Toxoplasma gondii establishes chronic infection in humans by forming thick-walled cysts that persist in the brain. Once host immunity wanes, cysts reactivate to cause severe, and often lethal, toxoplasmic encephalitis. There is no available therapy to eliminate cysts or to prevent their reactivation. Furthermore, how the cyst membrane and cyst wall structures develop is poorly understood. Here, we visualized and tracked the localization of Toxoplasma parasitophorous vacuole membrane (PVM) dense granules (GRA) proteins during cyst development in vitro. PVM-localized GRA5 and GRA7 were found at the cyst membrane and cyst wall region throughout cyst development, suggesting that the PVM remains intact and develops into the cyst membrane. In addition, our results show that genetic deletion of PVM GRAs reduced the rate of accumulation of cyst wall cargo at the cyst periphery and suggest that PVM-localized GRAs mediate the development and maturation of the cyst wall and cyst membrane. Toxoplasma gondii parasitophorous vacuole membrane (PVM) has been hypothesized to transition into the cyst membrane that surrounds the cyst wall and encloses bradyzoites. Here, we tracked the localization of two PVM dense granule (GRA) proteins (GRA5 and GRA7) after in vitro differentiation of the tachyzoite stage parasitophorous vacuole into the mature cyst. GRA5 and GRA7 were visible at the cyst periphery at 6 h and at all later times after differentiation, suggesting that the PVM remained intact as it transitioned into the cyst membrane. By day 3 postdifferentiation, GRA5 and GRA7 were visible in a continuous pattern at the cyst periphery. In mature 7- and 10-day-old cysts permeabilized with a saponin pulse, GRA5 and GRA7 were localized to the cyst membrane and the cyst wall regions. Cysts at different stages of cyst development exhibited differential susceptibility to saponin permeabilization, and, correspondingly, saponin selectively removed GRA5 from the cyst membrane and cyst wall region in 10-day-old cysts. GRA5 and GRA7 were localized at the cyst membrane and cyst wall region at all times after differentiation of the parasitophorous vacuole, which supports a previous model proposing that the PVM develops into the cyst membrane. In addition, evaluation of Δgra3, Δgra5, Δgra7, Δgra8, and Δgra14 mutants revealed that PVM-localized GRAs were crucial to support the normal rate of accumulation of cyst wall proteins at the cyst periphery.After differentiation is triggered, the tachyzoite-stage IMPORTANCEToxoplasma gondii establishes chronic infection in humans by forming thick-walled cysts that persist in the brain. Once host immunity wanes, cysts reactivate to cause severe, and often lethal, toxoplasmic encephalitis. There is no available therapy to eliminate cysts or to prevent their reactivation. Furthermore, how the cyst membrane and cyst wall structures develop is poorly understood. Here, we visualized and tracked the localization of Toxoplasma parasitophorous vacuole membrane (PVM) dense granules (GRA) proteins during cyst development in vitro. PVM-localized GRA5 and GRA7 were found at the cyst membrane and cyst wall region throughout cyst development, suggesting that the PVM remains intact and develops into the cyst membrane. In addition, our results show that genetic deletion of PVM GRAs reduced the rate of accumulation of cyst wall cargo at the cyst periphery and suggest that PVM-localized GRAs mediate the development and maturation of the cyst wall and cyst membrane. Toxoplasma gondii , which are part of an interferon gamma (IFN-γ)-induced mechanism that targets and eliminates foreign intracellular microbes –34. ROP5–39–44–N-acetylgalactosamine moieties that selectively bind to Dolichos biflorus agglutinin (DBA) stain  stain , 51, 52.A) stain . GRA proA) stain . RecentlA) stain . Deletioro cysts , 55. In ro cysts . Current2 and high-pH in vitro tachyzoite-to-bradyzoite differentiation method  to track the fate of the PVM and its relationship to the development of the cyst membrane and (ii) to test whether PVM GRA proteins influence the development of the cyst wall. Our findings reveal that deletion of PVM GRAs also significantly reduced the rate of accumulation of cyst wall proteins at the cyst periphery relative to that in the cyst interior, suggesting an important role for these PVM GRA proteins in the development of the cyst wall. In addition, our results support the current model proposing that the tachyzoite-stage PVM transitions into the cyst membrane that surrounds the cyst wall.The PVM is hypothesized to develop into the cyst membrane . A low-Cn method , 58 prodn method  that posted mice . Using tku80 genetic background (Dolichos biflorus agglutinin (DBA) fluorescence intensity at the cyst peripheries of Δgra3, Δgra5, Δgra7, Δgra8, and Δgra14 cysts   .The cyst membrane is hypothesized to originate from the PVM . GRA5 lo63–+) bradyzoites and cyst wall proteins stained by DBA were visible within the developing cyst 6 h after differentiation  microscopy and imaged by confocal microscopy. The presence of bradyzoites inside cysts was verified by locating parasite nuclei with 4′,6-diamidino-2-phenylindole (DAPI) staining and verifying that each parasite nucleus was surrounded by expression of cytosolic green fluorescent protein (GFP) (GFP+ bradyzoites). Cysts fixed in 4% paraformaldehyde were permeabilized with either a short pulse (P) of saponin or with continuous (C) exposure to Triton. Cysts were stained with DBA and (A) α-GRA5 or (B) α-GRA7 antibody. Panels show GFP and DAPI; GFP and DBA; DBA; α-GRA; DBA and α-GRA; and DIC. Number (n) of cysts analyzed, 9 to 18. Bar, 5 μm. (C) Infected HFFs on coverslips were infected with tachyzoites at a multiplicity of infection (MOI) of 0.5, permeabilized with 0.01% saponin, and stained with DBA. Vacuoles were located using DIC microscopy and imaged by confocal microscopy. DAPI-stained host and parasite nuclei. Panels show DAPI; DAPI and GFP; GFP; DBA; GFP and DBA; and DIC. Number (n) of vacuoles analyzed, 6. Bar, 5 μm. Download FIG S1, TIF file, 0.3 MB.Parasitophorous vacuole membrane (PVM)-associated GRAs are localized within the developing cyst in 6-hour-old cysts. (A to B) Infected human foreskin fibroblasts (HFFs) on coverslips were treated under bradyzoite-inducing conditions for 6 hours to differentiate Copyright © 2020 Guevara et al.2020Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the + bradyzoites were visible within a DBA-stained cyst that was permeabilized with Triton. In contrast, a pulse with saponin did not permeabilize the cyst membrane of 1-, 2-, and 3-day-old cysts to visibly DBA stain the developing cyst wall  and verifying that each parasite nucleus was surrounded by expression of cytosolic GFP (GFP+ bradyzoites). Cysts fixed in 4% paraformaldehyde were permeabilized with continuous (C) exposure to saponin. Cysts were stained with DBA and α-GRA5. Panels show GFP and DAPI; GFP and DBA; DBA; α-GRA5; DBA and α-GRA5; and DIC. The percent occurrence is shown for GRA5 at day 2  and at day 3 . Bar, 5 μm. Download FIG S2, TIF file, 0.1 MB.GRA5 is localized at the cyst periphery after saponin exposure of 2- and 3-day-old cysts. Infected HFFs on coverslips were treated under bradyzoite-inducing conditions for 2 or 3 days to differentiate Copyright © 2020 Guevara et al.2020Guevara et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the + region that defines bradyzoites was not readily apparent again, indicating that this compartment was not permeabilized -derived cholesterol increases tachyzoite-to-bradyzoite stage conversion . Our fin85–ost cell . Thus, wThe glycosylated cyst wall forms beneath the cyst membrane. How cyst wall cargo is delivered to the cyst periphery remains unknown. A favored hypothesis is that wall cargo is delivered in vesicles secreted by bradyzoite-stage parasites. Our data revealed bright puncta of GRA5 and GRA7 close to the cyst periphery at 6 h after differentiation. This phenotype is similar to previous findings that GRA1, GRA2, GRA4, GRA6, GRA9, and GRA12 were observed in puncta at the cyst periphery 6 h after differentiation . It is tTo assess the proximity of PVM GRAs, we used a previously developed quantitative method , which dThe initial PVM is hypothesized to undergo changes that culminates in a limiting cyst membrane. Cyst wall proteins congregate at the cyst periphery , presumaWhen mature 7- or 10-day-old cysts were permeabilized with Triton, GRA5 and GRA7 were localized in the inner layers of the cyst wall and in the cyst matrix as prominent puncta. Remarkably, saponin permeabilization caused differential localization of GRA5 and GRA7 selectively in 10-day-old mature cysts. GRA5 was localized to the inner layers of the cyst wall and to prominent puncta in the cyst matrix after continuous saponin permeabilization. In contrast, GRA7 was localized at the cyst membrane and cyst wall region. The simplest explanation for this differential localization is that GRA5 most likely resides in cholesterol-rich lipid rafts in the cyst membrane, whereas GRA7 does not. Alternatively, GRA7 may interact with a protein component of the cyst wall that stabilizes its cyst membrane localization after saponin permeabilization, but, as mentioned earlier, not after Triton permeabilization. Elucidating the functions of PVM GRAs during cyst development is necessary to define the mechanisms that regulate the development and maturation of the cyst wall and cyst membrane.Toxoplasma gondii parasites were maintained in vitro by serial passage of tachyzoites in human foreskin fibroblast (HFF) monolayers (ATCC SCRS-1041.1) cultured in Eagle’s modified essential medium  containing 1% fetal bovine serum , 2 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 36°C in 95% air and 5% CO2. HFF cells were maintained in EMEM, 10% FBS (HyClone), 2 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in 95% air and 5% CO2. The parental Pru strain PruΔku80 was previously made transgenic for green fluorescent protein (GFP) under the control of the LDH2 bradyzoite stage-specific promoter (ku80 knockout strain of the type II Pru strain using previously described methods (Type II Prugniaud (Pru) background promoter . Strains methods , 90.in vitro into bradyzoites within cysts essentially as previously and elegantly described by Knoll and colleagues (l-glutamine (HyClone), 20 mM HEPES-free acid (IBI Scientific), 1% XL-glutamine , 1% FBS, and 1% penicillin-streptomycin. The pH of differentiation medium was adjusted to 8.1 with sodium hydroxide and filter sterilized. HFF cells were cultured on circular micro cover glass until confluent (Electron Microscopy Sciences), and confluent monolayers were infected with type II Pru parasites at a multiplicity of infection (MOI) of ∼0.5. Infected cells were washed 3 h after infection once in Dulbecco’s phosphate-buffered saline (DPBS) supplemented with Ca2+ and Mg2+ and incubated in differentiation medium for 6 h, 1 day, 2 days, 3 days, 7 days, or 10 days at 37°C in ambient air. Medium was changed on days 3 and 7.Tachyzoites were differentiated lleagues . Differe2+ and Mg2+ and incubated for 1 h at RT with a 1:1,000 dilution of secondary goat anti-rabbit (H+L) (Thermo Fisher) and goat anti-mouse IgG (H+L) antibodies conjugated to Alexa Fluor 647 . All samples were incubated with a 1:250 dilution of rhodamine-labeled Dolichos biflorus agglutinin (Vector Laboratories) for 1 h at RT. The Pru background used in this study has a bradyzoite-specific gene, LDH2, under a GFP promoter  selected for quantification was from the middle of the cyst, where the cyst size is maximal. Raw .nd2 files of cyst images were imported into Fiji for processing. Images were minimally processed for brightness  in Fiji  0.01% saponin (Sigma) for 10 min, (ii) 3%FBS/0.01% saponin for 30 min at room temperature (RT), with this permeabilization solution used throughout the experiment, or (iii) 3%FBS/0.2% Triton X-100 for 30 min at RT, with this permeabilization solution used throughout the experiment. All samples were incubated with a 1:1,000 dilution of primary mouse monoclonal α-GRA5 antibody  or with promoter , which i in Fiji . The num2. Layers were created by dilation to measure the fluorescence intensity outside the cyst, which provided the background fluorescence intensity. All data were imported into Excel to be further analyzed, as previously described  for 1 h at RT. All samples were mounted in SlowFade Gold antifade with DAPI (Life Technologies) and imaged at ×100 with a Nikon A1Rsi confocal microscope . Vacuoles were located using differential interference contrast (DIC) microscopy. Confocal images as raw .nd2 files were imported and minimally processed for brightness in Fiji  for 10 min, and blocked with 10% FBS for 20 min. All samples were incubated with a 1:250 dilution of rhodamine-labeled  in Fiji .t tests were used to calculate P values. All calculations of average, plus or minus standard error of the mean (SEM), and P values were performed using GraphPad Prism software version 5.0c.Unpaired


We describe a patient with bilateral cystic tumors of the pelvis. The left one rapidly grew during pregnancy and combined with the right one, whose clinical course made diagnosis difficult. A pregnant woman with a history of laparotomy was referred to us due to suspected bilateral pelvic cysts. The left-sided cyst had rapidly grown to 27 cm in diameter and merged with the right cyst, forming a large cyst occupying the entire pelvic cavity in the third trimester. Considering this rapid growth, cesarean section and resection of the cyst were performed at 37th week. The resected cyst consisted of two components: a large unilocular cyst containing serous fluid and a multilocular cyst suggestive of ovarian mucinous cystadenoma in the right ovary. The wall of the former largely lacked lining epithelium, but it was partly continuous with the latter mucinous epithelium. Immunohistochemically, estrogen and progesterone receptors were focally positive in the cyst wall, suggesting that pregnancy-associated sex-hormones may have contributed to the rapid growth of the cyst. We diagnosed this condition as a peritoneal inclusion cyst margining with a right ovarian mucinous cystadenoma. Peritoneal inclusion cyst should be considered in the differential diagnosis of a rapidly growing pelvic mass during pregnancy. Large cystic tumors of the pelvis during pregnancy are rarely observed . TreatmeAn inclusion cyst refers to serous fluid inclusion in the enclosed peritoneal cavity. Laparotomy, endometriosis, and abdominal infection have been reported as causes of this cyst –5. The cWe experienced a case of rapidly growing pelvic cystic tumor during pregnancy. We speculate that the peritoneal inclusion cyst had grown rapidly during pregnancy under pregnancy-associated sex-hormones.A 24-year-old pregnant woman (G2P1) was referred to us due to suspected bilateral ovarian cysts at 8 weeks of gestation. She had undergone ovarian cystectomy twice under open surgery: left and right ovarian cystectomy for mature cystic teratoma and mucinous cystadenoma, respectively. She had no additional medical history or familial medical history. Transvaginal ultrasound and magnetic resonance imaging (MRI) Figures  revealedAlthough the serum levels of tumor markers  were normal for a pregnant woman, considering the large size of the cyst, cyst resection was attempted at 14 weeks; however, it was converted to probe laparotomy. Marked adhesion around the cysts, posterior uterus, and Douglas' pouch made cyst resection impossible as extensive adhesiolysis may cause uterine damage and also uterine contractions after surgery. Gross examinations revealed no metastatic lesions or lymph node swelling. Abdominal fluid cytology revealed no malignant cells.At 32 weeks of gestation, MRI revealed that the left-sided cyst size had increased to 27 cm in diameter Figures , althougThis large cyst showed no solid-part or papillary growth. The serum levels of tumor markers remained normal. Malignant ovarian tumor could not be ruled out but was considered less likely. We weighed merits and demerits between relaparotomy for tumor resection during pregnancy and a wait-and-see approach for several weeks; the former is likely to require extensive adhesiolysis and may cause preterm delivery. We decided on the latter strategy, since resection should be performed in the event of a size increase or images indicative of malignancy. The fetus normally developed without fetal growth restriction.Cesarean section and tumor resection were performed at 37+4 weeks of gestation, yielding 3,012-g male infant with Apgar score 8/9 at 1/5 minutes, respectively. The infant did not have congenital abnormalities. After the completion of cesarean section, we ruptured the wall of this large cyst, with care to avoid the cyst content entering into the abdominal cavity. A large amount of serous fluid was drained. This large cyst was a multicystic cyst (5 cm), considered to be the right multicystic ovarian cyst that had been observed from the first trimester. The wall of the large cyst showed marked adhesion to the peripheral peritoneal cavity. We resected it as widely as possible together with right salpingo-oophorectomy Figures . The lefHere, we report bilateral pelvic cysts, in which the left one grew rapidly during pregnancy and merged with the right cystic tumor. Histologic examination revealed a large cyst containing serous fluid and a mucinous cystadenoma, with both showing no malignant cells. To explain the clinical course and histogenesis of this tumor, we speculated as follows: originally, there was a left serous cyst (9 cm) (possibly an inclusion cyst) and a right ovarian mucinous cystadenoma (5 cm). The former inclusion cyst merged with the latter (a mucinous cystadenoma), resulting in this unique composite tumor.Inclusion cysts can be formed as follows: an enclosed cavity is formed following abdominal or pelvic surgery or its inflammation, fluid is secreted from the ovarian surface or cyst wall, and fluid production increases, exceeding its absorption, leading to a large fluid-containing cyst –7, 9, 10One inconsistency with this proposed scenario may be that we could not detect typical mesothelial cells in the cyst cavity. The cyst cavity was partly covered by mucin-producing cells. This may have resulted from mucin-producing cells being incorporated with the large inclusion cyst during the merging of the two masses. Adhesion of the large cyst wall was marked, and, thus, some of its parts could not be removed. This may have made it difficult to confirm typical mesothelial cells in the cyst wall. We believe that the lack of confirmation of histologically evident mesothelial cells in the cyst wall does not refute our suggested scenario, considering the difficulty of confirming the histological findings.A history of multiple abdominal surgeries and an increased secretion of sex-hormones during pregnancy are not specific to this patient. We do not know why this inclusion cyst showed rapid growth. One possible reason is that laparotomy during pregnancy may have caused additional inflammation at the site, further increasing the surrounding tissue tension and increasing fluid production. However, even if this was the case, laparotomy would remain unavoidable even though it may eventually lead to probe laparotomy. Findings based on a single case cannot lead to a conclusion. However, we suggest that peritoneal inclusion cyst should be considered in the differential diagnosis of a rapidly growing pelvic mass during pregnancy, especially when the patient has a risk associated with it such as a history of multiple abdominal surgeries or laparotomy during pregnancy.


Background: No universal  demarcation of gestational age  distinguishes miscarriage and stillbirth or extreme preterm birth (exPTB). This study provides a synopsis of outcome between 22 to <28 weeks gestation from a low resource setting.Methods: A retrospective record review of a population on the Thailand-Myanmar border was conducted. Outcomes were classified as miscarriage, late expulsion of products between 22 to < 28 weeks gestation with evidence of non-viability  prior to 22 weeks; or  exPTB (stillbirth/live born) between 22 to < 28 weeks gestation when the fetus was viable at ≥22 weeks. Termination of pregnancy and gestational trophoblastic disease were excluded.Results: From 1995-2015, 80.9%  of registered women had a known pregnancy outcome, of whom 99.8%  had a known gestational age. Delivery  between 22 to <28 weeks gestation included 0.9%  of pregnancies after removing 18 cases (3.8%) who met an exclusion criteria. Most  pregnancies had an ultrasound: 72.5% (n=329/454);  43.6% (n=197) were classified as  miscarriage and 56.4% (n=257) exPTB.  Individual record review of miscarriages estimated that fetal death had occurred at a median of 16 weeks, despite late expulsion between 22 to <28 weeks. With available data  the proportion of stillbirth was 47.6% (n=120), congenital abnormality 10.5%  and neonatal death was 98.5% . Introduction of ultrasound was associated with a 2-times higher odds of classification of outcome as exPTB rather than miscarriage.Conclusion: In this low resource setting few (<1%) pregnancy outcomes occurred in the 22 to <28 weeks gestational window; four in ten  were miscarriage (late expulsion) and neonatal mortality approached 100%.  In the scale-up to preventable newborns deaths  greater benefits will be obtained by focusing on the viable newborns of ≥ 28 weeks gestation. Without a universally accepted definition to distinguish fetal death from miscarriage (spontaneous abortion) versus stillbirth5 ; and stillbirth (expulsion of a fetus with no signs of life) classified as early fetal death ; late fetal death 9. These definitions overlap. For example, is a stillborn 1000 g fetus with accurately determined gestation born at 27+2 weeks+days, a late fetal death because of the birth weight or an early fetal death because of the gestation? For organizations that utilize the WHO definition of stillbirth recommended for global comparison i.e.: “a baby born with no signs of life at or after 28 weeks gestation”10 confusion arises with live newborns before 28 weeks. It is difficult to explain to health care workers thatstillbirth starts at 28 weeks butlive birth can start at <28 weeks.To determine progress towards the Sustainable Developmental Goal (SDG) 3.2 “by 2030, to end preventable deaths of newborns and children under 5 years of age”, standardized data collection is criticalbirth5  and the 12. Miscarriage rates have been stable over time at 10% (2257/23 118) from 1994 to 201313. The outcomes of pregnancies of 28 weeks or more gestation in this area have been published previously and are not the focus of this analysis15.On the Thailand-Myanmar border at Shoklo Malaria Research Unit (SMRU), the WHO definition of stillbirth based upon reaching 28 weeks gestation has been used for the past 32 years . Based oP. falciparum malaria16. The Thailand-Myanmar border was one of the first places in the world to introduce artemisinin-based combination therapy for treatment of malaria in the general population17. The artemisinin derivatives were also used initially for treatment of malaria in the 2nd and 3rd trimesters of pregnancy or to save the life of the mother with severe malaria in any trimester18. This class of drug distinguished itself in pregnancy as it was first reported by Chinese scientists to cause fetal resorption (or early miscarriage)19 so it was important to be able to determine gestational age during pregnancy and to have a clear definition of trimester and miscarriage. Quinine remained the WHO recommended drug for women in the first trimester of pregnancy and systematic data collection by local health workers has built an evidence base that has made a significant contribution to global knowledge about antimalarial drug use in pregnancy particularly in the first trimester20.One of the most significant factors in this border region has been the 3 decades long struggle with recurring loss of antimalarials to treat patients with multi-drug resistant22 or by last menstrual period (LMP), and from late 2001 by ultrasound23. In 1995 the only tools available in the field were pregnancy tests, symphysis fundal height measurement (SFH), pinnard and at best (late 1990’s) a small hand-held Doppler to detect fetal heart beat. SFH measurements were routinely carried out at antenatal care and quality control measurement exercises were conducted because it was so heavily relied upon. SFH was examined at every visit until the fundus of the uterus could be felt for the first time and thereafter approximately every 2–4 weeks unless more frequent repeated measures were indicated. In these circumstances and in the absence of bleeding or fetal movements, detection of loss of fetal viability was identified late. When ultrasound was introduced in the area, a first trimester or first ANC visit scan was followed up by a repeat scan initially at 18 weeks and later shifted to 22 weeks. Gestational age of a non-viable fetus could be more readily obtained by ultrasound but for SMRU data to be consistent with the former data, the end of gestation of pregnancy has remained constant, as the date of expulsion of the products of conception.In the 1990’s at SMRU, gestational age during pregnancy was estimated from fundal height by a formula developed for the study population24. In the guidelines the working definition of miscarriage is outcome of pregnancy before 28 weeks, while births (stillbirth and live birth) commence from 28 weeks, as does reporting of neonatal deaths. This definition has remained unchanged with the establishment of a special care baby unit in this resource limited setting where assisted ventilation of newborns is not available (i.e birth before 28 weeks is not viable)12.At the same time, work has been standardized for local health workers by use of obstetric guidelines9.The objective of this study is to bring clarity to the nature of the products of conception that are expulsed in the 22 to <28 weeks gestational age window in a low resource setting, highlighting operational issues and the role of ultrasoundSMRU is an operational field-based research unit uniquely combining humanitarian work with research of direct relevance to the local population. It is a limited-resource setting working with marginalized populations on the western border of Thailand in Tak Province. In this area, there are an estimated 140,000 refugees and 200,000 migrants from Myanmar. There have been decades of neglect of the health system in Myanmar and the government is currently trying to address this. The refugee situation on the border of Thailand and Myanmar is amongst the most protracted in Asia but it has set a scenario of how health, particularly among pregnant women and obstetric emergencies of Myanmar people are managed. Due to conflict in Eastern Kayin state, refugees obtained surgical care in Thailand hospitals via a system of referral from Community-Based- and Non-Government Organizations. Health care for migrant pregnant women was established in 1998 by SMRU as there were minimal services available for them .15. SMRU is staffed predominantly by locally trained workers for antenatal care and ultrasound23, child birth24 and emergencies in adults and neonates12. Local medics, midwives and nurses do the majority of the clinical work and expatriate doctors assist local staff with 24-hour back up. Ultrasound quality has been measured previously in this setting23 and the eight sonographers undergo a small quality control every 6 months by a clinician 24. Over 3,000 women register at SMRU antenatal clinics annually and these were well established before birth services were offered. The first birthing unit was opened at Shoklo refugee camp in 1986, with border skirmishes and closure of Shoklo, this was relocated to Maela Refugee camp in 1995, and two more units for marginalized migrant workers were opened in Wang Pha in Dec-2007 and in Maw Ker Thai in April 2010  and nifedipine at a gestation of at least 28 weeks. If extreme PTB (exPTB) occurs (<28 weeks) infants are provided with palliative care. Parents are involved and counseled in the process12. No resuscitative efforts are offered to live born infants of 22 to <28 weeks gestation as no further sophisticated care i.e. intubation, incubators, surfactant, parenteral nutrition etc. is available25. Women who need caesarean section or who have complex medical conditions are transferred by car to Thai hospitals (45 to 90 minutes away).The seven signal functions for Basic Emergency Obstetric and Newborn Care, including parenteral administration of an oxytocic, antibiotics and anticonvulsants, removal of retained products of conception, assisted vaginal birth including breech birth, resuscitation of the newborn using a bag and mask for infants ≥ 28 weeks gestation, and screened blood transfusions, are provided by local staff. A description of the special care baby unit for neonates has been detailed by Turner23. Ultrasound can be repeated at any time as required. For example, if a woman reported absence of fetal movement, or bleeding, or the fundal height did not increase, an ultrasound could be done to determine viability. Measurement of the fetus size at each scan was encouraged. Loss of fetal heart beat could also be an incidental finding when the woman attended for her second scan. Loss of viability with ultrasound could be confirmed by presence of a fetus and absent fetal heartbeat. In some cases, ultrasound confirmed pregnancies persisting to 22 weeks gestation, but a fetus was never observed, e.g. anovulatory gestation or non-classic gestational trophoblastic disease.Ultrasound scans have been performed by local health workers using various scanners, including Toshiba Powervision 7000, Dynamic Imaging (since 2001), Fukuda Denshi UF 4100, and General Electric Voluson-1. Since 2001 all women have been offered two scans: once at booking to determine viability, number of fetus and gestation, regardless of how far progressed the pregnancy is, but preferably between 8 and 14 weeks; and again at 22 (18–24) weeks to reassess viability, measure fetal biometry and major abnormalities and determine placental location26. Before ultrasound availability, loss of fetal viability could be confirmed by SFH measurements, which were increasing and then levelled off, or decreased with unexpected SFH results being confirmed by a clinician), complaints of loss of fetal movement or never feeling movement, bleeding episodes or expulsion of products. Quality control exercises of SFH involved comparing 20 women per month between SFH measurers – a difference of > 1cm was considered inacceptable and corrections of the firmness of applying the tape measure, correctly placing the end of the tape measure on the upper border of the symphysis pubis and identifying the fundus, were made during these sessions.In this setting SFH measurement has been important to determine whether a woman was in the first or second trimester which made the difference between being able to use quinine or artesunate to treat uncomplicated malaria. At antenatal care, pregnant women had abdominal palpation at each visit until the SFH was first palpable and measurable above the symphysis, then SFH was checked monthly up to 32 weeks and weekly from 36 weeks gestation. In case of doubt, urine (sometimes serum) pregnancy testing was available. It was and still is common to have the SFH measured multiple times in a single pregnancyManagement of fetal loss diverged from high income settings. Before misoprostol was available, induction of pregnancies with confirmed fetal loss was difficult and if there was no vaginal bleeding and the cervix was not open a conservative management style was adopted. Induction with syntocinon, the only available agent for many years, was frequently a prolonged and unsuccessful process. In this low resource context, only surgical emergencies were referred to tertiary hospitals, so non-viable pregnancy loss, with no imminent danger signs did not qualify for referral.All birth records at SMRU are computer based and paper-based records are archived at SMRU head office in Mae Sot. The original paper records initially matched data collection used by Médecins Sans Frontières (MSF) in humanitarian settings. In 1998 computer based registration commenced with adjustments to the MSF form, in late 2001 ultrasound commenced requiring further amendments, and in late 2007 all data was captured in real time in an application developed by Technology Sans Frontières (TSF) with data limitations set to alert users to inaccurate entry. Verification of paper and computer based data has taken place over decades to a computer based record system.All pregnancy records from 1995 until 2015 in the window period from 22 to <28 weeks gestation were selected and reviewed case by case by for the present study. The starting point of 1995 was selected, since the first local guideline for obstetrics was introduced at this time.For each record the following evaluation was conducted using a step-wise query process:a) Was there evidence of in utero fetal demise (no FHB) before 22 weeks? If yes, could the gestation of loss be estimated? For example, was fetal anthropometry measured by ultrasound when absence of fetal heart beat was confirmed; or did the SFH measurements stall or decrease (and at how many centimeters) before fetal heart beat could be heard or fetal movements felt, or did the mother report that she never felt fetal movements? Was there evidence that there was never a fetus i.e. that the pregnancy was never viable? Did ultrasound measure only annovulatory pregnancy (blighted ovum)?b) Was there evidence of in utero fetal viability at 22 to <28 weeks gestation ? If yes, what was the estimated gestation at loss of viability and were there any signs of life (clinic births-heart beat by auscultation and home births-respiratory effort/movement) at birth?c) If the outcome was a live birth, what was the neonatal outcome?9.Records with evidence of in utero fetal demise before 22 weeks but expulsion between 22 to <28 weeks were classified as miscarriage (late expulsion). Pregnancies with evidence of fetal viability at ≥22 weeks that were expulsed between 22 to <28 weeks were classified as exPTB (live or stillborn). Neonatal mortality was defined as death in the first 28 days of a live born neonate of 22 to <28 weeks gestation. Gestational trophoblastic disease and termination of pregnancy were excluded. Congenital abnormalities were coded using the ICD-10 criteria+0 to 22+6 weeks, (i.e 22 weeks plus 6 days) of pregnancy. Continuous normally distributed data, such as gestation and birth weight, were described using the mean, and standard deviation (SD) and compared with the Student’s t-test. Only the first born twin birth weight was retrieved from the electronic files. Non-parametric data, such as gravidity, were described using median and 25th-75th percentiles and compared with the Mann-Whitney U test. Proportions were compared using the Chi-squared test. To assess the role of ultrasound in the final classification as exPTB rather than miscarriage between 22 to <28 weeks gestation, univariable and multivariable logistic regression was used to determine the association between ultrasound use and outcome, adjusted for first ANC attendance in first trimester and delivering with a skilled attendant (confounders identifieda priori). Data was analysed using SPSS version 20  and Stata version 13 .Gestation was reported by week, for example 22 weeks included women from 22Ethical approval for retrospective analysis of pregnancy records was given by the Oxford Tropical Research Ethics Committee (OXTREC 28–09) and after discussion with the local Community Advisory Board (TCAB-4/1/2015)Between 1995 and 2015, 80.9%  of women registered to antenatal care had a known pregnancy outcome and were included in the present study. Only a small proportion, 0.2% , of these pregnancies could not be assigned a reliable gestational age . The proThere were 3.8% (18/372) excluded from analysis: termination of pregnancy involved 13 cases including: six ultrasound confirmed major fetal abnormality , two life-threatening maternal conditions both with uncontrollable severe pre-eclampsia, and five self-induced (one of whom was recently widowed); and five gestational trophoblastic disease. The demographic characteristics of the remaining 454 pregnancy outcomes are summarized inOf the 454 pregnancy outcomes from 22 to <28 weeks gestation 197 (43.5%) were miscarriage (loss of viability before 22 weeks) two of which were twin gestations (23 weeks) . More th+1 [IQR 4+5 to 11+0] weeks+days. (+3 (n=24) vs 7+4 weeks+days (n=88), p=0.084.Loss of viability was not possible to determine for 11 records (nine indicated spontaneous miscarriage and two records could not be located) leaving 186 records. Evidence of fetal demise  also known as fetal death in utero, occurred in 60.2% (112/186), with most of these determined by ultrasound . The pro+days. , and notThere were 56.4% (257/454) of women with an exPTB of which 89.9% (231/257) were singletons and 10.1% (26/257) were twins . The genAmongst the 257 pregnancies ending in exPTB, 1.9% (5/257) had missing data on whether the infant was still- or live born , and forThe birth weight measured in singletons was not available for 42.0% (108/257) of neonates. For the 75 homebirths this is not surprising. Birth weight of 17 congenitally abnormal infants was excluded from analysis. Birth weight in live born, normal singletons in the period before ultrasound (n=18) and when ultrasound was available (n=67) was similar: mean±SD (range): 817±253 (350–1300) and 875±231 (220–1500) g (p=0.380). The mean±SD (min-max) birth weight was higher in live born (n=85) than stillborn (n=27) normal singletons infants: 863±235 [220–1500] and 652±208 [400–1320] g, (p<0.001). Mean birth weights for singletons and first-born twins were summarized for each gestational age week after excluding birth weight of those with congenital abnormality .Congenital abnormality involved 10.6%  of exPTB and half of these congenital abnormality cases 50.0% (12/24) were stillborn .+5 weeks, and two ultrasounds, including an early scan at 8 weeks and a later scan at 18 weeks, assured the gestation. The mother was a 32-year-old refugee with a gravidity of two and parity of one, with no history of PTB, who went into spontaneous labour and received a single dose of nifedipine and dexamethasone less than one hour before delivery. Delivery was supervised by skilled birth attendants and after a normal vaginal birth of a 890g baby, the Apgar scores were six and seven, at one and five minutes, with no resuscitation necessary. The neonate was provided with supportive care  because that was all that was available, and discharged home after ten weeks at 1061g.Of the 132 liveborn exPTB, the fate of one neonate was unknown at one month. Of the remaining neonates 98.5% (128/131) had a neonatal death and 1.5% (2/131) survived the first 28 days. The median  age of neonatal deaths was 1  day, with 87.0% (114/131) by day 3 . One newUse of ultrasound was associated with an increased adjusted odd ratio (AOR) of being classified as exPTB rather than miscarriage: AOR 2.09 ; while attending in the first trimester for the first antenatal visit and delivery at SMRU clinics were not associated: AOR 1.29 , and AOR 0.93 , respectively .27. In the context of this post-conflict, cross border, low resource setting, the fact that 81% of outcomes were reported, with ultrasound available for nearly ¾, supports the use of the data to bring clarity to the nature of the products of conception expulsed in the 22 to <28 weeks gestational age window. As well the median number of six antenatal consultations . Ultrasound was associated with a 2 times higher odds of the outcome being classified as an exPTB rather than a miscarriage. This suggests that ultrasound adds clarity to the outcome of pregnancy in this 22 to <28 week window, mostly because it detects the presence/absence of a fetal heart beat or in this setting even the presence of annovulatory pregnancy – both of which are not easily identified with more limited tools . Under the WHO/ICD 10 definition these miscarriages could be (wrongly) defined as exPTB based on gestation alone, which would falsely inflate stillbirth rates4 .28. Two infants emerged from the neonatal period, one died at day 33 and one female of 27+5 weeks gestation survived infancy (end of the first year). Not only are the outcomes of pregnancies of 22 to <28 weeks gestation in this setting dismal, the proportion of pregnancies that are involved, relative to all pregnancies with a known outcome, is small (0.9%). It could be argued that if more than palliative care was offered to the newborns at SMRU, outcomes may have been different but with no means to provide additional support such as intubation, incubators, surfactant, parenteral nutrition etc, improvements would remain marginal. The high proportion of stillbirths (47.6% (120/252) contrasts with rates under 2% previously reported in this population in the 28 to <34 week’ gestation window12. Stillbirth rates may be inflated using gestational age rather than birthweight cut-offs29 and also because nearly all these infants were born vaginally, including breech births, whereas caesarean section may have been offered in HIC. These different proportions are important because they indicate that in low resource setting, the maximum benefits of interventions to prevent newborn death, is in the group of 28 weeks (and above) gestation30. Overall, this data supports the WHO definition of 28 weeks to define birth (live birth and stillbirth)31, and <28 weeks as a pragmatic definition of miscarriage32Amongst the outcomes classified as exPTB, nearly one-half were stillborn, 98.5% of the babies born alive were neonatal deaths of which two-thirds occurred on day one, and there was a high proportion (as expected) of congenital abnormalities. A high congenital abnormality rate is expected amongst this age group in a setting that does not actively screen for abnormalitiesfetus papyraceus, which, surprisingly, is culturally fortunate33.In low resource settings there are many reasons why women may have a late outcome (22 to <28 weeks gestation) of a miscarriage (non-viable before 22 weeks) . Coming 34 and dating pregnancies reliably a particularly challenging issue35. Some of the higher end birth weights may be due to inaccurate dating but meta-analysis14 also suggests 1000g as a cut-off misses gestations in the exPTB window. On the Thailand-Myanmar border, SMRU has integrated basic ultrasound delivered by local sonographers to routine ANC and this has been accepted by women36. Efforts have been directed towards having local staff skilled in routine gestational age scanning23, standard care at antenatal clinics and at birth15, and in newborn care12. Record keeping has been based on the cut-off point of 28 weeks gestation for birth and miscarriage and while that has been a strength in directing human resources in the delivery room and in special care baby unit to viable neonates it has also resulted in weaker reporting of pregnancy outcomes from 22 to <28 weeks, which is an obvious limitation of this data set. Another limitation of the analysis is the 21 year period of the cohort. This could however be viewed differently as it puts into perspective the small group of pregnancies involved: approximately six live born exPTBs per year compared to >2,000 births of 28 weeks or more per year. While there have been changes over time including the introduction of a special care baby unit and ultrasound, there has been no change in the assisted ventilatory support of newborns, which is not available.Registration of births is incomplete in many LICIn a low resource setting the outcome of pregnancy between 22 to <28 weeks gestation involves <1% of all outcomes, a high proportion of miscarriage (late expulsion), with one-in-two of the exPTBs being stillborn and amongst livebirths, a neonatal mortality approaching 100%. The distinction between miscarriage and exPTB in this gestational window is improved by ultrasound but this is unlikely to result in improvement in survival due to significant resource constraints. The WHO cut-point to define stillbirth and miscarriage is pragmatic and useful in low resource settings in the scale-up towards reducing preventable newborn deaths as it allows a greater focus on newborns more likely to survive with a gestation of 28 weeks or more.http://www.tropmedres.ac/data-sharing. The application form for datasets under the custodianship of MORU Tropical Network can be found inDue to ethical and security considerations, the data that supports the findings in this study can be accessed only through the Data Access Committee at Mahidol Oxford Tropical Medicine Research Unit (MORU). The data sharing policy can be found here: I reviewed the 3rd revision of the manuscript titled "Miscarriage, stillbirth and neonatal mortality in the extreme preterm birth window of gestation in a limited-resource setting on the Thailand-Myanmar border: a population cohort study’. I think the authors have addressed the concerns to the maximum of their capability within the confines of the study design and data set and I approve for publication.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The latest version of the paper "The overlap between miscarriage and extreme preterm birth in a limited-resource setting on the Thailand-Myanmar border; a population cohort study " version 3 is a significant improvement on previous 2 versions of this paper. However, I think it is worth mentioning that although it is sensible and logical to report as SBs those fetuses that are at least 28 weeks or 1000g, for international comparisons of PMRs, it is still worth gathering data for all fetuses I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Abstract The Methods section of the abstract does not distinguish well the classification used. It seems there were two groups but this is not clear from the structure of the sentence. How was non-viability versus viability assessed?  It is stated that non-viability was determined at a median GA of 16 weeks. It is unclear in what cases would non-viability of births at 22-28 weeks can be determined at 16 weeks.Methods What was the quality control for data collection? Were clinicians trained in data collection? Were data collectors prospectively assessing the quality of the data? What QI efforts were made to improve data collection? Were the definitions of stillbirth versus neonatal death prospectively applied throughout the whole period of data collection? What signs of live birth were assessed? How was HR assessed? What resuscitation measures used in HR was not audible? Were data systematically collected on bag and mask ventilation, incubator use, IV fluids, oxygen supplementation, etc?Results The outcome of 19% of booked pregnancies was not known. This is an important problem as pregnancies losses may be over-represented in these unknown outcome pregnancies. The stillbirth rate is very high especially at the lower gestations. For example, over 40% of births at up 26 weeks were assessed to be stillbirths. There is a frequent of limited assessment for live birth at the lowest gestations. It is common to assess that a newborn is stillbirth because resuscitation is not implemented when few signs of live birth are present. Signs of live birth are not reported. Resuscitation efforts are not reported. It is concerning that birth weight was not available for 42% of the births? Other basic data also were frequently not available. This raises concerns about the quality of the data. Gestational age may be underestimated as one SD is above 1000 g which is unlikely for a population of 22-27 week pregnancies. Data on provision of care are not provided well. It is unclear to what extent live births were provided full support (other than the therapies stated not to be available).Discussion In the Discussion, the authors appear to implicate that palliative care was generally provided. Details on this should be provided in Results. The Discussion does not address most major limitations including the 19% of the pregnancies without outcome data, the post-natal interventions rate of use, and the rate of provision of palliative care. The Discussion is lengthy and yet does not address critical aspects of the limitations so the study can be pit in a better perspective and better interpret its contribution to the literature.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Referee Report Version 2 01 Aug 2018 Waldemar A. Carlo, Department of Pediatrics, The University of Alabama at Birmingham, Birmingham, AL, USA  Approved with Reservationsin bold) has been provided (below), We would genuinely like to thank the reviewer Dr Waldemar A Carlo for his repeated review and acceptance to take time to understand this manuscript. The comments have helped to clarify the contents and limitations of the manuscript. A point – by – point response to each comment  between 22 to <28 weeks was observed with non-viability occurring at an estimated median gestation of 16 weeks.” …says miscarriage – not birth: and has now been amended “Individual patient analysis of all miscarriages, estimated non-viability to have occurred at a median of 16 weeks despite late expulsion between 22 to <28 weeks.” Details are provided in the methods but the space limitation of the abstract prevents further elucidation of the reviewers query scenario e.g. A woman may present for the first time at 6 months (24 weeks GA) by LMP, she is sure of her dates but the ultrasound shows absent FHB in a fetus with a FL of 16 weeks  e.g FDIU since 16 weeks or IUGR only growing to a 16 week size. The expulsion occurs from 22 to <28 weeks pregnant based on LMP.  Methods3.What was the quality control for data collection?  The records have been quality controlled for data entry since 1986 when the antenatal care program commenced, firstly by author FN and later by RM. The data has been quality controlled for completeness and correctness of entry as this has been the most intensely studied population (dataset) in terms of malaria in pregnancy worldwide.P.falciparum drug resistance is amongst the highest in the world. In addition significant efforts with gestational age were important to understand the association of artemisinin derivatives  with fetal resorption. Gestational age has been a significant issue for decades as antimalarial drug exposures and pregnancy outcomes have been so important in this population where4.Were clinicians trained in data collection?  Yes5.Were data collectors prospectively assessing the quality of the data?  Yes6.What QI efforts were made to improve data collection?  Amendment at methods: section of data extraction7.Were the definitions of stillbirth versus neonatal death prospectively applied throughout the whole period of data collection?  Yes. See supplementary file 1 for clarification. The introduction has also been modified to conform with the reviewers suggestion.8.What signs of live birth were assessed?  In clinic based birth heart rate was assessed. In clinic and home births respiratory effort, and movement were assessed (or asked to the birth attendant).9.How was HR assessed? Auscultation10.What resuscitation measures used in HR was not audible?  < 28 weeks none. This is not without reason in our experience: while the baby may live it does not live for very long as no accessory support can be provided..PLoS One 2018,13:e0190419 which is ref 45 in v2..0). ≥ 28 weeks: 5 inflation breaths and then action dependent on HR according to the neonatal drill which is published elsewhere  infants are provided with palliative care. Parents are involved and counseled in the process10 and Janet(ref 45 in version 2) (both open access) are important as they describe everything that does happen (e.g.bag and mask ventilation etc) when an infant is of 28 weeks’ gestation or more, in this low resource environment. The two references TurnerResults12.The outcome of 19% of booked pregnancies was not known. This is an important problem as pregnancies losses may be over-represented in these unknown outcome pregnancies.Amendment to the first sentence of the results (and abstract).This limitation of the data as the first paragraph to the discussion. However we do not think that this limitation detracts from our main conclusion i.e. the need to focus on viable fetuses that have reached a GA of 28 weeks. We agree with the reviewer and we have added13.The stillbirth rate is very high especially at the lower gestations. For example, over 40% of births at up 26 weeks were assessed to be stillbirths. There is a frequent of limited assessment for live birth at the lowest gestations. It is common to assess that a newborn is stillbirth because resuscitation is not implemented when few signs of live birth are present. the reviewers comment to further explain the high rate of stillbirth as many of these births would have been by emergency C.section in a HIC setting, not vaginal birth.   Thank you and this has been added as a limitation.Signs of live birth are not reported. nd bullet point. Amended, see methods, data extraction and data definitions; 2Resuscitation efforts are not reported. See point 11.14.It is concerning that birth weight was not available for 42% of the births? Other basic data also were frequently not available. This raises concerns about the quality of the data. We agree this is a limitation. As the 28 week cut-point has been used to define birth and miscarriage it has been an ongoing process to motivate staff to weigh infants born prior to this gestation. Parents also see no reason to bring a stillborn or perinatal death infant to the clinic for a weight.15.Gestational age may be underestimated as one SD is above 1000 g which is unlikely for a population of 22-27 week pregnancies. There are no SD above 1000g, only the upper range. These are possible from the local centiles produced for this population from ultrasound dated pregnancies  Quantifying Low Birth Weight, Preterm Birth and Small-for-Gestational-Age Effects of Malaria in Pregnancy: A Population Cohort Study. PLoS ONE 9(7): e100247. doi:10.1371/journal.pone.0100247). They may represent incorrect EGA but birthweight give lower stillbirth rates than gestational age cut-offs and this has been added to the discussion.14.Data on provision of care are not provided well. It is unclear to what extent live births were provided full support (other than the therapies stated not to be available). Amended. This is the same as point 9. There was no resuscitation, intubation, incubators, surfactant, parenteral nutrition etc.Discussion15.In the Discussion, the authors appear to implicate that palliative care was generally provided. Details on this should be provided in Results. Amended as same as point 12.16.The Discussion does not address most major limitations including the 19% of the pregnancies without outcome data, the post-natal interventions rate of use, and the rate of provision of palliative care. Amended as same as point 11 and 13. Limitations are addressed in paragraph 1, paragraph 3. In paragraph17.The Discussion is lengthy and yet does not address critical aspects of the limitations so the study can be pit in a better perspective and better interpret its contribution to the literature. Agree: Significantly reduced Thank you for the opportunity of reviewing this manuscript entitled “Miscarriage, stillbirth and neonatal mortality in the extreme preterm birth window of gestation in a limited-resource setting on the Thailand-Myanmar border: a population cohort study.” The work done is impressive and has the potential to provide insight in how we: (a) view and manage the ‘grey zone’ in low resource setting, (b) support mothers and their families who present for care when in this ‘grey zone’, and (c) should allocate the limited resources in reducing neonatal mortality in low resource setting.Title  The title is very long and needs to be shortened. Moreover, it needs to better capture the findings of the study.Abstract  Introduction: Clearly define WHO and study definitions – this is critical to understanding the study results. Objective: Associations were also examined (page 4) which is not captured here. Methods: a more comprehensive description is required. Results: please share the statistics related to the association found. Conclusion: bold and very relevant!Article Content:Introduction The definition of stillbirth, miscarriage, preterm birth, and neonatal death has been identified as problematic given overlaps in gestational cut-points. Hence, it is imperative that these terms be defined at the outset so that there is conceptual clarity both with respect to variables being examined and the intended purpose of the study. This would also help with interpretation of findings. There are discrepancies with respect to the definition of miscarriage in the introduction and methodology . Moreover, issues are highlighted about accuracy of gestational assessment, lack of ultrasound diagnosis of congenital abnormality which further impacts definitions of terms particularly with inclusion of data before 2001. It is therefore not clear what the study hopes to accomplish by examining pregnancy outcomes between 24 to < 28 weeks’ gestational age over a 20 year period.     The second paragraph: the information pertaining to preterm births, neonatal deaths, and rationale for the high rates seems misplaced. Explain the relevance of the information shared to the aims of the study. Furthermore, explain the consequence(s) of poor reporting of stillbirth and neonatal deaths. The third paragraph: relevance of statistics related to mortality secondary to malaria is not clear.  The introduction should more clearly articulate the conceptual approach , explain the rationale of the study, and situate information related to preterm birth, neonatal deaths, maternal mortality within the context of study aims.MethodologyData extraction and data definition  Data were reviewed from 1995 to 2015 which is problematic as care patterns have changed significantly in this time frame (details shared in second and third paragraph). For example, the authors explain that there has been a shift from home birth to hospital births and improvement in care during the antenatal period. These changes are likely to reduce fetal mortality, as well as maternal and neonatal mortality. Thus, it is not clear why this time span was used for the purpose of this study. Moreover, ultrasound was introduced in 2001 which means that accurate assessment of gestational age may be an issue prior to this time. Ultrasound has also changed the way in which loss of fetal viability was confirmed . Given the intent to describe viability and mortality in the window of 24 to <28 weeks’ gestational age it is important to have consistency in the way definitions are operationalized throughout the course of the study.  Data Quality – birth records are computer based; however is initial data recorded on paper and then transferred to the computer system? Please comment on the accuracy of data and strategies employed in ensuring quality data. Based on the results it appears that there are potential issues with data quality as in 4.4% of the 204 pregnancies between 24 and < 28 weeks’ gestational age there was missing data; birth weight data was available for 57.6% of neonates. This is a limitation and your discussion should explain how this impacts interpretation of findings. Ultrasound assessment – was this done by one person? If multiple assessors please comment on inter-rater reliability of ultrasound dating. Page 4 – please indicate the actual average of transporting women by car to the Thai hospital. Is this a potential limitation of the study?Statistical analysis “Univariable and multivariable logistic regression was used to assess the association between estimation of gestational age by ultrasound, homebirth and year of birth, and birth outcome (birth rather than miscarriage).” A better description needs to be provided about the analytic plan and how it relates to the objectives of the study. The analytic plan should identify how variable were identified to be important to include in the multivariable logistic regression analysis. How were variables entered in the multivariable logistic regression analysis? Explain the rationale for the decision(s). It appears that the outcome of interest is birth however only 57.8% of these were live birth. What outcome was used? Furthermore, only one infant survived. What is the minimal sample size required for your multivariable regression analysis?Results Pregnancy outcomes were unknown for 19.1% of the registered women – what was the issue? Only 50.6% of the women had their first antenatal care visit in trimester one which has implications for accuracy of gestational dating using ultrasound (most accurate in the first trimester). The inclusion of termination of pregnancy is confusing particularly since two were for maternal conditions at 24 and < 28 weeks’ gestation which in high income countries would be managed by inducing labour. Table 1 – Characteristic age, years – please indicate what the data represents (as you do for other variables). Figure 2 – study flow diagram can be expanded to more clearly display outcomes including stillbirths, live births, and survival. Figure 3 – Can be removed as I don’t think it provides any additional information.Discussion The results do not demonstrate the bias detailed in the opening paragraph. Is there an opportunity here to compare historical cohorts (before introduction of ultrasound and after introduction of ultrasound)?I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The study titled ‘Miscarriage, stillbirth and neonatal mortality in the extreme preterm birth window of gestation in a limited-resource setting on the Thailand-Myanmar border: a population cohort study’ is a well thought out and interesting study and has a lot of potential to be better and more useful to the reader. The following are some points I am hoping will be useful in the authors ’efforts to improve the manuscript.Recommendation 1: The main objectives of the study are not clear. If it is challenging the definition (or lack thereof) of the miscarriages and its overlap with extreme premature deliveries, please say so more clearly. Obviously, this overlap is very confusing and just to demonstrate the conceptual difficulties one faces when trying to analyze a data set like this, is a worthy cause. Spend more time with these conceptual inconsistencies so the readers who are not familiar with these concepts will have a better understanding of your objectives and results.Recommendation 2: Third paragraph in the Introduction: The background information given is interesting but very short. In addition, it is not clear what purpose it serves in the general context of the manuscript. Recommendation: It would be ideal to provide more detailed information that is pertinent to the etiology of maternal diseases, miscarriages, stillbirth etc. in that region to help the reader to better understand the characteristics of the patient population.Recommendation 3: It appears there is a discrepancy between the definition of miscarriages at the end of the first paragraph of page 4 and the definition given under the header of miscarriage in the second column. The first definition: When a fetus dies <24 weeks but is delivered between 24-28 weeks. The second definition is used in the first sentence just under the subtitle of ‘miscarriage’: 114 women out of 318 women miscarried from 24<28 weeks’ gestation and the distribution is given according to the gestational age. In this section it appears that for a fetus to be called he/she does not have to die<24 weeks and be delivered after 28 weeks. In addition, in our experience it not very common for the mother to keep the dead fetus for two or more weeks (in this case up to 4 weeks).Recommendation 4: Second column on page 6. The correct term for ‘surface’ examination is ‘external’ examination.Recommendation 5: I would recommend exclusion of terminations. That adds more confusion to the calculations and it is considered a very different group.Question 1: The true consequence of using different GA for the definition of fetal death/stillborn vs miscarriage (except causing confusion) is not clear.Question 2: Since you have the numbers, what happens if you analyze the data where a pregnancy loss up to 20 weeks gestational age will be classified as a miscarriage and any delivery ≥20 weeks is classified either as a liveborn or stillborn?I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Halit Pinar, Warren Alpert Medical School, Brown University, Providence, RI, USA  Approved with Reservations The study titled ‘Miscarriage, stillbirth and neonatal mortality in the extreme preterm birth window of gestation in a limited-resource setting on the Thailand-Myanmar border: a population cohort study’ is a well thought out and interesting study and has a lot of potential to be better and more useful to the reader. Agree with potential to be better.The following are some points I am hoping will be useful in the authors ’efforts to improve the manuscript.Recommendation 1:The main objectives of the study are not clear. If it is challenging the definition (or lack thereof) of the miscarriages and its overlap with extreme premature deliveries, please say so more clearly. Obviously, this overlap is very confusing and just to demonstrate the conceptual difficulties one faces when trying to analyze a data set like this, is a worthy cause. Thank you for your patience with the manuscript – we are trying to articulate i.e. that this window (22 to <28 weeks) can include miscarriage, a large proportion.Spend more time with these conceptual inconsistencies so the readers who are not familiar with these concepts will have a better understanding of your objectives and results. We agree that we did not succeed to give clarity on the main objective. We have rephrased the entire manuscript. The aim of this study is to contribute to the sparse body of evidence from limited resource settings that support the use of the 28 weeks’ gestational age cut-point for stillbirth. We also argue for the usefulness of <28 weeks’ gestational age to define miscarriage.Recommendation 2:Third paragraph in the Introduction: The background information given is interesting but very short. In addition, it is not clear what purpose it serves in the general context of the manuscript. Recommendation: It would be ideal to provide more detailed information that is pertinent to the etiology of maternal diseases, miscarriages, stillbirth etc. in that region to help the reader to better understand the characteristics of the patient population. Thank you for this comment – the introduction has been rewritten to address this point and Recommendation 1.Recommendation 3:It appears there is a discrepancy between the definition of miscarriages at the end of the first paragraph of page 4 and the definition given under the header of miscarriage in the second column. The first definition: When a fetus dies <24 weeks but is delivered between 24-28 weeks. The second definition is used in the first sentence just under the subtitle of ‘miscarriage’: 114 women out of 318 women miscarried from 24<28 weeks’ gestation and the distribution is given according to the gestational age. In this section it appears that for a fetus to be called he/she does not have to die<24 weeks and be delivered after 28 weeks. The definitions have been laid out more clearly. For this analysis the definitions of miscarriage is absence of viability before 22 weeks but delivery in the 22 to <28 week window and extreme preterm birth is presence of viability and delivery at 22 to <28 weeks. In addition, in our experience it not very common for the mother to keep the dead fetus for two or more weeks (in this case up to 4 weeks). This is a very helpful comment and it is exactly what we want to highlight for an in issue relevant to low resource settings. We have an average of 6 weeks before expulsion. We suspect this must be a source of confusion in other low resource settings as these types of pregnancy outcomes are hard to document without a reliable method of measuring pregnancy viability e.g. ultrasound or multiple. We are also trying to highlight that the experience in low resource settings may not match the bulk of published evidence available.Recommendation 4:Second column on page 6. The correct term for ‘surface’ examination is ‘external’ examination. Amended as suggestedRecommendation 5:I would recommend exclusion of terminations. That adds more confusion to the calculations and it is considered a very different group. Amended as suggestedQuestion 1:The true consequence of using different GA for the definition of fetal death/stillborn vs miscarriage (except causing confusion) is not clear. We have spent more time to explain this in the introduction. The practice of obstetrics in resource limited settings is not as efficient. Miscarriage can occur at 22-28 weeks (the window of extreme preterm birth) and in the setting described in this manuscript where significant effort has been placed on assessment of gestational age, we present data to prove that point. The WHO definition of 28 week cut-point that defines a stillbirth is attractive and pragmatic for low resource settings.Question 2:Since you have the numbers, what happens if you analyze the data where a pregnancy loss up to 20 weeks gestational age will be classified as a miscarriage and any delivery ≥20 weeks is classified either as a liveborn or stillborn? We have gone back to the records to classify outcomes from 22 to <28 weeks gestation as miscarriage, livebirth, stillbirth, neonatal mortality. The records of 20 and 21 weeks start to have too much missing data to contribute reliably to the argument.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Competing Interests: No competing interests were disclosed. Close


Birth asphyxia is a major contributor to neonatal mortality worldwide. In Ethiopia, birth asphyxia remains a severe condition that leads to significant mortality and morbidity. This study aims to assess the prevalence and contributing factors of birth asphyxia among the neonates delivered at the Nigist Eleni Mohammed Memorial Teaching Hospital, Southern Ethiopia.. Data were entered into EpiData (version 3.1) and analyzed using SPSS software (version 24). Multivariable regression analysis was used to identify the association between the independent variables and outcome variable with a 95% confidence interval (CI).This hospital-based cross-sectional study was carried out on 279 participants using the systematic sampling method during June 1–30, 2019. Data were collected using a pretested structured interviewer administered questionnaire, check list and chart review, which was used to retrieve medical information and mother’s test results that could not be captured by the interviewThe overall prevalence of birth asphyxia among newborns was found to be 15.1%. Factors that were significantly associated with birth asphyxia included mothers aged ≥35 , primigravida , prolonged second stage of labor , preterm birth , meconium stained amniotic fluid  and tight nuchal .Birth asphyxia is still prevalent in the study setting. The obtained findings indicated that the mothers aged ≥35, being primigravida, preterm birth, meconium stained amniotic fluid and tight nuchal were the factors associated with birth asphyxia. The results of this study show the need for better maternal care, creating awareness about contributing factors of birth asphyxia to the maternity health professionals, careful monitoring of labor, and identifying and taking proper measures that could help in reducing the occurrence of birth asphyxia. Birth asphyxia is defined by the World Health Organisation (WHO) as ‘the failure to initiate and sustain breathing at birth’ . It is aBirth asphyxia is a leading cause of brain damage. If severe, it can injure brain cells and cause potentially fatal conditions, including hypoxic-ischemic encephalopathy, brain injuries, autism, attention deficit hyperactivity disorder, seizures, and cerebral palsy . SurvivoBirth asphyxia is associated with a complex range of risk factors, which vary from industrialised and non-industrialised countries. These factors are grouped according to whether they are before birth (antepartum risk actors), during birth (intrapartum risk factors), or after birth (postpartum risk factors) . AnteparThe Federal Ministry of Health developed the first comprehensive National Child Survival Strategy (2015–2020) in 2015, aiming to reduce under-five mortality by two thirds. Since its development in 2015, several evidence-based interventions that need to be incorporated in the strategy have been initiated. These include community management of pneumonia through integrated community case management, community-based newborn sepsis management through community-based newborn care, newborn intensive care units, newborn corners, and the introduction of Haemophilus influenza, pneumococcal, and shifting PMTCT to ‘Option B+’ . The guiDespite this, a high number of newborn deaths due to birth asphyxia were reported in Ethiopia. From observations thus far, the issue of birth asphyxia remains unresolved. Ethiopia’s rate of neonatal mortality is still among the highest in sub-Saharan Africa , 28. In Analysis of birth asphyxia is important because it may reveal possible factors. Therefore, this study aims to assess the prevalence and contributing factors of birth asphyxia among neonates delivered in the Nigist Eleni Mohammed Memorial Teaching Hospital, Southern Ethiopia.This hospital-based cross-sectional study was undertaken at Nigist Eleni Mohammed Memorial Teaching Hospital in the Hossana Town, during June 1–30 in 2019. The single population proportion formula was used to determine the sample size with the following assumptions; the proportion of birth asphyxia was taken from the study conducted as Jimma Public Hospitals (12.5%) , with a Data were collected using a combination of a pretested structured interviewer-administered questionnaire, observation check list and client’s chart reviewed, which was used to retrieve medical information and mother’s test results that could not be captured by the interview. The questionnaire was developed based on the instrument that was applied in other related studies , 31, 32.To ensure the quality of data to be gathered from the study subjects, the questionnaire was translated first to the local language and then translated back to English by experts to check its consistency. The instruments were pretested on 5% of the sample size in similar settings, and necessary modifications were made based on the nature of gaps identified in the questionnaire. Additionally, data collectors and supervisors were trained for a day by the investigators on the content of the questionnaire and the ways to collect the data. Moreover, the supervisors and the investigators closely followed the day-to-day data collection process during the pretest and the actual data collection. Furthermore, the filled questionnaire was collected and signed by the supervisor after it was checked for any missing items and completeness. Moreover, cross check was done on 10% of the sample size.Birth asphyxia was considered when the Apgar score was < 7 at the 5th minutes. The trained data collectors (midwives) in the maternity ward and operating theatres performed the scoring and the information was documented on an Apgar score card.Prolonged if it exceeds 3 h with provision of regional anaesthesia, or 2 h in the absence of regional anaesthesia for nulliparas. It is multiparous if it exceeds 2 h with regional anaesthesia or 1 h without it. We have assessed this in numbers. Finally, the labor is classified as prolonged or not prolonged based on the definition.Nuchal cord was defined as a loop of umbilical cord ≥ 360 degree around the fetal neck. At the time of delivery, each birth was recorded as having a tight nuchal cord, or no tight nuchal cord. ‘Tight nuchal cord’ was defined as the inability to manually reduce the loop over the fetal head.p-value ≤ 0.25 in the bivariate logistic regression analysis. The degree of association between independent, and dependent variables were assessed using odds ratio with 95% confidence interval.The data were entered into EpiData software (version 3.1) and analyzed using SPPS software (version 24.0). Descriptive statistics, frequency, and proportions were computed to summarize the data. Logistic regression analyses were conducted to identify contributing factors to birth asphyxia. Initially, bivariate logistic regression analysis was performed on all independent variables. Multivariable logistic regression was then performed on variables that had a A total of 279 participants were involved in this study, wherein the response rate was 100%. Approximately, 83.9% of the participating mothers were aged 25–34 years. The majority of the participants were married 271 (97.1%), 224 (80.3%) of them were Hadiya ethnics, 209 (74.9%) were protestants in terms of their religion, and 185 (66.3%) were housewives. Regarding their educational status, 54 (19.4%) of them had pursued higher education, and 122 (43.7%) were rural residents  of them were multigravida and another 90 (32%) were primigravida. Twenty four (12.5%) participants had ever experienced abortion, while 23 (12.4%) of them had a history of still birth. The majority of the mothers, i.e. 252 (90.3%) participants, were at the gestational age of ≥37 weeks, while 157 (56.3%) had ≥ four antenatal care visits. Furthermore, 44 (15.8%) had faced pregnancy-related complications, wherein 13 (29.5%), 12 (27.3%), 11 (15%), 5 (11.4%), and 3 (6.8%) encountered antepartum hemorrhage, hypertensive disorders, premature rupture of fetal membranes, hyperemesis gravidarum, and other, respectively. In this study, the overall prevalence rate of birth asphyxia was found to be 42(15.1%)The outcome of the bivariate logistic regression analysis showed that, mothers aged ≥35, primigravida, pregnancy related complication, primigravida, prolonged labour, preterm birth, meconium stained amniotic fluid and tight nuchal cord were contributing factors of birth asphyxia. However, in multivariable logistic regression analysis, mothers aged ≥35, primigravida, prolonged second stage of labor, preterm birth, meconium stained amniotic fluid and tight nuchal cord were contributing factors of birth asphyxia. Mothers aged ≥35 were more or six times more likely to have experienced birth asphyxia respect to women in the age group between 20 and 34 years old . Similarly, the prolonged second stage of labor was 4.6 times more likely to have a birth asphyxia than their counterparts . Additionally, birth asphyxia was five times more likely to occur in primigravida in comparison to their counterparts . Furthermore, preterm birth was nearly 5 times more likely to develop birth asphyxia respect to their counterpart . Moreover, meconium stained amniotic fluid were 7.5 times more likely faced births asphyxia than their counterparts . Furthermore, tight nuchal cord were more or 3 times more likely to develop birth asphyxia respect to their counterpart  (Table In this study, the overall prevalence of birth asphyxia was found to be 15.1%. This is higher than seen in studies conducted in Jimma, Dire Dawa, and Tanzania which were12.5, 3.6, and 11.5%, respectively , 34. TheIn this study, mothers aged ≥ 35 were one of the contributing factors to birth asphyxia rates. The following discovery was very nearly found to be a universal fact: the increased age of the mother increased the risk of several adverse outcomes , 36, 37.Primigravida was found to be a contributing factor of birth asphyxia and this was consistent with the research conducted in India , EthiopiThis study also showed that mothers encountering a prolonged second stage of labor were at a greater risk for birth asphyxia with respect to their counterparts. These findings were similar to those of studies conducted in Ethiopia, Colombia and Pakistan , 16, 22.Meconium stained amniotic fluid was a contributing factor to birth asphyxia, similar to what was reported in Ethiopia , India , and PakBirth asphyxia was also found to be associated with a tight nuchal cord. This finding is similar to that of the studies conducted in Ethiopia  and PakiIn this study, the Apgar score was used to diagnose asphyxia. However, using the Apgar score alone to diagnose birth asphyxia has been found to be inappropriate as it is an expression of the infant’s physiological condition at one point in time, which includes subjective components. Also, the study relied on hospital deliveries. However, several deliveries occurred outside the hospital, making it difficult to capture data such as birth asphyxia, so the estimates contained in this study may have underreported the cases at the community level. Additionally, sometimes women who came to the hospital may encounter labor complications, compared to women that deliver out of hospital. Therefore, this may also cause overestimation of the prevalence of birth asphyxia.Birth asphyxia is still prevalent in the study setting. Primigravida, prolonged labour, preterm birth, and mothers aged ≥35 meconium stained amniotic fluid and tight nuchal cord were contributing factors of birth asphyxia. Results of this study show the need for the better maternal care, creating awareness about contributing factors of birth asphyxia to the maternity health professionals, careful monitoring of labor, identifying and taking proper measure could help in decreasing the occurrence of birth asphyxia. The results of the study will help policy-makers, program designers and Non-governmental organizations to support the study area. Additionally, the findings of the study can also help as a secondary data for further study in same area of inquiry.


Electric fields have been proposed as having a distinct ability to catalyze chemical reactions through the stabilization of polar or ionic intermediate transition states. Although field-assisted catalysis is being researched, the ability to catalyze reactions in solution using electric fields remains elusive and the understanding of mechanisms of such catalysis is sparse. Here we show that an electric field can catalyze the cis-to-trans isomerization of [3]cumulene derivatives in solution, in a scanning tunneling microscope. We further show that the external electric field can alter the thermodynamics inhibiting the trans-to-cis reverse reaction, endowing the selectivity toward trans isomer. Using density functional theory-based calculations, we find that the applied electric field promotes a zwitterionic resonance form, which ensures a lower energy transition state for the isomerization reaction. The field also stabilizes the trans form, relative to the cis, dictating the cis/trans thermodynamics, driving the equilibrium product exclusively toward the trans. Electric fields have been proposed as having a distinct ability to catalyse chemical reactions through stabilizing polar intermediates. Here, the authors show that electric fields can catalyse the cis-to-trans isomerization reactions of cumulenes in solution in a scanning tunnelling microscope In principle, electric fields can catalyze chemical transformations through stabilizing otherwise unfavorable electronic structures, enabling new transition states, modifying reaction barriers5, and altering reaction selectivity9. For example, studies in electrochemical environments suggest that interfacial electric fields can be exploited to control the selectivity of non-faradaic reactions11, whereas vibrational Stark effect spectroscopy4 and single-crystal diffraction experiments12 have been utilized to measure the magnitude of the electric field at enzyme active sites and cocrystal systems. Although it is conceptually appealing and, in principle, widely applicable, the use of oriented electric fields to alter the electronic structure of molecular reactants and transition states is beset with challenges14. In this regards, the scanning tunneling microscopy (STM) offers an ideal platform with which to apply a large external electric field over the small distance between two electrodes to induce chemical transformations that can be probed directly by measuring single-molecule junction conductance. Although a previous STM-based study has demonstrated a field-induced single-molecule reaction, it was limited in scope due to an ultra-low conversion rate (a few molecules per hour)15.Understanding and controlling electronic reorganizations in chemical reactions is at the heart of synthesis chemistry16, and follow the cis-to-trans isomerization reactions under an electric field. The experiments are performed using the STM-BJ technique18, which possesses the advantage of being able to apply a large  electric field, while at the same time being able to detect (through measurements of single-molecular junction conductance) the chemical products of its catalysis. We also analyze the reactants and products formed within the STM setup using high-performance liquid chromatography (HPLC) and confirmed that the reaction indeed occurs in the solution phase. We show that, at room temperature, an electric field accelerates the otherwise extremely sluggish cis-to-trans isomerization of the cumulene. Density functional theory (DFT)-based calculations confirm that the electric field alters the [3]cumulene charge distribution promoting an ionic resonance form, thereby decreasing the isomerization barrier. The applied field also alters the reaction thermochemistry, favoring the trans form and determining the ultimate distribution of the products to one that cannot be achieved by simply heating or irradiating the solution.The study based on STM-break junction (STM-BJ) technique described herein demonstrates that electric fields can direct and catalyze solution-phase reactions. We focus on [3]cumulene derivatives, which consists of chains of carbon atoms bound together to give contiguous π-bonds19. The synthesis, detailed in the cis[3] and trans[3]. We separated the isomers by fractional crystallization to isolate the pure cis[3] (23% yield) and trans[3] forms (28% yield), both of which are air stable as evidenced by 1H-nuclear magnetic resonance (NMR) experiments (see We design and synthesize an asymmetric [3]cumulene that has both an aurophilic (SMe) and an alkyl (t-Bu) substituent on each end. Each SMe terminal group binds to undercoordinated Au atoms on the STM tip and substrate, enabling single-molecule junction conductance measurements, whereas the t-Bu group provides steric bulk to prevent unwanted reactions of the cumulated double bonds to ensure air stabilityents see .20 that applies a large electric field to the molecular solution in the region between the STM tip  in tetradecane, a nonpolar solvent, at a tip bias of 0.1 V. The illustrative conductance traces  and show a molecular conductance plateau around 10−4 G0. Logarithmically binned one-dimensional (1D) conductance histograms of such traces, compiled without any data selection from thousands of traces, collected within a 2 h period are shown in the main panel. The peak around 10−4 G0 in the 1D histograms corresponds to the conductance of the single-molecule junctions. The peak for cis[3] is broad, whereas that for trans[3], at a slightly higher conductance, is more well-defined. We attribute the similar conductance value of the two isomers to their similar electronic structure, as can be deduced from their electrochemical and optical characterizations and their DFT-based frontier orbitals  are shown in Fig. cis[3] and trans[3] differs by about 0.6 nm. This results in the conductance plateaus having different lengths as seen in the inset of Fig. trans[3] junctions are roughly 0.6 nm longer than cis[3] ones. The experimental plateaus are shorter than the DFT-based junctions due to the gap that is formed after the Au-contact ruptures and relaxes20. Given the similar junction formation probability  show conductance peak heights and plateau lengths increase as the concentration of the trans isomer is increased  impact the entire sample. We deduce that the observed isomerization reactions are catalyzed by an electric field. To test whether this is indeed the case, we carry out control experiments with the SMe-terminated cis[3] in different solvents including 1,2,4-trichlorobenzene  and propylene carbonate  with a supporting electrolyte (tetrabutylammonium perchlorate), which screens the field. In TCB, again, we observed a cis-to-trans conversion, which takes ~20 h  and trans[3]  is >1 eV , has a lower energy in a field. This can be attributed to a more efficient charge delocalization over the longer length of the trans isomer compared with the cis. These effects result in a lower activation barrier for isomerization, which favors the trans product in an electric field , nanoscale techniques cannot be applied for large-scale catalysis.31. After the Au-point contact is broken, a molecule bridges the gap between the gold electrodes to form a single-molecule junction. Conductance is measured as a function of the tip-substrate displacement under an applied bias voltage, yielding traces that reveal molecular-dependent conductance plateaus. With these measurements, we obtain molecular junction-specific conductance by compiling measured traces into 1D conductance histograms and the molecular backbone length from 2D conductance-displacement histograms.We repeatedly form and break point contact between a gold STM tip and substrate in a dilute solution (0.1 mM) of the target molecule under ambient conditions at room temperature in a custom setup2Cl2/hexanes as the mobile phase, 1.00 mL/min flow rate, detection λ = 350 nm.All samples were resolved by an Agilent 1200 Series analytical HPLC equipped with a diode array detector (220–900 nm) on a CHIRALPAK IA-3 column  from Chiral Technologies, premixed 0.5% CH33, using DFT with generalized gradient approximations for exchange-correlations energy developed by Becke, 3-parameter, Lee-Yang-Parr. The calculations are performed without and with an electric field by determining the energy for optimized structures at a range of dihedral angles.The geometry optimization and calculation of molecular total energy was carried out using the Fritz Haber Institute ab initio molecular simulation (FHI-aims) packageSupplementary Information