External pH Variations Modify Proliferation, Erythrophagocytosis, Cytoskeleton Remodeling, and Cell Morphology of Entamoeba histolytica Trophozoites.

To infect the human host, Entamoeba histolytica carries out processes requiring cytoskeleton remodeling, which involves reorganizing the actin fibers. However, little is known about the external influence factors, e.g., the pH, on the parasite's cytoskeleton remodeling or cell morphology. Such influence becomes relevant given the pH gradient that the amoeba cope with when going through the human colonic mucus during infection. Therefore, we analyzed the proliferation, the reorganization of the actin fibers, and other actin structures and cell shape during adhesion to fibronectin and erythrophagocytosis in trophozoites at different external pH conditions (6.0, 6.5, 6.8, 7.5, 8.0). We found that the best condition of external pH to perform such functions was 6.8. At acid pH, the trophozoites presented better-defined actin fibers that formed a more compact network, while at alkaline pH, the fibers reorganized, forming a looser and less defined network. Similarly, the number of actin dots also changed from acid to alkaline pH. In conclusion, the external pH alters the proliferation of the amoebas and promotes the dynamic restructuration of their cytoskeleton, allowing them to carry out their functions.

Characterization of low molecular weight protein tyrosine phosphatases of Entamoeba histolytica.

Entamoeba histolytica is an intestinal protozoan parasite of humans and is endemic in developing countries. E. histolytica has two low molecular weight protein tyrosine phosphatase (LMW-PTP) genes, EhLMW-PTP1 and EhLMW-PTP2, which are expressed in cultured trophozoites, clinical isolates, and cysts. The amino acid sequences of proteins EhLMW-PTP1 and EhLMW-PTP2 showed only one amino acid difference between them at position A85V, respectively. Both genes are expressed in cultured trophozoites, mainly EhLMW-PTP2, and in trophozoites recovered from amoebic liver abscess, the expression of EhLMW-PTP1 is downregulated. We cloned the two genes and purified the corresponding recombinant (rEhLMW-PTPs) proteins. Antibodies anti-rEhLMW-PTP2 showed that during red blood cells uptake by E. histolytica, the EhLMW-PTPs were found in the phagocytic cups based on analysis of fluorescence signals. On the other hand, rEhLMW-PTPs showed an optimum phosphatase activity at pH 6.0 with p-nitrophenyl phosphate as the substrate. They dephosphorylate phosphotyrosine and 3-O-methylfluorescein phosphate, but not phosphoserine or phosphothreonine, and the enzymatic activity is inhibited by orthovanadate. rEhLMW-PTP1 and rEhLMW-PTP2 exhibited optimum temperatures of activities at 60 °C and 58 °C, respectively, with high thermal stability at 50 °C. Also, the rEhLMW-PTPs showed high specific activities and specific km value with pNPP or OMFP as the substrates at the physiological temperature (37 °C).

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract.

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

Transcriptional control of hyphal morphogenesis in Candida albicans.

Candida albicans is a multimorphic commensal organism and opportunistic fungal pathogen in humans. A morphological switch between unicellular budding yeast and multicellular filamentous hyphal growth forms plays a vital role in the virulence of C. albicans, and this transition is regulated in response to a range of environmental cues that are encountered in distinct host niches. Many unique transcription factors contribute to the transcriptional regulatory network that integrates these distinct environmental cues and determines which phenotypic state will be expressed. These hyphal morphogenesis regulators have been extensively investigated, and represent an increasingly important focus of study, due to their central role in controlling a key C. albicans virulence attribute. This review provides a succinct summary of the transcriptional regulatory factors and environmental signals that control hyphal morphogenesis in C. albicans.

Does acupuncture the day of embryo transfer affect the clinical pregnancy rate? Systematic review and meta-analysis.

The effects of acupuncture on IVF outcomes is still unknown. We carried out a systematic review and meta-analysis of RCT to determine whether acupuncture performed at the time of ET improves outcomes. We searched Medline and Embase from January 1990 to June 2017, for the following terms): (acupuncture; acupuncture therapy) and (reproductive techniques, assisted; in vitro fertilization; embryo transfer). We selected RCT that compared acupuncture with sham acupuncture or no treatment. We included only trials in which acupuncture involved the insertion of needles into traditional meridian points. We evaluated the methodological quality of the trials using the Cochrane risk of bias tool. The measure of treatment effect was the pooled odds ratio of achieving a clinical pregnancy, ongoing pregnancy, or live birth for women in the acupuncture group compared with women in the control group. For pooled data, summary test statistics were calculated using the Mantel-Haenszel method, using the Rev-Man software, version 5.1. We analyzed six studies, including 2,376. In all trials, there were no significant differences between the groups concerning the mean numbers of embryos transferred, the mean age of the women undergoing the procedure, diagnose and use of ICSI. Acupuncture performed the day of ET was associated with a reduced risk of clinical pregnancy (0.87, 95% confidence interval 0.77 to 0.98). The pooled rate difference was -0.06 (-0.12 to -0.01) for clinical pregnancy. None of the trials reported significant adverse effects of acupuncture.

DHEA use to improve likelihood of IVF/ICSI success in patients with diminished ovarian reserve: A systematic review and meta-analysis.

The aim of this review is to determine if the use of DHEA increases the likelihood of success in patients with POR. We searched MEDLINE and EMBASE using the terms "DHEA and diminished ovarian reserve", "DHEA and poor response", "DHEA and premature ovarian aging". A fixed effects model was used and Peto's method to get the odds ratio (OR) with 95% confidence intervals (CI 95%). For quantitative variables, Cohen's method was used to present the standardized mean differences (SMD) with their corresponding confidence intervals. Only five studied fulfilled the selection criteria. DHEA was administered in 25 mg doses, three times a day. In all studies, the authors corrected for the presence of confounding variables such as partner's age, infertility diagnosis and number of transferred embryos. The meta-analysis of the five selected studies assessed a total of 910 patients, who underwent IVF/ICSI, of which 413 had received DHEA. DHEA use was associated with a significant increase in pregnancy likelihood (OR 1.8, CI 95% 1.29 to 2.51, p=0.001). When analyzing the association between DHEA use and the likelihood of abortion, we found low heterogeneity between studies (I2=0.0%) and the use of DHEA to be associated to a significant reduction in the likelihood of abortion (OR 0.25, CI 0.07 to 0.95; p=0.045). Analysis of the association of DHEA with average oocyte retrieval showed high variability between studies (I2=98.6%), as well as no association between DHEA use and the number of oocytes retrieved (SMD -0.01, CI 95% -0.16 to 0.13; p<0.05).

Is the risk of preeclampsia higher in donor oocyte pregnancies? A systematic review and meta-analysis.

OBJECTIVE: Preeclampsia (PE) occurs in 4.6% of pregnancies worldwide. The social phenomenon of increasing maternal age has raised the demand for donor oocytes. Egg donation has allowed women with poor ovarian reserve, premature ovarian failure, genetic disorders or surgical menopause to get pregnant. Recipients provide a unique model of immune response because of the differences in the genetic makeup of mothers and fetuses. In PE, immune tolerance may be impaired as a result of having non-autologous eggs implanted. Egg donation is a highly successful assisted reproductive technology, despite the significant number of issues arising from the implantation of non-autologous eggs. This study aimed to determine whether there is an association between egg donation and preeclampsia. METHODS: A systematic review of the literature available in PubMed and Google Scholar was carried out from January of 1995 to August of 2016 using the terms 'oocyte donation, preeclampsia', 'oocyte donation, in vitro fertilization, preeclampsia', 'oocyte donation, preeclampsia, outcomes pregnancies', 'oocyte donation, obstetric outcome.' Only six retrospective cohort studies met the selection criteria. RESULTS: The meta-analysis revealed a statistically significant association between egg donation and onset of preeclampsia (OR 4.50; 95% CI: 3.28-6.19; p<0.0001). CONCLUSION: Oocyte donation is associated with increased risk of preeclampsia in singleton pregnancies. Therefore, it is crucial to properly record and assess this finding when egg donation is the chosen assisted reproductive technology to attain pregnancy.

There is no association between the presence of anti-thyroid antibodies and increased reproductive loss in pregnant women after ART: a systematic review and meta-analysis.

Women submitted to ART treatments represent a select subgroup of individuals. Several studies have described the relationship between TAI and pregnancy outcomes as a result of ART, with contradictory results. The purpose of this systematic review was to determine the association between TAI and the risk of miscarriage in pregnancies resulting from ART. MEDLINE via PubMed, LILACS and Embase were searched for studies published in peer-reviewed journals from 1999 to 2017. The studies were summarized using the fixed effects model and the Peto's method to calculate RR in order to flesh out the association between TAI and spontaneous abortion. Only four papers were included in this systematic review and meta-analysis. Thirty-one miscarriages were observed in 210 clinical pregnancies of women with antithyroid antibodies; and 158 miscarriages were seen in 1,371 pregnancies without antithyroid antibodies. The meta-analysis failed to find an association between TAI and higher risk of reproductive loss, RR=0.94 95% confidence interval: 0.71-1.24; p=0.879. In conclusion, the presence of antithyroid antibodies was not associated with increased reproductive loss in patients submitted to ART treatments. It is our opinion that the presence of antithyroid antibodies should be considered as a secondary biomarker of autoimmune disease, rather than an actual cause of miscarriage in patients undergoing ART. Due to the small amount of evidence on the matter, the determination of TAI before the initiation of ART should be limited to research contexts.

Are children born from singleton pregnancies conceived by ICSI at increased risk for congenital malformations when compared to children conceived naturally? A systematic review and meta-analysis.

Since 1992, the development of intra-cytoplasmic sperm injection (ICSI) has allowed infertile couples and couples affected by severe male factor infertility in particular, many of which with a history of failed traditional IVF, to become parents. This has generated considerable controversy over the safety of the procedure for the offspring. This systematic review seeks to determine whether evidence indicates that the use of ICSI increases the risk of congenital malformation in children born from singleton pregnancies versus naturally conceived children. Twenty-one of the 104 publications listed in the literature search were included in the analysis. Observational studies reported mostly an increased risk for congenital malformation; the risk of congenital malformations is 7.1% in ICSI and 4.0% in the general population (OR 1.99 (95% CI [1.87 - 2.11]). However, attributing higher risk solely to ICSI might seem far-fetched, as in vitro and simulation procedures, patient diseases, and ICSI indication may also be associated with higher risk of malformation.

Do women offered assisted reproduction technologies have a higher incidence of gynecologic cancer? A systematic review and meta-analysis.

The last two decades have seen an increase in the number of women diagnosed with infertility. The consequent growth in the use of assisted reproductive technologies (ART) calls for the determination of its long-term effects, including the risk of cancer. Many studies have attempted to answer this question, albeit with contradictory results. This review aimed to assess whether assisted reproductive technologies are associated with an increased risk of gynecological cancer. A search for papers in the literature was carried out on MEDLINE, TRIP DATABASE and NICE, resulting in 11 studies enrolling 3,900,231 patients altogether. Of these, 118,320 were offered ART. The incidence of gynecological cancer in the group offered ART was 0.6%, while the incidence in the group not offered ART was 2.1%. Taking all the studies into consideration, women offered ART were not at greater risk of having gynecological cancer; instead, a protective association was found.

Is there a reason to perform ICSI in the absence of male factor? Lessons from the Latin American Registry of ART.

STUDY QUESTION: Does the use of ICSI offer any outcome advantage over IVF in patients with non-male factor infertility? SUMMARY ANSWER: We did not find any outcome improvement that justifies the routine use of ICSI over IVF in non-male factor ART cycles. WHAT IS ALREADY KNOWN: Since its introduction in Latin America, the use of ICSI has increased substantially, even among patients without male factor infertility. However, it is not clear whether ICSI provides an advantage over IVF in non-male factor infertility. STUDY DESIGN SIZE DURATION: A retrospective cohort study of fresh cycles performed in 155 ART clinics located in 15 Latin American countries between 2012 and 2014. Records were assessed for 49,813 ART cycles (39,564 ICSI and 10,249 IVF) performed in infertile couples who did not have male factor infertility. Student's t-test was used to analyze normally distributed data, Wilcoxon test to analyze non-normally distributed data, and Fisher's exact test for categorical data. Logistic regression was used to quantify the effect of ICSI on delivery rate, adjusting for age of female partner, number of oocytes inseminated, number of embryos transferred, and transfer at blastocyst stage as possible confounding factors. Poisson regression analysis was used to quantify the effect of ICSI on fertilization rate, adjusting for age of female partner. PARTICIPANTS/MATERIALS SETTING METHOD: Cycles with the diagnosis of male factor and use of cryopreserved semen and with a freeze-all strategy were excluded. MAIN RESULTS AND THE ROLE OF CHANCE: After correcting for age of female partner, number of oocytes inseminated, number of embryos transferred and transfer at blastocyst stage, we found that the use of ICSI was associated with a significant decrease in the odds of delivery compared to IVF (odds ratio 0.88, 95% CI 0.84 to 0.93; P < 0.0001). LIMITATIONS REASONS FOR CAUTION: An important limitation of this study is the lack of randomization owing to its retrospective nature. This could result in selection bias, i.e. couples with the worst prognosis undergoing ICSI, or patients with a history of fertilization failure in IVF cycles undergoing ICSI. More than one cycle from the same couple may be included in the study. WIDER IMPLICATIONS OF THE FINDINGS: The lack of an outcome benefit-and, indeed, a reduced likelihood of delivery-following ICSI in non-male factor infertile couples suggests that ICSI may not be the most appropriate clinical approach in these patients. STUDY FUNDING/COMPETING INTERESTS: None.

[Live birth after the transfer of embryos obtained by fertilization of vitrified oocytes: report of one case]. / Parto tras la transferencia de embriones generados por fecundación de ovocitos vitrificados: Comunicación de un caso.

We report the first successful live birth after the transfer of embryos obtained by fertilization of vitrified oocytes. A couple with primary infertility due to teratozoospermy and chronic an ovulation decided to undergo assisted reproductive technology after 4 failed cycles of super ovulation and intrauterine insemination. The woman underwent a standard luteal phase agonist protocol, with controlled ovarian hyper stimulation with daily 150 IU recombinant gonadotropin and 75 IU urinary gonadotropin for 12 days. Due to the high risk of ovarian hyper stimulation syndrome the couple was advised to delay embryo transfer. Eighteen mature oocytes were recovered, eight were vitrified and 10 fertilized and cryopreserved as pro-nuclei. Two months later, four vitrified oocytes were thawed, and three morphologically-normal embryos were transferred to an estrogen-progesterone-primed uterus, obtaining triple clinical pregnancy. The pregnancy was uneventful until 32 weeks of amenorrhea, when pre-term delivery started. After inducing lung maturity, a cesarean section was performed and three healthy fetuses were delivered. Nowadays, the babies are two and a half years old, and have had a normal psycho-motor development.

Parto tras la transferencia de embriones generados por fecundación de ovocitos vitrificados: Comunicación de un caso / Live birth after the transfer of embryos obtained by fertilization of vitrified oocytes: Report of one case

We report the first successful live birth after the transfer of embryos obtained by fertilization of vitrified oocytes. A couple with primary infertility due to teratozoospermy and chronic an ovulation decided to undergo assisted reproductive technology after 4 failed cycles of super ovulation and intrauterine insemination. The woman underwent a standard luteal phase agonist protocol, with controlled ovarian hyper stimulation with daily 150IU recombinant gonadotropin and 75IU urinary gonadotropin for 12 days. Due to the high risk of ovarian hyper stimulation syndrome the couple was advised to delay embryo transfer. Eighteen mature oocytes were recovered, eight were vitrified and 10 fertilized and cryopreserved as pro-nuclei. Two months later, four vitrified oocytes were thawed, and three morphologically-normal embryos were transferred to an estrogen-progesterone-primed uterus, obtaining triple clinical pregnancy. The pregnancy was uneventful until 32 weeks of amenorrhea, when pre-term delivery started. After inducing lung maturity, a cesarean section was performed and three healthy fetuses were delivered. Nowadays, the babies are two and a half years old, and have had a normal psycho-motor development.

Genetic profiling of epithelial cells expressing E-cadherin repressors reveals a distinct role for Snail, Slug, and E47 factors in epithelial-mesenchymal transition.

The transcription factors Snail, Slug, and bHLH E47 have been recently described as direct repressors of E-cadherin and inducers of epithelial-mesenchymal transition (EMT) and invasion when overexpressed in epithelial cells. Although a role of those factors in tumor progression and invasion has been proposed, whether the different repressors play distinct or redundant roles in the tumorigenic process has not been established. To further investigate this important issue, we have analyzed the gene expression profiling of Madin-Darby canine kidney (MDCK) epithelial cells expressing the different repressors (MDCK-Snail, MDCK-Slug, and MDCK-E47 cells) versus control MDCK cells by cDNA microarrays. A total of 243 clones (228 genes and 15 expressed sequence tags) were found to be differentially expressed between either of the three MDCK-derived cell lines and control MDCK cells. Twenty two of the candidate genes were validated by Northern blot, Western blot, immunofluorescence, and promoter analyses in cell lines and by immunohistochemistry in xenografted tumors. Gene clustering analysis indicated that about a third of the 243 candidate genes were common to MDCK cells expressing Snail, Slug, or E47 factors, whereas the rest of the genes were regulated in only one or two cell types. Differentially regulated genes include those related to EMT (45 genes), transcriptional regulation (18 genes), cell proliferation and signaling (54 genes), apoptosis (12 genes), and angiogenesis (9 genes). These results indicate that Snail, Slug, and E47 transcription factors induce common and specific genetic programs, supporting a differential role of the factors in tumor progression and invasion.