A Prospective, Randomized Study of Endometrial Telomerase

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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.
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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)
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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-
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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.
This work was supported by an American College of Obstetricians
and Gynecologists/Solvay Pharmaceuticals, Inc., Research Award (to
C.W., J.B., and B.L.), NIH Grants HD-35041 and HD-34824 (to B.L.), and
in part by the National Cooperative Program on Markers of Uterine
Receptivity for Blastocyst Implantation.
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