ASRM CELL Module 1: Introduction to Male Reproductive Physiology and Endocrinology

Lecture 3 - The Male Factor in Reproductive Success (PDF) →

Lecture 4 - Infertility Diagnosis in Men (PDF) →

So it is my huge pleasure to introduce Dr. Dolores Lamb to you. She's an internationally recognized leader in andrology, male reproductive biology, and developmental biology.

Dr. Lamb has held faculty appointments at several leading academic institutions, including Baylor College of Medicine, Cornell University, and she currently serves as deputy director of the Children's Mercy Research Institute in Kansas City and a professor of urologic surgery and integrative molecular biology and physiology at the University of Kansas Medical Center and I think the Cancer Center as well. So Dr. Lamb's work actually has shaped our understanding of spermatogenesis and male infertility. She is one of the original greats.

She influenced national and international research priorities and trained generations of clinicians, clinician scientists, myself included, and we are extremely honored to welcome her today to our Cell School. And now starting with Joseph and then Leandro and Andrea, please introduce yourselves to Dr. Lamb and we'll go ahead and get started. All right, my name is Joseph Bird.

I'm currently practicing as a fertility specialist in the Chattanooga, Tennessee area, looking to get a little bit more technical experience in the clinical lab science arena, ultimately hoping to become HCLD certified eventually. Oh excellent, I chair the board too. Perfect.

Yeah, nice to meet you, Joseph, and I will tell you that I loved visiting Chattanooga years ago when I went through there. It's a great place. Okay, Leandro, you are muted.

There you go. Nice to meet you, Dr. Lamb. I'm an embryologist here in Florida with about 13 years of experience.

Ten of those are from the animal side and then I did my transition to the human side so and I'm here to learn as much as possible from you guys. It's a pleasure. Excellent, excellent.

What part of Florida? I'm in southwest Florida in Naples. Gotcha. Okay, I have friends there.

Very nice. Okay, Andrea. Hi, Dr. Lamb.

It's nice to meet you. Hi, Andrea. I graduated with my undergrad degree in microbiology and as soon as I graduated, I started at RMA as an embryology assistant and I worked my way up so I'm an entry-level embryologist now and I'm just looking to, you know, further my career in my company.

Perfect. Good, good, good. Nice to meet you all.

Okay, so I can start, Marina? Of course. Okay, so we're going to focus today a lot on the male factor and in this case in terms of reproductive success, which I spelled wrong there, I see contributions to fertilization, embryogenesis, and I added and beyond because really I'm going to give you kind of the whole story in terms of the male contribution and don't hesitate to ask me questions if you have some going along the way, okay? So these are my disclosures. None of them are relevant to the topic of this talk and so I essentially have no conflicts and I was supposed to give you a learning objective slide, which I only found out about five minutes ago or so and so I can add that and I think you all get a copy, don't they, Marina? Yes, yes.

So I'll add that to the slides. So I want to begin just by telling you what is known about kind of the things that can affect both sort of fertilization as well as embryogenesis and then fetal development as well and even the birth of a live baby. So I've expanded this a bit and here I'm showing you just a nice little slide where they're talking specifically on the female side about the maternal effects that can impact IVF outcomes and, you know, you see on the left side the hypertensive diseases of pregnancy and gestational diabetes and preterm delivery and then on the other side are the fetal effects that we're concerned about, which are the congenital birth defects and imprinting disorders, low birth weight babies, as well as having long-term cardiometabolic effects or neurodevelopmental defects and those are usually the things that people are focused on.

And when we think about IVF, there's a bunch of things that are different with IVF as compared to a natural conception and, you know, some of those have to do with the hormonal induction of the woman where there's very high levels of estradiol. There can be elevated VEGF as well as some epigenetic changes, which have been at least postulated and some data presented to support that, although I'm not sure it meets the level of evidence needed for, like, thinking of this medically. And then, of course, we know a lot about the multiple gestation problem, which can affect these outcomes.

And so, it turns out that just having a infertility diagnosis alone doesn't matter whether it's male infertility or female. We are going to focus on the male, but right now we're talking just about unknowns. Couples with an infertility diagnosis is associated with having a child, increased risk of having a child with birth defects, and that is regardless of whether or not ART was used for conception.

So, even if they manage a natural conception, they're at higher risk for a birth defect. And so, there's been some work done on looking again at some of these types of defects which can occur in couples seeking treatment, in this case for IVF, and here they're looking at birth defects and or cancer risk in children conceived by IVF. So, again, this is all IVF couples.

They weren't selected for by types of infertility or anything like that. And there is some degree of controversy when we look at all the ART procedures, but for IVF there is quite a bit of data, and some of it is strong, some of it is less strong. So, about 9% of babies conceived by ICSI with IVF, they have an increased rate of congenital anomalies compared to the rate which occurs in about 4.2% of control infants, and that's either naturally conceived or, you know, from fertile couples.

And some of these are caused by non-chromosomal defects, as well as there's a variety of different birth defects, which I list for you here. You wouldn't be required to remember this. I think it's kind of interesting, though, that they're affecting all different types of developmental processes, right, and developmental organ systems that can be affected that are increased in children conceived by ART, and in particular this is for the ICSI-conceived children.

And the risk for other birth defects after just ART in general varies by the type of defect which is being considered. Now, in terms of just IVF-conceived incidents, it's less clear because there's controversial papers in the literature. Some say there's increases of certain birth defects.

Some say there aren't. And I think probably a lot of this may have to do with the populations that are, and maybe patient bias for the evaluations, but understand that that is considered to be controversial. Now, there's conflicting data about whether IVF has an effect on neurodevelopmental disorders in offspring, and there is strong data showing that cerebral palsy is associated with IVF with and without ICSI, but as is autism spectrum disorders, which is, again, associated at a higher incidence with babies conceived by ICSI but not by just routine IVF.

And there's also a risk of autism and mental retardation, and I didn't use that word. It should be cognitive dysfunction, but that was a quote out of the paper, is associated with ICSI for male factor infertility as well. Now, when we look at the risk of birth defects and or cancer in the children conceived by ART in general, it varies, again, as I said, by the type of birth defect that's being looked at.

And there certainly seems to be an association between having any ART procedure that includes, you know, artificial insemination as well as in vitro fertilization and ICSI. There's an association with that and transposition of the great vessels. So this is a cardiovascular anomaly, which has been reported by several different…independently by several different groups.

And then there have been these huge meta-analyses where, you know, epidemiologists are taking a look at huge data sets of children conceived by ART. And in some of these cases, there's no significant difference between IVF and ICSI, but when you compare these offspring conceived to naturally conceived offspring, there was an increased risk of birth defects for all organ systems combined collectively. And predominantly, these affected the nervous system, the genitourinary system, digestive circulatory, musculoskeletal, eye, ear, face, and neck.

So that part seems very solid. Now, there are also retrospective studies of children conceived by IVF, and these children were looked at for both the presence of birth defects and the development of childhood cancers compared to a large cohort of naturally conceived children. So in this study, they looked at over a million children born to fertile couples, and then about almost 53,000 children born to women who required in vitro fertilization.

And so, although the risk of a cancer diagnosis was not increased among the children conceived by IVF that did not have a major birth defect, it was increased for the children who were born with a major birth defect. That's one requiring surgery. Now here, let me just try and close this window a bit so I can see a little better.

So here, we're looking again at the data that I just told you about. So again, it's stratified by maternal age here, as you can see, and paternal age. Some had missing data, and these were the cancer rates per 10,000 children and cancer rate per 10,000 person years, and those are two different ways that this data can be presented.

And so, you see that there's really, for the child with no birth defect, this was the percentage of them in each group, and the percentage of, excuse me, at the mother's age and the father's age. And you can see that when the children were conceived by ICSI, that that their, excuse me, by IVF, their cancer rate was similar to that of that from the fertile group. So, it's a little bit higher, but not nearly as significantly as when you look at the children that were born between the fertile and the IVF group, okay, where here you're seeing about a sevenfold increase in the incidence of these childhood, these babies with birth defects developing malignancies.

And when we look at this in terms of cancer rate per 10,000 years, again, it's about a, it's a little bit less than a, I guess it's about a tenfold increase, right, in the value. But the data is quite solid, and again, this was a huge study of large numbers of individuals. And it turns out that these ART offspring in general, again, not selecting by type of male or female infertility, just all comers, that the offspring at a higher risk of having a low birth weight and gestational age, premature delivery, and hospital admission.

And so, here you're looking at some of the data that was reviewed in a nice paper in 2013, where they showed a twofold increased risk of perinatal mortality, low birth rate, and preterm birth, as well as an increased risk, even if it was singletons that were considered, you know, because at first people thought that this was due to the fact of multiple gestations impacting the prevalence of these poor outcomes. But it wasn't just the increased number of gestations that were present, but rather, you know, it had to do with the actual ART itself. There was a larger study, again, which kind of confirmed this study.

And then, they also realized that there were also maternal problems, which were more prevalent in patients where the women had an increased risk of placenta previa, preterm delivery, and low term birth rate, regardless of the type of ART used. So, again, you know, if you think about it, we're kind of bypassing nature's natural barriers, right, to fertilization by defective gametes, whether it be on the female side or the male side. And so, the pregnancy outcomes and the effects on the actual pregnancy itself were more prevalent to have problems in the patients who had ART procedures, the couples.

So, what are the potential causes of these types of anomalies and others that I mentioned for the ART-conceived offspring? So, one of the things that people have looked at some in terms of the whole IVF procedure is, you know, we're doing all types of culturing. The embryos are in kind of a very foreign condition, right, you know, not being present in the fallopian tube and then in the uterus. And there's been concern that maybe there are epigenetic changes which are occurring because of all the treatments that are done in in vitro fertilization in general, and that this may impact normal development.

And I'm going to focus today just on talking about DNA hypermethylation, which is one major form of causing an epigenetic mark. But it's important to know that there are other types of modifications that can be present, histone modifications, DNA-binding proteins, and a lot of interest in recent years on these micro-RNAs impacting, again, the epigenetic changes which can occur to the chromatin in the…either in the oocyte or in the developing fertilized egg and embryo. And genomic imprinting is one specific type of epigenetic modification which is determined by paternal origin.

And so when this occurs, depending on the gene involved, either the maternal or the paternal gene only, right, so the one inherited from the mother or from the father, in this case is expressed in specific cells at specific times during development. And this is like a permanent type of imprint with the placement of this epigenetic mark, which then does not disappear over time. And just to show you what these marks look like, one that we're talking about right now is related CPGs in the DNA sequence.

You see these blue dots, so these are areas with repetitive repeats, CAG, CAG, CAG, or CG, CPG, which are along the DNA. And this is just the chromatin structure that you're seeing right here. And you see these methylations right here that are attached to these sites, these CPGs that are hypermethylated.

So you can see the methyl groups here. And so when there are methyl groups like at these CPGs, that actually prevents the gene from being transcribed or it diminishes the transcription of that gene. Whereas when the methyl groups are no longer frequent on the sequences, then it's active transcription that permits the gene to make the messenger RNA to encode the specific protein that's present.

And there's a whole series of these different imprinted genes, and these are involved not only in embryonic and fetal development, but also placental growth and neurodevelopment of the fetuses. And the process of imprinting these genes, putting these hypermethylation marks occurs during gametogenesis in both the males and the female. And the imprinting, it's thought, may not occur properly in men with either spermatogenic or oogenesis deficiencies.

And many of these genes cluster in specific regions on chromosomes 6, 7, 11, 14, and 20. And it's known that this process of imprinting is vulnerable to both physical and chemical stresses, again, raising concerns that the simple procedure of doing in vitro fertilization and also superovulation required for ART can disrupt this imprinting, in this case on the female side, resulting in severe but rare imprinted disorders. And there's many of these known, Beckwith-Wiedemann and Angelman syndrome and Prader-Willi, many of these syndromes are known.

So, when they looked at children conceived by ICSI with IVF with natural conceptions as compared to those conceived by ICSI-IVF, this was a large systematic review and, again, a meta-analysis of a number of different studies in the literature, all meeting certain criteria for quality that were combined to look at large numbers of these children. And they looked at whether or not imprinting disorders were increased in children conceived by IVF and ICSI. And it turned out that the odds ratio for having any imprinted disorder in children, which is a rare condition in humans, in the children conceived by ICSI-IVF was 3.67-fold compared to the children conceived by natural conception.

So, that's obviously a huge, very significant increase in this. But, you know, but then when they tried to break this down by the individual genes that, you know, have, you know, get hypermethylated with imprinting, where it's, when it's improperly done, where you end up with a disease state, a syndrome, they could, they didn't have enough data to be able to draw conclusions. Although Beckwith-Wiedemann, which is one of the more common ones, approached the limit of statistical significance, but they really didn't have enough samples to break out that data showing that almost four-fold increase in the odds ratio, because of not having enough in the individual groups of types of different imprinting disorders.

Sorry, quick question. Yeah. And they're subdividing those between infertile couples naturally conceiving and naturally conceiving couples without infertility? Or is this looking at the infertile couple IVF versus not? This is looking at IVF with ICSI versus, but it was compared to normal conception.

Within the, within the infertility patient population? No, within the population. So, as a whole. They took, they took and looked at babies conceived naturally with no ART treatment at all, right? And they, so, and again, remember like in places like Scandinavia and all, where they have socialized medicine, they have all this information to be able to know, you know, to stratify and know whether they had ART treatments or not.

So, it's basically the general population of people, perhaps we'll call them subfertile, as compared to the infertile couples who had to have ART to achieve a pregnancy. And the children conceived by ART seem to be at an increased risk of these epigenetic-related conditions, these imprinting disorders, right? Again, such as Beckwith-Wiedemann and these other ones in the long term. Did that answer that? It was Joseph.

Yeah, yeah, that was me. Because I know a lot of the discussions that I have with my colleagues is, you know, is it the infertility diagnosis that leads to some of these disorders, or is it the fact that they're undergoing ART therapy? And sometimes that's hard, at least in the literature that I've reviewed, to differentiate out sometimes. Right, right.

So, again, people have not been able to totally... Some of these ART patients certainly had, like, artificial insemination, you know. So, again, they are, you know, they may be removing the seminal fluid, right? I mean, it's things that are not usually done, right, in a natural conception. But I think that there's more concern that it's actually during the process of oogenesis and spermatogenesis, when that has gone awry or is perhaps less than perfectly controlled, that you may get these imprinting disorders.

It is certainly not acquired after fertilization. So, it's definitely in the gametes for these imprinting disorders, because that's where the imprinting occurs in the sperm and in the oocyte. All right.

Thank you. Great. Thank you.

Good question. So, you know, so if we think about what are sort of other potential inducers of adverse outcomes, we just sort of talked a little bit about the infertility itself, right, and hormonal induction of ovulation. And we mentioned about the, you know, the handling of the gametes, both male and female, but predominantly the concerns have always been on the female side with oocyte retrieval and ICSI embryo culture and then the embryo transfer.

We talked about, you know, the imprinting disorders and epigenetic mutations. We didn't mention structural chromosomal defects, and I'm going to only mention them briefly when we talk. And I'm also going to talk about paternal age.

But it's very clear that for all of these different types of possibilities, that really we need very good, strongly controlled prospective studies, right, to really try to determine what are the main inducers of these increased adverse outcomes, which are, you know, notable, right, I mean, and major, you know, with the mortality increase and the birth defects and the malignancies and so on. And again, I think that there's no doubt that some of those things could be the gamete problem itself, right, that may lead to it as well, say the birth defect as well as the associated malignancy. Hard to know.

And, you know, people also have been very interested in looking at genetic causes. These can be chromosomal defects, numerical or sex chromosome defects, structural defects, gene deletions or mutations, problems with gene expression. Years ago, oh, how did that go ahead? But whether or not people have done linkage analyses as well.

Now, I think we're all familiar with a normal karyotype. So here we're looking at a XY male. So you can see, you know, all the different chromosomes are displayed, right? These are isolated usually from white cells in the blood.

And you can see that they're all displayed. They're all about the proper size. And here's our poor little Y chromosome.

I think I'm on autopilot here. But here's the Y chromosome here. Now, there are different ways to do this.

Mostly it's done by a high resolution banding karyotype, which is what this is. Today, we also have spectral karyotypes where every chromosome is labeled with a different fluorescent probe. And usually this isn't done for like a routine karyotype.

This might be done more commonly for, say, a malignancy where you have all types of translocations and inversions and, you know, aneuploidies and so on being present. But I think the important thing for you to know is that normal men and women have 22 pairs of autosomes, right? So these would be the autosomes, right? And then the sex chromosomes here, in this case, the male has one X and one Y, with the Y being male determining and women have two X chromosomes. And I would just say this is a very superficial look at our genetic information.

And yet there are a lot of patients, as it turns out, who actually just have chromosome problems, not, you know, specific to like minute mutations. And if you think about this as like the Encyclopedia Britannica, with each chromosome, you know, being a volume of the encyclopedia, you know, you can sort of say, oh, well, all the volumes are present. They're in about the right size.

They look pretty good. Or maybe there's an extra volume, right? Or an extra chapter or paragraph or paragraphs switched between different chromosomes. All of these things can cause, you know, both either numerical or structural abnormalities.

So if you do a karyotype of, this was done here in a study, this was comparing fertile men. It's on autopilot. I don't know how that happened.

But so here we're comparing fertile men and infertile men. And all they did was give their blood for a karyotype analysis. There's no breakdown of what the causes of infertility are in these men.

But we see that the chromosome abnormalities are increased more than tenfold in infertile men as compared to fertile men. And you can see this is about 5.8%. Some studies say 6% of infertile men have a karyotype abnormality as compared to that seen in the normal population, which is 0.5%. Now, in my lab at Baylor, we did a study looking at the cytogenetic. So that's the cell complement of chromosomes in fertile men and compared them to actually to fertile men down here.

And here we have infertile men in general. And then they were broken down by their sperm count with azoospermic men, severely oligozoospermic men. So that was like under 5 million per mil.

And then oligozoospermic men, both of these should be zoospermic. And what you can see then is they're all color coded, right? And this is in purple are the fertile men. So you can see that a couple of them had some structural sex chromosome abnormalities here, right? But it was restricted to that, whereas the men who were infertile right here had varying degrees of types of chromosomal abnormalities.

Many of them, especially for the azoospermic men, had Klinefelter's syndrome, which I will show you about in a second, where these men have an extra or multiple X chromosomes. And that's a known cause of non-obstructive azoospermia. Here, this is infertile men in general, looking at all these men, not the fertile ones, but the infertile men in combination.

And these are the oligozoospermic and the severely oligozoospermic. So the structure, the numerical chromosomal disorders were most common in the azoospermic men. In the men with structural abnormalities, it was most prevalent in the severely oligozoospermic men.

And the sex chromosome abnormalities were actually fewer. But again, in this case, you know, a fair number of fertile men also had some sex chromosome structural abnormalities. But I think it gives you a good sense of kind of the types of abnormalities that are associated with different types of spermatogenic failure.

Now, importantly, it's not just the men who have these chromosomal abnormalities. This was a great study of a thousand couples seeking treatment for their infertility, asking for ICSI. They weren't necessarily treated with that.

But of these thousand couples, the incidence of chromosome abnormalities was similar in the males, right? 6.1% to what I just showed you in that previous study of 5.8 or something it was. And then in females, though, so here we see about 6% of men who are infertile or being seen for infertility. None of these couples had a diagnosis at all at this point, had a chromosomal abnormality, and almost 5% of the women.

So if we simply did a karyotype analysis of all infertile couples, we could account for about 11% of infertility. Now, that's not what is usually done, but it points out a need, right, for doing increased evaluation of the couples early on in their reproductive pathway to trying to achieve a pregnancy when there's a suspicion of infertility. So again, if we look at the chromosomal defects causing male infertility, I told you about Klinefelter syndrome, that's XX or multiple XY men.

It accounts for a very significant proportion of non-obstructive azoospermia. There can also be XX males, and this is a structural, actually, chromosomal defect, but they're listed here because they don't have their Y chromosome, and that's where a small piece of the testis-determining gene is transferred onto one of the X chromosomes, but they have no Y chromosome, but totally male development, but no active germ cells in the testis, so they have a totally cell-only testis. There can be structural defects and translocations, inversions, deletions, duplications.

The most common is this Robertsonian translocation, which is a major issue also for men with non-obstructive azoospermia and severe oligozoospermic men, and then gene dosage changes due to microdeletions or microduplications of regions of chromosomes, and again, the one that's most well-known, I think, to all of you are the Y chromosome microdeletions, although they can also occur on autosomes as well, resulting in either male infertility and are shockingly common in patients with genitourinary birth defects, many of which are associated also with male infertility, and there can be other, again, gene dosage changes on autosomes and the X chromosome that can affect men's fertility. Now, I talked to you a little bit about the structural chromosomal anomalies, and it's nearly 5% of oligozoospermic men have autosomal translocations or inversions, and this number goes up even higher to almost 14% of men with non-obstructive azoospermia who have autosomal translocations or inversions. These men frequently get a karyotype.

These men do not, even though it's quite prevalent in this population of men with low sperm count. Infertile women, again, have a somewhat higher incidence of these autosomal translocations, but really it's, and that's about a tenfold increase compared to the general population, but you see that it's very high, right, what, a hundredfold almost increased in men with non-obstructive azoospermia. Now, the concern is that these translocations or inversions will be transferred to the offspring using assisted reproductive technologies, and balanced translocations, right, may become unbalanced, right, when you have homologous recombination during meiosis, and this is one of the main dangers of having these types of translocations or inversions because you can get very serious birth defects or fetal loss, and so it's certainly an area that should be considered more seriously.

I don't know why that keeps getting bigger. This just shows you what the Robertsonian translocation looks like. That's when you have chromosomes where they have a short arm and a long arm, and they're usually between two different chromosomes that you get this translocation, so they're called, these are called acrocentric chromosomes for the, because the centromere is not in the middle of the chromosome, it's towards, you know, the tip, the short arm, and so when you go have homologous recombination in error, you lose the little fragment here, right, that is where there's rejoining, and then this Robertsonian translocation, you have a molded, right, chromosome.

This fragment's usually lost, and there's a whole variety of different syndromes that you can get with these translocations in the offspring or in individuals, so between 13 and 14, you can get three chromosome 13s. This is PAT2 syndrome when you have an unbalanced situation. 14 and 15, you know, parental disomy imprinting disorders, so again, this would be a cause of imprinting disorders between chromosomes 13 and 21.

You can get Down syndrome, trisomy 21, and 21, 22 can also lead to Down syndrome, trisomy 21, so again, you can see these are important when looking at these infertile couples to do assisted reproductive technologies that could lead to a bad outcome in terms of the birth. Now, as sperm count declines, the likelihood of these karyotypic abnormalities increases, and in men with normal semen parameters, these men can have these Robertsonian translocations that I just mentioned, but again, remember that when we look at all couples, men and women seeking treatment for infertility, about 11% of them will have some of these types of cytogenetic anomalies. Well, it turns out that one of the major contributors to effects on embryos, pregnancy, fetuses, and offspring are changes to male fertility, and women have known for many years that, you know, they have a biological clock, right, and there's a limit to their ability to have a child, and it's very obvious to them because they go through menopause, and at least classically through history, men seem to be untouched by the notion of this fertility precipice that they are at, and they're usually viewed as being kind of immune to the ravages of aging as it relates to fertility, and this is both in a physical sense, because they can still continue to make sperm, and a social one, but the reality is that scientists have been studying what's called the paternal age effect for more than a century, and today concerns about paternal age are rarely discussed with couples where there's an older male who is seeking to become parents, and there are lots of reasons for this, but throughout the world, there have been a lot of changes in the parental age at the time that they have a child born, and this really began with the development of contraceptives, right, the pill in particular, and you can see that over time, then the birth rate has been declining, and the age at which couples then have their first child has been increasing.

This is the maternal age here, and this is the age then, again, first birth for the male, so what are the reproductive changes that occur in the men, and why does this affect the offspring? Well, their semen quality declines. This is a very nice study from an IVF setting by Fraterelli where they looked at all of the men that they tested in general, it was over a thousand of them, and then they stratified, so this is all of them combined, so then they stratified these men by age, right, looking at five-year intervals, so we have less than 35, 36 to 40, and so on, up to being older than 55 years of age, and you can see that until you're about 40, everything seems to be pretty normal in terms of, say, sperm count, but here, by the time you're 41 to 45, you see a precipitous decline in sperm count, which, of course, is highly statistically significant and very reproducible in the literature by others. Now, if we look at the semen parameters themselves, we see that every parameter that's measured, you know, so here the volume of the semen goes down because of declining testosterone, here we're looking at the sperm concentration and sperm count, sperm motility goes down, as well as morphology, so every parameter that we measure in this part of the routine semen analysis declines, and so then the probability of producing an abnormal semen specimen increases with increasing paternal age, and this accounts for abnormalities in a variety of different parameters.

Now, interestingly, it turns out that the female partners of older men, and these are, you know, all fertile couples, and they've controlled for the age of the woman in all of these studies, but they showed that pregnancy complications in female partners of men over 45 years of age were more likely to develop all types of disorders, hypertensive disorders of pregnancy, placental abruption, intrauterine fetal demise, and preterm deliveries, and the age was also impacting the risk of late stillbirth, which went up in children, in pregnancies where the man had advanced paternal age. This also resulted in low birth rate, more preterm births, and increased risk of very preterm births, so it's affecting not only the female, but also the offspring and the outcome of the pregnancies. Now, to look a little closer, again, back with our friend Fratarelli, here he's looking at the same cohort of men that I showed you with the declining count, and you can see that even for the men over 55 years of age, that all of these individuals, there's no statistically significant difference between the implantation rate for the embryos that are obtained from these men.

Pregnancy rates are not statistically different, but when we start to look at pregnancy loss, we see a very significant increase from the age of the men 55, really, and above, and here we're looking at the live birth rate, and again, we see a dramatic decline in live birth rate controlling for the age of the women from the fetuses conceived by the older men. Now, just being of advanced paternal age also increases the miscarriage rate, and here's a study of over 5,000 natural conceptions, and they found that when the men were older than 35 years of age, that their younger wives were 1.26 times more likely to have a miscarriage than the partners of men where the males were younger than 35 years of age, always controlling for maternal age, and here's another study of 17,000 IUI cycles where they controlled for maternal age, and then again, they stratified the partners of these men who were older than 35 years of age as compared to the ones, the outcomes where the men were younger than 35 years of age, and again, in this case, the miscarriage rate for the pregnancies initiated by the older men were 32.4% as compared to 13.7% in the men younger than 35 years of age, and I can tell you that there are tons of fathers today who are well over 35 years of age having natural conceptions, but they're at higher risk, again, for the female having a miscarriage, so we talked a little bit about how paternal age has negative impacts on semen quality, and in some instances with a highly advanced paternal age, there can also be effects on implantation and pregnancy and live birth rates for sure, and it turns out that increased paternal age is also associated with DNA fragmentation, and this now has met the levels of evidence needed in medicine to say that DNA fragmentation is independently associated not only with infertility, but also with a lower live birth rate in the offspring conceived from these men, so the children born to fathers over 40 also have increased mortality, and this again is a large registry study in Scandinavia. It's very well known.

There are many of these studies, but this is really the one that's best done, where there's almost 1.6 million children where they stratified by age under the age of five years of age, and they found an increased risk of mortality due to congenital abnormalities, malignancies, and external causes, and this is for children born, depending on the age range tested, comparing the fathers age 40 to 44 or the fathers over 45, and all of them had an increased risk of having mortality due to these various types of problems, so the major contribution of the male gamete to the embryo are new de novo germline mutations. Now, it turns out that the concerns about paternal age effects were raised, actually first noticed about 150 years ago, and there was a family, there was a, excuse me, a doctor in Germany in a small town who realized that dwarf limbs occurred more commonly in the last-born child in the families that he cared for in his community someplace in Germany. He published this in 1912, and of course, the last-born child was conceived when the male was significantly older, as the woman was as well, but then 41 years later, another investigator published a study about children with dwarf limbs, which is today we know achondroplasia, and he was the first to use and propose a paternal age effect on the offspring and their healthy development, and so again, this was reported in 1955, and, you know, it's thought that repeated cycles of spermatogenesis probably increase to the likelihood of, like, having these de novo mutations that don't get fixed in the sperm of older men, and the mutations that are found and that are present in all of us from our fathers are generally single base pair substitutions in genes, so like spelling errors in genes that are random and sporadic in the genome.

Now, if we look at genetic conditions that are known to be associated with advanced paternal age, you can see all these different conditions here. I told you about achondroplasia. Apert, Pfeiffer, and Croussant syndrome are three due to problems.

They're all in a very close region on one of the chromosomes, but they're different genes that are encoding these syndromes, and, but you see there's a whole variety of different types of things, not just syndromes and birth defects, but also epilepsy and breast cancer, leukemia, and various types of malignancies, so there's a variety of different types of bad outcomes that could come with advanced paternal age, and so this is the frequency of these types of birth defects in the general population, and when we look specifically at the children conceived by men of advanced paternal age, we see basically an eightfold, almost tenfold increased risk for these two syndromes. These are still hugely increased, a six to eightfold increase in these other two syndromes, and fourfold, so you can see that these are huge increases from something that's quite rare to something that's really quite common, right, in the population of children born from older men, and, you know, I'm part of the American College for Medical Genetics and working on practice guidelines about advanced paternal age, and you can see that when we look at the levels of evidence, so these are medical descriptions, right, based on the quality of the studies that the data is derived from, we can see that, again, for this list, and this is not complete, this is just giving you an example, the levels of evidence are predominantly incredibly strong, or at least medium, which is, like, mind-boggling that the evidence is so strong for seeing these increased risks of these various syndromes, and so, just to emphasize that, you know, since Dr. Weinberg, you know, reported in Penrose the paternal age effect, that there have been literally hundreds, and this is, I'm sure not, it keeps increasing exponentially. There's so many different types of gene defects which are caused by advanced paternal age in the offspring, and in 2012, paternal age effect disorders is not now a standard diagnostic category in terms of diagnosing these children because there's a bias in the paternal origin of mutations and strong paternal age effect, and it's thought to be due to, again, the high mutation rate that occurs in the spermatogonia during spermatogenesis.

Well, next-generation sequencing has really revolutionized our ability to look more closely at what happens in the offspring conceived by the men with advanced paternal age, and so, the studies that I'm going to show you used whole-genome sequencing, where the entire genome is sequenced from the mother, the father, and the offspring to look at the increase, or whether there's an increase, in new mutations in the offspring conceived by the older men. So, in this study, which was in, I think it's Iceland, there were about 11,000 new mutations in the genomes from 250 families, so again, the mother, the father, here they're looking at new mutations in the offspring of all their offspring, and they showed that paternal age effect explains 95 percent of the variation of the global mutation rate in the human population. It's a staggering number, and these new mutations were more numerous in the children of older fathers, so we all get new mutations, more so from our fathers, but it goes up exponentially with age.

So, in this study, they looked at offspring born to 40-year-old fathers over the age range of about 18 to about 45, and you can see these are the new mutations which were present in the men's offspring spread throughout the genome. This was the non-coding region of the genome, but all of these kind of pink or peach spots here are new mutations found in the offspring conceived by the older men, and these were in the coding region, so these coding region mutations are more likely to have functional consequences for the offspring, and if we look at these new mutations, again, in the offspring, comparing the age of the father in the blue and the mother in the pink, we see that there's a natural stop here, you know, because most of these women were starting to be perimenopausal and their pregnancies were not occurring, whereas the older men with other partners were able to, you know, kept having increase in the new mutations in the offspring that were born as they got older and older, and so the majority of the new mutations either way come from the father, even from the child conceived by the couples where, you know, they're both 18 or 20 years of age, so again, the major contribution of the male gamete is new mutations that are germline, so in every cell of the body, and it's thought, again, that this hypermutation occurs in the spermatogonia, which have the highest rate of proliferation in the body of all cells, and normally, each one of us has about 44 to 82 new mutations, and only about one to six of them are in the coding region of the gene, but when we look, then, at the children conceived by older fathers, we see that this rate of new mutations increases about two new mutations per year of paternal age as the man gets older, and so that means that the rate of paternal mutations increases about four and a half percent per year, so it increases eightfold in, say, 50 years of a man's life, so in general, the paternal mutations that are inherited are four times more than the maternal ones, so again, most of our new mutations we get from our father, and again, this correlates well with the number of estimated spermatogonial replications that go on throughout a man's lifespan, and so you can see that there's pretty good alignment between this, so it's thought that the mutational load of offspring for each paternal year varies considerably between families, and we know that this occurs, and you know, there's some thought that maybe different people have different rates of mutagenesis, you know, some of this most likely is due to genetic variation and DNA repair, and it could be also that the spermatogonial stem cell divisions aren't dividing as frequently as they might between some different families. Just to know that not everyone agrees that all types of problems, you know, from advanced paternal age or that they occur from new mutations and their studies in psychiatric illnesses, some of them have shown that, you know, A, that there can be a cause of this from the older fathers because their children have higher risk of psychiatric disorders such as schizophrenia and autism, and some may be due to advanced paternal age, but then there are some credible explanations to how they may have other ways to get these increased new mutations, so just to understand that, you know, it's not to say that every single new mutation is due to paternal age, but certainly a great deal of them are, and I'm just going to finish quickly, and we talked about epigenetics, but here was a study where they looked at DNA methylation profiles of sperm during human aging, male aging, and there was a problem with the study because they looked at both fertile and infertile men, but it was mostly infertile, and the men were stratified as being younger men, 18 to 38 years of age as compared to men who were 46 to 71 years of age, and they looked at a large number of the CPG methylation sites and saw that they were differentially regulated, and you don't have to memorize this, but the pink dots are the methylation sites that are present in the same man's blood, CPGs at the same gene sequence, and you can see the great variation of all of the different methylation sites by chromosome that are present in the sperm by comparison, and so when they did studies of all this, they were able to look at the data and then group it by paternal age, and they found that by looking at the age by year, and here they're looking at the actual age in blue that they had, and then they were able to calculate and estimate the man's age based on these hypermethylation sites and how accurately they could determine how old the man was, and here we're looking between about 30-year-olds up to 60-year-old men and looking at their sperm, so it's pretty amazing that their sperm shows these kind of differences.

Things can be hypermethylated and hypomethylated, and people have tried to understand what kinds of genes were affected in the sperm by this, and they came up with studies that suggested that there's a link of these hypermethylation sites to the higher risk of the psychiatric and neurodevelopmental disorders in children of age fathers that I mentioned previously, so the problem of potential of advanced paternal age, you know, has many sort of issues, delayed parenthood, trophy wives, you know, younger women with older men, and then they have poor semen quality, some have male hypogonadism that we didn't talk about. We did talk about the paternal age effect and the obstetrical complications, but the take-home message is that there's increased de novo mutation rates in the offspring of older men, we know that, and this age-related decrease in DNA hypermethylation is also correlated with the increase of genetic syndromes that I mentioned for you as well, so the recommendations for couples with advanced paternal age, you know, really the couple should be counseled prior to conceiving a child so that they just know that their risks of having a less than perfect baby are increased, and they should be offered prenatal diagnosis up to 16 weeks of gestation, and that's what's done for every pregnancy, so the pregnancies are not treated any differently, there's no testing that can be done, and, you know, if there's a concern, they may look at ultrasounds at higher density, you know, a little later in gestation, in addition to the routine evaluations. There's no upper limit set.

American Society for Reproductive Medicine suggests an upper limit of 45, but that's not based on anything evidence-based, but that's for semen donors only, and, you know, it's very hard to predict patient outcomes in terms of the children that are born, you know, because there's such a wide range of effects on offspring, and, you know, and the assorted risk of aging do not necessarily simultaneously increase, so clearly older men, you know, who want to conceive a child should be informed that the risk of having an offspring with serious disorders increases continuously as he gets older and conceives more and more children. There's no diagnostic test that can be used, and once there's a pregnancy, it should be treated no different than any other pregnancy. So I don't know what time, we're five minutes over.

You guys have quick questions? Oh, and this is my new position where I work here. It's an amazing research institute in Kansas City. Questions? Marina, you're muted.

So sorry. I was saying it's an amazing building, Dory, and I think last time I heard your lecture, you actually marked your office for us. Yeah, yeah.

Can you put your, yeah, your mark? Yes, my office is right here, and then there's more of the building that goes beyond this, you know, hallway-wise, and it's amazing because at night, the lights, you see, can you see there's like some different colors, blue and white? So at night, it lights up to be a DNA sequencing. DNA, yes, absolutely. And then here we have stairwell for emergency exit, but it's the double helix that lights up at night.

So you see the double helix here and the DNA sequencing there. Is that cool? Amazing. It is the coolest, actually.

Let me know if you are hiring. I just come, I will just come for the building, if nothing else, the building only. Thank you, Dory, for this amazing lecture.

I heard some of this before, but I'm always grateful for the refresher, and I feel, you know, our CELL scholars are extremely lucky to have this opportunity to learn from the best. We are, I think we are very excited about your next lecture, but do we need a bio break? Important question. Oh, I'm fine.

I don't know about the rest. I'm just trying to figure out how it is that I, there, okay, because I need to. Okay, fine.

So should we continue, or we need a break? Yeah, we can, if everyone's okay with that. I'm okay with that. I did have a question, just because you sit sort of at the forefront of this.

If you think that testing male sperm for DNA fragmentation is going to become more routine, because it's definitely not a commonly practiced thing, at least where I'm at, partly because we don't have a lot of things that we can do for these men other than make lifestyle recommendation changes, but I know from what you presented that even, you know, slight discrepancies in male semen analysis parameters will see those genetic or fragmentation changes. So first of all, I'm one of the authors of the ASRM, AUA-ASRM practice guidelines for male infertility. I think there's like 14 of us, something like that, and so the lifestyle issues, and some of this has to do with quality of the studies that have been done in the male, with the exception of like really toxic occupational exposures and people exposed to things like DBCP and, you know, I'm saying pesticides, herbicides that, you know, have some endocrine disrupting effects like DDT and DDE.

With those exceptions, most of the studies in humans, not in mice, but the studies in humans are indeterminate, right? And even things like obesity, you know, things that we all agree are bad for our health, doesn't seem to have the effect on male fertility parameters the way that it does in women, and I think you can stay tuned. Obviously, it's an area of active investigation, and again, remember that the problem is in the quality of the studies, and it's not to say that there's no effect, it's just that the data is not strong to show it from the studies that have done to date. And, you know, think about it, like you could have an exposure, or you could, you know, you could have done something in your youth, right, or been, you know, if it was weight, maybe you were obese, and then you slimmed down, or, you know, I mean, you have no way of knowing, and things are self-reported, we don't know what people were eating in the environment, which I think adds to the complexity of trying to understand these kinds of data.

So the lifestyle part is on hold, I mean, we know that, like, anabolic steroids are bad, and, you know, smoking, but, you know, they're bad for you anyway. Yeah, did that? Yeah, I think, yeah, yeah, I think that's probably the hardest aspect of what I do is dealing with the male aspect, because I, despite, you know, what you said, the years of study, it just doesn't feel like there's a lot to tell them, unfortunately. Yeah, yeah, yeah.

Oh, it's truly lacking, you know, it's, it's not good. And, and of course, you have to understand that the men, the men are not always willing, or, like, enthusiastic participants of, you know, doing all of these reproductive studies. So we're good? Okay, I think we can go ahead and get started with the second lecture.

Okay, so, so I'm going to talk now about the infertility diagnosis in men, approaches and advances in diagnostic andrology. And nothing's changed with my disclosures, and we'll find some learning needs, you know, when you all get the slides, I apologize, I wasn't told that that was needed. So I wanted to just remind you about what the urologist or the, the andrologist does when they examine a male, potential male infertility patient.

And, you know, our, our AUA ASRM practice guidelines, which I've referenced below, we came out with a very strong statement saying that both male and female partners should undergo concurrent assessment. We know from what we see in looking at the male patients, that the woman seeks treatment, but the man is not seen for more than two years, on average, after the woman begins this fertility, you know, trip, and the man is not even looked at. And in many IVF labs, no one even cares exactly what the semen parameters are, because they shuttle people, you know, couples for IVF, and now pretty much exceed routinely, even though that was always restricted originally in the practice guidelines for male factor and fertility only, but the men are not even evaluated.

And there's lots of reasons why they should be. And, you know, this first evaluation should include like a reproductive history and physical exam, right, by someone who's very expert in doing this, not a gynecologist. And the initial evaluation in our practice guidelines, we said that there should be one routine semen analysis, and if everything is normal, that's sufficient.

Now, WHO recommends two, and that was usually the earlier recommendations, but it was the consensus of the group that, you know, if abnormal, a second semen analysis should be performed. And quite frankly, many, many times, we have four pages in WHO-6 on the collection problems that contribute to abnormal results, you know, and so there's all types of collection problems that lots of people don't ever worry about, but they have mega, mega effects on the semen analysis results and looking at the semen parameters. And if a man has no sperm in the eugenic glute, azospermic, then he needs more evaluation to determine whether he has a plumbing problem, which would be non-obstructive azospermia, excuse me, a plumbing problem, which is obstructive azospermia, and whether he's simply not making sperm or enough sperm to be in the ejaculate.

And so they're also going to take into effect semen volume. I would say we always ran fructose assays, right, because that's very informative, as well as measuring semen volume. So a low-volume fructose-negative semen sample is indicative of obstruction, but then they also do a physical exam, right, feeling the epididymis and so on, and measuring FSH levels.

Now, the urological assessment also depends usually on an endocrine evaluation, but it's thought that those endocrine evaluations should be dictated to what the symptoms are that are reported and the clinical findings. And so if a man is infertile and he has, say, deficient libido or ED or spermatogenic failure, you know, these would elicit different hormone panels to be to be evaluated. Most urologists just measure, you know, FSH, LH, testosterone, and prolactin in these men, but, you know, it was thought that you don't necessarily have to order a panel when you really do a good physical exam of the patient.

Now, remember that I know you all know semen analysis, but, you know, the macroscopic assessment, we always think of everything that's measured in the wet mount microscopy, but again, the macroscopic assessment is really very critical as well, as is the microscopic assessment, which is looking at the stained sperm for morphology. Now, when you take the semen analysis result together with the history and the physical, the semen analysis is really still sort of the cornerstone of the evaluation of male reproductive health, and there's a ton of articles in the literature that show how poorly semen analysis is performed. It's the most poorly performed test of all of clinical medicine in terms of reproducibility, variability of results on the exact same sample.

Most of the results are not accurate and or precise, which is hugely problematic, and there's lots of reasons for that, and it mostly has to do with people don't adhere to the established methods, or they've been poorly trained, or they keep switching and using different methods to perform the semen analysis, but the bottom line is that when a physician gets semen analysis result from, say, an outside lab, and it's no better in Quest than it is in many andrology labs or andrology labs in IVF clinics, no better, you know, and the results are incredibly variable and totally unrelated to what the proper answer is, and so I'm one of the 10, I think 10 or 12 were called editorial board, but were the authors of WHO-6, which is the laboratory manual for the evaluation and processing of human semen, and that is really the guideline that you should be using in your lab for how you perform a semen analysis, and it was actually edited to make it much easier for the laboratorian to be able to follow the instructions and do it accurately, and so it's felt that, you know, that this would also improve training. Well, why is it important to perform the routine semen analysis in the evaluation of male fertility? Well, I think you're all familiar with semen nomenclature, because I'm going to use this in what I'm going to tell you, but just remember that there are some hiccups here that people always get wrong. Aspermia is no ejaculate at all, all right? Hyperspermia is when you have a huge ejaculate, and we set the—I thought we set the record when I was at Baylor for a 27-milliliter ejaculate, which was all—it wasn't urine, it was truly an ejaculate, but while I was at Weill Cornell, we had a man with 32 milliliters, right? So that is hugely above what would be the normal average, right? The lower limit of normal is now 1.4 milliliters up to about 7 milliliters, so, you know, but there's huge ranges of what you may get in the ejaculate.

And again, we talked about azoospermia, oligospermia, and remember that, you know, some of these things, like oligospermia would be low semen volume, not low sperm count for the purest. So anyway, so, you know, when the urologist or physician gets the results of the semen analysis, if there's no ejaculate, so that would be aspermia or what's known as a dry ejaculate, that could be due to the man not having an orgasm. He might have retrograde ejaculation, so that's common in men with diabetes and neuropathies.

Lots of medications can cause failure of ejaculation, or there could be an ejaculatory duct obstruction. That's where the, remember, where the vas comes into the prostate, and you have a ejaculatory duct cyst that blocks the passage of the upper tract secretions and the sperm into the ejaculate. If men have had any type of neurogenic neurological injury down in the pelvis, such as retroperitoneal lymph node dissection or prostatectomy, that can all cause a dry ejaculate.

Low volume, again, oligospermia or hypospermia are the correct terms. There can be either weak ejaculation due to aging or sometimes weakened pelvic muscles, and also testosterone deficiency, right, because the seminal vesicles are exquisitely sensitive to testosterone levels. So if you have low T, you're going to have a low semen volume and other medications.

And again, volume can also vary with mood and sexual activity, and it's actually been shown that the level of sort of enjoyment or enthusiasm, you know, when the sample is collected can actually impact both the sperm count and the semen volume, and there are studies published on all of this. When you have low or no sperm in the ejaculate, so that would be oligospermia or asospermia. Remember, I told you there can be collection problems or abstinence problems.

There can also be various forms of spermatogenic failure. I just reviewed for you the histopathologies that are seen. There can be endocrine defects.

Anything affecting the hypothalamic, pituitary, gonadal axis, steroid biosynthesis, steroid metabolism, steroid hormone receptor action, downstream signaling. There's a whole plethora of these different problems. And I will tell you that the endocrine problems that I'm mentioning, they account for less than 1% of male infertility, even though we always emphasize the endocrine aspects.

Certainly, anabolic steroid abuse is very prevalent and very detrimental to male fertility, and some of it cannot be even corrected once the men are off taking these high doses of anabolic steroids. Genitourinary birth defects, especially cryptoarchidism, where the testes have not properly descended into the scrotum during development, can cause also spermatogenic failure, even when the men have had orgiopexy, putting the testes into the scrotum and attaching it there during infanthood, and other disorders of sexual differentiation. And then there's other things here which are less clearly understood.

Varicoceles, emission and ejaculation might be lacking. There can be infection tumors, poor health for sure, and things like celiac disease, right, which are, you know, very damaging to patients, as well as both prescription drugs, as well as social drugs and alcohol abuse. And I do say other lifestyle issues that we already talked about a few minutes ago.

But what do these results actually tell us? They tell us a lot about testicular function in terms of sperm production. They tell us a lot about genital tract secretions from the prostate seminal vesicle, from the proximal genital tract, right, from the testis and epididymis, and the patency of the tract. But it is not a test of fertility.

And it's very misused in reproductive medicine today as saying that a man is fertile or infertile based on deficient semen parameters. So, and why do I say it's not a test of fertility? This is a famous paper with a very large number of couples from all over the United States. This was part of the reproductive medicine, a large NIH-funded clinical trial of various sites where they simply took, in this case, fertile men and, you know, measured their sperm concentration.

And you can see that there is a few like little dips here and peaks and valleys. And this is the sperm concentration in millions per mil that were reported. And you can see that though, you know, the most common is probably about 90 or 100 million sperm per mil that's reported in fertile men.

But when you look at infertile men, all comers, you can see that the values are pretty much identical, right, for the infertile men. And yet, you know, we're thinking that this is a measure of their fertility. And I can tell you that studies of vasectomy patients in the late 1970s, like early 1980s, showed that about 11% of couples who had a natural conception and the man was seeking a vasectomy, 11% of those men had either oligozoospermic and about 5% of them had severely oligozoospermic counts.

And some of them good motility, some poor motility. But the fact remains that they were all able to achieve pregnancies with no assisted reproductive technology at all. And that wasn't available in any way, shape or form.

I'm talking about these vasectomy studies. And the lowest concentration of sperm in an ejaculate where a child was conceived was 50,000 sperm in the ejaculate. This was published by Rebecca Sokol years ago.

And again, it was a natural conception and paternity was documented, right? They did all the genetics and it was certainly the man's child. And again, no ART was needed. And I can tell you anecdotally that we had a patient who had non-obstructive azoospermia, multiple, multiple semen analyses with pellet counts with no sperm at all, with every single cell tested in the pellet.

And, you know, and he had a microtesi where they had some challenges to find sperm, but they found enough to get a few eggs injected. And they ended up having a child. And then the guy comes in to see me, he's a faculty member, and he told me that, you know, like he and his wife thought they didn't have to use contraception and she wasn't feeling well.

And she went to the doctor and it turned out she was pregnant. And he said to her like, honey, is there something here I should know? And she said, no, you know, like there's no one but you. And sure enough, the baby was born and it was his.

And, you know, so some rare sperm found its way, right, and fertilized the egg. So, you know, there's really no lower limit that you can be assured that you won't have a lucky day, right, and achieve a pregnancy for some of these men with really severely, even cryptozoospermia. So anyway, but the important thing from this is that when we look at the semen parameters, and this is the paper from Guzak that I just quoted, the individual parameters, motility, unless you have no motility at all, count and concentration, as well as even a morphology is not diagnostic of prediction of whether or not you can achieve a pregnancy.

And no matter what you do on an individual basis, you can't separate the fertile from the infertile individual to know from their semen parameters what, you know, whether or not they're going to be fertile, and the low numbers don't exclude a pregnancy. And that can be natural, IUI, or by IVF. Now, they also found that the more abnormalities you have, so let's say that, you know, this is your starting point and person has low sperm count, poor motility, and we'll say poor morphology.

So that, you know, increased your odds ratio of being infertile, but on a per patient basis, you still can't determine, you know, whether or not they're fertile or infertile at all. And that includes even for the infertile men that were tested. And so the semen analysis is not really a test of infertility.

You can only be diagnosed as being infertile when there's no sperm in the ejaculate, right, at the time of being examined. They have complete globosomal spermia, remember the round-headed sperm lacking the acrosome, necrosomal spermia, and also I would say asthenosomal spermia, where you have totally immodal sperm. And, you know, so they could not achieve a pregnancy without an assisted reproductive technology.

So I'm going to review for you the changes that are present in WHO-6, because we, again, made it very simple to follow the instructions of how to do all the assays, but we also added several new methods, right, which included both sperm aneuploidy and DNA fragmentation. And I happen to have been, we all wrote the whole thing, but I was the lead author for this chapter three, which has these new methods in it. And so one is DNA fragmentation, which was not included in the previous issues.

And, you know, we know that it's very important to have sperm DNA be totally, you know, protected and to have genomic integrity. And it's important because I told you about some of the chromosomal aberrations that can cause various poor outcomes for offspring. And in the patients who have them, we talked about epigenetic modifications and a little bit about mutations.

There can also be base pair oxidation due to like free radicals in the semen from inflammation. And many of these things may cause DNA fragmentation. And this is when you have either double strand or single strand breaks in DNA in the highly compacted sperm head.

And why this happens isn't clearly understood. You know, certainly there's a sense that there, and we know actually when there's protamine deficiencies and so on, remember when you're getting the condensation of the sperm head during spermiogenesis, if the chromatin is not properly packaged, you know, during ejaculation, there can be shearing forces, right, and exposure to free radicals and other things where the DNA is not properly protected, right, in the sperm head. There could be defective DNA repair that occurs during mitosis and meiosis in spermiogenesis.

Free radicals, again, which are generated at times of inflammation and by white cells and so on in the semen, and certainly exposures to either industrial or environmental toxicants. Now, how do we measure DNA fragmentation? There are two types of methods. One are the direct methods.

So these directly measure the breaks that are actually present in the sperm heads themselves. This is done by either a comet assay or a tunnel assay, and I'm going to show you examples of both. Tunnel is where you're labeling with a terminal deoxynucleotide transphrase mediated.

I'm just looking here at my screen here. I don't know why this is talking to me. But anyway, but then you label the DUTP, right, of the sequence that allows you then to identify it with things like horseradish peroxidase if you're using a horseradish peroxidase and type labeling system.

But there are also indirect methods, and many of you probably use the indirect methods because they're commercially available kits largely. And these are different because you're not measuring the damaged DNA that's present. You're measuring the susceptibility of sperm DNA to break after acid treatment.

So that is not the same kind of thing. And these, I think, are probably quite familiar to you. The sperm chromatin structure assay or acridine orange flow cytometry or sperm chromatin dispersion assay.

And, you know, these are using like chromatin or DNA intercalating dyes to try and differentiate double and single-stranded DNA breaks. And for the tunnel assay, so here you're measuring, again, existing DNA breaks, and you're looking at the sperm under the microscope. And they can be labeled with either fluorochromes or these biotin-tagged probes with streptavidin and horseradish peroxidase.

And you have a color-responsive substrate that you're using for the horseradish peroxidase. And there are kits available to do this. So this makes it very easy, and many labs do do a tunnel assay because of the simplicity of it.

There is a real tendency for the technicians counting the damaged versus not damaged sperm to kind of be biased, right, in terms of the sperm that they pick to count. And that is one problem with the assay. But it can work well, and it can be very reliable.

This just gives you an example using, here we're using a fluorescent probe for measuring the DNA damage in the tunnel assay, the same principle but with a fluorescent probe. So here is sperm from an individual that is normal. Here we're staining the heads of the sperm blue with DAPI, and here's the fluorescent green showing the cells which have extensive DNA damage in the brighter spots.

But you can see that there's a number of cells here, right, which are really not lighting up. There's some others over here. And this person has a normal analysis.

Here we have someone with more sperm, but you can see that virtually every sperm practically is hugely damaged. And, you know, again, very easy to detect those sperm when you're using, I think the fluorescent tests are really easier and more quantitative, easy to analyze. Now, people have also used variations of this where they're using flow cytometry, again, with the green fluorescence and using propidium iodide.

And so for the flow cytometry studies, you know, you're looking at the sperm and then you're sorting them based on here you're excluding the cells that have apoptotic bodies. And here you have the cells, right, that is a normal pattern, right, for both the propidium iodide. And here we're doing the green fluorescence, which is measuring the sort of the tunnel based nucleotides that are labeled.

And you can see that when you do the flow cytometry of someone who has high levels of DNA fragmentation, which is in this region right here, you can see, right, very easily that people with no DNA or low DNA damage, you see a few scatters here, you know, 99% of the cells are all in the normal area, not with DNA fragmentation, where this would be a man with high levels of DNA fragmentation present in the sample. So again, this is flow cytometry using just differential staining. Now, my lab always used the comet assay.

And this is not the comet assay, this is a real comet. But I think it shows you very easily what we think of when we look at the sperm head. So here the sperm are placed in agarose on a slide, they've been treated prior to placing them in electrophoretic field.

But the sperm head itself with undamaged DNA is very bright and compacted. And then the damaged DNA splays out behind the head of the sperm under the electrophoretic field. And so here we're looking on the bottom, this would be sperm with no evidence really of DNA fragmentation, you can see little shadows, right, which is normal with very low levels of background.

But here you can see that the sperm heads are smaller, right, because most of the DNA is in the damaged portion that have splayed out, this would be the comet tail here and here. And so it makes it very easy to visualize. And so here you could count simply the percentage of sperm that are intact, such as we see down here with the tight fluorescent heads, and then the sperm with the tails.

Now, with the addition of using image analysis, you can capture the image very easily and use actually a very free program on the web to quantify the amount of pixels, right, that you see the bright headed sperm, right, for the sperm head. And here you can see all the DNA, you know, which has migrated out in the electrophoretic field. And so you can actually quantify the relative amount based on these pixels here of the DNA in the sperm head compared to what's in the sperm tail.

So here you can not only guesstimate how, what percentage of sperm are damaged, but how damaged they are as well. And so in this case, this patient had 88% of his sperm DNA splayed out that was DNA damaged and broken, right, single and double strand breaks. So it can be highly quantitative and highly reproducible.

One of the indirect methods is the sperm chromatin dispersion assay. And this one I think you're very familiar with, and that's where you evaluate the, whoops, I'm just trying to, what happened there? I lost my view. Just a minute.

We can still see you, Dory. Yeah, but let me just see if I can lessen that. There, are we good still? All right.

So anyway, but it's looking at the susceptibility of the sperm to break when you denature the sperm with an acid treatment. And in this indirect method, this sperm chromatin dispersion assay, it's the opposite of what you see with the comet assay. So here you have the chromatin of the sperm, right? And the healthy DNA, then when you treat it with acid, you get loops of normal unbroken DNA that come out.

And so these forms sort of halos around the sperm head. So it's the opposite of what you're seeing in the comet assay where the damaged DNA is coming out. Here in this assay, you're looking at the intact DNA.

And this halo forms when, again, you've used acid and a lysing solution to relax the sperm head and have these DNA loops come out. And this is what this looks, oops, it's all done. Wow.

I don't know what happened there. This looked fine before, but essentially we can see at least two examples here. This would be a sperm with an intact DNA.

You can see the halo around the sperm. All right. And here's a sperm head where you have fragmented DNA.

So there's no halo. All of that is missing. All right.

I had other examples there, but this at least is giving you the take-home message here. These are sperm that have normal heads and normal, unfragmented DNA. And this would be a sperm where it did have DNA damage.

People have used also an acridine orange flow cytometry sort of version of doing this, this same assay, but they're using flow sorting again. And here they're using acridine orange, which if you have single strand breaks, it fluoresces red. And if you have double strand breaks where it binds, fluoresces green.

And so in the flow cytometry, you're basically analyzing the sperm for the number that have red fluorescence over the total fluorescence of the green and the red fluorescence. And this is commercially available. And the methods are all also published on how to do this.

And Dr. Evenson actually died last year who invented this, but the company is still in operation. But I'm saying, you know, again, this is a commercially available test that many, many IVF labs use and send their samples to. Well, why should we measure this? You know, as I mentioned earlier, it was not until recently that the level of evidence of studies that were prospective and well done of patients with where the men had high and low levels of DNA fragmentation were able to be looked at with large numbers of patients.

And what was found was that the men who had increased and high levels of DNA fragmentation, that adversely affected the outcomes of ART treatments, as well as natural fertility. It increased the miscarriage rate. And again, that was quite strong in terms of the level of evidence.

And it's also indicated for couples with recurrent pregnancy loss, which is an unrecognized or under-recognized problem, which again is paternally derived, but affecting pregnancy outcomes. Now, along those same lines, there is now a cytogenetic test. So this is where we're looking in the cells themselves, the sperm themselves at the chromosomes, which are present.

And this is a sperm aneuploidy test, sometimes called sperm fish for fluorescent and cytohybridization. So here we used probes that are specific for the Y chromosome. In this case, it's in green.

The X is in red for girls. And yellow is for control for an autosome, which is chromosome 18. And so here we're looking at a normal sperm, right, which is haploid, one chromosome 18, and one X chromosome.

So this is an X-bearing sperm. This is also an X-bearing sperm with one chromosome 18 and one X chromosome. And here is a Y-bearing sperm with one chromosome 18.

And here is our Y chromosome as well. And you have to focus up and down also to get the, you know, the right plane of focus, right, for where the chromosome is in the cell. But it's very clear and easy to distinguish the normal sperm that have normal complements as compared to the ones that have an extra chromosome or a missing chromosome.

And so when you have aneuploidy, that's when you have one or multiple chromosomes above or below the normal number, right? So if we had two X chromosomes, right, if that sperm fertilized the egg, you would have XXX female. Or if this chromosome had, I guess, two Ys, right, which can happen, then you would have an XYY male, which is also abnormal. That's Jacob's syndrome.

So normally, sperm should have a haploid complement to the 22 autosomes that I showed you earlier, and one of the sex chromosomes, right, the X or the Y. But when you have an aneuploid sperm, there's a loss of gain of one or more autosomes or sex chromosomes. So if you take unselected infertile men, again, all comers, and do sperm fish on the sperm in their ejaculate, you see a tenfold increased incidence in sperm chromosome aneuploidy. And this is despite them having a normal karyotype.

So what happens is during meiosis, when you have crossing over, remember during meiosis, you have to come together, you cross over, and then you have to segregate the chromosomes, and right, so then the crossover portions should segregate appropriately. But if they don't properly segregate, that's how you can end up with extra chromosomes or missing chromosomes. And in normal fertile men, you see very little of these aneuploid sperm.

And this is looking at five chromosomes, so 13, 18, 21, X, and Y. And that's because all of these genes, if you have an extra copy, result in a syndrome for 13, 18, 21, right, Down syndrome, Pateau and Edwards syndrome. And then, of course, the sex chromosome defects, which I alluded to before. So they use five-color fish instead of the more simple three that I showed.

But in this study, Dr. Templato did a great, very expensive study where she looked at many semen samples, and they analyzed every single chromosome practically. You see we're missing chromosome 10. There's a few that are missing, but the fact remains that the aneuploidy rates are incredibly low, all right? And even when you add them up together, it's still extremely low for normal fertile men.

But here we looked at, and this is my lab, sperm aneuploidy in couples where they had recurrent pregnancy loss. The male partners all had normal semen parameters, right? And what we found, and we also had a large number of fertile controls. So you can see the aneuploidy rates here, and it's by color for XY, you know, disomy, that would be sex chromosome disomy, chromosome 13, two copies, two copies of 18, and two copies of 21.

So this would be a Down syndrome child if it's conceived. And of course, we're only measuring five out of all the chromosomes, and that's because when you have aneuploidy of those other chromosomes, it's not consistent with a viable birth. And chromosome 16 is one that's common in terms of causing those abnormalities, but it can be any of them, because in some patients, it's a global event that occurs in the man in spermatogenesis.

And you can see that we see two things in particular. One is that when we look at the various men, obviously their rates of aneuploidy, when we add up these numbers, add up to very high amounts, some approaching 50% of the chromosomes, where we looked at 50% of the sperm, where we looked at these five chromosomes, are aneuploid. And what these men have, really all of them that I'm showing you here, is a global kind of problem with meiosis, where they had many, many chromosomes that didn't segregate properly.

Yes, there's some variation and preference between different patients right here. So they had high levels of chromosome 18 disomy. This one had a high level of 21 disomy.

But the fact remains that all of these are tremendously abnormal compared to our fertile controls, which are consistent with the values presented previously by Dr. Templato. And so there are some rare patients who have very specific chromosomes, like maybe just a disomy 21, right? And that's thought to be due to some structural motif that maybe predisposes just that one chromosome to not be segregating properly during meiosis. But more commonly, what we see in these recurrent pregnancy loss patients is this type of situation.

So why should we do this? I'm asked this by the IVF folks who say, oh, well, we can do all types of PGD, PGS, all the different varieties of pre-implantation genetic diagnosis. But the advantage of doing this is that, first of all, we were able to show that some people had even had one defect in all the sperm one man in his testes that was totally not in his somatic cells. And then all the embryos he was forming in IVF, and they had pregnancy loss and were having problems with birth defects and so on for the couple.

But it turned out that he had like a chimeric chromosome in the sperm where his somatic ones were totally normal. But for him, it was every cell that was affected that way. So you can do also custom kind of analyses of these couples.

But I think the important thing is that couples can make educated reproductive choices. You know, they can go for counseling. They can decide, do they want to keep going the course? Do they want to do PGD or whatever type of whole genome sequencing that you may be using on these embryos before transfer? They might choose to adopt.

They might choose to have donor sperm. They might choose to stay the course and hope that they have a lucky day, that they have a conception naturally. But at least they make an educated decision.

And it's before they have to make a decision when they've got embryos that they have to decide about. So I think that it's not perfect, but it's a very effective genetic screening tool. And we know very clearly what happens when we inject sperm or have oocytes that have aneuploidy.

We know what happens to the offspring, right? There's good data on that. And that's what's done in post-implantation genetic diagnosis as well. So now, there's also a big interest in Europe.

People are looking very closely at the assessment of interleukins in the genital tract inflammation. These are very simple assays that, you know, again, kits are available that you can adjust to use for looking at inflammation in the male genital tract, right? And this would be more for men who have, you know, irritative symptoms or chronic prostatitis, pelvic pain syndrome. But again, these are looking at evidence of inflammation in the male genital tract, very commonly done, especially in Italy.

There's also tests related to immunology that have been approved in this panel of different types of immunologic infertility assessments. These are not widely done in the United States, but again, other parts of the world, many people, many labs do these types of assays. We used to run a number of these ourselves in my large andrology lab at Baylor.

Now, in terms of the advanced examinations, these are assays that WHO-6 said are not ready for clinical use, but they can be used in research, right? And so we included them. And some of these, I can tell you, I do research also on non-hormonal male contraceptive development, and we certainly did studies of acrosome reaction. We looked at some ion flux and transport in sperm.

And it's important for you to know that according to WHO-6, computer-aided or computer-assisted semen analysis, CASA, was moved from the routine diagnostic test to the extended advanced examinations. And that is because CASA, you can have two instruments from the same vendor with very different results sitting side by side on the same semen sample. And then if you try to compare those results to the manual semen analysis or with a different vendor's CASA system, the numbers are totally incongruent.

And it was felt that it's useful, say, for me looking at, say, agents for non-hormonal male contraceptive where I'm trying to impact maybe the acrosome reaction or capacitation or something like that. And so those results can be quickly obtained, but the numbers are incongruent with the actual values and numbers that we all think of what describes normal semen parameters. It's not to say that these are not going to improve with time, and it's thought that there are emerging technologies that are going to improve CASA as well as other computational advances.

And I can tell you that there are companies working night and day to be the first to be coming out with many of these advanced technologies. And, you know, so stay tuned. But at the moment, this is not considered appropriate for routine semen analysis, even though IVF labs use them heavily.

We also do describe vitrification now in terms of cryopreservation of the sperm, and I know you do it all the time. And the results that you get are thought to be quite good. But, you know, the problem is that there is no sort of safety data that's been prospectively done to determine whether sperm vitrification has effects on the offspring health or the long-term development, you know, and growth of the fetus and so on.

And so it was considered an experimental procedure, knowing that IVF labs use it all the time, but it was not advised. I think it's very important to realize that our guidelines in WHO-6 also stressed that for sperm cryopreservation, it's best to use semen and not density gradients before you freeze the sperm. And that has to do with, first of all, all the free radicals that are formed when you're doing these density gradients, the poor yield of sperm that you get relative to the total concentration, and the fact that semen is actually preferable in terms of the thaw rates that you get doing sperm cryopreservation.

And I had a sperm bank with 76,000 patient samples, and I can assure you that we, as well as most standalone andrology labs, would not use density gradients. We use them sometimes, right, in patients for IUIs and things like that, depending on the semen sample and what we had to separate out, but certainly not for doing sperm cryopreservation. The appendices is really just, you know, information for you.

What was changed is that we had more data on coming up with the normal ranges, right, for what is normal in the population. So we had a large increase of about 3,500 men from 12 countries in continents that were not well represented in the first WHO-5 data. And really, there's no clinically important changes that came from it.

They're just relative small deviations that are not clinically significant. So at least the data is reproducible still. And again, you know, all of these men's samples were measured in different labs across the country, but ones that were very rigorous with following the protocols and the methodologies.

And then finally, and we're going to rush, but we talked about the karyotype analysis and other genetic and genomic tests that can be done for looking at male infertility. I have some of the syndromes that you'll see here. We've already discussed Robertsonian translocation and Kleinfelter and Jacobs syndrome.

I think you're all very familiar with Y-chromosome microdeletions. There are also some X-chromosome microdeletions which cause male infertility. And one is TEX11, which is on the female-specific X. And then there's also autosomal microdeletions and microduplications, some of which, in addition to the Y-chromosome, these other autosomal microdeletions can cause male infertility.

They certainly underlie a great deal of congenital genitourinary birth defects, upper and lower track birth defects, and things such as cryptoarchidism, hypospadias, and so on. But I just wanted to give you some examples where sperm is impacted by gene defects and their function is also impacted, and not just their function and morphology. So I want to give you first two examples of teratozoa of spermia.

You know, we've all seen all the different types of abnormal sperm morphology. And of course, this is the normal sperm of what we all, you know, seek to hopefully find in samples. But when we have patients with globozoa spermia, which is round-headed sperm, that's where the acrosome is either absent or atrophied and misplaced, and you end up with a round-headed sperm.

So this is a normal sperm by confocal microscopy. Here is the equatorial ridge and the acrosome right over that, so the acrosome is intact. This is electron microscopy, and here you can see another image of the sperm.

So here you can see the very round head compared to what we see here in the normal sperm. And here we have a misplaced acrosome where some of the acrosome material is here. But again, the round head is the prevalent thing that we see under the microscope.

And here, again, we have atrophied acrosome, right? But in all of them, we have round heads. Sometimes these heads are actually large for the size, but it turns out that we know a lot now about genes that are involved in the formation of the acrosome, and it turns out that two of these genes account for defects, account for many of the types of globozoa spermia. SPATA16 is spermatogenesis associated 16, highly expressed in the human testis.

This is one of the most commonly genes that has damaging mutations that represent a good deal of globozoa spermia. There's another gene called DPY19L2. Don't ask me why they picked all those letters, but that's the name of the gene.

And here you could have either copy number, right? Again, structural chromosomal variations or damaging mutations resulting in deficiency of that protein. And it's more common in some geographic and ethnic regions, including some U.S. and northern European men. And these men have mostly type 1 globozoa spermia, but a high percentage of abnormal sperm.

But it's important to know that they have these gene defects because in the IVF lab, right, they're going to just take those globozoa sperm and inject them into the oocyte. And yet the likelihood of achieving a pregnancy with these gene defects, or there's about seven others genes, gives them a very poor likelihood of achieving pregnancy and certainly not a live birth, even when you do all the oocyte activation and so on. And so it's important for couples to take this into consideration before they decide to go on this very expensive course of treatment with ICSI-IVF with egg activation when the odds of getting a child are very low.

There's another type of morphology defect, which is called macrozoa spermia. So you have the mega head, the macrozoa spermic sperm, and here you have four tails, right, flagella, with this large head. And this is normal, what happens during normal meiosis.

It's due to defects in aurora kinase C, which is involved in meiosis at the single spindle, at the meiotic spindle checkpoint, when you have the attachment of the two sort of divided chromosomes at the centromere. And when this protein is absent, you have a problem with this, so you never get cytokinesis to separate the individual sperm and cell division. So instead of having, you know, the first and the second meiotic divisions to get a functional sperm, you end up with one sperm with essentially four complements of chromosomes and four tails because you never had the appropriate cell divisions occur.

And again, when you see these large-headed sperm, these men have essentially no chance of achieving a pregnancy with doing ICSI. The mutation that causes this is common in men of European and northern African origin who have this macrozoospermia, the big-headed sperm with four tails. There are a lot of other gene defects which cause both globozoospermia and other head deformities, so it's important to realize that, you know, even when you have these abnormal shaped heads that you may see, there's a ton of different genes, some of which have lots of different functions.

This one in particular is involved in capacitation of sperm, but you can see that there's many different genes that are involved that can also cause these types of defects. And right now, there's like another 145 known and candidate genes associated with teratozoospermia. And so again, this just summarizes what I just told you in these slides, but it's important to know because mutations in these genes suggest that there's going to be very poor ICSI-IVF outcomes, and in some, no possibility of a live birth, depending on the defect and the genes involved.

And then finally, what about men who have either poor motility or poor motility with poor morphology as well? There is now a different, another category when we do morphology of sperm, and this is multiple morphologic anomalies of the sperm flagella, or MMAF. And so here, I'm just reminding you of, like, if we do cross sections, right, down the sperm flagella, right, we see different structures, right, going along. We see commonly, right, the dimer, right, here in the center, and then the microtubules, right, that are in between, and dienine arms, and, right, these are the various microtubules, right, that are causing the central pair, and then these peripheral set of structures that are required to be the motor to drive the flagella to get the sperm to swim.

And so in patients that have poor motility, there are a number of different mutations in DNA, H1, dienine, axoneal, heavy chain 1, and then another gene, cilia and flagella associated protein 44 and 43. These are the majority of cases that cause poor motility. And again, you see the defects of the centriole assembly and the periaxoneme structure, right, that I've just showed you that affect both the proximal centriole, right, as well as, you know, the 9 plus 2 pairs, this, right, they go down the inside of the flagella, and then you can also get, like, disruption of the fibrous sheath and outer dense fibers and all types of abnormalities.

There is one variation of this called primary ciliary dyskinesia, where, again, you have poor motility or absent motility, actually, in the sperm as well as every cilia in the individual's body. So they have airway disease, infertility, they have laterality defects where their body organs are reversed, right, so they have situs inversus where, you know, the stomach isn't here, it's over here, and so on, which is complicated, but again, it's due to loss of these inner dianine arms that I just showed you in the previous slide. And now, with whole exome and now whole genome sequencing, there's a lot of known about the causes of this primary ciliary dyskinesia, can be in men or women.

Clinical labs now assay about 89 different genes and their defects that affect all aspects of the development of these structures that I showed you before that result in the cilia not being able to beat and move, and there's huge numbers of additional genes. These are ones that are well known to cause poor motility and primary ciliary dyskinesia, but there's another 100,000, 245 known and candidate genes associated just with primary ciliary dyskinesia, and then 413 candidate genes for acenozoa spermia, and then genes, 49 genes that are known and candidate to cause acenoteratozoa spermia. So, stay tuned, because there's lots of genes and gene discoveries in the future, which really should be considered in the diagnosis of these patients, because it has significant effects on the offspring conceived.

And just to tell you that this is the tip of the iceberg, right? So, now I told you a little bit about genomics and epigenomics, but there's also transcriptomics, proteomics, metabolomics, lipidomics, and glycomics, right, for glycosylations, all of which tremendously affect functions of cells. So, you know, there's plenty more work for scientists like me to be able to look at these, and the thing that's really needed for us so seriously is phenomics, which is what are the patient phenotypes to really have really good, strong evaluation of patients, noting subtle dysmorphic features, right, that are frequently overlooked, you know, in the clinical exam. So, I hope I convinced you that, you know, that semen analysis is still important and that there are also other tests which are important to perform as well, given the clinical settings and the need and what's seen in the patient phenotype.

In WHO-6, again, there's new protocols for DNA fragmentation, sperm aneuploidy, and especially for inflammatory cytokines, and I think it's much easier to use it. And finally, I just want to stress that, you know, this application of what I'm going to call big omics, where we have whole genome sequencing on, you know, embryos, parents, you know, maternal, paternal sequences, you know, it's underutilized, but it probably holds the future for us diagnosing patients. So, in the future, eventually, we're probably going to do a quick look at the sperm and the semen and then do genetic testing to ask what are the abnormalities that these patients have, right, that are causing, in this case, either spermatogenic failure or sperm function deficiencies or problems with, you know, spermiogenesis and so on and flagella function.

So, I think that, you know, we're going to see that there's a plethora of causative genes, some of them more common than others that underlie these defects. Phew. Oh, and we're still late.

I'm sorry, but we're close because we started late. No problem. Yeah, no problem.

So, questions? Okay, guys, don't be shy. That's your chance. I do have one question.

In sort of the data that you presented, do we have any correlation, I guess, between some of these genetic abnormalities that we're seeing and the morphology that we're seeing? I mean, especially when we're deciding on, say, something like ICSI, and we find something that has a morphological normal head, are we seeing abnormalities in the morphologically normal sperm, especially when you're choosing sperm for ICSI, I guess is my question. Yeah, so, you know, what do you do, and at the magnification that you use for ICSI, and we were actually doing ICSI before Palermo in my lab years ago, and we were too gentle, right, and Marina was there, right, and she knows, right, and remembers all of that, but we were too gentle, right, because I never would have sort of popped the cytoplasm, you know, to activate the egg and so on, and once we knew to do that, you know, we were off and running, but I think that I can tell you that, for example, so we had a very fancy setup where we could screen like an entire, when we would do sperm fish, the instrument could screen an entire slide of sperm smeared on the slide for doing sperm fish, but in our study, we first did strict morphology on every single sperm, and there's a micro locator, right, so the computer can take the head of the scope, you know, and focus on sperm 523 or whatever, so we had strict morphology that we could correlate exactly with whether or not a sperm was aneuploid or not, all right, and we did it, you know, obviously for fertile controls and men who had high levels of aneuploidy, and strict morphology, at least, didn't show any difference between the aneuploid sperm heads and the not aneuploid sperm heads, and, you know, now, of course, and some of these defects are even smaller, right, where you're looking at like small gene defects. I can tell you, I have a paper coming out in, a review paper coming out in endocrinology, which talks, in this case, about infertile men's increased risk of a cancer diagnosis.

In general, they have a two-fold increased incidence for cancer diagnosis within five to seven years of their semen analysis, so young men, 20s, 30s, 40s, but then, as you stratify by count, it goes up to, depending on if it was our data, Baylor, it was like an eight-fold increased risk, and at Weill Cornell, it was about a 12-fold increased risk. I think that was a little biased due to the patients going to Cornell for microtessing, right, and severe kind of male infertility, but my point is that, you know, that paper talks about, like, some of these morphology defects and some of these gene defects, right, that we've identified, like, that I mentioned even today, with associations with cancer, even though they have nothing to do with DNA repair or DNA replication, right, and, but they're affecting other processes in the sperm themselves, so, you know, I think it's going to be interesting to see as work goes on, because people haven't focused on the cancer studies, but then the cancer patients had, say, damaging mutations like our patients, but were also, the malignancy had a high, say, expression rate or a low expression of the genes, right, depending on which they were, but my point is, I think we're still at an early stage to really know what is really a healthy sperm, right, we can't really tell by looking at it. I think that some of the, there are some very novel, totally, totally different principles being used for imaging sperm by various research groups, you know, in Harvard and several different places, and I think that that, together maybe with doing machine learning form of artificial intelligence, will help to perhaps pick out a healthier sperm, but it's still not going to tell you the one that has the gene defect, you know, causing, you know, flagella defects or, you know what I'm saying, so that's why I think that a lot of our diagnosis, even though WHO didn't want to hear this, they wouldn't let us talk about anything that wasn't done on sperm or semen, so even though, like, you know, we're looking at the cause of the sperm defect, it didn't matter, like, for the WHO manual, but I think eventually it's going to be a lot of it genetic rather than, you know, morphologic or even functional, and, but again, you know, it doesn't help you with picking the best sperm, right, so did that help? Yeah, I mean, I guess that's the ultimate question is, is there any sort of predictor on choosing the right sperm, but I guess at this point, with what we're discovering about genetics, there is no clear associated marker for that.

Right, and, you know, like, we've, like, if you look at consanguineous families or people living in small countries, right, where there's a lot of sort of close breeding, if you will, they have, like, what I'm going to call, what do they call it, but it's like a familial, right, so, like, they may have a defect in, say, a spermatogenesis gene, and it may be common just to that family or just to that neighborhood or whatever, but then, like, you look at the rest of the world and almost no one has it, you know, it just kind of perpetuates sometimes in certain regions, so I think that there's a lot of genes involved, but, you know, the reality is that probably some are going to be more common than others and looked at. Yeah, did that help? Yes, thank you so much. Yeah, yeah, yeah, but we're a long way from picking that perfect sperm, and I will tell you, you know, like, you all are using, like, all the microfluidics and so on.

In my mind, it's like a glorified swim up, right, so the good thing is that you're not doing, you're not doing centrifugation, right? Basically, so much. Yeah, so that's, that's the advantage, but, you know, when people are saying, oh, DNA damage is minimized and so on, there's no, there's no, if you think about physics, right, and fluid flow and, you know, and then the sperm swimming down, but there's no reason why they should have less DNA damage, right, that would be the basis for that separation. So I'm just saying, you know, with all these things, we don't totally understand good or bad, but certainly I think it's better than a, you know, centrifuging and doing a swim up, right, but yeah.

Dr. Lamb, I didn't get to introduce myself earlier. I'm not one of the students. I'm one of the faculty organizers along with Maria and Stephanie.

We have met in past, but, you know, in passing amongst many other people. I am glad you just touched on microfluidics because I think all of the students have also come across the use of that likely in their lab or consider the use of that. And, you know, at our, I'm at Yale and we don't routinely use that for the same reason you mentioned.

There's no real evidence to show that it does make an improvement. Yes, it makes the workload a little bit easier, but not significantly so. Just a plain old swim up is also, yeah, that could be considered as well.

And I just also want to point out there is actually centrifugation. If you use one of these microfluidics, if you're doing standard insemination, once you pull that sample off the chip, you still have to wash it once. And then it's, so there is, you know, a centrifugation step, if not.

Yeah. But it's still like one centrifugation, five minutes versus density gradient. That's true.

That's true. Almost half plus two. Isn't it a soft wash anyway? Don't you do a soft wash? I mean, like lower deforces.

Yeah. Yeah. Yeah.

That's yeah. That's similar to the Zymot. Yeah.

Zymot is one of the microfluidic devices. Zymot doesn't require any centrifugation though. Not for ICSI, but for IVF it does.

Oh, see, we never do. I know you don't. You always do it.

Right? I think you've done it like three times in the last four years. So that's, that's actually for the record, that's opposite to Dr. Lam's music to her ears. Yes.

We do a lot of ICSI and not a lot of conventional. Doing 100% ICSI on everybody. But, you know, we all know that the practices vary between between different programs.

Oh yeah. For sure. For sure.

And I mean, that's true for the semen analysis too, right? Which we know only too well. So yes. Yeah.

But I'm sorry we went over time a bit. Oh no, I think it's all for a good, good reason and good measure. And I mean, I personally am grateful for the time spent.

So I thank you very much, Dory, for accepting the invitation to be on our faculty. And I hope this collaboration continues for the next cohort and the one after and so on. But unless the students have any questions.

I just have a quick question. Yes. Talking about the DNA fragmentation.

And at what level of the sperm DNA fragmentation do you see a consistent impact in the embryo development? So, so every, so you have to figure out what the normal ranges are for the individual tests that you're using, right? And every lab needs to determine that themselves, right? In terms of the, where the cutoff is of normal versus abnormal. So, you know, you would do a comparison between say the infertile men and the fertile men, right? But even if you just took all of the men in the population, right, to begin with, you would come up with the, the percentage of the men, right? Who were, had normal, who, who had lower levels of DNA fragmentation, but those values range tremendously. So for example, in many labs, it's like 30, 31%, 32%, right? It would be the cutoff of the, the upper limit for the normal range, right? For the, for the undamaged, right? The fertile men.

But in other places, you know, say using, we used Comet, but for other places using like a tunnel assay, some people report like 4%. So then like everybody that they test has DNA fragmentation, and we know that that's not true, right? And it has to do with problems with the way they count and so on. But I, I'm just saying, when you, when you look at all these different tests and the varying controls, right? And the lower limits of normal.

So, you know, you need to apply the principles of clinical chemistry, right? To determine what that lower limit is. And, and that's what's done throughout WHO 6, right? But, but we can tell you how to do that, but every lab needs to validate their, their, their ranges, right? Of normality, regardless of what they're measuring. Did, did that answer the question? Yeah.

It's okay. Yeah. It's okay.

Okay. And we do talk a lot about, you know, how to, to calculate all of those things, because again, people do it wrong. I'm sure the two and 3%, as you know, the, the, anything over that is DNA fragmentation that they've going to be doing all these extreme things too.

So that like drums up a lot of business for them, right? You know and I don't know where they came up with those numbers, right? So it's hard to understand. Let's put it that way. So, so Dr. Lam, so in application, let's say we have a gentleman that comes through their RPL and they've undergone a sperm DNA fragmentation testing.

We've established our lower limits in-house and we determined that he has a, an abnormal percentage of DNA fragmentation. What next, what are we going to do for this male when he comes into the IVF lab for ICSI, which most, the physician will choose ICSI for sure. Yeah.

And as you said, there's no, there's no way for the embryologist to know this one versus this one, right? Well, you're just going to pick and hope that you're playing a numbers game essentially, right? You're, you're at the lottery with the correct one. Yeah, absolutely. So well, of course, the urologist, a bunch of them are going to give all types of supplements and things, right? And anti-oxidants, anti-inflammatories, you know, and so on.

And I'm not telling you that that's the right approach. And at least, you know, because we would see patients back, you know, after treatments and so on. And with the exception of men who had, you know, really big infections, you know, I'm saying genital tract infections.

But with that exception, we never saw anybody improve on any of the treatments that the urologist gave trying to diminish DNA fragmentation. And the numbers were quite consistent over time. And I think that, you know, but in the IVF lab, you know, the problem is that you really have no way of knowing which sperm is damaged and which isn't.

I mean, you might expect that if it's, you know, if it's like one out of three sperm are damaged, then maybe you would expect like 66%, right, of the sperm to, or embryos to be okay. But you don't really know that, right? You have no way to measure it. So, so therein lies the problem, I think.

I don't think that the only place, you know, like I lectured at ASRM, past president there, but I lectured at ASRM last year, on when is it justified to do a testing when a man has sperm in their ejaculate? All right. And, you know, and so DNA fragmentation was one example of that. And there is some evidence that for men who have high DNA fragmentation, that it is appropriate then to try to take sperm from the testis.

And, you know, and the studies have supported that, right, in terms of looking at it. I think, and also, you know, I helped with one of the urologists who had a patient actually with necrozoa of spermia, right? And he had great semen parameters, and, but everything was dead. And the couple was insistent that they wanted to try, right? And, and the urologist told them that, you know, well, we don't think insurance certainly is not going to pay for this because we can't justify this based on our practice guidelines.

And the guy wanted to pay cash. And, you know, and I, I said to him that, you know, conceivably the sperm at spermiation are alive, right? And here you're taking the testicular sperm, right? You're not even just taking epididymal, right? You're taking it fresh from the testis. And sure enough, they were able to get, and I forget the exact numbers.

It was like 10 embryos. I know they transferred one and froze the others. And the lady got pregnant and she should be delivering soon.

She was, she was like five months along at ASRM. So maybe she already delivered, but I'll touch base and find out. But, you know, at least in that case, it, it did make a difference, right? And of course it was Tessie Icksey, but they didn't have to look for rare sperm, although they were very careful with the ones that they picked and they did hypotonic, right? You know, tests like we used to do, Marina, to pick the viable sperm to make sure not to take a chance.

Wow. Yep. It's good.

It's good. But that's the thing. You, you, you, you came to that suggestion in a very, it made sense biologically speaking as well, right? I mean, it makes sense.

Absolutely. I am saying these ideas then are sort of heard by other people as well, and perhaps like morphed into something like, how can we make money on this? We'll suggest this. That's completely different from like translational approach.

Why is it happening? And then, you know, will this help or not? I actually, I actually had someone order just, just today, basically a peer physician orders oocyte activation on a patient who had almost a hundred percent fertilization in the previous IVF cycle. So, yeah. Yeah.

But I was going to say, this man had huge numbers of sperm in his... They were just all that. So something was happening in the epididymis there. Or someplace.

Yeah. Yeah. Oh, someplace.

Yeah. Who knows? His... But... Geminal vesicle fluid was toxic. It was just making... I still worry, you know, what if there's some major reason, right? Why the sperm all died, you know? And, but, you know, they were, I mean, they had a huge consent form, you know, I'm saying with like every possibility of things going wrong and the couple was determined to do it.

So, yeah. Which is a problem. Also, they're vulnerable, right? And desperate.

So... Of course. Of course. Up to them.

Of course, we hope for the best, but yeah, you do think about there's maybe an evolutionary reason why. Yeah. And maybe, like, what else is wrong with this man's health? I think he needs to be, you know... Oh, he was very healthy.

I mean, they did every test known to man on the guy, honestly. They were very cautious going forward. And again, the urologist said, you know, like, I'm not telling you to do this at all, right? Because nobody knows, right? In this, for necrosoaspermia, I think there was one report, but it was not particularly well done, you know what I'm saying, in the literature.

So... Right. And this hasn't been written up, but... Yeah. Hopefully they will write up a case report for all of us to use.

Or not. Or not. Yeah.

So... I think we'll just, we should let Dory go. Thank you so very much for today. And if our students have any questions going forward, we know where to find you.

Yeah, that's fine. Y'all can reach out to me. I get like a thousand emails a day.

So, like, if I don't answer, just do it again. You know, it's not on purpose. It's just that it's overwhelming.

Okay. You can also do it through me. I can text her directly.

So... Oh, yeah. It's nice. It's nice to be connected.

But no, I'm just kidding. We're not gonna abuse any connections or anything. Thank you all very much.

And we will all, unless you want to have any questions for me, and you can stay after Dr. Lam leaves, we will see each other on Sunday in person for on-site training. Good. Good.

Enjoy. Enjoy. You've got a great day.

Thank you. Thank you. Bye, Dory.