Phenotypic and transcriptomic analysis reveals early stress responses in transgenic rice expressing Arabidopsis DREB1a.

Overexpression of Arabidopsis dehydration response element binding 1a (DREB1a) is a well-known approach for developing salinity, cold and/or drought stress tolerance. However, understanding of the genetic mechanisms associated with DREB1a expression in rice is generally limited. In this study, DREB1a-associated early responses were investigated in a transgenic rice line harboring cold-inducible DREB1a at a gene stacked locus. Although the function of other genes in the stacked locus was not relevant to stress tolerance, this study demonstrates DREB1a can be co-localized with other genes for multigenic trait enhancement. As expected, the transgenic lines displayed improved tolerance to salinity stress and water withholding as compared with non-transgenic controls. RNA sequencing and transcriptome analysis showed upregulation of complex transcriptional networks and metabolic reprogramming as DREB1a expression led to the upregulation of multiple transcription factor gene families, suppression of photosynthesis, and induction of secondary metabolism. In addition to the detection of previously described mechanisms such as production of protective molecules, potentially novel pathways were also revealed. These include jasmonate, auxin, and ethylene signaling, induction of JAZ and WRKY regulons, trehalose synthesis, and polyamine catabolism. These genes regulate various stress responses and ensure timely attenuation of the stress signal. Furthermore, genes associated with heat stress response were downregulated in DREB1a expressing lines, suggesting antagonism between heat and dehydration stress response pathways. In summary, through a complex transcriptional network, multiple stress signaling pathways are induced by DREB1a that presumably lead to early perception and prompt response toward stress tolerance as well as attenuation of the stress signal to prevent deleterious effects of the runoff response.

Targeting TOR and SnRK1 Genes in Rice with CRISPR/Cas9.

Genome targeting with CRISPR/Cas9 is a popular method for introducing mutations and creating knock-out effects. However, limited information is currently available on the mutagenesis of essential genes. This study investigated the efficiency of CRISPR/Cas9 in targeting rice essential genes: the singleton TARGET OF RAPAMYCIN (OsTOR) and the three paralogs of the Sucrose non-fermenting-1 (SNF1)-related kinase 1 (OsSnRK1α), OsSnRK1αA, OsSnRK1αB and OsSnRK1αC. Strong activity of constitutively expressed CRISPR/Cas9 was effective in creating mutations in OsTOR and OsSnRK1α genes, but inducible CRISPR/Cas9 failed to generate detectable mutations. The rate of OsTOR mutagenesis was relatively lower and only the kinase domain of OsTOR could be targeted, while mutations in the HEAT region were unrecoverable. OsSnRK1α paralogs could be targeted at higher rates; however, sterility or early senescence was observed in >50% of the primary mutants. Additionally, OsSnRK1αB and OsSnRK1αC, which bear high sequence homologies, could be targeted simultaneously to generate double-mutants. Further, although limited types of mutations were found in the surviving mutants, the recovered lines displayed loss-of-function or knockdown tor or snrk1 phenotypes. Overall, our data show that mutations in these essential genes can be created by CRISPR/Cas9 to facilitate investigations on their roles in plant development and environmental response in rice.

Multigene Transformation Through Cre-lox Mediated Site-Specific Integration in Rice.

Plant transformation with multiple genes is a major challenge, rendering multi-trait engineering extremely difficult in crop plants. One of the hurdles in multigene transformation is the uncontrolled integration process that leads to low quality transgenic lines that are unsuitable for practical application. Recombinase-mediated site-specific integration has been tested and validated for developing high quality transgenic lines expressing one, two, or multiple genes. Of the numerous recombinase systems tested, Cre-lox and FLP-FRT show high efficiency in plants. Recently, Cre-lox system was successfully used to stack a set of 3 constitutive, 1 heat-induced, and 1 cold-induced gene. A number of transgenic lines were obtained through a relatively small effort, and the resulting transgenic lines all expressed the genes properly as determined by their promoter-specificity. Here, a method of Cre-lox mediated stacking of a multigene construct is described using rice as a model crop.

Recombinase-mediated integration of a multigene cassette in rice leads to stable expression and inheritance of the stacked locus.

Efficient methods for multigene transformation are important for developing novel crop varieties. Methods based on random integrations of multiple genes have been successfully used for metabolic engineering in plants. However, efficiency of co-integration and co-expression of the genes could present a bottleneck. Recombinase-mediated integration into the engineered target sites is arguably a more efficient method of targeted integration that leads to the generation of stable transgenic lines at a high rate. This method has the potential to streamline multigene transformation for metabolic engineering and trait stacking in plants. Therefore, empirical testing of transgene(s) stability from the multigene site-specific integration locus is needed. Here, the recombinase technology based on Cre-lox recombination was evaluated for developing multigenic lines harboring constitutively-expressed and inducible genes. Targeted integration of a five genes cassette in the rice genome generated a precise full-length integration of the cassette at a high rate, and the resulting multigenic lines expressed each gene reliably as defined by their promoter activity. The stable constitutive or inducible expression was faithfully transmitted to the progeny, indicating inheritance-stability of the multigene locus. Co-localization of two distinctly inducible genes by heat or cold with the strongly constitutive genes did not appear to interfere with each other's expression pattern. In summary, high rate of co-integration and co-expression of the multigene cassette installed by the recombinase technology in rice shows that this approach is appropriate for multigene transformation and introduction of co-segregating traits. SIGNIFICANCE STATEMENT: Recombinase-mediated site-specific integration approach was found to be highly efficacious in multigene transformation of rice showing proper regulation of each gene driven by constitutive or inducible promoter. This approach holds promise for streamlining gene stacking in crops and expressing complex multigenic traits.

Heat-shock-inducible CRISPR/Cas9 system generates heritable mutations in rice.

Transient expression of CRISPR/Cas9 is an effective approach for limiting its activities and improving its precision in genome editing. Here, we describe the heat-shock-inducible CRISPR/Cas9 for controlled genome editing, and demonstrate its efficiency in the model crop, rice. Using the soybean heat-shock protein gene promoter and the rice U3 promoter to express Cas9 and sgRNA, respectively, we developed the heat-shock (HS)-inducible CRISPR/Cas9 system, and tested its efficacy in targeted mutagenesis. Two loci were targeted in rice, and the presence of targeted mutations was determined before and after the HS treatment. Only a low rate of targeted mutagenesis was detected before HS (~16%), but an increased rate of mutagenesis was observed after the HS treatment among the transgenic lines (50-63%). Analysis of regenerated plants harboring HS-CRISPR/Cas9 revealed that targeted mutagenesis was suppressed in the plants but induced by HS, which was detectable by Sanger sequencing after a few weeks of HS treatments. Most importantly, the HS-induced mutations were transmitted to the progeny at a high rate, generating monoallelic and biallelic mutations that independently segregated from the Cas9 gene. Additionally, off-target mutations were either undetectable or found at a lower rate in HS-CRISPR/Cas9 lines as compared to the constitutive-overexpression CRISPR/Cas9 lines. Taken together, this work shows that HS-CRISPR/Cas9 is a controlled and reasonably efficient platform for genome editing, and therefore, a promising tool for limiting genome-wide off-target effects and improving the precision of genome editing.

Utility of I-SceI and CCR5-ZFN nucleases in excising selectable marker genes from transgenic plants.

OBJECTIVES: Removal of selection marker genes from transgenic plants is highly desirable for their regulatory approval and public acceptance. This study evaluated the use of two nucleases, the yeast homing endonuclease, I-SceI, and the designed zinc finger nuclease, CCR5-ZFN, in excising marker genes from plants using rice and Arabidopsis as the models. RESULTS: In an in vitro culture assay, both nucleases were effective in precisely excising the DNA fragments marked by the nuclease target sites. However, rice cultures were found to be refractory to transformation with the I-SceI and CCR5-ZFN overexpressing constructs. The inducible I-SceI expression was also problematic in rice as the progeny of the transgenic lines expressing the heat-inducible I-SceI did not inherit the functional gene. On the other hand, heat-inducible I-SceI expression in Arabidopsis was effective in creating somatic excisions in transgenic plants but ineffective in generating heritable excisions. The inducible expression of CCR5-ZFN in rice, although transmitted stably to the progeny, appeared ineffective in creating detectable excisions. Therefore, toxicity of these nucleases in plant cells poses major bottleneck in their application in plant biotechnology, which could be avoided by expressing them transiently in cultures in vitro.

Dual-targeting by CRISPR/Cas9 leads to efficient point mutagenesis but only rare targeted deletions in the rice genome.

The present study investigated the efficiency of CRISPR/Cas9 in creating genomic deletions as the basis of its application in removing selection marker genes or the intergenic regions. Three loci, representing a transgene and two rice genes, were targeted at two sites each, in separate experiments, and the deletion of the defined fragments was investigated by PCR and sequencing. Genomic deletions were found at a low rate among the transformed callus lines that could be isolated, cultured, and regenerated into plants harboring the deletion. However, randomly regenerated plants showed mixed genomic effects, and generally did not harbor heritable genomic deletions. To determine whether point mutations occurred at each targeted site, a total of 114 plants consisting of primary transgenic lines and their progeny were analyzed. Ninety-three plants showed targeting, 60 of which were targeted at both sites. The presence of point mutations at both sites was correlated with the guide RNA efficiency. In summary, genomic deletions through dual-targeting by the paired-guide RNAs were generally observed in callus, while de novo point mutations at one or both sites occurred at high rates in transgenic plants and their progeny, generating a variety of insertion-deletions or single-nucleotide variations. In this study, point mutations were exceedingly favored over genomic deletions; therefore, for the recovery of plant lines harboring targeted deletions, identifying early transformed clones harboring the deletions, and isolating them for plant regeneration is recommended.

Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane.

KEY MESSAGE: A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination. Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools.

Metabolic engineering of sugarcane to accumulate energy-dense triacylglycerols in vegetative biomass.

Elevating the lipid content in vegetative tissues has emerged as a new strategy for increasing energy density and biofuel yield of crops. Storage lipids in contrast to structural and signaling lipids are mainly composed of glycerol esters of fatty acids, also known as triacylglycerol (TAG). TAGs are one of the most energy-rich and abundant forms of reduced carbon available in nature. Therefore, altering the carbon-partitioning balance in favour of TAG in vegetative tissues of sugarcane, one of the highest yielding biomass crops, is expected to drastically increase energy yields. Here we report metabolic engineering to elevate TAG accumulation in vegetative tissues of sugarcane. Constitutive co-expression of WRINKLED1 (WRI1), diacylglycerol acyltransferase1-2 (DGAT1-2) and oleosin1 (OLE1) and simultaneous cosuppression of ADP-glucose pyrophosphorylase (AGPase) and a subunit of the peroxisomal ABC transporter1 (PXA1) in transgenic sugarcane elevated TAG accumulation in leaves or stems by 95- or 43-fold to 1.9% or 0.9% of dry weight (DW), respectively, while expression or suppression of one to three of the target genes increased TAG levels by 1.5- to 9.5-fold. Accumulation of TAG in vegetative progeny plants was consistent with the results from primary transgenics and contributed to a total fatty acid content of up to 4.7% or 1.7% of DW in mature leaves or stems, respectively. Lipid droplets were visible within mesophyll cells of transgenic leaves by confocal fluorescence microscopy. These results provide the basis for optimizations of TAG accumulation in sugarcane and other high yielding biomass grasses and will open new prospects for biofuel applications.

Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion.

BACKGROUND: Practical approaches for multigene transformation and gene stacking are extremely important for engineering complex traits and adding new traits in transgenic crops. Trait deployment by gene stacking would greatly simplify downstream plant breeding and trait introgression into cultivars. Gene stacking into pre-determined genomic sites depends on mechanisms of targeted DNA integration and recycling of selectable marker genes. Targeted integrations into chromosomal breaks, created by nucleases, require large transformation efforts. Recombinases such as Cre-lox, on the other hand, efficiently drive site-specific integrations in plants. However, the reversibility of Cre-lox recombination, due to the incorporation of two cis-positioned lox sites, presents a major bottleneck in its application in gene stacking. Here, we describe a strategy of resolving this bottleneck through excision of one of the cis-positioned lox, embedded in the marker gene, by nuclease activity. METHODS: All transgenic lines were developed by particle bombardment of rice callus with plasmid constructs. Standard molecular approach was used for building the constructs. Transgene loci were analyzed by PCR, Southern hybridization, and DNA sequencing. RESULTS: We developed a highly efficient gene stacking method by utilizing powerful recombinases such as Cre-lox and FLP-FRT, for site-specific gene integrations, and nucleases for marker gene excisions. We generated Cre-mediated site-specific integration locus in rice and showed excision of marker gene by I-SceI at ~20 % efficiency, seamlessly connecting genes in the locus. Next, we showed ZFN could be used for marker excision, and the locus can be targeted again by recombinases. Hence, we extended the power of recombinases to gene stacking application in plants. Finally, we show that heat-inducible I-SceI is also suitable for marker excision, and therefore could serve as an important tool in streamlining this gene stacking platform. CONCLUSIONS: A practical approach for gene stacking in plant cell was developed that allows targeted gene insertions through rounds of transformation, a method needed for introducing new traits into transgenic lines for their rapid deployment in the field. By using Cre-lox, a powerful site-specific recombination system, this method greatly improves gene stacking efficiency, and through the application of nucleases develops marker-free, seamless stack of genes at pre-determined chromosomal sites.

Neonatal Outcomes by Mode of Delivery in Preterm Birth.

OBJECTIVE: We set out to test the hypothesis that infants born vaginally at ≤ 30 weeks gestation have less respiratory distress syndrome (RDS) than those born by cesarean delivery. STUDY DESIGN: We conducted a retrospective cohort study of 652 infants born between 24 and 30 (6/7) weeks gestation from March 31, 1996 to May 31, 2014. Comparisons of neonatal outcomes by intended and actual mode of delivery were made using chi-square and t-tests (α = 0.05). Multiple logistic regression was performed to control for confounding variables. RESULTS: Neonates born by cesarean delivery were more likely to have RDS (odds ratio [OR], 1.79; 95% confidence interval [CI], 1.10-2.90), require intubation (OR, 1.80; 95% CI, 1.12-2.88), and have longer neonatal intensive care unit stay (70.0 ± 37.1 vs. 57.3 ± 40.1 days, p = 0.02). CONCLUSION: Compared with cesarean delivery, vaginal delivery is associated with a significant reduction in RDS among infants born at ≤ 30 weeks gestation.

Promoting utilization of Saccharum spp. genetic resources through genetic diversity analysis and core collection construction.

Sugarcane (Saccharum spp.) and other members of Saccharum spp. are attractive biofuel feedstocks. One of the two World Collections of Sugarcane and Related Grasses (WCSRG) is in Miami, FL. This WCSRG has 1002 accessions, presumably with valuable alleles for biomass, other important agronomic traits, and stress resistance. However, the WCSRG has not been fully exploited by breeders due to its lack of characterization and unmanageable population. In order to optimize the use of this genetic resource, we aim to 1) genotypically evaluate all the 1002 accessions to understand its genetic diversity and population structure and 2) form a core collection, which captures most of the genetic diversity in the WCSRG. We screened 36 microsatellite markers on 1002 genotypes and recorded 209 alleles. Genetic diversity of the WCSRG ranged from 0 to 0.5 with an average of 0.304. The population structure analysis and principal coordinate analysis revealed three clusters with all S. spontaneum in one cluster, S. officinarum and S. hybrids in the second cluster and mostly non-Saccharum spp. in the third cluster. A core collection of 300 accessions was identified which captured the maximum genetic diversity of the entire WCSRG which can be further exploited for sugarcane and energy cane breeding. Sugarcane and energy cane breeders can effectively utilize this core collection for cultivar improvement. Further, the core collection can provide resources for forming an association panel to evaluate the traits of agronomic and commercial importance.

Calcium dependent protein kinase (CDPK) expression during fruit development in cultivated peanut (Arachis hypogaea) under Ca²âº-sufficient and -deficient growth regimens.

Adequate soil calcium (Ca²âº) levels are crucial for sustained reproductive development of peanut (Arachis hypogaea). A role for calcium dependent protein kinase was evaluated during peanut fruit development under sufficient and deficient soil Ca²âº conditions. Quantitative RT-PCR and protein gel blot analyses confirmed transcriptional upregulation of CDPK in seeds developing under inadequate soil Ca²âº regimen, as well as spatiotemporal regulation of CDPK expression during early mitotic growth and later during the storage phase of seed development. However, a consistent basal level of CDPK was present during similar developmental stages of pod tissue, irrespective of the soil Ca²âº status. Immunolocalization data showed CDPK decoration primarily in the outer most cell layers of the pericarp and around vascular bundles linked by lateral connections in developing pods, as well as the single vascular trace supplying nutrients to the developing seed. Finally, carbohydrate analyses and qRT-PCR data are provided for peanut genes encoding enzymes involved in sucrose cleavage (orthologs of Vicia faba, VfCWI1 and VfCWI2) and utilization (AhSuSy and AhSpS), and oleosin gene transcripts (AhOleo17.8 and AhOleo18.5) validating a role for CDPK in the establishment and maintenance of sink strength, and subsequent onset of storage product biosynthetic phase during seed maturation.

Postoperative middle cerebral artery peak systolic velocity changes confirm physiological principles of the sequential laser technique for twin-twin transfusion syndrome.

INTRODUCTION: Treatment of the twin-twin transfusion syndrome (TTTS) via sequential selective laser photocoagulation of communicating vessels (SQLPCV) mandates ablation of donor-to-recipient arteriovenous anastomoses first. It is hypothesized that SQLPCV facilitates intraoperative transfusion to the donor, thereby minimizing donor hypovolemia and anemia. We sought to determine if postoperative changes in fetal middle cerebral artery-peak systolic velocities (MCA-PSV) support this hypothesis. MATERIALS AND METHODS: Patients undergoing preferential SQLPCV for TTTS had MCA-PSV measured 1 day before surgery and on postoperative day 1 (POD-1). Fetal anemia was defined as an MCA-PSV ≥1.5 multiples of the median (MoM). Exclusions included: POD-1 demise, missing MCA-PSV data, or gestational age <18 weeks. RESULTS: Study criteria were met by 139 patients. Mean MCA-PSV in recipients increased from 0.97 to 1.15 MoM postoperatively (p < 0.0001). Donor mean MCA-PSV remained stable at 1.00 MoM preoperatively and 0.98 MoM postoperatively (p = 0.272). Nine fetuses, 6 donors and 3 recipients, had preoperative anemia; SQLPCV was not attempted in the 3 anemic recipients. Postoperatively, the proportion of donors with anemia remained stable (increase 3.6%, p = 0.419), and the proportion of recipients with anemia increased (increase 12.2%, p = 0.009). DISCUSSION: Our findings confirm the presumed physiological basis for the SQLPCV treatment of TTTS.

The mechanical PR interval in fetuses of women with intrahepatic cholestasis of pregnancy.

OBJECTIVE: The purpose of this study was to evaluate the fetal mechanical PR interval in intrahepatic cholestasis of pregnancy (ICP). STUDY DESIGN: Fetal echocardiography was performed for women with ICP and control subjects. Clinical characteristics, total bile acids, and liver profile tests were compared between groups. RESULTS: Fourteen women with ICP and 7 control subjects were enrolled. Total bile acids (28.3 vs 6.2 µmol/L; P < .001), aspartate aminotransferase (53 vs 23 IU/L; P = .002), alanine aminotransferase (63 vs 19 IU/L; P = .002), and the PR interval (124 vs 110 msec; P = .006) were significantly higher in fetuses with ICP than in control fetuses. On multivariable linear regression analysis, only the presence of ICP was associated significantly with an increase in the PR interval (95% confidence interval, 4-24 msec; P = .01). CONCLUSION: The fetal cardiac conduction system is altered in ICP. Further investigation is needed to determine whether fetal echocardiography can help to predict which fetuses are at risk for death that is associated with ICP.

Cholestasis of pregnancy.

Intrahepatic cholestasis (ICP) of pregnancy is a disease that is likely multifactorial in etiology and has a prevalence that varies by geography and ethnicity. The diagnosis is made when patients have a combination of pruritus and abnormal liver-function tests. It is associated with a high risk for adverse perinatal outcome, including preterm birth, meconium passage, and fetal death. As of yet, the cause for fetal death is unknown. Because fetal deaths caused by ICP appear to occur predominantly after 37 weeks, it is suggested to offer delivery at approximately 37 weeks. Ursodeoxycholic acid appears to be the most effective medication to improve maternal pruritus and liver-function tests; however, there is no medication to date that has been shown to reduce the risk for fetal death.

Amniopatch as a treatment for rupture of membranes following laser surgery for twin-twin transfusion syndrome.

OBJECTIVE: The risk of iatrogenic rupture of membranes (IROM) is 5-30% after operative fetoscopy. The aim of this study was to describe outcomes of patients with IROM following selective laser photocoagulation of communicating vessels (SLPCV) for twin-twin transfusion syndrome (TTTS) who were subsequently treated with amniopatch therapy. METHODS: A review of patients who underwent treatment for mid-trimester TTTS between March 2006 and February 2008 with IROM within 7 days of SLPCV was performed. IROM patients without evidence of preterm labor or chorioamnionitis were offered expectant management, pregnancy termination, or amniopatch therapy. RESULTS: Ninety-three patients were treated with SLPCV, of which three (3.2%) had IROM within 7 days. All three opted for amniopatch therapy which was performed at 18 2/7, 23 1/7, and 22 6/7 weeks' gestation in patients 1, 2 and 3, respectively. In patients 1 and 2, amniopatch therapy sealed membranes within 7 days. A second amniopatch was required for patient 3 before IROM resolved. Gestational ages at delivery were 38 2/7, 37 5/7 and 30 2/7 weeks, respectively. CONCLUSION: Amniopatch is a viable treatment option for iatrogenic ROM following SLPCV.

Sudden fetal death in intrahepatic cholestasis of pregnancy.

BACKGROUND: Intrahepatic cholestasis of pregnancy is associated with an increased risk of fetal death. The mechanism of death is unknown. CASES: The first case involved a young primipara with pruritus and a bile acid concentration of 79 mumol/dL. While undergoing fetal heart rate monitoring, the fetus had a prolonged deceleration resulting in intrauterine death. The second case involved a young multipara with cholestasis who received ursodeoxycholic acid. Her bile acid concentration improved to13 micromol/dL. At 34 weeks of gestation, she had uterine contractions with prolonged decelerations resulting in delivery of her fetus with Apgar scores of 0, 0, and 5 at 1, 5, and 10 minutes, respectively. CONCLUSION: Fetal death from intrahepatic cholestasis of pregnancy can be abrupt and not reliably predicted by the characteristics of the fetal heart rate tracing.

Pregnancy outcomes during an era of aggressive management for intrahepatic cholestasis of pregnancy.

Our objective was to examine whether delivery at 37 weeks of gestation alters adverse pregnancy outcomes in Latina patients with intrahepatic cholestasis of pregnancy (ICP). We conducted a retrospective chart review of Latina patients who delivered at our institution coded with ICP between 2000 and 2007. During this time period it was our practice to offer delivery to patients with ICP at 37 weeks of gestation. Subjects were classified into three groups according to total bile acid (TBA) concentration: < 20 micromol/L (mild ICP), > or = 20 micromol/L and < 40 micromol/L (moderate ICP), and > or = 40 micromol/L (severe ICP). Meconium passage was observed in no births in patients with mild IC, but was found in 18% of deliveries with moderate/severe ICP. The risk of meconium passage increased linearly, with a 19.7% increased risk for each 10 mumol/L increase in TBA concentration ( P = 0.001). There was no association with higher TBA concentration and other adverse outcomes. There was no difference in adverse outcomes between moderate and severe ICP. We concluded that in our Latina population with ICP, an association existed between meconium passage and moderate/severe ICP. Delivering at 37 weeks was associated with a low risk of adverse outcomes due to ICP among all patients, including those with higher TBA concentrations.