145
Cryopreserved Embryo Transfer: Endometrial Preparation and Timing Richard Paulson, MD1
1 Division of Reproductive Endocrinology and Infertility, Department
of Obstetrics and Gynecology, Keck School of Medicine of the University of Southern California, Los Angeles, California Semin Reprod Med 2015;33:145–152
Abstract
Keywords
► cryopreserved embryo transfer ► frozen embryo transfer ► endometrial receptivity ► endometrial preparation
Address for correspondence Richard Paulson, MD, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Keck School of Medicine of the University of Southern California, 2020 Zonal Avenue, IRD Room 534, Los Angeles, CA 90033 (e-mail: [email protected]).
The objective of this article was to review and synthesize information from the scientific literature pertaining to the preparation of endometrium for cryopreserved embryo transfer. This article is a critical review of selected scientific literature, synthesis, and formulation of opinion. Estrogen and progesterone are necessary and sufficient to induce endometrial receptivity in cryopreserved embryo transfer cycles. A variety of regimens have been described including natural cycles using endogenous ovarian hormones and artificial or programmed cycles with exogenously administered steroid hormones. To achieve optimal synchrony between embryo and endometrium, the timing of progesterone administration needs to be adjusted to the developmental stage of the thawed embryos. There is currently no evidence that any single regimen or adjuvant substance results in superior outcomes in cryopreserved embryo transfer cycles, although timing of progesterone administration does matter. Although no single regimen of endometrial preparation for cryopreserved embryo transfer has been proven superior to the others, the relative convenience and ease of use do vary, depending on the route of administration chosen and any adjuvant added to the cycle.
Successful implantation following cryopreserved (or frozen) embryo transfer (FET) requires the ability to synchronize preimplantation embryo development with the period of peak endometrial receptivity, often termed the window of implantation (WOI). In a natural menstrual cycle, the timing of this window falls approximately 7 days after ovulation. To produce receptive endometrium, a period of endometrial proliferation stimulated by estradiol must be followed by progesterone exposure to the endometrium. Ovarian steroid hormones accomplish this in a natural menstrual cycle. In artificial cycles using exogenous hormones, estrogen and progesterone alone are sufficient to produce a receptive luteal phase.1 Even so, various adjuvant therapies designed to either suppress ovarian activity or support a thin endometrium have been utilized. Although no single regimen of endometrial preparation for cryopreserved embryo transfer has been proven superior to the others,2 the relative convenience and ease of use do vary, depending on the route of administration chosen and any adjuvant added to the cycle.
Issue Theme Best Practices in In Vitro Fertilization; Guest Editor, Bradley J. Van Voorhis, MD
Cycle regimens for FET overlap significantly with regimens used in recipients of oocyte donation and in women without intrinsic ovarian activity. The first pregnancy achieved in a woman with ovarian failure in 19833 demonstrated that endometrial receptivity could be generated artificially, and allowed separation of the oocyte source and the endometrium. In FET cycles, an ovulatory woman’s own ovarian activity can be utilized to produce receptive endometrium. However, a variety of artificial regimens have also been described that allow greater provider control of timing of embryo transfer. The various routes of administration of estrogen and progesterone are discussed here, as well as dosing principles and timing.
Estradiol: The Proliferative Phase In a natural menstrual cycle, the proliferative phase of the endometrium coincides with the follicular phase of the ovary. Granulosa cells of the dominant follicle produce estradiol (E2)
Copyright © 2015 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0035-1546302. ISSN 1526-8004.
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Heather Burks, MD1
Cryopreserved Embryo Transfer
Burks, Paulson
Fig. 1 Enzymatic metabolism of estradiol (E 2 ) to estrone (E 1) and estrone sulfate (E 1 S). This takes place in the liver as well as peripheral tissues including the endometrium.
in an escalating amount, increasing the serum estradiol up to a peak exceeding 200 to 300 pg/mL in the days just before ovulation.4 The serum level of estradiol has been shown to accurately reflect the amount of estradiol stimulation required to produce the desired proliferative effect in the endometrium.5 Estrogen also “primes” the endometrium for response to subsequent progesterone by inducing the expression of progesterone receptors. Early regimens for FET were designed to mimic this escalating exposure to estradiol.6–9 Available routes of E2 administration include oral, transdermal, intramuscular (IM), or vaginal.
Routes of Administration The oral route of administration is simple, well tolerated, and relatively inexpensive. There is, however, substantial intersubject variability in serum levels following administration.10 This is likely due to variable absorption of micronized estradiol as well as extensive first-pass metabolism, in the small intestinal mucosa followed by the liver. Estradiol is interconverted to estrone (E1) by 17β-hydroxysteroid dehydrogenase, with steady-state levels of E1 reaching three to six times higher than E2.11,12 Compared with E2, E1 is a weaker estrogen with lower binding affinity for both α and β receptors. Estrone is then converted to estrone sulfate (E1S) and back by sulfuryl transferase and sulfatase, respectively (►Fig. 1). This enzymatic activity also takes place in the endometrium, where it is modulated in part by progesterone.11 Despite these metabolic conversions, relatively normal circulating levels of E2 are achieved with oral administration. Parenteral routes of administration—transdermal, IM, or vaginal—circumvent the hepatic first-pass metabolism of E2. For example, transdermally administered E2 is converted less extensively to E1, with E1/E2 ratios near 1.0 to 2.0,13 similar to natural menstrual cycles (►Table 1). The transdermal route also achieves more stable steady-state levels than the other Seminars in Reproductive Medicine
Vol. 33
No. 2/2015
parenteral routes, although actual absorption can be highly variable. An E2 patch with 0.1-mg dosing is analogous to an oral dose of 2 mg. It has been suggested that transdermal is superior to oral administration for inducing endometrial proliferation,14 and may be a reasonable option for women who fail to achieve an adequate response with oral administration. Vaginally administered E2 is well absorbed not only into the circulation but also selectively by the endometrium. High serum levels—approximately eightfold higher than with oral administration—are achieved,15–17 but more notably this appears to be an extremely effective method to deliver E2 right to the tissue of interest,18,19 with even higher endometrial tissue levels observed (80-fold higher).16 Metabolism to the weaker E1 is even less pronounced than in transdermal administration, with E1/E2 ratios of 0.2 to 0.4.15 The vaginal route offers an additional method to achieve adequate proliferation, and is generally reserved for cases in which the oral or transdermal routes were ineffective (►Fig. 2).20
Duration of Administration The proliferative phase in an average natural menstrual cycle is around 14 days, but it can range widely from approximately 7 to 21 days and still result in a normal luteal phase. Likewise, Table 1 Comparison of E1/E2 ratios following various routes of estradiol administration13 Route of administration
E1/E2
Oral
3.0–6.0
Transdermal
1.0–2.0
Vaginal
0.2–0.4
Note: In the natural menstrual cycle, the E1/E2 ratio in the early follicular phase is 1.0 and in the late follicular phase it is 2.0.
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
146
Fig. 2 Relative concentrations of estradiol in serum and endometrium following oral or vaginal administration of 17β-estradiol. Serum levels are reported in pg/mL, whereas endometrial levels are reported in pg/mg protein.
there appears to be a wide range of duration of estrogen administration that can successfully prime the endometrium to respond to progesterone. Among normal responders, a relatively short period of E2 administration (5–7 days) appears to be sufficient to prime the endometrium.6 In contrast, the upper limit of the duration of estrogen administration is not established. Studies evaluating longer artificial follicular phase lengths have shown that up to 5 weeks of estrogen priming can result in optimal luteal phase histology6 and even pregnancy.20 In women with chronic anovulation, pregnancy rates have been shown to be equal, if not improved, when ovulation induction takes place without first inducing a progesterone withdrawal bleed.21 These patients have effectively been in the proliferative phase for any number of months or years. Therefore, women who fail to adequately respond to a short course of E2 may benefit from extending their course.
Assessment of Adequate Proliferation The true measure of an adequate proliferative phase is the successful induction of secretory histology in the secretory phase.8,9,22,23 However, this requires a “dry run” of the preparation regimen in use, with an endometrial biopsy performed around the WOI. This may be done clinically in some situations, but may not be necessary in all patients. An ultrasound measurement of endometrial thickness prior to the initiation of progesterone may have sufficient predictive value to be used as a predictor of adequate E2 priming.24
Progesterone: The Luteal Phase and Endometrial Receptivity Progesterone is a necessary component for achievement of endometrial receptivity. The preceding estrogenic stimulation results in endometrial proliferation and the induction of progesterone (P) receptors. Once exposed to P, either endogenous or exogenous, the endometrium undergoes histologic changes from proliferative to secretory. Structural changes include increased tortuosity of glands and intensified coiling of spiral arteries.25 During the WOI, endometrial expression of many proteins is altered. The loss of epithelial estrogen and progesterone receptors during the WOI is well described. With the advent
Burks, Paulson
of DNA microarray technology26 and proteomics,27 a greater appreciation of the vast number of gene alterations is emerging, but this has added to the difficulty in identifying any single best biomarker of endometrial receptivity. The bestdescribed candidate biomarkers include ανβ3 integrin28 and HOXA10.29 Other potential markers that have been less well studied include MUC1,30–35 trophinin,36 L-selectin ligand,37–40 and heparin-binding EGF-like growth factor (HBEGF).41–44 Currently, there is no single best biomarker for the WOI. Endometrial biopsy specimens exhibiting secretory histologic changes are sufficiently indicative of appropriate response to hormone administration for endometrial preparation in the clinical setting. Serum concentrations of P are higher than E2 throughout the menstrual cycle, even during the follicular phase when estrogen effects on the endometrium are dominant. Compared with serum E2 levels, which range from 20 to 300 pg/mL during the follicular phase, during this same time, serum P levels are approximately 300 to 500 pg/mL, typically reported as 0.3 to 0.5 ng/mL. The precise level of serum P necessary to induce endometrial luteinization has not been established, but it can be assumed to be higher than the 0.5 ng/mL seen in the follicular phase, during which no luteinization takes place. During the natural luteal phase, P levels tend to fluctuate with pulsatile release of luteinizing hormone (LH), but they are typically above 10 ng/mL at the midluteal peak.45 The threshold level of P necessary for luteinization therefore is likely higher than 1 ng/mL and lower than 10 ng/mL. Attempts have been made to experimentally identify the lowest level of P necessary to achieve endometrial luteinization. In one study,46 low levels of P in the range of 5 ng/mL were induced in the experimental group, who showed similar histologic changes to the control group with serum P levels of 19.2 6.6 ng/mL. Markers of endometrial receptivity were also examined, and found to have no difference in expression between the two groups. This study suggests that the threshold for luteinization might be between 1 and 5 ng/mL. A follow-up study by the same investigators47 found that with an even lower serum level of 3 ng/mL; while histologic changes progressed normally, gene expression was altered. Thus it appears that with currently available data, serum levels above 5 ng/mL can be considered sufficient to achieve orderly luteinization and endometrial receptivity, similar to those achieved with higher P levels.
Routes of Administration Because greater doses of P are needed to achieve the necessary serum levels compared with E2, the routes of administration are more limited. Transdermal preparations are not clinically feasible because a prohibitively large surface area would be necessary to absorb sufficient quantities of hormone. Furthermore, P can be metabolized in the skin by 5αreductase. Oral preparations are not absorbed well enough for use in assisted reproduction48 and result in extensive firstpass metabolism. Progesterone can also be administered via intranasal,49 rectal,50 or sublingual routes,51 but bioavailability is low in all cases and none have been shown to be useful in assisted reproduction. Seminars in Reproductive Medicine
Vol. 33
No. 2/2015
147
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Cryopreserved Embryo Transfer
Cryopreserved Embryo Transfer
Burks, Paulson
Vaginal and IM administration of P both result in adequate progesterone levels to accomplish endometrial luteinization and receptivity. The IM route results in higher serum levels, but endometrial tissue levels are higher with vaginal administration. This is because, as with E2, vaginally administered P is preferentially absorbed by the endometrium. No difference in assisted reproductive outcomes has been demonstrated between vaginal or IM progesterone use.
Timing of Administration In order for successful embryo implantation to take place, the embryo and endometrium must be sufficiently synchronized, so that the embryo has achieved the preimplantation stage during the endometrial window of receptivity.52,53 In natural menstrual cycles, implantation occurs on approximately the seventh day following ovulation. Theoretically, timing the administration of progesterone to coincide with embryo development should result in optimal synchrony. Serum P levels start to increase at the end of the follicular phase, reaching approximately 1 ng/mL on the day of ovulation.54 When using exogenous hormones to approximate natural cycles, it is reasonable to consider the first day of progesterone administration as the day ovulation would have occurred to produce the embryo(s) being utilized, or the day after at the latest. This approach results in day 3 embryos being transferred on day 3 or 4 of P, and blastocysts being transferred on day 5 or 6 of P. However, rigid adherence to this timing may not be entirely necessary, as successful pregnancies have resulted from the transfer of cleavage stage embryos on days 1 to 6 of progesterone administration.55,56 This suggests that the WOI in humans may be quite large, although the exact duration is not known. It does, however, seem reasonable to most closely approximate normal physiologic timing. Clinical evidence has yielded mixed results when investigating the exact timing of embryo transfer and subsequent outcomes. Transfer of cryopreserved blastocysts on day 5 of P achieved a reasonable pregnancy rate,57 as might be anticipated. Other contrasting evidence comes largely from oocyte donation programs utilizing endometrial preparation for fresh donor embryo transfer. One study in donor oocyte recipients transferred day 2 embryos on days 2, 3, 4, 5, or 6 of P administration. No pregnancies were seen on day 2 or 3 transfers, but rather optimal pregnancy rates were seen from transfers on day 4 or 5. The authors interpreted these findings to indicate that the WOI begins 48 hours after starting progesterone and extends for 4 days.58 A 2010 Cochrane review59 found that in oocyte donation cycles, pregnancy rates are best when P is begun on the day of oocyte retrieval or 1 day after, compared with cycles when P is initiated the day prior to oocyte retrieval. This would indicate that day 3 embryos likely have the best chance of implantation on day 3 or 4 of P, as would be anticipated by analogous timing in natural cycles.
Estrogen in the Luteal Phase In the natural luteal phase, the corpus luteum produces E2 in addition to P. When estrogen activity is antagonized in the Seminars in Reproductive Medicine
Vol. 33
No. 2/2015
luteal phase, disrupted luteinization occurs,60 so some E2 is likely necessary. However, comparing programmed cycles in women without endogenous ovarian function, normal histology was found in women who continued transdermal E2 through the luteal as well as those who did not.61 The women without luteal phase E2 did have earlier vaginal bleeding, but otherwise no differences were noted. A similar study62 using oral E2 found that despite significantly higher serum E2 levels among women randomized to continue E2 in the luteal phase, no differences in endometrial histology were noted. In normally cycling women with pituitary suppression using gonadotropin-releasing hormone (GnRH) agonists, similar findings were noted between groups using no E2, physiologic E2 supplementation, or supraphysiologic E2 supplementation alongside IM P in the luteal phase.63 No histologic differences were noted, nor were there differences in putative biomarkers of endometrial function. Additionally, the group receiving no exogenous E2 had a serum level of 21.9 pg/mL of E2 and 22 ng/mL of P. Taken together, these results indicate that very low levels of serum E2 are sufficient for normal luteinization of the endometrium. Given the presence of low levels of E2 among women receiving only P, exogenously administered P is likely metabolized to E2, at a conversion rate that can be calculated to approximately 0.1%.
Strategies for Inadequate Endometrial Response As previously mentioned, ultrasound measurement of endometrial thickness has been identified as a good predictor of adequate endometrial proliferation and priming. There is also some evidence that endometrial thickness correlates with outcomes in assisted reproduction, with thinner endometrium resulting in lower pregnancy rates.64,65 This evidence comes primarily from studies of FETs after controlled ovarian stimulation cycles or in recipients of oocyte donation,66,67 and has been extrapolated to FET cycles clinically. El Toukhy et al68 found that the highest FET cycle implantation and pregnancy rates are achieved at endometrial thicknesses of 9 to 14 mm, which was statistically significantly higher than cycles with thickness of 7 to 8 mm. Fresh cycle endometrial thickness has also been shown to predict subsequent endometrial thickness during endometrial preparation for FET,69 indicating that extrapolating IVF data to FET cycles may be reasonable. A recent systematic review and meta-analysis by Kasius et al investigated 22 studies reporting on the relationship between endometrial thickness and pregnancy rates in IVF.70 The authors concluded that although pregnancy rates were lower among women with endometrial thickness 7 mm or less, the ability of this parameter to identify women with a low chance of conception is limited. Supporting this conclusion are multiple reports in the literature of successful pregnancies and live births in women with thin endometrium. One woman gave birth to twins after oocyte donation and embryo transfer with endometrium less than 4 mm,71 whereas another gave birth after FET with endometrial thickness of 5 mm.72
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
148
Nonetheless, an endometrial thickness of 7 or 8 mm is frequently considered adequate for embryo transfer in clinical practice.24 One approach to improving endometrial thickness in inadequate responders is to extend the duration of E2 administration. Often, this is done through additional vaginal administration, to improve delivery of E2 to the endometrium.17 Tourgeman et al20 found that extending vaginal E2 administration 4 to 6 weeks increased endometrial thickness to at least 7 mm and achieved an ongoing pregnancy rate of 70%. Liao et al73 compared additional oral estradiol valerate to the addition of vaginal estradiol in patients planning FET whose endometrium was thinner than 8 mm on the 13th day of oral estradiol valerate, in a nonrandomized study. Vaginal administration increased the endometrial thickness to a greater extent, but both groups had improvement and were able to achieve comparable implantation rates (46 vs. 50%, not significant) (►Fig. 2). Low-dose aspirin has been proposed as a possible adjuvant therapy to improve endometrial thickness by improving blood flow to the endometrium. This is based on a study from 1994 by Wada et al,74 in which women found to have poor uterine perfusion by Doppler assessment were given aspirin in either 150- or 300-mg doses. Aspirin was found to improve uterine perfusion in these women and to improve pregnancy rates. Although this intervention has not been studied in FET cycles, Weckstein et al75 studied low-dose aspirin in 28 recipients of oocyte donation with a history of thin endometrium (
Cryopreserved Embryo Transfer: Endometrial Preparation and Timing Richard Paulson, MD1
1 Division of Reproductive Endocrinology and Infertility, Department
of Obstetrics and Gynecology, Keck School of Medicine of the University of Southern California, Los Angeles, California Semin Reprod Med 2015;33:145–152
Abstract
Keywords
► cryopreserved embryo transfer ► frozen embryo transfer ► endometrial receptivity ► endometrial preparation
Address for correspondence Richard Paulson, MD, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Keck School of Medicine of the University of Southern California, 2020 Zonal Avenue, IRD Room 534, Los Angeles, CA 90033 (e-mail: [email protected]).
The objective of this article was to review and synthesize information from the scientific literature pertaining to the preparation of endometrium for cryopreserved embryo transfer. This article is a critical review of selected scientific literature, synthesis, and formulation of opinion. Estrogen and progesterone are necessary and sufficient to induce endometrial receptivity in cryopreserved embryo transfer cycles. A variety of regimens have been described including natural cycles using endogenous ovarian hormones and artificial or programmed cycles with exogenously administered steroid hormones. To achieve optimal synchrony between embryo and endometrium, the timing of progesterone administration needs to be adjusted to the developmental stage of the thawed embryos. There is currently no evidence that any single regimen or adjuvant substance results in superior outcomes in cryopreserved embryo transfer cycles, although timing of progesterone administration does matter. Although no single regimen of endometrial preparation for cryopreserved embryo transfer has been proven superior to the others, the relative convenience and ease of use do vary, depending on the route of administration chosen and any adjuvant added to the cycle.
Successful implantation following cryopreserved (or frozen) embryo transfer (FET) requires the ability to synchronize preimplantation embryo development with the period of peak endometrial receptivity, often termed the window of implantation (WOI). In a natural menstrual cycle, the timing of this window falls approximately 7 days after ovulation. To produce receptive endometrium, a period of endometrial proliferation stimulated by estradiol must be followed by progesterone exposure to the endometrium. Ovarian steroid hormones accomplish this in a natural menstrual cycle. In artificial cycles using exogenous hormones, estrogen and progesterone alone are sufficient to produce a receptive luteal phase.1 Even so, various adjuvant therapies designed to either suppress ovarian activity or support a thin endometrium have been utilized. Although no single regimen of endometrial preparation for cryopreserved embryo transfer has been proven superior to the others,2 the relative convenience and ease of use do vary, depending on the route of administration chosen and any adjuvant added to the cycle.
Issue Theme Best Practices in In Vitro Fertilization; Guest Editor, Bradley J. Van Voorhis, MD
Cycle regimens for FET overlap significantly with regimens used in recipients of oocyte donation and in women without intrinsic ovarian activity. The first pregnancy achieved in a woman with ovarian failure in 19833 demonstrated that endometrial receptivity could be generated artificially, and allowed separation of the oocyte source and the endometrium. In FET cycles, an ovulatory woman’s own ovarian activity can be utilized to produce receptive endometrium. However, a variety of artificial regimens have also been described that allow greater provider control of timing of embryo transfer. The various routes of administration of estrogen and progesterone are discussed here, as well as dosing principles and timing.
Estradiol: The Proliferative Phase In a natural menstrual cycle, the proliferative phase of the endometrium coincides with the follicular phase of the ovary. Granulosa cells of the dominant follicle produce estradiol (E2)
Copyright © 2015 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0035-1546302. ISSN 1526-8004.
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Heather Burks, MD1
Cryopreserved Embryo Transfer
Burks, Paulson
Fig. 1 Enzymatic metabolism of estradiol (E 2 ) to estrone (E 1) and estrone sulfate (E 1 S). This takes place in the liver as well as peripheral tissues including the endometrium.
in an escalating amount, increasing the serum estradiol up to a peak exceeding 200 to 300 pg/mL in the days just before ovulation.4 The serum level of estradiol has been shown to accurately reflect the amount of estradiol stimulation required to produce the desired proliferative effect in the endometrium.5 Estrogen also “primes” the endometrium for response to subsequent progesterone by inducing the expression of progesterone receptors. Early regimens for FET were designed to mimic this escalating exposure to estradiol.6–9 Available routes of E2 administration include oral, transdermal, intramuscular (IM), or vaginal.
Routes of Administration The oral route of administration is simple, well tolerated, and relatively inexpensive. There is, however, substantial intersubject variability in serum levels following administration.10 This is likely due to variable absorption of micronized estradiol as well as extensive first-pass metabolism, in the small intestinal mucosa followed by the liver. Estradiol is interconverted to estrone (E1) by 17β-hydroxysteroid dehydrogenase, with steady-state levels of E1 reaching three to six times higher than E2.11,12 Compared with E2, E1 is a weaker estrogen with lower binding affinity for both α and β receptors. Estrone is then converted to estrone sulfate (E1S) and back by sulfuryl transferase and sulfatase, respectively (►Fig. 1). This enzymatic activity also takes place in the endometrium, where it is modulated in part by progesterone.11 Despite these metabolic conversions, relatively normal circulating levels of E2 are achieved with oral administration. Parenteral routes of administration—transdermal, IM, or vaginal—circumvent the hepatic first-pass metabolism of E2. For example, transdermally administered E2 is converted less extensively to E1, with E1/E2 ratios near 1.0 to 2.0,13 similar to natural menstrual cycles (►Table 1). The transdermal route also achieves more stable steady-state levels than the other Seminars in Reproductive Medicine
Vol. 33
No. 2/2015
parenteral routes, although actual absorption can be highly variable. An E2 patch with 0.1-mg dosing is analogous to an oral dose of 2 mg. It has been suggested that transdermal is superior to oral administration for inducing endometrial proliferation,14 and may be a reasonable option for women who fail to achieve an adequate response with oral administration. Vaginally administered E2 is well absorbed not only into the circulation but also selectively by the endometrium. High serum levels—approximately eightfold higher than with oral administration—are achieved,15–17 but more notably this appears to be an extremely effective method to deliver E2 right to the tissue of interest,18,19 with even higher endometrial tissue levels observed (80-fold higher).16 Metabolism to the weaker E1 is even less pronounced than in transdermal administration, with E1/E2 ratios of 0.2 to 0.4.15 The vaginal route offers an additional method to achieve adequate proliferation, and is generally reserved for cases in which the oral or transdermal routes were ineffective (►Fig. 2).20
Duration of Administration The proliferative phase in an average natural menstrual cycle is around 14 days, but it can range widely from approximately 7 to 21 days and still result in a normal luteal phase. Likewise, Table 1 Comparison of E1/E2 ratios following various routes of estradiol administration13 Route of administration
E1/E2
Oral
3.0–6.0
Transdermal
1.0–2.0
Vaginal
0.2–0.4
Note: In the natural menstrual cycle, the E1/E2 ratio in the early follicular phase is 1.0 and in the late follicular phase it is 2.0.
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
146
Fig. 2 Relative concentrations of estradiol in serum and endometrium following oral or vaginal administration of 17β-estradiol. Serum levels are reported in pg/mL, whereas endometrial levels are reported in pg/mg protein.
there appears to be a wide range of duration of estrogen administration that can successfully prime the endometrium to respond to progesterone. Among normal responders, a relatively short period of E2 administration (5–7 days) appears to be sufficient to prime the endometrium.6 In contrast, the upper limit of the duration of estrogen administration is not established. Studies evaluating longer artificial follicular phase lengths have shown that up to 5 weeks of estrogen priming can result in optimal luteal phase histology6 and even pregnancy.20 In women with chronic anovulation, pregnancy rates have been shown to be equal, if not improved, when ovulation induction takes place without first inducing a progesterone withdrawal bleed.21 These patients have effectively been in the proliferative phase for any number of months or years. Therefore, women who fail to adequately respond to a short course of E2 may benefit from extending their course.
Assessment of Adequate Proliferation The true measure of an adequate proliferative phase is the successful induction of secretory histology in the secretory phase.8,9,22,23 However, this requires a “dry run” of the preparation regimen in use, with an endometrial biopsy performed around the WOI. This may be done clinically in some situations, but may not be necessary in all patients. An ultrasound measurement of endometrial thickness prior to the initiation of progesterone may have sufficient predictive value to be used as a predictor of adequate E2 priming.24
Progesterone: The Luteal Phase and Endometrial Receptivity Progesterone is a necessary component for achievement of endometrial receptivity. The preceding estrogenic stimulation results in endometrial proliferation and the induction of progesterone (P) receptors. Once exposed to P, either endogenous or exogenous, the endometrium undergoes histologic changes from proliferative to secretory. Structural changes include increased tortuosity of glands and intensified coiling of spiral arteries.25 During the WOI, endometrial expression of many proteins is altered. The loss of epithelial estrogen and progesterone receptors during the WOI is well described. With the advent
Burks, Paulson
of DNA microarray technology26 and proteomics,27 a greater appreciation of the vast number of gene alterations is emerging, but this has added to the difficulty in identifying any single best biomarker of endometrial receptivity. The bestdescribed candidate biomarkers include ανβ3 integrin28 and HOXA10.29 Other potential markers that have been less well studied include MUC1,30–35 trophinin,36 L-selectin ligand,37–40 and heparin-binding EGF-like growth factor (HBEGF).41–44 Currently, there is no single best biomarker for the WOI. Endometrial biopsy specimens exhibiting secretory histologic changes are sufficiently indicative of appropriate response to hormone administration for endometrial preparation in the clinical setting. Serum concentrations of P are higher than E2 throughout the menstrual cycle, even during the follicular phase when estrogen effects on the endometrium are dominant. Compared with serum E2 levels, which range from 20 to 300 pg/mL during the follicular phase, during this same time, serum P levels are approximately 300 to 500 pg/mL, typically reported as 0.3 to 0.5 ng/mL. The precise level of serum P necessary to induce endometrial luteinization has not been established, but it can be assumed to be higher than the 0.5 ng/mL seen in the follicular phase, during which no luteinization takes place. During the natural luteal phase, P levels tend to fluctuate with pulsatile release of luteinizing hormone (LH), but they are typically above 10 ng/mL at the midluteal peak.45 The threshold level of P necessary for luteinization therefore is likely higher than 1 ng/mL and lower than 10 ng/mL. Attempts have been made to experimentally identify the lowest level of P necessary to achieve endometrial luteinization. In one study,46 low levels of P in the range of 5 ng/mL were induced in the experimental group, who showed similar histologic changes to the control group with serum P levels of 19.2 6.6 ng/mL. Markers of endometrial receptivity were also examined, and found to have no difference in expression between the two groups. This study suggests that the threshold for luteinization might be between 1 and 5 ng/mL. A follow-up study by the same investigators47 found that with an even lower serum level of 3 ng/mL; while histologic changes progressed normally, gene expression was altered. Thus it appears that with currently available data, serum levels above 5 ng/mL can be considered sufficient to achieve orderly luteinization and endometrial receptivity, similar to those achieved with higher P levels.
Routes of Administration Because greater doses of P are needed to achieve the necessary serum levels compared with E2, the routes of administration are more limited. Transdermal preparations are not clinically feasible because a prohibitively large surface area would be necessary to absorb sufficient quantities of hormone. Furthermore, P can be metabolized in the skin by 5αreductase. Oral preparations are not absorbed well enough for use in assisted reproduction48 and result in extensive firstpass metabolism. Progesterone can also be administered via intranasal,49 rectal,50 or sublingual routes,51 but bioavailability is low in all cases and none have been shown to be useful in assisted reproduction. Seminars in Reproductive Medicine
Vol. 33
No. 2/2015
147
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Cryopreserved Embryo Transfer
Cryopreserved Embryo Transfer
Burks, Paulson
Vaginal and IM administration of P both result in adequate progesterone levels to accomplish endometrial luteinization and receptivity. The IM route results in higher serum levels, but endometrial tissue levels are higher with vaginal administration. This is because, as with E2, vaginally administered P is preferentially absorbed by the endometrium. No difference in assisted reproductive outcomes has been demonstrated between vaginal or IM progesterone use.
Timing of Administration In order for successful embryo implantation to take place, the embryo and endometrium must be sufficiently synchronized, so that the embryo has achieved the preimplantation stage during the endometrial window of receptivity.52,53 In natural menstrual cycles, implantation occurs on approximately the seventh day following ovulation. Theoretically, timing the administration of progesterone to coincide with embryo development should result in optimal synchrony. Serum P levels start to increase at the end of the follicular phase, reaching approximately 1 ng/mL on the day of ovulation.54 When using exogenous hormones to approximate natural cycles, it is reasonable to consider the first day of progesterone administration as the day ovulation would have occurred to produce the embryo(s) being utilized, or the day after at the latest. This approach results in day 3 embryos being transferred on day 3 or 4 of P, and blastocysts being transferred on day 5 or 6 of P. However, rigid adherence to this timing may not be entirely necessary, as successful pregnancies have resulted from the transfer of cleavage stage embryos on days 1 to 6 of progesterone administration.55,56 This suggests that the WOI in humans may be quite large, although the exact duration is not known. It does, however, seem reasonable to most closely approximate normal physiologic timing. Clinical evidence has yielded mixed results when investigating the exact timing of embryo transfer and subsequent outcomes. Transfer of cryopreserved blastocysts on day 5 of P achieved a reasonable pregnancy rate,57 as might be anticipated. Other contrasting evidence comes largely from oocyte donation programs utilizing endometrial preparation for fresh donor embryo transfer. One study in donor oocyte recipients transferred day 2 embryos on days 2, 3, 4, 5, or 6 of P administration. No pregnancies were seen on day 2 or 3 transfers, but rather optimal pregnancy rates were seen from transfers on day 4 or 5. The authors interpreted these findings to indicate that the WOI begins 48 hours after starting progesterone and extends for 4 days.58 A 2010 Cochrane review59 found that in oocyte donation cycles, pregnancy rates are best when P is begun on the day of oocyte retrieval or 1 day after, compared with cycles when P is initiated the day prior to oocyte retrieval. This would indicate that day 3 embryos likely have the best chance of implantation on day 3 or 4 of P, as would be anticipated by analogous timing in natural cycles.
Estrogen in the Luteal Phase In the natural luteal phase, the corpus luteum produces E2 in addition to P. When estrogen activity is antagonized in the Seminars in Reproductive Medicine
Vol. 33
No. 2/2015
luteal phase, disrupted luteinization occurs,60 so some E2 is likely necessary. However, comparing programmed cycles in women without endogenous ovarian function, normal histology was found in women who continued transdermal E2 through the luteal as well as those who did not.61 The women without luteal phase E2 did have earlier vaginal bleeding, but otherwise no differences were noted. A similar study62 using oral E2 found that despite significantly higher serum E2 levels among women randomized to continue E2 in the luteal phase, no differences in endometrial histology were noted. In normally cycling women with pituitary suppression using gonadotropin-releasing hormone (GnRH) agonists, similar findings were noted between groups using no E2, physiologic E2 supplementation, or supraphysiologic E2 supplementation alongside IM P in the luteal phase.63 No histologic differences were noted, nor were there differences in putative biomarkers of endometrial function. Additionally, the group receiving no exogenous E2 had a serum level of 21.9 pg/mL of E2 and 22 ng/mL of P. Taken together, these results indicate that very low levels of serum E2 are sufficient for normal luteinization of the endometrium. Given the presence of low levels of E2 among women receiving only P, exogenously administered P is likely metabolized to E2, at a conversion rate that can be calculated to approximately 0.1%.
Strategies for Inadequate Endometrial Response As previously mentioned, ultrasound measurement of endometrial thickness has been identified as a good predictor of adequate endometrial proliferation and priming. There is also some evidence that endometrial thickness correlates with outcomes in assisted reproduction, with thinner endometrium resulting in lower pregnancy rates.64,65 This evidence comes primarily from studies of FETs after controlled ovarian stimulation cycles or in recipients of oocyte donation,66,67 and has been extrapolated to FET cycles clinically. El Toukhy et al68 found that the highest FET cycle implantation and pregnancy rates are achieved at endometrial thicknesses of 9 to 14 mm, which was statistically significantly higher than cycles with thickness of 7 to 8 mm. Fresh cycle endometrial thickness has also been shown to predict subsequent endometrial thickness during endometrial preparation for FET,69 indicating that extrapolating IVF data to FET cycles may be reasonable. A recent systematic review and meta-analysis by Kasius et al investigated 22 studies reporting on the relationship between endometrial thickness and pregnancy rates in IVF.70 The authors concluded that although pregnancy rates were lower among women with endometrial thickness 7 mm or less, the ability of this parameter to identify women with a low chance of conception is limited. Supporting this conclusion are multiple reports in the literature of successful pregnancies and live births in women with thin endometrium. One woman gave birth to twins after oocyte donation and embryo transfer with endometrium less than 4 mm,71 whereas another gave birth after FET with endometrial thickness of 5 mm.72
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
148
Nonetheless, an endometrial thickness of 7 or 8 mm is frequently considered adequate for embryo transfer in clinical practice.24 One approach to improving endometrial thickness in inadequate responders is to extend the duration of E2 administration. Often, this is done through additional vaginal administration, to improve delivery of E2 to the endometrium.17 Tourgeman et al20 found that extending vaginal E2 administration 4 to 6 weeks increased endometrial thickness to at least 7 mm and achieved an ongoing pregnancy rate of 70%. Liao et al73 compared additional oral estradiol valerate to the addition of vaginal estradiol in patients planning FET whose endometrium was thinner than 8 mm on the 13th day of oral estradiol valerate, in a nonrandomized study. Vaginal administration increased the endometrial thickness to a greater extent, but both groups had improvement and were able to achieve comparable implantation rates (46 vs. 50%, not significant) (►Fig. 2). Low-dose aspirin has been proposed as a possible adjuvant therapy to improve endometrial thickness by improving blood flow to the endometrium. This is based on a study from 1994 by Wada et al,74 in which women found to have poor uterine perfusion by Doppler assessment were given aspirin in either 150- or 300-mg doses. Aspirin was found to improve uterine perfusion in these women and to improve pregnancy rates. Although this intervention has not been studied in FET cycles, Weckstein et al75 studied low-dose aspirin in 28 recipients of oocyte donation with a history of thin endometrium (
