Characteristics of the IVF Cycle that Contribute to the Incidence of Mosaicism.

Highly sensitive next-generation sequencing (NGS) platforms applied to preimplantation genetic testing for aneuploidy (PGT-A) allow the classification of mosaicism in trophectoderm biopsies. However, the incidence of mosaicism reported by these tests can be affected by a wide number of analytical, biological, and clinical factors. With the use of a proprietary algorithm for automated diagnosis of aneuploidy and mosaicism, we retrospectively analyzed a large series of 115,368 trophectoderm biopsies from 27,436 PGT-A cycles to determine whether certain biological factors and in vitro fertilization (IVF) practices influence the incidence of overall aneuploidy, whole uniform aneuploidy, mosaicism, and TE biopsies with only segmental aneuploidy. Older female and male patients showed higher rates of high-mosaic degree and whole uniform aneuploidies and severe oligozoospermic patients had higher rates of mosaicism and only segmental aneuploidies. Logistic regression analysis identified a positive effect of female age but a negative effect of embryo vitrification on the incidence of overall aneuploid embryos. Female age increased whole uniform aneuploidy rates but decreased only segmental aneuploidy and mosaicism, mainly low-mosaics. Conversely, higher ovarian response decreased whole uniform aneuploidy rates but increased only segmental aneuploidies. Finally, embryo vitrification decreased whole uniform aneuploidy rates but increased mosaicism, mainly low-mosaics, compared to PGT-A cycles with fresh oocytes. These results could be useful for clinician's management of the IVF cycles.

Segmental aneuploidy in human blastocysts: a qualitative and quantitative overview.

BACKGROUND: Microarray-based and next generation sequencing (NGS) technologies have revealed that segmental aneuploidy is frequently present in human oocytes, cleavage-stage embryos and blastocysts. However, very little research has analyzed the type, size, chromosomal distribution and topography of the chromosomal segments at the different stages of development. METHODS: This is a retrospective study of 822 PGT-A (preimplantation genetic test for aneuploidies) performed on trophectoderm samples from 3565 blastocysts biopsied between January 2016 and April 2017. The cycles in question had been initiated for varying clinical indications. Samples were analyzed by next generation sequencing-based technology. Segmental aneuploidies were evaluated when fragment size was > 5 Mb. Blastocysts presenting a single segmental aneuploidy (SSA), without any additional whole-chromosome gain/loss, were statistically analyzed for incidence, type, size and chromosomal emplacement. Segment sizes relative to the whole chromosome or arm (chromosome- and arm-ratios) were also studied. RESULTS: 8.4% (299/3565) of blastocysts exhibited segmental aneuploidy for one or more chromosomes, some of which were associated with whole-chromosome aneuploidy while others were not. Nearly half of them (4.5%: 159/3565 of blastocysts) exhibited pure-SSA, meaning that a single chromosome was affected by a SSA. Segments were more frequent in medium-sized metacentric or submetacentric chromosomes and particularly in q-chrmosome arms, variables that were related to trophectoderm quality. SSA size was related to a greater extent to chromosome number and the arm affected than it was to SSA type. In absolute values (Mb), SSA size was larger in large chromosomes. However, the SSA:chromosome ratio was constant across all chromosomes and never exceeded 50% of the chromosome. CONCLUSIONS: SSA frequency is chromosome- and topographically dependent, and its incidence is not related to clinical or embryological factors, but rather to trophectoderm quality. SSA might be originated by chromosome instability in response to chromothripsis, bias introduced by the biopsy and/or iatrogenic effects. TRIAL REGISTRATION: Retrospectively registered.

Sperm chromosomal abnormalities and their contribution to human embryo aneuploidy.

In this work we reviewed 18 years of experience using fluorescence in situ hybridization (FISH) for sperm aneuploidy testing. We evaluated parameters associated with increased numerical sperm chromosome abnormalities and determined the male contribution to embryo aneploidies in terms of reproductive outcome by increased sperm aneuploidy. This retrospective study analyzed data from 2008 sperm samples of infertile males undergoing FISH analysis because of clinical history of repetitive implantation failure, recurrent miscarriage, impaired sperm parameters, or mixed causes. Sperm concentration was the only sperm parameter associated with FISH results-we observed a gradual increase of abnormal sperm FISH results in males with decreasing sperm concentration. However, a great proportion of normozoospermic males also showed increased sperm aneuploidies, suggesting that sperm parameters alone do not enable identification of a substantial proportion of infertile males at risk of sperm aneuploidies. Regarding reproductive outcomes, couples with normal sperm FISH results for the male had similar outcomes regardless of conventional in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or preimplantation genetic testing for aneuploidies (PGT-A). However, couples with abnormal sperm FISH results for the male showed better clinical outcomes after PGT-A, suggesting a potential contribution of sperm to embryo aneuploidy. Moreover, PGT-A cycles showed better clinical outcomes when 24 chromosomes were analyzed by array comparative genome hybridization (aCGH) or next-generation sequencing (NGS) instead of only nine chromosomes analyzed by FISH. In conclusion, sperm FISH analysis offers clinical prognostic value to evaluate reproductive possibilities in infertile couples. Therefore, couples with abnormal sperm FISH results should be offered genetic counseling and presented with clinical options such as PGT-A.

Clinical application of embryo aneuploidy testing by next-generation sequencing.

We review here the evolution in the field of embryo aneuploidy testing over the last 20 years, from the analysis of a subset of chromosomes by fluorescence in situ hybridisation to the transition toward a more comprehensive analysis of all 24 chromosomes. This current comprehensive aneuploidy testing most commonly employs next-generation sequencing (NGS). We present our experience in over 130 000 embryo biopsies using this technology. The incidence of aneuploidy was lower in trophectoderm biopsies compared to cleavage-stage biopsies. We also confirmed by NGS that embryo aneuploidy rates increased with increasing maternal age, mostly attributable to an increase in complex aneuploid embryos. In contrast, the number of MII oocytes retrieved or the use of oocyte vitrification did not affect aneuploidy rates. Similarly, neither maternal age, oocyte number, nor oocyte vitrification affected the incidence of mosaicism. Analysis of clinical outcomes, indications, and potential benefits of embryo aneuploidy testing revealed advanced maternal age as the most favored group, with some evidence of improved delivery rate per transfer as well as decreased miscarriage rates and time to pregnancy. Other indications are: recurrent miscarriage, repetitive implantation failure, severe male factor, previous trisomic pregnancy, and good prognosis patients mainly undergoing single embryo transfer, with the latter indication used to reduce the occurrence of multiple pregnancies without compromising cycle outcome. In conclusion, NGS has become the most appropriate technology for aneuploidy testing in trophectoderm biopsies, with accurate results, high throughput, and cost efficiency. This technology can be also applied to the analysis of the embryonic cell free DNA released to the culture media at blastocyst stage. This is a promising approach towards a non-invasive preimplantation genetic testing of aneuploidy.

Correlations between Synaptic Initiation and Meiotic Recombination: A Study of Humans and Mice.

Meiotic recombination is initiated by programmed double strand breaks (DSBs), only a small subset of which are resolved into crossovers (COs). The mechanism determining the location of these COs is not well understood. Studies in plants, fungi, and insects indicate that the same genomic regions are involved in synaptic initiation and COs, suggesting that early homolog alignment is correlated with the eventual resolution of DSBs as COs. It is generally assumed that this relationship extends to mammals, but little effort has been made to test this idea. Accordingly, we conducted an analysis of synaptic initiation sites (SISs) and COs in human and mouse spermatocytes and oocytes. In contrast to our expectation, we observed remarkable sex- and species-specific differences, including pronounced differences between human males and females in both the number and chromosomal location of SISs. Further, the combined data from our studies in mice and humans suggest that the relationship between SISs and COs in mammals is a complex one that is not dictated by the sites of synaptic initiation as reported in other organisms, although it is clearly influenced by them.

New tools for embryo selection: comprehensive chromosome screening by array comparative genomic hybridization.

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.

Use of array comparative genomic hybridization (array-CGH) for embryo assessment: clinical results.

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.

False positive rate of an arrayCGH platform for single-cell preimplantation genetic screening and subsequent clinical application on day-3.

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.

Chromosomal abnormalities in embryos from couples with a previous aneuploid miscarriage.

OBJECTIVE: To compare the incidence of chromosomal abnormalities in preimplantation embryos from couples undergoing preimplantation genetic screening (PGS) after previous aneuploid miscarriage after either natural conception (NC) or assisted reproductive technology (ART) versus fertile couples who underwent PGS for sex-linked diseases as a control group. DESIGN: Retrospective study. SETTING: IVF clinic. PATIENT(S): Patients with previous aneuploid conception undergoing PGS. INTERVENTION(S): Embryo biopsy, fluorescence in situ hybridization. MAIN OUTCOME MEASURE(S): Embryo aneuploidy rates and pregnancy and implantation rates in couples with a previous aneuploidy for autosomes or sex chromosomes. RESULT(S): The overall rates of chromosomal abnormalities in groups with previous autosomal aneuploidy were significantly higher compared with the control group (67.8% for those whose previous aneuploidy arose after NC and 65.8% for those previously arising after ART, vs. 34.0%). No significant differences were observed in those with previous sex chromosome abnormalities compared with control subjects. Within couples with previous aneuploidies after NC, no difference existed in the incidence of chromosomal abnormalities compared with the ART groups. Clinical outcomes were better (trend) in patients with previous autosomal aneuploidy after NC. CONCLUSION(S): In preimplantation embryos, the incidence of chromosomal abnormalities due to a previous aneuploid miscarriage after either NC or ART is significantly higher than in the control group. Furthermore, this incidence is higher when the previous aneuploidy was for autosomes; PGS is recommended in these couples.

Testicular sperm from patients with obstructive and nonobstructive azoospermia: aneuploidy risk and reproductive prognosis using testicular sperm from fertile donors as control samples.

OBJECTIVE: To establish a baseline incidence of chromosomal abnormalities in testicular sperm of fertile men and to determine the best control sample for comparisons with azoospermic males to estimate their reproductive prognosis. DESIGN: Prospective study. SETTING: Infertility clinic. PATIENT(S): Sixteen obstructive azoospermic (OA) and 19 nonobstructive azoospermic patients (NOA). Control samples were ejaculated sperm from ten fertile donors and testicular sperm from ten other fertile donors. INTERVENTION(S): Fluorescence in situ hybridization (FISH) in sperm. MAIN OUTCOME MEASURE(S): Sperm numerical abnormalities for chromosomes 13, 18, 21, X, and Y; ongoing implantation and pregnancy rates in intracytoplasmic sperm injection (ICSI) cycles. RESULT(S): In control samples, testicular sperm showed higher incidences of diploidy (0.27% vs. 0.10%) and disomy for chromosomes 13 (0.16% vs. 0.07%), 21 (0.25% vs. 0.12%), and sex chromosomes (0.34% vs. 0.21%) than ejaculated sperm. Comparisons with ejaculated control samples showed 12.5% OA and 68.4% NOA patients having significantly higher incidence of sperm chromosomal abnormalities. Compared with testicular control subjects, fewer OA (6.3%) and NOA (42.1%) patients had chromosomally abnormal sperm. NOA patients had lower ongoing implantation and pregnancy rates than OA patients, particularly those with abnormal FISH compared with testicular control samples. CONCLUSION(S): Sperm FISH analysis using testicular sperm control samples better identifies NOA patients with a lower likelihood of reproductive success.

Redefining advanced maternal age as an indication for preimplantation genetic screening.

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.

Aneuploidies in embryos and spermatozoa from patients with Y chromosome microdeletions.

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.

Impact of different patterns of sperm chromosomal abnormalities on the chromosomal constitution of preimplantation embryos.

OBJECTIVE: To evaluate the effect of sperm chromosome abnormalities--disomy for sex chromosomes and diploidy--in the chromosomal constitution of preimplantation embryos. DESIGN: Retrospective cohort study. SETTING: Infertility clinic. PATIENT(S): 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. INTERVENTION(S): Sperm collection for fluorescence in situ hybridization analysis. Embryo biopsy for preimplantation genetic screening. MAIN OUTCOME MEASURE(S): 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. RESULT(S): 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. CONCLUSION(S): A strong correlation between sperm and embryo chromosomal constitution has been shown in infertile men with 46,XY and 47,XYY karyotypes.

Prognostic factors for preimplantation genetic screening in repeated pregnancy loss.

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.