Implantation: The Challenge of Assessment and Treatment

by Harvey J. Kliman, MD, PhD, Yale University
David L. Keefe, MD, Brown University

The hurdle of implantation

Many couples must overcome many hurdles before they can have a successful pregnancy. These include the considerable journey the sperm must make to reach the egg, the penetration of the egg by one successful sperm, the division of cells to create an embryo, the journey of the embryo to the uterus and, possibly the most difficult step of all, the attachment of the embryo to the uterine lining (implantation). Any abnormalities in the process of implantation are believed to be the basis of many cases of unexplained infertility in women. The question that researchers are trying to answer is: Can we help women for whom implantation has not been successful?

In many assisted reproductive technology (ART) programs, fewer than 10% of embryos successfully implant. Predictors of implantation success are needed, both to better understand the causes of infertility in women and to improve the efficacy and reliability of ART. Despite almost two decades of assisted reproductive technologies, the precise mediators of human implantation remain unknown. As a corollary to this problem, up to now no markers have existed to predict adequately whether implantation will occur during any given assisted cycle.

Markers of endometrial receptivity

During each menstrual cycle, the human endometrium goes through dramatic changes necessary to prepare for implantation. A normal endometrium will express many different substances which promote implantation. One of these substances is termed MAG. The MAG test, done during an endometrial biopsy measures a sticky, mucinous substance that is secreted by glands in the endometrium before the implantation stage. The results of our studies have shown that over 85% of the endometrial biopsies from normal, fertile women express higher levels of MAG between days 5 and 18 of the menstrual cycle-with no expression after day 19 (Fig. 1). The results from our unexplained infertility patients, in contrast, revealed a very different pattern of MAG expression.

Implantation Issues Figure 1 

Figure 1. Normal pattern of MAG expression through the menstrual cycle. MAG is expressed within the endometrial glandular cells beginning at about day 5 of the cycle. On day 16, two days after ovulation, MAG begins to move out of the glandular cells. Between days 17 and 19 MAG appears to migrate to the uterine surface were it appears to play a role in the earliest attachment of the embryo to the endometrium. After day 19 there is normally no more expression of MAG in the endometrium.

Only 20% of our infertile patients revealed a normal MAG pattern. Approximately 70% of the patients with unexplained infertility showed abnormal MAG levels, i.e. MAG was inappropriately expressed after day 19 (Fig. 2). The remaining 10% of patients showed no expression of MAG at any time in their cycle. Interestingly, follow-up clinical data on a subset of patients undergoing donor oocyte transfer has revealed that 75% of these patients with normal MAG expression became pregnant, while only 18% of the patients with abnormal MAG levels became pregnant. None of the patients with absent MAG expression became pregnant.

Implantation Issues Figure 2 

Figure 2. Delayed pattern of MAG expression through the menstrual cycle. In the majority of infertile women with abnormalities of the endometrium, MAG appears to be expressed inappropriately in the menstrual cycle. The presence of MAG after day 19 is a marker of abnormal endometrial development.

Two other new markers of endometrial development, cyclin E and p27, parallel the normal and abnormal expression of MAG in endometrial biopsies (Figs. 3 and 4). Abnormalities in MAG, cyclin E and p27 collectively are indicative of abnormal development of the endometrium.

Implantation Issues Figure 3 

Figure 3. Normal expression of cyclin E and p27 through the menstrual cycle. Cyclin E normally first appears early in the proliferative phase and is not seen after day 19. p27, on the other hand, first appears around day 17, and remains strongly positive for the remainder of the cycle.

Implantation Issues Figure 4 

Figure 4. Delayed expression of cyclin E and p27 through the menstrual cycle. In this example, cyclin E expression persists through the luteal phase (labeled as secretory phase in this diagram). p27 is still present in the luteal phase, but at lower levels. The abnormal persistence of cyclin E staining in the luteal phase is another marker of abnormal endometrial development. This pattern of cyclin E staining is associated with unexplained infertility suggests that some form of medical and/or surgical intervention will be necessary to improve the chances for implantation to occur.

The goal of our research is to evaluate the use of MAG, cyclin E, p27 and other markers as the basis of an Endometrial Function Test (EFT)™ in order to differentiate implantation-receptive endometrium from non-receptive endometrium. Such an EFT can then be used by infertility specialists to assess the potential success of their treatment modalities.

Treatment options to improve endometrial receptivity

The two most rapidly evolving treatment strategies for implantation defects are optimization of steroid hormone treatments and elimination of extrauterine factors that interfere with implantation.

The hormones estrogen and progesterone always have been acknowledged as critical for implantation. Women undergoing infertility treatments which lower progesterone production, such as GnRH agonist (Lupron™) and egg retrieval for IVF, require progesterone supplementation to achieve pregnancies. More controversial has been the issue of whether subtle variations in the doses and/or duration and/or routes of administration influence implantation.

Studies comparing the effects of hormone preparations and routes of administration on markers of endometrial receptivity provide a sensitive indicator of the effects of hormones on the implantation process. Indeed, studies comparing vaginal versus intramuscular routes of progesterone administration have shown vaginal administration provides more normal structure of the endometrium, when examined under the microscope. The EFT is especially helpful in this regard since it examines endometrial functions which are specifically mediated by both estrogen and progesterone. Estrogen helps trigger endometrial cells to divide and proliferate. Progesterone halts endometrial proliferation and promotes transformation of the endometrium into a stable platform for implantation. (Using the EFT as a guide, therefore, we have been able to alter the doses and durations of the steroid hormones and hence individualize treatment for the specific patient. While systematic study of these treatment strategies still are under way, we have had promising early successes in some very challenging patients.)

Other factors may disrupt implantation by indirectly altering the uterine lining. One example of this is hydrosalpinx. When the ends of the Fallopian tubes become obstructed, e.g. from previous infection or endometriosis, normal secretions from cells lining the Fallopian tube accumulate, become stagnant, then leak back into the uterus, where they interfere with implantation. Removing or draining a hydrosalpinx may be a critical first step in promoting normal implantation in these patients. Like hydrosalpinx, there is also evidence that endometriosis disrupts endometrial development, and hence implantation. Medical and/or surgical treatment of endometriosis has been shown to improve pregnancy rates for some patients. Hopefully, as tests of endometrial receptivity, such as the EFT, become more available, the decision of who should undergo removal of their damaged Fallopian tubes or treatment for endometriosis will become a more precise and more individualized process.

Conclusions

Infertility is an emotionally devastating clinical problem for couples who wish to have children. In addition, infertility treatment demands significant time and financial resources from couples facing this disease. Currently, we can evaluate male fertility by testing semen function with direct microscopic examination and sperm function with in vitro fertilization testing. No such tests have been available for endometrial receptivity. Based on awarded and pending patents, the Endometrial Function Test (EFT)™ may be the most efficient way to directly assess endometrial receptivity prior to undergoing expensive assisted reproductive technology procedures.

For more information about EFT contact

harvey.kliman@yale.edu
dkeefe@smtp.wihri.org

or visit http://info.med.yale.edu/obgyn/kliman