The impact of uterine leiomyomas on reproductive outcomes.

Uterine leiomyomas (fibroids, myomas) are a common benign disease of the uterus with a prevalence of 8-18%. Prevalence rates vary with race, and fibroids are most common in African American women. Uterine leiomyomas can also be present during pregnancy, which may occur more frequently than previously suspected, with prevalence rates reported of up to 10%. Recent evidence has emerged to clarify the relationship of uterine fibroids on fertility and obstetrical outcomes. In this paper we review evidence that uterine fibroids, specifically submucosal and intramural myomas, negatively impact fertility and are associated with adverse obstetrical outcomes such as: pain, preterm labor, placental abruption, malpresentation, postpartum hemorrhage, and cesarean section. Myomectomy performed for submucosal and intramural fibroids significantly improves fertility outcome, and current evidence suggests myomectomy is the treatment of choice in women desiring to conceive. For women that do not desire surgery, medical management of myomas is available. Treatment with GnRH agonists may be considered, however newer medications with fewer side effects give practitioners and patients more options. Progesterone antagonists, selective progesterone receptor modulators, and aromatase inhibitors have all shown promise as effective therapies. Non-pharmacologic treatments such as uterine artery embolization and MRI-guided ultrasound have also emerged as effective treatments for uterine fibroids. With such a wide range of new and emerging treatment options, it is important for providers to understand which fibroids are likely to respond optimally to a specific treatment, in order to individualize appropriate and effective management for patients.

The presence of blood in the transfer catheter negatively influences outcome at embryo transfer.

BACKGROUND: Embryo transfer (ET) influences pregnancy rates in patients undergoing assisted reproduction. Data are conflicting as to which variables affect ET success. This study examines variables that may affect outcome after ET in assisted reproductive technology patients who had high-quality embryos transferred. METHODS: Over a 23 month period, 669 consecutive cycles were examined. Only patients having grade I and grade II embryos, or blastocyst transfers, were included in this retrospective analysis. A total of 584 consecutive cycles met study criteria. At the time of ET, the following variables were recorded: aborted first attempt at ET; presence of blood and/or mucus in or on the transfer catheter after ET; ease of ET as judged by provider; need for mock embryo transfer immediately before the actual transfer and retention of embryos in the transfer catheter. These variables were retrospectively analysed for their impact on implantation rate (IR) and clinical pregnancy rate (CPR). RESULTS: There were 290 gestations (49.7% CPR). Multiple attempts at ET, subjective difficulty of ET, performance of a sham pass immediately prior to embryo transfer, and presence of mucus on or in the catheter did not affect the CPR or IR. No difference was noted in the mean age of patients having or lacking any of these factors. There was a significant association between the presence of blood on or in the catheter and decreased IR (P = 0.015) and CPR (P = 0.004). Retained embryos also decreased IR (P = 0.03). Multivariable analysis confirmed that the presence of blood on the transfer catheter was the most important of these transfer characteristics in predicting IR (P = 0.042) and CPR (P = 0.018). CONCLUSIONS: These results suggest that when only high-grade embryos or blastocysts are transferred, the presence of blood on the catheter is associated with decreased IR and CPR in assisted reproduction.

Pseudo-isolated FSH deficiency caused by an inhibin B-secreting granulosa cell tumour: case report.

Isolated FSH deficiency due to a mutation in the FSHbeta subunit is characterized by an extremely low serum FSH concentration. We report a patient who presented with an FSH of 0.8 mIU/ml and infertility associated with anovulation. Endocrinological assessment and immunohistochemistry revealed that a granulosa cell tumour was secreting inhibin B and suppressing FSH; however, LH and estradiol were within their normal ranges. Upon removal of the tumour, inhibin B decreased and FSH levels rose to normal values. The patient subsequently conceived and delivered successfully. Based on this case and on those previously described in the literature, we suggest that inhibin B levels should be evaluated in anovulatory patients having a clinical presentation consistent with functional hypothalamic amenorrhoea and very low to normal values of FSH.

Endometrial cavity fluid is associated with poor ovarian response and increased cancellation rates in ART cycles.

BACKGROUND: Endometrial cavity fluid (ECF) is occasionally observed during assisted reproductive technology (ART) cycles. However, few reports have described its prevalence or significance. METHODS AND RESULTS: We examined the relationships between ECF, clinical pregnancy rate (CPR), tubal factor infertility and ultrasound-visible (USV) hydrosalpinges. In 843 ART cycles involving 721 patients, ECF was observed during stimulation in 57 cycles and after human chorionic gonadotrophin (HCG) administration in 12 cycles, with an overall incidence of 8.2% (69/843). When ECF was observed during stimulation, the cancellation rate due to poor ovarian response was significantly higher (29.8 versus 16.9%, P <0.05) and the CPR per started cycle was significantly lower (26.3 versus 42.4%, P <0.05) than cycles without ECF. When ECF developed after HCG administration, the CPR was similar compared with that of the group for which ECF was not observed. In the 327 cycles involving tubal factor infertility patients, USV hydrosalpinges were noted in 71 cycles (71/327; 21.7%), and ECF developed in five of those cycles (5/71; 7.0%). A total of 27 cycles during which ECF developed (27/57, 47.4%) involved non-tubal factor patients. CONCLUSIONS: ECF during stimulation was associated with increased cancellation rates and lower CPRs per started cycle, and was not associated with USV hydrosalpinges. Furthermore, ECF observed after HCG administration did not impact CPR and may represent a different clinical entity.

Human chorionic gonadotropin levels after blastocyst transfer are highly predictive of pregnancy outcome.

OBJECTIVE: To determine the predictive value(s) of beta-hCG serum levels for pregnancy outcome following blastocyst transfer. DESIGN: Retrospective review. SETTING: University-based assisted reproductive technology (ART) program. PATIENTS: All ART patients enrolled from January 1998 to December 1999. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Beta-hCG serum levels and pregnancy outcomes. RESULT(S): Of the 836 ART cycles initiated, 608 embryo transfers met study criteria and were assigned to one of two groups: 248 day 5 blastocyst transfers or 360 day 3 embryo transfers. In the day 5 blastocyst group, 147 pregnancies occurred (59.2%), and day 3 transfers resulted in 165 pregnancies (45.8%). For day 3 and day 5 transfers, mean values of beta-hCG on day 16 post-retrieval of spontaneous abortions were lower than ongoing pregnancies (P< .05). A beta-hCG value on day 16 of >300 mIU/mL predicted an ongoing pregnancy for day 5 transfer group in 97% of pregnancies compared with 92% for day 3 embryo transfers. A multiple gestation was observed in 70% of pregnancies with a beta-hCG level >400 mIU/mL in the day 5 group compared with 63% for the day 3 group. The incidence of higher-order multiple gestations was significantly lower in the day 5 blastocyst group (P< .05). CONCLUSION(S): Beta-hCG serum levels on day 16 post-retrieval were highly predictive of pregnancy outcome after a blastocyst transfer.

The proto-oncoprotein Brx activates estrogen receptor beta by a p38 mitogen-activated protein kinase pathway.

The estrogen receptors (ERs) are ligand-inducible transcription factors that play key roles in the control of growth and differentiation in reproductive tissues. We showed that the novel Dbl family proto-oncoprotein Brx enhances ligand-dependent activity of ERalpha via a Cdc42-dependent pathway. Brx also significantly enhances ligand-dependent activity of ERbeta. This enhancement is not affected by inhibition of p44/42 mitogen-activated protein kinase (MAPK) activation by PD98059. However, addition of the p38 MAPK inhibitor SB202190 abrogates the enhancement of ERbeta activity by Brx, showing that p38 MAPK activity is required for the enhancement of ERbeta function by Brx. In COS-7 cells, transfection of Brx leads to activation of endogenous p38 MAPK activity. Co-expression of the beta2 isoform of human p38 MAPK and a constitutively active form of the p38 MAPK kinase MKK6 (MKK6-EE) synergistically augments ligand-dependent activity of ERbeta. Our findings suggest that p38 MAPKs may be important regulators of ERbeta activity.

Training of providers in embryo transfer: what is the minimum number of transfers required for proficiency?

BACKGROUND: Embryo transfer represents one of the most critical procedures in the practice of assisted reproduction. The objective of this study was to identify retrospectively the minimum number of embryo transfers required to train providers properly in this skill. METHODS AND RESULTS: The study group consisted of 204 patients who received embryo transfers between January 1996 and March 2000 in a university-based programme of assisted reproduction. The main outcome measure was clinical pregnancies per embryo transfer. Five Fellow trainees performed a total of 204 embryo transfers for an overall pregnancy rate of 45.5% per embryo transfer (93/204). In comparison, the programme pregnancy rate per transfer for experienced providers was 47.3% (560/1179). A chronological graph of each individual trainee's experience for the first 50 embryo transfers performed suggested a lower initial pregnancy rate for three of the five trainees. To determine whether a learning curve might exist, results of the first 25 transfers were compared as a subgroup with the second 25 transfers. Pregnancy rates were lower for the 1-25 transfer subgroup than in the 26-50 subgroup for three of the five Fellow trainees, although the difference was not statistically significant. CONCLUSION: Clinical pregnancy rates of Fellows-in-training were indistinguishable statistically from those of experienced staff by 50 transfers.

Clitoroplasty with preservation of neurovascular pedicles.

BACKGROUND: Optimal sexual function after surgical correction of clitoral hypertrophy requires an adequate postoperative innervation and vascular supply to the glans clitoris. Several clitoroplasty methods have been reported, but few describe preservation of dorsal and ventral neurovascular bundles in sexually mature women. CASE: A 22-year-old woman with clitoromegaly caused by non-salt-wasting classic 21-hydroxylase deficiency presented for a second surgical procedure after an operation in her infancy. The erect clitoral length exceeded 7.5 cm. Clitoral reduction was done through a semicircular incision in the phallus, with preservation of dorsal and ventral neurovascular pedicles. CONCLUSION: Preservation of ventral and dorsal vascular pedicles at clitoroplasty had a satisfactory result in sexually mature women.

Intracytoplasmic sperm injection increases embryo fragmentation without affecting clinical outcome.

PURPOSE: To examine the effect of intracytoplasmic sperm injection (ICSI) on embryo fragmentation and implantation rates in those embryos chosen for transfer compared to conventional in vitro fertilization (IVF). METHODS: We compared 253 infertility patients (71 ICSI and 182 IVF) with respect to age, semen analysis, number of embryos transferred, embryo fragmentation, implantation rate, and pregnancy rate. Embryo fragmentation was determined by one observer at the same laboratory over the entire study period. RESULTS: A statistically significant difference was observed in mean embryo grade between IVF (2.2 +/- 0.84) and ICSI (2.5 +/- 0.77), P = 0.01. Additionally, the IVF patients had significantly more nonfragmented (grade I) embryos compared to the ICSI group, P < 0.01. CONCLUSIONS: These data suggest that ICSI, irrespective of semen parameters, may increase embryo fragmentation and produce fewer nonfragmented grade I embryos while maintaining implantation and pregnancy rates similar to conventional IVF.

Pregnancy rates after embryo transfer depend on the provider at embryo transfer.

OBJECTIVE: To evaluate the effect of individual providers on pregnancy outcome after embryo transfer. DESIGN: Retrospective data analysis. SETTING: University-based tertiary-care assisted reproductive technology program with 10 physician-providers. PATIENT(S): Six hundred and seventeen women who underwent 854 fresh embryo transfers between January 1996 and January 1999. INTERVENTION(S): Pregnancies after embryo transfer were recorded for each provider. MAIN OUTCOME MEASURE(S): Establishment of a clinical pregnancy. RESULT(S): Three hundred ninety-three clinical pregnancies resulted from 854 embryo transfers, for an overall clinical pregnancy rate of 46.0% per embryo transfer. Three hundred forty-seven (40.6%) pregnancies were ongoing. The clinical pregnancy rate varied significantly between providers: for example, 17.0% (47 transfers) vs. 54.3% (57 transfers) (P<.05). Similarly, the ratio of high-grade embryos required to produce a gestational sac differed between providers. The number or quality of embryos transferred did not differ significantly. CONCLUSION(S): Significant differences were observed in pregnancy rates after embryo transfer done by different providers, suggesting that embryo transfer technique may influence pregnancy outcome in assisted reproductive technology.

Expression of brx proto-oncogene in normal ovary and in epithelial ovarian neoplasms.

OBJECTIVE: We previously identified a protein, Brx, that interacted with estrogen receptor alpha. Sequence analysis determined that Brx is a novel member of the Dbl family of oncoproteins involved in signaling pathways that regulate cell growth. Because the Brx protein was found to be highly expressed in hormoneresponsive breast epithelium, the objective of this study was to determine whether Brx was expressed in both normal and neoplastic ovarian tissues. STUDY DESIGN: A polyclonal antiserum directed against the Brx protein was used to perform immunolocalization on sections from 5 normal ovaries and 20 ovarian neoplasms. Chromosomal localization of the brx gene was accomplished by means of fluorescence in situ hybridization. Northern and Western blot analyses were performed on extracts prepared from human ovaries. RESULTS: Brx protein was localized to the cytoplasm of granulosa cells from mature graafian follicles, the corpus luteum, and islands of hilar cells in normal ovaries. In tumors with low malignant potential and ovarian carcinomas the neoplastic epithelium stained strongly for Brx protein. Northern and Western blot analyses, respectively, confirmed expression of Brx messenger ribonucleic acid and protein in normal ovary. Finally, the brx gene was localized to 15q25. CONCLUSION: The proto-oncogene brx is expressed in specific normal human ovarian tissues and is also present in ovarian epithelial neoplasms.

Microdose follicular phase gonadotropin-releasing hormone agonist (GnRH-a) compared with luteal phase GnRH-a for ovarian stimulation at in vitro fertilization.

OBJECTIVE: To compare an ovarian stimulation protocol using microdose follicular phase GnRH agonist (GnRH-a) and oral contraceptive (OC) pills to a luteal phase GnRH-a protocol. DESIGN: Retrospective analysis. SETTING: University affiliated IVF program. PATIENT(S): One hundred seventy patients who underwent IVF and ET in 1996. INTERVENTION(S): Patients were assigned to either a midluteal start of leuprolide acetate (LA) 1 mg/d, reduced to 0.5 mg/d after addition of gonadotropins (LUT), or OC pills until cycle day 0 followed by 20 microg of LA every 12 hours on cycle day 3 with addition of gonadotropins on cycle day 5 (MICRO). MAIN OUTCOME MEASURE(S): Number of FSH ampules, days of stimulation, peak E2, and number of oocytes retrieved. RESULT(S): There were no statistically significant differences in the main outcome measures between the two groups using an age-matched ANOVA. Clinical pregnancy rate per cycle start was not statistically different (LUT = 54%, and MICRO = 37%). The cancellation rate was significantly higher in the MICRO group (22.5% vs. 8.2%). CONCLUSION(S): Given the higher cancellation rate in the microdose group, a randomized clinical trial is required to determine the possible benefit of a lower dose of GnRH-a in patients with normal ovarian function.

Characterization of Brx, a novel Dbl family member that modulates estrogen receptor action.

Regulation of gene activation by the estrogen receptor (ER) is complex and involves co-regulatory proteins, oncoproteins (such as Fos and Jun), and phosphorylation signaling pathways. Here we report the cloning and initial characterization of a novel protein, Brx, that contains a region of identity to the oncogenic Rho-guanine nucleotide exchange (Rho-GEF) protein Lbc, and a unique region capable of binding to nuclear hormone receptors, including the ER. Western and immunohistochemistry studies showed Brx to be expressed in estrogen-responsive reproductive tissues, including breast ductal epithelium. Brx bound specifically to the ER via an interaction that required distinct regions of ER and Brx. Furthermore, overexpression of Brx in transfection experiments using an estrogen-responsive reporter revealed that Brx augmented gene activation by the ER in an element-specific and ligand-dependent manner. Moreover, activation of ER by Brx could be specifically inhibited by a dominant-negative mutant of Cdc42Hs, but not by dominant negative mutants of RhoA or Rac1. Taken together, these data suggest that Brx represents a novel modular protein that may integrate cytoplasmic signaling pathways involving Rho family GTPases and nuclear hormone receptors.

Interaction between the retinoid X receptor and transcription factor IIB is ligand-dependent in vivo.

The retinoid X receptor (RXR) influences gene activation through heterodimeric and homodimeric association with DNA and associates with TATA binding protein, TAF110, and cAMP response element-binding protein-binding protein; yet the molecular mechanisms responsible for gene activation by RXRs remain incompletely defined. Since the general transcription factor IIB (TFIIB) is a common target of sequence-specific transcriptional activators, we suspected that RXR might regulate target genes via an interaction with TFIIB. Coimmunoprecipitation, far Western analysis, and glutathione S-transferase binding studies indicated that murine RXR beta (mRXR beta) was capable of binding to human TFIIB in vitro. Functional analysis with a dual-hybrid yeast system and cotransfection assays revealed the interaction of mRXR beta with TFIIB to be ligand-dependent in vivo. Truncation experiments mapped the essential binding regions to the carboxyl region of mRXR beta (amino acids (aa) 254-389) and two regions in the carboxyl region of TFIIB (aa 178-201 and aa 238-271). Furthermore, the delta 390-410 mRXR beta mutant bound to TFIIB in vitro but was not active in the dual-hybrid yeast system, suggesting that the extreme carboxyl region of RXR was required for in vivo interaction with TFIIB. These data indicate that interaction of mRXR beta with TFIIB is specific, direct, and ligand-dependent in vivo and suggest that gene activation by RXR involves TFIIB.

The tumor suppressor p53 is a negative regulator of estrogen receptor signaling pathways.

The estrogen receptor (ER) is a ligand-dependent transcription factor which regulates growth, development, differentiation and reproduction. To test the hypothesis that the diverse effects of the ER could be mediated by interacting with other transcription factors/oncogenes, the present study assessed its interaction with the tumor suppressor p53. p53 is a transcription factor which is involved in cell cycle regulation and apoptosis. We found that the wild-type p53 physically interacted with ER in vivo and repressed the estrogen-activated transcriptional activity. However, p53 mutants had no or reduced repression effect, depending on the sites of mutation. These findings suggest that p53 can cross talk with the ER in hormone-activated signaling pathways in cells.

Steroidogenic factor 1 messenger ribonucleic acid expression in steroidogenic and nonsteroidogenic human tissues: Northern blot and in situ hybridization studies.

Steroidogenic factor-1 (SF-1), a tissue-specific orphan nuclear receptor, regulates the genes of several steroidogenic enzymes, Mullerian inhibiting substance, and the gonadotrophins. Also, this transcription factor is crucial for hypothalamic, adrenal, and gonadal organogenesis in the mouse. We recently cloned the human SF-1 (hSF-1) complementary DNA (cDNA) and now report the distribution of this factor's messenger RNA (mRNA) in human tissues. Northern blot analyses of peripheral tissues revealed high hSF-1 mRNA expression in the adrenal cortex and the gonads, but no hSF-1 mRNA was detected in the placenta. High hSF-1 mRNA expression also was seen in the spleen. In this tissue, in addition to the main transcript of 3.5-4 kb seen in the adrenal and gonads, two additional transcripts of 4.4 kb and 8 kb were noted. The additional 4.4-kb transcript also was seen in several peripheral tissues and various components of the brain. However, adult liver and heart showed only the 4.4-kb transcript. In the human brain, hSF-1 mRNA expression was widespread, including several components of the limbic system. In situ hybridization studies confirmed the strong expression of hSF-1 mRNA in adrenal cortex, ovary, testis, and the spleen, primarily within reticuloendothelial cells. Thus, in the human, the hSF1 mRNA is present in both steroidogenic and nonsteroidogenic tissues, albeit not in the placenta. In the central nervous system, the expression of hSF-1 mRNA is widespread. It is composed of several different mRNA species distributed in a tissue-specific fashion. These findings suggest that hSF-1 may play a role in reticuloendothelial/immune cell maturation and/or function, as well as nervous system development and/or neurosteroid biosynthesis.

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

Tissue expansion vaginoplasty for treatment of congenital vaginal agenesis.

BACKGROUND: A patient with congenital vaginal agenesis was unsuccessful in the use of dilation to create a vaginal orifice and rejected the option of a buttock graft. CASE: Two tissue expanders were introduced beneath the labia majora bilaterally and slowly expanded over 4 weeks. Redundant labial tissue was advanced as a bipedicle flap to line the neovagina created intraoperatively. Postoperative stent placement and dilation resulted in a vaginal canal lined by full-thickness mucosa exceeding 8 cm in depth. CONCLUSION: A modified method of tissue expansion vaginoplasty using a bipedicle flap is an option for the surgical creation of a vaginal orifice.

Retinoic acid induction of major histocompatibility complex class I genes in NTera-2 embryonal carcinoma cells involves induction of NF-kappa B (p50-p65) and retinoic acid receptor beta-retinoid X receptor beta heterodimers.

Retinoic acid (RA) treatment of human embryonal carcinoma (EC) NTera-2 (NT2) cells induces expression of major histocompatibility complex (MHC) class I and beta-2 microglobulin surface molecules. We found that this induction was accompanied by increased levels of MHC class I mRNA, which was attributable to the activation of the two conserved upstream enhancers, region I (NF-kappa B like) and region II. This activation coincided with the induction of nuclear factor binding activities specific for the two enhancers. Region I binding activity was not present in undifferentiated NT2 cells, but binding of an NF-kappa B heterodimer, p50-p65, was induced following RA treatment. The p50-p65 heterodimer was produced as a result of de novo induction of p50 and p65 mRNAs. Region II binding activity was present in undifferentiated cells at low levels but was greatly augmented by RA treatment because of activation of a nuclear hormone receptor heterodimer composed of the retinoid X receptor (RXR beta) and the RA receptor (RAR beta). The RXR beta-RAR beta heterodimer also bound RA responsive elements present in other genes which are likely to be involved in RA triggering of EC cell differentiation. Furthermore, transfection of p50 and p65 into undifferentiated NT2 cells synergistically activated region I-dependent MHC class I reporter activity. A similar increase in MHC class I reporter activity was demonstrated by cotransfection of RXR beta and RAR beta. These data show that following RA treatment, heterodimers of two transcription factor families are induced to bind to the MHC enhancers, which at least partly accounts for RA induction of MHC class I expression in NT2 EC cells.