The Anatomy of adrenal gland was described almost 450 years ago in 1563 by Bartholomeo Eustacius and zonation of gland and its distinction from medulla were elucidated shortly thereafter. ⁽¹⁾

Steroid hormones produced by fetal adrenal cortex regulate intrauterine homeostasis, the maturation of fetal organ systems necessary for extra uterine life and in some species the timing of parturition. (Liggins G.C.1976). Appropriate development and function of fetal adrenal cortex therefore are critical for fetal maturation and perinatal survival. ⁽²⁾

The Development of human fetal adrenal is regulated primarily by adrenocorticotrophin (ACTH) secreted from the fetal pituitary gland. However, as ACTH is not a mitogen per se, its proliferative action on human fetal adrenal cortical cells is thought to be mediated by autocrine/ paracrine growth factors produced by adrenal cortical cells in response to ACTH. In addition, these growth factors appear to modulate the functional response of fetal adrenal cortical cells to ACTH. The role of several growth factors including the Insulin like growth factor I & II (IGF-I & IGF-II), Epidermal growth factor (EGF), Basic fibroblast growth factor (BFGF), Activin, Inhibin and Transforming growth factors alpha and beta (TGF- alpha & TGF- beta) have been studied. Thus, human adrenal development is primarily regulated by ACTH, but its actions appear to be mediated or modulated by cohort of locally expressed growth factors.  The net effect of which results in unique growth and steriodogenic activity of human fetal adrenal cortex. Also, one factor essential for adrenal development is Orphan Nuclear receptor steriodogenic factor (SF-1, AD4BP-NR5A1). ^(3,4)

The present was conducted to study the histogenesis of fetal adrenal gland at different stages of development in intra uterine life in Marathwada region of Maharashtra.

Objectives: 

To observe microscopic structure of suprarenal gland at various gestational age group.

MATERIALS AND METHODS

The present observational study conducted at Department of Anatomy and Obstetrics and Gynecology of Government Medical College and Hospital, Aurangabad. The study was done on 40 spontaneous abortuses and stillborns ranging from 12^th weeks to 36^th weeks of gestation. Twins and fetuses with gross anomalies were excluded.  The study was approved by the Ethical Committee of the Institute.

The gestational age, sex, weight crown-rump length was studied in detail. Weight of fetuses were measured in grams on double pan balance.

The fixation of fetuses was ensured by injecting them with 10 % formalin locally, at various sites with the help of a 10-ml. syringe and 20 number needle in cranial cavity, in the neck and subcutaneously in the upper and lower limbs. 

The suprarenal glands were obtained from fetuses by a midline incision over the anterior abdominal wall. Both glands were carefully dissected out, immediately weighed, and initially fixed in 10% formalin. Subsequently, the tissues were cut into small pieces and fixed in Bouin’s fluid for 24 hours. Post-fixation, the specimens were dehydrated through graded alcohols—70% alcohol for 6–8 hours, 90% alcohol overnight, followed by three changes of absolute alcohol for one hour each. Clearing was performed in xylene for approximately 30 minutes. The tissues were then infiltrated with paraffin wax at 56–60°C through three changes and embedded using L-shaped moulds. Paraffin blocks were sectioned at 5–7 µm thickness, and ribbons were floated on cold water to remove folds. Sections were mounted on egg albumin–coated slides and dried on a hot plate.

Routine haematoxylin and eosin (H&E) staining was carried out following dewaxing, rehydration, nuclear staining, differentiation, bluing, counterstaining, dehydration, clearing, and mounting with DPX. Selected sections were additionally stained using Masson’s trichrome technique to differentiate connective tissue components. The stained slides were examined under a light microscope and photomicrographs were obtained for histological analysis.  

OBSERVATIONS AND RESULTS:

In the present study, a total of 40 fetuses (19-males, 21-females) ranging from 12^th weeks to 36^th weeks of gestation were studied.

Table 1: Showing microscopic structure of Suprarenal Gland across gestational ages:

DISCUSSION

Human fetal growth is a net result of a complex interplay of genetic, hormonal and growth factor effects. Suprarenal gland is essential for intrauterine development of fetus. The fetal adrenal cortex is involved in maintenance of intrauterine homeostasis and in the preparation of fetus for extra uterine life, by regulating the maturation of essential organ systems including lungs, liver and gut.⁽³⁾

In the present study, comparison of histological structure of Suprarenal Glands at various stages of fetal life was done with the findings of other workers. It was observed that for definitive cortex of gland, throughout the gestational period definitive or permanent cortex forms narrow sub capsular zone. This finding was corroborative with the findings of Benirschke, Bloch and Hertig ⁽⁵⁾ (1956).  They found that definitive cortex formed 1/5^th of adrenal tissue. In present study it was found that definitive cortex is 1/10^th at 12 weeks a gestation, 1/8^th at 28 weeks of gestation and approximately 1/5^th of total cortex at 34-36 weeks of gestation. The cells of definitive cortex are small, polygonal with basophilic cytoplasm and euchromatic nuclei. The findings were similar to that of Bernirschke et al ⁽⁵⁾  (1956) and Turkel & Itabashi ⁽⁶⁾ (1974).

In the present study it was observed that the thickness of definitive cortex remains almost the same from 12 weeks to 22 weeks. There after it shows slight increase in thickness up to 28 weeks with a further faster growth. Similarly, according to “Keene & Hewer” that permanent cortex slowly increases in size throughout the intra uterine life. According to Damayanti N. et al ⁽⁷⁾ (2005), the cytoarchitecture of 12 weeks suprarenal shows a superficial zone and a larger deeper zone. The findings of present study were same as that of Damayanti et al. ⁽⁷⁾ Uotila ⁽⁸⁾ (1940) had also mentioned that the cells of definitive cortex did not show any regular arrangement but period of study was only 4-8 weeks of gestation. Bosian- Sobkowska J et al ⁽⁹⁾ (1993) have found that the zona glomerulosa and outer part of zona fasciculata begin to form by 20^th week of gestation. Mesiano & Jaffe ⁽¹⁰⁾ (1997) have described that the cells of definitive cortex took the appearance of adult zona glomerulosa by 30 weeks. Damayanti et al⁽⁷⁾ had mentioned that from 28 weeks the outer zone of cell organizes into an arcuate shape typical of the future zona glomerulosa.

In the present study it was observed that a larger part of fetal cortex is contributed by fetal cortex. This corroborated with the study of Bernivschke et al ⁽⁵⁾ (1956) who found that fetal cortex formed 4/5^th of adrenal cortex. The cells of fetal cortex were large, polyhydral with eosinophilic cytoplasm and round vesicular nuclei. These findings of present study were similar with findings of Bernirschke, Bluch & Hertig, Turkel & Itabashi and Mesiano & Jaffe. ^(5,6,10)

In the present study, it was observed that the cells in outer part of fetal cortex were arranged in the form of longitudinal columns separated by capillaries, whereas the cells in inner part were arranged in the form of a network with sinusoidal spaces in between. Because of this arrangement of fetal cortical cells Uotila ⁽⁸⁾ (1940) described the fetal cortex of suprarenal gland as fasciculo-reticular layer which he found from 7^th week onwards. According to Keene M.B & Hewer ⁽¹¹⁾ the fetal cortex degenerated progressively during the last 10 weeks of fetal life, but still contributing to the bulk of the gland till birth. 

McNutt and Jones ⁽¹²⁾ (1970) reported that the transitional zone appeared between permanent and fetal zone by 14^th week. Mesiano & Jaffe ⁽¹⁰⁾ had described the transitional zone as a zone of finger like columns of cells extending from definitive cortex into the fetal cortex. In the present study also, the transitional zone was seen throughout the studied period that is from 12 weeks to 36 weeks. According to Mesiano & Jaffe ⁽¹⁰⁾ by the 30^th week of gestation the definitive zone and transitional zone began to take on the appearance of zona glomerulosa and zona fasciculata respectively. In the present study also, it was observed that the zona glomerulosa and zona fasciculata appeared at 34 and 36 weeks.

In the present study these cells were seen scattered throughout the gland and at places, they form groups usually near the large primitive vessels in reticular layer of fetal cortex. This finding was same as that of Antti Hervonen ⁽¹³⁾ (1971), Turkel & Itabashi ⁽⁶⁾ (1974) and Bogdanova & Debelenko ⁽¹⁴⁾ (1989). These cells were small with round densely staining nuclei surrounded by a thin rim of basophilic cytoplasm. These cells were given different names by different scientists like neuroblasts by Turkel & Itabashi ⁽⁶⁾ (1974) and Cooper et al ⁽¹⁵⁾ (1990)

Phaeochromoblast by RE Coupland ⁽¹⁶⁾ (1952), primitive symphathetic cells by Antti Hervonen ⁽¹³⁾ (1971). A group of these neuroblasts was called a neuroblastic nodule by Turkel & Itabashi ⁽⁶⁾ (1974).  They described that these neuroblastic nodules were avascular and non-capsulated but they lie in contact with capillaries and fetal cortical cells. The appearance of neuroblastic nodules in the present study was similar to that found by Turkel & Itabashi. ⁽⁶⁾ However, Antti Hervonen ⁽¹³⁾ (1971) reported that these primitive symphathetic cell groups were avascular and partly capsulated.

In present study at 12^th week, neuroblastic nodules were seen invading the capsule of the gland and also the parenchyma. Solitary neuroblasts were also seen scattered throughout the gland. With increasing gestational age, the number of neuroblastic nodules increased reaching maximum at 22 weeks showing decline thereafter. They were absent at 34 and 36 weeks. These finding are corroborative with the study of Turkel & Itabashi ⁽⁶⁾ (1974) but they had found that the maximum number of neuroblastic nodules were observed between 17-20 weeks of gestation. However, other workers including Keene & Hewer ⁽¹¹⁾ (1927) and R.E. Coupland ⁽¹⁶⁾ (1965) had reported that after 30 weeks neuroblastic nodules slowly regressed so that few nodules remained by the time of birth.

In the present study solitary neuroblasts were observed from 12 weeks to 28 weeks of gestation, the number decreasing thereafter with very few neuroblasts being present at 36^th week. Antti Hervonen ⁽¹³⁾ (1971) and Turkel & Itabashi ⁽⁶⁾ (1974) had reported similar findings. But according to Cooper et al (1990) neuroblasts were absent after 28 weeks of gestation. Decrease in neuroblastic cell number in later fetal period was because of their conversion into other cells. According to R.E. Coupland ⁽¹⁶⁾ (1952) these neuroblasts were converted into chromaffin cells from 8^th week onwards.

According to Antti Hervonen ⁽¹³⁾ (1971) neuroblasts in peripheral part of the gland were converted into chromoffin cells whereas neuroblasts in the central region of nodule differentiated into sympathetic ganglionic cells from 11^th weeks onwards.

Turkel & Itabashi ⁽⁶⁾ (1974) had reported that the decline in neuroblastic nodules was due to the differentiation into chromaffin cells by apoptosis of neuroblasts.

In the present study it was observed that the number of neuroblastic nodules goes on increasing, but with the conversion of neuroblasts into chromoffin cells, nodules & solitary neuroblasts both decreased and chromaffin cells increased. These chromaffin cells have slightly basophilic cytoplasm with round nuclei, peripherally condensed chromatin and were intermediate in size between that of neuroblasts and fetal cortical cells. R.E. Coopland ⁽¹⁶⁾ (1952) and Antti Hervonen ⁽¹³⁾ (1971) have described the chromaffin cells similar to this.

In the present study, few chromaffin cells were seen at 22 weeks. The number of these chromaffin cells gradually increased upto 36 weeks forming the rudimentary medulla. Chromaffin cell were found at same stage of gestation i.e. 22 weeks by Keene & Hewer ⁽¹¹⁾ (1927). Chromaffin cells were found at an earlier stage than the present study by R.E. Coupland⁽¹⁶⁾ (1952) and Antti Hervonen ⁽¹³⁾ (1971), who found chromafin cells 8 weeks on wards and 11 weeks onwards respectively. Mesiano & Jaffe ⁽¹⁰⁾ (1997) had found few chromaffin cells at 30 weeks i.e. at a later stage than the present study.

In the present study rudimentary medulla was found at 34-36 weeks, whereas Bogdanova, Debelenko ⁽¹⁴⁾ (1989) and Cooper et al ⁽¹⁵⁾ (1990) had reported the presence of rudimentary medulla at 30^th week. Similarly, Mesiano & Jaffe ⁽¹⁰⁾ (1997) observed that medulla was absent at birth and rudimentary medulla was formed around central vein in the first postnatal week which became adult like by 12-18 months.

Capsule is seen at 12^th week of gestation which is very thin.  The thickness of the capsule increases by deposition of collagen fibers as the gestational age advances. Vascularity of the gland is found to be increased throughout the gestational period studied i.e. 12 weeks to 36weeks.

CONCLUSION

The study concludes that, histogenesis of the suprarenal gland demonstrates a progressive increase in gland size with advancing gestational age, accompanied by a gradual increase in capsular thickness and vascularity throughout fetal life. Histologically, the gland is organized into three distinct cortical zones—definitive zone, transitional zone, and fetal zone—whose relative proportions change with maturation. The definitive cortex constitutes approximately one-tenth of the total cortex between 12 and 20 weeks of gestation, increases to about one-eighth by 28 weeks, and further expands to nearly one-fifth by 36 weeks, reflecting progressive cortical differentiation and replacement of the fetal zone.

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