B. McCallie

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.

Altered microRNA and gene expression in the follicular fluid of women with polycystic ovary syndrome

PurposeTo determine if microRNAs are differentially expressed in the follicular fluid of women with PCOS compared to fertile oocyte donors and identify associated altered gene expression.MethodsWomen undergoing IVF who met Rotterdam criteria for PCOS or who were fertile oocyte donors were recruited from a private IVF center. Individual follicle fluid was collected at the time of oocyte retrieval. MicroRNA analysis was performed using microarray and validated using real-time PCR on additional samples. Potential gene targets were identified and their expression analyzed by real time PCR.ResultsMicroarray profiling of human follicular fluid revealed expression of 235 miRNAs, 29 were differentially expressed between the groups. Using PCR validation, 5 miRNAs (32, 34c, 135a, 18b, and 9) showed significantly increased expression in the PCOS group. Pathway analysis revealed genes involved in insulin regulation and inflammation. Three potential target genes were found to have significantly decreased expression in the PCOS group (interleukin 8, synaptogamin 1, and insulin receptor substrate 2).ConclusionsMicroRNAs are differentially expressed in the follicular fluid of women with PCOS when compared to fertile oocyte donors. There is also altered expression of potential target genes associated with the PCOS phenotype.

Impact of maternal aging on the molecular signature of human cumulus cells

OBJECTIVE To investigate the impact of maternal aging on the molecular signature of cumulus cells. DESIGN Experimental study. SETTING Research laboratory. PATIENT(S) Patients, young fertile oocyte donors (n = 40) and infertile women of advanced maternal age (40-45 years; n = 48), donated, with Institutional Review Board consent, cumulus cells during routine infertility treatment. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Proteomic and gene expression profiles of cumulus cells. RESULT(S) Proteomic analysis identified a total of 1,423 cumulus cell proteins. Statistical analysis revealed 110 (7.7%) proteins to be differentially expressed in relation to female aging (>1.5-fold change). Pathway annotation revealed significant involvement in metabolism (ACAT2, HSD17B4, ALDH9A1, MVK, CYP11A1, and FDFT1), oxidative phosphorylation (OP; NDUFA1, UQCRC1, MT-ATP6, ATP5I, and MT-ATP8), and post-transcriptional mechanisms (KHSRP, SFPQ, DDX46, SNRPF, ADAR, NHPL1, and U2AF2) relative to advanced maternal age. Gene expression analysis also revealed altered profiles in cumulus cells from women in their early to mid-40s. CONCLUSION(S) This novel study reveals that the cumulus cell molecular signature, at both the gene and protein level, is impacted by advanced maternal aging. A compromised follicular environment is evident with altered energy metabolism and post-transcriptional processes.

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.

Analysis of global gene expression following mouse blastocyst cryopreservation

BACKGROUND The aim of this study was to examine the effect of the cryopreservation procedure (slow freezing or vitrification) and cryoprotectants (1,2-propanediol or dimethylsulphoxide) on mouse blastocyst gene expression. METHODS Cultured mouse blastocysts were cryopreserved with different protocols. Following thawing/warming, total RNA from re-expanded blastocysts was isolated, amplified and then analyzed using mouse whole-genome microarrays. RESULTS Compared with non-cryopresevered control blastocysts, gene expression was only significantly altered by slow freezing. Slow freezing affected the expression of 115 genes (P < 0.05). Of these, 100 genes exhibited down-regulation and 15 genes were up-regulated. Gene ontology revealed that the majority of these genes are involved in protein metabolism, transcription, cell organization, signal transduction, intracellular transport, macromolecule biosynthesis and development. Neither of the vitrification treatment groups showed statistically different gene expression from the non-cryopreserved control embryos. Hierarchical cluster analysis, did however, reveal that vitrification using 1,2-propanediol could result in a gene expression profile closest to that of non-cryopreserved blastocysts. CONCLUSIONS Investigating the effects of cryopreservation on cellular biology, such as gene expression, is fundamental to improving techniques and protocols. This study demonstrates that of the cryopreservation regimens employed, slow freezing induced the most changes in gene expression compared with controls.

Human and mouse embryonic development, metabolism and gene expression are altered by an ammonium gradient in vitro

Ammonium is generated in culture media by the spontaneous deamination of amino acids at 37 °C and through the metabolism of amino acids by human embryos. The appearance of ammonium is a time-dependent phenomenon and can compromise embryo physiology, development and viability. In this study, the effects of a gradient of ammonium on the development, metabolism and transcriptome of human and mouse embryos were investigated. Pronucleate oocytes were cultured in the presence of an ammonium gradient that mimicked the spontaneous deamination of Eagles amino acids together with 1 mM glutamine. All embryos were cultured in sequential media G1/G2 at 5% O2, 6% CO2 and 89% N2. Human embryo metabolism was assessed through a non-invasive fluorometric analysis of pyruvate consumption. Transcriptome analysis was performed on the resultant blastocysts from both species using a microarray technology. Embryo development prior to compaction was negatively affected by the presence of low levels of ammonium in both species. Human embryo metabolism was significantly inhibited after just 24 and 48 h of culture. Transcriptome analysis of blastocysts from both species revealed significantly altered gene expression profiles, both decreased and increased. Functional annotation of the altered genes revealed the following over represented biological processes: metabolism, cell growth and/or maintenance, transcription, cell communication, transport, development and transcription regulation. These data emphasize the enhanced sensitivity of the cleavage-stage embryo to its environment and highlight the requirement to renew culture media at frequent intervals in order to alleviate the in vitro induced effects of ammonium build-up in the environment surrounding the embryo.

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.

Blastocysts from patients with polycystic ovaries exhibit altered transcriptome and secretome

Polycystic ovaries (PCO) is a common phenotype of women presenting for infertility treatment. This study investigated whether blastocysts derived from women with PCO have an altered molecular signature which could be a causative factor contributing to reproductive failure. Morphologically similar blastocysts derived from women with PCO and donor oocyte cycles were analysed for transcription and protein secretion. Unsupervised hierarchical clustering demonstrated that the transcriptome profiles of blastocysts derived from PCO patients and control blastocysts were markedly different with complete branch separation. Statistical analysis revealed 829 genes with significantly different expression: 784 decreased (94.6%) and 45 increased (5.4%) in blastocysts derived from women with PCO compared with controls (P<0.05). Functional annotation of these genes revealed predominant gene ontology biological processes including protein metabolism (30%), transcription (22%), signal transduction (15%), biosynthesis (15%) and cell cycle (14%). Proteomic profiling identified 12 biomarkers that displayed significant decrease in expression in blastocysts derived from women with PCO compared with controls (P<0.05). These data indicate molecular alterations in human blastocysts derived from PCO patients, potentially demonstrating for the first time a link between patient aetiology/phenotype and subsequent embryo development, which in part may explain the observed reduction in reproductive capacity.

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.