Sperm selection by birefringence: a promising non-invasive tool to improve ICSI outcomes.

OBJECTIVE: Despite higher sperm DNA fragmentation may affect intracytoplasmic sperm injection (ICSI) outcomes, sperm selection protocols do not evaluate this parameter. Therefore, sperm's head birefringence has been suggested as an adjuvant of seminal processing to select viable sperm for couples with severe male factor. Considering men with normal seminal parameters may also curse with DNA fragmentation, the aim of this study was to evaluate the impact of sperm selection by birefringence on ICSI outcomes in couples with different infertility factors compared to those submitted to conventional sperm selection. METHODS: In this case-control study, medical records from 181 couples who underwent ICSI from January 2018 to August 2020 (107 from the Conventional and 74 from the Birefringence group) were included in the study. Clinical characteristics and ICSI outcomes were compared between the groups using Student's t test or Chi-square test (p<0.05) and a multivariate logistic regression model was applied regarding clinical pregnancy. RESULTS: Despite the Birefringence group showed higher female age (p=0.01), lower seminal sperm concentration (p<0.01) and higher sperm DNA fragmentation (p<0.01), those patients cursed with both higher cleavage rate (p=0.04), clinical pregnancy rate per transfer (p=0.03) and clinical pregnancy rate per initiated cycle (p=0.02). The logistic regression showed a positive group effect on clinical pregnancy. CONCLUSIONS: The findings suggest a positive clinical impact of this cheap and easily reproducible adjuvant technique on ICSI outcomes in couples with different infertility factors. If confirmed by further methodologically appropriate studies, the sperm's head birefringence could be considered to improve the reproductive chances of those patients.

Screening of Variants in the Transcript Profile of Eutopic Endometrium from Infertile Women with Endometriosis during the Implantation Window / Rastreio de variantes no perfil de tanscritos do endométrio eutópico de mulheres inférteis com endometriose durante a janela de implantação

Abstract Objective Abnormalities in the eutopic endometrium of women with endometriosis may be related to disease-associated infertility. Although previous RNA-sequencing analysis did not show differential expression in endometrial transcripts of endometriosis patients, other molecular alterations could impact protein synthesis and endometrial receptivity. Our aim was to screen for functional mutations in the transcripts of eutopic endometria of infertile women with endometriosis and controls during the implantation window. Methods Data from RNA-Sequencing of endometrial biopsies collected during the implantation window from 17 patients (6 infertile women with endometriosis, 6 infertile controls, 5 fertile controls) were analyzed for variant discovery and identification of functional mutations. A targeted study of the alterations found was performed to understand the data into disease's context. Results None of the variants identified was common to other samples within the same group, and no mutation was repeated among patients with endometriosis, infertile and fertile controls. In the endometriosis group, nine predicted deleterious mutations were identified, but only one was previously associated to a clinical condition with no endometrial impact. When crossing the mutated genes with the descriptors endometriosis and/or endometrium, the gene CMKLR1 was associated either with inflammatory response in endometriosis or with endometrial processes for pregnancy establishment. Conclusion Despite no pattern of mutation having been found, we ponder the small sample size and the analysis on RNA-sequencing data. Considering the purpose of the study of screening and the importance of the CMKLR1 gene on endometrial

Resumo Objetivo Anormalidades no endométrio eutópico de mulheres com endometriose podem estar relacionadas à infertilidade associada à doença. Embora a análise prévia de sequenciamento de RNA não tenha evidenciado expressão diferencial em transcritos endometriais de pacientes com endometriose, outras alterações moleculares poderiam afetar a síntese de proteínas e a receptividade endometrial. Nosso objetivo foi rastrear mutações funcionais em transcritos de endométrios eutópicos de mulheres inférteis com endometriose e de controles durante a janela de implantação. Métodos Os dados do sequenciamento de RNA de biópsias endometriais coletados durante a janela de implantação de 17 pacientes (6 mulheres inférteis com endometriose, 6 controles inférteis, 5 controles férteis) foram analisados para a descoberta de variantes e a identificação de mutações funcionais. Um estudo direcionado das alterações encontradas foi realizado para compreender os dados no contexto da doença. Resultados Nenhuma das variantes identificadas foi comuma outras amostras dentro do mesmo grupo, assim como nenhuma mutação se repetiu entre pacientes com endometriose, controles inférteis e férteis. No grupo de endometriose, foram identificadas nove mutações deletérias preditas, mas apenas uma foi previamente associada a uma condição clínica sem impacto endometrial. Ao cruzar os genes mutados com os descritores endometriose e/ou endométrio, o gene CMKLR1 foi associado a resposta inflamatória na endometriose e a processos endometriais para estabelecimento da gravidez. Conclusão Apesar de nenhum padrão de mutação ter sido encontrado, ponderamos o pequeno tamanho da amostra e a análise dos dados de sequenciamento de RNA. Considerando o objetivo do estudo de triagem e a importância do gene CMKLR1 na modulação endometrial, este poderia ser um gene candidato para estudos adicionais que avaliem mutações no endométrio eutópico de pacientes com endometriose.

Ethiopathogenic mechanisms of endometriosis-related infertility.

Endometriosis is a highly prevalent disease among women of reproductive age and is frequently associated to infertility. However, the mechanisms underlying endometriosis-related infertility are still not completely known. Several studies have been conducted in order to elucidate this question. Besides anatomical changes that may impair gametes and embryo transport along the tubes; a smaller ovarian reserve due to advanced endometriosis and endometriomas; and a dysregulated hypothalamic-pituitary-ovarian axis, there are pieces of evidence suggesting that the peritoneal ectopic endometrial foci may induce a local inflammatory response, with the recruitment of macrophages, cytokine release, and reactive oxygen species generation, leading to a pro-oxidant peritoneal microenvironment. These alterations may be systemically reflected and also affect the follicular microenvironment. A harmful follicular fluid may disrupt cumulus cells functions and, consequently, compromise oocyte competence. There is also evidence suggesting that the peritoneal fluid of women with endometriosis may alter sperm function. Reduced endometrial receptivity is also pointed as a possible mechanism involved in endometriosis-related infertility, which needs further investigation.

Perfil diferencial de transcritos em endométrio eutópico de mulheres inférteis com endometriose e controles durante a janela de implantação / Differential profile of transcripts in eutopic endometrium of infertile women with endometriosis during the implantation window

Objetivos: Alterações moleculares no endométrio eutópico de mulheres com endometriose podem estar envolvidas na infertilidade associada à doença. Este estudo objetivou comparar os genes diferencialmente expressos (DEG) no endométrio eutópico de mulheres inférteis com endometriose, controles inférteis (CI; fator masculino e/ou tubário) e controles férteis (CF) durante a janela de implantação, através de RNA­Seq. Material e métodos: Biópsias endometriais foram obtidas de 17 pacientes (seis inférteis com endometriose, seis CI e cinco CF) durante a janela de implantação. O RNA total foi extraído e o RNA­Seq foi feito na plataforma Illumina HISEQ 2500, high output, paired end. A normalização dos dados e a expressão diferencial foram conduzidas no ambiente estatístico R através do pacote DESeq2. Resultados: Os grupos CI e CF foram semelhantes. Nenhum DEG foi identificado quando comparados os grupos CF e endometriose (independentemente do estágio da doença). Cinco DEGs (SCUBE1, CCL20, LGALS9C, TRIM 29 e WNT11) foram identificados no grupo endometriose avançada (EIII/IV) e um DEG (KANSL1­AS1) no grupo endometriose inicial (EI/II), quando comparados com o CF. Dois DEGs (KANSL1­AS1 e VGLL3) foram identificados com a comparação de EI/II e EIII/IV. Conclusões: Os dados sugerem que o endométrio eutópico de mulheres inférteis com endometriose, especialmente na doença avançada, seja molecularmente diferente do endométrio eutópico de mulheres férteis durante a janela de implantação.(AU)

Molecular alterations in the eutopic endometrium of women with endometriosis may be involved in the endometriosis­related infertility. This study aimed to compare the differentially expressed genes (DEG) in eutopic endometrium of infertile women with endometriosis, infertile controls (IC; male and/or tubal factor) and fertile controls (FC) through RNA­Seq. Material and methods: Endometrial biopsies were obtained from 17 patiens (6 infertile women with endometriosis, 6 IC and 5 FC) during the implantation window. The RNA was extracted and the RNA­Seq was performed at a HISEQ 2500 Illumina Platform, high output, paired end. Standardization and differential expression were conducted in the statistical R environment using DESeq2 package. Results: The groups IC and FC were similar. No DEG has been identified comparing CF and endometriosis groups. Five DEGs (SCUBE1, CCL20, LGALS9C, TRIM 29 e WNT11) were identified in the advanced endometriosis (EIII/IV) group, and 1 (KANSL1­AS1) in the initial endometriosis (EI/II) group compared to FC. Two DEGs (KANSL1­AS1 and VGLL3) were identified by comparing EI/II and EIII/IV groups. Conclusions: These data suggest that the eutopic endometrium of infertile women with endometriosis, especially those with advanced disease, may be molecularly different from those of fertile women during the implantation window.(AU)

Comparative Analysis of the Spindle of Fresh In Vivo-Matured Human Oocytes Through Polarized Light and Confocal Microscopy: A Pilot Study.

BACKGROUND: Considering the scarcity of literature data about the predictive capacity of polarization microscopy (PM) in identifying meiotic normality in human in vivo-matured oocytes, the main objective of the present study was to determine whether qualitative analysis of the spindle through PM can predict meiotic normality in fresh in vivo-matured human oocytes. METHODS: Infertile patients undergoing controlled ovarian stimulation to intracytoplasmic sperm injection (ICSI) were selected. Fresh in vivo-matured oocytes were evaluated by PM and, immediately afterward, fixed for confocal microscopy (CM) analysis for evaluation of the spindle and chromosome distribution. We evaluated the percentage of oocytes with meiotic normality and abnormality determined by CM among oocytes with visible and nonvisible spindle and between oocytes with the spindle located at 0° to 60° angle to the first polar body (PB; normal position) and at 60° to 90° angle to the first PB (risky position) according to PM. RESULTS: From 23 patients, 73 oocytes were analyzed. There were no significant differences in the percentage of oocytes with meiotic abnormalities among oocytes with visible and nonvisible spindles and among oocytes with the spindle in the normal and risky positions. CONCLUSIONS: Under the conditions tested, qualitative evaluation of the spindle through PM is not consistent with CM analysis and has limited predictive value of meiotic normality in fresh in vivo-matured human oocytes, which needs to be confirmed in larger studies. Our findings make questionable the usefulness of this methodology as a tool for noninvasive oocyte selection for ICSI.

Análise invasiva e não invasiva do fuso meiótico de oócitos humanos obtidos de ciclos estimulados: dados preliminares / Invasive and noninvasive analysis of the meiotic spindle of human oocytes obtained from stimulate cycles: preliminary results

OBJETIVOS: Avaliar a concordância entre as técnicas de microscopia de polarização e microscopia confocal na avaliação do fuso meiótico de oócitos humanos maturados in vivo. MÉTODOS: Estudo prospectivo que avaliou oócitos com o primeiro corpúsculo polar extruído obtidos de mulheres inférteis submetidas à estimulação ovariana para realização de injeção intracitoplasmática de espermatozoide. Os oócitos com o primeiro corpúsculo polar extruído foram avaliados por meio da microscopia de polarização e, imediatamente após, foram fixados e corados para avaliação dos microtúbulos e cromatina pela microscopia confocal de alto desempenho. Foram comparadas as técnicas de microscopia de polarização e confocal, de acordo com a visualização ou não do fuso meiótico pela microscopia de polarização e a presença ou não de anomalias meióticas à análise pela microscopia confocal. Foram calculados os intervalos de confiança, o índice de Kappa e a concordância entre as metodologias, considerando a análise da microscopia de imunofluorescência como padrão-ouro para avaliação de normalidade do fuso e distribuição cromossômica oocitária. RESULTADOS: Observou-se que 72,7% dos oócitos em metáfase II com fuso celular não visível à polarização apresentaram anormalidades meióticas à análise confocal e que 55,6% dos oócitos em metáfase II com fuso celular visível à polarização apresentaram-se como oócitos anormais à análise confocal. Somente 44,4% dos oócitos com fuso celular visível à polarização apresentaram-se como normais à análise confocal. A concordância entre os métodos foi de 51,1% (Kappa: 0,11; IC95% -0,0958 - 0,319). CONCLUSÕES: A baixa concordância entre a microscopia de polarização e a confocal na avaliação do fuso meiótico oocitário sugere que a visualização do fuso meiótico de oócitos humanos em metáfase II pela microscopia de polarização tem limitado o valor preditivo de normalidade meiótica oocitária.

PURPOSE: To evaluate the concordance between polarization microscopy and confocal microscopy techniques in the evaluation of the meiotic spindle of human oocytes matured in vivo. METHODS: Prospective study that evaluated oocytes with the first polar extruded body obtained from infertile women who had undergone ovarian stimulation for intracytoplasmic sperm injection. The oocytes with the first polar extruded body were evaluated by polarization microscopy and were then immediately fixed and stained for microtubule and chromatin evaluation by high-performance confocal microscopy. We determined the correlation of polarization microscopy with confocal microscopy in the detection of meiotic oocyte anomalies, and we also evaluated the percentage of oocytes with a visible and non-visible cell spindle by polarization microscopy and with meiotic normality and abnormalities by confocal microscopy. Confidence intervals, Kappa's index and concordance between the methodologies were calculated, considering immunofluorescence microscopy analysis as the golden-standard for evaluating normal spindle and oocyte chromosome distribution. RESULTS: We observed that 72.7% of metaphase II oocytes with a nonvisible meiotic spindle by polarization microscopy showed no meiotic abnormalities by confocal analysis and 55.6% of metaphase II oocytes with a visible meiotic spindle by polarization microscopy were found to be abnormal oocytes by the confocal analysis. Only 44.4% of oocytes with a visible meiotic spindle by polarization microscopy were found to be normal by confocal analysis. Concordance between the methods was 51.1% (Kappa: 0.11; 95%CI -0.0958 - 0.319). CONCLUSIONS: The low correlation between polarization microscopy and confocal microscopy in the assessment of oocyte meiotic spindle suggests that visualization of the meiotic spindle of human oocytes at metaphase II by polarization microscopy is not a good indicator of oocyte meiotic normality.