Patients with abnormal sexual differentiation and development are commonly seen for primary or secondary amenorrhea, external genital malformations, pubertal failure, or height abnormalities. Because of the complex and variable clinical manifestations and multiple etiologies, there are difficulties in their diagnosis and differential diagnosis. In addition to some specific manifestations that suggest diagnosis, the diagnosis and differential diagnosis depend on gynecological endocrine measurements, and some diseases can only be diagnosed through surgery and pathological diagnosis. Sexual differentiation and developmental abnormalities are classified as sex chromosome abnormalities, gonadal developmental abnormalities, and sex hormone and functional abnormalities according to three key components of sexual differentiation and development: sex chromosomes, gonads, and sex hormones [1]. In addition, the process of sexual differentiation and development is influenced by a variety of factors at different levels, and abnormalities in any one of them during the differentiation and development and interaction of hypothalamus-pituitary-target glands (ovaries, adrenal glands and thyroid) – target organs can lead to abnormal manifestations of sexual differentiation and development. In addition, endocrine hormones need to act through their specific receptors, and abnormalities in various receptors can also lead to different abnormalities in sexual differentiation and development. The measurement and analysis of gynecological endocrine of abnormal sexual differentiation and development are based on the two axes of sex chromosomes, gonads, sex hormones and hypothalamus, pituitary, target glands and target organs. 1.Sex chromosome determination The normal human karyotype is 46XX or 46XY. Sex chromosome abnormalities include abnormalities in the number and structure of sex chromosomes. 1.1 Sex chromosome number abnormalities: including increase or decrease in the number of X or Y chromosomes, such as 45X congenital ovarian hypoplasia (Turner syndrome) with one X chromosome missing, and superfemales with more than two X chromosomes, 47XXX, 48XXXX. in male karyotype, additional sex chromosomes have a greater impact, regardless of the increase of X or Y chromosomes, which may seriously affect testicular development and cause changes in sex characteristics and signs, such as 47XXY in Creutzfeldt-Jakob syndrome. 1,2 Chromosomal structural aberrations: chromosomes are affected by various factors during division and break, and the broken segments may be connected to each other in different ways, forming a variety of chromosomal structural aberrations. Chromosome structural aberrations are essentially additions, deletions, or positional changes of genetic material or genetic information, including various deletions and translocations. Abnormalities in the number and structure of sex chromosomes can exist alone, together, or in combination with normal ones, such as 45X/46XY gonadal dysgenesis and various chimeric types in Turner syndrome and true hermaphroditism. The determination of sex chromosomes is the key first step in identifying sexual differentiation and developmental abnormalities. 2.Hormone measurement The raw materials for the synthesis of sex hormones are derived from circulating or locally synthesized cholesterol. Patients with abnormal sexual differentiation and development mainly show a deficiency or excess of certain sex hormones, and the hormones generally measured include gonadotropins (mainly FSH and LH) and gonadotropins. The latter refers to estrogen, progesterone and androgens. Estrogen is mainly secreted by the ovaries, with small amounts secreted by the testes and adrenal glands. Androgens are mainly synthesized and secreted by the testes, with small amounts also secreted by the adrenal glands and ovaries. Therefore, after understanding the results of sex chromosomes, the abnormalities of sex hormones can be classified into hormone deficiency and hormone excess. (1) Androgen excess: refers to 46XX patients, commonly in congenital adrenocortical hyperplasia, true hermaphroditism and exogenous androgen excess. In congenital adrenocortical hyperplasia, 17 hydroxyprogesterone and androstenedione are significantly increased. (3) Estrogen deficiency: in 46XX patients, estrogen deficiency combined with increased gonadotropin levels suggests gonadal hypoplasia (e.g., XX simple gonadal hypoplasia); (4) Estrogen deficiency: in 46XY patients, estrogen deficiency combined with increased gonadotropin levels suggests gonadal hypoplasia (e.g., XX simple gonadal hypoplasia). (3) Estrogen deficiency: in patients with 46XX, estrogen deficiency combined with increased gonadotropin levels suggests hypothalamic-pituitary abnormalities; (4) Estrogen excess: rare, seen in patients with 46XY true hermaphroditism or androgen insensitivity syndrome; in addition, the differentiation and development of the external genitalia depends on the local conversion of testosterone to dihydrogenase by the enzyme 5a reductase. 5a reductase to dihydrotestosterone; therefore, abnormalities of 5a reductase can also lead to abnormal differentiation of the external genitalia. When androgen action is insufficient, the external genitalia will have only partial masculinization, such as small penis, hypospadias, partial fusion of scrotum, etc., and some may have blind end vagina, resulting in ambiguous external genitalia gender. In this case, some functional tests are needed to identify the cause. 3.Functional test Endocrine function test can reflect the functional status of endocrine gland, divided into stimulation or excitation test and inhibition test. The stimulation test observes whether the response of the stimulated gland is normal, while the inhibition test observes whether the gland with elevated function can be inhibited. The ability to stimulate and be inhibited indicates that there is normal positive and negative feedback function. (1) GnRH stimulation test: when LH, FSH and estrogen or androgen are low, it should be distinguished between hypothalamic GnRH deficiency or pituitary secretion deficiency, and intravenous GnRH injection test can be used. If the LH value rises 2-3 times, it is a normal response, which is seen in hypogonadotropic hypogonadism with loss of smell (such as Kallmann syndrome); if LH does not change or rises slightly and FSH changes little, it is no response or low response, which suggests abnormality of pituitary tissue itself (such as injury) or abnormal GnRH receptors. (2) ACTH excitation test and dexamethasone inhibition test: androgen excess, either from the ovaries or testes, or from the adrenal glands. ACTH excitation test and dexamethasone inhibition test can be used to identify the source of androgens. (3) hCG stimulation test: hCG has LH activity and can be used to test the function of testicular interstitial cells. It helps to understand the cause of sex hormone deficiency and can be used to identify 5a-reductase deficiency, androgen synthesis disorder and incomplete androgen insensitivity syndrome. (4) Androgen excitation test: It is mainly used to identify androgen synthesis disorder and incomplete androgen insensitivity syndrome. With the rapid development of molecular biology, some diseases can be diagnosed at the cellular and molecular levels by using specific diagnostic tools. (1) SRY test: There is a structural gene in the short arm of Y chromosome, called Sex-determining region of the Y (SRY), which is currently considered to be the best candidate gene for testicular determinants. The genomic DNA was extracted from peripheral blood by isolating leukocytes and from skin and bilateral gonadal biopsies of surgical patients, and the conserved sequence of SRY gene was amplified by polymerase chain reaction (PCR). The significance of SRY testing is that the SRY gene is the best candidate for testicular determinants, but not the sole determinant of testicular formation; peripheral blood sex chromosomes do not represent the sex of the gonads; and patients with abnormal sexual development should be routinely tested for SRY, even in the absence of Y If the SRY is positive, the gonads should be explored even if there is no Y chromosome [2]. (2) Androgen receptor binding and receptor gene assay: the binding of androgens to target tissues can be measured to identify the cause of 46XY external genital gender ambiguity, and the cause of androgen insensitivity syndrome can be understood by directly measuring the alteration of androgen receptor gene [3]. (3 ) 5a reductase activity assay: the determination of 5a reductase activity in target tissues is important for the diagnosis of the etiology of some external genital gender dysphoria [4]. (4 ) The most common form of congenital adrenocortical hyperplasia is 21 hydroxylase deficiency, which accounts for about 95% or more. 95% of patients with congenital adrenocortical hyperplasia have deletion of the 21 hydroxylase gene. Point mutations or deletions can now be diagnosed using their PCR probes [5].