Abstract
This study aimed to assess frozen-thawed embryo transfer (FET) outcomes in natural, hormone replacement treatment (HRT) and semi-HRT cycles. This was a retrospective cohort study of 5414 cycles of patients in an academic hospital. Patients were grouped as 2216 natural cycles, 1180 semi-HRT cycles, and 2018 HRT cycles. Primary outcome measures were implantation rate, clinical pregnancy rate and live birth rate. Other parameters, such as peak endometria-thickness, were also analyzed. Patients undergoing FET with HRT obtained higher implantation rate and clinical pregnancy rate than patients with natural or semi-HR cycles (29.3% vs. 21.5% vs. 25.6%, P=0.01, and 48.7% vs. 42.7% vs. 36.1%, P=0.01, respectively). This finding was not changed in patients with thin endometrium (≤8 mm). A Subanalysis in patients with HRT showed that the implantation and clinical pregnancy rate was higher in patients without ovulation than ovulatory patients (29.8% vs. 16.9%, P<0.01, and 49.5% vs. 26.3%, P<0.01, respectively). This study suggests that HRT increases the possibility of pregnancy. Further, our data showed that ovulation in HRT cycle has a detrimental effect on pregnancy. Therefore, we recommend that HRT should be used in FET cycles, and ovulation of patients should be evaluated during the treatment.
Keywords: Frozen-thawed embryo transfer, natural cycle, hormone replacement treatment, implantation rate, clinical pregnancy rate
Introduction
The first successful pregnancy after use of frozen-thawed embryo transfer (FET) was reported in 1983 [1]. Thereafter, FET treatment was rapidly expanded. FET can effectively prevent IVF associated complications, such as ovarian hyperstimulation syndrome and multiple births. In addition, FET serves as an safe and cost-effective way to increase cumulative pregnancy rate [2]. Further, FET enables a strategy of supporting an elective single embryo transfer policy, which has been adapted in many countries. In 2006, FET has accounted for around 20% of all assisted reproductive technology cycles in Europe [3].
Embryo implantation is an important step in human reproduction. A successful implantation depends on the reciprocal interaction between the embryo and receptive uterus [4,5]. Some parameters have been reported as prognostics in evaluating endometrial receptivity, such as endometrial volume, endometrial thickness, and artery blood flow [6]. Numerous biomarkers were also found to be associated with endometrial receptivity, including LIF, Integrin, and HOXA10 [7-9]. Due to the important role of receptive endometrium, various endometrial preparation protocols are applied, aiming to improve FET success. FET has been successfully performed in natural cycles after spontaneous ovulation and in hormone replacement treatment (HRT) cycles [10-12]. Previous studies were performed to compare FET outcomes in different endometrial preparation protocols. However, the results still remain conflicting. Therefore, the present study was conducted to explore the optimal endometrial preparation protocol in FET treatment.
Materials and methods
Patients
This was a retrospective, non-interventional, single-center cohort study of patients undergoing FET treatment at reproductive medicine center in Tongji hospital, Wuhan, China, between January 2008 and December 2011. A total of 5414 FET cycles were enrolled. In order to avoid potential bias, only the first two FET cycles of each patient was included. Non-autologous and cancelled cycles were excluded. Institutional Review Board (IRB) approval was not necessary, because all women in the present study underwent the routine FET treatment in our center and no additional intervention or sampling was performed.
Natural cycles
Transvaginal ultrasound scan and measurement of serum progesterone level were initiated from cycle day 10-12 to assess endometrial thickness, follicle growth, and ovulation. FET was planned 3 days after ovulation, indicated by a serum progesterone >5 ng/mL. Embryo transfer was performed 1 day after collapse, and progesterone administration was commenced as luteal support.
Semi-HRT cycles
Oral estradiol Valerate (Progynova, Bayer, Germany) were administrated from the cycle day 10-12, dosage of estradiol was adjusted according to the endometrial-thickness assessed by transvaginal ultrasound. Administration of 40 mg progesterone (progesterone injection, Sansheng, China) intramuscularly was given when the endometrium reached a thickness of 8 mm or maximum. Administration with 60 mg, 80 mg and 80 mg progesterone were used respectively in the following 3 days. Embryos were collapsed and transferred after 4 days of progesterone administration.
HRT cycles
Oral estradiol was taken at 2 mg/d from cycle day 1-4, 4 mg/d from day 5-8, 6 mg/d from day 9-12. Transvaginal ultrasound scan was performed to assess endometrial-thickness and ovulation from day 13 and estradiol dosage was adjusted based on the endometrial-thickness. Administration with 40 mg progesterone intramuscularly was given when the endometrium reached a thickness of 8 mm or maximum. Administration with 60 mg, 80 mg, and 80 mg progesterone were used respectively in the following 3 days. Embryo transfer was performed after 4 days of progesterone administration.
FET protocols
Day 3 embryos were classified as Grade 1 to Grade 4, according to cleavage stage, blastomere size and shape, and fragmentation [13]. The assessment criteria of blastocyst quality were based on blastocoele cavity, inner cell mass, trophectodermal cells, and volume of zona pellucida [14]. Surplus embryos in fresh cycles were cryopreserved for subsequent FET cycles. Cryopreserve and thawing of the embryos were implemented as previously published [15].
Pregnancy outcomes
In the present study, biochemical pregnancy was defined as a serum hCG >20 IU/L 2 weeks after transfer but then declined to negative. Pregnancy was defined as a serum hCG level >20 IU/L and confirmed by observation of gestational sac on transvaginal ultrasound scan 5-7 weeks after transfer. Implantation rate was defined as the number of gestational sacs present on ultrasound divided by the number of embryos transferred. All pregnant women were followed up to obtain the delivery data.
Statistical analysis
The continuous data were given as mean and SD. Groups were compared with one-way analysis of variance (ANOVA) with Bonferroni adjustment or student’s t-test as appropriate. Categorical variables were presented as number and percentage. Differences were evaluated with chi-square test and the Fisher exact test. A P value <.05 was considered statistical significant. SPSS version 13.0 (SPSS Inc.) was used for statistical analysis.
Results
Demographic data were presented in Table 1. The age and BMI were higher in natural cycle group than those in semi-HRT and HRT Group, whereas these differences were small. No differences were found in duration of infertility, infertility type, and basal FSH level.
Table 1.
Demographics
| Natural cycle | Semi-HRT cycle | HRT cycle | |
|---|---|---|---|
| No. of cycles | 2216 | 1180 | 2018 |
| Mean age (year) | 31.3±3.8a | 30.8±3.8 | 30.8±4.0 |
| BMI (kg/m2) | 22.6±3.3b | 22.3±3.7 | 22.4±3.5 |
| Duration of infertility (year) | 4.9±3.3 | 4.8±2.8 | 4.8±3.1 |
| Primary infertility (%) | 49.2 (1090/2216) | 48.9 (577/1180) | 50.1 (1011/2018) |
| Basal FSH (mIU/mL) | 5.7±2.2 | 5.6±2.2 | 5.8±2.0 |
P<0.01;
P=0.04.
FET parameters and outcomes were shown in Table 2. There were no differences in delay between cycles and the number of embryo transferred. The peak endometrial-thickness was higher in patients with natural cycles (10.4±4.2, P<0.01), as compared to patients with semi-HRT cycles (9.0±2.5) and HRT cycles (9.0±2.1). As for FET clinical outcomes, patients in the three group obtained comparable biochemical pregnancy rate. Patients undergoing HRT obtained higher implantation rate (29.3%, P=0.01) and clinical pregnancy rate (48.7%, P=0.01) than patients in natural cycle (21.5% and 36.1%, respectively) and semi-HRT (25.6% and 42.7%, respectively) group. No difference was found regarding live birth rate among the three groups. Similar results were found in patients with thin endometrial thickness ≤8 mm on the day of transfer (Table 3). Patients undergoing HRT obtained elevated implantation rate and clinical pregnancy rate than patients with natural cycles and semi-HRT cycles (27.8% vs. 22.1% vs. 19.9%, P<0.01, and 47.6% vs. 33.7% vs. 34.4%, P<0.01, respectively).
Table 2.
FET characteristics and outcomes
| Natural cycle | Semi-HRT cycle | HRT cycle | |
|---|---|---|---|
| No. of cycles | 2216 | 1180 | 2018 |
| Delay between cycles (months) | 5.6±2.7 | 5.2±2.0 | 5.3±2.5 |
| Peak endometria thickness (mm) | 10.4±4.2a | 9.0±2.5 | 9.0±2.1 |
| No. of embryo transferred | 2.1±0.5 | 2.0±0.5 | 2.0±0.5 |
| Biochemical pregnancy rate (%) | 3.3 (74/2216) | 3.6 (43/1180) | 3.9 (79/2018) |
| Implantation rate (%) | 21.5 (977/4552) | 25.6 (616/2408) | 29.3 (1197/4080)b |
| Clinical Pregnancy rate (%) | 36.1 (801/2216) | 42.7 (504/1180) | 48.7 (982/2018)b |
| Live birth rate (%) | 85.7 (837/977) | 84.3 (519/616) | 87.0 (1041/1197) |
P<0.01;
P=0.01.
Table 3.
FET results in patients with endometrial-thickness ≤8 mm
| Natural cycle | Semi-HRT cycle | HRT cycle | |
|---|---|---|---|
| No. of cycles | 199 | 302 | 502 |
| Delay between treatments (months) | 5.5±5.2 | 5.7±4.5 | 5.5±5.3 |
| Endometrial thickness (mm) | 7.4±0.7 | 7.4±0.6 | 7.4±0.7 |
| No. of embryo transferred | 2.0±0.5 | 2.0±0.5 | 2.0±0.5 |
| Implantation rate (%) | 22.1 (86/389) | 19.9 (123/617) | 27.8 (281/1012)a |
| Clinical pregnancy rate (%) | 33.7 (67/199) | 34.4 (104/302) | 47.6 (239/502)a |
| Live birth rate (%) | 77.9 (67/86) | 79.7 (98/123) | 86.5 (243/281) |
P<0.01.
A subanalysis was performed on patients undergoing HRT. Dividing patients into patients with and without ovulation, our data showed that implantation rate and clinical pregnancy rate in anovulatory patients were higher than patients with ovulation (29.8% vs. 16.9%, P<0.01 and 49.5% vs. 26.3%, P<0.01, respectively). Patients in the two groups obtained similar live birth rate (Table 4).
Table 4.
FET parameters and outcomes in patients undergoing HRT with or without ovulation
| With ovulation | Without ovulation | P value | |
|---|---|---|---|
| No. of cycles | 76 | 1942 | |
| Delay between treatments (months) | 5.8±3.4 | 5.2±4.5 | NS |
| Endometrial thickness (mm) | 9.1±1.8 | 8.9±2.1 | NS |
| No. of embryo transferred | 2.0±0.4 | 2.0±0.5 | NS |
| Implantation rate (%) | 16.9 (25/148) | 29.8 (1172/3932) | <0.01 |
| Clinical pregnancy rate (%) | 26.3 (20/76) | 49.5 (962/1942) | <0.01 |
| Live birth rate (%) | 84.0 (21/25) | 87.0 (1020/1172) | NS |
Note: NS=not significant.
Discussion
Our study found that HRT improved FET outcomes, in terms of implantation rate and clinical pregnancy rate. Furthermore, in HRT cycles, ovulation negatively affected the pregnancy results.
In the present study, HRT was applied without prior GnRHa down-regulation but still yielded favorable pregnancy results, which was consistent with the study by Queenan et al. [16]. They reported that prior suppression with a GnRH-a is not necessary for endometrial preparation. Multiple studies were performed to assess the efficacy of various endometrium preparation protocols. However, controversies still remain on the issue of optimal protocol in FET treatment. The study by Morozov et al. [17] showed that HRT was associated with decreased pregnancy rate in comparison to natural cycle. The alteration of implantation window span in FET cycles may contribute to the reduced pregnancy rate at supraphysiologic estrogen level [18]. In contrast, some studies reported superior pregnancy outcomes in HRT cycles [11,19]. Additionally, several studies suggested that patients in natural cycles and HRT cycles obtained comparable FET outcomes [10,20,21]. Our study found better FET outcomes in patients who underwent HRT, which may attribute to the following reasons. Firstly, scheduling the day of embryo thawing and transfer was difficult in natural cycles, because the length of follicular phases varies among patients. In contrast, in HRT cycles, the day of embryo transfer can be programmed in advance, which favors embryo implantation. Secondly, it was difficult to identify a positive serum LH surge as the ascending or the descending limb of the LH surge, and thus the planned day of embryo transfer may not match the implantation window in natural cycles. Lastly, it was a dilemma in some cases with natural cycles that the ovulation was completed, whereas the endometrium failed to reach suitable thickness.
Endometrial-thickness provides an effective approach to assess endometrial development and predict endometrial receptivity [6]. The study by Gonen et al. [22] suggested pregnancy was unlikely to occur when the endometrial-thickness was <6 mm. Other studies reported FET success rates were higher when endometrial-thickness was greater than 8 mm on the day of transfer [10,17]. In patients with poor endometrial response, exogenous estradiol treatment constitutes as a common practice to improve endometrial thickness. Our data showed that patients with thin endometrium undergoing different protocols had similar endometrial thickness, indicating that administration with estradiol is insufficient to increase endometrial thickness in patients with poor endometrial response. Indeed, it has been reported that aberrant expression pattern of estrogen receptors was associated with thin endometrium and infertility [23]. Interestingly, despite the lack of ability to increase endometrial thickness, HRT yielded better pregnancy results than other protocols. Therefore, it should be recommended that patients with thin endometrium take HRT.
HRT carries numerous advantages over natural cycles. Based on the results of our study, HRT increases the possibility of pregnancy. By using HRT, fewer times of visits were needed during the cycles, which considerably benefits the patients. The days of embryos thawing and transfer can be scheduled in advance. Treatment cancellation rate can be limited low in HRT, due to the programmed exogenous hormone replacement and suppression of ovulation. HRT constitutes a more suitable option for some specific patients, such as women with >40 age old, premature ovarian failure, and oligomenorrhea [12,24].
Whether an ovulatory HRT cycle should be cancelled constitutes a dilemma in practice of FET treatment. The unsuppressed follicle development and ovulation may alter the hormone profile, and thus impair the endometrial receptivity and pregnancy. In our study, we found decreased implantation rate and pregnancy rate in ovulatory patients. This finding has clinical implication that the HRT cycle with unsuppressed ovulation may be considered to be cancelled. However, it should be noted that the arm of ovulatory patients included only 76 cycles. A prospective observational study with large sample size is needed to confirm this finding.
The strength of the present study is the large size of the cohort: a total of 5414 FET cycles were analyzed. In addition, the patients data was collected in a relative short period of time (Jan. 2008 to Dec. 2011), during this period of time, the protocols of endometrium preparation, as well as FET, were relative consolidate, which favors intra-group homogeneity in our study. To discriminate the effect of embryo-quality on pregnancy, only the FET cycles with good quality embryo transferred were enrolled. To alleviate the confounding influence of the factor that the same patient with excessive embryos may underwent multiple FET cycles, only the first 2 FET cycles were analyzed. Limitation of the present study is its respective nature, and confounding factors may interfere with the results.
In conclusion, we found that HRT increases the possibility of pregnancy. This result was not changed in patients with thin endometrium. In addition, ovulation in HRT cycle may defer the FET success. Therefore, we recommended that HRT should be applied in patients who plan to take FET treatment, and ovulation in HRT cycle should be evaluated during the treatment.
References
- 1.Zeilmaker GH, Alberda AT, van Gent I, Rijkmans CM, Drogendijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril. 1984;42:293–296. doi: 10.1016/s0015-0282(16)48029-5. [DOI] [PubMed] [Google Scholar]
- 2.Lieberman BA, Troup SA, Matson PL. Cryopreservation of embryos and pregnancy rates after IVF. Lancet. 1992;340:116. doi: 10.1016/0140-6736(92)90438-9. [DOI] [PubMed] [Google Scholar]
- 3.de Mouzon J, Goossens V, Bhattacharya S, Castilla JA, Ferraretti AP, Korsak V, Kupka M, Nygren KG, Nyboe Andersen A. Assisted reproductive technology in Europe, 2006: results generated from European registers by ESHRE. Hum Reprod. 2010;25:1851–1862. doi: 10.1093/humrep/deq124. [DOI] [PubMed] [Google Scholar]
- 4.Lee KY, Jeong JW, Tsai SY, Lydon JP, DeMayo FJ. Mouse models of implantation. Trends Endocrinol Metab. 2007;18:234–239. doi: 10.1016/j.tem.2007.06.002. [DOI] [PubMed] [Google Scholar]
- 5.Wang H, Dey SK. Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet. 2006;7:185–199. doi: 10.1038/nrg1808. [DOI] [PubMed] [Google Scholar]
- 6.Schild RL, Knobloch C, Dorn C, Fimmers R, van der Ven H, Hansmann M. Endometrial receptivity in an in vitro fertilization program as assessed by spiral artery blood flow, endometrial thickness, endometrial volume, and uterine artery blood flow. Fertil Steril. 2001;75:361–366. doi: 10.1016/s0015-0282(00)01695-2. [DOI] [PubMed] [Google Scholar]
- 7.Xiong T, Zhao Y, Hu D, Meng J, Wang R, Yang X, Ai J, Qian K, Zhang H. Administration of calcitonin promotes blastocyst implantation in mice by up-regulating integrin beta3 expression in endometrial epithelial cells. Hum Reprod. 2012;27:3540–3551. doi: 10.1093/humrep/des330. [DOI] [PubMed] [Google Scholar]
- 8.Aghajanova L. Update on the role of leukemia inhibitory factor in assisted reproduction. Curr Opin Obstet Gynecol. 2010;22:213–219. doi: 10.1097/GCO.0b013e32833848e5. [DOI] [PubMed] [Google Scholar]
- 9.Fogle RH, Li A, Paulson RJ. Modulation of HOXA10 and other markers of endometrial receptivity by age and human chorionic gonadotropin in an endometrial explant model. Fertil Steril. 2010;93:1255–1259. doi: 10.1016/j.fertnstert.2008.11.002. [DOI] [PubMed] [Google Scholar]
- 10.al-Shawaf T, Yang D, al-Magid Y, Seaton A, Iketubosin F, Craft I. Ultrasonic monitoring during replacement of frozen/thawed embryos in natural and hormone replacement cycles. Hum Reprod. 1993;8:2068–2074. doi: 10.1093/oxfordjournals.humrep.a137983. [DOI] [PubMed] [Google Scholar]
- 11.Schmidt CL, de Ziegler D, Gagliardi CL, Mellon RW, Taney FH, Kuhar MJ, Colon JM, Weiss G. Transfer of cryopreserved-thawed embryos: the natural cycle versus controlled preparation of the endometrium with gonadotropin-releasing hormone agonist and exogenous estradiol and progesterone (GEEP) Fertil Steril. 1989;52:609–616. doi: 10.1016/s0015-0282(16)60973-1. [DOI] [PubMed] [Google Scholar]
- 12.Sathanandan M, Macnamee MC, Rainsbury P, Wick K, Brinsden P, Edwards RG. Replacement of frozen-thawed embryos in artificial and natural cycles: a prospective semi-randomized study. Hum Reprod. 1991;6:685–687. doi: 10.1093/oxfordjournals.humrep.a137407. [DOI] [PubMed] [Google Scholar]
- 13.Holte J, Berglund L, Milton K, Garello C, Gennarelli G, Revelli A, Bergh T. Construction of an evidence-based integrated morphology cleavage embryo score for implantation potential of embryos scored and transferred on day 2 after oocyte retrieval. Hum Reprod. 2007;22:548–557. doi: 10.1093/humrep/del403. [DOI] [PubMed] [Google Scholar]
- 14.Schoolcraft WB, Gardner DK, Lane M, Schlenker T, Hamilton F, Meldrum DR. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril. 1999;72:604–609. doi: 10.1016/s0015-0282(99)00311-8. [DOI] [PubMed] [Google Scholar]
- 15.Zhu L, Xi Q, Zhang H, Li Y, Ai J, Jin L. Blastocyst culture and cryopreservation to optimize clinical outcomes of warming cycles. Reprod Biomed Online. 2013;27:154–160. doi: 10.1016/j.rbmo.2013.04.006. [DOI] [PubMed] [Google Scholar]
- 16.Queenan JT Jr, Ramey JW, Seltman HJ, Eure L, Veeck LL, Muasher SJ. Transfer of cryopreserved-thawed pre-embryos in a cycle using exogenous steroids without prior gonadotrophin-releasing hormone agonist suppression yields favourable pregnancy results. Hum Reprod. 1997;12:1176–1180. doi: 10.1093/humrep/12.6.1176. [DOI] [PubMed] [Google Scholar]
- 17.Morozov V, Ruman J, Kenigsberg D, Moodie G, Brenner S. Natural cycle cryo-thaw transfer may improve pregnancy outcome. J Assist Reprod Genet. 2007;24:119–123. doi: 10.1007/s10815-006-9100-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Ma WG, Song H, Das SK, Paria BC, Dey SK. Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation. Proc Natl Acad Sci U S A. 2003;100:2963–2968. doi: 10.1073/pnas.0530162100. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Givens CR, Markun LC, Ryan IP, Chenette PE, Herbert CM, Schriock ED. Outcomes of natural cycles versus programmed cycles for 1677 frozen-thawed embryo transfers. Reprod Biomed Online. 2009;19:380–384. doi: 10.1016/s1472-6483(10)60172-1. [DOI] [PubMed] [Google Scholar]
- 20.Gelbaya TA, Nardo LG, Hunter HR, Fitzgerald CT, Horne G, Pease EE, Brison DR, Lieberman BA. Cryopreserved-thawed embryo transfer in natural or down-regulated hormonally controlled cycles: a retrospective study. Fertil Steril. 2006;85:603–609. doi: 10.1016/j.fertnstert.2005.09.015. [DOI] [PubMed] [Google Scholar]
- 21.Ghobara T, Vandekerckhove P. Cycle regimens for frozen-thawed embryo transfer. Cochrane Database Syst Rev. 2008:CD003414. doi: 10.1002/14651858.CD003414.pub2. [DOI] [PubMed] [Google Scholar]
- 22.Gonen Y, Casper RF. 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;7:146–52. doi: 10.1007/BF01135678. [DOI] [PubMed] [Google Scholar]
- 23.Wang A, Ji L, Shang W, Li M, Chen L, White RE, Han G. Expression of GPR30, ERalpha and ERbeta in endometrium during window of implantation in patients with polycystic ovary syndrome: a pilot study. Gynecol Endocrinol. 2011;27:251–255. doi: 10.3109/09513590.2010.487584. [DOI] [PubMed] [Google Scholar]
- 24.Queenan JT Jr, Veeck LL, Seltman HJ, Muasher SJ. Transfer of cryopreserved-thawed pre-embryos in a natural cycle or a programmed cycle with exogenous hormonal replacement yields similar pregnancy results. Fertil Steril. 1994;62:545–550. doi: 10.1016/s0015-0282(16)56943-x. [DOI] [PubMed] [Google Scholar]