Impact of COVID-19 on patients with metastatic breast cancer: REthink Access to Care and Treatment survey results.

Aim: Patients with metastatic breast cancer (MBC) may be vulnerable to changes in healthcare management, safety standards and protocols that occurred during the COVID-19 pandemic. Materials & methods: The REthink Access to Care & Treatment (REACT) survey assessed USA-based patient perspectives on COVID-19-related impacts to their MBC treatment experience between 27 April 2021 and 17 August 2021. Results: Participants (n = 341; 98.5% females, mean age 50.8 years) reported that overall oncology treatment quality was maintained during the pandemic. Delayed/canceled diagnostic imaging was reported by 44.9% of participants while telemedicine uptake was high among participants (80%). Conclusion: Overall, MBC care was minimally affected by the pandemic, possibly due to the expanded use of telemedicine, informing MBC management for future public health emergencies.

The COVID-19 pandemic has forced healthcare providers to change the way that healthcare is delivered. These changes could particularly affect people with metastatic breast cancer (MBC), an advanced stage of cancer that has spread to other parts of the body. The authors of this study used a web-based survey to ask 341 volunteers with MBC how the pandemic has affected their cancer treatment. The authors found that people with MBC thought that the quality of their care stayed the same during the pandemic. Most people (80%) surveyed were able to use telemedicine, the remote delivery of care by phone or computer, to replace in-person visits to their doctor. However, almost half of people surveyed reported delays or cancellation of their diagnostic imaging appointments. Overall, this study shows that the COVID-19 pandemic did not affect peoples' opinions of their MBC care. Increased use of telemedicine may have contributed to the lack of disruption in care. These findings will help guide MBC care during future public health emergencies.

Targeted BRCA1/2 population screening among Ashkenazi Jewish individuals using a web-enabled medical model: An observational cohort study.

PURPOSE: This study aimed to evaluate uptake and follow-up using internet-assisted population genetic testing (GT) for BRCA1/2 Ashkenazi Jewish founder pathogenic variants (AJPVs). METHODS: Across 4 cities in the United States, from December 2017 to March 2020, individuals aged ≥25 years with ≥1 Ashkenazi Jewish grandparent were offered enrollment. Participants consented and enrolled online with chatbot and video education, underwent BRCA1/2 AJPV GT, and chose to receive results from their primary care provider (PCP) or study staff. Surveys were conducted at baseline, at 12 weeks, and annually for 5 years. RESULTS: A total of 5193 participants enrolled and 4109 (79.1%) were tested (median age = 54, female = 77.1%). Upon enrollment, 35.1% of participants selected a PCP to disclose results, and 40.5% of PCPs agreed. Of those tested, 138 (3.4%) were AJPV heterozygotes of whom 21 (15.2%) had no significant family history of cancer, whereas 86 (62.3%) had a known familial pathogenic variant. At 12 weeks, 85.5% of participants with AJPVs planned increased cancer screening; only 3.7% with negative results and a significant family history reported further testing. CONCLUSION: Although continued follow-up is needed, internet-enabled outreach can expand access to targeted GT using a medical model. Observed challenges for population genetic screening efforts include recruitment barriers, improving PCP engagement, and increasing uptake of additional testing when indicated.

Challenges and Opportunities in Engaging Primary Care Providers in BRCA Testing: Results from the BFOR Study.

PURPOSE: Engaging primary care providers (PCPs) in BRCA1/2 testing and results disclosure would increase testing access. The BRCA Founder OutReach (BFOR) study is a prospective study of BRCA1/2 founder mutation screening among individuals of Ashkenazi Jewish descent that sought to involve participants' PCPs in results disclosure. We used quantitative and qualitative methods to evaluate PCPs' perspectives, knowledge, and experience disclosing results in BFOR. METHODS: Among PCPs nominated by BFOR participants to disclose BRCA1/2 results, we assessed the proportion agreeing to disclose. To examine PCP's perspectives, knowledge, and willingness to disclose results, we surveyed 501 nominated PCPs. To examine PCPs' experiences disclosing results in BFOR, we surveyed 101 PCPs and conducted 10 semi-structured interviews. RESULTS: In the BFOR study overall, PCPs agreed to disclose their patient's results 40.5% of the time. Two hundred thirty-four PCPs (46.7%) responded to the initial survey. Responding PCPs were more likely to agree to disclose patients' results than non-responders (57.3% vs. 28.6%, p<0.001). Among all respondents, most felt very (19.7%) or somewhat (39.1%) qualified to share results. Among PCPs declining to disclose, insufficient knowledge was the most common reason. In multivariable logistic regression, feeling qualified was the only variable significantly associated with agreeing to disclose results (OR 6.53, 95% CI 3.31, 12.88). In post-disclosure surveys (response rate=55%), PCPs reported largely positive experiences. Interview findings suggested that although PCPs valued the study-provided educational materials, they desired better integration of results and decision support into workflows. CONCLUSION: Barriers exist to incorporating BRCA1/2 testing into primary care. Most PCPs declined to disclose their patients' BFOR results, although survey respondents were motivated and had positive disclosure experiences. PCP training and integrated decision support could be beneficial. TRIAL REGISTRATION: ClinicalTrials.gov (NCT03351803), November 24, 2017.

Choline acts during preimplantation development of the bovine embryo to program postnatal growth and alter muscle DNA methylation.

The preimplantation period of embryonic development can be a key window for programming of postnatal development because extensive epigenetic remodeling occurs during this time. It was hypothesized that modification of one-carbon metabolism of the bovine embryo by addition of the methyl-donor choline to culture medium would change postnatal phenotype through epigenetic modification. Embryos produced in vitro were cultured with 1.8 mM choline chloride or control medium. Blastocysts were transferred into females and pregnancy outcomes and postnatal phenotype of the resultant calves determined. Exposure of embryos to choline increased gestation length and calf birth weight. Calves derived from choline-treated embryos were also heavier at weaning and had increased ratio of body weight to hip height than control calves. Choline altered muscle DNA methylation of calves 4 months after birth. A total of 670 of the 8149 CpG examined were differentially methylated, with the predominant effect of choline being hypomethylation. Among the genes associated with differentially methylated CpG were ribosomal RNAs and genes in AMPK, mTOR, integrin, and BEX2 canonical pathways and cellular functions involved in growth and proliferation. Results demonstrate that provision of the methyl-donor choline to the preimplantation embryo can alter its developmental program to increase gestation length, birth weight, and weaning weight and cause postnatal changes in muscle DNA methylation including those associated with genes related to anabolic processes and cellular growth. The importance of the nutritional status of the embryo with respect to one-carbon metabolism for ensuring health and well-being after birth is emphasized by these observations.

Effect of addition of ascorbate, dithiothreitol or a caspase-3 inhibitor to cryopreservation medium on post-thaw survival of bovine embryos produced in vitro.

Experiments were conducted to investigate whether supplementation of cryopreservation medium with ascorbate, dithiothreitol (DTT) or an inhibitor of caspase-3 (z-DEVD-fmk) could improve post-thaw survival of bovine embryos produced in vitro (IVP). For all experiments, embryos were harvested on day 7 after insemination and subjected to controlled-rate freezing in medium containing 1.5 M ethylene glycol and treatments as described below. In experiments 1-3, embryos were cryopreserved in freezing medium with ascorbate (0, 0.1, 0.3 or 0.5 mM), DTT (0, 50, 100 or 200 µM) and z-DEVD-fmk (0, 50, 100 or 200 µM), respectively. Post-thaw survival was assessed at 24, 48 and 72 h. For experiments 4-5, embryos were cryopreserved in freezing medium with or without 0.1 mM ascorbate. At 24 h post-thaw, embryo total cell number, DNA fragmentation and levels of reactive oxygen species (ROS) were evaluated. Embryos subjected to freezing and thawing in medium supplemented with 0.1 mM ascorbate had greater (p < .05) re-expansion rates at 24, 48 and 72 h and hatching rate at 72 h as compared to embryos not treated with ascorbate. Post-thaw cryosurvival was not affected by the addition of either DTT or z-DEVD-fmk to medium used for cryopreservation. Embryos cryopreserved in medium supplemented with 0.1 mM ascorbate had reduced (p < .001) levels of intracellular ROS and fewer (p < .001) cells with DNA fragmentation. In conclusion, post-thaw survival of bovine IVP embryos is enhanced by supplementation of freezing medium with ascorbate.

Experimentally Induced Endometritis Impairs the Developmental Capacity of Bovine Oocytes†.

Uterine infection is associated with infertility in women and dairy cows, even after the resolution of infection. However, the mechanisms causing this persistent infertility are unclear. Here, we hypothesized that induced endometritis in non-lactating dairy cows would reduce the developmental competence of oocytes. Non-lactating Holstein cows received an intrauterine infusion of endometrial pathogenic bacteria (Escherichia coli and Trueperella pyogenes; n = 12) or vehicle control (n = 11) on day 2 of the estrous cycle. Bacterial infusion increased expression of endometrial inflammatory mediators, and a mucopurulent discharge in the vagina confirmed the establishment of endometritis. Oocytes were collected by transvaginal ultrasound-guided ovum pickup on days 2, 24, 45, and 66 following infusion and subjected to in vitro fertilization and embryo culture. Bacterial infusion resulted in fewer cleaved oocytes developing to morulae compared to vehicle-infused controls (30.7 versus 45.0%), with the greatest effect observed in oocytes collected on day 24. Development to morula was inversely correlated with endometrial expression of IL6 on day 6. The expression of genes associated with embryo quality did not differ significantly between morulae from bacteria-infused and control cows. Artificial insemination 130 days after intrauterine infusion resulted in normal, filamentous embryos that produced interferon tau 16 days after conception in both infusion groups. This model of experimentally induced uterine infection successfully resulted in endometritis and a reduction in the proportion of oocytes that developed to morulae following in vitro fertilization. In conclusion, endometritis reduced the capacity of oocytes to develop to morulae.

Uterine infection alters the transcriptome of the bovine reproductive tract three months later.

Infection of the postpartum uterus with pathogenic bacteria is associated with infertility months later in dairy cattle. However, it is unclear whether these bacterial infections lead to long-term changes in the reproductive tract that might help explain this infertility. Here we tested the hypothesis that infusion of pathogenic bacteria into the uterus leads to changes in the transcriptome of the reproductive tract 3 months later. We used virgin Holstein heifers to avoid potential confounding effects of periparturient problems, lactation, and negative energy balance. Animals were infused intrauterine with endometrial pathogenic bacteria Escherichia coli and Trueperella pyogenes (n = 4) and compared with control animals (n = 6). Three months after infusion, caruncular and intercaruncular endometrium, isthmus and ampulla of the oviduct, and granulosa cells from ovarian follicles >8 mm diameter were profiled by RNA sequencing. Bacterial infusion altered the transcriptome of all the tissues when compared with control. Most differentially expressed genes were tissue specific, with 109 differentially expressed genes unique to caruncular endometrium, 57 in intercaruncular endometrium, 65 in isthmus, 298 in ampulla, and 83 in granulosa cells. Surprisingly, despite infusing bacteria into the uterus, granulosa cells had more predicted upstream regulators of differentially expressed genes than all the other tissues combined. In conclusion, there were changes in the transcriptome of the endometrium, oviduct and even granulosa cells, 3 months after intrauterine infusion of pathogenic bacteria. These findings imply that long-term changes throughout the reproductive tract could contribute to infertility after bacterial infections of the uterus.

Persistent effects on bovine granulosa cell transcriptome after resolution of uterine disease.

Metritis is associated with reduced fertility in dairy cows, but the mechanisms are unclear because the disease resolves several weeks before insemination. One hypothesis is that metritis causes persistent changes in granulosa cells during follicle development, which might be evident in the transcriptome of granulosa cells from dominant follicles weeks after parturition. To test this hypothesis, we collected the follicular fluid and granulosa cells from dominant follicles 63 days post partum from cows previously diagnosed with metritis, at least 6 weeks after resolution of the disease and from cows not diagnosed with metritis (control cows). Bacterial lipopolysaccharide was detected in follicular fluid, and concentrations were associated with follicular fluid IL-8 and glucose concentrations. Transcriptome analysis using RNAseq revealed 177 differentially expressed genes in granulosa cells collected from cows that had metritis compared with control cows. The most upregulated genes were ITLN1, NCF2, CLRN3, FSIP2 and ANKRD17, and the most downregulated genes were ACSM1, NR4A2, GHITM, CBARP and NR1I3. Pathway analysis indicated that the differentially expressed genes were involved with immune function, cell-cell communication, cell cycle and cellular metabolism. Predicted upstream regulators of the differentially expressed genes included NFκB, IL-21 and lipopolysaccharide, which are associated with infection and immunity. Our data provide evidence for a persistent effect of metritis on the transcriptome of granulosa cells in ovarian follicles after the resolution of disease.

Economic and genetic performance of various combinations of in vitro-produced embryo transfers and artificial insemination in a dairy herd.

The objective of this study was to find the optimal proportions of pregnancies from an in vitro-produced embryo transfer (IVP-ET) system and artificial insemination (AI) so that profitability is maximized over a range of prices for embryos and surplus dairy heifer calves. An existing stochastic, dynamic dairy model with genetic merits of 12 traits was adapted for scenarios where 0 to 100% of the eligible females in the herd were impregnated, in increments of 10%, using IVP-ET (ET0 to ET100, 11 scenarios). Oocytes were collected from the top donors selected for the trait lifetime net merit (NM$) and fertilized with sexed semen to produce IVP embryos. Due to their greater conception rates, first ranked were eligible heifer recipients based on lowest number of unsuccessful inseminations or embryo transfers, and then on age. Next, eligible cow recipients were ranked based on the greatest average estimated breeding values (EBV) of the traits cow conception rate and daughter pregnancy rate. Animals that were not recipients of IVP embryos received conventional semen through AI, except that the top 50% of heifers ranked for EBV of NM$ were inseminated with sexed semen for the first 2 AI. The economically optimal proportions of IVP-ET were determined using sensitivity analysis performed for 24 price sets involving 6 different selling prices of surplus dairy heifer calves at approximately 105 d of age and 4 different prices of IVP embryos. The model was run for 15 yr after the start of the IVP-ET program for each scenario. The mean ± standard error of true breeding values of NM$ of all cows in the herd in yr 15 was greater by $603 ± 2 per cow per year for ET100 when compared with ET0. The optimal proportion of IVP-ET ranged from ET100 (for surplus dairy heifer calves sold for ≥$300 along with an additional premium based on their EBV of NM$ and a ≤$100 embryo price) to as low as ET0 (surplus dairy heifer calves sold at $300 with a $200 embryo price). For the default assumptions, the profit/cow in yr 15 was greater by $337, $215, $116, and $69 compared with ET0 when embryo prices were $50, $100, $150, and $200. The optimal use of IVP-ET was 100, 100, 62, and 36% of all breedings for these embryo prices, respectively. At the input price of $165 for an IVP embryo, the difference in the net present value of yr 15 profit between ET40 (optimal scenario) and ET0 was $33 per cow. In conclusion, some use of IVP-ET was profitable for a wide range of IVP-ET prices and values of surplus dairy heifer calves.

The WNT signaling antagonist Dickkopf-1 directs lineage commitment and promotes survival of the preimplantation embryo.

Successful embryonic development is dependent on factors secreted by the reproductive tract. Dickkopf-1 (DKK1), an antagonist of the wingless-related mouse mammary tumor virus (WNT) signaling pathway, is one endometrial secretory protein potentially involved in maternal-embryo communication. The purpose of this study was to investigate the roles of DKK1 in embryo cell fate decisions and competence to establish pregnancy. Using in vitro-produced bovine embryos, we demonstrate that exposure of embryos to DKK1 during the period of morula to blastocyst transition (between d 5 and 8 of development) promotes the first 2 cell fate decisions leading to increased differentiation of cells toward the trophectoderm and hypoblast lineages compared with that for control embryos treated with vehicle. Moreover, treatment of embryos with DKK1 or colony-stimulating factor 2 (CSF2; an endometrial cytokine known to improve embryo development and pregnancy establishment) between d 5 and 7 of development improves embryo survival after transfer to recipients. Pregnancy success at d 32 of gestation was 27% for cows receiving control embryos treated with vehicle, 41% for cows receiving embryos treated with DKK1, and 39% for cows receiving embryos treated with CSF2. These novel findings represent the first evidence of a role for maternally derived WNT regulators during this period and could lead to improvements in assisted reproductive technologies.

Definition and classification of evaluation units for CASP10.

For the 10th experiment on Critical Assessment of the techniques of protein Structure Prediction (CASP), the prediction target proteins were broken into independent evaluation units (EUs), which were then classified into template-based modeling (TBM) or free modeling (FM) categories. We describe here how the EUs were defined and classified, what issues arose in the process, and how we resolved them. EUs are frequently not the whole target proteins but the constituting structural domains. However, the assessors from CASP7 on combined more than one domain into 1 EU for some targets, which implied that the assessment also included evaluation of the prediction of the relative position and orientation of these domains. In CASP10, we followed and expanded this notion by defining multidomain EUs for a number of targets. These included 3 EUs, each made of two domains of familiar fold but arranged in a novel manner and for which the focus of evaluation was the interdomain arrangement. An EU was classified to the TBM category if a template could be found by sequence similarity searches and to FM if a structural template could not be found by structural similarity searches. The EUs that did not fall cleanly in either of these cases were classified case-by-case, often including consideration of the overall quality and characteristics of the predictions.

Sexual dimorphism in developmental programming of the bovine preimplantation embryo caused by colony-stimulating factor 2.

Physiology of the adult can be modified by alterations in prenatal development driven by the maternal environment. Developmental programming, which can be established before the embryo implants in the uterus, can affect females differently than males. The mechanism by which sex-specific developmental programming is established is not known. Here we present evidence that maternal regulatory signals change female embryos differently than male embryos. In particular, actions of the maternally derived cytokine CSF2 from Day 5 to Day 7 of development affected characteristics of the embryo at Day 15 differently for females than males. CSF2 decreased length and IFNT secretion of female embryos but increased length and IFNT secretion of male embryos. Analysis of a limited number of samples indicated that changes in the transcriptome and methylome caused by CSF2 also differed between female and males. Thus, sex-specific programming by the maternal environment could occur when changes in secretion of maternally derived regulatory molecules alter development of female embryos differently than male embryos.

Consequences of gene editing of PRLR on thermotolerance, growth, and male reproduction in cattle.

Global warming is a major challenge to the sustainable and humane production of food because of the increased risk of livestock to heat stress. Here, the example of the prolactin receptor (PRLR) gene is used to demonstrate how gene editing can increase the resistance of cattle to heat stress by the introduction of mutations conferring thermotolerance. Several cattle populations in South and Central America possess natural mutations in PRLR that result in affected animals having short hair and being thermotolerant. CRISPR/Cas9 technology was used to introduce variants of PRLR in two thermosensitive breeds of cattle - Angus and Jersey. Gene-edited animals exhibited superior ability to regulate vaginal temperature (heifers) and rectal temperature (bulls) compared to animals that were not gene-edited. Moreover, gene-edited animals exhibited superior growth characteristics and had larger scrotal circumference. There was no evidence for deleterious effects of the mutation on carcass characteristics or male reproductive function. These results indicate the potential for reducing heat stress in relevant environments to enhance cattle productivity.

Structural studies and protein engineering of inositol phosphate multikinase.

Inositol phosphates (IPs) regulate vital processes in eukaryotes, and their production downstream of phospholipase C activation is controlled through a network of evolutionarily conserved kinases and phosphatases. Inositol phosphate multikinase (IPMK, also called Ipk2 and Arg82) accounts for phosphorylation of IP(3) to IP(5), as well as production of several other IP molecules. Here, we report the structure of Arabidopsis thaliana IPMKα at 2.9 Å and find it is similar to the yeast homolog Ipk2, despite 17% sequence identity, as well as the active site architecture of human IP(3) 3-kinase. Structural comparison and substrate modeling were used to identify a putative basis for IPMK selectivity. To test this model, we re-engineered binding site residues predicted to have restricted substrate specificity. Using steady-state kinetics and in vivo metabolic labeling studies in modified yeast strains, we observed that K117W and K117W:K121W mutants exhibited nearly normal 6-kinase function but harbored significantly reduced 3-kinase activity. These mutants complemented conditional nutritional growth defects observed in ipmk null yeast and, remarkably, suppressed lethality observed in ipmk null flies. Our data are consistent with the hypothesis that IPMK 6-kinase activity and production of Ins(1,4,5,6)P(4) are critical for cellular signaling. Overall, our studies provide new insights into the structure and function of IPMK and utilize a synthetic biological approach to redesign inositol phosphate signaling pathways.

Changes in the transcriptome of morula-stage bovine embryos caused by heat shock: relationship to developmental acquisition of thermotolerance.

BACKGROUND: While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3' tag digital gene expression (3'DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance. RESULTS: Using 3'DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (P<0.05) and tended to increase expression of HSP90AA1 (P<0.07) but had no effect on expression of SOD1 or CAT. CONCLUSIONS: Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that the increased resistance of morula-stage embryos to heat shock as compared to embryos at earlier stages of development is due in part to developmental acquisition of mechanisms to prevent accumulation of denatured proteins and free radical damage.

The Influence of Visual Provenance Representations on Strategies in a Collaborative Hand-off Data Analysis Scenario.

Conducting data analysis tasks rarely occur in isolation. Especially in intelligence analysis scenarios where different experts contribute knowledge to a shared understanding, members must communicate how insights develop to establish common ground among collaborators. The use of provenance to communicate analytic sensemaking carries promise by describing the interactions and summarizing the steps taken to reach insights. Yet, no universal guidelines exist for communicating provenance in different settings. Our work focuses on the presentation of provenance information and the resulting conclusions reached and strategies used by new analysts. In an open-ended, 30-minute, textual exploration scenario, we qualitatively compare how adding different types of provenance information (specifically data coverage and interaction history) affects analysts' confidence in conclusions developed, propensity to repeat work, filtering of data, identification of relevant information, and typical investigation strategies. We see that data coverage (i.e., what was interacted with) provides provenance information without limiting individual investigation freedom. On the other hand, while interaction history (i.e., when something was interacted with) does not significantly encourage more mimicry, it does take more time to comfortably understand, as represented by less confident conclusions and less relevant information-gathering behaviors. Our results contribute empirical data towards understanding how provenance summarizations can influence analysis behaviors.

Consequences of conceptus exposure to colony-stimulating factor 2 on survival, elongation, interferon-τ secretion, and gene expression.

Exposure of bovine conceptuses to colony-stimulating factor 2 (CSF2) from days 5 to 7 of development can increase the percentage of transferred conceptuses that develop to term. The purpose of this experiment was to understand the mechanism by which CSF2 increases embryonic and fetal survival. Conceptuses were produced in vitro in the presence or absence of 10  ng/ml CSF2 from days 5 to 7 after insemination, transferred into cows, and flushed from the uterus at day 15 of pregnancy. There was a tendency (P=0.07) for the proportion of cows with a recovered conceptus to be greater for those receiving a CSF2-treated conceptus (35% for control versus 66% for CSF2). Antiviral activity in uterine flushings, a measure of the amount of interferon-τ (IFNT2) secreted by the conceptus, tended to be greater for cows receiving CSF2-treated conceptuses than for cows receiving control conceptuses. This difference approached significance when only cows with detectable antiviral activity were considered (P=0.07). In addition, CSF2 increased mRNA for IFNT2 (P=0.08) and keratin 18 (P<0.05) in extraembryonic membranes. Among a subset of filamentous conceptuses that were analyzed by microarray hybridization, there was no effect of CSF2 on gene expression in the embryonic disc or extraembryonic membranes. Results suggest that the increase in calving rate caused by CSF2 treatment involves, in part, more extensive development of extraembryonic membranes and capacity of the conceptus to secrete IFNT2 at day 15 of pregnancy.

Programming of postnatal phenotype caused by exposure of cultured embryos from Brahman cattle to colony-stimulating factor 2 and serum.

Alterations in the environment of the preimplantation embryo can affect competence to establish pregnancy and phenotype of resultant calves. In this study, the bovine embryo produced in vitro was used to evaluate postnatal programming actions of the embryokine colony-stimulating factor 2 (CSF2) and serum, which is a common additive of culture media. Oocytes were collected by ovum pick up from Brahman donors and fertilized with semen from Brahman bulls. Embryos were randomly assigned to one of the three treatments: vehicle, CSF2 10 ng/mL, or 1% (v/v) serum. Treatments were added to the culture medium from day 5 to 7 after fertilization. Blastocysts were harvested on day 7 and transferred into crossbred recipients. Postnatal body growth and Longissimus dorsi muscle characteristics of the resultant calves were measured. The percent of cleaved embryos becoming blastocysts was increased by serum and, to a lesser extent, CSF2. Treatment did not affect survival after embryo transfer but gestation length was shortest for pregnancies established with serum-treated embryos. Treatment did not significantly affect postnatal body weight or growth. At 3 mo of age, CSF2 calves had lower fat content in the Longissimus dorsi muscle and less subcutaneous fat over the muscle than vehicle calves. There was a tendency for cross-sectional area of the muscle to be smaller for serum calves than vehicle calves. Results confirm the importance of the preimplantation period as a window to modulate postnatal phenotype of resultant calves. In particular, CSF2 exerted actions during the preimplantation period to program characteristics of accumulation of intramuscular and subcutaneous fat of resultant calves. The use of a low serum concentration in culture medium from day 5 to 7 of development can increase the yield of transferrable embryos without causing serious negative consequences for the offspring.

Effects of gamete source and culture conditions on the competence of in vitro-produced embryos for post-transfer survival in cattle.

One limitation to the use of in vitro-produced embryos in cattle production systems is the fact that pregnancy rates after transfer to recipients are typically lower than when embryos produced in vivo are transferred. Conceptually, the oocyte and spermatozoon from which the embryo is derived could affect competence for post-transfer survival. There are sire differences in embryonic survival after transfer, but there is little evidence that an embryo's ability to establish pregnancy is determined by sex sorting of spermatozoa by flow cytometry. The role of the source of the oocyte as a determinant of embryonic survival after transfer has not been examined carefully. Conditions for embryo culture after fertilisation can have an impact on the ability of the embryo to establish pregnancy following transfer. Among the specific molecules produced in the reproductive tract of the cow that have been shown to improve competence of in vitro-produced embryos for post-transfer survival are colony-stimulating factor 2, insulin-like growth factor-1 (for recipients exposed to heat stress) and hyaluronan (for less-advanced embryos). There is also a report that embryo competence for post-transfer survival can be improved by inclusion of a carbon-activated air filtration system in the incubator used to culture embryos. Progress in developing culture systems to improve embryonic competence for survival after transfer would be hastened by the development of in vitro assays that accurately predict the potential of an embryo to establish pregnancy after transfer. A group of 52 genes has been identified that are differentially expressed in embryos that developed to term v. embryos that did not establish pregnancy. Perhaps a gene microarray consisting of these genes, alone or in combination with other genes, could be used to screen embryos for competence to establish pregnancy.