M.G. Katz-Jaffe

Clinical application of comprehensive chromosomal screening at the blastocyst stage

OBJECTIVE To evaluate a new strategy for comprehensive chromosome screening at the blastocyst stage. DESIGN Clinical research study. SETTING An IVF clinic and a specialist preimplantation genetic diagnosis laboratory. PATIENT(S) Forty-five infertile couples participated in the study. The mean maternal age was 37.7 years, and most couples had at least one previous unsuccessful IVF treatment cycle (mean 2.4). INTERVENTION(S) This study used a novel chromosome screening approach, combining biopsy of several trophectoderm cells on day 5 after fertilization and detailed analysis of all 24 types of chromosome using comparative genomic hybridization. MAIN OUTCOME MEASURE(S) Proportion of embryos yielding a diagnostic result, aneuploidy rate, implantation rate, and pregnancy rate. RESULT(S) A diagnosis was obtained from 93.7% of embryos tested. The aneuploidy rate was 51.3%. The probability of an individual transferred embryo forming a pregnancy reaching the third trimester/birth was 68.9%, an implantation rate 50% higher than contemporary cycles from the same clinic. The pregnancy rate was 82.2%. CONCLUSION(S) The comprehensive chromosome screening method described overcomes many of the problems that limited earlier aneuploidy screening techniques and may finally allow preimplantation genetic screening to achieve the benefits predicted by theory. The high embryo implantation rate achieved is particularly encouraging and, if confirmed in subsequent studies, will be of great significance for IVF clinics attempting to reduce the number of embryos transferred or to implement single embryo transfer.

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.

Comprehensive molecular cytogenetic analysis of the human blastocyst stage

BACKGROUND The high frequency of chromosomal abnormalities observed in human gametes and embryos is unlike that seen in other mammalian species and is of great clinical significance, leading to high rates of pregnancy loss, and live-born children with aneuploid syndromes. Although much is known concerning the aneuploidy rates of oocytes, cleavage stage embryos and fetuses during pregnancy, the chromosomal status of blastocysts has been relatively little investigated. METHODS A total of 158 good quality blastocysts were examined using micromanipulation, whole genome amplification and comparative genomic hybridization. RESULTS From the obtained data, it was evident that the aneuploidy rate (38.8%) is significantly lower for blastocysts than for embryos at earlier stages (51%). However, in many cases, chromosome errors, including monosomy, imbalance affecting the larger chromosomes and complex aneuploidy persisted to this final stage of preimplantation development. CONCLUSIONS This study represents the first attempt to gain a detailed insight into the extent and type of chromosome errors seen at the blastocyst stage, using a comprehensive molecular cytogenetic method. Our data suggest that the blastocyst stage does not represent an absolute selective barrier, and that the majority of aneuploid embryos are lost at the time of implantation or shortly thereafter.

Comprehensive chromosome screening of polar bodies and blastocysts from couples experiencing repeated implantation failure.

OBJECTIVE To identify and transfer cytogenetically normal embryos after screening all chromosomes of first and second polar bodies (PBs) or trophectoderm samples with the use of comparative genomic hybridization. DESIGN Clinical research study. SETTING In vitro fertilization clinic referring samples to a specialist preimplantation genetic diagnosis laboratory. PATIENT(S) Thirty-two couples with repeated implantation failure. INTERVENTION(S) Zygotes from patients with repeated implantation failure and poor response to ovarian stimulation underwent PB biopsy. Patients with repeated implantation failure who were candidates for blastocyst transfer received trophectoderm biopsy. Zygotes or blastocysts were vitrified while chromosome analysis took place. Euploid embryos were transferred during a subsequent cycle. MAIN OUTCOME MEASURE(S) Cytogenetic status and implantation and pregnancy rates. RESULT(S) The oocyte and blastocyst aneuploidy rates were 65.5% and 45.2%, respectively. Abnormalities affecting all chromosomes were detected. Implantation and pregnancy rates for the patients with PB biopsy were 11.5% and 21.4%, respectively, whereas for patients receiving blastocyst analysis they were 58.3% and 69.2%. CONCLUSION(S) Initial results for patients of advanced maternal age (39.8 years) with repeated implantation failure and poor ovarian response were encouraging. However, further study is required to confirm whether or not screening is beneficial. Blastocyst analysis was associated with high pregnancy rates, suggesting that comprehensive chromosome screening may assist patients with repeated implantation failure capable of producing blastocysts in achieving pregnancies.

Preimplantation aneuploidy testing for infertile patients of advanced maternal age: a randomized prospective trial

OBJECTIVE To assess the potential benefit of preimplantation aneuploidy testing on the outcome of in vitro fertilization (IVF) for women of advanced maternal age (AMA). DESIGN Prospective randomized clinical trial. SETTING Private IVF clinic. PATIENT(S) Sixty-two infertile AMA couples undergoing fertility treatment. INTERVENTION(S) Fluorescent in situ hybridization (FISH) for chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22. MAIN OUTCOME MEASURE(S) Preimplantation aneuploidy testing of biopsied blastomeres on day 3 of development. RESULT(S) Fertilization and blastocyst developmental rates were similar for the test and control groups: 80% versus 77.4% and 49% versus 48.2%, respectively. The average number of embryos transferred was comparable at 2.2 for the test group and 2.7 for the control group. Implantation rates were also equivalent across the two groups: 37.3% in the control group versus 36.5% in the test group. Nevertheless, the spontaneous abortion rate was observed to be lower for the test group: 25.9% versus 32.26% in the control group. This resulted in an observed increase in delivery rates for the test group: 78% versus 67.74% in the control group. CONCLUSION(S) Preimplantation aneuploidy testing does not appear to statistically significantly improve outcome parameters in infertile AMA patients; however, a trend toward a decrease in the spontaneous abortion rate with a subsequent higher delivery rate was observed.

The role of proteomics in defining the human embryonic secretome

Non-invasive gamete and embryo assessment is considered an important focus in assisted reproductive technologies (ART). Currently, the selection of embryos for transfer is based on morphological indices. Though successful, the field of ART would benefit from a non-invasive quantitative method of viability determination. Omics technologies, including transcriptomics, proteomics and metabolomics, have already begun providing evidence that viable gametes and embryos possess unique molecular profiles with potential biomarkers that can be utilized for developmental and/or viability selection. Unlike the human genome that is relatively fixed and steady throughout the human body, the human proteome, estimated at over a million proteins, is more complex, diverse and dynamic. It is the proteins themselves that contribute to the physiological homeostasis in any cell or tissue. Of particular interest in ART is the secretome, those proteins that are produced within the embryo and secreted into the surrounding environment. Defining the human embryonic secretome has the potential to expand our knowledge of embryonic cellular processes, including the complex dialogue between the developing embryo and its maternal environment, and may also assist in identifying those embryos with the highest implantation potential. Advances in proteomic technologies have allowed the non-invasive profiling of the human embryonic secretome with ongoing research focused on correlation with outcome. From a clinical perspective, embryo selection based on morphological assessment and non-invasive analysis of the human embryonic secretome may improve IVF success and lead to routine single embryo transfers.

Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health

Bidirectional somatic cell-oocyte signaling is essential to create a changing intrafollicular microenvironment that controls primordial follicle growth into a cohort of growing follicles, from which one antral follicle is selected to ovulate a healthy oocyte. Such intercellular communications allow the oocyte to determine its own fate by influencing the intrafollicular microenvironment, which in turn provides the necessary cellular functions for oocyte developmental competence, which is defined as the ability of the oocyte to complete meiosis and undergo fertilization, embryogenesis, and term development. These coordinated somatic cell-oocyte interactions attempt to balance cellular metabolism with energy requirements during folliculogenesis, including changing energy utilization during meiotic resumption. If these cellular mechanisms are perturbed by metabolic disease and/or maternal aging, molecular damage of the oocyte can alter macromolecules, induce mitochondrial mutations, and reduce adenosine triphosphate production, all of which can harm the oocyte. Recent technologies are now exploring transcriptional, translational, and post-translational events within the human follicle with the goal of identifying biomarkers that reliably predict oocyte quality in the clinical setting.

Aberration of blastocyst microRNA expression is associated with human infertility

OBJECTIVE To examine human blastocyst microRNA (miRNA) expression in correlation with human infertility. MicroRNAs are small, noncoding RNA molecules that regulate gene expression via mechanisms such as degradation and translational suppression of targeted messenger RNAs. Recent data has pointed to the importance of miRNAs in disease states and during mouse embryo development. DESIGN Descriptive study. SETTING Nonprofit research foundation. PATIENT(S) Transferable quality human blastocysts donated with consent to research (n = 40). INTERVENTION(S) Quantitative real-time PCR. MAIN OUTCOME MEASURE(S) MicroRNA expression profile. RESULT(S) Morphologically similar blastocysts derived from patients with polycystic ovaries or male factor infertility exhibited a significant decrease in the expression of six miRNAs in comparison with donor fertile control blastocysts (P < 0.05). Annotation of predicted gene targets for these differentially expressed miRNAs included gene ontology (GO) biological processes involved in cell growth and maintenance and transcription as well as GO molecular functions implicated in nucleic acid binding and signal transducer activity. Three predicted miRNA target genes were selected for analysis and demonstrated significant altered expression consistent with aberrant miRNA profiles. CONCLUSION(S) This study describes for the first time that transferable quality blastocysts derived from infertile patients (male factor infertility and polycystic ovaries) possess aberrant miRNA profiles. With growing evidence indicating the importance of miRNAs during development, an association may exist with human infertility.

Transcriptome analysis of in vivo and in vitro matured bovine MII oocytes

In vitro maturation (IVM) of mammalian oocytes does not support the same rates of embryo development or pregnancy when compared to oocytes that have matured in vivo. Therefore, environment has a significant influence on the oocytes ability to complete maturation and acquire the mRNA and proteins required for successful fertilization and normal embryonic development. The aim of this study was to analyze the MII oocyte transcriptome between in vivo and in vitro conditions. Total RNA was extracted, processed and hybridized to the Affymetrix GeneChip Bovine Genome Array. Following normalization of the microarray data, analysis revealed 10 differentially expressed genes after IVM compared to in vivo matured controls, including Aqp3, Sept7, Abhd4 and Siah2 (P<0.05). K-means cluster analysis coupled with associated gene ontology, identified several biological processes affected by IVM, including metabolism, energy pathways, cell organization and biogenesis, and cell growth and maintenance. Quantitative real-time PCR validated the microarray data and also revealed altered expression levels after IVM of specific putatively imprinted genes, Igf2r, Peg3 and Snrpn (P<0.05). Distinct IVM transcription patterns reflected the oocytes response to its surrounding environment. Monitoring transcription levels of key oocyte maturation genes may subsequently assist in improving IVM success.

Comprehensive chromosome screening of trophectoderm with vitrification facilitates elective single-embryo transfer for infertile women with advanced maternal age

The universal goal of assisted reproduction technologies is a singleton delivery of a healthy full-term baby. For younger women (<35 years of age) single-embryo transfer is a viable option resulting in clinical success similar to multiple-embryo transfers. In contrast, older women have significantly lower pregnancy rates following single-embryo transfer. To provide effective single-embryo transfer options for older women, improved methods of embryo selection are required to overcome the marked differences in outcome of single- versus double-embryo transfer. With the development of comprehensive chromosome screening, blastocyst vitrification, and trophectoderm biopsy techniques, older women have the opportunity of elective single-embryo transfer with live birth rates as high as those reported for younger good-prognosis infertility patients.