J.C. Parks

Blastocyst gene expression correlates with implantation potential

OBJECTIVE To investigate the role of the blastocyst in implantation failure. This study examined trophectoderm (TE) gene expression relative to pregnancy outcome. DESIGN Retrospective experimental study. SETTING Nonprofit research foundation. ANIMAL(S) Six-week-old BDF1 female mice. INTERVENTION(S) Hatching blastocysts underwent trophectoderm biopsy before single blastocyst transfer (one per uterine horn). MAIN OUTCOME MEASURES Blinded gene expression analysis was performed on biopsied TE cells by quantitative real-time polymerase chain reaction (Q RT-PCR). Healthy placenta and absorption sites were biopsied on day 16 of fetal development for comprehensive transcriptome analysis with validation by Q RT-PCR. RESULT(S) Compared with blastocysts that resulted in healthy fetal development, blastocysts that failed to implant (negative) showed decreased B3gnt5 and Eomes gene expression, while blastocysts that resulted in spontaneous pregnancy loss (absorption) displayed decreased Wnt3a and Eomes gene expression. Comprehensive transcriptome analysis of biopsied absorption sites and healthy placenta revealed distinct gene expression signatures, with 5,918 significantly altered transcripts (greater than twofold). The predominantly altered pathways associated with spontaneous pregnancy loss were the complement and coagulation cascades. CONCLUSION(S) This study revealed for the first time that individual blastocyst gene expression profiles correlate with outcome, including successful implantation and pregnancy loss.

Aging sperm negatively impacts in vivo and in vitro reproduction: a longitudinal murine study

OBJECTIVE To investigate the impact of paternal aging on reproductive success. DESIGN Animal study. SETTING Research facility. ANIMAL(S) Outbred CF1 mice. INTERVENTION(S) Ten young male mice with proven fertility were mated routinely over 15 months with superovulated young females to assess in vivo and in vitro reproductive outcome. MAIN OUTCOME MEASURE(S) In vivo fertilization, in vivo fetal development, in vitro embryo morphology, and developmental outcome were assessed. RESULT(S) There were no differences observed for any reproductive end point until the paternal age of 12 months. At 12-15 months, in vivo fertilization was significantly decreased (35% vs. 78% at <12 months). Natural matings with males ≥ 12 months revealed significantly smaller fetuses (11.36 mm vs. 14.73 mm <12 months) and placental weight (0.10 g vs. 0.13 g at <12 months). In vitro blastocyst development showed a significant decline at ≥ 12 months, and in vitro blastocyst transfer resulted in a significant increase in pregnancy loss with males ≥ 12 months (61.5% vs. 0% at <12 months). In addition, the expression levels of Ace-1, Prm1, Prm2, and Smcp were observed to be decreased in sperm from males ≥ 12 months compared with young male control subjects. CONCLUSION(S) Results from this study indicate an abrupt reproductive deterioration at paternal midlife, with an adverse effect observed on natural conception, in vitro blastocyst development, implantation potential, and fetal viability.

Human blastocysts exhibit unique microrna profiles in relation to maternal age and chromosome constitution

PurposeTo determine microRNA (miRNA) expression in human blastocysts relative to advanced maternal age and chromosome constitution.MethodsCryopreserved human blastocysts were warmed and underwent a trophectoderm biopsy for comprehensive chromosomal screening. Select blastocysts were then lysed, reverse transcribed, and pre-amplified prior to running real-time PCR. Statistical analysis was performed using an internal constant housekeeping miRNA. Significant microRNA’s of interest were then analyzed for their predicted genes and biological pathways. Additional cryopreserved blastocysts were warmed and stained for the SIRT1 protein for validation.ResultsHuman blastocysts exhibit unique miRNA expression profiles in relation to maternal age and chromosome constitution. miR-93 was exclusively expressed in blastocysts from women in their forties and further up-regulated with an abnormal chromosome complement. Up-regulated miR-93 resulted in an inverse down-regulation of targets like SIRT1, resulting in reduced oxidative defense.ConclusionsMiRNAs play an important role in aging as well as chromosome constitution and have downstream effects that regulate proteins which can compromise embryonic development.

Hypomethylation and Genetic Instability in Monosomy Blastocysts May Contribute to Decreased Implantation Potential

DNA methylation is a key epigenetic mechanism responsible for gene regulation, chromatin remodeling, and genome stability, playing a fundamental role during embryonic development. The aim of this study was to determine if these epigenetic marks are associated with chromosomal aneuploidy in human blastocysts. Surplus, cryopreserved blastocysts that were donated to research with IRB consent were chosen with varying chromosomal aneuploidies and respective implantation potential: monosomies and trisomies 7, 11, 15, 21, and 22. DNA methylation analysis was performed using the Illumina Infinium HumanMethylation450 BeadChip (~485,000 CpG sites). The methylation profiles of these human blastocysts were found to be similar across all samples, independent of chromosome constitution; however, more detailed examination identified significant hypomethylation in the chromosome involved in the monosomy. Real-time PCR was also performed to determine if downstream messenger RNA (mRNA) was affected for genes on the monosomy chromosome. Gene dysregulation was observed for monosomy blastocysts within significant regions of hypo-methylation (AVEN, CYFIP1, FAM189A1, MYO9A, ADM2, PACSIN2, PARVB, and PIWIL3) (P < 0.05). Additional analysis was performed to examine the gene expression profiles of associated methylation regulators including: DNA methyltransferases (DNMT1, DNMT3A, DNMT3B, DNMT3L), chromatin modifying regulators (CSNK1E, KDM1, PRKCA), and a post-translational modifier (PRMT5). Decreased RNA transcription was confirmed for each DNMT, and the regulators that impact DNMT activity, for only monosomy blastocysts (P < 0.05). In summary, monosomy blastocysts displayed hypomethylation for the chromosome involved in the error, as well as transcription alterations of associated developmental genes. Together, these modifications may be contributing to genetic instability and therefore be responsible for the limited implantation potential observed for full monosomy blastocysts.

Infertility diagnosis has a significant impact on the transcriptome of developing blastocysts

STUDY QUESTION Is the human blastocyst transcriptome associated with infertility diagnosis, specifically: polycystic ovaries (PCO), male factor (MF) and unexplained (UE)? SUMMARY ANSWER The global blastocyst transcriptome was significantly altered in association with a PCO, MF and UE infertility diagnosis. WHAT IS KNOWN ALREADY Infertility diagnosis has an impact on the probability for a successful outcome following an IVF cycle. Limited information is known regarding the relationship between a specific infertility diagnosis and blastocyst transcription during preimplantation development. STUDY DESIGN, SIZE, DURATION Blastocysts created during infertility treatment from patients with specific infertility diagnoses (PCO, MF and UE) were analyzed for global transcriptome compared to fertile donor oocyte blastocysts (control). PARTICIPANTS/MATERIALS, SETTING, METHODS Surplus cryopreserved blastocysts were donated with patient consent and institutional review board approval. Female patients were <38 years old with male patients <40 years old. Blastocysts were grouped according to infertility diagnosis: PCO (n = 50), MF (n = 50), UE (n = 50) and fertile donor oocyte controls (n = 50). Pooled blastocysts were lysed for RNA isolation followed by microarray analysis using the SurePrint G3 Human Gene Expression Microarray. Validation was performed on significant genes of interest using real-time quantitative PCR (RT-qPCR). MAIN RESULTS AND THE ROLE OF CHANCE Transcription alterations were observed for all infertility etiologies compared to controls, resulting in differentially expressed genes: PCO = 869, MF = 348 and UE = 473 (P < 0.05; >2-fold). Functional annotation of biological and molecular processes revealed both similarities, as well as differences, across the infertility groups. All infertility etiologies displayed transcriptome alterations in signal transducer activity, receptor binding, reproduction, cell adhesion and response to stimulus. Blastocysts from PCO patients were also enriched for apoptotic genes while MF blastocysts displayed enrichment for genes involved in cancer processes. Blastocysts from couples with unexplained infertility displayed transcription alterations related to various disease states, which included mechanistic target of rapamycin (mTOR) and adipocytokine signaling. RT-qPCR validation confirmed differential gene expression for the following genes: BCL2 like 10 (BCL2L10), heat shock protein family A member 1A (HSPA1A), heat shock protein family A member 1B (HSPA1B), activating transcription factor 3 (ATF3), fibroblast growth factor 9 (FGF9), left-right determination factor 1 (LEFTY1), left-right determination factor 2 (LEFTY2), growth differentiation factor 15 (GDF15), inhibin beta A subunit (INHBA), adherins junctions associated protein 1 (AJAP1), cadherin 9 (CDH9) and laminin subunit alpha 4 (LAMA4) (P < 0.05; >2-fold). LARGE SCALE DATA Not available due to participant privacy. LIMITATIONS, REASONS FOR CAUTION Blastocyst samples for microarray analysis required pooling. While this allows for an overall average in each infertility etiology group and can reduce noise from sample-to-sample variation, it cannot give a detailed analysis of each blastocyst within the group. WIDER IMPLICATIONS OF THE FINDINGS Underlying patient infertility diagnosis has an impact on the blastocyst transcriptome, modifying gene expression associated with developmental competence and implantation potential. STUDY FUNDING AND COMPETING INTEREST(S) No conflict of interest or outside funding provided.

Corona cell RNA sequencing from individual oocytes revealed transcripts and pathways linked to euploid oocyte competence and live birth

Corona cells surround the oocyte and maintain a close relationship through transzonal processes and gap junctions, and may be used to assess oocyte competence. In this study, the corona cell transcriptome of individual cumulus oocyte complexes (COCs) was investigated. Isolated corona cells were collected from COCs that developed into euploid blastocysts and were transferred in a subsequent frozen embryo transfer. Ten corona cell samples underwent RNA-sequencing to generate unique gene expression profiles. Live birth was compared with negative implantation after the transfer of a euploid blastocyst using bioinformatics and statistical analysis. Individual corona cell samples produced a mean of 21.2 million sequence reads, and 307 differentially expressed transcrpits (P < 0.05; fold change ≥ 2). Enriched pathway analysis showed Wnt signalling, mitogen-activated protein kinases signalling, focal adhesion and tricarboxylic acid cycle to be affected by implantation outcome. The Wnt/beta-catenin signalling pathway, including genes APC, AXIN and GSK3B, were independently validated by real-time quantitative reverse transcription. Individual, corona cell transcriptome was successfully generated using RNA-sequencing. Key genes and signalling pathways were identified in association with implantation outcome after the transfer of a euploid blastocyst in a frozen embryo transfer. These data could provide novel biomarkers for the non-invasive assessment of embryo viability.

Epigenetic Dysregulation Observed in Monosomy Blastocysts Further Compromises Developmental Potential

Epigenetic mechanisms such as DNA methylation regulate genomic imprinting and account for the distinct non-equivalence of the parental genomes in the embryo. Chromosomal aneuploidy, a major cause of infertility, distorts this highly regulated disparity by the presence or absence of chromosomes. The implantation potential of monosomy embryos is negligible compared to their trisomy counterparts, yet the cause for this is unknown. This study investigated the impact of chromosomal aneuploidy on strict epigenetically regulated domains, specifically imprinting control regions present on aneuploid chromosomes. Donated cryopreserved human IVF blastocysts of transferable quality, including trisomy 15, trisomy 11, monosomy 15, monosomy 11, and donor oocyte control blastocysts were examined individually for DNA methylation profiles by bisulfite mutagenesis and sequencing analysis of two maternally methylated imprinting control regions (ICRs), SNRPN (15q11.2) and KCNQ1OT1 (11p15.5), and one paternally methylated imprinting control region, H19 (11p15.5). Imprinted genes within the regions were also evaluated for transcript abundance by RT-qPCR. Overall, statistically significant hypermethylated and hypomethylated ICRs were found in both the trisomy and monosomy blastocysts compared to controls, restricted only to the chromosome affected by the aneuploidy. Increased expression was observed for maternally-expressed imprinted genes in trisomy blastocysts, while a decreased expression was observed for both maternally- and paternally-expressed imprinted genes in monosomy blastocysts. This epigenetic dysregulation and altered monoallelic expression observed at imprinting control regions in aneuploid IVF embryos supports euploid embryo transfer during infertility treatments, and may specifically highlight an explanation for the compromised implantation potential in monosomy embryos.