Purpose: This study aimed to highlight the clinical features, diagnosis, and different modalities of the treatment of cesarean scar pregnancy (CSP).Methods: This study was done in the tertiary referral hospital of India for one year. A total of 11 cases were enrolled prospectively. In each case, the diagnostic ultrasonography and measurement of baseline beta-human chorionic gonadotropin (β-HCG) levels were done. The treatment was given based on the hemodynamic status of the patient and desire for future fertility. Various treatment modalities used were medical, surgical, or interventional digital subtraction angiography to control hemorrhage. Also, in some cases, ultrasound-guided methotrexate was injected into the scar ectopic. Medically treated cases were followed up until their β-HCG levels became normal.Results: Out of 11 patients, six had a history of two cesarean sections in the past, four patients had a history of one cesarean section and one patient with a previous three low segments cesarean section (LSCS). Seven out of 11 patients underwent medical management with either methotrexate with potassium chloride (KCl) or methotrexate alone. The success of the medical management was monitored by serial β- HCG values. The mean time for the resolution of these 10 patients was 86.7 ± 53.6 days. Three patients underwent emergency uterine artery embolization due to uncontrolled bleeding and one patient required laparotomy.Conclusion: CSP is a life-threatening condition that can be diagnosed with the help of transvaginal ultrasonography. The treatment, however, depends on the hemodynamic status of the patient and desire for future fertility. Well-defined diagnostic criteria coupled with structured management and follow-up protocol can help in treating this challenging form of ectopic pregnancy. Implantation of the pregnancy within the scar of the previous cesarean section can be a life-threatening condition. Cesarean scar pregnancy (CSP) was first described in 1978 by Larsen and Solomon . The mecThe patients with CSP can have variable clinical profiles. They may present as spontaneous abortion in the first trimester or with devastating complications like uterine rupture, adherent placenta, and hemorrhagic shock in the second or third trimester. The main objective of our study is to highlight the clinical features, diagnosis, and different modalities of the treatment of CSP with a background of our clinical experience. We describe the management of patients with CSP based on their varied clinical presentations.This study was carried out in the department of Obstetrics & Gynecology of Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India, for a total of one year. A total of 11 cases were enrolled, six prospectively and five retrospectively. The patients were diagnosed with CSP based on the grayscale transvaginal ultrasound scan. We analyzed the data from the inpatient records related to presenting complaints, maternal age, gravidity, gestational age (weeks), number of previous cesarean section, previous history of abortion, presenting complaints, presence of fetal cardiac activity in an ultrasound scan, history of medical termination of pregnancy (MTP) intake and history of dilatation and curettage in the current pregnancy. The diagnostic ultrasound and the measurement of baseline human chorionic gonadotropin (β-HCG) levels were done in each case. The treatment was given based on the hemodynamic status of the patient, gestational age, and desire for future fertility. The various treatment paradigms used were medical, surgical, and uterine artery embolization (UAE) via angiography. The medical options offered were dependent on the presence of fetal cardiac activity. In presence of fetal cardiac activity, injection potassium chloride (1-2 mEq) was instilled into the fetal heart using a 20 G, 15 cm long needle under transvaginal ultrasound guidance. This was followed by the administration of systemic methotrexate . Serial monitoring of β-HCG levels was done, on days one, seven, 14, 21, and further to document the subsequent fall in serum levels.In the absence of cardiac activity, the patient received a methotrexate injection. Subsequent monitoring of β-HCG levels was carried out till serum levels normalized. If the patient presented with hemodynamic instability, resuscitation was performed till adequate circulatory volume status was restored. A further decision was taken based on the patients’ wish to preserve fertility. A laparotomy along with a hysterectomy was performed if the patient did not wish to retain fertility.In the case of an actively hemorrhaging patient, angiographic assisted treatment is life- and time-saving. The embolization of the bilateral uterine arteries aided by digital subtraction angiography was done by the intervention radiologist. Clearance from the ethics committee of the hospital was taken.A total of 11 patients were included in the study. Table Table The patients receiving medical management were followed based on serum β-HCG levels measured on day four and day seven and then every week until the serum levels were undetectable. The patients undergoing UAE had a follow-up with weekly β-HCG levels. β -HCG value for each patient is listed in Table Overall in our study, seven patients (63.6%) underwent medical management with methotrexate with or without intracardiac injection of potassium chloride. Three patients (27%) underwent emergency uterine artery embolization and one (9%) underwent a hysterectomy.CSP is a life-threatening condition and carries a high risk of uterine rupture and massive hemorrhage. Despite the medical description of CSP as early as 1978, its diagnosis and management remain unclear. This is due to the rarity of the scar pregnancy. Our case series represents one of the largest case series from India. In our study, we found that the symptoms of cesarean scar ectopic pregnancy are like that of abortion. This frequently leads to misdiagnosing these patients as incomplete abortions and ultimately undergo blind procedures like dilatation and curettage.The reported mean age group in one of the largest case series reported from Israel was 35 years, whereas, in our study, it was 31.5 years . It may Apart from the age and previous cesarean section, the relationship between gravidity, parity, and previous abortion with CSP has been well-documented. A study conducted by Zhou et al. reported that gravidity and previous induced abortions were independent risk factors for developing CSP . Our stuUltrasound is considered the first line of diagnostic modality to diagnose CSP. There are four diagnostic features seen in the transvaginal ultrasound vis-a-vis (i) an empty uterus, (ii) an empty cervical canal, (iii) discontinuity in the anterior uterine wall, and (iv) the gestational sac located in the anterior part of the isthmic portion of the uterus with a diminished myometrial layer between the bladder and the sac -5. The sThe treatment of CSP depends on the clinical presentation of the patient. If the patient has massive bleeding or uterine rupture, then emergency embolization or laparotomy followed by scar excision or hysterectomy is indicated. The desire to retain fertility is of paramount importance here. In our study, four patients (36.3%) presented with life-threatening bleeding, where three underwent emergency bilateral uterine artery embolization and one needed an emergency hysterectomy. It is particularly important to mention that three out of these four patients who presented with acute bleeding were initially managed with dilatation and curettage (D&C). There could be a possibility that D&C not just failed to manage the initial disease but may have led to the development of arteriovenous malformation. The laparoscopic excision of the previous scar is a surgical option that needs to be considered in women without living issues. The main advantage of laparotomy or laparoscopy is quick normalization of β-HCG values and decreasing the risk of recurrence as it involves complete removal of the microtubular tract .The medical treatment is primarily indicated in hemodynamically stable patients. The aim of medical management is avoidance of laparotomy and preservation of fertility. However, close surveillance with β-HCG level is essential to monitor the response. The medical management includes the use of methotrexate either local or systemic with or without the use of potassium chloride. The rationale for injecting potassium chloride after the confirmation of the diagnosis is to immediately cause fetal death and prevent further invasion and proliferation of villi. Seow et al. have previously advocated the use of ultrasound-guided methotrexate in the successful management of scar ectopic . A lowerThe mean time for the resolution of β-HCG in these 10 patients was 86.7 ± 53.6 days. In the study by Timor-Tritsch et al., the mean resolution of β-HCG values excluding patients undergoing hysterectomy or embolization was 88.6 days [In the past, many investigators have advocated methotrexate as the first-line management of scar pregnancy. Previous studies have shown that 50% of patients with CSP initially managed with methotrexate would require additional intervention ,15. In oWhen uterine artery embolization was used as the primary modality of treatment in our study, no complications related to CSP were reported. Only one patient had gluteal skin necrosis, which was managed symptomatically. The viability of the post-embolised uterus is something that needs to be studied in detail since the option of fertility preservation also needs to be considered in patients who may be surgical candidates.In our series, life-threatening vaginal bleeding was documented in all patients who underwent dilatation and curettage. So, we believe that curettage should not be used as a primary modality of treatment in CSP. We observed that systemic methotrexate is an effective primary modality in the treatment of CSP with fewer complications. However, this seems to be acceptable only in the absence of fetal cardiac activity. The possible explanation could be slow absorption or activity of the drug which may fail to inhibit the vascularisation of the growing fetus resulting in secondary bleeding. In the presence of fetal cardiac activity, the beneficial role of intra-amniotic injection of methotrexate along with potassium chloride is well documented in the literature . TimmermAlso, we did not leave any patient for the expectant management, however, there have been reports in the past that this could be a viable option for the scar ectopic with no fetal cardiac activity . Given tThe higher β-HCG levels are found in most cases of cesarean scar pregnancy and this should not be considered as an inhibition to resort to the non-surgical methods in women desiring future fertility. The women should be counseled about the likely success of the procedure, the possibility of acute hemorrhage during the follow-up, and the slow resolution of the visible scar pregnancy on the ultrasound.The limitation of our study is that we did not use the doppler indices for predicting the outcome of the patient. The number of the recruited patients was less given the rarity of this condition. CSP is a rare yet life-threatening obstetric condition. It can be diagnosed early with the help of transvaginal ultrasonography complemented with doppler. The treatment is not well defined, yet we suggest the use of medical management as the first line of treatment in hemodynamically stable patients. Invasive procedures like laparotomy or embolization should be reserved for the acutely bleeding patient. β-HCG should not be the determining factor in the management of this rare form of ectopic pregnancy. Well-defined diagnostic criteria coupled with structured management and follow-up protocol can help in treating this challenging form of ectopic pregnancy.


A dynamic baseline tracking method and a range of calibration protocols to address system performance were explored under practical scenarios to assess the performance of the PEK in reducing the impact of rapid changes in the ambient environment in personal exposure assessment applications. The results show that the accuracy and stability of the ppb level gas measurement is enhanced even in heterogeneous environments, thus avoiding the need for data post-processing with mathematical algorithms, such as multi-linear regression. This establishes the potential for use in personal exposure monitoring, which has been difficult in the past, and for reporting more accurate and reliable data in real-time to support personal exposure assessment and portable air quality monitoring applications.Sensor technology has enabled the development of portable low-cost monitoring kits that might supplement many applications in conventional monitoring stations. Despite the sensitivity of electrochemical gas sensors to environmental change, they are increasingly important in monitoring polluted microenvironments. The performance of a compact diffusion-based Personal Exposure Kit (PEK) was assessed for real-time gaseous pollutant measurement (CO, O Assessments of exposure to air pollutants have often depended heavily on measurements from stationary reference instruments, but these may poorly represent individual exposure linked to the pattern of human activity. Conventional networks do not accurately represent concentrations in the microenvironments experienced by people. Such networks are costly, require considerable care in housing and maintenance and are not easily moved. They fail to provide data that reflect the high degree of spatial and temporal variation that contributes to personal exposure in urban microenvironments ,2,3. It In the last decade, the development of microsensor-based monitoring methods has shown potential to address many of the limitations in exposure estimation imposed by conventional monitoring networks. Sensor technology benefits from portability and low cost and can represent locations where individuals are exposed to air pollution ,10,11. IImproving sensor measurement is a key aspect for further research. Simple linear regression, which assumes the linearity of sensor responses and pollutant concentration from regular reference instruments, is not suitable for dynamic microenvironments with abrupt environmental variation. Mathematical algorithms to compensate for sensor response to environmental variation have used multi-linear regression ,15 and aThis study assessed the performance of a sensor-based personal exposure kit  in exposure applications. The kit directly outputs real-time pollutant concentrations, thus providing the ability to determine individual exposure. The concentration data from the PEK with its dynamic baseline tracking were compared with raw sensor output using commonly employed universal correction algorithms ,21 often2 and O3 simultaneously. Each gas is measured by a separate gas module based on electrochemical sensing and pair differential filter (PDF) technology (PCT patent pending) with real-time dual signal outputs of simultaneous raw signal and dynamic baseline signal for the individual gas sensors. The two signal outputs in volts, together with concentration outputs from the PEK in parts per billion (ppb), are examined here to determine the effectiveness of the method in reducing sensitivity to humidity and temperature. The sensor modules are comprised of raw sensor heads of A-Type 4-electrode electrochemical sensors from Alphasense  to provide the raw signal and corresponding proprietary baseline sensor heads to provide the baseline signal. The Ox-A431 sensor responds to oxidizing gases , while the NO2 sensor includes a filter that removes O3 such that the sensor responds to NO2, the difference giving an estimate of the O3 concentration. Temperature, humidity  and light intensity  are measured simultaneously to provide the environmental conditions to differentiate the microenvironments [The PEK  used forronments . The defSensor system performance traits were evaluated experimentally in a range of laboratory environments.2 and O3 were generated with a NO2/NO/O3 calibration source . During calibration, the gas flow rate was fixed at 3 L min−1 in all cases , and validated with a flow meter . Prior to the experiments, both the T700U and 2B 714 instruments were left to warm up for 30 min. In all cases, the kit was allowed to warm up for 48 h to assure a steady-state response. Temperature and humidity measurements from the chamber were refined beyond the standard configuration by including a Vaisala Temperature/Humidity probe . Data output was stored on an internal SD card during the experimental procedure. The laboratory setup consisted of several subcomponents: real-time data acquisition, an environmentally controlled chamber , and the kit which was placed inside the chamber. The flow-through calibration allowed concentrations of gases in the air to be set using a standard gas supply, diluted to the required concentration. Target CO standard gas concentrations were produced with a dynamic calibrator , which combined zero-gas generated using a zero-air generator  with compressed CO from a cylinder . NO2, as well as for O3; 0.2 ppm, 0.5 ppm, 1 ppm, 1.5 ppm and 2 ppm for CO. Stable values, at each test concentration, were maintained for 15 min. The PEK was placed in the calibration manifold kit connected to the gas supply with Teflon tubing. The tests were run one gas at a time, and to avoid interference NO2 was completed first followed by O3. It is important that gas sensors respond linearly over the concentration range of interest. The PEK sensor signals were recorded at a range of specific concentrations of the analyte gases  during calibration procedures. In this study, standard gas concentrations were 50 ppb, 100 ppb, 200 ppb for NO3 sensor output is also influenced by the environment [One of the concerns in gas monitoring using electrochemical sensors is the effect of environmental factors on the sensor baseline, humidity in particular . O3 sensironment , especiaironment , suggest90 or t50, is a performance characteristic of electrochemical sensors and measurement criterion [90 is defined as the time taken for the sensor response signal to reach 90% of its steady state value; similarly, the t50 time refers to the time to reach 50% of the value. Two Nafion tubes , each 60 cm long, were connected in parallel to the gas delivery tubing, located inside the chamber to ensure a controlled environment [−1. The concentrations used were zero air or 2 ppm, 500 ppb and 200 ppb corresponding to CO, NO2 and O3. Sensor response times and concentration measurements were evaluated at constant temperature and humidity once a stable environment was established in the chamber . Response time, typically as triterion . The t90ironment . This tuAnother key performance requirement is sensor response to rapid changes of temperature and humidity, which can distort the concentration measurements during real world exposure. Examples include entering and exiting air conditioning, heated spaces or transit systems. Two separate experiments explored PEK operation under sudden changes in temperature and humidity. The variable temperature scenario (T) used a decrease from 40 °C to 15 °C over an hour. For humidity variation, it was arbitrarily fixed at 47%, then varied from 65% to 15% for 7 min at a constant temperature of 25 °C. Although temperature and humidity variation can be effectively controlled in the laboratory, the ambient environment is more complex with different factors affecting sensor response. The study also included intensive field performance assessment. The PEK was deployed at three different government monitoring sites in Hong Kong: Tseung Kwan O ; Mong Kok  and Kwun Tong . The units were mounted on the railing on the opposite side of the road from the MK site, at the same height of its sampling inlet. All data from the conventional monitoring instruments as well as the PEK values were recorded at 1-min intervals. Laboratory calibration—to assess suitability for field use, a laboratory calibrated PEK was deployed at reference sites to evaluate the robustness of the calibration with respect to accuracy and precision.Field co-location calibration—to quantify sensor changes among different environments, calibration took place via co-location at the TKO site and was subsequently validated via co-location at the other two sites for three days.Sensor-based monitors used in the field are typically calibrated in the laboratory, using standard gases to establish basic sensor response, or via co-location with regular monitoring sites before and/or during use . HoweverRobustness of the measurements across dynamic and changing microenvironments was established by simulating daily routines to reflect typical individual exposure profiles. Initially, the PEK was co-located with a well characterized and calibrated mini air station  at Hong Kong University of Science and Technology which served as an indoor reference. Co-location initially took place from 06:00 to 08:00, as described in previous studies ,18. The Raw, (ii) output in volts tuned by the dynamic baseline as Baseline, (iii) concentration output in ppb (hereafter PEK measurement). The algorithm used in the PEK to convert from sensor signals to concentration is as follows and the calculation is performed onboard of PEK for real-time concentration output.The PEK has three separate outputs: (i) a raw sensor output in volts as Multi-linear regression models are commonly used in sensor adjustment algorithms. Previous researchers ,25,26 haV, which is equivalent to the raw signal output from the PEK. RH and T of the sampled air are measured directly from the PEK. The parameters a1, a2… are regression coefficients and b the intercept [A widely used approach for mathematical baseline correction with a 4-electrode sensor subtracts the working electrode output from the reference electrode output and yields a differential voltage Δntercept . Data wentercept .2 and O3) show strong linear relationships over the calibration range, indicating the reliability of both sensor outputs under laboratory conditions, which is consistent with other results [ results ,12,14. TR2 > 0.90) and with humidity (R2 > 0.65) for the three gas sensors. Temperature increases lead to a decrease in sensor voltage output and the output under RH increase shows a step-like character followed by an increase in voltage. The sensor voltage outputs for NO2 and O3 are more easily affected by environmental variables under high temperatures (T > 35 °C) or low humidity (RH < 20%) with piecewise linearity over the entire range (Sensor output (corrected output and raw sensor output) from the environmental chamber are shown in re range c–e. The 3, the t90 values were less than a minute at a flow of 3 L min−1 according to laboratory test , while the NO2 sensor took just a little longer to achieve stability (~1.5 min) due to the ozone absorbent filter on top of the NO2 sensor head that slightly reduces the air diffusivity. There was a slight time lag observed between the raw sensor output and corrected output for O3 and less being observed for CO and NO2. The lag may be due to the fact that the raw and corrected outputs come from two independent signals of raw and dynamic baseline tracked sensor heads with inherent difference of gas permeability from the surface to the electrode of individual sensors. However, the lag appears to be insignificant and no obvious discrepancies in data were observed for the three gases. 3 PEK output signals recover from this humidity shock to the almost stable original state, while the raw sensor signal recovers more slowly under the same conditions. For NO2, such a transient humidity change affects both sensor signals, with the NO2 sensor data from the PEK shows a smaller effect and recovers more quickly than the raw sensor signal. The PEK returns to stability within ~5 min following a RH drop. Overall, these results show that output adjusted using dynamic baseline correction can reduce the effect of rapid changes in temperature and humidity on signal output, as is evident in the effects on raw sensor output.The PEK output incompletely reduces this impact when humidity reduces 50% in 9 min. However, CO and ONote: The elapsed time refers to the time period counted from the beginning of the experiment and the zero-point shown in the x-axis refers to the experiment starting point. The experiment in At the MK roadside station, a large amount of sensor noise seems to arise from wind variation and local vehicle emissions . The und2. The measurements remain well-correlated even when the kit is moved from one site to another. According to a previous study [us study , sensor 3 measurements showed only fair correlation with fixed site data (R2MLR = 0.56) after one month of operation. It is unclear what factors contributed to this limited agreement.PEK measurements were compared with raw sensor output corrected using multi-linear regression with parameters derived from co-location at the TKO site as described in Methodology 2.3.1 (II). 2); see The PEK data is in better agreement than the multi-linear regression (MLR) adjusted data (across the period shown in The performance of the PEK at different reference sites in Hong Kong is shown in RMSEP1 = 57.4 ppb), while Protocol 2 is less effective, with an RMSE 47.7% higher than under Method 1. NO2 shows approximately equal RSME values of ~8.0 ppb, and for O3, the highest RMSE is seen under Method 1 (RMSEM1 = 8.9 ppb).For CO, Protocol 1 is the best choice as a routine calibration protocol (2 and O3 over longer periods (1 month). Overall, these three protocols are largely in agreement for the three pollutants over a one-month observation period at different monitoring stations. The laboratory calibration (Protocol 1) proves robust for the PEK across the monitoring sites. However, if conventional instruments are not available for laboratory calibration, field co-location (Protocol 2) aids data quality assurance. Although multi-linear regression has acceptable RMSE values for CO, the discrepancies were larger for NO2 and O3 measurements. There is no obvious difference between the PEK measurements and the multi-linear regression model under the initial indoor conditions. However, the influence of environmental factors is problematic in output corrected using the multi-linear regression model. When the kit was taken back to the identical indoor conditions, both NO2 and O3 algorithms continued to fail. The kit was trialed as a personal exposure monitor across a range of urban microenvironments. It was carried on a backpack into various indoor and outdoor locations, which represent notable variations in pollutant concentrations. The agreement at roadside station MK is not as convincing as ambient station TKO, which may be caused by instantaneous vehicle emissions. Such emissions have very dynamic changes in concentration over short distances, and the distance between the sensors and the station air inlet due to space constraints may play a role. Smaller discrepancies were observed at TKO. This reinforces a frequent concern over the accuracy of measurement when deploying electrochemical sensors at disparate locations or at the same site at different times. rPEK,TKO = 0.804; rMLR,TKO = 0.657; rPEK,MK = 0.261; rMLR,MK = 0.372). In general, the PEK is able to capture the short-period gaseous air pollutant concentrations experienced during personal exposure even when concentrations are highly variable. The multi-linear regression model is effective for CO transient concentration assessment but did not perform well for NO2 and O3 measurement in this case.3, NO2 and CO concentration measurement that appears to be largely free of temperature-humidity effects in the laboratory and under field conditions. The PDF enabled dynamic baseline tracking method overcomes the sensitivity to ambient conditions often found with electrochemical cell-based systems. This is especially useful when the kit is used under conditions where temperature and humidity vary on diurnal and shorter time scales. Results show good linearity in controlled environments and a more stable NO2 signal response when used in indoor-to-outdoor applications. The performance of the dynamic baseline tracking exceeds that of the prevalent multi-linear regression model under the same conditions. In addition, comparison between PEK output and concentrations from the regression model in calibration protocols and changing environments indicates the dynamic baseline correction performs better, particularly when measuring NO2 and O3 at low ppb concentrations.The personal exposure kit demonstrated an excellent ability to provide output for OAdditionally, validating the applicability of various calibration protocols, both in the laboratory and field, revealed that data from this kit shows acceptable results when deployed at a new site. Sensor-based monitors calibrated in the laboratory are not always easy to apply directly to field measurement, whereas the PEK calibrated system can provide reliable measurements in such cases. Our study shows that when standard gas calibration is not possible, co-located field calibration against regulatory monitors is valuable. Molecular diffusion delay via diffusion-based sampling impedes response times by seconds to a minute, so the capture of high-resolution measurements requires further work.Overall, the dynamic baseline correction offers a promising technology that is of benefit for measurements made at many locations, especially pollutant hotspots that might best be assessed real-time. It will thus contribute to epidemiology and urban air quality characterization. The kit has been tested in a reasonable range of environments. However, further studies might also cover pollutant concentrations, temperature and humidity to establish its performance characteristics under a wider range of conditions. It is likely that the dynamic baseline correction could be applied to an advantage with other pollutants measured using microsensors sensitive to temperature and humidity conditions. It offers the potential for direct reading and on-site reporting of data of high quality without post-processing using data correction algorithms.


Toluene, as one of the volatile organic solvents, has important industrial applications and can be used in a wide range of consumer and commercial products. It can be inhaled and absorbed easily by the human body and can therefore affect the individual’s health through damaging different body tissues, including the ovary. This organic solvent has been one of the most well-studied neurotoxins in recent decades. Studies reporting its effects on ovarian function are still limited. To advance our knowledge on the effect of toluene on female reproduction, an in vivo study using female Wistar rats was investigated. We found that toluene exposure affected ovarian structure and hormone balance by increasing progesterone and testosterone levels. In addition, it has disrupted most of the ovarian markers involved in granulosa cell proliferation and differentiation. Interestingly, Toluene exposure induced both apoptosis and autophagy, confirming the crosstalk between both mechanisms. The promising results of this study may contribute to the prevention of reproductive problems in society by raising the awareness about the use of this hydrocarbon.Insl3, ccnd2 and actb. The TUNEL assay showed that apoptosis occurred at the middle and high doses only , whereas no effect was observed at the low dose (2000 ppm). Interestingly, we showed that toluene exposure induced autophagy as LC3 protein and its encoding gene significantly increased for all doses of treatment. These results may suggest that the activation of autophagy at a low dose of exposure was to protect ovarian cells against death by inhibiting apoptosis, whereas its activation at high doses of exposure triggered apoptosis leading to cell death.Toluene has been shown to be highly toxic to humans and animals and can cause damage to various tissues. However, studies reporting its effects on ovarian function are still limited. In this study, we investigated the in vivo effect of toluene using female Wistar rats. We found that toluene exposure decreased ovarian weight and affected ovarian structure by increasing the number of abnormally growing follicles. Moreover, it significantly increased progesterone and testosterone levels. We also showed that toluene exposure decreased GDF-9 protein and its encoding gene. In addition, it inhibited the expression of most of the genes involved in granulosa cell proliferation and differentiation, such as  Volatile organic solvents have important industrial applications and can be used in a wide range of consumer and commercial products, such as paints, cleaners and adhesives. Toluene, as one of these volatile organic compounds, represents one of the major precursors of secondary organic aerosols ,2. It caToluene exposure can affect an individual’s health  and can Interestingly, toluene has been reported to cause renal and liver damage and toxicity in humans ,27,28. AThis study was approved by the Scientific Research Ethics Committee at King Saud University (Reference No: KSU-SE-20-76). The Animal Welfare Center provided us with forty female Wistar rats weighing 200–250 g. They were held in single cages (22 to 24 °C) with a 12 h light/dark cycle and free access to food and water. The females were randomly divided into four groups of ten rats that were exposed to different concentrations of toluene for 28 days by inhalation for 30 min per day. The first group is composed of females that did not receive any toluene treatment. Rats in Group 2 were exposed to 2000 ppm toluene; rats in Group 3 were exposed to 4000 ppm toluene and rats in Group 4 were exposed to 8000 ppm toluene. After 28 days of treatment, animals were transferred individually to a transparent plastic box connected to a carbon dioxide tube at a flow rate of 10 L/hour for ten minutes. Blood samples were drawn from the heart and transferred directly to tubes containing an anticoagulant (EDTA) to obtain the serum after centrifugation. The ovaries were removed carefully, cleaned of adherent tissue and weighed. The sampled ovaries were labeled according to their origin (groups), and a portion of them were fixed in neutral buffered formalin (NBF) for classic histological study, immunostaining and TUNEL assay. A second portion was collected and stabilized in RNA later and stored at −80 °C for the molecular assays. Ovarian samples were collected, weighed, and fixed in 10% neutral buffered formalin (NBF) for 24 h after 28 days of treatment. For classic histological analysis, samples were embedded in paraffin blocks that were serially cut into 5 µm-thick parts using a rotary microtome and stained with hematoxylin-eosin. For immunostaining and TUNEL assay, some blocks were cut into 3 µm-thick pieces. The follicular count for each ovary was calculated using the method mentioned in our previous research .To obtain plasma, blood samples were collected into lithium heparin tubes and centrifuged at 3000 rpm for 10 min. The levels of progesterone, estradiol, testosterone and IGF-1 were measured using competitive enzyme-linked immunosorbent assays (ELISAs) as directed by the manufacturer .The ovaries were fixed in 10% NBF for 24 h at room temperature for the TUNEL assay. Paraffin wax blocks were made, and 3 µm-thick paraffin parts were prepared and placed on coated slides. Dewaxed sections were rehydrated using normal methods and washed in phosphate-buffered saline (PBS). The tissue parts were then permeabilized in 0.1% Triton X-100 with 0.1% sodium citrate and incubated with a Proteinase K working solution at 37 °C for 15 min. TUNEL staining was performed according to the manufacturer’s instructions for the TMR red 12156792910 In Situ Cell Death Detection Kit . A positive control was created for one of the ovarian tissue sections, and a negative control was created by excluding the TdT from one of the other ovarian tissue sections. The nuclei were stained with Hoechst, and the labeled parts were examined with a Nikon TE 2000 fluorescence microscope using confocal microscopy  to look for DNA fragmentation, chromatin condensation, and other apoptotic changes. The signal for TMR-red was analyzed by Zen 3.1 service (ZEN lite) and quantified using GraphPad Prism 9 program .Slides containing tissue sections were placed on hotplates (60 °C) and dewaxed with xylene (2× 10 min). Then, they were rehydrated with decreasing concentrations of ethanol and washed twice with distilled water and with 1× PBS. The slides were removed from the washing process and dried by placing them diagonally on a surface containing dry tissues to ensure that the washing solution was removed. After drying, the slices were placed in a suitable container on the floor covered with layers of tissues moistened with water to reduce the rate of evaporation of the solution that the tissue sections would be treated with in the following stages. The tissue sections were then placed in 0.1% Triton X-100 with 0.1% sodium citrate. Blocking buffer (1% BSA in PBS) was used to block nonspecific staining (45 min at RT). The slides were placed into a humid box, and were then incubated with anti-GDF-9 (1:100 dilutions) and anti-LC-3 (1:100 dilutions) primary antibody. The humid box was closed carefully with tin paper and kept overnight at 4 °C on a flat balanced surface in the dark. On the second day, the slides were washed with 1× PBS, and were incubated with antirabbit FITC-conjugated secondary antibody  for 45 min at RT in the dark. During the next step, slides were washed with PBS and then TE buffer before adding Hoechst solution . Finally, the slices were washed, dried, and covered with cover slides, ensuring that the edges were covered with nail polish. Photomicrographs of GDF-9 and LC-3 specific signals and nuclear stains were captured with spinning disk confocal microscope from Zeiss (Germany). The immunofluorescence signal for protein expression was analyzed by Zen 3.1 service  and quantified using GraphPad Prism 9 program .RNA was extracted from ovarian tissues that had been previously stabilized with RNAlater . We used an RNeasy Mini Kit  with on-column DNase treatment with an RNase-Free DNase Package for RNA extraction (Qiagen). SYBR green and an Applied Biosynthesis 7500 Quick RT–PCR system  were used to perform real-time PCR (RT–PCR) with the gene-specific primers described in p value was less than 0.05.The mean and standard error of the mean were used to express the data (SEM). The statistical significance of the differences in mean values between the treatment and control groups was determined using GraphPad Prism version 5. For statistical comparisons, one-way analysis of variance (ANOVA) was used, followed by Tukey’s multiple comparison test. The difference was deemed statistically important when the The results showed a significant increase in body weight of the 2000 ppm-treated group compared to the control group, whereas no significant difference was found between the 4000 and 8000 ppm-treated groups compared to the control group A. Toluene exposure significantly reduced the ovarian weights of rats treated with doses of 4000 and 8000 ppm, whereas the dose of 2000 ppm did not affect ovarian weights B.The effects of toluene on the number of growing ovarian follicles and abnormal ovarian follicles are presented in While there was no significant effect on estradiol B, exposuInsl3, Cyp19, ccnd1, Igf-1, Actb, GDF-9 and Atg5 genes  compared to the control group .The green fluorescence intensity of LC3 was significantly increased in the ovaries of rats exposed to toluene at different concentrations  compared to the control group .Apoptosis was measured in ovarian tissues from various treatment groups . When coExposure to hazardous chemicals is currently the main occupational disease and the main clinical concern, especially when we know that the direct effect may affect the next generation ,43. AmonCyp17a is responsible for androgen production, including testosterone [Cyp17a mRNA levels shown by RT–PCR. The increase in progesterone release discords the results of our previous study in which we used cultured ovarian granulosa cells, in which we found that the addition of toluene resulted in suppression of progesterone secretion [In the present study, we identified the reproductive toxic effect of toluene exposure on ovarian function and follicle development potential and initially confirmed that this toxicity was derived from ovarian structure disruption, inhibition of folliculogenesis and steroidogenesis-related markers, induction of apoptosis and autophagy. We first observed a reduced ovarian weight in rats treated with doses of 4000 and 8000 ppm. This reduction was associated with an increase in the number of abnormal follicles, whereas the number of growing follicles was reduced at 8000 ppm. Our results are consistent with previous studies that reported that toluene exposure caused multiple injuries in adult female mouse ovaries, leading to the disruption of the follicular development process and altering their histological structure ,48. Baseosterone ,50, thisecretion . It has ecretion  and protecretion . Thus, tecretion . Indeed,ecretion , which mecretion  which maInsl3, Ccnd2, Actb and Gdf are essential for granulosa cell proliferation and differentiation [During follicular growth and development, ntiation . Our datntiation  and playntiation ,59. The ntiation . The knontiation , which intiation  by supprntiation . In othentiation ,67,68,69Autophagy is an evolutionarily conserved cellular mechanism that contributes to the protection of cells against a variety of intracellular and extracellular stressors . It is aWe conclude from our in vivo study that toluene disrupted ovarian function injury by significantly decreasing the number of growing follicles and increasing the number of abnormal follicles, progesterone and testosterone levels. In addition, it inhibited GDF-9 protein expression and mRNA levels of genes responsible of follicular growth and development. At lower concentrations of toluene exposure, autophagy was activated to prevent ovarian injury by inhibiting apoptosis. Middle and higher concentrations of toluene induced autophagy and apoptosis that eventually caused ovarian cell death. Our findings provide novel information on the mechanism of disruption of hydrocarbons, and the potential mutual interactions between autophagy and apoptosis in reproductive toxicity that will aid in future research. For future studies, it will be critical to determine the in utero impacts of toluene exposure in order to determine the alterations and potential consequences that could have a long-term impact on the health of future generations.


This article aims to report the results from past and ongoing investigations focused on the role of RANBP9 in the response to DNA damage, particularly in the context of NSCLC. This review concludes with future directions and speculative remarks which will need to be addressed in the coming years.Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths in the Western world. Despite progress made with targeted therapies and immune checkpoint inhibitors, the vast majority of patients have to undergo chemotherapy with platinum-based drugs. To increase efficacy and reduce potential side effects, a more comprehensive understanding of the mechanisms of the DNA damage response (DDR) is required. We have shown that overexpressby live cell imaging (Incuyion of the scaffold protein RAN binding protein 9 (RANBP9) is pervasive in NSCLC. More importantly, patients with higher levels of RANBP9 exhibit a worse outcome from treatment with platinum-based drugs. Mechanistically, RANBP9 exists as a target and an enabler of the ataxia telangiectasia mutated (ATM) kinase signaling. Indeed, the depletion of RANBP9 in NSCLC cells abates ATM activation and its downstream targets such as pby live cell imaging  kinase but not to ATM inhibition. The absence of RANBP9 renders cells more sensitive to drugs inhibiting the Poly(ADP-ribose)-Polymerase (PARP) resulting in a “BRCAness-like” phenotype. In summary, as a result of increased sensitivity to DNA damaging drugs conferred by its ablation  As an introduction, we will briefly mention the state-of-the-art strategies in the treatment of NSCLC, as well as the main molecular features of RANBP9. Moving on, we will then succinctly discuss RANBP9 in cancer generally, before focusing on the role of RANBP9 specifically in the cellular response to DNA damage of NCSLC cells. The standard of care for NSCLC during the last decades has been the use of combination chemotherapy, including that of platinum-based drugs,5. Significant progress has been made in the treatment of this devastating disease through the development of targeted therapies for tumors expressing oncogenic driver alterations (examples include that of EGFR mutations and ALK rearrangements)-8. Nevertheless, the vast majority of NSCLC patients treated with targeted therapy commonly exhibit de novo or acquired resistance.Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths in the developed world-14. However, for unknown reasons, an estimated half of the NSCLC patients are refractory to this new modality and the majority become resistant after an initial response-18.Treatment with immune checkpoint inhibitors (ICIs) has been transformational in the management of NSLCLC. These drugs have now set new standards to the point that ICIs evolved to become first-line treatment. It is known that tumors with high mutational burden tend to be immunologically “hot”, displaying favorable responses to treatment. Therefore, the specific mechanisms of the DNA damage response (DDR) causing the tumor to be more vulnerable to ICIs represent an active area of research and investigation. Clinical trials testing whether the use of low doses of DNA damaging agents may sensitize advanced NSCLC to targeted or immuno-therapies are ongoing-23. Therapeutic regimens include both platinum-based drugs and ICIs for advanced NSCLC, which have shown superior results compared to the use of chemotherapy alone,24.Upon first line or after failure of targeted- and/or immune therapies, the vast majority of patients will undergo treatment with platinum-based cytotoxic drugs. Hence, seeking out new modalities of DDR which can be used as biomarkers to better stratify patients, or as new therapeutic targets represents a valuable clinical and experimental goal,26.From a clinical and basic science perspective, the relationship between tumor mutational burden and response to ICIs is of great interest . -39. Nevertheless, after it has become clear that RANBP9 is linked to critical cancer-causative pathways and to hallmarks of cancer in general, its investigative interest has shifted towards tumor-related paradigms. RANBP9 has been shown to demonstrate tumor-suppressive effects in vitro. For example, when acutely over-expressed, RANBP9 is proapoptotic in nature,41. Further, it increases stability of other accepted tumor suppressors such as p73 or mammalian Lethal Giant Larvae-1,43. Reduced expression of RANBP9 has been associated with distant metastasis and chemo-resistance in gastric cancer. The silencing of RANBP9 in colorectal cancer cells also resulted in an increase of cell growth in vitro and tumorigenesis in vivo. However, a straightforward cancer suppressive role is clearly inconsistent with the ability of RANBP9 to enhance Receptor Tyrosine Kinases or MAPK signaling-49.With regard to human disease, RANBP9 has been initially studied for its potential involvement in abnormal brain and gonadal development, as well as in Alzheimer’s diseasein vitro, most studies agree that RANBP9 is over-expressed in a variety of highly prevalent tumor types including that of NSCLC,45,50-52.While it remains unclear whether RANBP9’s tumor suppressive effects is observed ,32,53. In order to elucidate the underlying biological mechanisms which could be relevant to the treatment of NSCLC, we focus on studying RANBP9 in the DNA damage response in NSCLC. Our specific interests arose from the observation that γH2AX staining lingered for longer periods of time in RANBP9 knockout (KO) mice testes,54. In addition, RANBP9 had been listed as putative target of the ATM kinase in a seminal study by Matsuoka et al.. In this regard, we previously demonstrated that ATM phosphorylates RANBP9 on at least three Serine residues following DNA damage in NSCLC cells. We also showed that the absence of RANBP9 blunts the effect of ATM signaling. This observation prompted us to test the sensitivity of NSCLC cells to DNA damaging agent such as ionizing radiation (IR) and Cisplatin (CDDP), which are frequently used on patients. Our results show that RANBP9 KO NSCLC cells exhibit increased sensitivity to both IR and CDDP.RANBP9 and the entire CTLH complex are constitutively expressed and respond to different modalities of cellular stress. As part of the same study, we also determined that the absence of RANBP9 in NSCLC cells caused an increase in the sensitivity to Poly-ADP ribose phosphorylase (PARP)-inhibitors. Recently, our findings were substantiated indirectly by an innovative high throughput CRISPR screening, where the combined ablation of MAEA, UBE2H, and WDR26  was found to be associated with an augmented sensitivity to PARP inhibitors. Hence, we speculate that the lack of RANBP9 alone or in combination with other members of the CTLH complex may result in a “BRCAness-like” phenotype in NSCLC. This could be clinically important in light of the active pursuit of biomarkers with “BRCAness-like” status in malignancies other than breast and ovarian cancer, including that of NSCLC,60.The significance of our findings was highlighted in a retrospective study where we observed that negative correlation existed between the levels of RANBP9 protein expression and response to platinum-based treatment in NSCLC patients.Herein, we show that RANBP9 KO NSCLC cells have increased sensitivity to ATR inhibitors while not having the same response to ATM inhibitors . In esse. However, we have found that the levels of protein expression may not necessarily correlate with the cellular transcription levels. We found this lack of correlation in commonly used NSCLC cell lines as well as in a limited number of freshly extracted NSCLC patient samples. This observation needs to be further substantiated and confirmed in other studies. However, it is not unusual for proteins which are part of macromolecular complexes to modulate other proteins’ stability,62. In addition, RANBP9 is involved in the response to stress and it is conceivable that protein levels are not always regulated by mRNA expression,62. If confirmed, the lack of correlation between RANBP9 protein and mRNA amounts will have profound implications. In fact, it indicates that only the study of protein levels can provide a reliable assessment of the expression of RANBP9.As a whole, RANBP9 is highly expressed in NSCLC compared to normal adjacent tissue,51. However, this does not preclude the untested possibility that RANBP9 may possess a tumor suppressive function during the initial phases of NSCLC tumorigenesis due to its role in promoting genomic stability. This hypothesis needs to be tested in relevant preclinical models. However, it is conceivable that RANBP9 opposes initiation but may later becomes advantageous for tumor progression, which is similar to TGF-β related signaling pathways,64.Taken together, RANBP9 is highly expressed in NSCLC cells as compared to normal lung tissue. The second relates to the mechanisms underlying the augmented sensitivity to DNA damaging drugs caused by the lack of RANBP9. Finally, we also consider the potential partial functional redundancy of RANBP10. Due to the presence of high homology, this second Scorpin (Spry-COntaining Ran binding ProteIN) and paralog cannot be ignored, and it is likely a major confounding factor in establishing the importance of RANBP9,67.Studying the role of RANBP9 in the context of cellular response to stress and DNA damage is a major focus of our group. We are currently exploring three specific aspects of RANBP9 biology in response to genotoxic stress. The first is the close association of RANBP9 with the “guardian of the genome” known as p53,56. The relationship between RANBP9 with p53 is likely more complex than anticipated and worthwhile to be further investigated. Although an interaction has been previously described between RANBP9 and a specific isoform of p73 by co-IP and colocalization, three groups including ourselves have failed to demonstrate a physical interaction between RANBP9 and p53 by co-IP,68 . Nevertheless, a high throughput study reported a co-IP between RANBP9 and the p53 R273H mutant that needs to be further validated. In summary, it appears that the effects of the absence of RANBP9 on p53 total and phosphorylation levels upon DNA damage are indirect. How the absence of RANBP9 negatively affects ATM-kinase remains to be clarified, but the blunted ATM activity could potentially explain the decrease in p53 levels. On the other hand, an impaired ATM signaling may be only one of the possible mechanisms through which RANBP9 affects p53 abundance and activity. Considering that p53 is degraded by MDM2, an unexplored explanation for our findings is that a potential functional link exists between RANBP9 and MDM2. Alternatively, RANBP9 has been reported to co-localize with Tip60 (a.k.a. KAT5). Tip60 mediates histone dynamics in conjunction with PARP1,73. Although the acetylation by Tip60 of ATM has recently been put into question, it is undisputed that Tip60 acetylates and stabilizes p53. It is therefore possible that the absence of RANBP9 further impinges on the stabilization of p53 operated by Tip60 [.As a consequence of impairment of ATM signaling in the absence of RANBP9, we have reported that phosphorylation of p53 on Serine 15 is severely compromised, affecting the total expression of p53 as shown in Figure 3by Tip60 . Tip60 a, causing phosphorylation of p53 on Serine 46 in situations where the DNA damage is beyond repair. Similarly to the K120 acetylation, the phosphorylation of this residue is particularly important in deciding the fate of the cell upon DNA damage and would be in line with the suggested role for RANBP9 in mediating apoptosis,78. To complicate the jigsaw regarding the relationship of RANBP9 with ATM, p53, and HIPK2, the latter kinase was degraded via a p53-controlled pathway during recovery from sub-lethal DNA damage.RANBP9 was also reported to have interaction with the homeodomain-interacting protein kinase 2 (HIPK2)Altogether, RANBP9 appears to be intricately linked to p53 on multiple levels following DNA damage. Therefore, RANBP9 levels play an important role in fine-tuning the activity of the guardian of the genome .From a clinical point of view, the absence of RANBP9 renders cells more sensitive to DNA damaging agents such as IR and CDDP, but also to inhibitors of ATR or PARP. This warrants a pre-clinical investigation of RANBP9 as a potential target of therapy which may serve to ameliorate the cancer cell response and resistance to these drugs. However, it is also worthwhile to systematically test RANBP9 KO cells for drug sensitivity and attempt to find additional vulnerabilities caused by the absence of this protein. In fact, RANBP9 is associated with pathways other than the DDR and consequently its absence might result in fatal damage to cancer cells.. The genetic deletion of these two proteins consequently results in two very different phenotypes,36,80. However, considering the similarity in protein sequences and genomic organization, they appear to have evolved as duplication of the ancestral yeast Gid1,29. We proposed that these proteins may have partially overlapping functions and RANBP10 has been found to be post-translationally modified following DNA damage,81-83.To date, the study of RANBP9 has largely ignored the existence of the highly homologous RANBP10, which shares four out of five protein-protein interaction domainsFrom an experimental perspective, it is conceivable that the similarities between the two Scorpins hinders a clear identification of RANBP9 and nullifies the functional effects of RANBP9 deletion. To test this hypothesis, it will be necessary to perform experiments by deleting both RANBP9 and RANBP10 simultaneously.Finally, a partial redundancy between the two Scorpins would also explain in part why these genes alone are rarely linked with susceptibility to DNA damaging agents.in vivo about the RANBP9-immunocoprecipated proteome, without the risk of using antibodies that may recognize RANBP10.Using CRISPR/Cas9, we have recently engineered a novel murine model in which RANBP9 is tagged at the C-terminus with both V5 and HA (RANBP9 with double Tag = RanBP9-TT). This model has been validated through the use of immunohistochemistry and by coimmunoprecipitation, showing that the expression and interactions of the tagged protein faithfully recapitulates those of the wild type RANBP9. This new murine strain will be instrumental in obtaining data ,85. In light of these multiple links with critical signaling pathways and critical biological processes, RANBP9 potentially provides a target to block important mechanisms of cell resistance to therapy. Therefore, our group and others have proposed RANBP9 as potential target for cancer therapy,56,85. However, this protein is ubiquitously expressed and additional work will be required to take this translational concept to the clinics.It is clear that treatment of cancer with modalities based on the administration of a single therapeutic agent is rarely successful, whilst combined therapies are more efficacious in the clinical setting. Rationalized drug combinations in anticipation for precision medicine should be developed based on the mechanisms which allow cancer cells to resist and thrive. In particular, RANBP9 has been linked to cell proliferation, cell death, cell adhesion and migration. It has been shown to interact with Receptor Tyrosine Kinases at the membrane, intracellular messengers, and nuclear transcription factors. Therefore, it is conceivable that it mediates the turnover of proteins that are directly involved in the repair of DNA that need to be disposed of according to the tightly concerted choreography of the DDR,88.Firstly, we have not fully elucidated the molecular role of RANBP9 is in the DDR. The nuclear accumulation within hours after genotoxic stress suggests the participation of RANBP9 in the resolution of the damage. Apart from being constitutively expressed in the CTLH E3 ligase complex, RANBP9 has also been physically linked to the proteasomein-vivo question.With regards to the subnuclear localization of RANBP9 during the DDR, a question which remains is whether RANBP9 is physically present at sites of damage. Novel tools such as the cell and mouse lines with the endogenous RANBP9 tagged may be instrumental in answering this ,90. Whether the absence of RANBP9 results in differences in histone acetylation should be a major topic of investigation.In addition, Tip60 modulates the acetylation of DNA following cellular damage. With regard to this, the p53 pathway is the main regulator of cell metabolism-94. Recently, RANBP9 has been shown to impinge on crucial metabolic nodes such as AMPK and MTOR signaling including processeses such as autophagy. Therefore, the metabolic consequences as a result of the absence of RANBP9 should be investigated to ascertain whether drugs targeting specific metabolic pathways should be used in combination with DNA damaging agents.For future clinical management and stratification of patients, a thorough investigation on the outcomes of treatment in the absence of RANBP9 should be conducted using cells containing different mutational status of p53In summary, RANBP9 is highly expressed in NSCLC, participating in critical signaling pathways. Therefore, targeting this specific protein may significantly weaken the ability of tumor cells to survive and proliferate when treated with DNA damaging or other types of drugs. Similar consideration may be made with other malignancies in which RANBP9 has been found to be highly expressed.


N = 201) completed self-report measures assessing experiences of negative interpersonal dependent events, negative non-interpersonal events, and negative independent events at two times. At the same time, they also responded to measures of aggressive behaviors, trait rumination, and depressive symptoms. Path analyses revealed that baseline aggressive behaviors were positively associated with an increase in subsequent negative interpersonal dependent events, even after controlling for the influences of negative interpersonal dependent events, rumination, and depressive symptoms at baseline. However, aggressive behaviors were not significantly associated with subsequent negative non-interpersonal dependent events or negative independent events. These findings suggest that aggressive behaviors may have been a factor leading to interpersonal stress generation. Furthermore, all categories of negative event experiences predicted an increase in subsequent depressive symptoms, but not subsequent rumination, and rumination was not significantly associated with subsequent depressive symptoms. This research extends previous studies on the causes and consequences of stress generation conducted in the US by using specific measures of aggressive behaviors and including a non-restricted sample of university students in Japan.The present study examined the causes and consequences of stress generation in university students in Japan. A two-wave longitudinal study with an 8- or 9-week interval was conducted in the fall of 2020. Undergraduate and graduate students at four universities in Japan (The online version contains supplementary material available at 10.1007/s12144-022-02859-9. Early studies on stress and depression have mainly focused on the effect of negative life events on the risk of developing depressive symptoms and depressive disorders  in these universities responded to a packet of questionnaires during September and October 2020 (Time 1). Most participants answered the questionnaires again 8 weeks later (Time 2) in the same classroom in which they had been recruited at Time 1. However, 25 participants answered the questionnaires 9 weeks after Time 1 because their class could not be held 8 weeks later due to the spread of COVID-19. In addition, 36 participants at Tokushima University and 119 undergraduate students at Tokai Gakuin University who participated at Time 1 could not respond to the questionnaires at Time 2 because their classes could not be conducted face-to-face on the day originally scheduled as Time 2, for the same reason. Although these classes were held online at Time 2, online surveys could not be conducted because the Japanese version of the Beck Depression Inventory-Second Edition, one of the scales used in this study, was prohibited from being used online by Nihon Bunka Kagakusha Co., Ltd. In total, 259 students answered the questionnaire 8 or 9 weeks after Time 1.Participants were recruited in classes at Hirosaki University, Senshu University, Tokai Gakuin University, and Tokushima University in Japan. Undergraduate and graduate students  at Time 1. All participants were Japanese, except for 3 participants .The data of participants who did not participate at both time points and those with missing data on any questionnaire at either time were excluded from the analyses. The final sample comprised 201 students . The mean age of the final sample was 20.20  and 5 (extremely true of me). The overt aggression subscale demonstrated sufficient internal consistencies at Time 1 and 2 .This scale is a measure of aggressive behaviors, which was developed in a study of Japanese university students. This study used only the items of the overt aggression subscale for assessing aggressive behaviors. This subscale is composed of 12 items having face validity assessing physical and verbal aggressive behaviors, including “sometimes I was violent unintentionally” and “sometimes I was sarcastic and said bad things to others’ faces.” This subscale showed good internal consistency  to 4 (often). Alpha coefficients at Time 1 and 2 were .91 and .92 for the negative interpersonal dependent events subscale, .82 and .83 for the negative non-interpersonal dependent events subscale, and .80 and .83 for the negative independent events subscale.This scale is a self-report measure assessing experiences of negative events in Japanese university students that was designed to test the stress generation hypothesis. The Negative Independent/Dependent Events Scale contains 25 items on the negative interpersonal dependent events subscale , 14 items on the negative non-interpersonal dependent events subscale , and 20 items on the negative independent events subscale . This scale showed acceptable construct validity in a study of a sample of Japanese university students  and 4 . Five items in the RRS assess brooding , five items assess reflection , and 12 depression-related items  in our sample.The BDI-II is a well-validated questionnaire assessing the severity of depressive symptoms experienced in the past two weeks. Participants respond to 21 items using a 0–3 scale, with higher scores indicating more severe depression. The Japanese translation of the BDI-II by Kojima and Furukawa  was usedStudents were given a description of the study prior to participation. Only the students who agreed to take part in the study responded to the questionnaires. Participants answered all scales described above at Time 1 and 2. We set 8 weeks as the interval between Time 1 and 2 because we considered that this interval would be required to detect the effect of the aggressive behaviors on subsequent negative interpersonal dependent events.Participants wrote their birthdays and the last four digits of their mobile phone numbers on spaces provided in the questionnaire packet, which were used to match the data from the surveys at the two times. The Ethics Committee of Tokai Gakuin University approved the study.t-tests were conducted using SPSS ver. 23 (IBM Corporation), and all other analyses were conducted using Mplus 8.3  = 233.58, p = .057), indicating that values were missing at random. Missing data were handled with multiple imputations using Bayesian analysis when conducting correlation analyses and path analyses. Imputation was conducted using age, gender, and all items of study variables, and 20 data sets were generated and used for analyses. In other analyses, missing data were handled with the pairwise method.Little’s MCAR test  yielded Because some studies have suggested that influences of psychological predictors on stress generation are stronger among females than males .Descriptive statistics of the final sample are shown in Table Table Supplementary analyses were conducted with brooding or reflection subscales as a rumination measure instead of total RRS scores. The results were largely the same as those in the models using total RRS scores, except for a nonsignificant association between depressive symptoms at Time 1 and reflection at Time 2 see  and S2.Consistent with Hypothesis 1, aggressive behaviors at baseline were related to an increase in negative interpersonal dependent events, but not negative non-interpersonal dependent events or negative independent events, assessed 8 or 9 weeks later. In addition, the longitudinal association of aggressive behaviors with subsequent negative interpersonal dependent events was significant even after controlling for baseline depressive symptoms and rumination. Students who frequently engage in aggressive behaviors are likely to be rejected by their counterparts and result in interpersonal stress generation because aggressive behaviors physically or psychologically harm others.The present result of a significant longitudinal association between baseline aggressive behaviors and future increase in negative interpersonal dependent events supports several findings


Growing evidence suggests an association of lumbar paraspinal muscle morphology with low back pain (LBP) and lumbar pathologies. Unilateral spinal disorders provide unique models to study this association, with implications for diagnosis, prognosis, and management. Statistical shape analysis is a technique that can identify signature shape variations related to phenotypes but has never been employed in studying paraspinal muscle morphology. We present the first investigation using this technique to reveal disease-related paraspinal muscle asymmetry, using MRIs of patients with a single posterolateral disc herniation at the L5-S1 spinal level and unilateral leg pain. Statistical shape analysis was conducted to reveal disease- and phenotype-related morphological variations in the multifidus and erector spinae muscles at the level of herniation and the one below. With the analysis, shape variations associated with disc herniation were identified in the multifidus on the painful side at the level below the pathology while no pathology-related asymmetry in cross-sectional area (CSA) and fatty infiltration was found in either muscle. The results demonstrate higher sensitivity and spatial specificity for the technique than typical CSA and fatty infiltration measures. Statistical shape analysis holds promise in studying paraspinal muscle morphology to improve our understanding of LBP and various lumbar pathologies. To improve the understanding of painful spinal disorders and their prognoses, as well as developing appropriate treatment strategies, there is a growing interest in their association with lumbar paraspinal muscle morphometry and composition, using magnetic resonance imaging (MRI). Commonly viewed as an indicator for muscle force production capacity, muscle cross-sectional area (CSA) is often used as a key measure to assess paraspinal muscle status, as is bilateral paraspinal muscle asymmetry. Paraspinal muscle composition  is also commonly used as an indicator of muscle health. In many existing case–control studies3 that compare patients with healthy controls, LBP and painful lumbar pathologies have been associated with smaller muscle size and/or greater fatty infiltration4. However, previous findings have not been consistent, which is likely at least in part due to variations in muscle measurement methodology across studies and differences in clinical presentation, as well as variable adjustment of possible confounding factors, such as age and sex5.Low back pain (LBP) is among the most common musculoskeletal problems in adults, yet the underlying source of pain most often remains unknown, hindering effective prevention and treatment efforts10 that examine disease-related muscle asymmetry have primarily focused on the multifidus muscle. Previously, with a porcine model, Hodges et al.11 demonstrated rapid level- and side-specific multifidus muscle atrophy and adipocyte enlargement resulting from anterolateral disc or nerve root lesions. In their study of multifidus asymmetry, Battie et al.7 found greater total multifidus CSA ipsilateral to the pathology at the level of disc herniation, and greater fat infiltration on the side of disc herniation both at and below the level of pathology. Later, Fortin et al.8 investigated the asymmetry of the multifidus and erector spinae for posterolateral disc herniation at L4-L5. Although no significant CSA asymmetry of the multifidus was found at spinal levels above, at, or below the disc herniation, greater fat infiltration was reported on the side of the pathology at the adjacent level. To date, reports9 regarding multifidus asymmetry have not been consistent, particularly for muscle size and location of findings with respect to the level of pathology. If multifidus asymmetry is side- and level-specific in patients with symptomatic disc herniations, characteristic muscle morphology  may serve as a helpful biomarker to localize less obvious disc pathologies responsible for disc-related symptoms.Asymmetry in paraspinal muscles has been linked with unilateral LBP and lumbar disorders, which provide a model to gain insights into the etiology and pathology of these disorders. Studies12 provide a compact description for the shapes of a collection of similar objects, by using data dimension reduction with principal component analysis (PCA). The technique allows extraction of uncorrelated shape variations (modes) from the population under study and quantifies their contributions to the total variation from the mean shape. Compared with CSA measures, this technique has two major advantages. First, it can identify detailed local and global geometric variations in a low-dimensional representation, which can be used to reveal phenotype-specific muscle shape characteristics. Second, the technique allows visualization of individual shape variations, which offer intuitive assessment of the morphometric properties. This technique has been successfully employed to study phenotype-related shape variations and alterations in various organs of the human body, including the heart13, brain15, and bones16, providing valuable biomarkers for diagnosis and prognosis purposes. More recently, Deane et al.17 investigated the link between intrinsic lumbar spinal bone shape and lumbar disc degeneration using this technique. Their study provides new evidence that intrinsic spinal shape phenotypes are associated with lumbar disc degeneration and quality of life in patients. However, to the best of our knowledge, statistical shape models have not been employed to study morphometric characteristics of lumbar paraspinal muscles in relation to pathology.Although quantitative measurements of muscle CSA are commonly used to examine their relationship with different lumbar pathologies that contribute to pain, these metrics fail to describe local variations in morphometric properties of the muscle groups beyond their sizes across targeted populations. Statistical shape models (SSMs)In this study, we present the first exploratory investigation using statistical shape analysis to reveal asymmetry of paraspinal muscles in a cohort of patients with single unilateral disc herniation at the L5-S1 level. More specifically, statistical shape models were constructed for the multifidus and erector spinae muscles segmented from axial MRI slices at the L5-S1 and S1 levels, and resulting shape variations were correlated with phenotypes, including disease status, sex and age. As a comparison, conventional measures of bilateral CSAs and level of fat infiltration were analyzed. We hypothesized that unilateral disc herniation is associated with asymmetry in paraspinal muscle shapes, and that statistical shape models would provide a more sensitive and detailed characterization of muscle morphometry in analyzing disease-related variations.In total, 24 patients  were retrospectively selected from the European research consortium project, Genodisc, on commonly diagnosed lumbar pathologies (physiol.ox.ac.uk/genodisc). Included subjects had a single level posterolateral disc herniation at L5-S1 with concomitant leg pain suggestive of radiculopathy on the side of the herniation. No other spinal conditions were noted and no patients received prior surgical treatments for disc herniation at the time of imaging. The herniation and leg pain were on the left in 15 of the 24 cases and the durations of leg pain and back pain were 10.7 ± 14.4 months and 14.2 ± 25.1 months, respectively. T2-weighted (T2w) MRI scans of the lumbar muscles were used for all patients, and axial slices of the L5-S1 and S1 levels were acquired. All MRI data were obtained with the patients in the supine position. The MRI was cropped at the S1 level for two subjects and due to the limited field of view, we obtained only 22 MRI slices at S1 for analysis, rather than the full 24 MRI slices as available for L5-S1. All patients provided informed consent, acknowledging that their data would be used for research to better understand and characterize common spinal disorders. The study was approved by the Ethics Research Board of Western University. The primary Genodisc data were obtained during the ECFP7 Project (HEALTH-F2-2008-201626). All methods were performed in accordance with the relevant guidelines and regulations.18. Then image intensity standardization was performed19 so that each scan exhibited a unified image contrast and intensity range. For each cross-sectional MRI, multifidus and erector spinae muscles were manually segmented bilaterally using the medical image segmentation software, ITK-SNAP (itksnap.org). Segmentation was conducted by JA, who was familiar with the relevant anatomy and had segmented over 300 cross-sectional MRIs of paraspinal muscles at different levels. A representation of the segmentation is shown in Fig. Supplement Information. Overall, the intra- and inter-rater variability was measured above 0.90 on average for all muscle groups at both L5-S1 and S1 levels, indicating excellent manual segmentation quality.All axial cross-sectional images were corrected for MRI intensity anomalies due to radiofrequency field inhomogeneities in the scanner20. The algorithm can automatically separate fat and lean muscle based on their intensity difference. Visual quality control for the results was performed by the author YX, and all results were deemed of good quality. Finally, fat % relative to CSA was computed for multifidus and erector spinae for all patients at the L5-S1 and S1 levels. A demonstration of the fat area segmentation is shown in Fig. Cross-sectional areas were computed using manual segmentation of the left and right multifidus and erector spinae muscles. By using manually segmented muscle CSA to constrain the regions of interest, fatty cross-sectional areas were segmented with the k-means algorithm12 can be used to characterize the morphological property of a muscle group among a target population. For this study, we built the SSMs for the multifidus, as well as for the erector spinae at the L5-S1 and S1 levels. They model intrinsic anatomical variations across the population and shape alterations that were correlated with disc herniation. The procedure of statistical shape modelling is demonstrated in Fig. 21. To normalize the global individual size differences and reveal localized shape variations, the muscle segmentations  were linearly registered to those of the respective MRI templates. With statistical shape analysis of the aligned muscle segmentations, the dominant principal shape variations (or modes) explaining 90% of the morphometric variations were extracted for further analysis. More specifically, the reconstruction coefficients for the dominant shape modes, which represent the magnitude of contribution from the modes to an individual shape, were obtained for all subjects with respect to the multifidus and erector spinae at both levels. The resulting coefficients were then correlated with the clinical and demographic factors to further identify associated trends in morphological variations.Statistical shape modelsp-value < 0.05 were considered statistically significant.To investigate the commonly used measures of muscle morphology and composition, Spearman partial correlation was computed to examine CSAs and fat infiltration  of the multifidus and erector spinae between the affected and unaffected sides at the L5-S1 and S1 levels for the entire cohort, while controlling for the effects of sex and age, which are known to affect paraspinal muscle morphometry. To verify the intrinsic impact from sex and age in the cohort, CSAs and muscle composition from both affected and unaffected sides were correlated with one of the factors while controlling for the effects of the other and the disease influence. For all tests, correlations with a As for statistical shape analysis, Spearman partial correlation was performed to compare the reconstruction coefficients for the muscle shapes between the affected and unaffected side. For each test, the effects of age and sex were set as covariates and controlled in the analysis. The modes with significant correlations  were identified, and their visual representations Figs. , 4 and 5p = 0.041) and greater fatty infiltration (p = 0.022) in erector spinae at L5-S1, as well as higher erector spinae fat% (p = 0.0093) at S1.The measured muscle CSAs and fat % on the affected and unaffected sides at different spinal levels are reported in Table Supplementary Information. With statistical shape analysis, the relevant modes that are associated with demographic and clinical factors are listed in Table Supplementary Information. Note that as SSMs offer statistically independent shape variations and partial correlation can remove the effects of covariates, each correlation in Table 17. However, we acknowledge that some of the discovered correlations with statistical significance may be subject to chance, and we will further validate these findings with more subjects in our future confirmatory studies. More specifically, disc herniation at L5-S1 was associated with Mode 4 (p = 0.032) and Mode 6 (p = 9.2 × 10–3) from the SSM of the multifidus at S1. These two modes are illustrated in Fig. The SSMs contain 6 modes that account for ~ 90% of the shape variations for the multifidus and erector spinae at the L5-S1 and S1 levels. For each SSM, the respective modes are ranked in a descending order of their contributions to the total shape variability . The top 6 modes for each muscle group at each level are demonstrated in Fig. 4 p = 0.02 and Mod10 on side-to-side differences of CSA in disc herniation have provided conflicting conclusions. Some10 reported smaller or larger CSA of the multifidus on the painful side, while others found no significant asymmetry4. Meanwhile, increased fatty infiltration in lumbar multifidus on the side of pathology has been demonstrated with relatively higher consistency in the literature10. Overall, we found no significant CSA asymmetry at L5-S1 and S1 or side-to-side differences in fatty infiltration. With respect to the effects of sex and age, previous studies22 have concluded that female sex and older age are associated with smaller CSA and higher fatty infiltration in lumbar paraspinal muscles. Our results are in agreement with these findings, regardless of the influence from disc herniation. In addition, at the S1 level, the ilium can constrain the geometries of the paraspinal muscles, and its shape and size can be influenced by sex23. Therefore, the geometry of the ilium can be a major contributor to the observed CSA and morphological differences  with respect to sex at the S1 level.In our sample of 24 patients with disc herniation and longstanding associated radiating leg pain, neither CSAs nor fatty infiltration showed significant dependence on the side of the disc herniation. So far, existing studies24.In contrast to the commonly employed paraspinal measures of CSA and fatty infiltration, where no significant results were found, statistical shape analysis identified significant shape variations in multifidus on the affected side. Among the dominant modes of the multifidus at S1, those that correspond to the influence of pathology account for 8.2% of the total variance. However, the modes corresponding to sex and age were responsible for higher contributions to the overall shape variance. The substantial influence of sex and age on muscle morphology may partially mask pathology-related CSA asymmetry in our study, and future investigations that place additional constraints in demographic factors  during cohort selection may be beneficialAs fatty infiltration may be indirectly associated with muscle morphology, an additional experiment was conducted to correlate fat% and the identified modes in Figs. 25, Macintosh et al.26 found that the principal fascicles of the lumbar multifidus are innervated by a single nerve root that stretches 3 ~ 4 vertebral levels caudally, implying that disease-related changes should be mainly detected at the levels below and on the side of the pathology. This was demonstrated in several studies27 that investigated muscle asymmetry related to disc herniation and injuries. From statistical shape analysis, we observed side- and level-specific asymmetry that was correlated with symptomatic disc herniation at L5-S1. More specifically, the identified variations at the level below the pathology  support previous reports27 and offer insights on the trend of local shape variations in addition to prior findings related to cross-sectional areas.Although different descriptions for lumbar multifidus morphology exist20 will be highly instrumental. Furthermore, intrinsic side-to-side anatomical differences may exist beyond the effects of sex and age and contribute to the identified shape asymmetry. A previous study28 reported that paraspinal muscle asymmetry greater than 10% is common in men without a history of LBP. Furthermore, behavioral, environmental, and genetic factors may also modulate muscle asymmetry24. These can further complicate the investigation, but in-depth analyses that de-couple these factors, as well as comparison of paraspinal muscle shapes between patients and healthy controls, are out of the scope of the present study. Future work will incorporate healthy participants in statistical shape modelling, as well as additional variates to further elucidate the effects of various factors on paraspinal muscle asymmetry, as well as the underlying interaction between lumbar pathologies, external factors, and muscle shapes.One major limitation of the presented study is that the included patient sample was relatively small. Therefore, we exclusively focused on one pathology at a single level. However, despite the limited sample, the exploratory investigation introducing statistical shape analysis to identify muscle morphological properties in lumbar pathology did reveal significant trends in multifidus shape asymmetry related to disc herniation. In the future, validation of this technique in paraspinal muscle morphometry with respect to larger patient populations, various lumbar pathologies, and additional spinal levels of pathology will be important. For this, automatic image segmentation algorithms based on deep learning techniquesIn conclusion, we present the first investigation using statistical shape analysis to study the morphology of paraspinal muscles. Using a patient cohort with symptomatic unilateral L5-S1 disc herniation, we identified statistically significant and specific morphological asymmetry in the multifidus at the S1 level. With potentially superior sensitivity and spatial specificity, statistical shape analysis has demonstrated promise beyond simple CSA measurements in studying paraspinal muscle morphology as possible diagnostic and prognostic biomarkers, to improve our understanding of LBP and various lumbar pathologies.Supplementary Information.


To report 1-year follow-up of a phase 1/2a clinical trial testing a composite subretinal implant having polarized human embryonic stem cell (hESC)-derived retinal pigment epithelium (RPE) cells on an ultrathin parylene substrate in subjects with advanced non-neovascular age-related macular degeneration (NNAMD)The phase 1/2a clinical trial included 16 subjects in two cohorts. The main endpoint was safety assessed at 365 days using ophthalmic and systemic exams. Pseudophakic subjects with geographic atrophy (GA) and severe vision loss were eligible. Low-dose tacrolimus immunosuppression was utilized for 68 days in the peri-implantation period. The implant was delivered to the worst seeing eye with a custom subretinal insertion device in an outpatient setting. A data safety monitoring committee reviewed all results.P = not significant) and a larger proportion of nonimplanted eyes demonstrated a >5-letter loss .The treated eyes of all subjects were legally blind with a baseline best-corrected visual acuity (BCVA) of ≤ 20/200. There were no unexpected serious adverse events. Four subjects in cohort 1 had serious ocular adverse events, including retinal hemorrhage, edema, focal retinal detachment, or RPE detachment, which was mitigated in cohort 2 using improved hemostasis during surgery. Although this study was not powered to assess efficacy, treated eyes from four subjects showed an increased BCVA of >5 letters (6–13 letters). A larger proportion of treated eyes experienced a >5-letter gain when compared with the untreated eye (27% vs. 7%; Outpatient delivery of the implant can be performed routinely. At 1 year, the implant is safe and well tolerated in subjects with advanced dry AMD.This work describes the first clinical trial, to our knowledge, of a novel implant for advanced dry AMD. It is a major unmet medical need that impacts millions of people in the Western world.,––In subjects with NNAMD and GA, macular translocation surgery,The study designAll surgeries were conducted at the Outpatient Surgery Center of the University of Southern California, Keck School of Medicine, in Los Angeles, CA. A data monitoring and safety committee was assigned for the study and reviewed all results and adverse events. The primary outcome measure of the study was safety, as assessed by clinical examination at 365 days after implantation. Predetermined secondary and exploratory endpoints included efficacy as assessed by visual acuity and visual function measures, including microperimetry. The stopping rules for the study were defined in the clinical protocol and included (1) development of an expanding mass; (2) accelerated loss of visual acuity in the implanted eye; (3) development of any serious adverse pathology associated with the delivery, immunosuppression, or use of the implant that warrants enucleation of the eye; and (4) surgical delivery-related events involving the device, implant, or surgical procedure that lead to failure of the implant delivery.Key inclusion criteria for subjects were previously described,Detailed surgical methods for all subjects were described in an earlier publication.,Details of the clinical evaluations and structural and functional imaging methods have been described previously and are summarized here for convenience.Fixation stability was performed on all subjects using the Nidek MP1 microperimeter  at baseline and at day 365 and analyzed as previously reported.,,Sixteen subjects were enrolled in the study, and 15 were implanted with the CPCB-RPE1 implant, with complete follow-up for at least 365 days. One enrolled subject was not implanted, as previously described.2; range, 6.0–46.4 mm2).Preoperative clinical examination and fundus photographs demonstrated that all implanted subjects had moderate to large areas of GA involving the fovea  dependent on the baseline size of its area.,P = 0.02, Fisher's exact test), suggesting that the better hemorrhage control observed in cohort 2 led to reduced ocular serious adverse events. In no case did fluorescein or indocyanine angiography demonstrate choroidal neovascularization. Nevertheless, site investigators were allowed to treat with anti–vascular endothelial growth factor agents at their own discretion. One subject was noted to have a pre-existing peripheral retinal schisis at the time of surgery with possible retinal detachment. The area of schisis and possible retinal detachment was treated with laser barricade without any subsequent sequelae. Notably, there were no reports of vitreous or aqueous cells (after postoperative month 1) and no report at all of vasculitis, choroiditis, or retinitis. In addition, no antibodies to HLA class I or II molecules unique to the implanted RPE cells and not found on the recipients cells were induced in the peripheral blood of the subjects after implantation (manuscript submitted).Adverse events in the perioperative period associated with the surgery or implant have been previously reported.Severe subject-related systemic adverse events were noted in five subjects. One subject was diagnosed with esophageal adenocarcinoma that was unrelated to the surgery, implant, or immunosuppression associated with this study. Another subject had an exacerbation of congestive heart failure requiring hospitalization which was also not associated with the surgery, implant, or immunosuppression associated with this study. A separate subject was diagnosed with pneumonia requiring hospitalization during the period of immunosuppression taper postoperatively. For this subject, immunosuppression was stopped before the full course was administered; however, there was no evidence that the pneumonia was associated with the surgery or implant. There was no clinical evidence of graft rejection in any subject.P = not significant, Fisher's exact test) at day 365. Early Treatment Diabetic Retinopathy Study (ETDRS) letter improvements ranged from 6 to 13 letters at 1 year. Similarly, more implanted eyes than non-implanted eyes had gained >5 letters BCVA or remained within 5 letters of baseline BCVA . Fewer implanted eyes than non-implanted, contralateral eyes lost >5 letters of BCVA at 1 year .The mean and median BCVA values of the treated eye at day 365 were logMAR 1.33 ± 0.42 and 1.24 , respectively. Although the severity of the baseline GA in the subjects in this study likely precluded significant improvement in visual acuity , more implanted eyes than non-implanted, contralateral eyes had gained >5 letters in BCVA .The data from this study also suggest that subjects who received the implant were more likely to improve by >5 letters, and non-implanted eyes were more likely to lose >5 letters. Although the number of subjects in this study does not provide power for a definitive conclusion about efficacy, this trend is encouraging for this advanced stage of the disease treated. Natural history studies of GA demonstrate that patients with a mean ETDRS letter score of 60 letters (20/63) lose an average of 12 letters by 2 years and 23 letters by 5 years.The documented safety of CPCB-RPE1 treatment supports recruitment of subjects with better visual acuity. Subjects with better visual acuity will have a shorter duration of disease, smaller region of GA, and enhanced visual potential attributable to a higher frequency of surviving photoreceptor cells able to benefit from the rehabilitating effects of the implanted RPE cells. Also, preliminary data in this study suggest that subjects exhibiting the most impressive improvement in visual acuity were those with the smallest areas of GA. Finally, our experience indicates that the surgical separation of the retina overlying the area of GA is easier and faster in less chronic cases.Additional improvements can be implemented in future trials to further improve the adverse event profile. For example, several subjects in the current study had evidence of macular edema not associated with choroidal neovascularization. A possible cause of this postoperative cystoid macular edema is long-term use of silicone oil.We believe the mechanism of action for the CPCB-RPE1 is the result of enhanced viability of surviving photoreceptor cells in the retina directly overlying or adjacent to the CPCB-RPE1 implant. This possibility is consistent with the limited evidence we have from microperimetric sensitivity studies of subject 216, but additional data are needed from definitive efficacy studies. It has been demonstrated that visual function in GA subjects is primarily derived from preferred retinal loci at the edge of the GA lesion.