Well, I'm Shez Basaria from Brigham and Women's Hospital. And it's a pleasure for me to chair this session with Dr. Hayes from across town. We have a very exciting session, a blend of basic science and clinical work in the area of spermatogenesis. And the title of this session is Spermatogenesis Targets for Fertility and Contraception. Some housekeeping items. After each talk, we'll have a brief session for questions and answers. So please come to the microphone so that the presenters can address your questions. Also, this QR code, which was there before, I'm sure you had time to scan because you would be able to ask questions via that as well. So our first speaker is Dr. Chris Gehr, who is from East Carolina University. And he will be talking about studying spermatogonial differentiation and meiotic initiation to identify putative male contraception targets outside the blood testis barrier. So please welcome Dr. Gehr. Thank you very much. And thank you to the organizers. I'm sorry about the title. My talk kind of got out of hand and just kept going and going and going. But I'm really excited to talk to you about some of the work that's been going on in my lab over the last couple of years. And this is the first time I'm going to be presenting most of this. So let's see how it goes. OK, so this slide is one that has been shown at most of the different contraceptive talks that you'll see. And I think this is pretty obvious. Most of us know this from following the news, is that planet Earth is getting more crowded. We are having an increase in the number of children such that estimates are that by the time we get out to 2050, we're going to have somewhere estimates range on the low end perhaps 8 billion people to a high end being as many as 10 billion people. And in extreme cases, maybe even more than that. And I think that because of all the issues that are going on with climate change, it's a great concern about our ability to support that many people on our planet effectively and safely without having things like famine and disease, which is obviously, we just got a very important reminder about that and how globalization affects disease spread. And so this growth in the population of our world is not taking place uniformly across the planet. And so this is a chart. This is from the United Nations. And so these are countries. And it has the number of live births per woman charted over here on the left, if you can see that. So in the yellow countries, it's less than 1 and 1 1, so that would be a declining population. And then as you get towards the darker blue, that's a growing population. So you could see where most of the growth in the world is taking place, which is in Africa. And there are a lot of countries now, Canada being a great example, where the population rate is actually shrinking. And so governments are doing things there to increase the population. But I think overall, as a planet, we need to think about ways to control and curb population growth. So that's what's going on in 2020. If you put this out to 2050, I think the trend is still largely the same in that the regions of Africa are going to still lead the world in number of children. Unfortunately, that also goes along with the highest infant mortality rates, the highest rates of disease, and things of that nature. But I think these graphs are just meant to highlight where, perhaps, areas of the world might benefit the most from means of population control. And so if we look at population control from the male side of the equation, what are our options? And so ways to control fertility on the male side for years really have been only two options. And one here is obviously the condom. This is a technology. I was looking into the history of the condom. And it led back to at least medieval times, if not earlier. They were using intestines. And really, I was not aware that those were the earliest condoms were made out of animal intestines. But the concept's been around forever. And in 2022, it's kind of sad that we're still relying on technology that's that old to provide a physical barrier. That's obviously very important for preventing disease. But that's an option. There's a relatively high, I guess, failure rate associated with that, and not a great, not as good of a usage across the board as we would like for a lot of reasons. The other is a much more permanent, which is obviously the vasectomy. So this is a surgical intervention. It's not completely unreversible. But it's an expensive and, I hear, painful and not always successful procedure to go through reversals of vasectomies. So that's sort of a, if you make that decision, most people are done having children. So the desire, when we're looking at population control, is to come up with something that can work reversibly. And so this is where coming up with male contraceptive drugs comes in, where we can have something that doesn't have some of the drawbacks of the condom, like reducing sensitivity and reducing the pleasure of intercourse, or vasectomies, which are fairly, I'd say, somewhat irreversible. And so if you want to develop a male contraceptive, and I took some of this from conversations with Dan Johnston and off of his website at the NICHD, but what are some of the things that we're looking for when we're thinking about designing a male contraceptive drug? So the main goal would be to give men a burden in this family planning, because right now, a lot of this is put on women. And not all women can tolerate hormonal contraceptives very well. Another benefit, it reduces unwanted pregnancies, reduces the number of abortions. Again, the third one with the sensation during intercourse versus the condom, and something that is reversible. And so what would this look like? So obviously, high efficacy. These drugs need to work. It needs to be reversible. It needs to not affect the hormonal access in the male. It needs to have minimal side effects. And I would say, since a lot of its use is probably going to be in third world countries, it needs to be affordable. And so here's the model system that we use in my lab, which is spermatogenesis. And I'm going to go through this fairly quickly. So this is the beginning of spermatogenesis with the spermatogonial stem cells, the end being sperm. And spermatogenesis can be broken into three categories. So this is the pre-miotic or mitotically dividing component, which is made up of spermatogonia, followed by the miotic spermatocytes, the post-miotic spermatids going through spermiogenesis to generate sperm. And one thing that I want to highlight here is the location of the blood testis barrier, which is right here as cells are entering the first phase of meiosis, because that's going to be important here in just a second. And so here's the blood testis barrier. It's made up by tight junctions. Sorry, I'm pointing this pointer right at y'all's heads. Sorry about that. I don't want to poke anybody with the laser pointer. So the tight junctions between adjacent sertoli cells make up the blood testis barrier. And it separates the seminiferous epithelium into two compartments. And this is the adluminal compartment up here. Here's the lumen, the flagella of the condensed spermatids that are getting ready to be released. And this is where meiosis takes place primarily. And it starts down here, but most of meiosis takes place in the adluminal compartment, and all of spermiogenesis takes place. And the blood testis barrier separates that from the basal compartment, which is where the mitotic spermatogonia and the earliest miotic cells, preleptotine spermatocytes, reside. And the blood testis barrier is an important immune barrier. Prevents the immune system, along with sertoli cells themselves, from seeing what's going on inside of this. Because as you know, self versus non-self is established very early in life. Puberty happens in humans at 13, 14 years old. All of a sudden, these new proteins get made. Can't let the immune system see what's going on largely inside the adluminal compartment. So if we focus on the basal compartment, this is what's going on. We have spermatogonial stem cells, which are a typical self-renewing population. They make more of themselves, as well as proliferate as progenitors that amplify the population before committing to meiosis. And they make the commitment to meiosis in response to retinoic acid, or RA. Retinoic acid then starts a process in mice that's 8.6 days long. In humans, it's 6.5 days. In humans, it's 16 days long. We think they're very similar. And it's during that time that is an irreversible march towards meiosis. There are no genetic models that I'm aware of where you have any retention stably of these cell types. So the way I sort of look at it is once they commit to meiosis, it's meiosis or die. They're either going to go into meiosis or they're going to die trying. And then the testis in lots of models will fill up with undifferentiated spermatogon. There's lots of examples of that, but not any that I can think of where you have a stable population of differentiating. So it's those two paths that they're headed towards. And so they go through in mice these different cell types. You don't need to worry about the names. That's for a few people in the world that care about such things, the different types of differentiating spermatogon. But they go through this largely poorly understood process that takes, again, 8.6 days in mice, and then enter meiosis as preleptotene spermatocytes. And so this is all going on in the basal compartment outside of the blood testis barrier. And so how do we manipulate the system to study each cell type? I'll go through that here. And the reason why manipulating the system is so important is because spermatogenesis is a mess. To the untrained eye, you look at the testis, and it's just a wild mixture of all the different germ cells types. It's fairly well organized in the mouse. In the human, it's patches of spermatogenesis scattered all over the place. And so it doesn't look like a very organized system. So you have here in yellow arrows are pointing to two spermatogonia in a sea of other more advanced germ cells. So how do we study these progenitors that are moving up towards meiosis when this is the system that you have to deal with? And the way we do this is by synchronizing spermatogenesis. So we go from an asynchronized system to a synchronized system. And the way that we do that was developed by Mike Griswold's lab at Washington State University. It's a really neat trick where you block retinoic acid synthesis with this WIN18446, also commonly known as BDAD. And you treat mice. We've adapted it a little bit from the way that Mike originally reported it for our purposes. But basically, we treat mice with BDAD or WIN18446 for 10 days. And we're blocking retinoic acid synthesis specifically so the testis fills up with undifferentiated spermatogonia. And those can be seen here. So this is at P11. So from P1 to P10, we block retinoic acid synthesis. And at P11, you can see here we're stained for two different markers, STRIATE, which is a marker of retinoic acid signaling, and KIT, which is a receptor tyrosine kinase that is the best bona fide marker of differentiating spermatogonia. And so you can see, the other thing that is the caveat is that interstitial cells also express KIT. So there's no KIT inside the tubules. There's KIT in the cells outside. And actually, when you block retinoic acid synthesis, KIT actually goes up in somatic cells. It's just an observation. We're not sure what that really means. And you add retinoic acid, and it goes down. It goes up in germ cells and down in the soma. And then we give a pulse of retinoic acid and then follow. And so you have all of these undifferentiated spermatogonia that have piled up. You give that pulse of retinoic acid. They all turn on stimulated by retinoic acid gene number 8. And then they march forward in synchrony. And so there are now KIT-positive germ cells that are expressing STRIATE. STRIATE's a transient protein. It comes on, then goes off. So if you go out to P17 in this model, these are what are called type B spermatogonia. They're still KIT-positive. They're now not STRIATE-positive. And then you go out to P19 in our model. And these are preleptotine spermatocytes. They're always STRIATE-positive. They express STRIATE much more than even spermatogonia. And they're just on the cusp and entering meiosis. And so preleptinemia is also a very long stage. It's 41 hours in the mouse. But it's during this time that they are actually entering meiosis. So if we follow this and then look at the meiotic cells in this system, so if we go out to P23, we have pacotine spermatocytes. You could see those with SYCP3 staining. And ZBTB16 or PLZF, which is an undifferentiated spermatogonia marker, this is the next generation of spermatogonia coming up behind the one that's synchronized. So you could see a few of those positive undifferentiated spermatogonia. Also, there are KIT-positive. Obviously, not these same cells. But you also have KIT-positive spermatogonia in these tubules. And if you go out to P30, based on the timing of spermatogenesis, you would expect to see haploid spermatids. And you can see those here stained with lectin, which stains the developing acrosome. And you can see all of these haploid spermatids here towards the adluminal side of the epithelium. And then just to show that these are the types that we think they are, we did ploidy analysis. So at P23, we have no haploid cells. This is whole testis. But we have 2C and 4C. So pacotine spermatocytes are 4C. All the other somatic cells and spermatogonia are 2C. If you go out to P30, you have a huge number of 1C, or haploid, cells in the middle. And you also have 2C, of course. And you also have the next generation of 4C spermatocytes starting to appear. If we follow this a little bit further into spermiogenesis, by P37, we have elongating spermatids showing up. Then we have condensing spermatids by P43. And if you go out to P50 and look in the cauda epididymis, it's full of epididymal sperm. So here we have a system where we've synchronized spermatogenesis. And we have huge cohorts of cells all at the same stage of development moving forward together on the same developmental timeline as adult steady state spermatogenesis. And that gives us a tool to start to look for genes and proteins that might be good contraceptive targets. And so to start to do that, how do we use this system to isolate spermatogonians and spermatocytes? We have testis full of them. None of the methods that are really available in the literature have been that powerful to isolate large numbers of them. And we wanted millions in order to do a lot of the biochemical assays that we're interested in. And so we identified this is a mouse that's in JAKS that was made. It's the UCHL1-EGFP mouse that was made to study motor neurons. As most of the people that work on spermatogenesis know, anything that's expressed in the test is also expressed in the brain and vice versa. So this was a mouse that was used to study motor neurons. And they also have green spermatogonia, which is great. And so here's the UCHL1-GFP testes. And you can see all of the spermatogonia. These are all type B spermatogonia or bright green. And then a cool feature that we had no idea was going to happen is that once they enter preleptonema, this EGFP level dropped dramatically, which was great. Because then it gave us the ability to isolate preleptotine spermatocytes away from the next generation of spermatogonia, because you have brights and dims. And with flow cytometry, we could separate those two. I don't know if the timer is going. I just wanted to see how it was going. OK. And so here's just an example of some of the flow sorting that we did. And then we verified the different sorts that we did during all of these different stages, because we were really interested in knowing not only that we could get millions of cells, but what the percent purity of these populations are. And so in all of ours, we had at least 92% to 93% purity, which we felt was reasonable to be able to use for a variety of different biochemical assays. And so the first thing that we used this system to do was to perform RNA-seq. And so our field has certainly cataloged the transcriptomes of testes in multiple ways over the last 30 years. So we started out with RT-PCR arrays, and then went to microarrays, and then went to RNA-seq. And we went to single-cell RNA-seq. And we were actually on with one of our collaborators, one of the earlier testes, single-cell RNA-seq papers. And the reason why we decided to do this with my collaborator Brian Herman, who is a big single-cell RNA-seq guy, is that the downside, I guess, to single-cell RNA-seq is that you don't know the identity of the cells. The computer is predicting it based on the transcriptome. Here, we know not only what the identity of the cell is. We know what it was yesterday. We know what it's going to be the next day. We know exactly the progression of these cells through a biological system. You also get more sequencing depth. And we felt like it was a great way to start to look at the different genes and proteins that are expressed in these cell types. And the reason why we're interested in spermatogonia and spermatocytes is because they're outside the blood testis barrier. The blood testis barrier, from what I understand from colleagues who work on drug development, is a major impediment to delivering drugs to the inside of the ad luminal compartment of the testis, which is where most of today's efforts for male contraceptive drugs are being pushed. And I'm not saying there's anything wrong with that. It's great that they're doing that. It's just this is a different way of thinking about it. So we're not targeting spermatocytes or spermatids. We're targeting cells in the basal compartment. And our hope is that drugs that come from this are going to be more readily able to not only get to these cells, but also hit these cells before they go through the large amplification that's going to take place as they get ready to go into meiosis. And so this is a heat map of clustering genes based on mRNA abundance from the different cell types. So here we have A-aligned, which are undifferentiated, early differentiating, mid-differentiating, and late-differentiating spermatogon. And these were all run in triplicate with three litters treated, three different sorts for each. So it's a very robust and very expensive data set to put together. And so what we did here was group these into seven clusters. And I think you can hopefully appreciate. So blue, we're saying these are lower levels. And the redder they are, the higher levels, basically. And so if you're looking at the undifferentiated population here on the left, this is lowly expressed. It goes up as they go into differentiation. And then it drops when they go into meiosis. This is one that would be very high in the undifferentiated population and then just progressively declines. And so I just put a few sort of representative genes that folks might recognize in each of these clusters. This is not an exhaustive. There are hundreds of genes in each of these clusters. But these are maybe ones that people will recognize. So here in cluster two, as you might suspect, if I had to ask people what they thought these genes are probably involved with, it's probably spermatogonial stem cells in the undifferentiated fate. And so you can see genes like IOMES that Bob Braun studied in his lab for its role in self-renewal. ID4 that John Oatley's lab worked on. ETV5 that John also worked on. Here we have OCT4. Nanos 2 and 3 that are Yumiko Saga in Japan that John works on. So these are classic genes that are involved in spermatogonial stem cell self-renewal. So if we look at cluster four, you might expect if I had to take a poll, these are mostly meiotic genes. So these are genes that are not expressed in spermatogonia and go up dramatically at meiosis. And so you can look in there if you study meiosis and you can recognize lots of different meiotic genes in here that are going up. CMC1, RAD21L, SYCP1 through 3, et cetera. SPO11's another one. So all the meiotic genes are in this cluster four. Cluster five are things that are involved in spermatogonia proliferation and differentiation and they drop dramatically at meiosis. And so these are genes like Shosei Yoshida's Neurogenin 3 is important for progenitor formation and proliferation is in that list. Also some of the Nuage components like PUE-Like-2 and MOV10L. And then in this cluster, this is low in the early stages of spermatogonial development and it starts to rise. And surprisingly, there were a lot of meiotic genes that go up two days prior to meiosis. So these are genes that are transcriptionally activated two days prior to the meiotic program. And so if you look in there, there are lots of genes that are recognizable as well, I think. MeioC, TEX11, RAD51, REC8. These are genes that are involved in meiosis but turned on much earlier than meiosis. And so when we're thinking about male contraception, one of the things we don't wanna target are the undifferentiated spermatogonia because if this is gonna be reversible, we can't design a drug that's going to target spermatogonial stem cells. That would obviously be a no-go. And so cluster two would obviously be one we wouldn't consider any candidates for male contraception in that cluster. And so what we're thinking about are these three clusters. So these are ones that go up during differentiation. These are going up right at onset of meiosis. And these are going on up at the prelude to meiosis, two to three days before as spermatogonia are completing differentiation. So these would be reasonable candidate lists to think about for male contraception. The other thing that we did quickly was we took advantage of being able to get millions of cells at these different stages and we did shotgun proteomics. And because I always need to remind myself even that mRNAs are simply blueprints for protein synthesis. They are not in and of themselves typically a product that's important. They are there to tell how much proteins. And studies have shown I think rather clearly by this point that sometimes levels of mRNA are very instructive, but often they're not. And there was a big paper in Nature that came out 2011, 2012 that showed that transcriptomes predict the proteome in maybe half of the time. So certainly things can be regulated transcriptionally, but there's certainly a lot of post-transcriptional control. So we were very curious about this. And so we have the ability of getting millions of cells to be able to do shotgun proteomics. And so here we just did this one on the last step of differentiation and the first step of meiosis. And so these are genes up here in this volcano plot that were differentially expressed. We got 70 differentially expressed proteins, which really bummed me out, but our collaborator said, this is great for proteomics to get 3,000 proteins and to get 70 differentially expressed. It's like, okay, I was expecting 10,000, but he brought me back down to earth. So here's an example of some of these. And so this is immunostaining for the marker here on the side. So you can't quite see it, but straw aid. So kit we know as a marker of differentiating spermatogonia just a pan germ cell marker. So look at the ones in green. And so here's a plot of the protein abundance and here's immunostaining showing that. So the plot is from the shotgun proteomics and numbers of peptides that were detected. And here's immunostaining to verify. And did this match the mRNA abundance? The answer was not. About half the time it did. And so with straw aid, it did not. This gene, DMRT1, it did in the same direction. And then in these two, it did not. And so to take all of this into the next step, we're not gonna do this by ourselves. We are mostly a basic science laboratory. And so I've gotten in touch with Marty Matsik and an old friend, Thomas Garcia at Baylor College of Medicine. I'm sure everybody in here who knows anything about contraception is very well aware of the amazing work Marty's doing at Baylor College of Medicine. And so we're working with Thomas on this project who works with Marty to start to think about how to mine these data sets to identify male contraceptive drugs. And so I should say we are in the first phase here which is the transcriptomics and proteomics and then validating. And then we're gonna move to the next phase and make wild type and knockout mice to validate that these are targets for infertility. And then move from there into drug discovery or drug development for anything that has a viable use. So with that, I wanna thank all the folks. Sasha here who just graduated last week who did the bulk of this work with Taylor, my technician, Brian Niedenberger, a collaborator at EHS, Guang Hu, my longtime collaborator and good friend Brian Herman, Karen Schindler who's at Rutgers and also Thomas Garcia. And I wanna thank the funding organizations. Thank you. Thanks Dr. Gehr for an excellent presentation. Chris had mentioned that he is showing these data for the first time and we are very happy that you chose this forum. In the interest of time, perhaps one or two short questions. Thank you. Thanks Chris, that was crystal clear. In your drug development pipeline, you're gonna move to making the knockout mice and I'm just wondering what criteria you're going to apply there. Are you looking for things that are sort of classically drugable targets versus some of the other genes you had there that maybe aren't classically drug targets? Sure, yeah, so I think it's gonna be a balanced negotiation between myself and Thomas in terms of what I wanna knock out and what he wants to knock out. But I think absolutely. So we're looking at enzymes, things with active sites that can be targeted by novel drugs and then run through Marty's DECTEC platform to identify small molecule inhibitors. So I think the focus is gonna be on novel genes that haven't been knocked out elsewhere and are drugable like enzymes probably be and receptors being probably the highest. But we're gonna certainly defer to Marty and Thomas on which ones to go after. Yeah, that was really fun to see that data after a long time. Joni Jorgensen, University of Wisconsin. Sort of along the same lines, I'm wondering if there's any strategy related to sort of the stage and how much machinery is actually active, transcription, translation and that kind of thinking. Yeah, I think that's gonna be one of the first things that I'm gonna need to convince Thomas that we probably need to do. Maybe we need to move that up the pipeline a little bit, which is validating the protein is there before we think about targeting. Because I suspect for a lot of these meiotic genes that are turned on two days earlier than meiosis, the proteins are not there until meiosis. So I think that's a huge concern. Last question, Dr. Page. That was great, Chris. One question, time and again we've been stymied by translating these mouse findings and these infertile mice into the human platform. So have you and Marty come up with something to, you're gonna have so many targets here and you're maybe gonna make a lot of infertile mice. But how do we move this into people? That's a great question. Yeah, Thomas' budget for making mice is not the same as Masa's and Marty's, I think. Couple a year. So I think it's much more reasonable and I think we need to be a little more strategic than just knocking out 100 a year like Marty's been doing with Masa. So when we had a Zoom call last week, one of the things that Thomas has done is pulled in multiple different bioinformatics analyses and bringing in the human transcriptome data. And I think that's gonna be one of our criteria is it has to be expressed in similar cell types in the humans and hopefully has an example of perhaps a human patient that has infertility that also has that. So we're gonna try to pull in multiple things but I think at the end of the day it's still gonna be a bit of a guess but we're gonna do our best using those approaches. Thanks, Dr. Deer. Thank you very much. So our next speaker is Dr. John Oatley. Dr. Oatley is a professor in the School of Molecular Biomedicine at Washington State University. Unfortunately, he cannot be with us in person today but has kindly prerecorded his talk. So we will not be able to take questions but he has offered to respond to any questions should you send them directly to him by email. Greetings to all of you in attendance at the meeting. I apologize for not being there with you in person. Unfortunately, I caught the SARS-CoV-2 bug while at another recent conference. In the interest of public health, I made the tough decision to sit out the end of meeting. I'd like to thank the organizers though for willingness to play a prerecorded talk so that I am still able to share with you some of the recent research. My lab has been conducting with germline ablated animal models to achieve fertility following spermatogonial stem cell transplantation. These are my disclosures. My lab is interested in studying mammalian sperm, how they are formed, how high levels of production are sustained in adulthood and with basic understanding of their biology and the molecules that control their formation, how we can diagnose and treat infertility as well as devise novel male contraceptives. Sperm are quite a remarkable and important cell type for many reasons. They are the only cell type that performs its function outside the body it was produced. Half of their genomic contribution to the next generation is delivered by sperm. Their genome is a major source of genetic variation and it establishes sex ratios. And the payload they have is required for fertilization and for proper egg activation. In higher order mammals, sperm are morphologically quite similar as can be seen in these images of sperm for male cattle, pigs and humans. However, the amount produced per gram of testicular tissue can vary quite a lot between species. Also, although sperm seem to be designed similarly in higher order mammals, there is a wide range in head shape and tail size. And body size is not correlated to sperm size as can be seen in this image from a recent review article from Yannick Maci. Mouse sperm is quite distinct in terms of head shape, tail length in comparison to both human sperm as well as sperm from domestic animals. Also rather strikingly, the size of the mouse sperm head is similar to whale sperm yet the body size of the organism of the animal is quite different. Regardless of differences in morphology, sperm are generated in largely the same way across mammalian species through the process of spermatogenesis which occurs within seminiferous tubules of testes. Seminiferous tubule contains a seminiferous epithelium that is made up of two major cell types, the spermatogenic germ cell population and the somatic support cells or toli cells. The germ cell lineage contains spermatogonia which are the mitotic amplifying population. The spermatocyte populations, both primary and secondary spermatocytes undergo mitotic divisions and the haploid spermatids that start out as round spermatids morphologically transform to create elongate spermatids that are then released from the apical surface of toli cells as spermatozoa into the seminiferous tubule lumen that then make their way into the epididymis and out the rest of the male reproductive tract upon ejaculation. Spermatogenesis is one of the most robust processes in the male body. Indeed, in adult male cattle, they produce roughly 37,000 sperm per heartbeat which is about half as much as an adult male pig produces and although humans pale in comparison to domestic animals at 1300 sperm per heartbeat is still one of the most robust cell producing, robust systems for producing differentiated cells. The body and these levels of sperm production are sustained from puberty to old age. The foundation for continual and robust spermatogenesis is provided by actions of the spermatogonial population. The majority of what we know about spermatogonia has come from studies of mice and although there may be some uniqueness to the mouse system, the basic biology and functional attributes are similar across mammalian species. All mammals spermatogonia can be broadly classified as undifferentiated and differentiating subtypes. It's largely accepted that the undifferentiated population contains the stem cell component with cells that can self-renew and out of this self-renewing reservoir arises trans and amplifying progenitors that build numbers via mitotic cell divisions until a differentiation signal induces an undifferentiated to differentiating transition. A multitude of studies in rodent models as well as experimental evidence in human have demonstrated that the differentiation signal for spermatogonia is retinoic acid. The mouse system, a retinoic acid pulse causes a 96% depletion of the undifferentiated population when the majority of the cells transition to differentiated state. It's the 4% that are left behind in that undifferentiated compartment that are tasked with regenerating the undifferentiated pool for preparation of the next retinoic acid pulse. So we define spermatogonial stem cells functionally based on their ability to regenerate the undifferentiated spermatogonial pool. In addition to supporting steady state spermatogenesis, spermatogonial stem cells also have the remarkable capacity to regenerate spermatogenesis following transplantation. They can either be isolated freshly from testicular tissue or put in vitro for a period of in vitro amplification and upon injection into the seminiferous tubules of a recipient male that has the endogenous germline depleted or ablated, colonies of regenerated spermatogenesis can arise from engrafted spermatogonial stem cells. The spermatogonial population, including the spermatogonial stem cell, is not present at birth. Rather, it's established during a period of, during the perinatal period of late fetal and early neonatal development. Studies with mouse models have demonstrated that subsets of the precursor pro-spermatogonial population that formed post-sex determination during fetal life give rise in neonatal development to either spermatogonial stem cell or directly differentiating spermatogonia that will give rise to the first round of speratogenesis. It's the spermatogonial stem cell pool that was formed from a subset of the precursor pro-spermatogonial population that sustains spermatogenesis into adulthood for successive rounds of spermatogenesis. So although spermatogenesis is a robust system and it's critical for persistence of a species, things can and do go wrong. Defects in perinatal spermatogonial establishment, defects in maintenance of the spermatogonial population, or defects in spermatogenic maturation can manifest as infertility. An extreme cause of male infertility is azolespermia, which is defined as absence of sperm in the ejaculate. And this is estimated to occur in one to 2% of men worldwide. There are several causes of azolespermia, but about 25% of azolespermia is due to either genetic deficiency that's idiopathic or as a collateral damage from cancer therapy. These instances are referred to as non-obstructive azolespermia and are often due to males having a Sertoli cell-only syndrome in which there's complete absence of the germline. And these cross-sections of testes illustrate a Sertoli cell-only syndrome, although they are from mice, similar phenotype occurs in humans in which the seminiferous tubules are devoid of spermatogenesis and contain Sertoli cells only. As you can imagine, this condition causes sterility. So unfortunately, options for fertility preservation of men with non-obstructive azolespermia and Sertoli cell-only syndrome are limited to unavailable. For cancer therapy patients, if the patient is an adult, sperm crop preservation is an option for fathering biological children later in life. But for prepubescent boys, this is not an option. In fact, there are no viable options for fertility preservation of prepubescent cancer patients. In the case of idiopathic or genetic cause of non-obstructive azolespermia and Sertoli cell-only syndrome, whether it's an adult or a prepubescent male, there are no viable options for fertility preservation. For many years, spermatogonial stem cell transplantation has been proposed as a means to preserve fertility in male cancer patients or attain fertility in cases of non-obstructive azolespermia that are caused by genetic deficiencies. For adult men with NLA, non-obstructive azolespermia, there's this idea that patient-specific induced pluripotent stem cells could be generated and they could be in vitro coaxed into a primordial or germ cell-like state in which those cells would have gametogenic potential and could be used in various strategies to generate sperm so a male could have biological children. For prepubescent cancer therapy patients, the idea that testicular tissue could be crowd-preserved and this testicular tissue contains spermatogonial stem cells, thus it could be thawed later in life and used as a cell suspension or just as tissue to derive sperm and that individual in adulthood could father biological children. Both use of pluripotent stem cells to create cells with gametogenic potential in vitro or from crowd-preserved testicular tissue would utilize spermatogonial stem cell transplantation to generate sperm that could be used to father biological children. So the ideal outcome of spermatogonial stem cell transplant in a non-obstructive azoospermia or sertoli cell-only patients would be natural fertility. And there's been several experimental animal models of both NOA and SCO to help advance this concept of stem cell transplantation to attain fertility. These models have both pluses and minuses for use in a biological proof concept. The classic genetic mutant mouse model is the WWB mouse, which was developed from a spontaneous DNA mutation and it results in germ cell depletion and arrest of spermatogenesis. But there is still some spermatogonial stem cell persistence. These animals also have other somatic cell diseases. Another model that's been devised is the sulfen-treated animal model that's been applied to mouse, domestic pig, as well as non-human primate. This is treatment of animals with a chemotoxic drug that leads to germ cell depletion and also induces levels of somatic cell damage. There is still spermatogonial stem cell persistence and this leads to varying degrees of endogenous regeneration of spermatogenesis. Another model that's been more recently developed are Nanos II knockout males that my research program has developed over the last several years. The Nanos II knockout originally developed in the mouse and we have now advanced that into large animal models using genetic engineering strategies to create animals that have complete germ cell ablation, including spermatogonial stem cell elimination, and they lack spermatogenesis throughout adult life, yet they have normalcy of the somatic cell populations. Nanos II is an evolutionarily conserved core regulator of germ cell survival. In lower order organisms, they have a single Nanos gene and knockout causes both male and female sterility. In the mouse, they possess three Nanos genes, knockout of Nanos I produces no phenotype, knockout of Nanos III causes both male and female sterility and in the original ES cell gene targeted model, knockout of Nanos II causes male specific sterility. We've shown that the Nanos II gene in domestic livestock, including cattle, pigs and goats, that knockout of the Nanos II gene by CRISPR-Cas9 gene editing also produces male specific sterility. So we were interested in addressing a question of whether spermatogenesis and fertility could be established in the Sertoli-Salomi model of a Nanos II knockout male. By spermatogonial stem cell transplantation. We started with a mouse model in all mammalian species, the Nanos II gene is a single exon. We designed a double guide RNA approach and use CRISPR-Cas9 gene editing to produce several lines of mice that had inactivated Nanos II alleles. And you can see that their testes are severely regressed in comparison to a normal wild type mouse and cross sections, some of their vestibules show complete genome manipulation. We performed spermatogonial stem cell transplantation to address whether the testes of these males, Nanos II knockout males, could harbor regeneration of spermatogenesis and whether they could sire offspring by natural mating. We collected spermatogonial stem cells from donor males that were of the C57 Black6 genetic background, so they have a black coat color, and they were the ROSA26-LAGZ line, so LAGZ transgene insertion of the ROSA26 login. In this way, we can track the donor cells by staining them blue, and also any offspring that might be produced, we can track by coat color, because the C57 Black6 mouse has a black coat color. We used a Nanos II knockout mouse that was on CD1 genetic background, so a white coat color. And several months after transplantation, we could observe high levels of donors for spermatogonial stem cell engraftment, regeneration of spermatogenesis, and I don't have to point out which testes received a stem cell transplantation and which didn't. We could also take males that were transplanted, Nanos II knockout males that were transplanted with donor stem cells, breed them by natural mating, and they sired offspring. And we could determine that the offspring were donor-derived based on black coat color, also taking tail tips or pieces of ear of the offspring and staining them for LAGZ. And through a number of experiments, we've now been able to show that an efficiency of roughly 75% of Nanos II knockout recipient males that have been transplanted with donors of spermatogonial stem cells attain natural fertility. And these males have produced over 600 offspring now, and 100% of them are derived. We then took this concept to the pig. Again, a single Nanos II, a single exon Nanos II gene, take a single guide RNA, use microinjection or electroporation into pig zygote stage embryos, perform embryo transfer, and generate knockout males like this pig, in which he had two different deletional alleles for the Nanos II gene, one that was 150 base pair deletion, and another one was a frame-shifting four base pair deletion. Take a biopsy of testicular tissue from Nanos II knockout male. We could show that they have some nephrostubules, but complete germline ablation. But we collect the ejaculate, conclude the azole sperm, no sperm at all. And that's in comparison to a wild-type male, in which cross-section of seminiferous tubules shows complete spermatogenesis, and they collect lots of sperm from the ejaculate. We would perform spermatogonial stem cell transplantation in these Nanos II knockout pigs when they were well into an advanced adult stage, 14-month stage. And this is one of our pigs that was able to have Arbor donor-derived spermatogenesis. So, at 110 and persisting out to 750 days post-transplantation, we could detect sperm in the ejaculate, and we could show by genotyping of that sperm for the Nanos II allele, that it was indeed donor-derived, because we can only pick up intact Nanos II allele, did not possess the 150 base pair or four base pair deletion. And we can take cross-sections of testicular tissue from a transplanted Nanos II knockout pig and see complete spermatogenesis, whereas a male pig that had not been transplanted with Nanos II knockout male pig that had been not transplanted with donor spermatogonia, we could never find seminiferous tubules that had germline or spermatogenesis. We next took this to another high-order, higher-order mammalian species in the goat. Again, a single exon Nanos II gene, single guide RNA. This time we used cloning by introducing the CRISPR into XY fibroblasts, used somatic cell nuclear transfer to produce a number of knockout males. These males had biallelic knockout inactivation mutations of Nanos II, 16 base pair and 23 base pair deletion levels. We could do testicular biopsy of these males when they were adults and find that they had complete germline population, which is in contrast to a wild-type male that had complete spermatogenesis. We then performed spermatogonial transplantation when these males were at the pubertal age of four months of age and let them age out until they were a year of age and collect ejaculates. We're able to find that at 135 and persisting out to 730 days post-transplantation, we could find sperm in the ejaculate of these Nanos II knockout males. And by an RFLP genotyping assay, could detect intact Nanos II allele, which confirmed that they were donor-derived spermatogenesis. Lastly, we took this to even another pyridomammalian model in male cattle. Designed a double guide RNA approach to mutate the single Nanos II exon, used electroporation of the CRISPR into zygostage boline embryos, form embryo transfer and generate male cattle, such as this guy that had a 160 base pair and 510 base pair Nanos II deletion alleles. So a true knockout. We take a test as biopsy at an early pre-pubertal age of four months and stain by immunohistochemistry for the germ cell marker DDX4 in the Nanos II knockout male. See complete germline ablation, no evidence of germ cells. Which is in contrast to the wild-type male in which there are a multitude of DDX4 stained germ cells within some of their vestibules. So we then performed spermatogonial transplantation by taking germ cells from wild-type donor male, transplanting them into a Nanos II knockout male when he was four months of age, and then letting him age out until he was a year old and collect ejaculate to see if there was presence of sperm. And beginning at 180 days and persisting out to 380 days post-transplantation. We can detect sperm in the ejaculate in these cattle. High level of donor sperm production at over 145 million per mil of ejaculate and a high level of motility at almost 90%. In order to determine whether this was donor-derived, we performed sperm genotyping analysis for horn versus pole allele. We knew that our wild-type donor male was homozygous for the horned alleles. And we could take eggs from cattle, female cattle, that are homozygous for pole allele. And we knew that our Nanos II knockout male was heterozygous for horned pole alleles. We could perform in vitro fertilization, take morula and early blastocyst stage embryos, perform genotyping analysis for horn versus pole allele. By Mendelian inheritance laws, if 100% of the embryos were horned pole, then that would be donor-derived. And indeed, that's what we observed by genotyping 100 embryos at this point and 100% were horned pole. Confirming that it is donor-derived spermatogenesis. So coming back to this question of whether spermatogenesis and fertility can be established in testes of Nanos II knockout males that have non-obstructive basal spermia via stem cell transplantation. Could we reestablish spermatogenesis and fertility by stem cell transplantation? The answer to that question is yes. But we did observe differences in the amount of spermatogenic regeneration among species. So we're left with wondering why. And I think the answer is that it's the recipient age of transplantation that influences the amount of stem cell, donor stem cell engraftment and regeneration of spermatogenesis. In our mouse studies, we found that recipient age was associated with whether the recipient male could reestablish, could attain natural fertility or not. We found that the younger the age of the recipient male, the better chance that he would become fertile. And we could only obtain fertility with males that were less than 25 days of age at the time of transplantation. And you can see the difference here at the age of transplant. The image here on the left of high level of donor engraftment and lots of sperm in the epididymis was a male at 21 days of prepubertal age when he was transplanted. And the testis on the right is from a male that was at 42 days, which would be a post-pubertal age point and the engraftment is less and there's fewer sperm in the epididymis. And so relating this to our large animal studies, in the pig, we transplanted males when they were 14 months of age, which is an advanced adult age. And with goats at four months of age, which is also well into the pubertal stage of development and both have low amounts of sperm production. Whereas in cattle, we transplant the recipient at four months of age, which is in early prepubertal development, almost a neonatal developmental period, we can achieve high levels of sperm production. And so what I think we've learned from these studies is that Nanos II is an evolutionarily conserved cross-mammalian species as a core regulator of male germ cell survival. The testes of males with non-obstructive azoospermia due to a germ cell genetic deficiency can harbor regeneration and spermatogenesis. And the spermatogonial stem cell transplantation in non-obstructive azoospermia males can lead to fertility, and that the age at the time of spermatogonial stem cell transplant in a non-obstructive azoospermia male is critical for attainment of natural fertility. So the implications and impact of these findings and this new knowledge is that I think it shows that species comparative analysis can yield important new information about the genetics and biology of spermatogenesis. I think it shows that mutations in nanos too could be an underlying monogenic cause of non-obstructive azoospermia across mammalian species. That attainment of natural fertility in men with genetic deficiency non-obstructive azoospermia by stem cell transplantation is unlikely without further refinement because these transplantations would likely happen when men are well into their adult stage of life. And that large animal models are invaluable for devising and refining fertility treatment strategies in humans. So lastly I'd just acknowledge our funding sources from the NICHD and the United States Department of Agriculture, the research team at Washington State University, my collaborators at Utah State University and the Paul Ojiba Lab that helped with the goat cloning and Bruce Whitelaw's lab at the Roslin Institute. So again I apologize for not being there in person and I thank you for the opportunity to present this recorded talk. So I'd like to say thank you to Dr. Oakley for recording a talk when he's not feeling 100% and as I said feel free to reach out to him directly with questions. The last talk of the day is by Dr. Stephanie Page from University of Washington. Dr. Page is Professor of Medicine and Chief of the Division of Endocrinology and today she'll be providing an update on male hormonal contraception, the contraceptive and metabolic effects of progesterone androgens. Thank you Dr. Page. Great, thank you for the introduction and thank you to the committee for inviting me. The work I'm gonna present today is really a team effort. I'll introduce the team members as we go along but really represents a collaboration between NICHD, a team led by Dr. Diana Blythe and our colleagues at the Lundquist Institute, Ron Swerdloff and Christina Wang. There's my disclosures. So we heard a little bit about some of the reasons that we need more contraceptives and I would also argue that we have an ongoing global public health crisis of unplanned pregnancy. Despite introduction of new female methods and really also in part because of difficulty with access, we haven't really made a big impact on about a 40% incidence of unplanned pregnancy across the globe and that has a disproportionate impact on low and middle income countries as well. And this really has important health risks for women. It has economic risks and consequences for all people and of course has climate implications. And I also would really say that here in the United States, we have an acute crisis and reason for introduction of new contraceptives with the siege on female reproductive agency and human rights. So lots of reasons. So why are we developing male methods? And I think one of the reasons is that we know that men are really engaged in contraception. So despite the paucity of available methods for them that Chris really outlined very nicely, men do participate in contraception. Between the use of condoms, withdrawal, and vasectomy, globally men really are responsible for almost 30% of contraception. But there is really a lot of cases where new novel contraceptives are really important. And these kinds of users could be couples where the woman has a contraindication to hormonal methods or has side effects with all the options that are available for her. There are many indications where dual methods would be very advantageous for effective contraception. More and more we're learning that young men in particular want to control their own fertility and want to share the burden of contraception with their female partners. We have lots of data from about 20 years ago across multiple nations as well as cultures indicating that men are quite willing to use novel contraceptives in all kinds of different forms and probably even more importantly that women in committed relationships will trust their male partners to do so. Less than 2% of women surveyed in these sorts of studies say they would not trust their partner to use a male contraceptive. And some of that work's been updated recently. A shout out to the Male Contraceptive Initiative who did a very nice market analysis of about 1,800 young men, again showing a real enthusiasm for novel methods of male contraception. So we think there's a market out there and there's a need. So we work on hormonal methods which interrupt spermatogenesis. I don't think that I need to remind too many people here at the Endocrine Society about how male hormonal contraception works but it really interrupts the naturally occurring negative feedback loop between the brain and the testis. So under normal circumstances, pulsatile GnRH from the hypothalamus regulates the pituitary and the production of gonadotropins, FSH and LH. These importantly interact on the sertoli cells and the leydig cells and are critical for the production of both testosterone and for spermatogenesis. So when we introduce exogenous hormones, initially this was done with testosterone only at superphysiologic doses and then the field realized that the addition of a progestin provided a much more potent clamp at the level of the hypothalamus and the pituitary. When we give testosterone and a progestin, we block the production of GnRH, gonadotropins and this results in the testis no longer making testosterone which is normally at incredibly high concentrations within the testis and those high concentrations are required for normal spermatogenesis. And so in so doing, we block the production of sperm. So because of this mechanism of action, any hormonal method does have some limitations in terms of on and off times. And so this is an example from a combination product that I'm gonna talk about later using the novel progestin, nesterone and testosterone. And in white, we see testosterone alone and in yellow and blue, the combination. And the point here of this slide here is not only that these combinations potently suppress spermatogenesis but also that it takes a little bit of time. So between about eight and 12 weeks to reach contraceptive levels of sperm suppression and similar timeframe for the recovery of spermatogenesis. So this field has been going on for over 60 years and there's been really a plethora of work done and I'm not going to summarize all of that. But many combinations of testosterone and various progestins have been trialed in men. And there's two key points that we have, three key points that we've learned from these studies. One is that these combinations are incredibly effective and that most men achieve contraceptive levels of sperm suppression with these combinations, about 90% across the board in these studies. Secondly, that this is entirely reversible. So we're asked this time and again and really it's 100% reversible. Some of the time when you hear case reports wherein you might think that it's not reversible, it's usually that there's either been a testicular injury in the interim or that a baseline sperm count was not adequately collected. And the last thing that we've learned is, sorry, is this critical point of how low do we need to go. And so the early studies really depended on markers of azospermia and the thinking was that all the sperm, since men make so many millions of sperm a day, that all the sperm had to be gone because you only needed one, obviously for successful reproduction. But in fact, we learned from these multitude of studies and the efficacy studies that have followed that getting men to a sperm concentration of less than a million sperm per milliliter is effective for effective contraception. So the studies I just alluded to were really just studies of sperm suppression. There have been six phase two clinical efficacy studies to date enrolling approximately 2,000 couples. So the original studies done by the WHO used high doses of testosterone and that's where we learned a lot about the levels that we needed of sperm suppression for effectiveness, but we also learned a lot about highly androgenic side effects. And so as the field developed, the introduction of progestins, as I mentioned, allowed for the physiologic dosing of testosterone and at a time also when, as endocrinologists, we became more and more aware of the need to keep hormones in the physiologic range if we're gonna be thinking about long-term use. So these studies combined injections, implants, and injections, all things that require people to come to the clinic for delivery. And most recently in work I'm gonna talk about next, we've developed a transdermal gel that is the first self-applied male contraceptive to be trialed in an efficacy study. Again, in these studies, these combinations are highly effective with a PERL index, so an index of pregnancy, very comparable to female oral contraceptives. So we know they work and we know that they are reversible. And really in the long run, we are going to need a large phase three study to get one of these products to market. So in the next few slides are about the ongoing trial we are doing again with NICHD led by Diana Blythe and including the Population Council who developed the progestin Nesterone and Regene Citric Wear. So why are we using Nesterone? I just showed you two slides ago that many different progestins have been used in male contraceptive trials, so why do we need a new one? So Nesterone is a really exciting progestin, again, developed by the Population Council, and it's really a pure progestin. So as you probably know, many of the side effects that women have from contraceptives come from the various activities of their progestin, also some of the benefits. And so Nesterone doesn't really interact with the androgen receptor or the estrogen receptor or the glucocorticoid receptor, and so it really has very pure progestogenic activity, and we think that that will help us in terms of side effects. As I mentioned, the gel is user-controlled, so the individual applies it every day. It won't require repeated visits to the clinic. We think that the shorter action time, so instead of a long-acting injection of two to three months in duration, the off rate may be actually accelerated, which may be important for couples that are using this as a temporary method for spacing or as a short gap method for contraception. So we think the recovery time may be accelerated after discontinuation. As I showed you a few slides ago in a six-month study of the combination gel, 90% of men reached this critical sperm threshold of less than a million per milliliter. And the guys really like it, actually. So we actually have been incorporating acceptability measures in all of our studies over the last five to seven years. And even in a clinical trial setting when they actually had to apply this gel using two different syringes with four milliliters of goop in their hands and rubbing on in these early studies, 60% of the men actually found it to be quite acceptable. And now that we've streamlined this into a single gel, I'm sure that will be much higher. So men like it, men use it, and it works. So now we need to actually prove that that's the case in the setting of couples. So we know it will suppress sperm. We know those sperm levels are where we need to get, but we need to actually do this as an efficacy study. So this is ongoing. We started this prior to COVID. And like any of you who are doing clinical trials or bench work, you know that things have really slowed down over the last couple of years. And so this trial's taking us a little longer than we'd expected. But nonetheless, we hope to finish up here in the next year or so. So the study, we plan to enroll about 400 couples. We hope we also will complete at least 200 of those. It's a huge commitment for these couples. They're actually enrolled in the study for approximately two years. We designed the study because it is phase two to minimize the risk of pregnancy for the partner. And what that really means is that we're monitoring sperm concentrations throughout the study. So the couple enters the study. The man starts to put the gel on every day. And when his sperm count reaches that threshold of less than a million per milliliter, the couple stops using their backup method of contraception. And we monitor her menstrual cycle as well as his sperm count throughout the study. And if there's a bump, we let them know and so forth. And then after 52 weeks, they begin to use a backup method if they don't wish to have pregnancies or they stop using contraception if they do. The primary endpoint, of course, is prevention of pregnancy. And we're looking at all kinds of different things, including safety laboratories, acceptability, markers of sexual function, and importantly, markers of mood. The study is going on all across the globe, which is actually very exciting. There's never been a male contraceptive efficacy study that's had sites in Africa. And we have a site in Kenya, and we're going to be opening a site in Zimbabwe. There is a site in Santiago, Chile, various sites across Europe, and various sites across the United States. So we're really hoping that this will allow us to have a really good swath of different types of couples that might be interested, and of course, different settings. So we expect to finish again in about 18 months. So what does the product look like? So in the era of COVID, everyone now, globally, knows a lot more about hand sanitizer than we ever did before. The consistency of the gel is very much like hand sanitizer. It was modeled on some of the testosterone gel products that are out on the market. And as I said, we've been able to combine the two steroids into a single gel, and it's much more easy to use, about half a teaspoon of gel. The man puts a single pump on each hand and rubs it on his shoulders, and does so after he showers. We're often asked about transfer. So that's not a problem you'd have with an injection or a pill, but we're rubbing testosterone, the man's rubbing testosterone and a progestinon, what about transfer to kids or to his partner? And so we modeled some clinical studies similarly to models of testosterone gel studies to look at transfer in a very intense clinical setting where we had enrolled couples and they actually did observed rubbing after application, and then after a shower or wearing a T-shirt. And so the instructions for use of the gel are very similar to the androgen products, gel products that are on the market, which is to put the gel on, to not shower for four hours, to allow for maximum absorption. And if there is going to be prolonged skin-to-skin contact with a child or the female partner to take a shower or use a T-shirt. And in those studies, you can see that when those precautions were taken, there was absolutely no transfer to the female partner. Importantly, we are actually evaluating this in the more real world setting of our current clinical trial, measuring nesterone levels in the female partner at intervals across our efficacy study. But we think that this will not be a major issue. So that's the nesterone study. Again, we're really excited to be showing those results, hopefully, maybe at the next Endocrine Society meeting, but maybe sometime thereafter. So I'm gonna switch gears and talk a little bit about dual-action androgens that we're also doing clinical trials with. The concept here is instead of having to use two products, can we modify an androgen to have both progestin and androgen-binding action? So why would we care about doing that? Well, first of all, we're always hampered by delivery and PK of the two components. If we could put it all in one molecule, we would potentially streamline that process. The field's been really stymied by not having oral steroids that are available, and I'll talk about that in a minute. There could be the potential for streamlined manufacturing with a single molecule, and these molecules could have dual uses potentially in androgen replacement therapy. So why do we care about having a pill? Well, most of the time when you ask men what they would want for a contraceptive, they say that they would like a daily pill followed by injections. That's kind of changing. Actually, some of the press that's out there about the gel study, I think people are becoming aware of that, and so some of the more contemporary surveys suggest that men are starting to be interested in gels, which is very interesting and encouraging. But by and large, men ask for pills, and probably that's based upon their knowledge of the female birth control pill. And again, these are hypotheticals, and so we all know how hard it is to take medicine. So we're trying to develop both injections and oral pills, trying to have something for everybody. So we're always asked, why don't we have a male pill? I mean, any cocktail party I go to, I've gotta tell you, people are first of all super excited about this, both men and women, and secondly, they can't really believe that we don't have a male pill yet. And one of the reasons is that oral testosterone delivery has been exceedingly challenging. So if you just give a man testosterone, the half-life in the bloodstream is about 15 minutes, and early modifications of the testosterone molecule that added a methyl group, while the methyl testosterone did make it to the market, is associated with hepatotoxicity, so not really ideal for a male contraceptive. Luckily, there has been really great strides in this field, so there are a few products now of oral testosterone undecanoate that appear to be safe and well tolerated without that liver toxicity. But it's hard enough to take a pill every day, and I think we can all appreciate that twice daily dosing is really not gonna work as a contraceptive. And again, testosterone alone is not gonna be effective as a contraceptive, so we needed something else. So NICHD has developed these progestogenic androgens that are modeled on 19-nor testosterone, so they have a few modifications, including the addition of a methyl group and the addition of a long-chain fatty acid. And the long-chain fatty acid allows the molecule to be more bioavailable and prolongs the half-life within the body. So these are pro-hormones, if you will, pro-drugs. Dimethandrolone, undecanoate, and 11-beta-MNTDC. I'm not gonna try and, I can never pronounce the whole name when I'm up on stage, so suffice it to say that these are both very similar molecules. 11-beta is only different from dimethandrolone at the 7-alpha methyl group. Dimethandrolone has an 11-chain fatty acid and 11-beta 12-chain fatty acid. And these fatty acid moieties are cleaved by naturally occurring esterases in the body to the active molecules DMA and 11-beta MNT. So the preclinical studies showed that both these molecules are effective activators at both the androgen receptor and the progestin receptor, so they have that dual action that we're looking for. And a very nice study by Barbara Tardy about 10 years ago now showed that oral dimethandrolone administered to rabbits every day was an effective and reversible contraceptive in rabbits. So we've taken these into clinical trials. I won't bore you with all of the early studies, but we were really pumped about these molecules because in single-dose studies, we actually, to our own surprise, found potent suppression of LH and testosterone, again, with just a single dose. And so this was dose finding of 200, 400, and 800 milligrams and you can see LH on the top. That's hours across the X-axis and testosterone on the bottom. And this really was exciting because it showed this single-dose pharmacodynamic effect. We also learned, not too much to our surprise, that these molecules need to be taken with food to maximize their absorption. So we went on to do 28-day studies. These are 28-day PK and effect, not efficacy, but effectiveness studies in that we monitored the hormone effects of these molecules as well as safety and tolerability. And I'd like you to focus on the green and the blue. And again, we have LH on the top and testosterone on the bottom. So the X-axis is a little more complex because we take these subjects, they get their first dose in the clinical research unit and have 24 hours of IV sampling. They go home, they take the drug every day. We see them twice a week in the clinic for safety and blood monitoring and then they come back for one more 24-hour PK at the end of the study. And you can see the blue and the green that the LH and the testosterone are markedly suppressed by day two of administration at these highest doses of 400 milligrams a day. And there is a dose response effect. The yellow is placebo. And in the far to your right panel, nice reversibility with normalization of hormones just a few days after discontinuation of the drug. So this was super exciting to us. And we have subsequently done a similar study with the other compound I mentioned, 11-beta-MNTDC and seen similar effects here. There's just two doses in the dark and light gray. It's the exact same study design. And you can see again compared to placebo, a marked suppression of testosterone and LH. And the one thing I really wanna point out here is the testosterone levels. So the X-axis here, the testosterone, normal testosterone levels I think you all know are about 300 to 1,000 nanograms per deciliter. And these testosterone levels are well below that. We think of castration as being less than 50 nanograms per deciliter. So these folks have no testosterone. So how did they feel? Well, remember, these compounds have androgenic activity. And so I think the most exciting thing about these studies besides the safety was that these guys really felt just fine. So we would expect to see at those testosterone levels hot flashes, decreases in energy, really limited, some real limitations in sexual function after four weeks. And we did not see any of that. So that shows us pharmacodynamically that the androgen activity of these compounds is potent in vivo. We really didn't see anything concerning with regards to safety, which we monitored very closely. A little bit of weight gain and as expected, some suppression of HDL in these guys. So we have actually gone on to do a three-month study of dimethandrone undecanoate to try and get to the holy grail of sperm suppression. We would not expect to see that in just one month of administration because as I mentioned at the outset, we need at least eight to 12 weeks for hormonal methods to reach the important endpoint of sperm suppression. So we've completed that study, but we're just again, the COVID delay has delayed us. And so I hope to present those results next year. So as I said, we have really been focused on acceptability. And part of that is that we're trying to address some of the concerns we feel there is in pharmaceutical industry and other potential funders about we're constantly told, well, men won't do this. They won't take this stuff. You know, you gotta be kidding. But actually, at least in the participants in our clinical trials, which of course is a biased sample, there's a great deal of enthusiasm for all of these methods. And so even in these studies of male, of oral male prototype contraceptives, when the men had to take four very large pills every day so we could do the appropriate dose comparisons, about 80% of the men said they were very satisfied with the method. They would use it as their primary method in most cases. They would recommend it to others and so forth. So a high degree of acceptability and interest in our study participants. And Tamara Jacobson, who's here in the audience, presented a poster today and just recently got a paper accepted to contraception examining who these guys want to prescribe these methods when they do get to the market. And despite their wonderful relationship with their endocrinologist in the study trial setting, they really wanna be able to just go to the pharmacy or their primary care doctor. So that'll be important as we move these products forward and think about access. We are also developing these novel androgens for as long-acting injectables, which we hope will address other things that men want in terms of a contraceptive, kind of a depo per vera, if you will, for men. And that dose finding study is underway. I can't say too much about it except that one of the reasons it's taking so long is that the product does last quite a long time. So that's really exciting and really helps us think about methods that could be used even every six months or a year. Again, looking, of course, reversible. So I just wanna talk, we're always asked about side effects. And the side effects with hormonal methods, because we hear so much about side effects in women. I think it's first of all important to remember that many women use hormonal contraceptives without side effects that they find bothersome enough to stop. So side effects are super important, but people do put up with side effects when they want the benefit of a medication. The side effects with high-dose androgens are what you would expect, acne, changes in hematocrit, changes in libido, maybe the positive changes in body composition. But as we've been able to titrate down the androgen dosing in these compounds, I think the androgenic side effects have really been minimized. And as with female methods, the side effects that we attribute to the progestin can really vary. And the one thing that we do see in many of our studies is a little bit of weight gain. And we are monitoring, as I said, very closely, changes in mood and libido, which are difficult to do in this setting when we don't have placebo, but are really important for us to do in our pre-efficacy studies. So men are probably gonna have a few side effects. Some men, a minority of men, that use these methods. And they're gonna be similar to what women experience. We haven't been able to use these methods for long enough to demonstrate the potential benefits, which we think might be there, in terms of bone density and body composition. The weight gain could very much be lean body mass, which could have beneficial metabolic effects. So they're probably not gonna be that different. And one of the issues has been, will men tolerate these side effects? But also, are regulators or the industry gonna be willing to tolerate them? And those are actually probably slightly different equations. It may actually be that men are fine with them, but the regulators are sort of shaking a finger at us. And that's partly because the risk-benefit calculation we usually think about as part of the equation for an individual. So it is true that men who use these methods are not preventing an adverse health consequence for themselves, like a woman is, who's using a contraceptive. So she's preventing a potentially life-threatening pregnancy. And he's not so much for himself. But I think, as a society, we are evolving. At least I like to think we are in some areas. And there's been some nice new work really trying to reframe this question in terms of a dyad. So the dyad is the couple. And what is the risk for the couple? And what is the benefit for the couple? And there are models of this in organ transplantation. So when we think about living donors giving organs to other individuals, they're taking on a risk without a benefit. So we do have these models in healthcare. They're just not that common. And so this is a nice paper that John Amory and some others at the Male Contraceptive Initiative did together, trying to look at this as an ethical model. And really, if you think about it that way, a male contraceptive does not have to be any better than a female contraceptive in terms of its risk. And even if there were very, very rare bad side effects for a man, the overall risk reduction for the couple could actually be pretty astonishing. So it's just a different way to think about it. And as we think about the evolving social context, hopefully this will help us to talk about this with regulators. So in summary, I hope that I've shown you that male hormonal contraceptives are effective and reversible. We've really done a lot, and we as a field, in minimizing the side effects with these more modern methods. We think that they're gonna be very acceptable and exciting to both partners. But we really need to convince the regulators and investors if we're gonna accelerate the timeline for two development. And finally, I really do want to acknowledge that all these studies are team efforts. We get all of our funding from NICHD, and a lot of our intellectual input from Diana Blyth and her colleague Min Li. Christina and Ron lead a group at the Lundquist Institute. Racine Citric Ware at the Population Council. And I'm privileged to work with an astonishing group of colleagues at the University of Washington, John Amory, Arthith Rumalai, Brett Anawalt, and Bill Bremner, who could not be here this time, but wants to say hello to everybody. And then these are our sites for our Nesterone Testosterone Gel Study. And I just want to acknowledge their contribution and the contribution of our study participants. Thank you. Thanks, Dr. Page, for a comprehensive update. Dr. Page's presentation is open to questions. Hi. Thank you for that wonderful presentation. So how long after stopping the contraceptives do you expect fertility to be resumed? Yeah, so we monitor that really closely. So a normal male sperm concentration is 15 million sperm per milliliter, and it goes up from there. So the normal range is between 15 and 200. So return to fertility, we use that as our gauge. And usually with the gel, it's a little bit shorter, we think, probably on the long lines of eight to 12 weeks. With the long-acting injections and implants, it can be longer, up to six months, and there's the occasional person that takes a lot longer than that. But the good news is that we have returned to fertility, and it's been fun to watch pregnancies that have happened in the context of the recovery period in various trials. So we know it works. Thank you. Thanks, a couple questions. The first one is your dual-acting molecules, do they have any cross-reactivity with the glucocorticoid receptor? They don't actually have much, so not in vitro. I mean, a little bit, but not anything like some of the other progestins that have been studied. And then in terms of effects of, I guess, any of these agents that are effective as contraceptives, and you're saying the men say they feel generally normal, have you looked at effects on athletic performance, and are there any concerns in competitive athletes about the implications in that context? Yeah, it's super important for some folks. We haven't looked at that because we haven't, that's not been our focus. You know, by minimizing, by keeping the androgen component in the physiologic range, depending on the regulations, it may be fine for competitive athletes. We'll wanna do, obviously do body composition, that's gonna be really important both for that, but more importantly to understand the metabolic effects or potential metabolic effects. You know, right now testosterone's a controlled substance. If we get to enough of a crisis in the population graph that Chris showed earlier, maybe there'll be some movement to say, look, we need to actually have a venue for these kinds of approaches and for testosterone as a contraceptive. And so then the rules will have to be rewritten. I mean, that's the way it goes. I don't think, much as I am interested in athletics and participate them, I mean, for God's sake, that can't be driving what we're doing when we're talking about a population crisis. Great, thank you. Thank you. Mike Schwartz, Seattle. Couple questions. One is, I noticed that with some of the treatments it took maybe 20 days or so for testosterone to recover. So do men have symptomatic hypogonadism during that period? Yeah, that's a great question. So, not when it's just a few days. When we get to three weeks, so some of the studies that there's been some mood issues with, there's been a question as to whether, when they were giving injections for progestin and testosterone, that if the progestin effect was lasting longer than the testosterone effect, perhaps exactly what you're saying was happening during that interval time was there hypogonadism that might have contributed to depressed mood and stuff. So we have to think a lot about that. We monitor the men. If they have any issues, we check their testosterone levels. You could always bridge with a testosterone gel product if that was needed. We don't really see symptoms of hypogonadism until men have it for at least two to three weeks in my clinical experience, and I think that that's really true across the board. But there is this sort of magical point at about two and a half weeks, and if their testosterone levels are really low, you know it, because they get hot flashes. But the testosterone's coming up, and so it's not usually down here at that point. And so we don't really see symptoms in there, but it's something to really think about. And that's again why having these dual action androgens, we hope that everything's kind of coming up and coming down together. So follow-up question is, we know if you treat a patient, for example, with thyroid hormone for a long time, and you suppress the TSH, and you get some atrophy of the thyroid gland, or you give prednisone, and you suppress the adrenal cortex, if you do that for a year or two, it can take a really long time for the gland to recover. So do you think that's gonna be an issue? Yeah, it's a great question, actually. Probably will be, it will take a little bit longer, I imagine, and exactly for those reasons. So when men use these, even after a year, we see testicular shrinkage. Most men don't care. But I think the length of use may, when we look at, there's been a meta-analysis of that not using these products. And it does suggest that the longer-acting compounds, again, there's a longer recovery time. So that is one of the benefits of the shorter-acting nestrome. But it is likely true if a man were to use these, just like we see with using any kind of steroid for a long time, that the recovery time will be a little bit more prolonged. Thanks. Stephanie, can I ask a question? So for female contraception, there are a number of clear contraindications. Do you see some similar ones for men? Well, one of the big reasons there's contraindications for women is the risk of thromboembolic disease. And at least with testosterone, although that's been controversial, we really haven't seen that, in my opinion, when we look at the large pharmacogen, pharmaco, the big drug registry studies and so forth. So I think that one is not gonna be as much of an issue. With oral, there could be, we know that HDL will go down. The clinical implications for that, we don't know. So if a man was at high risk for heart disease, perhaps we're gonna know more by the time this stuff gets to the market about that, I think. What about your progestin? Do you have any data on its thrombogenicity? Nestrone doesn't, well, not in people yet. We haven't seen anything bad in our studies. And nestrone is used in women. So there is a product that's been approved on the market, and there's no, as far as I'm aware, Diana, you can create, there's no, it's in a ring, a vaginal ring. So I think, yeah. So there may be contraindications, but we don't necessarily know what they are yet. Okay. Great, well, I'd like to thank our three speakers for a wonderful session. And thank you all for your attention. Thank you.

male contraception

spermatogonial differentiation

meiotic initiation

reversible methods

minimal side effects

genetic models

transcriptomics

proteomics analysis

male hormonal contraceptives

gels

injections

oral pills

efficacy