Four-dimensional (4D) printing is an innovative additive manufacturing technology used to fabricate structures that can evolve over time when exposed to a predefined environmental stimulus. 4D printed objects are no longer static objects but programmable active structures that accomplish their functions thanks to a change over time in their physical/chemical properties that usually displays macroscopically as a shapeshifting in response to an external stimulus. 4D printing is characterized by several entangled features , structure geometry, and applied stimulus entities) that need to be carefully coupled to obtain a favorable fabrication and a functioning structure. Overall, the integration of micro-/nanofabrication methods of biomaterials with nanomaterials represents a promising approach for the development of advanced materials. The ability to construct complex and multifunctional triggerable structures capable of being activated allows for the control of biomedical device activity, reducing the need for invasive interventions. Such advancements provide new tools to biomedical engineers and clinicians to design dynamically actuated implantable devices. In this context, the aim of this review is to demonstrate the potential of 4D printing as an enabling manufacturing technology to code the environmentally triggered physical evolution of structures and devices of biomedical interest. Additive manufacturing (AM) technologies  printing”) have been widely exploited in many interdisciplinary research fields  due to their excellent repeatability and capacity to construct complex structures with precise geometry control ,2. AM prD printinIn the last decades, different innovations have been introduced in AM, such as multi-material and multi-scale AM, in which different AM technologies are combined to fabricate heterogeneous structures ,5, in siMore in detail, 4D printing was introduced in 2013 by Dr. Skyler Tibbits  to denotTherefore, the key points of 4D printing are: (i) active materials  or combinations of different materials; (ii) external stimuli ; and (iii) AM technologies, which act as an enabling tool that allows the exact positioning of a precise quantity of one or more stimulus-responsive materials in predetermined locations without restrictions on the complexity of the geometry ,13,14. T−1. Moreover, laser printing technology has been used in synergy with 4D printing to convert graphene oxide into graphene-based composites [Using two dimensional (2D) nanomaterials , 4D-printed nanocomposites have been used to design shape-programming objects. For example, Wei et al.  used 4D mposites . This lamposites ,20.Using responsive materials and the related stimuli for their activation, it is possible to physically program many morphological transformations enabled by the proper organization guaranteed by AM. However, designing a new structure via 4D printing is a complex problem. Indeed, it is influenced by several variables  that need to be properly combined to achieve a functional structure. Moreover, the knowledge of the material’s behavior and interactions, the correct stimulus, and the printing parameters are fundamental elements that increase the complexity of the problem and that need to be considered and deeply understood. In this context, mathematical modeling is a very useful tool to determine the combination of variables that leads to the maximum and desired transformation of the 4D-printed structures ,22.Since 2013, when 4D printing was introduced, this fabrication strategy has seen fast growth in many sectors, including smart textiles, autonomous and soft robotics, biomedical devices, electronics, and tissue engineering (TE). This is due to its advantages over static AM, such as the possibility of an easier fabrication of complex 3D structures that are fabricated as flatted objects and then achieve their 3D conformation after actuation, thus reducing the encumbrance of the objects, and the exploitation of reliable alternatives to electrical energy , capable of remote control without the use of cumbersome wiring, thus leading to the use of 4D printed structures in severe environments .In this review, we aim at providing an overview of 4D printing as a new additive manufacturing technology in the biomedical field. In the first part, a brief introduction to stimuli-responsive materials with a special focus on natural polymers and nanomaterials will be provided, along with a description of the achievable transformations and mathematical modeling. Then, applications of 4D printing in bioengineering  will be discussed, reporting relevant examples from the recent literature.In many applications, 4D printing exploits active materials, namely materials that undergo useful, predictive, reproducible, and macroscopic physical or chemical changes in their properties as a consequence of an environmental change ,23. WhilWith the rapid progress of 4D printing, the prevalence of eco-friendly, sustainable smart polymers from natural sources is gaining great interest in the field ,25,26. TMoreover, exploiting natural biomaterials allows for the expansion of the library of possible bioinks/biomaterial inks, thanks to the variety of natural building blocks, such as peptides, amino acids, and deoxyribonucleic acid (DNA) sequences.In the following paragraphs, the active polymers, with a deep focus on natural polymers, used in 4D printing will be analyzed and classified in relation to the involved stimulus.Currently, one of the most investigated methods to achieve shape-changing in 4D printing is temperature responsiveness. A thermo-responsive material can employ exogenous temperature changes as stimulus to achieve a particular shape transformation or can be activated by heating through the Joule effect due to electrical current flow. The easiest form of temperature-responsiveness is thermal expansion, which leads to the volume expansion of a structure as a consequence of a temperature increase. In this context, there are also a few materials that exhibit the opposite behavior, undergoing contraction with an increase in temperature . Among tTaking a step forward, changes in temperature can induce variations in wettability and solubility alterations of materials, as for poly(N-isopropylacrylamide) (PNIPAAm), poly(methyl vinyl ether), and poly ,32.An easy method that fosters the temporal shape transition of 4D-printed structures is humidity responsiveness. The phenomenon refers to the inherent swelling feature observed in both synthetic and natural hydrogels . HydrogeAnother category of active materials are pH-responsive polymers. They can vary their rheological characteristics, such as viscosity and shear modulus, in response to changes in the pH or ion concentration of the surrounding environment ,23,38. EMoreover, pH can be utilized to promote the self-assembly of peptide hydrogels made of the alternation of natural amino acids, increasing the mechanical and rheological properties of the substance by promoting the creation of intra-molecular sheets or alpha-helices ,42.In light-sensitive materials, the applied optical stimulation , and near-infrared (NIR) light) is converted into other responses, usually mechanical ones . The chrIt is crucial to emphasize that in numerous works, light is employed as a substitute technique to heat the 4D structure in specific spots. Consequently, the rise in temperature brought on by the lights is the true stimulus that causes the shape-shifting ,49.Polythiophene and poly(2-hydroxyethyl methacrylate) are some examples of electrically responsive materials characterized by intrinsic electrical conductivity. Their shape and size can be regulated by the intensity and direction of an external electric field ,51.Coulombic, electrophoretic, electroosmotic, and piezoelectric processes are some examples of the electrical interactions that can take place in a material to produce electric field responsiveness . AdditioMagnetic fields, in addition to electrical fields, are frequently utilized for triggering shape changes in 4D-printed structures. Uncontactable remote manipulation using magnetic fields is effective and safe . SimilarSMPs are a widely utilized class of smart polymers that are crucial for 4D printing in the biomedical industry ,24. TheiPolylactic acid (PLA) and acrylonitrile butadiene styrene (ABS) are the most commonly used temperature-responsive SMPs ,25, wherIn the literature, there are different attempts to define a taxonomy of shape-changing structures that can be obtained with 4D printing. Here, starting from the classification provided by Nam and Pei , an advaFour-dimensional printing is influenced by several entangled variables, such as the entity of the applied stimulus, the spatial deposition of different material(s), and their properties. Those variables need to be carefully coupled to obtain a favorable fabrication and a functioning structure, making the design of a new structure via 4D printing a complex problem. In this contest, the development of an appropriate mathematical model is essential for the success of the 4D printing process ,79,80. MAn appropriate mathematical model for 4D printing comprises four main elements: (i) the final desired shape , dictated by the application of the 4D printed object; (ii) the spatial deposition of the involved material(s), i.e., the initial shape. This aspect closely depends on the employed AM technology and on the material processability; (iii) material properties and responsiveness to the applied stimulus; and (iv) entities of the applied stimulus.Mathematical models can be divided into two main categories: forward problems and inverse problems. The forward problems aim at determining the final shape of the structure and are more oriented toward discovering concepts. A glaring example of a forward problem is the use of lumped parameter models to study the shape evolution of SMPs. For instance, the standard linear solid model  was exploited by Yu et al. to quantitatively model the energy storage and release process achieved during the multiple shape transformation in SMPs .In inverse problems, the final state is known, and the user aims to find out how to obtain it by defining the initial geometries and spatial deposition of material(s) . Thus, inverse problems are more application-oriented. A brilliant example of an inverse problem can be found in the study of Gladman et al. , in whicAnalytical models represent the mathematical formulation of a certain mechanical or biophysical system or phenomenon, thus being extremely used in the mathematical modeling of 4D printing . ExampleSoft actuators, also referred to as bioactuators, are highly deformable structures characterized by ease of movement that can be activated by external stimuli to generate the desired motion and forces/torques . They arA milestone example of this use of 4D printing for soft actuators is provided by Liu et al. . The aut−1, thus possessing a highly efficient mechanism of locomotion. This was achieved using the cell contractive forces and the anisotropic friction of the supporting structure.Taking a step forward, Chan et al.  fabricatNative tissues exhibit complex 3D structures and microarchitectures, as well as unique functions that are achieved through dynamic changes in tissue conformation . TherefoThe 4D-bioprinted scaffolds can improve the functionality of the scaffold by simplifying its seeding and the fabrication of structures capable of adapting to the complex 3D shapes of the human body or providing appropriate stimuli to promote cell differentiation and activities ,101.For example, in the context of bone TE, it is difficult for traditional 3D static scaffolds to have a good match with the sharp edge shape of bone defects, thus often leading to incomplete edge and material absorption ,103. In Moving to a diverse anatomical district, Kim et al.  4D-bioprFocusing on the same tissue  and shape-morphing strategies, Ding et al.  fabricatTaking a step forward, Yang et al.  fabricatFinally, thinking about patient-adaptable soft tissues such as blood vessels, Luo and colleagues created an effective approach to manufacturing scaffolds with fine topologies combining 4D printing, fused deposition modeling (FDM), and crosslinkable shape memory linear copolyesters . These 4A medical device is any article manufactured to be used in human beings with the final aim of diagnosis, prevention, monitoring, prediction, prognosis, treatment, or alleviation of disease, injury, or disability, as proposed by the Global Harmonization Task Force and reported in official legislation as European Regulation 745/2017 ,107. 4D In previous works, we exploited the temperature responsiveness of regenerated silk (RS) for the fabrication of two devices to be used in intestinal surgery applications ,59. The Drug delivery systems play a critical role in the pharmaceutical industry by ensuring that medications are administered to patients effectively and with precise control. 4D printing can also be applied to drug delivery systems, where printed structures release drugs in a controlled manner. In this context, 4D printing possesses several advantages if compared with traditional manufacturing techniques . Indeed, 4D printing offers a number of benefits, including the possibility to obtain a controllable kinetic and to achieve a time- and/or site-dependent drug release based on the shape-shifting property of the device itself ,111.4D-printed structures for drug delivery systems should be biocompatible to avoid toxicity or irritation at the delivery site. In this case, 4D-printed drug delivery systems utilize biocompatible hydrogels or smart polymers that can change their properties in response to environmental factors. These materials are well-suited for controlled drug release applications. For example, a chitosan-pectin hydrogel was utilized by Long and colleagues to print a biodegradable wound dressing for the local anaesthetic medication lidocaine . The appIn smart drug delivery system fabrication, temperature-responsive materials are carefully chosen based on their ability to undergo a phase transition, typically a sol-gel transition, within a specific temperature range. Common materials include thermosensitive polymers like PNIPAAm and its derivatives . The druStaphylococcus aureus and Enterococcus faecalis [Moreover, 4D-printed hydrogels with antimicrobial properties have been receiving attention in the last decade, especially for wound closure. 4D hydrogels were functionalized with the cell-adhesive motif Arginine-Glycine-Asparagine (RGD) to enhance cell spreading , and at faecalis . In factfaecalis . Regenerfaecalis ,120.Other examples were represented by 4D-printed drug-eluting stents that could release medications gradually to prevent restenosis in blood vessels .Last, but not least, 4D printing enables the fabrication of patient-specific drug delivery systems . Each paUtilize 4D-printed materials to create drug delivery systems that can release chemotherapy agents with precise control by using the right quantity of the drug at the right time. One example is the prosthesis of paclitaxel and doxorubicin microspheres to prevent tumor recurrence and metastasis after breast-conserving surgery designed and prepared by Hao et al. in 2021 .Other examples were studied to implement real-time monitoring of treatment outcomes and patient responses to adjust drug delivery parameters as needed .For sure, to allow the use of 4D printing in clinical practice in an effectively integrated manner, we must create multidisciplinary care teams involving oncologists, pharmacists, nurses, and 4D printing specialists to facilitate patient-specific treatment planning and implementation.Finally, by designing drug delivery systems that release medication over extended periods, patients may require fewer doses, improving compliance and reducing the risk of medication errors. The potential for personalized drug delivery through 4D printing brings undoubtedly with it a host of ethical and regulatory challenges. Addressing these challenges is crucial to ensuring the safe and responsible use of this technology. First of all, personalized drug delivery may require the collection and storage of sensitive patient data, such as medical history and genetic information. For this reason, patients must understand and consent to the use of their data and the personalized drug delivery approach. On the other hand, one must implement strict data protection measures, secure storage and encryption, and ensure compliance with data privacy laws  or the Healthcare Insurance Portability and Accountability Act (HIPAA)).In conclusion, although several open challenges still must be faced , 4D (bio)printing is a breakthrough technology that, thanks to the constant progress in materials science, additive manufacturing, and biology, represents an enabling tool to address unsolved clinical needs in tissue engineering and medical device manufacturing. This review highlighted the importance and potentiality of 4D (bio)printing to tailor and customize the functionalities of a device by combining active materials with appropriate 3D architectures and advanced AM technologies.


MKL1 displayed ethnic stratification, with the variant being significantly more prevalent in the African cohort and resulting in reduced transcript levels as measured by qPCR. Functional validation of MKL1 by shRNA-mediated knockdown of MKL1 resulted in a more resistant phenotype. Gene expression analysis identified the developmentally associated TGFB1I1 as the most significant gene associated with sensitivity. qPCR among a panel of sensitive and resistant LCLs revealed immunoglobulin class-switching as well as differences in the expression of B cell activation markers. Flow cytometry showed heterogeneity within some cell lines relative to surface Ig isotype with a shift to more IgG+ cells among the resistant lines. Pretreatment with prednisolone could partly reverse the resistant phenotype. Results suggest that the efficacy of anti-CD20 mAb therapy may be influenced by B cell developmental status as well as polymorphism in the MKL1 gene. A clinical benefit may be achieved by pretreatment with corticosteroids such as prednisolone followed by mAb therapy.Monoclonal antibody (mAb) therapy directed against CD20 is an important tool in the treatment of B cell disorders. However, variable patient response and acquired resistance remain important clinical challenges. To identify genetic factors that may influence sensitivity to treatment, the cytotoxic activity of three CD20 mAbs: rituximab; ofatumumab; and obinutuzumab, were screened in high-throughput assays using 680 ethnically diverse lymphoblastoid cell lines (LCLs) followed by a pharmacogenomic assessment. GWAS analysis identified several novel gene candidates. The most significant SNP, rs58600101, in the gene  MS4A1 gene is a member of the membrane-spanning 4A gene family encoding the B-lymphocyte surface molecule (CD20). Its direct function is largely unknown, but it has been proposed to regulate calcium conductance and be involved in cellular activation [The lecule CD0. Its ditivation  with limtivation ,6,7,8. Itivation ,10, thustivation . This st+ B lymphocytes (LCLs), enabling genome-wide association studies (GWAS) and gene expression studies in large multi-ethnic cohorts. LCLs are thus ideally suited for studying the genetic determinants influencing sensitivity toward anti-CD20 therapies. The LCLs are prepared from peripheral blood B cells of donors immortalized by in vitro transformation with Epstein–Barr virus. Each cell line is thus a population of genetically matched cells, yet significant B cell heterogeneity may exist with respect to immunoglobulin isotypes as well as differences in activation and differentiation pathways [The lymphoblastoid cell lines (LCLs) used in this study were obtained from the 1000 Genomes Project , which ppathways .regs), or terminally differentiate into more metabolically active antibody-secreting plasma cells. Antigen engagement by the BCR is essential to B cell activation and differentiation, driving the clustering of BCR complexes along with co-receptors into microdomains organized around lipid rafts. Amplification of signaling occurs by the fusion of these microdomains. Fusion is mediated by the cytoskeleton and Src-family kinases to form larger signaling and endocytic regions on the cell surface [Gene expression analysis suggested differences in B cell developmental status differentiate sensitive from resistant cells. B cell development and maturation is a complex, multi-stage process, generating numerous B cell intermediates and subtypes reflected by a changing repertoire of cell surface markers and functionality. Circulating naïve B cells express surface-bound IgM/D. Cellular activation by antigenic binding through the IgM/BCR (B cell Receptor Complex), as well as cytokine stimulation and interaction with helper T cells, induces immunoglobulin class-switching to IgG/A/E with additional affinity maturation of the immunoglobulin through somatic hypermutation in germinal centers (GC) of secondary lymphoid tissue. GC-derived B cells then differentiate into either relatively quiescent memory B cells, regulatory B cells , associated with drug sensitivity. MKL1 is a transcriptional regulator controlling the expression of cytoskeletal genes. While a role for MKL1 in hematopoietic development is implicated by its involvement with the t translocation specific to acute megakaryoblastic leukemia, there is limited research examining an MKL1 role during B cell differentiation. B cells from a patient with an intronic deletion in MKL1 have been recently characterized and shown to have elevated levels of MKL1, which was associated with a number of phenotypic changes: increased actin content; hyperproliferation; genomic instability; decreased expression of CD11a integrin molecules; and decreased homotypic aggregation [Genome-wide association analysis identified a novel gene, regation .2 using RPMI medium 1640 and 15% fetal bovine serum .This study was performed using 680 lymphoblastoid cell lines (LCLs) from the 1000 Genomes Project  after re®; Genentech, Inc., San Francisco, CA, USA) and ofatumumab  were purchased through the North Carolina Cancer Hospital pharmacy. Obinutuzumab  was kindly provided by Angela Facul .Rituximab  or in the presence of 25% human serum (as a source of complement) and with or without 10 μg/mL of each antibody. In initial screening experiments, which were part of a larger drug screening project , pooled Gene expression analyses were conducted using previously established procedures . RNA-Seqi = β0 + Li*β + ϵi, ϵi ~ NFirst stage: GijY = β0 + β1*PC1i + β2*PC2i + β3*PC3i + β4*iSex + β5*ϵi + ije, eij ∼ N, where, in the first stage: Gi is the quality-controlled RNA-Seq read count of gene g for individual i, Li is a vector of indicator variables for the lab in which the RNA-Seq was conducted for individual i, β is a vector of the regression parameters, and ϵi are the residuals = observed read count for gene g-predicted gene read count for the gene for an individual I; in the second stage: Yij is the vector of normalized responses for the one concentration for the mAb for each individual i; the Eigenvalues for the first three principal components, PC1, PC2, and PC3 are calculated using EigenStrat [i is an indicator variable denoting gender.Second stage: genStrat ; and i ii is the quality-controlled RNA-Seq read count of gene g for individual i. Li is a vector indicating the lab in which the RNA-Seq was conducted for an individual. β is a vector of the regression parameters, and ϵi are the residuals . To minimize extreme residual values from having an excessive influence on parameter estimates, residuals were log-transformed and standardized prior to use in the second stage. In the second stage, linear regression was performed using the R function lm in package stats v3.4.0 [g. Yij is the vector of normalized responses for the six concentrations of the drug for individual i; the Eigenvalues for the first three principal components, PC1, PC2, and PC3, are calculated using EigenStrat [I as an indicator variable denoting gender. Significant results from the second stage were obtained after correcting for multiple testing on a per-drug basis using: the Bonferroni correction with a significance level of p < 0.05 for the gene expression analysis; and the Benjamini–Hochberg method [p.adjust , package stats v3.4.0) [In the first stage, linear regression is performed using the limma package  to removs v3.4.0  for eachgenStrat  with I ag method  with a f v3.4.0) .p-value < 0.05) associated with ofatumumab sensitivity were used to identify signaling pathways involved in the differences between LCLs sensitive and resistant to CD20 mAbs treatment.Unsupervised and supervised enrichment analyses using gene set enrichment analysis (GSEA) and gene ontology (GO) analysis were performed using the ClusterProfiler R package . The moshttps://www.internationalgenome.org/data-portal/data-collection/phase-1, accessed on 1 May 2015) and processed as described elsewhere [p-value < 1 × 10−6. After routine quality control, 1,510,701 SNPs were used for GWAS, as described in Abdo et al. [http://pngu.mgh.harvard.edu/purcell/plink/, accessed on 20 September 2019) under a genotypic model, adjusted for sex, temperature, growth, experimental batch, and the Eigenvalues from the first three principal components using EigenStrat [Genotype data for the 1000 Genome cell lines from the Illumina HumanOnmi2.5 platform was obtained from the 1000 Genomes database  . Control cell lines were prepared using a scrambled shRNA sequence. 1 μg/mL of puromycin was used to select positive cell lines. Knockdown was confirmed by Western blotting.Stable knockdown cell lines were prepared as previously described  by lenti−6 cells using Quick-RNA Microprep Kit . Purified RNA was then reverse-transcribed into cDNA using Verso cDNA Synthesis Kit . Conventional qPCR was performed using PowerUp SYBR Green Master Mix . Reactions were conducted in quadruplicate in 10 μL volumes using 500nM of forward and reverse primers prepared by Sigma Genosys  and run on the Applied Biosystems QuantStudio 6 Flex Real-Time PCR System. Primer pairs used are provided  within one field of view for each cell line.Homotypic aggregation or cell clump size was estimated from micrographs taken 30 min following the dispersion of cells by pipetting and pulse centrifugation. Images were captured using a Nikon Eclipse Ts2R microscope with a 10x objective and NIS-Elements software. 8-bit grayscale images were converted to binary then particles were defined using the best-fit ellipse selection tool. The particle areas were then estimated with ImageJ software  with a size defined as 2000–∞ ; αIgG-FITC (Invitrogen); and αIgM-BV711 . Antibodies were added to samples for an additional 45 min before adding an equal volume of buffer. Samples were centrifuged, washed, and pellets resuspended in 0.5 mL of the same buffer containing either TOPRO-3 (Invitrogen) or DAPI (Thermo Scientific) as a viability dye. Surface expression analysis was performed on an Attune NxT V6 flow cytometer (Thermo Scientific). Data analysis was performed with FlowJo software . For sorting experiments, 107 cells were stained as above with αIgG except omitting sodium azide from the staining buffer and sorted on a FACSAria III  within the UNC Flow Cytometry Core Facility.1.0 × 10−5 cells/well in 25ul media. The reaction was begun by the addition of an equal volume containing 8 μM EthD1 (Molecular Probes), ±20 μg/mL of anti-CD20 mAb, and containing 50% human serum. An Infinite F200 microplate reader (Tecan US) and iControl software (Version 1.6) were used to measure the fluorescence intensity of the dye at EX535nm and EM595nm at timed intervals. The fluorescence of untreated cells was subtracted as background from treated cells. Dye uptake is then presented as a percentage relative to a positive control using cells lysed with 0.1% saponin.Cells were plated into 384 well plates at 2.5 × 10g for 10 min to pellet cells. The supernatant was then centrifuged at 1500× g for 10 min to remove cell fragments, including apoptotic bodies. Exosomal material was collected in a subsequent 15,000xg centrifugation for 30 min. In some experiments, 10% (w/v) PEG-10000 (Sigma) in 0.3M NaCl was added to the 15,000× g supernatant overnight at −20 °C then centrifuged at 15,000× g for 30′ to confirm quantitative removal of CD20 in the preceding fractions. Pelleted fractions were resuspended in lysis buffer and analyzed by western blotting.Exosomes were isolated by centrifugation of cell cultures at 250× R2 = 0.51 , ofatumumab (ofat), and obinutuzumab (obin) using a single, saturating concentration of 10 μg /mL, were compared using 680 lymphoblastoid cell lines (LCLs) under both normal cell culture conditions (ofat and obin) as well as in the presence of human serum as a source of complement  as part of a larger, drug screening project . Alamar 2 = 0.51 . All thr2 = 0.51 . LCL res = 0.441 . There wtwo mAbs . The mintwo mAbs . Likewistwo mAbs . Direct ectively .p-value < 10−6). At the genome-wide suggestive threshold of p-value < 10−6, there were eight peak associations for ofatumumab and obinutuzumab under normal culture conditions measuring direct cell kill , the most significant SNPs were associated with obinutuzumab treatment (SCAMP5 gene). An additional SNP, rs60910940 (synonymous variant in SCAMP5), was also associated with obinutuzumab treatment.Using genotype data from the 1000 Genomes database, separate GWAS was performed using the response data from the three monoclonal antibodies: rituximab; ofatumumab; and obinutuzumab. ell kill . In the ell kill . At the reatment . They weMKL1 gene with ofatumumab treatment . MKL1 was therefore chosen as a candidate gene for functional validation to examine if its expression influenced sensitivity toward anti-CD20 mAbs using shRNA knockdown constructs.Under the more physiologic condition with complement present, the most significant association is for SNP, rs58600101, an intronic SNP within the reatment . The MKLMKL1 exons 14–15, which help comprise the transcriptional activation domain (TAD) of MKL1 [MKL1 transcript variant occurs within this region through the use of an alternate splice site, resulting in a frameshift and an early stop codon. For this reason, primers were designed spanning exons 13–15 for both the reference transcript as well as for the splice variant. A third pair of primers were included in a separate region of the gene spanning exons 11–12. MKL1 gene expression of either of the reference transcripts was reduced in the SNP variants to an average of 0.37 (stdev ± 0.114) relative to wildtype expression (MKL1 in the SNP variant averaged 0.34 (stdev ± 0.19) in comparison to wildtype. Therefore, SNP rs58600101 affects transcript levels but did not appear to influence the formation of splice variants.SNP, rs58600101, is an intronic SNP located between the last pair of  of MKL1 . qPCR wapression . The splMS4A1 (CD20), was examined by both qPCR and flow cytometry in these cell lines  of the nine cell lines, suggesting MKL1 is responsible for additional mechanisms of resistance. Further characterization of the MKL1 knockdown cells  and lymphoma lines  using shRNA constructs. Knockdown was confirmed by western blotting to average 87% . Transforming Growth Factor Beta 1 Induced Transcript 1  was the most significant gene correlating with sensitivity. Two genes were identified for rituximab  that were likewise significant for ofatumumab. Eight genes were identified for obinutuzumab . Five of these genes were likewise significantly associated with ofatumumab. The three remaining genes could thus be uniquely assigned to the type II antibody, obinutuzumab, and included: RSAD1 (p < 0.02), RP11-251G23.2 (p < 0.02), and PDCD4 (p < 0.04).Associations between basal gene expression and complement-mediated cytotoxic drug response were examined for these three anti-CD20 mAbs using publicly available expression data . The genp < 0.05 . The MS4MHCII-associated genes was associated with the resistant phenotype. SCAMP5 again appeared as a significant gene during this analysis. Its interaction with the cytoskeleton at the inner plasma membrane surface and involvement in calcium-triggered vesicle fusion followed by exocytosis likewise suggest potential protective mechanisms.The gene list generated by ofatumumab was examined en masse to identify biological processes that might distinguish sensitive from resistant cells using GSEA  and GO (Gene ontology) enrichment analyses . The GO-p ≤ 0.001). Since homotypic aggregation can be indicative of a more activated B cell, frequently as a consequence of CD40 signaling [IgM relative to IgG. In contrast, resistant cell lines expressed more IgG as well as markers associated with an activated B cell phenotype, such as IRF4, TNFSRF17, CD40, and CD23 (FcεRII) [PAX5 (p ≤ 0.0003).During the screening of LCLs in viability assays, increased homotypic aggregation was often observed in resistant cell lines relative to more sensitive lines. Therefore, the size of cell aggregate was measured in seven resistant and seven sensitive cell lines. The relative aggregate size of resistant cell lines averaged 778 μm (±101 sem) compared to 161μm (±45.8 sem) for sensitive cell lines  ,40,41,42+, and generally, sensitive cells were more IgM+. Some cell lines exhibited mixed populations of both IgG+ and IgM+ cells  higher in IgG+ cells relative to the IgM+ population when assayed against rituximab (p ≤ 0.01) higher in the IgG+ population when compared to IgM+ cells.Surface expression of IgG and IgM were then analyzed in 9 LCLs by flow cytometry. As expected, based on results from qPCR data, resistant lines were predominantly IgGM+ cells . One sucfor IGHG . The twoituximab . The difGiven that there appeared to be an association between mAb resistance and expression of activated B cell markers, a panel of B cell lines was pre-treated with the immunosuppressant, prednisolone for 10 days to essentially deactivate and thus sensitize the cell lines toward CD20 mAb treatment. While not all cell lines appeared responsive to prednisolone, the majority of cells were sensitized. Cell viability was reduced an additional 63% (±0.21 stdev) on average relative to cells not pre-treated with prednisolone following exposure to ofatumumab  following high-throughput in vitro screening of LCLs as an unbiased approach to identify genes affecting sensitivity toward anti-CD20 mAb therapy without reliance on a priori knowledge for potential gene candidates.MKL1, also referred to as Myocardin-related transcription factor A (MRTFA), as a significant determinant of sensitivity. MKL1 is a transcriptional co-regulator along with serum response factor (SRF) controlling the expression of cytoskeleton-related genes, including actin [MKL1 SNP, rs58600101, is an intronic SNP located in the 3′ end of the gene between the last pair of exons 14/15. The SNP variant was associated with increased resistance toward anti-CD20 mAbs during high-throughput screening. The variant could be associated with reduced MKL1 mRNA expression. This was consistent with subsequent shRNA knockdown experiments in which reduced MKL1 expression was accompanied by a more resistant phenotype in both lymphoblastoid as well as lymphoma cell lines. This SNP exhibited ethnic stratification with the minor allele found predominantly in the African population cohort (MAF = 0.170), with only one variant found within the non-African cohort of the 680 cell lines in this study sample. This result suggests that the efficacy of anti-CD20 mAbs may be diminished in patients of African descent.GWAS identified the SNP, rs58600101, in ng actin ,46. MKL1ng actin ,48,49. Tng actin ,51,52,53ng actin . The MKLTGFB1l1, as one of the most significant determinants of sensitivity. TGFB1l1 is a member of the paxillin superfamily of focal adhesion adaptor proteins [Gene expression results indicate that differences in B cell development or activation status likely affect sensitivity with potential involvement of the TGF-beta 1 signaling pathway. TGFb1 is an important regulator of B cell development, having been shown to interfere with the expression of cell surface λ light chains during in vitro differentiation of normal human pre-B cells  and to iproteins ,59 that proteins .CBLB, as a determinant of anti-CD20 sensitivity. Knockdown experiments resulted in increased CD20 clustering on the cell surface and increased resistance toward anti-CD20 mAbs [A previous, smaller study by this group relying on linkage analysis of pedigreed cell lines identified a similar gene, D20 mAbs . Like TGD20 mAbs ,62. TherD20 mAbs  characteD20 mAbs . Togethe+, while resistant lines were enriched in class-switched IgG+ cells. Other cell lines used in this study were observed to have a more evenly mixed population of IgM+ and IgG+ cells. While this Ig isotype-mixed population of cells within a cell line may interfere with the results obtained during gene expression analysis, it allowed for the separation of a single cell line, one individual, based on the surface expression of the Ig isotype. The cell line GM12876 was thus separated by flow cytometry based on Ig surface expression. Consistent with the results above, the IgG+ subline was more resistant toward anti-CD20 mAb treatment relative to the IgM+ subline.The developmental/activation status of LCLs was examined more closely for evidence of immunoglobulin class-switching among a group of sensitive versus resistant cell lines by flow cytometry. Sensitive lines were predominantly IgM+ cells [IgG-switched cells can be associated with a more activated status. In mice, IgG memory cells have been shown to be more likely to differentiate into antibody-secreting plasma cells following re-stimulation with antigen compared to IgM memory cells ,66. Memb+ cells ,70. HoweDifferences in gene expression, as well as differences in homotypic aggregation between resistant and sensitive cells, suggest that B cell activation status affects sensitivity to anti-CD20 mAbs. Based on this hypothesis, the glucocorticoid prednisolone was used as an immunosuppressant to deactivate and thus sensitize resistant cell lines in this study. Prednisone is presently used concurrently as a component of the cancer drug combination R-CHOP , prednisone) for treatment against B cell malignancies. This combination regimen is typically used for leukemia and lymphomas. Rituximab treatment may also be used for a variety of autoimmune conditions such as rheumatoid arthritis, myasthenia gravis, multiple sclerosis, antiphospholipid syndrome, and potentially, systemic lupus erythematosus . MKL1 gene, a transcriptional activator controlling the expression of cytoskeletal genes. A polymorphism in MKL1 was associated with mAb resistance and exhibited ethnic stratification suggesting the efficacy of anti-CD20 mAb therapy may be impacted by ethnicity. In addition, results suggested that B cell developmental/activation status likewise influences sensitivity toward anti-CD20 mAb therapy, with some increase in sensitivity occurring following extended pretreatment with the immunosuppressant prednisolone. Exploring potential protective mechanisms toward complement-mediated mAb therapy suggests that while CD20 is constitutively shed from cells, it is the kinetics of membrane repair following complement fixation that distinguishes sensitive from resistant cells.The purpose of this study was to pharmacogenomically assess several hundred ethnically diverse lymphoblastoid cell lines for response to anti-CD20 mAb therapy to identify genes affecting sensitivity or having prognostic value. Results provided several insights. Multiple gene candidates were identified with a focus on the


Reproductive coercion is a significant public health issue in Australia which has mainly been conceptualised as a form of violence at the interpersonal level. This limited scope ignores the role of the gendered drivers of violence and fails to encompass a socio-ecological lens which is necessary to consider the multiple interacting layers that create the context in which reproductive coercion occurs. The aim of the scoping review was to explore how the reproductive coercion is defined by international research. Specifically, how is reproductive coercion defined at the social-cultural-systems-structural levels, and are the definitions of reproductive coercion inclusive of the conditions and contexts in which reproductive coercion occurs?A scoping review was undertaken to explore existing definitions of reproductive coercion. Searches were conducted on Embase, Cochrane Library, Informit Health Collection, and the EBSCOHost platform. Google was also searched for relevant grey literature. Articles were included if they were: theoretical research, reviews, empirical primary research, grey literature or books; published between January 2018 and May 2022; written in English; and focused on females aged 18–50 years. Data from eligible articles were deductively extracted and inductively thematically analysed to identify themes describing how reproductive coercion is defined.A total of 24 articles were included in the scoping review. Most research defined reproductive coercion at the interpersonal level with only eight articles partially considering and four articles fully considering the socio-cultural-systems-structural level. Thematic analysis identified four main themes in reproductive coercion definitions: Individual external exertion of control over a woman’s reproductive autonomy; Systems and structures; Social and cultural determinants; and Freedom from external forces to achieve reproductive autonomy.We argue for and propose a more inclusive definition of reproductive coercion that considers the gendered nature of reproductive coercion, and is linked to power, oppression and inequality, which is and can be perpetrated and/or facilitated at the interpersonal, community, organisational, institutional, systems, and societal levels as well as by the state.The online version contains supplementary material available at 10.1186/s12889-023-16281-8. Reproductive coercion is a significant public health issue globally, driven by gender inequality and imbalances in power, with negative consequences for a wide range of sexual, reproductive, and mental health issues –2. It isGiven the historically hidden nature of reproductive coercion, limited Australian research exists to demonstrate its full extent. The data that does exist relates to women’s experiences of gender-based violence, which commonly includes reproductive coercion. Research exploring the prevalence of violence against women in Australia demonstrates that one in five women over the age of 15 will experience violence in their lifetime . FurtherThe categorisation of reproductive coercion as a form of violence , 18 has Reproductive coercion has historically been the responsibility of multiple, overlapping sectors. These include the domestic violence, sexual assault/harassment and sexual and reproductive health sectors which are informed by their own distinct approaches to prevention, policy reform and service delivery . A recenA socio-ecological approach can be utilised to move beyond the interpersonal level. This approach recognises the broader systems that individuals are embedded within and the changing nature of the social environment . Socio-eThe model highlights how violence against women is the outcome of interactions between various levels (particularly at the primary prevention level), therefore it uniquely moves beyond just looking at the interpersonal level (or tertiary prevention level). The model was developed to support public health practitioners and academics to approach violence against women from a primary prevention lens, which requires underlying drivers to be addressed across all socio-ecological levels. Like other forms of violence against women, we argue reproductive coercion is a complex public health issue that needs to be viewed and addressed from a socio-ecological lens, as opposed to only focusing on the interpersonal level.It is clear reproductive coercion within the Australian context is multi-layered, complex, gendered, driven by gender inequality and differentials in power, and rooted in historical experiences yet there has been no exploration of how definitions of reproductive coercion may be limiting the scope of work to prevent and address reproductive coercion. As such, the current scoping review explored existing definitions of reproductive coercion, drawing on international research due to the limited Australian research, and asked; how is reproductive coercion defined, how is reproductive coercion defined at the social-cultural-systems-structural levels, and are definitions of reproductive coercion inclusive of the conditions and contexts in which reproductive coercion occurs? This approach enabled an exploration of reproductive coercion in the broadest sense to enable alternative definitions and contexts to be considered in how we might better conceptualise reproductive coercion in Australia. The outcome of this review is a proposed definition of reproductive coercion that moves beyond the interpersonal and considers the role of gendered drivers at the societal, system, institutional, organisational and community levels is conceptualised, which may be applicable in other similar socio-cultural-politico contexts to Australia.A scoping review was undertaken following the process as described by Arksey and O’Malley , and advFive authors  developed the search strategy, search terms and eligibility criteria, which were refined after preliminary searches were completed (AD). The information sources searched included Embase, Cochrane Library, Informit Health Collection, and the EBSCOHost platform , PsycINFO (psychology), and SocINDEX (sociology)). Google was also searched for grey literature.Search terms covered two key concepts: 1) reproductive coercion; and (2) definition  and combined using the OR operator and repeated for concept 2 (definition). Concepts 1 and 2 were then combined using the AND operator to conduct the final search. For example, “reproductive coercion” OR “coercive reproduction” OR “reproductive pressure” OR “reproductive control” OR “reproductive sabotage” OR “reproductive autonomy” OR “reproductive agency” AND definition OR conceptualise OR framing OR theory OR defining OR theoretical OR lens OR positioning OR constructs OR elements OR attributes. Searches were set to identify key terms in the titles and abstracts.A search was also undertaken using Google to identify any grey literature. The Google search was restricted to .gov, .org, or .edu domains and was conducted using the same search terms Table . The firTo be eligible for inclusion articles had to be: theoretical research articles, reviews , empirical primary research, grey literature , books, commentaries or opinion pieces; published between January 2018 and May 2022; written in English; full-text available; focus on females of reproductive age (18 to 50 years); and include a definition of reproductive coercion. Dissertations and conference presentations were excluded. The search was limited to articles published between January 2018 and May 2022 to ensure contemporality of the evidence.While reproductive coercion is not limited to cis-gendered females of reproductive age, research focusing only on adolescents was excluded. However, research which included both adolescent and adult females was included. The reproductive age span selected (18 to 50 years of age) reflects when adult females are most likely to be directly impacted by and experience reproductive coercion. Reproductive coercion may also occur among transgender and gender diverse people. While non-cis-gendered female search terms were not included, the term female was not considered exclusively as meaning cis-gendered female and as such did not exclude adult female identifying populations. To this end, females were not considered exclusively as cis-gender, and reproductive age sometimes included females younger than 18 years alongside those aged 18 years and over.Results from the searches were uploaded into Covidence. All publications were screened for eligibility. Abstracts were reviewed against the eligibility criteria by any two of the researchers. In cases of a disagreement, a third researcher reviewed the abstract which was then discussed to reach a consensus. Publications which met the eligibility criteria based on abstract screening were then moved to full-text screening. As per abstract screening, the full-text of the publications were reviewed against the eligibility criteria by any two members of the research team, with a third reviewing the full-text when there was a conflict. The three researchers then discussed the publication to reach a consensus regarding its inclusion or exclusion. The main reason for conflict was whether key findings related to defining reproductive coercion.Data extraction was undertaken for each article including author(s), year of publication, publication type, aims/objectives, study design, sampling method(s) or eligibility criteria, data collection methods, study population (sample size and characteristics), key findings related to the research questions, conclusion/recommendations, and limitations. An abbreviated data extraction chart is provided in supplementary Table Data were thematically analysed to identify key ideas, concepts, and factors relevant to the research questions. These were then reviewed to identify any similarities and were merged to develop themes. While data extraction provided a way to draw out data, thematic analysis provided a more nuanced and contextual understanding of the ways in which reproductive coercion is defined and we believe, more informative insights than a profile of the included studies as a data chart , 28. ThrThe search strategy produced 239 articles once duplicates were removed. Of the 239 articles, title and abstract screening against eligibility criteria excluded 192 articles. At full-text screening, 47 articles were assessed against eligibility criteria and 23 were excluded. Of the 23 excluded articles, 22 had no key findings related to defining reproductive coercion, and one article was an excluded source type (conference abstract). A total of 24 articles were included in the scoping review. Figure All 24 articles included study populations that focused on the experiences of females. Ten studies included females aged between 15 and 49 years –40, threTwenty-one journal articles, two policy submissions , 46, andSixteen studies specified their data collection methods. Eight conducted interviews including semi-structured , 44, 48,Three studies specified their sample eligibility criteria , 51, 53.At the most basic level reproductive coercion can be understood as anything that may impact reproductive choices and autonomy. Most research defines reproductive coercion as ‘behaviour’ that occurs at the interpersonal level  ‘interfering’ with women’s reproductive ‘autonomy’ through acts such as contraceptive ‘sabotage’, ‘coercion’ to get pregnant, or ‘controlling’ the outcome of a pregnancy. Two articles , 47 specThematic analysis identified four main themes which describe the ways in which reproductive coercions is defined: Individual external exertion of control over a woman’s reproductive autonomy; Systems and structures; Social and cultural determinants; Freedom from external forces to achieve reproductive autonomy. The relation between these themes is depicted in Fig. Individual external exertion of control over a woman’s reproductive autonomy captured the interpersonal level of reproductive coercion. Most definitions of reproductive coercion (16 articles) were positioned at the interpersonal level [The theme al level , 51, 53.Within this frame, reproductive coercion is contextualised as a form of intimate partner violence, usually perpetrated by a man against a woman, with reproductive coercion just ‘one type of coercive behaviour used by some abusers’ (p248). AThe definitions that focus on the interpersonal level use words such as ‘control’ , 39, 48,These definitions suggest the goal of reproductive coercion is to ‘purposefully’  ‘interfeSystems and structures describes organisational, institutional, structural and systems level forms of reproductive coercion and relates to Freedom from external forces to achieve reproductive autonomy. Ten of the articles in this review [Individual external exertion of control over a woman’s reproductive autonomy) to acknowledge that reproductive coercion can also be perpetrated by ‘organised criminal activity’ [Systems and structures can also be ‘protective factors’ which contribute to obtaining reproductive autonomy free from coercion [The theme s review , 52, 53 ctivity’ , healthcctivity’ , 41, andctivity’ , 49, 52.ctivity’ . These ictivity’ , are desctivity’ . In doinctivity’ (p5). Concoercion .These understandings of reproductive coercion highlight that it is ‘bidirectional’, a ‘spectrum’ or continuum from ‘subtle to overt’, not a dichotomy, and that reproductive coercion is a ‘structural phenomenon rather than simply an interpersonal one’ (p8). ‘PoWords such as ‘power’ to ‘establish’ and ‘maintain’ ‘control’ of women’s reproduction are used in these definitions, particularly in reference to already marginalised and othered populations  highlighting the intersections of ‘multiple layers of structural oppression’ (p2). WorThese explanations of reproductive coercion draw attention to the inequities experienced by marginalised and othered populations which are exacerbated by ‘anti-choice reproductive health policies’, and can further reduce access to reproductive health services (p36). FoWithin healthcare institutions which are governed by not only their own policies but also the policies, laws, and legislation of the state, reproductive coercion is reproduced and enacted due to their coercive effect. This is also apparent in healthcare provider actions which reflect the coercive policies of the institution and state. For example, ‘downward coercion’ whereby ‘accessible and affordable contraception’ required for reproductive autonomy is not available and simultaneously ‘upward coercion’ (p3) whenThese descriptions of reproductive coercion suggest the goal is to give more attention to the ‘structural’ in addition to the ‘individual’ form of coercion, recognising that reproductive coercion is a continuum. Further, to better understand the ‘everyday ways that women’ (p3) see Social and cultural determinants which highlights the social and cultural conditions and contexts, including concepts of gender and cultural norms, and discrimination primarily driven by dominant ideologies and discourses including racism and patriarchy. Here it becomes apparent that denial of reproductive autonomy through coercive practices is shaped by ‘misogyny’, ‘white supremacy’ and ‘neoliberalism’ [Systems and structures, this theme necessitates the need for an intersectional lens in definitions of reproductive coercion. This theme demonstrates how reproductive coercion which manifests at the interpersonal level  is shaped by the ideologies underpinning and constituting reproductive coercion, and the social and cultural norms which act as ‘contributing factors’ to limit women’s autonomy [Nine articles −49, 52 cautonomy .Social and cultural understandings and practices ‘interfere with some of the most fundamental aspects of a woman’s reproductive autonomy and bodily integrity’ (p3) and Social and cultural norms and values regarding women’s role in society, ‘pregnancy, motherhood, and reproduction’ (p1403) nThe conditions in which a woman lives and experiences her everyday world provides the context in which reproductive decisions are made. While not necessarily direct coercive practices, these underlying conditions create indirect coercive control of a woman’s reproductive autonomy. For example, lack of access due to either resources  or structures, transport and healthcare institutions / providers, education, housing, social exclusion, or residency or citizenship status which may afford full or restricted rights and access to women , 33, shaFreedom from external forces to achieve reproductive autonomy, captured the broader reproductive rights, justice, and autonomy lens identified in four of the included articles [The final theme, articles , 43, 52,articles (p157) anIndividual external exertion of control over a woman’s reproductive autonomy, to more positive and empowering language. Further, these definitions tend to be underpinned by human rights, which include reproductive rights, for example, the right to free choice to have a child or not, and the right to have a child ‘in a safe and healthy environment which is free from individual and state violence’ [These explanations of reproductive coercion use words such as ‘freedoms’, ‘human rights’, ‘rights-based approach’, ‘gender-sensitive’, and ‘reproductive autonomy’, shifting away from the negative language of the definitions described in the theme iolence’ (p2), andiolence’ (p12). ThThese descriptions of reproductive coercion suggest the goal is to be inclusive of the intersectionalities of women’s lives and their position in society to demonstrate that choice is constrained, and that there is ‘a duty to respect and protect individual choice in matters relating to reproduction’ which ‘requires the state to take steps to render the choice meaningful for realizing reproductive health’ for example, through the ‘provision of means to exercise choice’ (p129).The findings of this scoping review reveal most definitions of reproductive coercion are situated within the interpersonal level, ignoring the interconnected structural, social, and cultural factors that create and perpetuate women’s reproductive coercion. This has led to a narrow definition of reproductive coercion, which does not take account of the individual within their broader setting. Defining reproductive coercion at the interpersonal level has been an important starting point in getting this public health issue recognised and on the broader health priority agenda. However, we now need to move beyond this conceptualisation to take account of the inter-related multi-level factors. Some articles included in this scoping review contend that the broader conditions beyond the interpersonal level contribute to reproductive coercion but do not constitute reproductive coercion, and thus should not be considered as part of the definition of the concept , 47. HowSocial and cultural determinants shape the constitution and practices of organisations, institutions and systems discussed in theme Systems and structures. These institutions and systems in-turn create the operational conditions including policies and services in which reproductive rights, justice and autonomy are enabled and enacted (or not). Through explanations of reproductive coercion, using a reproductive justice lens, ‘intersectionality and the language of human rights to address the power asymmetries that arise due to citizenship, gender, race, caste, ethnicity, class, abilities’ [The analysis revealed the themes operate in an inter-related way rather than being independent, emphasising and reifying the influence of the structural and systemic contexts of reproductive coercion. For instance, dominant societal ideologies and discourses discussed in theme ilities’ (p4) are Systems and structures in regards to ‘incentivizing’, commercial reproductive coercion was not explicit in any of the identified research included in this review. Commercial reproductive coercion may be perpetrated by healthcare providers whereby women feel coerced into agreeing to additional or more expensive treatments through fear or pressure. For example, women/couples seeking in vitro fertilisation (IVF) to conceive may feel pressured to sign up for additional treatments in fear of not conceiving if they do not. These IVF add-ons are procedures or medicines claiming to increase the chance of a pregnancy, mostly in countries where IVF is delivered in a commercial setting, including the USA and Australia [Missing from the research identified in this scoping review is reproductive coercion by commercial entities or interests. While this was implicitly discussed in ustralia . While wustralia .As with all scoping reviews, this review had its limitations. While scoping reviews do not formally appraise the quality of the evidence in the studies reviewed , the artThough a comprehensive and systematic search strategy was designed and undertaken by the research team, studies of relevance to the aim of this review may have been missed. As a result, the findings are a reflection of the analysis of the articles included in this review. However, the strengths of this review were the systematic approach taken and the use of multiple reviewers in the screening of title and abstracts and full-texts to ensure strict application of the eligibility criteria. Further, the review reported on a range of study types. Finally, this scoping review has provided important insights into the current state of how reproductive coercion is defined.The aim of this scoping review was to explore how reproductive coercion is defined, how it is defined at the social-cultural-systems-structural levels, and if the definitions of reproductive coercion are inclusive of the conditions and contexts in which reproductive coercion occurs. The identification of four interacting themes on current definitions of reproductive coercion suggests four key findings: (1) reproductive coercion is experienced in multiple ways within and across a continuum from the interpersonal to the systems, structural and societal; (2) is exercised at multiple interacting levels in an effort to diminish reproductive decision-making freedoms and choices by controlling or limiting access to reproductive options through policy, law, legislation, behaviours, attitudes and so forth; (3) is socially and culturally embedded systematic control and oppression of females reproductive rights enacted across multiple levels to hold power over female’s reproductive autonomy creating, maintaining, and reinforcing gender inequality and other intersectional forms of oppression ; and (4) manifests at the interpersonal level as an artifact of intersecting social, cultural, institutional systems and structures, and organisations and the state which set the necessary context within which reproductive coercion occurs.We argue for a more inclusive definition of reproductive coercion that recognises the gendered nature and female’s multiple oppression and inequality. Further, reproductive coercion is, and can be, perpetrated and/or facilitated by the state and that it is produced and enacted at the societal, institutional, systems, and structural levels. We propose the following definition of reproductive coercion to capture the multiple interacting layers within which reproductive coercion occurs:Reproductive coercion is the act of removing or limiting reproductive autonomy to control reproductive decision-making freedoms and choices to hold power over reproductive autonomy. It occurs through socially and culturally embedded systematic control and oppression of reproductive rights, beliefs, conceptualisations of gender roles, behaviours, attitudes, and actions, practices, policy, law, and legislation resulting in gender inequality and other intersecting forms of oppression . This manifests and is experienced at the interpersonal level in multiple ways as an artifact of interconnected and interacting forces across the social, cultural, institutional systems and structures, organisations, and the state which create the context within which reproductive coercion occurs.This definition is explicitly inclusive of the broader conditions and context in which reproductive coercion manifests which is required to effectively implement settings based, whole-of-population, primary prevention programs using an intersectional approach to reduce reproductive coercion and the subsequent negative health outcomes. Further research is required to explore how the ways in which reproductive coercion is defined impacts public health primary prevention approaches and outcomes, including if adopting a broader more inclusive definition of reproductive coercion would facilitate improved reproductive autonomy. This scoping review and the resulting definition have provided the important first step in situating reproductive coercion beyond the interpersonal level to allow for the primary prevention of this important public health issue.Below is the link to the electronic supplementary material.Supplementary Material 1


Reproductive function is critical for species survival; however, it is energetically costly and physically demanding. Reproductive suppression is therefore a physiologically appropriate adaptation to certain ecological, environmental, and/or temporal conditions. This ‘allostatic’ suppression of fertility enables individuals to accommodate unfavorable reproductive circumstances and safeguard survival. The mechanisms underpinning this reproductive suppression are complex, yet culminate with the reduced secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, which in turn suppresses gonadotropin release from the pituitary, thereby impairing gonadal function. The focus of this review will be on the role of RFamide-related peptide (RFRP) neurons in different examples of allostatic reproductive suppression. RFRP neurons release the RFRP-3 peptide, which negatively regulates GnRH neurons and thus appears to act as a ‘brake’ on the neuroendocrine reproductive axis. In a multitude of predictable  and unpredictable  situations in which GnRH secretion is suppressed, the RFRP neurons have been suggested to act as modulators. This review examines evidence for and against these roles. Reproductive function is fundamental for species survival. However, it is physiologically costly—especially in mammals—due to the high energetic demands of pregnancy, lactation and rearing of offspring. When environmental or physiological conditions are not conducive for reproduction, neuroendocrine pathways that can exert allostatic control over reproductive function cause acute reproductive suppression. This ‘allostatic’ reproductive suppression is adaptive, as it enables individuals to accommodate unfavorable changes in their environment and/or in their physiology in order to safeguard survival. According to McEwen and Wingfield , allostaThere are many examples of allostatic reproductive suppression. For example, in seasonal breeders, reproductive suppression is predictable, such that fertility is coordinated to ensure offspring are born when environmental conditions are most favorable for survival. During different life cycle stages, such as pre-puberty and post-menopause, reproductive function is also predictably suppressed. However, unpredictable events can also cause reproductive suppression, such as certain environmental and/or physiological states. During times of severe metabolic stress, for example due to famine, energy demand exceeds energy supply, which will initiate an allostatic response that directs an individual away from energetically costly breeding and into survival mode. During times of psychosocial trauma or severe illness, which are also unfavorable times to reproduce, sufficient energy may be present, but the activation of the stress systems can induce a facultative physiological and/or behavioral response that ultimately defers reproduction ,2. AllosTo understand the mechanisms underpinning allostatic reproductive suppression, it is necessary to first understand how the reproductive axis—the hypothalamo-pituitary-gonadal (HPG) axis—is regulated. The hypothalamic gonadotropin-releasing hormone (GnRH) neurons governing the HPG axis serve as the final integrators of a multitude of neuroendocrine and hormonal inputs . GnRH redecrease in RFRP neuronal activity is the preovulatory GnRH/LH surge [Rfrp gene expression, RFRP cell numbers, and RFRP + cFos coexpression  all decline on the afternoon of the surge in naturally cycling or surge-induced hamsters and mice [RFamides form a class of neuropeptides characterized by an arginine–phenylalanine–amide motif at their C terminus. They were first identified in 1977 in molluscan cerebral ganglia, but almost another decade passed before they were purified and chemically characterized in any mammalian species. There are now five RFamide families known to exist in the mammalian central nervous system, where their widespread fiber distribution implies varied roles including regulation of energy balance, behavior, and reproduction . The RFRLH surge . Hypothaand mice ,26. Thisand mice ,4.As mentioned previously, RFRP neurons appear to be involved in both the temporal regulation of reproductive function, which causes predictable cycles of reproductive activation and inactivation, as well as in stress-related reproductive regulation, which allows for facultative reproductive suppression. RFRP neurons are thus emerging as important integrators of environmental and physiological cues within the neuroendocrine reproductive axis, such that they release RFRP-3 peptide to act as a ‘gate’ or ‘brake’ on reproductive function when the afferent inputs they receive suggest conditions are not optimal see . HoweverMany studies have explored the impact(s) of exogenous central RFRP-3 administration on reproductive function. In contrast to the variable results obtained from peripheral administration, these studies mostly support the concept that RFRP-3 acts as a brake on GnRH-induced LH release and reproductive drive. For example, intracerebroventicular (ICV) injection of RFRP-3 to male rats significantly suppressed all facets of male sexual behavior and also reduced plasma levels of LH . ICV RFRRfrp gene silencing using targeted shRNA alleviated various stress-induced infertility measures in female rats [Rfrp, thereby inhibiting its effect, show elevated gonadotropin levels [To further investigate the in vivo role of RFRP-3 on the HPG axis, our lab generated a novel RFRP-Cre mouse line, which we then crossed with Cre-dependent designer receptor (DREADD)- or diphtheria toxin receptor-expressing mice to investigate the effects of endogenous RFRP-3 excitation and inhibition, as well as RFRP neuronal ablation . Chronicale rats , suggestale rats  . WhiSeasonal breeding is crucial to the survival and reproductive success of the vast majority of vertebrate species inhabiting temperate regions. Seasonal changes in environmental factors, such as photoperiod, lead to alternating periods of photosensitivity and photorefractoriness in small birds and short-lived mammals to bring about alternating periods of reproductive activity and quiescence, the latter being maintained by an increased potency of negative feedback regulation. In larger long-lived animals, the environmental cues interact with the underlying endogenous rhythm, producing the same reproductive outcome. The timing of reproductive activity, which precedes the birth period by the duration of gestation, ensures that the young are born during a season in which the environmental conditions most favor neonatal survival. Photoperiodic cues can act directly on photoreceptors on the GnRH neuronal network in birds, whereas in mammals they are encoded and communicated to reproductive (and other) brain control centers in the form of elevated secretion of the pineal hormone melatonin during the hours of darkness, which are extended in duration during winter and shorter in summer in temperate regions ,50.It would be reasonable to hypothesize that RFRP neuronal activity would be higher during reproductive quiescence than during the breeding season. Consistent with this hypothesis, more RFRP-3 immunoreactive cell bodies were observed during the non-breeding season  than the breeding season in male macaques  . SimilarRfrp mRNA and immunoreactive RFRP neurons compared to virgin diestrus controls, and this effect was attributable to the presence of elevated circulating prolactin concentrations in the lactating animals . In another study that employed a different experimental design (lactating rats with pups removed were used as controls), the suckling stimulus exerted the opposite effect: it increased Rfrp mRNA levels, and the authors suggested this might be a cause of lactational infertility [Rfrp expression.There are currently very few studies in which the role of RFRP-3 in lactational infertility has been tested. Conceptually, the output of RFRP neurons might increase to cause lactational amenorrhea since they can inhibit reproductive function. Alternatively, it might decrease since lactation is associated with stress hyporesponsiveness  and RFRPertility . ClearlyStress-related reproductive dysfunction is perhaps the most notable form of allostatic reproductive suppression. The reproductive impairments can be subtle, such as reduced hormone secretion, or severe, such as the complete suppression of puberty onset and/or fertility . While mRfrp gene expression in vitro [Rfrp gene expression in male and female rats [Rfrp gene silencing using a targeted shRNA prevented stress-induced infertility in female rats [Importantly, approximately half of RFRP neurons express GR , and corin vitro . Physiolale rats ,67, but ale rats . Data frale rats . Similarale rats . A singlale rats , indicatRfrp mRNA levels [Metabolic challenges, such as starvation, inhibit fertility and sexual behaviors. Many of the neurons that provide important input to the reproductive axis are also involved in control of metabolic functions, and this allows the availability of metabolic fuels to be coordinated with fertility . For exaA levels  or no deA levels  relativeA levels . TogetheIn Syrian hamsters, the percent of RFRP cells co-expressing c-Fos  was significantly increased at 8 and 12 days after food restriction . The effRfrp and Npffr1 mRNA levels, and these were negatively correlated with Gnrh mRNA and serum LH levels [Rfrp mRNA despite causing a reduction in serum LH levels [Immune stress, such as infection and inflammation, can also have a negative impact on fertility ,78,79. EH levels . In anotH levels . HoweverH levels , suggestH levels ,67,69, bRfrp mRNA, which correlates with a downregulation of serum LH in male rats [Rfrp mRNA, since it was prevented by adrenalectomy [Rfrp expression associated with suppressed sexual maturation in non-dominant naked mole rats, RFRP-3 administration was sufficient to sustain their suppressed sexual maturation, even when the individuals were removed from the dominance cues [As with metabolic and immune stress, which are physiological stressors, psychosocial stress  can likewise cause allostatic reproductive suppression. However, unlike metabolic and immune stress, which directly threaten an individual’s survival, psychosocial stress is purely a perceived threat. In humans, depression and other mood disorders are examples of psychosocial stress associated with reproductive dysfunction . One comale rats . GC relealectomy . We havealectomy . Furtheralectomy ,31,83,84alectomy  and othealectomy . Not onlnce cues . CollectThe mechanisms underpinning adaptive reproductive suppression are complex and involve a multitude of factors. The RFRP-3 neuropeptide system appears to play a modulatory role in integrating many of these factors and acting as a ‘brake’ on reproductive drive when conditions are unfavorable. RFRP neurons appear to contribute to stress-induced and (in some species) seasonal reproductive suppression, while their role in mediating changes in reproductive competency during different life cycle stages  is less clear and would benefit from functional data from manipulating RFRP neuronal activity see . With th


BRCA1/2-mutant (BRCAMUT) TNBC, durable responses will likely be achieved if PARPi are used in combination. It is plausible that sequential administration of a potent PARPi like talazoparib in combination with carboplatin can enhance primary tumour and metastasis inhibition in BRCAMUT and BRCA1/2 wild-type (BRCAWT) TNBCs, and decrease toxicity.Patients with triple-negative breast cancer (TNBC) develop early recurrence. While PARP inhibitors (PARPi) have demonstrated potential in We evaluated the impact of the concurrent combination of talazoparib and carboplatin on cell survival in 13 TNBC cell lines. We compared the concurrent and sequential combination upon fork replication, migration and invasion. We also used three orthotopic xenograft models to evaluate primary tumour growth, distant metastasis, and toxicity.BRCA1/2-mutation status. The sequential combination decreased fork speed in normal cells, but not in TNBC cells. The talazoparib-first sequential combination resulted in a strong reduction in migration , invasion , lung micrometastasis , and less toxicity in a BRCAWT model.Concurrent talazoparib and carboplatin was synergistic in 92.3% of TNBC cell lines, independent of The sequential combination of talazoparib and carboplatin is an effective approach to inhibit micrometastatic disease, providing rationale for the use of this combination in early TNBC patients. BRCA1/2 (gBRCAMUT). However, patients with gBRCAMUT constitute about 11–20% of all TNBCs   and Mouse anti-BrdU [clone B44] antibodies , overnight at 4 °C. Slides were incubated with anti-Rat AlexaFluor 568  and anti-Mouse AlexaFluor 488  antibodies, for 1 h at room temperature. Slides were mounted in ProLong™ Gold antifade Mountant  and analysed using a Zeiss Axio Observer Z1 microscope with ×63 oil objective. Replication fork speed was calculated using the CldU + IdU track length/60 min * 2.59 kb/µm.Cells were sequentially labelled with 25 µM of 5-Chloro-2′-deoxyuridine (CldU)  and then 250 µM of 5-Iodo-2’-deoxyuridine (IdU)  for 30 min each . Cells w3, mice were randomised into treatment groups based on tumour volume volumes and weight. Our sample size was based on the experiments previously described [All animal experiments were approved by the Institutional Animal Protection Committee (CIPA) of the Centre de Recherche de Centre hospitalier de l’Université de Montréal (CRCHUM) under protocol C17017SHs. Either 5 million MDAMB231 or 2 million MX1/HCC1806 cells were resuspended in 50% Matrigel Matrix Phenol Red Free , 25% PBS and 25% collagen type 1  solution . Using 74 or 5 × 104 cells were suspended in serum-free media in 8-μm transwell inserts , for 24-h migration or 48-h invasion assays, respectively. For the invasion assay, the upper chamber was pre-coated with 1/9 dilution of Matrigel . After incubation, inserts were washed with PBS. Migrated or invaded cells on the underside of the membrane were fixed with 4% paraformaldehyde and stained with hematoxylin . Migrated or invaded cells were counted from six representative fields from the inverted EVOS XL core microscope (Thermo Fisher Scientific).In all, 3 × 10In total, 200 μg of protein was extracted from cell lysates from MDAMB231 cells to detect the expression of 31 chemokines using the human chemokine array kit . Images of membranes were acquired with ChemiDoc (BioRad) and dots were quantified using QuickSpots (Western vision) software. At the same time point, supernatants of MDAMB231 and HCC1395 cells were collected to detect MCP-1 levels with the human CCL2/MCP-1 Immunoassay .See Supplementary Methods for details regarding RNA-seq analysis.P < 0.05 is considered statistically significant.Normality of each dataset was first verified before performing Kruskal–Wallis ANOVA or one-way ordinal ANOVA with multiple-comparison post test. Two-way ANOVAs with post-tests were performed for grouped conditions. Data are represented as mean + /− SEM, except for fork speed, in which median values with interquartile ranges are presented. Data were analysed and plotted using GraphPad Prism software 8. Further information on research design is available in the 50 values of talazoparib and carboplatin as single agents in a panel of 13 TNBC cell lines  values <1 in 92.3% (12/13) of cell lines  were calculated to determine the dose-fold reduction of each therapeutic agent to achieve synergy. DRI for talazoparib ranged from 1.5 to 3.8, while the DRI for carboplatin ranged from 2.1 to 7.1. This is suggestive that synergy can be accomplished with important dose reductions of both drugs, with greater reductions in carboplatin versus talazoparib commonly observed in 61.5% (8/13) of cell lines.We determined the IC μM Fig. . Using tnes Fig. . Stronge+ )  of talazoparib (T1-9), carboplatin (C1-9), or the concomitant combination of talazoparib and carboplatin (TC1-9) upon 53BP1 response and apoptosis using a 10-day chemosensitivity assay in 14 TNBC cell lines Fig. . DNA dam+ ) Fig. . While cWT cells, including two PARPi-resistant cell lines, HCC1143 and MDAMB231, a PARPi-sensitive yet carboplatin-resistant cell line, HCC1806; and one PARPi-sensitive cell line that is both BRCA1-deleted and BRCA2-mutated, MX1. In all four cell lines, cell survival was comparable between the concomitant and sequential combination approaches, with either talazoparib or olaparib as the PARPi backbone  and two sequential approaches: carboplatin first followed by talazoparib (seq.C→T), and talazoparib run-in approach followed by carboplatin (seq.T→C) upon cell survival using the 10-day chemosensitivity assay Fig. . We seleone Fig. .Fig. 3DiTo better understand the impact of the combination approaches on in vitro toxicity, we compared fork replication speed and DNA damage response between TNBC and normal cells. We used two TNBC cell lines, HCC1806 and MDAMB231, and two human normal cell lines including MCF10A  and BM-MSC  Fig. . We deteP < 0.0001), 48.9% (P < 0.0001), 59.2% (P < 0.0001), and 59.9% (P < 0.0001) in MDAMB231, HCC1806, MCF10A and BM-MSC cells, respectively  and 14.8% (P = 0.0005) reduction in median fork speed in comparison to the concomitant combination, in MCF10A and BM-MSC cells, respectively. Conc.T + C resulted in the highest proportion of γ-H2AX+ cells in MCF10A  and BM-MSC  in comparison to control , in comparison to conc.T + C. We also evaluated apoptosis in MCF10A and BM-MSC cells , and 31.7% (P < 0.0001) for MCF10A and BM-MSC, respectively.However, in normal cells, sequential treatments decreased fork speed in comparison to concomitant treatments. Seq.T→C demonstrated a 10.3% , 75.5% (P = 0.0001), 71.7% (P = 0.002), by the conc.T + C., seq.C→T, and seq.T→C strategies, respectively, in comparison to control  with conc. T + C, 46.6% (P = 0.04) with seq.C→T, and 62.0% with seq.T→C (P < 0.0001) in comparison to control , and 66.7% (P < 0.0001) reductions in tumour volume, respectively, which was more remarkable than the 51.4% reduction (P = 0.08) with conc.T + C, in comparison to control , while seq.C→T demonstrated the greatest gain in weight at 10.4%. In the HCC1806 cohort  Fig. .P = 0.002), 1.5 (P = 0.0003), and 1.7 (P = 0.0098), for conc.T + C, seq.C→T, and seq.T→C respectively, in comparison to control (mean 4.9) , seq.C→T  and seq.T→C , in comparison to control (mean 4.3)  Fig. . Similar.3) Fig. . No stat.3) Fig. .No changes in haemoglobin (Hgb) levels were observed with treatment in the MX1 cohort Fig. . While tP = 0.003) and seq.T→C led to a 56.4% (P < 0.0001) reduction in lung micrometastases, which was not observed with the concomitant combination  , and 36.9% in comparison to the concomitant approach (P < 0.05). Similar trends were also observed with liver micrometastasis  reduction in micrometastasis, which was similar to the 76.3% (P = 0.02) reduction with the seq. T→C combination.To further evaluate the impact of the combination of talazoparib and carboplatin on distant metastasis, we used an orthotopic xenograft model of MDAMB231, which is known for its high metastatic efficiency . In compNS) Fig. . The seqsis Fig. . In compP = 0.05) and 30.8% (P = 0.01) reduction in cell migration, respectively. Once again, the seq.T→C demonstrated the most striking inhibition in cell migration  reduction in comparison to control, 61.1% reduction (P < 0.0001) in comparison to talazoparib, and 39.0% reduction (P = 0.008) in comparison to conc.T + C. The other combination strategies also inhibited migration with 51.5% (P < 0.0001) reduction for the concomitant approach, and 38.9% (P < 0.0001) for the seq.C→T approach. The treatments as single agents or in combination demonstrated a similar trend with HS578T  reduction in cell migration.To further dissect the metastatic process, we evaluated the impact of talazoparib as monotherapy and in combination on cell migration and invasion in vitro Fig. . In MDAMion Fig. , with 70P < 0.0001), 47.0% (P < 0.0001), 47.5% (P < 0.0001), and 41.3% (P = 0.0005) reduction in comparison to control, talazoparib, carboplatin, seq.C→T, and conc.T + C, respectively  inhibition in cell invasion in comparison to control. In HS578T, the greatest inhibition was also demonstrated by seq.T→C with a 26.7% (P < 0.0001), 18.9% (P = 0.0002), and 21.3% (P < 0.0001) reduction in cell invasion in comparison to control, talazoparib, and carboplatin, respectively  reduction in cell invasion, in comparison to control. Similar results were obtained with other PARPi. Olaparib or niraparib resulted in the greatest reduction in migration or invasion when administered witih the sequential PARPi-first combination approach , 30.7% reduction in comparison to carboplatin (P < 0.0001), and a 11.8% reduction in comparison to conc.T + C (P = 0.03). In HCC1395, seq.T→C also reduced MCP-1 levels by 31.5% in comparison to control (P < 0.0001), and by 33.9% in comparison to carboplatin (P < 0.0001). Conc.T + C also reduced MCP-1 levels by 19.5% (P = 0.049), and seq.C→T reduced MCP-1 levels by 21.7% (P = 0.01). Since the greatest downregulation of MCP-1 levels was demonstration by seq. T→C in comparison to carboplatin, this is suggestive that the sequential combination of talazoparib and carboplatin may be an important approach to inhibit MCP-1 levels that may otherwise be upregulated with carboplatin.CCL2/MCP (Monocyte chemoattractant protein)-1 drives migration and invasion, and is implicated in cancer cell homing at the metastatic site , 39. PARTo better understand the underlying mechanisms for enhanced inhibition of metastasis, we also performed gene expression analysis of the metastatic lung tissue of the MDAMB231 xenograft to determine if there are differentially expressed pathways in the human cancer tissue between the treatment groups (Supplementary Figs. Altogether, the sequential administration of talazoparib and carboplatin resulted in a noteworthy reduction in distant metastasis, cell migration and invasion. Our results are suggestive that the PARPi-first combination may have a distinct mechanism of action in comparison to other combination approaches, which can significantly enhance the efficacy of each therapeutic agent.MUT models [MUT and BRCAWT subtypes. Talazoparib and carboplatin demonstrated a mean dose-reduction index of 2.8- and 3.7-fold, respectively. Similarly, sustained DNA damage and apoptosis responses were observed at lower drug concentrations of the combination, particularly in the PARPi-resistant cell lines. These results could partly explain the toxicity observed with the concomitant combination of talazoparib and carboplatin in a phase I trial, in which only a 0.25-fold reduction in talazoparib and carboplatin doses were utilised [Preclinical studies have commonly focused on the combination of PARPi and platinum-based therapy using BRCAT models , 44. To utilised .MUT model, MX1, the least weight gain and haematologic toxicity was demonstrated by the talazoparib-first approach in the BRCAWT model, MDAMB231. Since the same background strain was used, one plausible explanation for the differential toxicity could be different inflammatory responses induced by the BRCAMUT versus BRCAWT tumours [MUT context and talazoparib-first approach in the BRCAWT context may offer patients the greatest safety profiles.Sequential talazoparib and carboplatin was associated with a reduction in fork speed, DNA damage, and cell death in normal cells with, in comparison to TNBC cells. This is similar to what was previously reported: higher endogenous fork speeds correlated with lower endogenous replication stress in normal cells, which explained the reduced toxicity observed with the sequential PARPi combination approaches . In our  tumours , 46, whiFor our in vivo experiments, we based our dosing schedule of one dose of carboplatin and nine days of talazoparib on a xenograft model of subcutaneous tumour fragment implantation . Our resWT model with high metastatic efficiency. We showed comparable efficacy of the concurrent and sequential approaches for primary tumour inhibition. However, to our knowledge, we are the first to report that the sequential combination approaches strongly inhibit migration, invasion, and distant metastasis. The talazoparib-first combination demonstrated the greatest reduction in cell migration and invasion at 70.4% and 56.9%, respectively, with an enhanced effect in comparison to concurrent combination. In vivo, the talazoparib-first approach resulted in a 56.4% and 76.3% inhibition in lung and liver metastasis, respectively. Since we did not identify a differential impact on the tumour volumes with the sequential or concomitant combination approach, it is plausible that the impact upon migration and metastasis is independent of treatment effects on the primary tumour.While we understand some of the limitations of cells lines and their derived models , the strFurthermore, the talazoparib-first combination was associated with decreased expression of seven chemokines. The expression of one such chemokine, MCP-1, was downregulated the greatest with the talazoparib-first combination. Interestingly, chemotherapy including cisplatin has been shown to promote secretion of MCP-1, recruit inflammatory monocytes enriched with the receptor of MCP-1, CCR2, forming pre-metastatic niches, thus promoting distant metastasis in breast and lung cancer , 51. SucWT model.Finally, we evaluated the pathways that were differentially expressed by the different treatment groups using metastatic lung tissue. We found that DNA damage response pathways were distinctly downregulated with seq.T→C in comparison to control and other combination groups. We and others have previously shown that the downregulation of these pathways allow for enhanced sensitivity to PARPi , 52, sugTaken together, we have provided a comprehensive preclinical analysis of the combination of talazoparib and carboplatin in TNBC. We have demonstrated that the combination is synergistic in most TNBC cell lines. While the sequence of administration does not impact tumour proliferation or tumour growth, sequential administration of talazoparib and carboplatin can decrease toxicity and significantly inhibit migration, invasion, and distant metastasis. Therefore, our results lead the way to future clinical trials with the evaluation of the sequential combination of talazoparib and carboplatin in early breast cancer, potentially providing an effective approach to eradicate micrometastatic disease in TNBC patients.Supplementary MethodsSupplementary FiguresSupplementary Table 1ARRIVE ChecklistReporting Summary