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# The Effects of Perinatal Oxycodone Exposure on Behavioral Outcome in a Rodent Model

*Thitinart Sithisarn1 \*, Sandra J. Legan2 , Philip M. Westgate3 , Melinda Wilson2 , Kristen Wellmann4 , Henrietta S. Bada1 and Susan Barron4*

*1Division of Neonatology, Department of Pediatrics, College of Medicine, University of Kentucky, Lexington, KY, United States, 2Department of Physiology, College of Medicine, University of Kentucky, Lexington, KY, United States, 3Department of Biostatistics, College of Public Health, University of Kentucky, Lexington, KY, United States, 4Department of Psychology, University of Kentucky, Lexington, KY, United States*

#### *Edited by:*

*Joav Merrick, Ministry of Social Affairs, Israel*

#### *Reviewed by:*

*Norma Beatriz Ojeda, University of Mississippi Medical Center School of Dentistry, United States Cihad Dundar, Ondokuz Mayis University, Turkey*

*\*Correspondence:*

*Thitinart Sithisarn<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Child Health and Human Development, a section of the journal Frontiers in Pediatrics*

*Received: 12 July 2017 Accepted: 09 August 2017 Published: 25 August 2017*

#### *Citation:*

*Sithisarn T, Legan SJ, Westgate PM, Wilson M, Wellmann K, Bada HS and Barron S (2017) The Effects of Perinatal Oxycodone Exposure on Behavioral Outcome in a Rodent Model. Front. Pediatr. 5:180. doi: 10.3389/fped.2017.00180*

Opiate addiction is now a major public health problem. Perinatal insults and exposure to opiates such as morphine *in utero* are well known to affect development of the hypothalamic–pituitary–adrenal axis of the offspring adversely and are associated with a higher risk of developing neurobehavioral problems. Oxycodone is now one of the most frequently abused pain killers during pregnancy; however, limited data are available regarding whether and how perinatal oxycodone exposure (POE) alters neurobehavioral outcomes of the offspring. We demonstrated that exposure to 0.5 mg/kg/day oxycodone *in utero* was associated with hyperactivity in adult rats in an open field. No significant effects of POE were detected on isolation-induced ultrasonic vocalizations in the early postnatal period or on learning and memory in the water maze in adult offspring. Our findings are consistent with hyperactivity problems identified in children exposed to opiates *in utero*.

Keywords: prenatal opiate exposure, prenatal oxycodone exposure, behavior, hyperactivity, ultrasonic vocalization, learning and memory

# INTRODUCTION

The prevalence of opiates and prescription opioid abuse among pregnant women is a major public health concern. Non-prescription opioids are the second most abused illicit substance and one of the most commonly abused opiate pain relievers is oxycodone (1). In 2015, an estimated 10.4% of the population over 12 years of age used oxycodone products. Importantly, substance use in pregnant women and subsequent fetal exposure to drugs have been linked to adverse health effects for the maternal–fetal dyad. Opiates can affect the developing fetus directly or indirectly through various mechanisms. Opiates can cross the placenta (2–4) and act directly on fetal opioid receptors. Opiates can also enhance maternal secretion of cortisol in the mother or stimulate the secretion of stress hormones in the fetus (5), which can pose long-term effects to the developing fetus (6) and the hypothalamic–pituitary–adrenal (HPA) axis of the offspring (7, 8). In addition, because the HPA axis has important roles in programming neurobehavioral development (9) and dysregulation of the HPA axis has been linked to several neuropsychiatric disorders, such as anxiety (10), depression (11, 12), ADHD (13), and learning/memory deficits (14, 15, 16), any opioid-induced changes in the HPA-axis may also be associated with short- and long-term behavioral problems.

Both human and animal studies have shown that exposure to opiates has deleterious effects on neurodevelopmental outcomes. However, most of them used other opiates, including morphine and heroin, which are mu-opioid receptor agonists, rather than oxycodone, a semisynthetic putative kappa opioid receptor (KOR) and partial mu opioid receptor agonist. Because illegal oxycodone use during pregnancy continue to rise, and the effects of opiates on the HPA axis differ depending on the type of opiate (17, 18, 19), it is important to determine the effects of perinatal oxycodone exposure (POE) on the neurobehavioral outcome of the offspring.

The current study was designed to examine the potential effects of POE on three diverse behaviors; isolation induced ultrasonic vocalizations (USVs) in neonatal pups followed by activity levels in adolescent rats and spatial learning in young adult rats. Isolation-induced USVs are the responses of young rat pups when separated from their mothers (20, 21). USVs are considered an adaptive response of the pup and these USVs correlate directly with distress and/or anxiety in the rat pup (22). USVs elicit maternal behavior and play an important role in the interaction between the pup and the dam (21). USV cues may be comparable to the crying sounds of human infants (23), which have been used to identify infants at risk for poor neurobehavioral outcomes (23, 24). Although existing data on the effects of prenatal opiate exposure on isolation-induced USVs remain limited, neonatal exposure to alcohol (25) or other illicit drugs, such as cocaine, can alter many USV characteristics (26). Therefore, we hypothesized that rat pups prenatally exposed to oxycodone would have an increased latency to the first USV and decreased USV numbers per minute.

The second testing paradigm used was an open field test, which has been widely used to study hyperactivity, anxiety, and stress in animals (27, 28, 29, 30). Although prenatal exposure to morphine was not associated with increased locomotor activity in the open field in rats (31, 32), human studies have shown that children exposed to opiates *in utero* manifest hyperactivity, impulsivity, and attention problems, whether the mothers were in opiate maintenance therapy or were polysubstance users (33, 34). Therefore, we hypothesized that POE would be associated with hyperactivity in the open field in rat offspring.

Learning and memory were assessed in a water maze. Children prenatally exposed to opiates have learning problems and lower scores on neurodevelopmental tests (35, 36, 37). In rats, prenatal exposure to morphine is associated with learning and memory deficits (38, 39). Thus, we hypothesized that POE would impair learning and memory of the offspring. To the best of our knowledge, these studies are the first to look at the effects of POE on these behavioral measures.

# MATERIALS AND METHODS

## Experimental Design: Animals and Prenatal Treatments

The study protocol was approved by the University of Kentucky Institutional Animal Care and Use Committee. Virgin female Sprague Dawley rats (Harlan, Indianapolis, IN, USA) weighing 194–223 g were individually housed at 22–25°C and maintained in a 14L:10D photoperiod (lights on at 0500 h) room with regulated 30–70% humidity. Rat chow and water were provided *ad libitum*.

Once released from quarantine, the females were fitted with a right atrial cannula connected to a subcutaneous (S.C.) access port implanted between the shoulder blades. The rats were allowed to recover for 1 week. To determine estrous cycles, vaginal lavages were obtained daily. Each female was group housed with a proven breeder male for breeding 1 week after cannulation. Gestational day (GD) 0 was designated as the day that sperms were detected in the vaginal smear, and the females were individually housed thereafter. Foster dams were bred at the same time and remained untreated throughout their gestation.

The cannulae were flushed daily *via* the S.C. port with sterile heparinized saline (0.4 cc, 100 IU/ml) until GD 8. From GD 8–21, the experimental dams were divided into three treatment groups that received either oxycodone (Mallinckrodt, St. Louis, MO, USA) at a low (OXY-L, 0.5 mg/kg/day, *n* = 5) or high dose (OXY-H, 2.0 mg/kg/day, *n* = 12) or an equivalent volume of vehicle [control (CON), normal saline solution (NSS, 1.0 ml/kg/day), *n* = 12] from GD 8–21. These solutions were slowly injected i.v. over 10 min *via* the S.C. access port manually.

On postnatal day (PD) 1, the pups were counted and weighed. Oxycodone or NSS was also administered on PD 1, 3, and 5 to the dams because brain development during early postnatal life overlaps the human "third trimester" brain growth spurt and to prevent maternal withdrawal symptoms that might affect maternal nursing behavior. On PD 2 all litters were adjusted to contain 10–11 pups with equal numbers of male and female pups when possible. At 1700 h on PD 5, all pups in each litter were transferred to an untreated foster dam to minimize exposure to altered maternal rearing behavior that has been described in rat dams after exposure to opiates (40). To preclude potential litter effects, only one male and one female from each litter were included in the behavioral studies (41). The pups were weighed daily and weaned at PD 25, when they were separated by sex.

#### Experimental Design: Behavioral Tests Ultrasonic Vocalizations

Ultrasonic Vocalizations were determined according to published procedures (42). In brief, an ultrasonic bat detector (Ultra Sound Advice Model #S-25, UK—http://www.ultrasoundadvice.co.uk) set at 40 kHz with a condenser microphone (SM-1) set 21.5 cm above the test cage floor was used. The output was recorded on a SONY #WM-D8C Cassette Recorder using low noise cassette tapes. On PD 14, the pups in each litter were separated from the dams, remaining in their home cage and were brought to a neighboring test room one litter at a time. Their cages were placed halfway on a heating pad to provide warmth. During testing, the dam was placed in a new cage in the same test room. CON offspring (*n* = 6) and OXY-L (*n* = 4) and OXY-H (*n* = 8) rats underwent USV testing. The number of pups in the OXY-L group was smaller due to smaller numbers of dams in this group and limited numbers of pups available for all experiments that were performed. During USV testing, pups were isolated one at a time in a clean cage for 6 min during which USVs from the pup were recorded. A fan was used to provide white noise during testing. Upon completion of USV testing for the entire litter, the dam was returned to the home cage and the home cage was returned to their original animal room.

Assessment of USV: audio data for each 6-min test period were individually scored for latency to vocalization (seconds) and number of USVs per minute per test period. Scoring was performed independently by two experimenters who were blind to the treatment groups and used a stopwatch and clicker counter. Similarity of their scores was compared and indicated that the reliability between experimenters was greater than 90%.

#### Open Field Test

On PD 43, a group of offspring that had not undergone USV testing were transferred to another building, where they were housed for at least 7 days prior to subsequent behavioral testing. This was an independent group of subjects, i.e., not the same offspring that had previous USV testing. The open field test was preceded by 5 days of habituation to the new surroundings followed by 2 days of 3-min handling periods and weighing. CON offspring (*n* = 30, 19 males, 11 females) and OXY-L (*n* = 8, 4 males, 4 females) and OXY-H (*n* = 32, 17 males, 15 females) rats underwent open field tests on PD 50–51. The number of pups in the OXY-L group was smaller due to limited numbers available after other experiments (not described herein) were performed. The open field apparatus was a circular chamber 36 cm height and 58 cm diameter. This circular chamber was used to prevent thigmotaxis or excessive time in corners (43). In addition, the open field was divided into two zones, with the center zone representing 25% of the total area. All testing was conducted in a test room with white noise to reduce external distractions. On days 1 and 2 of testing, two rats were transferred in separate cages and brought into the test room for a 10 min habituation period. Each animal was then placed in a separate open field, and their activities were recorded with a Polytracker® (San Diego Instruments, San Diego, CA, USA) for 30 min. The animals were then returned to their home cages in the colony room upon completion of testing.

Activity was recorded in 5 min blocks across the 30 min test period for each day. Total distance traveled (cm), distance traveled in the center zone, and the ratio of distance traveled in the center zone to total distance traveled or to distance in the outer zone were determined.

#### Water Maze

On PD 55–56, two groups of offspring, CON (*n*= 20, 11 males, 9 females) and OXY-H (*n* = 20, 10 males, 10 females) were tested in the water maze. Rats from the OXY-L group were not tested due to the limited numbers of pups. This experiment was modified from a procedure previously described (43). The apparatus was a 130 cm × 90 cm × 40 cm black Plexiglas chamber, divided such that several divergent paths, each 18 cm wide, branched off from the central start area. The apparatus and methodology were modified from von Euler et al. (44). Water temperature was maintained at 76° ± 2°. In this test, rats must learn to swim and make three successive right/left choices to a platform that is submerged below the water level and invisible; the water was made black with the addition of non-toxic black tempera paint to obscure the submerged platform. A plastic sheet surrounded the maze, reducing extra-maze cues, including the experimenter. A major advantage of this maze is that control animals can learn the maze in a single day. Movement in the maze was recorded using a video tracking system (SMART program; Panlab, S.L.) (43).

On the first trial, the rat was placed in the maze and allowed to swim freely. If the animal did not reach the platform after 1 min, it was guided to the platform. After 5 s on the platform, the animal was transferred to a cage warmed by a heat lamp (25 W) for 30 s. Trials were repeated until the animal completed two consecutive trials without errors (wrong turns) or a total of three successful trials. The number of trials required to reach either criterion was recorded as the outcome measure. The next day the rats were tested identically for 24 h to determine retention.

#### Statistical Analyses

Statistical analyses were considered significant if *p* < 0.05. Multilevel linear regression models or linear mixed effects models were used to analyze USVs, open field and water maze data. Such models accounted for potential litter effects and correlation among outcomes from the same pup over time. The models were fitted using restricted maximum likelihood to test for the impact of treatment group, gender, time, and any potential interactions these variables have on the mean values for the data. Due to skewness and outliers of the data, a natural log transformation was applied when appropriate. The Kenward and Roger (45) approximation was used to estimate standard error and degrees of freedom. Variables (trial days, sex, and treatment groups) were treated as categorical and backward elimination at the 5% significance level was utilized. Kruskal–Wallis tests were applied to compare litter sizes and number of males per litter between treatment groups. A linear mixed-effects model was applied to compare differences in body weight between groups with treatment group, gender, PD, and the interaction of gender and PD included as predictors of weight. Tests were two-sided and were conducted in SAS version 9.4 (SAS Institute, Cary, NC, USA).

#### RESULTS

#### Litter Size and Body Weight

Perinatal oxycodone exposure did not affect litter size or number of males and females per litter (**Table 1**).

Although the average maternal weight gain during pregnancy was different between groups (*p* = 0.005), perinatal oxycodone treatment did not affect mean birth weight of male or female pups weighed within 24 h after birth (**Table 1**). Dams in the OXY-H group gained less weight than CON dams (*p* = 0.01) and OXY-L dams (*p* = 0.023) (**Table 1**). Oxycodone treatment did not affect weight gain of the pups; body weights up to PD 50 were not different between groups (*p* = 0.61). However, there was an interaction between gender and postnatal age (*p* < 0.0001); males and females had different growth trajectories over time, as expected (**Figure 1**).

Body weights of both male and females were similar across treatment groups in the rats tested in the open field and water maze (*p* = 0.43), the females weighing less than the males as expected (*p* < 0.0001) (**Table 1**). The body weight of the pups at the time of USV testing (PD14) (g ± SEM) were 30.9 ± 1.8 in male and 29.1 ± 1.6 in female CON group; 30.2 ± 1.9 in male and

TABLE 1 | Litter size, the number of male and female rat pups per litter, maternal weight gain during pregnancy and birth weight of the pups and weight of the young adult offspring at the neurobehavioral tests (g ± SEM).

*CON, control; OXY-H, oxycodone high dose; OXY-L, oxycodone low dose.*

*n* = 81), oxycodone low dose (OXY-L, open circles, *n* = 42) and oxycodone high dose (OXY-H, closed triangles, *n* = 76) groups. Right panel: body weight of female pups from birth through PD 50 in control (CON, closed circles, *n* = 65), OXY-L (open circles, *n* = 30) and OXY-H (closed triangles, *n* = 84) groups.

28.1 ± 1.9 in female OXY-L group; and 30.1 ± 1.4 in male and 28.7 ± 1.1 in female OXY-H group.

 There was no significant difference in the latency to the first vocalization between treatment groups; however, there was a trend for longer latencies in OXY-exposed rats, **Figure 2A**. Latency to the first USV as plotted in Kaplan–Meier Plots is shown in **Figure 2B**, a point on the plot denotes the estimated probability of having a first USV after the given time point. Although there was a trend for prenatal oxycodone-exposed pups to display longer latencies to their first USV after isolation, there were no significant differences between treatment groups (*p* = 0.25), likely due to small sample sizes, **Figure 2B**.

Total USVs across time also did not differ among the three treatment groups (*p* = 0.85), **Figure 2C**. The number of USVs increased across time in all treatment groups (*p* = 0.0004); however, there was no significant effect of perinatal OXY exposure (*p* = 0.32), **Figure 2D**.

#### Open Field Test Analysis of Total Distance Traveled in 30 Min

Analysis of the raw data indicated that there were no statistically significant differences in total distance traveled among rats in CON, OXY-L, or OXY-H groups (*p* = 0.26). There was also no effect of test day on the total distance traveled (*p* = 0.48). No interactions were significant. Female rats traveled a greater total distance compared to males [traveled 1,654 cm further than males, 95% CI: (539, 2,768), *p* = 0.004]. After a natural log transformation of the data, because of outliers and skewness, it was similarly determined that females traveled a greater total distance than males (*p* < 0.0001). In addition, this transformation of the data revealed that the OXY-L rats traveled farther than rats in both other treatment groups (vs OXY-H, *p* = 0.011; vs CON, *p* = 0.0004, **Figure 3**). This difference was likely largely driven by OXY-L-treated males on day 2.

There was no main effect of perinatal treatment or test day (*p* = 0.10 and *p* = 0.07, respectively) on the total distance per 5 min block. However, females travel 241 cm further than males in any given 5 min period (*p* = 0.003). The mean total per 5 min time block decreased over time (*p* < 0.0001), **Figure 3**.

#### Analysis of Total Distance Traveled in the Inner Zone, Outer Zone, and Mean Ratio of Distance Traveled in the Inner/Outer Zone

period in CON (closed circles), OXY-L (open circles), and OXY-H (closed triangles) groups.

#### *The Inner Zone*

Rats exposed to OXY-L traveled more in the inner zone than rats in the other two treatment groups (OXY-L vs OXY-H, *p* = 0.011; vs CON, *p* = 0.003), likely due to the travel of the OXY-L-treated males on day 2, **Figure 4**. The distance traveled in the inner zone did not differ between days (*p* = 0.61), and no interactions were significant.

Oxycodone treatment did not affect the distance traveled in the inner zone per 5 min block, **Figure 4**. The large standard errors were likely due to the small sample size.

#### *Outer Zone*

There were no significant interactions. Neither oxycodone treatment nor testing day impacted the mean total outer zone distance (*p* = 0.35 and *p* = 0.29, respectively), **Figure 5**. After natural log transformation and removal of two outliers, there were main effects of gender (*p* < 0.0001) and treatment group (*p* = 0.005). Namely, males traveled to the outer zone less than females (*p* = 0.003) and OXY-L rats traveled more in the outer zone than the CON group (*p* = 0.002, **Figure 5**). There was no difference in outer zone distance traveled between either CON or OXY-L compared to OXY-H, **Figure 5**.

There were no interactions in outer zone distance per 5 min and no effect of oxycodone treatment (*p* = 0.15). Females travel 194 cm further than males in the outer zone in any given 5 min period (*p* = 0.01), **Figure 5**. In addition, rats tended to travel 113 cm further on the first test day in any given 5 min time period (*p* = 0.032), **Figure 5**. The mean total distance traveled in the outer zone per 5 min period decreased over time as expected (*p* < 0.0001), **Figure 5**.

#### The Ratio of the Distance Traveled in the Inner Zone to Outer Zone

The ratio of activity in the center zone vs outer zone of the field is a potential marker of motor impulsivity and/or anxiety (46). There were no differences in mean ratios across test days (*p* = 0.18) but there was a treatment group × sex interaction (*p* = 0.003; sex × treatment group interaction). Specifically, the estimated mean ratio of the distance traveled in the inner to outer zone of male OXY-L rats which was 0.245, was 0.095 larger than that of the CON, *p* = 0.008, and 0.112 larger than for OXY-H, *p* = 0.002 (**Table 2**). This pattern differed across sex; no differences in the inner:outer ratio were observed in the females (estimated mean ± SEM for days 1 and 2 in CON: 0.13 ± 0.01, OXY-L: 0.10 ± 0.05, and OXY-H: 0.17 ± 0.02) (**Table 2**). The differences in the males persisted when the ratios of distance traveled in the inner to outer zone for each 5 min time block were

analyzed (OXY-L vs CON: *p* = 0.04; OXY-L vs OXY-H = 0.007), **Figure 6**.

oxycodone high dose (OXY-H, closed triangles; male *n* = 17, female *n* = 15) groups.

#### Water Maze

Using a multivariate repeated measures Gaussian linear model to analyze the data with trial days, treatment groups and sex as predictor categories, there was no effect of oxycodone or sex on the mean values of the average number of trials until criterion performance was reached on both days (*p* = 0.62). The expected decrease in the mean values of the average number of trials on day 2 was observed in all groups (*p* < 0.0001) (**Figure 7**).

# DISCUSSION

We explored the effects of POE on behavioral outcomes using three behavioral tests (USV, open field, and water maze) in juvenile, adolescent, and young adult rats. POE increased locomotion and preference for the center in the open field, consistent with hyperactivity. However, we did not find any effects of POE on USVs of rat pups when separated from their mothers or on learning and memory in the water maze.

# Open Field Test

Oxycodone low dose rats were hyperactive and traveled more in the inner zone of the open field compared to CON and OXY-H rats, resulting in a higher mean ratio of inner:outer zone distance traveled by the OXY-L males. Thus, perinatal exposure to oxycodone was associated with hyperactivity behavior in adolescence. In addition, the increase in the ratio of inner zone:total distance traveled suggests that perinatal oxycodone reduced anxiety-like behavior and decreased normal species-typical thigmotaxic behavior in a new environment. Alternatively, these differences might reflect overall hyperactivity with a failure to inhibit entries to the center. It remains to be determined whether the increased travel in the center is due to deficits in hyperactivity, impulse control, or reduction in anxiety.

Our finding that perinatal exposure to oxycodone was associated with hyperactivity in the offspring is in agreement with human studies that identify hyperactivity, impulsivity, and attention problems in children exposed to opiates *in utero* (33, 34). In animal models, however, the results are more conflicting due to differences in studied drugs, which were mainly morphine, as well as in paradigms, ages, instruments, and end

points. For example, in one study, postnatal handling but not prenatal morphine exposure increased locomotor activities in the open field in adult male and female rats (31). In contrast, others reported that postnatal stress but not prenatal exposure to morphine increased locomotor activity in the open field (32). Thus, morphine yields different results than oxycodone in the open field. However, it should be noted that the effects of morphine are contradictory among studies and differ among tests. Thus, prenatal exposure to morphine was associated with increased anxiety-like behavior in an elevated plus maze (EPM) and reduction in time spent in the lit side of a light/dark box (L/D box) (47). Others reported that rats prenatally exposed to morphine exhibited decreased anxiety-like behavior in the EPM and L/D box with no differences in distance traveled over 30 min in the open field (48). Another study reported no significant anxiogenic effect of prenatal morphine exposure determined in the EPM (49). Finally, prenatal exposure to several opiates, such as methadone, buprenorphine, and most effectively morphine, increased anxiety-like behaviors in the light–dark transition test, with no effect on locomotor activity in an open field (50).

A possible mechanism by which prenatal exposure to opiates could result in hyperactivity may involve changes in multiple neurotransmitter signaling pathways such as dopaminergic pathways and the HPA system. It is well described that opioids have a significant role in controlling the release of dopamine and acetylcholine in the key reward regions of the brain including the ventral tegmental area (VTA) and the nucleus accumbens (51, 52, 53, 54). Long-term exposure to opiates leads to both structural and biochemical changes in the mesolimbic dopaminergic system; for example, a reduction in the cell size of dopaminergic neurons in the VTA (55), and increased levels of tyrosine hydroxylase, which is the rate-limiting enzyme in the synthesis of dopamine in the VTA (56). In addition, convincing evidence suggests that the impairment of dopamine-mediated development and the monitoring of motivated behavior and reward-related memory formation might be associated with ADHD symptoms (57, 58). Thus, taken together, it is possible that perinatal exposure to opiates may disrupt the normal development of dopaminergic reward-related circuits leading to hyperactive behavior. However, this speculation remains to be further elucidated.

Changes in the HPA system have also been linked to ADHD symptoms. In children, both reduced basal cortisol secretion and cortisol hyporeactivity have been associated with

FIGURE 5 | Total distance traveled in outer zone in each 5 min time block in the open field on test day 1 (upper panel) and test day 2 (lower panel), by male (left panel) and female (right panel) rats in control (CON, closed circles), oxycodone low dose (OXY-L, open circles), and oxycodone high dose (OXY-H, closed triangles) groups.

*\*p* < *0.05 {the estimated mean ratio of distance traveled in the inner to outer zone of male OXY-L rats which was 0.245, was estimated to be 0.095 larger than that of the CON [95% CI: (0.027, 0.162), p* = *0.008], and 0.112 larger than for OXY-H [95% CI: (0.042, 0.1830, p* = *0.002)]}.*

*CON, control; OXY-H, oxycodone high dose; OXY-L, oxycodone low dose.*

hyperactivity/impulsivity or a combined type ADHD (59). An abnormal diurnal rhythm and less effective negative feedback mechanisms after a dexamethasone suppression test were also identified more frequently in the children with ADHD that were severely hyperactive compared to those with milder symptoms (60). In agreement with many community-based

FIGURE 6 | The ratio of the distance traveled in the inner/outer zone in 30 min in the open field (mean ± SEM) by male (left panel) and female (right panel) rats in control (CON, closed circles), oxycodone low dose (OXY-L, open circles), and oxycodone high dose (OXY-H, closed triangles) groups. Male OXY-L rats had overall higher ratios of distance traveled in the inner to outer zone for each 5 min time block across time compared to other treatment groups (OXY-L vs CON: *p* = 0.04; OXY-L vs OXY-H = 0.007).

studies, in which ADHD is more prevalent in males (61), in our study hyperactivity was more notable in the male OXY-L group. Interestingly, in adults with ADHD, although the basal salivary

cortisol levels were not different, cortisol levels 20-min after a mental cognitive stress test were higher than those in healthy adult controls (62). These findings are similar to our observations that POE is associated with increased CRH-induced ACTH release (8) and with increased corticosterone reactivity to restraint stress in adult female rats (unpublished). However, we did not measure corticosterone, the major circulating glucocorticoid in rats, during the behavioral tests in the current studies. The discrepancy between hyperactivity in males and increased levels of corticosteroids in females could be due to the methods used to produce the stress in each study, relatively small sample sizes, and/or the gender of the participants. Referral biases and methodological difficulties were present in other human studies (63, 64). Thus, the relationship between POE, hyperactive behaviors, and abnormal HPA axis function that may interact with gender warrants further investigations, which should include the measurement of corticosterone during and after neurobehavioral testing. The abnormal HPA axis response to stress may be a promising candidate for use as a biomarker for early detection and treatment of ADHD in infants prenatally exposed to oxycodone/opiates.

Data from both animal and human studies suggest that early nutritional stress and malnourishment are associated with anxiety, high impulsivity, and attention problems (65). Although the weight gain of the OXY-H dams was significantly lower than that in the other groups, the weight gain of the OXY-L dams was actually comparable to that in the CON group. In addition, birth weights of the pups were comparable in all groups. Therefore, the hyperactivity in the open field of the OXY-L group could not solely be explained by poor maternal nutritional status.

#### Ultrasonic Vocalizations

Although there was a trend toward increased latency to first vocalization, oxycodone treatment had no effect on this end point or on the number of vocalizations per minute. The lack of differences in these USVs parameters may be due in part to the relatively low sample size in the OXY-L group. To our knowledge, there are no reports thus far on the effects of perinatal oxycodone on USVs In contrast to our results, however, perinatal cocaine treatment decreased the number of USVs on PD 1 and on PD 21, but this effect was not observed on PD 14 (66). Therefore, it is possible that the lack of an effect of oxycodone in our study occurred because testing was limited to PD 14. In fact, there are other types of USVs based on frequency and duration that are elicited as the rat becomes more mature (67). These include a 22 kHz USV emitted by juvenile and adult rats in response to predators and pain indicating a negative affective state (68, 69), and a 50 kHz USV emitted in adult rats in response to rewarding stimuli, expressing a positive affective state (70, 71). So it is also possible that POE may affect these other types of USVs that were not tested at an older age.

#### Water Maze

We did not find any effects of POE on spatial learning and/or memory in the water maze test as hypothesized; this may have resulted from not including rats perinatally exposed to a lower dose of oxycodone in this experiment due to the small sample size of this group. Previous studies using prenatal morphine exposure models report conflicting results; memory and learning in rodents are either impaired or enhanced. Namely, juvenile rats prenatally exposed to morphine had impaired spatial memory in the Morris water maze or Y-maze test (38, 72, 73), but aberrant memories such as morphine reward memory in the conditioned place preference or forced swim tests were enhanced (49, 74–76). In contrast to our study, Davis et al. (39) found that prenatal exposure to oxycodone impaired spatial learning and memory in a battery of spatial tasks; in the Morris water maze, rats prenatally exposed to OXY had increased latency and greater distance traveled to find the platform when the intertrial interval was long, not short. Rats prenatally exposed to oxycodone also had a decreased use of spatial strategies and more use of non-spatial strategies such as wall-hugging. In addition, the retention of learning memory in the T-maze, assessed 5 days after acquisition of the training, was impaired. This finding is actually consistent with our report that although POE male rats were able to discriminate between the stress and non-stress cues during a classical conditioning paradigm on PD 40, they had impaired discrimination ability when retested on PD 75 (77). Moreover, rats prenatally exposed to oxycodone have more reference memory errors in the radial arm maze (39). These differences in results could be due to many reasons. In their study, the dose and route of administration of OXY were different than those used in the current experiment with escalating doses of OXY (10 mg/kg/day up to 15 mg/kg/day) *via* gavage for 28 days prior to breeding. In addition, the pups were reared by their biological mothers in their study, while surrogate fostering was used in the current study. A number of previous studies have shown that opiate administration negatively alters maternal rearing behavior toward the pups including measures such as cleaning of the pups, delay of maternal behaviors, and maternal aversion to pup odor (78, 79, 80). Neonatal rearing condition and neonatal maternal interaction such as maternal separation had long-term effects on the stress response of the offspring including an increase in restraint stressinduced norepinephrine release in the PVN in adult rats (81) and changes in the HPA axis at multiple levels that could be linked to epigenetic modification (82). Variations in maternal care also influence learning and behaviors of the offspring (83, 84). The adverse effects of neonatal maternal separation on the HPA axis were lessened by fostering the litters (85). Thus, fostering the pups when the dams were exposed to opiates could alter the HPA axis and possibly the neurobehavioral outcomes of the offspring. In addition, even though signs of withdrawal in the dams were monitored in the study by Davis et al., body weights of the OXY pups were approximately 10% lower than those of the controls, indicating possible neonatal opiate withdrawal and poorer nutritional status that can also affect long-term outcomes. In contrast, the body weights of the pups in our study were comparable in all groups.

In humans, prenatal exposure to opiates results in impairments in cognitive function and learning. Bunikowski et al. also reported that when evaluated at 1 year of age, children prenatally exposed to opiates had a mild psychomotor developmental impairment compared to the control group; these included impairments in "hearing and speech" and "intellectual performance" subscales (36). Guo et al. found that *in utero* opiate exposure was associated with impairments in the Auditory Rare Event Monitoring task and the Sternberg Memory task in children 7–12 years of age (35). More recently, Hunt et al. reported from their case–control study that infants prenatally exposed to opiates are more likely to experience neurodevelopmental impairments compared to healthy control infants, when assessed at 18 months and 3 years of age (37). The deleterious effects of prenatal opiate exposure on cognitive function persisted and did not decrease over time after controlling for permanent home placement and heroin use in the mother when children prenatally exposed to opiates were retested on the Wechsler Intelligence Scale for Children from 1 year old up to 8.5 years of age (86). These data suggest that prenatal exposure to opiates is associated with impaired cognitive functions. Therefore, although we did not detect any effects of POE on cognitive function, learning, and memory using a water maze as a paradigm, further investigation is warranted with different testing paradigms, testing at different ages, and with a lower dose of oxycodone.

Interestingly, we found that exposure to the lower dose of oxycodone of 0.5 mg/kg/day but not the higher dose of 2.0 mg/ kg/day was associated with hyperactivity in the offspring. This difference could be due to the development of tolerance to the

#### REFERENCES

higher dose of oxycodone. Opiate tolerance is characterized to be pharmacodynamic, time and dose-dependent, and opioid receptor specific (87). Tolerance can develop after exposure to a KOR agonist (88) even for as short as 5 days (17). Whether or not tolerance to the stimulatory effects on the HPA axis by KOR agonists is dose-dependent has not been well studied, but tolerance to MOR agonists was dose-related (89). It is possible that in our study, rat dams that were exposed to the higher dose of oxycodone (2 mg/kg/day) may have developed opioid tolerance leading to a decreased fetal CORT exposure, thus there were fewer effects on the developing HPA axis and the neurodevelopmental outcomes of the offspring.

In summary, POE was associated with hyperactivity in young adult rats. In these studies, perinatal oxycodone treatment had no effect on the responses of pups isolated from their dams, or in learning and memory deficits, which could be due to the small sample sizes, testing paradigms, or the doses of oxycodone tested. These issues remain to be resolved.

#### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of the University of Kentucky Institutional Animal Care and Use Committee. The protocol was approved by the University of Kentucky Institutional Animal Care and Use Committee.

# AUTHOR CONTRIBUTIONS

TS: designed and conducted the studies, analyzed the data, wrote the manuscript, PI of the funding. SL and MW: assisted in designing, conducting, and analyzing the data and edited the manuscript. PW: statistician, responsible for all data analysis. KW: conducted and analyzed the data. HB: assisted in designing and analyzing the data. SB: main assistant in designing, conducting, and analyzing the data and edited the manuscript.

# FUNDING

This study is funded by Children's Miracle Network Grant, #1012122240.

effect. *Psychoneuroendocrinology* (1986) 11(2):177–84. doi:10.1016/0306- 4530(86)90052-1

89. el Daly ES. Influence of acute and chronic morphine or stadol on the secretion of adrenocorticotrophin and its hypothalamic releasing hormone in the rat. *Life Sci* (1996) 59(22):1881–90. doi:10.1016/S0024-3205(96)00535-8

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Sithisarn, Legan, Westgate, Wilson, Wellmann, Bada and Barron. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Neonatal abstinence syndrome: twins Case series

*Rajesh Pandey <sup>1</sup> \*, Narmada Pandey Sapkota2 and Deepak Kumar <sup>3</sup>*

*1Department of Pediatrics, University of Texas, Health Science Center at Houston, Houston, TX, United States, 2Cleveland State University, Cleveland, OH, United States, 3Department of Pediatrics, MetroHealth Medical Center, Cleveland, OH, United States*

Background: Use or abuse of opioids and related drugs during pregnancy increases the risk of maternal and neonatal morbidities, including increased susceptibility to Neonatal Abstinence Syndrome (NAS). Severity of NAS is determined by both environmental and genetic factors, but the level of influence each one of them has in determining the severity of NAS is not fully understood. Since the incidence and severity of NAS vary a lot among susceptible infants, twin studies might give us valuable insights into understanding the relative roles of environmental and genetic factors at the onset of the disease and during its progression. Higher concordance of occurrence and severity of NAS in monozygotic twins compared to dizygotic twins would suggest a genetic role in the pathogenesis of NAS. However, comparable concordance suggests a non-genetic or an environmental basis. In this case series, we report neonatal outcomes including severity of NAS among monozygotic and dizygotic twins.

#### *Edited by:*

*Jean Marc Guile, University of Picardie Jules Verne, France*

#### *Reviewed by:*

*Paula G. Radmacher, University of Louisville, United States Eduardo Daniel Rosas-Blum, Texas Tech University Health Sciences Center, United States*

#### *\*Correspondence:*

*Rajesh Pandey<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Child Health and Human Development, a section of the journal Frontiers in Pediatrics*

*Received: 30 July 2017 Accepted: 24 October 2017 Published: 22 December 2017*

#### *Citation:*

*Pandey R, Pandey Sapkota N and Kumar D (2017) Neonatal Abstinence Syndrome: Twins Case Series. Front. Pediatr. 5:242. doi: 10.3389/fped.2017.00242*

Methods: A retrospective chart review was performed for all newborn twins who were at risk of developing NAS, were born in our institution between January 2006 and December 2014, and had a gestational age of 30 weeks or greater.

Results: During the study period, we identified seven sets (total of 14 infants) of eligible twins, comprising six dizygotic and one monozygotic twins, from a total of 550 infants who were at risk of developing NAS. Among the seven sets of twins, two sets were concordant for severe NAS and required pharmacological management, three sets of twins were concordant in not having severe NAS and did not require pharmacological management, and the remaining two sets were discordant, where one of the twins required pharmacological treatment.

Conclusion: Five of the seven sets of twins in our study exhibited concordance and two sets showed discordance in withdrawal severity. Larger studies may help in understanding the roles of genetic and environmental factors in determining the severity of NAS.

Keywords: neonatal abstinence syndrome, intrauterine opioid exposure, severity of neonatal abstinence syndrome, dizygotic twins, monozygotic twins, newborn, genetic, determinants of neonatal abstinence syndrome

# INTRODUCTION

Maternal use, misuse, and abuse of prescription and nonprescription opioids and other illicit drugs, including heroin and cocaine, are increasing in the United States (1–3). With increasing drug use, risks of various maternal and neonatal morbidities such as preterm birth, low birth weight, possible congenital anomalies (4), neonatal abstinence syndrome (NAS), and adverse neurodevelopmental outcomes have increased (5, 6).

The incidence and severity of NAS have been variably reported to occur in 50–90% of infants following intrauterine exposure to drugs that are known to cause NAS (5, 6). Among the infants with severe NAS, there is a wide variation in the length of their pharmacological treatment, duration of hospital stay, need for intensive care, and other morbidities associated with NAS (5, 6), but what determines the variations in outcome is not completely known.

Twin studies are a powerful tool in understanding the roles of genetic and environmental factors in determining the incidence and severity of diseases. Incidence of twins is about one in eighty in spontaneous pregnancy, and incidence of NAS is estimated to be 3.4–5.6 per 1,000 births, resulting in about 21,000 infants being susceptible to NAS (2). Based on these data, we calculated that, annually, about 260 pairs of twins born in USA are susceptible to NAS. Twins can be dizygotic (non-identical) or monozygotic (identical). Dizygotic twins share about 50% of the genetic material while monozygotic twins share almost 100%. The composition of placenta and membrane—amnion and chorion—helps in determining whether twins are monozygotic or dizygotic. All dizygotic twins have a dichorionic-diamniotic membrane. Monozygotic twins can be dichorionic-diamniotic (about a third of monozygotic twins), monochorionic-diamniotic, or monochorionicmonoamniotic, depending on the time of splitting of the fertilized egg. If dichorionic-diamniotic twins are of the same gender, have the same blood group, and appear identical, they are usually designated as monozygotic twins in twin studies. Higher concordance of incidence of disease in monozygotic twins compared to dizygotic twins suggests a genetic basis of the disease (7).

We hypothesized that twin studies might give us valuable information in understanding the relative roles of environmental and genetic factors in the onset and progression of NAS where incidence and severity of disease vary widely among susceptible infants. If concordance of occurrence of NAS or severity of NAS is greater in monozygotic twins than in dizygotic twins, it would suggest a genetic basis of pathogenesis of NAS. However, lack of higher concordance among monozygotic twins compared to dizygotic twins would suggest non-genetic or environmental basis in pathogenesis of NAS. In our study, we have defined "infants at risk of NAS" as infants with intrauterine exposure to opioids and related substances. We have defined "severe NAS" as NAS requiring pharmacological management. Also, we have classified twins having concordance pattern of NAS if both or neither of the twins showed severe NAS, and discordance pattern of NAS if one of the twins, but not the other, exhibited severe NAS.

# MATERIALS AND METHODS

We mined the database of MetroHealth Medical Center's Mother and Child Dependency Program—a multidisciplinary treatment program for drug-dependent mothers and their infants—to identify all twins born at a gestational age (GA) of 30 weeks or greater, who were at risk of developing NAS, and were born between January 2006 and December 2015. This study was approved by the Institutional Review Board. We collected the maternal and infants' demographics and other factors related to severity of NAS, including need and duration of pharmacological treatment, duration of hospitalization, mean NAS scores (modified Finnegan score) in the first, second, and third week of life, maximum NAS score, and cumulative dose of Morphine: the first-line medicine for treatment of NAS at MetroHealth. In demographics, we collected information including gender, birth weight, GA at birth, and types of twins: monozygotic or dizygotic. We also collected details about intrauterine exposure of the infants to methadone, buprenorphine, heroin, cocaine, marijuana, tobacco, and drugs used for treatment of underlying psychiatric conditions in the mother. Additionally, we collected information on the type of feeding: breast feeding or formula feeding. Amnionicity and chorionicity was determined based on obstetric ultrasound and, when necessary and available, infant's blood group and gender were also used to designate zygosity of twins.

# CHARACTERISTICS AND OUTCOME OF SEVEN SETS OF TWINS

#### First Set of Twins

This set of diamniotic-dichorionic (dizygotic) twins was born at 38 weeks of GA to a 30-year-old Caucasian woman through emergency cesarean section for non-reassuring fetal heart tracing. She had a history of heroin use (both snorting and intravenous routes), cocaine use, and tobacco smoking, as well as a history of anxiety and depression, which were not treated. She was in the Methadone maintenance program, taking 45 mg methadone daily at the time of delivery. Her last reported illicit drug use was more than 4 months prior to delivery. She tested negative for Hepatitis B, Hepatitis C, and human immunodeficiency virus (HIV). Both twin A and B were male, with a birth weight of 2,775 g (blood group, A positive) and 2,830 g (blood group, O positive), respectively. Both infants were predominantly formula fed. Their median NAS scores in the first week were 3 and 2, respectively. Neither of the twins required pharmacological management for NAS, and both were discharged home on day 8.

#### Second Set of Twins

Diamniotic-dichorionic (dizygotic) twins were born at 38 weeks of GA to a 34-year-old Caucasian woman by scheduled repeat cesarean section. The mother had a history of intravenous heroin and cocaine use, tobacco smoking, as well as a history of anxiety and depression, which were not treated. She was in the Methadone maintenance program, taking 45 mg methadone daily at the time of delivery. Her last reported illicit drug use was more than 4 weeks prior to delivery. She was tested positive for Hepatitis C but negative for HIV and Hepatitis B. Both twin A and B were male with a birth weight of 2,365 g and 2,025 g, respectively. The infants' blood type was not available in their medical records. Both infants were predominantly formula fed. Their median NAS scores in the first week were 3 and 4, respectively. Neither of the twins required pharmacological management of NAS and both were discharged home on day 8.

# Third Set of Twins

This set of diamniotic-monochorionic (monozygotic) twins was born at 38 weeks of GA by vaginal delivery to a 21-year-old Caucasian woman. She had a history of intravenous heroin and cocaine use, tobacco smoking, as well as a history of anxiety and post-traumatic stress disorders, which were not treated. She was in the Buprenorphine maintenance program, taking 8 mg buprenorphine daily at the time of delivery. Her last reported use of illicit drugs was more than 3 weeks prior to delivery. She was Hepatitis B, Hepatitis C, and HIV negative. Both twin A and B were female, with a birth weight of 2,861 g and 2,658 g, respectively. Both infants had O positive blood group and were exclusively formula fed. Their median NAS scores in the first week were 5 and 5, with the highest scores of 10 and 13 for twin A and B, respectively; both required NICU admission. Both infants required pharmacological treatment for NAS for 12 and 26 days and were discharged on day 26 and 36, respectively.

#### Fourth Set of Twins

This set of dichorionic-diamniotic (dizygotic) twins was born at 36 weeks of GA by vaginal delivery to a 29-year-old Caucasian woman. She had a history of heroin use (both snorting and intravenous routes), along with cocaine and marijuana, tobacco smoking, as well as a history of anxiety and post-traumatic stress disorder, which were not treated. She was in the Methadone maintenance program, taking 60 mg methadone daily at the time of delivery. Her last reported illicit drug use was more than 4 months prior to delivery. She was Hepatitis B, Hepatitis C, and HIV negative. Twin A was a female, with a birth weight of 2,121 g, and Twin B was a male, with a birth weight of 1,823 g. Both infants were exclusively formula fed. Twin A required pharmacological treatment for 9 days and twin B did not require any pharmacological treatment. Both infants were discharged home on day 19.

#### Fifth Set of Twins

This set of dichorionic-diamniotic (dizygotic) twins was born at 37 weeks of GA by vaginal delivery to a 32-year-old Caucasian woman. She had a history of using heroin, cocaine, marijuana and tobacco smoking as well as a history of unspecified mental health illness, which was not treated. She was not in any opioids assisted program. Her last reported illicit drug use was 4 weeks prior to delivery. She was Hepatitis C infected, but tested negative for HIV and Hepatitis B. Twin A was a female, with a birth weight of 2,410 g, and twin B was a male, with a birth weight of 2,505 g. Both infants were exclusively formula fed. Their median NAS scores in the first week were 2 and 3 for twin A and B, respectively, and they stayed in the normal newborn nursery (NBS). Neither of the infants required any pharmacological treatment and both were discharged home on day 8.

#### Sixth Set of Twins

This set of dichorionic-diamniotic (dizygotic) twins was born at 34 weeks of GA by vaginal delivery to a 27-year-old Caucasian woman. She had a history of using heroin (by snorting and intravenous route), cocaine, marijuana as well as tobacco smoking, but no history of mental health illnesses. She was in the Methadone maintenance program and was taking 120 mg methadone daily at the time of delivery. She tested negative for Hepatitis B, Hepatitis C, and HIV. Twin A was a female, with birth weight of 2,121 g, and twin B was a male, with birth weight of 2,105 g. Both infants were exclusively formula fed and both required pharmacological treatment for withdrawals for 32 days. Twin A was discharged on day 43, while twin B was discharged on day 41.

#### Seventh Set of Twins

This set of dichorionic-diamniotic (dizygotic) twins was born at 31 weeks of GA by vaginal delivery to a 31-year-old Caucasian woman. She had a history of heroin, cocaine, other opioids' (not specified in chart) abuse and tobacco smoking, as well as a history of anxiety and depression, which were not treated. She was in the Methadone maintenance program and was taking 50 mg methadone daily at the time of delivery. She was tested negative for Hepatitis B, Hepatitis C, and HIV. Twin A was a female, with a birth weight of 1,440 g (blood group, A positive) and twin B was also a female, with a birth weight of 1,570 g (blood group, O positive). Both infants were exclusively formula fed. Twin A required 14 days of pharmacological treatment, whereas no pharmacological treatment was required for twin B. Both infants were discharged home on day 49. Severe NAS is not very common among preterm infants born at 31 weeks, with a birth weight of about 1,400 g. We thus looked for alternative diagnosis that could explain the need of morphine but could not find anything other than NAS.

### DISCUSSION

Of the seven sets of twins, there was concordance of severity of NAS in five sets of twin siblings, which included a set of monozygotic twins, and discordance in two sets of twin siblings. Among the five pairs of twins with concordance of withdrawal severity, two pairs of twins developed severe NAS and three pairs did not develop severe NAS. Although the pair of identical twin siblings developed severe NAS and required pharmacological treatment, the length of treatment and hospital stay varied greatly among the twin siblings. Twin A was treated pharmacologically for 12 days and stayed in hospital for 26 days while B was treated for 26 days and stayed in hospital for 36 days. In the two pairs of dizygotic twins with discordance of NAS severity, one pair required medical treatment while the other did not. A total of 6 out of the 14 (42%) infants required treatment.

To the best of our knowledge, this is the first case series of twins with NAS. Among all twins, we found 70% concordance of severity for withdrawal as measured by requirement of pharmacological treatment and 30% discordance of severity. Adequately powered twin studies may help in delineating the influence of genetic and environmental factors in determining the severity of NAS.

# AUTHOR CONTRIBUTIONS

All authors were involved in conceptualizing and designing the work, RP prepared the initial draft and all authors revised the initial draft and agreed on the final version as submitted.

# REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Pandey, Pandey Sapkota and Kumar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Prenatal Opioid Exposure and Intermittent Hypoxemia in Preterm Infants: A Retrospective Assessment

*Elie G. Abu Jawdeh1 \*, Philip M. Westgate2 , Amrita Pant1 , Audra L. Stacy3 , Divya Mamilla4 , Aayush Gabrani5 , Abhijit Patwardhan6 , Henrietta S. Bada1 and Peter Giannone1*

*1Division of Neonatology, Department of Pediatrics, University of Kentucky, Lexington, KY, United States, 2Department of Biostatistics, College of Public Health, University of Kentucky, Lexington, KY, United States, 3College of Medicine, University of Kentucky, Lexington, KY, United States, 4Children's Hospital of Michigan, Detroit, MI, United States, 5Department of Pediatrics, New Jersey Medical School, Newark, NJ, United States, 6Department of Biomedical Engineering, College of Engineering, University of Kentucky, Lexington, KY, United States*

Introduction: Intermittent hypoxemia (IH) is defined as episodic drops in oxygen saturation (SpO2). Preterm infants are at increased risk for IH due to their immature respiratory control/apnea of prematurity. The clinical relevance of IH is a relatively new observation with rising evidence linking IH to neonatal morbidities and long-term impairment. Hence, assessing factors that influence IH in preterm infants is imperative. Given the epidemic of opioid misuse in the USA, there is an urgent need to understand the impact of prenatal opioid exposure on neonatal outcomes. Hence, we wanted to assess the relationship between isolated prenatal opioid exposure and IH in preterm infants.

#### *Edited by:*

*Po-Yin Cheung, University of Alberta, Canada*

#### *Reviewed by:*

*Christoph Martin Rüegger, University of Zurich, Switzerland Roland H. Hentschel, Universitätsklinikum Freiburg, Germany*

> *\*Correspondence: Elie G. Abu Jawdeh<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics*

*Received: 04 August 2017 Accepted: 10 November 2017 Published: 06 December 2017*

#### *Citation:*

*Abu Jawdeh EG, Westgate PM, Pant A, Stacy AL, Mamilla D, Gabrani A, Patwardhan A, Bada HS and Giannone P (2017) Prenatal Opioid Exposure and Intermittent Hypoxemia in Preterm Infants: A Retrospective Assessment. Front. Pediatr. 5:253. doi: 10.3389/fped.2017.00253*

Methods: In order to accurately calculate IH, SpO2 data were prospectively collected using high-resolution pulse oximeters during the first 8 weeks of life in preterm infants less than 30 weeks gestational age. Data related to prenatal opioid misuse were retrospectively collected from medical charts. Infants with tobacco or poly-drug exposure were excluded. The primary outcome measure is percent time spent with SpO2 below 80% (%time-SpO2 < 80). The secondary outcome measure is the number of severe IH events/week with SpO2 less than 80% (IH-SpO2 < 80).

Results: A total of 82 infants with isolated opioid exposure (*n* = 14) or who were unexposed (*n* = 68) were included. There were no significant differences in baseline characteristics between opioid exposed and unexposed groups. There was a statistically significant increase of 0.23 (95% CI: 0.03, 0.43, *p* = 0.03) in mean of the square root of %time-SpO2 < 80. The number of IH-SpO2 < 80 events was higher in the opioid exposed group (mean difference = 2.95, 95% CI: −0.35, 6.25, *p*-value = 0.08), although statistical significance was not quite attained.

Conclusion: This study shows that preterm infants prenatally exposed to opioids have increased IH measures compared to unexposed infants. Interestingly, the increased IH in the opioid exposed group persists beyond the immediate postnatal period.

#### Keywords: prenatal, opioid exposure, opiates, intermittent hypoxemia, apnea, preterm infants

**Abbreviations:** GA, gestational age; IH, intermittent hypoxemia; NICU, neonatal intensive care unit; SIDS, sudden infant death syndrome; SpO2, oxygen saturation; THC, tetrahydrocannabinol.

# INTRODUCTION

Intermittent hypoxemia (IH) is defined as brief, episodic drops in oxygen saturation (SpO2) (1, 2). Preterm infants are at increased risk for IH due to their respiratory control instability/apnea of prematurity superimposed on immature lung structure/function. IH in preterm infants can persist beyond discharge from the neonatal intensive care unit (NICU) (3). Brief episodes of oxygen desaturations may seem clinically insignificant, but these IH episodes, occurring up to hundreds of times per day, have a cumulative effect on neonatal morbidity and mortality. There is ample evidence showing a significant effect of IH on neurocognitive handicap, decreased neuronal integrity, increased inflammation and oxidative stress, and impaired growth (4, 5). Furthermore, IH has been linked to severe retinopathy of prematurity and long-term neurodevelopmental impairment such as worse language and motor outcomes (2, 6–9). The clinical relevance of IH is a relatively new observation with the advent of high-resolution pulse oximeters and assessing factors that influence IH is imperative.

There is a rise in substance misuse in the USA reaching a nationwide epidemic (10–15). There is an urgent need to understand the impact of prenatal opioid exposure on neonatal outcomes (5). Opioid exposure is associated with long-term neurobehavioral and developmental impairment in infants (16–23). Opioids are known to suppress breathing and respiratory effort especially in neonates (24). Since most mothers who misuse opioids have also been found to smoke and use poly-drugs that affect breathing pattern, it has been challenging to assess the isolated effect of prenatal opioid exposure on respiratory outcomes. Prenatal tobacco exposure alters respiratory control and worsens lung function (25–29). Prenatal exposure to other illicit drugs such as cocaine perturbs maturation of respiratory control, resulting in disruption of postnatal respiration (30). Only few studies were able to assess the effect of isolated opioid exposure on neonatal respiratory outcomes. However, these studies included mostly later preterm and term infants or were limited to short monitoring times and small sample sizes (31, 32). In this study, we utilize continuous high-resolution pulse oximeters to assess the relationship between isolated prenatal opioid exposure and IH in preterm infants during the first 2 months of life.

# MATERIALS AND METHODS

#### Study Design and Data Collection

Oxygen saturation data were prospectively collected from 130 preterm infants less than 30 weeks gestational age (GA) admitted to our level 4 NICU between November 2014 and April 2017. We used high-resolution pulse oximeters (Radical 7: Masimo, Irvine, CA, USA) set at 2 s averaging time and 1 Hz sampling rate to continuously monitor patients during the first 8 weeks of life. In order to differentiate intermittent from sustained hypoxemia, we included events between 4 and 180 s (1). The exact threshold below which IH is clinically significant is controversial. A drop in SpO2 to less than 80% is widely considered to be clinically relevant (1, 2, 6). Therefore, the primary outcome measure was defined as percent time spent with SpO2 below 80% (%time-SpO2 < 80). The secondary outcome measure was defined as the number of severe IH events with SpO2 less than 80% (IH-SpO2 < 80). Other outcome measures such as length of stay and neonatal morbidities were collected.

Pulse oximeters were equipped with serial data recorders (Acumen Instruments Corp.) for continuous data collection. Novel programs were utilized to filter and analyze data (Matlab, Natick, MA, USA) (1, 33). Data with artifacts were excluded. Only SpO2 data with good signal were included in the analyses. Preterm infants less than 30 weeks GA were included. Infants with major congenital malformations were excluded.

Data related to substance misuse and tobacco use were retrospectively collected from medical charts. If a mother chronically used prenatal opioids and/or the maternal/neonatal drug screens were positive for opioids, then the infant was considered for screening. Infants were then excluded from the study if the mother used tobacco, alcohol, or other drugs (such as cannabis); i.e., in order to assess for isolated opioid exposure, patients with any other exposure were excluded. Infants in our cohort who were not exposed to opioids, tobacco, or other drugs served as controls. Neonatal meconium or urine drug screens are performed in the immediate newborn period. Positive drug screens due to opioids and other medications used for pain or sedation during delivery were excluded, as they do not represent prenatal misuse. Tobacco and alcohol use were collected from mothers' medical records, as the toxicology screens at our hospital do not test for alcohol or tobacco exposure. The study was approved by the University of Kentucky Institutional Review Board, and informed consent was obtained prior to SpO2 data acquisition.

#### Statistical Analysis

Descriptive statistics for continuous variables are presented as either the mean with SD or median with interquartile range, and frequencies and percentages are given for categorical variables. Two-sample *t*-tests and Wilcoxon two-sample tests were used to compare opioid exposure to non-exposure with respect to continuous variables, and chi-square or Fisher's exact tests were used for categorical variables. To compare opioid exposure to non-exposure with respect to IH measures over time, we utilized multivariate Gaussian linear modeling in order to account for repeated measurements from subjects, and to adjust for the potential confounders of GA, birth weight, APGAR score at 5 min of life, gender, and the use of prenatal steroids. In order to meet statistical assumptions in these models, the square root of the IH measures was taken. Furthermore, weekly observations were weighted by the percentage of time IH was tracked during the given week. Analyses were conducted in SAS version 9.4 (SAS Institute, Cary, NC, USA), and all tests were two-sided with a 5% significance level.

# RESULTS

Of the 127 infants in our database with complete data sets, 19.7, 29.1, and 4.7% were prenatally exposed to opioids, tobacco, and cannabis, respectively. None were exposed to alcohol, cocaine, and other illicit drugs. Opioid exposed infants were positive for buprenorphine metabolites (64%), oxycodone (16%), and other opioids such as heroin and fentanyl (20%). A total of 82 infants qualified for analysis as they were either unexposed to any illicit drug/tobacco (*n* = 68) or exposed to opioids only (*n* = 14). **Figure 1** presents the flow diagram for patient eligibility and exclusion.

There were no significant differences in baseline characteristics as presented in **Table 1**. The mean GA was 27 weeks in both groups. There were no significant differences in birth weight, gender, and Apgar scores at 5 min of life. The vast majority of infants received prenatal steroids with no difference between groups. There were no significant differences in respiratory outcomes and neonatal morbidities between groups as presented in **Table 2**. Our cohort included preterm infants less than 30 weeks GA. Essentially all infants had respiratory distress syndrome and received surfactant. Severe bronchopulmonary dysplasia, postnatal steroids use for lung disease, and oxygen need at 28 days, 36 weeks postmenstrual age, and at discharge did not differ between opioid exposed and unexposed groups (all *p* = NS). Other neonatal morbidities such as patent ductus arteriosus, late onset sepsis, and necrotizing enterocolitis did not differ between groups (all *p* = NS). None of the exposed infants died versus nine deaths in the unexposed

TABLE 1 | Baseline characteristics.

*Mean* ± *SD, median (interquartile range).*

group (*p* = 0.35). The median length of stay was 17 days longer in the opioid group (85 days) compared to unexposed group (68 days); however, the results were not statistically significant (*p* = 0.32).

There was a statistically significant increase in our primary outcome measure, %time-SpO2 < 80, as represented in **Figure 2**. The estimated difference in the means of the square root of %time-SpO2 < 80 was 0.23 (95% CI: 0.03, 0.43, *p* = 0.03). The mean number of IH events was estimated to be 2.95 (95% CI: −0.35, 6.25, *p*-value = 0.08) higher in the opioid exposed group, as represented in **Figure 3**; however, this did not reach statistical significance. Note that these results represent the square root of means in order to meet statistical assumptions in these models; estimated medians for IH measures are calculated using our model results and are presented in **Figures 2B** and **3B**. Given increased death in the unexposed group, we then analyzed data excluding deaths, and results were similar. Specifically, there was a statistically significant increase in our primary outcome measure (%time-SpO2 < 80) in the opioid exposed compared to the unexposed group, with an estimated mean difference (square root) of 0.24 (95% CI: 0.05, 0.44, *p*-value = 0.02). Furthermore, the mean number of IH events was estimated to be 2.98 (95% CI: −0.20, 6.16, *p*-value = 0.07) higher in the opioid exposed group, not quite reaching statistical significance.

#### DISCUSSION

These results suggest that prenatal opioid exposure is associated with increased IH measures compared to unexposed preterm infants. This study has two main findings. First, interestingly, the increased IH measures in opioid exposed infants persisted beyond the early postnatal period. Preterm infants were continuously monitored with high-resolution pulse oximeters during the first 2 months of life. Second, we had the unique opportunity to assess the relationship between isolated opioid exposure and respiratory instability in preterm infants. It was challenging in the past to assess the relationship between isolated prenatal opioid

TABLE 2 | Neonatal morbidities and outcomes.

*Frequency (%), median (interquartile range).*

unexposed infants (*p* = 0.03). The model adjusted for gestational age, birth weight, gender, prenatal steroids, and Apgar scores at 5 min of life. (B) This figure demonstrates the estimated average %time-SpO2 < 80 medians in both groups calculated using the adjusted model results. Sqrt, square root.

model results. Sqrt, square root.

exposure and respiratory outcomes/IH, as the majority of women who use opioids also smoke or misuse poly-drugs. Given our cohort demographics, we had the ability to report this association in infants exposed to opioids only.

Another interesting secondary finding in our study is the steady increase in IH in the first month of life before plateauing and then decreasing. This natural progression of IH has been described before from another cohort of preterm infants less than 28 weeks GA (1, 2). Our study replicates this finding from a new cohort of preterm infants less than 30 weeks GA. The rise in IH may be related to peripheral chemoreceptor dysregulation and development of lung disease (34).

Patients in our opioid exposed and unexposed groups did not significantly vary in terms of baseline characteristics (such as age, weight, gender) and neonatal morbidities (such as lung disease, patent ductus arteriosus, late onset sepsis, and necrotizing enterocolitis). In addition, we adjusted in the model for factors that may influence oxygenation in preterm infants such as GA and prenatal steroids. The finding of nine deaths in the unexposed group compared to no deaths in the opioid exposed group may be due to chance. Secondary analyses excluding deaths showed similar results with increased IH in the opioid exposed group. A significant secondary finding in this study is the high prevalence of tobacco and drug exposure in our cohort of preterm infants. The frequency of opioid exposure in our preterm population is higher than previously reported, thus creating urgency toward addressing this significant problem in this vulnerable patient population (10, 12–15).

There are multiple proposed mechanisms by which prenatal opioid exposure may affect breathing patterns and subsequent persistent IH in preterm infants. Prenatal opioid exposure alters the response to carbon dioxide and depresses central respiratory control centers (31, 35–38); a main driver for respiratory output. Olsen and Lees demonstrated a blunted response to carbon dioxide in methadone exposed infants compared to controls (31). Ali et al. compared the response to hypercarbia among three groups of term patients who were exposed to tobacco/ substance misuse, tobacco alone, and unexposed controls. The authors showed a lower increase in central respiratory drive in response to hypercarbia in infants exposed to substance misuse as compared to tobacco alone and unexposed controls (35). Another mechanism that explains our results may be related to *in utero* hypoxia related to opioids. Prenatal opioids, especially street heroin, cause chronic intrauterine hypoxia leading to brainstem gliosis, resulting in injury to the central respiratory network. This may lead to respiratory instability and subsequent IH (36). Finally, data from animal models showed that exposure to opioid agonists caused downregulation of placental neurotransmitter receptors (39). Abnormalities or depletion of receptor sites, especially if the same process occurs in the fetal brain, could impair the function of the normal neonatal respiratory control network leading to frequent or prolonged apnea and subsequent IH.

Many studies have assessed the impact of prenatal opioid exposure on sudden infant death syndrome (SIDS) in infants with controversial results. This study does not address SIDS; rather, it focuses on IH, the end result of apnea of prematurity. However, the mechanism by which prenatal opioid exposure is associated with increased SIDS and IH may be similar. Although our study period focused on the inpatient setting, it is plausible that opioid exposed infants continue to have increased cardiorespiratory events/IH after discharge. Interestingly, compared to unexposed infants, opioid exposed infants had a trend toward longer length of stay (68 versus 85 days, *p* = NS), which may be related, in part, to persistent cardiorespiratory events.

A major limitation of this study is that data related to exposure were retrospectively collected. Another limitation is a lack of reporting daily caffeine use and daily respiratory support settings. At our center, virtually all infants with GA less than 30 weeks are started on caffeine therapy. Furthermore, our study focused on IH events and lacked reporting of apnea and bradycardia events. Lack of addressing heart rate is a limitation since bradycardia events may be associated with poor long-term outcomes (6). Another limitation is the small sample size; however, our sample size of isolated opioid exposure is relatively large compared to existing literature. This is a single center study; hence, our results may not be generalizable. Finally, we did not compare the long-term neurodevelopmental outcomes for exposed versus unexposed infants.

# CONCLUSION

There is rising evidence linking IH to neonatal morbidities and impairment. However, the exact threshold (frequency, duration, severity) by which IH leads to injury in preterm infants needs further investigation; i.e., any increase in IH may be associated with impairment in preterm infants. Furthermore, there is a need to understand factors, such as prenatal opioid exposure, that may influence IH and subsequently increase neonatal morbidities. In this study, we show an association between prenatal opioid exposure and increased IH measures in preterm infants. Studies to address the relationship between opioid exposures, IH, and long-term neurodevelopmental outcomes are imperative. Given the rising epidemic of opioid misuse in the USA, understanding the relationship between opioid exposure, IH and long-term impairment is imperative. A larger prospective study aimed at understanding these relationships may have a direct impact on short- and long-term management of preterm infants.

# ETHICS STATEMENT

This study was carried out in accordance with the recommendations of University of Kentucky Institutional Review Board (IRB). A written informed consent was obtained from all subjects included in this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the University of Kentucky IRB.

# AUTHOR CONTRIBUTIONS

EA designed and conceptualized the study and was actively involved in the enrollment, data collection, as well as analysis and interpretation of the results. He drafted the initial manuscript and wrote the final manuscript as submitted. PW was involved in conceptualization of the study and performed the statistical analyses. He also critically reviewed the manuscript and made the final approval manuscript as submitted. AP, AS, DM, and AG were involved in the conceptualization of the study, data collection, as well as analysis and interpretation of the results. They critically reviewed the manuscript and approved the final manuscript as submitted. AP was involved in the conceptualization of the study, the data acquisition software development, as well as in the analysis and interpretation of the results. He critically reviewed the manuscript and approved the final manuscript as submitted. HB and PG were involved in the conceptualization of the study as well as analysis and interpretation of the results. They critically reviewed the manuscript and approved the final manuscript as submitted.

# ACKNOWLEDGMENTS

This project was supported by funds from the Gerber Foundation, Children's Miracle Network and the National Center for Research Resources, UL1RR033173, and is now at the National Center for Advancing Translational Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The authors thank the support of Katrina Ibonia MD, Enrique Gomez-Pomar MD, Vicki Whitehead RN CCRC, Kimberly Walker BSN, Alisa (Beth) McKinney-Whitlock CCRP, Sean Carpenter BSBE, John Bauer PhD, NICU nurses, research nurses and staff, and neonatology faculty and fellows at the Division of Neonatology, University of Kentucky.

# FUNDING

The study was funded by: (1) The Gerber Foundation, (2) Children's Miracle Network, and (3) National Center for Research Resources, UL1RR033173, and is now at the National Center for Advancing Translational Sciences.

# REFERENCES

39. Perry BD, Pesavento DJ, Kussie PH, U'Prichard DC, Schnoll SH. Prenatal exposure to drugs of abuse in humans: effects on placental neurotransmitter receptors. *Neurobehav Toxicol Teratol* (1984) 6(4):295–301.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Abu Jawdeh, Westgate, Pant, Stacy, Mamilla, Gabrani, Patwardhan, Bada and Giannone. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

#### *Eric W. Reynolds1 \*, Debbie Grider2 and Cynthia S. Bell3*

*1 Division of Neonatology, Department of Pediatrics, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, United States, 2 Division of Neonatology, Department of Pediatrics, University of Kentucky, Lexington, KY, United States, 3 Department of Pediatrics, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, United States*

Background: The development of suck–swallow–breath rhythms during non-nutritive suck (NNS) may be an indicator of neurologic integrity. We have described swallow– breath (SwBr) interaction and phase of respiration (POR) with swallow during NNS in low-risk preterm (LRP) infants. NNS in infants with neonatal abstinence syndrome (NAS) has not been described with our method.

#### *Edited by:*

*Eugene Michael Dempsey, University College Cork, Ireland*

#### *Reviewed by:*

*María Gormaz, Agencia Valenciana Salud, Spain Jonathan Michael Davis, Tufts University, United States*

> *\*Correspondence: Eric W. Reynolds<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics*

*Received: 23 June 2017 Accepted: 21 September 2017 Published: 04 October 2017*

#### *Citation:*

*Reynolds EW, Grider D and Bell CS (2017) Swallow–Breath Interaction and Phase of Respiration with Swallow during Non-Nutritive Suck in Infants Affected by Neonatal Abstinence Syndrome. Front. Pediatr. 5:214. doi: 10.3389/fped.2017.00214*

Method: Suckle, swallow, thoracic motion, and nasal airflow were measured during NNS in 10 infants with NAS and 12 unaffected infants (control). Logistic regression models were fit to describe the three types of SwBr and five types of POR in terms of the independent variables (gender, gestational age, birth weight, postmenstrual age, weeks postfirst nipple feed and swallows per study). We also compared the NAS group to 16 LRP infants.

results: In the NAS group, there were 94 swallows in 18 studies. In the control group, there were 94 swallows in 12 studies. There were statistical differences between groups for all three types of SwBr. The distribution of SwBr in NAS was similar to LRP infants with NAS having fewer swallows with attenuated respiration and more with central apnea. For POR, there were few differences. Over time, the distribution of SwBr in NAS infants approaches that of control infants.

Discussion: Variability in SwBr and POR during NNS may represent neurologic dysfunction in infants with NAS. Specifically, term infants with NAS display an immature pattern of SwBr making them more similar to preterm infants, rather than a unique pathology. The distribution of SwBr and POR in NAS infants becomes more like term infants, possibly representing catch-up development as the NAS symptoms resolve.

conclusion: SwBr in babies with NAS is different from that of unaffected term infants, actually being similar to preterm infants. Infants with NAS exhibit a dysmature pattern of NNS development which resolves over time.

Keywords: neonatal abstinence syndrome, non-nutritive suck, suck–swallow–breath coordination, infant feeding, neonatology

# INTRODUCTION

Efficient suckle-feeding can be considered to be the most complex skill a newborn infant must master to attain independent survival. However, feeding problems are frequent in preterm infants (1) and can lead to prolonged hospital stays (2). Poor feeding in the neonatal period may be an early indicator of neurologic injury (3, 4) and has been linked to language delay later in life (5).

The development of efficient suckle-feeding is dependent on the maturation and coordination of neuronal central pattern generators controlling suck, swallow, and breath (SwBr) (6).  Thus, NNS may provide an earlier marker of intact neurodevelopment than nutritive feeding does.

We have previously used our method to study NNS in low-risk preterm (LRP) infants (7) and have shown that the interaction of SwBr and the phase of respiration (POR) incident to swallow develop in a predictable pattern in these infants. The progression of SwBr is influenced by increasing opportunities to practice the skill, or what can be considered "learning." The progression of POR was more affected by measures of maturation, indicating a developmental progression. Our method has not yet been applied to pathologic conditions.

Neonatal abstinence syndrome (NAS) is a constellation of withdrawal-like symptoms experienced by infants born to mothers who have been chronically taking opiates or other drugs/ medications during the pregnancy. Feeding difficulty and disruption of suck–swallow–breath rhythms have been reported in infants affected by NAS (8, 9). In general, abnormal sucking and feeding behaviors in infants affected by NAS include excessive sucking, inattention, fussiness, and decreased swallow efficacy. The specifics of suck–swallow–breath organization during NNS in infants affected by NAS have not been described.

# MATERIALS AND METHODS

We performed a prospective observational study comparing suck–swallow–breath coordination in infants with NAS and unaffected infants. The study participants included 10 term infants with NAS and 12 healthy term (control) infants. We also included a group of 16 LRP infants who were concurrently enrolled in the study and described in a previous manuscript (7). **Table 1** is a list of the abbreviations provided to help the reader understand the presented information.

The 10 infants in the NAS group were diagnosed by a combination of *in utero* exposure to substances known to result in NAS and postnatal symptoms consistent with the diagnosis. *In utero* exposures included benzodiazipines, methadone, cocaine, oxycotin, klonipin, fentanyl, THC, oxycodone, and buprenorphine. Five infants had poly-drug exposure. Five infants had single drug exposures (four methadone and one oxycodone). These included four mothers who were compliant with a methadone treatment program and another mother who was taking chronic pain medication. Finnegan scores at the time of enrollment ranged from 9 to 20. In our NICU at the time, babies who were at risk for NAS were monitored for symptoms of NAS and scored with the standard Finnegan Score tool (10). Per institutional norm, Table 1 | Abbreviations and acronyms used in this study.

infants who score 8 or more on 2 consecutive scores, or 12 or more once, were given a diagnosis of NAS and treated with opiate replacement therapy. At the time of this study, the treatment for NAS in our facility was not standardized. Infants were treated with morphine or methadone. The protocol for adjusting doses was not proscribed. Two infants required adjunctive therapy with phenobarbital. Infants were studied once per week from the time of enrollment until discharge from the hospital. There were 94 swallows collected from 18 studies in this group.

The control group included 12 babies born between 37 and 42 weeks of gestation, appropriate size for gestational age, 5-min Apgar score of 7 or more and with no congenital anomalies or metabolic disorders. These infants were studied once prior to discharge from the hospital. There were 94 individual swallow events collected over 12 studies from these infants.

We also compared the NAS group to a group of 16 LRP infants who were born at less than 36 weeks (mean gestational age at birth: 28 5/7, mean postmenstrual age at study: 35 3/7), appropriate size for gestational age (mean birth weight: 1,056), no congenital anomalies, no IVH of grade 3 or 4 and deemed to be "low risk" for bronchopulmonary dysplasia per the definition in our previous publication (7). There were 176 swallows in 35 studies in this group.

Because of potential statistical problems with repeated studies in the NAS group and not in control infants, we analyzed the data using only a single study for each NAS infant. This decreased the number of studies in this group (NAS1st) to 10 and the number of swallows to 49.

Informed consent was obtained from the parent(s) of each infant prior to the infant's participation in the study. The project complies with all applicable HIPAA standards and was approved by the Institutional Review Board of the University of Kentucky.

We have previously published the specific method and equipment for preparing the babies for the study and data collection (7). To summarize the salient portion of the study for this project, the infant study participants were prepared in the following manner:

The infants were offered a pacifier for 1-min of NNS just prior to a nutritive feeding. Data were collected and displayed as multichannel linear graphs, using the Windaq Waveform Browser (Dataq Industries, Akron, OH, USA). The entire 1-min sequence of NNS was canvassed for swallows, noted as deflections in the naso-pharyngeal pressure recording. The type of SwBr and POR were classified, as described below. During NNS, swallow occurs infrequently. When it does occur, the SwBr must abruptly interact for about 1–2 s, in what has been termed "deglutition apnea" (11), and should not be confused with apnea (AP) of prematurity which requires 15–20 s of no breathing to be defined as AP.

We can identify three types of SwBr interaction: central apnea (CA) (cessation of both nasal airflow and chest movement), obstructive apnea (OA) (cessation of nasal airflow but continued rhythmic chest movement), or attenuated respiration (AR) (a slight deflection of the slope of the respiratory line on the graph at the time of the swallow without disruption of the respiratory rhythm). Examples of each type of SwBr are shown in **Figure 1**.

The respiratory cycle can be divided into five phases (POR): beginning expiration (BE), mid-expiration (ME), end-expiration (EE), mid-inspiration (MI), and AP. Swallows occur at any of these PORs. It has been hypothesized that the most mature pattern exists when the swallow occurs at a point of minimal air movement (BE, EE, or AP) (11).

Independent variables for this analysis included gender, birth weight, gestational age, postmenstrual age, number of swallows in the study, and weeks postfirst nipple feed (time between first nipple feed and day of study). Day-of-life at the time of the study is used only for demographic descriptions and not for analysis because it would create a collinearity issue since postmenstrual age is a function of gestational age + day-of-life.

Infants were invited to return for developmental testing [Bayley-III and Preschool Language Scales version IV (PLS-IV)] at 12 and 24 months. 11 infants from the control group returned at 12 and 24 months. 7 infants in the NAS group returned at 12 months and 5 returned at 24 months. One of the NAS infants at 24 months could not complete all of the assessments.

We used Stata 13.1 (College Station, TX, USA) to calculate descriptive statistics on patient demographics and to construct logistic regression models relating the odds of each type of SwBr and POR to the independent variables defined above. Statistical inferences were made *via* generalized estimating equations (12) with an exchangeable structure to take into account the correlations inherent to repeated assessments on the same baby. The number of patients/studies came from a subset of a larger study of infant feeding. Power calculations were based on that study and these patients were selected because they had data that was useable for this analysis.

#### RESULTS

#### Demographics

Demographic characteristics of the infants in the study groups are shown in **Table 2**. Each group was approximately 60% female and 40% male. Mean birth weight and gestational age were similar for each group. Babies in the NAS group were studied weekly during their in-hospital treatment. Thus, day-of-life, postmenstrual age, and weeks postfirst nipple feed are greater in the NAS group. The number of swallows per study was not different between the two groups. Because of the bias caused by having repeated studies

#### Table 2 | Patient population characteristics.

*There are significant differences between NAS and control for day-of-life (p* < *0.0001), postmenstrual age (p* = *0.003), and weeks postfirst nipple feed (p* < *0.0001). There are significant differences between NAS1st and control for day-of-life (p* < *0.0001) and weeks postfirst nipple feed (p* < *0.0001).*

*\*indicates statistical significance.*

in the NAS group and not in the control group, we performed the analysis using only a single study for each baby in the NAS group (NAS1st). The significant differences for day-of-life and weeks postfirst nipple feed remained. This is because babies in the NAS group were enrolled in the study after their symptoms had progressed to the point of being diagnosed with NAS, which can take over a week, depending on the drug of exposure, timing of the last dose and specific genetic polymorphisms which affect NAS timing and severity. The difference in Postmenstrual Age was eliminated.

#### Distribution of SwBr and POR in NAS

The percent of swallows occurring at each type of SwBr and POR for the study groups are shown in **Table 3**. Section A shows the results of univariate analysis comparing NAS and control with differences for SwBr as follows. AR is less common in NAS than control (OR = 4.05, *p* = 0.0041). There was no difference for OA. CA was more common in NAS than control (OR = 0.212, *p* = 0.0353). For POR, there were no differences between NAS and control infants for BE, ME, EE, or AP. MI was more common in NAS than control (OR = 0.28, *p* = 0.0361).

Using multivariate analysis (**Table 3**, B) to control for differences in the independent variables, CA was predicted to be more common in NAS then control (OR = 0.1117, *p* = 0.0075). OA was more common in NAS then control (OR = 0.351, *p* = 0.0241). AR was less common in NAS than control (OR = 8.1, *p* = 0.0004). OA was more common in females (OR = 0.387, *p* = 0.0011) and AR was more common with advancing weeks postfirst nipple feed (OR = 1.64 per week, *p* = 0.0210). There were no predicted differences between NAS and control for ME, MI, or EE. BE was less common in NAS than control (OR = 4.60, *p* = 0.0026). AP was more likely in NAS than control (OR = 0.099, *p*= 0.0092). AP was less common with advancing weeks postfirst nipple feed (OR = 0.456 per week, *p* = 0.0115).

#### SwBr and POR with a Single NAS Study

Babies in the control group underwent one study prior to discharge from the hospital, while babies in the NAS group were studied weekly during their hospital stay. In order to evaluate the data without the inherent bias introduced by repeated measures on the

#### Table 3 | Distributions of SwBr and POR for each group.

*A: distribution of SwBr and POR for control vs. NAS. There are significant differences for CA and AR in SwBr and for MI in POR.*

*B: predicted distribution of SwBr and POR with multivariate analysis. There are significant differences for all three types of SwBr and BE and AP in POR. C: distribution of SwBr and POR for control vs. NAS1st. There are significant differences for CA and AR in SwBr and for BE in POR.*

*D: distribution of SwBr and POR for NAS vs. LRP. There are no significant differences for SwBr. There is a significant difference for EE in POR.*

*\*indicates statistical significance.*

same baby, the analysis was performed using only a single study from each baby in the NAS group (NAS1st). This decreased the number of swallows for analysis to 49. **Table 3** shows the results of univariate analysis of NAS1st vs. control. The statistical difference between NAS1st and control for CA and AR remained. CA was more common in NAS1st than control infants (OR = 0.146, *p* = 0.0042). AR was less common in NAS1st than control infants (OR = 4.401, *p* = 0.0022). For POR, the significant difference initially noted between NAS1st and control for MI was no longer present. However, BE was less common in NAS1st than control infants (OR = 2.565, *p* = 0.0451).

#### SwBr and POR in NAS and LRP

Having established that the distribution of SwBr is different between NAS and control, we were interested to determine if the NAS infants were similar to preterm infants. Thus, we compared the SwBr in NAS to a group of LRP infants. As shown on **Table 3**, the percentages of each type of SwBr were similar to that of the LRP babies, with no statistically significant differences for any SwBr. There were some differences noted for POR.

#### Developmental Follow-up

Study participants returned for developmental follow-up assessments with the Bayley Scales of Infant Development version III (Bayley-III) and the PLS-IV. **Table 4** shows the average scores for infants in the control and NAS groups. At 12-month follow-up, the average scores for Bayley-III and PLS-IV assessments were not different between the control and NAS infants. At 24 months, Bayley-III scores were not different between control and NAS infants. PLS-IV scores at 24 months were statistically lower in the NAS group, but the number lost to follow-up limits the ability draw conclusions from this data.

#### DISCUSSION

Successful newborn feeding requires coordination of the rhythms of suck, SwBr (13–16). Feeding problems have been identified as a sequelae of hypoxic–ischemic injury (17) and later neurologic injury such as cerebral palsy (16). Mild disruptions of the rhythmicity of suckle feeding can be linked to less severe injury (18) and may predict subsequent feeding and neurologic problems (19, 20). Abnormal newborn feeding behaviors have been linked to language delay at 18 months (5). The progression of normal rhythmic suckle feeding is dependent on the interaction of brainstem central pattern generators for suck, SwBr (6). These central pattern generators are also activated during NNS. Therefore, we hypothesize that the organization of suck, SwBr during NNS may be an early indication of the integrity of the neonatal CNS.

Babies affected by NAS are known to have disorders of the central nervous system which can manifest as excessive suck, abnormal breathing and feeding difficulty. Kamal et al. has shown that infants affected by NAS have an abnormal ventilatory response to hypercarbia in the newborn period and at 6–12 weeks of life that may increase their risk for SIDS (21, 22). Finnegan et al. included excessive suck, respiratory distress and other gastrointestinal disturbance among the symptoms of NAS included in their tool to track the severity of NAS disease (10). Most studies of abnormal feeding related to NAS have focused on behaviors of the infant or mother. Maguire et al. (23) found the infants with NAS exhibit characteristic behaviors such as tremors, hyperextension, altered muscle tone, and multiple transitions between behavioral states

*There were statistical differences for PLS scores at 24 months.*

*a One infant in the NAS group at 24 months did not complete all of the assessments. AC, auditory comprehension; EC, expressive communication; TLS, total language score.*

*Boldface indicates statistical significance.*

that contribute to difficult feeding. Furthermore, these infants spend an excessive amount of time fussing, including averting the face from the mother, pulling or turning away or otherwise resisting, hyperextension, flailing arms or vocal objections to feeding, "but not a robust cry." LaGasse et al. (8) found that opiate-exposed infants had prolonged sucking with fewer pauses, more feeding problems such as spitting up and refusal, and increased arousal. None of these studies looked at the development of suck–swallow–breath rhythms. Gewolb et al. (9) evaluated the integration of these rhythms during feeding in infants born to mothers with drug-abuse problems. They found subtle abnormalities of respiratory control and swallow rhythmicity in drug-exposed infants. Drug-exposed infants were less efficient feeders than control infants (decreased volume/swallow), but this decreased efficiency was offset by a faster swallow rate. The differences they noted at 3 days of life between drug-exposed and unaffected control infants were not present when the infants were studied at 1 month of life.

Very few studies of NAS have focused on NNS and none have looked at the integration of SwBr during NNS. Maone et al. (24) offered cocaine-exposed infants, and control babies, a standard pacifier and a sucrose flavored pacifier. The drug-exposed cohort had about the same suck rate as control infants when offered the unflavored pacifier. However, when offered a sucrose-flavored pacifier, the cocaine-exposed cohort increased their suckle rate in a statistically significant manner.

Our previous work evaluating NNS in LRP infants supports the idea that there is an interaction between SwBr that occurs during NNS, similar to deglutition AP during nutritive feeding and that this interaction, which we identified as SwBr and POR incident to swallow, or POR, progresses in a predictable and measurable fashion (7). We hypothesized that disruptions of this progression may correlate with abnormalities of central nervous system development.

In our primary analysis, we compared a group of term infants affected by NAS to a group of healthy infants delivered at term gestation. We found particularly pronounced differences between the infants with NAS and control infants, especially related to SwBr. The differences were less pronounced for POR. Babies with NAS had significantly more swallows occurring with CA and fewer occurring with AR than control babies. There was a nonsignificant trend toward more swallows with OA in the NAS group as well. When controlling for identified confounders with multivariate analysis, the differences in predicted distributions for CA and AR were still present. Additionally, the difference in predicted percentage of swallows at OA became statistically significant.

The only significant difference for POR was for more swallows occurring at MI. It may be more difficult to identify statistically significant differences in POR since there are more categories in which swallows are grouped, thus requiring more statistical power. Furthermore, in our previous study of LRP infants, we found that the progression of POR was affected mostly by postmenstrual age, suggesting a developmental process that was not affected by practice or learning (7). Since there is very little difference in the postmenstrual age for babies across this study, it is not surprising that we find only minor differences in POR.

Given that bias can be introduced into the analysis by the fact that some babies in the NAS group were studied weekly while in the hospital but control babies only had a single study, we sought to limit the effect of repeated measures by performing the analysis with a single study for each NAS baby. This decreased the number of swallows available for analysis and thus decreased the statistical power. However, the statistically significant differences for SwBr remained with more swallows from NAS infants occurring at CA and fewer occurring at AR. The difference noted in POR for MI was no longer present, but a statistical difference for BE was noted. It is possible that either, or both, of these associations may occur by chance, given the number of comparisons we are analyzing in this work. Even if these differences in the distribution of POR are real, it is easy to conclude that any variability in POR introduced by NAS is quite small compared to the differences in SwBr.

Having established that the distribution of SwBr is different for infants with NAS versus control infants, we were interested to determine if these differences were a reflection of immaturity of the suck–swallow–breath reflexes or a completely different pathological process. Thus, we compared the NAS infants to a group of LRP infants. These infants were relatively healthy aside from being born preterm, with no sepsis, no severe intraventricular hemorrhage and at low-risk for developing bronchopulmonary dysplasia. These babies were studied once per week from the onset of nipple feeding until discharge from the NICU. Interestingly, there were no statistically significant differences between the NAS and LRP groups for any of the SwBr types. There was a single significant difference in POR noted, with more swallows occurring at EE in the LRP group. Thus, it appears that the coordination of suck–swallow–breath rhythms in infants with NAS is immature, more like preterm infants, when compared to term infants.

Our previous work in preterm infants suggested that the distribution of SwBr was heavily influenced by the number of attempts the baby had to try nipple feeding, regardless of Gestational Age at delivery or Postmenstrual Age at the time of the study (7). Thus, it seems that the coordination of SwBr may be a learnable skill. Since babies with NAS do experience improved feeding over the course of their treatment, it is logical to assume that there will be an improvement in their SwBr distribution. In fact, comparing the distribution of SwBr in the total NAS group and the single-study NAS group, one can appreciate that the additional NAS studies are changing the SwBr distribution toward that of term infants. That is to say, the percentage of CA in NAS1st is 35%. With the added weekly studies, the percentage of CA is 22% in the NAS group. For control infants it is 5%. Similarly, AR changes from 39% in NAS1st to 57% in whole NAS group and 74% in control infants. Thus, we hypothesize that the distribution of SwBr in these NAS infants is approaching that of the control infants in a manner similar to that described by Gewolb et al. (9) for nutritive feeding. This also suggests that the differences noted in day-of-life, postmenstrual age, and weeks postfirst nipple feed does not account for the statistical differences in our results. Since NAS babies are older (day-of-life, postmenstrual age) and have been feeding longer (weeks postfirst nipple feed) we would predict that they would be more mature than the control group, rather than the immature pattern we have described.

Previous studies of feeding in infants with NAS have described excessive fussiness, inattention, spitting, and decreased efficiency. It is possible that the pathologic immaturity of suck–swallow– breath reflexes is contributing to the infants' overall state of dysfunction. It is unclear from this data if the immature suck– swallow–breath coordination we are describing is a function of *in utero* exposure to various toxins or occurs as a consequence of the treatment for NAS. This dataset does not allow us to answer these questions because there were variations in the specific exposures and the choice of treatment as well as the fact that all of the NAS babies were receiving medical treatment at the time of the study. If we will see a similar dysfunction with studies of nutritive feeding is also unclear.

# CONCLUSION

Infants affected by NAS have an immature pattern of SwBr during NNS when compared with healthy term infants, making them more like preterm infants in this respect. There is evidence to support the idea of "catch-up" development as the distribution of SwBr approaches that of healthy term infants as the affected infants are studied subsequently over time. This is interesting because it shows that infants with NAS are not affected by a unique pathology, but rather are showing an immature pattern which improves over time. It is unclear if the dysfunction of SwBr is a consequence of the initial exposure or due to the treatment. Likewise, the improvement in SwBr may be simply related to maturation or could be due to receiving appropriate treatment. The immature pattern of suck–swallow–breath reflexes may be a contributing factor to the overall feeding difficulty experienced by babies with NAS. Future studies can focus on improving the development of suck–swallow–breath integration in infants with NAS.

# ETHICS STATEMENT

Informed consent was obtained from the parent(s) of each infant prior to the infant's participation in the study. The project complies with all applicable HIPAA standards and was approved by the Institutional Review Board of the University of Kentucky.

# AUTHOR CONTRIBUTIONS

ER was responsible for the project design, data collection and interpretation, and manuscript preparation. DG was responsible for subject identification and enrollment and data collection. CB was responsible for statistical methods, data analysis, and manuscript preparation.

# FUNDING

This project was funded by the NIH through a K23 award K23HD050581-02.

# REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Reynolds, Grider and Bell. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Plasma Brain-Derived neurotrophic Factor levels in newborn infants with neonatal abstinence syndrome

*Lochan Subedi <sup>1</sup> , Hong Huang1 , Amrita Pant <sup>1</sup> , Philip M. Westgate2 , Henrietta S. Bada1 , John A. Bauer <sup>1</sup> , Peter J. Giannone1 and Thitinart Sithisarn1 \**

*1Department of Pediatrics, University of Kentucky, Lexington, KY, United States, 2Department of Biostatistics, College of Public Health, University of Kentucky, Lexington, KY, United States*

Background: Brain-derived neurotrophic factor (BDNF) is a type of growth factor that promotes growth and survival of neurons. Fetal exposure to opiates can lead to postnatal withdrawal syndrome, which is referred as neonatal abstinence syndrome (NAS). Preclinical and clinical studies have shown an association between opiates exposure and alteration in BDNF expression in the brain and serum levels in adult. However, to date, there are no data available on the effects of opiate exposure on BDNF levels in infant who are exposed to opiates *in utero* and whether BDNF level may correlate with the severity of NAS.

#### *Edited by:*

*Vineet Bhandari, Drexel University, United States*

#### *Reviewed by:*

*Subrata Sarkar, University of Michigan Health System, United States Karel Allegaert, University Hospitals Leuven, Belgium*

> *\*Correspondence: Thitinart Sithisarn<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics*

*Received: 31 July 2017 Accepted: 20 October 2017 Published: 03 November 2017*

#### *Citation:*

*Subedi L, Huang H, Pant A, Westgate PM, Bada HS, Bauer JA, Giannone PJ and Sithisarn T (2017) Plasma Brain-Derived Neurotrophic Factor Levels in Newborn Infants with Neonatal Abstinence Syndrome. Front. Pediatr. 5:238. doi: 10.3389/fped.2017.00238*

Objective: To compare plasma BDNF levels among NAS and non-NAS infants and to determine the correlation of BDNF levels and the severity of NAS.

Methods: This is a prospective cohort study with no intervention involved. Infants ≥35 weeks of gestation were enrolled. BDNF level was measured using enzyme-linked immunosorbent assay technique from blood samples drawn within 48 h of life. The severity of NAS was determined by the length of hospital stay, number of medications required to treat NAS.

results: 67 infants were enrolled, 34 NAS and 33 non-NAS. Mean gestational age did not differ between the two groups. Mean birth weight of NAS infants was significantly lower than the non-NAS infants (3,070 ± 523 vs. 3,340 ± 459 g, *p* = 0.028). Mean BDNF level in NAS group was 252.2 ± 91.6 ng/ml, significantly higher than 211.3 ± 66.3 ng/ ml in the non-NAS group (*p* = 0.04). There were no differences in BDNF levels between NAS infants that required one medication vs. more than one medication (254 ± 91 vs. 218 ± 106 ng/ml, *p* = 0.47). There was no correlation between the BDNF levels and length of hospital stay (*p* = 0.68) among NAS infants. Overall, there were no significant correlations between BDNF levels and NAS scores except at around 15 h after admission (correlation 0.35, *p* = 0.045).

conclusion: Plasma BDNF level was significantly increased in NAS infants during the first 48 h when compared to non-NAS infants. The correlations between plasma BDNF levels and the severity of NAS warrant further study. These results suggest that BDNF may play a neuromodulatory role during withdrawal after *in utero* opiate exposure.

# INTRODUCTION

Brain-derived neurotrophic factor (BDNF) is a member of neurotrophin family that is highly expressed in central and peripheral nervous system. The functional maintenance and survival of neurons depends on the availability of BDNF (1, 2). BDNF regulates neuronal survival, promotes neurite outgrowth, and maintains synaptic connectivity in the nervous system (3). BDNF has a neuromodulatory effect on learning and memory (4) and drug addiction (5, 6). BDNF plays significant role in brain synaptic plasticity (7) and locomotor sensitization (8) after opiate withdrawal. Furthermore, several studies demonstrated alterations of serum or plasma BDNF levels in drug abuser (9, 10) and implicated BDNF in the development of addiction (11).

Brain-derived neurotrophic factor is synthesized by neuronal and glial cell populations (12); however, BDNF is also expressed in several non-neuronal tissues such as immune cells and vascular endothelium (13–15). Altered levels and expression of BDNF may lead to abnormal fetal growth and brain development (16). Preclinical study reported increasing peripheral BDNF levels that were positively correlating with the cortical BDNF levels as the animal maturating from early postnatal period to young adults (17). Although there are no normative data for BDNF levels during infancy, study showed that plasma BDNF levels also increased during the early perinatal period in healthy full term infants (18). There was a wide range of peripheral BDNF levels in healthy adult human; however, BDNF levels decreased significantly with increasing age or weight and was affected by gender (19, 20). Expression of BDNF is influenced by many conditions including stress, cigarette smoking (21), alcoholic consumption (22), and depression (23). Decreased serum BDNF levels were found in adults with attention-deficit hyperactive disorder (24) and other neuropsychiatric disorders (23).

In the past decade, neonatal abstinence syndrome (NAS) has been a major health problem. There has been substantial increase in incidence of maternal opiate use and NAS leading to increased health related costs (25). NAS signs include poor sleep, high pitch cry, increased muscle tone, jitteriness, loose stool, poor feeding, etc. Prenatal opiate exposure also results in long-term deleterious consequences including behavioral problems, speech and cognitive deficits, poor social skills, anxiety, aggression, and poor fine and gross motor coordination (26, 27). To date, no reliable biomarker has been identified to predict the severity of NAS and long-term outcomes of children exposed to opiates *in utero*. However, BDNF may be a good candidate biomarker. Opiate exposure is known to induce apoptosis, downregulate cAMP response element binding expression, and decrease in dendritic branching and spine density, and BDNF protects neurons against these effects (28). Opiate withdrawal can affect serum BDNF level (29) and BDNF expression in the brain (30). Decreased BDNF expression and protein in the brain is associated with behavioral, learning, and memory problems (12, 24, 31). Previous study using a rat model found that opiate exposure compromised memory, increased anxiety levels, and decreased BDNF precursors in the hippocampus (31). Although these studies suggested the likely effects of *in utero* opiate exposure on the BDNF level and its role in long-term neurobehavioral outcome, to date, there has been no published study on the correlation of NAS/opiate exposure on the levels of BDNF in human infants. Therefore, in the present study, we aimed to assess possible changes in serum BDNF levels in opiates exposed infants compared to unexposed infants and to determine the correlation of BDNF levels with the severity of NAS.

### MATERIALS AND METHODS

This prospective cohort study was performed on 68 infants born at ≥35 weeks of gestation age (GA) and admitted to Kentucky Children's Hospital Neonatal Intensive Care Unit (NICU) and Newborn Nursery between 2015 and 2016 in Lexington, KY, USA. Inclusion criteria for NAS infants were infants born to mother with history of opiate intake or urine drug screen (UDS) test positive during pregnancy and were admitted in NICU for withdrawal symptoms. These infants were born in University of Kentucky or transferred from outside hospital within 1 week of life. Infants unexposed to opiates by history and/or UDS negative were enrolled in non-NAS group. The exclusion criteria were neonates with major congenital anomalies, infants of mothers <18 years of age, infant transferred from outside hospital after 1 week of life, infant born at GA < 35 weeks, infant who are critically ill, parental refusal to consent, and parents unavailable to consent. The study was approved by the University of Kentucky Institutional Review Board.

Parents of infants who met the inclusion criterion were identified and approached. The informed consent was obtained from the parents. Blood samples, 1.2 ml, were collected from all the subjects at 48 h of life in the non-NAS group and within the first 48 h after the admission to the NICU in the NAS group during the regular blood draw for lab work. Plasma was separated by centrifugation and stored in −80°C till all samples were collected. Measurement of plasma level of BDNF was performed by an enzyme-linked immunosorbent assay (ELISA) method using the human BDNF kit (RayBio Human BDNF ELISA, RayBiotech Inc., GA, USA), according to the manufacturer's instructions. Plasma samples were diluted 1:100 for BDNF measurement. All BDNF measurement was performed in duplicate. Both NAS and non-NAS were run together in the same plate. The BDNF content was expressed as nanogram (ng) of human recombinant BDNF protein per milliliter of plasma.

We followed the clinical practice guideline for NAS treatment for all NAS infants. NAS scoring using the Finnegan Scale was performed every 3 h after admission, equaled to total of 16 time points in the first 48 h after admission. Opiate replacement therapy with morphine sulfate was started at 0.05 mg/kg, q3 h was started when met the criteria: 3 consecutive scores each ≥9 or 2 consecutive scores ≥13. Morphine dose was increased by 25% of the prior dose if the consecutive scores still met the above criteria and symptoms not captured until reached the maximum dose of 0.12 mg/kg/dose. Second medications were added if the symptoms were still not under controlled with scores met the criteria despite being on the maximum morphine dose based on the history; clonidine for opiate use, phenobarbital for barbiturate use, and diazepam for benzodiazepine use. Morphine weaning was started if the scores remained at desired limits for at least 48 h by 0.02 mg per dose q 24–48 h. The infants were monitored for at least 48 h after morphine was discontinued before discharge. If the infants were also on second medications, they were discharged home on tapering doses with close follow-up at the NICU graduate clinic.

#### Statistical Analysis

An independent sample *t*-test was conducted to compare the BDNF levels between NAS group and non-NAS group. A value of *p* ≤ 0.05 was considered to indicate significance. We also studied the correlation between BDNF levels and the severity of NAS among infants exposed to opiates *in utero*. The severity of NAS was defined by the length of stay in hospital and whether more than one medicine was needed to treat withdrawal symptoms. Pearson correlation test was performed to study the possible association between BDNF level and length of stay. SAS version 9.4 (SAS Institute, Cary, NC, USA) was used for analyses.

# RESULTS

Total of 67 infants were enrolled, 34 in NAS group and 33 in non-NAS group. One infant from NAS group withdrew from study by the parents.

Mean gestational age did not differ between NAS (38.5 ± 1.3 weeks) and non-NAS groups (39 ± 1.2 weeks), *p*= 0.84 (**Table 1**). Mean birth weight of the NAS infants was significantly lower than the non-NAS infants, 3,070 ± 523 vs. 3,340 ± 459 g, *p* = 0.028 (**Table 1**). Mean BDNF level was significantly higher in NAS infants compared to non-NAS infants (252.2 ± 91.6 vs. 211.3 ± 66.3 ng/ml, difference 41 (2, 80) *p* = 0.04) (**Figure 1**).

The area under the receiver operating characteristic curve (ROC) curve is estimated to be 0.641. Based on the minimum Euclidean distance using the ROC curve, the estimated optimal cutoff BDNF level for predicting groups is 240. Using this cutoff value, estimated sensitivity is 61.7%, specificity is 60.6%, positive predictive value is 61.7%, and negative predictive value is 60.6%.

Among the NAS group, 29 infants required one medication whereas 4 infants required two medication for the treatment of NAS, 1 infant did not required any medication for the treatment. There were no differences in BDNF levels between NAS infants who required one medication vs. more than one medication

*Mean birth weight of the NAS infants was significantly lower than the non-NAS infants, 3,070* ± *523 vs. 3,340* ± *459 g, p* = *0.028. NS, not significant.*

[254 ± 91 vs. 218 ± 106 ng/ml, difference 36 (−64, 137), *p* = 0.47]. The correlation between the BDNF levels and length of hospital stay among NAS infants was not detected using Pearson Correlation (correlation 0.07, *p* = 0.68) (**Figure 2**).

In the first 48 h after admission for NAS, there were no significant correlations between the BDNF levels and NAS scores at 15 of the 16 time points (Spearman Correlation 0.31 to −0.21, *p* = 0.07–0.95). However, there was a marginally significant correlation between BDNF levels and NAS scores at time point 5 (around 15 h after admission) (Spearman correlation 0.35, *p* = 0.045). There were no significant correlations between BDNF levels and maximum scores (*p* > 0.05), and the means or the total scores in the first 48 h of admission (*p* = 0.43). The distribution of the NAS scores significantly changed over time based on Friedman's test (*p* < 0.0001) as depicted in **Figure 3**.

Infants in the NAS group were also exposed to other substances, which include cocaine, benzodiazepines, tetrahydrocannabinol (THC), tobacco, amphetamine, methamphetamine, and gabapentin. When BDNF levels among infants with NAS were compared across levels in the given variables, there was no significant difference (**Table 2**).

# DISCUSSION

To our knowledge, this is the first study on plasma BDNF level in infants exposed to opiates *in utero*. Our results showed that plasma BDNF level was significantly increased in NAS infants in early withdrawal phase compared to non-NAS infants. There were no statistically significant correlations between plasma BDNF levels and the severity of NAS based on the length of hospital stay or the number of medications needed for treatment. The BDNF levels did not correlate with NAS scores at any given time point except at around 15 h after admission for NAS, at which point, most of the infants had been started on morphine. The

NAS Scores distribution became less dispersed over time after being treated as could be expected. Based on the data, we did not find the cutoff BDNF level that will provide good sensitivity or specificity to predict NAS.

The lower birth weight in NAS infants was consistent with the effect of opiates on the birth weight previously described in the literature (32), but whether this factor may contribute to the higher BDNF levels in this group remains to be further

Table 2 | Brain-derived neurotrophic factor levels among infants with neonatal abstinence syndrome and comparisons across levels in the given variables.

*Mean* ± *SD (n).*

elucidated. The existing data regarding the effects of low birth weight/intrauterine growth restriction on the BDNF levels are still conflicting; at term, the BDNF levels of infants with IUGR were not different from those born appropriate for gestational age (AGA) (33) while the BDNF levels were lower in very preterm infants with severe growth restriction (34).