Effect of ormeloxifene, a selective estrogen receptor modulator, on biomarkers of endometrial receptivity and pinopode development and its relation to fertility and infertility in

CONTRACEPTION

Effect of ormeloxifene, a selective estrogen receptormodulator, on biomarkers of endometrial receptivityand pinopode development and its relation to fertilityand infertility in Indian subjectsAnnu Makker, Ph.D.,a Indu Tandon, M.D.,b Madhu Mati Goel, M.D.,c Mastan Singh, M.D.,d

and Man Mohan Singh, Ph.D., D.Sc.a

a Division of Endocrinology, Central Drug Research Institute, Lucknow, India; b Department of Obstetrics & Gynecology,c Department of Pathology, and d Department of Microbiology, CSM Medical University, Lucknow, India

Objective: To compare expression of endometrial estrogen (ER) and progesterone (PR) receptors, b3-integrin sub-unit, leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and pinopodes in Indian women using ormeloxifene asa contraceptive with fertile and infertile subjects during the window of implantation.Design: Controlled, prospective, clinical study.Setting: Hospital-based reproductive health unit and research laboratories.Patient(s): Thirteen fertile women, 6 using ormeloxifene (30 mg/week), 29 with primary and 10 with secondaryinfertility.Intervention(s): Transvaginal ultrasonography, midluteal endometrial biopsies, and blood samples.Main Outcome Measure(s): Histologic dating, endometrial thickness, immunohistochemical localization of ER,PR, b3-integrin subunit, LIF, and IL-6 and scanning electron microscopy.Result(s): Ormeloxifene significantly reduced endometrial thickness, pinopode density, and caused histologic de-lay with increased epithelial ER and PR and unaltered epithelial b3-integrin subunit expression. Appearance offully developed pinopodes, down-regulation of epithelial PR, and increased epithelial b3-integrin subunit expres-sion was observed in in-phase endometrium from fertile and infertile women. LIF and IL-6 expression and serumestradiol and progesterone levels remained unaltered between groups.Conclusion(s): Ormeloxifene-induced effects might produce asynchrony between endometrial and embryo devel-opment resulting in implantation failure. Based on unaltered luteal phase serum progesterone concentration, orme-loxifene did not appear to prevent ovulation in any participant. (Fertil Steril� 2009;91:2298–307. �2009 byAmerican Society for Reproductive Medicine.)

Key Words: Endometrial receptivity biomarkers, pinopode development, hormone levels, ormeloxifene, endome-trial thickness, histologic delay, contraceptive action, fertility and infertility

One of the major obstacles to reproductive success is the in-ability to diagnose or treat a nonreceptive endometrium. En-dometrial receptivity or implantation window is defined asthe period during which the endometrial epithelium and sub-sequently the stroma are susceptible to the adhesive anddegradative properties of the trophoblast during nidation

Received January 17, 2008; revised and accepted April 9, 2008; published

online August 4, 2008.

Financial support from the Ministry of Health and Family Welfare, Govern-

ment of India, New Delhi.

A.M. has nothing to disclose. I.T. has nothing to disclose. M.M.G. has

nothing to disclose. M.S. has nothing to disclose. M.M.S. has nothing

to disclose.

Reprint requests: Man Mohan Sing, Ph.D., D.Sc., Central Drug Research

Institute, Endocrinology Division, MG Marg, Lucknow, Uttar Pradesh

226001, India (FAX: þ91-522-2623405; E-mail: singhmm@rediffmail.

com).

Fertility and Sterility� Vol. 91, No. 6, June 2009Copyright ª2009 American Society for Reproductive Medicine

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(1). Recent studies suggest that in humans, the window ofreceptivity occurs from days 20 through 24 of the menstrualcycle (2).

The physiology of human endometrium is highly depen-dent on the action of estrogen and progesterone of ovarian or-igin (3), and thereby, on spatial and temporal expression oftheir specific receptors throughout the menstrual cycle (4).In addition, a large number of identified molecular mediators,under the influence of ovarian hormones, have been postu-lated to be involved in endometrial receptivity and earlyembryo–maternal interrelationship (5, 6). These include en-dometrial adhesion molecules (e.g., integrins), antiadhesionmolecules (e.g., mucin-1), cytokines (e.g., leukemia inhibi-tory factor [LIF], IL-6, and so forth), growth factors, immunemarkers, and certain other endometrial markers. Recent stud-ies show that expression of b3-integrin in endometrial

0015-0282/09/$36.00, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2008.04.018

epithelium increases during the putative window of implanta-tion, and is suppressed in patients with unexplained infertility(7). Leukemia inhibitory factor receptors are expressed by theblastocyst as well as the endometrium (8), with maximum ex-pression of LIF mRNA and protein occurring in endometrialepithelium during the implantation window. Targeted inacti-vation of LIF leads to failure of implantation that is rescuedafter LIF administration (9). Interleukin-6, a pleiotropic cyto-kine, in addition to playing an important role in regulation ofovarian steroidogenesis, folliculogenesis, and early implanta-tion, shows stronger immunoreactivity during the secretoryrather than the proliferative phase (10). Pinopodes are protru-sions from microvillous cells on the surface of midlutealphase endometrium. Although precise function of pinopodesin women is not clearly understood, timed correlation of pi-nopode expression, period of blastocyst hatching, and prefer-ence of human blastocyst to attach to pinopodes suggest thatpinopodes should be good indicator of receptive endome-trium (11).

This study was aimed to substantiate the precise role ofthese molecular and structural markers in relation to endome-trial receptivity in women using ormeloxifene (67/20; INN forcentchroman), a multifunctional nonsteroidal selective estro-gen receptor (ER) modulator developed by the Central DrugResearch Institute, Lucknow, India. It is a unique need-ori-ented contraceptive that is effective when taken immediatelyafter coitus or routinely as a weekly pill (12–14), and has theadvantage of less frequent administration. Laboratory animalstudies show that it interferes with the process of implantationpostfertilization by inhibiting endometrial receptivity (15, 16)without exerting any deleterious direct effect on preimplanta-tion development or viability of embryos up to blastocyst stage(12). To the best of our knowledge, this is the first reportcomparing expression of these markers during the windowof implantation in normoovulatory Indian women using orme-loxifene as a contraceptive with fertile and infertile states.

MATERIALS AND METHODS

Subjects and Study Cycle

The study included fertile women volunteers (n ¼ 13) under-going tubal sterilization, women with primary (n ¼ 29) andsecondary (n ¼ 10) infertility undergoing routine workup,and women on continuous ormeloxifene (30 mg weeklydose) use for 1 to 3 years (n ¼ 6). The use of human tissuefor research was based on informed consent, and was ap-proved by the ethics committee of CSM Medical University,Lucknow, India. The fertile control group included healthywomen volunteers aged between 24 and 35 years (mean �SEM: 29.5 � 3.5 years) with mean parity of 1.4 (range:1–4). The mean age of the infertile women and women usingormeloxifene as a contraceptive was 26.2 � 4.5 (range:22–40) and 31.0 � 3.2 (range: 28–35) years, respectively.All volunteers had regular menstrual patterns every 27 to32 days. The mean duration of infertility was 2.9� 2.5 years.The main cause of infertility was minimal to mild endometri-osis (n ¼ 13), tubal factors with no hydrosalpinges (n ¼ 7),

Fertility and Sterility�

nonabsolute male factors (sperm density >5 to <20 � 106/mL, <50% motile sperm grades ‘‘a’’ and ‘‘b’’ according tothe World Health Organization criteria; n¼ 6), immunologic(n ¼ 3; two seropositive for anticardiolipin antibodies andone seropositive for anticardiolipin and lupus anticoagulantantibodies), and/or unexplained etiology (n ¼ 10). Allwomen volunteers underwent transvaginal ultrasonographicmonitoring of ovulation. Follicular growth was evaluateddaily commencing on days 8 to 10 of menstrual cycle depend-ing on cycle length using a 3.5–8.5 MHz vaginal transducer(Shimadzu SBU 350, Japan). The day of ovulation was des-ignated as day of maximum follicular enlargement that wasfollowed by sudden disappearance or filling in of follicleshowing loss of clear demarcation of walls and intrafollicularechoes (17,18). midluteal (ovulation days þ6 to þ8) endo-metrial thickness and appearance was observed, endometrialbiopsy was performed, and �2-mL blood samples were ob-tained for determination of serum estradiol and progesteronelevels.

Endometrial Samples

Biopsies were obtained from the uterine fundus region usinga standard endometrial suction curette (Gynetics, Belgium).Endometrial samples were immediately divided into two por-tions. One portion was fixed in 4% buffered formaldehyde for24 hours, embedded in paraffin, and cut into 5 mm-thick sec-tions on a rotary microtome (Leica, Nussloch, Germany;Model RM 2165) for histologic and immunohistochemicalstudies. The second portion was fixed overnight at 4�C in0.1 M cacodylate buffer (pH 7.4) containing 2.5% (w/v) glu-taraldehyde and 0.5% paraformaldehyde, washed with caco-dylate buffer, and stored at 4�C in a refrigerator for 1 to 7 daysbefore processing for scanning electron microscopy (SEM).

Histologic Dating

Histologic dating of the endometrial samples was performedindependently by two investigators according to the standardcriteria (19). In each case, endometrial glandular and stromalelements were dated independently. Women with appropriatedating of both glandular and stromal elements were said tohave ‘‘in-phase’’ endometrium. Endometrial dyssynchronywas defined as endometrial glandular dating lagging bothchronologic and stromal dating by R3 days. Endometrial im-maturity was deemed when both glandular and stromal ele-ments were judged to be lagging chronologic date by R3days. Endometrial glandular dating exceeding chronologicdate by R3 days was described as advanced.

Immunohistochemical Detection

Immunohistochemical detection of steroid receptors and en-dometrial receptivity markers was conducted as describedpreviously with slight modifications (20–23). Briefly, endo-metrial sections were deparaffinized in xylene and rehydratedthrough various grades of ethanol. For antigen retrieval, sec-tions were placed in boiling sodium citrate buffer (10 mM,

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pH 6.0) for 30 minutes. Endogenous peroxidase activity wasquenched by incubation in 0.3% hydrogen peroxide (H2O2)for 30 minutes. Nonspecific antibody binding was preventedby incubation with 4% PBS–bovine serum albumin for 1hour. Primary antibody of ERs (mouse monoclonal; 1:100,Neomarkers, Fremont, CA), progesterone receptors (PRs)(mouse monoclonal; 1:50, Neomarkers), LIF (goat poly-clonal; 1:100, Santa Cruz, Santa Cruz, CA) or IL-6 (rabbitpolyclonal; 1:50, Santa Cruz) was applied to tissue sectionsand incubated overnight at room temperature. Sections withantibody for b3-integrin subunit (goat polyclonal; 1:100,Santa Cruz) were incubated at 37�C for 1 hour. For negativecontrol samples, primary antibody was replaced with homol-ogous nonimmune serum of equivalent concentration. Thesecond antibody used in all cases was a biotinylated anti-mouse, antirabbit, or antigoat immunoglobulin. The reactionwas developed by an extravidin–peroxidase system, and3,30-diaminobenzidine was used as chromogen. Stainingwas evaluated by two independent investigators at �200magnification using a semiquantitative subjective scoringsystem on a Leica light microscope. Observations weremade in 10 randomly selected fields of the sections to avoiderrors because of uneven staining. The staining was graded as0 (absent), 1 (faint), 2 (moderate) or 3 (strong) (24).

Scanning electron microscopy (SEM)

For SEM studies, fixed tissue samples kept at 4�C werewashed several times in cacodylate buffer, postfixed in 1%buffered osmium tetraoxide for 1 hour, and dehydrated by in-creasing concentrations of ethanol. Specimens were trans-ferred in absolute ethanol to Critical Point Dryer (ModelCPD 020, Balzers Union Ltd., Principality of Liechtenstein),dried using liquid carbon dioxide, mounted on to aluminiumscanning electron microscopy stubs using double-sidedtape, and sputter coated with gold-palladium (60:40) alloyto a thickness of 20 nm in a Hummer X Sputter Coater (Ana-tech, Ltd., Alexandria, VA). Scanning electron microscopywas performed on Philips Scanning Electron Microscope505 operating at 20 KeV. Scanning photomicrographs wereobtained from 12 randomly selected areas of each tissue spec-imen and were evaluated independently by two investigators.Pinopodes were defined as smooth apical protrusions from thesurface epithelium without microvilli (25–27). Endometrialsamples were considered as expressing pinopodes when theseprotrusions were detected at any stage of development viz.developing, fully developed and regressing. Pinopode expres-sion was scored as 0 (absence of pinopodes), 1 (covering<25%), 2 (covering 25-50%), or 3 (covering >50% of thesurface epithelium) (28).

Hormone Assays

Estradiol and progesterone levels in serum were measured bycommercially available competitive immunoenzymatic assaykits (DRG, Germany). Sensitivity of the assays was 10 pg/mLand 0.2 ng/mL, respectively, and interassay coefficient of var-iation was 5% and 6.7%, respectively. Blood samples were

2300 Makker et al. Ormeloxifene and endometrial recept

allowed to clot and the serum was separated and stored at�20�C until assayed. Samples from all subjects of eachgroup were analyzed in a single assay.

Statistical Analysis

Data analysis was performed using Minitab 13.20 statisticalsoftware (Minitab Inc., State College, PA). One-way ANOVA(unstacked) was performed to compare means between twogroups. A P-value of< .05 was considered as statistically sig-nificant.

Approval of the institutional review board has been ob-tained for communication of this manuscript for publication,and communication number (7430) has been allotted to thismanuscript.

RESULTS

Histologic Dating

The percent in-phase, immature, advanced, and asynchro-nous endometrial biopsies in different study groups areshown in Table 1. A total of 53.8% of fertile control, 34.5%of primary, and 11% of secondary infertile subjects showedin-phase endometrium. Delayed endometrial maturation ofR3 days was observed in all women using ormeloxifene,in comparison to 15.4%, 24%, and 33% in fertile controland primary and secondary infertile subjects, respectively(Table 1).

Endometrial Thickness

The mean endometrial thickness as measured by transvaginalsonography was not significantly different between the fer-tile control and the infertile women. Endometrium (4.67 �0.67 mm) in women using ormeloxifene as contraceptivewas, however, significantly (P<.01) thinner than that inwomen of fertile control (9.80 � 0.97 mm) or primary(8.86 � 0.64 mm) and secondary (9.00 � 0.84 mm) infertilegroups (Table 2).

Serum Hormone Levels

No significant difference in midluteal serum concentration ofestradiol or progesterone was detected among differentgroups (Table 2). Serum estradiol and progesterone concen-trations also did not vary significantly between women volun-teers of fertile control group and those using ormeloxifene asa contraceptive. Based on unaltered luteal phase serum pro-gesterone concentration, ormeloxifene did not appear to pre-vent ovulation in any participant.

Pinopodes

Pinopodes, when present on the endometrial surface, were indeveloping, fully developed, or regressing states. Classicfully developed pinopodes were observed in in-phase midse-cretory biopsies from both fertile control and infertile groups(Fig. 1A). The mean pinopode score (Table 2) in women with

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TABLE 1Histologic dating of the endometrium in fertile control, infertile, and ormeloxifene-treated subjects onovulation days D6 to D8 of the menstrual cycle.

Percent of total subjects

Infertile

Histologic statusof endometrium

Fertile control(n [ 13)

Primary(n [ 29)

Secondary(n [ 10)

Ormeloxifenetreated (n [ 6)

In phase 53.8 34.5 11 0Immature 15.4 24.0 33 100Advanced 15.4 10.5 22 0Dyssynchronous 15.4 31.0 34 0

Note: In phase endometrium is the endometrial state with appropriate dating of both glandular and stromal elements.Immature endometrium is the endometrial state with both glandular and stromal elements lagging chronological date by

R3 days.Advanced endometrium is the endometrial state with glandular dating exceeding chronological date by R3 days.Dyssynchronous endometrium is the endometrial state with glandular dating lagging both chronologic and stromal dating

by R3 days.Primary infertility is the term used to describe a couple that has never been able to conceive a pregnancy after at least 1

year of attempting to do so through unprotected intercourse.Secondary infertility is the term used when a couple has conceived previously, but is unable to conceive again, whether or

not the first pregnancy resulted in a live or still birth, miscarriage, or termination. This group also includes those who havehad a change of mind following sterilization.

Makker. Ormeloxifene and endometrial receptivity. Fertil Steril 2009.

primary infertility (1.71 � 1.21) was lower than normal fer-tile controls (2.46� 0.66), although it did not reach statisticalsignificance level (P¼.053). Delayed or advanced endome-trial maturation was accompanied by alterations in pinopodedensity and morphology (Fig. 1C and D). Retarded endome-trial maturation was observed in all women using ormeloxi-

Fertility and Sterility�

fene as an oral contraceptive. Less than 40% of theirendometrial surface was covered with pinopodes. The cellswere slightly bulging and were covered with short microvilli(Fig. 1B) and pinopode score (1.25 � 0.50) was significantly(P<.05) lower than that for normal fertile controls (2.46 �0.66, Table 2).

TABLE 2Endometrial thickness, serum estradiol, and progesterone levels and pinopode status in fertile control,infertile, and ormeloxifene treated subjects on ovulation days D6 to D8 of the menstrual cycle.

Subjects

Infertile

ParameterFertile control

(n [ 13)Primary(n [ 29)

Secondary(n [ 10)

Ormeloxifenetreated (n [ 6)

Endometrial thickness (mm) 9.80 � 0.97 8.86 � 0.64 9.00 � 0.84 4.67 � 0.67a

Estradiol (pg/mL) 88.36 � 20.39 97.94 � 20.34 81.51 � 13.22 111.22 � 36.67Progesterone (ng/mL) 14.31 � 0.84 11.59 � 0.96 12.58 � 2.02 13.67 � 1.39Pinopode score 2.46 � 0.66 1.71 � 1.21b 2.00 � 0.89 1.25 � 0.50c

Note: Values are mean � SEM.All other relevant comparisons were statistically nonsignificant.a P< .001; versus corresponding fertile control or primary and secondary infertile groups.b P¼ .053; versus corresponding fertile control group.c P< .05; versus corresponding fertile control group.

Makker. Ormeloxifene and endometrial receptivity. Fertil Steril 2009.

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FIGURE 1

Scanning electron microscopic pictures of the endometrial surface during the window of implantation in a fertilecontrol volunteer (A), a volunteer taking 30 mg weekly dose of ormeloxifene for the last 1 year (B), a primaryinfertility patient showing delayed endometrial maturation (C), and a primary infertility patient showingaccelerated endometrial maturation (D). Note classic fully developed pinopodes (P) covering almost the entireepithelial surface in the normal ‘‘in-phase’’ endometrium. Endometrial biopsy of woman using ormeloxifene asa contraceptive showed retarded maturation resembling late proliferative or early secretory endometrium with<40% of the endometrial surface covered with pinopodes. Luminal epithelial cells in these subjects are slightlybulging and covered with microvilli. A few smooth projections are also observed. Endometrial biopsy in a primaryinfertility patient with delayed maturation showed developing pinopodes (p), whereas those with acceleratedmaturation showed regressing pinopodes (R). Bar: 10 mm.

Makker. Ormeloxifene and endometrial receptivity. Fertil Steril 2009.

Immunohistochemistry

Immunohistochemically, nuclear expression of PR was sig-nificantly (P<.05) elevated in glandular as well as luminalepithelium in endometrial samples from women with primaryinfertility compared with fertile controls or women with sec-ondary infertility (Table 3). No statistically significant differ-ence in the expression of ERs was observed between thefertile and infertile subjects. When subjects in each groupwere stratified on the basis of histologic dating, an increasein stromal PR expression and down-regulation of epithelialPR expression was observed in in-phase endometrium fromfertile control (Fig. 2A) as well as infertile women (Table4). Histologic delay in biopsies from subjects using ormelox-ifene was associated with high levels of epithelial PR and ER,and the levels were significantly higher compared with fertilecontrol group (PR: P<.001, ER: P<.05, Table 3; Fig. 2A–D).

2302 Makker et al. Ormeloxifene and endometrial recept

b3-Integrin subunit expression in the glandular epitheliumof primary (Fig. 2F, inset) and secondary infertile subjectswas significantly lower relative to the fertile control group(Table 3 and Fig. 2E). No significant change in the expres-sion of the b3-integrin subunit, however, was observed inthe epithelial component of endometrium from women usingormeloxifene (Table 3 and Fig. 2F). A slightly increasedexpression (P<.05) of the b3-integrin subunit, however,was observed in the stromal component of these womencompared with fertile controls (Table 3). A strong stainingof the b3-integrin subunit was observed in the epitheliumof the in-phase endometrial tissue collected from fertile con-trol and infertile women during the midsecretory phase. Thestaining intensity for the b3-integrin subunit was lower in en-dometrial luminal and glandular epithelial cells from fertileand infertile women with out-of-phase biopsy (Table 4). No

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TABLE 3Immunostaining for endometrial ER, PR, b3-integrin subunit, LIF and IL-6 in fertile control, infertile, andormeloxifene-treated subjects on ovulation days D6 to D8 of the menstrual cycle.

Subjects

Infertile

Parameter Fertile control Primary Secondary Ormeloxifene treated

Progesterone receptorsLuminal epithelium 0.50 (0-1.5) 0.98 (0-3)a 0.44 (0-1) 1.67 (1.5-2.0)b

Glandular epithelium 0.58 (0-2) 1.22 (0-3)a 0.61 (0-2) 2.00 (1.5-2)b

Stroma 2.10 (1-3) 1.52 (0.5-3)a 1.39 (0-3) 1.83 (1.5-2)Estrogen receptors

Luminal epithelium 0.80 (0-2.5) 0.79 (0-2) 1.06 (0-2) 2.00 (1.5-2.5)a

Glandular epithelium 1.10 (0-3) 1.17 (0-3) 1.39 (0-3) 2.67 (2-3)a

Stroma 1.60 (1-3) 1.41 (0.5-3) 1.50 (1-3) 1.67 (1.5-2)b3-Integrin subunit

Luminal epithelium 1.59 (1-2) 1.40 (0.5-3) 1.28 (1-3) 1.83 (1.5-2.0)Glandular epithelium 2.04 (1-3.0) 1.43 (0.5-3)a 1.28 (1-3)a 2.17 (2.0-2.5)Stroma 0.92 (0.5-1.5) 1.22 (0.5-2.5) 1.27 (1-3) 1.33 (1.0-2.0)a

LIFLuminal epithelium 2.20 (1.5-2.5) 1.85 (1-2.5) 2.06 (1-3) 2.00 (1.5-2.5)Glandular epithelium 2.80 (2-3) 2.38 (1-3) 2.44 (2-3) 2.67 (2.5-3.0)Stroma 1.80 (1-3) 1.49 (1-3) 1.56 (1-3) 1.83 (1.5-2.5)

IL-6Luminal epithelium 2.30 (2-3) 2.25 (1-3) 2.29 (2-3) 2.33 (2-3)Glandular epithelium 2.60 (2-3) 2.62 (1.5-3) 2.50 (2-3) 2.50 (2-3)Stroma 2.10 (1.5-3) 1.89 (1-3) 2.00 (1.5-3) 1.67 (1-2)

Note: Values indicate median with range in parentheses.PR ¼ progesterone receptors; ER ¼ estrogen receptors; LIF ¼ leukemia inhibitory factor; IL-6 ¼ interleukin-6.All other relevant comparisons were statistically nonsignificant.a P< .05.b P< .001; versus corresponding fertile control group.

Makker. Ormeloxifene and endometrial receptivity. Fertil Steril 2009.

significant difference in the expression of LIF (Fig. 2G andH) and IL-6 (Fig. 2I and J) was observed between the variousstudy groups (Table 4).

DISCUSSION

The findings of this study clearly demonstrate that endome-trial maturity in women, irrespective of fertile or infertilestates, was associated with the appearance of fully developedpinopodes, increased expression of epithelial b3-integrin andstromal PR, and down-regulation of epithelial PR. Histologicdelay in ormeloxifene users, in comparison, was associatedwith significantly reduced endometrial thickness and pino-pode density and elevated epithelial ER and PR levels. Al-though elevated epithelial PR was also observed in primaryinfertile women, the epithelial b3-integrin subunit, which re-mained unaltered in ormeloxifene users, was low in fertileand infertile women with an out-of-phase biopsy. LIF andIL-6 expression and serum estradiol and progesterone levelsremained unaltered between different groups.

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Ormeloxifene has been reported to mediate its effects byantagonizing action of estrogen by high-affinity interactionwith ER in target tissues (12). Impaired endometrial matura-tion observed in ormeloxifene users in the present study at thetime when implantation normally ensues was accompaniedby endometrial parameters clearly different from those ofsubjects of fertile control group. Histologic delay in thesewomen was associated with high epithelial ER and PR levelsas observed in late proliferative to early secretory endome-trium. Abnormal abundance of PR in the epithelial compart-ment of the midsecretory endometrium in ormeloxifene usersmight indicate an alteration in uterine receptivity as reportedin women with a luteal phase defect (20). Although direct ev-idence of association between persistently high ER and fail-ure of implantation or infertility is not available, elevatedexpression of ERa during the midsecretory phase has beenreported in patients with polycystic ovarian syndrome (29)and endometriosis (30). These investigators have suggestedthat inappropriate estrogen action during maximal receptivitymight alter the normal receptive pattern of gene expression.

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It has also been contemplated that a less than optimal en-dometrial milieu might result in embryonic diapause (31)or physiologic arrest, well documented in certain lower mam-mals (32). The present study, however, does not provide anyevidence of a shift in the window of implantation in womenusing ormeloxifene as a contraceptive. In previous studiesfrom this laboratory, about a 40-hour displacement in pino-pode development on the endometrial surface was observedin ormeloxifene-treated rats. This, however, was not associ-ated with corresponding displacement of endometrial recep-tivity, because application of intraluminal peanut oil inparallel with pinopode development failed to elicit any decid-uomal response in these rats (15, 16, 33). These studies alsodemonstrated that inhibition of endometrial receptivity byormeloxifene in the rat was associated with altered perme-ability characteristics of uterine luminal epithelial cells, re-sulting in inhibition of endocytosis/pinocytosis of luminal

FIGURE 2

Makker. Ormeloxifene and endometrial receptivity. Fertil Steril 2009.

2304 Makker et al. Ormeloxifene and endometrial recept

fluid, luminal closure, apposition of blastocyst trophoblastto luminal epithelium, and implantation failure (15, 16, 34).

Pertinently, ormeloxifene did not influence plasma levelsof estradiol or progesterone in any participant, suggestinglack of effect on follicular maturation and ovulation. Reportsof lack of effect of prolonged ormeloxifene administration onfollicular maturation and ovulation in rats, rhesus monkeys,and humans are available (12, 35–37). Serum estradiol andprogesterone levels were also not statistically different be-tween fertile and infertile groups in this study, confirmingthe observations of Ordi et al. (37) that receptivity of theendometrium to progesterone can vary independent of serumprogesterone levels, and that histologic delay can occur evenwith physiologic progesterone levels or despite supraphysio-logic progesterone concentrations. In comparison, the role ofestradiol during the luteal phase in humans is believed to bepermissive rather than obligatory (38, 39).

The percentage of women with in-phase biopsies wasmarkedly low in the infertile group compared with the fertilegroup. Because a paracrine communication between epithe-lium and stroma is important at the beginning of implanta-tion, an out-of-phase endometrium may affect uterinereceptivity. A delay in maturation early in the secretory phasehas been suggested to prevent a perfectly normal embryofrom attachment and successful establishment of pregnancyeven though the endometrium may have caught up later inthe same cycle (40). The work by Castelbaum et al. (41)indicates the possibility that an out-of-phase early biopsy

FIGURE 2 Continued

Immunohistochemical localization of PR (A,B), ER(C,D), b3-integrin (E,F), LIF (G,H), and IL-6 (I,J) inendometrial biopsies obtained during the window ofimplantation from fertile control volunteers(A,C,E,G,I) and volunteers taking 30 mg weekly doseof ormeloxifene for the last 1 to 3 years (B,D,F,H,J).Note the absence of nuclear PR expression in theluminal (long arrow) as well as glandular (short arrow)epithelium of the normal ‘‘in-phase’’ endometrium,whereas intense staining was evident in the stromalregion (A). In comparison, in the ‘‘out-of-phase’’endometrium in woman using ormeloxifene, intensenuclear PR staining was seen in the luminal as well asglandular epithelium (B). Arrow shows high nuclearER in the glandular epithelium in an ormeloxifeneuser (D). Staining of the b3-integrin subunit wasalmost similar in volunteers of fertile control (E) andormeloxifene (F) groups. Diminished staining of b3-integrin subunit in the ‘‘out-of-phase’’ biopsy froma primary infertility patient is shown in the inset. Nosignificant difference in staining intensity wasobserved for LIF (G,H) and IL-6 (I,J) between fertilecontrols and ormeloxifene users. G, endometrialgland; S, Endometrial stroma; Lu, uterine lumen.

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TABLE 4PR and b3-integrin subunit expression in the endometrium from normal fertile and primary infertilesubjects stratified on the basis of histologic dating.

Endometrial status

Group In-phase Immature Advanced Dyssynchronous

Fertile control PRLuminal epithelium 0.21 � 0.15 1.75 � 0.25a 0.50 � 0.00 0.75 � 0.25Glandular epithelium 0.14 � 0.09 1.25 � 0.25c 0.75 � 0.25 1.25 � 0.25c

Stroma 2.43 � 0.20 1.25 � 0.25d 1.75 � 0.25 2.00 � 0.50b3-Integrin subunit

Luminal epithelium 1.86 � 0.09b,c,d 1.25 � 0.25 1.00 � 0.00 1.50 � 0.50Glandular epithelium 2.57 � 0.13b,c 1.25 � 0.25 1.00 � 0.00 2.00 � 0.00a

Stroma 0.93 � 0.13a,c,e 0.75 � 0.25 0.75 � 0.25 1.25 � 0.25Primary infertile PR

Luminal epithelium 0.30 � 0.11 1.71 � 0.18b 0.67 � 0.18 1.28 � 0.19b

Glandular epithelium 0.20 � 0.11 2.21 � 0.28c 0.50 � 0.16 1.83 � 0.19c

Stroma 1.75 � 0.17 0.86 � 0.08e 1.33 � 0.09 1.83 � 0.25b3-Integrin subunit

Luminal epithelium 1.85 � 0.21b,c 1.43 � 0.14d 1.00 � 0.14 1.33 � 0.11Glandular epithelium 1.80 � 0.21b,c 1.36 � 0.17d 1.00 � 0.16 1.22 � 0.11d

Stroma 1.20 � 0.11b,c,d 1.29 � 0.22 1.00 � 0.00 1.28 � 0.11

Note: Values are mean � SEM.PR ¼ progesterone receptors.a P< .05,b P< .001; versus PR expression level in luminal epithelium of the in-phase biopsy of the corresponding group.c P< .001; versus PR expression level in glandular epithelium of the in-phase biopsy of the corresponding group.d P< .05,e P< .001; versus PR expression level in stroma of the in-phase biopsy of the corresponding group.

Makker. Ormeloxifene and endometrial receptivity. Fertil Steril 2009.

during the window of implantation may represent a crypticform of luteal phase deficiency, because in these patientsa second, later (in the same cycle) biopsy showed entirelynormal maturation. In the present study, 31% of infertilewomen showed dyssynchronous endometrium comparedwith 15.4% fertile women. There is considerable evidencesuggesting that estrogen and progesterone may differentiallyaffect this dyssynchrony, and the disparity in maturation be-tween the stroma and the epithelium observed in histologycould compromise uterine receptivity or early trophoblasticinvasion (42).

Endometrial maturity in women in the present study, irre-spective of fertile or infertile states, was associated withdown-regulation of epithelial PR. The diminution in the ex-pression of steroid receptors in the endometrium during thesecretory phase of a normal menstrual cycle has been largelydocumented (20, 43). According to Musgrove et al. (44), lossof PR by glandular epithelium appears to be required forglandular epithelial morphogenesis and differentiated func-tion, as well as to prevent inhibition of these events by pro-gesterone. In the present study, nuclear expression of PRwas significantly (P<.05) elevated in glandular as well as lu-minal epithelium in endometrial samples from women with

Fertility and Sterility�

primary infertility compared with fertile controls or womenwith secondary infertility. However, no statistically signifi-cant difference in the expression of ER was observed betweenthe fertile and infertile subjects.

Expression of the b3-integrin subunit, in both infertile andfertile women in this study, was closely related to endometrialmaturation as evaluated by histologic dating. Although endo-metrium dated midsecretory (i.e., in-phase) in both fertile andinfertile women stained positively for the b3-integrin subunit,histologic delay (i.e., out-of-phase endometrium) in thesesubjects was associated with significant reduction in the ex-pression of the b3-integrin subunit. Although these findingsmight provide support to the reported suggestion on the useof b3-integrin expression as a marker of the luteal phase en-dometrial glandular maturity during the secretory phase (42),no change in expression of the b3-integrin subunit was ob-served in endometrial biopsies from ormeloxifene users de-spite significant histologic delay.

LIF and IL-6 belong to the group of cytokines that reactwith receptors associated with gp130, and appear to play im-portant role in implantation. In the present study, whereasstaining for of LIF and IL-6 were observed in all endometrial

2305

biopsies, no significant difference was evident among fertileand infertile groups and ormeloxifene users, suggesting thatexpression of these markers per se might not truly reflectthe period of high endometrial receptivity in women. Al-though there are reports that have suggested that productionof LIF and IL-6 is reduced in some infertile women (45),others have failed to confirm these findings (21, 22). In addi-tion, the LIF content in uterine secretion at the time of oocyteretrieval does not seem to be correlated to pregnancy rate(46). Despite high levels during midsecretory phase, func-tional significance of these proteins has been enigmatic.However, their facilitatory role in preparing the endometriumfor implantation cannot be entirely ruled out, because contin-ued perturbation in the expression of these molecules mightaffect endometrial growth, remodeling, and differentiation,which in turn, might render the endometrium nonreceptive(47). Pertinently, it has been reported that although blastocystimplantation is not impaired in IL-6�/�mice, development ofthe conceptus is compromised (48, 49). Thus, as suggested ina previous study (22), it may be necessary to examine signal-ing components together with the production of these cyto-kines.

Pinopodes or apical surface protrusions of the uterine lu-minal epithelium have in recent years been suggested as po-tential marker of endometrial receptivity (11). In the presentstudy, also, delayed or advanced endometrial maturation wasaccompanied by alterations in pinopode density and mor-phology. Histologic delay and reduced endometrial thicknessin all subjects using ormeloxifene was accompanied by<40% of endometrial surface covered with pinopodes andsignificantly lower pinopode score than fertile controls. Incomparison, classic fully developed pinopodes were ob-served in in-phase biopsies from both fertile and infertilegroups. The mean pinopode score in women with primary in-fertility also was lower compared with normal fertile con-trols. Results of this study, therefore, provide support to thepossible use of pinopodes as marker of endometrial receptiv-ity in women.

Findings of this study also demonstrate that the endometrialchanges such as histologic delay, reduced thickness and pino-pode density, elevated epithelial ER and PR levels, and unal-tered epithelial b3-integrin subunit expression observed inwomen on long-term ormeloxifene use are distinct from thoseobserved in infertile subjects. Moreover, the antifertility ef-fect of ormeloxifene is easily reversible, because most womenwho withdrew from phase II and III multicentric clinical trialsfor want of an issue became pregnant within 6 to 8 months anddelivered healthy babies (12). Further studies on reproductiveendocrine and endometrial effects of ormeloxifene in womenwith regular menstrual cycles have been proposed to confirmthese findings. This has also been necessitated in view of itsbetter antiresorptive (antiosteoporosis) activity in a numberof in vitro and in vivo test systems than raloxifene (50, 51).These studies, in addition to demonstrating potential of orme-loxifene in the management of postmenopausal osteoporosis,suggest its beneficial effect on BMD in women taking this

2306 Makker et al. Ormeloxifene and endometrial recepti

selective estrogen receptor modulator for contraception orany hormone-related clinical disorder.

CONCLUSIONS

In conclusion, our studies show direct deleterious effect oformeloxifene on the endometrial development that jeopar-dizes the chance of implantation due possibly to the lack ofsynchronization between the development of the endome-trium and the early embryo. Implantation failure has beensuggested to result from the disparity in maturation betweenendometrial stroma and epithelium observed in histology.Because a paracrine communication between epitheliumand stroma appears important at the beginning of implanta-tion, this disparity could compromise uterine receptivity orearly trophoblastic invasion.

Statistically significant changes in endometrial thickness,ER, PR, and pinopode expression were observed in womenusing ormeloxifene, suggesting that ormeloxifene-inducedendometrial changes resulting in failure of implantationmight be mediated via these mechanisms. No significantchange in epithelial and stromal expression of b3-integrinsubunit, LIF, and IL-6 was observed in women using orme-loxifene indicates that the expression of these proteins maynot be associated with anti-implantation action of ormeloxi-fene, and that ormeloxifene appears to alter endometrialreceptivity by mechanism(s) independent of expression ofthese proteins.

Acknowledgments: The authors thank Dr. Geetanjali Mishra for help in

statistical analysis of the data. A.M. thanks the Indian Council of Medical

Research, New Delhi for research fellowship and M.M.S. for appointment

as Emeritus Medical Scientist. CDRI communication no. 7430.

REFERENCES1. Tabibzadeh S, Babaknia A. The signals and molecular pathways in-

volved in implantation, a symbiotic interaction between blastocyst and

endometrium involving adhesion and tissue invasion. Mol Hum Reprod

1995;10:1579–602.

2. Wilcox AJ, Baird DD, Weinberg CR. Time of implantation of the con-

ceptus and loss of pregnancy. N Eng J Med 1999;340:1796–9.

3. Brenner RM, West NB. Hormonal regulation of the reproductive tract in

female mammals. Annu Rev Physiol 1975;37:273–302.

4. Kurita T, Young P, Brody JR, Lydon JP, O’Malley BW, Cunha GR. Stro-

mal progesterone receptor mediate mitogenic effects of progesterone on

estrogen-induced uterine epithelial cell deoxyribonucleic acid synthesis.

Endocrinology 1998;139:4708–13.

5. Giudice LC. Potential biochemical markers of endometrial receptivity in

clinical practice. Hum Reprod 1999;14(Suppl 2):3–16.

6. Makker A, Singh MM. Endometrial receptivity: clinical assessment in

relation to fertility, infertility and antifertility. Med Res Rev 2006;26:

699–746.

7. Lessey BA, Castelbaum AJ, Sawin SJ, Sun J. Integrins as marker of uter-

ine receptivity in women with primary unexplained infertility. Fertil

Steril 1995;63:1535–42.

8. Laird SM, Tuckerman EM, Dalton CF, Dunphy BC, Li TC, Zhang X. The

production of leukemia inhibitory factor by human endometrium: pres-

ence in uterine flushings and production by cells in culture. Hum Reprod

1997;12:569–74.

9. Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadil I, Kontgen F, et al. Blas-

tocyst implantation depends on maternal expression of leukemia inhibi-

tory factor. Nature 1992;359:76–9.

vity Vol. 91, No. 6, June 2009

10. Tabibzadeh S, Babaknia A, Liu R, Marconi D, Romanini C. Site and

menstrual cycle dependent expression of proteins of the TNF receptor

family and BCL-2 oncoprotein and phase specific production of TNF-

alpha in human endometrium. Hum Reprod 1995;10:277–86.

11. Bentin-Ley U, Sjogren A, Nilsson L, Hamberger L, Larsen JF, Horn T.

Presence of uterine pinopodes at the embryo-endometrial interface dur-

ing human implantation in vitro. Hum Reprod 1999;14:515–20.

12. Singh MM. Centchroman, a selective estrogen receptor modulator, as

a contraceptive and for the management of hormone-related clinical dis-

orders. Med Res Rev 2001;21:302–47.

13. Nityanand S, Anand N. Centchroman: a nonsteroidal antifertility agent.

FOGSI (Fed Obstet Gynaecol Soc, India) FOCUS 1996;8–10.

14. Macgregor JJ, Jordan VC. Basic guide to the mechanism of antiestrogen

action. Pharm Rev 1998;50:151–96.

15. Singh MM, Chauhan SC, Trivedi RN, Maitra SC, Kamboj VP. Correla-

tion of pinopod development on uterine luminal epithelial surface with

hormonal events and endometrial sensitivity in rat. Eur J Endocrinol

1996;135:107–17.

16. Singh MM, Trivedi RN, Chauhan SC, Srivastava VML, Kamboj VP.

Uterine estradiol and progesterone receptor concentration, activities of

certain antioxidant enzymes and dehydrogenases and histoarchitecture

in relation to time of secretion of nidatory estrogen and high endometrial

sensitivity in rat. J Steroid Biochem Mol Biol 1996;59:215–24.

17. Shoupe D, Mishell DR, Lacarra M, Lobo RA, Hornstein J, d’Ablaing G,

et al. Correlation of endometrial maturation with four methods of esti-

mating day of ovulation. Obstet Gynecol 1989;73:88–92.

18. Peters AJ, Lloyd RP, Coulam CB. Prevalence of out-of-phase endome-

trial biopsy specimens. Am J Obstet Gynecol 1992;166:1738–46.

19. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil

Steril 1950;1:3–25.

20. Lessey BA, Killam AP, Metzger DA, Haney AF, Greene GL,

McCarty KS Jr. Immunohistochemical analysis of human uterine estro-

gen and progesterone receptors throughout the menstrual cycle. J Clin

Endocrinol Metab 1988;67:334–40.

21. Sherwin JRA, Smith SK, Wilson A, Sharkey AM. Soluble sp130 is up

regulated in the implantation window and shows altered secretion in

patients with primary unexplained infertility. J Clin Endocrinol Metab

2002;87:3953–60.

22. Dimitriadis E, Sharkey AM, Tan YL, Salamonsen LA, Sherwin JRA.

Immunolocalisation of phosphorylated STAT3, interleukin 11 and leuke-

mia inhibitory factor in endometrium of women with unexplained infer-

tility during the implantation window. Reprod Biol Endocrinol 2007;5:

44–52.

23. Quezada S, Avellaira C, Johnson MC, Gabler F, Fuentes A, Vega M.

Evaluation of steroid receptors, coregulators, and molecules associated

with uterine receptivity in secretory endometria from untreated women

with polycystic ovary syndrome. Fertil Steril 2006;85:1017–26.

24. Sun XX, Gemzell-Danielsson K, Li HZ, Stabi B, Stavreus-Evers A. Ex-

pression of heparin-binding epidermal growth factor-like growth factor

and its receptors in the human fallopian tube and endometrium after

treatment with mifepristone. Fertil Steril 2006;85:171–8.

25. Nikas G. Pinopodes as markers of endometrial receptivity in clinical

practice. Hum Reprod 1999;14(Suppl 2):99–106.

26. Acosta AA, Elberger L, Borghi M, Calamera JC, Chemes H, Doncel GF,

et al. Endometrial dating and determination of the window of implanta-

tion in healthy fertile women. Fertil Steril 2000;73:788–98.

27. Creus M, Ordi J, Fa’bregues F, Casamitjana R, Ferrer B, Coll E, et al.

avb3 integrin expression and pinopod formation in normal and out of

phase endometria of fertile and infertile women. Hum Reprod

2002;17:2279–86.

28. Usadi RS, Murray MJ, Bagnell RC, Fritz MA, Kowalik A, Meyer WR,

et al. Temporal and morphologic characteristics of pinopod expression

across the secretory phase of the endometrial cycle in normally cycling

women with proven fertility. Fertil Steril 2003;79:970–4.

29. Gregory CW, Wilson EM, Apparao KBC, Lininger RA, Meyer WR,

Kowalik A, et al. Steroid receptor coactivator expression throughout

the menstrual cycle in normal and abnormal endometrium. J Clin Endo

Metab 2002;87:2960–6.

Fertility and Sterility�

30. Lessey BA, Palomino WA, Apparao KBC, Young SL, Lininger RA. Es-

trogen receptor-alpha (ER-alpha) and defects in uterine receptivity in

women. Reprod Biol Endocrinol 2006;4(Suppl 1):S9.

31. Tarin JJ, Cano A. Do human concepti have the potential to enter into dia-

pause? Hum Reprod 1999;14:2434–6.

32. Song JH, Houde A, Murphy BD. Cloning of leukemia inhibitory factor

(LIF) and its expression in the uterus during embryonic diapause and im-

plantation in the mink (Mustela vison). Mol Reprod Dev 1998;51:13–21.

33. Trivedi RN, Chauhan SC, Dwivedi A, Maitra S, Kamboj VP, Singh MM.

Time related effects of a triphenylethylene antiestrogen on estrogen in-

duced changes in uterine weight, estrogen receptors and endometrial sen-

sitivity in immature rats. Contraception 1995;51:367–79.

34. Bansode FW, Chauhan SC, Makker A, Singh MM. Uterine luminal epi-

thelial alkaline phosphatase activity and pinopod development in relation

to endometrial sensitivity in rat. Contraception 1998;58:61–8.

35. Rajan R. Contraceptive and non-contraceptive benefits of centchroman.

Asian J Obstet Gynaecol 1996;1:65–71.

36. Vaidya R, Joshi U, Meherji P, Rege N, Betrabet S, Joshi L, et al. Activity

profile of centchroman in healthy female volunteers. Indian J Exp Biol

1977;15:1173–6.

37. Ordi J, Creus M, Quinto L, Caamitjana R, Cordesa A, Balasch J. Within-

subject between-cycle variability of histological dating, avb3 integrin

expression and pinopod formation in the human endometrium. J Clin En-

docrinol Metab 2003;88:2119–25.

38. Edgar DH. Oestrogen and human implantation. Hum Reprod 1995;10:

2–4.

39. Zegers-Hochschild F, Altieri E. Luteal estrogen is not required for the es-

tablishment of pregnancy in the human. J Assist Reprod Genet 1995;12:

224–8.

40. Blasco L. Dyssynchrony in the maturation of endometrial glands and

stroma. Fertil Steril 1994;61:596–7.

41. Castelbaum A, Coutifaris C, Lessey BA, Mastroianni L, Wheeler J. Tim-

ing of the endometrial biopsy may be critical for the accurate diagnosis of

luteal phase deficiency. Fertil Steril 1994;61:443–7.

42. Damario MA, Lesnick TG, Lessey BA, Kowalik A, Mandelin E,

Seppala M, et al. Endometrial markers of uterine receptivity utilizing

the donor oocyte model. Hum Reprod 2001;16:1893–9.

43. Lessey BA, Ilesanmi AO, Castelbaum AJ, Yuan LW, Somkuti SG,

Satyaswaroop PG, et al. Characterization of the functional progesterone

receptor in an endometrial adenocarcinoma cell line (Ishikawa): proges-

terone-induced expression of the a1-integrin. J Steroid Biochem Mol

Biol 1996;59:31–9.

44. Musgrove EA, Swarbrick A, Lee CS, Cornish AL, Sutherland RL. Mech-

anisms of cyclin-dependent kinase inactivation by progestins. Mol Cell

Biol 1998;18:1812–25.

45. Hambartsoumian E. Endometrial leukemia inhibitory factor (LIF) as

a possible cause of unexplained infertility and multiple failures of im-

plantation. Am J Reprod Immunol 1998;39:137–43.

46. Olivennes F, Ledee-Bataille N, Samama M, Kadoch J, Taupin JL,

Dubanchet S, et al. Assessment of leukemia inhibitory factor levels by

uterine flushing at the time of egg retrieval does not adversely affect preg-

nancy rates with in vitro fertilization. Fertil Steril 2003;79:900–4.

47. Aghajanova L, Stavraeus-Evers A, Nikas Y, Hovatta O, Landgren B-M.

Coexpression of pinopodes and leukemia inhibitory factor, as well as its

receptor, in human endometrium. Fertil Steril 2003;79(Suppl 1):808–14.

48. Kopf M, Baumann H, Freer G, Fredenberg M, Lamers M, Kishimoto T,

et al. Impaired immune and acute-phase responses in IL-6 deficient mice.

Nature 1994;368:339–42.

49. Salamonsen LA, Dimitriadis E, Robb I. Cytokines in implantation. Sem

Repr Med 2000;18:299–310.

50. Narayana Murthy PS, Sengupta S, Sharma S, Singh MM. Ormeloxifene

prevents ovariectomy-induced bone loss by inhibition of osteoclastogen-

esis, apoptosis of mature osteoclasts and up-regulation of TGFb-3 ex-

pression in rat. J Steroid Biochem Mol Biol 2006;100:117–28.

51. Arshad M, Sengupta S, Sharma S, Ghosh R, Savlani V, Singh MM. Invitro

anti-resorptive activity and prevention of ovariectomy-induced osteopo-

rosis in female Sprague-Dawley rats by dl-ormeloxifene, a selective

estrogen receptor modulator. J Steroid Biochem Mol Biol 2004;91:67–78.

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