Noha A. Mousa

Droplet-Scale Estrogen Assays in Breast Tissue, Blood, and Serum

A digital electrode array, on which drops of solvent can be electronically manipulated, allows automated steroid extraction from tiny tissue samples for diagnosis or research. Extracting Estrogen from Small Samples Microfluidic devices, in which small samples are manipulated within narrow channels, have been profitably applied to clinical sample analysis and biomedical research. These devices have advantages over more standard analysis techniques because they require only limited amounts of reagents, a boon when reagents are expensive. In addition, microfluidic devices are easy to make and can handle multiple processes and multiple samples on a single platform, enabling automation and scale-up. In closely related digital microfluidic (DMF) devices, the usual channels through which liquids are pumped or electrically coaxed are replaced by arrays of flat electrodes. When an electrical potential is applied to an electrode, the surface becomes locally charged, such that the droplet moves to that region. When several electrodes are charged, one by one, droplets can be made to move across the surface (and dispense and mix) in a controlled fashion. To be moveable, liquids must be slightly conductive or have dipole moment > 1; fortunately, most liquids fall into this category. This DMF technology has now been harnessed by Mousa et al. for extraction of the steroid estradiol from very small tissue or blood samples for ultimate quantitation. Steroid hormones, which regulate essential reproductive and homeostatic functions in the human body, are routinely measured clinically in blood but not in tissue because of the large samples that would be required by current methods. Separation of steroids from other biological components is usually necessary for accurate measurements because these components often interfere with the assays. To enable measurement of estrogen in tissues or small samples of blood, Mousa et al. have used DMFs and built a device to extract estradiol from 1-μl samples. In their method, the biological sample is dried onto one of the electrodes and a lysing solvent is moved by application of electrical potential to the sample and dried. Then, a polar solvent moves to the sample, extracting soluble material, including steroids. The droplet containing the steroid (and other components) is then circulated within a pool of nonpolar solvent; many of the interfering compounds partition into the nonpolar liquid. The polar droplet, still containing the steroid, is moved out of the nonpolar solvent pool and dried on an electrode, ready for quantitation. For routine application, the extraction process will need to be linked to an automated measurement method. A convenient technique for measuring estrogen in small amounts of breast tissue or tumor, for example, would allow testing whether failure to respond to therapy with selective estrogen receptor modulators such as tamoxifen or aromatase inhibitors is a result of locally high estrogen concentrations in the tumor and whether breast cancer risk can be predicted by local estrogen concentrations. Further, androgen concentrations in the prostate could be measured to check their influences on cancer. Estrogen is a key hormone in human reproductive physiology, controlling ovulation and secondary sexual characteristics. In addition, it plays an important role in the pathogenesis of breast cancer. Indeed, estrogen receptor antagonists and aromatase inhibitors (which block estrogen biosynthesis) are primary drugs used for treatment and prevention in at-risk populations. Despite its importance, tissue concentrations of estrogen are not routinely measured because conventional techniques require large samples of biopsies for analysis. In response to this need, we have developed a digital microfluidic method and applied it to the extraction and quantification of estrogen in 1-microliter samples of breast tissue homogenate (as would be collected with fine-needle aspiration), as well as in whole blood and serum. This method may be broadly applicable to conditions requiring frequent analysis of hormones in clinical samples (for example, infertility and cancer).

Aromatase inhibitors in the treatment of severe endometriosis.

BACKGROUND: Endometriosis-associated chronic pelvic pain unresponsive to surgical menopause is a difficult clinical problem. CASE: A middle-aged woman presented with endometriosis and severe pelvic pain after hysterectomy and bilateral salpingo-oopherectomy. She was first treated with exemestane without improvement of symptoms. However, another aromatase inhibitor, letrozole, relieved her pain, and concomitant treatment with estrogen relieved hot flushes without pain reactivation. CONCLUSION: Letrozole was superior to exemestane in relieving the endometriosis-associated pain in this postmenopausal woman. This suggests that patients may respond variably to different aromatase inhibitors.

Aromatase inhibitors and mammographic breast density in postmenopausal women receiving hormone therapy.

Objective: One of the main concerns regarding long-term use of hormone therapy (HT) in symptomatic menopausal women is the perceived increased risk of breast cancer. A method to reduce breast cancer risk in this population of women is urgently needed. We hypothesized that adding aromatase inhibitors (AIs) to HT would reduce local breast estrogen exposure and breast cancer risk without altering the beneficial systemic effects of HT on menopausal symptoms or bone density. The aim of this study was to investigate the effect of AIs and HT on mammographic breast density (MBD) as a surrogate marker of breast cancer risk in postmenopausal women receiving low-dose HT. Design: This was a retrospective cohort study conducted at private clinics affiliated with a university hospital. One group of postmenopausal women (n = 28) received low-dose HT daily plus letrozole 2.5 mg three times weekly. Postmenopausal women receiving HT alone (n = 28) served as controls. MBD, the primary outcome, was measured using quantitative image analysis software as well as by visual analysis by a radiologist. Hypoestrogenic effects, adverse reactions, and bone mineral densities were secondary outcome measures. Results: The mammograms of 18 women in the study group and 22 women in the control group were suitable for comparison. A statistically significant reduction in MBD occurred in the women who received HT plus an AI, whereas no significant change was observed in the women receiving HT alone. There was no significant increase in hypoestrogenic symptoms during the use of AIs, and bone mineral densities were not significantly reduced. Conclusions: Adding an AI to HT may lower MBD in postmenopausal women. AIs could be good candidates for primary chemoprevention of breast cancer in postmenopausal women using HT.

The effect of acute aromatase inhibition on breast parenchymal enhancement in magnetic resonance imaging: a prospective pilot clinical trial.

ObjectiveThe breast is highly hormonally sensitive especially to the sex steroid hormone estrogen. Both physiological and iatrogenic steroid hormone modifications could affect how the breast tissue may appear in breast imaging techniques. We hypothesized that estrogen deprivation therapy could reduce breast nonspecific enhancement on magnetic resonance imaging (MRI). MethodsThis study was a prospective pilot phase II clinical trial. The study was approved by Health Canada and the institutional research ethics board, and participants signed informed consent forms. Sixteen healthy postmenopausal women were enrolled, and 14 completed the study. Baseline breast MRI was done followed 1 month later by administration of a high-dose aromatase inhibitor (letrozole 12.5 mg/day) for 3 successive days before a second breast MRI. Background breast parenchymal enhancement was compared between the pretreatment and posttreatment studies. ResultsThere was a statistically significant reduction of the average background breast enhancement after treatment with aromatase inhibitors compared with baseline MRI. Of particular interest, specific areas of benign breast enhancement were reduced after aromatase inhibitor treatment. No significant adverse effects were recorded using this relatively high dose of the aromatase inhibitors. ConclusionsThis preliminary study provided evidence that aromatase inhibitors could reduce the parenchymal background enhancement of benign breast tissue during MRI and may improve the specificity of the technique.

Hormonal, follicular and endometrial dynamics in letrozole-treated versus natural cycles in patients undergoing controlled ovarian stimulation

The objective of this study was to compare letrozole-stimulated cycles to natural cycles in 208 patients undergoing intrauterine insemination (IUI) between July of 2004 and January of 2007. Group I (n = 47) received cycle monitoring only (natural group), Group II (n = 125) received letrozole 2.5 mg/day on cycle days three to seven, and Group III (n = 36) received letrozole 5 mg/day on cycle days three to seven. There were no differences between the groups in endometrial thickness or P4 on the day of hCG. Estradiol levels had higher variation in the second half of the follicular phase in both letrozole-treated groups compared to the control group. Estradiol per preovulatory follicle was similar in both letrozole cycles to that observed in the natural cycles. LH was lower on the day of hCG administration in the letrozole 2.5 mg/day group vs. the natural group. In summary, letrozole results in some minor changes in follicular, hormonal and endometrial dynamics compared to natural cycles. Increased folliculogenesis and pregnancy rates were observed in the letrozole-treated groups compared to the natural group. These findings need to be confirmed in larger, prospective studies.

Aromatase inhibitors prevent the estrogen rise associated with the flare effect of gonadotropins in patients treated with GnRH agonists

This was a preliminary study to determine the effect of aromatase inhibitors in preventing the flare induced by GnRH agonist (GnRH-a) in women with endometriosis (n = 9) or leiomyomata (n = 4) who were given letrozole on the first 5 days of the GnRH-a therapy. Serum LH and FSH levels showed the typical flare 1 day after the injection of the GnRH-a; however, E(2) declined immediately after letrozole administration and was notably suppressed at 1, 2, and 4 days after GnRH-a. No patients complained of clinical symptoms typical of the GnRH-a flare phenomenon.

Aromatase Inhibitors: Potential Reproductive Implications

MEDLINE, EMBASE, Scopus, and Web of Science databases literature search from inception to March 2009 was performed to identify published clinical trials and cohort, observational, and in vitro studies that evaluated the use of aromatase inhibitors in reproductive medicine for indications other than ovulation induction. Aromatase inhibitors are currently being investigated for breast cancer prevention in women at high risk. Aromatase inhibitors may be used for treatment of symptomatic myomas and endometriosis as an alternative to surgical intervention. Current evidence does not support the routine use of aromatase inhibitors for these conditions without prospective controlled trials. Aromatase inhibitor cotreatment can be used to prevent the initial estrogen flare effect of gonadotropin-releasing hormone agonist treatment to offer flexibility in initiating this therapy.

A Cost-Effective Method to Assemble Biomimetic 3D Cell Culture Platforms

Developing effective stem cell based therapies requires the design of complex in vitro culture systems for more accurate representation of the stem cell niche. Attempts to improve conventional cell culture platforms include the use of biomaterial coated culture plates, sphere culture, microfluidic systems and bioreactors. Most of these platforms are not cost-effective, require industrial technical expertise to fabricate, and remain too simplistic compared to the physiological cell niche. The human amniotic membrane (hAM) has been used successfully in clinical grafting applications due to its unique biological composition and regenerative properties. In this study, we present a combinatorial platform that integrates the hAM with biomolecular, topographic and mechanical cues in one versatile model. Methods We utilized the hAM to provide the biological and the three dimensional (3D) topographic components of the prototype. The 3D nano-roughness of the hAM was characterized using surface electron microscopy and surface image analysis (ImageJ and SurfaceJ). We developed additional macro-scale and micro-scale versions of the platform which provided additional shear stress factors to simulate the fluid dynamics of the in vivo extracellular fluids. Results Three models of varying complexities of the prototype were assembled. A well-defined 3D surface modulation of the hAM in comparable to commercial 3D biomaterial culture substrates was achieved without complex fabrication and with significantly lower cost. Performance of the prototype was demonstrated through culture of primary human umbilical cord mononuclear blood cells (MNCs), human bone marrow mesenchymal stem cell line (hBMSC), and human breast cancer tissue. Conclusion This study presents methods of assembling an integrated, flexible and low cost biomimetic cell culture platform for diverse cell culture applications.

Tissue dissociation miniaturized platform for uterine stem cell isolation and culture

Endometrial stem cells provide a readily accessible source of adult stem cells suitable for various stem cell based therapies. The first essential step in the process of stem cell isolation, sorting and culture is tissue dissociation into single cell suspension. Tissue dissociation efficiency determines the number of resulting viable cells which in turn could affect the feasibility of the subsequent stem cell application. Microfluidic based platforms could provide several advantages over current conventional tissue dissociation techniques (e.g. mechanical or enzymatic or both) and help the optimization of the process towards a better reproducible cellular outcome. Objective: To develop a simple miniaturized platform for endometrial tissue dissociation which can be used to evaluate and optimize experimental conditions necessary for producing single cell suspension with minimal cell damage. Methods: A Poly-DiMethylSiloxane (PDMS) channel microfluidic platform was designed. The platform includes a tissue dissociation chamber where raw endometrial tissue was incubated. Trypsin was delivered through an inlet channel while another inlet enabled parallel delivery of a cell suspension medium and an outlet channel received the downstream dissociated endometrial cells. A cell size based filtration step is designed to trap large debris using two rows of posts. A viability stain is used to test the viability of cells produced at different enzyme-tissue contact durations. Conclusion: We present a simple microfluidics based platform for endometrial tissue dissociation that can be used for subsequent endometrial stem cell research applications and can be further applied to other tissues.