Endometrial stromal fibroblasts from women with polycystic ovary syndrome have impaired progesterone-mediated decidualization, aberrant cytokine profiles and promote enhanced immune cell migration in vitro.
OBJECTIVE
Do endometrial stromal fibroblasts (eSF) in women with polycystic ovary syndrome (PCOS) (eSFpcos) exhibit altered estrogen and/or progesterone (P4) responses, which may explain some of the adverse reproductive outcomes and endometrial pathologies in these women?


CONCLUSIONS
In vitro, eSF from women with PCOS exhibit an aberrant decidualization response and concomitant changes in pro-inflammatory cytokine, chemokine and matrix metalloproteinase (MMP) release and immune cell chemoattraction. In vivo these aberrations may result in suboptimal implantation and predisposition to endometrial cancer.


BACKGROUND
The endometrium in women with PCOS has several abnormalities including progesterone (P4) resistance at the gene expression level, likely contributing to subfertility, pregnancy complications and increased endometrial cancer risk in PCOS women.


METHODS
Prospective, university-based, case-control, in vitro study.


METHODS
Cultures of eSFPCOS (n = 12, Rotterdam and NIH criteria) and eSFControl (Ctrl) (n = 6, regular cycle length, no signs of hyperandrogenism) were treated with vehicle, estradiol (E2, 10 nM) or E2P4 (10 nM/1 μM) for 14 days. Progesterone receptor (PGR) mRNA was assessed with quantitative real-time PCR (qRT-PCR) and eSF decidualization was confirmed by insulin-like growth factor-binding protein-1 (IGFBP-1) transcript and protein expression. Fractalkine (CX3CL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL) 6, 8 and 11, macrophage chemoattractant protein (MCP) 1 and 3, CCL5 (RANTES) and MMPs (MMP1, 2, 3, 7, 9, 10 and 12) were measured in conditioned media by Luminex multiplex assays, and chemotactic activity of the conditioned media was tested in a migration assay using CD14+ monocyte and CD4+ T-cell migration assay. Effects of IL-6 (0.02, 0.2, 2 or 20 ng/ml) or IL-8 (0.04, 0.4, 4, or 40 ng/ml) or combination (0.2 ng/ml IL-6 and 4.0 ng/ml IL-8) on 14-d decidualization were also tested. ANOVA with pre-planned contrasts was used for statistical analysis.


RESULTS
Hormonal challenge with E2P4 to induce decidualization revealed two distinct subsets of eSFPCOS. Eight eSFPCOS (dPCOS) and all eSFCtrl (dCtrl) cultures showed a normal decidualization response to E2P4 as determined by morphology and IGFBP-1 secretion. However, 4 eSFPCOS cultures showed blunted decidualization (ndPCOS) in morphological assessment and low IGFBP-1 levels even though all three groups exhibited normal estrogen-mediated increase in PGR expression. Interestingly dPCOS had decreased IL-6 and GM-SCF secretion compared with dCtrl, whereas the ndPCOS cultures showed increased IL-6 and 8, MCP1, RANTES and GM-CSF secretion at base-line and/or in response to E2 or E2P4 compared with dCtrl and/or dPCOS. Furthermore, even though PGR expression was similar in all three groups, P4 inhibition of MMP secretion was attenuated in ndPCOS resulting in higher MMP2 and 3 levels. The conditioned media from ndPCOS had increased chemoattractic activity compared with dCtrl and dPCOS media. Exogenously added IL-6 and/or 8 did not inhibit decidualization in eSFCtrl indicating that high levels of these cytokines in ndPCOS samples were not likely a cause for the aberrant decidualization.


CONCLUSIONS
This is an in vitro study with a small sample size, utilizing stromal cell cultures from proliferative and secretory phase endometrium. The effect of PCOS on endometrial epithelium, another major histoarchitectural cell compartment of the endometrium, was not evaluated and should be considered in future studies. Furthermore, results obtained should also be confirmed in a larger data set and with mid/late secretory phase in vivo samples and models.


CONCLUSIONS
The alterations seen in ndPCOS may contribute to endometrial dysfunction, subfertility and pregnancy complications in PCOS women. The results emphasize the importance of understanding immune responses related to the implantation process and normal endometrial homeostasis in women with PCOS.


BACKGROUND
Sigrid Juselius Foundation, Academy of Finland, Finnish Medical Foundation, Orion-Farmos Research Foundation (to T.T.P.), the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) U54HD 055764-07 Specialized Cooperative Centers Program in Reproduction and Infertility Research (to L.C.G.), the NICHD the Ruth L. Kirschstein National Research Service Awards grant 1F32HD074423-03 (to J.C.C.). The authors have no competing interests.
Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA, USA Department of Obstetrics and Gynecology, Medical Research Center, University of Oulu, Oulu University Hospital, Oulu, Finland.
T T
TT
Piltonen
Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA, USA.
J C
JC
Chen
Department of Obstetrics and Gynecology, Medical Research Center, University of Oulu, Oulu University Hospital, Oulu, Finland.
M
M
Khatun
Department of Obstetrics and Gynecology, Medical Research Center, University of Oulu, Oulu University Hospital, Oulu, Finland.
M
M
Kangasniemi
Department of Pathology, Medical Research Center, University of Oulu, Oulu University Hospital, Oulu, Finland.
A
A
Liakka
Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA, USA.
T
T
Spitzer
Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA, USA.
N
N
Tran
Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA, USA.
H
H
Huddleston
Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA, USA.
J C
JC
Irwin
Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA, USA Linda.Giudice@ucsf.edu.
L C
LC
Giudice
D000328:Adult; D001706:Biopsy; D016022:Case-Control Studies; D002465:Cell Movement; D049109:Cell Proliferation; D016207:Cytokines / Q000378:metabolism; D003656:Decidua / Q000378:metabolism* / Q000473:pathology; D010064:Embryo Implantation; D016889:Endometrial Neoplasms / Q000378:metabolism; D004717:Endometrium / Q000166:cytology*; D004967:Estrogens / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000473:pathology*; D005786:Gene Expression Regulation; D020022:Genetic Predisposition to Disease; D005951:Glucose Tolerance Test; D006801:Humans; D008875:Middle Aged; D011085:Polycystic Ovary Syndrome / Q000175:diagnosis* / Q000378:metabolism*; D011247:Pregnancy; D011374:Progesterone / Q000378:metabolism*; D011446:Prospective Studies
D016207:Cytokines; D004967:Estrogens; D011374:Progesterone
MMP; PCOS; cytokines; decidualization; endometrium