Live birth after PGD with confirmation by a comprehensive approach (karyomapping) for simultaneous detection of monogenic and chromosomal disorders.

Preimplantation genetic diagnosis (PGD) for monogenic disorders has the drawback of time and cost associated with tailoring a specific test for each couple, disorder, or both. The inability of any single assay to detect the monogenic disorder in question and simultaneously the chromosomal complement of the embryo also limits its application as separate tests may need to be carried out on the amplified material. The first clinical use of a novel approach ('karyomapping') was designed to circumvent this problem. In this example, karyomapping was used to confirm the results of an existing PGD case detecting both chromosomal abnormalities and a monogenic disorder (Smith-Lemli-Opitz [SLO] syndrome) simultaneously. The family underwent IVF, ICSI and PGD, and both polar body and cleavage stage biopsy were carried out. Following whole genome amplification, array comparative genomic hybridisation of the polar bodies and minisequencing and STR analysis of single blastomeres were used to diagnose maternal aneuploidies and SLO status, respectively. This was confirmed, by karyomapping. Unlike standard PGD, karyomapping required no a-priori test development. A singleton pregnancy and live birth, unaffected with SLO syndrome and with no chromosome abnormality, ensued. Karyomapping is potentially capable of detecting a wide spectrum of monogenic and chromosome disorders and, in this context, can be considered a comprehensive approach to PGD.

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.

An algorithm for determining the origin of trisomy and the positions of chiasmata from SNP genotype data.

Trisomy causes mental retardation, pregnancy loss, IVF failure, uniparental disomy and several other pathologies, and its accurate detection is thus clinically essential. Most trisomies arise at meiosis I and are associated with increasing maternal age and reduction or alteration in recombination patterns. Investigations into the relationship between trisomy and meiotic recombination have used short tandem repeat markers; however, this approach is limited by the resolution with which the position of crossovers can identified. As cytogenetics enters the post-genomic era, recent work has used array comparative genomic hybridisation (aCGH) to screen for trisomy of all 24 chromosomes, determining chromosome copy number by dosage analysis. However, aCGH has a fundamental drawback for studying the aetiology of trisomy since neither the parent and phase of origin nor uniparental disomy can be ascertained. The development of SNP microarrays has made it possible to analyse multiple loci for sequence variation, and the proprietary software provided can determine the presence of aneuploidy by algorithms based on fluorescence intensity. To the best of our knowledge, however, such software is not equipped to determine the phase of origin of the error or the position of any chiasmata. In this study, therefore, we present an algorithm to determine the parent of origin, the phase of origin and the location of chiasmata in a series of nine "trisomy triplets" (i.e. samples derived from father, mother and their trisomic foetus). Novel adaptations of well-established principles are applied along with a simple algorithm written in Microsoft Excel for visualisation of the results. Such analysis has a range of applications in preimplantation and prenatal diagnosis.

Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes.

The use of genome wide single nucleotide polymorphism (SNP) arrays for high resolution molecular cytogenetic analysis using a combination of quantitative and genotype analysis is well established. This study demonstrates that by Mendelian analysis of the SNP genotypes of the parents and a sibling or other appropriate family member to establish phase, it is possible to identify informative loci for each of the four parental haplotypes across each chromosome and map the inheritance of these haplotypes and the position of any crossovers in the proband. The resulting 'karyomap', unlike a karyotype, identifies the parental and grandparental origin of each chromosome and chromosome segment and is unique for every individual being defined by the independent segregation of parental chromosomes and the pattern of non-recombinant and recombinant chromosomes. Karyomapping, therefore, enables both genome wide linkage based analysis of inheritance and detection of chromosome imbalance where either both haplotypes from one parent are present (trisomy) or neither are present (monosomy/deletion). The study also demonstrates that karyomapping is possible at the single cell level following whole genome amplification and, without any prior patient or disease specific test development, provides a universal linkage based methodology for preimplantation genetic diagnosis readily available worldwide.

Quantum dots as new-generation fluorochromes for FISH: an appraisal.

In the field of nanotechnology, quantum dots (QDs) are a novel class of inorganic fluorochromes composed of nanometre-scale crystals made of a semiconductor material. Given the remarkable optical properties that they possess, they have been proposed as an ideal material for use in fluorescent in-situ hybridization (FISH). That is, they are resistant to photobleaching and they excite at a wide range of wavelengths but emit light in a very narrow band that can be controlled by particle size and thus have the potential for multiplexing experiments. The principal aim of this study was to compare the potential of QDs against traditional organic fluorochromes in both indirect (i.e. QD-conjugated streptavidin) and direct (i.e. synthesis of QD-labelled FISH probes) detection methods. In general, the indirect experiments met with a degree of success, with FISH applications demonstrated for chromosome painting, BAC mapping and use of oligonucleotide probes on human and avian chromosomes/nuclei. Many of the reported properties of QDs (e.g. brightness, 'blinking' and resistance to photobleaching) were observed. On the other hand, signals were more frequently observed where the chromatin was less condensed (e.g. around the periphery of the chromosome or in the interphase nucleus) and significant bleed-through to other filters was apparent (despite the reported narrow emission spectra). Most importantly, experimental success was intermittent (sometimes even in identical, parallel experiments) making attempts to improve reliability difficult. Experimentation with direct labelling showed evidence of the generation of QD-DNA constructs but no successful FISH experiments. We conclude that QDs are not, in their current form, suitable materials for FISH because of the lack of reproducibility of the experiments; we speculate why this might be the case and look forward to the possibility of nanotechnology forming the basis of future molecular cytogenetic applications.

Molecular comparison of single cell MDA products derived from different cell types.

The quality of DNA obtained from single cells for molecular analysis is primarily dependent on cell type and cell lysis. Multiple displacement amplification (MDA) amplifies the DNA isothermally with the use of Phi29 polymerase and random hexamer primers. The efficiency and accuracy of MDA was assessed on different cell types (buccal cells, lymphocytes, fibroblasts) using two multiplex PCR reactions that have been applied in clinical preimplantation genetic diagnosis cases (DM triplex-DM1, APOC2, Dl9S112 and CF triplex-DF508del, IVS8CA, IVS17TA). These results were compared using the DM triplex with MDA products from single blastomeres. Cells were lysed using a modified protocol excluding dithiothreitol in the alkaline lysis buffer. The MDA amplification efficiency for buccal cells was 82.0% (41/50) compared with 96.0% (48/50) for lymphocytes and 100% (20/20) for fibroblasts. The average allele dropout (ADO) rates were 31.0% for buccal cells, 20.8% for lymphocytes and 20.0% for fibroblasts with high inter-locus variation across all cell types (5.0-45.5%). Overall, MDA on single lymphocytes and fibroblasts lysed using the modified protocol produced DNA of sufficient quantity and quality for subsequent molecular analysis by PCR and gave results comparable with MDA products from blastomeres, in contrast to buccal cells.

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.

Efficient isothermal amplification of the entire genome from single cells.

Preimplantation genetic diagnosis for single gene disorders is usually performed using polymerase chain reaction (PCR)-based methodologies modified for use in single cells. At present, single cell PCR tests require costly and time-consuming development and validation of highly sensitive amplification strategies to cover a growing number of mutations responsible for genetic disease. Whole-genome amplification (WGA) provides an opportunity to amplify the genome from a single blastomere to a level at which multiple tests can be performed on the same cell. Early WGA methods (primer extension preamplification and degenerate oligonucleotide-primed PCR) have not proved sufficiently accurate and reliable for routine clinical use. However, WGA using multiple displacement amplification (MDA) offers approx 5 million-fold amplification with fidelity, apparently sidestepping the limitation of a single cell, and is sufficient for use in most off-the-shelf molecular tests. This chapter describes an optimized MDA protocol for the preparation of genomic DNA from single fibroblasts.

The effect of cryopreservation on the genome of gametes and embryos: principles of cryobiology and critical appraisal of the evidence.

BACKGROUND: Cryopreservation has been extensively used in assisted reproductive technology, agriculture and conservation programmes for endangered species. The literature reports largely positive results regarding the survival of frozen-thawed cells and clinical outcomes. Nonetheless, it is unclear whether or not cryopreservation of sperm, oocytes and embryos causes any disruption in their genetic integrity. Drawing on the available published evidence, this review paper describes in detail the physical and biochemical factors of cryopreservation that could potentially affect genomic integrity. METHODS: A critical review of the published literature using PubMed with particular emphasis on studies which include assessment of genetic stability after cryopreservation of oocyte, sperm and embryos. The search was performed in 2014 and covered the period from the beginning of electronic records until July 2014. No language restrictions were applied. RESULTS: Cryopreservation is associated with extensive damage to cell membranes, and results in alteration of the functional and metabolic status of the cells and mitochondria. Some evidence suggests an increase in DNA single-strand breaks, and degree of DNA condensation or fragmentation in sperm after cryopreservation. The extent of these changes may vary between different individuals and different techniques. The addition of antioxidants to the cryopreservation media and the use of well-controlled cooling regimes could potentially improve such outcomes. Limited numbers of studies on oocytes provide controversial results regarding the effect on DNA fragmentation, sister chromatid exchange (SCE) and aneuploidy. The only study on human embryos suggested that vitrification affects DNA integrity to a much lesser extent than slow freezing. Animal studies show increases in mitochondrial DNA mutations in embryos after cryopreservation. The limited numbers of long-term follow-up studies in humans provide reassurance that derives mostly from retrospective studies with some methodological weaknesses. CONCLUSIONS: This review provides an overview of studies performed to date on the effect of cryopreservation on the oocyte, sperm and embryos. Controversy of the reported data has highlighted the gaps in our knowledge not only for clinical studies, but also for basic research in human embryos. New perspectives for future research are proposed.

Use of suboptimal sperm increases the risk of aneuploidy of the sex chromosomes in preimplantation blastocyst embryos.

OBJECTIVE: To compare autosomal and sex chromosome aneuploidy rates of embryos derived from sperm with abnormal and normal parameters. DESIGN: Retrospective cohort study. SETTING: Assisted reproduction center. PATIENT(S): Three thousand eight hundred thirty-five embryos generated from 629 couples undergoing IVF. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Incidence of aneuploidy in the trophectoderm of blastocyst embryos derived from standard IVF embryos and intracytoplasmic (ICSI) males with normal and oligozoospermic semen samples, in couples with donor eggs (mean maternal age, 25.0 years) and their own eggs (mean maternal age, 35.4 years). RESULT(S): The rate of sex chromosome aneuploidy was significantly (around threefold) higher in the oligozoospermic group compared with in both control groups (standard vs. ICSI insemination). This applied whether donor (young) or own (older) eggs were used. Significant differences were seen in the oligozoospermic samples for autosomes 1, 2, 11 (own eggs), and 18 (donor eggs) compared with both control groups; however, no significant difference was seen between each of the treatment groups for the overall rate of autosomal aneuploidy. No significant differences were seen between the two control groups (normozoospermic males, standard vs. ICSI insemination) in either of the egg group types for any chromosome pairs. CONCLUSION(S): Severe male factor infertility is associated with a significant increase in the occurrence of sex chromosome abnormalities in blastocyst embryos compared with in embryos derived from normal semen samples. Aneuploidy rates in embryos derived from sperm with normal parameters were not significantly different whether ICSI or standard insemination was used to achieve fertilization. These results highlight severe male factor infertility as a possible referral category for preimplantation comprehensive chromosomal screening.

Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection for maternal recombination rates.

Crossover recombination reshuffles genes and prevents errors in segregation that lead to extra or missing chromosomes (aneuploidy) in human eggs, a major cause of pregnancy failure and congenital disorders. Here we generate genome-wide maps of crossovers and chromosome segregation patterns by recovering all three products of single female meioses. Genotyping >4 million informative SNPs from 23 complete meioses allowed us to map 2,032 maternal and 1,342 paternal crossovers and to infer the segregation patterns of 529 chromosome pairs. We uncover a new reverse chromosome segregation pattern in which both homologs separate their sister chromatids at meiosis I; detect selection for higher recombination rates in the female germ line by the elimination of aneuploid embryos; and report chromosomal drive against non-recombinant chromatids at meiosis II. Collectively, our findings show that recombination not only affects homolog segregation at meiosis I but also the fate of sister chromatids at meiosis II.

Insulin and messenger ribonucleic acid expression of insulin receptor isoforms in ovarian follicles from nonhirsute ovulatory women and polycystic ovary syndrome patients.

Insulin action is mediated by two insulin receptor (IR) isoforms, differing in mitogenic and metabolic function. IR isoform expression might occur in human granulosa cells and could be altered in polycystic ovary syndrome (PCOS) from hyperinsulinemia. To determine the relationship between granulosa cell IR isoform expression and follicular fluid insulin concentration in individual follicles, 18 normal women and seven PCOS patients receiving gonadotropins for in vitro fertilization were studied. Glucose tolerance testing was performed before pituitary desensitization, and fasting serum insulin was measured at oocyte retrieval. Granulosa cells and fluid aspirated from the first follicle were used to determine IR isoform mRNA expression and insulin concentration, respectively. IR isoform A mRNA expression was greater than that of IR isoform B expression in normal mural granulosa and cumulus cells, without a cell type effect. Intrafollicular insulin levels increased with adiposity and serum insulin levels at oocyte-retrieval but did not predict IR mRNA expression. Total IR mRNA expression, but not intrafollicular insulin levels, was elevated in PCOS patients, whereas intrafollicular insulin levels were increased in women with impaired glucose tolerance. Granulosa cell IR heterogeneity, together with adiposity-dependent intrafollicular insulin availability, introduces a novel mechanism by which insulin may affect granulosa cell function within the follicle.

Equivalent blastocyst rates after freezing murine embryos in Cryo Bio System high security or standard instruments-medicine-veterinarian straws.

OBJECTIVE: To validate the Cryo Bio System (CBS) straw in our current cryopreservation system before using it in clinical practice. DESIGN: A prospective comparison of blastocyst development rates in 278 murine embryos after refreezing and thawing at the two-cell stage against the standard Instruments-Medicine-Veterinarian (IMV) straw used in our cryopreservation program. SETTING: Private IVF laboratory. PATIENT(S): No human subjects or material was used in this study. INTERVENTION(S): Frozen two-cell murine embryos were thawed and randomized into three treatments [1] refreezing in the CBS straws, [2] refreezing in IMV 0.25-mL straws, and [3] control embryos remaining in culture without refreezing. Embryos were refrozen using identical cryoprotectants and identical programmed controlled-rate freezers. After cryopreservation, straws were held in liquid nitrogen for a brief period before thawing and continued culture. MAIN OUTCOME MEASURE(S): Postthaw murine blastocyst development rate. RESULT(S): When the manufacturer's filling and loading protocol was used for the CBS straw there was no significant difference in the blastocyst development rate between CBS (75.0%) and IMV (76.4%) straws. CONCLUSION(S): The CBS straw may be a viable and potentially safer alternative for cryopreservation of human embryos, particularly for patients with known infections.

Body mass index and uterine receptivity in the oocyte donation model.

OBJECTIVE: To evaluate the relationship of body mass index (BMI) to uterine receptivity under conditions of programmed hormonal support and standardized embryo quality. DESIGN: Retrospective cohort study.A tertiary referral center. PATIENTS: Ninety-seven consecutive first-cycle recipients of anonymous oocyte donation. After programmed hormone replacement, recipients had transfer of embryos derived from oocyte donation. Anonymous oocyte donors received ovarian stimulation and underwent transvaginal ultrasound-guided oocyte retrieval. SETTING: A receiver operator characteristic (ROC) curve of implantation versus BMI. Area under the ROC curve was 0.51, 95% confidence interval (CI) 0.41-0.62, suggesting no relationship between BMI and implantation. There was no difference in implantation rates between obese (BMI >or=30) and nonobese (BMI <30) recipients, odds ratio 1.1, 95% CI 0.5-2.4. CONCLUSION(S): Uterine receptivity was unimpaired in women with increased BMI when hormonal support and embryo quality were standardized.

Cumulative first live birth after elective cryopreservation of all embryos due to ovarian hyperresponsiveness.

OBJECTIVE: To estimate cumulative chance for first live birth after elective pronuclear stage cryopreservation of all embryos due to ovarian hyperresponsiveness. DESIGN: Retrospective analysis with longitudinal follow-up. SETTING: Academic hospital. PATIENT(S): Thirty subjects with elective cryopreservation of all embryos due to ovarian hyperresponsiveness. INTERVENTION(S): Elective cryopreservation of all embryos at the pronuclear stage (n = 30) and subsequent cryopreserved-thawed ET (n = 51). MAIN OUTCOME MEASURE(S): Cumulative chance for first live birth. RESULT(S): Cumulative chance for first live birth was 77% when analyzed by intention to treat and 82% by treatment with ET. Nearly 40% of live births were multiple. CONCLUSION(S): Cumulative first live birth increased with repetitive ET after elective pronuclear stage cryopreservation of all embryos due to ovarian hyperresponsiveness. Multiple births, however, were frequent. In the context of initial ET attempts in young women, transfer of no more than two cryopreserved-thawed embryos is advised.

Human preimplantation embryo development in vitro: a morphological assessment of sibling zygotes cultured in a single medium or in sequential media.

A comparison was made of the development of human zygotes in either a one-step (Global® medium) or two-step culture system (Quinn's Advantage®). A total of 257 normally fertilized 2PN zygotes from 28 patients were used in the study. The study was broken down into two parts: the first concerned the development of embryos from Days 1 to 3 in Global® medium and Quinn's Advantage® cleavage medium; the second consisted of a comparison of the development of embryos from Day 3 to 5/6 in Global® medium and Quinn's Advantage® blastocyst medium. There were no significant differences between the two culture media with respect to embryo quality throughout the preimplantation phase of human embryo development as determined by the extent and variability of the cell counts, fragmentation, and nucleation. A difference was noted in the blastomere symmetry of Day 2 embryos in the two media, but was no longer apparent on examination of Day 3 embryos. No differences were noted in the rates of blastocyst development, inner cell mass (ICM), and trophectoderm (TE) scores in the two culture media. Finally, no significant differences were noted with either the proportion of blastocysts chosen for transfer or cryopreservation (vitrification). The findings support the view that two-step sequential media protocols are sufficient but not necessary to support the complete in vitro development of human preimplantation embryos.