**3.1.3 Immunohistochemical studies**

Immunohistochemical studies was performed on paraffin sections using TUNEL technique for detection of apoptosis and Ki-67 technique for proliferation assay. Both TUNEL positive and Ki-67 positive cells were distinct from nonpositive cells by their brownish color. In control group TUNEL positive cells were fewer and was limited to granulosa cells near the antrum of atretic follicles (Figure 11). Proliferative cells (Ki-67 positive cells), in this group, were observed in granulose layer close to the basement membrane.

In experimental group, exposed to EMF, apoptotic cells were mainly found near the antrum but also were present among cells close to basement membrane (Figure 12). TUNEL positive cells were not observed in thecal layers but were observed in ovarian stroma. The corpora lutea in the EMF-exposed group also contained numerous TUNEL positive cells. The result from apoptotic cells indicate that EMF exposure induces apoptosis not only in granulose cells but also in other parts of the ovary. Regarding Ki-67 assay, the number of Ki-67 positive cells in granulosa layer from EMF-exposed group were obviously fewer than that in control group meaning that EMF exposure inhibits proliferation.

In experimental group, exposed to EMF, apoptotic cells were mainly found near the antrum but also were present among cells close to basement membrane (Figure 12). TUNEL positive cells were not observed in thecal layers but were observed in ovarian stroma. The corpora lutea in the EMF-exposed group also contained numerous TUNEL positive cells. The result from apoptotic cells indicate that EMF exposure induces apoptosis not only in granulose cells but also in other parts of the ovary. Regarding Ki-67 assay, the number of Ki-67 positive cells in granulosa layer from EMF-exposed group were obviously fewer than that in control group meaning that EMF exposure inhibits proliferation.

Environmental Electromagnetic Field and Female Fertility 67

Different phases of the uterus is recognized according to the morphological characteristics of endometrium. The study was carried out in the proliferative phase. The phase was primarily selected based on estrous cycle and after sacrificing of the animals the morphological characteristics of the endometrium was used as the second criterion, otherwise the case was

In the control group, the endometrium is lined with simple columnar epithelium with nucleus located basally and contained limited uterine glands (Figure 13). In EMF-exposed group, the endometrial thickness was reduced and epithelial cells were smaller, shorter and had condensed nuclei (Figure 14). The difference between two groups was statistically significant (p<0.01). In experimental group, the cells in the stroma of the endometrium were smaller, contained condensed nuclei and blood vessels were less extensive. However,

Fig. 13. Photomicrograph of uterine endometrium from a control rat. Note, columnar cells of

endometrial lining (E). H &E staining, 200X.

**3.2 Effect of EMF on uterus** 

excluded from the study.

**3.2.1 Light microscopic studies** 

myometrium and perimetrium were similar in both groups.

Fig. 11. Photomicrograph of a section from control rat ovary. An antral follicle (A), containing apoptotic cells with golden brown color at luminal surface. TUNEL method, counterstained with toluidine blue. 300X.

Fig. 12. Photomicrograph of an antral follicle from EMF-exposed rat ovary. Note numerous apoptotic cells with golden brown color among granulosa cells (GC), at luminal surface. Techa interna (TC). TUNEL method, counterstained with toluidine blue. 700X.

#### **3.2 Effect of EMF on uterus**

66 From Preconception to Postpartum

Fig. 11. Photomicrograph of a section from control rat ovary. An antral follicle (A), containing apoptotic cells with golden brown color at luminal surface. TUNEL method,

Fig. 12. Photomicrograph of an antral follicle from EMF-exposed rat ovary. Note numerous apoptotic cells with golden brown color among granulosa cells (GC), at luminal surface.

Techa interna (TC). TUNEL method, counterstained with toluidine blue. 700X.

counterstained with toluidine blue. 300X.

Different phases of the uterus is recognized according to the morphological characteristics of endometrium. The study was carried out in the proliferative phase. The phase was primarily selected based on estrous cycle and after sacrificing of the animals the morphological characteristics of the endometrium was used as the second criterion, otherwise the case was excluded from the study.

#### **3.2.1 Light microscopic studies**

In the control group, the endometrium is lined with simple columnar epithelium with nucleus located basally and contained limited uterine glands (Figure 13). In EMF-exposed group, the endometrial thickness was reduced and epithelial cells were smaller, shorter and had condensed nuclei (Figure 14). The difference between two groups was statistically significant (p<0.01). In experimental group, the cells in the stroma of the endometrium were smaller, contained condensed nuclei and blood vessels were less extensive. However, myometrium and perimetrium were similar in both groups.

Fig. 13. Photomicrograph of uterine endometrium from a control rat. Note, columnar cells of endometrial lining (E). H &E staining, 200X.

Environmental Electromagnetic Field and Female Fertility 69

Fig. 15. Electron micrograph of uterine endometrial epithelium from control rat. Secretory

Fig. 16. Electron micrograph of uterine endometrial epithelium from EMF-exposed rat.

Nucleus (N), lipid droplet (L), and irregular basal lamina (arrow). 3500X.

cell (SC), ciliated cell (CC). 3500X.

Fig. 14. Photomicrograph of uterine endometrium from an EMF-exposed rat. Note flattened endometrial epithelium (arrow head). H&E staining, 200X.

Stereological studies showed that the V/v of the nucleus to cytoplasm and axial ratio of the nuclei in the epithelial cells from experimental group was significantly lower than the control group (p<0.01).

#### **3.2.2 Electron microscopic studies**

Ultrastructural studies revealed that epithelial lining of the endometrium consist of two types of cell; ciliated and nonciliated cells. The ciliated cells are randomly scattered among the nonciliated cells. The nonciliated cells undergo morphological changes during different uterine phases. Both cell types contain euchromatic nuclei, a relatively well developed rough endoplasmic reticulum and Golgi apparatus, some apical secretory granules and some fat droplets. The cells prosses small and slender mitochondria. The cells at their lateral interface close to the apex show junctional complex, desmosomes and in some cases an interdigitation attaches the neighbor cells together. In addition to changes that observed with light microscope the electron microscopy showed that in experimental group the nuclear heterochromatin was increased and their mitochondria were condenser and sometimes ruptured in comparison to control group. Furthermore, the secretory granules were dispersed in the cytoplasm while in the control group they were localized to apical area. The rouph endoplasmic epithelium in experimental group was dilated and had a cystic appearance. The number of microvilli in experimental group was reduced in comparison to control group (Figures 15 and 16).

Fig. 14. Photomicrograph of uterine endometrium from an EMF-exposed rat. Note flattened

Stereological studies showed that the V/v of the nucleus to cytoplasm and axial ratio of the nuclei in the epithelial cells from experimental group was significantly lower than the

Ultrastructural studies revealed that epithelial lining of the endometrium consist of two types of cell; ciliated and nonciliated cells. The ciliated cells are randomly scattered among the nonciliated cells. The nonciliated cells undergo morphological changes during different uterine phases. Both cell types contain euchromatic nuclei, a relatively well developed rough endoplasmic reticulum and Golgi apparatus, some apical secretory granules and some fat droplets. The cells prosses small and slender mitochondria. The cells at their lateral interface close to the apex show junctional complex, desmosomes and in some cases an interdigitation attaches the neighbor cells together. In addition to changes that observed with light microscope the electron microscopy showed that in experimental group the nuclear heterochromatin was increased and their mitochondria were condenser and sometimes ruptured in comparison to control group. Furthermore, the secretory granules were dispersed in the cytoplasm while in the control group they were localized to apical area. The rouph endoplasmic epithelium in experimental group was dilated and had a cystic appearance. The number of microvilli in experimental group was reduced in comparison to

endometrial epithelium (arrow head). H&E staining, 200X.

control group (p<0.01).

**3.2.2 Electron microscopic studies** 

control group (Figures 15 and 16).

Fig. 15. Electron micrograph of uterine endometrial epithelium from control rat. Secretory cell (SC), ciliated cell (CC). 3500X.

Fig. 16. Electron micrograph of uterine endometrial epithelium from EMF-exposed rat. Nucleus (N), lipid droplet (L), and irregular basal lamina (arrow). 3500X.

Environmental Electromagnetic Field and Female Fertility 71

Fig. 18. Photomicrograph of uterine luminal epithelium from EMF-exposed rat. Note numerous TUNEL positive cells stained as golden brown. Luminal face (L), uterine gland (G), and uterine stroma (S). TUNEL method, counterstained with toluidine blue. 700X.

The uterine tubes act as the site of fertilization and have a critical role in the conduction of zygote to the uterine cavity. Any changes in the structure and or function of uterine tubes would result in tubal pregnancies. The aim of the present study is evaluating the effect of EMF on uterine tubes by examining histological and morphological features using light and

Light microscopic studies of uterine tubes from control group showed that: uterine tubes are lined with ciliated simple columnar epithelium and their cilia formed a smooth and orderly arranged ribbon at luminal face. The nuclei of epithelial cells were light and euchromatic. The epithelium was rested on a highly vascularized loose connective tissue (Figure 19). In the EMF-exposed group, epithelial cells were low columnar and mostly had lost their cilia. The nuclei of the epithelial cells were condensed and heterochromatic (Figure 20). Morphometric studies, based on final magnification, showed that the height of epithelial cells were decreased after EMF exposure, it was 2.77±0.19 mm VS 2.83±0.46 mm (Figure 21),

**3.3 Effect of EMF on uterine tubes** 

**3.3.1 Light microscopic studies** 

electron microscopy and immunohistochemical techniques.

the difference between two groups is significant (p<0.05).

#### **3.2.3 Immunohistochemical studies**

Detection of apoptotic cells using TUNEL reaction technique showed that in control group the luminal epithelium appeared regular with some glycogen deposition but had almost no sign of apoptosis (Figure 17). In experimental group, the epithelium appeared irregular and there were several apoptotic cells in the luminal and glandular epithelium (Figure 18).

Fig. 17. Photomicrograph of a uterine section from control rat. Luminal epithelium (arrow), the uterine glands are seen. TUNEL method, counterstained with toluidine blue. 700X.

Detection of apoptotic cells using TUNEL reaction technique showed that in control group the luminal epithelium appeared regular with some glycogen deposition but had almost no sign of apoptosis (Figure 17). In experimental group, the epithelium appeared irregular and there were several apoptotic cells in the luminal and glandular epithelium

Fig. 17. Photomicrograph of a uterine section from control rat. Luminal epithelium (arrow), the uterine glands are seen. TUNEL method, counterstained with toluidine blue. 700X.

**3.2.3 Immunohistochemical studies** 

(Figure 18).

Fig. 18. Photomicrograph of uterine luminal epithelium from EMF-exposed rat. Note numerous TUNEL positive cells stained as golden brown. Luminal face (L), uterine gland (G), and uterine stroma (S). TUNEL method, counterstained with toluidine blue. 700X.
