Variable aneuploidy mechanisms in embryos from couples with poor reproductive histories undergoing preimplantation genetic screening

BACKGROUND: Preimplantation genetic screening (PGS) is used to determine the chromosome status of human embryos from patients with advanced maternal age (AMA), recurrent miscarriage (RM) or repeated implantation failure (RIF). METHODS: Embryos from 47 such couples were investigated for chromosomes 13, 15, 16, 18, 21 and 22 using fluorescence in situ hybridization with two rounds of hybridization. The investigation included parental lymphocyte work-up, the screening of blastomeres on day 3 and full follow-up on day 5/6 of untransferred embryos. RESULTS: The outcome of 60 PGS cycles is described, in which 523 embryos were biopsied; 91% gave results, of which 18% were diploid for all the chromosomes tested and 82% were abnormal. The pregnancy rate per cycle that reached the biopsy stage was 27%, and 30% per embryo transfer. Satisfactory follow-up was obtained from 353 embryos; all those diagnosed as abnormal were confirmed as such, although two false-positives were detected in relation to specific chromosome abnormalities. Meiotic errors were identified in 16% of embryos. Between the RM, AMA and RIF groups, there was a significant difference in the distribution of embryos that were uniformly abnormal and of those with meiotic errors; with an almost 3-fold increase in meiotic errors in the first two groups compared with the RIF group. CONCLUSIONS: This complete investigation has identified significant differences between referral groups concerning the origin of aneuploidy in their embryos.     

Molecular methods for selection of the ideal oocyte

Some recent strategies for identifying the ideal oocyte for insemination in assisted reproduction techniques are reviewed. Established methods of assessing the female gamete, such as morphological evaluation of oocytes and cytogenetic analysis of polar bodies using fluorescence in-situ hybridization, will soon be joined by more advanced cytogenetic methods such as the use of comparative genomic hybridization to improve understanding of oocyte genetics. It seems likely, however, that the greatest advances will originate from the evolution of molecular genetic technologies. The application of microarray technology to individual oocytes and their associated cumulus cells has recently been accomplished, providing a simultaneous assessment of activity for thousands of genes and revealing potential viability markers. Furthermore, improved equipment and optimized methods of mass spectrometry have provided sufficient sensitivity to allow proteomic profiles to be generated from single oocytes and embryos, while metabolomic investigations have searched for indicators of oocyte/embryo quality in spent culture medium. Techniques of this type may ultimately lead to non-invasive tests for oocyte quality revealing previously hidden information concerning both oocyte and embryo developmental competence. Once fully validated, these new approaches are expected to revolutionize oocyte and embryo selection, leading to improved implantation rates and higher probabilities of success using elective single embryo transfer.     

Preimplantation genetic diagnosis of chromosome abnormalities: implications from the outcome for couples with chromosomal rearrangements

OBJECTIVES: Chromosomal rearrangements can lead to infertility or repeated spontaneous or induced abortions. The use of preimplantation genetic diagnosis (PGD) allows the selected transfer of chromosomally balanced embryos. The aim of this study was to carry out detailed analysis of the outcome of 11 PGD cycles for 8 patients carrying various chromosomal rearrangements. METHODS: Patients underwent routine in vitro fertilisation with biopsy of embryos on day 3. Specific fluorescent in situ hybridisation protocols were developed for each couple. Embryo transfer was possible in all 11 cycles. RESULTS: The outcome was four pregnancies, leading to three live births and one biochemical pregnancy. Post-zygotic mosaicism was detected in 75% of untransferred embryos, the majority of which were chaotic. Detailed follow-up and analysis provided evidence for the co-existence of chromosomally balanced and abnormal cells in six embryos. The mechanisms involved included chromosome breakage and loss of material. CONCLUSIONS: Biopsy and analysis of two blastomeres, where possible, reduced the risk of misdiagnosis in cases of balanced/aneuploid mosaics. The three live births achieved for the eight couples treated in this series, despite the poor history in almost all cases, is further proof that a policy of biopsying two cells from embryos consisting of six or more cells and a single cell from four- or five-cell embryos is compatible with a positive outcome.     

Sequential FISH analysis of oocytes and polar bodies reveals aneuploidy mechanisms

OBJECTIVES: Constitutional aneuploidy occurs in at least 5% of recognised pregnancies, with apparent preferential involvement of the X chromosome and the smaller autosomes. Molecular cytogenetic investigations of cleavage-stage embryos have revealed anomalies affecting all sizes of chromosomes. The aim was to investigate the variety of anomalies arising during maternal meiosis I by analysis of unfertilised oocytes and polar bodies to gain insight into aneuploidy mechanisms. METHODS: Sequential FISH analysis was carried out with specific probes derived from eight chromosomes, representing all sizes. Only imbalance due to a gain of a whole chromosome or chromatid, represented by extra signals, was counted to avoid artefact. RESULTS: Data were obtained on 236 eggs from 124 patients of average age 32.5 years (range 22-44). Ten patients (average 32.6 years) had abnormal eggs. The abnormality rate for oocytes and for polar bodies was close to 4% for each. Fourteen hyperploidies were found, seven involving additional single chromatids. The abnormalities affected chromosomes 13,16,18, 21 and X but not chromosomes 1, 9 or 12. CONCLUSION: The data provide evidence for several mechanisms leading to aneuploidy, including classical non-disjunction of whole univalents; pre-division of chromatids prior to anaphase I, leading to imbalance detected at metaphase II; gonadal mosaicism for a trisomic cell line and preferential involvement of the smaller chromosomes. Monosomy for the large autosomes is not uncommon in cleavage-stage embryos and may additionally arise from anaphase lag preferentially affecting such chromosomes.     

Enhancement of soluble CD23 levels in the seminal plasma of infertile men with idiopathic testicular lesion.

Since increased levels of soluble CD23 (sCD23) were demonstrated in patients with autoimmune diseases, seminal plasma sCD23 levels were examined in 110 men divided into seven groups according to etiological diagnosis of infertility and two groups on the basis of normal or abnormal spermiogram. sCD23 was absent in the seminal plasma of normal men: According to the ANOVA results, all measurements were significantly different between the groups of patients examined (p<.01). Specifically, patients with idiopathic testicular lesion have a significantly higher mean value of sCD23 than all other patients. A statistically significant difference was noted in the sCD23 levels between men with normal and those with abnormal spermiograms, although with wide overlapping of the individual values. Men with idiopathic testicular lesion may be immunologically more active than other groups with subfertility.     

A clinical study of optical biopsy of the uterine cervix using a multispectral imaging system

OBJECTIVE: To present the clinical application of the multispectral imaging colposcopic system (MIS colposcopy). METHODS: MIS colposcopy was performed on 123 enrolled women. After a 3% acetic acid application, sequential images were captured, analyzed, and stored automatically. Directed biopsies were taken from distinct marked acetic acid-responsive tissue areas indicated on the monitor, while a real-time assessment of the curves of intensity of the backscattered light (IBSL) vs. time was performed. Blind biopsies were taken from non-acetowhitening areas. Histological findings were correlated with MIS colposcopy results and compared with conventional colposcopy and Pap test results. RESULTS: Acetic acid-tissue interaction resulted in temporal and spatial alterations to the light scattering properties of the abnormal tissue that was analyzed. The shape of IBSL curve and the "relaxation time" (the time it takes for IBSL to decay to 1/e of its peak value) changed in accordance with the underlying lesion. More severe CIN lesions lead to higher maximum IBSL; longer durations of acetowhitening lead to increasingly delayed exponential decay of IBSL curve. To compare with histological examination, MIS colposcopy had a 1.7% false-diagnostic rate, while PAP test and conventional colposcopy had 24.4% and 22% false-diagnostic rates, respectively. A triple exponential function created a "pseudocolor" image that comprised the grade map of the lesion, and this is frequently representative of the duration/degree of the induced alterations. CONCLUSION: Improved diagnostic information can be gained by recording the optical information in a narrow spectral range with high spatial resolution. MIS colposcopy can be used in the diagnosis of uterine cervix pathological conditions and in the differentiation between CIN lesions.     

Analysis of the evolution of chromosome abnormalities in human embryos from Day 3 to 5 using CGH and FISH

The use of interphase fluorescent in situ hybridization (FISH) has shown that a large number of human embryos exhibit chromosomal abnormalities in vitro. The most common abnormality is mosaicism which is seen in up to 50% of preimplantation embryos at all stages of development. In this study, comparative genomic hybridization (CGH) was used to analyse 1-2 cells biopsied on Day 3 of development while the rest of the embryo was cultured until Day 5. Embryos were spread on Day 5 and analysed by FISH using probe combinations that varied depending on the CGH result, to investigate the progress of any abnormalities detected on Day 3. A total of 37 frozen-thawed embryos were analysed in this study. One gave no CGH or FISH results and was excluded from analysis. Six embryos failed to give any FISH result as they were degenerating on Day 5. Thirty embryos provided results from both techniques. According to the CGH results, the embryos were divided into two groups; Group 1 had a normal CGH result (13 embryos) and Group 2 an abnormal CGH result (17 embryos). For Group 1, three embryos showed normal CGH and FISH results, while 10 embryos were mosaic after FISH analysis, with various levels of abnormalities. For Group 2, FISH showed that all embryos were mosaic or completely chaotic. The combination of CGH and FISH enabled the thorough investigation of the evolution of mosaicism and of the mechanisms by which it is generated. The main two mechanisms identified were whole or partial chromosome loss and gain. These were observed in embryos examined on both Day 3 and 5.     

Aneuploidy screening for embryo selection

Chromosome abnormalities are extremely common in human oocytes and embryos and are associated with a variety of negative outcomes for both natural cycles and those using assisted conception techniques. Embryos containing the wrong number of chromosomes (aneuploidy) may fail to implant in the uterus, miscarry, or lead to children with serious medical problems (e.g., Down syndrome). Preimplantation genetic screening (PGS) is a method that seeks to improve the outcomes of assisted reproductive treatments, such as in vitro fertilization (IVF), by ensuring that the embryos chosen for transfer to the uterus are chromosomally normal. Here we summarize published and novel data concerning the frequency and variety of chromosomal abnormalities seen in oocytes and embryos at the cleavage and blastocyst stages of development. Clinical outcomes of studies using PGS are presented, and the controversy over the use of chromosome screening as a tool for embryo selection is discussed. We describe validation and preliminary clinical data from the new generation of methods being used for PGS, including comparative genomic hybridization (CGH), microarrays (aCGH and single nucleotide polymorphism arrays), and quantitative polymerase chain reaction. These methodologies allow comprehensive chromosomal analysis, provide high accuracy, and have yielded encouraging preliminary clinical data. The combination of advances in genetics and embryology seems poised to usher in a new era in the treatment of infertility.