Human endometrial epithelial telomerase is important for epithelial proliferation and glandular formation with potential implications in endometriosis.
OBJECTIVE
How does regulation of telomerase activity (TA) in human endometrial epithelial cells (EEC) by ovarian hormones impact on telomere lengths (TL) and cell proliferation?


CONCLUSIONS
Healthy endometrial epithelial cell proliferation is characterized by high TA and endometrial TL changes according to the ovarian hormone cycle, with shortest TL observed in the progesterone dominant mid-secretory phase, when TA is lowest, implicating progesterone in the negative regulation of TA and TL.


BACKGROUND
Critical shortening of telomeres may result in permanent cell cycle arrest while the enzyme telomerase maintains telomere length (TL) and replicative capacity of cells. Telomerase expression and activity change in the human endometrium with the ovarian hormone cycle, however the effect of this on endometrial TL and cell growth is not known.


METHODS
A prospective observational study, which included endometrial and blood samples collected from 196 women.


METHODS
We studied endometrial samples from five different groups of women. Endometrial and matched blood TL and circulating steroid hormones were studied in samples collected from 85 women (Group 1). Fresh epithelial and stromal cell isolation and culture in vitro for TL and TA was done on endometrial biopsies collected from a further 74 healthy women not on hormonal therapy (Group 2) and from 5 women on medroxyprogesterone acetate (MPA) for contraception (Group 3). The epithelial TL and telomerase protein expression was examined in active, peritoneal, ectopic endometriotic and matched uterine (eutopic) endometrial samples collected from 10 women with endometriosis (Group 4); the in vivo effect of mifepristone on telomerase protein expression by immunohistochemistry (IHC) was examined in endometrium from 22 healthy women in mid-secretory phase before (n = 8), and after administering 200 mg mifepristone (n = 14) (Group 5). TA was measured by telomere repeat amplification protocol (TRAP) assay; TL by qPCR, and Q-FISH; cell proliferation was assessed by immunoblotting of histone H3 and 3D-culture to assess the ability of EECs to form spheroids; telomerase reverse transcriptase protein levels and Ki-67 (proliferative index) were assessed with IHC.


RESULTS
Endometrial TLs correlated negatively with serum progesterone levels (n = 58, r = -0.54) and were significantly longer than corresponding blood TLs (4893 ± 929 bp versus 3955 ± 557 bp, P = 0.002) suggesting a tissue-specific regulation. High TA and short TLs were observed in proliferating EECs in vivo and in vitro. During the progesterone dominant mid-secretory phase endometrial TL were significantly shorter compared with the proliferative phase (P = 0.0002). Progestagen treatment suppressed EEC TA in vivo and reduced endometrial TA in explant (P = 0.01) and in vitro cultures (P = 0.02) compared with untreated cells. Mifepristone (progesterone receptor antagonist) increased telomerase protein levels in vivo (P < 0.05). In 2D culture, Imetelstat inhibited EEC TA (P = 0.03), proliferation (P = 0.009) and in 3D culture disrupted endometrial glandular architecture (P = 0.03).


CONCLUSIONS
The in vitro telomerase inhibition data were tested in a mono-cellular system for a short-term. Further confirmation of the results in an in vivo model is necessary. The women in group 2 included a high proportion of women although with a regular menstrual cycle, with an increased BMI (>25) therefore this may affect extrapolation of data to other groups.


CONCLUSIONS
The observed effects of telomerase inhibition in vitro on epithelial cell proliferation, suggest that telomerase might be an attractive target in developing new therapies for proliferative disorders of the endometrium, such as endometriosis.
Department of Women's and Children's Health, Institute of Translational Medicine, University of Liverpool, Liverpool L8 7SS, UK.
A J
AJ
Valentijn
Institute for Cell and Molecular Biosciences and Institute for Ageing, Campus for Ageing and Vitality, Newcastle University, Newcastle upon Tyne NE4 5PL, UK.
G
G
Saretzki
Department of Women's and Children's Health, Institute of Translational Medicine, University of Liverpool, Liverpool L8 7SS, UK Liverpool Women's Hospital NHS Foundation Trust, Liverpool, UK.
N
N
Tempest
MRC Centre for Reproductive Health, University of Edinburgh, Edinburgh EH16 4TJ, UK.
H O D
HO
Critchley
Department of Women's and Children's Health, Institute of Translational Medicine, University of Liverpool, Liverpool L8 7SS, UK Liverpool Women's Hospital NHS Foundation Trust, Liverpool, UK dharani@liv.ac.uk.
D K
DK
Hapangama
D000293:Adolescent; D000328:Adult; D049109:Cell Proliferation / Q000187:drug effects / Q000502:physiology*; D004715:Endometriosis / Q000378:metabolism* / Q000473:pathology; D004717:Endometrium / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D008875:Middle Aged; D015735:Mifepristone / Q000494:pharmacology; D011374:Progesterone / Q000097:blood; D011446:Prospective Studies; D011980:Receptors, Progesterone / Q000037:antagonists & inhibitors; D017154:Stromal Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D019098:Telomerase / Q000378:metabolism*; D055815:Young Adult
D006727:Hormone Antagonists; D011980:Receptors, Progesterone; D015735:Mifepristone; D011374:Progesterone; D019098:Telomerase
3D culture; Imetelstat; cell culture; endometrial epithelium; endometriosis; endometrium; progesterone; proliferation; telomerase; telomere length