Diabetic retinopathy, which was primarily regarded as a microvascular disease, is the leading cause of irreversible blindness worldwide. With obesity at epidemic proportions, diabetes-related ocular problems are exponentially increasing in the developed world. Oxidative stress due to hyperglycemic states and its associated inflammation is one of the pathological mechanisms which leads to depletion of endogenous antioxidants in retina in a diabetic patient. This contributes to a cascade of events that finally leads to retinal neurodegeneration and irreversible vision loss. The xanthophylls lutein and zeaxanthin are known to promote retinal health, improve visual function in retinal diseases such as age-related macular degeneration that has oxidative damage central in its etiopathogenesis. Thus, it can be hypothesized that dietary supplements with xanthophylls that are potent antioxidants may regenerate the compromised antioxidant capacity as a consequence of the diabetic state, therefore ultimately promoting retinal health and visual improvement. We performed a comprehensive literature review of the National Library of Medicine and Web of Science databases, resulting in 341 publications meeting search criteria, of which, 18 were found eligible for inclusion in this review. Lutein and zeaxanthin demonstrated significant protection against capillary cell degeneration and hyperglycemia-induced changes in retinal vasculature. Observational studies indicate that depletion of xanthophyll carotenoids in the macula may represent a novel feature of DR, specifically in patients with type 2 or poorly managed type 1 diabetes. Meanwhile, early interventional trials with dietary carotenoid supplementation show promise in improving their levels in serum and macular pigments concomitant with benefits in visual performance. These findings provide a strong molecular basis and a line of evidence that suggests carotenoid vitamin therapy may offer enhanced neuroprotective effects with therapeutic potential to function as an adjunct nutraceutical strategy for management of diabetic retinopathy. Although half a billion individuals are estimated to be living with this condition globally, diabetes remains severely underdiagnosed, with one in every two individuals living with the disease unaware ,2,3. It The natural history of DR features retinal capillary degeneration and subsequent significant visual impairment , when poHyperglycemia-induced damage to other parts of the body has been shown to correlate with the severity of DR, including peripheral neuropathy, nephropathy and cardiovascular complications ,7,8,9,10meso-zeaxanthin ; the primary cause of significant vision loss in DR ,36. Signmeso-zeaxanthin [meso-zeaxanthin found in the retina is an outcome of biochemical conversion of lutein via RPE65 isomerase in the retinal pigment epithelium (RPE) [The yellow spot that is visible during ophthalmoscopy is due to macular pigment, which contains three carotenoids—(1) lutein, (2) zeaxanthin, and (3) a stereo isomer of zeaxanthin called axanthin ,44—whichaxanthin ,46,47. Laxanthin . Common axanthin ,49,50. Uum (RPE) ,52,53,54um (RPE) ,56. Suppum (RPE) ,52,57 anum (RPE) ,67,68.Clinical measurement of the macular pigment optical density (MPOD) is as close as we can get to quantification of macular carotenoids. The level of MPOD is indeed a biomarker and is strongly associated with maintenance of retinal health and optimal visual function in both health and disease ,46,50,59There are several techniques available to effectively quantify MPOD in vivo ,78,79,80Heterochromatic flicker photometry (HFP) is the most widely used technique to measure MPOD ,76,78,79Fundus reflectometry ,91,92,93This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) reporting guidelines .meso-zeaxanthin, macular pigment, macular pigment optical density, MPOD, diabetes, diabetic eye disease, and diabetic retinopathy. The database selection strategy was limited to records pertaining to macular carotenoids  and diabetic retinopathy only. Primary search results were identified for initial screening according to titles and abstracts available in English by PGD and DWL. Among the eligible records, full-text publications were retrieved and evaluated for study inclusion or exclusion criteria. To ensure all relevant studies were included in this review, we individually conducted backward and forward searches of the eligible publications by reviewing reference lists and cited references, respectively. All records retrieved in full text were individually screened and evaluated by two authors (PGD and DWL) for inclusion/exclusion and any discrepancies were resolved through discussion involving the third author (DLG). Selected publications were quantitative research articles evaluating the association between MPOD/carotenoids (including lutein and/or zeaxanthin and/or meso-zeaxanthin) and diabetic retinopathy. Additional records involving other forms of diabetes-associated ocular disease were not considered in this review . The full inclusion criteria for eligible publications from experimental and clinical studies are outlined below.Two authors (PGD and DWL) performed a wide-ranging search of the scientific databases National Library of Medicine and Web of Science to identify all relevant publications reporting on the association between macular carotenoids and DR until 21 December 2020. Under the guidance of the university librarian, the two authors conducted the full search strategy and data collection together using the following keywords and the combination of their variants during the search query: carotenoids, lutein, zeaxanthin, Leprdb model) in rodents only; and (4) experimental models of type 1 or type 2 diabetes in rodents were included.Experimental animal studies included in this review met the following criteria: (1) evaluating the effects of treatment with carotenoids  on outcomes of retinal neurodegeneration, such as markers of oxidative stress, cell viability and visual performance in murine models of DR; (2) carotenoid interventions include powder diet supplemented with L and/or Z only, nutraceutical diet containing L/Z, and powder diet supplemented with micronutrient formula containing L/Z; (3) presentation of DR pathology induced using standard induction methods  or genetic models (namely the Inclusion criteria for this systematic review were: (1) observational studies evaluating the association among macular xanthophylls and DR; (2) prospective randomized clinical trials assessing the benefits of carotenoid vitamin therapy in diabetic patients; (3) interventions include dietary carotenoid supplementation (containing L and/or Z) or in a multivitamin formula containing micronutrients and antioxidants; (4) assessment of macular carotenoid levels reported by serum/plasma concentrations of L/Z, or by validated MPOD measurement techniques; (5) cohorts of diabetic patients ; and (6) study cohorts of both T1DM and T2DM with either no retinopathy present or mild/moderate NPDR.Exclusion criteria were based on the following: (1) carotenoid treatment did not include either lutein and/or zeaxanthin in formulation/design; (2) carotenoid treatment included other types of carotenoids; (3) experimental diabetes pathology (as listed previously) were not standard methods of induction; (4) inclusion of adults with other forms of diabetes associated eye disease; and (5) publications were not available in English.The PRISMA reporting guidelines were carefully followed as closely as possible, as discussed previously . The risIn total, 397 studies were identified during the primary search from scientific databases . After rThere is an increasing amount of research and animal trials that substantiate the neuroprotective effects of carotenoids lutein and zeaxanthin in rodent models of DR using either chemical induction or genetic modes to engender diabetic state  102,103,107,108.The importance of macular carotenoid’s antioxidant properties is evident by their enhanced capacity to ameliorate the extent of oxidative injury caused by hyperglycemia in diabetic retina. Supplementation with lutein and/or zeaxanthin was shown to protect against measures of oxidative and nitrosative stress, marked by significant reductions in malondialdehyde, 8-OHdG (oxidatively-modified DNA), and nitrotyrosine, respectively ,122,123.Dietary augmentation of the compromised endogenous antioxidant defenses has been considered the key modulator in the pathogenesis of DR. Multiple studies found that lutein and zeaxanthin recovered enzymatic activity and expression levels of glutathione, glutathione peroxidase and manganese superoxide dismutase ,107,117;Macular carotenoids may further protect against retinal neurodegeneration by limiting activation of low-grade inflammatory pathways triggered by metabolic and oxidative insults concomitant with hyperglycemic conditions ,137,138.The neuroprotective potential of lutein and zeaxanthin positively influencing the pathogenesis of DR was most substantial preventing changes in retinal morphology as a consequence of accelerated capillary cell loss induced by hyperglycemia; regarded as hallmark features of early-stage retinopathy ,152,153.Experimental studies strongly suggest that carotenoids may sufficiently protect against the cumulative effect of hyperglycemic-induced retinopathy, or rather progressive neurodegeneration in retinal function made evident by abnormal or delayed response on electroretinogram (ERG). Studies found that lutein and zeaxanthin preserved measures of inner retinal function at the post-receptor level, attenuating DM-induced reduction in oscillatory potentials and the amplitudes of both a- and b-waves on ERG ,158,159.Although results from these animal models are promising, interpretation of the immediate translative potential for clinical application must be performed with prudence. Briefly, accumulation of carotenoids in the macula is unique to primate retinas, and therefore macular pigments cannot be fully studied using only rodent models of DR ,164,165.Clinical studies implicate MPOD depletion, as well as low serum levels of lutein and zeaxanthin, may represent a novel clinical feature of DR; one that is likely contingent upon several metabolic perturbations associated with chronic hyperglycemia in type 1 and type 2 diabetes. Reports from observational studies are consistent in demonstrating carotenoid levels (measured both in serum and the macular pigment) are further reduced among diabetic patients with clinically evident retinopathy  19,25,3,115,116.There is limited evidence of RCTs evaluating the benefits of carotenoids in management of diabetic retinopathy. We used the Cochrane Collaboration’s tool for assessing risk of bias, which covers the following domains—selection bias, performance bias, detection bias, attrition bias, reporting bias, and other bias . Figure It is well known that both type 1 and type 2 diabetic patients with mild NPDR or no retinopathy exhibit a notable range of visual function impairment, even in the absence of clinically relevant lesions of neurodegeneration in the retina ,172,173.Various reports seem to suggest these improvements in visual performance following increases in serum carotenoid levels and MPOD concentrations may be attributed, at least in part, to the enhanced functional capacity of the macular pigments to preferentially absorb short-wavelength blue light ,182,183.In view of these findings, available reports among diabetic patients with and without non-proliferative retinopathy are encouraging in demonstrating the potential for carotenoid supplementation as an adjunct nutraceutical approach to offer enhanced protection against further hyperglycemia-induced injury to the retina. While there are no established recommendations currently regarding daily intake levels of lutein and zeaxanthin consumption, oral supplementation with these carotenoids has a relatively high safety profile, with low risk for adverse effects and are appropriately considered by the US Food and Drug Administration to be Generally Regarded as Safe (GRAS) . Large-sIt is important to note that these clinically meaningful benefits in diabetic patients with or without DR were independent of any changes in hyperglycemic status or in relation to blood glucose control. Moreover, based on these results, there is a considerable body of preliminary evidence to substantiate the neuroprotective capacity of macular carotenoids to inhibit or reverse disease progression by ameliorating the metabolic correlates and comorbidities often seen in patients with type 2 or poorly controlled type 1 diabetes. Encouraging results from early interventional studies offer scientific justification for renewed clinical trials thereby corroborating the potential use of carotenoid vitamin therapy as an adjunctive therapeutic approach in the management of diabetic retinopathy for patients with either type 1 or type 2 diabetes.meso-zeaxanthin in diabetic patients with or without DR to date. Further investigations are required to better understand if the addition of meso-zeaxanthin in combination with lutein and zeaxanthin may offer greater benefit or ascertain whether formulations with the two dietary xanthophylls are sufficient to elicit protective effect in diabetic retina. One of the limitations of this systematic review is that the number of databases searched was limited to National Library of Medicine and Web of Science. Additionally, the articles evaluated were limited to those published in the English language.However, there are several limitations currently that must be addressed in future clinical studies should carotenoid supplementation be used for this purpose. First, there is a growing need for further studies to investigate the potential implications associated with long-term use of adjunctive carotenoid vitamin therapy in larger cohorts of individuals with T1DM and T2DM. Second, additional randomized placebo-controlled trials are needed to determine the optimal dosage of lutein and zeaxanthin necessary to achieve clinically meaningful benefits, in addition to whether all three xanthophyll carotenoids found in the retina should be included in formulation. A recent systematic review in healthy adult eyes, found that lutein and zeaxanthin intake of less than 5 mg per day  was insufficient dosage to significantly raise MPOD levels during trials up to six months . AdditioLastly, given the systemic etiopathogenesis of diabetes which can manifest in the eye as vascular endotheliopathy, future strategies may focus on ameliorating early microvasculature complications such as retinal vascular occlusion as a consequence of capillary nonperfusion. While experimental models have shown that lutein and zeaxanthin offer protection against retinal capillary degeneration triggered by ischemic-reperfusion injury, it is unclear whether these xanthophylls can prevent microvasculature alterations which ultimately lead to vascular dysregulation. However, oral supplementation with a similar xanthophyll carotenoid known as astaxanthin has been shown in healthy adults has shown to exert benefits on retinal hemodynamic measures including capillary blood flow and velocity of choroidal circulation ,223,224.meso-zeaxanthin, in addition to other potent antioxidants that are pertinent for maintaining retinal health as seen in various observational studies. MPOD measurements may also have a role to play in screening high-risk individuals prior to overt changes in retina due to DR pathology. Further randomized placebo-controlled trials are needed to support and solidify its use more universally as a first line of defense in combination with routine systemic management of diabetes and in susceptible individuals that are at risk of diabetes or pre-diabetics.Substantial efforts are necessary in developing early prophylactic measures that offer synergistic protection against several pathogenic mechanisms contributing to retinal neurodegeneration and subsequently preventing irreversible vision loss. To this accord, there is robust preclinical evidence and at least early clinical trials supporting the potential use of carotenoid vitamin supplementation in diabetics with and without retinopathy. Chronic hyperglycemia significantly compromises the endogenous defense systems in a diabetic individual. The metabolic changes due to diabetes possibly lead to depletion of macular carotenoids lutein, zeaxanthin, and


Developmental arrest of the preimplantation embryo is a multifactorial condition, characterized by lack of cellular division for at least 24 hours, hindering the in vitro fertilization cycle outcome. This systematic review aims to present the molecular drivers of developmental arrest, focusing on embryonic and parental factors. A systematic search in PubMed/Medline, Embase and Cochrane-Central-Database was performed in January 2021. A total of 76 studies were included. The identified embryonic factors associated with arrest included gene variations, mitochondrial DNA copy number, methylation patterns, chromosomal abnormalities, metabolic profile and morphological features. Parental factors included, gene variation, protein expression levels and infertility etiology. A valuable conclusion emerging through critical analysis indicated that genetic origins of developmental arrest analyzed from the perspective of parental infertility etiology and the embryo itself, share common ground. This is a unique and long-overdue contribution to literature that for the first time presents an all-inclusive methodological report on the molecular drivers leading to preimplantation embryos’ arrested development. The variety and heterogeneity of developmental arrest drivers, along with their inevitable intertwining relationships does not allow for prioritization on the factors playing a more definitive role in arrested development. This systematic review provides the basis for further research in the field. In vitro fertilization (IVF) is rapidly evolving, with research focusing on improving the probabilities of a live-birth. The focal point of IVF research is the developmental dynamic of the preimplantation embryo, which is clinically cultured in vitro for a maximum of 5–7 days . Only thVarious mechanisms leading to arrested development have been proposed coupled by numerous explanations on why these mechanisms may be instigated . One of Studying, understanding and predicting developmental arrest is at the spotlight of research. It has been established that the number of available embryos for transfer is positively associated with live-birth rate . Thus, aEmbryo developmental arrest is a multifactorial phenomenon subject to intrinsic and extrinsic parameters. Investigating the causative relationships ruling the developmental arrest event, it appears that the list of the usual suspects includes maternal or paternal factors, embryonic metabolism activity, impairment of crucial molecular pathways that ceases the developmental process and genetic abnormalities, or laboratory protocols and conditions that may serve as culprits. Surely, extrinsic factors may equally lead to developmental arrest through, trauma, stress, of the said gametes and embryos as data has shown. These extrinsic factors may be described as techniques employed in the IVF laboratory that gametes and embryos are subjected to, along with conditions of embryo culture This constitutes an equally interesting area of research. Two comprehensive reviews regarding developmental arrest and one systematic review focused on the embryonic abnormal developmental patterns have been recently published ,31,32, in = 149), all records were screened and full-text was sought and obtained for relevant articles. Relevant articles (n = 119) were identified following title and abstract screening, employing the flow chart of Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) as presented in A systematic search on PubMed/Medline, Embase and Cochrane Central Database was performed on 22 January 2021. The keywords employed and combined for the search strategy were “in vitro fertilization”, “IVF”, “assisted reproduction”, “intracytoplasmic sperm injection”, “ICSI”, “embryo arrest”, “embryonic arrest”, “arrested development” and “developmental arrest”. The original search yielded 771 studies from the three databases. Following removal of duplicate studies , mitochondrial DNA (n = 6), methylation patterns (n = 3), small noncoding RNAs (n = 1), chromosomal abnormalities (n = 7), embryonic metabolic profile (n = 9), while specific morphological characteristics (n = 2), namely vacuolization were found to be associated with developmental arrest.A total of 34 studies were identified evaluating embryonic factors leading to developmental arrest. The factors indicated as developmental arrest drivers were differential gene expression (DNMT3B), Histone Deacetylase 1 (HDAC1), Tet Methylcytosine Dioxygenase 3 (TET3) and Yin Yang 1 (YY1) may lead to higher rates of embryonic loss [ATG5), Cullin 3 (CUL3), Ubiquitin Specific Peptidase 11 (USP11) and Ubiquitin Specific Peptidase 2 (USP2), all instrumental in protein synthesis, ubiquitination and autophagy [A number of different genes with an essential role in pre-implantation development participating in crucial processes and molecular pathways have been reported to be implicated in developmental arrest ,34. Non-BRCA1), Telomeric Repeat Binding Factor 1 (TERF1), ERCC Excision Repair 1 (ERCC1), Endonuclease Non-Catalytic Subunit (ERCC1), X-Ray Repair Cross Complementing 6 (XRCC6) [ITPR1), a main receptor for initiating the Ca2+ oscillations that could damage cortical granule release and zona-hardening [BMP15), which is highly connected with the function of granulosa and theca cells plays a vital role in follicular growth and fertilization [BMP-15 was significantly higher and Cx43 significantly lower in normally developing embryos than in arrested [BMP-15 mRNA has been suggested as an indicator of embryo competence [Extensive DNA damage may be responsible for developmental arrest. Non-viable embryos express lower levels of genes which play a key role in DNA repair and telomere maintenance mechanisms including Breast Cancer 1 ( (XRCC6) . Compariardening . In the ardening . Bone molization ,36,37,38lization . Studiesarrested . The spempetence .It should be noted that data pertaining to the role of embryonic gene variation and expression in arrested development largely requires confirmation and retains an experimental status. However, examination of these genes in a clinical setting would be challenging as preimplantation genetic testing (PGT) may be required in order for the data acquired to hold any prognostic value. This fact poses two major concerns. Firstly, PGT being an invasive procedure would add another level of complexity that ultimately may compromise the embryo itself and one would have to weigh benefit versus detriment. Secondly, PGT is commonly performed on blastocysts, thus the embryos that present with differential expression levels will have already arrested failing to reach the biopsiable stage. It may be of importance to further investigate factors that may influence expression levels. These factors may range from maternal or paternal mutations on gene promoters or silencers to epigenetic modifications due to laboratory conditions. Hence, the intertwined nature of the relationships between the factors leading to developmental arrest is highlighted.COXIII), and NADH-CoQ oxidoreductase were differentially expressed due to mtDNA 4977-bp deletion [COXII) and ATP synthase subunit 6 (MTATP6) expression levels in cumulus cells (CCs) was detected in non-viable embryos, unveiling the importance of proper mitochondrial function at the earliest stages of development [HO-1) gene in arrested embryos as an extensive oxidative stress may be harmful for oocyte competence [BMP15, COXII, and MT-ATP6 observed in arrested embryos [A novel indicator to enhance identification and selection of embryos with the highest blastulation potential is mitochondrial DNA (mtDNA), evaluated in the culture medium ,41,42. Adeletion . This dedeletion . Additioelopment . A mitocmpetence ,47. A ne embryos .Mitochondrial DNA copy number has been explored as an option of a non-invasive biomarker on embryo competence. Although a number of studies have reported a correlation between mtDNA and IVF cycle outcome, there is controversy regarding the efficacy of mtDNA quantitation as a beneficial adjunct to embryo selection. Other studies suggest that mtDNA is a negligible predictor of implantation rates ,49,50. MDuring human development both paternal and maternal genome activation is required . ConsequMethylation patterns are an epigenetic modification regulating gene expression. Epigenetic modifications may be mainly attributed to environmental factors. These factors in the case of the preimplantation embryos are mainly culture conditions and techniques employed in the IVF laboratory. It should be noted that the role of methylation patterns in decoding arrested development has still a long way to go before formed hypotheses become widely accepted knowledge and a true “ally” in deciphering what leads to arrested development. Further studies should be designed to concur on the value of reporting on the methylome profile of the embryo in the context of the IVF laboratory as a predictive tool. Such a tool would enable identification of the actual factors that impact the methylome compromising the embryo.Only one study evaluated the expression of small noncoding RNAs (sncRNA) in preimplantation embryos . It was Chromosomal abnormalities are a common observation in pre-implantation embryos. Studies based on FISH technique observed that a high number of embryos presented with mosaicism and a significant correlation between aneuploidy and developmental arrest ,70,71,72A higher rate of nuclei with apoptotic chromatin has been observed in blastomeres of embryos of poor morphology and with extensive fragmentation compared to embryos with good morphology , while aSegmental aneuploidy is an outcome of DNA double stranded breaks (DSBs) derived from various factors such as oxidative stress . An inteDespite the undoubtable improvement in techniques employed to identify embryo ploidy status, the detection of mosaicism in embryos remains challenging, as a number of methods such as array-CGH and qPCR are insensitive in detecting low level mosaicism. This may lead to a considerable portion of euploid embryos being inaccurately evaluated as clinically unsuitable and becoming discard material ,86,87. AIt is undoubtable that the chromosomal complement of the embryo is the major driver that can dictate embryonic fate and the wealth of research on this topic confirms that. The role of aneuploidy and chromosomal abnormalities in embryo development has been thoroughly researched and discussed throughout decades. In more recent years the development of whole genome sequencing techniques enabled enhanced identification of the embryo ploidy status to the extent that on various levels this data is considered accepted rather than experimental knowledge. However, Nonetheless, there are aspects that still present as a mystery and merit further investigation. Mosaicism, and the “secret life” of the embryo towards self-correcting, along with factors affecting mitotic segregation patterns and the cell cycle certainly constitute areas that should be further delineated. Four possible models for embryonic self-correction , while oDuring the developmental process, embryos secrete into culture media several substances, which may reveal their developmental potential and may be considered as markers for identifying the optimal embryo to transfer. Caspase-3 is a protein with an established role in apoptosis . It beloAdditionally, a tendency towards higher levels of Interleukin 6 (IL-6) secretome of blastocyst-formed embryos compared to non-viable embryos has been observed . IL-6 haA recent study, attempted to analyze the peptides in embryo culture medium and assess their roles in the developmental competency . FindingCDK inhibitors such as p27, which is a member of Cip/Kip inhibitor family, may cause G1 arrest when it is overexpressed in transfected cells ,125. TheThere are several methods for the evaluation of apoptosis including terminal transferase-mediated DNA end labelling (TUNEL) and annexin V labelling ,141,142.Oxygen consumption has been proposed as a metabolic biomarker, which mirrors the synthesized levels of adenosine triphosphate (ATP) and therefore mitochondrial oxidative phosphorylation process . This biHigh concentration of reactive oxygen species (ROS) may be associated with embryonic developmental arrest ,156. A sIt may be safely concluded that the metabolic profile of an embryo is a non-invasive biomarker that may provide useful information regarding its developmental potential. To further this point, it has been reported that even in the later stages of the preimplantation embryonic development the metabolic profile may assist in predicting the cycle outcome ,160. EvaA casual association between multinucleation, delayed cleavage timing and extensive blastomere fragmentation has been established The origins of multinucleation still remain unclear, despite the fact that numerous explanations have been proposed . In factn = 15), types of female factor infertility (n = 6), as well as biomarkers in the follicular fluid (n = 6) have been associated with developmental arrest and are analyzed herein. Following identification of these studies employing systematic review methodology, the authors proceeded with investigating possible associations of infertility etiologies with genetic factors. Subsequently the authors proceeded to explore the connections and mechanisms between the identified genetic factors that were found to be implicated in developmental arrest. This ultimately led to the critical analysis presented herein regarding maternal factors leading to developmental arrest.A total of 27 studies identified maternal factors as causes of embryonic developmental arrest. Maternal factors describe factors related to the biochemical, genetic, and overall clinical and physical profile of the intended mother contributing the oocyte that forms the embryo studied. In this article the authors have divided maternal factors leading to embryonic arrested development in three distinct categories. Genetic factors (PADI6). PADI6 is a of a family of calcium-dependent cysteine hydrolase enzymes that includes five isotypes (PADI1-4 and PADI6) [PADI6 is mainly expressed in the oocyte, the ovary, the testis and the early embryo, and its target protein is protamine [PADI6 is related to different tissue-specific processes, namely PADI6 presents with a high expression on the oocyte, regulating its activation thus arising to be an important maternal infertility factor [PADI6 deficient mice are infertile due to arrested embryonic development at the 2-cell stage [PADI6 expression, result to embryo arrest between the 2-cell and 5-cell stages [PADI6 has been shown to participate in the subcortical maternal complex and through its recessive variants it has been demonstrated that, its misexpression was related with early embryonic arrest and infertility [Genetic factors represent by nature a multidimensional and vast field of investigation. It appears that a number of gene mutations and genomic alterations may contribute to particular phenotypes. One of the maternal genes associated with embryonic developmental arrest is Peptidyl Arginine Deiminase type-6 (d PADI6) . Their md PADI6) . PADI6 irotamine ,171. Thuy factor . In factll stage . The invll stage . These ol stages . PADI6 hertility .TUBB8). TUBB8 is a specific β-tubulin isotype, expressed in oocytes and during the first embryo divisions [TUBB8 is responsible for the expression of the primary beta-tubulin subunit of the oocyte and early embryo. TUBB8 mutations affect the microtubule behavior and oocyte meiotic spindle assembly, thus they are correlated with oocyte maturation [TUBB8 are determinant for the process of oocyte maturation and are associated with early embryo developmental arrest [TUBB8 mutations could be the cause of zygotic arrest in some embryos, by impairing the cell division process. These pathogenetic maternal and zygote patterns, are mainly observed in arrested embryos at the stage of the zygote, as well as at the 8-cell stage [TUBB8 as a marker of embryo arrest [Another gene that has been examined regarding its role in embryonic arrest is Tubulin B 8 chain  which permits embryonic development; nucleotide-binding leucine-rich-repeat (NLR) family pyrin domain containing 5 (NLRP5) also known as the maternal antigen that embryo requires (MATER); transducin-like enhancer of split 6 (TLE6) and K-Homology domain containing protein 3 (KHDC3) also known as FILIA [The subcortical maternal complex (SCMC) is a structure of multiple proteins which is uniquely expressed in early stages of embryo development and mammalian oocytes . This coas FILIA . The SCMas FILIA ,181. Theas FILIA ,183 and as FILIA ,185. Theas FILIA , withoutNLR gene family is characterized by a central nucleotide-binding domain, a C-terminal leucine rich repeats (LRR) domain, and an N-terminal effector domain. The N-terminus could be CARD, PYRIN, BIR, AD or X domain, which proves certain homology with CARD or PYRIN [NLRP2 and NLRP5 mutations which are illustrated as novel mutant genes [NLRP7 is a member of SCMC as well and has been demonstrated as a maternal factor involved in inflammation and apoptosis [NLRP5 induces primary infertility and Centromere protein H (CENPH) relates to embryonic arrest [The or PYRIN . NLR proor PYRIN ,189. NLRor PYRIN , inflammor PYRIN , carcinoor PYRIN  and autoor PYRIN . The NLRnt genes . NLRP7 ipoptosis . It appec arrest .TLE6 is a highly expressed oocyte protein and a main component of SCMC [TLE6 have been related with impaired phosphorylation as TLE6 is known to be phosphorylated by protein kinase A (PKA) [TLE6 mutations have been associated with early human embryonic lethality [TLE6 experience fertilization failure and early embryonic arrest setting TLE6 as a possible genetic diagnostic marker [ of SCMC . Mutatio A (PKA) . This af A (PKA) . Notablyethality . Patientc marker . Anotherc marker . PATL2 ic marker . PATL2 pc marker .TLE6, PADI6 and KHDC3L to patients exhibiting embryo arrest presenting from day 2 to day 7 [KHDC3L) is encoded in several tissues such as the epididymis, lung, lymphoid tissue, testis and is a subcortical maternal complex member [Further investigation of the subcortical maternal complex would provide a more comprehensive knowledge of the molecular pathways and a direct indication of the mechanism. There have been indicated novel biallelic mutations in the SCMC genes to day 7 . KH domax member .ZAR1), a gene contributing to the oocyte-to-embryo transition in mouse and in humans [ZAR1 could also express a determinant impact in the regulation of meiosis and post meiotic differentiation of male and female germ cells. This is also supported by its preserved expression among different species and its regulation may reveal its crucial role in early reproductive procedures. [ZAR1 is emerging the association with zygote arrest in humans enhancing the critical role in maternal fertility [Variation screening of additional genes has confirmed the impact of zygote arrest 1  mRNA has been identified abundantly in CCs of oocytes leading to embryos associated with live birth rate compared to immature oocytes and oocytes leading to arrested embryos. CCs of oocytes which led to euploid embryos, present with lower hydroxy-Δ-5-steroid dehydrogenase, 3 β- and steroid Δ-isomerase 1 (HSD3B1) mRNA levels compared to those leading to aneuploid blastocysts, and arrested embryos . Genes eIn addition, upregulation of prostaglandin-endoperoxide synthase 2 (PTGS2) gene has been associated with lower blastocyst formation rate and poor quality embryos . FollicuFollicular size has been associated with genetic expression in the CCs, thus serving as a developmental indicator. It has been reported that embryos originating from smaller follicles are more likely to arrest . FollicuApart from, the genetic and molecular causes of embryo arrest, the clinical pathogenetic diagnosis is of substantial value on the interpretation of the underlying female infertility status. Specific etiologies namely, advanced maternal age, endometriosis, poor ovarian response have been associated with developmental arrest.The incidence of embryonic chromosomal abnormalities is increased due to advanced maternal age. Respectively, women of advanced age are more likely to present with chromosomal abnormalities leading to embryonic arrest and to compromised fertility. The meiotic spindle area and major axis have a fundamental role in proper oocyte development. The expanded size of the meiotic spindle is correlated with embryo developmental arrest prior to reaching the blastocyst stage. Notably, there seems to be an underlining association between major axis of the meiotic spindle and patient age . The ageEndometriosis is one of the most crucial idiopathic pathologies that jeopardizes developmental process . An incrFMR1, and FMR2, along with polymorphisms in the BMP-15, GDF-9, in the gene encoding the receptor of FSH (FSHR) and in the NOBOX gene may present as a cause for POR [FMR1 gene is associated with fragile X syndrome in the offspring [BMP-15 and GDF-9, members of the TGF-b superfamily, along with NOBOX and have been associated with oogenesis, oocyte growth and maturation [BMP-15 mRNA levels in embryos have been positively associated with embryo viability. Furthermore, other candidate genes have also been investigated regarding their role in the pathophysiology of POR, including BRCA1 and BRCA2 [IGF-1, IGF-2, along with their respective receptors, AMH, LHCGR and GREM1 [Undoubtedly, patients of poor ovarian reserve (POR) are a group of patients presenting with challenges extending to a compromised embryo development. Exploring the impact of this situation on embryo arrest, it has published that POR follicles that lead to arrested embryos present with higher cell free DNA. What compromises DNA integrity and leads to developmentally arrested embryos is the increased necrosis and subsequent diminished steroid hormone levels in the follicular fluid . In orde for POR . The FMRffspring . BMP-15 turation . All theturation . As mentnd BRCA2 , IGF-1, nd GREM1 , howeverWhen considering the role of maternal factors leading to embryonic arrest, it becomes clear that besides the directly associated gene variations and follicular factors, infertility etiologies may indirectly influence embryonic development. Advanced maternal age was anticipated to be identified as a major cause of developmental arrest, as it has been associated with aneuploidy. Further to this, endometriosis and POR are two of the most prevalent female infertility etiologies, among females undergoing IVF, according to SART . Their rn = 2), metabolomic profile (n = 6), types of male infertility (n = 4) and genetic factors (n = 3). Following identification of these studies employing systematic review methodology, the authors proceeded with investigating possible associations of infertility etiologies with genetic factors. Subsequently, the authors proceeded to explore the connections and mechanisms between the identified genetic factors that were found to be implicated in developmental arrest. This ultimately led to the critical analysis presented herein regarding paternal factors leading to developmental arrest.A total of 15 studies investigated paternal factors leading to embryonic arrest. Through the examination of factors involved in developmental arrest, there emerge specific categories revealing contribution of the male factor. More specifically, etiologies include advanced paternal age , while the latter has been associated with autosomal translocations [Considering the impact of male factor in the developmental process, it should be mentioned that the types of male infertility may affect the developmental process differently. More than half of the embryos originating from azoospermic men arrest between the 2-cell stage and the 6-cell stage . Patientc arrest  and miRNc arrest . It has ocations . Howeverocations . Regardiocations . Thus, fDPY19L2 gene, while variations in the SPATA16, PICK1 and GGN have also been investigated as causes of globozoospermia [Another type of male infertility, globozoospermia has been related with the outcome of developmental arrest. Globozoospermia is a sub-type of teratozoospermia, where patients present with round-headed spermatozoa and malformation of the acrosome . It has ospermia ,275. MorA study has investigated the potential impact of intracytoplasmic sperm injection (ICSI) along with abnormal semen parameters on developmental potential, and more specifically on blastocyst formation. An increase of arrested embryos in days 3 and 4 was observed when male factor infertility cases where involved, indicating that paternal genomic activation is a determinant factor for embryo development. . It shou2+) ionophore, observed that the fertilization rate increased from 55% to 67.60%, and slightly less than half of the embryos managed to cleave successfully following application of Ca2+ ionophore [2+ may resolve the issue of inadequate signaling events which obstruct oocyte activation and assist in further development [In men with globozoospermia or other severe defects impairing fertilization potential, the method of artificial oocyte activation (AOA) has been proposed. A study comparing the outcomes of ICSI cycles without AOA to ICSI cycles with AOA using calcium (Caonophore . It shouelopment . In agreelopment . The stuelopment . Despiteelopment  and no selopment .Undoubtedly, age is a common infertility etiology denominator in both women and men ,286. In When considering the contribution of male infertility in developmental arrest, different etiologies appear to be hindering embryonic developmental potential. Abnormal chromosomal structure, increased DNA fragmentation and defective chromatin content have been associated with arrested development. Male types of infertility diagnosed according to abnormal semen parameters, may undermine fertilization rate suggesting an impaired developmental process. Despite the fact that limited genes were found to be directly linked with developmental arrest, it may be extrapolated that the genes and sncRNAs causing specific infertility etiologies may exert an indirect effect. However, in order to reach safe and robust conclusions, further research is required to directly associate paternal gene variations or differential expression levels of genes or sncRNAs with developmental arrest of the preimplantation embryo. It should be highlighted that research on the relationship between etiology of male factor infertility and developmental arrest provides verified evidence that could be regarded as accepted knowledge. This evidence pertains mainly to severe male factor infertility. On the other hand, regarding diagnosis of milder male infertility cases, the associations presented herein constitute indirect conclusions that could serve as guidance to mapping future research.The matter of arrested development has been well documented in literature and albeit there are numerous reports aiming to delineate it, surprisingly hitherto there is no collective, in-depth report on the drivers leading to this phenomenon. It is this gap in literature that fueled conduct of this study, aiming to assist clinical embryologists in better understanding the phenomenon while indicating the focus of future research that should be performed on preimplantation embryo arrested development. This is a unique and -in our opinion-long-overdue contribution to literature that for the first time presents an all-inclusive methodological report subject to the full proof nature of a systematic review on the drivers leading to the preimplantation embryo’s arrested development. What becomes apparent a priori is that the variety and heterogeneity of developmental arrest drivers, along with their inevitable intertwining relationships does not allow for prioritization on the factors playing a more definitive role in preimplantation embryo arrested development.Novel technologies introduced in the IVF laboratory have allowed for a better understanding of the developmental potential of the preimplantation embryo. The introduction of TLM has enabled continuous monitoring, thus abnormal developmental patterns are easily detectable . The devDevelopmental arrest is caused by a variety of reasons, that may originate from embryonic or parental factors. A summary of the causes of embryonic arrest is presented in Information on the environment and conditions that a gamete and an embryo may be exposed to provide useful insight regarding competence and viability. The numerous metabolites in the follicular fluid, as well as cumulus cells’ gene expression may indicate suitability of the environment of the oocyte, and its potential competence. Similarly, proteins in the seminal plasma may also provide insight on fertilization capability of the spermatozoa. When considering the embryo, the environment is of reciprocal significance, taking into account, the numerous and complex interactions during communication. Undoubtably, the metabolic profile of the embryo may provide a non-invasive biomarker regarding its viability. The conditions under which the embryo develops, ranging from the technique employed for fertilization to culture media and oxygen supplementation, may influence viability and ultimately its suitability for transfer in order to achieve a clinical pregnancy followed by a live-birth. The culture conditions imposed on the embryo may induce possible genetic or epigenetic alterations. Thus, it is of importance to conduct longitudinal studies evaluating the pediatric follow-up data of the offspring originating from IVF.Several infertility etiologies may be indicative of the outcome of developmental arrest. Endometriosis, and poor ovarian response constitute conditions that have been shown to be related to hindering developmental potential of the preimplantation embryos. Similarly, globozoospermia and azoospermia in men present as poor prognostic factors for embryonic viability. Parental age is one of the most common infertility etiologies. Following an exhaustive search according to the systematic methodology employed herein, it became clear that despite the fact that maternal age is extensively studied, little information is provided regarding the influence of paternal age. However, it has been observed that advanced paternal age has been associated with reduced blastocyst formation rate. Further studies should be conducted to enable a more in-depth understanding of the effect of paternal age. Moreover, it may be observed that while a number of maternal originating genetic causes of embryonic arrest have been investigated, the search yielded a limited number of results regarding the paternal genetic profile. It may be timely and essential to evaluate male infertility beyond the scope of a semen analysis and the common anatomical etiologies to a deeper genomic wide profiling of the intended father. While this may not be employed in clinical practice directly, research may reveal possible biomarkers which in turn may be indicative of the outcome. Perhaps the fact that a diagnosis of male infertility can be circumvented through employment on techniques such as ICSI to ensure fertilization, may explain the lack of incentive behind aspiring to treat some forms of male infertility. Enhancing the research field of male infertility may consequently lead to development of different approaches resulting to enhanced IVF cycle outcomes in terms of both safety and efficacy. A graphic illustration presenting parental infertility etiologies and suggested factors that may affect embryonic developmental potential is presented in The effects of DNA fragmentation have been studied in both males and females presenting as an apoptotic marker. In males it may been evaluated in the form of sperm DNA fragmentation. In women on the other hand identification of this marker is more complicated. Studies have investigated ALU fragments in follicular fluid of POR patients. It has been observed that the levels of these apoptotic factors may hinder the IVF cycle outcome. Apoptotic events in embryos hinder developmental potential. However, there is still lack of an all-inclusive study. According to the observations enabled by this systematic review it may be of importance to evaluate the possible apoptotic mechanisms by which parental DNA fragmentation may affect subsequent embryo development. Implementation of this non-invasive biomarker during the infertility evaluation may be of prognostic value. Investigation of the apoptotic pathways and the factors that may affect them, may provide personalized treatment for the infertile couple, which could in turn improve IVF outcomes.One would expect that oocyte morphology would be associated with embryonic developmental arrest. However, the majority of studies present with great heterogeneity and a relevant systematic review, performed a decade ago, was unable to reach robust conclusions . More reResults yielded herein following a systematic review methodology indicate the need for further studies on unveiling the molecular backbone of embryonic arrest, accounting for various sources from intrinsic genetic factors to the clinical profile of the intended parents. Future research should focus on genetic and epigenetic alterations that may influence developmental potential. It may be of interest to perform whole-genome sequencing in sibling embryos and evaluate possible loci that may be responsible for arrest. However, this type of research design may not yield specific results and may not be cost-effective. A targeted research evaluating variations as well as their expression levels in genes presenting with a correlation to a known infertility pathophysiology may yield more conclusive results. The fact that infertility pathophysiology seems to share a common ground with developmental arrest strengthens the proposal that these pathways should be considered for further research. This type of research design should be considered as a first step in investigating the origins of developmental arrest. Further to this, it has been established that miRNA expression levels are associated with infertility pathophysiologies ,292,293.Understanding the drivers of developmental arrest may assist in future attempts to enhance blastocyst formation rates, thus indirectly improving clinical pregnancy and live-birth rates. The promise of precision medicine relies on the concept that identification of the genetic or molecular causes of rare phenotypes will lead to adjusting respective treatments accordingly, to address the particular needs of each individual patient. In the case of arrested development of the preimplantation embryo the hypothesis is formed that understanding the molecular drivers behind the phenomenon can ascertain optimal treatment. However, hitherto, only a limited number of studies have focused on addressing this issue and improve developmental arrest rates. Recently a study on animal models, and more specifically mice, reported that the novel and promising technique of maternal spindle transfer may enable overcoming this phenomenon of in cases where developmental arrest is triggered by defective ooplasm . Since tIt should be mentioned that embryonic arrested development is not a primary outcome for all the studies in the field. Thus, it may not be directly mentioned in the studies. This may present as a limitation for the present systematic review. Moreover, conclusions pertaining to female infertility etiology and mainly to male infertility etiology are indirect, thus the results of the present study should be interpreted with a degree of caution. Hence, it is of importance to conduct further studies directly investigating the parental molecular pathways that lead to developmental arrest. It should be noted that along with the said intrinsic factors leading to arrested development, extrinsic factors present as an equal contributor. Albeit this review focuses strictly on the intrinsic factors, it should be stated that both elements can impact synergistically embryo potential development. When contemplating arrested development extrinsic factors ranging from laboratory conditions, techniques, operating protocols and the role of the practitioner should be taken into account. It has been well established that environmental conditions and culture media are of paramount importance, as poor culture conditions may restrict embryo developmental potential subjecting the embryo to stress that is unequivocally related to a compromised developmental outcome ,299. It This systematic review provides the basis for further research regarding embryonic developmental arrest. The goal of the study is to assist in understanding and set the fundamentals for further attempts to control and possibly avoid developmental arrest. Investigating the factors that may lead to developmental arrest will raise awareness on one of the most crucial and complicated processes in assisted reproduction that is still underexplored. Profiling of the intended parents along with elucidating on the factors that may cause genetic or epigenetic alterations during embryonic development may be equally vital to proper embryo profiling in the era where precision embryology is the future. Successful identification of the reasons causing developmental arrest may assist research focusing on development of improved technologies and optimizing IVF treatment. Albeit current options in addressing arrested development challenge clinical practice, this direction remains a noble goal fueling formation of hypotheses and development of novel approaches that merit validation. This would ultimately ascertain a greater number of embryos available for embryo transfer and higher percentage of live-births.


An analysis of the hybrid choice model and further insights are presented in the article “How landscape-technology fit affects public evaluations of renewable energy infrastructure scenarios. A hybrid choice model.”We present stated preference data based on a national representative Swiss online panel survey related to preference of mixed renewable energy infrastructure in landscapes. Data were collected between November 2018 and March 2019 via an online questionnaire and yielded 1026 responses. The online questionnaire consisted of two main parts – (1) questions covering meanings related to landscapes, nature and renewable energy infrastructure and questions regarding the “fit” of landscape/renewable energy infrastructure (REI) combinations and (2) a stated choice experiment. While in the first part of the questionnaire we asked respondents about their personal connection to certain landscapes, to nature and to specific REI, we also asked them to evaluate the fitting of seven different Swiss landscapes  with five different REI  combinations. In the second part of the questionnaire, the stated choice experiment confronted respondents with 15 consecutive choice tasks, with each task involving a choice between two “energy system transformation” options and an opt-out option (none). Each choice option (beside the opt-out option) included four unlabeled attributes  with varying levels. Due to data cleaning procedures (item nonresponse) the number of responses used within hybrid choice modeling and analysis was  They also provide a proof-of-concept for “landscape-technology fit” and contain information about predictors  of peoples’ preferences related to landscape developments. Also, the dataset highlights the interconnectedness of landscape and energy aspects in terms of the perceived landscape quality and its potential relevance for decision making processes.•The consideration of meanings for decision making processes and policy making  could be brought into all policy areas and technical decision-making tools, even those that are not landscape-oriented. During communication and planning residents of potential energy sites could be (1) informed early on and (2) invited to participatory workshops in which the meaning of landscape and REI is addressed in addition to usual visual scenarios and (3) discussing siting alternatives.•The dataset can be used to operationalize landscape-technology fit (LTF) concept which derived from place-technology fit (PTF). In particular, this dataset may be used as a base line for future LTF model improvements in alpine regions. They contain explicit information on meanings ascribed to alpine landscapes and to specific renewable energy infrastructures.1We conducted a representative online panel survey in Switzerland between November 2018 and March 2019 to elicit the preferences of Swiss residents for landscape oriented renewable energy infrastructure developments. The questionnaire was developed by WSL and operated by panel provider BILENDI GmbH. The survey is representative in language, age, gender, education and landscape.supplementary material of the present artice. Socio demographic items and respondent ID were provided by the panel provider (items 1 to 6). After starting the survey, respondents were first asked to select landscapes that most closely represent the landscape of their living, recreation and childhood environment (variables 160–162). In a next step, respondents were asked to evaluate (randomly presented) meanings ascribed to each of the seven landscapes presented. A generalized overview of the evaluation of landscape meaning items (variables 84 to 153) is provided in The questionnaire consisted of two major parts, where within the first part questions were related (1) to meanings ascribed to landscapes and renewable energy infrastructure, (2) to aspects of landscape-technology fit and (3) to exposure of people to landscapes and renewable energy infrastructures. Within the second part a stated choice model was presented. All respondents were designated to one of seven landscapes  according to the ZIP code of their origin. The landscape visualizations used in this study are illustrated in The second part of the online panel survey consisted of a discrete choice study in which respondents faced 15 consecutive choice tasks. Respondents were asked to choose among two landscape oriented renewable energy infrastructure alternatives and one opt-out option. Each of these alternatives (beside the opt-out option) had four attributes . Choice design, consecutive choice tasks and choice attributes are presented in https://doi.org/10.16904/envidat.206).For reasons of confidentiality we anonymized the data by removing all fields that would enable personal identification. The complete questionnaire, the dataset and data description are available on the Environmental Data Platform EnviDat of the Swiss Federal Institute for Forest, Snow and Landscape Research WSL , whereas option 3 described an opt-out (no-action). Relevant attributes and credible attribute levels were developed based literature research, project meetings and workshops with the project steering group from different disciplines We identified four relevant attributes and the respective levels. The choice design was generated with Ngene software The participation in the survey was operated and organized by a panel provider. Respondent participation was voluntary and respondents were informed that the data will be analyzed anonymously. Data collection and handling were implemented in accordance with the social data gathering ethics regulations of the institution conducting this research.Salak B.: Resources, Methodology, Conceptualization, Formal analysis, Investigation, Data Curation, Visualization, Writing - original draft; Lindberg K.: Methodology, Formal analysis, Writing - review & editing, Software, Validation; Kienast F.: Funding acquisition, Conceptualization, Writing - review & editing, Validation; Hunziker M.: Funding acquisition, Project administration, Conceptualization, Writing - review & editing, Validation, Supervision.The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.


Hanahan and Weinberg introduced the “hallmarks of cancer” and typified essential biological abilities acquired by human cancer. Since then, a growing understanding of hallmark principles associated with breast cancer has assisted knowledge-based therapeutics development; however, despite the rapidly increasing number of targeted therapeutics, enduring disease-free responses for most forms of breast cancer is rare. Invasion and metastasis are the most defining feature of breast cancer malignancy and the leading cause of patient mortality. Hence, we propose a modified hallmarks model adapted to breast cancer, in which invasion and metastasis are shifted to the center of attention, thereby emphasizing it as a potentially superior therapeutic target. Although the scientific community highly appreciates the importance of the invasion and metastasis hallmark, as can be demonstrated by the growing number of publications on breast cancer metastasis, very few clinical trials concentrate on testing anti-metastasis inhibitors and even fewer trials focus on inhibitors for breast cancer metastasis. Here, we discuss the obstacles of applying research on invasion and metastasis therapeutics into the clinic and present current developments that could provide a potential solution to this dilemma. Breast cancer is the most common neoplasm and the second leading cause of death in women ,2.It is a complex and highly heterogeneous disease, encompassing multiple tumor subtypes, each characterized by a different morphology, histopathological features and cellular markers, gene expression profiles, outcomes and response to systemic therapies ,4,5. Bec+/HER2+, tumors which are sensitive to either estrogen or progesterone and present HER2 receptors, (2) HR+\HER2−, tumors which are sensitive to either estrogen or progesterone but lack HER2 receptors, (3) HR−\HER2+, tumors which express HER2 receptors, but lack hormone receptors and (4) HR−\HER2−, also known as triple-negative breast cancer (TNBC).Breast tumor subtypes are classically defined according to hormonal and growth factor responsiveness. The main clinically relevant receptors in this regard are estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Breast tumors are thus divided into four main subtypes based on immunohistochemical properties and hormone receptor (HR) status : (1) HR+In 2000, Weinberg and Hanahan defined six hallmarks of cancer: sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis and activating invasion and metastasis . In 2011Normal tissues control the production and release of growth signals that regulate proper entry and progression through the cell cycle and regular tissue architecture and function. Cancer cells de-regulate these growth signals and, by doing so, acquire the capability to sustain proliferative signaling in several ways. For example, cancer cells can enhance autocrine stimulation ,11 or paSeveral key receptors and their downstream signaling pathways have emerged as critical drivers of breast cancer development and growth, as well as potential therapeutic targets. For example, growth factor receptor pathways, particularly receptor tyrosine kinases (RTKs), play a significant role in initiating proliferative and cell survival pathways that are normally tightly controlled ,14. AmonHER2 amplification or protein overexpression is found in 20–30% of invasive breast cancers and is associated with accelerated cell growth, proliferation and poor clinical outcomes [outcomes ,19. One outcomes . In addiElevated levels of insulin-like growth factor 1 (IGF1) and excessive IGF1R signaling have been implicated in an increased risk of breast cancer . IGF1R i+ subtypes, luminal A and ER+ luminal B. Trastuzumab, on the other hand, binds to HER2 and shows a highly beneficial effect in subtypes that overexpress HER2, such as HER2+ luminal B and HR−/HER2+. TNBC, which is the most aggressive breast cancer with the highest metastatic potential, has no targeted treatment at present and is usually treated by chemotherapy [+/HER2+ and AR+/TNBC [Determining tumor sensitivity to proliferative signaling is crucial in selecting appropriate breast cancer treatment. Indeed, several targeted therapies which aim to block breast tumor cell proliferation are currently being used at the clinic. Tamoxifen is an ER blocker that inhibits estrogen sensitivity and therefore is tailored for the ERotherapy ,25. AR hotherapy . Severalotherapy ,27,28,29AR+/TNBC .−/HR+ breast cancer patients [The success of the neoplastic process depends on high mutation rates of the genome of neoplastic cells. One way cancer cells increase their mutation rate is by disrupting one or several components of the genomic maintenance and repair machinery. Such alterations are selectively advantageous for tumor progression because they accelerate the rate at which pre-malignant cells can accumulate mutations and become cancerous ,9. Appropatients .BCL2, a well-recognized prognostic factor, is an anti-apoptotic  gene. Specifically, BCL2 is an estrogen-responsive gene and therefore tends to be overexpressed in primary ER+ tumors [+ breast cancer patients. In other words, the overall better prognosis of ER+ tumors outweighs the prospective damage promoted by BCL2. In recent studies, BCL2 inhibitors have been suggested as a possible therapeutic approach to breast cancer, both due to their apoptosis-enabling effect and their potential synergistic effect with tamoxifen as an ER inhibitor. One such inhibiting drug, venetoclax, is a selective BCL2 inhibitor used to treat leukemia but has not yet been evaluated in solid tumors. It has now undergone a phase Ib clinical trial in breast cancer patients to determine safety preparation for further clinical trials [Disruption of the apoptotic process allows tumor cells to evade their intrinsic self-destruction mechanism. The apoptotic trigger is controlled by counter-balancing pro- and anti-apoptotic members of the B-cell lymphoma 2 (BCL2) family of regulatory proteins. Pro-apoptotic genes are typically known to enhance tumor growth and increase resistance to breast cancer therapy . BCL2, a+ tumors . The corl trials . TherefoTelomers which protect the ends of chromosomes are essential for the capability of unlimited proliferation of cancer cells. Telomerase is almost absent in non-immortalized and normal cells but is highly expressed in most human cancer cells. Overexpression and high activation of telomerase in pre-malignant cells enable the maintenance of their telomeric DNA at lengths that are sufficient to avoid the triggering of senescence or apoptosis, leading to their immortalization and transformation into malignant cells. Telomerase is activated in 85% of all human cancers  and nearBreast cancer cell telomeres are much shorter than those of normal cells , suggest+ (SKBR-3). The combined treatment resulted in an over 90% reduction in telomerase activity and a decrease of more than 60% in cell viability, demonstrating a cumulative effect of chemotherapy. These promising findings may offer a potential new strategy for the inhibition of breast cancer [A recent study examined the inhibition of telomerase by small interference RNA (RNAi) combined with chemotherapy (doxorubicin) on breast cancer cell lines of three types: luminal A (MCF-7), TNBC (MDA-MB-468) and HER2t cancer .TP53 is a prototypical tumor suppressor gene that governs a wide range of cellular functions such as cell proliferation, apoptosis, DNA repair, gene expression and metabolism. The TP53 protein receives input from stress and abnormality sensors within the cell and decides, based on the levels of DNA damage caused in the cell genome, whether to halt the cell cycle and enable repair or to trigger apoptosis in cases where excessive damage that cannot be repaired exists [One of the mechanisms by which cancer cells maintain a high proliferation rate is by evading programs that negatively regulate cell proliferation. These programs, which exist in every normal cell, mainly depend on tumor suppressor genes. d exists .TP53 are observed in nearly 50% of all cancers and 25–30% of the basal-like subtype of breast cancer [TP53 germline mutation have an 85% chance of developing breast cancer by the age of 60 [TP53 mutation profile may influence the effectiveness of chemotherapy and hormonal therapy, which are used to treat breast cancer and are therefore examined before administration of such treatments [Inactivating mutations in t cancer . Moreovege of 60 . Thus, ieatments ,43,44,45TP53 [The uncontrolled cell proliferation that represents neoplastic disease involves, in addition to de-regulated control of cell proliferation, corresponding adjustments of energy metabolism to fuel cell growth and division. Thus, a common observation in neoplastic disease is a sharp increase in glucose consumption that accompanies a shift to anaerobic glycolysis instead of a more efficient aerobic mitochondria-mediated oxidative phosphorylation utilized by healthy nucleated cells. These changes are usually related to activation of RAS and MYC and inactivating mutations in TP53 , enhanci+, HR+ and TNBC tumors [In breast cancer, there is a difference in metabolic activity between the different subtypes, as both hormones and growth factors regulate glucose consumption and energy metabolism, leading to slightly different enzymatic profiles of ERC tumors . FurtherC tumors .Like normal tissues, tumors require nutrients and oxygen and the ability to evacuate metabolic waste and carbon dioxide. These needs are emphasized in tumors due to the rise in glucose consumption and the ever-increasing energy demands of the growing tumor. Tumor-associated neo-vasculature, generated by the process of angiogenesis, addresses these needs. Angiogenesis is a process that is transiently activated during embryogenesis and in normal physiological processes such as wound healing and the female reproductive cycle in the adult. During tumor progression, an angiogenic switch is activated and remains constitutively on, leading to continuous sprouting of new vessels which help to sustain expanding neoplastic growth ,9.− breast cancer and TNBC, which are highly resistant to standard breast cancer therapy [Bevacizumab (Avastin) is a monoclonal antibody that targets the vascular endothelial growth factor (VEGF), a prototypical promoter of angiogenesis. Bevacizumab is used to treat many types of cancer, such as non-small-cell lung cancer, glioblastoma, renal cell carcinoma, ovarian cancer and cervical cancer . Similar therapy . Today,  therapy ,49. The It has long been recognized that some tumors are densely infiltrated by immune cells, thereby mirroring inflammatory conditions in non-neoplastic tissues. Such tumor-associated immune responses were initially thought to reflect an attempt of the immune system to eradicate tumors. However, it is now known that inflammation can also contribute to multiple hallmark capabilities by supplying bioactive molecules such as growth and survival factors, pro-angiogenic factors and extracellular matrix modifying enzymes to the tumor microenvironment, which supports tumor progression and metastasis. Inflammatory cells can also release reactive oxygen species mutagenic for nearby cancer cells, accelerating their genetic evolution towards malignancy ,9.According to the immune surveillance theory, cells and tissues are constantly monitored by the immune system, which recognizes and eliminates most developing cancer cells and immature tumors that accidentally develop . AccordiUnderstanding the mechanisms used by cancer cells to evade immune destruction has led to the development of immunotherapy, which is based on stimulating a patient’s immune system to recognize and destroy cancer cells more effectively. An essential part of the immune system is its ability to keep itself from attacking normal cells in the body. To do so, it uses “checkpoints,” proteins on immune cells that need to be turned on or off to start an immune response. Breast cancer cells often use these checkpoints to avoid being attacked by the immune system. Therapies that target these checkpoint proteins assist in restoring the immune response against breast cancer cells. Today, immunotherapy is used to treat some types of breast cancer. Pembrolizumab (Keytruda) is a drug that targets programmed cell death 1 protein (PD-1), a protein on T cells that normally help keeps these cells from attacking other cells in the body. By blocking PD-1, this drug boosts the immune response against breast cancer cells and often leads to tumor shrinkage. Pembrolizumab is used in combination with chemotherapy to treat TNBC which expresses programmed cell death protein ligand 1 (PD-L1). Atezolizumab targets PD-L1, the ligand of PD-1, which is found on some tumor cells and immune cells. Blocking this protein can help boost the immune response against breast cancer cells and consequently shrink some tumors or slow their growth. Atezolizumab is used in combination with paclitaxel for advanced TNBC when the tumor expresses the PD-L1 protein .− and HER2+ tumors have shown evidence of immunogenicity, these types of inflammatory breast tumors are rare when compared to TNBC tumors, which are unique among breast cancer subtypes in having strong antigen expression and high stromal and tumor-infiltrating lymphocytes, parameters with a vital prognostic and predictive significance to immunotherapy and chemotherapy. Except for these immunogenic subtypes, breast tumors are usually considered immunologically silent or “cold” tumors, characterized by the presence of low mutation and neoantigen burden and few tumor-infiltrating lymphocytes. In addition, resistance to cancer immunotherapies often develops in breast cancer due to alternative immunosuppressive checkpoint pathways. Therefore, despite the use of combination immunotherapy in metastatic TNBC patients today, the immunologic treatment of breast cancer remains a significant challenge [The immunogenicity of breast cancer is heterogeneous and depends mainly on the specific subtype of breast cancer. Although ERhallenge .Metastatic dissemination of tumor cells, also known as the invasion-metastasis cascade , is a muFundamental understanding of the cancer hallmarks has facilitated the development of knowledge-based therapeutics. Regardless of the rapidly growing number of targeted therapies, their effectiveness is usually transient and prolonged progression-free survival and remedy are rare; the chances of survival usually increase if the tumor is detected early and surgically excised . Two majGene expression profiling of breast tumors has yielded signatures from which prognostic tests could be derived to facilitate clinical and therapeutic decision-making in breast cancer patients. Interestingly, although intended to predict the metastatic potential of breast tumors, most of these tests gain predictive and prognostic power from the quantification of genes that are involved in proliferation and are efficient for only specific subtypes of breast cancer . Using thi/VEGFhi) macrophage and an angiopoietin 2 (ANG2) secreting endothelial cell [Within primary breast tumors, tumor cell intravasation occurs at micro-anatomical doorways on blood vessels of the tumors, called Tumor Micro Environment of Metastasis (TMEM). Each functional TMEM doorway comprises three different cell types in direct and stable physical contact: a tumor cell highly expressing the actin-regulatory protein Mammalian-enabled (MENA), a tumor-associated macrophage expressing high levels of perivascular tyrosine kinase with immunoglobulin-like loops and epidermal growth factor homology domains 2 (TIE2) and VEGF (TIE2ial cell ,57. TMEMial cell ,59,60. BTo gain an insight into research efforts invested on each of the hallmarks, we analyzed the association between each of the ten hallmarks and relevant scientific research publications. As demonstrated in Translating research findings in invasion and metastasis into clinical applications is challenging for two major reasons. First, tumor size, which is affected by all other hallmarks of cancer besides invasion and metastasis, is considered an indicator for cancer progression mainly because it is a measurable endpoint and is constantly used in clinical trials. However, tumor size does not usually correlate with the invasion and metastasis potential of tumor cells and can sometimes even have an opposite correlation . Second,Even though the hallmarks model excellently illustrates what goes wrong in breast cancer cells, it fails to interrogate the hierarchy of the hallmarks in the broader aspect of therapeutic approaches. Being the most defining characteristic of malignancy, the invasion and metastasis hallmark should be placed in the center of attention and considered a potentially superior therapeutic target. Our analysis demonstrates that, while it is well recognized that invasion and metastasis is the most important hallmark of breast cancer, its prevention receives very little attention in the literature compared to preventing other hallmarks in primary or secondary breast tumors. These observations justify the necessity of shifting both scientific and clinical attention towards the invasion and metastasis hallmark as a potential therapeutic target in breast cancer. With new developments such as defining MFS as an endpoint in clinical trials and recognizing neoadjuvant treatment as a possible accelerator of breast cancer metastasis, this strategy becomes feasible. A successful application of this concept will depend on a collaborative effort of researchers, physicians and pharmaceutical scientists.


Seth Inzaule and co-authors discuss implications of the COVID-19 pandemic for health in African countries. Coronavirus Disease 2019 (COVID-19) and the mitigation measures taken to limit its spread have significantly disrupted other essential health services in Africa. This disruption has threatened the control of major high-burden diseases such as HIV, tuberculosis (TB), and malaria as well as the prevention of maternal and child mortality.While the 2020 WHO global reports for HIV, TB, and malaria show progress in control of these diseases in African countries, there are still significant gaps in meeting the global targets. Similarly, modeling studies predict that most African countries are unlikely to meet the Sustainable Development Goals (SDGs) 2030 targets for reductions in maternal and child mortality under the current rate of progress.Prediction models and emerging empirical data indicate that the implemented mitigation measures against COVID-19 such as travel restrictions and lockdowns as well as the repurposing of health resources and suspension of prevention programs such as immunizations will lead to an increase in new infections and deaths, significantly reversing the gains achieved in the control of these health challenges.A more comprehensive COVID-19 response that minimizes indirect deaths is therefore warranted in Africa. These include implementing WHO recommendations that limit contact with the clinic where possible, such as multimonths drug dispensing, self-testing, virtual platforms for case management, community- and home-based prevention, and care services such as home distribution of test kits, vaccines, treatment, and mosquito nets.This is in addition to ensuring effective implementation of COVID-19 infection prevention and control measures in healthcare facilities including providing healthcare workers with personal protective equipment and prioritizing them for COVID-19 immunization.There is also a need to incorporate aggressive recovery plans to reverse the lost gains in disease control efforts and put African countries back on course toward achieving the global targets. This includes leveraging on the wider COVID-19 response enablements such as the increased political will and global solidarity funding efforts to support a more comprehensive response that accounts for the indirect public health effects of the pandemic.In 2020, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus Disease 2019 (COVID-19) spread rapidly, resulting in a pandemic that is still ongoing in most countries. Africa’s response to the pandemic was largely decisive with various levels of implementation of nonpharmaceutical intervention measures .The drasExperience from past epidemics shows that disruptions in healthcare systems result in a significant number of indirect deaths –9. ModelCOVID-19 and the containment measures are also likely to impact the response to other epidemics. Between January and December 2020, an approximately 58 non-COVID-19 disease outbreaks were reported in Africa . RespondAfrica bears a disproportionate burden of HIV, TB, and malaria as well as unacceptable levels of child and maternal mortality –17. In 2The 2020 UNAIDS report showed that the interim target of having 73% of people living with HIV on treatment and virally suppressed was offtrack in Africa by 11.8 percentage points in 2019 . The repAlthough empirical data confirming the model predictions are largely missing, a few surveys and studies seem to indicate significant disruptions in health services.A survey by WHO conducted between April and June 2020 showed that 19 of 33 58%) African countries had a potential risk of antiretroviral (ARV) treatment service disruption 8% Africa had missWHO End TB strategy 2020 interim targets of reducing TB incidence by 20% and deaths by 35% from 2015 levels are also offtrack .–41.39–41Other partners like the African Society of Laboratory Medicine have also been instrumental in facilitating the sharing of best practices as well as innovative online training through digital platforms, which can further be expanded to support other disease control and health programs . In addiIn addition, as others have also recommended there is a need to integrate COVID-19 surveillance and response with the other major health threats in the continent . This inThe indirect socioeconomic effects of COVID-19 have further worsened existing health disparities, significantly affecting the ability of the most-at-need populations to access quality healthcare. As such, there is a critical need to accelerate the implementation of the 2030 universal health coverage (UHC) goal to cushion this population. Moreover, early findings indicate better COVID-19 pandemic responses in countries with good UHC such as Taiwan, Vietnam, and Singapore , suggestDespite the commitment made by most African countries including the wide integration of UHC into national health strategies, the actual progress has been dismal, and this may have affected the ability to mount an effective response against COVID-19 as well as maintain routine healthcare services. A 2021 report assessing the state of UHC in Africa shows that only 48% of the population receives the healthcare services that they need, while up to 97 million people (8.2%) experience catastrophic healthcare costs . It is fResource mobilization to support a more comprehensive and integrated strategy may however be challenging for most African countries due to shrinking economies, weakened local currencies, and potential budget cuts. Some countries have in fact already adjusted to COVID-19 by cutting health budgets ,52. As sThe COVID-19 pandemic presents multifaceted challenges to many healthcare programs: from reduced access to healthcare services including testing, treatment, and care support services, to structural impacts on drug stockouts, resource shortages, and malnutrition. While every effort must be put in place to contain the pandemic, neglecting endemic diseases and MCH is likely to lead to a greater loss in population health in African countries with comparatively more indirect deaths. An integrated pandemic response that ensures continuity of endemic disease control efforts and MCH services and catch-up campaigns for essential disease prevention and control activities is urgently needed. Post-pandemic recovery plans must also include structural changes in public health systems to ensure rebuilding resilient systems that are capable of supporting effective epidemic response while at the same time ensuring continuity of essential health services. Sustained international solidarity as observed during COVID-19 is required to support an integrated COVID-19 response that ensures the continent remains on course toward achieving the global targets for HIV, TB, and malaria control and MCH mortality reductions.The authors alone are responsible for the views expressed in this publication, and they do not necessarily represent the decisions or policies of the Africa CDC or ASLM.


Over the past two decades, several studies have positioned early-life microbial exposure as a key factor for protection or susceptibility to respiratory diseases. Birth cohorts have identified a strong link between neonatal bacterial colonization of the nasal airway and gut with the risk for respiratory infections and childhood asthma. Translational studies have provided companion mechanistic insights on how viral and bacterial exposures in early life affect immune development at the respiratory mucosal barrier. In this review, we summarize and discuss our current understanding of how early microbial–immune interactions occur during infancy, with a particular focus on the emergent paradigm of “innate immune training”. Future human-based studies including newborns and infants are needed to inform the timing and key pathways implicated in the development, maturation, and innate training of the airway immune response, and how early microbiota and virus exposures modulate these processes in the respiratory system during health and disease. The past 30 years have revolutionized our view of the early development and maturation of the immune system in humans. Early studies introduced the concept of the “hygiene hypothesis”, following the observation that extremely clean household environments during infancy may increase the risk of atopy, asthma, and other respiratory conditions ,2,3. Morn = 839) [n = 234 children) and the Growing Up in Singapore Towards Healthy Outcomes (GUSTO) birth cohort (n = 122) have reported similar results [Several studies have demonstrated that early-life microbial environments are critically associated with developing protection or susceptibility to respiratory diseases . Some ofn = 839) . Follow-n = 839) . The Aus results ,16. Colln = 662) showed that colonization with M. catarrhalis and H. influenzae induced a mixed Th1/Th2/Th17 response with high levels of IL-1β, TNF-α, and macrophage inflammatory protein-1β [Although the mechanism underlying the link between early pathogenic airway bacterial colonization and subsequent susceptibly to respiratory infections and asthma is unknown, current evidence suggests that a pro-inflammatory immune signature of the respiratory mucosa is implicated. A study examining cytokines and chemokines in the neonatal nasal airway  exhibit increased production of type III IFN-lambda during viral respiratory infections . Type IIAside from nasal mucosal responses, the in vitro systemic immune response to Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in the peripheral blood mononuclear cell of infants is also predictive of lower respiratory infection . The incBecause of its location, the nasal microbiome is likely to play a direct role in the development of immune responses in the respiratory tract. Nonetheless, the gut microbiota is much more diverse and complex, and there is strong evidence that is linked to distinct immunological responses during health and disease . The intn = 319) found that transient gut microbial dysbiosis in the first 100 days of life is linked to asthma risk. The CHILD study also demonstrated that Faecalibacterium, Lachnospira, Veillonella, and Rothia are protective against asthma [Some of the most compelling evidence or the gut–lung axis, driven by changes in the intestinal microbiota, comes from the Canadian Healthy Infant Longitudinal Development (CHILD) cohort. The CHILD cohort  identified that wheezing episodes were associated with both bacterial infection  and virus infection  [n = 234) and identified that temporary overabundance of Streptococcus, Moraxella, or Haemophilus occurred during viral respiratory infections [n = 842 infants hospitalized for bronchiolitis), established that the nasal microbiome enrichment of Moraxella or Streptococcus species after bronchiolitis hospitalization was associated with recurrent wheezing by the age of three [n = 74), the relative abundance of Haemophilus, Moraxella, and Klebsiella was linked to recurrent wheezing [n = 118) identified that the during RSV acute respiratory infection in infancy, nasopharyngeal identification and increased abundance of Lactobacillus were linked to a lower risk of childhood wheezing illnesses at age 2 [The traditional view that respiratory viruses are primary triggers of wheezing while bacteria are mostly linked to pneumonia is increasingly being re-evaluated. Several studies have shown the coexistence of virus and bacteria in the airways with important clinical and pathobiological implications . A study< 0.001) . In anotfections . Mansbacof three . Zhang eat age 2 . Thus, eMechanistic insights into the relationship between viruses and bacteria during respiratory infections in children have been examined using transcriptomic profiles and companion clinical outcomes. A translational study by de Steenhuijsen Piters  identifin = 429) demonstrated that a virus-targeted intervention shaped the airway microbiome [Although most studies have focused on how bacteria may influence the outcomes of viral infections, it is important to mention that there is also evidence of a bidirectional interaction. A multicenter, single-blind, randomized, placebo-controlled trial to evaluate RSV palivizumab prophylaxis  [All of the evidence from birth cohorts and companion mechanistic studies indicate that the early microbiome shapes immune system development, and may increase or reduce susceptibility to respiratory diseases, including childhood asthma. This new paradigm has been conceptualized under the emergent model of “Innate Immune Training (IIT)”, in which microbiota and epigenetic influences are convergent drivers and mediators of the asthma trajectory from pregnancy to childhood ,7,8,9,10n = 120) . In thisn = 120) . While tn = 120) . As cesan = 120) , novel an = 120) .Several human-based translational studies have provided strong support to the concept that microbial and other environmental exposures taking place during early life, even in utero, can affect the biology of the mucosal barriers and drive the development and maturation of immune responses in the respiratory system. Future human-based studies including newborns and infants are still needed to inform the timing and key pathways implicated in the development, maturation and innate training of the airway immune response, as well as how early microbiota and viruses exposure modulates these processes in the respiratory system during health and disease.