CSIRO PUBLISHING
Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD14419
Accuracy of preimplantation genetic diagnosis in equine in vivo-recovered and in vitro-produced blastocysts Y. H. Choi A, M. C. T. Penedo B, P. Daftari B, I. C. Velez A and K. Hinrichs A,C,D A
Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, College Station, TX 77843-4466, USA. B Veterinary Genetics Laboratory, University of California, Old Davis Road, Davis, CA 95616, USA. C Department of Large Animal Clinical Sciences, Texas A&M University, 4475 TAMU, College Station, TX, 77843-4475, USA D Corresponding author. Email: [email protected]
Abstract. Preimplantation genetic diagnosis has great potential in the horse, but information on evaluation of equine embryo biopsy samples is limited. Blastocysts were biopsied using a Piezo drill and methods for whole-genome amplification (WGA) investigated. Results for 33 genetic loci were then compared between biopsy samples from in vitroproduced (IVP) and in vivo-recovered (VIV) blastocysts. Under the experimental conditions described, WGA using the Qiagen Repli-g Midi kit was more accurate than that using the Illustra Genomiphi V2 kit (98.2% vs 25.8%, respectively). Using WGA with the Qiagen kit, three biopsy samples were evaluated from each of eight IVP and 19 VIV blastocysts, some produced using semen from stallions carrying the genetic mutations associated with the diseases hereditary equine regional dermal asthenia (HERDA), hyperkalemic periodic paralysis (HYPP) or polysaccharide storage myopathy 1 (PSSM1). Three of 81 biopsy samples (3.7%) returned ,50% accuracy. In the remaining 78 samples, overall accuracy was 99.3% (2556/2574 loci interrogated). Accuracy did not differ significantly between samples from IVP and VIV blastocysts. Allele drop-out in heterozygous loci was 1.6% (17/1035). Accuracy for sex determination was 100%; accuracy for heterozygosity for disease-causing mutations was 97.7% (43/44). In conclusion, Piezo-driven embryo biopsy with WGA has .95% overall accuracy in IVP and VIV embryos, and this technique is suitable for use in a clinical setting. Additional keywords: embryo biopsy, genetic screening, HERDA, HYPP, polymerase chain reaction (PCR), PSSM, whole genome amplification.
Received 31 October 2014, accepted 19 January 2015, published online 17 March 2015
Introduction Preimplantation genetic diagnosis (PGD) has the potential for extensive use in the horse. This technique may be used to screen for genetic diseases such as hereditary equine regional dermal asthenia (HERDA), hyperkalemic periodic paralysis (HYPP) or other devastating single-locus mutations (Rudolph et al. 1992; Bernoco and Bailey 1998; Ward et al. 2004; Brault and Penedo 2009; Herszberg et al. 2009; Tryon et al. 2009; Brooks et al. 2010) or to select embryos on traits such as embryo sex or coat colour before transfer. While use of PGD is not applicable to breeds that prohibit embryo transfer, including the Thoroughbred, the possibility of selecting embryos for desired traits may lead to increased importance of embryo transfer as an equine reproductive management tool in arenas in which embryo transfer is accepted. Biopsy of equine embryos, especially expanded blastocysts, was initially associated with poor pregnancy rates after transfer (Huhtinen et al. 1997; Seidel et al. 2010). Expanded blastocysts in the horse have an embryonic capsule, a glycoprotein envelope that surrounds the equine embryo after the zona pellucida has Journal compilation Ó CSIRO 2015
been shed, which makes some methods of biopsy, such as microblade dissection, problematic. We reported a technique for biopsy of equine embryos via micropipette aspiration under micromanipulation, which supports normal viability (rate of normal pregnancy with heartbeat) after biopsy of both morulae– early blastocysts and expanded blastocysts (Choi et al. 2010). Others have used this technique with modifications for biopsy of expanded equine blastocysts, finding similar normal viability after transfer of over 100 biopsied embryos (Herrera et al. 2014). There is increasing clinical demand for in vitro production (IVP) of equine embryos, via intracytoplasmic sperm injection (ICSI) and embryo culture (Hinrichs et al. 2014). Biopsy of in vitro-produced equine embryos appears straightforward, as there is no capsule, and intercellular junctions are relatively weak. In contrast, biopsy of in vivo-recovered (VIV) expanded equine blastocysts is complicated not only by the presence of the capsule but also by the tenacity of intercellular attachments among the trophoblast cells at this stage (Hinrichs and Choi 2012). However, the larger cell numbers in VIV embryos provides an opportunity www.publish.csiro.au/journals/rfd
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for larger biopsy samples. It is not known if these differences in biopsy sample characteristics affect the accuracy of PGD. Embryonic cells obtained by biopsy may be used for the interrogation of a single locus or a few loci (such as for embryo sexing) by direct or multiplex polymerase chain reaction (PCR; single-cell PCR; Hahn et al. 2000), and sex of horse embryos has been accurately determined by this method (Huhtinen et al. 1997; Troedsson et al. 2010; Herrera et al. 2014). However, for evaluation of multiple loci, direct or multiplex PCR is problematic, as primers may interfere with one another and no sample is left for additional or confirmatory tests. An alternative for the interrogation of multiple loci is performance of whole-genome amplification (WGA) followed by PCR, as has been done on human embryo biopsies (Handyside et al. 2004). We utilised WGA previously for genetic analysis of equine embryo biopsy samples for sex, HERDA and HYPP (Choi et al. 2010), but only two of 16 loci analysed were heterozygous for a disease-causing mutation and only one of these was accurately detected on analysis of the biopsy sample, thus the accuracy of biopsy– WGA for detection of disease-related genotypes (heterozygous or homozygous affected) is unknown. Interrogation of heterozygous genotypes is more challenging than is interrogation of homozygous genotypes, due to the occurrence of allele drop-out (failure of one allele to amplify). This results in the detection of only one allele, thus indicating homozygosity, at a given locus in a heterozygous individual. Allele drop-out is common in genetic analysis of samples with small cell numbers (reviews Piyamongkol et al. 2003; Spits and Sermon 2009). In horses, the accurate detection of both normal and disease-associated alleles in heterozygous animals and differentiation of heterozygous animals from either genotype of homozygous animals is important to breeders requesting PGD. Most sex, disease and coat-colour loci of interest to horse breeders are amenable to interrogation; however, some loci present challenges because causative mutations are located within GC-rich sequences or represent large or complex duplications. Thus, analysis of accuracy of interrogation needs to be determined separately for each locus. We hypothesised that the WGA method used would affect the accuracy of genetic evaluation of multiple loci in equine embryo biopsy samples and that biopsy samples from IVP equine embryos would allow more accurate genetic analysis than would those from VIV equine embryos. Thus, in this study, we investigated two methods for WGA and then compared the accuracy of WGA–multiplex PCR for interrogation of multiple loci (sex, disease-related mutation, coat colour and microsatellite identification markers) between biopsies from IVP and VIV equine blastocysts, to establish the efficiency of biopsy for genetic analysis in these two embryo types.
according to the United States Government Principles for the Utilisation and Care of Vertebrate Animals Used in Testing, Research and Training and were approved by the Institutional Animal Care and Use Committee at Texas A&M University. Briefly, immature oocytes were collected by aspiration and flushing of follicles $ 5 mm in diameter. The aspirated fluid was filtered through an embryo filter (EmCon filter; Immuno Systems Inc., Spring Valley, WI, USA) and the oocytes were recovered from the collected cellular material. Collected oocytes were held overnight (Choi et al. 2006) then cultured for maturation. Mature oocytes were subjected to ICSI, using frozen–thawed spermatozoa, via a Piezo drill as described previously (Choi et al. 2002, 2003). In Experiment 2, to increase the chance of production of embryos heterozygous for diseaserelated mutations and colour-related genes, spermatozoa from a stallion heterozygous N/Cr for the Cream dilution and heterozygous N/H for HYPP were used. Injected oocytes were cultured for embryo development in microdroplets of Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F-12) (Hinrichs et al. 2005) or a commercial human embryo culture medium (global medium (GB); LifeGlobal, Guilford, CT, USA), both with 10% fetal bovine serum (FBS), in an atmosphere of 5–6% CO2, 5% O2 and the remainder N2 at 38.28C for 7–10 days. Media were completely changed at Day 5 into the same medium (DMEM/F-12) or, for GB-cultured embryos, to GB containing 20 mM glucose. In the first experiment (comparison of WGA methods), IVP blastocysts were vitrified as we have described previously (Choi et al. 2011) then warmed and cultured for 18–30 h before being biopsied. In the second experiment (comparison of IVP and VIV embryos), IVP embryos were biopsied on the day they developed to blastocyst in culture.
Materials and methods In vitro embryo production For this study blastocysts were produced in vitro by culturing oocytes after ICSI. Immature oocytes were collected from abattoir tissue (Experiment 1) or from research mares by transvaginal ultrasound-guided follicle aspiration (Jacobson et al. 2010). All experimental procedures were performed
Embryo biopsy Embryo biopsy was performed as described in our previous study (Choi et al. 2010), with minor modifications. Briefly, embryos were placed in 50-mL droplets of Chatot-ZiomekBavister manipulation (CZB-M) medium (Choi et al. 2003) with 10% FBS at room temperature in a Petri dish on an inverted microscope. The embryos were held with a holding
Collection of in vivo-produced embryos In vivo-produced blastocysts (Experiment 2) were recovered by transcervical uterine flush from research mares 6–10 days after ovulation, as previously described (Choi et al. 2009). Briefly, mare ovarian activity was monitored by palpation and ultrasonography per rectum. When a dominant follicle reached over 30 mm in diameter, biorelease deslorelin (1.5 mg, intramuscular (i.m.); BET Pharm, Lexington, KY, USA) was administered and mares were artificially inseminated with fresh or cooled, extended semen from a fertile stallion, containing a minimum of 500 106 progressively motile spermatozoa. To increase the chance of production of embryos heterozygous for diseaserelated mutations, spermatozoa from stallions heterozygous N/HRD for HERDA or N/PSSM1 at the polysaccharide storage myopathy 1 (PSSM1) locus were used for some inseminations. After confirmation of ovulation by ultrasonography per rectum, mares were assigned for uterine flushing to recover embryos at various days after ovulation.
PGD in equine blastocysts
pipette and a pipette of 15 3 mm external diameter, attached to a Piezo drill, was used to perform the biopsy. We attempted to obtain 10–30 cells for each biopsy sample. The biopsied cells were expelled into a 3-mL droplet of CZB-M without calcium, glutamine, nonessential amino acids or FBS, but supplemented with 2% polyvinylpyrrolidone, then aspirated from the droplet with a fine-bore glass pipette and placed in a 0.2-mL PCR tube (VWR International, West Chester, PA, USA). After the completion of the biopsies, the remaining embryo was moved into a Petri dish containing CZB-M þ 10% FBS, then split approximately in half using 27-gauge needles and each half transferred into a separate PCR tube. All PCR tubes were stored at 208C until they were shipped to the Veterinary Genetics Laboratory at the University of California (Davis, CA, USA) for genomic analysis. For clarity in discussing these analyses, the original blastocyst was designated as such and the embryonic tissue remaining after biopsy was designated the embryo or the demi-embryo (after splitting). Experiment 1: evaluation of methods for whole-genome amplification This experiment was performed to determine if there was a difference in accuracy of genetic diagnosis related to the method used for WGA. For this study, three biopsy samples were analysed from each of eight IVP blastocysts. For the first blastocyst biopsied, the entire remaining embryo was submitted for analysis; after this, remaining embryos were split as described above and each demi-embryo submitted separately for analysis, to provide a reference for evaluation of accuracy of biopsy samples for that blastocyst. The biopsy samples, embryos and demi-embryos were subjected to WGA using either the Illustra Genomiphi V2 kit (GE Healthcare Bioscience Corp, Piscataway, NJ, USA; first four blastocysts) or the Qiagen Repli-g Midi kit (Qiagen Inc., Valencia, CA, USA; next four blastocysts). Under each amplification system, loci for two disease-related mutations (HERDA, HYPP), one sex (AME), one X-linked marker (LEX3) and 16 microsatellite identification markers (AHT4, AHT5, ASB17, ASB2, ASB23, HMS2, HMS3, HMS6, HMS7, HTG10, HTG4, LEX33, TKY333, TKY374, TKY394 and VHL20) were genotyped, for a total of 20 loci, for each biopsy sample and embryo or demi-embryo. The reference standard for assessment of accuracy of biopsy analysis for each blastocyst was generated by combining the analysis for the two demi-embryos for that blastocyst. In cases in which no signal for a given allele was detected in the embryo or demi-embryos but was present in one or more biopsy samples, this allele was included in the reference standard for that blastocyst. WGA was done in the 0.2-mL PCR tubes that contained the embryo or biopsy sample cells; when thawed, liquid media did not exceed 2 uL in volume. WGA with the Illustra Genomiphi V2 kit was performed according to the manufacturer’s protocol. WGA with the Qiagen Repli-g Midi kit was performed according to the manufacturer’s protocol for genomic DNA from blood or cells, with the following modifications: Buffer D2 was prepared with water instead of dithiothreitol solution and, after addition of Buffer D2 to sample tubes, cells were incubated at 608C for 5 min and then put on ice for at least 10 min. For all PCR reactions, 3 mL of DNA solution (WGA product) was used as template.
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DNA amplification for the specific loci to be interrogated was done in three multiplexed PCR reactions. Two multiplexed panels amplified sex and ID markers (Panel A: AME, AHT4, AHT5, ASB23, HTG4, HTG10, HMS2, TKY333, VHL20; Panel B: ASB2, ASB17, HMS6, HMS7, LEX33, TKY374, TKY394, HMS3, LEX3). One multiplexed panel amplified two diseaserelated loci (HERDA, HYPP). PCR was carried out in 25-mL total-volume reactions containing 3 mL DNA template, 1 PCR buffer IV (Denville Scientific, Metuchen, NJ, USA), 1 U Choice Taq (Denville Scientific), 200 mM dNTPs, 2.5 mM MgCl2 and fluorescencelabelled primers needed for each multiplex. Cycling conditions consisted of four cycles of 1 min at 948C, 30 s at 608C, 30 s at 728C, followed by 25 cycles of 45 s at 948C, 30 s at 608C, 30 s at 728C and a final extension at 728C for 30 min. PCR products were separated by capillary electrophoresis on an ABI 3730 DNA Analyser (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Fragment size analysis and genotyping were done with the computer software STRand (available at https://www.vgl. ucdavis.edu/informatics/strand.php, accessed 23 February 2015). Allele designations for microsatellite markers followed international standardised nomenclature defined by the International Society of Animal Genetics (ISAG). For diseaserelated loci, normal alleles were designated by the letter ‘N’ and alleles corresponding to disease-related mutations were assigned a descriptive code (HRD, HERDA; H, HYPP). Positive and negative control samples were included in all PCR reactions. Experiment 2: comparison of PGD results for in vitroproduced and in vivo-recovered embryos For this experiment, eight IVP blastocysts produced via ICSI using spermatozoa from a stallion heterozygous N/Cr for the Cream dilution and heterozygous N/H for HYPP, and 19 VIV blastocysts obtained on Day 6 (n ¼ 2), Day 7 (n ¼ 12), Day 8–9 (n ¼ 4) or Day 10 (n ¼ 1) after ovulation were analysed. Eight of the VIV blastocysts were sired by a stallion that was heterozygous N/HRD for HERDA and four were sired by a stallion that was heterozygous N/PSSM1 at the PSSM1 locus. Three biopsy samples were obtained from each blastocyst, then the remaining embryos were bisected and samples stored and shipped as for Experiment 1. The Qiagen Repli-g Midi kit was used for WGA. A total of 33 loci were evaluated; DNA amplification was performed in five multiplexed PCR reactions. Two multiplexed panels amplified sex and ID markers, as above but including SRY and excluding AHT5. One multiplexed panel amplified four disease loci (HERDA, HYPP, PSSM1 and MH1, a locus responsible for malignant hyperthermia that also modifies expression of PSSM1; McCue et al. (2009)). To increase the number of potential heterozygous protein-coding loci evaluated, 11 coat-colour loci were evaluated; two multiplexed panels amplified these loci (Panel C: Cream dilution, Lethal White Overo, Sabino-1, Pearl dilution, Tobiano; Panel D: Agouti, Extension, Champagne, Dun, Grey, Silver). Results for each biopsy sample were compared with results for the reference standard for that blastocyst, formulated as
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described above. The first biopsy sample from each of the first five blastocysts evaluated was tested with a different primer multiplex, which did not interrogate six loci (LEX3, HMS2, LEX33, TKY333, TKY374 and TKY394) and so these loci were excluded from the analysis of these biopsy samples. Allele dropout in biopsy sample and demi-embryo analyses was evaluated based on detection of both alleles in loci found to be heterozygous in the corresponding reference standard, disregarding sex and X-linked loci (AME, SRY and LEX3). For disease and coat-colour loci, normal and wild-type alleles were designated by the letter ‘N’. Alleles corresponding to disease and colour mutations were assigned a descriptive code, e.g. PSSM1; MH, MH1; E and e, Extension; A and a, Agouti; Cr, Cream dilution. Statistical analysis Differences in proportions of loci with signal and of accordant loci between WGA methods, and in proportions of nonperforming biopsy samples, loci with signal, accordant loci and heterozygous loci showing allele drop-out between biopsy
samples from IVP and VIV blastocysts, were evaluated via Chi square analysis, with Fisher’s exact test used when a value of less than 5 was expected in any cell, using the Epistat Statistical Package (Epistat Services, Richardson, TX, USA). The number of discordant loci per VIV demi-embryo was evaluated in relation to embryo diameter via Pearson’s product moment correlation using SigmaPlot Version 13.0 (Systat Software, San Jose, CA, USA). This analysis was also used to compare the number of discordant loci between paired demi-embryos. Results Experiment 1: evaluation of methods for whole-genome amplification The results of analysis with the two methods for whole-genome amplification are presented in Table 1. Illustra Genomiphi V2: demi-embryo analysis In the first blastocyst of this series, the complete remaining embryo was analysed to generate the reference standard;
Table 1. Comparison of accuracy of genetic analysis of embryo biopsy samples undergoing whole-genome amplification by two different methods before multiplex PCR GM, Illustra Genomiphi V2; QG, Qiagen Repli-g Midi. a,bWithin columns, values with different superscripts differ significantly (P , 0.0001) Amplification technique
GM
Embryo number
A
B
C
D
Biopsy
Signal obtained
Accurate
1 2 3 1 2 3 1 2 3 1 2 3
20 20 20 20 20 20 20 20 20 20 20 20 240
1 9 9 16 18 13 8 13 10 7 10 9 123a (51.3%)
0 5 7 6 12 7 2 6 4 1 6 6 62a (25.8% of total; 50.4% of loci with signal)
1 2 3 1 2 3 1 2 3 1 2 3
20 Vial broken 20 20 20 20 20 20 20 20 20 20 220
20
19
20 20 20 20 20 20 20 20 20 19 219b (99.5%)
19 20 20 20 20 20 20 20 20 18 216b (98.2% of total; 98.6% of loci with signal)
Total
QG
E
F
G
H
Total
Number of loci Interrogated
PGD in equine blastocysts
however, there was no signal for four of the 20 analysed loci. All missing results were from microsatellite markers. The subsequent three blastocysts were split after biopsy and the two demiembryos were analysed separately. In one of these cases, one demi-embryo provided a signal for only one of the 20 loci. In the two remaining cases, in which the analyses of the two demiembryos could be compared, 3/80 loci (3.75%) had no signal and there was a discrepancy at 6/38 loci (15.8%) for which there was a signal in both demi-embryos. In each case this was due to allele drop-out (one demi-embryo provided a heterozygous signal for the locus while the other yielded only one of the alleles). Three alleles in demiembryos appeared to be inaccurate (allele signal not compatible with the other demiembryos and biospies). At six loci, both demi-embryos appeared to have undergone allele dropout, as they were homozygous for one allele while one or more biopsy samples showed a second allele at that locus. Illustra Genomiphi V2: biopsy sample analysis For the analysis of the biopsy samples (n ¼ 12; three for each blastocyst, Table 1), interrogation of 20 loci in each sample allowed evaluation of 240 loci in comparison to the reference standard for the corresponding blastocyst. There was no signal for 117 of the possible 240 loci (48.8%). Of the 123 loci that provided a signal, 62 (50.4%) matched exactly with the genotype of the reference standard for that blastocyst. Of 61 loci in the biopsy sample analyses that did not match the reference standard, 59 (96.7%) were due to allele drop-out and two appeared to be inaccurate (allele signal present in the biopsy sample that was not compatible with the reference standard). Overall, when using the Illustra Genomiphi V2 kit for wholegenome amplification an accurate diagnosis was obtained from the biopsy samples at 62 of 240 possible loci (25.8%). Qiagen Repli-g Midi: demi-embryo analysis Two demi-embryos were analysed separately for each of the four blastocysts. Twenty loci were evaluated for each demiembryo, thus providing 80 comparisons (20 loci compared between demi-embryos, for each of the four blastocysts). For two blastocysts, both corresponding demi-embryos gave no signal at one locus (LEX33). For the comparisons for which a signal was obtained, results for paired demi-embryos matched at all compared loci (78/78). Qiagen Repli-g Mid: biopsy sample analysis One biopsy sample could not be evaluated due to cracking of the cryovial and loss of the contents; therefore a total of 11 biopsy samples was analysed in comparison to the reference standard for their corresponding blastocyst, allowing evaluation of 220 loci for accuracy. There was no signal at one locus (LEX33) for one biopsy sample (0.5%). Of the 219 loci in biopsy samples that provided a signal, 216 (98.6%) matched exactly with the genotype of the reference standard for that blastocyst. The three loci in the biopsy sample analyses that did not match the reference standard were due to allele drop-out. Overall, WGA of biopsy samples with the Qiagen Repli-g Midi kit yielded an accurate diagnosis result in 216 of 220 possible loci (98.2%); this was significantly higher than that for biopsy
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samples amplified using Illustra Genomiphi V2 (P , 0.0001). Because of the higher accuracy, the Qiagen Repli-g Midi kit was used for the subsequent experiment. Experiment 2: comparison of PGD results in in vitroproduced and in vivo-recovered embryos The results of genetic analysis of demi-embryos and biopsy samples from the in vitro-produced (IVP) and in vivo-recovered (VIV) blastocysts are presented in Table 2. Demi-embryo analysis All demi-embryos analysed provided a signal. For IVP embryos, of 16 demi-embryos analysed, 15 matched the genotype of the reference standard at all loci analysed. For VIV embryos, of 38 demi-embryos analysed, 25 matched the reference standard at all loci analysed; this difference in proportion of demi-embryos showing discordancy was significant (P , 0.05). In demi-embryos not matching the reference standard, the number of discordant loci was four (n ¼ 1) for IVP and one (n ¼ 6), two (n ¼ 6) and four (n ¼ 1) for VIV. Because discordancy was more common in VIV demi-embryos and VIV blastocysts are larger, more differentiated and contain more cells than do IVP blastocysts, we postulated that embryo size may be negatively correlated with accuracy under this system; however, there was no correlation between initial VIV blastocyst diameter and the number of discordant loci per demiembryo (r ¼ –0.16; P . 0.1). There was also no correlation in the number of discordant loci between the two paired demiembryos (r ¼ 0.20; P . 0.1). The overall accuracy of genetic analysis per interrogated locus in IVP and VIV demi-embryos was 524/528 (99.2%) and 1232/1254 (98.2%), respectively; these were not significantly different (P . 0.05). Biopsy sample analysis One biopsy sample from a VIV embryo did not provide a signal at any locus; the sample was retested with the same results. Two additional biopsy samples, one from an IVP embryo and one from a VIV embryo, provided an accurate signal at ,50% of the loci interrogated (nine and 14 accurate readings out of 33 loci). Notably, at the 15 microsatellite identification markers, one of these non-performing biopsy samples returned two signal failure/ one incorrect (non-matching) allele/11 homozygous/one heterozygous, and the other returned four signal failure/11 homozygous/none heterozygous. In both of the corresponding reference standards, 11 of the 15 identification marker loci were heterozygous. These three biopsy samples (3.7%) were designated ‘nonperforming’ and were considered to represent failure of the biopsy process or loss of material during handling of the biopsy sample. The proportion of non-performing biopsy samples was not different between IVP and VIV embryos (1/24, 4.2% vs 2/57, 3.5%, respectively; P . 0.1). The three non-performing biopsy samples were excluded from further data analyses. In the remaining 78 biopsy samples, there was no significant difference between IVP and VIV blastocysts in the proportion of biopsy samples that matched the genotype of the reference standard at all 33 analysed loci (20/ 23, 87.0% and 49/55, 89.1%, respectively; P . 0.1). The number
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Table 2. Results of genetic analysis of demi-embryos and biopsy specimens from in vivo-recovered and in vitro-produced equine embryos IVP, embryo produced by ICSI and in vitro embryo culture; VIV, embryo recovered ex vivo 6–10 days after ovulation. a,bWithin rows, values with different superscripts differ significantly (P , 0.05) Parameter
IVP
VIV
Embryos Demi-embryos analysed Demi-embryos with ,50% accordant loci Demi-embryos accordant at all loci (%) Demi-embryos total loci evaluated Loci discrepant with reference standard (%) No signal Allele drop-out Total heterozygous loci analysedA Allele drop-out (%)
8 16 0 15 (93.8%)a 528 4 (0.8%) 1 3 224 3 (1.3%)
19 38 0 25 (65.8%)b 1254 22 (1.8%) 18 4 504 4 (0.8%)
Biopsy samples analysed Biopsy samples with ,50% accordant loci (%) Performing biopsy samples Biopsies samples accordant at all loci (%) Biopsy samples total loci evaluated Loci discrepant with reference standard (%) No signal Allele drop-out Total heterozygous loci analysedA Allele drop-out (%)
24 1 (4.2%) 23 20 (87.0%) 759 7 (0.9%) 0 7 323 7 (2.2%)
57 2 (3.5%) 55 49 (89.1%) 1815 11 (0.6%) 1 10 712 10 (1.4%)
A
Not including gender and X-linked loci.
Table 3. Accuracy of genetic analysis of biopsy samples for specific loci of interest Embryo type
VIV IVP Total accurate readings
Genetic status
Homozygous Heterozygous Homozygous Heterozygous
Biopsy samples returning accurate readings at AME
SRY
HERDA
HYPP
PSSM1
MH
23/23 (XX) 32/32 (XY) 14/14 (XX) 9/9 (XY) 78/78
23/23 (SRY) 32/32 (SRYþ) 14/14 (SRY) 9/9 (SRYþ) 78/78
35/35 19/20 23/23
55/55
43/43 12/12 23/23
55/55 23/23
78/78
78/78
of discordant loci found in biopsy samples not matching the standard was one, two and four (n ¼ 1 each) for IVP and one (n ¼ 3), two (n ¼ 2) and four (n ¼ 1) for VIV. One of these discordances (VIV blastocyst) was due to no signal; the remainder were due to allele drop-out. When all loci interrogated in the 78 performing biopsy samples were considered, the accuracy per interrogated locus was 752/759 (99.1%) for biopsy samples from IVP blastocysts and 1804/1815 (99.4%) for biopsy samples from VIV blastocysts; these were not significantly different (P . 0.1). The incidence of allele drop-out on interrogation of heterozygous loci (not including sex and sex-linked loci) did not differ between biopsy samples from IVP and VIV blastocysts (7/323, 2.2% and 10/712, 1.4%, respectively; P . 0.1). The results of evaluation of specific loci of interest are presented in Table 3. Of the 27 embryos evaluated, 13 were female and 14 were male. All 78 performing biopsy samples returned accurate readings for the sex-determination loci AME and SRY. The 27 biopsy samples
77/78
11/11 12/12 78/78
returning a heterozygous reading for LEX3 (locus on the X chromosome) were all appropriately from XX embryos. For HERDA, seven VIV blastocysts were heterozygous HRD/N; of the 20 performing biopsy samples from these blastocysts, one showed allele drop-out, returning an N/N result. At the HYPP locus, four IVP blastocysts were N/H; all 12 biopsy samples from these blastocysts returned an accurate reading. At the PSSM1 locus, four blastocysts were PSSM1/N; all 12 biopsy samples from these blastocysts returned an accurate reading. All blastocysts were N/N at the MH locus. Overall accuracy for determination of heterozygosity at disease-related loci was 43/44 (97.7%). In the 11 coat-colour loci, heterozygosity or presence of a colour variant was found in at least one blastocyst for the Agouti, Cream, Dun, Extension and Tobiano loci. Accuracy of detection of the heterozygous colour state for these loci in biopsy samples from heterozygous blastocysts was 27/27 (100%), 25/26
PGD in equine blastocysts
(96.2%), 6/6 (100%), 24/25 (96.0%) and 6/6 (100%), respectively. Overall accuracy for determination of heterozygosity at coat-colour loci was 88/90 (97.8%). Discussion The results of this study demonstrate that biopsy of equine blastocysts via aspiration during micromanipulation results in samples that can be used effectively for interrogation of multiple loci of interest, including genotyping for disease-related mutations. Our findings indicate that the method used for WGA has a profound effect on the accuracy of the subsequent PCR analysis. In our previous study on genetic analysis of equine embryo biopsy samples (Choi et al. 2010), the Illustra Genomiphi V2 kit was used for WGA; the relatively low accuracy achieved on genetic analysis in that study may have been related to the use of this WGA method. The present data show that, under the testing conditions described here, use of the Qiagen Repli-g kit for WGA is compatible with a high rate of accurate analysis of multiple loci in equine biopsy samples by subsequent multiplex PCR. In these studies, the reference standard was generated from the combined results of the demi-embryos and biopsy samples, rather than tissue from corresponding foals, as done previously (Choi et al. 2010). Thus, the reference standard in the present studies had a small chance of being in itself inaccurate. The error rate of the reference standard can be estimated by multiplying the error rate in each embryo or demi-embryo with that for each of the three biopsy samples; this was highest for the samples evaluated by Illustra Genomiphi V2 (Experiment 1), due to both low accordance and failure of biopsies and demi-embryos to give adequate signal, but approached 0 in the samples evaluated by Qiagen Repli-g Midi (e.g. (0.018)2 (0.006)3 ¼ 7.0 1011 for VIV embryos in Experiment 2). We found that biopsy samples obtained from IVP and VIV blastocysts yielded equivalent high accuracy (.99% of interrogated loci) on genetic analysis. Interestingly, the accuracy of analysis of biopsy samples was equivalent to that for analysis of complete demi-embryos (98 to 99%), although the latter have hundreds to thousands more cells. Notably, three of the 81 biopsy samples obtained (4%) were not useable for analysis, as they returned ,50% accuracy for interrogated loci; this appears to represent the main potential source for error on genetic analysis of equine embryo biopsy samples obtained using the methods described herein. One of these non-performing biopsy samples had no signal at any locus, thus the sample itself appears to have been lost. The other two biopsy samples returned a combination of no signal, error and allele drop-out results, suggesting low fidelity of WGA, perhaps due to very low cell number. The findings at microsatellite markers in these embryos were detailed in the Results, as they suggest that evaluation of identification markers may be useful in determining the quality of the biopsy sample. The two non-performing biopsies provided heterozygous signals at only none and one of these commonly-heterozygous identification loci. In contrast, in the reference standards for the 27 blastocysts used in the study, the mean number of heterozygous identification loci (out of 15) was 11.7 0.3, ranging from nine to 14. This finding suggests that evaluation of biopsy samples at microsatellite identification markers, even if the actual genotype of the embryo is unknown,
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may provide an indication of the overall quality of a sample. This will help to validate the accuracy of homozygous findings on analysis of genes of interest. A second approach to reduce the possibility of non-performing sample error would be to take two biopsy samples from each embryo and analyse them separately. This approach, however, would double the expense of the genetic analysis and the effect of obtaining two biopsy samples on future embryo viability has not been studied. Until this approach has been validated, accuracy of the biopsy procedure should be considered to be subject to the quality of the biopsy obtained; in our hands this was ,95% overall. It is important to assess allele drop-out rates on interrogation of heterozygous loci, as these loci are more challenging than are homozygous loci in relation to accuracy of results. In our study, allele drop-out rates for performing biopsies were below 3% for both IVP and VIV embryos. When we looked specifically at loci that may be of special interest to horse owners, i.e. those for sex determination and presence of disease-associated mutations, sex was accurately detected in 78/78 biopsy samples using either AME or SRY. The accuracy of detection of the heterozygous state for disease-related mutations was also high (97.7%). Not all disease-causing mutations nor all coat-colour mutations could be formally evaluated in this study. Among these, glycogen-branching enzyme disorder (GBED) is potentially the most problematic important mutation, because of the rich GCsequence surrounding the mutation, and would merit validation for use in PGD testing in a future study. In conclusion, results obtained using Piezo-driven embryo biopsy and WGA followed by multiplex PCR can have high (,95%) accuracy for determination of embryonic sex and parentage and for genotyping at disease-associated and coatcolour loci. There was no difference in accuracy of biopsy for genetic diagnosis between IVP and VIV blastocysts. This technique appears to be efficient enough for use in a clinical setting. Acknowledgements This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University, the Veterinary Genetics Laboratory at UC Davis and by Ms. Kit Knotts.
References Bernoco, D., and Bailey, E. (1998). Frequency of the SCID gene among Arabian horses in the USA. Anim. Genet. 29, 41–42. doi:10.1046/J.13652052.1998.00237.X Brault, L., and Penedo, M. C. T. (2009). Refinement of the equine cerebellar abiotrophy locus on ECA2 by haplotype analysis. J. Equine Vet. Sci. 29, 318–319. doi:10.1016/J.JEVS.2009.04.025 Brooks, S. A., Gabreski, N., Miller, D., Brisbin, A., Brown, H. E., Streeter, C., Mezey, J., Cook, D., and Antczak, D. F. (2010). Whole-genome SNP association in the horse: identification of a deletion in myosin Va responsible for lavender foal syndrome. PLoS Genet. 6, e1000909. doi:10.1371/JOURNAL.PGEN.1000909 Choi, Y. H., Love, C. C., Love, L. B., Varner, D. D., Brinsko, S., and Hinrichs, K. (2002). Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilised by intracytoplasmic sperm injection with fresh or frozen–thawed spermatozoa. Reproduction 123, 455–465. doi:10.1530/REP.0.1230455 Choi, Y. H., Chung, Y. G., Walker, S. C., Westhusin, M. E., and Hinrichs, K. (2003). In vitro development of equine nuclear transfer embryos: effects
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of oocyte maturation media and amino acid composition during embryo culture. Zygote 11, 77–86. doi:10.1017/S0967199403001102 Choi, Y. H., Love, L. B., Varner, D. D., and Hinrichs, K. (2006). Holding immature equine oocytes in the absence of meiotic inhibitors: effect on germinal vesicle chromatin and blastocyst development after intracytoplasmic sperm injection. Theriogenology 66, 955–963. doi:10.1016/ J.THERIOGENOLOGY.2006.01.064 Choi, Y. H., Harding, H. D., Hartman, D. L., Obermiller, A. D., Kurosaka, S., McLaughlin, K. J., and Hinrichs, K. (2009). The uterine environment modulates trophectodermal POU5F1 levels in equine blastocysts. Reproduction 138, 589–599. doi:10.1530/REP-08-0394 Choi, Y. H., Gustafson-Seabury, A., Velez, I. C., Hartman, D. L., Bliss, S., Riera, F. L., Roldan, J. E., Chowdhary, B., and Hinrichs, K. (2010). Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis. Reproduction 140, 893–902. doi:10.1530/REP-10-0141 Choi, Y. H., Velez, I. C., Riera, F. L., Rolda´n, J. E., Hartman, D. L., Bliss, S. B., Blanchard, T. L., Hayden, S. S., and Hinrichs, K. (2011). Successful cryopreservation of expanded equine blastocysts. Theriogenology 76, 143–152. doi:10.1016/J.THERIOGENOLOGY.2011.01.028 Hahn, S., Zhong, X. Y., Troeger, C., Burgemeister, R., Gloning, K., and Holzgreve, W. (2000). Current applications of single-cell PCR. Cell. Mol. Life Sci. 57, 96–105. doi:10.1007/S000180050501 Handyside, A. H., Robinson, M. D., Simpson, R. J., Omar, M. B., Shaw, M. A., Grudzinskas, J. G., and Rutherford, A. (2004). Isothermal whole-genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease. Mol. Hum. Reprod. 10, 767–772. doi:10.1093/MOLEHR/GAH101 Herrera, C., Morikawa, M. I., Bello, M. B., von Meyeren, M., Centeno, J. E., Dufourq, P., Martinez, M. M., and Llorente, J. (2014). Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program. Theriogenology 81, 758–763. doi:10.1016/J.THERIOGENOLOGY.2013.12.013 Herszberg, B., McCue, M. E., Larcher, T., Mata, X., Vaiman, A., Chaffaux, S., Che´rel, Y., Valberg, S. J., Mickelson, J. R., and Gue´rin, G. (2009). A GYS1 gene mutation is highly associated with polysaccharide storage myopathy in Cob Normand draught horses. Anim. Genet. 40, 94–96. doi:10.1111/J.1365-2052.2008.01778.X Hinrichs, K., and Choi, Y. H. (2012). Equine embryo biopsy, genetic testing and cryopreservation. J. Equine Vet. Sci. 32, 390–396. doi:10.1016/ J.JEVS.2012.05.005 Hinrichs, K., Choi, Y. H., Love, L. B., Varner, D. D., Love, C. C., and Walckenaer, B. E. (2005). Chromatin configuration within the germinal vesicle of horse oocytes: changes post mortem and relationship to meiotic and developmental competence. Biol. Reprod. 72, 1142–1150. doi:10.1095/BIOLREPROD.104.036012
Hinrichs, K., Choi, Y. H., Love, C. C., and Spacek, S. (2014). Use of in vitro maturation of oocytes, intracytoplasmic sperm injection and in vitro culture to the blastocyst stage in a commercial equine assisted reproduction program. J. Equine Vet. Sci. 34, 176. doi:10.1016/J.JEVS. 2013.10.129 Huhtinen, M., Peippo, J., and Bredbacka, P. (1997). Successful transfer of biopsied equine embryos. Theriogenology 48, 361–367. doi:10.1016/ S0093-691X(97)00247-1 Jacobson, C. C., Choi, Y. H., Hayden, S. S., and Hinrichs, K. (2010). Recovery of mare oocytes on a fixed biweekly schedule and resulting blastocyst formation after intracytoplasmic sperm injection. Theriogenology 73, 1116–1126. doi:10.1016/J.THERIOGENOLOGY.2010. 01.013 McCue, M. E., Valberg, S. J., Jackson, M., Borgia, L., Lucio, M., and Mickelson, J. R. (2009). Polysaccharide storage myopathy phenotype in quarter horse-related breeds is modified by the presence of an RYR1 mutation. Neuromuscul. Disord. 19, 37–43. doi:10.1016/J.NMD.2008. 10.001 Piyamongkol, W., Bermudez, M. G., Harper, J. C., and Wells, D. (2003). Detailed investigation of factors influencing amplification efficiency and allele drop-out in single-cell PCR: implications for preimplantation genetic diagnosis. Mol. Hum. Reprod. 9, 411–420. doi:10.1093/ MOLEHR/GAG051 Rudolph, J. A., Spier, S. J., Byrns, G., Rojas, C. V., Bernoco, D., and Hoffman, E. P. (1992). Periodic paralysis in quarter horses: a sodium channel mutation disseminated by selective breeding. Nat. Genet. 2, 144–147. doi:10.1038/NG1092-144 Seidel, G. E., Jr, Cullingford, E. L., Stokes, J. E., Carnevale, E. M., and McCue, P. M. (2010). Pregnancy rates following transfer of biopsied and/or vitrified equine embryos: evaluation of two biopsy techniques. Anim. Reprod. Sci. 121S, 297–298 [Abstract]. Spits, C., and Sermon, K. (2009). PGD for monogenic disorders: aspects of molecular biology. Prenat. Diagn. 29, 50–56. doi:10.1002/PD.2161 Troedsson, M. H. T., Paprocki, A. M., Koppang, R. W., Syverson, C. M., Griffin, P., Klein, C., and Dobrinski, J. R. (2010). Transfer success of biopsied and vitrified embryos. Anim. Reprod. Sci. 121S, 295–296 [Abstract]. Tryon, R. C., Penedo, M. C. T., McCue, M. E., Valberg, S. J., Mickelson, J. R., Famula, T. R., Wagner, M. L., Jackson, M., Hamilton, M. J., Nooteboom, S., and Bannasch, D. L. (2009). Evaluation of allele frequencies of inherited disease genes in subgroups of American Quarter Horses. J. Am. Vet. Med. Assoc. 234, 120–125. doi:10.2460/JAVMA.234.1.120 Ward, T. L., Valberg, S. J., Adelson, D. L., Abbey, C. A., Binns, M. M., and Mickelson, J. R. (2004). Glycogen branching enzyme (GBE1) mutation causing equine glycogen storage disease IV. Mamm. Genome 15, 570–577. doi:10.1007/S00335-004-2369-1
www.publish.csiro.au/journals/rfd
Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD14419
Accuracy of preimplantation genetic diagnosis in equine in vivo-recovered and in vitro-produced blastocysts Y. H. Choi A, M. C. T. Penedo B, P. Daftari B, I. C. Velez A and K. Hinrichs A,C,D A
Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, College Station, TX 77843-4466, USA. B Veterinary Genetics Laboratory, University of California, Old Davis Road, Davis, CA 95616, USA. C Department of Large Animal Clinical Sciences, Texas A&M University, 4475 TAMU, College Station, TX, 77843-4475, USA D Corresponding author. Email: [email protected]
Abstract. Preimplantation genetic diagnosis has great potential in the horse, but information on evaluation of equine embryo biopsy samples is limited. Blastocysts were biopsied using a Piezo drill and methods for whole-genome amplification (WGA) investigated. Results for 33 genetic loci were then compared between biopsy samples from in vitroproduced (IVP) and in vivo-recovered (VIV) blastocysts. Under the experimental conditions described, WGA using the Qiagen Repli-g Midi kit was more accurate than that using the Illustra Genomiphi V2 kit (98.2% vs 25.8%, respectively). Using WGA with the Qiagen kit, three biopsy samples were evaluated from each of eight IVP and 19 VIV blastocysts, some produced using semen from stallions carrying the genetic mutations associated with the diseases hereditary equine regional dermal asthenia (HERDA), hyperkalemic periodic paralysis (HYPP) or polysaccharide storage myopathy 1 (PSSM1). Three of 81 biopsy samples (3.7%) returned ,50% accuracy. In the remaining 78 samples, overall accuracy was 99.3% (2556/2574 loci interrogated). Accuracy did not differ significantly between samples from IVP and VIV blastocysts. Allele drop-out in heterozygous loci was 1.6% (17/1035). Accuracy for sex determination was 100%; accuracy for heterozygosity for disease-causing mutations was 97.7% (43/44). In conclusion, Piezo-driven embryo biopsy with WGA has .95% overall accuracy in IVP and VIV embryos, and this technique is suitable for use in a clinical setting. Additional keywords: embryo biopsy, genetic screening, HERDA, HYPP, polymerase chain reaction (PCR), PSSM, whole genome amplification.
Received 31 October 2014, accepted 19 January 2015, published online 17 March 2015
Introduction Preimplantation genetic diagnosis (PGD) has the potential for extensive use in the horse. This technique may be used to screen for genetic diseases such as hereditary equine regional dermal asthenia (HERDA), hyperkalemic periodic paralysis (HYPP) or other devastating single-locus mutations (Rudolph et al. 1992; Bernoco and Bailey 1998; Ward et al. 2004; Brault and Penedo 2009; Herszberg et al. 2009; Tryon et al. 2009; Brooks et al. 2010) or to select embryos on traits such as embryo sex or coat colour before transfer. While use of PGD is not applicable to breeds that prohibit embryo transfer, including the Thoroughbred, the possibility of selecting embryos for desired traits may lead to increased importance of embryo transfer as an equine reproductive management tool in arenas in which embryo transfer is accepted. Biopsy of equine embryos, especially expanded blastocysts, was initially associated with poor pregnancy rates after transfer (Huhtinen et al. 1997; Seidel et al. 2010). Expanded blastocysts in the horse have an embryonic capsule, a glycoprotein envelope that surrounds the equine embryo after the zona pellucida has Journal compilation Ó CSIRO 2015
been shed, which makes some methods of biopsy, such as microblade dissection, problematic. We reported a technique for biopsy of equine embryos via micropipette aspiration under micromanipulation, which supports normal viability (rate of normal pregnancy with heartbeat) after biopsy of both morulae– early blastocysts and expanded blastocysts (Choi et al. 2010). Others have used this technique with modifications for biopsy of expanded equine blastocysts, finding similar normal viability after transfer of over 100 biopsied embryos (Herrera et al. 2014). There is increasing clinical demand for in vitro production (IVP) of equine embryos, via intracytoplasmic sperm injection (ICSI) and embryo culture (Hinrichs et al. 2014). Biopsy of in vitro-produced equine embryos appears straightforward, as there is no capsule, and intercellular junctions are relatively weak. In contrast, biopsy of in vivo-recovered (VIV) expanded equine blastocysts is complicated not only by the presence of the capsule but also by the tenacity of intercellular attachments among the trophoblast cells at this stage (Hinrichs and Choi 2012). However, the larger cell numbers in VIV embryos provides an opportunity www.publish.csiro.au/journals/rfd
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for larger biopsy samples. It is not known if these differences in biopsy sample characteristics affect the accuracy of PGD. Embryonic cells obtained by biopsy may be used for the interrogation of a single locus or a few loci (such as for embryo sexing) by direct or multiplex polymerase chain reaction (PCR; single-cell PCR; Hahn et al. 2000), and sex of horse embryos has been accurately determined by this method (Huhtinen et al. 1997; Troedsson et al. 2010; Herrera et al. 2014). However, for evaluation of multiple loci, direct or multiplex PCR is problematic, as primers may interfere with one another and no sample is left for additional or confirmatory tests. An alternative for the interrogation of multiple loci is performance of whole-genome amplification (WGA) followed by PCR, as has been done on human embryo biopsies (Handyside et al. 2004). We utilised WGA previously for genetic analysis of equine embryo biopsy samples for sex, HERDA and HYPP (Choi et al. 2010), but only two of 16 loci analysed were heterozygous for a disease-causing mutation and only one of these was accurately detected on analysis of the biopsy sample, thus the accuracy of biopsy– WGA for detection of disease-related genotypes (heterozygous or homozygous affected) is unknown. Interrogation of heterozygous genotypes is more challenging than is interrogation of homozygous genotypes, due to the occurrence of allele drop-out (failure of one allele to amplify). This results in the detection of only one allele, thus indicating homozygosity, at a given locus in a heterozygous individual. Allele drop-out is common in genetic analysis of samples with small cell numbers (reviews Piyamongkol et al. 2003; Spits and Sermon 2009). In horses, the accurate detection of both normal and disease-associated alleles in heterozygous animals and differentiation of heterozygous animals from either genotype of homozygous animals is important to breeders requesting PGD. Most sex, disease and coat-colour loci of interest to horse breeders are amenable to interrogation; however, some loci present challenges because causative mutations are located within GC-rich sequences or represent large or complex duplications. Thus, analysis of accuracy of interrogation needs to be determined separately for each locus. We hypothesised that the WGA method used would affect the accuracy of genetic evaluation of multiple loci in equine embryo biopsy samples and that biopsy samples from IVP equine embryos would allow more accurate genetic analysis than would those from VIV equine embryos. Thus, in this study, we investigated two methods for WGA and then compared the accuracy of WGA–multiplex PCR for interrogation of multiple loci (sex, disease-related mutation, coat colour and microsatellite identification markers) between biopsies from IVP and VIV equine blastocysts, to establish the efficiency of biopsy for genetic analysis in these two embryo types.
according to the United States Government Principles for the Utilisation and Care of Vertebrate Animals Used in Testing, Research and Training and were approved by the Institutional Animal Care and Use Committee at Texas A&M University. Briefly, immature oocytes were collected by aspiration and flushing of follicles $ 5 mm in diameter. The aspirated fluid was filtered through an embryo filter (EmCon filter; Immuno Systems Inc., Spring Valley, WI, USA) and the oocytes were recovered from the collected cellular material. Collected oocytes were held overnight (Choi et al. 2006) then cultured for maturation. Mature oocytes were subjected to ICSI, using frozen–thawed spermatozoa, via a Piezo drill as described previously (Choi et al. 2002, 2003). In Experiment 2, to increase the chance of production of embryos heterozygous for diseaserelated mutations and colour-related genes, spermatozoa from a stallion heterozygous N/Cr for the Cream dilution and heterozygous N/H for HYPP were used. Injected oocytes were cultured for embryo development in microdroplets of Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F-12) (Hinrichs et al. 2005) or a commercial human embryo culture medium (global medium (GB); LifeGlobal, Guilford, CT, USA), both with 10% fetal bovine serum (FBS), in an atmosphere of 5–6% CO2, 5% O2 and the remainder N2 at 38.28C for 7–10 days. Media were completely changed at Day 5 into the same medium (DMEM/F-12) or, for GB-cultured embryos, to GB containing 20 mM glucose. In the first experiment (comparison of WGA methods), IVP blastocysts were vitrified as we have described previously (Choi et al. 2011) then warmed and cultured for 18–30 h before being biopsied. In the second experiment (comparison of IVP and VIV embryos), IVP embryos were biopsied on the day they developed to blastocyst in culture.
Materials and methods In vitro embryo production For this study blastocysts were produced in vitro by culturing oocytes after ICSI. Immature oocytes were collected from abattoir tissue (Experiment 1) or from research mares by transvaginal ultrasound-guided follicle aspiration (Jacobson et al. 2010). All experimental procedures were performed
Embryo biopsy Embryo biopsy was performed as described in our previous study (Choi et al. 2010), with minor modifications. Briefly, embryos were placed in 50-mL droplets of Chatot-ZiomekBavister manipulation (CZB-M) medium (Choi et al. 2003) with 10% FBS at room temperature in a Petri dish on an inverted microscope. The embryos were held with a holding
Collection of in vivo-produced embryos In vivo-produced blastocysts (Experiment 2) were recovered by transcervical uterine flush from research mares 6–10 days after ovulation, as previously described (Choi et al. 2009). Briefly, mare ovarian activity was monitored by palpation and ultrasonography per rectum. When a dominant follicle reached over 30 mm in diameter, biorelease deslorelin (1.5 mg, intramuscular (i.m.); BET Pharm, Lexington, KY, USA) was administered and mares were artificially inseminated with fresh or cooled, extended semen from a fertile stallion, containing a minimum of 500 106 progressively motile spermatozoa. To increase the chance of production of embryos heterozygous for diseaserelated mutations, spermatozoa from stallions heterozygous N/HRD for HERDA or N/PSSM1 at the polysaccharide storage myopathy 1 (PSSM1) locus were used for some inseminations. After confirmation of ovulation by ultrasonography per rectum, mares were assigned for uterine flushing to recover embryos at various days after ovulation.
PGD in equine blastocysts
pipette and a pipette of 15 3 mm external diameter, attached to a Piezo drill, was used to perform the biopsy. We attempted to obtain 10–30 cells for each biopsy sample. The biopsied cells were expelled into a 3-mL droplet of CZB-M without calcium, glutamine, nonessential amino acids or FBS, but supplemented with 2% polyvinylpyrrolidone, then aspirated from the droplet with a fine-bore glass pipette and placed in a 0.2-mL PCR tube (VWR International, West Chester, PA, USA). After the completion of the biopsies, the remaining embryo was moved into a Petri dish containing CZB-M þ 10% FBS, then split approximately in half using 27-gauge needles and each half transferred into a separate PCR tube. All PCR tubes were stored at 208C until they were shipped to the Veterinary Genetics Laboratory at the University of California (Davis, CA, USA) for genomic analysis. For clarity in discussing these analyses, the original blastocyst was designated as such and the embryonic tissue remaining after biopsy was designated the embryo or the demi-embryo (after splitting). Experiment 1: evaluation of methods for whole-genome amplification This experiment was performed to determine if there was a difference in accuracy of genetic diagnosis related to the method used for WGA. For this study, three biopsy samples were analysed from each of eight IVP blastocysts. For the first blastocyst biopsied, the entire remaining embryo was submitted for analysis; after this, remaining embryos were split as described above and each demi-embryo submitted separately for analysis, to provide a reference for evaluation of accuracy of biopsy samples for that blastocyst. The biopsy samples, embryos and demi-embryos were subjected to WGA using either the Illustra Genomiphi V2 kit (GE Healthcare Bioscience Corp, Piscataway, NJ, USA; first four blastocysts) or the Qiagen Repli-g Midi kit (Qiagen Inc., Valencia, CA, USA; next four blastocysts). Under each amplification system, loci for two disease-related mutations (HERDA, HYPP), one sex (AME), one X-linked marker (LEX3) and 16 microsatellite identification markers (AHT4, AHT5, ASB17, ASB2, ASB23, HMS2, HMS3, HMS6, HMS7, HTG10, HTG4, LEX33, TKY333, TKY374, TKY394 and VHL20) were genotyped, for a total of 20 loci, for each biopsy sample and embryo or demi-embryo. The reference standard for assessment of accuracy of biopsy analysis for each blastocyst was generated by combining the analysis for the two demi-embryos for that blastocyst. In cases in which no signal for a given allele was detected in the embryo or demi-embryos but was present in one or more biopsy samples, this allele was included in the reference standard for that blastocyst. WGA was done in the 0.2-mL PCR tubes that contained the embryo or biopsy sample cells; when thawed, liquid media did not exceed 2 uL in volume. WGA with the Illustra Genomiphi V2 kit was performed according to the manufacturer’s protocol. WGA with the Qiagen Repli-g Midi kit was performed according to the manufacturer’s protocol for genomic DNA from blood or cells, with the following modifications: Buffer D2 was prepared with water instead of dithiothreitol solution and, after addition of Buffer D2 to sample tubes, cells were incubated at 608C for 5 min and then put on ice for at least 10 min. For all PCR reactions, 3 mL of DNA solution (WGA product) was used as template.
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DNA amplification for the specific loci to be interrogated was done in three multiplexed PCR reactions. Two multiplexed panels amplified sex and ID markers (Panel A: AME, AHT4, AHT5, ASB23, HTG4, HTG10, HMS2, TKY333, VHL20; Panel B: ASB2, ASB17, HMS6, HMS7, LEX33, TKY374, TKY394, HMS3, LEX3). One multiplexed panel amplified two diseaserelated loci (HERDA, HYPP). PCR was carried out in 25-mL total-volume reactions containing 3 mL DNA template, 1 PCR buffer IV (Denville Scientific, Metuchen, NJ, USA), 1 U Choice Taq (Denville Scientific), 200 mM dNTPs, 2.5 mM MgCl2 and fluorescencelabelled primers needed for each multiplex. Cycling conditions consisted of four cycles of 1 min at 948C, 30 s at 608C, 30 s at 728C, followed by 25 cycles of 45 s at 948C, 30 s at 608C, 30 s at 728C and a final extension at 728C for 30 min. PCR products were separated by capillary electrophoresis on an ABI 3730 DNA Analyser (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Fragment size analysis and genotyping were done with the computer software STRand (available at https://www.vgl. ucdavis.edu/informatics/strand.php, accessed 23 February 2015). Allele designations for microsatellite markers followed international standardised nomenclature defined by the International Society of Animal Genetics (ISAG). For diseaserelated loci, normal alleles were designated by the letter ‘N’ and alleles corresponding to disease-related mutations were assigned a descriptive code (HRD, HERDA; H, HYPP). Positive and negative control samples were included in all PCR reactions. Experiment 2: comparison of PGD results for in vitroproduced and in vivo-recovered embryos For this experiment, eight IVP blastocysts produced via ICSI using spermatozoa from a stallion heterozygous N/Cr for the Cream dilution and heterozygous N/H for HYPP, and 19 VIV blastocysts obtained on Day 6 (n ¼ 2), Day 7 (n ¼ 12), Day 8–9 (n ¼ 4) or Day 10 (n ¼ 1) after ovulation were analysed. Eight of the VIV blastocysts were sired by a stallion that was heterozygous N/HRD for HERDA and four were sired by a stallion that was heterozygous N/PSSM1 at the PSSM1 locus. Three biopsy samples were obtained from each blastocyst, then the remaining embryos were bisected and samples stored and shipped as for Experiment 1. The Qiagen Repli-g Midi kit was used for WGA. A total of 33 loci were evaluated; DNA amplification was performed in five multiplexed PCR reactions. Two multiplexed panels amplified sex and ID markers, as above but including SRY and excluding AHT5. One multiplexed panel amplified four disease loci (HERDA, HYPP, PSSM1 and MH1, a locus responsible for malignant hyperthermia that also modifies expression of PSSM1; McCue et al. (2009)). To increase the number of potential heterozygous protein-coding loci evaluated, 11 coat-colour loci were evaluated; two multiplexed panels amplified these loci (Panel C: Cream dilution, Lethal White Overo, Sabino-1, Pearl dilution, Tobiano; Panel D: Agouti, Extension, Champagne, Dun, Grey, Silver). Results for each biopsy sample were compared with results for the reference standard for that blastocyst, formulated as
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described above. The first biopsy sample from each of the first five blastocysts evaluated was tested with a different primer multiplex, which did not interrogate six loci (LEX3, HMS2, LEX33, TKY333, TKY374 and TKY394) and so these loci were excluded from the analysis of these biopsy samples. Allele dropout in biopsy sample and demi-embryo analyses was evaluated based on detection of both alleles in loci found to be heterozygous in the corresponding reference standard, disregarding sex and X-linked loci (AME, SRY and LEX3). For disease and coat-colour loci, normal and wild-type alleles were designated by the letter ‘N’. Alleles corresponding to disease and colour mutations were assigned a descriptive code, e.g. PSSM1; MH, MH1; E and e, Extension; A and a, Agouti; Cr, Cream dilution. Statistical analysis Differences in proportions of loci with signal and of accordant loci between WGA methods, and in proportions of nonperforming biopsy samples, loci with signal, accordant loci and heterozygous loci showing allele drop-out between biopsy
samples from IVP and VIV blastocysts, were evaluated via Chi square analysis, with Fisher’s exact test used when a value of less than 5 was expected in any cell, using the Epistat Statistical Package (Epistat Services, Richardson, TX, USA). The number of discordant loci per VIV demi-embryo was evaluated in relation to embryo diameter via Pearson’s product moment correlation using SigmaPlot Version 13.0 (Systat Software, San Jose, CA, USA). This analysis was also used to compare the number of discordant loci between paired demi-embryos. Results Experiment 1: evaluation of methods for whole-genome amplification The results of analysis with the two methods for whole-genome amplification are presented in Table 1. Illustra Genomiphi V2: demi-embryo analysis In the first blastocyst of this series, the complete remaining embryo was analysed to generate the reference standard;
Table 1. Comparison of accuracy of genetic analysis of embryo biopsy samples undergoing whole-genome amplification by two different methods before multiplex PCR GM, Illustra Genomiphi V2; QG, Qiagen Repli-g Midi. a,bWithin columns, values with different superscripts differ significantly (P , 0.0001) Amplification technique
GM
Embryo number
A
B
C
D
Biopsy
Signal obtained
Accurate
1 2 3 1 2 3 1 2 3 1 2 3
20 20 20 20 20 20 20 20 20 20 20 20 240
1 9 9 16 18 13 8 13 10 7 10 9 123a (51.3%)
0 5 7 6 12 7 2 6 4 1 6 6 62a (25.8% of total; 50.4% of loci with signal)
1 2 3 1 2 3 1 2 3 1 2 3
20 Vial broken 20 20 20 20 20 20 20 20 20 20 220
20
19
20 20 20 20 20 20 20 20 20 19 219b (99.5%)
19 20 20 20 20 20 20 20 20 18 216b (98.2% of total; 98.6% of loci with signal)
Total
QG
E
F
G
H
Total
Number of loci Interrogated
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however, there was no signal for four of the 20 analysed loci. All missing results were from microsatellite markers. The subsequent three blastocysts were split after biopsy and the two demiembryos were analysed separately. In one of these cases, one demi-embryo provided a signal for only one of the 20 loci. In the two remaining cases, in which the analyses of the two demiembryos could be compared, 3/80 loci (3.75%) had no signal and there was a discrepancy at 6/38 loci (15.8%) for which there was a signal in both demi-embryos. In each case this was due to allele drop-out (one demi-embryo provided a heterozygous signal for the locus while the other yielded only one of the alleles). Three alleles in demiembryos appeared to be inaccurate (allele signal not compatible with the other demiembryos and biospies). At six loci, both demi-embryos appeared to have undergone allele dropout, as they were homozygous for one allele while one or more biopsy samples showed a second allele at that locus. Illustra Genomiphi V2: biopsy sample analysis For the analysis of the biopsy samples (n ¼ 12; three for each blastocyst, Table 1), interrogation of 20 loci in each sample allowed evaluation of 240 loci in comparison to the reference standard for the corresponding blastocyst. There was no signal for 117 of the possible 240 loci (48.8%). Of the 123 loci that provided a signal, 62 (50.4%) matched exactly with the genotype of the reference standard for that blastocyst. Of 61 loci in the biopsy sample analyses that did not match the reference standard, 59 (96.7%) were due to allele drop-out and two appeared to be inaccurate (allele signal present in the biopsy sample that was not compatible with the reference standard). Overall, when using the Illustra Genomiphi V2 kit for wholegenome amplification an accurate diagnosis was obtained from the biopsy samples at 62 of 240 possible loci (25.8%). Qiagen Repli-g Midi: demi-embryo analysis Two demi-embryos were analysed separately for each of the four blastocysts. Twenty loci were evaluated for each demiembryo, thus providing 80 comparisons (20 loci compared between demi-embryos, for each of the four blastocysts). For two blastocysts, both corresponding demi-embryos gave no signal at one locus (LEX33). For the comparisons for which a signal was obtained, results for paired demi-embryos matched at all compared loci (78/78). Qiagen Repli-g Mid: biopsy sample analysis One biopsy sample could not be evaluated due to cracking of the cryovial and loss of the contents; therefore a total of 11 biopsy samples was analysed in comparison to the reference standard for their corresponding blastocyst, allowing evaluation of 220 loci for accuracy. There was no signal at one locus (LEX33) for one biopsy sample (0.5%). Of the 219 loci in biopsy samples that provided a signal, 216 (98.6%) matched exactly with the genotype of the reference standard for that blastocyst. The three loci in the biopsy sample analyses that did not match the reference standard were due to allele drop-out. Overall, WGA of biopsy samples with the Qiagen Repli-g Midi kit yielded an accurate diagnosis result in 216 of 220 possible loci (98.2%); this was significantly higher than that for biopsy
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samples amplified using Illustra Genomiphi V2 (P , 0.0001). Because of the higher accuracy, the Qiagen Repli-g Midi kit was used for the subsequent experiment. Experiment 2: comparison of PGD results in in vitroproduced and in vivo-recovered embryos The results of genetic analysis of demi-embryos and biopsy samples from the in vitro-produced (IVP) and in vivo-recovered (VIV) blastocysts are presented in Table 2. Demi-embryo analysis All demi-embryos analysed provided a signal. For IVP embryos, of 16 demi-embryos analysed, 15 matched the genotype of the reference standard at all loci analysed. For VIV embryos, of 38 demi-embryos analysed, 25 matched the reference standard at all loci analysed; this difference in proportion of demi-embryos showing discordancy was significant (P , 0.05). In demi-embryos not matching the reference standard, the number of discordant loci was four (n ¼ 1) for IVP and one (n ¼ 6), two (n ¼ 6) and four (n ¼ 1) for VIV. Because discordancy was more common in VIV demi-embryos and VIV blastocysts are larger, more differentiated and contain more cells than do IVP blastocysts, we postulated that embryo size may be negatively correlated with accuracy under this system; however, there was no correlation between initial VIV blastocyst diameter and the number of discordant loci per demiembryo (r ¼ –0.16; P . 0.1). There was also no correlation in the number of discordant loci between the two paired demiembryos (r ¼ 0.20; P . 0.1). The overall accuracy of genetic analysis per interrogated locus in IVP and VIV demi-embryos was 524/528 (99.2%) and 1232/1254 (98.2%), respectively; these were not significantly different (P . 0.05). Biopsy sample analysis One biopsy sample from a VIV embryo did not provide a signal at any locus; the sample was retested with the same results. Two additional biopsy samples, one from an IVP embryo and one from a VIV embryo, provided an accurate signal at ,50% of the loci interrogated (nine and 14 accurate readings out of 33 loci). Notably, at the 15 microsatellite identification markers, one of these non-performing biopsy samples returned two signal failure/ one incorrect (non-matching) allele/11 homozygous/one heterozygous, and the other returned four signal failure/11 homozygous/none heterozygous. In both of the corresponding reference standards, 11 of the 15 identification marker loci were heterozygous. These three biopsy samples (3.7%) were designated ‘nonperforming’ and were considered to represent failure of the biopsy process or loss of material during handling of the biopsy sample. The proportion of non-performing biopsy samples was not different between IVP and VIV embryos (1/24, 4.2% vs 2/57, 3.5%, respectively; P . 0.1). The three non-performing biopsy samples were excluded from further data analyses. In the remaining 78 biopsy samples, there was no significant difference between IVP and VIV blastocysts in the proportion of biopsy samples that matched the genotype of the reference standard at all 33 analysed loci (20/ 23, 87.0% and 49/55, 89.1%, respectively; P . 0.1). The number
F
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Table 2. Results of genetic analysis of demi-embryos and biopsy specimens from in vivo-recovered and in vitro-produced equine embryos IVP, embryo produced by ICSI and in vitro embryo culture; VIV, embryo recovered ex vivo 6–10 days after ovulation. a,bWithin rows, values with different superscripts differ significantly (P , 0.05) Parameter
IVP
VIV
Embryos Demi-embryos analysed Demi-embryos with ,50% accordant loci Demi-embryos accordant at all loci (%) Demi-embryos total loci evaluated Loci discrepant with reference standard (%) No signal Allele drop-out Total heterozygous loci analysedA Allele drop-out (%)
8 16 0 15 (93.8%)a 528 4 (0.8%) 1 3 224 3 (1.3%)
19 38 0 25 (65.8%)b 1254 22 (1.8%) 18 4 504 4 (0.8%)
Biopsy samples analysed Biopsy samples with ,50% accordant loci (%) Performing biopsy samples Biopsies samples accordant at all loci (%) Biopsy samples total loci evaluated Loci discrepant with reference standard (%) No signal Allele drop-out Total heterozygous loci analysedA Allele drop-out (%)
24 1 (4.2%) 23 20 (87.0%) 759 7 (0.9%) 0 7 323 7 (2.2%)
57 2 (3.5%) 55 49 (89.1%) 1815 11 (0.6%) 1 10 712 10 (1.4%)
A
Not including gender and X-linked loci.
Table 3. Accuracy of genetic analysis of biopsy samples for specific loci of interest Embryo type
VIV IVP Total accurate readings
Genetic status
Homozygous Heterozygous Homozygous Heterozygous
Biopsy samples returning accurate readings at AME
SRY
HERDA
HYPP
PSSM1
MH
23/23 (XX) 32/32 (XY) 14/14 (XX) 9/9 (XY) 78/78
23/23 (SRY) 32/32 (SRYþ) 14/14 (SRY) 9/9 (SRYþ) 78/78
35/35 19/20 23/23
55/55
43/43 12/12 23/23
55/55 23/23
78/78
78/78
of discordant loci found in biopsy samples not matching the standard was one, two and four (n ¼ 1 each) for IVP and one (n ¼ 3), two (n ¼ 2) and four (n ¼ 1) for VIV. One of these discordances (VIV blastocyst) was due to no signal; the remainder were due to allele drop-out. When all loci interrogated in the 78 performing biopsy samples were considered, the accuracy per interrogated locus was 752/759 (99.1%) for biopsy samples from IVP blastocysts and 1804/1815 (99.4%) for biopsy samples from VIV blastocysts; these were not significantly different (P . 0.1). The incidence of allele drop-out on interrogation of heterozygous loci (not including sex and sex-linked loci) did not differ between biopsy samples from IVP and VIV blastocysts (7/323, 2.2% and 10/712, 1.4%, respectively; P . 0.1). The results of evaluation of specific loci of interest are presented in Table 3. Of the 27 embryos evaluated, 13 were female and 14 were male. All 78 performing biopsy samples returned accurate readings for the sex-determination loci AME and SRY. The 27 biopsy samples
77/78
11/11 12/12 78/78
returning a heterozygous reading for LEX3 (locus on the X chromosome) were all appropriately from XX embryos. For HERDA, seven VIV blastocysts were heterozygous HRD/N; of the 20 performing biopsy samples from these blastocysts, one showed allele drop-out, returning an N/N result. At the HYPP locus, four IVP blastocysts were N/H; all 12 biopsy samples from these blastocysts returned an accurate reading. At the PSSM1 locus, four blastocysts were PSSM1/N; all 12 biopsy samples from these blastocysts returned an accurate reading. All blastocysts were N/N at the MH locus. Overall accuracy for determination of heterozygosity at disease-related loci was 43/44 (97.7%). In the 11 coat-colour loci, heterozygosity or presence of a colour variant was found in at least one blastocyst for the Agouti, Cream, Dun, Extension and Tobiano loci. Accuracy of detection of the heterozygous colour state for these loci in biopsy samples from heterozygous blastocysts was 27/27 (100%), 25/26
PGD in equine blastocysts
(96.2%), 6/6 (100%), 24/25 (96.0%) and 6/6 (100%), respectively. Overall accuracy for determination of heterozygosity at coat-colour loci was 88/90 (97.8%). Discussion The results of this study demonstrate that biopsy of equine blastocysts via aspiration during micromanipulation results in samples that can be used effectively for interrogation of multiple loci of interest, including genotyping for disease-related mutations. Our findings indicate that the method used for WGA has a profound effect on the accuracy of the subsequent PCR analysis. In our previous study on genetic analysis of equine embryo biopsy samples (Choi et al. 2010), the Illustra Genomiphi V2 kit was used for WGA; the relatively low accuracy achieved on genetic analysis in that study may have been related to the use of this WGA method. The present data show that, under the testing conditions described here, use of the Qiagen Repli-g kit for WGA is compatible with a high rate of accurate analysis of multiple loci in equine biopsy samples by subsequent multiplex PCR. In these studies, the reference standard was generated from the combined results of the demi-embryos and biopsy samples, rather than tissue from corresponding foals, as done previously (Choi et al. 2010). Thus, the reference standard in the present studies had a small chance of being in itself inaccurate. The error rate of the reference standard can be estimated by multiplying the error rate in each embryo or demi-embryo with that for each of the three biopsy samples; this was highest for the samples evaluated by Illustra Genomiphi V2 (Experiment 1), due to both low accordance and failure of biopsies and demi-embryos to give adequate signal, but approached 0 in the samples evaluated by Qiagen Repli-g Midi (e.g. (0.018)2 (0.006)3 ¼ 7.0 1011 for VIV embryos in Experiment 2). We found that biopsy samples obtained from IVP and VIV blastocysts yielded equivalent high accuracy (.99% of interrogated loci) on genetic analysis. Interestingly, the accuracy of analysis of biopsy samples was equivalent to that for analysis of complete demi-embryos (98 to 99%), although the latter have hundreds to thousands more cells. Notably, three of the 81 biopsy samples obtained (4%) were not useable for analysis, as they returned ,50% accuracy for interrogated loci; this appears to represent the main potential source for error on genetic analysis of equine embryo biopsy samples obtained using the methods described herein. One of these non-performing biopsy samples had no signal at any locus, thus the sample itself appears to have been lost. The other two biopsy samples returned a combination of no signal, error and allele drop-out results, suggesting low fidelity of WGA, perhaps due to very low cell number. The findings at microsatellite markers in these embryos were detailed in the Results, as they suggest that evaluation of identification markers may be useful in determining the quality of the biopsy sample. The two non-performing biopsies provided heterozygous signals at only none and one of these commonly-heterozygous identification loci. In contrast, in the reference standards for the 27 blastocysts used in the study, the mean number of heterozygous identification loci (out of 15) was 11.7 0.3, ranging from nine to 14. This finding suggests that evaluation of biopsy samples at microsatellite identification markers, even if the actual genotype of the embryo is unknown,
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may provide an indication of the overall quality of a sample. This will help to validate the accuracy of homozygous findings on analysis of genes of interest. A second approach to reduce the possibility of non-performing sample error would be to take two biopsy samples from each embryo and analyse them separately. This approach, however, would double the expense of the genetic analysis and the effect of obtaining two biopsy samples on future embryo viability has not been studied. Until this approach has been validated, accuracy of the biopsy procedure should be considered to be subject to the quality of the biopsy obtained; in our hands this was ,95% overall. It is important to assess allele drop-out rates on interrogation of heterozygous loci, as these loci are more challenging than are homozygous loci in relation to accuracy of results. In our study, allele drop-out rates for performing biopsies were below 3% for both IVP and VIV embryos. When we looked specifically at loci that may be of special interest to horse owners, i.e. those for sex determination and presence of disease-associated mutations, sex was accurately detected in 78/78 biopsy samples using either AME or SRY. The accuracy of detection of the heterozygous state for disease-related mutations was also high (97.7%). Not all disease-causing mutations nor all coat-colour mutations could be formally evaluated in this study. Among these, glycogen-branching enzyme disorder (GBED) is potentially the most problematic important mutation, because of the rich GCsequence surrounding the mutation, and would merit validation for use in PGD testing in a future study. In conclusion, results obtained using Piezo-driven embryo biopsy and WGA followed by multiplex PCR can have high (,95%) accuracy for determination of embryonic sex and parentage and for genotyping at disease-associated and coatcolour loci. There was no difference in accuracy of biopsy for genetic diagnosis between IVP and VIV blastocysts. This technique appears to be efficient enough for use in a clinical setting. Acknowledgements This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University, the Veterinary Genetics Laboratory at UC Davis and by Ms. Kit Knotts.
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