Mapping the expression of transient receptor potential channels across murine placental development.

Funder: Onderzoeksraad, KU Leuven; doi: http://dx.doi.org/10.13039/501100004497Funder: Centre of Trophoblast Research, University of CambridgeTransient receptor potential (TRP) channels play prominent roles in ion homeostasis by their ability to control cation influx. Mouse placentation is governed by the processes of trophoblast proliferation, invasion, differentiation, and fusion, all of which require calcium signaling. Although certain TRP channels have been shown to contribute to maternal-fetal transport of magnesium and calcium, a role for TRP channels in specific trophoblast functions has been disregarded. Using qRT-PCR and in situ hybridisation, the spatio-temporal expression pattern of TRP channels in the mouse placenta across gestation (E10.5-E18.5) was assessed. Prominent expression was observed for Trpv2, Trpm6, and Trpm7. Calcium microfluorimetry in primary trophoblast cells isolated at E14.5 of gestation further revealed the functional activity of TRPV2 and TRPM7. Finally, comparing TRP channels expression in mouse trophoblast stem cells (mTSCs) and mouse embryonic stem cells (mESC) confirmed the specific expression of TRPV2 during placental development. Moreover, TRP channel expression was similar in mTSCs compared to primary trophoblasts and validate mTSC as a model to study TRP channels in placental development. Collectivity, our results identify a specific spatio-temporal TRP channel expression pattern in trophoblasts, suggesting a possible involvement in regulating the process of placentation

Genome-wide haplotyping embryos developing from 0PN and 1PN zygotes increases transferrable embryos in PGT-M.

STUDY QUESTION Can genome-wide haplotyping increase success following preimplantation genetic testing for a monogenic disorder (PGT-M) by including zygotes with absence of pronuclei (0PN) or the presence of only one pronucleus (1PN)? SUMMARY ANSWER Genome-wide haplotyping 0PNs and 1PNs increases the number of PGT-M cycles reaching embryo transfer (ET) by 81% and the live-birth rate by 75%. WHAT IS KNOWN ALREADY Although a significant subset of 0PN and 1PN zygotes can develop into balanced, diploid and developmentally competent embryos, they are usually discarded because parental diploidy detection is not part of the routine work-up of PGT-M. STUDY DESIGN, SIZE, DURATION This prospective cohort study evaluated the pronuclear number in 2229 zygotes from 2337 injected metaphase II (MII) oocytes in 268 cycles. PGT-M for 0PN and 1PN embryos developing into Day 5/6 blastocysts with adequate quality for vitrification was performed in 42 of the 268 cycles (15.7%). In these 42 cycles, we genome-wide haplotyped 216 good quality embryos corresponding to 49 0PNs, 15 1PNs and 152 2PNs. The reported outcomes include parental contribution to embryonic ploidy, embryonic aneuploidy, genetic diagnosis for the monogenic disorder, cycles reaching ETs, pregnancy and live birth rates (LBR) for unaffected offspring. PARTICIPANTS/MATERIALS, SETTING, METHODS Blastomere DNA was whole-genome amplified and hybridized on the Illumina Human CytoSNP12V2.1.1 BeadChip arrays. Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the embryonic genome architecture. Bi-parental, unaffected embryos were transferred regardless of their initial zygotic PN score. MAIN RESULTS AND THE ROLE OF CHANCE A staggering 75.51% of 0PN and 42.86% of 1PN blastocysts are diploid bi-parental allowing accurate genetic diagnosis for the monogenic disorder. In total, 31% (13/42) of the PGT-M cycles reached ET or could repeat ET with an unaffected 0PN or 1PN embryo. The LBR per initiated cycle increased from 9.52 to 16.67%. LIMITATIONS, REASONS FOR CAUTION The clinical efficacy of the routine inclusion of 0PN and 1PN zygotes in PGT-M cycles should be confirmed in larger cohorts from multicenter studies. WIDER IMPLICATIONS OF THE FINDINGS Genome-wide haplotyping allows the inclusion of 0PN and 1PN embryos and subsequently increases the cycles reaching ET following PGT-M and potentially PGT for aneuploidy (PGT-A) and chromosomal structural rearrangements (PGT-SR). Establishing measures of clinical efficacy could lead to an update of the ESHRE guidelines which advise against the use of these zygotes. STUDY FUNDING/COMPETING INTEREST(S) SymBioSys (PFV/10/016 and C1/018 to J.R.V. and T.V.), the Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D., A.D. and M.Z.E. M.Z.E., T.V. and J.R.V. co-invented haplarithmisis (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’), which has been licensed to Agilent Technologies. H.M. is fully supported by the (FWO) (ZKD1543-ASP/16). The authors have no competing interests to declare

Parental genomes segregate into distinct blastomeres during multipolar zygotic divisions leading to mixoploid and chimeric blastocysts

BACKGROUND: During normal zygotic division, two haploid parental genomes replicate, unite and segregate into two biparental diploid blastomeres. RESULTS: Contrary to this fundamental biological tenet, we demonstrate here that parental genomes can segregate to distinct blastomeres during the zygotic division resulting in haploid or uniparental diploid and polyploid cells, a phenomenon coined heterogoneic division. By mapping the genomic landscape of 82 blastomeres from 25 bovine zygotes, we show that multipolar zygotic division is a tell-tale of whole-genome segregation errors. Based on the haplotypes and live-imaging of zygotic divisions, we demonstrate that various combinations of androgenetic, gynogenetic, diploid, and polyploid blastomeres arise via distinct parental genome segregation errors including the formation of additional paternal, private parental, or tripolar spindles, or by extrusion of paternal genomes. Hence, we provide evidence that private parental spindles, if failing to congress before anaphase, can lead to whole-genome segregation errors. In addition, anuclear blastomeres are common, indicating that cytokinesis can be uncoupled from karyokinesis. Dissociation of blastocyst-stage embryos further demonstrates that whole-genome segregation errors might lead to mixoploid or chimeric development in both human and cow. Yet, following multipolar zygotic division, fewer embryos reach the blastocyst stage and diploidization occurs frequently indicating that alternatively, blastomeres with genome-wide errors resulting from whole-genome segregation errors can be selected against or contribute to embryonic arrest. CONCLUSIONS: Heterogoneic zygotic division provides an overarching paradigm for the development of mixoploid and chimeric individuals and moles and can be an important cause of embryonic and fetal arrest following natural conception or IVF

Impact van de Belgische wetgeving op de uitkomst van medisch begeleide voortplanting in het Leuvens Universitair Fertiliteitscentrum.

In this research project we investigated the impact of the Belgian legislation on practice and outcome of assisted reproductive technology (ART) treatment. We also investigated the impact of these legislations from a health-economic point of view. How can we still improve reproductive outcome in the context of new legislation that forced us to change our daily clinical practice? In order to address these questions, several studies and randomized controlled trials were performed. To decrease multiple pregnancies and the associated perinatal costs, a new legislation was introduced in July 2003 (Belgisch Staatsblad) regulating the reimbursement of the laboratory costs of 6 in-vitro fertilization (IVF) cycles for Belgian women under the age of 43. This strategy was linked to a restriction of the number of transferable embryos, depending on the female age and the cycle ranking. In an initial retrospective cohort study, we compared the cumulative delivery rate (CDR) between a study group after introduction of new legislation (N=795 patients) and a control group before legislation (N=463 patients) within six ART cycles or 36 months. Based on realistic estimates, CDR within 36 months was 60.8% in the study group after introduction of new legislation and 65.6% in the control group before introduction. CDR within 6 ART cycles was also comparable between both groups. We concluded that Belgian public health policy combining reimbursement of six ART cycles with a legally enforced reduction in the number of embryos transferred, did not had a negative impact on the CDR in spite of a 50% reduction in multiple live birth rate (MLBR) from 24% to 12%. In an additional retrospective economic analysis, we reported that a 50% reduction in MLBR led to a 13% reduction in cost based on high quality real life data obtained in the Leuven University Fertility Center.the health-economic effect of the reduction of the multiple delivery rate with 50% since the introduction of Belgian legislation of July 1st 2003 the health-economic effect of the reduction of the multiple delivery rate with 50% since the introduction of Belgian legislation of July 1st 2003 the health-economic effect of the reduction of the multiple delivery rate with 50% since the introduction of Belgian legislation of July 1st 2003 (chapter 2) The substantial budget saved by this policy can be used to improve patient access to ART by selective reimbursement. These results have implications for public health policies worldwide with respect to quality, safety, regulation, and financial control of treatments with ART. From a public health point of view, the ‘Belgian model’ can now be considered by other policy makers for application worldwide. In September 2006, a law came into force regulating the reimbursement of gonadotrophins in the context of controlled ovarian stimulation (COS) with or without intrauterine insemination (IUI). Before this law, COS in combination with IUI could be performed with either clomiphene citrate (CC) or gonadotrophin. This law states that gonadotrophins are only refunded in selected cases. This decision, taken from a health economic concern, is opposed to observational and randomized studies that suggest that IUI after treatment with gonadotrophins results in a higher pregnancy rate than with CC. As expected, our RCT including 330 women scheduled for IUI during 657 IUI cycles to ovarian stimulation with low dose gonadotrophins (hMG) subcutaneous or CC orally administered showed that hMG was superior to CC with respect to the clinical pregnancy rate (12.4% versus 6.7%) and live birth rate (11.8% versus 6.4%) per started cycle. Additionally, the cancellation rate per started cycle was considerably lower when hMG is administered instead of CC (4.7% versus 15.4%). In an additional cost-effectiveness analysis (CEA) based on real life health care costs, we demonstrated that ovarian stimulation with hMG is associated with increased cost-effectiveness when compared to CC. The question remains whether luteal phase support (LPS) is needed in IUI cycles stimulated with gonadotrophins? In both the single center RCT and multicenter RCT performed, we found no significant difference in clinical pregnancy rate after HMG stimulated IUI cycles with or without LPS with vaginal progesterone. These results should be interpreted cautiously since sample size was never reached in both studies due to recruitment problems. However, in the multicenter RCT, there was an absolute risk difference for clinical pregnancy rate of 6% and for live birth rate of 5% in the treatment group with LPS. The clinical pregnancy rate, was 16.8% in the LPS group and 11% in the control group. Similarly, live birth rate was 14.9% in the LPS group and 9.4% in the control group. And, to the best of our knowledge, this RCT includes the highest number of patients (N=393) ever included in an RCT allowing only one cycle per patient, testing this hypothesis. In July 2007, a new legislation stated that supernumerary embryos created by ART need to be thawed and used before a new oocyte aspiration is allowed to create new embryos. Due to the limited number of reimbursed ART cycles in Belgium, the demand for a healthy child within these cycles has become very compelling. Therefore, it is important to continuously improve the implantation potential of embryos during freeze-thaw cycles. In a first RCT, the implantation rate (IR) per embryo transferred after an embryo transfer (ET) of frozen-thawed embryos with thinned zona pellucida (ZP) after assisted hatching (AH) by laser was compared to an ET of frozen-thawed embryos without AH. A total of 647 thawing cycles were randomized to either the AH group (N=324) or control group (N=323) and no significant difference in IR per embryo transferred was observed between the AH group (13.3%) and control group (15.6%). In a second RCT, the IR per frozen-thawed embryo transfer cycle (FET) stimulated with gonadotrophins (hMG) was compared to natural cycle (NC) FET cycles. To the best of our knowledge, no RCTs have been published testing this hypothesis in women with a regular ovulatory cycle. A total of 672 embryos were transferred during 434 cycles. In this study we demonstrated that the IR per embryo transferred, was not statistically different between the hMG FET group (16%) and the NC FET group (12%). However, there was a trend towards a slightly higher IR (4% higher) in the hMG FET group.status: publishe

Challenges in the development of novel therapeutic strategies for treatment of endometriosis

Endometriosis is an estrogen-dependent disease that results in pelvic pain and infertility. Its treatment is often frustrating due to limited medical treatment options, complex surgical treatment and high recurrence rates. Despite the advances in our understanding of the pathogenesis over the last decades and the consequent novel therapeutic strategies, no new drugs have been introduced in daily clinical practice. Areas covered: In the first part we present an overview of the pathogenesis of endometriosis. In the second part we discuss how new insights have led to the development of novel non-hormonal strategies for the treatment of endometriosis, focusing on anti-inflammatory and anti-angiogenic agents. In the third part we describe the problems encountered in the translation from experimental drugs to routine medicine for the treatment of endometriosis. Expert Opinion: Despite the multitude of agents that have been tested in pre-clinical trials, only few drugs have passed to the stage of clinical testing and none have been introduced into clinical practice. It is our opinion that the major challenges in the translation from novel agents for endometriosis is due to the use of inadequate rodent models and a lack of standardization in the design and reporting of preclinical endometriosis models.peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=iett20status: publishe

Follicular fluid biomarkers for human in vitro fertilization outcome: Proof of principle

Human follicular fluid (FF) is a unique biological fluid in which the oocyte develops in vivo, and presents an optimal source for non-invasive biochemical predictors. Oocyte quality directly influences the embryo development and hence, may be used as a predictor of embryo quality. Peptide profiling of FF and its potential use as a biomarker for oocyte quality has never been reported.status: publishe

Predicting the chance on live birth per cycle at each step of the IVF journey: external validation and update of the van Loendersloot multivariable prognostic model

Objective To study the performance of the ‘van Loendersloot’ prognostic model for our clinic’s in vitro fertilisation (IVF) in its original version, the refitted version and in an adapted version replacing previous by current cycle IVF laboratory variables. Methods This retrospective cohort study in our academic tertiary fertility clinic analysed 1281 IVF cycles of 591 couples, who completed at least one 2nd–6th IVF cycle with own fresh gametes after a previous IVF cycle with the same partner in our clinic between 2010 and 2018. The outcome of interest was the chance on a live birth after one complete IVF cycle (including all fresh and frozen embryo transfers from the same episode of ovarian stimulation). Model performance was expressed in terms of discrimination (c-statistics) and calibration (calibration model, comparison of prognosis to observed ratios of five disjoint groups formed by the quintiles of the IVF prognoses and a calibration plot). Results A total of 344 live births were obtained (26.9%). External validation of the original van Loendersloot model showed a poor c-statistic of 0.64 (95% CI: 0.61 to 0.68) and an underestimation of IVF success. The refitted and the adapted models showed c-statistics of respectively 0.68 (95% CI: 0.65 to 0.71) and 0.74 (95% CI: 0.70 to 0.77). Similar c-statistics were found with cross-validation. Both models showed a good calibration model; refitted model: intercept=0.00 (95% CI: −0.23 to 0.23) and slope=1.00 (95% CI: 0.79 to 1.21); adapted model: intercept=0.00 (95% CI: −0.18 to 0.18) and slope=1.00 (95% CI: 0.83 to 1.17). Prognoses and observed success rates of the disjoint groups matched well for the refitted model and even better for the adapted model. Conclusion External validation of the original van Loendersloot model indicated that model updating was recommended. The good performance of the refitted and adapted models allows informing couples about their IVF prognosis prior to an IVF cycle and at the time of embryo transfer. Whether this has an impact on couple’s expected success rates, distress and IVF discontinuation can now be studied.status: publishe

Selecting the embryo with the highest implantation potential using a data mining based prediction model

Embryo selection has been based on developmental and morphological characteristics. However, the presence of an important intra-and inter-observer variability of standard scoring system (SSS) has been reported. A computer-assisted scoring system (CASS) has the potential to overcome most of these disadvantages associated with the SSS. The aims of this study were to construct a prediction model, with data mining approaches, and compare the predictive performance of models in SSS and CASS and to evaluate whether using the prediction model would impact the selection of the embryo for transfer.status: publishe