Endometrial cancer accounts for 4-8% of all carcinomas, fourth position after breast, colon and lung cancers. It has become the most common invasive malignancy of female genital tract in the United States [1]. Risk factors of endometrial carcinoma are prolonged estrogens, early menarche or late menopause. Other risk factors are obesity, nulliparity, anovulation, polycystic ovary disease. Its incidence is higher in postmenopausal women.[2] It has been noted that many women remain asymptomatic in presence of premalignant conditions. So, it is necessary to identify and diagnose premalignant condition of endometrial carcinoma like EIN. [2] Endometrial Intraepithelial Neoplasia (EIN) is a clonal proliferation of architecturally and cytologically altered premalignant endometrial glands which are prone to malignant transformation to endometrioid (Type I) endometrial adenocarcinomas. EIN lesions are non-invasive genetically altered neoplasms which arise focally and may convert to malignant phenotype upon acquisition of additional genetic damage. [3] Due to lack of awareness and knowledge about the disease and also about the medical facilities, many of these patients visit our hospital at late stage of the disease. Endometrioid carcinoma and EIN have a common pathogenesis and thus share many overlapping risk factors. These risk factors are reduced in initial stage of the disease. [1] Diagnosis and management of EIN lesions is important to avoid developing endometrial carcinoma. Histopathological diagnosis of premalignant lesions of endometrioid endometrial cancer is best source to avoid late stage of the disease. [1]As the aim of current medicine era is to diagnose the disease at its early stage, the present study is undertaken with the rationale to assess the incidence of EIN, premalignant condition in the patients presenting with the abnormal uterine bleeding so as to avoid complications, improve the prognosis, change the treatment modalities as required. We further hypothesise that many cases of endometrial intraepithelial neoplasia may go undiagnosed due to inadequate screening and expertise diagnostic facility.

The study is of prospective and analytical type, conducted on 80 patients with history of abnormal uterine bleeding who attended outpatient and inpatient department of Obstetrics and Gynaecology, Acharya Vinoba Bhave Rural Hospital Sawangi (M), Wardha. The duration of study was of two years from 1st July 2014 to 31st July 2016.Endometrial Biopsy was taken from patients who presented with history of abnormal utrine bleeding. These biopsy specimens were then processed in the histopathology section, routine H&E staining was then performed on these biopsies. These cases were then analysed considering the criteria laid down for diagnosis and classification of endometrial hyperplasia by WHO classification of endometrial hyperplasia (1994). [4] After this, they were reclassified using the criteria for diagnosis of EIN. [5]After receiving the tissue specimen in the histopathology division, the specimen labelling and patient information such as patient’s name and Medical Records Department (MRD) number were verified. The type of surgery, type of specimen, detailed clinical history and previous treatment of patienit was reviewed. Tissue was then kept in 10% formalin solution overnight for fixation. Using rotary microtome, 5μm thickness sections were taken on the clean glass slides and then subjected to routine H & E staining.

Required for processing of tissue and Harris Haematoxylin and Eosin staining

• Automated Tissue processor (Leica TP 1020)
• Rotary microtome (Leica RM 2125RT)
• Compound light microscope
• Leukhart‟s moulds, Paraffin, Tissue float bath
• Hot plate, routine H & E stain, Scalpel and blade
• Gloves and mask Forceps
• Glass slides
• Cover slips
• Glass marker
• Distyrene Plasticizer Xylene (DPX) mountant.

Procedure of H & E staining on Paraffin Section [28, 29]:-

• Deparaffinize sections in xylene 3 changes 10 min each.
• Hydrate through graded alcohols to water.
• Running tap water for 1 min.
• Remove fixation stains if necessary
• Stain with Harris haematoxylin for 5-10 min
• Running tap water (Blueing) 5 min or less
• Differentiate in 1 % acid alcohol (1% HCL in 70% alcohol) 5-10 Sec
• Wash well in tap water until sections are again „blue‟ - 5minutes or less
• Stain in 1% Eosin Y for- 10 min
• Wash in running tap water 1-5 min
• Dehydrate through alcohols
• Clear in Xylene- 5 min
• Apply cover slip with mounting medium DPX

Interpretation:-

• Nuclei- Blue
• Cytoplasm- varying shades of Pink
• Paraffin section slides, stained with routine H & E were viewed under light microscope and the histopathological diagnosis was made.

These were then classified according to the WHO classification of Endometrial Hyperplasia. All these cases were then reclassified as Endometrial Intraepithelial Neoplasia using EIN diagnostic criteria.

EIN Diagnostic Criteria-

1) Architecture: Area of Glands greater than Stroma,

2) Cytology: Cytology differs between architecturally crowded focus and background, or clearly abnormal,

3) Size >1 mm, 4) Benign conditions with overlapping criteria: Basalis, secretory, polyps, repair, etc.,

4) Carcinoma if mazelike glands, solid areas, polygonal “mosaic-like” glands, myoinvasion or significant cribriforming.

By using above mentioned criteria, re-classification of all the cases of endometrial hyperplasiai.e simple hyperplasia without atypia, simple hyperplasia with atypia, complex hyperplasia without atypia and complex hyperplasia with atypia into endometrial intraepithelial lesion (EIN) was done.

Statistical analysis was carried out using descriptive and inferential statistics using chisquare test. Software used in the analysis was SPSS 17.0 version and Graph Pad 6.0 version and p<0.05 was considered as level of significance (S).

The study is of prospective and analytical type, conducted on 80 patients presented with history of abnormal uterine bleeding who attended outpatient and inpatient department of Obstetrics and Gynaecology, Acharya Vinoba Bhave Rural Hospital Sawangi (M), Wardha. All the results of histopathology were carefully noted, interpreted, and recorded in tabular form.

Table 1: Distribution of cases according to the age

A total of 80 cases were included in the present study. Of these, 35% cases were in premenopausal age group, 50 % in perimenopausal and 15 % in postmenopausal. Maximum patients were in perimenopausal age group as shown in table 1.

Table 2: Distribution of cases according to the types of abnormal uterine bleeding/ Pattern of bleeding

Cases were classified according to the pattern of bleeding. The most common presenting complaint was menorrhagia in 38 cases. The second most common presenting complaint was post-menopausal bleeding in 19 cases followed by 12 cases of polymenorrhoea, 6 cases of metrorrhagia and 5 of menometrorrhagia which is shown in above table 2.

Table 3: Correlation of clinical diagnosis with age group

Table 3 showed that the incidence of abnormal uterine bleeding was more in perimenopausal age group than postmenopausal age group. Most common type of bleeding was menorrhagia followed by post-menopausal bleeding. Menorrhagia was predominantly seen in perimenopausal age group.

Table 4: Distribution of cases according to the histopathological diagnosis

Histopathological pattern of all 80 cases were noted. It was observed that the most common histopathological pattern was simple hyperplasia without atypia in 48 cases followed by complex hyperplasia without atypia in 15 cases. 13 cases were of complex hyperplasia with atypia and 4 of simple hyperplasia with atypia which is shown in table 4.

Table 5: Endometrial histology in premenopausal, perimenopausal and postmenopausal age group

Table 5 shows, Out of total 80 cases, simple hyperplasia without atypia comprises of 48 cases, out of which 19 cases are in premenopausal age group, 23 are in perimenopausal age group and 06 cases are postmenopausal age group. Four cases were of simple hyperplasia with atypia out of which one case was in premenopausal age group, two cases were in perimenopausal age group and one case in postmenopausal age group. Out of 15 cases of complex hyperplasia without atypia, 04 cases were in premenopausal age group, 07 cases in perimenopausal age group and 04 cases in postmenopausal age group. Out of 13 cases of complex hyperplasia with atypia, 04 cases were in premenopausal age group, 08 cases of perimenopausal age group and 01 case in postmenopausal age group. The maximum cases were of simple hyperplasia without atypia seen predominantly in perimenopausal age group.

Table 6: Correlation of clinical presentation with histopathological diagnosis of cases

As shown in table 6, Out of 38 cases of menorrhagia, simple hyperplasia without atypia was the commonest histopathological finding in 25 cases (65.78%), followed by complex hyperplasia with atypia which was seen in 7 cases (18.42 %) of menorrhagia, complex hyperplasia without atypia was seen in 5 cases (13.16 %). Considering postmenopausal bleeding in 19 cases, simple hyperplasia with atypia was seen in 10 cases (52.62%), complex hyperplasia with atypia in 5 cases (26.32%) and simple hyperplasia with atypia in 2 cases(10.53), complex hyperplasia without atypia in 2 cases (10.53%) cases each. 12 cases with polymenorrhagia showed simple hyperplasia without atypia in 06 cases (50%), followed by complex hyperplasia without atypia in 04 cases (33.33%) and complex hyperplasia with atypia in 02 cases (16.66%). Out 6 cases of metrorrhgia, simple hyperplasia without atypia was seen in 5 cases (83.33%) whereas complex hyperplasia with atypia was seen in only 1 case (16.67%). In 5 cases who presented with menometrorrhgia, 2 cases (40%) revealed simple hyperplasia without atypia while other 2 cases (40%) revealed complex hyperplasia without atypia and 1case (20%) as simple hyperplasia with atypia.

Table 7: Re-classification of Endometrial hyperplastic lesions using EIN criteria

In the present study, 80 cases of various endometrial biopsies were classified as simple and complex, with and without atypia according to WHO. These cases were then further reclassified into EIN using EIN criteria. 12 (70.58%) out of 17 cases of atypical hyperplasia were reclassified as EIN. 5(33.33%) cases out of 15 cases of complex hyperplasia without atypia were reclassified as EIN. 3 (06.25%) cases out of 48 cases of simple hyperplasia without atypia of EIN. (As shown in table 7)

‘Endometrium’ is the mirror of hormonal status in females. Endometrium is most sensitive to ovarian hormones. The endometrium is a dynamic tissue that undergoes physiologic and characteristic morphologic changes during the menstrual cycle as a result of sex steroid hormones coordinately produced in the ovary. ^[6] Histopathological diagnosis of premalignant lesions of endometrioid endometrial cancer is best source to avoid late stage of the disease.^[1] Due to lack of awareness and knowledge about the disease and also about the medical facilities, many of these patients visit hospital at late stage of the disease. Endometrial Intraepithelial Neoplasia. (EIN) is a clonal proliferation of architecturally and cytologically altered premalignant endometrial glands which are prone to malignant transformation to endometrioid (Type I) endometrial adenocarcinoma. EIN lesions are non-invasive genetically altered neoplasms which arise focally and may convert to malignant phenotype upon acquisition of additional genetic damage.^[3] Endometrioid carcinoma and EIN have a common pathogenesis and thus share many overlapping risk factors. These risk factors are reduced in initial stage of the disease.^[1]

Abnormal uterine bleeding is considered one of the most common and challenging problems presenting to the Gynecologist, it is responsible for as many as one-third of all outpatient gynaecologic visits. With medical advancements combined with increasing awareness about gynecological problems, women gain access to most of the diagnostic and therapeutic modalities.^[6]

The most common presentations are menorrhagia, polymenorrhoea, metrorrhagia and instrumental bleeding. The endometrial biopsy is one of the various investigations chosen to evaluate the endometrial pathologies because it has several advantages over other diagnostic methods. The hormonal assay is very expensive and laboratories with hormonal assay are not available in rural areas^[6]. Thus, early accurate diagnosis and proper treatment of endometrial hyperplastic lesions are essential to prevent progress to endometrial carcinoma and preclude unwarranted hysterectomy without definitive diagnosis.  With this background, the present study assessed the endometrium in patients presenting with abnormal uterine bleeding and incidence of endometrial intraepithelial neoplastic lesion within these patients by reclassification using EIN diagnostic criteria.

In the present study, it was found that age range of patient was wide from 23 yrs to 68 yrs with a median age of 45.5 yrs. All these cases were then grouped in premenopausal, perimenopausal and post-menopausal. Of these, 28 (35%) cases were in premenopausal age group, 40 (50 %) were in perimenopausal and 12 (15 %) were in postmenopausal. Majority of patients were in perimenopausal age group. Findings of the present study were concordant with the results of studies by Rajshri Damle et al.^[7], Sharma Juhi et al.^[8], S Vaidya et al.^[9] and Bhatt S et al.^[10] which showed that maximum patients were in perimenopausal age group.

Cases were classified according to the pattern of bleeding. The most common presenting complaint was menorrhagia in 38 cases (47.50%) followed by post-menopausal bleeding in 19 cases (23.75%) then polymenorrhoea in 12 (15%) cases and metrorrhagia in 6(7.5%) cases and menometrorrhagia 5(6.25%) cases.

Table 8: Distribution of cases according to types of abnormal uterine bleeding/pattern of bleeding

As shown in table 8, Findings of the present study were concordant with the findings of studies carried out by Rujuta Prajapati et al.^[11] M. B. swami et al.^[12], Vijay kumar et al.^[6], Sharma Juhi et al.^[8], Nair et al.^[13], and Jairajpuri et al.^[14] which revealed that the most common symptom is menorrhagia .Postmenopausal bleeding was the second most common bleeding pattern. However, in a study conducted by Jairajpuri et al.^[14], metrorrhagia was the second most common presenting bleeding pattern. This difference may be attributed to the age of the patient included in the study group.

In the present study,      histopathological pattern of all 80 cases were noted. It was observed that amongst all types of hyperplasia, the most common histopathological pattern was simple hyperplasia without atypia in 48 cases (60.00%) followed by complex hyperplasia without atypia in 15 cases (18.75%). 13 cases (16.25%) were of complex hyperplasia with atypia while 4 (05.00%) cases were of simple hyperplasia with atypia followed by complex hyperplasia without atypia in 15 cases (18.75%). 13 cases (16.25%) were of complex hyperplasia with atypia while 4 (05.00%) cases were of simple hyperplasia with atypia.

Table 9: Distribution of cases according to histopathological diagnosis

Findings of present study are in concordance with the findings of studies conducted by Khanna R et al.^[15], Baak et al.^[16], Hech et al.^[17], Baak et al.^[18], Raychaudhary et al.^[43], Kurman et al.^[20], Supriya S et al.^[21] and Rujuta Prajapati et al.^[11] which showed that the most common histopathological pattern was simple hyperplasia without atypia .

In the present study, when histopathological pattern of endometrium was analysed with premenopausal age group, it was found that simple hyperplasia without atypia was the commonest pattern seen in 19 cases. This was followed by both complex hyperplasia with atypia and complex hyperplasia without atypia in 4 cases each. The least common pattern was simple hyperplasia with atypia seen in only 1 case.  When the present study was compared with other studies conducted by Lyla et al.^[22], S Vaidya et al.^[9], Gerald D et al.^[23], Rupal Shah et al.^[24], and Baral et al.^[25], findings were in concordance. (as shown in table 9)

In this study, when histopathological pattern of endometrium was analysed with perimenopausal age group, it was found that simple hyperplasia without atypia was the most common pattern seen in 23 patients followed by complex hyperplasia with atypia in 8 patients then complex hyperplasia without atypia in 7 patients. The least common pattern was simple hyperplasia with aytpia seen in 2 patients. Perimenopausal age group was the predominant age group for all types of endometrial hyperplasias.

When the present study was compared with other studies such as Gerald D et al.^[23], Rupal Shah et al.^[24], and Baral et al.^[25], findings were in concordance which showed that the most common endometrial pattern in perimenopausal age group was simple hyperplasia without atypia followed by complex hyperplasia with atypia.

In the present study, in post menopausal age group, out of 12 cases, the most common histopathological pattern was found out to be simple hyperplasia without atypia in 6 cases (50.00%), followed by complex hyperplasia without atypia in 4 cases (33.33%). Simple and complex hyperplasia with atypia was seen in 1 case each (08.33%).

When the findings of the present study were compared with other studies such as Gerald et al.^[23], Layla et al.^[22], Kairavi etal^[26], S Vaidya et al.^[9], results were in concordance showing simple hyperplasia without atypia was the most common histopathological pattern in endometrium of postmenopausal age group.

In the present study, when analysis of patients of hyperplasias with pattern of bleeding was done, it was found that maximum number of patients with simple hyperplasia had heavy bleeding (Menorrhagia) in 25(52.08%) whereas complex and atypical hyperplasias are associated with irregular bleeding (polymenorrhea) in 4 cases (30.70%).and 2 cases (10.52%)

Table 10: Correlation of histopathological diagnosis with clinical diagnosis

Findings of the present study were similar with the study conducted by S. Kayastha et al.^[27] which showed that menorrhagia was mostly seen in patients with simple hyperplasia without atypia. (As shown in table 10)

Reclassification of WHO Endometrial hyperplasia into EIN using EIN diagnostic criteria

In the present study, 80 cases of various endometrial hyperplastic lesions were examined and reclassified according to the EIN diagnostic criteria. 12 cases (70.58%) out of 17 cases of atypical hyperplasia were reclassified as EIN. 5 cases (33.33%) out of 15 cases of complex hyperplasia without atypia were reclassified as EIN. 3 cases (06.25%) out of 48 cases of simple hyperplasia without atypia of EIN. The incidence of EIN was seen maximum with simple and complex atypical hyperplasia.

Table 11: Reclassification of WHO Endometrial hyperplasia into EIN using EIN diagnostic criteria

Similar results were obtained in various studies done by Khanna R et al.^[15], Baak et al.^[16], Hech et al.^[17], Baak et al.^[18], and Supriya et al.^[21] which showed that the incidence of EIN was seen maximum with atypical hyperplasias. (as shown in table 11)

It has been concluded that majority of women with abnormal uterine bleeding presented in perimenopausal age group. Menorrhagia was the most common type of bleeding pattern followed by postmenopausal bleeding. The incidence of endometrial intraepithelial neoplasia which is a premalignant condition was more in atypical hyperplasia. This was more common in perimenopausal and postmenopausal age group. Study of endometrial histopathology in perimenopausal and post menopausal women with AUB is helpful to diagnose aypical hyperplasia in early stage.  Hence, histopathological evaluation of the endometrium is specially recommended in women with these age groups, presenting with abnormal uterine bleeding in order to rule out the possibility of premalignant condition.

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