Vitrification preserves murine ovarian follicular cell transcriptome in a 3D encapsulated in vitro follicle growth system

Vitrification is a method for long-term biological sample cryopreservation without causing intra- and extra-cellular ice formation. We recently established a novel closed vitrification system to cryopreserve mouse ovarian follicles. Using the 3D alginate hydrogel encapsulated in vitro follicle growth (eIVFG) method, we demonstrated that compared to freshly-harvested follicles, vitrified follicles had normal follicle and oocyte reproductive outcomes. However, it is unknown whether vitrification preserves molecular signatures of folliculogenesis, which is the primary research focus in this study. Six fresh and six vitrified antral follicles grown from eIVFG were collected on day 8 for the whole single-follicle RNA sequencing. Principal component analysis (PCA) and Pearson’s correlation analysis revealed that vitrified follicles had similar transcriptomic profiles to fresh follicles. There were 35 differentially expressed genes between vitrified and fresh follicles, however, none of those genes have been shown to be critical to folliculogenesis and oogenesis. Meanwhile, gene ontology (GO) and KEGG pathway analysis revealed that no GO terms or signaling pathways were significantly enriched. Furthermore, the expression of genes essential for the gonadotropin-dependent folliculogenesis and oogenesis were comparable between vitrified and fresh follicles. Taken together, these results demonstrate that vitrification preserves follicular cell transcriptome and molecular signatures of gonadotropin-dependent folliculogenesis in the eIVFG system, providing a robust model for fertility preservation, conservation of endangered species, and also establishing a high-content ovarian follicle biobank for studying ovarian biology and female reproductive toxicology.

Deleterious Mutations in the TPO Gene Associated with Familial Thyroid Follicular Cell Carcinoma in Dutch German Longhaired Pointers

Familial thyroid cancer originating from follicular cells accounts for 5–15% of all the thyroid carcinoma cases in humans. Previously, we described thyroid follicular cell carcinomas in a large number of the Dutch German longhaired pointers (GLPs) with a likely autosomal recessive inheritance pattern. Here, we investigated the genetic causes of the disease using a combined approach of genome-wide association study and runs of homozygosity (ROH) analysis based on 170k SNP array genotype data and whole-genome sequences. A region 0–5 Mb on chromosome 17 was identified to be associated with the disease. Whole-genome sequencing revealed many mutations fitting the recessive inheritance pattern in this region including two deleterious mutations in the TPO gene, chr17:800788G>A (686F>V) and chr17:805276C>T (845T>M). These two SNP were subsequently genotyped in 186 GLPs (59 affected and 127 unaffected) and confirmed to be highly associated with the disease. The recessive genotypes had higher relative risks of 16.94 and 16.64 compared to homozygous genotypes for the reference alleles, respectively. This study provides novel insight into the genetic causes leading to the familial thyroid follicular cell carcinoma, and we were able to develop a genetic test to screen susceptible dogs.

Prevalence of Malignancy in MNG: Final Histopathology Perspective

Objective: To determine the malignancy in multinodular goiter by doing final histopathology of specimen. Study Design: This is an observational study. Setting: Study carried out in the department of ENT, Head & Neck Surgery of Khairpur Medical College Hospital Khairpur, from August 2016 to July 2019. Materials and Methods: All those patients with MNG with or without thyrotoxicosis were selected and advised for Thyroid function tests, ultrasound thyroid and serum calcium level. FNAC was performed only in cases with suspicious nodule. All the patients under went total/near total thyroidectomy after all base line routine investigations along with thyroid function tests. Histopathological evaluation was also conducted. Results: Out of total 70 patients with MNG, 17 (24.3%) cases were suspected of malignancy. Out of 17 suspicious cases, FNAc showed colloid goiter in 8 (47%), follicular in 7 (41%) cases and papillary in 2 (12%) cases. Final histopathology showed total 5 (29%) cases as malignant and remaining 12 (71%) cases were benign. Out of 5 malignant cases, 4 (80%) cases were papillary and 1 (20%) cases were Follicular cell carcinoma. While other 53 (75.7%) cases under went for near total thyroidectomy and specimens sent for histopathology, among these only 1 (2%) case found as Papillary cell Carcinoma. Total 6 cases were malignant out of which in which 5 cases were Papillary cell Carcinoma and one was Follicular cell Carcinoma. Conclusion: We conclude that multinodular goiter is the most prevalent thyroid disease found in female. Follicular thyroid carcinoma is the most frequent cancer seen in this study.

Validation of Immunohistochemistry for Canine Proteins Involved in Thyroid Iodine Uptake and Their Expression in Canine Follicular Cell Thyroid Carcinomas (FTCs) and FTC-Derived Organoids

Thyrotropin receptor (TSHR), sodium iodide symporter (NIS), pendrin, and thyroid peroxidase (TPO) are essential for the uptake of iodine by follicular thyroid cells. The aim of this study was to establish immunohistochemistry (IHC) protocols for TSHR, NIS, pendrin, and TPO in canine tissues and characterize their expression in organoids derived from canine follicular cell thyroid carcinoma (FTC) and in the respective primary tumors. This constitutes a fundamental step to establish organoids as a model to study the uptake of iodine in canine FTC. Commercially available antibodies directed against human proteins were selected. Antibody specificity was confirmed by western blot using lysates of the HTori-3 human thyroid cell line and healthy canine thyroid gland. IHC was validated using HTori-3 cells and a set of canine normal tissues including healthy thyroid gland. The expression of TSHR, NIS, pendrin, and TPO was evaluated in 3 organoid lines derived from FTC and respective primary tumors. All 4 antibodies produced specific bands by western blot and cytoplasmic labeling in follicular cells by IHC in both human HTori-3 cells and canine thyroid gland. NIS also showed basolateral membrane immunolabeling in follicular cells. All 4 proteins were highly expressed in organoids derived from FTC. The expression was similar or higher compared to the primary tumors. The results of this study characterize organoids derived from canine FTC as a suitable in vitro model to investigate iodine uptake, opening new research possibilities in the field of canine thyroid cancer therapy.

DNA repair proteins may differentiate papillary thyroid cancer from chronic lymphocytic thyroiditis and nodular colloidal goiter

Malignant thyroid lesions are the most common malignancy of the endocrine glands with increasing rates in the last two decades. Papillary thyroid cancer is the most common thyroid malignancy. In our study, we aimed to quantitatively evaluate the levels of DNA repair proteins MSH2, MLH1, MGMT, which are representative blocks of patients diagnosed with papillary carcinoma, chronic thyroiditis, or colloidal goiter. Total or subtotal thyroidectomy material of 90 patients diagnosed with papillary carcinoma, nodular colloidal goiter, or chronic thyroiditis between 2009 and 2012 were retrospectively evaluated. Tissue samples obtained from paraffin blocks were stained with MGMT, MSH2, MLH1 proteins and their immunohistochemistry was evaluated. Prepared sections were examined qualitatively by an impartial pathologist and a clinician, taking into account the staining method under the trinocular light microscope. Although there was no statistically significant difference in MGMT, MSH2, MLH1, follicular cell positivity, staining intensity, and immunoreactivity values, papillary carcinoma cases showed a higher rate of follicular cell positivity, and this difference was more pronounced between papillary carcinoma and colloidal goiter. In the MSH2 follicular cell positivity evaluation, the difference between chronic thyroiditis and colloidal goiter was significant (p = 0.023). The difference between chronic thyroiditis and colloidal goiter was significant in the MSH2 staining intensity evaluation (p = 0.001). The difference between chronic thyroiditis and colloidal goiter was significant in MLH1 immunoreactivity evaluation (p = 0.012). Papillary carcinoma cases were demonstrated by nuclear staining only for MSH2 and MLH1 proteins as opposed to hyperplastic nodules. The higher levels of expression of DNA repair genes in malignant tumors compared to benign tumors are attributed to the functional activation of DNA repair genes. Further studies are needed for DNA repair proteins to be a potential test in the development and progression of thyroid cancer.

Familial thyroid follicular cell carcinomas in a large number of Dutch German longhaired pointers

Thyroid carcinomas originating from follicular cells of the thyroid gland occur in both humans and dogs and they have highly similar histomorphologic patterns. In dogs, thyroid carcinomas have not been extensively investigated, especially concerning the familial origin of thyroid carcinomas. Here we report familial thyroid follicular cell carcinomas confirmed by histology in 54 Dutch origin German longhaired pointers. From the pedigree, 45 of 54 histopathologically confirmed cases are closely related to a pair of first-half cousins in the past, indicating a familial disease. In addition, genetics contributed more to the thyroid follicular cell carcinoma than other factors by an estimated heritability of 0.62 based on pedigree. The age of diagnosis ranged between 4.5 and 13.5 years, and 76% of cases were diagnosed before 10 years of age, implying an early onset of disease. We observed a significant higher pedigree-based inbreeding coefficient in the affected dogs (mean F 0.23) compared to unaffected dogs (mean F 0.14), suggesting the contribution of inbreeding to tumour development. The unique occurrence of familial thyroid follicular cell carcinoma in this dog population and the large number of affected dogs make this population an important model to identify the genetic basis of familial thyroid follicular cell carcinoma in this breed and may contribute to the research into pathogenesis, prevention and treatment in humans.