a The Division of Disease Control and Prevention, and the Center for Human Nutrition (CHN), Department of International Health, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD, USA.
b National Society for the Prevention of Blindness, Kathmandu, Nepal.
Correspondence: Dr Joanne Katz, Johns Hopkins School of Hygiene and Public Health, Division of Disease Control and Prevention, Department of International Health, Room 5515, 615 N Wolfe Street, Baltimore, MD 212052103, USA. E-mail: jkatz{at}jhsph.edu
Abstract
Background Twin pregnancies are common but there are few data on rates of twinning or survival of liveborn twin infants in developing countries.
Methods The rates of multiple births were calculated in a population-based cohort of married women of childbearing age who were enrolled in a randomized community trial to assess the impact of vitamin A or beta-carotene on maternal and infant health and survival.
Results The rate of twinning was 16.1 per 1000 pregnancies (7.4 if only twin pregnancies resulting in two liveborn infants were used). The rate for triplets and quadruplets was 0.19 and 0.06 per 1000 pregnancies. Twinning rates were higher among women of higher parity, but were not associated with maternal age. Twinning rates among twins where at least one was live born (or increased in utero survival) were 30% (95% CI : 1%, 71%) and 44% (95% CI : 9%, 89%) higher among women receiving vitamin A and beta-carotene supplements than placebo, after adjusting for maternal age, gestational age, and parity. The perinatal mortality rate was 8.54 times higher for twins than singletons, 7.32 higher for neonatal mortality, and 5.84 higher for cumulative 24-week mortality. This difference was reduced but not erased by adjusting for gestational age. No difference in survival of liveborn twin infants was seen by supplement group. A higher mortality rate among male twins was largely explained by gestational age.
Conclusions Multiple births are relatively common occurrences in rural Nepal, and carry a much higher mortality risk for the infants than for singletons. Vitamin A or beta-carotene supplementation appeared to increase the rate of twinning, or improve the survival of twins in utero, but did not increase twin survival after birth.
Keywords Nepal, vitamin A, beta-carotene, infant mortality, neonatal mortality, perinatal mortality, pregnancy, twins
Accepted 21 September 2000
Twin pregnancies are relatively common, but few population-based data from developing countries exist regarding the rates and epidemiology of twinning, nor of the prognosis for survival of twins in an environment where obstetric and health care services are minimal. The most comprehensive population-based data on twinning come from the Matlab Demographic Surveillance System.1,2 Twinning rates varied by maternal age, parity and season, and the introduction of family planning increased the rate of twinning. Both neonatal and infant mortality rates were several times higher among twins than singletons, and gender differences in infant but not neonatal mortality were evident among twins of unlike gender.
As part of a randomized community trial assessing the impact of vitamin A or beta-carotene supplementation for women of childbearing age on survival of women and infants, data on the rates of multiple births and their survival through 24 weeks of age were available for analysis.3,4 These data provided a unique opportunity to estimate the rate and survival of multiple births and associated risk factors in a population with low contraception rates, high maternal and infant mortality, and very little access to any antenatal or obstetric services.
Methods
The population described in this study were participants in a randomized community trial conducted in Sarlahi district, Nepal.3,4 All married women of childbearing age living in 270 wards in the district were eligible to participate in the trial. Wards were randomly assigned to one of three treatment groups: placebo, vitamin A or beta-carotene. All eligible women within those wards received a capsule containing the assigned treatment each week. Local village-based women were trained to ask about menses in the past week, ascertain pregnancy status by history, provide the assigned capsule, and record compliance. Women who declared themselves pregnant were enrolled in a more detailed protocol that included a questionnaire completed around 4 months of pregnancy, that recorded their age, pregnancy history, date of last menstrual period, morbidity, physical work and diet in the past week. Another questionnaire ascertaining similar information was also completed around 7 months gestation. Each pregnancy was followed until an outcome of miscarriage or stillbirth, live birth, or maternal death occurred. For liveborn infants, vital status was ascertained at 3 months and 6 months of age. A verbal autopsy was done to obtain information on date and cause of death. All pregnancies that were reported on or after July 1994 and whose outcome occurred prior to March 1997 were included in this analysis. This allowed for 6 months of follow-up of all liveborn infants prior to the termination of the study.
For pregnancies that did not result in at least one live birth, there was no information about the multiplicity of the pregnancy, or of the gender of stillborn infants. For pregnancies that resulted in at least one live birth, the number of live and stillborn infants in each pregnancy was ascertained by interview with the mother at 3 months postpartum, but only the gender of liveborn children was recorded. Since the zygosity of twins was unknown in this study, the method described by Weinberg5 and verified by Potter,6 was used to estimate rates of mono- and dizygotic twinning among liveborn twins. This method doubles the observed number of pairs of twins of unlike gender and uses this number to estimate the rate of dizygotic twins, and the remaining number of pairs out of the total are classified as monozygotic. Gestational age was calculated using the date of last menstrual period or the menstrual data recorded weekly. If neither of these was available, the gestational age as determined by history on the second or third trimester questionnaire was used.
The rates of multiple births were estimated by taking the number of multiple births divided by the number of pregnancies, and expressed per 1000 pregnancies. The twinning rate was calculated as the number of pregnancies in which there were two fetuses, regardless of whether one was stillborn, divided by the number of pregnancies ascertained in the study. Perinatal deaths were defined as those occurring within 7 days of birth among liveborn infants, and neonatal deaths as those within 28 days of birth. The cumulative 24-week mortality rate was calculated by taking all deaths of liveborn infants occurring within 24 weeks of birth, divided by the number of live births, and expressed as deaths per 1000 live births.
Logistic regression was used to identify risk factors associated with twinning, and to estimate the impact of treatment on twinning rates adjusted by various factors that might be associated with twinning risk. Relative risks were used to compare mortality rates of singletons with those of twins where both were live born. Pairwise odds ratios (OR) were used to assess the association of mortality within liveborn twin pairs.7,8 This OR is obtained by taking all possible pairs of liveborn twins and constructing a 2 x 2 table with numbers of pairs that are concordant for mortality (or survival) in diagonal boxes and the number of discordant pairs divided evenly between the other two boxes. The OR calculated from this table is the pairwise OR.
Results
There were a total of 15 868 pregnancies that ended in a live birth of at least one child during the follow-up period. The multiplicity of pregnancies with at least one liveborn infant was available for 15 726 (99.1%) pregnancies of 15 845 liveborn infants (Table 1). There were 116 pregnancies that resulted in two liveborn infants, and 137 pregnancies where 1 infant was live born, and 1 was stillborn. There were three sets of triplets, one with a liveborn infant and two stillbirths, and two where two infants were born alive, and one was stillborn. There was also one quadruplet, of which two infants were born alive and two were stillborn. This resulted in a twinning rate of 16.1 per 1000 pregnancies if both live and stillborn infants were counted (7.4 per 1000 if only liveborn twins were counted). This translates to a rate of 1 twin in 62 pregnancies (or 1 in 135 for liveborn twins only). The triplet and quadruplet rates were 0.32 and 0.06 per 1000 pregnancies, respectively (1 in 3125 and 1 in 16 667).
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There were no significant differences in twinning rates (live and stillborn combined) by maternal age, but pregnancies of parity two or higher were 1.59 times (95% CI : 1.18, 2.14) more likely to produce twins (Table 2). The twinning rate was higher among women who received vitamin A (adjusted OR = 1.30, 95% CI : 0.99, 1.71) and beta-carotene (adjusted OR = 1.44, 95% CI : 1.09, 1.89) than women who received placebo during the trial. The findings regarding maternal age, parity, gestational age and treatment effects were similar for twinning rates that included only liveborn twins. Gestational age was strongly associated with twinning, with twins having a shorter gestational age, even after adjusting for maternal age, parity and treatment effect. Other risk factors found not to be associated with twinning after adjustment for maternal age, parity, treatment group and gestational age were maternal smoking during pregnancy (OR = 0.98, P-value = 0.89), maternal mid upper arm circumference (OR = 0.984, P-value = 0.08), and seasonality (OR = 1.20, P-value = 0.24 for pre-monsoon versus monsoon, and OR = 1.05, P-value = 0.71 for pre-monsoon versus post-monsoon). There was also no interaction between maternal age and parity where both were continuous variables in the regression model (OR = 1.00, P-value = 0.83).
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For singletons and liveborn twins, 07-day, neonatal, and 24-week cumulative mortality was slightly higher for males than for females (Table 4). However, the gender difference was larger for twins than for singletons, especially during the period between 7 and 28 days. For singletons, the relative risks of death for males versus females was 1.14, 1.15 and 1.04 during the 07-day, neonatal, and 24-week period, respectively. For twins, the relative risks were 1.17, 1.41, and 1.14 for each of these periods. None of these were statistically different from one.
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Twinning rates appear to vary substantially across geographical areas and by ethnicity.915 Some of this variation is due to differences in the definition of twinning, and whether stillborn infants are included or not. An additional problem is the higher rate of twinning in hospital-based studies than would be observed in the population, since twins would be more likely to deliver in hospitals. Rates of 10.6 and 14.9 per 1000 pregnancies were reported for whites and blacks, respectively, in California between 1922 and 1958.9 The rate in the state of Georgia was 9.5 per 1000 pregnancies between 1974 and 1978.11 These rates were population-based and included stillbirths. A large hospital-based study in California found a rate of 8.9 per 1000 deliveries in a predominantly Mexican-American population.12 Among Fijians of Indian ethnicity, the twinning rate was 6.2 per 1000 pregnancies,13 but the rate was 9.0 per 1000 in a rural area outside of Bombay15 and in Bangladesh.1 The twinning rate, inclusive of stillbirths in our population-based study in Nepal was 12.6 per 1000 pregnancies in the placebo group (95% CI : 9.55, 15.65). This is somewhat higher than reported by other studies, but it is difficult to compare our rate with these other rates because the confidence intervals for many of the other studies were not given. However, if only the liveborn twins are used, the rate is 7.4 per 1000 pregnancies. The higher rate of twinning may be due to overreporting of stillbirths that accompany live births in this population, or there may have been underreporting of stillbirths in other populations. Alternately, the specific probing for stillbirths in this population may have led to higher rates of twinning than in other studies that relied on self-report without additional probing. Other population differences may be related to maternal age, parity and contraceptive use. Oral contraceptive use has been thought to increase the rate of twinning.16 This was seen in Bangladesh where the rates of twinning were higher in a family planning intervention area than in a control area.1 The use of oral contra-ceptives is likely lower than in Bangladesh, and maternal age and parity are likely similar. Hence, these factors are unlikely to explain the differences in the twinning rates.
While the rates of twinning may have been higher in Nepal than in Bangladesh, the ratio of dizygotic to monozygotic twins was comparable in the two populations (the ratio in Nepal was based only on liveborn twins, since gender was not available for twins where one was stillborn). Since the same method was used to estimate the rate of twinning by zygosity, this similarity reflects a comparable ratio of like to unlike gender pairs. The monozygotic twinning rate in Nepal was 3.3 per 1000, which is comparable to the relatively constant rates found worldwide to vary between 3 and 4 per 1000.10,17 The rate was 2.8, 4.7, and 3.6 per 1000 among Indians in Fiji, in rural India, and in Bangladesh, respectively.1,13,15 Rates for whites, blacks, Chinese and Japanese in California varied from 3.9 to 4.8 per 1000.9,12
As was seen in Bangladesh, rural India, and Indians in Fiji, the rates of twinning in Nepal increased with increasing parity. In Bangladesh, the rate of twinning was highest among women 35 years and older, but the only difference in twinning by maternal age observed in Nepal was between women under versus over 20 years, and this difference disappeared when the twinning rate was adjusted for parity. The pattern of twinning by maternal age seen in Nepal is more similar to that seen among twins in California, where the rate increased until about age 37, after which it declined.9 The variation in twinning rates by maternal age and parity is largely due to variations in dizygotic twinning, and is thought to be associated with gonadotrophin production.9,17 Social factors such as smoking, maternal nutritional status, seasonality and oral contraceptive use may also be associated with dizygotic rather than monozygotic twinning rates, but we were unable to examine this issue. However, smoking during pregnancy, maternal mid upper arm circumference in the second trimester, and seasonality were not associated with overall twinning rates in this study. Oral contraceptive use was not assessed but is known to be very low in this population.
There was more than an 8-fold difference in the neonatal mortality between twins and singletons in Nepal. The neonatal mortality rate was five times higher for twins than singletons in Bangladesh.2 This increased neonatal mortality has also been seen in the US (4 times higher in California,12 6.6 times higher in Georgia).11 Since birthweight was not available for all liveborn infants, we were unable to see if this difference was entirely explained by birthweight, as has been seen in developed countries.11,18 Gestational age was available for a large proportion of pregnancies. After adjusting for gestational age, twins still had a higher mortality rate, except around full term (4042 weeks), where mortality rates were comparable for liveborn twins and singletons.
In Nepal female infants had slightly lower mortality rates in the peri and neonatal periods than male infants. This gender difference in survival was also seen among twins, and was larger than that seen among singletons. In Bangladesh, the mortality rates in the neonatal period were comparable among males and females, but this study only looked at twin pairs that were discordant for gender as a way to adjust for gestational age.2 In our data, only 32 pairs of twins were discordant for gender, making a comparable analysis difficult. However, adjustment for gestational age among all infants, and among twins alone, reduced the gender difference in mortality, making our findings comparable to those in Bangladesh and Georgia among the twin pairs that were discordant for gender.2,19
The very high pairwise OR seen among twins is an indicator of the likelihood of survival of both infants as compared with survival of at least one infant. This suggests that the probability of a mother having no surviving offspring of a twin pregnancy where both are born alive is much higher than if the pregnancy was a singleton. This likelihood diminishes as the infants get beyond the first month of life, but is not entirely gone by 6 months of age.
The impact of vitamin A or beta-carotene supplementation on the twinning rate has not been reported in any populations previously. Since supplementation started prior to conception in all these women, it is possible that vitamin A or beta-carotene may have acted to increase the rate of twinning. However, since we could not obtain information on pregnancies that were twins but did not result in a live birth, it is unclear whether the supplements increased the twinning rate, or improved survival in utero of twin infants. The increased twinning was seen for twins where both were live born, and for twins where only one was live born. This suggests that twinning rates may have been increased, rather than there being increased survival in utero. An analysis of five data sets in which multivitamin supplements were used found that four of the five studies showed a 30% to 60% greater prevalence of supplement use during the periconceptional period among those with multiple births and there was no association among pregnancies where supplementation started after embryogenesis was complete.20 Although specific vitamins cannot be identified from these studies, the results do lend support to the notion of micronutrient supplementation's association with twinning rather than improved survival of multiple fetuses. The fact that there was no impact of supplementation on fetal survival, survival of singleton live births, or neonatal survival among twins in our study further supports this.4 There was no difference in the fertility rates of singletons by supplement group. Hence, the impact of beta-carotene and vitamin A supplementation on fertility appears to be specific to twinning. If the impact of supplementation was to increase the twinning rate, rather than the survival of twins in utero, then the generalizability of these results to nutrition supplementation programmes in developing countries is likely limited when these are aimed at antenatal rather than pre or periconceptual supplementation. The increased perinatal mortality associated with twinning should not reduce the imperative for antenatal supplementation, and, since twinning rates are relatively low, should not reduce the imperative for pre or periconceptual supplements that can be demonstrated to have an impact on early infant mortality of singletons who constitute the vast majority of births.
In summary, the twinning rate was somewhat higher in this population than that reported in other developed and developing countries. Mortality was much higher among twins, as seen in other settings. A significantly higher twinning rate, or survival of twins in utero, was seen among women who had received vitamin A or beta-carotene supplements prior to conception and throughout pregnancy, although there was no impact of supplementation on survival of liveborn infants from twin pregnancies. Other planned trials of antenatal vitamin A supplementation may help answer the question of whether survival of twins in utero is increased by supplementation.
KEY MESSAGES
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Acknowledgments
This study was carried out under co-operative agreement No. DAN0045-A-5094 between the Office of Nutrition, US Agency for International Development (USAID), Washington, DC, and the Center for Human Nutrition (CHN). It was a joint undertaking between the CHN and the National Society for the Prevention of Blindness, Kathmandu, Nepal. The study was funded by USAID, with financial and technical assistance form Task Force Sight and Life (Roche, Basel), the United Nations Children's Fund (UNICEF), Nepal, and NIH grant no. RR04060.
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