Centre for Reproductive Medicine, Dutch-Speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium
1 To whom correspondence should be addressed. e-mail: stratis{at}easynet.be
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Abstract |
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Key words: assisted reproduction/ICSI/monozygotic triplets/monozygotic twins
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Introduction |
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A potential drawback that appears to accompany blastocyst transfer is an increased incidence of monozygotic twinning (Kolibianakis and Devroey, 2002). Several additional factors, however, have been implicated in the aetiology of monozygotic pregnancies, including manipulation of the zona pellucida, ovarian stimulation, in vitro culture and increased maternal age.
This is the first case report of a quintuplet pregnancy, consisting of a monochorionic biamniotic and a monochorionic triamniotic pregnancy, following transfer of two blastocysts in a 39-year-old woman who underwent ICSI.
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Case report |
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In her third attempt, she was stimulated with 150 IU of recombinant FSH (Puregon, NV Organon, Oss, The Netherlands) and 300 IU of Menopur (Ferring Pharma ceuticals A/S, Denmark) daily. Inhibition of premature LH surge was achieved using 600 µg daily of GnRH agonist (Buserelin, Suprerfact, Hoechst AG, Frankfurt, Germany) starting on day 1 of the treatment cycle (short protocol). After injection of 10 000 IU of HCG (Pregnyl, NV Organon), 10 cumulusoocyte complexes (COCs) were retrieved; eight metaphase II (MII) oocytes were injected, resulting in eight two-pronuclei oocytes. After 5 days of culture in sequential media (G1, G2; IVF Scandinavian, Gothenburg, Sweden), two grade B quality blastocysts (Gardner et al., 1998) were transferred (Figure 1), while one blastocyst was cryopreserved. The luteal phase was supported with 600 mg of vaginal progesterone daily (Utrogestan; Besins, Brussels, Belgium). Ultrasound performed at 7 weeks of gestation showed a quintuplet pregnancy consisting of a monochorionic biamniotic pregnancy and a monochorionic triamniotic pregnancy.
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Discussion |
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The incidence of monozygotic twinning after normal conception is estimated to be 0.4% of births (Derom et al., 1987; Saito et al., 2000
). Its frequency is reported to be significantly higher after ovulation induction (1.2%; Derom et al., 1987
) and IVF [3.2%, (Wenstrom et al., 1993
); 3.2% (Saito et al., 2000
); 1.2% (Sills et al., 2000
); 5% (Behr et al., 2000
); 3.2% (Milki et al., 2000
); 1.9% (Alikani et al., 2003
)]. The incidence of monozygotic triplets on the other hand, is much rarer (0.000023%; Imaizumi and Nonaka, 1997
). Monozygotic triplets could be formed by a first division of the embryo to provide a twin pregnancy and a second division of one of the twins (Belaisch-Allart et al., 1995
). High order multiple gestations consisting of a monozygotic triplet with biamniotic bichorionic twins or a monozygotic triplet with biamniotic twins have been reported to occur following transfer of three ICSI blastocysts (Yakin et al., 2001
) or four IVF embryos (Salat-Baroux et al., 1994
), respectively.
Several factors have been involved in the aetiology of monozygotic pregnancies, all of which appear to be present and potentially contributing to the occurrence of the quintuplet pregnancy in the current case report.
The effect of extended culture on the incidence of monozygotic pregnancies
It is not known whether blastocyst transfer is related to an increased occurrence of monozygotic triplets. However, such an association appears to be present between blastocyst transfer and the occurrence of monozygotic twins.
In a retrospective study, da Costa et al. (2001) observed a significantly higher incidence of monozygotic twinning in 129 pregnancies after blastocyst transfer as compared with 814 pregnancies after day 3 transfer (3.9% versus 0.7%, respectively).
In addition, Milki et al. (2000) in a retrospective study observed a significantly higher incidence of monozygotic twinning after blastocyst transfer (5.6%) as compared with day 3 transfer (2%), by analysing 197 and 357 pregnancies, respectively. Similar results have been reported by Sheiner et al. (2001
).
On the other hand, Cassuto et al. (2003), by analysing retrospectively 1263 pregnancies, suggested that culture conditions and not prolonged culture are responsible for monozygotic twinning in human IVF.
Several hypotheses have been proposed to explain the above findings, including anomalies in apoptosis-related remodelling of the inner cell mass during extended culture to the blastocyst stage using sequential media (Menezo and Sakkas, 2002). Using co-culture with feeder cells, no monozygotic twinning with blastocyst transfer has been reported in a series of 800 deliveries during a 10-year period (Menezo and Sakkas, 2002
). Co-culture using feeder cells might provide free radical scavengers, which may not be efficient when sequential media are used (Ouhibi et al., 1990
). Alternatively, an advantage of co-culture as compared with sequential media might reside in the presence of growth factors or cytokines (Wang et al., 2002
), which have been shown to inhibit apoptosis in mouse embryos (Milki et al., 2000
; Behr et al., 2002
).
Excessive growth of the inner cell mass, probably resulting from prolonged in vitro culture, has also been suggested to explain the formation of monozygotic twinning (Scott, 2002).
Zona pellucida manipulation and incidence of monozygotic pregnancies
As expected, and due to their rare occurrence, no data are available on the association of monozygotic triplets with zona manipulation. Zona manipulation procedures have been involved in the aetiology of monozygotic twins. Artificially induced damage to the zona pellucida may be associated with inner cell mass splitting during hatching, and thus may result in monozygotic twinning in the human.
Abusheikha et al. (2000) compared the incidence of monozygotic twins in multiple pregnancies achieved after ICSI (n = 56) or after conventional IVF (n = 662). The incidence of monozygotic twins was significantly higher after ICSI than IVF (8.9% versus 0.9%, respectively).
Similarly, Tarlatzis et al. (2002) reported a significantly higher incidence of monozygotic twins per started cycle after ICSI (n = 79) than after IVF (n = 102) (5.9% versus 0%, respectively).
In addition, Saito et al. (2000) examined the incidence of monozygotic twinning in pregnancies resulting from single embryo transfer. The monozygotic twinning rate was significantly higher after microinsemination (ICSI, subzonal insemination, partial zona dissection; n = 83) than after conventional IVF (n = 196) (7.2% versus 1.5%, respectively).
Alikani et al. (2003) suggested that zona pellucida disruption appears to increase the incidence of monozygotic twinning by analysing 81 consecutive cases after assisted conception.
Furthermore, an increased incidence of monozygotic twins has been reported in cycles in which assisted hatching (AH) was performed (Hershlag et al., 1999; Schieve et al., 2000
).
On the other hand, Schachter et al. (2001), by reviewing 602 pregnancies achieved after conventional IVF or ICSI/AH, observed a similar incidence of monozygotic twins (0.72% versus 0.86%, respectively). Similarly, Sills et al. (2000
) analysed 1674 IVF transfer cycles and observed that the frequency of monozygotic twins was not statistically different between zona-manipulated (1.03%) and zona-intact groups (2.5%).
In summary, although not supported by all studies, an association of manipulation of the zona pellucida and an increased incidence of monozygotic twins appears to exist in humans.
Age and incidence of monozygotic pregnancies
Following natural conception, the only factor found to affect an otherwise constant frequency of monozygotic twinning is maternal age (Bulmer, 1970). In assisted reproductive technology (ART) pregnancies, no data exist to support such a relationship at present. However, it has been proposed that a naturally thin zona pellucida may affect the hatching process as the blastocyst may protrude at multiple sites of zona lysis rather than at a single one (Abusheika et al., 2000
).
Ovarian stimulation/in vitro culture and incidence of monozygotic twins
Edwards et al. (1986) suggested that in vitro culture conditions are responsible for monozygotic twinning. Derom et al. (1987
), however, postulated that it is the ovulation induction itself rather than in vitro conditions that predisposes to zygotic division or enhanced survival of monozygotic twins. The increased incidence of monozygotic twins observed was assumed to be a result of a hardening of the zona pellucida (Derom et al., 1987
). If the hardening is not uniform, this could result in weak spots through which blastocyst might herniate.
It should be noted that all of the factors involved in the aetiology of monozygotic pregnancies were present in our patient (increased maternal age, ovarian stimulation, manipulation of the zona pellucida and use of extended culture). Moreover, the patient had already had a twin dizygotic pregnancy in a previous cycle, which indicates that her endometrium was receptive following ovarian stimulation.
The current case report indicates that monozygotic pregnancies consisting of both twins and triplets are possible after ART and that it is therefore necessary to identify predictive factors for their occurrence. This can be accomplished by performing prospective trials that will allow us to counsel patients appropriately. Besides identifying predictive factors for the occurrence of monozygotic pregnancies, the importance of transferring a single embryo in IVF is also obvious. Both approaches could increase the safety of IVF and help achieve its aim, the delivery of a healthy child after a singleton gestation.
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References |
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Submitted on September 9, 2003; accepted on October 23, 2003.