Gastrointestinal Coinfection Promotes Chlamydial Pathogenicity in the Genital Tract.

Sexually transmitted Chlamydia, which can cause fibrotic pathology in women's genital tracts, is also frequently detected in the gastrointestinal tract. However, the medical significance of the gastrointestinal Chlamydia remains unclear. A murine Chlamydia readily spreads from the mouse genital tract to the gastrointestinal tract while inducing oviduct fibrotic blockage or hydrosalpinx. We previously proposed a two-hit model in which the mouse gastrointestinal Chlamydia might induce the second hit to promote genital tract pathology, and we are now providing experimental evidence for testing the hypothesis. First, chlamydial mutants that are attenuated in inducing hydrosalpinx in the genital tract also reduce their colonization in the gastrointestinal tract, leading to a better correlation of chlamydial induction of hydrosalpinx with chlamydial colonization in the gastrointestinal tract than in the genital tract. Second, intragastric coinoculation with a wild-type Chlamydia rescued an attenuated Chlamydia mutant to induce hydrosalpinx, while the chlamydial mutant infection in the genital tract alone was unable to induce any significant hydrosalpinx. Finally, the coinoculated gastrointestinal Chlamydia failed to directly spread to the genital tract lumen, suggesting that gastrointestinal Chlamydia may promote genital pathology via an indirect mechanism. Thus, we have demonstrated a significant role of gastrointestinal Chlamydia in promoting pathology in the genital tract possibly via an indirect mechanism. This study provides a novel direction/dimension for further investigating chlamydial pathogenic mechanisms.

Oncogenic exon 2 mutations in Mediator subunit MED12 disrupt allosteric activation of cyclin C-CDK8/19.

Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at high frequency in uterine fibroids (UFs) and breast fibroepithelial tumors as well as recurrently, albeit less frequently, in malignant uterine leimyosarcomas, chronic lymphocytic leukemias, and colorectal cancers. Previously, we reported that UF-linked mutations in MED12 disrupt its ability to activate cyclin C (CycC)-dependent kinase 8 (CDK8) in Mediator, implicating impaired Mediator-associated CDK8 activity in the molecular pathogenesis of these clinically significant lesions. Notably, the CDK8 paralog CDK19 is also expressed in myometrium, and both CDK8 and CDK19 assemble into Mediator in a mutually exclusive manner, suggesting that CDK19 activity may also be germane to the pathogenesis of MED12 mutation-induced UFs. However, whether and how UF-linked mutations in MED12 affect CDK19 activation is unknown. Herein, we show that MED12 allosterically activates CDK19 and that UF-linked exon 2 mutations in MED12 disrupt its CDK19 stimulatory activity. Furthermore, we find that within the Mediator kinase module, MED13 directly binds to the MED12 C terminus, thereby suppressing an apparent UF mutation-induced conformational change in MED12 that otherwise disrupts its association with CycC-CDK8/19. Thus, in the presence of MED13, mutant MED12 can bind, but cannot activate, CycC-CDK8/19. These findings indicate that MED12 binding is necessary but not sufficient for CycC-CDK8/19 activation and reveal an additional step in the MED12-dependent activation process, one critically dependent on MED12 residues altered by UF-linked exon 2 mutations. These findings confirm that UF-linked mutations in MED12 disrupt composite Mediator-associated kinase activity and identify CDK8/19 as prospective therapeutic targets in UFs.

Reproductive Risk Factors and Coronary Heart Disease in the Women's Health Initiative Observational Study.

BACKGROUND: Reproductive factors provide an early window into a woman's coronary heart disease (CHD) risk; however, their contribution to CHD risk stratification is uncertain. METHODS AND RESULTS: In the Women's Health Initiative Observational Study, we constructed Cox proportional hazards models for CHD including age, pregnancy status, number of live births, age at menarche, menstrual irregularity, age at first birth, stillbirths, miscarriages, infertility ≥1 year, infertility cause, and breastfeeding. We next added each candidate reproductive factor to an established CHD risk factor model. A final model was then constructed with significant reproductive factors added to established CHD risk factors. Improvement in C statistic, net reclassification index (or net reclassification index with risk categories of <5%, 5 to <10%, and ≥10% 10-year risk of CHD), and integrated discriminatory index were assessed. Among 72 982 women (CHD events, n=4607; median follow-up,12.0 [interquartile range, 8.3-13.7] years; mean [standard deviation] age, 63.2 [7.2] years), an age-adjusted reproductive risk factor model had a C statistic of 0.675 for CHD. In a model adjusted for established CHD risk factors, younger age at first birth, number of still births, number of miscarriages, and lack of breastfeeding were positively associated with CHD. Reproductive factors modestly improved model discrimination (C statistic increased from 0.726 to 0.730; integrated discriminatory index, 0.0013; P<0.0001). Net reclassification for women with events was not improved (net reclassification index events, 0.007; P=0.18); and, for women without events, net reclassification was marginally improved (net reclassification index nonevents, 0.002; P=0.04) CONCLUSIONS: Key reproductive factors are associated with CHD independently of established CHD risk factors, very modestly improve model discrimination, and do not materially improve net reclassification.

The Chlamydia-Secreted Protease CPAF Promotes Chlamydial Survival in the Mouse Lower Genital Tract.

Despite the extensive in vitro characterization of CPAF (chlamydial protease/proteasome-like activity factor), its role in chlamydial infection and pathogenesis remains unclear. We now report that a Chlamydia trachomatis strain deficient in expression of CPAF (L2-17) is no longer able to establish a successful infection in the mouse lower genital tract following an intravaginal inoculation. The L2-17 organisms were cleared from the mouse lower genital tract within a few days, while a CPAF-sufficient C. trachomatis strain (L2-5) survived in the lower genital tract for more than 3 weeks. However, both the L2-17 and L2-5 organisms maintained robust infection courses that lasted up to 4 weeks when they were directly delivered into the mouse upper genital tract. The CPAF-dependent chlamydial survival in the lower genital tract was confirmed in multiple strains of mice. Thus, we have demonstrated a critical role of CPAF in promoting C. trachomatis survival in the mouse lower genital tracts. It will be interesting to further investigate the mechanisms of the CPAF-dependent chlamydial pathogenicity.

Intravenous Inoculation with Chlamydia muridarum Leads to a Long-Lasting Infection Restricted to the Gastrointestinal Tract.

Chlamydia has been detected in the gastrointestinal tracts of both animals and humans. However, it remains unclear whether the chlamydial organisms can be introduced into the gastrointestinal tract via pathways independent of the oral and anal routes. We have recently shown that Chlamydia muridarum spreads from the genital tract to the gastrointestinal tract potentially via the circulatory system. To test whether hematogenous C. muridarum can spread to and establish a long-lasting colonization in the mouse gastrointestinal tract, we inoculated mice intravenously with a luciferase-expressing C. muridarum strain and monitored its distribution. After tail vein inoculation, most luciferase-generated bioluminescence signals were detected in the mouse abdominal area throughout the experiment. The ex vivo imaging revealed that the abdominal signals came from the gastrointestinal tract tissues. Simultaneous monitoring of chlamydial organisms in individual organs or tissues revealed an initial stage of systemic spreading followed by a long-lasting infection in the gastrointestinal tract. A retro-orbital vein inoculation of the C. muridarum organisms at a lower dose in a different mouse strain also led to colonization of the gastrointestinal tract. We have demonstrated that intravenous C. muridarum inoculation can result in colonization of the gastrointestinal tract, suggesting that the chlamydial organisms may use the sexual behavior-independent circulation pathway to infect the gastrointestinal tract.

In Vivo and Ex Vivo Imaging Reveals a Long-Lasting Chlamydial Infection in the Mouse Gastrointestinal Tract following Genital Tract Inoculation.

Intravaginal infection with Chlamydia muridarum in mice can ascend to the upper genital tract, resulting in hydrosalpinx, a pathological hallmark for tubal infertility in women infected with C. trachomatis. Here, we utilized in vivo imaging of C. muridarum infection in mice following an intravaginal inoculation and confirmed the rapid ascent of the chlamydial organisms from the lower to upper genital tracts. Unexpectedly, the C. muridarum-derived signal was still detectable in the abdominal area 100 days after inoculation. Ex vivo imaging of the mouse organs revealed that the long-lasting presence of the chlamydial signal was restricted to the gastrointestinal (GI) tract, which was validated by directly measuring the chlamydial live organisms and genomes in the same organs. The C. muridarum organisms spreading from the genital to the GI tracts were detected in different mouse strains and appeared to be independent of oral or rectal routes. Mice prevented from orally taking up excretions also developed the long-lasting GI tract infection. Inoculation of C. muridarum directly into the upper genital tract, which resulted in a delayed vaginal shedding of live organisms, accelerated the chlamydial spreading to the GI tract. Thus, we have demonstrated that the genital tract chlamydial organisms may use a systemic route to spread to and establish a long-lasting infection in the GI tract. The significance of the chlamydial spreading from the genital to GI tracts is discussed.

Risk of cardiovascular disease among postmenopausal women with prior pregnancy loss: the women's health initiative.

PURPOSE: Metabolic, hormonal, and hemostatic changes associated with pregnancy loss (stillbirth and miscarriage) may contribute to the development of cardiovascular disease (CVD) in adulthood. This study evaluated prospectively the association between a history of pregnancy loss and CVD in a cohort of postmenopausal women. METHODS: Postmenopausal women (77,701) were evaluated from 1993-1998. Information on baseline reproductive history, sociodemographic, and CVD risk factors were collected. The associations between 1 or 2 or more miscarriages and 1 or more stillbirths with occurrence of CVD were evaluated using multiple logistic regression. RESULTS: Among 77,701 women in the study sample, 23,538 (30.3%) reported a history of miscarriage; 1,670 (2.2%) reported a history of stillbirth; and 1,673 (2.2%) reported a history of both miscarriage and stillbirth. Multivariable-adjusted odds ratio (OR) for coronary heart disease (CHD) for 1 or more stillbirths was 1.27 (95% CI, 1.07-1.51) compared with no stillbirth; for women with a history of 1 miscarriage, the OR=1.19 (95% CI, 1.08-1.32); and for 2 or more miscarriages the OR=1.18 (95% CI, 1.04-1.34) compared with no miscarriage. For ischemic stroke, the multivariable odds ratio for stillbirths and miscarriages was not significant. CONCLUSIONS: Pregnancy loss was associated with CHD but not ischemic stroke. Women with a history of 1 or more stillbirths or 1 or more miscarriages appear to be at increased risk of future CVD and should be considered candidates for closer surveillance and/or early intervention; research is needed into better understanding the pathophysiologic mechanisms behind the increased risk of CVD associated with pregnancy loss.

Conjugated equine oestrogen and breast cancer incidence and mortality in postmenopausal women with hysterectomy: extended follow-up of the Women's Health Initiative randomised placebo-controlled trial.

BACKGROUND: By contrast with many observational studies, women in the Women's Health Initiative (WHI) trial who were randomly allocated to receive oestrogen alone had a lower incidence of invasive breast cancer than did those who received placebo. We aimed to assess the influence of oestrogen use on longer term breast cancer incidence and mortality in extended follow-up of this cohort. METHODS: Between 1993 and 1998, the WHI enrolled 10,739 postmenopausal women from 40 US clinical centres into a randomised, double-masked, placebo-controlled trial. Women aged 50-79 years who had undergone hysterectomy and had expected 3-year survival and mammography clearance were randomly allocated by a computerised, permuted block algorithm, stratified by age group and centre, to receive oral conjugated equine oestrogen (0·625 mg per day; n=5310) or matched placebo (n=5429). The trial intervention was terminated early on Feb 29, 2004, because of an adverse effect on stroke. Follow-up continued until planned termination (March 31, 2005). Consent was sought for extended surveillance from the 9786 living participants in active follow-up, of whom 7645 agreed. Using data from this extended follow-up (to Aug 14, 2009), we assessed long-term effects of oestrogen use on invasive breast cancer incidence, tumour characteristics, and mortality. We used Cox regression models to estimate hazard ratios (HRs) in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT00000611. FINDINGS: After a median follow-up of 11·8 years (IQR 9·1-12·9), the use of oestrogen for a median of 5·9 years (2·5-7·3) was associated with lower incidence of invasive breast cancer (151 cases, 0·27% per year) compared with placebo (199 cases, 0·35% per year; HR 0·77, 95% CI 0·62-0·95; p=0·02) with no difference (p=0·76) between intervention phase (0·79, 0·61-1·02) and post-intervention phase effects (0·75, 0·51-1·09). In subgroup analyses, we noted breast cancer risk reduction with oestrogen use was concentrated in women without benign breast disease (p=0·01) or a family history of breast cancer (p=0·02). In the oestrogen group, fewer women died from breast cancer (six deaths, 0·009% per year) compared with controls (16 deaths, 0·024% per year; HR 0·37, 95% CI 0·13-0·91; p=0·03). Fewer women in the oestrogen group died from any cause after a breast cancer diagnosis (30 deaths, 0·046% per year) than did controls (50 deaths, 0·076%; HR 0·62, 95% CI 0·39-0·97; p=0·04). INTERPRETATION: Our findings provide reassurance for women with hysterectomy seeking relief of climacteric symptoms in terms of the effects of oestrogen use for about 5 years on breast cancer incidence and mortality. However, our data do not support use of oestrogen for breast cancer risk reduction because any noted benefit probably does not apply to populations at increased risk of such cancer. FUNDING: US National Heart, Lung, and Blood Institute; Wyeth.

Breast cancer after use of estrogen plus progestin in postmenopausal women.

BACKGROUND: Following the release of the 2002 report of the Women's Health Initiative (WHI) trial of estrogen plus progestin, the use of menopausal hormone therapy in the United States decreased substantially. Subsequently, the incidence of breast cancer also dropped, suggesting a cause-and-effect relation between hormone treatment and breast cancer. However, the cause of this decrease remains controversial. METHODS: We analyzed the results of the WHI randomized clinical trial--in which one study group received 0.625 mg of conjugated equine estrogens plus 2.5 mg of medroxyprogesterone acetate daily and another group received placebo--and examined temporal trends in breast-cancer diagnoses in the WHI observational-study cohort. Risk factors for breast cancer, frequency of mammography, and time-specific incidence of breast cancer were assessed in relation to combined hormone use. RESULTS: In the clinical trial, there were fewer breast-cancer diagnoses in the group receiving estrogen plus progestin than in the placebo group in the initial 2 years of the study, but the number of diagnoses increased over the course of the 5.6-year intervention period. The elevated risk decreased rapidly after both groups stopped taking the study pills, despite a similar frequency of mammography. In the observational study, the incidence of breast cancer was initially about two times as high in the group receiving menopausal hormones as in the placebo group, but this difference in incidence decreased rapidly in about 2 years, coinciding with year-to-year reductions in combined hormone use. During this period, differences in the frequency of mammography between the two groups were unchanged. CONCLUSIONS: The increased risk of breast cancer associated with the use of estrogen plus progestin declined markedly soon after discontinuation of combined hormone therapy and was unrelated to changes in frequency of mammography.

Aberrant R-loop-induced replication stress in MED12-mutant uterine fibroids.

Uterine fibroid (UF) driver mutations in Mediator complex subunit 12 (MED12) trigger genomic instability and tumor development through unknown mechanisms. Herein, we show that MED12 mutations trigger aberrant R-loop-induced replication stress, suggesting a possible route to genomic instability and a novel therapeutic vulnerability in this dominant UF subclass. Immunohistochemical analyses of patient-matched tissue samples revealed that MED12 mutation-positive UFs, compared to MED12 mutation-negative UFs and myometrium, exhibited significantly higher levels of R-loops and activated markers of Ataxia Telangiectasia and Rad3-related (ATR) kinase-dependent replication stress signaling in situ. Single molecule DNA fiber analysis revealed that primary cells from MED12 mutation-positive UFs, compared to those from patient-matched MED12 mutation-negative UFs and myometrium, exhibited defects in replication fork dynamics, including reduced fork speeds, increased and decreased numbers of stalled and restarted forks, respectively, and increased asymmetrical bidirectional forks. Notably, these phenotypes were recapitulated and functionally linked in cultured uterine smooth muscle cells following chemical inhibition of Mediator-associated CDK8/19 kinase activity that is known to be disrupted by UF driver mutations in MED12. Thus, Mediator kinase inhibition triggered enhanced R-loop formation and replication stress leading to an S-phase cell cycle delay, phenotypes that were rescued by overexpression of the R-loop resolving enzyme RNaseH. Altogether, these findings reveal MED12-mutant UFs to be uniquely characterized by aberrant R-loop induced replication stress, suggesting a possible basis for genomic instability and new avenues for therapeutic intervention that involve the replication stress phenotype in this dominant UF subtype.

Localization of Chlamydia trachomatis hypothetical protein CT311 in host cell cytoplasm.

The chlamydia-specific hypothetical protein CT311 was detected both inside and outside of the chlamydial inclusions in Chlamydia trachomatis-infected cells. The extra-inclusion CT311 molecules were distributed in the host cell cytoplasm with a pattern similar to that of CPAF, a known Chlamydia-secreted protease. The detection of CT311 was specific since the anti-CT311 antibody labeling was only removed by absorption with CT311 but not CPAF fusion proteins. In addition, both anti-CT311 and anti-CPAF antibodies only detected their corresponding endogenous proteins without cross-reacting with each other or any other antigens in the whole cell lysates of C. trachomatis-infected cells. Although both CT311 and CPAF proteins were first detected 12 h after infection, localization of CT311 into host cell cytosol was delayed until 24 h while CPAF secretion into host cell cytosol was already obvious by 18 h after infection. The host cell cytosolic localization of CT311 was further confirmed in human primary cells. CT311 was predicted to contain an N-terminal secretion signal sequence and the CT311 signal sequence directed secretion of PhoA into bacterial periplasmic region in a heterologous assay system, suggesting that a sec-dependent pathway may play a role in the secretion of CT311 into host cell cytosol. This hypothesis is further supported by the observation that secretion of CT311 in Chlamydia-infected cells was blocked by a C16 compound known to inhibit signal peptidase I. These findings have provided important molecular information for further understanding the C. trachomatis pathogenic mechanisms.

Identification and characterization of the mediator kinase-dependent myometrial stem cell phosphoproteome.

OBJECTIVE: To identify, in myometrial stem/progenitor cells, the presumptive cell of origin for uterine fibroids, substrates of Mediator-associated cyclin dependent kinase 8/19 (CDK8/19), which is known to be disrupted by uterine fibroid driver mutations in Mediator complex subunit 12 (MED12). DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S): Women undergoing hysterectomy for uterine fibroids. INTERVENTION(S): Stable isotopic labeling of amino acids in cell culture (SILAC) coupled with chemical inhibition of CDK8/19 and downstream quantitative phosphoproteomics and transcriptomic analyses in myometrial stem/progenitor cells. MAIN OUTCOME MEASURE(S): High-confidence Mediator kinase substrates identified by SILAC-based quantitative phosphoproteomics were determined using an empirical Bayes analysis and validated orthogonally by in vitro kinase assay featuring reconstituted Mediator kinase modules comprising wild-type or G44D mutant MED12 corresponding to the most frequent uterine fibroid driver mutation in MED12. Mediator kinase-regulated transcripts identified by RNA sequencing were linked to Mediator kinase substrates by computational analyses. RESULT(S): A total of 296 unique phosphosites in 166 proteins were significantly decreased (≥ twofold) upon CDK8/19 inhibition, including 118 phosphosites in 71 nuclear proteins representing high-confidence Mediator kinase substrates linked to RNA polymerase II transcription, RNA processing and transport, chromatin modification, cytoskeletal architecture, and DNA replication and repair. Orthogonal validation confirmed a subset of these proteins, including Cut Like Homeobox 1 (CUX1) and Forkhead Box K1 (FOXK1), to be direct targets of MED12-dependent CDK8 phosphorylation in a manner abrogated by the most common uterine fibroid driver mutation (G44D) in MED12, implicating these substrates in disease pathogenesis. Transcriptome-wide profiling of Mediator kinase-inhibited myometrial stem/progenitor cells revealed alterations in cell cycle and myogenic gene expression programs to which Mediator kinase substrates could be linked directly. Among these, CUX1 is an established transcriptional regulator of the cell cycle whose corresponding gene on chromosome 7q is the locus for a recurrent breakpoint in uterine fibroids, linking MED12 and Mediator kinase with CUX1 for the first time in uterine fibroid pathogenesis. FOXK1, a transcriptional regulator of myogenic stem cell fate, was found to be coordinately enriched along with kinase, but not core, Mediator subunits in myometrial stem/progenitor cells compared with differentiated uterine smooth muscle cells. CONCLUSION(S): These studies identify a new catalog of pathologically and biologically relevant Mediator kinase substrates implicated in the pathogenesis of MED12 mutation-positive uterine fibroids, and further uncover a biochemical basis to link Mediator kinase activity with CUX1 and FOXK1 in the regulation of myometrial stem/progenitor cell fate.

Association of tubal factor infertility with elevated antibodies to Chlamydia trachomatis caseinolytic protease P.

OBJECTIVE: The objective of the study was to assess antibodies against Chlamydia trachomatis heat shock proteins (HSP) in patients with tubal factor infertility (TFI), infertility controls (IFC), and fertile controls (FC). HSPs assist organisms in surviving caustic environments such as heat. STUDY DESIGN: Twenty-one TFI, 15 IFC, and 29 FC patients were enrolled after laparoscopic tubal assessment. The titers of antibodies against C trachomatis organisms and 14 chlamydial HSPs were compared among the 3 groups. RESULTS: TFI patients developed significantly higher levels of antibodies against C trachomatis and specifically recognizing chlamydial HSP60 and caseinolytic protease (Clp) P, a subunit of the ATP-dependent Clp protease complex involved in the degradation of abnormal proteins. CONCLUSION: In addition to confirming high titers of antibodies against C trachomatis organisms and HSP60 in TFI patients, we identified a novel link of TFI with anti-ClpP antibodies. These findings may provide useful information for developing a noninvasive screening test for TFI and constructing subunit anti-C trachomatis vaccines.

Estrogen plus progestin and breast cancer incidence and mortality in postmenopausal women.

CONTEXT: In the Women's Health Initiative randomized, placebo-controlled trial of estrogen plus progestin, after a mean intervention time of 5.6 (SD, 1.3) years (range, 3.7-8.6 years) and a mean follow-up of 7.9 (SD, 1.4) years, breast cancer incidence was increased among women who received combined hormone therapy. Breast cancer mortality among participants in the trial has not been previously reported. OBJECTIVE: To determine the effects of therapy with estrogen plus progestin on cumulative breast cancer incidence and mortality after a total mean follow-up of 11.0 (SD, 2.7) years, through August 14, 2009. DESIGN, SETTING, AND PARTICIPANTS: A total of 16,608 postmenopausal women aged 50 to 79 years with no prior hysterectomy from 40 US clinical centers were randomly assigned to receive combined conjugated equine estrogens, 0.625 mg/d, plus medroxyprogesterone acetate, 2.5 mg/d, or placebo pill. After the original trial completion date (March 31, 2005), reconsent was required for continued follow-up for breast cancer incidence and was obtained from 12,788 (83%) of the surviving participants. MAIN OUTCOME MEASURES: Invasive breast cancer incidence and breast cancer mortality. RESULTS: In intention-to-treat analyses including all randomized participants and censoring those not consenting to additional follow-up on March 31, 2005, estrogen plus progestin was associated with more invasive breast cancers compared with placebo (385 cases [0.42% per year] vs 293 cases [0.34% per year]; hazard ratio [HR], 1.25; 95% confidence interval [CI], 1.07-1.46; P = .004). Breast cancers in the estrogen-plus-progestin group were similar in histology and grade to breast cancers in the placebo group but were more likely to be node-positive (81 [23.7%] vs 43 [16.2%], respectively; HR, 1.78; 95% CI, 1.23-2.58; P = .03). There were more deaths directly attributed to breast cancer (25 deaths [0.03% per year] vs 12 deaths [0.01% per year]; HR, 1.96; 95% CI, 1.00-4.04; P = .049) as well as more deaths from all causes occurring after a breast cancer diagnosis (51 deaths [0.05% per year] vs 31 deaths [0.03% per year]; HR, 1.57; 95% CI, 1.01-2.48; P = .045) among women who received estrogen plus progestin compared with women in the placebo group. CONCLUSIONS: Estrogen plus progestin was associated with greater breast cancer incidence, and the cancers are more commonly node-positive. Breast cancer mortality also appears to be increased with combined use of estrogen plus progestin. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00000611.

CD44 variant 6 is involved in the attachment and invasion of endometrial cells to peritoneum.

OBJECTIVE: To study the effects of CD44 standard (CD44s), CD44v3, and CD44v6 overexpression (OE) on immortalized human endometrial epithelial (iEECs) and stroma cells (human endometrial stromal cells [hESCs]) using in vitro assays and a nude mouse xenograft model. Menstrual endometrial cells from women with endometriosis have increased adhesion and also express higher levels of CD44 variant 6 (v6), but not v3, compared to menstrual endometrial cells from women without endometriosis. DESIGN: In vitro studies and in vivo xenograft model. SETTING: Academic center. PATIENTS(S): Deidentified immortalized endometrial epithelial tissue samples of a reproductive-age woman. INTERVENTION(S): Overexpression of CD44s, CD44v3, and CD44v6 was carried out using lipofectamine, and their expression was verified with mRNA and protein in iEEC and hESCs. The OE cells were used in in vitro studies and an in vivo xenograft model compared to plasmid control. MAIN OUTCOME MEASURE(S): The effect of CD44s, CD44v3, and CD44v6 OE on attachment and invasion assays and a xenograft model with immortalized human stromal and epithelial cells. RESULT(S): Expression of mRNA and protein confirmed appropriate OE of CD44s, CD44v3, and CD44v6 in the different cell types. CD44v6 OE increased attachment of hESCs compared with controls. CD44v6 OE did not change the attachment of iEECs. There was no difference in attachment in iEECs or hESCs with OE of CD44s or CD44v3. CONCLUSION(S): Overexpression of CD44v6 increases attachment of hESCs to peritoneal mesothelial cells in an in vitro assay and an in vivo xenograft model. Menstrual endometrial cell type and CD44 variants play a complex role in the development of the early endometriotic lesion.

Inhibition of Hyaluronic Acid Synthesis Decreases Endometrial Cell Attachment, Migration, and Invasion.

To characterize the effects of 4-methylumbelliferone (4-MU) on expression of the hyaluronic acid (HA) system and on attachment, migration, and invasion of endometrial epithelial (EECs) and stroma cells (ESCs) to peritoneal mesothelial cells (PMCs), this in vitro study was performed in an Academic Center. De-identified endometrial tissue samples used were from reproductive-aged women. EECs and ESCs isolated from menstrual endometrial biopsies were treated with 4-MU or vehicle. Real-time polymerase chain reaction and western blot were used to assess expression of HA synthases (HAS), hyaluronidase, and standard CD44. Established in vitro assays were used to assess attachment, migration, and invasion with and without treatment with 4-MU. Chi square and Student's t-test were used to analyze the results as appropriate. The addition of 4-MU decreased mRNA and protein expression of HAS 2, HAS 3, and CD44 in EECs and ESCs compared to control. Treatment with 4-MU also decreased attachment, migration, and invasion of EECs and ESCs to PMCs compared to control. 4-MU decreases endometrial cell adhesion, migration, and invasion to PMCs. This effect appears to be mediated by a decrease in HAS 2, HAS 3, and CD44. 4-MU is a potential treatment for endometriosis. Future in vivo studies are needed to evaluate 4-MU as a therapeutic agent for endometriosis.

Induction of endometrial epithelial cell invasion and c-fms expression by transforming growth factor beta.

Transforming growth factor beta 1 (TGF-beta1) levels are increased in the peritoneal fluid of endometriosis patients, and endometrial cells express TGF-beta signaling components; however, little is known regarding the role of TGF-beta in endometriosis. Our objective was to examine the effects of TGF-beta1 on (i) the expression of macrophage colony-stimulating factor receptor encoded by the c-fms gene, (ii) transmesothelial invasiveness of endometrial cells, (iii) cellular proliferation and (iv) attachment to peritoneal mesothelial cells (PMCs). Effects of TGF-beta1 on c-fms mRNA expression were determined by real-time RT-PCR and c-fms cell-surface expression by flow cytometry. Effects of TGF-beta1 on the invasiveness of the immortalized endometrial epithelial cell (EEC) line EM42 and primary EECs were examined using a three-dimensional in vitro system modeling the peritoneum. Cellular proliferation and attachment to PMCs were also examined using established techniques. TGF-beta1 had little or no effect on cellular proliferation and endometrial cell attachment to PMCs. TGF-beta1 significantly induced the expression of c-fms mRNA and c-fms cell-surface expression. TGF-beta1 enhanced transmesothelial invasion by EM42 cells and EECs. Antagonists of TGF-beta1 signaling significantly inhibited both the induction of c-fms expression and cellular invasiveness, suggesting that additional studies are warranted to assess the therapeutic potential of TGF-beta antagonists in endometriosis.

EC313-a tissue selective SPRM reduces the growth and proliferation of uterine fibroids in a human uterine fibroid tissue xenograft model.

Uterine fibroids (UFs) are associated with irregular or excessive uterine bleeding, pelvic pain or pressure, or infertility. Ovarian steroid hormones support the growth and maintenance of UFs. Ulipristal acetate (UPA) a selective progesterone receptor (PR) modulator (SPRM) reduce the size of UFs, inhibit ovulation and lead to amenorrhea. Recent liver toxicity concerns with UPA, diminished enthusiasm for its use and reinstate the critical need for a safe, efficacious SPRM to treat UFs. In the current study, we evaluated the efficacy of new SPRM, EC313, for the treatment for UFs using a NOD-SCID mouse model. EC313 treatment resulted in a dose-dependent reduction in the fibroid xenograft weight (p < 0.01). Estradiol (E2) induced proliferation was blocked significantly in EC313-treated xenograft fibroids (p < 0.0001). Uterine weight was reduced by EC313 treatment compared to UPA treatment. ER and PR were reduced in EC313-treated groups compared to controls (p < 0.001) and UPA treatments (p < 0.01). UF specific desmin and collagen were markedly reduced with EC313 treatment. The partial PR agonism and no signs of unopposed estrogenicity makes EC313 a candidate for the long-term treatment for UFs. Docking studies have provided a structure based explanation for the SPRM activity of EC313.

The Hyaluronic Acid System is Intact in Menstrual Endometrial Cells in Women With and Without Endometriosis.

OBJECTIVE: To characterize the production and degradation of hyaluronic acid (HA) in menstrual endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) in women with and without endometriosis. To identify the presence of CD44, the primary receptor of HA, in menstrual EECs and ESCs in women with and without endometriosis. DESIGN: In vitro study. SETTING: Academic center. PATIENT(S): Deidentified patient samples from women with and without endometriosis. INTERVENTIONS: EECs and ESCs were isolated from menstrual endometrial biopsies performed on women with (N = 9) and without (N = 11) endometriosis confirmed by laparoscopy. MAIN OUTCOME MEASURE: Real-time polymerase chain reaction, Western blot, and immunohistochemistry were used to assess hyaluronic acid synthase (HAS) isoforms 1, 2, and 3; hyaluronidase (HYAL) isoforms 1 and 2; and standard CD44. Student t test was used to analyze the results. RESULTS: There was no significant difference in messenger RNA (mRNA) or protein expression of HAS2, HAS3, HYAL1, or HYAL2 in EECs or ESCs from women with or without endometriosis. HAS1 mRNA was variably detected, whereas HAS1 protein was similarly expressed in EECs and ESCs from women with and without endometriosis. Standard CD44 was expressed in both cell types, and expression did not differ in cells from women with or without endometriosis. CONCLUSIONS: The HA system is expressed in eutopic menstrual ESCs and EECs from women with and without endometriosis. There are no differences in expression in HA production or degradation enzymes in EECs or ESCs from women with and without endometriosis. Standard CD44 expression does not differ in eutopic menstrual endometrial cells from women with and without endometriosis.

Prewash total motile count is a poor predictor of live birth in intrauterine insemination cycles.

OBJECTIVE: To determine whether there is a relationship between prewash total motile count and live births in couples undergoing IUI. DESIGN: Retrospective review in a single academic center. SETTING: Not applicable. PATIENT(S): Couples with infertility undergoing ovulation induction with IUI between 2010 and 2014. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Live births. RESULT(S): Our cohort included 310 women who underwent 655 IUI cycles with a cumulative live birth rate (LBR) per couple of 20% and an LBR per cycle of 10%. A analysis yielded no correlation between prewash total motile count (TMC) and live births. No live births occurred with TMC <2 million sperms. Age had a significant negative relationship to LBR. A receiver operating characteristic analysis comparing age and live births indicated a significant decline in live births for women >37 years (90% sensitivity, 70% specificity). The LBR per couple was decreased to 7% in women >37 years compared with 25% in women <37 years. CONCLUSION(S): Prewash TMC is a poor predictor of live birth. There were no live births with prewash TMC <2 million sperms. The LBR for women >37 years with IUI was significantly lower than women <37 years.