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STUDY QUESTION Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? SUMMARY ANSWER AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. WHAT IS KNOWN ALREADY The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. STUDY DESIGN, SIZE, DURATION This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient’s sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm–oocyte-activating factor PLCZ1. MAIN RESULTS AND THE ROLE OF CHANCE We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient’s sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. LIMITATIONS, REASONS FOR CAUTION Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. WIDER IMPLICATIONS OF THE FINDINGS Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. TRIAL REGISTRATION NUMBER NCT03354013

STUDY QUESTION Can spindle transfer (ST) overcome inferior embryonic development of in vitro matured ovarian tissue oocytes (OTO-IVM) originating from testosterone-treated transgender men? SUMMARY ANSWER ST shows some potential to overcome the embryo developmental arrest observed in OTO-IVM oocytes from transgender men. WHAT IS KNOWN ALREADY OTO-IVM is being applied as a complementary approach to increase the number of oocytes/embryos available for fertility preservation during ovarian tissue cryopreservation in cancer patients. OTO-IVM has also been proposed for transgender men, although the potential of their oocytes remains poorly investigated. Currently, only one study has examined the ability of OTO-IVM oocytes originating from transgender men to support embryo development, and that study has shown that they exhibit poor potential. STUDY DESIGN, SIZE, DURATION Both ovaries from 18 transgender men undergoing oophorectomy were collected for the purposes of this study, from November 2020 to September 2022. The patients did not wish to cryopreserve their tissue for fertility preservation and donated their ovaries for research. All patients were having testosterone treatment at the time of oophorectomy and some of them were also having menses inhibition treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS Sibling ovaries were collected in either cold or warm medium, to identify the most optimal collection temperature. Cumulus oocyte complexes (COCs) from each condition were isolated from the ovarian tissue and matured in vitro for 48 h. The quality of OTO-IVM oocytes was assessed by calcium pattern releasing ability, embryo developmental competence following ICSI, and staining for mitochondrial membrane potential. In vitro matured metaphase I (MI) oocytes, germinal vesicle (GV) oocytes, and in vivo matured oocytes with aggregates of smooth endoplasmic reticulum (SERa) were donated from ovarian stimulated women undergoing infertility treatment and these served as Control oocytes for the study groups. ST was applied to overcome poor oocyte quality. Specifically, enucleated mature Control oocytes served as cytoplasmic recipients of the OTO-IVM spindles from the transgender men. Embryos derived from the different groups were scored and analysed by shallow whole genome sequencing for copy number variations (CNVs). MAIN RESULTS AND THE ROLE OF CHANCE In total, 331 COCs were collected in the cold condition (OTO-Cold) and 282 were collected in the warm condition (OTO-Warm) from transgender men. The maturation rate was close to 54% for OTO-Cold and 57% for OTO-Warm oocytes. Control oocytes showed a calcium releasing ability of 2.30 AU (n = 39), significantly higher than OTO-Cold (1.47 AU, P = 0.046) oocytes (n = 33) and OTO-Warm (1.03 AU, P = 0.036) oocytes (n = 31); both values of calcium release were similar between the two collection temperatures. Mitochondrial membrane potential did not reveal major differences between Control, OTO-Warm, and OTO-Cold oocytes (P = 0.417). Following ICSI, 59/70 (84.2%) of Control oocytes were fertilized, which was significantly higher compared to 19/47 (40.4%) of OTO-Cold (P LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Due to the limited access to human oocytes and ovarian tissue, our results should be interpreted with some caution, as only a limited number of human oocytes and embryos could be investigated. WIDER IMPLICATIONS OF THE FINDINGS The results of this study, clearly indicate that OTO-IVM oocytes originating from transgender patients are of inferior quality, which questions their use for fertility preservation. The poor quality is likely to be related to cytoplasmic factors, supported by the increased blastocyst numbers following application of ST. Future research on OTO-IVM from transgender men should focus on the cytoplasmic content of oocytes or supplementation of media with factors that promote cytoplasmic maturation. A more detailed study on the effect of the length of testosterone treatment is also currently missing for more concrete guidelines and guidance on the fertility options of transgender men. Furthermore, our study suggests a potentially beneficial role of experimental ST in overcoming poor embryo development related to cytoplasmic quality. STUDY FUNDING/COMPETING INTEREST(S) A.C. is a holder of FWO grants (1S80220N and 1S80222N). A.B. is a holder of an FWO grant (1298722N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) and 2018000504 (GOA030-18 BOF) funding. B.H. has additional grants from FWO-Vlaanderen (Flemish Fund for Scientific Research, G051516N and G1507816N) and Ghent University Special Research Fund (Bijzonder Onderzoeksfonds, BOF funding (BOF/STA/202109/005)), and has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

Background Bacterial vaginosis (BV) is a condition that, if symptomatic, is characterized by discharge and odor, with high recurrence rates even when treated. This study aims to review what literature exists on the association between BV and the emotional, sexual, and social health of women. Methods MEDLINE, Embase and Web of Science databases were searched from inception until November 2020. Studies reporting an association between women’s emotional, sexual and/or social health and symptomatic BV in a qualitative and/or quantitative manner were included. Selected studies were divided in three categories, i.e. reporting on the emotional, sexual and/or social association. All studies were critically evaluated and discussed. Results Sixteen studies were included. Concerning emotional health, we found eight studies that calculated the association between stress and BV, in four this was statistically significant. Four qualitative studies on emotional health showed that the severity of the symptoms influenced the impact on women’s lives. All studies on sexual health reported that many women experienced an impact on their relationship and sexual intimacy. Results for social life ranged from no association found to most of the study population showing avoidance behavior. Conclusion This review shows that symptomatic BV can be associated with diminished emotional, sexual, and social health, but there is too little evidence to state the extent of this association.

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.

Study question How do patients perceive the pre-IVF treatment communication by the nursing and midwifery team and how do they evaluate implemented optimization? Summary answer Patient satisfaction with the pre-IVF treatment communication by the nursing and midwifery team improved from 86% to 98% after implementation of standardized written patient information. What is known already Nurses are the main point of contact for patients undergoing medically assisted reproduction techniques (Applegarth et al., 2008). Morris (2001) suggested that infertility nurses could more specifically be involved in informative counselling in anticipation of the fertility treatment. Specific training for nurses and midwives would be required in order to achieve that central role in effective fertility counselling (Applegarth et al., 2012). However, little has been published in peer reviewed literature on how to assess and improve these pre-IVF treatment information sessions. Study design, size, duration The pre-IVF communication by the nurse/midwife was assessed by female patients with a self-developed questionnaire (cohort 1). The nursing and midwifery team was subsequently informed about the results of the questionnaire and efforts were undertaken to optimise future patient communication. Four years later, a follow-up questionnaire assessed patient satisfaction about the intervention (cohort 2). Each cohort comprised fifty patients and nurses and midwives were blinded for patient participation to the study. Participants/materials, setting, methods The study was performed at an academic fertility centre and was approved by the Investigational Review Board. Data were collected with SurveyMonkey (pre intervention) and REDCap (post intervention). A descriptive analysis of the cohorts and patient feedback was performed. The intervention consisted of (1) the optimization of patient information documents if needed (2) providing a training to the nursing and midwifery staff, and (3) a follow-up questionnaire in a second cohort of female fertility patients. Main results and the role of chance The first questionnaire revealed that overall, patients were satisfied with the informative counselling session in anticipation of the IVF treatment. However, some patients indicated that they received a lot of information at once and that specific information that was of relevance for the further treatment (e.g. oocyte pick-up) was sometimes missed. Also, the data showed that not all patients received the same information. Therefore, all information that patients needed during an IVF treatment was gathered into a patient binder. It was supplemented with a timeline of the whole IVF trajectory, QR codes to movies explaining the administration of medication, and answers to common patients’ questions. It was possible to individualize the content of the binder per patient. Ideally, patients received this binder before the informative counselling session so they could read the content in advance. The binder was developed by a member of the nursing and midwifery team and was reviewed by the medical, laboratory, and administrative staff. Then, the nursing and midwifery team was trained on the use of the patient binder. The follow-up questionnaire revealed that patient satisfaction with the pre-IVF treatment counselling was 98% after the optimization of patient documentation (compared to 86% before the optimization). Limitations, reasons for caution Eligible patients were recruited by the treating physician. As no records were kept of the number of solicited patients for this study, no assessment of the response rate is possible. The cohorts were self-selected and limited in size and could therefore not reflect the general patient population. Wider implications of the findings A patient questionnaire and follow-up is a useful tool for a centre specific assessment and improvement of pre-IVF nurse/midwife communication with patients. Clinics could make efforts to invest in complete and written information and ask colleagues of the nursing and midwifery team to be involved in its creation. Trial registration number NCT04420169

Study question Could collection temperature and spindle transfer (ST) potentially improve development of embryos derived from in vitro matured (IVM) ovarian tissue oocytes (OTO) of transgender men? Summary answer Spindle transfer, but not collection temperature, significantly improved embryo development of OTO-IVM oocytes from transgender men. What is known already For transgender men, the fertility preservation strategy of ovarian stimulation may interfere with the desired masculine characteristics and enhance gender dysphoria. Alternatively, ovarian tissue oocytes collected ex vivo could serve as potential gametes, not requiring ovarian stimulation. Oocytes can be collected during gender affirming surgery, matured, and vitrified. Ovarian tissue oocyte in vitro maturation (OTO-IVM) has successfully been used for cancer patients, as live births have been reported. OTO-IVM in transgender men demonstrated sufficient maturation rates and survival following vitrification. Nevertheless, a decreased fertilization potential of these oocytes and severely compromised embryonic development have been observed. Study design, size, duration Patients between 18-24 years were recruited for this study from November 2020 to September 2021. Ovaries from 14 transgender men were collected in either cold (4oC, OTO-Cold) or warm (37oC, OTO-Warm) collection medium, to verify the best collection method. Following ovarian manipulation, cumulus oocyte complexes (COCs) were harvested from spent medium and underwent maturation for 48hrs. ST was performed to overcome inferior fertilization and embryonic development. Participants/materials, setting, methods Injected IVM oocytes underwent calcium imaging or were monitored for embryonic developmental potential. In vitro matured GV (germinal vesicle), MI (metaphase I) and in vivo matured oocytes with clusters of smooth endoplasmic reticulum (SERa) served as controls and cytoplasmic recipients for ST. OTO-IVM or control oocytes were used as spindle donors (ST-OTO or Control-ST respectively). Genetic analysis was performed to detect chromosomal abnormalities in embryos from all groups. Main results and the role of chance In total, we collected 252 OTO-Cold and 230 OTO-Warm oocytes, showing similar maturation rates (53%). For calcium imaging, 39 control, 33 OTO-cold and 31 OTO-warm oocytes were analysed, determining the product of amplitude per frequency, in arbitrary units (AU). The average value for control oocytes was 2.30AU, significantly higher than OTO-Cold (1.47AU, p=0.046) and OTO-Warm oocytes (1.03AU, p=0.036). Calcium release was similar between OTO-Cold and OTO-Warm oocytes. Following ICSI, 19/47 OTO-Cold and 24/48 OTO-Warm oocytes normally fertilized, significantly lower than the control group (42/52) (p Limitations, reasons for caution A major limitation of our study is the lack of ovaries from cis women. Control oocytes used in this study originate from infertility patients that underwent ovarian stimulation. High abnormality rate in ST-OTO embryos might be concerning for the safety of ST, but the number of embryos analysed is limited. Wider implications of the findings Our data indicate that OTO-IVM oocytes from transgender men display poor cytoplasmic quality, demonstrated by embryonic arrest and calcium imaging. ST was able to overcome poor embryo development, and it could be of interest to use freshly donated oocytes as cytoplasmic recipients for this. Trial registration number Not applicable

Gender dysphoria, a discrepancy between gender identity and genetically determined sex, is encountered in approximately 0.5% of people uniformly across the world. In the case of transgender men, formerly called female-to-male transsexuals, the available gender-affirming measures, hormone therapy and possible surgical procedures, are multiple and discussed in detail in this series of articles.

Research question Is implantation impaired in patients with endometriosis undergoing IVF and intracytoplasmatic sperm injection (ICSI) cycles? Design A retrospective matched cohort study was carried out on IVF/ICSI cycles with fresh single embryo transfer at the Department of Assisted Reproductive Medicine, Ghent University Hospital, Belgium, between July 2015 and August 2017 (n = 1053). A total of 118 endometriosis cases were matched 1:1 to 118 couples diagnosed with male subfertility and stratified by embryo quality (identical ALPHA grading categories), female age (±1 year) and parity (±1 delivery). Transvaginal ultrasound, magnetic resonance imaging or laparoscopy was used to diagnosed endometriosis, and the revised American Society for Reproductive Medicine score was used to classify the endometriosis into grade I/II versus grade III/IV. Male subfertility was defined in accordance with World Health Organization criteria (fifth edition). Results Compared with endometriosis cases, control couples with male subfertility had significantly higher rates of positive HCG test on day 16 (P = 0.047, OR 2.077, CI 1.009 to 4.276), ongoing implantation (defined as a positive fetal heart rate on transvaginal ultrasound at a gestational age of at least 6.5–7 weeks) (P = 0.038, OR 2.265, CI 1.048 to 4.893), ongoing pregnancy (defined by a vital pregnancy at 11 weeks) (P = 0.046, OR 2.292, CI 1.016 to 5.173) and live birth (P = 0.043, OR 2.502, CI 1.029 to 6.087). Conclusions After matching for embryo quality, woman's age and parity, rates of positive HCG tests, ongoing implantation, ongoing pregnancy and live birth were more than twice as high in the control group compared with the endometriosis group.

INTRODUCTION: Emotions are theorized to contain the components of affect and action readiness. Affect guides behavior by causing an approach or withdrawal orientation. Action readiness is the individual's degree of willingness to interact with the environment. Emotions contribute to changes in behavior and physiological responses. AIM: The present study applied these notions to sexuality and examined the associations between affect, action readiness, and sexual functioning. METHODS: Participants were male patients with urologic condition (N = 70) with and without sexual problems. MAIN OUTCOME MEASURE: Affect and action readiness were jointly assessed using the latent factor of affective polarity of the Positive and Negative Affect Schedule. Trait affective polarity was assessed questioning generally experienced feelings. State affective polarity was assessed after exposure to an erotic stimulus and questioning momentaneously experienced feelings. Sexual functioning was assessed using the International Index of Erectile Functioning questionnaire. RESULTS: A significant increase of approach-oriented action readiness was found after erotic stimulation, relative to trait levels. In addition, significant associations were found between state approach-oriented action readiness and various aspects of sexual functioning. Interventions based on principles of positive psychology might be developed to reinforce action readiness in men with erectile dysfunction. The strength of the current research concerns the introduction of action readiness as a potential psychological factor implied in sexual functioning. Limitations pertain to the use of the algorithm used to calculate state approach-oriented action readiness and the use of the current sample of patients with urological conditions, limiting generalizability of findings. CONCLUSION: Action readiness was found to correlate positively with all aspects of sexual functioning. Further research into the role of action readiness in sexuality is recommended. Henckens MJMJ, de Vries P, Janssen E, et al. Associations of Affect, Action Readiness, and Sexual Functioning. Sex Med 2020;8:691-698. ispartof: SEXUAL MEDICINE vol:8 issue:4 pages:691-698 ispartof: location:England status: published

Background Advanced maternal age and obesity are associated with impaired female fertility. Moreover, fatty acids (FA) in follicular fluid (FF) play important roles in oocyte maturation and embryo development. However, the effects of body mass index (BMI), age, and FF FA composition on embryo development between days 3 and 5 and blastocyst stage on day 5 are still unclear. Methods This study included 138 patients undergoing assisted reproductive technology (ART), which were divided into three BMI groups (18.5–24.9 kg/m2 vs. 25.0–29.9 kg/m2 vs. ≥ 30.0 kg/m2) and three age-related groups (20–30 years vs. 31–34 years vs. ≥ 35 years) which were compared for ART outcomes. Further, observations were divided into quartiles based on either of three parameters related to embryo outcome, i.e. (i) embryos developing between days 3 and 5 (ED3-5) and (ii) expanded blastocysts on day 5 (EB5), both expressed proportionally to the number of oocytes with two pronuclei (2PN), as well as (iii) the embryo utilization rate (EUR). Proportions of FF FA were then compared between Q1 and Q4, representing the quartile with the worst vs. the best embryo outcome, respectively. Finally, regression models were created to assess the relationships between BMI, age, FF total FA (TFA) concentration, relative proportions of specific FA and embryo outcome. Results Patients of Q1 had higher proportions of FF C20:5n-3, C22:6n-3 and total n-3 PUFA than Q4 patients. Furthermore, Q4 patients tended to be younger than Q1 patients. Within the whole cohort, the proportion of C20:5n-3 negatively correlated with ED3-5/2PN and EUR, while EB5/2PN tended to be negatively correlated with age. Regression models within the overweight and obese group confirmed the negative relation between C20:5n-3 and ED3-5/2PN, but also indicated additional associations: C18:1n-9 and C20:4n-6 were positively associated with ED3-5/2PN and EUR, respectively while the proportion of C18:0 was negatively associated with EUR. Conclusion The proportions of n-3 PUFA, particularly C20:5n-3 and C22:6n-3 were reduced in the patients’ quartile with the best embryo outcome. This group of patients was also younger. However, the embryo quality parameters of overweight/obese patients were not associated with age but were positively associated with FF C18:1n-9 and negatively with the proportions of C18:0 or C20:5n-3. Trial registration This study’ registration number was B670201627735.

PURPOSE: Providing additional insights on the efficacy of human nuclear transfer (NT). Here, and earlier, NT has been applied to minimize transmission risk of mitochondrial DNA (mtDNA) diseases. NT has also been proposed for treating infertility, but it is still unclear which infertility indications would benefit. In this work, we therefore additionally assess the applicability of NT to overcome failed fertilization. METHODS: Patient 1 carries a homoplasmic mtDNA mutation (m.11778G > A). Seventeen metaphase II (MII) oocytes underwent pre-implantation genetic testing (PGT), while five MII oocytes were used for spindle transfer (ST), and one in vitro matured (IVM) metaphase I oocyte underwent early pronuclear transfer (ePNT). Patients 2–3 experienced multiple failed intracytoplasmic sperm injection (ICSI) and ICSI-assisted oocyte activation (AOA) cycles. For these patients, the obtained MII oocytes underwent an additional ICSI-AOA cycle, while the IVM oocytes were subjected to ST. RESULTS: For patient 1, PGT-M confirmed mutation loads close to 100%. All ST-reconstructed oocytes fertilized and cleaved, of which one progressed to the blastocyst stage. The reconstructed ePNT-zygote reached the morula stage. These samples showed an average mtDNA carry-over rate of 2.9% ± 0.8%, confirming the feasibility of NT to reduce mtDNA transmission. For patient 2–3 displaying fertilization failure, ST resulted in, respectively, 4/5 and 6/6 fertilized oocytes, providing evidence, for the first time, that NT can enable successful fertilization in this patient population. CONCLUSION: Our study showcases the repertoire of disorders for which NT can be beneficial, to overcome either mitochondrial disease transmission or failed fertilization after ICSI-AOA. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-022-02401-7.

PURPOSE: To examine the intra- and inter-individual variability in fatty acid composition of follicular fluid (FF) of 23 patients undergoing assisted reproductive treatment. METHODS: The average coefficient of variation within each patient (CV(w)) and intra-class correlation coefficient (ICC) values of FF fatty acid composition as well as correlation between the fatty acid composition of individual, pooled or first-punctured follicles, were assessed. RESULTS: The proportions of 16:0, 18:0, cis-9 18:1, 18:2n-6, 20:5n-3, total MUFA and n-3 PUFA showed good reproducibility (CV(w)

Research question Is there a difference in blastocyst formation between fresh and vitrified-warmed sibling oocytes and can this difference be attributed to changes in embryo morphokinetics? Design Between February 2016 and December 2017, 472 metaphase II (MII) oocytes in 67 donor-recipient cycles from 27 different healthy anonymous oocyte donors were allocated for fresh transfer (FSHO) (n = 220) to a synchronous recipient (n = 36) or vitrified (VITO) (n = 252) to be warmed and transferred to another recipient (n = 31). Embryos derived from the FSHO and their sibling VITO were analysed for morphokinetic development using time-lapse imaging, blastocyst formation and clinical outcome. Results Time-lapse analysis showed an overall delay in cleavage rate from the time of pronuclei disappearance up to the time of blastulation in the VITO compared with their sibling FSHO. Twelve morphokinetic variables were significantly different between the groups. On Day 5 significantly more FSHO embryos developed to blastocyst (expansion 1-6) and reached the full blastocyst stage (expansion 3-6) compared with the VITO embryos [53.2% (84/158) versus 40.0% (64/160); P = 0.0244 and 48.1% (76/158) versus 31.3% (50/160); P = 0.0028, respectively]. The embryo utilization rate was similar in both groups at the time of cryopreservation; 51.3% (FSHO) versus 45.0% (VITO) (P = 0.3124). The pregnancy rate per cycle was 47.2% (17/36) in FSHO patients and 48.4% (15/31) in VITO patients (P = 1). Limitations in this study: non-randomized, small study size and not powered to detect differences in clinical outcomes. Conclusions Timing of development is altered and blastocyst formation is delayed in embryos derived from vitrified-warmed donor oocytes compared with their fresh sibling counterparts. Although preliminary results suggest that the clinical impact of this delay may be limited, this needs further investigation in larger randomized studies.

Research question To what extent does vitrification affect the Ca2+-releasing and activation potential of mouse oocytes, which are commonly used to determine the oocyte activation potential of human spermatozoa? Design The effect of mouse oocyte vitrification on Ca2+ dynamics and developmental competence after oocyte activation was assessed and compared with fresh mouse oocytes. Moreover, the Ca2+ store content of the endoplasmic reticulum was determined at different time points during the vitrification–warming procedure. Finally, the Ca2+ pattern induced by cryoprotectant exposure was determined. Results After human sperm injection into mouse oocytes, Ca2+ dynamics but not fertilization rates were significantly altered by vitrification warming (P 0.05), whereas activation rates after ionomycin exposure were significantly lower in vitrified–warmed oocytes (P 0.05). Cryoprotectant exposure was associated with a strong drop in endoplasmic reticulum Ca2+ store content. Oocytes rapidly recovered during warming and recovery in Ca2+-containing media; a threshold area under the curve of Ca2+ dynamics to obtain activation rates above 90% was determined. Conclusions Vitrified–warmed mouse oocytes display reduced Ca2+-releasing potential upon oocyte activation, caused by cryoprotectant exposure. With adapted classification criteria, these oocytes could be used for diagnosing oocyte activation deficiencies in patients. Evaluating the Ca2+-signalling machinery in vitrified–warmed human oocytes is required.

Objective To investigate the extent to which assisted oocyte activation (AOA) improves clinical outcomes in patients diagnosed with oocyte activation deficiencies (OADs). Design Retrospective cohort study comparing AOA cycles and previous intracytoplasmic sperm injection (ICSI) cycles in couples experiencing low or total failed fertilization after ICSI. Importantly, the sperm-related oocyte-activating capacity was examined in all patients before AOA with the use of the mouse oocyte activation test (MOAT). Setting Infertility center at a university hospital. Patient(s) A total of 122 couples with a history of low or total failed fertilization after ICSI. Intervention(s) ICSI, MOAT, AOA, and embryo transfer. Main Outcome Measure(s) Fertilization, pregnancy, and live birth rates. Result(s) MOAT revealed 19 patients with a sperm-related OAD (MOAT group 1), 56 patients with a diminished sperm-related oocyte-activating capacity (MOAT group 2), and 47 patients with a suspected oocyte-related OAD (MOAT group 3). AOA (191 cycles) significantly improved fertilization, pregnancy, and live birth rates in all MOAT groups compared with previous ICSI attempts (243 cycles). Fertilization rates after AOA were significantly different among MOAT groups 1 (70.1%), 2 (63.0%), and 3 (57.3%). Between MOAT group 1 and 3, significant differences in pregnancy (49.0% vs. 29.4%) and live birth (41.2% vs. 22.1%) rates were observed. In total, 225 embryo transfers resulted in 60 healthy live births following AOA. Conclusion(s) Patients undergoing diagnostic testing before AOA show a significant improvement in clinical outcomes compared with previous cycles. Our findings highlight that AOA should be reserved for patients with clear OADs.

Purpose The importance of the surrounding ovarian stromal cells and extracellular matrix in the development and maturation of follicles has recently gained attention. An aberrant extracellular matrix has been described in ovaries of patients with polycystic ovary syndrome where a more rigid structural environment, possibly induced by endogenous testosterone, impairs normal folliculogenesis. In this context, we describe the textural parameters of the ovarian cortex of transgender men after prolonged testosterone administration compared to the textural parameters of the non-exposed ovarian cortex originating from female oncological patients. Methods Texture profile analysis (TPA) was performed on ovarian cortex (5 × 5 mm) of oncological and transgender patients in order to measure stiffness, hardness, cohesiveness, and springiness of the ovarian cortex (LRXplus universal testing system). Statistical analysis was performed using repeated measurements mixed models and the Spearman rank order correlation test (IBM SPSS Statistics 23). Results A total of 36 frozen-thawed cortical strips (5 × 5 mm) were subjected to TPA. The superficial part of cortex fragments originating from transgender persons (fragments < 1.4 mm; N = 10) appeared to be significantly stiffer compared to cortex derived from oncology patients (fragments < 1.4 mm; N = 7) (6.78 ± 1.38 N/mm versus 5.41 ± 0.9 N/mm respectively, p = 0.036). Conclusions This is the first application of TPA in ovarian cortex to study the physical properties. Comparing the physical properties, we objectively describe an increased cortical stiffness in the most outer part of the ovarian cortex following prolonged testosterone administration in transgender men compared to the ovarian cortex of oncological patients. This preliminary and novel approach could be the start of future research to understand the physical properties of ovarian tissue. Electronic supplementary material The online version of this article (10.1007/s10815-019-01513-x) contains supplementary material, which is available to authorized users.

Research question Can oocyte-related activation deficiencies be evaluated in oocytes that failed to fertilize after intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA)? Design Evaluation of the spindle–chromosome complexes and intracellular distribution of inositol trisphosphate type 1 receptors (IP3R1) in in-vitro matured (IVM) and failed-to-fertilize oocytes from patients undergoing AOA. Assessment of the oocyte-related Ca2+ releasing capacity in response to Ca2+ ionophores and sperm microinjection in oocytes that failed to fertilize after ICSI or ICSI-AOA. Results IVM oocytes from patients undergoing conventional ICSI (control) and ICSI-AOA (study group) revealed a similar normalcy of spindle–chromosome complexes and distribution patterns of IP3R1. Failed-to-fertilize oocytes from both groups showed significant differences in proportion of normal or abnormal spindle–chromosome complex conformations. However, migration of IP3R1 was identified in a higher proportion of failed-to-fertilize oocytes after ICSI-AOA than after conventional ICSI. It was further observed that oocytes which failed to fertilize, either after ICSI or ICSI-AOA, mostly retain their capacity to respond to stimuli such as exposure to Ca2+ ionophores or to sperm microinjection. Conclusions Evaluation of spindle–chromosome normalcy and distribution of IP3R1 does not help identify the presence of Ca2+ releasing deficiencies in these oocytes. However, oocyte Ca2+ analysis adds value in identifying Ca2+ releasing incapacity of oocytes that failed to fertilize after ICSI or ICSI-AOA. Some patients experiencing fertilization failure after ICSI-AOA present with a suspected activation deficiency downstream of the Ca2+ machinery, which cannot be overcome by ICSI-AOA based on the use of Ca2+ ionophores.

Objective: To develop a random forest model (RFM) to predict implantation potential of a transferred embryo and compare it with a multivariate logistic regression model (MvLRM), based on data from a large cohort including in vitro fertilization (IVF) patients treated with the use of single-embryo transfer (SET) of blastocyst-stage embryos. Design: Retrospective study of a 2-year single-center cohort of women undergoing IVF or intracytoplasmatic sperm injection (ICSI). Setting: Academic hospital. Patient(s): Data from 1,052 women who underwent fresh SET in IVF or ICSI cycles were included. Intervention(s): None. Main Outcome Measure(s): The performance of both RFM and MvLRM to predict pregnancy was quantified in terms of the area under the receiver operating characteristic (ROC) curve (AUC), classification accuracy, specificity, and sensitivity. Result(s): ROC analysis resulted in an AUC of 0.74 ± 0.03 for the proposed RFM and 0.66 ± 0.05 for the MvLRM for the prediction of ongoing pregnancies of ≥11 weeks. This RFM approach and the MvLRM yielded, respectively, sensitivities of 0.84 ± 0.07 and 0.66 ± 0.08 and specificities of 0.48 ± 0.07 and 0.58 ± 0.08. Conclusion(s): The performance to predict ongoing implantation will significantly improve with the use of an RFM approach compared with MvLRM.

Study question Can clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing result in the correction of a single base pair substitution that causes male infertility? Summary answer CRISPR/Cas9 administration during intracytoplasmic sperm injection (ICSI) leads to correction attempts of mutant phospholipase C zeta (PLCζ), howeverc loss-of-heterozygosity (LOH). What is known already Failed fertilization after ICSI can be caused by mutations in the sperm-related oocyte factor PLCζ which can be overcome by assisted oocyte activation (AOA). In this way, children may inherit the infertility-causing mutation. Mutation transmission can be overcome through CRISPR/Cas9 delivery during ICSI. In previous studies using CRISPR/Cas9 in the human germline for mutation correction, loss-of-heterozygosity (LOH, loss of the allele of one of the parents) was observed. Two different explanations were given, namely partial or complete paternal chromosomal loss or the correction of the mutation by using the maternal wild-type allele instead of the exogeneous supplied repair template. Study design, size, duration We injected a gRNA-Cas9 protein complex to target the PLCζ mutant allele, a repair template harboring the desired nucleotide substitution and an additional synonymous variant to track template usage, together with patient’s sperm. To overcome fertilization failure, AOA was applied during ICSI. After a culture period of maximal 6 days the embryos were collected. At day 3, some embryos were dissociated in individual blastomeres. The extracted DNA was analyzed through different genetic sequencing techniques. Participants/materials, setting, methods Donated sperm of a patient experiencing complete fertilization failure after routine ICSI, harboring a heterozygous base pair substitution in PLCZ1 (c.136-1G>C), was utilized. Sperm was injected in donated in vitro matured oocytes or in vivo matured oocytes containing clusters of smooth endoplasmic reticulum. Next-generation sequencing was used to assess correction potential. Short tandem repeat (STR) and single nucleotide polymorphism (SNP) assays were used to determine whether the sperm contained the mutation and to evaluate LOH. Main results and the role of chance CRISPR/Cas9 injections had no significant impact (p > 0.05) on embryonic development. Due to the heterozygous nature of the mutation, 47% (27/58) of the embryos originated from mutated sperm injection. The CRISPR components showed a high specificity with absence of insertions/deletions in 97% of the embryos originating from wild-type sperm (n = 31). Embryos originating from mutant sperm (n = 27) fall into three categories:(1) 22% showed the untargeted mutant allele, (2) 52% showed additional mutagenesis and (3) 26% showed the wild-type allele, which could be explained by correction. Mosaicism, defined as various editing events, was present in 17% (1), 21% (2) and 71% (3) of the embryos. The low occurrence of the synonymous variant, incorporated in the repair template, suggests that the template is not used during correction attempts. In only 29% (2/7) and 14% (1/7) of the ‘corrected embryos’, respectively long (>18Mb) or medium width LOH (4Mb) was observed through STR analysis. SNP analysis in closer proximity showed in 71% (5/7) of the embryos LOH, even in the absence of LOH through STR, suggesting also the occurrence of short width LOH. These results will be studied in more detail before definitive conclusions can be made. Chromosomal LOH will be studied by ddRADseq. Limitations, reasons for caution The occurrence of mosaicism and LOH might complicate the use of traditional CRISPR/Cas9 in human embryos and should be studied in detail to draw definite conclusions on its potential future use. To this end, genomic data have been produced from both individual blastomeres and whole-embryos which will be further analyzed. Wider implications of the findings Our findings demonstrate caution to use CRISPR/Cas9 to correct mutations in the germ line. They seem to contradict other reports that show predominant lack of mosaicism and presence of long width LOH. A deeper evaluation will be undertaken to define the length and type of LOH in this study. Trial registration number Not Applicable

Study question What is the main pathway regulating trophectoderm (TE) differentiation during pre-implantation development in mouse versus human embryos? Summary answer TEAD4 is acting upstream of CDX2 and is involved in TE differentiation, as TEAD4-null human embryos exhibit compromised TE lineage differentiation. What is known already TEAD4 is the earliest transcription factor during early embryo development, required for the expression of TE-associated genes leading to successful TE differentiation and subsequent blastocoel formation in mouse. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination have shown that Tead4-null embryos do not express TE specific genes, including Caudal-Type Homeobox Protein 2 (Cdx2) and GATA Binding Protein 3 (Gata3), but expression of inner cell mass (ICM)-specific genes, remains unaffected. Furthermore, ablation of Tead4 compromises embryonic development and subsequent blastocoel formation in mouse. The role of TEAD4, during human pre-implantation development has not been functionally characterized yet. Study design, size, duration CRISPR-Cas9 was introduced in mouse zygotes and editing efficiency was evaluated by next-generation sequencing (NGS) on 4.5dpc embryos (n = 55). Developmental kinetics were monitored in CRISPR-Cas9 targeted (n = 83), sham-injected (n = 26) and non-injected media-control (n = 51) mouse embryos. Immunofluorescence analysis was performed in Tead4 targeted (n = 57) and non-injected media-control embryos (n = 94). The same methodology was applied in human donated in vitro matured (IVM) metaphase-II (MII) oocytes, which were CRISPR-Cas9 targeted (n = 74) during ICSI or used as media-Control (n = 33). Participants/materials, setting, methods A gRNA-Cas9 mixture targeting exon 2 of Tead4/TEAD4 was microinjected in respectively mouse 2PN (pronuclear) stage zygotes, or human IVM MII oocytes along with the sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. Embryonic development and morphology were assessed daily, followed by a detailed scoring at the late blastocyst stage. Successful targeting following CRISPR-Cas9 introduction was assessed by immunostaining and NGS analysis of the targeted locus. Main results and the role of chance In mouse, we confirmed previous findings, as the developmental capacity of Tead4 targeted embryos was significantly reduced starting from the morula stage and blastocyst formation rates were 8.97% in the targeted group, compared to 87.23% in the control and 87.50% in the sham group, respectively. Immunofluorescence analysis of late morula and blastocyst stage embryos confirmed the absence of Tead4, Cdx2 and Gata3, resulting from the successful interruption of the Tead4 locus (n = 57). Exon 2 of TEAD4 was successfully targeted in human. In total, 21 embryos from various developmental stages were successfully NGS analyzed and 90,48% (19 out of 21) of the embryos carried genetic modifications as a result of CRISPR-Cas9 genome editing and seven blastocysts were identified carrying exclusively frameshift mutations. In contrast to mouse, the developmental capacity of human targeted embryos (25%) did not differ significantly from the control group (23%). However, the blastocyst morphology and quality were compromised in the targeted group showing mostly grade C TE scores, containing very few cells. Immunofluorescence analysis of targeted blastocysts (n = 6) confirmed successful editing by complete absence of TEAD4 and its downstream TE marker CDX2. Limitations, reasons for caution CRISPR-Cas9 germline genome editing results in multiple editing outcomes with variable phenotypic penetrance, the mosaic nature of which complicates the phenotypic analysis and developmental behaviour of the injected embryos. Wider implications of the findings Elucidation of the evolutionary conserved molecular mechanisms that regulate self-renewal of the trophoblast lineage can give us fundamental insights on early implantation failure. Trial registration number Not Applicable

Study question Can oocytes isolated from transgender men after oophorectomy support embryonic development? Summary answer Embryo developmental arrest at 4–8cell stage (day 3 embryos) indicates poor quality of in vitro matured oocytes from transgender men. What is known already Gender affirming surgery for transgender men leads to permanent infertility, as it involves bilateral oophorectomy. Current approaches for fertility preservation, such as oocyte freezing following ovarian hyperstimulation, may interfere with the wanted masculine characteristics and enhance gender dysphoria. In vitro matured oocytes (IVM) isolated following oophorectomy have been proposed as a source of potential gametes to ensure fertility preservation for transgender men with a child wish. From previous studies, it has been shown that these oocytes are able to undergo maturation and display normal spindles, but their competence to be fertilized and support embryonic development has not been addressed yet. Study design, size, duration We evaluated the quality of in vitro matured oocytes isolated from ovaries of transgender men by applying calcium imaging and monitoring fertilization and embryonic development following intracytoplasmic sperm injection (ICSI). Ovaries were collected in cold (4oC) or warm (37oC) medium, to investigate the best collection procedure. So far, results from four transgender men have been included. Participants/materials, setting, methods: Ovaries from four transgender men undergoing testosterone treatment were collected after oophorectomy in cold or warm medium. Cumulus oocyte complexes (COCs) were isolated and cultured in maturation medium for 48hrs. Mature oocytes were injected with donated sperm and assessed either by calcium imaging, measuring the total calcium release following injection, or following embryonic development. Donated in vitro matured oocytes, germinal vesicle(GV) or metaphase I(MI) origin, from other patients undergoing IVF treatment were used as controls. Main results and the role of chance In total, 179 COCs were collected from ovaries (n = 8) of four transgender men. From the COCs collected in warm medium, 73/105(69%) survived and 33/73(45%) reached metaphase II (MII). Of 21 MII injected with sperm, 13/21(62%) fertilized, 9/21(43%) formed 2 pronuclei (PN), 8/9(89%) reached the 2-cell stage, 3/9(33%) reached 4–8cell stage but arrested. From 74 COCs isolated in cold medium, 57/74(77%) survived and 28/57(49%) matured. Of the 11 MII injected with sperm, 7/11(64%) fertilized, 6/11(54%) formed 2PN, 6/6(100%) reached the 2-cell stage, 4/6(67%) reached 4–8cell but arrested. In the control group, 10/13 oocytes injected with the same sperm sample, were normally fertilized (77%), 8/10(80%) reached the 2-cell stage, 7/10(70%) reached the 4–8cell stage and 4/10(40%) became blastocysts. From the warm, cold and control conditions, respectively 12,14 and 17 MII oocytes were used for calcium imaging. The product of amplitude and frequency of calcium peaks, representing total calcium release, was calculated. Oocytes showed an average release of 0.66AU and 1.69AU for the warm and cold condition, respectively. The average value for control oocytes was 2.19AU. Limitations, reasons for caution One major limitation of our study is the lack of ovaries from cis women as control group. Our control oocytes originated from women undergoing IVF treatment and have undergone ovarian stimulation. Furthermore, the number of oocytes analysed and number of patients per group was limited and is being increased. Wider implications of the findings: Our data indicate that in vitro matured oocytes from transgender men ovaries display poor quality, as demonstrated by the poor embryonic development. In the future, we will apply nuclear transfer technology as a mean to overcome embryonic developmental arrest in this group of oocytes. Trial registration number Not applicable

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.

Objective To study the feasibility of in vitro maturation of ovarian tissue oocytes for fertility preservation in transgender men on testosterone treatment. Design Cross-sectional study Setting University hospital Patient(s) Eighty-three transgender men enrolled from November 2015 to January 2019 Intervention(s) In vitro maturation of cumulus-oocyte complexes (COCs) harvested at the time of gender confirmation surgery, and fertilization through intracytoplasmic sperm injection. Main Outcome Measure(s) In vitro maturation, fertilization, and blastulation rates; comparison of morphokinetics with vitrified-warmed oocytes; and analysis of the genetic profiles of embryos. Secondary outcomes: association between serum hormone levels; COCs’ morphologic characteristics, and vitrification rate. Result(s) All participants were on testosterone treatment for a median of 83 (64[Quartile 1]; 113.2[Quartile 2]) weeks. A total of 1,903 COCs (mean per participant, 23 ± 15.8) were collected. The in vitro maturation rate was 23.8%, vitrification rate was 21.5%, and survival rate after warming was 72.6% (n = 151). Intracytoplasmic sperm injection was performed in 139 oocytes. The rate of normal fertilized oocytes was 34.5%, and 25 (52.1%) embryos reached day 3. One blastocyst was achieved on day 5. Aberrant cleavage patterns and early embryo arrest were observed in 22 (45.8%) and 44 (91.7%) zygotes, respectively. Compared with vitrified-warmed donor oocytes, a delay was observed in pronuclei disappearance, t2 (time to reach 2 cell stage) timings, and CC1 (the duration of the 1st cell cycle) and SS3 (synchronization of cleavage pattern (calculated as t8-t5) time intervals. A normal genetic pattern was seen in 42% embryos. The proportion of vitrified oocytes was negatively associated with progesterone (odds ratio, 0.76) and positively associated with antimullerian hormone serum levels (odds ratio, 1.23). The highest vitrification rate was achieved by the morphologic characteristic 344 at day 0 and by 433 at day 2. Conclusion(s) Ovarian tissue oocytes matured in vitro show low developmental capacity in transgender men, when collected under testosterone treatment.

Mammalian fertilization encompasses a series of Ca2+ oscillations initiated by the sperm factor phospholipase C zeta (PLCζ). Some studies have shown that altering the Ca2+ oscillatory regime at fertilization affects preimplantation blastocyst development. However, assisted oocyte activation (AOA) protocols can induce oocyte activation in a manner that diverges profoundly from the physiological Ca2+ profiling. In our study, we used the newly developed PLCζ-null sperm to investigate the independent effect of AOA on mouse preimplantation embryogenesis. Based on previous findings, we hypothesized that AOA protocols with Ca2+ oscillatory responses might improve blastocyst formation rates and differing Ca2+ profiles might alter blastocyst transcriptomes. A total of 326 MII B6D2F1-oocytes were used to describe Ca2+ profiles and to compare embryonic development and individual blastocyst transcriptomes between four control conditions: C1 (in-vivo fertilization), C2 (ICSI control sperm), C3 (parthenogenesis) and C4 (ICSI-PLCζ-KO sperm) and four AOA groups: AOA1 (human recombinant PLCζ), AOA2 (Sr2+), AOA3 (ionomycin) and AOA4 (TPEN). All groups revealed remarkable variations in their Ca2+ profiles; however, oocyte activation rates were comparable between the controls (91.1% ± 13.8%) and AOA (86.9% ± 11.1%) groups. AOA methods which enable Ca2+ oscillatory responses (AOA1: 41% and AOA2: 75%) or single Ca2+ transients (AOA3: 50%) showed no significantly different blastocyst rates compared to ICSI control group (C2: 70%). In contrast, we observed a significant decrease in compaction (53% vs. 83%) and blastocyst rates (41% vs. 70%) in the absence of an initial Ca2+ trigger (AOA4) compared with the C2 group. Transcription profiles did not identify significant differences in gene expression levels between the ICSI control group (C2) and the four AOA groups.

Research question Does uterine activity differ in patients who have undergone successful IVF treatment compared with patients who have undergone unsuccessful IVF treatment? Design Prospective study of 16 women who underwent fresh single embryo transfer. All patients underwent transvaginal ultrasound in three phases of the IVF treatment: ovarian stimulation 1 h before embryo transfer (ET1) and 5–7 days after embryo transfer (ET5–7). Uterine motion analysis was implemented by a dedicated speckle tracking algorithm; frequency- and amplitude-related features were extracted from the derived signals to characterize the uterine activity in relation to ongoing implantation (positive HCG after 6 weeks) and ongoing pregnancy at 11 weeks. Results Uterine activity in terms of frequency (ovarian stimulation ET1, P = 0.04; ovarian stimulation ET5–7, P = 0.002) and amplitude (ovarian stimulation ET1, P = 0.0003; ovarian stimulation ET5–7, P = 0.000008) is significantly higher in the ovarian stimulation phase compared with ET1 and ET5–7. Women with ongoing pregnancies showed significantly higher uterine contraction frequency compared with those with no ongoing pregnancies in all phases (ovarian stimulation, P = 0.006; ET1, P = 0.015; ET5–7, P = 0.007). Uterine contraction amplitude was significantly lower (P = 0.037) in women at ET5–7 in women with ongoing pregnancies. Conclusions This study is a first step towards assessing uterine activity during IVF objectively and non-invasively. It is an essential step to understanding the previously suggested effect of contractions on IVF failure. Uterine activity after embryo transfer characterized by high frequency and low amplitude may favour embryo implantation. Research with larger patient cohorts is needed to build on current evidence and knowledge of uterine contractions during IVF.

Objective The aim of this study was to evaluate the predictive value of the dynamic morphological development process between cleavage-stage and blastocyst-stage embryos. Study design A retrospective study was executed between 2015 and 2017 at Ghent University Hospital. A total of 996 first fresh IVF/ICSI cycles resulting in a single embryo transfer on day 5 were included. Embryos were scored on day 3 and day 5 as excellent, good, moderate or poor based on Alpha/ESHRE guidelines and Gardner and Schoolcraft scoring-system. If embryos changed category between day 3 and 5, the number of steps (between excellent; good; moderate; poor) in positive and negative direction was expressed. Results On day 5, the ongoing pregnancy rate (OPR) of excellent embryos was 37.4 %. Univariate analyses showed that on day 5, both a higher cell stage, better inner cell mass and better trophectoderm were significantly associated with an ongoing pregnancy. In case of deterioration in quality of individual embryos between day 3 and day 5, the OPR was significantly lower. Conversely, improvement of embryo quality between day 3 and day 5 showed higher ongoing pregnancy rates (overall OPR of good day-3 embryos improving to excellent day-5 embryos: 30 %; moderate day 3 to excellent day 5: 50 %; poor day 3 to excellent day 5: 42 %; poor day 3 to good day 5: 20 %; poor day 3 to moderate day 5: 16 %). When embryos improved from poor on day 3 to excellent day 5 the OPR was significantly higher in comparison with embryos that did not change in quality scoring during development (steady embryos) (OR: 1.785, p Conclusion Our results suggest that it is more likely to achieve an ongoing pregnancy when transferring an embryo that has improved in quality between days 3 and 5 as opposed to one that has remained stable.

Oocyte activation is a calcium (Ca2+)-dependent process that has been investigated in depth, in particular, regarding its impact on assisted reproduction technology (ART). Following a standard model of signal transduction, Ca2+drives the meiotic progression upon fertilization in all species studied to date. However, Ca2+changes during oocyte activation are species specific, and they can be classified in two modalities based on the pattern defined by the Ca2+signature: a single Ca2+transient (e.g. amphibians) or repetitive Ca2+transients called Ca2+oscillations (e.g. mammals). Interestingly, assisted oocyte activation (AOA) methods have highlighted the ability of mammalian oocytes to respond to single Ca2+transients with normal embryonic development. In this regard, there is evidence supporting that cellular events during the process of oocyte activation are initiated by different number of Ca2+oscillations. Moreover, it was proposed that oocyte activation and subsequent embryonic development are dependent on the total summation of the Ca2+peaks, rather than to a specific frequency pattern of Ca2+oscillations. The present review aims to demonstrate the complexity of mammalian oocyte activation by describing the series of Ca2+-linked physiological events involved in mediating the egg-to-embryo transition. Furthermore, mechanisms of AOA and the limitations and benefits associated with the application of different activation agents are discussed.

Objective To describe the decisions lesbian couples make when creating a family through donor insemination [DI] and to explore the negotiations between the biological and the non-biological mother. Methods We included 18 lesbian parents (9 biological and 9 non-biological mothers) with at least one child (7–10 years old) conceived through anonymous DI. We conducted in-depth semi-structured couple interviews at the participants' homes or at the Department of Reproductive Medicine of Ghent University Hospital (Belgium) where participants were treated in the past. The data were analysed using step-by-step inductive thematic analysis based on Braun and Clarke. Results Lesbian couples were confronted with decisions related to two themes: (1) the fertility treatment and (2) the organisation of the family. In this paper we focused on three particular decisions: whether or not to go through treatment together, the acceptance of an anonymous sperm donor, and the celebration of Mother's and Father's Days. Several decisions were linked to the heteronormative social context. The lesbian couples seemed to want to adapt as much as possible to this frame. Conclusion Heteronormativity and the genetic link between parent and child influenced the decision making in lesbian couples creating a family through DI.

Our current knowledge of the mechanisms leading to human primordial germ cell (PGC) specification stems solely from differentiation experiments starting from human pluripotent stem cells. However, information regarding the origin of PGCs in vivo remains obscure. Here we apply an improved system for extended in vitro culture of human embryos to investigate the presence of PGC-like cells (PGCLCs) 12 days post fertilization (dpf). Good quality blastocysts (n = 141) were plated at 6 dpf and maintained in hypoxia, in medium supplemented with Activin A until 12 dpf. We primarily reveal that 12 dpf outgrowths recapitulate human peri-implantation events and demonstrate that blastocyst quality significantly impacts both embryo viability at 12 dpf, as well as the presence of POU5F1+ cells within viable outgrowths. Moreover, detailed examination of 12 dpf blastocyst outgrowths revealed a population of POU5F1+, SOX2– and SOX17+ cells that may correspond to PGCLCs, alongside POU5F1+ epiblast-like cells and GATA6+ endoderm-like cells. Our findings suggest that, in human, PGC precursors may become specified within the epiblast and migrate either transiently to the extra-embryonic mesoderm or directly to the dorsal part of the yolk sac endoderm around 12 dpf. This is a descriptive analysis and as such the conclusion that POU5F1+ and SOX17+ cells represent bona fide PGCs can only be considered as preliminary. In the future, other PGC markers may be used to further validate the observed cell populations. Overall, our findings provide insights into the origin of the human germline and may serve as a foundation to further unravel the molecular mechanisms governing PGC specification in human.

Despite many advancements in the past decades, we are currently being confronted with a threat of regression of SRHR. In several European countries, SRHR-related policies and laws have been made mo...

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 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 0.05). 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 0.05). 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

Introduction 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. Aim 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. Methods 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. Main Outcome Measure 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 Results 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 Clinical Implications 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. Conclusion 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.

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.

Introduction: A lot of attention has been given to the quest of parents, children and donors to find donor siblings (= half siblings who share the same donor gametes but who are born in different families). However, literature is scarce about the use of the same sperm donor for subsequent children in the same family.Methods: This study included 68 lesbian and heterosexual (aspiring) parents, recruited at the Department of Reproductive Medicine of Ghent University Hospital (Belgium). The in-depth semi-structured couple interviews were performed between October 2012 and October 2013. Data were analyzed through step-by-step inductive thematic analysis.Results: The couples showed a clear preference to use the same sperm donor for their children. The most common reasons for this preference were related to the family or sibling relationships and medical reasons. Uncertainty about the availability of the same donor over time seeped through in their stories. Most lesbian aspiring parents decided that both partners should have a genetic link with at least one child.Conclusion: The use of the same sperm donor for subsequent conceptions appeared quasi unambiguously in the interviews of the lesbian and heterosexual (aspiring) parents in our study.

Prevention of mitochondrial DNA (mtDNA) diseases may currently be possible using germline nuclear transfer (NT). However, scientific evidence to compare efficiency of different NT techniques to overcome mtDNA diseases is lacking. Here, we performed four types of NT, including first or second polar body transfer (PB1/2T), maternal spindle transfer (ST) and pronuclear transfer (PNT), using NZB/OlaHsd and B6D2F1 mouse models. Embryo development was assessed following NT and mtDNA carry-over levels were measured by next generation sequencing (NGS). Moreover, we explored two novel protocols (PB2T-a and PB2T-b) to optimize PB2T using mouse and human oocytes. Chromosomal profiles of NT-generated blastocysts were evaluated using NGS. In mouse, our findings reveal that only PB2T-b successfully leads to blastocysts. There were comparable blastocyst rates amongst PB1T, PB2T-b, ST and PNT embryos. Furthermore, PB1T and PB2T-b had lower mtDNA carry-over levels than ST and PNT. After extrapolation of novel PB2T-b to human in vitro matured (IVM) oocytes and in vivo matured oocytes with smooth endoplasmic reticulum aggregates (SERa) oocytes, the reconstituted embryos successfully developed to blastocysts at a comparable rate to ICSI controls. PB2T-b embryos generated from IVM oocytes showed a similar euploidy rate to ICSI controls. Nevertheless, our mouse model with non-mutated mtDNAs is different from a mixture of pathogenic and non-pathogenic mtDNAs in a human scenario. Novel PB2T-b requires further optimization to improve blastocyst rates in human. Although more work is required to elucidate efficiency and safety of NT, our study suggests that PBT may have the potential to prevent mtDNA disease transmission.

STUDY QUESTION: What are the transcriptional changes occurring during the human embryonic stem cell (hESC) derivation process, from the inner cell mass (ICM) to post-ICM intermediate stage (PICMI) to hESC stage, that have downstream effects on pluripotency states and differentiation? SUMMARY ANSWER: We reveal that although the PICMI is transcriptionally similar to the hESC profile and distinct from ICM, it exhibits upregulation of primordial germ cell (PGC) markers, dependence on leukemia inhibitory factor (LIF) signaling, upregulation of naive pluripotency-specific signaling networks and appears to be an intermediate switching point from naive to primed pluripotency. WHAT IS KNOWN ALREADY: It is currently known that the PICMI exhibits markers of early and late-epiblast stage. It is suggested that hESCs acquire primed pluripotency features due to the upregulation of post-implantation genes in the PICMI which renders them predisposed towards differentiation cues. Despite this current knowledge, the transcriptional landscape changes during hESC derivation from ICM to hESC and the effect of PICMI on pluripotent state is still not well defined. STUDY DESIGN, SIZE, DURATION: To gain insight into the signaling mechanisms that may govern the ICM to PICMI to hESC transition, comparative RNA sequencing (RNA-seq) analysis was performed on preimplantation ICMs, PICMIs and hESCs in biological and technical triplicates (n = 3). PARTICIPANTS/MATERIALS, SETTING, AND METHODS: Primed hESCs (XX) were maintained in feeder-free culture conditions on Matrigel for two passages and approximately 50 cells were collected in biological and technical triplicates (n = 3). For ICM sample collection, Day 3, frozen-thawed human embryos were cultured up to day five blastocyst stage and only good quality blastocysts were subjected to laser-assisted micromanipulation for ICM collection (n = 3). Next, day six expanded blastocysts were cultured on mouse embryonic fibroblasts and manual dissection was performed on the PICMI outgrowths between post-plating Day 6 and Day 10 (n = 3). Sequencing of these samples was performed on NextSeq500 and statistical analysis was performed using edgeR (false discovery rate (FDR) < 0.05). MAIN RESULTS AND THE ROLE OF CHANCE: Comparative RNA-seq data analysis revealed that 634 and 560 protein-coding genes were significantly up and downregulated in hESCs compared to ICM (FDR < 0.05), respectively. Upon ICM to PICMI transition, 471 genes were expressed significantly higher in the PICMI compared to ICM, while 296 genes were elevated in the ICM alone (FDR < 0.05). Principle component analysis showed that the ICM was completely distinct from the PICMI and hESCs while the latter two clustered in close proximity to each other. Increased expression of E-CADHERIN1 (CDH1) in ICM and intermediate levels in the PICMI was observed, while CDH2 was higher in hESCs, suggesting a role of extracellular matrix components in facilitating pluripotency transition during hESC derivation. The PICMI also showed regulation of naive- specific LIF and bone morphogenetic protein signaling, differential regulation of primed pluripotency- specific fibroblast growth factor and NODAL signaling pathway components, upregulation of phosphatidylinositol 3- kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway (PI3K/AKT/mTORC), as well as predisposition towards the germ cell lineage, further confirmed by gene ontology analysis. Hence, the data suggest that the PICMI may serve as an intermediate pluripotency stage which, when subjected to an appropriate culture niche, could aid in enhancing naive hESC derivation and germ cell differentiation efficiency. LARGE-SCALE DATA: Gene Expression Omnibus (GEO) Accession number GSE119378. LIMITATIONS, REASONS FOR CAUTION: Owing to the limitation in sample availability, the sex of ICM and PICMI have not been taken into consideration. Obtaining cells from the ICM and maintaining them in culture is not feasible as it will hamper the formation of PICMI and hESC derivation. Single- cell quantitative real- time PCR on low ICM and PICMI cell numbers, although challenging due to limited availability of human embryos, will be advantageous to further corroborate the RNA- seq data on transcriptional changes during hESC derivation process. WIDER IMPLICATIONS OF THE FINDINGS: We elucidate the dynamics of transcriptional network changes from the naive ICM to the intermediate PICMI stage and finally the primed hESC lines. We provide an in- depth understanding of the PICMI and its role in conferring the type of pluripotent state which may have important downstream effects on differentiation, specifically towards the PGC lineage. This knowledge contributes to our limited understanding of the true nature of the human pluripotent state in vitro. STUDY FUNDING/COMPETING INTEREST(S): This research is supported by the Concerted Research Actions funding from Bijzonder Onderzoeksfonds University Ghent (BOF GOA 01G01112). The authors declare no conflict of interest.

Objective To evaluate the multivariate embryo selection model by van Loendersloot et al. (2014) (VL) in a different geographical context. Design This is a retrospective external validation study of a 5-year cohort of women undergoing in vitro fertilization or intracytoplasmatic sperm injection. Setting Two outpatient fertility clinics. Patient(s) A total of 1,197 women who underwent 1,610 fresh in vitro fertilization or intracytoplasmatic sperm injection cycles with single embryo transfer were included. Intervention(s) None. Main Outcome Measure(s) The area under the receiver operating characteristics curve for diagnostic efficacy was used to assess the discriminative value of the model. Calibration for testing the validity of the VL model was performed using the Hosmer-Lemeshow goodness-of-fit test and a calibration plot. Result(s) Three hundred thirty-three patients (21%) achieved a viable pregnancy of at least 11 weeks. The area under the receiver operating characteristics curve using the VL model was 0.68. No significant difference between the predicted implantation rate and the observed implantation rates was showed using the Hosmer-Lemeshow (X2= 6.70). The calibration plot showed an intercept of the regression line of 0.34 and the estimated slope was 0.72. Conclusion The investigated VL model was able to distinguish between higher and lower implantation potential of embryos in our clinical setting.

Platelet-activating factor (PAF) is a well-known marker for embryo quality and viability. For the first time, we describe an intracellular localisation of PAF in oocytes and embryos of cattle, mice and humans. We showed that PAF is represented in the nucleus, a signal that was lost upon nuclear envelope breakdown. This process was confirmed by treating the embryos with nocodazole, a spindle-disrupting agent that, as such, arrests the embryo in mitosis, and by microinjecting a PAF-specific antibody in bovine MII oocytes. The latter resulted in the absence of nuclear PAF in the pronuclei of the zygote and reduced further developmental potential. Previous research indicates that PAF is released and taken up from the culture medium by preimplantation embryos invitro, in which bovine serum albumin (BSA) serves as a crucial carrier molecule. In the present study we demonstrated that nuclear PAF does not originate from an extracellular source because embryos cultured in polyvinylpyrrolidone or BSA showed similar levels of PAF in their nuclei. Instead, our experiments indicate that cytosolic phospholipase A2 (cPLA2) is likely to be involved in the intracellular production of PAF, because treatment with arachidonyl trifluoromethyl ketone (AACOCF3), a specific cPLA2 inhibitor, clearly lowered PAF levels in the nuclei of bovine embryos.

The derivation of gametes from patient-specific pluripotent stem cells may provide new perspectives for genetic parenthood for patients currently facing sterility. We use current data to assess the gamete differentiation potential of patient-specific pluripotent stem cells and to determine which reprogramming strategy holds the greatest promise for future clinical applications. First, we compare the two best established somatic cell reprogramming strategies: the production of induced pluripotent stem cells (iPSC) and somatic cell nuclear transfer followed by embryonic stem cell derivation (SCNT-ESC). Recent reports have indicated that these stem cells, though displaying a similar pluripotency potential, show important differences at the epigenomic level, which may have repercussions on their applicability. By comparing data on the genetic and epigenetic stability of these cell types during derivation and in-vitro culture, we assess the reprogramming efficiency of both technologies and possible effects on the subsequent differentiation potential of these cells. Moreover, we discuss possible implications of mitochondrial heteroplasmy. We also address the ethical aspects of both cell types, as well as the safety considerations associated with clinical applications using these cells, e.g. the known genomic instability of human PSCs during long-term culture. Secondly, we discuss the role of the stem cell pluripotency state in germ cell differentiation. In mice, success in germ cell development from pluripotent stem cells could only be achieved when starting from a naive state of pluripotency. It remains to be investigated if the naive state is also crucial for germ cell differentiation in human cells and to what extent human naive pluripotency resembles the naive state in mouse.

Research question Are there proteomic differences between endometrial stromal cells of repeated implantation failure (RIF), recurrent pregnancy loss (RPL) and normal fertile women, and is there differential protein expression upon decidualization? Design This exploratory study investigated the proteome of in-vitro cultured endometrial stromal cells of women with RIF (n = 4), women with RPL (n = 3) and normal fertile women (n = 4), comparing day 0 with 5 days of decidualization. Total proteins extracted from cell lysates were analysed by high-definition mass spectrometry. Data analysis was performed using significance analysis of microarray in R (P Results In the RIF group, ANXA6, PSMC5 and FSCN1 were up-regulated (1.9-fold, 2.5-fold and 1.9-fold, respectively), whereas PBXIP1 was down-regulated (7.7-fold) upon decidualization. In the RPL group, RPS25 and ACADVL were down-regulated (1.9-fold and 2.4-fold, respectively; FDR 10%) between the non-decidualized and the decidualized samples. In the normal fertile group VIM and RPL23A were down-regulated (1.9-fold and 2.4-fold, respectively). Comparing ratios of expression of decidualized over non-decidualized samples in the different groups revealed six differentially expressed proteins: DUX4L2, CNPY4, PDE7A, CTSK, PCBP2 and PSMD4. Comparison of RPL versus normal fertile in the decidualized condition revealed serotransferrin to be differentially expressed. The changes in expression levels for serotransferrin, ANX6, ACDVL and VIM were confirmed by western blot. Conclusions Results show a varying response of endometrial stromal cells in distinct clinical groups (RIF, RPL and normal fertile) upon in-vitro decidualization. Serotransferrin could serve as a marker for the aberrant decidualization process in RPL.

Introduction Heritable connective tissue disorders (HCTD) are a group of mostly autosomal dominant genetic diseases, characterized by cardiovascular involvement, tissue fragility, joint hypermobility and skin involvement. Some of them are associated with life threatening events such as aortic dissection and rupture, and a high mortality rate. We recently introduced Preimplantation Genetic Testing for monogenic disease (PGT-M) with a focus on HCTD and set up an explorative qualitative study to investigate the lived experiences and feelings of women, as well as their attitudes and psychological responses to PGT-M. Materials & Methods All couples are seen by a clinical geneticist and a psychologist, and blood samples of the couple are drawn for karyotyping, exclusion of carriership of cystic fibrosis, spinal muscular atrophy and fragile-X syndrome. Informative linked microsatellite markers flanking the causal (familial) mutation are identified for each couple. Once these parameters are defined, DNA, obtained from trophectoderm cells biopsied from a day 5-6 embryo, is genome-wide amplified. Subsequently, the informative linked microsatellite markers as well as the underlying disease causing mutation are analysed in each of these day 5-6 embryo's. In total, twelve HCTD couples have undergone the PGT-M procedure (FBN1: 4, SMAD3: 2, one each for TGFBR1, TGFB2, COL3A1, COL1A2, COL2A1 and WNT10A). In total, 52 embryos have been tested, 19 of them carried the healthy haplotype (microsatellite analysis) and did not carry the familial mutation (Sanger sequencing) and thus were suitable for transfer to the mother. Data of seven women, aged 26-39years, were collected by semi-structured interviews and interpreted by thematic analysis. At the time of the interviews, one participant had one child through PGT-M, two participants were pregnant after embryo transfer and four women did not have a successful embryo transfer leading to pregnancy yet. Results Our study shows that PGT-M is both physically and emotionally a demanding procedure. The main reason for choosing PGT-M is the severity of the disease. None of the couples wants to transmit the disease to their offspring. Since termination of pregnancy is not acceptable to them, PGT-M is preferred over prenatal diagnosis. The “drop-out race”, as all interviewed women call it, going from an encouraging number of available oocytes (after pick-up), to a limited number of useful embryos, is perceived as extremely stressful. Due to the frequent hospital visits and the prescribed strict timing of hormone injections, all women stated it is impossible to keep the PGT-M procedure hidden from their friends and colleagues. Women emphasize that receiving adequate information, during every step of the PGT-M process, is essential to learn more about the entire PGT-M trajectory. They also express the need of psychological support to help them in coping with this physical and psychological demanding technique. Conclusions PGT-M is a valuable option of reproductive technology for HCTD couples. Due to the significant emotional impact the PGT-M procedure causes, we advise that it should always go hand-in-hand with effective communication and psychological care in order to prevent distress in couples.

The aim of this qualitative study was to explore how recipients viewed payment of sperm donors. The study was conducted in Belgium, where, as in many countries, sperm donors receive recompense for their time and expenses. Face-to-face semi-structured interviews were conducted with 34 heterosexual and lesbian couples who, at the time of data collection, had at least one donor-conceived child aged 7–10 years or who were undergoing donor conception treatment. Although participants commonly described the issue of financial compensation as something that did not really concern them, all supported the idea that some level of payment was acceptable or even necessary. The participants also identified several ways in which donor payment offered advantages to their own position as (future) parents. Although the idea is commonly rehearsed that sperm donation is a gift and that monetary transaction for conception is demeaning, the participants of this study did not generally share this view. To them, a small financial return served as a symbolic acknowledgement of the donor's contribution and helped secure the type of relationship they expected from their donor. There was clearly concern, however, over high payments and the risk of attracting the wrong kind of donor.

Background: Fertility preservation before or during cancer treatment in young women has become an important health issue because of delayed motherhood and improved survival rates. This study evaluates the necessity and the efficacy of fertility preservation, with a focus on actual pregnancy wish and outcome after fertility preservation and cancer treatment. Patients and Methods: All consecutive patients who received fertility preservation in 2 university referral centers before or during cancer treatment were included. After a minimal follow-up of 3 years, pregnancy wish, pregnancy attempts and fertility outcome were assessed during a dedicated consultation or during a telephone interview. Results: A total of 159 patients received fertility preservation including hormonal protection with gonadotropin-releasing hormone agonist (n = 93, 58.5%), ovarian tissue cryopreservation (n = 44, 27.7%), and combined hormonal protection and ovarian tissue cryopreservation (n = 22, 13.8%). Among the 91 (57.2%) patients in remission after a mean follow-up of 61.5 months, 29 (31.9%) women actively attempted pregnancy. Patients who had received ovarian cryopreservation were more likely to attempt pregnancy (18/66) than those who only received hormonal protection (11/93, p = 0.02). Out of the 29 women who attempted pregnancy, 16 (55.2%) became pregnant, and most of them conceived spontaneously (87.5%, 14/16). Out of the 13 women who did not become pregnant, 1 patient adopted a child and 12 patients still wanted to become pregnant, including 1 patient who underwent a transplantation of her cryopreserved ovarian tissue without success. Conclusion: In one of the first studies reporting real-life experience in centers for fertility preservation, we found that, within 5 years following the end of cancer treatment, only one third of patients in remission attempted to become pregnant, with a pregnancy rate of 55%, mostly after spontaneous conception.

Patients presenting with abnormally high numbers of immature oocytes at retrieval are more likely to exhibit maturation resistant oocytes. However, the clinical relevance of such events remains unknown. We investigated nuclear maturation competence of immature oocytes from patients showing >40% of collected immature oocytes (Study group) and Controls, in which a normal number of mature oocytes (≥60%) was retrieved. Following in-vitro culture, oocytes were classified as maturation resistant or in-vitro matured (IVM). Treatment outcomes were evaluated in Study and Control groups based on presence of maturation resistant oocytes. Overall, similarly high spindle and chromosome abnormality rates were observed in maturation resistant oocytes from both Study and Control groups. IVM oocytes from the Study group revealed significantly higher percentages of misaligned chromosomes compared with Controls (P < 0.05). Remarkably, Study group patients with at least one maturation resistant oocyte showed significantly reduced cumulative pregnancy and live birth rates compared with Control group maturation resistant patients (P < 0.05). When further investigating the aetiology, a maturation resistant mouse model revealed defective Ca2+ signalling of maturation resistant oocytes at germinal vesicular breakdown and parthenogenetic activation. In conclusion, appropriate treatment strategies, including clinical utilization of IVM oocytes from Study group patients, warrant further investigation.

Background: Gonadotropin therapy and laparoscopic ovarian drilling (LOD) are treatment options for ovulation induction (OI) in clomiphene citrate (CC)-resistant polycystic ovary syndrome (PCOS) patients. The current evidence of the cost-effectiveness of both treatments is scarce, conflicting and performed from different health-economic perspectives. Methods: A retrospective health-economic evaluation was performed from a societal perspective in which human menopausal gonadotropin (hMG) therapy (n = 43) was compared with LOD (n = 35), followed by OI with CC and/or hMG if spontaneous ovulation did not occur within 2 months. Data were collected until the patients were pregnant, with a time limit of 6 months after the onset of treatment. Outcomes were expressed as ongoing pregnancy rate and number of live-born children. Results: The ongoing pregnancy rate was 21/35 (60%) after LOD and 30/43 (69.8%) after hMG treatment (relative risk 0.85, 95% CI 0.61-1.19). The societal cost per patient, up to an ongoing pregnancy, was significantly higher after LOD versus hMG treatment (adjusted mean difference EUR 1,073, 95% CI 180-1,967). Conclusion: This economic evaluation based on real-life data shows that the societal cost up to an ongoing pregnancy is less after hMG treatment when compared with LOD surgery in CC-resistant PCOS patients.

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.

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.

Female-to-male transgender people (trans men) are faced with the risk of losing their reproductive potential owing to gender-affirming hormone treatment and genital reconstructive surgery. This observational, prospective cohort study investigates the effect of prolonged androgen therapy on their ovarian histology and fertility preservation perspectives. Hormone serum levels, ovarian histology and cumulus-oocyte complexes (COC) of 40 trans men were analysed at the moment of hysterectomy with bilateral oophorectomy in the context of genital reconstructive surgery after testosterone treatment (58.18 ± 26.57 weeks). In the cortex, most follicles were primordial (68.52% total follicle count) compared with 20.26% intermediate and 10.74%primary follicles. Few secondary follicles (0.46%) and a single antral follicle were found in the sections analysed. In total, 1313 COC were retrieved from the medulla of 35 patients (37.51 ± 33.58 COC per patient). Anti-Müllerian hormone serum levels were significantly correlated with number of COC (Rs 0.787, P < 0.001). After 48 h in-vitro maturation, 34.30% metaphase II oocytes were obtained, with 87.10% having a normal spindle structure. In conclusion, the cortical follicle distribution in trans men, after more than a year of testosterone treatment, seems to be surprisingly normal. This work confirms the presence and in-vitro maturation potential of cumulus-oocyte complexes.

STUDY QUESTION: What progress has been made in fertility preservation (FP) over the last decade?SUMMARY ANSWER: FP techniques have been widely adopted over the last decade and therefore the establishment of international registries on their short- and long-term outcomes is strongly recommended.WHAT IS KNOWN ALREADY: FP is a fundamental issue for both males and females whose future fertility may be compromised. Reproductive capacity may be seriously affected by age, different medical conditions and also by treatments, especially those with gonadal toxicity. There is general consensus on the need to provide counselling about currently available FP options to all individuals wishing to preserve their fertility.STUDY DESIGN, SIZE, DURATION: An international meeting with representatives from expert scientific societies involved in FP was held in Barcelona, Spain, in June 2015.PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty international FP experts belonging to the American Society of Reproductive Medicine, ESHRE and the International Society of Fertility Preservation reviewed the literature up to June 2015 to be discussed at the meeting, and approved the final manuscript. At the time this manuscript was being written, new evidence considered relevant for the debated topics was published, and was consequently included.MAIN RESULTS AND THE ROLE OF CHANCE: Several oncological and non-oncological diseases may affect current or future fertility, either caused by the disease itself or the gonadotoxic treatment, and need an adequate FP approach. Women wishing to postpone maternity and transgender individuals before starting hormone therapy or undergoing surgery to remove/alter their reproductive organs should also be counselled accordingly. Embryo and oocyte cryopreservation are first-line FP methods in postpubertal women. Metaphase II oocyte cryopreservation (vitrification) is the preferred option. Cumulative evidence of restoration of ovarian function and spontaneous pregnancies after ART following orthotopic transplantation of cryopreserved ovarian tissue supports its future consideration as an open clinical application. Semen cryopreservation is the only established method for FP in men. Testicular tissue cryopreservation should be recommended in pre-pubertal boys even though fertility restoration strategies by autotransplantation of cryopreserved testicular tissue have not yet been tested for safe clinical use in humans. The establishment of international registries on the short- and long-term outcomes of FP techniques is strongly recommended.LIMITATIONS, REASONS FOR CAUTION: Given the lack of studies in large cohorts or with a randomized design, the level of evidence for most of the evidence reviewed was 3 or below.WIDER IMPLICATIONS OF THE FINDINGS: Further high quality studies are needed to study the long-term outcomes of FP techniques.STUDY FUNDING/COMPETING INTEREST(S): None.TRIAL REGISTRATION NUMBER: N/A.