Assessment of Endometrial Carcinoma Markers and Hormone Receptors Profile before and after Bariatric Surgery: A Clinico-Pathological-Immunohistochemical Study

Background: Obesity is a major risk factor for endometrial carcinoma, and we aim to 
assess markers of carcinogenesis including PTEN and Ki-67 and hormone receptors 
profile including ER, PR and AR before and after bariatric surgery to find out 
its effects in reducing endometrial carcinoma risk in morbid obese females. Patients 
and methods: The study included 80 females with morbid obesity (BMI > 40 
Kg/m2) who underwent bariatric surgery. All were sampled by Pipelle 
biopsy at baseline and 12 months after operation and examined 
histopathologically and immunohistochemically for Ki-67, PTEN, ER, PR and AR. Results: Sixty two out of 80 (62/80) females showed no pathological abnormalities; 4 had 
polyps; 7 had simple endometrial hyperplasia; 4 had atypical endometrial 
hyperplasia and 3 had endometrial carcinoma. In total, 34 females underwent 
gastric bypass operation (42.5%) and 46 underwent a sleeve gastrectomy 
operation (57.5%). There was a statistically significant difference between 
baseline weight and BMI before and after surgery (p Conclusion: Females with morbid obesity have a higher risk of 
harboring endometrial abnormalities even if asymptomatic. However, the 
endometrial pathology and the high ER and PR expression can be normalized 
within one year without medical treatment, signifying the role of bariatric 
surgery-induced weight loss in reducing the risk of endometrial neoplasia 
development. Also, the marked weight loss occurring after bariatric surgery 
induces highly significant endometrial change as resolution of atypical 
hyperplasia, and molecular changes as reduction of Ki-67 and restoration of 
PTEN that are associated with transition of endometrium from high to low risk.


Introduction
Endometrial carcinoma (EC) ranks as the fourth most common cancer in females in the United States with a rising incidence [1].
Obesity is considered as the strongest risk factor for EC and atypical hyperplasia (AH), the precursor lesion [2]. Every 5 kg/m 2 increase in body mass index (BMI) increases risk of EC by 1.6 folds. As a result, females with class III-IV obesity (BMI ≥ 40 kg/m 2 and ≥50 kg/m 2 , respectively) will have ten folds higher risk of EC compared to normal weight females [3].
It is estimated that 40% of EC cases are directly resulting from obesity [4].
Also, females with class III-IV obesity who showed long-term weight loss after bariatric surgery had reduction in risk of EC in both retrospective studies [5] [6] and the prospective Swedish Obesity Subjects study [7].
Bariatric surgery can be indicated for patients with clinically morbid obesity (BMI ≥ 40 mg/m 2 ) or these with BMI ≥ 35 mg/m 2 but having other co-morbidities as diabetes mellitus, obstructive sleep apnea, obesity-caused cardiomyopathy or severe joint disease [8].
The program of bariatric surgery should include physical activity, dietary counseling, and lifestyle changes after operation [9].
In females with severe obesity, bariatric surgery succeeds in marked weight loss and shows greater long-term benefits compared to non-surgical weight loss methods and it was found that surgical weight loss reduces obesity-related comorbidites and gets better outcomes in severe obese populations [10].
In addition to improved cardiovascular risk factors, there is also reduction on Open Journal of Obstetrics and Gynecology cancer risk after bariatric surgery. Studies found a statistically significant reduction in overall cancer risk in females after bariatric surgery [11].
So, there is link between obesity and EC; however, the mechanism is not well understood but may include changes in proliferation markers  or excess oestrogen unopposed by progesterone in noncycling premenopausal and postmenopausal endometrium.
The patterns of estrogen receptor (ER) and progesterone receptor (PR) expression in normal endometrial tissue, malignant and premalignant endometria are well characterized. However, their expression in obese women, implications of androgen receptor (AR) expression, and effects of weight reduction on these patterns remain unknown [12].
In this study we hypothesized that weight reduction following bariatric surgery is accompanied by improvement of endometrial morphology and molecular biomarkers involved in endometrial carcinogenesis including PTEN and Ki-67 and normalization of hormone receptors patterns by assessment of ER, PR and AR.

Endometrial Sampling and Clinical Management
Clinical measurements and endometrial samples were obtained at baseline, and 12 months after bariatric surgery. At baseline, we reported the last menstrual period (LMP), menstrual bleeding status. Postmenopausal status was defined as last menses occurring >1 year before baseline; otherwise cases were defined as premenopausal. Height and weight were measured using a stadiometer and electronic scales respectively after removal of bulky clothing, and BMI was calcu- Females who showed endometrial pathology were managed after being discussed by Gynecological Oncology Multidisciplinary Team (MDT). Endometrial carcinoma patients underwent total abdominal hysterectomy with bilateral salpingo-oopherectomy followed by adjuvant therapy. While females with atypical Open Journal of Obstetrics and Gynecology hyperplasia (AH) had repeated endometrial sampling after 6 -7 weeks. If abnormalities were persistent, they were treated with 6 months of intrauterine progestin. All women who had resolved endometrial abnormalities were followed by 3-to 6-monthly endometrial observation.

Histopathology and Immunohistochemistry
Endometrial tissue was obtained by pipellebiopsy, formalin-fixed, paraffin embedded, then sectioned and stained by hematoxylin and eosin. It was then examined and assessed by the histopathologist using the WHO classification system [13] [14].

Assessment of Immunohistochemistry
The Ki-67 score was considered by percentage of glandular cells having positive nuclear staining. Overview of the whole specimen was done to choose 3 representative fields. The final Ki-67 score was determined by >1000 nuclei noticed in 3 high-powered fields (×20) [15].

Statistical Analysis
Quantitative data were expressed as the mean ± SD & median (range), and qualitative data were expressed as absolute frequencies (number) & relative frequencies (percentage). Continuous variables were checked for normality by using

Bariatric Surgery and Changes in Weight
In total, 34 females underwent gastric bypass (42.5%) operation and 46 underwent a sleeve gastrectomy operation (57.5%). The mean weight after 12 months was 93.08 ± 14.22, mean BMI was 28.16 ± 4.64. There was a statistically significant difference between baseline weight and BMI before and after surgery (p < 0.001) ( Table 3).

Endometrial Pathology, Immunohistochemical Changes before and after Bariatric Surgery
Of the seven women with simple hyperplasia, resolution occurred in five women within 7 months of bariatric surgery after weight loss of between 12% and 20% of their total body weight. In three of them, resolution was accompanied by restoration of normal menstrual cycles. Three of 4 females with AH showed resolution after 9 months (Table 4).
The three females who showed pre-operative endometrial carcinoma, underwent total abdominal hysterectomy. So, on assessment of the Ki-67, PTEN, ER,   The median H-score of ER for patients with pre-operative hyperplasia was 220, which was significantly higher than for patients without pre-operative hyperplasia (165, p = 0.001). Patients without pre-operative pathology had no Open Journal of Obstetrics and Gynecology Totally, there was a significant reduction in ER score after surgery (p < 0.001).
PR H-scores were higher also for patients with pre-operative hyperplasia (me- AR H-scores were higher significantly in pre-operative biopsies than post-operative ones (median 70 vs 50, p < 0.001) ( Table 5).

Discussion
Bariatric surgery is associated with a considerable and a clinical significant reduction of uterine malignancy risk. Our study, in concurrence with the results of others, has revealed that the uterine malignancy risk increases with increasing BMI [18] [19] [20] [21]. So, our finding with previous ones, suggest that obesity can be considered as a modifiable risk factor for endometrial carcinoma.
The pathogenic mechanisms of obesity associated endometrial carcinoma include exogenous estrogen formation and insulin resistance. It is well recognized W. M. Elnagar et al.  that circulating estradiol levels are affected by the percentage of adipose tissue, as aromataseenzyme is found in the adipose tissue. Also, obesity induce a chronic Open Journal of Obstetrics and Gynecology inflammation state, with increasing levels of interleukin-6, C-reactive protein and TNF-α, leading to insulin resistance, which is linked to low levels of sex hormone binding globulin, with subsequent increasing in the bioavailability of estrogen, and is related to tumorogenesis and tumor spread [22].
The connection of cancer risk reduction with bariatric surgery has been already established [23] [24]. Bariatric surgery cause weight loss throughout decreased caloric intake and increased physical activity. It also decreases cancer risk by many weight loss independent pathways [25]. These mechanism include reorganization of gastrointestinal anatomy, decreased iinflammatory markers and inflammation, changes in bile and fat metabolism, gut hormone release, and alternation of intestinal gluconeogenesis [23].
We have found a high frequency of endometrial pathology in class III-IV obese females selected for gastric bypass or sleeve gastrectomy. Also, occult AH and EC were seen in a part of our participants at baseline, Bariatric surgery resulted in significant reduction in weight and fall in BMI and resolution of some AH cases. This finding support the causal association between obesity and endometrial carcinoma.
Our study revealed that class III-IV obesity is associated with high Ki-67, and loss of PTEN expression. This pre-proliferative profile, act as background for mutational changes that initiate endometrial carcinogenesis [26].
In addition, this combined expression profile of these biomarkers give a emolecular signature denoting the endometrium at risk, and supply a chance for screening, risky women for the prevention of EC.
In females with endometrial pathology, loss of glandular PTEN was common finding at baseline biopsy and its restoration accompanied resolution of atypical hyperplasia. In women with normal endometrial biopsy, baseline PTEN null persisted through weight loss, this suggests that bariatric surgery is effective in clearing these endometrial precursor lesions. Consequently, PTEN loss can be a reversible element of early endometrial carcinogenesis and its regain indicates the conversion to low risk endometrium [27].
A drop of Ki-67 was noticed at 12 months after bariatric surgery. This was noticed in most cases with endometrial abnormalities who performed repeat sampling, Ki-67 is a well known prognostic marker for EC, and it is suggested that a drop of Ki-67 is linked to EC risk reduction [28].
Another important finding in our study is that ER, PR and AR scores of females with hyperplasia dropped after bariatric surgery without any medications. This may indicate a direct consequence of weight loss on peripheral estrogen secretion, loss of gastrointestinal adipokines due to excision of part of intestine or the restoration of ovulation with subsequent effect of endogenous progesterone. However, morbidly obese females are relatively hyper estrogenic, so, the mechanism by which weight reduction act may simply be adjustment of the estrogen and progesterone balance via estrogen mitigation. Estrogen is recognized to persuade proliferation and increased of ER and PR expression, which are usually over-expressed in type I, obesity-related endometrial carcinoma; while PR un-Open Journal of Obstetrics and Gynecology dergo down-regulation [29].
We cannot exclude sampling errors as a source of errors. We decided the Pipelle biopsy method over curettage for our study, to minimize surgical risks inan volunteer asymptomatic females; nevertheless, this may not be the optimal method for evaluation, and despite that most studies found a high degree of correlation between its results and curettage results, there may be increasing discrepancies among abnormal specimens, which may be significant in a study as ours.
We also recommend further studies with larger sample size and use of control matched groups for better results evaluation.

Conclusion
Females with morbid obesity have a higher risk of harboring endometrial abnormalities even if asymptomatic. However, the endometrial pathology and the high ER and PR expression can be normalized within one year without medical treatment, signifying the role of bariatric surgery-induced weight loss in reducing the risk of endometrial neoplasia development. Also, the marked weight loss occurring after bariatric surgery induces highly significant endometrial change as resolution of atypical hyperplasia, and molecular changes as reduction of Ki-67 and restoration of PTEN that are associated with transition of endometrium from high to low risk.