|
 
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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 12
| Issue : 1 | Page : 9-14 |
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Development of the submucosa and musculature in the human fetal stomach: A microscopic study
Nivedita Roy1, Sagnik Roy2
1 Professor, Department of Anatomy, Gujarat Adani Institute of Medical Sciences, Bhuj, Gujarat, India
2 Professor and Head, Department of Anatomy, Gujarat Adani Institute of Medical Sciences, Bhuj, Gujarat, India
| Date of Submission | 15-Nov-2022 |
| Date of Decision | 01-Dec-2022 |
| Date of Acceptance | 05-Dec-2022 |
| Date of Web Publication | 21-Feb-2023 |
Correspondence Address:
Sagnik Roy
Department of Anatomy, Gujarat Adani Institute of Medical Sciences, Opposite Lotus Colony, Bhuj - 370 001, Kutch, Gujarat
India
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/NJCA.NJCA_227_22
Background: Development of the human fetal stomach starts at the 4th week of intrauterine life. The knowledge of histogenesis of the musculature of the stomach is important for understanding, diagnosis, and treatment of gastric adenomyoma, congenital anomalies like congenital hypertrophic pyloric stenosis, and abnormalities in the development of the enteric nervous system. The aim of this study was to find out the histogenesis of the submucosa (SM), muscularis mucosa (MM), and muscularis externa (ME) in the human fetal stomach. Methodology: Sixty-three freshly aborted fetuses which were free from any gross abnormality were collected from the obstetrics and gynecology department and studied for 2 years. The fetuses were dissected, the stomach was removed, and the tissue was processed for a histological study. Staining was done by hematoxylin and eosin, Masson's trichrome, and Van Gieson's stain. Slides were studied under Zeiss branded light microscope using ×10 and ×40 objectives. Results: MM was discontinuous initially and became well defined by 21 weeks in the fundus and body and by the 23rd week in the pyloric region. The density of collagen fibers increased gradually in SM and was seen in bundles from the 23rd week onward. The thickness of SM was more than ME till 22 weeks, and after 27 weeks, it was less than that of ME. In the fundus and body of the stomach, till 18 weeks, ME consisted of two layers, with the inner oblique layer being thicker than the circular layer. The circular layer thickened gradually, and the longitudinal layer appeared by the 25th week. In the pyloric region, initially, there was only a circular layer. The oblique and longitudinal muscle layers appeared at 17–18 weeks and 25th week, respectively. The thickness of the circular layer was found to be more than that of the body from the 21st week onward and became much thicker at around the 27th week. The histological feature of MM, ME, and SM of the stomach resembled an adult pattern by the 28th week of gestational age. Conclusion: The findings of the present study about the time of appearance and further development of MM and ME and the thickness and formation of SM of the human fetal stomach are quite comprehensive. These findings are expected to aid in the knowledge of histogenesis of the stomach in the human fetuses and help in the diagnosis and treatment of various congenital anomalies and clinical conditions involving gastric connective tissue and musculature.
Keywords: Circular layer, collagen fibers, longitudinal layer, muscularis externa, muscularis mucosa, oblique layer
How to cite this article:
Roy N, Roy S. Development of the submucosa and musculature in the human fetal stomach: A microscopic study. Natl J Clin Anat 2023;12:9-14 |
How to cite this URL:
Roy N, Roy S. Development of the submucosa and musculature in the human fetal stomach: A microscopic study. Natl J Clin Anat [serial online] 2023 [cited 2023 Mar 20];12:9-14. Available from: http://www.njca.info/text.asp?2023/12/1/9/370138 |
| Introduction | |  |
In the human embryo, the stomach starts developing as a fusiform dilatation at the 4th week of gestational age[1] and thereafter undergoes several morphological and microscopic changes during the process of development. The mucosal epithelium differentiates from the endodermal lining, and the mesodermal coating gives rise to the submucosal connective tissue and smooth muscle layer.[2]
The submucosa (SM) consists of a thick layer of dense connective tissue containing collagen fibers, elastic fibers, arterioles, venous plexus, lymphatics, and nervous plexus.[3] The muscularis mucosa (MM) of the stomach is thin and composed of two layers, whereas the muscularis externa (ME) has three layers from inside out, oblique, circular, and longitudinal, but unevenly located in various parts of the stomach. The circular layer is thickened at the distal pyloric antrum, whereas the longitudinal layer and oblique layer are most pronounced in upper two-third and lower half, respectively.[4]
In infantile hypertrophic pyloric stenosis, an abnormal thickening of the pyloric muscle can lead to gastric outlet obstruction. Exposure to antibiotics after birth, preterm birth, and children born to mothers who are heavy smokers can be among the few reasons which lead to this abnormality.[5]
The knowledge of histogenesis of the musculature and SM is needed for a better understanding and treatment of diseases like congenital hypertrophic pyloric stenosis, and abnormalities in the enteric nervous plexuses present in these layers.
There is no definite information available in textbooks regarding the histogenesis of different layers of the stomach. A very few studies have been conducted on the development of the human stomach. The result of those studies showed a large variability regarding the timing and pattern of the development of SM and different layers of the musculature of the stomach. Hence, the present study was carried out to find out the developmental pattern of the SM and musculature of the human fetal stomach in order to compare the results with that of previous studies and supplement information related to the histogenesis of these distinct gastric layers.
| Materials and Methods | | |
Sixty-three normal fresh fetuses (28 males and 35 females) were collected from the Department of Obstetrics and Gynecology, Regional Institute of Medical Sciences, Imphal, Manipur. The fetuses were products of terminated pregnancy under the Medical Termination of Pregnancy Act of India, 1971, and stillbirths. The age group of the fetuses was in the range of 15–40 weeks. Only those fetuses that were free from any gross anatomical abnormality were selected for the present study. Requisite clearance from the local ethical committee was obtained. The study was carried out after obtaining institution ethics committee approval. Study duration was 2 years.
The age of the fetuses was calculated from obstetrical history and crown-rump length (CRL).
The fetuses were divided into different groups according to their gestational age.
- Group 1: 15–18 weeks (CRL: 80 mm to 130 mm): 10 fetuses
- Group 2: 19–22 weeks (CRL: 131 mm to 175 mm): 21 fetuses
- Group 3: 23–27 weeks (CRL: 176 mm to 250 mm): 17 fetuses
- Group 4: 28–40 weeks (CRL: 251 mm to 450 mm): 12 fetuses.
Incision was given on the anterior abdominal wall along the subcostal margin and inguinal folds to open the abdominal cavity. The stomach was taken out after removing the anterior abdominal wall and parts of the liver. Tissue from different parts of the stomach was collected and fixed immediately in 10% formal saline. Some of the tissues were further fixed in Bouin's fluid for 1 day.[6]
After fixation, the tissues were subjected to dehydration, clearing, paraffin embedding, and paraffin block making. Five-micron thick sections were cut from the blocks using a rotary microtome. The sections were then mounted on egg albumin-coated glass slides and kept in an incubator overnight at room temperature.
The sections were stained with hematoxylin and eosin, using Harris's hematoxylin and 1% aqueous eosin. Some of the sections were stained with Van Gieson's stain for a selective demonstration of collagen fibers (CFs) and Masson's trichrome to differentiate the smooth muscle fibers from the connective tissue.[7]
The stained slides were studied under a Zeiss trinocular research light microscope using ×10 and ×40 objectives.
| Results | | |
The significant changes in histogenesis of SM and musculature of the stomach observed in the fetuses of different age groups are as follows:
Group 1 (15–18 weeks)
Fundus and body
In the fetuses of 15–16 weeks, all the layers of the stomach were present, but SM could not be demarcated clearly from the lamina propria because MM had not clearly differentiated.
SM was very thick, containing blood vessels, some stellate-shaped cells, and scanty connective tissue fibers.
In ME, a thick oblique layer was seen and outer to that patches of circular muscle fibers were seen at places [Figure 1]. | Figure 1: Human fetal stomach, body – 15 weeks (H and E, ×10). OML: Oblique muscle layer, CML: Circular muscle layer, SM: Submucosa
Click here to view |
The thickness of SM was much more than ME.
At 17–18 weeks, MM was still not well defined; only a few discontinuous fibers were present between the lamina propria and SM. SM could be differentiated from the lamina propria by less cellularity and pale staining. It contained fibroblasts, scanty collagen fibers, and blood vessels.
ME of the body of the stomach comprised two layers. The circular muscle layer (CML) was observed to have become continuous, but the thickness remained the same as that of the previous stage.
Pyloric part
SM was similar in appearance to the body. MM did not develop at this stage.
ME showed only the circular fibers at places at 15–16 weeks. At 17–18 weeks, the inner oblique layer started appearing, and the circular layer became continuous. The thickness of the circular layer was similar to that of the fundus and body.
Group 2 (19–22 weeks)
Fundus and body
The density of CFs and blood vessels increased in SM, and the thickness of the layer increased at places from the 21st week. Folding of SM was seen at some places at around the 22nd week.
The well-defined MM clearly dividing the lamina propria and SM was observed by 21 weeks [Figure 2]. | Figure 2: Human fetal stomach, body – 21 weeks (H and E, ×10). MM: Muscularis mucosa
Click here to view |
ME was more developed than before, entirely represented by an increase in the thickness of the circular layer. The longitudinal layer was still not developed.
SM was thicker than ME throughout this stage.
Pyloric part
SM was rich in CFs and blood vessels like that of the fundic part and body [Figure 3]. | Figure 3: Human fetal stomach, pylorus – 19 weeks (Van Gieson's stain, ×10). SM: Submucosa, CF: Collagen fiber
Click here to view |
The MM was not prominent and was discontinuous throughout the stage unlike that of the body.
The ME showed thicker circular fibers than that of the body from the 21st week onward and intermingling of the circular layer and oblique layer was seen at places.
Group 3 (23–27 weeks)
Fundus and body
The SM was characterized by an increased folding, and it formed the core of the rugae by the 26th week. Bundles of CFs were seen, and the thickness of the SM was almost equal to the ME [Figure 4]. | Figure 4: Human fetal stomach, body – 26 weeks (Masson's trichrome stain, ×40). SM: Submucosa, ME: Muscularis externa
Click here to view |
MM became more prominent and thicker compared to the previous stage.
ME showed a discontinuous coat of the longitudinal muscles outer to the circular layer at 25 weeks. By 27 weeks, the oblique layer was quite pronounced, especially along the greater curvature, but the circular layer still formed the thickest layer of ME.
Pyloric part
The well-defined MM was seen at the 23rd week, which further thickened and attained the adult pattern by the 26th week.
In ME, a marked increase in the thickness of the circular layer was noticed from the 24th week onward. The longitudinal layer appeared at places at the 25th week and became consistent by the 27th week [Figure 5]. The thickness of the circular layer was almost twice the thickness of the longitudinal layer at this stage. | Figure 5: Human fetal stomach, pylorus – 27 weeks (Masson's trichrome stain, ×40). CML: Circular muscle layer, LML: Longitudinal muscle layer
Click here to view |
Group 4 (28–40 weeks)
At this stage, the histological features of the fetal stomach largely resembled those in adults.
Fundus and body
SM showed bundles of CFs and numerous blood vessels. The thickness of this layer was less than ME at this stage.
MM was thick like in adults. The thickness of ME was comparable to that in adults and showed well-defined layers of oblique, circular, and longitudinal muscle fibers from the inside outward.
Pyloric part
The appearance and thickness of the SM were same as that of the fundic part and body. The histological feature resembled the adult pattern [Figure 6]. | Figure 6: Human fetal stomach, pylorus – 30 weeks (Masson's trichrome stain, ×10X). MM: Muscularis mucosa, SM: Submucosa, ME: Muscularis externa
Click here to view |
MM was very thick and wavy in appearance.
ME had three well-distinguished layers of muscles, and the circular layer was immensely thick compared to that of the body.
| Discussion | | |
There is a lot of difference in opinion as published in previous studies and information given on textbooks regarding the timing of appearance and further development of MM in the human fetal stomach.
According to Hamilton and Mossman,[8] MM appeared at the stage of 80–240 mm crown-rump (CR) length. Jit[9] observed that MM differentiated at 120 mm CR stage and at 135 mm CR stage in the body and pyloric parts, respectively. Arey[10] opined that MM appeared at 160 mm CR stage. Chimmalgi and Sant[11] observed MM at the end of the 22nd week of gestational age. Pangtey et al.[12] observed that MM was present at the 14th week of gestation. Ghosh et al.[13] observed that MM was present in the cardio-esophageal junction at 22–24 weeks but did not develop in the body and pyloric region at that stage. In the present study, it was found that in the fundus and body of the stomach, MM was sparse at 15–18 weeks, became well defined at around 21 weeks and thickened further in the following weeks, and finally attained the adult pattern at around 28 weeks. In the pyloric part of the stomach, MM was not seen at 15–18 weeks of gestational age. At 19–22 weeks, it was discontinuous and sparse, and at the 23rd week, the well-defined MM was observed which attained an adult pattern by the 26th week.
Chimmalgi and Sant[11] noted the folding of SM from the 25th week, and it showed well-formed CFs from the 28th week onward. According to the same study, SM was found to be the thickest layer at 15–20 weeks and 25–27 weeks, whereas the present study found the folding of SM from the 22nd week. The SM was very thick at 15–18 weeks, and the thickness increased up to 21 weeks. Thereafter, the thickness gradually diminished to become equal to ME, and it was in fact thinner than ME from the 28th week onward. The density of CFs increased gradually and was seen in bundles from the 23rd week onward. Bevelander and Ramaley[14] mentioned that SM does not contain any gland in any region of the stomach, whereas Bradbury[15] stated that the glands of the pyloric part may penetrate through the MM into the submucosal coat. In the present study, no gland was found in SM in any region of the stomach at any stage of development. According to Ham,[16] SM forms the core of the gastric rugae. Meschan[17] mentioned that the rugae of the fundus are arranged in a mosaic pattern and are more regular in the body of the stomach. Rose and Pawlina[18] opined that the longitudinal folds in the inner surface of an empty stomach are composed of mucosa and SM. In the present study, SM was found to form the core of the rugae in both body and pyloric regions at around the 26th week.
Hamilton and Mossman[8] opined that the differentiation of the inner circular layer of ME occurs at 10–42 mm CR stage. Byrne and D'Harlingue[19] stated that the circular layer in the human fetal stomach is present by the 9th week. Jit[20] observed that the circular layer first differentiated at 10 mm CR stage. According to Borley et al.,[21] the inner circular layer of ME starts developing at 8–9 weeks of gestational age, followed by the longitudinal layer after few weeks. Chimmalgi and Sant[11] observed that CML of ME was present from the 15th week. Pangtey et al.[12] observed a continuous inner circular layer in the fundus and body of the stomach at the 14th week and in the pyloric region from the 10th week of gestational age. They also noted that ME was found to be thicker in the pyloric region than that of other regions at all stages of development.
Munawara et al.[22] observed in their study that the thickness of the circular layer was equal to that of the oblique layer at the 12th week, and from the 14th week onward, the circular layer started to become thicker and showed a significant thickening at the pyloric end. Ghosh et al.[13] observed that ME started developing as bundles of fibers at 10–12 weeks. At 16–18 weeks, the circular layer became continuous and showed further thickening till the 28th week. They also observed that from 24 to 26 weeks, the circular layer became thicker in the pyloric region than in the body. In the present study, at the 15th week, the discontinuous CML was seen and became continuous by the 17th week in both body and pyloric regions. The oblique layer was found to be thicker than the circular layer in the body of the stomach at this stage. The circular layer became thicker at around the 19th week. The difference in the thickness of the circular layer of the body and pyloric part was seen from the 21st week onward, and it became very thick, especially toward the pyloric sphincter by the 27th week. Hollinshead[23] opined that the hypertrophy of the circular fibers at the pyloric end causing the obstruction of the pyloric orifice is the most frequent anomaly, and the thickness can be 5–10 mm. Pyloric atresia or the presence of membrane in the pylorus can also cause obstruction. The present study did not find any such anomaly in any of the fetal specimens.
Pangtey et al.[12] observed that the oblique layer started developing at 16 weeks in all parts of the stomach, and by the 26th week, three well-defined layers of ME comprising the inner oblique, middle circular, and outer longitudinal layers were seen. Munawara et al.[22] observed that the inner oblique layer became continuous and well defined by the 20th week. Ghosh et al.[13] observed the oblique layer as discontinuous fibers at 24–26 weeks in the fundus and body of the stomach. In the present study, the youngest fetus aged 15 weeks had the inner oblique layer which was thicker than the circular layer in the body of the stomach. By the 19th week, the circular layer became thicker. In the pyloric part, the inner oblique layer was first seen by the 17th–18th week.
Jit[20] observed that the longitudinal layer of the stomach appeared from 21 mm CR stage, Chimmalgi and Sant[11] observed that it developed by the 28th week of gestational age in the human fetuses, and Munawara et al.[22] observed that the longitudinal layer developed by the 20th week in the human fetal stomach. Ghosh et al.[13] noted that the stripes of the longitudinal muscles were seen at 18–20 weeks but were not continuous. It became well developed by 24–26 weeks in the body and fundus but was not found in the pyloric region even at 28 weeks. Esrefoglu et al.[24] reported that the longitudinal layer developed at the 28th week. In the present study, the longitudinal layer started developing from the 25th week and became consistent by the 27th week.
Field and Hillemeier[25] stated that pyloric motility and gastric emptying are developed by the 30th week in the human fetuses, whereas the present study has found well-developed three layers of the musculature in all the regions of the stomach with a very thick circular muscle toward the pyloric end by the 27th week itself.
Pedersen et al.[26] reported the incidence of infantile hypertrophic pyloric stenosis as 2/1000 live births in different regions of Europe. Another study conducted by Svenningsson et al.[27] reported that young maternal age, prematurity, and smoking were the risk factors for infantile hypertrophic pyloric stenosis. Ndongo et al.[28] observed that infantile hypertrophic pyloric stenosis predominantly occurred in males, and the male:female ratio of occurrence was 4.25:1. The present study did not find any abnormal thickening of any layer of ME at any of the gestational age groups. There was no appreciable difference in the thickness of ME between male and female fetuses.
Limitations
Fetuses younger than 15 weeks were not available for the study, so the time of appearance of the oblique layer in the fundus and body and the circular layer in the fundus, body, and pyloric region of the stomach could not be determined.
| Conclusion | | |
It was observed in the present study that the developing musculature of the stomach underwent several changes as the fetal age advanced. Some of the findings of this study are in accordance with other studies, while some findings were contradictory. Available literature has insufficient information as well as a lot of discrepancies regarding the time of appearance and thickness of MM and the layers of ME of the stomach. In our study, we clearly noted the time of appearance of MM at the 21st week in the body and at the 23rd week in the pyloric region. ME consisted of oblique and circular layers at the 15–18th week, whereas in the pyloric region, only the circular layer was present at the 15th week with the oblique layer making its appearance at around the 17th–18th week. The longitudinal layer appeared at around the 25th week and became well defined by the 27th week in all regions of the stomach. The thickness of SM was more than ME at early stages, but gradually, it became equal to ME and was overtaken by ME by the 28th week. The density of CFs increased gradually in SM and was seen in bundles in the later stage of development.
The findings of the present study are expected to supplement and fill any existing gap in the information and knowledge about the development of the musculature and SM in the human fetal stomach. This, in turn, can help in the diagnosis and treatment of various congenital anomalies and diseases involving gastric musculature and connective tissue.
Acknowledgment
The authors also sincerely thank those who donated their fetuses to science so that anatomical research could be performed. The results from such research can potentially increase mankind's overall knowledge which can then improve patient care. Therefore, these donors and their families deserve our highest gratitude.[29]
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
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