RESUMO
The mouse and human embryo gradually loses totipotency before diversifying into the inner cell mass (ICM, future organism) and trophectoderm (TE, future placenta). The transcription factors TFAP2C and TEAD4 with activated RHOA accelerate embryo polarization. Here we show that these factors also accelerate the loss of totipotency. TFAP2C and TEAD4 paradoxically promote and inhibit Hippo signaling before lineage diversification: they drive expression of multiple Hippo regulators while also promoting apical domain formation, which inactivates Hippo. Each factor activates TE specifiers in bipotent cells, while TFAP2C also activates specifiers of the ICM fate. Asymmetric segregation of the apical domain reconciles the opposing regulation of Hippo signaling into Hippo OFF and the TE fate, or Hippo ON and the ICM fate. We propose that the bistable switch established by TFAP2C and TEAD4 is exploited to trigger robust lineage diversification in the developing embryo.
Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição de Domínio TEA , Fator de Transcrição AP-2 , Fatores de Transcrição , Fator de Transcrição AP-2/metabolismo , Fator de Transcrição AP-2/genética , Animais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos , Humanos , Transdução de Sinais , Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Via de Sinalização Hippo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Desenvolvimento Embrionário/genéticaRESUMO
BACKGROUND: Freezing all embryos, followed by thawing and transferring them into the uterine cavity at a later stage (freeze-all), instead of fresh-embryo transfer may lead to improved pregnancy rates and fewer complications during in vitro fertilisation and pregnancies resulting from it. OBJECTIVE: We aimed to evaluate if a policy of freeze-all results in a higher healthy baby rate than the current policy of transferring fresh embryos. DESIGN: This was a pragmatic, multicentre, two-arm, parallel-group, non-blinded, randomised controlled trial. SETTING: Eighteen in vitro fertilisation clinics across the UK participated from February 2016 to April 2019. PARTICIPANTS: Couples undergoing their first, second or third cycle of in vitro fertilisation treatment in which the female partner was aged < 42 years. INTERVENTIONS: If at least three good-quality embryos were present on day 3 of embryo development, couples were randomly allocated to either freeze-all (intervention) or fresh-embryo transfer (control). OUTCOMES: The primary outcome was a healthy baby, defined as a live, singleton baby born at term, with an appropriate weight for their gestation. Secondary outcomes included ovarian hyperstimulation, live birth and clinical pregnancy rates, complications of pregnancy and childbirth, health economic outcome, and State-Trait Anxiety Inventory scores. RESULTS: A total of 1578 couples were consented and 619 couples were randomised. Most non-randomisations were because of the non-availability of at least three good-quality embryos (n = 476). Of the couples randomised, 117 (19%) did not adhere to the allocated intervention. The rate of non-adherence was higher in the freeze-all arm, with the leading reason being patient choice. The intention-to-treat analysis showed a healthy baby rate of 20.3% in the freeze-all arm and 24.4% in the fresh-embryo transfer arm (risk ratio 0.84, 95% confidence interval 0.62 to 1.15). Similar results were obtained using complier-average causal effect analysis (risk ratio 0.77, 95% confidence interval 0.44 to 1.10), per-protocol analysis (risk ratio 0.87, 95% confidence interval 0.59 to 1.26) and as-treated analysis (risk ratio 0.91, 95% confidence interval 0.64 to 1.29). The risk of ovarian hyperstimulation was 3.6% in the freeze-all arm and 8.1% in the fresh-embryo transfer arm (risk ratio 0.44, 99% confidence interval 0.15 to 1.30). There were no statistically significant differences between the freeze-all and the fresh-embryo transfer arms in the live birth rates (28.3% vs. 34.3%; risk ratio 0.83, 99% confidence interval 0.65 to 1.06) and clinical pregnancy rates (33.9% vs. 40.1%; risk ratio 0.85, 99% confidence interval 0.65 to 1.11). There was no statistically significant difference in anxiety scores for male participants (mean difference 0.1, 99% confidence interval -2.4 to 2.6) and female participants (mean difference 0.0, 99% confidence interval -2.2 to 2.2) between the arms. The economic analysis showed that freeze-all had a low probability of being cost-effective in terms of the incremental cost per healthy baby and incremental cost per live birth. LIMITATIONS: We were unable to reach the original planned sample size of 1086 and the rate of non-adherence to the allocated intervention was much higher than expected. CONCLUSION: When efficacy, safety and costs are considered, freeze-all is not better than fresh-embryo transfer. TRIAL REGISTRATION: This trial is registered as ISRCTN61225414. FUNDING: This project was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 26, No. 25. See the NIHR Journals Library website for further project information.
During in vitro fertilisation, eggs and sperm are mixed in a laboratory to create embryos. An embryo is placed in the womb 25 days later (fresh-embryo transfer) and the remaining embryos are frozen for future use. Initial research suggested that freezing all embryos followed by thawing and replacing them a few weeks later could improve treatment safety and success. Although these data were promising, the data came from small studies and were not enough to change practice and policy. We conducted a large, multicentre, clinical trial to evaluate the two strategies: fresh-embryo transfer compared with later transfer of frozen embryos. We also compared the costs of both strategies during in vitro fertilisation treatment, pregnancy and delivery. This study was conducted across 18 clinics in the UK from 2016 to 2019, and 619 couples participated. Couples were allocated to one of two strategies: immediate fresh-embryo transfer or freezing of all embryos followed later by transfer of frozen embryo. The study's aim was to find out which type of embryo transfer gave participants a higher chance of having a healthy baby. We found that freezing all embryos followed by frozen-embryo transfer did not lead to a higher chance of having a healthy baby. There were no differences between strategies in the number of live births, the miscarriage rate or the number of pregnancy complications. Fresh-embryo transfer was less costly from both a health-care and a patient perspective. A routine strategy of freezing all embryos is not justified given that there was no increase in success rates but there were extra costs and delays to embryo transfer.
Assuntos
Transferência Embrionária , Síndrome de Hiperestimulação Ovariana , Transferência Embrionária/métodos , Feminino , Fertilização in vitro/métodos , Congelamento , Humanos , Nascido Vivo , Masculino , Gravidez , Taxa de GravidezRESUMO
During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and imaging approaches, we uncover that in the mouse E6.5 epiblast, where apical tension is highest, ASPP2 safeguards tissue integrity. It achieves this by preventing the most apical daughter cells from delaminating apically following division events. In this context, ASPP2 maintains the integrity and organisation of the filamentous actin cytoskeleton at apical junctions. ASPP2 is also essential during gastrulation in the primitive streak, in somites and in the head fold region, suggesting that it is required across a wide range of pseudostratified epithelia during morphogenetic events that are accompanied by intense tissue remodelling. Finally, our study also suggests that the interaction between ASPP2 and PP1 is essential to the tumour suppressor function of ASPP2, which may be particularly relevant in the context of tissues that are subject to increased mechanical stress.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Epitélio/crescimento & desenvolvimento , Morfogênese , Proteínas Supressoras de Tumor/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Células CACO-2 , Polaridade Celular , Cães , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Epitélio/metabolismo , Feminino , Gastrulação , Camadas Germinativas , Humanos , Células Madin Darby de Rim Canino , Camundongos , Camundongos Transgênicos , Mutação , Linha Primitiva , Receptores de Neuropeptídeo Y/metabolismo , Estresse Mecânico , Junções Íntimas/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
STUDY QUESTION: Does a policy of elective freezing of embryos, followed by frozen embryo transfer result in a higher healthy baby rate, after first embryo transfer, when compared with the current policy of transferring fresh embryos? SUMMARY ANSWER: This study, although limited by sample size, provides no evidence to support the adoption of a routine policy of elective freeze in preference to fresh embryo transfer in order to improve IVF effectiveness in obtaining a healthy baby. WHAT IS KNOWN ALREADY: The policy of freezing all embryos followed by frozen embryo transfer is associated with a higher live birth rate for high responders but a similar/lower live birth after first embryo transfer and cumulative live birth rate for normal responders. Frozen embryo transfer is associated with a lower risk of ovarian hyperstimulation syndrome (OHSS), preterm delivery and low birthweight babies but a higher risk of large babies and pre-eclampsia. There is also uncertainty about long-term outcomes, hence shifting to a policy of elective freezing for all remains controversial given the delay in treatment and extra costs involved in freezing all embryos. STUDY DESIGN, SIZE, DURATION: A pragmatic two-arm parallel randomized controlled trial (E-Freeze) was conducted across 18 clinics in the UK from 2016 to 2019. A total of 619 couples were randomized (309 to elective freeze/310 to fresh). The primary outcome was a healthy baby after first embryo transfer (term, singleton live birth with appropriate weight for gestation); secondary outcomes included OHSS, live birth, clinical pregnancy, pregnancy complications and cost-effectiveness. PARTICIPANTS/MATERIALS, SETTING, METHODS: Couples undergoing their first, second or third cycle of IVF/ICSI treatment, with at least three good quality embryos on Day 3 where the female partner was ≥18 and <42 years of age were eligible. Those using donor gametes, undergoing preimplantation genetic testing or planning to freeze all their embryos were excluded. IVF/ICSI treatment was carried out according to local protocols. Women were followed up for pregnancy outcome after first embryo transfer following randomization. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 619 couples randomized, 307 and 309 couples in the elective freeze and fresh transfer arms, respectively, were included in the primary analysis. There was no evidence of a statistically significant difference in outcomes in the elective freeze group compared to the fresh embryo transfer group: healthy baby rate {20.3% (62/307) versus 24.4% (75/309); risk ratio (RR), 95% CI: 0.84, 0.62 to 1.15}; OHSS (3.6% versus 8.1%; RR, 99% CI: 0.44, 0.15 to 1.30); live birth rate (28.3% versus 34.3%; RR, 99% CI 0.83, 0.65 to 1.06); and miscarriage (14.3% versus 12.9%; RR, 99% CI: 1.09, 0.72 to 1.66). Adherence to allocation was poor in the elective freeze group. The elective freeze approach was more costly and was unlikely to be cost-effective in a UK National Health Service context. LIMITATIONS, REASONS FOR CAUTION: We have only reported on first embryo transfer after randomization; data on the cumulative live birth rate requires further follow-up. Planned target sample size was not obtained and the non-adherence to allocation rate was high among couples in the elective freeze arm owing to patient preference for fresh embryo transfer, but an analysis which took non-adherence into account showed similar results. WIDER IMPLICATIONS OF THE FINDINGS: Results from the E-Freeze trial do not lend support to the policy of electively freezing all for everyone, taking both efficacy, safety and costs considerations into account. This method should only be adopted if there is a definite clinical indication. STUDY FUNDING/COMPETING INTEREST(S): NIHR Health Technology Assessment programme (13/115/82). This research was funded by the National Institute for Health Research (NIHR) (NIHR unique award identifier) using UK aid from the UK Government to support global health research. The views expressed in this publication are those of the author(s) and not necessarily those of the NIHR or the UK Department of Health and Social Care. J.L.B., C.C., E.J., P.H., J.J.K., L.L. and G.S. report receipt of funding from NIHR, during the conduct of the study. J.L.B., E.J., P.H., K.S. and L.L. report receipt of funding from NIHR, during the conduct of the study and outside the submitted work. A.M. reports grants from NIHR personal fees from Merck Serono, personal fees for lectures from Merck Serono, Ferring and Cooks outside the submitted work; travel/meeting support from Ferring and Pharmasure and participation in a Ferring advisory board. S.B. reports receipt of royalties and licenses from Cambridge University Press, a board membership role for NHS Grampian and other financial or non-financial interests related to his roles as Editor-in-Chief of Human Reproduction Open and Editor and Contributing Author of Reproductive Medicine for the MRCOG, Cambridge University Press. D.B. reports grants from NIHR, during the conduct of the study; grants from European Commission, grants from Diabetes UK, grants from NIHR, grants from ESHRE, grants from MRC, outside the submitted work. Y.C. reports speaker fees from Merck Serono, and advisory board role for Merck Serono and shares in Complete Fertility. P.H. reports membership of the HTA Commissioning Committee. E.J. reports membership of the NHS England and NIHR Partnership Programme, membership of five Data Monitoring Committees (Chair of two), membership of six Trial Steering Committees (Chair of four), membership of the Northern Ireland Clinical Trials Unit Advisory Group and Chair of the board of Oxford Brain Health Clinical Trials Unit. R.M. reports consulting fees from Gedeon Richter, honorarium from Merck, support fees for attendance at educational events and conferences for Merck, Ferring, Bessins and Gedeon Richter, payments for participation on a Merck Safety or Advisory Board, Chair of the British Fertility Society and payments for an advisory role to the Human Fertilisation and Embryology Authority. G.S. reports travel and accommodation fees for attendance at a health economic advisory board from Merck KGaA, Darmstadt, Germany. N.R.-F. reports shares in Nurture Fertility. Other authors' competing interests: none declared. TRIAL REGISTRATION NUMBER: ISRCTN: 61225414. TRIAL REGISTRATION DATE: 29 December 2015. DATE OF FIRST PATIENT'S ENROLMENT: 16 February 2016.
Assuntos
Síndrome de Hiperestimulação Ovariana , Medicina Estatal , Transferência Embrionária/métodos , Feminino , Fertilização in vitro , Congelamento , Humanos , Recém-Nascido , Síndrome de Hiperestimulação Ovariana/epidemiologia , Síndrome de Hiperestimulação Ovariana/etiologia , Gravidez , Taxa de Gravidez , Reino UnidoRESUMO
In the United Kingdom, between 2012 and 2017 the annual number of frozen-thawed embryo replacement (FER) cycles doubled, while fresh cycles declined. With FER now accounting for 34% of IVF cycles, the aim of this UK-wide survey of IVF clinics was to determine current trends in the management of FER. A senior clinician in each of the 84 UK IVF clinics was asked to complete an online survey between September 2018 and February 2019 focussing on their clinic's first-line protocols for FER. Sixty-five clinics (77%) responded, accounting for approximately 24,419 FER cycles annually. In ovulatory women, 69% of clinics favour medicated, 26% natural cycle and 5% modified natural cycle FER. In medicated FER, 61% of clinics undertake blastocyst transfer on the sixth day of progesterone administration, 21% on the fifth, 13% on the seventh, 3% on the fourth and 2% on the third. The preferred route of progesterone in medicated FER is vaginal, favoured by 82% of clinics. For pituitary suppression, 55% of clinics favour GnRH-agonist, 11% GnRH-antagonist and 34% oestrogen-only. In natural cycle FER, 31% always, 44% sometimes and 25% never give supplementary luteal support. In summary, the results illustrate wide variation in practice and highlight key research priorities.
Assuntos
Transferência Embrionária , Progesterona , Estudos Transversais , Criopreservação , Transferência Embrionária/métodos , Feminino , Fertilização in vitro/métodos , Hormônio Liberador de Gonadotropina , Humanos , Gravidez , Taxa de Gravidez , Progesterona/uso terapêutico , Estudos RetrospectivosRESUMO
Phospholipase C zeta (PLCζ) is a sperm-specific protein that triggers oocyte activation. The analysis of PLCζ expression in human spermatozoa can be used as a diagnostic marker for oocyte activation deficiency. Our laboratory has previously optimized a standard "in-house" assay to determine PLCζ expression in human spermatozoa. However, one study has suggested that an antigen unmasking method (AUM) would be more efficient in visualizing PLCζ in human sperm. This study aimed to compare our established assay and AUM (involving HCl, acidic Tyrode's solution [AT], and heat). The mean relative fluorescence (RF) intensity of PLCζ in frozen-thawed spermatozoa from fourteen fertile donors stained with the in-house method was significantly higher than three other AUM groups (in-house [mean ± standard error of mean]: 18.87 ± 2.39 arbitrary units [a.u.] vs non-AUM: 11.44 ± 1.61 a.u., AT-AUM: 12.38 ± 1.89 a.u., and HCl-AUM: 12.51 ± 2.16 a.u., P < 0.05, one-way analysis of variance). The mean RF intensity of PLCζ in AT- and HCl-treated spermatozoa from 12 infertile males was not significantly different from that of the non-AUM group. However, the in-house method resulted in the highest RF intensity (12.11 ± 1.36 a.u., P < 0.01). Furthermore, specificity testing of antibody-antigen binding indicated that the in-house method showed more specific binding than spermatozoa treated by the AUM. In conclusion, our in-house method showed superior visualization and reliability than the AUM, thus supporting the continued use of our in-house assay for clinical research screening.
Assuntos
Fosfoinositídeo Fosfolipase C/metabolismo , Sêmen , Fosfolipases Tipo C , Humanos , Masculino , Oócitos/fisiologia , Reprodutibilidade dos Testes , Sêmen/metabolismo , Espermatozoides/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
Recurrent Pregnancy Loss (RPL) affects 2-4% of couples, and with increasing numbers of pregnancy losses the risk of miscarrying a euploid pregnancy is increased, suggesting RPL is a pathology distinct from sporadic miscarriage that is due largely to lethal embryonic aneuploidy. There are a number of conditions associated with RPL including unspecified "immune" pathologies; one of the strongest candidates for dysregulation remains T regulatory cells as depletion in the very early stages of pregnancy in mice leads to pregnancy loss. Human endometrial Treg and conventional CD4T cells were isolated during the peri-implantation period of the menstrual cycle in normal women. We identified an endometrial Treg transcriptomic signature and validated an enhanced regulatory phenotype compared to peripheral blood Treg. Parous women had an altered endometrial Treg transcriptome compared to nulliparity, indicating acquired immune memory of pregnancy within the Treg population, by comparison endometrial conventional CD4T cells were not altered. We compared primary and secondary RPL to nulliparous or parous controls respectively. Both RPL subgroups displayed differentially expressed Treg gene transcriptomes compared to controls. We found increased cell surface S1PR1 and decreased TIGIT protein expression by Treg in primary RPL, confirming the presence of altered Treg in the peri-implantation RPL endometrium.
Assuntos
Aborto Habitual/imunologia , Implantação do Embrião/fisiologia , Endométrio/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Movimento Celular , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Tolerância Imunológica , Paridade , Fenótipo , Receptores Imunológicos/genética , Receptores de Esfingosina-1-Fosfato/genética , Transcriptoma , Adulto JovemRESUMO
The human endometrium is the innermost mucosal membrane of the uterus and is the first point of contact for an implanting blastocyst. A wide variety of immune cells are found amongst the endometrial epithelial layers and stromal cells which both provide host immune responses against pathogens and also assist with placentation and pregnancy establishment, however, B cells have not been characterized, despite being a vital player in both adaptive and mucosal immunity. Through analysis of mid-luteal endometrial biopsies, we find 1-5% of endometrial immune cells are B cells, the majority were naïve or memory B cells, with few plasma cells. Compared with circulating B cells, endometrial B cells had an activated phenotype, with increased expression of CD69, HLA-DR, CD74, and CD83, and IL-10 production capacities. PD1+CXCR5+ICOS+ T follicular helper-like cells and FAS+IgD-BCL6+ germinal center B cells were also present in the endometrium, which may indicate that endometrial B cells are playing an active role through germinal center reactions in the human endometrial environment.
RESUMO
STUDY QUESTION: What is the clinical-effectiveness and safety of the endometrial scratch (ES) procedure compared to no ES, prior to usual first time in vitro fertilisation (IVF) treatment? SUMMARY ANSWER: ES was safe but did not improve pregnancy outcomes when performed in the mid-luteal phase prior to the first IVF cycle, with or without intracytoplasmic sperm injection (ICSI). WHAT IS KNOWN ALREADY: ES is an 'add-on' treatment that is available to women undergoing a first cycle of IVF, with or without ICSI, despite a lack of evidence to support its use. STUDY DESIGN, SIZE, DURATION: This pragmatic, superiority, open-label, multi-centre, parallel-group randomised controlled trial involving 1048 women assessed the clinical effectiveness and safety of the ES procedure prior to first time IVF, with or without ICSI, between July 2016 and October 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants aged 18-37 years undergoing their first cycle of IVF, with or without ICSI, were recruited from 16 UK fertility clinics and randomised (1:1) by a web-based system with restricted access rights that concealed allocation. Stratified block randomisation was used to allocate participants to TAU or ES in the mid-luteal phase followed by usual IVF with or without ICSI treatment. The primary outcome was live birth after completing 24 weeks gestation within 10.5 months of egg collection. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 1048 women randomised to TAU (n = 525) and ES (n = 523) were available for intention to treat analysis. In the ES group, 453 (86.6%) received the ES procedure. IVF, with or without ICSI, was received in 494 (94.1%) and 497 (95.0%) of ES and TAU participants respectively. Live birth rate was 37.1% (195/525) in the TAU and 38.6% (202/523) in the ES: an unadjusted absolute difference of 1.5% (95% CI -4.4% to 7.4%, P = 0.621). There were no statistical differences in secondary outcomes. Adverse events were comparable across groups. LIMITATIONS, REASONS FOR CAUTION: A sham ES procedure was not undertaken in the control group, however, we do not believe this would have influenced the results as objective fertility outcomes were used. WIDER IMPLICATIONS OF THE FINDINGS: This is the largest trial that is adequately powered to assess the impact of ES on women undergoing their first cycle of IVF. ES was safe, but did not significantly improve pregnancy outcomes when performed in the mid-luteal phase prior to the first IVF or ICSI cycle. We recommend that ES is not undertaken in this population. STUDY FUNDING/COMPETING INTEREST(S): Funded by the National Institute of Health Research. Stephen Walters is an National Institute for Health Research (NIHR) Senior Investigator (2018 to present) and was a member of the following during the project: National Institute for Health Research (NIHR) Health Technology Assessment (HTA) Clinical Trials and Evaluation Committee (2011-2017), NIHR HTA Commissioning Strategy Group (2012 to 2017); NIHR Programme Grants for Applied Research Committee (2020 to present); NIHR Pre doctoral Fellowship Committee (2019 to present). Dr. Martins da Silva reports grants from AstraZeneca, during the conduct of the study; and is Associate editor of Human Reproduction and Editorial Board member of Reproduction and Fertility. Dr. Bhide reports grants from Bart's Charity and grants and non-financial support from Pharmasure Pharmaceuticals outside the submitted work. TRIAL REGISTRATION NUMBER: ISRCTN number: ISRCTN23800982. TRIAL REGISTRATION DATE: 31 May 2016. DATE OF FIRST PATIENT'S ENROLMENT: 04 July 2016.
Assuntos
Fertilização in vitro , Injeções de Esperma Intracitoplásmicas , Coeficiente de Natalidade , Feminino , Humanos , Fase Luteal , Gravidez , Taxa de Gravidez , Resultado do TratamentoRESUMO
OBJECTIVE: To validate and apply a strategy permitting parallel preimplantation genetic testing (PGT) for mitochondrial DNA (mtDNA) disease and aneuploidy (PGT-A). DESIGN: Preclinical test validation and case reports. SETTING: Fertility centers. Diagnostics laboratory. PATIENTS: Four patients at risk of transmitting mtDNA disease caused by m.8993T>G (Patients A and B), m.10191T>G (Patient C), and m.3243A>G (Patient D). Patients A, B, and C had affected children. Patients A and D displayed somatic heteroplasmy for mtDNA mutations. INTERVENTIONS: Embryo biopsy, genetic testing, and uterine transfer of embryos predicted to be euploid and mutation-free. MAIN OUTCOME MEASURES: Test accuracy, treatment outcomes, and mutation segregation. RESULTS: Accuracy of mtDNA mutation quantification was confirmed. The test was compatible with PGT-A, and half of the embryos tested were shown to be aneuploid (16/33). Mutations were detected in approximately 40% of embryo biopsies from Patients A and D (10/24) but in none from Patients B and C (n = 29). Patients B and C had healthy children following PGT and natural conception, respectively. The m.8993T>G mutation displayed skewed segregation, whereas m.3243A>G mutation levels were relatively low and potentially impacted embryo development. CONCLUSIONS: Considering the high aneuploidy rate, strategies providing a combination of PGT for mtDNA disease and aneuploidy may be advantageous compared with approaches that consider only mtDNA. Heteroplasmic women had a higher incidence of affected embryos than those with undetectable somatic mutant mtDNA but were still able to produce mutation-free embryos. While not conclusive, the results are consistent with the existence of mutation-specific segregation mechanisms occurring during oogenesis and possibly embryogenesis.
Assuntos
Aneuploidia , Blastocisto/patologia , Análise Mutacional de DNA , DNA Mitocondrial/genética , Fertilização in vitro , Doença de Leigh/diagnóstico , Mutação , Diagnóstico Pré-Implantação , Adulto , Feminino , Predisposição Genética para Doença , Hereditariedade , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Doença de Leigh/genética , Doença de Leigh/patologia , Masculino , Pessoa de Meia-Idade , Linhagem , Valor Preditivo dos Testes , Gravidez , Reprodutibilidade dos TestesRESUMO
Precise patterning within the three-dimensional context of tissues, organs and embryos implies that cells can sense their relative position. During preimplantation development, outside and inside cells rely on apicobasal polarity and the Hippo pathway to choose their fate. Despite recent findings suggesting that mechanosensing might be central to this process, the relationship between blastomere geometry (i.e. shape and position) and the Hippo pathway effector YAP remains unknown. We used a highly quantitative approach to analyse information on the geometry and YAP localisation of individual blastomeres of mouse and human embryos. We identified the proportion of exposed cell surface area as most closely correlating with the nuclear localisation of YAP. To test this relationship, we developed several hydrogel-based approaches to alter blastomere geometry in cultured embryos. Unbiased clustering analyses of blastomeres from such embryos revealed that this relationship emerged during compaction. Our results therefore pinpoint the time during early embryogenesis when cells acquire the ability to sense changes in geometry and provide a new framework for how cells might integrate signals from different membrane domains to assess their relative position within the embryo.
Assuntos
Blastocisto/citologia , Blastocisto/metabolismo , Blastômeros/metabolismo , Animais , Blastômeros/citologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVE: To study fertility issues and pregnancy outcomes in Turner syndrome (TS). DESIGN: Retrospective cohort study. SETTING: Not applicable. PATIENT(S): One hundred fifty-six TS patients, median age 32 years, 23 mosaic 45,X/46,XX, 45,X/47,XXX, 45,X/46,XX/47,XXX. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Fertility choices, spontaneous pregnancy, and oocyte donation (OD) outcomes. Conditions associated with aortic dissection and poor pregnancy outcomes at preconception were considered. Pregnancy-related aortic dimension changes and the long-term impact of pregnancy on TS-related comorbidities were assessed. RESULTS(S): In all, 13.5% had spontaneous pregnancies, resulting in a pregnancy with live birth in 18 patients (37 newborns); 16% considered OD, one adopted, and one underwent fertility preservation. Spontaneous pregnancy predictive factors were a karyotype with a second or third cell line with more than one X and spontaneous menarche. In all, 47.6% had miscarriages, two experienced preeclampsia, and two had gestational diabetes. One daughter was diagnosed with TS in adulthood. Seven of 14 who attempted OD had a pregnancy with live birth; two of seven had gestational diabetes; 64.3% attempting OD had risk factors associated with poor pregnancy outcomes, including four who had double embryo transfer. Cardiac status at preconception was evaluated in 12 of 25 women who had a pregnancy. The aortic diameters during pregnancy increased. The aortic growth at sinuses was 0.51 ± 0.71 mm/year and at ascending aorta 0.67 ± 0.67 mm/year, reaching a significant difference at sinuses compared with the growth in nulliparous TS. Among women who had a pregnancy, none experienced aortic dissection during and in the years after pregnancy. CONCLUSION(S): This study highlights the importance of a TS-dedicated multidisciplinary management of pregnancy, before and during pregnancy and in the postpartum period.
Assuntos
Fertilidade , Infertilidade Feminina/terapia , Técnicas de Reprodução Assistida , Síndrome de Turner/complicações , Adolescente , Adulto , Comorbidade , Feminino , Humanos , Infertilidade Feminina/diagnóstico , Infertilidade Feminina/etiologia , Nascido Vivo , Gravidez , Resultado da Gravidez , Técnicas de Reprodução Assistida/efeitos adversos , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Resultado do Tratamento , Síndrome de Turner/diagnóstico , Síndrome de Turner/genética , Síndrome de Turner/fisiopatologia , Adulto JovemRESUMO
OBJECTIVE: To investigate the applicability of phospholipase C zeta (PLCζ) analysis in assisting the clinical decision-making process when considering artificial oocyte activation (AOA) for infertile males in assisted reproductive technology. DESIGN: Fifty-six males (43 infertile/13 fertile) were screened using our PLCζ assay. SETTING: Fertility unit/university laboratory. PATIENT(S): Infertile males with abnormal sperm morphology or total fertilization failure, low fertilization rate (<50%), or repeated fertilization failure in assisted reproductive technology. INTERVENTION(S): We analyzed PLCζ levels in sperm from fertile and infertile males. Eligible patients subsequently underwent intracytoplasmic sperm injection (ICSI)/artificial oocyte activation (AOA) with calcimycin (GM508). MAIN OUTCOME MEASURE(S): PLCζ localization and level and the proportion of sperm expressing PLCζ. Thresholds of PLCζ deficiency, fertilization rates, pregnancy rates, and live birth rates of AOA and non-AOA cycles. RESULT(S): Compared with 13 fertile controls, 34 of the 43 infertile males had significantly lower levels of PLCζ and/or a significantly lower proportion of sperm exhibiting PLCζ. Of these 34 patients, 15 showed a significant PLCζ reduction in both parameters, which we termed "PLCζ deficiency." Five PLCζ-deficient patients opted for AOA; all five achieved fertilization, and four achieved clinical pregnancies and live births. The fertilization rate improved significantly from 18.6% (ICSI) to 56.8% (ICSI/AOA). The clinical pregnancy rate and live birth rate with AOA were both 40% per initiated cycle. Youden index analysis revealed that the cutoffs below which infertile males were likely to benefit from AOA were 71% for the proportion of sperm expressing PLCζ and 15.57 arbitrary units for mean PLCζ level. CONCLUSION(S): PLCζ analysis is a useful diagnostic tool to determine patient eligibility for subsequent AOA treatment.
Assuntos
Algoritmos , Tomada de Decisão Clínica , Técnicas de Apoio para a Decisão , Infertilidade Masculina/terapia , Oócitos/fisiologia , Fosfoinositídeo Fosfolipase C/análise , Injeções de Esperma Intracitoplásmicas , Espermatozoides/enzimologia , Adulto , Biomarcadores/análise , Estudos de Casos e Controles , Transferência Embrionária , Feminino , Fertilidade , Humanos , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/enzimologia , Infertilidade Masculina/fisiopatologia , Masculino , Recuperação de Oócitos , Indução da Ovulação , Gravidez , Taxa de Gravidez , Injeções de Esperma Intracitoplásmicas/efeitos adversos , Resultado do TratamentoRESUMO
BACKGROUND: This study intends to present the role of rescue in vitro maturation (IVM) in polycystic ovarian syndrome (PCOS) patients undergoing in vitro fertilization (IVF) treatment who have inappropriate responses to ovarian stimulation. MATERIALS AND METHODS: This was a retrospective case series study of five PCOS patients undergoing IVF treatment considered for cycle cancellation due to increased risk of ovarian hyperstimulation syndrome (OHSS) as group A or poor response to ovarian stimulation as group B. Patients in group A had high oestradiol levels and recruitment of high numbers of small/intermediate sized follicles that did not meet the criteria for human chorionic gonadotropin (hCG) triggering. Patients in group B responded inadequately to hormonal stimulation despite high gonadotropin dosage. Treatment was changed to rescue IVM cycles after the patients provided consent. RESULTS: In group A, three IVF patients deemed to have high chances of developing OHSS as evidenced by high oestradiol levels were converted to IVM. A total of the 58/68 oocytes retrieved were mature or matured in vitro. There were 26 cleaving embryos obtained. Two patients had live births and one patient suffered a miscarriage. In group B, rescue IVM was implemented in two patients due to poor ovarian response (POR). A total of 22/26 oocytes retrieved were mature or matured in vitro. There were 13 cleaving embryos obtained. One patient had a live birth, whilst the other suffered a miscarriage. CONCLUSION: Rescue IVM could be a viable option in PCOS patients undergoing IVF treatment who are unable to safely meet the criteria for hCG triggering due to overresponse to ovarian stimulation or ovarian resistance to high doses of stimulation. Conversion to IVM can still result in reasonable oocyte retrieval and lead to clinical pregnancy and live births without the risks of OHSS.
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This review and meta-analysis aim to assess the effect of prolonged progesterone support on pregnancy outcomes in women undergoing fresh embryo transfer after IVF/intracytoplasmic sperm injection (ICSI). Two independent authors searched Embase, MEDLINE and grey literature from inception to January 2019 for randomized controlled trials (RCT) of prolonged progesterone support versus early cessation. Risk of bias was assessed. Outcome measures were live birth, miscarriage and ongoing pregnancy rate. The study was registered with PROSPERO (CRD42018088605). Seven trials involving 1627 participants were included: three reported live birth rate (672/830), seven the miscarriage rate (178/1627) and seven the ongoing pregnancy rate (1351/1627). Clinical outcomes were similar between early progesterone cessation versus progesterone continuation: live birth rate (risk ratio [RR] 0.94, 95% confidence interval [CI] 0.88-1.00), miscarriage rate (RR 0.91, 95% CI 0.69-1.20) and ongoing pregnancy rate (RR 0.98, 95% CI 0.91-1.05). Ongoing pregnancy rates were similar when analyses were restricted to those with cessation of progesterone on the day of a positive human chorionic gonadotrophin (RR 0.93, 95% CI 0.83-1.06). This meta-analysis suggests that prolonged progesterone support may be unnecessary after fresh embryo transfer. Further larger RCT would be useful to corroborate and lead to standardized duration of progesterone luteal phase support across IVF/ICSI centres.
Assuntos
Fertilização in vitro/métodos , Indução da Ovulação/métodos , Progesterona/administração & dosagem , Feminino , Humanos , Fase Luteal , Fatores de TempoRESUMO
STUDY QUESTION: What is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral presence and seroconversion in staff members in European fertility units prior to recommencement of clinical activity? SUMMARY ANSWER: A large proportion of fertility clinic staff remain susceptible to SARS-CoV-2 with no evidence of seroconversion, indicating that continued comprehensive risk mitigation strategies are essential. WHAT IS KNOWN ALREADY: In response to the coronavirus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2, routine fertility treatment was temporarily stopped in several European countries. The SARS-CoV-2 prevalence and seroconversion in fertility clinic staff, who are at potentially lower risk than routine healthcare workers, are unknown. STUDY DESIGN SIZE DURATION: This cross-sectional study included 554 staff in 16 European IVF clinics, 13 ultrasound clinics, one diagnostic laboratory and one head office in four European countries (Austria, Denmark, Germany and the UK) between 15 April and 30 June 2020. PARTICIPANTS/MATERIALS SETTING METHODS: There were 554 staff members returning for resumption of clinical activity. Paired nucleic acid amplification tests of oropharyngeal swabs for SARS-CoV-2 and serological testing for SARS-CoV-2 IgG were performed. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 554 staff members tested, 0.19% (95% CI 0.03, 1.10%) had evidence of SARS-CoV-2 as detected by RT-PCR. In contrast, 23 staff members, i.e. 4.15% (95% CI 2.78, 6.15%), had antibodies against SARS-CoV-2, with a wide range of antibody titres. There was no evidence of differences in seroconversion between countries with estimates ranging from 2.78% (95% CI 0.77, 9.58) in Austria to 6.75% (95% CI 4.46, 10.1) for the UK. There was no strong evidence of clustering within the clinics, with 21 of the 30 facilities having no staff members affected (prevalence estimates ranging from 0% to 35%), and one clinic having seven staff members affected (35% (95% CI 18.1%, 56.7%)). The single staff member who tested positive for SARS-CoV-2 virus was in the pre-symptomatic phase and was isolated, with no contacts having evidence of infection on repeat testing. LIMITATIONS REASONS FOR CAUTION: This was a cross-sectional study prior to resumption of clinical activity, with repeat testing not undertaken. WIDER IMPLICATIONS OF THE FINDINGS: The low prevalence of seroconversion of fertility clinic staff highlights the need for continued comprehensive risk mitigation strategies and engagement with national endeavours to identify and isolate new cases and their contacts as we embark on the resumption of fertility services. STUDY FUNDING/COMPETING INTERESTS: The Fertility Partnership funded the study. S.M.N. reports personal fees from Access Fertility, personal fees from Merck, personal fees from Ferring, grants and personal fees from Roche Diagnostics, personal fees from The Fertility Partnership and personal fees from Modern Fertility, outside the submitted work. T.C. reports personal fees from Merck and personal fees from Ferring, outside the submitted work. G.T. reports personal fees from Merck, personal fees from Ferring and personal fees from Roche Diagnostics, outside the submitted work. S.E. and P.S.G. report no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.
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OBJECTIVE: To evaluate the benefit of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) for embryo selection in frozen-thawed embryo transfer. DESIGN: Randomized controlled trial. SETTING: Not applicable. PATIENT(S): Women aged 25-40 years undergoing IVF with at least two blastocysts that could be biopsied. INTERVENTION(S): Randomization for single frozen-thawed embryo transfer with embryo selection based on PGT-A euploid status versus morphology. MAIN OUTCOME MEASURE(S): Ongoing pregnancy rate (OPR) at 20 weeks' gestation per embryo transfer. RESULT(S): A total of 661 women (average age 33.7 ± 3.6 years) were randomized to PGT-A (n = 330) or morphology alone (n = 331). The OPR was equivalent between the two arms, with no significant difference per embryo transfer (50% [137/274] vs. 46% [143/313]) or per intention to treat (ITT) at randomization (41.8% [138/330] vs. 43.5% [144/331]). Post hoc analysis of women aged 35-40 years showed a significant increase in OPR per embryo transfer (51% [62/122] vs. 37% [54/145]) but not per ITT. CONCLUSION(S): PGT-A did not improve overall pregnancy outcomes in all women, as analyzed per embryo transfer or per ITT. There was a significant increase in OPR per embryo transfer with the use of PGT-A in the subgroup of women aged 35-40 years who had two or more embryos that could be biopsied, but this was not significant when analyzed by ITT. CLINICAL TRIAL REGISTRATION NUMBER: NCT02268786.
Assuntos
Aneuploidia , Blastocisto/patologia , Criopreservação , Fertilização in vitro , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Infertilidade/terapia , Diagnóstico Pré-Implantação/métodos , Transferência de Embrião Único , Adulto , Austrália , Biópsia , Implantação do Embrião , Feminino , Fertilidade , Fertilização in vitro/efeitos adversos , Humanos , Infertilidade/diagnóstico , Infertilidade/fisiopatologia , América do Norte , Valor Preditivo dos Testes , Gravidez , Taxa de Gravidez , Fatores de Risco , Transferência de Embrião Único/efeitos adversos , Resultado do Tratamento , Reino UnidoRESUMO
BACKGROUND: Infertility affects one in seven couples; many of these need in vitro fertilisation (IVF). IVF involves external hormones to stimulate a woman's ovaries to produce eggs which are harvested surgically. Embryos, created in the laboratory by mixing eggs with sperm, are grown in culture for a few days before being replaced within the uterus (fresh embryo transfer). Spare embryos are usually frozen with a view to transfer at a later point in time - especially if the initial fresh transfer does not result in a pregnancy. Despite improvements in technology, IVF success rates remain low with an overall live birth rate of 25-30% per treatment. Additionally, there are concerns about health outcomes for mothers and babies conceived through IVF, particularly after fresh embryo transfer, including maternal ovarian hyperstimulation syndrome (OHSS) and preterm delivery. It is believed that high levels of hormones during ovarian stimulation could create a relatively hostile environment for embryo implantation whilst increasing the risk of OHSS. It has been suggested that freezing all embryos with the intention of thawing and replacing them within the uterus at a later stage (thawed frozen embryo transfer) instead of fresh embryo transfer, may lead to improved pregnancy rates and fewer complications. We aim to compare the clinical and cost effectiveness of fresh and thawed frozen embryo transfer, with the primary aim of identifying any difference in the chance of having a healthy baby. METHODS: E-Freeze is a pragmatic, multicentre two-arm parallel group randomised controlled trial where women aged ≥18 and < 42 years, with at least three good quality embryos are randomly allocated to receive either a fresh or thawed frozen embryo transfer. The primary outcome is a healthy baby, defined as a term, singleton, live birth with appropriate weight for gestation. Cost effectiveness will be calculated from a healthcare and societal perspective. DISCUSSION: E-Freeze will determine the relative benefits of fresh and thawed frozen embryo transfer in terms of improving the chance of having a healthy baby. The results of this pragmatic study have the potential to be directly transferred to clinical practice. TRIAL REGISTRATION: ISRCTN registry: ISRCTN61225414 . Date assigned 29/12/2015.
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Criopreservação/economia , Transferência Embrionária/métodos , Fertilização in vitro/métodos , Congelamento , Infertilidade Feminina/terapia , Nascido Vivo/epidemiologia , Adolescente , Adulto , Análise Custo-Benefício , Criopreservação/métodos , Implantação do Embrião , Embrião de Mamíferos , Feminino , Fertilização in vitro/legislação & jurisprudência , Humanos , Síndrome de Hiperestimulação Ovariana/epidemiologia , Síndrome de Hiperestimulação Ovariana/prevenção & controle , Indução da Ovulação , Gravidez , Complicações na Gravidez/epidemiologia , Resultado da Gravidez , Taxa de Gravidez , Adulto JovemRESUMO
Extracellular vesicles are highly abundant in seminal fluids and have a known role enhancing sperm function. Clinical pregnancy rates after IVF treatment are improved after female exposure to seminal fluid. Seminal fluid extracellular vesicles (SF-EVs) are candidate enhancers, however, whether SF-EVs interact with cells from the endometrium and modulate the implantation processes is unknown. Here, we investigated whether SF-EVs interact with endometrial stromal cells (ESCs) and enhance decidualisation, a requisite for implantation. SF-EVs, isolated from human seminal fluid (n = 11) by ultracentrifugation, were characterised by nanoparticle tracking analysis and Western blotting, and purified using size exclusion chromatography. Non-decidualised and decidualised primary ESCs (n = 5) were then treated with SF-EVs. Binding of bio-maleimide-labelled SF-EVs was detected by flow cytometry and fluorescence microscopy. Prolactin and IGFBP-1 protein levels in culture media were also analysed after single and multiple SF-EV exposure. SF-EVs size ranged from 50 to 300 nm, and they expressed exosomal markers (ALIX, SYNTENIN-1, CD9 and CD81). SF-EVs bound to non-decidualised and decidualised ESCs at similar levels. ESCs prolactin secretion was increased after single (p = 0.0044) and multiple (p = 0.0021) SF-EV exposure. No differences were found in IGFBP-1 protein levels. In conclusion, SF-EVs enhance in vitro ESC decidualisation and increase secretion of prolactin, an essential hormone in implantation. This elucidates a novel role of SF-EVs on endometrial receptivity. Abbreviations: ECACC: European Collection of Authenticated Cell Cultures; ESCs: endometrial stromal cells; EVs: extracellular vesicles; FCS: foetal calf serum; HRP: horse-radish peroxidase; IFNγ: interferon-gamma; IGF: insulin-like growth factor; IGFBP-1: insulin-like growth factor binding protein 1; IVF: in vitro fertilisation; MVB: multivesicular bodies; NTA: nanoparticle tracking analysis; PRLR-/-: homozygous prolactin receptor knockout; RT: room temperature; SF-EVs: seminal fluid extracellular vesicles; STR: short tandem repeat; TGFß: transforming growth factor ß; uNK: uterine natural killer.