M. Payson

Reduced Dermatopontin Expression Is a Molecular Link Between Uterine Leiomyomas and Keloids

Uterine leiomyomas are prevalent estrogen‐responsive clonal tumors, but the specific genetic alterations that contribute to their development have not been elucidated. To identify genes involved in the formation of leiomyomas, we used global expression profiling to compare clonal tumors with normal myometrium. Contrary to expectation, genes involved in estrogen action were not differentially expressed between leiomyoma and normal myometrium. Genes encoding extracellular‐matrix proteins were prominently featured, suggesting their involvement in formation of a myofibroblast phenotype. Analysis of the extracellular matrix in the leiomyomas revealed a disordered collagen fibril orientation. Expression of the collagen‐binding protein dermatopontin was found to be consistently decreased in leiomyoma by both reverse transcriptase–polymerase chain reaction (RT‐PCR) and real‐time RT‐PCR (mean underexpression = 9.41‐fold) regardless of leiomyoma size, leiomyoma location, patient race, and patient age. This expression pattern was observed in 11 subjects and a total of 23 leiomyoma:myometrium pairs. Decreased expression of dermatopontin was also associated with keloid formation, a fibrotic disease that shares epidemiologic similarities with leiomyoma. Immunohistochemical studies of leiomyomas and keloids demonstrated reduced levels of dermatopontin in both tissues. In addition, ultrastructural analysis revealed that the orientation of the collagen fibrils in the keloid tissues strongly resembled that in the leiomyomas. Reduction in dermatopontin was associated with an increase in transforming growth factor–β3 (TGFB3) mRNA levels in leiomyomas, whereas other genes involved in dermatopontin signaling were not differentially expressed. These findings suggest that leiomyoma development involves a myofibroblast cell phenotype characterized by dysregulation of genes encoding extracellular‐matrix proteins. In particular, decreased expression of dermatopontin represents a molecular link between the leiomyoma and keloid phenotypes.

Strategy for elucidating differentially expressed genes in leiomyomata identified by microarray technology

OBJECTIVE cDNA microarray technology identifies genes that are differentially expressed between tissues. Our previous study identified several genes that might contribute to the fibroid phenotype. We therefore sought to confirm genes involved in three distinct signal transduction pathways. DESIGN Evaluation of differential mRNA and protein expression of Dlk, Frizzled-2, and CD-24 in fibroids compared with adjacent myometrium. University hospital. PATIENT(S) Five women undergoing medically indicated hysterectomy for symptomatic fibroids. INTERVENTION(S) Microarray analysis of up to 33000 genes, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time RT-PCR, Western blot, and immunohistochemistry. MAIN OUTCOME MEASURE(S) Expression of mRNA transcripts and protein in fibroid compared with myometrium.A more extensive microarray confirmed differential expression of Frizzled-2 and CD-24 but did not confirm Dlk overexpression. RT-PCR and real-time PCR demonstrated equivalent Dlk mRNA expression between fibroid and myometrium (ratio, 1.02), a slight Frizzled-2 overexpression (ratio, 2.09), and robust CD-24 overexpression in fibroids (ratio, 12.35). Western blot and immunohistochemistry confirmed Frizzled-2 overexpression, but did not confirm Dlk overexpression. CONCLUSION(S) Microarray technology is the first phase of tissue evaluation, but changes in gene expression must be confirmed. Confirmed genes can then be used to generate hypotheses testing their involvement in fibroid development.

The pathophysiology of ovarian hyperstimulation syndrome: an unrecognized compartment syndrome

OBJECTIVE To compare and contrast the pathophysiology of ovarian hyperstimualtion syndrome (OHSS) with known syndromes of increased intraabdominal pressure (IAP), and to explore the relationship of increased IAP with symptom severity in OHSS. DESIGN Literature review. MAIN OUTCOME MEASURE(S) Correlation of OHSS symptoms with IAP; effects of paracentesis on IAP in patients with OHSS. SETTING Academic Research Institution. INTERVENTION(S) None. RESULT(S) OHSS involves a rapid accumulation of volume (from 1.5-17 liters) in the peritoneal cavity that can lead to organ dysfunction, including respiratory impairment and oliguria. In published reports of 20 moderate-to-severe OHSS patients in whom IAP was measured, IAP was found to be elevated to a pathologic range. The increased IAP indicates that OHSS may be considered a compartment syndrome and meets criteria for abdominal compartment syndrome in advanced cases. For this reason, management of OHSS should include reduction of pressure by paracentesis to avoid morbidity and syndrome progression. In addition, measurement of IAP may help to classify the stage of OHSS. CONCLUSION(S) IAP was found to be elevated in the few cases of OHSS in which it was measured, substantiating the conclusion that OHSS may be considered a compartment syndrome. An understanding of the pathophysiology of increased intrabdominal pressure is useful in the management of OHSS.

Curcumin, a nutritional supplement with antineoplastic activity, enhances leiomyoma cell apoptosis and decreases fibronectin expression.

OBJECTIVE To determine if curcumin has an antiproliferative effect on leiomyoma cells via apoptosis induction and whether curcumin impacts extracellular matrix (ECM) production by assessing the fibronectin expression in leiomyoma cells treated with curcumin. DESIGN Tissue culture study of immortalized human leiomyoma and patient-matched myometrial cells treated with curcumin. SETTING University hospital. PATIENT(S) Immortalized leiomyoma and myometrial cells from patients with symptomatic leiomyomata. INTERVENTION(S) Tissue culture, followed by proliferation studies, RNA, and protein analysis. MAIN OUTCOME MEASURE(S) Cell proliferation, alteration in apoptotic signaling pathways. RESULT(S) Curcumin demonstrated an antiproliferative effect on leiomyoma cell lines (IC50 = 20 muM). Importantly, no statistically significant inhibition of growth was observed when patient-matched myometrial cells were exposed to equivalent concentrations of curcumin. Curcumin stimulated caspase-3 and caspase-9 expression while inhibiting extracellular signal-regulated kinase 1 (ERK 1), ERK 2, and nuclear factor kappa B (NF-kappaB), suggesting regulation of leiomyocyte apoptosis. Finally, curcumin inhibited expression of fibronectin in leiomyoma cells. CONCLUSION(S) Our findings demonstrate that curcumin inhibited uterine leiomyoma cell proliferation via regulation of the apoptotic pathway, and inhibited production of the ECM component fibronectin. Curcumin provides a novel direction for leiomyoma therapies.

Utilization and success rates of unstimulated in vitro fertilization in the United States: an analysis of the Society for Assisted Reproductive Technology database

OBJECTIVE To examine the utilization and outcomes of natural cycle (unstimulated) IVF as reported to the Society of Assisted Reproductive Technology (SART) in 2006 and 2007. DESIGN Retrospective analysis. SETTING Dataset analysis from the SART Clinical Outcome Reporting System national database. PATIENT(S) All patients undergoing IVF as reported to SART in 2006 and 2007. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Utilization of unstimulated IVF; description of patient demographics; and comparison of implantation and pregnancy rates between unstimulated and stimulated IVF cycles. RESULT(S) During 2006 and 2007 a total of 795 unstimulated IVF cycles were initiated. Success rates were age dependent, with patients <35 years of age demonstrating clinical pregnancy rates per cycle start, retrieval, and transfer of 19.2%, 26.8%, and 35.9%, respectively. Implantation rates were statistically higher for unstimulated compared with stimulated IVF in patients who were 35 to 42 years old. CONCLUSION(S) Unstimulated IVF represents <1% of the total IVF cycles initiated in the United States. The pregnancy and live birth rates per initiated cycle were 19.2% and 15.2%, respectively, in patients <35 years old. The implantation rates in unstimulated IVF cycles compared favorably to stimulated IVF. Natural cycle IVF may be considered in a wide range of patients as an alternative therapy for the infertile couple.

Ovarian follicular flushing among low-responding patients undergoing assisted reproductive technology

A randomized comparison trial was performed to evaluate whether follicular reaspiration with use of a double-lumen retrieval needle improves oocyte recovery when compared with direct follicular aspiration among low-responding patients undergoing ART. There were no differences observed in the number of oocytes retrieved (single lumen: 6.5 +/- 2.2 oocytes, double lumen: 7.2 +/- 2.3 oocytes) whereas follicular reaspiration with the double-lumen retrieval needle resulted in a twofold increase in procedure time.

GnRH antagonist rescue in high responders at risk for OHSS results in excellent assisted reproduction outcomes

Gonadotrophin-releasing hormone (GnRH) antagonist rescue is performed by replacing a GnRH agonist with a GnRH antagonist in patients with rapidly rising serum oestradiol who are at risk of ovarian hyperstimulation syndrome (OHSS) during stimulation. It results in a rapid reduction in serum oestradiol, allowing for the avoidance of cycle cancellation and the continuation of exogenous gonadotrophin administration. A total of 387 patients who underwent GnRH antagonist rescue for ovarian hyperresponse were compared with 271 patients who did not receive GnRH antagonist rescue and had oestradiol concentrations >4000 pg/ml on the day of human chorionic gonadotrophin (HCG) administration. GnRH antagonist rescue decreased the mean oestradiol concentration by 35% on the first day of use. There was no difference in oocyte maturity (82% versus 83%) or fertilization rate (69% versus 67%) between the antagonist rescue and comparison groups, respectively. The percentage of high-grade embryos on day 3 and the blastocyst development rate were also similar between groups. The live-birth rate was 41.9% in the antagonist rescue group and 36.9% in the comparison group. GnRH antagonist rescue enabled cycle completion with high live-birth rates in patients at risk for OHSS. GnRH antagonist was associated with high oocyte quality, blastocyst development and pregnancy. Gonadotrophin-releasing hormone (GnRH) antagonist rescue is a protocol to reduce the risk of ovarian hyperstimulation syndrome (OHSS) in assisted reproduction treatment. Patients who have a hyperresponse to medication during their treatment cycle have their GnRH agonist discontinued and a GnRH antagonist started in its place. This causes a rapid reduction in oestrogen concentrations and allows for the continuation of stimulation medication. We evaluated the effectiveness of this protocol by comparing patients who had GnRH antagonist rescue against high-responding patients who did not receive GnRH antagonist rescue. GnRH antagonist rescue resulted in a 35% reduction in oestrogen concentration and only a 1.5% cycle cancellation rate. There were no differences in oocyte maturity or fertilization between the two groups. There were no differences in the quality of day-3 and day-5 embryos between the two groups. The live birth rate was 41.9% in the antagonist rescue group and 36.9% in the comparison group. GnRH antagonist rescue reduced serum oestradiol concentrations and enabled cycle completion with high live-birth rates in patients at risk for OHSS. GnRH antagonist was associated with high oocyte quality, blastocyst development and pregnancy.

Are there ethnic differences in pregnancy rates in African-American versus white women undergoing frozen blastocyst transfers?

OBJECTIVE To determine whether frozen-thawed blastocyst transfer pregnancy rates (PR) are lower in African-American compared with white women. DESIGN Retrospective review of frozen blastocyst cycles. SETTING University-based assisted reproductive technology (ART) program. PATIENT(S) All patients who underwent a frozen blastocyst transfer between 2003 and 2008. INTERVENTION None. MAIN OUTCOME MEASURE(S) Live birth rate. RESULT(S) One hundred sixty-nine patients underwent transfer of a frozen-thawed blastocyst. African-American women had a higher incidence of leiomyoma (40% vs. 10%) and tubal and uterine factor infertility. There was no difference in the live birth rate for African-American patients (28.0%) compared with white patients (30.2%). Of the patients who underwent a frozen-thawed blastocyst transfer, 58% (n=98) had their fresh, autologous IVF cycle, which produced the cryopreserved blastocyst, at Walter Reed Medical Center. A higher peak serum E2 level was noted in African-American patients (5,355 pg/mL) compared with white patients (4,541 pg/mL). During the fresh cycle, the live birth rates between African-American and white patients were significantly different at 16.7% versus 39.7%, respectively. CONCLUSION(S) Live birth rates after frozen blastocyst transfer are not different between African-American and white women despite a fourfold higher incidence of leiomyomas in African-American women.

Activating transcription factor 3 gene expression suggests that tissue stress plays a role in leiomyoma development.

OBJECTIVE To determine whether expression of the stress response gene ATF3 and related members of activator protein complex-1, cJun and cFos, were altered in leiomyoma compared with myometrium, and whether this difference might correlate with leiomyoma size or race. DESIGN Laboratory study. SETTING University hospital. PATIENT(S) Fifteen women undergoing hysterectomy for symptomatic leiomyoma. INTERVENTION(S) Tissue procurement, RNA isolation, reverse-transcriptase polymerase chain reaction, real-time reverse-transcriptase polymerase (RT-PCR) chain reaction, immunohistochemistry, Western blot. MAIN OUTCOME MEASURE(S) Expression of mRNA and protein in leiomyoma and patient-matched myometrium. RESULT(S) mRNA transcripts of ATF3 were decreased in leiomyoma compared with matched myometrium by both RT-PCR and real-time RT-PCR. The decrease was greater than fivefold in a majority of samples. The reduction seen in ATF3 mRNA expression did not show a correlation with race and leiomyoma size. Surprisingly, immunohistochemistry and Western blot analysis demonstrated an elevation of ATF3 protein expression by a mean of 2.9-fold. Transcripts of related AP-1 genes, cJun and cFos, were significantly decreased by a mean of -29.57 for cJun and -23.78 for cFos, but there was no significant change in protein expression of the two transcription factors. CONCLUSIONS Alterations in ATF3 gene expression resemble the response to mechanical and ischemic stress reported in other tissues. Results suggested that ATF3 protein expression was increased in leiomyoma, and may reflect increased tissue stress.

Balloon fluoroscopy as treatment for intrauterine adhesions: a novel approach

OBJECTIVE To report a unique fluoroscopically guided approach to treat severe intrauterine adhesions and cervical stenosis using balloon hysteroplasty. DESIGN Case report. SETTING Military-based fertility center. PATIENT(S) A 33-year-old woman undergoing assisted reproductive technology whose uterus could not be cannulated because of the development of intrauterine synechiae and cervical stenosis after a post-IUI infection that was further complicated by a prominent lower uterine segment-filling defect in the location of a prior cesarean delivery scar. INTERVENTION(S) Fluoroscopic cannulation and balloon uterine dilation. MAIN OUTCOME MEASURE(S) Resolution of synechiae by hysterosalpingogram and successful uterine cannulation. RESULT(S) A postprocedure hysterosalpingogram demonstrated a normalized uterine cavity with the exception of a persistent prominent lower uterine segment-filling defect from a prior cesarean delivery. A frozen ET cycle was performed successfully. CONCLUSION(S) Hysteroplasty, using standard interventional radiographic techniques, may provide an alternative treatment modality for patients with intrauterine adhesions and lower uterine defects from prior cesarean deliveries in select cases. While treating intrauterine adhesions improves pregnancy outcome, the effect of lower uterine segment-filling defects from cesarean deliveries on pregnancy outcome in assisted reproductive technology cycles warrants further investigation.