Immunohistochemical Expression of Endometrial L-Selectin Ligand Obtained During the Natural Menstrual Cycle From Women Undergoing Autologous Endometrial Coculture

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Immunohistochemical expression of endometrialL-selectin ligand is higher in donor egg recipientswith embryonic implantationMousa I. Shamonki, M.D.,a Isaac Kligman, M.D.,a Jaime M. Shamonki, M.D.,b

Glenn L. Schattman, M.D.,a Elizabeth Hyjek, M.D.,b Steven D. Spandorfer, M.D.,a

Nikica Zaninovic, M.S.,a and Zev Rosenwaks, M.D.a

a The Center for Reproductive Medicine, Weill Medical College of Cornell University, and b Department of Pathology,New York Presbyterian Hospital, New York, New York

Objective: To correlate L-selectin ligand (LSL) expression in human endometrium with embryonic implantation.Design: Retrospective cohort analysis.Setting: University-based fertility center.Patient(s): Donor egg recipients (DERs) who underwent programmed hormonal replacement for ET with priormock cycle luteal phase endometrial biopsy.Intervention(s): Immunohistochemical expression of LSL using MECA-79 antibody was examined. Slides werescored with a new scoring system, the IHC-Level (range 0–4) as follows: strength of staining—absent (0), weak(1), or strong (2); plus distribution of staining—absent (0), �50% of tissue (1), and �50% (2). Cellular apex andcytoplasm were scored independently in both the endometrial glandular and surface epithelium.Main Outcome Measure(s): Endometrial LSL expression in pregnant versus nonpregnant patients.Result(s): MECA-79 IHC-Level of the apex of surface epithelium was significantly higher for pregnant versusnonpregnant DERs (3.8 vs. 3.4). When controlling for embryo morphology, there continues to be a significantdifference in apex score on surface epithelium (3.8 vs. 3.3, respectively). The new scoring system resultscorrelated with an established scoring system, the HSCORE.Conclusion(s): We demonstrate significantly higher expression of LSL at the apex of human endometrial surfaceepithelium obtained from DERs with embryonic implantation. Furthermore, we present the IHC-Level, a methodof evaluating immunohistochemistry that may be applied to other markers of endometrial receptivity. (FertilSteril� 2006;86:1365–75. ©2006 by American Society for Reproductive Medicine.)

Key Words: L-selectin ligand, L-selectin, endometrium, embryo, infertility, immunohistochemistry, implantationwindow, HSCORE, IHC-Level, uterine receptivity

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mbryonic implantation is a relatively poorly understoodnd complex biological process in reproductive biology. Inumans, there is a distinct “window of implantation” duringhe midluteal phase, requiring an appropriately developedmbryo and an adequately hormonally primed endometrium1). In a 28-day cycle, this window has been described toccur as narrowly as cycle days 21–23 (2–5) and as broadlys cycle days 20–24 (6–9). During the past 25 years, greatdvances have been achieved in clinical reproductive med-cine; however, causes and treatments of infertility related tohe endometrium largely remain undiscovered.

Recently, Genbacev et al. (10) identify candidate mole-ules likely involved in the initial step of implantation,howing that L-selectin, expressed by human embryos at theatching blastocyst stage, initiates interactions with the uter-ne lining. L-selectin is a carbohydrate-binding protein (11),r lectin, that has been determined to enable circulatingeukocytes to bind to high endothelial venules through roll-

eceived April 20, 2005; revised and accepted April 9, 2006.eprint requests: Mousa I. Shamonki, M.D., Reproductive Medicine As-sociates of New Jersey, 111 Madison Avenue, Morristown, New Jersey

n07960 (FAX: 757-299-2558; E-mail: [email protected]).

015-0282/06/$32.00oi:10.1016/j.fertnstert.2006.04.035 Copyright ©2006 American Soc

ng adhesion in flowing blood (12). Genbacev et al. (10)escribe up-regulation of carbohydrate ligands, present onhe surface of the receptive endometrium, that are bound by-selectin. These L-selectin ligands (LSL) begin to appear inreater quantities on the surface of receptive endometrium,t greatest concentration in the midluteal phase, at the sameime the embryo at the blastocyst stage begins to hatch fromhe zona pellucida (ZP) and express L-selectin on the outerellular surface (10).

This ligand-receptor interaction may act as the initial stepn embryonic implantation at the maternal–fetal interface.herefore, if this interaction between embryo and endome-

rium is essential for implantation, then decreased ligand oreceptor expression may lower implantation rate. However,o study to date has demonstrated a direct relationship be-ween LSL expression and successful implantation in theuman, as examining endometrium during a pregnancy cycleoses an ethical impasse.

Examination of endometrial tissue obtained from donorgg recipients during the midluteal phase of a programmedmock” cycle might allow for comparison with the preg-
ancy outcome of an eventual ET cycle, as hormonal re-
1365Fertility and Sterility� Vol. 86, No. 5, November 2006iety for Reproductive Medicine, Published by Elsevier Inc.

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lacement during a mock cycle and ET cycle are hormonallydentical at our center. In addition, to our knowledge, thereave been no studies examining for the presence of the LSLn endometrium exposed to exogenous estrogen (E) and P, ass the case with women undergoing hormonally programmedycles in preparation to receive embryos conceived fromggs donated by other women. Donor egg recipients haveigh pregnancy rates (PRs) independent of age (13) be-ause oocyte donors are young, typically �36 years ofge, and as a result, donor egg recipients have a relativelyigh PR per ET secondary to the high quality of donormbryos (13–21). For these reasons, sensitivity to detectndometrium-associated implantation failure should bencreased in this population.

Our central hypothesis is that LSL is very important forndometrial receptivity and that it plays an integral role inhe apposition and attachment of the embryo to receptivendometrium leading to embryonic implantation in the hu-an. It is believed that some women have endometrium-

ssociated infertility, and therefore a subgroup of endome-rial abnormalities, may be related to a relative absence orysfunction of the L-selectin receptor–ligand interaction. Toxamine this hypothesis, we performed immunohistochemi-al (IHC) expression of LSL in endometrium obtained fromonor egg recipients and compared levels of expression withregnancy outcome. Furthermore, we present a newly devel-ped and reliable method for analyzing immunohistochem-cal markers for implantation in human endometrium.

ATERIALS AND METHODStudy Designll cycles of oocyte donation performed at The Center foreproductive Medicine and Infertility, Weill Medical Col-

ege of Cornell University and The New York Presbyterianospital from October 22, 2002 to June 23, 2003 were

eviewed. Approval for this research was obtained (Protocol903-936) from the Institutional Review Board of the Com-ittee for Human Rights in Research of the study center. For

tudy inclusion, only patients with archived endometrialissue obtained from a preceding midluteal (cycle days 21–3) mock treatment cycle endometrial biopsy at The Nework Presbyterian Hospital–Weill Medical College of Cor-ell University were selected. The archived endometrialissue was stained for endometrial dating and immunohisto-hemical analysis. Seventy-eight cases fit all inclusion cri-eria and were studied further.

ocyte Donorsll oocyte donors were 21–35 years of age with the excep-

ion of one sister donor who was 36 years old. Oocyte donorsere anonymous volunteers (n � 72) or sisters of recipients

n � 6). Every donor underwent careful screening, as de-cribed by Moomjy et al. (22), where medical, psychologi-al, and genetic evaluations were undertaken. In our center,
ocytes from a single anonymous donor are shared with two I
1366 Shamonki et al. L-selectin ligand expression and pregn

otential recipients. Shared oocyte donation was successfuln the majority of anonymous donation cases (23, 24). Eachecipient received an average of 8–9 oocytes, evenly dividedccording to the quality and maturity. A small minority ofnonymous donors (n � 6) were given a single recipientither because of a low number of oocytes retrieved, aistory of prior compromised fertilization, or lack of anotherhenotypically appropriate match.

onor Treatmentocyte donors underwent controlled ovarian hyperstimula-

ion using a step-down protocol, with a typical initiating dosef 150–225 IU of pure FSH or a combination of pure FSHnd hMG. During the midluteal phase of the previous men-trual cycle, GnRH agonist (GnRH-a) down-regulation usingeuprolide acetate (LA; Lupron; TAP Pharmaceuticals, Deer-eld, IL) was initiated at a dose of 1 mg in 0.2 mL given SCnd decreased to 0.5 mg daily on the third day of theenstrual cycle. Women at high risk for ovarian hyperstimu-

ation syndrome, or women with oligoamenorrhea, received8 days of oral contraceptives (OCs) overlapping for the lasteek with LA administration (25).

ecipientsatients who underwent hormonally programmed cycles re-ulting in donor ET from October 22, 2002 until June 23,003, were identified. Patients underwent a prior pro-rammed mock cycle using E2 patches and P injections withn endometrial biopsy in the midluteal phase for endometrialating as described later.

ecipient Treatmentach donor egg recipient underwent identical hormone re-lacement for the oocyte donation transfer and mock cycles.atients who were having regular spontaneous menses un-erwent luteal phase GnRH-a down-regulation with a dailyC injection of 1 mg in 0.2 mL of LA. Leuprolide acetateas continued daily until the initiation of P supplementation.n days 3–7 of their menses, each patient had a baseline

ransvaginal ultrasound examination and baseline E2 andSH level assays. Upon documentation of adequate hor-onal down-regulation (E2 �70 pg/mL, FSH �20 mIU/L), the recipient started with a 0.1-mg transdermal E2 patch

Climara; Berlex, Montville, NJ) placed every other dayypically for 5 days. Estradiol patch dosage was increased to.2 mg for 4 days, followed by 0.4 mg for 4 days, and finallyecreased to 0.2 mg during the mock luteal phase.

Serum hormone levels were checked on days 7, 11 (E2

nly), and 15 (E2 and P) of stimulation and E2 patching wasdjusted accordingly to maintain a serum level between 150nd 400 pg/mL. Transvaginal ultrasound imaging was thenerformed on replacement day 15. If the endometrial thick-ess was measured as adequate (typically �6 mm), 25 mg of
M P supplementation (50 mg/mL of oil) was initiated on
ancy Vol. 86, No. 5, November 2006

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ycle day 15 and increased to 50 mg daily beginning day 16f the replacement cycle. Endometrial biopsy was performedn mock cycle day 21, 22, or 23 for endometrial dating. Ifhe endometrial lining on day 15 was thin, E2 patching wasontinued for one additional week before P supplementation.

Hormonal replacement during a subsequent ET cycle waserformed in an identical manner to the mock replacementycle. The recipient began transdermal E2 replacement ap-roximately 1 week before the commencement of gonado-ropin stimulation of the donor. Embryo transfers were per-ormed on the fourth day (day 3 embryo) or the sixth dayday 5 embryo) of P supplementation.

Recipients who were having irregular menses were placedn OCs for 28 days while subcutaneous GnRH-a was ad-inistered for the last 7 days of OC treatment until a GnRH-

nduced menses was observed. Occasionally, OCs were usedo synchronize a recipient cycle with a donor cycle. Therotocol was then continued as described. Patients deemedenopausal with consistently elevated FSH levels and with-

ut menses for �1 year did not require GnRH-a down-egulation.

mbryo Grading Methodologymbryo morphology was graded according to criteria byeeck (26). Briefly, embryo morphology on day 3 receivedgrade between 1 and 5, with 1 indicating an embryo of theighest quality and 5 indicating an embryo of the poorestuality. Embryo grade on day 3 was related to the number,ize, shape, and cytoplasmic appearance of blastomeres inddition to percentage of embryonic fragmentation. Embryorading for a day 3 transfer only reflects the morphology ofhe finest embryo transferred and is not an average score forll embryos transferred. Embryo grade for day 5 embryosncluded three scores: a score for blastocyst expansion from

for least expanded to 3 for most expanded, a score forhe inner cell mass from A to D for highest quality toowest quality, respectively, and a similar letter score forhe trophectoderm.

ndometrial Biopsyndometrial biopsy was obtained on day 21, 22, or 23 ofxogenous hormonal replacement during a mock cycle usingrotating plastic pipelle-biopsy catheter (Pipelle de Cornier;rodimed, Neuilly-en-Thelle, France) after the cervix wasleansed with betadine. Tissue was fixed in formalin andmbedded into paraffin. Tissue sections of 5 �m in thicknessere placed on slides, and either stained with hematoxylin &

osin (H & E) in preparation for endometrial dating, orrepared for immunohistochemical reactivity for LSL asescribed later.

ndometrial Datingndometrial dating was performed by one experienced phy-
ician using the dating criteria of Noyes et al. (27). Endo- m
ertility and Sterility�

etrial stroma and glands were dated separately, and thehysician dating the slides was blinded to patient demo-raphics, specific day of endometrial biopsy, and pregnancyutcome.

mmunohistochemistrymmunohistochemical staining was performed on paraffinissue sections using a TechMate 500 automated immunos-ainer (Ventana Medical Systems, Tucson, AZ). The stainingas performed according to a modified MIP protocol usingChemMate ABC peroxidase secondary detection System

Ventana Medical Systems). Monoclonal rat IgM antibodieshat bind to L-selectin ligands: MECA-79 (dilution 1:100,oncentration 5 �g/mL) and HECA-452 (dilution 1:100,oncentration 5 �g/mL) (DB Biosciences Pharmingen, Saniego, CA) were used for staining. MECA-79 recognizes-selectin ligand sulfate and carbohydrate epitopes andECA-452 reacts with a carbohydrate domain shared by

ialyl Lewisx (sLex) and sialyl Lewisa (sLea) antigens. Forhe secondary antibodies, a biotinylated mouse antirat IgMclone G53–238) antibody (BD Biosciences Pharmingen)as added in dilution 1:200, concentration 2.5 �g/mL. Per-xidase reaction was developed using Liquid DAB Substratehromogen provided in the kit (Ventana Medical Systems).ections of endometrial biopsies were stained without anti-en retrieval. Negative isotypic controls with rat IgM at theame concentration as primary antibodies and positive con-rols with tonsil were run in parallel.

mmunohistochemical Analysiswo physicians evaluated all specimens and were blinded toatient demographics, cycle day of biopsy, and pregnancyutcome. All slides were evaluated on a Nikon microscopesing �40 to �400 magnification (Nikon Inc., Kawasaki,anagawa, Japan). Slides were scored using a new scoringethod (IHC-Level) as follows: strength of staining—absent

0), weak (1), or strong (2) (Fig. 1A,B), plus distribution oftaining—absent (0), �50% of tissue (1), and �50% (2).oth scores were added for a total score range from 0 to 4.ellular apex and cytoplasm were scored independently

Fig. 1C) in both the endometrial gland crypt and surfacepithelium (Fig. 1D).

The IHC-Level was used to simplify the reading process,s four different regions of the endometrial epithelial cellsere analyzed using two antibodies for a total of eight scoreser subject. Two months after the original readings, the samebservers independently scored each slide using the IHC-evel blinded to patient demographics, cycle characteristics,utcomes, and any prior readings.

The IHC-Level scoring system is a modification of theSCORE, which has been used clinically with low interob-

erver and intraobserver variation (28, 29). This method waslso previously used to evaluate donor egg recipient endo-
etria for glycodelin and osteopontin expression (24). For
1367

FIGURE 1

Example of MECA-79 intensity scores (range 0–2) for the endometrial surface cellular apex (A and B).(A) received a score of 1 and (B) received a score of 2 (magnification, �400). (C) Illustration showing thetwo regions (apex and cytoplasm) that receive immunohistochemical scores for the L-selectin ligand.(D) Illustration showing the two regions of endometrial epithelium (surface and gland crypt) that receiveimmunohistochemical scores for the L-selectin ligand. Comparison between HECA-452 (E) and MECA-79(F) expression in endometrial tissue obtained from the same patient. Note the greater cytoplasmicexpression with HECA-452 and greater apical expression with MECA-79.

Shamonki. L-selectin ligand expression and pregnancy. Fertil Steril 2006.

1368 Shamonki et al. L-selectin ligand expression and pregnancy Vol. 86, No. 5, November 2006

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he HSCORE, scores per slide range from 0 for no stainingo 4 for maximal staining. The HSCORE � � Pi(I � 1),here I is the intensity of staining with a value of 1, 2, or 3

weak, moderate, or strong, respectively), and Pi is theercentage of stained epithelial cells for each intensity, vary-ng from 0–100% and 1 is a correction for optical density24). The HSCORE is typically used to give one score for thentire slide. HSCOREs were performed on each slide by theame physicians while blinded to any patient descriptors,efore IHC-Level scores, and outcomes to correlate with theesults of the IHC-Level. To be consistent, four HSCOREsrather than one) were given to each slide, where cell apicalurface and cytoplasm were scored independently in both thendometrial glandular crypt and surface epithelium as withhe IHC-Level.

tatistical Analysistatistical analysis was performed using SPSS for windowsoftware (version 11.0.1, SPSS, Inc. and LEAD Technolo-ies, Inc., Chicago, IL). The IHC staining results, patientharacteristics, and pregnancy outcome were analyzed usinghe t-test, Mann-Whitney U test, Fisher’s exact test, or �2 testhere applicable, and correlations were performed using

he Pearson (parametric) and Spearman’s (nonparametric)orrelations.

TABLE 1Comparison of patient demographics, mock cycrecipients with positive (�) and negative (�) preg

Characteristic�Pr

(n

Recipient age (y) 40.Body mass index (kg/m2) 25.Prior deliveries 0.Successful IVF cycles 0.Cycle day of biopsy 21.Day 15 endometrial thickness (mm) 9.Day 21 E2 (pg/mL) 435.Day 21 P (ng/dL) 31.Stromal date 21.Donor age (y) 28.Oocytes retrieved 8.Mature oocytes 6.Fertilized oocytes 5.Embryos transferred 2.Cycle day of embryo transfer 3.Cycle endometrial thickness (mm) 10.Day 3 embryo transfer cycles (%) 83Blastocyst transfer cycles (%) 17Day 3 embryo grade (1–5) 1.Note: Values are means � SD. Values of P�.05 are cons



ESULTSatient Demographicsf the 78 patients who fit all inclusion criteria, the overallR was 75.6% (59/78), the clinical PR was 71.5% (55/78),nd the implantation rate was 41.5% (76/183). The clinicaliscarriage rate was 12.8% (10/78) and the live delivery rateas 57.7% (45/78) per ET. There were no differences be-

ween nonpregnant (n � 19) and pregnant (n � 59) donorgg recipients with regard to recipient age, gravida, parity,ody mass index, number of successful and failed IVFycles, day 3 FSH and E2 levels, cycle day of biopsy,hickness of the endometrial stripe during the mock and ETycles, levels of E2 and P on the day of endometrial biopsy,landular and stromal dating or percentage of gland/stromayssynchrony, egg donor age, number of oocytes retrieved,ature, fertilized, frozen, and transferred, blastocyst grade,

nd percentage of day 3 or blastocyst transfers (Table 1).here was a trend for better day 3 embryo grade in theregnant patients (P�.08), but this was corrected for, as wille described later.

ean Ligand Expression with MECA-79xpression of LSL was localized to endometrial epitheliumith no visible expression in the stroma. Slides stained withECA-79 showed much higher expression at the cellular

nd ET cycle characteristics between donor eggcy.

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�Pregnancy(n � 19) P value

3.8 39.5 � 3.7 .196.3 24.3 � 5.4 .670.5 0.3 � 0.6 .660.3 0.2 � 0.5 .300.8 21.6 � 1.0 .731.9 10.2 � 2.2 .37241.7 466.3 � 246.5 .6310.3 29.4 � 8.7 .551.0 21.8 � 0.8 .773.3 27.9 � 3.0 .933.5 8.6 � 4.4 .922.8 7.0 � 4.0 .732.4 5.3 � 3.5 .780.6 2.2 � 0.6 .180.7 3.3 � 0.8 .962.4 10.5 � 2.4 .9249) 78 (n � 15) NS10) 22 (n � 4) NS

0.4 2.1 � 0.7 .08d significant. NS � nonsignificant.

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pex as opposed to the cytoplasm, as compared with HECA-tained tissue where cytoplasmic expression was greater thanpical expression (Fig. 1E,F). When using the IHC-Levelcoring method, MECA-79 mean expression of the surfacepex was significantly higher in pregnant versus nonpreg-ant recipients (3.8 vs. 3.4, respectively, P�.003) as wasECA-79 overall apex score (glands � surface) (7.1 vs. 6.4,

espectively, P�.023). No differences were noted in theytoplasm scores (Table 2).

When controlling for embryo morphology by includingnly patients with day 3 transfers with embryo grade �2 orlastocyst transfers, there continues to be a significant dif-erence in MECA-79 surface apex expression in pregnantersus nonpregnant recipients (3.8 vs. 3.3, respectively,�.02) and MECA-79 overall apex score (7.0 vs. 6.3,

TABLE 2Comparison of MECA-79 IHC-Level between donopregnancy.

Parameter�Pregnancy

(n � 59)

Gland apex 3.3 � 0.7Gland cytoplasm 1.6 � 1.2Surface apex 3.8 � 0.4Surface cytoplasm 0.9 � 1.1Gland score 4.9 � 1.6Surface score 4.7 � 1.2Apex score 7.1 � 1.0Cytoplasm score 2.6 � 2.0Note: Values are means � SD. Values of P�.05 are cons


TABLE 3Comparison of MECA-79 IHC-Level between donopregnancy corrected for embryo quality.

Parameter�Pregnancy

(n � 53)

Gland apex 3.3 � 0.7Gland cytoplasm 1.5 � 1.2Surface apex 3.8 � 0.4Surface cytoplasm 0.9 � 1.1Gland score 4.8 � 1.7Surface score 4.7 � 1.2Apex score 7.0 � 1.0Cytoplasm score 2.4 � 2.0Note: Scores adjusted by excluding patients with day 3 e

refers to the best-quality embryo only, not the mean ofValues of P�.05 are considered significant.



�.03) (Table 3). Patients with a MECA-79 IHC-Level of 4t the apex of surface epithelium had a PR of 89.1% (41/46)ersus an IHC-Level of �3 where the PR was 63.2% (12/9), P�.025.

omparison, Correlation, and Validation of the IHC-Levelntraobserver and interobserver variability was minimal asndicated with a high degree of correlation between scoresor each observer (P�.001), and scores between observersP�.001) for all regions of tissue analyzed. The HSCOREethod showed statistically similar results when comparedith the new method. IHC-Level and HSCORE show a highegree of correlation in all areas of evaluated tissue, with�.001 (Table 4).

g recipients with positive (�) and negative (�)


3.1 � 0.7 .171.6 � 1.4 .993.4 � 0.6 .0031.0 � 1.3 .754.7 � 1.9 .574.4 � 1.5 .356.4 � 1.2 .0232.7 � 2.4 .84

d significant.

g recipients with positive (�) and negative (�)


3.0 � 0.7 .251.3 � 1.2 .603.3 � 0.6 .020.5 � 0.9 .304.3 � 1.9 .393.8 � 1.4 .056.3 � 1.3 .031.9 � 2.0 .39

yo grade �2 (scale: 1–5; 1 � best). Day 3 embryo gradembryos transferred. Stain score values are means � SD.

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ean Ligand Expression with HECA-452 andonparametric Analysis of Ligand Expressiony Both Antibodiesean HECA-452 IHC-Levels before and after correction for

mbryo quality showed no significant differences betweenregnant and nonpregnant recipients; however, when exam-ning the percentage of each score, gland apex score stained

TABLE 4Comparison of MECA-79 IHC-Level and HSCOREand negative (�) pregnancy corrected for embry

Region�Pregnancy

(n � 53)�Pregn

(n � 1

Gland apexIHC-Level 3.3 � 0.7 3.0 �HSCORE 1.9 � 0.9 1.6 �

Gland cytoplasmIHC-Level 1.5 � 1.2 1.3 �HSCORE 1.0 � 0.6 0.8 �

Surface apexIHC-Level 3.8 � 0.4 3.3 �HSCORE 3.2 � 0.6 2.6 �

Surface cytoplasmIHC-Level 0.9 � 1.1 0.5 �HSCORE 0.2 � 0.2 0.1 �

Note: The IHC-Level and HSCORE show similar results betissue scored. Values are means � SD. CorrelationsCorrelation coefficients reported as Pearson and Spear

a Values of P�.05 are considered significant.b Two-tailed test; values of P�.01 are considered signific


TABLE 5Nonparametric analysis between donor egg recipcorrected for embryo quality.

Stain�Pregnancy

(n � 53)

HECA-452Gland apex 28.3Gland cytoplasm 66.0Surface Apex 24.5Surface cytoplasm 24.5

MECA-79Gland apex 43.4Gland cytoplasm 0Surface apex 77.4Surface cytoplasm 0

Note: Values are percentages of highest IHC-Level (score



ith HECA-452, and surface apex scores stained with bothECA-79 and HECA-452 showed significantly higher per-

entages of maximal scores in pregnant versus nonpregnantecipients (Table 5). When examining the endometrial surfacepex for �50% versus �50% distribution of the ligand, 77.4%41/53) of pregnant versus 41.7% (5/12) of nonpregnant pa-ients showed uniform distribution of MECA-79 (P�.03).

tween donor egg recipients with positive (�)ality.

y Pvaluea

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CorrelationP valueb

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.15n pregnant and nonpregnant recipients for each region ofween MECA-79 IHC-Level and HSCORE stain scores.tests, respectively.

ts with positive (�) and negative (�) pregnancy


16.7 .0558.3 .688.3 .038.3 .32

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ISCUSSIONecent research has implicated embryo interaction through-selectin with endometrial LSL as the initiating event in

mplantation. Our study findings demonstrate for the firstime a direct relationship between LSL expression and theikelihood for embryonic implantation, albeit this tissue wasot obtained during the ET cycle due to ethical concerns, butather during a mock cycle. Although tissue was obtainedrom a cycle other than that of the ET where differences ofSL expression may exist, endometrial characteristics arexpected to be as similar as possible due to identical hor-onal stimulation.

Ligand expression detected by immunohistochemistryas significantly higher and more uniform on the apicalortion of endometrial surface epithelium in patients withmplantation despite no discernable differences in patientemographic, stimulation cycle, and endometrial datingharacteristics between pregnant and nonpregnant donor eggecipients. In a study examining discordant outcome fromhared donated oocytes, Garcia-Velasco et al. (30) found noifference in age, serum E2, endometrial thickness, indica-ions for oocyte donation, male semen parameters, numberf oocytes received, fertilization rate, intracytoplasmicperm injection rate, or embryo quality between recipientsith and without pregnancy, thus highlighting that otherore subtle factors for implantation failure require further

nvestigation. Although we detected LSL expression in bothhe apex and cytoplasm of the endometrial surface andlandular crypt epithelium, expression at the apex of endo-etrial surface epithelium showed the only consistently sig-

ificant relationship with overall PR. This finding supportsur understanding regarding the initial step of implantation,s the embryo makes contact with the apex of endometrialurface epithelium. Here the L-selectin receptor–ligand in-eraction is believed to initiate embryonic attachment to thendometrial surface.

Human endometrium is an exquisitely hormonally sensi-ive tissue that undergoes complex structural and biochem-cal changes during the menstrual cycle. These changes, nowppreciable at the molecular level, are essential for embry-nic implantation. Using electron microscopy, several stud-es have demonstrated formation of pinopodes, dome-shapedrojections at the apical portion of endometrial surface epi-helium, circa the window of implantation that are believedo be important markers for endometrial receptivity (31–37).iochemical markers with proposed or demonstrated impor-

ance include leukemia inhibiting factor (38, 39), Hoxa-1040), Hoxa-11 (41), osteopontin, and integrins (42–48). Likehe functional epitope of LSL, �v�3 integrin (49) is local-zed to the cellular apex of receptive endometrial surfacepithelium (pinopode region) where initial interaction withhe embryo occurs (46).

The L-selectin receptor and ligand interaction between thelastocyst and endometrium, respectively, precedes a well-
ocumented cascade of events leading to successful embry- t

nic implantation (10). Our immunohistochemical studiesave confirmed the presence of LSL resulting from exog-nous hormone exposure. Previous work has shown ele-ated expression of this ligand in midluteal endometriumbtained either during a natural menstrual cycle (10, 50)r after gonadotropin stimulation for IVF (10). Our studyemonstrates that up-regulation of LSL appears to beependent on adequate and sequential exposure to sexteroids. Endometrial exposure to increasing P leads touteal maturation and the up- or down-regulation of hun-reds of genes in the human as demonstrated by DNAicroarray technology (9, 51–53).

Before DNA microarray technology, most of these genesere previously unknown with regard to their intimate in-olvement with cyclic menstrual tissue changes. Kao et al.9) found up-regulation of messenger RNA coding for-acetylglucosamine-6-O- sulfotransferase-1 (GlcNAc6ST-), an enzyme that provides a sulfation modification on LSLllowing recognition by MECA-79 (54). MECA-79 (55), aidely used antibody, effectively blocks the tethering and

olling of lymphocytes along high endothelial venules56), and has the same sulfation-dependent recognition as-selectin (57). The function-blocking activity ofECA-79 is explained by overlap of its sulfated carbohy-

rate epitope with the L-selectin recognition determinant54). Future studies examining endometrial tissue can useaser capture microdissection (58) with DNA microarrayechnology so that changes in gene regulation can be local-zed to glandular epithelium or stroma.

Although it is clear that LSL is maximally expressed inhe midluteal phase secondary to P exposure, little is knownf the factors or conditions that may alter endometrial ligandxpression. Recent work using DNA microarray analysis hasemonstrated that infertile women with a history of endo-etriosis, relative to fertile controls, have lower levels oflcNAc6ST-1 during the implantation window (59), imply-

ng that endometriosis may affect implantation by decreasinghe level of functional LSL. However, studies comparingonor egg recipients with mild or severe endometriosis ver-us disease-free controls found no difference in implantationate or PR, suggesting that any potential negative effect ofndometriosis on the uterine environment is undetectable, ateast in the donor egg recipient population (60) and may, inact, have more of an effect on oocyte and embryo quality61). A study, examining women undergoing traditional IVF,emonstrated that the follicular hormonal milieu is altered inatients with endometriosis relative to disease-free controls.ollicular fluid P and interleukin-6 were higher, and vascularndothelial growth factor was lower in IVF patients withndometriosis, which may result in lower-quality embryoshat have a reduced ability to implant (62). In our study, thereere only two patients with endometriosis, both with aaximal MECA-79 IHC-Level of 4 at the surface apex. One

f the two women became pregnant, but there are far too fewubjects in this analysis to ascertain the impact of endome-
riosis on LSL expression in donor egg recipients.

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The process by which expression of functional LSL oc-urs is quite complicated. Although Kao et al. (59) foundecreased up-regulation of GlcNAc6ST-1 in patients withndometriosis relative to controls, this may not translate toecreased expression of functional LSL. There are at leasthree molecules from a family of sialomucins (glycoproteinsith multiple O-linked glycans) that are LSLs—endoglycan,odocalyxin, and CD34—the latter two are found in humans54). Furthermore, there are �10 enzymes implicated in keyosttranslation modifications on the precursor proteins nec-ssary for LSL functionality (63–69). Posttranslation modi-cations include galactosylation (69), fucosylation (67), sia-

ylation (70), and sulfation (66). There is tremendous overlapnd redundancy in the production of this ligand, and there-ore, a decrease in the level of a precursor molecule, or aecrease (but not elimination) in an enzyme responsible forosttranslation ligand modification, may not necessarily re-ult in a decrease in functional LSL expression (54). Basedn this evidence, we believe the best way to analyze ligandxpression is by detection of the end product. Thus, weecommend immunohistochemical analysis of tissue using

ECA-79, where the level detected should correlate to themount of functional LSL, assuming the tissue biopsy isepresentative of the entire endometrium.

Genbacev et al. (10) measured the most concentration ofSL in endometrial tissue obtained approximately 6 days after

he LH surge in both the natural cycle and gonadotropin-timulated, hCG-induced menstrual cycle. Spandorfer et al.71) reported an increase in PRs in women who previouslyad multiple pregnancy losses with assisted reproductionfter autologous embryo–endometrial coculture. The PRsere significantly higher when the embryos were coculturedith autologous endometrial tissue obtained �5 days after

he LH surge during a prior menstrual cycle. It is during thismplantation window that several markers for implantationre maximally expressed. Current studies, however, have yeto address the impact of preimplantation chemical interac-ion between embryo and endometrium with respect to al-ering LSL expression. In addition, this study does not ex-mine expression of L-selectin on transferred embryos, andherefore cannot assess the embryonic contribution to thiseceptor–ligand interaction.

When examining tissue immunohistochemical character-stics, we noted differences between reactivity in the endo-etrial surface epithelium versus the glandular crypt epithe-

ium. Furthermore, HECA-452-stained slides revealed farreater cytoplasmic expression of LSL relative to MECA-9-stained slides from the same patient, whereas MECA-79-tained slides demonstrated far greater expression on theellular apex, especially in surface epithelium (refer to Ta-les 2, 3, and 5). This difference indicated that HECA-452ntibodies are bound to a cytoplasmic epitope of the maturer precursor ligand, and less so to an epitope on the extra-ellular portion of the ligand, whereas MECA-79 antibodiesere more specifically targeted to an extracellular epitope.
o account for these differences, we chose to use four scores p

or each slide: [1] surface apex, [2] surface cytoplasm, [3]land crypt apex, and [4] gland crypt cytoplasm. By detail-ng our scoring system in this manner we were able todentify a significant difference in ligand expression at thepex of the endometrial surface epithelium between pregnantnd nonpregnant recipients.

This alternative scoring system served two purposes: [1]nlike the HSCORE system, designed to give one score perlide, the IHC-Level was used to score specific areas ofissue, and [2] because four scores were given for each slideeight scores per patient for HECA-452 and MECA-79 an-ibodies), a simple, easily reproducible, and less time con-uming scoring system was advantageous. Statistical com-arison of the two methods demonstrate that althoughbsolute values differ slightly between the IHC-level and theSCORE, the relative differences are very similar, and there

s a high degree of correlation between scoring systemsrefer to Table 4). We also demonstrate a low intraobservernd interobserver variability for the IHC-Level. Based onhese findings, we recommend scoring the surface epitheliumnd gland crypts separately, and if applicable, with separatecores for apical and cytoplasmic reactivity when examiningterine epithelium for immunohistochemical expression ofn epithelial marker of implantation. The IHC-level mayerve as an alternative to the HSCORE in this scenario.

Donor egg recipients were chosen for evaluation of endo-etrial LSL expression for three reasons: [1] direct access to

rchived endometrial tissue in patients with known cycleutcome, [2] identical or near-identical stimulation protocoletween mock and ET replacement cycles, and [3] lowerikelihood of embryo-associated implantation failure due tohe high quality of embryos obtained from young donors,ffectively increasing the sensitivity of detecting implanta-ion failure related to endometrial factors alone. Examiningatural cycle or gonadotropin-stimulated endometrium mayequire many more patients to detect the same relationshipetween ligand expression and implantation unless the sub-ects are of a young age, where embryo quality is maximized.

We demonstrate significantly higher immunohistochemi-al reactivity for the L-selectin ligand at the apex of endo-etrial surface epithelium obtained during the mock cycle

rom donor egg recipients who conceived. By comparingesults of this study with clinical features such as ultrasoundterine thickness and serum hormone levels, we may soondentify risk factors that are associated with the depletion orysfunction of endometrial L-selectin ligand. In the nearuture, we may predict which patients have endometrium-ssociated infertility related to L-selectin and its ligand, withhe hope that further scientific exploration will lead to suc-essful therapy in this population. Furthermore, we presentn alternative scoring system and methodology for evaluat-ng immunohistochemical expression in human endome-rium that may be applied to other markers of uterine recep-ivity, and potentially in other tissues. Hence, this study
resents a novel approach for evaluating the endometrium
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hat may have useful clinical applications. Future studies canocus on methods to alter expression of endometrial L-electin ligand, where maximization of expression may im-rove implantation rate, whereas minimization may serve asuseful contraceptive tool.

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Documents

Immunohistochemical Expression of Endometrial L-Selectin Ligand Obtained During the Natural Menstrual Cycle From Women Undergoing Autologous Endometrial Coculture