The patient was a 21-year-old woman who underwent bilateral ovarian cystectomies for bilateral OTs (5 × 5 cm on the left ovary and 6 × 6 cm on the right ovary). The tumors were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. Histologically, the left-sided tumor was diagnosed as an immature teratoma, grade 1, and was composed of three mature germ layers with small foci of neuroepithelial tubules; therefore, left oophorectomy was performed. In the resected left ovary (3 × 2 cm), another 2 × 0.8 mm tumor was detected in the cortex, away from the surgical margin, which appeared to be an OT bud. The right-sided tumor was a mature teratoma, and so the right ovary was preserved. In the > 3 years since, the patient has been disease-free.
The patient provided informed consent for the publication of her case. The study overall conformed the ethical guidelines of the Declaration of Helsinki (revised in 2013). Ethical approval for this case report was obtained from the Institutional Review Board of Musashino Red Cross Hospital, Shonan Kamakura General Hospital and Doai Memorial Hospital.
In the 3 × 2 cm left ovary, an OT bud (width, 2 mm; height, 0.8 mm) was found protruding on the inner surface of the atretic follicle, which was 9 mm in diameter (Figs. 1 and 2). The OT comprised two nodules, 0.6 × 0.8 mm and 1.3 × 0.8 mm in size, which touched each other. No tumor was found in the adjacent 5-µm section.
The OT bud exhibited several rudimentary organ-like features resembling those of an early human embryo (Fig. 2). Two primitive glandular structures were observed inside the tumor; the structure in the upper portion of the tumor had a cystic shape and consisted of dense and overlapping high columnar cells and one layer of flat cells, whereas the structure in the lower portion had an elongated, tubular shape with dense columnar cells, and the center was narrowed and bent. The area around these two structures was composed of small, mesoderm-like, spindle-shaped to cuboidal cells. These cells had densely and sparsely distributed parts. Cord-like arrangements were seen mixed with these mesoderm-like cells. Spindle cells forming sinus-like structures that contained blood cells were also observed. The surface of the two nodules was covered with one layer of cells that were flat-to-cuboidal. These tumor cells showed no evidence of nuclear atypia. Components of the three germ layers of the gonads, neuroepithelial tubules, trophoblast giant cells, and embryonal carcinoma cells were not observed in this OT.
RNA in situ hybridization
RNA in situ hybridization (ISH) revealed three representative genes (SHH, SOX2, and SOX9) involved in maturation and differentiation whose levels of expression differed (Figs. 3 and 4). SHH was highly expressed in approximately 33% of the upper primitive glandular structure and in 75% of the lower one. Cord-like arrangements also stained strongly in the gene probe. However, SHH expression was not confirmed in the mesoderm-like cells or in the flat-to-cuboidal surface cells (Fig. 3a–c). SOX2 was widely distributed in the two glandular structures with medium to high intensity (Fig. 4a–c). Although both SHH and SOX2 were co-expressed in parts of those structures, the right side of the lower structure, which showed high expression of SOX2, did not hybridize with the SHH probe (Figs. 3c and 4c). Low signals of SOX9 were detected on the flat-to-cuboidal surface cells and mesenchymal mesoderm-like cells (Fig. 4d–f). SOX9 was clearly detected in the sinus-like structures with medium intensity, which also hybridized with the SHH probe (Figs. 3b and 4d–f).
Signals of these three RNA probes were completely absent in the normal ovarian tissues around the OT. Immunohistochemistry results for neurofilament protein and glial fibrillary protein were negative (data not shown).
Review of Cases
For this report, we reviewed 1254 consecutive OTs diagnosed at Musashino Red Cross Hospital (Musashino, Japan), Shonan Kamakura General Hospital (Kamakura, Japan), and Doai Memorial Hospital (Tokyo, Japan), and we describe one that manifested as an OT bud, 2 × 0.8 mm in size. To characterize the OT bud, we performed RNA sequencing, and to evaluate the expression of genes involved in embryogenesis, we used RNA ISH.
The 2 × 0.8 mm tumor was embedded in paraffin after formalin fixation and cut serially once into 50 5-µm sections for histological observations, RNA extraction, and RNA ISH. These serial sections were labeled as slides 1–50 and preserved at 4°C until use (Figs. 2–4). The tumor components of three unstained formalin-fixed paraffin-embedded (FFPE) sections (slides 26–28) were scraped with a needle tip, and the RNeasy FFPE kit (QIAGEN, Hilden, Germany) with deparaffinization steps  was used to extract the RNA. For the experimental control, RNA was similarly extracted from the normal parts of the same slides where they did not contain oocytes (Fig. 1). The quality of RNA was assessed with Agilent Bioanalyzer High Sensivity RNA Analysis kit (Agilent Technologies, Santa Clara, CA, USA). Since the value of RNA integrity was low, the QIAseq Ultralow Input Library Kit (QIAGEN) was used for sequencing, and 30 million reads of RNA were obtained from both tissues.
To perform RNA sequencing (2 × 36 bases paired-end sequencing), we used NextSeq500 (Illumina, San Diego, CA, USA), which showed that 813 genes were expressed more strongly in the OT sample than in the surrounding ovarian tissue. Sixty genes involved in embryonic morphogenesis were identified by low-expression gene filtering and by Metascape [6, 7]. Three of these genes, SHH, SOX2, and SOX9, were selected for RNA ISH of the tumor. Each of these three genes is well known to be involved in the differentiation and maturation of the early human embryo [8–11]. SHH encodes Sonic hedgefog which is a key mediator of anterior–posterior embryonic structures and is expressed in several sites during embryogenesis, where it helps direct the development of neighboring tissues, including the primitive node, notochord, and the floor plate of the neural tube [8, 9, 11]. SOX2 and SOX9 encode important transcription factors that regulate the expression of genes that control cellular differentiation during early embryonic stages [9–11].
RNA ISH was performed to localize SHH, SOX9, and SOX2 in FFPE tissues of the OT. To obtain specific signals but not background noise from nonspecific hybridization, we used RNAscope™ (Advanced Cell Diagnostics, Newark, CA, USA) as described by Wang et al. . Theoretically, sequential hybridizations with the preamplifier, amplifier, and label probe can enable the visualization of single molecules within each RNA dot plot. The probes used were RNAscope™ Target Probe-Hs-SHH, RNAscope™ Target Probe-Hs-SOX2, and RNAscope™ Target Probe-Hs-SOX9.