Creating an Artificial 3-Dimensional Ovarian Follicle Culture System Using a Microfluidic System.

We hypothesized that the creation of a 3-dimensional ovarian follicle, with embedded granulosa and theca cells, would better mimic the environment necessary to support early oocytes, both structurally and hormonally. Using a microfluidic system with controlled flow rates, 3-dimensional two-layer (core and shell) capsules were created. The core consists of murine granulosa cells in 0.8 mg/mL collagen + 0.05% alginate, while the shell is composed of murine theca cells suspended in 2% alginate. Somatic cell viability tests and hormonal assessments (estradiol, progesterone, and androstenedione) were performed on days 1, 6, 13, 20, and 27. Confocal microscopy confirmed appropriate compartmentalization of fluorescently-labeled murine granulosa cells to the inner capsule and theca cells to the outer shell. Greater than 78% of cells present in capsules were alive up to 27 days after collection. Artificially constructed ovarian follicles exhibited intact endocrine function as evidenced by the production of estradiol, progesterone, and androstenedione. Oocytes from primary and early secondary follicles were successfully encapsulated, which maintained size and cellular compartmentalization. This novel microfluidic system successfully encapsulated oocytes from primary and secondary follicles, recapitulating the two-compartment system necessary for the development of the mammalian oocyte. Importantly, this microfluidic system can be easily adapted for sterile, high throughput applications.

Rapamycin Corrects T Regulatory Cell Depletion and Improves Embryo Implantation and Live Birth Rates in a Murine Model.

There are few treatments for patients with recurrent pregnancy loss (RPL) or recurrent implantation failure (RIF). Women with RPL and unexplained infertility have lower T regulatory cell (Treg) expression when compared to fertile controls. A murine model has been developed with depletion of regulatory T cells (DEREG) after administration of diphtheria toxin (DT), resulting in smaller litter sizes, secondary to embryo implantation failure. Numerous murine studies have shown that adoptive transfer of CD4+CD25+FoxP3+ Tregs from donors improves litter sizes in DEREG mice with depleted Tregs. Our hypothesis is that DEREG mice treated with a single dose of DT will deplete Tregs and subsequently decrease litter sizes and that treatment with rapamycin (sirolimus; Pfizer) during the time of embryo implantation will increase Tregs and restore litter sizes nearly back to normal levels. Syngeneic mating of DEREG mice after depletion of Tregs resulted in smaller litter sizes and this defect was reversed when these DEREG mice were treated with rapamycin at the time of embryo implantation. The importance of Tregs at the time of embryo implantation has been well established and immunotherapy treatments, such as rapamycin (mammalian target of rapamycin inhibitor), may prove to be an effective treatment for patients with RPL, RIF, or unexplained infertility with low Treg.

Oocyte cryopreservation for women with GATA2 deficiency.

PURPOSE: To describe controlled ovarian stimulation (COS) in a population of women with GATA2 deficiency, a genetic bone marrow failure syndrome, prior to allogeneic hematopoietic stem cell transplant METHODS: This is a retrospective case series of nine women with GATA2 deficiency who underwent oocyte preservation at a research institution. Main outcomes measured include baseline fertility characteristics ((antimullerian hormone (AMH) and day 3 follicle-stimulating hormone (FSH) and estradiol (E2)) and total doses of FSH and human menopausal gonadotropins (HMG), E2 on day of trigger, and total number of metaphase II oocytes retrieved. RESULTS: The mean age was 24 years [16-32], mean AMH was 5.2 ng/mL [0.7-10], and day 3 mean FSH was 5.1 U/L [0.7-8.1], and E2 was 31.5 pg/mL [< 5-45]. The mean dose of FSH was 1774 IU [675-4035], and HMG was 1412 IU [375-2925] with a mean E2 of 2267 pg/mL [60.7-4030] on day of trigger. The mean total of metaphase II oocytes was 7.7 [0-15]. One patient was diagnosed with a deep vein thrombosis (DVT) with pulmonary embolism (PE) during COS. CONCLUSION: This study is the first to analyze the outcomes of COS in women with GATA2 deficiency. The response to ovarian stimulation suggests that oocyte cryopreservation should be considered prior to gonadotoxic therapy. However, due to the risk of potentially life-threatening complications, it is prudent that patients are properly counseled of the risks and are evaluated by a multi-disciplinary medical team prior to COS.

Effects of hormones on skin wrinkles and rigidity vary by race/ethnicity: four-year follow-up from the ancillary skin study of the Kronos Early Estrogen Prevention Study.

OBJECTIVE: To measure skin wrinkles and rigidity in menopausal women of varying race/ethnicity with or without hormone therapy (HT) for up to four years. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: Academic medical centers. PATIENT(S): Women (42-58 years of age) within 36 months of last menstrual period and enrolled in the Kronos Early Estrogen Prevention Study (KEEPS). INTERVENTION(S): Treatment with 0.45 mg oral conjugated equine estrogens (CEE), transdermal E2 (50 µg/d) with micronized P (200 mg daily), or placebo for 4 years. MAIN OUTCOME MEASURE(S): Skin wrinkles were assessed at 11 locations on the face and neck, and skin rigidity was assessed at the forehead and cheek at baseline and yearly for 4 years. RESULT(S): Neither total wrinkle score nor total rigidity score was significantly different at baseline or over the 4-year follow-up among patients randomized to CEE, E2, or placebo. Skin wrinkle and rigidity scores were primarily affected by race/ethnicity, with scores being significantly different between races for almost all of the wrinkle parameters and for all of the rigidity measures. There was no association between race and response to HT for total wrinkle or rigidity scores. Black women had the lowest wrinkle scores compared with white women across all 4 years. In general, skin rigidity decreased in all groups over time, but black women had significantly reduced total facial rigidity compared with white women after 4 years. CONCLUSION(S): Race is the strongest predictor of the advancement of skin aging in the 4 years following menopause. HT does not appear to affect skin wrinkles or rigidity at most facial locations. CLINICAL TRIAL REGISTRATION NUMBER: NCT00154180.

Are intracytoplasmic sperm injection and high serum estradiol compounding risk factors for adverse obstetric outcomes in assisted reproductive technology?

OBJECTIVE: To evaluate whether intracytoplasmic sperm injection (ICSI) use and E2 on the final day of assisted reproductive technology (ART) stimulation are associated with adverse obstetric complications related to placentation. DESIGN: Retrospective cohort study. SETTING: Large private ART practice. PATIENT(S): A total of 383 women who underwent ART resulting in a singleton live birth. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Adverse placental outcomes composed of placenta accreta, placental abruption, placenta previa, intrauterine growth restriction, preeclampsia, gestational hypertension, and small for gestational age infants. RESULT(S): Patients with adverse placental outcomes had higher peak serum E2 levels and were three times more likely to have used ICSI. Adverse placental outcomes were associated with increasing E2 (odds ratio 1.36, 95% confidence interval 1.13-1.65) and ICSI (odds ratio 3.86, 95% confidence interval 1.61-9.27). Adverse outcomes increased when E2 was >3,000 pg/mL and continued to increase in a linear fashion until E2 was >5,000 pg/mL. The association of ICSI with adverse outcomes was independent of male factor infertility. Interaction testing suggested the adverse effect of E2 was primarily seen in ICSI cycles, but not in conventional IVF cycles. Estradiol >5,000 pg/mL was associated with adverse placental events in 36% of all ART cycles and 52% of ICSI cycles. CONCLUSION(S): ICSI and elevated E2 on the day of hCG trigger were associated with adverse obstetric outcomes related to placentation. The finding of a potential interaction of E2 and ICSI with adverse placental events is novel and warrants further investigation.

Oral prenylation inhibition with lonafarnib in chronic hepatitis D infection: a proof-of-concept randomised, double-blind, placebo-controlled phase 2A trial.

BACKGROUND: Therapies for chronic hepatitis delta virus (HDV) infection are unsatisfactory. Prenylation is essential for HDV and inhibition abrogates HDV production in experimental models. In a proof-of-concept study, we aimed to assess the effect on HDV RNA levels, safety, and tolerability of the prenylation inhibitor lonafarnib in patients with chronic delta hepatitis. METHODS: In this phase 2A double-blind, randomised, placebo-controlled study, patients aged 18 years or older with chronic HDV infection were randomly assigned (3:1 in group 1 and 2:1 in group 2) to receive lonafarnib 100 mg (group 1) or lonafarnib 200 mg (group 2) twice daily for 28 days with 6 months' follow-up. Participants were randomised by random-number tables blocked in groups of four without stratification. Both groups enrolled six treatment participants and two placebo participants. Group 1 placebo patients received open-label lonafarnib as group 2 participants. The primary therapeutic endpoint was a decrease in HDV RNA viral titre in serum and the primary safety endpoint was the ability to tolerate the drug at the prescribed dose for the full 4-week duration, defined as drug discontinuation due to intolerance or grade 3/4 adverse events. This trial is registered with ClinicalTrials.gov, number NCT01495585. FINDINGS: Between Jan 19, 2012, and April 28, 2014, 14 patients were enrolled, of whom eight were assigned to group 1 and six were assigned to group 2. At day 28, compared with placebo, mean log HDV RNA declines from baseline were -0·73 log IU/mL in group 1 (95% CI 0·17-1·31; p=0·03) and -1·54 log IU/mL in group 2 (1·21-1·93; p<0·0001). Lonafarnib serum concentrations correlated with HDV RNA change (r(2)=0·78, p<0·0001). Model fits show that hepatitis B surface antigen (HBsAg) remained stable after a short pharmacological delay (0·75 days [SE 0·24]), lonafarnib effectiveness in blocking HDV production was greater in group 2 than in group 1 (0·952 [SE 0·06] vs 0·739 [0·05], p<0·001), and the HDV half-life was 1·62 days (0·07). There was no evidence of virological resistance. Adverse events were mainly mild to moderate with group 1 patients experiencing diarrhoea in three patients (50%) and nausea in two patients (33%) and in group 2 with all patients (100%) experiencing nausea, diarrhoea, abdominal bloating, and weight loss greater than 2 kg (mean of 4 kg). No treatment discontinuations occurred in any treatment groups. INTERPRETATION: Treatment of chronic HDV with lonafarnib significantly reduces virus levels. The decline in virus levels significantly correlated with serum drug levels, providing further evidence for the efficacy of prenylation inhibition in chronic HDV. FUNDING: National Institute of Diabetes and Digestive and Kidney Diseases and National Cancer Institute, National Institutes of Health, and Eiger Biopharmaceuticals Inc.

Endometrial stem cell transplantation in MPTP- exposed primates: an alternative cell source for treatment of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra. Cell-replacement therapies have emerged as a promising strategy to slow down or replace neuronal loss. Compared to other stem cell types, endometrium-derived stem cells (EDSCs) are an attractive source of stem cells for cellular therapies because of their ease of collection and vast differentiation potential. Here we demonstrate that endometrium-derived stem cells may be transplanted into an MPTP exposed monkey model of PD. After injection into the striatum, endometrium-derived stem cells engrafted, exhibited neuron-like morphology, expressed tyrosine hydroxylase (TH) and increased the numbers of TH positive cells on the transplanted side and dopamine metabolite concentrations in vivo. Our results suggest that endometrium-derived stem cells may provide a therapeutic benefit in the primate model of PD and may be used in stem cell based therapies.

Ovarian reserve and subsequent assisted reproduction outcomes after methotrexate therapy for ectopic pregnancy or pregnancy of unknown location.

OBJECTIVE: To assess ovarian reserve after methotrexate treatment for ectopic pregnancy or pregnancy of unknown location after assisted reproductive technology (ART). DESIGN: Retrospective cohort study. SETTING: Large ART practice. PATIENT(S): Women receiving methotrexate or surgery after ART. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicle-stimulating hormone (FSH), antral follicle count (AFC), and oocyte yield compared between women treated with methotrexate or surgery, with secondary outcomes of clinical pregnancy and live birth. RESULT(S): There were 153 patients in the methotrexate group and 36 patients in the surgery group. Neither group demonstrated differences in ovarian reserve or oocyte yield in a comparison of the before and after treatment values. The change in ovarian reserve and oocyte yield after treatment were similar between the two groups. The number of doses of methotrexate was not correlated with changes in ovarian reserve, indicating no dose-dependent effect. Time between treatment and repeat ART was not correlated with outcomes. Live birth in subsequent cycles was similar in the two groups. CONCLUSION(S): Ovarian reserve and subsequent ART cycle outcomes were reassuring after methotrexate or surgical management of ectopic pregnancy. No adverse impact of methotrexate was detected in this large fertility cohort as has been previously described elsewhere.

Peripheral blood stem cell transplants do not result in endometrial stromal engraftment.

OBJECTIVE: To determine whether peripheral blood stem cell transplant (PBSCT) result in engraftment of donor stem cells in the recipient uterus. DESIGN: Prospective clinical and laboratory research. SETTING: Translational medicine research hospital. PATIENT(S)/ANIMAL(S): Macaque and human bone marrow transplant recipients. INTERVENTION(S): Rhesus macaques received autologous transduced immunoselected cytokine-mobilized CD34+ cells after total body irradiation. Vector constructs expressed green fluorescent protein. In the human subjects, prior PBSCT subjects underwent endometrial biopsy and bone marrow aspiration. Macaque and human endometrial and bone marrow cells were isolated and cultured. Fluorescent microscopy, flow cytometry, and polymerase chain reaction (PCR) were used to evaluate for the presence of donor-derived cells. MAIN OUTCOME MEASURE(S): Presence of donor cells in recipient endometrium and bone marrow stroma. RESULT(S): The macaque endometrial cells did not exhibit evidence of green fluorescent protein labeling. Human endometrial cells were cultured and the absence of donor blood contamination was verified. The PCR evaluation of the human endometrial cells did not demonstrate evidence of donor short tandem repeats. CONCLUSION(S): The PBSCT did not result in engraftment of donor-derived cells in the endometrium.

Aromatase inhibitor treatment of menorrhagia and subsequent pregnancy in a patient with familial hyperparathyroidism-jaw tumor syndrome.

OBJECTIVE: To describe the clinical management of menorrhagia in a woman with hyperparathyroidism-jaw tumor syndrome (HPT-JT). DESIGN: Case report. SETTING: Large translation research hospital. PATIENT(S): A 26-year-old nulligravid woman with familial HPT-JT presented with life-long menorrhagia resistant to progesterone intrauterine device (IUD) therapy and a desire for fertility. INTERVENTION(S): Aromatase inhibitor therapy. MAIN OUTCOME MEASURE(S): Clinical response to therapy and pregnancy. RESULT(S): Imaging demonstrated an enlarged endometrial lining and thickening of the junctional zone. At operative hysteroscopy, multiple atypical endometrial polyp-like lesions filled the entire uterine cavity and were removed. Histologic evaluation demonstrated the lesions to be adenomyomas with an abundance of aromatase expression. Postoperative treatment included an aromatase inhibitor. The patient's menorrhagia, which had previously been resistant to progesterone IUD therapy, resolved with the aromatase inhibitor. After 10 months of this treatment, the aromatase inhibitor was discontinued and a repeated hysteroscopy revealed a markedly improved uterine cavity. The patient subsequently became pregnant on her first natural cycle and delivered a healthy term infant. CONCLUSION(S): Aromatase inhibitors may represent a novel treatment for benign uterine pathology in HPT-JT.

Endometrial stem cell transplantation restores dopamine production in a Parkinson's disease model.

Parkinson's disease (PD) is a neurodegenerative disorder caused by the loss of dopaminergic neurons. Adult human endometrial derived stem cells (HEDSC), a readily obtainable type of mesenchymal stem-like cell, were used to generate dopaminergic cells and for transplantation. Cells expressing CD90, platelet derived growth factor (PDGF)-Rß and CD146 but not CD45 or CD31 were differentiated in vitro into dopaminergic neurons that exhibited axon projections, pyramidal cell bodies and dendritic projections that recapitulate synapse formation; these cells also expressed the neural marker nestin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Whole cell patch clamp recording identified G-protein coupled inwardly rectifying potassium current 2 channels characteristic of central neurons. A 1-methyl 4-phenyl 1,2,3,6-tetrahydro pyridine induced animal model of PD was used to demonstrate the ability of labelled HEDSC to engraft, migrate to the site of lesion, differentiate in vivo and significantly increase striatal dopamine and dopamine metabolite concentrations. HEDSC are a highly inducible source of allogenic stem cells that rescue dopamine concentrations in an immunocompetent PD mouse model.

Expression of benign and malignant thyroid tissue in ovarian teratomas and the importance of multimodal management as illustrated by a BRAF-positive follicular variant of papillary thyroid cancer.

BACKGROUND: The most common type of ovarian germ cell tumor is the teratoma. Thyroid tissue, both benign and malignant, may be a component of an ovarian teratoma. Here we review this topic and illustrate major features by presenting multimodal management of a patient with BRAF-positive disseminated follicular thyroid cancer arising in an ovarian teratoma. SUMMARY: Malignant thyroid tissue is often difficult to distinguish from benign thyroid tissue arising in ovarian teratomas. Preoperatively, an elevated thyroglobulin (Tg) level, laboratory or clinical evidence of hyperthyroidism, or ultrasonography appearance of "struma pearl" should prompt referral to oncologist for surgical management of a possibly malignant ovarian teratoma. Postoperatively, tumor tissue should be referred to pathologists experienced with differentiating benign from malignant struma ovarii. Once diagnosed, treatment of this rare condition should be handled by a team of specialists with combined treatment modalities. We cared for woman with disseminated thyroid cancer arising in an ovarian teratoma whose history illustrates the complexity of managing ovarian teratomas with malignant thyroid tissue. At age 33 she had an intraoperative rupture of an ovarian cyst, thought to be struma ovarii. During her next pregnancy, pelvic masses were noted; biopsies revealed well-differentiated papillary thyroid carcinoma, follicular variant. She was euthyroid, but had elevated serum Tg levels. Surgical staging demonstrated widely metastatic intraabdominal dissemination. A thyroidectomy revealed no malignancy. A post-(131)I treatment scan revealed diffuse uptake throughout the abdomen. She then developed abdominal pain and, on computed tomography, was found to have multiple intraabdominal foci of disease. Serum Tg was 264 ng/mL while on L-thyroxine for hypothyroidism and to obtain thyrotropin suppression. A 18 fluorodeoxyglucose positron emission tomography scan showed no pathological uptake. The tumor was found to be BRAF mutation positive (K601E). She underwent extensive secondary debulking and a second course of (131)I with lithium pretreatment. Posttreatment scan revealed diffuse abdominal uptake. Six months posttherapy, the patient is asymptomatic with a serum Tg of 18.1 ng/mL. CONCLUSIONS: Aggressive multimodal management appears to be the most promising approach for malignant thyroid tissue arising in ovarian teratomas.

Demonstration of multipotent stem cells in the adult human endometrium by in vitro chondrogenesis.

Stem cells are defined by their unique capacity for self-renewal and multilineage differentiation. Stem cells have been obtained from multiple extramedullary tissues. Recently, a population of progenitor cells have been identified in the endometrium. However, multilineage differentiation of endometrial stem cells has not been reported. Endometrial tissue was obtained from reproductive-aged women undergoing surgery for benign disease, from which monolayer endometrial stromal cell (ESC), myometrial, fibroid, fallopian tube, and uterosacral ligament tissue cultures were generated. Once confluent, cells were trypsinized and centrifuged in conical tubes to form a cell pellet. Cell pellets were cultured in a defined chondrogenic media (CM) containing dexamethasone and transforming growth factor (TGF)-beta2 or TGF-beta 3 for 3 to 21 days. Samples were analyzed for markers of human articular cartilage, including sulfated glycosaminoglycans and expression of type II collagen. ESC pellets cultured in CM were found to contain cells that resemble chondrocytes. These cells expressed sulfated glycosaminoglycans and type II collagen typical of human articular cartilage. Myometrial, fibroid, fallopian tube, and uterosacral ligament cells were unable to undergo chondrogenic differentiation using the pellet culture method. Cells derived from the endometrium were able to differentiate into a heterologous cell type: chondrocytes, thus demonstrating the presence of multipotent stem cells. Endometrium is a potential source of multipotent stem cells.

Long-term effects of hormone therapy on skin rigidity and wrinkles.

OBJECTIVE: To evaluate the effects of long-term hormone therapy (HT) on skin rigidity and wrinkling. DESIGN: Single blinded cross-sectional analysis. SETTING: Academic medical center. PATIENT(S): Sixty-five long-term HT users who underwent menopause at least 5 years before evaluation and who have either consistently used HT or have never used HT. INTERVENTION(S): Visual assessment of severity of wrinkles at 11 facial locations using the Lemperle scale by a plastic surgeon blinded to HT use. Measurement of skin rigidity at the cheek and forehead with a durometer. MAIN OUTCOME MEASURE(S): Lemperle wrinkle score and skin rigidity. RESULT(S): Twenty women met inclusion criteria. Eleven women who had not used HT were compared to nine long-term HT users. Demographics including age, race, sun exposure, sunscreen use, tobacco use, and skin type were similar. Rigidity was significantly decreased in HT users compared to nonusers at both the cheek (1.1 vs. 2.7) and forehead (20 vs. 29). Average wrinkle scores were lower in hormone users than in nonhormone users (1.5 vs. 2.2). CONCLUSION(S): Long-term postmenopausal HT users have more elastic skin and less severe wrinkling than women who never used HT, suggesting that hormone therapy may have cosmetic benefits.

Value of the day 3 follicle-stimulating hormone measurement.

Although elevated day 3 FSH is associated with diminished ovarian reserve, the predictive value is low in young women. Its use in this population as an exclusion criterion is unjustified.