Fertility Preservation to BRCA Mutations: A Path to Discovering Mechanisms and Manipulation of Ovarian Aging - Kutluk Oktay, MD, PhD, FACOG

Fertility Preservation to BRCA Mutations: A Path to Discovering Mechanisms and Manipulation of Ovarian Aging  - Kutluk Oktay, MD, PhD, FACOG

Endofound Medical Conference 2017
"Breast, Ovary and Endometriosis"
October 28, 2017 - Lotte New York Palace Hotel

Fertility Preservation to BRCA Mutations: A Path to Discovering Mechanisms and Manipulation of Ovarian Aging

Kutluk Oktay, MD, PhD, FACOG
Director, Laboratory of Molecular Reproduction & Fertility Preservation
Professor, OBGYN: Yale University School of Medicine

Thanks to Dr. Seckin and the Endometriosis Foundation for including me right here, so today we're going to talk about something that's not direct related to endometriosis, but we will have some common themes with that. I'm going to tell you a story about how we come in to a topic that relates to aging overall through dealing with cancer patients and going through the path of fertility preservation, but first of all, let's look at a few basic things.

Women are born with a set number of eggs, and these eggs that are inactive and which are like the Sleeping Beauty, you'd never know when they would wake up and start growing. We call them primordial follicles. There are about a million of these at birth, and let me just set up my pen here. There we go. There we go. All right, here we go. There are about a million of these, and they would start growing through an unknown process. Eventually, it may take about three months for a sleeping follicle to get to an ovulatory stage and be seen on an ultrasound, so we cannot directly measure these follicles which are considered the ovarian reserve, but we can use a marker, for example, called Anti-Müllerian hormone which indirectly reflects the ovarian reserve.

I started my early part of my career doing my fellowship studying these follicles, and the question was that how come they'd be sleeping for such a long time, and what were the mechanisms that would make them start growth and eventually die? Because that determines what happens to a women's reproductive lifespan and when she goes to menopause, so I started this research under a mentorship of an aging researcher, Jim Nelson, at the UT San Antonio — we're actually heading there tomorrow for a conference — and which led me to work with another pioneer in the field in Leeds. After my fellowship, I took a year to work with him, Roger Gosden. Those two guys actually connected me with Dr. Malcolm Faddy in Australia, who's a mathematician, so what is the connection here? We were actually, again, studying the mechanism of how these follicles are growing and eventually disappearing.

Here's a slide from a study that actually these two scientists did, and I kind of piggybacked on to that, looking at number of follicles from cadavers of different ages. At birth there are about a million of these eggs present, and as a woman ages there's less and less. Initially there's a steady decline in these numbers, but once you hit the age of about 37 and 38, there's a sharper decline. A couple of things here. Even though you're starting at a million, you're ovulating maybe 500 in a lifetime. By the time you get to about age 50, almost nothing is left, so about 99.9% are wasted. The second part is that these follicles, once you hit age 37, they're disappearing at a much faster rate, so for example, if you could keep this rate steady, menopause may happen in age 70 or so.

We were intrigued by these, and my earlier part of my career was focused on that. As a matter of fact, that parallels everything else that happens with the reproductive life. For example, if you look at natural fecundity rates, they also decline rather steadily upon till age 37 or so and there's a sharper decline. IVF success rates also start dropping sharply after that age, 36 or 37. If you look at oocytes that are ovulated and you check the chromosomes in them, and then plot the percentage of oocytes with normal chromosomes versus age, you will again see that once you hit 37, 38 or so there's a shaper decline, so there's definitely something happening, once you're in your late 30s, to oocytes. In a number of children born with chromosomal abnormalities, also sharply increase around that age.

That question intrigued us, and my goal in my early career was to study that, so when I went to England and worked with Roger Gosden, he was also working on fertility preservation research, specifically on ovarian freezing in animal models. That's where I picked the procedure, and I came back to the United States, did the first clinical application, and even though we did this quite secretly and quietly, my mentor in England leaked us to press and it turned into a big circus that what we did was actually reversing menopause. I don't know if you remember this morning. I told Dr. Gomez that I met you. Maybe you don't remember me. Actually, I met him when this happened and interviewed me. At that time, he looked at me and said, "Dr. Oktay, you look awfully young." I was young. That was about 18 years prior to this.

What happened, though, because of this attention, fertility preservation got more attention, and as a result we've started getting patients who wanted to present their fertility for various reasons under various case scenarios. One of the most common cancers that we would see in a reproductive-aged woman is breast cancer, so we have developed this algorithm that you're seeing here to help the women with various case scenarios, but for a woman with estrogen sensitive cancer, with breast cancer, about 10, 15 years ago nobody would do ovarian stimulation because breast cancer is in general estrogen sensitive, so we've come up with a protocol that uses aromatase inhibitors. Here's our connection, perhaps, with endometriosis, and that's a drug that's also used in endometriosis, as well in breast cancer, so by using that, we could stimulate these patients, keep their estrogen levels low. At the same time, obtaining oocytes and eggs for future childbearing.

Currently, we've completed this protocol, and success rates are looking good and lots of good outcomes and no recurrent rates from that, but the interesting thing that we've observed from this protocol was that since these are young patients and a lot of them had BRCA mutations, as many of you know, BRCA mutations are associated with a high risk of breast and ovarian cancers, so when we looked at how many eggs we were getting from BRCA1 patients and compared them to those that were negative, we realized that there were actually fewer. That made us to ask the question of why. There are actually two BRCA genes, and they're completely different genes on different chromosomes. One is on 17 and the other one is on 13, but what they do is actually repair DNA.

You and I, as we are sitting here, are probably going through thousands of DNA damage points in our DNA, especially double-strand DNA break, so if we didn't have a repair mechanism, we would just fall apart in a matter of minutes and so many mutations would happen, so these are genes are part of a pathway that constantly repairs DNA. There are different kinds of DNA damage. If you just had one strand of your DNA broken, that's called a single-strand break, and that's not a big problem because you have the other strand there and you can just copy from there and repair it. The problem happens when you lose both strands. That's called a double-strand DNA break, and that's a very severe form of mutation. You don't have the other copy of the gene there that you can copy and repair from.

For these conditions there are a couple of pathways that evolved, but for oocytes this is the most applicable pathway. You are using your sister chromatid, so you got a pair of chromosome from your father and your mother, and if one lost both copies of the genes, the other one is right next to it, especially in a certain part of the cell cycle, what we call late prophase, and then with this repair mechanism, you use that sister chromatid and then copy from that to repair that. That's called homologous recombination, but it has to be a very precise mechanism because, again remember, if you lose both copies of a gene or you don't repair properly, it's very severe mutation. Often leads to cancer and other severe conditions, so because of that, a particular pathway has evolved.

It's a complex pathway there because there are a lot of molecules, as you see here. They interact with each other and they each do different things. For example, this group here that includes MRE11 and other couple of genes there, we call them MRN complex. It actually senses the damage. "Oh, there is a break there and we have to act on it." Whereas other genes like BRCA1 and BRCA2, they work on repair. There's also an orchestrator here called ATM. It tells people, "You go there, you go there," and etc. Then there's what's called an histone H2AX protein there that actually marks where the damage is, so it goes and binds there, and we can use this to our advantage. We can use stains, especial stains, that bind to that H2AX and then we can count those sites and tell how much damage that cell has, so you'll see that in later data that I'm going to show, how we use that.

When we saw that, we put these two things together and said, "Is it possible that the DNA, this double-strand repair very important in oocyte aging, then is it also possible that this decline in repair efficiency with age could explain and maybe parallel this decline in ovarian reserve and reproductive efficiency and egg quality?" We started asking a number of questions, so we designed experiments involving mice, human patients, etc., and which was published a number of years ago in Science Translational Medicine. Without going into too much detail, these studies involved again mouse and human oocytes, patients, and as well as transgenic mice.

What did we find here? As I told that we could stain those H2AX molecules which correlate with DNA damage, so when we look at inhuman primordial follicles, those are the Sleeping Beauty follicles and count them, what we find is that compared to young, the older individuals, these are maybe 10 year older than the younger group, and there are tremendous increase in H2AX. For example, this is a ovarian biopsy from a two year old. We get these tissues because we have a lot of patients undergoing ovarian tissue freezing. A part of that is set aside for research. Almost no DNA damage in their follicles, but when you go to age 23, lots of follicles are lighting up. This is also true in the ovulated follicles. The same thing happens.

Perhaps the mouse data shows it better. The red stain indicates DNA damage, so the young mice here are maybe a couple of months old, they're like teenagers, and the old are more like a human in their late 30s, early 40s, and look at the amount of red in these older mice. These oocytes are, as we grow older, accumulating more and more double-strand DNA breaks, but we have all these enzymes in genes. They're supposed to be repairing them. Why are they accumulating them? We hypothesized that this is happening because the ability of the cell to repair these damages is going down. To be able to study that, we designed an approach where we would take a single oocyte and amplified messenger RNA. That's the DNA's way of telling the factories in the cell what molecules to make, so with that methodology, we could study from individual eggs their ability to repair DNA.

We did that, and we looked at the key genes that are involved in that pathway that I showed you in DNA repair, including BRCA1 and BRCA2. Again, these are oocytes from individual patients, aged somewhere from 23 to 41, and if you plot them against age, what we are finding is that there's a steady decline. There's a decline in BRCA1 and other key DNA repair genes, but not in BRCA2, not significantly if you look at it this way. This kind of parallels what's happening with the cancer risks as well, where this function of, for example, BRCA1 is much more evident early on. I'm not going to get into detail of that, but BRCA1 presents a more significant issue here.

Then we said, "Well, the decline in DNA repair ability, does it also go down faster after age 36 or so when all the reproductive functions decline?" It turns out that for ATM, MRE11 we're at 51. Again, these are key players. This is what happens. The ability to repair DNA in eggs drops five to six times once you hit about 35, 26, that range. Interesting. For BRCA1 that's pretty steady, but BRCA2, even though we did not see a significant decline, that change happens much later, perhaps a little past reproductive age. That's probably why we're not seeing any significant effect from BRCA2 mutations on reproduction.

We actually confirmed this in a transgenic mouse model where the mice are deficient for BRCA1 and BRCA2. Only BRCA1 mice that we would find that if you stimulate them, you would have fewer oocytes compared to controls. Actually, you make them. They will have fewer number of pops again, and they have fewer follicles in their ovaries and that's where it is, but not BRCA2. Over a lifetime, they would accumulate more damage in their ovaries compared to BRCA2 mice, so the physiology and pathophysiology parallels between mouse and human.

Then the next question that we asked. Could we actually manipulate this? You know, could we change the way the oocyte repairs DNA? You can actually inject chemicals into a cell, what we call interfering RNA, to block genes individually in each cell, in this case oocyte, or you could inject what's called cDNA or mRNA to push the cell to make more of those enzymes, in this case again, DNA repair. When we did that, injected into oocytes first the interfering RNA, which blocks the genes' function, and measured DNA damage, cell death, and their survival, what we are finding is striking. These two groups are controls, so when you inject and block those genes and expose these cells to genotoxic stress, in this case it's hydrogen peroxide, the DNA damage goes up tremendously within 16 hours. The cell, that goes up and their survival goes down.

What about the opposite? If you're BRCA deficient, can we regulate that and make that cell as good as the young cell? Here we took older oocytes, and this is a control old oocyte. You inject them with a cDNA construct and expose them to genotoxic stress, and what happens here is that the survival levels are now brought to the level of a young oocyte, so this indicates that the ability of these oocytes to survive can be manipulated to our advantage.

Finally, we wanted to see if this is true, then women with BRCA mutations or BRCA function deficient who have developed cancer at the same time might have lower ovarian reserve, so we measured their serum Anti-Müllerian hormone level, which as I explained you, an indirect way of measuring primordial follicles, so these are women with no BRCA mutations. These are with BRCA1 mutations and these with BRCA2, so again, only the ones with BRCA1 mutations had reduced serum AMH levels.

Recently, we also were able to collect ovarian tissue from women with BRCA mutations while they undergo prophylactic ovary removal, so we could count their follicles and compare them to controls. These are actual counts of those quiet follicles or the sleeping follicles in their ovaries. Look at the primordial follicle counts in control group and versus women with BRCA1 and 2 mutations. Not only that, when you plot their follicle numbers against age, what we are finding is that there's much faster decline in BRCA carriers, and if you take those older than 30, it diverges further. There's much faster loss. Top is control. You can see where this is going. They're also accumulating DNA damage at a much faster pace, so in the ovaries of women with BRCA mutations, the process of aging seems to be accelerated.

Putting all this together, it appears that what we call this homologous recombination DNA repair pathway might have a significant role in oocyte aging both in mouse and human, and this seems to be most significant in patients with BRCA1 mutations. Now what we have shown you has been now confirmed by many studies. There are at least four other studies that have shown that serum AMH levels are lower in women with BRCA mutations, that they experience earlier menopause. There are also large wide association studies. Now all of us have some letter variations in our DNA, and some of those letter variations can affect how the gene functions, so you can look at those variations and correlate them with certain outcomes. In this case, menopausal age. When they did that in 70,000 women, guess in what kind of genes the letter variation matters most in terms of determining menopausal age? That is again DNA repair genes. That's this big list here, so the way we repair DNA matters a lot in terms of reproductive lifespan.

Now what about egg quality? When we say quality, it's an ability of an egg to turn into a healthy baby, and that goes down with age. Does that play a role in that as well, the DNA repair? We're encountering patients like this. This is a patient with BRCA2 mutation. When she was 36, she had a perfect and normal spontaneous pregnancy. Then she developed breast cancer, found to have BRCA2 mutations. Next pregnancy was a trisomy 21. Then after that, she had nine IVF cycles. From eight to four oocytes only, three went to the last stage of embryo development called blastocyst. From those, we could only find one embryo that has normal number of chromosomes, which was BRCA positive, so if you look at the embryos, we are seeing embryos that fertilize with four or five different nuclei and abnormal embryo development, etc.

But this is not so surprising because in mice, if you knock, again, with one of those methods that I showed you, BRCA1 gene out of the egg, the chromosomes go all over the place and they become unemployed. I don't have time to go into details, but the same pathway that controls maybe eggs' faith in terms of survival also seems to control what happens with chromosomes, and as I showed you in the beginning of my talk, as women get older, their chromosomes tend to go astray and those oocytes will have more [unemployety 00:22:25] resulting in more abnormalities.

With that, we've come with this explanation as a theory that as women age, their DNA repair goes down, their BRCA function goes down, and that results in oocytes becoming more faulty and more liable to death, because if you have your DNA damage, the body has survival mechanisms, gets rid of the DNA damaged cells if it can't repair it, so at the older age, that's happening at a faster rate. At the same time, because DNA repair is also involving keeping chromosomes in check and it doesn't function as well, now the chromosomes are all over the place, so that explains why with aging reproductive quality goes down.

Now what about men? Now we are actually finding out that ... this is one of my past recently graduated PhD students. His thesis is not published yet. If you look at DNA damage, double-strand DNA damage in sperm, compare young mice to old mice, there's a striking increase. If you take BRCA1 mutant mice, male mice, if you look at their sperm compared to mice that don't have BRCA mutation, again DNA damage goes up strikingly. If you take sperm from a BRCA mutant mice and fertilize an egg from a normal mouse, and compare it to controls, the number of abnormal embryos goes up strikingly and you would get embryos like this, all with fragments and etc., and the implantation rate of these embryos, again this is if you fertilize with a BRCA mutant sperm versus normal sperm in normal mice, there's a tremendous decrease.

In summary, it appears that the way we repair DNA is very important in female, but possibly in male as well, so what does that mean? That means that if you have target that we can manipulate and that controls aging, that bodes well for future therapeutic applications, and there may be more implications of this. Now, humans don't die of aging. They actually die of the complication of the diseases of aging. Both neurodegenerative diseases, cardiovascular diseases, metabolic diseases, cancer, they're actually consequences of aging, so if we can enhance DNA repair, it might not only correct reproductive aging, but potentially we can slow down overall aging and perhaps we can discover the fountain of youth, and we can all live long and prosper. With that, I'd like to thank immediate members of my team involved in most of the work and my research laboratory RTL and other collaborators. This work is supported by National Institutes of Health. Thank you.