Journal Club Global LIVE at ANZSREI 2025

ANZSREI panel reviews a PGT-A study on male age and sperm factors in blastulation and euploidy, finding female age dominates and calling for better research.

Article discussed:  The influence of male age and sperm parameters on blastulation and euploidy rates

Discussants:

• Dr Derek Lok
• Dr Jeffrey Persson
• Prof John Aitken
• Dr. Yousif Alyousif
• Dr. Hossam Elzeiny 

So welcome everyone today to what has become an annual event, the Fertility and Sterility Podcast at ANZSREIconference. We as the Australian and New Zealand Society of Reproductive Endocrinologists and Infertility Subspecialists have the honour today to invite a panel of experts to discuss a paper published soon in Fertility and Sterility on the influence of male age and sperm parameters on blastulation and euploidy rates. I'll introduce you to our esteemed panel today.

I'm Dr. Aurelia Liu, Medical Director of Melbourne IVF and SREI subspecialist from Melbourne Victoria. On the panel we have Professor John Aitken who is a Distinguished Emeritus Laureate Professor on Biological Sciences from the University of Newcastle, New South Wales. John's research career began with a PhD in Reproductive Biology from the University of Cambridge.

Following post-doctoral positions in Edinburgh and Bordeaux and a year with the WHO in Geneva, he returned to Edinburgh to join the MRC Reproductive Biology Unit and establish a research program with clinical outreach into male infertility. In 1998 he moved to the University of Newcastle in New South Wales as Chair of Biological Sciences and later Director of the ARC Centre of Excellence in Biotechnology and Development and Pro Vice-Chancellor of the Faculty of Health and Medicine. He is currently the Distinguished Emeritus Laureate Professor of Biological Science.

We also have Dr. Jeff Perssons. Dr. Perssons trained and worked in Amsterdam before obtaining his Master in Reproductive Medicine. In 1995 he received his fellowship from the Royal Australian and New Zealand College of Obstetricians and Gynaecologists and his CREI with his thesis Genetic Aspects of Reproductive Failure.

He's the Clinical Director of IVF Australia's City Unit in Sydney, New South Wales. With research interests in genetics and male infertility he's a pioneer in the field of advanced embryo selection for chromosome analysis and digital high magnification to assist with male infertility issues. As an established expert in male infertility as well as female infertility he receives referrals for complex cases in Australia.

He helps men with azoospermia with using sophisticated microscopic sperm extraction procedures and is a leading expert in microsurgical vasectomy reversal. Next on the panel we have Dr. Yousif Alyousif. He is a Senior SREI trainee in Melbourne, Victoria at Melbourne IVF and he'll be presenting our paper today.

Comparing our panel we have Dr. Micah Hill. Micah is a board certified OBGYN and SREI. Dr. Hill completed his residency in OBGYN at Tripla Army Medical Centre Honolulu, Hawaii.

He then completed his fellowship in REI at the National Institutes of Health in Bethesda, Maryland. Prior to joining Shady Grove Fertility Dr. Hill retired from the US Army as a Colonel with his most recent position being the Medical Director for the Assisted Reproductive Technologies Program and the OBGYN Research Director at Walter Reed National Military Medical Centre. He has served as Media Editor for Fertility and Sterility, President for the Society of Reproductive Endocrinology and Infertility, President-elect and Quality Assurance Committee Chair for the Society for Assisted Reproductive Technology SART and on the Practice Committee for the American Society for Reproductive Medicine.

He served on the Board of Directors of the American College of Obstetricians and Gynecologists SART, SREI, PCRS and ASRM. As an avid researcher Dr. Hill has contributed to hundreds of publications and presentations exploring topics such as IVF protocols and outcomes from frozen embryo transfers among many others and we're very proud to have him today at ANZSREI. We have Dr. Hossam Elzeiny who is an Australian expert in male infertility, a SREI subspecialist and Medical Director of City Fertility.

He's also a Visiting Medical Specialist at the Royal Women's Hospital in Melbourne and has special and advanced interests and expertise in Andrology, Microteasy and Male Factor Infertility. And finally on our panel we have Dr. Derek Lok, Clinical Director of Genea Clinics Liverpool and Bella Vista, Obstetrician and Gynecologist, Fertility Specialist and Advanced Endoscopic Reproductive Microsurgeon in Sydney. Dr. Lok has major interests in different types of IVF treatment including embryo selection and pre-implantation genetic diagnosis, recurrent miscarriage and IVF treatment failure management.

Dr. Lok conducts microsurgical vasectomy reversal through small scrotal incision which results in rapid recovery. Derek is a Certified Level 5 Laparoscopic Surgeon specializing in fertility and gynecological surgeries including laparoscopic tubal reversal and tubal surgeries. He is also a Consultant Gynecologist at Royal Prince Alfred Hospital in Liverpool Hospital and Westmead Hospitals here in Sydney.

Welcome to our panel and I'll hand over to Dr. Micah Hill to compare. Great, thank you so much for the kind introduction and for welcoming Fertility and Sterility to your meeting. To our listeners around the world, we're delighted to be here in Sydney at this esteemed meeting of the Australia and New Zealand SREI and to have this expert panel to discuss this paper on male contribution to aneuploidy.

Yousif, I'm going to turn it over to you to tell us about the paper and then we'll dive into the discussion. Perfect, so Micah to the front. I've got a few slides.

So I will introduce the paper and set the scene and then hand over to the panel of experts. So the title of the article is the influence of male age and sperm parameters on blastulation and euploidy. The first author was Ibrahim Elkhatib and senior author Human Fatemi that was done at the Art Fertility Clinic in the United Arab Emirates.

The objective was to study the association between paternal age and sperm parameters on fertilization, blastulation, as well as euploidy rates and the sperm parameters they looked into was concentration, mortality, and morphology as well as sperm origin, whether it came from ejaculated or testicular sperm and they also looked at fresh and frozen sperm. The study design was retrospective cohort study. They included all PGT-A cycles performed in that one single center between July 2017 and August 2023.

They only included autologous cycles, both IVF and ICSI and they excluded PGT-M and PGT-SR. The control ovarian stimulation, sperm preparation, and insemination all done as per established protocols. In terms of blastocyst biopsy, this was done between day five and seven on embryo grade at least three cc or above.

The PGT-A analysis was done in two genetic labs. One was outsourced and one was in-house. It's not clear if there was a change during the study time frame or if they both ran in parallel.

They used next-generation sequencing for their PGT-A. They included for more than 47,000 metaphase 2m2 oocytes from 5,847 cycles. The study doesn't specify how many subjects or individuals were involved.

In terms of the statistical method, the clinical measures like normal fertilization and the euploidy rates per blastocyst were analyzed with what they called mixed effect log binomial regression model. For couples that had multiple cycles, they also introduced random intercepts to account for dependency structure. A lot of work is there, so we'll just unpack that a little bit.

First, imagine we're trying to figure out what affects whether an embryo is euploid or not. Yes, no, there's no answer. We want a test that will look at proportions and probabilities, not a linear model.

That's what the log binomial regression where it comes in. It lets you estimate risk ratios. Like saying men over the age of 40, for example, has 10% lower chance of having outcome X compared to men under the age of 30.

However, each couple within the study might have contributed to more than one cycle, and each cycle might have contributed more than one embryo. Those embryos are not completely independent. They are kind of like siblings in the same household, so we cannot treat them as independent or strangers.

That's where the random intercept comes in. In practical terms, the model says, let's have each couple have their own starting points, and then we will model the effects like age and sperm on top of that. They're not all starting from the same starting points, and this can account for unmeasured couple-specific variables that otherwise we might miss in the analysis.

My simplistic analogy, because I'm a simple-minded person, is you're not comparing apples and oranges. You're comparing apples and apples, but you're taking into account which three that apple has come from, kind of thing. Enough about statistics.

They also did the sensitivity analysis, specifically looking at the euploidy rate and the effect of male age, and they broke it down into two female age groups, the female age under 35 and female age between 35 and 37. The reason they chose the two groups, because of sample size issues, and the other reason is under the assumption that female age would have negligible effect within those brackets. The results were reported as risk ratios with 95 percent confidence intervals, and p-value less than 0.05 was considered statistically significant.

If we look at the results, specifically looking at the impact of sperm parameters, sperm concentration with severe oligospermia under one million per male seemed to reduce euploidy rate per biopsy with a relative risk of 0.92, confidence interval of 0.86 to 0.99, and a p-value of 0.029, which was statistically significant. It also reduced fertilization and euploidy per M2. When they looked at the stickler sperm, in similar fashion, it did reduce the euploidy per biopsy with a relative risk of 0.89 and confidence interval of 0.81 to 0.97, and a p-value of 0.014. It was also associated with reduced fertilization and euploidy per M2.

The other sperm parameter they looked into is sperm motility, and if we focus on the reduced motility less than 25 percent, there was reduced fertilization and blastulation, but not euploidy. And when they looked at frozen sperm, there was reduced euploidy per biopsy with relative risk of 0.94, confidence interval of 0.89 to 0.99, and a p-value of 0.035, and that was statistically significant, but there was no association between frozen sperm and fertilization or blastulation. Interestingly, this effect was not statistically significant if we limit the analysis to female age under 35 or between the ages of 35 to 37.

The main interest of the paper is the male age, so if we look at table two, which looked at the sperm parameters and outcomes stratified according to male age, and this is one of the sensitivity analyses looking at women younger than the age of 35, and if we specifically look at the euploidy per biopsy, we can see in the younger paternal age group under 30, between 20 to 30, the euploidy per biopsy was 58.5 percent, which drops to 52.7 percent in the paternal age group between 30 and 40, and further dropped to 49.1 percent in the paternal age between 40 and 50, which seems to go up slightly in the older age group to 51.6 percent. And if we look at table three, and that was the univariable and multivariable regression analysis, again looking at the same female age group under the age of 35, and the multivariable analysis took into account the sperm type, morphology, mortality, and sperm concentration, so if we look at the multivariable analysis compared to a paternal age of under 30 as the reference point, the relative risk of a euploid blastocyst per biopsy in the age group between 30 and 40 was 0.96, with a confidence interval of 0.92 to 0.99, and a p-value of 0.011, which was statistically significant, and also in the paternal age group of 40 to 50, the relative risk was 0.92, and a confidence interval of 0.86 to 0.99, and again the p-value was 0.041, which was statistically significant. We lose that statistical significance when we go to the older paternal age group above the age of 50.

The same analyses were then repeated in the second sensitivity analysis in the female age between 35 and 37, but none of the analyses achieved statistical significance, and the likelihood there is the female age is just a strong predictor of euploidy that overshadows the other factors. And we will look at figure one at the back of the paper. We have two graphs.

On the left-hand side, again, the sensitivity analysis look at female age under 35. On the right-hand side, female age between 35 and 37. On the y-axis, we have the euploidy per biopsy blastocyst, and the x-axis, we have the male age categories.

If we focus on the female age under 35, the reference paternal age is under the age of 30. The euploidy rate is around 65 percent and shows a steady drop as the paternal age increases to just under shy of 60 percent in the age group of 40 to 50, and there's an uptick when you get to the age above 50, but such a wide confidence interval because of fewer numbers in that age group. However, again, if we look at all the confidence intervals, there is significant overlap and the upper end of the confidence intervals approach 0.99 or the null value of one.

The female age between 35 and 37, there is significant overlap. There is no statistically significant finding there. If we go back to table one, and we just focus on the female age and look at the euploidy rate per biopsy, it remains as the strongest predictor or the strongest association is the female age.

If we use the under 35 as our reference group, as the female age increases, 35 to 37, the relative risk of euploidy per blastocyst drop to 0.83, further drop to 0.58 in the age group between 38 and 40, and by the time you get to more than 42, the relative risk is 0.14, and all of those were statistically significant differences. So male age and sperm quality, particularly extreme oligospermia and the use of testicular sperm and frozen sperm may influence embryo outcomes and euploidy rates, especially when in the female group under age of 35, but female age remains as the strongest association with euploidy rates, regardless of male factor contribution. One of the references they mentioned in their discussion, as it's actually a study that connects Australia and the US, the first author is based in Melbourne and association with Boston IVF.

They looked at 35,000 embryos, so a much larger study, and when they did a propensity score matching at the two extremes where you have 100% euploidy or 0% euploidy, there was no association even at the extremes between male age and sperm parameters with euploidy. So in terms of this study, it was a large study. They analyzed nearly 6,000 PGT-A cycles.

They used a modern PGT-A platform with NGS. They tried to use sophisticated statistical modeling to account for clustering of embryos from the same couple. They did sensitivity analysis to try and account for the female age under the age of 35, and they included a wide range of sperm parameters, including testicular and frozen sperm.

However, there are a few drawbacks. We know the retrospective nature of the study. It opens it up to confounding and biases.

The use of non-ejaculated sperm may reflect some underlying pathology that could introduce a confounding variable, and there could be potential selection bias, especially when we look at the biopsy per plasticist because we can miss some early developmental errors in poorer quality embryos that might have an association with other male factors and sperm parameters. And we are missing some functional sperm biomarkers like DNA fragmentation. We lack some follow-through outcomes like implantation, miscarriage rate, and live birth rate, which are outcomes of interest for all our patients.

And the female age was excluded from the models due to risk of this variance inflation, which might obscure the interaction with male factors because we know, especially as euploidy still varies within the tight female age groups under 35 and the 35 to 37. So even in the under 35, there is correlation between maternal age and paternal age. And as the female age gets closer to 35, the paternal age will also follow through and potentially could be older.

So we can't separate the paternal and maternal age. They are directly linked. And they did use two genetic providers.

And we know sometimes there is a variance in the euploidy rate in different labs, and it's not clear whether they used them both in parallel or there was a change during the study period. And knowing that an embryo is euploid doesn't tell us about the origin of the aneuploidy. Is it maternal? Is it paternal? Is it at the level of the embryo itself? A lot of the semen analysis parameters were taken from a single sample, and we know there is variability within samples within the same individual.

And many of the confidence intervals approached a null value of one, which tells us while the results might be statistically significant, the magnitude of the effect as bits is modest, and the biological clinical relevance may vary depending on the context in front of you and the patient and the couple in front of you. In terms of future direction, I was pleased to see there's more emphasis on male factor and male infertility papers, and I think we should see more. It's half of the embryo.

And larger prospective studies, I think, are warranted. And maybe to design a prospective study, we can add more advanced techniques like DNA fragmentation, epigenetic markers, which can add another dimension to assess why the outcomes that we're seeing might be associated with, say, a concentration or a morphology number. I think it's important to include pregnancy outcomes because that's an outcome of interest to our patients, but to power a study for those outcomes, we will need a much larger number and potentially a multi-center collaboration.

So now that we've set the scene, I'll open it to the panel of experts to take it further. Thank you so much, Yusuf. That's a wonderful summary of the study.

So as a senior fellow, just a quick question for you before we get to the panel. What did you think they did well from a methodologic and statistical standpoint? And if you were doing the study, is there anything that you might have done different? Do you think there was a limitation or something in their methodology or statistics that you might have approached differently? I think the numbers that they had in the study were high, and I think that gives them statistical power. But that's if we analyze it as per oocyte basis.

But when we go to per patient basis, I think we might lose that statistical power. And we made the assumption there that there is less variability with the euploidy rate in female age under 35. And I think that can be a dangerous assumption, because as you get closer to age 35, we can see an increase in the rate of aneuploidy.

And because of that link between the male and the female age, and if we just focus on the paternal age above 35, the female age is probably approximating 35 as well. And that will introduce a strong confounder that might explain some of the associations that we're seeing. And it's important to acknowledge the confidence intervals that are quite wide and approaching the null value of one.

Yeah. No, I think that's good. The study's been talked about on LinkedIn quite a bit, and some doctors from California, one in particular, Max Ezzati, has focused on what you said is one of the limitations in those subgroup analyses.

They chose not to account for maternal age because of what you described, but potentially that introduces some confounding bias in it. So I want to open it up to the panel. Do we believe this study? Do we think that male age is associated with aneuploidy? Nope.

No? Why not? Well, if you look at the numbers, it was only the groups from 30 onwards, right up to age 60, there was really insignificant difference. I'd fail the current CRA exam for statistics, but I can easily save the problems with this study. And it's just simple maths.

You had a 59% euploidy rate in the under 30s, but all the way from 30 up to 60, it went in those 10-year age groups, went 53, 49, 52. It might be statistically marginally significant, but clinically, it's insignificant. And if you understand that there's distinct biological differences, this is what I think this whole thing opens up.

You start to think more deeply into what happens with eggs and sperm. It's really interesting. The reality is that the biology of sperm, and you're the expert on that, is quite different.

It's got this explosive opportunity to do speed dating once, okay, and then you've got an egg that's sitting around like the possessor of the egg until age 35 for a sperm. And the meiotic processes are extended over that whole 35, say, year lifespan. And meiosis isn't really even kicked in until the processes of us injecting HCG.

And there's a super rapid process. So the frequency of euploidy, we all know from so much data in embryos, comes from eggs. That's my conclusion from this.

Professor, do you agree with him or do you think the paper's right? Well, I think my learned colleague is correct that we shouldn't overestimate the paternal impact on aneuploidy. As a generalization, I would say that in embryos, the mutational load, single point mutations, is largely paternally determined. That's where most mutations come from.

But aneuploidies, by and large, come from the female germline. But I wouldn't be totally denying that there's some possibility of aneuploidy being paternally derived. And what I think is really interesting is to go into the mechanisms.

What might explain this kind of thing? And it's one of the problems with a retrospective study is that you're wise after the event. If you had the opportunity to redesign that study, there are some things that you would want to have measured to see if they shed some light on the mechanisms. And the obvious things that I would like to have seen would be some measure of sperm DNA fragmentation and oxidative stress.

And I say that because the sperm delivers two things to the egg. It delivers the male genome, obviously, but it also delivers a number of epigenetic factors. And I'll talk about those a bit in a minute.

But the major factor, one of the major factors that introduces are the centrioles. Every cell division in your body is orchestrated by your father's centrioles. And we do have data that the centrioles are very vulnerable to oxidative stress.

So if the embryo inherits a damaged centriole, it's very likely to be associated with chromosome instability and an increased risk of aneuploidy. So I think there are plausible mechanisms there that might explain a paternal impact. And all the things we look at, whether it's low sperm count or cryo storage or male age, they're all associated with oxidative stress.

There's a plausible mechanism by which this aneuploidy rate might be paternally driven. But in general, it's single point mutations that are paternally derived. And aneuploidies are mostly maternally derived.

All right. You gave us a lot of stuff we can dive into there as we explore this more. Gentlemen, your overall thoughts, were you convinced that male age is associated with aneuploidy from the study? I'm not convinced and I agree with them.

For sure, paternal age is going to have an effect, but that effect more likely is not going to be clinically relevant. For example, we know changes that happen with paternal aging, lower sperm count, motility, and so on, higher DNA fragmentation. But if we look at someone who was in his thirties, a male who was in his thirties and his sperm count say average 75 millions, and then now he's 60 or 70 years old and it's went down to 16 millions.

So there is a significant difference here statistically, but more likely is not going to be clinically relevant. The other things when we are discussing the advanced paternal age, it's to look at the etiology, the etiological factors. As Professor Aitken says, DNA fragmentation play a big role here, but also we need to look at other things like we know that telomere shortening is a problem, but when you look at the sperm, there is actually lengthening of the telomere.

Although it can be associated with other medical problems, but usually we say telomere shortening has a negative effect. Also we may need to look at the mitochondrial effect as well. And if we go back to the study, actually when they say we looked at close to 60,000 oocytes, that's when you look there, you say, okay, that's going to be a big study.

And then when you look at table two, you look at patients who are of concern, like the age group, which is I believe from 50 to 60. If you go back to table two, they are actually only 21 patients. And we spoke yesterday about the sample size of older studies talking about the thyroid and if they go to the fertility and sterility now, they will be rejected.

And they should also exclude patients who are having azoospermia and they are going to do a testicular biopsy for because there is a huge difference according to the etiology between obstructive azoospermia and non-obstructive azoospermia. They have divided patients into two groups and they are wondering why we got these results that with the fine needle, they have better results with the TESE, the open TESE, they have different results obviously because these patients are just obstructive and these ones non-obstructive. And for non-obstructive, you actually don't go for TESE, you do for micro-TESE if you want to have better results.

And we know that the sperm from non-obstructive azoospermia patients are much worse than freezing. For example, they are using the slow freezing method and obviously, it is associated with a higher DNA fragmentation compared to the vitrification as well. Yeah, I think the point about losing power in the older men is as you say, it's you have to dig in to find the numbers in those but Yousif's slide that shows the confidence interval very clearly shows that as you get out to the older gentlemen, the confidence intervals become very wide.

We really lack any power to be able to detect anything. Sir, your thoughts, were you convinced by the paper? Yeah, well, it's certainly that we know that the paternal age will have some effect on the reproductive outcome. But I think that the study, we probably look at different, look at the wrong parameters to determine that.

And I think the gene cells in the myo, they have very high fidelity in the cell division, in the euploid right. And what's causing the issue is not so much of the euploidy. And interesting that Professor Atkin just mentioned about a centromere and those things that which may affecting the mitotic aneuploidy, for example.

And I think these studies, the deficiency is that they haven't presented the mosaic rate as well as the segmental mosaic rate. Because that will be interesting, because I mean, they look at pure, yes or no, purely euploid, pure aneuploid, there's no difference, no effect on age. So if there's anything there that my thinking is that probably the segmental mosaicism, and if you look at, and I just look at the paper they put up in the discussion about the Danish studies that were published a long time ago, about like 74,000 single birth, look at the increase in the complex, we're not talking about single gene mutation, talking about the complex and the syndrome type of anomalies, that's more than single gene mutation.

The increase in the rates are quite dramatic. I think from 15% in the late 30s, double to the early 30s, about 40% increased, and then double again in the late 40s to 70%. I mean, you get up to 50, over 50 is about 300 times higher.

So that's something that we are looking at the biological possibility, and certainly this study has shown that. So that there is a drop, but then there's nothing further, there may be issues there. So I think that we are probably looking at the wrong thing in this particular study, and more, I think more useful that as condition that we want to look at the reproductive outcome, because that you look at the mosaicism per cycle, or per embryo biopsy, is not a very good determinant of the reproductive outcome, because you can have embryo, which is not suitable for biopsy for whatever reason, you may be in a cell mass coming out first.

They're good embryos, they can lead to a baby, and then you'll miss that. And also that having a euploid embryo does not say that you're going to get a baby. Yeah, so there are variations in there as well.

So I think the studies up to about 2023 is a pity that they should have all the status. And if they, I believe that they have a percentage, probably that is no difference. No difference there, yeah.

All right. We've only got about 12 minutes to go. Audience, get your questions ready.

Get some hard ones that you want to ask this expert panel. Let's see what we can throw at them, and what they answer. Professor, come back to you real quick before we go to the audience questions.

Can you take a deeper dive into what you described with DNA fragmentation, oxidative stress, and is there anything practically that we can do to help improve that in our patients? Yeah. So, well, DNA in sperm cells is, generally speaking, very well protected against oxidative stress. It's very, very tightly compacted.

And the way that the sperm cell compacts its DNA is really quite unique. It wraps it up into little donuts-like structures called toroids. But there are pieces of DNA that link the toroids, that are attached to the nuclear membrane, that are very vulnerable to attack, and particularly to oxidative attack.

The guanine residues, in particular, are very vulnerable. And when that happens, you form a base adduct called 8-hydroxy-2-prime-deoxy-guanosine. And when that base adduct is formed, the DNA becomes destabilized, and they will then tend to fragment.

And this, you know, is responsible, I think, for a large amount of the DNA damage that we see in men, and particularly we see in aging men. Remember that aging itself is a free radical mediated event, right? So we're all peroxidizing a bit too fast. And so this is one of the reasons that underpins DNA damage in the germline.

It's definitely paternally age-dependent, and could be related to abnormalities in chromosome stability and aneuploidy, at least in theory. Yeah. Is there anything practically that we can do? Yeah, the obvious thing to do, and this is a really bugbear of mine, the obvious thing to do is if your patient's suffering from oxidative stress, you give them antioxidants, right? But people just hand out antioxidants like Smarties, you know, these are given to anybody who wants them.

And if you give antioxidants to people who are not antioxidant deficient, you create a new state of reductive stress. It's not oxidative stress, it's now reductive stress, and things get worse, not better. So what I would like to see people do is to monitor their patients for oxidative stress, and select those that have that condition for antioxidant therapy.

That should be beneficial. And in animal models, that definitely works. Yes, it's interesting.

They say that the Danish studies looking at most of them will be natural conceptions. So we can see that dramatic increase. But in the IVF setting, that paper may be true that somehow the IVF treatment per se, the sperm selection has dampened that effect.

If it is DNA fermentation, for example, that we know that people with the high DNA fermentation are less affected when you do IUI treatment, for example, they are washed to some degree, less IVF, and even less with the ICSI. So there's that sperm selection process. I'm not saying that the effect is not there, but that effect may be dampened, because this is IVF treatment.

So that leads to that, what can we do about the DNA fermentation? So if you've got a high DNA fermentation, and the oxidant is not sufficient, you look for sperms that have less DNA damaged. And then you mentioned in the paper about freezing and thawing, the frozen sperm do have less U-point, right? Yes, again, Professor Atkins would say that, would explain that freezing and thawing process is a stressful stress for the sperm DNA, and especially when you're taking from an older man. Now, the same, but I can't say that fresh is always better than a frozen, especially in the testicular sperm issues.

And certainly from our own experience that the same process of freezing and thawing, they actually serve as a selection process. Because the reasons that we have so many, we mix so many sperms that most sperms are not very good. Maybe you take a sperm from the testes, especially you take them from the biopsy, there's a mix up with a lot of things, a lot of young cells.

So that's why the finding aspiration actually give you better cells, better results, because you get actually more mature sperm that way. But the same process of freezing and thawing, from our own experience, we actually get equal or better results with the frozen sample testes from testicular sperm. Do you think though it matters then, what insemination procedure you use? Because in this study, as I understand it, both IVF and ICSI were used.

And the impact of sperm selection is going to be very different depending on the methodology. And the data were not stratified according to insemination. Yeah, but the data looking at the freezing and the frozen sperm, fresh sperm, only less than 5% of our testicular sperm.

So I can't say that you can conclude that for testicular sperm. That's why I just try to say that in the men who actually have testicular sperm, they may not be always a bad thing to use frozen sperm. And because they started with less DNA fermentation.

So that freezing and thawing process will not cause a lot of DNA damage to those sperm. So let's give our audience a chance for their input. Who has a question? I might kick it off with the first question.

Yeah, thank you. Obviously, sperm and egg have very different biology and sperm are haploid from the start of their reproductive journey. Can an aneuploid sperm look normal, swim normally, or fertilize an egg? We can extrapolate from studies about Klinefelter syndrome that there is always like a natural selection and mechanisms that happen that guarantee to some extent that the mature sperm is going to be euploid.

And if you look at all of the studies that looked at the outcome of Klinefelter syndrome, they are usually without doing PGT-A and doing PGT-A as well, they are always, always euploid. There is only one study that came from Israel and very early where there were triplets and there was Klinefelter syndrome with them and fetal reduction has been done. This was the only study.

Yeah, I think as far as Klinefelter is a good example, I think there is a difference from the people, what the question of biological questions or the micro-TESE sample. I think a lot of the micro-TESE, the reason you can get sperm is that because you've got a euploid and you put the most cells inside, that's why you get, and most of them will be normal. But interestingly, that we look at the people looking at a sperm DNA, aneuploidy, that as men's age increased, you have a dramatic increase in sex chromosome aneuploidy.

But then that hasn't been translated into the relationship between Klinefelter in relation to age people. So from that perspective, I think that yeah, there's a big selection out. Those aneuploid sperm does not get naturally inside that to create the problem.

I think your comments highlight for me another piece of information which is missing from the study, which is what were the aneuploid use? Are they monosomies? Are they trisomies? Are all chromosomes equally affected or are these all trisomy 21s? What are they? That would have been very interesting. It would have been very interesting to know whether there's differences between chromosomes and their susceptibility to aneuploidy. But I guess in response to the question, the fact that Down syndrome exists suggests that the aneuploid sperm can fertilise eggs.

We have a question from Dr Clare Boothroyd. I just want to ask the panel, just one, two, three, because I got the message that John believes that the sperm age or the age of the male who provides the sperm does contribute, in some cases, to embryo aneuploidy. But the rest of the panel said no.

Can I just get that consensus? No, what I intended to say was that it's at least theoretically possible. I'm not saying that the statistical evidence supports that. And as a generalisation, I tried to emphasise that if there is a contribution, it's a minor one to aneuploidy coming from the male side.

But the point I was trying to make is that conceptually, I could imagine how this might happen. Yeah. And am I right in thinking that the rest of the panel say no to that, that male age does not contribute to embryo aneuploidy? Not significantly at all.

But also for us to answer that question precisely, we need to look at the test that we are going to do. So you want to have a test that will tell you if this aneuploidy, which is there, is due to maternal or paternal, and if it is meiotic or mitotic in origin. And we have now tests developed that can tell us that.

So we are waiting for studies to look at this. Yeah, I think, again, going back to the Danish study, that you have these syndromes and multi anomalies that may be down to the aneuploidy, but those aneuploidies may not be down to the meiotic events, mitotic events, that you may have segmental or mosaicism in some of the tissues leading to that kind of syndromes. So again, that's interesting, as Professor Aitken mentioned about centromere and that interfere with the subsequent mitotic cell divisions.

And that's why it's interesting in the papers there, they haven't reported the mosaic rates inside that one, but for the pure aneuploid, no difference. We have time for one more question from the audience. I think we have a question from the floor, from Dr. Sebastian Leathersich.

Thank you very much. My question really reflects what Professor Aitken mentioned briefly, which was that both conventional IVF and ICSI were used in this study, but there's no information on the proportion of patients that had either method of insemination, it wasn't included in any of the statistical corrections. So I'm interested, and also it's strongly related with many of the factors that this study found were associated with aneuploidy, oligospermia, for example, testicular sperm.

So I'm interested in the panel's thoughts on how important that would have been as a potential confounder to be considered in this study. Who wants to take that one? Well, I think I raise it because I think it is an important confounder, absolutely. That's when you're using ICSI, you don't have those selective mechanisms we were talking about earlier, and so the danger that you inseminate the egg with a damaged DNA damage spermatozoa is obviously enhanced.

Great. So we're almost at the end of our time. I want to give each member of the panel 30 to 60 seconds to give us your high-level summary.

What's your take-home message from this? It can be what you learned, what you think, what the next steps are. We'll start with our senior fellow, and we'll come down the line. I think it's important to take into account male factors when it comes to fertility and IVF outcomes.

I'm not sure if this study will change my practice in terms of offering, for example, PGT-A for male factor. I don't think that's indicated based on this study, but I think it gives us some points to discuss and maybe better ways to design future studies to look at other male factors that might influence IVF and fertility outcomes, like sperm DNA fragmentation and epigenetic testing and those other factors. Sir? Yeah, I certainly totally agree that I don't think that aneuploidy is a big issue, but certainly that the male aging have significant impact on the sperm performance.

So that may be in the forms of gene expression, DNA damages, or protaminations, or lots of other issues, which are accounting for the obvious increase in the enamelity rates in offsprings with increasing male age. So the research is encouraged that we try to look into what's actually causing the problem and how we address it. So the paper will not change my clinical practice.

Definitely, there are changes happening to the sperm with aging. I do believe that the oocyte quality is the best treatment for male factor. And I still, even when I have my own patients who have sperm problems, I also try to get a good quality egg as well.

We are waiting for more studies to tell us if there is a significant impact of advanced paternal age on certain outcomes, including the live birth rate. And that will set the scene if we need to do certain interventions, like freezing sperm at a younger age, or do eggs for older men, and so on. I can just repeat what you guys all said.

I think the paper really failed to prove its hypothesis that there was a significant effect there from age, advancing paternal age. But what it did force me to do was think about other stuff, like what John's talked about. I do think we need to think more deeply about insemination methods and DNA fragmentation.

That's my take-home message from learning, reading. Very good. Well, I can only agree with everything my colleagues have said.

I think my passing comment is I'd like to congratulate the organisers for putting this podcast together, because I think the discussion has been very interesting and certainly stimulated me to think about other things related to male age and fertility. Wonderful. Well, if I can summarise what I'm hearing from the panel, the egg is still queen in this whole game, but there is probably some partial component that we're talking about here from the sperm and that we need more research in sperm, if anything, that's definitely an under-researched area within our field.

So thank you to our expert panel for your insights. Thank you to ANZSREI for, again, partnering with Fertility and Sterility to have this journal club and this podcast here. To our listeners around the world, please like and subscribe to Fertility and Sterility On Air.

You can listen to us wherever you listen to your podcasts. Thank you very much.