[Show abstract][Hide abstract] ABSTRACT: Study question: To evaluate the capability of next-generation sequencing (NGS) to detect pure and mosaic segmental aneuploidies in trophectoderm bi- opsies and the concordance rate with results from the current platform of array comparative genomic hybridization (aCGH).
Summary answer: NGS allows the detection of pure segmental aneuploidies in human blastocyst with the same efficiency as aCGH. NGS platform software could be trained to establish new thresholds for the detection of segmental an- euploidies in mosaic blastocysts.
What is known already: Comprehensive chromosomal screening (CCS) has become a must in every fertility center around the world. Nowadays, aCGH is the most used method for this purpose. Recently, NGS has emerged as a promis- ing platform for the aneuploidy detection in the human embryo.
Study design, size, duration: Amplified DNA from trophectoderm biopsies in which segmental aneuploidies (Range: 12.4-187.8Mb) were detected by aCGH in a CCS cycle were selected. A total of 50 segmental aneuploidies were reana- lyzed by NGS. In addition, blastocyst containing 17 segmental events were disas- sembled into single cells and analyzed by fluorescent in situ hybridization (FISH). Participants/materials, setting, methods: Samples from each embryo under- went whole genome amplification. For aCGH, DNA was labeled, co-hybridized in 24 sure arrays and analyzed by BlueFuse Multi software. For NGS, a library was generated from dsDNA and loaded into a MiSeq instrument. Finally, FISH analysis was performed by using telomeric probes for affected chromosomes. Main results and the role of chance: Segmental aneuploidies were classified into pure and mosaic according to log2 ratio values in the aCGH experiments. In pure segmentals a concordance rate of 97.1% (34/35) was found with NGS. In the mosaic ones the concordance rate was 80% (12/15). FISH validation for pure segmentals in disassembled blastocysts confirmed the results in 8 out of 10 cases (39.0 ± 18.7 analyzed cells per blastocyst). FISH was performed for 7 mosaic segmentals, showing a mosaic pattern in 4 of them (54.2 ± 34.5 cells), with an average of 39.7 ± 22.5% aneuploid cells per blastocyst.
In total, only 4 discrepancies out of 50 were observed between aCGH and NGS. FISH analysis was performed in 2 of them resulting in a concordance with aCGH in one of them, and with NGS in the other.
Limitations, reason for caution: This study was limited by the sample size. Beyond that, extremely low mosaicism levels in the blastocyst could have not been detected. Finally, we assumed that mosaicism degree in biopsied cells was the same that in whole embryo, but no necessarily.
Wider implications of the findings: Studies like this are essential for the de- velopment of appropriate software that allows the efficient translation of NGS into the CCS programs.
Study funding/competing interest(s): Funding by commercial/corporate company(ies), IVIOMICS S.L., ILLUMINA Inc.
Trial registration number: N/A.
30th Annual Meeting of ESHRE, Munich, Germany; 06/2014
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS) using array comparative genomic hybridization (aCGH). The study included 1420 CCS cycles for recurrent miscarriage (n = 203); repetitive implantation failure (n = 188); severe male factor (n = 116); previous trisomic pregnancy (n = 33); and advanced maternal age (n = 880). CCS was performed in cycles with fresh oocytes and embryos (n = 774); mixed cycles with fresh and vitrified oocytes (n = 320); mixed cycles with fresh and vitrified day-2 embryos (n = 235); and mixed cycles with fresh and vitrified day-3 embryos (n = 91). Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5-54.2%) and pregnancy rates per transfer (range: 46.0-62.9%) were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1%) due to the higher percentage of aneuploid embryos (85.3%) and lower number of cycles with at least one euploid embryo available per transfer (40.3%). We concluded that aneuploidy is one of the major factors which affect embryo implantation.
BioMed Research International 01/2014; 2014:517125. · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: To review clinical outcomes after preimplantation genetic screening. Most methods of embryo viability assessment involve morphologic evaluation at different preimplantation developmental stages. A weak association between blastocyst morphology and aneuploidy has been described, supporting the basis for preimplantation genetic screening (PGS) for assessment of embryo viability. The expected improvement in reproductive outcome rates has been reached with the application of microarrays based on comparative genomic hybridization (CGH) in clinical routine PGS. DESIGN: Review of published studies and own unpublished data. SETTING: University-affiliated private institution. PATIENT(S): IVF patients undergoing PGS at different stages. INTERVENTION(S): PGS with polar body, cleavage-stage, and blastocyst biopsies. MAIN OUTCOME MEASURE(S): Aneuploidy, implantation, and pregnancy rates. RESULTS: The clinical outcome after analysis of all 24 chromosomes improved pregnancy and implantation rates for different indications to a higher degree than the previously available technology, fluorescence in situ hybridization (FISH), in which only a limited number of chromosomes could be analyzed. CONCLUSION(S): Most of the data regarding the controversy of day-3 biopsy come from FISH cycles, and the utility of day-3 biopsy with new array-CGH technology should be further evaluated through randomized controlled trials. The current trend is blastocyst biopsy with a fresh transfer or vitrification for transfer in a nonstimulated cycle.
Fertility and sterility 02/2013; · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this work, false positive rate of an arrayCGH platform for its use in day-3 single-blastomere analysis was calculated. For this purpose, 38 embryos diagnosed as abnormal on day-3 by FISH were re-biopsied on day-4. Single-cell day-4 arrayCGH diagnosis was then performed. A successful amplification was obtained in 97.4 % (37/38) of the day-4 cells analysed by arrayCGH. Day-3 FISH and day-4 arrayCGH diagnosis were concordant in 35/37 cases. The two discordant embryos were spread and all the cells from each embryo were re-analysed by FISH on day 5. The same error rate (2.7 %) for day-3 FISH and day-4 arrayCGH was obtained when comparing day-5 FISH re-analysis. After this pre-clinical phase, the platform was used for day-3 arrayCGH clinical application in 320 patients (1,760 embryos). Day-3 amplification rate was 98.6 %. An optimal reproductive outcome was obtained when applying arrayCGH to a clinical program: clinical pregnancy rate per cycle of 38.4 % and 60.3 % per transference were obtained, with an implantation rate of 53.5 %. Overall miscarriage rate was 10.6 %. Additionally, day-5 FISH re-analysis was performed in 42 of the embryos from the clinical phase, obtaining a concordance rate of 97.6 % with day-3 arrayCGH.
Journal of Assisted Reproduction and Genetics 12/2012; · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In patients with Y chromosome microdeletions and high percentage of numeric chromosome abnormalities detected by fluorescence in situ hybridization on sperm, a high percentage of abnormal embryos was observed compared with oligozoospermic patients without Y chromosome microdeletions, with a significant increase in the percentage of embryos with monosomy X. Differences in fertilization rates between the different patient groups were not observed; however, blastocyst rates were significantly impaired in patients with Y chromosome microdeletions.
Fertility and sterility 12/2010; 94(7):2874-7. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this retrospective study, the utility of preimplantation genetic screening (PGS) in patients with advanced maternal age is evaluated. The patient population consisted of women aged 38-44years and included in a regular IVF programme with or without PGS analysis. Transfer rate, ongoing implantation rate and ongoing pregnancy rate were the main outcome parameters measured. A trend of better ongoing pregnancy rate per oocyte retrieval was observed in patients aged 38 and 39years in the non-PGS group when compared with PGS groups, but better ongoing pregnancy rate per oocyte retrieval was observed in patients 41-44years old in the PGS group. When patients with a low ovarian response accumulated oocytes in several stimulation cycles, clinical outcomes were comparable to those of normal-responder patients. These results show that, although PGS does not benefit patients less than 40years of age, reproductive success increases more than two-fold in patients over 40years, especially in patients with more than six metaphase II oocytes, as a result of a good ovarian response or gamete accumulation, suggesting a redefinition of advanced maternal age as indication for PGS. In this retrospective study, the utility of preimplantation genetic screening (PGS) in patients with advanced maternal age is evaluated. Patient population consisted of women aged 38-44 years and included in a regular IVF programme with or without PGS analysis. Transfer rate, ongoing implantation rate and ongoing pregnancy rate were the main outcome parameters measured. A trend of better ongoing pregnancy rate per ovarian stimulation cycle was observed in patients aged 38-39 years in the non-PGS group when compared with PGS groups, but better ongoing implantation rate was observed in patients aged 41-44 years old in the PGS group. When patients with a low ovarian response (low number of oocytes available for the IVF cycle) accumulated oocytes in several stimulation cycles, their reproductive possibilities were comparable to those of normal-responder patients. These results show that, although PGS does not benefit patients less than 40 years of age, reproductive success increases more than 2-fold in patients over 40 years, especially in patients with more than six metaphase II oocytes, as a result of a good ovarian response or gamete accumulation, suggesting a redefinition of advanced maternal age as indication for PGS.
[Show abstract][Hide abstract] ABSTRACT: In our routine programme of preimplantation genetic aneuploidy screening (PGS) by fluorescence in situ hybridization (FISH), nine chromosomes (13, 15, 16, 17, 18, 21, 22, X and Y) are analysed in two consecutive hybridization rounds. We also perform additional hybridization rounds for these chromosomes, using probes that bind to different loci, for non-conclusive results and for confirmation of certain aneuploidies. The aim of this study was to evaluate the impact of additional hybridization rounds on FISH accuracy.
This is a retrospective analysis of our FISH data from 1000 PGS cycles performed from December 2007 to December 2008 for various indications. In addition to the hybridization rounds described above, 132 of the embryos diagnosed as chromosomally abnormal were re-analysed on Day 5.
A total of 2477 embryos were re-hybridized, 1496 due to non-conclusive results and 981 to confirm observed aneuploidies. After re-hybridization, 882 embryos (59%) were then diagnosed as normal, 600 embryos (40.1%) had a clear abnormality and only 14 embryos (0.9%) remained non-informative. From the 981 embryos in the latter group, 890 embryos had monosomies and, after re-hybridization 174 embryos (19.6%) were normal and 716 (80.5%) had confirmed monosomies. In contrast, re-hybridization confirmed 90 (98.9%) of the 91 observed trisomies. In addition, Day-5 re-analysis of abnormal embryos showed a higher rate of concordant diagnosis between Day 3 and Day 5 when re-hybridizations had been included on Day-3 (95 versus 82.7%; P= 0.0443), especially for the confirmation of monosomies (82.8 versus 61.0%; P = 0.0087).
Our data indicate that additional hybridization rounds improve the accuracy of the diagnosis, increasing the number of chromosomally normal embryos available for transfer. Re-hybridization with additional probes as a standard approach to PGS could enhance the potential benefits of the technique.
Human Reproduction 07/2010; 25(7):1812-7. · 4.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the effect of sperm chromosome abnormalities--disomy for sex chromosomes and diploidy--in the chromosomal constitution of preimplantation embryos.
Retrospective cohort study.
Three groups: 46,XY infertile men with increased incidence of sex chromosome disomy in sperm; 46,XY infertile men with increased diploidy rates in sperm; 47,XYY infertile men with increased sex chromosome disomy and diploidy rates in sperm.
Sperm collection for fluorescence in situ hybridization analysis. Embryo biopsy for preimplantation genetic screening.
Frequencies of numerical abnormalities in sperm for chromosomes 13, 18, 21, X, and Y, and in embryos for chromosomes 13, 16, 18, 21, 22, X, and Y.
A significant increase of chromosomally abnormal and mosaic embryos was observed in the three study groups compared with controls. Those sperm samples with increased sex chromosome disomy rates produced significantly higher percentages of aneuploid embryos, with a threefold increase for sex chromosomes. Sperm samples with increased diploidy rates were mainly associated to the production of triploid embryos.
A strong correlation between sperm and embryo chromosomal constitution has been shown in infertile men with 46,XY and 47,XYY karyotypes.
Fertility and sterility 08/2009; 94(4):1380-6. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to identify specific subgroups of recurrent pregnancy loss (RPL) patients of unknown aetiology in whom the selection of chromosomally normal embryos for transfer improves reproductive outcome in preimplantation genetic screening (PGS). A total of 428 PGS cycles were included and chromosomes 13, 15, 16, 18, 21, 22, X and Y were evaluated. In RPL patients < or =37 years, a lower incidence of chromosomal abnormalities (P = 0.0004) and miscarriages (P = 0.0283) was observed, and there were significantly higher pregnancy (P < 0.0384) and implantation (P < 0.0434) rates than in patients >37 years. In the former subset, results showed: (i) significantly higher implantation rates (P = 0.0411) in couples that had experienced a previous aneuploid miscarriage; (ii) similar aneuploidy, pregnancy and implantation rates in couples suffering previous miscarriages during fertility treatments and in those with previous spontaneous pregnancies; (iii) no miscarriages after PGS in couples in whom a fluorescence in-situ hybridization assay showed the male partner's sperm to be abnormal; and (iv) lower implantation rates in couples with > or =5 previous miscarriages, associated with a lower percentage of chromosomally abnormal embryos. It is concluded that PGS is to be strongly recommended when RPL is associated with miscarriages during infertility treatments, chromosomopathy in a previous miscarriage, up to five previous miscarriages and a high incidence of chromosomal abnormalities in spermatozoa.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the influence of numerical chromosomal abnormalities on preimplantation embryo development.
This study includes 6936 embryos from 1245 women undergoing preimplantation genetic diagnosis (PGD). Indications for aneuploidy screening were: recurrent miscarriages, implantation failure, severe male factor, advanced maternal age, and mixed causes. Embryo biopsy was performed on day 3, and embryos were co-cultured until day 5, when embryo transfer was performed.
In the aneuploidy screening regimen, normal euploid embryos showed significantly higher blastocyst rates (68.2%) compared to chromosomally abnormal (42.8%, p < 0.0001) and mosaic (53.7%, p < 0.0001) embryos. Among aneuploid embryos for autosomes, higher blastocyst rates were observed in trisomies than monosomies, although only statistically significant in patients over 36 years of age (50.8 vs 38.9%; p < 0.0001). In contrast, in embryos with sex chromosomes aneuploidy, similar blastocyst rates were observed between trisomies and monosomy X.
Embryos with certain types of chromosomal abnormalities were negatively selected during preimplantation embryo development. Despite this selection, a remarkable percentage of chromosomally abnormal embryos can develop normally to blastocyst stage with high probability of implantation and pregnancy.
[Show abstract][Hide abstract] ABSTRACT: Preimplantation genetic diagnosis (PGD) has transformed the approach to the infertility patient in the IVF setting. Although the principal applications of PGD have been to prevent the transmission of sex-linked diseases, in time and with growing knowledge of the chromosomal abnormalities observed in preimplantation embryos, its applications have widened. Nowadays, apart from its implications in the prevention of transmission of chromosomal and genetic abnormalities, PGD is being used with increased frequency to improve the IVF outcome in patients with advanced maternal age (> or =38 years of age), recurrent miscarriage (> or =2 miscarriages), recurrent IVF failure (> or =3 failed IVF attempts) and severe male infertility. A high incidence of chromosomal abnormalities has been observed in these patient groups.
[Show abstract][Hide abstract] ABSTRACT: Previous observations have addressed a decreased male:female ratio associated with smoking. Our aim was to assess whether this effect is observed at the spermatozoa or at the early embryo development.
We retrospectively assessed smoking intake habits of 56 couples included in our preimplantation genetic diagnosis (PGD) program. Three groups were established according to male or female cigarette consumption per day: non-smokers, smokers (1-19 cigarettes per day) and heavy smokers (> or =20 cigarettes per day). Fluorescence in-situ hybridization (FISH) was performed on ejaculated sperm samples to analyse chromosomes X and Y. On day 3, embryos were also analysed. Additionally, sperm samples from four heavy smoking and four non-smoking donors were prospectively analysed before and after capacitation.
FISH on spermatozoa revealed no statistical differences in the Y:X ratio between the three groups. However, in the PGD study, in male heavy smokers, the XY:XX embryo ratio was decreased compared with non-smokers (22:47 versus 80:71; P = 0.0057). The smoking condition of the female partner had no significant effect on embryo XY:XX ratio, but for non-smoking females with a heavy smoking partner, the ratio was decreased (P = 0.0018) compared with non-smoking males. In heavy smoking donors a decreased of Y:X ratio was observed after swim-up with a statistically significant difference of ratios (P = 0.021).
Smoking habits of males do not have an effect on the percentage of X- and Y-bearing spermatozoa on ejaculated samples. However, male heavy smokers produce an increased incidence of female embryos that could be related to an enrichment of X spermatozoa after swim-up in patients with high tobacco consumption.
Human Reproduction 10/2005; 20(9):2517-22. · 4.67 Impact Factor