We are going to have three speakers. The first one is online. He's from Australia, Dr. Vincent Harley. He's the head of the Molecular and Genetics and Developmental Laboratory at the Hudson Institute of Medical Research, Melbourne, and professor in the Anatomy and Biochemistry Department of Monash University. He's going to talk about disorders of sex development, new genes, and new concepts. And we are going to have the tape because unfortunately, he couldn't make it. But kindly, he sent his presentation and his tape. Hello, and thank you for the opportunity to speak today. Today, I'll be talking about FGF signalling and information about its role in human sex determination, gleaned from the analysis of the genomes of intersex people, particularly XY females. First, I'll discuss FGF signalling in the XY gonad in two components, the receptor FGFR2C, where I'll be discussing a mouse knockout and a human mutation we've identified and a mouse knock-in model of that mutation, the ligand of the receptor called FGF9, mouse mutations in FGF9, and a human mutation from an XY female and a mouse knock-in to validate its function in sex determination. And secondly, I'll turn to the XX gonad in some very recent data showing a role for FGF signalling and particularly using a mouse knockout. And the two key outcomes are, as you can see in red, sex reversal in the XY gonad and germ cell loss in the XX gonad. So, beginning with the FGFR2C mouse knockout, some 20 years ago, Blanche Capel showed that when FGF9 is knocked out in a mouse, the gonads develop as ovaries, the XY gonads, so it's a complete sex reversal. We showed some years later that the receptor we think is FGFR2C because when we knock that out, we get a phenocopy of the FGF9 knockout, so we think FGFR2C is the receptor for FGF9. And here we're using markers of Sertoli cells, AMH, and granulosa cells, FOXL2, in green. I'll turn now to a human mutation that we identified in a mouse knock-in model. We were screening XY female patients and identified an FGFR2 mutation, which turned out to be a classic mutation found in synostosis patients, although this patient had very mild synostosis, overlooked initially. She presented with primary amenorrhea, 46XY gonadal dysgenesis with dysgermanoma, female genitalia, and the mutation is 342S, what's called a dominant activating mutation in the skeletal system. However, this mutation leads to loss of function of FGFR2C in the testis, where the homozygote missense mutation, knocked in a mouse, leads to ovotestes, as you can see with the marker of the testes and the ovaries in the XY ovary of the knockout, showing ovarian material at the poles and a little bit of testicular material left over. But it's essentially the shape of an ovary and it's almost a complete sex reversal. And as you probably know in this field, homozygous knock-in of a human mutation is needed, of a heterozygous human mutation is needed for the intersex gene to reveal itself in the mouse. That's common for many genes, including SOX9 and FGFR2 and FGF9. I'll turn now to FGF9 and the first thing we did is we looked at some classic synostosis models. When we analysed the gonads, the embryonic gonads, with markers of the testes and the ovaries, we found that the XY gonads showed partial sex reversal containing ovarian granulosa cells in both cases. And we published this a couple of years ago with the title ovotesticular disorders of sex development in FGF9 mouse models of human synostosis syndromes. So it's possible that in other synostosis syndromes, we think given the receptor and ligand mutations, that there could be reproductive gonadal phenotypes. So I'd like to turn now to a human mutation of mouse knock-in model and this is work. It's not published yet. An intersex screen with a panel identified a heterozygous mutation in FGF9 encoding the amino acid substitution D195N, aspartic acid to asparagine. The symptoms identified at puberty were primary amenorrhoea and little breast development in clitoromegaly and the mutation was inherited from her mother. Internal reproductive structures include female fallopian tubes and male mastepherins and the gonadal phenotype were gonads resembling small cysts with no ovarian or testicular tissue identified. And given the mutation, the patient was re-examined for skeletal changes in the light of the variant, but no synostosis was identified. So we started some analysis of this mutant protein to see if there's any functional changes compared to wild-type protein. The first thing we did, Makoto Ono did, was make the recombinant protein in bacteria and bound it to a heparin affinity chromatography column. The reason is that heparin sulfate proteoglycan is a co-receptor of FGFR2 and what it does is it brings dimer forms of FGF9 into proximity of the receptor and then the dimers dissociate to monomer and it's the monomer form that binds the receptor and signals into the cell. So when we looked at the ability of these proteins to bind to heparin, we found that with increasing salt concentration the mutant protein eluted earlier, which is a sign that it binds less strongly to the heparin. So this interaction is weaker in the mutant. The particular position at which the mid-sense mutation occurs is one of three residues that form a triad which is required for dimer stabilisation based on the three-dimensional structure of FGF9 solved by Musa Mohammadi at NYU. The D195 forms a crucial salt bridge to the arginine at 62 on the opposing dimer FGF ligand and we wondered what the effect of this variant of change from D to N at 195 would be on dimer formation. So to look at this, Brittany Croft in the lab did proximity ligation assays in hex cells where the FGF9 gene was either tagged with a hemagglutinin epitope tag or a flag epitope tag. When co-transfecting the hex cells, you can look at the proximity of the two molecules within a cell as a mark of its dimer formation. And when this is done, we found that mutant showed a dimerisation defect and we had some controls that others had published as showing defects before. Whereas when we did the same kind of assay with co-transfected ligand and receptor with different tags and different antibodies, we did not see a difference in the ability of the FGF9 mutant to bind to its receptor. So if I summarise quite a bit of work because we also, we and others have looked at the other known mutants, it seems as though defects can occur either in the heparin binding, the dimerisation, dimerisation or receptor binding would in this case not work by us, and receptor binding alone and all of these affect testis development. So we went on to examine in vivo the consequence of this D195N mutation by knocking in the variant using CRISPR. And what we saw was that the ovarian pathways normally repressed in the XY gynad were incompletely repressed. And so if I first take you to the purified recombinant protein I talked about earlier, this was applied to hex cells and Wnt4 levels were examined. And what we found is that the wild type FGF9 at low concentrations is able to repress Wnt4 activity, Wnt4 transcription, whereas the mutant protein was not able to repress it at these very low concentrations where dimer formation is normally happening at these KDs. If you push the concentration up, you lose the effect. So it seems as though a function of FGF9 is to repress Wnt4. We also wondered about the other ovarian pathway, which is the FOXL2 pathway. And so we looked at the fetal gonads of the knock-in mice and saw that there were granulosa cells at the poles of the XY knock-in gonad, suggesting that the XY gonad is being feminised by the variant. We looked at other genes and found that whereas Wnt4 was being derepressed, as I showed in a different way, in vitro, now in vivo, to intermediate levels. So these are the male levels, the female levels, and the XY knock-in levels. And here, again, for FOXL2, you can see not as strong of an effect, only an effect in the double mutant. You're seeing some derepression in the XY gonad. The effect on the male markers, which you might expect to be reduced, were not significantly so. So it seemed as though FGF signaling was having an anti-ovarian action rather than a pro-testis action on both the Wnt4 and the FOXL2 pathways. So here is a slide Emily Frost is making for a review that should be coming out shortly, I hope. The FGF9, FGFR2 signaling pathway seems to be one in males that is repressing Wnt4 and FOXL2 pathways. So in summary, for this first part of the talk, FGF signaling in the XY gonad, XY females carry FGF9 misense mutant, and another XY female carries the FGFR2C, C342S mutant, or variants. And that suggests a role for FGF signaling in human sex determination. These are the first FGF signaling variants to be described in intersex conditions, and why are they so rare? Well, even these variants that we've studied, we think show weak expressivity because the variants have been instances of these variants occur in the wild-type population of XY males with no gonadal phenotypes described. So we think these are very weakly expressing. And the same is true in the mice where the feminized phenotype only of the mouse knock-ins only is that highly strain dependent and the knockouts as well. So we think that we extrapolate to the human situation, why is it so rare? It suggests that the FGF signaling variants only manifest in certain rare sensitized genetic backgrounds. The next thing we went on to do is to look at whether FGF signaling could be important in the ovary. And I'll talk about receptor mouse knockouts. Here we knocked out FGFR2C in the XX gonads. But first we looked at the expression of the wild-type, and the wild-type XX gonads gonads shows expression in the somatic cells. The germ cells are shown in red. There's no overlap. In green is FGFR2, and you can see it's fairly evenly distributed throughout the XX gonad. It's well described already by several groups in the XY gonad, but not in the XX gonad. And it's predominantly in the somatic cells in the XX gonad by gene expression analysis by the Koopman Lab. So we knocked it out in the ovary, and here we have markers of granulosa cells and germ cells again. And you can see that the germ cell marker has completely disappeared in the knockout. So how do we interpret this? Firstly, there was no change if we look at these the gene expression, there's no change in Wnt4 or FOXCELL2 levels or Rsp1 and 1 levels, but there was a great change in a germ cell marker DDX4, which is consistent with the immunofluorescence data. So how do we explain this? Well, we think there are at least two sort of mechanisms that support germ cell survival from the supporting cells, or for factors from the supporting cells doing that. And these are probably independent pathways. Others that show Wnt4 is an essential factor. And we think FgfR2c is also an essential factor. And you need both. And if you don't have FgfR2c, loss of the Fgf signal leads to weakened overall survival and loss of germ cells. And we wondered about FOXCELL2. And so we crossed in the FOXCELL2 knockout into the FgfR2c knockout. And when we did this, to our surprise, we saw this is the wild type XX gonads. And this is the XX knockout gonads as shown before with the germ cell marker DDX4. You can see loss of the germ cells. And this is the FOXCELL2 knockout crossed into this FgfR2c knockout. And once again, we have the granulosa cells here, shown by GATA4 here in p27. You can see FOXCELL2 has gone from the granulosa cells, as you expect in the knockout. But the germ cells have come back. And so it seems as though, to our surprise, loss of FOXCELL2 leads to the rescue of the FgfR2c germ cell loss phenotype. So we're still struggling with this. And here is a kind of crude model of what we think is going on. So as I said before, FgfR2 and Wnt4 are are sort of promoting germ cell survival. But we speculate that FOXCELL2 is attenuating germ cell survival by some mechanism. So in the 2c knockout only, with no 2c, you've only got this sort of weakened situation where with a loss of Fgf signal, you've got a weakened overall survival signal because you've lost one of the two blue arrows. And that leads to loss of germ cells. But in the double knockout, if you lose both the Fgf signal plus the loss of the inhibitory FOXCELL2 signal, you still have the Wnt signal and the germ cells survive. So we seem to be seeing a sort of opposing action of Fgf signaling and FOXCELL2 in XX-GONAD germ cell survival. So in summary, FgfR2 knockout shows germ cell loss, leading to Fgf signaling from the somatic cells being necessary for survival of the germ cells. And in the context of Fgf signaling loss, germ cell loss is rescued by the loss of FOXCELL2 surprisingly, suggesting the antagonistic role. So I'd like to thank principally Brittany Croft, Stephan Begheri-Pham and Makoto Ono for doing the bulk of the work from my lab and my collaborators. Thank you. So we thank Dr. Harley for kindly sending us his video presentation. Unfortunately, we will not have a chance for questions and answers. My name is Rodolfo Rey from Buenos Aires Children's Hospital in Argentina. I thank Veronica Americ, University of Chile in Santiago for co-chairing this session with me. She took over because Dr. Jill Guerra from University of Campinas in Brazil could not come, unfortunately. And I have the pleasure of introducing our next speaker, who is Dr. Humphrey Yao. Humphrey is Senior Principal Investigator of Reproductive Development Biology Group at National Institute of Environmental Health Sciences NIH in Research Triangle Park, North Carolina. Thank you. Humphrey. Thank you. Thank you. First of all, I would like to thank the organizer for inviting me to this conference. And it's always been fun to be the last speaker of the section. And thank you all for coming. And I bet many of you are staying because you want to see the glowing penis, right? I guess for the last talk of the day, I better make the title a little sexier so some of you may be willing to stay here. So my name is Humphrey Yao. I'm the Senior Investigator at NIHS, which is one of the 27 institutes at NIH. And the main focus of my laboratory is to understand how embryos establish their sexually dimorphic reproductive system using the mouse as the model organism. Hopefully we can use that to understand the human intersex condition. So today I'm going to tell you two stories. One story is about the oocyte-producing testis. I bet you probably are very confused. How could a testis be able to produce oocyte? And I'm going to introduce to you a very interesting mouse model that we actually found that the male is able to produce oocyte. And the second project I'm going to save the best for last so that you can really appreciate that we can use the glowing penis to understanding the human situation of hypospadia, which is a very, very severe birth defect in the human population. So the first, and first of all, I have no financial relationship to disclose. So the first story actually started in the early 90s. That's when Robin Levelbatch, who is a professor in London, was the first to discover a single genes on the Y chromosome called SRY, or sex determining region on the Y chromosome. This gene is necessary and essential for the formation of the testis. So when he made the mouse model where the XX embryo is carrying the human SRY gene on the X chromosome, he was able to completely sex reverse the XX embryo into embryos that develop the testicle. On the other hand, if he mutate the SRY in the XY individual, that XY embryo will be completely sex reversed from the testis formation into ovary formation. Also, you can observe this kind of mutation in the human cases where there is a translocation of the SRY gene from the Y chromosome into the X chromosome, the XX individual that carry that translocated SRY gene will be completely sex reversed. So this really tells us the single gene on the Y chromosome, SRY, is necessary and essential for the testis formation. So this groundbreaking observation really break open the field for us to understand the mechanism underneath the sex determination process. And many, many years later, people start to ask what is the mechanism of SRY action? And of course, the first important question you must ask is which cell types actually produce SRY in the testis? So based on many, many research from our lab and other people's lab that we know, the function of the SRY is as a trigger in the Sertoli cell only manner. If you look at all the other cell type in the testis, the lytic cell, the germ cells, or the Sertoli cells, Sertoli cells is the only cell type that produce SRY. And we know also SRY function as a transcription factor. And one of the most direct downstream target of SRY is another transcription factor called SOX9. And the cascade of this signaling transduction pathway actually facilitate the establishment of the Sertoli cell fate. So we consider the establishment of the Sertoli cell fate is a cell autonomous event driven by the expression of the SRY and the downstream event that triggered by the SOX9. So this is basically lay the foundation for us to understand how the testis formation is established. And we know Sertoli cell fate is specified cell autonomously by the actions of this two transcription factor. But there is a case that always bug me, which when I took the reproductive biology 101 classes. So how many of you have ever actually heard about the case of Freemartin? Oh, many people nodding, but I noticed there's a generation gap. Many of the younger generation never heard of the Freemartin. But I do remember when I was a graduate student almost 30 years ago, that was one of the first thing I learned from the reproductive biology 101. So for those of you who don't know anything about the Freemartin, Freemartin cases was observed 100 years ago by farmers and by philosopher. What they found in the farm animal, particularly in the cow, when the cow is carrying XX and XY twin at the same time, more than 95% of the chances, this X embryo is completely sex reversed into an embryos that carry the testis-like structure in their ovary. So this is pretty interesting, right? If you remember what I just told you, testis formation is a cell autonomous event driven by a Y-linked gene, SRY. But in this case, this is an XX individual without the SRY gene. So how could this individual develop testis structure? So this is very confusing. So there are many, many hypothesis that being proposed. So one of the most interesting hypothesis that there are some diffusible factors coming from the XY individual that travel through the blood circulation through the shear placenta of the Freemartin model, and this diffusible factor get to the gonad of the XX individual, and then sex reverse that ovary and make it into a testis-like structure. So this is totally against what I just told you that the SRY function as a cell autonomous effect on the testis formation. So what we're thinking that maybe there are some hormone-like factors coming from the XY individual that actually can masculinize the ovary in this case. So that was one of the project that we started long time ago to look for. Maybe there are some hormone-like factor coming from the fetal testis that is able to facilitate testis formation in the XX individual. So we start to search for potential hormone-like factor produced by the XY gonad. So one of the first factor, I'm pretty sure many of you already know, the anti-malarian hormone, or malarian inhibiting substance, which was the old name. This was the first hormone that was identified produced by the fetal Sertoli cell. And the function of this hormone is to suppress the female reproductive tract, malarian duct. And the second hormone we actually found produced by the fetal Sertoli cell is Activin B. I'm pretty sure here many of you know Activin B is a very important hormones that control many aspect of the female and male reproductive system. And we also found that the Activin B is important for the testis vasculature formation in the mouse situation. So if you could think these two factors are free-margin factor, meaning they are important for testis formation, then if you remove these two factors, you will be able to see compromises in terms of the testis formation. So fortunately, the mouse model of these two knockout were generated. But very disappointingly, the single knockout of AMH or Activin B had no impact on the normal development of the testis. So what we're thinking here, maybe this two factor actually work together. This two factor all belongs to the TGF beta superfamily protein. So maybe they have some overlap function. So we make a very bold hypothesis that maybe this two factor work together to facilitate testis formation. And we need to remove both of them to see some phenotype. And Karina Rodriguez, which is a biology in the lab, she was brave enough to take over this project. So if these two factors are the free-margin factor, they must fall into the following criteria. First, they must be important for testis development. And second, they must be able to mesclonize the fetal ovary, just like I mentioned to you in the free-margin scenario. So we look at the first question, whether they are important for testis development. So what you are seeing here is a cross-section of the fetal mouse testis, stained with two marker. First is the SOX9, which is a stratolyte cell marker. Second is the FOXL2, which is a granulosa cell marker. So this is a normal wild-type testis, of course. You only find the normal stratolyte cell. And there's no FOXL2 staining at all. When we first look at the double knockout testis, remember, this is an XY individual. So what we are seeing here is a mixture of the stratolyte cell, SOX9-positive stratolyte cell, as well as the FOXL2-positive granulosa cell. So basically, this is the structure called the oval testis. Particularly, you will find the ovarian domain at the two poles of this XY gonad. So basically, this is a very confused XY gonad. If I look at the higher magnification, you can see the mixture of stratolyte cell and the FOXL2 cell. And in some cases, you can make up the yellow cell, which are the cells that express both SOX9 and the FOXL2, meaning those stratolyte cell are transitioned from the SOX9-positive cell into the FOXL2-positive granulosa cell. So in the lab, we call this very confused gonadal cell type. They are taking off their tuxedo and putting on their very nice dress at the same time. So this really tells us these two factors are very important for the maintenance of the stratolyte cell identity. If you remove these two factors together, you start seeing transdifferentiation of the stratolyte cell into the granulosa cell. So usually, in the mouse case of the oval testis I showed you will be resolved at the time of the birth. You seldom have this oval testis situation last into adulthood. Most of the case, the testicular domain will take over and the ovary domain will disappear. So we are asking the question, is possible that this oval testis structure is maintained to adulthood? So Karina was brave enough to let some of the animals to last to adult situation. You are looking at a six-month adult situation. So what you are going to see is the presence of folliculogenesis and spermatogenesis happen at the same time in this individual. So what you're seeing here are the follicle. This is like the secondary to pre-antral follicle on the two pole. And in the middle, you still can see some seminiferous tubule here. This is pretty, pretty wild, right? You don't really see this kind of situation very often. But what you can see here, there's no corpus luteum, meaning this individual was not able to ovulate. The reason being, we're thinking the hypothalamic and pituitary system is probably also very confused too. When we look at the hormone, you can see pretty substantial level of testosterone and as well as estrogen and maybe some level of progesterone. So we think that hypothalamic pituitary axis is probably not able to induce ovulation. But Karina was pretty wild. She was thinking, can we super ovulate this male to induce ovulation in this XY individual? This is exactly what she did. She was able to inject PMSG and the HCG and collect oocyte from this XY individual. You can see the well-developed oocyte. And at the same time, this individual is able to produce sperm. So basically, this is a true intersex individual capable of producing both gametes from the XY individual. So this is very wild. Of course, there are many questions remain. Can we actually self-fertilize this individual and produce the next generation? And hopefully in several months later, we'll be able to show you some of the crazy results here. But the most important take-home message is AMH and activin B double knockout. They can actually maintain their germline capacity here and eventually facilitate gametogenesis of both sexes. So of course, we are basic embryologists and developmental biologists. We want to know the mechanism. What do this factor do that in the fetal test is to maintain the identity? Is it important for the initial fate specification of the Sertoli cell? Or is it important for the maintenance of the cell fate? I hope you can understand the differences between the two. So first of all, we look at the gene expression between the knockout and the control animal and asking this two question. First, we look at the fate determination phases and compare the transcriptome between the two genotype. So what you're seeing here is on the x-axis, on the right-hand side, meaning upregulation of the gene, on the left-hand side, meaning the downregulation of the gene, and the y-axis representing the p-value. So what you're seeing here is the comparison between the wild type and the control at the fate determination phase. There's nothing to show because there's nothing different. The only differences is AMH and the ectobing B expression because they are knockout. So you can see there's two little dots right here showing you that indeed we were able to knock out this gene completely. But at this fate determination phases, the transcriptome is identical between the two sexes, meaning the fate determinations in the absence of these two hormones is not affected. When we look at the fate maintenance phases about three days later, you can appreciate there are much, much more genes are changing. There are many genes that are being upregulated and most of them are ovarian gene, meaning the tests start to pick up ovarian characteristics and many of the genes that are being downregulated are sertoli cell gene as well as the latex cell gene. In one of the gene that is, okay, so this tells us that the actions of these two hormones is to maintain the cell identity rather than inducing the specification of the cell identity. So this is a really interesting phase that no one really look at it. We always think that cell fate specification of the sertoli cell occur at the time when the SRY is expressed and once the cell fate is maintained, it's a point of no return. But what you are seeing here is sertoli cell fate is actually very plastic. In the absence of these two hormones, we can actually reverse the cell identity by removing them. And also another important gene I want to bring your attention is the most upregulated one is the granulosa cell gene, FOXL2. So then we really want to see how is FOXL2 really responsible for the fate changes. So we took advantage of the new single cell sequencing technology and we sequenced the wild-type testis, wild-type ovary as well as the double knockout gonad. So what you are seeing here, each little single dot representing the transcriptome profile of that particular cell. So what you are seeing here are the sertoli cell from the, sorry, granulosa cell from the control ovary, sertoli cell from the control testis, and those sex reverse cell from the double knockout XY gonad. So you can see their transcriptome actually shift from the control situation toward the female fate. This really tells us this is the gradual fate changing phases. In the absence of these two hormone, actually the sertoli cell started from here and gradually shift their pattern into the control granulosa cell. Actually FOXL2 completely mimic this completely change. So we're thinking that these two hormones are actually suppressing the FOXL2, which is a granulosa cell identity gene in the sertoli cell. And without these two hormone, the FOXL2 become upregulated, therefore changing the identity of the sertoli cell. So we also take advantage of the single cell sequencing. We can actually look at the cell interaction in that gonad. So what we are able to do is using a software to match ligand and receptor pair among different cell type and see the changes among them. So how do you read this? So basically, if you look at the interaction between ligand cell and germ cell, by looking at this receptor in the ligand between the two, this is what the numbers tells you. There are about 86 receptor ligand pair present between ligand cell and germ cell. So what you're seeing here, the most significant interaction is between sertoli cell and ligand cell. But then you probably will ask, so where is AMH and activine B fall into? You will find the ligand and receptor pair actually present only among sertoli cell. Ligand is expressed in the sertoli cell, and the receptor is expressed in the sertoli cell. So this tells us these two factors are serving as a paracrine and the ultracrine factor that maintain their own cell identity. So what we're proposing here is that a new mechanism, that the sertoli cell produce two hormone to secure their identity. And in the absence of the AMH and activine B, which function as a ultracrine, paracrine factor, the sertoli cell lose their identity by expressing FOXL2, which is the granulosa cell marker. And the FOXL2 cells eventually will start to promote ovarian pathway. And the presence of the granulosa cell facilitate the disintegration of the seminiferous tubule, and eventually you lose the seminiferous tubule. And because of this, those germ cell will also start to change their fate, and eventually they facilitate the formation of the follicle, and this phenotype is able to persist to adulthood. Eventually this individual is able to produce both oocyte and the sperm. But that was the first point I show you, that these two factors should be important for the testis formation. Then you probably will ask, Humphrey, how about the free martin phenomenon? Can you actually prove that these two factors are two free martin factors? So this will be very difficult to prove experimentally, right, because you could imagine. So Paula Brown in the lab, which was very brilliant, she looked at the literature and found a very interesting potential model for us to test this hypothesis. A Japanese group actually found that when they transplant the fetal ovary into a wild type normal XY adult male, and wait for three weeks, what they found is the fetal ovary is completely sex-reversed into a testis. So this tells us that whatever hormones present in the wild type XY adult male is able to masculinize the fetal ovary. So we're thinking, can we use this model by replacing the wild type XY male with the double knockout male, and transplant the fetal ovary into this animal, and see whether they still have the ability to masculinize the fetal ovary. So this is what we found. First, we did the control experiment by transplanting the fetal ovary into a control XY. What we found is only 14% of the ovary remain ovary, but most of the ovary were sex-reversed into the oval testis, or a complete sex-reversal into the testis. So this is completely identical to whatever that Japanese group identified. So we were very happy that we are able to reproduce that data. So then how about if we replace that wild type animal with the double knockouts, and you probably could expect, in this case, the double knockout lose the ability to masculinize the fetal ovary. 86% remain as the wild normal ovary, only 14% establish oval testis structure. So with this, we are confident to say that AMH and activating B probably are the free-marting factor. One function of them is to secure the sertori self-maintenance. The second is that they serve as the free-marting factor that you found in the cow situation. So sorry for keep you waiting that we finally get to the glowing penis part, and this is a completely unexpected. A postdoc zero, a model who came to the lab want to study the gonad development, and one of the model we use very frequently is the SF1. SF1, or serogenic factor one, or NR5A1, which is a very important orphan nuclear receptor that involve in many aspect of gonadal development and adrenal development and pituitary gland development. And we know this gene for sure is expressed in the gonad and the adrenal gland. But one day zero come into the lab, what you are seeing here is a reporter line to show you the red fluorescence showing the SF1 positive cells. And zero come into the lab saying, Humphrey, this is not quite right. I found the penis is completely red. It's glowing fluorescently red. I said, this is not real, because no one have ever report SF1 present in the penis. So we decided to use, I said, go back to do the experiment again. At least prove to me in two different separate model to show that indeed this is the case. So he look at another reporter line and using the protein staining and also the in-situ hybridization. And we were able to confirm the SF1 cell is actually present in the fetal penis. And the most strikingly, what he found is that those SF1 cells in the beginning, before the penis formation, is actually coming from the hind limb. So what you are seeing here is the ventral view of the mouse embryo that we remove the hind limb as well as the tail. What your penis is actually right here. So what you're seeing here, these cells are not present in the penis. They actually migrate through the hind limb centrally and eventually settle on the base of the penis and eventually colonize close to the urethra. So this is pretty exotic because no one ever report that the cell inside the penis is actually coming from outside of the penis. So this is pretty amazing. And he wants to see, is this really a migration event that push the formation of the penis? And one of the most important biological event during penis formation is the closure of the urethra. And in the male, the urethra has to close from the proximal to distal part to ensure the opening of the urethra is at the tip of the penis, compared to the female, which the urethra is close, very close to the proximal side. So this is very important for the male penis function. So to really show that this SF1 cell is really important for facilitate the closure of the urethra, Cyril was very smart. He actually used a cross-section culture system that we can actually visualize the closure of the urethra. So what you're going to see here is a movie of the urethra closure. Just to orient you, this is a cross-section of the penis and this is the ventral side. Here is the opening urethra. And you can actually see the SF1 positive cell. So I'm going to show you, this is the same cross-section. You can see the SF1 cell here. So I'm going to play the video here. So you can see the migration and movement of the cell. And the key point is, these cells are pushing. They are migrating. And their goal is to make sure the urethra is actually closed. You can see the movement of the cell. They are reaching very hard. And eventually, they actually meet together in the middle. And entirely, the urethra is right here. They are completely closed. So in the lab, we have a joke. This is just like the God and the David. They are trying to put their two fingers together. And eventually, they touch. So, and then, of course, what is the functional importance of it? So Ciro developed a model that we can actually remove the SF1 cell. So in this case, what you are seeing here is the control animal. The urethra is completely closed. When he removed the SF1 cell, we actually produced a very severe hypospadia cases. So this tells us the SF1 cell is very important for normal penis development, particularly for the closure of the urethra. So in conclusion, hopefully, you didn't waste your time sitting here for half an hour. I'm able to show you that the mouse genetics is very powerful. We can understand a lot of basic biology about male sexual differentiation, particularly the two factor I mentioned to you is important for the cell maintenance of the Sertoli cell. We also be able to show a novel cell population, the SF1 cell, coming from outside of the penis that is important for the normal penis formation. So with this, I would like to thank my wonderful lab people, particularly Ciro was the one did the amazing penis development project. And Paula and Karina are the one that did double knockouts project. And my lab is just, I'm very fortunate to have a very diverse lab. I come from all different parts of the world. And thank you very much for your attention. Sorry that I have to went over so long. Thank you. Thank you. Thank you, Humphrey, for this provocative talk on our paradigms. Thank you. And so please introduce yourselves. Hello, so Leticia Martinery from Paris, France. Thank you very much for this brilliant talk and amazing results. Thank you. I have two questions. One concerning the AMH and ATVB. Did you try conditional knockouts earlier in life to see if you could also reverse the phenotype after puberty? So the double knockout were not conditional knockout. Did you try to do conditional ones? No, they were able to survive actually pretty late. So we didn't have to do that. Oh, so you're saying, okay. Something that is only possible in fetal gonads are already differentiated. Yes, so the conditional will be just ablating than in the ovary, right, in the gonads, right. So unfortunately, the conditional model is not available. So you have to have the double flux allele for the AMH and the ATVB. So unfortunately, those two models are not available. And that will be really good model to do because then you don't affect the expression of those two genes in other parts of the tissue. I wish we could have those, but we don't. Thank you. And I have a second question. In humans, there are children and adults that have ovotestes. Usually they are XX. Yes. So if your model is transposable to humans, do you have a clue on a common regulator that could upregulate ATVB and AMH in ovaries? Yeah, so that's always a question. How do we apply that information to human? To my knowledge, the mutation for both factors in human, I don't think it's ever being reported because those are very important factor. It's quite seldom to have mutation present in both gene, but the single mutations are indeed present. And the human cases, they don't really have this severe scenario happen, but it's not to say that this is not possible. I would be interesting to see if we can actually discover a situation like that in the future. But of course, this will be important job for the human geneticist to screen the situation of the intersex to see whether, especially with the patient with the ovotestes structure, whether these two factors are involved in that situation. Definitely, I would love to see the human geneticist to look into that. Maybe they are common type one receptor. Yes, indeed, they do. Yes. Thank you. Hi, Ignacio Vergara from Argentina. Fascinating talk. Thank you. Just a question regarding the last part of the Freemartin experiment. Yes. I want to know if the adult mouse, XY, has low AMH circulating, has a human that has low AMH in circulation. So you are talking about the recipient that we put the double knockout, right? Yes. The double knockout, the AMH level is not detectable, as you could imagine, because it's a double knockout. We also measure activating B is not detectable, confirm that the double knockout efficiency. So basically, that XY recipient had no AMH in activating in the circulation. But the normal XY. The normal, yes. The normal XY. And you put the explant of the ovary and it turns the testicle. Yeah, we look at the literature. According to the literature, there should be no AMH production in the adult. To our surprise, we were able to measure AMH in the wild-type situation. So that really tells us in the normal circulation, at least in the mouse scenario, the Sertoli cells still be able to produce AMH to the level that we are able to detect. Thank you. So thank you, Humphrey, for a great talk. Thank you very much. Thank you. We will now move to the last talk of the session. We are very happy to have Dr. Krista Flug here. She's professor for pediatrics, endocrinology, and diabetology, and head of the division of pediatric endocrinology, diabetology, and metabolism at the Bern University Children's Hospital in Bern, Switzerland. Here, our talk is entitled Uncommon Steroidogenic Defects in Disorders of Sex Development. Hello, everybody. First of all, I would also like to thank the ENDO for inviting me here, and thank you all for making it up to the really last talk, even though I cannot present glowing penis. So we will move a little bit from sex determination to sex differentiation, where we know that steroids play a role. And we will move a little bit into a story which is less about genetics also, but also about stereogenesis, biochemistry, and what we have learned in recent years, after the genetics actually opened the whole field of all genes in the steroid pathway. So I have nothing to disclose. I would introduce a bit the newer stereogenesis for those who are not in the steroid community completely, and have seen all the androgen pathways lately which have evolved. And then I go and talk about the two different groups which affect TSD, the variants of sex development from steroids. One group which is actually affecting adrenal and gonadal stereogenesis with androgen excess or androgen deficiency, versus the defects which affect exclusively the gonadal stereogenesis, again, with androgen excess or deficiencies. And I think that's a good grouping, if you are coming and you have a view on your patients with a steroid disorder in TSD. And finally, actually, to make the story round, I didn't talk to my colleague over there, but I have also something to talk about stereogenic factor one, because we are running a bigger study now in my country with worldwide participation. So human steroid biosynthesis takes place in these organs, and of course, we are most interested here now about the testis, the ovary, and the adrenal. And a little bit, of course, also for the placenta and the fetus. So if we go through the organs which are actually participating in steroid biosynthesis, first of all, you see on the left-hand side the classic view, how an ovary produces the steroids in the granulosa and the tica cell, which collaborate perfectly together to take all androgens, preferably, to the next step and make out of them in final step the estrogens, and that only little of these androgens actually leave the ovary and go out into circulation to give also to a woman a little bit spice of androgens. Then on the other hand, we have the testicle, which in the classic view produces only testosterone through the classic pathway, but we've learned in recent years there are other androgenic pathways taking testosterone or DHT or BACTOR or 11-oxy, as I will see you in a few slides, I show you, but we don't know exactly what is known about these pathways in the testis so far because there is much work to be done. So what about the adrenal cortex? The adrenal cortex is actually also producing androgens and not only the classic pathway. We know now since 20 years there are other pathways, the 11-oxy pathway, which actually plays a pivotal role and that these androgens, the 11-oxy androgens are actually going out into the circulation and contribute the active androgen pool in the periphery. And on the other hand, also the BACTOR pathway where actually 17-OH progesterone is taking away through 3-alpha and 5-alpha to the BACTOR which end up to the dehydrotestosterone without going through testosterone and especially in diseases like CIH or POR deficiency are able to produce active androgens. And in a nice review, Andrina, maybe she's here, she has actually shown here that if such androgens from these organs are reaching the periphery that other organs help them to produce not only inactive metabolites, but reactivated metabolites which are as active at the androgen receptors as the well-known classic androgens. And that's very important to keep in mind as a clinician or if you look at steroid profiles that we see there are patterns of steroids which when you only do the routine labs, you might miss. So when it comes to the fetal placental unit and where actually a lot of things can happen in sex differentiation when something goes wrong because this metabolism which now gets much more complex can virilize your female fetus or undervirilize your male fetus. So on the left-hand side is a picture out of the Spurling, the recent, where we see the classic view that the fetal adrenal produces DHEA which actually goes either through the liver or the placenta to get, in terms of androgen production, inactivated and not harm the female fetus to become overvirilized. That's the view that in C21 deficiency, the virilization happens intrauterine in your fetus. On the right-hand side, you see a nice paper by O'Shaughnessy and Al where they show the work gets even more complex. You see that not only the fetal adrenal, the fetal liver, and the placenta work together, but there is a new view that actually more organs are participating when it comes to metabolite, that they circulate between these organs involving testes, adrenal, the liver, and that metabolize in the periphery might go back to these organs and create other androgens through pathways collaborating, which we yet have to really figure out which are the major roles in which organs. They had found out in this paper, looking at this fetal material, that androsterone is actually higher in these fetuses than testosterone. So this is not easy to actually find out because you imagine it's very difficult to get the material and then also difficult to find out how the circulation goes between the organs. So finally, what we end up now is we have at least four different pathways on the plate. You have the classic pathway, which you find in all textbooks. You have the back door, which we know now since 20 years from Wilson and Rich Aukes, which have first described it in Mouse and Tamar Wallaby. Then now we have the C11 oxy, which is important in the adrenal to produce new androgens. And lately, Amanda Swartz Group in South Africa has shown in cell culture that actually it's not about the back door or the C11 oxy. They can actually collaborate. If you do cell culture experiments, you can see on the far right-hand side that you reach from the C11 oxy or through the back door, you can combine them and you have the so-called C11 oxy back door, C19 back door pathway. So all of these pathway can actually feed in the periphery to the androgen pool and contribute to androgen action at your androgen receptors. So when does actually all these steroids are important in human sex development? So simply talking, it's at the very beginning of life. You see it on the far left. You have to be able to make steroids as a male fetus at least to get virilized around six to 12 weeks gestation. You have to safeguard the female fetus from that. There is mini-puberty soon after birth. The role of it is not completely clear. Then we go to a quiescent phase until puberty. Then you have to be able to rise your steroids again, be it for the female or the male, to get to the reproductive phase. And finally, you need the steroids to reproduce. So now, let's have a look with all that background. How are these steroids affecting in disorders which affect adrenal and gonads? So first of all, very far up in the steroid pathway, we know that the steroidogenic, the star protein is important to import the cholesterol to the mitochondria where all steroid production starts. In the 1950s, Prader and Zurich showed a patient where they found that the 46XY baby was manifesting completely as female. And then Walt Miller, at this time, brought forward the human genetics, saying that there is a two-hit to explain that, that first of all, you have genetically the star loss, and second of all, because of the lipid accumulation, the cell damage is done, and you are ending up as a complete sex reversal. Later, we found out that there is a founder mutation in Switzerland. We have quite some of these patients which have star deficiency, although it's, of course, a rare disease, and not like 21-hydroxylase deficiency, but we are not talking about that today, right? Similarly, later, others found out, together with Walt Miller's lab, that Cp11A1 has a very similar phenotype clinically, and those patients can also manifest as sex reversal. But fair enough, what we learned over the years, there is not only black and white. You don't have this loss of function, or you are functional. There is gray. And some of these patients don't have a gonadal phenotype. They only have an adrenal phenotype. When you have this function, partially absent. So that's important, which we have to keep in mind as clinicians. So we move on to 3-beta, which is a one step lower down in steroidigenesis. The enzyme system attached in the inner mitochondrial membrane, which converts all steroids to pregnenolone. 3-beta is also a very rare condition in human beings, and maybe in the magnitude of 1 in 100,000. What is special about 3-beta, it actually can produce a phenotype in both sexes, a 46XY DST in males, and a 46XX DST because of androgen overproduction in females. And that's only the case in one other sterogenic disorder, which is POR. When it comes to the gonade, when we go along now, because we know these genes longer, you see the first paper was published in 1992, we learn a lot by looking at these cohorts and patients. Also for questions these patients have now, they are having a diagnosis, but they want to know, is my gonade now going to be malignant, does it have to be removed? We have learned along the way that many of these steroid disorders, actually the gonade is actually not at big risk for malignancy, so we can leave these gonades in. And we have also learned going along the way, looking at more comprehensive steroid profiling to understand what's going on in this metabolism in the periphery. So in 3-beta type 2, type 1 enzyme will be responsible that some of these babies actually are catched by neonatal screening because of the type 1 enzyme, and they have elevated 17-OH-prog, and some of them are misdiagnosed as having CYP21 deficiency. So let's move to 11-hydroxylase deficiency, which is very similar to 21, but it was also first described 30 years ago. And the difference to 21 deficiency is that these persons with CIH, they develop hypertension not very early in life, but later on. And this is a mutation which now over the years, with people collecting big cohorts like in this nice paper to the right-hand side by Kotlub and all, they have collected over 200 patients and then genetically defined more than 100. You can actually do great work to get some better information to these patients and to see how they are doing in terms of growth for bone development, where they come from, and also what phenotype score they have in terms of severity of DSD. Also you find out which are the best markers for screening to put a diagnosis on them. So that's maybe new in the field because we know all genetic defects that we do cross-sectional cohort studies and longitudinal studies to learn how are the features in clinics going on in these patients. When it comes to CYP17 deficiency, you see it here in the middle, a very important enzyme for two steps in steroidigenesis, on one hand to go to the pathway of cortisol production and then also to the androgens. You see that first publications, again more than 30 years ago, but quite few mutations published and very, very rare mutations found. So you have again a CIH form with cortisol deficiency only by testing, but also hypertension and androgen deficiency in males. And of course the female phenotype in them will then be that they cannot go into puberty or just don't go through it. Finally, POR deficiency. You see on the left-hand side that Peterson, 1985, actually saw in the steroid profile this mixed oxidase deficiency where it appeared that 21 and C17 were not present, but when they were sequencing these genes, both were perfectly fine. And patients manifested with a craniosynostosis syndrome called Antlibixler's, and then they figured out that in a subset where there were genital anomalies, a DSD phenotype, then there were these POR gene mutations found. And you see here in the middle where POR actually plays an important role as a collaborator with the enzyme of C17, C21, and aromatase, and therefore you get a broad phenotype. What is very particular for this genetic mutation is that you have a huge and broad phenotype because the collaborators between the enzyme and the POR mutations, they are very specific how they dock together, and therefore you have to study them almost one by one. So what we found when we made the first description is that we had a female which was 20-year-old and manifested with a PCOS-like phenotype versus this very severe phenotype in babies with the Antlibixler's skeletal malformation syndromes. And what is also particular about POR, it is a partner for P450s of many metabolic and other pathways in biochemistry. But so far, we have not actually found a lot of other problems in these patients than DSD, subclinical adrenal deficiency, and virilizing of the mothers during pregnancy, what is actually a little bit astonishing. So let's move to the defects which affect the gonadal stereogenesis only. Here we have the exclusive lyase deficiency of CYP17 deficiency. There are maybe only a handful of mutations so far published, and you see them on the right-hand side, that those are specific mutations which actually hinder the substrate access, the redox partner site, or the binding. And actually, what we have learned from such mutation and deep studies is that how these collaborate with the substrates, and that we can learn how we might want to design compounds that hinder specifically lyase, like arbinaterone, which is now also up as a possible treatment for CIH. So studying such mutation makes sense, also to learn for basic biochemistry. Another set of genes which are involved in the so-called syndrome of isolated lyase deficiency are therefore the collaborator cytochrome B5, where also very, very few patients have been published. They all manifest in a 46XY DSD phenotype with micropenis and undervirilization at birth, or some very specific mutations of P450 oxidoreductase deficiency, which can be very broad. You see that the phenotype is completely the same, so you really need to go and have a look at several genes which are possible with that phenotype. Then the genes in the backdoor. I'm not sure whether this is real, whether we can say that these genes are up on the scene yet. So far, I was involved and we were able to find two families which had genes exclusively expressed and needed in the backdoor pathway, which actually had a severe 46XY DSD phenotype, and which were previously actually described as having lyase deficiency by Tzachman. But when we then sequenced these genes, we didn't find a CYP17 mutation. And when we sequenced the genes in the backdoor, we found mutation in the AKR1C2 and 4, as I said, which are exclusively used in this backdoor pathway. And the patients had, in both independent families, a severe phenotype. But by doing this work, actually there were, at the end, more questions on the plate than solved. So how can we explain if this is true, why is not compensatory the classic pathway rescuing these patients? And why is another pathway then needed? So there is a lot of unsolved in this family. But fair enough, these patients, they were already gonadectomized, so we cannot go back and study them. So we need to find other patients with mutations in these pathways to actually study that better. Then HST17B3, also a very known and old known gene, which can produce a 46XY DSD phenotype with a complete sex reversal. There are founder effects, there are not many patients out there, and you need to look at the steroid profile and you need to do the genetics after work. Because the steroid profile in the exclusive gonadal steroidogenesis, this is very difficult sometimes to really put the diagnosis only by the biochemistry. We have alpha reductase deficiency type 2, known since 1991. You see again on the left-hand side, the first paper described female external genitalia, virilization at puberty. And most patients nowadays, if we catch them at birth, we would let them grow up as males. Because we've learned that over the time by doing these cohort studies and going back to the patients, what they want. And I think that's a very important message. We shouldn't only do the genetics and then describe them. We should go on and follow them to look how they develop and how they age and what other medical problems they will have at the end of the day. To learn from them and go back to the patients. I think that's a big advantage, what we have done in the last 20 years. And the only one which affects the female, 46XX, DSD19A1 aromatase, also described 30 years ago. You have ambiguous genitalia at birth. You have primary amenorrhea. You have no pubertal development, PCOS, and maternal virilization during pregnancy. Maternal virilization during pregnancy. You have heard me before, this is shared by POR. There are quite a lot of mutations out there. Maybe now it's difficult from the literature to estimate the numbers of families. But between 50 and 80 mutations out there. And you see here representative one of the very severe virilized cases. And for this particular, these girls turn up again and again during their childhood years with polycystic ovaries. And they profit if you just give them a little bit low-dose estrogen to avoid that. So finally, let's go back to SF1. SF1, in my perspective, also a little bit belongs to the sterogenic disorders. Because it is a transcription factor which almost regulates all of the sterogenic enzymes. The first case described, again, 20 years ago. Sex reversal, adrenal insufficiency, and an extremely broad phenotype which partially is unexplained. Now we've just heard nicely and seen the pictures. It's involved also in the closure and hypospadias. And you see here on the right-hand side where SF1 actually plays a role in sex determination and sex differentiation. And now penis formation. So what we did now with a lot of collaboration worldwide, we are actually collecting all the data to do a cross-sectional study to learn how SF1 actually in this broad phenotype is behaving in terms of phenotype, whether we can form clusters, and whether we have second hits in other genes. And you can see here that without going into the whole exome sequencing data, which we will be able to maybe present in a year, that only by candidate approach and what people report to us, you see many genes which are actually second hits in these patients which have SF variants reported. And you also see that which was just presented, we have a lot of patients which have a severe phenotype, sex reversal, but others also with a mild phenotype, which actually then goes to the hypospadias. So there is a whole spectrum there. And I think we have to also think in genetics about background genes then. Of course, in early days, you had one hit of SF1. You just were happy and you gave the diagnosis to your patients. But nowadays, you have to say, maybe they have another hit, and that's maybe what is explaining the broad phenotype. And with that, I would like to end my story about steroidogenesis in sex development over the years. So I conclude that normal sex hormone biosynthesis and metabolism are essential for typical fetal sex development, especially differentiation, but also pubertal maturation and fertility. Autosomal recessive mutations are known now for almost all genes, cofactors, collaborators involved in sex development, production, and metabolism, and lead to different forms of 46X, XX, and XYDST, with and without adrenal insufficiency. P-allelic CYP21A2 mutations are, of course, the most common variants found in 46XY. In Switzerland, around 1 in 12,000. All other steroidogenic defects resulting in DSD are less common or, let's say, ultra-rare. The modulatory role of haploinsufficiency of steroidogenic genes and enzyme activities in combination with other DSD gene, like SF1, remain to be investigated, and I think that's the next step which we have to go. With that, I would like to thank my whole team. Okay, here it turned black. My whole team in Bern and the founders of all that work, and really last, we are going to run in four weeks the 9th IDSD Symposium in Bern. You're all welcome. Thank you very much for your attention. Thank you. Thank you, Krista. Thank you. Thank you, Krista. Now this paper is open for questions. I know you're very tired, but you are the survivors here, so I invite you to make your questions. I don't have any questions here by the web. While there are questions from the, please, go ahead. Hello. Beautiful talk. I'm Fei Zhao from Wisconsin Medicine. So can you comment about the potential environmental exposure on the DSD, and also potentially the environmental exposure on the sterogenesis pathway? That's a very tough question, right? Because of course there are environmental factors. If you are exposing a fetus to the steroids during pregnancy, you virilize, right, or you under-virilize. We know that. If you have to give mothers compounds which do that, you don't want to get them pregnant, right? I can just maybe say it from these sides, but I guess you allude more to the environmental factors otherwise around there, and me personally, we don't do these studies, but I know that it will have an influence on the levels of sex steroids, which we all have, right, our environmental factors, because the system is plastic. If you put in androgens, of course, through the regulatory circuits, which we all know we influence our systems, and be that during a pregnancy and having an effect on a fetus, or be it on our life as adults for reproduction, for sure, right? Just adjust this. Hi. I'm Priya Phulwani. I'm a pediatric endocrinologist, an adult endocrinologist, and my sub-areas of interest are variations in sex development as well as gender transition. So my question is, do you feel, and thank you for this amazing talk as an endocrinologist. I kept taking pictures. Do you feel that in terms of the future directions, are people really looking more at how these patients choose their gender identities over time and keeping more of an open mind to that aspect in terms of clinical care than we had perhaps done in the past? Thanks. Yeah. I mean, me, I really feel that this 20 year, we shift a bit, right? And I think what is really important that we work together is these patients and that we are registering, because this is a rare disease, to collaborate together. I mean, now that I was really thankful, I mean, I'm in Bern. It's a very small country. We collect now 164 as of yesterday. It was never possible. We have collaborators from the US to Australia. And if then we have this data and we can actually then see what's going on, and maybe also then have them in a registry like the IDST registry, as they are going to, we can go back tomorrow and we can see how now with these genetic defects, maybe with other collaborating genes, what's going on in a human being, right? I mean, of course, it's a lot doable, as you've heard, in a mouse, right? But we are not mice. So what's going on with us in 10 years and these people in 10 or 20 or 30 years, right? It's important to know. And we have, with CIH, a lot of this data available, but we cannot just deduct that this from 21 is the same now in the other defects. And we, in this perspective, to kind of care for the patient, to answer the questions, we really need to do this collaborative work. And for that, registries are important to convince the patients that it is for them, maybe not directly, but indirectly, for the next baby to come, that we can say, like nowadays, 5 alpha, even though they look female, please let them be growing up as a male. And if they come in late, of course, it's a different story. If they are 15 or so or not going into puberty and they were growing up as female, you have to do individualized medicine and let them be female if they want to be female. It's not for you as a doctor to decide. That's my opinion. You have to get the mental DSD team and work it out with them. It's their life. Hi. I'm Jay Whitehead, PSNDO in Chicago. Based on what we know so far about the SF1, do you recommend screening periodically for adrenal insufficiency, or does it depend on which particular variant they have? Now you have to repeat your question, sorry. Oh, sorry. Do you recommend screening for adrenal insufficiency in the SF1 patients? It's a good question. So far in this 164, we have five with adrenal insufficiency. They can have the same mutation. So actually there is no information that you can say this mutation makes the adrenal insufficiency. But we have to be cautious maybe as they age. So I would say at present, I would rather go for it, yeah. Okay. Thank you. So we thank you for your attendance, and we are very happy for the three speakers. And congratulations for being here, for being the last ones in the convention center. Happy return home for everyone, and see you next year, hopefully. Thank you.

disorders of sex development

FGF signaling pathway

human sex development

mouse models

FGFR2C mouse knockout

FGFR2 mutation

XY female

FGF9

activin B

Sertoli cell identity

testis development

Freemartin

AMH