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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2016 Sep 1;97(4):329–336. doi: 10.1111/iep.12196

Reversal of tubo‐ovarian atypical epithelial patterns after cessation of ovarian stimulation by letrozole

Ahmed AM Abdel‐Hamid 1,✉, Yaser Mesbah 2, Mona FM Soliman 1
PMCID: PMC5061763  PMID: 27581552

Summary

Letrozole (LTZ), one of the ovulation induction medications, is increasingly prescribed in various gynaecological conditions. Previous studies have demonstrated its potential hazardous effect on the ovarian surface epithelium (OSE) as well as on tubal epithelial cells (TEC). However, it is not clear whether this effect could be reversed by LTZ cessation. Therefore, the objective of our study was to investigate the effect of stoppage of LTZ on these cells after 12 cycles of ovarian stimulation. A total of 54 Sprague Dawley rats were used in this study, divided equally into control, LTZ12 and CES12 groups (received saline, 12 cycles of LTZ and 12 cycles of cessation post‐LTZ12 respectively). Samples from the ovaries as well as fallopian tubes (FTs) were studied histologically for the changes associated with LTZ12 and CES12 respectively. There was evident increase in the proliferative activity and Ki67 immunoexpression in the OSE of LTZ12. The OSE was hyperchromatic, and abnormally frequent deep invaginations, micropapillae and cortical cysts. Their TEC showed frequent multilayering, papillary projections and loss of cilia. Almost all these changes disappeared 12 cycles after LTZ cessation. While the tubal IL‐1β, IL‐6, TNF‐α and serum MCP‐1 levels significantly increased in the LTZ12 group compared with the control group, their levels decreased in the CES12 group compared with those of the control. Therefore, the abnormal tubo‐ovarian epithelial patterns may completely regress after cessation of LTZ stimulation for a reasonable duration. This is a potentially good omen and a positive indicator of the relatively safe use of LTZ after its intake has been stopped.

Keywords: letrozole cessation, epithelial atypia reversal, Ki67 proliferation index, cytokines, interleukins

Introduction

Fertility drugs induce incessant ovulation which may play a major role in the development of ovarian carcinoma (Gadducci et al. 2013). Their long‐term effects on the fallopian tubes (FTs) and ovaries after stopping ovarian stimulation need to be studied further (Lima et al. 2014).

Testosterone and androstenedione are converted to oestrogens by the aromatase enzyme. This aromatization is inhibited by aromatase inhibitors such as letrozole (LTZ) which is as effective as the first line fertility drug, clomiphene citrate (CC) (Usluogullari et al. 2015).

While some studies have revealed that ovulation‐inducing drugs can increase the incidence of ovarian carcinoma (Gadducci et al. 2013), others have reported that they do not increase the risk of this cancer (Ayhan et al. 2004; Diergaarde & Kurta 2014). The genesis of borderline ovarian epithelial dysplasia (OED) by the use of these drugs has been described in previous studies (Mahdavi et al. 2006; Dauplat et al. 2009; Chene et al. 2012) but the direct relationship of this to ovarian carcinoma remains uncertain.

The ovary and FT are contiguous at a narrow isthmus (Ng & Barker 2015) which is evident in both humans and rodents (Flesken‐Nikitin et al. 2013; Ng et al. 2014). The hypothesis that ovarian carcinoma originates from the distal end of the FT (Chene et al. 2013; Dietl 2014; Gilks et al. 2015) is supported by molecular and genetic studies (Reade et al. 2014).

Owing to lack of a definite ovarian carcinoma precursor lesion, it has been suggested that ovarian high‐grade serous carcinoma (HGSC) may arise from cortical inclusion cysts (CICs) (Vang et al. 2013; Ng & Barker 2015). The latter can develop by implantation of tubal epithelial cells (TEC) on the denuded ovarian surface at the site of ovulation rather than by metaplasia from ovarian surface epithelium (OSE) (Kurman 2013; Yang‐Hartwich et al. 2014).

Exposure of the implanted TEC to an incompatible ovary microenvironment leads to HGSC (Ng & Barker 2015). Therefore, removal of FTs can be considered as a primary preventive measure to reduce mortality from ovarian carcinoma (Chene et al. 2013; Dietl 2014; Reade et al. 2014).

Pro‐inflammatory cytokines are frequently detected in tumour microenvironments (Keibel et al. 2009). King et al. (2011) have demonstrated that superovulation increases pro‐inflammatory macrophages in the FTs which release cytokines such as interleukin (IL)‐65, interleukin (IL)‐6, tumour necrosis factor‐α (TNF‐α) and transforming growth factor‐β1 (TGF‐β1) that are associated with poor prognosis of ovarian tumours (Thibault et al. 2014). These cytokines can reach the adjacent OSE following breakdown of OSE basement membranes during ovulation (Ng & Barker 2015).

In this study, we hypothesized that reversibility of the expected tubo‐ovarian atypical epithelial patterns could be attained after a reasonable stoppage period of the LTZ stimulation. We also aimed to find out whether the LTZ cessation is associated with cytokine as well as hormonal changes.

Materials and methods

Experimental animals

Adult female Sprague Dawley rats (210 ± 40 g and with regular menstrual cycles) were obtained from Mansoura Faculty of Pharmacy animal house, where the experiment was performed. Animals (n = 54) were divided equally into three groups (n = 18 each) and were maintained on 12/12‐h light/dark cycle at 24 ± 2°C. The animals received standard laboratory animal's chow and water ad libitum during the period of the experiment.

Experimental design

The animals were divided equally (n = 18 each) into three groups: control, LTZ12 and CES12. The control group received normal saline solution by intraperitoneal injection (i.p) (2.5 ml/kg/day). In the LTZ12 group, ovulation was induced by LTZ which was diluted in normal saline solution (0.5 mg/kg/day, i.p, Femara; Novartis Pharma) on the first and second days of the menstrual cycle, which usually lasts for four days in rodents (Caligioni et al. 2009). Then, human chorionic gonadotropin (hCG) (Pregnil; Schering‐Plough, 100 UI/kg, i.p) was administrated on the third day of the cycle, while the fourth day remained free of any medications. This regimen was repeated for 12 cycles in the LTZ12 group. Likewise in the CES12 group, the same regimen of LTZ 12 stimulation was employed followed by a cessation of LTZ for a similar period. At the end of the experiment, salpingo‐oophorectomy was performed by a professional obstetrician (Y.M.) in each animal under ketamine anaesthesia and strict sterile conditions.

Histological procedure

The resected ovaries and FTs were immediately fixed in 10% formaldehyde solution, from which paraffin blocks were prepared. Sections from the latter were cut (4–5 μm thickness) for the haematoxylin and eosin (H & E) as well as for immunohistochemical staining.

The histological examination was performed by a light microscope (Olympus CX31, (Olympus, Tokyo, Japan)) mounted to digital camera connected to a computer.

We evaluated various histopathological parameters in the ovaries and fallopian tubes of all groups according to the previously described literature with slight modification (Celik et al. 2004; Dauplat et al. 2009; Lima et al. 2014). These parameters in the ovaries included cortical cysts, cysts with papillary projections, epithelial multilayering, epithelial proliferation, surface micropapillae, cortical invaginations, cellular pleomorphism, hyperchromatic nuclei of OSE, increased nuclear size of OSE, irregular nuclear contour of OSE, psammoma bodies and stromal proliferation. In addition, histopathological parameters of assessment in the fallopian tubes included the epithelial multilayering, tufting, increased nuclear density, loss of nuclear polarity, nuclear enlargement, nuclear atypia and loss of cilia.

Immunohistochemical study

Paraffin sections from specimens of the ovaries were stained immunohistochemically to detect Ki67 expression using a monoclonal anti‐rat Ki‐67 antibody at a dilution of 1/20 according to the guidelines supplied by the manufacturer (Clone MIB‐5, Dako, Glostrup, Denmark).

Biochemical assays

Tubal cytokine assessment

Tissue homogenates were prepared from the freshly resected FTs for detection of levels of IL‐1β, IL‐6, IL‐10, TNF‐α and TGF‐β1 by ELISA kits according to the instructions provided by the manufacture (eBioscience; Vienna, Austria).

Serum monocyte chemoattractant protein‐1 (MCP‐1) assessment

Serum was separated from the presacrified blood samples in each animal and stored at −80°C for the determination of MCP‐1 by ELISA kit purchased from RayBiotech Inc. (Norcross, USA).

Reproductive hormone assay

The isolated serum was also used for the detection of the reproductive hormones in each animal by rat‐specific ELISA kits. In the LTZ12 group, the assay was performed before and after hCG injection (6 and 12 cycles after stimulation).

Serum testosterone (T), oestrogen (E2) and progesterone (P4) assay

Serum T (DRG Instruments, Marburg, Germany), E2 (DRG Instruments) and P4 (USCN Life Science Inc., Wuhan, China) were evaluated according to the manufacturers’ instructions. The lower limit for the detection (LLD) of T is 0.083 ng/ml, for E2 is 9.714 pg/ml and for P4 is less than 2.52 ng/ml.

Serum follicle‐stimulating hormone (FSH) and luteinizing hormone (LH) assay

The serum levels of FSH and LH were evaluated by an ELISA kit purchased from Cusabio, Biotech. The LLD for FSH and LH detection is less than 0.2 mIU/ml and 0.5 mIU/ml respectively.

Statistical analysis

Values were presented as mean±SEM. One‐way anova test was employed for comparisons between groups followed by Tukey test using spss 16 software (Chicago, IL, USA). A value of P less than 0.05 was considered as statistically significant.

Ethical approval statement

The experimental techniques were approved by the Institutional Laboratory Animal Care and Use Committee of Mansoura Faculty of Medicine and were performed in accordance with their guidelines.

Results

Histological results

No frank malignant lesion was detected in the ovaries of either the LTZ6 or LTZ12 group. However, ovaries of the latter showed atypical changes in the form of frequent cortical cysts (Figure 1a,b), which sometimes acquired papillary projections (Figure 1a, Table 1) and epithelial lining (Figure 1b). Their surfaces showed numerous deep invaginations (Figure 1a,d) and occasional micropapillae (Figure 1c) which were increased significantly compared with the other groups (Table 1). On the other hand, after 12 cycles of LTZ cessation, the contour of the ovarian surface was completely smooth and there was complete disappearance of the cortical cysts and deep invaginations (Figure 1e) in the CES12 group. In addition, the OSE of the LTZ12 group was composed of abnormally high columnar cells (Figure 2b) with large atypical pleomorphic hyperchromatic nuclei compared to the flat cells covering the ovaries of the control (Figure 2a) or CES12 (Figure 2c) groups. The latter displayed obvious regression of the statistically significant increases in the abnormal histological criteria detected in the LTZ12 group (Table 1).

Figure 1.

Figure 1

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Representative photomicrographs of the ovaries in the LTZ12 and CES12 groups. Cortical cysts (*) (a, b) and micropapillae (arrow) (c) are frequently seen in the ovaries of the LTZ12 group along with the deep cortical invaginations (arrow head) (a, d) that dip from their surface. Sometimes, these cysts acquire papillary projections (a) and epithelial lining (b). However in the CES12 group (e), the surface of the ovary appears smooth with complete disappearance of these cysts, micropapillae as well as the deep cortical invaginations. (H&E a, d, e × 200, Scale bar = 40 μm, b, c × 400, Scale bar = 20 μm).

Table 1.

Quantitative histopathological assessment of the ovarian atypical changes occurring in various groups

Control (n = 18) (%) LTZ12 (n = 18) (%) CES12 (n = 18) (%)
Cortical cysts 5.6 77.8a 5.6b
Cysts with papillary projections 0 16.7a 0b
Epithelial multilayering 0 0 0
Epithelial proliferation 11.1 61.1a 16.6b
Surface micropapillae 0 22.2a 0b
Cortical invaginations 0 44.4a 5.6b
Cellular pleomorphism 0 0 0
Hyperchromatic nuclei of OSE 0 16.7a 0b
Large nuclei of OSE 5.6 66.7a 5.6b
Irregular nuclear contour of OSE 0 11.1a 0b
Psammoma bodies 0 0 0
Proliferative stromal cells 0 16.7a 0b
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Values are represented as percentages in each group (n = 18 rats).

P < 0.05 is significant

a

significant vs. the control

b

significant vs. the LTZ12

Figure 2.

Figure 2

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Representative photomicrographs of the ovaries in various groups. The surface of the ovary of the LTZ12 group (b) is covered by a columnar cells having abnormally large size, pleomorphic hyperchromatic nuclei compared with the control group (a). Similarly, hyperchromatic nuclei were seen in the underlying proliferative stromal cells in the LTZ12 group (b). On the other hand, the surface of the ovary of CES12 group (c) becomes smooth and is covered by flat cells nearly similar to that of the control group. (H&E a, b, c × 400, Scale bar = 20 μm).

Moreover, the epithelial lining of the FTs of the control group (Figure 3a) showed the typical simple columnar ciliated cells. However in the LTZ12 group, there was abnormal proliferation of the TEC, which frequently formed papillae (Figure 3b) and lost most of their cilia along with the appearance of atypical pleomorphic nuclei. After 12 cycles of the LTZ cessation, the TEC proliferation regressed and the typical simple columnar epithelium with numerous cilia became the lining of the FTs (Figure 3c) accompanied by the normalization of almost all disturbed histopathological criteria compared with the LTZ12 group (Table 2).

Figure 3.

Figure 3

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Representative photomicrographs of the FTs in various groups. The mucosal folds of the control are covered by simple columnar ciliated epithelium (a). Abnormally increased proliferation of the TEC is seen in the LTZ12, which frequently form multilayers and papilla (b) and loss most of their cilia (arrow) with the appearance of atypical pleomorphic nuclei. However after 12 cycles of LTZ cessation, the TEC in the CES12 group (c) showed the typically normal columnar epithelium covered by numerous cilia (arrow). (H&E a, b, c, d × 400, Scale bar = 20 μm).

Table 2.

Quantitative histopathological assessment of the atypical changes occurring in tubal epithelial cells (TEC) of various groups

Control (n = 18) (%) LTZ12 (n = 18) (%) CES12 (n = 18) (%)
TEC multilayering 11.1 88.9a 11.1b
Tufting 0 0 0
Increased nuclear density 0 5.6 5.6
Loss of nuclear polarity 0 0 0
Large nuclei 5.6 38.9a 11.1b
Nuclear atypia 0 33.4a 0
Loss of cilia 0 27.8a 5.6b
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Values are represented as percentages in each group (n = 18 rats).

P < 0.05 is significant

a

significant vs. the control

b

significant vs. the LTZ12

Immunohistochemical results

While an obvious increase in Ki67 immunoexpression was detected in the OSE of the LTZ12 group (Figure 4b), the ovaries of the CES12 group (Figure 4c) showed minimal Ki67 expression in their OSE, similar to that seen in the control group (Figure 4a).

Figure 4.

Figure 4

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Representative photomicrographs of the ovaries in various groups. An obvious increase in the proliferative marker, Ki67, expression is observed in the surface epithelium of the LTZ12 (b) group, unlike the CES12 group (c) where minimal Ki67 expression could be detected similar to that of the control group (a). (Anti‐Ki67 immunostain, a, b, c × 400, Scale bar = 20 μm).

Cytokine assay results

Significant increases in the tubal cytokines, IL‐1β, IL‐6 and TNF‐α, as well as serum levels of MCP‐1 (P < 0.05) were observed in FTs of the LTZ12 group compared with the control group (Figure 5a,b). However, after 12 cycles of LTZ cessation, the levels of these cytokines returned back to the control status. In addition, no significant changes were observed in the low ‐ barely detectable ‐ tubal IL‐10 or TGF‐β1 levels seen in the various groups (Figure 5a).

Figure 5.

Figure 5

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A graph of the ELISA assessments of the tubal IL‐1β, IL‐6, IL‐10, TNF‐α and TGF‐β1 (pg/g tissue) (a) as well as the serum level of MCP‐1 (pg/ml) (b) measured in various groups. P < 0.05 is significant asignificant vs. the control bsignificant vs. the CES12 Values are represented as mean ± SEM in each group (n = 18 rats).

Hormonal assay results

No significant changes in the serum level of all reproductive hormones, except E2 and LH, were observed among the various groups. The level of these two hormones increased significantly after hCG administration (6 and 12 cycles after LTZ stimulation) in the LTZ12 group. However on stoppage of LTZ for a similar period, the hormonal profile became comparable with that of the control (Table 3).

Table 3.

Measurements of the serum levels of T (ng/ml), E2 (pg/ml), P4 (ng/ml), FSH (mIU/ml) and LH (mIU/ml) in all groups

Control LTZ12 after 6 cycles of stimulation LTZ12 after 12 cycles of stimulation CES12
Before hCG After hCG Before hCG After hCG
T ng/ml 2.41 ± 0.28 2.39 ± 0.31 2.40 ± 0.24 2.38 ± 0.45 2.39 ± 0.26 2.40 ± 0.27
E2 pg/ml 53.12 ± 17.46 52.87 ± 16.94 432 ± 41.5 a , b 53.23 ± 18.41 435 ± 39.7 a , b 54.22 ± 19.28
P4 ng/ml 5.74 ± 1.63 5.62 ± 1.85 5.65 ± 1.27 5.81 ± 1.72 5.76 ± 1.54 5.73 ± 1.57
FSH (mIU/ml) 7.29 ± 0.48 7.18 ± 0.39 7.26 ± 0.40 7.24 ± 0.43 7.19 ± 0.47 7.25 ± 0.42
LH (mIU/ml) 4.05 ± 0.32 4.13 ± 0.38 7.46 ± 1.25 a , b 4.07 ± 0.41 7.52 ± 1.68 a , b 4.08 ± 0.41
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P < 0.05 is significant

a

significant vs. the control

b

significant vs. the CES12

Values are represented as mean ±SEM in each group (n = 18 rats)

Discussion

In the current study, there was increased proliferative activity of the OSE in the LTZ12 group accompanied with increased Ki67 expression. Moreover, atypical epithelial patterns were present in their ovaries in the form of high columnar OSE, hyperchromatic large nuclei, frequent deep surface invaginations, micropapillae and numerous CICs.

All these findings are considered in the context of the criteria for OED described in previous studies (Chene et al. 2011). Ozcan et al. (2009) have reported that ovulation induction medications, except hCG, can result in OED. In addition, expression of the marker for the increased proliferation Ki67, has been previously observed in ovaries stimulated by CC (Lacoste et al. 2013), tamoxifen and LTZ (Lima et al. 2014).

OED is considered to be a premalignant lesion of the ovary (Sabourin 2011). It has been demonstrated in the OSE by its characteristic histological features (Chene et al. 2008) and was first defined after prophylactic oophorectomy for genetic predisposition (BRCA mutation) (Chene et al. 2011). Furthermore other studies have demonstrated a possible association between OED and incessant ovulation induced by ovulation induction medications (Dauplat et al. 2009). The severity of OED has been reported to increase with prolongation of the duration of these medications and increasing their doses (Celik et al. 2004; Chene et al. 2009; Abdel‐Hamid et al. 2016).

The resultant CICs may acquire epithelial stratification on prolongation of stimulation (Celik et al. 2004). CICs can result from intraovarian endosalpingiosis of TEC (Li et al. 2011). Then, they may undergo malignant transformation (Kurman 2013) by being exposed to the incompatible stromal milieu (Auersperg 2013; Ng & Barker 2015).

Excessive gonadotropin stimulation and exposure to the inflammatory cytokines within follicular fluid can contribute to the development of ovarian carcinoma (Levanon et al. 2008). Moreover, ectopic expression of homeobox (Hoxa) genes induces OSE to differentiate along Müllerian lineages to the numerous histological varieties of ovarian carcinoma (Cheng et al. 2005). Therefore, the occurrence of OED despite the relatively unchanged blood levels of reproductive hormones may indicate a local interplay between hormones and cytokines in the tubo‐ovarian milieu rather than a systemic action.

Our findings revealed that in the LTZ12 group, there was abnormal proliferation of the TEC which frequently formed multilayers and papillae. These findings are considered from the criteria of TEC dysplasia described by Lima et al. (2014) who mentioned that the tubal dysplasia increases by ovulation induction medications whatever the dosage used. It can be manifested after only three cycles of stimulation by FSH and CC (Lacoste et al. 2013).

Moreover, loss of TEC cilia was frequently observed in the FTs of the LTZ12 group. The progressive decrease in the ciliated cells from CICs to serous carcinoma may indicate that the latter is a clonal expansion of secretory cells (Li et al. 2011). Secretory epithelial outgrowth is commonly found in the tubes of ovarian carcinoma, particularly HGSC (Wen et al. 2012). These cells have an intrinsic delayed response to DNA damage compared with the ciliated ones (Levanon et al. 2010), making them more susceptible to accumulation of the genetic mutations on exposure to the pro‐inflammatory microenvironment (Ng & Barker 2015). Additionally, King et al. (2011) have suggested that ovulation increases TEC proliferation by generating DNA damage and stimulating macrophage infiltration which secretes inflammatory cytokines.

In the current study, on cessation of the LTZ for 12 cycles after a similar period of stimulation, regression of the atypical epithelial patterns in OSE and TEC was observed. This might be attributable to the complete clearance of LTZ after approximately 10–12 days from its last dose (Usluogullari et al. 2015). However, it may take a longer time to downregulate the inflammatory cytokines produced in the tubo‐ovarian milieu.

Our data revealed that there were no significant changes in the serum level of all reproductive hormones except E2 and LH. Similar findings were demonstrated by Bedaiwy et al. (2011) who suggest that LTZ induces minor changes in follicular and hormonal dynamics compared with natural cycles. Parsanezhad et al. (2010) have demonstrated the advantageous effect of LTZ by producing a rapid onset of action and clearance of systemic gonadotropin flare. Taken together, our and previous findings suggest that the local tubo‐ovarian milieu plays the key role in the development of dysplasia.

The locally increased gonadotropins in the follicular fluid stimulate OSE proliferation (Burdette et al. 2006) by acting on the receptors located in OSE and TEC (Wright et al. 2011). The inhibition of aromatization by LTZ results in a local increase in androgens that potentiate the follicular sensitivity to FSH (Usluogullari et al. 2015). The latter receptors are upregulated in OSE overlying large follicles (Saddick 2012), suggesting that gonadotropins may have a differential local growth‐stimulating effect (Ng & Barker 2015).

Previous studies have correlated the prolonged presence of the inflammatory milieu with an increased risk of developing cancer (Keibel et al. 2009). IL‐6 is anti‐ and pro‐inflammatory (Naka et al. 2002) and pro‐angiogenic (Nilsson et al. 2005), and its expression associates with a poor prognosis of ovarian carcinoma (Garg et al. 2006) by increasing its resistance to chemotherapy (Wang et al. 2010).

In addition, TNF‐α enhances the formation of a protumoral microenvironment (Thibault et al. 2014) and increases the synthesis of MCP‐1 as well as levels of IL‐1β and IL‐6 that support ovarian carcinoma growth and progression (Charles et al. 2009; Subbaramaiah et al. 2011). The significant rise in the level of these cytokines in the LTZ12 group may indicate their role in the development of dysplasia particularly at the level of tubo‐ovarian microenvironment.

Moreover, IL‐10 activates type M2 macrophages that are predominantly found in neoplasia (Balkwill et al. 2005). TGF‐β is a paracrine niche signal candidate that has anti‐ and protumoral effects and is involved in the epithelial–mesenchymal transition as well as postovulatory wound repair (Ahmed et al. 2006; Sakaki‐Yumoto et al. 2013). Nonetheless, we did not observe any significant changes among various groups in the minimally detectable IL‐10 and TGF‐β which may signify a minor role played by both cytokines at least in the early dysplastic changes.

Conclusion

It could be concluded that cessation of letrozole for a reasonable period after ovarian stimulation can lead to regression of the atypical cytological patterns seen in the ovarian surface and tubal epithelial lining. This favourable effect is associated with the normalization of the tubal cytokine levels which flared up during the ovarian stimulation. Taken together, the wise prescription of letrozole for the shortest duration needed followed by its withdrawal may offer a natural built‐in protective effect without the need of prophylactic postreproductive salpingo‐oophorectomy for the prevention of tubo‐ovarian dysplasia and consequently cancer.

Conflict of interest

There is no conflict of interest.

Acknowledgements

The authors have not made any acknowledgments.

Funding source

We did not receive any funds for performing the current research.

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