Immunolocalization of stem/progenitor cell biomarkers Oct-4, C-kit and Musashi-1 in endometriotic lesions.

BACKGROUND: Human endometrium harbors stem/progenitor cells (SPCs) that may contribute to the establishment of endometriosis when seeded outside the uterus. Oct-4, C-kit and Musashi-1 are some of the many proteins used to characterize SPCs, but their association with endometriosis is uncertain. OBJECTIVE AND DESIGN: In this study, specimens of normal endometrium (n = 12), eutopic endometrium from women with endometriosis (n = 9), superficial peritoneal endometriosis (SUP, n = 12) and deep endometriosis (DE, n = 13) lesions were evaluated for localization and intensity of immunostaining for Oct-4, C-kit and Musashi-1. RESULTS: The three markers were abundantly expressed in normal endometrium, eutopic endometrium from endometriosis patients, SUP and DE specimens. Oct-4 and C-kit expression did not vary across groups as regards intensity or frequency. C-kit staining signal was seldom detected in vascular endothelium of normal or eutopic endometrium from endometriosis patients; however, it was positive in 67% of the SUP lesions and in 25% of the DE lesions (p = 0.042). Musashi-1 was expressed in some endometriotic glands as cell clusters, but its signal was similar between the four types of tissue (p = 0.971) CONCLUSION: The wide distribution of Oct-4, C-kit and Musashi-1 in endometria of patients with and without endometriosis and in SUP and DE endometriotic lesions suggests that these markers are not suitable for the in situ characterization of endometrial SPCs and should not be taken as surrogates for the study of SPCs in the pathogenesis of endometriosis.

Human oocyte morphometry before and after cryopreservation: A prospective cohort study.

The cryopreservation of human oocytes is an important strategy to spare fertility in women submitted to gonadotoxic therapy, ovarian surgery, or even to allow gestation by assisted reproduction technology after natural ovarian senescence. Methods to predict oocyte resistance to cryopreservation are still based on qualitative morphological assessment. In this study we evaluated whether morphometric characteristics of mature oocytes before vitrification and after warming are related to successful fertilization by intracytoplasmic sperm injection (ICSI). This was a prospective cohort study including 28 infertile women and 71 oocytes. Morphometric assessments included oocyte diameter, perivitelline space (PS), zona pellucida (ZP) and first polar body (PB). Out of 49 warmed oocytes, 27 (55%) survived cryopreservation and their pre-vitrification measures were similar to those of the 22 oocytes that perished. However, the oocytes that eventually failed to be fertilized had undergone more enlargement of the total diameter (p = 0.029) and shrinking of the PS (p = 0.033) after cryopreservation, compared to oocytes that were successfully fertilized. These findings suggest that the morphometric characteristics of fresh oocytes do not predict their survival to vitrification, while fertilization failure is associated with oocyte enlargement and PS shrinking after cryopreservation.

BMP-4 increases activin A gene expression during osteogenic differentiation of mouse embryonic stem cells.

Activin A is a growth factor released by mature osteoblasts that has a critical effect on bone formation. We investigated the effect of bone morphogenetic protein (BMP)-4 on activin A gene expression during in vitro osteogenic differentiation of mouse embryonic stem (ES) cells. Embryoid bodies were cultured in retinoic acid (RA) for three days and then without RA for two days. Seeded cells received osteogenic medium with ß-glycerophosphate, L-ascorbic acid 2-phosphate and dexamethasone during 19 days, with or without BMP-4. Six independent experiments were carried out. Real-time PCR was used to detect gene expression of activin A, Oct-4, Nanog, osteocalcin, RUNX2 and bone alkaline phosphatase. Immunofluorescence was used to co-localize activin A with the undifferentiation marker stage-specific embryonic antigen 1. Cells treated with BMP-4 had an increased gene expression of activin A, osteocalcin and bone alkaline phosphatase (p < 0.05). In conclusion, BMP-4 increases activin A gene expression during mouse ES cell differentiation into bone precursors.

Five-year follow-up of levonorgestrel-releasing intrauterine system versus thermal balloon ablation for the treatment of heavy menstrual bleeding: a randomized controlled trial.

BACKGROUND: The study was conducted to compare 5-year follow-up of levonorgestrel-releasing intrauterine system (LNG-IUS) or thermal balloon ablation (TBA) for the treatment of heavy menstrual bleeding (HMB). STUDY DESIGN: A prospective, randomized controlled trial comparing LNG-IUS (n=30) and TBA (n=28) was performed. Hysterectomy rates, hemoglobin level, bleeding pattern, well-being status and satisfaction rates were assessed. Comparisons between groups were performed by χ(2) test and by unpaired and paired t tests. RESULTS: After 5 years of follow-up, women treated with a TBA had higher rates of hysterectomy (24%) compared to the LNG-IUS group (3.7%) due to treatment failure (p=.039). Use of LNG-IUS resulted in higher mean hemoglobin (±SD) levels in comparison to the TBA group (14.1±0.3 vs 12.7±0.4 g/dL, p=.009). Menstrual blood loss was significantly higher in the TBA when compared to the LNG-IUS group (45.5% vs 0.0% p<.001). The psychological general well-being index scores were similar. Patient acceptability, perceived clinical improvement and overall satisfaction rates were significantly higher in women using LNG-IUS. CONCLUSION: Five-year follow-up of HMB treatment with LNG-IUS was associated with higher efficacy and satisfaction ratings compared to TBA.

Variation in neuronal differentiation of a newly isolated mouse embryonic stem cell line: a detailed immunocytochemistry study.

Neural precursor differentiation from mouse ES (embryonic stem) cells have been demonstrated using EB (embryoid body), co-culture on stromal feeder layers, and in the absence of external inducing signals. Most of available mouse ES cell original research articles have worked with only six different cell lines. Our goals were to isolate one new mouse ES lineage, and perform a detailed immunocytochemistry study during neural differentiation, making use of an EB strategy protocol following the generation of neural progenitors, glial cells and postmitotic neurons. The dynamics of differentiation of ES cell derived neuronal precursors into differentiated glia cells and neurons were followed in vitro and correlated to exposure to specific elements of feeder medium. Morphological aspects of generated cellular types, including its immunocytochemical expression of differentiation markers were studied. Immuno-positivity against ß-III tubulin, PGP and TH (tyrosine hydroxylase) was observed from stage I. Approximately 80% of cells were positive for TH at stage I. The first glial cell type appears in stage III. TH, PGP or ß-III tubulin-positive cells with neuronal typical morphology only being seen in stage III when TH-positive cells corresponded to approximately 12% of total cells. Variations among other literature findings can be explained by the choice we made to use a newly isolated ES cell line. As colonies may behave differently during neuronal differentiation, it reinforces the necessity of studying original ES cell lines.

Infusion of Sydenham's chorea antibodies in striatum with up-regulated dopaminergic receptors: a pilot study to investigate the potential of SC antibodies to increase dopaminergic activity.

BACKGROUND: Sydenham's chorea (SC) is a neurological manifestation of rheumatic fever. Autoimmune mechanism of SC is supported by clinical improvement with immunomodulatory therapy; presence of circulating serum anti-basal ganglia antibodies; increase in Th2 group of cytokines in serum and CSF of patients. However, a role of the antibodies in the pathogenesis can only be established by their passive transfer. Chorea is a manifestation clearly related to increased dopaminergic (DA) activity. The purpose of this study was to investigate the potential of antibodies from patients with Sydenham's chorea to cause behavior alterations on rats with unilateral post-synaptic dopamine receptor up-regulation. METHODS: Rats previously submitted to 6-hydroxidopamine (6-OH-DA) unilateral lesion of substantia nigra pars compacta (SNc) and tested with apomorphine to ensure DA receptors up regulation, received intrastriatal infusion of antibodies from SC patients (n=4) or healthy controls (n=3) during 48 h. 24h post infusion initiation (24PI) and 48 h post infusion initiation (48PI), we registered the occurrence of spontaneous contra lateral rotations (CLR). FINDINGS: SC group exhibited significantly higher number of CLR than control group at 24PI (p=0.049) and 48PI (p=0.048). CONCLUSION: The limited sample of the present study restricts us to affirm that SC is really an immune-mediated condition. However the significant result of this pilot study points to preliminary evidence that SC antibodies may affect DA activity in rats with up-regulated striatal DA receptors.

Dienogest: Uma nova opção terapêutica em endometriose / Dienogest: A new option in the treatment of endometriosis

Endometriose é uma doença crônica que afeta mulheres jovens em idade reprodutiva provocando dor, dispareunia, infertilidade e que também afeta a qualidade de vida das pacientes. Numerosos tratamentos existem, alguns clínicos e outros cirúrgicos. Entretanto, essa doença deve ser considerada como uma doença crônica que pode exigir muitos anos de tratamento, já que as recidivas são frequentes após as cirurgias ou após qualquer tratamento. Entre os novos tratamentos médicos, embora ainda não disponível no Brasil, está a administração oral de um novo progestagênio, Dienogest (DNG), o qual tem tido grande êxito no tratamento da endometriose, principalmente no tocante aos sintomas de dor associados. Esta revisão mostra os estudos mais relevantes realizados em diversos países com tratamentos em curto prazo - em geral 24 semanas - e em longo prazo - aproximadamente 53 semanas - e que mostraram alta eficácia, poucos eventos adversos e de pouca intensidade e alta satisfação das pacientes. A revisão também discute a relevância do DNG na prática clínica

Endometriosis is a chronic disease which affects young women at reproductive age and provokes pain, dyspareunia, infertility and which impaires patients' quality of life. Several treatments are available including medical and surgical. However, this disease must be considered as a chronic disease which could need many years of treatment because recurrences are common after surgery or any medical treatment. Among the new medical treatments, albeit not available in Brazil at the present time, there is the oral administration of a new progestin, Dienogest (DNG) which was successful in the treatment of endometriosis, mainly regarding pain-associated endometriosis. This review shows the main studies conducted in several countries with short term treatments - up to 24 weeks - as well as long-term treatments - in general up to 53 weeks - that showed high efficacy, few adverse events, and of middle or minimum intensity with high satisfaction of the patients. The review also discusses the relevance of DNG in the clinical practice

Telomerase activity alterations in sequential passages of mouse embryonic stem cells.

Telomerase is associated with cell proliferation capacity, protection and stabilization of chromosomes. TA (telomerase activity) can be detected in highly replicative cells, e.g. stem and cancer cells. Most available mESC (mouse embryonic stem cell) research is done with a few cell lines. The purpose of this study has been to evaluate the TA in different passages of newly isolated mESC. TRAP (Telomeric Repeat Amplification Protocol)-ELISA method was used in a semi-quantitative evaluation of TA. Three mESC lineages were investigated (CT2, CT3 and CT4) at three different passages (P13, P15 and P19). In contrast with previous studies, these mESC lines did not show the same TA throughout their passages, having initially low TA values, followed by a subsequent rise and stabilization.

Rapid thawing human sperm does not affect basic parameters in normozoospermic men: a double-blind prospective study.

PURPOSE: To compare sperm recovery from slow versus rapid thawing technique using thirty-eight normozoospermic human sperm samples, as follows. Twentyone samples from men taking part in routine infertility screening exams (infertile group) and seventeen from proven fertile volunteer men with at least one child (fertile group). MATERIALS AND METHODS: After analysis of motility, concentration, strict morphology and functional integrity of membranes, sperm was divided into two aliquots of 0.5 mL each and frozen in TyB-G medium. Samples were thawed at room temperature (25 ± 2° C) for 25 minutes (slow thaw) or in a water bath at 75° C for 20 seconds followed by water bath at 37° C for 3 minutes (rapid thaw). After thawing, motility, strict morphology and functional integrity of membranes were evaluated by a blinded investigator. The results were expressed as mean ± standard deviation for parametric variables and analyzed using Student's t-test. Data with unpaired non-parametric variables were expressed as median (interquartile range) and analyzed by the Mann-Whitney test. Wilcoxon test was used to analyze non-parametric paired variables. RESULTS: There was no significant difference between techniques for total and progressive motility, percentage of normal morphological forms, hypoosmotic swelling test. CONCLUSIONS: Although the rapid thawing protocol was completed in a shorter time (three minutes and 20 seconds versus 25 minutes, respectively), it wasn't harmful since both techniques showed comparable spermatozoa recovery. Additional research is needed to confirm its safety in clinical research before introducing this methodology in routine assisted reproduction.

Computer-assisted oocyte morphometry before ICSI: correlation of oocyte measurements with fertilization and embryo development.

The present study aimed to correlate morphometric parameters of the oocytes with the occurrence of fertilization following intracytoplasmic sperm injection (ICSI). In a prospective, controlled cohort design, women (n = 32) who were candidates for ICSI had oocytes (n = 258) collected and submitted to morphometric evaluation using the Cronus3 software program. The morphometric parameters obtained were oocyte diameter, perivitelline space width, zona pellucida thickness, and first polar body diameter. The median oocyte diameter was similar in cases in which fertilization occurred compared with those in which fertilization failed (75.2 and 75.9 µm, respectively; P = .218). The 2 groups also had similar measurements of perivitelline space, zona pellucida, and first polar body. However, the best quality zygotes identified by a morphological score resulted from oocytes with larger diameter (75.6 vs 74.0 µm; P < .01) and narrow perivitelline space (5.3 vs 7.1 µm; P < .01). Embryo development, as assessed by cleavage at second day of culture, was not significantly associated with oocyte morphometric parameters. These findings suggest that morphometric parameters of the oocytes do not correlate with the occurrence of fertilization following ICSI but may assist in selecting oocytes more likely to originate high-quality zygotes.

Rapid thawing human sperm does not affect basic parameters in normozoospermic men: a double-blind prospective study

PURPOSE: To compare sperm recovery from slow versus rapid thawing technique using thirty-eight normozoospermic human sperm samples, as follows. Twenty-one samples from men taking part in routine infertility screening exams (infertile group) and seventeen from proven fertile volunteer men with at least one child (fertile group). MATERIALS AND METHODS: After analysis of motility, concentration, strict morphology and functional integrity of membranes, sperm was divided into two aliquots of 0.5 mL each and frozen in TyB-G medium. Samples were thawed at room temperature (25 ± 2º C) for 25 minutes (slow thaw) or in a water bath at 75º C for 20 seconds followed by water bath at 37º C for 3 minutes (rapid thaw). After thawing, motility, strict morphology and functional integrity of membranes were evaluated by a blinded investigator. The results were expressed as mean ± standard deviation for parametric variables and analyzed using Student's t-test. Data with unpaired non-parametric variables were expressed as median (interquartile range) and analyzed by the Mann-Whitney test. Wilcoxon test was used to analyze non-parametric paired variables. RESULTS: There was no significant difference between techniques for total and progressive motility, percentage of normal morphological forms, hypoosmotic swelling test. CONCLUSIONS: Although the rapid thawing protocol was completed in a shorter time (three minutes and 20 seconds versus 25 minutes, respectively), it wasn't harmful since both techniques showed comparable spermatozoa recovery. Additional research is needed to confirm its safety in clinical research before introducing this methodology in routine assisted reproduction.

Development and prevention of postsurgical adhesions in a chimeric mouse model of experimental endometriosis.

OBJECTIVE: To examine the impact of a recent surgery on development of endometriosis-related adhesions in a chimeric model and to determine the therapeutic efficacy of pioglitazone (PIO). DESIGN: Human endometrial biopsies were maintained in E(2) with or without PIO for 24 h before intraperitoneal injection into immunocompromised mice also treated with or without PIO at multiple time points after peritoneal surgery. The presence and extent of adhesions were examined in animals relative to the initial establishment of experimental endometriosis. SETTING: Medical school research center. PATIENT(S): Endometrial biopsies for experimental studies were provided by normally cycling women without a medical history indicative of endometriosis or adhesions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Examination of the development of endometriosis-related adhesions in an experimental model. RESULT(S): Without therapeutic intervention, injection of E(2)-treated human endometrial tissue into mice near the time of peritoneal surgery resulted in multiple adhesions and extensive endometriotic-like disease. In contrast, PIO treatment reduced adhesive disease and experimental endometriosis related to surgical injury. CONCLUSION(S): The presence of human endometrial tissue fragments in the peritoneal cavity of mice with a recent surgical injury promoted development of both adhesive disease and experimental endometriosis. Targeting inflammation and angiogenesis with PIO therapy limited the development of postsurgical adhesions associated with ectopic endometrial growth.

Angiotensin-(1-7), its receptor Mas, and the angiotensin-converting enzyme type 2 are expressed in the human ovary.

OBJECTIVE: To investigate whether angiotensin (Ang)-(1-7), its receptor Mas, and angiotensin-converting enzyme type 2 (ACE2) are present in human ovary. DESIGN: Cross-sectional study. SETTING: Academic hospital. PATIENT(S): Twelve reproductive-age women and five postmenopausal women undergoing oophorectomy for nonovarian diseases and seven women having controlled ovarian hyperstimulation for IVF. INTERVENTION(S): Ovarian tissue was obtained from the reproductive-age women and postmenopausal women undergoing oophorectomy for nonovarian diseases. Follicular fluid (FF) samples were obtained from the women having controlled ovarian hyperstimulation for IVF. MAIN OUTCOME MEASURE(S): Localization of Ang-(1-7) and Mas by immunohistochemistry; measurement of Ang-(1-7) in ovarian FF by RIA; detection of messenger RNAs encoding Mas and ACE2 with use of real-time polymerase chain reaction; assessment of 125I-labeled Ang-(1-7) binding to ovarian sections with use of autoradiographic binding assay. RESULT(S): Angiotensin-(1-7) and the receptor Mas were localized to primordial, primary, secondary, and antral follicles, stroma, and corpora lutea of reproductive-age ovaries. Postmenopausal women expressed both the peptide and its receptor in the ovarian stroma. Angiotensin-(1-7) was detectable in FF (mean±SE: 191±54 pg/mL). Both Mas and ACE2 messenger RNAs were expressed in ovarian tissue, as revealed by real-time polymerase chain reaction, and ovarian binding sites for 125I-labeled Ang-(1-7) were identified by autoradiography. CONCLUSION(S): Angiotensin-(1-7), its receptor Mas, and ACE2 are expressed in the human ovary. The peptide is present in several ovarian compartments and can be quantified in FF.

Células-tronco: a resposta para os enigmas da patogênese da endometriose / Stem cells: the answer to the puzzles of the pathogenesis of endometriosis

A endometriose é uma doença crônica caracterizada pela presença de tecido endometrial ectópico. Esta doença frequentemente resulta em alta morbidade, incluindo dor pélvica crônica e infertilidade. A causa da endometriose é provavelmente multifatorial, sendo ainda objeto de muitos estudos. Fatores genéticos, ambientais e imunes estão possivelmente envolvidos na etiopatogenia desta doença, sendo especulada a associação com outros mecanismos. Nos últimos anos, alguns trabalhos têm demonstrado a presença de células-tronco/progenitoras no endométrio sadio; tais células possivelmente estão envolvidas na capacidade regenerativa desse tecido, assim como na patogênese de doenças ginecológicas proliferativas, como endometriose e carcinoma endometrial. Esta revisão avaliou as evidências disponíveis sobre a existência de células tronco/progenitoras endometriais e agrupou os resultados em uma hipótese de envolvimento dessas células na patogênese da endometriose. Foram selecionados os artigos mais relevantes sobre o tema. A identificação de células-tronco nos tecidos humanos e animais é presumida a partir da identificação de label retaining cells, células side population, marcadores de indiferenciação, potencial de clonogenicidade e diferenciação celular. A presença de supostas células-tronco no endométrio normal e ectópico foi demonstrada por alguns pesquisadores. Células endometriais no endométrio eutópico e em implantes endometrióticos, originadas a partir de células-tronco derivadas de medula óssea transplantada em humanos e de animais, também foram identificadas. Esses resultados sugerem que as células-tronco/progenitoras podem estar envolvidas na gênese da endometriose. No entanto, mais estudos são necessários para corroborar esta hipótese.

Endometriosis is a chronic disease characterized by the presence of ectopic endometrial tissue. This disease often results in high degree of morbidity, including chronic pelvic pain and infertility. Probably, the cause of endometriosis is multifactorial and it has been the object of many studies. Genetic, environmental and immune factors are possibly involved in the pathogenesis of this disease, and it is speculated that there are other mechanisms associated. In recent years, some studies have shown the presence of adult stem cells in the healthy endometrium. These cells are possible involved in the regenerative capability of endometrium and in the pathogenesis of proliferative gynecological diseases, such as endometriosis and endometrial carcinoma. This review evaluated the available evidence on the existence of stem/progenitor cells in the endometrium and gathered the results in an hypothesis of involvement of these cells in the pathogenesis of endometriosis. The most relevant articles about this subject were selected. The identification of stem cells in animal and human tissues is presumed from the identification of label retaining cells, side population cells, undifferentiation markers, besides the potential of clonogenesis and cellular differentiation. The presence of putative stem cells in the normal and ectopic endometrium was demonstrated by some researches. Endometrial cells in eutopic endometrium and endometriotic implants, originated from bone marrow-derived stem cells transplanted into humans and animals, have also been identified. These results suggest that stem/progenitor cells may be involved in the genesis of endometriosis. However, more studies are necessary to this hypothesis

Assessment of fertility protection and ovarian reserve with GnRH antagonist in rats undergoing chemotherapy with cyclophosphamide.

BACKGROUND: Reproductive function following chemotherapy is of increasing importance given that survival rates are improving. We assessed whether a gonadotropin-releasing hormone antagonist (GnRHant; cetrorelix) could promote ovarian protection against damage due to chemotherapy. METHODS: Forty-two female Wistar rats were used in this study. Animals were divided into four groups: group I (n=9) received placebo twice; group II (n=12) received placebo+cyclophosphamide (CPA); group III (n=12) received GnRHant+CPA; and group IV (n=9) received GnRHant+placebo. After medication, the estrous cycle was studied through vaginal smears. Rats were mated, pregnancy was documented and the number of live pups evaluated. Afterwards, rat ovaries were removed and prepared for histological studies. The ovarian cross-sectional area was measured and follicles were counted. RESULTS: Cyclic changes in vaginal smears were observed in all but one animal after treatment, but group II had a significantly lower rate of animals with proestrus or estrus (p<0.01). The offspring was markedly reduced by CPA treatment (group II, 3.00+/-1.33 pups vs. group I, 11.44+/-0.78 pups, p<0.01) and this effect was partly reversed by pre-treatment with GnRHant (group III, 7.00+/-1.31 pups). The ovarian cross-sectional area was not significantly different between groups, neither was the number of individual follicle types. However, rats in Group IV had a higher total number of ovarian follicles than those in the control group (17.1+/-1.22 vs. 10.9+/-0.70, p<0.05). CONCLUSION: The use of a GnRHant before CPA chemotherapy provided protection of fertility.