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The objective of this study was to evaluate the function, and usability of a novel automated software-guided cryostorage system in an active IVF laboratory setting. The investigational device (ID) was installed at 3 IVF laboratories (sites: α, ß, and γ). A total of 15 embryologists were trained to use the ID. Mock patient specimens containing mirrored live patient data were handled using the ID. Temperature readings were recorded every minute. Successful identification, storage, and retrieval of mock patient specimens by the ID were evaluated. To assess an LN2 pressure builder, the frequency of use and events of workflow interruption were logged. Student's t-test was used to determine statistical significance. The ID was in active use for 164 days total. During this time, 329 mock patient egg and embryo cohorts were handled by the ID. The mean ± SD temperatures during active use were: α, - 176.57 ± 1.83 °C; ß, - 178.21 ± 2.75 °C; γ, - 178.98 ± 1.74 and did not differ significantly. The highest recorded temperatures were: α, - 165.14 °C; ß, - 157.41 °C; γ, - 164.45 °C. A total of 1064 automation transactions on 409 specimen vessels were performed. Data was managed on 1501 eggs and embryos. The ID did not lose or misplace any specimen data or vessels, and no mock specimen was exposed to a detrimental (> - 150 °C) temperature excursion. Over the 25 LN2 pressure builder usages during 99 total days, there was 1 occurrence where usage interrupted workflow due to a lack of LN2 pressure. The ID has advantages over the current manual-based cryostorage systems, including radio frequency identification (RFID) tracking, automation of manual tasks, and software guidance to ensure accurate specimen storage and retrieval. The results of this study indicate that the ID can be integrated into active IVF laboratories.
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Criopreservación , Fertilización In Vitro , Humanos , Fertilización In Vitro/métodos , Criopreservación/métodos , Criopreservación/instrumentación , Femenino , Temperatura , Programas InformáticosRESUMEN
Mechanisms controlling trophoblast proliferation and differentiation during embryo implantation are poorly understood. Human trophoblast stem cells (TSC) and BMP4/A83-01/PD173074-treated pluripotent stem cell-derived trophoblast cells (BAP) are two widely employed, contemporary models to study trophoblast development and function, but how faithfully they mimic early trophoblast cells has not been fully examined. We evaluated the transcriptomes of trophoblast cells from BAP and TSC and directly compared them with those from peri-implantation human embryos during extended embryo culture (EEC) between embryonic day 8 to 12. The BAP and TSC grouped closely with trophoblast cells from EEC within each trophoblast sublineage following dimensional analysis and unsupervised hierarchical clustering. However, subtle differences in transcriptional programs existed within each trophoblast sublineage. We also validated the presence of six genes in peri-implantation human embryos by immunolocalization. Our analysis reveals that both BAP and TSC models have features of peri-implantation trophoblasts, while maintaining minor transcriptomic differences, and thus serve as valuable tools for studying implantation in lieu of human embryos.
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RESEARCH QUESTION: What is the clinical outcome of the first attempt at conception between two embryo selection methods, blastocyst morphology and preimplantation genetic testing for aneuploidies (PGT-A), chosen at the initial physician IVF consultation? DESIGN: In this prospective analysis, a clinical decision regarding embryo selection, blastocyst morphology (group A) or PGT-A (group B) was made during initial physician IVF consultation. Female infertility patients were matched based on maternal age (mean 32.6 ± 3.6 years; range 25-43 years) and a similar time frame of oocyte retrieval. The primary outcome was live birth rate from the initial consultation to the first conception attempt for all female patients and for a subset analysis of patients aged <35 years. RESULTS: The inclusion of PGT-A (group B) for embryo selection during the initial physician IVF consultation resulted in 23 additional women out of the total 100 achieving a healthy live birth following the first conception attempt in this maternally age-matched infertile population (group Bâ¯=â¯72.0% versus group Aâ¯=â¯49.0%; Pâ¯=â¯0.0014). This same benefit was observed for age-matched, younger infertility patients (<35 years), with live birth rates from the initial consultation being significantly higher when the upfront clinical decision included PGT-A for embryo selection (group Bâ¯=â¯76.7% versus group Aâ¯=â¯53.4%; Pâ¯=â¯0.0052). Interestingly, 17 women from group B would have received an aneuploid embryo transfer if selection had been determined by blastocyst morphology alone, as their best-grade embryo was aneuploid. CONCLUSIONS: This prospective analysis from the initial physician IVF consultation revealed that euploid embryo selection significantly improved live birth potential with the first conception attempt, even for younger women with infertility.
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Aneuploidia , Fertilización In Vitro , Infertilidad Femenina , Diagnóstico Preimplantación , Humanos , Femenino , Adulto , Infertilidad Femenina/terapia , Embarazo , Estudios Prospectivos , Índice de Embarazo , Edad Materna , Transferencia de EmbriónRESUMEN
BACKGROUND: Advanced paternal age (APA) is associated with adverse outcomes to offspring health, including increased risk for neurodevelopmental disorders. The aim of this study was to investigate the methylome and transcriptome of the first two early embryonic tissue lineages, the inner cell mass (ICM) and the trophectoderm (TE), from human blastocysts in association with paternal age and disease risk. High quality human blastocysts were donated with patient consent from donor oocyte IVF cycles from either APA (≥ 50 years) or young fathers. Blastocysts were mechanically separated into ICM and TE lineage samples for both methylome and transcriptome analyses. RESULTS: Significant differential methylation and transcription was observed concurrently in ICM and TE lineages of APA-derived blastocysts compared to those from young fathers. The methylome revealed significant enrichment for neuronal signaling pathways, as well as an association with neurodevelopmental disorders and imprinted genes, largely overlapping within both the ICM and TE lineages. Significant enrichment of neurodevelopmental signaling pathways was also observed for differentially expressed genes, but only in the ICM. In stark contrast, no significant signaling pathways or gene ontology terms were identified in the trophectoderm. Despite normal semen parameters in aged fathers, these significant molecular alterations can adversely contribute to downstream impacts on offspring health, in particular neurodevelopmental disorders like autism spectrum disorder and schizophrenia. CONCLUSIONS: An increased risk for neurodevelopmental disorders is well described in children conceived by aged fathers. Using blastocysts derived from donor oocyte IVF cycles to strategically control for maternal age, our data reveals evidence of methylation dysregulation in both tissue lineages, as well as transcription dysregulation in neurodevelopmental signaling pathways associated with APA fathers. This data also reveals that embryos derived from APA fathers do not appear to be compromised for initial implantation potential with no significant pathway signaling disruption in trophectoderm transcription. Collectively, our work provides insights into the complex molecular mechanisms that occur upon paternal aging during the first lineage differentiation in the preimplantation embryo. Early expression and epigenetic markers of APA-derived preimplantation embryos highlight the susceptibility of the future fetus to adverse health outcomes.
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Trastorno del Espectro Autista , Humanos , Masculino , Envejecimiento , Blastocisto/metabolismo , Epigénesis Genética , Padre , Persona de Mediana Edad , FemeninoRESUMEN
Profiling bovine blastocyst transcriptome at the single-cell level has enabled us to reveal the first cell lineage segregation, during which the inner cell mass (ICM), trophectoderm (TE), and an undefined population of transitional cells were identified. By comparing the transcriptome of blastocysts derived in vivo (IVV), in vitro from a conventional culture medium (IVC), and in vitro from an optimized reduced nutrient culture medium (IVR), we found a delay of the cell fate commitment to ICM in the IVC and IVR embryos. Developmental potential differences between IVV, IVC, and IVR embryos were mainly contributed by ICM and transitional cells. Pathway analysis of these non-TE cells between groups revealed highly active metabolic and biosynthetic processes, reduced cellular signaling, and reduced transmembrane transport activities in IVC embryos that may lead to reduced developmental potential. IVR embryos had lower activities in metabolic and biosynthetic processes but increased cellular signaling and transmembrane transport, suggesting these cellular mechanisms may contribute to improved blastocyst development compared to IVC embryos. However, the IVR embryos had compromised development compared to IVV embryos with notably over-active transmembrane transport activities that impaired ion homeostasis.
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Blastocisto , Linaje de la Célula , Técnicas de Cultivo de Embriones , Animales , Bovinos , Blastocisto/metabolismo , Blastocisto/citología , Técnicas de Cultivo de Embriones/veterinaria , Desarrollo Embrionario/fisiología , Femenino , Transcriptoma , Medios de CultivoRESUMEN
The refinement of embryo culture media is essential in improving embryo viability and in vitro production efficiency. Our previous work demonstrated that the nutrients (carbohydrates, amino acids, and vitamins) in traditional culture media far exceed the need for an embryo and producing developmentally competent embryos in a reduced nutrient environment is feasible. Here, we aim to evaluate the impact of exogenous lipid and L-carnitine supplementation on bovine blastocyst development and refine our RN condition further. Zygotes were cultured in the control medium (100% nutrients) and reduced nutrient media containing 6.25% of the standard nutrient concentrations supplemented with L-carnitine and lipid free or lipid rich BSA. Increased blastocyst development was observed in the reduced nutrient lipid rich medium compared to the other two groups. However, in both reduced nutrient conditions, blastocyst cell numbers were lower than those obtained in the control condition. We then examined the expression level of 18 transcripts correlated with lipid metabolism, glucose metabolism, redox balance, and embryo quality, along with mitochondrial DNA copy numbers, ATP productions, and lipid profile. The results indicated lipid metabolism, embryo quality, and redox enzyme related genes were upregulated while glucose related gene was downregulated in embryos derived from reduced nutrient lipid rich condition Finally, we identified that the lipid rich BSA has enriched linoleic, stearic, oleic, palmitic, and alpha-linoleic fatty acids, a lipid profile that may contribute to the increased lipid metabolism and improved blastocyst development of the bovine embryos under the reduced nutrient condition.
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Recent advances in human blastoids have opened new avenues for modeling early human development and implantation. One limitation of our first protocol for human blastoid generation was relatively low efficiency. We now report an optimized protocol for the efficient generation of large quantities of high-fidelity human blastoids from naive pluripotent stem cells. This enabled proteomics analysis that identified phosphosite-specific signatures potentially involved in the derivation and/or maintenance of the signaling states in human blastoids. Additionally, we uncovered endometrial stromal effects in promoting trophoblast cell survival, proliferation, and syncytialization during co-culture with blastoids and blastocysts. Side-by-side single-cell RNA sequencing revealed similarities and differences in transcriptome profiles between pre-implantation blastoids and blastocysts, as well as post-implantation cultures, and uncovered a population resembling early migratory trophoblasts during co-culture with endometrial stromal cells. Our optimized protocol will facilitate broader use of human blastoids as an accessible, perturbable, scalable, and tractable model for human blastocysts.
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Implantación del Embrión , Transducción de Señal , Humanos , Blastocisto , Supervivencia Celular , TrofoblastosRESUMEN
Reduced quality in oocytes from women of advanced maternal age (AMA) is associated with dysfunctional mitochondria. The objective of this study was to investigate the mechanisms controlling mitochondrial quality during maternal aging in mouse and human oocytes. We first evaluated the expression of proteins involved in the mitochondrial unfolded protein response (UPRmt) and mitophagy in in vivo matured metaphase II (MII) oocytes collected from young and aged mice. Expression of UPRmt proteins, HSPD1 and LONP1, and mitophagy proteins, total-PRKN and phosphorylated-PRKN, was significantly decreased in aged compared to young oocytes. Treatment of aged oocytes during in vitro maturation with the mitochondrially targeted antioxidant mitoquinone (MQ) specifically restored total-PRKN and phosphorylated-PRKN expression to levels seen in young oocytes. We next investigated whether maturing young oocytes under a high-oxygen environment would mimic the effects observed in oocytes from aged females. Phosphorylated-PRKN expression in oxidatively stressed young oocytes was reduced compared to that in oocytes matured under normal oxygen levels, and the mitochondrial DNA (mtDNA) copy number was increased. Treating oxidatively challenged young oocytes with MQ restored the phosphorylated-PRKN expression and mtDNA copy numbers. Treatment of oxidatively challenged oocytes with MQ also increased the co-localization of mitochondria and lysosomes, suggesting increased mitophagy. These data correlated with the developmental potential of the oocytes, as blastocyst development and hatching of oxidatively stressed oocytes were reduced, while treatment with MQ resulted in a significant increase in blastocyst development and hatching, and in the percentage of inner cell mass. Consistent with our results in mice, MII oocytes from women of AMA exhibited a significant decrease in phosphorylated-PKRN and total-PRKN compared to those of young women. Our findings suggest that the protein machinery to control the health of the mitochondria via UPRmt and mitophagy may be compromised in oocytes from aged females, which may result in inefficient clearance of dysfunctional mitochondria and reduced oocyte quality.
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Mitocondrias , Oocitos , Femenino , Humanos , Animales , Ratones , Anciano , ADN Mitocondrial , Envejecimiento/genética , Oxígeno , Proteínas Mitocondriales , Proteasas ATP-DependientesRESUMEN
Profiling transcriptome at single cell level of bovine blastocysts derived in vivo (IVV), in vitro from conventional culture medium (IVC), and reduced nutrient culture medium (IVR) has enabled us to reveal cell lineage segregation, during which forming inner cell mass (ICM), trophectoderm (TE), and an undefined population of transitional cells. Only IVV embryos had well-defined ICM, indicating in vitro culture may delay the first cell fate commitment to ICM. Differences between IVV, IVC and IVR embryos were mainly contributed by ICM and transitional cells. Pathway analysis by using the differentially expressed genes of these non-TE cells between groups pointed to highly active metabolic and biosynthetic processes, with reduced cellular signaling and membrane transport in IVC embryos, which may lead to reduced developmental potential. IVR embryos had lower activities in metabolic and biosynthetic processes, but increased cellular signaling and membrane transport, suggesting these cellular mechanisms may contribute to the improved blastocyst development compared to IVC embryos. However, the IVR embryos had compromised development when compared to IVV embryos with notably over-active membrane transport activities that led to impaired ion homeostasis.
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PURPOSE: Estrogen is well-known for preparing uterine receptivity. However, its roles in regulating embryo development and implantation are unclear. Our objective was to characterize estrogen receptor 1 (ESR1) in human and mouse embryos and determine the effect of estradiol (E2) supplementation on pre- and peri-implantation blastocyst development. METHODS: Mouse embryos, 8-cell through hatched blastocyst stages, and human embryonic days 5-7 blastocysts were stained for ESR1 and imaged using confocal microscopy. We then treated 8-cell mouse embryos with 8 nM E2 during in vitro culture (IVC) and examined embryo morphokinetics, blastocyst development, and cell allocation into the inner cell mass (ICM) and trophectoderm (TE). Finally, we disrupted ESR1, using ICI 182,780, and evaluated peri-implantation development. RESULTS: ESR1 exhibits nuclear localization in early blastocysts followed by aggregation, predominantly in the TE of hatching and hatched blastocysts, in human and mouse embryos. During IVC, most E2 was absorbed by the mineral oil, and no effect on embryo development was found. When IVC was performed without an oil overlay, embryos treated with E2 exhibited increased blastocyst development and ICM:TE ratio. Additionally, embryos treated with ICI 182,780 had significantly decreased trophoblast outgrowth during extended embryo culture. CONCLUSION: Similar ESR1 localization in mouse and human blastocysts suggests a conserved role in blastocyst development. These mechanisms may be underappreciated due to the use of mineral oil during conventional IVC. This work provides important context for how estrogenic toxicants may impact reproductive health and offers an avenue to further optimize human-assisted reproductive technology (ART) to treat infertility.
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Desarrollo Embrionario , Aceite Mineral , Humanos , Ratones , Animales , Fulvestrant , Desarrollo Embrionario/genética , Blastocisto , Estrógenos/farmacologíaRESUMEN
Ovarian aging precedes that of any other mammalian organ and is the primary cause of female age-related infertility. The biological mechanisms responsible for ovarian aging remain unclear. Previous studies have been limited by their use of bulk RNA-sequencing, which masks the dynamic and heterogeneous nature of the ovary. In this study, we spatially resolved the transcriptomic landscape of ovaries from young and aged outbred mice. In total, we defined eight main ovarian cell populations, all of which were characterized by significant transcriptomic changes between young and aged samples. Further sub-cluster analysis revealed separate transcriptomes for distinct granulosa cell populations found in young versus aged mice, in addition to an oocyte sub-cluster population completely absent from aged mouse ovaries. This study provides a new perspective on mammalian ovarian aging using spatial transcriptomics to achieve deeper understanding of the localization and cell-population-specific mechanisms underlying age-related fertility decline.
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OBJECTIVE: To determine what patient and embryo characteristics are correlated with the developmental potential of the peri-implantation embryo. DESIGN: Retrospective study. SETTING: Research laboratory. PATIENTS: Six hundred fifty-one cryopreserved human blastocysts donated for research with informed patient consent. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Blastocyst attachment to fibronectin-coated plates, trophectoderm outgrowth area, epiblast cell number, total cell number, human chorionic gonadotropin secretion. RESULTS: Patients' body mass index, age, follicle-stimulating hormone: luteinizing hormone ratio on menstrual cycle day 3, antral follicle count on menstrual cycle day 3, antimüllerian hormone level on menstrual cycle day 3, and blastocyst morphological grade were correlated with peri-implantation development outcomes. After controlling for good-quality morphological grades, blastocysts from patients of advanced maternal age developed fewer epiblast cells than blastocysts from younger patients. CONCLUSIONS: Extended embryo culture during the peri-implantation period mirrors several disparities in fertility treatment outcome that we see clinically, including those from patients with advanced maternal age, high body mass index, and low ovarian reserve and from embryos with lower-quality morphological grades. This model system may be useful by providing an alternative or more sensitive endpoint assessment in studying patient, clinical, or laboratory factors that may influence preimplantation embryo developmental potential.
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Aneuploidia , Blastocisto , Blastocisto/fisiología , Técnicas de Cultivo de Embriones , Implantación del Embrión/fisiología , Desarrollo Embrionario/fisiología , Femenino , Humanos , Estudios RetrospectivosRESUMEN
RESEARCH QUESTION: What is the reproductive potential of embryos that achieve blastulation on day 7 followed by preimplantation genetic testing for aneuploidies (PGT-A) for infertility patients with slow embryo development? DESIGN: This was a retrospective cohort study in a private IVF clinic of consecutive female infertility patients (nâ¯=â¯2966) aged 24-48 (36.3 ± 3.8) years who underwent frozen embryo transfer (FET) of a single euploid blastocyst. RESULTS: The women underwent single euploid FET of an embryo that achieved blastulation on day 5 (nâ¯=â¯1880), day 6 (nâ¯=â¯986) or day 7 (nâ¯=â¯100). Day 7 embryos resulted in lower implantation and live birth rates compared with both day 5 and day 6 embryos (P < 0.001). The day 5, day 6 and day 7 groups had 68.5%, 55.2% and 36.0% live birth rates, respectively. The day 7 group was older than the day 5 group (P < 0.001); comparing age-matched cohorts, the day 7 group still had lower implantation and live birth rates (P < 0.0001 and P < 0.001, respectively). Embryo grade was unrelated to live birth rates. Day 7 embryos of expansion grade 5 or 6 or trophectoderm grade A were more likely to be euploid compared with expansion grade 3 or trophectoderm grade B. CONCLUSIONS: Euploid day 7 embryos represented reduced implantation potential, even when controlling for maternal age. Of all day 7 embryos that underwent PGT-A, euploidy was associated with expansion grade 5 or 6 and trophectoderm grade A. These results can help providers manage patient expectations in cases where infertile women have slow embryo development.
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Infertilidad Femenina , Diagnóstico Preimplantación , Aneuploidia , Blastocisto , Implantación del Embrión , Desarrollo Embrionario , Femenino , Pruebas Genéticas , Humanos , Infertilidad Femenina/terapia , Embarazo , Diagnóstico Preimplantación/métodos , Estudios RetrospectivosRESUMEN
Ovarian aging is associated with elevated oxidative stress and diminished oocyte developmental competence. We aimed to determine the impact of systemic antioxidant treatment in aged mice. Female outbred CF-1 mice were aged for 9 months prior to an 8-week 45 mg Euterpe oleracea (açaí) daily supplement. The açaí treatment induced a threefold increase in serum antioxidant power (FRAP) compared to both young and aged mice (p < 0.0001). Compared to young mice, aged mice had fewer oocytes and reduced blastocyst development (p < 0.0001); açaí did not affect the oocyte numbers, but improved blastocyst formation (p < 0.05). Additionally, açaí alleviated the aging-related decrease in implantation potential (p < 0.01). The aged mice showed evidence of elevated ovarian ER stress (increased whole-ovary PDIA4 expression, granulosa cell and oocyte GRP78 expression, and oocyte PDIA4 protein), reduced oocyte mitochondrial quality (higher PRKN activation and mitochondrial DNA oxidative damage), and dysregulated uterine glandular epithelium. Antioxidant intervention was sufficient to lessen these effects of ovarian aging, likely in part by the upregulation of NRF2. We conclude that açaí treatment is a promising strategy to improve ER and mitochondrial function in the ovaries, thereby ameliorating the decreased oocyte competence that occurs with ovarian aging.
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Envejecimiento , Antioxidantes/metabolismo , Oocitos/metabolismo , Animales , Antioxidantes/química , Blastocisto/citología , Blastocisto/efectos de los fármacos , Blastocisto/metabolismo , Chaperón BiP del Retículo Endoplásmico/genética , Chaperón BiP del Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Euterpe/química , Euterpe/metabolismo , Femenino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oocitos/citología , Oocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/química , Extractos Vegetales/farmacología , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
RESEARCH QUESTION: Is there a risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exposure and potential cross-contamination from follicular fluid, culture media and vitrification solution within the IVF laboratory using strict patient screening and safety measures? DESIGN: This was a prospective clinical study. All women undergoing transvaginal oocyte retrieval were required to have a negative SARS-CoV-2 RNA test 3-5 days prior to the procedure. Male partners were not tested. All cases used intracytoplasmic sperm injection (ICSI). The first tube of follicular fluid aspirated during oocyte retrieval, drops of media following removal of the embryos on day 5, and vitrification solution after blastocyst cryopreservation were analysed for SARS-CoV-2 RNA. RESULTS: In total, medium from 61 patients, vitrification solution from 200 patients and follicular fluid from 300 patients was analysed. All samples were negative for SARS-CoV-2 viral RNA. CONCLUSIONS: With stringent safety protocols in place, including testing of women and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in follicular fluid, medium or vitrification solution. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.
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Medios de Cultivo/análisis , Fertilización In Vitro , Líquido Folicular/virología , Laboratorios , ARN Viral/aislamiento & purificación , SARS-CoV-2/aislamiento & purificación , Femenino , Humanos , Recuperación del Oocito , Seguridad del Paciente , Estudios Prospectivos , Inyecciones de Esperma Intracitoplasmáticas , VitrificaciónRESUMEN
PURPOSE: To investigate the biological networks associated with DOR in young women and the subsequent molecular impact on preimplantation embryos. METHODS: Whole peripheral blood was collected from patients: young women presenting with diminished ovarian reserve (DOR) and age-matched young women with normal ovarian reserve. Maternal exome sequencing was performed on the NovaSEQ 6000 and sequencing validation was completed using Taqman® SNP Genotyping Assays. Blastocyst global methylome and transcriptome sequencing were also analyzed. RESULTS: Exome sequencing revealed 730 significant DNA variants observed exclusively in the young DOR patients. Bioinformatic analysis revealed a significant impact to the Glucocorticoid receptor (GR) signaling pathway and each young DOR female had an average of 6.2 deleterious DNA variants within this pathway. Additional stratification based on patient age resulted in a cut-off at 31 years for young DOR discrimination. Embryonic global methylome sequencing resulted in only a very small number of total CpG sites with methylation alterations (1,775; 0.015% of total) in the DOR group. Additionally, there was no co-localization between these limited number of altered CpG sites and significant variants, genes, or pathways. RNA sequencing also resulted in no biologically significant transcription changes between DOR blastocysts and controls. CONCLUSION: GR signaling DNA variants were observed in women with early-onset DOR potentially compromising oocyte production and quality. However, no significant downstream effects on biological processes appear to impact the resulting blastocyst. The ability to forecast premature DOR for young women may allow for earlier identification and clinical intervention for this patient population.
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Infertilidad Femenina/genética , Reserva Ovárica/genética , Adulto , Blastocisto/fisiología , Islas de CpG , Epigenoma , Femenino , Predisposición Genética a la Enfermedad , Humanos , Enfermedades del Ovario/genética , Polimorfismo de Nucleótido Simple , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Secuenciación del ExomaRESUMEN
OBJECTIVE: To evaluate the epigenetic consequence of a prolonged disease state of infertility in euploid blastocysts. DESIGN: Methylome analysis as well as targeted imprinted methylation and expression analysis on individual human euploid blastocysts examined in association with duration of patient infertility and time to live birth. SETTING: Research study. PATIENT(S): One hundred four surplus cryopreserved euploid blastocysts of transferrable-quality were donated with informed patient consent and grouped based on time to pregnancy (TTP). INTERVENTION(S): None MAIN OUTCOME MEASURE(S): The Methyl Maxi-Seq platform (Zymo Research) was used to determine genome-wide methylation, while targeted methylation and expression analyses were performed by pyrosequencing and quantitative real-time polymerase chain reaction, respectively. Statistical analyses used Student's t test, 1-way ANOVA, Fisher's exact test, and pairwise-fixed reallocation randomization test, where appropriate. RESULT(S): The methylome analysis of individual blastocysts revealed significant alterations at 6,609 CpG sites associated with prolonged infertility (≥60 months) compared with those of fertile controls (0 months). Significant CpG alterations were localized to numerous imprinting control regions and imprinted genes, and several signaling pathways were highly represented among genes that were differentially methylated. Targeted imprinting methylation analysis uncovered significant hypomethylation at KvDMR and MEST imprinting control regions, with significant decreases in the gene expression levels upon extended TTP (≥36 months) compared to minimal TTP (≤24 months). CONCLUSION(S): The prolonged disease state of infertility correlates with an altered methylome in euploid blastocysts, with particular emphasis on genomic imprinting regulation, compared with assisted reproductive technologies alone.
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Blastocisto/metabolismo , Metilación de ADN , Impresión Genómica , Infertilidad/genética , Epigénesis Genética , Femenino , Humanos , Técnicas Reproductivas AsistidasRESUMEN
OBJECTIVE: To study messenger ribonucleic acid (mRNA) and protein expressions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry receptors (angiotensin 1-converting enzyme 2 [ACE2] and CD147) and proteases (transmembrane serine protease 2 [TMPRSS2] and cathepsin L [CTSL]) in human oocytes, embryos, and cumulus (CCs) and granulosa cells (GCs). DESIGN: Research study. SETTING: Clinical in vitro fertilization (IVF) treatment center. PATIENTS: Patients undergoing IVF were treated at the Colorado Center for Reproductive Medicine. INTERVENTIONS: Oocytes (germinal vesicle and metaphase II [MII]) and embryos (1-cell [1C] and blastocyst [BL]) were donated for research at the disposition by the patients undergoing IVF. Follicular cells (CC and GC) were collected from women undergoing egg retrieval after ovarian stimulation without an ovulatory trigger for in vitro maturation/IVF treatment cycles. MAIN OUTCOME MEASURES: Presence or absence of ACE2, CD147, TMPRSS2, and CTSL mRNAs detected using quantitative reverse transcription polymerase chain reaction and proteins detected using capillary Western blotting in human oocytes, embryos, and ovarian follicular cells. RESULTS: The quantitative reverse transcription polymerase chain reaction analysis revealed high abundance of ACE2 gene transcripts in germinal vesicle and MII oocytes than in CC, GC, and BL. ACE2 protein was present only in the MII oocytes, and 1C and BL embryos, but other ACE2 protein variants were observed in all the samples. TMPRSS2 protein was present in all the samples, whereas mRNA was observed only in the BL stage. All the samples were positive for CD147 and CTSL mRNA expressions. However, CCs and GCs were the only samples that showed coexpression of both CD147 and CTSL proteins in low abundance. CONCLUSIONS: CCs and GCs are the least susceptible to SARS-CoV-2 infection because of lack of the required combination of receptors and proteases (ACE2/TMPRSS2 or CD147/CTSL) in high abundance. The coexpression of ACE2 and TMPRSS2 proteins in the MII oocytes, zygotes, and BLs demonstrated that these gametes and embryos have the cellular machinery required and, thus, are potentially susceptible to SARS-CoV-2 infection if exposed to the virus. However, we do not know whether the infection occurs in vivo or in vitro in an assisted reproductive technology setting yet.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , ARN Mensajero , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Angiotensinas , Basigina/genética , Basigina/metabolismo , COVID-19/genética , COVID-19/metabolismo , COVID-19/virología , Catepsina L/genética , Catepsina L/metabolismo , Femenino , Humanos , ARN Mensajero/genética , SARS-CoV-2/genética , Serina Endopeptidasas/metabolismo , CigotoRESUMEN
OBJECTIVE: To investigate how endogenously elevated DNA fragmentation alters the human sperm proteome, and whether this fragmentation contributes to genomic deletions. DESIGN: Research study. SETTING: Commercial fertility clinic. PATIENT(S): Men with low (0%-4%, n = 7) or high (≥16%, n = 6) sperm DNA fragmentation, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Global sperm proteome, single-nucleotide polymorphism genotyping array. RESULT(S): A total of 78 significantly differentially abundant proteins (30 decreased, 48 increased) were observed in control vs. high DNA damage samples. DNA damage resulted in robust proteomic responses, including markers of oxidative stress and apoptosis, DNA damage repair proteins, and transcription/translation and protein turnover machinery. Several key sperm functional proteins were significantly decreased in ejaculates with high DNA damage. We were unable to substantiate a link between increased DNA fragmentation and genomic deletions in human spermatozoa. CONCLUSION(S): Developing human spermatozoa initiate an active transcriptional response to endogenous DNA damage, which manifests as alterations in the sperm proteome.