For the diagnosis of a baby born with an ambiguous genitalia or not clearly a boy or a girl, complementary clinical, genetic and hormonal diagnoses are needed first. To the best of our knowledge, this is the largest retrospective study on DSD patients in Turkey. In the present study, the sex ratios (male/female ratio) of the patients were equal. This shows that sex irregularities do not show phenotypic sex discrimination and affect male and female sex equally. 64.9% of the patients had normal karyotype, their genotype and phenotypic sex were compatible, but 35.1% had abnormal chromosomes (92.9% numerical and 7.1% structural). The condition in patients with a normal karyotype may be due to a difference in genes and how the body responds to sex hormones or both. In addition, DSD may occur as a part of some syndromes, or it may occur due to imbalances affecting autosome or X-linked genes involved in the sex development mechanism (Kim et al.,2015).
Patients Presenting With Ambiguous Genitalia
AG patients, who constitute the largest group (53.8%) of our patient population, are a heterogeneous group formed due to different chromosome damages. These account for over 50% of all cases of genital ambiguity in the neonatal period (Thyen et al.,2006). The incidence of mixed gonadal dysgenesis worldwide is 1:10,000, but varies considerably among different populations (Pang et al.,1988; Skakkebaek 2003). While it usually results in the presence of a missing or extra sex chromosome in patients with AG, the cause cannot be determined in some cases. On the other hand, 61.9% of our AG patients had normal karyotype, and CA was found in 38.1% of patients with AG. In a similar study conducted in Morocco, it was reported that 78.31% of patients with AG had normal karyotype, and 7.1% of patients had CAs (Elkarhat et al.,2019). In other studies, CA was reported to be 4.4% in Saudi Arabia, 10.1% in Oman and 13.6% in Turkey (Balkan et al.,2010; Husain and Zaki 1999; Al-Alawi et al.,2016). According to this ratios, according to the phenotypic sex of our patients, the male and female ratios were very close to each other and the CAs ratio was higher. The difference in these rates may have changed according to clinical diagnostic criteria and analysis methods. Unfortunately, we could not interpret the genetic sex of the newborn 5 patients as the phenotypic sex was uncertain. At the same time, phenotypic sex and genotypic sex were not compatible in 19.0% (de la Chapelle 1972) patients with AG. Incompatibility of genotypic and phenotypic sex is a very rare condition. It is estimated that 1 in every 20,000 to 30,000 men has the karyotype XX (Claire 2010; Witchel et al.,2019). It is known that chromosomal irregularities, hormonal deficiency, gene transfer and gene mutations are among the reasons for this. Sometimes, the cause of AG cannot be determined. Deficiency of male hormones in a genetically male fetus causes the disease, and a genetically female fetus that is exposed to male hormones during development also develops AG.
XX sex change is a rare condition in which an individual with the XX karyotype has phenotypically male characteristics. Translocation and expression of the SRY gene into female individuals with XX results in male or indeterminate genitalia. Mutations in the SRY gene are identified in approximately 15% XY females. Missense mutations lead to XY gonadal dysgenesis. Evaluation of those positive for SRY is more difficult due to the multitude of various etiologies. Congenital adrenal hyperplasia can also cause a fetus with an XY karyotype. Etiologies of such patients include 17 alpha-hydroxylase, 3 beta-hydroxysteroid dehydrogenase, steroid acute regulatory protein, P450 oxidoreductase, and P450 side chain degrading enzyme deficiencies. In some patients, the diagnosis can be made by measuring the concentration of the anti-Müller hormone. SRY-negative XX males, which can be caused by a mutation in a gene on the autosomal or X chromosome, are less common and the masculinization of these individuals can be variable (de la Chapelle 1972). XX males have normal internal and external genitalia, and males with external ambiguities can be males with both internal and external genital ambiguities. Most XX men have small testicles and more undescended testicles or urethral opening (hypospadias) on the underside of the penis than normal XY men. The degree of development of the male phenotype in XX males varies even among SRY-positive individuals. Sometimes the presence of the SRY gene may cause ambiguity in the internal and/or external genitalia (Kusz et al.,1999). It is thought that X inactivation in XX men may explain the genital ambiguities and incomplete masculinization observed in SRY positive XX men (Kusz et al., 1999; Bouayed et al.,2003). Masculinization of SRY-negative XX males depends on which genes are mutated and at what developmental stage these mutations occur (Rajender 2006). Some XX males lack the SRY gene and the male phenotype may be caused by another gene on autosomal chromosomes.
Fertilization errors can result in the formation of more than one cell line (mosaicism) in an individual. We found different sex chromosome mosaicism in 11 (17.5%) patients with AG. With this, 5 patients (5.9%) with AG, HG and IS had 46,XY/45,X karyotype, and one patient (1.2%) had 45,X/46,XX karyotype. The 46,XY/45,X karyotype is the most common karyotype in newborn children with mixed gonadal dysgenesis. Most of these patients have dysgenetic testes, contralateral striated gonads, and a 45,X/46,XY karyotype (Telvi et al.,1999). Its pathogenesis may possibly be related to the expression of a defective gene that controls testicular differentiation. Most 45,X/46,XY mosaic patients with mixed gonadal dysgenesis also have a structural abnormality of the Y chromosome. An increase in the long arm of the Y chromosome was detected in one of our patients with AG. In other studies, while 25% of patients with mixed gonadal dysgenesis have 45,X mosaicism and dicentric Y chromosome, high frequency Y chromosome microdeletions have been reported in male patients with 45,X/46,XY mosaic (Tuck-Muller et al.,1995; Leilei et al.,2020). This mosaicism is an important genetic factor for spermatogenesis failure, and the most common sex CA found in infertile men. Y chromosome mosaicism/chimerism can result in a broad range of reproductive phenotypes. Males with mixed gonadal dysgenesis have varying degrees of under-masculinization and infertility (Lee et al.,2006). In addition, these patients are at great risk for gonadal tumors. We reported 45,X/46,XX mosaicism in two (2.4%) female patients with AG and GH. In other studies, it was reported that 64.89% of patients with Turner syndrome (TS). TS had mosaicism (45,X/46,XX) with a rate of 15–65% (Rabah et al.,2012; Huang et al.,2002). Patients with mixed gonadal dysgenesis may have a normal female appearance or have the TS phenotype, males with hypospadias, and male or female pseudohermaphrodism findings.
In the present study, we found an unexpected sex chromosome mismatch chimerism (46,XX/46,XY) cytogenetically in 7 (6.0%) phenotypically abnormal individuals presenting to the clinic with AG and IS complaints. But we could not perform molecular genetic analysis. We also reported both 46,XY/46,XX/46,Xi(Xp) chimerism and short-arm isochromosome of the X chromosome in a male patient. Mosaicisms have various physiological roles and pathological consequences. The cell lines of most such cases show sex chromosome incompatibility with the 46,XX/46,XY karyotype. Sex chromosome mismatched chimerism 46,XX/46,XY is associated with mosaicism or chimerism and is rarely found in people with a phenotypically normal appearance. Chimera is defined as the fusion of two different zygotes in an embryo, while mosaic is caused by a mitotic error in a single zygote. It is very difficult to distinguish these incompatibles. The physical findings of patients with this condition are very variable and can range from normal male or female genitalia to ambiguous genitalia to varying degrees. Chimeric individuals with sex chromosome mismatch have been described mostly because of gonadal dysplasia and hermaphroditism and have been reported to be responsible for approximately 13% of true hermaphroditism cases (Danon and Friedman 1996; Schoenle et al.,1983). Same-sex chimeras (XX/XX and XY/XY tetragametik chimeras) are rarely detected because they are not usually sex-chromosome mismatches and do not usually show a clinical effect (Binkhorst et al.,2009). It is difficult to determine the exact incidence of these rare chimeric. The phenotypic ranges of these patients can range from normal male, female external genitalia and ambiguous genitalia, and these patients are usually infertile.
The physical findings of patients with chimerism are very variable because there are many different mechanisms involved in its formation. Some mechanisms have been suggested for whole-body chimerism; it can occur as postzygotic fusion (tetragametic) of two independently fertilized oocytes, fertilization of gametes undergoing parthenogenetic division, or fertilization of the first/second polar body of an oocyte by two sperm followed by fusion of two zygotes (Strain et al.,1998; Niu et al., 2002; Sudik et al.,2001; Strain et al.,1995; Gardner and Sutherland 2004). Moreover, it can also arise from a 46,XX/46,XY mosaic karyotype consisting of a single zygote, as well as from a diploid (46,XY) or an aneuploid cell (47,XXY or 48,XXYY) (Malan et al.,2006). Therefore, chimerism can be distinguished from mosaicism because of its genotypic differences. In some patients, potential chimerism can be determined cytogenetically, and it is possible to determine the mechanism of chimerism by analyzing genotype patterns. Chimeric individuals with sex chromosome mismatch have been described mostly because of gonadal dysplasia and hermaphroditism and have been reported to be responsible for approximately 13% of true hermaphroditism cases (Danon and Friedman 1996: Schoenle et al.,1983). It has been reported that 10–33% of patients with ovotesticular sexual development disorder have 46,XX/46,XY karyotype (Danon and Friedman 1996; Krob et al.,1994; Hughes 2008).
Y chromosome structural rearrangements are higher in newborns. In the present study, we reported an increase in the long arm of the Y chromosome in 2 male patients with AG and IS, and a short arm deletion of the Y chromosome in a female patient with HG. Structural abnormalities of the Y chromosome, usually deletions, can cause serious congenital anomalies and significant mental/physical defects. Since genes on the long arm of the Y chromosome are responsible for spermatogenesis, microdeletions are associated with the complete absence of spermatozoa and reduced sperm count (Ferlin 2006; Vogt et al.,1996). AG may also be associated with autosomal abnormalities. We also found both sex and autosomal structural anomalies in one patient with AG [46,XYqh+,22s+]. Double CAs containing one sex chromosome and one autosomal chromosome have been reported previously. Dyspermic chimerism was reported in two cases; one of them was a stillborn dysspermic male with a balanced translocation [t(14;20)] carrier (Tsuji et al., 1993). We also described Robertsion translocation (14;15) in one of our patients with HG. One of our male patients with AG had gonadal cancer. It is known that some sexual development disorders are associated with certain types of cancer. The expression of SRY in other somatic tissues such as adipose, esophagus, thymus, adrenal glands, kidneys, as well as in some cancer cell lines suggests that it has functions beyond sex determination (Maan et al., 2017; Meyfour et al.,2017). It is also known that patients with mixed gonadal dysgenesis and partial androgen insensitivity have a higher oncologic risk. The highest risk of gonadal tumors is found in testicular specific protein Y positive gonadal dysgenesis and partial androgen insensitivity of the internal gonads, while the lowest risk is found in ovotestis and complete androgen insensitivity syndrome (Scully 1970). Men with mixed gonadal dysgenesis are at risk for gonadal tumors.
Patients Presenting With Hypogonadism
Hypogonadism can occur as a result of either the testicles or ovaries not working properly, or problems with the pituitary and hypothalamus (when the sex glands produce little or no hormones). This condition is responsible for the signs and symptoms observed in both men and women (Qaseem et al.,2020). The most common genetic disorders that cause primary HG are TS and Klinefelter syndrome (KS). The activity of sex hormones can cause defective primary or secondary sexual development in adults. Clinical features of XX or XY dysgenesis include primary amenorrhea, little or no pubic hair, and absence of breast development. Both XX and XY gonadal dysgenesis can occur individually, they are genetically heterogeneous and in fact most cases remain unexplained. We found CAs in 3 (4.8%) of patients with HG. Two patients had structural irregularities of the sex chromosome and one patient had an autosomal Robersonian translocation. One patient also had the KS karyotype. Long-armed isochromosome of the X chromosome [46,X,i(Xq)] was found in one of our patients who was referred to us with the suspicion of HG. Also, both long arm isomer and long arm duplication of X chromosome were found in a 45,X/46,XX mosaic female [45,X/46,X,iso(Xq),dup(Xq11-q13)]. This karyotype is the most common of the Turner variants, and one of the most observed features of this condition is haploin-sufficiency of the SHOX gene (Gürsoy and Erçal 2017). Rarely, 0.3–0.9% of males with X chromosome polysomies or X-Y translocations cause KS with isochromosome Xqi(Xq) (Arps et al., 1996; Kalousek et al.,1978). KS is also the most common cause of HG and infertility in males. We reported a rob(14;15) translocation in a patient with HG. Robertsonian translocations occur with a prevalence of about 1/1000 in the general population (Gardner and Sutherland 1996). Individuals with reciprocal translocations have a significantly increased risk of infertility and miscarriage due to unbalanced gamete formation. The most common Robertsonian translocation observed in infertile males is t(13q;14q). Other D/D Robertsonians are much less frequent and rob(14;15) can be expected to have risks (Gardner and Sutherland 1996).
Patients Presenting With Intersex
Intersex is the condition of abnormally having both male and female characteristics of genital/sex organs and reproductive structures. In this disease, there is a mismatch between the appearance of the external genitalia and the internal sex organs or genetic sex due to the deterioration in the sex determination steps. We found CAs in 6 (60.0%) of 10 patients diagnosed as IS. Three of these patients had phenotypic gender and genotypic gender mismatch. In the other 3 patients; Yq + was found in one, mosaic 45,X/46,XY in one, and chimerism 46,XY/46,XX in one. 46,XX/46,XY karyotype was found in 10–33% of ovotesticular patients (Krob et al.,1994; Hughes 2008). This is a genetically heterogeneous clinical condition in which both ovarian and testicular tissue occur (Krob et al.,1994). IS is evaluated in four categories as 46,XX IS, 46,XY IS, true gonadal IS, and complex/ambiguous IS. One of our patients had XX intersex and two of them had XY IS. An XX IS person has female chromosomes and female ovaries, but has external genitalia that look like a male. This is usually due to exposure of a female fetus to excess male hormones before birth. The XY IS person has male chromosomes, but the external reproductive organs are not fully formed, indistinct, or clearly female, and the testicles may be normal, defective, or absent. This condition is also referred to as 46,XY with undervirilization. An IS person may appear to be female on the outside and be born with a mostly male anatomy inside. Or, as in our patients, it can be born with genitals that appear between normal male and female types. Sometimes, a person may be born with mosaic genetics, as in our two patients, some cells have XX chromosomes and some have XY chromosomes. Although IS is a congenital condition, its anatomy may not always be revealed at birth. Sometimes, the person is not found until they have IS anatomy, reach puberty, become a sterile adult, and die or have an autopsy performed. One of our patients was 30 years old and had just been discovered. In fact, some people unwittingly live and die with IS anatomy.
Patients Presenting With Hypospadias
Hypospadias is one of the most common genital malformations in male newborn children, affecting about one of every 250 males at birthand and results from an abnormal penile and urethral development (Snodgrass 2005). It is thought to be due to a combination of genetic, endocrine and environmental factors. In about 90% of cases, the urethral opening is located on or near the glans penis. In most cases, the foreskin is less developed, does not completely surround the penis, and exposes the underside of the glans (King and Beasley 2012). We found 45,X/46,XY mosaicism in one of the two patients with HS and inv(9)(p12;q13) in the other. It may occur as a result of mutations in the genes involved in the interactions between the mesenchyme and the urothelium. Mutations in the SOX9, DMRT1 and GATA4 genes can cause male sex differentiation disorders often associated with testicular dysgenesis (Leipoldt et al.,2007; Maciel-Guerra et al.,2008). The duplicated SOX9 gene can lead to the formation of penoscrotal HS in patients with mosaicism 46,XX and 46,XX dup(17q) (Huang et al.,1999). However, the finding of 46,XX male HS patients with or without the SRY gene suggests the presence of other virilizing genes. Gonosomal abnormalities have been found in approximately 7% of patients with HS. These include Klinefelter syndrome, 47,XXY, 48,XXYY and some mosaics (such as 45,X/46,XY; 45,X/46,XYq-; 45,X/46,X,idic(Yp) and 45,X/69,XXY). Abnormal genital development in these patients may be related to a dosage effect of the male gene (Moreno-Garcia and Miranda 2002; Sinisi et al.,2003). Autosomal dominant forms of syndromic HS may result from mutations in autosomal genes involved in early genital development. It can also be associated with various CAs such as gonosomal mosaicism and autosomal deletions.
Since there is neither gain nor loss of genetic material in inversions, they are said to have no pathological significance. The relationship between the inv(9) variant and clinical pathologies is still unclear. Some researchers believe that it is a normal variant, while others associate it with infertility and various pathologies in the obstetric history. However, inv(9)(p12;q13) karyotype was found in our patient, and we do not know whether this inversion causes a genetic anomaly. In a similar study, it was reported that inv(9)(p12;q13) causes HS, spontaneous abortion and infertility in couples (Vijay et al., 2016). Also, the most widely recognized reversal is 46,XY,inv(9)(p11;q13) in the oligozoospermic infertile male. inversion 9(p12;q13) causes spontaneous abortions and couple infertility (Vijay et al., 2016; Demirhan et al.,2008).
Patients Presenting With Testicular Feminization (TF)
In the present study, one of the two patients with TF was phenotypically female but with XY genotype, and the other was female in phenotype and genotype. TF is a rare disease in phenotypically normal women with adequate breast development, normal external genitalia, vagina of variable depth, uterus, absence of pubic and axillary hair, or infrequent. This syndrome occurs with a frequency of approximately one in 20,000–62,400 and occurs due to hormonal resistance due to androgen receptor dysfunction (Legato Marianne 2017; Gurer and Demirkiran 2000). These patients have a male karyotype and negative sex chromatin and may have undescended testicles, inguinal or labial (Zorlu et al., 2001). In most cases, XX or XY gonadal dysgenesis remains genetically unexplained. Although the absence of the SRY gene is known in approximately 20 percent of those with 46 XX testicular sex disorder, the cause of the disorder is unknown in these individuals. Individuals with SRY-negative 46,XX testicular DSD are more likely to have AG than SRY-positive individuals (Vorona et al.,2007). Female patients with XY have primary amenorrhea, sexual ambiguity, facial dysmorphism, or psychomotor retardation. The cause of gonadal dysgenesis in these patients may be either loss of SRY or a mutated SRY gene.
Patients Presenting With Vaginal Hypoplasia
We detected 46,XX/45,X mosaicism in a female patient with VH. This anomaly is a birth defect characterized by an underdeveloped or reduced size vagina, and occurs in about 1 in 5,000 to 7,000 women. Although this condition is present at birth, it often remains undiagnosed until a teenager reaches puberty and realizes they are not menstruating. Indeed, our patient was a 16-year-old girl. Among them, the frequency of menstruation reaches 20%, but this rate does not exceed 3% in pure Turner patients (Prior et al.,2000). Our patient did not show the Turner phenotype.