Hum. Reprod. Advance Access originally published online on June 30, 2009
Human Reproduction 2009 24(10):2642-2648; doi:10.1093/humrep/dep172
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Reduction of the multiple pregnancy rate in a preimplantation genetic diagnosis programme after introduction of single blastocyst transfer and cryopreservation of blastocysts biopsied on Day 3
1 Assisted Conception Unit and PGD Centre, Guy's and St Thomas' Hospital NHS Foundation Trust, 11th Floor Tower Wing, St Thomas Street, London SE1 9RT, UK 2 Division of Reproduction and Endocrinology, King's College London, St Thomas Street, London, UK
3 Correspondence address. Tel: +44-207-188-0497; Fax: +44-207-188-0490; E-mail: tarek.el-toukhy{at}gstt.nhs.uk
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Abstract |
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BACKGROUND: An elective single-embryo transfer (SET) policy has not been applied to preimplantation genetic diagnosis (PGD) for inherited genetic disorders because of concerns regarding post-thaw survival of biopsied embryos. Our objective was to evaluate the survival and pregnancy potential of embryos biopsied for PGD at the cleavage stage and cryopreserved at the blastocyst stage and its contribution to the overall success of an elective SET policy in a PGD programme.
METHODS: From January 2006, all couples who had two or more transferable PGD blastocysts biopsied on Day 3 of culture were offered single-blastocyst transfer (SBT) and cryopreservation of surplus blastocyst(s) using a slow-freezing technique. We compared the outcome of 32 cryo-thawed PGD cycles with that of 191 cryo-thawed conventional in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles performed between January 2006 and July 2008. We also compared the outcome of all fresh PGD cycles performed before and after January 2006.
RESULTS: The cryo-thawed blastocyst survival rate was similar between the PGD and IVF/ICSI groups (87% versus 88%, P = 0.94). There was no significant difference in the implantation and clinical pregnancy rates between the two groups (35% versus 29%, P = 0.45 and 34% versus 36%, P = 0.77, respectively). During the same period, the multiple pregnancy rate in the fresh PGD programme dropped from 36% to 10% (OR = 0.20, 95% CI 0.08–0.48, P < 0.001) with no reduction in pregnancy rates.
CONCLUSIONS: The survival and implantation potential of biopsied PGD embryos cryopreserved at the blastocyst stage is comparable to that of non-biopsied IVF/ICSI cryopreserved blastocysts. Elective SBT and cryopreservation of surplus blastocysts for later use should extend to include PGD for inherited genetic disorders.
Key words: PGD/embryo biopsy/blastocyst transfer/cryopreservation/implantation
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Introduction |
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Preimplantation genetic diagnosis (PGD) is an established alternative to prenatal screening and termination of affected pregnancies for couples at risk of transmitting serious genetic conditions to their children (PGDIS, 2008
). The most commonly used technique involves analysis of a single blastomere removed from the cleavage stage embryo on Day 3 of in vitro culture (Harper et al., 2006). Embryos diagnosed as being free of the genetic disorder under investigation using either fluorescence in situ hybridization for chromosomal abnormalities or polymerase chain reaction-based analysis for single-gene disorders are transferred to the uterus either on Day 4 (Pickering et al., 2003) or Day 5 (Palmer et al., 2002; Kokkali et al., 2007).
PGD is available in only a limited number of centres worldwide (Harper et al., 2008) because of its technical complexity and high cost. Furthermore, the number of embryos available for transfer is reduced compared with standard in vitro fertilization (IVF) cycles, with or without intracytoplasmic sperm injection (ICSI), due to the additional selection undertaken on the basis of genetic analysis (Harper et al., 2008). Thus, the imperative for maximizing the pregnancy potential of each PGD cycle has been perceived to be paramount.
Attempts to increase the chance of pregnancy in PGD cycles by replacing more than one embryo have led to a high multiple pregnancy rate (Grace et al., 2006; Goossens et al., 2008; Harper et al., 2008). This represents the single most significant iatrogenic complication of assisted reproductive technology (ESHRE Capri Workshop, 2000; Braude, 2006). Indeed, multiple pregnancy after PGD treatment could be relatively more problematic than after conventional IVF because of the increased technical difficulty and consequent complications in cases where prenatal testing is required.
A strategy of elective single-embryo transfer (SET) with cryopreservation of surplus embryos for later use has been shown to reduce the multiple pregnancy rate without compromising the overall pregnancy rate after conventional IVF/ICSI (Gerris et al., 2002; De Sutter et al., 2003; Thurin et al., 2004; Lukassen et al., 2005). We have demonstrated that elective single-blastocyst transfer (SBT) and cryopreservation of surplus blastocysts in good prognosis patients maintain a high pregnancy rate while reducing the multiple pregnancy rate (Khalaf et al., 2008). However, the effectiveness of applying this approach in PGD cycles for inherited genetic disorders has not yet been established.
The objective of the present study was to evaluate the survival and implantation potential of PGD embryos biopsied at the cleavage stage and cryopreserved at the blastocyst stage compared with that of blastocysts cryopreserved after conventional IVF/ICSI treatment and to determine the impact of applying an elective SBT strategy to fresh PGD cycles for inherited genetic disorders on the overall success of a PGD programme.
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Materials and Methods |
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From January 2006, all couples undergoing fresh PGD treatment at Guy's and St Thomas' Hospital Assisted Conception Unit and PGD Centre where two or more disease-free embryos were available for transfer were offered postponing embryo transfer from Day 4 to Day 5 of in vitro culture and having a single blastocyst replaced and supernumerary blastocyst(s) cryopreserved for future use.
Between January 2006 and July 2008, 32 PGD couples who had embryos biopsied on Day 3, two or more embryos available for transfer and at least one blastocyst cryopreserved on Day 5 returned for a cryo-thawed embryo transfer cycle and were included in this study. The indication for embryo biopsy in the fresh PGD cycles was chromosomal rearrangement in 4 cycles and single-gene disorder in 28 cycles.
No embryos were cryopreserved before biopsy.
During the same period, 191 cryo-thawed blastocyst transfer cycles after conventional IVF/ICSI treatment without embryo biopsy were performed.
Written informed consent was obtained from each couple upon entering our PGD or IVF programme and before starting a frozen–thawed embryo transfer cycle. Each disorder under investigation was licensed by the Human Fertilisation and Embryology Authority.
Fresh embryo cryopreservation
In the fresh PGD cycle, embryos that had five or more blastomeres on Day 3 of culture and showed no more than 20% fragmentation were biopsied by mechanical opening of the zona pellucida and removal of one nucleated blastomere (Pickering et al., 2003). Biopsied embryos were transferred to blastocyst culture medium (BM, SAGE, Rochford Medical, Oxford, UK) and cultured until Day 5 for transfer or cryopreservation. After embryo transfer, supernumerary disorder-free blastocysts were cryopreserved using a slow-freezing protocol, using 1,2-propanediol and 0.2 M sucrose (SAGE) as cryoprotectants (Lassalle et al., 1985; Kaufman et al., 1995). After conventional IVF/ICSI, supernumerary blastocysts were cryopreserved using the same protocol.
In both PGD and IVF/ICSI cycles, blastocysts were cryopreserved providing they had a quality score of at least 3, type B for the inner cell mass and type B for trophectoderm according to the blastocyst grading criteria suggested by Gardner and Schoolcraft (1999) and compared with diagrams by Stephenson et al. (2006) at the time of cryopreservation.
Blastocyst thawing
Blastocysts were thawed on the morning of Day 6 of progesterone therapy and transfer was carried out later the same day. Blastocysts were thawed rapidly by removal from liquid nitrogen and exposure to air for 45 s followed by immersion in a water bath at 30°C for 30 s. A two-step process of propanediol removal in the presence of 0.2 M sucrose ensued at room temperature for 10 min in each step until final re-hydration in a HEPES-buffered salt solution. Thawed blastocysts were then assessed for survival using an inverted microscope (Nikon UK Limited, Kingston, Surrey, UK) at a magnification of x200, before being transferred into culture medium at 37°C. Lysed or degenerate blastocysts and those that had lost >50% of their cells were considered damaged and excluded from transfer. Re-expansion was assessed 2–3 h after thawing and immediately before transfer. Blastocyst status at the time of the cryo-thawed transfer was recorded.
Endometrial preparation
Estradiol valerate 6 mg daily (Climaval, Novartis Pharmaceuticals, Surrey, UK) was commenced orally on Day 2 of menstruation after pituitary suppression and continued for 13–15 days, after which endometrial thickness was evaluated. If endometrial thickness was <7 mm, the dose of Climaval was increased to 8 mg/day for a further 7–12 days. Progesterone supplementation in the form of micronized progesterone pessaries (Cyclogest, Shire Pharmaceuticals Ltd, Hants, UK) 400 mg twice daily was commenced 6 days prior to transfer.
Frozen–thawed embryo transfer and hormonal support
One or two blastocysts were transferred to the uterus on Day 6 of progesterone administration using an Edwards–Wallace embryo transfer catheter (Sims Portex Ltd, Hythe, Kent, UK). After embryo transfer, hormonal supplementation was continued for 14 days until a urine pregnancy test was performed using commercially available kits. Patients with a positive test continued with estrogen and progesterone supplementation until 12-week gestation.
Effect of an elective SBT strategy on outcome of PGD programme
To examine the effectiveness of introducing a policy of elective SBT and cryopreservation of surplus blastocysts to couples undergoing PGD, we analysed the outcome of all fresh PGD cycles performed between January 2006 and July 2008 and compared it with that of fresh PGD cycles performed between January 1998 and December 2005 prior to implementation of the elective SBT policy. All fresh PGD cycles which had reached oocyte retrieval were included in the analysis with no age restriction. In order to obtain the true cumulative success rate of our PGD programme, we combined the outcome of cryo-thawed PGD cycles with that of the corresponding fresh PGD cycles performed after January 2006 (Fig. 1).
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Cycle outcome
Pregnancy was diagnosed by a positive urine test for human chorionic gonadotrophin 14 days after embryo transfer. A clinical pregnancy was defined as the observation of a gestational sac with fetal heart beat on ultrasound scanning between 4 and 5 weeks after the positive pregnancy test. An ongoing pregnancy was defined as a viable pregnancy beyond 12-week gestation. Implantation rate was defined as the number of gestational sacs observed on ultrasound compared with the number of embryos transferred. The multiple pregnancy rate was calculated according to the number of pregnancies with more than one fetal heart beat observed on ultrasound scanning per clinical pregnancy achieved. The number of miscarriages was calculated as the number of pregnancies minus the number of ongoing pregnancies (i.e. including pre-clinical and clinical pregnancy loss).
Statistical analysis
Data were collected for patients' demographics and fresh and cryo-thawed cycle characteristics and outcomes. Univariate analysis of the study outcome measures and associated clinical variables was performed using two-sample t-test, 
2 or Fisher's exact test where appropriate. Multiple logistical regression analysis was used to determine the impact of patient age and basal FSH level at the time of fresh treatment cycle, number of fertilized oocytes, blastomere biopsy for PGD, number of blastocysts cryopreserved, number of blastocysts thawed and replaced in the cryo-thawed cycle and blastocyst re-expansion on the cryo-thawed cycle outcome. Odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated using the exact method. Statview software package for Macintosh (Statview 4.1, Abacus Concepts Ltd, Berkeley, CA, USA) was used for statistical analysis. A P-value of <0.05 was considered statistically significant.
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Results |
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During the study period, 32 PGD and 191 IVF cryo-thawed blastocyst transfer cycles were performed and included in the analysis. In these cycles, 432 frozen blastocysts (52 PGD and 380 conventional IVF/ICSI blastocysts) were thawed (a mean ± SD of 2 ± 0.8 embryo/thaw). In total, 376 (88%) blastocysts survived the process of thawing (45 PGD and 331 conventional IVF/ICSI blastocysts) and 350 blastocysts (43 PGD and 307 conventional IVF/ICSI blastocysts) were transferred (a mean ± SD of 1.6 ± 0.5 cryo-thawed embryo/transfer). The overall pregnancy, clinical pregnancy and ongoing pregnancy rates per thaw were 48%, 35% and 34%, respectively. The implantation rate was 31%.
Outcome of cryo-thawed cycles
Table I shows the treatment outcome in the PGD and IVF/ICSI cryo-thawed blastocyst transfers. The two groups were comparable in their baseline and fresh treatment cycle characteristics. The PGD group had a significantly lower number of blastocysts cryopreserved in the fresh cycle and thawed in the frozen cycle (Table I). Only two thaw cycles (0.9%) did not reach embryo transfer (one cycle in each group). The survival and re-expansion rates of thawed blastocysts were comparable in the two studied groups. In addition, there was no difference in overall pregnancy, clinical pregnancy, ongoing pregnancy and implantation rates between the two groups, despite replacing fewer cryo-thawed blastocysts in the PGD group. One monozygotic ongoing twin pregnancy (10%) occurred in the PGD group after transfer of a single re-expanded blastocyst and seven dizygotic ongoing twin pregnancies (11%) occurred in the IVF/ICSI group after transfer of two re-expanded blastocysts in each of these seven cryo-thawed cycles.
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To examine the impact of important clinical and embryological confounding variables on the cryo-thawed cycle outcome, the occurrence of a clinical pregnancy after a cryo-thawed blastocyst transfer cycle was employed in a logistic regression model using the following factors as independent variables: patient age and basal FSH level at the time of the fresh cycle, number of fertilized oocytes, number of blastocysts cryopreserved, number of blastocysts thawed and replaced in the cryo-thawed cycle (as continuous variables), and blastomere biopsy for PGD and transfer of at least one re-expanded blastocyst (as discrete variables). After adjusting for these variables, the only factor that was significantly associated with the occurrence of a clinical pregnancy was the transfer of one or more re-expanded blastocyst (OR = 7.6, 95% CI 1.7–33.7, P = 0.008, Table II).
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Effect of the elective SBT strategy on the fresh PGD programme
In total, we examined 645 fresh PGD cycles performed between January 1998 and July 2008, of which 571 cycles reached oocyte retrieval and were analysed. Of the 571 fresh PGD cycles, 522 cycles (91%) had embryo biopsy and 437 (77%) reached embryo transfer.
Before January 2006, 309 fresh PGD cycles were performed for 199 couples (1.5 cycles per couple), of which 239 cycles (77%) reached embryo transfer. Since January 2006, 262 fresh PGD cycles have been performed for 188 couples (1.4 cycles per couple), of which 198 cycles (76%) reached embryo transfer. In 46 cycles of the latter group, one or more surplus blastocysts were cryopreserved (mean number of cryopreserved blastocysts 2.2 ± 1.3, range 1–6). Table III shows the outcome of fresh PGD cycles in the two groups. Despite replacing significantly fewer embryos per transfer since January 2006, the pregnancy rates were comparable between the two groups, whereas the multiple pregnancy rate was reduced by 72% in Group 2 compared with Group 1 (OR = 0.20, 95% CI 0.08–0.48, P < 0.001). The outcome of PGD cycles performed after January 2006 was further examined depending on the number of embryos replaced (Table IV). There was no statistically significant difference in the implantation and pregnancy rates between the single- and double-embryo transfer groups. After adding the 32 frozen PGD cycles to the corresponding fresh PGD cycles performed after January 2006 [32 frozen and 262 fresh PGD cycles performed for 188 couples (1.5 cycles per couple)], the cumulative ongoing pregnancy rate per couple was not different in the two groups (35% for the 199 couples treated before January 2006 versus 37% for the 188 couples treated since January 2006, P = 0.48).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
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In order to extend the policy of elective SET to include PGD treatment cycles, it is critical to ensure optimum utilization of all transferable PGD embryos (Kokkali et al., 2007
). Given the relative complexity of PGD treatment, uptake of elective SET by PGD patients and specialists is unlikely to expand in the absence of a demonstrable benefit. At present, data showing cumulative pregnancy rates after application of an elective SBT policy and cryo-thawed cycles are lacking (Donoso et al., 2007).
In conventional IVF/ICSI cycles, elective SBT followed by transfer of cryo-thawed blastocysts if necessary has yielded high pregnancy rates (Henman et al., 2005; Le Lannou et al., 2006; Khalaf et al., 2008). A similar strategy has not been widely adopted in PGD cycles because of concern regarding the fewer number of transferable embryos in PGD cycles (Goossens et al., 2008; Harper et al., 2008), the relatively lower success rate of PGD compared with conventional IVF/ICSI (Harper et al., 2006) as well as the compromised post-thaw survival of cryopreserved embryos which have been subjected to the stress of blastomere biopsy (Joris et al., 1999; Magli et al., 1999; Ciotti et al., 2000; Jericho et al., 2003; Zheng et al., 2005). Post-thaw embryo survival reported in these studies was relatively low and ranged between 33% and 65% of cryo-thawed biopsied embryos. However, the majority of the embryos used in these studies were not suitable for transfer (because they had either developed from abnormally fertilized oocytes or showed evidence of chromosomal aneuploidy or poor morphological quality). Additionally, the cryopreservation was performed on Day 3 or 4 of in vitro culture before the embryos had reached the blastocyst stage (Joris et al., 1999; Magli et al., 1999; Ciotti et al., 2000; Jericho et al., 2003; Zheng et al., 2005).
Satisfactory survival rate of cryopreserved blastocysts obtained after conventional IVF/ICSI treatment has been documented (Veeck et al., 2004). The present study provides evidence that survival and implantation potential of cryo-thawed PGD blastocysts, which were biopsied on Day 3, is similar to that of non-biopsied cryo-thawed IVF/ICSI blastocysts. Our results concord and compare favourably with those of the study of Magli et al. (2006) in which approximately half of the cryopreserved biopsied blastocysts did not survive thawing and 18 of 34 thaw cycles (53%) reached embryo transfer. Both studies demonstrate the significant impact of blastocyst re-expansion after thawing on the cryo-thawed cycle outcome. However, the improved blastocyst survival and treatment outcome in our study relative to that of Magli et al. (2006) could be explained by variation in the indication for embryo biopsy, which was mostly for aneuploidy screening in the study of Magli et al. (2006), whereas it was exclusively for avoidance of an inherited genetic disease in our study. The improved outcome could also be related to differences in selection of fresh PGD embryos for biopsy and cryopreservation, the biopsy technique used including the number of blastomeres removed per biopsy and the protocols employed during blastocyst freezing, thawing and transfer in both studies. Exploration of the role of these factors in enhancing the survival and pregnancy potential of cryo-thawed biopsied blastocysts warrants further study.
In the present study, we evaluated the success of cryopreservation of blastocysts following a single blastomere biopsy on Day 3 using a standard slow-freezing protocol. Our results show that using this protocol can yield high blastocyst survival and implantation rates, equivalent to those achieved after thawing of non-biopsied cryopreserved IVF/ICSI blastocysts. Although the standard slow-freezing protocol is not considered optimal for biopsied embryos cryopreserved at the cleavage stage, the successful use of this protocol in freezing of blastocysts biopsied on Day 3 could be related either to the thinning of the zona pellucida in the expanding blastocyst, thereby limiting its role as a water barrier and making the presence of a breach due to blastomere biopsy less detrimental in terms of water permeability (Youssry et al., 2008) or to the structure of the blastocyst itself, which could render it less vulnerable to the cryodamage induced by rapid rehydration (Magli et al., 2006). This postulation is supported by the results of the study of Kung et al. (2003), which demonstrated high survival and clinical pregnancy rates after thawing of cryopreserved blastocysts that had undergone quarter laser-assisted hatching on Day 3 of in vitro culture before slow freezing. However, in view of the paucity of published literature in this area, more studies depicting the experience of various PGD centres with survival and implantation of cryopreserved biopsied embryos using either the slow freezing or vitrification techniques are warranted.
The development of our PGD blastocyst cryopreservation programme was paralleled by an increase in the proportion of PGD cycles having elective SBT (Table III). This practice has resulted in a significant reduction in the multiple pregnancy rate from 36% to 10% (P < 0.001) without any reduction in the overall pregnancy rates. Donoso et al. (2007) demonstrated that compulsory SBT for women <36 years (mean age = 29.8 years) having their first PGD cycle after implementation of the new Belgian legislation reduced the multiple pregnancy rate without significantly affecting the delivery rate in that group of patients. However, the study did not report on the use of elective SBT in PGD patients who were not legally required to have an SET, nor on the overall pregnancy and multiple pregnancy rates within their entire PGD programme. Indeed, the authors (Donoso et al., 2007) called for more data assessing the cumulative pregnancy rates with fresh and cryo-thawed PGD cycles to provide much needed information on the optimum transfer strategy in PGD cycles. Thus, to the best of our knowledge, our study is the first to provide reassurance that a strategy of elective SBT in good prognosis PGD patients (Grace et al., 2006), backed by an efficient PGD blastocyst cryopreservation service, can empower fertility centres to include PGD cycles for inherited genetic disorders in their efforts to reduce the multiple pregnancy rate after various forms of assisted conception treatment. Given the increasing number of PGD cycles performed each year, the advantage of widespread application of this policy would be considerable.
In conclusion, the present study has demonstrated that the likelihood of survival and implantation of PGD embryos biopsied on Day 3 and cryopreserved at the blastocyst stage is comparable to that of non-biopsied conventional IVF/ICSI cryopreserved blastocysts, although a direct comparison of pregnancy rates might be clouded by the difference in background fertility potential between the two groups. Our results suggest that a policy of elective SBT and cryopreservation of surplus PGD blastocyst(s) biopsied on Day 3 for later use is successful in reducing the multiple pregnancy rate without compromising the overall success of PGD cycles performed for inherited genetic disorders. Confirmation of these results in larger series would be valuable in optimizing the use of disease-free PGD embryos.
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Funding |
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No financial support has been sought or granted for this study.
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References |
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Braude P. Report of the expert group on multiple births after IVF. One child at a time. (2006) www.hfea.gov.uk/docs/MBSET_report_final_dec_06.pdf (2 February 2007, date last accessed).
Ciotti P, Lagalla C, Ricco S, Fabbri R, Forabosco A, Porcu E. Micromanipulation of cryopreserved embryos and cryopreservation of micromanipulated embryos in PGD. Mol Cell Endocrinol (2000) 27:63–67.
De Sutter P, Van der Elst J, Coetsier T, Dhont M. Single embryo transfer and multiple pregnancy rate reduction in IVF/ICSI: a 5-year appraisal. Reprod Biomed Online (2003) 6:464–469.[Medline]
Donoso P, Verpoest W, Papanikolaou E, Liebaers I, Fatemi H, Sermon K, Staessen C, Van der Elst J, Devroey P. Single embryo transfer in preimplantation genetic diagnosis cycles for women <36 years does not reduce delivery rate. Hum Reprod (2007) 22:1021–1025.
ESHRE Capri Workshop. Multiple gestation pregnancy. Hum Reprod (2000) 15:1856–1864.
Gardner D, Schoolcraft W. Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol (1999) 11:307–311.[CrossRef][Web of Science][Medline]
Gerris J, De Neubourg D, Mangelschots K, Van Royen E, Vercruyssen M, Barudy-Vasquez J, Valkenburg M, Ryckaert G. Elective single embryo transfer halves the twinning rate without decrease in the ongoing pregnancy rate of an IVF/ICSI programme. Hum Reprod (2002) 17:2626–2631.
Grace J, El-Toukhy T, Scriven P, Ogilvie C, Pickering S, Lashwood A, Flinter F, Khalaf Y, Braude P. Three hundred and thirty cycles of preimplantation genetic diagnosis for serious genetic disease. I. Clinical considerations affecting outcome. Br J Obstet Gynaecol (2006) 113:1393–1401.
Goossens V, Harton G, Moutou C, Scriven PN, Traeger-Synodinos J, Sermon K, Harper JC, European Society of Human Reproduction Embryology PGD Consortium. ESHRE PGD Consortium data collection VIII: cycles from January to December 2005 with pregnancy follow-up to October 2006. Hum Reprod (2008) 23:2629–2645.
Harper J, Boelaert K, Geraedts J, Harton G, Kearns W, Moutou C, Muntjewerff N, Repping S, SenGupta S, Scriven P, et al. ESHRE PGD consortium data collection V: cycles from January to December 2002 with pregnancy follow-up to October 2003. Hum Reprod (2006) 21:3–21.
Harper J, De Die-Smulders C, Goossens V, Harton G, Moutou C, Repping S, Scriven P, SenGupta S, Traeger-Synodinos J, Van Rij M, et al. ESHRE PGD consortium data collection VII: cycles from January to December 2004 with pregnancy follow-up to October 2005. Hum Reprod (2008) 23:741–755.
Henman M, Catt J, Wood T, Bowman M, de Boer K, Jansen R. Elective transfer of single fresh blastocysts and later transfer of cryostored blastocysts reduces the twin pregnancy rate and can improve the in vitro fertilization live birth rate in younger women. Fertil Steril (2005) 84:1620–1627.[CrossRef][Web of Science][Medline]
Jericho H, Wilton L, Gook D, Edgar D. A modified cryopreservation method increases the survival of human biopsied cleavage stage embryos. Hum Reprod (2003) 18:568–571.
Joris H, Van den Abbeel E, De Vos A, Van Steirteghem A. Reduced survival after human embryo biopsy and subsequent cryopreservation. Hum Reprod (1999) 14:2833–2837.
Kaufman R, Menezo Y, Hazout A, Nicollet B, DuMont M, Servy E. Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles. Fertil Steril (1995) 64:1125–1129.[Web of Science][Medline]
Khalaf Y, El-Toukhy T, Coomarasamy A, Kamal A, Bolton V, Braude P. Selective single blastocyst transfer reduces the multiple pregnancy rate and increases pregnancy rates: a pre- and post-intervention study. Br J Obstet Gynaecol (2008) 115:385–390.
Kokkali G, Traeger-Synodinos J, Vrettou C, Stavrou D, Jones G, Cram D, Makrakis E, Trounson A, Kanavakis E, Pantos K. Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of β-thalassaemia: a pilot study. Hum Reprod (2007) 22:1443–1449.
Kung F, Lin Y, Tseng Y, Huang F, Tsai M, Chang S. Transfer of frozen-thawed blastocysts that underwent quarter laser-assisted hatching at the day 3 cleaving stage before freezing. Fertil Steril (2003) 79:893–899.[CrossRef][Web of Science][Medline]
Lassalle B, Testart J, Renard J. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril (1985) 44:645–651.[Web of Science][Medline]
Le Lannou D, Griveau J, Laurrent M, Gueho A, Veron E, Morcel K. Contribution of embryo cryopreservation to elective single embryo transfer in IVF-ICSI. Reprod Biomed Online (2006) 13:368–375.[Web of Science][Medline]
Lukassen HGM, Braat DD, Wetzels A, Zielhuis A, Scheenjes E, Krener J. Two cycles with single embryo transfer versus one cycle with double embryo transfer: a randomised controlled trial. Hum Reprod (2005) 20:702–708.
Magli MC, Gianaroli L, Fortini D, Ferraretti A, Munne S. Impact of blastomere biopsy and cryopreservation techniques on human embryo viability. Hum Reprod (1999) 14:770–773.
Magli MC, Gianaroli L, Grieco N, Cefalu E, Ruvolo G, Ferraretti A. Cryopreservation of biopsied embryos at the blastocyst stage. Hum Reprod (2006) 21:2656–2660.
Palmer G, Traeger-Synodinos J, Davies S, Tzetis M, Vrettou C, Mastrominas M, Kanavakis E. Pregnancies following blastocyst stage transfer in PGD cycles at risk for B-thalassaemic haemoglobinopathies. Hum Reprod (2002) 17:25–31.
Pickering S, Polidoropoulos N, Caller J, Scriven P, Ogilvie C, Braude P. Strategies and outcome of the first 100 cycles of preimplantation genetic diagnosis at the Guy's and St. Thomas' Centre. Fertil Steril (2003) 79:81–90.[CrossRef][Web of Science][Medline]
Preimplantation Genetic Diagnosis International Society (PGDIS). Guidelines for good practice in PGD. Programme requirements and laboratory quality assurance. Reprod Biomed Online (2008) 16:134–147.[Web of Science][Medline]
Stephenson E, Braude P, Mason C. Proposal for a universal minimum information convention for the reporting on the derivation of human embryonic stem cell lines. Regen Med (2006) 1:739–750.[CrossRef][Web of Science][Medline]
Thurin A, Hausken J, Hillensjo T, Jablonowska B, Pinborg A, Strandell A, Bergh C. Elective single-embryo transfer versus double-embryo transfer in in-vitro fertilization. N Engl J Med (2004) 351:2392–2402.
Veeck L, Bodine R, Clarke R, Berrios R, Libraro J, Moschini R, Zaninovic N, Rosenwaks Z. High pregnancy rates can be achieved after freezing and thawing human blastocysts. Fertil Steril (2004) 82:1418–1427.[CrossRef][Web of Science][Medline]
Youssry M, Ozmen B, Zohni K, Diedrich K, Al-Hasani S. Current aspects of blastocyst cryopreservation. Reprod Biomed Online (2008) 16:311–320.[Web of Science][Medline]
Zheng W, Zhuang G, Zhou C, Fang C, Ou J, Li T, Zhang M, Liang X. Comparison of the survival of human biopsied embryos after cryopreservation with four different methods using non-transferable embryos. Hum Reprod (2005) 20:1615–1618.
Submitted on January 1, 2009; resubmitted on April 8, 2009; accepted on April 14, 2009.
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