Molecular Human Reproduction, Vol. 5, No. 1, 52-56,
January 1999
© 1999 European Society of Human Reproduction and Embryology
X-chromosome inactivation patterns do not implicate asymmetric splitting of the inner cell mass in the aetiology of twin–twin transfusion syndrome
Institute of Obstetrics & Gynaecology, Imperial College School of Medicine, Queen Charlotte's & Chelsea Hospital, Goldhawk Road, London W6 OXG, UK
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Abstract |
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The aetiology of twin–twin transfusion syndrome (TTTS) is unclear. We investigated the hypothesis that monochorionic (MC) pregnancies with TTTS are associated with differences in the timing and symmetry of twinning compared to MC twin pregnancies without TTTS. DNA was extracted from the umbilical cord vessels of 26 female MC twins, 14 with and 12 without TTTS on serial antenatal ultrasound. X-inactivation patterns were determined by DNA digestion with HhaI and HpaII followed by polymerase chain reaction for a polymorphic trinucleotide repeat in the androgen receptor gene. Products were quantified by densitometry and results compared to those in peripheral blood samples of adult female controls. The median degree of non-random inactivation was similar in MC twins with TTTS, in MC twins without TTTS, and in adult controls. The percentage of individuals with skewed (
30/70%) inactivation patterns was no different in MC twins with TTTS compared to those without TTTS, and was similar to adult controls using either enzyme technique. In conclusion we found no difference in the degree or frequency of non-random X-inactivation patterns in TTTS. X-inactivation patterns do not appear to be a useful tool for studying the symmetry of inner cell mass splitting in monochorionic twins. inner cell mass/monochorionic twins/twin–twin transfusion syndrome/X-inactivation
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Introduction |
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Twin–twin transfusion syndrome (TTTS) complicates 10–15% of monochorionic (MC) twin pregnancies, and accounts for 17% of perinatal mortality in twins (Duncan et al., 1997
). TTTS presents in the mid-trimester with discordant amniotic fluid volume. One twin becomes growth-restricted, oliguric and develops oligohydramnios, whereas the other develops polyuria, severe hydramnios, and cardiac dysfunction which may progress to hydrops fetalis (Zosmer et al., 1994). Without treatment, perinatal mortality exceeds 80%, and there is a high incidence of morbidity in survivors (Denbow et al., 1997).
The pathophysiology of TTTS is poorly understood. It is attributed to net transfusion of blood from one twin, the donor, to the other, the recipient, along placental vascular anastomoses (Talbert et al., 1996). However, almost all MC twins have vascular anastomoses, and thus inter-twin transfusion. Recent data implicate asymmetric placental vascular anastomotic patterns in the pathophysiology of TTTS (Bajoria et al., 1995; Machin et al., 1996; Denbow et al., 1998). In particular, MC twins with TTTS are associated with a paucity of bidirectional superficial artery-to-artery and vein-to-vein anastomoses in the placenta compared to those without TTTS. The factors which determine vascular anastomotic patterns in TTTS are not known, and it remains possible that they arise secondary to asymmetric developmental or haemodynamic influences.
Several features implicate the timing and symmetry of inner cell mass (ICM) splitting in the aetiology of TTTS. First, among monozygous twins, TTTS only occurs in MC diamniotic sets, which arise 4–7 days after fertilization (Bardawil, 1988), and not in dichorionic (DC) diamniotic or MC monoamniotic twins, which arise <3 and 7–10 days after fertilization respectively (Duncan et al., 1997). Second, TTTS has been suggested to arise secondary to deficient placentation in the growth-restricted donor twin (Saunders et al., 1991). This is supported by the donor having (i) an unequal share of placental mass, usually <30% of the recipient's territory (Bajoria et al., 1995), (ii) an increased incidence of velamentous cord insertion (Fries et al., 1993), and (iii) a high incidence of absent end-diastolic frequencies in umbilical artery Doppler waveforms (Mari, 1998), indicative of increased downstream resistance secondary to placental atherosis. Unequal division of the ICM would result in one twin inheriting a smaller cell number than the other. It might be thought that this would lead to only transient developmental asymmetry with hypertrophy rapidly correcting the situation. However, the developmental signals acting on each twin will be simultaneous; therefore at any given moment of differentiation, one will have a smaller cell number compared to the other. Asymmetric splitting might also result in the vasculature from one twin populating the placental mass to a greater extent than that from its co-twin, eventually resulting in haemodynamic imbalance between the two circulations.
X-inactivation is the process of gene dosage compensation in females whereby every second X chromosome in a cell is randomly inactivated to create cellular mosaics of their paternal and maternal X chromosomes. Inactivation is considered to occur in approximately the same 4–7 day window after fertilization in which MC diamniotic twinning occurs (Tan et al., 1993). Non-random inactivation patterns have been found more frequently in some (Trejo et al., 1994; Goodship et al., 1996), but not all (Bamforth et al., 1996) studies of female monozygous twins. Numerous reports describe affected female carriers of X-linked disease in one but not the other of monozygous twins, with skewed inactivation of more paternally than maternally inherited X chromosomes in the affected twin (Richards et al. 1990; Lupski et al., 1991; Jorgensen et al., 1992; Kruyer et al., 1994). Since inactivation is usually a random process, skewed inactivation in MC twins could arise either (i) by chance due to X-inactivation of the smaller number of progenitor blastomeres resulting from ICM splitting or (ii) by asymmetric splitting of an ICM which has already undergone X-inactivation. Reduced size of the embryonic/progenitor cell pool has been postulated as the mechanism for the high incidence of skewed X-inactivation in singletons with confined placental mosaicism (Lau et al., 1997).
The aim of this pilot study was to explore, using X-inactivation patterns, the hypothesis that TTTS is associated with differences in the timing and symmetry of twinning compared to non-TTTS MC twins. Twin X-inactivation patterns should be random if ICM splitting occurs before inactivation (Nance, 1990). If conversely inactivation precedes twinning, inactivation patterns could be used to study the symmetry of ICM division. Symmetrical X-inactivation patterns, whether skewed or non-skewed, would indicate symmetrical splitting, and skewed asymmetrical patterns asymmetrical splitting.
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Materials and methods |
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Patients
The study population comprised seven MC twin pregnancies with female fetuses complicated by TTTS managed in three different centres. Entry criteria were presentation before 28 weeks with discordant amniotic fluid volume (Fisk, 1995
). Estimated fetal weight discordancy 15% was present in all cases. TTTS was managed by serial aggressive amnioreduction. Both infants were liveborn in each pregnancy, although three of the 14 babies died in the neonatal period. The control population comprised six MC twin pregnancies with female fetuses managed at Queen Charlotte's Hospital. These had monochorionicity determined on ultrasound in the mid trimester, and underwent fortnightly ultrasonic surveillance for signs of TTTS. Discordant amniotic fluid volume was defined on ultrasound as (i) an amniotic fluid index 40 cm and (ii) qualitative polyhydramnios in one sac and oligohydramnios in the other. Inclusion criteria for control pregnancies without TTTS were (i) absence of both the above features and (ii) amniotic fluid index <27 cm throughout the second and third trimesters. All control babies survived the neonatal period. Inspection of the placental membranes and/or histology confirmed monochorionicity in all cases. The study was approved by the institutional ethics committee and all patients gave informed consent to the collection of placental tissues at delivery. Twenty normal adult females were also studied to estimate the degree of skewed X-inactivation in the non-twin female population. These were RhD negative women with singleton pregnancies attending the antenatal clinic at Queen Charlotte's from whom blood had been collected and DNA extracted as part of another project.
Sample preparation
Fetally derived tissue was obtained from each twin's umbilical cord collected at delivery. Fetal blood was not collected because of circulatory chimerism in MC twins (Jorgensen et al., 1992; Trejo et al., 1994). Placental samples were also considered unsuitable because of preferential paternal X-inactivation in human trophoectodermally derived tissues (Harrison and Warburton, 1986; Harrison, 1989; Goto et al., 1997). A 5–10 cm segment was collected from the mid-portion of each cord in saline, transported to our laboratory within 2 days of delivery, and then stored at –20°C. A venous blood sample was collected from the mother and father where available by peripheral venepuncture and stored at –20°C.
DNA was prepared from dissected and homogenized umbilical cord vessels or umbilical cord vein epithelial cells and whole blood samples by standard proteinase K and phenol–chloroform extraction. DNA was dissolved in distilled water overnight at 4°C and the concentration of each sample determined by spectrophotometry.
Analysis of inactivation patterns
X-chromosome inactivation status was determined by the standard technique of DNA digestion with the methylation sensitive enzymes, HhaI and HpaII. This was followed by polymerase chain reaction (PCR) with primers flanking both restriction sites and a highly polymorphic trinucleotide repeat within the human androgen receptor (HUMARA) gene (Cutler Allen et al., 1992) (Figure 1). We validated this technique in our laboratory on DNA from hamster/human hybrid cell lines containing inactive or only active human X chromosomes (kind gift of Dr P.Subramanian, Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX, USA), using the methylation resistant isoschizomer MspI to control for complete digestion (Figure 2).
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Two µg of DNA were digested overnight at 37°C with the restriction enzymes HhaI and HpaII (Gibco BRL, Paisley, UK) respectively, in a 30 µl reaction consisting of 3 µl of the appropriate buffer, 1 µl spermidine and 30 U of restriction enzyme. Both digested and undigested DNA samples were amplified with oligonucleotide primers flanking the polymorphic CTG repeat within exon 1 of the HUMARA gene (Cutler Allen et al., 1992). Two µl of restriction enzyme digest mix or 100 ng of undigested DNA were amplified in a 12.5 µl reaction consisting of 1.25 µl Optiperform buffer (Bioline, London, UK), 200 µm each of dCTP, dGTP and dTTP; 40 µM [35S]dATP
S, 1 µl (100 ng) of each oligonucleotide primer, and 1U of Taq polymerase (Bioline). PCR conditions were as follows: initial denaturation step of 94°C for 5 min followed by 28 cycles of denaturation at 94°C for 45 s, annealing at 60°C for 30 s and extension at 72°C for 30 s, followed by a final extension step of 5 min at 72°C. PCR products were resolved by gel electrophoresis on 6% polyacrylamide gels at 70 W for 3 h. Gels were dried and exposed to X-ray film (Kodak XOMAT, USA) at room temperature for 16–48 h.
Densitometric analysis
Densitometric analysis of captured autoradiograph images was carried out using an ImageMaster visual display system (Pharmacia Biotech, St Albans, UK). The relative amount of DNA in both alleles from each informative twin pair was calculated as a percentage. This corresponds to the degree of inactivation of each parental X chromosome. Thus for each enzyme, the percentage inactivation for one parental allele in per cent (x) equalled the reciprocal percentage (y = 100 – x)% for the other parental allele. The percentage of skewing (0–100%) was then calculated as the absolute value of the percentage inactivation of one allele minus that of the other (% skewing = |x – y|, which also = 2 (|x – 50|). Skewed X-inactivation was defined as a percentage skewing >40% (i.e. a result more extreme than 30/70% inactivation). Results were analysed non-parametrically in view of their non-Gaussian distribution. Significance testing was by the Mann–Whitney U-test for unpaired, and the Wilcoxon test for paired data.
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Results |
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One of the TTTS twin pairs, and three of the adult controls were uninformative for separate parental alleles and were excluded from further analysis. Poor quality DNA from one of the TTTS and one of the MC twin control fetuses precluded meaningful analysis. Paired analysis of all specimens showed no significant difference in percentage skewing between the HpaII and HhaI techniques (median HhaI–HpaII = difference 1.8%, range –96.8 to +50.0).
Percentage skewing was similar in all three groups for both the HhaI [median in TTTS twins 18.8 (range 2.0–87.4), in MC twin controls 12.0 (1.0–47.6), and in adult females 34.6 (4.0–65.8)] and HpaII enzymes [median 20.0 (10.8–94.0) in TTTS twins, 18.8 (5.6–62.0) in MC twin controls, and 31.6 (3.2–51.8) in adult females].
Results for HpaII are shown in Figure 3. Skewed X-inactivation (>40%) was present in two out of 11 (18%) TTTS twins, three out of 11 (27%) MC twins without TTTS, and 3 of 17 (18%) adult controls with HpaII. The proportions with skewing in the three groups were also similar with HhaI. As can be seen from Figure 3, the highest values for percentage skewing (86 and 94%) with HpaII however occurred in two TTTS fetuses; these similarly had the highest values (84 and 73%) with HhaI. These came from a single MC twin pair, in which both twins showed preferential inactivation of the same maternal allele (Figure 4). One MC twin pair without TTTS similarly had skewed inactivation, again of the same parental allele in both twins.
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Discussion |
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This study shows that TTTS is not significantly associated with non-random X-inactivation patterns. Thus, the results do not support the hypothesis that X-inactivation during embryogenesis precedes monochorionic twinning, or that X-inactivation patterns can be used to study the symmetry of ICM splitting.
Research in this area is hampered by an understandable lack of knowledge of early human embryogenesis. The exact timing of monochorionic diamniotic twinning remains unclear (Fisk et al., 1996) although duplication of the ICM is presumed to occur in the morula to blastocyst stage 4–7 days after ovulation (Bardawil et al., 1988). Indeed, the splitting theory of monozygous twinning, which is supported by some animal studies and clinical observations, is not uniformly accepted. An alternate co-dominant axis theory explains monochorionic (but not monozygous dichorionic) twinning, and some of the duplication anomalies seen in later monoamniotic twinning (Baldwin, 1994). In addition, the exact timing of X-inactivation in human female embryos remains to be established. X-inactivation occurs at around day 5 in the mouse, when there are 10–20 embryonic precursor cells present in the ICM, and before the differentiation of embryonic tissues (Monk, 1992; Tan et al., 1993). Inactivation also occurs at different times in different tissues (Tan et al., 1993), at least in the mouse, although limited human data suggest that the cord, amnion and chorionic mesoderm have similar patterns (Bamforth et al., 1996).
We found no significant difference in the degree of skewing or the frequency of X inactivation in MC twins compared to controls. Goodship et al. (1996) suggested that the frequency of skewing was increased in monozygous twins. However, this was based on qualitative analysis of non-random patterns in six of 43 monozygous twin pairs of unknown chorionicity. In contrast, we used quantitative analysis of specimens from twins of known chorionicity. More recent studies have also suggested similar degrees of skewing in monozygous and control populations (Watkiss et al., 1994; Bamforth et al., 1996) although not all have used non-blood tissues to control for haemopoietic chimerism in MC twins (Trejo et al., 1994). Notwithstanding random variation of this stochastic variable, we found the two most extreme examples of skewing in MC twins with TTTS.
This is the first study to report numeric data on X-inactivation patterns in antenatally documented TTTS. In view of the inaccuracy of postnatal and histological criteria for TTTS (Fisk et al., 1990Fisk et al., 1995; Duncan et al., 1997), our group have shown the importance in aetiological studies of prospective ultrasonic surveillance in both TTTS and control MC pregnancies and of the application of antenatal diagnostic criteria (Bajoria et al., 1995; Denbow et al., 1998). A recent study showed qualitatively symmetric inactivation patterns in four sets of TTTS twins; no diagnostic or clinical details were provided, presumably as the samples were collected postnatally for routine zygosity analysis (Bamforth et al., 1996).
The numbers in this study are admittedly small, reflecting difficulties with acquisition from rare TTTS cases where both twins are female and survive to birth. Notwithstanding this, our study is otherwise robust, as we took care to collect non-blood non-placental tissue samples, to apply the modern diagnostic criteria for TTTS, to validate the technique in cell lines, to analyse each sample with two different digests in the presence of an undigested control, and to use densitometry for quantitative analysis. This controlled study involving six MC TTTS pairs is thus of sufficient size to rule out consistently aberrant X-inactivation patterns in association with TTTS.
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Acknowledgments |
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Supported by a grant from the Special Trustees of Queen Charlotte's and Hammersmith Hospitals. The authors thank Drs J.Tolosa, G.Rizzo, R.Bajoria and C.Hubinont for providing us with specimens.
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Notes |
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1 To whom correspondence should be addressed
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Submitted on June 17, 1998; accepted on September 30, 1998.
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