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Background The quantification of mitochondrial DNA heteroplasmy for the diagnosis of mitochondrial disease or after mitochondrial donation, is performed mainly using next-generation sequencing strategies (NGS). Digital PCR (dPCR) has the potential to offer an accurate alternative for mutation load quantification. Methods We assessed the mutation load of 23 low-input human samples at the m.11778 locus, which is associated with Leber’s hereditary optic neuropathy (LHON) using 2 droplet digital PCR platforms (Stilla Naica and Bio-Rad QX200) and the standard NGS strategy. Assay validation was performed by analyzing a titration series with mutation loads ranging from 50% to 0.01%. Results A good concordance in mutation rates was observed between both dPCR techniques and NGS. dPCR established a distinctly lower level of background noise compared to NGS. Minor alleles with mutation loads lower than 1% could still be detected, with standard deviations of the technical replicates varying between 0.07% and 0.44% mutation load. Although no significant systematic bias was observed when comparing dPCR and NGS, a minor proportional bias was detected. A slight overestimation of the minor allele was observed for the NGS data, most probably due to amplification and sequencing errors in the NGS workflow. Conclusion dPCR has proven to be an accurate tool for the quantification of mitochondrial heteroplasmy, even for samples harboring a low mutation load (

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.

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

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.

Successful pregnancy relies on the accurate regulation of the maternal-fetal immune system. Without enough tolerance in the uterine microenvironment, the mother and the hemiallogeneic fetus could not peacefully coexist. T cell immunoglobulin and mucin domain (Tim)-3 is a molecule originally regarded as to be expressed on terminally differentiated IFN-γ expressing CD4+ T cells (Th1). The engagement of Tim-3 with its ligand, galectin-9 (Gal-9) could induce the exhaustion or apoptosis of effector T cells, and thus might regulate the tolerance. Tim-3 pathway also participates in regulating the activities of CD4+ regulatory T cells, monocyte-macrophages, dendritic cells and natural killer cells. Dysregulation of Tim-3 expression can elicit excessive or inhibited inflammatory responses and ultimately result in autoimmune diseases, viral or tumor evasion and pregnancy complications. In this review, we will mainly focus on the expression of Tim-3 on local immune cells and its function in pregnancy. In addition, meaningful questions that need further investigation and the potential roles of Tim-3 in fetal tolerance will be discussed. Deeper understanding of the immune checkpoint receptor Tim-3 will shed new light on exploring the pathogenesis of some pregnancy complications, including pre-eclampsia, intrauterine growth restriction, recurrent spontaneous abortion and preterm birth. Tim-3 pathway might be a new target of immune therapy for pregnancy complications in the future.

To establish a mouse model for endometrial injury and determine the underlying mechanism regarding its favorable effect on embryo implantation. Female Kunming mice were randomly allocated into 4 groups: group I, normal control; group II, injury procedure control; and group III and group IV, the mice being scratched with a blunt syringe on the right uterine horn or both, respectively. All the mice were mated with the males during the next estrus phase. The number of implanted embryos on each side of uterus was calculated on day 8 of pregnancy. The endometrial samples were taken on day 4 of pregnancy, and the local morphological changes and cytokine expressions were examined. Compared to group II, our results showed that in group IV (1) there were significantly higher numbers of implanted embryos, (2) the endometrial glands and vasculatures in stroma were obviously increased and the pinopodes were abundant and well developed, and (3) the local levels of cytokines leukemia inhibitory factor (LIF) and oncostatin M (OSM) messenger RNA and protein expression were significantly increased. Local mechanical injury on mouse uteri enhanced endometrial receptivity and improved embryo implantation, which were correlated with the characteristic changes in endometrial morphology and the upregulation of LIF and OSM gene and protein expression.

PROBLEM We aim to investigate the proportion and absolute counts of peripheral blood monocyte subsets in women with normal pregnancy (NP) and pre-eclampsia (PE), and their correlation with the clinical manifestation and severity of PE. METHOD OF STUDY Peripheral blood was obtained from women with NP (n = 30), mild PE (MPE, n = 15) and severe PE (SPE, n = 30). The proportion and absolute counts of CD16(+) monocytes and the subsets including intermediate (CD14(++) CD16(+) HLA-DR(+) ) and non-classical (CD14(+) CD16(++) HLA-DR(+) ) monocytes were determined by flow cytometric analysis. RESULTS Women with MPE and SPE had significantly increased absolute count of CD14(++) CD16(+) HLA-DR(+) monocyte subsets (P < 0.01 each) as compared to NP women. In addition, there were significant differences in the absolute count of CD14(++) CD16(+) HLA-DR(+) monocyte subsets between MPE and SPE groups (P < 0.05). The proportion of CD14(++) CD16(+) HLA-DR(+) monocyte subsets was significantly increased in SPE compared to MPE and NP (P < 0.01 each). The absolute count (r = 0.332, P < 0.05) and proportion (r = 0.447, P < 0.01) of CD14(++) CD16(+) HLA-DR(+) monocytes were positively correlated with the severity of PE. Multivariate logistic regression analysis further revealed that the absolute count of CD14(++) CD16(+) HLA-DR(+) monocytes was a potential marker for PE (P < 0.01). CONCLUSION A preferential increase in peripheral blood CD14(++) CD16(+) HLA-DR(+) monocytes is quantitatively correlated with clinical manifestation of PE.

Pregnancy presents a great challenge to the maternal immune system. Given that maternal alloreactive lymphocytes are not depleted during pregnancy, local and/or systemic mechanisms have to serve a central function in altering the maternal immune responses. Regulatory T cells (Tregs) and the PD-1/PD-L1 pathway are both critical in controlling the immune responses. Recent studies have proved the critical function of the PD-1/PD-L1 pathway in regulating the T-cell homeostasis and the peripheral tolerance through promoting the development and function of Tregs, and inhibiting the activation of effector T cells. The function of the PD-1/PD-L1 pathway in feto-maternal interface and pregnancy has been investigated in human and animal models of pregnancy. In this review, we provide recent insight into the role of the PD-1/PD-L1 pathway in regulating T-cell homeostasis, maternal tolerance, and pregnancy-related complications as well as its possible applicability in clinical immunotherapy.

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

During pregnancy, the maternal immune system is challenged by the semi-allogeneic fetus, which leads to systemic and local immunity. Systemic immunity, including enhanced innate immunity with increased activation of monocytes, is induced by various placental factors. Maternal immune adaptations are most evident at the feto-maternal interface, where macrophages are enriched and communicate with various decidual leukocytes. These cells are not only contributing to the protection of the growing fetus from microorganisms, but also aiding placental development by promoting trophoblast invasion and spiral artery remodeling, and the parturition process. Thus, monocytes and macrophages concurrently play important roles throughout the trimesters. Dysregulation of these cells may thus lead to pregnancy complications, such as pre-eclampsia and preterm labor. In this review, monocytes and macrophage subsets and their roles in normal and pathological pregnancies are reviewed.