Magdy P. Milad

Promoter Methylation Regulates Estrogen Receptor 2 in Human Endometrium and Endometriosis

Abstract Steroid receptors in the stromal cells of endometrium and its disease counterpart tissue endometriosis play critical physiologic roles. We found that mRNA and protein levels of estrogen receptor 2 (ESR2) were strikingly higher, whereas levels of estrogen receptor 1 (ESR1), total progesterone receptor (PGR), and progesterone receptor B (PGR B) were significantly lower in endometriotic versus endometrial stromal cells. Because ESR2 displayed the most striking levels of differential expression between endometriotic and endometrial cells, and the mechanisms for this difference are unknown, we tested the hypothesis that alteration in DNA methylation is a mechanism responsible for severely increased ESR2 mRNA levels in endometriotic cells. We identified a CpG island occupying the promoter region (−197/+359) of the ESR2 gene. Bisulfite sequencing of this region showed significantly higher methylation in primary endometrial cells (n = 8 subjects) versus endometriotic cells (n = 8 subjects). The demethylating agent 5-aza-2′-deoxycytidine significantly increased ESR2 mRNA levels in endometrial cells. Mechanistically, we employed serial deletion mutants of the ESR2 promoter fused to the luciferase reporter gene and transiently transfected into both endometriotic and endometrial cells. We demonstrated that the critical region (−197/+372) that confers promoter activity also bears the CpG island, and the activity of the ESR2 promoter was strongly inactivated by in vitro methylation. Taken together, methylation of a CpG island at the ESR2 promoter region is a primary mechanism responsible for differential expression of ESR2 in endometriosis and endometrium. These findings may be applied to a number of areas ranging from diagnosis to the treatment of endometriosis.

Estrogen Receptor (ER) β Regulates ERα Expression in Stromal Cells Derived from Ovarian Endometriosis

CONTEXT Estradiol and its nuclear receptors, estrogen receptor (ER) alpha and ERbeta, play critical roles in endometrium and endometriosis. Levels of ERbeta, due to pathological hypomethylation of its promoter, are significantly higher in endometriotic vs. endometrial tissue and stromal cells, whereas ERalpha levels are lower in endometriosis. Estradiol regulates ERalpha gene expression via its alternatively used promoters A, B, and C. OBJECTIVE The aim of the study was to determine whether high levels of ERbeta in endometriotic stromal cells from ovarian endometriomas regulate ERalpha gene expression. RESULTS ERbeta knockdown significantly increased ERalpha mRNA and protein levels in endometriotic stromal cells. Conversely, ERbeta overexpression in endometrial stromal cells decreased ERalpha mRNA and protein levels. ERbeta knockdown significantly decreased proliferation of endometriotic stromal cells. Chromatin immunoprecipitation assays demonstrated that estradiol enhanced ERbeta binding to nonclassical activator protein 1 and specificity protein 1 motifs in the ERalpha gene promoters A and C and a classic estrogen response element in promoter B in endometriotic stromal cells. CONCLUSIONS High levels of ERbeta suppress ERalpha expression and response to estradiol in endometrial and endometriotic stromal cells via binding to classic and nonclassic DNA motifs in alternatively used ERalpha promoters. ERbeta also regulates cell cycle progression and might contribute to proliferation of endometriotic stromal cells. We speculate that a significantly increased ratio of ERbeta:ERalpha in endometriotic tissues may also suppress progesterone receptor expression and contribute to progesterone resistance. Thus, ERbeta may serve as a significant therapeutic target for endometriosis.

Limitations of the pelvic examination for evaluation of the female pelvic organs

To assess factors influencing an accurate pelvic examination under the best possible circumstances.

Risk factors for conversion to laparotomy during gynecologic laparoscopy.

STUDY OBJECTIVE To identify risk factors and short-term outcomes associated with conversion from laparoscopic surgery to laparotomy. DESIGN Case control study (Canadian Task Force classification II-2). SETTING Large urban, academic medical center. SUBJECTS All 2530 gynecologic laparoscopies, including those converted to laparotomy. INTERVENTION Laparoscopic surgery. MEASUREMENTS AND MAIN RESULTS Statistical analysis was performed to compare risk factors in converted versus non-converted cases. Multiple logistic regression analysis was performed using variables found to be significant on univariate analysis. Of 2530 laparoscopies identified, 159 (6.3%) were converted to laparotomy; 323 were selected by random number assignment from the remaining cases for the control group. The following factors were significantly associated with unintended laparotomy (OR; 95% CI): surgeon inexperience (2.43; 1.38, 4.17), level of laparoscopic complexity (3.19; 1.20, 5.10), body mass index greater than 30 kg/m(2) (2.45; 1.40, 4.41), suspicion of malignancy (17.45; 7.32, 43.57), history of laparotomy (1.34; 1.02, 1.78), presence of adhesions (2.30; 1.37, 3.76), and intraoperative technical difficulty (17.86; 7.32, 43.57). When stratified by level of complexity, experience in laparoscopy does not appear to confer protection during level 1 laparoscopic procedures (0.42; 0.12, 1.37) but does significantly reduce the frequency of conversion during level 2 procedures (0.39; 0.72, 0.93). Age, parity, bowel injury, vascular injury, and history of pelvic inflammatory disease and endometriosis did not increase the risk of conversion. Compared with controls, patients who were converted experienced significantly greater blood loss (p < 0.001), longer operating room time (p < 0.001), and longer hospital stay (p < 0.001). CONCLUSION All women undergoing laparoscopy should be counseled that unintended laparotomy is a known risk and has additional morbidity over laparoscopy alone. Less-experienced surgeons attempting complicated procedures significantly increase the risk of conversion.

A comparison of laparoscopic supracervical hysterectomy vs laparoscopically assisted vaginal hysterectomy.

BackgroundWe set out to compare the length of stay, costs, and morbidity associated with laparoscopic supracervical hysterectomy (LSH) with laparoscopically assisted vaginal hysterectomy (LAVH).MethodsWe performed a cohort analysis of consecutive patients at a university-based medical center from April 1997 through October 1999.ResultsA total of 145 patients were identified initially; however, 13 cases were excluded because of concomitant procedures (retropubic urethropexy, lymphadenectomy, paravaginal repair). Of the 132 patients included in the study, 27 underwent LSH and 105 underwent LAVH. The two groups were similar with respect to gravidity, parity, uterine weight, and preoperative diagnosis. Patients undergoing LSH had significantly shorter operating times (median, 181 vs 220 min, p = 0.007), briefer hospital stays (median, 1.0 vs 2.0 days, p = 0.0001), and less blood loss (median, 125 vs 400 ml, p = 0.0001). None of the patients submitted to LSH experienced morbidity, as compared with a 13% morbidity rate for LAVH (bladder injury, n = 3; blood loss >1000 ml, n = 7; vaginal cuff hematoma, n = 4; 0% vs 13%; p = 0.04).ConclusionsPatients undergoing laparoscopic supracervical hysterectomy had shorter operating times, shorter hospital stays, and less morbidity than those who underwent laparoscopically assisted vaginal hysterectomy. The practice of routine cervicectomy at laparoscopic hysterectomy should be reconsidered.

Relationship between peak serum estradiol levels and treatment outcome in in vitro fertilization cycles after embryo transfer on day 3 or day 5

OBJECTIVE To examine the relationships between peak serum estradiol (E(2)) levels and treatment outcome in in vitro fertilization (IVF) cycles after embryo transfer (ET) on day 3 or day 5. DESIGN Retrospective analysis of 697 IVF-ET cycles between January 1999 and December 2001. SETTING A university-affiliated assisted reproduction program. PATIENT(S) Infertile patients undergoing IVF-ET cycles. INTERVENTION(S) Peak E(2) concentration in serum was determined on the day of human chorionic gonadotropin (hCG) administration. The IVF-generated embryos were cultured for 2 days until transfer on day 3. If more than four 8-cell embryos were present on day 3, embryo culture was continued until day 5 for blastocyst transfer. MAIN OUTCOME MEASURE(S) Clinical pregnancy rates. RESULT(S) High peak E(2) levels did not adversely affect treatment outcome. After the cycles were divided according to the day of ET, high peak E(2) levels were associated with improved pregnancy rates after ET on day 5 but not on day 3. CONCLUSION(S) Increasing peak E(2) levels in IVF cycles are associated with improved pregnancy rates after ET on day 5.

Fertility preservation for social indications: a cost-based decision analysis

OBJECTIVE Age-related infertility remains a problem that assisted reproductive techniques (ART) have limited ability to overcome. Correspondingly, because an increasing number of women are choosing to delay childbearing, fertility preservation strategies, initially intended for patients undergoing gonadotoxic therapies, are being applied to this group of healthy women. Studies supporting the effectiveness of this practice are lacking. DESIGN Decision analytic techniques. SETTING We compared the cost-effectiveness of three strategies for women planning delayed childbearing until age 40: oocyte cryopreservation at age 25, ovarian tissue cryopreservation (OTC) at age 25, and no assisted reproduction until spontaneous conception had been attempted. PATIENT(S) Not applicable. INTERVENTION(S) Not applicable. MAIN OUTCOME MEASURE(S) Cost-effectiveness, which was defined as the cost per live birth. RESULT(S) In this analysis, the strategy of foregoing fertility preservation at age 25 and then choosing ART only after not spontaneously conceiving at age 40 was the most cost-effective option. OTC was dominated by the other strategies. Sensitivity analyses demonstrated the robustness of the model; no analysis existed in which OTC was not dominated by oocyte cryopreservation. Increasing the cost of an IVF cycle beyond

Steroidogenic factor-1 and endometriosis

22,000 was the only situation in which oocyte cryopreservation was the most preferred strategy. CONCLUSION(S) Neither oocyte cryopreservation nor OTC appear to be cost-effective under current circumstances for otherwise healthy women planning delayed childbearing. This analysis should give pause to the current practice of offering fertility preservation based only on the desire for delayed childbearing.

Infertility patients and their partners: Differences in the desire for twin gestations

Endometriosis is a common and chronic disease characterized by persistent pelvic pain and infertility. Estradiol is essential for growth and inflammation in endometriotic tissue. The complete cascade of steroidogenic proteins/enzymes including aromatase is present in endometriosis leading to de novo estradiol synthesis. PGE(2) induces the expression of the genes that encode these enzymes. Upon PGE(2) treatment, coordinate recruitment of the nuclear receptor SF-1 to the promoters of these steroidogenic genes is the key event for estradiol synthesis. SF-1 is the key factor determining that an endometriotic cell will respond to PGE(2) by increased estradiol formation. The presence of SF-1 in endometriosis and its absence in endometrium is determined primarily by the methylation of its promoter. The key steroidogenic enzyme in endometriosis is aromatase encoded by a single gene because its inhibition blocks all estradiol biosynthesis. Aromatase inhibitors diminish endometriotic implants and associated pain refractory to existing treatments in affected women.

Long-term outcome of nonconservative surgery (hysterectomy) for endometriosis-associated pain in women <30 years old.

OBJECTIVE To explore how the individuals within an infertile couple differ from one another in their attitudes toward the conception of twin gestations. METHODS From November 1999 through February 2000, consecutive couples undergoing treatment at a university-based infertility clinic were invited to participate in a face-to-face interview that ascertained their individual desires for singleton and twin gestations and their perception of the risks associated with these pregnancies. RESULTS Of the 94 couples approached, 90 (96%) agreed to participate in the study. Patients estimated the incidence of prematurity, low birth weight, preeclampsia, and postpartum depression in twin gestations to be significantly greater than their partners did (27% versus 17%, 26% versus 17%, 22% versus 16%, and 21% versus 16%, respectively). Despite these higher estimates, the desire for a twin gestation was similar. In 19 couples (21%), the patient and partner did not agree whether a singleton or twin pregnancy was the most preferred outcome. No predictive factor for this discordance could be identified. CONCLUSION Women undergoing infertility treatment are less risk averse than their partners. Additionally, a sizeable portion of couples do not align in their preference for a twin gestation. These differences should be recognized and addressed during the preconceptional period.