In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Objective To determine the risk of pre-eclampsia associated with factors that may be present at antenatal booking.

Design Systematic review of controlled studies published 1966-2002.

Data synthesis Unadjusted relative risks were calculated from published data.

Results Controlled cohort studies showed that the risk of pre-eclampsia is increased in women with a previous history of pre-eclampsia (relative risk 7.19, 95% confidence interval 5.85 to 8.83) and in those with antiphospholipids antibodies (9.72, 4.34 to 21.75), pre-existing diabetes (3.56, 2.54 to 4.99), multiple (twin) pregnancy (2.93, 2.04 to 4.21), nulliparity (2.91, 1.28 to 6.61), family history (2.90, 1.70 to 4.93), raised blood pressure (diastolic ≥ 80 mm Hg) at booking (1.38, 1.01 to 1.87), raised body mass index before pregnancy (2.47, 1.66 to 3.67) or at booking (1.55, 1.28 to 1.88), or maternal age ≥ 40 (1.96, 1.34 to 2.87, for multiparous women). Individual studies show that risk is also increased with an interval of 10 years or more since a previous pregnancy, autoimmune disease, renal disease, and chronic hypertension.

Conclusions These factors and the underlying evidence base can be used to assess risk at booking so that a suitable surveillance routine to detect pre-eclampsia can be planned for the rest of the pregnancy.

https://www.bmj.com/content/bmj/330/7491/565.full.pdf

Risk factors for pre-eclampsia at antenatal booking: systematic review of controlled studies

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Background. Maternal obesity, both in itself and as part of the insulin resistance syndrome, is an important risk factor for the development of preeclampsia. Accurately quantifying the relation between prepregnancy maternal body mass index and the risk of preeclampsia may better identify those at hi

Maternal body mass index and the risk of preeclampsia: a systematic overview.

This article provides an overview of the basic principles of percutaneous tumor ablation and describes the equipment and technologic modifications that have been developed to further improve clinical success of this therapy.

/pdf/principles-of-and-advances-in-percutaneous-ablation-2iegfo454b.pdf

Principles of and Advances in Percutaneous Ablation

Advanced maternal age and adverse perinatal outcomes in an Asian population.

Risk factors for pre-eclampsia in an Asian population.

Pregnancy is associated with increased oxidative stress, and exaggeration of oxidative damage is considered important in pregnancy complications such as preeclampsia. There is, however, only scattered information regarding the longitudinal change of oxidative stress during pregnancy. We surmised that the placenta is the cause of the increased oxidative stress during pregnancy and hypothesized that the amount of placenta-derived cell-free fetal DNA in maternal plasma is correlated with maternal oxidative stress status. Blood and urine samples were collected at different stages of gestation and 6 to 8 weeks postpartum from 105 women with uncomplicated pregnancies. Additionally, 40 healthy women of reproductive age, but not pregnant, were recruited as controls. All oxidative stress markers, including urinary 8-hydroxydeoxyguanosine (8-OHdG), plasma 8-isoprostane, total antioxidant capacity (TAC), and erythrocyte glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities, were increased in the third trimester, and most of them returned to nonpregnant levels postpartum. Furthermore, the DYS14 gene of the Y chromosome was quantified from plasma samples obtained at 26 to 30 weeks' gestation in 54 women who later delivered a male infant. Significant associations were noted between plasma concentrations of the DYS14 gene and the levels of urinary 8-OHdG and plasma 8-isoprostane, suggesting an association between the breakdown of syncytiotrophoblast and maternal oxidative stress during pregnancy.

A longitudinal study of oxidative stress and antioxidant status in women with uncomplicated pregnancies throughout gestation.

Background
Unexplained intrauterine growth restriction (IUGR) may be a consequence of placental insufficiency; however, its etiology is not fully understood. We surmised that defective placentation in IUGR dysregulates cellular bioenergic homeostasis, leading to increased autophagy in the villous trophoblast. The aims of this work were (1) to compare the differences in autophagy, p53 expression, and apoptosis between placentas of women with normal or IUGR pregnancies; (2) to study the effects of hypoxia and the role of p53 in regulating trophoblast autophagy; and (3) to investigate the relationship between autophagy and apoptosis in hypoxic trophoblasts.

/pdf/increased-autophagy-in-placentas-of-intrauterine-growth-9ydqwvil9i.pdf

Increased autophagy in placentas of intrauterine growth-restricted pregnancies.

https://www.fertstert.org/article/S0015-0282(08)00726-7/pdf

Liang-Ming Lo

Papers

Advanced maternal age and adverse perinatal outcomes in an Asian population.

Risk factors for pre-eclampsia in an Asian population.

A longitudinal study of oxidative stress and antioxidant status in women with uncomplicated pregnancies throughout gestation.

Increased autophagy in placentas of intrauterine growth-restricted pregnancies.

Effectiveness of ultrasound-guided aspiration and sclerotherapy with 95% ethanol for treatment of recurrent ovarian endometriomas.