The procedure was applied to 197 PGD cycles (150 for β-thalassaemia, 19 for ALL, 16 for DBA, 3 for Histiocytosis, 3 for Adrenoleukodystrophy , 2 for Duncan Sindrome, 1 for Chronic granulomatous disease, 1 for FANCG, 1 for Wiscott-Aldrich syndrome and 1 for Mannosidosis-Alpha) performed for 106 couples. A total of 1488 embryos were tested for HLA typing in combination with a genetic disease, and 329embryos for HLA typing only (Table 2). A total of 2492 blastomeres was analysed, in 2292 (92.0%) of which a successful amplification was obtained (Table 1). Two hundred blastomeres resulted in the amplification failure for all the markers/loci tested. The amplification efficiency of the individual STR markers on blastomeres ranged from 92.2 to 100%, with an overall amplification rate of 98.2%. The ADO rates varied between the different loci investigated, ranging from 0.0 to 10.8%

A reliable HLA haplotype was obtained in 2292/2292 (100%) of the blastomeres with positive PCR results.

Totally, 1704 (93.8%) embryos were diagnosed; however testing for chromosome 6 copy number revealed 187 (11.0%) embryos with aneuploidies, including a total of 38 (2.2%) trisomies, 143 (8.4%) monosomies and 6 (0.4%) uniparental dysomies, which affected the HLA matching diagnosis for these embryos (Table 4), leading to a conclusive diagnosis in only 1517 (89.0%) embryos.


In total, 311 (18.3%) embryos resulted to be HLA matched with the affected siblings. In the 159cycles of HLA matching combined a with single gene disorder, although the overall number of HLA-identical embryos was 259 (18.5%), only 196 (14.0%) resulted also in an unaffected genotype. In the couples undergoing preimplantation HLA typing without testing for a causative gene, 52 (17.2%) embryos appeared to be HLA compatible with the affected children. Recombination was found in 38 (2.2%) embryos.


Overall 211 embryos were transferred to the patients in 132 of the 197 cycles performed (1.6 + 0.7 on average), and 50 pregnancies were achieved. Eight pregnancies resulted only biochemical, 11 spontaneously miscarried and one resulted ectopic, and was then terminated.


Thirdy-one ongoing pregnancies were obtained. The ongoing pregnancy rate was 15.7% per cycle and 23.5% per transfer.


Fourteen pregnancies are still ongoing, while 3 twin and 14 singleton pregnancies, produced the birth of 20 healthy children, which were confirmed to be HLA identical to their affected sibling by blood samples HLA testing. Stem cells collected after delivery from the umbilical cords blood were transplanted to the affected siblings of 7 couples, resulting in a successful hematopoietic reconstruction for all patients.

Table 1: Multiplex PCR and HLA haplotyping results

 

Multiplex PCR results	HLA+PGD	%	HLA	%	Total	%
No. of cells analyzed	2077		415		2492
No. of cells with amplification failure of all markers (%)	163	7,8	37	8,9	200	8,0
No. of cells with a positive amplification of HLA markers(%)	1914	92,2	378	91,1	2292	92,0
HLA haplotyping results
No. of embryos analyzed	1488		329		1817
No. of embryos diagnosed (%) *	1402	94,2	302	91,8	1704	93,8
No. of embryos without HLA diagnosis (%) §	230	15,5	70	21,3	300	16,5
No. of embryos with conclusive HLA diagnosis (%) $	1258	89,7	259	85,8	1517	89,0
No of HLA identical embryos (%)	259	18,5	52	17,2	311	18,3
No of HLA identical healthy embryos (%)	196	14,0	52	17,2	248	14,6
No. of HLA non-identical embryos (%)	1143	81,5	250	82,8	1393	81,7
No. of embryos with recombination (%)	32	2,3	6	2,0	38	2,2
Embryos without amplification (%)	86	5,8	27	8,2	113	6,2
No. of abnormal embryos (%)	144	10,3	43	14,2	187	11,0
Monosomy chromosome 6  (%)	109	7,8	34	11,3	143	8,4
Trisomy chromosome 6 (%)	30	2,1	8	2,6	38	2,2
UPD (%)	5	0,4	1	1,9	6	0,4

*(No. of embryos analyzed) – (No. of embryos without diagnosis because of a total PCR failure on the single cell or both cells  biopsed) $(No. of embryos analyzed) – (No. of abnormal embryos) – (No. of embryos without diagnosis because of a total PCR failure on the unique cell biopsed) § (No. of abnormal embryos)+(No. of embryos w/o diagnosis because of PCR failure on both biopsied cells or on the single cell biopsied) 


Table 2: Results and outcome from 197 preimplantation HLA matching cycles 

 

Clinical results	HLA+PGD	%	HLA	%	Total	%
No. of couples treated	88		18		106
Maternal age + SD	31.8 ± 5.2		37.5 ± 4.3		32.8 ± 5.4
No. of cycles performed	159		38		197
No. of oocytes retrieved	2394		533		2927
	15,1		14,0		14,9
No. of oocytes injected (%)	1830	76,4%	413	77,5%	2243	76,6%
No. of oocytes fertilized (%)	1634	89,3%	358	86,7%	1992	88,8%
No. of embryos analyzed (%)	1488	91,1%	329	91,9%	1817	91,2%
Per cycle	9,4		8,7		9,2
No. of transfers	105		27		132
Per cycle	66,0%		71,1%		67,0%
No. of embryos transferred	165		46		211
Mean no. of embryos transferred	1.5 + 0.7		1.8 + 0.9		1.6 + 0.7
Outcome
No. of positive HCG pregnancies	41		9		50
UNEMBRYONIC	2		0		2
Biochemical	7		1		8
Clinical	34		8		42
Miscarried	8		3		11
Ectopic	1		0		1
Ongoing	26		5		31
Ongoing per cycle	16,4%		13,2%		15,7%
Ongoing per transfer	24,8%		18,5%		23,5%
No. of embryos implanted (fetuses)	38		8		46
Implantation rate	23,0		17,4		21,8
No. of pregnancies went to term	15		2		17
No. of babies born	18		2		20
No. of pregnancies still ongoing	11		3		14
No. of transplantations done	6		1		7

 

Table 3:
Number of families treated with distinct indications for PGD and/or HLA. ongoing pregnancies. life births and cord blood transplantations (CBTs).

Diseasese
Adrenoleukodystrophy	3	2	1	50,0	0	1	50,0	0	1	0	0
β-Thalassemia	150	81	38	38,8	7	31	31,6	8	10	13	15	5
Chronic granulomatous disease	1	1	1	100,0	0	1	100,0	0	0	1	2	0
Diamond Blackfan Anemia	16	3	4	36,4	1	3	27,3	2	1	0	0	0
Duncan Syndrome	2	1	0	0,0	0	0	0,0	0	0	0	0
FANCG	1	1	0	0	0	0	0	0	0	0	0	0
Histiocytosis	3	1	1	33,3	0	1	33,3	0	0	1	1	0
Leukemia	19	14	4	30,8	0	4	30,8	1	2	1	1
Mannosidosis-Alpha	1	1	0	0,0	0	0	0,0	0	0	0	0	0
Wiskott-Aldrich Syndrome	1	1	1	100,0	0	1	100,0	0	0	1	1	1
Total	197	106	50	37,9	8	42	31,8	11	14	17	20	7