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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2008-0460
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 10 4058-4064
Copyright © 2008 by The Endocrine Society

Endometrial Development and Function in Experimentally Induced Luteal Phase Deficiency

Rebecca S. Usadi, Jeremy M. Groll, Bruce A. Lessey, Ruth A. Lininger, Richard J. Zaino, Marc A. Fritz and Steven L. Young

Division of Reproductive Endocrinology and Infertility (R.S.U.), Carolinas Medical Center, Charlotte, North Carolina 28232; Division of Reproductive Endocrinology and Infertility (J.M.G.), United States Air Force Medical Center, Wright-Patterson Air Force Base, Dayton, Ohio 45433; Division of Reproductive Endocrinology and Infertility (B.A.L.), Greenville Hospital, Greenville, South Carolina 29605; Departments of Pathology (R.A.L.) and Obstetrics and Gynecology (M.A.F., S.L.Y.), Division of Reproductive Endocrinology and Fertility, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; and Department of Pathology (R.J.Z.), Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033

Address all correspondence and requests for reprints to: Rebecca S. Usadi, M.D., Carolinas Medical Center, Division of Reproductive Endocrinology and Infertility, P.O. Box 32861, Charlotte, North Carolina 28232-2861. E-mail: Rebecca.Usadi{at}carolinashealthcare.org.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: It is generally assumed that delayed endometrial development observed in luteal phase deficiency (LPD) is the result of abnormally low progesterone (P) levels. This hypothesis has never been tested by direct experiment.

Objective: Our objective was to evaluate the effects of P concentrations on human endometrium.

Design and Setting: A randomized trial was conducted at an academic medical center.

Subjects: Twenty-nine healthy, ovulatory 18- to 35-yr-old women participated.

Intervention: Endometrial samples were obtained from women in natural cycles and two groups of experimentally modeled cycles. Women undergoing modeled cycles were treated with GnRH agonist and a fixed physiological dose of transdermal estradiol, followed by randomization to 10 or 40 mg daily im P administration to achieve either normal circulating luteal P or 4-fold lower P concentrations, the latter representing an experimental model of LPD.

Main Outcome Measures: Tissue specimens, obtained after 10 days of P exposure, were analyzed by histological dating, immunohistochemistry, immunoblot, and real-time quantitative RT-PCR (qRT-PCR).

Results: Histological dating of endometrium, immunohistochemistry for endometrial integrins, and qRT-PCR analysis for nine putative functional markers showed no differences between the three groups. Preliminary data from Western analysis suggest that some proteins may be affected by low serum P concentrations.

Conclusions: Histological endometrial dating does not reflect circulating P concentrations and cannot serve as a reliable bioassay of the quality of luteal function. Assessment of selected functional markers by either immunohistochemistry or qRT-PCR is similarly insensitive to decreased circulating P. Preliminary evidence suggests that abnormally low luteal phase serum P concentrations may have important functional consequences not otherwise detected.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Embryo implantation occurs within a narrow window of time, determined by estradiol (E2) and progesterone (P) action on the endometrium (1, 2, 3, 4). P causes progressive structural changes in the endometrium as well as progressive functional changes that are essential to embryo implantation (4, 5, 6). Logically, there must be some minimal amount or duration of P exposure needed to induce secretory histological endometrial development and embryo receptivity. P exposure below this threshold is expected to result in abnormal endometrial maturation or function, a concept generally known as luteal phase deficiency (LPD) (7).

LPD is a controversial disorder, viewed as predisposing to failed or delayed implantation, infertility, and early pregnancy loss. The prevalence and clinical importance of LPD have not been established, because there is no validated diagnostic test for the disorder (8). Common measures of luteal function and endometrial receptivity, midluteal phase serum P concentrations and endometrial histological dating, have limitations. Serum P levels fluctuate due to pulsatile corpus luteum P secretion (9). Histological dating is subjective and lacks both precision (10, 11) and accuracy (12). Consequently, attention has focused on the expression of molecular markers of endometrial function, including the β3-integrin subunit and mRNA species identified by microarray (13, 14).

The fundamental concept of LPD, that low luteal phase P concentrations result in abnormal endometrial development, has never been tested by direct experiment. To test that hypothesis and to define the effects of low serum P concentrations on endometrial development and function, we compared the histological characteristics and expression of putative markers of embryo receptivity in endometrium obtained in natural cycles and in modeled cycles characterized by unvarying physiological serum E2 levels and either normal or low serum P concentrations.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Human subjects

Women (ages 18–35 yr) with regular menstrual cycles (25–35 d) were recruited to participate in two phases of the study and received compensation as approved by the institutional review board at the University of North Carolina at Chapel Hill. Subjects were evaluated by transvaginal ultrasonography, and those having any uterine abnormalities were excluded. Each subject was evaluated in a natural menstrual cycle. Urinary LH was monitored daily from cycle d 10 until detection of the midcycle LH surge (defined as luteal d 0), and endometrial biopsy was performed on luteal d 10. Subjects exhibiting abnormal endometrial dating (histological date ≥ cycle d 22) (6) or absent epithelial {alpha}vβ3 integrin expression (immunohistochemistry) (6) were excluded from the study.

In modeled cycles, summarized in Fig. 1Go, subjects received long-acting GnRH agonist [leuprolide acetate (Lupron; TAP Pharmaceuticals, Lake Forest IL); 1 mg/d sc] beginning in the late luteal phase of the natural cycle and for the duration of the study. Once menses occurred and leuprolide-induced down-regulation was confirmed (serum E2 < 40 pg/ml), subjects received a fixed dose of transdermal E2 (Vivelle Dot; Novartis, East Hanover, NJ; 0.2 mg/d) for a total of 20 d. Subjects were randomized to also receive P, 10 mg/d im (group I, n = 8) or 40 mg/d im (group II, n = 9), on d 11–20 of E2 treatment. Blood samples were obtained on alternate days during the study for later measurement of serum E2 and P concentrations. On the 10th day of combined E2/P treatment, endometrial thickness was measured by transvaginal ultrasonography (maximal two-layered thickness in the midsagittal plane), and endometrial biopsy was performed using a Pipelle (Milex Products Inc., Chicago, IL). A portion of each specimen was frozen in liquid nitrogen and the remainder processed for histopathological examination. Steady-state serum P concentrations were measured every other day starting on d 2 of P treatment.


Figure 1
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  		FIG. 1. Study protocol. The LH surge is defined as d 0.

 
To characterize serum P concentrations in the two experimental groups and to acquire sufficient tissue for molecular analysis, we conducted a second phase of the study for which 12 additional normally cycling women were recruited and randomized to receive the same experimental regimens described above; six subjects received P 10 mg/d, and the remaining six received P 40 mg/d. On four occasions between d 3 and 10 of P treatment, blood samples were obtained for measurement of serum P at times corresponding to the expected peak (2–3 h after injection) and trough (1–2 h before injection) concentrations. These additional 12 subjects underwent endometrial biopsy on the 10th day of combined E2/P treatment; tissue specimens were used for analysis by both histological dating and by quantitative real-time PCR (qRT-PCR). Endometrial specimens obtained from normally cycling women (ages 18–35 yr; n = 18) on luteal d 9–10 (n = 8) in monitored natural cycles were used as controls.

Serum E2 and P concentrations were measured using commercial immunoassay kits (Immulite DPC; Diagnostic Products Corp, Los Angeles, CA). Inter- and intraassay variability was less than 4 and 6%, respectively. P concentrations in the two groups were compared using a paired t test and repeated-measures ANOVA. Fixed specimens were assessed using standard histological dating criteria (6) by two gynecological pathologists blinded to intervention. Portions of the cryopreserved specimens were prepared for analyses of expression of putative markers of endometrial receptivity by immunohistochemistry, immunoblotting, and qRT-PCR as described below. Comparisons between groups were made using the nonparametric Wilcoxon signed rank sum test. Statistical calculations were performed with SAS software (SAS Institute, Inc., Cary, NC) or Prism (GraphPad Software, San Diego, CA). Statistical significance was set at a P value < 0.05.

Immunohistochemistry

Integrin expression was evaluated in cryopreserved specimens by immunohistochemistry using monoclonal antibodies directed against the {alpha}1 (TS2/7; 1:5000), {alpha}4 (B-5G10; 1:4000), and β3 (SSA6; 1:2000) integrin subunits as previously described (15). The resulting staining was evaluated by a single observer blinded to treatment. Staining intensity was assigned using a semiquantitative HSCORE as previously described (16). HSCORE was calculated using the following equation: HSCORE = {sum}Pi (i + 1), where i represents intensity of staining with a value of 1, 2, or 3 (corresponding to weak, moderate, or strong staining, respectively) and Pi is the percentage of stained epithelial cells for each intensity, varying from 0–100%.

Immunoblotting

Specimens were homogenized on ice using radioimmune precipitation assay extraction buffer [10 mM Tris HCl (pH 8.0), 10 mM EDTA (pH 8.0), 0.15 M NaCl, 1% Nonidet P-40, 0.5% sodium dodecyl sulfate (SDS)[ containing 250 µg/ml protease inhibitors, leupeptin and aprotinin (ICN Biomedicals, Inc., Aurora, OH). Specimens were pooled by each study condition. Protein concentrations were determined using the micro BCA protein assay kit (Pierce, Inc., Rockford, IL). Each lane was loaded with 100 µg protein, denatured by heating to 95 C in Laemmli buffer (0.25 M Tris, 1.92 M glycine, and 1% SDS) supplemented with 0.025 M dithiothreitol. Separation of proteins was achieved using a 7.5% one-dimensional SDS-polyacrylamide gel for all proteins except osteopontin, which required a 10% gel. The proteins were electroblotted to polyvinylidene difluoride membrane, which was blocked overnight at 4 C in TBST [20 mM Tris (pH 7.4), 483 mM NaCl, 0.5% Tween 20] buffer with 5% nonfat dry milk. Blots were then incubated with monoclonal antibodies against human β3 integrin subunit (SS-A6), osteopontin (OPN) (Mab53), and estrogen receptor-{alpha} (ER{alpha}) (6F11) for 1 h with rocking at room temperature. Blots were washed six times for 5 min with TBST and then incubated for 1 h at room temperature with peroxidase-conjugated goat, antimouse IgG while rocking. After washing with TBST, the immunoreactive protein complexes were detected using SuperSignal West Pico enhanced chemiluminescence (Pierce).

RNA isolation and quantification

Endometrial total RNA was isolated from frozen tissue samples using the RNAqueous-4PCR Kit (Ambion, Austin, TX) and conditions suggested by the manufacturer. RNA was quantitated using RiboGreen (Molecular Probes; Invitrogen, Carlsbad, CA) with a rRNA standard curve. First-strand cDNA was synthesized from 100 ng total RNA using avian myeloblastosis virus reverse transcriptase (Roche, Indianapolis, IN).

TaqMan primers and probes for putative markers of receptivity [OPN, β3 integrin subunit, P receptor (PR), ER{alpha}, Cyr61, cFos, CD55, FKB52, EGR-1, and the housekeeping control gene cyclophilin (PPIA)] were obtained in a predesigned mix (Applied Biosystems, Foster City, CA). Cyclophilin had the least variation among several housekeeping genes tested, and we have previously validated its use by comparing multiple housekeeping genes in other endometrial samples using geNorm software (17). Efficiency of each primer probe set was tested on the day of analysis. Reactions were performed in 96-well plates on a Stratagene MX3000 device (La Jolla, CA) for 40 two-step cycles, 95 C for 20 sec and then 60 C for 1 min. Cycle threshold (Ct) values were converted to relative expression using the {delta}-{delta} Ct method, allowing normalization to both the housekeeping gene (PPIA) as well as normalization within each experiment.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Our study was designed to achieve serum P levels between 3 and 10 ng/ml in group I, to reflect the lower range of concentrations regarded as consistent with ovulation (18, 19), and to achieve P levels between 10 and 20 ng/ml in group II, corresponding with established normal values during the midluteal phase of ovulatory cycles (20). Mean steady-state serum P concentrations in groups I and II are shown in Fig. 2AGo. On each day, P levels in group II subjects receiving the higher dose of exogenous P (40 mg/d) were significantly higher than those in women receiving the lower dose of P (10 mg/d). Mean (± SEM) P level in group II was 19.2 ± 6.6 ng/ml, compared with 5.5 ± 1.1 ng/ml in group I (P < 0.0001). Mean serum E2 concentration (± SEM) after 10 d of E2 treatment was 129 ± 95 pg/ml and did not differ between groups.


Figure 2
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  		FIG. 2. Comparison of P, histology, and molecular markers between groups I and II. A, Steady-state serum P concentration was determined in patients treated with either 10 mg (group I, represented by squares) or 40 mg (group II, represented by triangles) daily im P. Points represent means and bars represent ± SEM B, Histological dating in groups I and II. No patients were more than 1 d delayed in histological features. The box plot markings represent median, 25–50th percentile, and the range of all values. C, Immunohistochemical staining for β3 integrin subunit was evaluated using HSCORE (box plot as in C). D, Immunoblot results for {alpha}vβ3, OPN, ER{alpha}, and β-actin. Midluteal is from spontaneous cycles.

 
Mean endometrial thickness (± SEM) in group I (8.2 ± 2.7 mm) was lower but not significantly different from that in group II (10.9 ± 3.1 mm). Mean histological dates for groups I and II were not different (Fig. 2BGo). None of the endometrial samples in either group exhibited significant delay in maturation (≥3 d) by classical histological dating criteria (6).

Mean HSCORE determinations for the {alpha}1, {alpha}4, and β3 integrin subunits for groups I and II were not different. Comparison of HSCORE determinations for β3 is shown in Fig. 2CGo, and median HSCOREs for {alpha}1β1, {alpha}4β1, and {alpha}vβ3 are compared in Table 1Go. Lack of observed differences and variability of HSCORE values in both groups prompted a post hoc power analysis that yielded a 77% ({alpha}1), 72% ({alpha}4), and 95% (β3) power to detect a 1.5-point difference in HSCORE.


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  		TABLE 1. Immunohistochemical evaluation of endometrial integrins

 
Preliminary results of immunoblotting for the β3 integrin subunit, OPN, and ER{alpha} are shown in Fig. 2DGo. Due to low levels of recovered protein in individual endometrial specimens, each lane represents a pool of eight tissue specimens. β3 and OPN showed strong antibody staining, and ER{alpha} showed moderate staining in tissue specimens obtained during the midluteal phase in natural cycles. Band intensities for β3, OPN, and ER{alpha} in tissues obtained from group II were similar to those observed in tissues obtained during the midluteal phase of natural cycles, providing further validation for the experimental model. However, in tissues obtained from group I subjects having 4-fold lower serum P concentrations, staining for β3 and OPN appeared weaker, and ER{alpha} expression stronger than was observed in tissues obtained in the midluteal phase of natural cycles and in tissues obtained from group II subjects. Band intensities for β-actin, used as a loading control, were similar for tissues obtained in all conditions. Although differences in band intensities between lanes and the direction of change observed for the three markers suggest that the differing P levels resulted in significantly different amounts of β3, ER{alpha}, and OPN protein expression, our inability to assess intra- and interassay variability due to the small amounts of tissue available prevents confident conclusions.

The observation that groups I and II are indistinguishable by histological dating was unexpected. Given that the serum P levels generated by once-daily P administration is expected to vary with time between injections, and reasoning that scrutiny of serum P concentrations in the two groups might reveal less difference than was suggested by the comparison of mean P levels, we analyzed serum P concentrations at time points corresponding to the expected peak and trough levels across time in two additional groups of subjects randomly assigned to receive the same experimental treatment regimens as described earlier. Those data are summarized in Fig. 3Go and further confirm that our study design achieved the targeted serum P concentrations and clear separation between groups I and II. The peak and trough serum P concentrations (± SD) among women receiving the lower dose of exogenous P were 7.0 ± 3.0 and 3.4 ± 1.0 ng/ml, respectively.


Figure 3
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  		FIG. 3. Peak and trough levels of P in subjects treated daily with 10 or 40 mg im P injections. The data are represented as means ± SD. Comparisons were made using ANOVA with Tukey’s post hoc test. a, P < 0.01 for pairwise comparison; b–d, P < 0.001 for pairwise comparisons.

 
The tissue samples obtained from the additional two groups were processed for histological dating. Again, the histological dates (mean ± SD) for tissues from subjects receiving 10 mg/d (25.7 ± 0.8; n = 6) and 40 mg/d (24.4 ± 0.9; n = 6) P treatment were not different. Combining all histological dating data, the lack of any difference between the two experimental conditions is statistically robust, having greater than 99% power to detect a 2.5-d difference in histological dates.

To further evaluate the unconfirmed data from Western analysis that suggested differences in the expression of molecular marker proteins between groups, endometrial specimens obtained from the two groups of additional subjects and from another group of normally cycling women (ages 18–35, n = 8) on luteal d 10–11 of a natural cycle were stored for mRNA extraction and qRT-PCR to determine the relative amounts of ER{alpha}, PR, β3 integrin, OPN, EGR-1, Cyr61, cFos, and FKB52 mRNA (Fig. 4Go). There were no significant differences between the three groups for any of the molecular markers as judged by ANOVA and by separate comparison of the tissues derived from modeled cycles by t test. Interestingly, the overall variability in molecular marker expression as judged by range or SD was greatest in the natural cycles and least in those receiving the higher dose of exogenous P treatment.


Figure 4
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  		FIG. 4. Assessment of mRNA levels of markers of endometrial function using qRT-PCR. The bars represent the median relative expression. a, ER{alpha}; b, PR; c, β3 integrin subunit; d, OPN; e, Cyr61; f, EGR-1; g, FKBP52; h, c-fos.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
For more than 50 yr, endometrial dating was an accepted bioassay of the quality of luteal function, a diagnostic test for LPD, and a surrogate marker for endometrial receptivity. Recently, the accuracy, reproducibility, and clinical utility of endometrial dating have been challenged (10, 11, 12). Controlled studies revealed that endometrial dating lacks precision, primarily because the histological features of endometrial glands and stroma used for dating are less temporally discrete than originally described (11). A multicenter trial demonstrated that endometrial dating cannot distinguish reliably between fertile and infertile women (12).

It is assumed that morphological and functional endometrial maturation relate directly to circulating P concentrations. A study in fertile women found a modest correlation between endometrial histological dating and integrated urinary pregnanediol glucuronide levels (21), but the effect of varying serum P levels on secretory endometrial development never has been tested directly. Our experimental model of LPD investigates the effects of low P concentrations on secretory endometrial development and function. Pharmacokinetic data demonstrate that our study design achieved its purpose.

As anticipated, endometrial histological features obtained in modeled cycles characterized by normal midluteal phase P concentrations were indistinguishable from those obtained on luteal d 10 in natural cycles. However, our observation that endometrium obtained in experimentally induced LPD cycles could not be distinguished from those from either natural or modeled cycles characterized by higher serum P concentrations was unexpected. We previously demonstrated that endometrial dating cannot define a specific luteal day or narrow range of days in normally cycling fertile women (11). Nonetheless, we expected to observe an obvious delay in histological endometrial maturation in experimental LPD cycles, and did not. The results of our pharmacokinetic analysis of P concentrations in modeled cycles suggest that the observation cannot be attributed to transiently higher or normal serum P concentrations.

Traditionally, the diagnosis of LPD has been based on a 3-d or greater delay in histological endometrial maturation relative to that expected for the day of sampling. We observed identical mean and median histological dates for tissues obtained in our two experimental groups and had greater than 99% power to detect a 2.5-d difference. Moreover, none of the specimens from low-dose P cycles exhibited a 3-d or greater maturation delay. This demonstrates that endometrial dating does not accurately reflect circulating P concentrations and cannot serve as a reliable bioassay of the quality of luteal function.

Given the subjectivity and limited precision of histological dating, efforts to evaluate endometrial receptivity have focused on the expression of molecular markers of endometrial function, including the β3 integrin subunit and a variety of mRNA species identified by microarray (13, 14). We compared the immunohistochemical detection of endometrial integrins ({alpha}1, {alpha}4, and β3) in natural and in modeled cycles and observed no differences among the groups. Given that epithelial integrin expression correlates closely with histological dating (13, 14), the results of that analysis were not surprising. For all three proteins, the SD for HSCORE were least for tissues obtained from women receiving the higher dose of exogenous P treatment, suggesting that variations in expression may be minimized at higher P concentrations.

In contrast to the results obtained with immunohistochemistry, those obtained by immunoblot suggest that the expression of β3 integrin, OPN, and ER{alpha} proteins in experimentally induced LPD cycles differed from that in natural cycles and similarly modeled cycles characterized by normal circulating P concentrations. These data are preliminary because they were based on observations in pooled samples with each group represented by a single lane in the Western blot; inter- and intraassay variability therefore was not well controlled. Nonetheless, given that ER{alpha} is down-regulated by P (16) and that OPN is a P-induced endometrial protein (22), our observations of decreased β3 and OPN expression and increased ER{alpha} expression in tissues from experimental LPD cycles were entirely consistent with those expected. Our results suggest that low circulating P concentrations may have functional consequences with no morphological correlate or manifestation. Unfortunately, the limited amounts of tissue available prevented repeated Western analyses. Consequently, these observations were investigated further using qRT-PCR.

The abundance of mRNA coding for ER{alpha}, PR, the β3 integrin subunit, OPN, EGR-1, Cyr61, cFos, CD55, and FKB52 was similar in tissue specimens obtained in natural cycles and in modeled cycles characterized by normal or low circulating P concentrations. Our analyses of qRT-PCR data were sufficiently powered to detect a physiologically significant difference among groups, and we therefore can conclude that the dose of exogenous P administered had no apparent effect on expression of the genes we examined. Interestingly, the variability observed in molecular marker expression was again least for tissues obtained from women receiving the higher dose of exogenous P treatment. These observations suggest that high serum P concentrations yield the most consistent effects on gene expression

The absence of differences in mRNA expression may relate to a lack of precision in analyses of individual endometrial specimens (11) or to a number of other possible factors. Due to low levels of recovered protein in individual endometrial specimens, the immunoblot analysis was performed on pooled specimens. Amounts of β3, ER{alpha}, and OPN protein expression appeared different in the high and low P groups. The limited amounts of tissue did not allow assessment of intra- and interassay variability and prevented confident conclusions. A larger sample size may confirm a significant difference in protein expression related to variations in P concentration. Endometrial biopsy collects tissue from numerous locations within the uterine cavity, and RNA/protein expression may exhibit some degree of site-specific variation. A more uniform and predictable endometrial response may require the presence of an embryo. Although we observed no differences in the markers of endometrial receptivity we examined, it is possible that analyses of a wider array of genes may reveal differential effects. Studies investigating P resistance in endometrium of women with endometriosis demonstrate dysregulation in more than 60 P target genes (23, 24).

The differences observed between the results obtained with qRT-PCR and immunoblotting are intriguing. Whereas the similarities we observed in histological dating, immunohistochemical staining, and the gene expression of nine molecular markers suggest the endometrium in our experimental groups also had similar functional capacity, data from the Western analysis suggest otherwise. The changes in protein expression may reflect only assay variability. A similar discrepancy between protein and mRNA expression levels has been observed for an unrelated P-induced gene, p27Kip1, in endometrial cells (25).

Our results indicate that histological endometrial development is not sensitive to variations in circulating P concentrations and progresses normally at levels observed during the luteal phase in ovulating women. Whereas some minimal P concentration must be required to drive histological endometrial maturation, the threshold level in ovulating women appears much lower than previously thought. This is consistent with reports of pregnancies in women with abetalipoproteinemia lacking low-density lipoprotein (the primary substrate for corpus luteum P production) and exhibit luteal phase serum P concentrations at or below those observed in our low P group (26, 27). Our data further suggest that secretory histological endometrial development in normal ovulatory women is driven primarily by the duration of P exposure rather than by the P concentration. In women with reproductive abnormalities, altered sensitivity to P may raise the threshold required for normal endometrial function (23, 24).

Our study indicates that endometrial histological development is not a valid measure of the quality of luteal function or endometrial receptivity, but they do not invalidate the pathophysiological concept of LPD. Evidence indicates that implantation normally occurs within a narrow interval during the midluteal phase (1, 2, 3, 4), that a late human chorionic gonadotropin rescue from delayed implantation stimulates a reduced steroidogenic response from the corpus luteum (28, 29), and that late implantation predisposes to infertility or pregnancy loss (3).

Our data suggest that low luteal phase P concentrations may have functional and clinical consequences that merit further investigation. Applying our model in studies involving larger sample sizes and analyses of other genes and proteins may elucidate effects that variations in P concentration have on endometrial function.


   Footnotes
 
This work was supported by Nova Carta Foundation. This research was supported by the Eunice Kennedy Shriver NICHD/NIH through cooperative agreement U54HD035041-11 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research.

Disclosure Statement: R.U., J.G., B.L., R.L., R.Z., and S.Y. have nothing to declare. M.F. received grant support from Serono for prior research and royalties as a textbook coauthor.

First Published Online July 22, 2008

Abbreviations: E2, Estradiol; ER{alpha}, estrogen receptor-{alpha}; LPD, luteal phase deficiency; OPN, osteopontin; P, progesterone; PR, progesterone receptor; qRT-PCR, quantitative real-time PCR; SDS, sodium dodecyl sulfate.

Received March 7, 2008.

Accepted July 15, 2008.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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