March 3, 2017

Story and photo by David Edwards

February's featured paper is titled, "Functional human oocytes generated by transfer of polar body genomes," published in Cell Stem Cell. It was an extensive effort from researchers at OHSU's Center for Embryonic Cell and Gene Therapy, led by Shoukhrat Mitalipov, Ph.D., center director and professor of biomedical engineering, obstetrics and gynecology, pediatrics, and molecular and medical genetics, OHSU School of Medicine, in collaboration with researchers at the Salk Institute for Biological Studies in La Jolla, Calif., led by Joseph R. Ecker, Ph.D. Its co-first authors are Hong Ma, M.D., Ph.D., staff scientist, OHSU, and Ryan C. O'Neil, graduate student, Salk Institute.

Overcoming infertility

The birth of Louise Brown in 1978 represented a landmark achievement in reproductive medicine. She was the first child born from in vitro fertilization (IVF), the process of fertilizing an egg (also called an oocyte) outside the body and transplanting it back into the uterus. Since that time, IVF has become a commonplace practice, with over five million children born using IVF or other reproductive measures.

Although IVF has resulted in lower rates of infertility, in certain situations, there remain significant challenges. For example, because the number and quality of the oocytes available for IVF decreases over time, women of advanced maternal age have a reduced likelihood of having enough high-quality oocytes to give birth to healthy, genetically related children.

One way of overcoming this problem lies in the way oocytes develop and mature in the body. Before ovulation, the primary oocyte replicates its DNA and divides, giving rise to two daughter cells. Although both of these new cells contain the same genetic material, they divide unevenly: the larger daughter cell (called the secondary oocyte) continues the maturation process, while the much smaller daughter cell (called the first polar body, or PB1) undergoes a programmed cell death called apoptosis. This process happens again after fertilization with sperm. The secondary oocyte divides, forming a larger cell (the mature ovum) that continues along the fertilization process to become a zygote and a much smaller cell (the second polar body, or PB2), which also undergoes apoptosis.

Turning trash into treasure

In normal fertilization, the polar bodies (PB1 and PB2s) eventually die and disappear, never contributing to the development of the embryo. However, because the polar bodies contain the same genetic material, there has been increasing interest to harness these cells and use that genetic material to enhance the effectiveness of IVF.

The idea is simple: Scientists remove the nucleus (and thereby the genetic material), a process called enucleation, from the secondary oocyte of a healthy, younger donor. Then they extract the first polar bodies from the infertility patient oocyte and transfer it into an enucleated donor oocyte. Because of their mirror image genetic makeup, both the original patient oocyte and the newly reconstructed oocyte made from the polar body can be used as siblings for fertilization.

The strategy of using polar bodies to generate functional oocytes – also called Polar Body Nuclear Transfer or PBNT – has been borne out, pardon the pun, in previous studies involving mice. Researchers have showed that both PB1s and PB2s can be successfully transferred into enucleated oocytes, resulting in healthy offspring. However, the results from these two-decades-old studies had not been translated to other species, including primates and humans – until now.

Success in human cells

A team of researchers led by Dr. Mitalipov, in collaboration with researchers from the Salk Institute in La Jolla, California, recently tested this hypothesis in humans. They used secondary oocytes from 11 young, healthy donors (25-31 years old). Similar to previous studies, they extracted the PB1s from one donor and introduced them into enucleated oocytes from another donor. They measured oocyte development and evaluated the cells produced from PBNT-generated oocytes when compared to normal, healthy oocytes.

They found that the PBNT oocytes could form into complete, functional secondary oocytes, mirroring the normal development process. After fertilization with sperm, these oocytes successfully divided into two daughter cells, producing mature zygotes and the second polar bodies. They then allowed these zygotes to continue dividing, forming a 200-300 cell structure called a blastocyst, and found that the PBNT oocytes developed into blastocysts less often than mature zygotes.

Next, they evaluated the clinical applicability of PBNT by measuring the genomic integrity of the cells. (One common cause of oocyte maturation failure is an imbalanced transfer of genetic material during cell division, resulting in one daughter cell getting too many chromosomes and the other getting too few.) To do that, they generated embryonic stem cells (ESCs) from PBNT and control blastocysts. The genetic material isolated from PBNT-derived ESCs had a similar number of structural variations compared to normal ESCs. In addition, they shared similar patterns in gene regulation and expression, suggesting that the cells might function equivalently in the context of IVF.

PBNT in the clinic?

Their study demonstrated that, like previous studies have shown in mice, PBNT can successfully transfer genetic material from polar bodies to enucleated oocytes in humans. "What I like about this [study]," writes Mary Heinricher, Ph.D., associate dean for basic research, OHSU School of Medicine, "is the combination of basic cell biology and technology development to solve a specific clinical problem."

The PBNT technique can be used to potentially double the number of patient-derived oocytes and increase the rate of successful IVF treatments, helping women overcome fertility problems and become pregnant. Ultimately, the technique needs FDA approval before it can be used in fertility clinics, and Mitalipov's group is planning on conducting additional studies in nonhuman primates to evaluate its effectiveness.

"In addition to providing proof of principle for this process," the researchers wrote, "the approach could be helpful clinically for some forms of infertility and genetic disease." 

Specifically, it can be refined and expanded to overcome diseases caused by damaged mitochondria, small structures that produce energy in the cell. In theory, researchers could transfer healthy mitochondria from donor oocytes into mitochondria-deficient oocytes from the potential mothers. In other words, not only does this technique have the possibility of overcoming fertility problems, but also overcoming a broad category of diseases that affects around 1 in 4,000 people in the United States.

Resources

• Read the paper

Citation

Functional human oocytes generated by transfer of polar body genomes. Cell Stem Cell, 2017 Jan;20(1)112-9. Hong Ma, Ryan C O'Neil, Nuria Marti Gutierrez, Manoj Hariharan, Zhuzhu Z Zhang, Yupeng He, Cengiz Cinnioglu, Refik Kayali, Eunju Kang, Yeonmi Lee, Tomonari Hayama, Amy Koski, Joseph Nery, Rosa Castanon, Rebecca Tippner-Hedges, Riffat Ahmed, Crystal Van Dyken, Ying Li, Susan Olson, David Battaglia, David M Lee, Diana H Wu, Paula Amato, Don P Wolf, Joseph R Ecker, and Shoukhrat Mitalipov.

More Published Papers

• Previous School of Medicine Papers of the Month
• Recent OHSU published papers

Pictured above: 

Front Row from left to right: Diana Wu, Nuria Marti Gutierrez, Hong Ma, Crystal Van Dyken, Ying Li, Shoukhrat Mitalipov, David Lee

Back Row from left to right: David Battaglia, Paula Amato, Tomonari Hayama, Amy Koski, Riffat Ahmed, Susan Olson, Don Wolf