The successful strategy of comprehensive pre-implantation genetic testing for beta-thalassaemia-haemoglobin E disease and chromosome balance using karyomapping.

Thalassaemia is the commonest monogenic disease and causes a health and economic burden worldwide. Karyomapping can be used for pre-implantation genetic testing of monogenic disorders (PGT-M). This study applied karyomapping in two PGT-M cycles and made a comparison to polymerase chain reaction (PCR). Two families at risk of having beta-thalassaemia-haemoglobin E disease offspring decided to join the project and informed consent was obtained. Karyomapping results of family A (beta-thalassaemia (c.41_42delTCTT)-Hb E (c.26G>A) disease) revealed four normal, two beta-thalassaemia traits, one Hb E trait and six affected. Three embryos exhibited unbalanced chromosomes. One normal male embryo was transferred. Karyomapping results of family B (beta-thalassaemia (c.17A>T)-Hb E (c.26G>A) disease) revealed six Hb E traits and three affected. Three embryos were chromosomally unbalanced. One Hb E trait embryo was transferred. Two successful karyomapping PGT-M were performed, including deletion and single-base mutations. Karyomapping provides accuracy as regards the protocol and copy number variation which is common in pre-implantation embryos. Impact StatementWhat is already known on this subject? Thalassaemia syndrome is the commonest monogenic disease and causes a health and economic burden worldwide. Modern haplotyping using SNP array (aSNP) and karyomapping algorithms can be used for pre-implantation genetic testing of monogenic disorders (PGT-M). However, few clinical karyomapping PGT-M cycles have been done and validated so far.What do the results of this study add? Two successful clinical PGT-M cycles for beta-thalassaemia (c.41_42delTCTT and c.17A>T mutations)-haemoglobin E (c.26G>A) disease were performed using karyomapping. The outcome was two healthy babies. Multiplex fluorescent polymerase chain reaction (PCR) with mini-sequencing was also used for confirmation mutation analysis results. PCR confirmed haplotyping results in all embryos. Six embryos from both PGT-M cycles exhibited unbalanced chromosomes evidenced by aSNP.What are the implications of these findings for clinical practice and/or further research? Karyomapping provides accurate information quickly and the outcomes of the study will save time as regards protocol development, provide a usable universal PGT-M protocol and add additional copy number variation (CNV) information, chromosome number variation being a common issue in pre-implantation embryos.

No difference in high-magnification morphology and hyaluronic acid binding in the selection of euploid spermatozoa with intact DNA.

In this study, we compared conventional sperm selection with high-magnification morphology based on the motile sperm organellar morphology examination (MSOME) criteria, and hyaluronic acid (HA) binding for sperm chromosome aneuploidy and DNA fragmentation rates. Semen from 50 severe male factor cases was processed through density gradient centrifugation, and subjected to sperm selection by using the conventional method (control), high magnification at ×6650 or HA binding. Aneuploidy was detected by fluorescence in situ hybridization with probes for chromosomes 13, 18, 21, X and Y, and DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method. Spermatozoa selected under high-magnification had a lower DNA fragmentation rate (2.6% vs. 1.7%; P=0.032), with no significant difference in aneuploidy rate (0.8% vs 0.7%; P=0.583), than those selected by the HA binding method. Spermatozoa selected by both methods had much lower aneuploidy and DNA fragmentation rate than the controls (7% aneuploidy and 26.8% DNA fragmentation rates, respectively). In the high-magnification group, the aneuploidy rate was lower when the best spermatozoa were selected than when only the second-best spermatozoa were available for selection, but the DNA fragmentation rate was not different. In conclusion, sperm selection under high magnification was more effective than under HA binding in selecting spermatozoa with low DNA fragmentation rate, but the small difference (0.9%) might not be clinically meaningful. Both methods were better than the conventional method of sperm selection.

Slow programmable and ultra-rapid freezing of human embryos.

AIM: To compare the outcomes of slow freezing with ultra-rapid freezing (URF) of cleavage-stage human embryos on aluminum foil. METHODS: Two-cell mouse embryos were used to test our method of ultra-rapid freezing. The embryos were randomly allocated to a non-frozen control (208 embryos), and slow (204 embryos) or ultra-rapid freezing groups (204 embryos). Immediate survival rate, further cleavage and blastocyst formation were compared. After validating our ultra-rapid freezing method on mouse embryos, we applied a similar ultra-rapid freezing protocol to human embryos. Consecutive human frozen/thawed embryo transfer (FET) cycles from October 1998 to June 2005 were reviewed. The survival rate, further cleavage rate and the pregnancy outcomes were compared between the URF and slow programmable freezing. RESULTS: Mouse embryos in the URF group survived the freezing/thawing process better than those in the slow freezing group (93.1% vs 82.8%, P = 0.001). Blastocyst and hatching blastocyst formation of the surviving embryos were comparable in the URF and slow freezing group (59% vs 58.6%, P = 0.944 and 32.6% vs 42%, P = 0.066, respectively). There were 146 human FET cycles in the URF group and 28 cycles in the slow freezing group. The immediate survival of embryos was higher in the URF group than in the slow freezing group (87.9% and 64.3%, P < 0.001). There was no significant difference in the mean number of embryos per transfer (3.7 +/- 1.3 and 3.3 +/- 1.2, P = 0.178), clinical pregnancy rate per transfer (28.5% and 21.4%, P = 0.444) and implantation rate per embryo (10.98% and 10.9%, P = 0.974) in the URF or slow freezing groups. CONCLUSION: Our in-house URF method gave comparable results to slow programmable freezing. Although the risk of potential contamination is a major drawback of the present ultra-rapid freezing technique, future refinement will minimize or entirely eliminate this concern.