IVF and Infertility Unit, Assaf Harofeh Medical Center, Zerifin, The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
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Abstract |
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Key words: gonadotrophin therapy/micromanipulation/monozygotic twins/multiple pregnancy/zona pellucida
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Introduction |
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We report our experience with monozygotic twinning after ovulation induction or assisted reproduction treatment, from different clinical settings, and advance our hypothesis as to the evolution of this situation.
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Materials and methods |
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During the same time period, 602 pregnancies were achieved in our IVF programme. A total of 475 cycles of conventional IVF yielded 139 clinical pregnancies (29.3%). Conventional IVF was employed in patients with tubal factor infertility or unexplained infertility after gonadotrophin therapy with intrauterine inseminations had failed. Tubal factor infertility was determined by pathological hysterosalpingography or previous laparoscopy/laparotomy, and was so determined in 211 of these 299 patients, including 39 patients with endometriosis. Ninety-five patients were diagnosed as suffering from unexplained infertility. IVF was carried out after pituitary down-regulation with gonadotrophin-releasing hormone agonists (GnRHa, Decapeptyl® 3.75 controlled release; Ferring, Malmo, Sweden), or nasal Nafarelin® 400600 µg/day (Synarel, Delpharm, France) and HMG, using a standardized long protocol. The administration of HCG, oocyte aspiration, laboratory procedures, semen preparation, embryo handling and transfer have been described in detail (Ron-el et al., 1991). Embryo transfer was achieved in 92.4% of attempted cycles, the average number of embryos replaced was 2.4 ± 0.8 embryos per cycle. Identification of pregnancy and subsequent ultrasound examinations were as for the ovulation induction group.
Micromanipulation was employed in our IVF programme in 1438 cycles in the same time period. Intracytoplasmic sperm injection (ICSI) was used for patients with male factor infertilitydefined as severe oligoasthenoteratozoospermiaor in cases of failed fertilization in a previous conventional IVF cycle with a normal ovarian response. ICSI was also employed in cases of poor response on the part of the female partner (four oocytes or less aspirated per cycle) when two (or more) previous IVF attempts had been unsuccessful. Assisted hatching (AH) using acid Tyrode's solution (Irvine Scientific, Santa Anna, CA, USA) was employed in 327 of the 1438 ICSI cycles (22.7%). All of these treatment cycles were initiated using the same standard drug protocols, criteria for HCG administration, oocyte aspiration, laboratory procedures and semen preparation as previously described. The ICSI procedure was carried out as described by Van Steirteghem et al. (Van Steirteghem et al., 1993). Embryo transfer was achieved in 94% of attempted cycles, the average number of embryos replaced was 2.3 ± 1.2. Identification of pregnancy and subsequent ultrasound examination were as for the previously mentioned groups. Micromanipulation (ICSI and/or AH) in 1438 embryo transfer cycles yielded 463 clinical pregnancies (32.2%), the proportion of AH in conception and non-conception cycles was similar.
Statistical analyses
Variables relating to the patient or treatment characteristics were examined and compared with the overall IVF patient population treated in our centre, using Fisher's exact test to compare rates (drug type and dose distribution) and MannWhitney Rank Sum test, to compare non-parametric data (age, peak oestradiol and oocytes aspirated).
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Results |
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The multiple pregnancy rate in the conventional IVF arm was found to be 21.5% (30/139). Twins comprised 26 sets of 30 multiple pregnancies, all of which were dizygotic. Four sets of triplets were found in this groupthree were trizygotic and one dizygotic. The overall rate of MZT in this group was 0.72% (1/139).
The addition of micromanipulation (ICSI with or without AH) to IVF resulted in 463 pregnancies. Of these, 82 were multiple pregnancies (17.7%). Twins comprised 63 of the 82 multiple pregnancies, 62 were dizygotic and one was monozygotic. Triplets were diagnosed in 19 of these 463 pregnancies, 16 were trizygotic and three dizygotic. The overall rate of monozygotic twinning in the IVFmicromanipulation group was 0.86% (4/463). If the rate of monozygosity is calculated by dividing the number of monozygotic twins found by the number of implanted embryos (and not the number of pregnant patients), the `true' rate of monozygosity is slightly lower, as the denominator takes into account the dizygotic twins as two implantations, although this calculation was not made in previous studies (Table I).
Nearly 80% of the 126 sets of twins and triplets reached live delivery (99/126). Precise data on all placentae and membranes were not available.
Spontaneous reduction to singleton from twins or to twins from triplets occurred in five cases. Interventional reduction of triplets to twins in two cases of trizygotic triplets led to one late abortion of all three triplets, and to one ongoing twin pregnancy, which delivered at term. Analysis of the seven cases of MZT did not disclose any specific pattern, either in patient or in treatment variables, that could be construed as indicative of a monozygotic-prone profile. Patients' ages and indication for treatment were representative of the whole group, as were the type of medication used, dose of HCG, peak oestradiol levels, number of oocytes aspirated or number of embryos replaced. Monozygotic patients' demographic, treatment and outcome data are concluded in Table II.
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Discussion |
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Six of our cases showed a biamniotic monochorionic membrane configuration for the monozygotic twins and the seventh case was demonstrated to have a mono-amnioticmonochorionic membrane configuration for the twins, which demonstrates the time-frame of embryo splitting during hatching. Two of the seven cases demonstrated mono-amniotic twins in a dizygotic triplet (cases 2 and 3, Table II). This is rare event, and both patients had an extremely premature delivery.
Previous reports have described an increased incidence of MZT with assisted reproduction techniques (Table III). The incidence of MZT in the general population has been calculated to be ~1/250 (0.42%) live births, and could actually be higher if all clinical pregnancies were included (Bulmer, 1970
; Saito et al., 2000
).
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Alikani et al. (1994) reported their experience with MZT in 737 pregnancies achieved after IVFembryo transfer, ~75% of these arose after various forms of zonal manipulation (Alikani et al., 1994). These included subzonal insemination (SUZI), partial zona dissection (PZD), zonal drilling, or AH of embryos. Six sets of MZT or triplets were described, resulting in an incidence of 0.84%, approximately twice the expected frequency. Here, the authors propound zonal manipulation procedures as a possible cause of MZT.
Zonal manipulation was also cited (Slotnick and Ortega, 1996) as a major factor in the increased incidence of mono-amniotic twins in the IVFembryo transfer (micromanipulation) population. They found an incidence of 3.49% (5 /143), more than seven times the background rate of twinning. Hershlag and colleagues (1999) noted a significantly increased rate of MZT after mechanical assisted hatching after conventional IVF, which reached a rate of 1.2% per embryo transfer (Hershlag et al., 1999). Schieve et al. (2000) calculated the odds ratio of monozygotic twinning after assisted hatching in assisted reproduction pregnancies to be 3.23.8, and they considered the contribution of AH to be significantly beyond that of the ovulation induction drugs (Schieve et al., 2000
).
Wenstrom et al. (1993) counted seven monozygotic pregnancies of a total of 218 (3.2%). (Wenstrom et al., 1993) These constituted 9.8% of all multiple gestations, whereby the type of assisted reproduction treatment had no effect on the incidence of monozygotic twinning. The authors theorized that assisted reproduction has an effect on the timing of embryonic events that lead to an increased incidence of MZT, regardless of the specific assisted reproduction used.
Additional reports have highlighted the possibility of higher-order monozygous pregnancies after assisted reproduction treatment. Three cases of dizygotic triplet pregnancy, in addition to those reported here, have been described in detail (Avrech et al., 1993; Steiner and Ojakangas, 1994
; Inion et al., 1998
). All three arose after IVF without micromanipulation. Avrech et al. (1993) reported good obstetric results, all three babies delivered thrived, whereas Steiner and Ojakangas (1994) reported delivery at 28 weeks with subsequent neonatal death of two of the three infants. Inion et al. (1998) described monozygotic intrauterine twins and a tubal pregnancy after replacement of two embryos. This combination of heterotopic pregnancy and dizygotic triplets is extremely rare. A trizygotic quadruplet pregnancy has also been described (Biljan et al., 1995
) after transfer of three grade I embryos after IVF, again without micromanipulation. Salat-Baroux and colleagues (1994) described trizygotic quintuplets (mono-amniotic triplets with two additional sacs and embryos) after transfer of four grade I embryos after IVF without micromanipulation (Salat-Baroux et al., 1994
). This patient aborted after an attempt at fetal reduction at 13 gestational weeks. These cases demonstrate that higher-order MZT may also be independent of micromanipulation.
Multicentric experience with MZT after blastocyst transfer has been reported (Behr et al., 1999). The incidence of MZT in this specific setting was calculated to be 5% (10/199), which is higher than the background incidence of MZT by a factor of 10. Abusheikha et al. (2000) reported on 11 monozygotic multiple pregnancies from two assisted reproduction centres of 718 pregnancies (Abusheikha et al., 2000
). Six of the eleven occurred after ICSI.
Finally, Sills and colleagues reported on 23 sets of monozygotic twins from 1911 assisted reproduction pregnancies (1.2%) (Sills et al., 2000). Their data confirm that no correlation was found between zonal manipulative techniques and MZT. This group of investigators concluded that the most likely cause of MZT in the setting of assisted reproduction was the increased number of embryos transferred to the uterus. The rate of MZT was approximately three times the background rate, closely reflecting the number of embryos transferred per cycle.
A number of hypotheses may be advanced in an effort to explain the increased incidence of MZT in micromanipulation or blastocyst cycles. Alikani et al. (1994) suggested that artificially induced structural changes in the ZP may lead to complications in the normal process of zonal lysis (Alikani et al., 1994). The embryo might bypass its own mechanism of local zonal lysis and `choose' to escape through the already established path. The dimensions of this gap could restrict the emerging embryo and encourage splitting, leading to MZT. A naturally thin or irregular zona pellucida may affect the hatching process in the same way, if during the blastocyst stage the zona disintegrates at more than one spot. This could be one of the mechanisms to explain the age-related increase of MZT, as zona thickness is more irregular with age (Cohen and Feldberg, 1991
).
Edwards and colleagues discussed the incidence of MZT after IVF without zonal manipulation (Edwards et al., 1986). Importantly, they noted that most MZT arise spontaneously in vivo from a single embryo (monozygotic twins), conversely, many MZT arising from IVF were accompanied by one or more sibling embryos, i.e., monozygotic triplets or higher order twinning. They concluded that the nature of embryonic growth in vitro predisposes to twinning, as opposed to in-vivo growth after ovarian stimulation. A possible `hardening' of the human ZP in vitro, after exposure to artificial media, as opposed to salpingeal or uterine secretions, could lead to increased fragility or brittleness and thence `fracture' the ZP, or cell-to-cell adhesion might be disturbed after in-vitro culture. Sills et al. (2000) postulated that the increased rate of MZT is a simple reflection of an increase in the number of embryos transferred to the uterus, and that the rate of MZT per transferred embryo is not different from that found in the general population (Sill et al., 2000). This is not borne out by the data (Behr et al., 1999
; Blickstein et al., 1999
; Saito et al., 2000
) which described increased rates of MZT in single replaced embryos and blastocysts. Derom et al. (1987) hypothesized that ovarian stimulation itself might cause hardening of the ZP, as observed in animals by Lungo (Lungo, 1981
). If this hardening is not uniform, some weak spots could be formed through which the blastocyst might herniate. These authors also calculated a higher rate of monozygosity in ovulation induced triplets than in induced twins.
It is interesting that five of our cases were dizygotic triplets, whereas we identified in the same cohort of patients only two cases of monozygotic twins. Our literature search revealed 95 sets of monozygotic twins after assisted reproduction as opposed to 18 sets of dichorionic triplets or higher-order monozygotic pregnancies (Table III). The ratio of triplets to twins in nature is significantly less than that found in the setting of assisted reproduction. The mechanism responsible for the irregular distribution of MZT triplets after assisted reproduction is a matter of speculation, which warrants additional study.
Two possible biases limit our ability to draw firm conclusions from this study. Firstly, the low incidence of this phenomenon makes a large series difficult to accumulate. To be able to achieve conclusions with statistical significance, sample sizes of 10 00020 000 cases are needed. These requirements necessitate multicentric cooperation or meta-analysis. Secondly, because we relied on the early diagnosis of pregnancy sacs and membranes to predict zygosity accurately, we might be underestimating the true incidence of MZT in cases of dichorionic biamniotic monozygotic twinning; our ultrasound criteria might mistakenly label these pregnancies as non-MZT, and accurate post-natal follow-up data on all same-sex multiple pregnancy children was not available. However, if these data are inaccurate, they only underestimate the true higher incidence of MZT.
The cumulative combined MZT rate of all published series was 1.51%, approximately three times the background rate in the untreated population (Table III). It is significant that in all series reported, the incidence of MZT was increased, regardless of its being subsequent to ovulation induction, IVF and/or micromanipulationof any typemost often assisted hatching or ICSI (Edwards et al., 1986
; Wenstrom et al., 1993
; Alikani et al., 1994
; Slotnick and Ortega, 1996
; Hershlag et al., 1997
; Blickstein et al., 1999
; Saito et al., 2000
; Sills et al., 2000
)(Table III
). This finding supports the surmise that the ovulation induction stimulus could be a major influence promoting MZT in assisted reproduction, as opposed to the current thinking implicating zonal manipulation. The basic underlying common denominator of these three situations is ovulation induction therapy, which seems to predispose either to monozygotic twinning or enhanced survival of monozygotic twins after their formation. It is possible that improved endometrial conditions after gonadotrophin therapy encourage monozygotic implantation, or that the biochemical milieu of the uterine cavity after gonadotrophin therapy encourages asymmetrical ZP hatching, independently of zonal manipulation procedures done in vitro. Each of our cases represents a different arm of assisted reproduction therapies, gonadotrophin therapy, IVFembryo transfer and micromanipulation and embryo transfer. This leads us to the same conclusion as Derom and his associatesthat gonadotrophin treatment can increase the incidence of MZT in all patients so treated (Derom et al., 1987
). The phenomenon of MZT, although uncommon, is another factor to take into account during assisted reproduction treatment. It would appear that the dizygotic triplet combination is even more common than one would expect, respective to the monozygotic twin. Ovarian stimulation for ovulation induction or IVFembryo transfer should be undertaken carefully, and the number of induced follicles limited, or the number of embryos replaced limited, in order to minimize multiple pregnancies. Extra consideration should be applied to account for the increased multiple pregnancy rate, partially due to an increased rate of MZT (Derom et al., 1993
). The problems of fetal reduction are also amplified in cases of monozygosity, so that this modality may not always be available to fall back on. This short series also emphasizes the extremely poor obstetric outcome of monozygotic multiple pregnancies, another reason to exercise caution in decision-making in assisted reproduction treatment.
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Notes |
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References |
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Submitted on June 26, 2000; accepted on March 9, 2001.