I'm Dr. Erika Oikster, and it's my pleasure to chair this session along with my co-chair. So I am Maria Vogiaty from Children's Hospital of Philadelphia. And I'm at Riley Hospital for Children in Indianapolis. So we're in for a real treat this afternoon. We have three outstanding speakers. Each one will speak for 20 minutes, and that will be followed by 10 minutes for questions. So our first speaker is Professor Anders Juul. Professor Juul is clinical professor at the University of Copenhagen and head of Department of Growth and Reproduction at Rijkshospitalet Copenhagen. His research interests include growth and pubertal disorders, as well as genetic, epigenetic, and environmental influences on gonadal and adrenal function throughout life. And we're really excited to hear his talk today on premature theelarchy. Thank you very much, and thank you for the organizers for inviting me to give this talk on transient theelarchy. And I hope I'll provide you with some facts about whether or not it exists. And these are my disclosures. And in fact, I have one more disclosure, is that just a few weeks ago, I returned from walking the Camino from France to Spain, 800 kilometers. And you can see, and here you can see me with my wife at Finisterra, which is the world's end where you end the Camino walk. And while you walk the Camino, you are met with a lot of wisdom. And I'll just share one of these wisdoms with you, written here on a sign. And I don't know if you can see what it says, but actually when it says, there will always be more nipples than humans. I thought to myself, whoever wrote this thing? But it has grown on me, so now I'll share it with you. There will always be more nipples than humans. OK, and now back to the talk and some definitions. Now, thalachy is derived from the ancient Greek word thali, which means nipple, and archae, which means the beginning or the onset. So we talk about breast budding or onset of puberty when we talk about thalachy. Premature thalachy is when breast development starts before eight years of age. And we can perhaps just come back to that, whether or not that's an appropriate age. But we call that premature thalachy. And the topic of my talk is to talk about transient thalachy, which is the appearance, and then the regression, and then the reappearance of breast buds. So it's coming and going breasts. Is that a true phenomenon? And you know the traditional pubertal stages by Professor Jim Tanner, who had these five stages, B1 being the prepubertal state, and B2 the first breast budding state. And therefore, marking the onset of puberty together with pubic hair development, whereas menarche is, as you all know, a very late pubertal phenomenon. So it's very difficult to actually, Jim Tanner described his five pubertal stages based on photographs. He didn't palpate these kids in the 50s. But we know today that, of course, it's very difficult to visually inspect whether or not there's breast development, especially in an obese girl. So we did this study years back to look at how good are we at palpating palpable glandular breast tissue when we use MRI as the control. So we did this in some normal weight girls, healthy girls, and some girls with high BMI, as shown here on the right. And you can see that they have a lot more of the white subcutaneous fat. But you can clearly visualize by MRI the breast buds. And actually, when we compare to our manual palpation, we were quite good at doing it. But it is important to do palpation to be sure that there's breast tissue. I don't need to tell this audience that. But I think it's important to stress. Because when we look at available studies in the literature, and this is a medical student, Camilla, who I gave this assignment to look at all published papers on pubertal onset and timing of pubertal onset in healthy children, including the ones we did ourselves. And we included only studies where a medical health professional had been involved in the evaluation of breast budding. So it was not self-reported puberty. They were excluded. And this graph simply illustrates a year's work, showing a downwards trend in the age at thalachy with a decline of 0.24 years per decade. So there is a trend towards earlier breast budding in the general population. I think that's undistinguishable from the US first, and also from European and other places in the world. This is also reflected in more girls being referred for precocious puberty. And this is another study from our group. This is Elvira. She's a senior researcher. And she looked at a nationwide register-based study how the incidence of prevalence of precocious puberty in Denmark. Denmark is quite unique, because we are all given a unique ID when we are born. And then you can follow us for the rest of our lives when we have a police fine, or if we have a disorder or precocious puberty, and when we die. And actually, based on that, we can look here at the incidence and prevalence of precocious puberty based on these 7,000 girls that were diagnosed in this period. And we looked at this one doesn't, yeah, no, there it is. We looked at both premature adrenaline, premature celiac, and central precocious puberty. And this is the overall curve. And you see that they all increase over this time period. So there's an increase, a decrease in the age at pubertal onset in the general population. And that results also in more referrals. That's not magic. It's just a fact. Actually, when we looked years back at our population of girls referred with central precocious puberty, I asked another medical student, Sine, to look at 450 paper record files in my center. And when she did that, you don't need to understand this business slide, but actually, we were a little frustrated that 67 of these 400 and something girls, actually, when they came to us and were evaluated, they were described as being completely prepubertal. So this was 67 girls where the GP has said, clear breast spotting, central precocious puberty, look for that. And then when they came, it had gone away. So we thought, is that because the doctor didn't examine the child as well? Or is it a true phenomenon that breasts can disappear? So this is the aims of this talk is to describe if transient therapy is a true phenomenon. And if yes, at what ages does it occur? How frequent is it? And what characterizes girls with transient therapy? First, we have to look at hormonally active periods during a lifetime. And here, you see that there's a lot of this denotes gonadotropins or sex steroid levels in fetal life. And then you see that just after birth, we have this mini-puberty where the sex steroids are very active. And then by 6 or 12 months of age, sex steroids enter a quiescent period. Not much is going on for the next 10 years until this pituitary-gonadal axis is reactivated again during puberty. Some with average puberty, some with early puberty, and some with late puberty. So let's first look at the mini-puberty period. And we did this in a recent prospective cohort of healthy children. We call it Copenhagen Miniature Puberty. And these are all the PhD students involved in the study. And what we did was to study 200 kids and their parents. And we followed them for a year with six visits. So we recruited them during pregnancy and then followed them at either 1, 3, 5, 7, and 12 months, or 2, 4, 6, 8, and 12 months. And we did detailed examinations at all times to figure out when is the peak age at which hormones are high. We did anthropometric measures. We studied their genitals and ultrasound of testes in the boys. And we also took blood samples and urine samples in all 200 kids six times during the first year of life. And we studied the parents as well and looked at urine and breast samples, et cetera. And these are the kids. I have a poster of it in my office. I really enjoy looking at it every day because you get so happy. And that reminds me why I ended up being a pediatrician. From this study, we published a cohort paper last year and several other papers have followed on what happens in this period. And I'll just give you a brief example of this hormone peaks in relation to thalachy. First, the boys, you see here on the top. This was published earlier this year in JCNM. You see FSH and LH levels with a very early peak at one month of age. And you can see the blue lines are the top 50% of the boys that if you're high in FSH and LH during the first year of life, there's tracking, you remain to be high during the first year of life. And then here on the left side, you will notice the total cell markers inhibin B and AMH. And actually you can see that there is a peak at four or five months of age and a blunted rise as opposed to the lytic cell markers, testosterone and INSL3 that seems to mimic the very early LH peak occurring at one month of age. And this is just depicted here in a diagram where if you look at the bottom, you can see that testosterone and INSL3 has a distinct peak at one month of age as opposed to the total cell markers which peaks many months later. So mini puberty is not just mini puberty. It depends on which cell type that is the origin of the hormones. In girls, this was actually just accepted last week in JCNM. We looked at estradiol by mass spectrometry. And here you see in girls also that estradiol levels are quite high in the first three months of age. And when we do individual longitudinal modeling of this, just notice, look at the red line, that's estradiol. It looks like there is an early peak and then again a later peak, a biphasic pattern, fluctuating hormones, reproductive hormones in the first year of life. That probably influences when we look at breast development. So first we look at the boys, so this is not published yet. So each line is an individual child and here are the boys. And the dots are when the boy was examined for breast development. Filled circle means that there was breast tissue. So you can see at birth, breast tissue was present in all boys. And then around three to four to five months of age, when it opened circle, it disappears. And in this case, very few or none had actually breast tissue at one year of age. And these are the similar graph for girls. And actually, you can see that breast tissue remained for a longer period in girls. And actually, to sum up, at 12 months, 16% of girls still had breast tissue as opposed to one out of 75 boys who still had breast tissue. But this is breast tissue that is there and then goes away. So what about the transient coming and going breast? Well, that's also a phenomenon we see in mini puberty. So here, that's shown up here. Those boys who have breast tissue, breast tissue, and then no tissue, breast at all, and then breast tissue reappears. So that is a phenomenon we see both in boys as well as girls. And it actually, when you put numbers on it, it shows you that 25% of boys as well as girls have coming and going breast or transient telarchy during mini puberty. That was that period. Then now let's turn to puberty, where we also had the opportunity to study healthy children in a longitudinal setup. And we published this some years ago from the Copenhagen Puberty Study. And I'll share the results with you. This study is a longitudinal part of a big Copenhagen study, where here you see the height velocity curves in girls and in boys. And you get an idea about how frequent we measured these kids and studied their breast development. And also based on this, we constructed puberty nomograms, which is, I think, a neat way of transforming distinct pubertal stages prone to a lot of inter-observer variation or intra-observer as well into a continuous puberty SD score. So if you are 14.4 years of age and have a breast stage three, is that late or is it very late or? Well, this helps you to discuss this with the parents. And here you can see an example of a child where you can follow actually the progression through puberty. So this is a normal child with breast development one. This is the B1 line. And then breast stage two, denoted by this line, B2, and then B3, B4, B4, and then B5. So you can actually visualize the progression through puberty in this way. We also, in this cohort, then looked at those with transient thalachy. And these are just eight examples shown here. So this is the control example. You can just focus on one of them. B1, B1, and B2. And that next evaluation, there were no breasts again. And then at the next, again, there was breast tissue again. So that's why there are two lines here denoting the first transient thalachy and the second permanent thalachy. And you can see that sometimes first and second breast development occurs quite close to each other. And sometimes there are several years between them. So we looked at gonadotropins and sex steroid levels in these girls. And the red lines show individual curves for girls with transient thalachy. And it just shows you that this is where the first breast tissue is shown. And this is years from pubertal onset. And you can see that the red lines are lower than the blue ones. Gonadotropins are not elevated at the time of the first transient thalachy. I think that's the take-home message. And also estradiol levels at this time point is lower compared to those girls who enter puberty and progress through puberty. So we think it is a, most likely transient thalachy is a peripheral phenomenon. It's not a sign of central generates dependent pubertal onset, but rather some peripheral action of estrogen. So the conclusions for this Danish study is that we also looked at FSH genotypes and they were similarly distributed between the girls with or without transient thalachy. So it did not explain why some girls where their breasts went away again. We found that 50% of the girls with transient thalachy actually had the puberty as the first sign of pubertal onset. And I know it's a very small study. Here we only have six out of 12 girls. But actually in those control girls without transient thalachy, you can see that it's in Danish Caucasian population, absolutely the most common thing is to start with breast development and then later have pubic hair development as a pubertal sign. That is of course prone to a lot of ethnic differences. But in this context here, those with transient thalachy, they were much more likely to have pubic hair at that time. And that of course makes us speculate whether adrenal antigens may be involved in this phenomenon. We found that growth velocity was lower at first thalachy, and as I said, we believe it's a peripheral estrogen-dependent phenomenon. And then I'll just say that we are not the only one who studied this, and I'll just make some PR for a much larger and very interesting study from Veronica Marie, who's sitting here. And I had the fortune to visit Veronica and her group in Chile to see how they conducted their longitudinal puberty study, GOCS. And this is the design of the GOCS study where 500 girls or more have been followed sequentially for many years, and a lot of interesting data from that study. And in their study on transient thalachy that we sort of collaborated a little bit about, they found actually that 7% of their completely healthy girls from Santiago in Chile also had this transient thalachy, and they concluded that it was observed in 7%, as I said. And also here, there were no differences in the distribution of FSH genotypes, and there were some puzzling results regarding age at presentation of first transient thalachy that suggested perhaps age at presentation may give rise to different etiologies, but it's difficult to speculate, at least for me. And again, in Chile, those girls with transient thalachy were much more likely to have pubic hair development as the first sign of pubertal onset. So to conclude, I would like to say that what is the facts, I guess, in addition to the fact that there will always be more nipples than humans, I think I can say that transient thalachy does exist. The answer is yes, that is the take-home message. And it's frequent in mini-puberty, 25% of boys and girls have this phenomenon, and in girls, it's in puberty, it's 8% to 12%. And as we speculate, because of this association to puberty, we speculate that perhaps aromatization of adrenal androgens to estrogens could play a role. I mean, we're still left open with why do we have this phenomenon? I don't think, at least I can't answer you on that question, but thank you very much for your attention, and I'm happy to take questions. Thank you. So we would like to welcome anyone with questions to come to the microphone. Thank you. Hi, Anders, Rodolfo Rey, Buenos Aires Children's Hospital in Argentina. It's always a pleasure to hear, to listen to your work. So what you showed us is transient telearchy in normal girls. Can you comment on transient telearchy in precocious, I mean, precociously? How, if there is any particular evolution of transient telearchy in earlier ages? I think that was actually what the Chilean study suggested, that age at presentation of the transient telearchy showed different and rheumatic as well as hormonal differences, suggesting that perhaps those with early transient telearchy has another etiology compared to the later, but I can't speculate what these mechanisms were, but I think relating to the context of transient telearchy, whether or not it progresses into a true central generation dependent precocious puberty. There are some studies to suggest that, that's why some suggest that we should not just send them home and say, come back if you need it, but some follow-up may be required in some cases. César Camacho-Huebner, New York. Congratulations, beautiful studies. My question has to do with the anthropometric point that you just raised. With this trend of overweight in these girls, I know that in your center, and I think in Veronica, you can distinguish clearly where there is, you know, glandular tissue versus just fat tissue. So do you have that data to show that this is unrelated to the body weight? I mean, the phenomenon of transient telearchy, we cannot detect any differences in body composition, I mean, by BMI or skin folds, between those with transient and non-transient telearchy. Thank you. But of course, there were differences according to pubertal timing, the more fat, the earlier. So that was, but that's... Thank you very much for your talk. I have two quick questions. One is, did you study bone age in these girls? No, we didn't apply for ethics to do that, even though bone age is a very small radiation. I mean, we went out to public schools and started the kids out there. So we, I don't know, maybe we could have, we didn't do it. And my second question is, did you study any familial trends in sisters of these girls or brothers of these boys? I think actually we did report data on siblings whenever possible, whenever available, but the data was so inconsistent. We had mother's maternal, maternal menarche, menarche age, but I can't remember what actually, if there was an association. I don't think there was an association. Thank you. Miller from Minnesota, UNDER's beautiful studies. My question is, you mentioned adrenal androgens as potential for the transenthelarchy, both in the mini puberty and later. Did any of the studies measure them? Veronica says yes. She's shaking her head. She did it in a very much bigger study than ours. So I think, and I can't remember your results regarding the, you had DHEAS and interesting time. They were probably higher in the, she nods, they were higher. Okay, thank you. So we have one question from online. And the question is, is the growth velocity in transient telarchy higher than patients at the same age without telarchy? That's a good question. I think, I think what I, I remember specifically is that height velocity was lower at first transient telarchy as opposed to the height velocity observed when the second and permanent telarchy occurred, suggesting that they are less hormonally active at the first. That's my take on it. And I think, but I don't think I can remember the answer to that question. But I don't think I can remember if we looked at the height velocity compared to a similar age. And that's just a question. So sorry, I can't answer that. So Anders, why don't we see more infant boys referred for breast development if 25% have a return of telarchy? Well, to be honest, I think just preparing for this talk, I got several questions mostly related to the boys. I mean, we never looked at transient telarchy in pubertal boys. We know that 50% of them have short-term gynecomastia, but whether or not it's coming and going, I don't know. Maybe you did it in the Chilean. No, so we, but that was not your question, but that was one of the questions I had. Why don't we, why didn't we study the boys? And actually regarding the infant boys, I was a little surprised that they all had breast development and that 25% had transient. Yeah. I mean, it's clearly a manifestation of maternal estrogen in the newborn, but the fact that 25% had again, exactly, the population that I was surprised to hear about, and I wonder why we don't see that clinically. Yeah, I don't know. And one last question. Do you think that pubic hair of infancy in girls is a manifestation of the mini-puberty? That's okay, that's kind of out there, but thinking about... I don't know. I don't think we had, we did not have any any cases of that in this cohort, at least. That's an honest answer. It's a serious phenomenon in girls in particular, I think. Elizabeth Orozco from Argentina. My question is related, if you think that those girls who have transitional telartan might have higher risk of developing breast cancer in the future, since I suppose that aromatase expression in breast tissue is higher in comparison with those that do not have. Yeah, so do we know if transient telartan is associated with increased risk of adult breast cancer? Well, I would take it a step further. Do we know whether early breast development by itself is associated with increased breast cancer? We assume that it has to do with estrogen exposure, years of estrogen exposure. So from that point of view, I would say earlier breast development would be associated with a slightly increased breast cancer risk. But the studies are only relating to age of menarche, where cyclic fluctuations of estrogen levels are obtained. So we do not know if breast budding is associated with breast cancer risk, and we know even less about the risk that transient telarches should be a risk factor, to be honest. But I think from a biological point of view, I would say that compared to a prepubertal child, I would still suspect that they have more estrogen compared to a completely girl without breast development. So it would add in the direction towards increased risk, but I don't know that. I don't think we know that. Okay. Thank you so much. That was fantastic. Thank you. It is my absolute pleasure to introduce our next speaker, Dr. Sharon Oberfield. Dr. Oberfield is a professor of pediatrics and division chief of endocrinology, diabetes, and metabolism at Columbia University Medical Center. She has been engaged in patient-oriented research for her entire career with a particular focus on disorders of adrenal hormone synthesis, adrenarche, and PCOS. Dr. Oberfield is also the immediate past president of the Pediatric Endocrine Society, and we are excited to hear her talk about pubic hair. Thank you. I appreciate the invitation to speak from the Organizing Committee at the Endocrine Society, and thanks so much to our fellow colleagues, all the pediatric endocrinologists in the audience, thank you, all the fellows who are here for a first in-live meeting, and I hope we will have a lively discussion. As my husband says, unfortunately, I don't have any financial or other conflicts or presumed conflicts of interest, so, and thank you to the back for lowering the lights a little bit. It makes it easier. So, premature adrenarche is something that I've been involved with for a number of years. We know little about what triggers it. We know some more about the biochemical pathways leading to adrenarche. We know something about the long-term outcomes, and they certainly are variable, and we know treatment options are still controversial. The process of adrenarche actually is one of the least understood of human endocrine developmental events, and we really don't know why it's observed besides in humans, in chimpanzees, and gorillas only. Whenever you talk about premature adrenarche, we need to talk about a differential, and this is just as a very quick review. Isolated premature poo bark, up till now, was felt to be the same physical finding of pubic hair, but no elevation of DS, and now we think it's possibly related not only to increased androgen receptor sensitivity, but more recently, there's been discussion that it's likely due to an increase in 11 oxyandrogens. Precocious puberty, obviously, is a different kettle of fish, and here we have breast development, testicular enlargement, whereas in premature adrenarche, it is totally pre-pubital, we think. Non-classical adrenal hyperplasia is another differential, and obviously, there are elevations of 17 hydroxyprogesterone. To make that diagnosis, virilizing adrenal tumors can rarely present as premature pubarch or adrenarche, and of course, now we are very aware that many of the children who come to us with early pubic hair have exogenous hormonic exposures from their families compounds that are being taken. I've been involved, as I said, for a number of years. We've had a number of publications on this, including most recently, my graduate fellow, Brittany Weiss-Origin, really studied the unique role of the 11 oxy C19 steroids in both premature adrenarche and premature pubarch, and for those who want to review, there's an excellent review on normal and premature adrenarche by Robert Rosenfeld in endocrine reviews this past year. Eleven years ago, I gave a meet the professor here at the Endocrine Society in Boston. It was a Sunday, a little different amount of work. We had to do it twice then, and the learning objectives were pretty much the same. Understand the terms, recognize the common manifestations of premature adrenarche and its relationship to PCOS and metabolic syndrome in adolescence, and understand the current evolving areas of potential intervention and treatment for this condition. I gave a case then, and I've updated this case. A five-year-old Caucasian girl is referred for evaluation of body odor. She was born full-term, weighed seven pounds at birth, and I will say I will not be discussing small for gestational age individuals at this time who seem to have an increased incidence of premature adrenarche. She was born full-term, no complications, had a normal newborn screen, 17-hydroxyprogesterone was normal. She grew consistently along the 75th percent off her height, but recently had an increase in her growth velocity. She had always plotted on the 50th to the 75th percent off for weight, but now she plots at the 75th to 90th. Her parents are both Ashkenazi Jewish, again, maybe a non-classic variant. Mother is five feet seven inches, father six foot, so there's a tall mid-parent, sex-adjusted target height. The father had severe acne since age 14. We know that men also can have manifestation of classic PCOS, and balding is something we're interested in, and there's a family history of type 2 diabetes. On examination, she plotted at the 90th percent off for both height and weight. Now she had a BMI at the 83rd percent off, normal tensive, no glandular breast tissue by palpation, as per Dr. Jewel, and she had some peri-rectal hair and tanner two to early three pubic hair. She had a normal clitoris, her vaginal mucosa was shiny pink-red or pre-pubital, and she had no axillary hair, but did have adult apocrine-like body odor. She had a bone age that was a little ahead of her chronologic age, but very consistent with her height age. She had normal bloods drawn early in the morning with respect for 17 OHP, delta-4-androstenedione. Her DS was elevated to 80 microgram per deciliter, consistent with a tanner two level, and by convention, DS of either 45 or 50, depending on what criteria you use, is a cutoff. For tanner two, and consistent with the term adrenarch. She had no elevation of her gonadotropins. Fasting serum lipids demonstrated elevated triglyceride for age. Fasting glucose to insulin ratio was borderline. She had a fasting glucose in the upper range of normal, and a fasting insulin of 15. At the time, 10 years ago, I was asked to discuss the diagnosis, intervention, and follow-up. Here is her chart, her growth curve, and I'm not sure I can do this, but simply if you look, you can see she had a growth spurt, her height age was consistent with her bone age, and here you see her weight. She also had a recent progression in weight. So again, going through very quickly the background, pubarc is a term applied to the onset of sexual hair growth. In girls, the sexual hair usually appears first on the labia majora, and the hair gradually spreads upward into the moans. In some girls, it does begin in the axilla. Early or premature pubarc, by convention, is less than age 8 in girls, and 9 in boys. And as we said, it's accompanied sometimes by axillary body odor, a minor degree of acne. It's not accompanied by any other signs of puberty, frank virilization, or an abnormal advance in bone age. The sexual hair development increases slowly, and the bone age increases in proportion to linear growth. It is not synonymous with premature adrenarch. Adrenarch was first termed in 1940 by Fuller, Albright, and Nathan Talbot, who associated the developmental rise of adrenal androgens with the appearance of sexual hair. It's the term for the maturational increase in the secretion of DHEA and DS from the adrenal glands, and has classically been observed to start around age 6 to 8. Get this over here. Okay, both DHEA and DS. And here you see the sections of an adrenal gland on your right in a infant, six-month-old, and an adult male. And what you notice, the infant does not have the zona reticularis. And the age at which the focal islands of the reticularis is not cut-and-dry development. It's not cut-and-dry at 8 years of age. But as you see in the pink, there are little areas of the zona reticularis starting. And then later, by age 6, 7, 8 more so, until you have the development of the full gland. And these were done on autopsies of people, of youngsters who had died in an accident. So what actually triggers the initiation of adrenarch? And through the years, we've had many hypotheses. Extrapituitary factors, cortisol, paracrine hypothesis by the Boston group, primary shift of steroid biosynthesis, pituitary factors, LH prolactin, POMC-derived peptides, the pituitary adrenal androgen-stimulating hormone, ASH. And those of you who are of a certain vintage may remember MASH, the mysterious androgen-stimulating hormone. Premature, I got one left, but premature adrenal is the most common cause of premature pubic. It's interesting that it occurs 10 times as much in females as compared to males. There's a slightly advanced bone age, tall stature. It's associated, as stated, with decreased birth weight in some. And again, I underscore the adrenal androgens are elevated for age, but normal for pubic hair development. It's the term applied when the androgen profile in a child with premature pubic is in the early 10 or 2 adrenarchal range. I show you this steroid pathway slide to talk about the canonical pathway, the backdoor pathway. And now we know, if you look to your left, that there are a series of other steroids that in fact have been described, the 11 oxygenated pathway with various paths going downward towards the production of 11 ketotestosterone, 11 hydroxytestosterone, 11 hydroxydht, and 11 ketodht. And again, I'm trying to focus here. These now are probably related to the development of adrenarch. There's growing evidence for this. There have been comparative studies of the androgenic activities of T, DHT, 11 ketotestosterone, and 11 ketodihestosterone, and it's known that 11 ketotestosterone and 11 ketodihestosterone bind to the human androgen receptor similar to the affinity of T and DHT. The potencies and efficacies of 11KT and 11KDHT are comparable to testo and DHT respectively, and importantly, it's been confirmed by Reggie and Pretorius that 11 ketot and 11 ketodihestosterone were able to induce androgen receptor regulated gene expression and cell growth in two androgen dependent prostate cancer cell lines. Two years, three years ago actually, at the Endocrine Society, Dr. Reggie presented initial work that there's increased DHEAS, of course, 11 hydroxyandrostenedione and 11 ketotestosterone in children with premature adrenarch, and you can see that on the top panel. Subsequently, this paper was published, and there is a gradual rise in these hormones from early on in the ages of two, three, and four, going all the way through adrenarch and beyond. And we followed up on this initial study. My group actually very carefully characterized both the pubital development, the pubic hair development, in a group of young children presenting with pubarc, and we made the finding. Indeed, we compared the 11-oxo androgens and the conventional androgens in this group, and we determined that in this group, the children with pubarc and the children with adrenarch, that is, those that had elevated DS, the adrenarchs, and those that did not have elevated DS, all had elevated 11-oxy androgens. And you note that here, that both the adrenarch and the pubarc group had elevated oxy androgens, particularly the testosterone, 11 testosterone was elevated, so that indeed, the pubarc, although by convention, didn't have elevation of classic adrenal steroids, it did have elevation of the 11-oxy androgens. There were no significant difference in this group, in HOMA, hemoglobin A1c, other metabolic parameters, and the PA and the PP differed only by DS and not by 11-oxy androgens or insulin sensitivity, consistent with the 11-oxy androgens rather than the DS probably mediating the phenotypic changes of pubarc. So with that as a background, the crux of this talk, to treat or not to treat, to treat or not to treat want, a normal developmental process, to treat something that may develop into a problem. There are patients of various ethnicities that we know that do have more findings of adrenarch. We have children who have obesity, who may be at a cardiovascular risk if they present with precocious adrenarch. Do they develop metabolic syndrome? What's the story with the advanced bone age? Does this mean they're gonna have an early puberty or even develop into having PCOS? Are there particular genetics that we can look at that will say there are certain groups at risk for the development of long-term effects? We do know that there has been report in the past, not recently, followed up on that some of these young girls in particular have social anxiety. And now we are looking at neuroendocrine links to adrenarch. So I will try to go through very rapidly all of these newer findings in the past two or three years to say, now not the background, but really are there real evidence to suggest that we need to be concerned about this problem or not? So as far as ethnicity, it's been studied extensively in Hispanic and African American populations in the U.S., Chilean Indians, Northern Spanish populations. Obesity issue, cardiovascular risk, metabolic syndrome development. The exact relationship between obesity and premature adrenarch is not fully known. One should note that not all prepubertal children with obesity have elevated androgens and not all children diagnosed with premature adrenarch are overweight or obese. And a reasonable conclusion one might make is that obesity alone is not likely sufficient to produce PA. A very interesting study a while ago, actually, in young girls who were developing PCOS, demonstrated that for increased quartiles of circulating testosterone, there is an increase in metabolic syndrome. So if you have increased androgens early, are you more at risk for development of metabolic syndrome? There was a recent paper that looked at the evaluation of metabolic parameters and the aortic elasticity in norm-tensive children with premature adrenarch and being overweight or having a metabolic profile characterized by insulin-resistant and dyslipidemia were found to be major coexisting factors that were influencing the vactual structure rather than increased androgens and premature adrenarch. Early adiposity rebound has been reviewed and discussed in Greek children, in Chilean children. We'll be going through this a little bit in the next few slides. And in a very elegant study from George Kroosos' group a number of years ago, children with PA were found to be significantly taller and had more adipose than controls from the first year of life. Maybe I should put this down. And they actually appeared to have an earlier adiposity rebound, which is, as you know, the second coming of an increase in BMI in the early infant years. Early BMI gain and later high growth was found to predict higher DS concentration in seven-year-old Chilean children. This is part of the Chilean study that was referred to before. 972 term-born children with normal birth weight were recruited from the community. And what's important here, with the children who had higher DS levels, they reached higher weight SDS in four years when compared to children with lower DS levels. However, significantly higher height SDS was only detected at seven years. And you could see that over here, by seven years. Does this elevated DS at age seven determine a further increase in metabolic dysfunction beyond the physiologic insulin resistance that accompanies normal pubital development? And again, 504 girls were included in this study. Participants were followed at Thelarc, Breast 4, and one year post-Menarc. Interestingly, the prevalence of metabolic syndrome did not differ between the girls and the higher DHEA girls or the lower DHEA girls during early and mid-puberty. However, one year post-Menarc, the higher DHEA girls had a nearly five-fold greater prevalence of metabolic syndrome. And central obesity was also greater at B2 and one year post-Menarc in girls with the higher DHEA levels. Let's look at bone age. This is an old study from Sonia Pang, and this is a group of girls who had come to the Clinical Research Center for evaluation of Adrenarc. They are all tall, but they have a line that goes equally. Their height age and their bone age have a line of identity. So their height age is like their bone age. We and others have looked at actual height prediction and final height, and this study by Oren's group showed clearly that although they had advanced bone ages, they wound up ultimately actually some of them with a higher near-final height than their predicted family height. Now, there recently was a paper just in the past month about having models to include birth weight, target height, birth length, and height at diagnosis that might help you determine which, if any, of the children with Adrenarc might need some intervention if they had the appearance of having interruption or inability to reach their final height because of an advanced bone age. That was commensurate with their families. High DHEA level in girls is associated with earlier pubital maturation and mild increase in androgens throughout puberty without affecting postmenarcheal ovarian morphology. This was another study from the Chilean group, and what I would show you in this study was that there was an association found around age seven years with a slight younger age at telarch, menarch, and pubarch, just what was said in the previous lecture. But the conclusion was that in normal girls, pre-pubital higher amount of DHEA-S was associated with just a little bit earlier onset of telarch, menarch, and pubarch, as well as a mild increase in androgens throughout puberty, but likely of no significant consequences. This probably is the most important recent paper and takeaway done by Tenelia's group. And among women not using hormonal contraception who had a history of premature adrenarch, and there was a prevalence of hirsutism, but not acne, these women had lower sex hormone-binding globulin concentrations, resulting in a higher free androgen index, but there was no evident ovarian dysfunction in these women, and the only thing that was found was a decreased SHBZ, and they therefore probably had that as the cause of the increased bioavailability of circulating androgens. Now let's move quickly to genetics. There have been a number of studies over the year that talk about genetic gene polymorphism studies and trying to link variants that have been found with premature adrenarch. Unfortunately, none of these have really panned out. Recently, the Chilean group, and Dr. Marique's in the group to answer any questions here, had genome-wide genotyping in the children who participated in the group, and it does appear that one significant variant was identified at the genome-wide level in this cohort, which was close to the galanin receptor 1G. And these results reveal possibly with a larger group of study that one of these variants is associated with DS concentrations at the level of the genome-wide association study significance. But again, a lot of work to go forward. We know that ongoing studies regarding adrenarch and the brain, DHEA potentiates neurogenesis, DHEA promotes dendritic growth and branching, DHEA itself induces an increase of synaptic spine density in the hippocampus, and we are trying to look at whether or not the oxyandrogens may also be involved theoretically in brain activity and modulations. So getting back to the case and finishing up, leaving enough time for questions, this young lady had menarche that we talked about at a little over 10 years. After two years of persistently irregular cycles and slight hyperinsulinemia, she was started on metformin. She's followed a Mediterranean diet, low glycemic index, she does aerobic and resistant exercises. She reported continued acne and the presence of increased body hair and goes for laser hair removal on her face. She's reached the 50 to 75th percentile, perfectly normal for her family. She has reasonable blood pressure. She does have signs of hirsutism. She has slight elevation of some of her androgens that were measured. She has a slightly lower FGIR than before and has an element of increased body fat on dexa. However, she has normal menstrual cycles, and she has 10 cycles a year, six year post-menarche. To conclude, in the past, the early development of pubic hair related to modestly elevated levels of adrenal androgens before the onset of puberty was considered to be benign and a normal variant. Then we started to get concerned that some of these girls having premature adrenoid may have, in fact, later on, development of ovarian hyperandrogenism, including lipid abnormalities and insulin resistance. The risks may be increased with girls with a history of a low birth weight. This has been shown in males with respect to alteration in lipids. The issues regarding early intervention tactics and prevention measures are currently still under review. Once the diagnosis is made, follow-up usually is done every six months. Yearly fasting measures of insulin have been recommended, and glucose. Some clinicians follow adrenal androgens yearly. Most children do not demonstrate a rapid pubital progress or bone age advancement, and do well with lifestyle interventions. However, the rate of pubital changes, if it accelerates, you should reconsider further evaluations. But I want to conclude with most children will not need pharmacologic intervention, and can be managed with observation and guidance for healthy lifestyle choices. Thank you for your attention. Thank you. Thank you, Saron, for this wonderful, wonderful talk. We welcome now people to come to microphone and ask questions. Thank you. While we're waiting for people, let me take the opportunity and ask for one question. I was intrigued by this association between DHEA and CNS structure and hypothalamus in this relationship. So my question is, do you actually have assessed these children with premature adrenarche in terms of neurocognition? Or do you ever suggest going that route because of this link that you described? We were amongst the same group that reported white matter disease in the CAH group. We, at that point, attributed it to glucocorticoid therapy. I haven't gone, again, if you have a child who has a body of a nine, 10-year-old, and is five or six-year-old, it's a psychosocial issue. I'm not sure how much and what we should be further evaluating. Thank you. Buenos Aires, Argentina. Nice talk, Saron. I want to make a little comment, and I want your opinion. Because 11-ketoandrogens are produced mainly in the fasciculate, fasciculatosome. And the point is that we need to understand all the mechanism related to postnatal adrenal cortex donation, and it seems to be that early prognosis and production of 11-ketoandrogen represent that adrenal cortex donation is different in those children that they have prenatal adrenergic than those that they don't have. So I think your laboratory, thank you for that question, is very involved in zonation studies, and I think crossing over and where the hormones actually originate and their effectiveness is something certainly that we would need to look more into in this 11-hydroxysteroid group. Thanks so much. And we have one last question from online audience. What sort of intervention will you use for isolated precocious pubarchy to change the course of therapy? Is there anything I would do? I would, if it's an obese child with precocious adrenarch, I would certainly try to do effective lifestyle changes and a modification in diet. In someone who is thin, it is more difficult. I personally have not been using insulin sensitizers. Even in some instances where we have minor metabolic changes, most of the children do quite well without therapy. Okay, thank you so much, Dr. Overfield. Thank you. I'm delighted to introduce our last speaker this afternoon, Dr. Natalie Shaw. Dr. Shaw is a clinical investigator and PI of the Pediatric Neuroendocrinology Group at the National Institute of Environmental Health Sciences. Dr. Shaw's research focuses on the genetic and environmental determinants of pubertal timing with a particular focus on sleep and nutrition. She conducts reproductive physiological studies in normal girls complemented by genotypic and phenotypic studies in patients with rare syndromic forms of hypogonadism as well as functional studies in patient-derived neural stem cells. We're excited to hear her talk this afternoon. Dr. Shaw. All right, thank you very much for the opportunity to speak today. The title of my talk is The Female Reproductive Axis in the Early Postmenarchal Years. I have no disclosures. So I'm gonna start by reminding everyone about the ontogeny of the neuroreproductive axis. The gonadotropins are shown on the Y-axis and age is shown on the X-axis. And as you can see, following the mini-puberty of infancy, the axis is quiescent until it reactivates in early adolescence triggering puberty. And the question I have today is how does the body learn to go from the linear increase in LH that occurs during puberty to the regular monthly surges that characterize the menstrual cycles of a fertile woman? Or in other words, what is the major limiting factor in developing normal ovulatory cycles? I think this question is an important one because this process appears to go awry in conditions like PCOS, a major cause of infertility in women. I'd like to briefly remind everyone of the major components of a normal menstrual cycle. And I'm showing two cycles back to back to draw your attention. Is this the pointer? Yeah, okay, oh, okay. To the steady rise in FSH that occurs during the luteal follicular transition. And this rise in FSH drives the growth of follicles in the subsequent cycle. As these follicles grow, they secrete estradiol and inhibin B. Estrogen positive feedback at the brain then triggers the LH surge, culminating in ovulation and the formation of a corpus luteum. The corpus luteum secretes estradiol, progesterone, and inhibin A. Now in adult women, oops, sorry, a menstrual cycle length of 25 to 35 days is a standard marker of ovulatory cycles. A normal luteal phase length is 12 to 14 days, and a luteal phase length shorter than 10 days is defined as a short luteal phase. Now it's traditionally been taught that the hallmark of reproductive maturity is the development of estrogen positive feedback. And this is largely based on studies done in the 1960s in which large doses of estrogen were administered intramuscularly to girls ranging from pre-puberty to 10 or five breast development. And shown here are LH levels on the y-axis and time on the x-axis. And you can see that it's not until late in reproductive development that there's a robust and consistent rise in LH in response to intramuscular estrogen. But what about FSH? Well, as I've shown you, it has a very dynamic secretory profile rising nearly fourfold during the luteal follicular transition. We know that it's required for folliculogenesis, for inducing aromatase, and for inducing the expression of LH receptors on granulosa cells. So while the LH surge is important, ovulation very much requires the coordination of the LH surge with a follicle that is healthy and has reached an appropriate size, a process which largely depends on FSH. So to understand more about the developing reproductive axis, several years ago we launched the Adolescent MEN-C study. We enrolled 23 healthy early postmenarchal girls, obtained hormone measurements and transabdominal pelvic ultrasounds over the course of one and a half to two cycles. We then classified the menstrual cycles as ovulatory or anovulatory, and compared the adolescent cycles with those of historic adult controls. The 23 participants raged in age from 12.8 to 17.6 years old and were on average 1.7 years postmenarchal. About half were overweight or obese, and 70% were Caucasian. And beginning in the early to mid-follicular phase, subjects had reproductive hormones measured approximately every other day using a combination of dried blood spots, dried urine strips, and serum. They also underwent two to five transabdominal pelvic ultrasounds to monitor the growth of the dominant follicle and to document the presence of a corpus luteum. We compared these data with that of 65 historic adult controls. These were 18 to 34 year old women with regular ovulatory cycles who underwent daily blood sampling during the course of one menstrual cycle. And I should mention that the same serum assays were used in our adolescents as were used in these adults. The adults also underwent transvaginal pelvic ultrasounds approximately every other day in the week leading up to their predicted day of ovulation. We classified a adolescent cycle as ovulatory if it met the following criteria. So there had to be a mid-cycle LH surge, a mid-cycle estradiol surge, and if it met a specific progesterone cutoff. Now to determine an appropriate progesterone cutoff for adolescents, we looked at 37 of our monitored cycles with sonographic confirmation of either ovulation or anovulation. And the ovulatory cycles either showed a collapse of the dominant follicle, the presence of a corpus luteum, or free fluid in the posterior cul-de-sac which shows up as this, the black area beneath the uterus. We conducted ROC curve analysis to determine the urine pregnenol diol or serum progesterone value with the highest combined sensitivity and specificity. And these were determined to be a progesterone of 1.65 nanograms per ml and a urine pregnenol diol of 390.5 micrograms per gram creatinine. So for each cycle, we determined whether it was ovulatory or not and then looked at the length of the luteal phase and then classified it as either a normal ovulatory cycle or if the luteal phase was less than 10 days as a short ovulatory cycle. We compared our adolescent data with the adult data looking specifically at estrogen positive feedback. For example, the magnitude of the LH surge, the magnitude of the estradiol surge, and our primary index of positive feedback was the LH surge corrected for the peak estradiol. We also looked at FSH secretion. At the level of the ovary, we were interested in the dominant follicle growth rate, follicular steroid and peptide synthesis, and the function of the corpus luteum. So what did we find? Well, this table shows you the girls grouped according to their hormone pattern in the first cycle that was monitored. There were 11 girls with normal ovulatory cycles, five girls with short ovulatory cycles, and seven girls with anovulatory cycles. And what I hope you can see is that there was really no difference in these girls in any of the parameters that would be routinely available to us as clinicians. So there was no difference in BMI percentile, chronologic age, gynecologic age, or cycle length, although the cycles tended to be shorter in girls with short ovulatory cycles. When we looked at both of the monitored cycles for the girls, about 60% of girls had two ovulatory cycles, 21% had one of each, and 21% had two anovulatory cycles. So I think one of the take-home points here is that you really can't judge a girl in the early postmenarchal year by her cycle length, except perhaps at the extremes. And this is because all but one ovulatory cycle fell within the normal range for adolescents of 21 to 45 days, but this was also true for the anovulatory cycles. I'm now gonna show you some of the hormone data in these girls, and at the top is LH, then FSH, estradiol, and progesterone, and along the X-axis, the hormones are centered at day zero, which represents the day of ovulation, and then following the hatch marks, I'm showing the first week of the second menstrual cycle. So these, and the black circles and black lines represent mean plus or minus one standard error in the normal ovulatory girls, compared to the shaded backdrop of the adult women, mean plus or minus one standard D. So as you can see from these graphs, the girls had a normal rise in serum estradiol, a normal LH surge, and in fact, LH corrected for estradiol was no different than the adult women, consistent with mature estrogen-positive feedback. FSH, the second box down, was much lower across the luteal-follicular transition, as was LH, and the corpus luteum function was diminished relative to adults, there were lower estradiol and lower progesterone levels. The short ovulatory girls showed a very similar pattern, except here, the LH surge corrected for estradiol was lower than in adult women, suggestive of immature estrogen-positive feedback. At the level of the ovary, we observed that in the adult women, the dominant follicle grew at a rate of 2.2 millimeters per day. On the y-axis is dominant follicle diameter, and on the x-axis is cycle day relative to day zero, the day of ovulation. In girls, the follicles grew at a much slower rate of 1.5 millimeter per day, such that on the day of ovulation, the adults were more likely to ovulate a larger follicle of approximately 25 millimeters versus the girls who ovulated a smaller follicle of 20 millimeters. Now, the slower dominant follicle growth rate could be due to the diminished FSH stimulus. However, we also saw signs of what appeared to be intrinsic ovarian immaturity. So when we looked at estradiol levels, even after correcting for follicle size or lower FSH levels, the estradiol levels were significantly lower in adolescents than in adults. And the same was true for inhibin B. This is showing you inhibin B levels in the adults at the top, and in the adolescent girls at the bottom in the first week of cycle two. What about the anovulatory cycles? Well, these were kind of a mixed bag. So some girls showed absolutely no follicle growth. Others showed follicle growth, but with no LH surge. And in other girls, they had normal follicle growth and an LH surge, but ovulation did not occur. And instead, the follicle either regressed or we observed something called a luteinized unruptured follicle. I'll just give you one example of a representative subject. So this was a 14-year-old Caucasian girl with a gynecologic age of three years. She was obese, non-HERSUT, had normal screening labs, normal androgens, prolactin, thyroid. She reported having regular 25 to 30-day cycles. And in fact, the cycle that we monitored was 30 days. Here are her hormonal data. She had what appeared to be a normal preovulatory rise in estradiol and normal LH surge. But you can see that following the LH surge, the estradiol levels continued to climb. And the progesterone, which is the bottom right corner, increased just minimally to 1.3. So what we saw at the ovary was she had a dominant follicle of 18 millimeters six days before her LH surge. But the follicle just continued to grow. This is two days after the surge, it was 29 millimeters. And then five days after the surge, it grew to a whopping 39 millimeters before menses ensued three days later. So this is an example, again, of a luteinized unruptured follicle. And what it tells us is that in adolescents, and this has also been described in adults, that there can be an uncoupling of the intra-ovarian events triggered by the LH surge. So here we did not see follicle wall digestion and rupture, but there were signs of weak luteinization, the process whereby the granulosa cells are transformed into luteal cells. So in summary, we found that ovulatory cycles are quite common in the early postmenarchal years. About 75% of girls had at least one ovulatory cycle. However, these were not normal cycles by adult standards. We also found that acquisition of estrogen positive feedback was not synonymous with a mature GnRH secretory profile. FSH and LH levels remained much lower in adolescents, and the lower FSH levels led to a slower dominant follicle growth rate. Lastly, we found what appears to be intrinsic ovarian immaturity, such that the ovary, like the brain, must learn to ovulate. So taken together, these data suggest that it's the coordinated development of both brain and ovary that's required to achieve the mature ovulatory cycle of a fertile woman. Now, these cross-sectional data, like I've shown you, are certainly important, but what we really need to understand this developmental process is a longitudinal study, and so this motivated us to launch the Girls First Period Study in December of 2019. And this is going to allow us to determine the normal developmental trajectory from anovulatory to ovulatory cycles, and to one day be able to detect and treat girls with abnormal developmental pathways early on. The inclusion criteria for this longitudinal study, which is a two- to three-year study, consists of an age of 10 to 14 years old, within six months of menarche, a healthy weight, normal thyroid, prolactin, and androgens. We excluded girls taking oral contraceptive pills, those with a chronic medical condition, those who exercise excessively, and those with a first-degree relative with a reproductive condition. Although I should mention we're also enrolling a parallel cohort who are daughters of women with PCOS. The real meat of this study consists of these daily urine samples through dried urine strips, and this will allow us to measure creatinine-adjusted luteinizing hormone, and the main estradiol and progesterone metabolites. Additional components include a BOD pod, intensive monitoring of menstrual cycles every four to six months with blood draws and pelvic ultrasounds, vaginal and stool microbiome studies, wrist actigraphy to monitor sleep, wake, and activity, breast MRIs, and DNA collection. So we've, since December 2019, we've enrolled about 40 girls, 17 of whom have completed the first year of the study, and seven have completed the second year. And this just shows some of our preliminary data. So we used generalized estimating equations controlling for race and BMIZ score, and this is showing the predicted change in follicular phase length, luteal phase length, and total cycle length as a function of gynecologic age. And you can see that with each subsequent cycle, there was a decrease in the follicular phase length of .52 days, and a lengthening of the luteal phase by .39 days. And these two effects sort of canceled out so that we saw no change in total cycle length over time. This is a similar graph showing the hormonal data. So this shows predicted peak LH, peak estrone 3-glucuronide, and peak pregnenediol 3-glucuronide as a function of gynecologic age. And here I'm also showing adult data from a study by Dr. Janet Hall, in which about 20 women underwent daily urine sampling during the course of a menstrual cycle. And so the black lines represent the adult average plus or minus one standard deep. And I think what's interesting here is you can see in the adolescent girls shown in blue that there was really no change in peak LH, at least during the first two gynecologic years where most of our data lies. And the same was true for peak estradiol, or estrone 3-glucuronide. So those levels remained at the lower end of the adult range or below the adult range. And contrast that with the pregnenediol, in which we saw that levels increased 1.06 times with each subsequent cycle. And as expected, the probability of ovulation increased over time, with an odds of ovulation that increased 1.16 with each additional cycle. So in summary, a girl's first period study is going to be the largest study to date of intensive monitoring of reproductive hormones during the early postmenarchal years. And it's going to allow us to map normal physiology during this critical developmental stage that I would argue has not received adequate attention, and to identify pathology early on. Our preliminary findings are consistent with the cross-sectional studies, and still demonstrating this axis of maturity at the level of the brain and ovary in the first two gynecologic years. And I didn't have a chance to show some of the data, but we observed very heterogeneous cycle dynamics from normal ov, short ov, and ovulatory cycles, and even cycles with multiple LH surges. And while the probability of ovulation increases over time, it seems to do so in a stuttering fashion, with regression to anovulatory cycles being common. I want to thank and acknowledge the many people who've helped make this work possible, and happy to take any questions. Thank you. Thank you for sharing this wonderful work with us. Questions for the audience? Thank you. Veronica Marie from Chile. Very nice study, Natalie. Thank you. Congratulations. I was wondering whether you have measure, or you plan to measure some other markers of ovulatory functions, such as inhibin or MH in these girls to see whether those are better markers to predict. Yeah, that's a very good suggestion. We do have serum collected at screening, so we will be able to measure those. Hi, Tamar Jacobson from the NIH. I was wondering, in the cross-sectional study, did you look at numbers for progesterone to kind of create that cutoff for ovulation, because you expected it to be lower than adult women, rather than using kind of like the regular guidelines of three, I know they're argued, but. Yeah, yeah, we started to see a lot of these corpus lutei, and we were measuring the progesterone, and so it seemed to us that it would make more sense to use a lower cutoff in adolescent girls than what's reported in adults. Hi, Natalie. Marla Lutgen from Cornell. Really exciting work, so wonderful to see it all come together. Thank you. And to know that so much more exciting data to come. Can you tell us a little bit about the demographic of the participants? I'm particularly curious about race, ethnicity, and the distribution. Oh, okay. I don't have this at the tip of my finger. I would say we have a better representation of ethnicities in this NIH cohort than we did in Boston, and we definitely have a spectrum of body weights. Thank you. We don't have any other questions from the online. Yeah, so Natalie, in the cross-sectional studies, did you also see a correlation between time since menarche and ovulatory status? I assume you did. I don't know that we had the numbers, and we were only looking at two cycles. But yeah, I would expect that the probability of ovulation goes up over time, consistent with the longitudinal data. If there are no further questions, thank you so much for watching. Thank you.

premature adrenarche

sexual hair growth

children

adrenal gland

androgen levels

reproductive axis

postmenarchal girls

ovulatory cycles

FSH

LH

estradiol

inhibin B