RESUMEN
Meiotic maturation is a crucial step of oocyte formation, allowing its potential fertilization and embryo development. Elucidating this process is important for both fundamental research and assisted reproductive technology. However, few computational tools based on non-invasive measurements are available to characterize oocyte meiotic maturation. Here, we develop a computational framework to phenotype oocytes based on images acquired in transmitted light. We trained neural networks to segment the contour of oocytes and their zona pellucida using oocytes from diverse species. We defined a comprehensive set of morphological features to describe an oocyte. These steps were implemented in an open-source Fiji plugin. We present a feature-based machine learning pipeline to recognize oocyte populations and determine morphological differences between them. We first demonstrate its potential to screen oocytes from different strains and automatically identify their morphological characteristics. Its second application is to predict and characterize the maturation potential of oocytes. We identify the texture of the zona pellucida and cytoplasmic particle size as features to assess mouse oocyte maturation potential and tested whether these features were applicable to the developmental potential of human oocytes. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Células del Cúmulo , Oocitos , Animales , Femenino , Humanos , Aprendizaje Automático , Ratones , Oogénesis/genética , Zona PelúcidaRESUMEN
In humans, structural or functional defects of the sperm flagellum induce asthenozoospermia, which accounts for the main sperm defect encountered in infertile men. Herein we focused on morphological abnormalities of the sperm flagellum (MMAF), a phenotype also termed "short tails," which constitutes one of the most severe sperm morphological defects resulting in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we identified bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and identified five individuals with homozygous truncating variants in TTC29, a gene preferentially and highly expressed in the testis, and encoding a tetratricopeptide repeat-containing protein related to the intraflagellar transport (IFT). One individual carried a frameshift variant, another one carried a homozygous stop-gain variant, and three carried the same splicing variant affecting a consensus donor site. The deleterious effect of this last variant was confirmed on the corresponding transcript and protein product. In addition, we produced and analyzed TTC29 loss-of-function models in the flagellated protist T. brucei and in M. musculus. Both models confirmed the importance of TTC29 for flagellar beating. We showed that in T. brucei the TPR structural motifs, highly conserved between the studied orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that TTC29 mutations are a cause of male sterility due to MMAF.
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Astenozoospermia/etiología , Axonema/patología , Flagelos/patología , Infertilidad Masculina/etiología , Proteínas Asociadas a Microtúbulos/genética , Mutación , Animales , Astenozoospermia/metabolismo , Astenozoospermia/patología , Axonema/genética , Axonema/metabolismo , Evolución Molecular , Femenino , Fertilización In Vitro , Flagelos/genética , Flagelos/metabolismo , Humanos , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Ratones Endogámicos C57BL , Trypanosoma brucei brucei/fisiología , TripanosomiasisRESUMEN
Cyclic fertilin peptide (cFEE: phenylalanine, glutamic acid; glutamic acid) improves gamete interaction in humans. We investigate whether it could be via improvement of sperm movement parameters and their mitochondrial ATP production. Sperm movement parameters were studied using computer-assisted sperm analysis (CASA) in sperm samples from 38 patients with normal sperm in medium supplemented with cyclic fertilin against a control group. Sperm mitochondrial functions were studied using donor's sperm, incubated or not with cFEE. It was evaluated by the measurement of their ATP production using bioluminescence, their respiration by high resolution oxygraphy, and of mitochondrial membrane potential (MMP) using potentiometric dyes and flow cytometry. cFEE significantly improved sperm movement parameters and percentage of hyperactivated sperm. Impact of inhibitors showed OXPHOS as the predominant energy source for sperm movement. However, cFEE had no significant impact on any of the analyzed mitochondrial bioenergetic parameters, suggesting that it could act via a more efficient use of its energy resources.
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Mitocondrias/metabolismo , Péptidos Cíclicos/farmacología , Espermatozoides/fisiología , Adenosina Trifosfato/metabolismo , Metabolismo Energético , Humanos , Mediciones Luminiscentes , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Membranas/metabolismo , Mitocondrias/efectos de los fármacos , Fosforilación Oxidativa , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacosRESUMEN
Motile cilia and sperm flagella share an extremely conserved microtubule-based cytoskeleton, called the axoneme, which sustains beating and motility of both organelles. Ultra-structural and/or functional defects of this axoneme are well-known to cause primary ciliary dyskinesia (PCD), a disorder characterized by recurrent respiratory tract infections, chronic otitis media, situs inversus, male infertility and in most severe cases, hydrocephalus. Only recently, mutations in genes encoding axonemal proteins with preferential expression in the testis were identified in isolated male infertility; in those cases, individuals displayed severe asthenozoospermia due to Multiple Morphological Abnormalities of the sperm Flagella (MMAF) but not PCD features. In this study, we performed genetic investigation of two siblings presenting MMAF without any respiratory PCD features, and we report the identification of the c.2018T > G (p.Leu673Pro) transversion in AK7, encoding an adenylate kinase, expressed in ciliated tissues and testis. By performing transcript and protein analyses of biological samples from individual carrying the transversion, we demonstrate that this mutation leads to the loss of AK7 protein in sperm cells but not in respiratory ciliated cells, although both cell types carry the mutated transcript and no tissue-specific isoforms were detected. This work therefore, supports the notion that proteins shared by both cilia and sperm flagella may have specific properties and/or function in each organelle, in line with the differences in their mode of assembly and organization. Overall, this work identifies a novel genetic cause of asthenozoospermia due to MMAF and suggests that in humans, more deleterious mutations of AK7 might induce PCD.
Asunto(s)
Adenilato Quinasa/genética , Trastornos de la Motilidad Ciliar/genética , Homocigoto , Infertilidad Masculina/genética , Mutación Missense , Cola del Espermatozoide , Adenilato Quinasa/metabolismo , Adulto , Trastornos de la Motilidad Ciliar/enzimología , Trastornos de la Motilidad Ciliar/patología , Humanos , Infertilidad Masculina/enzimología , Infertilidad Masculina/patología , MasculinoRESUMEN
RESEARCH QUESTION: Is there a follicular fluid-specific metabolic profile in deep infiltrating endometriosis (DIE) depending on the presence of an associated ovarian endometrioma (OMA) that could lead to the identification of biomarkers for diagnosis and prognosis of the disease? DESIGN: In this prospective cohort study, proton nuclear magnetic resonance (1H-NMR) experiments were carried out on 50 follicular fluid samples from patients presenting with DIE, associated or not associated with an OMA, and 29 follicular fluid samples from patients with infertility caused by a tubal obstruction. RESULTS: Concentrations of glucose, citrate, creatine and amino acids such as tyrosine and alanine were lower in women with DIE than control participants, whereas concentrations of lactate, pyruvate, lipids and ketone bodies were higher. Metabolic analysis revealed enhanced concentrations of glycerol and ketone bodies in patients with OMA, indicative of an activation of lipolysis followed by beta-oxidation. Concentrations of lactate and pyruvate were increased in patients without OMA, whereas the concentration of glucose was decreased, highlighting activation of the anaerobic glycolysis pathway. Differences in concentrations of amino acids such as threonine and glutamine were also statistically relevant in discriminating between the presence or absence of OMA. CONCLUSIONS: Results indicate a mitochondrial dysregulation in endometriosis phenotypes, with a modified balance between anaerobic glycolysis and beta-oxidation in OMA phenotypes that could affect the fertility of women with endometriosis. As the composition of the follicular fluid has been shown to be correlated with oocyte development and outcome of implantation after fertilization, these findings may help explain the high level of infertility in these patients.
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Endometriosis/metabolismo , Líquido Folicular/metabolismo , Metaboloma , Adulto , Biomarcadores/metabolismo , Estudios de Casos y Controles , Estudios de Cohortes , Endometriosis/clasificación , Endometriosis/patología , Femenino , Líquido Folicular/química , Francia , Humanos , Infertilidad Femenina/etiología , Infertilidad Femenina/metabolismo , Infertilidad Femenina/patología , Metaboloma/fisiología , Persona de Mediana Edad , Enfermedades Peritoneales/clasificación , Enfermedades Peritoneales/metabolismo , Enfermedades Peritoneales/patología , Fenotipo , Estudios ProspectivosRESUMEN
Macrozoospermia is associated with severe male infertility. To date, the only gene implicated in this phenotype is the Aurora Kinase C gene. We report in this work the genetic screening of AURKC mutations in 34 patients with macrozoospermia among 3,536 Algerian infertile men. Nineteen patients (56%) were homozygotes for the c.144delC mutation, eight (23.52%) homozygotes for the c.744C>G (p.Y248*) mutation and two (5.88%) compound heterozygotes. No AURKC mutation was identified in five patients (14.7%). Interestingly and although it is generally accepted that nearly all positive mutated AURKC patients have close to 100% large-head spermatozoa, our results showed that 11 patients with AURKC mutations (32.35%) had large-headed spermatozoa lower than 70% (7 with c.144delC and 4 with p.Y248*), and no mutation was found in 2 patients who had >70% of macrocephalic spermatozoa. Twenty ICSI attempts were performed before genetic screening resulting in 39 embryos but no pregnancy was obtained. The sequencing of AURKC exons 3 and 6 is appropriate as a first-line genetic exploration in these patients to avoid unsuccessful ICSI attempts. A percentage of large head spermatozoa beyond 25% and a percentage of multiflagellar spermatozoa beyond 10% are predictive of a positive mutation diagnosis.
Asunto(s)
Infertilidad Masculina , Aurora Quinasa C/genética , Homocigoto , Humanos , Infertilidad Masculina/genética , Masculino , Mutación , EspermatozoidesRESUMEN
We have previously shown, using antibodies, that the sperm alpha6beta1 integrin is involved in mouse gamete fusion in vitro. Here we report the conditional knockdown of the sperm Itgb1 gene. It induced a drastic failure of sperm fusogenic ability with sperm accumulation in the perivitelline space of in vitro inseminated oocytes deleted or not for the Itgb1 gene. These data demonstrate that sperm, but not oocyte, beta1 integrin subunit is involved in gamete adhesion/fusion. Curiously, knockdown males were fertile in vivo probably because of the incomplete Cre-mediated deletion of the sperm Itgb1 floxed gene. Indeed, this was shown by Western blot analysis and confirmed by both the viability and litter size of pups obtained by mating partially sperm Itgb1 deleted males with females producing completely deleted Itgb1 oocytes. Because of the total peri-implantation lethality of Itgb1 deletion in mice, we assume that sperm that escaped the Itgb1 excision seemed to be preferentially used to fertilize in vivo. Here, we showed for the first time that the deletion, even partial, of the sperm Itgb1 gene makes the sperm unable to normally fertilize oocytes. However, to elucidate the question of the essentiality of its role during fertilization, further investigations using a mouse expressing a recombinase more effective in male germ cells are necessary.
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Adhesión Celular/genética , Células Germinativas/fisiología , Integrina beta1/genética , Subunidades de Proteína/genética , Animales , Adhesión Celular/fisiología , Fusión Celular/métodos , Femenino , Fertilización/genética , Fertilización/fisiología , Masculino , Ratones , Ratones Noqueados , Oocitos/fisiología , Interacciones Espermatozoide-Óvulo/genética , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/fisiologíaRESUMEN
Primary ciliary dyskinesia (PCD) is an autosomal-recessive disease due to functional or ultra-structural defects of motile cilia. Affected individuals display recurrent respiratory-tract infections; most males are infertile as a result of sperm flagellar dysfunction. The great majority of the PCD-associated genes identified so far encode either components of dynein arms (DAs), which are multiprotein-ATPase complexes essential for ciliary motility, or proteins involved in DA assembly. To identify the molecular basis of a PCD phenotype characterized by central complex (CC) defects but normal DA structure, a phenotype found in â¼15% of cases, we performed whole-exome sequencing in a male individual with PCD and unexplained CC defects. This analysis, combined with whole-genome SNP genotyping, identified a homozygous mutation in DNAJB13 (c.833T>G), a gene encoding a HSP40 co-chaperone whose ortholog in the flagellated alga Chlamydomonas localizes to the radial spokes. In vitro studies showed that this missense substitution (p.Met278Arg), which involves a highly conserved residue of several HSP40 family members, leads to protein instability and triggers proteasomal degradation, a result confirmed by the absence of endogenous DNAJB13 in cilia and sperm from this individual. Subsequent DNAJB13 analyses identified another homozygous mutation in a second family; the study of DNAJB13 transcripts obtained from airway cells showed that this mutation (c.68+1G>C) results in a splicing defect consistent with a loss-of-function mutation. Overall, this study, which establishes mutations in DNAJB13 as a cause of PCD, unveils the key role played by DNAJB13 in the proper formation and function of ciliary and flagellar axonemes in humans.
Asunto(s)
Trastornos de la Motilidad Ciliar/genética , Proteínas de Choque Térmico/genética , Infertilidad Masculina/genética , Mutación , Adolescente , Proteínas Reguladoras de la Apoptosis , Axonema/genética , Cilios/genética , Trastornos de la Motilidad Ciliar/patología , Exoma/genética , Femenino , Flagelos/genética , Flagelos/patología , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Homocigoto , Humanos , Infertilidad Masculina/patología , Síndrome de Kartagener/genética , Masculino , Persona de Mediana Edad , Chaperonas Moleculares , Mutación Missense/genética , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Empalme del ARN/genética , Semen , Espermatozoides/metabolismo , Espermatozoides/patologíaRESUMEN
STUDY QUESTION: Did the revised Alpha/ESHRE consensus (Vienna, 2017) bring a real answer on managing oocytes with aggregates of smooth endoplasmic reticulum (SERa)? SUMMARY ANSWER: According to the currently available literature, a case by case approach on the time of injecting/inseminating SERa+ oocytes may be not helpful for embryologists making a decision, so we suggest fertilizing both SERa+ and SERa- oocytes and prioritizing embryos derived from SERa- oocytes. WHAT IS KNOWN ALREADY?: In 2011, the Istanbul consensus recommended not to inject/inseminate SER+ oocytes due to adverse foetal outcomes reported in literature. At the end of 2017, a panel of experts reconsidered this recommendation and advised a case by case approach. Hence, with a lack of clear recommendations, in-vitro fertilization practitioners still have heterogeneous attitudes when managing SERa+ oocytes. In this context of controversy, an updated review could be helpful in (i) forming a common language for managing cases of SERa+ oocytes and (ii) offering the most ethical practice and best care for patients seeking infertility treatment or fertility preservation. STUDY DESIGN, SIZE, DURATION: This review (with a last literature search on 1 June 2018) evaluated the effect of the SER dysmorphism on embryological and neonatal outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Studies were considered for inclusion if they were prospective or retrospective cohort or case-control studies. Electronic searches of the Pubmed and Embase databases were done using the keyword combination: smooth endoplasmic reticulum, SER, oocyte and zygote. Abstracts and articles written in English and limited to humans were included. MAIN RESULTS AND THE ROLE OF CHANCE: The search returned a total of 726 studies among which 21 met the inclusion criteria. The literature does not unanimously support a negative association between SERa and embryogenesis, implantation or assisted reproductive therapy outcomes. The reviewed studies reported 112 neonatal outcomes after transfers where at least one embryo originated from oocyte affected by SERa. They included 101 healthy babies, three live births with malformations, three neonatal deaths, one stillbirth and four medical interruptions of pregnancy. After transfer of embryos exclusively derived from SERa+ oocytes, a total of 48 healthy newborns were reported along with four babies with perinatal complications (including one ventricular septal defect), one stillbirth, one neonatal death and one pregnancy termination for multiple malformations. LIMITATIONS, REASONS FOR CAUTION: As with any review, this review was limited by the quality of the included studies especially in terms of possible methodological limitations, the limited sample size and the retrospective aspect of the studies. Among the 21 selected studies, seven were abstracts and two were case reports. Of the remaining 14 studies, only three were prospective. The tools used in identifying SERa+ oocytes may have varied from one study to another and a consequent misclassification cannot be excluded. Considering the poor resolution of light microscopy in detecting SER aggregates, we are not sure that apparently SERa- oocytes do not really exhibit such a dysmorphism if they were analysed under electronic microscopy or a time lapse system. WIDER IMPLICATIONS OF THE FINDINGS: In the light of the existing data and the lack of a real link between fertilizing SERa+ oocytes and the occurrence of embryo aneuploidy/malformations, we think that discarding SERa+ oocytes may be not the most ethical approach even in patients with large cohorts on the day of oocyte retrieval. Avoiding the wastage of oocytes and embryos with respect to medical ethics remains a constant concern in daily IVF practice. Thus, we recommend that all mature oocytes could be fertilized and embryos originating from SERa- oocytes would be preferably transferred, even if they come from a cohort with SERa+ oocytes. The remaining embryos derived from SERa+ oocytes could be considered with a lower priority for transfer after obtaining consent from the couple if a strict follow-up of the pregnancy and the baby is performed. STUDY FUNDING/COMPETING INTEREST(S): We have no conflict of interest to declare and no funding was received. REGISTRATION NUMBER: N/A.
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Retículo Endoplásmico Liso , Fertilización In Vitro/métodos , Oocitos/citología , Toma de Decisiones Clínicas , Consenso , Implantación del Embrión/fisiología , Transferencia de Embrión/métodos , Femenino , Fertilización , Humanos , Recuperación del Oocito , Embarazo , Índice de EmbarazoRESUMEN
INTRODUCTION: The preservation of fertility is an integral part of care of children requiring gonadotoxic treatments for cancer or non-malignant diseases. In France, the cryopreservation of ovarian tissue has been considered and has been offered as a clinical treatment since its inception. The aim of this study is to review 20 years of activity in fertility preservation by ovarian tissue cryopreservation (OTC) for children and the feasibility of oocyte isolation and cryopreservation from the ovarian tissue at a single center. MATERIAL AND METHODS: Retrospective study including patients aged 15 years or younger who underwent OTC, combined for some with oocyte cryopreservation of isolated oocytes, before a highly gonadotoxic treatment for malignant or non-malignant disease was initiated. We describe the evolution of activities in our program for fertility preservation and patient characteristics at the time of OTC and follow up. RESULTS: From April 1998 to December 2018, 418 girls and adolescents younger than 15 years of age underwent OTC, representing 40.5% of all females who have had ovarian tissue cryopreserved at our center. In all, 313 patients had malignant diseases and 105 had benign conditions. Between November 2009 and July 2013, oocytes were isolated and also cryopreserved in 50 cases. The mean age of patients was 6.9 years (range 0.3-15). The most frequent diagnoses in this cohort included neuroblastoma, acute leukemia and hemoglobinopathies; neuroblastoma being the most common diagnosis in very young patients. During follow up, three patients requested the use of their cryopreserved ovarian tissue. All had undergone ovarian tissue transplantation, one for puberty induction and the two others for restoring fertility. So far, no pregnancies have been achieved. Eighty-four patients who had OTC died. CONCLUSIONS: Ovarian tissue cryopreservation is the only available technique for preserving fertility of girls. To our knowledge this is the largest series of girls and adolescents younger than 15 years so far reported on procedures of OTC before highly gonadotoxic treatment in a single center.
Asunto(s)
Antineoplásicos , Criopreservación , Preservación de la Fertilidad , Neoplasias , Ovario , Adolescente , Antineoplásicos/uso terapéutico , Antineoplásicos/toxicidad , Niño , Preescolar , Criopreservación/métodos , Criopreservación/estadística & datos numéricos , Femenino , Preservación de la Fertilidad/métodos , Preservación de la Fertilidad/estadística & datos numéricos , Francia/epidemiología , Humanos , Lactante , Neoplasias/epidemiología , Neoplasias/terapia , Recuperación del Oocito , Evaluación de Procesos y Resultados en Atención de Salud , Utilización de Procedimientos y Técnicas/estadística & datos numéricos , Estudios RetrospectivosRESUMEN
Ongoing progress in genomic technologies offers exciting tools that can help to resolve transcriptome and genome-wide DNA modifications at single-cell resolution. These methods can be used to characterize individual cells within complex tissue organizations and to highlight various molecular interactions. Here, we will discuss recent advances in the definition of spermatogonial stem cells (SSC) and their progenitors in humans using the single-cell transcriptome sequencing (scRNAseq) approach. Exploration of gene expression patterns allows one to investigate stem cell heterogeneity. It leads to tracing the spermatogenic developmental process and its underlying biology, which is highly influenced by the microenvironment. scRNAseq already represents a new diagnostic tool for the personalized investigation of male infertility. One may hope that a better understanding of SSC biology could facilitate the use of these cells in the context of fertility preservation of prepubertal children, as a key component of regenerative medicine.
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Medicina Regenerativa , Células Madre/metabolismo , Transcriptoma , Humanos , Infertilidad Masculina/diagnóstico , Infertilidad Masculina/terapia , Masculino , Análisis de la Célula Individual , Espermatogénesis , Espermatogonias/citología , Trasplante de Células Madre , Células Madre/citologíaAsunto(s)
Anemia de Células Falciformes/tratamiento farmacológico , Antidrepanocíticos/efectos adversos , Hidroxiurea/efectos adversos , Pubertad , Espermatogonias/patología , Factores de Edad , Anemia de Células Falciformes/patología , Antidrepanocíticos/uso terapéutico , Niño , Preescolar , Humanos , Hidroxiurea/farmacología , Hidroxiurea/uso terapéutico , Masculino , Tamaño de la Muestra , Espermatogonias/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/patologíaRESUMEN
STUDY QUESTION: The aim of this study was to evaluate the live birth rate (LBR) after frozen-thawed Day 5 (D5) and Day 6 (D6) blastocyst transfers. SUMMARY ANSWER: LBR following frozen-thawed blastocyst transfer is significantly lower with D6 than with D5 blastocyst regardless of embryo quality. WHAT IS KNOWN ALREADY: During fresh embryo transfer cycles, pregnancy rates (PR) are significantly higher when transferring blastocysts expanded on D5 compared with slow developing blastocysts (D6). In programmed thawed blastocyst transfer (TBT) cycles, the same clinical outcomes should be expected when transferring D5 or D6 blastocysts because of endometrial/embryonic synchronization due to hormonal priming of endometrial receptivity. However, the impact of delayed blastocyst expansion at D6 on clinical outcomes remains unclear. Some reports have shown higher PRs after D5 TBT compared with those of D6, while others have shown equivalent TBT outcomes after D5 and D6 cryopreserved blastocysts transfers. STUDY, DESIGN, SIZE, DURATION: This retrospective cohort follow-up study included 1347 single autologous frozen-thawed blastocyst transfers performed between January 2012 and December 2015 at a tertiary care university hospital. PARTICIPANTS/MATERIALS, SETTING, METHODS: All of the patients scheduled for TBT were allocated to two groups according to the day of blastocyst expansion: on D5 (n = 994) or on D6 (n = 353). The primary outcome was LBR per embryo transfer in the first blastocyst thawing cycle. Secondary outcomes were clinical pregnancy rate (cPR), early miscarriage rate and neonatal outcomes following TBT for the two groups. Statistical analyses were conducted using univariate and multivariate logistic regression model. MAIN RESULTS AND THE ROLE OF CHANCE: The LBR was significantly increased in the D5 group compared to the D6 group [294/994 (29.6%) versus 60/353 (17.0%); P < 0.001]. The cPR was also higher when blastocysts were vitrified on D5 compared with those vitrified on D6 [429/994 (43.2%) versus 95/353 (26.9%); P < 0.001]. No significant differences were found between groups in terms of early miscarriage rate (P = 0.862). More good-quality embryos (defined as an B3-B4 or B5 embryo ≥BB according to the grading scale proposed by Gardner) were transferred in the D5 group than in the D6 group [807 (81.2%) versus 214 (60.6%); P < 0.001]. However, a comparison of TBT cycles with equal embryo quality (good versus low) also supported the superiority of D5 blastocysts. Concerning neonatal outcomes, the D5 group infants had a lower mean birth weight compared to those of the D6 group (P = 0.001). In addition, a significantly shorter gestational age at birth is reported in the D5 blastocyst group as compared to the D6 group (P = 0.004). After multivariate logistic regression taking into account potential confounders such as the women's age, number of previous IVF/ICSI procedures, the day of the blastocyst vitrification (D5 or D6) and embryo quality, blastocyst expansion at D6 was independently associated with a significant decrease in LBR compared to D5 expanded-blastocysts (OR 0.52; 95% CI 0.38-0.72; P < 0.001). LIMITATIONS, REASONS FOR CAUTION: The poor predictive value of the morphological approach in embryo selection could constitute a limitation in this study. However, blastocyst quality was evaluated similarly in both groups. WIDER IMPLICATIONS OF THE FINDINGS: The LBR following frozen-thawed blastocyst transfer was significantly lower with D6 than with D5 blastocysts, regardless of their quality. These results could affect cryopreservation procedures as they suggest that the use of D5-expanded blastocysts for TBT may be preferred in order to shorten the time of conceiving. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was obtained for this study. None of the authors have any competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.
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Tasa de Natalidad , Transferencia de Embrión/métodos , Desarrollo Embrionario/fisiología , Adulto , Blastocisto , Criopreservación , Femenino , Humanos , Nacimiento Vivo , Inducción de la Ovulación , Embarazo , Índice de Embarazo , Estudios RetrospectivosRESUMEN
PURPOSE: The aims of this study were to investigate the possible benefits of extending the culture of poor-quality day-2 embryos (PQE) versus good-quality embryos (GQE) and to identify factors associated with pregnancy and live birth when transferring frozen-thawed blastocysts originating from GQE and PQE. METHODS: This is a retrospective cohort follow-up study performed between November 2012 and February 2015 at the IVF Laboratory Unit of Cochin University Hospital (Paris, France) including 3108 day-2 supernumerary embryos resulting from 1237 IVF/ICSI cycles. RESULTS: Total blastulation rate was 67.2% from GQE and 48.7% from PQE. Percentage of good-quality blastocysts was 60.7 and 47.9% respectively including 14.7 and 7.3% top-quality blastocysts. A total of 150 blastocysts originating from GQE and 729 from PQE were frozen, and then, 37 and 164 were thawed and transferred respectively resulting in 19 (51.4%) and 61 (37.9%) clinical pregnancies with 13 (35.1%) deliveries from GQE and 32 (19.9%) from PQE (p = 0.046) without any difference in neonatal outcomes. Quality of blastocysts that resulted in live birth was similar in the two groups. Women < 35 years old and day-5 blastocyst expansion were predictive of pregnancy and live birth. CONCLUSIONS: (i) PQE are able to reach the blastocyst stage, to implant, and to give healthy babies and (ii) women age and day of blastocyst expansion are predictive of pregnancy and live birth.
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Blastocisto/fisiología , Técnicas de Cultivo de Embriones/métodos , Fertilización In Vitro/métodos , Adulto , Tasa de Natalidad , Estudios de Cohortes , Criopreservación/métodos , Transferencia de Embrión/métodos , Femenino , Fertilización In Vitro/estadística & datos numéricos , Humanos , Recién Nacido , Nacimiento Vivo , Embarazo , Resultado del Embarazo , Índice de Embarazo , Estudios Retrospectivos , Inyecciones de Esperma Intracitoplasmáticas/métodos , Inyecciones de Esperma Intracitoplasmáticas/estadística & datos numéricosRESUMEN
PURPOSE: This study was designed to evaluate patient management and quality of information given by French oncologists to cancer women concerning fertility issues and possibilities of fertility preservation. METHODS: An online survey was sent to 1161 physicians in all major cancer centers throughout France between May 2012 and January 2013. RESULTS: A total of 102 responses were received and analyzed. Only 46% of all physicians surveyed reported discussing infertility risks with patients of reproductive age and 22% referred them to a fertility center before beginning treatments. Only 14% of practitioners considered themselves knowledgeable in FP techniques and ovarian transposition was the most frequently mentioned technique in consultation. CONCLUSION: This study is at the best of our knowledge the first nationwide survey to assess the state of the art in oncofertility management. It highlights inadequate management of fertility preservation for female patients in France. Physicians reported lacking knowledge and tools that would allow them to provide patients with appropriate information. A better collaboration between cancer and fertility centers needs to be organized in France as already organized in other countries.
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Preservación de la Fertilidad/métodos , Infertilidad Femenina/epidemiología , Neoplasias/epidemiología , Oncólogos/psicología , Adulto , Femenino , Preservación de la Fertilidad/psicología , Preservación de la Fertilidad/tendencias , Francia/epidemiología , Conocimientos, Actitudes y Práctica en Salud , Humanos , Infertilidad , Infertilidad Femenina/psicología , Neoplasias/fisiopatología , Neoplasias/psicología , Encuestas y CuestionariosRESUMEN
Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery.
Asunto(s)
Fertilización/fisiología , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Interacciones Espermatozoide-Óvulo/fisiología , Tetraspanina 29/metabolismo , Animales , Adhesión Celular/fisiología , Femenino , Humanos , Cinética , Masculino , Ratones , Microscopía ConfocalRESUMEN
X-chromosome inactivation (XCI) in female mammals allows dosage compensation for X-linked gene products between the sexes. The developmental regulation of this process has been extensively investigated in mice, where the X chromosome of paternal origin (Xp) is silenced during early embryogenesis owing to imprinted expression of the regulatory RNA, Xist (X-inactive specific transcript). Paternal XCI is reversed in the inner cell mass of the blastocyst and random XCI subsequently occurs in epiblast cells. Here we show that other eutherian mammals have very different strategies for initiating XCI. In rabbits and humans, the Xist homologue is not subject to imprinting and XCI begins later than in mice. Furthermore, Xist is upregulated on both X chromosomes in a high proportion of rabbit and human embryo cells, even in the inner cell mass. In rabbits, this triggers XCI on both X chromosomes in some cells. In humans, chromosome-wide XCI has not initiated even by the blastocyst stage, despite the upregulation of XIST. The choice of which X chromosome will finally become inactive thus occurs downstream of Xist upregulation in both rabbits and humans, unlike in mice. Our study demonstrates the remarkable diversity in XCI regulation and highlights differences between mammals in their requirement for dosage compensation during early embryogenesis.
Asunto(s)
Cromosomas de los Mamíferos/genética , Regulación del Desarrollo de la Expresión Génica/genética , Mamíferos/genética , Inactivación del Cromosoma X/genética , Cromosoma X/genética , Animales , Evolución Biológica , Blastocisto/metabolismo , Compensación de Dosificación (Genética)/genética , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Genes Ligados a X/genética , Impresión Genómica/genética , Histonas/metabolismo , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Masculino , Mamíferos/embriología , Ratones , Partenogénesis , ARN Largo no Codificante , ARN no Traducido/genética , Conejos , Especificidad de la Especie , Regulación hacia Arriba/genéticaRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is present in mature sperm and is required for sperm motility and capacitation. Both these processes are controlled by ions fluxes and are essential for fertilization. We have shown that SLC26A8, a sperm-specific member of the SLC26 family of anion exchangers, associates with the CFTR channel and strongly stimulates its activity. This suggests that the two proteins cooperate to regulate the anion fluxes required for correct sperm motility and capacitation. Here, we report on three heterozygous SLC26A8 missense mutations identified in a cohort of 146 men presenting with asthenozoospermia: c.260G>A (p.Arg87Gln), c.2434G>A (p.Glu812Lys), and c.2860C>T (p.Arg954Cys). These mutations were not present in 121 controls matched for ethnicity, and statistical analysis on a control population of 8,600 individuals (from dbSNP and 1000 Genomes) showed them to be associated with asthenozoospermia with a power > 95%. By cotransfecting Chinese hamster ovary (CHO)-K1 cells with SLC26A8 variants and CFTR, we showed that the physical interaction between the two proteins was partly conserved but that the capacity to activate CFTR-dependent anion transport was completely abolished for all mutants. Biochemical studies revealed the presence of much smaller amounts of protein for all variants, but these amounts were restored to wild-type levels upon treatment with the proteasome inhibitor MG132. Immunocytochemistry also showed the amounts of SLC26A8 in sperm to be abnormally small in individuals carrying the mutations. These mutations might therefore impair formation of the SLC26A8-CFTR complex, principally by affecting SLC26A8 stability, consistent with an impairment of CFTR-dependent sperm-activation events in affected individuals.