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Use of array comparative genomic hybridization (array-CGH) for embryo assessment: Clinical results
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Review of published studies and own unpublished data.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.
... [6][7][8] Embriões portadores de trissomias e monossomias representam mais de 10% das gestaç ões humanas e para as mulheres com idade materna avanç ada, acima dos 35 anos, a incidência pode exceder os 50%, 3,4 particularmente para os cromossomos 13, 18, 21, X e Y responsáveis pela maioria dos abortos espontâneos. 9,10 O aumento de erros de segregação durante a meiose não está relacionado apenas à idade materna avanç ada, mas também a uma interação entre as características originais da oogênese e uma série de fatores endógenos e exógenos. 4 A ideia pioneira do PGD foi elaborada por Edwards e Gardner em 1968 11 e a primeira aplicação clínica foi descrita em 1990 por Handyside et al., 12 na qual sequências específicas do cromossomo Y foram amplificadas com a reação em cadeia da polimerase (PCR, do inglês polymerase chain reaction) para determinar o sexo dos embriões originados de casais com risco de transmissão de doenças relacionas ao X. 13 O PGD deu origem ao screnning genético pré-implantacional (PGS, do inglês screening genetic preimplantation), um procedimento que identifica embriões com aberraç ões cromossômicas de mulheres com idade avanç ada, em casos de casais com cariótipo normal que sofreram abortos de repetição ou falha de implantação, assim como em casos de fator masculino de infertilidade grave, pois geram embriões com altas taxas de anormalidades cromossômicas. ...
... 46 Além do mais, estudos clínicos e de pesquisa também usam corpúsculos polares e a células da trofoectoderme para o diagnóstico dos embriões. 10,48,49 Para a aplicação da técnica primeiramente é preciso amplificar o DNA da amostra e do controle separadamente com o uso de uma amplificação genômica completa (WGA, do inglês whole genome amplification). Dentre as técnica de WGA, atualmente a mais usada em PGD é a amplificação por deslocamento múltiplo (MDA, do inglês multiple displacement amplification), por ter se mostrado muito mais eficaz na amplificação de quantidades mínimas de DNA. ...
... [38][39][40]46,53 Os amplificados então são marcados com fluoroforos, geralmente o controle com o vermelho e a amostra com o verde. 10,54 Esses então são misturados e adicionados ao chip de array para que aconteça a hibridação. Existem vários tipos de chips no mercado, contudo o mais usado em PGD é o que apresenta em sua superfície BAC's (cromossomos artificiais bacterianos). ...
O diagnóstico genético pré‐implantacional (PGD) é uma ferramenta que permite a seleção do embrião saudável por meio da análise gênica e cromossômica. Beneficia casais no grupo de risco, como, por exemplo, casais com histórico de aborto de repetição ou com histórico familiar de doença hereditária. Diante do avanço das metodologias empregadas no PGD, esta revisão tem como objetivo reunir as informações acerca das técnicas de biópsia, de biologia molecular e aspectos bioéticos dessa prática. Assim, foi possível agregar as informações como vantagens, desvantagens, restrições e indicações referentes ao estágio embrionário em que é retirado o material genético e as técnicas de biologia molecular usadas na análise genética. Além disso, foi possível especificar alguns questionamentos bioéticos que surgem com a prática do PGD, como, por exemplo, a possível eugenia. Concluiu‐se que a análise da trofoectoderme dos embriões e da aplicação da tecnologia de NGS é promissora para o futuro do PGD, bem como a primordialidade da criação de leis que regem e completam as lacunas no sentido ético e moral dessa prática.
... Preimplantation genetic testing for aneuploidy (PGT-A) informs the transfer of normal embryos based on chromosomal analysis [4] to avoid chromosomal aneuploidies, which can cause pregnancy loss and implantation failure [5]. Patients most likely to benefit from PGT-A are infertile women of advanced maternal age [6,7], while patients with a history of recurrent pregnancy loss or RIF may not benefit from PGT-A [8]. Although embryonic aneuploidy is likely a major contributor to human implantation failure, the proportion of euploid embryos failing to implant is approximately 30% [9,10], suggesting that endometrial Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10815-020-01948-7) ...
... Chromosomal analysis was performed by array comparative genomic hybridization (aCGH) or next-generation sequencing (NGS). Per iGenomix procedures and as specified by the manufacturer (Illumina), the 24sure aCGH platform has an effective 10-Mb resolution; therefore, only full chromosomal aneuploidies and segmental aneuploidies affecting chromosomal fragments larger than 10 Mb were identified [7,29]. Embryos were vitrified and transferred in subsequent natural or hormonal cycles. ...
Purpose:
To evaluate the clinical usefulness of the endometrial receptivity array (ERA) and the preimplantation genetic test for aneuploidy (PGT-A) in patients with severe and moderate recurrent implantation failure (RIF).
Design:
A retrospective multicenter cohort study was conducted in patients who failed to achieve implantation following transfer of 3 or more or 5 or more embryos in at least three single embryo transfers; patients were classified as moderate or severe RIF, respectively. Patients with previous RIF were compared based on the testing they received: PGT-A, ERA, or PGT-A+ERA versus a control group with no testing. Mean implantation rate and ongoing pregnancy rates per embryo transfer were considered primary outcomes. Multiple logistic regression analysis was performed and adjusted ORs were calculated to control possible bias.
Results:
Of the 2110 patients belonging to the moderate RIF group, those who underwent transfer of euploid embryos after PGT-A had a higher implantation rate than those who did not. Additionally, the PGT-A group had a significantly higher rate of ongoing pregnancy. The same outcomes measured for the 488 patients in the severe RIF group did not reveal any statistically significant improvements. The use of the ERA test did not appear to significantly improve outcomes in either group.
Conclusions:
PGT-A may be beneficial for patients with moderate recurrent implantation failure but not for severe cases. At its current level of development, ERA does not appear to be clinically useful for patients with RIF.
... 10 A partir de entonces este estudio se ha efectuado en numerosos centros de reproducción asistida alrededor del mundo; para el año 2010 habían nacido cerca de 10,000 seres humanos con estudio genético preimplantación. 11 Las técnicas de diagnóstico preimplantación han tenido una transición tecnológica que va desde la hibridación fluorescente in situ, [12][13][14][15] el estudio de microarreglos, [16][17][18][19][20] hasta la más reciente secuenciación de nueva generación. 21,22 La hibridación fluorescente in situ tiene la capacidad de establecer un diagnóstico específico para un número limitado de cromosomas; es una prueba rápida, capaz de detectar alteraciones numéricas y eliminaciones. ...
... Tiene una tasa de detección de mosaicismo de 4.8 a 32%, con más de 40% de células aneuploides. [16][17][18][19][20] El microarreglo de polimorfismos de secuencia única tiene la ventaja de detectar enfermedades monogénicas, triploidias y disomía uniparental además de variaciones en el número de copias. 23 Por último, el estudio de secuenciación de nueva generación tiene mayor resolución, detecta desequilibrios segmentarios entre 800 kb a 1 Mb. ...
OBJETIVO
Comunicar los resultados obtenidos del análisis del estudio genético preimplantación para aneuploidias en dos centros de reproducción asistida de México en un periodo de tres años, utilizando dos diferentes técnicas moleculares.
MATERIALES Y MÉTODOS
Estudio observacional, retrospectivo, en donde se reporta el resultado de blastocistos sometidos a preimplantación para aneuploidias durante 2014-2017, en dos centros de reproducción asistida (Ciudad de México y Guadalajara).
RESULTADOS
Se analizaron 404 blastocistos de 129 pacientes (edad promedio 39 ± 4 años). Los embriones se dividieron en dos grupos según la técnica aplicada: 76 por a-CGH y 328 por secuenciación de nueva generación. El porcentaje de embriones euploides fue de 33%. Las aneuploidias numéricas fueron las más frecuentes. Hasta la terminación del estudio se habían transferido 69 embriones euploides con tasas de implantación de 78% para secuenciación de nueva generación y de 57% para a-CGH.
CONCLUSIONES
La tasa de implantación reportada en este estudio fue mayor con el análisis de preimplantación para aneuploidias por secuenciación de nueva generación. Los resultados reportados en nuestra experiencia soportan la necesidad de favorecer una opción de transferencia de embrión único. Es importante reconocer los retos de las nuevas tecnologías y la necesidad de técnicas moleculares más sensibles.
... Also, the relationship between chromosomal abnormality and morphological grade is still not clear. For example, top quality embryos and blastocysts were sometimes genetically abnormal with the incidence of abnormalities as high as 50%, while poor grade embryos were characterized normal (12,13,19). Indeed, this confirms the limited prediction power of embryo morphology on implantation rate (20). ...
... Recently, comprehensive chromosomal screening for all chromosomes is suggested using single nucleotide polymorphism array, comparative genomic hybridization array or quantitative PCR which is more informative compared to the FISH techniques evaluated fewer than half of the chromosomes in each embryo (12)(13)(14)19). However, it was shown that the five-chromosome test detects 28-31%, nine probes detect 70-72% and the 12probe test detects 79-80% of chromosome abnormalities found in fetuses (9). ...
Background
Selection of the best embryo for transfer is very important in assisted reproductive technology (ART). Using morphological assessment for this selection demonstrated that the correlation between embryo morphology and implantation potential is relatively weak. On the other hand, aneuploidy is a key genetic factor that can influence human reproductive success in ART.
Objective
The aim of this lab trial study was to evaluate the incidence of aneuploidies in five chromosomes in the morphologically high-quality embryos from young patients undergoing ART for sex selection.
Materials and Methods
A total of 97 high quality embryos from 23 women at the age of 37or younger years that had previously undergone preimplantation genetic screening for sex selection were included in this study. After washing, the slides of blastomeres from embryos of patients were reanalyzed by fluorescence in-situ hybridization for chromosomes 13, 18 and 21.
Results
There was a significant rate of aneuploidy determination in the embryos using preimplantation genetic screening for both sex and three evaluated autosomal chromosomes compared to preimplantation genetic screening for only sex chromosomes (62.9% vs. 24.7%, p=0.000). The most frequent detected chromosomal aneuploidy was trisomy or monosomy of chromosome 13.
Conclusion
There is considerable numbers of chromosomal abnormalities in embryos generated in vitro which cause in vitro fertilization failure and it seems that morphological characterization of embryos is not a suitable method for choosing the embryos without these abnormalities.
... Conversely, with optimized embryo culture systems, appropriate biopsy procedures, and better patient inclusion criteria, there is a place for PGS in an IVF setting. In contrast to earlier data, PGS studies were conducted in advanced maternal age patients between 41 and 44 years of age, resulting in live birth rates of 32.3% compared with 15.5% in the blastocyst group 51 . Thus, if proper blastomere biopsy procedures and good laboratory conditions are applied, PGS with classic FISH is confirmed to be beneficial 52 . ...
... Therefore, an alternative to achieving an optimal number of embryos for biopsy is oocyte vitrification 55,57 or vitrification at the cleavage stage 58 . By using array-CGH, similar aneuploidy rates were observed in fresh cycles (72.6%) compared with mixed cycles with vitrified oocytes or cleavage-stage embryos (75.7% and 73.4%, respectively) 51 . Nevertheless, the potential increase in live birth rates with a more comprehensive aneuploidy screening must be tested. ...
Reduced maternal fertility is the consequence of depletion of follicles with maternal aging. In a 35-year-old woman, approximately 9.1% of the residual follicle pool disappears annually without entering into the growing stage, whereas, in a 45-year-old woman, this number triples. After the age of 35 years, the frequency of aneuploidies in oocytes increases sharply. Roughly 50-70% of mature oocytes from a 40-year-old woman have chromosomal abnormalities. The clinical pregnancy and implantation rates are lower in midlife women. Various controlled ovarian stimulation interventions have been suggested for the management of women in advanced age, most of whom are likely to be poor-responder patients. Currently, systematic reviews and meta-analyses suggest that there is insufficient evidence to recommend most of the treatments proposed to improve pregnancy rates in these poor responders. Minimal stimulation or natural cycle in vitro fertilization may be offered, without compromising the already existing pregnancy results.
... However, more and more recent studies had made us known that the ironic outcome revealed by that randomized study may be due to the limitations of the genetic tool used for PGS itself (FISH), as well as the timing of the biopsy was set at the cleavage stage embryos (Day 3). The outcomes of PGS, especially the most important livebirth rate, are at least greatly affected by three critical parameters: the timing of biopsy, the genetic tools used for PGS, and to freeze or not to freeze the biopsied embryos [8][9][10][11][12]. After 2007, since the emergence of many new technologies available for genetic investigation, researchers are keen to verify and validate the new tools when being used in PGS [5,[13][14][15]. ...
... Therefore in before for all amplification-based technologies, including aCGH and NGS, it is not feasible to do fresh embryo transfer if the timing of the biopsy was set at Day 5/6. Earlier studies did report some cycles based upon PGS by aCGH on Day 3 embryos [10,[22][23][24] , but very few at Day 5/6 because the protocol of aCGH usually needs at least 2–3 days, in which the time for analysis was not counted in [25]. To freeze the biopsied embryos sent for PGS, and to transfer the thawed euploid embryos in non-stimulated cycles, becomes the standard practice for most IVF-PGS centers. ...
Background:
Single embryo transfer (SET) has been utilized as a strategy to reduce the chance of multifetal gestations in in vitro fertilization (IVF) but lower pregnancy rate remains a concern. Recent studies showed that favorable outcome regarding SET can be achieved by selecting embryos with "more normal" genetic components. We explored the use of rapid array comparative genomic hybridization (aCGH) to select blastocysts for fresh SET and compared with the protocols adopting vitrified (ultrarapidly frozen) embryo transfer cycle. Validation of the rapid protocol of aCGH and comparison of the result with the regular protocol of aCGH and next generation sequencing (NGS) are also performed.
Results:
First-time IVF patients with normal karyotype (n = 21) were enrolled for elective fresh SET cycle (n = 8; designated as fresh SET group) or vitrified embryo transfer cycle (n = 13; designated as vitrified ET group) coupling with comprehensive chromosomal screening by a 9-h rapid aCGH from Day 5 trophectoderm (TE) biopsy. In fresh SET group, 86 blastocysts (10.8 blastocysts/patient) were biopsied and analyzed. Aneuploidy was detected in 53.5 % (46/86) of the biopsied blastocysts. All patients had a single embryo transferred on the following day. The clinical pregnancy rate was 87.5 % (7/8) and the ongoing pregnancy rate was 62.5 % (5/8). In vitrified ET group, 58 blastocysts (4.5 blastocysts/patient) were biopsied and 56 blastocysts were analyzed. Aneuploidy was detected in 39.3 % (22/56) of biopsies. The patients accepted for SET or double embryos transfer (DET) in non-stimulated cycles. The clinical pregnancy rate and the ongoing pregnancy rate was 76.9 % (10/13) and 53.8 % (7/13) respectively. Spontaneous abortions occurred in both of the two patient groups. In the series of fresh SET group, no twin pregnancy was noted and at least one healthy baby had been born at gestational age (GA) 37(+6) weeks when submission. The results of PGS by rapid aCGH, regular aCGH and NGS were comparable in most occasions.
Conclusion:
This study evaluates the use of rapid aCGH to select blastocysts for fresh SET and demonstrates its feasibility in a real clinical IVF program. A successful livebirth is achieved and the favorable outcome is superior to the protocol adopting vitrified ET cycle in our own setting. Additional studies are needed to verify this pilot data and validate its application in large randomized trials.
... PGT has undergone significant improvements with the addition of nested PCR, multiple displacement amplification, MALBAC, array comparative genomic hybridization, SNP arrays, and NGS [8,[17][18][19]. Our PGT methodology was formulated based on MARSALA, which was first reported in 2015 and demonstrated solid precision in linkage analyses [9,20]. ...
... There are reports showing that blastocyst implantation rates are higher compared to embryo transfer in the early stages (11). In addition, trophectoderm biopsy has significantly improved and become more popular in infertility centers (12). In vitro fertilization process enables infertility centers to successfully grow the embryo to the blastocyst stage, which is an appropriate stage for biopsy sampling (13). ...
Background: Preimplantation genetic diagnosis (PGD) is a diagnostic approach in assisted reproductive technology (ART) to detect and select unaffected embryos to be transferred. Obtaining biopsy samples from embryos (polar body, blastomere, or blastocyst) is a key step in preimplantation genetic testing (PGT), which has many technical issues. Objectives: This study aimed to evaluate the effect of biopsies from 3-day embryos (blastomere) on the quality of embryos and implantation success in couples who requested sex selection before embryo transfer. Methods: On the third day after fertilization, 352 high-quality embryos (> six cells on day third with < 10% fragmentation) were collected from 77 women and were tested for sex selection using FISH testing. A laser beam was used to obtain blastomere biopsies by removing a significantly small portion of the zona pellucida. One blastomere was gently biopsied by an aspiration pipette through its hole. After biopsy sampling, the embryo was immediately returned to the embryo scope until transfer. Embryos’ integrity and blastocyst formation were assessed on day 5. Results: A total of 595 embryos were studied, including 352 embryos that were biopsied on day 3 for gender selection (i.e., the intervention group) and 243 intracytoplasmic sperm injection (ICSI) embryos that did not undergo biopsy (i.e., the control group). Overall, 17.1% of the embryos were abnormal for X or Y chromosomes. Biopsy for PGD was performed 67 - 73 hours after ICSI. Blastomere biopsy taking was significantly associated with blastocyst quality and implantation success. Conclusions: In this study, after obtaining blastomere biopsies, we investigated the growth process of the embryos according to morphokinetic parameters. Our results showed that blastomere biopsy taking could affect the blastulation of embryos and decrease the success rate of implantation.
... PGT has been in development for almost 30 years, with the successful addition of techniques such as nested-PCR, multiple displacement amplification (MDA), MALBAC, array comparative genomic hybridization (CGH), SNP array and NGS [8,[32][33][34]. Routine PGT-M requires a complex process of preparatory work that incorporates linkage marker selection, primer design, development of amplification conditions, and single-cell validation [34]. ...
Purpose
To evaluate the clinical validity of preimplantation genetic testing (PGT) to prevent hereditary hearing loss (HL) in Chinese population.
Methods
A PGT procedure combining multiple annealing and looping-based amplification cycles (MALBAC) and single-nucleotide polymorphisms (SNPs) linkage analyses with a single low-depth next-generation sequencing run was implemented. Forty-three couples carried pathogenic variants in autosomal recessive non-syndromic HL genes, GJB2 and SLC26A4, and four couples carried pathogenic variants in rare HL genes: KCNQ4, PTPN11, PAX3, and USH2A were enrolled.
Results
Fifty-four in vitro fertilization (IVF) cycles were implemented, 340 blastocysts were cultured, and 303 (89.1%) of these received a definite diagnosis of a disease-causing variant testing, linkage analysis and chromosome screening. A clinical pregnancy of 38 implanted was achieved, and 34 babies were born with normal hearing. The live birth rate was 61.1%.
Conclusions and relevance
In both the HL population and in hearing individuals at risk of giving birth to offspring with HL in China, there is a practical need for PGT. The whole genome amplification combined with NGS can simplify the PGT process, and the efficiency of PGT process can be improved by establishing a universal SNP bank of common disease-causing gene in particular regions and nationalities. This PGT procedure was demonstrated to be effective and lead to satisfactory clinical outcomes.
... However, given the lack of any strong correlation between the embryo morphology and implantation success, the embryo transfer based on the morphology of embryo is still remains limited [7,8]. Furthermore, high-quality embryos may be genetically abnormal, while lowquality embryos are represented as normal [11]. Previous reports showed that the embryo quality has limited effects on implantation rate [12]. ...
Objective: To detect common chromosomal aneuploidy variations in embryos from couples undergoing assisted reproductive technology and preimplantation genetic screening and their possible associations with embryo quality.
Methods: In this study, 359 embryos from 62 couples were screened for chromosomes 13, 21, 18, X, and Y by fluorescence insitu hybridization. For biopsy of blastomere, a laser was used to remove a significantly smaller portion of the zona pellucida. One blastomere was gently biopsied by an aspiration pipette through the hole. After biopsy, the embryo was immediately returned to the embryo scope until transfer. Embryo integrity and blastocyst formation were assessed on day 5.
Results: Totally, 282 embryos from 62 couples were evaluated. The chromosomes were normal in 199 (70.57%) embryos and abnormal in 83 (29.43%) embryos. There was no significant association between the quality of embryos and numerical chromosomal abnormality (P=0.67).
Conclusions: Embryo quality is not significantly correlated with its genetic status. Hence, the quality of embryos determined by morphological parameters is not an appropriate method for choosing embryos without these abnormalities.
... Embryo biopsy was performed on day five at the blastocyst stage in all cases, and a single blastomere was withdrawn from each embryo with laser-assistance [22,23]. The most common PGT-A indications in this study were patients with a history of recurrent miscarriage, repetitive implantation failure, advanced maternal age, or male factor infertility. ...
Background
Infertility is defined as the inability to establish a pregnancy within 12 months of regular and unprotected
sexual intercourse. In response to these problems, assisted reproductive techniques (ARTs) have made
profound impacts on the therapeutic management of infertility. However, in-vitro fertilization (IVF) success
rates are confounded by several internal and external factors. A relatively new approach to embryo
assessment is known as MitoScore (Igenomix, Miami, USA). As a result, we sough to evaluate whether
MitoScore can help in predicting in IVF outcomes, and to assess the relationship between MitoScore, BMI,
and body fat percentage in determining the success of ARTs.
Methods
Using retrospective cohort, a study population consisting of 166 women aged 26-43 who were undergoing
ART with pre-implantation genetic testing for aneuploidy (PGT-A) was assessed to determine if MitoScore,
BMI, and body fat percentage impacted IVF outcomes.
Results
MitoScore, BMI, and body fat percentage were significantly lower in pregnant women as compared to nonpregnant
women. Furthermore, MitoScore was correlated with subclasses of IVF outcomes (delivery,
biochemical pregnancy, and spontaneous abortion) and was found to be positively correlated with BMI in
patients with biochemical pregnancies.
Conclusion
Our findings suggest that MitoScore, BMI, and body fat percentage could act as critical parameters in
determining the success of ART. However, the association between MitoScore, BMI, and body fat percentage
does not appear to be a significant confounding factor to determine pregnancy outcome at this stage. Still,
many factors need to be considered to establish the correlation reliably.
Categories: Urology
Keywords: body mass index, assisted reproductive technique, body fat, fertility, mitoscore
... When at least 2 follicles had reached R18 mm in diameter, human chorionic gonadotropin (hCG) and/or gonadotropin-releasing hormone agonist was administered, and oocyte retrieval was scheduled 35-36 hours later. Conventional insemination or intracytoplasmic sperm injection was performed, and embryo culture proceeded to the blastocyst stage, at which time embryo transfer, embryo cryopreservation, or embryo biopsy was performed, as previously described (20,21). Preimplantation genetic testing for aneuploidy was performed for different indications (recurrent miscarriage, repetitive implantation failure, advanced maternal age, or male factor infertility). ...
Objective
To study the beneficial effects of Thyroid replacement therapy (TRT) on pregnancy outcomes in patients with subclinical hypothyroidism (SCl hypoT) with respect to thyroid peroxidase autoantibodies (TPO).
Design
Retrospective study of 706 patients
Setting
Not applicable
Patients
Study evaluated 706 patients, which were defined into three cohorts: a) euthyroid, with pre-IVF thyroid stimulating hormone (TSH) levels < 2.5 uIU/mL, b) SCl hypoT defined as TSH levels > 2.5 uIU/mL and <4 uIU/mL who were not treated and c) SCl hypoT who received thyroid replacement therapy. The three cohorts were further subclassified into two groups each based on TPO antibody levels.
Interventions
The cohorts were compared for the effects of TRT on pregnancy outcomes.
Main Outcome Measures
Identification of effects of thyroid replacement therapy on ART outcomes
Results
Results showed that SCl hypoT patients had a significantly fewer positive pregnancy outcomes compared to euthyroid patients. Importantly, low dose TRT was found to be beneficial in improving IVF success rates and pregnancy outcomes in SCl hypoT patients. The original cohort of patients, further classified into two subgroups based on antithyroid (TPO) antibodies showed that low dose TRT was associated with improved pregnancy outcomes in women with SCl hypoT and TPO positive antibodies.
Conclusions
Our findings demonstrate that low dose TRT may be beneficial in improving IVF success and pregnancy outcomes in women with SCl hypoT and positive TPO antibodies.
... The evolution of ART and the advances of molecular biology have enabled the development of new techniques to analyze the number of chromosomes and establish the embryo ploidy, optimizing the transfer of chromosomally healthy embryos (Ebner et al., 2003;Harper et al., 2010). Several molecular techniques have been used in recent years to determine the embryo's genetic quality, including Fluorescent In Situ Hybridization (FISH) (Álvarez Sedó, 2018;Wilton, 2002), Array Comparative Genomic Hybridization (aCGH) (Ata et al., 2012;Rubio et al., 2013b;Rodrigo et al., 2014), quantitative real-time PCR (qPCR), Single Nucleotide Polymorphisms (SNP), and Next Generation Sequencing (NGS) Kung et al., 2015;Palmerola et al., 2019). The aCGH analyzes the entire genome with a high resolution; it is a powerful, agile, and highly specific tool suitable to diagnose chromosome gains; however, it cannot detect point mutations or balanced rearrangements (Shinawi & Cheung, 2008). ...
Objective:
To analyze the results of the preimplantation genetic testing for aneuploidy at the Instituto de Fertilidad Humana - Inser Bogotá, Colombia, from 2016 to 2020.
Methods:
This study is an observational, retrospective, and correlative analysis of biopsies from 319 embryos (from 54 patients) submitted to preimplantation genetic testing for aneuploidy by different molecular techniques.
Results:
Of the 54 patients included in the study, 42 provided their own oocytes, and 12 used donated oocytes. The main indication to perform the preimplantation genetic testing was advanced maternal age. We obtained 319 embryos: Ninety-one (28.5%) euploid, 197 (61.8%) aneuploid and 31 (9.7%) with no detectable DNA. The highest rate of aneuploid embryos was found in patients over 40 years (72.7%), and the euploidy rate in patients under 35 years was 37.1%. After the transfer of euploid embryos, the rates for implantation, ongoing pregnancy, live birth, and miscarriage were 40%, 50%, 40.6%, and 0%, respectively. Older maternal age correlated with higher numbers of aneuploid embryos and lower numbers of both euploid and 5-day embryos.
Conclusions:
There was a positive correlation between maternal age and aneuploidy rate. Complex chromosomal abnormalities were the most frequent aneuploidies, followed by mosaicism and double aneuploidies. The miscarriage rate after the transfer of euploid embryos was 0 %.
... 2g). aCGH provides a higher resolu tion than FISH as its probes are several orders of magni tude smaller than chromosomes 138 . Testing with aCGH showed that sperm from men undergoing chemother apy had a higher rate of chromosome aberration than healthy donors (23.8% versus 7.8%) 139 . ...
Infertility affects one in six couples worldwide, and fertility continues to deteriorate globally, partly owing to a decline in semen quality. Sperm analysis has a central role in diagnosing and treating male factor infertility. Many emerging techniques, such as digital holography, super-resolution microscopy and next-generation sequencing, have been developed that enable improved analysis of sperm motility, morphology and genetics to help overcome limitations in accuracy and consistency, and improve sperm selection for infertility treatment. These techniques have also improved our understanding of fundamental sperm physiology by enabling discoveries in sperm behaviour and molecular structures. Further progress in sperm analysis and integrating these techniques into laboratories and clinics requires multidisciplinary collaboration, which will increase discovery and improve clinical outcomes.
... Previous studies on patients with RIF have shown a strong skew toward down-regulation of the expression of endometrial receptivity genes [44,45]. Given the findings of the present study, it may be speculated that the genes were not normally up-regulated in the endometrial samples of RIF patients reported by Koler et al. [44] and Bastu et al. [45], possibly due to delayed transformation processes in the endometrium at the time of biopsy [46]. To support this hypothesis, it has been reported that endometrial samples from patients with RIF show a lack of typical secretory changes in comparison with fertile patients; this indicates an early secretory stage of the endometrium in patients with RIF and does not imply that they are unable to achieve mature endometrium with a receptive window of implantation [47]. ...
Purpose
Limited information is clinically available concerning endometrial receptivity; assessing endometrial transformation status is therefore an urgent topic in assisted reproductive technology. This study aimed to investigate individual endometrial transformation rates during the secretory phase in subfertile patients using personal endometrial transformation analysis.
Methods
Monitoring was carried out during the secretory phase to obtain endometrial receptivity profiles. For the investigation, two endometrial biopsies were taken within one menstrual cycle. The extended endometrial dating was based on the Noyes criteria, combined with immunohistochemical analyses of hormone receptors and proliferation marker Ki-67. Biopsies were taken mainly at days ovulation (OV, n = 76)/hormone replacement therapy (HRT, n = 58) + 5 and + 10.
Results
The results of the two biopsies were correlated with the clinically expected day of the cycle and showed temporal delays or hypercompensations, diverging from the expected cycle days by 0.5–5 days. In comparison with the first biopsies, the transformation rate in the second biopsies showed compensation, augmented delay, or constant transformation in 48.69, 22.37, and 28.94% of cases for ovulation in natural cycles and 56.89, 25.85, and 17.26% for HRT cycles, respectively.
Conclusion
The study revealed an individually dynamic transformation process of the endometrium, with the ability to compensate or enlarge an initial “delay”, which is now identified as a normal individual transformation process during the secretory phase. This information is of great importance for the scientific investigation of dynamic changes in endometrial tissue, as well as for the timing of embryo transfers.
... Much has been published concerning the advantages and disadvantages of sampling embryos at different stages, the different genetic testing techniques, which patient groups might benefit from testing embryos, which outcome measures are the most appropriate and what constitutes a well-designed clinical trial, and the current status of the clinical evidence available [11][12][13][14][15][16][17][18]. Whilst it is recognised that much of the published data does not meet the highest level of medical evidence, many eminent scientists and clinicians in the field have expressed the opinion in a recent statement that PGS should no longer be considered an experimental procedure and should be discussed with all patients considering assisted conception [19]. ...
A decision model was constructed to compare genetic testing and not testing, for the transfer of all suitable embryos, one at a time, from a cycle with up to ten embryos, until a first live birth was achieved or there were no more embryos available (a full cycle). Two strategies were investigated: (i) a fresh transfer with subsequent serial warmed cryopreserved embryo replacement, and (ii) freeze-all prior to serial embryo replacement. Sensitivity analyses were performed to assess the effect of embryo warming survival and diagnostic accuracy on cumulative rates. Cost-effectiveness was assessed using the incremental cost-effectiveness ratio for a live birth event, and a clinical miscarriage avoided. Reproductive outcome probabilities were obtained from published prospective non-selection studies, and costs from websites and publications.
Given 100% embryo warming survival and no false abnormal genetic test results, the live birth rate for a full cycle was the same with and without testing for both transfer strategies. Compared to not testing, it was theoretically possible for testing to be favoured for live birth only for the fresh and frozen transfer strategy, where more than one embryo was available, and dependent on the efficiency of warming survival and the positive predictive value of the test; however, this was unlikely to be cost-effective from a society perspective without a substantial reduction in genetic testing costs. For both transfer strategies, when more than one embryo was available, testing was more likely to achieve a live birth event following the first attempt with fewer attempts required overall. Testing was likely to be effective to avoid a clinical miscarriage but also to be expensive from a society perspective compared to the cost of dilation and curettage.
... Fertility treatment is stressful and is known to have significant psychological impact on couples. 1 Pre-implantation genetic testing (PGT) for aneuploidy (PGT-A) has been available for couples undergoing IVF for nearly 20 years. 2 PGT-A involves retrieving five to ten trophoectoderm cells from a blastocyst and screening for the presence of a normal number of chromosomes, with only euploid embryos selected for transfer. 3 Requests for PGT-A are now in demand worldwide and have presented both patients and clinicians with the decision of when to use PGT-A as an adjuvant treatment to standard IVF therapy. ...
Introduction:
Pre-implantation genetic testing for aneuploidy (PGT-A) is in high demand worldwide, with ongoing debate among medical societies as to which patient groups it should be offered. The psychological aspects for patients regarding its use, lag behind the genomic technological advances, leaving couples with limited decision-making support. The development of this technology also leads to the possibility for its utilization in gender selection. Despite the controversy surrounding these issues, very few studies have investigated the psychological aspects of patients using PGT-A.
Material and methods:
This systematic review provides an up-to-date analysis of the psychosocial aspects surrounding PGT for aneuploidy and sex selection, as well as decision-making factors. A systematic search of English peer-reviewed journals of three computerized databases were undertaken following PRISMA guidelines. The qualitative data were extracted using thematic analysis. PROSPERO Registration number: CRD42019126439.
Results:
The main outcome measures were patients' motivations, decision-making factors, attitudes and experiences surrounding the use of PGT for aneuploidy and sex selection. Ten studies were included, four for PGT-A and six for sex selection. Attitudes towards PGT-A were positive, with the main motivating factors being decreasing miscarriage rate, reducing the risk of termination of pregnancy and reducing the time to pregnancy. Consistently raised concerns regarding PGT-A were the financial burden and moral beliefs. The vast majority of patients felt sufficiently knowledgeable to make the decision; however, studies did reveal that a minority mis-interpreted certain potential benefits of PGT-A. Studies investigating PGT for sex selection predominantly reported the main motivation was to achieve gender balance within the family dynamic, with most studies finding no difference between couples using PGT for gender selection to have male or female offspring.
Conclusions:
Although this systematic review was limited by the small number of studies investigating this topic, a significant minority of patients appeared to misunderstand certain benefits and limitations of PGT-A. Fertility clinics must ensure they provide adequate counselling to all patients using PGT-A. With the use of PGT-A on the rise globally, there is a need to develop decision support tools for couples who have an increasing number of genetic testing options becoming available to them.
... Blastomere number, multinucleation, fragmentation of embryo, and blastocyst formation are the key factors associated with viability https://www.id-press.eu/mjms/index of embryo. The relationship between morphology and embryo aneuploidy was first evaluated with Fluorescence in situ hybridization (FISH) studies for 7-8 chromosomes [6]. Pellicer's experience showed that a remarkable rate of embryos with chromosomal abnormality was able to develop to the blastocyst with suitable morphology parameters; in fact, 42.8% of chromosomally abnormal embryos reached blastocyst stage [7]. ...
BACKGROUND: Aneuploidy is a major cause of miscarriages and implantation failure. Preimplantation genetic testing aneuploidy (PGT-A) by Next Generation Sequencing (NGS) is able to detect of the numeral and structural chromosomal abnormalities of embryos in vitro fertilization (IVF).
AIM: This study was aimed to assess the relationship between maternal age and chromosomal abnormalities NGS technology.
MATERIAL AND METHODS: 603 human trophectoderm (TE) biopsied samples were tested. RESULTS: Among the 603 TE samples, 247 samples (42.73 %) presented as chromosomal abnormalities. The abnormalities occurred to almost chromosomes, and the most popular aneuploidy observed is 22. Aneuploidy rate from 0.87% in chromosome 11 to 6.06% in chromosome 22. The rate of abnormal chromosome increased dramatically in group of mother's ages over 37 (54.17%) comparing to group of mother's ages less than 37 (38.05%) (p
... Сравнительная геномная гибридизация array-CGH оказалась надежным методом ПГТ и широко используется в клиниках репродукции по всему миру [14][15][16][17][18][19][20]. Преимуществом метода array-CGH на биологических чипах является способность выявлять как хромосомные нарушения (анеуплоидии, в том числе и мозаицизм, несбалансированные транслокации и маркерные хромосомы), так и микроделеционные/ микродупликационные синдромы, несбалансированные субтеломерные перестройки одномоментно во всем геноме. ...
... With an increase in the popularity of PGS, there is a growing need for concurrent PGD/PGS. Currently, the strategies used in PGS include array-based (either array-comparative genomic hybridization or single nucleotide polymorphism chromosomal microarray) techniques [55][56][57], qPCR-based techniques [58,59], and next-generation sequencing (NGS)-based techniques [26,42,60,61]; we are excluding the outdated fluorescence in situ hybridization (FISH)-based diagnostics [62]. Some of the techniques were reported to be successfully applied in concurrent PGD/PGS [21,26,63,64]. ...
Preimplantation genetic diagnosis (PGD) has become a crucial approach in helping carriers of inherited disorders to give birth to healthy offspring. In this study, we review PGD methodologies and explore the use of amplification refractory mutation system quantitative polymerase chain reaction (ARMS-qPCR) and/or linkage analysis for PGD in neurodegenerative diseases that are clinically relevant with typical features, such as late onset, and which are severely debilitating. A total of 13 oocyte retrieval cycles were conducted in 10 cases with various neurodegenerative diseases. Among the 59 embryos analyzed, 49.2% (29/59) were unaffected and 50.8% (30/59) were affected. Of the 12 embryo transfer cycles, three resulted in pregnancy, and all pregnancies were delivered. The implantation rate and livebirth rate were 23.1% (3/13) per oocyte retrieval cycle and 25.0% (3/12) per embryo transfer cycle. Allele dropout (ADO) was noted in two embryos that were classified as unaffected by ARMS-qPCR but were evidenced as affected after prenatal diagnosis, rendering the false negative rate as 6.3% (2/32). Four among the 13 cycles underwent PGD by ARMS-qPCR coupled with linkage analysis, and all were correctly diagnosed. We conclude that PGD by ARMS-qPCR and/or linkage analysis is a feasible strategy, whereas ADO is a concern when ARMS-qPCR is used as the sole technology in PGD, especially in autosomal dominant diseases.
... For the last 25 years, preimplantation genetic diagnosis was one of the major application of genomics in ART to diagnose chromosome and genome integrity abnormalities or specific gene disorders to eliminate the 2/3 Recently, Genomic or expression microarray technology, Comprehensive chromosome screening and new generation sequencing are becoming common tools for genome and gene investigation in research and development and in clinical diagnostic to predict and select the best gametes and embryo. For genome profiling and high resolution molecular karyotyping, array comparative genome hybridization and sequencing methods appears to be far better and have much higher sensitivity and specificity for subtle genomic changes [15][16][17][18][19][20]. In reproductive medicine and IVF, Nano genomics and omes technologies (genomic, expression or proteins arrays) will be an important clinical assay for genomics, transcriptomics and proteomics testing. ...
... Preimplantation genetic testing for aneuploidy (PGT-A) (previously termed preimplantation genetic screening (PGS)) has been available to patients undergoing the process of in vitro fertilization (IVF) to attempt to improve their reproductive outcomes for nearly 20 years [1]. The utilization of PGT-A was established due to the frequency of aneuploidy among human embryos, which often do not result in a successful outcome or live birth. ...
Purpose:
Embryo testing to improve pregnancy outcomes among individuals who are seeking assisted reproduction technologies is increasing. The purpose of this study was to assess decisional factors through in-depth interviews for why women would accept or decline preimplantation genetic testing for aneuploidy (PGT-A) with in vitro fertilization (IVF).
Methods:
Semi-structured telephone interviews were conducted with 37 women who were offered PGT-A with IVF during the summer 2017. Interviews lasted on average 40 min and were audio-recorded, transcribed, and analyzed using a content analysis.
Results:
Results identified a number of decisional factors related to values about conception, disability, and pregnancy termination, past pregnancy experiences, optimism toward technology, and cost. Other key issues that were identified include the use of expanded carrier screening prior to IVF, maternal age, and limited education about PGT-A due to the complexity about education for IVF alone.
Conclusion:
There is a need to develop decision support tools for the increasing choices of genetic testing options for patients seeking IVF. Including patients' values, past pregnancy experiences and attitudes toward science into the decision-making process may help promote a more informed decision.
... A recently published review by Simpson in 2012 suggested that array-CGH (aCGH) was the preferred approach for CCS in PGS [20]. Although aCGH for PGS has been well validated for the detection of aneuploidy or an abnormal number of chromosomes in embryos obtained during IVF treatment [21], this method is expensive and difficult to scale up to meet the increasing demand of IVF cycles. ...
Objective:
This study aims to validate the BACs-on-Beads (BoB) technology as a robust and high throughput method for pre-implantation genetic screening (PGS) for aneuploidy.
Material and methods:
The performances with respect to the sensitivity, specificity, success rate and detection rate of this technique from new BoBs technology and traditional array chromosomal genomic hybridization (aCGH) were compared. And the use of BoBs as a screening tool for euploid embryos in PGS was evaluated.
Result:
In the first part of validation study, there were total 75 embryos completed PGS by both BoBs and aCGH. The success rate of PGS was 97.4%, and the results showed 100% concordance between BoBs and aCGH for aneuploidy. In the second part, a total 219 embryos were involved. The success rate of PGS by BoBs was 100%. BoBs identified 28% (62/219) euploidy which were further confirmed to be euploidy by aCGH.
Conclusion:
This new strategic approach using BoBs as a first tier PGS screening tool and aCGH as a confirmatory tool can increase the throughput of PGS with a reduced cost and time to meet the demand in high volume units.
... In the post-genomic era, the high-resolution microarray platform has been progressively adopted for medical diagnostics which offers large-scale and in-depth screening of genomic imbalances. Ongoing validation efforts of this technique for the genetic testing of some pathogenic chromosomal disorders have been reported [Lee et al., 2007;Shearer et al., 2007;Yu et al., 2009;Slovak et al., 2010;Park et al., 2013;Peterson, 2013;Rubio et al., 2013;Mehrotra et al., 2014;Zahir, 2015;Pratte-Santos et al., 2016;Song et al., 2017]. Since the type and prevalence of structural and numerical imbalances are variable worldwide, a suitable validation and implementation of genome-wide screening approaches in a clinical setting is required. ...
DiGeorge syndrome (DGS) is a genetic disorder known as a clinically variable syndrome with over 180 associated phenotypic features. It is caused by a common human deletion in the 22q11.2 chromosomal region and currently is affecting approximately 1 in 4,000 individuals. Despite the prevalence of inherited diseases mainly due to consanguineous marriages, the current diagnosis of DGS in Saudi Arabia is mainly based on conventional high-resolution chromosome banding (karyotyping) and FISH techniques. However, advanced genome-wide studies for detecting microdeletions or duplications across the whole genome are needed. The aim of this study is to implement and use aCGH technology in clinical diagnosis of the 22q11.2 deletion in Saudi Arabian DGS patients and to confirm its effectiveness compared to conventional FISH and chromosome banding techniques. Thirty suspected DGS patients were assessed for chromosome 22q11.2 deletion using high-resolution G-banding, FISH, and aCGH. The aCGH results were compared with those obtained by the other 2 cytogenetic techniques. G-banding detected the 22q11.2 deletion in only 1 patient in the cohort. Moreover, it detected additional chromosomal aberrations in 3 other patients. Using FISH, allowed for detection of the 22q11.2 deletion in 2 out of 30 patients. Interestingly, the use of aCGH technique showed deletions in the chromosome 22q11.2 region in 8 patients, indicating a 4-fold increase in diagnostic detection capacity compared to FISH. Our results show the effectiveness of aCGH to overcome the limitations of FISH and G-banding in terms of diagnostic yield and allow whole genome screening and detection of a larger number of deletions and/or duplications in Saudi Arabian DGS patients. Except for balanced translocations and inversions, our data demonstrate the suitability of aCGH in the diagnostics of submicroscopic deletion syndromes such as DGS and most chromosomal aberrations or complex abnormalities scattered throughout the human genome. Our results recommend the implementation of aCGH in clinical genomic testing in Saudi Arabia to improve the diagnostic capabilities of health services while maintaining the use of conventional cytogenetic techniques for subsequent validation or for specific and known aberrations whenever required.
... However, the most detailed FISH-based studies evaluated fewer than half of the chromosomes in each embryo. As only limited numbers of chromosomes were examined, while comprehensive chromosomal screening for all the 24 chromosomes is more informative compared to the FISH techniques (Rubio et al., 2013;Scott et al., 2013). However, performing these techniques may not be always possible for all couples and embryos due to the number or quality restriction of oocytes/embryos, the availability of techniques for IVF centers, legal restrictions, and finally social or simply cost-related issues (Calabrese et al., 2012;Fragouli et al., 2011;Meseguer et al., 2011). ...
Aneuploidy is of great relevance to embryo selection, as it represents one of the important causes of implantation failure. Furthermore, immature oocytes, retrieved during gonadotrophin-stimulated IVF cycles, are generally discarded in clinics; whereas, there was no detectable comprehensive evidence on higher rates of aneuploidy based on maturity status on the day of oocyte retrieval. As well, the correlation between embryo morphology on aneuploidy remains unclear. The aim was to evaluate the developmental and genetic integrity of human preimplantation embryos from rescue in-vitro matured MII stage oocytes as well as in vivo matured oocytes. 541 rescue in-vitro matured oocytes as case as well as 659 in-vivo matured oocytes as control were used for the developmental assay. Finally, 121 cleaved embryos with good quality were analyzed by FISH technique for the detection of chromosomes X, Y, 13, 15, 16, 18, 21 and 22. The fertilization rates were 61.62% and 61.76% in case and control groups, respectively. Also, embryo formation rates of 89.1% vs. 92.2% were recorded for case and control groups, respectively. Good quality embryos on day 3 were 62.54% in case and 68.36% in control groups. There were insignificant differences in fertilization, embryo formation and quality between the groups. Total abnormality in 35 of the 60 embryos was 58.5% in case and 62.3% in control (p < 0.05). There were significant differences between aneuploidy rates of embryos using only sex chromosome preimplantation genetic screening (PGS) and sex chromosome in combination with autosomal chromosomes PGS in case (58.5% vs 28.3%, p = 0.000) and control groups (62.3% vs 21.3%; p = 0.000). The results demonstrated that a high proportion of good quality embryos were aneuploid in both patient groups with no obvious increase in aneuploidies as a result of rescue IVM application. Furthermore, the morphological characteristics of embryos do not completely consistent with chromosomal content. Despite the Rescue IVM is currently not a routine procedure in association with IVF, our finding suggested a viable option for young infertile women facing cancellation of their IVF treatment due to ovarian over-response or resistance factors as well as patients with low functional ovarian reserve considering good quality of embryos from rescue IVM-MII oocytes.
... However, the most detailed FISH-based studies evaluated fewer than half of the chromosomes in each embryo. As only limited numbers of chromosomes were examined, while comprehensive chromosomal screening for all the 24 chromosomes is more informative compared to the FISH techniques (Rubio et al., 2013;Scott et al., 2013). However, performing these techniques may not be always possible for all couples and embryos due to the number or quality restriction of oocytes/embryos, the availability of techniques for IVF centers, legal restrictions, and finally social or simply cost-related issues (Calabrese et al., 2012;Fragouli et al., 2011;Meseguer et al., 2011). ...
The purpose was to assess the developmental competence of the in vitro or in vivo matured human oocytes as well as the apoptotic genes expression of cumulus cells (CCs) regarding nuclear maturity status of associated oocytes retrieved from stimulated ICSI cycles. A total of 590 oocytes and the associated CCs were retrieved and divided into groups of test and control according to the nuclear maturity status in order to the developmental evaluation as well as expression patterns of apoptosis-related genes using real time PCR. The fertilization and embryo formation rates were 60.3% and 87.5% vs.69.1% and 92.8% in test and control groups, respectively. Good quality embryos on day 3 were 62.2% in test and 69.1% in control groups. There were significant differences in the rates of normal fertilized as well as unfertilized oocytes between the groups. Also, mRNA levels of some apoptotic genes were significantly higher in the CCs obtained from immature oocytes among patients with premature ovarian factors (POF) rather than other infertility etiologies (p<0.001). The data demonstrated the developmental competence of in vitro matured oocytes -even to good quality cleavage embryos- is not completely consistent with molecular integrity and well-mannered gene expression patterns resulting to ICSI success. It seems that using immature oocytes could be helpful for patients at risk of ovarian hyperstimulation syndrome (OHSS) as the same as patients with diminished ovarian reserve.
... A variety of platforms are used for downstream analysis of amplified DNA to obtain a comprehensive chromosome screening (CCS) diagnosis. Array comparative genomic hybridization (aCGH) has been the most widely used platform because of its commercial availability (4,7,8,16,18,23,25,33,42,44,45,53,55,56,68,90,95). The general strategy involves labeling WGA DNA from an embryo biopsy with one fluorophore and a control normal sample with a different fluorophore, then combining and hybridizing the sample to an array of bacterial artificial chromosomes containing DNA from across the human genome. ...
Genetic testing of preimplantation embryos promises to prevent monogenic disease in children born to at-risk couples, the transfer of unbalanced embryos to patients carrying a balanced translocation, and the use of aneuploid embryos created during in vitro fertilization. Technologies have evolved from fluorescence in situ hybridization to next-generation-sequencing-based aneuploidy screening and allow for simultaneous testing of multiple genetic abnormalities in a single biopsy. The field has also shifted away from polar body or blastomere biopsy and toward trophectoderm biopsy as the new standard. This review describes the multitude of available platforms and methodologies used in contemporary preimplantation genetic testing. Expected final online publication date for the Annual Review of Genomics and Human Genetics Volume 18 is August 31, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... 1 The aCGH technique enables us to screen all 24 chromosomes within 24 hours and makes fresh transfer possible after blastomere biopsy or trophectoderm biopsy. 15 A randomised study has shown that PGS by aCGH plus selection by morphology of blastocysts can significantly improve the ongoing pregnancy rate in patients with good prognosis when compared with selection of blastocysts by morphology alone. 16 Another randomised study also showed an improvement in the implantation rate after PGS by aCGH in addition to morphological assessment of embryos. ...
Introduction:
Preimplantation genetic screening has been proposed to improve the in-vitro fertilisation outcome by screening for aneuploid embryos or blastocysts. This study aimed to report the outcome of 133 cycles of preimplantation genetic diagnosis and screening by array comparative genomic hybridisation.
Methods:
This study of case series was conducted in a tertiary assisted reproductive centre in Hong Kong. Patients who underwent preimplantation genetic diagnosis for chromosomal abnormalities or preimplantation genetic screening between 1 April 2012 and 30 June 2015 were included. They underwent in-vitro fertilisation and intracytoplasmic sperm injection. An embryo biopsy was performed on day-3 embryos and the blastomere was subject to array comparative genomic hybridisation. Embryos with normal copy numbers were replaced. The ongoing pregnancy rate, implantation rate, and miscarriage rate were studied.
Results:
During the study period, 133 cycles of preimplantation genetic diagnosis for chromosomal abnormalities or preimplantation genetic screening were initiated in 94 patients. Overall, 112 cycles proceeded to embryo biopsy and 65 cycles had embryo transfer. The ongoing pregnancy rate per transfer cycle after preimplantation genetic screening was 50.0% and that after preimplantation genetic diagnosis was 34.9%. The implantation rates after preimplantation genetic screening and diagnosis were 45.7% and 41.1%, respectively and the miscarriage rates were 8.3% and 28.6%, respectively. There were 26 frozen-thawed embryo transfer cycles, in which vitrified and biopsied genetically transferrable embryos were replaced, resulting in an ongoing pregnancy rate of 36.4% in the screening group and 60.0% in the diagnosis group.
Conclusions:
The clinical outcomes of preimplantation genetic diagnosis and screening using comparative genomic hybridisation in our unit were comparable to those reported internationally. Genetically transferrable embryos replaced in a natural cycle may improve the ongoing pregnancy rate and implantation rate when compared with transfer in a stimulated cycle.
... First tests of this technique demonstrated a successful use of screening of all 24 chromosomes in human oocytes and embryos [10]. Today, array-CGH technology is accepted as a reliable and safe method for PGS and it is widely used in many IVF centers around the world [11,12]. ...
Chromosomal aneuploidies are known for being the main cause of abnormal development of embryos with normal morphology, their implantation failure and early reproductive losses in IVF treatments. Preimplantation genetic screening (PGS) allows selecting embryos with normal chromosomal content and increases IVF treatment efficiency due to higher implantation rates and less frequent early pregnancy losses. New technologies used for PGS allow making genome-wide analysis of the presence of all chromosomes in embryos. This article presents our study of evaluation of two techniques used for PGS: previously developed and used in our laboratory a-CGH assay based on Agilent technology and newly tested semi-conductive NGS technique (Torrent technology).
... Recently, it is noteworthy that because of the popularity of preimplantation genetic screening (PGS), there is a growing need of concurrent PGD/PGS. At the moment the strategies used in PGS, if we exclude the outdated FISH-based diagnostics [28], include array-based (either array comparative genomic hybridization or single nucleotide polymorphism chromosomal microarray) techniques [29][30][31], q-PCR based techniques [32,33], and next generation sequencing (NGS)-based techniques [34,35]. Some of the techniques had been reported to successfully being applied in PGD combined with PGS [36][37][38][39]. ...
Preimplantation genetic diagnosis (PGD) is a powerful tool to tackle the transmission of monogenic inherited disorders in families carrying the diseases from generation to generation. It currently remains a challenging task, despite PGD having been developed over 25 years ago. The major difficulty is it does not have an easy and general formula for all mutations. Different gene locus needs individualized, customized design to make the diagnosis accurate enough to be applied on PGD, in which the quantity of DNA is scanty, whereas timely laboratory diagnosis is mandatory if fresh embryo transfer is desired occasionally. Indicators for outcome assessment of a successful PGD program include the successful diagnosis rate on blastomeres (Day 3 cleavage-stage embryo biopsy) or trophectoderm cells (Day 5/6 blastocyst biopsy), the implantation rate per embryo transferred, and the livebirth rate per oocyte retrieval cycle. Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by various types of pathological defects in the factor VIII gene (F8). The mutation spectrum of the F8 is complex, according to our previous report, including large segmental intra-gene inversions, large segmental deletions spanning a few exons, point mutations, and total deletion caused by chromosomal structural rearrangements. In this review, the molecular methodologies used to tackle different mutants of the F8 in the PGD of HA are to be explained, and the experiences of successful use of amplification refractory mutation system-quantitative polymerase chain reaction (ARMS-qPCR) and linkage analysis for PGD of HA in our laboratory are also provided.
Cells are the fundamental units of biological systems and exhibit unique development trajectories and molecular features. Our exploration of how the genomes orchestrate the formation and maintenance of each cell, and control the cellular phenotypes of various organismsis, is both captivating and intricate. Since the inception of the first single-cell RNA technology, technologies related to single-cell sequencing have experienced rapid advancements in recent years. These technologies have expanded horizontally to include single-cell genome, epigenome, proteome, and metabolome, while vertically, they have progressed to integrate multiple omics data and incorporate additional information such as spatial scRNA-seq and CRISPR screening. Single-cell omics represent a groundbreaking advancement in the biomedical field, offering profound insights into the understanding of complex diseases, including cancers. Here, we comprehensively summarize recent advances in single-cell omics technologies, with a specific focus on the methodology section. This overview aims to guide researchers in selecting appropriate methods for single-cell sequencing and related data analysis.
OBJETIVO:
Analizar las tasas de concordancia, falsos positivos y negativos entre el ADN embrionario circulante en medio de cultivo y su relación con los reportes de la biopsia de trofoectodermo.
MATERIALES Y MÉTODOS:
Estudio observacional, prospectivo y comparativo, llevado a cabo en el Centro de Reproducción Arcos Nascere en noviembre 2018. Criterios: de inclusión: parejas en esquema de fertilización in vitro, con diagnóstico genético preimplantacional de aneuploidias. Criterios de exclusión: pacientes con anomalías estructurales o enfermedades monogénicas. Criterio de eliminación: blastocistos con eclosión asistida. Variables de respuesta: tasa de concordancia, falsos positivos y negativos entre las biopsias de trofoectodermo y los medios de cultivo. El análisis estadístico se realizó con SPSS 25.0, con pruebas t de Student y χ² con valor de p < 0.05 significativa.
RESULTADOS:
Se analizaron 20 blastocistos de 5 parejas y se obtuvieron resultados informativos de 17 (amplificación global exitosa); 70% en día 5 y 100% en día 6. La tasa general de concordancia entre las biopsias de trofoectodermo y los medios de cultivo fue de 68.7% (42.8% en día 5 y 88.8% en día 6). En cuanto a las discrepancias, solo se observaron 2 falsos negativos en los medios de cultivo vs la biopsia de trofoectodermo (14.2% en día 5 y 11.11% en día 6); hubo 3 casos de falsos positivos (la mitad en día 5 y ninguno en día 6-7).
CONCLUSIONES:
Con la prueba genética no invasiva de aneuploidias se alcanzaron altas tasas de concordancia, sobre todo en embriones en día 6.
Preimplantation genetic testing (PGT) is a practical option for couples at risk of having offspring with serious/fatal chromosomal or monogenic diseases. This chapter describes these important developments with the emphasis on addressing the problems of implementation of PGT into clinical practice. It contains tables that list conditions for which PGT was performed and PGT‐M outcome: 30 years of original experience. Initially, PGT was justified only for high‐risk pregnancies. The development and improvement of the methods for sampling and genetic analysis have made use of PGT for chromosomal disorders a reality. Considerations on ethical and legal issues are evolving, along with the evolution of the technology for the control of genetic diseases, and have become one of the key subjects in discussing the acceptability of preconception and preimplantation testing for genetic disorders.
Objectives:
To study the feasibility of using unbalanced embryos as a reference in distinguishing euploid carrier and non-carrier embryos by SNP array-based preimplantation genetic testing (PGT) for reciprocal translocations.
Methods:
After comprehensive chromosome screening (CCS), euploid embryos were identified as normal or carriers using a family member as a reference. Next, unbalanced embryos were used as a reference, and the results were compared with the previous ones. Karyotypes of transferred embryos were validated by prenatal diagnosis.
Results:
Of 995 embryos from 110 couples, 288 were found to be euploid. Using a family member as a reference, 142 and 144 embryos were tested to be euploid non-carrier and carrier respectively, and the remaining 2 embryos were undetermined. When unbalanced embryos were selected as references, all the results were consistent with the previous ones. A total of 107 embryos were transferred, resulting in 66 clinical pregnancies. Karyotypes of prenatal diagnosis were all in accordance with the results of tested embryos.
Conclusions:
SNP array-based haplotyping is a rapid and effective way to distinguish between euploid carrier and non-carrier embryos. In case no family member is available as a reference, unbalanced embryos can be used for identification of euploid carrier and non-carrier embryos. This article is protected by copyright. All rights reserved.
Klinefelter syndrome (KS) is the most common X chromosome abnormality encountered in men with infertility. The classic form 47,XXY accounts for 80–90% of cases. An error of nondisjunction during gametogenesis provides the extra X chromosome in KS men. The various karyotype variants of KS share the same features of hypergonadotropic hypogonadism but present with more distinct physical, medical and psychological features than the classic form. Spermatogenesis appears to be intact during infancy up to the prepubertal period; however, it progressively declines during adulthood. Learning and behavioural challenges are frequent at an earlier age, while androgen deficiency and infertility are usually encountered at an advanced age. Testosterone replacement therapy is the cornerstone to address hypogonadism to enhance the quality of life and prevent the long-term complications of the androgen-deficient state. Intracytoplasmic sperm injection (ICSI) is a major breakthrough in the treatment of infertility, particularly in KS men. Sperm can be found in the ejaculate in patients with KS and can be used for assisted reproductive technology. However, microdissection testicular sperm extraction (TESE) is the mainstay procedure of choice for sperm retrieval for ICSI, as most KS men present with non-obstructive azoospermia. This procedure provides significantly superior overall outcomes compared to other sperm retrieval techniques. Although men with sex chromosomal abnormalities have a low risk of producing offspring with the same abnormalities after ICSI, genetic counselling should be performed in a multidisciplinary approach to enhance the quality of life and overall health status of KS men.
Sperm aneuploidy testing has been proposed for clinical diagnosis of possible causes of male infertility. We describe the use of fluorescence in situ hybridization (FISH) for sperm aneuploidy testing and review causes and implications of sperm aneuploidy at clinical, embryo, and offspring levels. Due to infertility problems or genetic risk for offspring, genetic counseling and reproductive options, including preimplantation genetic testing for aneuploidy (PGT-A), prenatal testing, or sperm donation, should be offered to couples with increased sperm aneuploidy.
The aim of this study was to investigate the effects of zona drilling and biopsy on day 3 followed by vitrification on day 5 on the cytoskeleton and development of human embryos, by analysing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy in human biopsied and non-biopsied embryos. In total, 98 human blastocysts (50 non-biopsied and 48 following biopsy on day 3) were vitrified on day 5 using either a commercial dimethyl sulphoxide (DMSO)-free vitrification kit or increasing concentrations of DMSO/EG (5%/5–10%/10–20%/20%). Following warming, the blastocysts were allowed to recover in culture for 24 h and were immunostained with α-tubulin, acetylated tubulin, and/or γ-tubulin antibodies in combination with 4′,6-diamidino-2-phenylindole (DAPI). Labelled embryos were examined by both fluorescence and confocal laser scanning microscopy. The survival rates following warming (92% non-biopsied vs 83.3% biopsied) and the incidence of normal spindle chromosome configurations was not statistically different between the two groups (65.2% non-biopsied vs 59.2% biopsied, P >0.05). The incidence of spindle abnormalities including multipolarity, chromosome lagging, congression failure and chromosome bridging were also similar between the two groups ( P >0.05). This study is the first to compare the incidence of cytoskeletal abnormalities in biopsied and non-biopsied human embryos following vitrification. We conclude that there was no significant difference in the survival rates and the incidence of spindle abnormalities between the two groups.
The aim of embryo selection is to improve live birth rates per egg collection, by maximizing use of all embryos with implantation potential, but with correspondingly low multiple pregnancy rates. This includes selection of embryos for cryostorage for use in subsequent cycles to improve cumulative pregnancy rates. Routine or ‘conventional’ embryo assessment is currently focused mainly on morphological criteria and cleavage rate during development. may result in detrimental environmental stress and provides only a brief snapshot of embryo development. Furthermore, lack of standardized embryo morphology grading systems in in vitro fertilization clinics worldwide has made the definition of embryo ‘quality’ and comparison of studies very difficult. There has now been a move towards standardization of grading schemes internationally and the development of external quality assessment (EQA) schemes. However, while there is a certain amount of agreement on what constitutes a ‘top quality’ or a ‘poor’ embryo, it is more difficult to find a consensus between laboratories when attempting to evaluate intermediate quality embryos. The latter may be deselected from clinical use and yet may have implantation potential.
Objective:
To evaluate effects of autologous stem cell ovarian transplant (ASCOT) on ovarian reserve and IVF outcomes of women who are poor responders with very poor prognosis.
Design:
Prospective observational pilot study.
Setting:
University hospital.
Patient(s):
Seventeen women who are poor responders.
Intervention(s):
Ovarian infusion of bone marrow-derived stem cells.
Main outcome measure(s):
Serum antimüllerian hormone levels and antral follicular count (AFC), punctured follicles, and oocytes retrieved after stimulation (controlled ovarian stimulation) were measred. Apheresis was analyzed for growth factor concentrations.
Result(s):
The ASCOT resulted in a significant improvement in AFC 2 weeks after treatment. With an increase in AFC of three or more follicles and/or two consecutive increases in antimüllerian hormone levels as success criteria, ovarian function improved in 81.3% of women. These positive effects were associated with the presence of fibroblast growth factor-2 and thrombospondin. During controlled ovarian stimulation, ASCOT increased the number of stimulable antral follicles and oocytes, but the embryo euploidy rate was low (16.1%). Five pregnancies were achieved: two after ET, three by natural conception.
Conclusion(s):
Our results suggest that ASCOT optimized the mobilization and growth of existing follicles, possibly related to fibroblast growth factor-2 and thrombospondin-1 within apheresis. The ASCOT improved follicle and oocyte quantity enabling pregnancy in women who are poor responders previously limited to oocyte donation.
Clinical trial registration number:
NCT02240342.
Cells are a fundamental unit of life, and the ability to study the phenotypes and behaviours of individual cells is crucial to understanding the workings of complex biological systems. Cell phenotypes (epigenomic, transcriptomic, proteomic and metabolomic) exhibit dramatic heterogeneity between and within the different cell types and states underlying cellular functional diversity. Cell genotypes can also display heterogeneity throughout an organism, in the form of somatic genetic variation ‐ most notably in the emergence and evolution of tumours. Recent technical advances in single‐cell isolation and the development of omics approaches sensitive enough to reveal these aspects of cell identity have enabled a revolution in the study of multicellular systems. In this review, we discuss the technologies available to resolve the genomes, epigenomes, transcriptomes, proteomes and metabolomes of single cells from a wide variety of living systems.
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Preimplantation genetic diagnosis (PGD) is a clinically feasible technology to prevent the transmission of monogenic inherited disorders in families afflicted the diseases to the future offsprings. The major technical hurdle is it does not have a general formula for all mutations, thus different gene locus needs individualized, customized design to make the diagnosis accurate enough to be applied on PGD, in which the quantity of DNA is scarce, whereas timely result is sometimes requested if fresh embryo transfer is desired. On the other hand, preimplantation genetic screening (PGS) screens embryo with aneuploidy and was also known as PGD-A (A denotes aneuploidy) in order to enhance the implantation rates as well as livebirth rates. In contrasts to PGD, PGS is still under ferocious debate, especially recent reports found that euploid babies were born after transferring the aneuploid embryos diagnosed by PGS back to the womb and only very few randomized trials of PGS are available in the literature. We have been doing PGD and/or PGS for more than 10 years as one of the core PGD/PGS laboratories in Taiwan. Here we provide a concise review of PGD/PGS regarding its current status, both domestically and globally, as well as its future challenges.
Aneuploidies as quantitative chromosome abnormalities are a main cause of failed development of morphologically normal embryos, implantation failures, and early reproductive losses. Preimplantation genetic screening (PGS) allows a preselection of embryos with a normal karyotype, thus increasing the implantation rate and reducing the frequency of early pregnancy loss after IVF. Modern PGS technologies are based on a genome-wide analysis of the embryo. The first pilot study in Russia was performed to assess the possibility of using semiconductor new-generation sequencing (NGS) as a PGS method. NGS data were collected for 38 biopsied embryos and compared with the data from array comparative genomic hybridization (array-CGH). The concordance between the NGS and array-CGH data was 94.8%. Two samples showed the karyotype 47,XXY by array-CGH and a normal karyotype by NGS. The discrepancies may be explained by loss of efficiency of array-CGH amplicon labeling.
Preimplantation genetic diagnosis (PGD), first successfully carried out in humans in the early 1990s, initially involved the PCR sexing of embryos by Y- (and later also X-) chromosome specific detection. Because of the problems relating to misdiagnosis and contamination of this technology however the PCR based test was superseded by a FISH-based approach involving X and Y specific probes. Sexing by FISH heralded translocation screening, which was shortly followed by preimplantation genetic screening (PGS) for Aneuploidy. Aneuploidy is widely accepted to be the leading cause of implantation failure in assisted reproductive technology (ART) and a major contributor to miscarriage, especially in women of advanced maternal age. PGS (AKA PGD for aneuploidy PGD-A) has had a chequered history, with conflicting lines of evidence for and against its use. The current practice of trophectoderm biopsy followed by array CGH or next generation sequencing is gaining in popularity however as evidence for its efficacy grows. PGS has the potential to identify viable embryos that can be transferred thereby reducing the chances of traumatic failed IVF cycles, miscarriage or congenital abnormalities and facilitating the quickest time to live birth of chromosomally normal offspring. In parallel to chromosomal diagnoses, technology for PGD has allowed for improvements in accuracy and efficiency of the genetic screening of embryos for monogenic disorders. The number of genetic conditions available for screening has increased since the early days of PGD, with the human fertilization and embryology authority currently licensing 419 conditions in the UK [1]. A novel technique known as karyomapping that involves SNP chip screening and tracing inherited chromosomal haploblocks is now licensed for the PGD detection of monogenic disorders. Its potential for the universal detection of chromosomal and monogenic disorders simultaneously however, has yet to be realized.
Preimplantation genetic diagnosis (PGD) has been initiated to provide the option of avoiding the birth of an affected child without the need for abortion as an obligatory component in the prevention program. This chapter describes these important developments with the emphasis on addressing the problems of implementation of PGD into clinical practice. The following possibilities for PGD were mentioned: genetic analysis of the first or second polar bodies, and embryo biopsy at the cleavage or blastocyst stage. The majority of PGD cycles are done for preimplantation testing of chromosomal disorders. The chapter presents the application of PGD to a wider range of disorders, including conditions determined by de novo mutations (DNM), genetic predisposition for late-onset disorders, and preimplantation human leukocyte antigen (HLA) matching. The other emerging important PGD indication has been an inherited cardiac disease, for which there is no current prospect of effective treatment.
Preimplantation development comprises the initial stages of mammalian development, before the embryo implants into the mother's uterus. In normal conditions, after fertilization the embryo grows until reaching blastocyst stage. The blastocyst grows as the cells divide and the cavity expands, until it arrives at the uterus, where it “hatches” from the zona pellucida to implant into the uterine wall. Nevertheless, embryo quality and viability can be affected by chromosomal abnormalities, most of which occur during gametogenesis and early embryo development; human embryos produced in vitro are especially vulnerable. Therefore, the selection of chromosomally normal embryos for transfer in assisted reproduction can improve outcomes in poor-prognosis patients.
This chapter will cover the following topics:Introduction
Why develop PGD?
The impetus for its development
Research progress that made it possible
Legislation—the Warnock report
Early steps towards PGD
Sexing of embryos by DNA amplification
FISH comes into its own
Mosaicism and the extent of aneuploidy—implications for PGD
Aneuploidy screening begins
First single gene diagnosis
First cancer gene PGD
Development of single cell CGH for comprehensive chromosome analysis
PGS gets a bad name
Application of metaphase CGH
Development of array CGH and its application for PGS to select ‘the single euploid embryo’.
Significance
One missing or wrong nucleotide out of six billion in a human genome can cause a genetic disease. Detecting such a point mutation in a single human germ cell has been a daunting challenge in in vitro fertilization, yet one cannot afford to make any mistakes in selecting a viable embryo for transfer. Mutated allele revealed by sequencing with aneuploidy and linkage analyses (MARSALA) combines next-generation sequencing and single-cell whole-genome amplification methodologies, allowing embryo diagnosis with a single-molecule precision, significantly reducing false-positive or false-negative errors. MARSALA can benefit couples who desire to avoid transmitting their genetic diseases to their offspring.
What happens with our genome and epigenome in the first fundamental days of our development? How can this be analysed? What do we need to know when faced with patients' questions about their own infertility, or how to prevent the birth of affected children? For the first time, this book brings together both scientists' and clinicians' viewpoints on human reproductive genetics, making for a more comprehensive discussion of interest to ART professionals and developmental biologists. With worldwide leaders in this burgeoning field guiding the reader through from the basics to the most exciting recent discoveries, this book presents the wider picture of how reproductive medicine and biology links with genetics. The editors also address the new challenges raised in how to treat and counsel patients at fertility and genetic clinics, as well as eliciting vivid bioethical debates. This book brings together genetics, reproductive biology and medicine for practitioners and geneticists.
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.
STUDY QUESTION What is the optimal stage from oocyte through preimplantation embryo development for biopsy and preimplantation genetic screening
(PGS) to detect abnormal chromosome segregation patterns in eggs or embryos from advanced maternal age (AMA) patients?
Trisomic and monosomic (aneuploid) embryos account for at least 10% of human pregnancies and, for women nearing the end of their reproductive lifespan, the incidence may exceed 50%. The errors that lead to aneuploidy almost always occur in the oocyte but, despite intensive investigation, the underlying molecular basis has remained elusive. Recent studies of humans and model organisms have shed new light on the complexity of meiotic defects, providing evidence that the age-related increase in errors in the human female is not attributable to a single factor but to an interplay between unique features of oogenesis and a host of endogenous and exogenous factors.
To determine any beneficial effects of preimplantation genetic screening (PGS) of all chromosomes by array comparative genomic hybridization (aCGH), with either day 3 or blastocyst biopsy, for idiopathic recurrent pregnancy loss (RPL) patients compared with their expected loss rate.
Case series report.
Multiple fertility centers.
A total of 287 cycles of couples with idiopathic RPL (defined as two or more losses).
PGS was done with day 3 biopsy (n = 193) or blastocyst biopsy (n = 94), followed by analysis with aCGH.
Spontaneous abortion rate, euploidy rate.
A total of 2,282 embryos were analyzed, of which 35% were euploid and 60% were aneuploid. There were 181 embryo transfer cycles, of which 100 (55%) became pregnant with an implantation rate of 45% (136 sacs/299 replaced embryos) and 94 pregnancies (92%) were ongoing (past second trimester) or delivered. The miscarriage rate was found to be only 6.9% (7/102), compared with the expected rate of 33.5% in an RPL control population and 23.7% in an infertile control population.
Current PGS results with aCGH indicate a significant decrease in the miscarriage rate of idiopathic RPL patients and high pregnancy rates. Furthermore, this suggests that idiopathic recurrent miscarriage is mostly caused by chromosomal abnormalities in embryos.
We previously found that a normal karyotype in a previous miscarriage is a predictor of subsequent miscarriage. However, the prevalence of recurrent miscarriage caused by an abnormal embryonic karyotype has not yet been reported, since embryonic karyotype is not typically analyzed during conventional examinations.
A total of 482 patients who underwent both embryonic karyotype determination and conventional examinations for recurrent miscarriage were enrolled in this study. The distribution of the causes and the live birth rate for each cause were examined.
The total percentage of subjects in whom conventional causes of recurrent miscarriage could be detected was 29.5%. The prevalence of the abnormal embryonic karyotype was 41.1% in the subjects in whom no conventional causes of miscarriage could be identified. The prevalence of recurrent miscarriage of truly unexplained cause, that is, of subjects without conventional causes in whom the embryonic karyotype was ascertained to be normal, was 24.5%. Among the patients in whom the first determination revealed an abnormal embryonic karyotype, 76.2% (32/42) showed an abnormal embryonic karyotype in the repeat determination as well. The cumulative live birth rate (71.9%) in women with recurrent miscarriages caused by the abnormal embryonic karyotype was significantly higher than that (44.7%) in women with recurrent miscarriages associated with the embryonal euploidy.
An abnormal embryonic karyotype was found to represent the commonest cause of recurrent miscarriage, and the percentage of cases with recurrent miscarriage of truly unexplained cause was limited to 24.5%.The two groups should be distinguished for both clinical and research purposes.
Single embryo transfer (SET) remains underutilized as a strategy to reduce multiple gestation risk in IVF, and its overall lower pregnancy rate underscores the need for improved techniques to select one embryo for fresh transfer. This study explored use of comprehensive chromosomal screening by array CGH (aCGH) to provide this advantage and improve pregnancy rate from SET.
First-time IVF patients with a good prognosis (age <35, no prior miscarriage) and normal karyotype seeking elective SET were prospectively randomized into two groups: In Group A, embryos were selected on the basis of morphology and comprehensive chromosomal screening via aCGH (from d5 trophectoderm biopsy) while Group B embryos were assessed by morphology only. All patients had a single fresh blastocyst transferred on d6. Laboratory parameters and clinical pregnancy rates were compared between the two groups.
For patients in Group A (n = 55), 425 blastocysts were biopsied and analyzed via aCGH (7.7 blastocysts/patient). Aneuploidy was detected in 191/425 (44.9%) of blastocysts in this group. For patients in Group B (n = 48), 389 blastocysts were microscopically examined (8.1 blastocysts/patient). Clinical pregnancy rate was significantly higher in the morphology + aCGH group compared to the morphology-only group (70.9 and 45.8%, respectively; p = 0.017); ongoing pregnancy rate for Groups A and B were 69.1 vs. 41.7%, respectively (p = 0.009). There were no twin pregnancies.
Although aCGH followed by frozen embryo transfer has been used to screen at risk embryos (e.g., known parental chromosomal translocation or history of recurrent pregnancy loss), this is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients. The observed aneuploidy rate (44.9%) among biopsied blastocysts highlights the inherent imprecision of SET when conventional morphology is used alone. Embryos randomized to the aCGH group implanted with greater efficiency, resulted in clinical pregnancy more often, and yielded a lower miscarriage rate than those selected without aCGH. Additional studies are needed to verify our pilot data and confirm a role for on-site, rapid aCGH for IVF patients contemplating fresh SET.
An efficient method for cryopreservation of human oocytes may offer solutions to legal and ethical problems in routine infertility programs and may also be used for fertility preservation for medical and social reasons.
We conducted an observational longitudinal cohort multicentric study to investigate the efficacy and reproducibility of oocyte cryopreservation outcomes in IVF/ICSI cycles. Moreover, the effects of patient and cycle characteristics on the delivery rate (DR) were analyzed.
In 486 cycles performed in 450 couples, 2721 oocytes were warmed and 2304 of them survived cryopreservation (84.7%). Of the 2182 oocytes subjected to ICSI, the rates of fertilization and development to top-quality embryos were 75.2 and 48.1%, respectively. A total of 128 deliveries were obtained (26.3% per cycle and 29.4% per transfer) for 450 patients (28.4%) and 147 babies were live born from 929 embryos transferred (15.8%). The forward logistic regression analysis on a per patient basis showed that female age [odds ratio (OR): 0.93, 95% confidence interval (CI): 0.88-0.98], number of vitrified oocytes (OR: 1.08, 95% CI: 1.01-1.17) and the day of transfer (OR: 1.97, 95% CI: 1.14-3.42) influenced DR. By recursive partitioning analysis, it can be estimated that more than eight oocytes vitrified are required to improve the outcome (22.6 versus 46.4% DR, respectively). When fewer oocytes are available in women aged >38 years, results are dramatically reduced (12.6 versus 27.5% DR, respectively). Conversely, when >8 oocytes are available, blastocyst culture represents the most efficient policy (62.1% DR; data from one center only).
Oocyte vitrification is an efficient and reliable approach, with consistent results between centers and predictable DRs. It should be applied routinely for various indications. A predictive model is proposed to help patient counselling and selection.
Accumulation of oocytes from several ovarian stimulation cycles is currently possible using novel vitrification technologies. This strategy could increase the inseminated cohort, creating a similar situation to normoresponders. This study included 242 low-responder (LR) patients (594 cycles) whose mature oocytes were accumulated by vitrification and inseminated simultaneously (LR-Accu-Vit) and 482 patients (588 cycles) undergoing IVF/embryo transfer with fresh oocytes in each stimulation cycle (LR-fresh). Drop-out rate in the LR-fresh group was >75%. The embryo-transfer cancellation per patient was significantly lower in the LR-Accu-Vit group (9.1%) than the LR-fresh group (34.0%). Live-birth rate (LBR)/patient was higher in the LR-Accu-Vit group (30.2%) than the LR-fresh group (22.4%). Cumulative LBR/patient was statistically higher in the LR-Accu-Vit group (36.4%) than the LR-fresh group (23.7%) and a similar outcome was observed among patients aged ⩾40years (LR-Accu-Vit 15.8% versus LR-fresh 7.1%). The LR-Accu-Vit group had more cycles with embryo cryopreservation (LR-Accu-Vit 28.9% versus LR-fresh 8.7%). Accumulation of oocytes by vitrification and simultaneous insemination represents a successful alternative for LR patients, yielding comparable success rates to those in normoresponders and avoiding adverse effects of a low response. The accumulation of oocytes from several ovarian stimulation cycles is currently possible with the aid of novel vitrification technologies. This strategy could be useful for low-responder patients, contributing to increase the inseminated cohort and creating a similar situation as in normal responders. According to the results presented herein (higher live-birth rate per patient treated), this strategy represents a successful alternative for low-responder patients, yielding comparable success rates to those in normal responders and avoiding the adverse effects of a low response.
The emergence of the array comparative genomic hybridization technique (aCGH) is considered an advance in preimplantation
genetic testing. Analysis of the recently published pilot study using polar body aCGH indicates that the test accuracy compares
favourably with the fluorescence in situ hybridization technique although a substantial number of euploid zygotes are still likely to be excluded incorrectly. A sound
argument against selection in principle has recently been published, based on accumulating evidence that potentially all embryos
can now be cryopreserved and transferred in subsequent frozen replacement cycles without impairing pregnancy rates. We suggest
that vitrification and serial transfer without testing are likely to give patients the best chance for a successful pregnancy,
and avoid the use of an expensive technology.
To determine both the negative and positive predictive values of comprehensive chromosome screening (CCS) results for clinical outcome.
Data obtained from two prospective, double-blinded, nonselection studies.
Academic center for reproductive medicine.
One hundred forty-six couples with a mean maternal age of 34.0 ± 4.4 years and a mean paternal age of 37.3 ± 5.8 years.
Embryo biopsy for DNA fingerprinting and aneuploidy assessment.
Failure rate of embryos predicted aneuploid by CCS (negative predictive value) and success rate of embryos predicted euploid by CCS (positive predictive value).
A total of 255 IVF-derived human embryos were cultured and selected for transfer without influence from CCS analysis. Embryos were biopsied before transfer, including 113 blastomeres at the cleavage stage and 142 trophectoderm biopsies at the blastocyst stage. Comprehensive chromosome screening was highly predictive of clinical outcome, with 96% of aneuploid predicted embryos failing to sustain implantation and 41% sustained implantation from embryos predicted to be euploid.
These nonselection data provide the first prospective, blinded, clinical study directly measuring the predictive value of aneuploidy screening for clinical outcome. The clinical error rate of an aneuploidy designation is very low (4%), whereas implantation and delivery rates of euploid embryos are increased relative to the entire cohort of transferred embryos.
The purpose of this study was to assess the technical aspects related to polar body (PB) biopsy, which might have an influence on the results of the microarray comparative genomic hybridization analysis. Furthermore, a comparison was made between two biopsy methods (mechanical and laser).
Biopsy of the first and second PB (PB1 and PB2) was performed by mechanical- or laser-assisted biopsy in two different IVF centres. PBs were separately amplified by whole genome amplification.
The method of biopsy, mechanical or laser had no influence on the proportion of successfully biopsied oocytes. Especially, for the PB2, the timing of biopsy after ICSI was directly correlated to amplification efficiency.
Special care has to be taken with respect to the timing of biopsy of the PB2. Mechanical- and laser-assisted biopsy give the same performance in terms of diagnostic efficiency.
Several randomized controlled trials have not shown a benefit from preimplantation genetic screening (PGS) biopsy of cleavage-stage embryos and assessment of up to 10 chromosomes for aneuploidy. Therefore, a proof-of-principle study was planned to determine the reliability of alternative form of PGS, i.e. PGS by polar body (PB) biopsy, with whole genome amplification and microarray-based comparative genomic hybridization (array CGH) analysis.
In two centres, all mature metaphase II oocytes from patients who consented to the study were fertilized by ICSI. The first and second PBs (PB1and PB2) were biopsied and analysed separately for chromosome copy number by array CGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by array CGH for concordance analysis.
Both PBs were biopsied from a total of 226 zygotes from 42 cycles (average 5.5 per cycle; range 1-15) in 41 couples with an average maternal age of 40.0 years. Of these, the ploidy status of the zygote could be predicted in 195 (86%): 55 were euploid (28%) and 140 were aneuploid (72%). With only one exception, there was at least one predicted aneuploid zygote in each cycle and in 19 out of 42 cycles (45%), all zygotes were predicted to be aneuploid. Fresh embryos were transferred in the remaining 23 cycles (55%), and one frozen transfer was done. Eight patients had a clinical pregnancy of which seven were evolutive (ongoing pregnancy rates: 17% per cycle and 30% per transfer). The ploidy status of 156 zygotes was successfully analysed by array CGH: 38 (24%) were euploid and 118 (76%) were aneuploid. In 138 cases complete information was available on both PBs and the corresponding zygotes. In 130 (94%), the ploidy status of the zygote was concordant with the ploidy status of the PBs and in 8 (6%), the results were discordant.
This proof-of-principle study indicates that the ploidy of the zygote can be predicted with acceptable accuracy by array CGH analysis of both PBs.
We evaluated cytogenetic results occurring with first trimester pregnancy loss, and assessed the type and frequency of chromosomal abnormalities after assisted reproductive treatment (ART) and compared them with a control group. We also compared the rate of chromosomal abnormalities according to infertility causes in ICSI group.
A retrospective cohort analysis was made of all patients who were referred to the Genetics Laboratory of Fertility Center of CHA Gangnam Medical Center from 2005 to 2009 because of clinical abortion with a subsequent dilation and evacuation (D&E) performed, and patients were grouped by type of conception as follows: conventional IVF (in vitro fertilization) (n = 114), ICSI (intracytoplasmic sperm injection) (n = 140), and control (natural conception or intrauterine insemination [IUI]) (n = 128). Statistical analysis was performed using SPSS software.
A total 406 specimens were referred to laboratory, ten abortuses were excluded, and in 14 cases, we did not get any spontaneous metaphase, chromosomal constitutions of 382 specimens were successfully obtained with conventional cytogenetic methods. Overall, 52.62% of the miscarriages were found to be cytogenetically abnormal among all patients, the frequency was 48.4% in the control group, 54.3% of miscarriages after ICSI and 55.3% after conventional IVF (p = 0.503). The most prevalent abnormalities were autosomal trisomy, however, nine (11.69%) sex chromosome aneuploidy were noted in the ICSI group vs. four (6.45%) and two (3.23%) cases in the conventional IVF group and control group. We compared chromosomal abnormalities of miscarriages after ICSI according to infertility factor. 55.71% underwent ICSI due to male factors, 44.29% due to non-male factors. ICSI group having male factors showed significantly higher risk of chromosomal abnormalities than ICSI group having non-male factors (65.8% vs. 34.2%, p = 0.009, odds ratio = 1.529, 95% CI = 1.092-2.141).
There is no increased risk of chromosomal abnormalities due to ART was found with the exception of a greater number of sex chromosomal abnormalities in the ICSI group with male factor infertility. Therefore, these alterations could be correlated with the underlying parental risk of abnormalities and not with the ICSI procedure itself.
To validate and determine the best array-comparative genomic hybridization (aCGH; array-CGH) protocols for preimplantation genetic screening (PGS).
Embryos had one cell removed as a biopsy specimen and analyzed by one of two array-CGH protocols. Abnormal embryos were reanalyzed by fluorescence in situ hybridization (FISH).
Reference laboratory.
Patients donating embryos or undergoing PGS.
Embryo biopsy, array-CGH, FISH reanalysis.
Diagnosis, no result rate and error rate.
Method one produced 11.2% of embryos with no results and a 9.1% error rate compared with 3% and 1.9% for method two, respectively. Thereafter, only method two was used clinically. The aneuploidy rate for cleavage-stage embryos was 63.2%, significantly increasing with maternal age. The chromosomes most involved in aneuploidy were 16, 22, 21, and 15. We report the first live births after array-CGH combined with single blastomere biopsy.
Array-CGH is proved to be highly robust (2.9% no results) and specific (1.9% error rate) when applied to rapid (24-hour) analysis of single cells biopsied from cleavage-stage embryos. This comprehensive chromosome analysis technique is the first to be validated by reanalyzing the same embryos with another technique (e.g., FISH). Unlike some alternative techniques for comprehensive chromosome screening, array-CGH does not require prior testing of parental DNA and thus advance planning and careful scheduling are unnecessary.
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.
Many studies estimate that chromosomal mosaicism within the cleavage-stage human embryo is high. However, comparison of two
unique methods of aneuploidy screening of blastomeres within the same embryo has not been conducted and may indicate whether
mosaicism has been overestimated due to technical inconsistency rather than the biological phenomena. The present study investigates
the prevalence of chromosomal abnormality and mosaicism found with two different single cell aneuploidy screening techniques.
Thirteen arrested cleavage-stage embryos were studied. Each was biopsied into individual cells (n = 160). The cells from each embryo were randomized into two groups. Those destined for FISH-based aneuploidy screening (n = 75) were fixed, one cell per slide. Cells for SNP microarray-based aneuploidy screening (n = 85) were put into individual tubes. Microarray was significantly more reliable (96%) than FISH (83%) for providing an interpretable
result (P = 0.004). Markedly different results were obtained when comparing microarray and FISH results from individual embryos. Mosaicism
was significantly less commonly observed by microarray (31%) than by FISH (100%) (P = 0.0005). Although FISH evaluated fewer chromosomes per cell and fewer cells per embryo, FISH still displayed significantly
more unique genetic diagnoses per embryo (3.2 ± 0.2) than microarray (1.3 ± 0.2) (P < 0.0001). This is the first prospective, randomized, blinded and paired comparison between microarray and FISH-based aneuploidy
screening. SNP microarray-based 24 chromosome aneuploidy screening provides more complete and consistent results than FISH.
These results also suggest that FISH technology may overestimate the contribution of mitotic error to the origin of aneuploidy
at the cleavage stage of human embryogenesis.
Preimplantation genetic screening (PGS) has been used in an attempt to determine embryonic aneuploidy. Techniques that use new molecular methods to determine the karyotype of an embryo are expanding the scope of PGS.
We introduce a new method for PGS, termed 'parental support', which leverages microarray measurements from parental DNA to 'clean' single-cell microarray measurements on embryonic cells and explicitly computes confidence in each copy number call. The method distinguishes mitotic and meiotic copy errors and determines parental source of aneuploidy.
Validation with 459 single cells of known karyotype indicated that per-cell false-positive and false-negative rates are roughly equivalent to the 'gold standard' metaphase karyotype. The majority of the cells were run in parallel with a clinical commercial PGS service. Computed confidences were conservative and roughly concordant with accuracy. To examine ploidy in human embryos, the method was then applied to 26 disaggregated, cryopreserved, cleavage-stage embryos for a total of 134 single blastomeres. Only 23.1% of the embryos were euploid, though 46.2% of embryos were mosaic euploid. Mosaicism affected 57.7% of the embryos. Counts of mitotic and meiotic errors were roughly equivalent. Maternal meiotic trisomy predominated over paternal trisomy, and maternal meiotic trisomies were negatively predictive of mosaic euploid embryos.
We have performed a major preclinical validation of a new method for PGS and found that the technology performs approximately as well as a metaphase karyotype. We also directly measured the mechanism of aneuploidy in cleavage-stage human embryos and found high rates and distinct patterns of mitotic and meiotic aneuploidy.
The use of genome wide single nucleotide polymorphism (SNP) arrays for high resolution molecular cytogenetic analysis using a combination of quantitative and genotype analysis is well established. This study demonstrates that by Mendelian analysis of the SNP genotypes of the parents and a sibling or other appropriate family member to establish phase, it is possible to identify informative loci for each of the four parental haplotypes across each chromosome and map the inheritance of these haplotypes and the position of any crossovers in the proband. The resulting 'karyomap', unlike a karyotype, identifies the parental and grandparental origin of each chromosome and chromosome segment and is unique for every individual being defined by the independent segregation of parental chromosomes and the pattern of non-recombinant and recombinant chromosomes. Karyomapping, therefore, enables both genome wide linkage based analysis of inheritance and detection of chromosome imbalance where either both haplotypes from one parent are present (trisomy) or neither are present (monosomy/deletion). The study also demonstrates that karyomapping is possible at the single cell level following whole genome amplification and, without any prior patient or disease specific test development, provides a universal linkage based methodology for preimplantation genetic diagnosis readily available worldwide.
Many published papers suggest a favourable impact of preimplantation genetic screening (PGS) on implantation and pregnancy
rates, but more recent randomized studies have not confirmed, or could not conclude, that PGS actually improved implantation
rates. Team inexperience in embryo screening has been mentioned as the origin of the discrepancies; thus some clinicians allege
a need for more powerful, well-designed, randomized studies performed by specialized teams. However, what if all the contradictory
results about the benefits of PGS and implantation were not due to technical problems or team specialization but were biological
in origin? The developmental programme of an eight-cell embryo relies on signals of maternal origin retrieved from the cytoplasm
to initiate a new transcriptional network that will eventually serve as a filter (checkpoints and apoptosis) for aneuploidy.
Thus, the use of PGS with the objective of improving the likelihood of a successful pregnancy based only on nuclear abnormalities
(aneuploidies) in an early cleavage stage embryo could be invalid since the information (diagnosis) obtained at the moment
of biopsy could be overturned by the transcriptional machinery of the new zygote genome.
Recurrent IVF failure, implantation failure and early embryo demise can be attributed to the high frequency of chromosomal aneuploidy observed in human embryos. Preimplantation genetic screening (PGS) using multiple displacement amplifications (MDA) and array comparative genomic hybridization (aCGH) was successfully performed on eight patients with a minimum of seven recurrent IVF failures with the aim of detecting aneuploidy and ameliorating pregnancy rate. A total of 41 embryos with eight or more cells were biopsied by taking two blastomeres from each embryo. The DNA from these blastomeres were amplified and analysed by aCGH technology. The aCGH results showed a complex panel of chromosomal abnormalities in 60% of the diagnosed embryos. Some abnormalities could not be detected by the seven-probe panel (13, 16, 18, 21, 22, X and Y) used in fluorescence in-situ hybridization. Six out of eight patients had embryos for transfer with five out of those six showing positive pregnancy tests. As far as is known, this report is the first to show a pregnancy after PGS using the aCGH technology. The pregnancy rate obtained here is encouraging and will open the door for enrollment of more patients.
One of the most important factors influencing embryo viability is chromosome imbalance (aneuploidy). Embryos derived from aneuploid gametes have little potential for forming a viable pregnancy, but cannot be distinguished from normal embryos using standard morphological evaluation. For more than a decade, preimplantation genetic screening (PGS) has been used to assist in the identification of aneuploid embryos. However, current strategies, based upon cell biopsy followed by fluorescent in situhybridization, allow less than half of the chromosomes to be screened. In this review, we discuss methods that overcome the limitations of earlier PGS strategies and provide screening of the entire chromosome complement in oocytes and embryos. In recent months, there has been a rapid growth in the number of PGS cycles utilizing one such method, comparative genomic hybridization (CGH). Data from IVF cycles utilizing CGH must be considered preliminary, but appear to indicate a dramatic increase in embryo implantation following comprehensive chromosomal screening. It is expected that methods based upon microarrays will yield similar clinical results and may be sufficiently rapid to permit comprehensive screening without the need for embryo cryopreservation. Some microarray platforms also offer the advantage of embryo fingerprinting and the potential for combined aneuploidy and single gene disorder diagnosis. However, more data concerning accuracy and further reductions in the price of tests will be necessary before microarrays can be widely applied.
The recent debate on preimplantation genetic screening (PGS) has raised questions about its routine use in clinical practice.
It has been suggested that the most effective way to resolve the debate about the usefulness of PGS is to perform more well-designed
and well-executed randomized controlled trials (RCTs). However, in view of the lack of evidence for the effectiveness of PGS
and the accumulating evidence for its harmfulness, it is our opinion that it is unethical to perform additional RCTs for the
indication advanced maternal age using cleavage stage biopsy.
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.
The cause of non−disjunction of chromosome 21 remains largely unknown. Advanced maternal age is associated with both maternal meiosis I (MI) and meiosis II (MII) non−disjunction events. While reduced genetic recombination has been demonstrated in maternal MI errors, the basis for MII errors remains uncertain. We studied 133 trisomy 21 cases with maternal MII errors to test the hypothesis that segregation at MII may also be influenced by genetic recombination. Our data support a highly significant association: MII non−disjunction involves increased recombination that is largely restricted to proximal 21 q. Thus, while absence of a proximal recombination appears to predispose to non−disjunction in MI, the presence of a proximal exchange predisposes to non−disjunction in MII. These findings profoundly affect our understanding of trisomy 21 as they suggest that virtually all maternal non−disjunction results from events occurring in meioisis I.
To assess the outcomes achieved after Cryotop vitrification of both early cleavage and blastocyst-stage embryos and to determine whether the embryo developmental stage and embryo quality as well as the origin of the embryos (ovum donation cycles, patients' own oocytes) and the endometrial preparation for the embryo transfer had any effect on the final outcome.
Observational study.
Private university-affiliated IVF center.
Women undergoing 3,150 warming cycles whose embryos were vitrified due to various reasons.
Vitrification by the Cryotop open device.
Delivery rate (DR) per warming cycle.
Survival rate was 95% (5,722 out of 6,019 embryos). The percentage of intact embryos at warming showing 100% blastomere survival was 93% (95% CI 90.1%-95.3%) for day 2 and 95% (95% CI 94.3%-95.7%) for day 3; 3,057 embryo transfers were performed (3% cancellation rate). The DR/warming cycle was 32.5% (95% CI 30.9%-34.2%). Slight differences in survival rate were found [94.9% (95% CI 93.0%-96.8%) for day 2, 94.2% (95% CI 93.4%-94.9%) for day 3, 95.7% (95% CI 94.5%-96.9%) for day 5, and 97.6% (95% CI 96.9%-98.6%) for day 6]. Overall implantation, clinical pregnancy, ongoing pregnancy, and live birth rates per warming cycle were 35.5% (95% CI 33.5%-38.5%), 41.7% (95% CI 39.9%-43.4%), 32.6% (95% CI 31.0%-34.2%), and 38.1% (95% CI 36.4%-39.8%) respectively. The linear regression model considering embryo developmental stage, ovum donation or patient's own oocytes, and hormonal replacement therapy or natural cycle for endometrial preparation (odds ratio 1.179; 95% CI 0.912-1.277) showed no impact on the DR.
Highly successful cryopreservation of all embryo developmental stages is possible with the use of the Cryotop system. There are no variables clearly exerting a negative effect on the survival and delivery rates.
Pre-implantation genetic diagnosis provides prenatal genetic diagnosis before implantation, thus allowing detection of chromosomal abnormalities and their exclusion from embryo transfer in assisted reproductive technologies. Polar body, blastomere or trophectoderm can each be used to obtain requisite genetic or embryonic DNA. Pre-implantation genetic diagnosis for excluding unbalanced translocations is well accepted, and pre-implantation genetic diagnosis aneuploidy testing to avoid repeated pregnancy losses in couples having recurrent aneuploidy is efficacious in reducing miscarriages. Controversy remains about whether pre-implantation genetic diagnosis aneuploidy testing improves take home pregnancy rates, for which reason adherence to specific indications is recommended while the issue is being adjudicated. Current recommendations are for obligatory 24 chromosome testing, most readily using array comparative genome hybridisation.
Normally long-term depression (LTD) is difficult to be induced in naïve adult rats in vivo, but it can be induced in the juvenile females and acute-stressed adult males. Using these rats as LTD models, we find in our previous study that LTD induction by the classical low-frequency stimuli (LFS) may be associated with sleep. During sleep, endogenous field potential oscillations presented in the neocortical and hippocampal circuits play important roles in synaptic downscaling as well as memory consolidations. Generally, LTD can be considered as a special synaptic downscaling and the classical LFS is very similar to such endogenous oscillations. Thus, we speculate whether we can design a new LFS which is more similar to such oscillations and whether LTD can be induced by it in naïve adult rats? In this study, we found that in the naïve adult rats anesthetized in sleep stage, the classical LFS could not induce LTD, however, a low-intensity LFS, an endogenous oscillation-like one, could induce LTD. Furthermore, in the rats anesthetized in wakefulness stage, neither the classical nor the low-intensity LFS could induce LTD. Our study showed that in the naïve adult rats, LTD could be induced by the oscillation-like LFS in the sleep stage anesthesia, suggesting that LTD may physiologically occur during sleep and be inhibited in wakefulness stage. Our study suggested that in the hippocampus LTD may be a potential long-term synaptic plasticity underlying sleep-dependent memory consolidations.
Polyacrylonitrile (PAN) nanoparticles were successfully prepared by dispersion polymerization of acrylonitrile (AN) in water
using 10 and 20 wt% of the poly(ethylene oxide)-b-PAN macromolecular RAFT (PEO-b-PAN macro-RAFT) agent (M
n=5,600 g/mol, PDI=1.15). The degrees of polymerization of the PEO and PAN blocks were 113 and 16, respectively. The PAN nanoparticles
had a crumpled spherical appearance and their sizes ranged from 50–80 nm. The degree of crystallinity of the PAN particles
was 23 %. The M
n
values of the PAN nanoparticles prepared with 10 and 20 wt% of the PEO-b-PAN macro-RAFT agent were 33,900 and 25,800 g/mol, respectively. The existence of the PEO block on the surface of the PAN
nanoparticles was confirmed by 1H NMR spectroscopy and XPS.
KeywordsDispersion polymerization-Reversible addition fragmentation chain transfer (RAFT) polymerization-Macromolecular RAFT agent-Polyacrylonitrile-Nanoparticles
To assess the impact of oocyte vitrification on aneuploidy and reproductive potential by comparing vitrified and control oocytes from a single patient within a single cycle and a single fresh transfer.
Paired randomized controlled trial in which each patient's cohort of mature oocytes was divided into two even groups with half undergoing Cryotop vitrification and rapid warming and half serving as controls.
Academic center for reproductive medicine.
Forty-four patients with a mean age of 29.9 ± 2.3 years and normal ovarian reserve.
Cryotop vitrification of half of mature oocytes. Trophectoderm biopsy with single nucleotide polymorphism microarray analysis for ploidy and DNA fingerprinting.
Rate of aneuploidy (primary outcome), fertilization, cleavage, blastulation, and implantation in embryos derived from vitrified and control oocytes.
A total of 588 mature oocytes were randomized, with 240/294 (81.6%) surviving vitrification. Among surviving vitrified oocytes, there was a lower fertilization rate with intracytoplasmic sperm injection (77.9% vs. 90.5%; relative risk [RR], 0.86; 95% confidence interval [CI], 0.80-0.93), a lower cleavage rate (90.9% vs. 99.2%; RR, 0.92; 95% CI, 0.87-0.96), and a lower usable blastocyst formation rate per two pronuclei (34.8% vs. 50.8%; RR, 0.68; 95% CI, 0.54-0.86). There was no difference in the rate of embryonic aneuploidy (vitrified, 29.1% vs. control, 26.4%). In paired blastocyst transfers, the ongoing pregnancy rate per embryo transferred was similar (vitrified, 53.9% vs. control, 57.7%).
Although the IVF process is less efficient after oocyte vitrification, implantation rates are equivalent and there is no increased risk of aneuploidy. Given the lack of other viable options, this study provides great reassurance to patients and clinicians applying oocyte vitrification for fertility preservation.
This study retrospectively analysed array comparative genomic hybridization (CGH) results of 7753 embryos from 990 patients to determine the frequency of embryonic euploidy and its relationship with the cohort size (i.e. the number of embryos available for biopsy and array CGH analysis). Linear regression analysis was performed to assess the effect of cohort size on euploidy rate adjusted for the effect of female age. While increasing female age was associated with a significant decrease in euploidy rate of day-3 and day-5 embryos (P<0.001 for both groups), cohort size was not significantly associated with euploidy rate. Logistic regression analysis was performed to assess the effect of cohort size, adjusted for maternal age, on the likelihood of having at least one euploid embryo available for transfer. The odds of having at least one euploid embryo in an assisted cycle was significantly decreased by increasing female age (P<0.01 for both day-3 and day-5 embryos) and was significantly increased by every additional embryo available for analysis (P<0.001 for both day-3 and day-5 embryos).
To develop and validate a quantitative real-time polymerase chain reaction (qPCR)-based method for blastocyst trophectoderm comprehensive chromosome screening (CCS) of aneuploidy.
Prospective, randomized, and blinded.
Academic center for reproductive medicine.
Nine cell lines were obtained from a commercial cell line repository, and 71 discarded human blastocysts were obtained from 24 IVF patients that underwent preimplantation genetic screening.
None.
Consistency of qPCR diagnosis of aneuploidy compared with either conventional karyotyping of cell lines or microarray-based diagnoses of human blastocysts.
Samples from nine cell lines with well characterized karyotypes were diagnosed by qPCR with 97.6% (41/42) consistency. After applying a minimum threshold for concurrence, 100% consistency was achieved. Developmentally normal blastocysts designated as aneuploid or arrested blastocysts designated as euploid by single-nucleotide polymorphism microarray analyses were assigned identical 24 chromosome diagnoses by qPCR in 98.6% of cases (70/71). Overall euploidy (n = 37) and aneuploidy (n = 34) were assigned with 100% consistency. Data was obtained for both sample types in 4 hours.
These data demonstrate the first qPCR technology capable of accurate aneuploidy screening of all 24 chromosomes in 4 hours. This methodology provides an opportunity to evaluate trophectoderm biopsies with subsequent fresh euploid blastocyst transfer. Randomized controlled trials to investigate the clinical efficacy of qPCR-based CCS are currently underway.
To assess the presence of viral RNA or DNA sequences in spent culture media used after ovum pickup (OPU) or embryo culture and in liquid nitrogen (LN) used for oocyte or embryo vitrification in patients seropositive for human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) undergoing IVF cycles.
Descriptive study.
Private university-affiliated IVF center.
Twenty-four women who underwent controlled ovarian stimulation for oocyte vitrification or IVF/ET. A total of 6, 11, and 6 patients were seropositive for HIV, HCV, and HBV, respectively, whereas 1 patient showed a coinfection with HCV and HBV. Seven patients presented positive blood viral load (HIV, n = 1; HBV, n = 1; HCV, n = 5). Sixty-three samples were analyzed: follicular fluid, n = 3; spent culture media, n = 33 (20 after OPU and 13 after embryo culture); and LN, n = 27 (14 and 10 after oocyte and embryo vitrification; and 3 LN storage tank samples).
Ovum pickup, oocyte and/or embryo culture, and/or vitrification by the Cryotop open device. Reverse transcription-polymerase chain reaction analysis was performed for viral screening.
Detection of viral sequences of HIV, HCV, and HVB.
All the samples analyzed tested negative for the detection of viral RNA or DNA sequences.
We have not detected viral sequences after culture and vitrification of oocytes/embryos from HIV-, HBV-, and HCV-seropositive patients. These findings represent good evidence of the lack of risk of cross-contamination among seropositive patients, even using an open device for vitrification.
The combination of trophectoderm biopsy, blastocyst vitrification, and single-nucleotide polymorphism microarray-based technology for comprehensive chromosome screening results in high implantation and live birth rates that could contribute to the practical application of single embryo transfer for infertility patients.
To perform a systematic review of the literature to identify randomized controlled trials assessing the efficacy of oocyte vitrification in terms of oocyte survival, fertilization, embryo development, and pregnancy rates.
Systematic review and meta-analysis of randomized controlled trials.
Private university-affiliated IVF center, university-based hospital.
Patients recruited in randomized controlled trials considering oocyte vitrification as one of the experimental arms and slow freezing or fresh oocytes control as the other.
Vitrification of human oocytes vs. slow freezing or fresh oocytes.
Ongoing pregnancy rate; secondary outcomes were clinical pregnancy rate, implantation rate, embryo development, fertilization rate, and oocyte survival.
Five eligible studies were finally included. They involved 4,282 vitrified oocytes, 3,524 fresh oocytes, and 361 slow-frozen oocytes between 2005 and 2009. The rates of ongoing pregnancy, top-quality embryo, embryo cleavage, and fertilization did not differ between the vitrification and the fresh oocyte groups. The oocyte survival rate was higher in vitrified vs. slow-frozen oocytes (odds ratio [OR] 2.46, 95% confidence interval [CI] 1.82-3.32), although heterogeneity between studies was observed. The fertilization rate was higher in vitrified vs. slow-frozen oocytes (OR 1.50, 95% CI 1.07-2.11). Vitrification also resulted in a higher rate top-quality embryo (22.4% vs. 8.0%, OR 3.32, 95% CI 1.37-8.02) and embryo cleavage rate (day 2: 64.6% vs. 47.7%, OR 2.00, 95% CI 1.33-3.00; day 3: 53.0% vs. 33.3%, OR 2.25, 95% CI 1.32-3.85) as compared with slow freezing.
Vitrification is an efficient method to preserve oocytes, although more large controlled clinical trials are needed to strengthen this conclusion.
Preimplantation genetic screening (PGS) has increasingly been used in the past decade. Here we present a systematic review and meta-analysis of RCTs on the effect of PGS on the probability of live birth after IVF.
PubMed and trial registers were searched for RCTs on PGS. Trials were assessed following predetermined quality criteria. The primary outcome was live birth rate per woman, secondary outcomes were ongoing pregnancy rate, miscarriage rate, multiple pregnancy rate and pregnancy outcome.
Nine RCTs comparing IVF with and without PGS were included in our meta-analysis. Fluorescence in situ hybridization was used in all trials and cleavage stage biopsy was used in all but one trial. PGS significantly lowered live birth rate after IVF for women of advanced maternal age (risk difference: -0.08; 95% confidence interval: -0. 13 to -0.03). For a live birth rate of 26% after IVF without PGS, the rate would be between 13 and 23% using PGS. Trials where PGS was offered to women with a good prognosis and to women with repeated implantation failure suggested similar outcomes.
There is no evidence of a beneficial effect of PGS as currently applied on the live birth rate after IVF. On the contrary, for women of advanced maternal age PGS significantly lowers the live birth rate. Technical drawbacks and chromosomal mosaicism underlie this inefficacy of PGS. New approaches in the application of PGS should be evaluated carefully before their introduction into clinical practice.
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.
Prospective study.
Infertility clinic.
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.
Fluorescence in situ hybridization (FISH) in sperm.
Sperm numerical abnormalities for chromosomes 13, 18, 21, X, and Y; ongoing implantation and pregnancy rates in intracytoplasmic sperm injection (ICSI) cycles.
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.
Sperm FISH analysis using testicular sperm control samples better identifies NOA patients with a lower likelihood of reproductive success.
To assess correlation between blastocyst morphology and chromosomal status.
Observational research study.
An IVF clinic and a specialist preimplanation genetic diagnosis (PGD) laboratory.
Ninety-three couples undergoing IVF treatment in combination with chromosome screening of embryos.
Five hundred blastocysts underwent trophectoderm biopsy and comprehensive chromosome screening using comparative genomic hybridization (CGH). The morphology of the embryos was evaluated using standard methods.
Association of aneuploidy and morphologic score.
A total of 56.7% of blastocysts were aneuploid. One-half of the grade 5/6 blastocysts were euploid, compared with only 37.5% of embryos graded 1/2, suggesting an effect of aneuploidy on blastocyst development. Aneuploidy also had a negative effect on inner cell mass and trophectoderm grades. Morphologically poor blastocysts had a higher incidence of monosomy and abnormalities affecting several chromosomes. The gender ratio was significantly skewed in relation to morphology. A total of 72% of blastocysts attaining the highest morphologic scores (5AA and 6AA) were found to be male, compared with only 40% of grade 3 embryos.
Morphology and aneuploidy are linked at the blastocyst stage. However, the association is weak, and consequently, morphologic analysis cannot be relied on to ensure transfer of chromosomally normal embryos. A significant proportion of aneuploid embryos are capable of achieving the highest morphologic scores, and some euploid embryos are of poor morphology. Gender was associated with blastocyst grading, male embryos developing at a significantly faster rate than females.
To evaluate a new strategy for comprehensive chromosome screening at the blastocyst stage.
Clinical research study.
An IVF clinic and a specialist preimplantation genetic diagnosis laboratory.
Forty-five infertile couples participated in the study. The mean maternal age was 37.7 years, and most couples had at least one previous unsuccessful IVF treatment cycle (mean 2.4).
This study used a novel chromosome screening approach, combining biopsy of several trophectoderm cells on day 5 after fertilization and detailed analysis of all 24 types of chromosome using comparative genomic hybridization.
Proportion of embryos yielding a diagnostic result, aneuploidy rate, implantation rate, and pregnancy rate.
A diagnosis was obtained from 93.7% of embryos tested. The aneuploidy rate was 51.3%. The probability of an individual transferred embryo forming a pregnancy reaching the third trimester/birth was 68.9%, an implantation rate 50% higher than contemporary cycles from the same clinic. The pregnancy rate was 82.2%.
The comprehensive chromosome screening method described overcomes many of the problems that limited earlier aneuploidy screening techniques and may finally allow preimplantation genetic screening to achieve the benefits predicted by theory. The high embryo implantation rate achieved is particularly encouraging and, if confirmed in subsequent studies, will be of great significance for IVF clinics attempting to reduce the number of embryos transferred or to implement single embryo transfer.
To ascertain meiotic aneuploidy of the human egg using array comparative genomic hybridization to evaluate the 23-paired chromosome copy number of first polar body as an objective prognosticator of embryo viability for embryo transfer in the same cycle.
Case report.
Independent-sector IVF program.
A 41-year-old woman with a history of 13 failed cycles of IVF.
Polar body biopsy of metaphase II eggs.
Birth.
Two of the nine eggs were euploid, and the resulting embryos, although morphologically inferior to sibling embryos, were selected for transfer to the uterus, resulting in the birth of a normal healthy baby.
Selection of euploid eggs, as an objective parameter of subsequent embryo viability and with the opportunity to transfer embryos in the same cycle could maximise the opportunity for live birth after IVF even in cases with poor prognosis.
To evaluate the effect of sperm chromosome abnormalities--disomy for sex chromosomes and diploidy--in the chromosomal constitution of preimplantation embryos.
Retrospective cohort study.
Infertility clinic.
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.
