The impact of semen parameters on ICSI and pregnancy outcomes in egg recipient cycles with PGT-A.

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 × 106 /mL), accounting for 442 patients (27.8%); couples with mild-to-moderate oligozoospermia, with sperm number >5 × 106 and <15 × 106 /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.

Trophectoderm non-coding RNAs reflect the higher metabolic and more invasive properties of young maternal age blastocysts.

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.

The impact of preimplantation genetic testing for aneuploidies (PGT-A) on clinical outcomes in high risk patients.

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.

The Role of Interleukins in Recurrent Implantation Failure: A Comprehensive Review of the Literature.

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.

Investigating the Role of the microRNA-34/449 Family in Male Infertility: A Critical Analysis and Review of the Literature.

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.

The effects of aging on molecular modulators of human embryo implantation.

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.

PGT-A: who and when? Α systematic review and network meta-analysis of RCTs.

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; I2=75%). However, PGT-A lowered miscarriage rate in the general population (RR:0.45; 95%CI:0.25-0.80; n=912; I2=49%). Interestingly, the cumulative LBR per patient was improved by PGT-A (RR:1.36; 95%CI:1.13-1.64; n=580; I2=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; I2=0%), whereas it appeared to be ineffective in younger women (RR:0.92; 95%CI:0.62-1.39; n=666; I2=75%). Regarding optimal timing, only day-5 biopsy practice presented with improved LBR per ET (RR: 1.37; 95% CI: 1.03-1.82; I2=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.

ESHRE PGT Consortium good practice recommendations for the organisation of PGT.

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.

Reactivating Ovarian Function through Autologous Platelet-Rich Plasma Intraovarian Infusion: Pilot Data on Premature Ovarian Insufficiency, Perimenopausal, Menopausal, and Poor Responder Women.

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.

Couples with mild male factor infertility and at least 3 failed previous IVF attempts may benefit from laparoscopic investigation regarding assisted reproduction outcome.

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.

Can trophectoderm RNA analysis predict human blastocyst competency?

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.

A Case Series on Platelet-Rich Plasma Revolutionary Management of Poor Responder Patients.

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.

Preimplantation genetic testing for aneuploidy by microarray analysis of polar bodies in advanced maternal age: a randomized clinical trial.

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.

The why, the how and the when of PGS 2.0: current practices and expert opinions of fertility specialists, molecular biologists, and embryologists.

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.

Complex preimplantation genetic diagnosis for beta-thalassaemia, sideroblastic anaemia, and human leukocyte antigen (HLA)-typing.

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.

Polymerase chain reaction-based detection of chromosomal imbalances on embryos: the evolution of preimplantation genetic diagnosis for chromosomal translocations.

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.

Successful hematopoietic stem cell transplantation in 2 children with X-linked chronic granulomatous disease from their unaffected HLA-identical siblings selected using preimplantation genetic diagnosis combined with HLA typing.

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.

Monochorionic triplet and monoamniotic twins gestation after intracytoplasmic sperm injection and laser-assisted hatching.

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.

Conception and pregnancy outcome in a patient with 11-bp deletion of the steroidogenic acute regulatory protein gene.

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.