Introduction
The initial determination of gender occurs during fertilization and the phenotypic process of sex determination occurs during organellosis. Apart from sex-specific genes on chromosome X and chromosome Y, autosomal chromosome genes play a role in gender determination. Any change that occurs in these chromosomes as well as sex chromosomes results in genetic abnormalities and the sex change leads to hermaphroditis [1]. The importance of the Y chromosome has been identified in the determination of male sex since 1950.
The main factor determining the genetic status of a fetus is the presence or absence of Y chromosomes, and as a routine, XY fetus is a male and XX fetus is female. Information on the prevalence and influence of the causes of sexual obscurity and the reality of sexual evolutionary diseases is limited [2]. Due to complicated processes that occur casually between fetal weeks 6 to 14 to indicate sexual status, it is unlikely that an error will occur in this direction. Many of these errors lead to sexual ambiguity or, in spite of having a specific chromosomal system, the sex of the fetus would be of the other type. These diseases are sometimes referred to as intersex. Diseases of gender differentiation and sex determination are divided into four main groups, including dysgenesis of gonads, real hermaphrodite, false male hermaphrodite, and false female hermaphrodite [3].
Anomalies are relatively common in sexual differentiation, and it occurs in about one in every 4500 live births. A male with a 46xx karyotype was first described in 1964 by three groups of researchers. The external reproductive organs and small testicles are characteristic of this syndrome [2]. About one quarter of infants suffer from loss of salts in the second and third week of life with decreasing blood flow, decreased sodium and potassium levels, which in 90% of cases resulted from the deficiency of 21-hydroxylase enzyme and in very rare cases due to deficiency of 17- Alpha-hydroxylase, 17 and 20-lyase, and in some cases due to deficiency in the enzymes of 11-beta-hydroxylase or 3-beta-hydroxide hydrogenoase [4, 5]. Hence, a baby with sexual ambiguity needs a quick assessment of sex determination, which may threaten the baby's life with problems such as the loss of solubility in hyperplasia of adrenal glands [2]. Determining the chromosomal status requires the accurate diagnosis and counseling of each patient with sexual anomalies. This diagnostic action is necessary to determine the correlation between the genotype and the phenotype and the identification of the bases of the gender determination mechanism.
 
Patient and methods
In this paper, a chromosomal study was conducted on 4 neonates with sexual ambiguity referred to the cytogenetic laboratory of the Sarem Medical Genetics Department in the year 2011. The causes and characteristics of sexual ambiguity in these infants and chromosomal study were performed for all of these patients. Karyotype can help detect chromosomal abnormalities.
The structure of all 44 autosomal chromosomes and 2 sexual chromosomes were studied. For this purpose, the Banding High Resolution method was used. By this method, the cell cycle in the S stage is stopped by the use of thymidine and the chromosomes are collected for a short time in the pro-metaphase or early metaphase step using the calcimide; thus longer chromosomes that feature high-resolution strip-coloring (500-850 bands) are obtained which can be used to detect structural chromosomal abnormalities at the level of a strip, such as microdeletion. Peripheral blood was collected. At least 1cc of infants` blood were heparinized with a sterile syringe containing 0.1 cc antidiabetic heparin sodium at a concentration of 5000 units per ml, equivalent to 100 international units without any heparinized preservative and 0.2 ml of infant blood in 5 ml complete culture medium 1640 RPMI-Add at pH 7 and 1 serum FBS, 125 L of phytohemagglutinin, and 0.001 penicillin or streptomycin were added, and the contents of the culture tube was mixted a few times and it was placeed in a 37 ° C incubator at 30 °. After 48 hours, 0.1 ml of thymidine (10 mg / 150 ml PBS) was added to the culture and it was placed in incubator a 37 ° C for 16 to 17 hours. Then the tube was centrifuged for 8 minutes and the supernatant was removed, and after mixing of the precipitate, 5 ml complete culture medium (without phytoplasm) was added. the contents of the tube are mixed and placed in the incubator at 37 ° C for 4 hours and 45 minutes. 0.01 ml of calcimide is added and placed for 15 minutes in the incubator. the sample is harvested and then is placed on the slide. Then GTG Standard Bonding Method is used to analyze the chromosomes. In this method, the slides are stored at room temperature (20-23 ° C or at 40 ° C for at least one week. The slides are placed in trypsin 0.05% at room temperature for 10 to 60 seconds, and are washed inside of the physiological serum with 1% FBS serum, then it is stained with Giemsa [6].
For each patient, at least 15 metaphases were examined and 5 metaphases were analyzed for structural abnormalities. After performing the above steps and preparing and analyzing the karyotypes, it was found that two patients had the karyotype of 46XX and the two other cases had normal 46XY karyotype (Table 1). The first case was the result of a third-degree family marriage of daughter of aunt and son of uncle. This male infant had a vague phenotypic character, and had a micropenic effect, and the outcome of the karyotype was 46XY normal in this infant. Other phenotypic symptoms include multiple congenital anomalies and congenital heart abnormalities and loss of lung, and the reason of the sexual ambiguity is testicular feminization, which is a major cause of False male hermaphrodism.
       
The third case was sexual ambiguity with bilateral testis. resulting of karyotype was a normal 46xy, which is attributed to male false hermaphrodysm. In male false hermaphrodysm, there are only gonadal tissues of the one sex. In these people, the external genitalia is probably ambiguous or of opposite sex. Therefore, in male false hermaphrodysis, the karyotype is 46xY, but the genital system is feminine. The main cause of male false hormophidiosis is the irregularity of the androgen. These conditions are also known as testicidal femininity syndrome; the karyotype is male, but the person is feminine and completely normal. Their vagina is closed and there is no uterus and fallopian tubes. The testicles remain inside the abdomen or the inguinal canal and are confused with the hernia. The main reason for this situation is the lack of androgen receptors in target tissues, which is why, although testosterone is naturally produced, the effects that should be secreted should be created and blocked. Androgen receptors are coded by a X-linked gene, which stop the activity of the gene due to spot mutations or chromosomal removal. Some people have partial or complete irregularities in androgens, and they are diverted differently. Affected people tend to be feminine, but they will be clearly infertile. Their testicles need to be removed due to the risk of malignancy, and estrogen should be replaced in order to produce secondary sexual characteristics and prevent osteoporosis [4].
 
Discussion
The reasons for sexual ambiguity, variability and gender definition are very difficult and even impossible in some cases. Human sexual differentiation is a very complex process under the control of many genes and hormones [7]. The birth of a newborn with sexual ambiguity can be a big concern in the family. Proper and timely diagnosis is essential for physical well-being and the physiological evolution of children with sexual obscurity.    
The first patient suffered from multiple anomalies due to congenital heart disease and loss of lung in the past, and because the baby was the result of a third grade marriage. On the other hand, changing the male-dominated female phenotype and the range of male internal organs is an incomplete form of irregular androgenic insensitivity syndrome, and is also repeatedly embraced by the male phenotype with hypospadias, and the scrotum containing small testicles. In addition, in this group of patients, the closed vagina can appear without other female structures [2]. The second and fourth patients both had hyperplasia of the adrenal glands. Biochemical tests performed from two patients confirmed this. Hyperplasia of the adrenal glands (CAH) is an incident autosomal disease, which is due to the reduction of the activity of the steroid needed enzymes in the renal cortex, which is essential for the cortisol biosynthesis [8] (Figures 3 and 4).    
Conclusion
According to the study, all infants with ambiguous sexual and non-palpable testis, rapid determination of 17-hydroxyprogesterone and serum electrolytes is essential [5, 9]. Clinical emergence of all forms of hyperplasia of the adrenal glands is characterized by a false development of the specific characteristics of the female genital muscle, which is mildly likely to be enlarged with enlarged clitoris or a combination of lobes. It can also rarely show a male phenotype with bipolar testicles [6, 8]. The third patient was diagnosed with a testicular feminization syndrome, with a one-way testicle and the result of karyotype was 46xY and a normal male karyotype. Since identification of newborns with sexual ambiguity at birth is essential, clinical and chromosomal examination is important in order to determine the sex and shape of their sexual identity in the future. The use of molecular diagnostic methods along with cytogenetic methods will be very useful for the definitive and final detection of the cause of sexual obscurity.
 
Acknowledgments
The authors do acknowledge from the personnel of  Department of Genetic at Sarem Women’s  Hospital.
Ethical permissions
The case was not found by the authors.
Conflict of Interest
The case was not found by the authors.
Financial Support
This study was supported by Sarem Fertility and Infertility Research Center.
Contribution of authors:
Akram Abdi (First author), author of the article/main author/author of discussion (%15); Nesa Zarbati (Second author), author of the article/methodology/helper author (%10); Mehshad Asami(Third author), author of the article/methodology/helper author (%10); Iman Bagherizadeh (Fourth author), author of the article/methodology/helper author (%10); Fatemeh Hadipour (Fifth author), author of the article/helper author (%10); Zahra Hadipour (Sixth author), author of the article/helper author (%10); Yousouf Shafeghati (seventh author), author of the article/helper author/author of discussion (%15); Farkhondeh Behjati (eighth author), author of the article/methodology/main author/author of discussion (%20).