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Medium-based noninvasive preimplantation genetic diagnosis for human α-thalassemias-SEA.

Wu H, Ding C, Shen X, Wang J, Li R, Cai B, Xu Y, Zhong Y, Zhou C - Medicine (Baltimore) (2015)

Bottom Line: The diagnosis efficiency of medium-based α-thalassemias-SEA detection significantly increased compared with that of biopsy-based fluorescent gap PCR analysis (88.6% vs 82.1%, P < 0.05).There is no significant difference regarding ADO ratio between them.Medium-based α-thalassemias-SEA detection could represent a novel, quick, and noninvasive approach for carriers to undergo IVF and PGD.

View Article: PubMed Central - PubMed

Affiliation: From the Reproductive Medicine Center, First Af&filig;liated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China, Guangdong Provincial Key Laboratory of Reproductive Medicine.

ABSTRACT
To develop a noninvasive medium-based preimplantation genetic diagnosis (PGD) test for α-thalassemias-SEA. The embryos of α-thalassemia-SEA carriers undergoing in vitro fertilization (IVF) were cultured. Single cells were biopsied from blastomeres and subjected to fluorescent gap polymerase chain reaction (PCR) analysis; the spent culture media that contained embryo genomic DNA and corresponding blastocysts as verification were subjected to quantitative-PCR (Q-PCR) detection of α-thalassemia-SEA. The diagnosis efficiency and allele dropout (ADO) ratio were calculated, and the cell-free DNA concentration was quantitatively assessed in the culture medium. The diagnosis efficiency of medium-based α-thalassemias-SEA detection significantly increased compared with that of biopsy-based fluorescent gap PCR analysis (88.6% vs 82.1%, P < 0.05). There is no significant difference regarding ADO ratio between them. The optimal time for medium-based α-thalassemias-SEA detection is Day 5 (D5) following IVF. Medium-based α-thalassemias-SEA detection could represent a novel, quick, and noninvasive approach for carriers to undergo IVF and PGD.

Show MeSH
Characterization of cell-free DNA. Another set of integrated embryos without biopsy were subjected to quantification of cell-free DNA via medium-based detection of α-thalassemia-SEA. A. The standard curved generated by medium-based Q-PCR detection. B. Results summary.
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Figure 3: Characterization of cell-free DNA. Another set of integrated embryos without biopsy were subjected to quantification of cell-free DNA via medium-based detection of α-thalassemia-SEA. A. The standard curved generated by medium-based Q-PCR detection. B. Results summary.

Mentions: To confirm the new method of medium-based detection, an additional 61 integrated embryos that had not been biopsied were subjected to quantification of cell-free DNA in medium. As shown in Figure 3, the detectable ratio of the medium collected at D4 (D3→D4) after fertilization was 19.67% (12/61) with a concentration of 14.24 ± 4.76 pg/μL; this ratio dramatically increased to 90.16% (55/61) with a concentration of 48.78 ± 20.45 pg/μL at D5 and 88.46% (23/26) with a concentration of 54.35 ± 22.78 pg/μL at D6. No significant difference was identified between D5 and D6 (P > 0.05).


Medium-based noninvasive preimplantation genetic diagnosis for human α-thalassemias-SEA.

Wu H, Ding C, Shen X, Wang J, Li R, Cai B, Xu Y, Zhong Y, Zhou C - Medicine (Baltimore) (2015)

Characterization of cell-free DNA. Another set of integrated embryos without biopsy were subjected to quantification of cell-free DNA via medium-based detection of α-thalassemia-SEA. A. The standard curved generated by medium-based Q-PCR detection. B. Results summary.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554004&req=5

Figure 3: Characterization of cell-free DNA. Another set of integrated embryos without biopsy were subjected to quantification of cell-free DNA via medium-based detection of α-thalassemia-SEA. A. The standard curved generated by medium-based Q-PCR detection. B. Results summary.
Mentions: To confirm the new method of medium-based detection, an additional 61 integrated embryos that had not been biopsied were subjected to quantification of cell-free DNA in medium. As shown in Figure 3, the detectable ratio of the medium collected at D4 (D3→D4) after fertilization was 19.67% (12/61) with a concentration of 14.24 ± 4.76 pg/μL; this ratio dramatically increased to 90.16% (55/61) with a concentration of 48.78 ± 20.45 pg/μL at D5 and 88.46% (23/26) with a concentration of 54.35 ± 22.78 pg/μL at D6. No significant difference was identified between D5 and D6 (P > 0.05).

Bottom Line: The diagnosis efficiency of medium-based α-thalassemias-SEA detection significantly increased compared with that of biopsy-based fluorescent gap PCR analysis (88.6% vs 82.1%, P < 0.05).There is no significant difference regarding ADO ratio between them.Medium-based α-thalassemias-SEA detection could represent a novel, quick, and noninvasive approach for carriers to undergo IVF and PGD.

View Article: PubMed Central - PubMed

Affiliation: From the Reproductive Medicine Center, First Af&filig;liated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China, Guangdong Provincial Key Laboratory of Reproductive Medicine.

ABSTRACT
To develop a noninvasive medium-based preimplantation genetic diagnosis (PGD) test for α-thalassemias-SEA. The embryos of α-thalassemia-SEA carriers undergoing in vitro fertilization (IVF) were cultured. Single cells were biopsied from blastomeres and subjected to fluorescent gap polymerase chain reaction (PCR) analysis; the spent culture media that contained embryo genomic DNA and corresponding blastocysts as verification were subjected to quantitative-PCR (Q-PCR) detection of α-thalassemia-SEA. The diagnosis efficiency and allele dropout (ADO) ratio were calculated, and the cell-free DNA concentration was quantitatively assessed in the culture medium. The diagnosis efficiency of medium-based α-thalassemias-SEA detection significantly increased compared with that of biopsy-based fluorescent gap PCR analysis (88.6% vs 82.1%, P < 0.05). There is no significant difference regarding ADO ratio between them. The optimal time for medium-based α-thalassemias-SEA detection is Day 5 (D5) following IVF. Medium-based α-thalassemias-SEA detection could represent a novel, quick, and noninvasive approach for carriers to undergo IVF and PGD.

Show MeSH