Effect of Etofenamate on Midline Closure Defect in Early Chicken Embryos: An Experimental Study

Effect of Etofenamate on Midline Closure Defect in Early Chicken Embryos: An Experimental Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of Etofenamate on Midline Closure Defect in Early Chicken Embryos: An Experimental Study Recai Engin, Muhammet Kırkgeçit, Hasan Türkoğlu, Öykü Dila Gemci This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6571385/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose 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. Material and Methods 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 hours 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 (8, 16, 32 mg/kg/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. Results 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. Conclusion 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. Etofenamate Midline Closure Defect Figures Figure 1 Figure 2 1. 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 defect [ 1 ]. 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 cases [ 1 ]. NTDs cause approximately 88,000 deaths worldwide each year [ 2 ]. 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 valproate [ 3 ]. Nonsteroidal anti-inflammatory drugs (NSAIDs) including ibuprofen, aspirin, naproxen, and cyclooxygenase-2 (COX2) inhibitors are among the most commonly used medications during pregnancy [ 4 ]. Used to treat various conditions, NSAIDs reduce prostaglandins by blocking COX enzymes, thereby reducing pain, fever, and inflammation [ 5 ]. 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 trimester [ 6 ]. 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 development [ 7 ] 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 tissues [ 8 ]. 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. 2. Material and Methods Prior to this study, ethics committee approval was obtained from the local ethics committee for experimental animals of a university (Meeting date: 28.03.2025, Meeting Number: 2025/03). 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 hours. 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. Group 1a (Negative control group) : Only a 0.3 cm opening was made in the eggshell, without any injection (n = 10). Group 1b (Positive control group) : A 0.1 cc injection of physiological saline was administered (n = 10). Group 2 : Administered etofenamate at a dose of 1 mg/kg/day (embryonic dose: 1 µg/0.1 cc) (n = 20). Group 3 : Administered etofenamate at a dose of 2 mg/kg/day (embryonic dose: 2 µg/0.1 cc) (n = 20). Group 4 : Administered etofenamate at a dose of 4 mg/kg/day (embryonic dose: 4 µg/0.1 cc) (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. 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 (See Fig. 3). 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. 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. 3. Results 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. Table 1 Comparison of neural tube closure, developmental retardation, and early embryonic death among study groups Embryo Evaluatio 1 mg 2 mg 4 mg Control p No Development 3 5 5 1 .043 Developmental Retardation 2 0 2 0 .012 Neural Tube Open 1 0 0 0 .458 Neural Tube closed 14 15 15 19 .103 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. 4. 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 period [ 9 ]. 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 period [ 10 ]. 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 gestation [ 11 ]. 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 [ 12 – 15 ]. 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 pregnancy [ 16 ]. 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. This study specifically investigates the potential teratogenic effects of etofenamate, a topical NSAID, on fetal development. The study focuses on the risk of neural tube defects (NTDs) in a dose-dependent manner, examining whether even low doses of etofenamate could contribute to these malformations. Etofenamate is often preferred for its local anti-inflammatory and pain-relieving effects, especially in cases of localized pain, because it is believed to have minimal systemic absorption. However, there are growing concerns about the possibility of systemic absorption, which could potentially have toxic effects on the developing fetus [ 17 ]. 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 anencephaly [ 18 ]. 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 [ 19 , 20 ] 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. 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. Declarations Decleration of Interest The authors declare that they have no competing interests Financing statement All financing of the study was covered by the researchers. Author Contribution Authors’ contributionsConcept: R.E., M.K Design: R.E., M.K Data Collection or Processing: R.E., M.K, H.T, Ö.D.GAnalysis or Interpretation: R.E., M.K, H.T, Ö.D.GLiterature Search: R.E., M.K, H.T, Ö.D.GWriting: R.E., M.K, H.T, Ö.D.G References Brown E, Matthes JC, Bazan III. C, Jinkins JR (1994) Prevalence of incidental intraspinal lipoma of the lumbosacral spine as determined by MRI. Spine 19(7):833–836 Zaganjor I, Sekkarie A, Tsang BL, Williams J, Razzaghi H, Mulinare J, Rosenthal J (2016) Describing the prevalence of neural tube defects worldwide: a systematic literature review. PLoS ONE 11(4), e0151586 Avagliano L, Massa V, George TM, Qureshy S, Bulfamante GP, Finnell RH (2018) Overview on neural tube defects: From development to physical characteristics. Birth Defects Res 111(19):1455–1467 Mitchell AA, Gilboa SM, Werler MM, Kelley KE, Louik C, Hernández-Díaz S, Study NBDP (2011) Medication use during pregnancy, with particular focus on prescription drugs: 1976–2008. Am J Obstet Gynecol 205(1):51e1–51e8 Davis JS, Lee HY, Kim J, Advani SM, Peng HL, Banfield E, Frazier-Wood AC (2017) Use of non-steroidal anti-inflammatory drugs in US adults: Changes over time and by demographic. Open Heart 4(1):e000550 Hernandez RK, Werler MM, Romitti P, Sun L, Anderka M, National Birth Defects Prevention Study (2012) Nonsteroidal antiinflammatory drug use among women and the risk of birth defects. Am J Obstet Gynecol 206:228e1–228e8 Leathers TA, Rogers CD (2023) Nonsteroidal anti-inflammatory drugs and implications for the cyclooxygenase pathway in embryonic development. Am J Physiol Cell Physiol 9(2):C532–C539 Goldman AS, Baker L, Piddington R, Marx B, Herold R, Egler J (1985) Hyperglycemia-induced teratogenesis is mediated by a functional deficiency of arachidonic acid. Proc Natl Acad Sci USA 82:8227–8231 Ray-Griffith S, Wendel M, Stowe Z, Magann E (2018) Chronic pain during pregnancy: a review of the literature. Int J Women’s Health 10:153–164 Wang SM, Dezinno P, Maranets I, Berman MR, Caldwell-Andrews AA, Kain ZN (2004) Low back pain during pregnancy. Obstet Gynecol 104(1):65–70 Food US, Administration D (2020) : FDA recommends avoiding use of NSAIDs in pregnancy at 20 weeks or later because they can result in low amniotic fluid. FDA drug safety. Communication. 2020(10/15):1–9 Burdan F, Szumilo J, Klepacz R (2009) Maternal toxicity of nonsteroidal anti-inflammatory drugs as an important factor affecting prenatal development. Reprod Toxicol 28(2):239–244 Dathe K, Hultzsch S, Pritchard LW, Schaefer C (2019) Risk estimation of fetal adverse effects after short-term second trimester exposure to non-steroidal anti-inflammatory drugs: a literature review. Eur J Clin Pharmacol 75:1347–1353 Dathe K, Schaefer C (2019) The use of medication in pregnancy. Deutsches Ärzteblatt international 116(46):783 Hultzsch S, Paulus W, Padberg S, Fietz AK, Schaefer C, Dathe K (2021) First trimester naproxen exposure and outcome of pregnancy–A German case series. Reprod Toxicol 103:51–57 Desai RJ, Hernandez-Diaz S, Bateman BT, Huybrechts KF (2014) Increase in prescription opioid use during pregnancy among Medicaid-enrolled women. Obstetrics & Gynecology, 2014;123(5), 997–1002 Atkinson TJ, Fudin J (2020) Nonsteroidal antiinflammatory drugs for acute and chronic pain. Phys Med Rehabilitation Clin 31(2):219–231 Dheen ST, Tay SS, Boran J, Ting LW, Kumar SD, Fu J, Ling EA (2009) Recent studies on neural tube defects in embryos of diabetic pregnancy: an overview. Curr Med Chem 16(18):2345–2354 Black E, Khor KE, Kennedy D, Chutatape A, Sharma S, Vancaillie T, Demirkol A (2019) Medication use and pain management in pregnancy: a critical review. Pain Pract 19(8):875–899 Corley RA, Mast TJ, Carney EW, Rogers JM, Daston GP (2003) Evaluation of physiologically based models of pregnancy and lactation for their application in children's health risk assessments. Crit Rev Toxicol 33(2):137–211 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6571385","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":453733677,"identity":"5a8d1d7c-2114-41e9-95a0-37279c225e7e","order_by":0,"name":"Recai Engin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYNACA4Z6+/mHDz4AMnn4iNWSYCDBlmwA0sJGrD1ALTxmEiAWQS3mDczHHn4puJNnLt1gVvk1x06GjYH54aMbeLTIHGBLN5YxeFZsOedA2m3ZbclAh7EZG+fg0SLBwGMmLWFwmLHhQMKx25LbmIFaeNik8Wvh/wbVkthWLLmtnhgtPGySHwwOJ264kczG+HHbYSK0MLOZSTMYHDaW7DnGLM247TgPGzMhv7A3P5P88eewHD97/8ePP7dV2/OzNz98jE8LAzMQ8cDYPFARgoDxBzpjFIyCUTAKRgEyAADWjUIShT5gQQAAAABJRU5ErkJggg==","orcid":"","institution":"Samsun University","correspondingAuthor":true,"prefix":"","firstName":"Recai","middleName":"","lastName":"Engin","suffix":""},{"id":453733678,"identity":"a81b89a7-b378-43d4-b87e-a5db2640561b","order_by":1,"name":"Muhammet Kırkgeçit","email":"","orcid":"","institution":"Ministry of Health Elbistan State Hospital Neurosurgery Clinic","correspondingAuthor":false,"prefix":"","firstName":"Muhammet","middleName":"","lastName":"Kırkgeçit","suffix":""},{"id":453733679,"identity":"d87766cd-5027-4926-b336-0a6c426a5deb","order_by":2,"name":"Hasan Türkoğlu","email":"","orcid":"","institution":"Gaziantep City Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hasan","middleName":"","lastName":"Türkoğlu","suffix":""},{"id":453733680,"identity":"d3756cfa-f9e1-4c87-b79b-c2fcdd4282fd","order_by":3,"name":"Öykü Dila Gemci","email":"","orcid":"","institution":"Ministry of Health Elbistan State Hospital Pathology Clinic","correspondingAuthor":false,"prefix":"","firstName":"Öykü","middleName":"Dila","lastName":"Gemci","suffix":""}],"badges":[],"createdAt":"2025-05-01 11:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6571385/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6571385/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82474342,"identity":"db3a4056-6358-4ed7-9eb5-edebdc51a62c","added_by":"auto","created_at":"2025-05-12 01:04:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":610100,"visible":true,"origin":"","legend":"\u003cp\u003ea. Macroscopic image of embryo at stage 23 (3.5 days) according to Hamburger and Hamilton chicken embryo development staging. b. Macroscopic image of embryo at stage 24 (4 days) according to Hamburger and Hamilton chicken embryo development staging. c. Macroscopic image of the embryo at stage 26 (4.5-5 days) according to Hamburger and Hamilton chicken embryo development staging. d. Macroscopic image of the embryos at stage 31 (7 days) and stage 36 (10 days) according to Hamburger and Hamilton chicken embryo development stages.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6571385/v1/5c1c723186fda7075603f38d.png"},{"id":82474343,"identity":"339ba0a3-c744-44c7-a10a-05ae721d6aee","added_by":"auto","created_at":"2025-05-12 01:04:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":590536,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 3 a. Closed neural tube, normal embryonic development (x100, Hematoxylin\u0026amp;Eosin) (NL: Normal Lamina, MS: Medulla Spinalis, NTC: Notochord) b. Defective lamina and open neural tube (x100, Hematoxylin\u0026amp;Eosin) (DL: Defective Lamina, MS: Medulla Spinalis, NTC: Notochord)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6571385/v1/73f97662ab5b68715bb47795.png"},{"id":82474344,"identity":"7902837e-91b2-4ff5-828b-592b76b50c00","added_by":"auto","created_at":"2025-05-12 01:04:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1814240,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6571385/v1/460c06d7-5dbe-4259-8c53-2eab51674ec7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of Etofenamate on Midline Closure Defect in Early Chicken Embryos: An Experimental Study","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNeural 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 defect [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. 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\u0026ndash;6% of the general population, indicate that the neural tube is almost completely closed and are often incidentally detected during spinal imaging in many cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. NTDs cause approximately 88,000 deaths worldwide each year [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eKnown risk factors for spina bifida include family history, pre-pregnancy maternal diabetes, obesity, low socioeconomic status, hyperthermia, and exposure to certain medications such as valproate [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Nonsteroidal anti-inflammatory drugs (NSAIDs) including ibuprofen, aspirin, naproxen, and cyclooxygenase-2 (COX2) inhibitors are among the most commonly used medications during pregnancy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Used to treat various conditions, NSAIDs reduce prostaglandins by blocking COX enzymes, thereby reducing pain, fever, and inflammation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. 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 trimester [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. 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 development [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] 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 tissues [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe 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.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cp\u003ePrior to this study, ethics committee approval was obtained from the local ethics committee for experimental animals of a university (Meeting date: 28.03.2025, Meeting Number: 2025/03).\u003c/p\u003e \u003cp\u003eThis study was conducted in the Animal Laboratory of the Department of Animal Science, Faculty of Agriculture, Kahramanmaraş S\u0026uuml;t\u0026ccedil;\u0026uuml; 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\u0026thinsp;\u0026plusmn;\u0026thinsp;5 g) and incubated at 37.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C with 65\u0026ndash;75% relative humidity in an incubator equipped with an automatic turning system operating every two hours, for a period of 24 hours.\u003c/p\u003e \u003cp\u003eAt 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\u0026thinsp;=\u0026thinsp;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.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGroup 1a (Negative control group)\u003c/b\u003e: Only a 0.3 cm opening was made in the eggshell, without any injection (n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGroup 1b (Positive control group)\u003c/b\u003e: A 0.1 cc injection of physiological saline was administered (n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGroup 2\u003c/b\u003e: Administered etofenamate at a dose of 1 mg/kg/day (embryonic dose: 1 \u0026micro;g/0.1 cc) (n\u0026thinsp;=\u0026thinsp;20).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGroup 3\u003c/b\u003e: Administered etofenamate at a dose of 2 mg/kg/day (embryonic dose: 2 \u0026micro;g/0.1 cc) (n\u0026thinsp;=\u0026thinsp;20).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGroup 4\u003c/b\u003e: Administered etofenamate at a dose of 4 mg/kg/day (embryonic dose: 4 \u0026micro;g/0.1 cc) (n\u0026thinsp;=\u0026thinsp;20).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eFollowing the injection, the 0.3 cm windows were sealed using sterile tape. The eggs were then rotated 180\u0026deg; 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.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistopatoligical Examination\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDuring macroscopic evaluation, cases in which embryonic development was not observed were recorded, and no tissue sampling was performed from these embryos (See Fig.\u0026nbsp;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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Embryos that exhibited normal development were also documented (See Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eFrom 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\u0026amp;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.\u003c/p\u003e \u003cp\u003eThis methodological approach was designed to evaluate the effect of etofenamate on midline closure defects in early-stage chicken embryos.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe 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.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cstrong\u003eGroup 1 (Control Group)\u003c/strong\u003e \u003cp\u003eAmong 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.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGroup 2 (1 mg/0.1 cc Etofenamate)\u003c/strong\u003e \u003cp\u003eFollowing 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.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGroup 3 (2 mg/0.1 cc Etofenamate)\u003c/strong\u003e \u003cp\u003eNeural 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.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGroup 4 (4 mg/0.1 cc Etofenamate)\u003c/strong\u003e \u003cp\u003eSimilar 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.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of neural tube closure, developmental retardation, and early embryonic death among study groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmbryo Evaluatio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 mg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 mg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 mg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNo Development\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.043\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDevelopmental Retardation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNeural Tube Open\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.458\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNeural Tube closed\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.103\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA 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\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). 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.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe 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 period [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eFor 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 period [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eRegarding 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 gestation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. 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 [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNSAIDs 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\u0026rsquo;s organs during early pregnancy [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For this reason, it is essential to carefully assess the safety of any pharmacological agents, including NSAIDs, before incorporating them into a pregnant woman\u0026rsquo;s pain management plan.\u003c/p\u003e \u003cp\u003eThis study specifically investigates the potential teratogenic effects of etofenamate, a topical NSAID, on fetal development. The study focuses on the risk of neural tube defects (NTDs) in a dose-dependent manner, examining whether even low doses of etofenamate could contribute to these malformations. Etofenamate is often preferred for its local anti-inflammatory and pain-relieving effects, especially in cases of localized pain, because it is believed to have minimal systemic absorption. However, there are growing concerns about the possibility of systemic absorption, which could potentially have toxic effects on the developing fetus [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe 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 anencephaly [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurther 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.\u003c/p\u003e \u003cp\u003eThe 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 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLimitations\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe 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.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eDecleration of Interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFinancing statement\u003c/strong\u003e \u003cp\u003eAll financing of the study was covered by the researchers.\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthors\u0026rsquo; contributionsConcept: R.E., M.K Design: R.E., M.K Data Collection or Processing: R.E., M.K, H.T, \u0026Ouml;.D.GAnalysis or Interpretation: R.E., M.K, H.T, \u0026Ouml;.D.GLiterature Search: R.E., M.K, H.T, \u0026Ouml;.D.GWriting: R.E., M.K, H.T, \u0026Ouml;.D.G\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrown E, Matthes JC, Bazan III. C, Jinkins JR (1994) Prevalence of incidental intraspinal lipoma of the lumbosacral spine as determined by MRI. Spine 19(7):833\u0026ndash;836\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZaganjor I, Sekkarie A, Tsang BL, Williams J, Razzaghi H, Mulinare J, Rosenthal J (2016) Describing the prevalence of neural tube defects worldwide: a systematic literature review. PLoS ONE 11(4), e0151586\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvagliano L, Massa V, George TM, Qureshy S, Bulfamante GP, Finnell RH (2018) Overview on neural tube defects: From development to physical characteristics. Birth Defects Res 111(19):1455\u0026ndash;1467\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitchell AA, Gilboa SM, Werler MM, Kelley KE, Louik C, Hern\u0026aacute;ndez-D\u0026iacute;az S, Study NBDP (2011) Medication use during pregnancy, with particular focus on prescription drugs: 1976\u0026ndash;2008. 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Am J Physiol Cell Physiol 9(2):C532\u0026ndash;C539\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldman AS, Baker L, Piddington R, Marx B, Herold R, Egler J (1985) Hyperglycemia-induced teratogenesis is mediated by a functional deficiency of arachidonic acid. Proc Natl Acad Sci USA 82:8227\u0026ndash;8231\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRay-Griffith S, Wendel M, Stowe Z, Magann E (2018) Chronic pain during pregnancy: a review of the literature. Int J Women\u0026rsquo;s Health 10:153\u0026ndash;164\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang SM, Dezinno P, Maranets I, Berman MR, Caldwell-Andrews AA, Kain ZN (2004) Low back pain during pregnancy. Obstet Gynecol 104(1):65\u0026ndash;70\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFood US, Administration D (2020) : FDA recommends avoiding use of NSAIDs in pregnancy at 20 weeks or later because they can result in low amniotic fluid. FDA drug safety. Communication. 2020(10/15):1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurdan F, Szumilo J, Klepacz R (2009) Maternal toxicity of nonsteroidal anti-inflammatory drugs as an important factor affecting prenatal development. Reprod Toxicol 28(2):239\u0026ndash;244\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDathe K, Hultzsch S, Pritchard LW, Schaefer C (2019) Risk estimation of fetal adverse effects after short-term second trimester exposure to non-steroidal anti-inflammatory drugs: a literature review. Eur J Clin Pharmacol 75:1347\u0026ndash;1353\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDathe K, Schaefer C (2019) The use of medication in pregnancy. Deutsches \u0026Auml;rzteblatt international 116(46):783\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHultzsch S, Paulus W, Padberg S, Fietz AK, Schaefer C, Dathe K (2021) First trimester naproxen exposure and outcome of pregnancy\u0026ndash;A German case series. Reprod Toxicol 103:51\u0026ndash;57\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDesai RJ, Hernandez-Diaz S, Bateman BT, Huybrechts KF (2014) Increase in prescription opioid use during pregnancy among Medicaid-enrolled women. Obstetrics \u0026amp; Gynecology, 2014;123(5), 997\u0026ndash;1002\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtkinson TJ, Fudin J (2020) Nonsteroidal antiinflammatory drugs for acute and chronic pain. Phys Med Rehabilitation Clin 31(2):219\u0026ndash;231\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDheen ST, Tay SS, Boran J, Ting LW, Kumar SD, Fu J, Ling EA (2009) Recent studies on neural tube defects in embryos of diabetic pregnancy: an overview. Curr Med Chem 16(18):2345\u0026ndash;2354\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlack E, Khor KE, Kennedy D, Chutatape A, Sharma S, Vancaillie T, Demirkol A (2019) Medication use and pain management in pregnancy: a critical review. Pain Pract 19(8):875\u0026ndash;899\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorley RA, Mast TJ, Carney EW, Rogers JM, Daston GP (2003) Evaluation of physiologically based models of pregnancy and lactation for their application in children's health risk assessments. Crit Rev Toxicol 33(2):137\u0026ndash;211\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Etofenamate, Midline Closure Defect","lastPublishedDoi":"10.21203/rs.3.rs-6571385/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6571385/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThe 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.\u003c/p\u003e\u003ch2\u003eMaterial and Methods\u003c/h2\u003e \u003cp\u003eThe study was conducted at the Animal Laboratory of the Department of Animal Science, Faculty of Agriculture, Kahramanmaraş S\u0026uuml;t\u0026ccedil;\u0026uuml; İmam University. A total of 80 fertilized, pathogen-free, day-zero Broiler chicken eggs were used. The eggs were incubated for 24 hours at 37.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C with 65\u0026ndash;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 (8, 16, 32 mg/kg/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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn 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.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe 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.\u003c/p\u003e","manuscriptTitle":"Effect of Etofenamate on Midline Closure Defect in Early Chicken Embryos: An Experimental Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-12 01:04:21","doi":"10.21203/rs.3.rs-6571385/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c3160925-1bfd-4a01-a90e-7e366ed700b4","owner":[],"postedDate":"May 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T01:04:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-12 01:04:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6571385","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6571385","identity":"rs-6571385","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}