New in vitro maturation (IVM) technique from stem cell-derived ovarian support cells

A series of 2023 studies from Christian Kramme et al. demonstrate the effectiveness of a novel in vitro maturation (IVM) technique that uses human stem cell-derived ovarian support cells, which is both flexible and simple, enabling the maturation of eggs that would otherwise be discarded.

In vitro maturation (IVM) involves the retrieval of immature eggs that are then matured and fertilized in the IVF lab. However, eggs from traditional IVF procedures can be immature, and performing IVM on these eggs has its challenges.

In a series of papers published in March 2023, Christian Kramme et al. describe the development of ovarian support cells (OSCs) using human stem cells that can be used for a new type of IVM called OSC-IVM. The first paper shows how they developed these OSCs, and how they can go on to form artificial ovaries when mixed with germ cells. The second and third paper show that the OSCs can be mixed with immature eggs from IVF to mature them in the lab.

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Researchers create ovarian support cells from stem cells that can go on to form an artificial ovary

Stem cells are cells that are unspecialized and can be converted into a variety of different cell types, such as neurons or skin cells. Treating stem cells with transcription factors can cause the stem cell to differentiate into a particular cell type. Likewise, differentiated cells like skin cells can be “reprogrammed” to become stem cells once again. These are sometimes called “induced pluripotent stem cells.”

Pierson Smela et al. (2023) generated granulosa-like cells (or OSCs as they’re called in the next sections) by treating human induced pluripotent stem cells with the transcription factors NR5A1 and either RUNX1 or RUNX2. Granulosa cells are cells that surround and support the development of egg cells. They whittled down these 3 factors from a list of 35 that they suspected would help in producing granulosa cells.

They found that these granulosa-like cells were able to express genes that were characteristic of granulosa cells and stromal (theca) cells. So these granulosa-like cells were really a mix of granulosa-like cells and ovarian stroma-like cells.

Furthermore, the granulosa-like cells were also able to make estradiol from a precursor which was increased after stimulation with FSH. The cells were also able to make progesterone. These hormones are both produced by granulosa cells, so this is a good sign that these cells are functioning as granulosa cells.

While these granulosa-like cells were able to make estradiol and progesterone, they wanted to see if they could support the development of germ cells. Germ cells are cells that develop into reproductive cells, like egg cells. There are many steps from a germ cell to a mature egg, and in mouse models from previous studies, other scientists have been able to grow germ cells to a stage that resembles the oogonium, which is a very immature type of egg cell.

They combined these granulosa-like cells with human primordial germ cell-like cells (germ cells), to form ovarian organoids that they called “ovaroids.”

Note: As we’ll see in the next sections, immature egg cells will be added to these granulosa-like cells/OSCs instead of germ cells. They’re only making these ovaroids by adding germ cells in this study to see how far these granulosa-like cells can go in supporting egg development.

These ovaroids weren’t able to make egg cells, but they made empty follicles, which you can see below:

From Pierson Smela et al. (2023), CC BY-SA 4.0

And after 70 days, some of these follicles developed even further to make empty antral follicles (one of which is shown below). The granulosa-like cells are stained in green and formed layers around a central cavity. Cells outside of the granulosa-like cells (the stroma) resembled theca cells.

So there’s still work to be done to produce actual eggs from this artificial ovary, but it’s a step in the right direction!

While actual eggs weren’t made, these granulosa-like cells were able to function as granulosa cells to some degree, including producing hormones like estradiol and progesterone. Besides these hormones, it’s likely that these cells are producing other factors needed for egg development. So these granulosa-like cells (or OSCs), when mixed with immature eggs during IVF, may be a good strategy for making mature eggs in the lab.

In the next two papers, researchers looked at how these cells can be used in IVM.

OSC-IVM produces mature eggs that are comparable to normally developed mature eggs

Giovannini et al. (2023) tested the ability of ovarian supporting cells (OSCs) to mature immature eggs. They used donated immature eggs that were stripped of their cumulus cells.

Note: This study is not peer-reviewed (it was submitted to the biorxiv preprint server).

Let’s discuss the procedure a bit so all the embryologists/doctors out there can appreciate the simplicity of this system.

The morning of egg collection, OSCs were thawed. These were the cells that were made using the transcription factors that went on to form the artificial ovaries as described above.

The OSCs were thawed, washed, then added to culture drops overlaid with culturing oil. The immature and stripped eggs were added to these drops and left to culture for 24 to 28 hours.

After co-culture, the OSCs were washed off and the eggs were assessed for maturity.

The OSCs induced maturation more so than the control (62% vs 37%, p= 0.0138). The eggs had their morphology scored and there was no difference between the groups, suggesting that OSCs don’t affect morphological features of the eggs.

There was no difference in the angle between the meiotic spindle and the polar body in the eggs either, further suggesting the eggs matured normally.

They also found that gene expression profiles of OSC-IVM matured eggs was similar to controls.

OSC-IVM produces quality embryos that are euploid, comparable to traditional IVM

Piechota et al. (2023) compared OSC-IVM to a traditional IVM procedure. To do this, they used an abbreviated ovarian stimulation protocol, similar to what’s normally done with traditional IVM procedures.

Note: This study is not peer-reviewed (it was submitted to the biorxiv preprint server).

The OSCs were prepared like before and the eggs (with their cumulus cells still attached) were co-cultured for 24 to 28 hours with the OSCs.

The OSC-IVM group had a higher maturation rate compared to the traditional IVM group (68% vs 43%, p= 0.0349). Morphologically, the eggs were the same between the groups.

In terms of IVF outcomes, there were no statistically significant differences, however the groups were small (26 mature eggs from OSC-IVM vs 23 mature eggs from traditional IVM):

Maturation (61.5% vs 52.5%, p= 0.4247)
Fertilization (87.5% vs 75%, p= 0.3550)
Blastocyst rate (85.7% vs 55.6%, p= 0.1543)
Euploidy rate (57.1% vs 22.2%, p= 0.4130)

All the usable blastocysts developed by OSC-IVM were euploid (8 blastocysts: 8 euploid), compared to half of those by traditional IVM (4 blastocysts: 2 euploid).

Even though the results weren’t significant, it shows that OSC-IVM can produce quality euploid embryos.

Conclusions

The studies above show that OSCs function like granulosa- and ovarian stroma-like cells, and can produce the hormones estradiol and progesterone as well as gene expression patterns that are consistent with these cells.

The cells can also go on to form artificial ovaries when mixed with germ cells (an “ovaroid”) that was able to produce empty follicles. Egg development was limited in this system and more work needs to be done.

The OSCs, when mixed with immature eggs obtained during IVF, could cause the immature eggs to mature and develop into healthy blastocysts that were euploid. In terms of IVF outcomes, the OSC-IVM procedure was superior to traditional IVM by some metrics.

OSC-IVM represents a new way to perform IVM that’s much more flexible than traditional IVM procedures, which may involve a specialized stimulation protocol, a hCG trigger, and the presence of cumulus cells.

During a typical IVF cycle, eggs that are not mature are often discarded, but with OSC-IVM these immature eggs could be mixed with OSCs to mature them and potentially get more embryos for transfer.

This work is preliminary, and some of the studies have yet to be peer reviewed. The authors point out that more research is needed to ensure that healthy births result from these eggs.

About Embryoman

Embryoman (Sean Lauber) is a former embryologist and the founder of Remembryo, an IVF research and fertility education website. After working in an IVF lab in the US, he returned to Canada and now focuses on making fertility research more accessible. He holds a Master’s in Immunology and launched Remembryo in 2018 to help patients and professionals make sense of IVF research. Sean shares weekly study updates on Facebook, Instagram, and Reddit regularly. He also answers questions on Reddit or in his private Facebook group.