Developmental capacity is unevenly distributed among single blastomeres of 2-cell and 4-cell stage mouse embryos

During preimplantation development, mammalian embryo cells (blastomeres) cleave, gradually losing their potencies and differentiating into three primary cell lineages: epiblast (EPI), trophectoderm (TE), and primitive endoderm (PE). The exact moment at which cells begin to vary in their potency for multilineage differentiation still remains unknown. We sought to answer the question of whether single cells isolated from 2- and 4-cell embryos differ in their ability to generate the progenitors and cells of blastocyst lineages. We revealed that twins were often able to develop into blastocysts containing inner cell masses (ICMs) with PE and EPI cells. Despite their capacity to create a blastocyst, the twins differed in their ability to produce EPI, PE, and TE cell lineages. In contrast, quadruplets rarely formed normal blastocysts, but instead developed into blastocysts with ICMs composed of only one cell lineage or completely devoid of an ICM altogether. We also showed that quadruplets have unequal capacities to differentiate into TE, PE, and EPI lineages. These findings could explain the difficulty of creating monozygotic twins and quadruplets from 2- and 4-cell stage mouse embryos.

In Vitro Maturation of Oocytes Retrieved from Ovarian Tissue: Outcomes from Current Approaches and Future Perspectives

In vitro maturation (IVM) of transvaginally aspirated immature oocytes is an effective and safe assisted reproductive treatment for predicted or high responder patients. Currently, immature oocytes are also being collected from the contralateral ovary during laparoscopy/laparotomy and even ex vivo from the excised ovary or the spent media during ovarian tissue preparation prior to ovarian cortex cryopreservation. The first live births from in vitro-matured ovarian tissue oocytes (OTO-IVM) were reported after monophasic OTO-IVM, showing the ability to achieve mature OTO-IVM oocytes. However, fertilisations rates and further embryological developmental capacity appeared impaired. The introduction of a biphasic IVM, also called capacitation (CAPA)-IVM, has been a significant improvement of the oocytes maturation protocol. However, evidence on OTO-IVM is still scarce and validation of the first results is of utmost importance to confirm reproducibility, including the follow-up of OTO-IVM children. Differences between IVM and OTO-IVM should be well understood to provide realistic expectations to patients.

Human adipose-derived stromal cells transplantation prolongs reproductive lifespan on mouse models of mild and severe premature ovarian insufficiency

Background Although recent studies have investigated the ability of Mesenchymal Stromal Cells (MSCs) to alleviate short-term ovarian damage in animal models of chemotherapy-induced Premature Ovarian Insufficiency (POI), no data are available on reproductive lifespan recovery, especially in a severe POI condition. For this reason, we investigated the potential of MSCs isolated from human adipose tissue (hASCs), since they are easy to harvest and abundant, in ameliorating the length and performance of reproductive life in both mild and severe chemotherapy-induced murine POI models. Methods Mild and severe POI models were established by intraperitoneally administering a light (12 mg/kg busulfan + 120 mg/kg cyclophosphamide) or heavy (30 mg/kg busulfan + 120 mg/kg cyclophosphamide) dose of chemotherapy, respectively, in CD1 mice. In both cases, a week later, 1 × 106 hASCs were transplanted systemically through the tail vein. After four additional weeks, some females were sacrificed to collect ovaries for morphological evaluation. H&E staining was performed to assess stroma alteration and to count follicle numbers; immunofluorescence staining for αSMA was used to analyse vascularization. Of the remaining females, some were mated after superovulation to collect 2-cell embryos in order to evaluate their pre-implantation developmental capacity in vitro, while others were naturally mated to monitor litters and reproductive lifespan length. F1 litters’ weight, ovaries and reproductive lifespan were also analysed. Results hASC transplantation alleviated ovarian weight loss and size decrease and reduced alterations on ovarian stroma and vasculature, concurrently preventing the progressive follicle stockpile depletion caused by chemotherapy. These effects were associated with the preservation of the oocyte competence to develop into blastocyst in vitro and, more interestingly, with a significant decrease of chemotherapy-induced POI features, like shortness of reproductive lifespan, reduced number of litters and longer time to plug (the latter only presented in the severe POI model). Conclusion Human ASC transplantation was able to significantly reduce all the alterations induced by the chemotherapeutic treatment, while improving oocyte quality and prolonging reproductive functions, thus counteracting infertility. These results, strengthened by the use of an outbred model, support the potential applications of hASCs in women with POI, nowadays mainly induced by anticancer therapies. Graphic abstract

Fertilization and early embryonic development of in vitro matured metaphase I oocytes in patients with unexpected low oocyte maturity rate

Summary To determine the fertilization and embryonic potential of immature metaphase I (MI) oocytes in patients with low oocyte maturity rate in whom the percentage of mature oocytes obtained was less than 75% of the total retrieved ones. In vivo matured metaphase II (MII) oocytes (MII-ICSI, n = 244), and in vitro matured MI oocytes (MI-MII-ICSI, n = 202) underwent an intracytoplasmic sperm injection (ICSI) procedure. Maturation rate, fertilization rate and early embryonic development were compared in both groups. In total, 683 oocytes were collected from 117 ICSI cycles of 117 patients. Among them, 244 (35.7%) were mature MII and 259 (37.9%) were MI after the denudation process. Of those 259 MI oocytes, 202 (77.9%) progressed to MII oocytes after an incubation period of 18–24 h. The maturation rate was 77.9%. Fertilization rate was found to be significantly higher in the rescued in vitro matured MI oocyte group when compared with the in vivo matured MII oocyte group (41.6% vs 25.8%; P = 0.0006). However, no significant difference was observed in terms of cleavage rates on days 2 and 3 between the groups (P = 0.9126 and P = 0.5031, respectively). There may be unidentified in vivo factors on the oocyte maturation causing low developmental capacity in spite of high fertilization rates in the group of patients with low oocyte maturity rate. Furthermore, studies are needed to determine the appropriate culture characteristics as well as culture period and ICSI timing of these oocytes.

Talking to Children About Race, Gender, and Social Issues: Review and Recommendations

Children notice group conflict and societal injustices. Educators and caregivers sometimes shield children from challenging social issues because they think that children cannot understand complex topics or because they think learning the information will be harmful. By avoiding such conversations, educators and caregivers are ignoring societal structures that privilege some groups over others. Children are aware of current events, social issues, and differences between people. They come to their own conclusions about the observed differences and differential treatment but without the tools to challenge biases and inequities. This brief reviews research on children’s developmental capacity to understand discrimination, with a focus on early-to-middle childhood and topics related to race, gender, and immigration status. Implications for policy and practice appear alongside recommendations, with a particular focus on the benefits to having these challenging conversations in schools.

Delaying Effects of Prolactin and Growth Hormone on Aging Processes in Bovine Oocytes Matured In Vitro

Aging processes accelerate dramatically in oocytes that have reached the metaphase-II (M-II) stage. The present work aimed to study the patterns and intracellular pathways of actions of prolactin (PRL) and growth hormone (GH) on age-associated changes in bovine M-II oocytes aging in vitro. To this end, we analyzed spontaneous parthenogenetic activation (cytogenetic assay), apoptosis (TUNEL assay), and the developmental capacity (IVF/IVC) of in vitro-matured oocytes after prolonged culturing. Both PRL and GH reduced the activation rate of aging cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs), and their respective hormone receptors were revealed in the ova. The inhibitor of Src-family tyrosine kinases PP2 eliminated the effects of PRL and GH on meiotic arrest in DOs, whereas the MEK inhibitor U0126 only abolished the PRL effect. Furthermore, PRL was able to maintain the apoptosis resistance and developmental competence of aging CEOs. The protein kinase C inhibitor calphostin C suppressed both the actions of PRL. Thus, PRL and GH can directly support meiotic arrest in aging M-II oocytes by activating MAP kinases and/or Src-family kinases. The effect of PRL in maintaining the developmental capacity of aging oocytes is cumulus-dependent and related to the pro-survival action of the protein kinase C-mediated signal pathway.

O-099 TEAD4 regulates trophectoderm differentiation upstream of CDX2 in human preimplantation embryos

Study question What is the main pathway regulating trophectoderm (TE) differentiation during pre-implantation development in mouse versus human embryos? Summary answer TEAD4 is acting upstream of CDX2 and is involved in TE differentiation, as TEAD4-null human embryos exhibit compromised TE lineage differentiation. What is known already TEAD4 is the earliest transcription factor during early embryo development, required for the expression of TE-associated genes leading to successful TE differentiation and subsequent blastocoel formation in mouse. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination have shown that Tead4-null embryos do not express TE specific genes, including Caudal-Type Homeobox Protein 2 (Cdx2) and GATA Binding Protein 3 (Gata3), but expression of inner cell mass (ICM)-specific genes, remains unaffected. Furthermore, ablation of Tead4 compromises embryonic development and subsequent blastocoel formation in mouse. The role of TEAD4, during human pre-implantation development has not been functionally characterized yet. Study design, size, duration CRISPR-Cas9 was introduced in mouse zygotes and editing efficiency was evaluated by next-generation sequencing (NGS) on 4.5dpc embryos (n = 55). Developmental kinetics were monitored in CRISPR-Cas9 targeted (n = 83), sham-injected (n = 26) and non-injected media-control (n = 51) mouse embryos. Immunofluorescence analysis was performed in Tead4 targeted (n = 57) and non-injected media-control embryos (n = 94). The same methodology was applied in human donated in vitro matured (IVM) metaphase-II (MII) oocytes, which were CRISPR-Cas9 targeted (n = 74) during ICSI or used as media-Control (n = 33). Participants/materials, setting, methods A gRNA-Cas9 mixture targeting exon 2 of Tead4/TEAD4 was microinjected in respectively mouse 2PN (pronuclear) stage zygotes, or human IVM MII oocytes along with the sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. Embryonic development and morphology were assessed daily, followed by a detailed scoring at the late blastocyst stage. Successful targeting following CRISPR-Cas9 introduction was assessed by immunostaining and NGS analysis of the targeted locus. Main results and the role of chance In mouse, we confirmed previous findings, as the developmental capacity of Tead4 targeted embryos was significantly reduced starting from the morula stage and blastocyst formation rates were 8.97% in the targeted group, compared to 87.23% in the control and 87.50% in the sham group, respectively. Immunofluorescence analysis of late morula and blastocyst stage embryos confirmed the absence of Tead4, Cdx2 and Gata3, resulting from the successful interruption of the Tead4 locus (n = 57). Exon 2 of TEAD4 was successfully targeted in human. In total, 21 embryos from various developmental stages were successfully NGS analyzed and 90,48% (19 out of 21) of the embryos carried genetic modifications as a result of CRISPR-Cas9 genome editing and seven blastocysts were identified carrying exclusively frameshift mutations. In contrast to mouse, the developmental capacity of human targeted embryos (25%) did not differ significantly from the control group (23%). However, the blastocyst morphology and quality were compromised in the targeted group showing mostly grade C TE scores, containing very few cells. Immunofluorescence analysis of targeted blastocysts (n = 6) confirmed successful editing by complete absence of TEAD4 and its downstream TE marker CDX2. Limitations, reasons for caution CRISPR-Cas9 germline genome editing results in multiple editing outcomes with variable phenotypic penetrance, the mosaic nature of which complicates the phenotypic analysis and developmental behaviour of the injected embryos. Wider implications of the findings Elucidation of the evolutionary conserved molecular mechanisms that regulate self-renewal of the trophoblast lineage can give us fundamental insights on early implantation failure. Trial registration number Not Applicable

Telomere Length in Chromosomally Normal and Abnormal Miscarriages and Ongoing Pregnancies and Its Association with 5-hydroxymethylcytosine Patterns

The present study investigates telomere length (TL) in dividing chorionic cytotrophoblast cells from karyotypically normal and abnormal first trimester miscarriages and ongoing pregnancies. Using Q-FISH, we measured relative TLs in the metaphase chromosomes of 61 chorionic villous samples. Relative TLs did not differ between karyotypically normal samples from miscarriages and those from ongoing pregnancies (p = 0.3739). However, among the karyotypically abnormal samples, relative TLs were significantly higher in ongoing pregnancies than in miscarriages (p < 0.0001). Relative TLs were also significantly higher in chorion samples from karyotypically abnormal ongoing pregnancies than in those from karyotypically normal ones (p = 0.0018) in contrast to miscarriages, where relative TL values were higher in the karyotypically normal samples (p = 0.002). In the karyotypically abnormal chorionic cytotrophoblast, the TL variance was significantly lower than in any other group (p < 0.05). Assessed by TL ratios between sister chromatids, interchromatid TL asymmetry demonstrated similar patterns across all of the chorion samples (p = 0.22) but significantly exceeded that in PHA-stimulated lymphocytes (p < 0.0001, p = 0.0003). The longer telomere was predominantly present in the hydroxymethylated sister chromatid in chromosomes featuring hemihydroxymethylation (containing 5-hydroxymethylcytosine in only one sister chromatid)—a typical sign of chorionic cytotrophoblast cells. Our results suggest that the phenomena of interchromatid TL asymmetry and its association to 5hmC patterns in chorionic cytotrophoblast, which are potentially linked to telomere lengthening through recombination, are inherent to the development programme. The TL differences in chorionic cytotrophoblast that are associated with karyotype and embryo viability seem to be determined by heredity rather than telomere elongation mechanisms. The inheritance of long telomeres by a karyotypically abnormal embryo promotes his development, whereas TL in karyotypically normal first-trimester embryos does not seem to have a considerable impact on developmental capacity.

The global gene expression outline of the bovine blastocyst: reflector of environmental conditions and predictor of developmental capacity

Background Morphological evaluation of embryos has been used to screen embryos for transfer. However, the repeatability and accuracy of this method remains low. Thus, evaluation of an embryo’s gene expression signature with respect to its developmental capacity could provide new opportunities for embryo selection. Since the gene expression outline of an embryo is considered as an aggregate of its intrinsic characteristics and culture conditions, we have compared transcriptome profiles of in vivo and in vitro derived blastocysts in relation to pregnancy outcome to unravel the discrete effects of developmental competence and environmental conditions on bovine embryo gene expression outlines. To understand whether the gene expression patterns could be associated with blastocyst developmental competency, the global transcriptome profile of in vivo (CVO) and in vitro (CVT) derived competent blastocysts that resulted in pregnancy was investigated relative to that of in vivo (NVO) and in vitro (NVT) derived blastocysts which did not establish initial pregnancy, respectively while to unravel the effects of culture condition on the transcriptome profile of embryos, the transcriptional activity of the CVO group was compared to the CVT group and the NVO group was compared to the NVT ones. Results A total of 700 differentially expressed genes (DEGs) were identified between CVO and NVO blastocysts. These gene transcripts represent constitutive regions, indel variants, 3′-UTR sequence variants and novel transcript regions. The majority (82%) of these DEGs, including gene clusters like ATP synthases, eukaryotic translation initiation factors, ribosomal proteins, mitochondrial ribosomal proteins, NADH dehydrogenase and cytochrome c oxidase subunits were enriched in the CVO group. These DEGs were involved in pathways associated with glycolysis/glycogenesis, citrate acid cycle, pyruvate metabolism and oxidative phosphorylation. Similarly, a total of 218 genes were differentially expressed between CVT and NVT groups. Of these, 89%, including TPT1, PDIA6, HSP90AA1 and CALM, were downregulated in the CVT group and those DEGs were overrepresented in pathways related to protein processing, endoplasmic reticulum, spliceasome, ubiquitone mediated proteolysis and steroid biosynthesis. On the other hand, although both the CVT and CVO blastocyst groups resulted in pregnancy, a total of 937 genes were differential expressed between the two groups. Compared to CVO embryos, the CVT ones exhibited downregulation of gene clusters including ribosomal proteins, mitochondrial ribosomal protein, eukaryotic translation initiation factors, ATP synthases, NADH dehydrogenase and cytochrome c oxidases. Nonetheless, downregulation of these genes could be associated with pre and postnatal abnormalities observed after transfer of in vitro embryos. Conclusion The present study provides a detailed inventory of differentially expressed gene signatures and pathways specifically reflective of the developmental environment and future developmental capacities of bovine embryos suggesting that transcriptome activity observed in blastocysts could be indicative of further pregnancy success but also adaptation to culture environment.