Glycodelin and differentiation in endometrium and ovary

Glycodelin and differentiation in endometriumand ovary:

Clinical aspects related to reproduction and

epithelial ovarian cancer

Erik Mandelin

Academic Dissertation

To be publicly discussed with the permission of the Medical Faculty of theUniversity of Helsinki, in Auditorium 2, Biomedicum Helsinki, on October 3,

2003, at 12 noon.

Helsinki 2003Yliopistopaino

Department of Obstetrics and Gynecologyand

Department of Clinical Chemistry

Helsinki University Central Hospital and Biomedicum HelsinkiUniversity of Helsinki

Helsinki, Finland

Erik Mandelin

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SUPERVISED BY

Professor Markku Seppälä, M.D., Ph.D.Department of Clinical Chemistry

University of Helsinki

REVIEWED BY

Docent Seija Grénman, M.D., Ph.D.Department of Obstetrics and Gynaecology

University of Turku

Professor Jorma Isola, M.D., Ph.D.Institute of Medical Technology

University of Tampere

OFFICIAL OPPONENT

Professor Outi Hovatta, M.D., Ph.D.Department of Clinical Science

Division of Obstetrics and Gynaecology Karolinska Institute, Stockholm, Sweden

ISBN 952-91-5961-7ISBN 952-10-1237-4 (pdf)

Helsinki 2003Yliopistopaino

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Review of the literature

Contents

LIST OF ORIGINAL PUBLICATIONS ............................................................ 5

ABBREVIATIONS & DEFINITIONS .............................................................. 6

ABSTRACT....................................................................................................... 7

REVIEW OF THE LITERATURE1. Introduction .................................................................................................. 92. Glycodelin .................................................................................................... 9

2.1. Protein .................................................................................................. 92.2. Glycodelin gene and mRNA................................................................. 102.3. Temporal and spatial expression in normal tissues ................................. 10

2.3.1. Female reproductive tract ........................................................... 102.3.2. Male reproductive tract .............................................................. 112.3.3. Other cells and tissues ................................................................ 11

2.4. Regulation of glycodelin synthesis in endometrial and other cells ......... 122.4.1. Progesterone, progestogens, and antiprogestins .......................... 122.4.2. Estrogen ..................................................................................... 132.4.3. Relaxin ...................................................................................... 132.4.4. Other observations ..................................................................... 13

3. Glycodelin and reproduction ......................................................................... 143.1. Inhibition of gamete interactions .......................................................... 143.2. Clinical perspectives ............................................................................. 14

3.2.1. Fertilization window and glycodelin-induced contraceptiveactivity of the uterus .................................................................. 14

3.2.2. Glycodelin and oral contraceptives ............................................. 143.2.3. Emergency hormonal contraception ........................................... 153.2.4. In vitro fertilization ................................................................... 15

3.3. Reproductive immunology in relation to glycodelin ............................. 164. Glycodelin and cancer ................................................................................... 16

4.1. Epithelial cancer of the ovary ................................................................ 164.2. Endometrial adenocarcinoma ................................................................ 174.3. Glycodelin as a differentiation-related morphogen ................................ 19

AIMS OF THE STUDY .................................................................................... 20

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MATERIALS AND METHODS ........................................................................ 211. Samples ......................................................................................................... 212. Hormone treatments (III, IV)........................................................................ 213. Immunohistochemistry ................................................................................. 22

3.1. Glycodelin (I–V) ................................................................................... 223.2. αvβ3 integrin (IV) ................................................................................ 223.3. Progesterone receptors A and B (V) ...................................................... 223.4. Double stainings for glycodelin and progesterone receptor A or B (V) .. 23

4. RNA in situ hybridization (I, II) .................................................................. 235. CA-125 (V) ................................................................................................... 246. Statistical methods (I-V) ............................................................................... 24

RESULTS ........................................................................................................... 251. Endometrial glycodelin expression in the fertile midcycle in women

wearing the levonorgestrel-releasing intrauterine device (I) ........................... 252. Endometrial glycodelin expression of women using the subdermal

levonorgestrel-releasing contraceptive implant (II) ........................................ 253. Endometrial glycodelin expression in the luteal phase of stimulated ovarian

cycles (III) ..................................................................................................... 254. Expression of glycodelin and αvβ3 integrin at midluteal phase utilizing

the donor oocyte model (IV) ......................................................................... 265. Glycodelin in ovarian serous carcinoma: associations with differentiation

and survival (V) ............................................................................................ 275.1. Glycodelin expression in tumor tissue ................................................... 275.2. Glycodelin expression and clinical parameters ....................................... 275.3. Glycodelin and survival in ovarian cancer ............................................. 285.4. Expression of progesterone receptor A and B in tumor tissue in relation

to glycodelin expression ........................................................................ 325.5. Serum CA-125-levels and endometrial glycodelin expression ................ 32

DISCUSSION .................................................................................................... 331. The fertilization window (I,II) ...................................................................... 332. The implantation window (III, IV) ............................................................... 353. Glycodelin and differentiation (V)................................................................. 37

SUMMARY AND CONCLUSIONS.................................................................. 39

ACKNOWLEDGEMENTS ............................................................................... 40

REFERENCES................................................................................................... 42

ORIGINAL PUBLICATIONS ........................................................................... 51

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List of Original Publications

This thesis is based on the following original publications referred to in the text by theirRoman numerals.

I. Mandelin E, Koistinen H, Koistinen R, Affandi B, Seppälä M. Levonorgestrel-releasing intrauterine device-wearing women express contraceptive glycodelin Ain endometrium during midcycle: another contraceptive mechanism? Hum Reprod12:2671–2675, 1997.

II. Mandelin E, Koistinen H, Koistinen R, Arola J, Affandi B, Seppälä M. Endometrialexpression of glycodelin in women with levonorgestrel-releasing subdermalimplants. Fertil Steril 76:474–478, 2001.

III. Brown SE, Mandelin E, Oehninger S, Toner JP, Seppälä M, Jones HW Jr.Endometrial glycodelin-A expression in the luteal phase of stimulated ovarian cycles.Fertil Steril 74:130–133, 2000.

IV. Damario MA, Lesnick TG, Lessey BA, Kowalik A, Mandelin E, Seppälä M,Rosenwaks Z. Endometrial markers of uterine receptivity utilizing the donor oocytemodel. Hum Reprod 16:1893–1899, 2001.

V. Mandelin E, Lassus H, Seppälä M, Leminen A, Gustafsson J-Å, Cheng G, BützowR, Koistinen R. Glycodelin in Ovarian Serous Carcinoma: Association withDifferentiation and Survival. Cancer Res, in press.

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Abbreviations & Definitions

bp base pairscDNA complementary deoxiribonucleic acidCI confidence intervalCOH controlled ovarian hyperstimulationDa daltonFSH follicle-stimulating hormoneGnRH gonadotropin-releasing hormonehCG human chorionic gonadotropinIgG immunoglobulin GIUD intrauterine deviceIL interleukinIVF in vitro fertilizationLH luteinizing hormoneLNG levonorgestrelLNG-IUD levonorgestrel-releasing intrauterine devicemock cycle a control cycle preceding the actual embryo transfer cyclemRNA messenger ribonucleic acidNK cell natural killer cellpI isoelectric pointPBS phosphate buffered salinePRA progesterone receptor subtype APRB progesterone receptor subtype BRT-PCR reverse transcription-polymerase chain reactionSDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresisZIF-1 zona binding inhibitory factor 1

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Abstract

Glycodelin is the major progesterone-regulated lipocalin protein of the repro-ductive axis. It has contraceptive and immu-nomodulatory functions, and it may playan important role in differentiation andglandular morphogenesis. The aim of thisstudy was to investigate the expression ofglycodelin in three clinical settings: in thefertile window, in the window of implanta-tion, and in ovarian serous carcinoma, withspecial attention to changes in tissuedifferentiation.

Glycodelin is a uterine secretory glyco-protein that inhibits sperm-egg binding,and is not normally secreted during thefertile window at midcyle. This phase of themenstrual cycle was specifically addressedin respect to induced secretion of contra-ceptive glycodelin. Evidence showed that alevonorgestrel-releasing IUD is accom-panied by inappropriate expression ofglycodelin in endometrium between days7 and 16 of the menstrual cycle (6 of 6 cases).The same was also found in copper-IUD-wearing women, but less frequently (4 of11 cases, P < 0.0345). Experimentsemploying in situ hybridization localizedglycodelin mRNA into endometrial glandsin the midcycle endometrium, confirmingthe cellular site of synthesis.

Another group of contraceptive users, inwhom uterine glycodelin secretion wasstudied during the fertile window, werelevonorgestrel subdermal implant-wearingwomen. Of the endometrial specimens fromthese women, 80% stained positive forglycodelin. The endometrial morphology ofthese women showed proliferative (71%),inactive/weakly proliferative (19%),menstrual or regenerating (6.5%), and other

patterns (2.8%). Of these, 79%, 71%,100%, and 100% were glycodelin-positive.During the midcycle, when glycodelin isnot normally expressed, of 19 cases 89%showed glycodelin expression.

Another point of interest is the windowof implantation that in a normal menstrualcycle spans from LH+4/5 to LH+10. Factorsaffecting glycodelin expression during thephase of uterine receptivity were chosen inview of the immunosuppressive propertiesof glycodelin and its putative role in feto-maternal defense mechanisms. In womenwith COH (controlled ovarian hypersti-mulation), endometrium is exposed to highconcentrations of ovarian steroids and othersubstances. In such women, the proportionof glycodelin-positive endometrial cells waselevated above values in normal controls.This was evident throughout the windowof implantation. Glycodelin was present inthe endometrial glands, but not in thestroma or surface epithelium. A positivecorrelation appeared between glycodelinexpression and serum estradiol levels (r =0.5, P < 0.001) in normal menstrual cycles,and glycodelin and advanced histology inCOH cycles (r = 0.63, P = 0.01). NeitherLH nor progesterone serum levels werecorrelated with endometrial glycodelinexpression.

Because ethical constraints limit studieson endometrium during the peri-implantation phase of a fertile cycle, thedonor oocyte model was chosen forinvestigation of candidate endometrialmarkers in respect to uterine receptivity.Endometrial biopsies from cycle days 21 to23 of oocyte recipients undergoing mockhormonal treatment cycles were evaluated by

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standard histological criteria and byimmunohistochemical staining forglycodelin and αvβ3 integrin. Oocyterecipients underwent identical hormonalreplacement protocols for both the mocktreatment cycle and the actual oocytedonation cycle. Endometrial histology ofthese women showed 62 (61.3%) in-phase,34 (33.7%) dyssynchronous, 2 (2.0%)immature, and 3 (3.0%) advancedmaturation. The clinical outcomes of patientswith either in-phase or dyssynchronousendometria were similar. Strong correlationsexisted between endometrial glandulardating and either glycodelin- or αvβ3integrin-immunostaining intensity (P <0.001 for both). Glycodelin and αvβ3integrin-immunostaining intensities werealso highly correlated with each other (P <0.001).

In ovarian serous carcinomas from 460patients, we determined glycodelinexpression by immunohistochemistry oftissue microarrays, and analyzed the resultsin relation to the progesterone receptors PRA(progesterone receptor subtype A) and PRB(progesterone receptor subtype B), to clinicalparameters, and to survival. Glycodelin waslocalized in the cytoplasm of tumor cells,whereas vascular endothelium in tumor tissuewas glycodelin-negative. Glycodelinexpression was more frequent in well-differentiated (grade I, 79%) than in poorlydifferentiated carcinomas (grade III, 51%, P< 0.0001), and also more frequent in earlythan in advanced stage carcinomas (P =0.002). Cytoplasmic glycodelin was often co-expressed with nuclear PRA and PRB.Whereas this was not consistent in alltumors, a positive correlation existed in thetumor between the presence of glycodelinand progesterone receptors (P < 0.02), butnot between the presence or absence of

glycodelin in the tumor and the CA-125serum concentration. Although inmultivariate analysis glycodelin was not anindependent variable, those patients withglycodelin-expressing tumors showed ahigher 5-year overall survival than did thosewhose tumors were glycodelin-negative(55% vs. 39%, P < 0.0001, hazard ratio inunivariate analysis 0.57, CI 0.44-0.74). Inthe patients with grade I tumors or stageIII disease, this difference was pronounced.In the latter group, the 10-year survivalprobability of those with glycodelin-positive tumors was more than twice as highas that of those with glycodelin-negativetumors. This also occurred within well-defined clinical categories, e.g., in stage III/grade II and stage III/grade III carcinomas,in which patients with glycodelin-positivetumors exhibited a significantly better 10-year overall survival than did those withglycodelin-negative tumors.

Based on the potent inhibitory activity ofglycodelin on sperm-egg binding andsecretion into uterine fluid, in IUD andsubdermal-implant-wearing women,glycodelin secretion during the otherwisefertile window may thus lead, beforefertilization, to exposure of sperm tocontraceptive glycodelin. This maycontribute to the effectiveness of suchcontraceptive devices. Studies of the windowof implantation showed oocyte donorsundergoing COH treatment to havesignificantly higher endometrial glycodelinexpression throughout the implantationwindow than did women with natural cycles.Endometrial glycodelin expressionfrequently occurred in progesterone/estrogen- stimulated mock cycles. In ovarianserous carcinoma, glycodelin expression isrelated to differentiation and offers a betterprognosis.

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Review of the Literature

1. Introduction

In the 1970s and 1980s, several researcherswere involved in the identification andisolation of a protein in the amniotic fluid,human placenta, pregnancy decidua andseminal plasma, and gave it various names,independently of each other, according to thesource or physicochemical characteristics.Names suggested included α-uterine protein(Horne et al., 1982), placental α2-globulin(Petrunin et al., 1976), chorionic α2-microglobulin (Petrunin et al., 1980),placental protein 14 (Bohn et al., 1982),progesterone-associated endometrial protein(Kämäräinen et al., 1991), progestagen-dependent endometrial protein (PEP) (Joshiet al., 1980b), pregnancy-associated endo-metrial α2-globulin (α2-PEG) (Bell et al.,1985; Bell, 1986), and human β-lacto-globulin homolog (Bell et al., 1987; Huhtalaet al., 1987; Seppälä et al., 1987a).Subsequent studies revealed that all theseproteins were identical in respect to theirimmunological properties or primarystructure or both (Sutcliffe et al., 1982; Belland Bohn, 1986; Julkunen et al., 1986c; Bellet al., 1987; Huhtala et al., 1987; Julkunenet al., 1988; Garde et al., 1991; Seppälä etal., 1998a). The name glycodelin wasintroduced for all these proteins because noprevious names accurately reflected all thesites of glycodelin synthesis. The nameglycodelin also emphasizes the importanceof glycosylation for the biological activityof the protein (Seppälä et al., 2002).

2. Glycodelin

2.1. Protein

Glycodelin is a glycoprotein that belongs tothe lipocalin superfamily (Huhtala et al.,1987; Julkunen et al., 1988). Lipocalins aresmall, mostly extracellular proteins found invertebrates and invertebrate animals, plants,and bacteria. They perform transportfunctions and bind hydrophobic ligands suchas retinoids, steroids, and lipids. Lipocalinsalso have other functions includingmodulation of cell growth and metabolism,regulation of immune response, odorreception, tissue development, and animalbehavior (Åkerström et al., 2000).

Glycodelin consists of 180 amino acids,18 of which correspond to a signal peptide(Julkunen et al., 1988). Amniotic fluidglycodelin has a molecular mass of 28kDaby SDS-PAGE, and in gel filtration,glycodelin behaves as a homodimericcomplex with a molecular mass of 50 to 60kDa (Joshi et al., 1980a; Bohn et al., 1982;Joshi, 1983; Bell, 1986). The predictedmolecular mass for the mature polypeptideis 18 787 kDa (Julkunen et al., 1988).

Several differentially glycosylated gly-codelin isoforms are known. Amniotic fluidglycodelin-A and seminal fluid glycodelin-S, sharing the same protein core, are in-distinguishable from each other afterdeglycosylation and desialylation in bothSDS-PAGE and isoelectric focusing(Koistinen et al., 1996). Glycodelin-A and

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glycodelin-S contain 17.5% carbohydrates(Bohn et al., 1982), linked to two of the threepotential N-glycosylation sites atAsparagine-28 and Asparagine-63, whereasAsparagine-85 is not glycosylated (Julkunenet al., 1988; Dell et al., 1995; Morris et al.,1996).

Yao and coworkers recently (1998)characterized a factor in human follicularfluid which potently inhibited the bindingof spermatozoa to the zona pellucida.Comparative studies with glycodelin revealedthat this factor, zona binding inhibitoryfactor-1 (ZIF-1), is a glycodelin isoform (Chiuet al., 2003b). Glycodelin-A and ZIF-1 havethe same molecular mass in SDS-PAGE bothbefore and after deglycosylation, and the first25 N-terminal aminoacids of ZIF-1 areidentical to those of glycodelin-A. Proteolyticdigestion with trypsin and pepsin ofdeglycosylated ZIF-1 and glycodelin-Aresults in identical peptide patterns. Theisoelectric point of ZIF-1 is 4.2 to 4.8, i.e.,it is more acidic than glycodelin-A (pI = 4.5to 5.2) and glycodelin-S (pI = 4.9 to 5.6).Based on its follicular fluid origin, ZIF-1 hasbeen renamed glycodelin-F (Chiu et al.,2003b).

2.2. Glycodelin gene and mRNA.

The glycodelin gene is located onchromosome 9q34 (Van Cong et al., 1991).The gene consists of 5042 base pairs in sevenexons and six introns. The four putativeglucocorticoid/progesterone responseelements upstream in the promoter regionof the gene are located at positions –1799, -1071, -745, and –302, and two additional,downstream, at positions +1912 and +1965(Vaisse et al., 1990). The gene encodes a 900-bp long mRNA (Julkunen et al., 1988). Ofthe many mRNA splicing variants (Gardeet al., 1991; Morrow et al., 1994; Koistinenet al., 1997), some lack the sequences of theglycosylation sites or the Threonine-Aspartic

acid-Tyrosine sequence typical of proteins ofthe lipocalin family (Godovac-Zimmermann,1988) or lack both. Both single nucleotidepolymorphisms (Van Cong et al., 1991) andHinfI-restriction enzyme polymorphims areknown (Kämäräinen et al., 1991).

2.3. Temporal and spatial expression in normaltissues

2.3.1. Female reproductive tract

In normal menstrual cycles, the expressionof glycodelin in the endometrial glandsusually begins 4 to 5 days after ovulation (LH+4–5) (Seppälä et al., 1988b). Subsequently,its concentration in tissue and uterine fluidincreases until the end of the cycle (Julkunenet al., 1985; Julkunen et al., 1990; Li et al.,1993b) (Table 1). Glycodelin is normallyabsent from endometrium during theproliferative phase, except for the first daysof the cycle, when glycodelin remains in thebasal glands (Julkunen et al., 1985; Julkunenet al., 1986b; Waites et al., 1988b). Duringpregnancy, glycodelin is synthesized bydecidualized endometrium and secreted intothe amniotic fluid (Julkunen et al., 1986b;Julkunen et al., 1988; Julkunen et al., 1990).The tissue concentration of glycodelin inpregnancy decidua increases until the tenthweek. Thereafter, the synthesis decreasestowards the end of the pregnancy. Inmaternal serum, glycodelin concentrationsare lower and follow the same pattern as inendometrial tissue (Julkunen et al., 1985).

Glycodelin is present in the fallopian tubesin conformity with the Müllerian origin ofthe uterus and the fallopian tubes. No cycle-dependent differences in glycodelinconcentrations occur in the isthmic andampullary parts of the Fallopian tubes, butglycodelin concentrations in the fimbrial partvary in a cyclical manner, the concentrationbeing higher in the secretory phase than inthe proliferative phase (Julkunen et al.,1986d).

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In the normal ovary, glycodelin appearsin a variety of cells. In the follicular phase,immunoreactivity occurs in the ovariancortex, theca interna, and granulosa. Lutealphase cortical stroma is glycodelin-negativeor only weakly positive, whereas the thecainterna of the corpus luteum, corpus albicans,Leydig cells of the ovarian hilus, andluteinized granulosa cells are glycodelin-positive (Kämäräinen et al., 1996).Glycodelin mRNA appears in luteinizedgranulosa cells, but not in cumulus cells (Tseet al., 2002). Glycodelin is also present infollicular fluid (Seppälä et al., 1985;Chryssikopoulos et al., 1996) (Table 1).Glycodelin synthesized by granulosa cells isreleased into the follicular fluid, from whenceit is taken up by and partially modified bythe cumulus cells (Tse et al., 2002).

2.3.2. Male reproductive tract

Glycodelin protein and mRNA exist inseminal vesicles and the ampulla of the vasdeferens, but not in the prostate, testis, orepididymis. Glycodelin is present in seminalfluid at high concentrations (Petrunin et al.,1980; Bohn et al., 1982; Julkunen et al.,1984; Koistinen et al., 1997).

2.3.3. Other cells and tissues

Glycodelin mRNA and/or protein also occurin other glandular structures of the humanbody, in mammary glands, sweat glands, andbronchus epithelium (Kämäräinen et al.,1997; Kämäräinen et al., 1999). Glycodelinis present in megakaryocytes, platelets, andmegakaryotic cell lines (Morrow et al., 1994).Erythroid precursor cells also synthesizeglycodelin (Kämäräinen et al., 1994).

Table 1. Glycodelin in female reproductive tissues, fluids, and peripheral serum

Origin

EndometriumMid-proliferativeMid-secretoryLate secretoryDecidua

9 week40 week

Uterine flushingsProliferative phaseEarly secretory phaseMid-secretory phase

Fallopian tubeProliferative phaseSecretory phase

Amniotic fluid12 week16 week40 week

SerumMid-proliferative phaseMid-luteal phaseLate luteal phaseMenstrualPregnancy (12 weeks)Pregnancy (40 weeks)

Follicular fluid

Glycodelin concentration

<0.1 mg/g protein7.8 mg/g protein23 mg/g protein

160mg/g protein0.8 mg/g protein

Not detectableNot detectable12 mg/liter

4.3 µg/g protein16 µg/g protein

13 mg/liter125 mg/liter1 mg/liter

<20 µg/liter35 µg/liter47µg/liter74 µg/liter1200 µg/liter100 µg/liter7.9-122 µg/liter

Author(s) (Reference)

Julkunen et al., 1986bJulkunen et al., 1986bJulkunen et al., 1986b

Julkunen et al., 1985Julkunen et al., 1985

Li et al., 1993bLi et al., 1993bLi et al., 1993b

Julkunen et al., 1986dJulkunen et al., 1986d

Julkunen et al., 1985Julkunen et al., 1985Julkunen et al., 1985

Julkunen et al., 1986bJulkunen et al., 1986bJulkunen et al., 1986bJulkunen et al., 1986bJulkunen et al., 1985Julkunen et al., 1985Seppälä et al., 1985

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2.4. Regulation of glycodelin synthesis inendometrial and other cells

2.4.1. Progesterone, progestogens, andantiprogestins

Evidence indicates that glycodelin expressionis related to the action of progesterone (Table2). Glycodelin expression is confined to thesecretory and decidualized endometrium(Julkunen et al., 1985; Julkunen et al.,1986b), and its serum concentrations in-crease in ovulatory cycles only, after theprogesterone levels have risen. In anovulatorycycles, the glycodelin levels remain lowthroughout the cycle (Julkunen et al.,1986a). Micronized oral progesterone raisesthe luteal phase serum glycodelinconcentrations in women with unexplainedinfertility (Seppälä et al., 1987b). Thepromoter region of the glycodelin genecontains putative progesterone responseelements (Vaisse et al., 1990), and

Progesterone/

progestins

Estrogen

Evidence

+

+

+

+

-

+

+

-

-

Observation

Glycodelin expression is confined to the secretory and decidualized endometrium

Glycodelin serum levels increase only in ovulatory cycles - in anovulatory cycles glycodelin

levels remain low

Micronized oral progesterone increases the luteal phase glycodelin serum concentrations

in women with unexplained infertility

Postmenopausal women taking cyclical estrogen-progestogen replacement therapy have

elevated serum glycodelin levels at the end of the progesterone treatment

No correlation between serum glycodelin and mid- or late luteal phase progesterone levels

in infertile women

Estrogen serum levels in proliferative phase are correlated with glycodelin levels in the luteal

phase in women undergoing ovarian hyperstimulation for in vitro fertilization

Positive correlation between serum estrogen levels and endometrial glycodelin

immunostaining between cycle days 12 and 24

Proliferative endometrium is glycodelin negative or shows only a very low glycodelin

content

Patients receiving estrogen only for hormone replacement therapy do not show elevated

serum glycodelin levels

Reference(s)

Julkunen et al., 1986b

Julkunen et al., 1985

Julkunen et al., 1986a

Seppälä et al., 1987b

Seppälä et al., 1987a

Check et al., 1991

Seppälä et al., 1989

Waites et al., 1989

Julkunen et al., 1986b

Byrjalsen et al., 1989

Table 2. Evidence for (+) or against (-) stimulatory effects of progesterone/progestins and estrogen on glycodelin synthesis. Results from in vivo studies.

progestogens induce glycodelin inendometrial cell cultures (Taylor et al., 1998;Taylor et al., 2000). Ligand-activatedprogesterone receptors stimulate glycodelingene expression through two active Sp1 siteslocated in the glycodelin gene promoterregion (Gao et al., 2001).

In experimental settings in vitro, inter-estingly, the antiprogestin mifepristone hasa stimulatory effect on glycodelin synthesis(Taylor et al., 1998). Early pregnancyinterruption with mifepristone is associatedwith a small increase in glycodelin serumconcentrations (Howell et al., 1989). Lowdaily doses of mifepristone in women withnormal menstrual cycles, however, causeretarded endometrial development anddecreased endometrial glycodelin expression(Gemzell-Danielsson et al., 1996). Mife-pristone may have inappropriate agonistlikeactions in some tissues and tumors.Transcriptional coactivators, recruited to the

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transcription complex, may be responsiblefor this phenomenon (Jackson et al., 1997).

2.4.2. Estrogen

Proliferative endometrium is glycodelin-negative, and patients receiving estrogenonly for hormone replacement therapy showno increase in circulating glycodelinconcentrations (Julkunen et al., 1986b;Byrjalsen et al., 1989) (Table 2). In vitro,estradiol has no effect on the transcriptionalactivity of the glycodelin gene promoter(Taylor et al., 1998). Serum estrogen levelsin the proliferative phase are, however,correlated with glycodelin levels in the lutealphase, suggesting that estrogen priming mayenhance glycodelin synthesis (Seppälä et al.,1989; Li et al., 1992). Another study showeda strong positive correlation in natural cyclesbetween serum estrogen levels andendometrial glycodelin immunostainingbetween cycle days 12 and 24 (Waites andBell, 1989). In conclusion, estrogen does notdirectly induce glycodelin expression,although its stimulatory effect may bemediated by endometrial priming thatincludes up-regulation of progesteronereceptors (Seppälä et al., 2002).

Relaxin 2.4.3.

The secretion of relaxin precedes by 1 to 2days the secretion of glycodelin with a clearpositive correlation between them (Stewartet al., 1997). The blood circulation of womenwith no functional ovaries who becomepregnant through donated embryo transferlack relaxin (Emmi et al., 1991). In thesewomen, glycodelin levels are also low orundetectable (Critchley et al., 1990;Critchley et al., 1992; Johnson et al., 1993b).Endometrial epithelial cells cultured withporcine relaxin have shown a 2- to 6-foldincrease in glycodelin production rate, asmeasured by the solution hybridization and

ribonuclease protection assays. That glyco-delin mRNA concentration has increased2 to 11-fold in cells incubated with relaxinsuggests that relaxin activates glycodelintranscription (Tseng et al., 1999).

Although these studies indicate thatrelaxin stimulates glycodelin synthesis, notall studies support these observations.Relaxin not only fails to induce de novoproduction of glycodelin, it even repressesprogesterone-stimulated activation of theglycodelin promoter (Taylor et al., 2000).Intravaginal administration of humanrecombinant relaxin for induction of labordoes not affect glycodelin serum concen-trations (Critchley et al., 1994). Women whodemonstrated normal ovarian cyclicityshowed sustained elevation of glycodelinserum levels during a 28-day recombinanthuman relaxin treatment, whereas thosewithout ovarian cyclicity or placebo-treatedwomen showed no elevation (Stewart et al.,1997). Whereas the results of usingrecombinant human relaxin have beenneither confirmed nor contested, themechanism of relaxin action on glycodelinsynthesis remains to be elucidated.

2.4.4. Other observations

During pregnancy, circulating levels ofglycodelin and hCG show similar patterns(Seppälä et al., 2002). Investigations inhuman beings show no evidence for anystimulatory effect of hCG on endometrialglycodelin secretion (Ren and Braunstein,1990; Seppälä et al., 1991), whereas inintact ovariectomized baboons, exogenoushCG followed by progesterone and estrogentreatment resulted in up-regulation ofglycodelin secretion between post-ovulationdays 18 and 25. Baboons treated only withprogesterone and estrogen showed noincrease in endometrial glycodelin synthesis(Hausermann et al., 1998).

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3. Glycodelin and reproduction

3.1. Inhibition of gamete interactions

Glycodelin-A and glycodelin-F isoformspotently and dose-dependently inhibit thebinding of human sperm to the zonapellucida (Oehninger et al., 1995; Chiu etal., 2003a; Chiu et al., 2003b). Thecontraceptive activity of these glycodelinisoforms is mediated via uniquecarbohydrate structures not present inglycodelin-S (Morris et al., 1996; Chiu etal., 2003a; Chiu et al., 2003b). Thecontraceptive effects of glycodelin-A and -F are likely to result from interactionbetween glycodelin and the sperm ratherthan between glycodelin and the oocyte(Oehninger et al., 1995; Yao et al., 1998;Chiu et al., 2003b), although in thisinteraction zona pellucida proteins may alsobe involved (Chiu et al., 2003a).Glycodelin-F is a more potent inhibitor ofspermatozoa/zona pellucida binding than isglycodelin-A, and is suggested to have twobinding sites on the sperm surface, whereasglycodelin-A has only one (Chiu et al.,2003a). This must be related to differingglycodelin concentrations in uterine andfollicular fluids. Here, the glycodelinconcentration in uterine fluid exceeds byorders of magnitude the concentration ofglycodelin immunoreactivity in preovula-tory follicular fluid (Seppälä et al., 1985;Li et al., 1993b). Importantly, the highglycodelin concentrations such as in uterineflushings and uterine tissue during themidluteal phase of a normal menstrual cyclecan bring about a virtually completeinhibition of sperm-egg binding (Julkunenet al., 1986b; Li et al., 1993a; Li et al.,1993b; Morris et al., 1996).

Glycodelin-F and glycodelin-A fail toaffect the spontaneous acrosome reaction ofhuman spermatozoa (Dutta et al., 2001;Chiu et al., 2003a). Interestingly, afterprogesterone induction, acrosome reactionis inhibited by glycodelin-F, but not by

glycodelin-A (Chiu et al., 2003a). Non-glycosylated recombinant glycodelinimproves capacitation of humanspermatozoa, whereas glycosylatedrecombinant glycodelin inhibits both thecapacitation and the fertilization potentialof spermatozoa (Dutta et al., 2001).

3.2. Clinical perspectives

3.2.1. Fertilization window and glycodelin-induced contraceptive activity of the uterus

The likelihood of fertilization is highestduring a 6-day period ending on theestimated day of ovulation (Wilcox et al.,1995). In the postovulatory phase, thelikelihood of fertilization decreases due tochanges in the cervical mucus that disturbsperm motility. Glycodelin may also play arole here. In a normal menstrual cycle, thesynthesis of glycodelin-A begins 5 days afterovulation. Given its inhibitory effect onsperm-egg binding, during the latter halfof the secretory phase, glycodelin maycontribute to the uterine contraceptive mic-roenvironment. Here, absence of glycodelin-A would be compatible with the fertilewindow during the ovulatory midcycle toenable spermatozoa to maintain theirfertilization capacity (Seppälä et al., 1997;Seppälä et al., 1998b; Seppälä et al., 2002).

3.2.2. Glycodelin and oral contraceptives

The fact that glycodelin inhibits sperm-eggbinding has made glycodelin an interestingmodel for contraceptive research. In thiscontext, a relevant question would bewhether, during the fertile window, con-traceptives currently used induce inappro-priate glycodelin secretion. Glycodelin serumlevels and immunohistochemical localizationin endometrial tissues have been investigatedin women who use various combined oralcontraceptives (Wood et al., 1989). Theserum levels of the subjects remained at thesame low level throughout the cycle, similar

15


to levels in natural cycles, irrespective of thetype of progestagen in the pill. Thisphenomenon was noted in both serial andindividual samples from women takingeither monophasic or triphasic oralcontraceptives. However, immunohisto-chemical analyses showed evidence ofinduction of glycodelin synthesis.Apparently, local endometrial glycodelinsynthesis was not sufficient to be reflectedin serum levels of the protein.

3.2.3. Emergency hormonal contraception

Two reports on the Yuzpe regimen bothinvolved two subsequent cycles of the samewoman: a control cycle and a treatmentcycle (hormone intake). In the first study,ethinyl estradiol and norgestrel were givenon the ninth postovulatory day and resultedin a decrease in serum and uterine fluidglycodelin levels at day 11 postovulation(Young et al., 1994). In the second study,100µg ethinyl estradiol and 1mglevonorgestrel were given in the treatmentcycle on the day of the LH surge, and thetreatment was repeated 12 hours later.Endometrial biopsy was performed 8 to 10days after the LH surge. No differenceappeared between control and treatmentcycles, as assessed by immunohistoche-mistry, in glycodelin expression (Raymondet al., 2000).

3.2.4. In vitro fertilization

The first report on pregnancies after IVF (invitro fertilization) and embryo transferdemonstrated that glycodelin serum levelsincrease markedly after implantation(Julkunen et al., 1985). When the IVFstimulation is made with clomiphene andhuman menopausal gonadotropin, glyco-delin serum levels first decline, reaching theirlowest level at the moment of oocyteretrieval, and then rise again (Seppälä et al.,1988a). No difference appeared in womentreated by IVF between fertile and infertile

treatment cycles in serum samples taken at2- to 3-day intervals throughout the windowof implantation. Once pregnancy had beenestablished, however, glycodelin levelsincreased markedly (Edwards, 1988). Thesedata suggest that glycodelin is not useful asa predictor of implantation; rather, it reflectsendometrial response to implantation.

Another study compared late-luteal phaseserum glycodelin levels between conceiversand nonconceivers, with higher levels amongconceivers. The results were, however,inconclusive as to the greater likelihood ofthe pregnancy’s continuing in women withhigher luteal phase glycodelin levels (Checket al., 1992). Check and his co-workers(1991) also investigated whether subnormalglycodelin levels (serum levels and immuno-histochemical staining) could be improvedby treating patients with therapies normallyused to correct endometrial defects. Nocorrelations appeared (Check et al., 1991).These and other results indicate that becauseserum glycodelin measurements fail to reflectendometrial differentiation, they cannotreplace histological evaluation of the endo-metrium (Batista et al., 1993a; Batista et al.,1993b).

Serum glycodelin levels were investigatedduring the implantation phase in cycles, bothsuccessful and non-successful, during assistedreproduction. All women undergoingcontrolled ovarian hyperstimulation showeda rise in glycodelin serum levels, a rise thesame regardless of the implantation orinduction therapy used (Wood et al., 1990).These data suggest that glycodelin serumlevels fail to reflect local changes at theimplantation site. It is thus unlikely thatglycodelin serum measurements can providea tool for early detection of implantation orof pregnancy.

First-trimester glycodelin serum con-centrations have been compared betweenwomen with: 1) natural conception, 2)pituitary desensitization with buserelin andovarian stimulation with human menopausalgonadotrophin followed by IVF and embryo

Erik Mandelin

16

transfer, 3) ovarian stimulation with clomi-phene citrate and human menopausal gona-dotropin followed by IVF and embryotransfer. In normal pregnancies, glycodelinlevels increased 7- to 8-fold during weeks4 and 10. This increase was, however, earlierand less prominent in group 2 and wasabsent from group 3 (Johnson et al., 1993a).These findings suggest that followingovarian stimulation, glycodelin synthesis isaltered in those pregnancies which do occur,indicating changes in endometrial function.

3.3. Reproductive immunology in relation toglycodelin

The human conceptus is not subject to thelaws of classical immunity. Despite theirbeing semi-allografts, the maternal immunesystem does not reject embryos (Moffett-King, 2002). Glycodelin reacts with manycells of the immune system and may be oneof the factors contributing to the local

immunosuppressive microenvironment atthe fetomaternal interface. As immunologicalfactors appear to play a role in infertility andin many pregnancy disorders (Gleicher et al.,1989; Bowen et al., 2002), it is of interest tonote that women with a history of recurrentmiscarriages have lower glycodelin concen-trations in uterine flushings and in serum(Tulppala et al., 1995; Dalton et al., 1998).Another finding of interest is that latesecretory phase glycodelin levels in uterineflushings are significantly lower in patientswith unexplained infertility than in those ofnormal controls (Mackenna et al., 1993).Immunomodulatory functions of glycodelinare illustrated in Table 3.

4. Glycodelin and cancer

4.1. Epithelial cancer of the ovary

Approximately 190 000 new cases ofovarian cancer are diagnosed worldwide

Immunomodulatory functions of glycodelin

Inhibits thymidine uptake in mixed lymphocyte cultures and phytohemagglutinin-stimulatedlymphocytes

Decreases synthesis of IL-1 and IL-2 and release of soluble IL-2 receptors in mitogenicallystimulated lymphocytes and mononuclear cell cultures

Inhibits NK-cell cytotoxicity

Stimulates IL-6 production of secretory endometrial epithelial cells

Inhibits T cell proliferation

Binding of alpha2-macroglobulin to glycodelin potentiates the inhibitory effect of glycodelinin T cell proliferation assays

Induces apoptosis of T cells

Inhibits chemotaxis of monocyte-like U937 cells

Elevates T cell receptor activation thresholds

Facilitates dephosphorylation of T cell receptor-induced phosphoproteins

Alters the dynamic sequestration of CD45 tyrosine phosphatase from T cellreceptor-triggered phosphoproteins

Potently inhibits E-selectin mediated cell adhesion

Author (s) (reference)

Bolton et al., 1987Pockley et al., 1988

Pockley and Bolton, 1989and 1990

Okamoto et al., 1991

Laird et al., 1994

Rachmilewitz et al., 1999

Riely et al., 2000

Mukhopadhyay et al., 2001

Vigne et al., 2001




Jeschke et al., 2003

Table 3.

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each year, with about 114 000 womendying annually (Stewart and Kleihues,2003), a higher mortality rate than forany other gynecological malignancy. It isthe leading cause of death from cancerin women in the western world (Bealeand Friedlander, 1995a). In 2000, Finlandhad 436 new cases diagnosed (age-adjustedincidence 9.5/100.000), ranking carcinomaof the ovary the eighth most common femalecancer in the population. The predicted 5-year relative survival rate between the years1997 and 1999 was 42% (Finnish CancerRegistry, 2003, www.cancerregistry.fi).Centralization of surgical treatment mayfurther improve survival rates at the popu-lation level (Kumpulainen et al., 2002). Thehistological subtypes of ovarian carcinomaare serous, mucinous, endometrioid,transitional cell, clear cell, Brenner, andundifferentiated. Serous is the most commonsubtype, representing 40 to 70% of allovarian carcinomas (Beale and Friedlander,1995b).

Radical debulking surgery is the corner-stone of effective treatment of ovarian carci-noma, essential for accurate staging, histo-logical classification, grading, and riskestimation, and for adjuvant chemotherapy(Schilder et al., 1995). Whereas clinical stageand histological grade are the gold standardsfor clinical management, a great number ofdiagnostic and prognostic markers have beenexplored (Table 4). CA-125, the best-evaluated tumor marker in ovariancarcinoma, was found over 20 years ago (Bastet al., 1981; Bast et al., 1983). It hasdiagnostic (Bast et al., 1983; Jacobs et al.,1990) and prognostic (Nagele et al., 1995;Cooper et al., 2002) value, and aids in theevaluation of response to treatment anddetection of relapse. Due to its elevatedlevels in many benign conditions, CA-125is, however, unable to detect early stagecarcinomas and has low specificity (Mills etal., 2001; Guppy and Rustin, 2002). A needfor new, more powerful diagnostic andprognostic tools thus exists. This study

addressed the possible prognostic role ofglycodelin.

In ovarian carcinoma, glycodelin proteinand mRNA occur (Waites et al., 1990;Kämäräinen et al., 1996; Horowitz et al.,2001), but circulating levels of glycodelinin ovarian carcinoma patients do not differfrom those in healthy individuals (Than etal., 1988).

4.2. Endometrial adenocarcinoma

Although endometrial adenocarcinomas donot synthesize glycodelin, treatment withmedroxyprogesterone acetate causesglycodelin protein to appear in the regionsof normal histology among specimens fromendometrial cancer (Wood et al., 1988). Inkeeping with the results concerningadenocarcinoma tissue, glycodelin serumlevels in patients with endometrialadenocarcinoma are not elevated (Than et al.,1988). The Ishikawa endometrial cancer cellline does not express glycodelin understandard cell-culture conditions (Chatzaki etal., 1994; Taylor et al., 2000).

The endometrial cancer cell line MFE-280expresses both glycodelin protein andmRNA (Hackenberg et al., 1998). RL95-2endometrial cancer cells react with apolyclonal antibody raised against a 16 aminoacid-long synthetic peptide (Gp) from aminoacids 69 to 83 of the glycodelin sequence(Poddar et al., 1998; Horowitz et al., 2001;Song et al., 2001). By use of this antibody,increased Gp serum levels could be found inendometrial, ovarian, and cervical cancerpatients (Horowitz et al., 2001). GlycodelinmRNA and Gp appear in endometrial andovarian tumor tissues. The endothelium ofblood vessels of endometrial cancer showspositive staining with the anti-Gp antibody,whereas only very low immunoreactivity isvisible in normal endometrial tissues.Immunosuppressive properties of glycodelinare suggested to facilitate tumor growth ingynecological malignancies (Song et al.,2001).

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Diagnostic and prognostic biomarkers in ovarian carcinoma

Established biomarkersCA 125

Carcinoembryonic antigen (CEA)

Free beta subunit of human chorionic gonadotropin

Tumor-associated trypsin inhibitor (TATI)

Potential biomarkersOvarian cystadenocarcinoma-associated antigen (OCAA)

Ovarian Cancer Antigens OvC1 and OvC6

Tissue polypeptide antigen (TPA)

HER-2/neu (p105)

Immunosuppressive acid protein (IAP)

Cancer-associated serum antigen (CASA)

AKT-2 gene

Cytokeratin fragment 21-1 (CYFRA 21-1)

Interleukin-6 (IL-6)

D-dimer

Lysophosphatidylcholine (lysoPC)

Plasminogen activator inhibitor 2 (PAI-2)

alpha-catenin

pKi67

Plasminogen activator inhibitor 1 (PAI-1)

Lysophosphatic acid (LPA)

Carboxyterminal telopeptide of type I collagen (ICTP)

Inhibin A

Matrix metalloproteinase-2 (MMP-2)

p53

Soluble members of the Mesothelin/ megakaryocyte potentiating

factor family

AIB1 gene amplification

Bax and Bcl-2

Estrogen and progesterone receptors

Soluble Fas

Colony stimulating factor-1 (CSF-1, M-CSF)

Human kallikrein 4

Human kallikrein 5

Prostasin

Cyclooxygenase-2 (COX-2)

Osteopontin

Proteomic patterns

Aminoterminal and carboxyterminal propeptides of type I collagens

Cyclin E

Human kallikrein 6

Human kallikrein 10

Vascular endothelial growth factor-D

Reference

Bast et al., 1983

Tuxen et al., 1995

Vartiainen et al., 2001

Stenman, 2002

Bhattacharya and Barlow, 1973

Imamura et al., 1978

Inoue et al., 1985

Slamon et al., 1989

Castelli et al., 1991

Ward et al., 1993

Bellacosa et al., 1995

Inaba et al., 1995

Scambia et al., 1995

Gadducci et al., 1996

Okita et al., 1997

Chambers et al., 1997

Anttila et al., 1998

Anttila et al., 1998

Chambers et al., 1998

Xu et al.,1998

Santala et al., 1999

Frias et al., 1999

Westerlund et al., 1999

Baekelandt et al., 1999

Scholler et al., 1999

Tanner et al., 2000

Baekelandt et al., 2000

Münstedt et al., 2000

Hefler et al., 2000

Xu et al., 1991;

Van Haaften-Day et al., 2001

Dong et al., 2001

Kim et al., 2001

Mok et al., 2001

Denkert et al., 2002

Kim et al., 2002

Petricoin et al., 2002

Simojoki et al., 2003

Farley et al., 2003

Diamandis et al., 2003

Luo et al., 2003

Yokoyama et al., 2003

Table 4.

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4.3. Glycodelin as a differentiation-relatedmorphogen

The endometrium undergoes monthly cyclesof proliferation and secretory activitycontrolled by ovarian steroids. Estrogeninduces proliferation and hyperplasia of theendometrial epithelium, and progesteronetransforms the endometrium from proli-ferative into secretory, thereby creating asuitable environment for embryo implanta-tion. Glycodelin is a product of highly differ-entiated endometrial secretory epithelium(Julkunen et al., 1986b). Interestingly, theovarian surface epithelium in which approx-imately 90% of human ovarian cancers arebelieved to arise (Auersperg et al., 2001) isglycodelin-negative (Kämäräinen et al.,1996).

Regarding the wide glandular expressionof glycodelin, its role has been studied as adifferentiation marker and in glandularmorphogenesis. MCF-7 breast cancer cells donot express glycodelin under normal cellculture conditions, but transfection withglycodelin cDNA causes dramatic changesin cell growth behavior by suppression ofproliferation and formation of gland-likestructures. The transfected cells, due toapoptosis, lose their ability to grow onsemisolid media and to express markers of

organized epithelia such as E-cadherin andcytokeratins 8 and 18. Other alterations inthe transfected cancer cells include redistrib-ution of β-catenin, upregulation of the α2integrin subunit, and loss of the α6 integrinsubunit (Kämäräinen et al., 1997). Insummary, these results indicate that transfec-tion of glycodelin cDNA can cause changesresulting in less aggressive cell growth andmore advanced differentiation, suggestingthat glycodelin may play an active role as adifferentiation-related glandular morphogen.

Nor do Ishikawa endometrial cancer cellssynthesize glycodelin in normal cell cultureconditions (Chatzaki et al., 1994; Taylor etal., 2000). When cocultured in the presenceof stromal cells and basement membranecomponents, however, the cancer cellsresume concomitant differentiation andglycodelin secretion (Arnold et al., 2002).

Glycodelin cannot be detected in normalK562 erythroleukemia cells, whereastreatment of the cells during 2 to 3 days withtetradecanoylphorbol acetate brings aboutdifferentiation and expression of bothglycodelin message and the protein(Kämäräinen et al., 1994; Morrow et al.,1994). Interestingly, during induceddifferentiation, K562 cells become resistantto NK-cell-mediated lysis (Gidlund et al.,1981).

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Aims of the Study

Given the contraceptive, immunosuppressive, and differentiative associations of glycodelin,this study was designed to address the following questions:

1) Does the levonorgestrel-releasing intrauterine system induce glycodelin synthesis inthe endometrium during the period of the fertile window?

2) Can the levonorgestrel-releasing subdermal implant induce endometrial glycodelinexpression during the fertile window?

3) Does controlled ovarian hyperstimulation influence glycodelin expression during theimplantation window?

4) How is glycodelin expressed during the implantation window in the oocyte recipients´mock cycles?

5) Is glycodelin expression in ovarian serous carcinoma associated with differentiationand prognosis?

21

Materials and Methods


1. Samples

Studies I, II, and V were approved by theEthics Committee of the Department ofObstetrics and Gynecology, HelsinkiUniversity Hospital, Study III by theInstitutional Review Board of the EasternVirginia Medical School, Norfolk, Virginia,USA, and Study IV by the InstitutionalReview Board of the New York PresbyterianHospital-Cornell University MedicalCollege, New York, New York, USA.

The tissue samples in Studies I to IV andV originated from endometrium and fromovary, respectively. All tissue samples wereformalin-fixed and paraffin-embedded. InStudies I to III, specimens were takenprospectively, studies IV and V involvedarchival samples (Table 5). Because endo-metrial adenocarcinomas do not appear tosynthesize glycodelin (Wood et al., 1988),we decided to investigate glycodelin expres-

sion in a clinical series of glycodelin-positiveovarian serous carcinomas (Kämäräinen et al.,1996).

2. Hormone treatments (III, IV)

The treatments were performed at the JonesInstitute for Reproductive Medicine(Norfolk, VA, USA) and the Center forReproductive Medicine and Infertility at theNew York Presbyterian Hospital-CornellMedical College (New York, NY, USA). TheCOH program of oocyte donors includedpituitary down-regulation with a GnRHagonist (Lupron; TAP Pharmaceuticals,Deerfield, IL, USA) starting in the mid-lutealphase of the preceding natural cycle or 5 daysbefore the end of a cycle of oral contraceptives(Damario et al., 1997), followed by admi-nistration of gonadotropins FSH (Gonal-F;Serono Laboratories, Norwell, MA, USA) or

Study

I

II

III

IV

V

Number ofpatients

611

10819

1519

101101

460

therapy/disease

LNG-IUDcopper-IUD

subdermal LNG-implantpostmenopausal controls

COH (oocyte donors)normal controls

COH (oocyte donors)mock cycles (oocyte recipients)

ovarian serous carcinoma

Patients treated at

Family Planning Unit, University of Jakarta, Jakarta, Indonesiaand Femeda Gynecological Center, Helsinki, Finland

Family Planning Unit, University of Jakarta, Jakarta, IndonesiaHelsinki University Central Hospital, Helsinki, Finland

The Jones Institute for Reproductive Medicine, Norfolk,Virginia, USA

The New York Presbyterian Hospital – Cornell UniversityMedical College, New York, USA

Helsinki University Central Hospital, Helsinki, Finland

Table 5. Subjects

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human menopausal gonadotropin (Pergonal;Serono) or both. Ovarian response wasmonitored by serial serum hormone meas-urements and transvaginal ultrasound exami-nation, with dosage of gonadotropins ad-justed appropriately. Human chorionicgonadotropin (Profasi; Serono) at a dose of5 000 to 10 000 IU was given to each patientto induce ovulation.

Oocyte recipients underwent a similarprogram. Women with ovarian functionreceived leuprolide acetate for down-regulation beginning in the mid-luteal phaseof the preceding menstrual cycle. Womenwith no ovarian function received no GnRH-agonist. Estrogen (Estraderm; Ciba Pharma-ceutical Co., Summit, NJ, USA) andprogesterone (Progesterone-in-oil; SterisLaboratories, Phoenix, AZ, USA) replace-ment therapies were initiated on day 1 andon day 15 of the mock cycle, respectively. Inthe actual oocyte transfer cycle, progesteronereplacement therapy was initiated one daybefore the donor’s oocyte retrieval, per-mitting embryo transfer on cycle day 18.

3. Immunohistochemistry

3.1. Glycodelin (I–V)

The immunoperoxidase stainings were madeat the Helsinki University Central Hospitalaccording to Kämäräinen et al. (Kämäräinenet al., 1996), with the only modification,the inactivation of endogenous peroxidase,being performed after incubation with thesecondary antibody. Briefly, the sections(5µm) were deparaffinated and rehydratedand then washed and subjected tomicrowave heat treatment to enhanceimmunoreactivity. Rabbit anti-glycodelinIgG from two rabbits (rabbit #32 in StudyI and rabbit #1 in Studies II–V) served asthe primary antibody and preimmunerabbit IgG as a negative control. Biotinyl-ated porcine anti-rabbit immunoglobulins(Dako A/S, Glostrup, Denmark) served assecondary antibodies. Endogenous per-

oxidase activity was blocked by incubationof sections in 0.6% perhydrol in methanol.Immunostaining was then carried out withthe Vectastain ABC kit (Vector Labora-tories, Burlingame, CA, USA), with 3-amino-9-ethyl-carbazole as a substrate. Allsections were counterstained with hema-toxylin. Secretory endometrium served inthe analyses as a positive control. Stainingspecificity of rabbit #1 antibody wasmonitored by absorbtion of the glycodelinantibody with purified glycodelin (16 µg/ml, overnight at +4º). No staining wasobservable in control experiments. That bothantibodies #32 and #1 reacted identicallywith purified glycodelin and amniotic fluidin Western blot analysis indicated that theantibodies were highly specific.

3.2. αvβ3 integrin (IV)

Immunoperoxidase stainings for the β3integrin subunit were performed in theUSA at the University of North Carolinaat Chapel Hill with the SSA6 monoclonalantibody (Lessey et al., 1994). Briefly, 8µmthick formalin-fixed, paraffin-embeddedsections were dewaxed and rehydrated andthen blocked with 2% normal goat serumto avoid eventual unspecific binding of thesecond antibody. Overnight at +4ºC, thesesections were then incubated with the firstantibody (SSA6). Careful rinsing wasfollowed by incubation with a secondbiotinylated goat anti-mouse antibody(Vector). Immunoperoxidase staining wasperformed with the Vectastain Elite ABCkit (Vector), diaminobenzidine serving asthe substrate. Finally, all sections weredehydrated and coverslips placed for micro-spcopic examination.

3.3. Progesterone receptors A and B (V)

Stainings of PRA and PRB were performedat Karolinska Institute in Stockholm, withprimary antibodies, generated in mice,purchased from Novocastra (Newcastle

23


upon Tyne, UK). PGR-312 antibodyrecognizes PRA and PGR-AB-SAN27antibody recognizes PRB (Mote et al.,2001).

Paraffin sections (4 µm) were dewaxedand rehydrated by standard procedures.Endogenous peroxidase was blocked byincubation in 1% hydrogen peroxide for 30minutes, and antigen demasking performedby microwaving (800 watts) sections in 0.01M citrate buffer, pH 6.0, for 20 min. Thesections were incubated for 1 h at +4°C withnormal goat serum (1:10 in PBS). Primaryantibodies were diluted 1:100 in PBScontaining 3% bovine serum albumin andincubated on the sections overnight at+4°C. For negative controls, the primaryantibody was replaced with PBS alone. Afterwashing, the sections were incubated witha biotinylated goat anti-mouse antibody(1:200 dilution, Vector) at room tempera-ture for 2 hours, followed by washing andincubation with avidin-biotin-horseradishperoxidase for one hour (Vectastain ABC-kit, Vector). Diamino-benzidine (Dako)served as substrate-chromogen for horsera-dish peroxidase. Finally, the sections werecounterstained with Mayer’s hematoxylinand dehydrated with graded ethanolconcentrations, followed by exposure toxylene and mounting.

3.4. Double stainings for glycodelin andprogesterone receptor A or B (V)

At the Helsinki University Central Hos-pital, stainings were carried out on 66tumors with the PicTure™-Doublestaining kit (Zymed Laboratories Inc., SanFrancisco, CA, USA) according to themanufacturer’s instructions. Briefly, thesections were deparaffinized, rehydrated,and subjected to microwave treatment toenhance immunoreactivity. Endogenousperoxidase activity was blocked with per-hydrol in methanol. Nonspecific binding

of secondary antibodies was eliminated bythe kit’s own blocking solution. Primaryantibodies against glycodelin and PRA andPRB were the same as above. The proge-sterone receptor antibodies were diluted1:300 in PBS. Anti-glycodelin IgG wasused at a concentration of 2.5 µg/ml (inPBS). The first antibodies were incubatedconcomitantly on tissue sections for 1.5hours at room temperature. The twosecondary antibodies used according to kitinstructions were a horseradish peroxidase-conjugated goat anti-mouse antibody forthe progesterone receptors and an alkalinephosphatase-conjugated goat anti-rabbitantibody for glycodelin. Substrate-chromo-gens for horseradish peroxidase and alkalinephosphatase were, respectively, diamino-benzidine and Fast-Red. The sections werecounterstained with hematoxylin.

4. RNA in situ hybridization (I, II)

The RNA in situ hybridization was doneat the Helsinki University Central Hospitalaccording to a protocol adapted from Kois-tinen et. al (1997). Briefly, the deparaffi-nized tissue sections (5µm) were treatedwith proteinase K and fixed with 4%paraformaldehyde in PBS. In vitro trans-cripts of sense and antisense probes wereprepared with a 413 base-pair cloned glyco-delin cDNA serving as the template. Afterthe acetic anhydride treatment, hybridi-zation was carried out with the use of adigoxigenin-labeled RNA probe. Afterwashing, immunologic detection of thedigoxigenin-labeled RNA probe by meansof antidigoxigenin phophatase Fab frag-ments (Boehringer-Mannheim, Mannheim,Germany) was done according to the manu-facturer’s instructions with Fast Red tablets(Boehringer-Mannheim). Sections werecounterstained with hematoxylin.

Erik Mandelin

24

5. CA-125 (V)

Until 1995, CA-125 measurements werecarried out by routine immunoradiometricassay (Centocor, Malvern, PA, USA), butlater, CA-125 levels were determined byimmunoenzymatic assay (Immuno1, Bayer,Tarrytown, NY, USA), with a cut-off valueof 35 kU/L. At this level and higher thetwo methods’ results were similar.

6. Statistical methods (I–V)

By the Kaplan-Meier method, survivalprobabilities were calculated, and survivalcurves between groups with differentglycodelin staining compared by log-ranktest. The Pearson Chi-Square test, Fisher’sexact test, and the Mantel-Haenszel chi-square test served for testing associationsbetween categorical variables, and Student’st-test for significance testing of normally

distributed data. Spearman rank correlationand the Mann-Whitney U-test served forsignificance testing of continuous non-parametric data. The Wilcoxon rank sum andKruskal-Wallis tests served to comparecontinous variables with categorical clinicaloutcome. Binary logistic regression was usedfor multivariate modeling of the correlationbetween explanatory variables and theglycodelin staining status, and analysis ofcovariance to evaluate the association ofglycodelin with serum hormone levels. TheCox proportional hazards model was usedin creating multivariate survival models. Inthis case, the backward stepwise methodwas used to select important variables. Forestimations of differences in 5- and 10-yearsurvival between the glycodelin-positiveand -negative groups, standard errors andp-values were calculated for differences insurvival probabilities. A value of P < 0.05was considered statistically significant.

25


Results

1. Endometrial glycodelin expressionin the fertile midcycle in womenwearing the levonorgestrel-releasingintrauterine device (I)

Endometrium from six LNG-IUD users wasanalyzed by immunohistochemistry and insitu hybridization during days 7 to 16, whichis the most fertile period of the menstrualcycle. Endometrium from all the six LNG-IUD-wearing women contained bothglycodelin protein and mRNA duringmidcycle, regardless of the duration of IUDuse. Glycodelin was localized in the endo-metrial glands, with only sporadic patchesin the stroma. Between different samples, thestaining intensity varied slightly. Glycodelin-positive endometria was less frequent amongcopper-IUD users (4 of 11 or 36%, P < 0.05).In keeping with earlier results, theendometrium of LNG-IUD-wearing womenshowed epithelial atrophy and stromaldecidualization (Silverberg et al., 1986).

2. Endometrial glycodelin expressionof women using the subdermallevonorgestrel-releasing contraceptiveimplant (II)

Of the total of 108, endometrial glycodelinstaining was evident in 80%. Of 82specimens from women with menstrualperiods, 69 (84%), and of 11 women witholigomenorrhea, 7 (64%) stained positive forglycodelin. Of the 12 women with levon-orgestrel amenorrhea, 7 (58%) expressedglycodelin in the endometrium. Glycodelinexpression was more common in womenwith menstrual cycles than in those with

implant amenorrhea (P < 0.05). Of the 19endometrial samples taken at fertilemidcycle, 89% stained glycodelin-positive.

Endometrial morphology showed prolifer-ative (71%), inactive/weakly proliferative(19%), menstrual or regenerating (6.5%), orother patterns (2.8%). In these groups glyco-delin was expressed in 79%, 71%, 100%,and 100% of the cases. Among the fourgroups of women, no significant differenceswere apparent between morphologic patterns.

Glycodelin immunostaining score wasunrelated to age or to duration of implantuse. Women with irregular bleeding had usedthe implant for a shorter time than had thosein other groups and were also older than thosewomen with menstrual periods or oligome-norrhea.

None of the 19 samples from postmeno-pausal women stained positive for glycodelin.The difference in glycodelin staining betweenthe women with levonorgestrel amenorrheaand hypoestrogenic amenorrhea was striking,with 58% of the samples from the formerstaining glycodelin-positive (P < 0.001).

By mRNA in situ hybridization, four ofthe five specimens studied from the midcycleof the women with levonorgestrel implantsshowed positive staining for glycodelin. ThemRNA was localized to the glandularepithelium only.

3. Endometrial glycodelin expressionin the luteal phase of stimulatedovarian cycles (III)

Endometrial glycodelin expression wasinvestigated in 15 oocyte donors under-going COH cycles and in 19 natural-cycle

Erik Mandelin

26

control patients; glycodelin was visualizedon day 16 of the menstrual cycle. Thereafter,in both cycle types, glycodelin expressionincreased, but with a relatively greaterproportion of cells staining in stimulatedcycles. A significant difference wasobservable between natural and stimulatedcycles with respect to glycodelin stainingover standardized cycle days throughout thewindow of embryo implantation (days 17–24, P < 0.001). Confidence intervals nolonger overlapped at around days 20 to 21.At that time, glycodelin scored on average3.5 (range, 51–75% of cells staining) inCOH cycles, whereas the average glycodelinscore in natural cycles was about 2.5 (range,26–50% of cells staining). Women under-going COH showed advanced endometrialhistology (≥ 1day) in 42% of their glandsand in 70% of their stroma. A positivecorrelation was demonstrable betweenglycodelin and advanced glandular dating(r = 0.63, P = 0.01).

Glycodelin was localized in endometrialglands, not in the stroma or in the surfaceepithelium. In natural cycles, endometrialglycodelin showed a strong positivecorrelation with serum estradiol (r = 0.5, P< 0.001), but glycodelin and progesteronedid not correlate with each other. Nor didany correlation appear in COH cyclesbetween glycodelin and either estradiol orprogesterone levels. No correlation wasevident between COH cycle biopsies andprogesterone/estradiol serum levels.Luteinizing hormone serum levels correlatedwith no other parameter analyzed.

4. Expression of glycodelin and αvβ3integrin at midluteal phase utilizingthe donor oocyte model (IV)

The study’s 101 oocyte recipients hadundergone a mock hormonal treatment andat least one oocyte recipient embryo transfer,with hormonal protocols in mock andtreatment cycles being identical. An

endometrial biopsy was taken from all mockhormonal-treated patients during cycle days21 to 23. Mean age of the oocyte recipientswas 40.2 years. The mean number of oocytesdonated was 9.3 and those normallyfertilized, 6.0; and the mean number oftransferred embryos was 3.2. Overallimplantation rate per embryo transferredwas 30.9%, and clinical pregnancy anddelivery rates per transfer were 66.3 and56.4%. Residual ovarian function existedin 72 women, and these women weretreated with GnRH agonist in both mockand actual treatment cycles. The other 29women with documented ovarian failurerequired no GnRH agonist down-regulation. Clinical outcomes betweenovarian function and ovarian failure patientsdid not significantly differ, except that thelatter were significantly younger and had ahigher mean number of normally fertilizedoocytes (P < 0.01 for both). Interestingly,ovarian-failure patients seemed to exhibitbetter implantation, pregnancy, anddelivery rates than did the ovarian functionsubgroup, although none of thesedifferences reached statistical significance.

Histopathological analysis of the biopsiesfrom mock-treated cycles revealed 62(61.3%) in-phase, 34 (33.7%) dyssynchro-nous, 2 (2.0%) immature, and 3 (3.0%)advanced endometria. The clinical outcomesof in-phase and dyssynchronous endome-trium were almost the same, includingimplantation rate per embryo transferred(32.5 vs. 29.9%), clinical pregnancy (66.1vs. 67.6%), and delivery rates (56.5 vs.55.9%). However, immunohistochemicalscores for glycodelin and αvβ3 differedsignificantly (P < 0.001, for both) in thetwo groups. Both glycodelin and αvβ3integrin correlated strongly with endo-metrial glandular dating (P < 0.001). Thiswas seen in the whole series and in thesubgroup of only cycle-day 22 and –23patients. Immunohistochemical stainings ofboth glycodelin and αvβ3 also correlatedwith each other (P < 0.001).

27

Results

A lag in endometrial glandulardevelopment, which usually is associatedwith normal stromal development, wasrelatively common on cycle day 21, but wasrare on day 23. On day 21, glycodelin andαvβ3 integrin staining was often relativelyweak or absent. A considerable increase inthe immunohistochemical staining ofglycodelin and αvβ3 integrin was evidentover this time interval (P = 0.0001 forboth). No associations existed betweenglycodelin/αvβ3 integrin immunohisto-chemical scores and clinical outcomeparameters (implantation rate, clinicalpregnancy and delivery rates) by univariatemethodology. Because immunohistochem-ical staining was fairly restricted and notevenly distributed on day 21, a subset ofimmunohistochemical staining results fromcycle days 22 and 23 were also analyzed bythe same methodology. These analyses,however, failed to find any significantassociations between clinical outcome andthe two endometrial markers. Absence ofstatistical power restricted the use ofstatistical methods in the 23-day grouponly. Neither the generalized estimatingequation models nor logistic regressionspredicting clinical outcomes usingendometrial markers and histologicalcharacteristics showed associations for eitherglycodelin or αvβ3 integrin.

5. Glycodelin in ovarian serouscarcinoma: associations withdifferentiation and survival (V)

5.1. Glycodelin expression in tumor tissue

The flat surface epithelium of normalpostmenopausal ovary (n = 5) was glyco-delin- negative in all cases but one, whereas

metaplastic cuboidal and columnar cells onthe ovarian surface and in the inclusioninvaginations and cysts stained weaklypositive for glycodelin. On the array slides,specimens from 522 patients were initiallyavailable. Due to loss of tissue during thestaining procedure, 460 patients remainedfor the analysis. Glycodelin was localized inthe cytoplasm of malignant epithelial cells,with vascular endothelium in tumor tissuebeing glycodelin-negative. Of all tumors,301 (65%) were glycodelin-positive and 159(35%) glycodelin-negative. In the glyco-delin-positive group, 214 (71%) showedweak and 87 (29%) showed strong staining.Evaluation of all immunohistochemicalstainings was based on the same criteria andmade by a single investigator. When lessthan 50% of the cancer cells stained positivefor glycodelin, staining was considered weakand when over half showed intense staining,was considered strong.

5.2. Glycodelin expression and clinicalparameters

Glycodelin expression correlated withtumor grade, so that percentages of glyco-delin- positive tumors were, overall: gradeI, 79%; grade II, 63%; grade III, 51%(Figure 1). Strong glycodelin staining wasless frequent among those with grade IIor III disease than with grade I tumors(P < 0.0001). A similar trend toward adecrease in glycodelin expression wasevident in tumors of advancing clinicalstage (P = 0.002, Figure 1). When bothgrade and stage were included in the logisticregression model to predict glycodelinexpression, grade remained the significantvariable (P < 0.0001).

Erik Mandelin

28

No difference was observable in initialtumor size, age, or presence of ascitesbetween glycodelin-positive and -negativesubjects. After primary debulking surgery,residual tumors were more frequently (P <0.001) small (<1cm) among glycodelin-positive (148 of 250 or 59%) than amongnegative patients (54 of 132 or 41%).

5.3. Glycodelin and survival in ovariancancer

Overall, patients with glycodelin-positivetumors had a higher survival rate than didthe glycodelin-negative (Table 6, Figures 2and 3), a difference notable for grade Itumors (P = 0.0196) or stage III disease (P= 0.0015) in which the 10-year survivalprobability of the patients with glycodelin-positive was more than twice that of thepatients with glycodelin-negative tumors (P< 0.001, Table 6, Figure 3). Within certain

*

Tumor stage

I(n=97)

II(n=63)

III(n=243)

IV(n=54)

Rel

ativ

e fr

equ

ency

(%

)

ns

**

0

10

20

30

40

50

60

70

80

0

10

20

30

40

50

60

70

80R

elat

ive

freq

uen

cy (

%)

Tumor grade

**BA

***

I(n=177)

II(n=117)

III(n=158)

Figure 1. Relative frequencies of glycodelin-positive tumors in respect to A) tumor grade. Significantdifferences observed between grade I tumors and grade II or III tumors (P < 0.01**, P < 0.0001***).B) tumor stage. Differences calculated by comparing stage II, III, and IV tumors each with stageI tumors (ns=not significant, *P < 0.05, **P < 0.01).

well-defined clinical groups belonging tothe same clinical stage and differentiationgrade category, patients with glycodelin-positive carcinoma showed, at certain timepoints, better overall survival than did thosewith glycodelin-negative carcinoma. Thiswas seen in the women with stage II/gradeIII carcinoma at 5 years (P = 0.003), stageIII/grade II carcinoma at 10 years (P =0.0001), stage III/grade III carcinoma at 10years (P = 0.04), and stage IV/grade IIIcarcinoma at 5 years (P = 0.02).

In the whole series, survival in eitherweakly or strongly staining groups wasbetter than that in the group with noglycodelin staining (P < 0.001), whereasbetween weakly and strongly staininggroups no difference appeared. Furtheranalyses were thus performed between theglycodelin-positive and negative-groups,without regard to staining intensity. Whenthe glycodelin expression status was entered

29

Results

Grade III

Gd +ve

Gd -ve

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

No. at risk Gd +ve 80 11 4 1 0 0 0 0

Gd -ve 78 15 4 1 1 1 1 0

35302520151050

p = 0.3626

Grade II

Gd +veGd -ve

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

Figure 2.Cumulative survival of patients with glycodelin-positive vs. -negative tumors, including all subjects andsubjects stratified by histological grade. Gd = glycodelin.

No. at riskGd +ve301 132 64 30 16 9 3 1 0

Gd -ve159 41 18 4 4 4 3 0 0

Years from diagnosis

All 460 patients

Gd +ve

Gd -ve

p < 0.0001

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

00 5 10 15 20 25 30 35 40

No. at risk Gd +ve 74 24 10 5 1 0 0

Gd -ve 43 8 4 0 0 0 0


p = 0.1054

302520151050

No. at risk

Gd +ve140 93 48 23 14 8 3 1 0

Gd -ve 37 19 10 3 3 3 2 0 0


1.0

0.9

0.8

0.7

0.6

0.5

Grade I

p = 0.0196

0 5 10 15 20 25 30 35 40

Gd +ve

Gd -ve


Cu

mu

lativ

e s

urv

iva

lC

um

ula

tive

su

rviv

al

into the univariate Cox multiple hazardsmodel, the hazard ratio was 0.57 (CI 0.44-0.74) for patients with glycodelin-positivetumors. In the backwards stepwise selectionof multiple variables, glycodelin did notemerge as an independent variable.

The possibility was considered that,during long storage of tissue specimens,intensity of immunohistochemical stainingmay decrease. We controlled for this factorby comparing staining intensities ofspecimens taken during different periods.

Erik Mandelin

30

Stage I

Gd +ve

Gd -ve

No. at riskGd +ve 74 52 27 16 11 8 2 0

Gd -ve 23 14 9 2 2 2 1 0

35302520151050

Cum

ulat

ive

surv

ival

p = 0.0995

0.7

0.6

1.0

0.9

0.8

Stage III

Gd +ve

Gd -ve

Cum

ulat

ive

surv

ival

2520151050

No. at risk Gd +ve 148 52 22 6 1 0

Gd -ve 95 19 5 0 0 0

p = 0.0015

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

0.9

1.0

Stage II

Gd +ve

Gd -ve

4035302520151050

No. at risk Gd +ve 46 26 15 8 4 1 1 1 0

Gd -ve 17 6 4 2 2 2 2 0 0

p = 0.2003

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

0.9

1.0

Stage IV

Gd +ve

Gd -ve

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

0.9

1.0

No. at risk Gd +ve 31 6 2 0 0 0 Gd -ve 23 6 2 1 1 0

1086420

p = 0.2315

Figure 3. Cumulative survival of patients with glycodelin-positive vs. -negative tumors stratified by clinicalstage. Gd = glycodelin.


Years from diagnosis Years from diagnosis


Comparison of the first 230 patients treatedfrom 1964 to 1988 and the subsequent 230patients treated from 1988 to 1999 showedno significant difference in glycodelinstaining intensity. Staining was weak in 110of 158 tumors (69.6%) and strong in 48 of158 tumors (30.4%) in the earlier group,and weak in 104 of 143 (72.7%) and strongin 39 of 143 (27.3%) in the latter (Pearson

chi square test, P = 0.553). As with theintensity, no significant difference appearedin the relative number of glycodelin-positive tumors between these two groups(158 of 230 vs. 143 of 230; chi square test,P = 0.141). In both groups, survival of thosepatients with glycodelin-positive tumorswas higher than for those glycodelin-negative (log rank test, P = 0.0001 and P =

31

Results

Cancer

All subjects

Grade I

Grade II

Grade III

Stage I

Stage II

Stage III

Stage IV

AllGd +veGd -ve

AllGd +veGd -ve

AllGd +veGd -ve

AllGd +veGd -ve

AllGd +veGd -ve

AllGd +veGd -ve

AllGd +veGd -ve

AllGd +veGd -ve

N

460301159

17714037

1177443

1588078

977423

634617

24314895

543123

95% CI (%)

45-5550-6130-47

74-8678-9051-82

26-4528-5212-43

16-318-26

20-43

82-9685-9964-98

50-7549-7836-86

33-4637-5419-39

0-170-130-33

95% CI (%)

35-4641-5417-34

69-8373-8840-75

14-3214-364-33

6-193-202-23

74-9278-9647-90

36-6438-7012-69

22-3527-447-26

---

c

c

a

a

nsns

ns

ns

b

b

ns

ns

ns

ns

a

a

nsns

c

c

ns

ns

ns

ns

ns

ns

ns

ns

b

b

d

d

TABLE 6. 5- and 10-year overall survival rates of patients with glycodelin (Gd) positive and negative ovarian serouscarcinoma (ns=not significant, a) p<0.05, b) p<0.01, c) p<0.001, d) p<0.0001).

5-year overall survival (%)

505539

808466

364027

241731

899281

626361

404629

95

15

10-year overall survival (%)

404726

768057

232519

121213

838768

505441

293516

000

0.0117, respectively). In backwards step-wise selection of multiple variables, timeof diagnosis (binary variable: 1964-1988 or1988–1999) remained an independentprognostic factor (hazard ratio = 0.63; CIlow = 0.46, CI high = 0.85; P = 0.003),showing that in the latter group the risk ofdeath was significantly smaller.

Regarding treatments that took placebefore or after cisplatin, no difference existedin intensity of glycodelin staining betweenthese groups. Among glycodelin-expressingtumors treated before cisplatin, staining wasweak in 57 of 81 (70.4%) and strong in 24of 81 (29.6%), and during cisplatin, wasweak in 157 of 220 (71.4%) and strong in

63 of 220 (28.6%, Pearson chi square test,P = 0.866). By log rank test, higher survivalremained significant for patients withglycodelin-positive tumors, both before andafter cisplatin (P = 0.0113 and P < 0.0001).During the cisplatin period, survival signi-ficantly improved (multivariate analysis,hazard ratio = 0.54; CI low 0.38, CI high0.77; P < 0.001).

Glycodelin-positive tumors were lessfrequent after adoption of paclitaxel (53 of97; 54.6%) than before it (248 of 363;68.3%) (Pearson chi square test, P = 0.012),with no significant difference in stainingintensity. Before paclitaxel, glycodelin-positive staining was weak in 178 of 248

Erik Mandelin

32

(71.8%) and strong in 70 of 248 (28.2%)tumors, and during paclitaxel staining wasweak in 36 of 53 (67.9%) and strong in 17of 53 (32.1%) tumors (Pearson chi squaretest, P = 0.575). Unlike in the other groups,the paclitaxel group (n = 97), showed nodifference in survival between glycodelin-positive and -negative tumors (P = 0.3498),although the difference was highlysignificant for the group before paclitaxel(n = 363; P < 0.0001). In the multivariatemodel, time of diagnosis (binary variable,before or after adoption of paclitaxel) wasnot an independent prognostic factor in thepaclitaxel group, probably because of thesmall number of patients.

Despite improved diagnostic facilities,carcinomas at a relatively more advancedstage were referred for treatment at theHelsinki University Hospital during theperiod of more advanced chemotherapy.This is illustrated by mean stage of diseaseon admission, when the patients weredivided into various treatment categories.Thus, in the first group of 230 patientstreated until 1988, the mean figure for stagewas 2.41 compared to 2.71 in the lattergroup of 230 (Mann-Whitney test, P =0.00035). The same trend appeared with

respect to adoption of cisplatin (2.35 vs.2.63, P = 0.005) and paclitaxel treatments(2.5 vs.2.78, P = 0.007)

5.4. Expression of progesterone receptor A and Bin tumor tissue in relation to glycodelinexpression

In the 460 ovarian serous tumors, PRA wasfound in 33% and PRB in 14%. Wherepresent, both receptors were localized innuclei of the malignant cells. PRA waspresent in 37% of the glycodelin-positiveand 26% of the -negative tumors (P < 0.02),and PRB in 17% and 8% (P < 0.02). AllPRB-positive tumors also stained for PRA,whereas only 44% of the PRA-positivespecimens were PRB-positive. In doublestaining (n = 66), glycodelin and PRA/PRBexpression appeared in the same as well asin different cells.

5.5. Serum CA-125-levels and endometrialglycodelin expression

No correlation existed between serum CA-125 levels and glycodelin immunostainingstatus in tumors (P > 0.05, for all).

33

Results

Discussion

This study addresses three major areas inwhich the differentiative association ofglycodelin appears to be important. Thefirst is the fertile window, in which absenceof contraceptive glycodelin is relevant forfertilization, and induction of glycodelinsecretion would enhance the contraceptivemicroenvironment within the uterus. Thesecond is the window of implantation, inwhich the immunosuppressive effects ofglycodelin are likely to contribute to thefetomaternal defense mechanisms. Thethird area of the differentiative associationis related to cancer.

1. The fertilization window (I, II)

The fertilization window comprises the 5days preceding ovulation and the day ofovulation itself. During these 6 days,intercourse is most likely to result inpregnancy (Wilcox et al., 1995). Themechanisms of action for IUDs can beclassified as occurring before or afterfertilization. Possible prefertilization me-chanisms for IUDs include inhibition ofsperm migration and of viability (Moyer etal., 1970; Sagiroglu, 1971; Croxatto et al.,1973; Settlage et al., 1973; Tredway et al.,1975; Koch, 1980), interference with ovumdevelopment and transport (Ortiz andCroxatto, 1987), and damage to or de-struction of the ovum prior to fertilization(Alvarez et al., 1988). The possible post-fertilization mechanisms include destructionor damage of the early embryo before or afterit has reached the uterine cavity and theprevention of implantation (Ortiz andCroxatto, 1987).

This study addressed another potentialprefertilization mechanism. Both glycodelinprotein and mRNA appeared in theendometrium of LNG-IUD users in themidcycle, when glycodelin is normallyabsent (Julkunen et al., 1986b; Seppälä etal., 1988b; Waites et al., 1988a). Thisindicates that the glycodelin was inducedlocally by levonorgestrel, by the IUD itselfas a foreign body, or by both. Of greatinterest is the finding that all the LNG-IUDwearers had glycodelin in their endo-metrium throughout the otherwise fertilephase of the cycle: the six-day period endingin ovulation (Wilcox et al., 1995). Some ofthe copper-IUD-wearing women also hadinappropriate glycodelin expression in theendometrium. That this was less frequentthan among LNG-IUD-wearers suggeststhat local progestogen can induce or enhanceendometrial glycodelin synthesis. Theeffects of copper on induction of glycodelinsecretion remain to be elucidated.

Considering the potential contraceptiveeffects of glycodelin (Oehninger et al., 1995;Morris et al., 1996), these results aresignificant because at the time whenspermatozoa migrate into the Fallopiantubes, the uterine cavity is expected to befree of contraceptive activity. Prior exposureto endometrial glycodelin preventsspermatozoa from binding to the zonapellucida, with no effect from prior exposureof the zonae to endometrial glycodelin(Oehninger et al., 1995). Prior exposure toendometrial glycodelin may thus provide amechanism by which in LNG-IUD-wearingwomen, and also in some copper-IUD-wearing women the fertilizing capacity ofthe spermatozoa is reduced. This was not

Erik Mandelin

34

totally unexpected, because in post-menopausal women cyclical oral estrogen/progestagen replacement therapy raisesserum glycodelin in those whose uterus isintact, but not in those with a hysterectomy(Seppälä et al., 1987a).

Although we studied few women, ourtracking of glycodelin mRNA and proteinduring the menstrual cycle’s periovulatoryphase anticipates larger clinical studies onthe consistency of glycodelin expressionoutside its normal cyclical expression inwomen wearing a LNG-IUD and othertypes of IUD. Our study also raises thepossibility of a new contraceptive substancethat can inhibit fertilization.

Given the induction of glycodelin bylocal application of levonorgestrel (I), wehypothesized that levonorgestrel releasedfrom subdermal contraceptive implants mayalso cause inappropriate endometrial glyco-delin expression. To evaluate this possibleeffect, we studied endometrial biopsies fromour implant-wearing women. Endometrialglycodelin expression appeared in 80% ofthem. Of the specimens, 19 were takenduring the critical fertile 6-day period whenfertilization normally takes place (Wilcoxet al., 1995). Of these, 89% stained positivefor glycodelin. Because glycodelin is notnormally expressed in the endometriumduring this time of the cycle (Julkunen et

al., 1986b; Seppälä et al., 1988b; Waites etal., 1988a), and considering the potentialcontraceptive activity of glycodelin (Oeh-ninger et al., 1995; Morris et al., 1996), theappearance of endometrial glycodelin maybe yet another mechanism by which, inwomen using levonorgestrel implants, thefertilizing capacity of spermatozoa isreduced.

Contrary to the case of women with LNG-IUDs, not all of the specimens from LNG-releasing subdermal implant-wearingwomen contained glycodelin, perhaps be-cause LNG from subdermal implants reachesthe endometrium systemically. EndometrialLNG concentration is much lower after syste-mic administration than is levonorgestreldelivered locally from an LNG-IUD(Croxatto et al., 1981; Nilsson et al., 1982).

In subdermal implant-wearing women,serum levonorgestrel levels decrease withincreasing periods of use (Diaz et al., 1987),so glycodelin expression was analyzed withrespect to length of implant use. Thatglycodelin was not significantly affected bylength of use indicates that the LNGconcentration in long-term users must havegenerally been adequate to induceexpression of endometrial glycodelin. Serumlevels of LNG and progesterone in LNG-IUD- and subdermal implant-wearingwomen are illustrated in Table 7.

Normal menstrual cycle

Postmenopausal women

COH-treatment cycles

LNG-IUD-wearing women

Subdermal LNG-implant-wearing women

Steroid

Progesterone

Progesterone

Progesterone

Progesterone

Levonorgestrel

Progesterone

Levonorgestrel

Specifications

Follicular phaseLuteal phase

Follicular phase (prior to hCGadministration)

Ovulatory cycles (luteal phase)Anovulatory womenDuring the first 3 months of treatmentAfter one year of treatment

Ovulating women (average peak value)Anovulatory women1-12 month of treatment49-60 month of treatment

(nmol/l)

<6.06.0-64.0

<6.0

<2.9

>15.9<4.80.80.4

29.6<3.21.10.9

Reference(s)

Larsen et al., 2003

Wilson and Foster, 1985

Hofmann et al., 1993Copperman et al., 1995

Nilsson et al., 1980Nilsson et al., 1980Nilsson et al., 1980Nilsson et al., 1980

Croxatto et al., 1982Croxatto et al., 1982Croxatto et al., 1981Croxatto et al., 1981

Table 7. Serum progesterone and levonorgestrel concentrations in different clinical contexts.

35

Discussion

Yet another interesting observation wasthat the endometrium from women withLNG-associated amenorrhea was morelikely to be glycodelin-positive; in hypo-estrogenic amenorrhea the endometriumwas always glycodelin-negative, a differenceprobably resulting from differing endocrinemicroenvironments within the uterus.What accounts for the difference observedin glycodelin expression is certainly theremaining ovarian activity in implant-related amenorrhea (Xiao et al., 1995).

Levonorgestrel influenced endometrialdifferentiation. In keeping with earlierreports (Silverberg et al., 1986), irrespectiveof duration of LNG-IUD use, majormorphological changes in endometriumfrom LNG-IUD wearers were epithelialatrophy and stromal decidualization. Inwomen wearing subdermal implants, thedifference between glycodelin-positive andglycodelin-negative specimens was notreflected in endometrial changes such asthese. Interestingly, unlike in normal cycles,glycodelin staining was also evident in thespecimens with proliferative/inactiveendometrium from LNG implant users.

2. The implantation window (III, IV)

Of the two hormonally differing conditionsstudied, the first (III), during theimplantation phase, was related to theexcessive hormonal microenvironmentfollowing controlled ovarian hyperstimul-ation of women with functional ovaries. Thisis usually associated with multiple folliclessecreting estradiol and with multiple corporalutea following oocyte retrieval or ovulation.In addition to estradiol and progesterone, thecorpus luteum also secretes relaxin. Theeffects on glycodelin expression of such ahigh- endocrine microenvironment wasanother point of interest during theimplantation phase of the cycle.

The second study (IV) involving theimplantation phase addressed relative or

absolute ovarian failure as the cause ofinfertility. This condition was treated by IVFusing donor oocytes, and it required estrogen/progesterone replacement treatment in orderto achieve uterine receptivity. In the absenceof ovarian function, there existed none of thewoman’s own corpus luteum to secreteprogesterone and relaxin. Only progesteronewas replaced. Glycodelin expression duringsuch a low-endocrine microenvironment wastherefore of particular interest during theimplantation phase of the cycle, due to theabsence of ovarian relaxin secretion.

According to immunohistochemicalstudies, endometrial glycodelin expressionbegins in natural cycles 4 to 5 days afterovulation (Seppälä et al., 1988b). Thiscorresponds to the glycodelin concentrationmeasured in endometrial tissue (Julkunenet al., 1986b) and uterine fluid (Li et al.,1993b). In this study, glycodelin expressionwas evident in natural cycles as early as oncycle day 16 (LH+3). Previous studiesestimated the phase of the menstrual cycleby histological maturation, whereas thepresent study employed accurate timing ofovulation (LH surge). The start of glycodelinsecretion parallels the process ofimplantation, in which the human embryopenetrates the lining of the endometrialepithelium sampled at LH +4/5 (Landgrenet al., 1996).

Some controversy exists as to the role ofrelaxin in progesterone-regulated glycodelinexpression (Seppälä et al., 2002). This studyshowed no correlation between glycodelinand progesterone, which again indicatestemporal differences in secretion and a poss-ible role for other factors. Our observation ofa strong positive correlation between serumestradiol and glycodelin is an example of theeffects of estrogen priming, a fact inagreement with earlier reports (Seppälä et al.,1989; Li et al., 1992).

Endometrial glandular-stromal dyssyn-chrony is relatively frequent in the mid-lutealphase endometrium of women undergoingmock cycles (Droesch et al., 1988). In this

Erik Mandelin

36

study, endometrial sampling in mock-treated women was performed during themenstrual cycle days 21 to 23, the periodgenerally considered to be the most repre-sentative of the window of implantation(Sarani et al., 1999; Acosta et al., 2000). Asin previous studies (Droesch et al., 1988),oocyte recipients with glandular-stromaldyssynchrony showed no disadvantagecompared with those with in-phaseendometria. For women with in-phase anddyssynchronous endometria, clinicalpregnancy-, implantation-, and live-birthrates were fairly similar. Curiously, succesfulpregnancies also occurred in women whoseendometrium showed retarded or advancedglandular elements.

The clinical outcomes of women with atleast some residual ovarian function werecompared with outcomes of those withovarian failure, because only women in theformer group were treated with a GnRHagonist. No differences appeared betweenclinical outcomes of these two groups otherthan a younger mean age and a greater meannumber of normally fertilized oocytes amongthe ovarian failures, observations compatiblewith earlier ones demonstrating thattreatment with a GnRH analog does notaffect implantation rates in women receivingoocyte donation (Remohi et al., 1994).

Many putative markers of uterine re-ceptivity have been explored (Lindhard etal., 2002; Nikas and Aghajanova, 2002;Stavreus-Evers et al., 2002; Aghajanova etal., 2003). Two of them, glycodelin andαvβ3 integrin, in the present study weregood markers of luteal phase glandularmaturity, with a strong correlation betweenthe immunostaining results of either markerand endometrial glandular maturation.Values for glycodelin and αvβ3 integrinimmunostaining also strongly correlatedwith each other. These results are in linewith earlier ones suggesting thatdiminished expression of αvβ3 integrinand/or glycodelin is associated with retarded

endometrial differentiation (Klentzeris etal., 1994; Krasnow et al., 1996; Meyer etal., 1999).

None of the statistical models we usedwas able to detect any association betweenimmunostaining intensities of glycodelin/αvβ3 integrin and clinical outcomevariables (implantation rate, clinicalpregnancy, or live-birth rates). It would havebeen of interest to focus on the chronologicalsubgroup with limited endometrial-glandular dyssynchrony (cycle-day 23group), but this was impossible due to lackof statistical power.

One may speculate on why retarded en-dometrial development – plus its associatedlimited or absent expression of putativemarkers of implantation – do not influenceoutcome in mock treatment cycles. Oneobvious explanation is the normal variationbetween menstrual cycles, even in the sameindividual. Another may be that in exo-genous hormonal replacement cycles, thewindow of implantation is simply shifted.Retarded endometrial glandular histologyis often apparent in the mock cycles, andwhen women receive oral micronizedestradiol instead of transdermal estradiol,glandular histology is delayed even further(Krasnow et al., 1996). Yet another possibleexplanation may be that, due to a suboptimalendometrial milieu, the embryo undergoesdiapause (Bergh and Navot, 1992; Tarin andCano, 1999), a physiological arrest welldescribed in other mammals (Spindler et al.,1995; Song et al., 1998).

An alternative hypothesis is that endo-metrial maturation, receptivity, and implan-tation are all primarily determined by adialogue between the embryo and theendometrium. Although this study wasunable to address the possible contributionof embryo-derived signals, results from invivo and in vitro models suggest that hCGcan up-regulate both glycodelin and αvβ3integrin (Simon et al., 1997; Hausermannet al., 1998).

37

Discussion

3. Glycodelin and differentiation (V)

The postmenopausal ovary does not usuallyexpress glycodelin, unlike the fertile phaseovary, which has glycodelin present inseveral cell types such as luteinized gra-nulosa cells (Kämäräinen et al., 1996; Tseet al., 2002). This difference may be relatedto the postmenopausal ovary´s decreasedhormonal activity.

In ovarian carcinoma, glycodelin waslocalized not in the vascular endotheliumof tumor tissues, but in the cytoplasm ofmalignant cells. Studies employing anti-glycodelin peptide (Gp) antibodies, whichreact with vascular endothelial cells, showa different pattern (Zhou et al., 2000; Songet al., 2001). Because of the lack of glycodelinmRNA in umbilical cord vein endothelialcells, glycodelin peptide immunoreactivityappears to result not from synthesis butfrom uptake (Zhou et al., 2000). In ovariancumulus cells, similarly, glycodelin proteinexists but not its corresponding mRNA (Tseet al., 2002). Regardless of differences inimmunolocalization, immunologicalmimicry of the two types of antibodies isobvious. The similarities of anti-Gpantibodies and the conformational anti-glycodelin antibodies need investigation toreconcile their differentiative (glycodelin)vs. angiogenic (Gp) associations (Song et al.,2001; Arnold et al., 2002).

Of the 460 ovarian cancer patients in ourstudy, 65% of their tumors containedglycodelin, its expression being morecommon in the well-differentiated carci-nomas that showed papillary or glandularstructures. One would have expected immu-nohistochemical staining intensity to havedecreased during long storage of tissuespecimens as in this study, but this was notthe case, perhaps because glycodelin is stableand well protected by its high carbohydratecontent. The proportion of glycodelin-positive and -negative tumors remainedroughly similar over the long period of timeexcept that, after the adoption of paclitaxel

treatment, the frequency of glycodelin-positive tumors was lower than for thetumors treated before paclitaxel. This maybe related to the increased proportion ofadvanced stage carcinomas referred fortreatment as the new treatment modalitiesbecame available in the tertiary referralcenters. Paclitaxel is a cell-cycle-specificdrug that has dramatically improved theprognosis of ovarian carcinoma. Itsefficiency may well be the reason why, inthis study, no difference appeared betweenthe survival of patients with glycodelin-positive or -negative tumors in thepaclitaxel subgroup.

Changes in tumor grade are certainlysecondary to genetic and biochemicalchanges that have taken place in the cellsundergoing neoplastic transformation.Contact with the basement membraneregulates epithelial cell growth and normalorganization (Capo-Chichi et al., 2002).Both the basement membrane and thestromal cells are required in this process sothat, prior to its emergence, the tumorigenicphenotype must overcome the suppressiveeffects of the surrounding microenviron-ment (Roskelley and Bissell, 2002). Loss ofthe basement membrane componentscollagen IV and laminin may be an earlyevent in the pre-neoplastic transformationof ovarian surface epithelium (Capo-Chichiet al., 2002). The importance of the micro-environment has been demonstrated byexperiments in which, when coculturedwith normal stromal cells and basementmembrane components, endometrial adeno-carcinoma cells resume glycodelin secretionand concomitant differentiation (Arnold etal., 2002). Experiments on breast cancercells in which transfection of glycodelincDNA is followed by retarded growth, for-mation of gland-like structures, and expres-sion of epithelial cytokeratins (Kämäräinenet al., 1997) provide further evidence forthe differentiative association of glycodelin,suggesting that glycodelin is not only amarker of epithelial differentiation but may

Erik Mandelin

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play a fundamental role in glandularmorphogenesis. Reduced glycodelinexpression in poorly differentiated ovariancancer, unlike the case in well-differentiatedserous carcinomas, may be caused by de-differentiation or loss of genetic material.Genetic changes – both gains and losses –are frequent in ovarian tumors (Pere et al.,1998) and include a high frequency ofdeletions at chromosome 9 (Devlin et al.,1996) that harbors the glycodelin gene (VanCong et al., 1991).

Glycodelin and PRA/PRB frequentlyoccurred in the same malignant cells, butnot always. It is difficult to claim functionalassociation between the two, although thiscannot be ruled out because the glycodelingene consists of four putative gluco-corticoid/progesterone response elements(Vaisse et al., 1990). Both PRA and PRBfunction as ligand-activated transcriptionfactors, but the two isoforms have differingfunctional characteristics (Vegeto et al.,1993; McDonnell and Goldman, 1994).Ligand-activated PRA and PRB bothinduce glycodelin expression in endometrialadenocarcinoma cells in vitro (Gao et al.,2001). Our positive correlation betweenglycodelin and PRA and PRB in ovariancancer showed that both types of receptorsappeared in these glycodelin-expressingcancers, even in the same cells. Becausemany glycodelin-positive ovariancarcinomas were progesterone-receptor-

negative, however, glycodelin expressionmust also involve regulatory factors otherthan progesterone.

Glycodelin expression was not related toinitial tumor size, but glycodelin-expressingresidual tumors were smaller. As this was aretrospective observation, interpretation ofthis observation remains speculative. Asimple interpretation would be betteroperability of glycodelin-expressing tumors,and this is also supported by survival data.The finding that glycodelin does notcorrelate with CA-125 levels was not totallyunexpected, as earlier studies indicate noassociation between serum CA-125 levelsand histological grade of differentiation inovarian carcinoma (Tholander et al., 1990).

Concerning this association betweenglycodelin and differentiation, notsurprisingly, those patients with glycodelin-expressing tumors survived longer. This wasespecially noticeable in patients withhistological grade I or clinical stage IIIcancer. Trends were best seen in grade I andstage III tumors probably because thesegroups were sufficiently large to haveadequate power to detect differences unlikeother stages and grades with smallernumbers of subjects which were thereforemuch less likely to reveal any differences.Whether glycodelin expression is primaryor secondary to the changes in tumor gradeemerging as an independent variable is asyet undetermined.

39

Discussion

Summary and Conclusions

This study addressed the differentiation-related propensity of glycodelin in threeclinically relevant contexts, the fertilewindow, the window of implantation, andovarian carcinoma.

In studies on the fertile window, bothglycodelin protein and mRNA appeared inthe endometrium of users of the levo-norgestrel-releasing IUD and subdermalimplant. This is the phase when glycodelinis normally absent, indicating that glycodelinexpression was induced by levonorgestrel.Appearance of endometrial glycodelin duringthe fertile window may be one of themechanisms by which, in women using thesecontraceptives, the fertilizing capacity ofspermatozoa is reduced. These results shedlight on the changes in endometrial micro-environment during certain forms of contra-ception, and potentially they offer new leadsfor studies on other substances that mayreduce fertility by inducing inappropriateendometrial glycodelin secretion.

In studies addressing the window ofimplantation, oocyte donors who underwentCOH treatment had significantly higherendometrial glycodelin expressionthroughout the implantation window thanwomen with natural cycles. Endometrialglandular-stromal dyssynchrony was rela-tively common in the mock cycles of oocyterecipients but did not affect clinical outcome.Interestingly, in progesterone/estrogen-stimulated mock cycles, endometrialglycodelin expression was frequently present.These results lend further support to theimportance of progesterone for glycodelinsynthesis.

In serous ovarian carcinoma, glycodelinexpression was more frequent in well

differentiated than in poorly differentiatedcarcinomas and was also more frequent inearly- than in advanced-stage carcinomas.Although in multivariate analysis glycodelinwas not an independent variable, patientswith glycodelin-expressing tumors showeda higher 5-year overall survival than didthose with glycodelin-negative tumors, adifference notable in patients with grade Itumors or stage III disease. In the lattergroup, the 10-year survival probability ofpatients with glycodelin-positive tumors wasmore than twice that of women withglycodelin-negative tumors. This was alsotrue within well-defined clinical categories,e.g., in stage III/grade II and stage III/gradeIII carcinomas, in which patients withglycodelin-positive tumors had a signi-ficantly better 10-year overall survival thandid those with glycodelin-negative tumors.It can therefore be concluded that in ovariancancer, glycodelin is a differentiation-relatedglycoprotein predicting better prognosis.

Earlier in vitro studies have revealed aclear association between glycodelin andcellular differentiation. The present studyshows how crucial this association can bein a clinical context. It will be importantto investigate the cellular mechanisms un-derlying glycodelin’s differentative actions.Data concerning the effects of glycodelinon expression of other genes and proteinswithin a cell are still limited. Anotherimportant issue is that signals from adjacentcells and tissues can influence epithelialglycodelin synthesis. For this purpose,three-dimensional cell culture modelsmimicking the in vivo endometrialmicroenvironment may provide usefulinformation.

Erik Mandelin

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Acknowledgements

All my friends and colleagues from theresearch laboratory, thank you for creating afantastic working atmosphere. It has beengreat to work with you: Can Hekim, JariLeinonen, Tiina-Liisa Erkinheimo, HeiniLassus, Laura Sarantaus, Kristiina Nokelai-nen, Henrik Alftan, Susann Karlberg,Annikki Löfhjelm, Gynel Arifdshan, AnittaTamminen, Jakob Stenman, Anu Harju,Kristiina Hotakainen, Piia Vuorela, PiaVahteristo, Annukka Paju, Oso Rissanen,Meerit Kämäräinen, Marianne Niemelä,Kirsi Saukkonen, Ari Ristimäki, MaaritLeinimaa, Taina Grönholm, Helena Taski-nen, Anne Ahmanheimo, Liisa Airas, Jo-hanna Tapper, Maija Harrela, Susanna Lin-tula, Eira Halenius, Tarja Kalme, HeliNevanlinna, Outi Kilpivaara, Katja-AnneliWathén, Reetta Jalkanen, Sari Tuupanen,Ping Wu, Wang-Ming Zhang, Lei Chu, andSirpa Stick, as well as Patrik Finne, who isalso warmly acknowledged for his advice instatistics.

Mirva Hatakka, Leena Vaara, Laila Selki-nen, and Raili Alanne for helping me withall practical concerns.

Carol Norris for skillful author-editingof the language of this thesis.

Clinical research would not be possiblewithout volounteers. I want to express mywarmest thanks to all the patients whoparticipated in these studies.

All my friends outside the laboratory forjoyful moments together, accompanied withsong, schnaps, and laughter.

My parents Christina and Matti and mysisters Maria and Helena for their love, care,and encouragement throughout my life.

This study was carried out at the Depart-ment of Obstetrics and Gynecology underthe former head Professor Markku Seppäläand present head Professor Olavi Ylikor-kala, and at the Department of ClinicalChemistry headed by Professor Ulf-HåkanStenman. All three I sincerely thank forproviding me with excellent workingfacilities.

I wish to express my warmest gratitudeto all those who have made this studypossible, with special reference to thefollowing:

Professor Markku Seppälä, my supervisor,for introducing me to glycodelin andguiding me through the demanding worldof science. I deeply admire his creativity,expertise, and efficiency. I am most gratefulfor the opportunity of sharing insights withsuch an experienced and internationallyrenowned scientist.

Docent Seija Grénman and ProfessorJorma Isola, the official referees of thisthesis, for their careful review and valuablecomments on the manuscript.

Docent Riitta Koistinen and Dr. HannuKoistinen, the contribution of whom wascrucial for this project. They also receive mythanks for their friendship and support.

Torsten Wahlstöm, Leif Andersson, OlliCarpén, Johanna Arola, Arto Leminen,Mervi Halttunen, Jan-Åke Gustafsson,Guojun Cheng, Ralf Bützow, Biran Affandi,Sam Brown, Sergio Oehninger, BruceLessey, Zed Rosenwaks, Mark Damario, andHoward Jones Jr. for fruitful collaboration.

Professor Ulf-Håkan Stenman for hiskind and keen interest in my work.

41

Acknowledgements

My wife Johanna, to whom I dedicate thiswork, for her love and continuous supportduring this project and for bringing somuch happiness into my life.

This study was financially supported byFinska Läkaresällskapet, K. AlbinJohanssons stiftelse, Nylands Nation, theAcademy of Finland, the Federation of

Finnish Pension and Life InsuranceCompanies, the Finnish Medical SocietyDuodecim, the Helsinki University CentralHospital Research Funds, the BiomedicumHelsinki Foundation, the University ofHelsinki, the Finnish Society of Sciences andLetters, and the Finnish Cancer Foundation.

Nagu, July 2003

Erik Mandelin

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Erik Mandelin

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Glycodelin and differentiation in endometrium and ovary