 Yeah, so our next speaker is Tom Spencer. He's a professor at the University of Missouri He's a national Academy member who's well known for his work in livestock especially on the function of the uterus and placenta and the molecular pathways that specify those organ systems All right, so I really appreciate the opportunity to come and talk to you about Some work that's basically done in large animal models that's centered around developmental biology and reproductive Sciences Maybe it's gonna be a very short talk Which some of you may appreciate so I just wanted to start out and Well, this is it's hold on I Don't know why it's not showing the same. I need to back up Here we go All right, so I just wanted to think that this is a large team of people that do this work I'm not done yet I usually use this as my first slide and that this has been funded by the USDA NIFA as well as the NIH and some Other places like science Foundation Ireland and select sires All right, so I think it's pretty obvious that if you measure fertility Which is conception rates to a single insemination that in some of the species that are poly Ovulatory you have pretty high fertility rates like in sheep and pigs But if you go to a mon ovulatory species like horse Cattle as well as humans It's pretty clear that there's an overt problem in terms of for subfertility and infertility and That pregnancy loss is also a major limitation to production efficiency and adoption and efficiency of assisted reproductive technologies That we use to treat human fertility as well as domestic animal infertility So particularly today, we're going to talk about cattle and if you look at pregnancy loss in cattle The majority of pregnancies are lost during the first two months and this is exactly what you see in terms of humans as well and so there's a lot of developmental processes that occur during early pregnancy in terms of the what I would call the first 60 days and that includes ovulation obviously Fertilization in the overduct creation of a Blasicist that then enters the uterus and undergoes at least in cattle this tremendous elongation process Which occurs right before establishment of the placenta. So this all occurs within the first 60 days so the determinants of pregnancy success really rely on both the developmental competence of the embryo this includes the genetics of paternal contributions in terms of the sperm maternal Contributions such as the OSI and there are some distinct epigenetic influences on this process that we really don't fully understand at this point It also involves the reproductive tract environment and that would be the environment of the vagina the overduct and the uterus And we're going to talk more about the uterus in terms of how it impacts subfertility and infertility and cattle today But the overall impacts are the success of pregnancy is dependent on this is the origin of pregnancy Complications and as well accepted in humans now that the majority of later pregnancy complications like preeclampsia feel growth retardation As well as other syndromes really originate during the first two months of pregnancy And this of course can negatively Program or possibly program off-spring health So I got into this area because I was working with a group at the University of College Dublin and we had shown that there's some distinct alterations in terms of the gene expression profile of the uterus during early pregnancy so you can see Over here that days 5 and 7 of pregnancy are very similar in days 13 and 16 So you get this dramatic shift in gene expression that is really driven by the effects of ovarian progesterone And then we also showed that around day 16 the concept is begins to influence in the mutual gene expression But the problem is that you know of the 18,000 genes are expressing in the metrium which are the most important Right, which are the driver genes that are important for pregnancy establishment? And we knew that there were distinct gene expression differences and that actually the embryo the uterus could sense differences in the embryo whether they were derived in vivo and Or by in vitro means or nuclear transfer And so we just basically asked the question What are the most important genes in terms of this and we use basically natural variation in pregnancy rates to answer this question so we essentially took a large group of animals and turn them into a human fertility clinic and So we synchronized them we gave them one in vitro produced embryo on Day seven of high quality and then we asked are they pregnant on day 28? Biotro sound are they pregnant on day 44 and then essentially we terminated their pregnancies and did this over and over and over and And so what we are able to diverge out of this population are a population of animals that always got pregnant a Population of animals that never got pregnant and then a population of animals that would get pregnant But only 25 to 33 percent of the time and it turns out that if you look at Most objuan clinics or in the human population This is exactly what you see in terms of the human population about five to ten percent of females are Completely infertile due to female factor infertility Rather than male and this also holds true in terms of most production agriculture systems So we asked the question, you know, what is wrong with these animals? Can we understand the genetic basis for their fertility problems using this natural variation in pregnancy rates? And to kind of make a long story short You know, we found that there were no differences In their pregnancies on day 14 when you normally find a Small embryo within the uterus of about two to four millimeters in size And so we did what we call the money experiment in which we slaughtered We made all these animals pregnant and we slaughtered them on day 17 and day 17 is a very interesting time point of pregnancy and cattle because the the embryo goes from about a one or two millimeters in size and actually elongates Tremendously over a period of two to three days and it actually looks like a really thick piece of dental floss or umbilical tape if you've Ever entered the obstectures and gynecology world And so this is very important because this is when the embryo produces signals that cause the mother to know that she's pregnant And so this period really is very interesting not only because of this tremendously elongation It's also when you have a different trophoblasts development Cures in terms of the differentiation of bi-nucleid cells that produce factors that are involved in maternal adaptation of pregnancy You also have a origin of the allen toys and yolk sac placenta, which is important for embryo genesis So there's a lot of things that are occurring During this time point and kind of one important note. Maybe this is for the breakout session tomorrow This is actually an area that we simply don't know much about We don't know understand transcriptional regulation of these processes We don't really understand anything this remains a black box of pregnancy and actually I have one of these slides in the lower left is from some really nice work that Harris loon published a couple of years ago illustrating the dramatic Embryology type changes that you see during this a period of pregnancy establishment And so we essentially took all these a subset of these animals We gave them to in vivo produced embryos, and then we looked on day 17 And kind of surprisingly we found that there's really no difference in the high fertile and sub fertile animals That's there's no difference in their pregnancy rate. However, they are classified infertile Actually, you found only one conceptus and that conceptus was severely growth retarded Whereas this is what we found in all the other types of high fertile and sub fertile animals If you measure the length of the conceptus during this time period You'll note that the high fertile animals on average had about a two-fold longer Conceptus link than the sub fertile animals. There's a lot of variation In this developmental process, and we still understand if you're in a longer embryo Do you have a better chance of survivability? Because as you know size does not always equate in terms of actual matter so we did a lot of transcriptional Profiling of both the conceptus and the endometrium from these day 17 animals and just to remember that, you know We know that the infertile animals did not have a conceptus on day 17 Well, we know that the sub fertile animals are going to go on and lose their pregnancies by day 30 So if you look at the conceptus transcriptome, we found about 1,300 genes that were differentially expressed About 558 genes that are increased in the high fertile animals Many of them encoded known secreted trophoblast proteins as well as factors involved in lipid metabolism And you might imagine that if you're going to grow this tremendously long conceptus and you're going to establish a placenta that lipid Metabolism is very important for this process and genes that are decreased in the sub fertile animals included well known trophoblast differentiation factors as well as those that encodes secreted proteins as well as lipid metabolism and Many of the decreased genes and the sub fertile conceptuses are lethal and mice When you mutate them and these are due to defects in embryogenesis or placental development So we went on to profile the endometrium from both the open as well as the pregnant Heifers and here is a summary of the data and basically if you're a Normal animal that's high fertile. You always establish pregnancy. There's a tremendous response to the endometrium to pregnancy so you have about 4,000 genes that respond to pregnancy If you're a sub fertile animal, you'll note that there's a disruption in the response of the endometrium to pregnancy So you have about a fourfold decrease in the number of differentially expressed genes But most important a lot of the genes that are down regulated normally in pregnancy are not down regulated in the sub fertile animals So it turns out that basically what we think is that the endometrium of these animals is Responding inappropriately to the conceptus and that's causing a layer of pregnancy loss And this is exactly what you see in terms of a lot of pregnancies that if you look at the differences between An vivo derived pregnancy or one that's carrying a nuclear transfer derived clump If you go on and use a phantom 5 database and you map map all the known Either ligands or receptors present in the conceptus or the endometrium There's ample evidence that there's disruption in a large number of critical signaling pathways In which you have signaling pathways that are involved between this developing conceptus And the endometrium that are known or surmised to be important for pregnancy establishment Now what's interesting if you look at only the open animals Then you find that there's almost no gene expression differences whether they were high fertile sub fertile or infertile and Actually, this is well known in the human literature that you can't take a biopsy of an endometrium from a sub fertile or infertile Patient and determine whether or not they're going to have an enhanced ability to get pregnant So obviously if they're just missing their endometrium, they aren't going to get pregnant but really in terms of Translational diagnostics just looking at the endometrium or assessing a few Transcriptional regulation genes is not sufficient to basically determine whether or not a woman is going to get pregnant or not and IVF clinic so we also Genotype a subset of these animals with genus high 30 high fertile and 55 Subfertile and fertile animals and we found several low side that were either strongly or moderately associated with fertility What's interesting in many of these were actually located on the X chromosome? And currently we're finishing up studies where we've done whole genome sequencing on all these animals We're undergoing analysis of copy number variation in collaboration with John Cole at the USDA ARS We've also extended these studies to dairy cattle. So originally we did this work in beef cattle We've extended these studies at dairy cattle. We're essentially If we work with the right facilities given that very large numbers of heifers are raised or females are raised in the dairy industry Because they're the ones that produce milk we've also taken Done fertility phenotyping by artificial insemination We've characterized DNA from 500 high fertile or 500 sub fertile animals high fertile conceived on the first service The sub fertile animals only conceived after the forest service or were cold Due to a failure to conceive and also we found an association in terms of this genotyping array Where we found about 34 SNPs Representing about 26 QTLs were strongly associated with heifer fertility So our conclusions are that cattle having innate differences in uterine competence to support concept of survival and development Important biology of mechanisms underlying sub fertility infertility Manifest between days 14 and 17 of pregnancy This involves altered individual response to the conceptus Ultimately a program survival of the conceptus as well as development of the placenta And it results in early embryo loss And of course this is also what's been championed in terms of the origin of later pregnancy complications in humans That if you have problems essentially during the first two or three months during the implantation and placentation phase of pregnancy That you end up with these severe pregnancy problems that endanger health of the mother as well as babies such as preeclampsia And feel growth retardation or preterm labor But the good news is that the majority of animals in our studies Actually have a good uterus that means they have the ability to get pregnant the first time Or after two to three matings So then you would postulate that there are intrinsic differences in the conceptus that underlie pregnancy laws And so that's essentially what my laboratory as well as many others across the united states and world are now working on Is essentially working on this phase of pregnancy and trying to understand early And late embryo loss, but really the origin of pregnancy loss due to defects in either the embryo Or extra embryonic structures such as the placenta And one area that is particularly unique in terms of domestic animals is that there's a real advantage in terms of using dairy cattle for this work So in the dairy industry worldwide, there's about 1000 only 1200 active sires Most of these sires have been genotyped or fully sequenced And there's a council on dairy cattle breeding that actually nominates these individuals and and there's a consortium that pays for their sequencing And you have a large number of sires that are mated to A large numbers of females so For each sire we can get data on either from between a thousand to a hundred thousand matings to determine How fertile they are These records are curated by all the farms in the united states as well as a national database Which is housed near here in beltsville, maryland Called the usd8 ars agile facility And in terms of actual genotyping there are millions of cattle that have been genotyped worldwide Dairy and beef cattle in the u.s. There's over 3.5 million that have been genotyped with either a low or a high density array In ireland actually all dairy cattle and all beef cattle have been now been genotyped on the entire island Which is kind of a phenomenal resource so currently what People are trying to understand or what are the primary influencers of pregnancy establishment That is where their genetic mechanisms such as transposable elements or mitochondria long non-coding RNA small RNAs As well as if we know that because we have inbreeding problems within each of these breeds There are Recessive embryonic lethal alleles that have actually some quite high penetrance in terms of 15 to 20 percent of the population or carriers for these We know that there's also epigenetic influences in terms of sperm and egg quality Then involve dna and histone methylation. We've heard a lot about that from other talks today But essentially this kind of black box of pregnancy is real ripe for trying to understand this Early and late pregnancy loss So current foci that our laboratory as well as others are working on or you know, you can phenotype populations these animals essentially using collaborations with producers See very easy to hold genome sequencing now You can study in vivo pregnancy establishment as well as Several labs have developed in vitro models of tropholast differentiation Uh our lab is doing a lot of single cell RNA seek right now to try to understand You know, what are the differences In the placenta as it develops between the mononuclear and binucleate and multi-nucleated cells This is very similar to what you see And other species such as humans and mice And we're using a lot of sequencing technologies to try to understand different transcription factors and how they develop during this time period I think on a more global level what we're talking about in terms of genetic mechanisms would be There's undoubtedly some polymorphisms that are involved in this I would not be surprised if there aren't a lot of de novo mutations that are involved in the loss of the embryo Or primary extra embryonic structures such as the yolk sac or alan toys that cause some of this pregnancy loss We know from our own preliminary data as well as others that there Is a significant influence of copper number variants and they likely are probably more important Than single nucleotide polymorphisms in terms of some of this pregnancy loss We know from a lot of other people's work that these homozygous receptors or embryonic lethal alleles are also Segregating in the population And again, I think it's this area is real ripe in terms of transposable elements and how they influence Ultimately embryogenesis using this approach But I think this area is real fascinating because I mean you can really get at the heart of the primary embryonic And personal development that occurs in all species And use a comparative approach to really understand an important loss in terms of dairy and beef cattle as well Other domestic animals and really fill in kind of a black box of pregnancy that we don't understand In terms of domestic animals and it is completely inaccessible in terms of the human Particularly during my state, there's no way that you're going to be getting any early human embryos And so this is what's required in terms of comparative biology So what I end with is that animal models for reproduction and development of biology research are are very important So in sheep has been a predominant model for maternal fetal medicine as well as neotinotology and pediatrics Pigs are a great translational biomedical research model particularly for nutritional programming and neonatology Horses are absolutely fascinating in which they have an invasive placenta that survives immunological rejection by the mother and still functions to create a foal at the term of pregnancy And really cattle were the origin of many of the assisted reproductive technology procedures like in vitro fertilization maturation XC as well as embryo transfer And of course animals have an essential role in human health via nutrition with meat and milk So with that I look forward to the Session later in terms of any questions that you might have. So thank you very much