- Email: [email protected]
Freeze-all can be a superior therapy to another fresh cycle in patients with prior fresh blastocyst implantation failure
Accepted Manuscript Title: Freeze-all is a superior therapy to another fresh cycle in patients with prior fresh blastocyst implantation failure Author: Bruce S. Shapiro, Said T. Daneshmand, Forest C. Garner, Martha Aguirre, Cynthia Hudson PII: DOI: Reference:
S1472-6483(14)00242-9 http://dx.doi.org/doi:10.1016/j.rbmo.2014.04.009 RBMO 1143
To appear in:
Reproductive BioMedicine Online
Received date: Revised date: Accepted date:
28-10-2013 24-4-2014 29-4-2014
Please cite this article as: Bruce S. Shapiro, Said T. Daneshmand, Forest C. Garner, Martha Aguirre, Cynthia Hudson, Freeze-all is a superior therapy to another fresh cycle in patients with prior fresh blastocyst implantation failure, Reproductive BioMedicine Online (2014), http://dx.doi.org/doi:10.1016/j.rbmo.2014.04.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fresh versus frozen embryo transfer in patients with prior implantation failure
Freeze-all is a superior therapy to another fresh cycle in patients with prior fresh blastocyst implantation failure Authors: Bruce S. Shapiro, M.D., Ph.D.a,b Said T. Daneshmand, M.D. a,b Forest C. Garner, M.Sc. a,b Martha Aguirre, Ph.D. a Cynthia Hudson, M.S. a Authors’ affiliations: a Fertility Center of Las Vegas, 8851 W. Sahara Avenue, Las Vegas, NV 89117 USA. b Department of Obstetrics and Gynecology, University of Nevada, School of Medicine, 2040 W. Charleston Blvd Ste. 200, Las Vegas, NV 89102, USA. Corresponding author: Bruce Shapiro M.D., Fertility Center of Las Vegas, 8851 W. Sahara Avenue, Las Vegas, NV 89117 USA. Telephone: +1(702)-254–1777 Fax: +1-(702)-254–1213, e-mail: [email protected] Highlights
Studied patients with prior implantation failure with fresh blastocysts
Patients elected either another fresh transfer or else freeze-all
Freeze-all was associated with greater live birth rate with the first transfer
Freeze-all was associated with greater cumulative live birth rate
Abstract Implantation failure has various causes, including impaired uterine receptivity following ovarian stimulation. This retrospective cohort study compared outcomes in patients with prior implantation failure who elected to undergo another fresh cycle with those who opted for embryo cohort cryopreservation (freeze-all) and subsequent thaw. There were 269 patients with implantation failure following fresh autologous blastocyst transfer opting to undergo a subsequent cycle, with 163 choosing another fresh cycle and 106 electing freeze-all and subsequent thaw. Multiple logistic regression analysis indicated that cohort cryopreservation was
Page 1 of 13
associated with greater chance of live birth when compared with another fresh cycle (P < 0.0001). The odds ratio for live birth with freeze-all relative to a fresh cycle was 3.8 (95% CI 2.1–7.2). A second analysis was then performed using cumulative live birth rate as the outcome measure. Multiple logistic regression indicated freeze-all was associated with greater cumulative live birth rate than was a fresh cycle (OR 1.9, 95% CI 1.1–3.3, P = 0.0287). These findings suggest that, following implantation failure with fresh blastocysts, patients have a significantly greater chance of live birth with freeze-all and subsequent thaw than with another fresh cycle. Keywords: embryo cryopreservation, endometrial receptivity, implantation failure, IVF, ovarian stimulation
Introduction Despite numerous advances in assisted reproduction, implantation failure remained the most common outcome following embryo transfer in 2011 (Society for Assisted Reproductive Technology, 2011). Most fresh autologous embryo transfers in 2011 failed to result in live birth, and national average implantation rates ranged from 4–36%, depending on maternal age. Therefore, a large proportion of patients undergoing IVF experience at least one fresh embryo transfer cycle resulting in implantation failure. Furthermore, a history of just one failed fresh blastocyst transfer has been found to be prognostic of significantly increased risk of implantation failure in subsequent fresh autologous blastocyst transfers. Reported clinical pregnancy and implantation rates were found to decline from 50.3% and 30.1% in first fresh transfer to 31.1% and 18.0% in second fresh transfer, respectively (Shapiro et al. 2001). One possible cause of implantation failure is impaired endometrial receptivity following ovarian stimulation (Fatemi and Popovic-Todorovic 2013; Shapiro et al. 2011). The endometrial effects of ovarian stimulation include advanced histology, genomic dysregulation and biochemical abnormalities (Horcajadas et al. 2007). Histological advancement, premature progesterone elevation late in the follicular phase and slow embryo development are each associated with implantation failure, apparently due to embryo–endometrium asynchrony following ovarian stimulation (Shapiro et al. 2008; Murata et al. 2005; Pantos et al. 2004; Nikas et al. 2002; Nikas et al. 1999). The therapeutic options for subsequent IVF cycles in patients with a history of fresh blastocyst implantation failure include repeating another cycle of fresh transfer and embryo cohort cryopreservation (freeze-all) followed by thaw and transfer, among others. One randomized trial found significantly greater implantation and clinical pregnancy rates per transfer with frozen–thawed embryos compared with fresh embryos in normal responders (Shapiro et al. 2011). These patients were undergoing their first IVF cycles and the conclusions were based only on cycles with embryo transfer. However, studies of first-time IVF patients may not be applicable to patients with prior implantation failure, because of the poorer prognosis in those with just a single prior failure (Shapiro et al.
Page 2 of 13
2001). Also, a full assessment of therapeutic efficacy requires consideration of cancellations. A comparison of freeze-all versus fresh cycles in patients with prior fresh blastocyst implantation failure based on live birth per retrieval would more effectively assess these therapeutic options. This retrospective study compared live birth rates and cumulative live birth rates in patients who opted for freeze-all with those in patients who opted for another fresh cycle. The study population consisted of patients with prior implantation failure with fresh autologous blastocysts.
Materials and methods This study included patients with at least one prior fresh autologous transfer that failed to achieve ongoing pregnancy (fetal heart beat at 10 weeks of gestation) Fertility Center of Las Vegas in 2004–2012, whose next retrieval cycle was either an intended fresh autologous transfer (fresh group, serving as controls) or else cryopreservation of an entire cohort of autologous two pronuclear (2PN) oocytes followed by thaw (freeze-all group). Patients were excluded if their subsequent cycle used an oocyte donor, preimplantation genetic screening, a gestational carrier, multiple banking cycles or if only gonadotrophin-releasing hormone (GnRH) agonist was used for final oocyte maturation. Cancellations for lack of post-thaw survival or lack of viable transferable blastocysts in the subsequent cycle were included.
Comment [KMT1]: Author: please confirm edit or amend.
Ovarian stimulation was performed with daily exogenous FSH and human menopausal gonadotrophin in combination under a GnRH antagonist protocol described previously (Shapiro et al. 2011). Final oocyte maturation was achieved with human chorionic gonadotrophin or GnRH agonist in combination with low-dose human chorionic gonadotrophin as described previously (Shapiro et al. 2011). The standard of care throughout the study period was blastocyst transfer, regardless of cohort size. Following culture in sequential media (Quinn’s Advantage Protein Plus, Sage, prior to August 2011 or single-step media, LifeGlobal after Augist 2011), blastocysts were selected for transfer based on morphology only. Cohort cryopreservation was recommended to patients with prior implantation failure. Entire cohorts were cryopreserved at the 2PN stage using conventional slow freezing (Shapiro et al. 2011). Entire cohorts were thawed and cultured to the blastocyst stage in the same media as used for fresh culture, and the best blastocysts were selected for transfer based on morphology only. The cryopreservation technique was not changed during the study period. Whether following culture of fresh or thawed 2PN oocytes and subsequent transfer, supernumerary blastocysts of sufficient quality for transfer were frozen for potential future use. Throughout the study period, blastocyst cryopreservation was by conventional slow freezing as described previously (Shapiro et al. 2008).
Page 3 of 13
Patients in the freeze-all group received exogenous oestradiol (typically 6 mg daily; Estrace; Warner Chilcott, Rockaway, NJ, USA) for 10–14 days before starting progesterone (typically 100 mg daily i.m.; Actavis; Corona, CA, USA) on the day before 2PN oocyte thaw. Oestradiol and progesterone supplements were continued to sustain serum concentrations of 200 pg/ml and 15 ng/ml, respectively, until negative pregnancy test, pregnancy loss or rising serum concentrations confirming adequate placental production, typically at about 10 weeks of gestation. Patients receiving transfer of fresh blastocysts received similar progesterone supplements starting 1–2 days after retrieval and oestradiol supplements as needed to sustain the aforementioned serum concentrations. The total number of blastocysts formed was the number transferred plus the number cryopreserved. Cancelled cycles were those with no blastocysts of transferable quality. Implantation rate was the ratio of fetal hearts observed on ultrasound examination to the number of blastocysts transferred. Live births were defined by the birth of at least one live infant. Cumulative live birth rate was the proportion of retrievals that had at least one live birth, whether from the first transfer attempt or subsequent transfers of frozen–thawed supernumerary blastocysts. Statistical comparisons were performed with Fisher’s exact test (nominal variables) and Wilcoxon’s test (numeric variables). Logistic regression was used to compare cohort cryopreservation with fresh culture, while accommodating numerous potential confounding variables. Logistic regression models were constructed with forward stepwise selection followed by stepwise backward elimination (P < 0.25 was required for a variable to enter the model, while P < 0.05 was required for a variable to remain in the model). Statistical analyses were performed with JMP version 5 (SAS Institute, Cary, NC, USA). P-values <0.05 were considered significant. Institutional Review Board approval was obtained (protocol number SAIRB-07-0003, approved 18 July 2013).
Results This study included all 163 fresh cycles and all 106 freeze-all cycles meeting inclusion and exclusion criteria in the 9-year study period. The general parameters and outcomes of the two groups are described in Table 1. The freeze-all group had a significantly greater live birth rate per retrieval than the fresh group (46.2% versus 21.7%, P < 0.0001). However, there was a significant difference in the number of 2PN oocytes, with the freeze-all cycles having more 2PN oocytes (P < 0.0001), which confounded the direct outcome comparison. Logistic regression was used to compare the potential effects of cohort cryopreservation and fresh culture, while also considering the simultaneous effects of potential confounding variables. Selection of variables for inclusion in the model was achieved with stepwise forward selection followed by stepwise backward elimination. Such regression was first performed using live birth as the dependent variable, then a second regression was performed using cumulative live birth as the dependent variable. In each case, the independent variables were the type of cycle
Page 4 of 13
(fresh or freeze-all), age at retrieval in that cycle and the number of 2PN oocytes obtained. The number of blastocysts transferred was available as an independent variable for the first logistic regression (live birth through the first transfer), with the number transferred equal to zero in cancelled cycles. The logistic regression of live birth through the first transfer (including any cancelled transfers) versus the aforementioned variables resulted in a logistic model with cycle type (P < 0.0001), age at retrieval (P < 0.0001) and the number of blastocysts transferred (P = 0.0002) selected as significant predictors of live birth in the study cycle (Table 2). The odds ratio for cycle type was 3.8 (95% CI 2.1–7.2), in favour of the freeze-all group. The number of 2PN oocytes was not a significant predictor of live birth. The cumulative success rates are described in Table 3. The implantation rates in the subsequent thaw transfers were similar, but the cumulative birth rate was greater in the freeze-all group (P = 0.0025). The logistic regression of cumulative live birth using the same independent variables resulted in a logistic model with cycle type (P = 0.0287), number of 2PN oocytes (P = 0.0082) and age at retrieval (P < 0.0001) selected as significant predictors of cumulative live birth (Table 4). The odds ratio for cycle type was 1.9 (95% CI 1.1–3.3), in favour of the freeze-all group.
Discussion The patients considered here had a history of at least one failed fresh blastocyst transfer, representing a population with poor prognosis relative to new IVF patients. Each returned for another retrieval, opting for either another fresh transfer or else freeze-all followed by subsequent thaw. Those who opted for freeze-all were nonsignificantly younger and had significantly larger cohorts of 2PN oocytes. These differences made direct comparison inconclusive due to confounding effects of treatment protocol and cohort size. Therefore, logistic regression was applied to compare the two groups, with the treatment protocol (cycle type) available as one of the independent variables. The treatment protocol was a significant predictor of live birth and cumulative live birth rate. This is the first report to assess cohort cryopreservation as a therapy following implantation failure with fresh blastocyst transfer. These results suggest that patients with a history of fresh autologous implantation failure who elect freeze-all in their subsequent retrieval cycle followed by thaw will have an increased chance of live birth when compared with the option of another fresh cycle. Endometrial nonreceptivity is among the numerous factors implicated in implantation failure (Cakmak and Taylor 2011; Simon and Laufer 2012; Liu et al. in press). The current findings suggest many implantation failures following fresh autologous blastocyst transfer may be due to impaired endometrial receptivity following ovarian stimulation, and that this impairment may be avoided through cryopreservation followed by thaw in a subsequent cycle without ovarian
Page 5 of 13
stimulation. Patients with a history of fresh blastocyst implantation failure may be a population that may include increased risk of endometrial impairment from ovarian stimulation. The implantation rates with thawed supernumerary blastocysts (Table 3) were similar, despite those in the freeze-all group having been frozen twice. However, there were too few supernumerary blastocysts transferred (four blastocysts transferred in three patients) in the freeze-all group for meaningful comparison. The only patient in the freeze-all group to have a live birth with frozen–thawed supernumerary blastocysts already had live birth with her study cycle, so this additional live birth did not affect the cumulative live birth rate. The blastulation rate in the freeze-all group was 61% of that in the fresh group (22.4% versus 36.6%, respectively). This reduction is more than would be expected based on the 85% survival rate of 2PN oocytes alone (Table 1) and may reflect additional latent damage that impaired subsequent development. Post-thaw survival alone does not preclude cryopreservation damage (Shapiro et al. 2010). In one study of vitrified– warmed blastocysts, 100% of warmed blastocysts were assessed as surviving, but 36% of those degenerated and another 18% arrested within 24 h, and only the remaining 46% resumed development (Yamanaka et al. 2011). The transfer of thawed embryos that are briefly assessed for survival therefore risks the transfer of embryos that have been critically damaged and cannot resume development. The cryopreservation strategy employed here, freezing entire cohorts at the 2PN stage followed by thaw of the entire cohort and post-thaw extended culture, does not eliminate embryo cryodamage, but it does reduce the chance of transferring a damaged embryo (Shapiro et al. 2010). Another advantage over typical frozen–thawed embryo transfer cycles is that the current study employed entire frozen and thawed cohorts rather than the more typical second-best embryos cryopreserved after the best of the cohort was transferred fresh. Another interesting potential method to improve endometrial receptivity is endometrial scratching (Fatemi and Popovic-Todorovic, 2013). In one study (Nastri et al. 2013), 158 women, most of whom with one or more prior failed embryo transfers, were randomized to receive endometrial scratching or a sham treatment in the luteal phase of the prior cycle. Live birth rates were approximately doubled in the treatment arm (scratching) when compared with the control group (sham treatment), a degree of benefit strikingly similar to the current study. A limitation of this study is the use of retrospective analysis, with the attendant potential biases, although the study was designed to allow for the potential effects of confounding variables. A prospective randomized trial with intent-to-treat analysis is needed, particularly with cumulative success rates across all subsequent transfers using any frozen supernumerary blastocysts.
References Cakmak H, Taylor HS. Implantation failure: molecular mechanisms and clinical treatment. Hum Reprod Update. 2011;17:242–53.
Page 6 of 13
Fatemi HM, Popovic-Todorovic B. Implantation in assisted reproduction: a look at endometrial receptivity. Reprod Biomed Online. 2013;27:530–8. Horcajadas JA, Dıaz-Gimeno P, Pellicer A, Simon C. Uterine receptivity and the ramifications of ovarian stimulation on endometrial function. Semin Reprod Med 2007;25:454–60. Liu L, Zhou F, Lin X, Li T, Tong X, Zhu H, Zhang S. Recurrent IVF failure is associated with elevated progesterone on the day of hCG administration. Eur J Obstet Gynecol Reprod Biol. In press. Murata Y, Oku H, Morimoto Y, Tokuda M, Murata T, Sugihara K, Nagata F, Nakaoka Y, Fukuda A. Freeze–thaw programmes rescue the implantation of day 6 blastocysts Reprod BioMed Online 2005;11:428–433. Nastri CO, Ferriani RA, Raine-Fenning N, Martins WP. Endometrial scratching performed in the non-transfer cycle and outcome of assisted reproduction: a randomized controlled trial. Ultrasound Obstet Gynecol. 2013;42:375–82. Nikas G, Develioglu OH, Toner JP, Jones HW Jr. Endometrial pinopodes indicate a shift in the window of receptivity in IVF cycles. Hum Reprod. 1999;14:787–92. Nikas G, Aghajanova L. Endometrial pinopodes: some more understanding on human implantation? Reprod Biomed Online. 2002;4 Suppl 3:18–23. Pantos K, Nikas G, Makrakis E, Stavrou D, Karantzis P, Grammatis M. Clinical value of endometrial pinopodes detection in artificial donation cycles. Reprod Biomed Online. 2004;9:86–90. Shapiro BS, Richter KS, Harris DC, Daneshmand ST. Dramatic declines in implantation and pregnancy rates in patients who undergo repeated cycles of in vitro fertilization with blastocyst transfer after one or more failed attempts. Fertil Steril. 2001;76:538–42. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Thomas S. Large blastocyst diameter, early blastulation, and low preovulatory serum progesterone are dominant predictors of clinical pregnancy in fresh autologous cycles. Fertil Steril. 2008;90:302–9. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C, Thomas S. Embryo cryopreservation rescues cycles with premature luteinization. Fertil Steril. 2010;93:636–41. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C, Thomas S. 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. 2011;96:344–8. Simon A, Laufer N. Assessment and treatment of repeated implantation failure (RIF). J Assist Reprod Genet. 2012;29:1227–39. Society for Assisted Reproductive Technology. IVF success rate reports: 2011. www.SART.org. Yamanaka M, Hashimoto S, Amo A, Ito-Sasaki T, Abe H, Morimoto Y. Developmental assessment of human vitrified-warmed blastocysts based on oxygen consumption. Hum Reprod. 2011;26:3366–71. Declaration: The authors report no financial or commercial conflicts of interest.
Page 7 of 13
Table 1. Characteristics of retrievals and outcomes of the first attempted transfer. Characteristic
Fresh cycles (n = 163)
Freeze-all cycles (n = 106)
P-value
34.3 5.0 22–44 10.6 5.4
NS
Survival rate (%)
35.3 4.6 24–43 8.1 5.7 –
Cancellations (n, %) Blastocysts formed (n)
24 (14.7) 2.8 2.5
85.0 17.3 12 (11.3) 2.3 1.8
NS NS
Blastulation rate per 2PN oocyte (%) Transfers (n) Blastocysts transferred (n) Implantation rate (%)
36.6 25.8
22.4 14.1
<0.0001
139 2.0 0.7
94 1.8 0.5
0.0193
18.5 32.7 35 25.2 21.5
44.0 41.8 49 52.1 46.2
Maternal age (years) Mean SD Range 2PN oocytes (n)
Live births (n) Per transfer (%) Per retrieval (%)
<0.0001
<0.0001 <0.0001 <0.0001
Values are mean SD unless otherwise stated. NS = not significant.
Comment [KMT2]: Author: please confirm deletion of asterisks or add a table note.
Page 8 of 13
Table 2. Logistic regression of live birth through the first transfer versus available parameters. Parameter
P-value
Odds ratio (95% CI)
Intercept Cycle type Age at retrieval No. of blastocysts transferred No. of 2PN oocytes
0.0068 <0.0001 <0.0001 0.0002 NS
– 3.8 (2.1–7.2) 31.6 (7.6–144.3) 22.3 (4.6–124.4)
Page 9 of 13
Table 3. Cumulative live birth analysis, including study cycle and transfers of frozen–thawed supernumerary blastocysts. Parameter
Fresh cycles (n = 163)
Freeze-all cycles (n = 106)
Live births in first transfer attempt Implantation rate with thawed supernumerary embryos Retrievals resulting in at least one live birth Cumulative live birth rate per retrievala
35 20/83 (24.1)
49 1/4 (25.0)
45
49
45/163 (27.6)
49/106 (46.2)
Values are n or n (%). a
P < 0.05.
Page 10 of 13
Table 4. Logistic regression of cumulative live birth versus available parameters. Parameter
P-value
Odds ratio (95% CI)
Intercept Cycle type Number of 2PN oocytes Age at retrieval
0.0019 0.0287 0.0082 <0.0001
– 1.9 (1.1–3.3) 8.7 (1.8–45.3) 25.8 (6.4–113.4)
Page 11 of 13
Dr Shapiro earned his MD from the University of Nevada, Reno, and his PhD from the University of Amsterdam. He completed his residency in obstetrics and gynaecology and his fellowship in reproduction endocrinology at Yale-New Haven Hospital. He is chief of the Division of
Page 12 of 13
Reproductive Endocrinology and Infertility in the University of Nevada School of Medicine’s Department of Obstetrics and Gynecology. He is also a high-complexity laboratory director. He is the founder and medical director of the Fertility Center of Las Vegas. His recent research has included endometrial receptivity, embryo cryopreservation and alternative ovulatory triggering techniques.
Page 13 of 13
S1472-6483(14)00242-9 http://dx.doi.org/doi:10.1016/j.rbmo.2014.04.009 RBMO 1143
To appear in:
Reproductive BioMedicine Online
Received date: Revised date: Accepted date:
28-10-2013 24-4-2014 29-4-2014
Please cite this article as: Bruce S. Shapiro, Said T. Daneshmand, Forest C. Garner, Martha Aguirre, Cynthia Hudson, Freeze-all is a superior therapy to another fresh cycle in patients with prior fresh blastocyst implantation failure, Reproductive BioMedicine Online (2014), http://dx.doi.org/doi:10.1016/j.rbmo.2014.04.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fresh versus frozen embryo transfer in patients with prior implantation failure
Freeze-all is a superior therapy to another fresh cycle in patients with prior fresh blastocyst implantation failure Authors: Bruce S. Shapiro, M.D., Ph.D.a,b Said T. Daneshmand, M.D. a,b Forest C. Garner, M.Sc. a,b Martha Aguirre, Ph.D. a Cynthia Hudson, M.S. a Authors’ affiliations: a Fertility Center of Las Vegas, 8851 W. Sahara Avenue, Las Vegas, NV 89117 USA. b Department of Obstetrics and Gynecology, University of Nevada, School of Medicine, 2040 W. Charleston Blvd Ste. 200, Las Vegas, NV 89102, USA. Corresponding author: Bruce Shapiro M.D., Fertility Center of Las Vegas, 8851 W. Sahara Avenue, Las Vegas, NV 89117 USA. Telephone: +1(702)-254–1777 Fax: +1-(702)-254–1213, e-mail: [email protected] Highlights
Studied patients with prior implantation failure with fresh blastocysts
Patients elected either another fresh transfer or else freeze-all
Freeze-all was associated with greater live birth rate with the first transfer
Freeze-all was associated with greater cumulative live birth rate
Abstract Implantation failure has various causes, including impaired uterine receptivity following ovarian stimulation. This retrospective cohort study compared outcomes in patients with prior implantation failure who elected to undergo another fresh cycle with those who opted for embryo cohort cryopreservation (freeze-all) and subsequent thaw. There were 269 patients with implantation failure following fresh autologous blastocyst transfer opting to undergo a subsequent cycle, with 163 choosing another fresh cycle and 106 electing freeze-all and subsequent thaw. Multiple logistic regression analysis indicated that cohort cryopreservation was
Page 1 of 13
associated with greater chance of live birth when compared with another fresh cycle (P < 0.0001). The odds ratio for live birth with freeze-all relative to a fresh cycle was 3.8 (95% CI 2.1–7.2). A second analysis was then performed using cumulative live birth rate as the outcome measure. Multiple logistic regression indicated freeze-all was associated with greater cumulative live birth rate than was a fresh cycle (OR 1.9, 95% CI 1.1–3.3, P = 0.0287). These findings suggest that, following implantation failure with fresh blastocysts, patients have a significantly greater chance of live birth with freeze-all and subsequent thaw than with another fresh cycle. Keywords: embryo cryopreservation, endometrial receptivity, implantation failure, IVF, ovarian stimulation
Introduction Despite numerous advances in assisted reproduction, implantation failure remained the most common outcome following embryo transfer in 2011 (Society for Assisted Reproductive Technology, 2011). Most fresh autologous embryo transfers in 2011 failed to result in live birth, and national average implantation rates ranged from 4–36%, depending on maternal age. Therefore, a large proportion of patients undergoing IVF experience at least one fresh embryo transfer cycle resulting in implantation failure. Furthermore, a history of just one failed fresh blastocyst transfer has been found to be prognostic of significantly increased risk of implantation failure in subsequent fresh autologous blastocyst transfers. Reported clinical pregnancy and implantation rates were found to decline from 50.3% and 30.1% in first fresh transfer to 31.1% and 18.0% in second fresh transfer, respectively (Shapiro et al. 2001). One possible cause of implantation failure is impaired endometrial receptivity following ovarian stimulation (Fatemi and Popovic-Todorovic 2013; Shapiro et al. 2011). The endometrial effects of ovarian stimulation include advanced histology, genomic dysregulation and biochemical abnormalities (Horcajadas et al. 2007). Histological advancement, premature progesterone elevation late in the follicular phase and slow embryo development are each associated with implantation failure, apparently due to embryo–endometrium asynchrony following ovarian stimulation (Shapiro et al. 2008; Murata et al. 2005; Pantos et al. 2004; Nikas et al. 2002; Nikas et al. 1999). The therapeutic options for subsequent IVF cycles in patients with a history of fresh blastocyst implantation failure include repeating another cycle of fresh transfer and embryo cohort cryopreservation (freeze-all) followed by thaw and transfer, among others. One randomized trial found significantly greater implantation and clinical pregnancy rates per transfer with frozen–thawed embryos compared with fresh embryos in normal responders (Shapiro et al. 2011). These patients were undergoing their first IVF cycles and the conclusions were based only on cycles with embryo transfer. However, studies of first-time IVF patients may not be applicable to patients with prior implantation failure, because of the poorer prognosis in those with just a single prior failure (Shapiro et al.
Page 2 of 13
2001). Also, a full assessment of therapeutic efficacy requires consideration of cancellations. A comparison of freeze-all versus fresh cycles in patients with prior fresh blastocyst implantation failure based on live birth per retrieval would more effectively assess these therapeutic options. This retrospective study compared live birth rates and cumulative live birth rates in patients who opted for freeze-all with those in patients who opted for another fresh cycle. The study population consisted of patients with prior implantation failure with fresh autologous blastocysts.
Materials and methods This study included patients with at least one prior fresh autologous transfer that failed to achieve ongoing pregnancy (fetal heart beat at 10 weeks of gestation) Fertility Center of Las Vegas in 2004–2012, whose next retrieval cycle was either an intended fresh autologous transfer (fresh group, serving as controls) or else cryopreservation of an entire cohort of autologous two pronuclear (2PN) oocytes followed by thaw (freeze-all group). Patients were excluded if their subsequent cycle used an oocyte donor, preimplantation genetic screening, a gestational carrier, multiple banking cycles or if only gonadotrophin-releasing hormone (GnRH) agonist was used for final oocyte maturation. Cancellations for lack of post-thaw survival or lack of viable transferable blastocysts in the subsequent cycle were included.
Comment [KMT1]: Author: please confirm edit or amend.
Ovarian stimulation was performed with daily exogenous FSH and human menopausal gonadotrophin in combination under a GnRH antagonist protocol described previously (Shapiro et al. 2011). Final oocyte maturation was achieved with human chorionic gonadotrophin or GnRH agonist in combination with low-dose human chorionic gonadotrophin as described previously (Shapiro et al. 2011). The standard of care throughout the study period was blastocyst transfer, regardless of cohort size. Following culture in sequential media (Quinn’s Advantage Protein Plus, Sage, prior to August 2011 or single-step media, LifeGlobal after Augist 2011), blastocysts were selected for transfer based on morphology only. Cohort cryopreservation was recommended to patients with prior implantation failure. Entire cohorts were cryopreserved at the 2PN stage using conventional slow freezing (Shapiro et al. 2011). Entire cohorts were thawed and cultured to the blastocyst stage in the same media as used for fresh culture, and the best blastocysts were selected for transfer based on morphology only. The cryopreservation technique was not changed during the study period. Whether following culture of fresh or thawed 2PN oocytes and subsequent transfer, supernumerary blastocysts of sufficient quality for transfer were frozen for potential future use. Throughout the study period, blastocyst cryopreservation was by conventional slow freezing as described previously (Shapiro et al. 2008).
Page 3 of 13
Patients in the freeze-all group received exogenous oestradiol (typically 6 mg daily; Estrace; Warner Chilcott, Rockaway, NJ, USA) for 10–14 days before starting progesterone (typically 100 mg daily i.m.; Actavis; Corona, CA, USA) on the day before 2PN oocyte thaw. Oestradiol and progesterone supplements were continued to sustain serum concentrations of 200 pg/ml and 15 ng/ml, respectively, until negative pregnancy test, pregnancy loss or rising serum concentrations confirming adequate placental production, typically at about 10 weeks of gestation. Patients receiving transfer of fresh blastocysts received similar progesterone supplements starting 1–2 days after retrieval and oestradiol supplements as needed to sustain the aforementioned serum concentrations. The total number of blastocysts formed was the number transferred plus the number cryopreserved. Cancelled cycles were those with no blastocysts of transferable quality. Implantation rate was the ratio of fetal hearts observed on ultrasound examination to the number of blastocysts transferred. Live births were defined by the birth of at least one live infant. Cumulative live birth rate was the proportion of retrievals that had at least one live birth, whether from the first transfer attempt or subsequent transfers of frozen–thawed supernumerary blastocysts. Statistical comparisons were performed with Fisher’s exact test (nominal variables) and Wilcoxon’s test (numeric variables). Logistic regression was used to compare cohort cryopreservation with fresh culture, while accommodating numerous potential confounding variables. Logistic regression models were constructed with forward stepwise selection followed by stepwise backward elimination (P < 0.25 was required for a variable to enter the model, while P < 0.05 was required for a variable to remain in the model). Statistical analyses were performed with JMP version 5 (SAS Institute, Cary, NC, USA). P-values <0.05 were considered significant. Institutional Review Board approval was obtained (protocol number SAIRB-07-0003, approved 18 July 2013).
Results This study included all 163 fresh cycles and all 106 freeze-all cycles meeting inclusion and exclusion criteria in the 9-year study period. The general parameters and outcomes of the two groups are described in Table 1. The freeze-all group had a significantly greater live birth rate per retrieval than the fresh group (46.2% versus 21.7%, P < 0.0001). However, there was a significant difference in the number of 2PN oocytes, with the freeze-all cycles having more 2PN oocytes (P < 0.0001), which confounded the direct outcome comparison. Logistic regression was used to compare the potential effects of cohort cryopreservation and fresh culture, while also considering the simultaneous effects of potential confounding variables. Selection of variables for inclusion in the model was achieved with stepwise forward selection followed by stepwise backward elimination. Such regression was first performed using live birth as the dependent variable, then a second regression was performed using cumulative live birth as the dependent variable. In each case, the independent variables were the type of cycle
Page 4 of 13
(fresh or freeze-all), age at retrieval in that cycle and the number of 2PN oocytes obtained. The number of blastocysts transferred was available as an independent variable for the first logistic regression (live birth through the first transfer), with the number transferred equal to zero in cancelled cycles. The logistic regression of live birth through the first transfer (including any cancelled transfers) versus the aforementioned variables resulted in a logistic model with cycle type (P < 0.0001), age at retrieval (P < 0.0001) and the number of blastocysts transferred (P = 0.0002) selected as significant predictors of live birth in the study cycle (Table 2). The odds ratio for cycle type was 3.8 (95% CI 2.1–7.2), in favour of the freeze-all group. The number of 2PN oocytes was not a significant predictor of live birth. The cumulative success rates are described in Table 3. The implantation rates in the subsequent thaw transfers were similar, but the cumulative birth rate was greater in the freeze-all group (P = 0.0025). The logistic regression of cumulative live birth using the same independent variables resulted in a logistic model with cycle type (P = 0.0287), number of 2PN oocytes (P = 0.0082) and age at retrieval (P < 0.0001) selected as significant predictors of cumulative live birth (Table 4). The odds ratio for cycle type was 1.9 (95% CI 1.1–3.3), in favour of the freeze-all group.
Discussion The patients considered here had a history of at least one failed fresh blastocyst transfer, representing a population with poor prognosis relative to new IVF patients. Each returned for another retrieval, opting for either another fresh transfer or else freeze-all followed by subsequent thaw. Those who opted for freeze-all were nonsignificantly younger and had significantly larger cohorts of 2PN oocytes. These differences made direct comparison inconclusive due to confounding effects of treatment protocol and cohort size. Therefore, logistic regression was applied to compare the two groups, with the treatment protocol (cycle type) available as one of the independent variables. The treatment protocol was a significant predictor of live birth and cumulative live birth rate. This is the first report to assess cohort cryopreservation as a therapy following implantation failure with fresh blastocyst transfer. These results suggest that patients with a history of fresh autologous implantation failure who elect freeze-all in their subsequent retrieval cycle followed by thaw will have an increased chance of live birth when compared with the option of another fresh cycle. Endometrial nonreceptivity is among the numerous factors implicated in implantation failure (Cakmak and Taylor 2011; Simon and Laufer 2012; Liu et al. in press). The current findings suggest many implantation failures following fresh autologous blastocyst transfer may be due to impaired endometrial receptivity following ovarian stimulation, and that this impairment may be avoided through cryopreservation followed by thaw in a subsequent cycle without ovarian
Page 5 of 13
stimulation. Patients with a history of fresh blastocyst implantation failure may be a population that may include increased risk of endometrial impairment from ovarian stimulation. The implantation rates with thawed supernumerary blastocysts (Table 3) were similar, despite those in the freeze-all group having been frozen twice. However, there were too few supernumerary blastocysts transferred (four blastocysts transferred in three patients) in the freeze-all group for meaningful comparison. The only patient in the freeze-all group to have a live birth with frozen–thawed supernumerary blastocysts already had live birth with her study cycle, so this additional live birth did not affect the cumulative live birth rate. The blastulation rate in the freeze-all group was 61% of that in the fresh group (22.4% versus 36.6%, respectively). This reduction is more than would be expected based on the 85% survival rate of 2PN oocytes alone (Table 1) and may reflect additional latent damage that impaired subsequent development. Post-thaw survival alone does not preclude cryopreservation damage (Shapiro et al. 2010). In one study of vitrified– warmed blastocysts, 100% of warmed blastocysts were assessed as surviving, but 36% of those degenerated and another 18% arrested within 24 h, and only the remaining 46% resumed development (Yamanaka et al. 2011). The transfer of thawed embryos that are briefly assessed for survival therefore risks the transfer of embryos that have been critically damaged and cannot resume development. The cryopreservation strategy employed here, freezing entire cohorts at the 2PN stage followed by thaw of the entire cohort and post-thaw extended culture, does not eliminate embryo cryodamage, but it does reduce the chance of transferring a damaged embryo (Shapiro et al. 2010). Another advantage over typical frozen–thawed embryo transfer cycles is that the current study employed entire frozen and thawed cohorts rather than the more typical second-best embryos cryopreserved after the best of the cohort was transferred fresh. Another interesting potential method to improve endometrial receptivity is endometrial scratching (Fatemi and Popovic-Todorovic, 2013). In one study (Nastri et al. 2013), 158 women, most of whom with one or more prior failed embryo transfers, were randomized to receive endometrial scratching or a sham treatment in the luteal phase of the prior cycle. Live birth rates were approximately doubled in the treatment arm (scratching) when compared with the control group (sham treatment), a degree of benefit strikingly similar to the current study. A limitation of this study is the use of retrospective analysis, with the attendant potential biases, although the study was designed to allow for the potential effects of confounding variables. A prospective randomized trial with intent-to-treat analysis is needed, particularly with cumulative success rates across all subsequent transfers using any frozen supernumerary blastocysts.
References Cakmak H, Taylor HS. Implantation failure: molecular mechanisms and clinical treatment. Hum Reprod Update. 2011;17:242–53.
Page 6 of 13
Fatemi HM, Popovic-Todorovic B. Implantation in assisted reproduction: a look at endometrial receptivity. Reprod Biomed Online. 2013;27:530–8. Horcajadas JA, Dıaz-Gimeno P, Pellicer A, Simon C. Uterine receptivity and the ramifications of ovarian stimulation on endometrial function. Semin Reprod Med 2007;25:454–60. Liu L, Zhou F, Lin X, Li T, Tong X, Zhu H, Zhang S. Recurrent IVF failure is associated with elevated progesterone on the day of hCG administration. Eur J Obstet Gynecol Reprod Biol. In press. Murata Y, Oku H, Morimoto Y, Tokuda M, Murata T, Sugihara K, Nagata F, Nakaoka Y, Fukuda A. Freeze–thaw programmes rescue the implantation of day 6 blastocysts Reprod BioMed Online 2005;11:428–433. Nastri CO, Ferriani RA, Raine-Fenning N, Martins WP. Endometrial scratching performed in the non-transfer cycle and outcome of assisted reproduction: a randomized controlled trial. Ultrasound Obstet Gynecol. 2013;42:375–82. Nikas G, Develioglu OH, Toner JP, Jones HW Jr. Endometrial pinopodes indicate a shift in the window of receptivity in IVF cycles. Hum Reprod. 1999;14:787–92. Nikas G, Aghajanova L. Endometrial pinopodes: some more understanding on human implantation? Reprod Biomed Online. 2002;4 Suppl 3:18–23. Pantos K, Nikas G, Makrakis E, Stavrou D, Karantzis P, Grammatis M. Clinical value of endometrial pinopodes detection in artificial donation cycles. Reprod Biomed Online. 2004;9:86–90. Shapiro BS, Richter KS, Harris DC, Daneshmand ST. Dramatic declines in implantation and pregnancy rates in patients who undergo repeated cycles of in vitro fertilization with blastocyst transfer after one or more failed attempts. Fertil Steril. 2001;76:538–42. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Thomas S. Large blastocyst diameter, early blastulation, and low preovulatory serum progesterone are dominant predictors of clinical pregnancy in fresh autologous cycles. Fertil Steril. 2008;90:302–9. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C, Thomas S. Embryo cryopreservation rescues cycles with premature luteinization. Fertil Steril. 2010;93:636–41. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C, Thomas S. 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. 2011;96:344–8. Simon A, Laufer N. Assessment and treatment of repeated implantation failure (RIF). J Assist Reprod Genet. 2012;29:1227–39. Society for Assisted Reproductive Technology. IVF success rate reports: 2011. www.SART.org. Yamanaka M, Hashimoto S, Amo A, Ito-Sasaki T, Abe H, Morimoto Y. Developmental assessment of human vitrified-warmed blastocysts based on oxygen consumption. Hum Reprod. 2011;26:3366–71. Declaration: The authors report no financial or commercial conflicts of interest.
Page 7 of 13
Table 1. Characteristics of retrievals and outcomes of the first attempted transfer. Characteristic
Fresh cycles (n = 163)
Freeze-all cycles (n = 106)
P-value
34.3 5.0 22–44 10.6 5.4
NS
Survival rate (%)
35.3 4.6 24–43 8.1 5.7 –
Cancellations (n, %) Blastocysts formed (n)
24 (14.7) 2.8 2.5
85.0 17.3 12 (11.3) 2.3 1.8
NS NS
Blastulation rate per 2PN oocyte (%) Transfers (n) Blastocysts transferred (n) Implantation rate (%)
36.6 25.8
22.4 14.1
<0.0001
139 2.0 0.7
94 1.8 0.5
0.0193
18.5 32.7 35 25.2 21.5
44.0 41.8 49 52.1 46.2
Maternal age (years) Mean SD Range 2PN oocytes (n)
Live births (n) Per transfer (%) Per retrieval (%)
<0.0001
<0.0001 <0.0001 <0.0001
Values are mean SD unless otherwise stated. NS = not significant.
Comment [KMT2]: Author: please confirm deletion of asterisks or add a table note.
Page 8 of 13
Table 2. Logistic regression of live birth through the first transfer versus available parameters. Parameter
P-value
Odds ratio (95% CI)
Intercept Cycle type Age at retrieval No. of blastocysts transferred No. of 2PN oocytes
0.0068 <0.0001 <0.0001 0.0002 NS
– 3.8 (2.1–7.2) 31.6 (7.6–144.3) 22.3 (4.6–124.4)
Page 9 of 13
Table 3. Cumulative live birth analysis, including study cycle and transfers of frozen–thawed supernumerary blastocysts. Parameter
Fresh cycles (n = 163)
Freeze-all cycles (n = 106)
Live births in first transfer attempt Implantation rate with thawed supernumerary embryos Retrievals resulting in at least one live birth Cumulative live birth rate per retrievala
35 20/83 (24.1)
49 1/4 (25.0)
45
49
45/163 (27.6)
49/106 (46.2)
Values are n or n (%). a
P < 0.05.
Page 10 of 13
Table 4. Logistic regression of cumulative live birth versus available parameters. Parameter
P-value
Odds ratio (95% CI)
Intercept Cycle type Number of 2PN oocytes Age at retrieval
0.0019 0.0287 0.0082 <0.0001
– 1.9 (1.1–3.3) 8.7 (1.8–45.3) 25.8 (6.4–113.4)
Page 11 of 13
Dr Shapiro earned his MD from the University of Nevada, Reno, and his PhD from the University of Amsterdam. He completed his residency in obstetrics and gynaecology and his fellowship in reproduction endocrinology at Yale-New Haven Hospital. He is chief of the Division of
Page 12 of 13
Reproductive Endocrinology and Infertility in the University of Nevada School of Medicine’s Department of Obstetrics and Gynecology. He is also a high-complexity laboratory director. He is the founder and medical director of the Fertility Center of Las Vegas. His recent research has included endometrial receptivity, embryo cryopreservation and alternative ovulatory triggering techniques.
Page 13 of 13