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.

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.

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.

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.

Mediator Kinase Disruption in MED12-Mutant Uterine Fibroids From Hispanic Women of South Texas.

Context: Mutations in the gene encoding Mediator complex subunit MED12 are dominant drivers of uterine fibroids (UFs) in women of diverse racial and ethnic origins. Previously, we showed that UF-linked mutations in MED12 disrupt its ability to activate cyclin C-CDK8/19 in Mediator. However, validation of Mediator kinase disruption in the clinically relevant setting of MED12-mutant UFs is currently lacking. Objective: The objective of this study was twofold. First, to extend the ethnic distribution profile of MED12 mutations by establishing their frequency in UFs from Hispanic women of South Texas. Second, to examine the impact of MED12 mutations on Mediator kinase activity in patient-derived UFs. Methods: We screened 219 UFs from 76 women, including 170 tumors from 57 Hispanic patients, for MED12 exon 2 mutations, and further examined CDK8/19 activity in Mediator complexes immunoprecipitated from MED12 mutation-negative and MED12 mutation-positive UFs. Results: MED12 exon 2 mutations in UFs from Hispanic women are somatic in nature, predominantly monoallelic, and occur at high frequency (54.1%). We identified a minimal cyclin C-CDK8 activation domain on MED12 spanning amino acids 15 through 80 that includes all recorded UF-linked mutations in MED12, suggesting that disruption of Mediator kinase activity is a principal biochemical defect arising from these pathogenic alterations. Analysis of Mediator complexes recovered from patient UFs confirmed this, revealing that Mediator kinase activity is selectively impaired in MED12-mutant UFs. Conclusions: MED12 mutations are important drivers of UF formation in Hispanic women of South Texas. MED12 mutations disrupt Mediator kinase activity, implicating altered CDK8/19 function in UF pathogenesis.

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.

Assisted hatching and live births in first-cycle frozen embryo transfers.

OBJECTIVE: To assess the effect of assisted hatching (AH) on live-birth rates in a retrospective cohort of patients undergoing first-cycle, autologous frozen embryo transfer (FET). DESIGN: Longitudinal cohort using cycles reported to the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System between 2004 and 2013. SETTING: Not applicable. PATIENT(S): Women who underwent first-cycle, autologous FET with (n = 70,738) and without (n = 80,795) AH reported from 2004 to 2013. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Live births. RESULT(S): Propensity matching was used to account for confounding covariates, and a logistic regression model was constructed to identify the predictors of live-birth rates in relationship to AH. In all first-cycle FETs, there was a slight but statistically significant decrease in the live-birth rate with AH compared with no AH (34.2% vs. 35.4%). In older patients and in the years 2012-2013 AH was associated with decreased live births. Live-birth rates and the number of AH cycles performed before FET vary by the geographic location of clinics. CONCLUSION(S): Assisted hatching slightly decreases the live-birth rate in first-cycle, autologous FET. Its use should be carefully considered, especially in patients 38 years old and older. Prospective, clinical studies are needed to improve our knowledge of the impact of AH.

A Combination of a GnRH Antagonist and Agonist for Fertility Preservation in an Adolescent Female Murine Model.

Recent studies have suggested that GnRH agonists (GnRHags) protect ovarian function following chemotherapy. Here, we study the effect of a combination of GnRH antagonist (GnRHan) and GnRHag for gonadal protection from gonadotoxic chemotherapy in adolescent female rats. Cycling Sprague Dawley rats were treated at adolescent age. Thirty female rats were randomized to 5 treatment groups (n = 6/group): (1) placebo, (2) cyclophosphamide (CPA) alone, (3) GnRHan followed by GnRHag with placebo, (4) GnRHan followed by GnRHag with CPA, and (5) GnRHag with CPA. The main outcome measure was live birth rate (LBR), and secondary measures included rat weight, ovarian volume, and follicles. Group 2 had decreased LBR compared to all other groups. Group 4 and 5 had LBR similar to placebo. There was no difference in the ovarian volume. The CPA-alone group had decreased number of antral follicles compared to control. These studies demonstrate that the combination of GnRHan and GnRHag and GnRHag alone preserved fertility in female adolescent rats following gonadotoxic chemotherapy treatment. The addition of a GnRHan to a GnRHag does not confer a greater protective effect.

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.

Sperm deoxyribonucleic acid fragmentation assessment in normozoospermic male partners of couples with unexplained recurrent pregnancy loss: a prospective study.

OBJECTIVE: To determine whether sperm DNA integrity in normozoospermic male partners plays a role in idiopathic recurrent pregnancy loss (RPL). DESIGN: Prospective, cohort study. SETTING: Academic tertiary care center. PATIENT(S): Group I: 26 male partners of women with unexplained RPL. Group II: 31 normozoospermic males with proven fertility. INTERVENTION(S): Semen samples were collected by masturbation after 48-72 hours of abstinence. After liquefaction at room temperature, semen analysis was performed according to World Health Organization standards. Only samples with >20 × 10(6) spermatozoa/mL with at least 50% progressive sperm motility and 30 % normal morphology were selected for the study. DNA fragmentation of the sperm was assessed with TUNEL assay followed by flow cytometric analysis. MAIN OUTCOME MEASURE(S): Sperm DNA fragmentation in both groups. RESULT(S): Mean DNA fragmentation (mean ± SD) was significantly more in men with RPL (36.8 ± 5) compared with controls (9.4 ± 2.7). CONCLUSION(S): Sperm DNA fragmentation may play a role in unexplained RPL despite normal semen analysis parameters.

Impaired Development of Early Endometriotic Lesions in CD44 Knockout Mice.

Previous studies have shown endometrial cell (EC) CD44 and peritoneal mesothelial cell (PMC)-associated hyaluronan (hyaluronic acid [HA]) are involved in the attachment of endometrial stroma and epithelial cells to peritoneal mesothelium. Here we assess the CD44-HA interaction in the formation of the early endometriotic lesion using CD44(-/-) (knockout) mice. Using an established murine model and crossover technique, endometrial tissue from donor mice (wild type [WT] and CD44(-/-)) was used to induce endometriosis in recipient mice (WT and CD44(-/-)). Endometriotic lesions were visualized by fluorescent microscopy and confirmed by hematoxylin and eosin staining. Early endometriotic lesions were decreased when CD44(-/-) endometrium was placed in WT recipients and when WT endometrium was placed in CD44(-/-) recipients (P = .002). Early endometriotic lesions were also significantly decreased when both peritoneal and endometrial tissues lacked CD44 expression (P < .01). These studies demonstrate that both EC and PMC CD44 play a role in the development of early endometriotic lesion.

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.

Raf-1, a potential therapeutic target, mediates early steps in endometriosis lesion development by endometrial epithelial and stromal cells.

Endometriosis is a hormone-sensitive gynecological disorder characterized by the benign growth of endometrial-like tissue in the pelvic cavity. Endometriotic lesions composed of endometrial stromal cells (ESC) and glandular epithelial cells (EEC) are thought to arise from menstrual endometrial tissue reaching the pelvic cavity via retrograde menstruation. The cause of endometriotic lesion formation is still not clear. Recent evidence suggest that cytokines may play a role in the early development of endometriosis lesions. Because cytokines and growth factors signal via the v-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase pathway, we have examined the role of Raf-1 in early steps of endometriosis lesion formation, specifically attachment of endometrial cells to peritoneal mesothelial cells (PMC) and invasion of endometrial cells through PMC (trans-mesothelial invasion). Raf-1 antagonist GW5074 decreased attachment to PMC and trans-mesothelial invasion by primary EEC and ESC. Raf-1 also mediated TGFß-induced trans-mesothelial invasion by the established, low-invasive EEC line EM42. TGFß treatment of EEC resulted in Raf-1 phosphorylation at S338 and phosphorylation of ERK, suggesting that TGFß activates Raf-1 signaling in these cells. GW5074 had little effect on ESC proliferation but inhibited EEC growth significantly under reduced serum conditions. Antagonizing Raf-1 activity and expression via GW5074 and specific Raf-1 small interfering RNA, respectively, did not alter EEC resistance to growth inhibition by TGFß. Raf-1 inhibition blocked induction of EEC growth by epidermal growth factor. Our data suggest that Raf-1 may mediate pathologic steps involved in early endometriosis lesion formation and may be a mediator of TGFß and epidermal growth factor actions in endometriosis.

Chlamydia trachomatis antigens recognized in women with tubal factor infertility, normal fertility, and acute infection.

OBJECTIVE: To identify Chlamydia trachomatis antigens associated with tubal factor infertility and acute infection. METHODS: A C trachomatis proteome array was used to compare antibody profiles among women with tubal factor infertility, normal fertility, and acute C trachomatis infection. RESULTS: Thirteen immunodominant antigens reacted with 50% or more sera from all women (n=73). Six C trachomatis antigens were uniquely recognized in women with tubal factor infertility. Combining fragmentation of the six antigens with serum sample dilution, chlamydial antigens HSP60, CT376, CT557, and CT443 could discriminate between women with tubal factor infertility and women with normal fertility with a sensitivity of 63% (95% confidence interval [CI] 0.41-0.77) and specificity of 100% (95% CI 0.91-1), respectively. These antigens were designated as tubal factor infertility-associated antigens. However, these tubal factor antigens were unable to distinguish tubal factor infertility patients from those with acute infection. A combination of CT875 and CT147 distinguished women with acute infection from all other C trachomatis-exposed women with a detection sensitivity of 63% (95% CI 0.41-0.77) and specificity of 100% (95% CI 0.95-1), respectively. Thus, CT875 and CT147 were designated as acute infection-associated antigens. CONCLUSION: A sequential screening of antibodies against panels of C trachomatis antigens can be used to identify women with tubal factor infertility and acute C trachomatis infection. LEVEL OF EVIDENCE: II.

Increased expression of macrophage colony-stimulating factor and its receptor in patients with endometriosis.

OBJECTIVE: To investigate the expression and regulation of colony-stimulating factor 1 (CSF-1) and its receptor, C-FMS, in endometriosis. DESIGN: In vivo and vitro study. SETTING: University-based academic medical center. PATIENT(S): Reproductive-age women undergoing surgery for benign conditions. INTERVENTION(S): Peritoneal and endometrial tissue samples were obtained. MAIN OUTCOME MEASURE(S): CSF-1 and C-FMS expression. RESULT(S): Significantly higher CSF-1 levels were found in peritoneal fluid of patients with endometriosis compared with control subjects. Ectopic endometriotic tissue had 3.5-fold and 1.7-fold increases in CSF-1 and C-FMS expression, respectively, compared with eutopic tissue. Coculture of endometrial cells from either established cell lines or patient samples with peritoneal mesothelial cells (PMCs) led to increased expression of CSF-1 and C-FMS. A higher but nonsignificant increase in levels of C-FMS and CSF-1 was found in cocultures of endometrial epithelial cells from patients with endometriosis compared with those without endometriosis. CONCLUSION(S): Increased CSF-1 levels may contribute to endometriosis lesion formation and progression. Elevation in CSF-1 after coculture of endometrial cells with PMCs suggests that endometrial tissue may be a source of peritoneal CSF-1. Increased C-FMS expression in endometrial cells from women with endometriosis cocultured with PMCs suggests that endometrial tissue involved in lesion formation is highly responsive to CSF-1 signaling.

Serum beta human chorionic gonadotropin levels can inform outcome counseling after in vitro fertilization.

The predictive value of serum beta hCG level for fetal cardiac motion and pregnancy outcome after IVF was evaluated. The serum hCG level 12 days after ET is a useful predictor of subsequent presence of fetal cardiac activity and live birth and may assist clinicians in counseling patients regarding their IVF outcome.

Genome-wide identification of Chlamydia trachomatis antigens associated with tubal factor infertility.

OBJECTIVE: To identify Chlamydia trachomatis antigens that can be used to differentially diagnose tubal factor infertility in comparison with previously reported heat shock protein 60. DESIGN: In vitro study. SETTING: Academic medical center. PATIENT(S): Infertile women with and without tubal pathology diagnosed laparoscopically. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Antibody responses to C. trachomatis in infertile women with or without tubal pathologies using a C. trachomatis genome-wide proteome array. RESULT(S): Comparison of the antibody profiles revealed 30 C. trachomatis antigens that were preferentially recognized in women with tubal factor infertility, with a detection sensitivity and specificity of 80.6% and 56.5%, respectively, 10 of which showed 100% specificity. A combination of CT443 and CT381 antigens yielded the highest detection sensitivity (67.7%) while maintaining 100% specificity. CONCLUSION(S): These findings have demonstrated that antibodies to CT443 and CT381, when used in combination, have higher sensitivity and specificity in predicting tubal factor infertility than other indicators for tubal factor infertility, such as heat shock protein 60 antibodies (35.5%, 100%) or hysterosalpingogram (65%, 83%). Using a panel of C. trachomatis antigens to serologically diagnose tubal factor infertility can save the patients from undertaking expensive and invasive procedures for determining tubal pathology and choosing treatment plans.

In vitro fertilization outcomes in Hispanics versus non-Hispanic whites.

The in vitro fertilization (IVF) outcomes, including clinical intrauterine gestation rate and live birth rate, between Hispanic and non-Hispanic white women were compared, and there were no differences. Hispanics were more likely to have a diagnosis of tubal factor infertility, whereas non-Hispanic white women were more likely to have endometriosis as their infertility diagnosis.

Colony-stimulating factor-1 exerts direct effects on the proliferation and invasiveness of endometrial epithelial cells.

Although macrophage colony-stimulating factor (CSF-1) has been suggested to play a role in maintaining the chronic inflammatory response in endometriosis, our data suggest that CSF-1 may also play a role in early endometriosis lesion formation. We have shown that CSF-1, in an autocrine fashion, has a direct effect on endometrial epithelial cell proliferation and attachment to peritoneal mesothelial cells, early steps in endometriosis lesion formation on the peritoneum.

Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.