In this webinar, Dr Francisco Anaya Blanes, Gynaecologist at UR Vistahermosa, Alicante, Spain, has talked about endometrial assessment procedures.
The human endometrium is a unique tissue in many ways. No other tissue proliferates, differentiates, sloughs over and over again, cycle after menstrual cycle for up to 40 years.
The endometrium is a hormone-dependent tissue that forms the mucosal lining of the inside of the uterus. It’s not only an interface between the internal and external environment of a woman’s body, but philosophically speaking, it is the interface to our existence as all of us require the endometrium to begin our journey as human beings. This complex transformation is the direct result of the endometrium’s multifunctional task to be receptive to the developing embryo in cycles with successful fertilization.
On one hand, allow for the attachment of the blastocyst, and on the other hand, prevent overly aggressive incursion by the invasive trophoblasts which if not checked could penetrate and rupture the uterus. The final faction is to shed when there is no implantation, only to start over again in the next cycle.
Another issue in our species has the task of radically changing its structure, biology, endocrinology, and function, in this way repeatedly. It’s very important the fact that endometrial function is not only allowing embryo implantation but also does it with selectivity. If not, any embryo entering the uterine cavity would implant resulting in a pregnancy whatever the quality of the embryo or the state of the woman. It’s just this complex tissue that has the selective potential, which in turn, allows the development of a competent embryo when the woman is most able to support a successful pregnancy.
The endometrium consists of two basic layers:
There are two main facets in the endometrial cycle:
This is the first half of the menstrual cycle. During this, the main feature of the endometrial tissue is active proliferation and angiogenesis to ensure nutrition for the developing new tissue while suppressing apoptotic factors.
The key player in this proliferative phase is estrogen. This estrogen induces the proliferation of cells, stimulates the expression of steroid receptors, and acts as a cell survival factor. The secretory phase.
During this phase, morphological changes that characterize decidualization are taking place independently whether it’s conception or not. This phase takes place from the14th to 28th day of the natural cycle. Progesterone, which suppresses proliferation and induces cell differentiation, is the major player during this second half of the menstrual cycle. The endometrium is infiltrated by immune cells and is prepared then for the implantation of the blastocyst.
At this stage, the outer shell of the womb and the pellucid zone break down, and the embryo attaches to the wall of the uterus and grows there. This acceptance of the embryo is called endometrial receptivity and the period in which the uterus accepts the embryo is called the window of implantation.
We can define the window of implantation as the period when the endometrium is receptive to the embryo every month. Both actors, embryo and endometrium, must interact with each other in order to achieve implantation that continues until a live birth. In a natural conception, sperm and oocyte must find each other in the fallopian tube where the conception should occur, and then the embryo generated travels during three or four days along the tube to the uterus. The embryo should achieve to the endometrial cavity at its stage of the blastocyst and then the chemical process of implantation. There are many genes and many molecules involved in this complex event.
We start with the hatching of the embryo which, with the lp of some adhesion molecules, starts the apposition phase. After this, with the collaboration of pinopodes, it produces adhesion and finally, if the embryo finds a receptive endometrium, it produces an invasion of the endometrial wall.
The role of the embryo in this attachment may be the most important factor in the implantation rate. As we already know, if you have the potential of transferring three euploid blastocyst of high quality, you reach an accumulative rate of success of 95%. The endometrium is very important as well, and his task is double, it must be receptive and selective.
We have been studying for many years the characteristics of the embryo and we have them classified by quality.
We know that blastocyst is the best embryo to transfer in the uterus, and we even perform genetic testing in order to transfer only euploid embryos, improving our success rates and decreasing the risk of a miscarriage. We even use time-lapse system as a way of knowing more about our embryos development, not only in the morphology but also about its kinetics.
With these new incubators, we also improve the rate of embryos achieving the state of the blastocyst. Under all these circumstances, we could arrive to a 95% of success rate, but if we want to make higher rates, nowadays we should be studying the endometrium, which is the other main actor of implantation.
We have many ways of studying the endometrium. Fortunately, the endometrium is one of the most accessible of all tissues and can be monitored by biopsy, ultrasound, MRI, to understand its status. Endometrial assessment is essential in different moments of a woman’s life, first periods, reproductive age, peri and menopausal bleedings.
Assessment of the endometrium as part of infertility remains a very active area for research nowadays. It has been reported that the endometrial integrins expression can predict not only endometriosis but also success as part of IVF. Lots of integrins appear to be associated with overexpression of endometrial aromatase that has also been associated with poor IVF outcomes.
The blockade of this aromatase improves IVF success, suggesting the primary importance of this aberrant expression of aromatase. Interestingly, endometrial biopsy before IVF has been shown by systematic review to be beneficial for implantation, suggesting that the endometrial injury sustained during biopsy activates the endometrium and restores its function.
Histological methods:
Historically, much of the basis for endometrial assessment originated in the 1940s and 50s, when endometrial dating was established based on histological characteristics. The fact that the endometrium develops in a cyclic and organized way, provides a roadmap to what is considered normal development. Jones described the luteal phase defect in 1949 based on endometrial biopsies carried in different moments of the cycle. This is still nowadays an issue of debate and is considered controversial after 60 years.
Techniques to improve based on histological dating criteria have evolved, including the use of progesterone in the blood, a surrogate market, the use of endometrial adhesion molecules to date the window of implantation, and most recently, DNA microarrays that examine the entire genome to dissect and divide endometrial development each month into its four stages: proliferative, early, mid, and late secretory.
We advance in the study of endometrium and we have less invasive methods for endometrial assessment, for example, the most important is the transvaginal sonography that has become widely accepted as a tool for high resolution imaging of the uterus and endometrium.
Sonography.
The growth of the endometrium can be easily measured using ultrasound. The endometrial thickness and texture can be indicators of normal development or be used as a sign of disease. In the early proliferative phase, immediately following menses, it’s typically thin. In response to estrogen, the endometrium thickens and becomes thicker in appearance, growing between 0.1 and 0.5 millimetres daily. Following ovulation, the endometrium becomes hyperechoic and secretory changes.
Transvaginal pulse Doppler ultrasound measures uterine artery blood flow expressed as the pulsatility index. This varies along the cycle and may be used to predict implantation after an assisted reproduction technique. Aside from endometrial thickness and morphology, blood flow and uterine artery pulsatility have all been examined as possible markers of a receptive endometrium. Moreover, increased pulsatility has been associated with elevated markers of pregnancy, such as anticardiolipin antibodies.
An interesting future area of research will be the correlation between pulsatility index and other ultrasound characteristics with traditional and advanced assessment of endometrial biomarkers. We have recently incorporated three-dimensional ultrasound to our daily practice in fertility. It’s used to get a better assessment of uterine cavity, shape of uterus, and volume measures of both endometrium and follicles.
Sono-Hysterography.
Here we can see very clearly some polyps of the endometrium. We can see a clean cavity here, also we can see how you can define the polyps with three-dimension Sono-Hysterography.
It’s a very useful technique in some cases.
Magnetic Resonance Imaging (MRI)
In this picture, we can see some resonance imaging of the uterus. Nowadays, with the quality of our ultrasound, we don’t usually need magnetic resonance, but it provides a clear view of the uterine anatomy. This is especially useful in the evaluation of tumours.
MRI demonstrates the endometrial myometrial interface, so it can be used to assess an endometrial invasion by endometrial carcinoma, and we use it sometimes to get more information about our myomas.
Hysteroscopy
It’s an endoscopic technique that allows the study and treatment of the pathologies of the uterine cavity. We can use it just to see the endometrial cavity to remove polyps, myoma, septum, or just to take specific endometrial samples.
We can see how we perform the hysteroscopy. We can see a normal cavity with the ostium. We can see a septate uterus, which we should remove before transferring embryos. We can see a huge polyp making most of the cavity and some pathologies.
It’s now considered the gold standard technique to study the endometrial cavity, and we perform it as part of the study of implantation failure. Depending on the purpose:
These tests are some genetic molecular tests to study endometrium.
The endometrial receptivity analysis ERA is a molecular based test designed today for the window of implantation in every single woman.
In order to achieve this accuracy, the ERA requires an endometrial biopsy on day seven in a natural cycle or five of progesterone in a hormone replacement cycle. It shows, taking into account, 248 genes, if the endometrium is receptive in that moment, so we can talk about pre-receptive endometrium, receptive or poor receptive. It allows us to make the embryo transfer exactly during the window of implantation of the woman.
There is no histologic assessment of the sample, as we’re going to just study the genes in that sample.
The main critics to this technique are that we perform it during the cycle, previous to the transfer cycle which may be different from the real one, and results are not consistent in all the medical studies; however, it may be helpful in some cases of implantation failure and we may use it as part of our different strategies to help couples to conceive.
EMMA test is a molecular test that studies the microbiota of the uterus and is also helpful in some cases.
ALICE is used to discard chronic endometritis, a situation that could decrease our success rates. They are also carried out from an endometrial biopsy during the secretory phase of the cycle, in the cycle before the embryo transfer.
There are many other studies that, on some occasions, we carry out to investigate implantation failures, such as:
There are ways to improve embryo implantation from the point of view of endometrial receptivity.
Sometimes we apply treatments to our patients not sufficiently based on scientific studies. All the techniques we use in the clinic should have medical evidence of improving our results. If we don’t carry out further investigation, we will be part of a race to nowhere. These are some of the treatments frequently used to improve our implantation rates:
There are many other techniques, acupuncture, vitamins, probiotics, and more, but what is a good practice and we’re all nowadays trying to perform is an individualized treatment from the beginning and a personalized embryo transfer adapting our protocols to the characteristics of a concrete patient, attending her special circumstances and trying to get the best pregnancy rate possible.
Well, about this question, about the triple lining in the proliferative phase, we said before the first endometrial phase of the cycle, the endometrium is very thin at the beginning of the cycle and usually gets thick under the estrogen effect, and in most of the patients the endometrium shows a triple lining. That means that the endometrium is getting thicker and that it’s under the fact of estrogen activity. We used to think that it was an unreceptive endometrium, as we have said before, it doesn’t always mean that, but is the endometrium we like the most to make our transfers before adding progesterone.
This is a very difficult question. We’re always discussing the T-shaped uterus. I’m very involved in this because I’m used to doing the surgery at my clinic, so we’re very involved with this subject.
Yes, in some cases T-shaped uterus we consider it a cause of implantation failure, but considering it the main factor, we should, firstly, have transferred some euploid and good quality blastocyst as many times we’re thinking of problems with the endometrium, problems with the uterus, and what finally we find is that we have not had good quality and genetic tested embryos.
If we have an implantation failure in a uterus, after transferring good, healthy embryos, we used to correct it with hysteroscopy.
Well, with that mutation, we consider Clexane. It’s also a discussion thing because sometimes our team of haematology doesn’t agree with the heterozygote cases, but we used to add it because we’re trying to avoid implantation failure and to avoid miscarriages, and we think that it avoids it.
About the doses, it depends on the weight. It should be a milligram per kilogram, but most of the time we use 40 milligrams as a standard dose. We never use 20 milligrams, rarely. I’d like to see your weight and your height. We would probably use 40 milligrams; it may be enough.
If you have had just 1 ectopic pregnancy, I would not recommend doing an ERA test. The ectopic pregnancy is not only about endometrial receptivity, but about migration of the embryo and sometimes about problems with the mobility of the tubes, so I don’t think the ERA is the solution for one ectopic pregnancy.
We suggest doing three transfusions of intralipids. We used to make the first one around the day of the transfer, the second one with a positive test, and the third one in the 12th week of pregnancy. That’s our protocol.
It’s not the same. An endo culture is a common culture to see that there’s no infection about the endometrium and the EMMA is studying molecularly the endometrium. So, we shouldn’t assume that. There are different ways of studying the endometrium.
The EMMA biopsy, the same as ALICE biopsy, is performed in the second half of the menstrual cycle, maybe around the day 18, 20, 24, or less. In fact, we used to perform all 3 together, ERA, EMMA, and ALICE on the same day. Just with one biopsy, we can do the three of them.
In fact, we don’t advise doing them usually. We advise doing them on various special occasions. They are part of our study of implantation failure which we nowadays are considering after two or three embryos, euploid or good quality blastocysts transferred, so we do not perform them in a normal patient.
In fact, the last study I’ve been reading a summary of studies showed that after the ERA test, just when the endometrium was receptive, the rate of implantation was 50%, and when the endometrium was pre-receptive or post-receptive, using the ERA dates they achieved a 33% of implantation and those are similar rates to a patient without any of these tests. It’s not so clear the use of them. We of course use them, but in special occasions.
That’s a perfect question because I asked that question also for me. We don’t really know how long it is. In fact, our doubts, our concerns about these tests are that we sometimes have doubts, if it’s the same the cycle we perform them then the next cycle, and which we’re transferring the embryos. We have many doubts about this.
Some studies say they are valid for four months, maybe 4 to 6 months, but I wouldn’t say that. That’s the main critic to all these techniques that are not made in the same cycle that we’re transferring the embryos, so we’re not sure about this.
Yes, we would prescribe steroids and, depending on your medical history, maybe we would think of making intralipids infusion also.
the myomas are 7, 7, and 10cm. When you have subserosal fibroids, usually they don’t affect much about the endometrial cavity, but these sizes are very big. Two myomas of 7cm and one of 10cm, I think we would advise removing them before an embryo transfer. We would make surgery before transferring because they’re too big.
It’s possible to try one embryo transfer, but if you’ve gone into an implantation failure or a miscarriage, we would advise surgery for these myomas. They’re too big.
That’s what I was saying before. Sometimes, we focus our problems on the uterus, in the endometrium, and I don’t know about the embryos, if they were genetically tested, about the age, about the quality of the embryos, if they were blastocysts. These are the main things, in my opinion, the main facts that are influencing the rates of pregnancy.
Of course, it’s better not to have myomas, but maybe we should take a look at all your history and decide what is affecting the implantation.
Maybe you have a very difficult uterus, and it’s not easy to get pregnant, sometimes even removing the myomas because when we remove the myomas the uterus is still not normal. So, sometimes it affects implantation. Maybe it’s what is affecting you, I’m not sure about it. As I said before, with donor eggs and good sperm, if they are genetically tested, the embryos, should implant but even with donor eggs and good sperm, we should see if the sperm has the FISH test done or genetic tested. If then it is an implantation failure, we should consider every reason, maybe the uterus.
Yes, of course. Omega-3 is never going to harm, not in the beginning, it has nothing to do with the other. You can take Omega-3 and Clexane the same. It’s a different way of treating different things, I don’t think they’re related.
