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PGD is a well accepted reproductive choice for couples at genetic risk and involves the diagnosis and transfer of unaffected IVF embryos. PGD for monogenetic diseases is most commonly accomplished by the biopsy of one or two blastomeres from cleavage stage embryos, followed by PCR-based protocols. However, PCR-based DNA analysis of one or two cells is subject to several problems, including total PCR failure, or failure of one allele to amplify. Trophectoderm biopsy at the blastocyst stage enables the removal of more than two cells for diagnosis while being non-invasive to the inner cell mass which is destined for fetal development. The aim of this study was to develop a safe, reliable technique for the biopsy of trophectoderm cells from human blastocysts. This case report demonstrates that removal of trophectoderm cells prior to blastocyst transfer is compatible with implantation and development to term. Here we report successful PGD for β-thalassaemia following trophectoderm cell biopsy from blastocysts and the birth of a healthy infant. [ABSTRACT FROM AUTHOR]

: BACKGROUND Trophectoderm biopsy at the blastocyst stage is an emerging approach in preimplantation genetic diagnosis (PGD). This study aimed to compare genotyping success and implantation rates in PGD cycles for β-thalassaemia following biopsy at the cleavage versus the blastocyst stage, with transfer of blastocysts. : METHODS This pilot study included 20 cycles: Group A: 10 cycles, day 3 blastomere biopsy, day 5 transfer; Group B: 10 cycles, day 5 trophectoderm biopsy, day 6 transfer. Standard-assisted reproduction and laser biopsy procedures were used. Biopsied cells were genotyped using real-time PCR multiplexed with fluorescent microsatellite analysis. : RESULTS In Group A, 131 fertilized eggs developed to 101 embryos suitable for single blastomere biopsy; 76/101 blastomeres were diagnosed (75.2%), 30 unaffected blastocysts were transferred resulting in six pregnancies (eight fetal hearts, 26.7% implantation rate). In Group B, 128 fertilized eggs developed to 53 blastocysts for trophectoderm biopsy (four to five cells), with 50/53 blastocysts diagnosed (94.3%), 21 unaffected blastocysts transferred and 6 pregnancies initiated (10 fetal hearts, 47.6% implantation rate). Overall, nine pregnancies reached >10 weeks gestation and were confirmed unaffected by prenatal diagnosis, with 12 healthy babies born. : CONCLUSIONS This pilot study suggests that trophectoderm biopsy and blastocyst transfer may be more advantageous than cleavage stage biopsy with respect to outcome of PGD for monogenic diseases. [ABSTRACT FROM AUTHOR]

Background: The egg donation model offers an opportunity to isolate the male factor and evaluate its impact on IVF-intracytoplasmic sperm injection and pregnancy outcomes., Objective: To study the effect of non-obstructive azoospermia on intracytoplasmic sperm injection and pregnancy outcomes compared with severe oligozoospermia and mild-to-moderate oligozoospermia in egg recipient cycles., Materials and Methods: This is a retrospective longitudinal cohort study, including 1594 patients who underwent intracytoplasmic sperm injection in egg recipient cycles with preimplantation genetic testing for aneuploidies. The cohort was divided into three groups: couples with non-obstructive azoospermia accounting for 479 patients (30%); couples with severe oligozoospermia (sperm number <5 × 10 6 /mL), accounting for 442 patients (27.8%); couples with mild-to-moderate oligozoospermia, with sperm number >5 × 10 6 and <15 × 10 6 /mL, accounting for 673 patients (42.2%)., Results: The fertilisation rate was significantly reduced in the non-obstructive azoospermia group as compared with the severe oligozoospermia and the mild-to-moderate oligozoospermia group: 30.3% versus 63% and 77.3% (p < 0.05). Logistic regression analysis adjusted for confounders highlighted non-obstructive azoospermia as a negative predictor of obtaining a euploid blastocyst both per injected oocyte and per obtained blastocyst. The miscarriage rate in the non-obstructive azoospermia group was 11.8%; higher than the severe oligozoospermia and mild-to-moderate oligozoospermia groups (7% and 2.7%) (p < 0.05). The live birth rate per embryo transfer (ET) was significantly lower in the non-obstructive azoospermia group compared with the severe oligozoospermia and the mild-to-moderate oligozoospermia group (20.4% vs. 30.3% and 35.4%, p < 0.05). The risk of preterm labour was significantly higher in the non-obstructive azoospermia group, compared with the severe oligozoospermia and mild-to-moderate oligozoospermia group (55.1% vs. 46.8% and 16.1%, p < 0.001), and this difference was observed in both singleton and twin pregnancies., Discussion and Conclusion: In our retrospective comparative study, non-obstructive azoospermia significantly affects early embryonic potential and live birth rates per cycle and per embryo transfer. It is also associated with higher risk of preterm birth. Future prospective multi-centre studies are needed to highlight the effect of sperm quality on ART and pregnancy outcomes., (© 2023 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Increasing female age is accompanied by a corresponding fall in her fertility. This decline is influenced by a variety of factors over an individual's life course including background genetics, local environment and diet. Studying both coding and non-coding RNAs of the embryo could aid our understanding of the causes and/or effects of the physiological processes accompanying the decline including the differential expression of sub-cellular biomarkers indicative of various diseases. The current study is a post-hoc analysis of the expression of trophectoderm RNA data derived from a previous high throughput study. Its main aim is to determine the characteristics and potential functionalities that characterize long non-coding RNAs. As reported previously, a maternal age-related component is potentially implicated in implantation success. Trophectoderm samples representing the full range of maternal reproductive ages were considered in relation to embryonic implantation potential, trophectoderm transcriptome dynamics and reproductive maternal age. The long non-coding RNA (lncRNA) biomarkers identified here are consistent with the activities of embryo-endometrial crosstalk, developmental competency and implantation and share common characteristics with markers of neoplasia/cancer invasion. Corresponding genes for expressed lncRNAs were more active in the blastocysts of younger women are associated with metabolic pathways including cholesterol biosynthesis and steroidogenesis.

Purpose: To investigate whether preimplantation genetic testing for aneuploidy (PGT-A) improves the clinical outcome in patients with advanced maternal age (AMA), recurrent miscarriages (RM), and recurrent implantation failure (RIF)., Methods: Retrospective cohort study from a single IVF center and a single genetics laboratory. One hundred seventy-six patients undergoing PGT-A were assigned to three groups: an AMA group, an RM group, and a RIF group. Two hundred seventy-nine patients that did not undergo PGT-A were used as controls and subgrouped similarly to the PGT-A cohort. For the PGT-A groups, trophectoderm biopsy was performed and array comparative genomic hybridization was used for PGT-A. Clinical outcomes were compared with the control groups., Results: In the RM group, we observed a significant decrease of early pregnancy loss rates in the PGT-A group (18.1% vs 75%) and a significant increase in live birth rate per transfer (50% vs 12.5%) and live birth rate per patient (36% vs 12.5%). In the RIF group, a statistically significant increase in the implantation rate per transfer (69.5% vs 33.3%) as well as the live birth rate per embryo transfer (47.8% vs 19%) was observed. In the AMA group, a statistically significant reduction in biochemical pregnancy loss was observed (3.7% vs 31.5%); however, live birth rates per embryo transfer and per patient were not significantly higher than the control group., Conclusion: Our results agree with recently published studies, which suggest caution in the universal application of PGT-A in women with infertility. Instead, a more personalized approach by choosing the right candidates for PGT-A intervention should be followed., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Recurrent implantation failure (RIF) is a multifactorial condition affecting 10-15% of in vitro fertilization (IVF) couples. Data suggest that functional dysregulation of the endometrial immune system constitutes one of the main pathophysiological mechanisms leading to RIF. The aim of this article is to provide a thorough presentation and evaluation of the role of interleukins (ILs) in the pathogenesis of RIF. A comprehensive literature screening was performed summarizing current evidence. During implantation, several classes of ILs are secreted by epithelial and stromal endometrial cells, including IL-6, IL-10, IL-12, IL-15, IL-18, and the leukemia inhibitory factor. These ILs create a perplexing network that orchestrates both proliferation and maturation of uterine natural killer cells, controls the function of regulatory T and B cells inhibiting the secretion of antifetal antibodies, and supports trophoblast invasion and decidua formation. The existing data indicate associations between ILs and RIF. The extensive analysis performed herein concludes that the dysregulation of the ILs network indeed jeopardizes implantation leading to RIF. This review further proposes a mapping of future research on how to move forward from mere associations to robust molecular data that will allow an accurate profiling of ILs in turn enabling evidence-based consultancy and decision making when addressing RIF patients.

Study Question: Are there age-related differences in gene expression during the germinal vesicle (GV) to metaphase II (MII) stage transition in euploid human oocytes?, Summary Answer: A decrease in mitochondrial-related transcripts from GV to MII oocytes was observed, with a much greater reduction in MII oocytes with advanced age., What Is Known Already: Early embryonic development is dependent on maternal transcripts accumulated and stored within the oocyte during oogenesis. Transcriptional activity of the oocyte, which dictates its ultimate developmental potential, may be influenced by age and explain the reduced competence of advanced maternal age (AMA) oocytes compared with the young maternal age (YMA). Gene expression has been studied in human and animal oocytes; however, RNA sequencing could provide further insights into the transcriptome profiling of GV and in vivo matured MII euploid oocytes of YMA and AMA patients., Study Design, Size, Duration: Fifteen women treated for infertility in a single IVF unit agreed to participate in this study. Five GV and 5 MII oocytes from 6, 21-26 years old women (YMA cohort) and 5 GV and 6 MII oocytes from 6, 41-44 years old women (AMA cohort) undergoing IVF treatment were donated. The samples were collected within a time frame of 4 months. RNA was isolated and deep sequenced at the single-cell level. All donors provided either GV or MII oocytes., Participants/materials, Setting, Methods: Cumulus dissection from donated oocytes was performed 38 h after hCG injection, denuded oocytes were inserted into lysis buffer supplemented with RNase inhibitor. The samples were stored at -80°C until further use. Isolated RNA from GV and MII oocytes underwent library preparation using an oligo deoxy-thymidine (dT) priming approach (SMART-Seq v4 Ultra Low Input RNA assay; Takara Bio, Japan) and Nextera XT DNA library preparation assay (Illumina, USA) followed by deep sequencing. Data processing, quality assessment and bioinformatics analysis were performed using source-software, mainly including FastQC, HISAT2, StringTie and edgeR, along with functional annotation analysis, while scploid R package was employed to determine the ploidy status., Main Results and the Role of Chance: Following deep sequencing of single GV and MII oocytes in both YMA and AMA cohorts, several hundred transcripts were found to be expressed at significantly different levels. When YMA and AMA MII oocyte transcriptomes were compared, the most significant of these were related to mitochondrial structure and function, including biological processes, mitochondrial respiratory chain complex I assembly and mitochondrial translational termination (false discovery rate (FDR) 6.0E-10 to 1.2E-7). These results indicate a higher energy potential of the YMA MII cohort that is reduced with ageing. Other biological processes that were significantly higher in the YMA MII cohort included transcripts involved in the translation process (FDR 1.9E-2). Lack of these transcripts could lead to inappropriate protein synthesis prior to or upon fertilisation of the AMA MII oocytes., Large Scale Data: The RNA sequencing data were deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo), under the accession number: GSE164371., Limitations, Reasons for Caution: The relatively small sample size could be a reason for caution. However, the RNA sequencing results showed homogeneous clustering with low intra-group variation and five to six biological replicates derived from at least three different women per group minimised the potential impact of the sample size., Wider Implications of the Findings: Understanding the effects of ageing on the oocyte transcriptome could highlight the mechanisms involved in GV to MII transition and identify biomarkers that characterise good MII oocyte quality. This knowledge has the potential to guide IVF regimes for AMA patients., Study Funding/competing Interest(s): This work was supported by the Medical Research Council (MRC Grant number MR/K020501/1)., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Purpose: Wide controversy is still ongoing regarding efficiency of preimplantation genetic testing for aneuploidy (PGT-A). This systematic review and meta-analysis, aims to identify the patient age group that benefits from PGT-A and the best day to biopsy., Methods: A systematic search of the literature was performed on MEDLINE/PubMed, Embase and Cochrane Central Library up to May 2020. Eleven randomized controlled trials employing PGT-A with comprehensive chromosomal screening (CCS) on Day-3 or Day-5 were eligible., Results: PGT-A did not improve live-birth rates (LBR) per patient in the general population (RR:1.11; 95%CI:0.87-1.42; n=1513; I 2 =75%). However, PGT-A lowered miscarriage rate in the general population (RR:0.45; 95%CI:0.25-0.80; n=912; I 2 =49%). Interestingly, the cumulative LBR per patient was improved by PGT-A (RR:1.36; 95%CI:1.13-1.64; n=580; I 2 =12%). When performing an age-subgroup analysis PGT-A improved LBR in women over the age of 35 (RR:1.29; 95%CI:1.05-1.60; n=692; I 2 =0%), whereas it appeared to be ineffective in younger women (RR:0.92; 95%CI:0.62-1.39; n=666; I 2 =75%). Regarding optimal timing, only day-5 biopsy practice presented with improved LBR per ET (RR: 1.37; 95% CI: 1.03-1.82; I 2 =72%)., Conclusion: PGT-A did not improve clinical outcomes for the general population, however PGT-A improved live-birth rates strictly when performed on blastocyst stage embryos of women over the 35-year-old mark., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Study Question: What are the trends and developments in pre-implantation genetic testing (PGT) in 2016-2017 as compared to previous years?, Summary Answer: The main trends observed in this 19th and 20th data set on PGT are that trophectoderm biopsy has become the main biopsy stage for PGT for aneuploidies (PGT-A) and that the implementation of comprehensive testing technologies is the most advanced with PGT-A., What Is Known Already: Since it was established in 1997, the ESHRE PGT Consortium has been collecting and analysing data from mainly European PGT centres. To date, 18 data sets and an overview of the first 10 years of data collections have been published., Study Design Size Duration: The data for PGT analyses performed between 1 January 2016 and 31 December 2017 with a 2-year follow-up after analysis were provided by participating centres on a voluntary basis. Data were collected using a new online platform, which is based on genetic analysis as opposed to the former cycle-based format., Participants/materials Setting Methods: Data on biopsy method, diagnostic technology and clinical outcome were submitted by 61 centres. Records with analyses for more than one PGT for monogenic/single gene defects (PGT-M) and/or PGT for chromosomal structural rearrangements (PGT-SR) indication or with inconsistent data regarding the PGT modality were excluded. All transfers performed within 2 years after the analysis were included enabling the calculation of cumulative pregnancy rates. Data analysis, calculations, figures and tables were made by expert co-authors., Main Results and the Role of Chance: The current data collection from 2016 to 2017 covers a total of 3098 analyses for PGT-M, 1018 analyses for PGT-SR, 4033 analyses for PGT-A and 654 analyses for concurrent PGT-M/SR with PGT-A.The application of blastocyst biopsy is gradually rising for PGT-M (from 8-12% in 2013-2015 to 19% in 2016-2017), is status quo for PGT-R (from 22-36% in 2013-2015 to 30% in 2016-2017) and has become the preferential biopsy stage for PGT-A (from 23-36% in 2013-2015 to 87% in 2016-2017). For concurrent PGT-M/SR with PGT-A, biopsy was primarily performed at the blastocyst stage (93%). The use of comprehensive diagnostic technology showed a similar trend with a small increased use for PGT-M (from 9-12% in 2013-2015 to 15% in 2016-2017) and a status quo for PGT-SR (from 36-58% in 2013-2015 to 50% in 2016-2017). Comprehensive testing was the main technology for PGT-A (from 66-75% in 2013-2015 to 93% in 2016-2017) and for concurrent PGT-M/SR with PGT-A (93%)., Limitations Reasons for Caution: The findings apply to the data submitted by 61 participating centres and do not represent worldwide trends in PGT. Details on the health of babies born were not provided in this manuscript., Wider Implications of the Findings: Being the largest data collection on PGT in Europe/worldwide, the data sets provide a valuable resource for following trends in PGT practice., Study Funding/competing Interests: The study has no external funding and all costs are covered by ESHRE. There are no competing interests declared., Trial Registration Number: N/A., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

There is a great body of evidence suggesting that in both humans and animal models the microRNA-34/449 (miR-34/449) family plays a crucial role for normal testicular functionality as well as for successful spermatogenesis, regulating spermatozoa maturation and functionality. This review and critical analysis aims to summarize the potential mechanisms via which miR-34/449 dysregulation could lead to male infertility. Existing data indicate that miR-34/449 family members regulate ciliogenesis in the efferent ductules epithelium. Upon miR-34/449 dysregulation, ciliogenesis in the efferent ductules is significantly impaired, leading to sperm aggregation and agglutination as well as to defective reabsorption of the seminiferous tubular fluids. These events in turn cause obstruction of the efferent ductules and thus accumulation of the tubular fluids resulting to high hydrostatic pressure into the testis. High hydrostatic pressure progressively leads to testicular dysfunction as well as to spermatogenic failure and finally to male infertility, which could range from severe oligoasthenozoospermia to azoospermia. In addition, miR-34/449 family members act as significant regulators of spermatogenesis with an essential role in controlling expression patterns of several spermatogenesis-related proteins. It is demonstrated that these microRNAs are meiotic specific microRNAs as their expression is relatively higher at the initiation of meiotic divisions during spermatogenesis. Moreover, data indicate that these molecules are essential for proper formation as well as for proper function of spermatozoa per se. MicroRNA-34/449 family seems to exert significant anti-oxidant and anti-apoptotic properties and thus contribute to testicular homeostatic regulation. Considering the clinical significance of these microRNAs, data indicate that the altered expression of the miR-34/449 family members is strongly associated with several aspects of male infertility. Most importantly, miR-34/449 levels in spermatozoa, in testicular tissues as well as in seminal plasma seem to be directly associated with severity of male infertility, indicating that these microRNAs could serve as potential sensitive biomarkers for an accurate individualized differential diagnosis, as well as for the assessment of the severity of male factor infertility. In conclusion, dysregulation of miR-34/449 family detrimentally affects male reproductive potential, impairing both testicular functionality as well as spermatogenesis. Future studies are needed to verify these conclusions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pantos, Grigoriadis, Tomara, Louka, Maziotis, Pantou, Nitsos, Vaxevanoglou, Kokkali, Agarwal, Sfakianoudis and Simopoulou.)

Advancing age has a negative impact on female fertility. As implantation rates decline during the normal maternal life course, age-related, embryonic factors are altered and our inability to monitor these factors in an unbiased genome-wide manner in vivo has severely limited our understanding of early human embryo development and implantation. Our high-throughput methodology uses trophectoderm samples representing the full spectrum of maternal reproductive ages with embryo implantation potential examined in relation to trophectoderm transcriptome dynamics and reproductive maternal age. Potential embryo-endometrial interactions were tested using trophectoderm sampled from young women, with the receptive uterine environment representing the most 'fertile' environment for successful embryo implantation. Potential roles for extracellular exosomes, embryonic metabolism and regulation of apoptosis were revealed. These biomarkers are consistent with embryo-endometrial crosstalk/developmental competency, serving as a mediator for successful implantation. Our data opens the door to developing a diagnostic test for predicting implantation success in women undergoing fertility treatment., Competing Interests: Author P.N. has a pending patent application (application number: GB 2107813.4). This does not alter the authors’ adherence to the journal’s policies on sharing data and materials. All remaining authors declare no conflicting interests., (© 2021 The Authors.)

Study Question: What are the trends and developments in preimplantation genetic testing (PGT) in 2013-2015 as compared to previous years?, Summary Answer: The main trends observed in the retrospective data collections 2013-2015, representing valuable data on PGT activity in (mainly) Europe, are the increased application of trophectoderm biopsy at the cost of cleavage stage biopsy and the continuing expansion of comprehensive testing technology in PGT for chromosomal structural rearrangements and for aneuploidies (PGT-SR and PGT-A)., What Is Known Already: Since it was established in 1997, the ESHRE PGT Consortium has been collecting data from international PGT centres. To date, 15 data sets and an overview of the first 10 years of data collections have been published., Study Design Size Duration: Collection of (mainly) European data by the PGT Consortium for ESHRE. The data for PGT cycles performed between 1 January 2013 and 31 December 2015 were provided by participating centres on a voluntary basis. For the collection of cycle, pregnancy and baby data, separate, pre-designed MS Excel tables were used., Participants/materials Setting Methods: Data were submitted by 59, 60 and 59 centres respectively for 2013, 2014 and 2015 (full PGT Consortium members). Records with incomplete or inconsistent data were excluded from the calculations. Corrections, calculations, figures and tables were made by expert co-authors., Main Results and the Role of Chance: For data collection XVI/XVII/XVIII, 59/60/59 centres reported data on 8164/9769/11 120 cycles with oocyte retrieval: 5020/6278/7155 cycles for PGT-A, 2026/2243/2661 cycles for PGT for monogenic/single gene defects, 1039/1189/1231 cycles for PGT-SR and 79/59/73 cycles for sexing for X-linked diseases. From 2013 until 2015, the uptake of biopsy at the blastocyst stage was mainly observed in cycles for PGT-A (from 23% to 36%) and PGT-SR (from 22% to 36%), alongside the increased application of comprehensive testing technology (from 66% to 75% in PGT-A and from 36% to 58% in PGT-SR)., Limitations Reasons for Caution: The findings apply to the 59/60/59 participating centres and may not represent worldwide trends in PGT. Data were collected retrospectively and no details of the follow-up on PGT pregnancies and babies born were provided., Wider Implications of the Findings: Being the largest data collection on PGT worldwide, detailed information about ongoing developments in the field is provided., Study Funding/competing Interests: The study has no external funding and all costs are covered by ESHRE. There are no competing interests declared., Trial Registration Number: N/A., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Intraovarian platelet-rich plasma (PRP) infusion was recently introduced in the context of addressing ovarian insufficiency. Reporting on its effectiveness prior to adopting in clinical routine practice is imperative. This study aims to provide pilot data regarding PRP application for ovarian rejuvenation. Four pilot studies were conducted on poor ovarian response (POR), premature ovarian insufficiency (POI), perimenopause, and menopause, respectively. Each pilot study reports on thirty patients, 120 participants were recruited in total. All participants provided written informed consent prior to treatment. Primary outcome measures for the POR pilot study were levels of anti-müllerian hormone (AMH), antral follicle count (AFC) and oocyte yield. For the POI, perimenopausal and menopausal pilot studies primary outcome measures were restoration of menstrual cycle, and Follicle Stimulating Hormone (FSH) levels. A significant improvement on the hormonal profile and the ovarian reserve status was noted, along with improved intracytoplasmic sperm injection (ICSI) cycle performance concerning POR participants. Menstruation recovery was observed in 18 out of 30 POI patients, along with a statistically significant improvement on levels of AMH, FSH, and AFC. Similarly, 13 out of 30 menopausal women positively responded to PRP treatment. Finally, menstruation regularity, improved hormonal levels and AFC were reported for 24 out of 30 perimenopausal women. To conclude, PRP infusion appears to convey promising results in addressing ovarian insufficiency.

The field of preimplantation genetic testing (PGT) is evolving fast, and best practice advice is essential for regulation and standardisation of diagnostic testing. The previous ESHRE guidelines on best practice for preimplantation genetic diagnosis, published in 2005 and 2011, are considered outdated and the development of new papers outlining recommendations for good practice in PGT was necessary. The current updated version of the recommendations for good practice is, similar to the 2011 version, split into four documents, one of which covers the organisation of a PGT centre. The other documents focus on the different technical aspects of embryo biopsy, PGT for monogenic/single-gene defects (PGT-M) and PGT for chromosomal structural rearrangements/aneuploidies (PGT-SR/PGT-A). The current document outlines the steps prior to starting a PGT cycle, with details on patient inclusion and exclusion, and counselling and information provision. Also, recommendations are provided on the follow-up of PGT pregnancies and babies. Finally, some further recommendations are made on the practical organisation of an IVF/PGT centre, including basic requirements, transport PGT and quality management. This document, together with the documents on embryo biopsy, PGT-M and PGT-SR/PGT-A, should assist everyone interested in PGT in developing the best laboratory and clinical practice possible., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

The aim of this study is to assess the value of laparoscopy for couples diagnosed with mild male factor infertility and at least three previous failed In-Vitro Fertilization (IVF) attempts. A total of 169 couples were included in this prospective cohort study. Patients were presented with the option of being subjected to laparoscopic investigation for correction of previously unidentified endometriosis or pelvic adhesions. The outcome measures were Live Birth/Ongoing Pregnancy, clinical pregnancy and positive hCG rate. One-hundred and one of them opted for, whereas 68 opted against laparoscopic investigation. All patients proceeded with a single ICSI cycle. Following laparoscopic investigation, 43 patients were diagnosed with endometriosis, 22 with adhesions, while for 36 patients laparoscopic investigation provided no further diagnosis. No statistically significant differences were observed regarding baseline hormonal levels and other characteristics between the two groups and the three subgroups. When compared to the no-laparoscopy group, women subjected to laparoscopy presented with a higher clinical pregnancy and ongoing pregnancy/live birth rate. Following endometriosis correction, a marginally non-statistically significant trend was observed regarding a decrease in poor-quality blastocysts (p = 0.056). A statistically significant higher clinical pregnancy (p = 0.03) and ongoing pregnancy/live birth rate was observed in the endometriosis group when compared to male factor infertility only (p = 0.04). Laparoscopic identification and correction of undiagnosed endometriosis in couples initially diagnosed with male infertility and at least 3 failed previous IVF attempts, appears to be a promising approach efficiently addressing infertility for these patients while avoiding IVF overuse.

A systematic review of the literature showed that trophectoderm biopsy could assist in the selection of healthy embryos for uterine transfer without affecting implantation rates. However, previous studies attempting to establish the relationship between trophectoderm gene expression profiles and implantation competency using either microarrays or RNA sequencing strategies, were not sufficiently optimized to handle the exceptionally low RNA inputs available from biopsied material. In this pilot study, we report that differential gene expression in human trophectoderm biopsies assayed by an ultra-sensitive next generation RNA sequencing strategy could predict blastocyst implantation competence. RNA expression profiles from isolated human trophectoderm cells were analysed with established clinical pregnancy being the primary endpoint. Following RNA sequencing, a total of 47 transcripts were found to be significantly differentially expressed between the trophectoderm cells from successfully implanted (competent) versus unsuccessful (incompetent) blastocysts. Of these, 36 transcripts were significantly down-regulated in the incompetent blastocysts, including Hydroxysteroid 17-Beta Dehydrogenase 1 ( HSD17B1 ) and Cytochrome P450 Family 11 Subfamily A Member 1 ( CYP11A1 ), while the remaining 11 transcripts were significantly up-regulated, including BCL2 Antagonist/Killer 1 ( BAK1 ) and KH Domain Containing 1 Pseudogene 1 ( KHDC1P1 ) of which the latter was always detected in the incompetent and absent in all competent blastocysts. Ontological analysis of differentially expressed RNAs revealed pathways involved in steroidogenic processes with high confidence. Novel differentially expressed transcripts were also noted by reference to a de novo sequence assembly. The selection of the blastocyst with the best potential to support full-term pregnancy following single embryo transfer could reduce the need for multiple treatment cycles and embryo transfers. The main limitation was the low sample size (N = 8). Despite this shortcoming, the pilot suggests that trophectoderm biopsy could assist with the selection of healthy embryos for embryo transfer. A larger cohort of samples is needed to confirm these findings. Abbreviations: AMA: advanced maternal age; ART: assisted reproductive technology; CP: clinical pregnancy; DE: differential expression; FDR: false discovery rate; IVF: in vitro fertilization; LD PCR: long distance PCR; qRT-PCR: quantitative real-time PCR; SET: single embryo transfer; TE: trophectoderm.

Poor responders are described as those In Vitro Fertilization (IVF) patients who are failing to respond to controlled ovarian stimulation protocols. Extensive research has focused on crafting the optimal treatment. However, it appears that each approach fails to be established as effective or guaranteed towards successful management. Platelet-Rich Plasma (PRP) is a novel, highly promising approach that has been successfully applied for an array of medical issues. In this case series, we present 3 poor responder patients with the common denominator of: failed IVF attempts, poor oocyte yield, and poor embryo quality. The option of oocyte donation was rejected. All patients were treated with autologous PRP ovarian infusion following written consent. Within a 3-month interval, follicle-stimulating hormone decreased by 67.33%, while Anti-Müllerian hormone increased by 75.18%. These impressive results on the biochemical infertility markers alone are classified as a complete biological paradox, coupled by improved embryo quality. Results report a natural conception at 24 weeks, an uncomplicated healthy pregnancy at 17 weeks and a successful live birth. To our knowledge, this is the first time such an approach and results are reported, where PRP treatment on poor responders lead to overcoming their challenging reproductive barrier., (© 2018 S. Karger AG, Basel.)

Study Question: Does preimplantation genetic testing for aneuploidy (PGT-A) by comprehensive chromosome screening (CCS) of the first and second polar body to select embryos for transfer increase the likelihood of a live birth within 1 year in advanced maternal age women aged 36-40 years planning an ICSI cycle, compared to ICSI without chromosome analysis?, Summary Answer: PGT-A by CCS in the first and second polar body to select euploid embryos for transfer does not substantially increase the live birth rate in women aged 36-40 years., What Is Known Already: PGT-A has been used widely to select embryos for transfer in ICSI treatment, with the aim of improving treatment effectiveness. Whether PGT-A improves ICSI outcomes and is beneficial to the patients has remained controversial., Study Design, Size, Duration: This is a multinational, multicentre, pragmatic, randomized clinical trial with intention-to-treat analysis. Of 396 women enroled between June 2012 and December 2016, 205 were allocated to CCS of the first and second polar body (study group) as part of their ICSI treatment cycle and 191 were allocated to ICSI treatment without chromosome screening (control group). Block randomization was performed stratified for centre and age group. Participants and clinicians were blinded at the time of enrolment until the day after intervention., Participants/materials, Setting, Methods: Infertile couples in which the female partner was 36-40 years old and who were scheduled to undergo ICSI treatment were eligible. In those assigned to PGT-A, array comparative genomic hybridization (aCGH) analysis of the first and second polar bodies of the fertilized oocytes was performed using the 24sure array of Illumina. If in the first treatment cycle all oocytes were aneuploid, a second treatment with PB array CGH was offered. Participants in the control arm were planned for ICSI without PGT-A. Main exclusion criteria were three or more previous unsuccessful IVF or ICSI cycles, three or more clinical miscarriages, poor response or low ovarian reserve. The primary outcome was the cumulative live birth rate after fresh or frozen embryo transfer recorded over 1 year after the start of the intervention., Main Results and the Role of Chance: Of the 205 participants in the chromosome screening group, 50 (24%) had a live birth with intervention within 1 year, compared to 45 of the 191 in the group without intervention (24%), a difference of 0.83% (95% CI: -7.60 to 9.18%). There were significantly fewer participants in the chromosome screening group with a transfer (relative risk (RR) = 0.81; 95% CI: 0.74-0.89) and fewer with a miscarriage (RR = 0.48; 95% CI: 0.26-0.90)., Limitations, Reasons for Caution: The targeted sample size was not reached because of suboptimal recruitment; however, the included sample allowed a 90% power to detect the targeted increase. Cumulative outcome data were limited to 1 year. Only 11 patients out of 32 with exclusively aneuploid results underwent a second treatment cycle in the chromosome screening group., Wider Implications of the Findings: The observation that the similarity in birth rates was achieved with fewer transfers, less cryopreservation and fewer miscarriages points to a clinical benefit of PGT-A, and this form of embryo selection may, therefore, be considered to minimize the number of interventions while producing comparable outcomes. Whether these benefits outweigh drawbacks such as the cost for the patient, the higher workload for the IVF lab and the potential effect on the children born after prolonged culture and/or cryopreservation remains to be shown., Study Funding/competing Interest(s): This study was funded by the European Society of Human Reproduction and Embryology. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. M.B.'s institution (UZBrussel) has received educational grants from IBSA, Ferring, Organon, Schering-Plough, Merck and Merck Belgium. M.B. has received consultancy and speakers' fees from Organon, Serono Symposia and Merck. G.G. has received personal fees and non-financial support from MSD, Ferring, Merck-Serono, Finox, TEVA, IBSA, Glycotope, Abbott and Gedeon-Richter as well as personal fees from VitroLife, NMC Healthcare, ReprodWissen, BioSilu and ZIVA. W.V., C.S., P.M.B., V.G., G.A., M.D., T.E.G., L.G., G.Ka., G.Ko., J.L., M.C.M., M.P., A.S., M.T., K.V., J.G. and K.S. declare no conflict of interest., Trial Registration Number: NCT01532284., Trial Registration Date: 7 February 2012., Date of First Patient’s Enrolment: 25 June 2012.

Study Question: Has PGD-HLA been successful relative to diagnostic and clinical efficacy?, Summary Answer: The diagnostic efficacy of PGD-HLA protocols was found lower in this study in comparison to published PGD-HLA protocols and to that reported for general PGD by ESHRE (78.5 vs 94.1% and vs 92.6%, respectively), while the clinical efficacy has proven very difficult to assess due to inadequate follow-up of both the ART/PGD and HSCT procedure outcomes., What Is Known Already: The first clinical cases for PGD-HLA were reported in 2001. It is now a well-established procedure, with an increasing number of cycles performed every year. However, PGD-HLA is still offered by relatively few PGD centres, the currently available data is fragmented and most reports on PGD-HLA applications are limited in number and scope. Published systematic details on methodology, diagnostic results, overall ART success and haematopoietic stem cell transplantation (HSCT) outcomes are limited, precluding an evaluation of the true clinical utility of PGD-HLA cycles., Study Design, Size, Duration: This retrospective multi-centre cohort study aimed to investigate the diagnostic and clinical efficacy of the PGD-HLA procedure and the aspects of PGD-HLA cycles influencing positive outcomes: birth of genetically suitable donor-baby (or babies) and HSCT. In April 2014, 32 PGD centres (Consortium members and non-members) with published/known PGD-HLA activity were invited to participate. Between February and September 2015, 14 centres submitted their data, through a custom-designed secure database, with unique login access for each centre. Data parameters covered all aspects of PGD-HLA cycles (ART, embryology and genetic diagnosis), donor-babies born and HSCT., Participants/materials, Setting, Methods: From 716 cycles submitted by 14 centres (performed between August 2001 and September 2015), the quality evaluation excluded 12 cycles, leaving 704, from 364 couples. The online database, based on REDCap, a free, secure, web-based data-capture application, was customized by Centre for Clinical Epidemiology and Outcomes Research (CLEO), Athens. Continuous variables are presented using mean, standard deviation, median and interquartile range, and categorical variables are presented as absolute and relative frequencies., Main Results and the Role of Chance: The data included 704 HLA-PGD cycles. Mean maternal age was 33.5 years. Most couples (81.3%) requested HLA-typing with concurrent exclusion of a single monogenic disease (58.6% for beta-thalassaemia). In 92.5% couples, both partners were fertile, with an average 1.93 HLA-PGD cycles/couple. Overall, 9751 oocytes were retrieved (13.9/cycle) and 5532 embryos were analysed (7.9/cycle). Most cycles involved fresh oocytes (94.9%) and Day 3 embryo biopsy (85.3%). In 97.5% of cycles, the genotyping method involved PCR only. Of 4343 embryos diagnosed (78.5% of analysed embryos), 677 were genetically suitable (15.4% of those analysed for HLA alone, 11.6% of those analysed for HLA with exclusion of monogenic disease). Of the 364 couples, 56.6% achieved an embryo transfer (ET) and 598 embryos were transferred in 382 cycles, leading to 164 HCG-positive pregnancies (pregnancy rate/ET 41.3%, pregnancy rate/initiated cycle 23.3%) and 136 babies born (live birth rate/ET 34.3%, live birth rate/initiated cycle 19.3%) to 113 couples. Data analysis identified the following limitations to the overall success of the HLA-PGD procedure: the age of the mother undergoing the treatment cycle, the number of oocytes collected per cycle and genetic chance. HSCT was reported for 57 cases, of which 64.9% involved combined umbilical cord-blood and bone marrow transplantation from the HLA-identical sibling donor; 77.3% of transplants reported no complications., Limitations Reasons for Caution: The findings of the study may be limited as not all PGD centres with PGD-HLA experience participated. Reporting bias on completion of the online database may be another potential limitation. Furthermore, the study is based on retrospective data collection from centres with variable practices and strategies for ART, embryology and genetic diagnosis., Wider Implications of the Findings: This is the first multi-centre study evaluating the clinical utility of PGD-HLA, indicating variations in practice and outcomes throughout 15 years and between centres. The study highlights parameters important for positive outcomes and provides important information for both scientists and couples interested in initiating a cycle. Above all, the study underlines the need for better collaboration between all specialists involved in the ART-PGD/HLA procedure, as well as the need for comprehensive and prospective long-term data collection, and encourages all specialists to aim to properly evaluate and follow-up all procedures, with the ultimate aim to promote best practice and encourage patient informed decision making., Study Funding/competing Interest(s): The study wishes to acknowledge ESHRE for funding the customization of the REDCap database. There are no competing interests., Trial Registration Number: N/A.

Study Question: How does the data collection XIV-XV of the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium compare with the cumulative data for data collections I-XIII?, Summary Answer: The 14th and 15th retrospective collection represents valuable data on PGD/PGS cycles, pregnancies and children: the main trend observed is the increased application of array technology at the cost of FISH testing in PGS cycles and in PGD cycles for chromosomal abnormalities., What Is Known Already: Since 1999, the PGD Consortium has collected, analysed and published 13 previous data sets and an overview of the first 10 years of data collections., Study Design, Size, Duration: Data were collected from each participating centre using a FileMaker Pro database (versions 5-12). Separate predesigned FileMaker Pro files were used for the cycles, pregnancies and baby records. The study documented cycles performed during the calendar years 2011 and 2012 and follow-up of the pregnancies and babies born which resulted from these cycles (until October 2013)., Participants/materials, Settings, Method: Data were submitted by 71 centres (full PGD Consortium members). Records with incomplete or inconsistent data were excluded from the calculations. Corrections, calculations and tables were made by expert co-authors., Main Results and the Role of Chance: For data collection XIV-XV, 71 centres reported data for 11 637 cycles with oocyte retrieval (OR), along with details of the follow-up on 2147 pregnancies and 1755 babies born. A total of 1953 cycles to OR were reported for chromosomal abnormalities, 144 cycles to OR for sexing for X-linked diseases, 3445 cycles to OR for monogenic diseases, 6095 cycles to OR for PGS and 38 cycles to OR for social sexing. From 2010 until 2012, the use of arrays for genetic testing increased from 4% to 20% in PGS and from 6% to 13% in PGD cycles for chromosomal abnormalities; the uptake of biopsy at the blastocyst stage (from <1% up to 7%) was only observed in cycles for structural chromosomal abnormalities, alongside the application of array comparative genomic hybridization., Limitations, Reasons for Caution: The findings apply to the 71 participating centres and may not represent worldwide trends in PGD., Wider Implications of the Findings: The annual data collections provide an important resource for data mining and for following trends in PGD/PGS practice., Study Funding/competing Interest(s): None., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Study Question: We wanted to probe the opinions and current practices on preimplantation genetic screening (PGS), and more specifically on PGS in its newest form: PGS 2.0?, Study Finding: Consensus is lacking on which patient groups, if any at all, can benefit from PGS 2.0 and, a fortiori, whether all IVF patients should be offered PGS., What Is Known Already: It is clear from all experts that PGS 2.0 can be defined as biopsy at the blastocyst stage followed by comprehensive chromosome screening and possibly combined with vitrification. Most agree that mosaicism is less of an issue at the blastocyst stage than at the cleavage stage but whether mosaicism is no issue at all at the blastocyst stage is currently called into question., Study Design, Samples/materials, Methods: A questionnaire was developed on the three major aspects of PGS 2.0: the Why, with general questions such as PGS 2.0 indications; the How, specifically on genetic analysis methods; the When, on the ideal method and timing of embryo biopsy. Thirty-five colleagues have been selected to address these questions on the basis of their experience with PGS, and demonstrated by peer-reviewed publications, presentations at meetings and participation in the discussion. The first group of experts who were asked about 'The Why' comprised fertility experts, the second group of molecular biologists were asked about 'The How' and the third group of embryologists were asked about 'The When'. Furthermore, the geographical distribution of the experts has been taken into account. Thirty have filled in the questionnaire as well as actively participated in the redaction of the current paper., Main Results and the Role of Chance: The 30 participants were from Europe (Belgium, Germany, Greece, Italy, Netherlands, Spain, UK) and the USA. Array comparative genome hybridization is the most widely used method amongst the participants, but it is slowly being replaced by massive parallel sequencing. Most participants offering PGS 2.0 to their patients prefer blastocyst biopsy. The high efficiency of vitrification of blastocysts has added a layer of complexity to the discussion, and it is not clear whether PGS in combination with vitrification, PGS alone, or vitrification alone, followed by serial thawing and eSET will be the favoured approach. The opinions range from in favour of the introduction of PGS 2.0 for all IVF patients, over the proposal to use PGS as a tool to rank embryos according to their implantation potential, to scepticism towards PGS pending a positive outcome of robust, reliable and large-scale RCTs in distinct patient groups., Limitations, Reasons for Caution: Care was taken to obtain a wide spectrum of views from carefully chosen experts. However, not all invited experts agreed to participate, which explains a lack of geographical coverage in some areas, for example China. This paper is a collation of current practices and opinions, and it was outside the scope of this study to bring a scientific, once-and-for-all solution to the ongoing debate., Wider Implications of the Findings: This paper is unique in that it brings together opinions on PGS 2.0 from all different perspectives and gives an overview of currently applied technologies as well as potential future developments. It will be a useful reference for fertility specialists with an expertise outside reproductive genetics., Large Scale Data: none., Study Funding and Competing Interests: No specific funding was obtained to conduct this questionnaire., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Cryopreservation, the use of very low temperatures to preserve structurally intact living cells and tissues, has seen exponential growth in the field of in vitro fertilization (IVF). In the last decade, cryopreservation of embryos and freeze-all protocols have become an essential aspect and a prerequisite for a successful IVF outcome. Moreover, vitrification, which is a fast and safe cryopreservation method, has proved to be an effective choice for cryopreserving gametes and embryos. The increasing number of cryopreserved embryos worldwide in cryobanks and IVF clinics is an undisputable fact that raises important physiological, ethical, and moral considerations that merit careful examination and discussion. Many couples utilizing assisted reproduction will have a surplus of cryopreserved embryos, in other words they already have completed their family without exhausting all the embryos that were created and cryopreserved during the process. Additionally, the global IVF market has also experienced significant growth due to various factors, including advancements in technology, increased awareness about infertility treatments, and changing societal norms towards delayed parenthood. Thus, for the foreseeable future the number of cryopreserved embryos, and the phenomenon of surplus embryos will likely remain unresolved. In the present review, following a description of the cryopreservation method and the physiological changes during the cryopreservation of embryos, the bioethical issues raised by the surplus cryopreserved embryos will be discussed alongside possible solutions for resolving this phenomenon.

Preimplantation genetic diagnosis (PGD) to select histocompatible siblings to facilitate curative haematopoeitic stem-cell transplantation (HSCT) is now an acceptable option in the absence of an available human leukocyte antigen (HLA) compatible donor. We describe a case where the couple who requested HLA-PGD, were both carriers of two serious haematological diseases, beta-thalassaemia and sideroblastic anaemia. Their daughter, affected with sideroblastic anaemia, was programmed to have HSCT. A multiplex-fluorescent-touchdown-PCR protocol was optimized for the simultaneous amplification of: the two HBB-gene mutated regions (c.118C> T, c.25-26delAA), four short tandem repeats (STRs) in chr11p15.5 linked to the HBB gene, the SLC25A38 gene mutation (c.726C > T), two STRs in chr3p22.1 linked to the SLC25A38 gene, plus eleven informative STRs for HLA-haplotyping (chr6p22.1-21.3). This was followed by real-time nested PCR and high-resolution melting analysis (HRMA) for the detection of HBB and SLC25A38 gene mutations, as well as the analysis of all STRs on an automatic genetic analyzer (sequencer). The couple completed four clinical in vitro fertilization (IVF)/PGD cycles. At least one matched unaffected embryo was identified and transferred in each cycle. A twin pregnancy was established in the fourth PGD cycle and genotyping results at all loci were confirmed by prenatal diagnosis. Two healthy baby girls were delivered at week 38 of pregnancy. The need to exclude two familial disorders for HLA-PGD is rarely encountered. The methodological approach described here is fast, accurate, clinically-validated, and of relatively low cost.

Background: Fluorescence in situ hybridization (FISH) is the most widely used method for detecting unbalanced chromosome rearrangements in preimplantation embryos but it is known to have several technical limitations. We describe the clinical application of a molecular-based assay, array comparative genomic hybridization (array-CGH), to simultaneously screen for unbalanced translocation derivatives and aneuploidy of all 24 chromosomes., Methods: Cell biopsy was carried out on cleavage-stage embryos (Day 3). Single cells were first lysed and DNA amplified by whole-genome amplification (WGA). WGA products were then processed by array-CGH using 24sure + arrays, BlueGnome. Balanced/normal euploid embryos were then selected for transfer on Day 5 of the same cycle., Results: Twenty-eight consecutive cycles of preimplantation genetic diagnosis were carried out for 24 couples carrying 18 different balanced translocations. Overall, 187/200 (93.5%) embryos were successfully diagnosed. Embryos suitable for transfer were identified in 17 cycles (60.7%), with transfer of 22 embryos (mean 1.3 ± 0.5). Twelve couples achieved a clinical pregnancy (70.6% per embryo transfer), with a total of 14 embryos implanted (63.6% per transferred embryo). Three patients delivered three healthy babies, during writing, the other pregnancies (two twins and seven singletons) are ongoing beyond 20 weeks of gestation., Conclusions: The data obtained demonstrate that array-CGH can detect chromosome imbalances in embryos, also providing the added benefit of simultaneous aneuploidy screening of all 24 chromosomes. Array-CGH has the potential to overcome several inherent limitations of FISH-based tests, providing improvements in terms of test performance, automation, sensitivity and reliability.

Preimplantation genetic diagnosis (PGD) for β hemoglobinopathies has become the most common application among monogenic disorders. We present the identification of microsatellite markers [short tandem repeats (STRs)] closely linked to the β-globin gene for incorporation within PGD protocols, with the aim of increasing the number of transferable embryos. Nine candidate STRs were identified in-silico, of which three were selected based on rate-of-heterozygosity, polymerase chain reaction (PCR) efficiency and size. The multiplex reaction (β-globin gene and selected STRs, all within <0.4 Mb from the β gene) was optimized in single lymphocytes, and subsequently applied in 38 PGD cycles in couples at-risk for transmitting β hemoglobinopathies. In conclusion, incorporation of closely linked polymorphic microsatellite markers <0.4 Mb from the β-globin gene, facilitates robust assignment of β hemoglobinopathy genotypes, increasing the number of transferrable embryos otherwise rejected due to allele-drop-out (ADO), at the mutation-specific locus, compared to results based on disease-mutation genotyping alone (p < 0.001).

Objective: To develop and assess a polymerase chain reaction (PCR)-based preimplantation genetic diagnosis (PGD) approach for detection of chromosomal imbalances in embryos., Design: A prospective study of embryos derived from chromosome translocation carriers that have undergone PGD using a novel molecular-based approach., Setting: A reference molecular genetics laboratory specialized in the provision of transport PGD services and a private IVF clinic., Patient(s): Twenty-seven couples carrying 12 different reciprocal translocations and 2 Robertsonian translocations., Intervention(s): Preimplantation genetic diagnosis from chromosome translocation carriers on blastomeres biopsied from cleavage stage embryos., Main Outcome Measure(s): Embryo diagnosis rate, pregnancy rate (PR), implantation rate, take-home-baby rate., Result(s): Overall, 241/251 (96.0%) embryos were successfully diagnosed for chromosome rearrangements. Preimplantation genetic screening was included in the protocol of 12 couples, involving analysis of 90 embryos, 84 (93.3%) of which were successfully diagnosed and 53 (63.1%) showed aneuploidies. Embryos suitable for transfer were identified in 24 cycles. Eighteen couples achieved a clinical pregnancy (75.0% PR/embryo transfer), with a total of 31 embryos implanted (59.6% implantation rate). Ten patients (1 triplet, 1 twin, and 8 singleton pregnancies) have delivered 13 healthy babies, and the other patients (3 twins and 5 singletons) have currently ongoing pregnancies., Conclusion(s): The PCR-based PGD protocol for translocations has the potential to overcome several inherent limitations of fluorescence in situ hybridization-based tests, providing potential improvements in terms of test performance, automation, turnaround time, sensitivity, and reliability., (Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

We report 2 children with X-linked chronic granulomatous disease (X-CGD) who underwent hematopoietic stem cell transplantation (HSCT) using grafts from their siblings selected before implantation to be both unaffected and HLA-matched donors. Preimplantation genetic diagnosis (PGD) along with HLA-typing were performed on preimplantation embryos by single-cell multiplex polymerase chain reaction using informative short tandem repeat markers in the HLA locus together with the gene region containing the mutations. Two singleton pregnancies resulted from the intrauterine transfer of selected embryos; these developed to term, producing 1 healthy female and 1 X-CGD carrier female, which are HLA-identical siblings to the 2 affected children. Combined grafts of umbilical cord blood (UCB) and bone marrow (BM) stem cells were administered to the recipients after myeloablative (MA) conditioning at the ages of 4.5 years and 4 years, respectively. Both patients are well, with complete donor hematopoietic and immunologic reconstitution, at 18 and 13 months posttransplantation, respectively. This report demonstrates that HSCT with HLA-matched sibling donors created by PGD/HLA typing of in vitro fertilized embryos is a realistic therapeutic option and should be presented as such to families with children who require a non-urgent HSCT but lack an HLA-genoidentical donor., (Copyright (c) 2010 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

X-linked genetic diseases include a wide range of disorders such as the dystrophinopathies. Additionally in some rare genetic diseases, severity of expression is gender dependent. Prevention of such disorders usually involves prenatal diagnosis and termination of affected pregnancies, while preimplantation genetic diagnosis (PGD) represents a specialized alternative that avoids pregnancy termination. To preclude the rejection of unaffected male embryos that cannot be differentiated from those affected when using fluorescence in-situ hybridization, a flexible protocol based on multiplex fluorescence polymerase chain reaction (PCR) was standardized and validated for gender determination in single cells, which can potentially incorporate any disease-specific locus. The final panel of nine loci included four loci on the Y chromosome, two on the X chromosome plus up to three microsatellite markers to either support the gender diagnosis or to further monitor extraneous contamination. The protocol, standardized on single lymphocytes, established a PCR efficiency of >93% for all loci with maximum allele dropout rates of 4%. Microsatellite analysis excluded external contamination and confirmed biallelic inheritance. Proof of principle for the simplicity and flexibility of the assay was demonstrated through its application to clinical PGD cycles for lipoid congenital adrenal hyperplasia, which presents a more severe clinical course in males, and Duchenne muscular dystrophy.

Objective: To report the pregnancy outcome of a patient with congenital lipoid adrenal hyperplasia (CLAH) due to an 11-bp deletion of the steroidogenic acute regulatory protein (StAR) gene., Design: Case report., Setting: University-based pediatric endocrinology unit and private IVF clinic., Patient(s): A 24-year-old woman homozygous for a StAR gene deletion, married to a man heterozygous for the same molecular defect., Intervention(s): Ovarian stimulation, oocyte retrieval followed by IVF, blastomere biopsy, preimplantation genetic diagnosis, and additional estrogen support until placental function initiation., Main Outcome Measure(s): Normal pregnancy outcome and delivery of a healthy newborn., Result(s): A female patient with CLAH gave birth to a normal newborn after IVF and preimplantation genetic diagnosis., Conclusion(s): Pregnancy is feasible in patients with StAR gene mutations, provided that extra estrogens are offered until placental function ensues.

Objective: To report on a patient with a quintuplet pregnancy consisting of a monochorionic triamnionic triplet pregnancy and one case of monoamniotic twins after intracytoplasmic sperm injection (ICSI), laser-assisted hatching, and day 4 embryo transfer., Case Report: A 34-year-old woman who underwent ICSI due to male infertility. She underwent standard controlled ovarian hyperstimulation. Retrieved oocytes were fertilized in vitro, and 3 embryos were transferred on day 4 after laser-assisted hatching. Transvaginal ultrasound revealed a monoamniotic twin pregnancy and a monochorionic-triamniotic triplet pregnancy. A second ultrasound, 1 week later, showed three distinct foci of cardiac motion in one sac, while the sac of the monoamniotic twins had one embryo with heart beat. After extensive counseling with perinatologists about pregnancy complications, the patient elected to performed fetal reduction on the third sac. A healthy male infant was delivered by Caesarean section at 38 weeks of gestation., Conclusions: The exact mechanism of monozygotic multiple gestation is still poorly characterized. Procedures that modify the zona pellucida (e.g. ICSI and assisted embryo hatching) have been suggested as important in the monozygotic multiple gestation hypothesis, yet a definitive relationship between any clinical intervention during in vitro fertilization and the subsequent development of multiple monozygotic gestation remains speculative., ((c) 2009 S. Karger AG, Basel.)

Background: Currently there are no markers fully predictive of developmental competence of human IVF embryos. The present study investigated a novel strategy involving blastocyst biopsy and DNA fingerprinting to link developmental competence with gene expression patterns., Methods: Patient's blastocysts were biopsied to remove 8-20 trophectoderm (TE) cells for molecular analysis prior to transfer. Biopsy samples were amplified and gene expression was evaluated using microarrays. Sibling TE biopsies and cells from resulting offspring were subjected to DNA fingerprinting to identify which blastocyst(s) in the transfer cohort developed to term., Results: Blastocyst biopsy did not appear to impair developmental competence. Comparative microarray analysis of cDNA from pooled 'viable' and 'non-viable' TE samples identified over 7000 transcripts expressed exclusively in 'viable' blastocysts. The most significant of these included transcripts involved in cell adhesion and cell communication, key processes that have been associated with mammalian implantation. DNA fingerprinting of three cohorts of sibling blastocysts identified those blastocyst(s) that produced term pregnancies., Conclusions: The combination of blastocyst biopsy, microarray gene expression profiling and DNA fingerprinting is a powerful tool to identify diagnostic markers of competence to develop to term. This strategy may be used to develop a rapid diagnostic assay or for refining existing criteria for the selection of the single most viable blastocyst among a cohort developing in vitro.