A STUDY OF HISTOLOGY & HISTOGENESIS OF FETAL SPLEEN AT DIFFERENT GESTATIONAL AGES

Introduction: The spleen is a largest collection of lymphoid tissue with peculiar anatomical and physiological features. Spleen plays an important role in fetal hematopoiesis and immunomodulation. The aim of the study is to perform detailed histological analysis of human fetal splenic specimens of various gestational ages and compare the findings with earlier studies. Material and Methods: The present study included 40 fetal cadaveric spleen and morphometric features i.e., weight was measured and the sections of the spleen were stained with Haematoxylin and Eosin stain and were observed under compound light microscope Results: In the present study the histology of spleens of prenatal group showed the well defined red pulp, venous sinusoids and diffusely spread lymphocytes at 18 weeks of gestation and the organization of lymphoid follicles was noticed at 24-28 weeks. At 32 weeks well defined white pulp was observed and the microscopic architecture of the spleen was similar to the histology of adult spleen. Conclusion: A detailed knowledge of splenic morphometric dimensions, Histology and Histogenesis is crucial in deciphering the role of spleen in fetal development and fetal wellbeing.


Introduction
The spleen is a largest secondary lymphoid organ which plays an important role in fetal hematopoiesis and immunomodulation. The spleen arises from a mesenchymal proliferation in dorsal mesentery or mesogastrium during 5 th week of gestation [1,2]. The spleen assumes its definitive morphological structure during the 3rd month, the size gradually increases during the fetal period as mentioned by Sir Henry Gray in 1854 in the book "The structure and the use of spleen" [2].
Spleen by tissue nature belongs to the lymphoreticular organs but unlike the 'white' lymph nodes it is not included in the lymph but in the blood circulation [3]. In the antenatal period, the spleen plays an important role in the immunomodulation, acts as a primary haemopoietic centre until late in the fetal period, also plays and important role in apoptotic cell clearance, immune tolerance, efficient removal of blood-borne microorganisms, lymphocyte differentiation and activation [4]. These functions are carried out by the two main components of the spleen, the white pulp (including the marginal zone) and the red pulp, which are vastly different in their microscopic architecture, vascular organization, and cellular composition [5,6]. The white pulp is subdivided into periarteriolar lymphoid sheath (PALS), the follicles, and the marginal zone.
In the antenatal period the histogenesis of spleen occurs in three stages : 1. Preliminary stage also called the "primary vascular reticulum," lasts up to the 14th gestational week. The primordium of the spleen appears in the embryos of size 6-7 mm in the 5 th gestational week, and the hematopoietic cells were observed in the vascular lumen in 10th week [7].
Numerous erythrocytes, normoblasts and macrophages are seen among a network of mesenchymal cells and argyrophilic fibers.

Stage of transformation:
The characteristic structure of the organ becomes established during this stage beginning with the 15 th gestational week.
Splenic lobules begin to form during the 15th to 17th gestational week.

Stage of lymphoid colonization :
The development of the white pulp is correlated with the stage of lymphoid colonization around the central arteriole occurs during the 18-20 weeks. Around the 23rd week the aggregation of primary follicles is discernible at the periphery of the PALS and by 30-33 weeks distinct red and white pulp will be formed [4,7].
However there is limited research regarding the histology and histogenesis of spleen at different gestational ages which justifies the purpose of this study.

AIMS & OBJECTIVES:
The aim of the study is to perform detailed histological analysis of human fetal splenic specimens of various gestational ages and compare the findings with earlier studies.

MATERIAL AND METHODS
The present study is a prospective type of study conducted in the department of Anatomy, S.V.Medical College, Tirupati with cooperation of Government Maternity Hospital, Tirupati and Narayana Medical College, Nellore. Medically terminated fetuses of both sexes and relevant obstetric data were collected from Government Maternity Hospital, Tirupati which includes 40 dead aborted fetuses of both sexes ranging from 16 weeks of gestation to term. The ethical committee approval and consent of the relatives were obtained.
In the department of anatomy the collected fetuses were observed for congenital anomalies and they were preserved in 10% formalin. After one week of preservation, the abdomen was opened by using routine dissection method and the morphological observations were made insitu. Later spleen were removed by using routine dissection method and outer surface of fetal spleens were dried with blotting paper and then weighed by using digital weighing balance (SHIMADZU-ATY224-UNIbloc).
For microscopic observation, the antenatal specimens were broadly categorized into 5 groups. Representative samples from each group were subjected to routine histological processing for H & E and reticulin stains. The sections were observed under 10x and 40x objective piece of microscope and representative fields were photographed by using photomicrographic equipment and the results were analyzed.

Reticulin stain to demonstrate reticular fibres.
The collected data was subjected to statistical analysis by computing the mean of each parameter with respect to the gestational age-wise groups by using SPSS 20 version.

RESULTS:
The Crown-rump length of all the fetuses were initially measured and fetal gestational age is calculated in weeks and based on the age in weeks the fetal specimens were categorized into 5 groups i.e., 16-20 weeks, 21-24 weeks, 25-28 weeks, 29-32 weeks and 33 weeks to Term. The largest group was fetuses with gestational age 16-20 weeks with 10 specimens. The gender-wise distribution was 57.5% and 42.5 % for male and female groups respectively. The splenic weight is maximal in 29-32 weeks group with mean 3.33 ± 1.01 gm. The weight of the spleen ranged from 0.1 gm to 4.9 gm. The average weight of the fetal spleen is 1.98 ± 1.22 gm.

Histology of spleens:
In the present study the earliest specimen observed was that of fetal spleen of 16 weeks gestational age. The observations made in prenatal group were as follows.

Group A (16-20 weeks):
Fetal spleen sections showed thickened capsule and dilated sinusoids filled with blood cellular elements, diffuse aggregations of lymphoblasts. Well defined lymphoid follicles central arterioles and white pulp were conspicuously absent. Megakaryocytes seen with evidence of extramedullary hematopoiesis.

Group B (20-24 weeks):
The spleen sections showed distinct fibrous capsule, accumulation of lymphoid follicles around arteriole. aggregation of lymphocytes to form lymphoid follicles was noticed. Sinusoids, capillaries with thin endothelial lining were observed. Reticular network, Sinusoids and blood vessels increased in number. Extramedullary hematopoiesis was noticed. Clear demarcation between red pulp wand white pulp was absent.

Group C& D (25-32 weeks):
The spleen sections showed white pulp formation in the form of well defined lymphoid follicles and trabeculae were present. There was a well defined evolution of Red pulp with RBC. Periarteriolar lymphoid aggregation was noticed. Well developed lymphoid follicles with central arteriole were seen.

Group E (33 weeks -Term):
The spleen sections showed well fibrous capsule, numerous sinusoids, aggregation of the lymphoid follicles with an eccentric arteriole. Increased size of the white pulp was noticed. Well differentiated Malpighian corpuscles were present. In this group at term the splenic tissue showed well defined white pulp with dense aggregation of lymphocytes with clear demarcation from the surrounding tissue. White pulp was sparsely associated with collagen fibers. The red pulp showed collagen network of fibers in continuance with trabeculae. It also showed sinusoids filled with RBC, occasional fibroblasts and the microscopic structure at term resembled that of adult spleen architecture.

DISCUSSION:
In the present study the average weight of the prenatal spleen was 1.98 gm. The splenic weight ranged from 0.1 gm to 4.9 gm which gradually increased with gestational age significantly till 38 weeks of gestation. The findings regarding the weight of the spleen in relation to gestational age were in agreement with the observations of Gruenwald P & Minh HN [9] (1960) and with the descriptions of Sir Henry Gray [2] (1854) till 32 weeks of gestation and not in agreement afterwards.
On comparison of findings of present study with existent literature, the body weight in relation to gestational age was in agreement with findings of Parulekar etal [1995] [8], and was more when compared to the findings of Gruenwald et al (1960)[9], Schulz et al (1962) [10].

Histology of fetal spleen:
Vellguth et al (1985) [12] proposed three stages of spleen development from 14 th -24 th week of gestation 1. Thickened capsule, dilated sinusoids, lymphoblasts and few blood vessels were seen.

Evidence of extramedullary hematopoiesis was noted
At 20-24 weeks: aggregation of lymphocytes to form lymphoid follicles around an arteriole was noticed.
After 33 weeks -Well differentiated Malpighian corpuscles were present, structure resembled that of adult spleen. These findings were in agreement with the observations of Mrinmoy pal etal [15], Merida-Velasco JA [16].

Conclusion:
The current study was conducted to analyze the histological development of spleen at various gestational ages. We hope a detailed knowledge of Histology and Histogenesis may provide an insight in understanding the role of spleen in fetal development and fetal wellbeing. However further research directed at molecular level and immunobiology is required in elucidating the actual role of spleen in human beings in immunity & autoimmunity.