Is the profile of transcripts altered in the eutopic endometrium of infertile women with endometriosis during the implantation window?

STUDY QUESTION: Compared to healthy women, is the profile of transcripts altered in the eutopic endometrium of infertile women with endometriosis during the implantation window (IW)? SUMMARY ANSWER: The eutopic endometrium of infertile women with endometriosis seems to be transcriptionally similar to the endometrium of infertile and fertile controls (FC) during the IW. WHAT IS KNOWN ALREADY: Endometriosis is a disease related to infertility; nevertheless, little is known regarding the ethiopathogenic mechanisms underlying this association. Some studies evaluating the eutopic endometrium of endometriosis patients suggest there is an endometrial factor involved in the disease-related infertility. However, no study to date has evaluated the endometrial transcriptome (mRNA and miRNA) by next generation sequencing (NGS), comparing patients with endometriosis as the exclusive infertility factor (END) to infertile controls (IC; male and/or tubal factor) and FC. STUDY DESIGN, SIZE, DURATION: From November 2011 to November 2015 we performed a case-control study, where 17 endometrial samples (six END, six IC, five FC) were collected during the IW. PARTICIPANTS/MATERIALS, SETTING, METHODS: All endometrial samples had the RNA extracted. Two libraries were prepared for each one (mRNA and miRNA), which were sequenced, respectively, at HISEQ 2500 (RNA-Seq) and MiSeq System (miRNA-Seq), Illumina. The normalization and differential expression were conducted in statistical R environment using DESeq2 package. qPCR was used for data validation, which were analyzed by Kruskal-Wallis test and Dunn posttest (P < 0.05). MAIN RESULTS AND THE ROLE OF CHANCE: RNA-Seq revealed no differentially expressed genes (DEG) among END, IC and FC groups. miRNA-Seq revealed three differentially expressed miRNAs (has-27a-5p, has-miR-150-5p, has-miR-504-5p) in END group compared to FC group. However, none of the miRNAs identified in the sequencing was validated by qPCR. LIMITATIONS, REASONS FOR CAUTION: The main limitation of this study was the small sample size evaluated as a result of the restrictive eligibility criteria adopted, limiting the generalization of the results obtained here. On the other hand, strict eligibility criteria, which eliminated factors potentially related to impaired endometrial receptivity, were required to increase the study's internal validity. WIDER IMPLICATIONS OF THE FINDINGS: This study brings new perspectives on the mechanisms involved in endometriosis-related infertility. The present findings suggest the eutopic endometrium of infertile women with endometriosis, without considering the disease's stage, is transcriptionally similar to controls during the IW, possibly not affecting receptivity. Further studies are needed to evaluate endometrial alterations related to endometriosis' stages. STUDY FUNDING/COMPETING INTEREST(S): This study received financial support from the Sao Paulo Research Foundation (FAPESP-Fundação de Amparo à Pesquisa do Estado de São Paulo; fellowship 2011/17614-6, MGB) and from the National Council for Scientific and Technological Development (CNPq-Conselho Nacional de Desenvolvimento Científico e Tecnológico; INCT-National Institutes of Hormones and Woman's Health, grant 471 943/2012-6, 309 397/2016-2, PAN; fellowship 140 137/2015-7, MGB). The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

An update on the aspects of Zika virus infection on male reproductive system.

Zika virus (ZIKV) is mainly transmitted through Aedes mosquito bites, but sexual and post-transfusion transmissions have been reported. During acute infection, ZIKV is detectable in most organs and body fluids including human semen. Although it is not currently epidemic, there is a concern that the virus can still reemerge since the male genital tract might harbor persistent reservoirs that could facilitate viral transmission over extended periods, raising concerns among public health and assisted reproductive technologies (ART) experts and professionals. So far, the consensus is that ZIKV infection in the testes or epididymis might affect sperm development and, consequently, male fertility. Still, diagnostic tests have not yet been adapted to resource-restricted countries. This manuscript provides an updated overview of the cellular and molecular mechanisms of ZIKV infection and reviews data on ZIKV persistence in semen and associated risks to the male reproductive system described in human and animal models studies. We provide an updated summary of the impact of the recent ZIKV outbreak on human-ART, weighing on current recommendations and diagnostic approaches, both available and prospective, with special emphasis on mass spectrometry-based biomarker discovery. In the light of the identified gaps in our accumulated knowledge on the subject, we highlight the importance for couples seeking ART to follow the constantly revised guidelines and the need of specific ZIKV diagnosis tools for semen screening to contain ZIKV virus spread and make ART safer.

Influence of follicular fluid and cumulus cells on oocyte quality: clinical implications.

An equilibrium needs to be established by the cellular and acellular components of the ovarian follicle if developmental competence is to be acquired by the oocyte. Both cumulus cells (CCs) and follicular fluid (FF) are critical determinants for oocyte quality. Understanding how CCs and FF influence oocyte quality in the presence of deleterious systemic or pelvic conditions may impact clinical decisions in the course of managing infertility. Given that the functional integrities of FF and CCs are susceptible to concurrent pathological conditions, it is important to understand how pathophysiological factors influence natural fertility and the outcomes of pregnancy arising from the use of assisted reproduction technologies (ARTs). Accordingly, this review discusses the roles of CCs and FF in ensuring oocyte competence and present new insights on pathological conditions that may interfere with oocyte quality by altering the intrafollicular environment.

Magnetic-activated cell sorting before density gradient centrifugation improves recovery of high-quality spermatozoa.

Recent studies have evaluated the use of magnetic-activated cell sorting (MACS) to reduce apoptotic spermatozoa and improve sperm quality. However, the efficiency of using MACS alone, before or after sperm processing by density gradient centrifugation (DGC) has not yet been established. The purpose of this study is to determine the optimal protocol of MACS in assisted reproduction techniques (ART). Thus, we compared sperm quality obtained by DGC alone (DGC), DGC followed by MACS (DGC-MACS), MACS followed by DGC (MACS-DGC), and MACS alone (MACS), and found that the combined methods (MACS-DGC and DGC-MACS) led to retrieval of less spermatozoa with fragmented DNA compared to the single protocols. However, MACS-DGC protocol led to a significantly higher percentage of spermatozoa with progressive motility and normal morphology than DGC-MACS protocol. These findings suggest the potential clinical value of using MACS-DGC to improve sperm quality in seminal preparation for ART.

GnRH agonist during luteal phase in women undergoing assisted reproductive techniques: systematic review and meta-analysis of randomized controlled trials.

OBJECTIVE: To identify, evaluate and summarize the available evidence regarding the effectiveness and safety of administering a gonadotropin releasing hormone (GnRH) agonist during the luteal phase in women undergoing assisted reproductive techniques. METHODS: In this systematic review and meta-analysis, we searched for randomized controlled trials (RCTs) comparing the addition of a GnRH agonist during the luteal phase, compared with standard luteal-phase support. We searched seven electronic databases and hand-searched the reference lists of included studies and related reviews. Our primary outcome was live birth or ongoing pregnancy per randomized woman. Our secondary outcomes were clinical pregnancy per randomized woman, miscarriage per clinical pregnancy, adverse perinatal outcome and congenital malformations. RESULTS: The evidence from eight studies examining 2776 women showed a relative risk (RR) for live birth or ongoing pregnancy of 1.26 (95% CI, 1.04-1.53; I(2) = 58%). Sensitivity analysis when excluding the studies that did not report live birth and those at high risk of bias resulted in one study examining 181 women with an RR of 1.07 (95% CI, 0.73-1.58). Subgroup analysis separating the studies by single/multiple doses of GnRH agonists or by ovarian stimulation with GnRH agonist/antagonist was unable to explain the observed heterogeneity. The quality of the evidence was deemed to be very low: it was downgraded because of the limitation of the included studies, imprecision, inconsistency across the studies' results, and suspicion of publication bias. None of the included studies reported adverse perinatal outcomes or congenital malformations. CONCLUSIONS: There is evidence that adding GnRH agonist during the luteal phase improves the likelihood of ongoing pregnancy. However, this evidence is of very low quality and there is no evidence for adverse perinatal outcome and congenital malformations. We therefore believe that including this intervention in clinical practice would be premature.

Dydrogesterone vs progesterone for luteal-phase support: systematic review and meta-analysis of randomized controlled trials.

OBJECTIVES: To compare the effects of dydrogesterone and progesterone for luteal-phase support (LPS) in women undergoing assisted reproductive techniques (ART). METHODS: We performed a systematic review to identify relevant randomized controlled trials (RCTs) by searching the following electronic databases: Cochrane CENTRAL, PubMed, Scopus, Web of Science, ClinicalTrials.gov, ISRCTN Registry and WHO ICTRP. RESULTS: The last search was performed in October 2015. Eight RCTs were considered eligible and were included in the review and meta-analyses. There was no relevant difference between oral dydrogesterone and vaginal progesterone for LPS with respect to rate of ongoing pregnancy (risk ratio (RR), 1.04 (95% CI, 0.92-1.18); I(2) , 0%; seven RCTs, 3134 women), clinical pregnancy (RR, 1.07 (95% CI, 0.93-1.23); I(2) , 34%; eight RCTs, 3809 women) or miscarriage (RR, 0.77 (95% CI, 0.53-1.10); I(2) , 0%; seven RCTs, 906 clinical pregnancies). Two of the three studies reporting on dissatisfaction of treatment identified lower levels of dissatisfaction among women using oral dydrogesterone than among women using vaginal progesterone (oral dydrogesterone vs vaginal progesterone capsules: 2/79 (2.5%) vs 90/351 (25.6%), respectively; oral dydrogesterone vs vaginal progesterone gel: 19/411 (4.6%) vs 74/411 (18.0%), respectively). The third study showed no difference in dissatisfaction rate (oral dydrogesterone vs vaginal progesterone capsules: 8/96 (8.3%) vs 8/114 (7.0%), respectively). CONCLUSIONS: Oral dydrogesterone seems to be as effective as vaginal progesterone for LPS in ART cycles, and appears to be better tolerated . Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.

Ovarian response is a better predictor of clinical pregnancy rate following embryo transfer than is thin endometrium or presence of an endometrioma.

OBJECTIVES: To examine whether endometrial thickness and the presence of endometrioma are independent predictors of clinical pregnancy rate or simply associated with poor ovarian response (POR). METHODS: This was a retrospective cohort study assessing the first cycle of all women undergoing in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) in a university hospital in Brazil between January 2011 and December 2012. Only the first cycle of each woman within the study period was considered. Women over 40 years of age and those who used clomiphene citrate during controlled ovarian stimulation (COS) or did not undergo embryo transfer were excluded from analysis. POR was defined as ≤ three oocytes retrieved and a thin endometrium was defined as endometrial thickness ≤ 7.0 mm on the day of human chorionic gonadotropin (hCG) administration. We performed a multiple regression analysis to identify which of the following parameters were independent predictors of clinical pregnancy: age, number of oocytes retrieved, endometrial thickness or the presence of endometrioma. RESULTS: Within the study period, 787 women began COS, but 270 were excluded from analysis. Among the 517 women analyzed, those who achieved pregnancy were younger and yielded more oocytes. The proportion of POR was higher in women with a thin endometrium (17/57 (29.8%) vs 80/460 (17.4%); P = 0.03) and in women with endometrioma (15/39 (38.5%) vs 82/478 (17.2%); P = 0.002). The results of regression analysis showed that only age and the number of oocytes retrieved were independent predictors of pregnancy. Additionally, we observed higher clinical pregnancy rates in women with a thin endometrium from whom ≥ seven oocytes were retrieved (11/25 (44.0%)) compared to women with normal endometrial thickness (99/241 (41.1%)). Considering only women from whom ≥ four oocytes were retrieved, we observed reasonable pregnancy rates in those with a thin endometrium (14/40 (35.0%)) and in those with endometrioma (9/24 (37.5%)). CONCLUSION: Both a thin endometrium and the presence of endometrioma are associated with POR but are not important independent predictors of clinical pregnancy. Good pregnancy rates can be observed when these conditions are present in women with a good ovarian response.

Direct actions of androgens on the survival, growth and secretion of steroids and anti-Müllerian hormone by individual macaque follicles during three-dimensional culture.

STUDY QUESTION: What are the direct effects of androgens on primate follicular development and function at specific stages of folliculogenesis? SUMMARY ANSWER: Androgen addition altered primate follicle survival, growth, steroid and anti-Müllerian hormone (AMH) production, and oocyte quality in vitro, in a dose- and stage-dependent manner. WHAT IS KNOWN ALREADY: Androgens have local actions in the ovary, particularly in the developing follicles. It is hypothesized that androgen promotes early follicular growth, but becomes detrimental to the antral follicles in primates. STUDY DESIGN, SIZE, DURATION: In vitro follicle maturation was performed using rhesus macaques. Secondary (125-225 µm) follicles were mechanically isolated from 14 pairs of ovaries, encapsulated into alginate (0.25% w/v), and cultured for 40 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Individual follicles were cultured in a 5% O2 environment, in alpha minimum essential medium supplemented with recombinant human FSH. Follicles were randomly assigned to experiments of steroid ablation by trilostane (TRL), testosterone (T) replacement and dihydrotestosterone (DHT) replacement. Follicle survival and growth were assessed. Follicles with diameters ≥500 µm at Week 5 were categorized as fast-grow follicles. Pregnenolone (P5), progesterone (P4), estradiol (E2) and AMH concentrations in media were measured. Meiotic maturation and fertilization of oocytes from recombinant human chorionic gonadotrophin-treated follicles were assessed at the end of culture. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with controls, TRL exposure reduced (P < 0.05) follicle survival, antrum formation rate and follicle diameters at Week 5. While P5 concentrations increased (P < 0.05) following TRL treatment, P4 levels decreased (P < 0.05) in fast-grow follicles at Week 5. Few healthy oocytes were retrieved from antral follicles developed in the presence of TRL. T replacement with TRL increased (P < 0.05) follicle survival and antrum formation at Week 5, compared with TRL alone, to levels comparable to controls. However, high-dose T with TRL decreased (P < 0.05) diameters of fast-grow follicles. Although P4 concentrations produced by fast-grow follicles were not altered by T in the presence of TRL, there was a dose-dependent increase (P < 0.05) in E2 levels at Week 5. High-dose T with TRL decreased (P < 0.05) AMH production by fast-grow follicles at Week 3. More healthy oocytes were retrieved from antral follicles developed in TRL+T compared with TRL alone. DHT had the similar effects to those of high-dose T, except that DHT replacement decreased (P < 0.05) E2 concentrations produced by fast-grow follicles at Week 5 regardless of TRL treatment. LIMITATION, REASONS FOR CAUTION: This study reports T and DHT actions on in vitro-developed individual primate (macaque) follicles, which are limited to the interval from the secondary to small antral stage. WIDER IMPLICATION OF THE FINDINGS: The above findings provide novel information on the role(s) of androgens in primate follicular development and oocyte maturation. We hypothesize that androgens promote pre-antral follicle development, but inhibit antral follicle growth and function in primates. While androgens can act positively, excess levels of androgens may have negative impacts on primate folliculogenesis. STUDY FUNDING/COMPETING INTERESTS: NIH U54 RR024347/RL1HD058294/PL1EB008542 (Oncofertility Consortium), NIH U54 HD071836 (SCCPIR), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), NIH FIC TW/HD-00668, ONPRC 8P51OD011092. There are no conflicts of interest.

Assessment of ovarian reserve by antral follicle count in ovaries with endometrioma.

OBJECTIVE: To evaluate whether the antral follicle count (AFC) is underestimated in the presence of an endometrioma. METHODS: This was a retrospective cohort study assessing all women undergoing in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) at our clinic between January 2011 and December 2012 who had both ovaries and unilateral endometrioma. The primary outcome of the study was the difference between AFC and the number of oocytes retrieved per ovary. RESULTS: Within the study period 787 women underwent IVF/ICSI at our clinic. Sixty of these women had at least one endometrioma, but 23 were excluded from the analysis as six had only one ovary and 17 had bilateral endometriomas. Therefore a total of 37 women were included in this study and analysis. Compared with the contralateral ovaries, ovaries with an endometrioma were significantly larger in volume (median, 10.3 (interquartile range (IQR), 4.7-18.9) cm(3) vs median, 3.6 (IQR, 2.7-6.5) cm(3); P < 0.001) and presented a significantly lower AFC (median, 3.0 (IQR, 1.0-6.0) vs median, 5.0 (IQR, 2.0-6.5); P = 0.001). However, the median number of oocytes retrieved was similar (P = 0.60) between ovaries with an endometrioma (2.0 (IQR, 0.5-5.0)) and the contralateral ovaries (2.0 (IQR, 0.0-4.0)). Accordingly, the median difference between AFC and number of oocytes retrieved was significantly smaller (P = 0.005) for ovaries with an endometrioma (0.0 (IQR, -1.0 to 1.5) than for those without (2.0 (IQR, 0.0-4.0)). CONCLUSIONS: Although the AFC is reduced in ovaries with an endometrioma, the number of oocytes retrieved is similar, suggesting that the AFC is underestimated in such ovaries. We believe that this is a consequence of an impaired ability to detect small follicles in the presence of an endometrioma.

Time-lapse embryo imaging for improving reproductive outcomes: systematic review and meta-analysis.

OBJECTIVE: To identify, appraise and summarize the available evidence regarding the effectiveness and safety of time-lapse embryo monitoring on the main outcomes of assisted reproductive techniques. METHODS: In this systematic review and meta-analysis, we included only randomized controlled trials (RCTs) comparing time-lapse embryo imaging with standard embryo monitoring. Our primary outcomes were live births (efficacy) and congenital abnormalities (safety). The secondary outcomes were clinical pregnancy, ongoing pregnancy and miscarriage. RESULTS: Two RCTs were considered eligible, and their data were extracted and included in a meta-analysis. In both studies embryos were transferred at the blastocyst stage. No studies reported rates of live birth or congenital abnormalities. Our estimates were not sufficiently precise to identify whether time-lapse monitoring provided a small benefit, no effect or minor harm on rates of clinical pregnancy (relative risk (RR), 1.05 (95% CI, 0.80-1.38)) or ongoing pregnancy (RR, 1.05 (95% CI, 0.76-1.45)), based on two studies involving 138 women with moderate-quality evidence. Considering the available data, we were unable to determine whether the intervention poses substantial benefit, no effect or substantial harm in the risk of miscarriage (RR, 0.95 (95% CI, 0.30-2.99)), based on two studies involving 76 clinical pregnancies with low-quality evidence. CONCLUSIONS: Time-lapse embryo imaging is unlikely to have a large effect on the chance of achieving clinical and/or ongoing pregnancy when transferring embryos at the blastocyst stage. More studies are required to improve the quality of the current evidence and also to examine whether this intervention is useful when transferring embryos at the cleavage stage.

Impact of endometriosis and its staging on assisted reproduction outcome: systematic review and meta-analysis.

OBJECTIVE: To evaluate whether the presence or severity of endometriosis affects the outcome of assisted reproductive techniques (ART). METHODS: In this systematic review, all studies comparing the outcome of ART in women with and those without endometriosis, or at different stages of the disease, were considered eligible. We used either risk ratio (RR) or mean difference (MD) and their 95%CIs for comparisons. The primary outcome was live birth; the secondary outcome was clinical pregnancy. Miscarriage and the number of oocytes retrieved were examined as additional outcomes. RESULTS: We included 92 studies in the review and 78 in the meta-analysis: 20,167 women with endometriosis were compared with 121,931 women without endometriosis, and 1703 women with Stage-III/IV endometriosis were compared with 2227 women with Stage-I/II endometriosis. The following results were observed for the comparison of women with endometriosis vs women without endometriosis: live birth, RR = 0.99 (95%CI, 0.92-1.06); clinical pregnancy, RR = 0.95 (95%CI, 0.89-1.02); miscarriage, RR = 1.31 (95%CI, 1.07-1.59); number of oocytes retrieved, MD = -1.56 (95%CI, -2.05 to -1.08). The following results were observed for the comparison of women with Stage-III/IV vs Stage-I/II endometriosis: live birth, RR = 0.94 (95%CI, 0.80-1.11); clinical pregnancy, RR = 0.90 (95%CI, 0.82-1.00); miscarriage, RR = 0.99 (95%CI, 0.73-1.36); number of oocytes retrieved, MD = -1.03 (95%CI, -1.67 to -0.39). CONCLUSIONS: Women with endometriosis undergoing ART have practically the same chance of achieving clinical pregnancy and live birth as do women with other causes of infertility. No relevant difference was observed in the chance of achieving clinical pregnancy and live birth following ART when comparing Stage-III/IV with Stage-I/II endometriosis. The quality of the evidence for the additional examined outcomes was very low, not allowing meaningful conclusions to be drawn.

Follicular fluid from infertile women with mild endometriosis may compromise the meiotic spindles of bovine metaphase II oocytes.

STUDY QUESTION: What is the potential impact of follicular fluid (FF) from infertile women with mild endometriosis (ME) on oocyte quality, especially on nuclear maturation and the meiotic spindle? SUMMARY ANSWER: FF from infertile women with ME may compromise nuclear maturation and the meiotic spindles of in vitro matured bovine oocytes. WHAT IS KNOWN ALREADY: Controversial studies have suggested that impaired oocyte quality may be involved in the pathogenesis of endometriosis-related infertility. Moreover, some studies have demonstrated alterations in the composition of FF from infertile women with endometriosis. However, to date no study has evaluated the effect of FF from infertile women with ME on the genesis of meiotic oocyte anomalies. STUDY DESIGN, SIZE, DURATION: We performed an experimental study. Samples of FF were obtained from February 2009 to February 2011 from 22 infertile women, 11 with ME and 11 with tubal or male factors of infertility (control group), who underwent ovarian stimulation for ICSI at our university IVF Unit. From March 2011 to February 2012 we performed in vitro maturation (IVM) experiments using immature bovine oocytes as described below. PARTICIPANTS/MATERIALS, SETTING, METHODS: FF free of blood and containing a mature oocyte was obtained from 22 infertile women during oocyte retrieval for ICSI. Immature bovine oocytes underwent IVM in the absence of FF (No-FF) and in the presence of four concentrations (1, 5, 10 and 15%) of FF from infertile women without endometriosis (C-FF) and with ME (ME-FF). Eleven replicates were performed, each one using FF from a control patient and a patient with ME. Each FF sample was used in only one experiment. After 22-24 h of IVM, oocytes were denuded, fixed and immunostained for morphological visualization of microtubules and chromatin by confocal microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1324 cumulus-oocyte complexes were matured in vitro. Of these, 1128 were fixed and 1048 were analyzed by confocal microscopy. The percentage of meiotically normal oocytes was significantly higher for oocytes that underwent IVM in the absence of FF (No-FF; 76.5%) and in the presence of 1% (80.9%), 5% (76.6%), 10% (75%) and 15% (76.2%) C-FF than in oocytes that underwent IVM in the presence of 1% (44.4%), 5% (36.7%), 10% (45.5%) and 15% (51.2%) ME-FF (P < 0.01). No differences were observed among FF concentrations within each group. When the four concentrations from each group were pooled, the number of oocytes in metaphase I stage was significantly higher in the ME-FF (50 oocytes) than in the C-FF (29 oocytes) group and the percentage of meiotic abnormalities was significantly higher when oocytes were matured with ME-FF (55.8%) than with C-FF (23.1%), P < 0.01. LIMITATIONS, REASONS FOR CAUTION: Owing to the strict selection criteria for FF donors, this study had a small sample size (11 cases and 11 controls), and thus further investigations using a large cohort of patients are needed to confirm these results. In addition, data obtained from studies using animal models may not necessarily be extrapolated to humans and studies evaluating in vivo matured oocytes from infertile women with ME are important to confirm our results. WIDER IMPLICATIONS OF THE FINDINGS: Our results open new insights into the pathogenic mechanisms of infertility related to mild endometriosis, suggesting that FF from infertile women with mild endometriosis may be involved in the worsening of oocyte quality of these women. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Council for Scientific and Technological Development (CNPq), Brazil. The authors declare no conflicts of interest.

Cytoplasmic maturation of bovine oocytes: structural and biochemical modifications and acquisition of developmental competence.

Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3' terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development.

Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules.

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured in TCM plus FSH and 10% estrous cow serum. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P<0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless, there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. Similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis.

Association of the HLA-DRB1*0301 and HLA-DQA1*0501 alleles with Graves' disease in a population representing the gene contribution from several ethnic backgrounds.

Graves' disease (GD) is the most frequent cause of hyperthyroidism. Although the etiology is not completely elucidated, there are several lines of evidence suggesting multifactorial mechanisms. Genetic, constitutional, and environmental factors are involved in its pathogenesis. Major histocompatibility complex (MHC) class II alleles have been associated with GD in several populations of distinct ethnic backgrounds and there is increasing evidence supporting an association between GD and HLA-DR3 in Caucasian populations. The MHC class II alleles were evaluated in 75 Brazilian patients presenting with GD and in 166 control individuals from the same geographic area. HLA-DRB, DQB, and DQA alleles were identified using polymerase chain reaction (PCR)-amplified DNA hybridized with sequence-specific probes. The HLA-DRB1*0301 allele was significantly increased in patients (34/75, 45.3%) as compared with controls (37/166, 22.3%, p = 0.009), conferring a relative risk (RR) of 2.8 and an etiologic fraction (EF) of 0.287. The HLA-DQA1*0501 allele was also overrepresented in patients (48/71, 67.6%) in relation to controls (24/71, 33.8%; p = 0.004), conferring an RR of 3.74 and an EF of 0.351. The susceptibility conferred by HLA-DQA1*0501 was independent of the HLA-DRB1*0301 allele. On the other hand, the HLA-DQB1*0602 allele was significantly decreased in patients (6/75, 8.0%) in relation to controls (53/166, 31.9%, p = 0.0008), conferring an RR of 0.18 and a preventive fraction of 0.267. Although the Brazilian population comprises individuals of several ethnic backgrounds, these results corroborate the participation of the HLA-DRB1*0301 and HLA-DQA1*0501 alleles as susceptibility markers for GD, and emphasize the participation of the HLA-DQB1*0602 allele as conferring protection against the development of the disease.

Hypogonadotropic hypogonadism: retrospective analysis of 19 cases.

OBJECTIVES: To survey the clinical data of patients with isolated gonadotropin deficiency. METHODS: We retrospectively surveyed the medical records of 19 patients with isolated gonadotropin deficiency aged 16-31 years (mean: 20 years). The major complaint was primary amenorrhea in 100% of the patients, with 42.1% of them also reporting absence of secondary sex traits, and 10% reporting anosmia or hyposmia. Seventy-four percent of the patients had been submitted to hormonal replacement therapy. RESULTS: Bone densitometry was determined in 5 patients and revealed lumbar spine osteopenia in 3 patients and femoral osteopenia in 1. An association with urologic malformations was detected in 10.5% of cases and an association with gynecologic malformations was detected in 31.6%. CONCLUSIONS: Isolated gonadotropin deficiency can be easily diagnosed but requires early estrogen replacement therapy because of a higher risk of osteopenia and consequently of osteoporosis. Concomitant urogenital malformations are frequent and should be investigated.

Hamartoma esplénico sintomático / A symptomatic splenic hamartoma

No disponible

[Feeding of low-birth-weight infants]. / Alimentación en niños de peso bajo al nacimiento

A child with low birth-weight is defined. Physiological and biochemical bases for the nutrition of these babies are given. Breast-feeding is recommended describing advantages and disadvantages compared with other milk formulas. Some artificial milk formulas in the national market are analyzed and modified milk or protein-modified milk are recommended as substitutes for mother's milk.