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.

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.

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.

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.

Haplotyping-based preimplantation genetic testing reveals parent-of-origin specific mechanisms of aneuploidy formation.

Chromosome instability is inherent to human IVF embryos, but the full spectrum and developmental fate of chromosome anomalies remain uncharacterized. Using haplotyping-based preimplantation genetic testing for monogenic diseases (PGT-M), we mapped the parental and mechanistic origin of common and rare genomic abnormalities in 2300 cleavage stage and 361 trophectoderm biopsies. We show that while single whole chromosome aneuploidy arises due to chromosome-specific meiotic errors in the oocyte, segmental imbalances predominantly affect paternal chromosomes, implicating sperm DNA damage in segmental aneuploidy formation. We also show that postzygotic aneuploidy affects multiple chromosomes across the genome and does not discriminate between parental homologs. In addition, 6% of cleavage stage embryos demonstrated signatures of tripolar cell division with excessive chromosome loss, however hypodiploid blastomeres can be excluded from further embryo development. This observation supports the selective-pressure hypothesis in embryos. Finally, considering that ploidy violations may constitute a significant proportion of non-viable embryos, using haplotyping-based approach to map these events might further improve IVF success rate.

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.

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.

Effect of day 3 embryo morphometrics and morphokinetics on survival and implantation after slow freezing-thawing and after vitrification-warming: a retrospective cohort study.

BACKGROUND: Morphometric and morphokinetic evaluation of in vitro cultured human embryos allows evaluation without time restriction and reduces intra- and inter-observer variability. Even though these technologies have been reported to improve the quality of cleavage stage embryo evaluation during fresh culture, possible advantages in the evaluation of cryopreserved embryos have been scarcely explored. This study aims to compare morphometric and morphokinetic parameters between slow frozen and vitrified embryos and to determine their relationship to embryo survival and implantation rate (IR) after thawing/warming. METHODS: During fresh culture, morphometric characteristics (Total Cell Volume (TCV), symmetry, fragmentation and number of blastomeres) were measured in 286 thawed/warmed embryos. Likewise, after thawing/warming, similar morphometric characteristics were measured in 135 survived embryos. Moreover, morphokinetic parameters (time to mitosis resumption and time to compaction) were measured in 90 embryos after thawing/warming. Then, using linear regression, we investigated the differences between vitrified and slow frozen embryos and the relation of the measured characteristics to embryo survival and IR. Statistical corrections were applied to account for data clustering and for multiple testing. RESULTS: Vitrified embryos resume mitosis and start compaction significantly earlier than slow frozen embryos. Mitosis resumption rate was 82% for vitrified and 63% for slow frozen embryos and median time to mitosis resumption was 7.6 h and 13.1 h (p = 0.02), respectively. Compaction rate was 62% in vitrified and only 23% in slow frozen embryos. Median time to compaction was 18.1 h for vitrified embryos but, for slow frozen could not be computed since less than half of the slow frozen embryos reached compaction (p = 0.0001). Moreover, intact embryos resume mitosis significantly earlier than not intact ones regardless of the freezing method (rate: 79% vs. 66%, median time: 7.6 h vs 14.6 h, respectively, p = 0.03). Regarding morphometrics, slow frozen embryos showed lower TCV and higher blastomere symmetry after thawing than vitrified embryos despite having similar blastomere number. IR was related to blastomere number at cryopreservation in slow frozen embryos, but not in vitrified ones. CONCLUSIONS: Interestingly, vitrified/warmed embryos undergo mitosis resumption and compaction significantly earlier than slow frozen/thawed embryos. However, the clinical use of this morphokinetic parameters still remains to be investigated in larger studies. TRIAL REGISTRATION: Retrospectively registered on December 15, 2015 NCT02639715 .

Human embryo research in Belgium: an overview.

OBJECTIVE: To present an overview of the numbers and types of human embryos used in research projects in Belgium from 2007 to 2015. DESIGN: Analysis of all research proposals approved by the Federal Commission for Medical and Scientific Research on Embryos In Vitro. SETTING: Not applicable. PATIENT(S): Not applicable. MAIN OUTCOME MEASURE(S): Number of embryos used for research, number of embryos created for research, and areas of embryo research. RESULT(S): Since 2007, 15,811 embryos were used for 36 research projects. In total, 10,492 (66%) fresh supernumerary embryos (unfit for transfer or freezing) were used, 4,083 (26%) frozen supernumerary embryos (donated by parents whose child wish was completed or abandoned), and 1,236 (8%) embryos created for research. Most projects focused on research into embryo development. Fresh supernumerary embryos were mainly used for human embryonic stem cell (hESC) research. Frozen supernumerary embryos were almost exclusively used for research into embryo development and for hESC research. Embryos created for research were used for research into embryo development, oocyte research, research into cryopreservation of oocytes, and hESC research. CONCLUSION(S): Having concrete data on embryo research is crucial for an informed debate. Moreover, these data are necessary to find out trends in research such as the numbers of embryos needed and the areas of research. Data collection requires a sufficiently clear definition of "research" and "embryo." These conceptual questions frequently reveal lack of clarity in legislation.

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%.

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.

Effect of embryo morphology and morphometrics on implantation of vitrified day 3 embryos after warming: a retrospective cohort study.

BACKGROUND: Characteristics routinely used to evaluate embryo quality after thawing include number of blastomeres survived and presence of mitosis resumption after overnight culture. It is unknown to which extent symmetry and fragmentation affect implantation after warming and whether application of stricter criteria either before vitrification or after warming would improve implantation rate (IR) of vitrified/warmed embryos. This study aimed to find new parameters to improve selection criteria for vitrification and for transfer after warming. METHODS: Firstly, we evaluated standard morphological characteristics (intact survival, mitosis resumption, number of blastomeres, symmetry and fragmentation) of 986 warmed day 3 embryos and, from a subset of 654, we evaluated morphometric characteristics (fragmentation, symmetry and volume change). Secondly, we tested the hypothesis that IR of day 3 vitrified/warmed embryos is influenced by morphometric characteristics. IR per embryo transferred was calculated using embryos that were transferred in a single embryo transfer (SET) or a double embryo transfer (DET) with either 0 or 100 % implantation (830/986). We investigated the significant differences in IR between the different categories of a specific characteristic. These categories were based on our standard embryo evaluation system. The statistical tests Chi-square, Fisher's exact or Cochrane-Armitage were used according to the type and/or categories of the variable. RESULTS: The 986 embryos were transferred in 671 FET cycles with 16.9 % (167/986) IR. After exclusion of DET with 1 embryo implanted, IR per embryo transferred was 12.4 % (103/830). Embryo symmetry, fragmentation and volume change in vitrified/warmed day 3 embryos were not associated with IR. However, when mitosis resumption was present after overnight culture, intact embryos reached significantly higher IR than non-intact embryos and only when the embryo compacted after overnight culture the number of cells damaged after warming had no effect on IR. Concretely, embryos with 8 cells after warming or >9 cells after overnight culture-including compacted embryos-reached the highest IR (>15 %) while embryos with <6 cells after warming or with ≤6 cells after overnight culture had extremely low IR (<1 %). CONCLUSIONS: IR of vitrified embryos is determined by the number of cells lost, by the occurrence of mitosis resumption, and by the specific number of blastomeres present but not by fragmentation, blastomere symmetry or volume change. Unselecting embryos for cryopreservation because of fragmentation >10 % and/or symmetry < 75 % only leads to unwanted loss of embryos with acceptable implantation potential. TRIAL REGISTRATION: Retrospectively registered NCT02639715 .

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.

Revised guidelines for good practice in IVF laboratories (2015).

STUDY QUESTION: Which recommendations can be provided by the European Society of Human Reproduction and Embryology Special Interest Group (ESHRE SIG) Embryology to support laboratory specialists in the organization and management of IVF laboratories and the optimization of IVF patient care? SUMMARY ANSWER: Structured in 13 sections, the guideline development group formulated recommendations for good practice in the organization and management of IVF laboratories, and for good practice of the specific procedures performed within the IVF laboratory. WHAT IS KNOWN ALREADY: NA. STUDY DESIGN, SIZE, DURATION: The guideline was produced by a group of 10 embryologists representing different European countries, settings and levels of expertise. The group evaluated the document of 2008, and based on this assessment, each group member rewrote one or more sections. Two 2-day meetings were organized during which each of the recommendations was discussed and rewritten until consensus within the guideline group was reached. After finalizing the draft, the members of the ESHRE SIG embryology were invited to review the guideline. PARTICIPANTS/MATERIALS, SETTING, METHODS: NA. MAIN RESULTS AND THE ROLE OF CHANCE: The guideline provides recommendations on the general organization of an IVF laboratory (staffing and direction, quality management, laboratory safety), and on the specific aspects of the procedures performed in IVF laboratories (Identification of patients and traceability of their reproductive cells, consumables, handling of biological material, oocyte retrieval, sperm preparation, insemination of oocytes, scoring for fertilization, embryo culture and transfer, and cryopreservation). A last section provides recommendations regarding an Emergency plan for IVF laboratories. LIMITATIONS, REASONS FOR CAUTION: Evidence on most of the issues described is scarce, and therefore it was decided not to perform a formal search for and assessment of scientific evidence. However, recommendations published in the EUTCD and relevant and recent documents, manuals and consensus papers were taken into account when formulating the recommendations. WIDER IMPLICATIONS OF THE FINDINGS: Despite the limitations, the guideline group is confident that this document will be helpful to directors and managers involved in the management and organization of IVF laboratories, but also to embryologists and laboratory technicians performing daily tasks. STUDY FUNDING/COMPETING INTERESTS: The guideline was developed and funded by ESHRE, covering expenses associated with the guideline meetings. The guideline group members did not receive payment. Dr Coticchio reports speaker's fees from IBSA and Cook, outside the submitted work; Dr Lundin reports grants from Vitrolife, personal fees from Merck Serono, non-financial support from Unisense, outside the submitted work; Dr. Rienzi reports personal fees from Merck Serono, personal fees from MSD, grants from GFI, outside the submitted work; the other authors had nothing to disclose. TRIAL REGISTRATION NUMBER: NA.

Is There a Correlation between the Number of Follicular Flushings, Oocyte/Embryo Quality and Pregnancy Rate in Assisted Reproductive Technology Cycles? Results from a Prospective Study.

OBJECTIVE: To determine the mean number of follicular flushings needed to retrieve the maximal number of oocytes and to investigate the correlation between the number of flushings and oocyte/embryo quality as well as reproductive outcome. METHODS: This prospective study included 154 oocyte retrieval cycles in 138 patients undergoing assisted reproductive technology treatment. During oocyte retrieval, aspirate and flushes were collected in four separate collections (A, F1, F2, F3) and inspected for the presence of an oocyte-cumulus complex (OCC). Outcome variables included oocyte recovery rate, percentage of mature oocytes, fertilization rate, day 2/day 3/day 5 embryo quality and clinical pregnancy rate/live birth rate. RESULTS: Out of 1,495 OCCs collected, 91% were obtained in collections A or F1. Significantly more mature oocytes were obtained in collection A (p = 0.03). The fertilization rate was similar for oocytes obtained in the four separate collections (p = 0.50). The proportion of good-quality day 2/day 3/day 5 embryos was similar (p = 0.93, p = 0.85 and p = 0.14, respectively). Clinical pregnancy rate and live birth rate were not significantly affected by the first two flushes. No live birth emanated from oocytes obtained from the third flush onwards. CONCLUSION: More than 90% of all recovered OCCs were obtained after follicular aspiration followed by follicular flushing up to two times (collections A and F1). Follicular flushing may increase the oocyte recovery rate and does not have a negative influence on fertilization rate, day 2/day 3/day 5 embryo quality or pregnancy rate.

Frozen-thawed embryo transfer in a natural or mildly hormonally stimulated cycle in women with regular ovulatory cycles: a RCT.

STUDY QUESTION: Can ovarian stimulation with low dose hMG improve the implantation rate (IR) per frozen-thawed embryo transferred (FET) when compared with natural cycle in an FET programme in women with a regular ovulatory cycle? SUMMARY ANSWER: Both IR and live birth rate (LBR) per FET were similar in the group with mild ovarian stimulation and the natural cycle group. WHAT IS KNOWN ALREADY: Different cycle regimens for endometrial preparation are used prior to FET: spontaneous ovulatory cycles, cycles with artificial endometrial preparation using estrogen and progesterone hormones, and cycles stimulated with gonadotrophins or clomiphene citrate. At present, it is not clear which regimen results in the highest IR or LBR. More specifically, there are no RCTs in ovulatory women comparing reproductive outcome after FET during a natural cycle and during a hormonally stimulated cycle. STUDY DESIGN, SIZE, DURATION: A total of 410 women scheduled for FET during 579 cycles (December 2003-September 2013) were enrolled in an open-label RCT to natural cycle (NC FET group, n = 291) or to a cycle hormonally stimulated with s.c. gonadotrophins (hMG FET group, 37.5-75 IU per day, n = 288). A total of 672 embryos were transferred during 434 cycles (332 embryos and 213 cycles in the NC FET group; 340 embryos and 221 cycles in the hMG FET group). Assuming a = 0.05 and 80% power, it was calculated that 219 frozen-thawed embryos were required for transfer in each group to demonstrate a difference of 10% in IR. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women were eligible according to the following inclusion criteria: regular ovulatory cycle, female age ≥21 years and ≤45 years, informed consent. FET cycles with preimplantation genetic screening were excluded. The primary outcome was IR per embryo transferred. Secondary outcomes included IR with fetal heart beat (FHB), LBR per embryo transferred and endometrial thickness on the day of hCG administration. Statistical analysis was by intention to treat and controlled for the presence of multiple measures, as eligible women could be randomized in more than one cycle. Chi-square and independent t-test were used to compare categorical and continuous variables. The relative risk (RR) was estimated using a Poisson model with log link. Hierarchical models with random intercepts for patient and cycle were considered to account for clustering of cycles within patients and of embryos within cycles. MAIN RESULTS AND THE ROLE OF CHANCE: The primary outcome, IR per embryo transferred, was not statistically different between the NC FET group (41/332 (12.35%)) and in the hMG FET group (55/340 (16.18%)) (RR 1.3 (95% confidence interval (CI) 0.9-2.0), P = 0.19). Similarly, the secondary outcome, IR with FHB per embryo transferred, was 34/332 (10.24%) in the NC FET group and 48/340 (14.12%) in the hMG FET group (RR 1.4 (95% CI 0.9-2.1), P = 0.15). The LBR per embryo transferred was 32/332 (9.64%) in the NC FET group and 45/340 (13.24%) in the hMG FET group (RR 1.4 (95% CI 0.9-2.2), P = 0.17). Endometrial thickness was also similar in both groups [8.9 (95% CI 8.7-9.1) in the NC FET group and 8.9 (95% CI 8.7-9.1) in the hMG FET group]. The duration of the follicular phase was significantly shorter (P < 0.001) in the hMG FET group [13.7 days (95% CI 13.2-14.2)] than in the NC FET group [15.4 days (95% CI 14.8-15.9)]. LIMITATIONS, REASONS FOR CAUTION: Randomization of cycles instead of patients; open-label design; relatively long period of recruitment. WIDER IMPLICATIONS OF THE FINDINGS: Our observation that the IR per embryo transferred is not significantly increased after FET during natural or gonadotrophin stimulated cycle, suggests that the effect of mild hormonal stimulation with gonadotrophins is smaller than what was considered clinically relevant with respect to reproductive outcome after FET. These data suggest that endometrial receptivity is not relevantly improved, but also not impaired after hormonal stimulation with gonadotrophins. Since FET during a natural cycle is cheaper and more patient-friendly, we recommend this regimen as the treatment of choice for women with regular cycles undergoing FET. TRIAL REGISTRATION NUMBER: clinicaltrials.gov NCT00492934. TRIAL REGISTRATION DATE: 26 June 2007. DATE OF FIRST PATIENT'S ENROLMENT: 1 December 2003.

Concurrent whole-genome haplotyping and copy-number profiling of single cells.

Methods for haplotyping and DNA copy-number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required, but it substantially distorts the frequency and composition of the cell's alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP genotypes (AA, AB, BB) and DNA copy-number profiling remains difficult because true DNA copy-number aberrations have to be discriminated from WGA artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by employing phased parental genotypes and deciphering WGA-distorted SNP B-allele fractions via a process we coin haplarithmisis. We demonstrate that the method can be applied as a generic method for preimplantation genetic diagnosis on single cells biopsied from human embryos, enabling diagnosis of disease alleles genome wide as well as numerical and structural chromosomal anomalies. Moreover, meiotic segregation errors can be distinguished from mitotic ones.

Low-dose human menopausal gonadotrophin versus clomiphene citrate in subfertile couples treated with intrauterine insemination: a randomized controlled trial.

STUDY QUESTION: Can controlled ovarian stimulation with low-dose human menopausal gonadotrophin (hMG) improve the clinical pregnancy rate when compared with ovarian stimulation with clomiphene citrate (CC) in an intrauterine insemination (IUI) programme for subfertile couples? SUMMARY ANSWER: Ovarian stimulation with low-dose hMG is superior to CC in IUI cycles with respect to clinical pregnancy rate. WHAT IS KNOWN ALREADY: IUI after ovarian stimulation is an effective treatment for mild male subfertility, unexplained subfertility and minimal-mild endometriosis, but it is unclear which medication for ovarian stimulation is more effective. STUDY DESIGN, SIZE, DURATION: A total of 330 women scheduled for IUI during 657 cycles (September 2004-December 2011) were enrolled in an open-label randomized clinical trial to ovarian stimulation with low-dose hMG subcutaneous (n = 334, 37.5-75 IU per day) or CC per oral (n = 323, 50 mg/day from Day 3-7). Assuming a difference of 10% in 'clinical pregnancy with positive fetal heart beat', we needed 219 cycles per group (alpha-error 0.05, power 0.80). PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied subfertile couples with mild male subfertility, unexplained subfertility or minimal-mild endometriosis. Further inclusion criteria were failure to conceive for ≥12 months, female age ≤42 years, at least one patent Fallopian tube and a total motility count (TMC) ≥5.0 million spermatozoa after capacitation. The primary end-point was clinical pregnancy. Analysis was by intention to treat and controlled for the presence of multiple measures, as one couple could have more randomizations in multiple cycles. Linear mixed models were used for continuous measures. For binary outcomes we estimated the relative risk using a Poisson model with log link and using generalized estimating equations. MAIN RESULTS AND THE ROLE OF CHANCE: When compared with ovarian stimulation with CC, hMG stimulation was characterized by a higher clinical pregnancy rate (hMG 48/334 (14.4%) versus CC 29/323 (9.0%), relative risk (RR) 1.6 (95% confidence interval (CI) 1.1-2.4)), higher live birth rate (hMG 46/334 (13.8%) versus CC 28/323 (8.7%), RR 1.6 (95% CI 1.0-2.4)), low and comparable multiple live birth rate (hMG 3/46 (6.5%) versus CC 1/28 (3.6%), P > 0.99), lower number of preovulatory follicles (hMG 1.2 versus CC 1.5, P < 0.001), increased endometrial thickness (hMG 8.5 mm versus CC 7.5 mm, P < 0.001), and a lower cancellation rate per started cycle (hMG 15/322 (4.7%) versus CC 46/298 (15.4%), P < 0.001). LIMITATIONS, REASONS FOR CAUTION: We randomized patients at a cycle level, and not at a strategy over multiple cycles. WIDER IMPLICATIONS OF THE FINDINGS: This study showed better reproductive outcome after ovarian stimulation with low-dose gonadotrophins. A health economic analysis of our data is planned to test the hypothesis that ovarian stimulation with low-dose hMG combined with IUI is associated with increased cost-effectiveness when compared with ovarian stimulation with CC. STUDY FUNDING/ COMPETING INTERESTS: T.M.D. and K.P. were supported by the Clinical Research Foundation of UZ Leuven, Belgium. This study was also supported by the Ferring company (Copenhagen, Denmark) which provide free medication (Menopur) required for the group of patients who were randomized in the hMG COS group. The Ferring company was not involved in the study design, data analysis, writing and submission of the paper. TRIAL REGISTRATION NUMBER: NCT01569945 (ClinicalTrials.gov).