A model of the cyst wall interactome was constructed using proteins identified through BioID. The proteins within this cyst wall interactome model encompass several proteins identified in a prior characterization of the cyst wall proteome. This model provides a more comprehensive understanding of the composition of the cyst wall and may lead to insights on how the cyst wall is formed. Toxoplasma gondii is a thick cyst wall that forms underneath the membrane of the bradyzoite vacuole. Previously, our laboratory group published a proteomic analysis of purified in vitro cyst wall fragments that identified an inventory of cyst wall components. To further refine our understanding of the composition of the cyst wall, several cyst wall proteins were tagged with a promiscuous biotin ligase (BirA*), and their interacting partners were screened by streptavidin affinity purification. Within the cyst wall pulldowns, previously described cyst wall proteins, dense granule proteins, and uncharacterized hypothetical proteins were identified. Several of the newly identified hypothetical proteins were validated to be novel components of the cyst wall and tagged with BirA* to expand the model of the cyst wall interactome. Community detection of the cyst wall interactome model revealed three distinct clusters: a dense granule, a cyst matrix, and a cyst wall cluster. Characterization of several of the identified cyst wall proteins using genetic strategies revealed that MCP3 affects in vivo cyst sizes. This study provides a model of the potential protein interactions within the cyst wall and the groundwork to understand cyst wall formation.A characteristic of the latent cyst stage of  Toxoplasma gondii, an opportunistic pathogen, infects more than 1 billion people worldwide. Most infected individuals harbor this intracellular parasite as a latent infection with cysts (containing bradyzoites) being found in the central nervous system. For the most part, those with a healthy immune system do not present with symptoms of toxoplasmosis; however, these cysts can be reactive in immunocompromised hosts, leading to active retinochoroiditis or encephalitis  was completed using tandem mass spectrometry of immunoprecipitated in vitro cyst wall membrane fragments  Immunofluorescence images showing that BirA*-tagged parasites  localize correctly to the cyst wall as seen with colocalization with CST1. (B) Immunofluorescence assay of Pru and BirA*-tagged parasites stained with anti-HA antibody and Alexa Fluor 488-conjugated streptavidin under pH 8 conditions without exogenous biotin added to the medium. Endogenously biotinylated proteins within the apicoplast are observed within these parasite strains. (C) Western blot of lysates corresponding to Pru and BirA*-tagged parasites differentiated in pH 8 supplemented with and without 150 μM biotin was probed with streptavidin-horseradish peroxidase (HRP). ALD1 was used as the loading control. Download Copyright © 2020 Tu et al.2020Tu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the After confirming the activity of BirA* at the cyst wall, the targets labeled by each BirA* transgene were affinity captured with streptavidin beads and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After accounting for host cell proteins, for well-characterized and predicted nonsecreted proteins, and for endogenously biotinylated proteins from the Pru sample, a network graph was constructed showing the potential interacting proteins of each BirA*-tagged cyst wall protein . Each BiTo assess whether hypothetical proteins TgME49_204340, TgME49_310790, TgME49_312875, and TgME49_258870  localizOf these group 2 BioID tagged constructs , only the CST2-BirA*, CST4-BirA*, CST9-BirA*, and MCP3-BirA* parasites showed additional biotinylation signals at the cyst wall when these parasites were differentiated with exogenous biotin supplemented in the high alkaline medium . The Bir10.1128/mBio.02699-19.2FIG S2FIG S2, TIF file, 2.8 MB.Intracellular and extracellular immunofluorescence of BirA*- and HA-tagged cyst wall proteins. (A) Immunofluorescence assay of BirA*-tagged parasites differentiated to bradyzoites without exogenous biotin added to the induction medium. Fixed cells containing cyst vacuoles were stained with anti-HA antibody and Alexa Fluor 488-conjugated streptavidin, which labels the endogenously biotinylated proteins within the apicoplast. (B) Immunofluorescence micrographs of extracellular BirA*- (CST9) or HA-tagged (CST10) tachyzoites stained with anti-HA (red), anti-GRA1 (green), and DAPI . Download Copyright © 2020 Tu et al.2020Tu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Since only CST2-BirA*, CST4-BirA*, CST9-BirA*, and MCP3-BirA* parasites showed biotin ligase activity at the cyst wall, streptavidin affinity capture of the potential interacting partners of these cyst wall strains was performed and processed by LC-MS/MS. The proteins identified from this subsequent round of BirA* pulldowns contained well-characterized cyst wall proteins, recently validated cyst wall proteins, dense granule proteins, and additional hypothetical proteins . GRA36 wTo assess whether hypothetical proteins (HP) TgME49_313080 and TgME49_312330 localized to the cyst wall, they were tagged endogenously on their C termini with a 1×HA tag. IFA analysis showed that TgME49_313080 localized within tachyzoites around the nucleus and also extended into the basal end of the parasite that seems to connect the parasites together, reminiscent of the intravacuolar connection between tachyzoites . After bIn the group 1 BirA* pulldowns with CST1-BirA*, BPK1-BirA*, MAG1-BirA*, MCP4-BirA*, and GRA6-BirA* parasites, CST1 was the most prominent protein identified among all five BirA* cyst wall proteins . FollowiFor the group 2 BirA* pulldowns, CST1, MAG1, and MAG2 were identified in the CST2-BirA*, CST4-BirA*, CST9-BirA*, and MCP3-BirA* data sets . On the By analyzing the proteins identified between each pulldown, some reciprocal protein pairs between baits were identified. These include CST1-MCP4, CST1-MAG1, CST1-CST9, MCP4-CST4, MCP4-MCP3, and MCP3-CST4 pairings. Interestingly, while CST2-BirA* identified CST1, MAG1, MCP3, MCP4, and CST4 within its pulldown, peptides from CST2 were found only within its own data set. The absence of CST2 within other BirA* pulldowns may be due to a detection limit of CST2 if this protein is not very abundant within the cyst wall or if CST2 only interacts with the N termini of these other bait proteins, which may be too far from the C-terminally located BirA* tag.To incorporate all these pulldowns together, a network graph was constructed using the normalized spectral abundance factor (NSAF) values obtained from each BirA* bait’s prey . Nodes CT. gondii cyst wall and matrix proteins are most homologous to their Hammondia hammondi counterparts  targeting the N termini of CST4, CST8, CST9, and MPC3 BirA* parasites were cotransfected with donor DNA oligonucleotides into their respective endogenously BirA*-tagged parental strains using a deletion strategy described previously . Prematu10.1128/mBio.02699-19.4FIG S4FIG S4, JPG file, 2.0 MB.Sanger sequencing results of the loci targeted by Cas9 of ΔCST4, ΔCST8, ΔCST9, and ΔMCP3 parasites. Chromatograms showing the sequencing results of the N termini of ΔCST8 and ΔCST9 parasites demonstrating the inserted premature stop codons. ΔCST4 and ΔMCP3 parasites had the targetable stop PAM sequence (TSPS) inserted further downstream from the start codon. The red boxes depict the DNA sequences where stop codons or TSPSs were introduced to generate the knockout strains. Download Copyright © 2020 Tu et al.2020Tu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02699-19.8TABLE S2Table S2, DOCX file, 0.1 MB.Oligonucleotides used as donor DNA to generate HA-tagged, knockout, and complement strains. Mutations are designated in red, with stop codons designated as lowercase letters and the HA sequence in green. Download Copyright © 2020 Tu et al.2020Tu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vivo cyst growth  of these cyst wall protein knockouts was assessed by IFA. Within ΔCST4, ΔCST8, ΔCST9, and ΔMCP3 in vitro cysts, no mislocalization or abnormal staining of CST1, MAG1  Survival curve of C57BL/6 mice challenged with 100,000 Pru, ΔCST4, ΔCST8, ΔCST9, and ΔMCP3 parasites (n = 10). Download FIG S5, TIF file, 1.8 MB.Additional characterization of cyst wall proteins. (A) Quantification of the plaque size from each parasite strain. The number of plaques analyzed per strain is provided above each violin plot. No significant differences in plaque size between the parental, knockout, and complement strains were observed for each cyst wall protein. Immunofluorescence images of cyst wall protein knockout parasites stained for CST1 and MAG1 (B) or CST, DBA, and MCP4 (C) under bradyzoite conditions Copyright © 2020 Tu et al.2020Tu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro cysts of the respective knockout parasites were prepared for transmission electron microscopy (EM). No distinguishing differences in cyst morphology were observed within in vitro cysts of ΔCST4, ΔCST8, ΔCST9, and ΔMCP3 parasites compared to those of the Pru grand parental strain  showing labeling with the anti-HA antibody. White arrows point to particles from the gold-conjugated anti-rat antibody. Download FIG S6, TIF file, 2.6 MB.Ultrastructural localization of cyst wall proteins. Immunoelectron micrographs of Copyright © 2020 Tu et al.2020Tu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To further refine the model of the cyst wall proteome, a network of potential cyst wall-interacting components was constructed using BioID through various cyst wall proteins. By tagging well-characterized cyst wall proteins with BirA*, a network of cyst wall protein pulldowns was generated  which waT. gondii structures , CST8 (TgME49_204340), CST9 (TgME49_310790), and MCP3 (TgME49_208740). Of these proteins, CST8, CST9, and MCP3 also appear within tachyzoite GRAomes , 21. CSTT. gondii AP2 transcription factors revealed that the expression of CST4 and MCP3, among other cyst wall and cyst matrix proteins such as BPK1, MCP4, and MAG2, was altered when the AP2 bradyzoite activators and repressors were disrupted. For example, parasites with deleted bradyzoite activators AP2XI-4 and AP2IV-3 showed a downregulation of CST4 (Studies on  of CST4  and MCP3 of CST4  compared of CST4  represse of CST4  upregula of CST4 . Other gHammondia hammondi is split into two coding sequences (HHA_261650 as the upstream coding sequence and HHA_461840 as the downstream coding sequence). Analysis of the mass spectrometry data on TgME49_261650 revealed no peptide sequences that mapped to the N-terminal regions of this protein. In addition, immunoblot analysis (data not shown) of CST4-BirA* parasites showed that the endogenously tagged CST4 has a lower molecular weight than its predicted gene model after accounting for the BirA* and 3×HA tags. These data support that TgME49_261650 is likely two genes and that the CST4-BirA* transgene corresponds to the downstream gene.Initial attempts to target CST4 and MCP3 at their N termini and insert stop codons to stop their protein expressions were unsuccessful. Therefore, further downstream regions of CST4 and MCP4 were targeted using the TSPS which allows for accessible complementation at the site where stop codons were introduced. This method circumvents the previous issues of locating two adjacent PAM sites that could be targeted by efficient sgRNAs for deletion and complementation. Since downstream targeting of CST4 and MCP3 were successful in deleting these two genes, the actual start codons for these genes are likely downstream. The current gene model for CST4 (TgME49_261650) is likely two separate genes, as the ortholog equivalent of this gene in After generating the deletion and complementation strains for CST4, CST8, CST9, and MCP3, these parasites were assayed for parasite and cyst biology. Plaque assays show that these genes were not crucial for parasite replication , and surWhile CST4, CST8, and CST9 do not seem to play a role in determining cyst size, a lack of MCP3 resulted in statistically smaller cysts . The redin vitro bradyzoite culture differentiation. While this time point was sufficient in achieving >90% bradyzoite induction, longer differentiation periods may reveal different compositions of proteins within the cyst wall. In addition, proteins within the cyst wall interactome model may vary depending on the parasite strain used. Future studies on this interactome model will require using the ME49 strain which can generate more cysts within mice, which will allow for the characterization of proteins within an in vivo cyst wall. In addition, crystal structure characterization of cyst wall proteins, super resolution microscopy of the localization of the various cyst wall proteins, and cryoEM analysis of the cyst wall complex will aid in understanding the formation of the cyst wall. With modern cryoEM techniques, macro structures of hundreds of mega Daltons have been solved  wild-type parasites expressing superfolder green fluorescent protein (GFP) under the LDH2 promoter  containing 50 mM HEPES without NaHCO3, supplemented with 1% fetal bovine serum (FBS), penicillin, and streptomycin. Following the medium change, parasites were incubated in a humid 37°C incubator without CO2 for 3 days with the bradyzoite differentiation medium being changed every other day.Pru Δpromoter  and all promoter . For braDolichos biflorus agglutinin (DBA)-fluorescein isothiocyanate (FITC) (1:500), or anti-MAG1 mouse monoclonal antibody (1:500). CF405M-conjugated anti-rabbit, Alexa Fluor 594-conjugated anti-rat or anti-mouse, or Alexa Fluor 488-conjugated anti-rabbit or anti-mouse antibodies were used as secondary antibodies (1:500). For dense granule staining, extracellular parasites were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 before incubation with anti-HA rat monoclonal antibody 3F10 (1:250) and anti-GRA1 mouse monoclonal antibody 92.10B (1:500) (HFF monolayers were infected with epitope-tagged parasite tachyzoites and cultured for 24 h before fixing or were induced to become bradyzoites for 3 days before fixing. Fixed cells were stained by immunofluorescence as described previously . HA-tagg (1:500) . MountedThe genomic locus containing the promoter region (1 kb to 2 kb upstream of the start codon) and the coding sequence (excluding the stop codon) of BPK1, MAG1, MCP4, and GRA6 was cloned upstream of the BirA* sequence  in frameFor genes encoding CST1, CST2, CST3, CST4, CST7, CST8, CST9, and MCP3, 0.4 kb to 1 kb of each gene’s C-terminal sequence was cloned in frame upstream of the BirA* sequence followed by the 3×HA epitope tag, HXGPRT 3′ untranslated region (UTR), dihydrofolate reductase (DHFR) selectable marker, and 0.4 kb to 1 kb of the gene’s 3′ UTR by HiFi assembly (NEB). Forty micrograms of the resulting construct was cut with SwaI to remove the pUC19 backbone before being transfected into Pru tachyzoites to endogenously tag the C termini of the genes through homologous recombination. Transfected parasites were selected with 1 μM pyrimethamine before being subcloned by limiting dilution as described elsewhere . A list 10.1128/mBio.02699-19.7TABLE S1Table S1, DOCX file, 0.1 MB.Primers used in this study. Overhanging regions for Gibson assembly or KLD reaction are designated in green. Download Copyright © 2020 Tu et al.2020Tu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Pulldown of the biotinylated proteins using high-capacity streptavidin agarose resins (Pierce) was performed as previously described . Briefly18 tips (Thermo Fisher) and separated by liquid chromatography , and peptide masses and sequences were determined with the Thermo Scientific LTQ Orbitrap Velos mass spectrometer (nanoLC-MS/MS). Peptide masses were determined from the MS1 survey scan , and the top 5 most abundant ions with charge states greater than 2 were selected for fragmentation using high-energy collisional dissociation (HCD). Peptides/proteins were identified using Mascot (version 2.5) against T. gondii (type II ME49 strain genome from ToxoDB version 12) and a human protein database (UniProt) with search parameters of peptide and product mass tolerances of 50 ppm and 0.4 Da, fixed modifications of carbamidomethyl cysteines, and variable modifications of deamidation (Asn and Gln), oxidation (Met), and phosphorylation . Scaffold (version 4) was used for peptide and protein validation.Biotinylated proteins on the streptavidin beads were reduced, alkylated, and digested by Lys-C and trypsin proteases. The peptide mixture was desalted using CIndividual rounds of BirA* network graphs were generated in R using the igraph package. Weighted edges for the network were normalized using the NSAF values for each BirA* pulldown. Duplicate data for the group 1 BirA* pulldown were pooled. The combined interactome network was generated using the program Cytoscape. Communities of the network were analyzed by the Louvain method of community detection, which uses a greedy optimization method to optimize the modularity of the network. This analysis was performed on an undirected graph with self-loops removed. Betweenness and in-degrees of a node within the combined interactome network were plotted using ggplot2. Venn diagrams were plotted with the package VennDiagram in R. Protein identities between orthologous cyst wall proteins across cyst-forming coccidians were obtained using blastp. The identity percentage between different cyst-forming coccidians was graphed as a heat map within R using the package ggplot2. Protein alignment of cyst wall and cyst matrix proteins was also performed in R using the msa, ape, and ggtree packages.For CST8 and CST9, these genes were knocked out and complemented using methods mentioned elsewhere . For CSTTo complement the genes back into the knockout parasites, a sgRNA targeting the TSPS was cloned into p-HXGPRT-Cas9-GFP, and the resulting plasmid was cotransfected with donor DNA oligonucleotides containing synonymous mutations of the original coding sequence to reverse the premature stop codons. After transfection, transgenic parasites were selected with 25 μg/ml mycophenolic acid and 50 μg/ml xanthine for 10 days before subcloning immediately. Lists of primers and oligonucleotides used are available in A 27-gauge needle was used to lyse parasites from host cells, which were filtered through a 5-μm filter to remove host cell debris. Fifty parasites of the respective strains were added to three wells in six-well plates containing confluent HFFs. After 14 days, the infected monolayer was fixed and stained with a 20% methanol-0.5% crystal violet solution. Plaque sizes were imaged and determined using ImageJ.3 or 105 tachyzoites of the appropriate strains intraperitoneally (i.p.) and monitored every day. After 30 days, infected mice were sacrificed for brain cyst counts. Harvested brains were homogenized in PBS using a Wheaton Potter-Elvehjem tissue grinder with a 100- to 150-μm clearance (Thermo Fisher). The homogenate was observed under a Microphoto-FXA epifluorescence microscope (Nikon), and images of GFP fluorescent cysts were captured with a 60× lens objective for cyst size analysis using ImageJ. To calculate the size of a cyst, a microscope stage micrometer was imaged with a 60× lens objective, and the distance per pixel was determined with ImageJ, which was used to set the scale. Fluorescent cyst images were then converted to 32-bit images, and a threshold of the fluorescence from the whole cyst was applied. Then, the area of the threshold was measured to obtain the area of a cyst.Six-week-old female C57BL/6 mice (The Jackson Laboratory) were infected with 10in vitro cysts from Pru Δku80 Δhxgprt, ΔCST4, ΔCST8, ΔCST9, and ΔMCP3 parasites were prepared by incubating under bradyzoite-inducing conditions for 7 days. Cysts were fixed with 2.5% glutaraldehyde-2% paraformaldehyde in 0.1 M sodium cacodylate buffer, postfixed with 1% osmium tetroxide followed by 2% uranyl acetate, dehydrated through a graded series of ethanol, and embedded in LX112 resin . Ultrathin sections were cut on a Leica Ultracut UC7, stained with uranyl acetate followed by lead citrate, and viewed on a JEOL 1400EX transmission electron microscope at 120 kV.For ultrastructural analyses under transmission electron microscopy, in vitro Pru Δku80 Δhxgprt, CST4-BirA*, CST8-BirA*, CST10-BirA*, and MCP3-BirA* parasites were differentiated to bradyzoites for 7 days before fixing and were stained for immunofluorescence as described elsewhere  test. The R packages survminer and survival were used to compare the survival curves of mice infected with different parasite strains. The Gehan-Wilcox test was used to look for significance between the survival curves.Cyst numbers and cyst sizes were graphed in R as violin plots using ggplot2. To compare multiple groups, one-way analysis of variance (ANOVA) was performed followed by All mouse experiments were conducted according to guidelines from the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals. Animals were maintained in an AAALAC-approved facility, and all protocols were approved by the Institutional Care Committee of the Albert Einstein College of Medicine, Bronx, NY .The proteomic data from this paper have been deposited at ToxodB.org (EuPathdB.org).


Quadrigeminal arachnoid cyst (QAC) associated with encephalocele is rare; and while some treatments have been developed in recent years, no definite therapeutic approach for QAC has been established. Endoscopic treatment for arachnoid cyst is gaining popularity because it is relatively less invasive to the normal brain tissues.The patient, a 4-year-old girl, presented with QAC associated with congenital occipital encephalocele. At the age of 1 month, repair of the perinatal encephalocele had been performed at another institute. An asymptomatic arachnoid cyst remained in the posterior fossa, which was closely monitored with follow up. At age 4 years, the patient started to complain of headache, which gradually increased in both strength and frequency. Magnetic resonance imaging (MRI) revealed cerebellar compression due to cyst enlargement. We performed neuroendoscopic cyst fenestration with an occipital bone approach. Post-operative MRI showed reduced size of the cyst, and the headache dramatically improved and resolved.The standard treatment of QAC is still controversial; however, our successful use of endoscopic fenestration toward the third ventricle indicates its efficacy and safety. QACs have been classified into 3 types based on their expansion mechanisms; our case might suggest another possible mechanism of QAC development.In our case, endoscopic cyst fenestration was successful for QAC with perinatal encephalocele. However, long-term follow-up and analysis of similar cases are needed to determine its effectiveness. Arachnoid cysts are pouch-like intra-arachnoid masses filled with cerebrospinal fluid (CSF). They account for 1% of all intracranial lesions and 2.6% of intracranial lesions among those aged below 18 years –5. QACs Several surgical procedures have been reported for QACs, including stereotactic aspiration, craniotomy and cyst excision or fenestration, ventriculoperitoneal or cystoperitoneal shunting, and combined procedures –10. ThesHere, alongside our case report, we suggest possible mechanisms involved in the development of QACs and discuss the important role of neuroendoscopic surgery.The patient was a 4-year-old Asian girl, with a history of congenital encephalocele of the occipital region that was operated on at another hospital when she was 1 month old. The intraoperative findings after skin incision showed that there was a lack of galea on the defect of occipital bone that appeared as a small hole with a maximal diameter of 5 mm. The defect of the occipital bone was located near the midline occipital fissure. The intralumen of encephalocele sac contained encephalocele and stiff lipid-like tissues. The neck of the encephalocele sac seemed to continuously extend toward the subarachnoid space via the small defect of the galea and occipital bone. After ligation of the neck of the encephalocele, the ligated remnant tissue was automatically pulled down under the bone. Although the dura was not primarily sutured, the defect of the galea was sutured. An asymptomatic arachnoid cyst in the posterior fossa extending from quadrigeminal cistern to infratentorial supracerebellar space remained after the operation. During follow-up, imaging revealed which was closely monitored Fig. .Fig. 1PrWe made a diagnosis of enlarged QAC after the encephalocele removal.We performed endoscopic cyst fenestration under general anesthesia. We made 1 burr hole on the left side of the posterior bone and used a rigid endoscope to carefully observe the intracystic lumen. The cyst membrane was visible just below the dura, and we were able to directly approach the normal subarachnoid space through the cyst lumen. There were multi-layers of arachnoid membranes without any sign of previous hemorrhage, which we fenestrated toward the conventional subarachnoid space, and we confirmed the pulsatile to-and-fro movement of the CSF Fig. . The patAfter the operation, the headache subsided and eventually disappeared. One year after surgery, the headache did not recur, and MRI showed no further cyst enlargement Fig. .Fig. 3PoArachnoid cyst is often associated with congenital abnormalities; a retrospective analysis showed that 25.41% of patients with arachnoid cyst had some anomaly when they were born . Some paThere are four hypothesis regarding the mechanism of arachnoid cyst development, which are still being debated: (1) a ball-valve mechanism; (2) an osmotic gradient between the intra- and extra-cystic medium; (3) primary malformation of the arachnoid membrane or cerebral lobe agenesis; and (4) fluid hypersecretion by the lining cells of the cyst wall. Cine MRI or endoscopic procedure can be used to visualize unidirectional pulsation of the CSF flow , 18. In QACs appear close to the dorsal midbrain and compress midbrain aqueduct at an early stage of development. As a result, symptoms such as hydrocephalus , paralysis of upward gaze, and visual deficit can occur. Posterior cranial fossa arachnoid cysts cause dizziness, nystagmus, nausea, headache, mental deficiency, and gait disturbance. In our case, the patient had escalatory headache and a history of encephalocele, which led to the final diagnosis.Type 1: The most common type, with extension to both the supratentorial (at the level of the trigone) and infratentorial regions (in the supracerebellar cistern);Type 2: A rarer type, with infratentorial extension to the supracerebellar or supraretrocerebellar regions; andType 3: Which has lateral extension to the ambient cisterns toward the temporal lobe Fig.  7]..7].A prospective study classified the QAC into three types based on their expansion mechanisms :Type 1: In our case, the arachnoid cyst did not conform to any these three extension types; rather, it extended from dorsal cerebellar region for compressing cerebellar hemisphere. Compared with QAC type 2, which tend to extend toward anterior supracerebellar regions, the cyst in our case extended toward the posterior supracerebellar space. Generally, encephalocele forces the arachnoid membrane to extricate from the intracranial space and resulting in malformation. In our case, intraoperative findings showed that the outer arachnoid membrane was intact. We hypothesize that, in our case, an arachnoid cyst in posterior cranial space remained after the encephalocele operation, then, CSF malabsorption occurred in the arachnoid membrane resulting in the slow extension of the cyst. This mechanism is different from other congenital neurological abnormalities, such as holoprosencephaly and Chiari malformation type II, that do not involve extrication of the arachnoid membrane.Multiple surgical strategies have been developed for the management of QACs. These include endoscopic resection of the cyst wall with establishment of continuous communication between the cyst lumen and the normal surrounding CSF pathways ; craniotomy with microsurgical fenestration; and cyst-peritoneal shunting. Endoscopic treatment of QACs has been reported in retrospective studies, which have include isolated larger series’ and case reports, with encouraging results, especially when double fenestrations have been performed –10. For In our patient, we performed cyst fenestration toward the third ventricle via a suboccipital supracerebellar approach, because, prior to the procedure, we confirmed by MRI that there were no vital structures between dura and cyst membrane, such as brain tissue, arteries, or venous sinus. We chose a posterior approach to access the cyst lumen because it was easier and less invasive for the surrounding normal cerebral tissue than frontal ETV. The post-operative course has been good; however, we are not yet able to report long-term outcomes. Furthermore, it may be that the procedure used in our patient may be suitable only in particular cases; therefore, further studies are needed to verify the long-term prognosis and to analyze a larger number of similar cases.There are few reports of QAC associated with encephalocele. The uniqueness of the QAC in our case suggest there is a possibly another type of expanding of arachnoid cysts. We performed neuroendoscopic multiple fenestration via a suboccipital supracerebellar approach because this procedure is simple, minimally invasive, and associated with low morbidity and mortality. In our case, the prognosis is good, but long-term follow-up and the accumulation of similar cases are necessary to assess the applicability of this procedure.


Twins suffering a co-twin loss at birth have reported feelings of loneliness and grief while it remains unexplored if they suffer increased risk of psychiatric disorders. We contrasted rate of first-onset psychiatric disorders among all Swedish-born twins whose co-twin died within 60 days after birth between 1973 and 2011 (n = 787) to that of 3935 matched unexposed twins, 3935 matched singletons , and 880 full siblings of the exposed twins. During a median of 19-year follow-up, exposed twins were at increased risk of first-onset psychiatric disorders  compared with unexposed twins. We observed the strongest association for emotional disorders and for psychiatric disorders diagnosed before the age of 25. Comparisons with matched singletons and the twin’s full siblings rendered similar results, corroborating an association of loss of a co-twin at birth with subsequent risk of psychiatric disorders. Because of the sharp rise in medically assisted reproduction and delayed childbearing during the last decades, the twinning rate has increased dramatically in all developed countries . This riPrevious studies indicate that loss of a co-twin by death in childhood or adulthood is associated with considerable mental morbidities among the surviving twins . Our recIn a population-based matched cohort, we included in the study all Swedish-born twins that lost a co-twin at birth, defined as a death of the co-twin within 60 days after birth, between 1973 and 2011- , together with two reference groups. The first reference group included 3935 sex-, birth year-, and gestational age (GA)-matched unexposed twins randomly selected from the Swedish twin population that did not experience such a loss, to control for twin pregnancy and twin birth. Because the exposed twins grew up on their own, as singletons, which differs from the social conditions of a twin life, we also included 3935 singletons randomly selected from the singleton population that were individually matched to the exposed twins on sex, birth year, and birth factors  as the second reference group. In addition to the population-based matched cohort, to address the concern of familial factors such as genetic background and environmental factors during childhood shared within a family , we compared 569 exposed twins to their full siblings (n = 880) in twin-sibling family cohort. The study entry was the 60th day after birth of the exposed twin . We then followed all individuals from the index date until the first diagnosis of any psychiatric disorder, emigration, death, or the end of 2013, whichever occurred first  of 12.08, 7.76, and 9.33 per 1000 person-years, respectively . CompareAlthough not statistically significant, we observed higher HRs for emotional disorders such as depression and anxiety, compared with neurodevelopmental disorders and other psychiatric disorders . SubgrouIn sensitivity analyses, we found no significant effect modification by the presence of psychiatric disorders among parents -Table 2,To our knowledge, this is the first nationwide population-based and sibling-matched cohort study exploring the association between loss of a co-twin at birth or shortly after birth and the subsequent risk of psychiatric disorders. Compared to matched unexposed twins or singletons, as well as their non-twin full siblings, twins exposed to a co-twin loss at birth were at considerably elevated risk of psychiatric disorders, especially emotional disorders, such as depression and anxiety, before age of 25. Notably, this association was independent of multiple important confounders, including birth characteristics and childhood social conditions (by comparing bereaved twins to singletons), and other familial factors (by comparing bereaved twins to their full siblings), indicating that increased clinical alertness of the mental health of surviving twins after very early co-twin loss is warranted. In addition, although the excess risk was not modified by parent’s psychiatric disorder nor the surviving twin’s congenital or other severe diseases diagnosed during follow-up, it seemed more pronounced among twins exposed to early loss of a same-sex co-twin and among twins without family history of psychiatric disorders.While accumulating evidence supports that both childhood and adult twin loss are associated with increased risk of psychiatric morbidity among the surviving twins , no prevThe major concern in studies of this kind is that, the death of a co-twin at birth may be an indicator of a poor pregnancy or birth conditions or congenital defects that are shared within a twin pair, and hence vulnerability of the surviving twin to various diseases  during their later lives. In present analysis, we indeed attempted to have a control for birth characteristics, yet we cannot rule out the possibility that both physical vulnerabilities of the surviving twins and their close contacts with health care due to these somatic problems have contributed to a detection of psychiatric disorders in this population. However, similar results were obtained in our sensitivity analyses where the presence of congenital abnormalities and the diagnosis of severe somatic conditions during follow-up were taken into account by subgroup analyses or by additional adjustments, suggesting a minor influence of these factors. Another possible explanation for the observed increased risk of psychiatric disorders among the surviving twins could be altered parenting of the grieving parents. Indeed, given that we also observed a heightened risk of psychiatric disorder in full siblings, especially younger full siblings, of the bereaved twins in our twin-sibling family analysis, altered parenting style among bereaved individuals and its impact on offspring’s mental health need further investigation. Our additional analyses taking into account clinically confirmed psychiatric disorders of the bereaved parents during the follow-up suggest limited mediating role of clinically confirmed parental psychiatric disorders in the association between early co-twin loss and risk of psychiatric disorder. Nevertheless, severe mental illness requiring a clinical diagnosis affects a relatively small proportion of the bereaved parents.The major merit of our study is the use of population-based cohort design, including 787 exposed twins with a complete follow-up of up to 41 years. In addition to a full consideration of birth factors, the possible influence of twin pregnancy and twin birth was controlled through the twin-twin comparison, while the social conditions during childhood was taken into consideration in the twin-singleton comparison. Through the twin-full sibling comparison, we were further able to control for potential familial confounders, as well as explore possible underlying mechanisms related to parenting. As the largest cohort study on co-twin loss at birth to date, we had sufficient statistical power to perform most of the planned subgroup analyses. Information bias was minimized since the registration and diagnosis of exposure and outcome was compiled prospectively and independently. Furthermore, the availability of rich information on sociodemographic and medical conditions for both the study participants and their parents enabled considerations of a wide range of important confounding factors.Limitations include the late establishment of outpatient care records in the Swedish National Patient Register (2001-), which may have rendered an underestimated number of psychiatric disorder diagnoses, especially the milder ones. In addition, individuals were relatively young at the end of follow-up . Therefore, the study focused mainly on early-onset psychiatric disorders. The association between loss of a co-twin at birth and psychiatric disorders beyond early adulthood needs to be addressed in further studies. Furthermore, because we identified individuals through the unique personal identification numbers assigned at birth to each Swedish-born person, our study involved merely twins who lost their co-twin at or shortly after delivery. The loss of a co-twin during the early pregnancy period  or due to stillbirth, which might also have psychological effects on the surviving twin , is therIn conclusion, in the Swedish population, exposure to death of a co-twin at birth was associated with a subsequently elevated risk of psychiatric disorders among the surviving twin. These findings call for medical and scientific attention of the mental health of this bereaved population and further exploration of the underlying mechanisms.Based on the Swedish Total Population Register, we identified all individuals born in Sweden between 1973 and 2011. Utilizing the personal identification numbers that are uniquely assigned to all Swedish residents, we linked the data to the Multi-Generation Register and identified all twin pairs , excluding multiple births) and singletons . We obtained prospective information about the study population through cross-linkage to the Medical Birth Register (MBR), the Causes of Death Register, and the National Patient Register (NPR). In order to enter the study, all included individuals had to have survived at least 60 days after birth and have available information in MBR, which has collected nationwide information on deliveries in Sweden since 1973.Because twins have considerably elevated mortality rate during the first and second months after birth and to maximize the sample size of the exposed twins in our study, we defined loss of a co-twin at birth as a death of the co-twin within 60 days after birth, according to information obtained from the Causes of Death Register, which is available electronically for register-based research since 1952. As shown in We included two reference groups. First, with the aim of controlling for twin pregnancy and twin birth, five unexposed twins (with a co-twin that survived at least 60 days after birth) per exposed twin were randomly selected from the twin population on the index date of the exposed twin . They were individually matched to the exposed twin by birth year, sex, and GA . Second, as the exposed twins grew up on their own, as singletons, in order to control for such social conditions, five singletons per exposed twin were also selected from the singleton population, on the index date of the exposed twin . Because we had a larger pool for selection of matched singletons, compared with selection of unexposed twins, we were able to use more matching variables, including sex, birth year, GA , birth weight for GA , and birth order , to ensure the comparability between twins and singletons with regard to birth characteristics.To address the concern about familial confounders, such as genetic background and environmental factors during childhood shared within a family, we further constructed a twin-sibling family cohort where non-twin full siblings of the exposed twins, if any, identified through the Multi-Generation Register, were included on the index date of the exposed twins or 60 days after their own birth, if born later than the twins .Follow-up of all study participants started from the index date and lasted until the occurrence of any or a specific type of psychiatric disorders, death, emigration, or the end of follow-up , whichever occurred first.Any first-ever inpatient or outpatient hospital visit with a psychiatric disorder as one of the registered diagnoses, primary or secondary, during the follow-up was identified from the NPR . For sub-analyses on the three categories of psychiatric disorders, including neurodevelopmental disorders , emotional disorders , and other psychiatric disorders, first-ever diagnosis of each specific group was also extracted from the NPR, according to corresponding ICD codes shown in Information on birth factors of the study participants, including GA, birth weight, and Apgar score, as well as maternal characteristics, including maternal age, educational level, and cohabitation status at childbirth, was extracted from the MBR. Specifically, to calculate the birth weight for gestational age percentiles, we generated birth weight z-score for GA, sex, and twin/singleton-specific populations and categorized it into <10th, 10–30th, 31–50th, 51–70th, 71–90th, and >90th percentile. We considered a poor neonatal condition as the presence of low Apgar score (≤7) at 5 or 10 min after birth. Family history of psychiatric disorders was defined as any diagnosis of or death due to psychiatric disorder or suicide among the first-degree relatives  of the study participants, according to the NPR or Causes of Death Register. In sensitivity analyses, to detect the potential role of declined mental health among the bereaved parents on the association of interest, we extracted diagnoses of psychiatric disorders among parents of study participants during follow-up from the NPR. Furthermore, to take into consideration the possible physical weakness of the surviving twins compared with their reference individuals, we collected data on the presence of congenital abnormalities and severe somatic diseases during follow-up from the NPR see -Table 1.We estimated the association between loss of a co-twin at birth and risk of psychiatric disorders using HRs with 95% CIs, derived from conditional Cox regression models where the attained age was applied as the underlying time scale.In the population-based matched cohort, exposed twins were compared to matched unexposed twins and matched singletons. In addition to the matching variables, the models were further adjusted for birth weight for GA , maternal age at childbirth , low Apgar score (≤7) at 5/10 min , maternal educational level at childbirth , maternal cohabitation status during pregnancy , and family history of psychiatric disorders (yes or no). In subgroup analyses, we calculated the HRs by sex , sex difference of the twin pair (same-sex or opposite-sex), and family history of psychiatric disorders (yes or no). We also subgrouped exposed twins by survival days of the deceased twins . In addition to considering all psychiatric disorders as one group, we did sub-analyses for three categories of psychiatric disorders, that is neurodevelopmental disorders, emotional disorders, and other psychiatric disorders. We further visualized the change of HR by attained age using flexible parametric models.Next, we repeated the main analyses in the twin-sibling family cohort. Cox models were stratified by family identifiers, and adjusted for birth year, sex, GA, as well as all covariables used in the population-based matched cohort. In addition to a comparison between the exposed twins and all their full siblings, we also compared the exposed twins with their older or younger full siblings, separately.In sensitivity analyses, we explored the effect of mental health of parents on the observed association by considering the occurrence of psychiatric disorders among parents diagnosed during the follow-up (yes or no) in a subgroup analysis. In addition, to alleviate concerns that the observed associations were mainly attributed to the suboptimal somatic conditions of the surviving twins than their matched individuals, we did subgroup analyses by, or additionally adjusted for, the presence of congenital abnormalities or the diagnosis of severe somatic conditions during follow-up. Lastly, to test the robustness of the observed association to the definition of ‘death at birth,’ we re-ran the analyses by using 28 days , instead of 60 days, after birth for the definition of loss of a co-twin at birth. All analyses were conducted in SAS statistical software, version 9.4  and STATA 15 (StataCorp LP). In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Song and colleagues estimate the risk of psychiatric disorders in the surviving twin following the death of a co-twin at birth. The study leverages the excellent Swedish registry system, it includes informative sample groups that are large in size and examines a considerable number of background characteristics that have possible associations with psychiatric symptoms and disorders in surviving twins. The major finding was an excess of psychiatric disorders in twins who co-twins died at a very early age, relative to twins from intact twin pairs and full siblings. Overall, the study is a valuable addition to the literature on the mental health of bereaved twins.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Maria H Chahrour as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Eduardo Franco as the Senior Editor.Thank you for submitting your article "Loss of a co-twin at birth and subsequent risk of psychiatric disorders" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:eLife earlier this year, reporting an increased risk of psychiatric disorders in twins who had lost a co-twin during childhood or adulthood. In the current study, the group analyzed data from a Swedish cohort of 787 twins who lost their co-twins at birth (defined within 60 days from birth). They compared the rate of psychiatric disorders in these twins to 3,935 matched unexposed twins, 3,935 matched unexposed singletons, and 880 full sibs of the exposed twins. They found that the exposed twins were at an increased risk of developing psychiatric disorders compared to unexposed twins with an HR=1.56. Compared to matched singletons, the HR was 1.41. The study leverages the excellent Swedish registry system, it includes informative sample groups that are large in size and examines a considerable number of background characteristics  that have possible associations with psychiatric symptoms and disorders in surviving twins. The major finding was an excess of psychiatric disorders in twins who co-twins died at a very early age, relative to twins from intact twin pairs and full siblings. Overall, the study is a valuable addition to the literature on the mental health of bereaved twins. The strength of the study is the population-based cohort design with a 41-year follow-up and the consideration of multiple confounders in their analyses. However, there are several issues that need to be addressed.In this manuscript, Song and colleagues estimate the risk of psychiatric disorders in the surviving twin following the death of a co-twin at birth. The same group had published a study in Essential revisions:1) It was not clear to me how the authors controlled for social conditions and familial factors? Especially the influence of bereaved parents on the mental health and well-being of the surviving twin. Since this is a measure variable, please elaborate on this in the Materials and methods.2) The authors state that: "Nevertheless, our further analyses by clinically confirmed psychiatric disorders of the bereaved parents during the follow-up found limited effect of this factor on the observed associations."The bereaved parents do not necessarily need to have a formally diagnosed psychiatric disorder in order to influence the mental health of the exposed surviving twin. The authors should acknowledge this in the Discussion and discuss the influence of bereaved parents on the mental health and well-being of the surviving twin as a confounding factor.3) The part of the Discussion which starts: "In addition to the genetic relatedness" and ends with "genetic background on the formation of such a twinship bond." is inappropriate and out of place. The whole section should be removed. It's based on speculations, not scientific data, and does not constitute a discussion of the results or data presented in this manuscript.4) Please clarify this sentence in the Discussion "Finally, although we made every effort to control for all relevant confounders, we cannot exclude the possibility that residual confounding."eLife). The authors have failed to convince the reviewers that this additional complementary analysis is innovative enough to contribute to a manuscript; what additional information does this paper bring to the literature? Also, the rationale leading to the research question in not well articulated, i.e., the knowledge gap leading to the research question  needs to be clearly stated.5) One of the main concerns of this study is the similarity of the current manuscript to a recently published work in the same journal  The authors stated that they want "to explore a potential rise in rate of psychiatric disorders among surviving twins after loss of a co-twin at birth". Ideally, when writing the aim of epidemiological analytical studies, one should state the direction of the association they want to test as well as to use verbs that somehow represent the proposed analytical strategy. For example, using verbs such as “explore” is vague and may give the sense of fishing, that is, looking for statistically significant results. This type of verbs is more appropriate for qualitative research. One way to rephrase this aim would be something along the lines of “to estimate the extent to which loss of a co-twin at birth is associated with rate of psychiatric disorders among surviving twins.”7) Another issue that needs to be addressed is the definition of exposed twins, which is those who lost a co-twin within 60 days after birth. This long timespan might bias the results; an exposed twin having lost their co-twin immediately at birth might be substantially different than an exposed twin having lost their co-twin 60 days after birth. This is because the time span of 60 days might allow emotional bonds to form between twins. The authors should justify their choice of 60 days after birth as a cut off. Similarly, exposure to a grieving parent during the first 60 years of life may have a confounding effect. Although this was accounted for by recruitment of the twins' full siblings as control group, exposure of full siblings to parental grief may not necessarily impose the same risk as in the surviving twins. This is because the siblings might be in different developmental stages with different needs and dependency levels. Moreover, the manuscript would benefit from an explanation on how missing data were managed.8) The authors could add the clinical implications of the study to the Discussion section to improve the strength of their arguments. For instance, this study can inform future interventions to support the surviving twins to alleviate the mental health impact of the loss of a co-twin.9) There is no mention of zygosity, a variable that is understandably missing from the sample except in the case of opposite-sex twins. It is possible that surviving MZ twins would feel the loss more strongly than surviving DZ twins, mirroring the relationships we see in the adult twin population. This issue should at least be addressed. Of course, twine would only know their zygosity if they learned it from a reliable medical record.10) There are relevant books and research papers that have not been referenced: Woodward, 2010; Woodward, 1988; Segal, 2019.11) There is a passage in the author's paper suggesting that twins bond in the womb;“It has indeed been proposed that twins actually begin their co-twin identity formation in the womb, so called “in-utero bond” (21). This notion gains support by the fact that twins share cellular origins and the womb environment during the fetal development period, and the interactive patterns of behaviors in womb have been observed and documented by researchers using ultrasound .”The presence in this passage in the present paper is unfortunate because it only supports a baseless and romantic notion. This must be rephrased or omitted. Many reared-apart twins have no awareness of being a twin. Many of these reared-apart twins are adoptees and their feelings of loneliness or emptiness are better explained by lack of resemblance between themselves and their family members-many non-twin adoptees also express such feelings.See this from a recent book, Twin Mythconceptions:Prenatal twins' interactive behaviors do not appear to be expressed with any intention or awareness of the other. Low oxygen tension in fetal blood, as well as pregnanolone and prostaglandin D2 that are provided by the placenta, keep the fetus sedated [19]. If prenatal cotwins' interactive activities influence the nature of their postnatal relationship, then identical twins should show more sustained coordinated behaviors in the womb than fraternal twins, but that is not the case. Research conducted in 2012 found no evidence that fetal dichorionic twins' body movements and rest-sleep cycles are coordinated, challenging some previous reports. It seems, instead, that any synchronized behaviors displayed by twins are infrequent, brief, and unintentional [20].12) In my experience, some parents report that young singleton twins seem to crave physical contact. Perhaps they miss the tactile sensations that the company of the other twin provides, but that is also highly speculative. It seems more likely that surviving twins' increased psychiatric disorders may be linked to parenting issues , even when twins are not told they had a twin, or to whatever physical factor was responsible for the demise of the co-twin. Finally, we sometimes hear stories from therapists and adults that current depression of some clients is linked to a lost twin, but there is no evidence that the person was a twin. I believe that, as scientists, the authors of this paper would be likely to dismiss such stories; to link depression with loss of a twin at birth or soon after is irresponsible on the part of therapists. At best we can say that more work in this area needs to be done. Essential revisions:1) It was not clear to me how the authors controlled for social conditions and familial factors? Especially the influence of bereaved parents on the mental health and well-being of the surviving twin. Since this is a measure variable, please elaborate on this in the Materials and methods.Thank you for your comments. We did not have a lot of information on social conditions and familial factors because of the register-based nature of the study. But we did consider several main characteristics, including maternal age, educational level, and cohabitation status at childbirth as well as birth order of the index child. Furthermore, the control of social conditions and familial factors was mainly achieved by contrasting the exposed twins to a reference group of individuals with reasonably comparable conditions on these factors. For instance, one distinct social condition of a twin life is that they grow up with the close company of their twin partner. This is however not the case for twins who lost their co-twin at birth—they grow up like singletons. Therefore, in addition to having a reference group of matched twins who didn’t experience a twin loss at birth  to control for twin pregnancy and twin birth, we also compared the exposed twins with a group of singletons matched by age, sex, and birth characteristics .Similarly, we did not have detailed information on various familial factors. However, the family-related conditions, as a whole, were taken into consideration through the comparison between exposed twins and their full siblings , since it’s reasonable to assume that, to a large extent, full siblings  should have been equally influenced by factors that cluster within a family, such as genetic background and environmental factors during childhood (including the parenting of the bereaved parents). To clarify this further, we highlighted the aims, as well as the controlled factors, in each planned comparison, in the revised manuscript.Results section:“The first reference group included 3,935 sex-, birth year-, and gestational age (GA)-matched unexposed twins randomly selected from the Swedish twin population that did not experience such a loss, to control for twin pregnancy and twin birth. Because the exposed twins grew up on their own, as singletons, which differs from the social conditions of a twin life, we also included 3,935 singletons randomly selected from the singleton population that were individually matched to the exposed twins on sex, birth year, and birth factors  as the second reference group. In addition to the population-based matched cohort, to address the concern of familial factors such as genetic background and environmental factors during childhood shared within a family , we compared 569 exposed twins to their full siblings (n=880) in twin-sibling family cohort.”Discussion section:“Notably, this association was independent of multiple important confounders, including birth characteristics, childhood social conditions (by comparing bereaved twins to singletons), and other familial factors (by comparing bereaved twins to their full siblings), indicating that increased clinical alertness of the mental health of surviving twins after a very early co-twin loss is warranted.”Materials and methods section:“We included two reference groups. First, with the aim of controlling for twin pregnancy and twin birth, five unexposed twins (with a co-twin that survived at least 60 days after birth) per exposed twin, were randomly selected from the twin population on the index date of the exposed twin . They were individually matched to the exposed twin by birth year, sex, and GA . Second, as the exposed twins grew up on their own, as singletons, in order to control for such social conditions, five singletons per exposed twin were also selected from the singleton population, on the index date of the exposed twin .”Materials and methods section:“To address the concern about familial confounders, such as genetic background and environmental factors during childhood shared within a family, we further constructed a twin-sibling family cohort where non-twin full siblings of the exposed twins….”2) The authors state that: "Nevertheless, our further analyses by clinically confirmed psychiatric disorders of the bereaved parents during the follow-up found limited effect of this factor on the observed associations."The bereaved parents do not necessarily need to have a formally diagnosed psychiatric disorder in order to influence the mental health of the exposed surviving twin. The authors should acknowledge this in the Discussion and discuss the influence of bereaved parents on the mental health and well-being of the surviving twin as a confounding factor.We agree with the reviewer that by identifying the clinical diagnosis of psychiatric disorders among bereaved parents alone, we cannot fully address the influence of altered parenting by the bereaved parents on the studied association, as the parents do not need to have mental problems to give unfavorable parenting.However, we consider the altered parenting to be more likely a mediator, rather than a confounder, in the studied association. Namely, it is possible that altered parenting style resulting from the bereavement may be one of the reasons why surviving twins suffer increased risk of psychiatric disorders after early co-twin loss. In the revised manuscript, we have now extended a discussion on this in the Discussion.Discussion section:“Another possible explanation for the observed increased risk of psychiatric disorders among the surviving twins could be altered parenting of the grieving parents. Indeed, given that we also observed a heightened risk of psychiatric disorder in full siblings, especially younger full siblings, of the bereaved twins in our twin-sibling family analysis, altered parenting style among bereaved individuals and its impact on offspring’s mental health needs further investigation. Our additional analyses taking into consideration clinically confirmed psychiatric disorders of the bereaved parents during the follow-up suggest limited mediating role of clinically diagnosed parental psychiatric disorders in the association between early co-twin loss and risk of psychiatric disorder. Nevertheless, severe mental illness requiring a clinical diagnosis affects a relatively small proportion of the bereaved parents.”3) The part of the Discussion which starts: "In addition to the genetic relatedness" and ends with "genetic background on the formation of such a twinship bond." is inappropriate and out of place. The whole section should be removed. It's based on speculations, not scientific data, and does not constitute a discussion of the results or data presented in this manuscript.Thank you for this important comment. We agree and have now removed these speculative statements.4) Please clarify this sentence in the Discussion "Finally, although we made every effort to control for all relevant confounders, we cannot exclude the possibility that residual confounding."We have clarified the sentence as below:Discussion section:“Finally, although we made every effort to control for important confounders such as birth characteristics, social and familial conditions, and shared genetic background, we cannot exclude the possibility of residual confounding.”5) One of the main concerns of this study is the similarity of the current manuscript to a recently published work in the same journal . The authors have failed to convince the reviewers that this additional complementary analysis is innovative enough to contribute to a manuscript; what additional information does this paper bring to the literature? Also, the rationale leading to the research question in not well articulated, i.e., the knowledge gap leading to the research question  needs to be clearly stated.Thank you for your comments. We believe that the present study adds important new knowledge to our previous work. Below are our two main arguments.First, although bereavement due to a co-twin loss and its impact on risk of psychiatric disorders has been reported among adult twins, including our own work (as the reviewer pointed out), there is a clear lack of knowledge concerning the potential impact of exposure to a co-twin death at birth (before any memory of the deceased twin can be consolidated) on the risk of psychiatric disorders among the surviving twins.Second, the absence of such knowledge is mainly due to the complexity of the research question and scarcity of rigorous, informative data. In addition to the unique co-twin loss experience at birth, the surviving twins may be exposed to many other factors, such as the possible genetic vulnerability to diseases, suboptimal birth characteristics (due to the twin birth), a singleton-like life, and living in a family with experience of bereavement, etc. which might all influence their risk of future psychiatric disorders. Therefore, with the Swedish nationwide data containing more than 800 exposed twins and detailed information on birth characteristics and family structure, we have a unique opportunity to address this question.eLife paper.As most of co-twin loss occur at an older age (median age at loss=59 year), the majority of the study population in our previous study was born before 1973 and therefore had no detailed information from the Medical Birth Register. We therefore could not appropriately address and discuss such a question in our last In the revised manuscript, we have now emphasized the motivation and novelty of the present study, in both the Introduction and Discussion.Introduction section:“In the total absence of data on the rate of psychiatric disorders among twins who lost a co-twin at birth, we conducted a nationwide population- and sibling-matched cohort study to estimate the extent to which loss of a co-twin at birth is associated with the incidence of psychiatric disorders among surviving twins, after carefully controlling for important confounders such as birth characteristics and familial factors.”Discussion section:“Notably, this association was independent of multiple important confounders, including birth characteristics, childhood social conditions (by comparing bereaved twins to singletons), and other familial factors (by comparing bereaved twins to their full siblings), indicating that increased clinical alertness of the mental health of surviving twins after a very early co-twin loss is warranted.”Discussion section:“While accumulating evidence supports that both childhood and adult twin loss are associated with increased risk of psychiatric morbidity among the surviving twins , no previous study has addressed whether such emotional reactions can be observed after a very early co-twin loss where limited twin relationship, perception or memory from the loss could be expected. The absence of evidence is mainly due to the complexity of the research question and lack of high-quality data to address potential confounding by multiple factors, such as twin pregnancy and birth  but a singleton-like life, familial factors, and genetic susceptibility to diseases. Therefore, with the unique Swedish nationwide data sources, which provide a substantial sample size of exposed twins with detailed data on birth characteristics and familial information, we conducted the present study. By contrasting the rate of psychiatric disorders among surviving twins who were exposed to a co-twin loss at birth with that of several comparison groups, including matched unexposed twins and singletons as well as the full siblings of the exposed twins, our assessment demonstrates a robust association between early loss of a co-twin and subsequent risk of psychiatric disorders.”6) The authors stated that they want "to explore a potential rise in rate of psychiatric disorders among surviving twins after loss of a co-twin at birth". Ideally, when writing the aim of epidemiological analytical studies, one should state the direction of the association they want to test as well as to use verbs that somehow represent the proposed analytical strategy. For example, using verbs such as “explore” is vague and may give the sense of fishing, that is, looking for statistically significant results. This type of verbs is more appropriate for qualitative research. One way to rephrase this aim would be something along the lines of “to estimate the extent to which loss of a co-twin at birth is associated with rate of psychiatric disorders among surviving twins.”Thank you for the comment. The request changes have been made. Please see the new statement below:Introduction section:“In the total absence of data on the rate of psychiatric disorders among twins who lost a co-twin at birth, we conducted a nationwide population- and sibling-matched cohort study to estimate the extent to which loss of a co-twin at birth is associated with the incidence of psychiatric disorders among surviving twins, after carefully controlling for important confounders such as birth characteristics and familial factors.”7) Another issue that needs to be addressed is the definition of exposed twins, which is those who lost a co-twin within 60 days after birth. This long timespan might bias the results; an exposed twin having lost their co-twin immediately at birth might be substantially different than an exposed twin having lost their co-twin 60 days after birth. This is because the time span of 60 days might allow emotional bonds to form between twins. The authors should justify their choice of 60 days after birth as a cut off. Similarly, exposure to a grieving parent during the first 60 years of life may have a confounding effect. Although this was accounted for by recruitment of the twins' full siblings as control group, exposure of full siblings to parental grief may not necessarily impose the same risk as in the surviving twins. This is because the siblings might be in different developmental stages with different needs and dependency levels. Moreover, the manuscript would benefit from an explanation on how missing data were managed.Thank you for this important comment. We agree with the reviewer that the choice of “60 days after birth” needs justifications. Neonatal death is usually defined within 28 days after birth. We used slightly longer time window in the study, because: 1) there was a significantly higher mortality during 28-60 days after birth, compared to thereafter, among twins  and 2) by extending with 28 days to 60 days after birth, we increased the sample size of the exposed twins by 10%. We have now clarified this in the revised manuscript:Materials and methods section:“Because twins have considerably elevated mortality rate during the first and second months after birth and to maximize the sample size of the exposed twins in our study, we defined loss of a co-twin at birth as a death of the co-twin within 60 days after birth, according to information obtained from the Causes of Death Register, which is available electronically for register-based research since 1952.”In addition, to test the impact of different exposure windows on our estimates, we performed subgroup analysis by survival days of the deceased twin  The authors could add the clinical implications of the study to the Discussion section to improve the strength of their arguments. For instance, this study can inform future interventions to support the surviving twins to alleviate the mental health impact of the loss of a co-twin.Thank you for this important comment. The request changes have been made.Discussion section:“Notably, this association was independent of multiple important confounders, including birth characteristics, childhood social conditions (by comparing bereaved twins to singletons), and other familial factors (by comparing bereaved twins to their full siblings), indicating that increased clinical alertness of the mental health of surviving twins after a very early co-twin loss is warranted.”9) There is no mention of zygosity, a variable that is understandably missing from the sample except in the case of opposite-sex twins. It is possible that surviving MZ twins would feel the loss more strongly than surviving DZ twins, mirroring the relationships we see in the adult twin population. This issue should at least be addressed. Of course, twine would only know their zygosity if they learned it from a reliable medical record.As the reviewer correctly guessed, information on zygosity is not available for all twins apart from the opposite-sex twins. To clarify this, we have added a statement in the Discussion part of the revised manuscript.Discussion section:“Particularly, despite of the lack of information on zygosity, the higher relative risk observed after early loss of a same-sex co-twin, compared with a loss of opposite-sex co-twin, may indicate the importance of shared genetic background on the formation of such a twinship bond. This is similar to the greater grief intensity reported among monozygotic twins who experienced an adult loss of co-twin compared with dizygotic twins, and consistent with the evolutionary theory suggesting a role of genetic relatedness in the bereavement process .”10) There are relevant books and research papers that have not been referenced: Woodward, 2010; Woodward, 1988; Segal, 2019.Thank you for the information. We have added these references to the revised manuscript.Introduction section:“Previous studies indicate that loss of a co-twin by death in childhood or adulthood is associated with considerable mental morbidities among the surviving twins .”Introduction section:“However, several scientific and media accounts describe unexpected lingering sorrow among twins who lost their co-twin at or shortly after birth, even among the twins that didn’t know they were born as twins .”Discussion section:“This is similar to the greater grief intensity reported among monozygotic twins who experienced an adult loss of co-twin, compared with dizygotic twins, and consistent with the evolutionary theory suggesting a role of genetic relatedness in the bereavement process .”11) There is a passage in the author's paper suggesting that twins bond in the womb;“It has indeed been proposed that twins actually begin their co-twin identity formation in the womb, so called “in-utero bond” (21). This notion gains support by the fact that twins share cellular origins and the womb environment during the fetal development period, and the interactive patterns of behaviors in womb have been observed and documented by researchers using ultrasound .”The presence in this passage in the present paper is unfortunate because it only supports a baseless and romantic notion. This must be rephrased or omitted. Many reared-apart twins have no awareness of being a twin. Many of these reared-apart twins are adoptees and their feelings of loneliness or emptiness are better explained by lack of resemblance between themselves and their family members-many non-twin adoptees also express such feelings.See this from a recent book, Twin Mythconceptions:Prenatal twins' interactive behaviors do not appear to be expressed with any intention or awareness of the other. Low oxygen tension in fetal blood, as well as pregnanolone and prostaglandin D2 that are provided by the placenta, keep the fetus sedated [19]. If prenatal cotwins' interactive activities influence the nature of their postnatal relationship, then identical twins should show more sustained coordinated behaviors in the womb than fraternal twins, but that is not the case. Research conducted in 2012 found no evidence that fetal dichorionic twins' body movements and rest-sleep cycles are coordinated, challenging some previous reports. It seems, instead, that any synchronized behaviors displayed by twins are infrequent, brief, and unintentional [20].Thank you for pointing out this important issue. In the revised manuscript, we have tried to avoid the speculation of “in-utero bond”, in line with the comments from the reviewers and editors, by removing all related statements and references.12) In my experience, some parents report that young singleton twins seem to crave physical contact. Perhaps they miss the tactile sensations that the company of the other twin provides, but that is also highly speculative. It seems more likely that surviving twins' increased psychiatric disorders may be linked to parenting issues , even when twins are not told they had a twin, or to whatever physical factor was responsible for the demise of the co-twin. Finally, we sometimes hear stories from therapists and adults that current depression of some clients is linked to a lost twin, but there is no evidence that the person was a twin. I believe that, as scientists, the authors of this paper would be likely to dismiss such stories; to link depression with loss of a twin at birth or soon after is irresponsible on the part of therapists. At best we can say that more work in this area needs to be done.Thank you for sharing these experiences and we agree. In the revised manuscript, we emphasize now the currently limited evidence for the link between co-twin loss at birth and future psychiatric disorders and its underlying mechanisms and call for further research in this field.Discussion section:“Given the scarcity of existing data within this area of research, our findings call for further investigation on the possible underlying mechanisms linking the experience of a co-twin loss at birth to mental health decline during adulthood.”Discussion section:“These findings call for medical and scientific attention of the mental health of this bereaved population and further exploration of the underlying mechanisms.”* Cox regression models were stratified by matching identifiers , and adjusted for birth weight for gestational age, maternal age at childbirth, low Apgar score (≤7) at 5/10 min, maternal educational level at childbirth, maternal cohabitation status during pregnancy, and family history of psychiatric disorders.† Cox regression models were stratified by matching identifiers , and adjusted for maternal age at childbirth, low Apgar score (≤7) at 5/10 min, maternal educational level at childbirth, maternal cohabitation status during pregnancy, and family history of psychiatric disorders.£ Cox regression models were stratified by family identifiers, and adjusted for sex, birth year, gestational age, birth weight for gestational age, low Apgar score (≤7) at 5/10 min, maternal educational level at childbirth, and maternal cohabitation status during pregnancy.