Fertility and Sterility On Air - Unplugged: December 2024

In this month's Fertility & Sterility: Unplugged, we take a look at articles from F&S's sister journals! Topics this month include: a review of segmental aneuploidies and mosaicism (2:00), early removal of zona pellucida to improve embryo fragmentation (18:12), inflammatory markers in female infertility (31:53), and debating the role of adult content in collection rooms (44:51).

F&S Reviews: https://www.fertstertreviews.org/article/S2666-5719(24)00039-2/abstract

F&S Reports: https://www.fertstertreports.org/article/S2666-3341(24)00092-8/fulltext

F&S Science: https://www.fertstertscience.org/article/S2666-335X(24)00078-8/abstract

Consider This: https://www.fertstert.org/news-do/providing-adult-material-fertility-clinics-antiquated-and-nonessential

View the sister journals at:

• https://www.fertstertreviews.org
• https://www.fertstertreports.org
• https://www.fertstertscience.org

Welcome to Fertility and Sterility On Air, the podcast where you can stay current on the latest global research in the field of reproductive medicine. This podcast brings you an overview of this month's journal, in-depth discussion with authors, and other special features. FNS On Air is brought to you by the Fertility and Sterility Family of Journals in conjunction with the American Society for Reproductive Medicine.

Hello and welcome to another episode of FNS Unplugged. I am your co-host Pietro Bortoletto and joined by the triumvirate, the occasional Molly Kornfeld, the always present Daylon James, and the hero in whatever we invite him, Blake Evans. Wow.

I'm always here, Pietro. That is tough. Wow.

Same here. I don't really know where that came from. I like that I get always present because I am present.

I'm here in this moment. I've really missed you guys. This is the highlight of my... What are we doing this, on a monthly? We got to do this more often.

It's been a couple of months. I missed you guys. Well, I was going to say that, but then you said that, Pietro.

I'm a little offended. Well, I'll give you a little bit of backstory to our listeners. The reason I brought up attendance is that our own Molly Kornfeld is in the early term stage of her pregnancy and as of the time of this recording, contracting irregularly.

So, potentially very soon to be without child in utero and hopefully bringing in baby number two to the family. So, you may be missing out on Molly for the next couple of weeks to months as she takes time on her maternity leave. Did I get that right, Molly? Yeah.

We probably shouldn't have my article last today, just in case. Well, we'll have to see how it pans out. There's no tocolytic like discussing science, so you will be fine.

And if I were trying to get someone's uterus to stop contracting, I would also be starting with an F&S Reviews article, which is where we're starting today. Blake, tell us a little bit about the chromosomal challenge of human embryos. Ouch.

Thank you for that intro. But yeah, I'm going to be discussing a rather complex, but very common topic entitled, The Chromosomal Challenge of Human Embryos, Prevalence of Aneuploidy and Mosaicism by first author, Ilya Volodyaev. I apologize if I mispronounced that, and senior author, Ilya Mazunin of Moscow.

Diving into a little bit of background here. So, terms that we know very commonly, whole chromosome aneuploidy, segmental aneuploidies, whole chromosome mosaicism, segmental mosaicism, all terms becoming more and more common in the IVF clinics, and are key factors that are influencing of embryonic development. These four types of chromosomal imbalances do differ significantly in origin, including parental origin, maternal origin, meiotic or mitotic spindle errors, cell cycle checkpoint failures.

And as we well know, chromosomal aberrations are the primary cause of declining female fertility as associated with age. This also is associated with implantation failures and pregnancy losses. So, this review aims to clarify the current understanding of chromosomal aberrations in human embryos and their impact on reproductive health.

And just before I dive into this, a little poll for our audience here. Do your labs report all of these different aberrations? Do they just report normal, abnormal, segmental aneuploidies, segmental mosaicism? Just curious. So, it's a good question.

We actually use two PGT-A providers. One is within our network called Juno that will report segmental mosaicism, and the other one is called Genomic Predictions, which doesn't. So, depending on who you're speaking with and who you're asking information from, you get slightly different information.

Yeah, I was just curious because after reading through this, and I'm going to touch on the high points of this review article, but this is a pretty complex topic and having discussion points with patients when you get some of these results can be very complex on what to do. So, I'm going to go through each of these different aberrations.

So, as I mentioned, this is a complex topic. I did really enjoy reading it, learned a lot. As I always do, I encourage the listeners to go back and read through this paper and go through it.

So, a couple of things of special note, the authors did discuss just first and foremost technical artifacts and noisy profiles that can occur in these PGT profiles. This does further complicate the analysis and may introduce some PGT errors. So, primarily, they discuss false positives when the artifacts are mistaken for aberrations, but also false negatives when true, for example, true mosaicism is missed amid background noise.

The data can even be interpreted as multiple aneuploidies, also known as chaotic profiles, which some laboratories will report in their biopsy results. And such errors are a significant factor contributing to discordant results in repeat biopsies even, and reported rates have been shown to vary quite a bit between laboratories, which we'll get into. May also explain at least some cases of how a healthy life births result after a transfer of mosaic embryos, for example, or abnormal or chaotic embryos that are transferred.

So, first, we're going to discuss whole chromosomal aneuploidies. This is an imbalance affecting an entire chromosome and all cells of the embryo. Touching on the high points with this topic, they talk about PGT-detected whole chromosome aneuploidies are strongly correlative with the women's age, which we know.

This is no surprise to us. This ranges from about 20 to 30 percent in women ages 25 to 35, to up to 80 to 90 percent in women aged 42 to 44 and beyond. After 35 years of age, there's a nice little graph in this paper, and it shows that there's a near-linear increase of whole chromosome aneuploidy, about six percent per year.

They do, of course, you know, try not to put all of the blame on the female patient or on the egg, but that is the large puzzle piece, or a large part of this equation. They discuss that only about two to three percent of whole chromosome aneuploidies are sperm-derived meiotic aberrations. This is why PGT is not recommended based off of how old is the paternal patient.

They discuss about 50 to 70 percent of these issues arise from oocytes from meiosis one, about 20 to 30 percent from meiosis two, and about 5 to 15 percent from errors from the first mitotic phase. They talk about previous studies, how the authors overall say that using PGT with next-generation sequencing to detect whole chromosome aneuploidies is quite reliable. They talk about previous studies that rate one out of over a thousand prenatal tests that were done and undergone PGT had false negative values, so pretty reliable there.

And then they talk, this is an interesting study that they cite as well by Tiegs et al. from F&S in 2021. It's a unique approach to this concept, but they disclose the PGT results after they did a transfer, which I thought was interesting.

And they clarify clinical outcomes, and they report zero false negatives out of 122 pregnancies that occurred, and there are zero false negatives in those that had whole chromosome aneuploidies, so none of these patients were pregnant. And looking back at the transfers that had euploid embryos, about 65 percent of those resulted in a pregnancy. So again, the authors, they discuss that PGT using trophectoderm biopsy for a whole chromosome aneuploidy is quite reliable.

Then just briefly touching on these other aberrations here, segmental aneuploidies. So these, on the contrary to whole chromosome aneuploidy, the authors discuss that this is independent of age, but there are very diverse estimates. They do quote a very well-known clinic.

I'll let the readers go back and look at this, but they say as low as three percent report of segmental aneuploidies, up to as even high as 30 percent in different laboratories, some of which are even from the same biopsy embryos, so a very high discordance rate in these segmental aneuploidies. So kind of concerning to get these results, but also conflicting what you do with these when you get them, and how do you counsel the patient. They discuss how this wide range in aneuploidies could be due to artifacts introduced into sample preparation or DNA degradation, maybe issues in transport, and there's DNA damage or loss, and then how the bioinformatics and analytical methods are done too could result in this wide range there.

As I mentioned, concordance rate could be quite low, 40 to 50 percent concordance rate even in the same biopsy from the same embryo, and so they say that these aneuploidies, segmental aneuploidies, are associated with poor embryo morphology and arrest, which is likely why there's a decrease in these segmental aneuploidies during embryo development. So they say that the bottom line with these aneuploidies, segmental aneuploidies, is they suspect the real biological etiology causing these is about five percent at the blastocyst stage, but the true rate remains to be elucidated. And then lastly, looking at mosaic aberrations.

Mosaicism appears largely, also they say, independent of age too, and whole chromosome aneuploidy, it's independent of that. They do say there are some studies that say that there's a slight positive association. Do you all typically say mosaicism associated with age? I found it interesting that they said that this is not associated with age at all, but they do say, well, there's some studies that say it does, so.

Last year, one of the papers we reviewed definitely associated mosaicism with age, so I do counsel patients on that. I typically do too, I'll be honest, and when I read this, I was like, huh, it's like, maybe I should change how I counsel, but again, they kind of go back into saying, well, there are some studies that show this rate is quite a bit higher with younger patients with the mosaicism rate, and therefore able to self-correct. And they, similar to segmental aneuploidies, they're as low as 3% to about 35% range of mosaicism that's reported in these different laboratories.

And again, the concordance rate from the same biopsy is quite low, 27% to 43% concordance rate. They discussed that ultimately about true biological mosaicism affects probably less than 15% of embryos, with even a lower estimate of around 5%. They again discussed the artifact versus true mosaicism, and discerning the two is difficult to do, and that there are more advanced computational approaches that are being developed, so that way, how do we come out between these two? Again, if we get a mosaic report back on a biopsy, do we transfer it? Do we feel comfortable transferring it? With also knowing that there are more and more mounting studies showing that these mosaic embryos do lead to normal pregnancies, and even testing on the children after delivery, these turning out to be very reassuring data.

So in conclusion with all of this, so whole chromosome aneuploidies are predominantly maternal in origin. Again, this does increase with maternal age. Segmental aneuploidies and chromosomal mosaicism are largely age independent, widely variable in reported prevalence, and probably due to technical artifacts from what the authors are saying.

True biological mosaicism affects about 5% to 15% of blasts, with this developmental dynamics still unclear. Again, probably due to technical artifacts. So there are, now these studies show a lot of agreement in some areas, like whole chromosome aneuploidy, statistics, and age dependence, but they diverge on a lot of other issues, especially mosaicism and segmental aneuploidy.

So although these disagreements are not trivial, they impact two main questions that I'll kind of end with. One is whether and what extent do human embryos undergo self-correction, and the other question is what circumstances do mosaic or aneuploid embryos can be transferred? Do we feel comfortable transferring? A lot to unpack from this. Again, this is a super complex topic, but a lot to chew on.

So what do you guys think about this? How are you counseling your patients with these results? How do we think this is going to change our counseling for our patients? Before we jump in to the patient counseling, I just want to come with, from a more conceptual standpoint, my takeaway from listening to you there, and from my cursory review of the review, is that it's a real moving target, right? And the jury's out. And I think that's, at once, what's so frustrating about these reviews. I really feel your pain there when I was reading this, Blake.

And like every month, you really have to take this synthesis of so many diverging views. But at the same time, I think it justifies why these reviews are so important, is because every now and then, we have to kind of have a review, right? Take the temperature of the field. And in this case, I think it's particularly salient, because the technology is advancing at such a rapid pace that you really need to check in periodically.

Because as the resolution increases now, we're, I think, kind of transcending this linkage and concordance type stuff, and maybe starting to answer the question of causality or etiology to get finer granularity within these groups. You know, I'm talking about, you know, next-generation sequencing, I'm talking about single-cell resolution. And on the other side, from a purely experimental standpoint, I think we have a nice experimental platform in human embryo modeling, where we can start to kind of test these theories of self-correction and pre-implantation embryo development and the cell biology.

I mean, less so the maternal-fetal interface, less so the trophectoderm implantation, but I think to some degree that as well. So I think it's really murky, right? But I think that we're at the beginning of a really long and really beautiful ascent into clarity on these questions. I thought the review was really great.

My main takeaways, I didn't realize that segmental aneuploidies are mostly paternal in origin, and I didn't realize that they probably aren't age-related. And the review didn't give me an answer on whether I'm comfortable transferring these embryos or how I'm going to counsel my patients about it, but it gave me a little bit more information so that I can hopefully come to those conclusions maybe in the next few years, because I think I just need more information first. I think it made me, our lab personally, we report mosaicism, aneuploidy, or normal, but our lab does not report segmental aneuploidies, and this review made me thankful that it doesn't.

So it just seems like so complex, especially with this wide range of error, too, with these, and it just makes it so much harder to transfer, and it's just fewer and fewer embryos available to transfer. So I don't, this is a topic that's like a boxing match at ASRM, you know, when people go back and forth on this, but a lot of stuff to think about for sure. I'd be curious to see what you guys think about just the whole idea that we're always going to be sampling the trophectoderm.

We're never going to be sampling the inner cell mass, or at least not currently. And while the technology may continue to improve, we're still collecting an indirect assessment of the chromosomal makeup of what we expect to be the fetus down the road. Is this just going to always be a limitation of pre-implantation genetic testing? Well, I think it forced you to reframe and reconsider this, as you guys are discussing, the whether or not you want to transfer these mosaics, because the studies that have been done show that the trophectoderm is at higher risk of aneuploidy fundamentally.

So you really are not measuring the thing. You're just, you know, getting an indication that you can draw inferences from, but whether or not we're going to ever directly, you know, measure the thing, it beats me on how we're going to get there. And when we're comparing that this is an accurate test because it's compared to another trophectoderm biopsy of the same embryo, we're not that great at biopsying inner cell mass, even for, obviously for embryos we're not going to use, but the technology still isn't quite there to get a really good inner cell mass only biopsy that's not contaminated with trophectoderm cells.

So a ways to go. I'm sure that in 20 years we'll look back and think we were all so stupid at this point. Probably so.

Thanks, Blake. Let's move over to F&S Reports now. Molly, why don't you tell us what you have this month from reports? So I'm very excited about this article for reports this month.

It's called Artificial Removal of the Zona Pellucida at the Pronuclear Stage, an Exploratory Study to Improve Embryo Fragmentation. First author is Keitaro Yumoto and last author is Yasuyuki Mio. So as we all know, we've made some really incredible strides in our field towards optimizing our ability to obtain high quality embryos from IVF that lead to live births.

I think those of us who watched Joy recently on Netflix were like in awe of, oh my goodness, I can't believe we can do this now. This is so cool and how far the field has come. But there's still a lot of work to be done, particularly for our patients with really low oocyte yield and for our patients with advanced maternal age.

So the authors start off by citing time lapse data that supports that one of the contributors to poor pregnancy rates is massive fragmentation at the early cleavage stage. And then using this association, they focus their work and their research on obtaining embryos that have as few fragments as possible at the cleavage stage. So that's sort of their trail of logic.

The authors propose that fragmentation that occurs along the cleavage furrow leads to loss of cytoplasm and other important organelles, which then increases your risk of embryo arrest and then developing a poor quality embryo. So that makes sense to me. And then the authors propose that removing the zona pellucida prior to first cleavage could then reduce fragmentation.

So my initial reaction to this concept is, whoa, what? Isn't the zona pellucida protective? Wouldn't it prevent more insults to embryo development and quality? And should we be trying to stay more in vivo if we can, rather than completely exposed to this culture media, which is different than the in vivo environment? But the authors paint a pretty convincing and interesting argument. They say that the zona pellucida is essential in vivo for embryo development due to immunological attacks on the zygotes and also to preserve embryonic development as the embryo migrates down the fallopian tube to the uterus. So that kind of bouncy path it may be taking, but that these insults are not present in culture conditions.

So the authors explore removing the zona pellucida before the first cleavage to try to then improve development and reduce fragmentation. The authors initially completed a pilot study that they cite in their introduction on embryonic development after zona pellucida removal, and they looked specifically at three PN abnormally fertilized oocytes just to pilot this and see how it worked, as since those would otherwise be discarded in their clinic. And they found that in those three PNs, blastocysts that had zona pellucida removed maintained adhesion without the zona pellucida present and also had lower rates of fragmentation.

And so then they applied this to a select group of patients in their clinic that had specific challenges developing morphologically good quality blastocysts, or who had repeated severe fragmentation of these early cleavage stage embryos, and they decided to try zona pellucida removal for these patients. So the patients they selected for their study were 161 patients who had not become pregnant and had a morphologically good quality blast development rate of less than 10 percent due to massive fragmentation at the first or second cleavage in at least two prior IVF or ICSI cycles. So this is a really poor prognosis group, 10 percent.

So this was not a randomized control trial, just to highlight that, but it was rather just an exploratory study. And they then performed zona pellucida removal on 34 of the patients, so 34 of the 161 who then consented for this, and then the remainder did not have zona pellucida removal. Of the 34 consenting patients, I believe, based on reading it, the patients were getting to decide how many embryos got the zona pellucida removed versus how many remained intact.

And interestingly, the patients who wanted to be in the study requested for more of their embryos to have zona pellucida removal, ZP removal, than not. So it wasn't a true one-to-one allocation. They might end up with more with removal.

And so they had, for the whole study, 101 2PNs that had removal versus 72 that did not. Their STIM protocol seemed pretty standard to me. Fertilization was both included conventional and ICSI for this study.

And then another difference in the zona pellucida removal group was that they used a hypertonic solution with heaps medium that contained sucrose. So of note, in addition to the changes in the zona pellucida status where they removed it, this hypertonic solution was an additional intervention that was just in the zona pellucida removal group. So I just cite that as a possible confounder to the findings as well.

And the zona pellucida intact group had standard media that was just one-third of the osmotic pressure of the hypertonic solution. Then they did their zona pellucida removal procedure. So they first exposed the embryo to that hypertonic solution.

That created more of a gap between the 2PN and the surrounding zona pellucida. And then they used a laser to partially remove it. And then the time to remove the zona pellucida was about two minutes and required five minutes outside of the incubator per the authors.

Imagine if your lab was just starting to do this for the first time. It might take longer than that. And then the embryos underwent continuous culture for up to seven days.

And the zona pellucida-free embryos were either transferred or frozen when a good quality blastocyst was obtained. Of note, the zona pellucida-free embryos were always cultured to the blastocyst stage. So they didn't do any early fresh transfers at the forest cell stage because they were worried that the four blastomeres, or however many, would split apart.

But they wouldn't be held intact without the zona pellucida. In the zona pellucida intact group, they either did fresh embryo transfers or cryopreserved at either the forest cell or blastocyst stage. So they kind of had the whole gamut, which is a potential confounder as well.

But I think it may not play into things looking at the results, which are really interesting. So in terms of the results, they did a very interesting comparison. They compared the 2PN zygotes to the outcomes of zygotes in prior cycles in the same patient population.

So it's sort of this retrospective matching what they did. And then they also compared the zona pellucida-free to zona pellucida intact within the 34 patients and the 173 2PN zygotes. So focusing just on the zona pellucida-free to zona pellucida intact for this study, they found a greater proportion of grade 1 zona pellucida-free embryos, grade 1 being the best, at 58.4% compared to only 23.6% in the zona pellucida intact group.

Of the poorer quality embryos, those were as expected higher in the intact group or as consistent with their hypothesis. There were also more day 5 good quality blasts, and this is even more remarkable. It was 48.5% rate of good quality blasts in the zona pellucida-free group compared to only 6%, so only 4 embryos in the zona pellucida intact groups.

And after thawing, interestingly, 100% of embryos actually survived the thaw and broke both groups. And they did follow this out to clinical pregnancy, which I think is really important. So clinical pregnancy rate was 8.3 in the zona pellucida-free group and 6.7 in the zona pellucida intact groups.

But specifically looking at frozen embryo transfers, clinical pregnancy was much higher at 32% in the zona pellucida-free groups and then 0% in the zona pellucida intact groups. So looking at this really select group of very poor prognosis patients, it seems like in looking at this data, removing the zona pellucida may improve embryo morphology, reduce fragmentation, and even improve pregnancy outcomes. So just a few things I wanted to highlight with this paper.

So I think this data is very exciting, pretty compelling. I think the authors appropriately acknowledge the challenging of setting a new lab technique that's not established. And they have promising but very early data and pretty limited evidence of efficacy and safety as well.

I think it is appropriate to offer this to the population that you did, as long as they're adequately counseled. They're very poor prognosis. Otherwise, they've already had two failed cycles.

Otherwise, they'd be pursuing donor oocyte or embryo at this point in their journey. And it's also not a randomized control trial, which they're upfront about. So I thought it kind of funny how people wanted more embryos than the zona pellucida free group.

You'd want to be more experimental. I think there's a lot of challenges doing a study of this type in IVF research in general, where you're combining all these different factors with fresh and frozen transfer. You want to do what's right for your patient.

It's not an RCT, but still be able to study what you're doing. So what I don't know is how the embryologist allocated the 2PNs to either group. So if a patient said, I want 50-50 or 75-25, are the embryologists consciously or unconsciously selecting the best-looking 2PNs, however much you can tell at that stage, to be zona pellucida free? Or the ones that have just the thickest zona, the zona that at baseline has the least amount of debris or material in it.

That's a huge point of contention with this non-randomized, small cohort study. It's like, what was the embryologist picking for or against? Because I imagine it's technically pretty hard for an embryologist to remove zona without damaging an embryo, having never done it myself. And you'd probably want to make sure you pick the one where you think you have the best shot at doing it successfully.

Yep. Yep. And they really just wanted the best thing for the patient.

So that's the right thing for this embryologist to do, but it makes it a little flawed when you're trying to study it. I'd also love to have one of the statistics gurus with the FNS. I feel like the FNS on-air podcast has the really statistically gifted people, not that there's anything wrong with you guys.

But my statistician always totally cringes when I want to combine embryos to compare within a study, because she says, a patient cannot contribute that many embryos because the patient is the independent variable, not the embryo. And so, of course, the numbers were really small. They offer additional controls, but I had some hesitation about the statistics, especially with the mixed allocations and the variability there.

I think the last question is, what is the actual reason removing the zona pellucida helps? It seems so counterintuitive because it's something so protective, I would think. But they really propose that there are these perivitilating threads between the zona pellucida and the embryonic membrane that are inducing fragmentation, and that if we disrupt this, that may help. And it makes me wonder if it may not be beneficial for all patients, but maybe some poor prognosis patients have really strong perivitilin threads.

I don't know, and that's why. And then lastly, whenever we make a change to culture conditions or add a new procedure, we just have to be concerned whether there's effects on future offspring. We just don't know at this point.

And the authors of the study responsibly did create a birth registry from children born from zona pellucida-free embryos, so they're going to follow that. And that's really important. And there have been a small number of cases published in which unintentionally zona pellucida-free embryos led to healthy children.

But we know ICSI, which involves a very small disruption of zona pellucida is associated with a small but persistent increase in congenital anomalies. And we still don't really understand why that is or why that happens. And so I think we need to be really stringent and make sure our patients have been totally counseled if they're having this procedure.

But what do you guys think of this? Are you going to have only naked embryos in your labs going forward? Well, although I'm certainly not claiming to be Micah Hill, and I know you just think less of all of us. I'm just kidding. Having trained under him, I know that I would be remiss if I didn't at least say, because I'm certain he would say something about how we have to be cautious of regression to the mean here.

As you mentioned, this is not an RCT, not randomized. So these patients had failures in the past, you know, a couple of failed cycles. And doing another cycle of IVF, is it a matter of taking off the entire zona pellucida? What really had the outcomes become beneficial? Or is it just a matter of doing it again? And so being cautious of regression to the mean in this case, in this study certainly would be cautious of when we interpret these results.

But big ups to the embryologists who are trying to think outside the box and trying to figure out, how do we move the needle for patients? And well, this is cool. This is cool, I think, for innovation's sake, but I think also it lends itself to a much cooler, bigger, more well-controlled study to see if they're onto something. So love to see it published.

Yeah, this is a case of, if it's, we haven't hacked it enough, let's break the system even more and maybe it'll come back around to fix. But just a question here, technically or statistically, is there, is there, is it true that there's an increased risk of ectopic if you're transferring embryos like later stage like this without a zona? And if so, or if not, it's moot, but if so, is that like justified, I guess, in a patient with a poor prognosis like that? What do you guys say about that? I would think it's justified, but I wasn't aware of that data. Have you guys heard of that data, the ectopic risk? Maybe I made it up.

That's homework for all you listeners. Get out there and check it out. Yeah, I'll take some ectopic risks because we can, we can manage ectopics, so we catch them early in our field.

Have to look that up. All right, Dale, and let's go over to you and tell us what you got from FNS Science this month. Well, I think what we're dancing around a bit here with the first two stories is a little bit of murk in between cause and effect, right? And we got a lot of linkage, but we don't got a lot of good reason.

The etiology remains unclear, right? But infertility is on the rise. That's one thing that there really is no question about. And of course, there's a lot of numbers here, but they vary in terms of the relative attribution to male versus female factor in terms of etiology.

But I would argue that the onslaught of environmental exposure combined with the extended period of exposure as women delay childbearing means that the risk and incidence of female cause infertility, male too probably, but female certainly, I would argue, is on an increasingly upward trajectory. But again, the mechanistic links between aging or environmental exposure and result in reduced egg quality, not well fleshed out. One link that's common to both aging, ovarian aging, and environmental exposures is inflammation, right? Chronic inflammation is a driving component of diverse pathologies, including stroke, cancer, cardiovascular disease, autoimmunity, the whole gamut, right? And of course, infertility among those where chronic inflammation is observed in the context of PCOS, pelvic inflammatory disease, and endometriosis, and is linked to ovarian or uterine dysfunction.

Even more generally speaking, many infertility specialists, if you look online, but amongst the more, I don't want to call it pseudoscience, but more like, you know, infertility opinion havers versus like true experts. But there's all types that speak to the importance of diet, right? Including the corresponding author of the article I'm covering today talk about anti-inflammatory diet and other lifestyle changes for optimization of fertility in women. For you guys, I mean, I'm sure patients now they're very informed.

I'm sure patients have inquired as to the link between fertility and diet. Do you guys have an opinion or any counsel on the benefit of dietary modification beyond just weight loss, of course, but like incorporation of Mediterranean diet, for example, or perhaps other lifestyle changes for reduction of inflammation specifically as it pertains to fertility? What's your guys' counsel along those lines? That's a great question and one that I actually get quite often here in Boston. I point to them towards data from the EARTH study, which is a study that's been going on at the Mass General Fertility Center for a decade now that's produced tons of observational data looking at individual behaviors, couples' behaviors, food intake supplements, and has actually come up with a wealth of data to kind of answer some of these kinds of questions.

There's a great book called The Fertility Diet that's written by first author Dr. Jorge Chavarro, who's a MPH, PhD from the Harvard Public Health School that actually kind of takes a deep dive into this and looks at some of the associations. I think there's some data, but not a ton of data. But really one of the areas that I interface more with diet is actually for patients who are dealing with endometriosis.

There's a growing body of literature that anti-inflammatory diets in patients with known pro-inflammatory conditions like endometriosis can be symptom-modifying for these patients. It can actually make them feel a heck of a lot better. So there's something there for sure.

If patients are interested, I'll spend some time on it. But as Pietro said, observational, even our best studies on diet. And so I wouldn't have someone totally overhaul their life unless there are other health benefits for it.

Yeah, agreed. Very similarly, I'll have the same conversation, but rather than fixating on just completely revamping your diet and fixating on every single morsel of food that's put in your body, more so focus on just being healthy, exercise regularly. Just kind of leave it at that, to be honest.

Yeah, as you're saying that, Blake, I'm realizing that all this information can sometimes be a source of stress in itself, which is a known link to challenges with infertility beyond inflammation alone. So yeah, it's complicated. And the benefits as well as the risk of poor diet, but the benefits certainly of dietary modification are really tough to prove, right? Of course, intuition, right? Just looking at the long history, looking at these geographical differences, also observational studies have suggested the influence of lifestyle and diet in these so-called blue zones.

And even prospective RCTs like this seminal study of cardiovascular disease risk reduction in the New England Journal of Medicine in 2018 have supported the benefit of diet. But these studies are a monumental lift and they still don't incorporate that mechanistic link between inflammation and pathology. That's all inferred, but the link isn't there.

But here, work from senior authors Kushan De Silva and Aya Mousa at Monash tried to back into the problem, digging deep into the existing genome-wide association study data to determine a link between inflammatory signaling and infertility in female patients. And the hook here is their application of this statistical methodology called Mendelian randomization that uses genetic variation to infer the influence of modifiable exposures on different outcomes. And they had big numbers from the GWAS database to draw on, and those were stratified into three groups, right? They had anovulatory patients, of which there were 1,054, and then they had infertility or other unspecified origin.

That was 5,600-plus patients. And then they had patients that were treated medically for infertility, 2,700-plus patients. And all of those were compared to roughly 120,000 control patients.

So as I said, big numbers, GWAS information from all these. And in these groups, they focused on the coding sequences and single nucleotide polymorphisms or SNPs and inflammatory markers that are implicated in infertility. They've been widely published, like C-reactive protein, a bunch of interleukins, CCL2, tumor necrosis factor alpha, and interferon gamma.

There were 18 in total that they looked at. And among these increased interleukin, they found four hits, right? There was increase of interleukin 8 and CCL2 were associated with the anovulatory infertility group. And there was an inverse relationship.

So increased IL-12 and IL-18 were associated with a lower medical treatment and lower infertility of unspecified origin, respectively, in those groups. So a little bit divisive results. And I'll be honest, I'm generally wary of studies, one, that exist entirely in silico, if not a pipette, nary a pipette to be seen.

Because you do want to have that kind of confirmation, a kind of in vitro validation of something. So I get a little wary there. And also I get nervous when I see results like this, where inflammatory markers show both a positive and a negative association with the pathology.

Here out of 18 that they looked at, there were two positive, two negative. But, you know, inflammation is a bit of a yin-yang kind of situation, right? So the reciprocity there is absolutely plausible. And there's so much noise in an outbred, willful, stubborn model organism like humans that the emergence of any significant correlation in a massive data set like this one, I think is notable.

At the very least, I think that this, as part of a growing body of recent studies that, Pietro, you alluded to, and that I think there's a growing body of studies like that, it strengthens the rationale for more direct experimental, perhaps even clinical interventional, study of inflammation's contribution to ovarian dysfunction. I'll take your guys' thoughts on this. Yeah.

Is this the kind of line of inquiry that lends itself to an animal model? I know it's hard to draw conclusions from animal models for a lot of things, but do you think this is the kind of thing where you could really expose a mouse model to pro-inflammatory diet or have it overexpress these interleukins and study impact on egg quality, fertilization, blastulation rates, and come up with something meaningful? Absolutely, Pietro. As usual, you're way ahead of the curve here. And that, I think, as I mentioned there, this outbred human condition, right, that's the challenge.

So, getting this both in a system where there is much less variability, like the inbred mouse model, but also that can be very carefully controlled in terms of the exposure, obviously, that would be really a robust system. I think a lot of people would argue about the clinical relevance of polyovulatory model and whether or not the follicular cycle is conducive to modeling human, but I disagree with that. I think that exactly what you're saying is the case, and funny you should mention it, because one of the major reasons why this was brought to my attention, even though I read all of the wonderful FNS articles very closely, is because I do have an interest in this very model.

I think inflammation is really underappreciated as contributing to ovarian pathology. I've studied it in the context of kind of a PCOS model, and right now I'm kind of scooping myself here, but we've had abstracts that are published that are focused on the role of inflammation downstream of chemotherapeutic treatment, so iatrogenic infertility and the causal link of inflammation in mediating that. So, yes, the short answer is I think mice are a wonderful model for this, and we're trying to leverage mouse models as well as human xenograft models to color in a bit on this idea.

I take all this away as, you know, for our fellow listeners out there, I think we've just unpacked probably three thesis projects for you, so you're welcome, fellows, and we all expect to be co-authors on your thesis project, but this does, I think, you know, these GWAS studies, you know, there's so much that they look into, and they're intimidating at times, and I commend the authors for this study. This is a pretty cool study, but, you know, you piece out these different interleukins or inflammatory markers that you're looking at, and at least there's a direction to go towards. So, okay, now we see there's possible association, so now let's do an animal model.

Okay, now let's transition that to translational science here, do it in the human model. So, commend the authors. I think this is very interesting stuff, and fellows, remember, give us a shout out.

Yes, Blake, to your point, I guess the metaphor is the deus ex machina here, in silico giving birth to actual real-life results that could change the landscape for patients, and I think that that's exactly what this is. As you said, identifying targets that we can go out there and test. That was a lot of Latin all together in one sentence.

Et tu, Daylon? Et tu, my gladiator two? Still riding the high. Are you not entertained? Oh, I'm entertained. That was quite good.

Well, speaking on the theme of entertainment, let's talk about adult entertainment for a second. That's an unusual topic for the podcast. So, I have a paper from Consider This this month entitled Providing Adult Material in Fertility Clinics, Antiquated and Nonessential.

This is by first author Christopher Arkfeld and senior author Charles Bormann out of the MGH Fertility Center down the street from us. So, the whole premise of this article is that for the longest time, we've typically provided print materials, such as magazines or videos for patients who are coming into fertility centers to produce sperm. Now, that may have worked in the 70s, 80s, and 90s in the pre-internet era, the pre-smartphone era, but we've come a long way, baby.

There's a lot of stuff that's changed in the intervening time. For one, the population's changed. It's estimated about 7% of the U.S. population identifies as LGBTQ+, and in this younger generation, the Gen Z generation, that number can be as nearly as high as 20%.

So, that means we're probably doing a lot of our patients a disservice by providing pretty heteronormative materials that are not very inclusive. And for some people, that may provide problems because it may conflict with religious beliefs, personal comfort or preference. And for our younger patients, including fertility preservation patients, sometimes providing such materials may be uncomfortable or just, frankly, unnecessary.

Now, enter this study, which was a small retrospective cohort study performed at a single academic fertility center. They wanted to look at what was the impact of removing adult materials from production rooms. So, they looked at 15,378 patients who were producing samples on-site between January 2019 and April 2024, and nearly 8,900 of these patients had materials available for production, and 6,400 did not have adult material available with them.

Now, they wanted to do some forward statistics, but also wanted to look at logistic regression models, adjusting for age, ethnicity, time of day appointment, if it was AM or PM, and look at the indication for the semen sample production. And what they were ultimately trying to decide was, does the ability to produce a semen sample on-site have any relationship with the providing of adult material? So, I'm going to give you the punchline. No, it doesn't.

Duh, it's 2024. People are probably bringing their own material. Why? Well, people probably have preferences.

Two, they probably don't like what you're providing, and three, it's honestly just, frankly, a little weird in 2024 to have a stack of old magazines that are used by a bunch of people in the same collection room over and over and over again. And I really like this paper because I think it raises a why-do-we-do-what-we-do question, and I'm actually going to pose the question to the group here since we have three or four fertility centers represented. Do you guys provide any materials in your collection rooms at Boston IVF? We do not.

We do. I believe we had magazines and videos in our old collections. I'm not sure how the new ones are stocked.

I don't know. I think the clinics are being a little cheap. Not everyone's going to be comfortable using their phone.

They might not bring an iPad or a laptop into the collection room, and so I think they should still provide things. They should try to provide for a diversity of interests, and we really should be as encompassing of different sexual orientations and gender identities, obviously, in what we're providing. And then beyond that, people can bring, if they have a very specific preference that they don't think we're going to have or they don't like what we have, then they can bring their own as well, and that's really easy to do.

We should make sure we have good Wi-Fi for that, but I think some of this is the clinics don't want to pay for it anymore. I'm not totally sure. Let me let you in on a little secret.

They're not that expensive as a light item on the budget. I think a stack of magazines is not going to be a huge cost savings for people, but I would... I disagree, because I'll tell you what, I don't know what goes on in the collection rooms at most, but I would like to believe that that magazine has never been looked at before, has been handled very minimally, if at all, and if it's an iPad, that thing better be fresh out of the box. I would argue that the modern approach to providing adult materials probably means you should have a screen, headphones, a wireless mouse, a wireless keyboard, and a comfortable chair.

Give people access to the internet where you have an unlimited amount of adult material that will suit any personal, religious, ethical preference, and I think will accomplish the goal without having a more limited selection of material that's used over and over again by multiple people that has to be updated and has to be constantly revalued to make sure that it's inclusive. Just to clarify here, is this like an actual problem in ART? I feel like this is on the list of problems. This might be the very last one.

You know, I think if we're paying attention to the patient experience through ART, the often forgotten patient is the male partner who's producing the sperm. I think they get forgotten from a psychological impact of fertility treatment. I think they often get forgotten about from a patient experience over the course of the journey, and I think if we can make this part of the experience a little bit better without spending a lot of money, without spending a lot of time and effort, so we can really spend time, money, and effort on the bigger things that really move the I also think if it's a comfortable environment, sperm quality and quantity will be a little bit better.

I know one of our fertility urologists says, oh, this guy had to collect at 7 30 in the morning. This is a much worse sample than usual. Maybe later in the day it would have been better, and so I don't know what the data is on that, but it seems like a good idea, and furthermore, this would be a great fellow survey project too, actually, because they, the authors of this study, had a great outcome.

They used data they already had, but I think it'd be pretty easy to do a survey and see what people want, and I think for adolescents, I mean, if their parents don't give them access to internet at home, I guess, and this is all something they've never seen before, they can request there's no iPad in that room. I agree, no print materials, but an iPad can easily be wiped down and sterilized between visits. I think this is a great study, thought-provoking, and maybe bring it back to your next division meeting or practice meeting and talk about what are we doing, and could we be doing it a little bit better, so thanks for publishing this in Consider This.

That's all the time we have for today. We've covered a wide range of topics. This could not have been a more heterogeneous group of topics, but thrilled to see them coming out and being presented today.

I'm happy to reinforce that Molly is still with us. She has not given birth during the time of this recording, and hopefully by the time we record this next podcast, where she'll be around, what, 44 weeks gestation, she'll already be with child. This concludes our episode of Fertility and Sterility on Air, brought to you by Fertility and Sterility in conjunction with the American Society for Reproductive Medicine.

This podcast is produced by Dr. Molly Kornfield and Dr. Adriana Wong. This podcast was developed by Fertility and Sterility and the American Society for Reproductive Medicine as an educational resource and service to its members and other practicing clinicians. While the podcast reflects the views of the authors and the hosts, it is not intended to be the only approved standard of practice or to direct an exclusive course of treatment.

The opinions expressed are those of the discussants and do not reflect Fertility and Sterility or the American Society for Reproductive Medicine.