Embryonic development through in vitro fertilization using high-quality bovine sperm separated in a biomimetic cervix environment.

This study is the first to identify bovine blastocysts through in vitro fertilization (IVF) of matured oocytes with a large quantity of high-quality sperm separated from a biomimetic cervix environment. We obtained high-quality sperm in large quantities using an IVF sperm sorting chip (SSC), which could mimic the viscous environment of the bovine cervix during ovulation and facilitates isolation of progressively motile sperm from semen. The viscous environment-on-a-chip was realized by formulating and implementing polyvinylpyrrolidone (PVP)-based solutions for the SSC medium. Sperm separated from the IVF-SSC containing PVP 1.5% showed high motility, normal morphology and high DNA integrity. As a result of IVF, a higher rate of hatching blastocysts, which is the pre-implantation stage, were observed, compared to the conventional swim-up method. Our results may significantly contribute to improving livestock with superior male and female genetic traits, thus overcoming the limitation of artificial insemination based on the superior genetic traits of existing males.

Risk of pancreatic cancer after acute pancreatitis: A population-based matched cohort study.

BACKGROUND: We investigated the short- and long-term risks of pancreatic cancer after the diagnosis of acute pancreatitis. METHODS: This population-based matched-cohort study used data from the Korean National Health Insurance Service database. Patients with acute pancreatitis (n = 25,488) were matched with the control group (n = 127,440) based on age, sex, body mass index, smoking status, and diabetes. We estimated the hazard ratios for developing pancreatic cancer in both groups using Cox regression analysis. RESULTS: During a median follow-up of 5.4 years, pancreatic cancer developed in 479 patients (1.9%) in the acute pancreatitis group and 317 patients (0.2%) in the control group. Compared with the control group, the risk of pancreatic cancer in the acute pancreatitis group was very high within the first 2 years, which gradually decreased over time. The hazard ratio for the risk of developing pancreatitis was 8.46 (95% confidence interval, 5.57-12.84) at 1-2 years, and then decreased to 3.62 (95% confidence interval, 2.26-4.91) at 2-4 years. However, even after 8-10 years, the hazard ratio was still statistically significantly increased to 2.80 (95% confidence interval, 1.42-5.53). After 10 years, there was no significant difference in the risk of pancreatic cancer between the two groups. CONCLUSIONS: The risk of pancreatic cancer increases rapidly after acute pancreatitis diagnosis, gradually declines after 2 years, and remains elevated for up to 10 years. Further studies are needed to determine the long-term effects of acute pancreatitis on the risk of pancreatic cancer.

Impact of COVID-19 on ophthalmic medical access during successive waves: Demographics, disease factors, and wave locations.

BACKGROUND: The coronavirus disease (COVID-19) pandemic significantly impacted medical care, including ophthalmology. This study aimed to identify factors associated with reduced ophthalmic medical access during the pandemic. METHODS: This nationwide population-based cohort study analysed South Korean health insurance claims data from January 2019 to November 2021. Outpatient visits and surgeries for age-related macular degeneration, cataract, diabetic retinopathy, glaucoma, and retinal detachment during the two pandemic waves were compared with those in the non-pandemic period. Poisson regression was used to estimate incidence rate ratios and changes in outpatient visits and surgeries between waves concerning patient age, sex, residential location, and health insurance type. RESULTS: Outpatient visits for five eye diseases decreased in the first wave (0.733-0.985, P < 0.001). In the second wave, only outpatient visits for age-related macular degeneration, cataract, and glaucoma decreased (0.754, 0.878, and 0.874, respectively, all P < 0.001). Age-related macular degeneration, cataract, and glaucoma surgeries were significantly reduced in the first wave (0.829, P < 0.001; 0.836, P < 0.001; 0.904, P = 0.030, respectively). Age-related macular degeneration (0.852) and cataract (0.716) surgeries dropped in the second wave. Women and elderly (>65 years) patients curtailed outpatient visits and surgeries more throughout the pandemic and were less resilient during the second wave. Wave location was also related to outpatient visits and surgeries. CONCLUSIONS: The COVID-19 pandemic decreased outpatient visits and surgeries for eye diseases, with more significant impact on women, older patients, and those residing near wave locations. These findings can inform healthcare policies to minimise future pandemic impacts on healthcare delivery.

Lifestyle, body mass index, diabetes, and the risk of pancreatic cancer in a nationwide population-based cohort study with 7.4 million Korean subjects.

BACKGROUND: Large-scale epidemiological studies on pancreatic cancer in non-Western populations are insufficient. We investigated the risk factors for pancreatic cancer. METHODS: Using the Korean National Health Insurance database, subjects who participated in the health examination program between 2005 and 2006 were identified and followed up until 2017. Adjusted hazard ratios (HRs) for pancreatic cancer risk were estimated using a Cox proportional hazards model. RESULTS: During 11.5 years follow-up, 22,543 of 7,445,947 participants were newly diagnosed with pancreatic cancer. Compared with normal-weight subjects, pancreatic cancer risk was increased in those with severe obesity (BMI ≥ 28 kg/m2) (HR = 1.16; 95% CI, 1.11-1.23). Subjects with diabetes had an increased risk compared with those without diabetes (HR = 1.48; 95% CI, 1.43-1.53). Current smokers had a higher risk than never smokers (HR = 1.43; 95% CI, 1.38-1.48). Current smoking combined with diabetes increased the risk compared with never smokers without diabetes (HR = 2.13; 95% CI, 2.00-2.28). Current smoking combined with BMI ≥ 25 kg/m2 had an increased risk compared with never smokers with BMI < 23 kg/m2 (HR = 1.55; 95% CI, 1.46-1.65). CONCLUSION: Smoking, obesity, and diabetes are significant risk factors for pancreatic cancer in Koreans. Lifestyle modifications for smoking and obesity would be beneficial for pancreatic cancer prevention.

A simple and efficient cryopreservation method for mouse small intestinal and colon organoids for regenerative medicine.

Organoid cryopreservation method is one of key step in the organoid culture. We aimed to establish a simple and efficient cryopreservation method for mouse small intestinal organoids (MIOs) and colon organoids (MCOs) using various concentrations of cryoprotectant. Based on the theoretical simulation, we optimized the dimethyl sulfoxide (DMSO) concentration by pretreating the organoids with 5, 7.5, and 10% DMSO for 30 min at 4 °C to allow penetration into the organoids and evaluated their viability, proliferation, and function after cryopreservation. Gene expression in the MIOs and staining of lineage markers were examined real-time PCR. The organoids in the DMSO-treated groups as well as the control, expressed ChrgA, Ecad, Muc2, Lyz, villin, and Lgr5, and there are no significant. A forskolin-induced swelling assay for MIOs was performed to confirm normal cystic fibrosis transmembrane conductance regulator (CFTR) activity. Similar forskolin-induced swelling was observed in the DMSO-treated groups and the control. In addition, MCOs were transplanted into mouse colon for confirmation of regeneration therapy efficacy. Thawing organoids were cultured for two and four sequential passages after cryopreservation with 5% DMSO to confirm any changes in the gene expression of lineage markers after subculture. We developed a simple and efficient organoid freezing method using 5% DMSO with low potential toxicity and validated our findings with theoretical simulation.

The effect of tonsillectomy with adenoidectomy on medical services used in association with otitis media based on Korean national sample cohort data.

BACKGROUND: The effect of tonsillectomy with adenoidectomy (T&A) on otitis media has been investigated, but there have been no reports of the relationship between T&A and medical services used in association with otitis media. We investigated the effect of T&A on otitis media with regard to the number and cost of medical services used. METHODS: From the National Health Insurance Service National Sample Cohort data in Korea, we selected patients 7 years old or younger in 2002 who had T&A in 2005 while between the ages of three and ten. A control group was established matching the patient group with similar propensities of demographic characteristics. The number and cost of medical services used in association with otitis media were analyzed for 3 years before T&A through 8 years after T&A. RESULTS: The total number of patients was 1,338, with 227 in the T&A group and 1,111 in the non-T&A group. The number of medical services used was not significantly different between the T&A and non-T&A groups before and after surgery. The cost of medical services used was significantly higher in the T&A group than in the non-T&A group one year before surgery. The cost of medical services used was not significantly different between the two groups after surgery. CONCLUSIONS: There were no significant differences between the T&A and non-T&A groups in the number and cost of medical services used in association with otitis media after surgery.

Application of an S-layer protein as a self-aggregating tag for cost-effective separation of recombinant human and yeast D-amino acid oxidases in the aqueous two-phase system.

OBJECTIVE: To evaluate whether the surface layer (S-layer) protein of Lactobacillus brevis serves as a self-aggregating protein tag for cost-effective separation of human and yeast D-amino acid oxidases (hDAAO and yDAAO) expressed in E. coli. RESULTS: In aqueous two-phase (PEG-phosphate) system, the S-layer:DAAO fusion proteins (shDAAO and syDAAO) were separated at the interface with a recovery of 82 ± 10.6% for shDAAO and 95 ± 1.9% for syDAAO. Some shDAAO proteins were separated as precipitates with a recovery of 41 ± 0.5% in phosphate (9%, w/w) using PEG 3000 and PEG 4000 (16%, w/w), while some syDAAO proteins were also isolated as precipitates with a recovery of 75 ± 17.5% in phosphate (9%, w/w) using PEG 4000 and PEG 8000 (16%, w/w). CONCLUSIONS: The S-layer of L. brevis was applied to a self-assembled protein tag to enable cost-effective separation of human and yeast D-amino acid oxidases expressed in E. coli cells. Because of the self-assembling properties of S-layer proteins, human and yeast D-amino acid oxidases fused with S-layer proteins could be easily separated by aggregates at the interface and/or in a few conditions by precipitates to the bottom of the PEG-phosphate aqueous system.

Effect of periodontitis on the development of osteoporosis: results from a nationwide population-based cohort study (2003-2013).

BACKGROUND: The prevalence of osteoporosis associated with the aging process is anticipated to increase along with the rising aging population. Periodontitis that the most common chronic infections of humankind is considered the risk factor for osteoporosis. The aim of this study was to identify the association between osteoporosis and periodontitis using a population-based cohort. METHODS: The case group was defined as patients diagnosed with periodontitis and treated with subgingival curettage, root conditioning, periodontal flap operation, bone grafting for alveolar bone defects, and guided tissue regeneration. Case and control groups matched for gender, age, household income, type of social security, disability, and residential area were generated. A Cox proportional hazard model was constructed to examine the difference in the development of osteoporosis between the case and control groups. The final sample included 13,464 participants. RESULTS: The incidence of osteoporosis was 1.1% in males and 15.8% in females during a 10-year period. The risk factors for osteoporosis in males were increasing age and Charlson Comorbidity Index score. Periodontitis was not associated with the development of osteoporosis in males. The risk factors for osteoporosis in females were increasing age, body mass index, Charlson Comorbidity Index score, diabetes, and periodontitis. Women with periodontitis were more likely to also develop osteoporosis (HR: 1.22, 95% CI: 1.01-1.48). CONCLUSIONS: Periodontitis has an effect on the development of osteoporosis in females. Managing good teeth is required for the prevention and delay of osteoporosis. This includes dental examinations, regular cleanings and gum treatment.

The Impact of In-Home Services Utilization on the Institutionalization of Older People in Korea.

As a result of aging populations, institutionalization of older people is creating an increasing financial burden in many countries. The purpose of the present study was to explore the impact of in-home service utilization on institutionalization. The subjects were newly certified as eligible for long-term care insurance between January and February 2009 in Korea. The follow-up period was 40 months, to April 2012. We used logistic regression models to identify factors influencing the transition to institutional service, adjusting for gender, age, living status, income level, activities of daily living, and chronic disease. The institutionalization rate was estimated to be 17.3% over 40 months. The interval from transitional living to institutionalization was 36 ± 8 months. Risk factors of transition to institutional services are being female, having advanced age, living with friends or cousins, getting dementia, and not using in-home services. We determined that institutionalization is mitigated by use of in-home services. Therefore, supplying appropriate in-home services to current nonusers would help to greatly reduce the rate of institutionalization of older people.

National dental policies and socio-demographic factors affecting changes in the incidence of periodontal treatments in Korean: A nationwide population-based retrospective cohort study from 2002-2013.

BACKGROUND: Dental insurance coverage has recently expanded in Korea. The number of patients diagnosed with periodontal disease (PD), and the actual number of periodontally compromised patients has increased. However, few studies have investigated the relationships between the prevalence of periodontal disease and the incidence of PD treatment, dental insurance policies, and socio-demographic factors. To determine the incidence of periodontal treatments required, the comprehensive longitudinal data of the National Health Insurance Service were used. This study evaluated changes in the incidence of periodontal treatments, using data from the Korean National Health Insurance Cohort Database. METHODS: A random stratified sample of 1,025,340 Korean patients was selected from National Health Insurance database, using 1,476 multistage samplings (of sex, age, and income level) for 12 years from 2002 to 2013. Chi-square analysis, and univariate, and multivariate logistic regression were used to evaluate the association of socio-demographic factors with the prevalence of PD and the incidence of periodontal treatment. RESULTS: The incidence of periodontal treatment steadily and significantly increased, in both male and female participants, from 2002 to 2013. The increase was associated with socio-demographic factors and changes in national dental insurance policies. The incidence of periodontal treatment evaluated by age is influenced by the changes in national dental policies. These results suggest that the increase in patients diagnosed with PD reflects changes in dental policies and insurance benefits. CONCLUSIONS: This study confirms that national dental policies and socio-demographic factors are related to the incidence of periodontal treatments. The incidence of periodontal treatment is significantly related to the expansion of insurance coverage in South Korea.

Alginate Hydrogel Microencapsulation Inhibits Devitrification and Enables Large-Volume Low-CPA Cell Vitrification.

Cryopreservation of stem cells is important to meet their ever-increasing demand by the burgeoning cell-based medicine. The conventional slow freezing for stem cell cryopreservation suffers from inevitable cell injury associated with ice formation and the vitrification (i.e., no visible ice formation) approach is emerging as a new strategy for cell cryopreservation. A major challenge to cell vitrification is intracellular ice formation (IIF, a lethal event to cells) induced by devitrification (i.e., formation of visible ice in previously vitrified solution) during warming the vitrified cells at cryogenic temperature back to super-zero temperatures. Consequently, high and toxic concentrations of penetrating cryoprotectants (i.e., high CPAs, up to ~8 M) and/or limited sample volumes (up to ~2.5 µl) have been used to minimize IIF during vitrification. We reveal that alginate hydrogel microencapsulation can effectively inhibit devitrification during warming. Our data show that if ice formation were minimized during cooling, IIF is negligible in alginate hydrogel-microencapsulated cells during the entire cooling and warming procedure of vitrification. This enables vitrification of pluripotent and multipotent stem cells with up to ~4 times lower concentration of penetrating CPAs (up to 2 M, low CPA) in up to ~100 times larger sample volume (up to ~250 µl, large volume).

Improved low-CPA vitrification of mouse oocytes using quartz microcapillary.

Cryopreservation by low-cryoprotectant (CPA) vitrification has the potential to combine all the advantages of the conventional high-CPA vitrification and slow-freezing approaches while avoiding their drawbacks. However, current low-CPA vitrification protocol for cryopreservation of oocytes requires a lengthy and multi-step procedure for unloading CPAs. In this study, we report a much-simplified procedure of using quartz microcapillary (QMC) for low-CPA vitrification of mouse oocytes with only one step for unloading CPAs. The immediate viability of oocytes after the improved low-CPA vitrification was determined to be more than 90%. Moreover, no significant difference was observed in terms of embryonic development from the two-cell to blastocyst stages between the fresh and vitrified oocytes after in vitro fertilization (IVF). This improved low-CPA vitrification technology has the potential for efficient cryopreservation of oocytes to preserve the fertility of mammals including humans for assisted reproductive medicine, maintenance of animal resource and endangered species, and livestock management.

The crucial role of zona pellucida in cryopreservation of oocytes by vitrification.

Mammalian oocytes have a proteinaceous hydrogel-like outer shell known as the zona pellucida (ZP) that semi-encloses their plasma membrane and cytoplasm. In this study, we cryopreserved mouse oocytes either with or without ZP by vitrification. Our results show that the presence of an intact ZP could significantly improve the post-vitrification survival of oocytes to 92.1% from 13.3% for oocytes without ZP. Moreover, there was no significant difference in embryonic development between fresh and cryopreserved oocytes with ZP after in vitro fertilization (IVF). Further atomic force microscopy (AFM) analysis showed that the intact oocytes with ZP have an elastic modulus that is more than 85 times higher than that of oocytes without ZP. This may partially explain the important role of ZP in protecting the oocytes by resisting the mechanical stress due to possible ice formation during cryopreservation by vitrification. Collectively, this study reveals a new biophysical role of ZP during vitrification of oocytes and suggests microencapsulation of the many mammalian cells without a ZP in ZP-like hydrogel is an effective strategy to improve their survival post cryopreservation by vitrification.

Ovotestis Isolation and Cryopreservation of Nesiohelix samarangae (Oriental Snail) as a Snail Model for Conserving Other Endangered Snail Species.

This study aimed to develop a cryopreservation system for the reproductive organs of Nesiohelix samarangae (oriental snail) to support the conservation of their species. The reproductive glands of N. samarangae are divided into numerous acini by acinar boundaries. Within each acinus, the presence of spermatogonia, spermatocytes, spermatids, and sperm were observed, indicating various stages of sperm development. The spermatocytes were irregular in shape and possessed large nuclei. Spermatids, on the other hand, were predominantly located within the lumen of the tissue and exhibited densely packed nuclei. Furthermore, sperm with tails attached were observed within the tissue. In order to preserve the oriental snail species, we utilized the vitrification method to freeze the reproductive organs. Comparing the two methods, it was observed that cryopreservation of ovotestis using 2% alginate encapsulation exhibited superior viability following thawing, surpassing the viability achieved with the non-encapsulated approach. In this study, the establishment of a cryopreservation system for the reproductive organs of the oriental snail not only contributes to the genetic conservation of the endangered snail species but also plays a role in maintaining genetic resources and diversity.

Highly sensitive and selective spectroscopic detection of mercury(II) in water by using pyridylporphyrin-DNA conjugates.

Single-labeled pyridylporphyrin-DNA conjugates are reported as highly sensitive and selective spectroscopic sensors for mercury(II) ions in water. The effects of chemical structure (thymine versus adenine), number of nucleotides (monomer versus octamer), and porphyrin metalation (Zn versus free base) on the sensitivity and selectivity of mercury(II) detection are explored. The results indicated that pyridylporphyrin rather than the nucleobase plays a crucial role in mercury(II) sensing, because porphyrin conjugates with both adenosine and thymidine exhibited excellent mercury(II) detection. Mercury(II) recognition was shown in emission quenching, as well as in a redshift of the porphyrin Soret band absorption. The limit of detection (LOD, 3σ/slope) of zinc(II) pyridylporphyrin-5'-oligodeoxythymidine (ZnPorT8) obtained by fluorescence quenching was calculated to be 21.14 nM. Other metal cations (Zn(2+), Cd(2+), Pb(2+), Mn(2+), Ca(2+), Ni(2+), Mg(2+), Fe(2+), Cu(2+), and Na(+)) did not interfere with the emission and absorption sensing of mercury(II). Free-base porphyrin-oligothymine conjugate 2HPorT8 displayed similar sensitivity to ZnPorT8 but different selectivity. The results also implied that the sensing properties of porphyrin-deoxythymidine conjugates could potentially be tuned by porphyrin metalation.

Control of the growth and development of murine preantral follicles in a biomimetic ovary using a decellularized porcine scaffold.

This study aimed to derive mature oocytes from murine preantral follicles cultured in a biomimetic ovary with a porcine scaffold using decellularization technology. We evaluated the DNA content and the presence of cell and extracellular matrix (ECM) components, including collagen, elastin, and glycosaminoglycans (GAGs), in decellularized (decell) porcine ovaries. The DNA content inthe decell ovarian tissues was approximately 94 % less than that in native tissues (66 ± 9.8 ng/mg vs. 1139 ± 269 ng/mg). Furthermore, the ECM component integrity was maintained in the decell ovarian tissue. The soluble collagen concentration of native ovarian tissue (native) was 195.34 ± 15.13 µg/mg (dry wt.), which was less than 878.6 ± 8.24 µg/mg for the decell ovarian tissue due to the loss of cellular mass. Hydrogels derived from decell porcine ovaries were prepared to develop an in vitro biomimetic ovary with appropriate ECM concentration (2-6 mg/mL). Scanning electron microscope (SEM) imagining revealed that the complex fiber network and porous structure were maintained in all groups treated with varying ECM concentration (2-6 mg/mL). Furthermore, rheometer analysis indicated that mechanical strength increased with ECM concentration in a dose-dependently. The preantral follicles cultured with 4 mg/mL ECM showed high rates of antral follicle (66 %) and mature oocyte (metaphase II) development (47 %). The preantral follicles cultured in a biomimetic ovary with a decell porcine scaffold showed a higher rate of antral follicle and mature oocytes than those cultured in other biomaterials such as collagen and Matrigel. In mature oocytes derived from antral follicles, meiotic spindles and nuclei were stained using a tubulin antibody and Hoechst, respectively. Two-cell embryos were developed from MII oocytes following parthenogenetic activation. Preantral follicles were cultured in a biomimetic ovary derived from the ECM of a decell porcine ovary, and embryos were generated from MII oocytes. This biomimetic ovary could contribute to restoring fertility in infertile women with reduced ovarian function, benefit mating efforts for endangered species, and maintain animals with valuable genetic traits.

Trends in treatment patterns and survival outcomes in pancreatic cancer: A nationwide population-based study in Korea.

BACKGROUND: The treatment of pancreatic cancer has evolved; however, real-world data on treatment trends remain scarce. This study investigated treatment patterns for pancreatic cancer and patient survival using a nationwide population-based study in Korea. METHODS: Using the National Health Insurance database, data from 78,920 patients diagnosed with pancreatic cancer in Korea between 2006 and 2019 were extracted. Treatment patterns and survival by age group and year of diagnosis were examined. RESULTS: Primary treatment was delivered as follows: 16,562 patients (21.0%) underwent surgery, 20,998 patients (26.6%) received chemotherapy, 1332 patients (1.7%) received chemoradiotherapy, and 40,040 patients (50.7%) received supportive care only. The proportion of patients undergoing surgery or chemotherapy increased gradually in all age groups over time. The commonly used anti-cancer drugs have changed from gemcitabine±erlotinib to gemcitabine+nab-paclitaxel and FOLFIRINOX. Survival improved mainly in patients who underwent surgery or chemotherapy. Median overall survival for all patients improved from 5.5 months in 2006-2008 to 9.8 months in 2018-2019, with greater improvement observed in younger age groups: 8.8-18.8 months, age ≤59 years, 6.8-14.6 months, age 60-69 years, 4.2-8.3 months, age 70-79 years and 2.4-3.4 months, age ≥80 years (all p < 0.0001). CONCLUSIONS: An increase in the rates of surgery and chemotherapy for pancreatic cancer was observed. Survival rates for pancreatic cancer have generally improved over the past decade and are greater in patients undergoing surgery or chemotherapy and in younger age groups.

Germ Cell Isolation and Cryopreservation from Reproductive Organs of Brown Mealworm.

This study aimed to isolate and freeze germ cells from the superior brown mealworm. Styrofoam diet changes were observed for 20 days to determine whether mealworms were useful insects for decomposing Styrofoam. The average weight of mealworms before the Styrofoam diet was 500 mg, which decreased to 336 mg at D20 after their diet. To preserve mealworms with excellent Styrofoam-degrading ability, we first isolated the reproductive organs of mealworms, testes, ovaries, sperms, and ovarioles. Morphologically, male and female adult brown mealworms were distinguished according to the presence or absence of a protrusion at the tip of the fifth segment of the abdomen. Sperms and ovarioles were observed in anatomically isolated testes and ovaries. We compared mechanical and enzymatic (collagenase I) methods to effectively isolate ovarioles from adult female brown mealworms. For the enzymatic method, most were torn and burst as the membrane of the ovarioles was damaged by collagenase I, unlike the mechanical method. To preserve the superior genetic resources of mealworms, we cryopreserved the ovaries of female brown mealworms using slow-freezing and vitrification. Histological analysis showed that the yolk sac was completely damaged in the ovaries after slow-freezing. However, only partial damage was achieved in the vitrification group compared to the control group (no freezing). The newly developed vitrification method with alginate-encapsulated ovarioles maintained the yolk sac in the ovarioles but was evenly distributed. These results provide basic data for reproductive studies of other useful insects and contribute to the biobanking and fertility preservation of superior mealworm germ cells and endangered insects.

Impact of intensive rehabilitation on long-term prognosis after stroke: A Korean nationwide retrospective cohort study.

An increasing number of patients are receiving rehabilitation after stroke. But the impact of intensive rehabilitation on the long-term prognosis of patients with stroke remains to be elucidated. The purpose of this study was to identify the impact of intensive rehabilitation on the long-term prognosis of patients with stroke using data from the National Health Insurance Service database. This is a register-based, retrospective cohort study. Using data from the National Health Insurance Service database, we included the patients who received rehabilitation for stroke from 2006 to 2013. Of the 14,984 patients diagnosed with stroke, 2483 died within 1 year, and 2866 did not receive rehabilitation; hence, they were also excluded. The final sample included 9635 (49.2% men, 50.8% women) patients. After correcting for covariates, the Cox model was used to evaluate the effects of physical therapy (PT) and occupational therapy (OT) on survival. We estimated the independent contribution of each factor to the risk of death from the initiation of rehabilitation. Significant differences in mortality were observed according to age, Charlson comorbidity index (CCI), income level, and stroke type. Patients with stroke who received both PT and OT had a better long-term prognosis than those who received either treatment alone. Therapy performed by a physical therapist with more than 120 hours of training effectively improved the patients' long-term prognosis. Intensive PT and OT will help improve the long-term prognosis of patients with stroke. This study emphasizes the importance of intensive rehabilitation in these patients.