Objective This study aimed to explore the regulatory role of autophagy in acute kidney injury(AKI)-induced acute liver injury(ALI).Methods Forty-eight male Sprague-Dawley rats were randomly divided into four groups:sham operation group,IRI group,3-MA group and RA group.Except for the sham operation group,a rat model of AKI induced by IRI was established in all groups.The AKI model was established by removing the right kidney,separating the left renal artery,and clamping the left renal artery,followed by reper-fusion for 12,24,48,or 72 h.The 3-MA and RA groups were intraperitoneally injected with 3-MA(15 mg/kg,1 mL)or RA(2 mg/kg,1 mL)12 h before and after IRI treatment.The structure and function of the rat lung and kidney tissues were evaluated,and the expression levels of autophagy-related proteins,oxidative stress,and apoptosis were measured.Results Renal IRI led to ALI after AKI,and the levels of blood urea nitrogen,creatinine,tumor necrosis factor-α,and interleukin-1βwere all significantly increased.In addition,compared to the IRI group,the expression levels of P62 and caspase-3 significantly decreased in the RA group,whereas the expression levels of LC3-Ⅱ/LC3-Ⅰ,Beclin-1,Bcl-2,and ULK1 increased.Autophagy reduced pathological damage to kidney and lung tissues by inhibiting inflammation and oxidative stress and effectively ameliorated AKI-induced ALI.Conclusion Autophagy plays an important role in the regulation of ALI induced by AKI and can be used as a new target for AKI treatment and to reduce complication-related mortality.

Objective To construct and validate a risk prediction model for poor inhalation in chronic obstructive pulmonary disease(COPD)patients receiving inhaler therapy,providing a decision support tool for personalized prevention of poor inhalation.Methods A cross-sectional study was conducted to collect data related to COPD patients receiving inhaler therapy,forming a dataset.The dataset was randomly divided into a training set and a test set in a ratio of 4∶1.Four different methods for missing value imputation,3 methods for variable feature selection,and 18 machine learning algorithms were employed to successfully construct 216 models on the training set.The monte carlo simulation method was used for resampling in the test set to validate the models,with the area under curve(AUC),accuracy,precision,recall,and F1 score used to evaluate model performance.The optimal model was selected to build the poor inhalation prediction platform.Results A study involving 308 patients with COPD found that 135(43.8%)were at risk of adverse inhalation.Using 33 predictor variables,216 risk prediction models were developed.Of these models,the ensemble learning algorithm yielded the highest average AUC of 0.844,with a standard deviation of 0.058[95%CI=(0.843,0.845)].The differences in predictive performance among the 216 models were statistically significant(P＜0.01).Under the ensemble learning algorithm,adherence to inhaler use(38.087 4%),inhaler satisfaction(25.680 1%),literacy(24.031 3%),number of inhalers(5.482 3%),age(4.204 5%)and number of acute exacerbations in the past year(2.184 7%)contributed most to the predictive model.The model exhibited superior performance,with an AUC of 0.869 3,an accuracy of 83.87%,a precision of 86.96%,a recall of 74.07%,and an F1 score of 0.8.Conclusion This study has developed a predictive model for poor inhalation risk in COPD inhaler therapy patients using machine learning algorithms,which exhibits strong predictive capabilities and holds potential clinical application value.

Background::Heterogeneity of tumor cells and the tumor microenvironment (TME) is significantly associated with clinical outcomes and treatment responses in patients with urothelial carcinoma (UC). Comprehensive profiling of the cellular diversity and interactions between malignant cells and TME may clarify the mechanisms underlying UC progression and guide the development of novel therapies. This study aimed to extend our understanding of intra-tumoral heterogeneity and the immunosuppressive TME in UC and provide basic support for the development of novel UC therapies.Methods::Seven patients with UC were included who underwent curative surgery at our hospital between July 2020 and October 2020. We performed single-cell RNA sequencing (scRNA-seq) analysis in seven tumors with six matched adjacent normal tissues and integrated the results with two public scRNA-seq datasets. The functional properties and intercellular interactions between single cells were characterized, and the results were validated using multiplex immunofluorescence staining, flow cytometry, and bulk transcriptomic datasets. All statistical analyses were performed using the R package with two-sided tests. Wilcoxon-rank test, log-rank test, one-way analysis of variance test, and Pearson correlation analysis were used properly.Results::Unsupervised t-distributed stochastic neighbor embedding clustering analysis identified ten main cellular subclusters in urothelial tissues. Of them, seven urothelial subtypes were noted, and malignant urothelial cells were characterized with enhanced cellular proliferation and reduced immunogenicity. CD8 + T cell subclusters exhibited enhanced cellular cytotoxicity activities along with increased exhaustion signature in UC tissues, and the recruitment of CD4 + T regulatory cells was also increased in tumor tissues. Regarding myeloid cells, coordinated reprogramming of infiltrated neutrophils, M2-type polarized macrophages, and LAMP3 + dendritic cells contribute to immunosuppressive TME in UC tissues. Tumor tissues demonstrated enhanced angiogenesis mediated by KDR + endothelial cells and RGS5 +/ACTA2 + pericytes. Through deconvolution analysis, we identified multiple cellular subtypes may influence the programmed death-ligand 1 (PD-L1) immunotherapy response in patients with UC. Conclusion::Our scRNA-seq analysis clarified intra-tumoral heterogeneity and delineated the pro-tumoral and immunosuppressive microenvironment in UC tissues, which may provide novel therapeutic targets.

Objective:To investigate the expressions of miR-20a-5p and lysine (K) demethylase 6B (KDM6B) in osteosarcoma tissues and the effects of miR-20a-5p targeting KDM6B on the proliferation, migration and invasion of osteosarcoma cells and tumor growth.Methods:The clinicopathological and paracancerous tissues of 20 patients with osteosarcoma admitted to the First Affiliated Hospital of Chinese Medical University from January 2017 to March 2019 were collected. Quantitative real-time PCR (qRT-PCR) was used to detect the expression levels of miR-20a-5p and KDM6B mRNA in tissues. The osteosarcoma MG63 cells were divided into control group, mimic NC group, miR-20a-5p mimic group, and NC+ empty vector group, miR-20a-5p+ empty vector group, miR-20a-5p+ KDM6B group. The expression levels of miR-20a-5p and KDM6B mRNA of all groups were detected by qRT-PCR. Western blotting was used to detect the expression level of KDM6B. CCK-8 assay, cell scratch test and Transwell test were used to detect cell proliferation, migration and invasion ability. According to the random number table method, nude mice were divided into NC+ empty vector group, miR-20a-5p+ empty vector group and miR-20a-5p+ KDM6B group, with 5 mice in each group. Tumor growth ability was detected by tumor xenograft nude mouse models.Results:The relative expression level of miR-20a-5p mRNA in osteosarcoma tissues was 0.55±0.27, and that in paracancerous tissues was 1.22±0.28, with a statistically significant difference ( t=7.701, P<0.001). The relative expression level of KDM6B mRNA in osteosarcoma tissues was 1.66±0.19, and that in paracancerous tissues was 1.00±0.15, with a statistically significant difference ( t=12.219, P<0.001). After transfection of miR-20a-5p, KDM6B mRNA and protein expression levels decreased with the increase of miR-20a-5p expression level. After miR-20a-5p transfection for 48 h, the cell proliferation abilities of the blank control group, mimic NC group and miR-20a-5p mimic group were 0.83±0.04, 0.81±0.03 and 0.52±0.01 ( F=89.655, P<0.001), compared with the blank control group and mimic NC group, the cell proliferation ability was significantly inhibited in the miR-20a-5p mimic group (both P<0.001). The cell proliferation abilities of NC+ empty vector group, miR-20a-5p+ empty vector group and miR-20a-5p+ KDM6B group were 0.83±0.05, 0.52±0.01 and 0.67±0.05 ( F=43.919, P<0.001), compared with the NC+ empty vector group, the cell proliferation ability was significantly inhibited in the miR-20a-5p+ empty vector group ( P<0.001); compared with the miR-20a-5p+ empty vector group, the cell proliferation ability of miR-20a-5p+ KDM6B group increased significantly ( P<0.001). The scratch healing rates of the blank control group, mimic NC group and miR-20a-5p mimic group were (32.51±2.73)%, (30.26±3.22)% and (13.52±1.77)% ( F=46.314, P<0.001), compared with the control group and the mimic NC group, the scratch healing rate of the miR-20a-5p mimic group was significantly decreased (both P<0.001). The scratch healing rates of NC+ empty vector group, miR-20a-5p+ empty vector group and miR-20a-5p+ KDM6B group were (31.34±3.11)%, (12.15±1.64)% and (28.93±2.89)% ( F=47.511, P<0.001), compared with the NC+ empty vector group, the scratch healing rate of the miR-20a-5p+ empty vector group was significantly decreased ( P<0.001); compared with the miR-20a-5p+ empty vector group, the scratch healing rate of miR-20a-5p+ KDM6B group was significantly increased ( P=0.001). The numbers of transmembrane cells in the blank control group, mimic NC group and miR-20a-5p mimic group were 114±16, 108±11 and 42±6 ( F=36.282, P<0.001), compared with the control group and mimic NC group, the number of transmembrane cells of the miR-20a-5p mimic group was significantly decreased (both P<0.001). The numbers of transmembrane cells in the NC+ empty vector group, miR-20a-5p+ empty vector group and miR-20a-5p+ KDM6B group was 143±11, 39±4 and 139±12 ( F=112.120, P<0.001), compared with the NC+ empty vector group, the number of transmembrane cells of the miR-20a-5p+ empty vector group was significantly decreased ( P<0.001); compared with the miR-20a-5p+ empty vector group, the number of transmembrane cells of the miR-20a-5p+ KDM6B group was increased significantly ( P<0.001). The tumor volumes of mice for 21 d in the NC+ empty vector group, miR-20a-5p+ empty vector group and miR-20a-5p+ KDM6B group were (1 667.50±250.40) mm 3, (129.20±21.00) mm 3 and (775.41±77.51) mm 3 respectively, with a statistically significant difference ( F=77.651, P<0.001). The tumor weights of the 3 groups were (1.35±0.18) g, (0.12±0.01) g and (0.61±0.03) g respectively, with a statistically significant difference ( F=104.191, P<0.001). Conclusion:The expression of miR-20a-5p is significantly decreased in osteosarcoma tissues, and the expression of KDM6B is significantly increased in osteosarcoma tissues. Overexpression of miR-20a-5p may inhibit the proliferation, migration and invasion of osteosarcoma cells and tumor growth by targeting to reduce the expression of KDM6B.

Cardiopulmonary resuscitation (CPR) guidelines emphasize that external chest compression should be started as early as possible during CPR. In addition to manual hand compressions, the machine for CPR can be used instead. However traditional machines for CPR take a lot of time toinstall, and is not conducive to the early initiation of chest compressions. So the medical staff of the emergency department of Mindong Hospital Affiliated to Fujian Medical University designed a new type of machine for CPR based on scissor lift principle without back plate. The design has obtained the national utility model patent. This patented product with flexible lifting and accurate placement is easy to install and fix, and can overcome the drawbacks of conventional machines that are uneasy to install, thus ensuring the implementation of high quality CPR.

Objective: To evaluate the feasibility and safety of transanal lateral lymph node dissection for mid-low rectal cancer. Methods: A descriptive case series research method was used. Clinical and pathological data of 5 mid-low rectal cancer patients who underwent transanal lateral lymph node dissection at Department of Colorectal Surgery, the Sixth Affiliated Hospital of Sun Yat-sen University from November 2018 to May 2019 were retrospectively collected and analyzed. Of 5 cases, 4 were male and 1 was female with mean age of (43.2±13.2) years and mean body mass index of (21.2±2.6) kg/m²; the mean diameter of tumor was (3.2±2.4) cm; the mean distance between tumor and anus was (6.3±2.5) cm; 3 received preoperative neoadjuvant chemotherapy. In preoperative TNM staging, 2 cases were T3N1M0, 1 was T3cN2aM0, 1 was T3cN2bM0, and 1 was T2N1M0. All the patients had no intestinal obstruction before operation. Surgical methods: (1) total mesorectal excision: using general transanal and transabdominal methods to mobilize and resect total mesorectum, and dissect No.252, No.253 lymph nodes; (2) transanal lateral lymph node dissection: dissect the adipose lymphoid tissue on the surface of the iliococcygeal muscle, the coccygeal muscle, and the obturator muscle (the No.283 lymph nodes) upward, and dissect No.263d and No.263p lymph nodes with fat tissue sequentially till the bifurcation of the internal and external iliac artery; (3) take out the specimen from anus, and make anastomosis between proximal colon and anal canal. Intraoperative and postoperative variables was observed. Results: All the 5 patients completed surgery successfully, and no patient needed to convert to open approach. The mean operative time was (295.6±97.7) minutes, and the median intraoperative blood loss was 70 (50-500) ml. The mean length of specimen was (12.9±3.0) cm, and the mean number of harvested lymph node was 30.4±9.9. The positive lateral lymph nodes were founder in 4 patients. The median distance between tumor and distal resection margin was 1.5 (1.2-8.0) cm. The resection margin in all the patients was negative. The mean time to postoperative flatus was (4.2±1.6) days, the mean postoperative spontaneous urination was (3.0±1.9) days, time to drainage tube removal was (5.6±1.9) days, and the mean postoperative hospital stay was (9.4±2.1) days. The postoperative TNM staging by pathology was 1 case with T1N0M0, 1 with T2N1M0, 1 with T3N2bM0, and 2 with T3N2M0. Five patients were moderately differentiated adenocarcinoma. Only 1 patient developed postoperative abdominal bleeding, who was healed after conservative treatment. The other 4 patients did not develop any perioperative complications, such as incision infection, presacral abscess, pelvic abscess, anastomotic leakage, or anastomotic stricture. Four patients underwent postoperative chemotherapy. All the patients were followed up for 2 to 28 weeks after surgery and they all felt well. The patients with stoma had fluent bowel. Conclusions Transanal lateral lymph node dissection is feasible and safe in the treatment of mid-low rectal cancer, which can achieve the purpose of extended radical resection of mid-low movement rectal cancer. Moreover, this procedure is a new method to treat rectal cancer patients with lateral lymph node metastasis.

Objective To investigate the long-term efficacy of pure transanal total mesorectal excision (PtaTME) for middle-low rectal cancer.Methods The retrospective descriptive study was conducted.The clinicopathological data of 18 patients with middle-low rectal cancer who were admitted to the Sixth Affiliated Hospital of Sun Yat-sen University from July 2014 to August 2016 were collected.There were 7 males and 11 females,aged (58±13) years,with a range from 40 to 84 years.The body mass index was (22±3) kg/m2.All the 18 patients underwent PtaTME.Observation indicators:(1) surgical and postoperative conditions;(2) postoperative pathological examination;(3) follow-up and survival.Follow-up using inpatient reexamination,outpatient examination,and telephone interview were performed to detect anastomotic complications,anal function,urinary retention,sexual dysfunction,survival and tumor recurrence and metastasis once every 3 months within postoperative 6 months,once every 6 months from 6 months to 3 years,and once a year after 3 years up to June 2019.The measurement data with normal distribution were represented as Mean±SD,and the measurement data with skewed distribution were represented as M (range).Count data were expressed as percentages.Survival rates were calculated by the Kaplan-Meier method.Results (1) Surgical and postoperative conditions:18 patients successfully underwent PtaTME,without conversion to open surgery.The operation time,volume of intraoperative blood loss,distance between anastomosis and anal verge,time to first flatus,time to urinary catheter removal,and duration of postoperative hospital stay were (202±68) minutes,50 mL (range,20-400 mL),(4.5± 2.0)cm,2 days (range,2-7 days),3 days (range,2-5 days),and 7 days (range,5-10 days) in the 18 patients,respectively.There was no perioperative complication.Among 18 patients,4 underwent preventive ileostomy.(2) Postoperative pathological examinations:the length of surgical specimens,the number of lymph node dissection,distance from tumor to the distal margin were (11.0±3.0)cm,12±6,and 1.0 cm (range,0.8-3.7 cm),respectively.The 18 patients had complete mesorectal membrane excision,with negative proximal margin,distal margin,and circumferential margin.Tumor pathological staging:there were 2 cases in Tis stage,4 in T1 stage,7 in T2 stage,and 5 in T3 stage;16 in N0 stage,1 in N1 stage,and 1 in N2 stage.Tumor histological classification:2 patients had carcinoma in situ,9 had moderately differentiated adenocarcinoma,and 7 had high-differentiated adenocarcinoma.(3) Follow-up and survival:18 patients were followed up for 34.0-59.0 months,with a median follow-up time of 57.5 months.During the follow-up,4 patients developed grade B anastomotic leakage and were cured after conservative treatment.One patient developed anastomotic recurrence at 2 years after surgery,and no recurrence was found after surgical resection of the recurrent lesion.Four patients with prophylactic ileostomy had the stoma closured,and the anus function was satisfactory after surgery.There was no urinary retention or sexual dysfunction in the 18 patients.Of the 18 patients,17 had tumor free survival after surgery.The 3-year disease-free survival rate was 94.4％,and the 3-year overall survival rate was 100.0％ in 18 patients.Conclusion PtaTME can achieve high quality of specimen,which is safe and feasible for the treatment of rectal cancer.

Objective To evaluate the feasibility and safety of transanal lateral lymph node dissection for mid?low rectal cancer. Methods A descriptive case series research method was used. Clinical and pathological data of 5 mid?low rectal cancer patients who underwent transanal lateral lymph node dissection at Department of Colorectal Surgery, the Sixth Affiliated Hospital of Sun Yat?sen University from November 2018 to May 2019 were retrospectively collected and analyzed. Of 5 cases, 4 were male and 1 was female with mean age of (43.2±13.2) years and mean body mass index of (21.2± 2.6) kg/m2; the mean diameter of tumor was (3.2±2.4) cm; the mean distance between tumor and anus was (6.3 ± 2.5) cm; 3 received preoperative neoadjuvant chemotherapy. In preoperative TNM staging, 2 cases were T3N1M0, 1 was T3cN2aM0, 1 was T3cN2bM0, and 1 was T2N1M0. All the patients had no intestinal obstruction before operation. Surgical methods: (1) total mesorectal excision: using general transanal and transabdominal methods to mobilize and resect total mesorectum, and dissect No.252, No.253 lymph nodes;(2) transanal lateral lymph node dissection: dissect the adipose lymphoid tissue on the surface of the iliococcygeal muscle, the coccygeal muscle, and the obturator muscle (the No. 283 lymph nodes) upward, and dissect No. 263d and No. 263p lymph nodes with fat tissue sequentially till the bifurcation of the internal and external iliac artery; (3) take out the specimen from anus, and make anastomosis between proximal colon and anal canal. Intraoperative and postoperative variables was observed. Results All the 5 patients completed surgery successfully, and no patient needed to convert to open approach. The mean operative time was (295.6±97.7) minutes, and the median intraoperative blood loss was 70 (50?500) ml. The mean length of specimen was (12.9±3.0) cm, and the mean number of harvested lymph node was 30.4±9.9. The positive lateral lymph nodes were founder in 4 patients. The median distance between tumor and distal resection margin was 1.5 (1.2?8.0) cm. The resection margin in all the patients was negative. The mean time to postoperative flatus was (4.2±1.6) days, the mean postoperative spontaneous urination was (3.0±1.9) days, time to drainage tube removal was (5.6±1.9) days, and the mean postoperative hospital stay was (9.4± 2.1) days. The postoperative TNM staging by pathology was 1 case with T1N0M0, 1 with T2N1M0, 1 with T3N2bM0, and 2 with T3N2M0. Five patients were moderately differentiated adenocarcinoma. Only 1 patient developed postoperative abdominal bleeding, who was healed after conservative treatment. The other 4 patients did not develop any perioperative complications, such as incision infection, presacral abscess, pelvic abscess, anastomotic leakage, or anastomotic stricture. Four patients underwent postoperative chemotherapy. All the patients were followed up for 2 to 28 weeks after surgery and they all felt well. The patients with stoma had fluent bowel. Conclusions Transanal lateral lymph node dissection is feasible and safe in the treatment of mid?low rectal cancer, which can achieve the purpose of extended radical resection of mid?low movement rectal cancer. Moreover, this procedure is a new method to treat rectal cancer patients with lateral lymph node metastasis.

Objective To evaluate the feasibility and safety of transanal lateral lymph node dissection for mid?low rectal cancer. Methods A descriptive case series research method was used. Clinical and pathological data of 5 mid?low rectal cancer patients who underwent transanal lateral lymph node dissection at Department of Colorectal Surgery, the Sixth Affiliated Hospital of Sun Yat?sen University from November 2018 to May 2019 were retrospectively collected and analyzed. Of 5 cases, 4 were male and 1 was female with mean age of (43.2±13.2) years and mean body mass index of (21.2± 2.6) kg/m2; the mean diameter of tumor was (3.2±2.4) cm; the mean distance between tumor and anus was (6.3 ± 2.5) cm; 3 received preoperative neoadjuvant chemotherapy. In preoperative TNM staging, 2 cases were T3N1M0, 1 was T3cN2aM0, 1 was T3cN2bM0, and 1 was T2N1M0. All the patients had no intestinal obstruction before operation. Surgical methods: (1) total mesorectal excision: using general transanal and transabdominal methods to mobilize and resect total mesorectum, and dissect No.252, No.253 lymph nodes;(2) transanal lateral lymph node dissection: dissect the adipose lymphoid tissue on the surface of the iliococcygeal muscle, the coccygeal muscle, and the obturator muscle (the No. 283 lymph nodes) upward, and dissect No. 263d and No. 263p lymph nodes with fat tissue sequentially till the bifurcation of the internal and external iliac artery; (3) take out the specimen from anus, and make anastomosis between proximal colon and anal canal. Intraoperative and postoperative variables was observed. Results All the 5 patients completed surgery successfully, and no patient needed to convert to open approach. The mean operative time was (295.6±97.7) minutes, and the median intraoperative blood loss was 70 (50?500) ml. The mean length of specimen was (12.9±3.0) cm, and the mean number of harvested lymph node was 30.4±9.9. The positive lateral lymph nodes were founder in 4 patients. The median distance between tumor and distal resection margin was 1.5 (1.2?8.0) cm. The resection margin in all the patients was negative. The mean time to postoperative flatus was (4.2±1.6) days, the mean postoperative spontaneous urination was (3.0±1.9) days, time to drainage tube removal was (5.6±1.9) days, and the mean postoperative hospital stay was (9.4± 2.1) days. The postoperative TNM staging by pathology was 1 case with T1N0M0, 1 with T2N1M0, 1 with T3N2bM0, and 2 with T3N2M0. Five patients were moderately differentiated adenocarcinoma. Only 1 patient developed postoperative abdominal bleeding, who was healed after conservative treatment. The other 4 patients did not develop any perioperative complications, such as incision infection, presacral abscess, pelvic abscess, anastomotic leakage, or anastomotic stricture. Four patients underwent postoperative chemotherapy. All the patients were followed up for 2 to 28 weeks after surgery and they all felt well. The patients with stoma had fluent bowel. Conclusions Transanal lateral lymph node dissection is feasible and safe in the treatment of mid?low rectal cancer, which can achieve the purpose of extended radical resection of mid?low movement rectal cancer. Moreover, this procedure is a new method to treat rectal cancer patients with lateral lymph node metastasis.

OBJECTIVETo report on the result of thalassemia screening and genetic diagnosis for pregnant women from Guiyang region.

METHODSPrenatal screening for thalassemia was carried out based on erythrocyte parameters and hemoglobin electrophoresis. Single-tube multiplex GAP-PCR and PCR-reverse dot blot hybridization were performed on suspected cases to identify common alpha- and beta- thalassemia mutations, and direct sequencing was used for identifying rare mutations.

RESULTSAmong 13 738 pregnant women, 1745 (12.70%) were suspected as thalassemia. In terms of native place, the provinces with highest screening-positive rates were Guangxi, Guangdong, Jiangxi and Guizhou. And the ethnic groups with highest screening-positive rates were Zhuang, Li, and Buyi. Among 801 women subjected to genetic testing, 457 (57.05%) were diagnosed with thalassemia. In total 9 genotypes of alpha- thalassemia were detected, with the most common genotypes being --/alpha alpha (63.35%), - alpha/alpha alpha (19.37%) and - alpha/alpha alpha (8.90%). Eleven genotypes of beta- thalassemia were detected, with the most common genotypes being CD17/N (42.91%), CD41-42/N (32.46%) and IVS-II-654/N (11.94%). Two cases were detected with rare beta-thalassemia mutations (CD54-58/N and IVS-I-130/N).

CONCLUSIONThe screening-positive rate of thalassemia among pregnant women in Guiyang region is relatively high. The rates have shown substantial difference in terms of native place and ethnic group. Thalassemia-related mutations in Guizhou region have a diverse spectrum, which showed certain difference from those of other regions.

Adolescent ; Adult ; Female ; Genotype ; Humans ; Middle Aged ; Pregnancy ; Prenatal Diagnosis ; methods ; Thalassemia ; genetics ; Young Adult