Preimplantation Genetic Diagnosis Embryos Research Articles

Оцінка впливу гінекологічної патології на шанси живонародження серед жінок із повторними невдачами імплантації

Aim - to study the prognostic impact of the presence of gynecological pathology on live birth in women with multiple negative implantation attempts in the context of the proposed methods of examination and treatment, such as preimplantation genetic diagnosis of embryos and individualization of the endometrial preparation protocol, taking into account the results of the assessment of the implantation window. Materials and methods. The prospective cohort study included 93 women with infertility treated by in vitro fertilization (IVF). Patients had repeated failed implantation attempts and were divided into three groups: the Group 1 - patients who were treated with genetically untested embryos according to a standard fixed stimulation protocol, the Group 2 - patients who were treated with euploid embryos after preimplantation genetic screening according to a standard fixed protocol; the Group 3 - patients treated with euploid embryos after preimplantation genetic screening and determination of the implantation window with subsequent modification of the stimulation protocol, according to the results of the endometrial examination. The implantation window was determined by three-time aspiration biopsy of the endometrium during the luteal phase of the menstrual cycle. The samples were analyzed by scanning electron microscopy. Based on the results, the endometrial preparation protocol was individualized for the next IVF attempt. Preimplantation diagnostics of embryos was performed using the next generation method. Statistical analysis was performed using IBM SPSS V25.0 for Windows software. Results. According to the results obtained, the presence of genital pathology reduced the chance of live birth by 30% in patients with repeated failed implantation attempts. Pathologies such as endometriosis, including endometrioid ovarian cysts, benign breast disease and Asherman syndrome have a negative effect on the chance of live birth among patients who have undergone infertility treatment with IVF. Conclusions. The presence of gynecologic pathology reduces the chances of live birth in patients with repeated failed implantation attempts undergoing IVF treatment. The study was conducted in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee for all participants. Informed consent was obtained from the patients. No conflict of interests was declared by the authors.

Assessment of the implantation window and embryonic factor impact to the treatment of recurrent implantation failure (RIF). A prospective study

The aim: to study of the prognostic value of endometrial receptivity and preimplantation genetic diagnosis of embryos, and their influence on the effectiveness of in vitro fertilization (IVF) programs. We also evaluate the importance of this factor in comparison with other potential causes of infertility. Materials and methods: This prospective cohort study included 123 infertile women who underwent in vitro fertilization (IVF) treatment. 93 patients had repeated unsuccessful implantation attempts and were divided into three groups: group 1 - patients who were treated using genetically untested embryos according to a standard fixed stimulation protocol, group 2 - patients who were treated using euploid embryos after preimplantation genetic screening according to standard fixed protocol; group 3 - patients who underwent treatment using euploid embryos after pre-implantation genetic screening and determination of the implantation window with subsequent modification of the stimulation protocol, according to the endometrial examination result. 30 patients had a first attempt at IVF, which was carried out using genetically untested embryos, according to a standard fixed protocol, and made up the control group (CG). Determination of the window of implantation was carried out by triple aspiration biopsy of the endometrium during the luteal phase of the menstrual cycle since the endometrium is most susceptible to implantation during this period. Samples were analyzed using scanning electron microscopy. Based on the results obtained, the endometrial preparation protocol was individualized for the next attempt. preimplantation genetic testing (PGT) of embryos was carried out by the next generation (NGS) method. Statistical analysis was performed using IBM SPSS V25.0 for Windows software. Results: According to the obtained results, patient characteristics, screening rates, IVF cycle characteristics, and the number, quality, and stage of transferred embryos were compared between groups. The rate of clinical pregnancy was 46.7 % among patients of group 1.70 % among patients of group 2, 82.8 % among patients of group 3 and 50.0 % of the control group and statistically significantly different between groups (χ2=10.955, p= 0.012). The rate of live birth was 43.3 % among patients of group 1, 53.3 % among patients of group 2, 72.4 % among patients of group 3 and 43.3 % - of the control group, however, it did not differ statistically significantly between groups (χ2=6,639, р=0,084) Conclusions: The unique window of implantation and the embryonic factor are among the main reasons for multiple failed implantation attempts. Personalization of the endometrial preparation protocol and preimplantation embryo diagnosis are effective methods to improve IVF outcomes

GENETIC SEGREGATION OF MITOCHONDRIAL 3697>GA MUTATION IN PGD CYCLES

To explore the dynamics and inheritance of m.3697G>A mutation and the performance of preimplantation genetic diagnosis (PGD) for mutant carriers. To analyze whether the cumulus cells surrounding oocytes can be used as a noninvasive PGD indicator for maternal inherited mtDNA mutation. The study encompassed a pedigree of four m.3697G>A carriers, including one asymptomatic PGD patient and one LS patient. Next generation sequencing (NGS) was used to detect m.3697G>A heteroplasmy in blood samples from the pedigree. Twelve cumulus oocyte complexes (COCs) were collected in the first PGD cycle and 11 COCs in the second cycle from the patient. Cumulus cells were isolated from COCs. Trophectoderm (TE) cells were biopsied from blastocysts on day 5 or day 6. The mutation load of TE biopsies, arrested embryos, blastomeres, polar bodies, the degenerate oocyte and corresponding cumulus cells were analysed via NGS. From 23 COCs, 20 oocytes were fertilized successfully via intracytoplasmic sperm injection. On day 5 or day 6 post-fertilization, 15 blastocysts were obtained and biopsied. The mutation load of the 15 TE biopsies (ranged from 15.2% to 100%), allowing selection of embryos for implantation. In the first cycle, a blastocyst with mosaic chromosomal copy number and mutation load of 31.7% was opted for transfer. However, the patient failed to yield a clinical pregnancy. In the second PGD cycle, a euploid blastocyst with mutation load of 53.9% was selected to transfer, which enable her to carry a successful pregnancy. Now she is in the stage of second trimester. Mutation load of cumulus cells may not correlate well with TE mutation load. This study first reported that m.3697G>A mutation showed a random pattern of inheritance in PGD embryos, it is almost impossible to produce zero or very low heterogenous embryos. But PGD could be applied to reduce risk of inheritance of a high mutation load and to yield successful pregnancy. Cumulus cells are not a suitable predictor of late-stage embryo mutation load.

Effects of religious orientation and state secularism on pre-implantation genetic diagnosis

This study aimed to test the associations of religious orientation (extrinsic, intrinsic, and quest) and secularism of state with individuals' attitudes towards the pre-implantation genetic diagnosis of embryos. Moreover, we tested the mediating effects of secularism of state on the relationship between religious orientations and attitudes towards this issue related to embryos. Participants were 312 Catholic Italians that completed a questionnaire containing measures of investigated constructs. Results showed that attitude towards pre-implantation genetic diagnosis negatively correlated with intrinsic religious orientation and positively with extrinsic religious orientation and secularism. Moreover, results indicated that secularism mediated the relationship between extrinsic and intrinsic orientation and attitude towards pre-implantation genetic diagnosis. Taking together these results indicate that real endorsement with religion is associated with the refusal of pre-implantation genetic diagnosis because intrinsic religious orientation is related to the desire for state laws to follow religious principles; on the other hand, the use of religion for utilitarian reasons is associated to the acceptance of pre-implantation genetic diagnosis in order to have a religious state and then maintain Catholics’ privileges.

The Effect of Values and Secularism on Attitude towards Pre-Implantation Genetic Diagnosis of Embryos

In this study we tested the associations of four high-order values (openness to change, self-transcendence, conservation, and self-enhancement, devised according to Schwartz’s model) and secularism of state with individuals’ attitude towards pre-implantation genetic diagnosis of embryos. Moreover, we tested the mediating effects of secularism of state on the relationship between values and attitude towards this issue related to embryos. Participants were 289 Spaniards who completed a questionnaire. Results showed that attitude towards pre-implantation genetic diagnosis was negatively affected by conservation and positively by self-transcendence. Moreover, results indicated that attitude towards a secular state positively correlates with attitude towards pre-implantation genetic diagnosis. Finally, results showed that secularism mediated the effects of conservation and self-transcendence, but not the effect of openness to change and self-enhancement on attitude towards pre-implantation genetic diagnosis. Taken together, results of this study suggest that people adopting values emphasizing the defence of the tradition reject pre-implantation genetic diagnosis because they want state laws to represent religious traditional values; on the other hand, people endorsing values emphasising the welfare of all accept pre-implantation genetic diagnosis because they want state laws to be free from religious values.

The tyranny of choice: reproductive selection in the future.

This article explores the enormous challenges to reproductive decision making that could result from two emerging technologies: the potential capacity to create vast numbers of embryos for preimplantation genetic diagnosis and the ability to obtain ever more predictive information about the embryo. Together these technologies could change our reproductive experience, exacerbate existing inequities, and profoundly affect reproductive decision making. Simply comprehending the dizzying amounts of predictive information about the health and traits of future children will overwhelm future parents. But trying to choose embryos with the ‘best’ combination of genetic variants could be paralyzing. Nevertheless, numerous pressures will make this technology alluring, compelling providers to develop remedies to assist future parents with these difficult reproductive decisions. The remedies, however, will create their own challenges. Some might test the limits of reproductive autonomy and heighten social inequities. A particularly vexing remedy would be the development of algorithms for embryo selection, which could routinize reproductive decisions, reduce societal diversity, exacerbate ‘choice overload’ effects, challenge professional norms, and raise the specter of eugenics. Ultimately, this article is a cautionary tale urging circumspection as technological advancements seem to propel us inevitably toward a reproductive future that could create a tyranny of choice.

Moral challenges of research on embryos in pre-implantation genetic diagnosis and IVF

Purposes of pre-implantation genetic diagnosis (PGD) include screening for single gene mutations for late onset disorders or susceptibility to cancer. The problematic issue is that PGD is to produce a healthy baby, causing the destruction of some embryos that have been transferred by in vitro fertilization (IVF). Some PGD embryos may be discarded because they are excess to the woman or couples’ goal for family creation, and have been diagnosed as being affected by a particular genetic condition that woman/couple wish to avoid. So, the controversial issue is the destruction of embryos as a consequence of fertility treatment that raises questions on whether the moral status of an embryo of 3 days is the same as that of a born, living adult human being.

Natural selection between day 3 and day 5/6 PGD embryos in couples with reciprocal or Robertsonian translocations.

For translocation carriers, preimplantation genetic diagnosis (PGD) provides the opportunity to distinguish between normal/balanced and unbalanced embryos prior to implantation and, as such, increases the likelihood of a successful ongoing pregnancy. The data presented here compares autosomal reciprocal and Robertsonian translocation segregation patterns in day 3 versus day 5/6 IVF-PGD embryos to determine if there is a difference in the chromosome segregation patterns observed at these developmental time points. A retrospective analysis on PGD translocation carriers at Monash IVF was performed. Segregation patterns were compared between day 3 and day 5/6 embryos to ascertain whether selection against malsegregants exists. For reciprocal translocations, 1649 day 3 embryos (139 translocations) from 144 couples and 128 day 5/6 embryos (59 translocations) from 60 couples were analysed. Day 3 segregation analysis showed that 22.3% of embryos were normal/balanced (consistent with 2:2 alternate segregation) and 77.7% were unbalanced (malsegregation). Day 5/6 segregation analysis showed that 53.1% of embryos were normal/balanced and 46.9% were unbalanced. For Robertsonian translocations, 847 day 3 embryos (8 translocations) from 54 couples and 193 day 5/6 embryos (6 translocations) from 31 couples were analysed. Day 3 segregation analysis showed that 38.7% of embryos were normal/balanced (consistent with 2:1 alternate segregation) and 61.3% were unbalanced. Day 5/6 segregation analysis showed that 74.1% of embryos were normal/balanced and 25.9% were unbalanced. This data demonstrates an increase in the proportion of genetically normal/balanced embryos at day 5/6 of development. This suggests a strong natural selection process between day 3 and day 5/6 in favour of normal/balanced embryos. These findings support performing PGD testing on day 5/6 of embryo development.

Abstract PD6-03: Preserving fertility in young women undergoing chemotherapy for early breast cancer; the Maastricht experience

Abstract Purpose This study aimed to evaluate the uptake of fertility preservation, rate of pregnancy, pregnancy outcome and breast cancer outcome after diagnosis of early breast cancer in young women who were referred to Maastricht University Medical Center, from the regional hospitals in the Southeast part of the Netherlands. Patients and methods We prospectively registered the demographics of patients, who visited our university hospital for counseling on fertility preservation at diagnosis of stage I-III invasive breast cancer in the years 2008-2015. At baseline, tumor and treatment characteristics were registered. During follow-up information on fullfilled childwish and disease status was collected. To compare the fertility preservation group and the non-fertility preservation group independent samples Student t-tests and Chi-square tests were conducted. Results In total 128 women with a median age of 32 years (19 – 43) were referred for fertility preservation counseling before start of chemotherapy, with an increase in referral in the more recent years. Thirty-nine (30.5%) women chose for fertility preservation: in 26 patients embryos were frozen, in seven oocytes, and in one both embryos and oocytes. In four patients the procedure was not succesfull. Patients who had chosen for fertility preservation more often had a male partner (87.2% vs 70.8%, P = 0.05) and had a smaller tumor size (median 19 versus 23 mm, P = 0.04) at the time of diagnosis compared to those who did not chose for fertility preservation. During a median follow-up of 30.3 months (range 0 – 96.9), 27 (21.1%) patients had tried to conceive: 14 (35.9%) women in the fertility versus 13 (14.6%) in the non-fertility preservation group. All of these had recovery of ovarian function after chemotherapy-induced ovarian failure. Only two women used the cryopreserved embryos, both succesfully and combined with preimplantation genetic diagnosis of the embryos because of germline mutations in BRCA1-gene. Eight patients in the fertility preservation group and seven patients in the non-fertility preservation group became at least once pregnant. In the fertility preservation group, eight healthy babies were born, one baby had Morbus Hirschsprung, one women is pregnant at this moment and one woman had a miscarriage. Of the eleven pregnancies in the non-fertility preservation group, nine healthy babies were born and one woman had two miscarriages. Of the referred 128 women, nine (7.0%) had breast cancer recurrence, three in the fertility preservation group versus six in the non-fertility preservation group. Conclusion One third of referred patients choose for fertility preservation before start of chemotherapy. In all of these patients, the ovarian function recovered. However, two couples used their cryopreserved embryos for preimplantation genetic diagnosis and both became pregnant. Since the follow-up time is relatively short, more data are mandatory to make a statement on the effectiveness of fertility preservation techniques in young breast cancers patients. Citation Format: Vriens IJ, Butalid EM, Schepers-van der Sterren EE, van der Poel MH, Jansen-Engelen SL, van Riel A-MM, van de Wouw YJ, Vriens BE, van Haaren ER, Lemaire BM, Dercksen WW, Luiten EJ, de Boer M, de Die-Smulders CE, Derhaag JG, van Golde RJ, Tjan-Heijnen VC. Preserving fertility in young women undergoing chemotherapy for early breast cancer; the Maastricht experience [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD6-03.

Number of embryos biopsied as a predictive indicator for the outcome of preimplantation genetic diagnosis by fluorescence in situ hybridisation in translocation cases.

This study aimed to investigate the optimum number of embryos to be biopsied in order to increase the likelihood of obtaining a balanced/normal embryo following preimplantation genetic diagnosis (PGD) by fluorescence in situ hybridisation (FISH) for translocation carriers. Patients with low number of fertilised oocytes (≤5) or low number of embryos available for PGD (<7) underwent multiple hormonal stimulation cycles and their embryos from each cycle were vitrified and accumulated to obtain at least three embryos for PGD. Fifty-seven PGD cycles were performed for translocation carriers by FISH on day 3 of embryo development. PGD and pregnancy outcomes were examined according to the number of embryos biopsied. The cancellation rates of embryo transfer for the reciprocal translocation carriers were 40% when more than eight embryos were biopsied and it was as high as 78% when low number of embryos (less than nine) were biopsied. For Robertsonian translocation carriers, when more than eight embryos were biopsied, there were no embryo transfer cancellations. This study showed that when there are more than nine embryos biopsied for PGD, the likelihood of obtaining a balanced embryo and positive pregnancy outcome is significantly higher (P < 0.05) in such the overall pregnancy rate was 63% for reciprocal and 86% for Robertsonian carriers. This was reduced to only 7% for reciprocal and 14% for Robertsonian translocation carriers when less than nine embryos were biopsied. One of the limitations of this study was that the analysis was performed by FISH and more studies should investigate the outcomes of embryo accumulation following comprehensive chromosome analysis.

Validation of copy number variation sequencing for detecting chromosome imbalances in human preimplantation embryos.

Chromosome aneuploidies commonly arise in embryos produced by assisted reproductive technologies and represent a major cause of implantation failure and miscarriage. Currently, preimplantation genetic diagnosis (PGD) is performed by array-based methods to identify euploid embryos for transfer to the patient. We speculated that a combination of next-generation sequencing technologies and sophisticated bioinformatics would deliver a more comprehensive and accurate methodology to improve the overall efficacy of embryo testing. To meet this challenge, we developed a high-resolution copy number variation (CNV) sequencing pipeline suitable for single-cell analysis. In validation studies, we showed that CNV-Seq was highly sensitive and specific for detection of euploidy, aneuploidy, and segmental imbalances in 24 whole genome amplification samples from PGD embryos that were originally diagnosed by gold standard array comparative genomic hybridization. In addition, CNV-Seq was capable of detecting, mapping, and accurately quantifying terminal chromosome imbalances down to 1 Mb in size originating from abnormal segregation of translocation chromosomes. These validation studies indicate that CNV-Seq displays the hallmarks of an accurate and reliable embryo test with the potential to further improve the overall efficacy of PGD.

Aberrant Epigenetic Modification in Murine Brain Tissues of Offspring from Preimplantation Genetic Diagnosis Blastomere Biopsies1

Preimplantation genetic diagnosis (PGD) has been prevalent in the field of assisted reproductive technology, yet the long-term risks of PGD to offspring remain unknown. In the present study, the early development of PGD embryos, postimplantation characteristics, and birth rate following PGD were determined. Moreover, the behavior of the offspring conceived from the biopsied embryos was evaluated with the Morris water maze and pole climbing tests. Finally, the epigenetic modification of the global genome and methylation patterns for the H19, Igf2, and Snrpn imprinted genes were identified. The results indicated a significant delay in the blastocoel formation of PGD embryos and a decrease in the implantation ability of these embryos, which was related to the decreased number of cells in the PGD blastocysts. The PGD mice spent more time on both the nontrained quadrant of the water maze and climbing down the pole. Furthermore, the 5-hydroxymethylcytosine content in the brain tissues of PGD mice was significantly increased, but no difference was found in 5-methylcytosine content. The differentially methylated regions of H19/Igf2 exhibited decreased methylation patterns, but that of Snrpn was normal, compared to the control group. Quantitative RT-PCR indicated that Igf2 mRNA expression was significantly decreased but that H19 and Snrpn mRNAs were expressed normally. In conclusion, blastomere biopsies in PGD procedures carry potential risks to embryo development and the behavior of resulting offspring; these risks may arise from aberrant epigenetic modification and methylation patterns in brain tissues. Further studies are needed to better understand the risks associated with PGD.

Evolution of aneuploidy up to Day 4 of human preimplantation development

What is the incidence of aneuploidy and mosaicism in all cells of top-quality Day-4 embryos analysed by array-based comparative genomic hybridization (array CGH)? Our data show extensive abnormalities in Day-4 embryos. Numerous studies on human embryos at Day 3 and Day 5 of development show that they frequently contain aneuploid cells and are mosaic, although Day-5 embryos contain proportionally more normal cells than at Day 3. In contrast, only limited data exist on Day 4 of preimplantation development, despite the fact that it is the suggested stage for the initiation of the process of self-correction. Thirteen embryos were analysed: four fresh good-quality preimplantation genetic diagnosis (PGD) embryos and nine good-quality surplus embryos cryopreserved on Day 3 and donated for research. On Day 4, following removal of the zona pellucida, all blastomeres were disaggregated and collected. The genomic DNA of 283 single blastomeres from disaggregated embryos was amplified. Array CGH was carried out using 24SureTM Cytochip microarrays. After scanning of the microarray slides, the images were analysed using BlueFuse Software (BlueGnome). Combined with selective microsatellite analysis, hypothetical reconstructions of embryo chromosome complements were made following each of the first four cleavage divisions. No chromosome imbalance was detected for one PGD embryo, the other three were mosaic containing between 16 and 75% abnormal cells. All nine frozen-thawed embryos were abnormal. Six were mosaic with between 30 and 100% abnormal cells; three had abnormalities of meiotic origin, two of which displayed mitotic abnormalities. Evidence was also found of mitotic unbalanced structural chromosome rearrangements. The higher rate of abnormality of frozen-thawed embryos is based on a small number of embryos and cannot be tested statistically. The aneuploidy can mostly be explained by anaphase lag and non-disjunction. In some cases, we hypothesize endoreduplication followed by a cellular division with multipolar spindles to explain the results. Array CGH technology determines relative quantification of chromosomal domains but does not allow for the visualization of chromosomal rearrangements, assessment of ploidy or detection of uniparental isodisomy. Conclusions drawn on segmental abnormalities should be treated with caution. The division trees presented are hypothetical models projecting back in time that try to explain observations in single blastomeres of Day 4 embryos. The limited number of embryos analysed does not allow drawing firm conclusions, but nevertheless provides valuable data on the origin of aneuploidy in human embryos. Our data show extensive abnormalities in Day-4 embryos. We found no evidence of self-correction at this stage of development, suggesting that this process may start at a later stage of development.

An ethical view on the initial clinical application of the new type of preimplantation genetic diagnosis of embryos in Japan

An ethical view on the initial clinical application of the new type of preimplantation genetic diagnosis of embryos in Japan

Bioethical dilemmas of assisted reproduction in the opinions of Polish women in infertility treatment: a research report

Infertility Accepted treatment is replete with bioethical dilemmas regarding the limits of available medical therapies. Poland has no legal acts regulating the ethical problems associated with infertility treatment and work...

Analysis using fish of sperm and embryos from two carriers of rare rob(13;21) and rob(15;22) robertsonian translocation undergoing PGD

The majority of fluorescence in situ hybridization (FISH) studies on the meiotic segregation of Robertsonian translocations focus on the most common types, rob(13; 14) and rob(14; 21). Here we report the first study for carriers of rare Robertsonian translocations rob(13; 21) and rob(15; 22) combining analysis of meiotic segregation in sperm and blastomeres following pre-implantation genetic diagnosis (PGD). Dual-colour FISH was applied to nuclei from spermatozoa and blastomeres from PGD embryos using two subterminal contig probes for each translocation, and a second round with probes for chromosomes 16 and 18. Patient 1 had a rob(13; 21) and patient 2 had a rob(15; 22), and 86.3% and 87.5% of gametes respectively were consistent with meiotic segregation resulting in a normal or balanced chromosome complement. Analysis of embryos showed that for patient 1 and 2 respectively, 25% and 46% were balanced, and of the unbalanced embryos, 50% and 31% were mosaic or chaotic. Our patients with a rob(13; 21) and rob(15; 22) were found to have a similar meiotic segregation pattern to that for male carriers of the common Robertsonian translocations. The observed rate in unbalanced embryos being mosaic or chaotic may result in an increased risk of chromosomal abnormalities. Our results may help to improve the genetic counseling for carriers of rare Robertsonian translocations.

My Views on the Extention of Application of Preimplantation Genetic Diagnosis of Embryos (PGD) by JSOG, and on the Future Course and the Potential Problems of PGD Due to the Wide Application

My Views on the Extention of Application of Preimplantation Genetic Diagnosis of Embryos (PGD) by JSOG, and on the Future Course and the Potential Problems of PGD Due to the Wide Application

Miscarriage karyotype and its relationship with maternal body mass index, age, and mode of conception

This study investigated the association between miscarriage karyotype and body mass index, maternal age, and mode of conception. Miscarriages after IVF and/or intracytoplasmic sperm injection were less frequently aneuploid; advanced maternal age was associated with an increase in aneuploid products of conception; overweight and obese women aged <35 years were less likely to have aneuploid miscarriages than women in a healthy weight range, suggesting alternate mechanisms for miscarriage in this population.

Derivation of embryonic stem cells from pre-implantation genetic diagnosis embryos: possible but inefficient

OBJECTIVE: Disease specific embryonic stem cells (ESC) have been derived from affected embryos undergoing pre-implantation genetic diagnosis (PGD). However, the likelihood of creating an ESC line from a potential donor is unknown. Factors throughout the process limit the efficiency of ESC generation. Techniques to optimize ESC derivation from non-PGD embryos have been established, though whether they apply to PGD embryos is unclear. This information would be useful to patients considering donation as well as their clinicians. We report on our experience deriving ESC from our PGD program. DESIGN: Descriptive study. MATERIALS AND METHODS: Patients undergoing PGD were recruited to donate affected embryos. Embryos were graded and cultured until day 6. The inner cell mass (ICM) was isolated by laser and plated on feeder cells. Cultures were assessed regularly for adherence and growth. hESC were characterized using standard techniques. RESULTS: From 9/08 to 6/09, we initiated 140 PGD cycles: 39 for single gene disorders, 22 for translocations, and 79 for aneuploidy screening. 12 patients consented to participate and 4 ultimately donated. Consented subjects who did not donate had cancelled cycles or no viable embryos. 36 embryos were obtained ranging in grade from cleavage stage arrest to 6AA blastocysts. 30 ICMs were plated, of which 12 adhered. 3 ESC lines were derived, 2 from a patient with Charcot Marie Tooth and 1 from a patient with a translocation. CONCLUSION: ESC lines can be derived from PGD embryos, though with limited efficiency. In this study, 3 ESC lines were derived from a large potential group of donors. While our embryo-specific derivation rates are lower than those reported for non-PGD embryos, whether this is operator or embryo dependent is unclear. ESC yield from PGD cases may be limited by other factors such as, PGD indication, willingness to donate to research, and cycle outcome. Further study is needed to clarify these issues and optimize the creation of ESC lines.

Average size of blastomere should be biopsied from day 3 embryos to chose mononucleate cell suitable for PGD

OBJECTIVE: A single blastomere is usually biopsied from day 3 embryos for preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridization (FISH). In the preparation of blastomeres, specimen without nucleus is often encountered. Not only the loss of cells during fixation process, but also the fixation of anucleate blastomere causes this problem. The aim of the present study was to determine the relationship between the incidence of mononucleate blastomeres and the size of blastomeres in day 3 embryos. DESIGN: Experimental laboratory study. MATERIALS AND METHODS: Totally 60 day 3 embryos (448 blastomeres) to be discarded were applied for the present study under informed consent with patients after approval of IVF JAPAN IRB committee. Zona pellucida of the embryo was removed by acid Tyrode's solution and blastomeres were separated from each other. The average diameters of the blastomere were measured using a micrometer attached to inverted microscope. The presence of nucleus in each blastomere on the slide was assessed by fluorescent microscope, after staining with Hoechst 33342 for 10 minutes in incubator. RESULTS: The presence of mononucleate blastomeres in 8 cell embryos was 0% from the blastomeres less than 35μm in diameter, 40.0% between 35μm and 40μm, 42.9% between 40μm and 45μm, 82.6% between 45μm and 50μm, 94.3% between 50μm and 55μm, 94.4% between 55μm and 60μm, 100% between 60μm and 65μm and 33.3% over 65μm in diameter. The blastomeres between 45μm and 65μm in diameter had mononucleus significantly higher to the blastomeres either less than 45μm or over 65μm. The rates of mononucleate blastomere in 7 cell and 9 cell embryos were similar to that in 8 cell embryos. However, the size of blastomeres with mononucleus shifted to the larger in 6 cell embryos and smaller in 10 cell embryos. CONCLUSIONS: The results of the present study indicate that average size of blastomeres should be selected for biopsy in day 3 embryos to select mononucleate blastomere efficiently in PGD procedure.