Invasive Testing Methods for Fetal Aneuploidy - SD

Introduction

Hi, I'm Ron Wapner, a director of Maternal Fetal Medicine and Reproductive Genetics at Columbia University in New York City.

Today I'll be discussing invasive prenatal diagnostic procedures.

I'll be talking predominantly about amniocentesis and chorionic villus sampling.

Trying to give the equivalent risks and benefits of each of those.

Evolution of Prenatal Diagnosis

Prenatal diagnosis has changed dramatically over the past 20 years or so.

Certainly the candidates for prenatal diagnosis have been more selectively chosen based on marked improvements of screening from triple screening through quad screening through now first trimester screening.

In addition to changes in how we choose our patients invasive testing methods have also moved from the second trimester to the first trimester.

Obviously, the most important questions are what are the relative safety and advantages and disadvantages of procedures performed at different times in pregnancy gestation.

As illustrated here, our gold standard by which we evaluate other new and emerging technologies has already, has always been amniocentesis and, in addition to being our gold standard.

One must realize however, that this technology has changed.

Originally, amniocentesis was performed without ultrasound guidance, was, performed, in, using much larger needles.

But most recently, all operators do the procedure under direct ultrasound guidance, usually using a 20 gauge, needle or so.

So when we compare amnios to other procedures, that will be the technique that we'll be talking about.

Amniocentesis

History and Technique

Although amniocentesis has been with us for now almost 30 years, the complications of the procedure are still not as well defined as we would like them to be.

Complications

However, the common complications are listed here.

Infection still occurs in a very small percentage of cases, approximately one in a thousand to, eight and 10,000, procedures.

Most of the infections are not secondary to ascending infection coming up from the vagina.

Most infections nowadays are from e coli and come from inadvertent puncture of the intestines during the procedure.

Again, it occurs very rarely.

If one pays significant attention with ultrasound and make sure that there's no bowel in the, needle insertion, pathway, one can minimize the probability of this occurring.

It's also important to remember that an infection or a chorioamnionitis following genetic amniocentesis frequently presents more subtly than one might anticipate.

It'll present with sometimes with fever and chills.

Patient feels like they have a flu-like syndrome.

Rarely does it present as a typical pain in the uterus or severe cramping or bleeding.

So if one's going to recognize, this complication, which as I mentioned occurs very frequently, one has to realize sometimes the subtlety with which it can present.

However, if ignored, this can lead to a life-threatening complication.

And unfortunately, most studies have demonstrated that an infection following the procedure cannot successfully be treated with antibiotics and unfortunately must lead to termination of the pregnancy.

The second complication that occurs following, second trimester amniocentesis is leakage of amniotic fluid, surprisingly occurs in a higher percentage of women than we normally would've anticipated, and most studies have shown that about 2% of women who undergo a second trimester amniocentesis will leak a little bit of fluid following the procedure for a day or two.

However, this almost always stops and leads to no significant complications.

Prolonged rupture, which the fluid may, leak down the legs and continue to leak for a period of time.

Sometimes associated with oligohydramnios on an ultrasound, occurs in much less than 1% of cases.

Fortunately, as opposed to a spontaneous rupture of membranes in which pregnancy loss is frequently associated leakage, even significant leakage of fluid following second trimester, amnio will usually stop after the patient spends a couple days off their feet and result in an excellent prognosis for the pregnancy.

This difference between induced or iatrogenic, rupture and spontaneous rupture probably has to do that.

Most spontaneous ruptures are also associated with actually pre rupture chorioamnionitis or low grade infections.

So again, leakage of fluid occurs, not uncommonly in about 2% of patients, but prolonged rupture, leading to the necessity for treatment is less than 1% of cases.

Fetal maternal hemorrhage can also occur following second trimester amnio.

But nowadays, almost all operators administer RhoGAM if the woman is RH negative, and that complication is exceedingly rare.

There also continue to be cases of fetal trauma prior to ultrasound guidance of second trimester amnio.

Significant, complications from this have been reported.

There have been injuries to the legs.

There have been CNS injuries.

There have been a number of, cord injuries reported, however, now with ultrasound guidance, the risk of injury to the fetus has been significantly minimized, but patients still need to be informed that that remains a possible complication.

Miscarriage Risk

The most concerning complication following second trimester amniocentesis is the risk of miscarriage or procedure or pregnancy loss following the procedure.

And I've illustrated on, this slides all a number of national studies that have looked at the incidents of post amniocentesis fetal loss.

Most studies have looked at the incidences of loss from the procedure until 28 weeks gestation.

And the reason people have stuck with 28 weeks gestation is that that allows us to compare one study to other studies.

So just needed to do it during the same, interval of time.

In the studies performed pre 1980, the incidences of loss of pregnancy from the time of a second trimester amnio to 28 weeks gestation has been about 2.5%.

More recently, studies performed in the nineties have demonstrated over a 1% improvement in the National Canadian study, the total post-procedure loss rate was about one point a half percent, and most other studies have been consistent with this.

So we can demonstrate, or at least have a strong suggestion that with the use of ultrasound guidance for the procedure and with the tendency in many countries to only have a few very experienced operators performing the procedure, rather than all obstetricians, we have seen significant improvement in the total post post-procedure loss rate.

However, one must realize that this is the total post-procedure loss rate.

That's all patients that have an amnio and all losses until 28 weeks gestation.

What our patients are most interested in is what is the procedure induced additive, risk of of loss that doing an amniocentesis presents.

And to look at this, there has been only one randomized controlled study.

This study performed by Ann Tabor and her group in, Copenhagen, Copenhagen took all women under the age of 35.

That is women that had no indication whatsoever for having an invasive amniocentesis performed and offered them participation in the study and then randomize those willing women willing to participate, half having an invasive amnio, half having no procedure.

So it was truly a prospective randomized trial to look at the additive procedure induced risks of amniocentesis.

Surprisingly, in this study, we saw that there was a significant increased risk of spontaneous, loss following genetic apnea.

Over 4,500 women, agreed to participate in this study.

With 2300 being randomized to each group.

One can see that in the group that had amniocentesis performed, there was a 1%, a one in a hundred greater risk of pregnancy loss in women that had a second trimester amnio than in women who did not.

The control group risk of loss is a little lower than many people had expected to see, and there have been individuals who have questioned whether or not these are the most accurate representation of invasive second trimester apnea.

But again, it stands as the only prospective randomized trial, and it shows approximately a 1% additive risk of the procedure.

If one looks at the 95% confidence intervals, the risk is somewhere.

The true risk is somewhere between approximately one in 30 or so to approximately one in 300.

So it's from this study that the standard risk that we give patients of approximately one in 200 to one in 300, for pregnancy loss induced by amnio comes from.

Now, this study was done exactly as stud as individuals do amnios nowadays.

It was a, 20 gauge needle.

It was done by only three, experienced operators, and it was done under continuous ultrasound guidance.

There have been some individuals that have questioned, that whether or not the risk of amnio may be lower nowadays, and an excellent, meta-analysis has been performed by, SEEDS and published in the obstetrics in gynecology, and that confirmed the risk being approximately one in 300.

However, most recently a style A study using information from the first trimester screening study of called Faster that suggested that the risk may be significantly lower than that.

This study published by Edelman at all, suggested that the post that the amniocentesis induced rate may be as low as one in 1200 to one in 1600.

However, there was a significant statistical flaw in the way the study, was performed in that, in the group that had amniocentesis and had an abnormal result.

Patients with Cara typically abnormal pregnancies and who elected to terminate the pregnancy were excluded from the study.

Alternatively, in the control group that didn't have any procedure performed, there was no way to know which of the background losses or which of their losses were due to chromosomal or other genetic abnormalities.

So indeed, you couldn't compare these two risks because there was a hidden bias, in favor in this particular case of amnios.

When one, however looks at the, studies and includes, these terminated pregnancies for genetic abnormalities, the dramatic, safety of, amniocentesis disappears.

And you can see that those groups that had an amnio, if one includes second trimester terminations had a loss that was almost three times higher, than those that didn't have a procedure.

They also separated this from women that were screened positive and women that were screened negative.

And one would anticipate that in the screen positive group, there'd be a high, number of abnormal pregnancies lost in the no procedure group.

And indeed, that's exactly what's seen in screen positive patients.

The risk of, spontaneous loss was actually three times higher than in the amnio group, demonstrating that in indeed not that it's actually safer to have an amniocentesis than to have no procedure, but demonstrating the increased losses of chromosomally abnormals, which were biasing against the, no procedure group.

So I believe that although this study adds additional, information, most individuals will say the risk of second trimester amniocentesis.

The procedure induced risk of pregnancy loss is approximately one in 200 or one in 300 or so.

Timing and Early Amniocentesis

Amniocentesis is best performed after the 15th week of pregnancy.

About 10 years ago, there was a major emphasis predominantly to compete with, CVS, which was an emerging technology at that time.

That was a major emphasis to move amniocentesis earlier and earlier there became a procedure called early amniocentesis, and those were procedures performed under 14 weeks Gestation illustrated on these slides are some, results from the Canadian randomized trial between early amnio performed, as I said, under 16 weeks gestation and compared it to standard amnio, which in this trial, was procedures performed at 16 weeks or further.

As one can see, there was a significant, and in this particular case, taking into consideration, both electively terminated and spontaneously lost, pregnancies to eliminate the bias of termination of abnormal pregnancies in the procedure group.

There was a statistically greater risk of pregnancy loss in the group undergoing early amniocentesis compared to that undergoing amniocentesis at what had been the standard time period, which was approximately 16 weeks gestation.

And if we look at the predominant cause of loss in the early amnio studies, one can see that it was leakage of amniotic fluid.

If one had an early amniocentesis performed and leaked amniotic fluid following the procedure, their risk of loss was 15%.

If they had the procedure and didn't leak fluid, they still had a slightly higher risk of lost than one would anticipate, but was dramatically lower.

So indeed there was little question that the additive risk presented by early amnio, at least a significant contribution of that came from leakage of amniotic fluid not anticipated.

But a fairly dramatic finding from the study of early amnios was that they also found a significant incidence of talipes, equinovarus or club feet in patients that underwent early amniocentesis Club feet occur in approximately one per thousand to about three per thousand pregnancies in the general population.

Three studies are illustrated here, one from Canada, one from Denmark, and one from the United Kingdom.

And in their, patients that underwent early amniocentesis, they had a tenfold greater incidence of talipes equinovarus in the Canadian study, almost one in a hundred, over one in a hundred actually, patients had, severe club feet in the Danish study, almost 2%.

And in the United Kingdom study about one point a half percent.

So there's no question that early amnio, an amniocentesis performed under 16 weeks has a dramatic, almost tenfold increased risk of clubfoot compared to amnios performed in the standard window of 15 or 16 weeks and beyond.

The correlate here also appeared to be amniotic fluid leakage.

If you had an early amniocentesis and you leaked amniotic fluid, the frequency of club foot was a dramatic 15%, which is almost unheard of level of this particular complication.

The club feet that occurred following early amniocentesis were not just minor, positional club feet.

These were very severe club feet, many of which needed surgical repair and, took a long time.

So there was no question that early amnios are associated with this particular birth defect retrospectively, because hindsight is always 2020, we should have known that this complication could occur.

And the reason is we should have appreciated the difference in embryology or embryologic anatomy at that gestational age.

This is approximately a nine or 10 week pregnancy, and we see the outer chorionic membrane, and then we see the large space, which is called the extra embryonic coelom, and then we see the amniotic membrane by 15 weeks, actually by 14 weeks, the amount of amniotic fluid increases.

The membrane grows out to the chorion and fuses.

So the standard amnio would be inserting a needle into a fused chorio amniotic membrane.

Earlier amniocentesis actually were puncturing this kind of free floating amnion, making it much more vulnerable to, leakage and probably accounting for the leakage of fluid.

And then the leakage of fluid, as we all know, is associated with fetal constriction and complications such as club feet.

So I think with this in mind, one can clearly make the recommendation that amniocentesis for genetic diagnosis should almost never, if not never be performed under 14 weeks gestation.

Most people now will recommend waiting at least until 15, and many operators, will wait until the 16th week of gestation for safety reasons.

Chorionic Villus Sampling (CVS)

Procedure Description

The other technique that has led to earlier invasive testing has been chorionic villus sampling.

Chorionic villus sampling is performed approximately in the 11th and 12th week of gestation.

And this illustrates a pregnancy at that gestational age.

Again, we see the fetus inside the amniotic membrane.

We see the large space in this case, not as large as in the other example of the extra embryonic coelom.

And this is the chorionic membrane as opposed to amniocentesis that would enter the amniotic sac chorionic sampling samples.

The chorion frondosum, which is the embryologic name for what ultimately will become the placenta, the sampling, the placenta or the chorion frondosum, I tend to use those two words interchangeably, is not like sampling the placenta at term.

At term, we think of the placenta as being that firm hard organ.

Early in gestation, we can still see the individual chorionic villi.

So when we insert a catheter or a needle, the, the villi just kind of spread out of the way, kind of like a seaweed would when floating on the bottom of, the ocean, then you put your needle or your catheter into the villi, apply suction.

And some of these individual villi are then aspirated.

So we're not digging a hole in the placenta.

We're actually aspirating those free floating, villi at that gestational age.

Approaches to CVS

There are two approaches to performing CVS.

One is transcervical sampling, and we have two illustrations of that here on the left side, we see the, uterus with the cervix right here, and we see a posterior placenta.

So one would insert the catheter obviously under continuous ultrasound guidance through the cervix into the bulk of the placenta.

And again, as I had mentioned, the villi themselves are moving out of the way.

So rather than just insert the catheter into a small portion of the villi, one can safely insert it all the way to the distal, area without causing any major disruption.

Also can see how easily visualized the catheter is In this particular case.

On the right we have an anterior, placenta, and the catheter is inserted through the cervix.

One usually pulls down on the speculum that will direct the catheter into the anterior portion of the uterus.

The other technique that has been utilized for CVS sampling developed a few years later, and it was developed because some people had concerns that if one samples through the vagina, there may be a greater risk of infection and contamination.

That concern proved absolutely, to be, not to be, founded, but it did lead to a very important technique or transabdominal CVS.

In this particular example, we have biopsy guides on the screen.

One can see the anterior placenta here, and it's important to note with transabdominal sampling, one does not insert the needle.

And indeed with transabdominal, you would use a 20 gauge needle.

One does not insert it perpendicular to the chorion, but you must insert it parallel.

That decreases the chance of inadvertent entry through the chorion into the amniotic cavity.

But more importantly, this gives you this whole area with suction, from which to aspirate the villi.

The needle is inserted, it's actually moved up and down a few times and then removed under continuous suction.

One technique of sampling is no better than the other.

Actually, for some placenta, location is better for other, locations, abdominal is better.

But what's most important is the centers that have the lowest loss rates and those that have, the are able to sample.

All patients use a combination, and I, the operators are skilled in both, and then they choose whichever is most appropriate for that particular pregnancy.

In our own center where we're still doing over a thousand or so CVS a year, about half of 'em are done cerv, and about half of them are done abdominally.

Laboratory Issues and Considerations

Although CVS has been with us for almost 20 years now, there still remain a number of controversies and a number of questions.

And these must be answered if CVS is going to be used as a major clinical tool in all centers.

And it absolutely has to because with first trimester screening, it is unfair to ask patients who are screened positive in the first trimester to wait four or five weeks to have an amnio.

So what are these questions and what are the answers?

First of all, there are a number of lingering laboratory issues.

Number one is their maternal cell contamination in which, the sample that is sent to the lab analyzes maternal cells rather than fetal cells.

And that did used to be a problem when operators were giving a laboratories very, very small samples.

However, with experienced operators giving labs larger samples, they now can separate the villi, which are easily identifiable, compared to decidua, they can separate the villi and give, and, and maternal cell contamination should almost never, ever be a problem.

And this is a picture of the chorionic, villi from a procedure, one can see the typical frond like appearance.

Decidua is a much more, amorphous appearing.

You can also see the blood vessels that are coursing through the center of the villi that also help identify them.

They wouldn't be there in, decidua either.

So contamination with maternal tissue was a problem.

Before operators were able to give the labs adequate sized samples, we would beg the labs to try with whatever tissue they had.

And unfortunately that did lead to maternal cell contamination.

There's now been studies that have shown that if the villi are appropriately identified and clean, you can even use them for biochemical testing and DNA analysis that showed zero maternal contamination in most cases of adequately processed villi.

The chorionic villi also are composed of three different tissue sources, and this is just a slightly higher magnification, and one can see that there are the outside cytotrophoblastic buds.

Here we have what's called the mesenchymal core or the inside of the villi.

And we can see the blood vessels that I showed you on the last slide.

When one does what's called a rapid prep or a direct prep, one can take off these spontaneously dividing trophoblastic buds.

And you can actually see the mitosis taking place within some of these, and you could get results in only a couple of hours.

This represents embryologically, predominantly the placental tissue.

The core of the villi.

The mesenchymal core actually has to be processed in tissue culture and takes about five days or so, but embryologically, as we'll talk about in a few minutes, has slightly closer lineage to the fetus.

And finally, these blood vessels inside the villi can, if necessary be dissected free and tests on the fetal blood can be performed.

And we used to do this quite frequently for fetal blood typing, but now with DNA testing, it's much less necessary to, use this.

But it's important to illustrate that in these tissues that we're retrieving, there are separate, tissues that are processed separately.

In addition to maternal cell contamination.

One needs to confirm that tissue taken from the placenta.

And again, we'll talk a little bit about a tissue taken from each of those tissue sources is equally reliable to the karyotype that we would get from an amniocentesis or the karyotype that we would get if we directly tested the pregnancy itself.

We have learned with experience that 98 to 99% of the time the karyotype from the chorionic villi is an exact reflection of the karyotype from the fetus.

However, in about 1% of cases, we get what's called confined placental mosaicism or CPM.

As I said, this occurs in about one to 2% of CVS.

And what it is is it's a mosaicism in which the fetus itself has entirely normal karyotype, but the placenta is mosaic.

By mosaic we mean has two cell lines, has both a normal disomic cell line, but frequently we'll have a second abnormal cell line and is illustrated.

Here we have a trisomic cell line.

So if we didn't know about this and understand this, these could potentially be false positive CVS in which we would report an abnormal cell line in the placenta despite the fact that the fetus is completely normal.

Mechanisms of Confined Placental Mosaicism

Well, how does this occur?

Well, there's two, possibilities, that can lead to this.

One is that we can have mitotic, non-disjunction.

And what occurs is we start with entirely normal haploid gametes and they meet and they form a normal zygote.

The cells in the zygote then continue to develop, but we have a mitotic error in some of those cells so that some of the cells remain disomic.

But with the non-disjunction occurring, we will have a trisomy, also could have some monosomy cells.

So we have basically a mosaic morula.

Then the next step from conversion of the morula to the blastocyst, some of the cells go to make the inner cell mass, which will become the embryo.

But most of these cells, will go, to the placenta.

And where these trisomic cells go is just by random assortment.

But to give you an example of how of the distribution of cells, if you started with 64 cells, which is about what a morula contains, about 32 to 64, only four cells go to make the embryo proper, and the other 60 cells go to make the placenta and the amnion and the fluid and everything.

So when we have random distribution, it shouldn't be surprising that if there's only a few trisomic cells, in the morula, that they may go to the placenta leaving only normal cells in the embryo.

And that's the mitotic non-disjunction way of getting a mosaic blastocyst with only the abnormal mosaic cells in the precursors of the placenta, then leading to an entirely normal embryo, but a, placenta that is mosaic.

So one way is mitotic non-disjunction.

The more common way, which was totally unexpected and unknown at the time, was that we could have trisomic rescue in which we have a meiotic error leading to an abnormal gamete, usually an abnormal egg so that we start with a trisomic zygote.

Most trisomic zygotes ultimately wind up being miscarried.

However, as second genetic error of anaphase lag occurs so that some of these precursor cells go back to being normal disomic cells, again, that would leave us with a morula that would have some of the trisomic cells, but some that have been rescued from trisomy by a second genetic error leaving us a mosaic morula.

And again, the distribution of whether the trisomy cells go to the placenta or whether the disomic cells wind up in the fetus is important.

So those are the two mechanisms so that when we see mosaic results in CVS, we have an absolute biologic mechanism of that can be causing that.

Implications of Confined Placental Mosaicism

Well, the real question is what does it mean to have an abnormal placenta?

And actually it's not a good thing.

So that seeing confined placental mosaicism is way more than laboratory artifact.

About 15 to 20% of cases with confined placental mosaicism will have poor perinatal outcomes leading to miscarriage fetal death.

IUGR about 80% will still have a good outcome.

What determines whether or not the, placental abnormal, the abnormal placental cell line, will be pathologic or not is which chromosome is involved.

And some chromosomes are known to be more pathologic in a mosaic cell line in the placenta than others.

What percentage of the cells are abnormal?

Which tissues are involved, whether it involves, the cultured mesenchymal core or just the cytotrophoblast buds and whether or not the cell line continues to be present throughout the rest of gestation?

Here's an example of, probably the most well known and pathologic extra cell line in the placenta, and that's a trisomy 16 mosaic.

And here we have, 67 cases of pregnancies in which if you did a CVS, the villi would have two cell lines, a normal cell line and a trisomy 16.

The cell line, the fetus itself, if you did an amniocentesis, the results would be a hundred percent normal.

But just by having that abnormal Trisomy 16, cell line in the placenta, two-thirds of those pregnancies will either lead to fetal death, preterm delivery, an anomaly, or IUGR, and only one third will be normal surviving pregnancies.

So this just illustrates that despite the fact that an amnio would be normal in these cases by doing CVS and identifying confined placental mosaicism, one has an a new and unexpected cause of perinatal morbidity and mortality so that, again, the, results from CVS can, give us information that we might not get from other tests.

Biochemical Testing with CVS

One of the other things in addition to chromosomes that we will identify by CVS is we can do biochemical testing.

And this is a child with severe Tay Sachs disease.

The child's got a large head, he's blind, he's, deaf.

And about two months after this picture succumbed to his disease.

Tay-Sachs disease is an autosomal recessive disease.

These parents have in all subsequent pregnancies, a 25% chance of having another affected child with CVS.

One can analyze tissue from the villi for Tay Sachs disease.

And this is just an example of the laboratory test in 30 to 45 minutes.

So we have a disease with a 25%, recurrence rate that we can diagnose in the first trimester in a very short period of time.

Yet a recent study presented at the Society for Maternal Fetal Medicine showed that about 80% of women who were at risk for these biochemical high risk abnormalities were still waiting to have amnios.

Well, there's absolutely almost no test of biochemical or metabolic or molecular, causes that can't be identified in the first trimester.

And this should become, and in many places has become the prenatal diagnostic procedure of choice for those cases.

Safety and Success Rates

Well, how easy is, amnio? I'm sorry. How easy is CVS?

And in order, to, look at this, we looked at the number of insertions of the catheter or the needle at our center during our first just about 15,000 procedures.

And you can see that of 15,000 procedures, we've only had eight women that, basically were unable to be sampled, and all the other 15,000 got their diagnosis at their visit.

Of those eight in which we failed to retrieve tissue, seven of them were in our first a hundred procedures.

So in the last 14,900 or so procedures, we had only one patient who was unable to be sampled.

So the ability to retrieve tissue is almost absolute, and that is able to be accomplished because all of our operators are skilled in both transc, cervical and transabdominal.

And I really believe that any centers that's gonna do CVS should know how to do both.

And I would say the gold standard is that you should be able to sample all patients successfully on their visit.

What about pregnancy loss?

Following amnio here, I've given you our, again, our example on our first about 12, just over 12,000, CVS cases and our total post procedure, not procedure induced loss rate, the total post procedure loss rate was 2.3% and it didn't matter.

There was no statistical difference whether we did it abdominally or cervical.

Now, again, don't mix up apples and oranges.

This is the total loss rate after CVS and you can see that it's about 2.3%.

And if I can remind you of the more contemporary amniocentesis procedures, their total loss rate was about 1% less.

So does that say that CVS has a 1% greater pregnancy loss rate than does second trimester amnio?

Absolutely not, because the CVS is performed about four weeks earlier in gestation than amnio and illustrated in gray.

Here is the total background loss rate of unsampled pregnancies.

So if CVS and amniocentesis had absolutely equal procedure induced loss rates, you would still expect if you looked at total loss rates to have a 1% or so higher total loss rate after CVS just because it's done at a time in pregnancy when the background loss rates are higher.

But again, that's not the best way to compare total loss rates.

The real question is what about studies that have compared CVS to amniocentesis in single center studies?

And there have been as listed here, five studies that have done this.

Four studies have shown no statistical difference in post procedure pregnancy losses, in CVS compared to amnio.

Now it's important to point out that these patients were all enrolled in the study at the time you would do CVS and then all losses after that time period, were included for evaluation.

So that eliminated, any differences in, the period between CVS and amnio of, chromosomally abnormal losses occurring while people were waiting to have their amniocentesis performed.

So all patients enrolled at 11 weeks and then all losses looked at again.

Four studies showed no difference.

Two studies showed slightly higher losses after CVS.

Some studies showed higher pregnancy loss rates after amnio.

But when put together, there's no difference.

There's only one study that showed any increased risk with CVS, and that's the European study that was performed about 12 or 15 years ago and they showed about a three or 4% greater loss rate with CVS.

What's unique about this studies is that you had operators who were very, very experienced in doing amnio and you had operators who, some of which had never done CVS and as compared to all the other studies in which a limited number of centers were included, this study included over 30 inexperienced operators.

So many people, if not all, believe the difference in loss rate.

And what this study demonstrates is if you're good at one procedure and not good at another or experienced at one procedure and not experienced at another one, you're gonna do the one more safely that you're experienced at.

This is a study from France where they looked at a new operator's experience with transabdominal sampling his first 200 procedures, his next 400.

And, clearly you can see that there is a learning curve and it takes about four or 500, procedures to become very, very proficient and to not have any incremental loss.

So again, the studies that have compared the two techniques head to head using experienced operators have shown no difference in pregnancy loss.

Another way to say that is first trimester CVS and second trimester amniocentesis are equally safe procedures from the standpoint of inducing pregnancy loss.

Another way to look at the impact of experience has been the United States has had three, three, studies that have included the same operators.

The first study was a comparison in 85 to 87 of CVS versus amnio.

The second study was transabdominal versus transcervical CVS from 87 to 89 and 97 to 2000 ones was early amnio compared to CVS.

And as you look at how this, particular group of operators improved over the decade, and that's not just the operators themselves learning, but the community learning some of the tricks of doing the procedure.

You see that CVS has continued to get better until we have a procedure loss rate exactly what we would anticipate for amnio.

So I think I can say with absolute certainty that in the hands of people that do both procedures, they are equally safe.

Techniques to Reduce Risks in CVS

There are, however, a few issues that, will make an operator more successful and things that we've learned along the way that, we can transmit to other individuals.

And one of the points is that if you look at the loss rate by the number of needle and or catheter insertions, you can see that one or two has a relatively low loss rate, but once you do three or more, your loss rate dramatically increases.

So how can we keep down the insertion rate and how can we keep down the loss rate?

One of the big issues are uterine contractions.

And until we started doing CVS, we really didn't appreciate how frequently they occurred.

Here we have a first trimester pregnancy, we have a large contraction here, and we have an operator attempting to insert the catheter and he can't get the catheter to the placenta, can't even see the placenta, because of this contraction.

30 minutes later, this is the exact same pregnancy as you see illustrated here.

30 minutes later, and I apologize, these are not the highest quality scans.

30 minutes later the contraction is gone and he was easily able to identify the anterior placenta and to insert the catheter all the way through the chorion frondosum.

So number one is learn that there are contractions, learn how they will affect the way you pass the catheter and just wait until they go away and it will make it for a much safer procedure.

So number one way to improve your safety is to learn about contractions and to learn what they will do to your catheter, what they will do to the placenta location and how to, operate around them.

The second piece of information that will reduce losses is to make sure you have the needle in the proper tissue plane.

This is a first trimester pregnancy and you can see here's the chorion frondosum that we wish to sample so that if we're inserting a catheter or a needle, we would wanna put it in this area if our catheter is just a slightly bit too close to the decidua or the myometrium, right beneath the chorion frondosum.

Doum is the decidua basalis where there's giant blood vessels and with experience one learns the feel, there's absolutely no resistance required to insert a catheter for CVS.

If you're in this area of decidua, you get kind of a gritty feeling.

So by experience you learn exactly the right tissue plane in which to insert the instrument that will decrease bleeding.

And here is an immediate post CVS hematoma when the operator had the needle.

Here we see a posterior placenta that had the needle too far inserted, into the decidua.

So again, experience will teach you the appropriate feel for the tissue plane.

So CVS is equally safe.

We can dramatically decrease the risks with operators learning the best techniques to do.

Limb Reduction Defects Controversy

That leaves us with one final question and that is what about the controversy that existed about CVS and limb reduction defects?

This is from the original concern that was raised in Lancet back in 1991 in Firth.

And their group of individuals at, Oxford, at had done 539 CVS, of which 290, just under two 90 were under 66 days.

And of those CVS performed under 66 days, they had five.

So it's about one in 60 out of that group had severe limb defects.

Four of them had oral mandibular limb hypogenesis, small mouth, small tongue, small mandible, severe limb.

Under development, the incidence of oral mandibular limb hypogenesis in the general population is one in 175,000 and they saw one in 60.

So that really begged the question of whether or not CVS could be causing this problem.

Here is a child not after a CVS, but that has the very small jaw, the very severely deformed arm.

This is an example of oral mandibular limb hypogenesis.

And I illustrate this not because it was after CVS, but we've known about oral mandibular limb hypogenesis for decades.

And what was of most concern?

Oral mandibular limb hypogenesis syndrome was felt to be secondary to vascular disruption or disruption of the blood supply to the fetus.

So now we had biologic plausibility.

We had an increased incidence of a birth defect that was caused by vascular disruption following a procedure which was intended to sample the vascular organ of the fetus.

So really there was much concern and actually very justified concern.

Now, vascular disruption can cause an abnormality by causing hypoperfusion for even a short period of time of the fetus, then following hypoperfusion, which decreases the blood flow, particularly to the peripheral organs like the distal limbs and the jaw and the tongue.

Then there's reperfusion and reperfusion winds up causing edema and tissue necrosis so that we really had significant biologic plausibility by that mechanism.

However, most patients that were having CVS weren't having, this particular abnormality and it raised the question of what were some people doing compared to others that was leading to these abnormalities.

'cause many centers weren't seeing it.

So to answer this Quintero in the group where he was at Yale at the time, went to the operating room and did Embry, they inserted a small scope in which they could actually look at the fetus and then they took a sound and tried to disrupt the placenta.

And they found that when they just passed a CVS catheter, they could not disrupt the placenta and they couldn't cause the findings that others were finding in clinical care.

However, when they used the larger sound, which I described and severely disrupted the majority of the placenta, they started to see these hypoperfused areas exactly in the areas that one would anticipate.

So they did demonstrate that minor trauma couldn't cause this, but severe trauma could raising the specter of whether some operators were doing more traumatic procedures.

And that may be part of the component.

But what really gave us the information was a study that was done by Bruno Brambati in Milan, Italy.

And he looked at the gestational age that CVS was done and he started doing CVS rather than at the nine to 12 window that he had been doing it in earlier and earlier, trying to give patients a result as early in pregnancy as possible.

And what he illustrated was if you did CVS under the ninth week, you began to see an increased incidence of these defects from six per 10,000 in the normal nine to 12 week background group to one in a thousand.

And when he did it in the sixth to seventh week, he found the risk of limb defects exactly what they found at Oxford when they were doing very early procedures.

So it's now felt certain that this incidence or of limb defects following CVS was secondary to people doing sampling too early, under 70 days of gestation.

In other words, there's a window of vulnerability in which CVS can cause limb defects.

And I just wanna remind you that we saw the absolute same with a window of vulnerability for amniocentesis when it was performed under 14 weeks gestation.

So for any prenatal diagnostic procedure, we should perform it in the safest period of gestation.

Now, there've been over 300 procedures evaluated after the ninth week of gestation with no increased incidence of limb reduction defects identified.

Conclusion

So I think that for a number of reasons, first trimester screening and first trimester prenatal diagnosis is the way to go.

We get patients earlier reassurance in most cases.

We identify abnormalities at a time in pregnancy when it's psychologically and medically safer to terminate the, pregnancy so that, CVS has that advantage, particularly as first trimester screening has become an increasing part of obstetrical care.

And I'd like to thank everybody for, their attention to this, discussion.

Thank you.