Pronuclear check (PN check) during the first 24 hours of IVF, which assesses fertilization success by examining pronuclei number, size, and alignment, has significant limitations in predicting genetic normality of embryos. Studies show that approximately 50-60% of embryos initially classified as abnormal (0PN, 1PN, or 3PN) based on microscopic analysis are actually genetically normal (euploid) when tested with advanced molecular techniques like SNP-enhanced NGS. This highlights the importance of combining morphological assessment with genetic testing to rescue viable embryos that would otherwise be discarded, potentially improving cumulative live birth rates.
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Day Ones: the first 24 hours
Added:Thank you everyone for joining and welcome to this special I3 session. My name is Bert Ununes and this webinar is kindly sponsored by thermofisher scientific.
Today we are talking about pronuclear check. We know that pronuclear checks are a key step in assessing fertilization success in invitro fertilization.
Under the microscope, embryologists look for the number, size, alignment, and appearance of this pronuclei to judge whether fertilization occurred normally or not. However, while proni check provide useful early information, they are limited in predicting whether the resulting embryo is genetically normal.
A zygote may appear typical under the microscope, but still carry chromosomeal abnormalities. that only genetic testing like PGTA can detect.
Likewise, an abnormal looking pronuclei arrangement 1pn or multiple may still produce a Ullead embryo.
New data puts the humble day one check is at risk in favor of complete PGT at the transfer stage. This webinar hope to explore more this idea. I'd like to start introducing my first co-host, Balsam Al-Hashimi, which she is a PhD researcher in reproductive genetics, University College of London. Balsam also serves as a deputy laboratory manager and lead embryologist in genetics at the London Woman Clinic where she plays a central role in advancing reproductive science and supporting innovative clinical practice.
Balsam, it's my pleasure to pass it over to you.
>> Thank you so much, Beth. To reciprocate, Dr. Ununice is an experienced embryologist and currently serves at as the Emma market development manager for reproductive health portfolio at thermopisher scientific. Drives the Emma marketing strategy and support visionary life science organization in expanding their lab genetic testing capabilities.
We are joined by the one and only Prof. Darren Griffin, a professor for of genetics for over 20 years. He holds honorary position at University College London, the Royal Veterinary College and Kazard University. He now operates as a freelance professor sitting on advisory board of several companies. He's a fellow of Royal College of Pathology, the Royal Society of Biology and the Royal Society of Art and is of International Chromosome and Gene Society.
A world leader in cytogenetics, he performed the first successful cytogenetic pre-implantation genetic diagnosis and played a significant role in development of carrier mapping, a universal genetic diagnostic test for lab embryos nowadays. He is prolific science communicator, a part-time TV presenter and enthusiastic proponent in interdisciplinary research endeavor. He has supervised over around 50 PhD students and I'm one of them to completion and his work appears consistently in the media. Darren, over to you.
>> Wow, I'm blushing. I'm blushing. Thank you for that introduction and thank you all for being here. It's an absolute delight. Now, I'm afraid I have some ground rules for you. So, uh, behave yourselves. Um, we'll keep these bios short today, uh, for our speakers, but their full resumes as well as the 150 plus webinars can be found on www.ivvfmemeing.com.
That's www.ivfmeing.com.
Also, please be interactive and place your questions in the question sections of the portal. Don't use the chat function. You've been using the chat function to say hello. We're delighted to speak to you. We It's great that you're here. But for the questions, please use the Q&A uh because that's where we'll pick them up, otherwise we won't see them. So, without further ado, I've been practicing my Turkish all morning. And I will introduce our first speaker, Dr. Jenis Jingi Olu. is a a globally recognized expert in human genetics and founder of Luminary Genetics. He holds a PhD in forensic science and genetics from Istanbul University and began his career there before before joining Stanford's human population genetics lab under Dr. Lucas Cavali.
He has published in leading journals such as nature and fertility and sterility and he regularly presents at international conferences. So it's delight to have him here. Jenis holds a New York State Laboratory director license in areas including PGTA and cytogenetics and he's a member of ASRM and the PGD international society.
Jenis, it is fantastic uh to have you here. Please take it away.
>> Thank you so much for the kind invitation Darren. I really appreciate it. And uh and I want to start actually Perfect. again uh I assume everyone can see my slide and I would like to actually start by thanking the um international IVF initiative for the kind invitation and this is actually my the title for the today topic pre-implantation genetic testing and viability of plastic stage embryos developed from abnormally fertilized European and wino sites Okay. And this is my dis disclaimer and my disclosure. I'm a founder and gen general manager at luminary genetic.
Um today those are the topic and learning objective that I would like to cover. I would like to just start with the evolution of pre-implantation genetic testing and then I would like to quickly go over the fertilization check how we do the during the init fertilization. And there are few papers that I would like to go over and they talk about the 0 PM 1 PN 2.1 PN and 3P and towards the end we have some research that we have been performing at luminary genetic for the last few years.
Uh one of them is recently got published in fertility and sterility. The other one is actually we get u first price outstanding paper award at PCRS last year and at the end I would just would like to talk about how the improved PGT technology such as single nucleated polymorphism with next generation sequencing would help to rescue some of the embryos otherwise we discard it.
Okay, how how we started um many years ago probably many of you don't even remember uh we used to use fluorosan initiative hybridization analysis this is microscopic testing and we were able to screen only few chromosome because of the color limitation and under the microscope we count those chromosomes obviously I don't want to go over all the every single uh the testing platform that we use in the past uh but some of them is going to be relevant to our discussion that's why I kind of focused a little bit on the fish analysis but we didn't like the fish technology for a lot of different reason one of the main reason was microscopic testing subjective then we moved to CG and snip array those two technology came pretty much at the same time and uh it was a game changer because we were not only doing chromosome analysis we were able to screen all chromosome in 24 hours turnaround time even from single cell uh biopsy qPCR also came at the same time for as a comprehensive chromosome screening But um after using those technologies and they were the game changers but we still probably didn't really like them a lot and we actually switch to next generation sequencing and I call it version one because uh we had a lot of limitations with array platform and I don't think next generation sequencing by itself was able to overcome all the limitation that we were facing in the past. Um and I put this slide just to give you an idea what NGS were able to do and uh and the limitation are listed in here and what single nucleotided polymorphism NGS could actually provide. So this is a little bit long list but today I would like to focus only on this two topics which is hloy and polyployy and those are the one of the major actually um obstacle and limitation uh with next generation sequencing when it's used a lot and now the snip technology become very actually common um in many actually countries and um and I would like to just discuss about how those technologies can help um to uh in vitro fertilization field.
So, um I would like to start actually with the fertilization check. Um so I actually studied myself. Obviously I'm not an expert when it comes to fertilization check but I studied and tried to understand how uh the fertilization check it works. So my understanding is um once you have the all site injected with the sperm whether or not with conventional IVF or Ixie following day in the morning embryologist would wake up and first thing one of the first thing that they do uh under the microscope they look at if they can see two proni which is a normal fertilization or if they don't see any actually proni that's called like zeropian where if you see one proniclei this is also considered abnormal fertilization when you see three also this is also abnormal fertilization. So this is uh my understanding from fertilization check and the 2PN is normal filterization and any deviation from this would be considered abnormal fertilization and those are typically discarded ambience generally speaking. So I always kind of use the same analogy uh with the fish analysis because myself I did it a lot in the past many many years ago. Um microscopic analysis uh has some limitation. Uh for instance, if two signals are overlap, as you can see in here, we used to color code for each chromosome. Uh if you have we count the green, if you have two greens, that means those are actually we have two copy of chromosome 21. If you have two reds, we have two copies of 18. And based on those color counting, we would be able to see the unemploy.
And uh but again because of this is microscopic analysis in case two signal are sitting on top of each other we would be counting as a one uh which because of this is not three dimensional analysis we would be missing. So again microscopic analysis is a great technology but at the same time has some limitation and I think this is also applied to the fertilization check as well. So um one of the first paper that I'm aware of um that they tried to actually address one of the question about uh those fertilization check and this paper published in 2017 by Kabalobo and his co-workers um they had a little bit over 5,000 metaphase 2 um also injected with the sperm and out of those 5.2% 2% um were called one proniclei um with the during the fertilization check and 0.7% actually called 2 2.1 proni and if you look at the paper um try to understand what does it mean 2.1 so basically if you look at the paper they see two nice proni and there is one small one and they didn't want to say three they didn't want to say two it's kind of in between so when they did further investigation on those embryos because because the technology that they used was not able to differentiate between hloy versus deploy versus triploy and they used single lid polymorphism markers as well as the SDR markers and what they found is majority of the one pair of pronici embryos turn out to be actually deployed and some of them were actually still hloid and even some of them one some of the one proni embri actually they were even triploid so uh it is not a large data set but this is one of the first initial paper that they tried to address if those fertilization check with the microscopic analysis um it was really was making the right calls or not. um even when they look at two 2.1 pronuclei and they what identified is majority of them also turn out to be actually uh deployed in the past we thought that the one proni embryos they would be probably arrest at some point they would be never actually even reach out to day five or day six embryo development but um a lot of things is improved in in the invitro fertilization clinics so a lot of actually one chronic lay embryos are surviving to day five and day six and they look like a really beautiful glasses.
Um this is another paper published uh in in uh 2020. Um so one case report it's interesting in my opinion and that's why I put it actually in this slide. Um so 42 years old patient had no duploid embryos other than there was one called 46xx with the testing was done with next generation sequencing and that actually embryo was marked as a one pronei uh in the biopsy worksheet. Um since the patient did not have any uploid embryo to transfer um she was considering to transfer this embryo and knowing there are some limitation with this technology and the patient was consult and then they mentioned about this and alone cannot differentiate between hloid versus deploy. So at the end the patient decided to get this embryo with transfer. She wanted to take the risk and the the embryo implanted and unfortunately there was a miscarriage and the products of products of product of conception results showed with the SDN analysis it was a molar pregnancy and the paternal drive al was confirmed by using this short pandm repeat. So again there was an add-on uh um testing on the product of conception besides the NGS just to confirm that that was the initial analysis with microscopic testing was correct and but again patient want to take the risk and uh but unfortunately ended up with miscarriage.
So this is another nice paper that I would like to review because this is a little bit actually more larger data set compared to initial papers. Um it's the idea is the same and um 0 p.m. drive blast assist 100 p.m. drive blast assist and 3pm drive blast assist were uh tested further to understand uh out of those actually um fertilization check which one is actually confirmed and which one is actually show that that was a normal fertilization. So if you look at the first one 0PN there were 291 zerop embryos based on the microscopic analysis 56% were confirmed to be deployed which is half of them and the other half of them almost they were confirmed to be uneployed and some of them actually even they were hloid. uh when it comes to one proni there was 217 embryos and what they found is actually almost like one/ird confirmed that they were actually real hloid and that was kind of like a in an agreement with the fertilization check with the microscopic analysis and the other oneird turn out to be actually is a multiple antiploes and the other oneird actually they confirmed to be deployed and they were normally fertilized. So this is cut large number of percentage of embryos uh basically turn out to be completely uploid and normal normal fertilized embryos. When it come to 3pn, half of the 3PN that they were called during the fertilization check were confirmed, which is nice, but other half turned out to be unemployed. And 8.1% of the 3pn that they were called 3pn and again some of them could be even maybe 2.1. So I don't know. Um, but they were also confirmed to be deployed and normally fertilized.
Well, when you look at most of the 2PN when they did the fertiliz when they did the actually further testing, majority of the 2PN that they were called 2PN during the fertilization check actually they were confirmed with a few exception which is 0.63% of them turn out to be also triploid.
So if you look at uh so those um analysis already said um also we want to do also the clinical outcome because now we do the testing but what about the clinical outcome and out of those actually um further testing they transferred 74 of the ember that were previously initially called zero uh p p.m. or 1 p.m. or even 3 p.m. And out of those they got 16 ongoing pregnancy, 32 healthy live births. Um so when you look at the total of live births and ongoing pregnancy rate is about 64.8% which is very comparable to duplo embryo transfer. So their conclusion is that um with um accurate identification by using snip markers you can definitely identify the hloid and triplody which basically those are uh it's very critical to prevent implantation failure and pregnancy loss.
So after those papers many more actually uh came out and basically they would want to do the same investigation just to make sure that the fertilization check is was done correctly in the first place. Now after with this quick introduction I would like to actually talk about our research at luminary genetic which we uh had a couple collaboration we started few years ago just to answer the same question.
So before we actually start the study we did a survey that was in 2001 and we basically asked the participant we said what do you current to do with zero pep one embryos that continue to develop.
What we found is majority of the actually participant I want to say majority 36% of the participant said we continue with the PGTA knowing that NGS alone there are some some limitation and the 34% of the participants said we simply discard them which is quite larger actually percentage uh that actually the people who participate this um survey said that okay we discard this risk but uh you will see in the upcoming slide like discarding those zombies probably may not be a good idea.
So when we actually had this NGS alone um and we actually wanted to do further testing on some of those European 1pm and we actually decided to use short tandem repeat um and that's why I put this slide just to give you an idea shortandm repeat as you can see from the name there are some repeats actually happen to be in intron they don't really code any region um and we don't know why they exist but we are using those uh repeat markers for parentage human identification even in my laboratory we are using for product of conception testing to detect or roll out maternal cell contamination for mer pregnancy in embrenic level uh we are also using to detect hloides deploy and even we have also noninvasive PGTA effort in our laboratory in order to detect or rule out SUS contamination we are using also those STR markers so STR markers is a a wide application in our field. Um and when we did the genetic testing with NGS and we used the SDR markers just to identify uh those supplies and triploides. Um so this is a kind of a a screenshot how basically the DNA DNA fingerprinting it work with STR markers. So as we all know embriionic DNA get half of the DNA from maternal side and other half is for paternal side. And this is again a quick screenshot and from the SDR markers and basically each peaks is representing one al. So one al comes from mom one al come from the mom dad and um any deviation from this we know that this embionic DNA is not belong to those actually parents um then the testing by looking those informative markers. So those circles that I've just showing you, it represent uh one actually um markers uh from the STR and those two picss underneath is basically showing the al. So based on homozygosity and hetererozygosity, we will be a able to identify which homal would be going to the embryionic level. So again those are more markers uh to kind of to show and when we actually did those testing with no samples we wanted to make sure that if this STR markers would help us to also identify uh to look at the hloid and deploy and this is what we did the validation. So we had a maternal DNA and paternal DNA just because of the study um and then we had also embinic samples that they were initially called one pronuclei or zero pronuclei. What we found is actually um in some cases we have only one alil coming only from maternal site across the board and that was basically indication for hlo um and this is a table version of showing uh this STR markers uh here in the table what you're seeing in here a one case report from uh the the study where we get patternal and maternal samples and one embryo actually called one pronuclei in that cohort. And when we did further investigation by um profiling maternal sample and paternal sample with embryionic sample, what we found is embryo actually get both paratal contribution. So I just kind of mark one just to give you an idea how actually we are able to really do the analysis. So for instance for that specific markers we call it D16 5539. That's the name of the the SDR markers. The father happens to be hetererozygous has 10 and 12 13 repeat for that alil. Mother has 11 and 13.
When you look at embic sample has 11 13.
So we know that uh the 11 is definitely coming from maternal side and 13 is coming from paternal side. So we know that there is both parental contribution in embry level. This is another example also show that the embryo is getting both parental contribution. So when you look at across the board for every single STR markers that we did the analysis you would be able to see hetererozygosity or homozygousity or both parental contribution which that's indication uh deployed embryos but that embryo was called um one pronuclei during the fertilization check.
This is another example and in this case we have actually two embryos. Both of them are actually called one pronici. I would like to start with number three.
As you can see in here, um there is actually one parental contribution and all is coming from maternal site. Um which is clear indication from one proni results and the fertilization check was done definitely correctly. Um but when you look at the embryo number one, you see um hetererozygousity pretty much across the board and there's both parental contribution. So number three the fertilization check was definitely was the the correct call. But when you look at embryo number one actually that one pronuclei turn out to be actually both parental contribution which is true pronuclei.
Now the question is do we really need even parental samples to do hloid and triploidity? The short answer is no.
Those are very highly poly polymorphic markers. When you see homozygousity across the board like as you can see in embryo number three this is clear indication from pronlei but when you see a lot of hetererozygosity we know that there's both parental contribution and that would be the indication for deployed results now after this all this um introduction about the technology so I would like to just give you the outcome data from our study so the we started in 2021 we had two collaborators And during the one and a half two years um time we were able to get 432 embryos that they were initially called 0 PN 1 PN during the fertilization check and when we did NGS only uh PGTA what we found is 63.2% of those embryos turn out to be uploid and six uh 36.8% they were either mosaic or anoid. So we've only wanted to focus on only onlo one which is 63.3%.
Since we know that NGS alone cannot really differentiate the hloid versus duploate versus even triploid. So we did further testing. We did not have single nucleotided polymorphism markers spiked in into uh our platform. So we did further testing with SDR and what we found is 96.1% of the 0PN which is pretty much like almost all 0PN turn out to be actually biparital contribution so they were actually 2pn and when you look at uh the 1pn results 42.1% of the one pronuclei they were correctly called with the under the microscope but when you look at the other half which is a little bit more than half 56 6.8% of the W proni also turned out to be actually deployed both parental contribution. So they were actually the embers that they were rescued.
So finally those data has been finally published in fertility and sterility and um and I would like to just quickly go over about our conclusion with this paper. Secured HRA guideline recommend against using ambulance dried from zero or 1pn zygots. In this study, many zero and one PN zygos that went under PGTA and SDR analysis confirmed deployed and by parent of deploy and significant number of uh this genetically healthy embryos would have been discarded based on current ASA guideline and our recommendation is probably those guidelines should be really changed if the right tool is utilized for the genetic testing.
Now obviously we did all the testing now a lot of actually clinic and embryologist and patient asking us like then what about the clinical outcome?
Yeah, you did the testing. We saw WPN and you were telling us this is actually deployed. Um do you have any clinical outcome? And finally our collaborator um because of some of the patient did not have any normal embryo or any newloit and 2PN embryo did not have um um available to transfer. So they decided actually to go back and transfer those embryos that they were called initially 0 p.m. 1 PN but our further testing showed that they are completely deployed and deployed normal fertilized embryos.
And here is the data in here. So we actually did 228 analysis. Half of them came back through deployed with the further testing by using SDR markers.
And among those again majority of the n European again turn up to be steel deployed and almost half of the one pron to be also uh deployed and then out of those actually um uh 28 uh transfer because there is a 28 transfer out of those 0 p.m. 1 p.m.
which is not a large data set but that's what we have at this point.
14 of them actually were coming from 0 p.m. transfer and one of them exactly was coming from um from 1 p.m. answer and we got uh 53.57% live birth rate which is also comparable played ambria transfer and uh and our con conclusion is u although uh the data is limited uh but transferring those embryos resulted in live birth which is very similar to uh or even better than the current start data and we were able to rescue uh 124 embryos it's only from single centers and after actually we did all the study almost one and a half years ago um we decided actually to spike single nucleated polymorphism into NGS and with the collaboration of the thermofishure now this product line uh finally is launched and uh but with we still actually ask our collaborator to help us uh with the uh fertilization check analysis. So what we did was we actually asked the clinic although we will we will be able to actually identify zero one pian even though um they don't really have to tell us what they are during the fertilization check but we asked actually the collaborator to put notes on the biopsy worksheet when they sees European warp and what we found is uh 15% of the time the the clinic or the embryologist at the clinic is whether or not they are debating or whether or not they are actually kind of calling uh those embryos either 0 p.m. 1:00 p.m.
which is quite larger of uh embryos every day to be debating and what we found is among those 15% that they were initially marked as a zero pen one pen turned out to be transferable meaning that they were either completely or they were mosaic which is still transferable.
So with this uh single nucleotid polymorphism and justest that we spike in the our platform that gave us an opportunity to rescue embryos uh that would be discarded um or is not even transferred.
So again this is kind of like a quick summary about what NGS could do or cannot and what the single nucleotated polymorphism can provide and those are the two things that I just wanted to mark because those are relevant to our discussion today. hloid and triplo polylo become very relevant to rescue some of those embryos um that will be otherwise discarded during the fertilization check again this is my last slide um I think there's definitely some a lot of advantage of using single nucleotide polymorphism based NGS uh but the whole point of today discussion is is not about how often we see 0 p.m. 1 p.m. or 3 p.m. The question is what are we going to do when do when do when do we see them? Simply discarding those embryos probably is not right way to do it because we know that PGTA with NGS alone is not a solution and detection of 0 p.m. 1 PN 3 PM still limitation with NGS platform.
Um but by using those advanced technologies such as snip NGS can detect by parental deploy which would ultimately eliminate the need of fertilization check with that I would like to thank my old co-workers and happy to take any answer that you may have.
>> Thank you so much very very informative.
Um as you can imagine there'll be a lot of questions towards the end uh but I'm tasked with just asking one now. There are a few in the chat, but I'm gonna take chair's privilege and ask you one of my own. Um, so one of the criticisms of PGTA in general is that uh it doesn't improve um uh the live birth rate per treatment cycle. Now, do you think by using the approach that you uh that you outline here um and essentially that's creating more embryos than you would have normally? So do you think that it might have a significant uh enough effect to actually improve the cumulative live birth rate?
Yes. Uh definitely because um that's the reason I put one of the slide about like uh 15% of the embryos that are basically kind of debated during the fertilization check would probably never come to us if they were discarded in the first place.
So now having those regardless of what the fertilization check coming to us and we would be able to do the molecular testing and give the ultimate answer which based on the data that I show majority of them turn out to be actually G plate and normal fertilization and those are the embryos would kind of like will be saved and rescued and they will those are would probably help with the cumulative life birth for sure.
>> Yeah. I mean and the opposite argument uh which does appear in the chat is that isn't it overkill? Aren't you just charging patients too much?
>> Um, so if you look at the data, if you think about like majority of the European, which is pretty much 96% of the European turn out to be actually deployed. I don't think this is about really charging the patient is about like really rescuing the those embryos.
Um because again I trust the microscopic analysis, I trust the experience of the embryologist but at the same time and this is molecular approach. Um and like I probably you also heard about the same discussion at PGDIS like what does it means European like there's no such a thing there's definitely it's just like probably a window is missed so I think we're helping probably the clinic and embryologist to take that burden u we can do the molecular approach and we can identify whether or not they were like biparental or actually hloid embryos and I don't think again those are it's kind of like we're not charging extra to the patient we're just trying to rescue those embryos otherwise will be just discarding like healthy embryos.
>> Fantastic. Right, Jenis, thank you so much. Please stay around for the questions at the end. Um, very very grateful for all of that. Um, and it is time now to introduce our next speaker.
So, Dr. Anel Birich has over 25 years of experience in reproductive genetics. He is the scientific director of the IVFPGT unit at Europen's genoma group Italia.
He earned his bachelor's and his masters in medical biology and genetics and complete his PhD at Sabenza University of Rome with a focus on the genetics of endometriosis.
Anil has authored over 30 peer-reviewed publications in molecular biology and reproductive genetics and his current research focuses on the genetics of infertility and innovation in PGT. He's a member of Essher. He's a member of PGDIS and the Gen QA special advisory group. So it gives me the great pleasure to introduce Dr. Anil Birishek and he's is the talk of his the talk of his title or indeed the title of his talk is a snip in time genotyping jubiously fertilized embryos. Anil could you please take the stage?
>> Thank you very much. Thank you very much Darren for your uh kind introduction and I'm very honored to be invited to speak at this I3 webinar which covers one of the IVF hot topics. So my talk will be about the current application of molecular detection of ploy abnormalities in IVF embryos and our experience applying these method uh on a card of embryos with different fertilization pattern. So first I would like to share my disclaimer and my uh disclosure and I'm a full-time employee of European genoma group which provides a laboratory service in the field of reproductive genetics. So as a previous uh speaker Dr. Jangi Ginolu explained very well the different applications of pre-implantation genetic testing for an employes. So I would like to just uh start with the current uh gold standard method and adopt the screening in human pre-implantation embryo is a common practice now in IVF applications and which requires the surfect biopsy on day five or six IVF embryos and following comprehensive chromosome screening by CGH or NGS as a current gold standard uh methods and after processing the sequencing data by specific algorithms we obtain these uh NJS plots like these images are now very well known very common and very easy to interpret the uh with the chromosomal copy number changes with the balanced chromosomeal copy number for oploid embryos and some deviations like gains and losses like a triome or monomis in the anoid embryos.
So um but other than single or multiple copy number variations uh some forms of fertilization may affect entire set of chromosomes. Fertilization pattern of a normal zygote requires a conception of two games both with a balanced chromosome set and as a normal fertilization we expect a deployed set of chromosomes which are coming from 23 from father and 23 from uh mother. But in some forms of fertilization it may happen that one of the games loses a complete set of chromosome and at the end we have two zygots with maternal or paternal hoid and which is not a viable form for this kind of zygot and or as an alternative one gamut with an extra set of chromosome entering conception with a gamut with gamut with normal set of chromosome and at the end we have a we have a zygote with the maternal or patternal triploid which again is not viable. Or in some rare forms of fertilization one gamut may lose entire set while other may have an extra set and in these conditions we expect patternal or patternal origin of uniparental deploy based on the origin of is coming from oite or from the sperm and again these forms are not viable. So in the literature uh we now it is very well known that u the clinical importance of these kind of ploy ploy abnormalities as triploides are estimated to account about 1 to 3% of natural natural pregnancies and the frequency of triploides in spontaneous abortions rises uh to around 8%.
And if we look at the spontaneous pregnancy losses with chromosomal abnormalities triploides are found in about are found in about 15% and these forms of ploy abnormalities are not expected to be compatible with life but in some cases uh already reported the tripes arrive to term with severe congenital abnormalities.
So we expect that such kind of um severe chromosomal abnormalities would have an impact on embryo development and which has already shown in several studies at the end of 80s um boyers colleagues um showed that embryo cleavage rate and they were uh just um uh eval numbers as a indicator of a ploy status as an prediction of deployed status embryo cleavage level they absorb higher and 2pm group than those have more than 2pn like 3p so in 2p they absorb 92.7% of cleavage rate and in 3pm 65% of cleavage rate and um in another study by a zoo at all morphocinetic observation observation of abnormally fertilized oite sites uh shows significant uh delay in blastoid formation in one PN u and 3PN compared to 2PN groups. Another similar interesting study has been recently presented during the last ASHA congress two weeks ago uh by Marcia Barbarian and colleagues from Rome and uh they observed that in their study the the development delay in the for the to reach the blastois stage compared to 2PN embryos that all 1pn embryos were having uh the uh blastosis level as 76 6% at day six and only 24 uh% at day five.
However, uh the chromosomal rescue of the embryos deriving from abnormally fertilized oytes were already shown by the studies of like Kapalo and colleagues and Gerard and colleagues and some of these cases were also reported with a healthy live births like Brad Becca at all as show one pregnant and healthy live birth from 4 p.m. Dive embryo.
So the pronunc scoring I think this will be explained very well with the next uh speaker Dr. Paris uh but uh uh scoring after 16 of or 18 hours of fertilization is usually done for ploy prediction as a routine practice in IVF embryos and we expect uh that embryos growing from 3pm uh potentially result with triploides whereas others like 1 PN result as hloides and embryos coming from 2pn we expect dloy with different aloid or an employee.
uh and based on this approach um all embers from abnormal are not considered clinically usable and they are not considered more or less usually they are not considered for the biopsy but now uh we know that a potential ploy rescue may save these embryos if their ploy status can be confirmed with correct genetic uh testing strategies or vice versa.
uh a correct testing strategy allow us to confirm if an embryo from 2pm is really deployed.
So how can we detect uh polyloy with current standard comprehensive chromosome methods based on copy number variation. uh some forms of polyades like 69xy can be revealed by NGS copy number analysis uh with the intermediate log ratio profiles of sex chromosomes as we can see here in this image but some other forms uh like um uh 69 x66 or hloid 23x we cannot uh detect discriminate easily from an aloid female profile like the 46 XX as you can see in the second image and also in some challenging uh samples some challenging NGS plots it can be difficult to interpret even 69 X6Y genotype for example in the last bottom image we can easily interpret this profile as a chromosomeal mosaicism uh with the intermed intermediate chromosomal copy numbers and including the set chromosomes. But if we detect detection by molecular gening, we can see that this embryo is 69xy.
So all these data shown that additional test methods are needed to reveal all types of ployy abnormalities.
So single nucleotided polymorphism genotyping allow us uh the molecular detection of polyploides in an accurate way. Snip genotyping applications can be done by snip array or by NGS. A large number of snip panels which scattered on 24 chromosomes can show us the hetererozygosity frequencies of each chromosome. In DL frequency of snip array profile we observe three different genotype combination in uh deployed genotypes with the hetererozygous snip combinations in the intermediate level. You can see here whereas we obser whereas we observe only two homozygous gen genotypes with complete loss of hetererozygosity in hloid samples. Uh in triploid samples instead we observe two hetererozygous uh genotype combination coming from four different uh genotype combination.
Similarly in NGS snip panels we see the uh very similar PL profiles with the in the hloy with the complete loss of hetererozygosity in the intermediate level and in deploy one hetererozygous snip genotype combination uh in the middle and in triploid samples we see two different hetererozygous snip genotype combination.
So uh in our laboratory we investigated deployed incidents in different pian embryo groups by snip genotyping technology.
We used an NGS based PGTA methodology which with a combination of um copy number variation and snip analysis together and we applied a poly deployed panel with more than 500 snips uh included and the copy number analysis by um after standard trophy five um copy number analysis by low pass sequencing has been applied by [Music] some PGCA methodology and in parallelly on the same uh biopsy sample by snip genotyping to discriminate deployed uploaded and triploid genotyping approach uh provide us also some other genotype um extra genotype information like sibling QC. So in this profile is a heat map profile.
As you can see for instance the combination and the comparison of uh snip genotypes between the embryo pool give us if the embryos are coming from the same cohort. So means that this is coming from the same parents uh and in this for this information we don't need any additional DNA samples from the parents only the uh polytoid algorithm can compare the syn genotypes of the embryos between them as you can see here like white and gray uh dots are showing the uh the compatibility between the same cord. So these four embryos coming from the same father and the blue dots are showing the the individual side genotype of each single embryo and additionally we can also detect easily if there's an external or maternal contamination when they studied samples by snip genotyping. So before the application of this cine panel on M biopsy samples, we tested it on some known uh polyloit and aloid cell samples or whole genomic amplification products just to test the uh validation of this uh profile. So we used differently uh different ham amplification product which was which we expected 69xy profile uh from copy number an analysis and we also used some uh few cells isolated from a product of conception samples like 69 x66 or 69 x6y and also some other good genomic amplification product products coming from deployed aloid sample and deployed an employee sample like transm 21 and deploy the status of all these samples on uh detected uh correctly and uh with the high confidence of employee call.
So uh after uh this validation uh 196 samples uh they evaluated by PN scoring and 74 of them coming from 2PN and uh 12 of two of them were coming from abnormal fertilization including 0PN 1 PN 2.1 1 PN 3 PN 3.1 PN and 4p as uh previously demonstrated in the the diagram Four beaced arm biopsy has been applied on day five six and the parallel low pass and copy number analysis and targets the NGS for synapping has been applied and finally 100% of composite data has been obtained from the two PN embryos and uh 97.5% [Music] composite data has been observed from uh abnormal PN embryos.
The samples in this study were classified according to their pronuclear profiles. But we look at the PGT indications as well. And we see that among the indications like uh recurrent implantation failure, advanced maternal age and recurrent miscarriages. We observe that there was a strong dominance of uh advanced maternal in age indication uh in our cohort.
So these are the results we obtained after parallel analysis of coping analysis and snip genotyping and they these were some examples of confirmation of clo status by molecular methods. So one embryo coming from one pian fertilization were show a totally normal aloid like balanced profile by copy number analys analysis but the snip genotyping showed a hloid with complete loss of hetererozygosity. So this was a 23x embryo hloid embryo. Another uh pattern from coming from 2PN deriving from the 2p fertilization show a normal male profile in copy number analysis uh and again with normal hetererozygosity pattern for um for bison genotyping. It was a 46xy male embryo transferable and another sample was coming from 3pn embryo uh which was showing completely like a balanced profile in copy number analysis but uh is snip genotyping show of the two hetererozygous frequency uh peaks and this was again a 69 x6x embryo and uh if we see uh some embryos with the ploy rescue Uh here is one example coming from one PN embryo and it was a normal balanced female profile and hetererozygos allowed frequency by snip genotyping also confirmed that the hetererozygosity and this was a 46 x6 embryo. It was a ploy rescue and similarly another embryo coming from the 3PM fertilization showed a normal profile normal male embryo profile by copra analysis and hetererozygos frequency also was confirming this 46xy genotype of this embryo.
So if we look at the rate of ploy abnormalities in normal fertilization group it was a very small number. It was only 74 samples we analyzed and we defected 1.4% 4% of holy and 16 uh 2% of triploid rate in this group and the rest of the embryos were deployed what several uh deployed unloed deployed deployed results has been observed but interestingly this triploid rate is very very high than we expected even in the literature. So this was uh we should uh deeply investigate these samples. Of course this is a very small cohort but uh at least if you look at the indication uh 11 of uh these 12 samples were coming from advanced panel indication with 37 39.7 years old and one occasion from the recurrent miscarriage indication and uh if we look at the rate of ploy abnormalities in abnormal fertilization group we did not observe any uh employed abnormalities in 0PN, 3.1 p.m. and 4p groups. Uh these are very small numbers but especially zero zero group 13 samples were showed no ploy abnormality. uh and as we know uh it has been discussed also during the ASHRA and we know the guidelines in zero PN actually is been is suggested to use the PN not observed category so we cannot ex be sure if there is really there were really zero PM but in this category we didn't absor observe any ploy abnormalities but uh significantly there were a triploid rate highest triploid rate in PPang group like 76.9%.
And um and if we look at the overall deployed and employee uh deployed aloit rate in this abnormal fertilization group uh we observed 12.6% of these embryos coming from abnormal fertilization were deployed a so the this was not uh shouldn't be underestimated. It's a good percentage of samples can be rescued by correct molecular genetic testing by unemployed detection. So if we look at the deployed rates in different type of fertilization pattern uh we didn't see many difference between uh 0 p.m. 1 PN 2.1 PN but as we expected uh there were very very very low amount of fertilization uh deployed rate in 3PN fertilization group as 23% only for uh in our cohort in a small group of samples we also investigated if there is a difference uh if there's an effect of PN scoring method on polyploity detection rates So comparing the time-lapse PN scoring and based they deployed based on the conventional PN scoring 101 samples from time-lapse and 44 samples from conventional uh PN scoring observation scoring methodology. So there was no very statistically enough number of samples but at least I can give you the some percentages that especially in this group with the time-lapse group we see significantly rates were really high in one pianlo rates are really high inp group as we expected. So this is also showing that the correct observation strategy may help also the correct prediction prediction of the uh polyployed abnormalities.
So uh as my previous slide uh we I showed that we find 15 transferable embryos in abnormal fertilization group which resulted deployed aloid and among them five embryos were five single embryos were transferred resulting with one beta hg negative results and two beta hg positive results one from 1p one from 2.1 PN group and one ongoing pregnancy has been achieved from uh 1 PM group and uh one spontaneous mascarage came from 0PN group.
So in our study there were of course uh some limitations. Uh first of all we had a limited number of samples. Uh in this study and embryos from uh the develop from 74 from normally fertilized oides and 120 from abnormal fertilized oides were investigated. Observation standards and PN scoring parameters between I centers may introduce bias. So we couldn't get the total uh information about their method but we are we we will investigate the prospectively especially on these parameters.
We understand that this is very important parameter to observation and scoring uh method which method is used as a time lapse or as a conventional method. This creates a strong bias. Male factor indications were not investigated in this study. So we cannot understand if there the ploy abnormalities also coming from the male factor indications and no parental samples were included.
No parental DNA samples were included.
So we cannot also rule out the eventual uniparental deployes from maternal or paternal origin.
As a conclusion, our study confirms the importance of comprehensive molecular techniques like snip genotyping to reveal deployed status of the embryos.
And in our cohort uh 12.6% of embryos driving from abnormally fertilized oats show the rescue and resulted suitable for transfer. And this is very uh good number uh to uh showing that the correct genotyping strategy method can save this kind of embryos and high purity rate 16.2% was observed in two PM group. This may due to the patient's PGT indication.
But we cannot underestimate also that the limits of PN observation and classification. So we don't know if these two pans maybe classifi classified not with the correct like the timelapse method. So uh we need to also investigate this parameter high deployed rate uh observed in 0 p.m. 1 p.m. and 2.1 uh 2.1 p.m. groups. uh as 100% 71% and uh 86% respectively. So we can uh absolutely recommend that if the embryos uh growing from this kind of uh fertilization pattern biops is strongly recommended because they are really high uh percentage of uh ploy rescue and rescue rates and the trip embryos show highly triploid rate like 76.9% and only 2.6% 6% of this group was suitable for transfer like typtoid fluid. So this is also showing us that embryo biopsy and genetic analysis option should be discussed should be always suggested. Of course even 2.6% is a chance but uh this uh possibility also should be discussed during the genetic counseling before to decide the biopsy of these embryos. So finally I would like to thank uh all the participants and um I would like to thank my team in Europe's general laboratory and especially a big thanks to the uh collaborating IVF centers professor manogma [Music] from the extra fertility Niktos Ditra and Dr. Seon Biani and Georgief and thank you for your attention. Thank you.
Thank you Anil as always a great presentation. It's always nice to hear how controversial it could be. So uh based on your data I will just like follow up on your conclusion. We see advanced maternal age we know it's an indication that there's something that you cannot compare it like with younger age. And from your data I was like looking at it attentively 20% only 20% from the 2PN were deployed deployed >> whereas for the abnormal PN check you saw like 12% were deployed deployed so should we assume or is it legitimate to say like or should we consider when we have a woman with advanced maternal age that we skip the pr-uclear checks and we go directly to PGT.
Yeah, actually the indication I think one of the most important factors. Again I repeat that our cohort is very small numbers but uh we saw this significantly higher unemployed and the poly employee rates and advanced maternal age group and again for this uh this study we didn't have a study designed with the equal numbers of patient indications. So this was an overall distribution of the samples. uh we basically focused on the molecular uh detection by snip genotyping and during both uh our validation study and even during the clinical applying on the clinical samples we showed a really strong um correlation between advanced maternal age and these abnormalities. So I think uh my opinion is uh independent from the also the the the the the patient's indication uh polyploy analysis with the molecular testing of polyploy by snip genotyping uh bring us a big advantage also to to classify the correct detection of uh the genetic profile of these samples and uh we know we still know the limits of the copy number analysis uh which cannot detect all these kind of forms. But uh I think uh technology now is allowing us to combine all these uh genetic information to select the best embryo. But still of course we have to uh at least we know that the the the the despite the the the the the differences in the patient indications at least for example embryos coming also the the scientific data the the literature showing that the 3PN embryos we expect a high triploid rates and this is also the confirmation uh by the molecular technique. So I think we should consider all these specifications, all these parameters to decide the conselling of the patient and for the embryo biopsy.
>> Thank you Aniel. Thank you. I think it's always to find the balance depending on each so >> and please stay with us for the Q&A because we have a lot of questions that we will be answering shortly. But before we move to it, let me introduce for you our last speaker, George Lee Paris.
George is a consultant embryologist, founder and scientific director of embryoen and honorary senior lecturer at the University of Sydney. Certified by Eshre as a senior clinical embryologist, he combines clinical practice, teaching and research focused on improving assisted conception methods. He served on editorial boards for human reproduction, reproduction and fertility and focus on reproduction and is an ASHRA assessor for ART center certification and training programs. He has authored book chapters and numerous peer-reviewed publication and is a frequent speaker at international conferences. George, it's a great pleasure to have you here.
Please take over.
Thank you ver for the nice uh introduction. Thank you to i3 team for inviting me to be part of this uh exciting webinar. So the first two speakers Dr. Sigin Soglu and Dr. Nanil Bik did a great job in eloquently explaining the genetic constitution of what we see. But what happens in the embryology lab? What do we see? What are the first moves we observe during the first 24 hours?
First of all, I have um no conflict of interest or financial relationships to disclose relevant to this uh presentation.
And during this presentation, I'm going to first um define the fertilization events that take place during the first 24 hours. Talk about the features that we observe in the zygote. Do what do we see and what value do they have? Can they help us um select embryos? Can they serve as markers? Which of these characteristics can we use as markers?
Starting off with the fertilization events. Of course, the zygote is defined as the fertilized oven. It results from the union of the female gamut and the male gamut. And the fertilization involves several steps starting from the sperm penetration, the fusion with the oite oite activation that is followed by the calcium oxilations from the sperm that results in meiosis resumption. And there are key events that follow and those have to do with the P and formation their migration alignment and singam and of course it all starts with the oside and sperm but the key events of sperm passage in nature are a lot different when we consider art technologies and here we can see that even though in the natural all of these processes are conserved when it comes to assisted reproduction techniques we pi bypass certain aspects of it. With IVF, we bypass the vaginal disposition and the cervical and mucus passage. With Ixie, we bypass um further steps such as the oven recognition that happens.
So what are the fertilization events that take place during the first 24 hours? The first step is the sperm capacitation that entails secretion of molecules from the oite that orient and stimulate the sperm. that is followed by the acrosome reaction which involves release of hydraytic enzymes.
The fusion of the sperm with plasma membrane then um of the oite then follows and several proteins are involved in that process both from the egg and the sperm and that is followed by the cortical reaction and the calcium oxilations that lead to the formation of the fertilization cone and then those the release of cortical granules um result in the block of polyperine.
The sperm chromatin then decontens to form the male pronucleus. The oite exclude extrudes the second polar body and the female pr-ucleus also forms.
However, these fertilization events do differ based on the origins of fertilization. As we have said in natural conception and conventional IVF, there are differences compared to ixie.
In the first two we have the steps that um entail sperm capacitation as we explained acrosome reaction and the fusion of sperm with the egg membrane whereas in Ixie those steps are bypassed and only the last two two steps of cortical reaction and pronleia formation take place.
So what does the zygote tell us? What morphology can we observe? First of all, we have to observe the PNS and uh following the experimentary we have the habloid chromosome set that enters the oite. This is condensed and has to be unpacked and the patternal pronucleus expands and the nucleic membrane encloses the the condensing DNA. So this forms the patternal um pronucleus that is usually first. So when the paternal pronucleus forms then the maternal pronucleus also forms and this follows the polar body extrusion and again the chromosomes of the o of the oites uh are the condensed and enclosed by a nucleic membrane and this is necessary for the subsequent synthesis phase in which the DNA is duplicated. Of course what we want to see is this the two pronuclei but the other thing that we have to observe is the presence of the two polar bodies.
So in the embryology lab what we do is that we have to rely on morphological evaluations and the first step of evaluations we do in lab after fertilization is determining the number and shape of pronuclei. Do we have PNS and we usually expect to see two PNS.
However, not all zygot uh may have two pronuclei at fertilization check. As the previous figures have uh very nicely said, we sometimes see one PN and this can be seen both in conventional IDF ori. In some cases um when we only see one polar body, this may be due to pathogenic origins. However, when we see two polar bodies, this can be caused by errors in fertilization process or a synchrony in PN formation or fusion that is unseen.
So the resulting embryo can be deployed and we saw these studies that uh show that embryos can be deployed and this is what we see when we have 1 PN we still have the 1 PN and usually the two polar bodies when we have three PNS of course again we can see them both with conventional IVF and Ixie when we're doing conventional IVF it usually the cause is diri so more than one sperm entering the oite soplas however in IVF and this can also be caused by failed cytokinesis or fertilization or fertilization by bucleate sperm and this is a 3pn again we have to observe that we have the two polar bodies so this is a prerequisite and again the 2.1 it was explained earlier that we have two normal size PN and a smaller pronucleus the smaller pronucleus is also referred to as a micropronucleus and the exact cause for this is not fully understood but it could be linked to various factors including including incomplete extrusion of the second polar body during meiosis and here is a 2.1 uh PN. So what does the consensus tell us on PN number which is the first thing we observe the number of PNS? First of all, pronucle may not be seen at fertilization check when we're doing static observations. So we might miss them and time-lapse data show that a significant proportion of those two P and zygot can go undergo a pronuclear breakdown at earlier times than the suggested and uh recommended original instantul consensus which is 17 hours plus minus one. So in such cases we should always observe to see the second polar body to accompany the 2pn fertilization and that to be used as a scoring criterion additionally to the observation of PN also when we're seeing zero PN um the term that we were previously used using as unfertilized or zero PN should not be used and it should be replaced by PN not observed as these zero PN or unfertilized would lead and sometimes lead to normal development without confirmation of fertilization.
So the first instantul consensus uh that we all used from 2011 onwards suggested that we should check the PNS uh at 17 hours plus minus one with uh post IVF expected to be observed about 1 hour later due to the reasons that we explained earlier of the additional steps involved. However, the new instabble consensus published this year suggests that the PN check should be carried out a little bit earlier between 16 to 17 hours in both conventional IVF and exit cases. And this is to minimize the probability of missing uh PN due to relatively early pronuclear breakdown and um and therefore incorrectly classifying unfertilized oites. This is the comparison between the two consensus and as we can see um with the using the timelines of the of the latest consensus we expect to catch 98% of the pronuclei.
So therefore only 2% are not observed when we're using um the correct time frame indicated by the consensus.
So other than the number pronuclear PNS have also morphology and we can categorize the morphology based on their size on their position on the presence of nuclear precursor bodies or on the presence or absence of cytoplasmic halo and pronuclear scoring considers the symmetry and alignment of the pruclei and involves also the assessment of the number and relative position of the NPBs which are within the pronuclei And for those of you that have been in the field for more than a couple of decades, you might remember the zed score that was used to score pronuclei with scores zed one and zed 2 characterizing uh better quality PN having equally aligned PNS or equally not aligned PN followed by the second category of Z3 one and two that uh having a mixed scattered large and small and aligned and not aligned. followed by the third category of unequal numbers and many small scattered nucleoli or the fourth category of separated nuclei or of very different sizes.
Of course the literature there is um some discussion whether this type of scoring is beneficial in terms of clinical outcomes and the first instable consensus also shared a similar scoring and gave three categories for PNS the symmetrical the non-ymmetrical and the abnormal with the symmetrical being the equivalent to the higher scores of the Z scoring noty symmetrical having other arrangements and the abnormal having those ones that have the ghost pronuclei so pronuclei not seen or the one npb that is called otherwise the bullseye pronuclei other than that what we can see in uh in in PN is the size and earlier studies and uh we can see that there is evolution between the earlier studies that had uh no use of time-lapse technologies and the later studies that we're going to discuss in the later section the earlier studies showed that the size of the PNS depends on the time of observation and we expect to see an increase in the size of close to 50% from the time of formation to the stage of fertilization check and the presence of PN with a diameter smaller than normal at the at the time of fertilization check was also shown to be an indicator of delayed fertilization and this is possibly due to oxide immaturity when we're using conventional IVF or defects in the gamuts.
Another thing that was suggested in earlier studies was the alignment and uh the exclusion of the second polar body that establishes the polar axis. Um had to correlate with the alignment of piano onto the same axis and this was considered to be necessary for the formation of the polar axis at singami and the completion of the first cleavage division.
At a position, the chromadin of both PN begins to polarize and rotate to face each other with the longitudinal axis of PN being parallel to the plane of polar bodies. And further rotation, as we can see in the image here, brings the PN aligned onto the polar axis with the position of the of the second polar body defining the plane of the first division.
Other than that, earlier studies also explored uh the angle of the polar bodies and they suggested that not aligned polar bodies with respect to PN um to be resulting in sub-optimal development also the same for large angles between polar body at the time of fertilization check and they explored and they identified this effect to be due to suboptimal orientation of PN affecting the cytoplasm leading to an in cleavage and perhaps fragmentation.
How close are the PN? PN separation was another uh factor that was considered in earlier studies and control of the opposition of PN is regulated by the aster of the sperm centromeir something necessary for the distribution of organels in the cytolasp and failed progression to the opposition and singami therefore depends on the sperm centrosome activity. So we therefore know that the sperm also has a role.
Observation of zygot with separated PNA fertilization check has shown in earlier studies to severely delay or have arrested development in in many cases.
And where should PNS located? Is there a difference between if they are centrally located or peripherally located? Again earlier studies showed that uh the cleavage plane um is affected by the position of the PNS. And when PNS are positioned centrally, the first cleavage occurs regularly giving rise to normally developing embryos. And when PNS are positioned peripherally, cleavage occurs according to the pronuclear axis resulting in abnormal morphology.
However, this can lead to normal implantation.
How about morphology? General cytoplasmic morphology. Similar to the oite, we do expect to have a homogeneous cytoplasm in zygos.
uh but this is this predictive of developmental competence. Some studies have reported that if we have severe um cytoplasmic anomalies in the zygote this can affect the developmental outcomes and implantation and clinical outcomes of the embryos but there is no clear evidence to support that. Therefore um the recording of cytoplasmic morphology was shown in earlier studies to have some several uh relevant links to the developmental implantation potential. So therefore do we have a happy face or a sad face.
So nowadays we have this technology and we heard all about this genetic technology that can help shed light in towards the genetic composition of PN.
What about the time lapse and these technologies and whether it they can be used for embryo selection?
So when it comes to marker of fertilization, we have the molecular markers that um consist of uh molecules that cannot be identified in culture. We have the genetic markers and we have heard a lot about them and we have the morphological markers of fertilization and as embryologist we rely solely on morphological markers in order to assess fertilization and devel and and development.
So starting with the zygote size. So newer studies now show that fertilized oi normally undergo progressive and moderate shrinkage during fertilization.
This is also as a result of the extrusion of the polar body and loss of some cytoplasm through that. And there has also been shown that uh a negative correlation between the zygote diameter and cytoplasmic volume observed at the time of fertilization also linked to blastosis polity. However, there is insufficient and inconclusive evidence on the hypothesis that zygote size can be a predictive parameter for embryo developmental potential.
So what does timelapse uh studies show?
These studies show that offcenter PN position before breakdown is linked to abnormal cleavage.
But if we if we locate this offc center position at 8 to 9 hours, this is correlated with lower life birth rates and this is undetectable in static observation. So if we were only going to do a fertilization check at 16 to 17 hours, we would have missed that. Also the absence of PN juaposition is associated with the reduced cleavage, morula and blastosis rates.
In addition, newer studies show that males male PN are generally larger and sizes equalizes over time whereas smaller size difference near pronuclear breakdown is seen in embryos can lead to live birth. So therefore smaller PNS can also have clinical uh utility and no conclusing evidence has been shown for the size of PN as an independent outcome predictor. Again, in conclusion, the abnormalities in PN position, jtoposition, and size are rare and hard to detect via static assessment. This is according to the new stumble consensus.
What about the nuclear precursor bodies?
Most timelapse studies have found no consistent predictive value of NPVS patterning for implantation and live birth with the exception of a of an of a study that has used advanced computational tracking on on NPB speed.
Also, clustering has been associated with better blastosis development in some studies.
However, the kinetics differ between male and female PN and clustering can reverse before PN breakdown with these dynamics limiting reliability of static or simple um single observation. Again, in conclusion, MPB patterning is dynamic and variable making it an unreliable standalone biomarker of embryo quality.
What about the halo?
Absence of the halo has also been linked to abnormal cleavage and early embryo attrition and halo position whereas being symmetric or asymmetric has shown no impact on clinical outcomes being similar between u halo positive and halo negative blastoyst.
In conclusion again in in regards to the halo while early halo presence may reflect normal development in the predictive value diminishes if the embryo manages to reach the blastosis stage.
And these are the studies that have have been incorporated in the new consensus.
You can uh revisit them at your own leisure if you want to find out more about the indications and clinical um outcomes based on these different uh morphological features of the zygote.
So in summary, what does um this new um technology bring?
Socrates has said that the only true wisdom is in knowing that you know nothing. So do we know less now? No. On the contrary, we have learned that there is considerable plasticity regarding human fertilization.
We have also learned that unless we're using time-lapse and genetic analysis, the deployed status of an embryo cannot be validated to the full extent. And also we have learned that several zygotic characteristics ranging from zygote size, PN size, position, NPB patterning may be associated with embryo quality and clinical outcomes. but their use as biomarkers is hindered by insufficient evidence and when we're not using time lapse. Thank you very much for listening.
>> Perfect. Uh thank you so much George for this very informative presentation.
Um I'll sneak and have a question for you. So between these parameters that you present uh for morphological zygote check in your opinion which one is more correlated with upuploid and which one is more um correlated to um um successful outcome. I know you presented v various form of uh morphological characteristics but on daily life for embryologist to select uh the best uh embryo to choose.
>> Mhm.
>> Look the most important starting from all the features that we observe must be the number. So when it comes to 2PN the first thing that we observe is the number. We will always prioritize um 2p and embryos to be first regardless of other features or other morphological features um coming first. But as we have learned from the other speakers as well um all other embryos especially if we're using genetic testing can also have utility and therefore should not be excluded. And having in mind that the zygot and and uh thereafter embryos have a lot of plasticity, we should never um um disqualify all sides based on solely morphological features because it could be an outcome of uh our observations in time especially when we're not using time lapse and whether we cannot be sure if what we're seeing is uh is the whole story.
Thank you very much. Uh now I would like to thank uh all my um uh presenters and the moderators and I'll ask everyone to uh put their camera and speaker on and this is the time for uh Q&A and uh I'll start with the first >> I think your sound is cutting a little bit.
Sorry, this is from Karima and she is asking do you which evaluated to M2 can be exposed to give and PN uh embryos.
sorry for the challenges that Balsam is is facing. So I I would ask the question for you Balsam if you may because we are having a problems in the connection I'm afraid to. So um the question comes from Karima from Algeria. In cases of zero PN observation there is two probability.
One fusion of two PNS and followed by cleavage and blastation and the second not fertilized but can be followed by cleavage and blastation via an overight activation. I think this question will go to you George. Do you have a comment on it?
>> Um sorry Berta can you repeat the question? So um Karima is mainly asking in cases of zero PN or when we are not seeing a pronuclear uh there is two probability she's saying first is the fusion of the 2PN and followed by cleavage and blasulation or the second will be not fertilized and can be can be followed by cleavage and blasolation via anite activation. Could you please comment on it? What do we see? Um so those are called also ghost uh ghost PN sometimes we might uh not see them and this is also uh in fact uh the case when we're using time lapse so there are cases where we have time-lapse image from the beginning we don't see PNS and then we see normal division and sometimes these embryos can reach to the blastoyst I think the thing to point out um to make sure that um the first thing is to assess the status of the second polar body being extruded This is the first thing to observe. Um and sometimes um the PN can just be ghosted but uh those embryos again as we have seen from the genetic studies which have been previously characterized as zero PN can in fact be um deployed.
>> Thank you George.
>> So we've got one uh I think this is probably for you and Neil. Um this is from a very well-known name Gerald Geraldine Hartzson all the way from Warrick. Um so these studies obviously they analyze blastoysts um derived from unusually fertilized forms um and you find that many are normally diploloyed.
Do you consider that the genotype can change due to errors or corrections or cell losses uh during growth of the blastis or do you think that the PNS appear similar in size um and vary in their chromosome content? So how do you think that arises?
Yeah, basically the main concern about the pronuclear scoring is that the correct classification of the pronuclear. So if we are talking about the correct observation of the pron like 3PN of course we have we expect the resets of the chromosomes and which confirmed by the molecular methods. So we can see on the other hand the ploy rescue mechanism can be also uh started in the early developmental phase and the we expect that as like mosaism as we see the aneploy rescue that also some uh initial chronicle the samples can be um excluded during the development are balanced. So that's why we expect especially the the the samples are arriving to blast stage they may have also this possibility to to to to rescue. So I think so that's why our data and the the data of the other groups is strongly confirming that the P check should be also confirmed by the molecular tests.
>> Brilliant. Thank you Bam. Are you back with us? Can we hear you?
>> I think. Can you hear me well now?
>> Yes.
>> Oh, perfect. Um, okay. So, I can run this. Marina Agot from Dupin. Uh, she's asking if there is any news for non-invasive DNA testing for uh PN check. And that's probably to um any of the presenters.
>> Jagus, why don't you do why don't you jump in?
>> Yeah, sure. No. Yeah, I don't think um as far as I know I mean um there's no non-invasive analysis when it comes to pronlei check but that's all I know.
Yeah, I think yeah I think as well if I may come it's NIP NIPGT it's still a challenge for uh TPN embryos and uh therefore I don't think there is sufficient data for 2PN to introduce it to pr-uclei checkp yet to come in future but lots of to implement in in the technology and uh there is another question I may ask you as Well, Jenkins um they they one of the uh audience asking shall we implement 100% PGT uh and uh if so why do we do P and check >> um let me answer the first question should we do 100% for uh all the patient PGT probably this is something that the clinic have to have their own policy um but if you ever do PGTA then yes I recommend recommendation is um regardless of what the fertilization check says molecular approach probably eventually and give the ultimate answer.
>> Yeah. Add also uh one comment to the Jangis. Yeah. Basically especially PN check is still I think valuable but as we see especially for the trip embryos we see high percentage of triploidity.
All studies is showing that the high percentage of triploidity. So at least for as an indicator the first indicator it should be also constant but we cannot exclude even the embryos coming from 3PN uh for the biopsy and the confirmation of check even if we know that they have the less less possibility of deploy rates than the other ones.
>> But then I have a question for you. So if let's say you have uh implement molecular check and then you have upuploid embryo coming from 2PN and then another uploid coming from 3pn and they both have like uploid status and um one is uh less morphology than the the 3pn like the diploid it's 4 BC and the 3pn is 4a. So what would you recommend for uh for for us as clinics which want to transfer?
>> Basically this is a common the issue if we have many aloid embryos what would be the criteria for the priotization prioritization. So basically now if if this is the uh issue probably I would suggest that of course the for prioritization from the coming from 2PN and deploy embryo but we cannot exclude one embryo coming from abnormal PN and if there is no any other embryo to transfer it can also be transferred but of course we need also much more clinical data on this. Yeah, thank you.
>> And if I may, I can maybe add one more thing to this um morphology. Um we have seen a lot of like great great morphology um embryos where the unemployed result came back 21. So a lot of actually those down syndrome embryos are actually morphologically beautiful but that doesn't mean that they are actually the embryo that will be capable have a live birth. uh whereas um there are some actually morphologically relatively less good quality embryos that they are actually huploid and those are the one that actually implants and have a live birth. So my point is like obviously morphology is very very important but at the end of the day the the chromosomal makeup is the most important thing that we need to consider rather than morphology. And when we do the fertilization check again those are observational microscopic analysis and those are unfortunately missed uh during the check and the molecular approach will probably give the ultimate answer and I again very positive that eventually the guidelines will change and if you ever do PGTA testing uh probably you should kind of like go what the genetic testing says not necessarily what the fertilization check says. I know this frunk kind of like a open opium but that's what kind of I wanted to >> Thank you. I have I have a question actually. So, uh, have you ever um checked because I know in in Lepra's data he doesn't he didn't have um um um access to the parental swaps but I think in your data you had but did you observe any in sort of increase in in your UPD in your in your samples and if yes is there any specific uh type of uh PNS that associated with with UPD? Yeah, good question. So, unfortunately because of I don't have like high density of single nucleotided polymorphism, uh this is still a limitation with my platform to detect uniparalies.
So that's why I wasn't able to kind of do those checks because of the number of snips that I use in my testing.
>> That's perfect. Thank you.
>> Moving now back to you, George. Uh we have a question. Um thanks George coming from Karima. Thanks George for your excellent and practical presentation. Do you think that the M1 o site at denudation which is evaluated to M2 phase can be more exposed to give 0 PN or 1 PN I I don't have any answer for that in terms of studies that um have shown um genetic um genetic constitution maybe um anal or singis nose in terms of late maturity video sites whether they will be leading more to one PN or zero PN if they matured a little bit later.
is >> I sorry. Yeah, >> perhaps on the observation phase.
Perhaps on the observation phase, especially if we were using conventional IVF, um that could be a likely scenario, but in terms of the genetic constitution, I'm not too sure. Um whether that has been studying specifically for delayed mature doites.
uh basically I don't have data on this but I think even if the uh delayed method or sites if the P check has been done in a correct way maybe it should be and if they the development on the blasto stage should also be observed to uh as an indicator of a correct fertilization.
Yeah, >> the issue the issue with this Berta is that with conventional IVF we don't know which oides were um mature a little bit later during the fertilization process with Ixie also um we usually wait for maturation and then once they fertilize we categorize them as normally fertilized so um I I don't think there are many studies that have looked exclusively at 1p formation from delayed mature doites from what I know.
>> Thank you, Josh. I I think you you just like the question is like uh also another do we see if we want to rephrase it, do we see more abnormal PN when we do ixie convert compare compared to normal IVM maturation.
>> So I compared to IVF. No, when we do Ixie compared to traditional IVF where we just like let according to the Vienna consensus on on KPIs we do expect to have um if I'm not mistaken a higher um rate in or similar rates um when it comes to conventional IVF because of the simple reason of uh of poler me not being blocked. So usually we we do observe to have a higher um uh triploid rate with conventional IVF I would think.
>> Thank you. Thank you so much.
>> One um all the way from Turkey. This is from Charur. Um it specifically relates to the um the snipbased uh NGS uh and the poly priority rate of one of the studies up to about 16%.
Um a lot of the literature reports it much lower than that. Could this discrepancy be a possible over calling due to the technical limitations of this NGS process? Uh Bertie you're also com asked to comment on it but the the question is for George or Neil if you want to jump in either.
No, basically it was from my data we observed very high triplo rate. So probably the question is directly for me. So thank you for this question char.
But yeah is speaking uh in our data we showed very small court of the the 2pn samples. Uh we observed the 16% of triple is a quite height and I explained it's quite height compared to the literature. So we observe it technically I don't expect any NGS technical issue of the NGS applications because the validation of the test was quite uh quite good and I'm uh thinking about the the retrospective the piano observation can be one of the important points. So we are investigating these samples if they really which kind of observation system they used and also we are also observating if the uh PN is correctly indicated in the report and on the other hand as I showed that the patient the maternal age is quite high in this almost 40 40 years old patients but we are talking about again like 70 samples 74 samples so we should increase the more data after this uh checking of these parameters but I don't expect any issue from the technical uh overalling of NGS data.
>> Perfect. Thank you so much. And this question from Sophia Makiva from Zur to George. Uh she's asking in the lab that uh vitrify zygoods uh would you uh suggest freezing zore z4 the worst quality zygote should be avoided due to the low chances of further development.
>> I don't think we would ever exclude any oides from the freezing cohort regardless of any quality characteristics. Um the zygote scoring um has been some studies have shown that it has validity some others have shown no validity in their scoring in terms of uh clinical utility and uh and and outcomes. So therefore no sites are to spare when it comes to freezing. Perhaps if we're freezing at oides, um categorization of oites could be used and they're now AI platforms that can also categorize uh oites as an additional tool and give scores based on those morphological features. Um so these are just options to have for the patients but if there is no limit and there shouldn't be any limit um all oides um that have potential for uh making it to the blastosis and making a healthy uh baby should not be excluded.
>> Perfect. I think it's it's depends on the uh the the lab practice. if uh they need to to to do sort of develop sort of pathway if they freeze at the zygote stage prioritize those zygot but um based on our um research um I don't think morphologically it it can indicate any oruploy but it could give you some insights or if if you're a lab just doing um morphological check you can make your um um assumption on or priority with based on your data on which zygote to freeze or which embryo to transfer or which uh um blastosis to like yeah so that's I think it's lab based not rather than we make it as a general criteria for all uh labs for instance quite people asking whether time lapse was used for these studies. Um I won't name all the questions. We got we've got it three or four times. Uh uh obviously there's quite a difference if you just make a a single uh observation compared to time lapse. So could you maybe all comment on that very briefly?
>> Um in my study actually there wasn't any time-lapses used. So I saw the question.
Yeah. So the answer is was not in my case.
>> So one no >> one no J is um Anil rather.
>> Yeah actually it's like we are still checking the that the observation methods but the 100 samples were observed when time lapse and in my one of my slides I also showed that the strong correlation between the monopen and the hloid and tripen and the triploid in the time-lapse observation samples. Yes.
I would comment also that if we do the observations at the right time, we should pick up uh the PN most accurately um and we we show the study from Barry that uh 98% of the PN can be accurately be detected if done uh at the right interval for fertilization between 16 and 17 hours. So there's only a small proportion of those PN that are um not uh accurately categorized and characterized um even with uh static observations.
>> Just to say everyone um if you put questions in the chat we'll not be reading them. We're only reading them from the Q&A. So please put them there.
Um who's uh Bert Bam you got one ready?
Um perhaps uh we can summarize uh all the questions for um what we had u from the uh lovely uh speakers. Um we understand that uh time lapse is important element when we uh try uh to do P checks. uh in general I I understand there are some uh countries they have limitation using uh time-lapse uh incubators um and um especially with PN uh personally from our experience um we time-lapse is very important uh to detect certain parameters uh especially when you do PN uh implementing genetic testing for those uh embryos at the moment where we are is very important Um and thank you so much for all the speakers and my uh co-odderator and uh this uh webinar was sponsored by thermopisher scientific and we would like to thank all who took part in uh this webinar my cohort Dr. uh Bertis Ununes uh Prof. Darren Griffin uh Dr. Jenis Yunlo and Anil Berserk and of course George Lepris.
>> Thank you to Balam. It has been a pleasure and not forgetting the team from I3 the organizing committee of this initiative who are Jack Cohen, Peter Nars, Marianne Cvez, Thomas Elliot, Fran Farley, Violet Sura and the one and only J Palmer.
Uh and goodness me, I get the last word.
I am so flattered. So the i3 team will take a short break. They apparently have batteries to recharge over the summer, but they will be back in force with the new webinars and new topics in the fall or the autumn if you like. So keep those ideas, webinars, titles, and talks coming. We look forward to hear hearing from you and we shall see you soon.
Goodbye.
>> Bye.
>> Goodbye everyone. Thank you. Goodbye everybody. Bye-bye.
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