Wednesday, October 7, 2015

Twin Sons Of Different Mothers…… Or Fathers

Biology concepts – twins, superfecundity, superfetation, hormones, reproduction

Best In Show is one of the great movies; it also gives you
a good idea how crazy some people get about their dogs.
I applaud that they love them that much, but they expect
you to love them that much as well. I like old movies too,
but I don’t need to see dogs recreating the iconic scenes.
When it comes to their pets, some people can really come unglued. People have had them stuffed after death so they can pet them forever. There has even been paternity suits concerning the offspring of said pets.

True, in some cases a lot of money might be involved in stud fees and in selling purebred pups, but that just goes to show how crazy things can get when pets are involved. One case concerned a female Shih Tzu was bred to two different males in the same estrus cycle – why they did that I have no idea. What was dumber, the two male dogs, a Shih Tzu and a Coton de Tulear, look very similar.

When the pups were born, each owner claimed that they were the pups of his male dog. A DNA fingerprinting method called barcoding was new at the time, and was used to determine that one pup came from each male.

Giving birth at one time to offspring fathered by more than one male is called superfecundation (super = beyond, and fecund= fruitful). Dogs, cats, and many other mammals that have litters are capable of superfecundity; although it is usually seen in stray animals that may mate several time in a single day. Raccoons have had superfecundation litters, and I’m sure it has happened with other animals as well, but whose watching.

With superfecundity in dogs as an example, let's ask our last two questions concerning the definition of twins. We saw in the last two posts that twins can be born months apart and don’t even need to be of the same "race." Now let’s ask – do twins have to be conceived at the same time, or even by the same father?

A litter of puppies where one looks a lot different than the
others. Different breeds mating will give something in the
middle, and even pure breds can have puppies of different
colors, but I don’t think a mom and dad hound will ever
produce a huskie (right) on their own. This is definitely
a superfecundation litter.
The definition of superfecundity is two or more eggs released in the same estrus cycle fertilized by one or more males and implanted and developed in the uterus. Superfecundity can be seen in two different situations. The first is homopaternal superfecundation, when the eggs are fertilized by the same male, but at different times of the same cycle.

The question that immediately popped into my mind when read about homopaternal superfecundation was – how would you know? Run of the mill dizygotic twins would be from the same cycle, the same father, delivered at the same time (usually). How would homopaternal superfecundation twins look any different? How could you tell the two situations apart? And if you can’t tell them apart, how do you know if they can happen in humans?

Consider the following possibility: a husband and wife undergo in vitro fertilization because they have not been able to have a baby in several attempts. Her eggs and his male gametes are used. Two embryos are transferred and a several weeks later they do ultrasound to see if they implanted and are developing. Low and behold – there are five fetuses in there!

Is this proof of superfecundation? No, but the couple did attempt to make a baby the natural way again after the embryos were transplanted – no, that’s not proof. Maybe the two transferred embryos split into a set of twins and a set of triplets. We know that it is possible from our discussions of monozygotic twinning rates during assisted reproductive therapies (see this post).

If you isolate and amplify DNA from several different areas
of the genome (loci, each is a locus), then mom will have
two alleles and dad will have two alleles. For the child, every
allele should match one from mom and/or dad. If not, you
may have the wrong guy.
The only way to tell would be to test the DNA of the parents and all five kids. If the results say that the husband is the father of all of them – is that evidence of superfecundity? No – they could still be monozygotic twins and triplets. But what if the genetic profiles of all the kids are different; what would this mean?

Dizygotic twins and trizygotic triplets don’t have the same chromosome profiles, just like regular siblings do not. Random assortment in the production of gametes means that the odds of two fetuses having exactly the chromosomes is millions to one (see this post). Monozygotic twins or triplets will have the same genetic profile – that’s sort of the definition of monozygotic multiples (unless they are chimerics, see this post and this post).

So, if all the kids have the same mother and father and none are monozygotic multiples, then where did the other three come from? Yep, a fertilization by the father separate from the IVF procedure. This is proof of homopaternal superfecundity. Do you think this is just a hypothetical case and the odds are too long to ever have it ever happen? Well it did, not once, but twice in the literature of the last 15 years, once in 2001 and once in 2011.

Remember that IVF isn’t just an egg harvest and return. Lots of hormones have to be given to the woman to make her ovulate several eggs and to prepare the uterus for implantation after the embryo transfer. This makes it possible for her to ovulate again, and makes it more probable that any fertilized eggs released later might implant and develop as well. And there you have it.

FSH rises just before ovulation and is the reason women
may ovulate more than one egg. Progesterone is produced by
the follicle left from the egg (corpus luteum) and keeps FSH,
LH and estrogen lower, so no other eggs will be released. If the
embryo implants (right side of chart) then progesterone would
be produced by the placenta and stay high – this would prevent
cycling until the pregnancy is over.
A study in 1993 suggested that superfecundity might be responsible for up to 0.5% of dizygotic twins, but that would be a hard thing to prove. The cases that come to light are usually when a question of paternity arises, and that arises much too often.

The second possibility is that eggs can be fertilized by gametes of two different males; the term is heteropaternal superfecundation. There are several cases where this has been proven by genetic testing (1997 and 2000), and in ancient Greek accounts, it has been cited as the reason for any set of twins – they were an untrusting, and apparently, philandering lot.

But you can see how one might assume that there are two dads – remember our discussion of different "race" twins a few weeks back – one paper states that in the case of black and white twins, heteropaternal superfecundity has to be ruled out before a case of different race twins can be proposed. And why does it matter – paternity suits show that heteropaternal superfecundation is present in 2.4 % of the cases that come to court concerning multiples.

Romulus and Remus were raised by wolves – their dad
(Zeus) had sent someone to kill them, but they banished
them to the wild instead. They were twins, and twins in
Rome and Greece were admired and feared. They might be
offspring of the gods, and they might be a sign of infidelity.
If your definition of twins includes a condition that they have to be conceived at one time or by one father – then your definition just got shot down. But don’t feel bad, I’m getting the idea that it’s impossible to pin down just what the true definition of twinning might be. Case in point – we haven’t even gotten to the weird exception yet.

Have you ever heard of superfetation? It is like superfecundity, but stretched out through time. To define it sounds like a riddle – can a pregnant woman get pregnant? Believe it or not, the answer is yes.

After an egg is released from the ovary, the follicle becomes the corpus luteum (see this post). This structure releases progesterone hormone which acts on the hypothalamus, so does the progesterone released from the placenta. In both cases, the progesterone down-regulates the action of the hypothalamaus on the pituitary, so the pituitary releases less hormone that stimulates ovulation in the ovary.

Basically, the reproductive system is telling the brain, "Wait a minute, we may have an implanted embryo, don’t release any eggs in the next estrus cycle." This is why women who are pregnant more often have eggs and will ovulate longer in their life; they go nine months without releasing any eggs every time they are pregnant.

But if there is an over abundance of a hormone called human chorionic gonadotropin (hCG), the signal from the brain may get overridden. HCG is the only hormone known to stimulate ovulation in a woman who might be is releasing progesterone from the placenta and follicle.

Several types of fish, like this black molly, are the first
animals to produce a placenta and give birth to live offspring.
They lost and regained the placenta many times, so some
fish have it and some don’t. Even in the same family, so
species will give birth to live young (viviparity), and some
will scatter eggs to be fertilized outside.
If the first embryo isn’t taking up too much room, the egg released the next month is capable of being fertilized and implanting in the wall of the uterus. Now you have two developing fetuses that may differ in gestational age by as much as five weeks! You can imagine that this might be mistaken for growth discordance (see last week’s post) or growth discordance might be mistaken for superfetation.

Superfetation occurs in other mammals. Placental fish – yes, some fish have placentas and give birth to live young instead of releasing eggs into an underwater nest – are notorious for having immature eggs fertilized while they are carrying a brood.

In brown hares (Lepus europaeus), the same thing occurs; the hypothesis is that it is a way to increase reproductive success in one breeding season for animals that don’t know how long they have before something big might eat them. Still, it has been hard to document superfetation in humans.

In 2007 there was a case of growth discordant twins, but it was the smaller one that was the right size for the gestational age – sounds promising. And in 1999 there was a case where the ultrasound early in the first trimester shows very different sized embryos. Usually growth discordance wouldn’t be seen until much later. The predicted gestational ages for the two embryos differed by four weeks. hmmmm

One big sign that our species may survive – reality shows that
announced paternity testing results used to be a big deal, but
we moved past them. Finally, humans show some good taste.
I wonder if any ever tackled the problem of superfecundity or
superfetation? I think the Jerry Springer’s head would
explode when he contemplated the ratings.
I ask you one last time – how do you define dizygotic twins? Same father, same conception time, same delivery, same gestational age, same size, same "race"? It seems none of them apply concretely. The next time someone tells you that they are a twin, you’ll have a lot to talk about.

And just to blow your mind a bit more – consider a case of heteropaternal superfecundation where the two embryos merge either totally or partially to create chimeric(s). Could you actually end up with a person who has two biological fathers?

In the next weeks, we go way back into the vault to look at a couple of posts about Halloween. It turns out that nearly everyone is a vampire - and then we'll see all the different ways one might end up being buried alive. 

Pollux, B., Meredith, R., Springer, M., Garland, T., & Reznick, D. (2014). The evolution of the placenta drives a shift in sexual selection in livebearing fish Nature, 513 (7517), 233-236 DOI: 10.1038/nature13451

Peigné, M., Andrieux, J., Deruelle, P., Vuillaume, I., & Leroy, M. (2011). Quintuplets after a transfer of two embryos following in vitro fertilization: a proved superfecundation Fertility and Sterility, 95 (6), 2147483647-2147483647 DOI: 10.1016/j.fertnstert.2011.01.029

James WH (1993). The incidence of superfecundation and of double paternity in the general population. Acta geneticae medicae et gemellologiae, 42 (3-4), 257-62 PMID: 7871943

Baijal N, Sahni M, Verma N, Kumar A, Parkhe N, & Puliyel JM (2007). Discordant twins with the smaller baby appropriate for gestational age--unusual manifestation of superfoetation: a case report. BMC pediatrics, 7 PMID: 17239246

Claas, M., Timmermans, A., & Bruinse, H. (2010). Case report: a black and white twin Journal of Perinatology, 30 (6), 434-436 DOI: 10.1038/jp.2009.156

For more information or classroom activities, see:

Superfecundity –

Superfetation –


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