Good morning, good afternoon, and good evening, everyone. It is my pleasure to deliver this lecture today, which is about the endocrinology of testicular function in childbirth, wrong or future fulfilled. So hopefully, today we will get an understanding of the testicular function in childbirth, and also of how much it is important to face the current issue of male infertility that, as we will see, is very highly prevalent worldwide. We will also get a few insights into the needs for neon matinees currently for further research, and also about how much, I mean, how we can translate current available concepts into the clinical practice. But before we delve into the focus of this lecture, I'd like to briefly introduce my references. I am an endocrinologist working at the University of Catania in Sicily, in the south of Italy, and particularly in the endocrinology unit at the Department of Clinical and Experimental Medicine, under the guidance of my mentor, Professor Calogero. So we deal with general endocrinology, we deal with diabetology, but we have a special focus and interest for the management of the male infertility, which is also the focus of our research. So from our practice, we get the sense of how widespread this issue is, but also of the importance of the endocrinological management of the infertile male, which is often counseled by urologists, or even by the gynecologists in the IVF centre, and so does not usually get the proper management. If there is little information regarding the endocrinological management of male infertility, the function, the testicular function of the child with testes is totally overlooked. And therefore, there is a huge need for, you know, educating the endocrinological community, and also to understanding how, you know, what and in which ways we can face this problem. So in today's lecture, we will focus on the endocrinological function of the testicular, of the testes in childhood. And before doing this, we will deal about the epidemiology of male infertility. So we will see how common this problem is, why this issue really matters to the endocrinologist and not to other professionals, like urologists or other professionals. We will try to figure out if this is really a huge un-matter that needs to be faced in a timely manner. And then we will go into the matter, into the heart of this lecture, by talking about the endocrinology of the child's testes, which conditions can influence precovertal testicular function, and if there are any kind of actions that the endocrinologist can take to allow a primary prevention of male infertility. So let's start with the epidemiology of infertility. The WHO defines infertility as a dysfunction of the endocrinological of the male or of the female endocrinological apparatus, characterised by the failure to achieve pregnancy after 12 months or more of consistent, unprotected sexual intercourse. But the latest report from the WHO, dated April 4, 2023, has actually some concerning data because they claim that up to 17.5% of the general population, of the average general population, so roughly one in six, suffer from infertility. The reason behind couple infertility recognises female factor infertility, male factor infertility, or what. This pie chart that you can see enlarged in these slides, basically shows the data coming from the Italian IEF centre register, and here you can find specifically the data coming from the second level techniques procedure. And so for the couples attending, doing, basically undergoing to in vitro fertilisation and intracytoplasmic sperm injections. This data actually includes 59,000 cycles and 39,000 couples. So they provide a fair, you know, picture of the epidemiology of infertility. And according to this correlation, the 44% of couples recognise female factor infertility, the 20% of couples and male factor infertility, in 18% we have both a male and a female factor infertility, while in 16% of cases there is apparently no cause, no cause, no clear etiology. And so this slide is lauded as idempotent infertility. What strikes me about this chart is something that is, you know, can be translated not only, it's not typical of only Italian centres, but is, you know, a worldwide issue that accommodates several centres all over the world. So look at how much detailed is the etiology of the female infertile. In this picture, you can get a very clear understanding of the reason for the female infertility factor. But if you look on the other hand, to the male fertility factor, you don't get any idea regarding the reason for the infertility of the male partner. And this reflects what we were seeing before, that is that the male factor, the male partner does not always get the proper counselling. The reasons behind this are basically historical, because when the IVF procedure gets started, it was believed that only a single spermatozoa was needed to achieve a pregnancy, no matter the quality of the sperm, no matter the health of the male partner. But this is not really the case. Nowadays we know that the integrity of the sperm DNA is a very important factor to achieve a viable embryo. And also, we know that the sperm is not just a carrier of the paternal DNA, it carries thousands of different other molecules, like RNA, like proteins, which are involved in the fertilisation process, in pre-implantation embryo development, in post-implantation embryo development, and even, according to some animal models, with the offspring neurological, cardiovascular, and even metabolic health. So we will not delve into this topic, because this is not the focus of our lecture today, but you can get the sense of how fascinating this field is, and how impressive and important it is. Therefore, quality of the spermatozoa is important, not only for the achievement of the pregnancy, but likely even for, you know, impacting future generation health, and therefore finding professionals that are capable of understanding the reason for mating fertility, and applying a targeted approach, and so improving the quality of the sperm is very important. Now, why does all of this matter to the endocrinologist? So, there are a couple of reasons that I would like to discuss with you today. First of all, spermatogenesis is a hormone-regulated process. We know that it is overall a big, I mean, a really complex process, which is supported by three main phases. We have the proliferation of spermatogonia. Then we have the meiotic phase, from which the spermatogonia differentiates in spermatocytes, and then in spermatids, and then we have the spermeoestrogenesis, from which the spermatids differentiate themselves into mature spermatozoa. These processes are hormone-dependent. And the first hormone that I'd like to introduce is the follicle-simulating hormone that triggers its receptors that are on sartorius cells. Sartorius cells are really fascinating cells that are capable to respond to the FSH stimulation in a differential manner based on their degree of migration. So, when they are mature, which happens in the adult testes, they produce factors that are involved in the proliferation and, to some extent, even in the differentiation of the germ cells. But when they are immature, what happens in the childhood testes, they proliferate, so the FSH stimulates their mycosis. But also, they release factors like the antemullerian hormone, the antinepinephrine hormone, which are, as we will see later, important markers of the testicular function in childhood phase. Another hormone that is important is the luteinizing hormone, which triggers the Leydig cells, induces the steroidogenesis in these cells, and so they produce testosterone. Intratubular levels of testosterone are from 25 to 125 times higher than serum concentration of testosterone. And testosterone at the intratubular level is very important to trigger that differentiation from the late spermatocyte stage to the stage of the spermatozoa. So, intratubular testosterone levels are important for spermatogenesis, and some groups use serum concentration of hydroxyprogesterone level as a kind of diagnostic marker to somehow understand the levels of intratubular testosterone. And this may represent a reasonable approach, an interesting approach, for those males that have an apparently idiopathic oligodospermia, which may be supported by a low intratubular level of testosterone. There are several other factors and molecules and, you know, hormones that influence this very, very complicated process. But what I'd like you to focus on today is the importance of growth hormone like IGF-1 and insulin. Something that is already known, well known, in the granulosa cells is that these cells express the IGF-1 receptor and that this receptor, when that's stimulated by the IGF-1, is capable of sensitizing these cells to the FSH action. These molecular findings already find clinical application. Let's think to the use of the growth hormone for some of the protocols prescribed for ovarian hyper-stimulation, for controlled ovarian hyper-stimulation for the women undergoing IVF procedure that are prescribed especially in case of FSH or responsiveness. But in recent years, this finding has been, you know, confirmed also at the level of the sertoli cells. Sertoli cells do express the insulin receptor, the IGF-1 receptor, and there is a crosstalk between the IGF-1 receptor and the FSH-dependent pathway. This explains the reason why when we culture the sertoli cells and we up the FSH, we will get an increased phosphorylation in targets like the ERK, the AKT, which are tyrosine kinase-dependent targets. This understanding is very important because hypothetically, IGF-1 serum levels, hyperinsulinemia, insulin resistance can potentially interfere with sertoli cell metabolism or with their responsiveness to FSH. Another reason why all of these fall into the competence of the endocrinologists is that the sertoli cell metabolism and spermatogenesis are glucose-dependent. We know that sertoli cell metabolism is very important to support spermatogenesis. These cells express in an insulin-dependent fashion the GLUT1 and GLUT3, which allow the glucose uptake inside the cells. Then the glucose is used for the production of lactate, and in turn, the lactate is used by the germ cells to support their proliferation, their metabolism in general. Based on this molecular evidence, it has already been shown that diabetes or insulin deregulation are capable to interfere with the finally regulated glucose-dependent sertoli cell metabolism. Thus, there is no better professional as the endocrinologist capable of understanding the diabetes-dependent dysregulation in spermatogenesis. Another reason is that several endocrinologists are able to damage spermatogenesis. We have already mentioned about diabetes, but what about obesity? We know that adipokines are capable to interfere with spermatogenesis, but indirectly, by targeting the hypothalamus and the pituitary, but even directly, by targeting the leading cells, the sertoli cells, and the spermatozoa. Not to talk about the obesity-related gut dysbiosis, which according to recent evidence seems to play a role also in spermatogenesis dysregulation. So, a lot of research is going on right now confirming and supporting the importance of gut and also semen microbiota in the normal spermatogenesis. Last, but not least, lots of medical therapies that actually are available for the management of the infertile male are hormones themselves or act in a hormone-regulated process. I'm not talking only of patients with hypogonadism, but also to those drugs that are prescribed to patients with idiopathic oligozoaspermia, like the selective estrogen receptor modulators or the aromatase inhibitors. But what I want you to focus on with this slide is that, even according to evidence, the novel anti-diabetic drugs seem to have an impact on spermatogenesis itself. In fact, major human spermatozoa do express the receptor for GLP-1, as well as the Sertoli cells, and their incubation with the GLP-1 is associated with an improvement in mitochondrial metabolism and, in general, in the production of energy and so support a better spermatility. So, human research has already demonstrated the beneficial efficacy of the GLP-1 receptor agonist for supporting the obesity-dependent abnormal spermatogenesis that we found in some patients with obesity. So, as you can see, these are topics on the border between specialities that are such risks to be overlooked. Despite this, this affects a large part of the general population. But is the management of male infertility really a huge matter and why? So, there are several reasons to think that it is important to face this matter timely and one is this, as presented in these slides. So, many of you may already be familiar with this paper and this evidence, in general, which suggests that sperm count and sperm concentration both have halved in the last 40 years, with a higher slope from 2000. So, some researchers do not believe this is true because they address this, you know, declining to different, you know, the different rules over time that came up to semen analysis that may make the semen analysis, the results of the semen analysis, comparable among each other over time. But other researchers believe that this may be true and they make a point by saying that the environmental conditions have completely and dramatically changed in the last 40 years. Let's think of the climate change, global warming and the higher exposure to the endocrine disruptors or even to the higher prevalence of obesity or diabetes, even in childhood. Therefore, the environment to which the test is exposed to during its development, either both in fetal life or in postnatal life, in early postnatal life, is totally different compared to 40 years ago and this may play a role in determining the inability of the test to acquire its functional competence in childhood to support a normal spermatogenesis. Another piece of evidence that I'd like to show today regards the important high prevalence of apparently idiopathic oligospermia in males. So these are the data from the center in Neustadt in Germany, which is a very well-known center, and this picture actually presents the data coming from 26,000 infertile males, and despite a careful workup, diagnostic workup, what the authors claim is that up to 72% of patients show, you know, apparently unexplained reason for the oligozole spermia. This is concerning. This may be exaggerated on one side, on one hand, because the prevalence of adiposity is related to, you know, the completeness of the diagnostic workup, what you are supposed to listen to, but in our practice, we can also confirm that the prevalence of adiposity is very concerning, and this needs huge research. Something that is very interesting comes from this Italian survey that was conducted on 10,000 final year male high school students aged 18 years old, so these are healthy young males of 18 years old, and of which less than 4,000 underwent physical examination, basically for the evaluation of the genitals and of testicular volume. What is really concerning is that of these 4,000 males, up to 14%, so one in seven, show a bilateral testicular hypochondria. For those of you who are not familiar with that, testicular volume is a very important marker of the capability of the testis to promote a normal spermatogenesis, so a testicular hypotrophy speaks of an oligozole spermia, which is very concerning. The somehow positive news coming from this research is that the reduction in testicular volume came from a modifiable lifestyle behaviour, and therefore this makes for us some room to approach these guys and to avoid their exposure to harm factors. In this study, for example, alcohol intake, cigarette smoking, use of drugs, low physical activity were considered as predictors of testicular hypotrophy, and so cancelling our patients can represent an initial good strategy to avoid the exposure of our children or adolescents to this risky lifestyle and avoid testicular hypochondria. Last, but not least, there is the so-called Eisenberg hypothesis. Basically, there is a good amount of research outlining that the main infertility seems to be associated with a poor general health, and there is evidence suggesting that people with oligozole spermia show higher odds for hospitalisation later in life, but also for the development of cardiovascular diseases or of metabolic diseases, and it is very interesting. This research, in particular, shows the link between infertility and the risk of developing cancer later on. Therefore, approaching infertility is something that is not confined to the reproductive health of the individual, but that may be very important also for the general health of the subject. So, now that we have a sense of the epidemiology of infertility and the importance for the role of the endocrinologist in this, and why all of this is really urgent and needs to be faced, let's delve into the heart of the matter by discussing about the endocrinology of the childhood testis. So, the adult and the childhood testis basically are made of the same compartments. In both testis, indeed, we can find the sertoli cells, the germ cells, and the lytic cells, but these compartments vary over time qualitatively and quantitatively. So, for example, the sertoli cells in the adult testis made only the minority of the volume of the testis, and also after FSH stimulation. Basically, they are not able to proliferate, but release factors capable to support the spermatogenesis. The sertoli cells in the childhood testis are totally different. First of all, the testis in childhood is made mainly of sertoli cells. Second, the sertoli cells in childhood are completely immature. This means that they are capable of proliferation, and following FSH stimulation, they are able to release hormones. The first is the antemullerian hormone, whose levels, as we will see, represent an important marker of the vesicular function in pre-puberty. But something that is interesting about these cells is that they are unable, they do not express the androgen receptor until the one year of age. This is important because this makes these cells insensitive to their intratesticular levels, the increase in the intratesticular levels of testosterone during fetal life and even during mini-puberty. Testosterone, in turn, is able to allow for the switch of the sertoli, of the immature sertoli cells, from the immature stage to the mature stage. So, this is the reason why during mini-puberty they are unable to support spermatogenesis. They do not mature, do not sense the testosterone, do not have the receptor for the androgens, and therefore they are not exposed, basically, they are not respondent to testosterone levels of mini-puberty. They only are sensitive to FSH in this stage, and so during the mini-puberty, when the levels of FSH are high, they proliferate. After mini-puberty, and particularly during the time span that range from mini-puberty to puberty, the vestibular volume still increases from, let's say, 0.5 to 3 ml, and this increase is supported by the sertoli cell proliferation, but this is an FSH-independent sertoli cell proliferation, since the ASPG axis is quiescent, and so it is likely supported by other hormones that we will see later on. During puberty, what happens is that the levels of FSH increase, and so the sertoli cells proliferate again, but after even the LH levels increases, this in turn associates with an increase of infratubular levels of testosterone, and now the sertoli cells do express the androgen receptor, and therefore they switch from an immature state to a mature state, losing the capability to proliferate and acquiring the capability to support the spermatogenesis. Now, the testis will be capable of increasing its volume, mainly based on the start of spermatogenesis, so now the volume increases with a really big extent due to the increase in the volume of the tubers where the spermatogenesis takes place. Why is that so important? Because the sertoli cells are capable of supporting the differentiation of 18 to 20 gene cells. Therefore, all the factors that are capable during fetal life, during minipuberty, or after minipuberty, from minipuberty to puberty, to affect the rate of proliferation of the immature sertoli cells will result in a depletion of the compartment of immature sertoli cells. Therefore, reduction in the number of mature sertoli cells in the adult testis will result in a reduction in the number of germ cells that will be supported, and therefore in lower sperm alveols. So, this will determine an apparently idiopathic, irreversible oligodospermia, something that we define idiopathic, I mean, because it's always diagnosed in the adult stage, but that, I mean, idiopathic, it's not really idiopathic because it is supported by these mechanisms. Now, let's try to, you know, review which are the main hormones that are physiologically involved in the process that we described. The first hormone is the follicle-simulating hormone. So, this is a really landmark trial that has been conducted on 13 patients with congenital hypogonadotropic hypogonadus, and the aim of this study is to try to figure out which can be the best therapeutic approach to induce the puberty in these patients. So, these 13 recuperative patients in this study were randomized into Group 1, which was subjected to pre-treatment with recombinant FSH for four months, and then with GnRH therapy for two years. Group 2, indeed, was subjected directly to GnRH therapy for two years. What is nice of this study is that the authors provide histological data before and after treatment, so we can get information regarding the effect histologically, so inside the testes, of these hormones. Compared to baseline, you can see that the treatment with FSH resulted in an increase in the number of the immature sertori cells. So, there was an expansion of the pool of immature sertori cells, while treatment with GnRH by resulting in an increase in infratubular testosterone concentration associates with the acquisition of the maturity by the sertori cells. The increase in the amount of the, you know, the expansion of the immature sertori cell pool in turn associates, I mean, explains the higher inibin B in Group 1 compared to Group 2, since inibin B is produced by sertori cells. The higher testicular volume at time zero, when the groups receive the GnRH, and importantly, this higher volume is supported only by the immature sertori cells, and of course, an increase in the number of immature sertori cells will result in an increase in the number of the squamatozoa that the patients in Group 1 will be able to produce. This knowledge has been classically being used to support the efficacy of FSH over ACG or testosterone for puberty induction protocols, but in general, this data gives us the sense of the importance of FSH to allow a proliferation of immature sertori cells. This knowledge is very, very actual, and just one month ago, this review from Professor Howard's group has been published in Endocrine Reviews, and in this review, the authors suggest the use of gonadotropins, and therefore of FSH also, for the treatment of micropenis and the understanding testis. The rationale would be that 50% of these patients with micropenis or understanding testis do not experience micro puberty, and therefore, the sertori cells are not exposed to the physiological FSH increase that all the children experience in that phase, and so they do not proliferate. Therefore, treatment with gonadotropins would allow for an exposure to EFSH, and so forth, in increasing the vestibular growth, and particularly of the expansion of the immature growth, and they suggest the GnRVH approach over testosterone, ACG, or tetrotestosterone, which are the conventionally used treatment that are good to promote the phenyl growth or the adhesion of the testis, but do a bad job overall in the testis, because the testis decreases its volume, and this therapy does not allow for a proliferation of the sertori cells. So, another hormone that I'd like to discuss today is the role, the effect, of the insulin ligrofactor on testicular physiology during the childhood phase, and the reason why I'd like to discuss this hormone with you is that, as I was saying earlier, in the time span ranging from mini-puberty to puberty, the testicular volume increases, and this is supported by an expansion, a proliferation of the immature sertoid cells, which is, in this case, FSH-independent, because the ACG axis is squashed. So, it has been suggested that the increase, the levels, of the serum levels of IGF-1 may play a role in that, since they are high in childhood, and to support, of course, the growth of the children, and also the increase in peripubertal phase, reaching even acromegaloid levels, overlapping, levels overlapping those of the patients suffering from acromegaly, and so some authors suppose that this hormone may play a role in general, in the pubertal process. This is true, and I will not even, I mean, this topic, but IGF-1 receptors are present in GnRH neurons, and here IGF-1 support the migration of this neuron, but also the separation of GnRH, but what I want you to focus on is the importance, the direct importance of IGF-1, direct effect of IGF-1 on the gonads, and particularly on testicular function during the fetal life, and during the peripubertal life. So, I want to show this study that was published in Nature on 2003, and basically, in this study, the authors used triple knockout mice models, where they induced a constitutive knockout for the insulin receptor gene, the insulin-related receptor, and the IGF-1 receptor gene. What they did was to evaluate the degree of differentiation of the fetal gonad into testis or ovary, respectively in male and female mice. So, let's start with the female mice. These are the female mice with the triple knockout, and as you can see, the gonad is positive for the staining or ovarian-specific markers, like it happens in the female white-type mice, like FIG-alpha and BUNP-4. But if we look at the male testis of the triple knockout mice, what happens is that the gonad is negative for the staining for the testis-specific markers, like INS3 or SOX9, which are present in the testis of the white-type animal, of the male white-type animal. So, the authors conclude by saying that the knockout for these receptors basically interferes with the capability of the undifferentiated gonad to differentiate itself into testis, which is a process that is certainly self-dependent. To further try to understand the impact of these mutations in the adult life, I mean, that model could not be used because it's not compatible with life, and so the author created some models using the selective knockout for these receptors, specifically at the germ cells level or at the sertoli cells level. Basically, they induced a knockout for the IGF1 receptor, or for the insulin receptor, or for both. In this work, they demonstrated that the knockout, the single and the double knockout, at the level of germ cells is dispensable for spermatogenesis. So, there was any effect on spermatogenesis, meaning that the expression of these genes in the germ cells do not interfere with the normal development of the testis. But the opposite was found for the constitutive knockout at the level of the sertoli cells. Particularly here, you can see the volume and the morphology of the testis at the 180 postnatal day. So, these are adult mice and you can see here the testis of the wild-type mouse, the testis of the mouse with the selective knockout for insulin receptor at the level of the sertoli cell. Here is the testis of the mouse with the knockout selectively at the sercels for the insulin from the IGF-1 receptor. And here is the double, the effect of the double knockout, so IGF-1 receptor gene, insulin receptor gene on sertoli cell selectively. And so, you can see that there was a 70% degree of reduction compared in the double knockout experiment compared to the wild-type. If we look at the panel G, you can see also the impact of the knockout on the sperm out. And basically, you can see that the single knockout for the IGF-1 receptor impaired the sperm count. And even more dramatic was the impact on the sperm count in the double knockout moment. So, what the authors conclude with this experimental design is that, histologically, they could see that there wasn't a normal expansion of the immature sertoli cell group and that these receptors basically are involved in the process of sertoli cell proliferation in fetal life and early postnatal life. And so, the inability of sertoli cell to proliferate due to the absence of this receptor basically led to an inefficient and insufficient sertoli cell compartment in the adult asses, which explains the low volume and also the poor sperm count. So, the IGF-1 serum levels can play a role in the proliferation of the sertoli cell in the prepubertal phases and studies in the human needs to be carried out to elucidate this very interesting line of research. Before moving forward, I'd like to briefly talk about the antemullarian hormone. So, the function of the antemullarian hormone, as we all know, is to promote the regression of the mullerian ducts. So, the antemullarian hormone levels, which are high in the fetal life of the male fetus, basically allow for a normal differentiation of the male sexual apparatus. But this, so the role of antemullarian hormone has been so far confined only to prenatal life. But this does not explain the reason why the levels of antemullarian hormone are still high in males before puberty and during childhood phase. So, receptors for antemullarian hormone more recently have been found in GnRH neurons. And this has been reported in mice, but also in humans. And in vitro research indicates that incubation of antemullarian hormone of immature GnRH receptor, sorry, immature GnRH neurons with antemullarian hormone, leads to stimulation of the migration of these neurons, while the incubation of mature GnRH neurons with antemullarian hormone leads to increase in the production of GnRH itself. Also, there is evidence supporting that the intraventricular injection of antemullarian hormone in mice allow for the acquiring of GnRH neurons. And finally, in humans, severe mutation of antemullarian hormone gene and of its receptors have been found in patients with congenital hypogonadal hypogammas. We have no time to focus on this kind of research, but you can find this in the supporting references at the end of this presentation. But all of this points towards a role of the sertolian cell secreted antemullarian hormone in Fab13 induction. So, a better understanding of the physiology of vesicular function in childbirth could allow even to rewrite or understand better some chapters of the endocrinology, I mean some endocrinological chapters, like some of the facts of disorders of the onset of puberty. What I want you to consider here is that the amount of antemullarian hormone that you can measure in pre-pubertal life reflects the capability, the functional competence of the sertor cells. Basically, their capability to proliferate, their number in the testis, and their function. Therefore, lower number of antemullarian hormone will speak of a dysfunction of sertoli cells, of the immature sertoli cells. And this is the reason why antemullarian hormone can represent a very important and precious marker to heavily detect a sertoli cell dysfunction in pre-pubertal phase. This is why we can detect a vesicular dysfunction, you know, before even the puberty starts. Here you can find some reference ranges for antemullarian hormone during postnatal life, of course, during the first two years of life. And then you can see that the reference rates are stratified according to the Tanner stage. This reference range needs to be validated, you know, on large chord size of different races, but this represents a good starting point to orient our clinical activity. Now, what are the conditions that are capable to influence pre-pubertal vesicular function? So, from what we have said so far, we got that the sertoli cells express the insulin receptor, the IGF-1 receptor, and that so they are capable to, you know, they can be influenced by the serum levels of IGF-1, of insulin, and these levels of the presence of insulin resistance may even influence the capability of cells to respond to FSH simulation, which is very interesting and represents the, you know, the first reason to study, to investigate the impact of metabolic disorders on vesicular function in childhood and in adolescence. Another reason for which this line of research has been, is being studied actually, is that the downward trend for the sperm counts that we discussed about earlier, associated with a parallel increase of childhood diabetes and childhood obesity. And therefore, this higher prevalence of obesity in childhood, compared to 40 years ago, may represent something that may support, you know, the downward trend of sperm count. Of course, this is a hypothesis, but there is some research that is trying to see if there is, you know, an association, link between these two factors. So, in this study, what the authors did was to enroll, basically, patients from the RAIN court, which is an Australian court assessed longitudinally, and from the birth, basically, to now. And these patients at 17 years old, received a complete assessment, a complete metabolic assessment, which allowed the court to be divided into those with and without NASH at 17 years old, with and without insulin resistance. At 20 years old, the same authors evaluated these patients for the vesicular volume with ultrasound and the semen analysis. What they found is that at 20 years old, patients with NASH showed a 50% reduction in total sperm count, compared to those without NASH, and that also at 20 years of age, those with insulin resistance had a 20% reduction in vesicular volume, compared with those without insulin resistance. And so, the authors conclude, suggesting that the presence of insulin resistance, for example, can interfere with, you know, physiological proliferation, FSH-induced proliferation of certain cells during perituberty, and this may, you know, determine a reduction of the vesicular volume and of its competence to support spermatogenesis. There is very few line, you know, few papers investigating the impact of obesity in childhood on vesicular function. So, we tried to answer this question with a cross-sectional study designed involving 268 main children, and particularly, we stratified their vesicular volume based on their BMI. And as you can see here, in the time range from 9 to 14 years old, we found that those with normal BMI show higher vesicular volume compared with those with overweight, obesity, and severe obesity. This needs to be confirmed, but also with longitudinal study designs assessing whether these children have any difference in their sperm outputs during the adult age, and this is going on. Another factor that is important to consider is the exposure to endocrine-disrupting chemicals. We know that fetal exposure is associated with hyposcadia, cryptarchidism, but in the long term, it is also associated with infertility. There is a lot of research incubating mature sperm with endocrine-disrupting chemicals and proving the negative harmful effect on sperm function, but there is less research conducted on cervical cells. This is one of the two papers, basically, on this topic. Here, adult mice were exposed to a mixture of EDC in a dose-dependent manner, and basically, the mixture of EDC was realized to mimic the exposure to the several EDC to which adults are exposed nowadays, humans are exposed nowadays, and they found something interesting because the exposure to EDC basically interfered with the metabolism of certain cells which did not, I mean, became incapable of secreting lactate and thus to support the germ cell proliferation and metabolism in general, and this explains the lower total sperm count of these mice. This study is interesting because it supports the negative impact of EDC on certain cells, but this study on certain cell metabolism, but this study is on mature certain cells, but allowed to suppose that exposure to EDC may even impact the metabolism of immature certain cells, and this is something that needs to be studied. This other study, finally, has been realized on porcine prepubertal cervical cells, which is an interesting model because it's prepubertal, so these are immature cervical cells, and not porcine, so they have a really, you know, they are very similar to the human cervical cells, and the authors incubated these cells to nicotine, and they found that nicotine was able to decrease that cervical cell competence, basically, so the concentration of EDC that these cells were able to secrete was lower, as well as anti-mullerian hormones. After nicotine exposure, also their capability to, you know, make their blood vessels fire, we know that certain cells play a role in making their blood vessels fire, so their capability in that was reduced, and also the rate of apoptosis of these certain cells was increased. Therefore, this represents some evidence that may allow us to think that maybe exposure to fascism in children can interfere with that, can have a negative impact on the proliferation of immature certain cells, and this is aligned with the survey that we have discussed earlier, where cigarette smoke was one of the factors that was able to induce that hypertrophy in those 18-year-old mates. So, from the discussion that we had today, we get the sense that there is a lot going on, but also that a lot of research still needs to be done to translate many of the evidence, that there is much of the evidence in the animal model, to the human. In the meantime, in the meantime, is there anything that the endocrinologist can do in his clinical practice to prevent the onset of male hypertrophy? So, there are some very easy steps that the endocrinologist can take. The first is measurement of the vesicular volume over time in children, in adolescents, and in the adults, of course. We all use percentiles for the measurement of the weight of the children, of the height of the children, of the road velocity, etc., but using percentiles for the assessment of the vesicular volume and to see if the vesicular volume is growing poorly is something that very easily can allow us to understand if there is a low rate of proliferation of immature cells in these children. One can question if this should be done using the bureaucrasome, which would be a burden on the economy of the patients of our health system, but this has been already investigated by this group from the Nationwide Childhood Hospital, the Columbus University in Ohio, where they found a good degree of overlap between the bureaucrasome-measured vesicular volume and those measured at the physical examination. They basically used a ruler for the measurement of the wing of the testis and scrotum and used it to, with the aid of a specific formula, calculate the volume of the testis. So, this is something that can very easily be incorporated in our clinical practice. Secondly, we can ask for a semen analysis to our adolescents. So, during adolescence, the girls are used to being counseled by the gynecologist, but this practice is not done by the males, which often become aware of the impurity when they are adults and in the IVF centres where they get the first semen analysis. One can ask what is the right timing to ask for a semen analysis and this group actually conducted this research on 50 healthy subjects and they demonstrated that at 16 years old, more than 94% of adolescents already had their first ejaculation and already masturbated. So, this is the right timing to ask for a first semen analysis and so get an early understanding of what their testicular function is. Finally, there are several risk factors or risky lifestyles that can be easily identified. Microphenies and cryptorchidism are very interesting, very important because they, in 50% of the case, highlight an absence of mini-puberty and even hypogonadotropic hypogonadism and I suggest you to refer to Howard's paper for getting a clearer understanding of the path that we can take to make a diagnosis in these patients. But also, we can counsel the patients to identify and avoid them to expose themselves to passive or active cigarette smoking, use of drugs, alcohol intake, try to fight in the presence of overweight, obesity. This is important also so that the lack of use of barrier contraception because this determines a high frequency of urogenital infections that have a detrimental effect on sperm quality in general, and also try to avoid or reduce, take steps to reduce the EDC exposure. So, with this link or this QR code, you can find a link to the complete reference list that is associated to this lecture. You will find the references that we discussed of and those that I don't have the time to discuss today. And this is all of what I have. I thank you so much for your attention and I'm ready to take any questions if you have time now or feel free to reach me out. Thank you so much. Yes, hi, thank you so much for your talk. If you'd like to begin the Q and a, you can start with the 1st question in the box. Sure. So, let's see. So, the 1st question is, it is known that the use of clomiphene citrate in idiopathic mating fertility can lead to an increase in testosterone, and at the same time, an increase in estradiol. In such cases, is it worth using aromatase inhibitor? What is your aptitude to use aromatase inhibitor in men, despite the fact that this is an off-level therapy? Okay. So, basically, I suppose that who is asking this question is talking about adult, because of course the scenario is completely different. If we are talking of a mature, I mean of pre-pubertal testis or post-pubertal testis. So, in the scenario of a testis that is made of mature, certain cells, so the adult testis, certainly clomiphene citrate is used. We have written a meta-analysis on the use of CIRM and aromatase inhibitors, and they represent one of the strategies that can be used in patients with idiopathic corticosteroids. And in general, I mean, their off-level use depends on the country, basically. In the US, for example, clomiphene citrate is very, very, very often used by the urologist in case, for example, of patients with obesity, and it is useful. But my personal point of view is that what we need to do is to do a careful diagnostic workup. And, for example, if the problem is obesity, the patient should not be offered with CIRM and that's it, but we should work on its obesity, because this is the problem of the poor spermatogenesis. And giving him some support in therapy too, but we should, as endocrinologists, work on the heart of the problem and try to solve it. Second question, do you envision that in the near future, unexplained infertility may become explained by using genomic analysis? Thanks for this question. This is very, very interesting, because as I think you are referring to, NGS approach and WESS approach is becoming more and more used in research papers that are aimed at understanding the monogenic form of spermatogenesis. And there is a lot of, you know, interesting research on this. For example, a lot of genetic mutations are being found in patients with azoospermia or with severe oligozoospermia. And, of course, the odds for finding a genetic cause is higher, the higher is the confirmation of the testis. But say you have, in this mutation, not always impact on the testicular volume, because it depends on the stage of spermatogenesis they affect. If the mutation is post-meiotic, you will find the testis with a meiotic arrest at the histology, but the volume is not affected. If the mutation is a result in a certainly cellular syndrome, the volume is affected. Why am I saying that? Because for a problem that we think may be referred to the focus of this talk, so insufficient proliferation of the major compartment in childhood, we would expect a lower testicular volume. So, it's important to consider that to make a differential diagnosis. But for sure, genetics is very, very important for understanding the diagnosis, and you need also to consider something. When you do NGS and when you find a mutation, you know, it's not black or white. And the results of some mutation, the effect of specific mutation on the gene function may be partial. And so how to interpret that? If the gene is the function of a gene that is known to play a role in spermatogenesis is dramatically affected, we can say, okay, it's genetic. But if there is something, you know, it's impaired mildly, you can think that maybe the patient has that mild mutation, and maybe there is also a concomitant environmental factor, obesity, exposure to EDC, smoking, and so the combination of the two can lead to the phenotype. So this is a very amazing field that needs, I mean, requires our investigation and studies, but sure, genetics will for sure play a role into that. So, the other question is, thank you. What is your attitude toward the use of FSH in the therapy of idiopathic corticosteroid autospermia? So thanks for this question because, I mean, general attitude is very, very different about that. As you said, as you heard, I come from Italy, and in general in Italy there is a great attention toward the use of FSH for idiopathic corticosteroid autospermia, and personally I believe that is very interesting. I mean, its use is useful, but that Italian rules allow us to use the FSH only for really restricted timing for four months, which is not, you know, does not allow us to see an effect because, you know, spermatogenetic cycle lasts only for two months and a half. And so it is important to, you know, have a prolonged exposure, but we are not supported by the Italian law, and so this is restricted research. I can see that in the States FSH is not used at all. I mean, most of centres do not use it, mainly for economic issues, but there is a multi-centre trial from Europe and US going on on the use of FSH, which hopefully will, you know, allow us to get more insights about the efficacy of FSH for the treatment of idiopathic oligospermia. So, next question. Sorry. So, in the presence of hypogonadism and non-reproductive plants, should testosterone replacement therapy be combined with the use of ACG, gonadotropin? What regimen do you usually use in practice in men who are indicated for testosterone replacement therapy and who have non-reproductive plants in the near future? Next question. So, basically, so we are talking of major hypogonadism, because, you know, there are several kinds of hypogonadism. There is the so-called decompensated or metabolic or functional. Okay, the latest way of referring to it is functional, for example, those that have obesity and have borderline low testosterone. The intratubular testosterone levels are low and so spermatogenesis is affected. But, you know, we can give them short-acting testosterone to help their spermatogenesis, but the main approach should be on the weight. For those that have hypogonadism and that do not, of course, desire fertility, testosterone is a proper approach. Also, because if you use ACG, you expose him to, you know, the compliance is lower. With testosterone, you can, you know, safely manage the suspect. So, thanks for, I'm trying not to miss messages. Sorry if I'm doing. So, thanks for the message. If in childhood, there was surgical treatment of correct orchidism, in adult or adulthood oligodospermia, is it possible to use CIRM FSH therapy? If the state is normal gonadotropic, what do you think about perspective on this therapy? Okay. So, thanks for this question. Correct orchidism so far is a black hole because it is managed mainly by urologists right now. And as I was saying in this very, very interesting paper from Howard's group, the, you know, the treatment is most of patients get testosterone or ACG and then surgery. And so, most likely, they can have, in the adulthood, low testicular volume and oligodospermia. In this scenario, I mean, no matter, there is whatever therapy you prescribe, you cannot change the histology of the testis because the certain cells cannot maturate anymore. So, you can try with selective estrogen receptor regulators, you can try with FSH therapy. So, if the patient is normal gonadotropic. So, testicular volume can indicate reduced sperm count. Does it also correlate with the quality of the spermatogenesis? Yes, yes. So, in general, a low testicular volume in adulthood is associated with a poor process, a poor spermatogenesis, both qualitative and quantitative. Thank you. Will high IGF-1 levels in growth hormone treatment be responsible for the early onset of tuberculosis? Okay. So, generally, the patients that are exposed to GH treatment are GHT deficient patients. We have an expertise on this. We have published on that because GH deficient people represent a great model to see the effect of low IGF-1 levels and the effect of replacement on testicular volume growth and their efficacy in supporting spermatogenesis. So, it is a great question. In general, GH deficiency is a great model, but usually the GHT people have a delay in puberty. So, it's really rare to see precocious puberty in disguise. Usually, tract treatment normalizes the timing of puberty on itself. So, let's…oh, you want to…I showed the square, the QR code. I think you were referring to that. Just one sec. I think you're on the question, what is the influence of cannabis on testicular development and function during fetal life, infancy, puberty, and adulthood? Yeah, okay. So, basically, there is…I mean, in the evidence I showed, it seems that exposure to drugs in childhood and in adolescence associates with lower testicular volume. In vitro, there is an effect on exposure to TAC on mature spermatobzoa, but I'm not aware of any study that so far has evaluated the effect of exposure to TSHC in mature or mature tertiary cells. But, it's reasonable to think that if the exposure to drug has been associated with a lower testicular hypotrophy than the survey has shown, it's reasonable to think that there is an effect, a negative impact, basically, on immature tertiary cell function and competence, and this is a field that needs to be researched. Do you have a protocol for ACG and FSH administration for mini puberty? So, we don't see, actually, right now, patients with…I mean, chiropractors in our university, in the current standing of the field, basically. I mean, they are counselled by the urologist. What can be done is to apply to…in the Hogwarts paper, basically, there was a call to action. So, there was a call to register to an online, basically, multi-centre register where one can, you know, upload all the data on their patients with chiropractors and see, you know, if the approach with ACG testosterone with gonadotropins can influence the testicular volume. We, I mean, not yet prescribed gonadotropins because this also needs, you know, approval by IRB, but of course, we are willing to go in that path, because this would be the right path for good septory cell maturation. There are only less than 10 case reports on that, if you see our paper, and this is really actual and very interesting. Progesterone is commonly used in early pregnancy in obstetric clinics. What could be the effects on new fetus on aspect of your presentation? This is also a great question. So, basically, it needs to be responded by, you know, culturing septal cells and see the effect of progesterone, but I'm not aware of studies on that. Because, you know, in fact, the function of childbirth test is, as we know, is something that is not currently, you know, it's overlooked. So, going into this fascinating field requires also to understand treatment at which female, you know, mothers are exposed to during their, you know, their pregnancy. So this is for sure, needs to be researched. What is the cutoff value of PNPB to differentiate hypogonadism versus constitutional delay that you use in your clinical practice? Okay, so there is some research. I mean, there is a lot actually of research. So, basically, we use a cutoff of 150, which is a good cutoff. Lower than that, you can suppose that there is hypogonadism. Higher, you can think that there is, you know, delay in childbirth. So, my question regarding treating main patients with hypogonadic testosterone of 5 and normal other pituitary function. His BMI is 38. Patients' concerns regarding, maybe for clinical cases, you can just email me. It will be maybe easier to, you know, discuss and right now I will, you know, follow up just the brief questions if you don't mind, just for time reasons. So, what is known about the mechanism of XX epsilon genome effect on spermatogenesis? Okay, so basically, what is known about the, and I think you are referring to the childhood aspects of that, and this is interesting. So, what it seems is that there is apoptosis of the tertiary cells in a clinifelter, histologically, and that the pool of germ cells is preserved until a specific time. After that, it, I mean, it undergoes apoptosis. Of course, in the adult clinifelter patients, what you will find more likely in the adult stage is a serotonin cell-only syndrome picture. There is a half percentage of, you know, odds in sperm retrieval in these patients. But what is interesting about this field is that, like the timing, when there is this general, you know, disruption in histology, which can represent interesting knowledge, also to get the sense of when we can intervene. When should we retrieve the germ cells? Because, you know, there is research going on, I'm referring to Howard's research about in vitreous spermatogenesis. So, we would be able to retrieve the germ cells of these children, because in children in the pre-ubertal age, they still have germ cells, then we can, you know, work with in vitreous spermatogenesis and make a sperm. And this is something which people are working on. What is, you know, difficult is that clinifelter syndrome is diagnosed only lately. In children, there are poor signs of, you know, clinifelter is always, is usually missed. And this is what makes this kind of research difficult. What is the optimal treatment of an adolescent with hypogonadotropic hypogonadism? Okay, so this is interesting, because there is an open debate about the use of gonadotropins over the use of testosterone. The best would be to replace the normal physiological process. So, usually what we have, we have an increase in FSH, and after, physiologically, there is an increase in NIH. So, the best would be to start with the FSH, and after that, to add also the NIH. This would be important to support properly the circulatory cell compartment, because otherwise, if we start with NIH, or we prescribe the gonadotropins simultaneously, what will happen is that we induce an increase in the intratubular testosterone, sorry, an increase in intratubular testosterone concentration. And so, the circulatory cell, which still need to proliferate, switch in a mature state, lose the ability to proliferate. And so, it, you know, the patient will experience an oligospermia. So, in adolescence with an hypo, I would first prescribe FSH, then I will add ELH until the volume of the test is, and the sperm output is fine. After that, I can propose the option of sperm cryopreservation, and then just, you know, just that I have option B, and then I can prescribe them testosterone, because anyway, the compliance is better, and we avoid many injections. But at least we have a good, you know, we have preserved the physiological development of both compartments, of all the compartments in the testis. So, there are cases where the use of chromyphancy trait led to development of a zoosperm. What did you think? So, you need to consider that when you think about the effect of a treatment in general, you need to consider all the picture. So, majority of the evidence suggests a positive effect. There may be some cases, because we always talk of the chances of success, but we, you know, don't have, cannot ensure that maybe in that patient there is something that causes a reaction to the drug, or, you know, you think about the polymorphism, a lot of stuff. But this is not the rule. I mean, usually, some do not cause a zoosperm. Thoughts on sperm quality and certainly cell maturation. When patients are given GnRH agonist at different age or turner stages, any insight on fertility lifespan? What question do we still need to answer the use of this drug in fertility for PNG patients? Okay, so, GnRH determines an increase in both the gonadotropins, and it is important in general to replace the physiological process. So, we should, as much as possible, replace the physiology of the testis, and so provide the right amount of FSH and then the right amount of LH. So, what can be done is pre-treatment, as we saw in the nice paper with FSH, and then expose these patients to GnRH. In what cases were we using estradiol in men? Okay, so, estradiol generally is a result of obesity, and we don't always look at that as a target. We don't, I mean, we mainly consider as a target the gonadotropins and the estrogen also in the testosterone levels. Estrogen, of course, is important. Estrogenism, of course, gives us a sense of, you know, how the living cells are working, but they are not our main, you know, main marker when we cancel a patient. We try to have a good sense of the metabolic background of the patients and work on that, and then look at the general pictures. So, would you think there will be a place for GH and IGF-1 as part of the treatment? Really great question. So, we have a really active interest for this aspect. But right now, I mean, this requires early approval, because how can you justify the treatment with GH in a patient that do not suffer from GHT? So, practically, a study design on that is difficult, but I would absolutely support a design like that, because it may be very useful. We, as I said before, are working on the GHT model to see the effect of treatment and the absence or low levels of IGF-1 on testicular development. But for sure, I mean, I think replacing IGF-1 levels, seeing in general how the blood levels of IGF-1 is important, and looking at those patients with underlying low levels under a different perspective. So, not GHT, you know, patients without classic signs of GHT, but with underlying low levels of IGF-1 is interesting. So, for transgender patients, when posing puberty, how do we maintain gamete function without causing dysphoria when other hormone production can cause? How do you counsel patients? This is a very difficult question. What steps can be taken? Okay, so there is research to try to maintain the fertility of these patients, basically by cryopreserving their tissue before they undergo therapy. Because, I mean, there is a lot, really a lot of evidence that in vitro spermatogenesis can become, you know, can be applicable in this kind of clinical scenario. So, on a therapeutic point of view, I mean, the reason now, because if they want to change their gender, you cannot preserve the functionality of the gonads, but you can cryopreserve it and see what research will allow us to do when the patients will be actively looking for fertility. For hypo-hypo, how is FSH administered and how often it is given? I've never done it to patients. Okay, so if you are looking for inducing a pregnancy, you can start with 75 units, three times a week. And you can do this for three months. And then you look at the vesicular volume of the patient. Then you can add ACG with 500 units, two times a week. And then you progressively increase the dosage of blood until you reach the full dosage of 150 FSH, three times a week, and of 2000 units of ACG, one or two times a week. It depends on how the patient responds in terms of the vesicular volume, in terms of sperm output, in terms of androgenization. I think we are done. I really thank you so much for your questions, your attention. I hope the connection was good. And for any kind of question, please reach out to my email address. I would love to chat more about that. Thank you so much. Dr. Canarella, before you leave, can you put the QR code back on the screen? Can you see that? Yes. Thank you so much. We'll just leave this there for a couple of moments. I really appreciate your talk and taking all the time to answer the questions. You did an excellent job. Okay, I'm going to close out the webinar now. This talk will be available in the Center for Learning in about a few days. Maybe by next week, we'll have it available and posted in the Center for Learning along with this reference code. Thank you again so much for your presentation. Thank you all for your attendance and have a wonderful day. Bye. Thank you so much. Bye-bye.

testicular function

childhood

male infertility

prevalence

research

endocrinology

hormones

spermatogenesis

early detection

gonadotropin therapy

genetics

reproductive health