Differences/disorders of sex development (DSD) is the term used for individuals with congenital discrepancies between the sex chromosomes, gonadal tissue, and external genitalia. The medical approach to DSD depends on both the genital appearance and the specific type of DSD. Mixed gonadal dysgenesis (MGD) is a form of DSD characterized by atypical genital appearance and chromosomal mosaicism, arising when an individual possesses more than one chromosomally distinct cell line due to mitotic nondisjunction during the post-zygotic period. Because of the diversity in phenotypic presentation, the true prevalence of MGD remains unclear [1].

Notably, 45,X/46,XY mosaicism is a rare chromosomal abnormality, occurring in approximately 1.5 per 10,000 newborns, and is a significant contributor to ambiguous genitalia [2]. The clinical presentation of individuals with 45,X/46,XY mosaicism is highly variable, spanning from females (with or without Turner syndrome [TS]) to phenotypically normal males who exhibit some degree of genital ambiguity. Gender assignment at birth can be male or female, depending on the external appearance of the genitals [3]. Other uncommon mosaic karyotypes have also been reported, such as 45,X/47,XYY, 45,X/46,X,r(Y), 45,X/46,X,idic(Y), 45,X/46,Xdel(Y), 45,X/45,Y/46,XY/46,YY/47,XYY, among others [3-5]. Each of these mosaic patterns is associated with unique phenotypic features, including genital ambiguity, which can pose significant challenges for clinicians. In this context, we describe an 18-year-old female with primary amenorrhea and delayed puberty, who was found to have the exceedingly rare mosaic karyotype 46,XY/45,X/46,X,r(Y)(p?11.2q?12).

An 18-year-old phenotypically female patient was referred to our clinic with a complaint of primary amenorrhea. Clinical, physical, and histopathological assessments were performed by experienced clinicians, surgeons, and pathologists. Cytogenetic analysis was also conducted. Metaphase chromosomes from peripheral lymphocytes were analyzed using standard trypsin and GTG-banding techniques, achieving 500 to 550 band resolution for cytogenetic analysis. As per standard protocol, 50 random metaphase spreads were evaluated for each patient. Karyotypes were verified in accordance with the latest International System for Human Cytogenetic Nomenclature (ISCN 2020) guidelines for human chromosomes. Additionally, the presence of the Y chromosome sex-determining region Y protein (SRY) gene was tested by polymerase chain reaction (PCR) amplification on blood DNA extracted using specific primers. The patient provided informed consent for the use of her disease information, and the study was approved by the Ethics Committee of the Iran University of Medical Sciences, Tehran, Iran (IR.IUMS.REC.1403.341).

An 18-year-old female was referred to our endocrine clinic for the evaluation of primary amenorrhea. She was born at term via vaginal delivery. During the perinatal period, there was no history of maternal medication use. She is the only daughter of parents in a non-consanguineous marriage (Figure 1). There is no family history of irregular menstruation or premature ovarian failure. She experienced no significant medical issues or hospitalizations during infancy. After completing her education without interruption, she currently works as an English teacher. Socially, she maintains harmonious relationships with colleagues and students.

Pubarche began at the age of 9. Approximately 3 years ago, due to delayed puberty, she was evaluated by a gynecologist who initiated estrogen therapy to promote breast development. After 1 year, treatment was switched to a combination of estrogen and progesterone, resulting in regular menstrual cycles, which occur only with consistent hormonal therapy. She also has a history of primary hypothyroidism, which is controlled with levothyroxine.

On examination, her weight was 55 kg and height was 157 cm, with a mid-parental height of 162 cm. There were no dysmorphic features or prominent stigmata of TS, such as webbed neck or cubitus valgus. The external genitalia appeared predominantly female, with a normal clitoral size (approximately 14 mm) and absent labia minora. There was no evidence of virilization, hirsutism, or acne.

Hormonal blood testing performed after discontinuation of hormonal therapy revealed hypergonadotropic hypogonadism (Table 1). Ultrasonography showed a small uterus measuring 28×8×5 mm. Gonads were not visualized, and no renal abnormalities were detected. Magnetic resonance imaging confirmed the presence of a small uterus and gonads (Figure 2). Echocardiography, performed to screen for cardiac malformations, identified a bicuspid aortic valve (BAV) without evidence of aortic root dilatation. Audiometric evaluation demonstrated normal hearing.

The patient identified as female and expressed no concerns regarding her gender identity. Following thorough counseling with her and her parents about infertility and the risk of malignancy due to the presence of Y chromosomal material, she underwent laparotomic gonadectomy. Hormonal therapy was discontinued approximately 3 months before surgery to reduce thromboembolic risk. Intraoperatively, streak gonads were resected. Pathological examination revealed a portion of fallopian tube and remnants of epididymis with foci of gonadoblastoma (GB) and dysgerminoma-like tissue, with a tumor size of 1 cm within the dysgenetic gonad (Figure 3). On the contralateral side, fragments of fallopian tube with isolated primitive epididymis-like tissue within a fibrotic stroma were noted. Pathological findings were compatible with gonadal dysgenesis.

Despite the presence of primitive gonads, this unique mosaic karyotype could explain the observed female phenotype. Following surgery, hormone replacement therapy (HRT) was resumed. Tumor markers including human chorionic gonadotropin, alpha-fetoprotein, and lactate dehydrogenase were undetectable. She remains under regular follow-up for HRT and monitoring of the aortic root dimension (current aortic size index: 1.5 cm/m²). She was also counseled that in vitro fertilization with donor oocytes could be considered if she wishes to pursue pregnancy in the future.

Chromosomal analysis revealed three distinct cell types by GTG-banding (Figure 4). Fifty metaphase spreads were examined. The karyotype was designated as 46,XY (68%)/45,X (18%)/46,X,r(Y)(p?11.2q?12) (14%). The presence of the SRY gene was confirmed by gene analysis.

MGD characteristics include individuals with chromosomal mosaicism, atypical gonads, and a diverse range of internal and external reproductive organs. This diversity can result in a wide spectrum of phenotypes, ranging from males with normal appearances to females with TS-like features [6].

Among individuals with a 45,X/46,XY karyotype, phenotypic presentations include hormonal imbalances and gonadal developmental abnormalities, such as sexual ambiguity, which constitute approximately 60% of cases [5,7,8]. In males, phenotypic variability may extend from infertile men with otherwise normal external genitalia to those presenting with genital ambiguity and short stature [9]. Females with this mosaic pattern frequently present with gonadal dysgenesis and somatic dysmorphic features. Some cases lack a uterus and ovaries, whereas others, despite hypogonadism, may retain a uterus [10,11].

Based on previous studies, the phenotype of patients with sex chromosome mosaicism depends on several factors: (1) the distribution of mosaicism across various tissues, including both blood and gonads. Gonadal karyotyping is crucial for determining sexual differentiation status, regardless of the mosaicism percentage or the predominance of the 46,XY cell line with SRY present in lymphocytes [12,13]. (2) The structure of the rearranged Y chromosome and the presence of Y-chromosomal materials, including the SRY gene and other spermatogenesis-related genes, can significantly impact gonadal development [14,15]. (3) Mutations affecting the SRY gene or other essential testicular differentiation genes. The presence of the SRY gene alone does not guarantee function, as its expression and activity can be impaired by mutations, potentially preventing testicular development and resulting in phenotypic variability [15-17]. Additionally, certain challenges must be addressed due to discrepancies such as varying proportions of sex chromosomal cell lines in different tissues. Some studies suggest that the percentage of the 45,X cell line may influence gonadal determination, though this correlation remains inconsistent in several cases of 45,X/46,XY mosaicism [7,18-22]. Moreover, while the structure of the rearranged Y chromosome could theoretically affect phenotypic outcomes, one study in patients with a 45,X/46,XY mosaic pattern found that the sex of rearing was independent of the specific Y breakage site [23].

In this report, we present an 18-year-old female with primary amenorrhea, delayed puberty, and a hormonal profile indicative of hypergonadotropic hypogonadism, as well as a rare mosaic karyotype comprising a high percentage of 46,XY cells and minor populations of 45,X and 46,X,r(Y) cells. This rare karyotype (mos45,X/46,X,r(Y)/46,XY) has previously been reported in females with Turner features during the neonatal period [24]. Notably, our patient did not display dysmorphic or apparent Turner features.

The formation of male gonads is determined by genes on the Y chromosome, including the sex-determining gene (SRY) and spermatogenesis genes. Loss of the Y chromosome in the X monosomy cell line can lead to streak gonads [12]. Streak gonads are incapable of secreting testosterone, anti-Müllerian hormone (AMH), and α-inhibin [25]. In our patient, insufficient testosterone secretion likely resulted in undeveloped epididymis and Wolffian duct, as well as female-appearing external genitalia. Furthermore, a lack of AMH secretion may have permitted development of Müllerian duct structures, such as the fallopian tubes and uterus.

Our patient’s karyotype demonstrates that male sexual differentiation did not predominate, despite a higher proportion of cells carrying Y chromosomal material (46,XY and 46,X,r(Y)) in lymphocytes. Several explanations may underlie this discrepancy. Importantly, the karyotype and SRY gene presence were determined from blood rather than gonadal tissue. Existing research indicates no direct correlation between the distribution of cell lines and the diversity of gender phenotypes [7,18-22].

The risk of GB in patients with 45,X/46,XY mosaicism appears to be associated with female phenotypes, and is higher in such individuals. Given the malignancy risk in these patients, early prophylactic gonadectomy is recommended [26]. Accordingly, gonadectomy was performed for our patient. Histopathology confirmed the presence of GB and dysgerminoma. Systematic reviews indicate that germ cell tumors (GCT) in patients with DSD are more common when gonadectomy occurs after age 15. Although five-year recurrence-free and overall survival rates for GCT in DSD patients remain good, these outcomes are worse than for ovarian or testicular GCTs [27].

A second concern in our case was the presence of a BAV, a condition that can progress to aortic aneurysm and valvular dysfunction. TS is the genetic syndrome most frequently associated with BAV. Reduced X chromosome copy number may predict BAV formation. While our patient did not display other TS features, the reduced number of X chromosomes may explain this cardiac finding [28]. BAV, hypertension, aortic coarctation, and aortic dilation are cardiovascular anomalies observed in approximately 20% to 40% of people with MGD [29]. Since it is reported that patients with 45,X/46,XY karyotypes may develop complications similar to those seen in TS [1], we recommended repeat cardiology evaluation and cardiac imaging every 5 to 10 years, following TS management guidelines [30]. As mortality is higher in patients with 45,X/46,XY karyotypes, especially in females and those with a Y chromosome [31], our patient needs long-term follow-up. The most common causes of mortality in individuals with this karyotype include congenital abnormalities, cardiovascular problems, and endocrinopathies.

Our study is limited by the absence of karyotyping in gonadal tissue to determine the percentage of mosaic cell lines and SRY distribution. Although the SRY gene was detected, full gene sequencing would be required to identify possible mutations. To detect minor genetic abnormalities in the Y chromosome, karyotyping is insufficient; PCR assays and molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) or comparative genomic hybridization (CGH) arrays are necessary for precise delineation of Y chromosome breakpoints. These limitations preclude definitive correlation between chromosomal mosaicism and gender phenotype.

In summary, complex cases with mosaic patterns may display a broad array of phenotypic features, from apparently normal males with variable external genitalia to females with or without TS characteristics. These discrepancies are not determined solely by the number of mosaic cells or the site of Y chromosome breakage. Therefore, in cases of primary amenorrhea with discordant genotype and phenotype, a multidisciplinary approach is crucial for accurate sex determination and patient management.