Why do Chromosomally Normal Embryos Not Implant in the Uterus?

Human reproduction is an inefficient process.  Naturally, only about a third of our initial conceptions implant in the uterus and 15% of those are lost in the first trimester of pregnancy.  A significant proportion of our inefficiency can be attributed to embryonic aneuploidy, i.e.  The embryo having an abnormal chromosome number. Most of us have 46 chromosomes. Most of our embryos do not.  Embryos end up with an abnormal number of chromosomes because the egg has abnormal chromosomes, or the sperm has abnormal chromosomes or during the first cell division after fertilization, the chromosomes are inappropriately separated.

Despite our ever-improving approaches to care, embryonic issues still create inefficiency in all of our treatments.  Over the last two decades, various approaches to try and address this problem have been developed.  All of these approaches have utilized in vitro fertilization with the embryos being tested. The testing we currently use is called Pre-implantation Genetic Diagnosis with Comprehensive Chromosomal Screening (PGD/CCS). Another name for this is PGS, Pre-implantation Genetic Screening. For many patients, the testing allows us to accurately address the problem of embryos having abnormal chromosome numbers. Yet, about 1/3 of the chromosomally normal (euploid) embryos that are transferred to the uterus fail to implant in the lining.  How can this be?

Today, we are going to explore why chromosomally normal embryos may not implant and how we try to address the problems when we can.  Failure to achieve a successful pregnancy with euploid embryos can be generally grouped into three major areas: embryonic, uterine, and systemic.

EMBRYONIC

The development of an early embryo   is a complex and highly synchronized series of events to prepare it for attaching to and invading the lining of the uterus.  The process starts at fertilization with single cell called a zygote, which develops over 5 days into a 100-120 cell hollow ball, called a blastocyst.  It is a blastocyst that will attach to and implant in the lining of the uterus.  Our 46 chromosomes contain about 25,000 genes that have to function properly to have the embryo develop successfully. I think of it like a symphony of the genes being turned on and off at specific times.  If the timing of this is off, just like an orchestra, things will go badly. We think that gene malfunction is a very common cause of “good” embryos not implanting, but we have no way of testing this clinically. Basic science studies suggest that energy function (metabolism) of the embryo is also a likely cause of failure, but this too cannot be practically measured in the clinical lab.

Basic science studies and our clinical experience suggest that early embryo development can be altered by our clinical and laboratory environment.  It is clear that the type of follicular stimulation in an IVF cycle will impact the oocyte and embryo.  For example, excessive stimulation of women who can respond normally to medications is detrimental to the embryo, so that even “normal” embryos are not metabolically sound. The correct use of supporting medications (LH or low dose-hCG, growth hormone, androgens) may be beneficial to development of the growing follicle and egg for some individuals. However, incorrect use of these medications can be detrimental.  The type of trigger shot used to prepare for egg retrieval (hCG or GnRH agonist) may have an impact on egg quality also, but who may benefit from which drug and to what degree there is an advantage remain unclear.

As you can imagine, the laboratory environment plays a huge role in how the embryos will perform. Culture media has evolved tremendously over the last 30 years, starting from simple media adequate for most cells to the variety of high quality embryo-specific, commercially made media that we currently use.  There are several high quality culture media available that work well however, constant quality testing is essential to make sure a chosen media performs optimally. The incubators that the embryos grow in and their function are clearly a major factor. Most high quality practices are using the small bench top incubators which do a better job of maintaining the chemical environment and temperature stability for the embryos to grow in. However, these high-tech devices must be constantly monitored, as subtle changes can impact embryo performance.  Simple things like how often the embryos are evaluated, under what conditions, and the time it takes to do so can tremendously impact embryo performance.  The trend is clearly towards evaluating the embryos less frequently.

It should not go without saying that technical competence of the people in the laboratory is essential for good outcomes. However, those of us who have done inspections have seen a wide array of skill levels. All of our laboratory skills have a “user signature”. In ICSI (intracytoplasmic sperm injection- injecting the sperm in to the egg) for example: was the procedure done at the correct time? What kind of sperm was selected?  Was the sperm inserted in the correct place in the egg? How long was the oocyte (egg) out of the incubator?  Embryo biopsy for testing the chromosomes is a very demanding technique that requires a great deal of practice and experience.  Vitrification (very rapid freezing of embryos and eggs) has been a huge benefit to us, but the technique is subtle and not everyone gets the same results. Clearly, monitoring of laboratory personnel is vital to a high quality embryology laboratory.

UTERINE CAUSES

Quite honestly, the uterus is my best friend on a day-to-day basis.  That is because it usually works.  I say this because over the last 26 years, I have seen large numbers of sub-optimal appearing embryos make beautiful children. They were suboptimal often because of patient parameters, but also because our treatments and our labs were less sophisticated, and the uterus rescued them.  In addition, if one currently looks at good prognosis patients in high quality programs with mild stimulations and good labs, live birth rates over 70% are achieved in fresh cycles.   Since we know the general frequency of embryos with abnormal chromosomes in this population, the high birth rate demonstrates that uterine/endometrial issues are uncommon causes of failure. But there are clearly uterine issues that contribute to failure, some of our creation.

Structural Problems. Uterine structural defects are frequent problems, but these are usually found and corrected prior to treatment.  However, when IVF failures do occur, it must be accounted for. Almost everyone should have a hysterosalpingogram (HSG) as part of their evaluation to look for blocked, fluid filled fallopian tubes (hydrosalpinges). The fluid from these much damaged fallopian tubes can prevent an embryo from implanting in the uterus.  Infrequently, hydrosalpinges are missed because they are not seen (tube not seen on HSG) or the test is inaccurately interpreted.  Fibroids are common benign tumors of the muscle of the uterus and for some women can cause problems in achieving pregnancy because they can alter the blood supply to the embryo. However, it is a debate about when they are a problem. We all agree that fibroids that alter the inside of the uterine cavity are a problem and get them corrected. Where we have a problem is in trying to determine when intramural fibroids (fibroids that are buried in the muscle of the uterus but do not alter the uterine cavity) pose a problem.  Some studies suggest fibroids as small as 2.5 cm are a problem, while others can say it takes much larger ones (up to 4-5 cm) to cause a problem. On occasion, we will see seemingly insignificant fibroids grow during treatment, which can create problems, but this is rare. Uterine anomalies (birth defects of the uterus) do not prevent pregnancies, but contribute to miscarriages. Endometrial polyps (outgrowths of the lining of the uterus) are something that we are always on the watch for, but they can grow even during a treatment cycle.  Scar tissue in the lining of the uterus (Asherman’s syndrome) should be an infrequent problem, as pre-cycle testing should detect this accurately.

Endometrial Problems. Problems with the lining of the uterus are uncommon but can have a huge influence on whether or not an embryo implants. The lining of the uterus is receptive to the embryo for only a brief time, called the Window of Implantation. It is possible that a perfectly normal embryo might not implant because the lining was not ready for it.   In natural cycles, the window may be 4-5d wide, but in our treatments in can be only 12-48h long.  This window is controlled by the hormone progesterone.  The timing, dose and route of administration of progesterone can impact this. A new test called the Endometrial Receptivity Assay appears to accurately identify this window and in patients with multiple IVF failures, 25% are not receptive at time of transfer, most with delayed development. This provides us with a tool to work with in this specific group, and we have done well with our patients with abnormal testing.   However, the ERA has not yet been proven to be useful for patients just starting their evaluation, undergoing simple treatments or just starting IVF.  Hopefully, over the next couple of years, we will have information as to whether it is helpful in those situations.

The lining normally needs to reach a certain thickness (about 7 mm) to give an embryo a good chance of implanting. Pregnancies can occur with thinner linings, but they are much less likely to occur and the resulting pregnancies can be more complicated. In some women, thin linings occur due to the lining being damaged surgically, but in many women, we have no obvious reason for the thin lining.   For these women, a variety of treatments have been tried (estrogen, aspirin, sildenafil, pentoxifylline, Vitamin E and gCSF), but unfortunately they do not have significant impact for most patients.

Chronic endometritis is a disorder where there is inflammation in the lining of the uterus. Endometritis is very uncommon and thus we do not subject our patients to painful biopsies to detect it routinely.  However, when we have not been successful, it is something to consider looking for.  Treatment with antibiotics is usually effective when endometritis is found.

Evidence continues to mount that the lining of the uterus in endometriosis patients is different and may well be contributing to infertility beyond the clear anatomic issues seen at surgery with advance stages of endometriosis.  Since we are no longer performing laparoscopy on everyone, this is something that can be missed.  The mechanism appears to be related to progesterone resistance at a molecular level. Fortunately, it appears that the significant doses of progesterone used in ART treatments overcome this.

Technical Issues.  Embryo transfer is a subtle technique that not everyone can do.  The procedure needs to be atraumatic to avoid uterine contractions and minimize disrupting the endometrium. This requires good knowledge of the patient’s anatomy and good ultrasound visualization. Transfers need to be done quickly, so that the embryos are not subject to metabolic stress while awaiting transfer. The embryos need to be placed in the proper position in the uterus for optimal implantation and to avoid tubal and cervical pregnancies.  It takes very little to sabotage this.

SYSTEMIC CAUSES

Well, it is the usual suspects here. Over 1/3 of reproductive age women are obese and it impacts their reproductive function at all levels, including the oocyte quality and endometrial function.  Pregnancy rates also appear to be lower when the man is obese, even with normal sperm parameters.  Of course, pregnancies can be substantially more risky as our weight increases. Cigarette smoking is another modifiable factor that appears to have its primary manifestation through altering ovarian function and oocyte quality. However, in studies of oocyte donation recipients, the clinical pregnancy rate was reduced by a third, clearly indicating an effect on the lining of the uterus as well.  Secondhand smoke has also been shown to reduce the chances of a successful pregnancy by over 50%.

Thyroid dysfunction, certain antibodies and elevated prolactin levels do not appear to affect chances of pregnancy through assisted reproductive technologies such as in vitro fertilization. Vitamin D deficiency is common and is important to look at since the active form of vitamin D is secreted by the endometrium and impacts genes essential for implantation. However, the two largest studies (n=267, n=517) demonstrate no impact of vitamin D supplementation in oocyte donation cycles and euploid embryo transfers, respectively.

Summary

Embryos with abnormal chromosome numbers (embryonic aneuploidy) are clearly the most common reason for IVF failure, and has led to the development of our current embryo testing approach. However, there are a variety of other issues that impact egg development and embryo performance that can lead to the failure of implantation of a chromosomally normal embryo.  While some of these problems are currently beyond our control, we continually work to address them through comprehensive knowledge and application of the literature, detailed patient evaluation and unending surveillance of our laboratories and technical skills.