In recent years, numerous countries have witnessed a rise in the incidence of preterm births. About 15 million preterm neonates (less than 37 weeks of gestation) are born each year worldwide [1]. Substantial evidence indicates that prolonged exposure to painful stimuli in neonates before the maturation of the nervous system can result in enduring behavioral changes. Given the ongoing development of the central nervous system in preterm neonates, they exhibit heightened susceptibility to pain. Repetitive exposure to painful stimuli has the potential to modify physiological processes and stress hormone levels, activate the hypothalamic–pituitary–adrenal axis, induce alterations in pain sensitivity, and disrupt the structural and functional organization of the developing brain [2].

In this condition, unresolved pain may result in both short-term and long-term adverse effects [2-4]. Persistent exposure to painful procedures affects the neonate's nervous system development and may cause behavioral changes. In other words, uncontrolled pain may have both short and long-term effects [2, 5]. For example, the short-term effects of pain include decreased oxygenation, hemodynamic instability, increased HR, and elevated intracranial blood pressure [6-9]. The long-term effects include excessive anxiety and pain sensitivity, irritability, sleep problems, reduced nutrition, and attention deficiency, and may even result in the neonate's death [10-12].

To reduce the negative effects of necessary painful procedures on preterm infants, both drug-based and non-drug interventions are used [13]. However, due to possible side effects from medications, it is recommended to focus on non-pharmacological methods for managing pain in these infants. These non-pharmacological methods include kangaroo mother care, non-nutritive sucking (NNS), breastfeeding, sucrose consumption, massage, facilitated tucking, and exposure to the scent of breast milk [12, 14-16].

Non-nutritive sucking refers to placing a pacifier in a newborn's mouth to encourage sucking without delivering breast milk or formula for nutrition [13]. This technique is thought to provide pain relief by stimulating oral mechanoreceptors, which alter how pain signals are transmitted or processed through the body's natural non-opioid pain control system [17]. The results of some clinical trials showed that NNS was effective in reducing the pain of procedures such as heel stick, venipuncture, blood sampling, and vaccination [7, 18]. However, Perroteau et al. found that facilitated tucking and NNS were ineffective during heel stick [1].

Although some reviews have summarized techniques for acute pain reduction during procedures in neonates and young children, ours is the first meta-analysis to isolate the effect of pure non-nutritive sucking during heel lance specifically in preterm neonates. This is due to the limited availability of high-quality evidence and the absence of a validated pain assessment tool for preterm neonates [19]. Therefore, the present systematic review and meta-analysis was conducted to evaluate randomized clinical trials (RCTs) investigating the effect of NNS on heel stick pain in preterm neonates.

A total of 6,216 articles were retrieved through database and manual searches, with an additional 28 references identified through manual searching. After removing duplicates and excluding irrelevant studies based on title and abstract screening, eight full-text articles were assessed and selected for meta-analysis. Furthermore, the reference lists of the included studies were manually reviewed, but no additional relevant papers were identified (Fig. 1).

These studies included 225 newborns, with 116 in the NNS group and 109 in the control group. Male neonates comprised 58.5 percent of the subjects (n = 131). The mean gestational age (GA) was 31.6 ± 2.02 weeks, and the mean birth weight was 1632.2 ± 322.8 g.

The characteristics of the NNS interventions were reported in most of the studies (Table 2). Eight randomized clinical trials were included in the systematic review. One study compared NNS with oral sucrose and the combination of both techniques. A second study compared a combination of music, NNS, facilitated tucking, and holding versus a combination of facilitated tucking and holding. A third study compared sucking + breast milk and sucking + breast milk + tucking versus routine care. A fourth study compared a combination of NNS and facilitated tucking with NNS alone. A fifth study compared combined NNS and facilitated tucking with routine care. A sixth study compared NNS with routine care. A seventh study compared pharmacologic measures with non-pharmacologic measures. The final study compared NNS and swaddling with routine care.

In one study, the control group received no intervention. In the remaining studies, the control group received NNS, facilitated tucking, or routine care. Facilitated tucking, holding, and routine comfort through gentle touch were considered inactive control interventions.

Neonates, particularly preterm infants, exhibit heightened sensitivity to pain [1, 22]. Consequently, effective pain management is a critical component of care in the Neonatal Intensive Care Unit (NICU). Several studies have demonstrated the effectiveness of Non-Nutritive Sucking (NNS) in pain control [14-23]. The proposed analgesic effect is thought to involve stimulation of oral mechanoreceptors, which may modulate nociceptive transmission or perception through endogenous non-opioid pathways. This intervention should be used only when neuromuscular coordination has sufficiently developed to initiate oral feeding [17].

Heel stick is one of the most common procedures in NICUs [7, 15, 17], yet the analgesic effects of NNS in neonates, especially preterm infants, remain unclear. Therefore, this meta-analysis was conducted to evaluate the effectiveness of NNS in reducing heel-stick pain severity among preterm neonates.

The present systematic review and meta-analysis found that NNS is an effective method of managing pain in preterm neonates. A previous systematic review examining distraction techniques for young children undergoing medical procedures reported that toy-based distraction did not reduce post-needle pain scores. This may be attributed to inconsistent implementation and the fact that some children perceive physical contact from medical staff as intimidating; initiating distraction only at the moment of contact may have limited its effectiveness. In contrast, the present review focused exclusively on preterm infants and used NNS to minimize the influence of physical contact on outcomes [24].

Another systematic review on oral sucrose for neonatal pain relief showed that sucrose effectively reduces procedural pain. However, the optimal dosage was not established due to variability across studies. The present review focused solely on NNS as a safer and more accessible technique that does not require administration of additional substances or dose adjustments [23]. A similar systematic review on full-term neonates also confirmed the analgesic benefits of NNS during heel stick procedures [13]. However, unlike that review, our study specifically targeted preterm neonates, who are more sensitive to pain and whose pain management needs are especially critical.

Other evidence supports the analgesic role of NNS in various neonatal procedures. For example, NNS has been shown to significantly reduce pain during retinopathy screening [25] and to have a longer duration of action than sucrose [26]. Haixia Gao et al. [27] reported that while both NNS and sucrose reduced the duration of crying during heel stick in preterm neonates, neither intervention reduced the physiological response to pain. Other studies have suggested that NNS and sucrose have comparable effects on heel stick pain reduction.

Overall, there is strong evidence that NNS reduces pain behaviors during procedures such as heel stick and injections [28]. Although combined interventions such as NNS plus sucrose are effective, sucrose intake is restricted in preterm neonates [23]. Therefore, identifying methods that minimize pain with minimal risk is a major priority for clinicians. According to the Gate Control Theory, the use of a pacifier may inhibit transmission of pain impulses along the spinothalamic tract, thereby reducing pain perception. NNS may further decrease pain-related behaviors and mitigate associated complications [8]. Additionally, it is cost-effective, requires no advanced technology, and can be administered by parents.

The present meta-analysis also found that NNS was relatively effective in stabilizing heart rate (HR) during the recovery phase following heel stick compared with routine care. In three included studies, no baseline differences were observed between groups, and both groups demonstrated HR increases during the procedure due to pain. However, our findings differed from those of Yi Liu et al. [22], who reported no effect of NNS on HR after painful interventions. This discrepancy may be explained by the combined use of sucrose and NNS in their study. The limited number of comparative studies available in the literature highlights the need for further research. Hiroko Uematsu et al. [29] found that NNS combined with music (Brahms’ lullaby) decreased HR following heel stick in preterm neonates and resulted in fewer abnormal HR fluctuations.

Regarding oxygen saturation, baseline levels did not differ significantly between groups. Consistent with Yi Liu et al. [22], our meta-analysis found no significant difference in oxygen saturation between the NNS and control groups during the recovery period after heel stick.

This meta-analysis is the first comprehensive synthesis focusing exclusively on the effects of pure NNS interventions for reducing the severity of heel-stick pain in preterm neonates. However, the findings highlight a significant shortage of high-quality research in this area. Several limitations should be acknowledged. First, most included studies had relatively small sample sizes, which may influence effect size estimates, the statistical power of findings, and the methodological rigor required to minimize bias. Second, the presence of single-center studies limits the generalizability of the results, particularly in the context of small study populations. Third, the potential influence of confounding variables—such as neonatal hunger or discomfort, and prior exposure to painful procedures—may affect behavioral and physiological pain responses, underscoring the need for future research to monitor and control these factors. Finally, because this review included only English-language publications, relevant studies in other languages may have been inadvertently excluded.

Recognizing the heightened sensitivity to pain among preterm neonates, effective pain management is essential for this vulnerable population. Findings from this systematic review and meta-analysis indicate that NNS may be effective in reducing heel-stick–related pain behaviors; however, the limited number of available studies and their relatively small sample sizes restrict the generalizability of these results. Therefore, future research should prioritize larger, multicenter randomized controlled trials to validate and further elucidate the comparative effectiveness of non-pharmacological interventions for procedural pain in preterm infants. Overall, these findings offer meaningful guidance for nurses and clinicians, supporting the use of NNS as a practical, accessible strategy to minimize repeated procedural pain in preterm neonates.