Clinical and Genetic Study of a XX (SRY negative) Male.

Rima Dada, M. E. Ahmad, Rashmi Talwar and Kiran Kucheria*

Genetics Division, Dept. of Anatomy,
All India Institute of Medical Sciences, New Delhi-110029, India

*Corresponding Author:
Dr. Kiran Kucheria
Professor and Head, Dept. of Anatomy
All India Institute of Medical Sciences
New Delhi-110029, INDIA
Phone: 91-11-6593489/6593216/6589089
Fax; 91-11-6862663


The 46, XX male syndrome is a rare sex chromosomal disorder in man [1]. It mostly occurs due to unequal crossing over between X and Y chromosomes during meiosis [2]. A 32 year old XX male with normal male phenotype and normal adrenarchal hair pattern, bilateral atrophic testes, normal phallus presented with azoospermia and infertility. The gonadotrophins were elevated. Cytogenetic and molecular analysis using FISH with X and SRY probe showed a 46,XX (SRY negative) chromosomal constitution.

Key Words: Sex determining region on Y chromosome (SRY), Fluorescent in situ hybridization (FISH), Azoospermia, Male infertility, XX Male.


Genetic analysis of sex reversed subjects with a phenotypic sex different from that in the karyotype has been important in the identification and characterization of the sex determining region on the Y chromosome (SRY). SRY is the candidate gene for the testis determination. It resides within 35 Kilobase region of the Y chromosome immediately proximal to the Pseudoautosomal region I (PAR I). When the SRY gene is present, testes are formed and in its absence gonads develop into ovaries.
46, XX males are distinct from XX true hermaphrodite and have a male habitus, small testes, azoospermia with no evidence of uterus or ovaries [3,4]. The incidence is about 1 in 20,000 - 25,000 male births. Different pathogenic mechanism have been suggested which can lead to 46,XX sex reversal. These may be recombination between X and Y chromosomes [2], mosaicism with a prevalent XX lineage and a hidden cryptic lineage containing a Y chromosome [5], and mutation in genes other than TDF can trigger testis determination in XX (SRY negative) males [6]. We describe here a case of 46,XX infertile azoospermic male with normal male phenotypes showing the absence of SRY region on Fluorescent In Situ Hybridization (FISH) analysis. We have done molecular analysis using FISH technique, which not only detects the SRY region but also identifies the site of chromosome translocation if any.


A 32 year old male was referred to us for cytogenetic analysis from the infertility clinic at All India Institute of Medical Sciences (AIIMS), New Delhi. At the time of presentation he had been married for 4 years and cohabitation since then. He was a product of full term normal vaginal delivery from a non-consanguineous marriage. His childhood development was normal and uneventful. There was no history of any major illness like TB, mumps or injury to testis. There was no family history of sex reversal.
The patients height was 5 feet 4" and had normal facial, axillary hair and scant pubic hair.


Figure 1A: Phenotype of 46, XX Male - removed
Figure 1b and 1c: Metaphase spread and karyotype showing 46,XX chromosomal complement.

The patient's penile length was normal but both testes were soft, small and atrophic. Ultrasonogram showed the right testis as 23x17x12mm and left testis as 24x19x14mm in volume. Semen analysis was done which showed complete absence of sperms in semen confirming azoospermia [7]. The semen volume was 3ml and was fructose positive.

The gonadotrophin levels were markedly elevated, FSH levels were 70mIU/ml and LH level were 84mIU/ml (Normal: FSH 1.2-5mIU/ml; LH 2-9.8 mIU/ml). Chromosomal analysis performed on 50 G-banded metaphases showed 46, XX chromosomal complement (Figure 1 b & c). Fluorescent in situ hybridization (FISH) analysis was done using commercially available centromeric probes for chromosome X and Y (Vysis CEPX/CEPY) to rule out low level mosaicism. All the 200 cells scored showed XX complement (Figure 2a).


Figure 2: FISH images of interphase nuclei and metaphase chromosomes obtained from peripheral blood sample of 46,XX male patient:

(a) Interphase nuclei and metaphase chromosomes hybridized with CEP X/CEP Y probe showing two red signals (chromosome X) representing XX constitution.

(b) Metaphase chromosomes hybridized with LSI SRY/CEP X probe showing only two green signals (chromosome X) indicating the absence of SRY region.

Further, FISH was done using probe specific for the sex determining region (SRY) (Vysis CEPX/LSI SRY) to identify any cryptic rearrangement resulting in translocation of SRY region on to the chromosome other than Y or to analyse the presence of SRY region even in absence of Y chromosome. A total of 200 metaphases and interphase cells were scored which revealed the absence of SRY region (Figure 2b).


XX males were recognized for the first time by de La Chapelle et al, in 1964 [8]. In 1966, Ferguson Smith [9] and his colleagues proposed that this condition results from translocation of Y material including sex determining region (SRY) on Y chromosome to the X chromosome. Berkovitz [10] and Vilain et al. [11] reported 46,XX males which were negative for SRY using PCR. McElreavey et al. [6] proposed that SRY has been postulated to repress an inhibitor of male testicular development. SRY negatively regulates an autosomal locus, Z, that switches off male determining genes. In normal males SRY situated on the short arm of Y chromosome just proximal to pseudoautosomal region I negatively regulates Z locus. It is possible that in the present case XX male (SRY negative) may have a recessive mutation in the Z locus, so that it cannot switch off the male determining genes [11] and therefore the present case has normal male phenotypes and testicular development. 46,XX males lack the long arm of the Y chromosome (Yq). Which contains the Azoospermia factor (AZF) loci and has gene complex critical for germ cell development and differentiation [12]. Absence of AZF region in our case also explains for the infertile phenotype and azoospermia. Numabe et al. [13] reported that 46,XX males without Y sequences were hypospadiac but this was not seen in the present case. FSH and LH levels were elevated in the present case as seen in cases with severe testiculopathy and complete spermatogenic arrest leading to testicular failure.
Thus, for the complete workup of XX males, it is important that conventional cytogenetic analysis be followed by FISH analysis to determine the presence of SRY gene. These investigations indicate that the amount of Y specific material contributes to the phenotype heterogeneity. Inspite of the fact that SRY is the testis determining factor (TDF), it is likely that other genes are also involved in pathway of sex determination and differentiation process as both upstream and downstream regulator genes.


The authors are grateful to The Indian Council of Medical Research (ICMR), New Delhi for financial support, project no. 54/1/98.




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First Published: September 2002

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