Cultural problems, Part I

Introduction

According to the general assumption, laboratory embryology is one of the most advanced branches of science, comparable to space research, molecular genetics, computer and nuclear technology. We, embryologists,  do not strive to refute this view for obvious reasons. However, at closed informal discussions after conference dinners, most of us agree with the immortal statement of Rodney Wade, my respected American friend: "High technology? forget it. IVF is low technology...". He said it twenty years ago, but his words are more valid today than ever.

Do not get offended; it is not personal. Just, unfortunately, true.

Mammalian embryology had a very bad start. Sister branches, including invertebrate or reptilian developmental biology, were much more successful - by making nuclear transfer cloned offspring far before the First World War (no misprint - even in the 19th century). But these results could not be reproduced with mammals for one hundred years!

An even more shocking fact. In the fifties of the last century, we had already the nuclear bomb, the sputnik, the television, the first digital computers - but not a single mammalian embryo produced in vitro. Why?

Today, if you ask a primary school student what the most important thing to protect a mammalian embryo to avoid damages outside the body is, I am sure he would say: to keep it warm. We are not talking about reptiles or amphibians but warm-blooded animals, aren't we?

No doubt, an elementary student would have said the same one hundred years ago, too. But - was not asked. Retrospectively, it seems absurd, but the respected big-bearded academics disregarded the obvious fact that - unlike those of frogs and salamanders - oocytes and embryos of mammals are extremely sensitive to temperature changes and do not tolerate exposition to room temperature, not even for a short period. They are also seriously affected by seemingly slight differences between the core temperatures of various mammalian species. An optimal temperature for humans is devastating to cattle and pig embryos and vice versa, although the difference is an almost negligible 1.5 to 2.0 degrees.

As you see, incubation and incubators were crucial elements in our science and remain so today. In this blog, I try to summarise the story that played a decisive role in advancing laboratory embryology. Decisive but controversial, both positive and negative.

The creation of cell/tissue culture incubators

Although incubators (or rather incubator rooms) heated by fire were already used in China and Egypt for chicken eggs thousands of years ago, the system was largely forgotten in the Middle Ages. After introducing an accurate thermometer (Réaumur, 1730) and more or less proper heating, first in dry ovens, then with kerosene lamps in the late 19th century, incubators became suitable for laboratory use. Before the closing of the door, a candle was lit to produce the elevated carbon dioxide level.

Curiously, the first electrically heated incubators were used not for tissue culture but for babies - for transportation of premature infants (Hess, 1922). Afterwards, electric heating has become the standard in all laboratory incubators. Finally, 60 to 50 years ago, gas mixtures or mixers were also applied to maintain the required carbon dioxide level.

That was the time when mammalian embryology was born.

(You may say now: "Then, how could you blame embryologists for being late? They did not have the fundamental instrument that is absolutely indispensable for their work!"

Well, if they knew what was needed, they could make it. They could construct a highly accurate, perfect incubator not 100, but even 200 years ago - by using a little less technology and a bit more creativity and manual work.

Just imagine a water bath that may host a small airtight box. Then hot water and cold (well, room temperature) water, in separate buckets. Keep the water in the bath at 37 degrees by adding hot and cold water, respectively. Put the sample inside the box, close it, then blow in through a small hole your expired air. It contains almost precisely as much carbon dioxide as required, decreased oxygen concentration and close to 100% humidity. It is pre-warmed and even pre-filtered by your lung. Close the hole, and submerge the box into the water bath. Subsequently, you only need to add hot or cold water repeatedly to maintain the temperature, maybe in rotation with others, for 5-7 days.

Believe it or not, with a close analogue of this system, you can achieve as high as 50% blastocyst rates in cattle and pigs. I did it; I can prove it anytime, anywhere.)

Anyway, with the electric heating, with more or less accurate temperature controls, and standard 5% carbon dioxide supplementation, we had the convenient way to culture mammalian oocytes and embryos under laboratory conditions. All initial human, domestic animal and experimental embryology successes were achieved in these large box-type incubators.

However, very soon, in the early '90s, we realised that mammalian oocytes and embryos might require a different in vitro environment.

Attempts to make a proper embryo culture system in vitro

Quod licet bovi - licet Iovi? 

(if you need help, contact me)

In the first years - with few patients, few programs, and short incubations before transfer - limits and handicaps of traditional incubators were not obvious. Curiously, these problems were realised first not by human but bovine embryologists,  for three reasons.

1. Transvaginal ultrasound-guided ovum-pick up from unstimulated animals provided access to a large number of immature oocytes from highly valuable females, promising another breakthrough in cattle breeding, comparable to artificial insemination.

2. In parallel, abattoir-derived ovaries and billions of frozen semen samples offered a practically unlimited source for experiments without any bioethical concerns. Accordingly, efficient techniques for in vitro maturation of cattle oocytes were established with a 90 to 95% success rate and a biological value comparable with the in vivo counterparts. Sperm capacitation and in vitro insemination was also highly successful with an 80 to 90% cleavage rate and development to the 8-cell stage.

3. However, the once legendary "8-cell block" required an additional decade to overcome. Unfortunately, in contrast to humans, development to the blastocyst stage was the condition of non-surgical embryo transfer in cattle. The few embryos that escaped the block and grew to "blastocysts" were visibly handicapped, had fewer cells, smaller inner cell mass and ugly, dark cytoplasm full of lipid drops. Frustrating embryo transfer results discouraged animal breeding companies, and their financial support was reconsidered.

Moreover, these were the years when bovine spongiform encephalopathy (BSE; the cattle COVID of the '90s) decimated the stock, caused more suicides among breeders than infected human victims, and discouraged the remaining professionals from introducing any novel technologies - with long-lasting effect, up till today. 

However, decimated groups of bovine embryologists eventually discovered the reasons for bad development in vitro. They established the principles that - after decades (!)  - have also been accepted and adopted in human IVF. 

These principles included 

- Instead of using complex tissue culture media like TCM-199, restrict the components to those essential for embryo development by using various approaches but eventually converging to similar constituents, primarily but not entirely based on the in vivo situation.

- Establish a highly consistent environment during the whole period female gametes and embryos spend in the laboratory, including all essential parameters, especially temperature.

- As part of the above attempt, modify the standard "5% carbon dioxide in air" mixture with that containing less oxygen, according to the in vivo situation.

The second and third principle was found to be valid in all domestic species. However, it was pure luck - or generous and rare support of Nature? - that embryo culture media developed for cattle embryos were much more suitable for humans than those successful in other mammals, including pigs.

Also, dozens of disappointed and starving bovine embryologists continued their careers in the human field  - although very few fully accepted the related discipline -, and the fresh blood and novel mentality helped human IVF find the proper alternative.

First of all, in cryopreservation (vitrification)  and embryo culture.

Development of an incubator for mammalian embryology

The task was seemingly simple. ...    (to be continued)