Structural and biophysical analysis of cytochrome P450 2C9*14 and *27 variants in complex with losartan.

The human cytochrome P450 (CYP) 1, 2 and 3 families of enzymes are responsible for the biotransformation of a majority of the currently available pharmaceutical drugs. The highly polymorphic CYP2C9 predominantly metabolizes many drugs including anticoagulant S-warfarin, anti-hypertensive losartan, anti-diabetic tolbutamide, analgesic ibuprofen, etc. There are >80 single nucleotide changes identified in CYP2C9, many of which significantly alter the clearance of important drugs. Here we report the structural and biophysical analysis of two polymorphic variants, CYP2C9*14 (Arg125His) and CYP2C9*27 (Arg150Leu) complexed with losartan. The X-ray crystal structures of the CYP2C9*14 and *27 illustrate the binding of two losartan molecules, one in the active site near heme and another on the periphery. Both losartan molecules are bound in an identical conformation to that observed in the previously solved CYP2C9 wild-type complex, however, the number of losartan differs from the wild-type structure, which showed binding of three molecules. Additionally, isothermal titration calorimetry experiments reveal a lower binding affinity of losartan with *14 and *27 variants when compared to the wild-type. Overall, the results provide new insights into the effects of these genetic polymorphisms and suggests a possible mechanism contributing to reduced metabolic activity in patients carrying these alleles.

Impact of Combinatorial Histone Modifications on Acetyllysine Recognition by the ATAD2 and ATAD2B Bromodomains.

The ATPase family AAA+ domain containing 2 (ATAD2) protein and its paralog ATAD2B have a C-terminal bromodomain (BRD) that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2/B BRDs to select acetylated lysine among multiple histone post-translational modifications. The ATAD2B BRD can bind acetylated histone ligands that also contain adjacent methylation or phosphorylation marks, while the presence of these modifications significantly weakened the acetyllysine binding activity of the ATAD2 BRD. Our structural studies provide mechanistic insights into how ATAD2/B BRD-binding pocket residues coordinate the acetyllysine group in the context of adjacent post-translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands impact the recognition of an adjacent acetyllysine residue. Our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2/B BRDs, resulting in distinct binding modes.

peaksat: an R package for ChIP-seq peak saturation analysis.

BACKGROUND: Epigenomic profiling assays such as ChIP-seq have been widely used to map the genome-wide enrichment profiles of chromatin-associated proteins and posttranslational histone modifications. Sequencing depth is a key parameter in experimental design and quality control. However, due to variable sequencing depth requirements across experimental conditions, it can be challenging to determine optimal sequencing depth, particularly for projects involving multiple targets or cell types. RESULTS: We developed the peaksat R package to provide target read depth estimates for epigenomic experiments based on the analysis of peak saturation curves. We applied peaksat to establish the distinctive read depth requirements for ChIP-seq studies of histone modifications in different cell lines. Using peaksat, we were able to estimate the target read depth required per library to obtain high-quality peak calls for downstream analysis. In addition, peaksat was applied to other sequence-enrichment methods including CUT&RUN and ATAC-seq. CONCLUSION: peaksat addresses a need for researchers to make informed decisions about whether their sequencing data has been generated to an adequate depth and subsequently sufficient meaningful peaks, and failing that, how many more reads would be required per library. peaksat is applicable to other sequence-based methods that include calling peaks in their analysis.

Structural insights into acetylated histone ligand recognition by the BDP1 bromodomain of Plasmodium falciparum.

Plasmodium falciparum requires a two-host system, moving between Anopheles mosquito and humans, to complete its life cycle. To overcome such dynamic growth conditions its histones undergo various post-translational modifications to regulate gene expression. The P. falciparum Bromodomain Protein 1 (PfBDP1) has been shown to interact with acetylated lysine modifications on histone H3 to regulate the expression of invasion-related genes. Here, we investigated the ability of the PfBDP1 bromodomain to interact with acetyllsyine modifications on additional core and variant histones. A crystal structure of the PfBDP1 bromodomain (PfBDP1-BRD) reveals it contains the conserved bromodomain fold, but our comparative analysis between the PfBDP1-BRD and human bromodomain families indicates it has a unique binding mechanism. Solution NMR spectroscopy and ITC binding assays carried out with acetylated histone ligands demonstrate that it preferentially recognizes tetra-acetylated histone H4, and we detected weaker interactions with multi-acetylated H2A.Z in addition to the previously reported interactions with acetylated histone H3. Our findings indicate PfBDP1 may play additional roles in the P. falciparum life cycle, and the distinctive features of its bromodomain binding pocket could be leveraged for the development of new therapeutic agents to help overcome the continuously evolving resistance of P. falciparum against currently available drugs.

Epigenetic-Mediated Regulation of Gene Expression for Biological Control and Cancer: Fidelity of Mechanisms Governing the Cell Cycle.

The cell cycle is governed by stringent epigenetic mechanisms that, in response to intrinsic and extrinsic regulatory cues, support fidelity of DNA replication and cell division. We will focus on (1) the complex and interdependent processes that are obligatory for control of proliferation and compromised in cancer, (2) epigenetic and topological domains that are associated with distinct phases of the cell cycle that may be altered in cancer initiation and progression, and (3) the requirement for mitotic bookmarking to maintain intranuclear localization of transcriptional regulatory machinery to reinforce cell identity throughout the cell cycle to prevent malignant transformation.

Epigenetic-Mediated Regulation of Gene Expression for Biological Control and Cancer: Cell and Tissue Structure, Function, and Phenotype.

Epigenetic gene regulatory mechanisms play a central role in the biological control of cell and tissue structure, function, and phenotype. Identification of epigenetic dysregulation in cancer provides mechanistic into tumor initiation and progression and may prove valuable for a variety of clinical applications. We present an overview of epigenetically driven mechanisms that are obligatory for physiological regulation and parameters of epigenetic control that are modified in tumor cells. The interrelationship between nuclear structure and function is not mutually exclusive but synergistic. We explore concepts influencing the maintenance of chromatin structures, including phase separation, recognition signals, factors that mediate enhancer-promoter looping, and insulation and how these are altered during the cell cycle and in cancer. Understanding how these processes are altered in cancer provides a potential for advancing capabilities for the diagnosis and identification of novel therapeutic targets.

Functional networks of the human bromodomain-containing proteins.

Background: Bromodomains are a structurally conserved epigenetic reader domain that bind to acetylated lysine residues in both histone and non-histone proteins. Bromodomain-containing proteins (BRD proteins) often function as scaffolding proteins in the assembly of multi-protein complexes to regulate diverse biological processes. BRD proteins have been classified based on biological and functional similarity, however the functions of many BRD proteins remains unknown. PPI network analysis is useful for revealing organizational roles, identifying functional clusters, and predicting function for BRD proteins. Results: We used available data to construct protein-protein interaction networks (PPINs) to study the properties of the human bromodomain protein family. The network properties of the BRD PPIN establishes that the BRD proteins serve as hub proteins that are enriched near the global center to form an inter-connected PPIN. We identified dense subgraphs formed by BRD proteins and find that different BRD proteins share topological similarity and functional associations. We explored the functional relationships through clustering and Hallmark pathway gene set enrichment analysis and identify potential biological roles for different BRD proteins. Conclusion: In our network analysis we confirmed that BRD proteins are conserved central nodes in the human PPI network and function as scaffolds to form distinctive functional clusters. Overall, this study provides detailed insight into the predictive functions of BRD proteins in the context of functional complexes and biological pathways.

Surgeon and Patient Reports of Fertility Preservation Referral and Uptake in a Prospective, Pan-Canadian Study of Young Women with Breast Cancer.

BACKGROUND: Prompt referral by their surgeon enables fertility preservation (FP) by young women with breast cancer (YWBC) without treatment delay. Following a FP knowledge intervention, we evaluated surgeon and patient reports of fertility discussion, FP referral offer and uptake, and FP choices and reasons for declining FP among patients enrolled in the Reducing Breast Cancer in Young Women, prospective pan-Canadian study. METHODS: Between September 2015 and December 2020, 1271 patients were enrolled at 31 sites. For each patient, surgeons were sent a questionnaire inquiring whether: (1) fertility discussion was initiated by the surgical team; (2) FP referral was offered; (3) referral was accepted; a reason was requested for any "no" response. Patients were surveyed about prediagnosis fertility plans and postdiagnosis oncofertility management. RESULTS: Surgeon questionnaires were completed for 1068 (84%) cases. Fertility was discussed with 828 (84%) and FP consultation offered to 461 (47%) of the 990 YWBC with invasive disease. Among the 906 responding YWBC, referral was offered to 220 (82%) of the 283 (33%) with invasive disease who stated that they had definitely/probably not completed childbearing prediagnosis. Of these, 133 (47%) underwent FP. The two most common reasons for not choosing FP were cost and unwillingness to delay treatment. CONCLUSIONS: Although the rates of surgeon fertility discussion and FP referral was higher than most reports, likely due to our previous intervention, further improvement is desirable. FP should be offered to all YWBC at diagnosis, regardless of perceived childbearing intent. Cost remains an important barrier to FP uptake.

Insights into the Genetic Variations of Human Cytochrome P450 2C9: Structural Analysis, Characterization and Comparison.

Cytochromes P450 (CYP) are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics. The CYP2C9 enzyme is responsible for the metabolism of a wide range of clinical drugs. More than sixty genetic variations have been identified in CYP2C9 with many demonstrating reduced activity compared to the wild-type (WT) enzyme. The CYP2C9*8 allele is predominantly found in persons of African ancestry and results in altered clearance of several drug substrates of CYP2C9. The X-ray crystal structure of CYP2C9*8, which represents an amino acid variation from arginine to histidine at position 150 (R150H), was solved in complex with losartan. The overall conformation of the CYP2C9*8-losartan complex was similar to the previously solved complex with wild type (WT) protein, but it differs in the occupancy of losartan. One molecule of losartan was bound in the active site and another on the surface in an identical orientation to that observed in the WT complex. However, unlike the WT structure, the losartan in the access channel was not observed in the *8 complex. Furthermore, isothermal titration calorimetry studies illustrated weaker binding of losartan to *8 compared to WT. Interestingly, the CYP2C9*8 interaction with losartan was not as weak as the CYP2C9*3 variant, which showed up to three-fold weaker average dissociation constant compared to the WT. Taken together, the structural and solution characterization yields insights into the similarities and differences of losartan binding to CYP2C9 variants and provides a useful framework for probing the role of amino acid substitution and substrate dependent activity.

Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain.

The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four α-helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up-regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the "RVF" shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.

Functional Roles of Bromodomain Proteins in Cancer.

Histone acetylation is generally associated with an open chromatin configuration that facilitates many cellular processes including gene transcription, DNA repair, and DNA replication. Aberrant levels of histone lysine acetylation are associated with the development of cancer. Bromodomains represent a family of structurally well-characterized effector domains that recognize acetylated lysines in chromatin. As part of their fundamental reader activity, bromodomain-containing proteins play versatile roles in epigenetic regulation, and additional functional modules are often present in the same protein, or through the assembly of larger enzymatic complexes. Dysregulated gene expression, chromosomal translocations, and/or mutations in bromodomain-containing proteins have been correlated with poor patient outcomes in cancer. Thus, bromodomains have emerged as a highly tractable class of epigenetic targets due to their well-defined structural domains, and the increasing ease of designing or screening for molecules that modulate the reading process. Recent developments in pharmacological agents that target specific bromodomains has helped to understand the diverse mechanisms that bromodomains play with their interaction partners in a variety of chromatin processes, and provide the promise of applying bromodomain inhibitors into the clinical field of cancer treatment. In this review, we explore the expression and protein interactome profiles of bromodomain-containing proteins and discuss them in terms of functional groups. Furthermore, we highlight our current understanding of the roles of bromodomain-containing proteins in cancer, as well as emerging strategies to specifically target bromodomains, including combination therapies using bromodomain inhibitors alongside traditional therapeutic approaches designed to re-program tumorigenesis and metastasis.

Human umbilical cord perivascular cells maintain regenerative traits following exposure to cyclophosphamide.

Chemotherapies can cause germ cell depletion and gonadal failure. When injected post-chemotherapy, mesenchymal stromal cells (MSCs) from various sources have been shown to have regenerative effects in rodent models of chemotherapy-induced gonadal injury. Here, we evaluated two properties of a novel source of MSC, first trimester (FTM) human umbilical cord perivascular cells (HUCPVCs) (with increased regenerative potential compared to older sources), that may render them a promising candidate for chemotherapeutic gonadal injury prevention. Firstly, their ability to resist the cytotoxic effects of cyclophosphamide (CTX) in vitro, as compared to term HUCPVCs and bone marrow cells (BMSCs); and secondly, whether they prevent gonadal dysfunction if delivered prior to gonadotoxic therapy in vivo. BMSC, FTM HUCPVC, term HUCPVC, and control NTERA2 cells were treated with moderate (150 µmol/L) and high (300 µmol/L) doses of CTX in vitro. Viability, proliferative capacity, mesenchymal cell lineage markers and differentiation capacity, immunogenicity, and paracrine gene expression were assessed. CTX was administered to Wistar rats 2 days following an intra-ovarian injection of FTM HUCPVC. HUCPVC survival and ovarian follicle numbers were assessed using histological methods. We conclude that FTM HUCPVC maintain key regenerative properties following chemotherapy exposure and that pre-treatment with these cells may prevent CTX-induced ovarian damage in vivo. Therefore, HUCPVCs are promising candidates for fertility preservation.

Human umbilical cord perivascular cells prevent chemotherapeutic drug-induced male infertility in a mouse model.

OBJECTIVE: To study whether intratesticular (IT) administration of 2 sources of human umbilical cord perivascular cells (HUCPVC), rich and potent sources of mesenchymal stromal cells (MSC), before chemotherapy can prevent infertility in a mouse model. DESIGN: Two control groups of CD1 male mice without busulfan (BUS) administration (untreated and IT media injection groups) were included. Experimental groups included IT administration of media, first trimester (FTM) HUCPVCs or term HUCPVCs (n = 5 each) injected 3 days before BUS treatment (20 mg/kg). All groups were included in a mating time course study over 6 months. SETTING: Preclinical study in a fertility center research laboratory. PATIENTS: Not applicable. INTERVENTION: IT delivery of FTM or term HUCPVC before BUS treatment. MAIN OUTCOME MEASURES: Pregnancies, litter sizes, and gross morphology of offspring were monitored. Caudal epididymal sperm concentration, motility, and progressive motility were assessed by computer-assisted sperm analysis. Spermatogenesis was also assessed histologically in testicular tissue sections. RESULTS: FTM and term HUCPVC displayed an MSC-associated immunophenotype and expressed transcripts encoding paracrine factors known to regulate the testicular cell niche. IT administration of FTM and term HUCPVC before chemotherapy promoted the recovery of spermatogenesis and fertility compared with BUS-treated animals that received a media injection. Although the total number of pups sired over 6 months by males treated with FTM or term HUCPVC was reduced compared with untreated or media-injected controls, litter size and sperm parameters in fertile animals did not differ between control and cell-treated groups. CONCLUSION: HUCPVC represent a promising source of MSC-based therapy to prevent gonadotoxic chemotherapeutic drug-induced infertility.

Structural Insights into the Recognition of Mono- and Diacetylated Histones by the ATAD2B Bromodomain.

Bromodomains exhibit preferences for specific patterns of post-translational modifications on core and variant histone proteins. We examined the ligand specificity of the ATAD2B bromodomain and compared it to its closely related paralogue in ATAD2. We show that the ATAD2B bromodomain recognizes mono- and diacetyllysine modifications on histones H4 and H2A. A structure-function approach was used to identify key residues in the acetyllysine-binding pocket that dictate the molecular recognition process, and we examined the binding of an ATAD2 bromodomain inhibitor by ATAD2B. Our analysis demonstrated that critical contacts required for bromodomain inhibitor coordination are conserved between the ATAD2/B bromodomains, with many residues playing a dual role in acetyllysine recognition. We further characterized an alternative splice variant of ATAD2B that results in a loss of function. Our results outline the structural and functional features of the ATAD2B bromodomain and identify a novel mechanism regulating the interaction of the ATAD2B protein with chromatin.

Structure of Cytochrome P450 2C9*2 in Complex with Losartan: Insights into the Effect of Genetic Polymorphism.

The human CYP2C9 plays a crucial role in the metabolic clearance of a wide range of clinical therapeutics. The *2 allele is a prevalent genetic variation in CYP2C9 that is found in various populations. A marked reduction of catalytic activity toward many important drug substrates has been demonstrated by CYP2C9*2, which represents an amino acid variation at position 144 from arginine to cysteine. The crystal structure of CYP2C9*2 in complex with an antihypertensive drug losartan was solved using X-ray crystallography at 3.1-Å resolution. The Arg144Cys variation in the *2 complex disrupts the hydrogen-bonding interactions that were observed between the side chain of arginine and neighboring residues in the losartan complex of CYP2C9 and the wild-type (WT) ligand-free structure. The conformation of several secondary structural elements is affected, thereby altering the binding and orientation of drug and important amino acid side chains in the distal active site cavity. The new structure revealed distinct interactions of losartan in the compact active site of CYP2C9*2 and differed in occupancy at the other binding sites previously identified in the WT-losartan complex. Furthermore, the binding studies in solution using losartan illustrated lower activity of the CYP2C9*2 compared with the WT. Together, the findings yield valuable insights into the decreased hydroxylation activity of losartan in patients carrying CYP2C9*2 allele and provide a useful framework to investigate the effect of a single-nucleotide polymorphism that leads to altered metabolism of diverse drug substrates. SIGNIFICANCE STATEMENT: The *2 allele of the human drug-metabolizing enzyme CYP2C9 is found in different populations and results in significantly reduced activity toward various drug substrates. How the CYP2C9*2 variant induces altered drug metabolism is poorly understood given that the Arg144Cys variation is located far away from the active site. This work yield insight into the effect of distal variation using multitude of techniques that include X-ray crystallography, isothermal titration calorimetry, enzymatic characterization, and computational studies.

Safety and Assisted Reproductive Technology Outcomes of Hysteroscopic Tubal Microinserts Versus Laparoscopic Proximal Tubal Occlusion or Salpingectomy for Hydrosalpinges Treatment.

OBJECTIVE: This study sought to answer the following question: What are the complications and assisted reproductive technology outcomes among women with hydrosalpinges managed by hysteroscopic microinsert tubal occlusion compared with women with hydrosalpinges managed by laparoscopic proximal tubal occlusion or salpingectomy? METHODS: This was a retrospective cohort study conducted from January 2009 to December 2014 at two academic, tertiary care, in vitro fertilization centres in Toronto, Ontario. All patients (n = 52) who underwent hysteroscopic tubal occlusion for hydrosalpinges were identified. Patients who proceeded with embryo transfer cycles after hysteroscopic microinsert (n = 33) were further age matched to a cohort of patients who underwent embryo transfer after laparoscopic proximal tubal occlusion or salpingectomy (n = 33). Main outcome measures were clinical pregnancy rate per patient and per embryo transfer cycle. RESULTS: Among 33 patients, there were 39 fresh and 37 frozen embryo transfer cycles in the hysteroscopic group (group A); among 33 patients in the laparoscopic group (group B), there were 42 fresh and 29 frozen embryo transfer cycles. The cumulative clinical pregnancy rate in group A and group B was similar (66.7% vs. 69.7%, respectively; P = 0.8). The clinical pregnancy rate per embryo transfer cycle was also similar in both groups (28.9% in group A vs. 32.4% in group B; P = 0.6). There were two incidents of ectopic pregnancy in the laparoscopic group and no ectopic pregnancy in the hysteroscopic group. There were three major complications: tubo-ovarian abscess, distal migration of the coil after microinsert placement, and an acute abdomen following the hysteroscopic procedure. CONCLUSION: Pregnancy outcomes after hysteroscopic placement of a microinsert for hydrosalpinx management before embryo transfer were comparable to those following laparoscopic proximal tubal occlusion or salpingectomy. However, caution is advised regarding microinsert placement for hydrosalpinges before proceeding with assisted reproductive technology.

The BRPF1 bromodomain is a molecular reader of di-acetyllysine.

Bromodomain-containing proteins are often part of chromatin-modifying complexes, and their activity can lead to altered expression of genes that drive cancer, inflammation and neurological disorders in humans. Bromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ (monocytic leukemic zinc-finger protein) HAT (histone acetyltransferase) complex, which is associated with chromosomal translocations known to contribute to the development of acute myeloid leukemia (AML). BRPF1 contains a unique combination of chromatin reader domains including two plant homeodomain (PHD) fingers separated by a zinc knuckle (PZP domain), a bromodomain, and a proline-tryptophan-tryptophan-proline (PWWP) domain. BRPF1 is known to recruit the MOZ HAT complex to chromatin by recognizing acetylated lysine residues on the N-terminal histone tail region through its bromodomain. However, histone proteins can contain several acetylation modifications on their N-terminus, and it is unknown how additional marks influence bromodomain recruitment to chromatin. Here, we identify the BRPF1 bromodomain as a selective reader of di-acetyllysine modifications on histone H4. We used ITC assays to characterize the binding of di-acetylated histone ligands to the BRPF1 bromodomain and found that the domain binds preferentially to histone peptides H4K5acK8ac and H4K5acK12ac. Analytical ultracentrifugation (AUC) experiments revealed that the monomeric state of the BRPF1 bromodomain coordinates di-acetylated histone ligands. NMR chemical shift perturbation studies, along with binding and mutational analyses, revealed non-canonical regions of the bromodomain-binding pocket that are important for histone tail recognition. Together, our findings provide critical information on how the combinatorial action of post-translational modifications can modulate BRPF1 bromodomain binding and specificity.

Effect of a Knowledge-Translation Intervention on Breast Surgeons' Oncofertility Attitudes and Practices.

BACKGROUND: The breast surgeon, generally the first oncology specialist consulted, is ideally suited to offer fertility preservation (FP) referral to young women with breast cancer (YWBC). In the authors' 2015 survey of 84 surgeons participating in the pan-Canadian RUBY study of YWBC, oncofertility knowledge and rates of FP referral were suboptimal. The authors designed an oncofertility knowledge-translation intervention for surgeons. METHODS: A customized oncofertility toolbox was created including a seminar/webinar, an option grid, a three-question FP survey sent upon registration of each RUBY patient, and a management checklist. In 2018, the 28 site lead surgeons were re-invited to participate in a follow-up telephone interview, and 85 non-site lead surgeons were invited to complete a follow-up online questionnaire. RESULTS: A total of 27 site lead surgeons consented to be re-interviewed. After the intervention, 85% indicated that they "routinely" initiated a fertility discussion compared with 54% at baseline (p < 0.005), with 56% stating that the toolbox had been helpful for making positive changes in their practice regarding oncofertility, and 44% stating that they found it easier to initiate a fertility discussion. Among the 55 non-site lead surgeons who completed the questionnaire, a significant improvement in oncofertility knowledge was found. The percentage reporting "rarely" or "never" discussing FP options decreased from 41 to 14% (p < 0.005), and 84% stated that they referred patients who had not completed their families and were at risk for infertility to FP consultation compared with 32% before the intervention (p < 0.001). CONCLUSIONS: A multi-pronged but simple knowledge-translation intervention improved the attitudes, knowledge, and FP practice of Canadian breast surgeons.