AMH and Kisspeptin Receptor Expression in Rare Hydropic Leiomyoma: A Case Study

Introduction

Leiomyomas, the most common monoclonal benign uterine tumors (BUMTs), arise due to complex interactions between genetic predisposition and environmental factors [1]. In certain genetic contexts, hormonal and growth factor crosstalk, along with an increased number of estrogen receptors (ERs) in the myometrium, contribute to their development [1]. While many BUMTs remain asymptomatic, symptomatic cases often present with uterine enlargement, excessive menstrual bleeding leading to secondary anemia, and pressure-related symptoms affecting adjacent organs [2]. Additionally, these tumors can lead to infertility and obstetric complications, further complicating patient management [3]. Although various treatment options exist, each has limitations. Surgical interventions, including myomectomy, uterine artery embolization, and high-frequency magnetic resonance-guided focused ultrasound surgery, can negatively impact fertility and pregnancy outcomes [3–5]. Hormonal therapies, while effective, are associated with significant adverse effects, necessitating additional therapeutic strategies [2,6,7]. Diagnosing leiomyomas relies on a combination of gynecological examinations, ultrasonography, hysteroscopy, and magnetic resonance imaging (MRI) [2,8,9]. A promising advancement in this field is ultrasound-guided preoperative biopsy, which has demonstrated potential in improving diagnostic accuracy [10]. BUMTs exhibit significant heterogeneity in terms of location, biology, and histopathology. The FIGO classification categorizes them based on location (eg, pedunculated, intracavitary, submucosal, intramural, subserosal) and the degree of endometrial or intramural involvement [2,11]. The unusual biology of BUMTs is also reflected in parasitic leiomyomas, which can even develop at laparoscopic trocar sites [12]. Additionally, histopathological subtypes include cellular, bizarre nuclear, fumarate hydratase deficient, mitotically active, hydropic (HLM), apoplectic, lipoleiomyoma, epithelioid, myxoid, dissecting leiomyoma, and diffuse leiomyomatosis [13].

Hormonal activity plays a crucial role in BUMT pathophysiology. The presence of ERα and ERβ has been well-documented [14], and recent positron emission tomography (PET) imaging studies using 18F-FES (18F-labeled estradiol) have further confirmed ER expression in these lesions [15]. Additionally, progesterone receptors (PR-A and PR-B) and gonadotropin-releasing hormone receptors (GnRHRs) are found in BUMTs, with GnRHR expression varying between leiomyomatous and normal myometrial cells [14,16]. Other regulatory proteins involved in reproductive function, such as kisspeptin (KISS1) and anti-Müllerian hormone (AMH), have been implicated in leiomyoma pathophysiology. While AMHR2 expression in BUMTs has been confirmed in a study, the role of KISS1 and its receptor (KISS1R) remains unexplored [17]. KISS1, encoded on chromosome 1, is a key regulator of gonadotropin-releasing hormone (GnRH) secretion, thereby influencing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release [18,19]. It is naturally expressed in various tissues, including the placenta, pancreas, liver, and central nervous system [20–22]. KISS1 and KISS1R play critical roles in puberty onset, menstrual cycle regulation, and placental implantation [23,24]. Moreover, their anti-metastatic properties have been observed in ovarian, colorectal, and nasopharyngeal cancers, prompting interest in KISS1 analogs as potential therapeutic agents for both malignant and benign gynecological conditions, including BUMTs and endometriosis [25–27]. Similarly, AMH, a glycoprotein in the transforming growth factor-β (TGF-β) superfamily, exhibits anticancer properties in vitro and in animal models [28–30]. It plays a crucial role in embryonic Müllerian duct regression and is involved in GnRH neuron activation, gonadotropin secretion, puberty, menstrual cycle regulation, and polycystic ovary syndrome pathophysiology [30–32]. Beyond reproductive functions, AMH contributes to memory, osteoclast inhibition, and neuronal protection [33–35].

Given their regulatory roles in reproductive endocrinology and tumor suppression, modulating AMHR2 and KISS1R activity through targeted analogs may offer a novel approach to BUMTs treatment. Investigating AMH and KISS1 expression in rare, anatomically challenging leiomyomas, such as hydropic leiomyomas (HLMs) in the retroperitoneal space, may provide new insights into alternative treatment strategies. This case report highlights the importance of exploring these atypical molecular pathways in BUMTs, particularly in tumors that are difficult to resect surgically due to their location.

Case Report

PATIENT HISTORY AND TIMELINE:

A 44-year-old woman was admitted to the Clinical Department of Gynecological Oncology due to lower abdominal pain that had been intensifying over the previous hours. Her medical history included a laparotomy at the age of 13 due to acute appendicitis. At the age of 26, she underwent laparoscopic enucleation of an endometrial cyst of the right ovary. When she was 40 years old, she underwent another laparotomy due to a pedunculated cervical myoma, whose histopathology turned out to be a partially cellular leiomyoma with interstitial edema and chronic inflammation. At the age of 35, she gave birth vaginally to a healthy daughter at term. The course of pregnancy was uncomplicated. Since the age of 40, she has been using hormonal contraception, which is a levonorgestrel-releasing intrauterine device (LNG-IUD). Her family oncological medical history included her grandmother’s breast and colon cancer.

CLINICAL EXAMINATION AND INITIAL DIAGNOSTICS:

The patient reported that her abdominal pain had begun approximately 12 hours before admission and progressively worsened. Physical examination revealed a tender right part of the abdomen, with no Blumberg’s sign. Behind the uterus and on the right side, there was a hard, tender, and immovable lump, approximately 70 mm in diameter. A differential diagnosis was considered, including benign or malignant tumor of the uterus or right adnexa, endometriosis-related masses, and adnexal torsion.

LABORATORY EVALUATION AND ULTRASOUND EXAMINATION:

Initial laboratory tests, including a full blood count and inflammatory marker – C-reactive protein (CRP) – as well as laboratory parameters of the coagulation system and common electrolytes (sodium, potassium and chloride), were within normal limits. The carbohydrate antigen-125 (Ca 125) level was 28.0 U/ml, while the carbohydrate antigen 19-9 (Ca 19-9) level was not tested.

Ultrasound examination revealed a normal shape and size of the uterus, with a correctly located LNG-IUD and a pedunculated lesion extending probably posteriorly from the border of the body and the cervix of the uterus (BBCxU), with outlines and dimensions difficult to clearly estimate. Both ovaries had normal structure and size. Due to difficulties in interpreting the ultrasound image and the need to exclude malignancy, an MRI was performed.

MRI FINDINGS AND DIFFERENTIAL DIAGNOSIS:

MRI revealed a well-circumscribed, heterogeneous pathological mass located in the retroperitoneal space, posterior to the right ovary (between the lateral wall of the BBCxU, the vaginal fornix, and the lateral wall of the rectum) and medially to the internal iliac vessels and the piriformis muscle. The dimensions of the mass were 71×37×67 mm. The mass had clear outlines and appeared to be pedunculated, connecting a vascularized peduncle with the posterior wall of the BBCxU. The lesion on T2-weighted images (T2WI) was heterogeneous, with dominant hyperintense areas (Figure 1A, 1B). Elements with intermediate and low signal intensity with muscle tissue (MT) were also present in the form of septum and scattered foci. On T1-weighted images (T1WI) the mass was mostly isointense with MT, with small foci of low signal intensity (Figure 1C). There were no areas suggesting hemorrhagic changes or fatty elements. After administration of the contrast agent (CA), Clariscan™ gadoterate meglumine, intense enhancement was present; only small fluid zones of the tumor were not enhanced (Figure 1D). The signal on the diffusion-weighted imaging (DWI) was heterogeneously increased (Figure 2A). The apparent diffusion coefficient (ADC) was heterogeneous; solid elements showed lower values, but the signal of the lesion was generally higher, which may indicate lower cellularity (Figure 2B). ADC values of the tumor from the individual examined regions of interest (ROI) were from 2.979×10−3 s/mm2 to 1.306×10−3 s/mm2, mean 2.258×10−3 s/mm2 (Figure 2B). No infiltration of the surrounding structures was visible (Figure 1A–1D). The examination did not reveal enlarged lymph nodes.

DECISION FOR SURGICAL INTERVENTION:

Given the persistent abdominal pain, the MRI findings, and the uncertain etiology of the lesion, a differential diagnosis was considered, including BUMTs such as cellular leiomyoma, mitotically active leiomyoma, HLM, and bizarre leiomyoma, as well as malignancies like leiomyosarcoma (LMS). LMS was considered due to the heterogeneous T2WI hyperintensity and the presence of scattered areas with lower ADC values, although the absence of significant necrosis and hemorrhagic changes made this diagnosis less likely. We decided to proceed with exploratory laparotomy and intraoperative histopathological examination. After opening the retroperitoneal space, the pedunculated tumor extending from the BBCxU was excised. Macroscopically, an encapsulated polycyclic tumor measuring 90×80×14 mm was found, with a locally encrusted outer surface. In cross-sections, the tumor was cream-grayish in color. It was heterogeneous, with myxoid areas and hemorrhages.

HISTOPATHOLOGICAL FINDINGS:

Microscopically, the material was a mesenchymal tumor composed of nests, trabeculae, and cords of cells with oval nuclei without distinct nucleoli and dispersed chromatin. The nuclear membrane was highlighted, with occasional grooves. The cell population, despite the described nuclear features, was quite homogenous, without atypia. The architectural structures created by tumor cells were separated by a swollen stroma with features of focal hyalinization. Focally, small clusters of lymphocytes were visible. The vessels were mostly of medium and small caliber, straight, and non-branching. No necrosis was detected (Figure 3A, 3B).

The immunophenotype of the cells was: Vimentin /+/; Desmin /+/ (Figure 3C); SMA /+/ (Figure 3D); CD 56 /+/; CD 99 /+/−/; CK Pan /−/; CK 7 /−/; CK 20 /−/; Chromogranin /−/; Synaptophysin /−/. The Ki-67 proliferation index was 2% and the mitotic index was: 3/10 high-power fields (HPFs) (~1/mm2). The final diagnosis was HLM.

ADDITIONAL IMMUNOHISTOCHEMICAL ANALYSIS:

Additionally, HLM tissue was tested with non-routine immunohistochemical reactions. Rabbit polyclonal antibodies against AMH (1: 100, ab84952, Abcam, Cambridge, UK), AMHR2 (ab197148; 1: 1600, Abcam), KISS1 (1: 200, orb10955, Biorbyt Ltd., Cambridge, UK) and KISS1R (1: 100, orb522544, Biorbyt Ltd., Cambridge, UK) were used. Additional immunohistochemical reactions were assessed by 2 pathologists, and an immunoreactive score (IRS) of Remmele and Stegner was applied [36]. The immunohistochemical analysis revealed strong protein expression in the tumor cells: AMH (9 points in the IRS) (Figure 4A), AMHR2 (9 points) (Figure 4B), KISS1 (9 points) (Figure 4C), and KISS1R (9 points) (Figure 4D). All analyzed proteins indicated nuclear/cytoplasmic expression in tumor cells.

POSTOPERATIVE COURSE AND FOLLOW-UP:

The postoperative course was uneventful. The patient was discharged in stable condition. A 2-year follow-up showed no recurrence of HLM.

Discussion

Although ultrasonography, due to its widespread use and low cost, is a first-line imaging method for BUMTs, it may be insufficient for evaluating retroperitoneal lesions. In such cases, MRI provides a more reliable diagnostic tool, offering superior soft tissue contrast and detailed anatomical visualization. The retroperitoneal space can contain a variety of solid and cystic lesions, including benign and malignant tumors, as well as congenital or developmental anomalies [37–39]. The World Health Organization describes over 100 histological solid lesion types at this site [37].

Differentiation of BUMTs from LMS, a tumor with poor prognosis, is critical [40]. MRI features that help distinguish these entities include signal intensity, border definition, and contrast enhancement patterns. LMS typically exhibits strong or intermediate signal intensity with indistinct borders on T2WI and often shows hemorrhagic necrosis, leading to a high signal on T1WI [8,41]. LMS also has a lower ADC value and increased cellular density, making DWI a useful diagnostic adjunct [41]. However, degenerative changes in leiomyomas can complicate differentiation [41]. Dynamic contrast-enhanced MRI has been reported to distinguish LMS from degenerated leiomyomas with 100% sensitivity and 93.8% specificity [42,43]. Additionally, risk assessment models incorporating patient age, T2WI signal type, DWI signal, and ADC values have demonstrated 92.4% accuracy in differentiating LMS from benign myomas [43,44].

In the presented case, the hyperintense T2WI signal of HLM resembled that of a rapidly growing BUMT. However, the latter is additionally characterized by decreasing ADC [45]. The imaging findings shared also similarities with a leiomyoma with bizarre nuclei, mitotically active leiomyomas and cellular leiomyomas [45]. In the described HLM case, oncological disturbing facts, apart from the patient’s age (44 years old), were a heterogeneous and hyperintense signal on T2WI, the presence of scattered foci of intermediate and low signal intensity with MT and, to a lesser extent, a strong DWI signal. Comparing HLM cases described in the literature shows that other HLMs can present a lower T2WI signal, a more solid character, and indistinct borders of the lesion [46–48].

Pathologically, most leiomyomas are well-demarcated tumors composed of intersecting fascicles of spindle cells with eosinophilic cytoplasm, cigar-shaped nuclei, and rare mitoses [49]. Necrosis is generally limited to ischemic areas [50]. The classification into a group of smooth muscle tumors of uncertain malignant potential (STUMP) or LMS relies on 3 criteria: mitotic count, nuclear atypia, and tumor cell necrosis [49,51,52]. Our patient’s HLM lacked these features, confirming its benign nature. Notably, this case demonstrated strong expression of KISS1, KISS1R, AMH, and AMHR2, which has not been previously reported in HLMs. Most HLMs show overexpression of HMGA2 protein (high mobility group AT-hook 2), which is involved in mesenchymal development and is usually not present on mature cells. The present case of HLM was not analyzed for HMGA2 expression [53].

Serum Ca 125 is occasionally elevated in BUMT cases, typically due to tumor irritation of the peritoneal cavity, large tumor size, or coexisting adenomyosis or endometriosis [54–57]. Elevated Ca 125 and Ca 19-9 levels have been associated with LMS but are less common in benign BUMTs [55,58]. In the present case, Ca 125 levels were within the normal range, and Ca 19-9 was not measured. There are medical reports that in very rare cases, some BUMTs are responsible for hypercalcemia [59]. In this case, calcium level was not assessed. Some cases are also complicated by anemia [53]. In our patient’s HLM, hemoglobin level was 14 g/dL before the operation.

Treatment decisions for BUMTs depend on patient symptoms and reproductive goals. Hysterectomy is an option for patients with symptomatic BUMTs who do not desire future fertility [60]. Myomectomy is preferred for symptomatic patients wishing to preserve reproductive potential. In our case, surgical treatment was chosen due to persistent and worsening abdominal pain, the concerning MRI findings, and the atypical location of the pedunculated lesion. The risk of torsion in pedunculated subserosal leiomyomas further justified surgical intervention [61]. In the context of possible causes of pelvic pain, it is worth remembering that there are also described cases of HLM leading to iliac vein thrombosis due to compression of the tumor mass [48]. Although medical therapies such as gonadotropin-releasing hormone (GnRH) agonists, GnRH antagonists, and selective progesterone receptor modulators are available for BUMTs management, they have adverse effects, contraindications, and limitations on long-term use [60].

Future research should explore novel, less toxic therapeutic strategies for BUMTs. The role of AMH in HLMs remains unclear, but AMHR2 expression suggests potential applications. AMH inhibits aromatase, an enzyme with increased activity in BUMTs, that enhances tumor proliferation [1]. At supraphysiological concentrations, AMH has been shown to induce apoptosis in endometriosis and various cancer cells [28,29]. Additionally, the monoclonal antibody murlentamab, targeting AMHR2, has anticancer potential [62]. KISS1, known for its anti-metastatic properties, also influences GnRH neurons [63,64]. Investigating KISS1 antagonists as potential modulators of hypothalamic and pituitary function presents an intriguing avenue for future research [19]. It is important to search for new therapy methods tailored to the locus and biology of BUMTs with acceptable adverse effects and improving the fertility and live birth rate.

Conclusions

Our findings indicate that certain BUMTs, including those of exceptionally rare histological subtypes and atypical locations that pose surgical challenges, can exhibit high expression of receptors for key natural proteins, such as AMH and KISS1. Considering their biological roles, these proteins and their agonists and antagonists warrant further investigation as potential therapeutic agents, either adjuncts or alternatives to conventional pharmacological treatments.