How to demonstrate that eSET does not compromise ... - Oxford Journals

Download PDF
0 downloads 0 Views 236KB Size Report
Comment
Sep 13, 2009 - Seven hundred and twenty-six couples were divided into two groups. ... results: The probability of a woman having at least one live-born infant was ... Key words: elective single embryo transfer / cryopreservation / IVF/ICSI ...
Human Reproduction, Vol.24, No.12 pp. 3073–3081, 2009 Advanced Access publication on September 13, 2009 doi:10.1093/humrep/dep321

ORIGINAL ARTICLE Infertility

How to demonstrate that eSET does not compromise the likelihood of having a baby? S. Bechoua 1,2,6, K. Astruc 3, S. Thouvenot 4, S. Girod4, A. Chiron1, C. Jimenez 1,2, and P. Sagot 2,4,5 1

Service de Biologie de la Reproduction, CECOS, CHU de Dijon, France 2Faculte´ de Me´decine de Dijon, Universite´ de Bourgogne, France Service d’Epide´miologie et Hygie`ne hospitalie`res, CHU Dijon, France 4Service de Gyne´cologie Obste´trique, CHU de Dijon, France 5Equipe d’accueil EA 4184, Centre d’e´pide´miologie de populations (CEP), Dijon, France 3

6

Correspondence address. E-mail: [email protected]

background: In several randomized studies, elective single embryo transfer (eSET) has proven its effectiveness in reducing twin pregnancy rates while obtaining acceptable overall pregnancy rates. However, there is no outcome measurement consensus to evaluate the effectiveness of eSET versus double-embryo transfer (DET). methods: This study evaluated whether or not adopting an eSET strategy instead of a DET strategy lowers the probability of having at least one live-born infant in good prognosis couples. Seven hundred and twenty-six couples were divided into two groups. The retrospective arm of the study was undertaken on the first group of couples (n ¼ 483, DET group) and the prospective arm performed on the second group of couples (n ¼ 243, SET group). In these specific populations, the probability of a woman having at least one live-born infant and the probability that one embryo utilized leads to a child were the main outcome measures. results: The probability of a woman having at least one live-born infant was 60.5% in the DET group compared with 60.8% in the SET group. The probability of a live-born child per embryo utilized was not significantly different between the SET and the DET groups, 18.9% and 17.6%, respectively. In addition, the cumulative multiple live birth rate was significantly lower in the SET compared with the DET group.

conclusions: In this observational study, using appropriate cryopreservation techniques, the chance of delivering a live baby, per utilized embryo, in an elective SET strategy is as good as that for DET. Key words: elective single embryo transfer / cryopreservation / IVF/ICSI outcome / multiple pregnancy

Introduction During recent decades, the multiple pregnancy rate, especially the twin pregnancy rate, has increased markedly due to assisted reproduction treatment. Multiple pregnancies are considered high-risk pregnancies for both the mother and infants, due to the relative high incidence of maternal, perinatal and neonatal morbidity and mortality (Land and Evers, 2003). In France, according to Blondel et al. (2005), the probability of a woman undergoing a multiple pregnancy being admitted to an intensive care unit is seven times higher than a woman undergoing a single pregnancy. In addition, the C-section rate is higher in the multiple pregnancy population (48% versus 17%). Finally, the mortality rate for the mother is three times higher in multiple versus single pregnancies. In several randomized studies, the elective single embryo transfer (eSET) strategy, which consists of transferring a single embryo with cryopreservation of spare embryos (Tiitinen et al., 2001; Thurin

et al., 2004; Tiitinen, 2004), has proven its effectiveness in reducing twin pregnancy rates while obtaining acceptable overall pregnancy rates in selected patients (Bergh, 2005). On the basis of these studies, in several clinics, a transfer policy was introduced in which relatively young patients with good quality embryos received eSET and the remaining patients received DET (referred to as e-SET versus DET policy). In our in vitro fertilization centre in Dijon, we performed DET from 1 January 2001 to the end of August 2005. In this population, for a specific type of patients (first cycle, age , 35 and three top quality embryos), both the pregnancy rates and the rates of multiple pregnancies were high. In addition, in the frozen and thawed embryos cycles, the pregnancy rate was above 20%. On the basis of these observations, we decided at the beginning of September 2005 to apply an eSET policy to this specific patient population in order to minimize the risk of multiple pregnancies.

& The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

3074 At present, the prevalent evaluation criteria described in the literature are delivery rates and pregnancy rates. However, only delivery rate, when used as the primary clinical outcome, measures the cumulative live birth rate per woman (Veleva et al., 2009). Until now, there has been no consensus on the definition of the most appropriate outcome measure of an IVF/ICSI programme. The aims of this study were 2-fold: first, to determine the appropriate outcome measure to evaluate eSET versus DET strategy, and second, to verify that eSET can be performed without compromising the chances of having at least one baby. Hence, the results of the two different embryo transfer strategies (e-SET versus DET) performed in our IVF centre on two similar patient populations at two different time periods (from 1 January 2001 to 31 August 2005 for the retrospective study and from 1 September 2005 to 31 December 2007 for the prospective study) were compared. Two ways of looking at the results were envisaged: (i) we considered only the patients who had terminated their first IVF/ICSI attempt; and (ii) we took into account all the patients involved in the study. The IVF/ ICSI attempt was considered over when the woman had at least one live-born infant, or when all the embryos were utilized if there was not a live birth. Utilized embryos were defined as the fresh embryos plus the intact and lysed frozen –thawed embryos. The outcome measures we used were the probability for a woman to have at least one live-born infant and the probability of a live birth per embryo utilized (PLBEU) in two populations (SET and DET) at two

Bechoua et al.

different time periods (from 1 January 2001 to 31 August 2005: retrospective study; and from 1 September 2005 to 31 December 2007: prospective study).

Materials and Methods Patients Since 2005, in Dijon Hospital Fertility Centre, eSET has been performed on patients ,35 years, when at least three top quality embryos are present and if it is the first IVF cycle. Before 2005, a DET was performed for the same patient population. Our study was performed on 726 couples who, from 1 January 2001 to 31 December 2007, underwent a first IVF protocol (conventional IVF and ICSI) at the Centre Hospitalier Universitaire of Dijon. The cohort (n ¼ 726) was divided in two groups based on the protocol used. Two groups were considered (Fig. 1): the first group received DET (n ¼ 483; from 1 January 2001 to 31 August 2005) and the second received eSET (n ¼ 243; from 1 September 2005 to 31 December 2007). The groups were comparable. Women were eligible if they were ,35 years of age at the time of the transfer of fresh embryos, were undergoing their first IVF cycle and had at least three embryos of good quality available for transfer or freezing. All the patients for whom eSET was requested for medical reasons were excluded from the analysis. The patient’s characteristics are shown in Table I. The analyses were performed on both the initial population (243 and 483 patients in the SET and DET groups, respectively) and all the patients

Figure 1 Flow chart showing the distribution of our populations. Patients included: either one live birth or the depletion of the frozen – thawed embryos if there was not a live birth. Patients excluded: who had not delivered and still had frozen embryos.

3075

eSET and the probability of having a baby

Table I Characteristics of the SET and DET groups of patients SET

DET

P-value

........................................................................................ # of patients

243

483

Age (mean + SD)

29.7 + 3.2

29.6 + 3.1

# of oocytes

2892

5560

# of embryos

2184

4068

# of top quality embryos 1445

0.78

who had terminated their IVF/ICSI attempt (189 and 423 patients in the SET and DET groups, respectively) (Fig. 1). The results reported are from one fresh cycle and additional frozen/thawed cycles. Excluded from the analysis were: (i) patients who did not pursue their attempt, who had not delivered and still had frozen embryos (divorce, separation, death or other causes); and (ii) patients who were still in the process, had not delivered and still had frozen embryos. These patients represent 12.4% and 22.3% of the initial population in the DET and SET groups, respectively. For these patients, a sensitivity analysis was performed.

3155

# of oocytes/patient

11.9 + 6.1

11.5 + 5.7

0.31

Ovarian stimulation and oocyte retrieval

# of embryos/patient

8.9 + 4.4

8.4 + 4.3

0.10

# of top quality embryos/patient

5.9 + 2.9

6.5 + 3.1

0.01

The same biological and clinical IVF and ICSI techniques were used for both strategies. All the patients involved in our study underwent ovarian stimulation after pituitary suppression with a daily injection of 0.1 mg of decapeptyl (Ipsen Pharma, Paris, France). After down-regulation was verified, ovarian stimulation was performed with recombinant FSH (Puregon, Schering Plough, Courbevoie, France or Gonalf, Merck Serono, Lyon, France). Follicular development was monitored by vaginal ultrasound measurements of follicles combined with blood samples for estradiol analysis. On the day for oocyte retrieval (when three follicles with a diameter 17 mm were visible), 5000 UI of human chorionic gonadotrophin was given (hCG, Ovitrelle, Merck Serono, Lyon, France). Thirty-six hours later, oocyte retrieval was performed under general anaesthesia by transvaginal ultrasound-guided follicle aspiration. All the monitoring was performed by the same gynaecologist at Dijon Hospital Fertility Centre. Progesterone was administrated vaginally daily, from the time of the oocyte retrieval until either a negative pregnancy test or up to 8 weeks of gestation.

Fresh cycles # of fresh transfers

243

483

# of fresh embryos transferred

243

966

# of frozen embryos

1202

2189

# of fresh embryos/ transfer

1

2

# of embryos frozen/ 4.9 + 2.9 patient

4.5 + 3.1

Pregnancies

78

238

Gestational sacs

80

299

Deliveries

72

198

Babies

74

259

Singleton

70

137

Twin

2

61

0.08

Embryo culture and transfer

Thawing cycles # of women having at 134 least one thawing cycle

Oocytes and embryos were cultured in a ready-to-use commercially available medium: IVF medium (Fertipro, Belgium). After incubation for 18 – 20 h, the oocytes were checked for the presence of pronuclei as a proof of fertilization. On the second day after oocyte recovery, developmental stages and morphological aspects of all embryos originating from normally fertilized oocytes were assessed under an inverted microscope with 200 magnification. Embryo transfers were all performed under ultrasound guidance on Day 2 after fertilization. eSET was carried out exclusively on the condition that the morphology of the transferred embryo corresponded to strict morphological criteria: four to five blastomeres with ,20% fragmentation on Day 2 and total absence of multinucleated blastomeres on Day 2. Cryopreservation of supernumerary embryos was performed on the same day as the fresh embryo transfer, depending on their morphological aspect.

243

# of thawing cycles

193

359

# of embryos thawed

400

892

# of transfers

190

354

# of embryos transferred

306

677

Pregnancies

46

71

Gestational sacs

54

82

Deliveries

43

58

Babies

51

69

Singleton

36

47

Twin

6

11

Triplet

1

0

Embryo cryopreservation

Cumulative rates # of embryos utilized

643

# of embryos utilized 122 that led to at least one baby

1858 328

Probability of having at 18.9 (122/643), 17.6 (328/ least one baby per 95% CI (16.0– 1858), 95% CI embryo utilized (%) 22.2) (15.9–19.4)

P ¼ 0.45

Cryopreservation was performed on Day 2 embryos. The criteria for cryopreservation of embryos were at least 4 cells, fragmentation ,20% and no multinucleated blastomeres. The freezing procedure was performed following a slow freeze protocol (28C/min from 208C to 278C, manual seeding, 0.38C/min to 2308C, and 358C/min from 230 to 21508C). An adaptation of a freezing thawing procedure (FREEZE-KIT 1TM and THAW KIT 1TM , Vitrolife, Go¨teborg, Sweeden) was set up in our centre. Briefly, the straws were loaded successively with: sucrose 0.2 M, air (1 cm), the embryo in 1.5 M propanediol þ 0.1 M sucrose, air (1 cm) and 1.5 M propanediol þ 0.1 M sucrose. The straws were stored in

3076 liquid nitrogen until thawed. At the time of thawing, the straws were kept at RT for 2 min, at 37oC for 3 min and at RT for 1 min and the straw contents were then mixed gently for 10 s. The embryo, in a solution of propanediol and sucrose, was then transferred to IVF medium. Thawed embryos were evaluated under 200 magnification for: (i) total blastomere survival; (ii) partially damaged (50% cells survived) or degenerated (,50% cells survived). The frozen embryo transfers were performed using only embryos with 50% of intact blastomeres.

Frozen–thawed embryo transfer All the transfers of frozen and thawed embryo(s) were performed on stimulated cycle which consisted in injection of 75 UI of recombinant FSH, starting Day 2. Ultrasound monitoring was performed on Day 9 to ensure follicle growth. Human chorionic gonadotrophin (5000 UI) was injected when a follicle reached 17 mm in diameter. The embryo(s) were transferred 4 days after the hCG injection only if the level of progesterone in the plasma was 8 ng/ml on the day of the transfer. Cryocycles following eSET were most frequently performed with two embryos, as eSET is not popular in France and couples are reluctant to accept the transfer of only one embryo.

Statistical analysis The characteristics and the fresh embryo outcome of our initial populations were compared using x2 test and t-tests with two-tailed P , 0.05 as the limit of significance. For the two groups of patients, the cumulative rates we measured were: implantation rate per utilized embryo (IR); multiple live birth rate (MLBR); PLBEU; probability for a woman to have at least one live birth (.25 gestational weeks) at the end of the attempt. Those rates were compared using the x2 test with two-tailed P , 0.05 as the limit of significance. Utilized embryos were defined as fresh embryos plus all the frozen/thawed embryos (intact and lysed) utilized in order for the attempt to be over.

Logistic regression analyses In order to estimate the adjusted effect of eSET versus DET strategy on the two main outcome measures, multivariate analyses were performed using logistic regression models. First, regarding the probability of having at least one baby per utilized embryo, adjustment was performed for age, time period and group. Indeed for this analysis, data comprised two levels: level one relating to the embryo and level two relating to the woman within a given group. Second, regarding the probability for a woman to have at least one live birth when the attempt is over, the adjustment was performed for age, time period and number of top quality embryos. After assessing normality and linearity assumptions, age, period and number of top quality embryos were included in models as continuous covariates. The limit of significance was P , 0.05. Data analyses were performed using StataTM version 10.0 statistical analyses software.

Sensitivity analysis We undertook a sensitivity analysis for the excluded group of patients. For these excluded patients who still have frozen embryos at the end of the study, an estimation of the expected number of additional women who would have delivered a live-born infant if they had achieved their attempt is proposed. These patients did not have any delivery from the fresh transfer and only deliveries from cryopreserved embryos could be expected. The probability pi of success (at least one baby) for these attempts is related to the number n of potential thawing cycles left and on the probability of success for each cycle, pc. Assuming pc constant whatever the thawing cycle rank considered, the probability of failure qi (no baby and all the embryos utilized) for our excluded patients with n

Bechoua et al.

potential thawing cycles left is the probability that all potential cycles fail: qi ¼ (1 2 pc)n. Then the probability pi of success for this attempt is: pi ¼12qi ¼12(12pc)n. For these excluded patients, three hypotheses (low, medium and high) were assumed for pc, in order to calculate the expected number of additional women who would have delivered a live-born infant if they had used all their embryos. For the low, medium and high hypothesis, pc is assumed to be equal to 16.15%, 18.3% and 22.2%. These rates correspond, respectively, to the delivery rate per thawing cycle observed (i) in our DET population, (ii) in our initial population and (iii) in our SET population. For the high assumption, we chose the SET group as the reduced pregnancy rates in fresh eSET may be partly compensated by a higher number of good quality embryos available for cryopreservation resulting in higher delivery rate per thawing cycle in the SET compared with the DET group (22.2% versus 16.15% in the SET and DET groups, respectively). The potential number of thawing cycles, n, was defined upon the number of embryos cryopreserved and the mean number of thawed embryo per patient we usually transfer. For each patient with one, two or three embryo(s) cryopreserved left, one potential thawing cycle was considered. The number of potential thawing cycles considered were, respectively, 2 (four or five frozen embryos left), 3 (six or seven embryos left), 4 (eight or nine frozen embryos left) and so on. Then, the probability of success of the attempt, pi, is calculated for each excluded patient of both groups. The sum of these probabilities will give us the final expected number of additional women who would have delivered a live-born infant if they had used all their embryos.

Results Initial population From 1 January 2001 to 31 December 2007, a total of 726 couples were included in our study. Of those, 483 patients underwent DET and 243 underwent eSET (Fig. 1) (i.e. there were at least three good quality embryos to choose from). The groups were not significantly different in terms of age, number of oocytes per patients, number of embryos and number of frozen embryos per patient (Table I). The groups did differ significantly in the mean number of top quality embryos, 5.9 + 2.9 and 6.5 + 3.1 in the eSET and DET groups, respectively (P ¼ 0.01). No significant difference in the implantation rate per fresh embryo transfer was noted between the two groups (32.9 and 30.9% in the eSET and DET groups, respectively). However, the pregnancy rate per transfer was significantly higher in the DET group when compared with the eSET group (49.3% versus 32.1% in the DET and eSET groups, respectively, P , 0.001). In addition, in the cycle in which a single embryo was transferred, the delivery rate was significantly lower than in the DET group (29.6% versus 41% in the SET and DET groups, respectively, P ¼ 0.003), data not shown. Comparing the global effectiveness of the two strategies, the first outcome measure studied was the PLBEU in our initial population (Table I). This probability is not significantly different between the eSET and the DET groups (18.9% versus 17.6% in the SET and DET groups, respectively, P ¼ 0.45). The univariate logistic regression model (adjusted for clustering) indicated that when the patient’s age increases the probability that one embryo utilized leads to at least one baby decreases significantly (OR: 0.96, P ¼ 0.03). However, the group or time period factors did not

3077

eSET and the probability of having a baby

have a significant impact. When adjusting for age and period using a multivariate regression model, neither the group (eSET/DET) nor the time period had a significant effect (ORa: 0.97, P ¼ 0.9 and ORa: 1.04, P ¼ 0.54, respectively) and the age is the only variable that has a significant and independent effect on our measurement criteria (Table II).

Patients who had terminated their attempt The second way to approach the global effectiveness of the two strategies was the study of the cumulative rates of the patients who had terminated their attempts.

The characteristics of the two groups of patients who had terminated their attempt (either one live birth or the depletion of the frozen–thawed embryos if there was not a live birth) are presented in Table III. The number of women responding to this criteria was 423 in the DET group and 189 in the eSET group which corresponds, respectively, to 87.5% (423 out of 483) and 77.7% (189 out of 243) of initial groups. The probability of a woman having at least one live-born infant at the end of the attempt was 60.8% and 60.5% in the SET and DET groups, respectively (P ¼ 0.93). In addition, adopting an eSET strategy lowered significantly the cumulative MLBRs by a factor of 3.6 (28.1% in the DET group

Table II Probability of having at least one baby per embryo utilized in the initial population: result of logistic regression models Univariate analysis

Multivariate analysis

........................................................... OR

95% CI

.............................................................

P-value

ORa

95% CI

P-value

............................................................................................................................................................................................. Age (years)

0.96

0.92– 0.99

0.03*

0.96

0.92–0.99

Time period (years)

1.03

0.96– 1.10

0.37

1.04

0.92–1.16

0.03* 0.54

Group (reference¼DET group)

1.09

0.83– 1.44

0.53

0.97

0.61–1.54

0.90

*Significant.

Table III Characteristics of the patients who had terminated their attempt SET

DET

P-value

............................................................................................................................................................................................. # of patients

189

423

Age (mean + SD)

29.7 + 3.3

29.7 + 3.1

# of embryos

1686

3512

# of top quality embryos

1115

2740

# of embryos/patient

8.9 + 4.4

8.3 + 4.3

0.10

# of top quality embryos/patient

5.9 + 2.8

6.5 + 3.1

0.03

0.98

Fresh cycles # of fresh transfers

189

423

# of fresh embryos transferred

189

846

# of frozen embryos

926

1894

# of fresh embryos/transfer

1

2

# of frozen embryos/patient

4.9 + 2.8

4.5 + 3.1

# of women having at least one thawing cycle

115

272

# of thawing cycles

172

328

# of embryos thawed

362

814

# of transfers

169

323

# of embryos transferred

271

618

IR (per embryo utilized) (%)

24.3 (134/551)

22.9 (381/1660)

0.55

Probability for a woman to have at least one live born infant at the end of the attempt

60.8 (115/189), 95% CI (53.5–67.8)

60.5 (256/423), 95% CI (55.7–65.2)

0.93

MLBR (%)

7.8 (9/115)

28.1 (72/256)

Probability of having at least one baby /embryo utilized %

22.1 (122/551), 95% CI (18.7–25.8)

19.8 (328/1660), 95% CI (17.9– 21.7)

0.11

Thawing cycles

Cumulative rates

IR, implantation rate; MLBR, multiple live birth rate.

,0.001 0.23

3078

Bechoua et al.

Table IV Probability for a woman to have at least one live born infant at the end of the attempt: result of logistic regression models Univariate analysis

........................................................... OR

95% CI

P-value

Multivariate analysis

.............................................................

ORa

95% CI

P-value

............................................................................................................................................................................................. Age (years)

0.96

0.91–1.00

0.11

0.96

0.90– 1.01

0.10

Number of top quality embryos

1.23

1.15–1.33

,0.001

1.24

1.16– 1.34

,0.001

Time period (years)

1.01

0.93–1.10

0.74

1.09

0.94– 1.27

0.24

Group (reference¼DET group)

1.01

0.71–1.44

0.94

0.85

0.47– 1.55

0.60

versus 7.8% in the eSET group, P , 0.001). When considering the cumulative rates, the PLBEU was not significantly different between the two groups (22.1% and 19.8% in the SET and DET groups, respectively, P ¼ 0.23). The multivariate logistic regression modelling the probability for a woman to have at least one live-born infant at the end of the attempt according to age, number of top quality embryos, time period and group (SET versus DET) show that the number of top quality embryos has an independent effect on this probability. The probability of success increases when the number of top quality embryos obtained increases. Neither the age nor the time period nor the group has a significant effect on this probability (Table IV).

Excluded patients One hundred and fourteen patients (60 and 54 patients in the DET and SET groups, respectively) were excluded as they did not deliver and still had frozen embryos. Their characteristics were not significantly different compared with the patients included in the study (data not shown). The distribution of the reasons for exclusion was the following: (i) treatment finished (16.7% and 43.3% in the SET and DET groups, respectively), (ii) treatment in progress (77.8% and 38.3% in the SET and DET groups, respectively), (iii) lost to follow-up (5.5% and 18.4% in the SET and DET groups, respectively).

Sensitivity analysis results In the group of patients excluded from the study, using our model, the expected numbers of additional women who would have delivered if they had used all their embryos, assuming the low, medium and high hypothesis, are, respectively, 15, 17 and 20 in the DET group compared with 15, 16 and 19 in the SET group. In the initial population, according to the low (a), medium (b) and high (c) hypotheses for pc, the projected final cryoaugmented numbers of women who would have delivered are: (i) in the DET group, 271, 273 and 276 out of 483 women [(a) 56.1%, CI 51.6– 60.6; (b) 56.5%, CI 52.0 –61.0; (c) 57.1%, CI 53.6 –61.6]; (ii) in the SET group, 130, 131 and 134 out of 243 [(a) 53.5%, CI 47.0 –59.9; (b) 53.9%, CI 47.4–60.3; (c) 55.1%, CI 48.6 –61.5]. No significant difference was observed between the two groups, P ¼ 0.51, 0.50 and 0.61, respectively.

Discussion Patients under 35 years of age were good responders to DET, with a favourable prognosis for success, yet this patient population was at high risk for multiple pregnancies. Therefore, in September 2005, our centre moved from a DET to an eSET policy for this specific patient’s population, with the condition that at least two additional embryos could be available for cryopreservation and later transferred in the case of an unsuccessful initial fresh eSET outcome. Hence, an ambispective study was undertaken (retrospective from 1 January 2001 to 31 August 2005; prospective from 1 September 2005 to 31 December 2007) on this specific population in order to (i) determine the most appropriate outcome measure to evaluate the practices, (ii) verify whether adopting an eSET strategy lowers the probability of having a live-born infant. It should be noted that these results were obtained from only a single fresh cycle, and additional frozen –thawed cycles. Thus far, several different outcome measures have been used in studies comparing eSET with DET strategy. Some studies defined the outcome in terms of pregnancy rate per cycle (Martikainen et al., 2001; Lukassen et al., 2005; van Montfoort et al., 2006), others in terms of cumulative live birth rates per cycle (Lundin and Bergh, 2007). The cumulative live birth rate per woman was used as the clinical outcome, for the first time by Veleva et al. (2009). However, no consensus has yet been adopted, most probably because the evaluation criteria are not completely satisfying. For instance, when the results are expressed in cumulative rate per woman, all the potential pregnancies the women who still have frozen embryos might have are excluded from the analysis. Indeed, for multiple reasons (divorce, separation, death or other causes), some women might either postpone or not pursue their attempt. One way to circumvent this problem is to consider the results when the attempt is over, although some bias remains because of the time period effect. This explains why, in our study, the proportion of women who had terminated their attempt was higher in the DET group comparatively to the SET group. Other data-handling possibilities are either using the cryoaugmentation approach developed by Jones et al. (1997) or in performing a sensitivity analysis. We chose to apply a sensitivity analysis to our excluded group of patients. These patients were excluded as they had not delivered and still had frozen embryos. Assuming that the excluded patients would have terminated their attempt, the theoretical model we proposed to calculate the projected final cryoaugmentation number of women

eSET and the probability of having a baby

who would have delivered a live-born infant if they had used all their embryos does not reveal any significant difference between the two groups. Nevertheless, in our opinion, the most appropriate outcome measure is the PLBEU. By expressing the results per embryos utilized (fresh; frozen – thawed: intact and lysed), we take into account the possible deleterious effect of the cryopreservation process. Indeed, in our study, the percentage of non-surviving embryos among all frozen and thawed embryos represents 25% of thawed embryos. Five and three women in the SET and DET groups, respectively, did not receive a thawed-embryo transfer because they had no surviving embryos after thawing. A question remains: does the freezing process have a selective effect on the embryos? In other words, are the embryos that do not survive the freezing process the ones that do not have the capability to implant and develop? Similar questions have been raised in the literature. Do frozen embryos have the same developmental potential as fresh embryos? What is the impact of cryopreservation on a population of frozen embryos? The difficulties inherent in providing a simple answer to these questions have been extensively discussed (Ludwig et al., 1998; Speirs, 1998; Testard, 1998). The studies undertaken by Edgar et al. (2000a, b) indicate that intact thawed embryos have the same implantation potential as equivalent fresh embryos and that the impact of cryopreservation is limited to blastomere loss which is directly related to loss of implantation potential. Other studies, in which all the embryos were cryopreserved because of impending ovarian hyperstimulation syndrome, show satisfactory results (Bergh et al., 1995a; Frederick et al., 1995; Queenan et al., 1997; Horwarth et al., 2005), some in the known range for fresh embryo transfers (Urman et al., 2007). However, mostly these studies are retrospective analyses, and therefore are only partially able to answer to these questions. Prospective studies reported in the literature are few, and concern small sample sizes (Selick et al., 1995). One of the most appropriate approaches is similar to the one proposed by Thurin et al. (2004) with three additional conditions: (i) that the embryo chosen for cryopreservation and subsequent transfer in the SET strategy would have been the one selected to perform DET, (ii) that the embryos in SET intended to be either transferred or cryopreserved should be randomly assigned and (iii) that the loss of embryos during the thawing process should be considered. We did not perform a prospective study; however, we took into account in our calculation all frozen –thawed embryo utilized (included lysed embryos). This component (embryos utilized) has never been considered in studies comparing eSET with DET, and we feel it represents a good and accurate way to evaluate the results of an IVF/ ICSI attempt as it includes the potentially deleterious effects of the cryopreservation process. In our opinion, the outcome measures we used in our study are the most appropriate ones. In order to offer the eSET strategy to couples, it is crucial to know if the PLBEU is the same as when performing DET. Discrepancies in terms of pregnancy rates have been published on the eSET versus DET. Indeed, observational studies on SET and DET have focused mainly on fresh embryo transfers and indicate that similar ongoing pregnancy rates can be achieved using either eSET or DET (Gerris et al., 2002; Martikainen et al., 2004; Saldeen and Sundstro¨m,

3079 2005; van Montfoort et al., 2005). However, a number of studies have shown that in randomized settings, DET results in higher pregnancy rate and live birth rate than SET (Gerris et al., 1999; Thurin et al., 2004; Lukassen et al., 2005; van Montfoort et al., 2006). In other randomized controlled trials (Martikainen et al., 2001; Gardner et al., 2004), no significant difference was found between SET and DET. Our results on fresh embryo transfer outcome of our initial population indicate a similar implantation rate (SET: 32.9% versus 30.9%; P ¼ 0.61) and a pregnancy rate in favour of the DET (49.3% versus 32.1% in the SET group, P , 0.001), data not shown. In our initial population, our data indicated that the PLBEU was not significantly different between the eSET and the DET groups (18.9% and 17.6% in the SET and DET groups, respectively, P ¼ 0.45). When considering the women who had terminated their attempt (either one live birth or the depletion of all the embryos if there was not a live birth), we found that this probability was also not significantly different (22.1% versus 19.8% in the SET and DET groups, respectively, P ¼ 0.23). The number of women who had terminated their first IVF attempt was, respectively, 423 in the DET group and 189 in the eSET group which corresponds to 87.5% (423 out of 483) and 77.7% (189 out of 243) of our initial population. The probability for a woman to have at least one live-born infant was not significantly different between the eSET and the DET groups (60.5% in the DET group compared with 60.8% in the SET group). Thus, moving from a DET to a SET strategy in a selected population (women under 35, first cycle, at least three top quality embryos) would maintain the same probability of having at least one live-born infant, and simultaneously decrease the total multiple birth rate by a factor of 3.6 (28.1% in the DET group and 7.8% in the eSET group). By reducing the number of embryos transferred, delivery and multiple birth rates also decrease. These observations have been reported in other countries (Gordts et al., 2005; Karlstro¨m and Bergh, 2007). In our study, we also identified whether or not factors such as the age, the group and the time period had an impact on the PLBEU. To do so, univariate and multivariate logistic regression analyses were performed, indicating that the age was the only significant and predictive factor of diminished probability of having at least one baby. This factor was independent from the group and time period factors. Overall, these results could be achieved only if eSET is applied along with a sound embryo cryopreservation programme. Our data highlight the value of cryopreservation when applying eSET strategy (Martikainen et al., 2001; Tiitinen et al., 2001, Thurin et al., 2004; Veleva et al., 2009). The contribution of embryo cryopreservation following IVF/ICSI provides further possibilities of pregnancy in addition to transfer in fresh cycle (Thurin et al., 2004; Lundin and Bergh., 2007). The contribution of cryopreservation to pregnancy has been reported to increase the baby take-home rate by 5.2% (Kahn et al., 1993) to 11% (Wang et al., 1994) or even 19% (Bergh et al., 1995b). In our study, cryopreservation increases the baby take-home rate by 23% and 14% in the SET and DET groups, respectively. Unlike the study of Veleva et al. (2009), which indicates the superiority of eSET over DET, our study reveals that eSET is as effective as the DET strategy. Our results are in agreement with the randomized studies of Thurin et al. (2004) and Lundin and Bergh (2007) which indicate that the live birth rate for SET was not substantially or significantly lower than that of DET. A randomized controlled trial is a robust

3080 method available to evaluate the equivalence of SET and DET strategies. Unfortunately, our study was not randomized; however, our cumulative outcome measurements were done on a selected patients population and after taking into account all the embryos utilized (fresh, frozen/thawed intact and lysed) which is not the case in other studies which used cumulative outcome measures (Thurin et al., 2004; Tiitinen, 2004; Veleva et al., 2009). In conclusion, here we define appropriate approaches to evaluate the outcomes of a SET versus DET. We assert that the eSET strategy does not compromise the likelihood of having a baby, only if we take into account all the embryos utilized including the lysed embryos after thawing. Our observational study indicates that in a selected population, using appropriate cryopreservation techniques, the chances of obtaining a baby (per utilized embryo) in an eSET strategy are as good as following DET.

Supplementary data Supplementary data are available at http://humrep.oxfordjournals. org/.

Acknowledgements The authors gratefully acknowledge Professor Larry Daniel (Wake Forest University, NC, USA) for helpful suggestions.

References Bergh C. Single embryo transfer: a mini-review. Hum Reprod 2005; 20:323 – 327. Bergh C, Werner C, Nilsson L, Hamberger L. Cumulative birth rates following cryopreservation of all embryos in stimulated in vitro fertilization (IVF) cycles. J Assist Reprod Genet 1995a;12:191– 194. Bergh C, Josefsson B, Nilsson L, Hamberger L. The success rate in a Swedish in-vitro-fertilization unit: a cohort study. Acta Obstet Gynecol Scand 1995b;74:446– 450. Blondel B, Supernant K, du Mazaubrun C, Breart G. Enqueˆte Nationale Pe´rinatale. Situation en 2003 et Evolution depuis 1998. 2005. Edgar D, Bourne H, Jericho H, McBain JC. The developmental potential of cryopreserved human embryos. Mol Cell Endocrinol 2000a;169:69 – 72. Edgar D, Bourne H, Speirs A, McBain JC. A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod 2000b;15:175 – 179. Frederick J, Ord T, Kettel L, Stone S, Balmeceda J, Asch R. Successful pregnancy outcome after cryopreservation of all fresh embryos with subsequent transfer into unstimulated cycle. Fertil Steril 1995; 64:987 – 990. Gardner DK, Surrey E, Minjarez D, Leitz A, Stevens J, Schoolcraft WB. Single blastocyst transfer: a prospective randomized trial. Fertil Steril 2004;81:551 – 555. Gerris J, De Neubourg D, Mangelschots K, Van Royen E, Van de Meerssche M, Valkenburg M. Prevention of twin pregnancy after in vitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized clinical trial. Hum Reprod 1999;14:2581 – 2587. Gerris J, De Neubourg D, Mangelschots K, Van Royen E, Vercruyssen M, Barudy-Vasquez J, Valkenburg M, Ryckaert G. Elective single day 3 embryo transfer halves the twinning rate without decrease in the

Bechoua et al.

ongoing pregnancy rate of an IVF/ICSI programme. Hum Reprod 2002; 17:2626 – 2631. Gordts S, Campo R, Puttemans P, Brosens I, Valkenburg M, Norre J, Renier M, Coeman D, Gordts S. Belgian legislation and the effect of elective single embryo transfer on IVF outcome. Reprod Biomed Online 2005;10:436 – 441. Horwarth D, Check J, Choe J, Wilson C, Amui J. Frozen embryo transfer according to reason for freezing the embryos. Clin Exp Obstet Gynecol 2005;32:19 – 20. Jones HW, Jones D, Kolm P. Cryopreservation: a simplified method of evaluation. Hum Reprod 1997;12:548 – 553. Kahn J, von During V, Sunde A, Sordal T, Molne K. The efficacy and efficiency of an in-vitro fertilization programme including embryo cryopreservation: a cohort study. Hum Reprod 1993;8:247 – 252. Karlstro¨m PO, Bergh C. Reducing the number of embryos transferred in Sweden—impact on delivery and multiple birth rates. Hum Reprod 2007;22:2202 – 2207. Land JA, Evers JLH. Risks and complications in assisted reproduction techniques: report of an ESHRE consensus meeting. Hum Reprod 2003;18:455 – 457. Ludwig M, Al-Hasani S, Felberbaum R, Diedrich K. No impact of cryopreservation and thawing on embryo developmental potential— one more example for the problems of retrospective, non-controlled data. Hum Reprod 1998;13:786 – 787. Lukassen HG, Braat DD, Wetzels AM, Zielhuis GA, Adang EM, Scheenjes E, Kremer JA. Two cycles with single embryo transfer versus one cycle with double embryo transfer: a randomized controlled trial. Hum Reprod 2005;20:702– 708. Lundin K, Bergh C. Cumulative impact of adding frozen– thawed cycles to single versus double fresh embryo transfers. Reprod Biomed Online 2007; 15:76 – 82. Martikainen H, Tiitinen A, Toma´s C, Tapanainen J, Orava M, Tuomivaara L, Vilska S, Hyde´n-Granskog C, Hovatta O, and the Finnish ET Study Group. One versus two embryo transfer after IVF and ICSI: a randomized study. Hum Reprod 2001;16:1900– 1903. Martikainen H, Orava M, Lakkakorpi J, Tuomivaara L. Day 2 elective single embryo transfer in clinical practice: better outcome in ICSI cycles. Hum Reprod 2004;19:1364 – 1366. Queenan JT, Veeck LL, Toner JP, Oehninger S, Muasher SJ. Cryopreservation of all prezygotes in patients at risk of severe hyperstimulation does not eliminate the syndrome, but the chances of pregnancy are excellent with subsequent frozen – thawed transfers. Hum Reprod 1997;12:1573 – 1576. Saldeen P, Sundstro¨m P. Would legislation imposing single embryo transfer be a feasible way to reduce the rate of multiple pregnancies after IVF treatment? Hum Reprod 2005;20:4 – 8. Selick CE, Hofmann GE, Albano C, Horowitz GM, Copperman AB, Garrisi GJ, Navot D. Embryo quality and pregnancy potential of fresh compared with frozen embryos—is freezing detrimental to high quality embryos? Hum Reprod 1995;10:392– 395. Speirs AL. Aspects of epigenetic factors in artificial procreation. Hum Reprod 1998;13:787 – 788. Testard J. Episcinetific aspects of the epigenetic factors in artificial procreation. Hum Reprod 1998;13:783– 785. Thurin A, Hausken J, Hillensjo¨ T, Jablonowska B, Pinborg A, Standell A, Bergh C. Elective single-embryo transfer versus double-embryo transfer in in vitro fertilization. N Engl J Med 2004;351:2392 – 2402. Tiitinen A. Toward single embryo transfer policy. Acta Obstet Gynecol Scand 2004;83:1009 – 1111. Tiitinen A, Halttunen M, Ha¨rkki P, Vuoristo P, Hyde´n-Granskog C. Elective single embryo transfer: the value of cryopreservation. Hum Reprod 2001; 16:1140 – 1144.

eSET and the probability of having a baby

Urman B, Balaban B, Yakin K. Impact of fresh-cycle variables on the implantation potential of cryopreserved-thawed human embryos. Fertil Steril 2007;87:310 –315. van Montfoort AP, Dumoulin JC, Land JA, Coonen E, Derhaag JG, Evers JL. Elective single embryo transfer (eSET) policy in the first three IVF/ICSI treatment cycles. Hum Reprod 2005;20:433 – 436. van Montfoort AP, Fiddelers AA, Janssen JM, Derhaag JG, Dirksen CD, Dunselman GA, Land JA, Geraedts JP, Evers JL, Dumoulin JC. In unselected patients, elective single embryo transfer prevents all multiples, but results in significantly lower pregnancy rates compared

3081 with double embryo transfer: a randomized controlled trial. Hum Reprod 2006;21:338– 343. Veleva Z, Karinen P, Tomas C, Tapanainen JS, Martikainen H. Elective single embryo transfer with cryopreservation improves the outcome and diminishes the costs of IVF/ICSI. Hum Reprod 2009;1:1 – 8. Wang XJ, Ledger W, Payne D, Jeffrey R, Matthews CD. The contribution of embryo cryopreservation to in-vitro fertilization/gamete intrafallopian transfer: 8 years experience. Hum Reprod 1994;9:103– 109. Submitted on June 26, 2009; resubmitted on August 7, 2009; accepted on August 14, 2009