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Purpose: To describe the implementation of an in-house genotyping program to detect genetic variants linked to impaired dihydropyrimidine dehydrogenase (DPD) metabolism at a large multisite cancer center, including barriers to implementation and mechanisms to overcome barriers to facilitate test adoption., Summary: Fluoropyrimidines, including fluorouracil and capecitabine, are commonly used chemotherapy agents in the treatment of solid tumors, such as gastrointestinal cancers. DPD is encoded by the DPYD gene, and individuals classified as DPYD intermediate and poor metabolizers due to certain genetic variations in DPYD can experience reduced fluoropyrimidine clearance and an increased risk of fluoropyrimidine-related adverse events. Although pharmacogenomic guidelines provide evidence-based recommendations for DPYD genotype-guided dosing, testing has not been widely adopted in the United States for numerous reasons, including limited education/awareness of clinical utility, lack of testing recommendations by oncology professional organizations, testing cost, lack of accessibility to a comprehensive in-house test and service, and prolonged test turnaround time. Based on stakeholder feedback regarding barriers to testing, we developed an in-house DPYD test and workflow to facilitate testing in multiple clinic locations at Levine Cancer Institute. Across 2 gastrointestinal oncology clinics from March 2020 through June 2022, 137 patients were genotyped, and 13 (9.5%) of those patients were heterozygous for a variant and identified as DPYD intermediate metabolizers., Conclusion: Implementation of DPYD genotyping at a multisite cancer center was feasible due to operationalization of workflows to overcome traditional barriers to testing and engagement from all stakeholders, including physicians, pharmacists, nurses, and laboratory personnel. Future directions to scale and sustain testing in all patients receiving a fluoropyrimidine across all Levine Cancer Institute locations include electronic medical record integration (eg, interruptive alerts), establishment of a billing infrastructure, and further refinement of workflows to improve the rate of pretreatment testing., (© American Society of Health-System Pharmacists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Appropriate gene expression is vital for the regulation of developmental processes. Despite this fact there is a remarkable paucity of information concerning gene activity during preimplantation development., Methods: We employed reverse transcription and real-time fluorescent PCR to quantify the expression of nine genes (BRCA1, BRCA2, ATM, TP53, RB1, MAD2, BUB1, APC and beta-actin) in oocytes and embryos. A full characterization of all genes was achieved in 42 embryos and four oocytes. The genes analysed have a variety of important cellular functions., Results: Oocytes displayed relatively high levels of mRNA transcripts, while 2-3-cell embryos were seen to contain very little mRNA from any of the genes examined. Recovery of expression levels was not seen until the 4-cell stage or later, with the presumptive activation of the embryonic genome. Some genes displayed sharp increases in expression in embryos composed of 4-8 cells, but, for most, maximum expression was not achieved until the blastocyst stage., Conclusions: Our data show that it is possible to define characteristic gene expression profiles for each stage of human preimplantation development. The identification of genes active at defined preimplantation phases may provide clues to the cellular pathways utilized at specific stages of development. Expression of genes that function in DNA repair pathways indicate that DNA damage may be common at the cleavage stage. We suggest that specific patterns of gene expression may be indicative of embryo implantation potential.

Nitric oxide (NO) production plays an important role in regulating preimplantation embryo development. NO is produced from l-arginine by the enzyme nitric oxide synthase (NOS), which has three isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). It has been previously shown that inhibition of NO production by NG-nitro-l-arginine (l-NA) inhibits the development of two-cell embryos to the four-cell stage. However, excess NO also halts embryo development, possibly through the production of free radicals. We hypothesize that multiple NOS isoforms are expressed in order to ensure normal preimplantation embryo development and that, in this process, NO acts through the cGMP pathway. Using reverse transcription-polymerase chain reaction, mRNA for all three NOS isoforms was amplified from two-cell, four-cell, morula, and blastocyst embryos. However, blastocyst-stage embryos isolated midmorning on Day 4 of pregnancy expressed only nNOS and eNOS, whereas those isolated midafternoon again expressed all three NOS isoforms. Culture of one-cell embryos in various concentrations of Whitten (positive control), S-nitroso-N-acetylpenicillamine (SNP, a NO donor), l-NA, and/or 8-Br-cGMP demonstrated that NO is acting, at least in part, through cGMP in preimplantation embryo development. In addition, we determined that a critical concentration of NO and cGMP is required for normal embryo development and deviations from this concentration lead to developmental arrest and/or apoptosis of the embryo. This data provides support for a requirement of NO in preimplantation embryo development and one mechanism through which it regulates mitotic division in these embryos.

A study was conducted on patients who had attempted and failed previous in-vitro fertilization (IVF) procedures an average of 3.8 times following the application of assisted hatching with conventional culture systems. The aim of this investigation was to determine if addition of co-culture methodologies could reduce embryonic abnormalities and thus improve the prognosis for pregnancy. The study population consisted of 123 patients, subdivided into three patient categories. Previous IVF results from conventional culture were used to evaluate any potential benefits derived from the present co-culture application. Following co-culture, the rate of blastomere development was increased and the rate of fragmentation decreased. An increased rate of blastomere development was most noticeable in the patients aged < or = 39 years with no male factor as well as the intracytoplasmic sperm injection (ICSI) subgroup. Similarly, the rate of fragmentation was significantly reduced in the aforementioned subgroups. The most pronounced impact of co-culture was on pregnancy and implantation rates. The overall clinical and ongoing pregnancy rates were 38% (47/123) and 36% (44/123) respectively. The corresponding implantation rate was 17% (72/ 412) as shown by embryonic cardiac activity. The ongoing pregnancy rates in the < or = 39 years no male factor, > or = 40 years no male factor and ICSI no age limit patient subgroups were 41% (21/51), 30% (8/27) and 33% (15/45) respectively. The results indicate that addition of co-culture to the IVF procedure for poor-prognosis patients may be advisable.

A retrospective study was undertaken to determine if initial culture conditions and embryo quality had an effect on subsequent blastocyst development in co-culture for cryopreservation. The apparent effects of freeze-thawing on blastocysts at the ultrastructure level were also observed. On day 3 of culture, embryos were categorized into two groups based on their morphological attributes. Results suggest that the initial culture environment of embryos up to day 3 (5- to 8-cell stage) did not affect the subsequent rate of blastocyst formation in co-culture. However, the initial embryo quality had an impact on blastocyst formation and quality. On day 5.5, 90% (60/67) of the optimal quality embryos (six to eight blastomeres with minimal or no fragmentation on day 3) had attained the blastocyst stage, which was greater (P < 0.01) than the 55% (31/56) observed with the sub-optimal embryos (five to eight blastomeres with 30-50% fragmentation on day 3). Furthermore, 66% (44/67) of embryos initially graded as optimal were suitable for cryopreservation, which was greater (P < 0.01) than attained with embryos of lesser quality (22/56; 39%). At the ultrastructural level, the polarized distribution of plasma membrane microvilli was retained, as was the integrity of the nuclear membrane following thawing.

Videocinematography and image analysis procedures were utilized to evaluate the effect of conventional and coculture methodologies on morphological parameters in human embryos derived from in-vitro fertilization (IVF). Following 24-30 h of in-vitro development, cocultured embryos had more acceptable morphological features and less fragmentation present than embryos cultured in medium alone. Cocultured embryos were more advanced at the time of replacement when compared with conventionally cultured embryos. Zona pellucida variation (> or = 20%) also occurred more frequently in cocultured embryos. The morphological characteristic most enhanced after coculture was blastomere expansion. Patients who became pregnant across both culture treatments had a higher proportion of morphologically normal embryos replaced than patients who failed to achieve an ongoing pregnancy. Clinical pregnancy rate for patients following coculture was 49%, which was greater (P < 0.05) than the 29% detected for patients with embryos in the conventional culture group.