Practices and ethical concerns regarding preimplantation diagnosis. Who regulates preimplantation genetic diagnosis in Brazil?

Preimplantation genetic diagnosis (PGD) was originally developed to diagnose embryo-related genetic abnormalities for couples who present a high risk of a specific inherited disorder. Because this technology involves embryo selection, the medical, bioethical, and legal implications of the technique have been debated, particularly when it is used to select features that are not related to serious diseases. Although several initiatives have attempted to achieve regulatory harmonization, the diversity of healthcare services available and the presence of cultural differences have hampered attempts to achieve this goal. Thus, in different countries, the provision of PGD and regulatory frameworks reflect the perceptions of scientific groups, legislators, and society regarding this technology. In Brazil, several texts have been analyzed by the National Congress to regulate the use of assisted reproduction technologies. Legislative debates, however, are not conclusive, and limited information has been published on how PGD is specifically regulated. The country requires the development of new regulatory standards to ensure adequate access to this technology and to guarantee its safe practice. This study examined official documents published on PGD regulation in Brazil and demonstrated how little direct oversight of PGD currently exists. It provides relevant information to encourage reflection on a particular regulation model in a Brazilian context, and should serve as part of the basis to enable further reform of the clinical practice of PGD in the country.

Histopathological analysis of pre-implantation donor kidney biopsies: association with graft survival and function in one year post-transplantation

Introduction: Pre-implantation kidney biopsy is a decision-making tool when considering the use of grafts from deceased donors with expanded criteria, implanting one or two kidneys and comparing this to post-transplantation biopsies. The role of histopathological alterations in kidney compartments as a prognostic factor in graft survival and function has had conflicting results. Objective: This study evaluated the prevalence of chronic alterations in pre-implant biopsies of kidney grafts and the association of findings with graft function and survival in one year post-transplant. Methods: 110 biopsies were analyzed between 2006 and 2009 at Santa Casa de Porto Alegre, including live donors, ideal deceased donors and those with expanded criteria. The score was computed according to criteria suggested by Remuzzi. The glomerular filtration rate (GFR) was calculated using the abbreviated MDRD formula. Results: No statistical difference was found in the survival of donors stratified according to Remuzzi criteria. The GFR was significantly associated with the total scores in the groups with mild and moderate alterations, and in the kidney compartments alone, by univariate analysis. The multivariate model found an association with the presence of arteriosclerosis, glomerulosclerosis, acute rejection and delayed graft function. Conclusion: Pre-transplant chronic kidney alterations did not influence the post-transplantation one-year graft survival, but arteriosclerosis and glomerulosclerosis is predictive of a worse GFR. Delayed graft function and acute rejection are independent prognostic factors.

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

Abstract In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.

Sexual behavior and fertility of male rats submitted to prolonged immobilization-induced stress

In order to investigate whether prolonged stress interferes with the onset of sexual behavior at puberty and with fertility at adulthood, prepubertal male Wistar rats (40 days of age) were immobilized 6 h a day for 15 days (up to early puberty) or for 60 days (until sexual maturity). Pubertal stressed rats showed a two-fold increase in the latency for the first mount (probably due to repeated aversive experience in which a change of environment was always followed by immobilization) and a 2.5-fold increase in the frequency of thrusting (indicative of enhanced sexual performance). The apparently stimulatory effect of prolonged stress on the onset of sexual behavior is discussed in terms of increased testosterone level and interference with the complex interchanges between the neurotransmitters/neuropeptides involved in the central control of male sexual activity. Adult stressed animals were mated with normal females, which became pregnant but exhibited a more than two-fold increase in both pre-implantation and post-implantation loss, probably due to a smaller rate of fertilization and/or fertilization with damaged spermatozoa.

Blastocyst-endometrium interaction: intertwining a cytokine network

The successful implantation of the blastocyst depends on adequate interactions between the embryo and the uterus. The development of the embryo begins with the fertilized ovum, a single totipotent cell which undergoes mitosis and gives rise to a multicellular structure named blastocyst. At the same time, increasing concentrations of ovarian steroid hormones initiate a complex signaling cascade that stimulates the differentiation of endometrial stromal cells to decidual cells, preparing the uterus to lodge the embryo. Studies in humans and in other mammals have shown that cytokines and growth factors are produced by the pre-implantation embryo and cells of the reproductive tract; however, the interactions between these factors that converge for successful implantation are not well understood. This review focuses on the actions of interleukin-1, leukemia inhibitory factor, epidermal growth factor, heparin-binding epidermal growth factor, and vascular endothelial growth factor, and on the network of their interactions leading to early embryo development, peri-implantatory endometrial changes, embryo implantation and trophoblast differentiation. We also propose therapeutical approaches based on current knowledge on cytokine interactions.

No evidence of association of MUC-1 genetic polymorphism with embryo implantation failure

Pregnancy loss can be caused by several factors involved in human reproduction. Although up to 50% of cases remain unexplained, it has been postulated that the major cause of failed pregnancy is an error of embryo implantation. Transmembrane mucin-1 (MUC-1) is a glycoprotein expressed on the endometrial cell surface which acts as a barrier to implantation. The gene that codes for this molecule is composed of a polymorphic tandem repeat of 60 nucleotides. Our objective was to determine if MUC-1 genetic polymorphism is associated with implantation failure in patients with a history of recurrent abortion. The study was conducted on 10 women aged 25 to 35 years with no history of successful pregnancy and with a diagnosis of infertility. The control group consisted of 32 patients aged 25 to 35 years who had delivered at least two full-term live children and who had no history of abortions or fetal losses. MUC-1 amplicons were obtained by PCR and observed on agarose and polyacrylamide gel after electrophoresis. Statistical analysis showed no significant difference in the number of MUC-1 variable number of tandem repeats between these groups (P > 0.05). Our results suggest that there is no effect of the polymorphic MUC-1 sequence on the implantation failure. However, the data do not exclude MUC-1 relevance during embryo implantation. The process is related to several associated factors such as the mechanisms of gene expression in the uterus, specific MUC-1 post-translational modifications and appropriate interactions with other molecules during embryo implantation.

Antemortem diagnosis of human rabies in a veterinarian infected when handling a herbivore in Minas Gerais, Brazil

The Ministry of Health's National Human Rabies Control Program advocates pre-exposure prophylaxis (PEP) for professionals involved with animals that are at risk of contracting rabies. We report an antemortem and postmortem diagnosis of rabies in a veterinarian who became infected when handling herbivores with rabies. The antemortem diagnosis was carried out with a saliva sample and a biopsy of hair follicles using molecular biology techniques, while the postmortem diagnosis used a brain sample and conventional techniques. The veterinarian had collected samples to diagnose rabies in suspect herbivores (bovines and caprines) that were subsequently confirmed to be positive in laboratory tests. After onset of classic rabies symptoms, saliva and hair follicles were collected and used for antemortem diagnostic tests and found to be positive by RT-PCR. Genetic sequencing showed that the infection was caused by variant 3 (Desmodus rotundus), a finding confirmed by tests on the brain sample. It is essential that professionals who are at risk of infection by the rabies virus undergo pre-exposure prophylaxis. This study also confirms that molecular biology techniques were used successfully for antemortem diagnosis and therefore not only allow therapeutic methods to be developed, but also enable the source of infection in human rabies cases to be identified accurately and quickly.

Dried blood spots collected on filter paper: an international resource for the diagnosis and genetic characterization of human immunodeficiency virus Type-1

The collection of dried blood spots (DBS) on filter paper provides a powerful approach for the development of large-scale, population-based screening programs. DBS methods are particularly valuable in developing countries and isolated rural regions where resources are limited. Large numbers of field specimens can be economically collected and shipped to centralized reference laboratories for genetic and (or) serological analysis. Alternatively, the dried blood can be stored and used as an archival resource to rapidly establish the frequency and distribution of newly recognized mutations, confirm patient identity or track the origins and emergence of newly identified pathogens. In this report, we describe how PCR-based technologies are beginning to interface with international screening programmes for the diagnosis and genetic characterization of human immunodeficiency virus type 1 (HIV-1). In particular, we review recent progress using DBS specimens to resolve the HIV-1 infection status of neonates, monitor the genetic evolution of HIV-1 during early infancy and establish a sentinel surveillance system for the systematic monitoring of HIV-1 genetic variation in Asia.

Molecular paleoparasitological diagnosis of Ascaris sp. from coprolites: new scenery of ascariasis in pre-Colombian South America times

Paleoparasitological studies using microscopy showed that Ascarisand Trichuris trichiura are the human intestinal parasites most found in archaeological sites. However, in pre-Columbian South American archaeological sites, Ascaris is rare. In this work we standardized a molecular methodology for Ascaris diagnosis directly from ancient DNA retrieved from coprolites. Using cythochrome b gene (142 bp) target, ancient DNA sequences were retrieved from South American samples, negative by microscopy. Moreover, the methodology applied was sensitive enough to detect ancient DNA extracted from 30 Ascaris eggs from an European coprolite. These results revealed a new scenery for the paleodistribution of Ascaris in South America.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Effectiveness of see-and-treat for approaching pre-invasive lesions of uterine cervix

OBJECTIVE: To compare the effectiveness between the see-and-treat (S&T) approach and the conventional one (with prior biopsy) for squamous intraepithelial lesions of uterine cervix. METHODS: A cross-sectional study was conducted with 900 nonpregnant women with cytology suggestive of high grade squamous intraepithelial lesions in the city of Rio de Janeiro, Southeastern Brazil, between 1998 and 2004. The S&T approach consists of a large loop excision of the transformation zone procedure and is recommended when cytology is suggestive of high grade squamous intraepithelial lesion, satisfactory colposcopy with abnormalities compatible with the suspected cytological results, and the lesion is limited to the ectocervix or extends up to one centimeter of the endocervical canal. A subgroup of 336 patients whose colposcopy was considered satisfactory was analyzed, and they were divided into two groups for comparison: patients treated without prior biopsy (n = 288) and patients treated after a biopsy showing high grade squamous intraepithelial lesions (n = 48). Patients who were not treated or only treated more than a year later after recruitment at the colposcopy unit were considered dropouts. RESULTS: Of patients recruited during the study period, 71 were not treated or were only treated for at least a year. The overall dropout rate was 7.9% (95% CI: 6.1;9.7). Mean time elapsed between patient recruitment and treatment was 17.5 days in the S&T group and 102.5 days in the prior biopsy group. Dropout rates were 1.4% (95% CI: 0.04;2.7) and 5.% (95% CI: 0;12.3), respectively (p=0.07). The proportion of overtreated cases (negative histology) in the S&T group was 2.0% (95% CI: 0.4;3.6). CONCLUSIONS: The difference in the mean time elapsed between patient recruitment and treatment indicates that S&T is a time-saving approach The proportion of negative cases from using the S&T approach can be regarded as low.

The importance to update the guidelines for the use of genetic testing in noncancer patients in Brazil

The Brazilian National Regulatory Agency for Private Health Insurance and Plans has recently published a technical note defining the criteria for the coverage of genetic testing to diagnose hereditary cancer. In this study we show the case of a patient with a breast lesion and an extensive history of cancer referred to a private service of genetic counseling. The patient met both criteria for hereditary breast and colorectal cancer syndrome screening. Her private insurance denied coverage for genetic testing because she lacks current or previous cancer diagnosis. After she appealed by lawsuit, the court was favorable and the test was performed using next-generation sequencing. A deletion of MLH1 exon 8 was found. We highlight the importance to offer genetic testing using multigene analysis for noncancer patients.

SIMPLIFIED DIAGNOSIS OF MALARIA INFECTION: GFM/PCR/ELISA A SIMPLIFIED NUCLEIC ACID AMPLIFICATION TECHNIQUE BY PCR/ELISA

We report an adaptation of a technique for the blood sample collection (GFM) as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.

PRELIMINARY REPORT ON THE PUTATIVE ASSOCIATION OF IL10 -3575 T/A GENETIC POLYMORPHISM WITH MALARIA SYMPTOMS

Only a small percentage of individuals living in endemic areas develop severe malaria suggesting that host genetic factors may play a key role. This study has determined the frequency of single nucleotide polymorphisms (SNPs) in some pro and anti-inflammatory cytokine gene sequences: IL6 (-174; rs1800795), IL12p40 (+1188; rs3212227), IL4 (+33; rs2070874), IL10 (-3575; rs1800890) and TGFb1 (+869; rs1800470), by means of PCR-RFLP. Blood samples were collected from 104 symptomatic and 37 asymptomatic subjects. Laboratory diagnosis was assessed by the thick blood smear test and nested-PCR. No association was found between IL6 (-174), IL12p40 (+1188), IL4 (+33), IL10 (- 3575), TGFb1 (+869) SNPs and malaria symptoms. However, regarding the IL10 -3575 T/A SNP, there were significantly more AA and AT subjects, carrying the polymorphic allele A, in the symptomatic group (c2 = 4.54, p = 0.01, OR = 0.40 [95% CI - 0.17- 0.94]). When the analysis was performed by allele, the frequency of the polymorphic allele A was also significantly higher in the symptomatic group (c2 = 4.50, p = 0.01, OR = 0.45 [95% CI - 0.21-0.95]). In conclusion, this study has suggested the possibility that the IL10 - 3575 T/A SNP might be associated with the presence and maintenance of malaria symptoms in individuals living in endemic areas. Taking into account that this polymorphism is related to decreased IL10 production, a possible role of this SNP in the pathophysiology of malaria is also suggested, but replication studies with a higher number of patients and evaluation of IL10 levels are needed for confirmation.