Extracellular embryo genomic DNA and its potential for genotyping applications

Figures & data

Figure 1.  Representative standard curves constructed with 1:10 scalar dilutions (5000 to 5 pg per reaction tube) of purified human genomic DNA.

Curve (A) (Y = -3.24X + 31.32; R2 = 0.996) was constructed with human gDNA only; curve (B) (Y = -3.27X + 34.69; R2 = 0.995) and curve (C) (Y = -3.34X + 33.59; R2 = 0.998) were constructed with the addition of 2 µl of medium G-1 PLUS and G-2 PLUS, respectively.

Ct: Threshold cycle; gDNA: Genomic DNA.

Table 1.  Detection and quantification of genomic DNA in spent medium samples at day 3 and day 5/6 through TBC1D3 amplification.

Samples	Samples (n)	TBC1D3 positive (%)	Ct (mean ± SD)	Total gDNA content (pg; mean ± SD)
Spent medium day 3	32	30 (93.7)	31.47 ± 1.67	80 ± 70
Spent medium day 5/6	54	51 (94.4)	30.11 ± 1.78	99 ± 113

Ct: Threshold cycle; gDNA: Genomic DNA; SD: Standard deviation.

Table 2.  TBC1D3 and/or TSPY1 amplification from different matrices of the same embryos.

Embryo ID	Medium day 3		Medium day 5/6		Trophectoderm biopsy
	TBC1D3	TSPY1	TBC1D3	TSPY1	TSPY1
1_1	+	–	+	–	NA
2_1	+	–	+	–	NA
3_1	+	–	+	+	NA
4_1	+	–	+	–	NA
5_1	+	–	+	–	NA
6_1	+	–	+	+	NA
1_2	–	–	+	+	NA
2_2	–	–	+	–	NA
3_2	+	–	+	–	NA
4_2	+	–	+	–	NA
5_2	+	–	+	–	NA
6_2	+	–	–	–	NA
7_2	+	–	+	–	NA
B1	+	+	+	+	+
B4	+	–	+	+	+
B8	+	–	+	–	NA

‘+’ and ‘–’ represent positive and negative results, respectively.

NA: Not available.

Table 3.  MTHFR C677T genotyping results from medium samples.

Embryo ID	Sample amplified by WGA	MTHFR amplification	C677T genotype
1IB	Day 5/6 medium	+	C
	Trophectoderm biopsy	+	C/T
2IB	Day 5/6 medium	+	C
3IB	Day 5/6 medium	+	C/T
	Trophectoderm biopsy	+	C/T
4IB	Day 5/6 medium	–	NA
	Trophectoderm biopsy	+	T
3	Day 5/6 medium	–	NA
5	Day 5/6 medium	–	NA
6	Day 5/6 medium	+	C
7	Day 5/6 medium	+	C/T

NA: Not available; WGA: Whole-genome amplification.

Table 4.  MTHFR C677T genotyping results from blastocoele fluid samples.

Embryo ID	Sample amplified by WGA	MTHFR amplification	C677T genotype
1	Blastocoele fluid	–	NA
2	Blastocoele fluid	+	C/T
1IB	Blastocoele fluid	–	NA
	Trophectoderm biopsy	+	C/T
2IB	Blastocoele fluid	–	NA
3IB	Blastocoele fluid	+	C/T
	Trophectoderm biopsy	+	C/T
3	Blastocoele fluid	+	C/T
5	Blastocoele fluid	+	T
6	Blastocoele fluid	–	NA
7	Blastocoele fluid	–	NA

NA: Not available; WGA: Whole-genome amplification.