Abstract
Objective:
To determine the ratio of dichorionic (DC) to monochorionic (MC) twins by maternal age.
Methods:
We reviewed all twin pregnancies undergoing first trimester screening (FTS) with nuchal translucency from April 2009 to December 2012 with sonographic determination of chorionicity. Cases were linked to newborn screening (NBS) results and zygosity estimated based on rates of fetal sex discordance. The ratio of DC to MC placentation by maternal age was calculated.
Results:
We identified 11,351 twin pregnancies with FTS and documented chorionicity. Among these, 7,861 (64.2%) had linked data on FTS and NBS to allow estimation of zygosity based on neonatal sex. Of these, 1,464 (18.6%) were MC and 6,406 (81.4%) DC. The MC twin rate remained constant while the DC twin rate increased with maternal age until 40y. At < 20y, 55% of twin pregnancies were monozygotic (MZ), as compared to 29% at ≥ 40y. Of MZ twins, 38% were DC at < 20y, while 53% were DC at ≥ 40y.
Conclusions:
Our data suggest a relationship of both zygosity and chorionicity with maternal age. DZ twinning increased with maternal age, while among MZ twins, the proportion that were DC also increased with maternal age.
Keywords: twins, zygosity, chorionicity, maternal age
Introduction:
Twin pregnancies represent approximately 3% of live births in the United States1. Thirty percent of twin pregnancies are reported to be monozygotic (MZ), arising from division of a single embryo very early in development, with the remaining 70% being dizygotic (DZ), resulting from dual ovulation with fertilization of two ova by two sperm. Essentially all DZ twins are dichorionic diamniotic (DC-DA), whereas MZ twins may be dichorionic diamniotic (DC-DA, 25-30%), monochorionic diamniotic (MC-DA, 70-75%) or monochorionic monoamniotic (MC-MA, 1-2%) depending on the timing of twinning2. Ultrasound determination of chorionicity can be achieved in over 90% of cases, with greatest accuracy when examined by 14 weeks gestation3–5.
A number of factors may influence the incidence of twin pregnancies in the population, as well as the proportion that are MZ versus DZ. An increasing incidence of twins is seen with maternal age, with a greater proportion of DZ twins occurring among spontaneously conceived pregnancies with advancing maternal age 6, 7. Twinning is also more common with assisted reproductive technology (ART), and a greater proportion of MZ twins are seen with these pregnancies8–10. The incidence of DZ twinning is also increased with maternal family history, race, and geographic location2, 11.
It is well established in the literature that twin gestations are at increased risk of adverse pregnancy outcome compared to singleton gestations, with higher rates of congenital anomalies, preterm birth, fetal growth restriction, intrauterine fetal demise, and neonatal death12–15. These risks are increased further in MC twin pregnancies. Given the differential risk profile for MC and DC twin pregnancies, early determination of chorionicity is important both for counseling patients and planning appropriate pregnancy management. Currently though, sparse literature exists regarding the impact of maternal age on the proportions of twins by zygosity and chorionicity. Our objective was to determine the incidence of MC versus DC twin pregnancies, as well as the estimated incidence of MZ versus DZ twin pregnancies, across maternal ages in a large state-wide cohort.
Methods:
This is a retrospective cohort study of all twin pregnancies undergoing first trimester screening (FTS) as part of the California Prenatal Screening Program (PNS) through the California Department of Public Health from April 2009 to December 2012. California state regulations require that healthcare providers offer prenatal screening to all women seen before the 20th week of gestation. Medi-Cal (California’s Medicaid program serving low-income individuals) and almost all insurers cover the cost of this screening. Since April 2009, the PNS has provided first- and second-trimester serum screening with integration of nuchal translucency (NT) ultrasound measurements into the risk algorithm. The NT and chorionicity are assessed by an NT-certified obstetrician or radiologist for fetuses with a crown rump length between 45-84 mm at the time of ultrasound (US)16. FTS uses maternal pregnancy-associated plasma protein-A (PAPP-A) and total human chorionic gonadotropin (hCG). Patients who have an NT measurement performed are provided first trimester risk assessment for trisomy 21 (Down syndrome) and trisomy 18. In women with twin gestations, assessment and reporting of chorionicity is requested for interpretation of NT, therefore chorionicity is determined for most twin pregnancies participating in the FTS program. We included all women participating in the California PNS with data for FTS, NT, and linked data for NBS. Pregnancies with missing data for FTS, NT, or NBS were excluded.
In the PNS, data regarding use of ovum donation are collected but information on other forms of ART are not. As a result, we analyzed data for pregnancies resulting from ovum donation separately, as ART is known to influence rates of DC versus MC twinning. Data on chorionicity were linked to newborn screening results (NBS) that were collected 12-32 weeks after the FTS in order to confirm sex for estimation of zygosity. This range was selected to capture all viable deliveries after 22 weeks’ gestational age (as FTS is drawn between 10+0 to 13+6). Zygosity was then estimated based on probabilities for fetal sex discordance by zygosity. To estimate rates of MZ versus DZ twins, MC twins were considered to be MZ, while DC twins discordant for fetal sex were considered to be DZ. Among the DC pregnancies with concordant fetal sex, there are both MZ and DZ pregnancies. The methods of Weinberg were used to estimate the expected number of concordant male, concordant female, and discordant sex in dizygotic twins.
Calculations were done using the Weinberg equation and using the sign test to determine whether twin groups were equally sized2. This calculation applies at each year of maternal age, so we are able to estimate the number of DC pregnancies that are DZ (as twice the number of discordant pregnancies) and the MZ pregnancies as the remainder. The ratio of DC to MC placentation in all twins, and specifically in MZ twins, by maternal age was calculated.
The study was determined to be exempt from review by the Committee for the Protection of Human Subjects within the Health and Human Services Agency of the State of California.
Results:
Over the study period, a total of 539,372 women underwent FTS, 12,243 of these had twin pregnancies (2.27%) (Table 1). Of the 12,243 twin pregnancies with FTS results, 11,351 had reported chorionicity (92.7%) and 10,139 (89.3%) of these were not to be the result of ovum donation. Overall rates of twinning were higher in Black women compared to Asian women (Table 1). Newborn records could be linked to FTS results, and newborn sex was known for both infants in 7861 (64.2%) of pregnancies not resulting from ovum donation (Table 2). Of the 7861 twin pregnancies with complete data, 1464 (18.6%) were MC and 6406 (81.5%) were DC (Figure 1).
Table 1:
Cohort Demographics*
| Singleton | (%) | Twin | (%) | ||
|---|---|---|---|---|---|
| Total | 527129 | 12243 | |||
| Age Group | < 20 | 10204 | 1.9% | 76 | 0.6% |
| 20-24 | 61168 | 11.6% | 759 | 6.2% | |
| 25-29 | 110221 | 20.9% | 1849 | 15.1% | |
| 30-34 | 171850 | 32.6% | 3887 | 31.7% | |
| 35-39 | 132772 | 25.2% | 3760 | 30.7% | |
| > 40 | 38170 | 7.2% | 1445 | 11.8% | |
| Ethnicity | Asian | 75331 | 14.3% | 1614 | 13.2% |
| Black | 22282 | 4.2% | 665 | 5.4% | |
| Hispanic | 211814 | 40.2% | 3318 | 27.1% | |
| White | 164948 | 31.3% | 5248 | 42.9% | |
| Smoking | No | 518337 | 98.3% | 12052 | 98.4% |
| Yes | 5240 | 1.0% | 124 | 1.0% | |
| Diabetes | No | 514884 | 97.7% | 11930 | 97.4% |
| Yes | 5353 | 1.0% | 113 | 0.9% |
Missing and unknown values not presented but included in totals. Multiple and other ethnicities not presented
Table 2:
Twin pregnancies with known chorionicity and sex, with proportion of cases included with each successive condition (Step) or overall (Cum).
| Singleton | Twins | |||||
|---|---|---|---|---|---|---|
|
| ||||||
| N | Step (%) | Cum (%) | N | Step (%) | Cum (%) | |
| Has first trimester screening | 527,129 | -- | -- | 12,243 | ||
| With known chorionicity | -- | -- | -- | 11,351 | 92.7 | 92.7 |
| Not ovum donation | 524,336 | 99.5 | 99.45 | 10,139 | 89.3 | 82.8 |
| Linked to one NBS | 445,329 | 84.9 | 84.5 | 8,708 | 85.9 | 71.1 |
| Linked to two NBS | -- | -- | -- | 7,939 | 91.2 | 64.8 |
| With known sex(es) | 443,480 | 99.6 | 84.1 | 7,861 | 99.0 | 64.2 |
Figure 1: Zygosity and chorionicity for twin pregnancies with known chorionicity and sex.
Flow diagram showing number (N) of pregnancies stratified by zygosity and chorionicity for no ovum donor and ovum donor. Complete data includes linkage to two newborn screen results and sex for both babies.
Maternal age in our cohort ranged from 16 to 50 years of age for women with pregnancies that did not result from ovum donation. For all twin pregnancies (MZ and DZ), the proportion of twins that were DC increased from 66.2% for women < 20y to 86.2% for ≥ 40y (p=0.07 for zero slope of the regression of the rate against maternal age) (Figure 2). At age < 20y, 31.7% of all DC pregnancies were estimated to be MZ, while at age ≥ 40 this number decreased to 11.8%. For MZ twins, the rate of DC placentation increased with maternal age from 38.2% for women < 20y to 53.0% for women ≥ 40y (p=<0.001 for zero slope of the regression of the rate against maternal age) (Figure 2). We also saw an increase in DZ-DC twins with maternal age until age 38 years. Table 3 contains the distribution of twins as MZ-MC, MZ-DC or DZ-DC in 5-year age groups, with Table 4 providing the details of the estimation of the numbers of DC twins that are DZ or MZ.
Figure 2: Rates of twin pregnancy by zygosity and chorionicity by maternal age.
Graph showing rates of twin pregnancy by zygosity and chorionicity (Y-axis) by maternal age (X-axis).
Table 3:
Rates of zygosity and chorionicity by maternal age for twins (not donor ovum)
| MZ-MC N (%) |
MZ-DC N (%) |
DZ-DC N (%) |
Total | |
|---|---|---|---|---|
| < 20 Y | 21 (33.9) | 13 (21.0) | 28 (45.2) | 62 |
| 20-24 Y | 158 (29.3) | 88 (16.3) | 294 (54.4) | 540 |
| 25-29 Y | 319 (23.4) | 182 (13.4) | 860 (63.2) | 1361 |
| 30-34 Y | 526 (18.7) | 169 (6.0) | 2110 (75.2) | 2805 |
| 35-39 Y | 353 (14.3) | 249 (10.1) | 1868 (75.6) | 2470 |
| ≥ 40 Y | 87 (13.8) | 98 (15.5) | 446 (70.7) | 631 |
MZ-MC is monozygotic, monochorionic; MZ-DC is monozygotic, dichorionic, DZ-DC is dizygotic, dichorionic. The MZ total is the sum of the first and second columns, the DC total is the sum of the second and third columns
Table 4:
Calculation of Zygosity in Dichorionic Twin Pregnancies Not Known to be the Result of Ovum Donation
| Sex (A) / Sex (B) | |||||||
|---|---|---|---|---|---|---|---|
| Age | F / F | F / M | M / F | M / M | Total | Est DZ 2*(F/M+M/F) |
Est MZ (Total-DZ) |
| <20 | 16 | 4 | 10 | 11 | 41 | 28 | 13 |
| 20-24 | 108 | 67 | 80 | 127 | 382 | 294 | 88 |
| 25-29 | 303 | 222 | 208 | 309 | 1042 | 860 | 182 |
| 30-34 | 585 | 513 | 542 | 639 | 2279 | 2110 | 169 |
| 35-39 | 595 | 469 | 465 | 588 | 2117 | 1868 | 249 |
| 40-44 | 165 | 110 | 100 | 142 | 517 | 420 | 97 |
| > 45 | 9 | 7 | 6 | 5 | 25 | 26 | −1 |
This methodology can be verified further by applying it to pregnancies that result from ovum donation. The number of concordant pregnancies and discordant pregnancies is approximately equal. For these pregnancies, of the 36 ages between 23 and 59, there were 6 where the two groups were equal, and 15 with excess concordant and 15 with excess discordant, and the total excess of 4 sex-concordant twin pregnancies.
For the 793 twin pregnancies resulting from ovum donation, maternal age ranged from 23 to 58 years of age. The hypothesis is that all twin pregnancies resulting from ovum donation are the result of transfers of two ova. The basic method of comparing sex-concordant and sex-discordant twin pregnancies yielded an excess of only 4 more sex-concordant twin pregnancies, implying a rate of 0.5% MZ twins. A more powerful statistical test of this hypothesis is given by applying the sign-test to the data grouped by maternal age. For these pregnancies, of the 36 ages between 23 and 58, there were 6 ages where the two groups were equal, and 15 ages with excess concordant and 15 ages with excess discordant, which is consistent with the null hypothesis. Table 5 provides a summary of these data grouped into five-year age groups and showing the same trends.
Table 5:
Calculation of Estimates of DZ-DC and MZ-DC in Ovum-Donor Twin Pregnancies
| Sex (A) / Sex (B) | |||||||
|---|---|---|---|---|---|---|---|
| Age | F / F | F / M | M / F | M / M | Total | Est DZ 2*(F/M+M/F) |
Est MZ (Total-DZ) |
| < 25 | 0 | 1 | 0 | 3 | 4 | 2 | 2 |
| 25-29 | 4 | 2 | 2 | 3 | 11 | 8 | 3 |
| 30-34 | 11 | 14 | 12 | 12 | 49 | 52 | −3 |
| 35-39 | 28 | 25 | 37 | 38 | 128 | 124 | 4 |
| 40-44 | 81 | 76 | 71 | 82 | 310 | 294 | 16 |
| 45-49 | 43 | 56 | 64 | 64 | 227 | 240 | −13 |
| > 50 | 7 | 7 | 12 | 7 | 33 | 38 | −5 |
DISCUSSION:
In this large cohort of twins with first trimester sonographic determination of chorionicity on NT ultrasound, we found that there were differences in chorionicity as well as zygosity associated with maternal age. Our data confirm prior studies reporting that DZ twinning increases with maternal age, leading to a greater proportion of twin pregnancies that are DZ and DC with increasing maternal age. Further, we found that among MZ twins, the proportion that are DC increases with maternal age.
Our data confirm the known association between advancing maternal age and increased risk of twinning17, 18. This increase is largely due to an increase in DZ twins (against a relatively stable background rate of MZ twins). A large contribution to this increase in DZ twinning is likely due to multiple ovulations in the setting of increasing FSH with decreasing ovarian reserve. Our study adds novel information to the literature regarding trends in MZ twins with maternal age, including the finding that MZ twins are more likely to be DC as maternal age increases.
The etiology for this increase in MZ-DC twins with advancing maternal age is unknown. There is a known increase in MZ twinning with IVF, a finding that has been hypothesized to be due to intracytoplasmic sperm injection, assisted hatching (i.e. disruption of the zona pellucida), and time in culture medium among other factors. Additionally, the percentage of MZ twins that are DC is higher with IVF2, 10. Given the greater use of ART among women of advancing maternal age, it is possible that IVF plays at least some role in this observed increase in MZ-DC twins with maternal age. Another possibility is that advancing maternal age is associated with aging ova, which have a more brittle or fragile zona pellucida that is prone to breakage and division of the zygote with subsequent development of two embryos with two placentas (i.e., DC placentation). We know that with MZ-DC twins, cell division occurs earlier (days 0-3) than with their MZ-MC counterparts (days 4-13); it follows that whatever event is leading to increasing rates of dichorionicity with advancing maternal age, it is happening by the morula stage very early in embryogenesis. In their study of rates of Down syndrome in twin pregnancies Sparks et al noted fewer MC twins at older maternal ages, indicating the possibility is that MZ-MC twins have a high rate of miscarriage secondary to aneuploidy19.
These data fill an important gap in the literature on zygosity and chorionicity by maternal age and provide contemporary data on twin epidemiology. As accurate knowledge of chorionicity is important for guiding counseling and management these data contribute to our understanding of the proportion of MZ twins at a given maternal age that are DC (and lower risk of adverse pregnancy outcome than MC).
Our study provides contemporary information on zygosity and chorionicity for a large and diverse cohort of women. Our study is not without limitations, however. Use of ART is known to increase rates of both MZ and DZ twinning and is also known to increase the rate of MZ-DC twins in particular. The PNS collects data on whether ovum donation was used but does not provide information on the use of other types of ART (such as medications to promote ovulation induction, IVF without ovum donation, or number of embryos transferred for patients using donor ova). Since our analysis included older women who are more likely to have used ART to achieve pregnancy, we may be overestimating the number of twin pregnancies at a given maternal age beyond that which would be seen with spontaneous conceptions. In addition, this observed increase in MZ-DC twins may be in part due to IVF and not maternal age. In order to mitigate confounding from ART, data were analyzed separately for pregnancies resulting from known ovum donation, where we found that almost all twin pregnancies resulting from ovum donation were DZ and DC. However, even for these pregnancies, we are unable to determine whether a single embryo or multiple embryos were transferred. An additional limitation is the fact that calculations using sex ratio estimates are used to provide approximate rates for zygosity for DC twins. While MC twins are assumed to be MZ, the rates of MZ-DC twins are based on population estimates using sex ratios. Historically, the gold standard of pathologic evaluation to evaluate for chorionicity (and amnionicity) has not been performed on a population level to confirm these estimates.
This new information on chorionicity for a contemporary cohort provides a more detailed description of twin epidemiology and fills a gap in the literature with regards to rates of zygosity and chorionicity by maternal age, providing previously unappreciated data about the age-associated increased rate of twinning. As chorionicity is known to be more predictive than zygosity in determining the risk of adverse perinatal outcome, having current information on population rates of chorionicity at a given maternal age has implications for counseling and antepartum surveillance. Specifically, for women with dichorionic placentation who have questions about the likelihood that their fetuses are monozygotic, these data indicate that the chance of monozygosity is higher with younger maternal age, but that if twins are monozygotic, the likelihood of dichorionicity is greater in older women. This is important as the risk of adverse perinatal outcome is higher for monochorionic compared to dichorionic twins.
Bulleted Statements:
What is known about this topic?
The overall rate of twin pregnancy is 3% with roughly 30% being monozygotic and 70% being dizygotic
While the overall incidence of twin pregnancy increases with maternal age, the proportional zygosity and chorionicity by maternal age is not well established
What does this study add?
Using data from the California Prenatal Screening Program, we were able to estimate the ratio of dichorionic to monochorionic placentation by maternal age
We found that both zygosity and chorionicity vary by maternal age, with an increase in the proportion of monozygotic dichorionic twins
Funding Statement:
This work was supported in part by the University of California, San Francisco California Preterm Birth Initiative (LJP). Dr. Sparks is supported by grant 5K12HD001262-18 from the National Institutes of Health (NIH). The contents of the publication are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
Footnotes
Conflict of Interest Statement: The authors report no conflicts of interest
Data Availability Statement:
The data that support the findings of this study are available from The California Department of Public Health - California Prenatal Screening Program. Restrictions apply to the availability of these data, which were used under license for this study. Data are available from the authors with the permission of The California Department of Public Health - California Prenatal Screening Program.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from The California Department of Public Health - California Prenatal Screening Program. Restrictions apply to the availability of these data, which were used under license for this study. Data are available from the authors with the permission of The California Department of Public Health - California Prenatal Screening Program.