{"paper_id":"861a5000-08fd-4024-9178-adc058fb9445","body_text":"An ectopic pregnancy (EP) is defined as the development of the fertilized egg outside the endometrial cavity. This anomaly can occur in about 2% of all pregnancies, but the real incidence may be higher due to scarce reporting. EP may present as a high-risk medical condition, being the most frequent cause of first-trimester mortality, accounting for approximately 4% of all pregnancy-related deaths [ 1 ].\nRisk factors for EP include a previous EP, fallopian tube damage (like pelvic infections or prior pelvic surgery), smoking, use of an intrauterine device (IUD), infertility, and age above 35 years. However, more than 50% of EPs develop in women without risk factors. Therefore, keeping a high level of clinical suspicion during the initial assessment is fundamental for ensuring early detection [ 1 ]. EPs are most commonly located in the fallopian tube, typically in the ampulla, accounting for about 90% of cases. However, in around 1–3% of cases, EPs can occur in the ovary, cervix, and within the abdomen [ 1 ]. Ovarian ectopic pregnancy (OEP) is a rare occurrence, accounting for 1–3% of EPs and 0.03–0.09% of all pregnancies [ 2 ]. A primary OEP ensues when the gestational sac implants directly into the ovary. A secondary OEP occurs when the egg is fertilized in the fallopian tube and then expelled backward into the ovary, where it embeds within the ovarian stroma [ 3 ]. The main risk factor is the use of IUD, but a previous EP, endometriosis, and pelvic inflammatory disease may also be causative. OEP can be due to In Vitro Fertilization (IVF) in 28.5% of procedures, as tubal reflux may facilitate the implantation of the fertilized egg into the ovary [ 3 ]. A hydatidiform mole (HM) occurring in an OEP is almost exceptional, with only 14 cases of complete mole described in the literature [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ].\nTo the best of our knowledge, an ovarian partial hydatidiform mole (OPHM) has never been documented. Herein, we present a case of an OPHM developed in a follicular cyst/early corpus luteum (CL) with thorough investigations including histology and immunohistochemistry for p57, ploidy demonstration with HER2 fluorescent in situ hybridization (FISH) [ 18 ], and STR-based (Short Tandem Repeat) molecular technique for determination of autosomal and sex chromosome number. The case is presented and discussed.\n\nA 39-year-old woman, gravida 2 para 1, presented at 6 weeks + 2 days of amenorrhea to the emergency department complaining of abdominal pain and vaginal bleeding, which had started 3 h prior to the admission. During the gynecological examination with a speculum, bloody brownish traces were observed in the vagina, but there was no active bleeding. Transvaginal sonography (TVS) showed a proliferative endometrial lining measuring 13.5 mm in maximum thickness, but no intrauterine gestational sac. Both fallopian tubes were not dilated. The left ovary presented an anechoic cystic formation of 18 × 10 mm. The right ovary showed a superior round formation of 21 × 20 mm with peripheral vascularization. The content appeared heterogenous with an internal avascular component compatible with blood collection (“Swiss cheese” appearance). Inferiorly, there was the ovarian parenchyma, unremarkable ( Figure 1 ).\nNo pelvic effusions were detected. A diagnosis of probable right ovarian ectopic pregnancy was made. Beta-hCG levels were 6723 IU/L and 5924 IU/L three and one days before the visit. As the patient was clinically stable, she was discharged with a follow-up US in two days. Subsequent beta-hCG levels were 6703 IU/L and 7189 IU/L after one and two days.\nIn the following days the beta-hCG levels were 6703 U/L and 7189 U/L, after one and two days, respectively. Two days after the initial visit, TVS still showed no intrauterine gestational sac and the same right ovarian cyst. The patient underwent laparoscopic removal and intrauterine curettage the same day. Both samples were sent for histology. Three days after surgery beta-hCG levels were decreased to 800 U/L.\nThe patient’s medical history included IVF three years prior with intrauterine embryo implantation. This led to a full-term pregnancy of a healthy male infant approximately two and a half years before the ovarian cyst removal.\nThe samples sent for histological examination were the endometrial curettage and the ovarian cyst formation.\nThe endometrium was hypersecretory with decidualization of the stromal cells. No villous structures were detected. The morphological changes were compatible with early pregnancy, though ectopic.\nThe ovarian cyst was composed of villous structures, irregularly shaped, with focal trophoblast proliferation and focal hydropic changes. Only one villous showed abnormal inner cistern formation with patchy trophoblast atypical proliferation ( Figure 2 ).\nExtravillous implantation trophoblast and arterioles were present. No fetal red blood cells or fetal tissues were detected. All these elements were encapsulated and surrounded by a marked eosinophilic cellular layer compatible with a follicular cyst/early corpus luteum ( Figure 3 ).\nImmunohistochemistry for p57 kip2  showed nuclear positivity in the cytotrophoblast and stromal cells ( Figure 4 ).\nThese features arose the suspicion of an OPHM. Therefore, HER2 FISH and STR analyses were carried out.\nTo evaluate ploidy, HER2 FISH was performed according to the manufacturer’s instructions. A 4 μm thick slide from one FFPE block, the one with the suspicious villous, was pretreated with a Vysis IntelliFISH kit (Abbott, Abbott Park, IL, USA) and hybridized with a PathVysion HER-2 DNA probe (Abbott, Abbott Park, IL, USA). Analysis was performed using a Leica DM5500 fluorescent microscope (Leica Microsystem, Milan, Italy) [ 18 ]. To determine ploidy, signals referring to the centromere region of chromosome 17 (which is used as reference when evaluating HER2 amplification status) were counted in single, complete, non-overlapping nuclei. In our specific case, on 50 nuclei in interphase only 2 (1%) presented three copies of chromosome 17. As the result was still doubtful, molecular analysis was performed.\nDNA was extracted from 3 sliced sections of 5 μm thickness using a QIAamp DNA FFPE Tissue Kit. In the first step, we used an AmpFLSTR ®  Identifiler ®  Plus kit (ThermoFisher Scientific, Waltham, MA, USA) for determining the ploidy of autosomal chromosomes and then a Devyser Compact v3 kit (Devyser Devyser AB, Årsta, Sweden) for determining the number of X chromosomes, according to the manufacturer’s instructions for both. PCR products were run on a 3500 Genetic Analyze (ThermoFisher Scientific, Waltham, MA, USA) and analyzed using GeneMapper v2.0 Software.  Table 1  shows the results obtained with the AmpFLSTR ®  Identifiler ®  Plus kit, while  Table 2  shows the results obtained with Devyser Compact v3 kit.\nAll STR markers agreed with the presence of a female triploid genomic assortment (69,XXX) ( Table 1  and  Table 2 ).\nAfter the histological diagnosis of OPHM, a strict follow-up was planned to detect gestational trophoblastic disease (GTD) with periodic measurements of beta-hCG levels. One month after the surgery, beta-hCG levels were below the threshold (<2 U/L). The test was repeated every 30 days for one year, and the results were always negative.\nThoracic X-rays were performed one month after surgery, and they were normal.\nAfter one year of follow-up, the patient was declared disease-free. After six months, the patient was still in good health and underwent another advanced reproductive technology (ART) procedure with IVF.\n\nEP is estimated to have a global prevalence of approximately 9.69 cases per 100,000 women, based on the latest assessments from 1990 to 2019 [ 19 ]. In recent decades, there has been an increase in EPs mostly due to ART. Although early recognition of EP has dramatically changed maternal morbidity and mortality, epidemiologic data mainly comes from high income countries, underestimating the real incidence in middle and low income nations [ 19 ]. Tubal EP is the most common location, and OEP is a rare occurrence [ 1 , 2 ].\nTVS detection of a ring structure with the yolk sack (+YS), embryo (+EMB), or embryo with heart action (+HA) beyond the uterine cavity was considered as the  specific ,  sure , or  proving marker  of EP. Recently, the blob and bagel signs have been added. The blob sign refers to an irregular but capsulated well-defined ellipsoidal mass. The bagel sign indicates an echogenic ring of variable thickness encircling a unilocular, round cyst, either centrally or eccentrically located, devoid of content [ 20 ]. In EP, urgent intervention depends on the amount and location of abdominal free blood assessed by TVS [ 21 ]. A HM can occur in any kind of pregnancy, even ectopic. HMs, complete and partial (CHM, PHM), are part of the gestational trophoblastic disease (GTD), a variety of placental pathologies. HMs are classified as benign placental neoplasms at risk of malignant transformation. GTD also includes the gestational trophoblastic neoplasia (GTN) group, placental tumors exhibiting malignant behavior, including post-molar gestational trophoblastic neoplasia, invasive mole, gestational choriocarcinoma, placental site trophoblastic tumor, and epithelioid trophoblastic tumor [ 22 , 23 ]. HMs result from abnormal gametogenesis or fertilization, leading to placental overexpression of paternally derived genes [ 22 , 23 ].\nCHMs are characterized by a chromosomal asset entirely derived from the paternal genome. The most frequent karyotype is 46,XX, resulting from endoreduplication of a single sperm’s haploid genome following fertilization of an enucleated ovum, with complete exclusion of the maternal DNA. Approximately 5–10% of complete moles exhibit a 46,XY karyotype due to dispermic fertilization, which accounts for 10–20% of cases. Fetal tissue, vasculature, and red blood cells are typically absent in complete moles as fetal demise and resorption occurs early, prior to the establishment of the circulatory system [ 23 ]. Grossly, the specimen shows vesicular structures, also called “bunch of grapes”, appearing as “snowstorm” at US. Histologically, the villi exhibit a bulbous, “cauliflower-like” appearance with internal cisterns and widespread circumferential trophoblastic hyperplasia involving cytotrophoblast and syncytiotrophoblast. The implantation site and the extravillous trophoblast typically show marked atypia [ 23 ].\nPHMs typically exhibit a complete triploid chromosomal set composed of two paternal and one maternal haploid genomes. The most common karyotypes are 69,XXX or 69,XXY, arising from fertilization of a haploid ovum either by a single sperm followed by duplication of its genome or, less commonly, by dispermic fertilization. Triploidy with a 69,XYY karyotype is rare, and a 69,YYY karyotype has never been detected. A distinguishing pathological feature of partial moles is the presence of gross or histological evidence of fetal development, such as amniotic tissue or fetal vessels containing red blood cells [ 22 ]. Macroscopically, the specimen presents minimal vesicular degeneration. Microscopically, there are two groups of villi, one composed of larger villi with irregular shape and outline and cisterns formation. Circumferential trophoblastic hyperplasia is usually focal, mild to moderate. Implantation site and extravillous trophoblast atypia can be present and focal [ 23 ].\nImmunohistochemistry for p57 kip2 , can be helpful in distinguishing complete and partial moles. In CHM, nuclear expression of p57 kip2  is restricted to the intermediate trophoblast and decidual cells, with absence of staining in villous cytotrophoblast nuclei. Conversely, in PHM, p57 kip2  demonstrates strong nuclear positivity in villous cytotrophoblast, in addition to the previously mentioned sites [ 23 ].\nThis expression is due to the nature of the  p57KIP2  gene, a cyclin-dependent kinase inhibitor located on chromosome 11p15.5, subjected to strong paternal imprinting and expressed exclusively from the maternal allele [ 24 ].\nGenerally speaking, with no reference to a uterine or ectopic pregnancy, differentiating between CHM and PHM is crucial as CHM is associated with a greater risk of maternal complications and a higher likelihood of progressing to GTN. Post-molar GTN develops in approximately 15–20% of CHMs, compared to about 1.5% in PHMs [ 25 ].\nMolar degeneration in OEP has been described in few cases in the literature, but histology, US, and molecular STR analysis always matched an OCHM [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ].\nIn our case, the ovarian cyst presented only one villous suspicious for molar degeneration. Immunohistochemistry for p57 kip2  showed nuclear positivity in the cytotrophoblast and stromal cells, indicating OPHM. However, FISH analysis was not conclusive as on 50 nuclei in interphase only 2 (1%) presented three copies of chromosome 17. This result might have reflected the coexistence of maternal decidual diploid cells., then STR analysis demonstrated a female triploid genome.\nTriploid pregnancies can be classified as digynic, containing two sets of maternal chromosomes and one paternal, or diandric, comprising two paternal sets and one maternal. Karyotype can be 69,XXX; 69,XXY; and 69,XYY [ 26 ].\nTriploidy can be identified in 1% of conceptions, most of them result in spontaneous abortion in the first trimester with an incidence of almost 20%. Fetal development and survival beyond the first trimester in triploid pregnancies are quite rare, with an estimated occurrence of one triploid fetus per 5000 pregnancies between the 16th and 20th weeks of gestation [ 26 ].\nIn our specific case, in which triploidy 69,XXX was identified, no fetal tissues or nucleated red blood cells were observed. We therefore assumed that the PHM could have been diandric, but the genetic parental contribution was not pursued.\nOEP can be classified as intrafollicular (occurring in a corpus luteum) or extrafollicular (embedded in the ovarian stroma) [ 2 ]. OEP-intrafollicular type is quite rare, being misdiagnosed as a CL. A CL is usually a unilocular cyst less than 3 cm which can be anechoic or hemorrhagic with a thick, sawtooth wall. Internal echoes due to internal hemorrhage may mimic a solid mass without internal blood flow. Typically, blood flow surrounds the wall of the CL (ring of fire) and may be used to distinguish CL from OEP. The OEP is usually located within the ovarian stroma and cannot be separated from the ovary by pressure from the probe. The presence of a gestational sac or embryo can facilitate the diagnosis [ 2 ]. PHM may present with abnormal, heterogeneous, and large placental tissue for gestational age. Compared to CHM, it often presents greater vascularization and embryonic and extraembryonic structures, although altered, and less frequently presents cystic placental tissue (“Swiss cheese”) [ 27 ]. In our case, neither CHM nor PHM were suspected, and the ovarian lesion was misdiagnosed with a CL: although the ring of fire was not found and the Swiss cheese modification was not so evident. Moreover, no embryonic and extraembryonic structures were detected. However, it is known that diagnosing a PHM can be more difficult than a complete one, because rigorous diagnostic criteria are often not available [ 27 ].\nIn OEP, symptoms may include abdominal pain and/or vaginal bleeding, often alongside elevated beta-hCG levels.\nMaternal morbidity and mortality are associated with OEP rupture, hemoperitoneum, and hemodynamic instability [ 2 ]. As soon as the OEP is detected, surgical treatment is mandatory to prevent fatal outcomes.\nIn our case, the patient presented with vaginal bleeding, but no intrauterine pregnancy was detected at TVS. However, a suspicious cystic formation of 21 × 20 mm was found in the right ovary. The lesion was anomalous as the outer layer was vascularized and the content was compatible with blood. Beta-hCG levels were 7189 U/L at surgery. After the OEP removal, beta-hCG levels progressively decreased. Histological examination was tricky as the tissue available was scarce and only one villous raised the doubt of an OPHM. FISH analysis and immunohistochemistry for p57 kip2  oriented for an OPHM, but only STR technique identified a triploid chromosomal asset. PHM and CHM have a different prognosis, as in the latter there is a higher occurrence of GTN [ 25 ]. Therefore, in ambiguous cases, when histology, immunohistochemistry, and FISH analysis are not diriment, STR can represent a reliable and useful diagnostic tool. Early hydatidiform moles and hydropic abortion can be histologically similar and the usual ancillary tools might not be sufficient to discriminate them. Instead, this technique is highly sensitive and specific and can be used in formalin-fixed, paraffin-embedded tissue, providing the exact chromosomal asset [ 28 ].\n\nOEP is a rare occurrence, and its molar degeneration is almost exceptional. To the best of our knowledge, this was the first case of a PHM in an OEP. The main difficulty of this case was to detect histologically the PHM in the scarcity of the tissue submitted. Ancillary techniques, such as simple morphology, immunohistochemistry, and FISH, might not be sufficient to confirm the diagnosis. STR has been demonstrated an effective tool in defining the chromosomal asset, even in paraffin-embedded tissues.","source_license":"CC-BY-4.0","license_restricted":false}