European Journal of Obstetrics & Gynecology and Reproductive Biology 169 (2013) 261–267

Birth of a healthy infant after preimplantation genetic diagnosis bysequential blastomere and trophectoderm biopsy for b-thalassemiaand HLA genotyping

Tanya Milachich a,*, Tanya Timeva a, Cumhur Ekmekci b, Cagri Beyazyurek b,Huseyin Avni Tac b, Atanas Shterev a, Semra Kahraman b

a SAGBAL Dr. Shterev, IVF Unit, Hristo Blagoev 25-31, Sofia 1330, Bulgariab Istanbul Memorial Hospital, ART and Reproductive Genetics Institute, Piyalepasa Bulvari, 34385 Okmeydani, Istanbul, Turkey

A R T I C L E I N F O

Article history:

Received 31 August 2012

Received in revised form 7 April 2013

Accepted 11 April 2013

Keywords:

Preimplantation genetic diagnosis

b-Thalassemia

Preimplantation human leukocyte antigen

typing

Hematopoietic stem cell transplantation

IVS-I-6 (T!C) (HBB:c.92+6T>C) mutation

Codon 39 (C!T) (HBB:c.118C>T) mutation

A B S T R A C T

Background: Preimplantation genetic diagnosis (PGD) is a widely used technique for couples at genetic

risk and involves the diagnosis and transfer of unaffected embryos generated through in vitro

fertilization (IVF) techniques.

Study design: For those couples who are at risk of transmitting a genetic disease to their offspring,

preimplantation embryos can be selected according to their genetic status as well as human leukocyte

antigen (HLA) compatibility with the affected child. Stem cells from the resulting baby’s umbilical cord

blood can be used for transplantation to the affected sibling without graft rejection.

Results: Here we report successful hematopoietic stem cell transplantation (HSCT) after the birth of a

healthy infant, who was born after successful PGD testing with both cleavage stage and blastocyst stage

biopsy for the purpose of diagnosis of b-thalassemia and HLA compatibility.

Conclusion: The specific feature of this work is not only to have the first successful HSCT achieved

in Bulgaria after using preimplantation HLA typing technique, it also demonstrates how to

accomplish this success via cross-border collaboration of different units, which makes the application

of these sophisticated methods possible in hospitals not having the necessary equipments and

expertise.

� 2013 Elsevier Ireland Ltd. All rights reserved.

Contents lists available at SciVerse ScienceDirect

European Journal of Obstetrics & Gynecology andReproductive Biology

jou r nal h o mep ag e: w ww .e lsev ier . co m / loc ate /e jo g rb

1. Introduction

Since its first application for an X-linked disorder in 1990 [1],more than 6000 healthy children have already been born afterpreimplantation genetic diagnosis (PGD) techniques [2]. Sex-linked disorders, monogenic diseases, chromosomal abnormalitiesand the presence of translocations in either partner are some of themajor indications for PGD. Later, PGD has also been expanded forcancer predisposition disorders, rhesus incompatibility, mitochon-drial disorders and human leukocyte antigen typing [3–8].Nowadays, PGD can be carried out for any disorder for whichmolecular testing could be performed.

b-Thalassemia is a recessively inherited monogenic disorderwhere the affected individuals require blood transfusions on aregular basis. The incidence is quite high in certain parts of the

* Corresponding author. Tel.: +359 29200901; fax: +359 29201827.

E-mail addresses: tanya_ivf@yahoo.com, mcrz@mail.bg (T. Milachich).

0301-2115/$ – see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.ejogrb.2013.04.005

world, such as the Mediterranean region [9,10]. The carrierfrequency is around 4%, but this rate can be up to 14% in someareas where consanguineous marriage is common. Unfortunately,the only possibility of a cure lies in hematopoietic stem celltransplantation (HSCT). Stem cells of the umbilical cordblood from an HLA-compatible newborn have a great therapeuticvalue, as they can be used for transplantation without graftrejection, thus saving an affected sibling’s life [11]. The PGDtechnique together with HLA typing was first applied as atherapeutic tool in 2001 [12]. This technique could be used notonly to avoid the birth of an affected child but also to conceivehealthy children who may be HLA identical donors for HSCT[13,14].

Here we report the first successful HSCT after PGD for b-thalassemia and HLA typing in Bulgaria. The specific feature ofthis work is not only to achieve a live birth and a successfulHSCT after rigorous and difficult clinical and laboratoryprocedures but also to achieve this via cross-border collabora-tion.

T. Milachich et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 169 (2013) 261–267262

2. Materials and methods

2.1. Couple information

A b-thalassemia carrier couple (39 year old female and 39year old male), who have a son (aged 2 years) suffering from b-thalassemia major, were referred to the In Vitro FertilizationUnit at OB/GYN Hospital Dr. Shterev in Sofia, Bulgaria, to discussthe possibility of PGD and HLA typing in order to have a healthyand HLA compatible child. The couple also have a healthydaughter (11 years), who is HLA non-identical with her brother.Molecular analysis revealed that the affected son is a compoundheterozygote and inherited the IVS-I-6 (T!C) (HBB:c.92+6T>C)mutation from his mother and Codon 39 (C!T) (HBB:c.118C>T)mutation from his father. The couple indicated that an HLAcompatible donor could not be found after an extensive searchfrom national and international sources. Preimplantation HLAtyping technique was the only option for the possible treatmentof the affected boy, who has to receive blood transfusions every20–30 days.

The couple were provided with proper genetic counselingabout PGD with HLA typing processes and information regardingthe success rates, the risk of misdiagnosis and possible genetic,clinical and social outcomes. A written consent form wasobtained, in which a possible risk of misdiagnosis was statedand confirmatory prenatal diagnosis was recommended for anypregnancy achieved after PGD.

2.2. Preclinical setup study

Before initiating ovarian hyperstimulation, DNA samples fromthe parents and children were sent to the Reproductive GeneticsInstitute (RGI), Chicago, USA, in order to confirm the mutations inthe b-globin gene and to design appropriate primers to amplifyand select informative short tandem repeat (STR) markers for usein PGD and preimplantation HLA typing of single cells. An STRmarker is considered to be informative for HLA matching if bothpartners of the couple are heterozygote for that marker, so thatsegregation of each allele can be determined. It is also necessarythat individuals who are heterozygote for a specific STR marker donot present the same allele combination for that locus, so thatallele drop out (ADO) could be detectable [4]. The list of markersand the positions on each gene are shown in Figs. 1 (HLA) and 2(HBB, beta-globin).

2.3. IVF treatment and blastomere biopsy

A long GnRH down-regulation protocol with gonadotropinanalog (Lucrin) was initiated on day 21 of the menstrual period(0.1 ml - subcutaneus application). Gonadotropin (rFSH – Gonal F)with a daily dosage of 225 IU was administered. The total dose ofrFSH was 3375 IU, and to induce the final oocyte maturation asingle dose of 10 000 IU s.c. hCG (Pregnyl) was injected on the 11thday of stimulation. Controlled ovarian stimulation includingregular ultrasound checks and hormonal monitoring wereperformed in OB/GYN Hospital Dr. Shterev, Sofia.

The oocyte pick-up, intracytoplasmic sperm injection (ICSI),and embryo transfer procedures were all carried out in theIstanbul Memorial Hospital IVF Laboratory and EndocrinologyUnit in Turkey. A total of 17 oocytes were collected, 12 ofthem were mature (MII) and all of them were fertilized followingICSI. Cleavage-stage biopsy was performed on all embryos, sincethey had more than 6–8 cells on day 3 of embryonic development.One blastomere was removed from each embryo by using anon-contact laser system (Saturn3 Laser System, ResearchInstruments, UK), and then the cells were placed immediately

in RNase–DNase-free 0.2 ml PCR tubes containing 5 ml of lysisbuffer (0.5 ml 10� PCR buffer, 0.5 ml 1% Tween 20, 0.5 ml 1%Triton-X-100, 3.5 ml H2O and 0.05 ml proteinase K 20 mg/ml).

Trophectoderm biopsy was performed on only one embryoaccording to the results obtained after the blastomere biopsy (seeSection 3). On the 103rd hour after the ICSI, several hatchedtrophectoderm cells (4–5 cells) were aspirated from trophecto-derm tissue smoothly using a biopsy pipette (inner diameter40 mm, MBB-FP-M-30, Humagen Pipets, ORIGIO). Precautionswere taken to avoid harming the inner cell mass during thebiopsy procedure. While a holding pipette was fixing the embryo, asmall amount of budding cells were aspirated into the biopsypipette. Then 4 laser pulses of 0.7 ms of duration were applied tobreak down the tight junctions between the trophectoderm cells.The cells on the weak spot were mechanically compressedbetween the biopsy and holding pipette. The cells were placedimmediately in the tubes containing the same contents asdescribed above for the blastomere samples.

3. Results

3.1. PGD and preimplantation HLA matching study

Following the biopsy, the tubes containing the cells werereceived by the Genetics Laboratory in Istanbul MemorialHospital, where all the molecular analysis took place aspreviously described [12–15]. According to the b-thalassemiamutation analysis only one embryo was identified both as ahealthy carrier of IVS-I-6 (T!C) and its HLA matched with theaffected child’s (embryo no. 4) based on 3 fully informative STRmarkers (RING1, 2447 and CAR) and 2 semi-informative STRmarkers (1583 and 273) in the HLA region (Tables 1 and 2). Allother remaining embryos were not HLA identical with theaffected child: two embryos were identified as unaffected, oneembryo was identified as affected (compound heterozygote forIVS-I-6 (T!C), Codon 39 (C!T) and 8 embryos were heterozy-gote carriers IVS-I-6 (T!C) or Codon 39 (C!T) without HLAcompatibility (Tables 1 and 2).

Amplification failure was observed for 3 STR markers (510, RF,MOG) left in embryo no. 4, leaving only 3 informative and 2 semiinformative markers indicating compatibility. To increase theconfidence of the result based on the HLA markers and to rule outany possibility of recombination in the HLA region, a secondbiopsy from the same embryo in the blastocyst stage wasperformed. The cells obtained after the second biopsy gave abetter amplification of those markers previously not amplified,increasing the number of informative markers to six. This secondstudy also reduced the possibility of any recombination eventsthat might have taken place within the tested HLA region(Table 2).

On day 5, the re-biopsied embryo, which was at that time re-expanded and herniated through the biopsy hole, was transferredto the patient’s uterus using a Wallace catheter (Smiths MedicalInternational Ltd., Hythe, UK). Ten days after transfer, the serum b-HCG levels were positive (236 IU/ml). A singleton intrauterinepregnancy with single sac and fetal cardiac activity was confirmedby ultrasound at the 6th week of gestation at OB/GYN Hospital Dr.Shterev.

3.2. Confirmation by amniocentesis

For the purpose of diagnosis confirmation, amniocentesis wasperformed on the 16th gestational week (gw) at the IstanbulMemorial Hospital. Molecular analysis confirmed the previousresults; both the mutation and HLA status of the fetus which washeterozygote and HLA matched with the affected sibling.

Fig. 1. The short tandem repeat (STR) markers and their positions on the human leukocyte antigen (HLA) region on 6p21 locus.

T. Milachich et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 169 (2013) 261–267 263

Furthermore, karyotype analysis showed that the fetus had anormal female karyotype (46,XX).

3.3. Pregnancy outcome

A healthy female baby girl (3200 g, 49 cm) was delivered bycesarean section in the 40th gw in Bulgaria. Umbilical cord bloodwas collected and mononuclear cells were frozen as a source of

stem cells. The number of cells, however, turned out to beinsufficient for a complete treatment of the affected child and HSCTtook place at a later period.

3.4. HSCT outcome

When the donor reached the appropriate weight, the affectedson received a bone marrow transplant (BMT) from his

Table 1Results from molecular testing of STR markers in b-thalassemia locus.

Embryo numbers Family members

1 2 3 4 5 6 7 8 9 10 11 12 Father Mother Affected childMarker

4181 BA BA 145 136 142 142 145 136 142 136 145 142 142 142 145 136 136 142 142 142 145 136 142 142 145 142 136 142 145 142

1871 160 173 160 173 160 162 160 173 160 173 160 162 160 ADO 160 173 173 160 160 162 160 173 160 162 160 160 173 162 160 162

BSTR BA BA BA BA BA BA 143 137 BA BA BA BA BA BA BA BA BA BA BA BA BA BA BA BA 143 139 137 139 143 139

MUTATIONIVS1-6(T->C) N

IVS1-6(T->C) N N

Codon 39(C->T)

IVS1-6(T->C) N N N

IVS1-6(T->C)

Codon 39(C->T) N

Codon 39(C->T)

IVS1-6(T->C) N N N N

Codon 39(C->T)

IVS1-6(T->C) N N

Codon 39(C->T)

IVS1-6(T->C) N N

Codon 39(C->T)

IVS1-6(T->C)

Codon 39(C->T)

1760 BA BA BA BA BA BA 115 108 BA BA BA BA BA BA BA BA BA BA BA BA BA BA BA BA BA 133 108 115 115 115

1241 BA BA 116 116 150 167 116 116 150 116 BA BA 150 ADO 116 116 116 150 150 167 116 116 150 167 116 150 116 159 116 159

1323 BA BA BA BA BA BA 113 108 BA BA BA BA BA BA BA BA BA BA BA BA BA BA BA BA 113 106 108 108 113 108

1997 124 128 124 128 120 120 124 128 ADO 128 124 120 120 120 124 128 ADO 120 120 120 124 128 120 120 124 120 128 120 124 120

Red colored alleles are linked to the mutant gene; black colored alleles are linked to the normal gene.

Table 2Results from molecular testing of STR markers in HLA region.

Embryo numbers Famil y members

Mark ers 1 2 3 4 5 6 7 8 9 10 11 12 Father Mother Affec ted chil d

1618 ADO 137 127 ADO 123 ADO 123 ADO 127 ADO 127 ADO 127 ADO 127 137 127 141 123 141 127 137 127 ADO 123 127 141 137 123 137

1583 153 153 ADO 153 153 158 153 153 149 153 149 158 149 153 149 ADO ADO 158 153 158 149 158 ADO 153 153 149 158 153 153 153

RIN G1 BA BA 160 158 156 166 156 158 160 158 160 166 160 158 160 ADO ADO 166 156 166 160 166 160 158 156 160 166 158 156 158

2447 ADO 147 162 147 170 151 170 147 162 147 162 151 162 151 162 147 ADO 151 170 151 162 151 162 147 170 162 151 147 170 147

CAR 140 159 171 159 140 155 140 159 ADO 159 171 ADO 171 155 ADO 159 ADO 155 140 ADO 171 ADO ADO ADO 140 171 155 159 140 159

273 257 257 250 257 257 252 257 257 250 257 250 252 250 252 250 257 ADO ADO 257 252 250 252 250 257 257 250 252 257 257 257

510 148 156 ADO 148 148 156 148* 148* 167 148 167 156 167 156 167 148 ADO 156 148 156 167 156 167 148 148 167 156 148 148 148

RF 249 308 273 249 249 308 249* 249* 273 249 273 308 273 308 273 249 ADO ADO 249 308 273 308 273 ADO 249 273 308 249 249 249

MOG 185 ADO 168 ADO 185 158 185* 185* BA BA 168 158 168 158 168 ADO ADO ADO ADO 158 ADO ADO 168 ADO 185 168 158 185 185 185

Red colored alleles are linked to maternal HLA identical haplotype; blue colored alleles are linked to paternal HLA identical haplotype.

*The results of the three markers (MOG, RF, 510) in embryo 4 could be obtained after a second biopsy has been performed.

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D11S41 46 (3.69 Mb)

D11S236 2 (4,86 Mb)

D11S418 1 (4,72 Mb)

D11S175 8 (4,69 Mb)

D11S99 8 (4,49 Mb)

BSTR (5.2 Mb)

D11 S187 1 (5.08 Mb)

BRSA (5.2 Mb)

BTG (5.2 Mb)

HPFH (5.2 Mb)

HBG (5.2 Mb)

D11S199 7 (6.31 Mb)

D11 S132 3 (6.23 Mb)

D11 S133 8 (5.94 Mb)

HBB GENE

Mutat ion Site ( 5.21Mb)

11p1 5.5

Human Bet a Globin

3.7 - 6.3 Mb

D11S176 0 (5.34 Mb)

Fig. 2. STR markers and their location on the human beta-globin gene on chromosome 11.

T. Milachich et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 169 (2013) 261–267 265

HLA-matched sister. Engraftment was achieved 14 days after theBMT and the hematological reconstitution was completewithout any signs of acute or chronic graft-versus-host disease.Ten months after the transplantation, the patient and donor arein excellent general condition and the boy remains disease-freeand in good clinical condition (Table 3).

4. Discussion

Allogeneic HSCT is the only curative treatment available for b-thalassemia. Unfortunately, in most cases, there is only a limitedpossibility of finding an HLA-matched donor among relatives and/or from national and international sources. HSCT from an

Table 3Characteristics of patient, donor and HSCT outcome.

Recipient age at transplantation 4.5 years

Donor age at transplantation 14 months

Sex, donor/recipient Female/male

Weight, donor/recipient (kg) 12/20

Transplant graft content BM

BM nucleated cells/kg 2.58 � 108/kg

BM CD34+ cells/kg 10.6 � 106/kg

Neutrophil engraftment (>500 ml) Day +14

Platelet engraftment (>20 000 ml) Day +13

GvHD No

Complications CMV reactivationa

Chimerism at last follow up 100% donor

Posttransplantational time 10 months

BM, bone marrow; GvHD, graft versus host disease; CMV, Cytomegalovirus.a CMV reactivation was successfully treated with antiviral therapy.

T. Milachich et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 169 (2013) 261–267266

HLA-identical sibling provides the best treatment option, whichreduces the incidence of graft rejection and other seriouscomplications associated with transplantation, and nearly 90%of the cases can be cured successfully [23]. Therefore, PGD incombination with HLA typing provides the best treatment optionfor those couples searching for an HLA-matched donor. At the timeof delivery of the newborn, cord blood hematopoietic stem cellscan be collected and used later on to treat the affected sibling [10].For this purpose, the number of cycles initiated for preimplanta-tion HLA typing related to life-threatening inherited disorders(such as b-thalassemia, Fanconi anemia, Wiskott–Aldrich syn-drome), as well as for acquired disorders (such as leukemias) hasincreased gradually since its first introduction [4,5,12,14,16–22].

There exist, however, certain medical, ethical and socialparameters that should be considered and extensively discussedwith the couple before starting the treatment. The low probabilityof finding an HLA matched embryo should be discussed beforeinitiating the IVF cycle. Amniocentesis after achieving pregnancyshould be recommended. Furthermore, special attention should bepaid to the prospective child and the psychosocial environmentshould be scrutinized.

The legislation and regulation on PGD and HLA typing differfrom country to country and ethical debates on this subject areongoing. In many countries, however, such as the USA, UnitedKingdom, Italy, Turkey and Bulgaria, PGD and HLA matching withor without mutation analysis are ethically accepted. Although theEuropean Union and the Council of Europe have some argumentsagainst the use of PGD/HLA matching, the international opinionrepresented by ESHRE (ESHRE Ethics Taskforce on PGD 2003)considers these procedures as morally acceptable. Some Europeancountries such as France and Denmark consider the use of PGD toobtain a child solely on the basis of HLA compatibility unaccept-able. Finally, the World Medical Association (WMA) has no explicitopinion on PGD/HLA matching. Nevertheless, there is a lack ofregulation of this technology in some countries, and there is stillneed for national and international consensus regarding theappropriate uses of the PGD technique.

PGD can be performed at three different stages of embryodevelopment: (i) polar body biopsy [24] before and afterfertilization, (ii) blastomere biopsy [25] at cleavage stage, and(iii) trophectoderm tissue biopsy in the blastocyst stage [26].There are certain pitfalls related to the genetic diagnosis of singlecells such as amplification failure, preferential amplification,allele drop out (ADO) and contamination with extraneous DNA[27,28]. Polar body or blastomere cells are more prone to theseproblems since they contain a limited amount of materialavailable for genetic analysis, but trophectoderm tissue biopsyat the blastocyst stage has the advantage of removing more cells(5–10) which potentially reduces the occurrence of these risks[26,29]. In addition to the technical advantages, blastocyst stage

biopsy has the advantage of selecting developmentally morecompetent embryos for diagnosis, which improves pregnancyrates while decreasing the cost of the PGD study [29]. In thepresent study, cleavage stage and blastocyst stage biopsymethods were used in combination to increase the accuracy ofthe results without reducing the implantation potential of theembryo.

The most challenging part of the preimplantation HLA typingmethod is the low probability of transfer in these cycles. Thegenetic counselors should provide the patient with the informa-tion that there is an 18.7% (3/16) chance of finding both an HLA-identical (1/4 probability) and disease free embryo (3/4 probabil-ity), especially for autosomal recessive diseases such as b-thalassemia. For this reason, considerable numbers of embryosare necessary in order to select disease-free together with HLA-compatible embryos for transfer. Once suitable embryosare found, the clinical pregnancy rates and live birth ratesare acceptable [14,21]. In the present case, 12 embryos weresuitable for biopsy and among them one embryo was transferred,which resulted in clinical pregnancy. The most important factorwhich increased the chance of having a transfer was the goodovarian reserve of the patient, although she was considered to beof advanced maternal age.

According to worldwide experience, the procedures of PGD andHLA typing are reliable and provide couples with a realistic optionto treat an affected sibling. Radical treatments using PGD for HLAhave success in treating not only b-thalassemia but other diseasesas well, including chronic myeloid leukemia, X-linked chronicgranulomatous and Fanconi anemia, among others [30–36]. Thiscase also shows that, once an HLA-compatible and healthy embryois found, full term pregnancy and delivery can be obtained. Withthe availability of new molecular techniques, embryo freezing andmanipulation methods, it is now possible for fertility clinics andPGD laboratories to collaborate well, even if they are in differentcountries, in order to offer patients genetic services such aspreimplantation HLA typing.

5. Conclusion

A successful pregnancy was achieved in a family with a childaffected by b-thalassemia, and a healthy girl HLA compatible withthe affected brother was born at term. Successful HSCT wasperformed with no complications in both of the children, resultingin full recovery of the b-thalassemia with full allogeneicengraftment in bone marrow and peripheral blood. Thus, wecan conclude that HSCT from an HLA-matched related donor as aresult of PGD coupled with HLA matching is an alternativetreatment in patients with b-thalassemia when no HLA-matcheddonor is available. Other than being the first case of its kind inBulgaria, this success also demonstrates the feasibility of aninternational collaboration for the application of sophisticatedtechniques in hospitals which do not have the necessaryequipment and expertise.

Acknowledgements

The authors gratefully acknowledge the contributions of LanaRechitsky and the laboratory staff in the Reproductive GeneticsInstitute, Chicago, USA for the design of the PGD studies. Wegratefully thank the clinical, scientific and laboratory staff of theMemorial Hospital IVF and Genetics Center, Istanbul, Turkey fortheir extensive work, especially Huseyin Avni Tac and Nese Gulumfor their extensive efforts in PGD testing. We thank the clinical andlaboratory staff of OB/GYN Hospital Dr. Shterev, IVF unit, Sofia,Bulgaria for their extensive collaboration and contribution. Lastbut not least; we gratefully thank Prof. Dr. Guido Lucarelli and his

T. Milachich et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 169 (2013) 261–267 267

team in the International Center for Transplantation in Thalasse-mia and Sickle Cell Anemia in the Mediterranean Institute ofHematology, Rome, Italy, for their extensive efforts to treatpatients in need of transplantation.

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