European Journal of Cardiovascular Medicine

Endometrial hyperplasia is an abnormal thickening of the endometrium, primarily due to unopposed estrogen stimulation in the absence of sufficient progesterone. This hormonal imbalance leads to excessive proliferation of endometrial glands and stroma, increasing the risk of neoplastic transformation.¹

There are two main types: hyperplasia without atypia, which has a low risk of progressing to cancer, and atypical hyperplasia (or endometrial intraepithelial neoplasia), which carries a significantly higher risk of developing into endometrial carcinoma if left untreated.¹

In India, the incidence of endometrial carcinoma is about 4.3 per 100,000 women. It is broadly classified into Type 1 tumors (endometrioid and mucinous, estrogen-dependent, and genetically linked to PTEN, PAX2, and K-RAS mutations) and Type 2 tumors (serous, clear cell, and carcinosarcoma, which are aggressive and often TP53-mutated).²

Risk factors include obesity, PCOS, diabetes, unopposed estrogen therapy, early menarche, and late menopause. The most common clinical symptom is abnormal uterine bleeding, which requires timely evaluation to rule out precancerous or malignant changes^.3.

This study was conducted in the Histopathology Department at MGM Medical College and Hospital, Navi Mumbai, over an 18-month period (June 2023 to December 2024). With ethical approval and support from the Gynaecology Department, endometrial biopsy samples were collected, processed, and analyzed using standard H&E staining and immunohistochemistry.

Endometrial samples collected via D&C or biopsy curettes were fixed in formalin, processed routinely, and embedded in paraffin. Sections of 4 µm were stained with H&E for histological evaluation, while 3 µm sections on Poly-L-Lysine slides were used for immunohistochemistry.

A total of 30 well-preserved cases of endometrial hyperplasia (with or without atypia) and carcinoma were included, while normal or poorly processed samples were excluded to maintain data quality. The setting, equipped with experienced faculty and advanced lab facilities, provided a strong foundation for evaluating the diagnostic value of p53, Ki-67, and PTEN in endometrial pathology.

Evaluation of p53 Immunostaining: The assessment of p53 expression was carried out following the methodology outlined by Quin et al. (2002)^4. Tumor cells exhibiting p53 positivity were evaluated and graded on a scale from 0 to 3+, based on the percentage of positively stained cells. A score of 0 was assigned when fewer than 10% of tumor cells showed p53 positivity. A score of 1+ was given when 10–30% of the cells were stained, 2+ when 31–50% were positive, and 3+ when more than 50% of tumor cells demonstrated p53 expression.

Evaluation of Ki-67 Immunostaining: Ki-67 positive cells were identified by the presence of brown nuclear staining. The proportion of Ki-67 expression in glandular epithelial cells was evaluated semi-quantitatively across the entire tissue section under 40x magnification. Based on the extent of nuclear positivity, the staining was categorized into one of the predefined scoring groups.⁵

Table I: Scoring System for Ki-67 Expression

Tumors exhibiting no staining or with less than 5% positive nuclei were considered negative for Ki-67 expression.

Evaluation of PTEN Immunostaining:

PTEN immunoreactivity was interpreted as positive when a distinct brown staining was observed within the nuclei or cytoplasm of glandular epithelial cells. This pattern of staining indicates the presence of PTEN protein, reflecting its functional expression within the cellular compartments responsible for maintaining regulatory control over cell proliferation and survival. Both nuclear and cytoplasmic localization were taken into account, as PTEN can exert its tumor suppressor functions in multiple cellular domains. The intensity and distribution of this brown immunostaining served as critical parameters in assessing PTEN positivity during immunohistochemical evaluation.

Table II: Scoring system for PTEN expression

In this study, the average age of participants was 48.6 years, with most cases occurring between 35 and 50 years—peaking in the 46–50 age group. This aligns with the known trend of increased endometrial pathology around the perimenopausal period. Fewer cases were seen after age 60, likely due to declining hormonal activity with age.

Table III: Age descriptive statistics among study cases

Menopausal status among study participants

Among the 30 cases in this study, 16 participants (53.33%) were premenopausal, while 14 (46.67%) were postmenopausal, indicating a slight predominance of premenopausal women. This distribution is noteworthy, as hormonal variations between these two groups can significantly influence the expression of key immunohistochemical markers such as Ki-67, p53, and PTEN in endometrial tissue. Understanding this hormonal context is essential when interpreting marker expression and its implications in endometrial pathology.

Table IV: Menopausal status among study cases

Obstetric history among study cases

The obstetric history in this study showed varied reproductive backgrounds among the 30 cases. The most common pattern was P1L1A0 (36.67%), followed by P2L2A0 (26.67%) and P3L3A0 (20%). Less frequent histories included P2L2A1 and P1L1A1. These differences in pregnancies, live births, and abortions may influence the expression of Ki-67, p53, and PTEN in endometrial tissue, as reproductive events are closely tied to hormonal and physiological changes affecting endometrial biology.

Table V: Obstetric history among study cases

Endometrial thickness among study cases

The study revealed a diverse range of endometrial thickness (ET) among the 30 cases. The most frequent measurements were 12 mm (16.67%) and 13 mm (30%), followed by 15 mm (13.33%) and 9 mm, 10 mm, and 14 mm (each 10%). Less common values included 13 mm in 2 cases (6.67%) and 16 mm in 1 case (3.33%). This variation reflects the heterogeneity of the study group and may impact the expression of Ki-67, p53, and PTEN, given the association between endometrial thickness and underlying hormonal or pathological changes.

Table VI: Endometrial thickness among study cases

p53 Expression Scoring

In this study, p53 expression was mostly low or absent, with 80% of cases showing negative staining and 16.67% showing mild positivity. Only one case (3.33%) had moderate expression. No cases showed strong positivity, which is often linked to high-grade malignancies. These findings suggest a predominance of low-risk lesions and support the role of p53 in distinguishing benign from potentially neoplastic endometrial changes.

Table VII: P53 Expression score

0/Negative p53 expression ->10%, 1+ p53 expression -10-30%, 2+ p53 expression – 31-50%, 3+ p53 expression - >50%.

KI67 Expression Score

In this study of 30 cases, Ki-67 expression was most commonly mild (1+ in 36.67% of cases), indicating low proliferative activity. Minimal or no staining (score 0) was seen in 30%, often associated with benign lesions. Higher scores (2+ and 3+) were each observed in 16.67% of cases, suggesting increased proliferation and possible malignant potential.

Table VIII: KI67 Expression Score

ki67 scoring, O:<5%, 1+:5-25%, 2+: 25 - 50%, 3+: 50-100%

PTEN Expression Score

In this study of 30 endometrial samples, PTEN expression was evaluated using immunohistochemistry and classified as negative (0), mild (1+), or moderate (2+). A striking 76.67% showed complete loss of PTEN, indicating its early inactivation in endometrial tumorigenesis. Mild expression was noted in 20%, suggesting partial protein retention, while only one case (3.33%) showed moderate staining. These findings underscore PTEN's crucial role in endometrial pathology and its value, along with p53 and Ki-67, in diagnosis and prognosis.

Table IX: PTEN Expression Score

Histopathology Diagnosis Among Study Participants

The histopathological findings in this study of 30 cases revealed a predominance of endometrial hyperplasia without atypia, accounting for 23 cases (76.67%). Hyperplasia with atypia was identified in 2 cases (6.67%), while endometrial carcinoma was diagnosed in 5 cases (16.67%). This distribution underscores the high frequency of non-atypical hyperplasia within the cohort, reflecting the common occurrence of less aggressive endometrial alterations. Top of Form

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Table X: Histopathology diagnosis among study groups (On H&E, and early diagnosis)

Immunohistochemistry Diagnosis Among Study Participants

In this study of 30 endometrial cases, hyperplasia without atypia was the most common finding (17 cases), followed by atypical hyperplasia (8 cases) and endometrial carcinoma (5 cases). This pattern highlights the predominance of non-atypical lesions and provides a valuable framework for understanding how markers like p53, Ki-67, and PTEN vary across different stages of endometrial pathology, offering insights into their diagnostic and prognostic relevance.

Table XI: Diagnosis among study groups (On IHC and later diagnosis)

In this study, immunohistochemistry was employed to assess p53, Ki-67, and PTEN expression across various endometrial conditions, including hyperplasia (with and without atypia), endometrial carcinoma, mixed cell carcinoma, and mucinous adenocarcinoma. This approach enabled the identification of distinct marker expression patterns, underscoring their diagnostic value in differentiating endometrial lesions.

The age distribution in this study closely reflects patterns reported in earlier research on endometrial pathology. Zulfiqar et al.⁶ found that endometrial hyperplasia without atypia was most common in women aged 31 to 45, with a mean age of 36 years—slightly younger than the present study’s mean of 46.48 years. For atypical hyperplasia, both Zulfiqar et al.⁶ and our study show similar findings, with mean ages of 46 and 48.44 years, respectively. Regarding endometrial carcinoma, Emons et al.⁷ reported a mean age of 51 years, while Matsuo et al.⁸ noted 54 years; our study showed a comparable mean age of 52 years. These findings affirm the consistency of age-related trends across studies and reinforce age as a key factor in the development and progression of endometrial conditions.

In this study of 30 endometrial cases, 53.33% of participants were premenopausal and 46.67% postmenopausal, with ages ranging from 35 to 70 years. This menopausal distribution aligns with previous research, reflecting the hormonal shifts that influence endometrial pathology. Zulfiqar et al.⁶ reported that 80% of endometrial hyperplasia without atypia occurred in premenopausal women, a finding consistent with our study’s 53.33%. Conversely, studies by Emons ^7, Cakmak ^9, and others noted a predominance of postmenopausal women in cases of endometrial carcinoma, supporting our observation of 80% postmenopausal prevalence in carcinoma cases.

Immunohistochemically, p53 expression in our study was low in 80% of cases, suggesting wild-type TP53 and less aggressive lesions. This mirrors findings by Akiyama ^10,, Stavropoulos ^11, and Sahin ¹²who also noted minimal p53 expression in hyperplasia without atypia and higher expression in carcinoma among postmenopausal women. Our findings further confirmed the absence of high p53 expression, typically seen in more aggressive subtypes.

Ki-67 expression, a marker of proliferation, was mostly low to moderate in our cases, particularly in premenopausal women, indicating a hormonal influence on cellular activity. These results align with Allithy et al. ^13, who reported higher Ki-67 levels in hyperplasia without atypia, and with Ray ¹⁴ and Sangwan ^15, who found reduced Ki-67 in postmenopausal carcinomas—paralleling our finding of <5% expression in carcinoma cases.

PTEN analysis showed complete loss in 76.67% of cases, especially among postmenopausal women, highlighting its role in early endometrial tumorigenesis. While Allithy et al.¹³ reported over 80% PTEN positivity in hyperplasia without atypia, our study observed 50–70%. In carcinoma cases, PTEN expression was <6%, supporting earlier studies by Ray ¹⁴ and Sangwan ¹⁵ who also documented significant PTEN loss in postmenopausal women with endometrial carcinoma.

The present study revealed a broad range of endometrial thickness (ET) measurements, spanning from 9 mm to 16 mm, with a diagnostic threshold set at 8 mm. The most common thicknesses observed were 13 mm in 9 cases (30%) and 12 mm in 5 cases (16.67%), while less frequent values included 15 mm, 9 mm, 10 mm, 14 mm, 11 mm, and 16 mm. This variability underscores the importance of ET assessment in evaluating endometrial abnormalities. Elevated ET is well recognized as a potential marker for endometrial hyperplasia and carcinoma, making it a crucial component in diagnostic protocols.

Supporting this, Park et al.¹⁶ proposed an 8 mm cut-off as effective in detecting endometrial pathology, a finding closely reflected in our study’s 9 mm threshold. Similarly, Su et al.¹⁷ found a significant risk associated with ET above 5 mm in asymptomatic postmenopausal women. In terms of malignancy, the average ET in our carcinoma cases was 15 mm, which aligns with the >11 mm average reported by Park et al. ¹⁶ though slightly lower than the 20 mm mean noted by Krishnamoorthy et al. ^19.These findings collectively highlight the consistent association between increased endometrial thickness and the risk of hyperplastic or malignant transformation, reinforcing its diagnostic significance in endometrial pathology.

Obstetric history, especially parity, plays a vital role in influencing the risk of endometrial pathology. Numerous epidemiological studies have shown that a higher number of full-term pregnancies tends to lower the risk of endometrial hyperplasia and carcinoma. In the present study involving 30 cases, the obstetric profiles of patients diagnosed with non-atypical hyperplasia, atypical hyperplasia, and carcinoma were analyzed to explore this relationship.

In our study of 30 cases initially diagnosed on H&E staining, 76.67% were classified as hyperplasia without atypia, 6.67% as atypical hyperplasia, and 16.67% as carcinoma. After immunohistochemical evaluation with p53, Ki-67, and PTEN, diagnoses shifted: 56.67% remained non-atypical, 26.67% were upgraded to atypical hyperplasia, and carcinoma cases remained unchanged. My study is not in concordance with any of the study. This highlights the valuable role of IHC in improving diagnostic accuracy.

This study emphasizes the value of combining clinical, histological, and immunohistochemical findings for accurate diagnosis. While most cases initially appeared as non-atypical hyperplasia on H&E, IHC with p53, Ki-67, and PTEN revealed diagnostic upgrades, particularly to atypical hyperplasia. Notably, low p53 and Ki-67 were seen in benign cases, while PTEN loss was prominent in carcinomas, especially in postmenopausal women.

CONFLICT OF INTEREST: There are no conflicts of interest.

FINANCIAL SUPPORT AND SPONSORSHIP: Nil.

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