinical pregnancies (e.g. trisomy 21) were indistinguishable from euploid embryos. The sex ratio was seen to be equal throughout preimplantation development. Interestingly, however, females were overrepresented amongst the fastest growing cleavage-stage embryos, whereas a sex-related skew in the opposite direction was noted for the most rapidly developing blastocysts. In summary, this study confirms that, at the cleavage stage, chromosome abnormalities have little if any effect on morphological scores assigned using traditional criteria. At the blastocyst stage some forms of aneuploidy begin to affect microscopic appearance, but in most instances the impact is subtle. In the case of the most clinically relevant aneuploidies (those capable of forming a pregnancy) there was no detectable effect on morphology at any preimplantation stage.

The incidence and origin of segmental aneuploidy in human oocytes and preimplantation embryos.

STUDY QUESTION What is the incidence, origin and clinical significance of segmental aneuploidy in human oocytes and preimplantation embryos? SUMMARY ANSWER Segmental aneuploidy occurs at a considerable frequency in preimplantation embryos with a majority being mitotic in origin. WHAT IS KNOWN ALREADY In recent years, accurate techniques for the detection of aneuploidy in single cells have been developed. Research using such methods has confirmed that aneuploidy is a common feature of human oocytes and preimplantation embryos. However, thus far research has mainly focused on loss or gain of whole chromosomes. We utilized sensitive molecular methods to study another important form of cytogenetic abnormality at the earliest stages of human development, namely segmental aneuploidy. STUDY DESIGN, SIZE, DURATION Chromosomal copy number data was obtained from oocytes and embryos of 635 IVF patients, who requested chromosome screening for various reasons, most commonly for advanced maternal age or previously unsuccessful IVF treatments. A total of 3541 samples comprising of 452 human oocytes, 1762 cleavage stage and 1327 blastocyst stage embryos were investigated in the present study. PARTICIPANTS/MATERIALS, SETTING, METHODS Whole genome amplification (Sureplex, Illumina) was performed on cells biopsied from oocytes and embryos of IVF patients who requested chromosome screening. The samples were subsequently processed and analyzed for their chromosome complement using microarray comparative genomic hybridization (aCGH), (Illumina, Cambridge, UK). MAIN RESULTS AND THE ROLE OF CHANCE Segmental abnormalities, involving loss or gain of chromosomal fragments in excess of 15 Mb, were found to occur at a high frequency. The incidence of such abnormalities was 10.4% in oocytes, but this increased dramatically during the first 3 days of embryonic development (24.3%), before starting to decline as embryos reached the final (blastocyst) stage of preimplantation development (15.6%). While some segmental errors were clearly of meiotic origin, most appear to arise during the first few mitoses following fertilization. The reduction in frequency at the blastocyst stage suggests that many cells/embryos affected by segmental abnormalities are eliminated (e.g. via arrest of the affected embryos or apoptosis of abnormal cells). Interestingly, sites of chromosome breakage associated with segmental aneuploidy were not entirely random but tended to occur within distinct chromosomal regions. Some of the identified hotspots correspond to known fragile sites while others may be considered novel and may be specific to gametogenesis and/or embryogenesis. LIMITATIONS REASONS FOR CAUTION The cytogenetic analysis was performed on biopsies of embryos, which might not be representative of the true incidence of mosaic segmental aneuploidy of the entire embryo. WIDER IMPLICATIONS OF THE FINDINGS The findings of this study are valuable for understanding the origin of subchromosomal duplications and deletions, a clinically important class of abnormalities that are a common cause of congenital abnormalities and miscarriage. Furthermore, the results provide additional evidence that control of the cell cycle is more relaxed during the first few mitotic divisions following fertilization, permitting DNA double-strand breaks to occur and persist through cell division. The data are also of great relevance for preimplantation genetic testing, where the detection of segmental aneuploidy is currently considered problematic for embryo diagnosis and patient counseling. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by institutional funding (Reprogenetics UK). Additionally, DW is supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. DB was supported by the University of Oxfords Clarendon funding. No conflict of interests to declare.

A skewed sex ratio following blastocyst culture is a consequence of embryo grading systems that prioritise male embryos for transfer

Sir, The recent study by Dean et al. has provided the strongest evidence yet that assisted reproductive technology procedures affect the sex ratio at birth (secondary sex ratio). Several aspects of treatment were shown to affect the proportion of males and females, the most prominent being the use of extended embryo culture, with transfer at the blastocyst stage. This was associated with a significant increase in the frequency of male births (54.1% of babies born were male). We suggest that the skewed sex ratio is a consequence of the methods used for choosing which blastocyst will be given priority for transfer to the uterus. In a recent study of 500 blastocysts, we demonstrated an equal frequency of male and female embryos, suggesting that the primary sex ratio is 1:1. However, male embryos achieved significantly better morphological scores than equivalent female embryos and were therefore more likely to be selected for transfer. Blastocyst morphology was graded with the most widely used system, devised by Gardner and Schoolcraft. This strategy involves assignment of a number representing the degree of development (one for least advanced, early blastocyst; six for most advanced, hatched blastocyst) and two letters, one denoting the quality of the inner cell mass and the second signifying the quality of the trophectoderm (C being lowest quality; A being highest quality). Of the most advanced blastocysts (grades 5 or 6), 72% were found to be male versus only 28% female, whereas only 40% of the slowest growing embryos (grades 1–3) were male, compared with 60% female. The sex-related difference was highly significant (Fisher’s exact test P = 0.001), male embryos being 2.6 times more likely to produce a grade 5 or 6 blastocyst. The disparity in embryo score between male and female embryos is unlikely to reflect any difference in competence, but conclusively proves that male embryos reach the final stages of preimplantation development at a faster rate than female embryos. Similar observations have been reported in other species. The underlying biological cause remains unclear, but may be related to differences in gene dosage compensation associated with X chromosome inactivation during preimplantation development, or to the presence of growth factor genes on the Y chromosome. Given the substantial skew in the sex ratio, we suggest that women having blastocyst transfer should be counselled that embryo selection based upon traditional blastocyst scoring systems is likely to result in a reduced probability of achieving a pregnancy with a female fetus. Indeed, where only 5AA and/or 6AA embryos are transferred, over 70% of children born are expected to be male. Conversely, the transfer of only 3BB/3BC/3CB embryos is expected to shift the sex ratio in the opposite direction, resulting in ~66% female births. Morphological differences between male and female embryos could represent an inexpensive way for parents undergoing in vitro fertilisation to increase the chances of obtaining a child of a particular sex. This may be an attractive possibility in countries where sex selection is permitted (e.g. the USA), but could present ethical and legal problems in parts of the world where selection is not allowed (e.g. the UK and much of Europe). In addition, there is a risk that parents attempting to select for females by preferentially transferring grade-3 embryos, might experience a reduced pregnancy rate. This is because a third of the 3BB/ 3BC/3CB embryos are actually slow-growing males, which probably have reduced developmental competence. j