Pregnancies following blastocyst stage transfer in PGD cycles at risk for beta-thalassaemic haemoglobinopathies.

BACKGROUND: Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy 3 days post-insemination (p.i.), followed by genetic analysis and transfer of unaffected embryos later on day 3 or 4. We evaluate a strategy involving embryo biopsy on day 3 p.i., genetic analysis on day 4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on day 5 p.i. METHODS: PGD cycles were initiated in 15 couples at risk of transmitting beta-thalassaemia major. Oocyte retrieval and ICSI were performed according to standard protocols. Embryo culture used blastocyst sequential media. Embryos were biopsied on day 3 p.i. using acid Tyrode's for zona drilling, and the single blastomeres were genotyped by a protocol involving nested polymerase chain reaction and denaturing gradient gel electrophoresis analysis. RESULTS: Forty of 109 (37%) embryos biopsied on day 3 p.i. developed to blastocysts by day 5 p.i., with at least one blastocyst available for transfer in 12 cycles (80%). Genotype analysis characterized 51/109 (47%) embryos unaffected for beta-thalassaemia major, of which 28 were blastocysts. Transfer of 37 day 5 p.i. embryos (blastocysts and non blastocysts) initiated eight clinical pregnancies. Implantation rate per embryo transferred was 12/37 (32%). CONCLUSIONS: Embryo biopsy on day 3, followed by delayed transfer until day 5 p.i. offers a novel and effective strategy to overcome the time limit encountered when performing PGD, without compromising embryo implantation.     

Reduced survival after human embryo biopsy and subsequent cryopreservation.

Preimplantation genetic diagnosis (PGD) is performed in couples at risk of genetic disease, so as to avoid transfer of embryos which are affected by a monogenic disease or which carry chromosomal aberrations. As in all in-vitro fertilization (IVF) cycles, supernumerary non-affected good-quality embryos may be available after PGD. These embryos can be cryopreserved. So far, limited data on survival after cryopreservation of biopsied human embryos are available. In this study, human embryos of good morphological quality derived from abnormal fertilization were used to evaluate the influence of the embryo biopsy procedure on survival after cryopreservation. Embryos were allocated to three different groups: control (n = 20), drilling-only (n = 16), and biopsy (n = 29). After freezing and thawing, a significantly lower number of blastomeres was intact in the drilling-only group (46/118, i.e. 39.0%, P < 0.01) and in the embryo biopsy group (46/156, i.e. 29.5%, P < 0.0001) than in the control group (85/151, i.e. 56.3%). This difference was reflected in survival rates of embryos. Fifty-five per cent of the control embryos, 37.5% of the drilling-only group, and 33.3% of the biopsy group had at least 50% of their blastomeres intact. After further in-vitro culture, four blastocysts, three from the drilling-only group and one from the biopsy group, developed from the surviving embryos. From this study it can be concluded that current cryopreservation procedures are less successful when biopsied human embryos are cryopreserved, but that surviving embryos can develop to the blastocyst stage and thus may have the potential to develop to term.     

Cryopreservation of biopsied embryos at the blastocyst stage

BACKGROUND: The availability of an efficient cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for PGD. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. In this study, embryos diagnosed as normal after PGD were grown to the blastocyst stage, frozen and thawed for successive frozen embryo transfer. METHODS: A total of 34 patients performed a thawing cycle in which 47 blastocysts were thawed. The cryopreservation solutions were based on HEPES-buffered medium supplemented with human serum albumin (HSA), sucrose and 1,2-propanediol. The same protocol was applied to embryos from 88 IVF/ICSI patients, which underwent 92 thawing cycles with 150 thawed blastocysts. RESULTS: The survival rate was similar in the two groups (53% after PGD and 58% in IVF/ICSI cycles), as well as the cumulative pregnancy rate per patient (59% after PGD versus 47% in IVF/ICSI cycles), despite a higher maternal age and a lower proportion of embryos available for transfer or cryopreservation in the PGD group. CONCLUSIONS: Neither the survival rate nor the subsequent development and chances of implantation, differed between embryos frozen at the blastocyst stage following biopsy and those frozen intact.     

Preimplantation development of mouse and human embryos biopsied at cleavage stages using a modified displacement technique

A modified embryo biopsy method was tested on four- and eight-cell stage mouse embryos and used on human embryos to obtain blastomeres for preimplantation genetic diagnosis. The biopsy method tested combines zona drilling and fluid displacement to force one or two cells through an opening in the zona pellucida of the cleavage-stage embryo. Rates of cell division and the percentage of mouse embryos forming blastocysts following biopsy at the eight-cell stage were not significantly different from those observed in unoperated control embryos. The percentage blastocyst formation was not significantly different in embryos biopsied at the four-cell stage and in control embryos, although cell division was significantly retarded following biopsy. 96% of the mouse blastomeres isolated at the eight-cell stage were recovered intact and 96% of those placed in culture underwent cell division. Survival and division of cells isolated at the four-cell stage were 92 and 84% respectively. Most of the cultured blastomeres cleaved several times and formed small trophoblast vesicles. Chromosomes were observed in 59% of blastomeres incubated in the presence of colcemid. In the initial use of this biopsy technique for human preimplantation genetic diagnosis, blastocyst formation was observed in 9 of 13 human embryos biopsied at the 7- to 10-cell stage. These findings support the use of this biopsy method as an alternative to aspiration techniques.      

Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of beta-thalassaemia: a pilot study

BACKGROUND: Trophectoderm biopsy at the blastocyst stage is an emerging approach in preimplantation genetic diagnosis (PGD). This study aimed to compare genotyping success and implantation rates in PGD cycles for beta-thalassaemia following biopsy at the cleavage versus the blastocyst stage, with transfer of blastocysts. METHODS: This pilot study included 20 cycles: Group A: 10 cycles, day 3 blastomere biopsy, day 5 transfer; Group B: 10 cycles, day 5 trophectoderm biopsy, day 6 transfer. Standard-assisted reproduction and laser biopsy procedures were used. Biopsied cells were genotyped using real-time PCR multiplexed with fluorescent microsatellite analysis. RESULTS: In Group A, 131 fertilized eggs developed to 101 embryos suitable for single blastomere biopsy; 76/101 blastomeres were diagnosed (75.2%), 30 unaffected blastocysts were transferred resulting in six pregnancies (eight fetal hearts, 26.7% implantation rate). In Group B, 128 fertilized eggs developed to 53 blastocysts for trophectoderm biopsy (four to five cells), with 50/53 blastocysts diagnosed (94.3%), 21 unaffected blastocysts transferred and 6 pregnancies initiated (10 fetal hearts, 47.6% implantation rate). Overall, nine pregnancies reached >10 weeks gestation and were confirmed unaffected by prenatal diagnosis, with 12 healthy babies born. CONCLUSIONS: This pilot study suggests that trophectoderm biopsy and blastocyst transfer may be more advantageous than cleavage stage biopsy with respect to outcome of PGD for monogenic diseases.     

Fertilization and early embryolgoy: A comparison of the effects of different biopsy strategies on the post-thaw survival of 8-cell-stage mouse embryos: implications for preimplantation diagnosis

The aim of this study was to investigate the effects of twodifferent biopsy strategies, zona slitting and zona piercing,on the post-thaw survival and subsequent in-vitro developmentof 8-cell mouse embryos. From control experiments it was determinedthat neither biopsy by zona slitting nor zona piercing adverselyaffected embryo development in vitro, as similar rates of blastocystformation and hatching were found between biopsied and zona-intactembryos; there was, however, a trend towards a lower rate ofblastocyst hatching in embryos biopsied by zona piercing (78.3%compared with 91.9% of zona-intact embryos). When biopsy wasfollowed by cryopreservation a different picture was seen: similarrates of freeze—thaw survival were found for zona-slit,zona-pierced and zona-intact embryos (84.2, 88.5 and 87.2% respectively),but this was superseded by a significant (P < 0.05) reductionin the blastocyst formation rates (61.4% zona-slit and 63.9%zona-pierced versus 78.7% zona-intact) and hatching rates (51%zona-slit and 52.5% zona-pierced versus 72.3% zona-intact) ofthe biopsied embryos. When the effects of zona slitting andpiercing were considered in isolation, i.e. without performingbiopsy, it was found that the larger holes produced by zonaslitting rendered embryos more susceptible to freeze-thaw damage.Significant (P < 0.05) reductions occurred in the blastocystformation rates (63.7% zona-slit versus 78.7% zona-intact embryos)and hatching rates (52.3% zona-slit versus 72.3% zona-intactembryos) of the zona-slit embryos.     

Impact of assisted hatching on ART outcome in women with endometriosis

BACKGROUND: Assisted hatching can improve the implantation rate in cycles with poor outcome. The impact of assisted hatching in embryos from women with endometriosis is not known. Therefore, the hypothesis that the implantation potential of embryos obtained from women with endometriosis can be improved with assisted hatching was tested. METHODS: In a prospective randomized study, transfer embryos obtained from 60 women with endometriosis were hatched using a laser system and compared to embryos obtained from patients with the same diagnosis which were left intact (n = 30). RESULTS: The characteristics of cycles were similar between groups. The pregnancy (40% zona intact, 28.3% assisted hatching), and implantation rates (19.4% zona intact, 17.8% assisted hatching) did not differ in endometriosis cycles regardless of assisted hatching. CONCLUSION: Assisted hatching does not improve outcome in women with endometriosis undergoing assisted reproduction.     

Strategies and clinical outcome of 250 cycles of Preimplantation Genetic Diagnosis for single gene disorders

BACKGROUND: We report on our experience with preimplantation genetic diagnosis (PGD) for single gene disorders (SGDs), from 1999 to 2004, describing strategies and overall clinical outcome of 250 cycles in 174 couples for 23 different genetic conditions. METHODS: PGD cycles included 15 for autosomal dominant, 148 for autosomal recessive and 19 for X-linked SGDs. In addition, 68 cycles of PGD for SGDs were performed in combination with HLA matching. The strategy in each case used an initial multiplex PCR, followed by minisequencing to identify the mutation(s) combined with multiplex PCR for closely linked informative markers to increase accuracy. Linkage analysis, using intragenic and/or extragenic polymorphic microsatellite markers, was performed in cases where the disease-causing mutation(s) was unknown or undetectable. RESULTS: In 250 PGD cycles, a total of 1961 cleavage stage embryos were biopsied. PCR was successful in 3409 out of 3149 (92.4%) biopsied blastomeres and a diagnosis was possible in 1849 (94.3%) embryos. Four hundred and twenty-seven embryos were transferred in 211 cycles, resulting in 71 pregnancies (33.6% per embryo transfer), including 15 biochemical pregnancies, six spontaneous miscarriages, two ectopic pregnancies, which were terminated, and nine pregnancies which are still ongoing. The remaining pregnancies were confirmed to be unaffected and went to term without complications, resulting in the birth of 35 healthy babies. CONCLUSIONS: Minisequencing for mutation detection combined with multiplex fluorescence PCR for linkage analysis is an efficient, accurate and widely applicable strategy for PGD of SGDs. Our experience provides a further demonstration that PGD is an effective clinical tool and a useful option for many couples with a high risk of transmitting a genetic disease.     

A prospective randomized study to assess the benefit of partial zona pellucida digestion before frozen-thawed embryo transfers

BACKGROUND: Assisted hatching (AH) in fresh embryo transfer (ET) could be associated with increased implantation rates. However, very few prospective randomized studies have specifically addressed the issue of AH during frozen-thawed embryo transfers (FET) cycles, those that have reported controversial results. The aim of this study was to evaluate the benefit of an enzymatic zona pellucida treatment of frozen-thawed embryos before transfer. METHODS: This was a prospective study including 125 non-donor FET cycles from 125 infertile couples. FETs were randomly allocated into AH group (n = 61, embryos pretreated with pronase 5 IU/ml for 1 min at 37 degrees C) or control group (n = 64, untreated embryos). Zona pellucida thickness was measured for each transferred embryo. The main outcome parameters were clinical pregnancy and implantation rates. RESULTS: The two groups were comparable regarding mean women's age, duration and indications of infertility, IVF outcome after fresh ETs, numbers and quality of fresh and frozen embryos, frozen-thawed embryo survival rates and blastomeres survival indexes. Despite a statistically significant decrease of zona pellucida thickness after pronase treatment [(mean +/- SD) 18.5 +/- 2.25 versus 14.5 +/- 2.75 microm; P < 0.0001], implantation (9.6 versus 9.2%) and clinical pregnancy rates (18.0 versus 17.2%) were not statistically different after FETs, with a similar mean number of embryos transferred between AH and control groups, respectively. CONCLUSION: Within the constraints of our protocol, partial enzymatic digestion of zona pellucida by pronase was not related with any benefit of the FET outcome especially concerning the implantation ability of frozen-thawed embryos.     

A comparison of four different techniques of assisted hatching

BACKGROUND: Assisted hatching (AH) has been proposed as a means to increase the implantation rate in patients with poor prognosis for pregnancy. The procedure appears to be effective when used selectively. Several different methods for AH have been introduced over the years but comparative studies are lacking. The aim of the current study was to compare retrospectively the efficacy of AH performed with four different methods in patients undergoing IVF or ICSI. METHODS: AH was performed prior to day 3 embryo transfer in 794 IVF/ICSI cycles. Indications for AH were females aged >35 years and/or elevated follicular phase FSH levels, previous failed IVF/ICSI cycles, poor embryo quality, and thick zona pellucida (>15 microm). Assignment to one of the four methods of AH was according to the availability of the particular method during the study period. The study was not randomized. RESULTS: Partial zona dissection was used in 239, acid Tyrode in 191, diode laser in 219 and pronase thinning of the zona pellucida in 145. Mean female age, mean number of previous failed IVF/ICSI cycles, number of oocytes retrieved, fertilization and cleavage rates, good quality embryos and zona thickness on day 3 did not differ between groups. Mean number of embryos transferred, implantation rate, clinical pregnancy rate, and abortion rates were likewise similar. CONCLUSIONS: Selective AH using four different methods yields similar implantation and pregnancy rates.     

Impact of blastomere biopsy and cryopreservation techniques on human embryo viability

The purpose of the present study was to evaluate the effect of cryopreservation on 55 embryos which had one blastomere biopsied for preimplantation genetic diagnosis of aneuploidy before freezing. The thawing outcome was compared to that obtained in 94 embryos which derived from our conventional freezing programme in patients with comparable characteristics who were treated in the same period. Their embryos were morphologically similar but the incidence of aneuploidy was 100% in the biopsy group and unknown in the controls. The percentage of embryos which survived intact after thawing was significantly lower in the biopsied group compared to the controls (9 versus 25% respectively; P < 0.025), whereas the rate of lysis was superior among biopsied embryos (34 versus 13% in the controls; P < 0.001). Similarly, the survival index was higher in the frozen-intact embryos than in the embryos which were frozen after biopsy (61 versus 38%; P < 0.001). No empty zonae resulted in the control group, while six were found after thawing biopsied embryos. In the second part of the study, blastomere biopsy was implemented on 102 thawed embryos generated by 16 patients. The chromosomal analyses revealed that 49 were normal, leading to the transfer of 2.5 +/- 0.8 embryos per patient. Only three clinical pregnancies were obtained, and are presently ongoing. In conclusion, the present findings discourage the use of conventional cryopreservation protocols in strategies involving preimplantation genetic diagnosis in human reproductive medicine. Adequate protocols are required for freezing and thawing embryos which have been subjected to biopsy procedures.     

Parameters guiding selection of best embryos for transfer after cryopreservation: a reappraisal

BACKGROUND: The purpose of this study was to evaluate the respective influences of blastomere survival and resumption of mitosis on the outcome of frozen-thawed embryos. METHODS: A retrospective analysis was performed in our centre on 363 thawing cycles, involving 4-cell day 2 grade 1 embryos with <10% fragmentation. RESULTS: A higher implantation rate per transferred embryo was observed when all transferred embryos were characterized by fully intact blastomeres (100% blastomere survival) as compared with damaged embryos (50 or 75% blastomere survival) (22.0 versus 7.2%; P < 0.0001). Moreover, the implantation rate per transferred embryo was significantly higher for cleaved embryos compared with uncleaved embryos (19.7 versus 3%; P < 0.0001). Transfer of fully intact, cleaved embryos resulted in the highest implantation rates compared with transfer of damaged and uncleaved embryos (27.4 versus 0%; P < 0.0001). Intermediate implantation rates were observed when only one of the two criteria was fulfilled (13 versus 11% respectively; P > 0.05). Multivariate analysis showed that the clinical pregnancy rate was influenced by both criteria (odds ratio = 3.4 for transfer of embryos with six or more cells versus embryos with less than six cells. CONCLUSION: The results of our study suggest that the most important factor to predict further embryo development is the total number of blastomeres in transferred embryos, however they are obtained (good survival and/or resumption of mitosis).     

A modified cryopreservation method increases the survival of human biopsied cleavage stage embryos

BACKGROUND: The relatively poor survival rate of human biopsied cleavage stage embryos following cryopreservation is a significant obstacle in the application of preimplantation genetic diagnosis (PGD). We have attempted to improve cryosurvival of biopsied embryos by modifying the standard embryo cryopreservation technique. METHODS: Biopsied embryos were cryopreserved in 1.5 mol/l 1,2-propanediol in the presence of an elevated concentration of sucrose (0.2 mol/l) and human serum albumin was replaced by maternal serum (20% vol:vol). An additional initial thawing step in the presence of 0.3 mol/l sucrose was also included. RESULTS: The proportion of biopsied embryos which survived cryopreservation with > or =50% of their blastomeres intact was significantly higher using the modified method (138/185; 75%) than that observed using the standard propanediol method (20/46; 43%; P = 0.022). Total blastomere survival was also significantly increased as a result of the modifications (1010/1513; 67% versus 177/385; 46%; P < 0.001). Six fetal hearts have been detected to date following replacement of biopsied embryos cryopreserved with the modified method. CONCLUSIONS: Survival of human biopsied cleavage stage embryos can be restored to a level similar to that of non-biopsied controls by modification of the cryopreservation procedure. Embryos which have been cryopreserved using the modified method can implant following replacement in utero.     

Preimplantation genetic diagnosis of spinocerebellar ataxia 3 by (CAG)(n) repeat detection

Spinocerebellar ataxia 3 (SCA3) is an autosomal dominant neurodegenerative disorder characterized by variable expression and a variable age of onset. SCA3/MJD (Machado-Joseph disease) is caused by an expansion of a (CAG)(n) repeat in the MJD1 gene on chromosome 14q32.1. A single cell PCR protocol has been developed for preimplantation genetic diagnosis (PGD) of SCA3 to select unaffected embryos on the basis of the CAG genotype. Single leukocytes and blastomeres served as a single cell amplification test system to determine the percentage of allelic drop-out (ADO) and PCR efficiency. Out of 105 tested heterozygous single leukocytes, 103 (98.1%) showed a positive amplification signal, while five cells (4.9%) showed ADO. Amplification in single blastomeres was obtained in 13 out of a total of 14, and ADO was observed in two out of the 13 single blastomeres. PGD of SCA3 was performed in a couple with paternal transmission of the SCA3 allele. Seven embryos were available for biopsy, all biopsied blastomeres showed amplification and no ADO occurred. One embryo was diagnosed as affected whereas six embryos were diagnosed as unaffected. Two unaffected embryos were transferred and resulted in a singleton pregnancy and the birth of a healthy girl.     

An evaluation of PGD in clinical genetic services through 3 years application for prevention of beta-thalassaemia major and sickle cell thalassaemia

PGD represents an alternative within prenatal diagnosis services, which avoids terminating affected on-going pregnancies. In Greece, prevention programmes for haemoglobinopathies, including the option of prenatal diagnosis, are well established. Following optimization of a single-cell genotyping strategy (designed to be applicable for the majority of beta-thalassaemia major or sickle thalassaemia genotype interactions) along with close collaboration with an IVF unit, we integrated the option of PGD for at-risk couples with a problematic reproductive history. A total of 59 couples requesting PGD were counselled, of whom 41 initiated 63 PGD cycles. Following standard assisted reproduction treatment for oocyte retrieval, 20 cycles were cancelled (too few oocytes and/or poor quality embryos), but in 43 cycles single blastomeres were biopsied from 3 day embryos and genotyped (total 302). Diagnosis was achieved for 236 embryos, and 100 of 125 unaffected embryos were transferred. Sixteen pregnancies were established, although six were lost within the first trimester. Ten pregnancies underwent second trimester prenatal diagnosis, with nine pregnancies (13 babies: six singletons, two twins and one triplet) confirmed unaffected, although one singleton was a PGD misdiagnosis and terminated. The triplet pregnancy was selectively reduced to twins, and nine pregnancies went to term, with 12 healthy babies born. This report highlights advantages, limitations and approaches towards improvement when incorporating PGD within genetic services for a common recessive disease.     

Genetic diagnosis using polymerase chain reaction and fluorescent in-situ hybridization analysis of biopsied cells from both the cleavage and blastocyst stages of individual cultured human preimplantation embryos

Cultured human preimplantation embryos have been used to developmethods which allow preimplantation genetic diagnosis (PGD)analyses by polymerase chain reaction (PCR) and fluorescentin-situ hybridization (FISH) on biopsied blastomeres and trophectodermcells from the same embryo. An experimental design is describedand experiments undertaken, which demonstrate the feasibilityof extending biopsy and PGD procedures currently in use. Wehave shown that dual-stage biopsies are possible, and that thePCR and FISH analyses of the biopsied cell samples are effective.One to two blastomeres were biopsied from an 8- to 10-cell embryoand processed for the simultaneous PCR amplification of a -globinand a cytosine adenine (CA) repeat sequence, or a Y chromosomesequence. FISH procedures were also used to detect the presenceof Y chromosome markers. The biopsied cleavage-stage embryocan be cultured to the blastocyst stage, where the serial biopsyof three to five mural trophectoderm cells provides two furthercell samples. These can be used to repeat and/or undertake additionalPGD analyses. The biopsied blastocyst is either used to confirmearlier diagnoses, or placed in culture for a further 4–24h. Maintenance of a blastocoele cavity, hatching and formationof an outgrowth demonstrates continuing viability followingthe dual-stage biopsy procedures. The PCR DNA amplificationprocedures are effective at the cellular level for both biopsiedblastomeres and mural trophectoderm cells. The FISH techniqueshave shown a definitive Y signal in 50% (one out of two) and100% (two out of two) of the biopsied blastomeres and 72% (twoout of three, four out of five and 7 out of 10) for the trophectodermcell nuclei. Preliminary experiments have demonstrated thatthe FISH preparations can be re-amplified to improve the signal,and dual fluorescent procedures using the X and Y probes areeffective. A retrospective PCR analysis has also been undertakenon preparations of biopsied cells which were previously usedfor PGD analysis by FISH.     

Preimplantation diagnosis: The in-vitro and in-vivo developmental potential of frozen and non-frozen biopsied 8-cell mouse embryos

We investigated the in-vitro and in-vivo development of cryopreservedand non-cryopreserved biopsied 8-cell mouse embryos, from whichone to seven blastomeres were removed by micromanipulation.The results clearly indicate that the in-vitro and in-vivo developmentof biopsied 8-cell mouse embryos depended on the number of blastomeresremoved: the more blastomeres removed, the greater the effect.When five, six, or seven blastomeres were removed, fewer blastocystswere formed. Furthermore, when these blastocysts were transferredto pseudopregnant females, no living young were formed, indicatingthe abnormality of these blastocysts. When up to three blastomereswere removed, there was no significant effect on the rate ofin-vitro blastocyst formation. Living young were found evenafter the biopsy of four blastomeres, and after biopsy of onlyone or two blastomeres, the same percentage of living youngwas obtained as in the non-biopsied control embryos. Biopsied8-cell mouse embryos were frozen and thawed in straws with 1,2-propanediol (1.5 M) and sucrose (0.1 M) with slow-freezingand rapidthawing protocols. The survival after cryopreservation,defined as the percentage of embryos with the same number ofblastomeres intact as the number of blastomeres before freezing,was excellent and no different from non-biopsied embryos, independentof the number of blastomeres biopsied. Furthermore, cryopreservationhad no further impact on the in-vitro and in-vivo developmentof the biopsied embryos.     

The combination of polar body and embryo biopsy does not affect embryo viability

BACKGROUND: The biopsy of both polar bodies and a blastomere from the same embryo was investigated as an approach aimed at increasing the quantity of DNA available for genetic analysis in preimplantation embryos. METHODS: In 113 cycles, preimplantation genetic diagnosis (PGD) was performed for aneuploidy: 19 cycles underwent polar body biopsy, 32 cycles had both polar body and blastomere biopsy done, and the remaining 62 cycles underwent blastomere biopsy. The chromosomal analysis was performed in a two-round fluorescence in situ hybridization (FISH) protocol with probes specific for the chromosomes X, Y, 13, 15, 16, 18, 21 and 22. RESULTS: The morphological evaluation of the analysed embryos demonstrated similar rates of development irrespective of the biopsy procedure. Accordingly, the implantation rate did not differ significantly in the three biopsy groups and was 15% after polar body biopsy, 26% after the combined biopsy procedures of polar bodies and blastomeres, and 25% after blastomere biopsy. CONCLUSIONS: The removal of a blastomere subsequent to polar body biopsy does not seem to have negative effects on embryo viability. This approach could be especially valuable for a combined diagnosis of aneuploidy and single-gene disorders in preimplantation embryos generated by couples at high reproductive risk.     

Transfers of frozen-thawed human embryos in cycles stimulated by HMG

A total of 130 transfers of frozen-thawed (F-T) human embryos was carried out after moderate ovarian stimulation with human menopausal gonadotrophin (HMG). Embryos were replaced 3 days after the spontaneous luteinizing hormone (LH) surge or 4 days if ovulation was induced by human chorionic gonadotrophin (HCG). Embryos were thawed a few hours prior to transfer. One-hundred-and-twenty-three transfers were effective and 23 pregnancies were achieved. The rate of ongoing pregnancies per transfer was 17.9% (22/123). The survival rate of embryos originating from cycles stimulated by a combination of an LHRH analogue and HMG in a long protocol (LA-HMG protocol) was significantly lower when compared with the rate of embryos retrieved from clomiphene citrate-HMG (CC-HMG protocol) stimulated cycles (52 versus 67%, P less than 0.05). When fresh embryos originated from cycles stimulated with an LHRH analogue and HMG in a short protocol (SA-HMG protocol), the survival rate was not affected (59 versus 67%, NS). Although the difference was not significant, the ongoing pregnancy rate per transfer according to the three protocols from which the embryos originated seemed to be better with the SA-HMG protocol: 16% with the CC-HMG protocol, 14.5% with the LA-HMG protocol versus 27.6% with the SA-HMG protocol. The success rate was independent of the number of F-T transferred embryos if at least one embryo with 100% intact blastomeres was replaced.     

Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis

BACKGROUND: Preimplantation genetic diagnosis or screening (PGD, PGS) involves embryo biopsy on Day 3. Opting for one- or two-cell biopsy is a balance between the lowest risk for misdiagnosis on the one hand and the highest chance for a pregnancy on the other hand. METHODS: A prospective controlled trial was designed and 592 ICSI cycles were randomly assigned to the one-cell (group I) or the two-cell group (group II). Primary outcomes were diagnostic efficiency and embryonic development to delivery with live birth (analysed by cycle). The false-positive rate for the PCR cycles is presented as a secondary outcome (analysed by embryo). RESULTS: A strong significant correlation was observed between embryonic developmental stage on Day 3 and post-biopsy in vitro development on Day 5 (P < 0.0001). The influence of the intervention on Day 3 was less significant (P = 0.007): the biopsy of one cell is less invasive than the biopsy of two cells. PCR diagnostic efficiency was 88.6% in group I and 96.4% in group II (P = 0.008). For the fluorescence in situ hybridization (FISH) PGD cycles no significant difference in efficiency was obtained (98.2 and 97.5% in group I and II, respectively). Similar delivery rates with live birth per started cycle were obtained [58/287 or 20.2% in group I versus 52/303 or 17.2% in group II, P = 0.358; the absolute risk reduction = 3.05%; 95% confidence interval (CI): -3.24, 9.34]. Post-PGD PCR reanalysis showed six false positives in 97 embryos (6.2%) in group II and none in group I (91 embryos reanalysed). No false negatives were found. CONCLUSIONS: While removal of two blastomeres decreases the likelihood of blastocyst formation, compared with removal of one blastomere, Day 3 in vitro developmental stage is a stronger predictor for Day 5 developmental potential than the removal of one or two cells. The biopsy of only one cell significantly lowers the efficiency of a PCR-based diagnosis, whereas the efficiency of the FISH PGD procedure remains similar whether one or two cells are removed. Delivery rates with live birth per started cycle were not significantly different.