Today’s Journal club Mechanistic analysis of sperm selection with hyaluronic acid and treatment outcomes This based upon an article published in Human Reproduction with Professor Jackson Kirkman-Brown MBE PhD, as one of the authors, please scan the QR code here with your phone to open the article

Sperm selection with hyaluronic acid improved live birth outcomes among older couples and was connected to sperm DNA quality potentially affecting all treatment outcomes Jackson is internationally recognized for his work in Fertility and Andrology. He strongly believes that research into diagnostics and therapies in the area can have a rapid and direct impact not only on the care and therefore birth of the next generation but also quality of live for the patients and children involved.

As director of the Center for Human Reproductive Science he leads an interdisciplinary team with clinical trials and basic science collaborations across the UK and around the globe
Jackson holds a dual role alongside his academic work. He is also director, HFEA person responsible and Science lead at Birmingham’s Women’s Fertility Center which includes coordinating one of the largest regional preimplantation genetic diagnosis services.

Jackson has also been privileged to be able to have the opportunity to set up, develop and create the lab techniques and services for fertility preservation in the event of catastrophic genital injury. This work was undertaken with the Royal Center for Defense Medicine and recognized with him being conferred an MBE and most recently being named as the Chief Scientific Offices Healthcare Scientist of 2014 by Professor Sue Hill.

So now it’s over to you Jackson…

Thank you Steven, and thank you for giving me the opportunity to give this talk today. So I’m going to talk to everybody about advanced sperm selection methods and with this particular emphasis around PICSI. I do have a number of conflicts of interest but I don’t think any of them affect today’s presentation and they are there for people if they want to check on them.

So what I’m hoping to talk about today is something about optimization of the male but also the importance of delays in that why we should care about sperm how we can prepare and select sperm why PICSI the best evidence based technology out there for us at the moment and what the future holds in the area. I think in this it’s really important we recognize end points.

So, many studies with sperm have used surrogate end points that aren’t actually the things patients particularly care about. So couples, individuals turn up wanting a healthy live birth and preferably that child to be able to have its own healthy offspring. They are not particularly affected by earlier events like virtualization rates embryo grades biochemical or even clinical pregnancies. Yet these surrogates have been used in many of the studies we look at to derive whether male factors are a problem.

So I’m going to ask everybody to keep their eyes on looking at healthy live births and healthy offspring as really our endpoint aim. If we’re thinking about optimizing the male infertility before semen analysis and certainly after it there are various things that everybody could look at around diet and lifestyle. And there’s great data out there from Josh Schvarro and Albert Salisswervos around diet and Mediterranean diet and then other things about lifestyle that we know routinely are recommended to patients maybe slightly less. Obvious for some We know that protein shakes gym supplements are generally bad for male fertility and the vulnerable and there’s an argument out there, go away and read the systematic reviews with regard to whether there’s evidence yet for vitamin supplements to help certain subcategories of men with their fertility.

Similar situation for varicocele I think what we must remember is all of these things take time and very often if there isn’t a strong evidence base we have to think about whether the couple have that time If you’re looking at something that will take a minimum of three months and may be six months to a year to actually impact on the potential outcome.

If you’re looking at older the couples the decrease in fertility and potential outcome for the female in that time may far outweigh the decision to do something around diets and lifestyle or perhaps take a vitamin supplement. So we need a careful balance. What can we suggest without delay? Well optimizing the man making sure there is no sexually transmitted infections there, prostatitis.

Yes reducing abstinence And I think that’s often one of the most important things that people forget, W.H.O diagnostic guidelines may say 2 to 5 days but in actual fact daily ejaculation is much better with that reduced abstinence for the samples we’re going to use in treatment that ensures that the on demand secretion of seminal plasma is fresh and we’ve also got the freshest sperm sample.

If we’re thinking that people need to use a lubricant to help produce a sample what is that lubricant? Certainly most sperm friendly lubricants aren’t in our labs hands in Birmingham. That would always be that We’re giving them the same oil that we use to to culture our embryos in because that’s the thing we know that has been checked for toxicity.

And finally, getting sperm out of semen as fast as possible. The idea from maybe a few decades ago that we must leave semen to liquefy is actually a bit misjudged. So, as liquefaction and those processes start to happen, the osmolarity is changing and getting more different from the media. We’re going to put the sperm in, so as soon as you can after ejaculation, whether it’s gradient or other preparation form, you should be getting the semen on there and starting the preparation.

But, I do think perhaps the most important thing to remind the patients and give them some buy in is this thing about abstinence. It’s a simple way to increase the quality of the sperm that we have available that we’re going to be selecting and choosing from. But, what else affects success? Well, really interesting data here that was published late 2019 from Australian teams looking at how male factors in terms of male age affect success.

So, here you’ve got female age along the bottom axis with under 30, 30 to 34, 35 to 39, and over 40 for the females. And, you can see the impinging ages illustrated in colour, female age of 40 upwards to the top one being over 55. What this shows us is the thing we know, predicted live birth goes down by female age, but also very dramatic effect for male age within that, and in certain categories.

That’s more than halving the chance of a live birth. So, it may be a decade or so later, but male age is very strongly impacting. We need to also remember that we know about paternal age effects beyond pregnancy. And, as men age, we get increasing De novo mutations and epigenetic alterations that are changing the sperm that are there. But, there is very little evidence to suggest that’s changing all of them terribly.

It’s just changing some of them. Beyond that, why do we care about sperm? Certainly, many people will tell us it’s because of sperm DNA damage, and this meta analysis was performed by our team back in 2012 and was one of the first things out there to note the relationship between sperm DNA damage assays and chance of miscarriage.

I do think it’s important to note that, at this point, you can clearly see not all sperm DNA damage assays are strong for predicting miscarriage. And, it’s worth noting that the W.H.O., in the semen analysis manual, do clearly tell us that semen analysis, when it’s sperm DNA damage assays, should only be based on the evidence for the assay that might be most profoundly shown in the data that was available back in 2016.

For the systematic review by Cissen against clinical outcomes, where the different tests show quite different relationships to outcome. And, maybe none of them have the sensitivity or specificity that we’d like. What’s happening more recently is people saying, ‘Oh, you’ve got bad sperm DNA damage result. I’d like to step to SSR,’ and maybe surgical sperm retrieval is the way ahead.

I think that, although one day we may see the evidence for that, it’s very difficult to presume on the basis of sperm DNA damage testing that we should immediately jump to surgical sperm retrieval before trying things like lowering the ejaculatory interval, maybe even trying two ejaculations in a row from a man. Usually, they’re going to prefer that to having surgery, in terms of trying to look at improving the quality of the sperm.

So, what else can we do? Well, this is really where advance preparation comes in. We’re looking to improve selection. There is a concept of the things being physiological, though I may question sometimes why we’d aim for a natural idea. What we’re trying to do is ensure that the fertilizing sperm is a happy one, one of high genetic quality that gives us the very best chance of a healthy baby.

We know that from the female. We will get hopefully some eggs, but we can’t do a lot about those. We can’t do a lot to change the quality of them. So, it makes natural sense to pick the best sperm possible for each oocyte. And, that really brings me round to the HABSelect trial. So, this initial paper on the trial was published back in 2019 in The Lancet.

And, for those of you not familiar with the trial, there were over 2700 couples randomized in across 16 different assisted conception units in the UK. Those units were NHS and private sector parts of different groups. So, it really did include the full breadth of practice. And, this was blinded randomized controlled. So, is the best standard of evidence that we’ve got for whether an intervention makes a difference.

Now, if you’re not familiar with PICSI, it is based upon data initially generated by Gabor Huizar, looking at whether there was a difference with sperm that have this Hyaluronic binding capacity. So, you’re introducing sperm to a dish in these motile sperm, not all sperm combined. And, so you’re then only selecting from the bound sperm that are motile to do your injections in the main trial data.

Then, perhaps the headline outcome that was noted in the table is directly from the Lancet paper, is that there was no significant effect on live birth. That, if you can read the numbers, we saw a 27.4% live birth rate in PICSI and a 25.2% in ICSI. So, PICSI was about 2.2% higher. Live birth rate, which some in any case may consider a marginal gain, but it did not reach statistical significance, which would have had to be a 5% difference between the two.

What was, perhaps, more interesting to me when we look to more depth at the data was the secondary outcome of miscarriage. Now, this is confusing as presented in The Lancet because it had to be presented per person randomized, but we all know you can’t miscarry unless you’re clinically pregnant. So, if you look at the clinical pregnancy numbers, what you see is a 14% miscarriage rate in PICSI versus a 22%.

Rate in ICSI. So, an 8% difference in miscarriage. Right. Or, if you wanted and were looking at PICSI as a new intervention, you could say it reduces the miscarriage rate by 38%. That was clearly an interesting finding and it’s one that we decided to look at more as we moved into the mechanistic arm of the trial. So, the trial had this initial clinical trial setting and then, following on from that, a number of mechanistic aims was set down to try and understand how PICSI may work.

If it did. Some of that’s represented here in figure one from the paper, which has the consort diagram. If we look at panel B, what you see is that after ICSI or PICSI had been performed, the residual samples were all transferred to biobanking from the Biobank. We then selected a number of samples to study further the outcomes, looking at samples from biochemically pregnant, clinically pregnant couples, as well as those that had live births.

And, these were assayed by a number of assays within the assay set. We’d Comet TUNEL orange on a slide base format, sperm chromatin dispersion. So, the halo test, Hyaluronan binding as well was in alongside. Now, what was notable in this, and you can see that in the background information in the paper, was really that these assays didn’t correlate with each other in terms of outliers or any thresholds.

So, again it reinforces the observation that they shouldn’t be used interchangeably. And, if you’re going to use one of these assays to make a decision, then you really must do it on the basis of the evidence that you have for that specific assay, going on through the paper. We looked at different measures and how these correlated, whether they were identifying the same things, but I’m not really going to concentrate on that today because I want us to talk in more detail about the outcomes that maybe everybody will understand.

So, first of all, fertilization rates following ICSI and PICSI. So, what you can see here is that if we look at predicted fertilization rates, there is an expected difference. Panel B and panel C against female and male age. So, the fertilization rate does appear higher in ICSI, but you can see that in the overall data in that box whiskers plot, that could lead you to expect that we then see a ramification of that initial fertilization rate passing on through the trial to increasingly negative outcomes.

But, that actually doesn’t seem to be the case. So, when we look at biochemical pregnancy rate, then the biochemical pregnancy rates are pretty much the same against female age or male age. And, we did see in those predictive rates some relationship to halo. You can see a very clear scatter there in panel C. So, it isn’t a strong relationship.

So, at this stage of biochemical pregnancy, yes, there is a difference by age, but the two interventions aren’t showing any difference between them. Stepping on into conversion from biochemical to clinical. Again, we see no difference between the two interventions. So, similar numbers converting, no effect on the chance between one or the other. So, things at this stage are progressing very similarly for the two interventions.

However, once you have a clinical pregnancy, looking at predicting live birth, we certainly see a dramatic difference. Panel A and B here. So, this is predicted birth rate. Given a clinical pregnancy, what you can see is for female on the left or male on the right. There is a dramatic difference between PICSI and ICSI in our female graph, from around 33 years old, you see the ICSI rate begin to plunge beneath the PICSI.

Rate. And, similarly for the men, maybe seeing a difference around 35. So, what’s this telling us? How do we understand this graph? Well, maybe it’s helpful starting from the familiar graphs that we all know from START and others around female age miscarriage. So, we know that live birth rates decrease from the mid-thirties, but miscarriage rates start to increase from the later thirties.

And, generally, if we look at PGT-A data, this seems to be related to embryo aneuploidy. I can flip the west data to show it in a similar pattern. We were looking previously at the live birth, but we can look at probability of miscarriage, and you can see the probability of miscarriage taking off, and PICSI attenuating that. And, indeed, if we look at it on top of this ART data, you can see that the ICSI miscarriage line is following what we may expect it to from the start data in terms of a curve.

It also follows the matched HFEA data that’s available from that time, and the PICSI is eliminating that. So, what can we conclude? Well, best conclusion after females are going into the age range of 33 to 35 and beyond, it does appear that PICSI works as well or better. And then, maybe it also tells us that there is something there around risk, if we try and put some numbers on it to understand it.

If we look at a lady who’s 37 years old in these two graphs, if she was having PICSI at the stage she’s got a clinical pregnancy established, she’s an approximate 15% chance of miscarriage, if she’s having normal PVP ICSI at the age of 37, she’s got about a 40% chance of miscarrying after that clinical pregnancy. So, a really dramatic difference there.

Like I say, this is blinded randomized controlled. So, it’s the best standard of data that we could have for seeing whether this intervention makes a difference, and it appears to be doing that in this most advanced age group of females that are expected to have a slightly lower oocyte quality. How about a few words end about the future and maybe where we could go with these things in the future?

Well, one thing that our team in Birmingham have been doing is looking at flagella analysis. So, those of you that have used cases will know, it tracks a sperm head. And, although that’s maybe useful data, we’ve not seen big advances where that’s gone in the last few decades. And, that’s perhaps because it’s a bit like looking at someone’s head moving in a swimming pool, and you can’t tell what they’re doing to be able to make that movement.

So, we know the flagellum drives the sperm, and it gives you a readout of metabolism. It can tell you whether all the signaling things are there or functional in the sperm. And, tell us a little bit more about what’s going on. So, is there hope for that kind of technology? Well, we’ve already seen some advances this last year.

We’ve seen the publication of the paper on SiD, and that’s identifying again just from a sperm head, things about motility, and suggesting which sperm perhaps you pick for using ICSI, and that’s only using head movement. I think, if we think about PICSI and these real differences that we see in data in PICSI, and we think about AI, then the logical thing is to perhaps think about the flagellum as well.

So, this little video is sperm bound on an HA surface. And actually, you don’t just have to say we’ll pick a motile sperm. You can start to say we’ll pick a sperm that’s motile with a different flagella beat. Maybe that’s the next level, but you’re reading out something like a heart rate from those different sperm. And combining things.

I think that could maybe step even further our abilities in this field to improve things for patients. And certainly, if you look at flagella beat and sperm bound on to HA, you start to be able to see different things there and different patterns. But stepping back to the main trial data and what we’re talking about today, I think we now have to care about sperm.

There are lots of studies out there that say selecting sperm makes no difference. And generally, they’ve been done with younger females with donor eggs and not found a difference. We can see why from the HABselect trial. That isn’t where picking a sperm shows you the big clinical signal. It is arguable though, that when we think about potentially inherited risks, maybe it is not just about the younger females being able to correct things.

Perhaps there is a longer-term worry about whether those corrections result in healthy children that can go on to have healthy children. But that’s an argument for another day. For now, the data says that we have something for females. Certainly, beyond 35 gives a better chance of an improved outcome and reduced miscarriage versus conventional ICSI, and that is PICSI.

So, how we choose the sperm needs evidence and careful interpretation of a couple thinking about maybe the combined effects of male age that we can see that an intervention like this can change the outcome for individuals and it might change everything for them. What they argue for now is PICSI isn’t an add-on anymore. If we look at this data, the data supports its use.

It doesn’t say ‘Oh well maybe use it.’ It doesn’t say ‘Oh well if a patient feels like it, use it.’ It would support its routine use. If I was going to say oh ICSI was the new trial intervention and we’ve come up with data that said ICSI, we don’t quite get to a significant difference in live birth but it’s a little bit easier.

We should use it. People would be shouting at me and saying, ‘Well, we can’t use it because the miscarriage rate’s nearly 40% higher and that’s very worrying.’ We can see those miscarriages, they are a reason not to do conventional ICSI anymore. In certain groups of people, I’d argue for females over 35. All what we know is low ovarian reserve.

To do the best thing now means that we could do PICSI. So, in answer to the question of advanced methods, are there better than conventional ones? I think my answer would be yes. There may be other methods that are emerging that are out there that can equal these results, but as yet the evidence isn’t there to support that use.

So, thank you very much. And I look forward to taking questions. Thank you for this excellent and insightful presentation, Jackson. Let’s now open up the Q&A session. I’m going to take moderator’s privilege and ask the first question if I may, really just for clarification for all the listeners out there, would you describe your mechanistic analysis of the HABselect trial as merely a hypothetical model?

So, my simple answer to that is no, it is not a hypothetical model. So, Robert West, who is the very, very talented medical statistician who analyzed this data with us, has specialized in his career looking at data from clinical trials and interpreting them. And so these things that we read as probability of live birth, probabilities of miscarriage, and things are not generated or analyzed with any kind of bias, and they are based directly on the data in the trial.

So, as such, they are not a hypothesis. They are a result that’s found from the trial data, and if they were a hypothesis, we’d be saying ‘maybe,’ but they’re actually there in the trial data. So, no, I don’t think they’re a hypothesis. I think they generate a hypothesis, which is that in future, when we’re looking at trials around sperm selection, we need to not be thinking about things like donor eggs as our model, but using randomization of females in an advanced age group to look and see a signal that we can then maybe think about extrapolating backwards into the other groups for its effects.

Right, I’m going to concentrate mainly on the questions that have been submitted. Actually, during the webinar, we did actually have other questions submitted prior to the webinar. And just to reminder to everyone, don’t raise your hand, please put your questions in the Q&A box. So, there’s a group of three questions from one person, which I think are quite nice really to sort of understand the use of PICSI better.

So, they asked, ‘What are the criteria for doing PICSI, is it necessary to test hyaluronan binding first with a hyaluronan binding assay? And, is PICSI a time-consuming procedure?’ Okay, you might have to remind me as I’m going through, answering the criteria for doing PICSI, first of all. Criteria for doing PICSI, I think the data says the criteria for doing PICSI are certainly females over 35,

where we’re expecting lower ovarian reserve or where we’ve got an older male. And so, those are the criteria. There are lots of reasons that people have specified in the past, and you can look to things like the Warrillow trial which used HBA to decide, but in actual fact, we need to think through what we’re doing. So, the trial says if we pick sperm off HBA, we have this dramatic reduction in the chance of miscarriage.

So, if we saw somebody who has HBA binding of 90%, that’s still a 10% chance that you pick a sperm that’s of lower quality. If you see somebody with HBA binding of 5%, that’s a 95% chance that you pick a sperm. That’s of lower quality. So, I would argue, wherever you can pick a sperm by PICSI, you should be doing PICSI, then if you have no binding at all, you can revert to your other methods of selection of sperm.

But where you’ve got that sperm that is bound, that you can choose. The data says choose that one. Don’t, don’t leave it some other random chance. So, I think, you know, we can, we can group whether it’s females or females over 35 or whatever. We can argue about what a borderline is. But definitively, the data supports PICSI for everyone.

Basically. And the last part of that question, I’ll actually combine with another question that we have prior to the webinar. So, the last part of that question is, was this PICSI a time-consuming procedure? And the other question was, how to use PICSI effectively? Because this particular operator is always struggling with sperm getting stuck inside the injection pipette when handling sperm in the droplets of hyaluronan.

And so, maybe just about the technique of a PICSI, if you could comment on that. So, I think the simple answer is, we all know that as an individual, somebody trying to do ICSI, it takes them a reasonable amount of time. And as they train up and they do more, they get faster. So, the same is true with PICSI, if you’ve never done it before, when you’re starting to do it, you are going to find that it’s a bit slower, in the hands of experienced people,

It certainly doesn’t take a huge amount more time. Some people talk about a minute more, maybe some people talk about 2 minutes more. I think it’s worth remembering that usually in the patients when we’re talking about patients over 35, we’re not expecting that those patients are usually delivering us sets of thirty oocytes or something, where that’s certainly going to become a lot, lot more time.

We’re talking about people where their ovarian reserve is a little bit more limited, and so actually, that that time impact, if you’re just doing it in those cohorts, will be less. In terms of catching the sperm, there are a few different ways, obviously they’re bound on. I know one of the discussions we’ve had before is, it’s easier to suck them head first.

So, maybe people are used to trying to get the sperm and they’re doing some thing where they’re breaking its tail, catching it, and then getting it. You know, sucking a sperm head first into the pipette is possible. With PICSI, you can try and strike the tail and break it as it’s bound on the dish as well. And I think as people start, was there was mention of a droplet.

It’s worth saying sperm slow isn’t the same thing as binding on a dish, but a place that you could use Spermslow is to totally eliminate that potential risk of PVP. So, if you’ve got a sperm and you’ve picked it off the dish, and you’re wanting something that will slow it down a bit, then you can have Spermslow there as your medium that’s going to slow it down and enable you to do any of your other manipulation with it before injection.

Because I do think part of it is, you know, we’ve all accepted for decades that we use PVP. And, maybe different laboratories have different protocols for trying to wash it away, but there will be real traces of PVP in sperm that are injected. And it’s not a nice chemical when you look at the safety stuff on your thing,

for if you get PVP on your skin, the idea that we start life with it is a little bit strange. And so, I guess my other, my other thought in there relates, perhaps, back to those fertilization rate differences that we saw with ICSI. And, I do wonder whether sometimes some of that difference in fertilization rate actually stems from having a greater PVP contamination in there that’s doing something to the egg, rather than being something that is otherwise,

kind of a good thing about ICSI, because as you follow through the data in the trial and look through things, there weren’t cumulatively more high-quality embryos in the ICSI arm or anything else. So, there isn’t something else beneficial that happens later. So, so what you’ve got is a slightly higher fertilization rate, but that’s effectively dud. It’s not giving you any benefit through the trial.

It’s not doing anything good later. So, there’s something that actually, you’re not benefiting as a patient, and it’s something else that’s happening. Hmm, very interesting observation, that actually answers one of the other questions that I have since you mentioned Spermslow. So, one of the other questions was, would we, would you expect the same results that you achieve with PICSI with, we had you at the trial been conducted with Spermslow?

I don’t think anybody knows that answer. So, that is a very good question. And Spermslow is a different mechanism. So, we know that viscosity is very highly selective for sperm, and viscosity as a mechanism is selecting different things to relate to tail beat. So, I think what we can say at the moment is you can’t interchangeably use spermslow and say is doing the same thing.

It may be in the end that somebody could run a trial of PICSI against Spermslow, for instance, and find out whether one was better than the other. But, but the data from this trial really just supports HA binding on to a fixed surface and selection of sperm there. So, so I would discourage people from saying, ‘Oh, they’ve done Spermslow and got the same result.’

And on the subject of the differential results between ICSI and PICSI, there’s a couple of questions that have come in asking why it is that under the age of 37, the results

in terms of live birth rates and miscarriage rates are better with ICSI than with PICSI? Yeah, yeah. So, that’s that’s kind of a $10,000 question, isn’t it?

Why does it appear that so, so that’s really in women? If you look at the data, it’s probably women under the age of 30 that that really happens. So, it’s kind of it’s a group that are reasonably well represented in the trial is disproportionately younger women did say yes to the trial, but actually has very low representation. I’m sure most of us realize in our patient populations.

So, if we look at the trial, actually, 60% of the women were beneath 35. But if we look at patient populations, at least in the UK data, more than 60% of the women are over 35. And if you read those kind of numbers into the trial that matched our patient populations, we’d have probably reached a live birth significance.

But, obviously, nobody can. Nobody can really know what you can see. That’s all it is, is some data that appears real, but without explanation. I wonder whether we’ve got some complex interaction of things going on, because those much younger females are in a situation where usually there was a more severe male factor for them to have been attending at that stage, and they’re actually quite a different population.

So, they appear in a continuum of the data but are quite a different population in terms of causes and what’s going on in the background. So, yes, there appears to be something that I wouldn’t necessarily advocate that that for people in that younger age group should have PICSI, but I think it’s difficult to understand what’s happening, and it’s not something that’s part of the same strong signal if you want to look at it like that.

Okay, yes, it is interesting. And there was another interesting question that came in in terms of maybe better understanding what PICSI is actually selecting for in its entirety, if you like. And this was, is PICSI possibly selecting sperm based upon a combination of factors related to their ability to undergo capacitation? So, is there a link between the capacity and the binding to hyaluronan?

Yeah, so, I think that there’s still a lack of data in the field, but what’s clear is that sperm get their hyaluronan binding ability added right at the very end of spermatogenesis. So, one of the things in Gabor’s early work, looking at when this happened, would be to say that maybe sperm that have had some problem in spermatogenesis,

don’t end up with this as a marker on the surface. So, it’s coming from spermatogenesis into the system, and that means it’s not really related to capacitation. But what you can say is that sperm can lose the ability to bind HA. So, if you give sperm an oxidative insult, which equally might cause sperm DNA damage in certain cells, you can reduce their ability to bind,

HA, so there is a timeline of things that can happen in sperm, where obviously people associate sperm timeline with capacitation. But really, a sperm timeline ends with death from a sperm, and capacitation is just something on that journey. So, there is something very natural, such as sperm burn out the lose HA binding ability because they’re becoming more and more damaged.

And that protein that’s binding it appears to be more prone to oxidative damage and possibly also nitro occilation damage than certain other proteins. So, it’s doing two things. It’s being there functionally from the start on a good sperm that had the correct kind of spermatogenesis. And then, at the end, it can also get damaged and therefore remove a good sperm from being in the population.

So, yes, that’s very true. Now, there’s a number of questions that relate to how the study was conducted. So, you know, I don’t think everyone necessarily as I had a chance to read the article, so maybe could just clarify some of them. So, these include, was PGT-A selection included, was it all single embryo transfer? Did you correct for the number of eggs injected,

Was PICSI done 30 degrees C on the micro manipulator? These, these kinds of things. So, you know, if you could just address some of those. Yes, on the first. Yeah, easy ones first. The PICSI was generally actually being done at 37 in the systems, quite simply because people had to be able to be randomized between PICSI or ICSI.

And we all know that we’re very worried about a lab measurement. So, switching regs on and off to do things, and getting temperatures right would have been impossible. So, but so the majority of that balance was that that’s 37. So, we can put that aside, and lots of people out there are using PICSI at 37. I think it’s

just the initial studies that first talked about PICSI, use this 30 degrees, so it’s there in the stuff as you warm the dish up.

Actually, all that happens is sperm swim a bit more vigorously as they are a bit warmer. And so, if anything, you might be just improving the selection. It begs that that be less likelihood of one lazy sperm binding effectively. So, so I don’t think that’s a worry. And in terms of pgta, no, these were all non-pgta, so everybody in the trial was getting embryos put back without genetic testing.

And, I think it’s a very interesting question of if we had done pgta, what would we have seen? And you may predict that what we would have seen is more embryos that were euploid in those older females, but this was separate from pgta. I think thinking about that mechanism, there is a lot of emerging data, mainly being done in animal systems, but a lot of emerging data that says correcting a sperm is something the egg struggles with.

We know from various things that eggs and obviously the early embryo can try and correct certain things about ploidy. They can try and correct themselves and move on through. And that’s obviously often one of the arguments that’s leveled against technologies such as Pgta. But if the egg is having to deal with serious problems from the sperm, that is obviously where actually cumulatively, it may fail in its ability to correct things.

So, I mean, I’d love to see some studies of Pgta with ICSI and see whether that the numbers are different. But, but they’re not there yet. And what was the other bit? Stephen, sorry, there was a range of stuff you know, did you correct for the number of eggs injected on each arm of the study, that sort of stuff.

So, so things like that around the arms should really be taken care of by randomization. You’re not talking about a very small trial. So, correcting for things like that is something that you might do if you were doing a trial of sort of you know, in a hundred people, this is 2772. So, it’s one the most substantial trials a blinded randomized control that’s happened in our field, and in that that randomization really takes care of all those effects.

So, you can look back to the Lancet paper and see, and we do check that in that initial data. But, but actually, that’s something that randomization and correct randomization takes care of, rather than being something that you post hoc start having to correct for. Yeah, well, you answered one of the other questions about whether PICSI is selecting for more anuploid sperm.

Sorry, more euploid sperm. So, some other interesting questions in here. Well, some based upon experience. So, this one’s limited experience with PICSI shows that some sperm bound so the hyaluronan have terrible morphology, and sperm with good morphology don’t bind. So, would you still pick these sperm with terrible morphology, and another question that’s kind of related to that is, are there any situations in which you wouldn’t recommend the use of PICSI?

So, and morphology is always a difficult one for us, isn’t it? And I think partly that depends what morphology we’re seeing. So, you know, a sperm with horrible tail morphology, sperm with a strange cytoplasmic droplet, something like that actually probably, that’s not something that we are needing to worry about in the trial. We always said try to go for the normal morphology as well, and that’s what most people would be expecting to do.

I think if you only had sperm that had a gross, grossly abnormal head morphology and like you saw all of those bound, and there were lots of that were beautiful normal ones that were swimming, that would probably give you cause to think about whether PICSI was right in that situation. I think we have to remember that all of us have a few patients with very rare phenotypes, that they may be the only person with the genetic condition that causes that in the world due to a certain situation.

So, so we can always find an example of you know, this patient that had this, and I saw this, and and that’s really about tailoring care on a personalized medicine basis. In general, in routine use, I wouldn’t expect that you were finding that PICSI was always selecting lots of abnormal sperm, and or lots of normal sperm swimming. So, it could be that there’s something about the way that this is being set up or something else.

But in general, in normal use, I’d expect that to be a very, very rare situation. It will occur sometimes because there’s always a patient with something strange. Maybe then in that situation decide to split your case half half and see what happens. But I think it’s, it’s difficult because we all coincidence detect that we see something, and we think,

‘oh but this is happening.’

And actually, every patient’s different. This data sets routinely for most patients, ICSI is the answer. So, it should be a standard answer, if there is something that makes you deviate away from it, that’s a deviation for that individual patient. And yeah, and again, relating to that, do you actually have details? Sperm morphology data from the study? So, there was some sperm morphology data that was taken into the study, that was from the diagnostic Andrology on the original samples.

But to be honest, as everybody will know, there’s a great variance in diagnostic morphology results, both done by the old W.H.O. methods and when you look in the EQA schemes. So, I think the power is there in that morphology data to say, from a diagnostic sample, you can use it to make any decision. And I think within the redundant sperm that we had from PICSI, we didn’t pick up any great signal there for any morphological thing being a significant signal that you could tell anything about. Okay, well, I’m going to take moderators privilege again and ask the last question, which is, do you happen to know whether the HFEA are reconsidering their traffic light grading,

of PICSI in light of the HABselect trial and your subsequent mechanistic analysis? So, I think that they are, that we’re aware that the HFEA are also relooking at their entire traffic light system, so that they’re going to change the whole system. And, I would hope within that, that we see something more favorable about PICSI. Basically, at the moment, something has to work for everybody to give an improvement in live birth, to be something that looks green, and that’s quite an unrealistic hurdle for most things versus a kind of personalized medicine approach of certain groups will disproportionately benefit.

So, I’d, I’d hope that shortly, we will see that changing for PICSI and for other things with the HFEA. Wonderful. Okay, sadly, we’ve, we’ve reached the end of the session. I’d like to thank you again, Jackson Kirkman-Brown, as well as those of you attending this webinar, for all the really interesting topics that you’ve raised.