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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 (<1%). In addition, this alternative technique holds multiple benefits compared to NGS (e.g., less hands-on time, more straightforward data-analysis, and a lower up-front capital investment)., (© American Association for Clinical Chemistry 2021.)

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

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., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Objective: To establish a mouse model for endometrial injury and determine the underlying mechanism regarding its favorable effect on embryo implantation., Study Design: 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., Results: 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., Conclusions: 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., (© The Author(s) 2015.)

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., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)