External genital gender ambiguity will affect the correct gender determination and cause severe psychological trauma. External genital gender dysphoria is mainly related to androgen abnormalities, and its clinical manifestations are diverse and the clinical diagnosis and differential diagnosis are complicated. Based on years of clinical practice, this data presents some diagnostic and differential diagnostic clues through the analysis of the etiology of external genital gender ambiguity, and briefly describes the progress of the relevant treatment methods. Data and methods I. Sources The root cause of external genital gender ambiguity lies in androgen abnormalities, i.e. androgen excess, androgen deficiency and abnormal gonadal differentiation (external genitalia appear female when androgen action is completely lacking, which is not external genital gender ambiguity). 450 patients with various sexual developmental abnormalities were admitted to the Reproductive Endocrine Unit of Peking Union Medical College Hospital from 1976 to 1996, among which there were The total number of cases with unclear sex of external genitalia was 105, accounting for 23.3%. In addition to the general medical history of the patients, attention should be paid to the history of the mother’s medication during pregnancy and the developmental and family history of the patients. In addition to the general body examination and pelvic examination, we should pay attention to the abnormalities of vulva and the development of height and breast in adults. Luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone, estradiol, and 17a-hydroxyprogesterone will be measured, and peripheral blood karyotype will be checked. Other tests include peripheral blood and gonadal SRY gene tests, vulvar skin 5a reductase and androgen receptor binding assay and androgen receptor gene analysis. Results and analysis Sexual developmental anomalies leading to external genital gender ambiguity were, in order of prevalence, congenital adrenocortical hyperplasia (52.4%), incomplete androgen insensitivity syndrome (26.7%), true hermaphroditism (12.4%), 45,X/46,XY gonadal hypoplasia (4.8%), testicular degeneration (2.9%) and exogenous androgen excess during early pregnancy ( 1.0%). (Table 1) Table 1: Classification of 105 cases of external genital gender ambiguity: Number of cases (N) Percentage (%) Androgen excess Congenital adrenocortical hyperplasia 55 52.4 Exogenous androgen excess during early pregnancy 1 1.0 Androgen deficiency Incomplete androgen insensitivity syndrome 28 26.7 Testicular degeneration 3 2.9 Abnormal gonadal differentiation True hermaphroditism 13 12.4 45,X/46,XY Gonadal hypoplasia 5 4.8 Total 105 100.0 I. Androgen excess 1. Congenital adrenocortical hyperplasia: It is the most common cause of female external genital gender ambiguity, and there were 55 cases in this group, which is an autosomal recessive disorder with sex chromosome 46,XX and normal ovaries with uterus and fallopian tubes in gonads. The main cause is the lack of enzymes in the synthesis of steroid hormones in the adrenal cortex and the production of excessive androgens, including 21 and 11 hydroxylase deficiency. The external genitalia show varying degrees of masculinization, and Prader classified the vulva into five types according to the degree of masculinization; mild cases have only a slightly enlarged clitoris, while severe cases may have a developed penis and scrotum that resemble a male, but no testes in the scrotum [1]. In males, congenital adrenocortical hyperplasia manifests as homosexual precocious puberty. 2. Excessive exogenous androgens during early pregnancy: In this case, the chromosome of the patient was 46,XX, and the mother took high doses of androgens during early pregnancy because she was eager for a male baby [2]. It showed a short penis, fused labia and no gonads in the scrotum (Figure 1). The degree of external genitalia masculinization is related to the timing, type, dose and duration of medication during pregnancy. External genitalia will be masculinized before fetal external genitalia differentiation, and androgen action is strong and the dose is high, masculinization is also serious. Androgen deficiency 1. Incomplete androgen insensitivity syndrome: This group of 28 cases is a common male monogenic sex development abnormality with karyotype 46,XY, bilateral gonads are testes, testosterone secretion is normal, but the normal effect of androgen is completely or partially lost due to androgen receptor abnormalities. The total loss of androgenic function manifests as female external genitalia but no uterus; partial loss of external genitalia may have stages similar to congenital adrenocortical hyperplasia (Figure 2). The androgen receptor gene is located on the long arm of the X chromosome (Xq11-12). Various deletions and mutations in the DNA-binding or androgen-binding regions of the androgen receptor gene have been found to be the main cause of the complete androgen insensitivity syndrome [3], whereas in the incomplete androgen insensitivity syndrome, most molecular defects in the coding region of the androgen receptor are not found, and it is possible that the alterations are mainly in the non-coding promoter region, the 3′ untranslated region or other It is possible that the alterations are mainly in the non-coding promoter region, 3′ untranslated region or other related transcriptional regulators. The diagnosis of incomplete androgen insensitivity syndrome depends on the determination of androgen receptor binding in vulvar skin [4, 5] and the analysis of androgen receptor genes and related transcriptional regulators [6]. In addition, males with a complete deficiency due to defective androgen synthesis will show a completely female external genitalia; if partially deficient, they will show a deficiency in masculinization and may have five enzymes involved in the synthesis of testosterone from cholesterol. We have collected 7 patients with 17α-hydroxylase deficiency, all with external genitalia presenting as female and without masculinization, all with complete deficiency [7]. There were no cases of partial deficiency of androgen synthesis in our group, which may be related to the fact that there is still no specific identification method for certain enzymes. 2. Testicular degeneration: it is relatively rare, and the three cases in this group, with chromosome 46,XY, showed external genitalia that had been affected by testosterone, with fusion of the labia into the scrotum, slightly enlarged clitoris, and urethral opening at the root of the clitoris, which is a manifestation of early male embryo, and its etiology is due to testicular degeneration during the embryonic period, which no longer secretes testosterone without further development of external genitalia. Any abnormalities leading to delayed, incomplete or asymmetrical testicular differentiation can lead to unclear gender of external genitalia. 1. True hermaphroditism: There are 13 cases in this group, which are characterized by gonads with both ovarian and testicular gonadal tissues. The gonads can be either ovaries or testes alone or ovaries and testes in the same gonad (omo-testis). In true hermaphroditism, the ovaries and testes are usually differentiated and functional at the same time, while the testes only affect the differentiation of the ipsilateral genitalia. If the gonad is ovotestis, the Mullerian ducts are mostly not inhibited. The morphology of the external genitalia is highly variable and generally presents as a dysplastic male with hypospadias and a unilateral scrotum and gonads (Figure 3). If there is testicular action during embryonic period, most of them live according to male life after birth due to the presence of penis, but most of them have breast development in adulthood and some of them may have menstruation or blood in urine according to month. The karyotype of true hermaphroditism is 46,XX in 90% of cases, but there may be 46,XY or other chimeric types. The etiology of true hermaphroditism is unknown, but mutations or translocations in the SRY gene have been suggested as a possible cause, but most tests have not confirmed this hypothesis. Some studies suggest that the nature of the gonads may be determined by the gonads rather than by the peripheral blood Y chromosome or SRY [8]. Definitive diagnosis relies on the finding of both gonadal tissues by dissection or laparoscopy. 2. 45,X/46,XY gonadal hypoplasia: In five cases with chromosome 45,X/46,XY, the patients had hypoplastic testes and striated gonads with typical Turner’s syndrome and, in many cases, an enlarged clitoris [9] (Figure 4). Such patients often have the product of Mullerian ducts, which may be due to Mullerian duct inhibitor (MIS) insufficiency or delayed secretion. Although these patients may have testicular tissue, it is not clear why testosterone and MIS are not sufficient at this time to cause normal external genital masculinization and cause Mullerian duct degeneration. One possibility is that when there is sex chromosome chimerism, the induction of differentiation in the testes is so delayed that sufficient testosterone and MIS are not produced, thus missing the optimal sensitive period for internal and external genital differentiation. Discussion Normal sexual differentiation is initiated by the SRY gene on the Y chromosome, which is located at the end of the short arm 1A1 of the Y chromosome, is present on the Y chromosome in all mammals, and is expressed in a tissue-specific pattern at the urogenital crest at the time of gonadal differentiation. Transfer of the SRY gene into female rats leads to the development of male somatic traits. Another important factor affecting sexual differentiation is MIS, a large molecular weight glycoprotein secreted by early differentiated testis support cells, and MIS causes Mullerian duct degeneration. transcription of the MIS gene is initiated by tissue-specific (urogenital crest) and properly expressed SRY protein, which binds to the promoter region of the MIS gene in a sequence-specific manner [10]. Thus SRY can be compared to a master switch for sexual differentiation and MIS is one of the early target genes of SRY. The presence of MIS and testosterone, as well as the action of dihydrotestosterone converted by 5a-reductase, are necessary for male genital differentiation. In turn, MIS, testosterone and dihydrotestosterone all need to pass through their receptors to act. Impairment of any of these components will result in varying degrees of abnormal external genital differentiation. If the diagnosis is unclear, the newborn should be referred to an experienced hospital as soon as possible so that the diagnosis can be confirmed as soon as possible. The history of medication use during pregnancy and family history should be carefully inquired. The size of the clitoris, the degree of labial fusion, and the location of the gonads should be particularly noted during the physical examination. The presence or absence of breast development and normal height in adult patients are important differentiating factors. The development of male external genitalia depends on local dihydrotestosterone, which causes the enlargement of the genital nodes into the penis, the fusion of the labia into the scrotum and the formation of the penile urethra, and the differentiation of the urogenital sinuses into the prostate. If androgen action is incomplete, the external genitalia will show only partial masculinization, such as a small penis, hypospadias, partially fused scrotum, or still have urogenital sinuses. An enlarged clitoris suggests that there has been androgenic influence in the body. In contrast, the degree of labial-scrotal fusion is related to the duration of androgenic action. If the female fetus is affected by increased androgens before 10-12 weeks of gestation, the external genitalia will be masculinized and may have a male penis with the vagina and urethra opening at the base of the penis and a partially fused scrotum. If androgens are elevated after 20 weeks of gestation, the external genitalia will have completed differentiation and the only masculine manifestation will be an enlarged clitoris. The site of the gonads is also helpful in the diagnosis. Since the ovaries do not descend below the external inguinal ring, if the gonads are found below the external inguinal ring, the gonads are either testicular or oviductal. In congenital adrenocortical hyperplasia, the ovaries do not enter the scrotum, which helps in the differential diagnosis. In adults with indistinct external genitalia, if there is breast development and the karyotype is 46,XX, it is suggestive of estrogenic effects from ovarian tissue and is indicative of congenital adrenocortical hyperplasia or true hermaphroditism; if the karyotype is 46,XY, it may be an incomplete androgen insensitivity syndrome. If the patient’s height is <150 cm, the presence of 45,X is suggestive and the diagnosis of 45,X/46,XY gonadal dysgenesis should be highly suspected. Chromosomal examination plays a key role in the differential diagnosis, e.g., testicular degeneration presents almost identically to external genital gender dysphoria due to exogenous androgen excess during early pregnancy, and karyotyping is the only way to differentiate. In addition, measurement of gonadotropins, testosterone/dihydrotestosterone, 17-hydroxyprogesterone, and electrolytes can assist in the diagnosis. The human chorionic gonadotropin (HCG) stimulation test helps to identify the diagnosis of 5a-reductase deficiency, androgen synthesis disorder, and incomplete androgen insensitivity syndrome. Dexamethasone test helps to differentially diagnose congenital adrenocortical hyperplasia. Ultrasound of the abdomen and scrotum helps to understand the nature and location of the genitalia. Testing and analysis of SRY, MIS and MIS receptors, androgen receptors, 5a-reductase, 21-hydroxylase and androgen synthase can be performed if available to detect mutations in genes to understand the molecular biological basis of the disease, and prenatal diagnosis can be made by molecular biology techniques for those with a family history. Laparoscopy and dissection combined with pathological examination can clarify the nature of the gonads and are of irreplaceable value in the diagnosis of true hermaphroditism and other diseases with unclear diagnosis. In addition, in addition to abnormal sexual development, attention should be paid to the differentiation from androgen-secreting tumors, which secrete significantly higher levels of androgens. The location and nature of the tumor can be determined by chromosomal examination, testosterone determination, pelvic examination, ultrasound and various imaging examinations, as well as laparoscopy or dissection. The purpose of treatment of unclear gender of external genitalia is to give the patient the most suitable social gender, to achieve the ability to marry and have sex, and to assist in completing childbirth as much as possible if there is a possibility of pregnancy. For newborns, the appropriate gender is chosen according to the nature of the cause and the specific situation of the external genitalia after the cause is clarified. For adults, the treatment is generally based on maintaining the previous social gender. The most common form of congenital adrenocortical hyperplasia is 21 hydroxylase deficiency, which accounts for more than 95% of cases [11]. 21 hydroxylase gene has been cloned and localized to the human leukocyte antigen locus on chromosome 6 [12], and 95% of patients with congenital adrenocortical hyperplasia have 21 hydroxylase gene deficiency. The point mutation or deletion can now be diagnosed as early as the blastocyst stage using its polymerase chain reaction (PCR) probe; early confirmation is important, as otherwise patients with the salt-losing phenotype can have a life-threatening condition. Definitive diagnosis in patients with a family history can begin in early pregnancy, and intrauterine steroid hormone injections are very effective in preventing external genital masculinization in the female fetus if started at 6 weeks of gestation. In the future, it will be possible to use normal embryo selection and implantation techniques as a treatment strategy. After birth, the secretion of ACTH can be suppressed by supplementing with adequate amounts of adrenocorticotropic hormones to inhibit excessive androgen production by the adrenal glands, maintain electrolyte balance and prevent premature epiphyseal healing. Growth and bone age should be noted during treatment. Surgical correction is required for external genital deformities, including clitoral reduction with preservation of clitoral vessels and nerves, and vaginal and vulvoplasty [13] (Figure 5). Pregnancy can be achieved in adulthood by regulating the dose of adrenocorticotropic hormone to induce ovulation. In our group of 32 patients with congenital adrenocortical hyperplasia, 26 cases were operated on, 19 had menstruation, and of the 9 married cases, 5 were pregnant and delivered 3 women and 2 men in normal health [1]. Abnormally positioned and underdeveloped testes are prone to tumors and should be removed once diagnosed, followed by hormone replacement therapy. In true hermaphroditism, normal gonads of the same sex as the social sex should be preserved during surgery, and the external genitalia can be considered orthopedic according to the social sex. In our hospital, 13 patients with true hermaphroditism lived as women, and after removal of testes and preservation of ovaries, 3 cases were married, 2 cases had children, and the rest had not yet reached childbearing age. For patients with androgen synthesis deficiency or incomplete androgen insensitivity syndrome, the choice of gender needs to be decided according to the degree of external genital malformation and the degree of response to androgens. In the stimulated state, the mean length of the penis in term neonates is 3.5 ± 0.5 cm, 2.8 ± 0.5 cm at 34 weeks, and the mean width is 1.1 ± 0.2 cm [14]. If the penis is <1.5 cm X 0.7 cm it is generally advocated to be raised as female. If the penis of the child is smaller than the standard and does not respond to androgens, it is advocated to live as a female, with early clitoral reduction, vulvoplasty and bilateral orchiectomy, estrogen replacement therapy starting at puberty, and vaginoplasty if necessary in adulthood. In contrast, children with a certain length of penis and responsive to large doses of androgens can live as males and be given androgens to enlarge the penis, while understanding the degree of sensitivity to androgens, and later need to correct cryptorchidism and external genitalia plastic surgery. In conclusion, for patients with unclear external genitalia gender, we should strive for early and accurate etiological diagnosis and timely treatment to prevent the occurrence and development of complications. Attention should also be paid to the protection of patients' privacy and the psychological treatment of patients and their families.