Effect of etofenamate on midline closure defect in early chicken embryos: an experimental study

Abstract

The aim of this study is to investigate the effect of etofenamate, a nonsteroidal anti-inflammatory drug, on the development of midline closure defects in early chick embryos. The study was conducted at the Animal Laboratory of the Department of Animal Science, Faculty of Agriculture, Kahramanmaraş Sütçü İmam University. A total of 80 fertilized, pathogen-free, day-zero Broiler chicken eggs were used. The eggs were incubated for 24 h at 37.8 ± 0.2 °C with 65–75% humidity in an incubator with an automatic turning system. Subsequently, a 0.3 cm window was made in the eggs, and the eggs were randomly divided into four main groups and administered different doses of etofenamate (1, 2, 4 mg/day). On the 10th day of incubation, embryos were evaluated macroscopically, and those showing developmental delay or being unfertilized were excluded. Histological samples were taken from the brain and spinal cord regions of developed embryos to assess neural tube formation. In the control group, neural tube closure occurred in 95% of the embryos, while one embryo (5%) in the low-dose etofenamate group exhibited a neural tube defect. No neural tube defects were observed in the medium and high-dose groups; however, both groups showed a 25% rate of early embryonic death. The results indicate that etofenamate may negatively affect neural tube development at low doses, with the effect varying depending on the dose, and that higher doses increase the risk of embryonic death.

Introduction

Neural tube defects (NTDs) are the most common birth anomalies of the central nervous system (CNS) and encompass defects of varying severity. NTDs can occur anywhere along the neural axis and present with a wide range of clinical severity. The subtypes are named according to the anatomical region and the severity of the defect¹. The most severe forms of NTDs are anencephaly and craniorachischisis, where the forebrain or the entire CNS, respectively, fail to transition from the neural plate to the neural tube. The least severe forms, such as spinal lipoma or spina bifida occulta, which are seen in 4–6% of the general population, indicate that the neural tube is almost completely closed and are often incidentally detected during spinal imaging in many cases¹. NTDs cause approximately 88,000 deaths worldwide each year².

Known risk factors for spina bifida include family history, pre-pregnancy maternal diabetes, obesity, low socioeconomic status, hyperthermia, and exposure to certain medications such as valproate³. Nonsteroidal anti-inflammatory drugs (NSAIDs) including ibuprofen, aspirin, naproxen, and cyclooxygenase-2 (COX2) inhibitors are among the most commonly used medications during pregnancy⁴. Used to treat various conditions, NSAIDs reduce prostaglandins by blocking COX enzymes, thereby reducing pain, fever, and inflammation⁵. A survey conducted in the United States on over 20,000 women found that 22.6% of pregnant women reported using NSAIDs during the first trimester⁶. Although there is no FDA guideline against NSAID use in the first trimester, some studies suggest that NSAIDs may negatively affect embryonic processes occurring in the first 12 weeks of pregnancy. These processes include implantation, decidualization, neurulation, neural crest migration and differentiation, cardiogenesis, and nephrogenesis. The potential effects of developmental exposure are particularly concerning given that some NSAIDs, including ibuprofen and naproxen, are available over-the-counter. NSAIDs inhibit cyclooxygenase (COX) isoenzymes and thereby prevent the synthesis of prostanoids. Although not fully understood, studies have shown that prostanoids play significant roles in early development⁷. Previous in vivo studies using pregnant diabetic rats have shown that injecting arachidonic acid during the organogenesis period significantly reduces neural tube closure defects and abnormalities in neural crest-derived tissues⁸.

We hypothesize that etofenamate administration during early embryonic development leads to midline closure defects and embryonic death in a dose-dependent manner. The aim of this study is to investigate whether etofenamate, an NSAID, has an effect on the development of midline closure defects in early chick embryos.

Materials and methods

Prior to this study, ethics committee approval was obtained from the Kahramanmaraş Sütçü İmam University Experimental Animals Local Ethics Committee (Meeting date: 28.03.2025, Meeting Number: 2025/03). In this experimental study, we confirm that all experiments were carried out in accordance with relevant guidelines and regulations. This experimental study was reported in accordance with ARRIVE guidelines.

This study was conducted in the Animal Laboratory of the Department of Animal Science, Faculty of Agriculture, Kahramanmaraş Sütçü İmam University. A total of 80 freshly fertilized, pathogen-free, day-zero white Broiler chicken eggs were used. The eggs were weighed (average weight: 65 ± 5 g) and incubated at 37.8 ± 0.2 °C with 65–75% relative humidity in an incubator equipped with an automatic turning system operating every two hours, for a period of 24 h.

At the 24th hour of incubation, all eggs were sterilized, and a 0.3 cm window was carefully created at the location corresponding to the air sac. The eggs were then randomly divided into four main groups (n = 20). Etofenamate was dissolved under sterile conditions in physiological saline, and stock solutions of 0.1 cc at predetermined concentrations were prepared. Each group received 0.1 cc of the prepared solution via injection using an insulin syringe.

The Group 2 dose in the study was determined as the base dose corresponding to human exposure. The dose-response relationship was investigated by applying two times this dose to Group 3 and 4 times this dose to Group 4. Thus, the effects of different doses were examined systematically.

In this study, a power analysis was conducted to determine the difference in the incidence of neural tube defects (NTDs) among four groups (control and three different etofenamate dose groups). The expected incidence of NTDs was assumed to be 1% in the control group and 5%, 10%, and 15% in the groups receiving low, medium, and high doses of etofenamate, respectively. To detect these differences with a significance level of 5% and a power of 75–80%, approximately 20 embryos were planned for each group, resulting in a total sample size of 80 embryos. The power analysis was performed using the G*Power 3.1 software.

• Group 1a (Negative control group): Only a 0.3 cm opening was made in the eggshell, without any injection (n = 10).

• Group 1b (Saline-Injected Control Group): A 0.1 cc injection of physiological saline was administered (n = 10).

• Group 2: Administered etofenamate at a dose of 1 mg/day (n = 20).

• Group 3: Administered etofenamate at a dose of 2 mg/day (n = 20).

• Group 4: Administered etofenamate at a dose of 4 mg/day (n = 20).

Following the injection, the 0.3 cm windows were sealed using sterile tape. The eggs were then rotated 180° and returned to the incubator. On the 10th day of incubation, the eggs were opened for embryological evaluation. Macroscopic examination was conducted to assess embryonic development, and unfertilized embryos were excluded from the study. Histological examination under a light microscope was performed to determine whether the neural tube along the craniospinal axis was open or closed, which served as the basis for classifying the embryos. The collected data were subjected to statistical analysis.

Injections were performed at 24 h of incubation, corresponding to stages 7–8 according to the Hamburger-Hamilton classification. This timing was chosen based on literature review^9–16. The injections were carried out in the Animal Laboratory of Kahramanmaraş Sütçüimam University, Faculty of Agriculture, Department of Animal Husbandry.

Ethophenamate was administered to the embryo in the air sac at 24 h of incubation. A single dose was administered. The air chamber is considered to be the ideal injection site because of the ease of application of the air chamber injection method, the minimal risk of infection of the eggs, the homogeneous and rapid diffusion of the solution given, and the elimination of mechanical damages that may occur in the embryo due to the increase in intra-ovarian pressure in other methods^12,17,18.

In the studies performed with the subblastoderm technique, it is difficult to examine the samples taken on the 3rd day when neurulation is completed in chick embryos under the light microscope, it is difficult to take pathological preparations. In addition, due to the very low survival rate on the 4th and 5th days in this technique, the difficulty of macroscopic evaluation, gastroschisis, anencephaly, developmental retardation, etc. findings that may occur cannot be evaluated.

In the studies performed with pregabalin shown below, it was reported that pregabalin caused midline closure defect in the study performed with subblastodermal injection technique¹⁵while this effect was not observed in the study performed by Seçinti et al.¹². This suggests that the reason for this may not be a midline closure defect but a delay, and examining embryos on the 8th–10th day, when both the technique and the organogenesis process are completed, provides a more objective study and provides the advantage of evaluating other organ damages that may develop^12,13,19,20.

Histopatoligical examination

During macroscopic evaluation, cases in which embryonic development was not observed were recorded, and no tissue sampling was performed from these embryos (see Fig. 1). On the 10th day of incubation, embryos were evaluated according to the Hamburger and Hamilton (HH) scale. Embryos that did not reach the expected developmental stage for day 10, as defined by the HH scale, were interpreted as showing signs of developmental delay (see Fig. 2). Embryos that exhibited normal development were also documented. The reason for evaluating embryos on day 10 is that examining embryos on days 8–10, when the organogenesis process is completed, is a more objective study and provides the advantage of evaluating other organ damage that may develop^12,13,19,20.

Fig. 1.

Macroscopic images of embryos in 4 groups at day 10 after drug administration. (a) Macroscopic image of the embryo of group 1 (control group) at stage 36 (10 days) according to the Hamburger and Hamilton chicken embryo development stages. (b) Macroscopic image of embryo of group 2 (1 mg) at stage 23 (3.5 days) according to Hamburger and Hamilton chicken embryo development staging. (c) Macroscopic image of embryo of group 2 ( 1 mg) at stage 24 (4 days) according to Hamburger and Hamilton chicken embryo development staging. (d) Macroscopic image of the embryo of group 4 (4 mg) at stage 26 (4.5-5 days) according to Hamburger and Hamilton chicken embryo development staging. (e) Macroscopic image (white arrow) of embryos at stage 31 (7 days) in group 4 (4 mg) and macroscopic image (black arrow) of embryos at stage 36 (10 days) in group 2 according to Hamburger and Hamilton chicken embryo developmental stages.

Fig. 2.

(a) Microscopic image of a normally developing embryo in group 2 (1 mg). Closed neural tube, normal embryonic development (x100 magnification, Hematoxylin&Eosin) (NL: Normal Lamina, MS: Medulla Spinalis, NTC: Notochord). (b) Microscopic image of embryo with neural tube defect in group 2. Defective lamina and open neural tube (x100 magnification, Hematoxylin&Eosin) (DL: Defective Lamina, MS: Medulla Spinalis, NTC: Notochord). (c) Microscopic image of a normally developing embryo from the control group (group 1). Closed neural tube, normal embryonic development. (d) Microscopic image of a normally developing embryo from group 3 (2 mg). Closed neural tube, normal embryonic development. (e) Microscopic image of a normally developing embryo in group 4 (4 mg). Closed neural tube, normal embryonic development. (100x magnification, Haematoxylin and Eosin) (DL: Defective Lamina, MS: Medulla Spinalis, NTC: Notochord).

From embryos that had reached an adequate developmental stage for sampling, tissue samples approximately 2 mm thick were collected from two cranial and three spinal regions. Standard histological tissue processing protocols were followed, and sections were stained with Hematoxylin and Eosin (H&E). The sections were evaluated by a pathologist. Neural tube formation was histologically traced along the craniospinal axis, and embryos were classified into two categories based on whether the neural tube was open or closed.

This methodological approach was designed to evaluate the effect of etofenamate on midline closure defects in early-stage chicken embryos.

Tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 3 μm and stained with hematoxylin and eosin using a standard protocol²¹.

Histological images were captured using a Nikon Eclipse Ni-U upright microscope equipped with a Nikon DS-Fi3 digital camera. Images were acquired under bright-field illumination at 100× magnification.

Statistical analysis

The data obtained from the study were analysed with SPSS 21.0 software. The data from the study were analyzed to evaluate the incidence of neural tube closure, early embryonic death (EED), developmental delay, and neural tube defects (NTDs) across four different groups with varying doses of etofenamate. A comparison between the groups was conducted using descriptive statistics and chi-square tests to assess differences in the occurrence of these outcomes.

Results

Although the study included two separate control groups, no statistically significant difference was observed between these groups. Therefore, both control groups were combined and treated as a single control group in the subsequent analyses. The p-values presented in Table 1 indicate the results of the chi-square test used for multiple group comparisons.

Table 1.

Comparison of neural tube closure, developmental retardation, and early embryonic death among study groups.

Embryo evaluatio	n	1 mg	2 mg	4 mg	Control	p
No development	14	3	5	5	1	0.043
Developmental retardation	4	2	0	2	0	0.012
Neural tube open	1	1	0	0	0	0.458
Neural tube closed	63	14	15	15	19	0.103

Group 1 (Control Group)

Among the total of 20 embryos, neural tube closure was observed in 19 embryos (95%). One embryo (5%) exhibited no development and was classified as early embryonic death (EED). No embryos with neural tube defects were identified in this group.

Group 2 (1 mg/0.1 cc Etofenamate)

Following administration, neural tube closure was completed in 14 embryos (70%). Early embryonic death (EED) was detected in 3 embryos (15%), while 2 embryos (10%) exhibited notable developmental delay. Neural tube defect was observed in 1 embryo (5%). Tissue sections were taken from both embryos with developmental delay for histological analysis.

Group 3 (2 mg/0.1 cc Etofenamate)

Neural tube closure was confirmed in 15 embryos (75%) out of 20. No development (EED) was noted in 5 embryos (25%). No neural tube defects were detected in this group.

Group 4 (4 mg/0.1 cc Etofenamate)

Similar to Group 3, neural tube closure was observed in 15 embryos (75%). Five embryos (25%) exhibited early embryonic death, and 2 embryos (10%) showed signs of developmental delay. No neural tube defects were observed in this group either (Fig. 3).

Fig. 3.

Comparison of neural tube closure, developmental retardation, and early embryonic death among study groups.

A statistically significant difference was observed in the rate of early embryonic death between the control group and the group administered 2 mg/0.1 cc of etofenamate (p < 0.05) (Table 1). The incidence of early embryonic death was markedly higher in the etofenamate-treated group compared to the control group. These findings suggest that even moderate doses of etofenamate may adversely affect embryonic viability during early development.

In the study, normal development was observed in the embryos of the control group (Group 1), the neural tube was completely closed and normal embryonic structures were preserved (Fig. 2c). Similarly, in Group 3, which received 2 mg/kg/day etofenamate, the neural tube was closed and embryonic development was normal (Fig. 2d). Again, in Group 4, which received 4 mg/kg/day etofenamate, embryonic structures were normal and neural tube closure was completed (Fig. 2e). In all histologic sections, the lamina, medulla spinalis (MS) and notochord (NTC) were anatomically distinguished and no evidence of defective lamina (DL) was observed in these regions. Sections were stained with Hematoxylin-Eosin (H&E) and evaluated at 100x magnification.

Discussion

The perinatal period, which encompasses the time just before and after childbirth, is an especially delicate time for women who experience chronic pain disorders. This period brings with it a range of physiological and psychological challenges that can make managing chronic pain particularly difficult. Despite the fact that chronic pain conditions are common among pregnant women, there is still a lack of comprehensive data about the prevalence, progression, and best ways to manage these disorders during the perinatal period²². Treatment for chronic pain during this time typically involves both pharmacological and non-pharmacological strategies. Non-pharmacological options, like physical therapy, are often combined with medications to help relieve pain and enhance overall well-being. However, choosing the best pain management strategy is complicated by ongoing uncertainties about the safety and effectiveness of various treatments, which presents a significant challenge for both patients and healthcare providers.

For example, a study of pregnant women experiencing low back pain found that a significant portion of the patients (75%) did not receive any advice or recommendations on how to manage their symptoms, even though only 32% of these women reported their pain to their doctors during the antenatal period²³. This disconnect between patients and healthcare providers highlights a major issue in managing chronic pain during pregnancy, where concerns about the safety of treatments often lead to delays in intervention. Additionally, another challenge arises from the limited and often conflicting data about the pharmacological treatments available for chronic pain during pregnancy. This lack of clarity has resulted in a cautious, sometimes overly restrictive approach to pain management during pregnancy, leaving both patients and healthcare providers with little clear guidance on how to proceed.

Regarding pharmacological treatment, the U.S. Food and Drug Administration (FDA) has issued guidelines on the use of nonsteroidal anti-inflammatory drugs (NSAIDs) during pregnancy, advising against their use after the 20th week of gestation²⁴. This advice is based on the potential risks these medications pose to both the mother and fetus, particularly when they are not used properly during pregnancy. Pain management during pregnancy is important not only for the comfort and quality of life of the mother but also for maintaining her overall health. However, the decision to use pain-relieving medications must carefully balance the need for relief with the safety of the developing baby. While NSAIDs are commonly used in the general population for various pain conditions, their use during pregnancy presents significant risks, especially during the early stages of pregnancy^25–28.

NSAIDs work primarily by inhibiting the production of prostaglandins, which are essential for regulating various physiological functions, including the inflammatory response. However, when prostaglandin production is suppressed, adverse outcomes can occur, especially in the early stages of fetal development. For instance, the use of NSAIDs has been linked to a variety of harmful effects on the fetus, including premature closure of the ductus arteriosus, oligohydramnios (low amniotic fluid), kidney dysfunction, and malformations. These risks are particularly concerning given the rapid and sensitive development of the fetus’s organs during early pregnancy²⁹. For this reason, it is essential to carefully assess the safety of any pharmacological agents, including NSAIDs, before incorporating them into a pregnant woman’s pain management plan.

The findings obtained in this study indicate that etofenamate may exert adverse effects on embryonic development even at low doses. Notably, the administration of etofenamate at a dose of 1 mg/0.1 cc resulted in a 5% incidence of neural tube defects (NTDs), suggesting the possibility of a direct teratogenic effect on neural tube closure, which is a critical stage in normal embryonic development. Although no distinct NTDs were detected at higher doses (2 mg and 4 mg), the significant increase in early embryonic mortality and the rates of developmental delay at these doses is noteworthy. This suggests that etofenamate may exert dose-dependent toxic effects at different stages of embryonic development³⁰.

Similarly, previous research on flunixin meglumine (FM), another potent NSAID, demonstrated that FM exposure during early incubation significantly increased embryonic mortality and caused marked reductions in embryo weight and crown–rump length, particularly at higher doses, accompanied by structural anomalies³¹. These findings align with the present study’s observation that etofenamate may elevate the risk of neural tube defects even at low doses, reinforcing evidence that NSAIDs can exert embryotoxic effects during critical developmental windows. Given that neural tube formation is highly sensitive to teratogenic insults, these parallel results suggest that the systemic absorption of NSAIDs — including those administered topically — may pose underestimated risks. Therefore, the perceived safety of local NSAID use in early pregnancy warrants careful re-evaluation, especially for agents like etofenamate.

The study showed that, in the control group (Group 1), which did not receive any treatment with etofenamate, neural tube closure occurred successfully in 95% of cases, with no NTDs detected. However, in Group 2, which received a dose of 1 mg/0.1 cc of etofenamate, 5% of cases showed neural tube defects, along with increased rates of developmental delays and early embryonic death. This suggests that even low doses of etofenamate could have teratogenic potential, disrupting normal embryonic development and leading to significant developmental abnormalities. Neural tube formation occurs during the third and fourth weeks of embryonic life, and any toxic influence during this critical period could lead to conditions such as spina bifida or anencephaly³².

Further examination of higher-dose groups (Groups 3 and 4) revealed that while no NTDs were observed, the incidence of early embryonic death increased significantly to 25%, and the rate of developmental delay remained around 10%. This suggests that higher doses of etofenamate may interfere with embryonic development at even earlier stages, possibly causing embryonic loss before malformations can be detected. These findings emphasize the need for careful monitoring of topical NSAID dosages, even when it is assumed that systemic absorption is minimal.

The results from this study indicate that etofenamate has the potential to cause both developmental delays and embryonic death in a dose-dependent manner. More importantly, even low doses of etofenamate appear capable of causing neural tube defects, raising serious concerns about its safety during pregnancy, particularly in the first trimester. While topical NSAIDs are generally considered safe due to their localized effects and minimal systemic absorption, this study challenges that assumption, suggesting that the pharmacokinetics of topical NSAIDs and their potential for systemic absorption requires further investigation^33,34.

In the control group of our study, complete neural tube closure was observed in 95% of the embryos, with an early embryonic death (EED) rate of only 5%, and no neural tube defects were detected. In contrast, the groups treated with etofenamate showed a clear dose-dependent increase in EED rates, with the 2 mg/0.1 cc dose in particular demonstrating a statistically significant elevation in EED compared to the control group (p < 0.05). These findings suggest that even moderate doses of etofenamate may compromise embryonic viability.

A comparable study conducted with diclofenac sodium reported that increasing doses of this NSAID led to midline closure defects in early chick embryos, thereby impairing neural tube closure. Additionally, that study documented significant reductions in crown-rump length and somite number in the higher dose groups, further indicating adverse effects on embryonic growth at both morphological and histopathological levels³⁵.

When considered together, the results of both studies underscore the potential for non-steroidal anti-inflammatory drugs (NSAIDs) such as etofenamate and diclofenac sodium to interfere with neural tube development. Although both belong to the same pharmacological class, their teratogenic effects appear to differ in mechanism and presentation. In the etofenamate-treated groups, neural tube closure was morphologically complete even at higher doses; however, the increased rates of EED and occasional developmental delays imply that etofenamate may exert embryotoxic effects through pathways that do not necessarily involve gross neural tube defects. In contrast, the diclofenac sodium study demonstrated clear midline closure failures, suggesting a direct impact on neurulation.

Furthermore, histological examinations in the etofenamate groups revealed that the anatomical structures of the spinal cord and notochord remained intact, with no evidence of lamina defects. This indicates that while the neural tube may appear structurally normal, viability can still be compromised. On the other hand, embryos exposed to diclofenac sodium showed both morphological disruptions in tube closure and overall developmental retardation. These contrasting patterns imply that diclofenac sodium may disrupt neural tube formation through both mechanical and potentially cellular pathways.

In conclusion, the use of NSAIDs, including topical formulations like etofenamate, for pain management during pregnancy should be approached with extreme caution. This study stresses the importance of a thoughtful, informed approach when selecting analgesics, especially during the early stages of pregnancy. The findings contribute crucial insights into the potential risks of NSAID use during pregnancy, showing that even low doses of topical NSAIDs like etofenamate can lead to teratogenic effects such as neural tube defects, developmental delays, and early embryonic death. This research calls for more studies to better understand the pharmacokinetic properties of topical NSAIDs and their impact on fetal development. It also urges healthcare providers to exercise greater caution when recommending pain management options for pregnant women, particularly when using pharmacological treatments like NSAIDs. To clearly determine whether etofenamate has an effect on NTDs at higher doses, further large-scale studies with larger sample sizes should be conducted.

Limitations

The primary limitations of this study include the use of a single animal model (chicken embryos), the lack of long-term post-hatching follow-up, and the absence of pharmacokinetic measurements of etofenamate in embryonic tissues. No different measurements were made in this study. Measurements such as craniocaudal length, mean head diameter, thoracic and lumbar diameter can be done. In our next study, a comprehensive study will be conducted with an emphasis on histometric measurements.

Author contributions

Concept: R.E., M.K; Design: R.E., M.K; Data Collection or Processing: R.E., M.K, H.T, Ö.D.G; Analysis or Interpretation: R.E., M.K, H.T, Ö.D.G; Literature Search: R.E., M.K, H.T, Ö.D.G; Writing: R.E., M.K, H.T, Ö.D.G.

Funding

All financing of the study was covered by the researchers.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.