The process of in-vitro fertilization, the scientific procedure developed 40 years ago that has resulted in more than 8 million babies born to previously infertile women, may be more complicated than has been perceived to date.

Scientists in Japan have discovered that even when only one embryo is fertilized outside of the womb and implanted, multiple births can still occur. Depending on the age of the mother, a number of embryos may be transplanted into the womb during the IVF process, in order to increase the chances of success. However, implanting two or more embryos has resulted in more higher risk multiple births and the associated complications, including pre-eclampsia, gestational diabetes and birth defects. In a bid to prevent these multiple births occurring, a process called single embryo transfer, or SET, was introduced.

But the largest ever study to investigate the prevalence of multiple pregnancies after a single embryo transfer found that the methods used during IVF — including the use of thawed embryos, maturing the fertilized egg in the laboratory for up to six days before implanting it, and other assisted hatching methods — could themselves increase the risk of multiple births.

Researchers in Japan looked at nearly a million cycles of IVF to investigate why this happens and published their findings in the journal Human Reproduction.

Factors that could increase the risk of zygotic splitting, which results in multiple embryos in the womb, include using frozen-thawed embryos, maturing the fertilized egg in the lab for five or six days before implantation, and assisted hatching — a process where clinicians create a small hole in the layer of proteins surrounding the embryo to help it attach itself to the wall of the woman's womb.

In an email exchange with S&P Global Market Intelligence, Keiji Kuroda, one of the authors of the study, said basic procedures surrounding IVF are the same worldwide. "In most countries, embryo transfer using multiple embryos is common, but in recent years, the technology of cryopreservation has been progressed," he wrote in a response to questions. "Pregnancy outcomes using frozen embryos are better than those with fresh embryos. Therefore, all freezing of embryos and frozen-warmed selected single embryo transfer has been increased."

Kuroda, who is also a doctor at the Sugiyama Clinic Shinjuku and Juntendo University Faculty of Medicine in Japan, added that clinicians are performing embryo biopsies and preimplantation genetic screening at higher rates in order to improve the quality of the embryos that are selected and that these practices may play a part in the higher rate of zygotic splitting.

A zygote is the fertilized egg cell, and it contains all the genetic information from both parents. It soon starts to divide and subdivide into many more cells called blastomeres, which eventually form the embryo. Zygotic splitting occurs between days two and six when the zygote divides, and each zygote develops into an embryo, leading to identical twins, or triplets if it divides into three. These are known as monozygotic twins or triplets.

Kuroda and his colleagues looked at nearly a million cycles of SET carried out in Japan between 2007 and 2014. After SET using fresh or frozen and then thawed embryos, 29.5% resulted in pregnancies, of which 1.56% or 4,310 were twins and 109 were triplets. The prevalence of true zygotic splitting was 1.36%. Using frozen-thawed embryos increased the risk of zygotic splitting by 34%, maturing the blastocysts in the lab for a few days before embryo transfer increased the risk by 79%, and assisted hatching by 21%, when compared with single pregnancies, according to the study.

Kuroda said possible solutions to lowering the risk of multiple births include embryo selection using a computer-automated time-lapse image analysis test and transferring zygotes when they are just starting to divide.

Still, he pointed out that although the use of single embryo transfer has increased worldwide, the prevalence of zygotic splitting pregnancies has not. He attributes this to a possible improvement in IVF techniques, and also in the cultures in which blastocysts are matured in the lab, which reduces the stress on embryos and leads to a decrease in the risk of zygotic splitting.

"In fact, the risk of zygotic splitting from blastocyst culture was lower between 2010 and 2014 than between 2007 and 2014 — 79% and 120%, respectively, although the reason for this is unknown. So, there may be no need to avoid embryo manipulations, such as blastocyst culture, in order to select the single most viable embryo," he added.

Limitations of the study include the fact that the Japanese data did not include information regarding ovarian stimulation and fertilization methods. Furthermore, information on assisted hatching was not included in the registry until 2010 and as the study is observational, it cannot prove that IVF procedures cause zygotic splitting, Kuroda cautioned.