Strengths and limitations of diagnostic tools for endometriosis and relevance in diagnostic test accuracy research.

Endometriosis is a chronic systemic disease that can cause pain, infertility and reduced quality of life. Diagnosing endometriosis remains challenging, which yields diagnostic delays for patients. Research on diagnostic test accuracy in endometriosis can be difficult due to verification bias, as not all patients with endometriosis undergo definitive diagnostic testing. The purpose of this State-of-the-Art Review is to provide a comprehensive update on the strengths and limitations of the diagnostic modalities used in endometriosis and discuss the relevance of diagnostic test accuracy research pertaining to each. We performed a comprehensive literature review of the following methods: clinical assessment including history and physical examination, biomarkers, diagnostic imaging, surgical diagnosis and histopathology. Our review suggests that, although non-invasive diagnostic methods, such as clinical assessment, ultrasound and magnetic resonance imaging, do not yet qualify formally as replacement tests for surgery in diagnosing all subtypes of endometriosis, they are likely to be appropriate for advanced stages of endometriosis. We also demonstrate in our review that all methods have strengths and limitations, leading to our conclusion that there should not be a single gold-standard diagnostic method for endometriosis, but rather, multiple accepted diagnostic methods appropriate for different circumstances. © 2022 International Society of Ultrasound in Obstetrics and Gynecology.

Brain-derived neurotrophic factor (BDNF) expression and function in the mammalian reproductive Tract.

BACKGROUND: Neurotrophins of the nerve growth factor family are soluble polypeptides that are best known for their role in nerve growth, survival and differentiation in the central nervous system. A growing body of literature shows that neurotrophins and their receptors are also expressed throughout the reproductive tract. OBJECTIVE AND RATIONALE: Neurotrophins are key regulatory proteins in reproductive physiology during development and throughout adult life. Of the neurotrophins, the literature describing the expression and function of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, neurotrophin receptor kinase-2 (NTRK2), has been expanding rapidly. We therefore conducted a systematic inductive qualitative review of the literature to better define the role of the BDNF in the reproductive tract. We postulate that BDNF and NTRK2 are central regulatory proteins throughout the reproductive system. SEARCH METHODS: An electronic search of Medline (PubMed) and Web of Science for articles relating to BDNF and the reproductive system was carried out between January 2018 and February 2019. OUTCOMES: In the ovary, BDNF expression and levels have been linked with follicle organisation during ovarian development, follicle recruitment and growth and oocyte maturation. In the endometrium, BDNF is involved in cell proliferation and neurogenesis. In contrast, literature describing the role of BDNF in other reproductive tissues is sparse and BDNF-NTRK2 signalling in the male reproductive tract has been largely overlooked. Whilst estradiol appears to be the primary regulator of BDNF expression, we also identified reports describing binding sites for glucocorticoid and myocyte enhancer factor-2, a calcium-response element through activation of an N-methyl-D-aspartate (NMDA) receptor, and aryl hydrocarbon receptor nuclear transporter protein-4 (ARNT) response elements in promoter regions of the BDNF gene. Expression is also regulated by multiple microRNAs and post-translational processing of precursor proteins and intracellular shuttling. BDNF-NTRK2 signalling is modulated through tissue specific receptor expression of either the full-length or truncated NTRK2 receptor; however, the functional importance remains to be elucidated. Dysregulation of BDNF expression and circulating concentrations have been implicated in several reproductive disorders including premature ovarian failure, endometriosis, pre-eclampsia, intra-uterine growth restriction (IUGR) and several reproductive cancers. WIDER IMPLICATIONS: We conclude that BDNF and its receptors are key regulatory proteins central to gonadal development, ovarian regulation and uterine physiology, as well as embryo and placenta development. Furthermore, dysregulation of BDNF-NTRK2 in reproductive diseases suggests their potential role as candidate clinical markers of disease and potential therapeutic targets.

Hydroxychloroquine attenuates cigarette smoke induced autophagic signaling in the mouse ovary.

We previously demonstrated that Cigarette Smoke (CS) induces autophagy in the ovary. Therefore we aimed to determine if chloroquine (CQ) could inhibit CS-induced autophagy in the ovary. Eight week old mice were implanted with CQ pellets; 0, 25, and 50mg CQ/kg. Half of the animals in each group were exposed to room air and the other half were exposed to CS twice daily for 8 weeks. Ovaries were harvested for electron microscopy, gene and protein expression analysis. There was a significant increase in the production of autophagosomes in granulosa cells of mice exposed to CS (p=0.0297). However 25 and 50mg/kg CQ treatment significantly decreased the CS-induced autophagosomes (p=0.0505; p=0.0065) and attenuated the effects of CS on LC3B and BECN1 expression. In summary, this suggests that CQ attenuates CS-induced autophagy in the ovary and that ovarian protection from toxic insult is potentially feasible.

Are pharmacological interventions between conception and birth effective in improving reproductive outcomes in North American swine?

The objective of this review is to evaluate the effectiveness of using pharmacological compounds on reproductive outcomes, particularly litter size, in North American swine. While the opportunity to improve reproduction in North American pigs exists, numerous hurdles need to be overcome in order to achieve measureable results. In the swine industry, the majority of piglet losses are incurred during pregnancy and around farrowing. Over the last 20 years, a reduction in losses has been achieved through genetic selection and nutritional management; however, these topics are the focus of other reviews. This review will evaluate attempts to improve litter size by reducing losses at various stages of the reproductive process, from the time of conception to the time of farrowing, using pharmacological compounds. Generally, these compounds are used to either alter physiological processes related to fertilization, embryonic attachment or uterine capacity, etc., or to facilitate management aspects of the breeding females such as inducing parturition. Although some of the pharmacological agents reviewed here show some positive effects on improving reproductive parameters, the inconsistent results and associated risks usually outweigh the benefits gained. Thus, at the present time, the use of pharmacological agents to enhance reproduction in North American swine may only be recommended for herds with low fertility and presents an avenue of research that could be further explored.

Clinical markers of endometriosis: have we been too quick to judge?

Numerous biochemical differences have been documented in women with endometriosis compared to controls; however, identification of a clinically useful marker of endometriosis remains elusive. We postulate that the diversity of clinical presentations, patient objectives, and complexity of the pathophysiology of endometriosis mandates rigorous attention to study design and standardization of procedures and questionnaires that has heretofore been overlooked in the pursuit of clinical markers of this enigmatic disease. We further propose that it is premature to conclude that clinical markers of endometriosis brought forward in the literature lack clinical value in the diagnosis of endometriosis. To address this hypothesis we reviewed the literature and assessed papers according to a modified version of the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) criteria from which 55 high quality papers were reviewed. While pelvic inflammation and pain is a known significant component of endometriosis, control group definitions were widely divergent and included healthy women through to women with other inflammatory conditions. Although pain is a common presenting complaint in women with endometriosis, it was assessed in only 4 of 55 studies (7.3%) whereas infertility was documented in 34/55 studies (61.8%). Disease severity was assessed in 44 of 55 studies (80%) whilst the association between active vs. inactive disease was attempted in only 2 of the studies reviewed (3.6%). We conclude that experimental design criteria are inconsistently applied making comparisons across studies difficult. Thus, the clinical utility of previously described diagnostic markers of endometriosis remains uncertain.

An overview of molecular and cellular mechanisms associated with porcine pregnancy success or failure.

Prenatal mortality remains one of the major constraints for the commercial pig industry in North America. Twenty to thirty per cent of the conceptuses are lost early in gestation and an additional 10-15% is lost by mid-to-late gestation. Research over the last two decades has provided critical insights into how uterine capacity, placental efficiency, genetics, environment, nutrition and immune mechanisms impact successful conceptus growth; however, the exact cause and effect relationship in the context of foetal loss has yet to be determined. Similar to other mammalian species such as the human, mouse, rat, and primates, immune cell enrichment occurs at the porcine maternal-foetal interface during the window of conceptus attachment. However, unlike other species, immune cells are solely recruited by conceptus-derived signals. As pigs have epitheliochorial placentae where maternal and foetal tissue layers are separate, it provides an ideal model to study immune cell interactions with foetal trophoblasts. Our research is focused on the immune-angiogenesis axis during porcine pregnancy. It is well established that immune cells are recruited to the maternal-foetal interface, but their pregnancy specific functions and how the local milieu affects angiogenesis and inflammation at the site of foetal arrest remain unknown. Through a better understanding of how immune cells modulate crosstalk between the conceptus and the mother, it might be possible to therapeutically target immune cells and/or their products to reduce foetal loss. In this review, we provide evidence from the literature and from our own work into the immunological factors associated with porcine foetal loss.

Cellular and molecular events in early and mid gestation porcine implantation sites: a review.

Commercial, North American pork breeds (Sus scrofa) experience significant loss of genetically-normal conceptuses during the peri-implantation (attachment) period and at mid-gestation (day 50 to 90 of the 114 day porcine gestation interval). Although exact causes for these losses are not defined, asynchronous in-utero development and deficits in vascularization of the endometrium and placenta appear to be involved. Understanding of normal maternal-fetal dialogue is critical to develop breeding or therapeutic strategies that improve fetal health and overall litter size in commercial pigs. The non-invasive, epitheliochorial porcine placenta permits investigation of maternal or fetal compartments without cross contaminating cells. We developed and use protocols to capture single, homogenous populations of porcine cells (endometrial lymphocytes, dendritic or endothelial cells) from histological sections using laser capture microdissection (LCM), a powerful tool for study of gene expression that reflects the in vivo environment. These data are compared with gene expression in biopsies of endometrium and of trophoblast from the same, attachment sites. Here we review justifications for selection of the genes we have studied and our published and in progress work. These data provide new insights into the roles of the endometrial immune environment in the regulation of the success and failure of porcine conceptuses.

Investigation of lipid peroxidation in liposomes induced by heavy ion irradiation.

Lipid peroxidation induced by heavy ion irradiation was investigated in 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) liposomes. Lipid peroxidation was induced using accelerated heavy ions that exhibit linear energy transfer (LET) values between 30 and 15000 keV/microm and doses up to 100 kGy. With increasing LET, the formation of lipid peroxidation products such as conjugated dienes, lipid hydroperoxides, and thiobarbituric acid-reactive substances decreased. When comparing differential absorption spectra and membrane fluidity following irradiation with heavy ions and x-rays (3 Gy/min), respectively, it is obvious that there are significant differences between the influences of densely and sparsely ionizing radiation on liposomal membranes. Indications for lipid fragmentation could be detected after heavy ion irradiation.

Gamma-irradiation and UV-C light-induced lipid peroxidation: a Fourier transform-infrared absorption spectroscopic study.

Fourier transform-infrared spectroscopy of dry, multibilayer films has been used to study gamma-radiation and UV-C light induced lipid peroxidation in 1,2-dilinoleoyl-sn-glycero-3-phosphocholine liposomes. The observed spectral changes were compared with the results obtained from measurement of hydroperoxides, conjugated dienes and to the formation of thiobarbituric acid reactive substances, such as malondialdehyde (MDA) or MDA-like substances. Upon irradiation a decrease in intensity of the asymmetric C - H stretching vibration (va(CH2)) of the isolated cis C = C - H groups (3010 cm-1) was observed. Directly correlated with the decrease of the va(CH2) absorption was a shift of the asymmetric phosphate ester stretching vibration (va(P = O)) towards smaller wavenumbers (1260-->1244 cm-1), indicating that the lipid peroxidation induced molecular alterations in the fatty acid chains influence the packing of the phospholipids in dry multibilayer films. In addition, the formation of a new absorption band at 1693 cm-1 could be detected, the intensity of which was comparable with the formation of thiobarbituric acid reactive substances and, therefore, attributed to the (C = O) stretching of alpha, beta unsaturated aldehydes. Dose-dependent studies using ionizing radiation showed that the decrease of va(CH2) was directly correlated with an increase in absorption of the conjugated dienes at 234 nm and with the formation of hydroperoxides suggesting that the absorption at 3010 cm-1 is solely due to isolated cis C = C - H groups and hence subject to the early stages of the radical chain reaction. UV-C light induced lipid peroxidation revealed a non-linear decrease of I3010, which was directly correlated with the formation of hydroperoxides. The observed early saturation of the conjugated dienes was attributed to an early photodecomposition of the conjugated double bonds.

Photooxidation of tryptophan: O2(1 delta g) versus electron-transfer pathway.

Tris (2,2'-bipyridyl)ruthenium(II)chloride hexahydrate (Ru[bpy]3(2+)) free in solution and adsorbed onto antimony-doped SnO2 colloidal particles was used as a photosensitizer for a comparison of the O2(1 delta g) and electron-transfer-mediated photooxidation of tryptophan (TRP), respectively. Quenching of excited Ru(bpy)3(2+) by O2(3 sigma g-) in an aerated aqueous solution leads only to the formation of O2(1 delta g) (phi delta = 0.18) and this compound was used as a type II photosensitizer. Excitation of Ru(bpy)3(2+) adsorbed onto Sb/SnO2 results in a fast injection of an electron into the conduction band of the semiconductor and accordingly to the formation of Ru(bpy)3(2+) and was used for the sensitization of the electron-transfer-mediated photooxidation. The Ru(bpy)3(3+) is reduced by TRP with a bimolecular rate constant kQ = 5.9 x 10(8) M-1 s-1, while O2(1 delta g) is quenched by TRP with kt = 7.1 x 10(7) M-1 s-1 (chemical + physical quenching). Relative rate constants for the photooxidation of TRP (kc) via both pathways were determined using fluorescence emission spectroscopy. With Np, the rate of photons absorbed, being constant for both pathways we obtained kc = (372/Np) M-1 s-1 for the O2(1 delta g) pathway and kc > or = (25,013/Np) M-1 s-1 for the electron-transfer pathway, respectively. Thus the photooxidation of Trp is more than two orders of magnitude more efficient when it is initiated by electron transfer than when initiated by O2(1 delta g).

Singlet oxygen luminescence spectra: a comparison of interferometer- and grating-based spectrometers.

The current trend in methodology for determining IR and near-IR absorption spectra is to employ interferometer-based instruments to replace the monochromator-based devices used heretofore. As a dispersion element, the interferometer offers major improvements in spectral resolution (Connes advantage), light throughput (Jacquinot advantage) and data acquisition through multiplexing (Felgett advantage). We have compared signal-to-noise (S/N) ratios of grating-based and interferometer-based instruments for making spectral determinations of near-IR luminescence. Our results show that under identical excitation and detector conditions the interferometer instrument easily outperforms the grating, giving a 10-fold improvement in S/N at high signal amplitude (A488nm = 0.97) and a 20-fold improvement when the signal amplitude is low (A488nm = 0.06). Although some spectral resolution is sacrificed when scan times on the Fourier transform (FT)IR are significantly shortened, the S/N ratio was found only to decrease by a factor of 2 for a 10-fold decrease in scan time. This adds to the advantages of the FTIR technique because the S/N will thus improve for the same total acquisition time.

Flow cytometric detection of micronuclei by combined staining of DNA and membranes.

A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane-specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2-hydroxyethyl-7,12,17-tris(methoxyethyl)porphycene (HEPn) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN from debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH-3T3 cells by UV-B illumination was studied with this new staining technique. UV-B illumination (280-360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB and DPH were comparable to the results obtained with the combination of EB and HO.

Photodynamic antitumor agents: beta-methoxyethyl groups give access to functionalized porphycenes and enhance cellular uptake and activity.

Porphycene photosensitizers bearing two or four methoxyethyl side chains were synthesized in nine steps from commercially available starting materials. Ether cleavage led to (hydroxyethyl)- and (bromoethyl)porphycenes that were converted to vinyl and benzo derivatives. Five of the side chain-functionalized porphycenes were biologically studied in comparison with two tetra-n-propylporphycenes. Porphycenes were incorporated in small unilamellar liposomes and incubated with cultivated SSK2 murine fibrosarcoma cells. Cellular uptake and phototoxicity 24 h after 5 J/cm2 laser light treatment were determined. The porphycenes tested were between 17 and 220 times more photodynamically active than the currently clinically used sensitizer Photofrin, although extinction coefficients of the porphycenes' irradiated bands are only approximately 10-fold higher. The LD50 concentration for SSK2 cells in the incubation medium was as low as (8.5 +/- 2.8) x 10(-9) M for tetrakis(methoxyethyl)porphycene. Two methoxy or hydroxy groups enhanced cellular uptake, three or four methoxy groups both enhanced and accelerated cellular uptake of tetraalkylporphycenes. Half-life times of the uptake processes varied between (0.14 +/- 0.04) and (14 +/- 4) h and cellular saturation levels between (1.2 +/- 0.2) and (26 +/- 3) pmol/10(5) cells. When individual uptake rates were accounted for, all porphycenes had a similar "cellular" phototoxicity, pointing toward a common mechanism of action. Evidence is presented for the assumption that cell membranes are the primary targets of the tested porphycenes and that membrane solubility may play a critical role in their photodynamic efficiency. The results show that nonionic polar side chain functionalities can strongly enhance cellular uptake and antitumor activity of lipophilic porphyrinoids and thus that the known lipophilicity/activity relationship can be reversed for very hydrophobic sensitizers.

Cell cycle kinetics of hematopoiesis before and after in vivo administration of GM-CSF in refractory anemia: evidence for a shortening of the granulocyte release time.

GM-CSF administration to patients with refractory anemia (RA) induces an increase in neutrophils and eosinophils. We studied cell kinetic mechanisms underlying this observation using clonogenic assays and in vivo iododeoxyuridine labeling of bone marrow cells. Cell cycle kinetics were studied in three patients before and during GM-CSF administration (two daily subcutaneous injections of 54 or 108 micrograms). No consistent effect on the relative number of bone marrow CFU-GM was noticed. The DNA synthesis time and potential doubling time of low-density bone marrow cells remained essentially the same. A slight decrease (1.5-3.7%) in labeling index was found, originating from the myelo(-mono)cytic lineage. In all three patients the release time of labeled granulocytes from the bone marrow into the peripheral blood was shortened (before GM-CSF treatment 5-7 days and during GM-CSF 3-4 days). Cell cycle kinetics of CD34+ cells were studied in order to obtain kinetic information on immature precursor and progenitor cells. The DNA synthesis time of the CD34+ cells was shortened during GM-CSF therapy, resulting in a shorter potential doubling time. GM-CSF administration to patients with RA results in a rise in granulocytes that might be due partly to an accelerated release of granulocytes from the bone marrow compartment into the circulating blood and partly to an increased proliferative activity of the immature precursor and progenitor cells.

Photodegradation of protoporphyrin-dimethylester in solution and in organized environments.

The degradation of sensitizers used in photodynamic therapy (PDT) involves photooxidation either by molecular oxygen or by oxygen intermediates which leads to hydroxyaldehyde and formyl products or to ring opening. Our investigations focused on the spectroscopic changes which protoporphyrin-dimethylester (PP) exhibits upon irradiation. As the microenvironment strongly influences the effects, we used an aprotic organic solvent, L-alpha-phosphatidylcholine dioleoyl (DOPC) liposomes and isogenic fibrosarcoma cells (SSKII) as carriers for PP. Hydroxyaldehyde product isomers develop a new absorption band centred around 670 nm and a new emission band at 676 nm. These characteristics can be used to discriminate them from formyl products and intact PP. In organic solvents, the formation of the hydroxyaldehyde products dominates. In DOPC liposomes and cells, the hydroxyaldehyde yield drops and photooxidation results in attack of the macrocycle. Time-resolved fluorescence spectroscopy of monomeric PP in an organic solvent gives a monoexponential decay time tau of 10.1 +/- 1.3 ns. Upon irradiation a second component with a decay time of 4.9 +/- 0.6 ns, resulting from the hydroxyaldehyde product, was detected. In liposomes and cells the monomeric decay time was significantly longer (15 ns) due to the altered microenvironment. Additionally, we observed in liposomes and in cells a small contribution of a short component (1 ns) which is attributed to an aggregated sensitizer species. In irradiated cells the aggregated fraction doubles, indicating a change in the microenvironment caused by the photodynamic action of the sensitizer.

Phonotactic knowledge of word boundaries and its use in infant speech perception.

The development of a lexicon critically depends on the infant's ability to identify wordlike units in the auditory speech input. The present study investigated at what age infants become sensitive to language-specific phonotactic features that signal word boundaries and to what extent they are able to use this knowledge to segment speech input. Experiment 1 showed that infants at the age of 9 months were sensitive to the phonotactic structure of word boundaries when word-like units were presented in isolation. Experiments 2 to 5 demonstrated that this sensitivity was present even when critical items were presented in context, although only under certain conditions. Preferences for legal over illegal word boundary clusters were found when critical items were embedded in two identical syllables, keeping language processing requirements and attentional requirements low. Experiment 6 replicated the findings of Experiment 1. Experiment 7 was a low-pass-filtered version of Experiment 6 that left the prosody of the stimulus items intact while removing most of the distinctive phonotactic cues. As expected, no listening preference for legal over illegal word boundary clusters was found in this experiment. This clearly suggests that the preferential patterns observed can be attributed to the infants' sensitivity to phonotactic constraints on word boundaries in a given language and not to suprasegmental cues.

Sensitivity to inflectional morphology in aphasia: a real-time processing perspective.

The present study investigates Broca's aphasics' sensitivity to morphological information in an on-line task. German is used as the test language because it is highly inflected. Results from two word monitoring experiments show first that Broca's patients like normal controls are sensitive to the presence of a contextually incorrect inflection. Contrary to normals, they are, however, not sensitive to the absence of an obligatory inflection even when its presence is syntactically highly constrained. Second, they reveal that Broca's aphasics are only sensitive to the presence of an incorrect inflection when it functions as a marker of lexical category (noun vs. verb) and not when it functions as a diacritical marker (second person singular vs. third person singular). The results are taken as evidence for the claim that Broca's aphasics are impaired in the ability to process the full syntactic information encoded in closed class elements in a fast, automatic, and obligatory way.

Chemosensitivity testing of xenografted squamous cell carcinomas of the head and neck region.

Eight squamous cell carcinomas from the head and neck region were established as xenograft lines in nude mice and tested for their sensitivity to the antineoplastic drugs bleomycin and cisplatin. Tumor volume, histology, DNA flow cytometry and mitotic activity were used as parameters. One out of the 8 tumours appeared to be highly sensitive to bleomycin, while three other tumours were sensitive to both bleomycin and cisplatin. These observations are in good correlation with the reported data in patients. All chemosensitive tumours showed regrowth after the cytotoxic drug treatment had been completed. No change was seen in the chemosensitivity of other features of the regrown tumours, not even after repeated exposure to the drugs. Comparison of the tumour volume with the other parameters applied indicated that the tumour volume of squamous cell carcinomas was not always a reliable parameter for testing chemosensitivity, because of the important contribution of keratin to the tumour volume. It is concluded that additional parameters such as histological examination, DNA flow cytometry or mitotic activity are necessary in order to draw reliable conclusion on xenografts with a large avital component. In addition, DNA flow cytometry has proved to be very useful for the rapid screening of drug sensitivity.