Functional canonical RNAi in mice expressing a truncated Dicer isoform and long dsRNA.

Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer's adaptation to produce another small RNA class-microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in DicerΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. DicerΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo.

Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant.

miRNAs, ~22nt small RNAs associated with Argonaute (AGO) proteins, are important negative regulators of gene expression in mammalian cells. However, mammalian maternal miRNAs show negligible repressive activity and the miRNA pathway is dispensable for oocytes and maternal-to-zygotic transition. The stoichiometric hypothesis proposed that this is caused by dilution of maternal miRNAs during oocyte growth. As the dilution affects miRNAs but not mRNAs, it creates unfavorable miRNA:mRNA stoichiometry for efficient repression of cognate mRNAs. Here, we report that porcine ssc-miR-205 and bovine bta-miR-10b are exceptional miRNAs, which resist the diluting effect of oocyte growth and can efficiently suppress gene expression. Additional analysis of ssc-miR-205 shows that it has higher stability, reduces expression of endogenous targets, and contributes to the porcine oocyte-to-embryo transition. Consistent with the stoichiometric hypothesis, our results show that the endogenous miRNA pathway in mammalian oocytes is intact and that maternal miRNAs can efficiently suppress gene expression when a favorable miRNA:mRNA stoichiometry is established.

Preimplantation genetic testing for aneuploidies does not increase success rates in fresh oocyte donation cycles: a paired cohort study.

PURPOSE: To determine whether in vitro fertilization cycles using fresh oocyte donations benefit from preimplantation genetic testing for aneuploidies. METHODS: A paired cohort study compared 44 fresh oocyte donation cycles with or without preimplantation genetic testing for aneuploidy (PGT-A). The sibling oocyte study analyzed fertilized oocytes, blastocyst development, and euploidy rate. Only frozen embryo transfers were performed. Pregnancy, implantation, biochemical pregnancy, miscarriage, stillbirth, live birth, and twin pregnancy rates were analyzed between groups. RESULTS: Fresh oocyte donation cycles between PGT-A and non-PGT-A groups were similar in all laboratory and clinical outcomes. A euploidy rate of 74.2% was observed in the PGT-A group. Although a slight trend was observed for implantation rate in the PGT-A group, it was not statistically significant. No difference was observed for live birth between groups. CONCLUSION: PGT-A associated with fresh oocyte donation cycles does not improve clinical outcomes and can be seen as over-treatment for patients.

Morphology-based selection from available euploid blastocysts induces male-skewed sex proportion in the offspring.

PURPOSE: To determine whether blastocyst morphology has an impact on sex proportion at pre-implantation and birth in PGT-A and non-PGT-A cycles. METHODS: A total of 1254 biopsied blastocysts from 466 PGT-A cycles were analyzed for sex proportion, day of biopsy, degree of expansion, inner cell mass (ICM), and trophectoderm (TE) morphology. From these, 197 frozen single embryo transfers (SET) were assessed for clinical outcomes and sex proportion of ongoing pregnancies and deliveries. In addition, we evaluated the day of vitrification/embryo transfer, degree of expansion, and TE morphology in a group of 229 births (217 cycles) from frozen or fresh transfers of non-biopsied blastocysts. RESULTS: Sex proportion was impacted by day of biopsy and TE morphology, but not by ICM morphology, in PGT-A cycles. Therefore, biopsy on day 5 and TE "A" shifted the sex proportion towards males. Interestingly, we noted that our morphology-based embryo selection for SET of euploid blastocysts has favored the choice for XY embryos, generating a 54.3% XY proportion at transfer and 56.1% XY proportion at ongoing pregnancy/delivery. Our models indicate a weaker association between blastocyst morphology parameters and sex proportion of babies in non-PGT-A cycles. CONCLUSION: Blastocyst features associated with a skewed sex proportion towards XY embryos, such as biopsy on day 5 and top quality TE, are also parameters used for selecting euploid embryos for SET. Therefore, our data suggest that morphology-based embryo selection represents a strong factor responsible for a skewed male sex proportion at birth in PGT-A cycles.

Multicenter prospective study of concordance between embryonic cell-free DNA and trophectoderm biopsies from 1301 human blastocysts.

BACKGROUND: The recent identification of embryonic cell-free DNA in spent blastocyst media has opened a new era of possibilities for noninvasive embryo aneuploidy testing in assisted reproductive technologies. Yet, previous studies assessing a limited number of embryos reported variable concordance between embryonic cell-free DNA and trophectoderm biopsies, thus questioning the validity of this approach. OBJECTIVE: This study aimed to evaluate the concordance and reproducibility of testing embryonic cell-free DNA vs trophectoderm DNA obtained from the same embryo in a large sample of human blastocysts and to assess the contribution of the inner cell mass and trophectoderm to embryonic cell-free DNA released to the culture media. STUDY DESIGN: This is an interim analysis of a prospective, observational study among 8 in vitro fertilization centers in 4 continents to assess consistency between noninvasive embryo aneuploidy testing of embryonic cell-free DNA and conventional trophectoderm biopsy. The analysis included 1301 day-6/7 blastocysts obtained in 406 in vitro fertilization cycles from 371 patients aged 20-44 years undergoing preimplantation genetic testing for aneuploidy. Fresh oocytes underwent intracytoplasmic sperm injection or in vitro fertilization. No previous assisted hatching or vitrification was allowed before media collection. Individual spent blastocyst medium was collected from embryos cultured at least 40 hours from day 4. After media collection, conventional preimplantation genetic testing for aneuploidy, comprising trophectoderm biopsy and blastocyst vitrification, was performed. Embryonic cell-free DNA was analyzed blindly after embryo transfer. Inner cell mass and trophectoderm biopsies were also performed in a subset of 81 aneuploid blastocysts donated for research. RESULTS: Embryonic cell-free DNA analyses were 78.2% (866/1108) concordant with the corresponding trophectoderm biopsies. No significant differences were detected among centers ranging from 72.5% to 86.3%. Concordance rates exceeded 86% when all defined steps in the culture laboratory were controlled to minimize the impact of maternal and operator contamination. Sensitivity per center ranged from 76.5% to 91.3% and specificity from 64.7% to 93.3%. The false-negative rate was 8.3% (92/1108), and false-positive rate was 12.4% (137/1108). The 2 fertilization techniques provided similar sensitivity (80.9% vs 87.9%) and specificity (78.6% vs 69.9%). Multivariate analysis did not reveal any bias from patient clinical background, ovarian stimulation protocols, culture conditions, or embryo quality on testing accuracy of concordance. Moreover, concordances of embryonic cell-free DNA with trophectoderm and inner cell mass suggest that the embryonic cell-free DNA originates from both compartments of the human embryo. CONCLUSION: Noninvasive analysis of embryonic cell-free DNA in spent blastocyst culture media demonstrates high concordance with trophectoderm biopsy results in this large multicenter series. A noninvasive approach for prioritizing embryo euploidy offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population.

Molecular detection and characterization of BK and JC polyomaviruses in urine samples of renal transplant patients in Southern Brazil.

The human polyomaviruses JC (JCPyV) and BK (BKPyV) are widespread in the human population. Following the primary infection, virus reactivation may lead to nephropathy and graft rejection in renal transplant patients. This study was carried out to access the presence of BKPyV and JCPyV DNA in urine samples collected from renal transplant patients (n = 92) and healthy individuals (n = 88) in Porto Alegre, Rio Grande do Sul. The samples were submitted to a nested PCR. A significantly higher frequency (P < 0.001) of BKPyV was found in renal transplant patients (65.2%) in comparison to the control group (32.9%). JCPyV was detected equally in both groups. Phylogenetic analysis of both BKPyV and JCPyV amplicons demonstrates the presence of the BKPyV subtypes I and II, whereas for JCPyV, four different groups are found (1, 2, 3, and 4).

Pronuclear and blastocyst morphology are associated age-dependently with embryo ploidy in in vitro fertilization cycles.

This retrospective study aimed to assess the relationship between standard markers of embryo morphology, maternal age and blastocyst ploidy determined by trophectoderm (TE) biopsy and Next-generation Sequencing (NGS). A total of 774 oocytes and embryos from 288 PGT-A cycles were scored for pronuclear, cleavage stage and blastocyst morphology. Pronuclear oocytes aligned between the nuclei and presenting equal number of nucleolus precursor bodies (NPBs) were designated Z1, oocytes showing equal number of NPBs, but not aligned, as Z2 while Z3 oocytes had an unequal number of NBPs between the nuclei or NPBs aligned in one nucleus and non-aligned in the other. Pronuclear oocytes with unequal-sized or non-aligned nuclei were designated Z4. Blastocysts were graded as BL1 (AA, AB or BA), BL2 (BB or CB) and BL3 (BC or CC) based on the combination of inner cell mass (ICM) and TE scores. Pronuclear and blastocyst morphology were correlated with aneuploidy in a < 40-year-old group (p < 0.01 and p < 0.05, respectively), but not in those ≥40 years. Interestingly, BL3 blastocysts classified as Z1 or Z3-Z4 on day-1 had different aneuploidy rates (BL3/Z1 = 46.7% vs. BL3/Z3-Z4 = 90.0%, p < 0.05). In summary, our data showed that pronuclear and blastocyst morphology are associated with blastocyst ploidy in younger patients. This may help embryo selection for embryo transfer and decision-making on which blastocysts should be biopsied in PGT-A cycles.

First Baby Born in Brazil after Simultaneous Diagnosis through Non-Invasive and Conventional PGT-A.

Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) aiming to assess cell-free embryonic DNA in spent culture media is promising, especially because it might overcome the diminished rates of implantation caused by the inadequate performance of trophectoderm (TE) biopsy. Our center is part of the largest study to date assessing the concordance between conventional PGT-A and niPGT-A, and we report here the delivery of the first baby born in Brazil using niPGT-A. The parents of the baby were admitted to our center in 2018. They did not present history of infertility, and they were interested in using in vitro fertilization (IVF) and PGT-A in order to avoid congenital anomalies in the offspring. A total of 11 (3 day-5 and 8 day-6) expanded blastocysts were biopsied, and the spent culture media (culture from day-4 to day-6) from 8 day-6 blastocysts were collected for niPGT-A. Overall, 7 embryos yielded informative results for trophectoderm (TE) and media samples. Among the embryos with informative results, 5 presented concordant diagnosis between conventional PGT-A and niPGT-A, and 2 presented discordant diagnosis (1 false-positive and one false-negative). The Blastocyst 4, diagnosed as 46, XY by both niPGT-A and conventional PGT-A, was warmed up and transferred, resulting in the birth of a healthy 3.8 kg boy in February 2020. Based on our results and the recent literature, we believe that the safest current application of niPGT-A would be as a method of embryo selection for patients without an indication for conventional PGT-A. The approximate 80% of reliability of niPGT-A in the diagnosis of ploidy is superior to predictions provided by other non-invasive approaches like morphology and morphokinetics selection.

Abordagens para o teste genético pré-implantacional não-invasivo para aneuploidias (non-invasive preimplantation genetic testing for aneuploidies, niPGT-A, em inglês) com o objetivo de avaliar o DNA embrionário livre são promissoras, especialmente porque estas podem reverter as menores taxas de implantação causadas por inadequada biópsia de trofectoderma (TE). Nesse contexto, nosso centro é parte do maior estudo atual que avalia as taxas de concordância entre PGT-A convencional e niPGT-A, e relatamos aqui o nascimento do primeiro bebê brasileiro após niPGT-A. Os pais do bebê foram admitidos no nosso centro em 2018. Eles não apresentavam histórico de infertilidade, e estavam interessados em utilizar os tratamentos de fertilização in vitro (FIV) e PGT-A para evitar anomalias congênitas na progênie. Um total de 11 blastocistos expandidos (3 do dia-5 e 8 do dia-6) foram submetidos a biópsia, e os meios de cultivo condicionados (cultivo do dia-4 ao dia-6) de 8 blastocistos do dia-6 foram coletados para niPGT-A. No total, resultados informativos para as amostras de TE e dos meios foram obtidos para sete embriões. Entre os embriões com resultado informativo, 5 apresentaram diagnóstico concordante entre PGT-A convencional e niPGT-A, e 2 apresentaram diagnóstico discordante (1 falso positivo e 1 falso negativo). O Blastocisto 4, diagnosticado como 46, XY por ambos niPGT-A e PGT-A convencional, foi desvitrificado e transferido, o que resultou no nascimento de um menino saudável, que pesava 3,8 kg, em fevereiro de 2020. Com base em nossos resultados e literatura contemporânea, acreditamos que a aplicação atual mais segura do niPGT-A seria como método de seleção embrionária para pacientes sem indicação ao PGT-A convencional. A confiabilidade aproximada de 80% do niPGT-A para determinação da ploidia ainda é superior àquela obtida com abordagens não invasivas, como seleção morfológica ou morfocinética.

Platelet-Rich plasma as an effective alternative approach for improving endometrial receptivity - a clinical retrospective study.

OBJECTIVE: The aim of the present case series was to describe our experience with the use of PRP on patients with refractory thin endometria. METHODS: This retrospective analysis included 24 IVF cycles in which patients presenting different infertility factors received intrauterine PRP infusion prior to embryo transfer. Outcomes of interest were: clinical and ongoing pregnancies, miscarriages, and births. RESULTS: 54% of the cycles in which PRP was employed resulted in ongoing gestation or birth; 12.5% of the cycles ended in miscarriages. CONCLUSION: Our data suggest that PRP improves intrauterine receptivity to embryo implantation, regardless of whether the endometrium reached the appropriate growth for embryo transfer.

The Effects of Overweight and Obesity on Assisted Reproduction Technology Outcomes.

The aim of the present study was to assess the impact of professional nutrition assistance on assisted reproduction technology (ART) outcomes in overweight or obese patients with polycystic ovarian syndrome (PCOS). The study represents a retrospective analysis of fertilization rates, embryo quality and gestations after ART in seven PCOS patients, five obese and two overweight. The women attended a private Fertility Center in Brazil between the years 2010 and 2016. Out of the seven patients, the three that reached a successful gestation were the ones that underwent comprehensive lifestyle changes, taking care of their diet for a more prolonged period of time and reached an ideal weight loss during the nutrition counseling period.

First Baby Born in Brazil after Simultaneous Diagnosis through Non-Invasive and Conventional PGT-A / Primeiro bebê nascido no Brasil após diagnóstico simultâneo por PGT-A não-invasivo e convencional

Abstract Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) aiming to assess cell-free embryonic DNA in spent culturemedia is promising, especially because it might overcome the diminished rates of implantation caused by the inadequate performance of trophectoderm (TE) biopsy. Our center is part of the largest study to date assessing the concordance between conventional PGT-A and niPGT-A, and we report here the delivery of the first baby born in Brazil using niPGT-A. The parents of the baby were admitted to our center in 2018. They did not present history of infertility, and they were interested in using in vitro fertilization (IVF) and PGT-A in order to avoid congenital anomalies in the offspring. A total of 11 (3 day-5 and 8 day-6) expanded blastocysts were biopsied, and the spent culture media (culture from day-4 to day-6) from 8 day-6 blastocysts were collected for niPGT-A. Overall, 7 embryos yielded informative results for trophectoderm (TE) and media samples. Among the embryos with informative results, 5 presented concordant diagnosis between conventional PGTA and niPGT-A, and 2 presented discordant diagnosis (1 false-positive and one falsenegative). The Blastocyst 4, diagnosed as 46, XY by both niPGT-A and conventional PGTA, was warmed up and transferred, resulting in the birth of a healthy 3.8 kg boy in February 2020. Based on our results and the recent literature, we believe that the safest current application of niPGT-A would be as a method of embryo selection for patients without an indication for conventional PGT-A. The approximate 80% of reliability of niPGT-A in the diagnosis of ploidy is superior to predictions provided by other noninvasive approaches like morphology and morphokinetics selection.

Resumo Abordagens para o teste genético pré-implantacional não-invasivo para aneuploidias (non-invasive preimplantation genetic testing for aneuploidies, niPGT-A, em inglês) com o objetivo de avaliar o DNA embrionário livre são promissoras, especialmente porque estas podem reverter as menores taxas de implantação causadas por inadequada biópsia de trofectoderma (TE). Nesse contexto, nosso centro é parte do maior estudo atual que avalia as taxas de concordância entre PGT-A convencional e niPGT-A, e relatamos aqui o nascimento do primeiro bebê brasileiro após niPGT-A. Os pais do bebê foram admitidos no nosso centro em 2018. Eles não apresentavam histórico de infertilidade, e estavam interessados em utilizar os tratamentos de fertilização in vitro (FIV) e PGT-A para evitar anomalias congênitas na progênie.Umtotal de 11 blastocistos expandidos (3 do dia-5 e 8 do dia-6) foram submetidos a biópsia, e os meios de cultivo condicionados (cultivo do dia-4 ao dia-6) de 8 blastocistos do dia-6 foram coletados para niPGT-A. No total, resultados informativos para as amostras de TE e dos meios foram obtidos para sete embriões. Entre os embriões com resultado informativo, 5 apresentaram diagnóstico concordante entre PGT-A convencional e niPGT-A, e 2 apresentaram diagnóstico discordante (1 falso positivo e 1 falso negativo). O Blastocisto 4, diagnosticado como 46, XY por ambos niPGT-A e PGT-A convencional, foi desvitrificado e transferido, o que resultou no nascimento de ummenino saudável, que pesava 3,8 kg, em fevereiro de 2020. Com base em nossos resultados e literatura contemporânea, acreditamos que a aplicação atualmais segura do niPGT-A seria como método de seleção embrionária para pacientes sem indicação ao PGT-A convencional. A confiabilidade aproximada de 80% do niPGT-A para determinação da ploidia ainda é superior àquela obtida com abordagens não invasivas, como seleção morfológica ou morfocinética.

Effects of zinc transporters on Cryptococcus gattii virulence.

Zinc is an essential nutrient for all living organisms because it is a co-factor of several important proteins. Furthermore, zinc may play an essential role in the infectiousness of microorganisms. Previously, we determined that functional zinc metabolism is associated with Cryptococcus gattii virulence. Here, we characterized the ZIP zinc transporters in this human pathogen. Transcriptional profiling revealed that zinc levels regulated the expression of the ZIP1, ZIP2 and ZIP3 genes, although only the C. gattii zinc transporter Zip1 was required for yeast growth under zinc-limiting conditions. To associate zinc uptake defects with virulence, the most studied cryptococcal virulence factors (i.e., capsule, melanin and growth at 37 °C) were assessed in ZIP mutant strains; however, no differences were detected in these classical virulence-associated traits among the mutant and WT strains. Interestingly, higher levels of reactive oxygen species were detected in the zip1Δ and in the zip1Δ zip2Δ double mutants. In line with these phenotypic alterations, the zip1Δ zip2Δ double mutant displayed attenuated virulence in a murine model of cryptococcosis. Together, these results indicate that adequate zinc uptake is necessary for cryptococcal fitness and virulence.