ORIGINAL ARTICLE: ASSISTED REPRODUCTION

Freeze-all policy: fresh vs. frozen-thawed embryo transfer ~es, B.S.,a Marcos Sampaio, M.D., Ph.D.,b Matheus Roque, M.D.,a Marcello Valle, M.D.,a Fernando Guimara and Selmo Geber, M.D., Ph.D.b,c a ORIGEN, Center for Reproductive Medicine, Rio de Janeiro; b ORIGEN, Center for Reproductive Medicine, Belo Horizonte; and c Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Objective: To compare in vitro fertilization (IVF) outcomes between fresh embryo transfer (ET) and frozen-thawed ET (the ‘‘freeze-all’’ policy), with fresh ET performed only in cases without progesterone (P) elevation. Design: Prospective, observational, cohort study. Setting: Private IVF center. Patient(s): A total of 530 patients submitted to controlled ovarian stimulation (COS) with a gonadotropin-releasing hormone– antagonist protocol, and cleavage-stage, day-3 ET. Intervention(s): None. Main Outcome Measure(s): Ongoing pregnancy rates. Result(s): A total of 530 cycles were included in the analysis: 351 in the fresh ET group (when P levels were %1.5 ng/mL on the trigger day); and 179 cycles in the freeze-all group (ET performed after endometrial priming with estradiol valerate, at 6 mg/d, taken orally). For the fresh ET group vs. the freeze-all group, respectively, the implantation rate was 19.9% and 26.5%; clinical pregnancy rate was 35.9% and 46.4%; and ongoing pregnancy rate was 31.1% and 39.7%. Conclusion(s): The IVF outcomes were significantly better in the group using the freeze-all policy, compared with the group using fresh ET. These results suggest that even in a select group of patients that underwent fresh ET (P levels %1.5 ng/mL), endometrial receptivity may have been impaired by COS, and outcomes may be improved by using the freeze-all policy. Use your smartphone (Fertil SterilÒ 2015;-:-–-. Ó2015 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Freeze-all, frozen-thawed embryo transfer, delayed frozen-thawed embryo and connect to the transfer, embryo cryopreservation Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/roquem-fresh-frozen-thawed-embryo-transfer/

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mplantation is one of the most important steps to ensure in vitro fertilization (IVF) cycle success (1); its effectiveness depends on embryo quality, the embryo-endometrium interaction, and endometrial receptivity (ER) (2). Growing evidence in the literature shows that controlled ovarian stimulation (COS), with its supraphysiologic hormonal levels, may decrease ER (3, 4). Moreover, endometrial development can be controlled more precisely during its priming for frozen-thawed embryo transfer (ET) vs. for COS (5). Therefore,

as the COS may have adverse effects on ER (6), and embryo cryopreservation has become a routine procedure in IVF labs (7), the ‘‘freeze-all’’ policy has emerged as an alternative to fresh ET to improve IVF outcomes (8–10). With the freeze-all policy, the entire cohort of embryos is cryopreserved, and the ET is performed later in a natural cycle, or in a cycle with hormonal replacement for endometrial priming. The potential advantage of this method is that it provides a more physiologic environment in which ET can occur; this advantage could lead

Received November 20, 2014; revised January 27, 2015; accepted January 30, 2015. M.R. has nothing to disclose. M.V. has nothing to disclose. F.G. has nothing to disclose. M.S. has nothing to disclose. S.G. has nothing to disclose. Reprint requests: Matheus Roque, M.D., ORIGEN, Center for Reproductive Medicine, Avenida Rodolfo de Amoedo, 140, Barra da Tijuca, Rio de Janeiro, Brazil (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.01.045 VOL. - NO. - / - 2015

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to better pregnancy rates and decrease maternal and perinatal morbidity (11). However, controversies remain regarding patient selection and the threshold at which a cycle becomes supraphysiologic (11). To date, no effective noninvasive clinical tools are available to evaluate ER that can be used during a fresh cycle as well. The best way to select patients for the freeze-all policy seems to be by assessing progesterone (P) levels on the day of final oocyte maturation, as P-level elevation is related to decreased pregnancy rates in fresh cycles (12–14). To our knowledge, no published studies have used this method to select patients for fresh or frozen ET. Therefore, we performed this study to compare IVF outcomes for patients using fresh ET, performed when P levels were 15 follicles on the trigger day. Pregnant patients were followed for %12 weeks of pregnancy, via an ultrasound scan to confirm ongoing pregnancy. Fresh ET was performed only if P was %1.5 ng/mL on the trigger day. The freeze-all strategy was implemented in cases in which P was >1.5 ng/mL on the trigger day.

Study Protocol Patients underwent COS with recombinant folliclestimulating hormone (FSH) (Gonal-f; Merck Serono) starting on day 2 or 3 of menses, with doses ranging from 150 to 450 international units per day, according to the patient's age, in a step-down protocol. A GnRH antagonist (cetrorelix; Cetrotide, Merck Serono) was used for pituitary suppression when a leading follicle achieved 14 mm. Final oocyte maturation was induced with a ‘‘dual trigger’’: 250 mcg of recombinant human chorionic gonadotropin (hCG) (Ovidrel, Merck Serono) and 0.2 mg of triptorelin (Gonapeptyl Daily; Ferring Pharmaceuticals) when R2 follicles reached a diameter of 18 mm (15). The patients underwent transvaginal ultrasound-guided oocyte retrieval 35 hours after the trigger, followed by intracytoplasmic sperm injection (16). On the third day after oocyte retrieval, embryo quality was evaluated, and 1–4 good-quality embryos (6–10 cells with %20% fragmentation, and equal blastomere size) were transferred (fresh cycles). In the freeze-all group, the entire cohort of good-quality embryos was cryopreserved on day 3. Luteal phase support started on the day of oocyte retrieval. All patients received vaginal micronized P in gel form (Crinone 8%, Merck Serono) in a single daily administration. Progesterone was used for R13 days, when a pregnancy test was performed, and until 9 weeks if pregnancy was confirmed (17).

Frozen-Thawed Cycles All embryos were cryopreserved on day 3, by vitrification using an open system. The endometrial priming started on

2

the 2nd day of each patient's menstrual cycle, when E2 valerate was orally administered at a dose of 6 mg/d. After at least 12 days of E2 replacement, an ultrasound scan and hormone-level measurements were performed. If the endometrium was R7 mm, and the P level was %1.5 ng/mL, the frozen-thawed ET was scheduled. The P replacement with vaginal micronized P in gel (Crinone 8%; Merck Serono) for a single daily administration started 3 days before ET. Estradiol valerate and P were continued until the 9th week of pregnancy. If the endometrium was 1.5 ng/mL after endometrial priming, the ET was canceled.

Hormone Analysis Blood samples were collected on the day of the ovulation trigger, and serum P levels were measured using a chemiluminescent immunoassay for quantitative determination of the hormone (Diagnostics Biochem Canada Inc), with a sensitivity of 0.1 ng/mL.

Outcomes Pregnancy was determined by hCG levels measured 11 days after ET. Clinical pregnancy was defined by the observation of intrauterine embryo heart motion by 7 weeks of gestation. Ongoing pregnancy was defined as pregnancy proceeding beyond the 12th week of gestation. The implantation rate was calculated as the ratio of the number of observed embryo heart beats to the number of transferred embryos. The main outcome measure was ongoing pregnancy rate, which was shown to be comparable to that for live births, as a measure of efficacy (18). Implantation, pregnancy, and clinical pregnancy rates were the secondary outcome measures.

Statistical Analysis The data are presented as the mean
standard deviation (SD), or as a percentage. A comparison of the quantitative variables was performed using Student's t-test for independent samples. For a comparison of the categoric data, c2 analysis was performed. Differences were considered significant if P< .05. A logistic regression analysis was performed to determine the variables that could be independently associated with ongoing pregnancy, and could affect outcomes. Age, basal FSH level, antral follicle count, number of retrieved oocytes and mature eggs, number that were bipronuclear (2 PN), number of transferred embryos, and cycle type (freeze-all vs. fresh) were included in the analysis. Statistical analysis was done with SPSS version 19.0 for Windows (SPSS, Inc).

RESULTS During the study period, 1,357 oocyte retrievals were performed, and 530 patients fulfilled the inclusion criteria and agreed to participate in the study. The women's characteristics are shown in Table 1. A logistic regression analysis was performed, and the variables that were found to be independently associated with VOL. - NO. - / - 2015

Fertility and Sterility®

TABLE 1 Clinical characteristics of the patients who were using freeze-all cycles vs. fresh embryo transfer cycles. Characteristic Patient age (y) FSH levels (day 2 or 3) (mIU/mL) Antral follicle count (n) Days of stimulation (n) E2 trigger (pg/mL) P4 trigger (ng/mL) Retrieved oocytes (n) Mature eggs (n) Fertilization rate (%) Post-thaw survival rate (%)

Freeze-all cycles (n [ 179)

Fresh cycles (n [ 351)

P value

35.59
3.46 7.96
2.17 11.46
4.18 10.89
1.09 1,636.28
571.85 1.66
0.14 8.87
4.14 6.28
3.22 76.78
17.10 94.9
9.42

35.83
4.88 8.44
2.56 9.23
3.88 10.94
1.18 1,567.88
649.63 0.70
0.27 7.38
3.94 5.39
3.10 80.12
16.97

.398 .023 .001 .614 .215 .001 .001 .002 .042

Note: Values are mean
SD. E2 ¼ estradiol; FSH ¼ follicle-stimulating hormone; P4 ¼ progesterone. Roque. Freeze-all policy. Fertil Steril 2015.

ongoing pregnancy were age, number of embryos transferred, and cycle type. All other variables were not independently associated with IVF outcomes. The odds ratio (OR) for age was 0.90, with a 95% confidence interval (CI) of 0.858– 0.943 (P¼ .001); the OR for number of embryos transferred was 1.78, with a 95% CI of 1.35–2.35 (P¼ .0001); and the OR for cycle type was 1.73 with a 95% CI of 1.16–2.58 (P¼ .008), favoring the freeze-all group. All measured IVF outcomes (implantation, clinical pregnancy rate, and ongoing pregnancy rate) were higher in the freeze-all group (Table 2).

DISCUSSION To our knowledge, this is the first study comparing the freezeall policy to fresh ET when fresh ET is performed only if P%1.5 ng/mL on the trigger day. The results suggest that the freeze-all policy was advantageous in IVF outcomes, compared with fresh ET. Implantation failure remains an unsolved problem in assisted reproductive technology cycles, and 1 factor responsible for this problem could be poorer ER resulting from COS (2). In the past few years, much effort has been focused on embryo quality and selection. On the other hand, no effective noninvasive clinical tools are available to evaluate ER, to select patients for fresh or frozen ET. To date, using the P levels on the day of final oocyte maturation has seemed to be the best strategy for selecting patients. However, our results suggest that, even among patients who had fresh ET performed only if P was %1.5 ng/mL on the trigger day, the

freeze-all policy has advantages. We observed an increase of 33% and 28% in implantation and ongoing pregnancy rates, respectively. Embryo cryopreservation is a routine procedure in most IVF centers, and it is associated with good outcomes when frozen-thawed ET is performed (7, 19). Therefore, the freezeall policy can be implemented as an alternative, to avoid the deleterious effects of COS on embryo-endometrium synchrony (8, 9). With this strategy, the best embryos can be cryopreserved and transferred into a more receptive endometrium, compared with a fresh cycle, as COS may jeopardize ER (20). Our results showed good post-thaw survival rates (>90%), which are in accordance with those found in previous studies (7, 21). Although we used vitrification for all patients, and given that cryopreservation was performed on day 3, a point that remains unclear is which developmental embryo stage yielded better results (22, 23). The ovarian response differed in the 2 groups; however, this difference fell within a range observed in previous studies. In this range, for 1 ET, IVF outcomes did not differ. In these studies, patients that obtained either between 6 and 15 oocytes (24) or 6 and 13 oocytes (25) had the same pregnancy rates. Furthermore, we performed logistic regression analyses, which showed that the number of oocytes retrieved, the number of mature eggs, and the number of fertilized eggs, were not independently associated with outcomes. In conclusion, IVF outcomes can be improved using the freeze-all policy. Even in a group of patients that was selected for fresh ET (P%1.5 ng/mL), ER may be impaired by COS, and

TABLE 2 In vitro fertilization outcomes for freeze-all cycles and fresh ET cycles. Outcome Embryos transferred, mean
SD Implantation rate Pregnancy rate Pregnancy Clinical pregnancy Ongoing pregnancy

Freeze-all cycles (n [ 179)

Fresh ET cycles (n [ 351)

P value

Relative risk (95% CI)

2.07
0.65 98/370 (26.5)

2.27
0.79 159/797 (19.9)

.003 .011

1.33 (1.07–1.65)

90 (50.3) 83 (46.4) 71 (39.7)

144 (41) 126 (35.9) 109 (31.1)

.038 .017 .044

1.23 (1.01–1.49) 1.29 (1.05–1.59) 1.28 (1.01–1.62)

Note: Values are n (%), unless otherwise indicated. Roque. Freeze-all policy. Fertil Steril 2015.

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ORIGINAL ARTICLE: ASSISTED REPRODUCTION outcomes may be improved by using the freeze-all policy. Further randomized clinical trials are needed to confirm the advantage of this strategy and determine the population for which it would be most beneficial.

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