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Study Question: What is the role of transcriptional-enhanced associate (TEA) domain family member 4 (TEAD4) in trophectoderm (TE) differentiation during human embryo preimplantation development in comparison to mouse?Summary Answer: TEAD4 regulates TE lineage differentiation in the human preimplantation embryo acting upstream of caudal-type homeobox protein 2 (CDX2), but in contrast to the mouse in a GATA-binding protein 3 (GATA3)-independent manner.What Is Known Already: Tead4 is one of the earliest transcription factors expressed during mouse embryo preimplantation development and is required for the expression of TE-associated genes. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination, have shown that Tead4-targeted embryos have compromised development and expression of the TE-specific Cdx2 and Gata3 is downregulated. Cdx2 and Gata3 act in parallel pathways downstream of Tead4 to induce successful TE differentiation. Downstream loss of Cdx2 expression, compromises TE differentiation and subsequent blastocoel formation and leads to the ectopic expression of inner cell mass (ICM) genes, including POU Class 5 homeobox 1 (Pou5f1) and SRY-box transcription factor (Sox2). Cdx2 is a more potent regulator of TE fate in mouse as loss of Cdx2 expression induces more severe phenotypes compared with loss of Gata3 expression. The role of TEAD4 and its downstream effectors during human preimplantation embryo development has not been investigated yet.Study Design, Size, Duration: The clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (CRISPR-Cas9) system was first introduced in pronuclei (PN)-stage mouse zygotes aiming to identify a guide RNA (gRNA), yielding high editing efficiency and effective disruption of the Tead4 locus. Three guides were tested (gRNA1-3), each time targeting a distinct region of Exon 2 of Tead4. The effects of targeting on developmental capacity were studied in Tead4-targeted embryos (n = 164-summarized data from gRNA1-3) and were compared with two control groups; sham-injected embryos (n = 26) and non-injected media-control embryos (n = 51). The editing efficiency was determined by next-generation sequencing (NGS). In total, n = 55 (summarized data from gRNA1-3) targeted mouse embryos were analysed by NGS. Immunofluorescence analysis to confirm successful targeting by gRNA1 was performed in Tead4-targeted embryos, and non-injected media-control embryos. The downregulation of secondary TE-associated markers Cdx2 and Gata3 was used as an indirect confirmation of successful Tead4-targeting (previously shown to be expressed downstream of Tead4). Additional groups of gRNA1 Tead4-targeted (n = 45) and media control (n = 36) embryos were cultured for an extended period of 8.5 days, to further assess the developmental capacity of the Tead4-targeted group to develop beyond implantation stages. Following the mouse investigation, human metaphase-II (MII) oocytes obtained by IVM were microinjected with gRNA-Cas9 during ICSI (n = 74) to target TEAD4 or used as media-control (n = 33). The editing efficiency was successfully assessed in n = 25 TEAD4-targeted human embryos. Finally, immunofluorescence analysis for TEAD4, CDX2, GATA3 and the ICM marker SOX2 was performed in TEAD4-targeted (n = 10) and non-injected media-control embryos (n = 29).Participants/materials, Setting, Methods: A ribonucleoprotein complex consisting of a gRNA-Cas9 mixture, designed to target Exon 2 of Tead4/TEAD4, was microinjected in mouse PN stage zygotes or human IVM MII oocytes along with sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. In mouse, an additional group of Tead4-targeted and media-control embryos was cultured in vitro for an extended period of 8.5 days. Embryonic development and morphology were assessed daily, during culture in vitro of mouse and human embryos and was followed by a detailed scoring at late blastocyst stage. Targeting efficiency following gRNA-Cas9 introduction was assessed via immunostaining and NGS analysis.Main Results and the Role Of Chance: NGS analysis of the Tead4-targeted locus revealed very high editing efficiencies for all three guides, with 100% of the mouse embryos (55 out of 55) carrying genetic modifications resulting from CRISPR-Cas9 genome editing. More specifically, 65.22% (15 out 23) of the PN zygotes microinjected with gRNA1-Cas9, which exhibited the highest efficiency, carried exclusively mutated alleles. The developmental capacity of targeted embryos was significantly reduced (data from gRNA1), as 44.17% of the embryos arrested at the morula stage (2.5 days post coitum), coincident with the initiation of TE lineage differentiation, compared with 8.51% in control and 12.50% in sham control groups. High-quality blastocyst formation rates (Grade 3) were 8.97% in the gRNA1-targeted group, compared with 87.23% in the media-control and 87.50% in the sham group. Immunofluorescence analysis in targeted embryos confirmed downregulation of Tead4, Cdx2, and Gata3 expression, which resulted from successful targeting of the Tead4 locus. Tead4-targeted mouse embryos stained positive for the ICM markers Pou5f1 and Sox2, indicating that expression of ICM lineage markers is not affected. Tead4-targeted embryos were able to cavitate and form a blastocoel without being able to hatch. Extended embryo culture following zona pellucida removal, revealed that the targeted embryos can attach and form egg-cylinder-like structures in the absence of trophoblast giant cells. In human embryos, Exon 2 of TEAD4 was successfully targeted by CRISPR-Cas9 (n = 74). In total, 25 embryos from various developmental stages were analysed by NGS and 96.00% (24 out of 25) of the embryos carried genetic modifications because of gRNA-Cas9 editing. In the subgroup of the 24 edited embryos, 17 (70.83%) carried only mutant alleles and 11 out of these 17 (64.70%) carried exclusively frameshift mutations. Six out of 11 embryos reached the blastocyst stage. In contrast to mice, human-targeted embryos formed blastocysts at a rate (25.00%) that did not differ significantly from the control group (23.81%). However, blastocyst morphology and TE quality were significantly compromised following TEAD4-targeting, showing grade C TE scores, with TE containing very few cells. Immunofluorescence analysis of TEAD4-targeted embryos (n = 10) confirmed successful editing by the complete absence of TEAD4 and its downstream TE marker CDX2, but the embryos generated retained expression of GATA3, which is in contrast to what we have observed and has previously been reported in mouse. In this regard, our results indicate that GATA3 acts in parallel with TEAD4/CDX2 towards TE differentiation in human.Large Scale Data: N/A.Limitations, Reasons For Caution: CRISPR-Cas9 germline genome editing, in some cases, induces mosaic genotypes. These genotypes are a result of inefficient and delayed editing, and complicate the phenotypic analysis and developmental assessment of the injected embryos. We cannot exclude the possibility that the observed differences between mouse and human are the result of variable effects triggered by the culture conditions, which were however similar for both mouse and human embryos in this study. Furthermore, this study utilized human oocytes obtained by IVM, which may not fully recapitulate the developmental behaviour of in vivo matured oocytes.Wider Implications Of the Findings: Elucidation of the evolutionary conservation of molecular mechanisms that regulate the differentiation and formation of the trophoblast lineage can give us fundamental insights into early implantation failure, which accounts for ∼15% of human conceptions.Study Funding/competing Interest(s): The research was funded by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01) and Ghent University (BOF.BAS.2018.0018.01). G.C. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 11L8822N). A.B. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 1298722 N). We further thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Background: Primary health care providers (PHCPs) are assumed to be at high risk of a COVID-19 infection, as they are exposed to patients with usually less personal protective equipment (PPE) than other frontline health care workers (HCWs). Nevertheless, current research efforts focussed on the assessment of COVID-19 seroprevalence rates in the general population or hospital HCWs.Objective: We aimed to determine the seroprevalence in PHCPs during the second SARS-CoV-2 wave in Flanders (Belgium) and compared it to the seroprevalence in the general population. We also assessed risk factors, availability of PPE and attitudes towards the government guidelines over time.Methods: A prospective cohort of PHCPs (n = 698), mainly general practitioners, was asked to complete a questionnaire and self-sample capillary blood by finger-pricking at five distinct points in time (June-December 2020). We analysed the dried blood spots for IgG antibodies using a Luminex multiplex immunoassay.Results: The seroprevalence of PHCPs remained stable between June and September (4.6-5.0%), increased significantly from October to December (8.1-13.4%) and was significantly higher than the seroprevalence of the general population. The majority of PHCPs were concerned about becoming infected, had adequate PPE and showed increasing confidence in government guidelines.Conclusions: The marked increase in seroprevalence during the second COVID-19 wave shows that PHCPs were more at risk during the second wave compared to the first wave in Flanders. This increase was only slightly higher in PHCPs than in the general population suggesting that the occupational health measures implemented provided sufficient protection when managing patients. [ABSTRACT FROM AUTHOR]

Study Question: What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model?Summary Answer: POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation.What Is Known Already: Clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage.Study Design, Size, Duration: The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain-B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos.Participants/materials, Setting, Methods: A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing.Main Results and the Role Of Chance: Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts.Limitations, Reasons For Caution: One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes.Wider Implications Of the Findings: Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis.Study Funding/competing Interest(s): The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Can pronuclear transfer (PNT) or maternal spindle transfer (ST) be applied to overcome poor embryo development associated with advanced maternal age or early embryo arrest in a mouse model?Summary Answer: Both PNT and ST may have the potential to restore embryonic developmental potential in a mouse model of reproductive ageing and embryonic developmental arrest.What Is Known Already: Germline nuclear transfer (NT) techniques, such as PNT and ST, are currently being applied in humans to prevent the transmission of mitochondrial diseases. Yet, there is also growing interest in the translational use of NT for treating infertility and improving IVF outcomes. Nevertheless, direct scientific evidence to support such applications is currently lacking. Moreover, it remains unclear which infertility indications may benefit from these novel assisted reproductive technologies.Study Design, Size, Duration: We applied two mouse models to investigate the potential of germline NT for overcoming infertility. Firstly, we used a model of female reproductive ageing (B6D2F1 mice, n = 155), with ages ranging from 6 to 8 weeks (young), 56 (aged) to 70 weeks (very-aged), corresponding to a maternal age of <30, ∼36 and ∼45 years in humans, respectively. Secondly, we used NZB/OlaHsd female mice (7-14 weeks, n = 107), as a model of early embryo arrest. This mouse strain exhibits a high degree of two-cell block. Metaphase II (MII) oocytes and zygotes were retrieved following superovulation.Participants/materials, Setting, Methods: Ovarian reserve was assessed by histological analysis in the reproductive-aged mice. Mitochondrial membrane potential (△Ψm) was measured by JC-1 staining in MII oocytes, while spindle-chromosomal morphology was examined by confocal microscopy. Reciprocal ST and PNT were performed by transferring the meiotic spindle or pronuclei (PN) from unfertilised or fertilised oocytes (after ICSI) to enucleated oocytes or zygotes between aged or very-aged and young mice. Similarly, NT was also conducted between NZB/OlaHsd (embryo arrest) and B6D2F1 (non-arrest control) mice. Finally, the effect of cytoplasmic transfer (CT) was examined by injecting a small volume (∼5%) of cytoplasm from the oocytes/zygotes of young (B6D2F1) mice to the oocytes/zygotes of aged or very-aged mice or embryo-arrest mice. Overall, embryonic developmental rates of the reconstituted PNT (n = 572), ST (n = 633) and CT (n = 336) embryos were assessed to evaluate the efficiency of these techniques. Finally, chromosomal profiles of individual NT-generated blastocysts were evaluated using next generation sequencing.Main Results and the Role Of Chance: Compared to young mice, the ovarian reserve in aged and very-aged mice was severely diminished, reflected by a lower number of ovarian follicles and a reduced number of ovulated oocytes (P < 0.001). Furthermore, we reveal that the average △Ψm in both aged and very-aged mouse oocytes was significantly reduced compared to young mouse oocytes (P < 0.001). In contrast, the average △Ψm in ST-reconstructed oocytes (very-aged spindle and young cytoplast) was improved in comparison to very-aged mouse oocytes (P < 0.001). In addition, MII oocytes from aged and very-aged mice exhibited a higher rate of abnormalities in spindle assembly (P < 0.05), and significantly lower fertilisation (60.7% and 45.3%) and blastocyst formation rates (51.4% and 38.5%) following ICSI compared to young mouse oocytes (89.7% and 87.3%) (P < 0.001). Remarkably, PNT from zygotes obtained from aged or very-aged mice to young counterparts significantly improved blastocyst formation rates (74.6% and 69.2%, respectively) (P < 0.05). Similarly, both fertilisation and blastocyst rates were significantly increased after ST between aged and young mice followed by ICSI (P < 0.05). However, we observed no improvement in embryo development rates when performing ST from very-aged to young mouse oocytes following ICSI (P > 0.05). In the second series of experiments, we primarily confirmed that the majority (61.8%) of in vivo zygotes obtained from NZB/OlaHsd mice displayed two-cell block during in vitro culture, coinciding with a significantly reduced blastocyst formation rate compared to the B6D2F1 mice (13.5% vs. 90.7%; P < 0.001). Notably, following the transfer of PN from the embryo-arrest (NZB/OlaHsd) zygotes to enucleated non-arrest (B6D2F1) counterparts, most reconstructed zygotes developed beyond the two-cell stage, leading to a significantly increased blastocyst formation rate (89.7%) (P < 0.001). Similar findings were obtained after implementing ST between NZB/OlaHsd and B6D2F1 mice, followed by ICSI. Conversely, the use of CT did not improve embryo development in reproductive-age mice nor in the embryo-arrest mouse model (P > 0.05). Surprisingly, chromosomal analysis revealed that euploidy rates in PNT and ST blastocysts generated following the transfer of very-aged PN to young cytoplasts and very-aged spindles to young cytoplasts were comparable to ICSI controls (with young mouse oocytes). A high euploidy rate was also observed in the blastocysts obtained from either PNT or ST between young mice. Conversely, the transfer of young PN and young spindles into very-aged cytoplasts led to a higher rate of chromosomal abnormalities in both PNT and ST blastocysts.Large Scale Data: N/A.Limitations, Reasons For Caution: The limited number of blastocysts analysed warrants careful interpretation. Furthermore, our observations should be cautiously extrapolated to humans given the inherent differences between mice and women in regards to various biological processes, including centrosome inheritance. The findings suggest that ST or PNT procedures may be able to avoid aneuploidies generated during embryo development, but they are not likely to correct aneuploidies already present in some aged MII oocytes.Wider Implications Of the Findings: To our knowledge, this is the first study to evaluate the potential of PNT and ST in the context of advanced maternal age and embryonic developmental arrest in a mouse model. Our data suggest that PNT, and to a lesser extent ST, may represent a novel reproductive strategy to restore embryo development for these indications.Study Funding/competing Interest(s): M.T. is supported by grants from the China Scholarship Council (CSC) (Grant no. 201506160059) and the Special Research Fund from Ghent University (Bijzonder Onderzoeksfonds, BOF) (Grant no. 01SC2916 and no. 01SC9518). This research is also supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051017N, G051516N and G1507816N). The authors declare no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

This study examines the effectiveness of integrating mindfulness-based techniques within therapy for women suffering to achieve orgasm. Although widely applied in psychotherapy, this approach has only recently been introduced in the treatment of female sexual dysfunction. To compare the effectiveness of a video-based self-administered treatment, rooted within the cognitive behavioral treatment (CBT) framework, with a video-based self-administered mindfulness treatment applying cognitive behavioral sexual therapy (mindfulness-based cognitive therapy), the latter of which was specifically created to increase women's ability to achieve orgasm. A convenience sample of 65 women suffering from difficulties to achieve orgasm, aged 18 to 58 years (mean = 32.66, standard deviation = 9.48), were randomly allocated using a randomization procedure to either a mindfulness-based cognitive therapy (N = 35) or CBT (N = 30) group. Each participant completed questionnaires before and after the start of treatment and 2 months after its completion. We applied repeated-measure general linear models to compare the 2 groups (ie, between participant factor) on each dependent variable across time (ie, the within-participant factor). Compare mean analyses for paired samples were only conducted when the interaction effect between condition and time was significant (ie, P <.05). Statistical analyses show that women in both groups presented increased sexual functioning (P =.001) and decreased sexual distress (P <.001), as well as improved desire, arousal, orgasm, and sexual satisfaction (P <.05) after their respective treatments. Contrary to our hypothesis, significant reductions in sexual pain were only observed in CBT participants. To the best of our knowledge, this is the first study to apply a randomized allocation procedure to evaluate the effectiveness of a video-based mindfulness intervention for women struggling to achieve orgasm. These results should guide clinicians' decisions with respect to evaluating the relevance and the real added value of proposing mindfulness exercises to their patients with such difficulties. When women suffering from difficulties to achieve orgasm are randomly assigned to a mindfulness group or an active control, improvements in sexual functioning and reductions in sexual distress can be observed after both treatments. Adam F, De Sutter P, Day J, et al. A Randomized Study Comparing Video-Based Mindfulness-Based Cognitive Therapy With Video-Based Traditional Cognitive Behavioral Treatment in a Sample of Women Struggling to Achieve Orgasm. J Sex Med 2020;17:312–324. [ABSTRACT FROM AUTHOR]

Study Question: What is the accuracy of preimplantation genetic testing for aneuploidies (PGT-A) when considering human peri-implantation outcomes in vitro?Study Answer: The probability of accurately diagnosing an embryo as abnormal was 100%, while the proportion of euploid embryos classified as clinically suitable was 61.9%, yet if structural and mosaic abnormalities were not considered accuracy increased to 100%, with a 0% false positive and false negative rate.What Is Already Known: Embryo aneuploidy is associated with implantation failure and early pregnancy loss. However, a proportion of blastocysts are mosaic, containing chromosomally distinct cell populations. Diagnosing chromosomal mosaicism remains a significant challenge for PGT-A. Although mosaic embryos may lead to healthy live births, they are also associated with poorer clinical outcomes. Moreover, the direct effects of mosaicism on early pregnancy remain unknown. Recently, developed in vitro systems allow extended embryo culture for up to 14 days providing a unique opportunity for modelling chromosomal instability during human peri-implantation development.Study Design, Size, Duration: A total of 80 embryos were cultured to either 8 (n = 7) or 12 days post-fertilisation (dpf; n = 73). Of these, 54 were PGT-A blastocysts, donated to research following an abnormal (n = 37) or mosaic (n = 17) diagnosis. The remaining 26 were supernumerary blastocysts, obtained from standard assisted reproductive technology (ART) cycles. These embryos underwent trophectoderm (TE) biopsy prior to extended culture.Participants/materials, Setting, Methods: We applied established culture protocols to generate embryo outgrowths. Outgrowth viability was assessed based on careful morphological evaluation. Nine outgrowths were further separated into two or more portions corresponding to inner cell mass (ICM) and TE-derived lineages. A total of 45 embryos were selected for next generation sequencing (NGS) at 8 or 12 dpf. We correlated TE biopsy profiles to both culture outcomes and the chromosomal status of the embryos during later development.Main Results and the Role Of Chance: Of the 73 embryos cultured to 12 dpf, 51% remained viable, while 49% detached between 8 and 12 dpf. Viable, Day 12 outgrowths were predominately generated from euploid blastocysts and those diagnosed with trisomies, duplications or mosaic aberrations. Conversely, monosomies, deletions and more complex chromosomal constitutions significantly impaired in vitro development to 12 dpf (10% vs. 77%, P < 0.0001). When compared to the original biopsy, we determined 100% concordance for uniform numerical aneuploidies, both in whole outgrowths and in the ICM and TE-derived outgrowth portions. However, uniform structural variants were not always confirmed later in development. Moreover, a high proportion of embryos originally diagnosed as mosaic remained viable at 12 dpf (58%). Of these, 71% were euploid, with normal profiles observed in both ICM and TE-derived lineages. Based on our validation data, we determine a 0% false negative and 18.5% false positive error rate when diagnosing mosaicism. Overall, our findings demonstrate a diagnostic accuracy of 80% in the context of PGT-A. Nevertheless, if structural and mosaic abnormalities are not considered, accuracy increases to 100%, with a 0% false positive and false negative rate.Limitations Reasons For Caution: The inherent limitations of extended in vitro culture, particularly when modelling critical developmental milestones, warrant careful interpretation.Wider Implications Of the Findings: Our findings echo current prenatal testing data and support the high clinical predictive value of PGT-A for diagnosing uniform numerical aneuploidies, as well as euploid chromosomal constitutions. However, distinguishing technical bias from biological variability will remain a challenge, inherently limiting the accuracy of a single TE biopsy for diagnosing mosaicism.Study Funding, Competing Interest(s): This research is funded by the Ghent University Special Research Fund (BOF01D08114) awarded to M.P., the Research Foundation-Flanders (FWO.KAN.0005.01) research grant awarded to B.H. and De Snoo-van't Hoogerhuijs Stichting awarded to S.M.C.d.S.L. We thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: To what extent does a trophectoderm (TE) biopsy reliably reflect the chromosomal constitution of the inner cell mass (ICM) in human blastocysts?Summary Answer: Concordance between TE and ICM was established in 62.1% of the embryos analysed.What Is Known Already: Next generation sequencing (NGS) platforms have recently been optimised for preimplantation genetic testing for aneuploidies (PGT-A). However, higher sensitivity has led to an increase in reports of chromosomal mosaicism within a single TE biopsy. This has raised substantial controversy surrounding the prevalence of mosaicism in human blastocysts and the clinical implications of heterogeneity between the TE and ICM.Study Design, Size, Duration: To define the distribution and rate of mosaicism in human blastocysts, we assessed chromosomal profiles of the ICM and multiple TE portions obtained from the same embryo. We evaluated donated embryos with an unknown chromosomal profile (n = 34), as well as PGT-A blastocysts, previously diagnosed as abnormal or mosaic (n = 24). Our intra-embryo comparison included a total of 232 samples, obtained from 58 embryos.Participants/materials, Setting, Methods: Four embryo samples, including the ICM and three distinct TE portions, were acquired from good quality blastocysts by micromanipulation. Whole genome amplification (WGA), followed by NGS was performed on all embryo segments. Profiles were compared between samples from the same embryo, while the results from pretested blastocysts were further correlated to the original report. The embryos investigated in our untested group were obtained from good prognosis patients (n = 25), with maternal age ranging from 23 to 39 years. For the pretested embryo group, maternal age ranged from 23 to 40 years (n = 18).Main Results and the Role Of Chance: We uncover chromosomal mosaicism, involving both numerical and structural aberrations, in up to 37.9% of the blastocysts analysed. Within the untested group, the overall concordance between the ICM and all TE portions was 55.9%. A normal ICM was detected in 20.6% of blastocysts for which at least one TE portion showed a chromosomal aberration. Conversely, 17.6% of embryos presented with mosaic or uniform abnormalities within the ICM, while showing normal or mosaic TE profiles. For the pretested blastocysts, the overall concordance between the ICM and all TE samples was 70.8%. However, 50% of embryos previously diagnosed with mosaicism did not confirm the original diagnosis. Notably, 31.3% of embryos with a mosaic aberration reported in the original TE biopsy, revealed a euploid profile in the ICM and all three TE samples. Taken together, concordance between the ICM and all TE portions was established in 62.1% of blastocysts, across both embryo groups. Finally, we could not observe a significant effect of age on embryo mosaicism (P = 0.101 untested group; P = 0.7309 pretested group). Similarly, ICM and TE quality were not found to affect the occurrence of chromosomal mosaicism (P = 0.718 and P = 0.462 untested group; P = 1.000 and P = 0.2885 pretested group).Large Scale Data: All data that support the findings of this study are available online in Vivar (http://cmgg.be/vivar) upon request.Limitations, Reasons For Caution: Evaluating biological variation in some instances remains challenging. The technological limitations of sampling mitotic errors that lead to mosaicism, as well as WGA artefacts, warrant careful interpretation.Wider Implications Of the Findings: Our results highlight the complex nature of genetic (in)stability during early ontogenesis and indicate that blastocysts harbour a higher rate of chromosomal mosaicism than may have been anticipated. Moreover, our findings reveal an overall high diagnostic sensitivity and relatively low specificity in the context of PGT-A. This suggests that a considerable proportion of embryos are potentially being classified as clinically unsuitable. Ultimately, more precise quantification will benefit the clinical management of embryo mosaicism.Study Funding/competing Interest(s): M.P. is supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF01D08114). J.T. and L.D. are supported by the agency for innovation through science (131673, 141441). B.H. and this research are supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF15/GOA/011). The authors declare no competing interests.Trial Registration Number: Not applicable. [ABSTRACT FROM AUTHOR]

Study Question: Can human oocyte calcium analysis predict fertilization success after assisted oocyte activation (AOA) in patients experiencing fertilization failure after ICSI?Summary Answer: ICSI-AOA restores the fertilization rate only in patients displaying abnormal Ca2+ oscillations during human oocyte activation.What Is Known Already: Patients capable of activating mouse oocytes and who showed abnormal Ca2+ profiles after mouse oocyte Ca2+ analysis (M-OCA), have variable responses to ICSI-AOA. It remains unsettled whether human oocyte Ca2+ analysis (H-OCA) would yield an improved accuracy to predict fertilization success after ICSI-AOA.Study Design, Size, Duration: Sperm activation potential was first evaluated by MOAT. Subsequently, Ca2+ oscillatory patterns were determined with sperm from patients showing moderate to normal activation potential based on the capacity of human sperm to generate Ca2+ responses upon microinjection in mouse and human oocytes. Altogether, this study includes a total of 255 mouse and 122 human oocytes. M-OCA was performed with 16 different sperm samples before undergoing ICSI-AOA treatment. H-OCA was performed for 11 patients who finally underwent ICSI-AOA treatment. The diagnostic accuracy to predict fertilization success was calculated based on the response to ICSI-AOA.Participants/materials, Setting, Methods: Patients experiencing low or total failed fertilization after conventional ICSI were included in the study. All participants showed moderate to high rates of activation after MOAT. Metaphase II (MII) oocytes from B6D2F1 mice were used for M-OCA. Control fertile sperm samples were used to obtain a reference Ca2+ oscillation profile elicited in human oocytes. Donated human oocytes, non-suitable for IVF treatments, were collected and vitrified at MII stage for further analysis by H-OCA.Main Results and the Role Of Chance: M-OCA and H-OCA predicted the response to ICSI-AOA in 8 out of 11 (73%) patients. Compared to M-OCA, H-OCA detected the presence of sperm activation deficiencies with greater sensitivity (75 vs 100%, respectively). ICSI-AOA never showed benefit to overcome fertilization failure in patients showing normal capacity to generate Ca2+ oscillations in H-OCA and was likely to be beneficial in cases displaying abnormal H-OCA Ca2+ oscillations patterns.Limitations, Reasons For Caution: The scarce availability of human oocytes donated for research purposes is a limiting factor to perform H-OCA. Ca2+ imaging requires specific equipment to monitor fluorescence changes over time.Wider Implications Of the Findings: H-OCA is a sensitive test to diagnose gamete-linked fertilization failure. H-OCA allows treatment counseling for couples experiencing ICSI failures to either undergo ICSI-AOA or to participate in gamete donation programs. The present data provide an important template of the Ca2+ signature observed during human fertilization in cases with normal, low and failed fertilization after conventional ICSI.Study Funding/competing Interest(s): This work was supported by the Flemish fund for scientific research (FWO-Vlaanderen, G060615N). The authors have no conflict of interest to declare. [ABSTRACT FROM AUTHOR]

The presumed superiority of renin-angiotensin-aldosterone system (RAAS)-blocking agents over other antihypertensive agents in patients with diabetes to delay development of endstage kidney disease (ESKD) has recently been challenged. In addition, there is ongoing uncertainty whether RAAS-blocking agents reduce mortality and/or delay ESKD in patients with diabetes and chronic kidney disease (CKD) stages 3-5. In this subgroup, there might be an expedited need for renal replacement therapy (RRT) when RAAS-blocking agents are used. We conducted a meta-analysis of randomized controlled trials (RCTs) of at least 6-months duration in adult patients with diabetes who also have non-dialysis CKD stages 3-5. RCTs comparing single RAAS-blocking agents to placebo or alternative antihypertensive agents were included. Outcomes of interest were allcause mortality, cardiovascular morbidity, progression of renal function, ESKD and adverse events. A total of nine trials (n=9797 participants with CKD stages 3-5) fit our inclusion criteria. There was no difference between the RAAS group and control group regarding all-cause mortality {relative risk [RR]=0.97 [95% confidence interval (CI) 0.85-1.10]}, cardiovascular mortality [RR=1.03 (95% CI 0.75-1.41)] and adverse events [RR=1.05 (95% CI 0.89-1.25)]. There was a trend for a favourable effect for non-fatal cardiovascular events [RR=0.90 (95% CI 0.81-1.00)] and a lower risk of the composite endpoint need for RRT/doubling of serum creatinine [RR=0.81 (95% CI 0.70-0.92)] in the RAAS-blocking agents group versus the control group. We found evidence that in patients with diabetes mellitus and CKD stages 3-5, treatment with RAAS-blocking agents did not result in a clear survival advantage. The effect on renal outcomes did depend on the selected outcome measure. However, we did not find evidence that the use of RAASblocking agents expedited the need for RRT in patients with CKD stages 3-5. [ABSTRACT FROM AUTHOR]

Background: The use of nuclear transfer (NT) has been proposed as a novel reproductive treatment to overcome the transmission of maternally-inherited mitochondrial DNA (mtDNA) mutations. Pathogenic mutations in mtDNA can cause a wide-spectrum of life-limiting disorders, collectively known as mtDNA disease, for which there are currently few effective treatments and no known cures. The many unique features of mtDNA make genetic counselling challenging for women harbouring pathogenic mtDNA mutations but reproductive options that involve medical intervention are available that will minimize the risk of mtDNA disease in their offspring. This includes PGD, which is currently offered as a clinical treatment but will not be suitable for all. The potential for NT to reduce transmission of mtDNA mutations has been demonstrated in both animal and human models, and has recently been clinically applied not only to prevent mtDNA disease but also for some infertility cases. In this review, we will interrogate the different NT techniques, including a discussion on the available safety and efficacy data of these technologies for mtDNA disease prevention. In addition, we appraise the evidence for the translational use of NT technologies in infertility.Objective and Rationale: We propose to review the current scientific evidence regarding the clinical use of NT to prevent mitochondrial disease.Search Methods: The scientific literature was investigated by searching PubMed database until Jan 2017. Relevant documents from Human Fertilisation and Embryology Authority as well as reports from both the scientific and popular media were also implemented. The above searches were based on the following key words: 'mitochondria', 'mitochondrial DNA'; 'mitochondrial DNA disease', 'fertility'; 'preimplantation genetic diagnosis', 'nuclear transfer', 'mitochondrial replacement' and 'mitochondrial donation'.Outcomes: While NT techniques have been shown to effectively reduce the transmission of heteroplasmic mtDNA variants in animal models, and increasing evidence supports their use to prevent the transmission of human mtDNA disease, the need for robust, long-term evaluation is still warranted. Moreover, prenatal screening would still be strongly advocated in combination with the use of these IVF-based technologies. Scientific evidence to support the use of NT and other novel reproductive techniques for infertility is currently lacking.Wider Implications: It is mandatory that any new ART treatments are first adequately assessed in both animal and human models before the cautious implementation of these new therapeutic approaches is clinically undertaken. There is growing evidence to suggest that the translation of these innovative technologies into clinical practice should be cautiously adopted only in highly selected patients. Indeed, given the limited safety and efficacy data, close monitoring of any offspring remains paramount. [ABSTRACT FROM AUTHOR]

Background: The subject of epigenetic risk of assisted reproduction treatment (ART), initiated by reports on an increase of children with the Beckwith-Wiedemann imprinting disorder, is very topical. Hence, there is a growing literature, including mouse studies.Methods: In order to gain information on transgenerational epigenetic inheritance and epigenetic effects induced by ART, literature databases were searched for papers on this topic using relevant keywords.Results: At the level of genomic imprinting involving CpG methylation, ART-induced epigenetic defects are convincingly observed in mice, especially for placenta, and seem more frequent than in humans. Data generally provide a warning as to the use of ovulation induction and in vitro culture. In human sperm from compromised spermatogenesis, sequence-specific DNA hypomethylation is observed repeatedly. Transmittance of sperm and oocyte DNA methylation defects is possible but, as deduced from the limited data available, largely prevented by selection of gametes for ART and/or non-viability of the resulting embryos. Some evidence indicates that subfertility itself is a risk factor for imprinting diseases. As in mouse, physiological effects from ART are observed in humans. In the human, indications for a broader target for changes in CpG methylation than imprinted DNA sequences alone have been found. In the mouse, a broader range of CpG sequences has not yet been studied. Also, a multigeneration study of systematic ART on epigenetic parameters is lacking.Conclusions: The field of epigenetic inheritance within the lifespan of an individual and between generations (via mitosis and meiosis, respectively) is growing, driven by the expansion of chromatin research. ART can induce epigenetic variation that might be transmitted to the next generation. [ABSTRACT FROM AUTHOR]

Study Question: Does prospective embryo selection using the results from the Eava Test (Early Embryo Viability Assessment) in combination with standard morphology increase the pregnancy rate of IVF and ICSI patients compared to embryo selection based on morphology only?Summary Answer: Embryo selection using the Eeva Test plus standard morphology on Day 3 results in comparable pregnancy rates as conventional morphological embryo selection.What Is Known Already: Time-lapse monitoring of embryo development may represent a superior way to culture and select embryos in vitro. The Eeva Test records the development of each embryo with a cell-tracking system and predicts the likelihood (High, Medium or Low) that an embryo will form a blastocyst based on an automated analysis of early cell division timings.Study Design, Size, Duration: This trial was designed as a prospective, observational, two-center pilot study with a propensity matched control group. The analysis involved 280 of 302 enrolled patients who were included in the Eeva Test group in 2013 and 560 control patients who were treated in the years 2011-2013. The majority of transfers (98%) were single embryo transfers.Participants/materials, Setting, Methods: Two academic hospitals (VUmc Amsterdam and UZ Gent) enrolled patients <41 years old, with <3 previous attempts and ≥5 normally fertilized eggs. Propensity matching was used to identify a propensity matched control group from a cohort of 1777 patients based on age, cycle number, oocyte number and number of fertilized oocytes.Main Results and the Role Of Chance: There was no difference in patient baseline characteristics between the two groups. The ongoing pregnancy rate (OPR) of patients enrolled in the Eeva Test group (34.3%; 96/280) did not differ significantly from the OPR in the propensity matched control group (34.6%, 194/560; P = 0.92). However, significantly less top quality embryos (eight-cell embryos with ≤25% fragmentation) were transferred in the Eeva Test group compared to the propensity matched control group (70.4% vs. 82.3%; P < 0.001). The transfer of Eeva High and Medium embryos resulted in a significantly higher OPR of 36.8% (89/242) compared to 18.4% (7/38) for Eeva Low embryos (P = 0.02).Limitation, Reasons For Caution: This pilot study is limited by its nonrandomized design with a concurrent and historical control.Wider Implications Of the Findings: Our pilot data did not reveal significant differences between time-lapse based and conventional embryo selection. Interestingly, the pregnancy rates were comparable in both groups even though the morphological quality of the transferred embryos was significantly lower in the Eeva Test group compared to the propensity matched control group. A sufficiently powered three-armed randomized controlled trial (RCT) with a solid design should be performed to generate decisive evidence in the future.Study Finding/competing Interests: Progyny Inc., formerly Auxogyn provided the Eeva scopes, software and technical support for this study. The funding sources did neither influence data collection, management, analysis and interpretation of the data, nor the preparation of the manuscript.Trial Registration Number: ClinicalTrials.gov: NCT01671644. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made to trigger naïve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene-free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation. S tem C ells 2015;33:2686-2698 [ABSTRACT FROM AUTHOR]

Background: Until recently, the temporal events that precede the generation of pluripotent embryonic stem cells (ESCs) and their equivalence with specific developmental stages in vivo was poorly understood. Our group has discovered the existence of a transient epiblast-like structure, coined the post-inner cell mass (ICM) intermediate or PICMI, that emerges before human ESC (hESCs) are established, which supports their primed nature (i.e. already showing some predispositions towards certain cell types) of pluripotency. Methods: The PICMI results from the progressive epithelialization of the ICM and it expresses a mixture of early and late epiblast markers, as well as some primordial germ cell markers. The PICMI is a closer progenitor of hESCs than the ICM and it can be seen as the first proof of why all existing hESCs, until recently, display a primed state of pluripotency. Results: Even though the pluripotent characteristics of ESCs differ from mouse (naïve) to human (primed), it has recently been shown in mice that a similar process of self-organization at the transition from ICM to (naïve) mouse ESCs (mESCs) transforms the amorphous ICM into a rosette of polarized epiblast cells, a mouse PICMI. The transient PICMI stage is therefore at the origin of both mESCs and hESCs. In addition, several groups have now reported the conversion from primed to the naïve (mESCs-like) hESCs, broadening the pluripotency spectrum and opening new opportunities for the use of pluripotent stem cells. Conclusions: In this review, we discuss the recent discoveries of mouse and human transient states from ICM to ESCs and their relation towards the state of pluripotency in the eventual stem cells, being naïve or primed. We will now further investigate how these intermediate and/or different pluripotent stages may impact the use of human stem cells in regenerative medicine and assisted reproductive technology. [ABSTRACT FROM AUTHOR]

background: Until recently, the temporal events that precede the generation of pluripotent embryonic stem cells (ESCs) and their equivalence with specific developmental stages in vivo was poorly understood. Our group has discovered the existence of a transient epiblast-like structure, coined the post-inner cell mass (ICM) intermediate or PICMI, that emerges before human ESC (hESCs) are established, which supports their primed nature (i.e. already showing some predispositions towards certain cell types) of pluripotency. methods: The PICMI results from the progressive epithelialization of the ICM and it expresses a mixture of early and late epiblast markers, as well as some primordial germ cell markers. The PICMI is a closer progenitor of hESCs than the ICM and it can be seen as the first proof of why all existing hESCs, until recently, display a primed state of pluripotency. results: Even though the pluripotent characteristics of ESCs differ frommouse (naıve) to human (primed), it has recently been shown in mice that a similar process of self-organization at the transition fromICM to (naıve) mouse ESCs (mESCs) transforms the amorphous ICM into a rosette of polarized epiblast cells, a mouse PICMI. The transient PICMI stage is therefore at the origin of both mESCsand hESCs. In addition, several groups have now reported the conversion from primed to the naıve (mESCs-like) hESCs, broadening the pluripotency spectrum and opening new opportunities for the use of pluripotent stem cells. conclusions: In this review, we discuss the recent discoveries of mouse and human transient states from ICM to ESCs and their relation towards the state of pluripotency in the eventual stemcells, being naïve or primed. We willnowfurther investigatehowthese intermediate and/or different pluripotent stages may impact the use of human stem cells in regenerative medicine and assisted reproductive technology. [ABSTRACT FROM AUTHOR]

STUDY QUESTION How are objective characteristics of polycystic ovary syndrome (PCOS) and PCOS-related concerns associated with the sexual and relational satisfaction of PCOS women and their partners? SUMMARY ANSWER Both objective PCOS characteristics (parity, women's body mass index (BMI) and current unfulfilled wish to conceive) and PCOS-related concerns (women's infertility-related and acne-related concerns) were associated with sexual and/or relational satisfaction, although some associations differed for PCOS women and their partners. WHAT IS KNOWN ALREADY There is some evidence indicating an association between objective PCOS characteristics and sexual satisfaction of PCOS women, but this evidence is conflicting, scarce, and often validated questionnaires have not been used to evaluate sexual satisfaction. No evidence is available about the association of: (i) PCOS with relational satisfaction; (ii) PCOS-related concerns with sexual and relational satisfaction; and (iii) PCOS with sexual and relational satisfaction as experienced by partners of PCOS women. STUDY DESIGN, SIZE, DURATION We set up a cross-sectional study from April 2007 until April 2009, including 31 overweight (BMI ≥ 25 kg/m2) women with PCOS and at a reproductive age as well as their partners with whom they had a committed intimate relationship at the time of recruitment. PARTICIPANTS/MATERIALS, SETTING, METHODS The study was performed at the fertility center of the Ghent University Hospital. Objective PCOS characteristics were registered and PCOS-related concerns were evaluated by the PCOS Questionnaire. Sexual (SS) and relational (RS) satisfaction were measured by the Maudsley Marital Questionnaire (MMQ). Dyadic statistical analyses were performed using linear mixed models (α < 0.05). MAIN RESULTS AND THE ROLE OF CHANCE A lower parity tended to be associated with higher levels of sexual and relational satisfaction, with a significantly stronger association in PCOS women than in their partners (p(SS) = 0.015 and p(RS) = 0.009). A higher BMI tended to be associated with lower and higher satisfaction levels (sexual and relational) in PCOS women and their partners, respectively, with a significantly stronger association in the partners (p(SS) = 0.029 and p(RS) = 0.021). The presence of a current unfulfilled wish to conceive and a higher level of infertility-related concerns was significantly more strongly associated with a higher level of relational satisfaction for PCOS women than for their partners (p(RS) = 0.021 and p(RS) = 0.011, respectively). Higher levels of acne-related concern were significantly associated with lower levels of sexual satisfaction in both PCOS women (p(SS) = 0.025) and their partners (p(SS) = 0.002). LIMITATIONS, REASONS FOR CAUTION The fact that this study was performed in a sample of PCOS women who were all overweight and the small sample size are important limitations. Data were partially missing in some couples but this limitation was dealt with by using linear mixed models. WIDER IMPLICATIONS OF THE FINDINGS Our results suggest a differential association of PCOS with sexual and relational satisfaction between PCOS women and their partners. This should be kept in mind during the psychological guidance of couples dealing with PCOS. STUDY FUNDING/COMPETING INTEREST(S) V.D.F. is holder of a Special PhD Fellowship of the Flemish Foundation for Scientific Research (FWO-Vlaanderen). P.D.S. is holder of a fundamental clinical research mandate of the Flemish Foundation for Scientific Research (FWO-Vlaanderen). This research also received financial support by Merck Serono and Artevelde University College Ghent. There are no competing interests. [ABSTRACT FROM PUBLISHER]

Couples dealing with microdeletions of the Y chromosome have to make decisions about their reproductive future. Do they opt for intracytoplasmic sperm injection (ICSI), artificial insemination with donor insemination (AID) or no treatment? We analysed this decision in 28 couples and investigated the role of the counsellor and the counselling process on the final decision of the couple. Ten counsellors from six fertility clinics in The Netherlands and Belgium were interviewed about their genetic counselling of couples dealing with microdeletions. The answers to the questionnaire were converted to 11 dichotomous variables. Of the 1627 tested men in the six centres, 37 (2.3%) had a microdeletion in the AZFc region, a subregion of the AZF region on the Y chromosome important for normal spermatogenesis. The decisions of 28 of them could be analysed. Most couples chose ICSI (79%). The remaining couples chose donor insemination (7%) or refrained from treatment (14%). Several variables, including the counselling procedure, the counsellor and the available treatments in the fertility centre, influenced the decision of the couple. In conclusion, most couples dealing with microdeletions in the AZF region choose ICSI. Several aspects of the process of genetic counselling appear to be related to the final decision. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Do overweight women with polycystic ovary syndrome (PCOS) have a higher risk of perinatal complications than normal weight women with PCOS? SUMMARY ANSWER Overweight women with PCOS with an ongoing singleton pregnancy have an increased risk of preterm birth as well as an increased risk of giving birth to a baby with a higher birthweight than normal weight women with PCOS. WHAT IS KNOWN ALREADY There is evidence that overweight (BMI > 25 kg/m²) has a negative influence on the prevalence of gestational diabetes mellitus and fetal macrosomia in women with PCOS. STUDY DESIGN, SIZE, DURATION We set up a retrospective comparative cohort study of 93 overweight (BMI ≥ 25 kg/m²) and 107 normal weight (BMI < 25 kg/m²) women with PCOS who were scheduled for fertility treatment between January 2000 and December 2009 and achieved a pregnancy as a result of a treatment cycle, or spontaneously before or between treatment cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS All data (patient characteristics, medical information, pregnancy, delivery and neonatal outcome) were retrieved from patient medical files. All pregnancy, delivery and neonatal outcome parameters were adjusted for age and pre-pregnancy smoking behaviour. The neonatal outcome parameters were additionally adjusted for gestational age. MAIN RESULTS AND THE ROLE OF CHANCE The median BMI in the overweight and normal weight women was, respectively, 30.8 kg/m² [interquartile quartile range (IQR) 5.8] and 20.9 kg/m² (IQR 2.3) (P < 0.001). Baseline characteristics did not differ between groups, except for free testosterone and fasting insulin levels, which were higher, and sex hormone-binding globulin, which was lower, in overweight versus normal weight women (all P < 0.001). The time-to-pregnancy was significantly higher in the overweight group (P = 0.01). Multivariate analyses of the ongoing singleton pregnancies showed significantly more preterm births in overweight (10/61) versus normal weight (2/71) women [adjusted odds ratio 0.1, 95% confidence interval (CI) 0–0.6, P = 0.01]. The mean birthweight of newborns was significantly higher in overweight (3386 ± 663 g) than in normal weight (3251 ± 528 g) women (adjusted mean difference 259.4, 95% CI 83.4–435.4, P = 0.004). LIMITATIONS, REASON FOR CAUTION Our results only represent the pregnancy, delivery and neonatal outcome of ongoing singleton pregnancies. The rather small sample size and observational nature of the study are further limitations. WIDER IMPLICATIONS OF THE FINDINGS Our results suggest the importance of pre-pregnancy weight loss in overweight women with PCOS in order to reduce the risk of adverse perinatal outcomes. STUDY FUNDING/COMPETING INTEREST(S) Veerle De Frène is holder of a Special PhD Fellowship by the Flemish Foundation for Scientific Research (FWO-Vlaanderen). Petra De Sutter is holder of a fundamental clinical research mandate by the Flemish Foundation for Scientific Research (FWO-Vlaanderen). There are no competing interests. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Does self-operated endovaginal telemonitoring (SOET) of the ovarian stimulation phase in IVF/ICSI produce similar laboratory, clinical, patient reported and health-economic results as traditional monitoring (non-SOET)? SUMMARY ANSWER SOET is not inferior to traditional monitoring (non-SOET). WHAT IS KNOWN ALREADY Monitoring the follicular phase is needed to adapt gonadotrophin dose, detect threatening hyperstimulation and plan HCG administration. Currently, patients pay visits to care providers, entailing transportation costs and productivity loss. It stresses patients, partners, care providers and the environment. Patients living at great distance from centres have more difficult access to treatment. The logistics and stress during the follicular phase of assisted reproduction treatment (ART) is often an impediment for treatment. STUDY DESIGNS, SIZE, DURATION The study was a non-inferiority RCT between SOET and non-SOET performed between February 2012 and October 2013. Sample size calculations of number of metaphase II (MII) oocytes (the primary outcome): 81 patients were needed in each study arm for sufficient statistical power. Block randomization was used with allocation concealment through electronic files. The first sonogram was requested after 5 days of stimulation, after that mostly every 2 days and with a daily sonograms at the end. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Inclusion criteria were age <41 years, undergoing ICSI, no poor response and having two ovaries. We used a small laptop with USB connected vaginal probe and developed a specific web site application. Sonographic training was given to all women at the initiation of a treatment attempt at the centre. The website contained demonstration material consisting of still images and video sequences, as well as written instructions regarding the use of the instrument and probe handling. In total, 185 eligible patients were recruited in four centres: 123 were randomized; 121 completed SOET (n = 59) or non-SOET (n = 62), and 62/185 (33%) eligible patients declined participation for various reasons. MAIN RESULTS AND THE ROLE OF CHANCE Patient characteristics were comparable. The clinical results showed similar conception rates (P = 0.47) and ongoing pregnancy rates (SOET: 15/59 = 25%; non-SOET: 16/62 = 26%) (P = 1.00) were obtained. Similar numbers of follicles >15 mm diameter at oocyte retrieval (OR), ova at OR, MII oocytes, log2 MII oocytes, embryos available at transfer, top quality embryos and embryos frozen were obtained in the two groups, indicating non-inferiority of SOET monitoring. Regarding patient-reported outcomes, a significantly higher contentedness of patient and partner (P < 0.01), a higher feeling of empowerment, discretion and more active partner participation (P < 0.001) as well as a trend towards less stress (P = 0.06) were observed in the S versus the NS group. In the economic analysis, the use of SOET led to reduced productivity loss, lower transportation costs, and lower sonogram and consultation costs (all P < 0.001 but higher personnel cost than NS). LIMITATIONS, REASONS FOR CAUTION The study was stopped (no further funding) before full sample size was reached. There were also a few cases of unexpected poor response, leading to a wider SD than anticipated in the power calculation. However, although the study was underpowered for these reasons, non-inferiority of SOET versus non-SOET was demonstrated. WIDER IMPLICATIONS OF THE FINDINGS Home monitoring using SOET may provide a patient-centred alternative to the standard methods. ART sonograms can be made, and then sent to the care provider for analysis at any appropriate time and from anywhere if an internet connection is available. This approach offers several advantages for ... [ABSTRACT FROM PUBLISHER]

BACKGROUND In women with vaginal hypoplasia, such as in Mayer–Rokitansky–Küster–Hauser syndrome (MRKH) and in Complete Androgen Insensitivity Syndrome (CAIS), surgical vaginoplasty and non-surgical self-dilation treatments are available to lengthen the vagina and facilitate sexual intercourse, but the best treatment remains controversial. Vaginal dilation has been recommended as a first-line treatment, because of its less invasive character and high success rate. However, the exploration of factors associated with compliance and long-term outcome is incomplete, including whether psychological counselling needs to be embedded in treatment to maximize efficacy. It is not known if failed vaginal dilation therapy jeopardizes further surgical success outcomes, especially because in a number of these procedures ongoing vaginal dilation is required. In addition, if surgery is needed, there is a lack of evidence to inform physicians regarding the optimum surgical technique to use. Also, it is unclear whether maintenance dilation therapy in case of sexual inactivity is crucial to ensure functional success. METHODS In view of this ongoing debate, we performed a search of all published literature (English language only) restricted to the management of vaginal hypoplasia in patients with MRKH or CAIS from 1898 to March 2013 using Pubmed, Cochrane Library and Web of Science. Of the 6700 articles initially identified, a total of 190 studies are analysed. More specifically, by establishing the risk/efficacy profile (vaginal capacity, complications and long-term durability in terms of sexual function) of the different surgical and non-surgical reconstruction techniques, we evaluate if vaginal dilation proposed as the first-line technique is justified based on the evidence. RESULTS When anatomical success was defined as a length of ≥7 cm and functional success as coitus, all vaginoplasty techniques yielded significantly higher success rates (>90 versus 75% after vaginal dilation), irrespective of underlying diagnosis or start vaginal length. When functional success was defined as ‘satisfaction with sex’, including non-genital sex, differences disappeared. Failed dilation therapy does not preclude anatomical (nor functional) success if vaginoplasty afterwards is necessary. Traction vaginoplasty seems to have the highest anatomical (99%) and functional success rates (96%), whereas both split- and full-thickness skin graft procedures and intestinal procedures have the lowest successful outcomes (83–95%). Overall, complication rates were significantly lower within the vaginal dilation groups when compared with the different vaginoplasty techniques. Although no randomized control data exist regarding maintenance dilation, the available evidence suggests that continued dilation is needed to maintain patency in periods of coital inactivity. Despite the expectancy that the probability of further positive outcomes is maximized with psychological counselling, this could not be confirmed. CONCLUSIONS As the medical literature lacks high-quality comparative outcome studies and prospective, longitudinal studies are scarce, no evidence-based treatment guidelines can be provided. However, because of the physically low complication rate and an overall success chance of 75%, vaginal dilation as first choice treatment seems to be justified. Overall, the laparoscopic Vecchietti procedure, becoming more and more available in specialized centres, is considered an appropriate surgical option in patients who are poorly compliant and failed dilation therapy, or for those who do not want to start with vaginal dilation therapy. Future approaches need to raise a wider range of psychosexually oriented questions, elucidate the relationship between vaginal depth and ... [ABSTRACT FROM AUTHOR]

Study Question: Can Sphingosine-1-phosphate (S1P), a ceramide-induced death pathway inhibitor, prevent cyclophosphamide (Cy) or doxorubicin (Doxo) induced apoptotic follicle death in human ovarian xenografts?Summary Answer: S1P can block human apoptotic follicle death induced by both drugs, which have differing mechanisms of cytotoxicity.What Is Known Already: S1P has been shown to decrease the impact of chemotherapy and radiation on germinal vesicle oocytes in animal studies but no human translational data exist.Study Design, Size, Duration: Experimental human ovarian xenografting to test the in vivo protective effect of S1P on primordial follicle survival in the chemotherapy setting. The data were validated by assessing the same protective effect in the ovaries of xenografted mice in parallel.Participants/materials, Setting, Methods: Xenografted mice were treated with Cy (75 mg/kg), Cy+S1P (200 μM), Doxo (10 mg/kg), Doxo+S1P or vehicle only (Control). S1P was administered via continuous infusion using a mini-osmotic pump beginning 24 h prior to and ending 72 h post-chemotherapy. Grafts were then recovered and stained with anti-caspase 3 antibody for the detection of apoptosis in primordial follicles. The percentage of apoptotic to total primordial follicles was calculated in each group.Main Results and the Role Of Chance: Both Cy and Doxo resulted in a significant increase in apoptotic follicle death in human ovarian xenografts compared with controls (62.0 ± 3.9% versus 25.7 ± 7.4%, P < 0.01 and 76.7 ± 7.4% versus 25.7 ± 7.4%, P < 0.01, respectively). This chemotherapy-induced apoptotic death was reduced both in the Cy+S1P (32.7 ± 4.4%, P < 0.01) and the Doxo+S1P group (27.1 ± 7.6%, P < 0.01) compared with Cy and Doxo groups, respectively. In the Doxo+S1P and Cy+S1P groups, the percentages of apoptotic follicles were similar to those of vehicle-treated controls (P > 0.05). The findings from the ovaries of the severe combined immunodeficient mice mirrored the findings with human tissue.Limitations, Reasons For Caution: The functionality of the rescued human ovarian follicles needs to be evaluated in future studies though the studies in rodents showed that rescued oocytes can result in healthy offspring. In addition, the impact of S1P on cancer cells should be further studied.Wider Implications Of the Findings: S1P and its future analogs hold promise for preserving fertility by pharmacological means for patients undergoing chemotherapy.Study Funding/competing Interest(s): This research is supported by NIH's NICHD and NCI (5R01HD053112-06 and 5R21HD061259-02) and the Flemish Foundation for Scientific Research (FWO-Vlaanderen, grant number FWO G0.065.11N10). The authors have no conflicts of interest to disclose. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What are the precise patterns of calcium oscillations during the fertilization of human oocytes matured either in vivo or in vitro or aged in vitro and what is the effect of cryopreservation? SUMMARY ANSWER Human oocytes matured in vivo exhibit a specific pattern of calcium oscillations, which is affected by in vitro maturation, in vitro ageing and cryopreservation. WHAT IS KNOWN ALREADY Oscillations in cytoplasmic calcium concentration are crucial for oocyte activation and further embryonic development. While several studies have described in detail the calcium oscillation pattern during fertilization in animal models, studies with human oocytes are scarce. STUDY DESIGN, SIZE, DURATION This was a laboratory-based study using human MII oocytes matured in vivo or in vitro either fresh or after cryopreservation with slow freezing or vitrification. Altogether, 205 human oocytes were included in the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS In vivo and in vitro matured human oocytes were used for this research either fresh or following vitrification/warming (V/W) and slow freezing/thawing (F/T). Human oocytes were obtained following written informed consent from patients undergoing ovarian hyperstimulation. For the calcium pattern analysis, oocytes were loaded with the ratiometric calcium indicator fluorescent dye Fura-2. Following ICSI using sperm from a single donor, intracellular calcium was measured for 16 h at 37°C under 6% CO2. The calcium oscillation parameters were calculated for all intact oocytes that showed calcium oscillations and were analyzed using the Mann–Whitney U-test. MAIN RESULTS AND THE ROLE OF CHANCE Human in vivo MII oocytes display a specific pattern of calcium oscillations following ICSI. This pattern is significantly affected by in vitro ageing, with the calcium oscillations occurring over a longer period of time and with a lower frequency, shorter duration and higher amplitude (P < 0.05). In vitro matured oocytes from the GV and MI stage exhibit a different pattern of calcium oscillations with calcium transients being of lower frequency and shorter duration compared with in vivo matured MII. In MI oocytes that reached the MII stage within 3 h the calcium oscillations additionally appear over a longer period of time (P < 0.05). In vivo MII oocytes show a different calcium oscillation pattern following V/W with calcium oscillations occurring over a longer period of time, with a higher amplitude and a lower frequency (P < 0.05). In vitro matured oocytes, either from the GV or the MI stage, also display an altered pattern of calcium oscillations after V/W and the parameters that were similarly affected in all these oocyte groups are the frequency and the amplitude of the calcium transients. Slow freezing/thawing differentially affects the calcium oscillation pattern of in vitro matured and in vitro aged oocytes. LIMITATIONS, REASONS FOR CAUTION The relationship between a specific pattern of calcium oscillations and subsequent human embryonic development could not be evaluated since the calcium indicator used and the high-intensity excitation light impair development. Furthermore, all oocytes were derived from stimulated cycles and immature oocytes were denuded prior to in vitro maturation. WIDER IMPLICATIONS OF THE FINDINGS Our data show for the first time how calcium signalling during human fertilization is affected by oocyte in vitro maturation, in vitro ageing as well as V/W and slow freezing/thawing. The analysis of calcium oscillations could be used as an oocyte quality indicator to evaluate in vitro culture and cryopreservation techniques of human oocytes. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by a clinical research mandate from the Flemish Foundation of ... [ABSTRACT FROM PUBLISHER]

STUDY QUESTION Is there an effect of the TGFβ inhibitor SB431542 (SB) on the epiblast compartment of human blastocysts, and does it affect subsequent human embryonic stem cell (hESC) derivation? SUMMARY ANSWER SB increases the mean number of NANOG-positive cells in the inner cell mass (ICM), and allows for subsequent hESC derivation. WHAT IS KNOWN ALREADY It is known that inhibition of TGFβ by SB has a positive effect on mouse ESC self-renewal, while active TGFβ signalling is needed for self-renewal of primed ESC. STUDY DESIGN, SIZE, DURATION From December 2011 until March 2012, 263 donated spare embryos were used from patients who had undergone IVF/ICSI in our centre. PARTICIPANTS/MATERIALS, SETTING, METHODS Donated human embryos were cultured in the presence of SB or Activin A, and immunocytochemistry was performed on Day 6 blastocysts for NANOG and GATA6. Moreover, blastocysts were used for the derivation of hESC, with or without exposure to SB. MAIN RESULTS AND THE ROLE OF CHANCE Immunocytochemistry revealed a significantly higher number of NANOG-positive ICM cells in the SB group compared with the control (12.0 ± 5.9 versus 6.1 ± 4.7), while no difference was observed in the Activin A group compared with other groups (6.7 ± 3.7). The number of GATA6-positive ICM cells did not differ between the SB, Activin A and control group (8.8 ± 4.3, 8.0 ± 4.6 and 7.2 ± 4.0, respectively). Blocking TGFβ signalling did not prevent subsequent hESC line derivation. LIMITATIONS, REASONS FOR CAUTION The number of human blastocysts available for this study was too low to reveal if the observed increase in NANOG-positive epiblast cells after exposure to SB affected the efficiency of hESC derivation (12.5% compared with 16.7%). WIDER IMPLICATIONS OF THE FINDINGS This work can contribute to the derivation of naive hESC lines in the future. STUDY FUNDING/COMPETING INTEREST(S) M.V.d.J. is holder of a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT, grant number SB093128), Belgium. G.D. and this research are supported by the Research Foundation Flanders (FWO), grant number FWO-3G062910) and a Concerted Research Actions funding from BOF (Bijzonder Onderzoeksfonds University Ghent, grant number BOF GOA 01G01112). S.M.C.d. S.L. is supported by the Netherlands Organization of Scientific Research (NWO) (ASPASIA 015.007.037) and the Interuniversity Attraction Poles (PAI) (no. P7/07). P.D.S. is holder of a fundamental clinical research mandate by the FWO. We would like to thank Ferring Company (Aalst, Belgium) for financial support of this study. The authors do not have any competing interests to declare. TRIAL REGISTRATION NUMBER Not applicable. [ABSTRACT FROM PUBLISHER]

Background. Public Health England has reported a decrease of up to 20.8% in new diagnoses of external genital warts (GWs) among women aged <19 years since the national vaccination program with the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine began in 2008. A post hoc analysis of the phase III PATRICIA (PApilloma TRIal against Cancer In young Adults) trial (NCT00122681) was performed to ascertain whether protection against low-risk HPV types was apparent.Methods. Vaccine efficacy (VE) at 48 months was assessed against 6-month persistent infection (6MPI) with low-risk HPV types in the total vaccinated cohort (TVC) and in the TVC naive (for 25 HPV types tested) populations.Results. In the TVC naive cohort, VE against 6MPI (95% confidence interval) was 34.5% (11.3 to 51.8) for HPV-6/11, 34.9% (9.1 to 53.7) for HPV-6, 30.3% (−45.0 to 67.5) for HPV-11, and 49.5% (21.0 to 68.3) for HPV-74.Conclusions. The HPV-16/18 AS04-adjuvanted vaccine appears to have moderate efficacy against persistent infections with a number of low-risk HPV types (HPV-6/11/74), which are responsible for the majority of external GWs, and recently, antibody and cell-mediated immune response to HPV-6/11 have been observed. These findings may help to explain the decrease in external GW diagnoses seen in England. [ABSTRACT FROM PUBLISHER]

STUDY QUESTION Does the application of three different artificial activating stimuli lead to a difference in pre- and post-implantation embryo development in the wobbler mouse, a mouse model with oocyte activation deficient round-headed sperm cells similar to human globozoospermia? SUMMARY ANSWER No gross differences were found between strontium chloride, electrical pulses or ionomycin with respect to the pre- and post-implantation development in the wobbler mouse. WHAT IS KNOWN ALREADY Fertilization failure following intra-cytoplasmic sperm injection (ICSI) occurs in 1–3% of the ICSI cycles in human assisted reproduction technology (ART) and has been successfully overcome by different artificial activating stimuli. No comparison has been made yet in terms of their efficiency and safety. STUDY DESIGN, SIZE, DURATION Calcium release and embryo development were compared between oocytes fertilized by wobbler and wild-type (WT) sperm following ICSI with or without three different artificial activating agents. Preimplantation development was assessed on 70 injected oocytes on average per group. On average, 10 foster mothers were used per activating group to compare post-implantation development. PARTICIPANTS/MATERIALS, SETTING, METHODS We used the wobbler mouse model that possesses oocyte activation deficient round-headed sperm cells. First, the calcium release following ICSI using wobbler sperm was compared with that of WT sperm. Outcome measures were the percentage of oocytes that showed calcium release and their mean amount of calcium rises. Secondly, the pre- and post-implantation development was assessed following ICSI with wobbler sperm plus artificial oocyte activation using either: (i) strontium chloride (Wob-Sr), (ii) electrical pulses (Wob-E) or (iii) ionomycin (Wob-I). Outcome measures were the activation, cleavage and blastocyst rates and the assessment of blastocyst quality by differential staining. Following mouse embryo transfer, pregnancy and birth rates as well as mean litter sizes were examined. Finally, pups were followed up until 8 weeks of age and then mated with fertile controls to assess their fertility. MAIN RESULTS AND THE ROLE OF CHANCE The percentage of oocytes showing calcium rises as well as the number of calcium rises per oscillating oocyte were significantly lower in the wobbler group when compared with the WT group (9.3 versus 96% and 2.1 calcium rises versus 31 calcium rises) (P < 0.001). The fertilization rate was significantly lower in the wobbler group (11.4%) when compared with the WT group (92.1%) and the artificial activation groups (strontium chloride: 99%, electrical pulses: 99% and ionomycin: 81%, respectively) (P < 0.001). Post-implantation development did not differ significantly between the WT and artificial activation groups, with pregnancy rates in favor of strontium chloride and electrical pulses. The weight of the male pups did not differ between the study groups, whereas the weight of the female pups originating from Wob-Sr embryos was significantly lower at weeks 2, 3 and 4 when compared with female pups originating from WT embryos. However, the latter difference was not observed at later time points, nor in the other artificial activating groups. All offspring mated successfully with fertile controls. LIMITATIONS, REASONS FOR CAUTION Results in animal models should be extrapolated with caution to a subfertile human population. Also, ionomycin is currently the most widely used artificial oocyte activating agent in human ART. WIDER IMPLICATIONS OF THE FINDINGS The low frequency of observed calcium rises and the low activation rate make the wobbler mouse a highly suitable model to study oocyte activation deficiency. Strontium chloride and electrical pulses were more efficient means to ... [ABSTRACT FROM PUBLISHER]

STUDY QUESTION Does calcium oscillatory pattern analysis following heterologous intra-cytoplasmic sperm injection (ICSI) of human sperm into mouse oocytes lead to diagnostic and prognostic information for patients suffering from ICSI fertilization failure? SUMMARY ANSWER We found that calcium oscillatory pattern analysis following heterologous ICSI has the strength to reveal, for the individual patient, the most probable underlying reason for low or failed fertilization after conventional ICSI. WHAT IS KNOWN ALREADY Fertilization failure occurs in 1–3% of the couples undergoing conventional ICSI, for whom the mouse oocyte activation test (MOAT) or a similar heterologous ICSI model is the only diagnostic test available to evaluate the oocyte-activating capacity of human sperm cells. The MOAT classifies the patients into three groups: a low (group 1), an intermediate (group 2) and a high (group 3) activating group. In MOAT group 1 patients, a sperm-related deficiency is likely to be the cause of previous fertilization failures, while in MOAT group 3 patients a sperm-related deficiency can most probably be refuted. For MOAT group 2 patients, the result is called inconclusive; hence, both sperm and oocyte deficiencies may still contribute to the previous ICSI fertilization failure. STUDY DESIGN, SIZE, DURATION The calcium-releasing ability of sperm from 26 MOAT patients with a history of zero or low fertilization following conventional ICSI was compared with the calcium-releasing ability of sperm from 4 control patients, with proven oocyte activation potential. Per case an average of 19 mouse oocytes were injected. Calcium imaging started within 5–10 min after ICSI and continued for 2 h. PARTICIPANTS/MATERIALS, SETTING, METHODS Human sperm were demembranated with 0.02% lysolecithin for 1 min immediately before heterologous piezo-driven ICSI. For calcium imaging, metaphase II oocytes from B6D2/F1 mice were loaded with fura-2 acetoxymethyl ester. The calcium oscillatory patterns following heterologous ICSI were scored per oocyte and per patient individually based on the presence of calcium spikes and their frequency and amplitude. MAIN RESULTS AND THE ROLE OF CHANCE For patients with low or high MOAT activating capacity (MOAT group 1 or 3, respectively), calcium analysis confirmed the MOAT result. For patients with a former inconclusive intermediate MOAT activating capacity result (MOAT group 2), no or strongly dissimilar calcium oscillatory patterns were seen, with significantly lower amplitude and frequency compared with control sperm. When the product of the amplitude and the frequency of the calcium traces was compared between the groups, MOAT group 1 and 2 cases differed significantly from MOAT group 3 cases and the control sperm (P < 0.01). LIMITATIONS, REASONS FOR CAUTION The results of the calcium analysis in mouse oocytes should not be directly extrapolated to human oocytes, since it is well known that human spermatozoa exhibit a greater activating potency in mouse oocytes compared with mouse spermatozoa. Furthermore, not much is known yet about the influence of aberrant calcium oscillatory patterns, such as found in MOAT group 2 patients, on pre- and post-implantation embryo development in the human. WIDER IMPLICATIONS OF THE FINDINGS Based on the current calcium oscillatory pattern analysis, we found that the product of calcium spike amplitude with its frequency allowed us to create a new threshold value, which can assist in confirming or refuting, on a single patient base, a sperm-borne activation deficiency. The latter is especially interesting for patients with a former intermediate inconclusive MOAT result (MOAT group 2 patients), for whom calcium oscillatory pattern analysis should be considered. STUDY FUNDING/COMPETING ... [ABSTRACT FROM PUBLISHER]

BACKGROUND As pregnancy and childbirth have long been considered women's issues and male infertility has long been surrounded by taboo, limited research has focused on the experience of infertile men. The purpose of this study was to compare male motives for parenthood, male well-being and disclosure patterns concerning the method of conception among men from couples starting treatment with IUI using their own sperm (autologous sperm recipient, ASR) or donor sperm (donor sperm recipient, DSR). METHODS This prospective study included 46 DSR- and 151 ASR-couples. Self-report questionnaires assessing parenthood motives, well-being and disclosure patterns concerning the method of conception were administered to both men and women prior to treatment. Unpaired T-tests and Fisher's exact tests were used to test for significant differences. RESULTS When compared with ASR-men, DSR-men expect more positive effects from parenthood on relationships and feelings of fulfilment, and report less negative effects of infertility on sexuality, but a lower self-image and more guilt. DSR-men plan to disclose the method of conception less frequently and have so far disclosed to a lesser degree than ASR-men. CONCLUSIONS DSR-men feel differently about parenthood and infertility compared with ASR-men, and their higher expectations combined with lower self-esteem need (more) attention during counselling. [ABSTRACT FROM AUTHOR]

BACKGROUND Male factor and idiopathic infertility contribute significantly to global infertility, with abnormal testicular gene expression considered to be a major cause. Certain types of male infertility are caused by failure of the sperm to activate the oocyte, a process normally regulated by calcium oscillations, thought to be induced by a sperm-specific phospholipase C, PLCzeta (PLCζ). Previously, we identified a point mutation in an infertile male resulting in the substitution of histidine for proline at position 398 of the protein sequence (PLCζH398P), leading to abnormal PLCζ function and infertility. METHODS AND RESULTS Here, using a combination of direct-sequencing and mini-sequencing of the PLCζ gene from the patient and his family, we report the identification of a second PLCζ mutation in the same patient resulting in a histidine to leucine substitution at position 233 (PLCζH233L), which is predicted to disrupt local protein interactions in a manner similar to PLCζH398P and was shown to exhibit abnormal calcium oscillatory ability following predictive 3D modelling and cRNA injection in mouse oocytes respectively. We show that PLCζH233L and PLCζH398P exist on distinct parental chromosomes, the former inherited from the patient's mother and the latter from his father. Neither mutation was detected utilizing custom-made single-nucleotide polymorphism assays in 100 fertile males and females, or 8 infertile males with characterized oocyte activation deficiency. CONCLUSIONS Collectively, our findings provide further evidence regarding the importance of PLCζ at oocyte activation and forms of male infertility where this is deficient. Additionally, we show that the inheritance patterns underlying male infertility are more complex than previously thought and may involve maternal mechanisms. [ABSTRACT FROM AUTHOR]

Background: Mammalian oocyte activation occurs via a series of intracellular calcium (Ca(2+)) oscillations thought to be induced by a sperm-specific phospholipase C zeta (PLCζ). There is now strong evidence to indicate that certain types of human male infertility are caused by failure of the sperm to activate the oocyte in an appropriate manner. Molecular analysis of the PLCζ gene of a male patient with oocyte activation deficiency has previously identified a point mutation causing a histidine to proline substitution at PLCζ residue 398 (PLCζ(H398P)), leading to abnormal Ca(2+) release profiles and reduced oocyte activation efficiency.Methods and Results: In the present study, we used HEK293T cells to produce recombinant human wild-type PLCζ (PLCζ(WT)) protein which, upon microinjection into mouse oocytes, induced Ca(2+) oscillations characteristic of oocyte activation. Injection of recombinant PLCζ(H398P) was unable to elicit Ca(2+) oscillations in mouse oocytes. Loss of activity mutations, such as PLCζ(H398P) and an artificially induced frameshift mutation (PLCζ(ΔYC2)) did not affect Ca(2+) release when over-expressed in HEK293T cells, whereas PLCζ(WT) inhibited adenosine triphosphate-activated Ca(2+) release. Confocal imaging of fluorescently tagged PLCζ isoforms in HEK293T cells suggested a cytoplasmic pattern of localization, while quantitative analysis of fluorescence levels showed that PLCζ(WT) > PLCζ(H398P) > PLCζ(ΔYC2), indicating that loss of activity mutations may lead to protein instability. This was further indicated by the low proportion of sperm and the lower levels of total PLCζ immunofluorescence from the patient exhibiting PLCζ(H398P) compared with fertile controls.Conclusions: We demonstrate, for the first time, the production of active recombinant human PLCζ protein which retained the ability to elicit characteristic Ca(2+) oscillations in mouse oocytes, an ability which was eliminated by an infertility-linked mutation. These findings advance our understanding of PLCζ, and provide a critical step forward in obtaining purified PLCζ protein as a potential therapeutic agent for oocyte activation deficiency. [ABSTRACT FROM AUTHOR]

OBJECTIVE Cryopreservation of supernumerary embryos resulting from IVF treatment offers extra chances to conceive. The objective of this study is to describe patients’ decisions to continue or discontinue storage of their embryos after a minimum storage period of 2 years. METHODS Female patients who had embryos stored at the Infertility Centre of the Ghent University Hospital (Belgium) were sent a mail questionnaire to be completed anonymously. RESULTS The questionnaire had a response rate of 79% (326/412). After an embryo storage period of at least 2 years, 40% of the couples who were still together wished to continue storage of their embryos. Half of these had no concrete plans for a transfer and wanted to postpone the decision or keep all options open. For those who decided to discontinue storage (60%), the main reason was the completion of their families. Despite the fact that the patients’ child wish was the main factor in their storage decision, two groups of patients with distinct profiles made decisions that were inconsistent with their child wish: those who wanted to continue storage while not wanting a(nother) child (7% of those with no child wish), and those who wanted a(nother) child but decided to discontinue storage (25% of those with a child wish). Overall, these patients more often expressed emotional difficulties regarding this decision. CONCLUSIONS This study demonstrates the importance of gaining more insight into patients’ embryo storage decisions (along with their embryo disposition decisions) and into the emotional factors playing a role in patients’ decision-making. [ABSTRACT FROM AUTHOR]

BACKGROUND In some couples, not all retrieved oocytes mature, even after prolonged in vitro culture. The underlying mechanisms are not known, although ionophore treatment may alleviate metaphase I (MI) arrest in some mouse strains. We attempted to induce first polar body (PB) extrusion and fertilization using assisted oocyte activation (AOA) after ICSI in maturation-resistant human MI oocytes. METHODS Four ICSI patients are described in this retrospective study. A pilot study tested the calcium ionophore ionomycin (10 µM) on donated MI oocytes from patients with a normal number of metaphase II (MII) oocytes. Subsequently, ionomycin was used to induce first PB extrusion in two patients showing maturation-resistant MI oocytes. AOA, by calcium injection and ionomycin exposure, was applied when mature oocytes were available. Oocytes were analysed by polarized microscopy and immunostaining. RESULTS Ionomycin induced the first PB extrusion in MI oocytes from patients with a normal number of retrieved MII oocytes, while extended in vitro culture failed to achieve the MII stage. Similarly, ionomycin induced first PB extrusion in one of two patients with recurrent maturation-resistant MI oocytes. Use of ICSI combined with AOA on MII oocytes matured in vitro or in vivo resulted in failed or abnormal fertilization with no further embryo cleavage potential. Highly abnormal spindle and chromosome configurations were observed in MI maturation-resistant oocytes, in contrast to control MI oocytes. CONCLUSIONS Ionophore induced first PB extrusion in MI oocytes from patients without maturation arrest but to a lower extent in maturation-resistant MI oocytes. Immunofluorescence staining and confocal analysis revealed, for the first time, highly abnormal spindle/chromosomal structures that may be responsible for this maturation arrest. [ABSTRACT FROM AUTHOR]