Gaurang S. Daftary

Transcriptional Repression of Peri-Implantation EMX2 Expression in Mammalian Reproduction by HOXA10

ABSTRACT HOXA10 is necessary for mammalian reproduction; however, its transcriptional targets are not completely defined. EMX2, a divergent homeobox gene, is necessary for urogenital tract development. In these studies we identify and characterize the regulation of EMX2 by HOXA10. By using Northern analysis and in situ hybridization, we found that EMX2 is expressed in the adult urogenital tract in an inverse temporal pattern from HOXA10, suggestive of a negative regulatory relationship. Constitutive expression of HOXA10 diminished EMX2 mRNA, whereas blocking HOXA10 through the use of antisense resulted in high EMX2 mRNA expression. Deletional analysis of the EMX2 5′ regulatory region revealed that a 150-bp element mediated transcriptional repression when cotransfected with pcDNA3.1/HOXA10 in transient-transfection assays. Binding of HOXA10 protein to this element was demonstrated by electrophoretic mobility shift assay and further localized to a consensus HOXA10 binding site within this element by DNase I footprinting. Site-directed mutagenesis abolished binding, as well as the negative transcriptional regulation. Transcriptional activation of empty spiracles, the Drosophila ortholog of EMX2, by Abdominal-B (HOXA10 ortholog) has been previously demonstrated. These findings demonstrate conservation of the transcription factor-target gene relationship, although the direction of regulation is reversed with possible evolutionary implications.

Hydrosalpinx fluid diminishes endometrial cell HOXA10 expression

OBJECTIVE To determine the effect of hydrosalpinx fluid on the expression of HOXA10, an essential regulator of endometrial receptivity. DESIGN In vitro study. SETTING Academic medical center. PATIENT(S) Patients with unilateral or bilateral hydrosalpinx. INTERVENTION(S) Hydrosalpinx fluid was aspirated from 10 patients at laparoscopy. The fluid was serially diluted in minimum essential medium. Ishikawa cells (an endometrial adenocarcinoma cell line, representative of endometrial epithelium) were incubated with this fluid at concentrations of 10% and 50% for 48 hours. Cells were also incubated in undiluted minimum essential medium (MEM) and in 10% serum as controls. After incubation, the cells were lysed in Trizol, and total RNA was extracted and analyzed by Northern blot using a 32P-labeled HOXA10 riboprobe. A 32P-labeled G3PDH probe was used as a control for loading. MAIN OUTCOME MEASURE(S) HOXA10 mRNA expression. RESULT(S) HOXA10 mRNA expression in endometrial cells decreased with increasing concentrations of hydrosalpinx fluid. Densitometric analysis of the northern blot revealed that HOXA10 mRNA expression was different from control at both concentrations (P<.007). CONCLUSION(S) HOXA10 is necessary for implantation in the murine model. HOXA10 expression is diminished by hydrosalpinx fluid. This effect on HOXA10 is a potential molecular mechanism by which implantation rates are diminished in women with hydrosalpinges.

Efficient liposome-mediated gene transfection and expression in the intact human uterus

Although gene therapy has been used for correction of metabolic defects in diseases such as cystic fibrosis, as adjuvant treatment in cancer, and in the treatment of infectious diseases, there has been no report of gene transfer to the intact female reproductive tract. We assessed the ability to transfect the human uterus ex vivo and thereby evaluate the applicability of gene therapy to gynecology. The uterine lumen was accessed transcervically, using an intrauterine insemination catheter. pcDNA3.1 plasmid containing the Escherichia coli lacZ reporter gene was delivered to each uterus via liposome-mediated transfection. Control uteri were transfected with empty pcDNA3.1. Immunohistochemical analysis revealed beta-galactosidase expression in the lacZ-treated uteri in endometrial epithelial cells, endometrial stromal cells, and myometrium to a depth of 1.75 cm from the endometrial-myometrial junction. Highest expression was seen in endometrial glandular epithelial cells, with significant expression in the stroma and adjacent myometrium. Each of these cell types in the control uteri showed no beta-galactosidase expression. Successful gene transfection and expression in the intact human uterus can be accomplished easily, rapidly, and efficiently. Gene therapy may have wide applicability in the treatment and study of gynecologic disease.

Prenatal prediction of neonatal outcome in the extremely low-birth-weight infant☆☆☆★

OBJECTIVE Our goal was to identify prenatally available parameters that correlate with neonatal outcome and could be used for predicting such outcome in the extremely low-birth-weight pregnancy. STUDY DESIGN From 1990 through 1995, obstetric and neonatal data of live-born nonanomalous singleton infants with birth weights between 400 and 1000 gm were reviewed. Only cases in which ultrasonographic biometry, including biparietal diameter, abdominal circumference, and femur length, was performed < or =3 days before delivery were included. Overall survival (defined as alive at discharge) and survival without specific severe neonatal morbidities (namely, retinopathy of prematurity [stage 3 or 4], intraventricular hemorrhage [grade 3 or 4], periventricular leukomalacia, chronic lung disease, and deafness) were ascertained. The best combination of prenatal parameters for the prediction of overall survival and survival without severe morbidity was determined by backward stepwise logistic regression analyses. RESULTS The most significant prenatal predictors of overall survival were the obstetric estimate of gestational age and the abdominal circumference (chi2 = 11.8036, p = 0.0006 and chi2 = 8.1862, p < 0.005, respectively). Survival without severe morbidity was also predicted by the same combination of parameters (chi2 = 21.9079, p = 0.0001 and chi2 = 6.538, p = 0.01, respectively). The estimated fetal weight was not a significant independent predictor of either category of outcome (chi2 = 0.1249, p = 0.72 and chi2 = 0.0361, p = 0.85, respectively). On the basis of the regression formulas, curves displaying the probabilities of overall survival and survival without severe morbidity with any combination of gestational age and abdominal circumference were developed. CONCLUSION The combination of gestational age and the abdominal circumference measurements appears to be superior to any combination that included estimated fetal weight data for predicting neonatal outcome in the neonates weighing < or =1000 gm. We developed a mechanism for predicting neonatal outcome in this weight category on the basis of prenatally available parameters. This information could prove useful for both parental counseling and obstetric decision making.

Molecular markers of implantation: clinical implications

The endometrium has been conventionally studied using histologic criteria. Our understanding of endometrial physiology has been advanced tremendously by research into the molecules that mediate its development and function. These molecules demonstrate a dynamic expression pattern through the menstrual cycle and have been implicated in endometrial growth, differentiation, and receptivity. These molecules include secreted proteins (endometrial bleeding-associated factor, glycodelin-A, insulin-like growth factor binding protein-1), cell-surface receptors (integrins), and nuclear transcription factors (HOXA10 and HOXA11). The homeobox genes Hoxa10 and Hoxa11 are necessary for implantation because mice with mutations in these genes exhibit a failure of implantation. HOXA10 and HOXA11 have been shown to be important for implantation in humans as well. Knowledge of endometrial molecular dynamics may now be used to enhance our ability to diagnose implantation defects. It may soon be possible to treat individual molecular defects by protein supplementation or gene therapy.

Reproductive tract gene transfer

OBJECTIVE Gene therapy is a rapidly evolving novel treatment for human disease. This review discusses the latest development in gene transfer technology and its potential use in the female reproductive tract. METHODS A comprehensive search using the MEDLINE database was performed to review current, innovative trends in gene transfer technology. In addition, articles on reproductive tract gene transfer were reviewed. CONCLUSION(S) Recent developments, such as the Human Genome Project, have generated great interest in the genetic basis of human health and disease. Gene therapy is a rapidly evolving field that uses gene transfer to treat disease. Ongoing research in the field focuses on improving vector technology to enable efficient in vivo gene transfer. Although multiple techniques for gene transfer have been described, no single technique can be used in all instances. The human female reproductive tract is easily accessible and can be readily transfected. In vivo gene transfer has resulted in successful alteration of implantation rates and has demonstrated potential for use in treatment of ovarian cancer.

Endometrial HOXA10 expression after controlled ovarian hyperstimulation with recombinant follicle-stimulating hormone

OBJECTIVE To determine the effects of controlled ovarian hyperstimulation and resultant high levels of E(2) on endometrial HOXA10 expression (a marker of endometrial receptivity). DESIGN Prospective study. SETTING University academic medical center. PATIENT(S) Twenty-five women undergoing controlled ovarian hyperstimulation with recombinant FSH and 30 fertile controls. INTERVENTION(S) Endometrium was obtained by Pipelle endometrial biopsy on cycle days 21-25. In addition, Ishikawa cells (a well-differentiated endometrial adenocarcinoma cell line) were treated with either E(2), recombinant FSH, GnRH agonist, or GnRH antagonist. RNA was extracted and analyzed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). MAIN OUTCOME MEASURE(S) HOXA10 expression. RESULT(S) Endometrial HOXA10 expression in women undergoing controlled ovarian hyperstimulation (COH) with recombinant FSH was not different from that in fertile controls. Estradiol increased HOXA10 expression in Ishikawa cells in a dose-dependent manner from 10(-9) to 10(-7) M. Neither recombinant FSH, GnRH agonist, nor GnRH antagonist altered HOXA10 expression in these cells. CONCLUSION(S) Controlled ovarian hyperstimulation did not inhibit endometrial HOXA10 expression in vivo. In addition, in vitro endometrial cell HOXA10 expression was not altered by either recombinant FSH, GnRH agonist, or GnRH antagonist. COH is unlikely to adversely impact endometrial receptivity.