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Print version ISSN 0120-4157On-line version ISSN 2590-7379

Biomed. vol.40 no.4 Bogotá Oct./Dec. 2020  Epub Dec 09, 2020 

Case report

Twins from different fathers: A heteropaternal superfecundation case report in Colombia

Gemelos de diferentes padres: un caso de superfecundación heteropaternal en Colombia

Fernanda Mogollón1 

Andrea Casas-Vargas1 

Fredy Rodríguez1 

William Usaquén1  * 

1 Grupo de Genética de Poblaciones e Identificación, Instituto de Genética, Universidad Nacional de Colombia, Bogotá, D.C., Colombia


Heteropaternal superfecundation is an extremely rare phenomenon that occurs when a second ova released during the same menstrual cycle is additionally fertilized by the sperm cells of a different man in separate sexual intercourse.

In August, 2018, the Grupo de Genética de Poblaciones e Identificación at Universidad Nacional de Colombia received a request to establish the paternity of a pair of male twins with genetic markers. The following analyses were performed: amelogenin gene, autosomal short tandem repeat (STR), and Y-STR analyses by means of human identification commercial kits, paternity index, and the probability of paternity calculation and interpretation. A paternity index of 2.5134E+7 and a probability of paternity of 99.9999% for twin 2 were obtained while 14 out of 17 Y-chromosome markers and 14 out of 21 autosomal short tandem repeats were excluded for twin 1. The results indicated that the twins have different biological fathers.

Although heteropaternal superfecundation is rarely observed among humans given its low frequency, in paternity disputes for dizygotic twins it is mandatory to demand the presence of the two twins in the testing to avoid wrong conclusions.

Keywords: DNA fingerprinting; paternity; twins, dizygotic; fertilization; microsatellite repeats


La superfecundación heteropaternal es un fenómeno extremadamente raro que se produce cuando un segundo óvulo, liberado durante el mismo ciclo menstrual, es fertilizado por un espermatozoide de un hombre diferente en relaciones sexuales separadas. En agosto de 2018, el Grupo de Genética de Poblaciones e Identificación de la Universidad Nacional de Colombia recibió una solicitud para establecer la paternidad mediante marcadores genéticos de un par de mellizos varones, en quienes se hizo el análisis del gen de amelogenina, el análisis de repeticiones cortas en tándem (Short Tandem Repeats, STR) autosómicas y del cromosoma Y (Y-STR) mediante kits comerciales de identificación humana y cálculos e interpretación del índice de paternidad y probabilidad de paternidad. Se obtuvo un índice de paternidad de 2,5134E+7 y una probabilidad de paternidad de 99,9999 % para el gemelo 2, en tanto que en el gemelo 1 se excluyeron 14 de los 17 marcadores del cromosoma Y y 14 de los 21 sistemas STR autosómicos evaluados. Los resultados indicaron que los gemelos tienen diferentes padres biológicos. A pesar de que la superfecundación heteropaternal rara vez se observa en humanos debido a su baja frecuencia, en las disputas de paternidad para los gemelos dicigóticos, es obligatorio exigir en la prueba la presencia de los dos gemelos para evitar conclusiones incorrectas.

Palabras clave: dermatoglifia del ADN; paternidad; gemelos dicigóticos; fertilización; repeticiones de microsatélite

Heteropaternal superfecundation is an extremely rare phenomenon that occurs when a second ova released during the same menstrual cycle is additionally fertilized by the sperm cells of a different man in separate sexual intercourse taking place within a short period of time from the first one 1-4. Wenk, et al., found three cases in 39,000 records of a paternity-test database and showed a frequency of 2.4% heteropaternal superfecundation among dizygotic twins whose parents were involved in paternity disputes 5,6. Nevertheless, the frequency of these cases may vary depending on the population's coital rates and double ovulation rates 3.

This phenomenon was initially presented by Archer in 1810 7; he showed the phenotypic differences between a white female twin and the other mulatto female twin and his study was later followed by Terasaki, et al., in 1978 by typing histocompatibility antigens 8. Today, about 19 heteropaternal superfecundation cases worldwide have been reported 2,4,7-23. It has been suggested that this will cease to be a rare event, as cases are being and will be described more frequently thanks to the current availability of molecular methods and the popularity and increasing number of paternity tests 2-4,14,22.

In Colombia, Bravo-Aguilar presented a case of heteropaternal superfecundation as an example in the book "The genetic truth of the paternity" where two cases detected in the laboratory are mentioned, but no further information is given 24.

Here we present a case of dizygotic twins from different fathers detected in Colombia as evidenced by autosomal short tandem repeat (STR) DNA markers.

Case presentation

In August, 2018, the Grupo de Genética de Poblaciones e Identificación at Universidad Nacional de Colombia received a request to establish the paternity of a pair of male twins with genetic markers from the alleged father, who suspected the children's kinship and requested paternity testing. Both twins were males, born after 35 weeks of gestation; the first male was 1,700 g and the second one, 2,380 g. The mother denied receiving blood transfusions during pregnancy, but she mentioned oral contraceptive intake before conception and antecedents of twins on her mother's side. The study was conducted with written consent from both the twins' mother and the alleged father. They also gave their consent for the publication of this work.

Genetic profiles

Blood samples were collected from the twins, their mother, and the alleged father on FTA cards. We obtained two independent samples for each person taken at different times and each sample was investigated in duplicate.

DNA was extracted using two punches per sample and it was washed using FTA Reagent Purification buffer. To define the genetic profiles, the samples were typed for 15 autosomal STRs (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA) and amelogenin as the sex marker in the AmpFlSTR Identifiler Amplification Kit™ (Applied Biosystems).

To confirm and extend this analysis, we used 16 autosomal STRs (D10S1248, vWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D22S1045, D19S433, TH01, FGA, D2S441, D3S1358, D1S1656, D12S391, SE33) and amelogenin as the sex marker of the AmpFlSTR NGM SElect Kit™ (Applied Biosystems).

In another reaction, 17 Y-chromosome STR markers (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, GATA H4, DYS437, DYS438, DYS448) were amplified simultaneously with the AmpFlSTR Yfiler PCR Amplification Kit™ (Applied Biosystems).

These amplifications were done in an Applied Biosystems 2720 Thermal Cycler™. Capillary electrophoresis running and detection of amplified products were conducted with the ABI PRISM 310™ genetic analyzer. Samples were analyzed with GeneMapper ID™ software, version 3.2 (Applied Biosystem).

Alleged father - twin 1

Discrepancies in 14 autosomal STR markers and in 14 STR loci for Y chromosome (table 1) between the alleged father and twin 1 were observed.

Alleged father - twin 2

Results favored the paternity of the alleged father for only one of the twins whom we labeled as 'twin 2. All 21 autosomal and 17 Y-chromosomal markers were in concordance with paternity (table 1). The paternity index was 2.5134E+7.

Table 1 Genotypes of the mother, the alleged father, and the twins for 21 autosomal loci and genotypes of the alleged father and twins in 17 Y-chromosome loci 

Autosomal STR Y-STR
System Alleged father Mother Twin 1 Twin 2 Y-STR Loci Alleged father Twin 1 Twin 2
D8S1179 14 / 15 11 /13 11 / 12 13 / 15 DYS456 17 16 17
D21S11 32.2 / 32.2 30 / 30.2 27 / 30.2 30.2 / 32.2 DYS389I 13 12 13
D7S820 11 / 12 8 / 10 8/8 8/12 DYS390 24 21 24
CSF1PO 10 12 12 / 12 10 / 12 DYS389II 29 28 29
D3S1358 15 / 18 17 15 / 17 17 / 18 DYS458 16 15 16
TH01 6 / 7 6 / 9.3 7 / 9.3 7 / 9.3 DYS19 14 13 14
D13S317 12 / 13 10 / 11 10 / 11 10 / 13 DYS385a/b 11 / 14 13 / 16 11 / 14
D16S539 10 / 12 12 9 / 12 12 DYS393 13 14 13
D2S1338 17 / 23 17 / 22 22 / 22 22 / 23 DYS391 11 10 11
D19S433 13 / 14 13.2 / 13.2 12.2 / 13.2 13.2 / 14 DYS439 12 11 12
VWA 16 / 18 16 / 18 16 / 18 18 DYS635 23 22 23
TPOX 8 8 / 12 11 / 12 8 / 12 DYS392 13 15 13
D18S51 14 / 18 12 / 16 14 / 16 12 / 14 GATA H4 11 11 11
D5S818 11 11 11 11 DYS437 14 13 14
FGA 22 / 24 22 21 / 22 22 / 24 DYS438 12 12 12
D10S1248 15 / 16 14 / 15 13 / 15 14 / 16 DYS448 19 19 19
D22S1045 15 / 16 15 / 16 16 15 / 16
D2S441 11 / 14 10 / 11 10 / 11 11
D1S1656 11 / 12 17 / 17.3 17.3 / 17.3 12 / 17
D12S391 16 17 / 26 17 / 18 16 / 17
SE33 19 / 22.2 16 / 28.2 28.2 / 28.2 16 / 22.2
Amelogenin XY XX XY XY

As the genetic profile of the undoubted biological mother was available, the autosomal genotyping results indicated that the two twins had different fathers, which was confirmed with Y STR markers.


Few cases of heteropaternal superfecundation have been reported worldwide possibly because not all the cases have been involved in paternity disputes, which decreases the probability of their reporting 3,5,22. However, the frequency of cases will probably rise with time due to the availability of molecular techniques that serve as a tool for the correct resolution of such cases, as well as the recent increase in twin births 2.

In this case, the two brothers participated in the paternity study in response to a preview communication from the laboratory support service explaining that the test required the presence of both twins to study each of them separately, as well as the importance of including the mother. We observed 14 alleged father-twin 1 incompatibilities for Y-STRs loci (table 1) while no discrepancies between the alleged father and twin 2 were found allowing us to link twin 2 to the same paternal linage of the alleged father and to exclude twin 1 relationship to him. On the other hand, the analysis of the autosomal markers enabled us to establish the biological paternity of the alleged father in regard to twin 2 while excluding him as the father of twin 1. We also confirmed the biological maternity thus excluding any possibility of an interchange of the twins.

The difference between the birth weight of twin 1 and twin 2 was remarkable (680 g). This has also been mentioned by other authors 4,10,12,22 and is explained by the different gestational age of the children as a consequence of the time elapsed between one fertilization and the second one 21. Some authors assume that the time between the first and the second fertilization is about three or four days, although this time can be even longer (up to 14 days) 1,5,6.

Finally, this outcome allowed us to detect a case of dizygotic twins with different fathers, which is a rare and infrequent event reported in the literature. This case reiterates the need for the participation of both twins in paternity testing as these events happen although their frequency is not high.


The results of the present study confirmed that these twins have different biological fathers.


The authors would like to thank the mother and father for giving their written authorization for the publication of this case report. Finally, we would like to express our gratitude to Diana Roa for the support given to the parents and twins, to Claudia Bonilla for her support in carrying out the DNA extraction and amplification, and to Dayana Suárez for her comments.


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Citación: Mogollón F, Casas-Vargas A, Rodríguez F, Usaquén W. Twins from different fathers: A heteropaternal superfecundation case report in Colombia. Biomédica. 2020;40:604-8.

Contribución de los autores: Fernanda Mogollón: Conception, design and analysis, and interpretation of data, drafting of the article, and final approval of the version to be published. Andrea Casas-Vargas and William Usaquén: drafting of the article, critical revision of the manuscript for important intellectual content, and final approval of the version to be published Fredy Rodríguez: Genotyping analyses for autosomal STRs and Y chromosome, and final approval of the version to be published

Financiación: This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.

Conflicto de intereses: None declared.

Received: June 17, 2019; Accepted: July 21, 2020

*Correspondencia: William Usaquén, Grupo de Genética de Poblaciones e Identificación, Instituto de Genética, Universidad Nacional de Colombia, Carrera 30 N° 45-03, edificio 426, oficina 101, Bogotá, D.C, Colombia Telephone: (571) 316 5000, extension 11635

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