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Frozen embryo transfer: evidence-based markers for successful endometrial preparation

2020; Elsevier BV; Volume: 113; Issue: 2 Linguagem: Inglês

10.1016/j.fertnstert.2019.12.008

1556-5653

Robert F. Casper,

Prenatal Screening and Diagnostics

Endometrial receptivity appears to be as important as chromosomal normality in determining embryo implantation and pregnancy outcome, because the ongoing pregnancy rate with transfer of preimplantation genetic testing for aneuploidy euploid blastocysts is ∼50%. This Views and Reviews article focuses on our current knowledge of the biology of endometrial receptivity and on the role of both invasive (endometrial biopsy) and noninvasive (ultrasound) assessment in successful endometrial preparation for frozen-thawed embryo transfer. Endometrial receptivity appears to be as important as chromosomal normality in determining embryo implantation and pregnancy outcome, because the ongoing pregnancy rate with transfer of preimplantation genetic testing for aneuploidy euploid blastocysts is ∼50%. This Views and Reviews article focuses on our current knowledge of the biology of endometrial receptivity and on the role of both invasive (endometrial biopsy) and noninvasive (ultrasound) assessment in successful endometrial preparation for frozen-thawed embryo transfer. Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/56967-29432 Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/56967-29432 For a healthy baby to be born, a chromosomally and otherwise normal embryo needs to implant in a receptive endometrium. Up until a few years ago, the major focus of fertility research was on improving embryo quality and identifying a healthy embryo. However, even with the latest new technology for comprehensive chromosome screening, manuscripts published this year show an ongoing pregnancy rate with euploid embryo transfer of ∼45% for a general infertility population (1Cimadomo D. Soscia D. Vaiarelli A. Maggiulli R. Capalbo A. Ubaldi F.M. Rienzi L. Looking past the appearance: a comprehensive description of the clinical contribution of poor-quality blastocysts to increase live birth rates during cycles with aneuploidy testing.Hum Reprod. 2019; 34: 1206-1214Crossref PubMed Scopus (26) Google Scholar, 2Munné S. Kaplan B. Frattarelli J.L. Child T. Nakhuda G. Shamma F.N. et al.Preimplantation genetic testing for aneuploidy versus morphology as selection criteria for single frozen-thawed embryo transfer in good-prognosis patients: a multicenter randomized clinical trial.Fertil Steril. 2019; 112: 1071-1079.e7Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) and 60% for donor-egg embryos (3Masbou A.K. Friedenthal J.B. McCulloh D.H. McCaffrey C. Fino M.E. Grifo J.A. Licciardi F. A comparison of pregnancy outcomes in patients undergoing donor egg single embryo transfers with and without preimplantation genetic testing.Reprod Sci. 2019; 26: 1661-1665Crossref PubMed Scopus (18) Google Scholar). That means that factors other than chromosomal abnormality are responsible for only 40%–55% of failed pregnancies. More recently, attention has been directed to the endometrium in an attempt to optimize the chance of the embryo adhering to, and implanting in, the uterus. The so-called "window of implantation" (WOI) is a concept that has been proposed to define the period of receptivity of the endometrium for embryo implantation (4Bergh Paul A. Navot D. The impact of embryonic development and endometrial maturity on the timing of implantation.Fertil Steril. 1992; 58: 537-542Abstract Full Text PDF PubMed Google Scholar). To date, the timing, duration, and molecular basis for the WOI remains unclear, although it is thought to last from 2 to 4 days, opening and closing in the midluteal phase (5Wilcox A.J. Baird D.D. Weinberg C.R. Time of implantation of the conceptus and loss of pregnancy.N Engl J Med. 1999; 340: 1796-1799Crossref PubMed Scopus (734) Google Scholar), and several markers associated with a receptive endometrium have been proposed. Endometrial receptivity is a relative concept, with an absent or abnormal WOI associated with recurrent implantation failure and infertility and an altered WOI associated with poor implantation that leads to spontaneous pregnancy loss or possibly preeclampsia. The fact that there may be recurrent failure of the embryo to implant in the endometrium, but pregnancies in ectopic locations (tubal, ovarian, and peritoneal) can occur, suggests that the endometrium may actively block implantation in some cases (6Lessey B.A. Young S.L. What exactly is endometrial receptivity?.Fertil Steril. 2019; 111: 611-617Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). A flurry of recent research has suggested that the timing of the WOI may be shifted in some patients, resulting in the need for individualized embryo transfer. This suggestion is based on endometrial biopsies examining morphologic appearance of the endometrium, biochemical markers of receptivity, and gene microarrays. However, it remains a possibility that in some conditions associated with infertility, the WOI, rather than being advanced or delayed, is actually not present at all, thus making interpretation of these proposed tests for a shift in the timing of the WOI problematic. In the present Views and Reviews article, I examine some of the evidence for and against our present ability to determine when the WOI is actually present. From donor-egg cycles in women with no ovarian function, it is obvious that only sequential estrogen and progesterone is required for the endometrium to be receptive to embryo implantation (7Paulson R.J. Hormonal induction of endometrial receptivity.Fertil Steril. 2011; 213: 530-535Abstract Full Text Full Text PDF Scopus (135) Google Scholar). The methodology for achieving estrogen and progesterone levels adequate for implantation can be quite variable but all appear to be equally effective for pregnancy outcome (8Ghobara T. Gelbaya T.A. Ayeleke R.O. Cycle regimens for frozen-thawed embryo transfer.Cochrane Database Syst Rev. 2017; 7: CD003414PubMed Google Scholar). During the estrogen phase, the endometrium demonstrates linear growth of endometrial glands and blood vessels resulting in the typical trilaminar appearance of the full-thickness endometrium on ultrasound (9Gonen Y. Casper R.F. Prediction of implantation by the sonographic appearance of the endometrium during controlled ovarian stimulation for in vitro fertilization (IVF).J In Vitro Fert Embryo Transf. 1990; 7: 146-152Crossref PubMed Scopus (291) Google Scholar). The proliferation phase ends 2–3 days after ovulation, but there is continuing growth of endometrial glands and vessels under the influence of progesterone within the endometrium, resulting in coiling of the glands and vessels (10Noyes R.W. Hertig A.T. Rock J. Dating the endometrial biopsy.Fertil Steril. 1950; 1: 3-25Abstract Full Text PDF Google Scholar) and glycogen secretion into the glandular lumen. These changes are accompanied by increased proliferation of T-cells, macrophages, and lymphoid nodules (11Tabibzadeh S. Proliferative activity of lymphoid cells in human endometrium throughout the menstrual cycle.J Clin Endocrinol Metab. 1990; 70: 437-443Crossref PubMed Scopus (106) Google Scholar), and all of these effects result in a homogeneous hyperechoic pattern on ultrasound associated with increased endometrial density (9Gonen Y. Casper R.F. Prediction of implantation by the sonographic appearance of the endometrium during controlled ovarian stimulation for in vitro fertilization (IVF).J In Vitro Fert Embryo Transf. 1990; 7: 146-152Crossref PubMed Scopus (291) Google Scholar). The importance of growth in endometrial thickness during the estrogen phase is quite clear. A recent publication by Liu et al. (12Liu K.E. Hartman M. Hartman A. Luo Z.C. Mahutte N. The impact of a thin endometrial lining on fresh and frozen-thaw IVF outcomes: an analysis of over 40 000 embryo transfers.Hum Reprod. 2018; 33: 1883-1888Crossref PubMed Scopus (123) Google Scholar) demonstrated that optimal clinical and ongoing pregnancy rates after in vitro fertilization occurred with endometrial thickness of ≥8 mm at the end of the estrogen phase in more than 24,000 fresh embryo transfer cycles and ≥7 mm in more than 20,000 frozen-thawed embryo transfer (FET) cycles. There was a significant decline in pregnancy rates for each mm decrease of thickness below 8 mm in fresh embryo transfers and below 7 mm in the FETs. The findings of that study are consistent with many other publications on endometrial thickness. Therefore, it appears that any route of administration of estrogen that results in an endometrial thickness of ≥8 mm with a trilaminar appearance on ultrasound will be optimal for embryo implantation. In contrast, the progesterone phase of endometrial receptivity is teeming with controversy. There has been uncertainty regarding the route of progesterone administration (vaginal vs. intramuscular), serum concentrations of progesterone associated with the best pregnancy outcome, and timing of embryo transfer during the progesterone phase. It is surprising that this area is so contentious, because it has been known for more than 30 years that progesterone down-regulates estrogen receptors in the endometrium and that absence of estrogen receptors is required for endometrial receptivity (13Lessey B.A. Killam A.P. Metzger D.A. Haney A.F. Greene G.L. McCarty Jr., K.S. Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle.J Clin Endocrinol Metab. 1988; 67: 334-340Crossref PubMed Scopus (587) Google Scholar). Both increased osteopontin and integrin αvβ3 (14Lessey B.A. Adhesion molecules and implantation.J Reprod Immunol. 2002; 55: 101-112Crossref PubMed Scopus (143) Google Scholar) and decreased cyclin E in the endometrial function test (15Dubowy R.L. Feinberg R.F. Keefe D.L. Doncel G.F. Williams S.C. McSweet J.C. Kliman H.J. Improved endometrial assessment using cyclin E and p27.Fertil Steril. 2003; 80: 146-156Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) reflect absence of estrogen receptor activity and are markers of endometrial receptivity. In some infertile women, it appears that estrogen receptors remain present or that their down-regulation is delayed. Both situations could represent progesterone resistance, which now is one of the principal areas of research into receptivity markers. As discussed in an excellent review article by Lessey and Young (6Lessey B.A. Young S.L. What exactly is endometrial receptivity?.Fertil Steril. 2019; 111: 611-617Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), inflammation is a prime candidate for inducing progesterone resistance. For example, the inflammatory milieu, especially the increase in cyclooxygenase associated with endometriosis, has been shown to increase endometrial aromatase expression with increased estrogen activity and increases in SIRT-1 and Bcl-6, both of which are molecular mediators of progesterone resistance. Estrogen receptors in the endometrium are often not down-regulated completely in women with endometriosis. Thus, inflammation may shift the receptor balance in the endometrium to a more estrogen receptor–positive state, similar to progesterone withdrawal, which would have an anti-implantation effect. Endometrial biopsy with assessment of Bcl-6 and SIRT-1 expression has now been proposed as a test to determine the likelihood of endometriosis or of implantation failure in unexplained infertility as a result of progesterone resistance (16Almquist L.D. Likes C.E. Stone B. Brown K.R. Savaris R. Forstein D.A. et al.Endometrial BCL6 testing for the prediction of in vitro fertilization outcomes: a cohort study.Fertil Steril. 2017; 108: 1063-1069Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Of interest, medical suppression with the use of a GnRH agonist or surgical treatment of endometriosis normalized Bcl-6 expression in the endometrium and improved pregnancy outcomes in these women (17Likes C.E. Cooper L.J. Efird J. Forstein D.A. Miller P.B. Savaris R. Lessey B.A. Medical or surgical treatment before embryo transfer improves outcomes in women with abnormal endometrial Bcl6 expression.J Assist Reprod Genet. 2019; 36: 483-490Crossref PubMed Scopus (14) Google Scholar). A recent development in assessment of the WOI is a multigene microarray together with bioinformatics that has been proposed to identify genetic alterations associated with endometrial receptivity in an endometrial biopsy (18Dıaz-Gimeno P. Horcajadas J.A. Martínez-Conejero J.A. Esteban F.J. Alama P. Pellicer A. Simon C. A genomic diagnostic tool for human endometrial receptivity based on the transcriptomic signature.Fertil Steril. 2011; 95: 50-60Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). The endometrial receptivity array (ERA) test examines the expression of 238 genes thought to be involved in implantation. The goal of this test is to enable customized FET based on the determination of a personalized WOI. In a mock cycle, endometrial biopsy is performed on the 7th day after an LH surge or on the 6th day of progesterone during a hormone replacement (HRT) cycle. Results are expressed as prereceptive, receptive, or postreceptive. If the result is nonreceptive, for example, prereceptive, the embryo replacement timing is delayed in a subsequent cycle, thereby enabling personalized embryo transfer (19Ruiz-Alonso M. Blesa D. Díaz-Gimeno P. Gómez E. Fernández-Sánchez M. Carranza F. et al.The endometrial receptivity array for diagnosis and personalized embryo transfer as a treatment for patients with repeated implantation failure.Fertil Steril. 2013; 100: 818-824Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). The effectiveness of this strategy looks promising based on a small number of samples, but the test has yet to be properly validated in a large randomized study. We performed a study to determine the percentage of good-prognosis infertility patients (0–2 previous failed FETs) who had a nonreceptive endometrium according to the ERA test, and to determine whether adjusting the day of transfer according to the ERA result would improve the pregnancy rate compared with a control group without ERA testing (20Bassil R. Casper R. Samara N. Hsieh T.B. Barzilay E. Orvieto R. Haas J. Does the endometrial receptivity array really provide personalized embryo transfer?.J Assist Reprod Genet. 2018; 35: 1301-1305Crossref PubMed Scopus (54) Google Scholar). We included 53 good-prognosis patients who had a mock cycle with endometrial biopsy and ERA test before their frozen single-blastocyst embryo transfer cycle. In the next cycle, an FET was performed in the study group with the timing of the FET adjusted according to the ERA results. The control group consisted of 503 patients who underwent FET cycles at our clinic during the same period without undergoing the endometrial biopsy and ERA testing. In contrast to the previous publication regarding ERA testing (19Ruiz-Alonso M. Blesa D. Díaz-Gimeno P. Gómez E. Fernández-Sánchez M. Carranza F. et al.The endometrial receptivity array for diagnosis and personalized embryo transfer as a treatment for patients with repeated implantation failure.Fertil Steril. 2013; 100: 818-824Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar), rather than 12% of good-prognosis patients with an altered WOI, 64% of the women in the study group had pre- or postreceptive endometrium on ERA testing. After an adjustment in timing of their actual FET according to the ERA test result, these women with personalized FET had a 39.3% ongoing pregnancy rate, which was not different from the 35.2% ongoing pregnancy rate of the control group. Our results suggested that the ERA test in a mock cycle before an FET did not seem to improve the ongoing pregnancy rate in good-prognosis patients (20Bassil R. Casper R. Samara N. Hsieh T.B. Barzilay E. Orvieto R. Haas J. Does the endometrial receptivity array really provide personalized embryo transfer?.J Assist Reprod Genet. 2018; 35: 1301-1305Crossref PubMed Scopus (54) Google Scholar). Another small study of euploid or donor-egg embryo transfers with or without ERA testing also found no benefit of the test on implantation or pregnancy rates (21Neves A.R. Devesa M. Martínez F. Garcia-Martinez S. Rodriguez I. Polyzos N.P. Coroleu B. What is the clinical impact of the endometrial receptivity array in PGT-A and oocyte donation cycles?.J Assist Reprod Genet. 2019; 36: 1901-1908Crossref PubMed Scopus (25) Google Scholar) A recent study, however, did find an effect of personalized embryo transfer following ERA testing (22Tan J. Kan A. Hitkari J. Taylor B. Tallon N. Warraich G. et al.The role of the endometrial receptivity array (ERA) in patients who have failed euploid embryo transfers.J Assist Reprod Genet. 2018; 35: 683-692Crossref PubMed Scopus (73) Google Scholar). From 2014 to 2017, 48 patients who had failed at least one good-quality euploid embryo transfer underwent ERA testing. About one-fourth of these women were diagnosed as having a nonreceptive endometrium and underwent subsequent adjusted euploid FET based on the ERA result. The implantation rate and ongoing pregnancy rate after personalizing FET in these women were compared with the pregnancy rates in patients who had a receptive endometrium based on ERA testing and underwent standard-timing euploid FET. That study found that implantation and ongoing pregnancy rates were higher (73.7% vs. 54.2% and 63.2% vs. 41.7%, respectively) in women with adjusted timing of FET compared with the women diagnosed as receptive by the ERA test, although the differences were not statistically significant. The authors concluded that those women with failed euploid FET and a receptive endometrium according to ERA testing may have had other undiagnosed causes for their implantation failure. Certainly, more research is needed to validate the usefulness of the ERA test for improving clinical pregnancy rates. The problems with endometrial biopsy tests such as histologic dating, the ERA test, Bcl-6 and SIRT-1 measurement, and the endometrial function test are that they are invasive and the results need to be extrapolated to a subsequent cycle in which the embryo transfer will actually occur. This brings us back to ultrasound, which is noninvasive and can be used in the cycle of interest. Is there some way to improve the effectiveness of ultrasound imaging in detecting endometrial receptivity? There is already evidence that endometrial thickness has good negative predictive ability. That is, an endometrium of <8 mm thickness on the day of hCG trigger in fresh cycles, or <7 mm at the end of the estrogen phase in FETs, is associated with an incremental decrease in ongoing pregnancy for each mm decrease in thickness (12Liu K.E. Hartman M. Hartman A. Luo Z.C. Mahutte N. The impact of a thin endometrial lining on fresh and frozen-thaw IVF outcomes: an analysis of over 40 000 embryo transfers.Hum Reprod. 2018; 33: 1883-1888Crossref PubMed Scopus (123) Google Scholar). In contrast, endometrial thickness measured in the progesterone phase before embryo transfer had no significant value in predicting pregnancy outcome (23Griesinger G. Trevisan S. Cometti B. Endometrial thickness on the day of embryo transfer is a poor predictor of IVF treatment outcome.Hum Reprod Open. 2018; : 1-8Google Scholar, 24Barker M.A. Boehnlein L.M. Kovacs P. Lindheim S.R. Follicular and luteal phase endometrial thickness and echogenic pattern and pregnancy outcome in oocyte donation cycles.J Assist Reprod Genet. 2009; 26: 243-249Crossref PubMed Scopus (58) Google Scholar). However, the change in endometrial thickness between the day of the hCG trigger and the day of embryo transfer has not been examined. We hypothesized that serial ultrasound tests to determine the change in endometrial thickness between the end of the estrogen phase and the time of embryo transfer may be more important to predict pregnancy outcome than the absolute measure of endometrial thickness at either time point alone. Specifically, we hypothesized that the endometrial thickness should decrease in the natural or artificial luteal phase as the endometrium becomes denser (hyperechoic on ultrasound) as a result of the secretory changes that are induced by progesterone. We performed a single-center retrospective observational cohort study of 274 FET cycles in which hormonal preparation with the use of exogenous estrogen and progesterone was used to prepare the endometrium for single warmed blastocyst transfer (25Haas J. Smith R. Zilberberg E. Nayot D. Meriano J. Barzilay E. Casper R.F. Endometrial compaction (decreased thickness) in response to progesterone results in optimal pregnancy outcome in frozen-thawed embryo transfers.Fertil Steril. 2019; 112: 503-509Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). We found that about one-third of women demonstrated a reduction of endometrial thickness from the end of the estrogen phase to the day of embryo transfer (6th day of progesterone). We termed this decrease in thickness "endometrial compaction." In about two-thirds of the cycles, the endometrial thickness increased or stayed the same. Our results demonstrated that patients whose endometrium compacted by 10% after the administration of progesterone had a significantly higher ongoing pregnancy rate (52%) with the use of FET than patients whose endometrium became thicker or didn't change (24%). The data also showed an increase in ongoing pregnancy rates with each quartile increase in the change of endometrial thickness. That is, the greater the degree of compaction, the higher the ongoing pregnancy rate. Continuing growth of the luteal-phase endometrium as observed in the majority of these patients may be caused by a poor response to progesterone. This may not reflect the serum concentration of progesterone, because secretory changes in the endometrium have been found to be equivalent to normal or presumed abnormally low serum progesterone levels (26Usadi R.S. Groll J.M. Lessey B.A. Lininger R.A. Zaino R.J. Fritz M.A. et al.Endometrial development and function in experimentally induced luteal phase deficiency.J Clin Endocrinol Metab. 2008; 93: 4058-4064Crossref PubMed Scopus (68) Google Scholar). A potential cause for the lack of endometrial compaction may be endometrial progesterone resistance (6Lessey B.A. Young S.L. What exactly is endometrial receptivity?.Fertil Steril. 2019; 111: 611-617Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and may perhaps be explained by chronic endometrial inflammation from endometriosis with overexpression of Bcl-6 and SIRT-1 (27Yoo J.-Y. Kim T.H. Fazleabas A.T. Palomino W.A. Ahn S.H. Tayade C. Schammel D.P. et al.KRAS activation and over-expression of SIRT1/Bcl6 contributes to the pathogenesis of endometriosis and progesterone resistance.Sci Rep. 2017; 7: 6765Crossref PubMed Scopus (63) Google Scholar), progesterone receptor gene polymorphisms (28Hu M. Li J. Zhang Y. Li X. Brannstrom M. Shao L.R. et al.Endometrial progesterone receptor isoforms in women with polycystic ovary syndrome.Am J Transl Res. 2018; 10: 2696-2705PubMed Google Scholar), or altered microRNA expression with epigenetic modifications to progesterone receptors (29Patel B.G. Rudnicki M. Yu J. Shu Y. Taylor R.N. Progesterone resistance in endometriosis: origins, consequences and interventions.Acta Obstet Gynecol Scand. 2017; 96: 623-632Crossref PubMed Scopus (125) Google Scholar). It is also possible that the ratio of estrogen to progesterone has an important impact on endometrial growth, especially if shifted to estrogen excess. An alteration in this ratio because of supraphysiologic estrogen levels in stimulated cycles may explain why fresh IVF cycles have lower implantation and pregnancy rates than FET cycles. One could speculate that the high estrogen-to-progesterone ratio in stimulated cycles could prevent endometrial compaction. At present, our studies of endometrial compaction are continuing with inclusion of data from euploid-only FET cycles, natural or modified natural FET cycles, and fresh embryo transfers in stimulated IVF cycles. We have observed the same pattern of response with all of these different treatments, i.e., significantly increased pregnancy rates with endometrial compaction compared with noncompaction. We are now in the process of performing a blinded prospective study in more than 300 cycles of FET. A vaginal ultrasound measurement of endometrial thickness on day 4 of progesterone in HRT-prepared cycles will be performed, but the result will not be disclosed to the physician or patient and the FET will not be performed dependent on the estrogen phase thickness, as is standard practice. This study will be used to validate our results from retrospective studies. In addition, the findings of the new study should provide evidence of whether compaction can be detected before blastocyst warming so that the cycle can be cancelled in the absence of compaction. The observation that endometrial compaction appears to occur in a minority of cycles raises questions of how to improve the endometrium in the majority of patients without endometrial compaction. Possibilities could involve increasing the amount or duration of progesterone, reducing inflammation with the use of a GnRH agonist or other suppression of ovarian function, or blocking estrogen production. Much more research is needed into all of these areas. In summary, endometrial receptivity and its management in assisted reproduction is now a major focus of research interest. Genetic biomarkers and bioinformatic assessment of gene expression microarrays are the current methods of determining the molecular basis for human implantation. Ultrasound imaging still appears to have a role in determining the probability of implantation and ongoing pregnancy. Perhaps the use of artificial intelligence and machine learning applied to imaging or molecular markers will also play a role in future assessment of endometrial receptivity. The information garnered from all of these techniques will lead to informed changes in clinical management that should allow us to reduce the remaining 40%–55% deficit in live birth rates that currently exists with the transfer of euploid embryos.