Semen was collected by masturbation and after one hour of liquefaction, parameters were established according to WHO semen guide for semen analysis version 2010. Morphology was assessed by using Kruger criteria [9].

Chromosomes were obtained after culturing lymphocytes from peripheral blood by adding appropriated media culture and phytohemagglutinin according standard techniques.

R banding was performed and metaphases were selected and analyzed using a LEICA DM2500 microscope equipped with a camera and Cytovision CW4000 image software analysis.

A genome-wide analysis was performed using Human CytoSNP-12v2.1 Analysis BeadChip (Illumina) according to the adequate protocol with an input of genomic DNA. Scan data were visualized using iScan system (Illumina). Analysis was performed with Illumina Genome Studio V2001.1 and Illumina Karyostudio1.4.3.0. according to UCSC Genome browser. Results were confirmed by the collaboration with an external lab (Eurofins Biomnis).

The operative pieces were obtained after resection of an hemi-uterus associated with a tube and gonad, Operative pieces were fixed on formalin, dehydrated and paraffin-embedded. Slides were prepared after obtaining Samples of 4 μm thickness, and after deparaffinization and routine staining (HE) Histopathologic analysis was performed.

Sperm analysis revealed severe oligoasthenoteratozoospermia. Semen parameters were the following: 1.5 mL of semen, a concentration of 1.8 million spermatozoa/mL, 15% mobility for mobile progressive spermatozoa, 20% of vitality, and 2% of typical forms (Figure 2(B)).

R-band karyotyping, performed using the above-mentioned technique, revealed a chimeric formula Chi 46,XX/46,XY. The first classification of 30 metaphases allowed the detection of a 46,XX clone with 26 cells (Figure 1(A)) and a second clone containing 4 cells of type 46,XY (Figure 1(B)). We extended then the classification to 95 cells, confirming a percentage of 90 % for 46,XX clone and 10% for 46,XY cell clone.

A second blood sample collected in EDTA was analyzed by DNA chip (SNP-array), in a reference laboratory (Eurofins Biomnis). This examination formally excluded the diagnosis of mosaicism and confirmed the diagnosis of a chimera. Thanks to the molecular karyotype, the presence of 2 distinct cell populations was identified, including a majority population, 46,XX estimated at around 90% and a minority population 46,XY estimated at around 10%. The formula, arr(1-22)x2,(X)x2[0.90],(X,Y)x1[0.90], was established according to the nomenclature of the ISCN 2016 guide.

(A)

(B)

Figure1. Karyotype results with two sets; (A) 46,XY formula and (B) 46,XX formula.

After exploration of the pelvis, a hemi-uterus was identified as well as a tube and a whitish structure with an ovarian appearance. This ovarian-looking gonad was lateralized to the left in the pelvis.

At the patient’s request, a mastectomy and removal of the female-type internal organs were performed. The patient received hormone therapy with the administration of androgens.

The anatomopathological examination revealed macroscopically the presence of a tube with a hemi-uterus and an ovary, confirmed microscopically by the presence of a parenchyma containing ovarian follicles at varying degrees of maturation (Figure 2(A)).

Figure2. (A) Ovarian tissue with oocytes at different stages of maturation; (B) Spermatozoa morphology after semen analysis.

Ovotesticular disorder of sex development (OT-DSD) replaced the terminology of true hermaphrodite in 2006, during the consensus of Chicago [10]. This entity constitutes 3% to 10% of the total DSD and presents significant diagnostic and therapeutic challenges [11]. The cytogenetic aspects of OT-DSD vary depending on geographic location. In South Africa, the most common karyotype in OT-DSD is 46,XX (88%), followed by 46,XY (8%), 46,XY/45,X (3%), and 46,XX/46,XY (1%) [6]. On the other hand, in Japan, according to Matsui [12], the 46,XY karyotype was the most common form. In Europe, the most common karyotype would be 46,XX (53%) followed by cases of mosaicism and chimerism (40%) [13].

The genetic basis of specific types of disorders of sex development (DSD) remains partly unexplained [2] Identification of the presence of chimerism can be relevant in the context of elucidation of the genetic mechanism underlying ovotesticular-DSD (OT-DSD) knowing that OT DSD is defined by both presence of ovarian et testicular tissue. Indeed, at cytogenetic level, the presence of 2 cells lines as 46,XX and 46,XY able to induce respectively female and male differentiation of gonads could explain the presence of the two tissues. Nevertheless, about 60% of patients with OT DSD have 46,XX chromosomal formula [14]. This suggests that this phenotype could be explained by other variations at molecular level, as suggested by (Bashamboo etal.) [15].

A chimera is defined by the fusion product of two different zygotes in a single embryo, and its incidence is still unknown [16]. It is a rare condition. In 2020, Madan et al. [7] reviewed 52 cases collected since 1963. Since then, to our knowledge, six more cases have been reported in the NCBI PubMed base (Table1). The prevalence of natural human chimeras is hypothesized to be as high as 10%, and as far as it is probably completely unknown for humanity [7]. Suspicion of Chimera comes to medical attention when 46,XX/46,XY formula is found after karyotyping, but in some cases, normal karyotype was reported. Even with some clinical signs like Blashko line [17], the suspicion diagnosis is not evident, and the real percentage is probably underestimated. Chimerism may concern cases in phenotypically normal women [18] or normal men [19]. Some cases of normal pregnancy have been reported [20]. Thus, Bottega et al. [21] suggest that this phenomenon might be probably underdiagnosed, as all chimeric individuals would not be detectable by standard cytogenetic technology.

Table 1. Cases repertoried in the NCBI PubMed database since 2020.

Historically and classically, chimerism diagnosis is confirmed after short tandem repeat (STR) analysis using PCR and quantification of specific genetic differences (i.e., polymorphic markers) and classical cytogenetics (Karyotyping) [22]. Later, with the development of molecular cytogenetics, detection of chimerism has become feasible with the application of targeted tests as well as genome-wide single nucleotide polymorphism (SNP) analyses [23]. In the case of our patient, the analysis of the B-allele frequency of autosomes and the X chromosome revealed the presence of two different genomes, excluding mosaicism. Indeed, in the case of chimerism, SNP array results show a diffusely altered pattern of B allele frequencies (BAF) along all the autosomes that is consistent with the coexistence of two different genotypes with an altered ratio between the two haplotypes. Bottega et al. [21] suggest that the routine use of genotyping SNP array analysis would be helpful for the identification of “hidden” chimerism in patients presenting a few clinical clues such as Blashko lines, et cetera. According to Sheets et al. [24] SNP-based microarrays, also have limitations, as the lower limits of detection of chimeras using this platform are currently unknown. Conlin etal. [23] were able to detect a chimeric XX/XY individual using SNP-array technology with a sample estimated to have between 20% to 45% chimeric cells present. The author predicts SNP-based assays to be capable of detecting samples with as low as 5% chimerism.

On the other hand, STR would also have limitations as a test when performed to study the mechanism of the chimera by identifying the origin of the alleles inherited from the parents. Indeed, STR has been in the past responsible for false negative parental tests, precisely when the excluded parent was a chimera [24][25]. As suggested by Sheets et al. [24], a better understanding of the detection limits of STR, SNP-microarrays and other DNA tests utilized in diagnosing and detecting chimerism is needed. Indeed, single-cell sequencing could be a clue, allowing a better understanding of chimera and giving an idea about its real prevalence among humans.

In the case of our patient, the proband’s parents were not available to discriminate the parental allelic contribution and assess the mechanism of the chimera. Three main mechanisms are described It concerns tetragametic chimera, andro or gynogentic chimera and pathogenetic chimera [7]. Tetragametic chimeras are known as the most common subtype and are formed by fusion of two fertilized ova or by fusion of a fertilized 2nd polar body with a fertilized ovum Androgenetic chimera and gynogenetic chimera. Androgenetic chimeras are formed by duplication of the male pronucleus, fertilization of the ovum by one male pronucleus, and diploidization of the other. Gynogenetic chimera are formed by parthenogenetic division of the female pronucleus, fertilization of one daughter cell by a sperm, and diploidization of the other. Parthenogenetic chimera are formed by parthenogenetic division of the ovum duplication of the female pronucleus, followed by fertilization of the two female pronuclei by two spermatozoa [23].

In the case of association with OT DSD, patients are at a high risk of malignancy, including gonadoblastoma, dysgerminoma/seminoma, and yolk sac tumors. Due to the risk of malignant degeneration, monitoring is recommended with early recourse to surgery if necessary to avoid malignant degenerescence. Nevertheless, tumoral risk is reported to be lower compared to other cases of DSD [26] with an estimated prevalence of 2.5 - 4 [27].