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Luteal Progesterone Relates to Histological Endometrial Maturation in Fertile Women

Department of Obstetrics, Gynecology, and Women’s Health, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103-2757

Address all correspondence and requests for reprints to: Dr. Nanette Santoro, Department of Obstetrics, Gynecology, and Women’s Health, Division of Reproductive Endocrinology, Albert Einstein College of Medicine, Mazer 316, 1300 Morris Park Avenue, Bronx, New York 10461.

	Abstract

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To examine the relationship between endometrial histological maturation and reproductive hormones, we studied 11 fertile women, aged 18–37 yr. All participants had had at least 1 previous pregnancy and cycled regularly, every 25–35 days. Women collected daily, first morning voided urine for measurement of estradiol and progesterone metabolite excretion, estrone conjugates (E1c), and pregnanediol glucuronide (Pdg), respectively, throughout the cycle of study. Hormones were normalized for creatinine. Between 7–9 days after home detection of a LH surge (Sure Step), participants underwent an endometrial biopsy using a small bore (Pipelle) catheter. Tissue was prepared for histological and biochemical analyses. The histological analysis is reported herein. Endometrium was dated by 3 authors (N.S., D.H., and S.P.), all of whom were blinded to the participant’s identity or timing of biopsy within her cycle. Final dating was agreed upon based upon the method of Noyes et al. E1c and Pdg were integrated throughout the cycle using the trapezoidal rule, and correlations were sought between deviation from expected histology (based upon urinary hormones and LH surge) and integrated hormone values.

E1c varied over a 2-fold range in these normal women, from 1196–2040 ng/cycle. Pdg excretion was much more variable, ranging from 22–119 µg/cycle. No relationship could be found between histological lagging of endometrial maturation and lower excretion of E1c. A moderate correlation was observed (Spearman’s r = 0.6; P < 0.05) between degree of histological maturation and integrated Pdg. Of two women with evidence of a disparity between gland and stromal development (glands lagging behind stroma by >2 days), one excreted 24 µg Pdg/cycle, the next to lowest value.

We conclude that normal fertile women experience a wide range of hormone concentrations in the face of normal endometrial maturation. Progesterone appears to exert a dose-related effect on endometrial maturation, and the techniques we used, although relatively crude clinical measures, appeared to be sufficient to detect this relationship.

	Introduction

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ENDOMETRIAL HISTOLOGY varies across the normal menstrual cycle and is due to stimulation by estradiol and progesterone. Estradiol stimulation results in proliferation of the basalis and functionalis, whereas progesterone promotes glandular development and secretion and is believed to initiate the changes necessary for implantation. Progesterone appears essential for the process of normal endometrial maturation, and a window of implantation, approximately 6–8 days after ovulation, has been defined in the human (1, 2, 3).

The clinical concept of luteal phase insufficiency presumes that subthreshold progesterone stimulation of the uterus can cause failure of endometrial development. Moreover, clinical criteria for endometrial inadequacy have been defined, albeit arbitrarily, by inspection of histological specimens of endometrial biopsies taken from the mid to late luteal phase of the menstrual cycle (4, 5). In infertile women or those with recurrent miscarriage (6), luteal progesterone has been shown to be decreased. A dose-response relationship between histological maturation and progesterone production has been presumed to exist.

Detection of luteal inadequacy has been traditionally performed by late luteal endometrial biopsy (4, 5). Dating the endometrium histologically is customarily accomplished using standards set by Noyes et al. in the 1950s (7). Recent reevaluation of the best time for luteal phase biopsy has resulted in the recommendation that the implantation window (i.e. 6–8 days after ovulation) may provide greater detection of histological abnormalities (8) and, hence, greater sensitivity of the clinical test.

We evaluated the ability of the endometrial biopsy to detect luteal adequacy in a small population of fertile, midreproductive-aged women. We collected daily hormonal information on the women to allow us to determine a relationship between hormones and histology, should it exist.

	Materials and Methods

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This protocol was approved by the institutional review board of the New Jersey Medical School, and informed consent was obtained from all participants before their entry into the study.

Eleven women, aged 18–37 yr, participated in the protocol. All women met the following criteria: 1) regular menstrual cycles every 25–35 days, 2) normal PRL and TSH, 3) evidence of prior fertility (i.e. at least one previous term pregnancy or induced abortion), 4) no excessive exercise (defined as >6 h per week), 5) weight greater than or equal to 90% of normal weight for height (9), and 6) nonsmoking status.

Protocol

Women were given polypropylene test tubes containing glycerol and marked with a fill line that resulted in a 7% concentration (vol/vol) of glycerol when the tube was filled. They were instructed to collect their first morning voided urine upon awakening each day, to label each tube with their names and the date, and to freeze the tube within 2 h of voiding. Based upon each woman’s usual cycle length, they were instructed to start urinary home monitoring of their LH surge using the Sure Step ovulation predictor kit (Applied Biotech, Inc., San Diego, CA). Participants notified the research nurse upon detection of the LH surge, and an endometrial aspiration biopsy was scheduled 6–8 days later.

Biopsies were performed by two of the authors (N.S. and P.M.) using standard sterile technique. After preparation of the cervix and application of local anesthesia as needed, a Pipelle catheter (Unimar, Bridgeport, CT) was placed beyond 5 cm into the uterine cavity (up to the fundus when possible) and rotated gently 270° as it was withdrawn. Technique was geared toward obtaining a cylinder of intact tissue; therefore, a back and forth motion was not used. Tissue sampling was accomplished in a single pass in most cases. Sampling of the lower uterine segment was avoided when it was possible for the operator to appreciate the full dimensions of the uterus. If less than a 5-cm-long cylinder of tissue appeared to have been obtained, and the participant agreed, a second pass was made with the Pipelle to assure that adequate tissue was obtained. Tissue was apportioned into thirds. One third was immediately placed into formalin for fixation and histological evaluation. The remaining two thirds were divided equally, placed into nalgene tubes, and snap-frozen in liquid nitrogen for biochemical and genetic studies (not reported herein). The cylinder of tissue was apportioned randomly; that is, the top piece was not routinely the piece sent for pathological evaluation.

Histological evaluation of the endometrial biopsies was carried out by three authors (N.S., S.P., and D.H.) using the criteria of Noyes et al. The mean of the three observations was taken as the final dating. There was agreement among all three observers to within 2 days in most cases. When disparity was observed between glands and stroma, each component was dated separately, and the overall histological date was taken as the mean. The date of ovulation was defined hormonally as the day pregnanediol glucuronide (Pdg) rose beyond 1 ng/mg creatinine, coinciding with the day of or up to 2 days after the follicular phase estrone conjugate (E1c) peak.

Hormonal analyses of E1c and Pdg were carried out using previously described methods (10, 11). Interassay coefficients of variation for the E1c and Pdg assays were 19% and 15%, respectively. Corresponding intraassay coefficients of variation were 6% and 8%. Individual cycles were run in the same assay to avoid interassay variation. All hormonal data were corrected for glycerol (7%) and normalized for creatinine (12).

To determine the ability of the endometrial biopsy to detect delayed endometrial maturation, we designated the hormonally determined postovulatory date of the biopsy as the expected date and the histologically determined postovulatory date of the tissue as the observed date. We then subtracted the expected date from the observed date to derive a measure of the degree of maturation.

Data analysis

E1c and Pdg concentrations were integrated throughout the cycle using the trapezoidal rule. Correlation between the degree of endometrial maturation (the deviation of the histologically observed day from the expected postovulatory day based upon E1c and Pdg patterns) and the integrated E1c and Pdg was evaluated using Spearman’s nonparametric rank correlation coefficient (13). The criterion for statistical significance was = 0.05 in one-tailed tests.

	Results

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All cycles were ovulatory, based on a Pdg elevation to at least 3 µg/mg creatinine for at least 3 days (14). All 11 women completed the protocol with complete hormone collections and sufficient tissue for histological analysis.

Hormonal patterns

E1c concentrations were variable among women and demonstrated an overall 2-fold variation in whole cycle integrated values from 1196–2040 ng/cycle. The variation in luteal Pdg among women was much greater and ranged from a low of 22 µg per cycle to 119 µg/cycle, a 5-fold range.

Endometrial biopsies

Tissue was adequate for histological analysis in all cases. Hematoxylin and eosin preparations revealed good full layer thickness tissue cuts that revealed basalis in 9 of 11 specimens. Intact areas of surface epithelium were also observed in all specimens. A review of the anticipated postovulatory date expected for each biopsy revealed that the Sure Step ovulation predictor kit did not agree with the E1c and Pdg patterns in all cases.

Instead of all biopsies being prospectively timed to the implantation window (postovulatory days 6–8), they ranged from postovulatory days 3–10. We therefore relied upon the E1c and Pdg patterns, rather than the urinary LH surge, to determine most precisely the day of ovulation. A histogram showing the distribution of biopsy dating by anticipated window is given in Fig. 1. Of the 11 biopsies, 4 occurred within the anticipated window (6–8 days after ovulation), 4 were early (1 on day 3 and 3 on day 5), and 3 were later (2 on day 9 and 1 on day 10).

View larger version (21K): [in this window] [in a new window]   Figure 1. Histogram depicting the distribution of the timed endometrial biopsies based upon their relation to the implantation window (postovulatory days 6–8). Timing of the biopsies was confirmed retrospectively by E1 conjugate and Pdg patterns (see text). n, Number of specimens obtained on a given postovulatory day (POD).

View larger version (45K): [in this window] [in a new window]   Figure 2. Endometrial histology in tandem with cycle hormones from a normal volunteer (no. 1 from Table 1). Biopsy was taken from within the implantation window, 6 days after ovulation based upon E1c and Pdg pattern. The specimen was read as day 24 according to the criteria reported by Noyes et al. (7 ). The hormone pattern can be seen in the bottom panel. Data are standardized to day 0, the day of ovulation (see text). The day of the biopsy is highlighted (day 6). The shaded background is the mean ± SEM of 11 normal, midreproductive-aged women previously reported (11 ).

A complication of our study was the finding in two women of a disparity between stromal and glandular maturation. The Noyes method of endometrial dating does not provide a way to account for this finding. Such a specimen is shown in Fig. 3, with the corresponding hormonal pattern in the lower panel.

View larger version (57K): [in this window] [in a new window]   Figure 3. Endometrial histology from a normal volunteer (no. 10 from Table 1) demonstrating a gland-stromal disparity in maturation. The hormone pattern is depicted as in Fig. 2. Note the lower Pdg excretion curve.

	Discussion

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These assumptions have never been validated, but are supported by several lines of logic. Firstly, there is evidence in one study that women with recurrent pregnancy loss have decreased progesterone secretion in nonpregnancy cycles (6). Persistent evidence of delayed endometrial maturation may also predict persistent infertility, at least according to some investigators (4, 5). Correction of delayed endometrial maturation has also been demonstrated, in one study only, to predict subsequent pregnancy (21).

Luteal dysfunction has been considered a reflection of underlying subclinical hypothalamic-pituitary insufficiency. In this paradigm, inadequate progesterone production is a sequela of inadequate folliculogenesis brought on by partial hypothalamic-pituitary impairment. The typical inciting event for this chain reaction is stress. Behaviors such as acute weight loss and strenuous exercise have been shown to cause reduced progesterone production, and these hormonal abnormalities can be detected in urine samples (22).

Despite the logic underlying these ideas and their partial proof, much information is lacking about the validity of the endometrial biopsy as a sufficiently sensitive tool to detect syndromes of progesterone deficiency. Sampling of the endometrium using methods such as a Pipelle catheter, although effective for cancer screening, do not provide comprehensive assessment of the uterine cavity (23). Moreover, technique is operator dependent. The best time of the cycle for sampling is also controversial, with some investigators recommending a late luteal biopsy to take into account all of the antecedent hormonal stimulation of the endometrium across the entire luteal phase (7). Others have pointed out that the most critical time for biopsy should more properly be considered during the implantation window, because pregnancy may alter hormone production by the corpus luteum in the midluteal phase of the cycle, and a biopsy at that time will reflect the critical events around implantation (8).

The technique of endometrial dating is somewhat arbitrary and is based upon a set of morphological criteria (7). To assess the consistency of our methods of dating, including tissue preparation, one of the authors (D.H.) performed recuts of four specimens and redated them, blinded to their origin. In three of the four recuts, repeat dating was within 1 day of the original diagnosis; in the fourth instance, repeat dating was within 2 days (1.5 days, to be exact). This is consistent with the original morphologist’s performance (7).

In the present study we attempted to define the ability of a well timed midluteal endometrial biopsy, targeted during the implantation window, to reflect the hormonal stimulation in 11 nonpregnancy cycles from fertile women. Although we observed a wide range of Pdg excretion curves, a modest correlation was observed between maturation and progesterone stimulation. We collected daily hormonal information for each participant to provide a careful profile of her cycle, a technique that has been validated by us and others (10, 11, 21, 24). We exercised great care in timing the biopsy to the implantation window. We used standardized technique in biopsy acquisition and obtained adequate specimens on all women.

The fact that we were able to demonstrate a significant relationship between progesterone production and endometrial histological maturation supports the idea of a dose-response relationship between progesterone and endometrial secretory transformation. That this relationship can be demonstrated in normal fertile women has clinical implications. Firstly, as has been demonstrated in normally cycling women, there appear to be characteristics of the urinary reproductive hormonal patterns that are predictive of pregnancy (24). Interestingly, greater urinary Pdg production was not as significant a factor in the study by Baird et al. (24) as was urinary estrogen. Predicting pregnancy on the basis of hormone patterns can reflect the fact that in an optimal cycle follicle function was simply following a pattern more consistent with pregnancy or that circulating hormones exerted a salutary effect on the hypothalamic-pituitary axis or the uterus. Our data provide some support for the idea that hormonal patterns that are optimal for endometrial maturation exist.

Secondly, our findings support the association of endometrial maturation with progesterone stimulation and thereby lend credibility to the concept of the endometrial biopsy as a clinical test. As we only used normal fertile volunteer women, the performance of the endometrial biopsy as a clinical test cannot be inferred directly from this work. However, our data support the idea that current techniques, despite their limitations, are adequate to detect relatively small changes in endometrial maturation related to hormonal stimulation.

On the other hand, the strength of the dose-response relationship between luteal Pdg and endometrial histological maturation was relatively weak. This finding may indicate an inherent weakness of the true linkage between these two events (progesterone production and endometrial maturation), or it may be the result of key limitations of our study. The first limitation is our reliance on the histology alone as the sine qua non of endometrial maturation. Such a reliance may be unwarranted or insufficient, as biochemical markers of endometrial maturation and the identification of key markers necessary for implantation are currently underway (1, 2). We did not test for such markers. Second, our ability to time the biopsy procedure to the implantation window was less than expected. This problem may have been due to the relatively poor performance of the LH surge detection kit we used, which agreed poorly with our urinary steroid measurements. Concomitant markers of ovulation, such as concurrent use of ultrasound or prospective serum hormone measurements, might have improved the timing of the biopsy to the implantation window.

The usefulness of the criteria of Noyes et al. (7) may require reevaluation. With careful attention to sampling and interpretation of biopsies, we still observed specimens taken within the implantation window that did not correspond to the appropriate expected postovulatory date. This finding might represent true biological variability between women or within women. It may also represent sampling error (23). Furthermore, the finding of significant stromal-glandular disparity in 2 of the 11 women studied is a situation not accounted for by the original endometrial dating paradigm.

In summary, we have described a relationship between luteal progesterone metabolite excretion and histological appearance of the endometrium in a small group of fertile women. These data indicate that progesterone is related, in a dose-duration fashion, to the processes that effect endometrial transformation. Further refinements of hormonal measurement, endometrial morphological evaluation, and the use of additional markers of endometrial maturation may serve to strengthen this observed association. Our preliminary findings indicate that such an undertaking might be worthwhile and could lead to the evolution of the endometrial biopsy, appropriately timed and interpreted, as a useful clinical test of reproductive competency.

Received February 29, 2000.

Revised August 1, 2000.

Accepted August 10, 2000.

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