Key words
sex discordance - gemini - ART - sonography
Schlüsselwörter
Chimären - Geschlechtsdiskrepanz - Geminigravidität - ICSI - Ultraschalldiagnostik
Introduction
Ultrasound examination of twin pregnancies can reliably distinguish between monochorionic
and dichorionic placentation. Dichorionic twins have separate placentas and amniotic
cavities and in 90 % of cases arise from two oocytes. In contrast monochorionic twins
originate strictly from one oocyte (monozygote) and have identical sex. With monozygotic
twins one of three types of placentation and fetal membrane setup occur depending
on the timing of separation into two individuals: One third are dichorionic and diamniotic,
occurring when separation takes place during the cleavage or morula stages. In two
thirds of monozygotic twin pregnancies separation of the embryoblast occurs during
the blastocyst stage, resulting in monochorionic diamniotic placentation. When separation
happens only after formation of the amniotic cavity, monozygotic twins with monochorionic,
monoamniotic placentation occur (1 % of monozygotic twins).
We present a case of monochorionic, diamniotic twins following in vitro fertilisation
with intracytoplasmic sperm injection (IVF/ICSI), in which discordant sex was detected
on detailed antenatal ultrasound.
Case Presentation
A 36-year-old patient underwent long protocol IVF/ICSI in view of primary male factor
infertility. 16 oocytes were inseminated by intracytoplasmic sperm injection (ICSI).
Intrauterine transfer of two embryos at 8 cell stage and one at 6 cell stage was performed
on the third day after follicle puncture ([Fig. 1]).
Fig. 1 Three embryos at 6 and 8 cell stages before transfer (culture day 3).
Cleavage of the three transferred embryos was timely and the cumulative embryo score
(CES) after Steer [1] of 44 (16 + 16 + 12) was reduced since embryonic morphology was limited (blastomeres
of differing size and fragmentation). Laser treatment of the zona pellucida was carried
out on all the embryos before transfer to support hatching for successful implantation
(“assisted hatching”).
The first ultrasound examination, at 5 + 3 weeks gestation, documented a monochorionic
twin pregnancy ([Fig. 2]).
Fig. 2 Image from the first ultrasound performed at 5 + 3 weeks gestation showing one chorion
and two yolk sacs.
On detailed transabdominal and transvaginal ultrasound of the twins at 13 + 3 weeks
gestation discordant sex was suspected ([Figs. 3] and [4]). This was confirmed on subsequent examinations at 17 + 5 and 21 + 6 weeks gestation.
Fetal anatomy was otherwise normal.
Fig. 3 Ultrasound image of the first fetus in the sagittal plane showing the penis (arrow).
Fig. 4 Ultrasound image of the second fetus in the sagittal plane showing the clitoris (arrow).
The patient decided against further invasive investigation for lack of clinical consequence.
The pregnancy was uneventful apart from autoimmune thyroiditis, which was diagnosed
before pregnancy and treated with thyroxin.
The twins were delivered by primary caesarean section at 36 + 6 weeks gestation with
mild polyhydramnios. Apgar scores and pH values were normal. Post partum both twins
were phenotypically normal. One twin had normal male external genitalia with bilateral
descended testes. The other had normal female external genitalia and a uterus and
left ovary measuring 9 mm were demonstrated on abdominal ultrasound; the right ovary
could not be visualised. In addition, a double renal pelvis was found on the right.
Placental histology confirmed the monochorionic, diamniotic situation.
Cytogenetic analysis on heparinised blood was performed on the first day of life for
the female twin, and on the 6th day of life for the male twin. EDTA blood, urine and
oral mucosal cells were taken on the 21st day of life from both twins (for results
see [Table 1]).
Table 1 Results of karyotyping and fluorescent in situ hybridisation (FISH) analysis
|
Cell type
|
Method
|
Result
|
|
Mosaic of the phenotypic female twin:
|
|
Blood lymphocytes (Mesenchyme)
|
Conventional chromosomal analysis
|
46,XY[18]:46,XX[12]
|
|
Blood lymphocytes
|
FISH
|
X0[3]:XXY[3]:XXYY[1]:XY[155]:XX[138]
|
|
Oral mucosa (Ectoderm)
|
FISH
|
XY[0]:XX[200]
|
|
Urothelium (Endoderm)
|
FISH
|
XY[5]:XX[95]
|
|
Mosaic of the phenotypic male twin:
|
|
Blood lymphocytes
|
Conventional chromosomal analysis
|
46,XY[19]:46,XX[11]
|
|
Blood lymphocytes
|
FISH
|
XY[201]:XX[99]
|
|
Oral mucosa
|
FISH
|
XY[200]:XX[0]
|
|
Urothelium
|
FISH
|
XY[47]:XX[0]
|
A lineage analysis was performed for both twins on EDTA blood from the 21st day of
life to exclude a chimerism. This showed an identical signal pattern for both children:
More than two alleles were found in 4 of the 10 short tandem repeat (STR) systems
examined proving the diagnosis of chimerism, at least for blood lymphocytes.
Discussion
Chimerism is defined as genetically different cells/tissues from more than one zygote,
occurring in one individual [2]. To date 15 cases of monochorionic, dizygotic twins with chimerism have been described
[3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. Most of these cases were blood chimerisms due to twin-twin transfusion in the presence
of a common placenta [3], [5], [6], [7], [8], [10], [11], [12], [13]; in this case chromosomal analysis of mucosa or skin biopsy is consistent with phenotypic
sex.
In the current case FISH was carried out on urothelium from both twins. The female
twin was found to have a gonosomal constellation of XY[5],XX[95], so that a gonosomal
mosaic could not be excluded.
Only five cases of monochorionic, dizygotic twins following spontaneous conception
have been described in the literature to date. These include two sets of twins with
differing sex [7], [15], two sets with one trisomy 21 each [10] and one case with trisomy 13 in one twin [5].
As in our case, the majority of those described previously have occurred in the context
of infertility treatment with ovarian stimulation and artificial insemination i.e.
IVF or IVF + ICSI [3], [6], [8], [9], [11], [12], [13], [14], [16]. Typically numerous embryos were transferred simultaneously. It is thought that
fusion/amalgamation of the outer cells is possible at the late morula stage (from
day 4) with the inner cells remaining unchanged [13], [14], [15]. This type of fusion has been described in vitro in mouse blastocysts [17]. Procedures such as “assisted hatching”, where the zona pellucida is opened artificially,
and other factors including culture environment and simultaneous intrauterine transfer
of multiple embryos may be further predisposing factors for embryo amalgamation [9], [14], [18].
When oocyte insemination is by ICSI, simultaneous fertilisation of the oocyte with
two different spermatozoa before disintegration of the second polar body with subsequent
amalgamation followed by separation into two individuals is almost impossible. As
part of IVF/ICSI treatment correct oocyte insemination following microinjection with
one sperm cell is documented, as is confirmation of both pronuclei and embryo development.
Thus embryo amalgamation with subsequent twin separation is only possible after embryo
transfer. Separation of fused embryos can only occur after implantation (at least
5 days after fertilisation), as is the case with monochorionic, diamniotic twins,
and only after development of the trilaminar germ disc in the second week after fertilisation.
Both lymphocytes and cells of the urinary tract originate from mesenchyme and endoderm
and are therefore tissues originating from the trilaminar germ disc. In the current
case the gonosomal mosaic constellation was found in these two tissues of the female
infant, and in the male infantʼs lymphocytes. The finding could not be demonstrated
on oral mucosal cells, which originate from ectoderm.
Such chimerisms may occur more often than is commonly assumed since similar constellations
in same sex twins will go undetected, leading to underestimation of monochorionic,
dizygotic pregnancies. The possibility of discrepant cytogenetic findings in monochorionic
twins should be taken into consideration when performing invasive antenatal investigation.
It is almost impossible to assign karyotype or molecular genetic results from villus
sampling of a monochorionic placenta to a particular twin. The same is true for fetal
blood and blood samples from newborns in the first weeks of life because of twin-twin
transfusion, which occurs commonly with monochorionic placenta. Amniocentesis therefore
appears more reliable for allocating genetic results to specific twins in this context.
Apart from chimerism, the differential diagnosis of discordant sex on antenatal ultrasound
includes endocrine causes including virilisation of a female fetus, e.g. adrenogenital
syndrome. Disorders of sexual development such as complete androgen insensitivity
syndrome (CAIS) can result in a cytogenetically male fetus having a female phenotype
[19], [20], [21], [22].
Lineage tracing was repeated on both infants in order to differentiate between blood
chimerism – due to mixing of blood via twin-twin transfusion – and true chimerism.
An identical signal pattern was found in 4 of the 10 STR systems analysed confirming
chimerism at least for blood lymphocytes.
Consent
The patient gave written consent for the publication of this case report and relevant
images.
