title: Storage time does not modify the gene expression profile of cryopreserved human metaphase II oocytes.
abstract: OBJECTIVE
  Does storage time have any impact on the transcriptome of slowly frozen cryopreserved human metaphase II (MII) oocytes?
  
  
  CONCLUSIONS
  The length of cryostorage has no effect on the gene expression profile of human MII oocytes.
  
  
  BACKGROUND
  Oocyte cryopreservation is a widely used technique in IVF for storage of surplus oocytes, as well as for fertility preservation (i.e. women undergoing gonadotoxic therapies) and oocyte donation programs. Although cryopreservation has negative impacts on oocyte physiology and it is associated with decrease of transcripts, no experimental data about the effect of storage time on the oocyte molecular profile are available to date.
  
  
  METHODS
  This study included 27 women, ≤38 years aged, without any ovarian pathology, undergoing IVF treatment. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed.
  
  
  METHODS
  Pools of ≈10 oocytes for each group were prepared. Total RNA was extracted from each pool, amplified, labeled and hybridized on oligonucleotide microarrays. Analyses were performed by R software using the limma package.
  
  
  RESULTS
  Comparison of gene expression profiles between surviving thawed oocytes after 3 and 6 years of storage in liquid nitrogen found no differently expressed genes. The expression profiles of cryopreserved MII oocytes significantly differed from those of non-cryopreserved oocytes in 107 probe sets corresponding to 73 down-regulated and 29 up-regulated unique transcripts. Gene Ontology analysis by DAVID bioinformatics resource disclosed that cryopreservation deregulates genes involved in oocyte function and early embryo development, such as chromosome organization, RNA splicing and processing, cell cycle, cellular response to DNA damage and to stress, DNA repair, calcium ion binding, malate dehydrogenase activity and mitochondrial activity. Among the probes significantly up-regulated in cryopreserved oocytes, two corresponded to ovary-specific expressed large intergenic noncoding (linc)RNAs.
  
  
  CONCLUSIONS
  Data validation in a larger cohort of samples would be beneficial, although we applied stringent criteria for gene selection (fold-change >3 or <1/3 and FDR < 0.1). Further research should be undertaken to verify experimentally that the length of cryostorage has no effect on gene expression profile of vitrified/warmed MII oocytes, as well as to include in analyses 'older' frozen oocytes.
  
  
  CONCLUSIONS
  Confirmation that the length of storage does not alter the gene expression profile of frozen oocytes is noteworthy for the safety issue of long-term oocyte banking, i.e. fertility preservation, gamete donation.
  
  
  BACKGROUND
  This study was supported by a grant of the Italian Ministry of Health (CCM 2012) and by Ferring Pharmaceutical company. The authors have no conflicts of interest to declare.
authors.affiliation: U.O.S. Physiopathology of Human Reproduction, IRCCS A.O.U. San Martino-IST, Genoa, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Stigliani
authors.affiliation: CNRS UMR7243, Université Paris-Dauphine, Paris, France.
authors.forename: Stefano
authors.initials: S
authors.lastname: Moretti
authors.affiliation: U.O.S. Physiopathology of Human Reproduction, IRCCS A.O.U. San Martino-IST, Genoa, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Anserini
authors.affiliation: U.O.S. Physiopathology of Human Reproduction, IRCCS A.O.U. San Martino-IST, Genoa, Italy.
authors.forename: Ida
authors.initials: I
authors.lastname: Casciano
authors.affiliation: U.O.S. Physiopathology of Human Reproduction, IRCCS A.O.U. San Martino-IST, Genoa, Italy University of Genoa, Genoa, Italy.
authors.forename: Pier Luigi
authors.initials: PL
authors.lastname: Venturini
authors.affiliation: U.O.S. Physiopathology of Human Reproduction, IRCCS A.O.U. San Martino-IST, Genoa, Italy paola.scaruffi@hsanmartino.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Scaruffi
mesh_terms: D000328:Adult; D015925:Cryopreservation / Q000592:standards*; D005260:Female; D005307:Fertilization in Vitro / Q000592:standards*; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D008677:Metaphase / Q000502:physiology*; D009865:Oocytes / Q000502:physiology*; D013997:Time Factors
keywords: gene expression; human MII oocyte; microarray; slow freezing cryopreservation; storage time