Naturally conceived twins with monochorionic placentation have the highest risk of fetal loss

Authors


Abstract

Objective

The aim of this study was to estimate the rate of fetal loss in dichorionic (DC) and monochorionic (MC) twin pregnancies stratified according to zygosity and method of conception.

Methods

In a prospective multicenter observational study women with a twin pregnancy had an ultrasound scan before 14 + 6 weeks' gestation in order to determine chorionicity. The fetal loss rate, the perinatal, neonatal and infant mortality rates and the frequency of very preterm labor were estimated for the different types of twin.

Results

Among the 495 pregnancies (421 DC and 74 MC) 229 (46%) were conceived naturally and 266 (54%) by assisted reproduction (AR). Outcome data for 945 liveborn babies were obtained. The spontaneous miscarriage rate before 24 weeks' gestation was 10.9% (7/64) among naturally conceived MC compared to 3.0% (5/165) for naturally conceived DC twins (P < 0.05). For twins conceived by AR the corresponding figures were 0% (0/10) and 0.4% (1/256). The odds ratio (OR) for very preterm birth—before 28 weeks' gestation—was 4.2 for MC twins compared to DC twins. The relative risk of fetal loss or death among DC twins was 20% of the risk for MC twins.

Conclusion

The risk of fetal loss, very preterm delivery and neonatal/infant death is significantly higher among twins with MC compared to DC placentation. Twins conceived by AR have a much lower risk of MC placentation. The risk of losing one or both twins seems higher among naturally conceived twins compared to twins conceived by AR, despite the fact that the maternal age was higher among the mothers of the AR twins. Copyright © 2006 ISUOG. Published by John Wiley & Sons, Ltd.

Introduction

During the last decade the proportion of twin pregnancies has risen, primarily because of delayed childbearing and the use of assisted reproductive technologies. However, twin pregnancies have a higher incidence of complications than do singletons, from the first trimester until the delivery of the second fetus. There are two major types of twin gestation: dizygotic (DZ) and monozygotic (MZ). MZ twins occur sporadically, while the incidence of DZ twins increases with advancing age, parity and with induction of ovulation. Chorionicity denotes the type of placentation. In the monochorionic–diamniotic (MC–DA) placenta the septum consists of two layers of amnion, while in the dichorionic (DC) placenta it has two layers of both chorion and amnion. Monochorionic (MC) twins are always MZ, while DC twins can be MZ or DZ.

The perinatal, neonatal and infant mortality in twins seems to be 2–4-fold higher than in singletons1–3. These figures come from registry studies, and therefore they do not allow discrimination between the different types of twin. The differences between perinatal and neonatal mortality rates and preterm birth between twins conceived naturally or by assisted reproduction (AR) have been evaluated in different studies without taking chorionicity into account1, 2, 4. In a recent review by Helmerhorst et al.2, data from 25 different studies were pooled. The perinatal mortality was 40% lower among AR twins compared to that in twins conceived naturally. None of these studies was, however, stratified according to chorionicity. A prospective study5 reported that the rate of miscarriage as well as the rate of perinatal mortality was higher in twins than in singletons, but especially high among MC twins. Although Baghdadi et al.6 did not find any difference in the overall fetal loss rate between MC and DC twins in a prospective study of 238 consecutive twins from one department, they found different patterns of fetal loss according to chorionicity.

The aim of this study was to estimate the difference in rates of fetal loss between DC and MC twin pregnancies stratified according to zygosity and method of conception.

Patients and Methods

From November 1999 to May 2003 women with twin pregnancies were included in a prospective multicenter observational study at five university centers of fetal medicine (four in Denmark and one in Sweden). The women were invited to join the study when a twin pregnancy was diagnosed before 14 + 6 weeks' gestation as estimated from the crown–rump length (CRL) or biparietal diameter (BPD). Only 3.2% were included before 11 weeks. Half of these twins were conceived naturally, the other half by AR. Exclusion criteria were maternal age below 18 years, or the woman was not fluent in Danish. The Scientific Ethics Committee and the Data Protection Agency approved the study protocol, and written informed consent was obtained from all participants. At the time of inclusion a transabdominal or transvaginal ultrasound scan was done to determine chorionicity. The pregnancy was classified as DC if two separate placentae were seen or the intertwin membrane (twin peak or lambda sign) was seen. In MC pregnancies, there was a single placenta and no lambda sign7. A nuchal translucency (NT) scan was done if the gestational age was below 13 + 6 weeks at the time of inclusion and the woman had not had chorionic villus sampling (CVS) according to the regulations of the Danish National Board of Health from 1994. The risk assessment was done according to the guidelines of The Fetal Medicine Foundation (FMF)8. The MC pregnancies were seen at 16 weeks' gestation in order to rule out early twin-to-twin transfusion syndrome (TTTS). All pregnancies had an anomaly scan including biometry in week 19, fetal echocardiography in week 21 and a cervical assessment scan in week 23 with new biometries. Further observation during pregnancy was done according to the departments' guidelines, which in most cases included an ultrasound scan every 2–3 weeks.

To assess zygosity we analyzed DNA from all twin pairs with the same sex, using eight highly polymorphic microsatellites: tumor necrosis factor-alpha (TNF-α), D21S11, D21SS1412, D18S535, D13S258, D18S386, D21S1411 and D13S631. Primers can be obtained from the Human Genome Database (GDB) (http://gdbwww.gdb.org/). Polymerase chain reaction (PCR) products were analyzed on an ABI PRISM® 310 Genetic Analyzer using GENESCAN software (ABI) and GeneScanTM Size standard (PE Applied BioSystems, Foster City, CA, USA) as a size marker. All the markers had a heterozygosity level greater than 0.80. If twins shared alleles for all eight markers, they were classified as MZ twins9. DNA was isolated from cord blood by the salting out method. If the procedure failed, a filter blood spot was obtained after birth and a 3 mm-blood spot was punched out. The blood spot was soaked in 20 µL 0.2 M sodium hydroxide. The tubes were incubated at 75 °C for 5 min and neutralized by the addition of 10 µL 40 mM Tris, pH of 7.5. A sample of 5 µL was used for PCR amplification.

The obstetric history and information about whether the twin pregnancy was conceived naturally or by AR were obtained at inclusion. Information about the fetal outcome (mode of delivery, presentation, birth weight, Apgar score, pH and BE in the umbilical artery and admission to the neonatal or pediatric department) was retrieved from the files. The participants were also contacted by mail or by telephone 8 months or more after delivery. In those cases where it was not possible to reach the family, the babies' personal registration numbers were checked for admittance to any Danish hospital in the National Hospital Registry. In case of hospitalization, discharge reports were sought. In Denmark all admissions to hospital are reported to the National Hospital Registry.

The sample size was determined as 484 twin pregnancies in order to detect a difference in fetal loss rate between MC and DC pregnancies of 7% using a level of significance (P) of 0.05 and a power of 0.90. We estimated the sample size using an expected ratio between MC and DC twins of 0.20 (402 DC and 82 MC). We expected the proportion of AR pregnancies to be 20% with 5% MC10, while among the naturally conceived twins 20% were expected to be MC11. In Denmark the prevalence of twins was 2.1% in the study period.

The fetal loss rate (before 24 weeks' gestation), perinatal mortality (PM—number of intrauterine deaths, intrapartum deaths and neonatal deaths before 7 days per 1000 children born with a gestational age of 24 weeks) and neonatal mortality (NM—number of deaths within 28 days after birth per 1000 children born with a gestational age of 24 weeks) and infant mortality (death of liveborn babies within 1 year) were determined in twins with MC and DC placentation and in twins conceived naturally and by AR, and compared using the Chi-square test. A Kaplan Meier survival plot with logrank test was used to compare the survival curves for DC and MC twins. The null hypothesis was that the risk of death was the same in the two groups. The groups were compared with the Chi-square test and the hazard ratio was calculated. Student's t-test was used to analyze differences in mean and SD values. A P of less than 0.05 was regarded as being statistically significant. Odds ratio (OR) was used to determine how many times the ratio of response and non-response in one group is greater or smaller than in the other group.

Results

Five hundred and twenty-four pregnant women were initially enrolled in the study, but 29 women were excluded for different reasons (wish to drop out or failure to show for the booked appointment). In the remaining 495 (421 DC and 74 MC) pregnancies 46% were conceived naturally, while 54% had AR either as in-vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), egg donation or intrauterine insemination with or without stimulation of the ovaries. Outcome data for 945 liveborn babies were obtained and all had a direct or indirect follow-up. Information on zygosity was obtained in 98% of the twins.

Chorionicity, selective termination and maternal age

The ultrasound scan at inclusion assessed 64 (28%) of the naturally conceived twins and 10 (4%) of the twins conceived by AR to be MC–DA, and 1% of all the twins to be monoamniotic (MA). Among the 10 MC twins conceived by AR three were conceived with ICSI, one by egg donation and six by IVF. In four pregnancies classified by ultrasound as MC, the zygosity test showed DZ twins. These cases were classified as DC. Twenty-four (27%) of the naturally conceived MZ twins had a DC placentation. Four (1.5%) of the DC twins conceived by AR were MZ. Selective termination was carried out in four cases among the DC twins. All had a screen positive NT scan (risk of trisomy 21 above 1 : 300 based on maternal age and NT), and subsequently CVS diagnosed a trisomy. Two selective terminations were done among the MC twins by laser coagulation of the umbilical cord because of TTTS.

The mean maternal age was 31.2 ± 6.3 years among the women with naturally conceived twins compared to 33.3 ± 4.1 years for the AR twins (P < 0.0005; Table 1, Table 2), and 32.6 ± 5.0 years among DC twins compared to 30.9 ± 6.8 (P < 0.05; Table 3). Among the DC as well as among the MC twins 62% were nulliparous, but among the twins conceived by AR, 76% were nulliparous compared to 54% among the naturally conceived twins.

Table 1. Fetal loss rate, perinatal, neonatal and infant mortality, sex distribution and birth weight data among twins conceived naturally with dichorionic and monochorionic placentation
 DichorionicMonochorionicPOdds ratio (95% CI)Total
No of pregnancies (% of total no of pregnancies)16572.1%6427.9%  229 
No of fetuses at inclusion330 128   458 
Spontaneous miscarriages53.0%710.9%<0.053.9 (1.2 to 12.9)125.2%
Induced abortions10.6%21.6%  31.3%
Selective terminations10.6%23.1%0.4 30.7%
Spontaneous reductions before 24 weeks10.6%11.6%  20.4%
Fetal loss before 24 weeks (% of fetuses at inclusion)144.2%2116.4%<0.0014.4 (2.2 to 9.0)357.6%
Spontaneous reductions after 24 weeks0 10.9%  1 
No of liveborn babies316 106   422 
No of deliveries159 55   214 
Perinatal death (up to 6 days)51.6%21.9%  71.7%
Neonatal death (7–28 days)00.0%00.0%  00.0%
Death after day 29 to follow-up at 8 months or more00.0%32.8%  30.7%
Perinatal mortality (‰of born babies)515.8328.00.9 818.9
Neonatal mortality (‰of liveborn babies)515.8218.9  716.6
Infant mortality (‰of liveborn babies)412.7547.20.093.0 (0.8 to 10.9)921.3
No of babies alive after 8 months or more (% of fetuses at inclusion)31194.2%10179%<0.001 41290%
No of boys173 56   22954%
No of girls143 50   19346%
Pregnancies with same sex94 55   14970%
Maternal age ± SD (years)31.6 ± 6 30 ± 7 0.13 31.2 ± 6.3 
Mean birth weight of twin 1 ± SD (g)2608 ± 628 2336 ± 664 0.01   
Mean birth weight of twin 2 ± SD (g)2510 ± 627 2279 ± 675 0.03   
Mean gestational age ± SD at delivery (weeks)37 ± 3 35 ± 3 0.01   
Table 2. Fetal loss rate, perinatal, neonatal and infant mortality, sex distribution and birth weight data among twins conceived by assisted reproduction with dichorionic and monochorionic placentation
 DichorionicMonochorionicPOdds ratio (95% CI)Total
No of pregnancies (% of total no of pregnancies)25696.2%103.8%  266 
No of fetuses at inclusion512 20   532 
Spontaneous miscarriages10.4%0   10.4%
Induced abortions00.0%0   0 
Selective terminations31.2%0   30.6%
Spontaneous reductions before 24 weeks31.2%0   30.6%
Fetal losses before 24 weeks (% of fetuses at inclusion)81.6%0   81.5%
Spontaneous reductions after 24 weeks10.2%0 
No of liveborn babies503 20   523 
No of deliveries255 10   265 
Perinatal death (up to 6 days)20.4%15.0%0.4 30.6%
Neonatal death (7–28 days)20.4%0   20.4%
Death after day 28 to follow-up at 8 months or more0 0   00.0%
Perinatal mortality (‰of born babies)36.0150.0  47.5
Neonatal mortality (‰of liveborn babies)48.0150.0  59.6
Infant mortality (‰of liveborn babies)48.0150.0 6.6 (0.7 to 61.6)59.6
No of babies alive after 8 months or more (% of fetuses at inclusion)49997.5%1995%0.93 51897%
No of boys265 8   27352%
No of girls238 12   25048%
Pregnancies with same sex137 10   14755%
Maternal age ± SD (years)33 ± 4 35 ± 4 0.1 33.3 ± 4.1 
Mean birth weight of twin 1 ± SD (g)2542 ± 542 2179 ± 779 0.04   
Mean birth weight of twin 2 ± SD (g)2464 ± 549 2136 ± 681 0.07   
Mean gestational age at delivery ± SD (weeks)36 ± 3 34 ± 4 0.03   
Table 3. Fetal loss rate, perinatal, neonatal and infant mortality, sex distribution and birth weight data among all twins conceived with dichorionic and monochorionic placentation
 DichorionicMonochorionicPOdds ratio (95% CI)Total
No of pregnancies (% of total no of pregnancies)42185.1%7414.9%  495 
No of fetuses at inclusion842 148   990 
Spontaneous miscarriages61.4%79.5%<0.00057.2 (2.4 to 22.2)132.6%
Induced abortions10.2%22.7%  30.6%
Selective terminations41.0%22.7%0.52 61.2%
Spontaneous reductions before 24 weeks41.0%11.4%0.77 51.0%
Fetal losses before 24 weeks (% of fetuses at inclusion)222.6%2114.2%<0.016.1 (3.3 to 11.5)434.3%
Spontaneous reductions after 24 weeks10.2%1   2 
No of liveborn babies819 126   945 
No of deliveries414 65   479 
Perinatal death (up to 6 days)70.9%32.4%  101.1%
Neonatal death (7–28 days)20.2%0   20.2%
Death after day 28 to follow-up at 8 months or more0 32.4%  30.3%
Perinatal mortality (‰of born babies)89.8431.50.08 1212.1
Neonatal mortality (‰of liveborn babies)911.0323.80.44 1212.7
Infant mortality (‰of liveborn babies)911.0647.6<0.014.4 (1.6 to 12.8)1515.9
No of babies alive after 8 months or more (% of fetuses at inclusion)81196.3%12282.4%<0.015.5 (3.2 to 9.7)93394%
Twins with unknown zygosity5 2   71.4%
No of boys438 64   50253%
No of girls381 62   44347%
Pregnancies with same sex231 65   29662%
Maternal age ± SD (years)33 ± 5 31 ± 7 0.01   
Mean birth weight of twin 1 ± SD (g)2568 ± 577 2311 ± 679 <0.01   
Mean birth weight of twin 2 ± SD (g)2482 ± 580 2256 ± 672 <0.01   
Mean gestational age at delivery ± SD (weeks)36.4 ± 3 35 ± 4 <0.01   

Fetal loss

The spontaneous miscarriage rate was 9.5% (n = 7) among the MC compared to 1.4% (n = 6) for the DC twins (P < 0.0005), with OR = 7.2 (Table 3). For the naturally conceived twins the figures were 10.9% (n = 7) for MC and 3.0% (n = 5) for DC (P < 0.05), with OR = 3.9 (Table 1), as compared to 0% and 0.4% (n = 1), respectively, among the twins conceived by AR (Table 2). Among the DC twins the fetal loss rate before 24 weeks was 4% (n = 14) among the naturally conceived twins compared to 1.6% (n = 8) for the twins conceived by AR (P = 0.05) giving an OR of 2.8 (Table 4). Among the 28 MZ–DC pregnancies there were no fetal losses and only one perinatal death. The fetal loss rate was 14% for the MZ–MC compared to 0% among the MZ–DC twins. Two of the four MA twins were lost before 24 weeks' gestation, one spontaneously and one by induced abortion because of cord entanglement (Table 5).

Table 4. Fetal loss rate, perinatal, neonatal and infant mortality rates, sex distribution and birth weight data among dichorionic (DC) twins conceived by assisted reproduction or naturally
 Assisted reproductionNatural conceptionPOdds ratio (95% CI)MonozygoticAll
AllMonozygoticUnknown zygosityAllMonozygoticUnknown zygosity
No of pregnancies (% of total no of DC pregnancies)25660.8%41416539.2%2412  28 
No of fetuses at inclusion512   330 56 
Spontaneous miscarriages10.4% 153.0% 50.088.0 (0.9 to 68.8)  
Induced abortions0   10.6% 1 
Selective terminations31.2% 310.6% 1 
Spontaneous reductions before 24 weeks31.2% 310.6% 1 
Fetal losses before 24 weeks (% of fetuses at inclusion)81.6% 814 4% 140.052.8 (1.2 to 6.7)0 
Spontaneous reductions after 24 weeks10.4%  0 
No of liveborn babies503  12316  8  56 
No of deliveries255  6159  4  25 
Perinatal death (up to 6 days)20.4%0151.6%12  1 
Neonatal death (7–28 days)20.4%02  00 
Death after day 28 to follow-up at 8 months or more0 0  0.0% 0 
Perinatal mortality (‰of born babies)36.00 515.8  0.36 117.9
Neonatal mortality (‰of liveborn babies)48.0  515.8  0.51 117.9
Infant mortality (‰of liveborn babies)48.0  515.8 117.9
No of babies alive after 8 months or more (% of fetuses at inclusion)49997.5%  31194.2%  0.02   
Twin pregnancies with unknown zygosity   3   2 
No of boys265   173 
No of girls238   143 
Twin pregnancies with same sex137   94 
Maternal age ± SD (years)33 ± 4 34 ± 3 32 ± 6 36 ± 3 <0.01  29.3 ± 7.3 
Mean birth weight of twin 1 ± SD (g)2542 ± 542 2306 ± 191 2608 ± 628 2538 ± 522 0.27 2504.3 ± 484 
Mean birth weight of twin 2 ± SD (g)2464 ± 549 2214 ± 201 2510 ± 627 2388 ± 567 0.36 2358.1 ± 522 
Mean gestational age at delivery ± SD (weeks)36 ± 3 36 ± 1 37 ± 3 36 ± 3 0.15  36.0 ± 2.6 
Table 5. Fetal loss rate, perinatal, neonatal and infant mortality, sex distribution and birth-weight data among monochorionic twins conceived by assisted reproduction or naturally
 Assisted reproductionNatural conceptionTotal
AllMAUnknown zygosityAllMAUnknown zygosityPAllMAUnknown zygosity
  1. MA, monoamniotic.

No of pregnancies (% of total no of pregnancies)10 (13.5)1164 (86.5%)316 74417
No of fetuses at inclusion20  128   1480 
Spontaneous miscarriages (%)0  7 (10.9)170.57 (9.5)1 
Induced abortions (%)0  2 (3.1)12 2 (2.7)1 
Selective terminations (%)0  2 (3.1)02 2 (2.7)0 
Spontaneous reductions before 24 weeks (%)0  1 (1.6)01 1 (1.4)0 
Fetal losses before 24 weeks (% of fetuses at inclusion)0  21 (16) 21 21 (14)  
Spontaneous reductions after 24 weeks0  1 1 1  
No of liveborn babies20  106 6 126  
No of deliveries10  55 3 65  
Perinatal death (up to 6 days) (%)1 (5.0)012 (1.9)01 3  
Neonatal death (7–28 days)0 0  
Death after day 28 to follow-up at 8 months or more0  3 (2.8)00 3  
Perinatal mortality (‰of born babies)1 (50.0)  3 (28.0)  0.94 (31.5)  
Neonatal mortality (‰of liveborn babies)1 (50.0)  2 (18.9)  0.93 (23.8)  
Infant mortality (‰of liveborn babies)1 (50.0)  5 (47.2)   6 (47.6)  
No of babies alive after 8 months or more (% of fetuses at inclusion)19 (95.0)  101 (78.9)  0.3122 (82.4)  
No of boys8  56   64  
No of girls12  50   62  
Twin pregnancies with same sex10  55   65  
Maternal age ± SD (years)35.5 ± 4  30.2 ± 7  0.0230.9 ± 6.8  
Mean birth weight of twin 1 ± SD (g)2178.7 ± 779  2336 ± 664  0.512311.1 ± 678.8  
Mean birth weight of twin 2 ± SD (g)2136.2 ± 681  2279 ± 675  0.542255.7 ± 672.4  
Mean gestational age at delivery ± SD (weeks)34.4 ± 4  35 ± 3  0.4135.3 ± 3.5  

The perinatal mortality was 9.8 (n = 8) per 1000 liveborn babies for DC twins and 31.5 (n = 4) per 1000 liveborn babies for MC twins (P = 0.08). The neonatal mortality was 11.0 (n = 9) per 1000 liveborn babies for the DC twins compared to 23.8 (n = 3) per 1000 liveborn for the MC twins, and infant mortality was 11.0 (n = 9) per 1000 liveborn for DC and 47.6 (n = 6) per 1000 liveborn MC twins (P = 0.01) (Table 3). The infant mortality was 47.6 per 1000 liveborn babies for MC twins compared to 11 per 1000 liveborn DC twins, OR = 4.4 (95% CI, 1.6–12.8).

Figure 1 shows a Kaplan Meier plot of the survival according to chorionicity. The rate of fetal loss or death rose steeply from 13 to 40 weeks' gestation in MC twins, while the rate of fetal loss or death increased slightly until 37 weeks' gestation for DC twins (P < 0.0005). The relative risk of fetal loss or death among DC twins was 20% of the risk for MC twins (hazard ratio = 0.20).

Figure 1.

Survival curves for monochorionic (solid line) and dichorionic (dashed line) twins from 15 weeks' gestation to the age of 8 months.

Preterm delivery

The rate of very preterm delivery (VPTD)—before 28 weeks' gestation—was 8% (n = 5) for MC twins and 2% (n = 8) for DC twins (P < 0.05), with an OR of 4.2, while 17% (n = 11) of the MC twins were delivered before 32 weeks compared to 8% (n = 29) (P < 0.05) for the DC twins with an OR of 2.6 (data not shown). There were no significant differences between VPTD and preterm delivery (PTD) rates in twins conceived by AR compared to twins conceived naturally in any of the two placentation groups. Figure 2 shows that the rate of birth before 32 weeks' gestation was 2–3 times higher among MC compared to DC twins for a given gestational age.

Figure 2.

Rates of preterm birth for all the twins for a given gestational age according to placentation. equation image, monochorionic twins; equation image, dichorionic twins.

Birth weight

Twins with MC placentation had a lower birth weight for both naturally conceived twins and twins conceived by AR (Table 1, Table 2), but there were no differences in birth weight between MC (Table 5) and DC (Table 4) twins in either those conceived by AR or those conceived naturally, and no differences in birth weight between the MZ–DC and the DZ–DC. There was no difference in weight discordance between the groups.

Discussion

This study demonstrated that twins with MC placentation have an increased risk of preterm birth compared to twins with DC placentation. Our data further show that the fetal loss rate among naturally conceived twins with MC placentation is significantly higher than in twins with DC placentation, with an OR of 4.4 (95% CI, 2.2–9.0; Table 1). This difference was not seen among the twins conceived by AR, but the number of MC pregnancies in the AR group was small (Table 2). Our results thus show that naturally conceived MC twins seem to be the group of twins at highest risk of fetal demise. There does not seem to be a biologically plausible explanation for the difference between naturally conceived and AR MC twins, and it could be due to a type 1 error.

The perinatal, neonatal and infant mortalities in our prospective observational study were comparable to those in a recent Danish registry study12 and to a previous Danish registry study13. Pinborg et al. found—among 3393 ICSI/IVF twins and 10 239 control twins—a rate of infant mortality of 10.6 and 15.4 per 1000 compared to our finding of 9.6 and 21.3 per 1000 liveborn among twins conceived by AR and naturally conceived twins, respectively. The figures of Westergaard et al., among 1285 twins conceived by AR and 1291 naturally conceived twins, were 7.0 compared to 24.6 infant deaths per 1000 liveborn (P = 0.0069)13. Our figures are comparable with these registry-based studies, but are much lower than data from the Danish birth registry of 2003, where the neonatal mortality rate for twins was 29 per 1000 liveborn twin babies compared to 13 per 1000 liveborn twins in this study (Table 3). The differences in the infant mortality rate must be expected to be even higher3. A possible explanation could be that our multicenter study was carried out in university centers with intensive neonatal care units, while the two figures from the registry studies cover all departments in the country. Other studies from other countries have shown the same tendency2.

The differences in fetal loss and infant death among MZ twins with MC compared to MZ with DC placentation are similar to the results Machin et al.14 obtained in a study of 42 naturally conceived MZ–DC twins, which they compared to both DZ–DC and MZ–MC twins. From the East Flanders Prospective Twin Survey15 of 4767 twins with known zygosity there was also a significantly higher perinatal and neonatal mortality among MZ–MC compared to both MZ–DC and DZ twins.

Our results are in accordance with the two studies mentioned above14, 15 and with those of Sebire et al.5. They recruited women from one center in London, while our study is a multicenter Scandinavian prospective study. Our results therefore seem to confirm the statement that it is the chorionicity and not the zygosity that determines the outcome for twins. The distribution between DC and MC placentation was comparable to that of the study of Sepulveda et al.7, in which 30% (28% in our study) and 5% (4% in our study) were MC among the naturally conceived twins and the twins conceived by AR, respectively. They calculated the rate of identical twins from Weinberg's formula: number of identical twins = total numbers of twin − 2 × (total number of unlike sex pairs).

We classified zygosity in 98% of the same-sex twins with PCR. Using the Weinberg calculation would have given for the naturally conceived twins 65 identical twins compared to 88 after the zygosity test, and for the AR twins 29 identical twins compared to 14 after the zygosity test. This formula therefore seems to have major limitations for both naturally conceived and AR twins.

In our study the lambda sign had a high sensitivity to correctly classify chorionicity, confirming the findings of others16. The four misclassified cases are remarkable because, to our knowledge, this is the only study using zygosity with PCR to verify chorionicity. Our study showed that absence of the twin-peak sign does not exclude dichorionicity. We were able to obtain a zygosity test in as many as 98% of the included twin pregnancies, even though some experienced fetal loss, spontaneous reduction after 24 weeks' gestation or death shortly after birth. Unfortunately it was not possible to check the chorionicity in the placentae, thus we cannot be certain that some of the 28 MZ–DC twins were not in fact MC. None of the 28 cases did, however, show any signs of TTTS.

Maternal age was—not unexpectedly—statistically lower among the women with twin pregnancies who had conceived naturally compared to women who had AR, but there was no age difference between women with MC and DC placentation who conceived naturally or by AR. This is contrary to expectation, as DC placentation would be expected to arise more frequently in women of higher age, and MC placentation more frequently in women of younger age17.

We had a significantly higher PTD and VPTD rate among the MC compared to the DC twins, with an OR between 2.6 and 4.2. Sebire et al.5 also found a higher rate of PTD in MC (9.2%) compared to DC (5.5%) twins, although the difference was not significant.

Our overall 8% rate of PTD was comparable to the results of other studies2, and we found no difference between twins according to method of conception.

In conclusion, the risk of fetal loss, very preterm delivery and neonatal/infant death is significantly higher among twins with MC placentation than those with DC placentation. Twins conceived by AR have a much lower risk of MC placentation. The risk of losing one or both twins seems higher among naturally conceived twins compared to twins conceived by AR, despite the fact that the maternal age is higher among the mothers of AR twins. In future we will have to focus on twins with MC placentation, especially on those conceived naturally, in order to diminish the rates of fetal loss and infant mortality.

Ancillary