ABSTRACT
There is no consensus regimen for the optimal endometrial preparation for cryopreservation and vitrified-thawed embryo transfer cycles. This is largely caused by the lack of sufficient investigation and analyses on the respective pregnancy and perinatal outcomes by different regimens. This study aimed to compare both pregnancy and perinatal outcomes between the modified natural and artificial cycles in vitrified-thawed day three embryo transfer for women with regular menstruation. A total of 1,482 vitrified-thawed day three embryo transfer cycles were reviewed including 427 modified natural cycles (NC), 132 ovulation induction cycles (OC), 794 artificial cycles (AC), and 129 GnRH agonist artificial cycles (GAC). The primary outcome that was evaluated was live birth rate. The NC regimen demonstrated a higher rate of ongoing pregnancy (43.8% vs. 30.2%, P = 0.002) and a lower rate of late abortion (2.8% vs. 14.0%, P = 0.003) than the GAC regimen as well as a higher implantation rate (31.9% vs. 27.1%, P = 0.008) and live birth rate (43.1% vs. 34.1%, P = 0.002) than the AC regimen. A significantly higher peak endometrial thickness before transfer was observed in patients using the NC and GAC regimens (10.0 ± 1.7, 9.9 ± 2.4) compared to the AC regimens (9.2 ± 1.5, P = 0.000). Multivariate logistic regression showed that the NC protocol was associated with a higher live birth rate. There were no significant differences in rates of pregnancy complications, neonatal mortality, birth defects, mean birth weight, and other perinatal outcomes among the regimens. Modified natural cycle endometrial preparation regimen for vitrified-thawed day three embryo transfer is associated with superior live birth pregnancy outcomes compared to artificial cycles. Future studies are warranted to investigate the underlying biologic mechanisms of these findings.
Abbreviations ART: assisted reproductive technology; BMI: body mass index; FET: frozen-thawed embryo transfer; HCG: human chorionic gonadotropin; IVF: in-vitro fertilization; IVF-ET: in-vitro fertilization and embryo transfer; OHSS: ovarian hyperstimulation syndrome; RCTs: randomized controlled trials
Introduction
Since the first report of a successful human pregnancy following frozen-thawed embryo transfer (FET), embryo cryopreservation has been progressively used in assisted reproductive technology (ART) [Trounson and Mohr Citation1983]. Embryo cryopreservation after in vitro fertilization (IVF) is a cost-effective method to optimize cumulative pregnancy rates following oocyte retrieval, and to avoid the risk of ovarian hyperstimulation syndrome (OHSS) [Lieberman et al. Citation1992]. The introduction of vitrification, a rapid and an effective alternative to traditional slow-freezing, using a high concentration of cryoprotectant, has significantly improved the survival of the frozen embryo and success rates of FET cycles in the U.S. [Centers for Disease Control and Prevention ASRM, Society for Assisted Reproductive Technology Citation2007; de Mouzon et al. Citation2010]. Although inferior pregnancy rates were previously observed in FET cycles compared to fresh IVF embryo transfer (IVF-ET) cycles, continued refinements in vitrification methods have resulted in similar pregnancy success in FET cycles and fresh IVF-ET cycles [Roque et al. Citation2013; Roque et al. Citation2015; Shapiro et al. Citation2011; Wada et al. Citation1994].
Besides embryo survival and quality, successful vitrified-thawed embryo transfer also depends upon endometrial receptivity at the time of transfer. Several regimens can be used for endometrial preparation. The respective efficacy of different cycles has been greatly debated and remains controversial [Hancke et al. Citation2012]. For example, natural cycles with or without human chorionic gonadotropin (HCG) induced ovulation requires minimum medication and typically carried the least risk for adverse effects and is the least costly. Several groups have reported superior results for this regimen [Morozov et al. Citation2007; Xiao et al. Citation2012]. For the reason of convenience, artificial cycles are often applied to patients with regular menstrual intervals. Artificial cycles apply exogenous administration of estrogen and progesterone, and allow for a predictable monitoring of endometrial development and the advanced scheduling of embryo thaw and transfer. Some investigators have suggested that artificial cycles may optimize endometrial receptivity with a more consistent interval, duration, and concentration of estrogen and progesterone compared to the intrinsic variability of the LH surge, ovulation, and follicular/corpus luteum steroidogenesis of the natural cycle [Ghobara and Vandekerckhove Citation2008; Zheng et al. Citation2013]. In addition, the introduction of GnRH agonist suppression to prevent spontaneous ovulation in artificial cycles [Dal Prato et al. Citation2002; Gelbaya et al. Citation2006; Hil et al. 2010], has added a special type for artificial cycles.
Unfortunately, FET protocols have not been standardized and are dependent upon the discretion of the medical care provider, patient preferences, and acceptability of the costs. Due to the differences in exogenous hormone usage, it is reasonable to suspect that natural and artificial cycles may differentially affect the maintenance of healthy pregnancy after implantation, and on the long term outcomes at perinatal and even later stages. The impact of these regimens on pregnancy success rates and/or perinatal outcomes remains elusive. Focusing on the group with regular menstrual cycles, this study was designed to compare both live birth pregnancy and perinatal outcomes between the modified natural and artificial cycles of vitrified-warmed day three embryo transfer.
Results
A total of 1,482 vitrified-thawed embryo transfer cycles were analyzed, including 29% modified natural cycles (group NC, n=427), 9% ovulation induction cycles (group OC, n=132), 53% artificial cycles (group AC, n=794), and 9% GnRH agonist artificial cycles (group GAC, n=129). Except for higher age and body mass index (BMI) in women under the GAC regimen, no other significant differences were detected between women in the different regimen groups (). The average numbers of embryos transferred and embryo quality were also similar between groups (). Women that underwent AC (9.2 ± 1.5 mm) and OC (9.3 ± 1.8 mm) had thinner endometrium compared to those that underwent NC (10.0 ± 1.7 mm, P = 0.000) before transfer, indicating a better endometrial preparation of NC.
Table 1. Patient demographics in different frozen-thawed embryo transfer (FET) cycles and FET cycle characteristics.
No significant difference was detected in rates of clinical pregnancy, early abortion, and ectopic and multiple pregnancies among the four regimen groups (). The rate of perinatal complications, including preeclampsia and hypertensive disorders, placental vascular disease, and gestational diabetes, did not differ among the groups. However, implantation rates were significantly higher in women under NC compared to those under AC regimens (31.9% vs. 27.1%; P = 0.008). The rate of ongoing pregnancy was also significantly higher for NC regimens compared to GAC (43.8% vs. 30.2%; P = 0.006). In addition, the NC group showed a significantly lower rate of late abortion rate than those in GAC (2.8% vs.14.0%, respectively; P = 0.003). The perinatal and neonatal outcomes between the four types of cycles are shown in . The results showed that live birth rates per transfer were significantly higher in the NC group compared to the AC group (43.1% vs. 34.1%, respectively; P=0.002).
Table 2. The pregnancy outcomes in different frozen-thawed embryo transfer (FET) cycles.
Table 3. The perinatal and neonatal outcomes in different frozen-thawed embryo transfer (FET) cycles.
Results of multivariate logistic analysis of factors associated with live birth are shown in . Age, basal FSH, and BMI showed no independent association with live birth rate. The duration of infertility was negatively associated and reduced live birth rates (odds ratio 0.918, 95% confidence interval 0.885-0.952), while the number of embryos transferred (odds ratio 1.367, 95% confidence interval 1.130-1.654), and the endometrial thickness of the patients in FET (odds ratio 1.104, 95% confidence interval 1.036-1.176) were positively associated with live birth. A reduced likelihood for live birth was observed in both patients treated with the AC (odds ratio 0.753, 95% confidence interval 0.586-0.968) and GAC (odds ratio 0.579, 95% confidence interval 0.586-0.968) regimens compared to those treated with the NC regimen.
Table 4. Multivariate logistic regression analysis for live birth rate.
Discussion
Vitrified-thawed embryo transfer has emerged as an effective approach by the virtue of significantly improved embryo survival following thaw and increased cumulative pregnancy rates following oocyte retrieval and IVF. However, the concurrent pregnancy success and perinatal outcomes among different regimens of endometrial preparation has not been carefully compared. In the present study, we evaluated the outcomes of vitrified-thawed embryo transfers from four different endometrial preparation regimens in women with regular menstrual intervals, comparing modified natural cycles to induction and artificial cycles with and without GnRH agonist. This study showed the most favorable pregnancy and perinatal outcomes in women undergoing the modified natural cycles, including a higher rate of implantation, ongoing pregnancy rate, and live birth rate as well as a lower rate of late abortion in women with regular menstrual cycles. Furthermore, these results were unchanged after adjustments for confounders. These results suggest that pregnancy outcomes following modified natural cycles and vitrified-thawed embryo transfer cycles in woman with regular menstrual interval may be the most ideal compared to other regimens.
Comparisons between natural and artificial cycles in FET have shown conflicting results. In agreement with our findings, some previous studies observed superior outcomes in terms of implantation, clinical pregnancy, and lower miscarriage rates when natural cycles were used [Morozov et al. Citation2007; Orvieto et al. Citation2016; Xiao et al. Citation2012]. Moreover, since FET during a natural cycle is less expensive and more patient-friendly, Peeraer et al. [Citation2015] recommended NC regimen as the treatment of choice for women with regular cycles undergoing FET. In contrast, other investigators reported that patients undergoing artificial cycles had higher implantation and clinical pregnancy rates than patients undergoing natural cycles [Givens et al. Citation2009; Zheng et al. Citation2013]. Other studies have reported comparable clinical pregnancy and delivery rates between natural or artificial cycles [Tomas et al. Citation2012]. Hancke et al. [Citation2012] demonstrated that the live birth rates after natural and artificial cycles were not statistically different, although the data did suggest a trend towards higher pregnancy and live birth rates when natural cycles were used. It is widely known that the outcomes in IVF practices are affected by factors in multiple stages, including the baseline characteristics of patients, etiology and history of infertility, as well as the effectiveness during the process of ovulation, in vitro fertilization, implantation as well as successfully maintaining during pregnancy. Therefore, the different results among studies may be due to numerous confounding factors. In the current study, we focused on subjects with regular menstrual cycles, a group most likely associated with better ovary and endometrial functions, and confirmed the better outcomes by this group.
Optimal endometrial receptivity is essential for success of implantation [Casper and Yanushpolsky Citation2016]. It is generally considered that an ultrasonographic endometrium cross-section measurement of 7 mm is the minimum thickness required for successful implantation. A thicker endometrium (9-14 mm) is associated with a higher rate of successful implantation than a thinner endometrium (7-8 mm) [Adams et al. Citation2004]. Some studies have suggested that administration of exogenous E2 may lead to lower expression of endometrial receptivity markers in regularly menstruating patients [Ma et al. Citation2003]. It has also been noted in a recent study that oral contraceptive pills, in which the main effective substance is E2, can potentially affect optimal endometrium growth [Talukdar et al. Citation2012]. Therefore, artificial cycles in which exogenous estrogen and progesterone were administrated may interfere with endometrium readiness. Indeed, although our results showed that all groups got the satisfactory endometrium growth, just focusing on the patients with regular menstrual cycles, the NC group had a significantly thicker endometrium.
In an artificial cycle, administration of exogenous estrogen and progesterone does not guarantee complete pituitary suppression. Luteinization can occur in 5% of patients using artificial cycles [Bagis et al. Citation2010]. Pituitary suppression before the commencement of artificial cycles (i.e., with GAC) reduces the rate of cycle cancellation by suppressing spontaneous ovulation. In previous studies, the use of GAC resulted in similar implantation and clinical pregnancy rates [Gelbaya et al. Citation2006; Glujovsky et al. Citation2010; Tanos et al. Citation1996] as well as higher live-birth rates compared to the modified natural cycle approach [Hill et al. Citation2010]. However, with increased sample size, our data was able to show that modified natural cycles resulted in a higher ongoing pregnancy rate and lower late abortion rate than patients receiving GAC. In agreement with the study by Dal Prato et al. [Citation2002], we reported similar pregnancy outcomes with or without GnRH treatment in the artificial cycle approach.
The pregnancy and perinatal outcomes observed in all four groups of our study, as a whole, are consistent with previous studies of day 3 vitrified-thawed embryo transfer [Liu et al. Citation2013; Rama Raju 2009; Shi et al. Citation2012]. In those studies, as in ours, no significant differences between regimens were observed in the mean gestational week, birth weight, ratio for singleton and twins, congenital birth defects rate, neonatal mortality rate, still birth rate, or cerebral palsy rate in singletons. Therefore, long-term follow-up needs to be performed. Nevertheless, to the best of our knowledge, this is the first analysis to date that compares standard FET regimens in terms of both perinatal and neonatal outcomes.
Despite the large sample size of 1,482 FET cycles, some limitations deserve attention. The study design and multiple confounding factors of IVF outcomes make it difficult to draw definitive conclusions on the influence of any individual factor. Since the clinical choices of cycles were determined considering the monitored characteristics of patients, differences among patients in different groups were observed in baseline data. For example, although the overall study subjects were in the younger range for IVF patients, women undergoing with GAC cycles were apparently older than those under other regimens. The AC group had a higher mean BMI than the NC group. Women undergoing modified natural cycles also appeared to be with the lowest duration of infertility and less female-related infertility, which may contribute to final outcomes. Nevertheless, multivariate regression analysis suggested that NC correlated with higher live birth rate even after adjusting for the confounders. This suggests that the better outcomes in the NC group are most likely to be true although further studies with larger sample size and more thorough analysis is required for further verification. Future studies are warranted to investigate the underlying biologic mechanisms of these findings.
Conclusion
The present study demonstrates that a modified natural cycle approach appears to be more effective than other endometrial preparation regimens for achieving successful pregnancy with vitrified-thawed embryo transfer in women with regular menstrual cycles. The methods for endometrial preparation do not seem to differ in the pregnancy and perinatal outcomes. Whether these outcomes translate into clinical and cost-efficient benefits remain to be assessed in relation with the increased hospital visits, cost associated with natural cycle regimens, and the increased monitoring and follow-up required by artificial cycles. Therefore, future prospective, randomized, controlled trials (RCTs) should not only focus on the perinatal and neonatal outcomes through long-term follow-up, but should also evaluate its impact on patient convenience and cost-efficiency.
Material and methods
Study design
A retrospective cohort was conducted in consecutive vitrified-warmed day three embryo transfer cycles at the Center for Reproductive Medicine in the Third Affiliated Hospital of Zhengzhou University, Henan, China between January 2012 and July 2013. The study was approved by the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University and the written informed consents were obtained from all patients. Included patients were with regular menstrual intervals. Patients with a history of recurrent implantation failure or abortion were excluded. A normal endometrial cavity was confirmed via saline sonohysterogram, hysterosalpingogram, or office hysteroscopy within 12 months of FET. Demographic and reproductive characteristics, endometrial preparation cycle details, and pregnancy and perinatal outcomes were extracted from the clinical records. Pregnancy and perinatal outcomes were compared between subjects with natural and artificial endometrial preparations.
Embryo vitrification and thawing
The assessment criteria for embryo quality and the programs for embryo vitrification and thawing in our center have been previously published [Wang et al. Citation2012]. The ultrasound-guided embryo transfers were all conducted on day 4 after the first administration of supplemental progesterone, and refered to the same vitrified-thawing protocol. The embryo transfer procedures were applied to all patients, irrespective of endometrial preparation regimen.
Endometrial preparation protocols
The choice of endometrial preparation was dependent upon clinical discretion and patient preferences. Generally, follicle development in the natural cycle had been monitored in each patient before endometrial preparation in FET cycle. For those with predictable follicle development based upon regular menstrual intervals, modified natural cycle was the preferred choice. However, artificial cycles might be chosen due to convenience. In circumstances with unpredictable dominant follicle selection was observed, ovulation induction cycles, or the artificial cycles were chosen. Moreover, the GnRH agonist artificial cycles were chosen for patients with endometriosis or spontaneous ovulation during hormone treatment.
Modified natural cycles
Patients under NC were monitored by transvaginal ultrasound, urine LH, serum E2, LH levels to determine endometrial thickness, dominant follicle growth, and ovulation. Transvaginal ultrasound evaluation as well as urine and serum tests were repeated every 2-3 days until follicle maturation (diameter ≥18-20 mm). Ovulation was then stimulated with 10,000 IU of HCG (HCG, Livzon Pharmaceuticals, China).
Artificial cycles
Patients on artificial cycle regimens were administered 4-6 mg/day of oral estradiol (estradiol valerate, Progynova, Bayer HealthCare, Germany) starting on day 2-4 of the natural menstrual cycle. The endometrial pattern and thickness were monitored by transvaginal ultrasound. Serum E2 levels was measured every 5-7 days. The estradiol dosage was adjusted based on the endometrial thickness and level of serum E2. After adequate endometrial proliferation (diameter ≥8 mm) and serum E2 concentration (200-300 ng/L) were documented, intramuscular progesterone administration was commenced. Both estradiol and progesterone were administered until 10 weeks of gestation.
GnRH agonist artificial cycles
Patients on GAC was administered either 1.875 mg or 3.75 mg of a long-term GnRH agonist (Diphereline, Epsen Inovation for Patients Care, France), intramuscularly starting in the early follicular phase (day 2-4) of the menstrual cycle. Twenty-eight days after the injection, serum LH, FSH, P, and E2 levels were measured to confirm down-regulation, and a transvaginal ultrasound scan was performed to determine endometrial thickness. Endometrial preparation was initiated, as described above for the artificial cycle, when E2 levels were greater than 30ng/L, progesterone was <1µg/L, LH and FSH levels were <5 IU/L, and endometrial thickness was <5 mm. In all patients, intramuscular administration of progesterone (Progesterone Injection, XianJu, Pharmaceuticals, China, 60 mg/day, in oil) was started as common luteal support after ovulation was confirmed (for natural and induction cycles) or after endometrial proliferation was considered adequate (for artificial cycles).
Ovulation induction cycles
Patients under OC were administrated 2.5 to 5 mg of letrozole (Letrozole, Hengrui Pharmaceuticals, China) daily for five days (from day 3 to day 7 or from day 5 to day 9). Transvaginal ultrasound scan, urine LH, serum E2, LH, and P levels were monitored starting on day 10 of the menstrual cycle. If the diameter of the dominant follicle was ≥10 mm on day 10, then ultrasound and serum tests were repeated every 2-3 days until follicle maturation (diameter ≥18-20 mm). Ovulation was then stimulated with 10,000 IU of hCG .
Diagnosis of pregnancy and follow-up
Pregnancy was determined based on serum hCG levels greater than 30 IU/L. A clinical pregnancy was diagnosed by detection of fetal heart activity under transvaginal ultrasound scanning at approximately 4 weeks after the positive pregnancy test. Abortions were defined as ’early’ if they occurred within the first 12 weeks of pregnancy, and defined as ’late’ for those that occurred between 12 and 28 weeks. Pregnancies reaching over 24 weeks of gestation were considered as ongoing pregnancies. All pregnant women were followed up until 6 months after delivery to obtain data on pregnancy and perinatal outcomes.
Statistical analysis
Categorical variables were expressed as percentages. All other variables were presented as means with their associated standard deviations (SD). Comparative statistics were performed with chi-squared test or one-way analysis of variance (ANOVA) with Bonferroni post-hoc analysis. All P values presented were two-tailed. A P value < 0.05 was defined as statistically significant.
Multivariate logistic regression analysis was performed to determine the associating variables with live birth. Age, basal FSH level, BMI, duration of infertility, indication for treatment, number of embryos transferred, endometrial thickness, and endometrial preparation protocols were included as covariates. The Statistical Package for Social Sciences (SPSS; version 17.0, IBM, Chicago, USA) was used for statistical analyses.
Declaration of interests
This work was supported in part by grants from the Scientific and Technological Project of Henan Province (NO. 201203049) and the Merck Serono China Research Fund for Fertility. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
Additional information
Notes on contributors
Xingling Wang
Conceived and designed the experiments: ZZ, XW, YG; Perform the study: YG; Acquisition of data: HF, ZX, ZL; Writing or revision of the manuscript: YG, AKS; Administrative, technical, or material support: YG, JZ, LS; Study supervision: ZZ, XW.
Zhan Zhang
Conceived and designed the experiments: ZZ, XW, YG; Perform the study: YG; Acquisition of data: HF, ZX, ZL; Writing or revision of the manuscript: YG, AKS; Administrative, technical, or material support: YG, JZ, LS; Study supervision: ZZ, XW.
References
- Adams, S.M., Terry, V., Hosie, M.J., Gayer, N. and Murphy, C.R. (2004) Endometrial response to IVF hormonal manipulation: comparative analysis of menopausal, down regulated and natural cycles. Reprod Biol Endocrinol 2:21.
- Bagis, T., Haydardedeoglu, B., Kilicdag, E.B., Cok, T., Simsek, E. And Parlakgumus, A.H. (2010) Single versus double intrauterine insemination in multi-follicular ovarian hyperstimulation cycles: a randomized trial. Hum Reprod 25:1684–1690.
- Casper, R.F. and Yanushpolsky, E.H. (2016) Optimal endometrial preparation for frozen embryo transfer cycles: window of implantation and progesterone support. Fertil Steril 105:867–872.
- Centers for Disease Control and Prevention ASRM, Society for Assisted Reproductive Technology (2007) 2005 Assisted Reproductive Technology Success Rates: National Summary and Fertility Clinic Reports. Atlanta: Centers for Disease Control and Prevention.
- Dal Prato, L., Borini, A., Cattoli, M., Bonu, M.A., Sciajno, R. and Flamigni, C. (2002) Endometrial preparation for frozen-thawed embryo transfer with or without pretreatment with gonadotropin-releasing hormone agonist. Fertil Steril 77:956-960.
- De Mouzon, J., Goossens, V., Bhattacharya, S., Castilla, J.A., Ferraretti, A.P., Korsak, V., et al. (2010) Assisted reproductive technology in Europe, 2006: results generated from European registers by ESHRE. Hum Reprod 25:1851-1862.
- Gelbaya, T.A., Nardo, L.G., Hunter, H.R., Fitzgerald, C.T., Horne, G., Pease, E.E., et al. (2006) Cryopreserved-thawed embryo transfer in natural or down-regulated hormonally controlled cycles: a retrospective study. Fertil Steril 85:603-609.
- Ghobara, T. and Vandekerckhove, P. (2008) Cycle regimens for frozen-thawed embryo transfer. Cochrane Database Syst Rev. 2008:CD003414.
- Givens, C.R., Markun, L.C., Ryan, I.P., Chenette, P.E., Herbert, C.M. and Schriock, E.D. (2009) Outcomes of natural cycles versus programmed cycles for 1677 frozen-thawed embryo transfers. Reprod Biomed Online 19:380-384.
- Glujovsky, D., Pesce, R., Fiszbajn, G., Sueldo, C., Hart, R.J. and Ciapponi, A. (2010) Endometrial preparation for women undergoing embryo transfer with frozen embryos or embryos derived from donor oocytes. Cochrane Database Syst Rev 2010:CD006359.
- Hancke, K., More, S., Kreienberg, R. and Weiss, J.M. (2012) Patients undergoing frozen-thawed embryo transfer have similar live birth rates in spontaneous and artificial cycles. J Assist Reprod Genet 29:403-407.
- Hill, M.J., Miller, K.A. and Frattarelli, J.L. (2010) A GnRH agonist and exogenous hormone stimulation protocol has a higher live-birth rate than a natural endogenous hormone protocol for frozen-thawed blastocyst-stage embryo transfer cycles: an analysis of 1391 cycles. Fertil Steril 93:416-422.
- Lieberman, B.A., Troup, S.A. and Matson, P.L. (1992) Cryopreservation of embryos and pregnancy rates after IVF. Lancet 340:116.
- Liu, S.Y., Teng, B., Fu, J., Li, X., Zheng, Y. and Sun, X.X. (2013) Obstetric and neonatal outcomes after transfer of vitrified early cleavage embryos. Hum Reprod 28:2093-2100.
- Ma, W.G., Song, H., Das, S.K., Paria, B.C. and Dey, S.K. (2003) Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation. Proc Natl Acad Sci U S A 100:2963-2968.
- Morozov, V., Ruman, J., Kenigsberg, D., Moodie, G. and Brenner, S. (2007) Natural cycle cryo-thaw transfer may improve pregnancy outcome. J Assist Reprod Genet 24:119-123.
- Orvieto, R., Feldman, N., Lantsberg, D., Manela, D., Zilberberg, E. and Haas, J. (2016) Natural cycle frozen-thawed embryo transfer-can we improve cycle outcome? J Assist Reprod Genet 33:611-615.
- Peeraer, K., Couck, I., Debrock, S., De Neubourg, D., De Loecker, P., Tomassetti C., et al. (2015) Frozen-thawed embryo transfer in a natural or mildly hormonally stimulated cycle in women with regular ovulatory cycles: a RCT. Hum Reprod 30:2552-2562.
- Rama Raju, G.A., Jaya Prakash, G., Murali Krishna, K. and Madan, K. (2009) Neonatal outcome after vitrified day 3 embryo transfers: a preliminary study. Fertil Steril 92:143-148.
- Roque, M., Lattes, K., Serra, S., Sola, I., Geber, S., Carreras, R., et al. (2013) Fresh embryo transfer versus frozen embryo transfer in in vitro fertilization cycles: a systematic review and meta-analysis. Fertil Steril 99:156-162.
- Roque, M., Valle, M., Guimaraes, F., Sampaio, M. and Geber, S. (2015) Freeze-all policy: fresh vs. frozen-thawed embryo transfer. Fertil Steril 103:1190-1193.
- Shapiro, B.S., Daneshmand, S.T., Garner, F.C., Aguirre, M., Hudson, C. and Thomas, S. (2011) Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfer in normal responders. Fertil Steril 96:344-348.
- Shi, W., Xue, X., Zhang, S., Zhao, W., Liu, S., Zhou, H., et al. (2012) Perinatal and neonatal outcomes of 494 babies delivered from 972 vitrified embryo transfers. Fertil Steril 97:1338-1342.
- Talukdar, N., Bentov, Y., Chang, P.T., Esfandiari, N., Nazemian, Z. and Casper, R.F. (2012) Effect of long-term combined oral contraceptive pill use on endometrial thickness. Obstet Gynecol 120:348-354.
- Tanos, V., Friedler, S., Zajicek, G., Neiger, M., Lewin, A. and Schenker, J.G. (1996) The impact of endometrial preparation on implantation following cryopreserved-thawed-embryo transfer. Gynecol Obstet Invest 41:227-231.
- Tomas, C., Alsbjerg, B., Martikainen, H. and Humaidan, P. (2012) Pregnancy loss after frozen-embryo transfer–a comparison of three protocols. Fertil Steril 98:1165-1169.
- Trounson, A. and Mohr, L. (1983) Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 305:707-709.
- Wada, I., Macnamee, M.C., Wick, K., Bradfield, J.M. and Brinsden, P.R. (1994) Birth characteristics and perinatal outcome of babies conceived from cryopreserved embryos. Hum Reprod 9: 543–546.
- Wang, X.L., Zhang, X., Qin, Y.Q., Hao, D.Y. and Shi, H.R. (2012) Outcomes of day 3 embryo transfer with vitrification using Cryoleaf: a 3-year follow-up study. J Assist Reprod Genet 29: 883–889.
- Xiao, Z., Zhou, X., Xu, W., Yang, J. and Xie Q. (2012) Natural cycle is superior to hormone replacement therapy cycle for vitrificated-preserved frozen-thawed embryo transfer. Syst Biol Reprod Med 58: 107–112.
- Zheng, Y., Li, Z., Xiong, M., Luo, T., Dong, X., Huang, B., et al. (2013) Hormonal replacement treatment improves clinical pregnancy in frozen-thawed embryos transfer cycles: a retrospective cohort study. Am J Transl Res 6: 85–90.