0013-7227/01/$03.00/0 Printed in U.S.A. The Journal of Clinical Endocrinology & Metabolism 86(8):3912–3917 Copyright © 2001 by The Endocrine Society A Prospective, Randomized Study of Endometrial Telomerase during the Menstrual Cycle CHRISTOPHER D. WILLIAMS, JOHN F. BOGGESS, L. ROBERT LAMARQUE, WILLIAM R. MEYER, MICHAEL J. MURRAY, MARC A. FRITZ, AND BRUCE A. LESSEY Department of Obstetrics and Gynecology, Divisions of Reproductive Endocrinology and Fertility (C.D.W., L.R.L., W.R.M., M.A.F., B.A.L.) and Gynecologic Oncology (J.F.B.), University of North Carolina, Chapel Hill, North Carolina 27599; and Kaiser Permanente (M.J.M.), Sacramento, California 95815 The purpose of this study was to characterize telomerase activity during the menstrual cycle, focusing on the luteal phase. A total of 84 endometrial biopsy samples were obtained from 72 participants. Daily urinary LH testing (OvuQuick, Quidel) was used to establish the day of the LH rise, and participants were randomized to return during the secretory phase. Twelve women returned on the identical day during the luteal phase of a subsequent cycle to allow intercycle comparisons of telomerase activity. Telomerase activity was evaluated using a modified TRAP-eze (Intergen) detection protocol. At the time of each endometrial biopsy, serum estrogen and progesterone were measured. Proliferative phase endometrium showed high telomerase activity. At the onset of the luteal T ELOMERES OCCUPY the distal ends of all chromosomes and are comprised of repeat sequences of TTAGGG (1, 2). With each mitotic division some of the tandem repeats [(TTAGGG)n] of the telomere are lost, resulting in telomere shortening. Once the telomere is reduced to a critically shortened length the cell undergoes apoptosis, or programmed cell death (3, 4). Therefore, the telomere is an important determinant of a cell’s life span. Telomerase is a ribonuclear DNA polymerase found in most tumors (5–9), but in only a limited number of benign somatic tissues, such as bone marrow, epidermis, and endometrium (5). Telomerase functions to maintain telomere length and therefore prevents its otherwise progressive abbreviation (10). Telomerase activity is differentially expressed in the normal endometrium during the menstrual cycle (11–15). Endometrial telomerase activity is high during the proliferative phase of the endometrial cycle and rapidly declines after ovulation; information regarding the activity of this enzyme during the secretory phase is limited and inconclusive. A more thorough characterization of endometrial telomerase activity in cycles of normal fertile women might reveal evidence for hormonal regulation and would provide the basis for comparisons with disease states, such as endometrial hyperplasia or endometriosis, where an abnormal constitutive expression of telomerase activity could play a role in pathogenesis. In efforts to better establish the pattern of endometrial telomerase activity across the menstrual cycle, we undertook a detailed analysis focusing on the secretory phase of the cycle. We examined the expression of telomerase activity in Abbreviations: TPG, Total products generated. phase telomerase activity was high, but it decreased during the early luteal phase, disappeared by the midluteal phase (6 d after LH surge detected), and then rose to moderate levels in the late luteal phase beginning on luteal d 10. Serum progesterone levels were inversely related to telomerase activity. In conclusion, endometrial telomerase activity is dynamic: high during the proliferative phase but inhibited during the midsecretory phase of the menstrual cycle. The timing of expression coincides with the rise and fall of progesterone levels and the time period of maximal uterine receptivity for embryo implantation. This supports a relationship between sex steroid levels and telomerase regulation. (J Clin Endocrinol Metab 86: 3912–3917, 2001) a large series of carefully defined endometrial tissue specimens and correlated these observations to the concentrations of estradiol and progesterone in blood specimens obtained on the day of endometrial sampling. Subjects and Methods Eighty-four endometrial biopsy samples were prospectively obtained from 72 volunteer participants with a mean age of 30.6 yr. Eighty of the specimens and concurrently drawn serum samples were obtained from 68 normally menstruating, proven fertile women during the secretory phase of the menstrual cycle. The remaining 4 tissue specimens were obtained by excision of endometrium from fresh hysterectomy specimens that were confirmed to be in the proliferative phase by histology. Informed consent was obtained from all of the subjects enrolled in our study, which was approved by the committee on the protection of the rights of human subjects at University of North Carolina (Chapel Hill, NC). All women who participated in this study were between 18 –35 yr of age and reported having regular menstrual cycles at 25- to 35-d intervals. Women with uterine leiomyomata or any other endometrial cavitydeforming masses were excluded. Subjects used nonhormonal methods of contraception during the cycle of biopsy. The cycles of the 68 women sampled during the secretory phase of the cycle were monitored with daily urinary LH surges (OvuQuick, Quidel, San Diego, CA) from cycle d 10 until the midcycle LH surge was identified (luteal d 0). Subjects were randomized, using a computer-generated random number table, to undergo endometrial biopsy on luteal d 1–14. To investigate intercycle variations in endometrial telomerase activity, 11 subjects underwent a second endometrial sampling in a subsequent cycle on the same luteal day as in the initial cycle, defined in the same manner. Endometrial tissue specimens were immediately snap-frozen in liquid nitrogen and subsequently stored at ⫺80 C until assayed. Analysis of tissue was performed within 6 months from the time of biopsy. Blood samples were obtained on the day of endometrial biopsy in all subjects, and the serum was stored at ⫺80 C until assayed for estradiol and progesterone concentrations at the conclusion of the study. 3912 Williams et al. • Endometrial Telomerase The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3912–3917 3913 Telomerase detection assay Endometrial tissue samples were first weighed and then suspended in lysis buffer [10 mm Tris-HCl (pH 7.5), 1 MMMgCl2, 1 mm EGTA, 0.1 mm gensamidine, 5 mm -mercaptoethanol, 0.5% CHAPS, 10% glycerol, and 200 U/ml ribonuclease inhibitor (anti-Rnase, Ambion, Inc. Austin, TX]. Tissues were then homogenized using a sterile needle or a manual plastic cone homogenizer. Protein concentrations were determined using a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA) (16). The remaining extract was then stored at ⫺80 C. Telomerase activity was analyzed with a version of the original PCRbased method (5) using a modified TRAP-eze (Intergen, Purchase, NY) telomeric repeat amplification detection kit (17). The sensitivity, reproducibility, linearity. and reliability of the TRAP-eze kit were confirmed by other investigators (18). In brief, the primer mix (RP primer, K1 primer, and TSK1 template) was placed in a test tube, and a paraffin bead was melted over its surface to effectively separate the reverse primer during the telomerase extension step (hot start PCR). The remaining reagents of the 50-l reaction mix consisting of 2 l cell extract (10 –750 ng protein/l), the 5⬘-end-labeled TS primer (AATCCGTCGAGCAGAGGT) with [␥-32P]ATP, 50 m deoxy-NTP mix, and 2 IU Taq polymerase in 1 ⫻ PCR buffer were then added on top of the paraffin layer. The mixture was incubated at 30 C for 60 min to allow the telomerase enzyme in the cell extract to add the nucleotides to the primer (telomerase extension) followed by PCR performed at 94 C for 30 sec and at 59 C for 30 sec for a total of 30 cycles. The paraffin interface melts during the beginning of the first 94 C step to allow proper mixture of the reverse primer in the 50-l reaction. The PCR products were then resolved on a 12% polyacrylamide gel and visualized by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). As positive and negative controls, a cell extract was prepared from a known quantity of HeLa cells and diluted with lysis buffer to a concentration of 5 HeLa cell equivalents/l. Two microliters of the cell extract were used as a positive control, and 2 l lysis buffer alone were used as a negative control to determine the presence of primer-dimer contamination. For each tissue sample a ribonuclease-inactivated negative control lane was also prepared. Amplification efficiency in each reaction was determined using internal control oligonucleotides that form a 36-bp band, provided in the TRAP-eze assay kit. A semiquantitative method of densitometry, using phosphorimaging (PhosphorImager, Molecular Dynamics, Inc.) and computer-generated, gray scale comparisons (ImageQuant, Molecular Dynamics, Inc.), was used to evaluate the concentrations of telomerase in each tissue sample. Each TRAP-eze kit includes a standardized quantitation control (TSR8) that is used for comparisons to assayed results. Telomerase activity was expressed in total products generated (TPG) per g protein. The sensitivity, reproducibility, linearity, and reliability of the TRAPeze kit have been confirmed by other investigators (18). The quantitation control lane of each assay confirms intraassay reliability and allows quantitative assessment of each sample. To confirm the interassay precision of the modified TRAP-eze assay and phosphorimaging in this investigation, nine randomly selected biopsy samples (11% of the total) were evaluated using two separate TRAP-eze assay kits. Correlation coefficients (r) were calculated to assess the precision of the methodology. Daily secretory phase serum estrogen and progesterone levels and telomerase activity were plotted against the day of the secretory phase using STATA 6.0 (College Station, TX). Curves were fitted to demonstrate the mean daily values with 2 sd above and below the mean values. Intercycle variations in telomerase activity were also assessed, and correlation coefficients (r) were calculated. The data were analyzed to evaluate whether progesterone levels vary predictably across categories of telomerase activity or if the reverse is true, that telomerase activity varies across categories of progesterone. A univariate analysis was performed using SAS (Research Triangle Park, NC) to produce quartiles of progesterone and telomerase activity. A test of trend was performed using a linear model with a continuous dependent variable (the outcome) and a categorical independent variable (the predictor). Statistical significance was determined at ␣ ⬍ 0.05. RIAs for estradiol and progesterone RIAs for estradiol and progesterone were performed using unextracted human serum and commercial solid phase kits (Coat-a-Count) from Diagnostic Products (Los Angeles, CA). Assay performance was monitored by including three quality control samples (representing low, medium, and high values, respectively) in each assay. Tubes were counted in an LKB 1272 CliniGamma counter and were analyzed using StatLIA software from Grendan Scientific (Grosse Pointe, MI). Intraassay variations were 6.1% for estradiol and 5.4% for progesterone; interassay variations were 7.5% and 5.1%, respectively. Results Demographic characteristics of the study subjects are summarized in Table 1. Telomerase activity in the four proliferative phase endometrial tissue specimens was 138.5 TPG/g protein, with a range of telomerase activity from 50.17–318.53 TPG/g protein assayed. Figure 1 illustrates a sample electrophoretic gel with the characteristic laddering of telomerase products indicating the samples containing the telomerase enzyme. A total of 75 of the 80 (94%) secretory phase endometrial biopsy samples were quantifiable (Fig. 2). Levels of telomerase activity varied widely, ranging from undetectable to 234.14 TPG/g protein (median, 26.3 TPG/g protein). Results obtained for each of these specimens are demonstrated in Fig. 3. Levels of telomerase activity were highest in the early secretory phase (luteal d 1–5), were at or below the limits of detection during the midsecretory phase (luteal d 6 –9), and rose again moderately during the late secretory phase (luteal d 10 –14). The modified TRAP-eze telomerase detection assay was inhibited by an unidentified factor (PCR inhibitor) in 4 of the 12 (33%) tissue specimens obtained between luteal d 12–14 and in 1 of 68 (1.5%) of the tissue specimens collected earlier in the secretory phase (luteal d 1–11); these 5 specimens are therefore not represented in Fig. 3. Nine of the 80 secretory phase endometrial samples were randomly selected for repeat assay. Seven of the nine samples yielded analyzable data. Two did not as they were among those specimens exhibiting evidence for the presence of an intrinsic inhibitor of the assay system (PCR inhibitor; Fig. 2). The comparison of duplicate sample results had a correlation coefficient (r) of 0.99. Twelve of the secretory phase endometrial tissue specimens resulted from repeat samplings in 11 individuals on the same luteal day to which the subject was originally randomized for the first study cycle, thereby allowing an evaluation of intercycle differences in telomerase activity. Two of these 12 matched pairs of specimens were not included this analysis. One pair (obtained on luteal d 5) yielded no data due to inhibition of the detection assay in both of the 2 tissue specimens. A second pair (obtained on luteal d 13) also TABLE 1. Demographic characteristics of study population (excludes four participants with endometrial biopsies obtained during the proliferative phase) Age (yr) Gravidity Parity Race, no. (%) White Black Hispanic Asian Mean wt (lbs) Mean body mass index 30.6 (range, 20 –35) 2.3 1.7 53 (78) 8 (12) 5 (7) 2 (3) 149.1 (range, 105–300) 25.3 (range, 19 –55) 3914 The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3912–3917 Williams et al. • Endometrial Telomerase FIG. 1. Modified TRAP-eze assay. Lane 1, Control cell pellet; lane 2, proliferative phase sample; lane 3, ribonuclease-inactivated proliferative phase control; lane 4, secretory phase d 2 sample; lane 5, ribonuclease-inactivated sample from lane 4; lane 6, secretory phase d 5 sample of endometrium; lane 7, ribonuclease-inactivated sample from lane 6; lane 8, secretory phase d 8 sample; lane 9, ribonuclease-inactivated sample from lane 8; lane 10, secretory phase d 13 sample; lane 11, ribonuclease-inactivated sample from lane 10; lane 12, quantitation control (TSR8; 2 l; 0.2 amol); lane 13, lysis control. FIG. 2. Endometrial biopsy samples assayed for telomerase activity for each day of the luteal phase. yielded no analyzable data; telomerase activity was undetectable in the first tissue specimen, and there was evidence of assay inhibition in the repeat sample. Results of the analysis for the remaining 10 pairs of had a correlation (r) of 0.08. Secretory phase serum estrogen and progesterone concentrations were uniformly within the expected range for normally cycling ovulatory women and are shown in Fig. 4, A and B, respectively. Progesterone levels vary across categories of telomerase activity (P ⫽ 0.03), but the reverse is not true (P ⫽ 0.29). Discussion The telomerase enzyme can be identified in most carcinomas and a limited number of somatic tissues and is thought to play a vital role in cellular immortality, aging, and oncogenesis (5– 8). The endometrium is one of the few normal adult tissues that expresses telomerase. It is thought that endometrial telomerase accounts for the remarkable ability of this tissue to repeatedly proliferate during the prolonged period of time from menarche and menopause. Indeed, available evidence suggests that at least in the reproductive context, the endometrium does not age (19). However, unlike other benign tissues that express telomerase, endometrial telomerase is not constitutively active. Rather, it exhibits a dynamically changing pattern of activity during the menstrual cycle (11–13, 15). Previous studies have examined telomerase expression in the normal endometrium. Shroyer et al. demonstrated Williams et al. • Endometrial Telomerase The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3912–3917 3915 FIG. 3. Telomerase activity during the luteal phase. n, Number of samples; thick line, mean telomerase activity; thin line, 2 SD above/ below the mean. telomerase activity in 13 of 14 patients with proliferative endometrium and in 7 of 12 cases of secretory phase endometrium, but found no evidence of activity in specimens of atrophic endometrium (11). Kyo et al. (12) reported similar observations, finding consistent evidence of telomerase activity during the proliferative phase, but detecting activity in less than half of secretory phase endometrial specimens examined. Saito et al. (20) observed that telomerase expression increases as the proliferative phase advances, declines significantly with the onset of the secretory phase, and falls to undetectable levels as the secretory phase progresses. Our data clearly offer a plausible explanation for these inconsistent observations of telomerase activity in secretory phase endometrium. The previous studies are limited by their generalized categorization of luteal phase samples. Consequently, they have not been able to fully evaluate whether telomerase exhibits a temporally dynamic pattern of expression across the secretory phase, in much the same way that endometrial histology varies predictably with the cyclic changes in the steroid hormone milieu that characterize the luteal phase of the menstrual cycle. Additionally, this is the first study to correlate serum estrogen and progesterone measurements with telomerase activity and assess the intercycle variations in telomerase activity. Using a prospective, randomized approach in proven fertile women, we determined the levels of endometrial telomerase activity in a large series of carefully defined secretory phase tissue specimens. Our results confirm earlier reports that endometrial telomerase activity declines rapidly after ovulation. More importantly, our observations significantly expand the understanding of telomerase activity during the secretory phase of the endometrial cycle. We demonstrated that telomerase activity declines during the early secretory phase, becoming undetectable by luteal d 6 and remaining so until rising again to moderate levels during the late secretory phase, beginning on luteal d 10. The modified TRAP-eze telomerase detection assay was inhibited by and yielded no data for five tissue specimens, four obtained during the late secretory phase (luteal d 12–14). The absence of both TRAP-eze products and the internal control band in these samples after repeated assays demonstrates PCR inhibition rather than telomerase inhibition alone. Because the normal secretory phase lasts 14 ⫾ 2 d, necrosis and breakdown of the uterine lining would be expected to coincide with all but one of the PCR-inhibited samples, and it is likely that an unidentified byproduct of tissue breakdown was responsible. Due to PCR inhibition of some of the samples, the data describing luteal d 12–14 are limited and may preclude a confident interpretation of the telomerase activity in the late secretory phase. The sensitivity, reproducibility, linearity, and reliability of the TRAP-eze telomerase detection assay have been confirmed by other investigators (18). Precision of the assay detection system in our own hands was evaluated by reassay of nine randomly selected tissue specimens, and the results of the two determinations correlated very closely. Our comparison of results obtained for nine pairs of tissue specimens collected from individuals who were sampled on the same luteal day in two separate menstrual cycles suggests that the overall pattern of telomerase expression is consistent across cycles, although daily levels of telomerase activity may vary significantly. Our study is the first to examine the relationship between secretory phase endometrial telomerase activity and circulating concentrations of estradiol and progesterone during the luteal phase of the menstrual cycle. Earlier studies have suggested the possibility that telomerase expression might be influenced or regulated by steroid hormones (12). Kyo et al. observed that low or undetectable levels of telomerase activity in atrophic postmenopausal endometrium increased after treatment with estrogen or tamoxifen. Subsequent investigations suggested that estrogen exerts both direct and indirect effects on the human telomerase enzyme reverse transcriptase subunit promoter of the telomerase gene (21). The absence of a progesterone response element from the telomerase promoter may indicate that progesterone suppression of telomerase activity is indirect through its well established antiestrogenic actions. The patterns of steroid hormone production by the corpus luteum across the luteal phase of the menstrual cycle and the associated sequence of changes in secretory endometrial histology are well defined (22). Changes in circulating estrogen and progesterone concentrations are temporally associated with the production of a variety of endometrial proteins that may play a functional role in the implantation process (23– 25). Evidence of a cycle-dependent pattern of endometrial telomerase expression suggests that this enzyme may represent yet another endometrial protein subject to such steroid hormone regulation. In a manner similar to endometrial ER and PR concentrations and expression of certain integrins, telomerase appears to exhibit differential expression during the time period of maximal uterine receptivity for embryo implantation (luteal d 6 –10). It is interesting to note that telomerase activity falls during the early secretory phase as progesterone concentrations rise, becomes undetectable during the midsecretory phase when progesterone levels are peaking, and rises again during the late secretory phase as progesterone concentrations again fall. This inverse temporal relationship between endometrial telomerase activity and serum levels of progesterone suggests that telomerase ex- 3916 The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3912–3917 Williams et al. • Endometrial Telomerase FIG. 4. Luteal phase estrogen levels. n, Number of samples; thick line, mean estrogen level; thin line, 2 SD above/below the mean. pression may be in some way suppressed by progesterone. Although serum estradiol concentrations parallel the rise and fall of progesterone during the luteal phase, available evidence suggests that estrogen increases telomerase activity. The fact that telomerase activity falls to undetectable levels at a time when both estradiol and progesterone concentrations are peaking suggests that any such action of estrogen may be blocked or overwhelmed by an opposite action of progesterone. The inability to demonstrate that telomerase levels vary across categories of progesterone (P ⫽ 0.29) supports the hypothesis that endometrial telomerase activity is governed by a variety of different, possibly competing, regulatory mechanisms. Intercycle variations in telomerase activity may relate at least in part to similar variations in steroid hormone concentrations observed among cycles. Analysis of concurrently obtained serum estrogen and progesterone measurements indicates that the intercycle hormonal variations do not consistently explain the changes seen in telomerase activity, but the limited data may be insufficient to permit any confident interpretation. Yokoyama et al. (26) showed that endometrial telomerase Williams et al. • Endometrial Telomerase The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3912–3917 3917 activity can be identified in the epithelium, but not in the stroma. The endometrium contains a number of different cellular elements, including glandular epithelial cells, stromal cells, lymphocytes, erythrocytes, and endothelial cells. Among these, lymphocytes and endothelial cells are known to express telomerase activity (27, 28). Evidence that endometrial lymphocytes proliferate during the secretory phase (29) introduces the possibility that the pattern of telomerase expression we observed in the endometrium as a whole may reflect enzyme activity in other cells besides the epithelium. Preliminary studies in our own laboratory have demonstrated that when endometrial glands and stroma are separated by filtration and grown in vitro, telomerase activity continues to be measurable in endometrial glands for at least 21 d. In vitro culturing of endometrial glands on Matrigel (Paragon Bioservices, Baltimore, MD) for a prolonged period of time would effectively exclude the presence of telomerase-contaminating cells such as lymphocytes and endothelial cells. Current evidence suggests that endometrial telomerase may be vital to the ability of this tissue to regularly and repeatedly proliferate over a reproductive life that spans approximately 4 decades. Our study offers an explanation for previous observations of inconsistent levels of telomerase activity in secretory phase endometrium by providing evidence for a dynamic pattern of expression, the temporal characteristics of which suggest the regulatory influence of sex steroids and are coincident with the timing of implantation. The disappearance of measurable telomerase activity from the endometrium at the onset of the period of peak serum progesterone levels and maximal uterine receptivity probably reflects the shift in cellular production of proteins away from those with proliferative roles to maximize cellular differentiation to promote receptivity, implantation, and early invasion of the embryo. Our study provides the foundation for subsequent investigations comparing the features of telomerase activity in normal and abnormal endometrium and in related tissues, including endometriosis. Additional studies in cultured endometrium will be needed to confirm our hypothesis of steroid hormone regulation and hold promise as a means to better define the mechanisms that govern endometrial telomerase activity. Acknowledgments We thank Robert Strauss, M.D.; Michael McMahon M.D., M.P.H.; and R. B. Balu, M.S., for their assistance with the statistical methods and analysis, and Dr. Peter Petrusz and Catharina Weaver from the Immunotechnology and Histochemistry Core of the Laboratories for Reproductive Biology at the University of North Carolina (Chapel Hill, NC) for analyzing the serum samples. Also, we appreciate the assistance of Katherine Garver, R.N., B.S.N., with study coordination. Received November 28, 2000. Accepted April 30, 2001. Address all correspondence and requests for reprints to: John F. Boggess, M.D., Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, The University of North Carolina at Chapel Hill, Campus Box 7570, Chapel Hill, North Carolina 27599-7570. 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