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Comparable clinical outcomes and live births after single vitrified–warmed and fresh blastocyst transfer
Reproductive BioMedicine Online (2012) 25, 466– 473
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ARTICLE
Comparable clinical outcomes and live births after single vitrified–warmed and fresh blastocyst transfer Guixue Feng, Bo Zhang *, Hong Zhou, Jinhui Shu, Xianyou Gan, Fangrong Wu, Xihe Deng Reproductive Medicine center, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning 530003, China * Corresponding author. E-mail address: [email protected] (B Zhang). Guixue Feng, Ph.D. is an associate researcher and an associate director of the Center of Reproductive Medicine. After award of his doctorate from Guangxi University, he worked in an assisted reproduction laboratory for 9 years and gained his skills in techniques such as IVF, intracytoplasmic sperm injection, preimplantation genetic diagnosis, vitrification of embryos/oocytes and selective single-blastocyst transfer. He is proficient in a variety of laboratory and management skills and has published more than 10 papers in the field of reproduction medicine.
Abstract Selective single-blastocyst transfer (SBT) in fresh cycles has been effective in reducing multiple pregnancies. However,
we do not know whether this successful strategy of fresh transfer cycles is suitable for cryopreserved cycles. The present study was undertaken to evaluate the feasibility and value of SBT in vitrified–warmed cycles. Clinical pregnancy rate (CPR) was similar with vitrified and fresh SBT (46.61% versus 52.15% respectively). Of the pregnant patients, monozygotic twin, miscarriage and ectopic pregnancy rates were similar with vitrified and fresh SBT. For the newborns, no significant difference was observed in live birth, low birthweight, premature delivery and birth defects rates between vitrified and fresh SBT. With respect to the quality of transferred blastocysts (from BB to AA), a similar CPR and miscarriage rate was obtained for both vitrified and fresh SBT when a similar blastocyst cohort graded 3BB was transferred. The data show that vitrified SBT is an effective means of reducing multiple pregnancy and that comparable clinical outcomes and live births are achieved if single blastocysts graded 3BB are transferred for both vitrified and fresh SBT. These data should encourage clinics to evaluate their embryo transfer policy and adopt vitrified SBT as everyday practice. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: clinical pregnancy, implantation, single-blastocyst transfer, vitrification
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.07.008
Single vitrified–warmed and fresh blastocyst transfer
Introduction The goal of assisted reproduction technology is the birth of a healthy singleton infant. However, in 2004–2005, twins accounted for approximately 30% of assisted reproduction-related live births in the USA (Luke et al., 2010) and approximately 21% in Europe (Nyboe Andersen et al., 2009), in comparison with 1.6% of births from naturally conceived pregnancies worldwide (ESHRE, 2000). Multiple pregnancies are associated with unwanted morbidity risks to the mother and fetus as well as creating additional costs related to prenatal and post-partum health care in assisted reproduction (Fiddelers et al., 2007; Kjellberg et al., 2006; Martin et al., 2007). Multiple pregnancies, therefore, are defined as a common complication but not a successful pregnancy outcome (ESHRE, 2003). More recently, increased pregnancy rates, coupled with concerns about maternal and perinatal morbidity associated with multiple pregnancy, have led to attempts to restrict the number of embryos transferred, especially for good-prognosis patients. It has been admitted that the most effective method to reduce the multiple pregnancy rate after IVF is to adopt a policy of single-embryo transfer (SET) (Lukassen et al., 2005; Thurin et al., 2004). However, in many countries concerns remain that SET is less effective and more expensive than conventional IVF, which has resulted in SET not being widely adopted. With the improvement of blastocyst culture techniques, a number of investigators have reported superior clinical results with blastocyst transfer than transfers using cleavage-stage embryos (Barrenetxea et al., 2005; Levitas et al., 2004; Schwarzler et al., 2004). It has been suggested that the extra time needed to culture embryos to the blastocyst stage helps to distinguish the viability of potential embryos for eventual transfer. Further studies have demonstrated that single-blastocyst transfer (SBT) has been the most effective strategy to reduce multiple pregnancies while maintaining the overall success rate of the fresh transfer cycles (Criniti et al., 2005; Khalaf et al., 2008; Ryan et al., 2007; Stillman et al., 2009; Styer et al., 2008). Based on the trend to reduce multiple pregnancies, cryopreservation of embryos has become a necessary tool in assisted reproductive technology, leading to an increased cumulative outcome while decreasing costs (Herrero et al., 2011; Veleva et al., 2009). The literature contains a number of papers which compare the clinical outcomes of vitrification of blastocysts to that of the slow-cooling method. The single clear conclusion derived from these studies is that vitrification is a more effective cryopreservation tool for blastocyst when compared with the slowcooling method (Hong et al., 2009; Stehlik et al., 2005; Youssry et al., 2008). The increasing experience and success with cryopreservation of blastocysts has prompted an interest in decreasing the number of embryos transferred during cryopreserved cycles. But owing to concerns regarding the decreased implantation potential of cryopreserved blastocysts, IVF institutions may hesitate to apply the same criteria of fresh SBT to cryopreserved cycles because there have been only a few studies which addressed vitrified–warmed SBT (Yanaihara et al., 2008; Berin et al., 2011), especially with respect to comparisons of clinical outcomes and live births between vitrified and fresh SBT. Within the study
467 centre’s IVF unit, an elective SBT programme for fresh transfer cycles was established in 2009 and this resulted in favourable pregnancy rates, which encouraged the evaluation of the feasibility of SBT in cryopreserved cycles. The present study was undertaken to investigate whether vitrified SBT compromised outcomes of the IVF protocol compared with fresh SBT.
Materials and methods Patient cycle selection From January 2009 to December 2010, a total of 604 cycles (560 patients) of fresh SBT and 384 cycles (350 patients) of vitrified SBT were involved in this study. The indications of IVF were tubal infertility, mild male factor infertility, unexplained infertility, early stage endometriosis and polycystic ovary syndrome. In fresh SBT cycles, patients with a good prognosis and a high risk of multiple pregnancy (i.e. patients in the first or second assisted reproduction cycle, who were younger than 38 years old, with more than three good embryos on day 3 and a normal endometrium were advised to undergo SBT. Cleavage embryos were transferred if patients refused to receive SBT and the surplus embryos were cultured to the blastocyst stage. On day 5 or 6, all blastocysts were scored by the grading system established by Gardner and Schoolcraft (Gardner et al., 2000) and then were vitrified one by one for the first three vials if blastocysts 3BB were obtained. In vitrified SBT cycles, the patients younger than 38 years old, who did not become pregnant from fresh SBT (n = 231), who did not become pregnant after transfer of fresh early cleavage stage embryos (n = 132) and in whom fresh embryo transfer were cancelled (n = 21), were included. The patients included in the study in the vitrified SBT group were those who had a transfer between January 2009 and December 2010. Also included were those who had a fresh transfer before 2009 but did not become pregnant and subsequently underwent vitrified–warmed blastocyst transfer after January 2009. After warming, blastocysts were scored again and blastocysts scored 3BB were transferred singly. The clinical outcomes and live births of vitrified SBT were compared with fresh SBT. Both vitrified and fresh SBT cycles were further subdivided into three groups according to the quality of transferred blastocysts based on trophectoderm and inner cell mass quality scores (AA, AB or BA, and BB) and the clinical pregnancy and miscarriage rates compared. The study was approved by the reproductive ethical committee of the Maternal and Child Health Hospital of Guangxi Zhuang (reference number 2008001, approval granted on 6 March 2008).
Ovarian stimulation protocols, oocytes collected, oocyte insemination and embryo culture Ovulation induction was carried out after down-regulation with leuprolide acetate (Lupron; TAP Pharmaceuticals, Lake Forest, IL, USA). FSH (Follistim; Organon, USA; or Gonal F; Serono, Italy) was administered at a dose of 225 IU per day, unless previous stimulations indicated otherwise. When dominant follicles reached a diameter of 18 mm, ovulation
468 was induced by an intramuscular injection of 5000–10,000 IU human chorionic gonadotrophin (HCG; Serono, Switzerland; Livzon, China). Oocytes were recovered by transvaginal aspiration of follicles under ultrasound guidance. Oocytes were fertilized with the partner‘s spermatozoa 2–6 h after aspiration either conventionally or by intracytoplasmic sperm injection. Fertilization was confirmed and pronuclei were evaluated at 16–18 h after insemination and only zygotes displaying two pronuclei were selected to further culture for subsequent transfer. The embryos were cultured in Quinn’s 1026 medium (Sage, USA) supplemented with 10% synthetic serum substitute (SSS; Sage) from day 0–3 and then were removed into Quinn‘s 1029 medium (Sage) supplemented with 10% SSS for another 48 h, within a humidified incubator set at 37C, with an environment of 5.6% CO2 and atmospheric O2 tension.
Cryopreservation protocols Blastocysts were artificially shrunk before vitrification using a glass micropipette, as described in a previous study (Feng et al., 2010), and were vitrified one by one for the first three vials if blastocysts graded 3BB were obtained. The vitrification procedure was performed on a heating stage maintained at 37C. The blastocyst was first rinsed for 30 s in 200 ll HEPES-buffered culture medium (Sage) supplemented with 20% human serum albumin (Sage). Then, the blastocyst was enclosed in 200 ll equilibration solution containing 10% (v/v) ethylene glycol (EG; Sigma, USA) and 10% (v/v) dimethylsulphoxide (DMSO; Sigma) and incubated for 1 min. The blastocysts were then transferred to the vitrification solution consisting of 20% EG, 20% DMSO and 0.3 mol/l sucrose (Sigma) for 30 s. A microdroplet (<0.5 ll) containing one blastocyst was sucked into the narrowest end of the glass micropipette and quickly plunged into liquid nitrogen. On the day of transfer, blastocysts were warmed one by one according to their quality, which was estimated prior to vitrification and graded from high to low. Blastocysts were warmed by quickly immersing the capillary end of the glass micropipette into 200 ll warming solution (HEPES-buffered culture medium supplemented with 20% human serum albumin (HSA, Sage) and 0.6 mol/l sucrose for 2 min and subsequently transferred to 0.5 and 0.25 mol/l of sucrose for 3 min and 5 min, respectively, at 37C. Then the blastocysts were transferred to HEPES-buffered culture medium with 20% HSA for 20 min. Finally, the blastocysts were transferred to blastocyst medium (Sage, USA) in a 5.6% CO2 and atmospheric O2 tension incubator for 1–4 h and then were graded before transfer.
Embryo transfer procedure Patients were prepared for vitrified–warmed or fresh SBT using hormone replacement therapy consisting of increasing doses of oestrogen and progesterone (Progynova; Byer, Germany; Utogestan, Laboratoires Besins International, France) starting on day 3. The only monitoring in the replacement cycle was the evaluation of the endometrium prior to progesterone administration. Warming cycles were cancelled if the endometrial thickness was <7 mm. Embryo
G Feng et al. transfer was performed under ultrasound guidance using a Wallace Sure View catheter. Pregnancy testing was performed 14 days after the embryo transfer. Clinical pregnancy was confirmed by the presence of gestational sacs on ultrasound examination at 6–8 weeks in the hospital and miscarriage was defined as the loss of an embryo or fetus before the 28th week of pregnancy. The information regarding the delivery and children were collected by questionnaires to the parents. Implantation rates were defined as the number of gestational sacs observed on ultrasound scanning divided by the number of embryos transferred.
Data analysis The statistical analysis was performed using Statistical Package for Social Sciences version 13.0 (SPSS, USA). Values were reported as mean ± SD or frequencies. Means in groups were compared by Student0 s t-test and frequencies in groups were compared by chi-squared test. Statistical significance was attributed to two-tailed P < 0.05.
Results As shown in Table 1, there were no differences in patient age, method of fertilization and primary/secondary infertility between fresh and vitrified SBT. Endometrial thickness on HCG day was lower with fresh SBT than vitrified SBT (P < 0.01) and there was a significant difference in the rate of day-5 or day-6 blastocyst transfer between fresh and vitrified SBT (P < 0.01). A total of 405 vitrified blastocysts in 384 cycles were warmed with a 94.81% (384/405) survival rate. All surviving blastocysts were graded 3BB and singly transferred, which resulted in 46.61% (179/384) implantation/clinical pregnancy rate. A total of 604 fresh blastocysts graded 3BB were singly transferred, which resulted in 52.15% (315/604) implantation/clinical pregnancy rate. No statistically significant difference was observed in implantation/clinical pregnancy rate and live birth rate between these two groups. Of the pregnant patients, the monozygotic twin, miscarriage and ectopic pregnancy rates in vitrified SBT also showed no statistically significant difference compared with fresh SBT (2.79% versus 3.17%; 16.20% versus 13.02%; 2.79% versus 4.44%). In vitrified SBT, there were 142 singleton and one monozygotic twin live births, resulting in 144 healthy newborns, and in fresh SBT, 252 singleton and seven monozygotic twin live births, resulting in 266 healthy newborns. This study only compared the singleton live births between fresh and vitrified SBT because the number of newborns resulting from twin pregnancies was very small. As shown in Table 2, the mean gestational age at delivery was 38.61 ± 1.64 weeks in vitrified SBT and 38.17 ± 4.60 weeks in fresh SBT, which were not significantly different. Eleven of the 142 gestations (7.75%) were delivered between 34 and 37 weeks in vitrified SBT and 18 of 252 gestations (7.14%) were delivered in fresh SBT. Three of 142 gestations (2.11%) delivered at less than 34 weeks in vitrified SBT while seven of 252 gestations (2.78%) delivered in fresh SBT. The mean birthweights of vitrified and fresh SBT were 3155.35 ± 590.09 g and 3103.81 ± 515.55 g, respectively, which means that the
Single vitrified–warmed and fresh blastocyst transfer
469
Table 1 Comparison of patient characteristics and clinical outcomes between vitrified and fresh singleblastocyst transfer (SBT). SBT cycles
P-value
Vitrified (n = 384)
Fresh (n = 604)
Protocol IVF ICSI
328/384 (85.42) 56/384 (14.58)
524/604 (86.75) 80/604 (13.25)
NS NS
Primary infertility Secondary infertility Age (years) Endometrial thickness on HCG day (mm)
133/384 (34.64) 251/384 (65.36) 31.60 ± 3.56 9.34 ± 1.78
203/604 (33.61) 401/604 (66.39) 31.02 ± 3.69 11.20 ± 2.30
NS NS NS <0.01
Cryopreservation Vitrified blastocysts Survival rate
405 (1.07 ± 0.28) 384/405 (94.81)
– –
– –
Blastocyst transfer Day 6 Day 5
21/384 (5.47) 363/384 (94.54)
6/604 (0.99) 598/604 (99.01)
<0.01 <0.01
Implantation/clinical pregnancy Monozygotic twins Miscarriages Ectopic pregnancies Stillbirths
179/384 (46.61) 5/179 (2.79) 29/179 (16.20) 5/179 (2.79) 2/179 (1.11)
315/604 (52.15) 10/315 (3.17) 41/315 (13.02) 14/315 (4.44) 1/315 (0.32)
NS NS NS NS NS
Live births Singletons Monozygotic twins
143/384 (37.24) 142 1
259/604 (42.88) 252 7
NS
Values are n/total (%) or mean ± SD. ICSI = intracytoplasmic sperm injection; NS = not statistically significant.
babies were slightly heavier for vitrified SBT when compared with fresh SBT, but the difference did not reach statistical significance. The rates of low birthweight (1500–2500 g) and very low birthweight (<1500 g) in vitrified SBT were not significantly different when compared with fresh SBT (5.63% versus 7.94%, and 1.41% versus 0%, respectively). Birth defects were found in 1.41% (2/142) of the children in vitrified SBT and 0.79% (2/252) of the children in fresh SBT, which means that no difference was also observed between these two groups. Of 384 cycles in vitrified SBT, the rates of transferred blastocysts graded AA, AB or BA, and BB in vitrified SBT were 3.13%, 40.63% and 56.25% respectively, and of 604 cycles fresh SBT, 35.10%, 52.48% and 12.42% respectively, which were significantly different between the SBT groups (P < 0.05) (Table 3). With the improvement of transferred blastocyst quality from BB to AA, there was a non-significant trend of a gradual improvement of the clinical pregnancy rate from 94/216 (43.52%) to 8/12 (66.67%) in vitrified SBT and 30/75 (40.00%) to 119/212 (56.13%) in fresh SBT. Similar implantation/clinical pregnancy and miscarriage rates were obtained for vitrified SBT compared with fresh SBT when a similar cohort of blastocysts graded 3BB was transferred (Figures 1 and 2).
Discussion This study found that similar clinical outcomes and live births were achieved after single vitrified–warmed and fresh blastocyst transfer. A new embryo transfer strategy of SBT in vitrified–warmed cycles is therefore encouraged as everyday practice. Multiple pregnancy remains the foremost complication of IVF therapy. There has recently been an effort to decrease the number of embryos transferred in cryopreserved cycles in order to reduce the incidence of multiple pregnancy. Since the success and experience with extended culture has allowed for better selection methods, there has been increasing interest in assessing the efficacy of transferring a single cryopreserved blastocyst. Studies have demonstrated that SBT has been the most effective strategy in reducing multiple pregnancies while maintaining the overall success rate in fresh transfer cycles (Criniti et al., 2005; Khalaf et al., 2008; Ryan et al., 2007; Stillman et al., 2009; Styer et al., 2008). However, owing to a lack of adequate studies, it is not known whether this successful criteria of fresh SBT is suitable for cryopreserved cycles. Major problems with blastocyst culture have been the somewhat unpredictable results obtained with regard to
470
G Feng et al.
SBT singleton pregnancies
Birth sex ratio (male:female) Gestational age
Vitrified (n = 142)
Fresh (n = 252)
76:66 (1:0.87)
142:110 (1:0.77)
38.61 ± 1.64
38.17 ± 4.60
100.0%
fSBT
90.0%
Clinical pregnancy rate 1
Table 2 Comparison of the live births from singleton pregnancies between vitrified and fresh single-blastocyst transfer (SBT).
80.0%
vSBT 66.67%
70.0% 60.0%
56.13%
11 (7.75) 3 (2.11)
18 (7.14) 7 (2.78)
Birthweight (g) Low birthweight (1500 to 2500 g) Very low birthweight (<1500 g)
3155.35 ± 590.09 8 (5.63)
3103.81 ± 515.55 20 (7.94)
2 (1.41)
0
Birth defects
2 (1.41)
49.35%
43.52% 40.00%
40.0% 30.0% 20.0% 10.0% 0.0%
AA
Time of delivery 34–37 weeks <34 weeks
52.36%
50.0%
AB or BA
BB
Figure 1 Comparison of clinical pregnancy rates in vitrified and fresh single-blastocyst transfer (vSBT and fSBT) when a similar cohort of blastocysts graded 3BB was transferred. No statistically significant differences were found between the transfer groups for each grading category.
30.0%
fSBT vSBT
27.5% 25.0%
Values are n (%) or mean ± SD. There were no statistically significant differences between the two groups.
Miscarriage rate
22.5%
2 (0.79)
20.0% 17.5%
Grade
AA AB or BA BB
SBT cycles
P-value
Vitrified (n = 384)
Fresh (n = 604)
12 (3.13) 156 (40.63) 216 (56.25)
212 (35.10) 317 (52.48) 75 (12.42)
<0.01 <0.01 <0.01
Values are n (%)
survival of embryos and the pregnancy rate after blastocysts are frozen with the traditional slow-freezing methods (Liebermann and Tucker, 2006). The explanation may be technical, owing to the size, multicellular structure and the presence of the blastocoele within the blastocyst (Kader et al., 2009). A more reliable and consistent method than conventional cryopreservation of blastocysts (slow method) seems to be vitrification (Hong et al., 2009; Stehlik et al., 2005; Youssry et al., 2008), which has been associated with higher implantation rates. However, concerns have been raised with regard to possible toxic effects of the high concentrations of cryoprotectants (Vajta and Nagy, 2006). Many published studies have found that the pregnancy and live delivery rates are higher for the transfer of fresh compared with frozen–thawed embryos (mainly employing the slow-freezing method). Contrary conclusions have been made by studies using the transfer of vitrified–warmed blastocysts, which yielded higher implantation and clinical pregnancy rates when compared with fresh blastocyst transfer as a result of better endometrial receptivity and synchronization and the weeding out of poor-quality embryos through
12.50%
12.5% 10.0% 7.5%
Table 3 Comparison of blastocyst quality between vitrified and fresh single-blastocyst transfer (SBT).
15.96%
15.0%
13.33%
12.65% 11.69%
7.56%
5.0% 2.5% 0.0%
AA
AB or BA
BB
Figure 2 Comparison of miscarriage rates in vitrified and fresh single-blastocyst transfer (vSBT and fSBT) when a similar cohort of blastocysts graded 3BB was transferred. No statistically significant differences were found between the transfer groups for each grading category.
the act of cryopreservation itself (Zhu et al., 2011). As far as is known, there are very few studies associated with single cryopreserved blastocysts that have compared the clinical outcomes of one with two cryopreserved blastocysts. Berin et al. (2011) found that the pregnancy rate (34.7%) of single frozen blastocyst transfer were lower compared with double transfers (50.4%) (Berin et al., 2011) while Yanaihara et al. (2008) achieved similar clinical pregnancy with both types of transfer (Yanaihara et al., 2008). However, a significantly higher twinning rate was seen when two blastocysts were transferred in these studies. It is well established that blastocyst quality is an important factor for the clinical outcomes and that blastocyst quality conspicuously decreases with the increasing age of patients. It is reported that blastocysts graded 3AA are appropriate for fresh SBT and achieve desirable clinical outcomes. However, not all patients are able to obtain such high-quality blastocysts (Gardner et al., 2000). Teranishi reported that 3BB blastocysts are suitable for fresh SBT with 41.9% pregnancy rate (Teranishi et al., 2009). Yanaihara reported that more than 3BB blastocysts were frozen and these resulted in a 40.7% pregnancy rate after single vitrified-warmed blastocyst, which was not significantly different to that compared
Single vitrified–warmed and fresh blastocyst transfer with the double vitrified–warmed blastocysts transfer (46%) (Yanaihara et al., 2008). Korosec reported that the pregnancy rate was similar between SBT and double-blastocyst transfer in frozen–thawed transfer cycles when the embryo transfer solution contained a high concentration of hyaluronic acid (Korosec et al., 2007). In the present study, a 46.61% clinical pregnancy rate and a 37.24% live birth rate in vitrified SBT was achieved, which is slightly lower than that observed in fresh SBT (52.15% and 42.88%, respectively), but this did not reach statistical significance. This may be explained by the higher quality of blastocysts in fresh SBT observed when compared with vitrified SBT. Two reasons can be considered as explanations for this finding: (i) after warming, blastocysts were damaged during the procedure of vitrification and warming, which caused the lower scores when compared with fresh blastocysts; (ii) although the transferred blastocysts were scored 3BB and the age of patients was younger than 38 years old in both fresh and vitrified SBT, the patients of fresh SBT only included young, good-prognosis patients, while the patients of vitrified SBT included those who did not became pregnant from fresh SBT, those who did not became pregnant after transfer of fresh early cleavage stage embryos and those for whom fresh embryo transfers were cancelled. However, the mean age of patients between the two groups was not significantly different. With the improvement of transferred blastocyst quality from BB to AA, there was a trend of gradual improvement in the clinical pregnancy rate from 43.52% to 66.67% in vitrified SBT and from 40.00% to 56.13% in fresh SBT. Similar implantation/clinical pregnancy and miscarriage rates were obtained when transferring a similar cohort of blastocysts between the fresh and vitrified SBT groups. There is increasing interest in the safety of human assisted reproduction. During cryopreservation, embryos are at risk for cryodamage, resulting in lower clinical pregnancy outcomes. However, in relation to baby outcomes, recent evidence suggests that better perinatal outcomes follow the transfer of frozen–thawed embryos rather than fresh embryos (Shih et al., 2008; Wang et al., 2005). Babies conceived by the transfer of thawed embryos may have significantly lower rates of preterm birth and perinatal mortality than those following the transfer of fresh embryos (Shih et al., 2008). Live-born babies are also less likely to be low birthweight following the transfer of thawed compared with fresh embryos (Wang et al., 2005). Takahashi et al. (2005) reported a 4-year follow-up study of children born after transfer of vitrified blastocysts. They found no differences in obstetric outcomes for children born after vitrified blastocysts as compared with children born after fresh blastocysts with an 18.5% rate of preterm birth and a 43.5% low birthweight rate. In the present study, 16.20% (29/179) of pregnant patients in vitrified SBT suffered clinical miscarriage, with no difference observed compared with 13.02% (41/315) in fresh SBT, which agrees with spontaneously conceived pregnancies where there is a 10–15% rate of clinical miscarriage. No difference was observed in the incidence of delivery before 37 weeks in vitrified SBT compared with fresh SBT (9.86% versus 9.92%). The incidences of low birthweight (<2500 g) and very low birthweight (<1500 g) babies were 5.63% and 1.41%, respectively, which are similar to the rates of these outcomes in fresh SBT (7.94% versus 0%). It is well known that the number of previous children born can
471 affect both pregnancy length and birthweight. However, these data are not available, which is a limitation of this study. An increasing number of papers have compared assisted reproduction babies with those spontaneously conceived (Klemetti et al., 2005; Olson et al., 2005) and there have been several systematic reviews (Hansen et al., 2005), which show an increase in the risk of congenital abnormality associated with assisted reproduction technology. It is generally accepted that each step of in-vitro manipulation may compromise the viability of zygotes/embryos or cause chromosomal abnormality. Based on the present data, although blastocysts of vitrified SBT underwent the cryopreservation procedure, which may cause damage to blastocysts, no difference was observed in the rate of birth defects compared with fresh SBT. This is in line with the results of Wikland et al. (2010), who observed no adverse neonatal outcomes in children born after transfer of vitrified blastocysts as compared with fresh blastocysts. A systematic review documented that blastocyst transfer results in high implantation rates and high pregnancy rates when compared with cleavage embryo transfer while accompanying an increased rate of monozygotic twinning (Chang et al., 2009). Another meta-analysis included 27 studies and found blastocyst transfer to be associated with a 4-fold higher risk of monozygotic twinning as compared with spontaneous conception (1.7% versus 0.4%; Vitthala et al., 2009). The risks of monozygotic twins to both mother and unborn babies are much larger when compared with dizygotic twin pregnancy (Duncombe et al., 2003). However, the results of Papanikolaou et al. (2010) showed that the monozygotic twin rate was similar between the transfer of fresh blastocysts and fresh cleavage embryo transfers (Papanikolaou et al., 2010). Yanaihara (2008) found that, regardless of the transfer of one or two frozen–thawed blastocysts, there was no significant difference in the miscarriage rate, but there were significant differences in both the ectopic pregnancy incidence rate and monozygotic twin rate (Yanaihara et al., 2008). Compared with Yanaihara’s results for the monozygotic twin rate (2.3%), the present study found that the monozygotic twin rate (2.79%) of vitrified SBT was similar when transferring one vitrified–warmed blastocyst and there was no difference observed when compared with monozygotic twin rate of fresh SBT (3.17%). In conclusion, acceptable pregnancy outcomes were achieved after single vitrified blastocyst transfer (grade 3BB) and no adverse effects on neonatal outcome were observed compared with fresh SBT. These data should encourage clinicians to evaluate their embryo transfer policy and adopt vitrified SBT as their everyday practice. Continuous follow up of obstetric and neonatal outcomes as well as long-term follow up of children is recommended until several larger series have been evaluated.
Acknowledgements This work was supported by Guangxi Zhuang Autonomous Region Natural Science Foundation of China (Grant nos. 0897007, 0832183 and 0542058) and the Health Department of Guangxi Zhuang Autonomous Region (Grant nos. 200947, 2011063 and 2011065). The authors wish to thank the
472 clinicians, clinical embryologists, laboratory technologists and nurses of the Centre for Reproductive Medicine. They are also grateful to Dr S Sooranna, Imperial College London, for editing this manuscript.
References ESHRE, 2000. Multiple gestation pregnancy. The ESHRE Capri Workshop Group. Hum. Reprod. 15, 1856–1864. ESHRE, 2003. 6. Ethical issues related to multiple pregnancies in medically assisted procreation. Hum. Reprod. 18, 1976–1979. Barrenetxea, G., Lopez de Larruzea, A., Ganzabal, T., Jimenez, R., Carbonero, K., Mandiola, M., 2005. Blastocyst culture after repeated failure of cleavage-stage embryo transfers: a comparison of day 5 and day 6 transfers. Fertil. Steril. 83, 49–53. Berin, I., McLellan, S.T., Macklin, E.A., Toth, T.L., Wright, D.L., 2011. Frozen–thawed embryo transfer cycles: clinical outcomes of single and double blastocyst transfers. J. Assist. Reprod. Genet. 28, 575–581. Chang, H.J., Lee, J.R., Jee, B.C., Suh, C.S., Kim, S.H., 2009. Impact of blastocyst transfer on offspring sex ratio and the monozygotic twinning rate: a systematic review and meta-analysis. Fertil. Steril. 91, 2381–2390. Criniti, A., Thyer, A., Chow, G., Lin, P., Klein, N., Soules, M., 2005. Elective single blastocyst transfer reduces twin rates without compromising pregnancy rates. Fertil. Steril. 84, 1613–1619. Duncombe, G.J., Dickinson, J.E., Evans, S.F., 2003. Perinatal characteristics and outcomes of pregnancies complicated by twin-twin transfusion syndrome. Obstet. Gynecol. 101, 1190–1196. Feng, G.X., Zhang, B., Shu, J.H., Zhou, H., Zhou, L., Gan, X.Y., Liu, Y., 2010. Effects of artificial shrinkage of blastocoeles before vitrification on pregnancy outcome. Zhonghua Fu Chan Ke Za Zhi 45, 838–842. Fiddelers, A.A., Severens, J.L., Dirksen, C.D., Dumoulin, J.C., Land, J.A., Evers, J.L., 2007. Economic evaluations of singleversus double-embryo transfer in IVF. Hum. Reprod. Update 13, 5–13. Gardner, D.K., Lane, M., Stevens, J., Schlenker, T., Schoolcraft, W.B., 2000. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil. Steril. 73, 1155–1158. Hansen, M., Bower, C., Milne, E., de Klerk, N., Kurinczuk, J.J., 2005. Assisted reproductive technologies and the risk of birth defects – a systematic review. Hum. Reprod. 20, 328–338. Herrero, L., Martinez, M., Garcia-Velasco, J.A., 2011. Current status of human oocyte and embryo cryopreservation. Curr. Opin. Obstet. Gynecol. 23, 245–250. Hong, S.W., Sepilian, V., Chung, H.M., Kim, T.J., 2009. Cryopreserved human blastocysts after vitrification result in excellent implantation and clinical pregnancy rates. Fertil. Steril. 92, 2062–2064. Kader, A.A., Choi, A., Orief, Y., Agarwal, A., 2009. Factors affecting the outcome of human blastocyst vitrification. Reprod. Biol. Endocrinol. 7, 99. Khalaf, Y., El-Toukhy, T., Coomarasamy, A., Kamal, A., Bolton, V., Braude, P., 2008. Selective single blastocyst transfer reduces the multiple pregnancy rate and increases pregnancy rates: a pre- and postintervention study. BJOG 115, 385–390. Kjellberg, A.T., Carlsson, P., Bergh, C., 2006. Randomized single versus double embryo transfer: obstetric and paediatric outcome and a cost-effectiveness analysis. Hum. Reprod. 21, 210–216. Klemetti, R., Gissler, M., Sevon, T., Koivurova, S., Ritvanen, A., Hemminki, E., 2005. Children born after assisted fertilization have an increased rate of major congenital anomalies. Fertil. Steril. 84, 1300–1307.
G Feng et al. Korosec, S., Virant-Klun, I., Tomazevic, T., Zech, N.H., Meden-Vrtovec, H., 2007. Single fresh and frozen–thawed blastocyst transfer using hyaluronan-rich transfer medium. Reprod. Biomed. Online 15, 701–707. Levitas, E., Lunenfeld, E., Har-Vardi, I., Albotiano, S., Sonin, Y., Hackmon-Ram, R., Potashnik, G., 2004. Blastocyst-stage embryo transfer in patients who failed to conceive in three or more day 2–3 embryo transfer cycles: a prospective, randomized study. Fertil. Steril. 81, 567–571. Liebermann, J., Tucker, M.J., 2006. Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil. Steril. 86, 20–26. Lukassen, H.G., Braat, D.D., Wetzels, A.M., Zielhuis, G.A., Adang, E.M., Scheenjes, E., Kremer, J.A., 2005. Two cycles with single embryo transfer versus one cycle with double embryo transfer: a randomized controlled trial. Hum. Reprod. 20, 702–708. Luke, B., Brown, M.B., Grainger, D.A., Cedars, M., Klein, N., Stern, J.E., 2010. Practice patterns and outcomes with the use of single embryo transfer in the United States. Fertil. Steril. 93, 490–498. Martin, J.A., Hamilton, B.E., Sutton, P.D., Ventura, S.J., Menacker, F., Kirmeyer, S., Munson, M.L., 2007. Births: final data for 2005. Natl. Vital Stat. Rep. 56, 1–103. Nyboe Andersen, A., Goossens, V., Bhattacharya, S., Ferraretti, A.P., Kupka, M.S., de Mouzon, J., Nygren, K.G., 2009. Assisted reproductive technology and intrauterine inseminations in Europe, 2005: results generated from European registers by ESHRE: ESHRE. The European IVF Monitoring Programme (EIM), for the European Society of Human Reproduction and Embryology (ESHRE). Hum. Reprod. 24, 1267–1287. Olson, C.K., Keppler-Noreuil, K.M., Romitti, P.A., Budelier, W.T., Ryan, G., Sparks, A.E., Van Voorhis, B.J., 2005. In vitro fertilization is associated with an increase in major birth defects. Fertil. Steril. 84, 1308–1315. Papanikolaou, E.G., Fatemi, H., Venetis, C., Donoso, P., Kolibianakis, E., Tournaye, H., Tarlatzis, B., Devroey, P., 2010. Monozygotic twinning is not increased after single blastocyst transfer compared with single cleavage-stage embryo transfer. Fertil. Steril. 93, 592–597. Ryan, G.L., Sparks, A.E., Sipe, C.S., Syrop, C.H., Dokras, A., Van Voorhis, B.J., 2007. A mandatory single blastocyst transfer policy with educational campaign in a United States IVF program reduces multiple gestation rates without sacrificing pregnancy rates. Fertil. Steril. 88, 354–360. Schwarzler, P., Zech, H., Auer, M., Pfau, K., Gobel, G., Vanderzwalmen, P., Zech, N., 2004. Pregnancy outcome after blastocyst transfer as compared to early cleavage stage embryo transfer. Hum. Reprod. 19, 2097–2102. Shih, W., Rushford, D.D., Bourne, H., Garrett, C., McBain, J.C., Healy, D.L., Baker, H.W., 2008. Factors affecting low birthweight after assisted reproduction technology: difference between transfer of fresh and cryopreserved embryos suggests an adverse effect of oocyte collection. Hum. Reprod. 23, 1644–1653. Stehlik, E., Stehlik, J., Katayama, K.P., Kuwayama, M., Jambor, V., Brohammer, R., Kato, O., 2005. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. Reprod. Biomed. Online 11, 53–57. Stillman, R.J., Richter, K.S., Banks, N.K., Graham, J.R., 2009. Elective single embryo transfer: a 6-year progressive implementation of 784 single blastocyst transfers and the influence of payment method on patient choice. Fertil. Steril. 92, 1895–1906. Styer, A.K., Wright, D.L., Wolkovich, A.M., Veiga, C., Toth, T.L., 2008. Single-blastocyst transfer decreases twin gestation without affecting pregnancy outcome. Fertil. Steril. 89, 1702–1708. Takahashi, K., Mukaida, T., Goto, T., Oka, C., 2005. Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study. Fertil. Steril. 84, 88–92.
Single vitrified–warmed and fresh blastocyst transfer Teranishi, A., Kuwata, A., Fumino, T., Hamai, H., Shigeta, M., 2009. A theoretical model for single blastocyst transfer. J. Assist. Reprod. Genet. 26, 327–334. Thurin, A., Hausken, J., Hillensjo, T., Jablonowska, B., Pinborg, A., Strandell, A., Bergh, C., 2004. Elective single-embryo transfer versus double-embryo transfer in in vitro fertilization. N. Engl. J. Med. 351, 2392–2402. Vajta, G., Nagy, Z.P., 2006. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod. Biomed. Online 12, 779–796. Veleva, Z., Karinen, P., Tomas, C., Tapanainen, J.S., Martikainen, H., 2009. Elective single embryo transfer with cryopreservation improves the outcome and diminishes the costs of IVF/ICSI. Hum. Reprod. 24, 1632–1639. Vitthala, S., Gelbaya, T.A., Brison, D.R., Fitzgerald, C.T., Nardo, L.G., 2009. The risk of monozygotic twins after assisted reproductive technology: a systematic review and meta-analysis. Hum. Reprod. Update 15, 45–55. Wang, Y.A., Sullivan, E.A., Black, D., Dean, J., Bryant, J., Chapman, M., 2005. Preterm birth and low birth weight after assisted reproductive technology-related pregnancy in Australia between 1996 and 2000. Fertil. Steril. 83, 1650–1658.
473 Wikland, M., Hardarson, T., Hillensjo, T., Westin, C., Westlander, G., Wood, M., Wennerholm, U.B., 2010. Obstetric outcomes after transfer of vitrified blastocysts. Hum. Reprod. 25, 1699–1707. Yanaihara, A., Yorimitsu, T., Motoyama, H., Ohara, M., Kawamura, T., 2008. Clinical outcome of frozen blastocyst transfer; single vs. double transfer. J. Assist. Reprod. Genet. 25, 531– 534. Youssry, M., Ozmen, B., Zohni, K., Diedrich, K., Al-Hasani, S., 2008. Current aspects of blastocyst cryopreservation. Reprod. Biomed. Online 16, 311–320. Zhu, D., Zhang, J., Cao, S., Heng, B.C., Huang, M., Ling, X., Duan, T., Tong, G.Q., 2011. Vitrified–warmed blastocyst transfer cycles yield higher pregnancy and implantation rates compared with fresh blastocyst transfer cycles–time for a new embryo transfer strategy? Fertil. Steril. 95, 1691–1695. Declaration: The authors report no financial or commercial conflicts of interest. Received 29 February 2012; refereed 30 June 2012; accepted 11 July 2012.
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ARTICLE
Comparable clinical outcomes and live births after single vitrified–warmed and fresh blastocyst transfer Guixue Feng, Bo Zhang *, Hong Zhou, Jinhui Shu, Xianyou Gan, Fangrong Wu, Xihe Deng Reproductive Medicine center, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning 530003, China * Corresponding author. E-mail address: [email protected] (B Zhang). Guixue Feng, Ph.D. is an associate researcher and an associate director of the Center of Reproductive Medicine. After award of his doctorate from Guangxi University, he worked in an assisted reproduction laboratory for 9 years and gained his skills in techniques such as IVF, intracytoplasmic sperm injection, preimplantation genetic diagnosis, vitrification of embryos/oocytes and selective single-blastocyst transfer. He is proficient in a variety of laboratory and management skills and has published more than 10 papers in the field of reproduction medicine.
Abstract Selective single-blastocyst transfer (SBT) in fresh cycles has been effective in reducing multiple pregnancies. However,
we do not know whether this successful strategy of fresh transfer cycles is suitable for cryopreserved cycles. The present study was undertaken to evaluate the feasibility and value of SBT in vitrified–warmed cycles. Clinical pregnancy rate (CPR) was similar with vitrified and fresh SBT (46.61% versus 52.15% respectively). Of the pregnant patients, monozygotic twin, miscarriage and ectopic pregnancy rates were similar with vitrified and fresh SBT. For the newborns, no significant difference was observed in live birth, low birthweight, premature delivery and birth defects rates between vitrified and fresh SBT. With respect to the quality of transferred blastocysts (from BB to AA), a similar CPR and miscarriage rate was obtained for both vitrified and fresh SBT when a similar blastocyst cohort graded 3BB was transferred. The data show that vitrified SBT is an effective means of reducing multiple pregnancy and that comparable clinical outcomes and live births are achieved if single blastocysts graded 3BB are transferred for both vitrified and fresh SBT. These data should encourage clinics to evaluate their embryo transfer policy and adopt vitrified SBT as everyday practice. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: clinical pregnancy, implantation, single-blastocyst transfer, vitrification
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.07.008
Single vitrified–warmed and fresh blastocyst transfer
Introduction The goal of assisted reproduction technology is the birth of a healthy singleton infant. However, in 2004–2005, twins accounted for approximately 30% of assisted reproduction-related live births in the USA (Luke et al., 2010) and approximately 21% in Europe (Nyboe Andersen et al., 2009), in comparison with 1.6% of births from naturally conceived pregnancies worldwide (ESHRE, 2000). Multiple pregnancies are associated with unwanted morbidity risks to the mother and fetus as well as creating additional costs related to prenatal and post-partum health care in assisted reproduction (Fiddelers et al., 2007; Kjellberg et al., 2006; Martin et al., 2007). Multiple pregnancies, therefore, are defined as a common complication but not a successful pregnancy outcome (ESHRE, 2003). More recently, increased pregnancy rates, coupled with concerns about maternal and perinatal morbidity associated with multiple pregnancy, have led to attempts to restrict the number of embryos transferred, especially for good-prognosis patients. It has been admitted that the most effective method to reduce the multiple pregnancy rate after IVF is to adopt a policy of single-embryo transfer (SET) (Lukassen et al., 2005; Thurin et al., 2004). However, in many countries concerns remain that SET is less effective and more expensive than conventional IVF, which has resulted in SET not being widely adopted. With the improvement of blastocyst culture techniques, a number of investigators have reported superior clinical results with blastocyst transfer than transfers using cleavage-stage embryos (Barrenetxea et al., 2005; Levitas et al., 2004; Schwarzler et al., 2004). It has been suggested that the extra time needed to culture embryos to the blastocyst stage helps to distinguish the viability of potential embryos for eventual transfer. Further studies have demonstrated that single-blastocyst transfer (SBT) has been the most effective strategy to reduce multiple pregnancies while maintaining the overall success rate of the fresh transfer cycles (Criniti et al., 2005; Khalaf et al., 2008; Ryan et al., 2007; Stillman et al., 2009; Styer et al., 2008). Based on the trend to reduce multiple pregnancies, cryopreservation of embryos has become a necessary tool in assisted reproductive technology, leading to an increased cumulative outcome while decreasing costs (Herrero et al., 2011; Veleva et al., 2009). The literature contains a number of papers which compare the clinical outcomes of vitrification of blastocysts to that of the slow-cooling method. The single clear conclusion derived from these studies is that vitrification is a more effective cryopreservation tool for blastocyst when compared with the slowcooling method (Hong et al., 2009; Stehlik et al., 2005; Youssry et al., 2008). The increasing experience and success with cryopreservation of blastocysts has prompted an interest in decreasing the number of embryos transferred during cryopreserved cycles. But owing to concerns regarding the decreased implantation potential of cryopreserved blastocysts, IVF institutions may hesitate to apply the same criteria of fresh SBT to cryopreserved cycles because there have been only a few studies which addressed vitrified–warmed SBT (Yanaihara et al., 2008; Berin et al., 2011), especially with respect to comparisons of clinical outcomes and live births between vitrified and fresh SBT. Within the study
467 centre’s IVF unit, an elective SBT programme for fresh transfer cycles was established in 2009 and this resulted in favourable pregnancy rates, which encouraged the evaluation of the feasibility of SBT in cryopreserved cycles. The present study was undertaken to investigate whether vitrified SBT compromised outcomes of the IVF protocol compared with fresh SBT.
Materials and methods Patient cycle selection From January 2009 to December 2010, a total of 604 cycles (560 patients) of fresh SBT and 384 cycles (350 patients) of vitrified SBT were involved in this study. The indications of IVF were tubal infertility, mild male factor infertility, unexplained infertility, early stage endometriosis and polycystic ovary syndrome. In fresh SBT cycles, patients with a good prognosis and a high risk of multiple pregnancy (i.e. patients in the first or second assisted reproduction cycle, who were younger than 38 years old, with more than three good embryos on day 3 and a normal endometrium were advised to undergo SBT. Cleavage embryos were transferred if patients refused to receive SBT and the surplus embryos were cultured to the blastocyst stage. On day 5 or 6, all blastocysts were scored by the grading system established by Gardner and Schoolcraft (Gardner et al., 2000) and then were vitrified one by one for the first three vials if blastocysts 3BB were obtained. In vitrified SBT cycles, the patients younger than 38 years old, who did not become pregnant from fresh SBT (n = 231), who did not become pregnant after transfer of fresh early cleavage stage embryos (n = 132) and in whom fresh embryo transfer were cancelled (n = 21), were included. The patients included in the study in the vitrified SBT group were those who had a transfer between January 2009 and December 2010. Also included were those who had a fresh transfer before 2009 but did not become pregnant and subsequently underwent vitrified–warmed blastocyst transfer after January 2009. After warming, blastocysts were scored again and blastocysts scored 3BB were transferred singly. The clinical outcomes and live births of vitrified SBT were compared with fresh SBT. Both vitrified and fresh SBT cycles were further subdivided into three groups according to the quality of transferred blastocysts based on trophectoderm and inner cell mass quality scores (AA, AB or BA, and BB) and the clinical pregnancy and miscarriage rates compared. The study was approved by the reproductive ethical committee of the Maternal and Child Health Hospital of Guangxi Zhuang (reference number 2008001, approval granted on 6 March 2008).
Ovarian stimulation protocols, oocytes collected, oocyte insemination and embryo culture Ovulation induction was carried out after down-regulation with leuprolide acetate (Lupron; TAP Pharmaceuticals, Lake Forest, IL, USA). FSH (Follistim; Organon, USA; or Gonal F; Serono, Italy) was administered at a dose of 225 IU per day, unless previous stimulations indicated otherwise. When dominant follicles reached a diameter of 18 mm, ovulation
468 was induced by an intramuscular injection of 5000–10,000 IU human chorionic gonadotrophin (HCG; Serono, Switzerland; Livzon, China). Oocytes were recovered by transvaginal aspiration of follicles under ultrasound guidance. Oocytes were fertilized with the partner‘s spermatozoa 2–6 h after aspiration either conventionally or by intracytoplasmic sperm injection. Fertilization was confirmed and pronuclei were evaluated at 16–18 h after insemination and only zygotes displaying two pronuclei were selected to further culture for subsequent transfer. The embryos were cultured in Quinn’s 1026 medium (Sage, USA) supplemented with 10% synthetic serum substitute (SSS; Sage) from day 0–3 and then were removed into Quinn‘s 1029 medium (Sage) supplemented with 10% SSS for another 48 h, within a humidified incubator set at 37C, with an environment of 5.6% CO2 and atmospheric O2 tension.
Cryopreservation protocols Blastocysts were artificially shrunk before vitrification using a glass micropipette, as described in a previous study (Feng et al., 2010), and were vitrified one by one for the first three vials if blastocysts graded 3BB were obtained. The vitrification procedure was performed on a heating stage maintained at 37C. The blastocyst was first rinsed for 30 s in 200 ll HEPES-buffered culture medium (Sage) supplemented with 20% human serum albumin (Sage). Then, the blastocyst was enclosed in 200 ll equilibration solution containing 10% (v/v) ethylene glycol (EG; Sigma, USA) and 10% (v/v) dimethylsulphoxide (DMSO; Sigma) and incubated for 1 min. The blastocysts were then transferred to the vitrification solution consisting of 20% EG, 20% DMSO and 0.3 mol/l sucrose (Sigma) for 30 s. A microdroplet (<0.5 ll) containing one blastocyst was sucked into the narrowest end of the glass micropipette and quickly plunged into liquid nitrogen. On the day of transfer, blastocysts were warmed one by one according to their quality, which was estimated prior to vitrification and graded from high to low. Blastocysts were warmed by quickly immersing the capillary end of the glass micropipette into 200 ll warming solution (HEPES-buffered culture medium supplemented with 20% human serum albumin (HSA, Sage) and 0.6 mol/l sucrose for 2 min and subsequently transferred to 0.5 and 0.25 mol/l of sucrose for 3 min and 5 min, respectively, at 37C. Then the blastocysts were transferred to HEPES-buffered culture medium with 20% HSA for 20 min. Finally, the blastocysts were transferred to blastocyst medium (Sage, USA) in a 5.6% CO2 and atmospheric O2 tension incubator for 1–4 h and then were graded before transfer.
Embryo transfer procedure Patients were prepared for vitrified–warmed or fresh SBT using hormone replacement therapy consisting of increasing doses of oestrogen and progesterone (Progynova; Byer, Germany; Utogestan, Laboratoires Besins International, France) starting on day 3. The only monitoring in the replacement cycle was the evaluation of the endometrium prior to progesterone administration. Warming cycles were cancelled if the endometrial thickness was <7 mm. Embryo
G Feng et al. transfer was performed under ultrasound guidance using a Wallace Sure View catheter. Pregnancy testing was performed 14 days after the embryo transfer. Clinical pregnancy was confirmed by the presence of gestational sacs on ultrasound examination at 6–8 weeks in the hospital and miscarriage was defined as the loss of an embryo or fetus before the 28th week of pregnancy. The information regarding the delivery and children were collected by questionnaires to the parents. Implantation rates were defined as the number of gestational sacs observed on ultrasound scanning divided by the number of embryos transferred.
Data analysis The statistical analysis was performed using Statistical Package for Social Sciences version 13.0 (SPSS, USA). Values were reported as mean ± SD or frequencies. Means in groups were compared by Student0 s t-test and frequencies in groups were compared by chi-squared test. Statistical significance was attributed to two-tailed P < 0.05.
Results As shown in Table 1, there were no differences in patient age, method of fertilization and primary/secondary infertility between fresh and vitrified SBT. Endometrial thickness on HCG day was lower with fresh SBT than vitrified SBT (P < 0.01) and there was a significant difference in the rate of day-5 or day-6 blastocyst transfer between fresh and vitrified SBT (P < 0.01). A total of 405 vitrified blastocysts in 384 cycles were warmed with a 94.81% (384/405) survival rate. All surviving blastocysts were graded 3BB and singly transferred, which resulted in 46.61% (179/384) implantation/clinical pregnancy rate. A total of 604 fresh blastocysts graded 3BB were singly transferred, which resulted in 52.15% (315/604) implantation/clinical pregnancy rate. No statistically significant difference was observed in implantation/clinical pregnancy rate and live birth rate between these two groups. Of the pregnant patients, the monozygotic twin, miscarriage and ectopic pregnancy rates in vitrified SBT also showed no statistically significant difference compared with fresh SBT (2.79% versus 3.17%; 16.20% versus 13.02%; 2.79% versus 4.44%). In vitrified SBT, there were 142 singleton and one monozygotic twin live births, resulting in 144 healthy newborns, and in fresh SBT, 252 singleton and seven monozygotic twin live births, resulting in 266 healthy newborns. This study only compared the singleton live births between fresh and vitrified SBT because the number of newborns resulting from twin pregnancies was very small. As shown in Table 2, the mean gestational age at delivery was 38.61 ± 1.64 weeks in vitrified SBT and 38.17 ± 4.60 weeks in fresh SBT, which were not significantly different. Eleven of the 142 gestations (7.75%) were delivered between 34 and 37 weeks in vitrified SBT and 18 of 252 gestations (7.14%) were delivered in fresh SBT. Three of 142 gestations (2.11%) delivered at less than 34 weeks in vitrified SBT while seven of 252 gestations (2.78%) delivered in fresh SBT. The mean birthweights of vitrified and fresh SBT were 3155.35 ± 590.09 g and 3103.81 ± 515.55 g, respectively, which means that the
Single vitrified–warmed and fresh blastocyst transfer
469
Table 1 Comparison of patient characteristics and clinical outcomes between vitrified and fresh singleblastocyst transfer (SBT). SBT cycles
P-value
Vitrified (n = 384)
Fresh (n = 604)
Protocol IVF ICSI
328/384 (85.42) 56/384 (14.58)
524/604 (86.75) 80/604 (13.25)
NS NS
Primary infertility Secondary infertility Age (years) Endometrial thickness on HCG day (mm)
133/384 (34.64) 251/384 (65.36) 31.60 ± 3.56 9.34 ± 1.78
203/604 (33.61) 401/604 (66.39) 31.02 ± 3.69 11.20 ± 2.30
NS NS NS <0.01
Cryopreservation Vitrified blastocysts Survival rate
405 (1.07 ± 0.28) 384/405 (94.81)
– –
– –
Blastocyst transfer Day 6 Day 5
21/384 (5.47) 363/384 (94.54)
6/604 (0.99) 598/604 (99.01)
<0.01 <0.01
Implantation/clinical pregnancy Monozygotic twins Miscarriages Ectopic pregnancies Stillbirths
179/384 (46.61) 5/179 (2.79) 29/179 (16.20) 5/179 (2.79) 2/179 (1.11)
315/604 (52.15) 10/315 (3.17) 41/315 (13.02) 14/315 (4.44) 1/315 (0.32)
NS NS NS NS NS
Live births Singletons Monozygotic twins
143/384 (37.24) 142 1
259/604 (42.88) 252 7
NS
Values are n/total (%) or mean ± SD. ICSI = intracytoplasmic sperm injection; NS = not statistically significant.
babies were slightly heavier for vitrified SBT when compared with fresh SBT, but the difference did not reach statistical significance. The rates of low birthweight (1500–2500 g) and very low birthweight (<1500 g) in vitrified SBT were not significantly different when compared with fresh SBT (5.63% versus 7.94%, and 1.41% versus 0%, respectively). Birth defects were found in 1.41% (2/142) of the children in vitrified SBT and 0.79% (2/252) of the children in fresh SBT, which means that no difference was also observed between these two groups. Of 384 cycles in vitrified SBT, the rates of transferred blastocysts graded AA, AB or BA, and BB in vitrified SBT were 3.13%, 40.63% and 56.25% respectively, and of 604 cycles fresh SBT, 35.10%, 52.48% and 12.42% respectively, which were significantly different between the SBT groups (P < 0.05) (Table 3). With the improvement of transferred blastocyst quality from BB to AA, there was a non-significant trend of a gradual improvement of the clinical pregnancy rate from 94/216 (43.52%) to 8/12 (66.67%) in vitrified SBT and 30/75 (40.00%) to 119/212 (56.13%) in fresh SBT. Similar implantation/clinical pregnancy and miscarriage rates were obtained for vitrified SBT compared with fresh SBT when a similar cohort of blastocysts graded 3BB was transferred (Figures 1 and 2).
Discussion This study found that similar clinical outcomes and live births were achieved after single vitrified–warmed and fresh blastocyst transfer. A new embryo transfer strategy of SBT in vitrified–warmed cycles is therefore encouraged as everyday practice. Multiple pregnancy remains the foremost complication of IVF therapy. There has recently been an effort to decrease the number of embryos transferred in cryopreserved cycles in order to reduce the incidence of multiple pregnancy. Since the success and experience with extended culture has allowed for better selection methods, there has been increasing interest in assessing the efficacy of transferring a single cryopreserved blastocyst. Studies have demonstrated that SBT has been the most effective strategy in reducing multiple pregnancies while maintaining the overall success rate in fresh transfer cycles (Criniti et al., 2005; Khalaf et al., 2008; Ryan et al., 2007; Stillman et al., 2009; Styer et al., 2008). However, owing to a lack of adequate studies, it is not known whether this successful criteria of fresh SBT is suitable for cryopreserved cycles. Major problems with blastocyst culture have been the somewhat unpredictable results obtained with regard to
470
G Feng et al.
SBT singleton pregnancies
Birth sex ratio (male:female) Gestational age
Vitrified (n = 142)
Fresh (n = 252)
76:66 (1:0.87)
142:110 (1:0.77)
38.61 ± 1.64
38.17 ± 4.60
100.0%
fSBT
90.0%
Clinical pregnancy rate 1
Table 2 Comparison of the live births from singleton pregnancies between vitrified and fresh single-blastocyst transfer (SBT).
80.0%
vSBT 66.67%
70.0% 60.0%
56.13%
11 (7.75) 3 (2.11)
18 (7.14) 7 (2.78)
Birthweight (g) Low birthweight (1500 to 2500 g) Very low birthweight (<1500 g)
3155.35 ± 590.09 8 (5.63)
3103.81 ± 515.55 20 (7.94)
2 (1.41)
0
Birth defects
2 (1.41)
49.35%
43.52% 40.00%
40.0% 30.0% 20.0% 10.0% 0.0%
AA
Time of delivery 34–37 weeks <34 weeks
52.36%
50.0%
AB or BA
BB
Figure 1 Comparison of clinical pregnancy rates in vitrified and fresh single-blastocyst transfer (vSBT and fSBT) when a similar cohort of blastocysts graded 3BB was transferred. No statistically significant differences were found between the transfer groups for each grading category.
30.0%
fSBT vSBT
27.5% 25.0%
Values are n (%) or mean ± SD. There were no statistically significant differences between the two groups.
Miscarriage rate
22.5%
2 (0.79)
20.0% 17.5%
Grade
AA AB or BA BB
SBT cycles
P-value
Vitrified (n = 384)
Fresh (n = 604)
12 (3.13) 156 (40.63) 216 (56.25)
212 (35.10) 317 (52.48) 75 (12.42)
<0.01 <0.01 <0.01
Values are n (%)
survival of embryos and the pregnancy rate after blastocysts are frozen with the traditional slow-freezing methods (Liebermann and Tucker, 2006). The explanation may be technical, owing to the size, multicellular structure and the presence of the blastocoele within the blastocyst (Kader et al., 2009). A more reliable and consistent method than conventional cryopreservation of blastocysts (slow method) seems to be vitrification (Hong et al., 2009; Stehlik et al., 2005; Youssry et al., 2008), which has been associated with higher implantation rates. However, concerns have been raised with regard to possible toxic effects of the high concentrations of cryoprotectants (Vajta and Nagy, 2006). Many published studies have found that the pregnancy and live delivery rates are higher for the transfer of fresh compared with frozen–thawed embryos (mainly employing the slow-freezing method). Contrary conclusions have been made by studies using the transfer of vitrified–warmed blastocysts, which yielded higher implantation and clinical pregnancy rates when compared with fresh blastocyst transfer as a result of better endometrial receptivity and synchronization and the weeding out of poor-quality embryos through
12.50%
12.5% 10.0% 7.5%
Table 3 Comparison of blastocyst quality between vitrified and fresh single-blastocyst transfer (SBT).
15.96%
15.0%
13.33%
12.65% 11.69%
7.56%
5.0% 2.5% 0.0%
AA
AB or BA
BB
Figure 2 Comparison of miscarriage rates in vitrified and fresh single-blastocyst transfer (vSBT and fSBT) when a similar cohort of blastocysts graded 3BB was transferred. No statistically significant differences were found between the transfer groups for each grading category.
the act of cryopreservation itself (Zhu et al., 2011). As far as is known, there are very few studies associated with single cryopreserved blastocysts that have compared the clinical outcomes of one with two cryopreserved blastocysts. Berin et al. (2011) found that the pregnancy rate (34.7%) of single frozen blastocyst transfer were lower compared with double transfers (50.4%) (Berin et al., 2011) while Yanaihara et al. (2008) achieved similar clinical pregnancy with both types of transfer (Yanaihara et al., 2008). However, a significantly higher twinning rate was seen when two blastocysts were transferred in these studies. It is well established that blastocyst quality is an important factor for the clinical outcomes and that blastocyst quality conspicuously decreases with the increasing age of patients. It is reported that blastocysts graded 3AA are appropriate for fresh SBT and achieve desirable clinical outcomes. However, not all patients are able to obtain such high-quality blastocysts (Gardner et al., 2000). Teranishi reported that 3BB blastocysts are suitable for fresh SBT with 41.9% pregnancy rate (Teranishi et al., 2009). Yanaihara reported that more than 3BB blastocysts were frozen and these resulted in a 40.7% pregnancy rate after single vitrified-warmed blastocyst, which was not significantly different to that compared
Single vitrified–warmed and fresh blastocyst transfer with the double vitrified–warmed blastocysts transfer (46%) (Yanaihara et al., 2008). Korosec reported that the pregnancy rate was similar between SBT and double-blastocyst transfer in frozen–thawed transfer cycles when the embryo transfer solution contained a high concentration of hyaluronic acid (Korosec et al., 2007). In the present study, a 46.61% clinical pregnancy rate and a 37.24% live birth rate in vitrified SBT was achieved, which is slightly lower than that observed in fresh SBT (52.15% and 42.88%, respectively), but this did not reach statistical significance. This may be explained by the higher quality of blastocysts in fresh SBT observed when compared with vitrified SBT. Two reasons can be considered as explanations for this finding: (i) after warming, blastocysts were damaged during the procedure of vitrification and warming, which caused the lower scores when compared with fresh blastocysts; (ii) although the transferred blastocysts were scored 3BB and the age of patients was younger than 38 years old in both fresh and vitrified SBT, the patients of fresh SBT only included young, good-prognosis patients, while the patients of vitrified SBT included those who did not became pregnant from fresh SBT, those who did not became pregnant after transfer of fresh early cleavage stage embryos and those for whom fresh embryo transfers were cancelled. However, the mean age of patients between the two groups was not significantly different. With the improvement of transferred blastocyst quality from BB to AA, there was a trend of gradual improvement in the clinical pregnancy rate from 43.52% to 66.67% in vitrified SBT and from 40.00% to 56.13% in fresh SBT. Similar implantation/clinical pregnancy and miscarriage rates were obtained when transferring a similar cohort of blastocysts between the fresh and vitrified SBT groups. There is increasing interest in the safety of human assisted reproduction. During cryopreservation, embryos are at risk for cryodamage, resulting in lower clinical pregnancy outcomes. However, in relation to baby outcomes, recent evidence suggests that better perinatal outcomes follow the transfer of frozen–thawed embryos rather than fresh embryos (Shih et al., 2008; Wang et al., 2005). Babies conceived by the transfer of thawed embryos may have significantly lower rates of preterm birth and perinatal mortality than those following the transfer of fresh embryos (Shih et al., 2008). Live-born babies are also less likely to be low birthweight following the transfer of thawed compared with fresh embryos (Wang et al., 2005). Takahashi et al. (2005) reported a 4-year follow-up study of children born after transfer of vitrified blastocysts. They found no differences in obstetric outcomes for children born after vitrified blastocysts as compared with children born after fresh blastocysts with an 18.5% rate of preterm birth and a 43.5% low birthweight rate. In the present study, 16.20% (29/179) of pregnant patients in vitrified SBT suffered clinical miscarriage, with no difference observed compared with 13.02% (41/315) in fresh SBT, which agrees with spontaneously conceived pregnancies where there is a 10–15% rate of clinical miscarriage. No difference was observed in the incidence of delivery before 37 weeks in vitrified SBT compared with fresh SBT (9.86% versus 9.92%). The incidences of low birthweight (<2500 g) and very low birthweight (<1500 g) babies were 5.63% and 1.41%, respectively, which are similar to the rates of these outcomes in fresh SBT (7.94% versus 0%). It is well known that the number of previous children born can
471 affect both pregnancy length and birthweight. However, these data are not available, which is a limitation of this study. An increasing number of papers have compared assisted reproduction babies with those spontaneously conceived (Klemetti et al., 2005; Olson et al., 2005) and there have been several systematic reviews (Hansen et al., 2005), which show an increase in the risk of congenital abnormality associated with assisted reproduction technology. It is generally accepted that each step of in-vitro manipulation may compromise the viability of zygotes/embryos or cause chromosomal abnormality. Based on the present data, although blastocysts of vitrified SBT underwent the cryopreservation procedure, which may cause damage to blastocysts, no difference was observed in the rate of birth defects compared with fresh SBT. This is in line with the results of Wikland et al. (2010), who observed no adverse neonatal outcomes in children born after transfer of vitrified blastocysts as compared with fresh blastocysts. A systematic review documented that blastocyst transfer results in high implantation rates and high pregnancy rates when compared with cleavage embryo transfer while accompanying an increased rate of monozygotic twinning (Chang et al., 2009). Another meta-analysis included 27 studies and found blastocyst transfer to be associated with a 4-fold higher risk of monozygotic twinning as compared with spontaneous conception (1.7% versus 0.4%; Vitthala et al., 2009). The risks of monozygotic twins to both mother and unborn babies are much larger when compared with dizygotic twin pregnancy (Duncombe et al., 2003). However, the results of Papanikolaou et al. (2010) showed that the monozygotic twin rate was similar between the transfer of fresh blastocysts and fresh cleavage embryo transfers (Papanikolaou et al., 2010). Yanaihara (2008) found that, regardless of the transfer of one or two frozen–thawed blastocysts, there was no significant difference in the miscarriage rate, but there were significant differences in both the ectopic pregnancy incidence rate and monozygotic twin rate (Yanaihara et al., 2008). Compared with Yanaihara’s results for the monozygotic twin rate (2.3%), the present study found that the monozygotic twin rate (2.79%) of vitrified SBT was similar when transferring one vitrified–warmed blastocyst and there was no difference observed when compared with monozygotic twin rate of fresh SBT (3.17%). In conclusion, acceptable pregnancy outcomes were achieved after single vitrified blastocyst transfer (grade 3BB) and no adverse effects on neonatal outcome were observed compared with fresh SBT. These data should encourage clinicians to evaluate their embryo transfer policy and adopt vitrified SBT as their everyday practice. Continuous follow up of obstetric and neonatal outcomes as well as long-term follow up of children is recommended until several larger series have been evaluated.
Acknowledgements This work was supported by Guangxi Zhuang Autonomous Region Natural Science Foundation of China (Grant nos. 0897007, 0832183 and 0542058) and the Health Department of Guangxi Zhuang Autonomous Region (Grant nos. 200947, 2011063 and 2011065). The authors wish to thank the
472 clinicians, clinical embryologists, laboratory technologists and nurses of the Centre for Reproductive Medicine. They are also grateful to Dr S Sooranna, Imperial College London, for editing this manuscript.
References ESHRE, 2000. Multiple gestation pregnancy. The ESHRE Capri Workshop Group. Hum. Reprod. 15, 1856–1864. ESHRE, 2003. 6. Ethical issues related to multiple pregnancies in medically assisted procreation. Hum. Reprod. 18, 1976–1979. Barrenetxea, G., Lopez de Larruzea, A., Ganzabal, T., Jimenez, R., Carbonero, K., Mandiola, M., 2005. Blastocyst culture after repeated failure of cleavage-stage embryo transfers: a comparison of day 5 and day 6 transfers. Fertil. Steril. 83, 49–53. Berin, I., McLellan, S.T., Macklin, E.A., Toth, T.L., Wright, D.L., 2011. Frozen–thawed embryo transfer cycles: clinical outcomes of single and double blastocyst transfers. J. Assist. Reprod. Genet. 28, 575–581. Chang, H.J., Lee, J.R., Jee, B.C., Suh, C.S., Kim, S.H., 2009. Impact of blastocyst transfer on offspring sex ratio and the monozygotic twinning rate: a systematic review and meta-analysis. Fertil. Steril. 91, 2381–2390. Criniti, A., Thyer, A., Chow, G., Lin, P., Klein, N., Soules, M., 2005. Elective single blastocyst transfer reduces twin rates without compromising pregnancy rates. Fertil. Steril. 84, 1613–1619. Duncombe, G.J., Dickinson, J.E., Evans, S.F., 2003. Perinatal characteristics and outcomes of pregnancies complicated by twin-twin transfusion syndrome. Obstet. Gynecol. 101, 1190–1196. Feng, G.X., Zhang, B., Shu, J.H., Zhou, H., Zhou, L., Gan, X.Y., Liu, Y., 2010. Effects of artificial shrinkage of blastocoeles before vitrification on pregnancy outcome. Zhonghua Fu Chan Ke Za Zhi 45, 838–842. Fiddelers, A.A., Severens, J.L., Dirksen, C.D., Dumoulin, J.C., Land, J.A., Evers, J.L., 2007. Economic evaluations of singleversus double-embryo transfer in IVF. Hum. Reprod. Update 13, 5–13. Gardner, D.K., Lane, M., Stevens, J., Schlenker, T., Schoolcraft, W.B., 2000. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil. Steril. 73, 1155–1158. Hansen, M., Bower, C., Milne, E., de Klerk, N., Kurinczuk, J.J., 2005. Assisted reproductive technologies and the risk of birth defects – a systematic review. Hum. Reprod. 20, 328–338. Herrero, L., Martinez, M., Garcia-Velasco, J.A., 2011. Current status of human oocyte and embryo cryopreservation. Curr. Opin. Obstet. Gynecol. 23, 245–250. Hong, S.W., Sepilian, V., Chung, H.M., Kim, T.J., 2009. Cryopreserved human blastocysts after vitrification result in excellent implantation and clinical pregnancy rates. Fertil. Steril. 92, 2062–2064. Kader, A.A., Choi, A., Orief, Y., Agarwal, A., 2009. Factors affecting the outcome of human blastocyst vitrification. Reprod. Biol. Endocrinol. 7, 99. Khalaf, Y., El-Toukhy, T., Coomarasamy, A., Kamal, A., Bolton, V., Braude, P., 2008. Selective single blastocyst transfer reduces the multiple pregnancy rate and increases pregnancy rates: a pre- and postintervention study. BJOG 115, 385–390. Kjellberg, A.T., Carlsson, P., Bergh, C., 2006. Randomized single versus double embryo transfer: obstetric and paediatric outcome and a cost-effectiveness analysis. Hum. Reprod. 21, 210–216. Klemetti, R., Gissler, M., Sevon, T., Koivurova, S., Ritvanen, A., Hemminki, E., 2005. Children born after assisted fertilization have an increased rate of major congenital anomalies. Fertil. Steril. 84, 1300–1307.
G Feng et al. Korosec, S., Virant-Klun, I., Tomazevic, T., Zech, N.H., Meden-Vrtovec, H., 2007. Single fresh and frozen–thawed blastocyst transfer using hyaluronan-rich transfer medium. Reprod. Biomed. Online 15, 701–707. Levitas, E., Lunenfeld, E., Har-Vardi, I., Albotiano, S., Sonin, Y., Hackmon-Ram, R., Potashnik, G., 2004. Blastocyst-stage embryo transfer in patients who failed to conceive in three or more day 2–3 embryo transfer cycles: a prospective, randomized study. Fertil. Steril. 81, 567–571. Liebermann, J., Tucker, M.J., 2006. Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil. Steril. 86, 20–26. Lukassen, H.G., Braat, D.D., Wetzels, A.M., Zielhuis, G.A., Adang, E.M., Scheenjes, E., Kremer, J.A., 2005. Two cycles with single embryo transfer versus one cycle with double embryo transfer: a randomized controlled trial. Hum. Reprod. 20, 702–708. Luke, B., Brown, M.B., Grainger, D.A., Cedars, M., Klein, N., Stern, J.E., 2010. Practice patterns and outcomes with the use of single embryo transfer in the United States. Fertil. Steril. 93, 490–498. Martin, J.A., Hamilton, B.E., Sutton, P.D., Ventura, S.J., Menacker, F., Kirmeyer, S., Munson, M.L., 2007. Births: final data for 2005. Natl. Vital Stat. Rep. 56, 1–103. Nyboe Andersen, A., Goossens, V., Bhattacharya, S., Ferraretti, A.P., Kupka, M.S., de Mouzon, J., Nygren, K.G., 2009. Assisted reproductive technology and intrauterine inseminations in Europe, 2005: results generated from European registers by ESHRE: ESHRE. The European IVF Monitoring Programme (EIM), for the European Society of Human Reproduction and Embryology (ESHRE). Hum. Reprod. 24, 1267–1287. Olson, C.K., Keppler-Noreuil, K.M., Romitti, P.A., Budelier, W.T., Ryan, G., Sparks, A.E., Van Voorhis, B.J., 2005. In vitro fertilization is associated with an increase in major birth defects. Fertil. Steril. 84, 1308–1315. Papanikolaou, E.G., Fatemi, H., Venetis, C., Donoso, P., Kolibianakis, E., Tournaye, H., Tarlatzis, B., Devroey, P., 2010. Monozygotic twinning is not increased after single blastocyst transfer compared with single cleavage-stage embryo transfer. Fertil. Steril. 93, 592–597. Ryan, G.L., Sparks, A.E., Sipe, C.S., Syrop, C.H., Dokras, A., Van Voorhis, B.J., 2007. A mandatory single blastocyst transfer policy with educational campaign in a United States IVF program reduces multiple gestation rates without sacrificing pregnancy rates. Fertil. Steril. 88, 354–360. Schwarzler, P., Zech, H., Auer, M., Pfau, K., Gobel, G., Vanderzwalmen, P., Zech, N., 2004. Pregnancy outcome after blastocyst transfer as compared to early cleavage stage embryo transfer. Hum. Reprod. 19, 2097–2102. Shih, W., Rushford, D.D., Bourne, H., Garrett, C., McBain, J.C., Healy, D.L., Baker, H.W., 2008. Factors affecting low birthweight after assisted reproduction technology: difference between transfer of fresh and cryopreserved embryos suggests an adverse effect of oocyte collection. Hum. Reprod. 23, 1644–1653. Stehlik, E., Stehlik, J., Katayama, K.P., Kuwayama, M., Jambor, V., Brohammer, R., Kato, O., 2005. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. Reprod. Biomed. Online 11, 53–57. Stillman, R.J., Richter, K.S., Banks, N.K., Graham, J.R., 2009. Elective single embryo transfer: a 6-year progressive implementation of 784 single blastocyst transfers and the influence of payment method on patient choice. Fertil. Steril. 92, 1895–1906. Styer, A.K., Wright, D.L., Wolkovich, A.M., Veiga, C., Toth, T.L., 2008. Single-blastocyst transfer decreases twin gestation without affecting pregnancy outcome. Fertil. Steril. 89, 1702–1708. Takahashi, K., Mukaida, T., Goto, T., Oka, C., 2005. Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study. Fertil. Steril. 84, 88–92.
Single vitrified–warmed and fresh blastocyst transfer Teranishi, A., Kuwata, A., Fumino, T., Hamai, H., Shigeta, M., 2009. A theoretical model for single blastocyst transfer. J. Assist. Reprod. Genet. 26, 327–334. Thurin, A., Hausken, J., Hillensjo, T., Jablonowska, B., Pinborg, A., Strandell, A., Bergh, C., 2004. Elective single-embryo transfer versus double-embryo transfer in in vitro fertilization. N. Engl. J. Med. 351, 2392–2402. Vajta, G., Nagy, Z.P., 2006. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod. Biomed. Online 12, 779–796. Veleva, Z., Karinen, P., Tomas, C., Tapanainen, J.S., Martikainen, H., 2009. Elective single embryo transfer with cryopreservation improves the outcome and diminishes the costs of IVF/ICSI. Hum. Reprod. 24, 1632–1639. Vitthala, S., Gelbaya, T.A., Brison, D.R., Fitzgerald, C.T., Nardo, L.G., 2009. The risk of monozygotic twins after assisted reproductive technology: a systematic review and meta-analysis. Hum. Reprod. Update 15, 45–55. Wang, Y.A., Sullivan, E.A., Black, D., Dean, J., Bryant, J., Chapman, M., 2005. Preterm birth and low birth weight after assisted reproductive technology-related pregnancy in Australia between 1996 and 2000. Fertil. Steril. 83, 1650–1658.
473 Wikland, M., Hardarson, T., Hillensjo, T., Westin, C., Westlander, G., Wood, M., Wennerholm, U.B., 2010. Obstetric outcomes after transfer of vitrified blastocysts. Hum. Reprod. 25, 1699–1707. Yanaihara, A., Yorimitsu, T., Motoyama, H., Ohara, M., Kawamura, T., 2008. Clinical outcome of frozen blastocyst transfer; single vs. double transfer. J. Assist. Reprod. Genet. 25, 531– 534. Youssry, M., Ozmen, B., Zohni, K., Diedrich, K., Al-Hasani, S., 2008. Current aspects of blastocyst cryopreservation. Reprod. Biomed. Online 16, 311–320. Zhu, D., Zhang, J., Cao, S., Heng, B.C., Huang, M., Ling, X., Duan, T., Tong, G.Q., 2011. Vitrified–warmed blastocyst transfer cycles yield higher pregnancy and implantation rates compared with fresh blastocyst transfer cycles–time for a new embryo transfer strategy? Fertil. Steril. 95, 1691–1695. Declaration: The authors report no financial or commercial conflicts of interest. Received 29 February 2012; refereed 30 June 2012; accepted 11 July 2012.