Non-invasive cell-free DNA-based approach for the diagnosis of clinical miscarriage: A retrospective study.

OBJECTIVE: To evaluate cell-free DNA (cfDNA) testing as a non-invasive approach to detecting aneuploidies in clinical miscarriages. DESIGN: A retrospective cohort study of women with pregnancy loss. SETTING: Hospitals and genetic analysis laboratories. POPULATION OR SAMPLE: Pregnancy losses in the period 2021-2022. METHODS: Results derived from non-invasive cfDNA testing (Veriseq NIPT Solution V2) of maternal blood and invasive analysis of products of conception (POC) (Ion ReproSeq) compared in 120 women who suffered a miscarriage. MAIN OUTCOME MEASURES: Concordance rate results, cfDNA testing performance, non-informative rate (NIR) and fetal fraction (FF). RESULTS: We found no significant differences in the NIR between invasive (iPOC) and non-invasive (niPOC) analysis of POC (10.0% [12/120] versus 16.7% [20/120]). Of 120 samples, 90 provided an informative result in iPOC and niPOC groups (75%). cfDNA analysis correctly identified 74/87 (85.1%) samples (excluding triploidies). Sensitivity and specificity were 79.4% and 100%, respectively; all discordant cases were female. A binomial logistic model suggested fetal sex as the only variable influencing the concordance rate (P = 0.035). A Y-chromosome-based FF estimate allowed the optimal reclassification of cfDNA of non-informative male fetuses and a more accurate evaluation of cfDNA testing performance. The difference between the two FF estimates (native algorithm and Y-chromosome-based) suggests that female non-concordant cases may represent non-informative cases. CONCLUSIONS: Cell-free DNA-based testing provides a non-invasive approach to determining the genetic cause of clinical miscarriage.

Label-free electrochemical immunosensing of glial fibrillary acidic protein (GFAP) at synthesized rGO/MoS2/AgNPs nanocomposite. Application to the determination in human cerebrospinal fluid.

An electrochemical bioplatform involving screen-printed carbon electrodes modified with rGO/MoS2/AgNPs nanocomposites, the covalent immobilization of the specific capture antibody, and label-free detection has been developed for the determination of Glial Fibrillary Acidic Protein (GFAP). The resulting immunosensor profits the benefits of the rGO high conductivity, the pseudo-peroxidase activity of MoS2 and the electrocatalytic effect provided by AgNPs for improving the reduction current responses of hydrogen peroxide at the electrode surface. GFAP is a biomarker of central nervous system injuries has been proposed for the detection and monitoring of neurological diseases as epilepsy, encephalitis, or multiple sclerosis. For the first time, amperometric detection of the immunosensing event was performed by measuring the electrocatalytic response of hydrogen peroxide reduction at the modified electrode. Several techniques including scanning (SEM) and transmission (TEM) electron microscopies were used for the characterization of the synthesized composite whilst electrochemical impedance spectroscopy (EIS) using the redox probe Fe(CN)63-/4- was employed to evaluate the success of the steps implied in the fabrication of the immunosensor. After optimization of the involved experimental variables, a linear calibration plot for GFAP was constructed over the 0.6-100 ng mL-1 range, and a detection limit of 0.16 ng mL-1 was achieved. The developed immunosensor was successfully applied to the determination of GFAP in human cerebrospinal fluid (CSF) of patients diagnosed with encephalitis.

Inherited unbalanced reciprocal translocation with 3q duplication and 5p deletion in a foetus revealed by cell-free foetal DNA (cffDNA) testing: a case report.

BACKGROUND: Since 2011, screening maternal blood for cell-free foetal DNA (cffDNA) fragments has offered a robust clinical tool to classify pregnancy as low or high-risk for Down, Edwards, and Patau syndromes. With recent advances in molecular biology and improvements in data analysis algorithms, the screening's scope of analysis continues to expand. Indeed, screening now encompassess additional conditions, including aneuploidies for sex chromosomes, microdeletions and microduplications, rare autosomal trisomies, and, more recently, segmental deletions and duplications called copy number variations (CNVs). Yet, the ability to detect CNVs creates a new challenge for cffDNA analysis in couples in which one member carries a structural rearrangement such as a translocation or inversion. CASE PRESENTATION: We report a segmental duplication of the long arm of chromosome 3 and a segmental deletion of the short arm of chromosome 5 detected by cffDNA analysis in a 25-year-old pregnant woman. The blood sample was sequenced on a NextSeq 550 (Illumina) using the VeriSeq NIPT Solution v1 assay. G-band karyotyping in amniotic fluid only detected an abnormality in chromosome 5. Next-generation sequencing in amniocytes confirmed both abnormalities and identified breakpoints in 3q26.32q29 and 5p13.3p15. The foetus died at 21 weeks of gestation due to multiple abnormalities, and later G-band karyotyping in the parents revealed that the father was a carrier of a balanced reciprocal translocation [46,XY,t(3;5)(q26.2;p13)]. Maternal karyotype appeared normal. CONCLUSION: This case provides evidence that extended cffDNA can detect, in addition to aneuploidies for whole chromosomes, large segmental aneuploidies. In some cases, this may indicate the presence of chromosomal rearrangements in a parent. Such abnormalities are outside the scope of standard cffDNA analysis targeting chromosomes 13, 18, 21, X, and Y, potentially leading to undiagnosed congenital conditions.

The morphokinetic signature of mosaic embryos: evidence in support of their own genetic identity.

OBJECTIVE: To provide full morphokinetic characterization of embryos ranked with different degrees of chromosomal mosaicism. DESIGN: Retrospective cohort study. SETTING: University-affiliated private in vitro fertilization clinic. PATIENT(S): We analyzed 1,511 embryos from 424 intracytoplasmic sperm injection cycles by culturing embryos in a time-lapse imaging system and performing next-generation sequencing. We assessed 106 mosaic embryos. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Comparison of chromosomal, morphological, and morphokinetic characteristics of blastocysts classified as euploid, aneuploid, low-degree mosaic (30% to <50% aneuploid cells in trophectoderm biopsy), and high-degree mosaic (50% to <70% aneuploid cells in trophectoderm biopsy). Statistical analysis was performed using χ2, Kruskal-Wallis, or analysis of variance tests according to data type and distribution. A two-way random effects model was used to calculate interoperator correlation of annotations, and a logistic mixed effects model was performed to evaluate the effect of confounders on morphokinetic timing. RESULT(S): The mosaicism rate was ∼7% regardless of parental age. Mosaicism and uniform aneuploidies were not evenly distributed across chromosomes. The percentage of high-quality blastocysts significantly decreased from euploid (66.9%) to mosaic (52.8%) and aneuploid (47.7%). Aneuploid blastocysts significantly delayed development compared with euploid blastocysts in start of compaction (median, 84.72 hours postmicroinjection [hpm], interquartile range [IQR], 13.2; vs. median, 82.10 hpm, IQR, 11.5), start of blastulation (median, 101 hpm; IQR, 11.7; vs. median, 98.29 hpm, IQR, 10.5), and timing of blastocyst (median, 108.04 hpm, IQR, 11.50; vs. median, 104.71 hpm, IQR, 11.35). However, embryo morphokinetics were not correlated to the degree of mosaicism or to a mosaicism configuration that was apt for embryo transfer. CONCLUSION(S): Morphokinetic timing of mosaic embryos overlaps with that of euploid and aneuploid embryos, which may reflect their unique genetic and developmental identity. Although this suggests mosaic embryos are not simply a misdiagnosis by-product, further studies are needed to reveal the true identity of this particular type of embryo.

The impact of patient, embryo, and translocation characteristics on the ploidy status of young couples undergoing preimplantation genetic testing for structural rearrangements (PGT-SR) by next generation sequencing (NGS).

PURPOSE: To evaluate the factors that affect the incidence of euploid balanced embryos and interchromosomal effect (ICE) in carriers of different structural rearrangements. METHODS: This retrospective study includes 95 couples with reciprocal translocations (RecT) and 36 couples with Robertsonian translocations (RobT) undergoing Preimplantation Genetic Testing for Structural Rearrangements (PGT-SR) between March 2016 and July 2019. Next-generation sequencing (NGS) was the technique used coupled with trophectoderm (TE) biopsy. Only cases with females under 38 years were included. A total of 532 blastocysts were evaluated. RESULTS: The euploidy rate was similar in RobT when compared with RecT carriers [57/156 (36.5%) vs. 112/376 (29.8%), p = 0.127]. The pure ICE rate was significantly higher in RobT carriers [48/156 (30.8%) vs. 53/376 (14.1%), p < 0.001] than it was in RecT carriers. Female age was the independent factor for the probability of obtaining a euploid embryo in RecT and RobT carriers, and increasing female age decreases the probability of obtaining a euploid embryo. In RecT carriers, no significant differences were observed in euploidy rates, pure ICE, or combined ICE according to the length of the translocated fragment and the chromosome group. However, total ICE was significantly lower when there was a breakpoint in the short chromosome arm together with a breakpoint in the long arm [(44/158 (27.8%) for pq or qp, 51/155 (32.9%) for pp and 30/63 (47.6%) for qq; p = 0.02]. CONCLUSION: The incidence of euploid/balanced blastocysts was similar in both types of translocations. However, there was a significant increase in pure ICE in RobT compared to RecT carriers. In RecT carriers, the presence of the breakpoints in the long arm of the chromosomes involved in the rearrangement resulted in a higher total ICE.

Sperm genetic abnormalities and their contribution to embryo aneuploidy & miscarriage.

Sperm genetic testing has been proposed for clinical diagnosis of possible causes of male infertility. We reviewed the most remarkable publications of sperm DNA integrity and sperm aneuploidy as they relate to clinical outcomes, and the relationship between both genetic defects, and its association to embryo aneuploidy and recurrent pregnancy loss.

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.

Optimized NGS Approach for Detection of Aneuploidies and Mosaicism in PGT-A and Imbalances in PGT-SR.

The detection of chromosomal aneuploidies and mosaicism degree in preimplantation embryos may be essential for achieving pregnancy. The aim of this study was to determine the robustness of diagnosing homogenous and mosaic aneuploidies using a validated algorithm and the minimal resolution for de novo and inherited deletions and duplications (Del/Dup). Two workflows were developed and validated: (a,b) preimplantation genetic testing for uniform whole and segmental aneuploidies, plus mixtures of euploid/aneuploid genomic DNA to develop an algorithm for detecting mosaicism; and (c) preimplantation genetic testing for structural rearrangements for detecting Del/Dup ≥ 6 Mb. Next-generation sequencing (NGS) was performed with automatic library preparation and multiplexing up to 24-96 samples. Specificity and sensitivity for PGT-A were both 100% for whole chromosomes and segmentals. The thresholds stablished for mosaicism were: euploid embryos (<30% aneuploidy), low mosaic (from 30% to <50%), high mosaic (50-70%) or aneuploid (>70%). In the PGT-SR protocol, changes were made to increase the detection level to ≥6 Mb. This is the first study reporting an accurate assessment of semiautomated-NGS protocols using Reproseq on pools of cells. Both protocols allow for the analysis of homogeneous and segmental aneuploidies, different degrees of mosaicism, and small Del/Dup with high sensitivity and specificity.

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.

In vitro fertilization with preimplantation genetic diagnosis for aneuploidies in advanced maternal age: a randomized, controlled study.

OBJECTIVE: To determine the clinical value of preimplantation genetic diagnosis for aneuploidy screening (PGD-A) in women of advanced maternal age (AMA; between 38 and 41 years). DESIGN: This was a multicenter, randomized trial with two arms: a PGD-A group with blastocyst transfer, and a control group with blastocyst transfer without PGD-A. SETTING: Private reproductive centers. PATIENT(S): A total of 326 recruited patients fit the inclusion criteria, and 205 completed the study (100 in the PGD-A group and 105 in the control group). INTERVENTION(S): Day-3 embryo biopsy, array comparative genomic hybridization, blastocyst transfer, and vitrification. MAIN OUTCOME MEASURE(S): Primary outcomes were delivery and live birth rates in the first transfer and cumulative outcome rates. RESULT(S): The PGD-A group exhibited significantly fewer ETs (68.0% vs. 90.5% for control) and lower miscarriage rates (2.7% vs. 39.0% for control). Delivery rate after the first transfer attempt was significantly higher in the PGD-A group per transfer (52.9% vs 24.2%) and per patient (36.0% vs. 21.9%). No significant differences were observed in the cumulative delivery rates per patient 6 months after closing the study. However, the mean number of ETs needed per live birth was lower in the PGD-A group compared with the control group (1.8 vs. 3.7), as was the time to pregnancy (7.7 vs. 14.9 weeks). CONCLUSION(S): Preimplantation genetic diagnosis for aneuploidy screening is superior compared with controls not only in clinical outcome at the first ET but also in dramatically decreasing miscarriage rates and shortening the time to pregnancy.

Type of chromosome abnormality affects embryo morphology dynamics.

OBJECTIVE: To study the differences in the cleavage time between types of embryo chromosomal abnormalities and elaborate algorithm to exclude aneuploid embryos according to the likelihood to be euploid. DESIGN: Retrospective cohort study. SETTING: University affiliated private center. PATIENT(S): Preimplantational genetic screening patients (n = 112) including cases of advanced maternal age, repeated implantation failure, and recurrent miscarriage. A total of 485 embryos were analyzed. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): All biopsied embryos were cultured in an incubator with time-lapse technology, cleavage timing from insemination to day 3 and all kinetic parameters that have been described in previous studies by our group. RESULT(S): Logistic regression analysis were used to identify morphokinetic parameters and some were strongly associated with complex aneuploid embryos; t3 (odds ratio = 0.590, 95% confidence interval 0.359-0.971) and t5-t2 (odds ratio = 0.151, 95% confidence interval 0.082-0.278). CONCLUSION(S): Embryo morphokinetics are affected by chromosome aneuploidy and further analysis of the chromosome content reveals higher differences when the complexity in the chromosome disorders is increased. The use of time-lapse monitoring, although not able to detect an abnormal embryo, may be potentially useful to discard those embryos with high risk of complex chromosomal abnormalities.

Distribution patterns of segmental aneuploidies in human blastocysts identified by next-generation sequencing.

OBJECTIVE: To evaluate the ability of next-generation sequencing (NGS) to detect pure and mosaic segmental aneuploidies in trophectoderm biopsies and to identify distribution patterns in whole blastocysts. DESIGN: Validation study. SETTING: Reference laboratory. PATIENT(S): Seventy couples with known karyotypes who had undergone preimplantation genetic screening with diagnoses at the blastocyst stage using array comparative genomic hybridization (aCGH). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Concordance rates for segmental and whole-chromosome aneuploidies determined between aCGH and NGS, and estimates of mosaicism levels of segmental aneuploidies in fixed blastocysts. RESULT(S): We used NGS with amplified DNA from trophectoderm biopsies in which segmental aneuploidies had been previously detected by array comparative genomic hybridization (aCGH). Single-cell fluorescent in situ hybridization (FISH) was then used as an independent form of analysis. The concordance rate between NGS and aCGH was 124 (98.4%) of 126 for the detection of segmental aneuploidies, and 48 (96.0%) of 50 for whole-chromosome aneuploidies. The overall concordance rate was 99.8% (2,276 of 2,280 chromosomes assessed). After FISH analyses with 41.4 ± 24.3 cells per blastocyst, 26 (92.9%) of 28 segmentals detected by aCGH and NGS were confirmed. The FISH analysis did not detect the segmentals in two blastocysts, in which all cells analyzed were euploid. CONCLUSION(S): This is the first report analyzing distribution patterns of segmental aneuploidies in trophectoderm biopsy by NGS. We have demonstrated that NGS allows the detection of pure and mosaic segmental aneuploidies with the same efficiency as aCGH. The FISH analysis confirmed the existence of these events in the trophectoderm and the inner cell mass.

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.

Increasing the probability of selecting chromosomally normal embryos by time-lapse morphokinetics analysis.

OBJECTIVE: To study the differences in the cleavage time between chromosomally normal and abnormal embryos and to elaborate an algorithm to increase the probability of noninvasively selecting chromosomally normal embryos. DESIGN: Retrospective cohort study. SETTING: University-affiliated infertility center. PATIENT(S): Preimplantation genetic screening patients (n = 125; n = 77 with ET), including cases of repeated implantation failure or recurrent miscarriage. A total of 504 embryos were analyzed. INTERVENTION(S): Embryo culture within a time-lapse system. MAIN OUTCOME MEASURE(S): Kinetic variables included the time to 2 (t2), 3 (t3), 4 (t4), and 5 (t5) cells as well as the length of the second (cc2 = t3 - t2) and third (cc3 = t5 - t3) cell cycle, the synchrony in the division from 2 to 4 cells (s2 = t4 - t3), and the interval t5 - t2. Implantation and clinical pregnancy rates were also analyzed. RESULT(S): A logistic regression analysis identified t5 - t2 (odds ratio [OR] = 2.853; 95% confidence interval [CI], 1.763-4.616), followed by cc3 (OR = 2.095; 95% CI, 1.356-3.238) as the most relevant variables related to normal chromosomal content. On the basis of these results, an algorithm for embryo selection is proposed to classify embryos from A to D. Each category exhibited significant differences in the percentage of normal embryos (A, 35.9%; B, 26.4%; C, 12.1%; D, 9.8%). CONCLUSION(S): Chromosomally normal and abnormal embryos have different kinetic behavior. On the basis of these differences, the proposed algorithm serves as a tool to classify embryos and to increase the probability of noninvasively selecting normal embryos.

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.

Preimplantation genetic screening using fluorescence in situ hybridization in patients with repetitive implantation failure and advanced maternal age: two randomized trials.

OBJECTIVE: To evaluate the usefulness of preimplantation genetic screening (PGS) using fluorescence in situ hybridization (FISH) for two different indications: repetitive implantation failure (RIF) and advanced maternal age (AMA). DESIGN: Two prospective, randomized controlled trials with patients allocated in two arms: blastocyst transfer on day 5 (group A) or PGS with transfer on day 5 (group B). SETTING: University-affiliated private clinics. PATIENT(S): The RIF study included women <40 years with three or more failed IVF cycles without other known causal factors (91 patients). The AMA study included intracytoplasmic sperm injection patients aged between 41 and 44 (183 patients). INTERVENTION(S): In the PGS group, single-cell day 3 biopsy was performed with aneuploidy screening for chromosomes 13, 15, 16, 17, 18, 21, 22, X, and Y. In both the blastocyst transfer group and the PGS group, ET was performed on day 5. MAIN OUTCOME MEASURE(S): Live-birth rate per patient and per started cycle. RESULT(S): A significant increase in live-birth rates per patient was found in the PGS group compared with the blastocyst group for the AMA study (30/93 patients [32.3%] vs. 14/90 patients [15.5%]; odds ratio, 2.585; confidence interval, [1.262-5.295]). In the RIF study no significant differences were observed (23/48 patients [47.9%] vs. 12/43 patients [27.9%]). CONCLUSION(S): PGS with FISH was shown to be beneficial for the AMA group.

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.

Moderate ovarian stimulation does not increase the incidence of human embryo chromosomal abnormalities in in vitro fertilization cycles.

CONTEXT: A high chromosomal abnormalities rate has been observed in human embryos derived from in vitro fertilization (IVF) treatments. The real incidence in natural cycles has been poorly studied, so whether this frequency may be induced by external factors, such as use of gonadotropins for ovarian stimulation, remains unknown. DESIGN: We conducted a prospective cohort study in a University-affiliated private infertility clinic with a comparison between unstimulated and stimulated ovarian cycles in the same women. Preimplantation genetic screening by fluorescence in situ hybridization was performed in all viable d 3 embryos. OBJECTIVE: The primary objective was to compare the incidence of embryo chromosomal abnormalities in an unstimulated cycle and in an ulterior moderate ovarian stimulated cycle. Secondary outcome measures were embryo quality, blastocyst rate of biopsied embryos, number of normal blastocysts per donor, type of chromosomal abnormalities, and clinical outcome. RESULTS: One hundred eighty-five oocyte donors were initially recruited for the unstimulated cycle, and preimplantation genetic screening could be performed in 51 of them, showing 35.3% of embryo chromosomal abnormalities. Forty-six of them later completed a stimulated cycle. The sperm donor sample was the same for both cycles. The proportion of embryos displaying abnormalities in the unstimulated cycle was 34.8% (16 of 46), whereas it was 40.6% (123 of 303) in the stimulated cycle with risk difference=5.8 [95% confidence interval (CI)=-20.6-9.0], and relative risk=1.17 (95% CI=0.77-1.77) (P=0.45). When an intrasubject comparison was made, the abnormalities rate was 34.8% (95% CI=20.5-49.1) in the unstimulated cycle and 38.2% (95% CI=30.5-45.8) in the stimulated cycle [risk difference=3.4 (95% CI=-17.9-11.2); P=0.64]. No differences were observed for embryo quality and type of chromosomal abnormalities. CONCLUSIONS: Moderate ovarian stimulation in young normo-ovulatory women does not significantly increase the embryo aneuploidies rate in in vitro fertilization-derived human embryos as compared with an unstimulated cycle. Whether these results can be extrapolated to infertile patients is still unknown.

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.