1 Centre for Human Reproduction, Genesis Hospital and 2 Laboratory of Medical Genetics, Athens University, St Sophia's Children's Hospital, Athens, Greece, 3 Monash Immunology and Stem Cell Laboratories, Monash University and 4 Monash IVF, Melbourne, Australia
5 To whom correspondence should be addressed at: Centre for Human Reproduction, Genesis Hospital, Papanikoli Avenue 1416, Halandri, Athens 152-32, Greece. Email: georgiakokkali{at}mail.com
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words:
biopsy/blastocyst/-thalassaemia/laser/PGD
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Once the genetic diagnosis has been completed, the embryo transfer may be performed the same day (Boada et al., 1998) or delayed, and pregnancies have resulted from embryo transfer on day 4 or later (Grifo et al., 1998
; Palmer et al., 2002
). Since the development of sequential media (Gardner and Lane, 1998
; Jones et al., 1998a
), reports indicate improved pregnancy rates following blastocyst stage transfers (Gardner et al., 1998a
,b
; Jones et al., 1998a
,b
; Rijnders and Jansen, 1998
; Pantos et al., 2001
). Trophectoderm biopsy at the blastocyst stage enables the removal of more than two cells for diagnosis while being non-invasive to the inner cell mass which is destined for fetal development (Dokras et al., 1990
; Veiga et al., 1997
; de Boer et al., 2004
). This case report describes the first live birth following trophectoderm biopsy at the blastocyst stage of development for the PGD of
-thalassaemia. Another facet of this success is that the blastocysts were derived from thawed embryos previously cryopreserved at the pronucleate stage.
![]() |
Case report |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
On day 5, three early blastocysts had developed and a hole was made in the zona pellucida directly opposite the inner cell mass of each blastocyst, using the lowest setting of the ZILOS-tk non-contact laser (Hamilton Thorne Biosciences, Beverley, MA). Blastocysts were incubated for a further 4 h to allow further growth and herniation of the trophectoderm cells from the zona. After 4 h in culture, the blastocysts had grown and were expanding but no significant herniation was observed. A single cell had herniated from one blastocyst whereas no herniation was observed for the other two blastocysts. The blastocysts were placed individually in 20 µl of G-MOPS medium (Vitrolife, Goteborg, Sweden) under oil for biopsy. Applying gentle suction with the biopsy pipette (Cook Australia, Eight Mile Plains, Qld, Australia), trophectoderm cells were encouraged to herniate from the zona. Four to six trophectoderm cells were dissected from each of the blastocysts using four laser pulses of 3 msec duration. The biopsied cells were placed immediately in RNaseDNase-free 0.2 ml PCR tubes exactly as for biopsied blastomeres. -Globin gene mutation analysis for the maternal IVS1-110 G>A and paternal cd39 C>T mutations was performed by the protocol for genotyping blastomeres as described by Vrettou et al. (2004)
, which applies real-time multiplex PCR. Briefly, first round multiplex PCR was carried out directly in the tube containing the biopsied samples in a final volume of 50 µl containing primers for the
-globin gene and the two hypervariable microsatellite markers, GABRB3 and D13S314 (Table II), the latter to monitor extraneous DNA contamination in the PCRs. All PCR and cycling conditions were as previously reported (Vrettou et al., 2004
). Nested PCR amplifications for the
-globin gene mutation analysis were carried out on the LightCyclerTM (Roche Diagnostics GmbH, Manheim, Germany) using 0.5 µM of each nested
-globin gene PCR primer (Table II) and 0.15 µM of the relevant fluorescent mutation detection probes (TIB Molbiol, Berlin, Germany) as follows: Ac IVSI-110, 5'-tct gcc tat tgg tct att ttc cc-3', LC Red 640; Ac Cd39, LC Red 705 5'-acc ctt gga ccc aga ggt tct t-3' P; donor set C, FITC 5'-ccc tta ggc tgc tgg tgg tc-3' FITC (Vrettou et al., 2003
, 2004
). Immediately following the amplification stage, the
-globin gene alleles were assigned by melting curve analysis.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There are three potential sources of embryonic genetic material for preimplantation genetic analysis. In clinical practice, polar body biopsy has been used primarily for aneuploidy screening (Verlinsky et al., 1996) and also for the detection of maternal single gene defects (Verlinsky et al., 1997
) such as
-thalassaemia (Kuliev et al., 1998
), but cleavage stage biopsy and aspiration of the blastomere(s) remains the most commonly used source of embryonic genetic material for PGD (ESHRE PGD Consortium Steering Committee, 2002
). A number of modifications have been incorporated into the biopsy procedure for cleavage stage embryos. These include non-contact laser drilling of the zona pellucida instead of chemical digestion using acidified Tyrodes solution, which avoids exposure of embryos to low pH (Boada et al., 1998
; Joris et al., 2003
), and the use of Ca2+/Mg2+-free medium for pre-biopsy incubation to reverse intercellular adhesion which makes the removal of blastomeres less difficult (Dumoulin et al., 1998
). These modifications to the biopsy procedure have made cleavage stage biopsy highly efficient. According to the ESHRE PGD Consortium report, cleavage stage biopsy was applied with 99% efficiency in embryos biopsied for chromosomal and single gene defect analysis. A diagnosis was successful in 85% of the embryos biopsied in PGD cycles for single gene defects (ESHRE PGD Consortium Steering Committee, 2002
). PCR-based genetic analysis in one or two cells is not 100% efficient as it is subject to several problems, including sample contamination with extraneous DNA, PCR amplification failure or failure of one allele to amplify (allele drop-out, ADO) (ESHRE PGD Consortium Steering Committee, 2002
). Biopsy of the embryo at the blastocyst stage has the advantage that more cells may be removed and be available for genetic analysis, which potentially reduces the occurrence of amplification failure and ADO. In this case report, 46 cells were biopsied from each blastocyst and a diagnosis was available in each case, achieving a diagnosis for all three embryos with no ADO. The biopsy of several trophectoderm cells allows the possibility of making a diagnosis on duplicate samples. However, in our experience, the separation of biopsied trophectoderm cells appears to be technically difficult, due to strong cellcell contacts between individual cells. In addition, further manipulation of the sample to achieve duplicates runs the risk of introducing extraneous DNA contamination. In our opinion, the risks in relation to creating duplicate samples far outweigh any potential benefit.
Extended culture of cleavage stage embryos to the blastocyst stage has the advantage of selecting developmentally competent embryos for diagnosis. Reports of extended culture using more complex media or new generation sequential media have reported high rates of development to the blastocyst stage, regardless of whether all embryos or only surplus embryos were cultured to the blastocyst stage (3666%) (Muggleton-Harris et al., 1995; Scholtes and Zeilmaker, 1996
; Desai et al., 1997
; Gardner et al., 1998a
,b
; Jones et al., 1998a
,b
; Rijnders and Jansen, 1998
). The ability of the zygote to develop to the blastocyst stage may not necessarily reflect the viability of the embryo (Bolton et al., 1991
; Winston et al., 1991
). Embryo biopsy on day 3 allows selection of embryos that have at least demonstrated the potential of continued development under embryonic genomic control. However, it is not rare that embryos of a patient following day 3 biopsy and diagnosis are normal with respect to the genetic defect but fail to develop to blastocysts. Trophectoderm biopsy from day 5 blastocysts and subsequent genetic diagnosis results in transferring genetically tested and developmentally competent embryos to the uterus.
The success of PGD at the blastocyst stage is dependent on the capability of the biopsied blastocysts to produce pregnancies and the suitability of the biopsied material for analysis. The ability of blastocysts to implant after trophectoderm biopsy has been reported in animal studies (Gardner and Edwards, 1968; Gardner, 1971
; Betteridge et al., 1981
; Monk et al., 1988
; Summers et al., 1988
). Trophectoderm biopsy has been performed on human blastocysts, and sufficient extra-embryonic material can be obtained for preimplantation diagnosis of genetic disorders (Dokras et al., 1990
, 1991
; Veiga et al., 1997
). Furthermore, the biopsy of up to 10 trophectoderm cells from human blastocysts has been demonstrated to have no impact on the amount of HCG secreted by the surviving blastocyst (Dokras et al., 1991
), indicating that biopsied blastocysts may remain viable. Recently, de Boer et al. (2004)
reported 25 fetal hearts resulting from the transfer of biopsied human blastocysts that had 25 trophectoderm cells removed for PGD for genetic indications such as balanced translocations, aneuploidy and familial genetic disease. However, the genetic indications for the single gene disorders were not described in any detail.
In the present report, a singleton pregnancy resulted from the transfer of two blastocysts biopsied for the PGD of -thalassaemia. All three blastocysts were at the early blastocyst stage prior to zona drilling, and after 4 h of incubation they had grown to expanding blastocysts. No cells were herniating out of the zona at the time of biopsy; however, application of gentle suction on the trophoectoderm cells through the zona hole was effective to encourage trophectoderm cells to herniate outside the zona pellucida. With a few pulses of short duration, it was possible to dissect 46 trophectoderm cells for genetic analysis without any obvious signs of damage. This is further evidenced by the subsequent re-establishment of the blastocoel cavity and the continued development and expansion of two of the three biopsied blastocysts until the time of transfer. It is interesting to note that the blastocyst responsible for the pregnancy was the least advanced of the cohort at the time of embryo transfer and the lack of expansion following biopsy was not indicative of a loss of viability.
Patients undergoing assisted reproduction treatment and PGD procedures invest both financially and emotionally in the expected outcome of both a pregnancy and a healthy offspring. With the availability of new embryology and molecular techniques, it is now possible for PGD laboratories to offer patients at genetic risk the transfer of developmentally competent embryos unaffected by genetic disease. In the future, trophectoderm cells could potentially provide sufficient material for multiple genetic tests, allowing the simultaneous diagnosis of more than one genetic defect. The diagnosis of trophectoderm cells destined to the placenta could now be considered the earliest form of prenatal diagnosis.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boada M, Carrera M, De La Iglesia C, Sandalinas M, Barri PN and Veiga A (1998) Successful use of a laser for human embryo biopsy in preimplantation genetic diagnosis: a report of two cases. J Assist Reprod Genet 15, 302307.[CrossRef][ISI][Medline]
Bolton VN, Wren ME and Parsons JH (1991) Pregnancies after in vitro fertilization and transfer of human blastocysts. Fertil Steril 55, 830832.[ISI][Medline]
Chamayou S, Alecci C, Ragiola C, Giambona A, Siciliano S, Maggio A, Fichera M and Guglielmino A (2002) Successful application of preimplantation genetic diagnosis for -thalassaemia and sickle cell anaemia in Italy. Hum Reprod 17, 11581165.
de Boer KA, Catt JW, Jansen RPS, Leigh D and McArthur S (2004) Moving to blastocyst biopsy for preimplantation genetic diagnosis and single embryo transfer at Sydney IVF. Fertil Steril 82, 295298.[CrossRef][ISI][Medline]
Desai N, Kinzer D, Loeb A and Goldfarb J (1997) Use of synthetic serum substitute and -minimum essential medium for the extended culture of human embryos to the blastocyst stage. Hum Reprod 12, 328335.[Abstract]
Dokras A, Sargent IL, Ross C, Gardner RL and Barlow DH (1990) Trophectoderm biopsy in human blastocysts. Hum Reprod 5, 821825.[Abstract]
Dokras A, Sargent IL, Gardner RL and Barlow DH (1991) Human trophectoderm biopsy and secretion of chorionic gonadotropin. Hum Reprod 6, 14531459.[Abstract]
Dumolin JC, Bras M, Coonen E, Dreesen J, Geraedts JP and Evers JL (1998) Effect of Ca2+/Mg2+-free medium on the biopsy procedure for preimplantation genetic diagnosis and further development of human embryos. Hum Reprod 13, 28802883.
El-Hashemite N, Wells D and Delhanty JD (1997) Single cell detection of beta-thalassaemia mutations using silver stained SSCP analysis: an application for preimplantation diagnosis. Mol Hum Reprod 3, 693698.[Abstract]
ESHRE PGD Consortium Steering Committee (2002) ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium: data collection III (May 2001). Hum Reprod 17, 233246.
Gardner RL (1971) Manipulations on the blastocyst. Adv Biosci 6, 279296.
Gardner RL and Edwards G (1968) Control of the sex ratio at full term in the rabbit by transferring sexed blastocysts. Nature 218, 346349.[ISI][Medline]
Gardner DK and Lane M (1998) Culture of viable human blastocysts in defined sequential media. Hum Reprod 13 (Suppl 3), 148159.[Medline]
Gardner DK, Vella P, Lane M, Wagley L, Schlenker T and Schoolcraft WB (1998a) Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 69, 8488.[CrossRef][ISI][Medline]
Gardner D, Schoolcraft W, Wagley L, Schlenker T, Stevens J and Hesla J (1998b) A prospective randomised trail of blastocyst culture and transfer in in vitro fertilisation. Hum Reprod 13, 34343440.[Abstract]
Grifo JA, Giatras K, Tang YX and Krey LC (1998) Successful outcome with day 4 embryo transfer after preimplantation diagnosis for genetically transmitted diseases. Hum Reprod 13, 16561659.[Abstract]
Handyside AH, Pattinson JK, Penketh RJ, Delhanty JD, Winston RM and Tuddenham EG (1989) Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet i, 347349.
Hussey ND, Davis T, Hall JR, Barry MF, Draper R, Norman RJ and Rudzki Z (2002) Preimplantation genetic diagnosis for -thalassaemia using sequencing of single cell PCR products to detect mutations and polymorphic loci. Mol Hum Reprod 8, 11361143.
Jiao ZX, Zhuang GL, Zhou CQ, Shu YM, Li J and Liang XY (2004) Birth of healthy children after preimplantation diagnosis of -thalassemia. Chin Med J 117, 483487.[ISI][Medline]
Jones GM, Trounson AO, Gardner DK, Kausche A, Lolatgis N and Wood C (1998a) Evolution of a culture protocol for successful blastocyst development and pregnancy. Hum Reprod 13, 169177.[Abstract]
Jones GM, Trounson AO, Lolatgis N and Wood C (1998b) Factors affecting the success of human blastocyst development and pregnancy following in vitro fertilisation and embryo transfer. Fertil Steril 79, 10221029.[CrossRef]
Joris H, De Vos A, Janssens R, Devroey P, Liebaers I and Van Steirteghem A (2003) Comparison of the results of human embryo biopsy and outcome of PGD after zona drilling using acid Tyrode medium or a laser. Hum Reprod 18, 18961902.
Kanavakis E, Traeger-Synodinos J, Vrettou C, Maragoudaki E, Tzetis M and Kattamis C (1997) Prenatal diagnosis of the thalassaemia syndromes by rapid DNA analytical methods. Mol Hum Reprod 6, 523528.
Kanavakis E, Vrettou C, Palmer G, Tzetis M, Mastrominas M and Traeger-Synodinos J (1999) Preimplantation genetic diagnosis of 10 couples at risk for transmitting beta-thalassaemia major: clinical experience including the initiation of six singleton pregnancies. Prenat Diagn 19, 12171222.[CrossRef][ISI][Medline]
Kuliev A, Rechitsky S, Verlinsky O, Ivakhnenko V, Evsikov S, Wolf G, Angastiniotis M, Georghiou D, Kukharenko V, Strom C et al. (1998) Preimplantation diagnosis of thalassemias. J Assist Reprod Genet 15, 219225.[CrossRef][ISI][Medline]
Loukopoulos D (1996) Current status of thalassaemia and the sickle cell syndromes in Greece. Semin Hematol 33, 7686.[ISI][Medline]
Monk M, Muggleton-Harris A, Rawlings E and Whittingham DG (1988) Preimplantation diagnosis of HPRT-deficient male and carrier female mouse embryos by trophectoderm biopsy. Hum Reprod 3, 377381.[Abstract]
Muggleton-Harris AL, Glazier AM and Wall M (1995) A retrospective analysis of the in-vitro development of spare human in-vitro fertilization preimplantation embryos using in-house prepared medium and Medi-cult commercial medium. Hum Reprod 10, 29762984.[Abstract]
Palmer GA, Traeger-Synodinos J, Davies S, Tzetis M, Vrettou C, Mastrominas M and Kanavakis E (2002) Pregnancies following blastocyst stage transfer in PGD cycles at risk for -thalassaemic haemoglobinopathies. Hum Reprod 17, 2531.[CrossRef][ISI][Medline]
Pantos K, Meimeth-Damianaki T, Vaxevanoglou T and Kapetanakis E (1994) Prospective study of a modified gonadotropin-releasing hormone agonist long protocol in an in vitro fertilization program. Fertil Steril 61, 709713.[ISI][Medline]
Pantos K, Stavrou D, Pichos I, Grammatis M, Pappas K, Dafereras A and Tzigounis V (2001) The successful use of hatched blastocysts in assisted reproductive technology. Clin Exp Obstet Gynecol 28, 113117.[Medline]
Piyamongkol W, Harper JC, Delhanty JD and Wells D (2001) Preimplantation genetic diagnosis protocols for alpha and beta-thalassaemias using multiplex fluorescent PCR. Prenat Diagn 21, 753759.[CrossRef][ISI][Medline]
Ray PF, Kaeda JS, Bingham J, Roberts I and Handyside AH (1996) Preimplantation genetic diagnosis of -thalassaemia major. Lancet 347, 1696.
Rijnders PM and Jansen CAM (1998) The predictive value of day 3 embryo morphology regarding blastocyst formation, pregnancy and implantation rate after day 5 transfer following in-vitro fertilisation or intracytoplasmic sperm injection. Hum Reprod 13, 28692873.
Scholtes MCW and Zeilmaker GH (1996) A prospective, randomised study of embryo transfer results after 3 or 5 days of embryo culture in in vitro fertilisation. Fertil Steril 65, 12451248.[ISI][Medline]
Schoolcraft WB and Gardner DK (2001) Blastocyst versus day 2 or 3 transfer. Semin Reprod Med 19, 259268.[CrossRef][ISI][Medline]
Summers PM, Campbell JM and Miller MW (1988) Normal in vivo development of marmoset monkey embryos after trophoectoderm biopsy. Hum Reprod 3, 389393.[Abstract]
Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhorn JD and Frydman R (1986) High pregnancy rate after early human embryo freezing. Fertil Steril 46, 268272.[ISI][Medline]
Traeger-Synodinos J, Vrettou C, Palmer G, Tzetis M, Mastrominas M, Davies S and Kanavakis E (2003) An evaluation of preimplantation genetic diagnosis in clinical genetic services through three years application for prevention of -thalassaemia major and sickle cell thalassaemia. Mol Hum Reprod 9, 301307.
Veiga A, Sandalinas M, Benkhalifa M, Boada M, Carrera M, Santalo J, Barri PN and Menezo Y (1997) Laser blastocyst biopsy for preimplantation diagnosis in the human. Zygote 5, 351354.[ISI][Medline]
Verlinsky Y, Cieslak J, Freidine M, Ivakhnenko V, Wolf G, Kovalinskaya L, White M, Lifchez A, Kaplan B, Moise J et al. (1996) Polar body diagnosis of common aneuploidies by FISH. J Assist Reprod Genet 13, 157162.[ISI][Medline]
Verlinsky Y, Cieslak J, Ivakhnenko V, Wolf G, Lifchez A, Kaplan B, Moise J, Walle J, White M, Ginsberg N et al. (1997) Preimplantation diagnosis of single gene disorders by two-step oocyte genetic analysis using first and second polar body. Biochem Mol Med 62, 182187.[CrossRef][ISI][Medline]
Vrettou C, Palmer G, Kanavakis E, Tzetis M, Antoniadi T, Mastrominas M and Traeger-Synodinos J (1999) A widely applicable strategy for single cell genotyping of -thalassaemia mutations using DGGE analysis: application to preimplantation genetic diagnosis. Prenat Diagn 19, 12091216.[CrossRef][ISI][Medline]
Vrettou C, Traeger-Synodinos J, Tzetis M, Malamis G and Kanavakis E (2003) Rapid screening of multiple -globin gene mutations by real-time PCR on the Lightcycler: application to carrier screening and prenatal diagnosis of thalassaemia syndromes. Clin Chem 49, 769776.
Vrettou C, Traeger-Synodinos J, Tzetis M, Palmer G, Sofocleous C and Kanavakis E (2004) Real-time PCR for single-cell genotyping in sickle cell and thalassaemia syndromes as a rapid, accurate, reliable and widely applicable protocol for preimplantation genetic diagnosis. Hum Mutat 23, 513521.[CrossRef][ISI][Medline]
Winston NJ, Braude PR, Pickering SJ, George MA, Cant A, Currie J and Johnson MH (1991) The incidence of abnormal morphology and nucleocytoplasmic ratios in 2-, 3- and 5-day human pre-embryos. Hum Reprod 6, 1724.[Abstract]
Submitted on December 10, 2004; resubmitted on February 22, 2005; accepted on March 1, 2005.
|