The overall success of human reproduction, either spontaneously or after IVF, is highly dependent upon maternal age. The main reasons for age-related infertility include reduced ovarian reserve and ...decreased oocyte/embryo competence due to aging insults, especially concerning an increased incidence of aneuploidies and possibly decreased mitochondrial activity. Age-related chromosomal abnormalities mainly arise because of meiotic impairments during oogenesis, following flawed chromosome segregation patterns such as non-disjunction, premature separation of sister chromatids, or the recent reverse segregation. In this review, we briefly discuss the main mechanisms putatively impaired by aging in the oocytes and the deriving embryos. We also report the main strategies proposed to improve the management of advanced maternal age women in IVF: fertility preservation through oocyte cryopreservation to prevent aging; optimization of the ovarian stimulation and enhancement of embryo selection to limit its effects; and oocyte donation to circumvent its consequences.
STUDY QUESTION
Does conventional blastocyst morphological evaluation correlate with euploidy (as assessed by comprehensive chromosome screening (CCS) of trophectoderm (TE) biopsies) and implantation ...potential?
SUMMARY ANSWER
A moderate relation between blastocyst morphology and CCS data was observed but the ability to implant seems to be mainly determined by the chromosomal complement of preimplantation embryos rather than developmental and morphological parameters conventionally used for blastocyst evaluation.
WHAT IS KNOWN ALREADY
Combined with improving methods for cryopreservation and blastocyst culture, TE biopsy and CCS is considered to be a promising approach to select euploid embryos for transfer. Understanding the role of morphology in blastocyst stage preimplantation genetic screening (PGS) cycles may help in further optimizing the cycle management and clinical outcomes.
STUDY DESIGN, SIZE, DURATION
This is a multicenter retrospective observational study performed between January 2009 and August 2013. The study includes the data analysis of 956 blastocysts with conclusive CCS results obtained from 213 patients following 223 PGS cycles. Single frozen embryo transfer (FET) cycles of 215 euploid blastocysts were performed where it was possible to track the implantation outcome of each embryo transferred.
PARTICIPANTS/MATERIALS, SETTING, METHODS
PGS was offered to infertile patients of advanced maternal age (>35 years) and/or with a history of unsuccessful IVF treatments (more than two failed IVF cycles) and/or previous spontaneous abortion (more than two spontaneous miscarriages). Prior to TE biopsy for CCS, blastocyst morphology was assessed and categorized in four groups (excellent, good, average and poor quality). The developmental rate of each embryo reaching the expanded blastocyst stage was defined according to the day of biopsy post-fertilization. Day 5 and Day 6 biopsied blastocysts were defined as faster and slower growing embryos, respectively. A novel blastocyst biopsy method, not requiring the opening of the zona pellucida at the cleavage stage of embryo development, was used. Linear regression models were used to test the relationship between blastocyst morphology and developmental rate CCS data and FET cycle outcomes of euploid blastocysts.
MAIN RESULTS AND THE ROLE OF CHANCE
Among the embryological variables assessed (morphology and developmental rate), only blastocyst morphology was predictive of the CCS data. The euploidy rate was 56.4, 39.1, 42.8 and 25.5% in the excellent, good, average and poor blastocyst morphology groups, respectively. A diagnosis of complex aneuploidy was also associated with blastocyst morphology (P < 0.01) with 6.8, 15.2, 17.4 and 27.5% of excellent, good, average and poor quality embryos, respectively, showing multiple chromosome errors. Faster and slower growing embryos showed a similar aneuploidy rate. Regression logistic analysis showed that none of the parameters used for conventional blastocyst evaluation (morphology and developmental rate) was predictive of the implantation potential of euploid embryos. The implantation potential of euploid embryos was the same, despite different morphologies and developmental rates.
LIMITATIONS, REASONS FOR CAUTION
The study is limited by its retrospective nature. A higher sample size or a prospective randomized design could be used in future studies to corroborate the current findings.
WIDER IMPLICATIONS OF THE FINDINGS
This study provides knowledge for a better laboratory and clinical management of blastocyst stage PGS cycles suggesting that the commonly used parameters of blastocyst evaluation are not good enough indicators to improve the selection among euploid embryos. Accordingly, all poor morphology and slower growing expanded blastocysts should be biopsied and similarly considered for FET cycles. This knowledge will be of critical importance to achieve similar cumulative live birth rates in PGS programs compared with conventional IVF, avoiding the potential for exclusion of low quality but viable embryos from the biopsy and transfer procedures. Future research to identify non-invasive biomarkers of reproductive potential may further enhance selection among euploid blastocysts.
STUDY FUNDING/COMPETING INTEREST(S)
No funding was obtained for the study. All authors have no conflicts to declare.
TRIAL REGISTRATION NUMBER
None.
Chromosome errors, or aneuploidy, affect an exceptionally high number of human conceptions, causing pregnancy loss and congenital disorders. Here, we have followed chromosome segregation in human ...oocytes from females aged 9 to 43 years and report that aneuploidy follows a U-curve. Specific segregation error types show different age dependencies, providing a quantitative explanation for the U-curve. Whole-chromosome nondisjunction events are preferentially associated with increased aneuploidy in young girls, whereas centromeric and more extensive cohesion loss limit fertility as women age. Our findings suggest that chromosomal errors originating in oocytes determine the curve of natural fertility in humans.
Objective To compare the euploid blastocyst formation rates obtained after follicular phase (FP) versus luteal phase (LP) stimulation performed in the same menstrual cycle in a preimplantation ...genetic diagnosis for aneuploidy testing (PGD-A) program in patients with reduced ovarian reserve. Design Prospective paired noninferiority observational study. Setting Private infertility program. Patient(s) Forty-three reduced ovarian reserve patients undergoing a PGD-A. Intervention(s) Both FP and LP stimulations using follicle-stimulating hormone and luteinizing hormone in combination with gonadotropin-releasing hormone (GnRH) antagonist starting on day 2 of the cycle and 5 days after the first oocyte retrieval, respectively, where GnRH agonist was used for both FP and LP ovulation triggering; a trophectoderm biopsy quantitative polymerase chain reaction–based PGD-A strategy; and single euploid blastocyst transfers during a subsequent natural cycle. Main Outcome Measure(s) Primary outcome measure: euploid blastocyst rate per injected metaphase 2 (MII) oocyte; secondary outcome measures: number of cumulus-oocyte complexes (COCs), MII oocytes, and blastocysts. Result(s) Patients with an antimüllerian hormone level of <1.5 ng/mL, antral follicle count of <6 follicles, and/or <5 oocytes retrieved in a previous cycle were included. No statistically significant differences were found in the number of retrieved COCs (5.1 ± 3.4 vs. 5.7 ± 3.3), MII oocytes (3.4 ± 1.9 vs. 4.1 ± 2.5), or biopsied blastocysts per stimulated cycle (1.2 ± 1.2 vs. 1.4 ± 1.7) from FP versus LP stimulation, respectively. No differences were observed in the euploid blastocyst rate calculated either per biopsied blastocyst (46.9% vs. 44.8%) or injected MII oocyte (16.2% vs. 15.0%). Conclusion(s) Stimulation with an identical protocol in the FP and LP of the same menstrual cycle resulted in a similar number of blastocysts in patients with reduced ovarian response. The LP stimulation statistically significantly contributed to the final transferable blastocyst yield, thus increasing the number of patients undergoing transfer per menstrual cycle.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
To study whether embryonic cell-free DNA (cfDNA) in spent blastocyst media is representative of the chromosomal constitution of a blastocyst.
Pilot prospective blinded study.
In vitro fertilization ...center and genetics laboratory.
A total of 115 trophectoderm (TE) biopsies and spent blastocyst media (SBM) from 46 patients with ages ranging from 32 to 46 years, whose indications for preimplantation genetic testing of aneuploidy (PGT-A) were advanced maternal age, recurrent miscarriage, or recurrent implantation failure.
Spent blastocyst media collection and TE biopsy.
Concordance rates, sensitivity, and specificity between TE biopsies and SBM. Clinical outcomes in cases with euploid TE biopsies and euploid SBM compared with cases with euploid TE and aneuploid SBM.
In general, the total concordance rate for ploidy and sex was 78.7%, and sensitivity and specificity were 94.5% and 71.7%, respectively. A significant increase for all parameters was observed for day 6/7 samples compared with day 5 samples, with day 6/7 samples showing total concordance for ploidy and sex of 84%, and sensitivity and specificity of 95.2% and 82.1%, respectively. Ongoing implantation rates in euploid TE/euploid SBM showed a threefold increase compared with euploid TE/aneuploid SBM (52.9% vs. 16.7%, respectively), without reaching significant differences. Interestingly, no miscarriages were observed when TE and SBM were euploidy concordant.
These results offer a better understanding of the dynamics of cfDNA during embryo development and despite more basic research being needed, they are reassuring to consider in the future this noninvasive approach as an alternative to TE biopsy for PGT-A.
Adn embrionario libre comparado con biopsia de trofectodermo para análisis de aneuploidía: tasa de concordancia e implicaciones clínicas
Estudiar si el ADN embrionario libre (cfDNA) en medio de cultivo de blastocisto es representativo de la constitución cromosómica de un blastocisto.
Estudio ciego prospectivo piloto.
Centro de fecundación in vitro y laboratorio de genética.
Un total de 115 biopsias de trofectodermo (TE) y medio de cultivo de blastocisto (SBM) de 46 pacientes con edades entre 32 y 46 años, cuyas indicaciones para diagnóstico genético preimplantacional para aneuploidía (PGT-A) eran edad materna avanzada, aborto de repetición, o fallo de implantación recurrente.
Obtención de embriones tras biopsia de TE.
Tasa de concordancia, sensibilidad y especificidad entre biopsia de TE y SBM. Resultados clínicos en casos de biopsia de TE euploide y SBM euploide comparados con casos de TE euploide y SBM aneuploide.
En general, la tasa de concordancia total en ploidía y sexo fue de 78.7% y la sensibilidad y especificidad fueron 94.5% y 71.7%, respectivamente. Se observó un aumento significativo en todos los parámetros al comparar las muestras de días 6 ó 7 con las muestras de día 5, con las muestras de días 6 ó 7 mostrando una concordancia total de ploidía y sexo de 84%, y una sensibilidad y especificidad de 95.2% y 82.1%, respectivamente. La tasa de implantación en TE euploide/SBM euploide fue tres veces superior a la de TE euploide/SBM aneuploide (52.9% vs 16.7%, respectivamente), sin llegar dicha diferencia a ser significativa. Interesantemente, no se observaron abortos en casos en que el TE y el SBM eran concordantes.
Estos resultados ofrecen un mejor entendimiento de las dinámicas del cfDNA durante el desarrollo embrionario y, a pesar de necesitarse más investigación básica, son alentadores para el futuro a la hora de considerar este método no invasico como alternativa a la biopsia de TE para PGT-A.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected ...and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential.
Full text
Available for:
DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
The term “cryopreservation” refers to the process of cooling cells and tissues and storing them at subzero temperatures in order to stop all biological activity and preserve their viability and ...physiological competences for future use. Cooling to subzero temperatures is not a physiological condition for human cells; this is probably due to the high content of water in the living matter, whose conversion to ice crystals may be associated with severe and irreversible damage. Among reproductive cells and tissues, metaphase II oocytes are notably vulnerable to cryopreservation, mainly because of their large size, low surface area to volume ratio, relatively high water content and presence of the meiotic spindle. As human biological systems lack efficient internal defense mechanisms against chilling injuries, it is of the utmost importance to supply adequate external support, in terms of cryoprotectant additives, appropriate cooling/warming rates, and suitable long‐term storage. Over the years, scientists have proposed different cryopreservation strategies in the effort to achieve an optimized recipe ensuring cell survival and, at the same time, maintenance of the physiological functions and abilities necessary to continue life. However, despite the first success obtained in the 1980s with frozen oocytes, it was not until recently that notable improvements in the cryopreservation technique, thanks to the advent of vitrification, allowed a breakthrough of this fine procedure.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Objective To assess whether extracellular microRNAs (miRNAs) can be accurately profiled from spent blastocyst culture media (SBM) and used as embryonic biomarkers. Design Prospective cohort study. ...Setting Private and academic in vitro fertilization centers. Patient(s) Inner cell mass-free trophectoderm (TE) samples and their relative SBM from five good-quality human blastocysts. Intervention(s) Protocol for miRNA purification and analysis based on quantitative polymerase chain reaction set and validated on human embryonic stem cells (hESCs) and on SBM with and without biological variability. Main Outcomes Measure(s) Analysis of miRNAs in culture media in relation with TE cells and comparison of miRNA profiles between implanted and unimplanted euploid blastocysts. Result(s) Culture media from embryos in the cleavage, morula, and blastocyst stages were collected to investigate the presence of miRNAs. The SBM were prospectively collected from euploid implanted (n = 25) and unimplanted blastocysts (n = 28) for comparison. We hypothesized that human embryos secrete miRNAs in culture media that can be used as biomarkers. The comparative analysis of TE and SBM samples revealed that 96.6% (57 of 59; 95 CI, 88.3–99.6) of the miRNAs detected in the SBM were expressed from TE cells, suggesting a TE origin. The culture media collected from cleavage and morula stage embryos showed a pattern similar to blanks, suggesting that miRNAs profiling from spent culture media applies only for blastocysts. MicroRNAs analysis of SBM from euploid implanted and unimplanted blastocysts highlighted two miRNAs (miR-20a, miR-30c) that showed increased concentrations in the former and were predicted in silico to be involved in 23 implantation-related pathways. Conclusion(s) MicroRNAs secreted from human blastocysts in culture media can be profiled with high reproducibility, and this approach can be further explored for noninvasive embryo selection.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
To determine whether blastocoel fluid (BF) or spent blastocyst medium (SBM) is a suitable template for genotype and/or karyotype assessment of in vitro fertilization–generated embryos.
Prospective ...blinded study.
Genetic laboratory.
From 26 patients undergoing preimplantation genetic testing (PGT) treatments, 103 trophectoderms (TE), 92 BF samples, and 72 SBM samples.
The BF and SBM were retrieved at the time of TE biopsy. Two DNA extraction strategies were evaluated on independent BF and SBM samples. Further enrolled samples were processed using next-generation sequencing and quantitative polymerase chain reaction for assessment of monogenic disorders (PGT-M) or aneuploidy (PGT-A).
DNA amplification and concordance rates across BF, SBM, and TE to assess diagnostic efficiency.
No differences were detected among the DNA extraction methods tested. In PGT-M tests, for BF and SBM, 2.9% and 20.8% of all samples, respectively, produced a diagnosis concordant with the corresponding TE (n = 2 of 69 and 15 of 72, respectively). The SBM samples were associated with higher discordance rates and higher artifacts/contamination detection compared with BF. In multiple occasions, the maternal mutated variant was detected in the SBM of homozygous wild-type embryos, showing evidence of maternal DNA persistence in culture medium. In PGT-A tests, BF analysis showed high amplification failure rates (65.2%) and an overall concordance rate of 37.5% among amplified samples.
Based on current methodologies, BF and SBM genetic analyses do not provide sufficiently reliable results to be employed clinically. Until the risk of maternal contamination can be properly prevented, SBM should not be used for PGT-M purposes.
Eficacia diagnóstica del fluido del blastocele y del medio usado como fuentes de ADN para el examen genético preimplantacional en condiciones clínicas estándar
Determinar si el fluido del blastocele (BF) o el medio usado en el blastocisto (SBM) son modelos adecuados para la evaluación del genotipo y/o cariotipo de embriones generados por fecundación in vitro.
Estudio prospectivo ciego.
Laboratorio de genética.
103 trofoectodermos (TE), 92 muestras BF y 72 muestras SBM de 26 pacientes sometidas a tratamientos de examen genético preimplantacional (PGT).
El BF y el SBM se recuperaron en el momento de la biopsia de TE. Se evaluaron dos estrategias para la extracción de ADN en muestras independientes de BF y SBM. Las muestras adicionales incluidas se procesaron usando secuenciación de nueva generación y cuantificación de la reacción en cadena de la polimerasa para la evaluación de los trastornos monogénicos (PGT-M) o aneuploidías (PGT-A).
Tasas de amplificación del ADN y concordancia en BF, SBM y TE para evaluar la eficacia del diagnóstico.
No se detectaron diferencias entre los métodos de extracción de ADN evaluados. En las pruebas PGT-M para BF y SBM, 2.9% y 20.8% de todas las muestras, respectivamente, produjeron un diagnóstico concordante con el TE correspondiente (n=2 de 69 y 15 de 72, respectivamente). Las muestras de SBM se asociaron con mayores tasas de discordancia y más artefactos/contaminaciones comparados con BF. En muchas ocasiones, la variante materna mutada se detectó en el SBM de embriones homocigóticos de tipo silvestre, mostrando evidencia de la persistencia del ADN materno en el medio de cultivo. En las pruebas PGT-A, el análisis del BF mostró altas tasas de fallo en la amplificación (65.2%) y un total de tasa de concordancia del 37.5% entre las muestras amplificadas.
Con base en las metodologías actuales, los análisis genéticos BF y SBM no proporcionan resultados suficientemente fiables para ser empleados clínicamente. Hasta que no se pueda prevenir adecuadamente el riesgo de contaminación materna, el SBM no se debe utilizar con fines PGT-M.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Despite next-generation sequencing, which now allows for the accurate detection of segmental aneuploidies from in vitro fertilization embryo biopsies, the origin and characteristics of these ...aneuploidies are still relatively unknown. Using a multifocal biopsy approach (four trophectoderms TEs and one inner cell mass ICM analyzed per blastocyst; n = 390), we determine the origin of the aneuploidy and the diagnostic predictive value of segmental aneuploidy detection in TE biopsies toward the ICM’s chromosomal constitution. Contrary to the prevalent meiotic origin of whole-chromosome aneuploidies, we show that sub-chromosomal abnormalities in human blastocysts arise from mitotic errors in around 70% of cases. As a consequence, the positive-predictive value toward ICM configuration was significantly lower for segmental as compared to whole-chromosome aneuploidies (70.8% versus 97.18%, respectively). In order to enhance the clinical utility of reporting segmental findings in clinical TE biopsies, we have developed and clinically verified a risk stratification model based on a second TE biopsy confirmation and segmental length; this model can significantly improve the prediction of aneuploidy risk in the ICM in over 86% of clinical cases enrolled. In conclusion, we provide evidence of the predominant mitotic origin of segmental aneuploidies in preimplantation embryos and develop a risk stratification model that can help post-test genetic counseling and that facilitates the decision-making process on clinical utilization of these embryos.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
PDF
