Background
Prenatal alcohol exposure is the most common cause of birth defects and intellectual disabilities and can increase the risk of stillbirth and negatively impact fetal growth.
Objective
To determine the effect of early prenatal alcohol exposure on nonhuman primate placental function and fetal growth. We hypothesized that early chronic prenatal alcohol would alter placental perfusion and oxygen availability that adversely affects fetal growth.
Study Design
Rhesus macaques self-administered 1.5 g/kg/d of ethanol (n=12) or isocaloric maltose-dextrin (n=12) daily before conception through the first 60 days of gestation (term is approximately 168 days). All animals were serially imaged with Doppler ultrasound to measure fetal biometry, uterine artery volume blood flow, and placental volume blood flow. Following Doppler ultrasound, all animals underwent both blood oxygenation level–dependent magnetic resonance imaging to characterize placental blood oxygenation and dynamic contrast-enhanced magnetic resonance imaging to quantify maternal placental perfusion. Animals were delivered by cesarean delivery for placental collection and fetal necropsy at gestational days 85 (n=8), 110 (n=8), or 135 (n=8). Histologic and RNA-sequencing analyses were performed on collected placental tissue.
Results
Placental volume blood flow was decreased at all gestational time points in ethanol-exposed vs control animals, but most significantly at gestational day 110 by Doppler ultrasound ( P <.05). A significant decrease in total volumetric blood flow occurred in ethanol-exposed vs control animals on dynamic contrast-enhanced magnetic resonance imaging at both gestation days 110 and 135 ( P <.05); moreover, a global reduction in T 2 ∗ , high blood deoxyhemoglobin concentration, occurred throughout gestation ( P <.05). Similarly, evidence of placental ischemic injury was notable by histologic analysis, which revealed a significant increase in microscopic infarctions in ethanol-exposed, not control, animals, largely present at middle to late gestation. Fetal biometry and weight were decreased in ethanol-exposed vs control animals, but the decrease was not significant. Analysis with RNA sequencing suggested the involvement of the inflammatory and extracellular matrix response pathways.
Conclusion
Early chronic prenatal alcohol exposure significantly diminished placental perfusion at mid to late gestation and also significantly decreased the oxygen supply to the fetal vasculature throughout pregnancy, these findings were associated with the presence of microscopic placental infarctions in the nonhuman primate. Although placental adaptations may compensate for early environmental perturbations to fetal growth, placental blood flow and oxygenation were reduced, consistent with the evidence of placental ischemic injury.
Introduction
Alcohol freely crosses the placenta and can accumulate in the fetus at levels comparable with maternal blood alcohol concentrations. Prenatal alcohol exposure increases the risk of preterm birth, stillbirth, decreased fetal growth, and fetal alcohol spectrum disorder (FASD), the most common nongenetic cause of cognitive impairment in the United States. , Currently, there is no approved drug to treat FASD or established tool to prevent adverse outcomes. Among pregnant women in the United States, approximately 10% have consumed alcohol in the past 30 days, resulting in more than three-quarters of a million alcohol-exposed fetuses.
Why was this study conducted?
This study was conducted to determine the effects of chronic maternal first-trimester binge drinking on placental function and fetal growth.
Key findings
Early chronic prenatal alcohol exposure significantly diminished placental perfusion at middle and late gestation and significantly decreased the oxygen supply to the fetal vasculature throughout pregnancy in the rhesus macaque.
What does this add to what is known?
This study established correlations concerning reduced comprehensive placental blood flow and oxygen availability on in vivo magnetic resonance imaging, increased placental microinfarctions on histology, and up-regulated inflammatory and extracellular matrix remodeling pathways by RNA sequencing in pregnancies with early alcohol exposure.
The placenta occupies a central role in supporting normal fetal growth and development during pregnancy. Previous studies have suggested that placental dysfunction may contribute to intrauterine growth restriction in FASD, but the mechanisms and specific vasoactive effects of alcohol linking placental dysfunction to disrupted fetal growth remain an area of ongoing scientific exploration. Antenatal ethanol exposure has been previously shown to induce apoptosis in human placental trophoblast cells, disrupt trophoblast cell motility, and potentially affect uterine spiral artery remodeling. Previous studies on pregnant animals using ovine and baboon models have demonstrated abnormal uterine and cerebral blood flow following acute ethanol exposure. Furthermore, a rat model has demonstrated impaired uterine artery vasodilation from chronic binge drinking.
Nonhuman primate (NHP) fetal ontogeny, placental structure, and ethanol absorption and metabolism more closely resemble that of humans than other animal models. Recently, we developed an NHP model of first-trimester ethanol consumption that generated pregnancies from control animals and dams that drank 1.5 g/kg of ethanol daily (∼6 standard drinks for humans). Fetal brain maturation was characterized with in utero magnetic resonance imaging (MRI) and ex vivo slice electrophysiology, which revealed that cerebellar and brainstem fetal growth are diminished, in this model of FASD. Moreover, fetal diffusion MRI indicated an altered maturation of motor-related white matter fiber systems, and these findings were corroborated with altered synaptic development in cortical and striatal regions determined using electrophysiology. Previously, a pilot investigation demonstrated the feasibility and greater sensitivity of in utero MRI directed at the placenta to detect the effects of early-pregnancy drinking on placental function and fetal development over standard clinical Doppler ultrasound (Doppler-US). Placental blood flow was measured using dynamic contrast-enhanced MRI (DCE-MRI) and oxygen exchange was quantified through analysis of water T 2 ∗ values via the blood oxygen level–dependent (BOLD) effect.
Using these in vivo MRI methods at gestational days 110 (G110, term is ∼168 days) and 135 (G135), we observed that early-pregnancy alcohol exposure decreased both placental perfusion and fetal oxygen supply in midgestation and was associated with a decrease in both fetal and brain weight. However, the underlying mechanisms contributing to the observed altered placental function and fetal development were not explored in this work. The primary objective of this study was to utilize the complete set of animals in which placental tissue is available, including at an additional gestational time point (gestational day 85 [G85]), to evaluate the adverse effects of early, chronic prenatal alcohol exposure on placental outcomes and fetal growth. Our second objective was to study the effects of prenatal ethanol exposure on placental histology and gene expression to identify mechanisms underlying placental dysfunction detectable using noninvasive imaging approaches.
Materials and Methods
Experimental design
All protocols were approved by the Institutional Animal Care and Use Committee of the Oregon National Primate Research Center, and guidelines for humane animal care were followed. The generation of pregnancies in the NHP FASD model has been previously published. This study focused on the subset of time-mated pregnant rhesus macaques (n=24) consisting of 12 control and 12 ethanol-exposed animals that underwent placental collection at the time of delivery. Dams were trained to orally self-administer daily either 1.5 g/kg/d of 4% ethanol solution (∼6 drinks per day) or an isocaloric control fluid with training being initiated at least 4 months before undergoing time-mated breeding with plasma estradiol sampling ( Figure 1 ). The day of peak plasma estradiol was defined as gestational day 0 (G0). Each pregnant animal continued daily drinking of 1.5 g/kg/d ethanol ad lib until gestational day 60 (G60), at which point access to ethanol, or isocaloric solution, was removed ( Figure 1 ). All animals underwent Doppler-US followed by MRI consisting of T 2 ∗ and DCE measurements and immediate cesarean delivery with placenta collection and fetal necropsy after imaging at G85 (n=8), G110 (n=8), or G135 (n=8) ( Figure 1 ). Collected placental tissue was processed in liquid nitrogen for molecular analysis, RNAlater (Thermo Fisher Scientific, Waltham, MA) for RNA sequencing (RNA-Seq), and formalin fixation for histology.
Imaging
Doppler ultrasound
All ultrasounds were performed by a single sonographer (J.O.L) using image-directed pulsed and color Doppler equipment (GE Voluson 730; GE Healthcare, Chicago, IL) with a 5- to 9-MHz sector probe. Animals were sedated by intramuscular administration of 10 mg/kg ketamine (Henry Schein Animal Health, Dublin, OH) and maintained on a portable anesthesia delivery system providing O 2 with 1.5% isoflurane. Doppler waveform measurements for the uterine artery and umbilical artery were performed using machine-specific software. The following measurements were obtained: pulsatility index (PI), velocity time integral (VTI), and fetal heart rate (HR) to calculate uterine artery blood flow (cQ Uta ) and placental volume blood flow (cQ UV ) as previously described. , cQ Uta was calculated and corrected by maternal weight as follows: cQ Uta =VTI×CSA (cross-sectional area of the uterine artery)×HR. Placental volume blood flow (cQ UV ) was calculated as follows: mean velocity (V mean )×CSA×60.
Placental magnetic resonance imaging
Immediately following ultrasound, MRI studies were performed on a 3T Siemens TIM Trio scanner (Siemens Medical Solutions, Erlangen, Germany) as previously published. Following localization of the placenta and acquisition of anatomic images in the coronal and axial planes, axial 2-dimensional multislice spoiled multiecho gradient-echo images (repetition time [TR]=418 ms; flip angle=30°; 256×72 matrix; 96 slices; 1.5-mm isotropic spatial resolution) spanning the entire uterus were acquired at 6 in-phase echo times (time to echo [TE]=4.92, 9.84, 19.68, 29.52, 36.90, and 44.28 ms) for T 2 ∗ measurements, and <SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='T1′>𝑇1T1
T 1
was measured with the variable flip angle (VFA) method. After the acquisition of VFA data, 150 3-dimensional spoiled gradient recalled acquisitions in steady-state images were acquired for DCE-MRI (TR=2.00 ms; TE=0.72 ms; flip angle=20°; acquisition time of 3.64 s), with a field of view and resolution matched to the VFA images. Moreover, 10 baseline images were acquired before intravenous injection of 0.1 mmol/kg of gadoteridol contrast reagent (ProHance; Bracco Diagnostics Inc, Princeton, NJ) at a rate of 30 mL/min using a syringe pump (Harvard Apparatus, Holliston, MA). Anatomic and multiecho imaging were performed during expiratory breath-holding, whereas DCE-MRI data were acquired during ventilated breathing. Physiological monitoring of pulse rate, arterial blood oxygen saturation, and end-tidal CO 2 partial pressure was performed throughout, with no deviation from normal ranges observed in these parameters. BOLD and DCE-MRI analyses were performed as previously described.
Placental histology
Formalin-fixed paraffin-embedded histologic sections were stained with hematoxylin and eosin and reviewed by a single placental pathologist (T.K.M.) blinded to exposure and outcomes. Tissue sections were scored for any signs of infection and classic histologic features of maternal vascular malperfusion, including infarctions and accelerated villous maturation.
Statistical analysis
Data are expressed as mean±standard deviation. All animals (n=24) were analyzed, and differences between ethanol-exposed animals and controls at G85, G110, and G135 were tested by a 2-way analysis of variance with a post hoc Tukey comparison; significance was designated at P <.05. The T 2 ∗ results were evaluated using a 2-sample Kolmogorov-Smirnov test.
Gene expression
RNA isolation and quality assessment
Dissected placenta tissue samples (n=24) in RNAlater were delivered to the Oregon Health & Science University Gene Profiling Shared Resource, where phenol-chloroform extraction was performed followed by RNA isolation using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA integrity and size distribution were assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Moreover, 4 samples were excluded because of suboptimal RNA yield.
RNA sequencing
RNA-Seq libraries were prepared using the TruSeq with a ribosomal depletion kit (Illumina, San Diego, CA). The amplified product was profiled on the TapeStation (Agilent Technologies). Libraries were quantified using real-time polymerase chain reaction (Kapa Biosystems Inc, Wilmington, MA) and run on a HiSeq 2500 (Illumina). The resulting base call files were converted to fastq files using bcl2fastq (Illumina).
Gene-level differential expression analysis
Differential expression analysis used standard operating procedures established by the Oregon National Primate Research Center Bioinformatics and Biostatistics Core. The quality of the raw sequencing files was evaluated using FastQC combined with MultiQC ( http://multiqc.info/ ). Trimmomatic was used to remove any remaining Illumina adapters. Reads were aligned to the Ensembl mmul8 in addition to its corresponding annotation, release 87. The program STAR (version 2.7.3a) was used to align the reads to the genome, and RNA-SeQC was used to ensure alignments were of sufficient quality.
The differential expression analysis was performed in the open-source software R (R Foundation for Statistical Computing, Vienna, Austria). Gene-level raw counts were filtered to remove genes with extremely low counts in many samples following the published guidelines, normalized using the trimmed mean of M-values method, and transformed to log counts per million with associated observational precision weights using the voom method. Gene-wise linear models with primary variables treatment group, gestational stage, and their interaction, adjusting for sex and technical factors (RNA processing and sequencing batch), were employed for differential expression analyses using limma with empirical Bayes moderation and false discovery rate (FDR) adjustment.
RNA pathway analysis
A total of 12,879 genes were considered expressed in the 18 placental samples dataset after filtering. To adjust for multiple comparisons, an FDR-adjusted P value of <.2 was used to test for significance because of the small sample size and degree of biologic variation. As is common in longitudinal NHP studies with relatively high variability and small group sizes, few genes were identified as differentially expressed at an FDR of <0.2 in the comparisons between the ethanol and control groups or comparisons among gestational time points, except in ethanol-exposed samples at G135 vs G85. To overcome this limitation, pathway-based patterns were explored to look for effects on gene networks, pathways, and biologic processes because of maternal ethanol consumption. Pathway analysis was performed in Ingenuity Pathway Analysis (IPA). (Qiagen, https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis ). The core analysis option in IPA was used to perform pathway analysis using an unadjusted P value of <.05 and fold change of >1.5 for all pairwise post hoc comparisons.
Results
Growth parameters
Ultrasound measurements of fetal biometry were not significantly different between fetuses exposed to ethanol and controls at all gestational ages ( Table 1 ). There was no significant difference in fetal birthweight at time of delivery in ethanol-exposed fetuses vs control animals at G85 ( P =.5), G110 ( P =.07), and G135 ( P =.1) ( Table 2 ). Maternal weights and fetal sex ratios were not significantly different across treatment groups ( Table 2 ). All major fetal organs were examined by a veterinary pathologist, and no gross structural anomaly was noted in ethanol-exposed pregnancies.
| Parameter | Gestational day 85 | Gestational day 110 | Gestational day 135 | |||
|---|---|---|---|---|---|---|
| Control (n=4) | Ethanol (n=4) | Control (n=4) | Ethanol (n=4) | Control (n=4) | Ethanol (n=4) | |
| BPD (mm) | 29.4±1.4 | 28.3±1.6 | 38.0±1.7 | 36.7±3.5 | 44.5±0.8 | 42.6±2.6 |
| AC (mm) | 9.0±0.3 | 8.9±0.5 | 12.0±0.4 | 11.6±0.7 | 13.7±0.4 | 12.9±1.4 |
| FL (mm) | 17.9±1.2 | 6.8±0.9 | 28.5±0.9 | 27.3±1.2 | 36.6±1.9 | 36.0±0.4 |
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
