127Z.P. Nagy et al. (eds.), Practical Manual of In Vitro Fertilization: Advanced Methods and Novel Devices,DOI 10.1007/978...
128 D. WellsCCs are known to metabolize the bulk of glucose con-sumed by the COC, supplying metabolic intermediates likepy...
12915 Cumulus Cell Gene Expression in Assessment of Oocyte Quality(involved in gluconeogenesis) and downregulation of NFIB...
130 D. Wellsis generally considered the most accurate method forquantifying gene expression but only allows analysis ofsma...
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Practical manual of in vitro fertilization


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Practical manual of in vitro fertilization

  1. 1. 127Z.P. Nagy et al. (eds.), Practical Manual of In Vitro Fertilization: Advanced Methods and Novel Devices,DOI 10.1007/978-1-4419-1780-5_15, © Springer Science+Business Media, LLC 2012AbstractCumulus cells originate from granulosa cells and surround oocytes from the time offollicular antrum formation until after fertilization. The cumulus cells have essential func-tions in the ovary, mediating transmission of endocrine signals and supporting oocytegrowth and maturation. The relationship between oocytes and their associated cumuluscells is extremely intimate. Cytoplasmic projections extend from the innermost layer ofcumulus cells, penetrating the zona pellucida and forming gap junctions at the oocyte sur-face. This allows for direct exchange of macromolecules, a bidirectional communicationessential for the production of competent oocytes. The fact that cumulus cells are so closelyassociated with the oocyte, and share the same microenvironment within the ovary, has ledto suggestions that information concerning oocyte quality might be obtained by analyzingthem. Several studies focused on cumulus cell gene expression have now been published,indicating that a noninvasive assessment of oocyte potential, based upon analysis of the sur-rounding cumulus cells, may indeed be possible.KeywordsHuman • Oocyte • Cumulus cells • Transcriptome • Gene expressionCumulus Cell Gene Expressionin Assessment of Oocyte QualityDagan Wells15D. Wells, PhD, FRCPath (*)Nuffield Department of Obstetrics and Gynaecology,John Radcliffe Hospital, University of Oxford,Level 3, Women’s Centre, Oxford OX3 9DU, UKe-mail: Dagan.Wells@obs-gyn.ox.ac.ukCommentaryCumulus Cell Biologyand the Cumulus–Oocyte RelationshipThe mature cumulus–oocyte complex (COC) is composed ofthe secondary oocyte, arrested at metaphase II followingextrusion of the first polar body (PB), and surrounding cumu-lus cells (CCs). A unique characteristic of CCs is the pres-ence of highly specialized cytoplasmic projections thatpierce through the zona pellucida and form gap junctions attheir tips with the oocyte [1]. This intimate association allowsCCs to fulfill vital roles, supporting the maturation of theoocyte and relaying endocrine and other environmentalsignals.Cumulus cells (CCs) originate from granulosa cells, theprimary type of somatic cell in the follicle. Initiation of GCdifferentiation occurs upon follicular antrum formation, cor-responding approximately to the end of the oocyte growthphase. In humans and other mammals, two anatomically andfunctionally distinct lineages are generated—mural GCs thatline the wall of the follicle with primarily a steroidogenicrole and the CCs, which encircle the oocyte [2, 3].Cumulus cells undergo extensive proliferation prior toLH surge, and following the preovulatory LH surge, a cas-cade of events is initiated that leads to further proliferationand expansion [4, 5]. The competence to undergo expansionis a unique characteristic of CC differentiation [6], which hasbeen shown to be critical for normal oocyte development,ovulation, and fertilization [7–9].
  2. 2. 128 D. WellsCCs are known to metabolize the bulk of glucose con-sumed by the COC, supplying metabolic intermediates likepyruvate, mainly via glycolysis, to the oocyte (for a detailedreview on role of glucose metabolism in the oocyte, refer toSutton-McDowall [10]). Other substrates of low molecularweight such as amino acids and nucleotides are passed to thegrowing oocyte for its own synthesis of macromolecules aswell as ribosomal and messenger RNA from the GCs/CCs.The nutritional support, trafficking of macromolecules,and dissemination of endocrine signals that this systemallows may be particularly important for oocytes due to theavascular nature of the granulosa layer (reviewed by Johnsonand Albertini [1, 11]). This communication is so crucial thatgenetic deletion of the oocyte specific gap junctional sub-unit, connexin-37, leads to female sterility in mice, resultingfrom a lack of mature follicles, failure to ovulate, and devel-opment of numerous inappropriate corpora lutea.The role of CCs in supporting in vivo oocyte develop-ment as well as IVM [12, 13] has led increasing numbers ofresearchers to study these cells. Not only do investigations inthis area promise to shed light on the biology of the follicleand the mechanisms promoting oocyte competence, but it isalso possible that new biomarkers of oocyte potential may beidentified.One important component of the follicular environment,relevant to oocyte viability, is oxidative stress. Antioxidantsproduced by CCs, such as superoxide dismutases (SOD), arepostulated to protect the oocyte from damage caused by reac-tive oxygen species. SOD levels in CCs have been noted todecrease with advancing female age, and higher SOD activi-ties were associated with successful outcomes in assistedreproduction techniques [14]. Glutathione S transferases areanother class of enzymes known to protect cells from reac-tive oxygen species. In a study by Ito et al. [15], GSTT1(glutathione S transferase theta 1) was shown to be a goodindicator of age-related infertility. Not only does this dataemphasize the influence of oxidative stress on oocyte viabil-ity, it also suggests that SOD and GSTT1 might serve aspotential biomarkers of prognostic significance.Other studies have indicated that apoptosis rates are ele-vated for CCs associated with morphologically abnormaloocytes [16]. An increase in CC apoptosis has also beenassociated with immaturity of human oocytes, impairedfertilization [17], suboptimal blastocyst development [18],and poor IVF outcomes [19, 20]. It may be that abnormal/poor-quality oocytes induce apoptosis in the associatedCCs. Alternatively, CCs with high levels of apoptosis, per-haps symptomatic of a suboptimal follicular environment,may lead to impaired oocyte development. Whatever theexplanation, these observations highlight the interdepen-dence of the oocyte and its CCs and suggest that certainelements of CC biology may serve as indicators of oocyteviability.Gene Expression Studies of HumanCumulus Cells: Identifying New Biomarkersof Oocyte QualityWith appropriate methods, it may be possible to detect anddecode molecular alterations in the CCs associated with dif-ferences in oocyte viability [21]. For example, patterns ofgene expression reflect processes occurring within a cell at agiven moment in time, including the cell’s responses to envi-ronmental challenges. Thus, patterns of gene activity in CCsmay reveal much concerning the conditions within the folli-cle during the final stages of oocyte maturation. Severalgroups have utilized emerging transcriptomic techniques togain a better understanding of follicle biology and to try andidentify novel biomarkers of oocyte competence [22–29](see Glossary).CCs are constantly responding to the intrafollicular envi-ronment to ensure optimal oocyte development, adjustinggene expression in order to maximize oocyte support andminimize damage caused by extrinsic factors (e.g., reactiveoxygen species). An ongoing study of the CC transcriptomein our laboratory has indicated that the follicular microenvi-ronment might even play a role in the origin of oocyte mei-otic chromosome abnormality, one of the main causes ofoocyte incompetence. The study revealed that cumulus cellsassociated with aneuploid oocytes have characteristic devia-tions in their gene expression profile [29] (Fragouli, Wells,and Patrizio, unpublished data).Abnormally expressed genesinclude several involved in pathways related to cellular stress(e.g., hypoxia, nutritional deprivation), suggesting an asso-ciation between aneuploidy and suboptimal environment.Some genes involved in hormonal response also displayedabnormal expression, potentially providing a link betweenthe increased frequency of aneuploidy and the altered hor-mone levels seen with advancing age. Furthermore, a num-ber of genes with roles in apoptotic pathways showeddistorted expression levels, in keeping with previous studiessuggesting an association between CC proliferation and/orapoptosis and poor IVF outcomes [17–19, 30].Gasca and colleagues [23] attempted to identify potentialregulators and marker genes involved in oocyte maturationby screening human oocytes and CCs using microarrays[23]. Their study identified a number of potentially signifi-cant genes involved in processes such as cell cycle check-points and DNA repair, including BARD1, RBL2, RBBP7,BUB3 and BUB1B. Appropriate expression of these genesmay have relevance to oocyte quality, although this remainsto be conclusively proven.Another microarray study, conducted by Assou et al.reported patterns of CC expression associated with embryomorphology and pregnancy outcome. These included upreg-ulation of BCL2L11 (involved in apoptosis) and PCK1
  3. 3. 12915 Cumulus Cell Gene Expression in Assessment of Oocyte Quality(involved in gluconeogenesis) and downregulation of NFIB(a transcription factor). The researchers proposed that thesethree genes might be useful biomarkers for the prediction ofpregnancy [28].Feuerstein et al. [25] assessed the expression of six geneschosen because their expression is induced by the LH peak(STAR, COX2 and AREG) or because of known roles inoocyte lipid metabolism (SCD1 and SCD5) or in gap junc-tions (Cx43). With the exception of Cx43, all of the genesdisplayed increased expression in CCs after resumption ofmeiosis. Nuclear maturation of the oocyte was associatedwith increased expression of STAR, COX2, AREG, SCD1and SCD5 in CCs. Interestingly, mRNA transcript levels ofthese genes were lower and distributed over a narrower rangein CCs enclosing oocytes, achieving blastocyst developmentat day 5/6 than in CCs enclosing oocytes unable to developbeyond the embryo stage.Further potential markers of oocyte competence identifiedby gene expression studies include PTGS2 (prostaglandin-endoperoxide synthase; cyclooxygenase), HAS2 (hyaluronicacid synthase 2), and GREM1 (gremlin 1) [31]. CCs associ-ated with oocytes that produced high-quality cleavage-stageembryos were found to have greater numbers of transcriptscompared to CCs from oocytes that produced poor-qualityembryos (expression of PTGS2 and HAS2 was sixfold higher;GREM1 was 15-fold higher). Complementary results wereobtained for GREM1 and HAS2 by Cillo and colleagues,suggesting that the measurement of transcripts from thesegenes in CCs might complement morphological evaluationand provide a useful tool for selecting oocytes with greaterchances of fertilization and development in vitro [24].Altered expression of several genes has been correlatedwith early cleavage postfertilization, a feature generally con-sidered to be a positive indicator of IVF outcome. The func-tion of these genes suggest a role for hypoxic conditions(CXCR4, GPX3, DVL3, HSPB1) or delayed oocyte matura-tion (CCND2, TRIM28, DHCR7, CTNND1) in non-earlycleavage embryos [26]. Not only do these results shed light onaspects of follicle biology that might predispose to late/earlycleavage, but as with the studies discussed above, they alsoprovide a set of markers that might assist oocyte selection.A further set of markers of oocyte/follicle competencewere reported by Hamel et al. [27] who performed experi-ments with the aim of identifying cumulus/granulosa cellgenes specifically expressed in follicles that produced a preg-nancy. They created a DNA microarray composed of cumu-lus/granulosa cell expressed sequence tags from subtractedlibraries (cumulus/granulosa cells from women who becamepregnant versus cells from those who did not). Alteredexpression of the CDC42, 3bHSD, SERPINE2, FDX1 andCYPA191 genes was significantly associated with competentfollicles that resulted in pregnancies. These correlations wereconfirmed with quantitative PCR analyses [27].ConclusionThe various studies seeking to characterize the CC transcrip-tome have yielded a wealth of novel data, including a detailedcatalog of the genes expressed in CCs. Importantly, a num-ber of genes displaying differential activity, apparentlyrelated to oocyte competence, have been identified.Quantification of the mRNA transcripts from such genes, orthe proteins they produce, may provide new insights intooocyte (and embryo) competence, not possible using con-ventional techniques. Clinical trials aimed at assessing thepotential of CC-based strategies of oocyte quality assess-ment are now underway. In the near future, diagnosticapproaches based upon analyses of cumulus cells may revo-lutionize the way in which oocytes and embryos are selectedfor uterine transfer during IVF treatments, potentially lead-ing to increases in fertilization, implantation, and clinicalpregnancy rates. If markers of aneuploidy can also be identi-fied, as initial data suggests, a reduction in the rates of mis-carriage and aneuploid syndromes (e.g., Down syndrome)are also anticipated. A noninvasive preconception test foraneuploidy would overcome some of the most importanttechnical and ethical difficulties facing preimplantationgenetic screening [29].Acknowledgment Dagan Wells is funded by NIHR BiomedicalResearch Centre Program.GlossaryDownregulated/underexpressed Cases where fewer mRNAtranscripts are found. Gene expression is reduced (i.e., thegene is less active).Gene expression A complete set of all of the genes (i.e.,the entire genome) is present in all cells. However, onlya fraction of these genes are active in a cell at any givenmoment. Genes which are being actively transcribed,producing mRNA and ultimately proteins, are said to be“expressed.”Microarray A method for simultaneously quantifyingthe number of transcripts from large numbers of genes(typically thousands or tens of thousands of genes simul-taneously assessed).mRNA transcripts The molecules that serve as interme-diates between genes (made of deoxyribonucleic acid,DNA) and the proteins they produce. The DNA sequenceof a gene is transcribed into a messenger RNA (ribonu-cleic acid) copy, which is subsequently translated into apolypeptide.Real-time PCR A method of quantifying the number ofmRNA transcripts from individual genes. Real-time PCR
  4. 4. 130 D. Wellsis generally considered the most accurate method forquantifying gene expression but only allows analysis ofsmall numbers of genes at a time.Transcriptome The sum total of all mRNA transcriptsfound within an individual cell or tissue. The charac-terization of the transcriptome reveals all of the genesexpressed (i.e., active).Upregulated/overexpressed When two different samplesare compared, some genes may be found to have dif-ferences in the number of mRNA transcripts. If a sam-ple contains a greater number of mRNA transcripts thanexpected, the gene is said to be upregulated or overex-pressed (i.e., it is more active).References1. Albertini DF, Combelles CM, Benecchi E, Carabatsos MJ. Cellularbasis for paracrine regulation of ovarian follicle development.Reproduction. 2001;121:647–53.2. Chian RC, Lim JH, Tan SL. State of the art in vitro oocyte matura-tion. Curr Opin Obstet Gynecol. 2004;16:211–9.3. Gilchrist RB, Lane M, Thompson JG. Oocyte-secreted factors:regulators of cumulus cell function and oocyte quality. Hum ReprodUpdate. 2008;14:159–77.4. Scherzer J, Ghuman SPS, Pope M, Routly JE, Walter I, Smith RF,Dobson H. Follicle and oocyte morphology in ewes after treatmentwith insulin in the late follicular phase. Theriogenology. 2009;71:817–28.5. Lin YH, Hwang JL, Seow KM, Huang LW, Chen HJ, Tzeng CR.Effects of growth factors and granulosa cell co-culture on in-vitromaturation of oocytes. Reprod Biomed Online. 2009;19:165–70.6. Diaz FJ, O’Brien MJ, Wigglesworth K, Eppig JJ. The pre-granulosacell to cumulus cell transition in the mouse ovary: development ofcompetence to undergo expansion. Dev Biol. 2006;299:91–104.7. Elvin JA, Clark AT, Wang P, Wolfman NM, Matzuk MM. Paracrineactions of growth differentiation factor-9 in the mammalian ovary.Mol Endocrinol. 1999;13:1035–48.8. Chang H, Brown CW, Matzuk MM. Genetic analyses of the mam-malian transforming growth factor beta superfamily. Endocr Rev.2002;23:787–823.9. 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Identification of differentially expressed markers inhuman follicular cells associated with competent oocytes. HumReprod. 2008;23:1118–27.28. Assou S, Haouzi D, Mahmoud K, Aouacheria A, Guillemin Y,Pantesco V, Rème T, Dechaud H, De Vos J, Hamamah S. A non-invasive test for assessing embryo potential by gene expressionprofiles of human cumulus cells: a proof of concept study. MolHum Reprod. 2008;14:711–9.29. Wells D, Fragouli E, Bianchi V, Borini A, Patrizio P. Identificationof novel non-invasive biomarkers of oocyte aneuploidy. Fertil Steril.2008;90 Suppl 1:S35.30. Gregory L. Ovarian markers of implantation potential in assistedreproduction. Hum Reprod. 1998;4:117–32.31. McKenzie LJ, Pangas SA, Carson SA, Kovanci E, Cisneros P,Buster JE, Amato P, Matzuk MM. Human cumulus cells granulosacell gene expression: a predictor of fertilisation and embryo selec-tion in women undergoing IVF. Hum Reprod. 2004;19:2869–74.