Microarray analysis in gynaecology and its findings: a systematic review

Reproductive BioMedicine Online (2011) 22, 569– 582

www.sciencedirect.com www.rbmonline.com

REVIEW

Microarray analysis in gynaecology and its findings: a systematic review I Verginadis a, IP Kosmas a,c,*, Y Simos a, A Velalopoulou a, I Korkontzelos c, A Anogeianaki b, K Charalampopoulos d a Department of Physiology, Medical School, University of Ioannina, 45110 Ioannina, Greece; b Department of Physiology, University of Thessaloniki, Medical School, Greece; c Department of Obstetrics and Gynecology, Ioannina State General Hospital ‘‘G. Chatzikosta,’’ Ioannina, Greece; d Department of Physiology, Dimokrition University of Thrace, Medical School, Greece

* Corresponding author. E-mail address: [email protected] (IP Kosmas). Dr Verginadis Ioannis is a biologist and biotechnologist at the Laboratory of Physiology, Faculty of Medicine, University of Ioannina, Greece, where he earned his degree in Applied Biology and Biotechnology in 2006 and his PhD in 2010. His main research interests include cancer treatments with organometallic compounds and oocyte cryopreservation in cancer patients.

Abstract Microarray technology is a promising method for investigating gynaecological benign pathology. This systematic review

examined various parameters of the design of these studies, the methods used and the gene outcome in these diseases. Electronic searches were performed in Medline (up to April 2009). An overall representation of important genes for each disease detected was performed. The results showed genes were up-regulated or down-regulated. However, studies suffer from several flaws in their design, the sample size employed and the reporting method. In conclusion, a significant amount of work has been performed on benign gynaecological diseases using microarray technology. New trial designs need to be employed that incorporate microarray reporting standards. New research directions should evolve based on these results. RBMOnline ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: benign gynaecological diseases, gene expression, microarray technology

Introduction Benign gynaecological diseases are characterized by a variety of factors. At different stages of disease, different factors are involved, with the hormonal factor playing the major role. Until now, pharmaceutical interventions have been based on hormonal regulation. On the other hand, microarray

technology has been incorporated into the study of these benign conditions, with controversial results. Different sets of genes and molecules have emerged while attempts have been made to describe the novel pathways involved and to develop potentially new therapies based on these findings. The majority of microarray studies have been considered as experimental, but there is a small number that have been

1472-6483/$ - see front matter ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2011.03.014

570 performed prospectively. Studies have used different animal types with different methodologies and some studies have evaluated different molecules within a tissue type for a particular disease. Clinical outcomes are not mentioned in most of the studies and the choice of microarray technology also varies between studies. These discrepancies between studies and also the small sample sizes do not allow for safe results. Although new pathways have been proposed in benign gynaecological diseases, at present there is no clear picture of the genes and pathways involved. The purpose of this study is to systematically evaluate microarray studies performed for benign gynaecological conditions and infertility and find common genes that are up-regulated or down-regulated.

Materials and methods Identification and eligibility of relevant studies Medline searches (up to April 2009) were performed using various combinations of terms: microarray analysis, cDNA or oligonucleotide microarray, tissue microarray, RNA microarray, single-nucleotide polymorphism microarray, Affymetrix, molecular profiling, prognosis, gynaecology and infertility. The search was complemented with perusal of the bibliographies of retrieved papers and review articles. This review included studies that evaluated the use of microarrays in benign gynaecological diseases like leiomyomata and endometriosis, infertility and IVF, even male factor studies in the context of infertility. Gynaecological cancer studies with microarrays were excluded. The number of tested samples was not an exclusion criterion, nor was the number of tested genes. All studies including human, animals and cell cultures were included.

Data extraction For each study, whenever possible, information was obtained on authors, journal, year of publication, country and years of study, enrolment and type of study design, disease model (animal or human), number of tested samples, tissue, disease and grade, molecules researched and pathways involved and tissue biopsy. Also, age of the subjects (mean) and the clinical outcome were noted. For microarrays, the type of microarray technology for each disease and the number and type of probes was recorded. Also, information on the use of laser microdissection, sample processing, disease distribution, and biopsy characteristics and tissue size was collected. Data extraction was performed independently by two investigators and conflicts were resolved after discussion.

Statistical analysis Frequencies of all parameters were calculated and analysed. Statistical analyses were performed in using the Statistical Package for Social Sciences version 12.0 (SPSS, Chicago, IL, USA).

I Verginadis et al.

Results Baseline data A total of 208 abstracts were retrieved and further screened. Cancer studies were excluded because the complexity of the issue was beyond the scope of this study. From these, all articles that used microarray technology to compare patterns of gene expression between experimental groups and controls were retrieved for full review (n = 201). A further two articles were excluded because they were reviews. Out of the 199 included studies (the full reference list is available as Supplementary material online only), 85 (42.71%) were performed in USA, 14 (7.04%) in France, 12 in Korea (6.03%), 11 in Japan (5.53%), nine in China and Australia (4.52%), eight in Taiwan (4.02%), seven in Germany (3.52%), six in Netherlands (3.02%), five in Spain and UK (2.51%), four in Brazil (2.01%), three in Belgium, Italy, Israel, Finland and Sweden (1.51%), two in Canada and Austria (1.01%) and one in Denmark, Czech Republic, Switzerland, New Zealand and India (0.5%). Of the total 199 studies, 152 (76.38%) considered themselves experimental, 28 (14.07%) prospective, nine (4.52%) retrospective, three (1.51%) as pilot studies, two (1.01%) as case–control studies, one (0.50%) as a case report and one (0.50%) as a descriptive study and three as other descriptions: one double-blind (0.50%), one explorative (0.50%) and one longitudinal (0.50%). The percentages of the types of study initiated in each country can be seen in Figure 1. The majority of studies used human tissue as material to be examined (n = 166; 83.42%), while 28 (14.07%) used mouse/rat, three studies used monkey and one used the bovine model. One study did not mention the study model. The percentages of the animal models used for each experimental model can be seen in Figure 2. Regarding journal of publication, 56 (28.14%) were published in Human Reproduction, 98 (49.25%) in Fertility and Sterility (either as papers or abstracts) and 45 (22.61%) as European Society of Human Reproduction and Embryology abstracts. The majority of prospective studies were published in Fertility and Sterility while the majority of studies published in Human Reproduction considered themselves experimental. The distribution of type of study per journal is seen in Figure 3. Also, 116 (58.29%) studies did not mention selection of cases while the remainder (n = 83) did. All microarray technology studies were included. The majority used DNA microarray (n = 75; 37.69%), 60 (30.15%) used cDNA microarrays, 19 (9.55%) used oligonucleotide microarrays, 16 used tissue microarrays, two used RNA microarray and cRNA microarray, one used chromosome microarrays, one study used protein chip microarray and one study used single-nucleotide polymorphism microarray. Twenty-four studies did not mention the type of microarray used. Nine studies used radioactive probes and 24 used fluorescent probes. Sixty-three (31.66%) studies used amplification, and 136 (68.34%) studies did not report on amplification. Various controls were used, with 39 studies using paired normal tissue as a control. The number of probes used varied from four to 370,000. The majority of

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Figure 1

Distribution of the type of study according to country of enrolment.

Figure 2

Distribution of experimental model used according to type of study.

Figure 3 Distribution of journal of publication according to type of study. ESHRE = European Society of Human Reproduction and Embryology.

studies (n = 145; 72.86%) did not mention tissue processing while four studies used formalin-fixed paraffin-embedded tissue, three studies used paraffin-embedded tissue, 31 used frozen tissue, three used fresh–frozen specimens

and 13 used fresh material. Only nine (4.52%) studies used laser capture microdissection. The sample sizes examined ranged from one to 300. Only 16 studies had a sample size over 100. Disease distribution

572

Table 1

Studies in which changes in gene expression were found according to disease.

Publication

Disease

Type of study

Up-regulated or over-expressed genes

Down-regulated or under-expressed genes

Presentation type

Spiess et al. (2007)

Azoospermia

Experimental

ND

OA

Yasuo and Kitaya (2009)

Cervical carcinomaa

Experimental

PTGS2, TCEA2, LEFTY1, NOVA1, CCL21 (by 17b-oestradiol), CD44, SLC6A11, MMP26, ADAM17, TNC (by progesterone), TNFRSF4, ADAM17, WISP2, SPN, DES, FOS, TWIST1, FOLR1 (by oestradiol + progesterone)

OA

Hamamah et al. (2008) Nguyen et al. (2009)

COS Cryptorchidism

Experimental Prospective study

CPA3, CALB2, LDLR, CLEC2B, TPSAB1, SAMD9L, DPT, RBP1, CTSC, ADAMTS5, TNFSF13B, FAM129A CYB5R4, CXCL12, DOM3Z, SOD2, CD54, ICAM1, CXCL1, A2MP (by oestradiol), CEACAM1, CXCL9, VCAM1, VCAN, PTGES, AQP2, KDR, IL1RL1, MMP14, IL15, SLC3A1, COL1A2, GJA4, IFNGR1 (by progesterone), EGFR, VCAM1, FGF2, CXCL9, PDGFRB, FGA, IL15, CXCL9, MAP3K4, VWF, FGF13, NCR2, ACTGP4, WAS, IL15RA, NPPA CYP11A1, HSD3B2, CYB5B, and CYP1B1, out of 118 TNPAIP3, UBN1, SCAMP2, HLA-DRB3

P OA

Rozovski et al. (2007)

Down syndrome

Experimental

Baidya et al. (2008) Chen et al. (2002a)

Endometriosis Endometriosis

Experimental Pilot study

ND EEF1A1, CDC27, EIF4G2, HSPCD35, PRM1, TAF10, ATF2, CUL3, AGP-1, SUI1, FKBP3, PMS1, ODF1, NUP155, VDAC3, out of 38 CALCA, MAT2A, SSR1, BANF1, GLUL, PABPN1, BAT1, RBM5, NSDHL, KIAA0152, COPS6, ARPC3, IMPDH2, XPO7 SPP1, CD86, CCR1, out of 27 ND

Chen et al. (2002b)

Endometriosis

Experimental

ND

P

Chen et al. (2007)

Endometriosis

82 (not specified)

P

Cook et al. (2005)

Endometriosis

ND

IGF-BP1, MMPs1, 3, 10 and 12

P

Eyster et al. (2007)

Endometriosis

717, for both up- and down-regulation (not specified)

Endometriosis

Flores et al. (2006)

Endometriosis

Experimental

717, for both up- and down-regulation (not specified) Insulin-like growth factor binding protein 5, tyrosine 3-monooxygenase, tryptophan 5-monooxygenase activation protein and epsilon polypeptide 45 (not specified)

OA

Ezeh et al. (2003)

Prospective study Prospective study Prospective study Experimental

OA

Giudice et al. (2003)

Endometriosis

Experimental

115 (not specified)

P

MEST, SNF1LK, LOX, TRIM33, KIAA0179, PSCD1, FAM50A, BTG3, GPR137B, PLD3, APP, EIF4EBP2, CDC34, ITM2B, HMGN1, out of 51 12 (not specified) Bcl-2, cyclin B1, VCAM-1, BMP-4, TGFb receptor, GSTA2, and CD36, out of 177 EGF, IGF-II, Thy-1 antigen, integrin b 4 binding protein, ras-related oncoprotein, death-domain related genes, EGF, TGFb, neuropeptide Y receptor, NK cells-stimulated proliferative factor, keratin 18, out of 214 145 (not specified)

Stefin B, heat shock 70 kDa protein 1B and enolase 1

P P

P

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123 (SRPRB, P4HA1, LOXL1, IL2RG, LRP5, MBP, TNF, MAN2A2, PDGFD) 91 (not specified)

OA

(continued)

Publication

Disease

Type of study

Up-regulated or over-expressed genes

Down-regulated or under-expressed genes

Presentation type

Kao et al. (2001)

Endometriosis

Prospective study

ND

P

Lee et al. (2003)

Endometriosis

Experimental

Pregnancy-associated endometrial a2 globulin/ glycodelin, insulin-like growth factor/IGF-2, IGF binding proteins/BP-1, 2, and 4, plasminogen activator inhibitor, urokinase receptor, tissue inhibitor of metalloproteinases/TIMP-3, prostaglandin/PG-E receptor, integrin a, osteopontin, neural cell adhesion molecule/N-CAM, intercellular adhesion molecule/ICAM-2, secretory mucin/MUC-6, zona-pellucida-binding protein, chloride channel, calcium channel, out of 24 Glyceraldehyde-3-phosphate dehydrogenase, eukaryotic translation initiation factor, serine proteinase inhibitor

P

Matsuzaki et al. (2005)

Endometriosis

Print et al. (2004)

Endometriosis

Prospective study Experimental

Gap junction protein, ARP2 homologue, EIF2b, tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase, CGI-44 protein, calumenin Does not refer to genes, out of 84

Sherwin et al. (2008) Yanaihara et al. (2005)

Endometriosis Endometriosis

Experimental Pilot study

Haouzi et al. (2009) Assou et al. (2006)

ICSI IVF

Experimental Experimental

Bersinger et al. (2008)

IVF

Experimental

RON, uPAR, 14-3-3 protein eta, SOS, KSR, PI3K p85, out of 78 ADP ribosylation factor-like 2, collagen IV a6, CXCreceptor-4, cystatin C, EST DKFZP56600424, endoglin, fibronectin-1, glutathione S transferase p1, glyceraldehyde-3-phosphate dehydrogenase, insulin-like growth factor binding protein-2, interferon c receptor-2, interleukin 1 receptor like1, mitogen-activated protein kinase-12, matrix metalloproteinase-2, matrix metalloproteinase-3, out of 23 91 (not specified) WFDC2, HOX5, matrilysin, HOX1, proenkephalin A, ID2, serine proteinase inhibitor, CDC28 protein kinase 2, microsomal glutathione S-transferase 2, heat shock 27 kDa protein 1, tuberin, TFIID, MARCKSrelated protein, cellular retinoic acid-binding protein 2, cyclin A1, out of 28 945 (not specified) DAZL, DDX4/VASA, DPPA3/STELLA, CCNB1, CCNB2, CDC2, CDC25A, CDC25B, CDC25C, BUB1, BUB1B/ BUBR1, CENPA, CENPE, CENPH, MAD2L1/MAD2, out of 3152 Somatostatin, PLAP-2, mucin 4, CD163, out of 1875

Microarray analysis in gynaecology

Table 1

OA OA

115 (not specified) ND

OA OA

67 (not specified) 2328 (not specified)

OA OA

Glycodelin, IL-24, CD69, leukaemia inhibitory factor, prolactin receptor, out of 1807

P

(continued on next page)

573

Angiopoetin 1, c-Jun N-terminal kinase 3, Ryanodine receptor-1

574

Table 1

(continued)

Publication

Disease

Type of study

Up-regulated or over-expressed genes

Down-regulated or under-expressed genes

Presentation type

Fragouli et al. (2007) Gasca et al. (2007) Jones et al. (2008)

IVF IVF IVF

Experimental Case report Experimental

185 (not specified) 1214 (not specified) ND

P P OA

Loup et al. (2006)

IVF

Experimental

ND

P

Madar et al. (2008)

IVF

Experimental

ND

P

De Vos et al. (2005) Arslan et al. (2005)

IVF Leiomyomas

Experimental Experimental

Leiomyomas

Experimental

ND ADH1B, DPT, FY, APM2, HBB, FOS, ATF3, KLF4, CYR61, KCNB1, TPSB2, PIAS3, SELE, IGFBP6, EFEMP1, out of 66 Hexokinase 1, fumarate hydratase

P OA

Catherino et al. (2007)

Catherino et al. (2003) Dimitrova et al. (2008) Hoffman et al. (2004)

Leiomyomas Leiomyomas Leiomyomas

Experimental Experimental Experimental

153 (not specified) 1716 (not specified) Homophilic cell adhesion, calcium-independent cell adhesion, neuron adhesion, calcium-dependent cell adhesion, cell–cell signalling, synaptic transmission, nerve ensheathment, signal transduction, adenylate cyclase activation, G-protein signalling, transmembrane receptor protein tyrosine kinase activation, acetylcholine receptor signalling, glutamate signalling pathway, cell surface receptorlinked signal transduction, activation of MAPK activity, positive regulation of interleukin-13 biosynthesis, positive regulation of interleukin-6 biosynthesis, alanyl-tRNA aminoacylation, cyclic nucleotide metabolism, defence response to bacteria SOX2OT, DMBX1/OTX3, TGFB3, BCL2L11, CAS2, HIC2, CDC25A, CCNJ, CCNG2, SOCS7 and CUL5, out of 444 Granulocyte and granulocyte-macrophage colony stimulating factor (GCSF, GM-CSF), IL-1a, IL-2, IL-6, IL-7, IL-8, MCP-1 and MCP-2), group II: IL-6, IL-10 CD24, activin A, TNFSF11, PAPPA, BMP2 MEST, MMP11, TMSNB, CD24, SFRP1, HTR2B, QPRT, GAGEC1, IGF2, PTK7, CSPG2, NEGF2, PEMT, FLJ20550 Phosphofructokinase, aldolase, triosephosphate isomerase, phosphoglycerate kinase, enolase, pyruvate kinase, M-type FOS, ATF-3, ZFP, Jun 197 (not specified) ND

P OA OA

Malik and Catherino (2007) Marsh et al. (2008)

Leiomyomas Leiomyomas

Experimental Experimental

Versican, TGFb3, CYP26A1 19 (not specified)

ND 619 (not specified) GSN, SFRP1, SFRP4, MAP3K5, MST4, GADD45B, TNFSF10, ITGB3, CD59, THBD, SERPINE1, NCAM1, CD44, TYMS, ESR1, out of 226 Dermatopontin 27 (not specified)

OA

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OA OA

(continued)

Publication

Disease

Type of study

Up-regulated or over-expressed genes

Down-regulated or under-expressed genes

Presentation type

Okada et al. (2002)

Leiomyomas

Experimental

Leiomyomas Leiomyomas Leiomyomas

Experimental Experimental Experimental

Vollenhoven et al. (2002) Wang et al. (2003)

Leiomyomas Leiomyomas

Experimental Prospective study

11 (not specified) Glutamate receptor 2, thymidylate syntase, UDPgalactose translocator, hypothetical protein 384D8_2, retinoic acid-binding protein II, insulin-like growth factor II, KIAA0007, PEG3, mesoderm-specific transcript, preproinsulin-like growth factor II, transcription factor hGATA-6, CD97, prepro-a 1(I) collagen, corticotrophin-releasing factor, c-glutamyl hydrolase, out of 23

Wei et al. (2006)

Leiomyomas

Experimental

IGF2, MIB1, AIB1, CD24, SRC, RXR, HMGA2, BCL2, PR, ER, RAR, IGF1Rb, hamartin, EGFR, Ps6p, PDGF, tuberin, GCR

Wei et al. (2005)

Leiomyomas

Experimental

Zaitseva et al. (2008)

Leiomyomas

Experimental

Gasca et al. (2008)

PCOS

Experimental

HMGA2, sex-steroid receptor cofactors, proteins in insulin pathway, CD24 CDH2, CDKN1A, COL1A1, COL1A2, COL4A1, COL4A2, CRABP1, CRABP2, CTNNB1, PYCR2, GSTA4, HOXD4, IGF1, IGF2, IGFBP5, out of 26 RPS11, RPL23, EIF5A, ZNF548, out of 16

Insulin-like growth factor binding protein-5, heparin-binding epidermal growth factor-like growth factor, IL-13 receptor a2, out of 27 ND 43 (not specified) ADH1 a, b, c, tryptase, dermatopontin, thrombospondin, Ig rearranged c chain, chemokine CCL21, mast cell carboxypeptidase A, c-FOS, keratin 19, ALDH1, tenascin-XB, cadherin-13, ATF3, ATP-binding cassette subfamily A, GTPase Cdc42, out of 78 ND Class I alcohol dehydrogenase, chemokine HCC-1, secondary lymphoid tissue chemokine, NF-IL6b, heparinbinding EGF-like growth factor, MAP kinase, extracellular protein, microsomal glutathione transferase, dermatopontin, cytosolic aldehyde dehydrogenase, TNFrelated apoptosis inducing ligand TRAIL, osteoprotegerin, phospholipase D, AHreceptor, complement component C1r, out of 45 IGF2, MIB1, AIB1, CD24, SRC, RXR, HMGA2, BCL2, PR, ER, RAR, IGF1Rb, hamartin, EGFR, Ps6p, PDGF, tuberin, GCR Tuberin and glucocorticoid receptor

P

Peng et al. (2009) Roth et al. (2007) Tsibris et al. (2002)

Interleukin 1 receptor type 1, fibulin-1, fibulin-2, microsomal glutathione S-transferase 1, fumarylacetoacetate hydrolase, orphan G proteincoupled receptor IGF-2, p-AKT 30 (not specified) Dlk, doublecortin, JM27, ionotropic glutamate receptor, apolipoprotein E3, IGF2, semaphorin F homologue, IGFBP5, myelin proteolipid protein 1, MEST/PEG1, thymidylate synthetase, kinesin KIF5B, frizzled-2, CD24 signal transducer, PCP4, out of 67

ADH1, ALDH1A1, APOD, APP, BIRC3, CD44, CD59, HBEGF, EGR1, EMP1, FOS, GATA2, HOXA5, HOXC5, ITGB3, out of 43 ZNF718, inhibitor of IL1R, IL1RN, SLC2A6, ADLICAN/MXRA5, TIMP3, COL6A2, CTHRC1, COL1A1, SFRP1, SFRP4, out of 177

OA OA OA

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Table 1

P OA

OA

OA OA

P

575

(continued on next page)

576

Table 1

(continued)

Publication

Disease

Type of study

Up-regulated or over-expressed genes

Down-regulated or under-expressed genes

Presentation type

Oksjoki et al. (2005)

PCOS

Experimental

PCOS PCOSb

Experimental Experimental

Centlow et al. (2008)

Pre-eclampsia

Chen et al. (2006)

Stress urinary incontinence

Prospective study Experimental

Cell division protein kinase 6, mitoticspecific cyclin G1, CDC27HS protein, CDC37 homologue, PCNA (cyclin), growth factor receptorbound protein, MCL1, PIG7, GADD153, GADD45, UBE2A, plexin A3, trk-T3, COL6A3, entactin, out of 30 TM4SF4, ENTPD3, CTNNA2 MSEn versus PCOScc (388 genes), MSEn versus PCOSp (153 genes) not specified ND

OA

Qiao et al. (2007) Savaris et al. (2008)

notch2, Wnt-5A, Wnt-13, RPS30, IGF-binding protein 5, PDGFRb, TGFb receptor III, retinoic acid receptor b, a1 catenin, hepatoma-derived growth factor, gliaderived neurite-promoting factor, PDGF-associated protein, stromal cell-derived factor 1, IFN-c receptor 2 ND MSEn versus PCOScc (223 genes), MSEn versus PCOSp (94 genes) not specified ND Protease inhibitor 3 – skin-derived, splicing factor – arginine/serine-rich 2, non-metastatic cells 1 protein, FXYD domain containing ion transport regulator 3, creatine kinase –mitochondrial 1, MAD2 mitotic arrest deficient-like 1, deiodinase – iodothyronine type II, minor histocompatibility antigen HA-8, CDC28 protein kinase regulatory subunit 2, zinc finger protein, SMC2 structural maintenance of chromosomes 2-like 1, S100 calcium binding protein A2, Dickkopf homologue 1, collagentype XVII-a1, out of 39

Natriuretic peptide receptor A/guanylate cyclase A, cold-inducible RNA binding protein, ubiquitin-like 3, nischarin, growth arrest-specific 7, S100 calciumbinding protein A13, CD97 antigen, calpain 6, transducin-like enhancer of split 1, zinc finger protein 148, forkhead box O3A, KIAA0537 gene product, ubiquitin specific protease 13, potassium intermediate/small conductance calcium-activated channel-subfamily Nmember 3, prostaglandin E receptor 2, out of 39

P P OA OA

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ICSI = intracytoplasmic sperm injection; ND = not determined; OA = original article; P = poster; PCOS = polycystic ovary syndrome; COS=Controlled Ovarian hyperstimulation. a Uterine specimens were obtained from women with proven fertility undergoing hysterectomy for cervical carcinoma in situ or cervical dysplasia. b MSEn: normal mid-secretory endometrium; PCOScc: polycystic ovary syndrome in subjects treated with clomiphene citrate; PCOSp: polycystic ovary syndrome in subjects treated with progesterone.

Pathways involved and molecules studied.

Publication

Pathways involved

Molecules studied

Spiess et al. (2007) Hamamah et al. (2008) Baidya et al. (2008) Chen et al. (2007)

ND ND SPP1, CD86, CCR1 Oestrogen, gonadotropin releasing hormone analogue

Matsuzaki et al. (2005) Print et al. (2004) Sherwin et al. (2008) Haouzi et al. (2009) Bersinger et al. (2008) Catherino et al. (2007)

MAPK PKCa, RYR3 ND Mapk, phospholipids as signalling intermediaries pathway, toll-like receptor signalling pathway, focal adhesion signalling pathway, Wnt signalling pathway, Jak-STAT signalling pathway, CXCR4 signalling pathway, EGF signalling pathway, VEGF, hypoxia and angiogenesis pathways Elastin degradation pathway WNT1 inducible signalling pathway protein 1, WNT1 inducible signalling pathway protein 2 RAS/RAF/MAPK, PI3K ND ND Phosphatidylinositol 3-kinase/Akt ND Glycolysis pathway, Krebs cycle pathway

Dimitrova et al. (2008) Hoffman et al. (2004) Malik and Catherino (2007) Peng et al. (2009) Tsibris et al. (2002) Wang et al. (2003) Wei et al. (2006) Wei et al. (2005) Zaitseva et al. (2008) Gasca et al. (2008) Oksjoki et al. (2005) Savaris et al. (2008) Taniguchi et al. (2008)

WNT1 inducible signalling pathway FAS-FASL, MAPK, P38/JNK Extracellular matrix, TGFb/SMAD, retinoic acid IGF signalling PPARc/RXRa, Wnt/frizzled, cadherin-dependent ND ND IGF1, ILG1R, EGFR, PI3K, Ser473 Retinoic acid pathway Wnt signalling, IL1-R ND ND NFkB

Chen et al. (2006) Eyster et al. (2007)

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Table 2

Elafin, keratin 16, collagen type XVII, plakophilin 1, IL-1RA, RAMP-1 PLA2 group ND VEGF-A PCDH17, PTPRR, IL6ST ND GAPDH, b-actin Phosphofructokinase, triosephosphate isomerase, aldolase, enolase, fumarate hydratase DSG2, MST4, PKCb1, MMPs ND Dermatopotin, versican, TGFb3, CYP26A1 p-AKT, p-S6K, TSC-1, TSC-2, IGF-1, IGF-2 ND MAPKKK5, MAP kinase, NF-IL6b Progesterone receptor A, retinoid acid receptor a Steroid hormone receptors Aldehyde dehydrogenase, RA binding protein 2 FSH, LH a3(VI) collagen, CD9 antigen, entactin, a6 and b8 Insulin, progesterone TNFa, cIAP-2, IL-8 (continued on next page)

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was presented as: leiomyomas (n = 30; 15.08%); endometriosis (n = 26; 13.07%); polycystic ovary syndrome (n = 5; 2.51%); premature ovarian failure (n = 3; 1.51%); pre-eclampsia (n = 1; 0.5%); infertility and IVF/intracytoplasmic sperm injection (n = 26; 13.07%); chromosomal abnormalities (n = 1; 0.5%); and male factor diseases (n = 4; 2.01%). Normal tissue was studied in 42 (21.11%) cases while it was not mentioned in 51 (25.63%). All studies had a single disease in focus. The majority of studies did not mention disease grade or stage. Of the 199 studies, 154 examined extracted tissue. Tissue size extracted was mentioned in 15 studies. In the majority of studies, the tissue size extracted ranged from 0.2 to 6.8 cm3. Tissues examined included: amniotic fluid (n = 1; 0.5%); embryos (n = 14; 7.04%); blastomeres (n = 2; 1.01%); blastocysts (n = 3; 1.51%); endometrium (n = 52; 26.13%); embryonic stem cells (n = 5; 2.51%); follicular fluid and follicles (n = 1; 0.5% and n = 3; 1.51%, respectively); granulosa cells (n = 1; 0.5%); oocytes (n = 15; 7.54%); leiomyoma tissue (n = 20; 10.05%); normal myometrial tissue (n = 12; 6.03%); ovarian tissue (n = 4; 2.01%); spermatozoa (n = 6; 3.02%); testicular tissue (n = 9; 4.52%); uterine fluid sample (n = 1; 0.5%); white blood cells (n = 1; 0.5%); adipose tissue (n = 2; 1.01%); and specific cell lines (n = 2; 1.01%). A minority of studies (n = 10) mentioned the site where the tissue was extracted (applicable for solid tissue) and even fewer studies (n = 3) mentioned comparative sites. Only 40 studies mentioned the time of the cycle or another specific time point when tissue was collected, while two studies (1.01%) mentioned tissue collection at all times.

DAF, OPN, IL-15, a v b3 integrin, C3-Ligand of DAF, receptor of IL-15Ra Hba2, Hbc, tryptophanyl-tRNA synthetase, glutamate-ammonia

Outcomes The majority of studies (n = 180) had as an outcome the over-expression or under-expression of genes while time for outcome was mentioned in only 12 (6.03%) studies. A second outcome was reported in 111 studies while a third outcome in only 42 studies. Disease-specific outcomes were reported in 147 studies (73.87%). These are classified as shown below. Genes finally selected from the microarray analyses are presented in Table 1. Molecules studied and pathways involved are presented in Table 2. Endometriosis Some of the up-regulated genes were for matrix metalloproteinases and tissue inhibitor of metalloproteinases, insulin-like growth factor binding protein, heat shock proteins (70 kDa, 27 kDa) and integrin. Some of the down-regulated genes were for MAPK signalling pathway, WNT signalling pathway and Jak-STAT pathway.

ND = not determined.

Franchi et al. (2008) Centlow et al. (2008)

Gielchinsky et al. (2008) Meng et al. (2008) Rho et al. (2006) Van Vaerenbergh et al. (2008) Wells et al. (2005) Serafica et al. (2005) Sha et al. (2007)

MATER, ZAR1, NPM2, FIGLA

MOS-MPF, TGFb superfamily, WNT signalling pathway, NOTCH signalling pathway Apoptosis, ubiquitination, and energy production pathways ND BMP4, TGFb, FGF-4, Wnt, Hh, Notch ND WNT signal transduction pathway UPP, MAPK, p38/JNK, GPCR, Wnt, NF-kB, OXPHOS ECM, focal adhesion, gap junction, GREM1–TGFb signal, Cell growth, MAPK-Erk1/Erk2, MAPK-p38 ND ND Zhang et al. (2007)

Molecules studied Pathways involved Publication

Table 2

SERPINB2, PAI2, IGF1R, IGF-1/GH1, HSPA8, HSPD1 ERa, ERb, PR-B, PR-A + B, AR Oct3/4, Smad4, ERK1, b-catenin SERPINB6, SOX17, CDC42, CDC42, FOXO3A BRCA1, RB1, BRCA2, MAD2, ATM, BUB1, TP53, APC, b-actin ND ND

I Verginadis et al.

(continued)

578

Leiomyoma Some of the up-regulated genes were for enolase, sex-steroid receptor cofactors, proteins in insulin pathways and CD24 and CD97. Some of the down-regulated genes were dermatopontin, glucocorticoid receptor and CD4. Polycystic ovary syndrome Some of the up-regulated genes were for Wnt-5A and Wnt-13, IGF-binding protein 5 and retinoic acid receptor

Microarray analysis in gynaecology b. Some of the down-regulated genes were for cell division protein kinase 6, mitotic-specific cyclin G1, CD97 antigen and zinc finger protein 148.

Infertility Infertility (and IVF) was mentioned as a cause for investigation in 26 studies (13.07%), while 25 (12.56%) mentioned the tissue investigated: embryos (n = 3; 12%); blastocysts (n = 1; 4%); endometrium (n = 3; 12%); follicular fluid and follicles (n = 1; 4%); granulosa cells (n = 1; 4%); oocytes (n = 6; 24%); leiomyoma tissue (n = 1; 4%); ovarian tissue (n = 2; 8%); spermatozoa (n = 3; 12%); testicular tissue (n = 3; 12%), and placenta tissue (n = 1; 4%).

Discussion This review of microarray studies for benign gynaecological diseases showed basic flaws in the design of these studies. The selection of studies was exhaustive, although certain studies might have escaped attention. A small number of studies considered themselves prospective, double-blind or longitudinal. There was a great variance on the number of explored genes where reported. Many studies (33.3%) limited the number of explored genes to below 2000, but 50% of the studies used more than 12,000 probes. Although the diseases examined represented a large share of female reproductive tract pathology, the sample sizes used were minimal. Although a commentary about the strengths and weaknesses of this investigation cannot be given because of the great variability of the studies, it is obvious that general recommendations should be taken into account. Recommendations as described by Ntzani and Ioannidis (2003) and Dupuy and Simon (2007) could be incorporated in the design of the studies and rationale must be presented for not following them. In contrast to hypothesis-driven research, microarray investigation has been defined as discovery-based research. However, clear objectives are needed even with this kind of research: instituting clear objectives facilitates patient selection and avoids analysing heterogeneous groups of patients with different stages of disease. The choice of analysis methods should be made according to the objective of the study but sample size is also important. Small studies can give inflated and overpromising results, even in molecular medicine. Aiming for many independent studies with a total of several thousand patients is not the ultimate solution. Small sample sizes are not the only problem in these studies: there are also more general problems such as a lack of standard methods for design, data analysis and performance assessment according to clinical aims. On the other hand, clinical trials for multiple biomarkers pose certain challenges (Pusztai and Hess, 2004). Different trial designs may be used to assess the clinical utility of a certain predictive marker. A recent article has attempted to answer the question as to what is the appropriate design for a microarray clinical trial (Kim et al., 2010).

579 Most diseases even at the same stage have a high degree of inter-individual heterogeneity due to multiple genetic pathways, genetic polymorphisms and epigenetic influences. Combined genomic and proteomic approaches may offer better insights on disease specification and have to be seen in the clinical context. Different microarray platforms have been used to identify gene datasets. More challenges are posed in making general comparisons between different datasets, although such methods have already been developed. On the other hand, combining data for the same disease from different species or organisms also poses significant challenges. One-to-one relationships do not exist for many transcripts across species. Even identical cell lines may express different genes under the influence of experimental variation. Co-operative efforts between multidisciplinary research networks are needed to address the above problems and funding must preferably be issued to such teams. The study has to state clear objectives. Research strategies have to take priority over microarray technique implementation without clear objectives. When it comes to study design, it is essential to use masking and careful definitions of eligibility criteria and outcomes and to give attention to the standardized recommendations for laboratory techniques (as proposed by the Minimum Information About a Microarray Experiment group; Brazma et al., 2001). Unless there is a specific rationale to limit the number of explored genes, the use of comprehensive microarrays with a larger number of gene probes maximizes the yield of information (Ioannidis et al., 2003). Randomized studies are preferable with meticulous use and report of statistical analysis. Eventually all studies need to conform to established guidelines (Dupuy and Simon, 2007). In order to increase reliability, reproducibility and comparability of microarray studies, standardized reporting of these data is needed. The Standard Microarray Reporting Template will greatly help to this direction (Cahan et al., 2007). The clinical utility of these data will be in pharmacogenetics for maximizing the chances of benefiting from a treatment or avoiding adverse effects, a process that has resulted in a small number of practical applications so far. Genetic tests can be adopted before they prove their cost-effectiveness. That means that the population that will benefit the most from genetic testing need to be identified first. Until that time, transparent reporting of data on genetic association and careful grading of the evidence is needed (Ioannidis, 2007; Ioannidis and Contopoulos-Ioannidis, 1998).

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I Verginadis et al. Declaration: The authors report no financial or commercial conflicts of interest. Received 25 October 2010; refereed 24 February 2011; accepted 10 March 2011.