title: Assessing embryo quality by combining non-invasive markers: early time-lapse parameters reflect gene expression in associated cumulus cells.
abstract: OBJECTIVE
  Are there associations between early time-lapse parameters, expression of candidate embryo viability genes in cumulus cells and embryo quality on Day 5?
  
  
  CONCLUSIONS
  Early time-lapse parameters correlate to the expression levels of candidate embryo viability genes in cumulus cells but a combined analysis including both time-lapse and candidate gene expression did not identify significant predictors of embryo quality on Day 5.
  
  
  BACKGROUND
  Recent evidence suggests that early time-lapse parameters are predictive of blastocyst development. Similarly, a number of candidate genes in cumulus cells have been identified as potential markers of embryo viability. Relationships between time-lapse parameters and candidate gene expression in cumulus cells have not been investigated, and a combined analysis of these markers has not been attempted in relation to embryo quality.
  
  
  METHODS
  A total of 78 embryos obtained by ICSI from 22 patients were studied by time-lapse and measurement of cumulus cell gene expression of known markers of embryo viability. Time-lapse and cumulus cell gene expression data were assessed in relation to embryo quality on Day 5.
  
  
  METHODS
  All women, aged 32-40 years, underwent ICSI treatment for male infertility. Embryos with annotatable time to pronuclear breakdown (tPNB), division to two cells (t2C), three cells (t3C), four cells (t4C) and five cells (t5C) were included in the study. Expression levels of 27 candidate genes for embryo viability were measured in 78 associated cumulus cell masses using quantitative real-time PCR.
  
  
  RESULTS
  Cumulus cell expression of 11 candidate genes involved in energy metabolism (ATPase, H+ transporting, lysosomal 70 kDa, V1 subunit A (ATP6V1A), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1, 7.5 kDa (NDUFA1), lactate dehydrogenase A (LDHA), phosphofructokinase platelet (PFKP) and solute carrier family 2 member 4 (SLC2A4), mitochondrial biogenesis (DNA directed RNA polymerase, mitochondrial (POLRMT) and transcription factor A, mitochondrial (TFAM), signalling (prostaglandin-endoperoxide synthase 2), steroidogenesis (cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) and cell stress (heat shock 70 kDa protein 5 (HSPA5) and peroxiredoxin 3 (PRDX3)) correlated to time-lapse parameters of the developing embryo, largely for t3C onwards (all P < 0.05). Expression of ATP synthase, H+ transporting, mitochondrial Fo complex, subunit E (ATP51), HSPA5, PFKP, PRDX3 and versican (VCAN) and the parameter t4C were also related to embryo quality on Day 5 (all P < 0.05). Ordinal logistic regression, where gene expression and time-lapse parameters were combined, did not identify any significant predictors of embryo quality on Day 5.
  
  
  CONCLUSIONS
  Data are from a preliminary study, limited by a small sample size and using more than one ovarian stimulation protocol. A possible limitation is that each follicle was treated as an independent observation, although a considerable fraction of embryos were from the same patient.
  
  
  CONCLUSIONS
  Results presented in this study suggest that some of the variation of time-lapse parameters may be related to cumulus cell gene expression and thus the ovarian microenvironment in which the oocyte developed. Although the current study did not identify significant predictors of embryo quality on Day 5, investigation in a larger cohort may determine whether cumulus cell gene expression and time-lapse parameters can be combined to predict embryo quality.
  
  
  BACKGROUND
  Funding was provided by Fertility Associates Ltd, the Auckland Medical Research Foundation and the University of Auckland. J.C.P. has a 0.5% shareholding in Fertility Associates. All other authors of this manuscript have nothing to declare and no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1023, New Zealand.
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Hammond
authors.affiliation: Fertility Associates, Greenlane, Auckland 1051, New Zealand.
authors.forename: Bert
authors.initials: B
authors.lastname: Stewart
authors.affiliation: Fertility Associates, Greenlane, Auckland 1051, New Zealand.
authors.forename: John C
authors.initials: JC
authors.lastname: Peek
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1023, New Zealand.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Shelling
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1023, New Zealand Fertility Associates, Greenlane, Auckland 1051, New Zealand l.cree@auckland.ac.nz.
authors.forename: Lynsey M
authors.initials: LM
authors.lastname: Cree
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D054885:Cumulus Cells / Q000378:metabolism*; D047108:Embryonic Development / Q000235:genetics*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005260:Female; D015870:Gene Expression; D006801:Humans; D007248:Infertility, Male / Q000628:therapy*; D008297:Male; D020554:Sperm Injections, Intracytoplasmic; D059008:Time-Lapse Imaging
chemical_list: D015415:Biomarkers; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human
keywords: blastocyst; cumulus cells; gene expression; oocyte quality; time-lapse