Transgender (TG) is a broad term, which describes an individual whose gender self-identification is different from the assigned sex at birth. When the latter becomes overwhelming and causes a significant psychological impact, the term gender dysphoria (GD) is used thereby presenting itself as an extreme case of TGism. The individuals with GD often seek a medical intervention, surgical or therapeutic, to correct the existing discordance between their gender self-identification and physical appearance .
As of 2016, approximately 1.4 million adults identify as TG in the United States . The TG biological underlining is not well understood. Genetically, it appears that interplay between sex steroid receptor polymorphisms, particularly those of androgen and estrogen, which are associated with TG . Yet the RYR3 variant was identified in the Han Chinese population with GD  and CYP17 single nucleotide polymorphism was linked to female-to-male TGism  whereas a common SRD5A2 polymorphism has been shown recently to be benign in TGism (ClinicalTrials.gov Identifier: NCT00435513). Chromosomal aberrations, such as in Klinefelter syndrome (47, XXY), could also be responsible for GD in some individuals since a 1.13% frequency of patients with Klinefelter syndrome was found in GD population .
Metabolically, GD has been linked to androgen insensitivity in biological males. Individuals with complete androgen insensitivity syndrome (CAIS) have a 46, XY karyotype, bilateral testes, and female external genitalia . These individuals tend to be psychosocially female and oriented toward men . Individuals with partial androgen insensitivity syndrome (PAIS) also have a 46, XY karyotype, but may have penoscrotal hypospadias, micropenis, bifid scrotum, and undescended testes .
Most recently, a large cohort association study involving 380 transgender females (TFs) and 344 control male subjects revealed an over-representation in TFs of several allele combinations including androgen receptor (AR) that were proposed to feminize male individuals thereby leading to TGism .
Therefore, the main objective of this study was to provide in-depth examination of the current TF body to gain additional important insights into the respective biological underlinings through a systematic approach including gross anatomical dissection, histological analysis, and genetic screen using NGS technology.
2.1. Human Cadaveric Body Procurement
An 83-year-old TF cadaver was received through Saint Louis University (SLU) School of Medicine Gift of Body Program from an individual who had given his written informed consent. The body was embalmed through the right femoral artery with a mixture of water and a solution (2:1) containing 33.3% glycerin, 28.8% phenol, 4.6% formaldehyde, and 33.3% methanol.
2.2. Anatomical Dissection
The cadaveric body was dissected according to .
2.3. Histological Analysis
Testicular tissue was procured from the embalmed body. Tissue fixation, paraffin embedding, sectioning, and staining with hematoxylin & eosin were performed by Research Microscopy and Histology Core, Department of Pathology, Saint Louis University (SLU) School of Medicine according to the standardized procedures. Images were captured on Olympus 41BX-EPI microscope equipped with the 10× UPlan FL N, 20× LUCPlan FL, and 40× UPlan FL N objectives. The data acquisition and image analysis were performed by using CellSens Standard software.
2.4. Bone Densitometry
Bone density of the lumbar spine and left femoral neck of the embalmed body was measured in triplicates using certified Hologic QDR-4500 X-ray Bone Densitometer in the dual energy X-ray absorptiometry (DEXA) mode following the manufacturer’s protocol. The respective average T-score values were used throughout the text.
2.5. Genetic Analysis
The Next Generation Sequencing (NGS) and bioinformatics analysis were performed as previously described   with the following modifications. DNA extracted from the tibia specimen procured from the embalmed TF body was sequenced to 30× depth of coverage (~4.5 Gb) on the Illumina HiSeq 2500 NGS platform in the 2 × 100 base read format. DNA extraction was performed by Paleo-DNA Laboratory (Lakehead University, Canada) and exome sequencing was conducted by Omega Bioservices (Norcross, GA). The cumulative exome coverage for > 25× depth of coverage was 93% indicating that almost all of the exome was accessible for probing. The variant call and annotation were performed by Genome Technology Access Center (GTAC, Washington University in St. Louis) using SnpSift varType and ANNOVAR. The resultant data were converted into the Microsoft Excel format and pathologic (deleterious) variants were identified through the five consecutive filtering steps described in  . Functional annotation of the remaining variants was performed using UniProtKB Protein, Google Scholar, and PubMed database searches.
3.1. Anatomical Characterization
A body of an 83-year-old individual with the male sex assigned at birth was received through the Gift of Body Program at the Center for Anatomical Science and Education (CASE) of the SLU School of Medicine, USA. The records indicated that this individual self-identified as a female by legally changing his first and middle names to female ones later in his life and his death certificate listed his sex as a female. It was also self-reported by the body donor that in the adulthood he/she underwent a complete penectomy without further specification. The body was hairless with an exception of a sparse hair on the scalp (Figure 1(A)), had a large scrotum with no penis observed (Figure 1(B)), and the urethral orifice was identified posterior to the scrotum (Figure 1(C)). A scrotum dissection revealed that the ductus deferens, epididymis, and testes were all intact (Figure 2(A)). However, the latter were much smaller than normal with the volume ~3.3 cm3 (Figure 2(A)) versus ~7.5 cm3 for the age matched normal individuals . Dissection of the pelvic cavity revealed normal male anatomy with the seminal vesicles and ductus deferens located posterior to the bladder (Figure 2(B)). The prostate was also intact, positioned inferiorly to the bladder (Figure 2(C)).
3.2. Histological Analysis
Histological analysis of testicular tissue revealed abnormal seminiferous tubule epithelium. As compared to the age matched control body, the epithelium was devoid of germ and Sertoli cells and Leydig cells were not observed in the interstitium (Figure 2(D)).
3.3. Bone Densitometry
Because the results of histological analysis were indicative of testicular malformation and, consequently, hormone deficiency with the latter being commonly linked to osteoporosis, the bone mineral density in the cadaveric body was assessed by dual-energy X-ray absorptiometry (DEXA) scanning of the lumbar spine and left femoral neck yielding the respective T-score values of −3.1 and −3.4. These values are characteristic of osteoporosis (http://www.bones.nih.gov) and may serve as an indirect measure of the hormone deficiency in the examined body.
3.4. Genetic Analysis
The assessment of this individual karyotype was impossible due to incompatibility of the available methodologies with the biological material procured from the embalmed cadaveric body. However, the measured individual’s height of 1.73 m was significantly lower as compared to that of 1.83 m average for the Klinefelter syndrome patients , which along with the absence of other phenotypic characteristics such as gynecomastia  (Figure 1(A)) allows, although incomplete, exclusion of the respective 47, XXY chromosomal aberration from further consideration.
The putative genetic underlining of the present TF case was addressed by sequencing the entire coding regions of DNA (exome) extracted from the embalmed cadaveric tissue using NGS Illumina platform as described previously  . The rare genetic variants (minor allele frequency, MAF ≤ 0.01) linked to deleterious (pathological) amino acid substitutions in the mutant proteins were identified through the five sequential, stringent filtering steps described in  . In total, the genetic screen yielded 144 deleterious (pathological) variants of 137 genes with none of them associated with either penile or urethral cancer, or the genes known to regulate the androgen hormone axis (Table S1).
Figure 1. Physical examination of the TF body. (A): The torso view shows hairless body was with no evidence of gynecomastia. Pelvic area view demonstrates large scrotum with no penis (B) and transposed urethral orifice located posterior to the large, reflected scrotum (C).
However, six genetic variants, TPO, BBS12, DNAH9, ITF81, OFD1, and TAPT1 could be linked directly to our case (Table 1) due to their involvement in the regulation of a thyroid hormone production (TPO) and male sex organ development through the control of cilia function (BBS12, DNAH9, ITF81, OFD1, and TAPT1) .
The condition presented in the current report could be described as TGism with its progression to GD. The latter conclusion was based on the absence of genetic variants known to be associated with either penile or urethral cancer which puts forward an elective surgery as a primary cause for the reported complete penectomy.
Figure 2. Gross dissection of the pelvic area and histology of the seminiferous tubules of the TF body. (A): Scrotum dissection revealed intact ductus deferens, epididymis, and small testes. Pelvic dissection revealed normal location of seminal vesicles and ductus deferens (B) as well as intact but small prostate (C). (D): Abnormal seminiferous tubule epithelium lacking germ and Sertoli cells. Leydig cells were not observed in the interstitium. Boxed area is magnified in the upper right corner.
Table 1. Selected deleterious (pathological) variants associated with the current TF.
It is hypothesized herein that one of the important underlinings of the above condition could be linked to the impaired T3 and T4 thyroid hormone production which could negatively affect a cross-talk between the thyroid and androgen hormone axes   thereby potentially leading to anatomical and physiological alterations similar to those in PAIS . This hypothesis is based on: 1) a presence of TPO deleterious (pathological) variant that control the T3 and T4 production and the absence of the variants that regulate the androgen hormone axis (Table 1); 2) the observed testicular malformation at both the anatomical and histological levels (Figure 2); and 3) the T-score values derived for the lumbar spine (−3.1) and femoral neck (−3.4) being comparable to those of patients with PAIS  while being much worse than those of older male subjects with low testosterone levels, respectively −0.48 and −1.56 . Importantly, the T3 and T4 thyroid deficiency has been recently reported in a patient with GD and normal, 46, XX, female karyotype . With this regard, the data presented in the current report raise a very important question is to whether the thyroid hormone deficiency could be viewed as one of the important biological underlinings of TGism and GD. It should also be noted, that prenatal maternal thyroid deficiency has been previously linked to impacting sexuality  .
The detected deleterious (pathological) BBS12, DNAH9, ITF81, OFD1, and TAPT1 variants known to negatively affect cilia development and function could serve as a prerequisite for other male sex organ malformations ambiguously present in the current case, such as hypospadias (Figure 1(B))  and complete penoscrotal transposition . Intriguingly, in the model organisms, cilium has also been shown to be a major regulator of male sexual behavior such as mate searching and selection   .
The results reported in the current study provide unique information for the hypothesis that thyroid hormone deficiency could be one of the important biological underlinings of TGism and GD that merits its further evaluation in a clinical setting.
We gratefully acknowledge Dr. Paul Cliften (GTAC, Washington University in St. Louis, St. Louis, MO, USA) for his invaluable assistance with the bioinformatics analysis as well as Caroline Murphy and Barbara Nagel (SLU) for their skillful help with the histology slides preparation. Dr. Maria Teresa Tersigni-Tarrant is acknowledged for her assistance with the anatomical dissection.
The deleterious (pathologic) genetic variants were identified by the exome sequencing of a single proband. The high scientific value of a single proband approach to unique human cases has been recently demonstrated .
AF designed the study, coordinated genetics data collection and bioinformatics analysis, analyzed the data, wrote and revised the manuscript. LP participated in designing the study, collected and analyzed the anatomical and bone densitometry data, and performed functional annotation of genetic variants. CL analyzed the histological data. YT assisted with the anatomical data collection and analysis. JRM designed and supervised the study, analyzed the data, wrote and revised the manuscript.
This study was supported by the Center for Anatomical Science and Education, SLU School of Medicine.
These data were presented in part at the Annual Experimental Biology Meeting (FASEB J. (2019), 33: Suppl. 1, Abstract lb109).
The file contains Table S1 (Complete list of deleterious (pathologic) genetic variants associated with the current TF).
Table S1. Complete list of deleterious (pathologic) genetic variants associated with the current TF.
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