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.

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.

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.

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.

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.

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.

Estrogen plus progestin and risk of benign proliferative breast disease.

Women with benign proliferative breast disease are at increased risk of subsequent breast cancer. Estrogens and progesterone exert proliferative effects on mammary epithelium, and combined hormone replacement therapy has been associated with increased breast cancer risk. We tested the effect of conjugated equine estrogen plus progestin on the risk of benign proliferative breast disease in the Women's Health Initiative (WHI) randomized controlled trial. In the WHI trial of estrogen plus progestin, 16,608 postmenopausal women were randomly assigned either to 0.625 mg/day of conjugated equine estrogen plus 2.5 mg/day of medroxyprogesterone acetate or to placebo. Baseline and annual breast exams and mammograms were required. The trial was terminated early (average follow-up, 5.5 years). We identified women who had had a biopsy for benign breast disease, and subjected histologic sections from the biopsies to standardized review. Overall, 178 incident cases of benign proliferative breast disease were ascertained in the estrogen plus progestin group and 99 in the placebo group. The use of estrogen plus progestin was associated with a 74% increase in the risk of benign proliferative breast disease [hazard ratio, 1.74; 95% confidence interval (CI), 1.35-2.25]. For benign proliferative breast disease without atypia the hazard ratio was 2.00 (95% CI, 1.50-2.66), while for atypical hyperplasia it was 0.76 (95% CI, 0.38-1.52). The risk varied little by levels of baseline characteristics. The results of this study suggest that the use of estrogen plus progestin may increase the risk of benign proliferative breast disease.

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.

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.

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.

Effects of conjugated equine estrogens on breast cancer and mammography screening in postmenopausal women with hysterectomy.

CONTEXT: The Women's Health Initiative Estrogen-Aone trial comparing conjugated equine estrogens (CEE) with placebo was stopped early because of an increased stroke incidence and no reduction in risk of coronary heart disease. Preliminary results suggesting possible reduction in breast cancers warranted more detailed analysis. OBJECTIVE: To determine the effects of CEE on breast cancers and mammographic findings. DESIGN, SETTING, AND PARTICIPANTS: Following breast cancer risk assessment, 10,739 postmenopausal women aged 50 to 79 years with prior hysterectomy were randomized to CEE or placebo at 40 US clinical centers from 1993 through 1998. Mammography screenings and clinical breast examinations were performed at baseline and annually. All breast cancers diagnosed through February 29, 2004, are included. INTERVENTION: A dose of 0.625 mg/d of CEE or an identical-appearing placebo. MAIN OUTCOME MEASURES: Breast cancer incidence, tumor characteristics, and mammogram findings. RESULTS: After a mean (SD) follow-up of 7.1 (1.6) years, the invasive breast cancer hazard ratio (HR) for women assigned to CEE vs placebo was 0.80 (95% confidence interval [CI], 0.62-1.04; P = .09) with annualized rates of 0.28% (104 cases in the CEE group) and 0.34% (133 cases in the placebo group). In exploratory analyses, ductal carcinomas (HR, 0.71; 95% CI, 0.52-0.99) were reduced in the CEE group vs placebo group; however, the test for interaction by tumor type was not significant (P = .054). At 1 year, 9.2% of women in the CEE group had mammograms with abnormalities requiring follow-up vs 5.5% in the placebo group (P<.001), a pattern that continued through the trial to reach a cumulative percentage of 36.2% vs 28.1%, respectively (P<.001); however, this difference was primarily in assessments requiring short interval follow-up. CONCLUSIONS: Treatment with CEE alone for 7.1 years does not increase breast cancer incidence in postmenopausal women with prior hysterectomy. However, treatment with CEE increases the frequency of mammography screening requiring short interval follow-up. Initiation of CEE should be based on consideration of the individual woman's potential risks and benefits. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00000611.

Should symptomatic menopausal women be offered hormone therapy?

UNLABELLED: Many physicians remain uncertain about prescribing hormone therapy for symptomatic women at the onset of menopause. The American Society for Reproductive Medicine (ASRM) convened a multidisciplinary group of healthcare providers to discuss the efficacy and risks of hormone therapy for symptomatic women, and to determine whether it would be appropriate to treat women at the onset of menopause who were complaining of menopausal symptoms. MAJOR FINDINGS: Numerous controlled clinical trials consistently demonstrate that hormone therapy, administered via oral, transdermal, or vaginal routes, is the most effective treatment for vasomotor symptoms. Topical vaginal formulations of hormone therapy should be preferred when prescribing solely for the treatment of symptoms of vulvar and vaginal atrophy. Data from the Women's Health Initiative indicate that the overall attributable risk of invasive breast cancer in women receiving estrogen plus progestin was 8 more cases per 10,000 women-years. No increased risk for invasive breast cancer was detected for women who never used hormone therapy in the past or for those receiving estrogen only. Hormone therapy is not effective for the treatment of cardiovascular disease and that the risk of cardiovascular disease with hormone therapy is principally in older women who are considerably postmenopause. CONCLUSIONS: Healthy symptomatic women should be offered the option of hormone therapy for menopausal symptoms. Symptom relief with hormone therapy for many younger women (at the onset of menopause) with menopausal symptoms outweighs the risks and may provide an overall improvement in quality of life. Hormone therapy should be individualized for symptomatic women. This involves tailoring the regimen and dose to individual needs.

Should symptomatic menopausal women be offered hormone therapy?

UNLABELLED: Many physicians remain uncertain about prescribing hormone therapy for symptomatic women at the onset of menopause. The American Society for Reproductive Medicine (ASRM) convened a multidisciplinary group of healthcare providers to discuss the efficacy and risks of hormone therapy for symptomatic women, and to determine whether it would be appropriate to treat women at the onset of menopause who were complaining of menopausal symptoms. MAJOR FINDINGS: Numerous controlled clinical trials consistently demonstrate that hormone therapy, administered via oral, transdermal, or vaginal routes, is the most effective treatment for vasomotor symptoms. Topical vaginal formulations of hormone therapy should be preferred when prescribing solely for the treatment of symptoms of vulvar and vaginal atrophy. Data from the Women's Health Initiative indicate that the overall attributable risk of invasive breast cancer in women receiving estrogen plus progestin was 8 more cases per 10,000 women-years. No increased risk for invasive breast cancer was detected for women who never used hormone therapy in the past or for those receiving estrogen only. Hormone therapy is not effective for the treatment of cardiovascular disease and that the risk of cardiovascular disease with hormone therapy is principally in older women who are considerably postmenopause. CONCLUSIONS: Healthy symptomatic women should be offered the option of hormone therapy for menopausal symptoms. Symptom relief with hormone therapy for many younger women (at the onset of menopause) with menopausal symptoms outweighs the risks and may provide an overall improvement in quality of life. Hormone therapy should be individualized for symptomatic women. This involves tailoring the regimen and dose to individual needs.

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.

An in vitro model to study the pathogenesis of the early endometriosis lesion.

OBJECTIVE: To determine if whole fragments of endometrium can adhere to peritoneum with intact mesothelium. DESIGN: Tissue culture and immunohistochemical study. SETTING: University Medical Center. PATIENTS: Reproductive-age women undergoing surgery for benign conditions. INTERVENTIONS: Whole explants of human peritoneum from the anterior abdominal wall and the posterior surface of the uterus were cultured with whole fragments of mechanically dispersed endometrium. MAIN OUTCOME MEASURES: Adhesion of endometrial fragments to the surface of the peritoneum was evaluated. Adherent fragments of endometrium were identified using the dissecting microscope and by performing serial sections of the peritoneum explants for light and confocal laser-scanning microscopy. Immunohistochemical staining of the mesothelium with antibodies to cytokeratin and vimentin was used to ensure an intact layer of mesothelium beneath the endometrial implants. Transmission electron microscopy was also used to evaluate the adhesion of endometrium to the mesothelium. RESULTS: Endometrium was identified attached to the surface of the peritoneum. After 18-24 hours of culture, the majority of implants did not have identifiable mesothelium beneath them, but most had intact mesothelium running up to the point of attachment. Approximately 10% of the endometrial implants had intact mesothelium at the site of attachment. After 1 hour of culture, both endometrial stromal and epithelial cells were attached to intact mesothelium in nearly all cases. Early transmesothelial invasion involves endometrial stromal cells. CONCLUSIONS: Endometrial stromal and epithelial cells can attach to the intact mesothelial surface of the peritoneum. Endometrial stromal cell invasion through the mesothelium occurs in less than 18-24 hours.

The alpha(2)beta(1) and alpha(3)beta(1) integrins do not mediate attachment of endometrial cells to peritoneal mesothelium.

OBJECTIVE: To evaluate the possible role of mesothelial alpha(2)beta(1) and alpha(3)beta(1) integrins in the attachment of endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs). DESIGN: In vitro study. SETTING: University medical center. PATIENT(S): Women of reproductive age (n = 26). MAIN OUTCOME MEASURE(S): Mesothelial cells were grown on collagen IV. Endometrial stromal cells and EECs were plated on mesothelial cells for 1 hour. Before plating, mesothelial cells or endometrial cells were incubated with antibodies to alpha2, alpha3, and beta1 integrin subunits. The effect of these antibodies on ESC and EEC binding to collagen IV and collagen I was also examined. The expression of collagen I, collagen IV, fibronectin, and laminin by cultured ESCs and EECs was examined. RESULT(S): The anti-integrin antibodies had no effect on endometrial binding to mesothelium. The beta1 integrin antibody decreased binding of ESCs and EECs to the collagen matrices. In culture, ESCs and EECs expressed collagen I, collagen IV, fibronectin, and laminin to varying degrees. CONCLUSION(S): The initial adhesion of ESCs and EECs to mesothelium is not mediated by beta1 integrins. In contrast, ESC and EEC attachment to collagen IV and collagen I, which are present in the submesothelial extracellular matrix, is mediated by beta1 integrins.