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IVF often requires embryo cryopreservation through vitrification. During the vitrification process, the embryos can be collapsed by withdrawing the blastocoele fluid. The metabolomic profile of blastocoele fluid has been recently investigated by high-performance liquid chromatography-electrospray ionization-mass spectrometry to provide metabolite information that can help estimations of implantation efficiency. However, the presence of embryo DNA in blastocoele fluid has not been reported to date. This study shows using real-time PCR that genomic DNA was present in about 90% of blastocoele fluid samples harvested during the vitrification procedure. Moreover, the potential for determining embryo sex directly from blastocoele fluid is demonstrated by amplifying the multicopy genes TSPY1 (on the Y chromosome) and TBC1D3 (on chromosome 17). This opens up the possibility of screening embryos from couples carrying an X-linked disorder to identify male embryos at high risk of disease. The application of whole-genome amplification technologies to fluid samples is also shown to be feasible, potentially allowing more comprehensive genetic tests. As proof of principle, microarray comparative genomic hybridization was attempted to confirm the sex of embryos as well as detect several aneuploidies. However, further studies are needed to validate this approach and confirm that the accuracy is sufficient for diagnostic purposes.
IVF offers personal approaches to couples according to their specific infertility problems. IVF includes a preimplantation genetic diagnosis, single-embryo transfer, blastocyst transfer and optimization of uterine receptivity. These procedures require a valid embryo cryopreservation system. Vitrification is a cryopreservation method that offers superior survival and pregnancy rates performing an artificial shrinkage of a fully expanded blastocyst. This procedure can be performed with different methods, including laser-pulse, repeated micropipetting, microneedle puncture or microsuction, and there is a significant improvement in survival rates when blastocysts were collapsed. With microsuction, the blastocoele fluid is withdrawn and (typically) discarded. The aim of this study was to investigate the presence of DNA in blastocoele fluid. Recently, the metabolomic profile of blastocoele fluid was characterized, but to date, no studies have reported evidence of DNA. Here, using a real-time PCR-based approach, we showed that genomic DNA was amplifiable in about 90% blastocoele fluid samples analysed. Moreover, we demonstrated the potential for monitoring sex-linked diseases directly from blastocoele fluid, by amplifying the multicopy genes TSPY1 (on the Y chromosome) and TBC1D3 (on chromosome 17). The application of whole-genome amplification technologies to fluid samples has also shown to be feasible, potentially allowing more comprehensive genetic tests (i.e. microarray comparative genomic hybridization). However, further studies are needed to validate this approach for reliable diagnostic purposes.
IVF offers personal approaches to couples according to their specific infertility problems, allowing preimplantation genetic diagnosis (PGD), single-embryo transfer, blastocyst transfer and optimization of uterine receptivity.
PGD is conducted for couples at risk for specific inherited disorders. After the first report of PGD by
). PGD requires a biopsy of one or more cells from the human embryo; therefore, it is important to monitor the use and long-term safety of this technique. In cases of rescue PGD, day-5 PGD or other conditions (e.g. poor endometrial receptivity or ovarian hyperstimulation syndrome), the transfer can be delayed and embryos may be cryopreserved with vitrification. The vitrification process may also provide higher pregnancy and implantation rates compared with fresh blastocyst transfer cycles (
Vitrified-warmed blastocyst transfer cycles yield higher pregnancy and implantation rates compared with fresh blastocyst transfer cycles – time for a new embryo transfer strategy?.
). Therefore, this procedure is widely applied in IVF centres around the world. Vitrification is preceded by artificial shrinkage of a fully expanded blastocyst. The collapse can be performed with different methods, including laser-pulse, repeated micropipetting, microneedle puncture or microsuction.
Microsuction of blastocoelic fluid before vitrification increased survival and pregnancy of mouse expanded blastocysts, but pretreatment with the cytoskeletal stabilizer did not increase blastocyst survival.
reported significant improvement in survival rates when blastocysts were treated with blastocoelic fluid microsuction prior to vitrification. In that procedure, the blastocoele fluid (0.3–0.5 nl) was withdrawn and (typically) discarded.
The aim of this study was to investigate the presence of embryo DNA in the blastocoele fluid and evaluate its potential clinical use. Recently, the metabolomic profile of this fluid was characterized with high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS;
) with the purpose to provide metabolite information, in addition to morphology scores, to support estimations of implantation efficiency. However, the presence of embryo DNA in blastocoele fluid has not been reported to date.
This study therefore investigated, using a real-time PCR-based approach, the presence of genomic DNA in blastocoele fluid harvested during the vitrification process and its potential use in PGD applications (i.e. monitoring sex-linked diseases) by amplifying the multicopy genes TSPY1 (on the Y chromosome) and TBC1D3 (on chromosome 17). Moreover, whole-genome amplification (WGA) technology was also attempted and microarray comparative genomic hybridization (CGH) was performed to confirm the reliability of this approach.
Materials and methods
Blastocoele fluid collection
Blastocoele fluid was collected at the Cervesi Hospital (Cattolica, Italy), upon approval of the local Ethics Committee. The methods for blastocyst micropuncture and aspiration of blastocoele fluid have been described previously (
). Air particulate and microbiological checks were routinely performed in the IVF unit in the room and the hoods used for embryo culture, always conforming to national regulations concerning maintenance of sterility. The operators during the recovery and storage of fluid samples wore gloves, masks, caps and lab coats. Each culture dish was prepared in a sterile hood by overlaying medium droplets with paraffin oil (Ovoil; Vitrolife) and the dishes, the tips and the pipette for fluid aspiration were changed for every sample. The retrieved fluid (0.3–0.5 nl) was expelled into 4 μl of 1 mmol/l Tris–HCl and 0.1 mmol/l EDTA (pH 8.0) and stored at −20°C.
Target genes and primer design
Primers for amplifying GAPDH were described elsewhere (
). Primers for amplifying TSPY1 (NG_027958) and TBC1D3 (NW_003315949) were designed with Primer Express software (Applied Biosystems) to generate amplicons of 60 bp and 66 bp, respectively. The primer sequences were as follows: TSPY1_F 5′-TGTAAGTGACCGATGGGCAG-3′ and TSPY1_R 5′-AACTCCCCTTTGTTCCCCAA-3′; and TBC1D3_F 5′-GGGCAAGAGGTCATCTGAGC-3′ and TBC1D3_R 5′-TGCTTCCTTAATGTCCCGCT-3′. The sequence specificity was confirmed in silico with a blast search (http://blast.ncbi.nlm.nih.gov/) against the human genomic plus transcript database, and in vitro by amplifying human genomic DNA. Primers were purchased from Sigma–Aldrich.
Real-time PCR assays
Real-time PCR was performed in a final volume of 20 μl with the SYBR Green PCR master mix (Diatheva, Italy) with 200 nmol/l primers in a RotorGene 6000 instrument (Corbett Life Science, Australia). The amplification conditions for GAPDH were 95°C for 10 min and 45 cycles of 95°C for 10 s and 60°C for 1 min. The amplification conditions for TSPY1 and TBC1D3 were 95°C for 10 min and 40 cycles of 95°C for 10 s and 60°C for 35 s. For GAPDH amplification, the entire volume of the blastocoele fluid sample (∼4 μl) was added to each reaction tube as the source of template DNA. For TSPY1 and TBC1D3 amplification, approximately half of the blastocoele fluid volume (∼2 μl) was used. A non-template control was included in each PCR run. Moreover, negative controls consisting of TE buffer, empty culture medium and medium droplets covered by paraffin oil were also tested for TBC1D3 amplification to exclude contamination during the fluid withdrawal process. Human genomic DNA (1 ng) or human cDNA (equivalent to 25 ng total RNA) were used as positive amplification controls. At the end of each run, a melting curve analysis was performed from 60°C to 95°C to monitor primer dimers or non-specific products. Moreover, PCR products were analysed on a 4% (w/v) MetaPhor agarose gel (Cambrex, USA) stained with GelRed (Sichim, Italy). Serial 10-fold dilutions (5–0.0005 ng/reaction tube) of reference genomic DNA were prepared to evaluate PCR efficiency and sensitivity. Each dilution was amplified in triplicate.
Whole-genome amplification of blastocoele fluid
Whole-genome amplification (WGA) of blastocoele fluid was performed on five samples using the REPLI-g Mini Kit (Qiagen) according to the manufacturer’s instructions with slight modifications. Briefly, 2.5 μl of each sample were incubated in a total volume of 25 μl at 30°C for 16 h and heated to 65°C for 3 min to inactivate the DNA polymerase. The amplified DNA (referred to hereafter as WGA DNA) was visualized on 0.8% (w/v) agarose gel and its quantification was performed using a Qubit fluorometer (Life Technologies). As negative control, 2.5 μl of Milli-Q water were used in WGA reaction. In order to test the WGA outcome, the WGA DNA was diluted 1:100 to be used as a template in PCR reactions targeting genes on two different chromosomes (i.e. MAP1LC3B and TBC1D3, on chromosome 16 and 17, respectively).
Microarray comparative genomic hybridization
Microarray CGH analysis involved the use of 24Sure Cytochip microarrays (BlueGnome, Cambridge, UK) according to the method described in
. Briefly, WGA products were fluorescently labelled (Fluorescent Labelling System; BlueGnome) and then mixed with a reference DNA (46,XY or 46,XX) that had been labelled with a fluorochrome of a different colour. The labelled DNA was denatured and then applied to a microarray. After hybridization overnight, microarrays were washed and scanned (InnoScan 710; Innopsys, Carbonne, France) and the data were analysed with BlueFuse software (BlueGnome). This method allowed determination of embryo sex (X and Y chromosome copy number) and detection of aneuploidy.
Statistical analysis
Statistical analysis was performed with GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA).
Ethical approval
Approval of the study was obtained on 28 July 2011 from the local ethical committee (‘Analisi Blastocele procreatica in vitro Umana’, CEAV and IRST, no. 2945/2011/I.5/126).
Results
Blastocoele fluid contains PCR-amplifiable DNA
To detect the presence of genomic DNA and its ability to be amplified in blastocoele fluid, this study initially targeted the single-copy gene, GAPDH (
Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions.
), for real-time PCR amplification. This produced 183- or 91-bp products from genomic DNA or cDNA, respectively. This study also tested for potential PCR inhibitors in diluted blastocoele fluid samples by amplifying GAPDH cDNA with or without 4 μl blastocoele fluid. The resulting Ct values were similar (27.19 and 27.15), indicating the absence of PCR inhibitors. Next, the presence of DNA in blastocoele fluid was investigated by amplifying the endogenous GAPDH target sequence in 16 blastocoele fluid samples. Only nine samples showed an amplified product (Table 1). A melting curve analysis revealed that, of the nine PCR products, six had an average Tm of 91.15 ± 0.12°C and the other three had an average Tm of 83.27 ± 0.25°C. Gel electrophoresis confirmed the presence of two amplicons; the one with the higher Tm was the expected 183-bp product and the one with the lower Tm corresponded to a product with electrophoretic mobility below 100 bp (data not shown). A sequence similarity search indicated that the small PCR product was derived from non-specific amplification of the GAPDH pseudogene (GAPDHP1). In fact, the forward primer had only two mismatches at the 5′-end and the reverse primer perfectly matched the GAPDHP1 sequence (Supplementary Figure 1, available online). The calculated amplicon size was 91 bp. Therefore, it is possible that this small, non-specific PCR product was amplified in the absence (or in the presence of low amounts) of DNA fragments that encompassed the 183-bp length of the specific amplicon. Contamination with the cDNA used as the template in the positive controls (also 91 bp) was excluded, because the Tm were significantly different (P < 0.01; control 84.67 ± 0.15°C versus small amplicon 83.27 ± 0.25°C). This difference could be explained by the presence of eight mismatches in the sequences of the GAPDH cDNA and the GAPDHP1 pseudogene (Supplementary Figure 1). Nonetheless, these results indicate that nine out of 16 (56%) blastocoele fluid samples contained PCR-amplifiable DNA and that, in at least three samples, this DNA was fragmented below 183 bp.
Table 1Results of GAPDH amplification in blastocoele fluid.
The 91-bp products amplified from blastocoele fluid and from human cDNA (positive control) had average Tm values of 83.27 ± 0.25°C and 84.67 ± 0.15°C, respectively.
To improve the sensitivity of the assay, a new PCR assay was designed for targeting the multicopy gene TBC1D3, located on chromosome 17. This gene is moderately repeated in the human genome: approximately five to 53 copies are present in humans (
). Moreover, to facilitate amplification despite the possible DNA fragmentation, the target amplicon was 66 bp long. MetaPhor agarose gel analysis of the PCR products allowed the resolution of 66-bp amplicons (Supplementary Figure 2A). A total of 31 blastocoele fluid samples were tested. Furthermore, blastocyst classification scores (
) were available for these samples. Out of the 31 samples, 26 showed TBC1D3 amplicons with an average Tm of 85.3 ± 0.2°C (Table 2). Sample ED1 was accidentally overdiluted, and sample LE1 was collected from an abnormal blastocyst that lacked an inner cell mass. After excluding these two samples from the analysis, the detection rate was 89.7% (26/29). Absence of specific amplicons from negative controls (no template control, TE buffer, empty medium as it is or covered by paraffin oil) excluded DNA environmental contamination of tested samples.
Table 2Results from TBC1D3 and TSPY1 amplifications in blastocoele fluids.
DNA in blastocoele fluid could be used to detect male embryos in families carrying X-linked disorders
This study designed a PCR assay that targeted TSPY1 on the Y chromosome to be used together with the TBC1D3 PCR assay as proof-of-concept that PGD could be performed directly using blastocoele fluid. TSPY1 is present in approximately 35 (range 20–76) copies per human genome (
) and the amplicon was 60-bp long (Supplementary Figure 2B). To assess the PCR efficiency and sensitivity for TBC1D3 and TSPY1 amplification, 10-fold serial dilutions of genomic DNA were tested. The calibration curves and equations are shown in Figure 1. The PCR amplification efficiencies were 95% and 98% for TBC1D3 and TSPY1, respectively. The PCR sensitivity was defined as the lowest amount of template that consistently gave a specific amplicon in triplicate samples. The PCR sensitivity was 0.005 ng genomic DNA for both targets. Both TSPY1 and TBC1D3 are multicopy genes, with some variability among individuals; thus, the sensitivity might change with different genomic DNA samples.
Figure 1Calibration curves generated for estimating gene content. TBC1D3 (a) and TSPY1 (b) target sequences were amplified using 10-fold dilutions (5–0.005 ng/reaction tube) of human male genomic DNA. Each dilution was amplified in triplicate. The curve equations and R2 values are presented on each panel. The calculated amplification efficiencies were 95% and 98% for TBC1D3 and TSPY1, respectively.
TBC1D3 and TSPY1 had comparable copy numbers, PCR efficiencies and PCR sensitivities. Therefore, each blastocoele fluid sample was diluted and divided into two PCR tubes for amplification of the TBC1D3 and TSPY1 targets. TBC1D3 was considered a positive control because it is present in male and female genomes. Thus, samples were considered male or female according to whether two PCR products or only the TBC1D3 product were detected, respectively. Among the 26 samples that showed the TBC1D3 product, 17 samples also showed amplification of TSPY1, with an average Tm of 84.5 ± 0.1°C (Table 2).
Due to the gender imbalance (65.4% males and 34.6% females), the PCR results were double-checked in a subset of six samples (NO1, AL1, FA1, GR1, GR3, GR4) by amplifying blastocyst cells that were lost during the vitrification procedure or by amplifying embryo culture medium; in fact, a recent report showed that 18S rDNA could be detected in embryo culture media (
). Using these samples as a source of DNA, the results obtained with blastocoele fluid were confirmed (data not shown).
Estimation of DNA amount in the blastocoele fluid
The amount of DNA in the blastocoele fluid was estimated with RotorGene 6000 software, where the positive control for each run was the reference for calibration curve adjustments. Based on the TBC1D3 calibration curve, this study roughly estimated that 0.8–55 pg (median 7.3 pg) of genomic DNA were present in the blastocoele fluid samples. A similar range (3–85 pg; median 12.2 pg) was obtained with the TSPY1 calibration curve (Supplementary Table 1). Based on an unpaired t-test with the Welch correction, the mean values from the two calibration curves did not differ significantly. The combined results showed a total median value of 9.9 pg of genomic DNA per sample.
Whole-genome amplification of blastocoele fluid
Five blastocoele fluid samples were subjected to WGA. To confirm the WGA success, DNA was quantified and tested for amplification of two autosomal targets (MAP1LC3B and TBC1D3 on chromosomes 16 and 17, respectively). Moreover, the samples showing positive amplification were also tested for TSPY1 amplification. The WGA procedure was successful in four samples; two samples (FOS2 and VAN5) also showed amplification of TSPY1 target (Table 3). The WGA DNA quantification showed similar results for all samples, including sample VAN2 and the negative control (Table 3). This was probably due to random extension of primer dimers, a well-known artefact of WGA using multiple displacement amplification, since the PCR results were negative, indicating an absence of human DNA.
Table 3DNA quantification, PCR results and microarray CGH results of blastocoele fluid samples subjected to WGA.
To investigate whether WGA products were a suitable template for subsequent comprehensive chromosome analysis, amplified samples from FOS2 and VAN5 were labelled and tested using a well-established microarray CGH approach. Chromosome screening was accomplished in both cases, producing data that suggested that both embryos were male (i.e. a normal number of copies of the X and Y chromosomes detected). These results were concordant with data from the same two WGA samples obtained using PCR amplification of TSPY1 on the Y chromosome. Additionally, microarray CGH results indicated that both DNA samples were aneuploid. The predicted karyotype was 47,XY,+22 (male with trisomy 22) in one case and 46,XY,−1,−10,+11,+16 (male with complex aneuploidy) in the other (Table 3).
Discussion
This study confirmed that amplifiable DNA fragments were present in about 90% of blastocoele fluid samples harvested during the vitrification process. Moreover, it demonstrated the possibility of determining embryo sex directly from blastocoele fluid, which is of potential value for the avoidance of male embryos at high-risk of inheriting sex-linked disorders. Embryo sex was revealed by amplifying the multicopy genes TSPY1 (on the Y chromosome) and TBC1D3 (on chromosome 17). The median genomic DNA content in blastocoele fluid was estimated to be 9.9 pg per sample. However, since TBC1D3 and TSPY1 copy numbers can vary among individuals and robust quantifications of contents less than 5 pg were not feasible, a reliable quantification was not possible.
The TSPY1 amplicon was revealed in 17 out of 26 samples, showing an excess of male embryos. This gender imbalance may be due to: (i) the small number of samples processed; or (ii) the fact that gender can be associated with blastocyst grading, male embryos developing at a significantly faster rate than female embryos (
Previous reports showed that normally fertilized blastocysts on day 5 had a total of 58.3 ± 8.1 cells (trophectoderm 37.9 ± 6.0 cells; inner cell mass 20.4 ± 4.0 cells) and that cell death, shown by fragmented nuclei, occurred in both the trophectoderm and the inner cell mass (
). This cell death could result in release of fragmented DNA, which could then be detected in the blastocoele cavity. It is reasonable to exclude a DNA contribution derived from damaged cells during the process of blastocoele fluid aspiration, since microsuction is carefully performed under high magnification and the blastocyst is constantly monitored during this procedure (Figure 2).
Figure 2Blastocoele fluid aspiration. Expanded day-5 blastocysts were removed from culture and transferred to a 25 μl droplet of blastocyst medium under a paraffin oil underlay. The blastocysts were immobilized by a holding pipette connected to an oil-filled syringe and mounted on a micromanipulator. A finely pulled, oil-filled pipette was introduced through the mural trophectoderm to avoid damaging the inner cell mass and blastocoele fluid was aspirated gently until the blastocyst had fully collapsed around the pipette.
As far as is known, this is the first report of the presence of amplifiable DNA in blastocoele fluid. Although results were obtained from the majority of samples, the diagnosis rates using blastocoele fluid were lower compared with the current techniques involving the biopsy of one or more cells (which have an accuracy around 98–99%;
). Moreover, the need to target multicopy genes (an appropriate strategy for embryo gender determination in families known to be at risk for an X-linked disease, but unsuitable for diagnosis of most other inherited disorders) could limit the usefulness of this approach.
In an effort to enrich the sample obtained from blastocoele fluid and to assess whether DNA from all chromosomal regions was present, five additional samples were subjected to WGA. The DNA from four samples was successfully amplified as revealed by PCR results for loci on chromosomes 16 and 17, showing an 80% success rate (Table 3). Two of the WGA DNA samples were also tested using microarray CGH. This yielded data of sufficient quality to allow gender determination and the detection of apparent aneuploidies (Table 3). The microarray CGH results demonstrated the presence of a Y chromosome in both samples, concordant with data previously obtained using the PCR assay and showed several chromosome abnormalities, indicating also that WGA DNA was from embryonic origin. The fact that microarray CGH was successful may suggest that much of the DNA in the blastocoele is still reasonably intact. Furthermore, hybridization was seen across all chromosomal regions. At the resolution of the microarray (∼5 Mb) there were no obvious missing parts of the genome, indicating that the amount of DNA present is likely to be equivalent to at least one cell, if not several cells. These experiments should be considered as a simple proof-of-principle and not a definitive indication of accuracy. The proportion of blastocoele samples that will yield a microarray CGH result and the accuracy of such data remains unknown at this time and will require a carefully controlled analysis of a much larger number of samples.
The data presented in this manuscript indicate that amplifiable DNA exists in the blastocoele fluid. The suitability of this DNA for PCR, WGA and microarray CGH is demonstrated. However, its usefulness for diagnostic purposes still needs to be proven. In fact, further studies are required to validate this approach and to evaluate its diagnosis rate, with respect to current techniques involving embryo biopsy. If this approach turns out to be feasible, potential risks associated with embryo biopsy (
) could be reduced. Furthermore, genetic information of relevance to embryo viability or health could be obtained without disruptions to the workflow of a laboratory pipeline that includes the vitrification process.
Acknowledgements
The authors wish to thank Mariangela Primiterra, Dora Prota, Valentina Ditroilo and Anna Grottoli for technical help. Funds used to support the authors throughout the study period and manuscript preparation were from the Department of Biomolecular Sciences of University of Urbino and the IVF unit of Cervesi Hospital. Dagan Wells is funded by the Oxford National Institute of Heath Research Biomedical Research Centre.
Microsuction of blastocoelic fluid before vitrification increased survival and pregnancy of mouse expanded blastocysts, but pretreatment with the cytoskeletal stabilizer did not increase blastocyst survival.
Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions.
Vitrified-warmed blastocyst transfer cycles yield higher pregnancy and implantation rates compared with fresh blastocyst transfer cycles – time for a new embryo transfer strategy?.
Declaration: The authors report no financial or commercial conflicts of interest.
Footnotes
Simone Palini was the laboratory director of the IVF unit at ‘Cervesi’ hospital in Cattolica until 2004. He obtained a degree in Biology at Urbino University (1998) and a speciality in molecular genetics at Bologna University (2000) with a thesis on PGD. In 2008 he obtained ESHRE certification as senior clinical embryologist. He has worked in different public and private laboratories in Italy, Europe and the UK. His major interests are embryo/gamete vitrification, PGD and gene expression profile in infertility. Simone has reported as a speaker at numerous international conferences and Master degree programmes.
Simone Palini was the laboratory director of the IVF unit at ‘Cervesi’ hospital in Cattolica until 2004. He obtained a degree in Biology at Urbino University (1998) and a speciality in molecular genetics at Bologna University (2000) with a thesis on PGD. In 2008 he obtained ESHRE certification as senior clinical embryologist. He has worked in different public and private laboratories in Italy, Europe and the UK. His major interests are embryo/gamete vitrification, PGD and gene expression profile in infertility. Simone has reported as a speaker at numerous international conferences and Master degree programmes.
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