In the center of one laboratory dish is a white film that can only be seen when light is shined on it. When reproductive biologist Ayse Nihan Kilinc placed the dish under a microscope, an image appeared on a connected screen. When he focuses the microscope, he sees a cluster of drop-shaped spheres with translucent interiors bordered by black. In the microscope’s magnified field, these spheres ranged in size from a quarter coin to a golf ball. Of course, its actual size is only about the size of a grain of sand.
“The shape of the plump spheres is a good sign,” Killinck explained. “These are organoids and are currently growing in very good condition.”
Killinck works in the laboratory of bioengineer Linda Griffith at MIT. She is one of the few scientists who uses new methods, such as organ analogs, to study menstruation, a human physiological phenomenon that is little known and causes difficulties for many people. At least one-third of people who menstruate experience excessive menstrual blood or weakness. This causes her to miss several weeks each year from work or school, putting her social status at risk. About two-thirds of people with excessive menstrual blood have symptoms of anemia. And when menstrual blood flows through the fallopian tubes into the body cavity, it sometimes forms painful lesions. Due to the nature of the disease called endometriosis, multiple surgeries may be required for treatment.
No one knows how or why the human body coordinates the process of cell birth, maturation, and death each month. Many people desperately need treatment to deal with menstruation. However, if scientists do not properly understand this process, developing drugs will not be easy.
Now, thanks to endometrial organoids, scientists can better understand menstruation. Endometrial organoids are biomedical tools made from tissue from the endometrium, the inner mucosa of the uterus. To create endometrial organoids, scientists take cells from a volunteer, place them in a laboratory dish, and allow them to organize themselves. This research is still in its early stages. But organoids have already provided insight into how cells in the endometrium communicate and cooperate with each other, and why menstruation is a routine for some but a problem for others. Researchers see these early results as a positive sign of ushering in a new era. “Organoids will revolutionize the public understanding of reproductive health,” said Juan Gnecco, a reproductive engineer at Tufts University.
Menstruation is a rare phenomenon in the animal world. The human body menstruates to prepare the uterus for the presence or absence of a fetus. However, in most animals, the uterus is primed only when a fetus is present.
Menstruation shows a pattern of repeated damage and recovery. Menstruation begins when levels of a hormone called progesterone drop, which means that a baby will not grow in the womb that month. A decrease in progesterone triggers a response similar to that when the body fights an infection. The endometrium is damaged due to inflammation, and over the next five days, the damaged tissue peels off and flows out of the body.
The endometrium begins to recover as soon as menstruation begins. And in about 10 days, the thickness of this tissue increases four-fold. No other human tissue grows so quickly and extensively. “Even aggressive cancer cells fall short of this,” explains Jan Brosens, an obstetrician and gynecologist at the University of Warwick in the UK. Eventually, the endometrium recovers without a single scar (a rare occurrence in human tissue), creating an environment that can protect the embryo, which is a kind of foreign substance, from the immune system, which was originally trained to reject foreign substances.
After decades of research, scientists have a rough idea of what menstruation is, but many details remain mysterious. It is not clear exactly how the endometrium repairs itself so extensively. Why some people have more menstrual bleeding than others still remains a mystery. And why humans excrete unused endometrial tissue through menstruation instead of reabsorbing it like other mammals is a hot topic of debate among biologists.
Scientists are trying to find treatments for people whose pain is too severe to rely on regular painkillers or whose menstrual bleeding is too heavy for sanitary pads or tampons. However, many people still suffer as the lack of understanding about menstruation acts as an obstacle. A study conducted in the Netherlands found that women lose an average of one week of productivity each year due to menstrual-related symptoms, such as abdominal pain. Hilary Critchley, a gynecologist and reproductive biologist at the University of Edinburgh, said: “Many of my patients who come to my clinic complain that they need two or three days off work each month.”
People with heavy menstrual blood experience difficulties even in everyday tasks. For example, even when you get up from a chair, you have to worry about leaving marks. Menorrhagia can be a generational problem because mothers with low iron levels tend to give birth to babies with low birth weight or other health problems. Brosens criticized, “Even those who explore tissue regeneration, such as organ research, are neglecting the uterus,” adding, “This is very wrong.”
Why do menstruation remain such a mysterious topic? If you ask this question to researchers, you will get a variety of answers. Most agreed that there is not enough funding to attract the researchers this field needs. This is a common occurrence when studying health problems that primarily affect women. Another obstacle is the reality that menstruation is considered a taboo. But some researchers said it was difficult to find suitable means to study menstruation.
Scientists usually conduct research on other organisms, such as mice, fruit flies, and yeast, and then apply the knowledge they gain to humans. These so-called ‘model systems’ reproduce rapidly, are genetically modifiable, and do not pose the ethical or procurement challenges of testing on humans. However, because menstruation is so rare in the animal world, it has been difficult to find research subjects other than humans. “Honestly, I think the limitation of menstruation research is the lack of appropriate model systems,” said Julie Kim, a reproductive biologist at Northwestern University.
In the 1940s, Dutch zoologist Cornelius Jan van der Horst was one of the first scientists to use animal models to study menstruation. Van der Horst went to South Africa, fascinated by unusual and unstudied animals. There he captured elephant shrews and began studying them. With their long snouts reminiscent of elephant trunks and opossum-like bodies, elephant shrews were already unusual enough even before van der Horst discovered that they were one of the few animals that menstruated. “The fact that they menstruate was probably discovered by accident,” said Anthony Carter, a developmental biologist at the University of Southern Denmark who reviewed van der Horst’s work.
However, elephant shrews were not cooperative research subjects. I did not adapt well to being confined and having my period only at certain times of the year. Catching them wasn’t easy either; van der Horst and his colleagues used hand nets to catch them. But because the elephant shrews were so agile, he wrote, “this sport was sometimes exciting, but most of the time the results were disappointing.”
Around the same time, Harvard University biologist George WD Hamlett discovered an alternative to the elephant shrew. While examining preserved samples of long-tongued bats, which feed mainly on nectar, he found evidence that they menstruate. However, long-tongued bats mainly live in Central and South America, so access was not easy. For the next several decades, his discoveries remained little more than interesting facts to be found in the scientific literature.
And in the 1960s, John J. Rasweiler IV, an enthusiastic graduate student, entered Cornell University. He wanted to study animals with reproductive systems similar to humans, and his advisor told him about Hamlet’s discoveries. Lasweiler wanted to know what he could do with the long-tongued bat.
With their long snouts reminiscent of elephant trunks and opossum-like bodies, elephant shrews were already unusual enough even before van der Horst discovered that they were one of the few animals that menstruated.
“It was a very challenging task. “Everything had to be redesigned from start to finish.” Lasweiler recalled. First, he went to Trinidad and Colombia to capture bats. The next problem was transporting the bats to the United States without them being crushed or overheated by the intense heat. (Putting the bats in food containers, tying them together, and then placing them in a large box turned out to be the most effective method.) Once we had succeeded in transporting the bats to the lab, we now had to figure out a way to study them while keeping them from escaping. . After careful consideration, he built a special cage with wheels and used the method of moving the bats by rolling the wheels.
“The long-tongued bat is a really cool animal,” said Rasweiler, a retired reproductive physiologist at the State University of New York. “I really enjoyed the research process,” he said. But other researchers were not as excited about studying flying animals as Lasweiler.
In 2016, the spiny mouse joined the list of animals that menstruate. The spiny rat is a type of rodent that lives in dry environments in the Middle East, South Asia, and parts of Africa. Spiny mice can play an important role in menstruation research because they can be bred in the laboratory. But Brosens points out that because humans and mice evolved in different directions over millions of years, the genes that make up the uterus may be quite different.
Most basic research on menstruation has been conducted on macaque monkeys. However, primates are expensive to raise, and unlike other general laboratory animals, they are restricted by animal welfare laws. With a few tweaks, scientists have been able to make lab mice experience a phenomenon similar to menstruation. Although this model was useful, it was still only an artificial replica of human menstruation.
Researchers needed to conduct menstruation research on humans. However, in addition to obvious ethical issues, conducting actual research is not easy. “The endometrium is a very dynamic tissue that grows very quickly and changes in cellular responses and functions can be observed every hour,” said Aleksandra Tsolova, a cell biologist at the University of Calgary. In order to study the endometrium inside the human body, invasive tests must be continuously performed. Even so, it is nearly impossible to genetically modify or chemically change the endometrium.
However, in the early 1900s, solutions to the problem began to appear. It was not an animal from the jungle or the African grasslands that laid the foundation for the study, but an organism from the bottom of the ocean floor.
The emergence of organoids
The basic foundation for today’s organoids was laid in the 1910s. Henry Van Peters Wilson, a zoologist of the time, discovered that sponge cells retain a kind of ‘memory’ of their previous arrangement even after they are separated. When he separated the sponge by squeezing it into a mesh net and mixing the cells back together, the original shape formed again. Research in the mid-20th century also found that certain cells in chick embryos had similar abilities.
In 2009, a study explaining how to apply these functions to human organs was published in the academic journal Nature. This research team collected adult stem cells that can grow into intestinal cells from the intestines of mice and placed them in a gel-like material. Then, as the cells divided and grew, a simplified miniature version of the mouse’s intestinal mucosa was created. This was the first study to suggest a method of creating organoids from human tissue, and could be attempted in most laboratories and applied to other organs.
Since then, scientists have expanded this approach to create dozens of human tissue organoids, including intestines, kidneys, and brains, and at the end of 2010, uterine organoids were also created.
Endometrial organoids were created through serendipity. Scientists have been conducting research for several years by culturing endometrial cells in laboratory dishes. This method was effective for stromal cells, which structurally support endometrial tissue and play an important role in pregnancy. Stromal cells secrete substances that stick to each other, and they stuck to the laboratory dish just as well. But the epithelial cells, another major component of the endometrium, were different. The epithelial cells in the experimental dish did not respond to hormones and their morphology was different from those in the human body.
Later, reproductive biologist Margherita Turco discovered something unusual while mixing human placenta and endometrial tissue to create organoids from the placenta. When molecules derived from the human body were mixed in appropriate amounts and grown in a gel state rather than a liquid, endometrial epithelial cells were created like a three-dimensional miniature version of the endometrium. “The endometrial organoids grew well and there were a lot of them,” explained Turco. Around the same time, another research team also published similar research results.
Today, placental and endometrial organoids are valuable research tools in Turco’s laboratory at the Friedrich Miescher Institute for Biomedical Research in Basel, Switzerland. Through research published in 2017, Turco proposed a method of creating endometrial organoids from tissues collected from biopsies rather than stem cells. Some laboratories also use tissue obtained from people who have had hysterectomies. However, Turco’s research team recently demonstrated that endometrial fragments found in menstrual blood are also effective. In other words, it is now possible to create new endometrial organoids without biopsy or surgery.
Based on these advances, researchers were able to create a miniature version of the human uterus. Cholova said each organoid looks like a tiny bubble inside a jelly dessert. And they provide a special opportunity to understand menstruation, which has been neglected by the scientific community.
Menstruation recreated in the lab
For the small number of researchers studying the uterus, endometrial organoids have become a very important tool. Since 2017, many laboratories have been utilizing this new tool.
Julie Kim’s lab added stromal cells to the epithelial cells that make up a typical endometrial organoid. They mixed the two types of cells and observed what actions ‘happened on their own’. The result was a chocolate-coated cookie-like shape with stromal cells on the inside and epithelial cells on the outside.
In 2021, a research team led by Brosens created a similar structure and named it ‘assembloid’. However, instead of mixing the two types of cells, they created organoids from epithelial cells and then added a stromal cell layer on top. Through Assembleloid, they learned that disintegrating cells play a key role in the implantation of embryos into the uterus. Because endometrial cells repeatedly collapse and grow, the tissue is very flexible and can change shape, Brosens explained. These characteristics help with pregnancy. “The maternal cells grab the embryo and pull it into the endometrial tissue,” Brosens added.
A recently released video by Brosens shows assembleloids being reorganized around an embryo five days after fertilization. Before starting the study, his research team believed that the endometrium was a passive tissue that was invaded by embryos, as was commonly known. But Brosens insisted: “That was a completely wrong idea.” Understanding how embryos implant can improve existing in vitro fertilization methods and explain why some people easily miscarry.
Critchley hopes that one day scientists will be able to develop treatments that will allow people to choose when to menstruate or not to menstruate. Although hormonal birth control pills are useful for some, they can cause unexpected bleeding, make menstruation more difficult to regulate, and can have serious side effects in some people.
To provide better options, scientists need to understand how normal menstruation occurs. Research into inducing menstruation in organoids in a laboratory dish could go a long way toward achieving this goal, and many researchers are attempting it.
Gneco’s research team succeeded in reproducing the one-month cycle of the endometrium by administering hormones to organoids. They were able to observe that as the cycle progressed, the organoid cells regulated the combination of genes, just like in the human body. The shape of the organoids also showed a similar pattern to the real thing. The glands, which are junctions of cells that secrete mucus and other substances, changed from a smooth tube shape to a jagged structure as the virtual menstrual cycle progressed.
“It’s amazing that we can conduct research at a very close level to the patient, but without having to use the actual patient’s body as the subject. Organoids have tremendous potential.”
Alexandra Cholova, cell biologist, University of Calgary
Now that organoids are available, the next step is to identify problems that cause the endometrium not to function properly. Gneko expressed her excitement, saying, “I’m really looking forward to it.” In the first step of his research, he applied an inflammatory molecule called IL-1β, a characteristic lesion of endometriosis, to organoids. IL-1β led to rapid organoid growth, but only when stromal cells were mixed with epithelial cells. These results allowed Gneko to guess that stromal cells were responsible for causing endometriosis to be a painful condition.
Meanwhile, Killinck is trying to figure out why some people have excessive menstrual bleeding. When endometrial tissue grows in the muscles on the surface of the uterus, it causes lesions, which can cause excessive menstrual bleeding. To find out how these lesions are formed, Killinck observed how endometrial organoids responded when placed in a hard gel that mimicked the texture of muscle.
In the soft gel, the endometrial organoids maintained a smooth and round structure. However, it was completely different in the hard gel. A video of Killinck’s latest experiment shows the organoids pulsing and writhing, like a pot overflowing with water. Eventually, some cells broke off and formed an appendage-like structure that punctured the hard gel. This experiment led Killinck to consider the possibility that contact with muscle may be the cause of scarring of the endometrial tissue and causing severe bleeding. However, she added, “It is not yet a definite fact and we are investigating the cause.”
rapid progress in science
Currently, endometrial organoids are not fully tested on animals. For example, it does not contain blood vessels and immune cells, which are key components of menstruation. Additionally, it is unknown how distant tissues, such as the brain, affect the uterus. However, because organoids are made from human tissue, they have a high correlation with the strange and special process of human menstruation and are of high value in themselves. “It’s amazing that you can do research at a very close level to the patient, but you don’t have to use the actual patient’s body,” Cholova said. “Organoids have tremendous potential,” he emphasized.
In addition to organoid research, scientists are developing an ‘organ on a chip’ that mimics the endometrium. Small tubes attached to a flat surface mimic the flow of blood or hormones from other parts of the body and transport them to the endometrial tissue. In the future, the ideal experimental model will be one that combines the natural arrangement of endometrial cells, such as organoids, and the flow of blood and hormones, such as an organ chip.
Organoids have helped researchers solve an old mystery. For example, researchers in Vienna, Austria used organoids to determine which genes cause endometrial cells to grow into cilia. These cilia are responsible for moving fluid, mucus, and embryos inside the uterus. Some researchers have used organoids to understand how endometrial cells mature during the menstrual cycle. Julie Kim’s research team also used organoids to study how the endometrium responds to abnormal hormone levels, which can cause endometrial cancer.
Many people who menstruate have been waiting for a long time for full-scale research from scientists. Menstrual difficulties are often considered a ‘women’s problem’. But Cholova disagrees. This is because they have forgotten that people who experience menstrual problems cannot fully contribute their talents to society. Cholova said, “Menstruation is more of a social problem than an individual one. “Because it affects everyone in every way,” he emphasized.
Saima Sidik, who wrote this article, is a freelance science journalist based in Somerville, Massachusetts, USA.