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CONTROVERSY: ENDOMETRIAL BIOPSYAND INFERTILITY STATUS?

FERTILITY AND STERILITY�VOL. 82, NO. 5, NOVEMBER 2004

Copyright ©2004 American Society for Reproductive MedicinePublished by Elsevier Inc.

Printed on acid-free paper in U.S.A.

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eceived December 3,003; revised andccepted March 29, 2004.upported by National

nstitutes of Health/Nationalnstitute of Child Health anduman Development (NIH/ICHD) grants U10D27049 (C.C.), U01D38997 (E.M.), U10D27039 (D.G.), U10D39005 (M.D.), U10D27011 (S.C.), U10D33172 (M.S.), U10D38988 (B.C.), U10D38992 (R.L.), U10D38998 (W.S.), U10D38999 (P.McG.), U10D26981HD (J.O.), and U10D33173 (J.H.).eprint requests: Christosoutifaris, M.D., Ph.D.,ivision of Reproductivendocrinology and

nfertility, University ofennsylvania School ofedicine and Healthystem, Suite 860, Thecience Center, 3701arket Street, Philadelphia,A 19104 (FAX: 215-349-512; E-mail: ccoutifaris@bgyn.upenn.edu).Department of Obstetricsnd Gynecology, Universityf Pennsylvania School ofedicine.Department of Obstetricsnd Gynecology, Dukeniversity Medical Center.Department of Obstetricsnd Gynecology, Universityf Rochester.Department of Obstetricsnd Gynecology, Waynetate University.Department of Obstetricsnd Gynecology, Baylorollege of Medicine.Department of Obstetricsnd Gynecology,ennsylvania Stateniversity.Department of Obstetricsnd Gynecology, Universityf Medicine and Dentistryf New Jersey.

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264

istological dating of timed endometrialiopsy tissue is not related to fertilitytatus

hristos Coutifaris, M.D., Ph.D.,a Evan R. Myers, M.D., M.P.H.,b,k

avid S. Guzick, M.D., Ph.D.,c Michael P. Diamond, M.D.,d Sandra A. Carson, M.D.,e

ichard S. Legro, M.D.,f Peter G. McGovern, M.D.,g William D. Schlaff, M.D.,h

ruce R. Carr, M.D.,i Michael P. Steinkampf, M.D.,j Susan Silva, Ph.D.,k

onna L. Vogel, M.D., Ph.D.,l and Phyllis C. Leppert, M.D., Ph.D.,l for the NICHDational Cooperative Reproductive Medicine Network

ICHD National Cooperative Reproductive Medicine Network: University of Pennsylvania School of Medicine,hiladelphia, Pennsylvania; Duke University Medical Center, Durham, North Carolina; Duke Clinical Research

nstitute, Duke University Medical Center, Durham, North Carolina; Baylor College of Medicine, Houston,exas; Pennsylvania State University, Hershey, Pennsylvania; University of Medicine and Dentistry of Newersey, Newark, New Jersey; University of Colorado, Denver, Colorado; University of Texas—Southwesternedical Center, Dallas, Texas; University of Alabama, Birmingham, Alabama; Wayne State University, Detroit,ichigan; University of Rochester, Rochester, New York; University of Pittsburgh, Pittsburgh, Pennsylvania;righam and Women’s Hospital, Boston, Massachusetts; University of California, Davis, Sacramento,alifornia; University of Louisville, Louisville, Kentucky; and the Reproductive Sciences Branch, National

nstitute of Child Health and Human Development, Bethesda, Maryland

bjective: To assess the ability of histological dating to discriminate between women of fertile and infertileouples. The utility of histological dating of endometrium in the evaluation of infertile couples is uncertain.

esign: Prospective multicenter study, with subjects randomly assigned to biopsy timing. Criterion standardor infertility was 12 months of unprotected, regular intercourse without conception and for fertility at leastne live birth within 2 years.

etting: University-based infertility practices.

atient(s): Volunteer subjects (847) recruited at 12 clinical sites participating in the National Institutes ofealth-funded Reproductive Medicine Network. Inclusion criteria included ages 20–39 years, regular men-

trual cycles, and no hormonal treatment or contraceptive use for 1 month before the study. Fertile controlsere excluded if they had a history of infertility, recurrent pregnancy loss, or recent breastfeeding.

ntervention(s): Subjects underwent daily urinary LH testing. After detection of the LH surge, subjects wereandomized to biopsy in the mid (days 21–22) or the late (days 26–27) luteal phase. Pathologists at each sitestimated the cycle day based on standard criteria. For the primary analysis, an out-of-phase biopsy wasefined as a greater than 2-day delay in the histological maturation of the endometrium.

ain Outcome Measure(s): The proportion of out-of-phase biopsies in fertile and infertile women wasompared using logistic regression models with age at randomization as a covariate. Comparisons were alsoade between fertile vs. infertile at the midluteal or late luteal phase time points.

esult(s): Biopsies were evaluated (301 mid and 318 late; N � 619). Out-of-phase biopsy results poorlyiscriminated between women from fertile and infertile couples in either the midluteal (fertile: 49.4%,nfertile: 43.2%) or late luteal phase (fertile: 35.3%, infertile 23.0%). Results did not substantially differ usinglternative definitions of “out-of-phase” or standardized cycle day.

f Colorado.Department of Obstetrics and Gynecology, University of Texas—Southwestern Medical Center.Department of Obstetrics and Gynecology, University of Alabama.Department of Obstetrics and Gynecology, Duke Clinical Research Institute, Duke University Medical Center.

Department of Obstetrics and Gynecology, Reproductive Sciences Branch, National Institute of Child Health and Humanevelopment.

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onclusion(s): Histological dating of the endometrium does not discriminate between women of fertile and infertile couples and should note used in the routine evaluation of infertility. (Fertil Steril� 2004;82:1264–72. ©2004 by American Society for Reproductive Medicine.)

ey Words: Infertility, endometrial biopsy, luteal phase defect, endometrium, uterine receptivity

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One in seven couples of reproductive age in the Unitedtates faces infertility, defined as inability to achieve preg-ancy after 12 months of unprotected, regularly timed inter-ourse. Extrapolating from regional data obtained from thensurance industry (Aetna/USHealthcare; personal commu-ication) it is estimated that approximately 600,000 coupleseek infertility evaluation and treatment in the United Statesach year. The current standard of care, as suggested byajor textbooks of Gynecology and of Reproductive Endo-

rinology and Infertility, includes the routine evaluation ofhe luteal phase in the screening of infertile couples andecommends the histological evaluation of the endometriumi.e., an endometrial biopsy) as the test of choice.

There is excellent rationale for the evaluation of thendometrium of the female partner in a couple presentingith infertility, first as a method to document ovulation and

econd to evaluate the organ participating in the process ofmplantation. It should be noted that a receptive endome-rium is essential for successful embryo implantation (1–4).uring the menstrual cycle, the sequential action of estrogen

E) derived from the developing follicle(s) followed by theombined action of E and P on the endometrium aftervulation causes morphological and molecular changes inndometrial cells rendering them receptive to the adhesivend migratory properties of the trophoblast cells of themplanting embryo. Thus, it has long been advocated that thelinical evaluation of the infertile couple includes an evalu-tion of the quality of the luteal phase (5–21).

During the luteal phase, the morphological changes ob-erved in the endometrium occur in a predicticable pattern22). Traditionally, the endometrium is considered “out-of-hase” if the standardized menstrual cycle date based onorphological criteria assigned by a pathologist lags byore than 2 days than the “actual” standardized cycle date

etermined by a physiological marker such as the urinary orerum LH surge, ovulation, or the date of the onset of theext menstrual cycle. The endometrial biopsy, with evalua-ion of morphological changes, has been considered superioro alternatives such as serum P measurements, because of theulsatile nature of P secretion and a belief that these mor-hological changes better represent the cumulative effect ofycle-specific patterns of ovarian hormone secretion (9).

The endometrial biopsy has been used for decades in thecreening evaluation of the infertile couple to confirm ovu-ation and to also evaluate the histological maturation of thendometrium. Frequently, such evaluation is followed byrescription of “corrective” hormonal treatments. Neverthe-ess, the surprising fact is that the ability of the histologicalvaluation of the endometrium to distinguish between

omen of fertile and infertile couples has not been rigor- c

ERTILITY & STERILITY�

usly ascertained and thus, prescription of treatment basedn histological evaluation of the endometrium may be inap-ropriate. In addition, the prevalence of “out-of-phase” bi-psies in the fertile population has been reported to rangeetween �5% to as high as 50%, thus making this testifficult to interpret (1, 18, 23, 24). This high variability ofesults casts doubt on the validity of the histological datingf the endometrium as a clinical tool for the evaluation of theuteal phase in women of infertile couples. Furthermore,ven among those who advocate the routine use of thendometrial biopsy, there is controversy over the optimaliming of the procedure.

In the human, it is currently accepted that the window ofmplantation, the time during which the endometrium is mostonducive to trophoblast–endometrial interactions, is re-tricted to days 20–24 of an idealized 28 day cycle, consid-ring day 14 the day of the LH surge (25, 26). Somenvestigators have advocated performing the biopsy duringhe window (27), whereas other researchers have advocatederforming the biopsy late in the luteal phase (approximatelyays 26–27) to assess the cumulative effect of P on thendometrium (9).

Given the controversy surrounding the utility of the bi-psy, the National Institutes of Health/National Institute ofhild Health and Human Development (NIH/NICHD)-

unded National Cooperative Reproductive Medicine Net-ork performed the current study to determine whetheristological dating of the endometrium is a clinically usefulest for discriminating between women of infertile couplesnd fertile controls. In comparing infertile to fertile couples,wo questions were addressed: [1] Is there a difference in therevalence of an out-of-phase endometrial biopsy in womenf infertile couples as compared to fertile controls? and [2]hat is the most appropriate time during the luteal phase for

he endometrial biopsy to be performed?

MATERIALS AND METHODSA multicenter randomized prospective study was per-

ormed, as shown in Figure 1. This study was reviewed andpproved by the Network Data and Safety Monitoring Com-ittee (DSMC), and by each participating institution’s In-

titutional Review Board. In addition, there were no conflictsf interest of any of the investigators. Briefly, women ofnfertile couples and fertile controls were recruited and afteroluntary written informed consent, they were screened and,f they satisfied the inclusion criteria (see Table 1), werenrolled in the study. Patients with either primary or second-ry infertility were included. Etiology of infertility was not

ategorized because the study was designed to include all

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ouples seeking infertility evaluation and determine thealue of the endometral biopsy in the screening of couplesresenting with infertility.

Subjects were instructed in the use of home urinary LHits, were given a kit, and were asked to contact the sitetudy coordinator when the urinary LH surge was detected.

e chose the LH surge as the point of reference because instudy by Shoupe and co-workers (28), ovulation most

F I G U R E 1

chematic representation of the study design. Randomizatio

outifaris. Endometrial biopsy and infertility. Fertil Steril 2004.

T A B L E 1

nclusion criteria for women of infertile couples and fertileontrols.

roup Inclusion criteria

omen of infertile couples1. No pregnancy after 12 months of attemptedconception.

2. Regular menstrual cycles, every 24–34 days3. Aged 20–39 years.4. No hormonal treatment in the last menstrual

cycle.omen of fertile couples 1. At least one live born child.

2. Last delivery within 2 years of biopsy.3. Last pregnancy resulted in a live born or

was not a spontaneous abortion.4. Regular menstrual cycles every 24–34 days.5. No history of infertility (i.e., a 12-month

period of attempted conception withoutsuccess) or recurrent pregnancy loss.

6. Aged 20–39 years.7. Willing to discontinue hormonal

contraceptives for 1 month before study.8. If postpartum, no breast feeding for 2

months before study.

ote: There were no exclusion criteria.

boutifaris. Endometrial biopsy and infertility. Fertil Steril 2004.

266 Coutifaris et al. The endometrial biopsy and infertility

ccurately correlated with endometrial maturation. In addi-ion, the original studies by Noyes and co-workers con-tructed their dating tables based on the day of ovulationstimated by an increase in the basal body temperature (22,9). At the time of the urinary LH surge, subjects wereandomized to undergo the endometrial biopsy either 7–8ays (standardized cycle days 21–22) or 12–13 days (stan-ardized cycle days 26–27) after the urinary LH surgestandardized cycle day 14). A stratified randomization withandom permuted blocking within each stratum was con-ucted by the Data Coordinating Center at Duke University.tratification variables were site, fertility status (fertile ver-us infertile), and age subgroup (aged 20–34 years vs. 35–39ears). The randomization was successful in terms of bal-ncing the number of women from infertile couples andertile controls in the mid and late condition: In women fromnfertile couples, 139 were assigned in the midluteal biopsyroup and 148 in the late luteal group; as for fertile controls,62 were assigned to the midluteal biopsy group and 170 tohe late group.

The endometrial biopsies were performed using soft en-ometrial biopsy catheters (e.g., Pipelle). Tissue was imme-iately placed in fixative and processed for histological eval-ation. Slides for subjects enrolled in the study werenterpreted by the same pathologists reading clinical endo-etrial biopsy slides at each participating institution. Pathol-

gists were blinded to the purpose of the study, includingertility status and menstrual day the biopsy was performed.he criteria of Noyes et al. (22) were used to date theiopsies. An “out-of-phase” biopsy was defined as a morehan 2-day difference between the actual luteal day as de-ermined by the detection of the LH surge in urine andounting it as day 14 and the pathologist’s determination

s performed at the time of the urinary LH surge.

n wa

ased on the histological criteria of Noyes et al. (22).

Vol. 82, No. 5, November 2004

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The primary analysis compared the prevalence of anut-of-phase biopsy in women of infertile couples vs. fertileontrols. The analysis plan included three prespecified setsf infertile vs. control comparisons designed to evaluaterevalence of an out-of-phase: [1] regardless of the day inhe luteal phase of the cycle, [2] in the midluteal phase, and3] in the late luteal phase. Logistic regression model adjust-ng for age was used to test for a relationship betweenertility status and presence/absence of an out-of-phase bi-psy for each comparison set (30, 31). The logistic regres-ion model for the primary analysis included one predictorariable (fertility status), one explanatory variable (covari-te; age), and the response variable (out-of-phase determi-ation). This prespecified model was designed to evaluatehether fertility status, after adjusting for age, significantlyredicted the biopsy finding and what was the relative risk ofaving an out-of-phase biopsy. The model was double-pro-rammed by two independent statisticians as part of theuality control process required at the Duke Data Coordi-ating Center and was specified in the primary analysis planor study and approved before study enrollment. The predic-or (fertility status), explanatory (age), and response (out-of-hase determination) variables were specified in the statisti-al analysis plan approved before conducting the primarynalysis as per guidelines recommended for clinical trials.onfirmatory analyses using �2 tests were conducted toxamine the association between fertility status and propor-ion of out-of-phase biopsies, without adjusting for age. Inddition, given the hormonal responsiveness of the endome-rium, analyses were also performed using the recent use oformonal contraception and smoking as other confoundingariables.

Power and sample size calculations conducted beforetudy initiation indicated that a total of 880 patients would beequired to achieve 80% or greater power and an overalllpha of 0.05 (two-tailed tests; 32, 33). For this poweralculation, we assumed that the prevalence of an out-of-hase biopsy in the fertile population would be 4%, and weypothesized a threefold increase in the prevalence in thenfertile population. The required sample size was estimatedo be 400 fertile controls and 400 infertile patients, with 200ssigned to the midluteal phase biopsy and 200 to the lateuteal phase biopsy for each group. To account for a 10%ropout rate, we planned to enroll 220 patients in each of theour subgroups. To achieve an overall alpha of 0.05, the levelf significance for each of the three preplanned comparisonsas set at 0.0125 to adjust for the multiple comparisons asell as one preplanned interim analysis conducted at theidpoint of the study.

Applying an O’Brien–Fleming adjustment for multipleata looks, an interim analysis of the out-of-phase data wasonducted on the first 440 patients to complete the study.he results of this confidential analysis were reviewed by the

SMC only so that a recommendation for study continua- c

ERTILITY & STERILITY�

ion, discontinuation, or modification could be made. Afterhe interim analysis by the DSMC, the network was advisedo stop recruitment and proceed with final analysis of theata given the high proportion of out-of-phase biopsies ob-erved in the fertile population (discussed later). At the timef the DSMC decision, more than 800 subjects had beennrolled in the study. Specifically, the database included 847omen with 736 of them having undergone biopsies on the

ppropriate days, either days 21–22 for those randomized tohe midluteal phase biopsy or days 26–27 for those random-zed to the late biopsy group. Forty-six of the biopsies wereead as “proliferative” endometrium and thus were excludedrom the analysis. Although this could be considered toepresent extreme cases of “out-of-phase” endometrium, fur-her analysis of the menstrual history in these study subjectsndicated that no ovulation had occurred. This is the subjectf a companion article in this issue of Fertility and Sterilityealing with false-positive urinary LH testing. Furthermore,nclusion of these patients in the analysis, assuming that thendings represented severe out-of-phase endometrium, didot affect the results because the distribution between theifferent experimental groups was equivalent. In addition, 62iopsies yielded insufficient tissue for diagnosis and 9 biop-ies had missing end point data and were not interpretable.hus, there were 619 biopsies that were evaluable and used

n the analysis.

The primary analysis was conducted using the definitionsf out-of-phase as outlined previously. Additional secondarynalyses were conducted using definitions of 3 and 4 daysifference. In addition, alternative methods for defining thetrue” cycle day (counting the date of the urinary LH surges day 13 or counting the date of the next menstrual periods day 28 or day 29) were also used.

RESULTSThe study subjects enrolled in this multicenter investiga-

ion were of similar age. Women of infertile couples were2.6 � 3.7 years old (mean � SD) and fertile controls were1.4 � 3.9 years old. A total of 619 women (287 women ofnfertile couples and 332 fertile controls) had evaluable dataor analysis.

Table 2 shows the prevalence of an out-of-phase biopsy inertile women regardless of whether the biopsy was per-ormed during the midluteal or late luteal phase. The prev-lence of an out-of-phase biopsy, if more than 2 days ofifference between the histological and the postovulatoryay were observed, was 42.2% in fertile controls vs. 32.7%n the women of infertile couples (P�.0248). Even if moretringent criteria were used of more than 3 or 4 days, therevalence of an out-of-phase biopsy in these fertile womenas 33.2% and 19.9%, respectively. Interestingly, if theseore stringent criteria were used, the prevalence of out-of-

hase biopsies in women of infertile couples was signifi-

antly lower than the prevalence in fertile controls (Table 2;

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�.0011 for a more than 3-day maturational delay and�.0027 for a more than 4-day delay).

Tables 3 and 4 show the results comparing endometrialiopsies from women of fertile and infertile couples per-ormed during the midluteal phase (window of implantation)r the late luteal phase. There were no statistically significantifferences (P�.0125) when the primary end point of morehan a 2-day difference in the histological vs. postovulatoryating was used in either the midluteal or late luteal phaseiopsy groups (both P�.0125). If more stringent criteriaere used, no statistical significance was observed with the

xception of the late biopsies evaluated for more than 3 daysf difference in the histological vs. postovulatory datingP�.0023). However, even in this comparison, the preva-ence of out-of-phase biopsy was higher in fertile womenhan in women from infertile couples. For each analysis, theesults of the supplemental �2, which did not adjust for ageonfirmed the results from the logistic regression analyses.

We considered that the very high prevalence of an out-f-phase biopsy in fertile controls may have been secondaryo a recent discontinuation of hormonal medications, such asral contraceptives, in this group of patients. A total of 11omen of the 619 women (1.8%) reported the use of suchormonal preparations. Interestingly, 8 of these 11 had out-

T A B L E 2

revalence of an out-of-phase endometrial biopsy in fertile wr late luteal phase.

VariablesWomen of fertilecouples (n � 332)

Women of infertilecouples (n � 287)

OP�2 42.2% 32.7%OP�3 33.2% 20.9%OP�4 19.9% 11.2%

ote: P value for logistic regression, adjusting for age.OOP�2, OOP�3, and OOP�4 denotes more than 2, more than 3, and moresing the Noyes criteria. To adjust for preplanned multiple comparisons, f

outifaris. Endometrial biopsy and infertility. Fertil Steril 2004.

T A B L E 3

revalence of out-of-phase endometrial biopsy in women oferformed during the window of implantation (days 22–23).

VariablesWomen of fertilecouples (n � 162)

Women of infertilecouples (n � 139)

OP�2 49.4% 43.2%OP�3 42.0% 30.9%OP�4 22.2% 13.3%

ote: P value based on logistic regression, adjusting for age. OOP�2, Oifference between the postovulatory day and the histological dating using tere considered significant if P�.0125.

outifaris. Endometrial biopsy and infertility. Fertil Steril 2004.

268 Coutifaris et al. The endometrial biopsy and infertility

f-phase endometrial biopsies (3 of 5 women of infertileouples and 5 of 6 fertile controls). Although the prevalenceppears to be high, given the small numbers and the propor-ional distribution, further analysis indicated that this factorid not significantly influence the study results or contributeo the fertility group differences.

It should also be noted that one of the specific histologicalndings evaluated in this study was the presence or absencef gland–stromal dysynchrony. This finding may indicateither a pathological entity related to fertility or sampling ofhe lower uterine segment. The results indicated that gland–tromal dysynchrony was observed extremely infrequentlynd its presence did not differ between groups.

Because practitioners frequently use the date of the onsetf the subsequent menstrual period to evaluate the results ofhe histological dating, we analyzed the data using thispproach and considering day 28 the day of the onset of theubsequent menses and counting backward to determine theenstrual date of the performance of the biopsy. Using this

pproach, the proportion of out-of-phase biopsies was al-ays higher in fertile women than in women from infertile

ouples. Findings were similar using other, commonly used,lternative criteria for the chronological dating of the endo-etrial biopsy, such as assigning day 13 to the urinary LH

en and in women of infertile couples at either the midluteal

Odds ratiopoint estimate

Odds ratio estimates95% Wald CL P value

0.68 0.49–0.95 .02480.54 0.37–0.78 .0011a

0.49 0.31–0.78 .0027a

4 days difference between the postovulatory day and the histological datinggroup differences were considered significant if P�.0125.

rtile couples and fertile controls if the biopsy was

Odds ratiopoint estimate

Odds ratio estimates95% Wald CL P value

0.79 0.50–1.26 .33430.65 0.40–1.05 .07550.55 0.30–1.02 .0595

, and OOP�4 denotes more than 2, more than 3, and more than 4 daysyes criteria. To adjust for multiple comparisons, fertility group differences

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urge (thus assigning ovulation to day 14) or assigning theay of the subsequent menses as cycle day 29 and countingackward.

Figure 2 depicts the performance of the endometrial bi-psy as a diagnostic test for infertility on a receiver operatingharacteristic (ROC) curve. Sensitivity is defined as the

T A B L E 4

revalence of out-of-phase endometrial biopsy in women oferformed during the late luteal phase (days 26–27).

VariablesWomen of fertilecouples (n � 170)

Women of infertilecouples (n � 148)

OP�2 35.3% 23.0%OP�3 24.7% 11.5%OP�4 17.6% 8.8%

ote: P value based on logistic regression, adjusting for age.OOP�2, OOP�3, and OOP�4 denotes more than 2, more than 3, and moresing the Noyes criteria. To adjust for preplanned multiple comparisons, f

F I G U R E 2

eceiver operating characteristic curve for endometrial datindays 21–22) phase (green line), and biopsies obtained in theepresents the proportion of women in each group correctly c�), by more than 2 days (□), by more than 3 days (�), anroportion of women in each group incorrectly classified as i

hat does not discriminate between true and false positives.

outifaris. Endometrial biopsy and infertility. Fertil Steril 2004.

outifaris. Endometrial biopsy and infertility. Fertil Steril 2004.

ERTILITY & STERILITY�

roportion of infertile women with an out-of-phase biopsyased on definitions of 1, 2, 3, or 4 days discrepancy,hereas specificity is defined as the proportion of fertileomen with an in-phase biopsy based on each definition.urves are presented for all patients, and for patients sam-led in the midluteal and late luteal phase. Each curve runs

rtile couples and fertile controls if the biopsy was

Odds ratiopoint estimate

Odds ratio estimates95% Wald CL P value

0.55 0.34–0.92 .02180.38 0.20–0.71 .0023a

0.42 0.21–0.86 .0166

4 days difference between the postovulatory day and the histological datinggroup differences were considered significant if P�.0125.

r all biopsies (black line), biopsies obtained in the midlutealluteal (days 26–27) phase (red line). The y-axis of the graph

ified as infertile by a biopsy out-of-phase by more than 1 dayy more than 4 days (X), whereas the x-axis represents theile in each group. The dotted diagonal line represents a test

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oughly parallel to the line connecting 0% sensitivity and00% specificity, and 100% sensitivity and 0% specificity,ndicating poor discrimination between fertile and infertiletatus. Results did not differ when alternative criteria, suchs “LH surge on day 13” or “next menstrual period” countings cycle day 28 or 29, were used to assign true cycle days.

DISCUSSIONEvaluation of the luteal phase and its relation to infertility

ontinues to engender controversy in the management of thenfertile couple (13, 14, 34–36). Emphasis is often placed onhe methods used for the diagnosis of luteal phase defectsLPD) as a means to evaluate histologically the endometrialvents that serve as prerequisites for implantation. It isssumed that deficiencies in P production or endometrialaturation would result in implantation failure and hence in

nfertility. The hypothesis that an inadequately developedndometrium (by whatever validated or hypothesized crite-ia) during the window of implantation may prevent implan-ation and thus result in infertility is appropriate. It can alsoe hypothesized that factors that adversely affect the implan-ation process may consequently lead to early pregnancy loss37). Thus, at least theoretically, an inadequately developedndometrium can contribute to both infertility (lack of im-lantation) and recurrent pregnancy loss. In most discussionsf “unexplained” infertility, the possibility of an implanta-ion-related cause is raised, and clinicians are advised tovaluate the patient with particular reference to the presencef LPD (13, 24). However, it should not be forgotten thattudies have shown successful implantation during cycleshat the endometrium had exhibited maturational delay asssessed by histological criteria (38, 39).

The purpose of this investigation was not to question thexistence of LPD or define how to diagnose this condition.iven that the prevalence and clinical relevance of LPD areuestionable, we chose to evaluate the clinical tool, thendometrial biopsy, most commonly used for the evaluationf the quality of the luteal phase in screening of couplesresenting with infertility, that is, histological evaluation ofhe timed endometrial biopsy. In addition, given that a pre-ious study (27) has shown that biopsies performed in theame woman during the midluteal and the late luteal phaseossibly uncover cryptic forms of inadequate endometrialevelopment presenting as out-of-phase biopsies in the win-ow of implantation, but which were in-phase in the lateuteal phase, we also assessed whether the ability of the testo discriminate between women of infertile couples andertile controls differed during the window of implantationnd the late luteal phase.

The results, with respect to the usefulness of the endo-etrial biopsy as a screening tool for all couples presentingith infertility, are conclusive. First, the prevalence of anut-of-phase biopsy in the fertile population is extremely

igh. And it remains high, even if very stringent criteria of o

270 Coutifaris et al. The endometrial biopsy and infertility

ore than 3 or 4 days of difference in the histological vs.ostovulatory dating were used. Even more strikingly, theest failed to discriminate between women from infertileouples and fertile controls no matter what criteria were usedo define out-of-phase biopsies. If anything, fertile womenere more likely to have an out-of-phase biopsy than women

rom infertile couples. The timing of the biopsy did notffect the usefulness of the test either. Although the preva-ence of an out-of-phase biopsy appeared to be lower in theate luteal phase than in the midluteal phase, there were stillo differences between women of fertile and infertile cou-les. These observations, together with the results of previ-us smaller studies (36, 40–44), indicate that histologicalating of the endometrium based on tissue obtained throughn endometrial biopsy is not a useful test in the initialvaluation of all couples presenting with infertility.

There have been multiple reports of significant inter- andntraobserver variability in the histological evaluation forating of the human endometrium (24, 29, 43–45). Givenhat patients were randomly assigned to timing of the biopsy,hat the pathologist who read a given slide at a given site wasssigned at random, that these pathologists routinely pro-ided clinical services to busy infertility practices, and thathe sample size was large, it is unlikely that these resultseflect the effects of poor test reproducibility or limitedathologist experience on the variance of results. We con-rmed this in a substudy, which found that results fromandomly selected slides did not substantially differ whenead by a panel of expert gynecological pathologists (46).

It is also possible that if couples with male factor as thenly cause of infertility were excluded, some differencesay have been uncovered. However, given that, if anything,

ertile women had consistently a higher prevalence of out-f-phase biopsies, it seems likely that exclusion of couplesith male factor infertility would have led to an even lowerrevalence of out-of-phase biopsies in the infertile group. Inddition, it could be hypothesized that there may be someubgroups of infertile patients with specific diagnoses, suchs endometriosis, hyperprolactinemia, or unexplained infer-ility, in which an endometrial biopsy may be useful. Al-hough plausible, the extremely high prevalence of out-of-hase biopsies, both in the midluteal and late luteal phase inhe fertile population casts doubt as to whether any of theseossibilities would yield clinically relevant results.

Therefore, does this study put in question the existence ofhe diagnosis of LPD as a cause of infertility? The answer isno.” It should be made clear that the design of this study didot specifically address this question. Traditionally, the di-gnosis of LPD is made if two consecutive biopsies areound to be out-of-phase (9). This was not done in theresent investigation. However, the very high prevalence ofut-of-phase endometrial biopsies in fertile women we un-overed puts in question the use of this test in the diagnosis

f LPD. For example, if the random per-cycle probability of

Vol. 82, No. 5, November 2004

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n out-of-phase biopsy is 40%, then 16% of fertile womenill have two consecutive out-of-phase biopsies and beiven the diagnosis of LPD. This means that, at best, thisiagnostic tool would have a specificity of 84%. Even if abest case scenario for LPD” was considered by taking theower bound of the 95% confidence intervals for fertiles andpper bound for infertiles, only the midluteal biopsy resultedn greater frequency of two consecutive out-of-phase biop-ies in infertiles (23.6% vs. 19.0% for fertiles). If the prev-lence of “true LPD” is less than 6%, then the calculatedrequency of consecutive out-of-phase biopsies is more than00%. If the prevalence is 10%, then this calculated fre-uency drops to 65%. This means that at a prevalence of 6%,00% of women with true LPD would have two consecutiveut-of-phase biopsies. This is equivalent to a sensitivity of00%. In this situation, the specificity is 100% minus 19% or1%. However, at a prevalence of 6%, a sensitivity of 100%nd a specificity of 81%, the positive predictive value of twoonsecutive out-of-phase biopsies would be 7%. At a prev-lence of 10%, the positive predictive value would be 8%nd at a prevalence of 25%, it would be 13%. It is clear thatven in the best possible interpretation of the data as it relateso the diagnosis of LPD, the positive predictive value of thendometrial biopsy as the diagnostic test of choice wouldikely be between 7% and 10%. This is clearly suboptimal.

Therefore, although theoretically LPD may be a patho-ogic entity leading to infertility, the endometrial biopsy mayot be the test of choice for its diagnosis. The specificuestion needs to be addressed in a follow-up study, whichill evaluate alternative methods, such as measures of Putput by the corpus luteum or emerging endometrial mo-ecular markers in fertile and infertile women.

It should be stressed that this study only focused on theistological dating of the endometrium in the screening ofouples presenting with infertility. Certainly, if endometrialathology is suspected by the patient’s history or clinicalresentation, such as hyperplasia, cancer, or chronic endo-etritis, the endometrial biopsy is a very useful diagnostic

ool.

Finally, the cost of the procedure should also be consid-red. If an endometrial biopsy were included in the evalua-ion of all infertile couples, the national cost would rangeetween $210 and $300 million annually. In recent years,nd in spite of textbook recommendations and other pub-ished reports, the use of the endometrial biopsy appears toave decreased mainly driven by insurance industry recom-endations and partly by an empiric belief that results frombiopsy would not change the subsequent treatment plan. Toate, this has clearly not been based on any scientific evi-ence. Consistent with this trend, the 1999 and 2000 calen-ar year data from Aetna/USHealthcare indicate that in ap-roximately 25% of the couples evaluated for infertility, theemale partner actually underwent an endometrial biopsy.

hus, at present, the actual national cost can be conserva- Y

ERTILITY & STERILITY�

ively estimated to range between $53 and $75 million perear. In spite of the decreased utilization of the test, this islearly significant. Furthermore, when luteal phase defi-iency is diagnosed after an endometrial biopsy and byhatever histological criteria, treatments of questionable ef-cacy are instituted, leading to additional expenditure ofesources and repeat biopsies to confirm efficacy of treat-ent (5, 6).

In conclusion, the timed endometrial biopsy followed byistological dating of the endometrium provides no clinicallyseful information as a screening test. Given the data fromhis study and the high cost of the procedure, it is stronglyecommended that the histological evaluation of the endo-etrium be abandoned as a diagnostic tool in the routine

valuation of the infertile couple. Although the $53 to $75illion annual savings can be considered modest, the chan-

eling of these funds to support infertility treatments of highfficacy, such as IVF, can be of great benefit to infertileouples. In parallel, continued research on the emergingolecular markers of endometrial development should be

ncouraged. However, as proven in this study, it is clear thatigorous evaluation of any tests based on new parameterseeds to be undertaken before their routine application tolinical practice and their proclamation as “gold standards”or the evaluation of the luteal phase.

cknowledgments: Additional co-authors and their institutional affilia-ions: Kurt Barnhart, M.D., M.S.C.E. (University of Pennsylvaniachool of Medicine, Philadelphia, PA); Richard E. Leach, M.D. (Waynetate University, Detroit, MI); Judy Albert, M.D. (University of Pitts-urgh, Pittsburgh, PA); Joseph A. Hill, M.D. and Elizabeth Ginsburg,.D. (Brigham and Women’s Hospital, Boston, MA); James W. Over-

treet, M.D., Ph.D. (University of California, Davis, Sacramento, CA);teven T. Nakajima, M.D. (University of Louisville, Louisville, KY);inda C. Giudice, M.D., Ph.D. (Stanford University, Palo Alto, CA).

In addition to the authors and co-authors, other investigators of theational Cooperative Reproductive Medicine Network were as follows:niversity of Pennsylvania School of Medicine, Philadelphia, PA: L.astroianni Jr., M.D., V. LiVolsi, M.D., L. Martino, M.S.N., C.R.N.P.,. Timbers, M.S.N., C.R.N.P., R. Brown, M.S.N., C.R.N.P.; Dukeniversity Medical Center, Durham, NC: L. Lambe, R.N., E. Martinez,.N., C. Bodine, R.N., R. Brown, R.N., R. Oliverio, R.N.; Wayne Stateniversity, Detroit, MI: E. Puscheck, M.D., K. Ginsburg, M.D., K.ollins, M.S., N. Angel, R.N., M. Brossoit; Baylor College of Medicine,ouston, TX: P. Amato, M.D., M. Suzi Lindsay, R.N.; Pennsylvaniatate University School of Medicine, Hershey, PA: C. Gnatuk, M.D.,an Johnson-Rollings, R.N.; University of Medicine and Dentistry ofew Jersey, Newark, NJ: D. Heller, M.D., J. Colon, M.D., G. Weiss,.D., A. Solnica, R.N.; University of Colorado School of Medicine,enver, CO: K. Gattin, R.N., S. Hahn, R.N.; University of Texas-outhwestern School of Medicine, Dallas, TX: M. Roarck, R.N.C.;niversity of Alabama, Birmingham School of Medicine, Birmingham,L: V. Willis, R.N., L. Love, B.S.N., R.N.; Columbia University, New

ork, NY: R. E. Canfield, M.D., P. Factor-Litvak, Ph.D.

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