Single-cell genome-wide concurrent haplotyping and copy-number profiling through genotyping-by-sequencing.

Single-cell whole-genome haplotyping allows simultaneous detection of haplotypes associated with monogenic diseases, chromosome copy-numbering and subsequently, has revealed mosaicism in embryos and embryonic stem cells. Methods, such as karyomapping and haplarithmisis, were deployed as a generic and genome-wide approach for preimplantation genetic testing (PGT) and are replacing traditional PGT methods. While current methods primarily rely on single-nucleotide polymorphism (SNP) array, we envision sequencing-based methods to become more accessible and cost-efficient. Here, we developed a novel sequencing-based methodology to haplotype and copy-number profile single cells. Following DNA amplification, genomic size and complexity is reduced through restriction enzyme digestion and DNA is genotyped through sequencing. This single-cell genotyping-by-sequencing (scGBS) is the input for haplarithmisis, an algorithm we previously developed for SNP array-based single-cell haplotyping. We established technical parameters and developed an analysis pipeline enabling accurate concurrent haplotyping and copy-number profiling of single cells. We demonstrate its value in human blastomere and trophectoderm samples as application for PGT for monogenic disorders. Furthermore, we demonstrate the method to work in other species through analyzing blastomeres of bovine embryos. Our scGBS method opens up the path for single-cell haplotyping of any species with diploid genomes and could make its way into the clinic as a PGT application.

Exploring attitudes and experiences with reproductive genetic carrier screening among couples seeking medically assisted reproduction: a longitudinal survey study.

PURPOSE: This study aimed to assess the attitudes and experiences of subfertile couples applying for medically assisted reproduction (MAR) using their own gametes towards reproductive genetic carrier screening (RGCS) for monogenic conditions. METHODS: A prospective survey study was conducted where subfertile couples were recruited from the fertility centre of a university hospital in Flanders, Belgium. Participants were offered RGCS free of charge and completed self-administered questionnaires at three different time points. RESULTS: The study sample consisted of 26 couples. Most participants had no children, did not consider themselves as religious, and had some form of higher education. Overall, attitudes towards RGCS were mostly positive and the intention to participate in RGCS was high. Anxiety scores were only elevated and clinically relevant for a limited number of participants. A large proportion of participants would consider preventive reproductive options like prenatal diagnosis or in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) combined with pre-implantation genetic testing for monogenic conditions (PGT-M) in the event of an increased likelihood of conceiving a child with a hereditary condition. Participants were satisfied with their decision to undergo RGCS, and the majority would recommend RGCS to other couples. CONCLUSION: Our study findings suggest that subfertile couples applying for MAR using their own gametes find RGCS acceptable and have a positive attitude towards it. This study provides valuable insights into the perspectives of these couples, highlighting the need for appropriate counseling and timely information provision.

Preclinical workup using long-read amplicon sequencing provides families with de novo pathogenic variants access to universal preimplantation genetic testing.

STUDY QUESTION: Can long-read amplicon sequencing be beneficial for preclinical preimplantation genetic testing (PGT) workup in couples with a de novo pathogenic variant in one of the prospective parents? SUMMARY ANSWER: Long-read amplicon sequencing represents a simple, rapid and cost-effective preclinical PGT workup strategy that provides couples with de novo pathogenic variants access to universal genome-wide haplotyping-based PGT programs. WHAT IS KNOWN ALREADY: Universal PGT combines genome-wide haplotyping and copy number profiling to select embryos devoid of both familial pathogenic variants and aneuploidies. However, it cannot be directly applied in couples with a de novo pathogenic variant in one of the partners due to the absence of affected family members required for phasing the disease-associated haplotype. STUDY DESIGN, SIZE, DURATION: This is a prospective study, which includes 32 families that were enrolled in the universal PGT program at the University Hospital of Leuven between 2018 and 2022. We implemented long-read amplicon sequencing during the preclinical PGT workup to deduce the parental origin of the disease-associated allele in the affected partner, which can then be traced in embryos during clinical universal PGT cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: To identify the parental origin of the disease-associated allele, genomic DNA from the carrier of the de novo pathogenic variant and his/her parent(s) was used for preclinical PGT workup. Primers flanking the de novo variant upstream and downstream were designed for each family. Following long-range PCR, amplicons that ranged 5-10 kb in size, were sequenced using Pacific Bioscience and/or Oxford Nanopore platforms. Next, targeted variant calling and haplotyping were performed to identify parental informative single-nucleotide variants (iSNVs) linked to the de novo mutation. Following the preclinical PGT workup, universal PGT via genome-wide haplotyping was performed for couples who proceeded with clinical PGT cycle. In parallel, 13 trophectoderm (TE) biopsies from three families that were analyzed by universal PGT, were also used for long-read amplicon sequencing to explore this approach for embryo direct mutation detection coupled with targeted long-read haplotyping. MAIN RESULTS AND THE ROLE OF CHANCE: The parental origin of the mutant allele was identified in 24/32 affected individuals during the preclinical PGT workup stage, resulting in a 75% success rate. On average, 5.95 iSNVs (SD = 4.5) were detected per locus of interest, and the average distance of closest iSNV to the de novo variant was ∼1750 bp. In 75% of those cases (18/24), the de novo mutation occurred on the paternal allele. In the remaining eight families, the risk haplotype could not be established due to the absence of iSNVs linked to the mutation or inability to successfully target the region of interest. During the time of the study, 12/24 successfully analyzed couples entered the universal PGT program, and three disease-free children have been born. In parallel to universal PGT analysis, long-read amplicon sequencing of 13 TE biopsies was also performed, confirming the segregation of parental alleles in the embryo and the results of the universal PGT. LIMITATIONS, REASONS FOR CAUTION: The main limitation of this approach is that it remains targeted with the need to design locus-specific primers. Because of the restricted size of target amplicons, the region of interest may also remain non-informative in the absence of iSNVs. WIDER IMPLICATIONS OF THE FINDINGS: Targeted haplotyping via long-read amplicon sequencing, particularly using Oxford Nanopore Technologies, provides a valuable alternative for couples with de novo pathogenic variants that allows access to universal PGT. Moreover, the same approach can be used for direct mutation analysis in embryos, as a second line confirmation of the preclinical PGT result or as a potential alternative PGT procedure in couples, where additional family members are not available. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by KU Leuven funding (no. C1/018 to J.R.V.) and Fonds Wetenschappelijk Onderzoek (1241121N to O.T.). J.R.V. is co-inventor of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 'Methods for haplotyping single-cells' and ZL913096-PCT/EP2014/068315-WO/2015/028576 'Haplotyping and copy number typing using polymorphic variant allelic frequencies' licensed to Agilent Technologies. All other authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Protease secretions by the invading blastocyst induce calcium oscillations in endometrial epithelial cells via the protease-activated receptor 2.

BACKGROUND: Early embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive. METHODS: To identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression. RESULTS: We showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IP3R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the 'Window of implantation' markers in human endometrial epithelial cells. CONCLUSIONS: These findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst.

Impact of Indication for Oocyte and Embryo Reception on Reproductive and Obstetric Outcomes.

OBJECTIVES: This study aimed to assess if the indication for oocyte reception (OR) or embryo reception (ER) impacts the reproductive and obstetric outcomes by evaluating our experience at a tertiary fertility centre and by performing a literature review on this subject. Several previous studies have reported that, in contrast to other types of fertility treatment, the indication for OR/ER seems to have little impact on the outcomes. However, the compared indication groups vary considerably between these studies, and some data indicates worse outcomes in patients who developed premature ovarian insufficiency (POI) due to Turner syndrome or treatment with chemotherapy/radiotherapy. DESIGN: A retrospective analysis of all cases of OR/ER at a tertiary fertility centre from 2001 until 2020 was conducted. We analysed 584 cycles from 194 individual patients. A literature review on the impact of indication on reproductive or obstetric outcomes of OR/ER was performed using the following databases: PubMed/MEDLINE, Embase, and the Cochrane Library. A total of 27 studies were included and analysed. PARTICIPANTS, SETTING, METHODS: For the retrospective analysis, patients were divided into three major indication groups: failure of autologous assisted reproductive technology, POI, and genetic disease carrier. To assess reproductive outcomes, we determined pregnancy rate, implantation rate, miscarriage rate, and live birth rate. For comparing obstetric outcomes, we reviewed the term of birth, mode of delivery, and birthweight. Outcomes were compared using Fisher's exact test, χ2 test, and one-way ANOVA utilizing the GraphPad tool. RESULTS: There were no significant differences in reproductive and obstetric outcomes between the three major indication groups in our population, in line with the findings reported by existing literature. Data on impaired reproductive outcomes in patients with POI after chemotherapy/radiotherapy are conflicting. Obstetrically, these patients are at higher risk of preterm birth and possibly also low birthweight, especially after abdomino-pelvic or total body irradiation. For patients with POI due to Turner syndrome, most data suggest similar pregnancy rates but a higher rate of pregnancy loss, and obstetrically an increased risk of hypertensive disorders and caesarean section. LIMITATIONS: The small number of patients in the retrospective analysis resulted in low statistical power when evaluating differences between smaller subgroups. There were some missing data on the occurrence of complications during pregnancy. Our analysis covers a period of 20 years, during which several technological innovations have also been made. CONCLUSIONS: Our study shows that the important heterogeneity in couples treated with OR/ER does not significantly impact their reproductive or obstetric outcomes, except for POI due to Turner syndrome or treatment with chemotherapy/radiotherapy, where there seems to be an important uterine/endometrial component that cannot be entirely overcome by providing a healthy oocyte.

The Impact of Chronic Endometritis on Infertility: Prevalence, Reproductive Outcomes, and the Role of Hysteroscopy as a Screening Tool.

OBJECTIVES: The objective of this study was to examine the prevalence of chronic endometritis (CE) in infertile women, its impact on reproductive outcomes, and the accuracy of hysteroscopy as a screening tool for CE. DESIGN: This was a prospective observational study. PARTICIPANTS: Participants involved in this study were 514 asymptomatic patients with infertility. SETTING: The review was conducted in a tertiary care center. METHODS: The participants underwent a hysteroscopy and endometrial biopsy (EMB). Antibiotics were given for cases of CE. We investigated the prevalence of CE in patients starting assisted reproductive technologies (ART) as a primary outcome. Secondary outcomes were the clinical pregnancy rate (CPR) in the ART cycle after hysteroscopy, EMB, and antibiotic treatment in cases of CE; the cumulative CPR in the subsequent 2 years after hysteroscopy and EMB; the sensitivity and specificity of hysteroscopy as a screening tool compared to EMB as the "gold standard" for diagnosing CE. RESULTS: CE was identified in 2.8% of patients starting ART (11/393). CPRs did not differ significantly between patients with CE and the entire cohort of patients without CE in the subsequent ART cycle (OR: 0.43; 95% CI: 0.09-2.02) or in the 2 years after EMB (OR: 0.56; 95% CI: 0.16-1.97). In a matched control comparison (with matching for age, basal FSH, and cause of infertility), CPR in patients with CE did not differ in the subsequent ART cycle (OR: 0.39; 95% CI: 0.09-1.61); however, their CPR in the 2 years after EMB was significantly lower (OR: 0.22; 95% CI: 0.13-0.38). The sensitivity and specificity of hysteroscopy as a screening tool for diagnosing CE were 8.3% and 90.1%, respectively. LIMITATIONS: Due to our cohort's low CE prevalence, we could not detect significant differences in CPRs. CONCLUSION: CE is rare in our studied population of asymptomatic patients starting ART. Hysteroscopy cannot replace EMB for diagnosing CE.

A Randomized Trial of Endometrial Scratching before In Vitro Fertilization.

BACKGROUND: Endometrial scratching (with the use of a pipelle biopsy) is a technique proposed to facilitate embryo implantation and increase the probability of pregnancy in women undergoing in vitro fertilization (IVF). METHODS: We conducted a pragmatic, multicenter, open-label, randomized, controlled trial. Eligible women were undergoing IVF (fresh-embryo or frozen-embryo transfer), with no recent exposure to disruptive intrauterine instrumentation (e.g., hysteroscopy). Participants were randomly assigned in a 1:1 ratio to either endometrial scratching (by pipelle biopsy between day 3 of the cycle preceding the embryo-transfer cycle and day 3 of the embryo-transfer cycle) or no intervention. The primary outcome was live birth. RESULTS: A total of 1364 women underwent randomization. The frequency of live birth was 180 of 690 women (26.1%) in the endometrial-scratch group and 176 of 674 women (26.1%) in the control group (adjusted odds ratio, 1.00; 95% confidence interval, 0.78 to 1.27). There were no significant between-group differences in the rates of ongoing pregnancy, clinical pregnancy, multiple pregnancy, ectopic pregnancy, or miscarriage. The median score for pain from endometrial scratching (on a scale of 0 to 10, with higher scores indicating worse pain) was 3.5 (interquartile range, 1.9 to 6.0). CONCLUSIONS: Endometrial scratching did not result in a higher rate of live birth than no intervention among women undergoing IVF. (Funded by the University of Auckland and others; PIP Australian New Zealand Clinical Trials Registry number, ACTRN12614000626662 .).

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

Transient 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.

TRP channel expression correlates with the epithelial-mesenchymal transition and high-risk endometrial carcinoma.

Transient receptor potential (TRP) channels excel in cellular sensing as they allow rapid ion influx across the plasma membrane in response to a variety of extracellular cues. Recently, a distinct TRP mRNA expression signature was observed in stromal cells (ESC) and epithelial cells (EEC) of the endometrium, a tissue in which cell phenotypic plasticity is essential for normal functioning. However, it is unknown whether TRP channel mRNA expression is subject to the phenotypic switching that occurs during epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET), and whether TRP channel mRNA expression is associated with aggressive phenotypes in endometrial cancer (EC). Here, we induced EMT and MET in vitro using in primary EEC and ESC, respectively, and analyzed expression and functionality of TRP channels using RT-qPCR and intracellular Ca2+ imaging. The outcome of these experiments showed a strong association between TRPV2 and TRPC1 mRNA expression and the mesenchymal phenotype, whereas TRPM4 mRNA expression correlated with the epithelial phenotype. In line herewith, increased TRPV2 and TRPC1 mRNA expression levels were observed in both primary and metastatic EC biopsies and in primary EC cells with a high EMT status, indicating an association with an aggressive tumor phenotype. Remarkably, TRPV2 mRNA expression in primary EC biopsies was associated with tumor invasiveness and cancer stage. In contrast, increased TRPM4 mRNA expression was observed in EC biopsies with a low EMT status and less aggressive tumor phenotypes. Taken together, this dataset proved for the first time that TRP channel mRNA expression is strongly linked to cellular phenotypes of the endometrium, and that phenotypic transitions caused by either experimental manipulation or malignancy could alter this expression in a predictable manner. These results implicate that TRP channels are viable biomarkers to identify high-risk EC, and potential targets for EC treatment.

Reasons affecting the uptake of reproductive genetic carrier screening among nonpregnant reproductive-aged women in Flanders (Belgium).

Reproductive genetic carrier screening (RGCS) allows to identify couples who have an increased likelihood of conceiving a child affected with an autosomal recessive or X-linked monogenic condition. Multiple studies have reported on a wide and fragmented set of reasons to accept or decline RGCS. Only a few studies have been performed to assess the uptake of RGCS. Nonpregnant women visiting their gynecologist were invited to complete a questionnaire assessing perceived susceptibility, the acceptability of offering RGCS, attitudes, the intention to participate in RGCS, reasons to accept or decline RGCS, and sociodemographic characteristics. Women who showed the intention to have RGCS were asked to consider a free RGCS offer. Most women (n = 127) were between 25 and 34 years old (60%), in a relationship (91%), and wanted to have children (65%). Study participants had positive attitudes towards RGCS and the intention to consider RGCS in the future. Reasons to accept RGCS were being able to share genetic information with children or relatives (n = 104/127, 82%), to prevent the birth of a child affected with a hereditary condition (n = 103/127, 81%), and/or to know the chance of conceiving a child with a hereditary condition (n = 102/127, 80%). Reasons for declining RGCS were the possible concerns that could arise when receiving test results (n = 27/127, 21%), having no family history of hereditary disorders (n = 19/127, 15%), and not wanting to take action based on test results (n = 13/127, 10%). Among test intenders that met the inclusion criteria, 53% decided to participate in RGCS together with their male reproductive partner. More in-depth research on the decision-making process behind the choice to accept or decline an RGCS offer would be highly valuable to make sure couples are making informed reproductive choices.

Identity-by-state-based haplotyping expands the application of comprehensive preimplantation genetic testing.

STUDY QUESTION: Is it possible to haplotype parents using parental siblings to leverage preimplantation genetic testing (PGT) for monogenic diseases and aneuploidy (comprehensive PGT) by genome-wide haplotyping? SUMMARY ANSWER: We imputed identity-by-state (IBS) sharing of parental siblings to phase parental genotypes. WHAT IS KNOWN ALREADY: Genome-wide haplotyping of preimplantation embryos is being implemented as a generic approach for genetic diagnosis of inherited single-gene disorders. To enable the phasing of genotypes into haplotypes, genotyping the direct family members of the prospective parent carrying the mutation is required. Current approaches require genotypes of either (i) both or one of the parents of the affected prospective parent or (ii) an affected or an unaffected child of the couple. However, this approach cannot be used when parents or children are not attainable, prompting an investigation into alternative phasing options. STUDY DESIGN, SIZE, DURATION: This is a retrospective validation study, which applied IBS-based phasing of parental haplotypes in 56 embryos derived from 12 PGT families. Genome-wide haplotypes and copy number profiles generated for each embryo using the new phasing approach were compared with the reference PGT method to evaluate the diagnostic concordance. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included 12 couples with a known hereditary genetic disorder, participating in the comprehensive PGT program and with at least one parental sibling available (e.g. brother and/or sister). Genotyping data from both prospective parents and the parental sibling(s) were used to perform IBS-based phasing and to trace the disease-associated alleles. The outcome of the IBS-based PGT was compared with the results of the clinically implemented reference haplotyping-based PGT method. MAIN RESULTS AND THE ROLE OF CHANCE: IBS-based haplotyping was performed for 12 PGT families. In accordance with the theoretical prediction of allele sharing between sibling pairs, 6 out of 12 (50%) couples or 23 out of 56 embryos could be phased using parental siblings. In families where phasing was possible, haplotype calling in the locus of interest was 100% concordant between the reference PGT method and IBS-based approach using parental siblings. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Phasing of parental haplotypes will only be possible when the disease locus lies in an informative region (categorized as IBS1). Phasing prospective parents using relatives with reduced genetic relatedness as a reference (e.g. siblings) decreases the size and the occurrence of informative IBS1 regions, necessary for haplotype calling. By including more than one extended family member, the chance of obtaining IBS1 coverage in the interrogated locus can be increased. A pre-PGT work-up can define whether the carrier couple could benefit from this approach. WIDER IMPLICATIONS OF THE FINDINGS: Phasing by relatives extends the potential of comprehensive PGT, since it allows the inclusion of couples who do not have access to the standard phasing references, such as parents or offspring. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the KU Leuven grant (C14/18/092), Research Foundation Flanders (FWO; GA09311N), Horizon 2020 innovation programme (WIDENLIFE, 692065) and Agilent Technologies. J.R.V., T.V. and M.Z.E. are co-inventors of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 'Methods for haplotyping single-cells' and ZL913096-PCT/EP2014/068315-WO/2015/028576 'Haplotyping and copy number typing using polymorphic variant allelic frequencies' licensed to Agilent Technologies. The other authors have no conflict of interest to declare.

Multi-centre evaluation of a comprehensive preimplantation genetic test through haplotyping-by-sequencing.

STUDY QUESTION: Can reduced representation genome sequencing offer an alternative to single nucleotide polymorphism (SNP) arrays as a generic and genome-wide approach for comprehensive preimplantation genetic testing for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR) in human embryo biopsy samples? SUMMARY ANSWER: Reduced representation genome sequencing, with OnePGT, offers a generic, next-generation sequencing-based approach for automated haplotyping and copy-number assessment, both combined or independently, in human single blastomere and trophectoderm samples. WHAT IS KNOWN ALREADY: Genome-wide haplotyping strategies, such as karyomapping and haplarithmisis, have paved the way for comprehensive PGT, i.e. leveraging PGT-M, PGT-A and PGT-SR in a single workflow. These methods are based upon SNP array technology. STUDY DESIGN, SIZE, DURATION: This multi-centre verification study evaluated the concordance of PGT results for a total of 225 embryos, including 189 originally tested for a monogenic disorder and 36 tested for a translocation. Concordance for whole chromosome aneuploidies was also evaluated where whole genome copy-number reference data were available. Data analysts were kept blind to the results from the reference PGT method. PARTICIPANTS/MATERIALS, SETTING, METHODS: Leftover blastomere/trophectoderm whole genome amplified (WGA) material was used, or secondary trophectoderm biopsies were WGA. A reduced representation library from WGA DNA together with bulk DNA from phasing references was processed across two study sites with the Agilent OnePGT solution. Libraries were sequenced on an Illumina NextSeq500 system, and data were analysed with Agilent Alissa OnePGT software. The embedded PGT-M pipeline utilises the principles of haplarithmisis to deduce haplotype inheritance whereas both the PGT-A and PGT-SR pipelines are based upon read-count analysis in order to evaluate embryonic ploidy. Concordance analysis was performed for both analysis strategies against the reference PGT method. MAIN RESULTS AND THE ROLE OF CHANCE: PGT-M analysis was performed on 189 samples. For nine samples, the data quality was too poor to analyse further, and for 20 samples, no result could be obtained mainly due to biological limitations of the haplotyping approach, such as co-localisation of meiotic crossover events and nullisomy for the chromosome of interest. For the remaining 160 samples, 100% concordance was obtained between OnePGT and the reference PGT-M method. Equally for PGT-SR, 100% concordance for all 36 embryos tested was demonstrated. Moreover, with embryos originally analysed for PGT-M or PGT-SR for which genome-wide copy-number reference data were available, 100% concordance was shown for whole chromosome copy-number calls (PGT-A). LIMITATIONS, REASONS FOR CAUTION: Inherent to haplotyping methodologies, processing of additional family members is still required. Biological limitations caused inconclusive results in 10% of cases. WIDER IMPLICATIONS OF THE FINDINGS: Employment of OnePGT for PGT-M, PGT-SR, PGT-A or combined as comprehensive PGT offers a scalable platform, which is inherently generic and thereby, eliminates the need for family-specific design and optimisation. It can be considered as both an improvement and complement to the current methodologies for PGT. STUDY FUNDING/COMPETING INTEREST(S): Agilent Technologies, the KU Leuven (C1/018 to J.R.V. and T.V.) and the Horizon 2020 WIDENLIFE (692065 to J.R.V. and T.V). H.M. is supported by the Research Foundation Flanders (FWO, 11A7119N). M.Z.E, J.R.V. and T.V. are co-inventors on patent applications: ZL910050-PCT/EP2011/060211- WO/2011/157846 'Methods for haplotyping single cells' and ZL913096-PCT/EP2014/068315 'Haplotyping and copy-number typing using polymorphic variant allelic frequencies'. T.V. and J.R.V. are co-inventors on patent application: ZL912076-PCT/EP2013/070858 'High-throughput genotyping by sequencing'. Haplarithmisis ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies') has been licensed to Agilent Technologies. The following patents are pending for OnePGT: US2016275239, AU2014345516, CA2928013, CN105874081, EP3066213 and WO2015067796. OnePGT is a registered trademark. D.L., J.T. and R.L.R. report personal fees during the conduct of the study and outside the submitted work from Agilent Technologies. S.H. and K.O.F. report personal fees and other during the conduct of the study and outside the submitted work from Agilent Technologies. J.A. reports personal fees and other during the conduct of the study from Agilent Technologies and personal fees from Agilent Technologies and UZ Leuven outside the submitted work. B.D. reports grants from IWT/VLAIO, personal fees during the conduct of the study from Agilent Technologies and personal fees and other outside the submitted work from Agilent Technologies. In addition, B.D. has a patent 20160275239 - Genetic Analysis Method pending. The remaining authors have no conflicts of interest.

The relative importance of genetic parenthood.

RESEARCH QUESTION: How much do patients with severe infertility and their gynaecologists value genetic parenthood relative to other key treatment characteristics? DESIGN: A discrete choice experiment included the following treatment characteristics: genetic parenthood, pregnancy rate, curing infertility, maternal health, child health and costs. The questionnaire was disseminated between 2015 and 2016 among Dutch and Belgian patients with severe infertility and their gynaecologists. RESULTS: The questionnaire was completed by 173 patients and 111 gynaecologists. When choosing between treatments that varied in safety, effectiveness and costs, the treatment's ability to lead to genetic parenthood did not affect the treatment preference of patients with severe infertility (n = 173). Genetic parenthood affected the treatment preference of gynaecologists (n = 111) less than all other treatment characteristics. Patients indicated that they would switch to a treatment that did not enable genetic parenthood in return for a child health risk reduction of 3.6%, a cost reduction of €3500, an ovarian hyperstimulation risk reduction of 4.6%, a maternal cancer risk reduction of 2.7% or a pregnancy rate increase of 18%. Gynaecologists made similar trade-offs. CONCLUSIONS: While awaiting replication of this study in larger populations, these findings challenge the presumed dominant importance of genetic parenthood. This raises questions about whether donor gametes could be presented as a worthy alternative earlier in treatment trajectories and whether investments in novel treatments enabling genetic parenthood, like in-vitro gametogenesis, are proportional to their future clinical effect.

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.

Cost-effectiveness of ovarian stimulation with gonadotrophin and clomiphene citrate in an intrauterine insemination programme for subfertile couples.

Ovarian stimulation with low-dose human menopausal gonadotrophin (HMG) is superior to clomiphene citrate in intrauterine insemination (IUI) cycles with respect to clinical pregnancy rate, but it is unclear whether HMG is also the more cost-effective option. The aim of this study was to compare the cost-effectiveness of ovarian stimulation with low-dose subcutaneously administred HMG (37.5-75 IU per day) to orally administred clomiphene citrate (50 mg/day from day 3-7) in an IUI programme for subfertile couples. A cost-effectiveness analysis was conducted using the results of a randomized trial, including 620 IUI cycles. The primary outcome was the incremental cost-effectiveness ratio (ICER) of using HMG versus clomiphene citrate. Results are presented from the healthcare payer perspective. The total cost per patient associated with one IUI treatment with HMG is €764, whereas it is €558 if clomiphene citrate is used, resulting in an incremental cost of €206 for HMG per treatment. The incremental clinical pregnancy rate of using HMG instead of clomiphene citrate, however, is also 5.7 percentage points higher, resulting in an ICER of HMG versus clomiphene citrate of €3615 per additional clinical pregnancy achieved. On average, HMG was found to be more cost-effective than clomiphene citrate.

Evaluation of Total, Active, and Specific Myeloperoxidase Levels in Women with and without Endometriosis.

Myeloperoxidase (MPO) is a proinflammatory enzyme and a marker for neutrophil activation and oxidative stress. Since oxidative stress and inflammation are linked to the pathogenesis of endometriosis, we hypothesized that the total, active, and specific (active/total) MPO levels were significantly different in plasma of women with and without endometriosis. Samples were selected from our biobank from women with endometriosis (n = 212) and controls without endometriosis (n = 121) across the menstrual cycle. Total MPO plasma levels were measured by immunoassay and MPO activity by enzymatic assay. Total and active MPO levels did not differ significantly among endometriosis cases and controls, whereas the specific MPO activity was significantly lower in women with endometriosis than that in controls (p = 0.0159). After the subdivision of control patients into women with a normal pelvis and women with other benign gynecological disorders, a significant difference was observed only between women with endometriosis and women with other benign gynecological disorders (p = 0.0266). In conclusion, systemic MPO levels may not be suited as a single biomarker for endometriosis. Our data support the involvement of MPO in other gynecological disorders but do not provide any evidence for an association with endometriosis.

Principles guiding embryo selection following genome-wide haplotyping of preimplantation embryos.

STUDY QUESTION: How to select and prioritize embryos during PGD following genome-wide haplotyping? SUMMARY ANSWER: In addition to genetic disease-specific information, the embryo selected for transfer is based on ranking criteria including the existence of mitotic and/or meiotic aneuploidies, but not carriership of mutations causing recessive disorders. WHAT IS KNOWN ALREADY: Embryo selection for monogenic diseases has been mainly performed using targeted disease-specific assays. Recently, these targeted approaches are being complemented by generic genome-wide genetic analysis methods such as karyomapping or haplarithmisis, which are based on genomic haplotype reconstruction of cell(s) biopsied from embryos. This provides not only information about the inheritance of Mendelian disease alleles but also about numerical and structural chromosome anomalies and haplotypes genome-wide. Reflections on how to use this information in the diagnostic laboratory are lacking. STUDY DESIGN, SIZE, DURATION: We present the results of the first 101 PGD cycles (373 embryos) using haplarithmisis, performed in the Centre for Human Genetics, UZ Leuven. The questions raised were addressed by a multidisciplinary team of clinical geneticist, fertility specialists and ethicists. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sixty-three couples enrolled in the genome-wide haplotyping-based PGD program. Families presented with either inherited genetic variants causing known disorders and/or chromosomal rearrangements that could lead to unbalanced translocations in the offspring. MAIN RESULTS AND THE ROLE OF CHANCE: Embryos were selected based on the absence or presence of the disease allele, a trisomy or other chromosomal abnormality leading to known developmental disorders. In addition, morphologically normal Day 5 embryos were prioritized for transfer based on the presence of other chromosomal imbalances and/or carrier information. LIMITATIONS, REASONS FOR CAUTION: Some of the choices made and principles put forward are specific for cleavage-stage-based genetic testing. The proposed guidelines are subject to continuous update based on the accumulating knowledge from the implementation of genome-wide methods for PGD in many different centers world-wide as well as the results of ongoing scientific research. WIDER IMPLICATIONS OF THE FINDINGS: Our embryo selection principles have a profound impact on the organization of PGD operations and on the information that is transferred among the genetic unit, the fertility clinic and the patients. These principles are also important for the organization of pre- and post-counseling and influence the interpretation and reporting of preimplantation genotyping results. As novel genome-wide approaches for embryo selection are revolutionizing the field of reproductive genetics, national and international discussions to set general guidelines are warranted. STUDY FUNDING/COMPETING INTEREST(S): The European Union's Research and Innovation funding programs FP7-PEOPLE-2012-IAPP SARM: 324509 and Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D. and M.Z.E. J.R.V., T.V. and M.Z.E. have patents ZL910050-PCT/EP2011/060211-WO/2011/157846 ('Methods for haplotyping single cells') with royalties paid and ZL913096-PCT/EP2014/068315-WO/2015/028576 ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies') with royalties paid, licensed to Cartagenia (Agilent technologies). J.R.V. also has a patent ZL91 2076-PCT/EP20 one 3/070858 ('High throughout genotyping by sequencing') with royalties paid. TRIAL REGISTRATION NUMBER: N/A.

A 50% reduction in multiple live birth rate is associated with a 13% cost saving: a real-life retrospective cost analysis.

Belgian legislation limiting the number of embryos for transfer has been shown to result in a 50% reduction of the multiple live birth rate (MLBR) per cycle without having a negative impact on the cumulative delivery rate per patient within six cycles or 36 months. The objective of the current study was to evaluate the cost saving associated with a 50% reduction in MLBR. A retrospective cost analysis was performed of 213 couples, who became pregnant and had a live birth after one or more assisted reproductive technology treatment cycles, and their 254 children. The mean cost of a singleton (n = 172) and multiple (n = 41) birth was calculated based on individual hospital invoices. The cost analysis showed a significantly higher total cost (assisted reproductive technology treatment, pregnancy follow-up, delivery, child cost until the age of 2 years) for multiple births (both children: mean €43,397) than for singleton births (mean: €17,866) (Wilcoxon-Mann-Whitney P < 0.0001). A 50% reduction in MLBR resulted in a significant cost reduction related to hospital care of 13%.

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

BACKGROUND: 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. METHODS: A total of 871 single transferred embryos between 2008 and 2013 were included and evaluated with two scoring systems: SSS and CASS. Prediction models were developed using multivariable logistic regression (LR) and multivariate adaptive regression splines (MARS). The prediction models were externally validated with a test set of 109 single transfers between January and June 2014. Area under the curve (AUC) in training data and validation data was compared to determine the utility of the models. RESULTS: In SSS models, the AUC declined significantly from training data to validation data (p < 0.05). No significant difference was detected in CASS derived models. Two final prediction models derived from CASS were obtained using LR and MARS, which showed moderate discriminative capacity (c-statistic 0.64 and 0.69 respectively) on validation data. CONCLUSIONS: The study showed that the introduction of CASS improved the generalizability of the prediction models, and the combination of computer-assisted scoring system with data mining based predictive modeling is a promising approach to improve the selection of embryo with the highest implantation potential.

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

BACKGROUND: 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. METHODS: This study screened FF for peptide biomarkers that predict the outcome of in vitro fertilization (IVF). Potential biomarkers were discovered by investigating 2 training datasets, consisting both of 17 samples and validating on an independent experiment containing 32 samples. Peptide profiles were acquired by nano-scale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). RESULTS: From the training datasets 53 peptides were found as potential biomarker candidates, predicting the fertilization outcome of 24 out of the 32 validation samples blindly (81.3% sensitivity, 68.8% specificity, AUC = 0.86). Seven potential biomarker peptides were identified. They were derived from: insulin-like growth factor binding protein-5, alpha-2-antiplasmin, complement component 3, inter-alpha-trypsin inhibitor heavy chain H1, serum albumin, protein diaphanous homolog 1 and plastin-3. CONCLUSIONS: The MS-based comprehensive peptidomic approach carried out in this study, established a novel panel of potential biomarkers that present a promising predictive accuracy rate in fertilization outcome, and indicates FF as an interesting biomarker resource to improve IVF clinic routine.