Gestational carriers: A viable alternative for women with medical contraindications to pregnancy*

Objective: Compare the efficacy of surrogate or gestational carrier (GC) cycles to that of autologous in vitro fertilization (IVF)/intracytoplasmic sperm injections (ICSI) in patients with gynecologic or medical co-morbidities contraindicative to pregnancy. Design: Retrospective cohort study. Setting: Infertility patients from a single university hospital-based program from 1998-2009. Intervention(s) 128 GC cycles from 80 intended parents were identified and compared with 15,311 IVF or ICSI cycles. Main Outcome Measure(s) The peak estradiol (E2), number of oocytes retrieved, cycle cancellation, ongoing pregnancy, and live-birth were compared between GCs and autologous IVF carriers. Indications for GC use were also identified. Multiple cycles contributed by the same patient were accounted for using multivariable generalized estimating equations and two-sided Wald p-values. Results: Uterine factors (67%) was the most common indication for using a GC, followed by non-gynecologic medical conditions including coagulopathies (13%), end stage renal disease (10%), cardiovascular disease (5%) and cancer (5%). Adjusting for age, ovulation induction in GC cycles had similar peak E2 levels and number of oocytes retrieved relative to IVF cycles (p = 0.23 and 0.43, respectively). Clinical pregnancy (49% vs. 42%, p = 0.28) and live-birth rates (31% vs. 32%, p = 0.74) were also comparable. A sub-analysis of GC cycles in those women with uterine factor indications, demonstrated significantly higher clinical pregnancy rates (OR = 2.0; CI = 1.2 - 3.5) with 60% greater odds of live-birth relative to IVF/ICSI cycles, however this odds was not statistically significant for differences in live-birth (CI = 0.9 - 2.9). Conclusions: GCs are a viable alternative to start families for patients with medical co-morbidities precluding pregnancy

Pax6- and Six3-Mediated Induction of Lens Cell Fate in Mouse and Human ES Cells

Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development

Hereditary leiomyomatosis and renal cell cancer: Cutaneous lesions & atypical fibroids

Objective: To report a diagnosis of hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome following initial presentation with multiple cutaneous lesions. Design: Case report. Design classification N/A. Setting: Academic tertiary care center. Patient(s) 27-year-old nulligravid woman who presented with multiple red-brown lesions on her skin found to have cutaneous and uterine leiomyoma. Intervention(s) Biopsy of cutaneous lesions and fertility sparing robot-assisted laparoscopic myomectomy (RALM). Main outcome measures(s) Histological assessment of uterine leiomyoma. Results(s) Pathologic examination of uterine leiomyoma revealed diffuse atypia and fumarate hydratase loss phenotype concerning for genetic syndrome. Follow-up DNA sequencing via Sanger sequencing confirmed a pathogenetic R2333H mutation consistent with HLRCC. Conclusion(s) Consideration of HLRCC on differential diagnosis when patients present with cutaneous nodules and atypical or early onset uterine leiomyoma provides opportunity for early surveillance, family member testing, and more thoughtful surgical planning. Precis 27-year-old woman with multiple cutaneous lesions is found to have uterine leiomyomas and undergoes robotic myomectomy. Genetic testing of uterine leiomyomas reveals mutation in fumarate hydratase, etiologic in hereditary leiomyomatosis and renal cell cancer (HLRCC)

Amniocytes can serve a dual function as a source of iPS cells and feeder layers

Clinical barriers to stem-cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem-cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short- or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5–7 days with 0.5% efficiency. This efficiency is greater than that reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4+ or c-Kit+ amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human embryonic stem (ES) cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture

Pelvic and pulmonary benign metastasizing leiomyoma: A case report

Seven years after she had a total abdominal hysterectomy for benign leiomyomas, a 46-year-old woman presented with a pelvic mass and multiple pulmonary nodules. She underwent resection of the mass and core needle biopsy of a pulmonary lesion. Histopathologic analysis revealed that both the pelvic and the pulmonary lesions were consistent with benign leiomyomas. Benign metastasizing leiomyoma should be considered if a woman of reproductive age and with a history of leiomyomas presents with extrauterine nodules without evidence of malignancy. The final diagnosis should be based on histopathological examination. Treatment depends on tumor size, location, receptor positivity, and disease progression

Deformation of a single mouse oocyte in a constricted microfluidic channel

© 2015, Springer-Verlag Berlin Heidelberg.Single oocyte manipulation in microfluidic channels via precisely controlled flow is critical in microfluidics-based in vitro fertilization. Such systems can potentially minimize the number of transfer steps among containers for rinsing as often performed during conventional in vitro fertilization and can standardize protocols by minimizing manual handling steps. To study shape deformation of oocytes under shear flow and its subsequent impact on their spindle structure is essential for designing microfluidics for in vitro fertilization. Here, we developed a simple yet powerful approach to (1) trap a single oocyte and induce its deformation through a constricted microfluidic channel, (2) quantify oocyte deformation in real time using a conventional microscope and (3) retrieve the oocyte from the microfluidic device to evaluate changes in their spindle structures. We found that oocytes can be significantly deformed under high flow rates, e.g., 10 μL/min in a constricted channel with a width and height of 50 and 150 μm, respectively. Oocyte spindles can be severely damaged, as shown here by immunocytochemistry staining of the microtubules and chromosomes. The present approach can be useful to investigate underlying mechanisms of oocyte deformation exposed to well-controlled shear stresses in microfluidic channels, which enables a broad range of applications for reproductive medicine

Hereditary leiomyomatosis and renal cell cancer:Cutaneous lesions & atypical fibroids

Objective: To report a diagnosis of hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome following initial presentation with multiple cutaneous lesions. Design: Case report. Design classification: N/A. Setting: Academic tertiary care center. Patient(s): 27-year-old nulligravid woman who presented with multiple red-brown lesions on her skin found to have cutaneous and uterine leiomyoma. Intervention(s): Biopsy of cutaneous lesions and fertility sparing robot-assisted laparoscopic myomectomy (RALM). Main outcome measures(s): Histological assessment of uterine leiomyoma. Results(s): Pathologic examination of uterine leiomyoma revealed diffuse atypia and fumarate hydratase loss phenotype concerning for genetic syndrome. Follow-up DNA sequencing via Sanger sequencing confirmed a pathogenetic R2333H mutation consistent with HLRCC. Conclusion(s): Consideration of HLRCC on differential diagnosis when patients present with cutaneous nodules and atypical or early onset uterine leiomyoma provides opportunity for early surveillance, family member testing, and more thoughtful surgical planning. Precis: 27-year-old woman with multiple cutaneous lesions is found to have uterine leiomyomas and undergoes robotic myomectomy. Genetic testing of uterine leiomyomas reveals mutation in fumarate hydratase, etiologic in hereditary leiomyomatosis and renal cell cancer (HLRCC)

Functional Maintenance of Differentiated Embryoid Bodies in Microfluidic Systems: A Platform for Personalized Medicine

Hormone replacement therapies have become important for treating diseases such as premature ovarian failure or menopausal complications. The clinical use of bioidentical hormones might significantly reduce some of the potential risks reportedly associated with the use of synthetic hormones. In the present study, we demonstrate the utility and advantage of a microfluidic chip culture system to enhance the development of personalized, on-demand, treatment modules using embryoid bodies (EBs). Functional EBs cultured on microfluidic chips represent a platform for personalized, patient-specific treatment cassettes that can be cryopreserved until required for treatment. We assessed the viability, differentiation, and functionality of EBs cultured and cryopreserved in this system. During extended microfluidic culture, estradiol, progesterone, testosterone, and anti-mullerian hormone levels were measured, and the expression of differentiated steroidogenic cells was confirmed by immunocytochemistry assay for the ovarian tissue markers anti-mullerian hormone receptor type II, follicle-stimulating hormone receptor, and inhibin p-A and the estrogen biosynthesis enzyme aromatase. Our studies showed that under microfluidic conditions, differentiated steroidogenic EBs continued to secrete estradiol and progesterone at physiologically relevant concentrations (30-120 pg/ml and 150-450 pg/ml, respectively) for up to 21 days. Collectively, we have demonstrated for the first time the feasibility of using a microfluidic chip system with continuous flow for the differentiation and extended culture of functional steroidogenic stem cell-derived EBs, the differentiation of EBs into cells expressing ovarian antigens in a microfluidic system, and the ability to cryopreserve this system with restoration of growth and functionality on thawing. These results present a platform for the development of a new therapeutic system for personalized medicine

Functional Maintenance of Differentiated Embryoid Bodies in Microfluidic Systems: A Platform for Personalized Medicine

Hormone replacement therapies have become important for treating diseases such as premature ovarian failure or menopausal complications. The clinical use of bioidentical hormones might significantly reduce some of the potential risks reportedly associated with the use of synthetic hormones. In the present study, we demonstrate the utility and advantage of a microfluidic chip culture system to enhance the development of personalized, on-demand, treatment modules using embryoid bodies (EBs). Functional EBs cultured on microfluidic chips represent a platform for personalized, patient-specific treatment cassettes that can be cryopreserved until required for treatment. We assessed the viability, differentiation, and functionality of EBs cultured and cryopreserved in this system. During extended microfluidic culture, estradiol, progesterone, testosterone, and anti-müllerian hormone levels were measured, and the expression of differentiated steroidogenic cells was confirmed by immunocytochemistry assay for the ovarian tissue markers anti-müllerian hormone receptor type II, follicle-stimulating hormone receptor, and inhibin β-A and the estrogen biosynthesis enzyme aromatase. Our studies showed that under microfluidic conditions, differentiated steroidogenic EBs continued to secrete estradiol and progesterone at physiologically relevant concentrations (30–120 pg/ml and 150–450 pg/ml, respectively) for up to 21 days. Collectively, we have demonstrated for the first time the feasibility of using a microfluidic chip system with continuous flow for the differentiation and extended culture of functional steroidogenic stem cell-derived EBs, the differentiation of EBs into cells expressing ovarian antigens in a microfluidic system, and the ability to cryopreserve this system with restoration of growth and functionality on thawing. These results present a platform for the development of a new therapeutic system for personalized medicine