Oncological microdissection testicular sperm extraction(onco-micro-TESE)represents a significant breakthrough for patients with nonobstructive azoospermia(NOA)and a concomitant in situ testicular tumor,to be managed a...Oncological microdissection testicular sperm extraction(onco-micro-TESE)represents a significant breakthrough for patients with nonobstructive azoospermia(NOA)and a concomitant in situ testicular tumor,to be managed at the time of sperm retrieval.Onco-micro-TESE addresses the dual objectives of treating both infertility and the testicular tumor simultaneously.The technique is intricate,necessitating a comprehensive understanding of testicular anatomy,physiology,tumor biology,and advanced microsurgical methods.It aims to carefully extract viable spermatozoa while minimizing the risk of tumor dissemination.This review encapsulates the procedural intricacies,evaluates success determinants,including tumor pathology and spermatogenic tissue health,and discusses the implementation of imaging techniques for enhanced surgical precision.Ethical considerations are paramount,as the procedure implicates complex decision-making that weighs the potential oncological risks against the profound desire for fatherhood using the male gametes.The review aims to provide a holistic overview of onco-micro-TESE,detailing methodological advances,clinical outcomes,and the ethical landscape,thus offering an indispensable resource for clinicians navigating this multifaceted clinical scenario.展开更多
The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6V...The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the 'single tube', all the subsequence steps were conducted. After two round of LA (Linker adaptor)_PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600-1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with 32 P as probe. The PCR products were purified and ligated into clone vector-pGEM_T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl 2. It was estimated that there were more than 17 000 white clones in the library. The size of insert fragments distributed from 100-1 500 bp, with average of 600 bp. Using H. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H. villosa_specific clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.展开更多
Objective： Laser capture microdisection has become indispensable to the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma （BTCC）. However,...Objective： Laser capture microdisection has become indispensable to the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma （BTCC）. However, to obtain sufficient RNA from laser-capture microdissected cells is quite difficult. The study was designed to determinc a feasible technical routine to isolate transitional cells from bladder membrane, separate carcinoma cclls from stromal cells and to amplify the RNA isolated from laser-capture microdissected cells. Methods： Bladder transitional cell were obtained from frozen sections of bladder membrane applying LCM, by the same token, BTCC cells from frozen sections of BTCC tissue. Then RNA was extracted and linearly amplified in vitro. The expression levels of β-actin in primary total RNA and amplified RNA were detected using RT-PCR. Results： That RNA integrity was good after LCM was confirmed by control experiment Ⅰ; By control experiment Ⅱ, the correlation between the number of LCM-shooting and RNA quantity undcr arranged conditions was preliminarily confirmed. About 0.5-2.5kb RNA fragments were obtained after RNA amplification and β-actin levels were integral. Conclusion： Laser capture microdissection combined with RNA linear amplification in vitro can be successfully applied to obtain pure objective cells for research. The integrity of the amplified RNA is good and can be employed in further research.展开更多
Background:Hormonal treatment and response as a predictor of sperm retrieval prior to microdissection testicular sperm extraction(micro-TESE)are not well established in the current literature.This study aimed to inves...Background:Hormonal treatment and response as a predictor of sperm retrieval prior to microdissection testicular sperm extraction(micro-TESE)are not well established in the current literature.This study aimed to investigate the hormonal response as a predictor of sperm retrieval among men with nonobstructive azoospermia(NOA).Methods:Seventy-seven consecutive patients who had testosterone levels≤14 nmol/L were treated medically with an aromatase inhibitor or recombinant human chorionic gonadotropin(rec-hCG)prior to micro-TESE and were included.Thirty-four(44.2%)had unexplained NOA(UNEX),25(32.5%)had Klinefelter syndrome(KS),8(10.4%)had a history of cryptorchidism(UDT),4(5.2%)had microdeletion of the Azoospermia factor C(AZFc),and 6(7.8%)were treated previously with chemotherapy.Baseline and post-treatment serum hormonal levels were documented.Pre-op testosterone levels were entered into binary logistic regressions with age,Follicle-stimulating hormone(FSH),and Luteinizing hormone(LH)levels to test for significance with sperm retrieval.We then built logistic regression models to identify predictors of successful surgical sperm retrieval(SSR).Results:Forty-five patients(58%)had successful retrieval.In 32 patients(42%),no sperm was retrieved.Both the mean pre-op testosterone and the mean testosterone change between the two groups were significant(p=0.02 and p=0.011,respectively).Receiver operating characteristic(ROC)analysis demonstrated an area under the curve(AUC)of 0.785(95%CI=0.685-0.886,p<0.001).The Youden index coefficient was calculated for KS and UNEX.The cut-off point for KS was established at 0.764(sensitivity=0.875,false positive rate[FPR]=0.111),and 0.215 for UNEX(sensitivity=0.438,FPR=0.222).We also observed a correlation between age and SSR(p=0.05).In KS patients,SSR was determined by pre-op testosterone levels irrespective of age.Conclusion:Pre-operative hormonal response is a predictor for SSR in NOA patients who were treated medically.This data may help during pre-operative counselling.展开更多
The aim of our study was to compare the sperm retrieval rates(SRRs)and clinical outcomes of patients with different causes of azoospermia who underwent microdissection testicular sperm extraction-intracytoplasmic sper...The aim of our study was to compare the sperm retrieval rates(SRRs)and clinical outcomes of patients with different causes of azoospermia who underwent microdissection testicular sperm extraction-intracytoplasmic sperm injection(micro-TESE-ICSI).We conducted a retrospective study at the Reproductive Medicine Center of Peking University Third Hospital in Beijing,China,from January 2014 to December 2017.This study examined 769 patients with nonobstructive azoospermia who underwent 347 cycles of micro-TESE-ICSI.Patients with azoospermia were classified into Group A(Klinefelter syndrome,n=284,125 cycles),Group B(azoospermia Y chromosome factor c[AZFc]microdeletion,n=91,64 cycles),Group C(cryptorchidism,n=52,39 cycles),Group D(previous mumps and bilateral orchitis,n=23,23 cycles),and Group E(idiopathic azoospermia,n=319,96 cycles).Clinical characteristics,SRR,embryonic development,and pregnancy outcomes of the patients were compared between all groups.Patients in Group D had the highest and most successful SRR.The average SRR for all patients was 46.0%.The rates of clinical pregnancy,implantation,and live birth in Group D were 78.3%,65.0%,and 74.0%,respectively,which were higher than those in all other groups(P<0.05).Group B patients had the lowest clinical pregnancy,implantation,and live birth rates of all groups(P<0.05).No differences were found in the miscarriage rate or birth defects among the groups(P>0.05).Patients with orchitis had the highest SRR and best clinical outcomes.Although AZFc microdeletion patients had a higher SRR,their clinical outcomes were worse.展开更多
We performed this meta-analysis to evaluate the predictive value of different parameters in the sperm retrieval rate （SRR） of microdissection testicular sperm extraction （TESE） in patients with nonobstructive azoo...We performed this meta-analysis to evaluate the predictive value of different parameters in the sperm retrieval rate （SRR） of microdissection testicular sperm extraction （TESE） in patients with nonobstructive azoospermia （NOA）. All relevant studies were searched in PubMed, Web of Science, EMBASE, Cochrane Library, and EBSCO. We chose three parameters to perform the meta-analysis： follicle-stimulating hormone （FSH）, testicular volume, and testicular histopathological findings which included three patterns： hypospermatogenesis （HS）, maturation arrest （MA）, and Sertoli-cell-only syndrome （SCOS）. If there was a threshold effect, only the area under the summary receiver operating characteristic curve （AUSROC） was calculated. Otherwise, the pooled sensitivity, specificity, positive likelihood ratio （PLR）, negative likelihood ratio （NLR）, and the diagnostic odds ratio （DOR） were also calculated. Twenty-one articles were included in our study finally. There was a threshold effect among studies investigating FSH and SCOS. The AUSROCs of FSH, testicular volume, HS, MA, and SCOS were 0.6119, 0.6389, 0.6758, 0.5535, and 0.2763, respectively. The DORs of testicular volume, HS, and MA were 1.98, 16.49, and 1.26, respectively. The sensitivities of them were 0.80, 0.30, and 0.27, while the specificities of them were 0.35, 0.98, and 0.76, respectively. The PLRs of them were 1.49, 10.63, and 1.15, respectively. And NLRs were 0.73, 0.72, and 0.95, respectively. All the investigated factors in our study had limited predictive value. However, the histopathological findings were helpful to some extent. Most patients with HS could get sperm by microdissection TESE.展开更多
AIM. To study the differential gene expression profiles of target cells in primary gastric cancer and its metastatic lymph nodes using laser microdissection （LMD） in combination with cDNA microarray. METHODS： Norma...AIM. To study the differential gene expression profiles of target cells in primary gastric cancer and its metastatic lymph nodes using laser microdissection （LMD） in combination with cDNA microarray. METHODS： Normal gastric tissue samples from 30 healthy individuals, 36 cancer tissue samples from primary gastric carcinoma and lymph node metastasis tissue samples from 58 patients during gastric cancer resection were obtained using LMD in combination with cDNA microarray independently. After P27-based amplification, aRNA from 36 of 58 patients （group 1） with lymph node metastasis and metastatic tissue specimens from the remaining 22 patients （group 2） were applied to cDNA microarray. Semiquantitative reverse transcriptase polymerase chain reaction （RT-PCR） and imrnunohistochemical assay verified the results of microarray in group 2 and further identified genes differentially expressed in the progression of gastric cancer. RESULTS： The expression of 10 genes was up-regulated while the expression of 15 genes was down-regulated in 22 gastric carcinoma samples compared with that of genes in the normal controls. The results were confirmed at the level of mRNA and protein, and suggested that four genes （OPCML, RNASE1, YES1 and ACK1） could play a key role in the tumorigenesis and metastasis of gastric cancer. The expression pattern of 3 genes （OPCML, RNASE1 and YES1） was similar to tumor suppressor genes. For example, the expression level of these genes was the highest in normal gastric epithelium, which was decreased in primary carcinoma, and further decreased in metastatic lymph nodes. On the contrary, the expression pattern of gene ACK1 was similar to that of oncogene. Four genes were further identified as differentially expressed genes in the majority of the cases in the progression of gastric cancer. CONCLUSION： LMD in combination with cDNA microaro ray provides a unique support foe the identification of early expression profiles of differential genes and the expression pattern of 3 genes （OPCML, RNASE1 and YES1） associated with the progression of gastric cancer. Further study is needed to reveal the molecular mechanism of lymph node metastasis in patients with gastric cancer.展开更多
In 220 consecutive patients with non-obstructive azoospermia, sperm retrieval was attempted by a combination of conventional and microdissection testicular sperm extraction （TESE）. For sperm retrieval, 2-3 conventio...In 220 consecutive patients with non-obstructive azoospermia, sperm retrieval was attempted by a combination of conventional and microdissection testicular sperm extraction （TESE）. For sperm retrieval, 2-3 conventional biopsies were performed followed by a microdissection TESE in cases of negative conventional biopsies. During the surgery, the vasculature of the testis was assessed using the operative microscope, and the location of positive biopsies was registered in relation to the blood supply. The overall sperm retrieval rate was 58.2%. From the initial conventional biopsies, sperm could be retrieved in 46.8% of the patients. With microdissection TESE, sperm could be retrieved from an additional 11.4% of the patients. The further use of microdissection TESE improved the sperm retrieval rate significantly （P=0.017）. No significant accumulation of positive biopsies was found towards the rete testis or the main testicular vessels.展开更多
Microdissection testicular sperm extraction(micro-TESE)is widely used to treat nonobstructive azoospermia.However,a good prediction model is required to anticipate a successful sperm retrieval rate before performing m...Microdissection testicular sperm extraction(micro-TESE)is widely used to treat nonobstructive azoospermia.However,a good prediction model is required to anticipate a successful sperm retrieval rate before performing micro-TESE.This retrospective study analyzed the clinical records of 200 nonobstructive azoospermia patients between January 2021 and December 2021.The backward method was used to perform binary logistic regression analysis and identify factors that predicted a successful micro-TESE sperm retrieval.The prediction model was constructed using acquired regression coefficients,and its predictive performance was assessed using the receiver operating characteristic curve.In all,67 patients(sperm retrieval rate:33.5%)underwent successful micro-TESE.Follicle-stimulating hormone,anti-Miillerian hormone,and inhibin B levels varied significantly between patients who underwent successful and unsuccessful micro-TESE.Binary logistic regression analysis yielded the following six predictors:anti-Mullerian hormone(odds ratio[OR]=0.902,95%confidence interval[Cl]:0.821-0.990),inhibin B(OR=1.012,95%Cl:1.001-1.024),Klinefelter’s syndrome(OR=0.022,95%Cl:0.002-0.243),Y chromosome microdeletion(OR=0.050,95%Cl:0.005-0.504),cryptorchidism with orchiopexy(OR=0.085,95%Cl:0.008-0.929),and idiopathic nonobstructive azoospermia(OR=0.031,95%Cl:0.003-0.277).The prediction model had an area under the curve of 0.720(95%Cl:0.645-0.794),sensitivity of 65.7%,specificity of 72.2%,Youden index of 0.379,and cut-off value of 0.305 overall,indicating good predictive value and accuracy.This model can assist clinicians and nonobstructive azoospermia patients in decision-making and avoiding negative micro-TESE results.展开更多
Spatial omics technologies have become powerful methods to provide valuable insights into cells and tissues within a complex context,significantly enhancing our understanding of the intricate and multifaceted biologic...Spatial omics technologies have become powerful methods to provide valuable insights into cells and tissues within a complex context,significantly enhancing our understanding of the intricate and multifaceted biological system.With an increasing focus on spatial heterogeneity,there is a growing need for unbiased,spatially resolved omics technologies.Laser capture microdissection(LCM)is a cutting-edge method for acquiring spatial information that can quickly collect regions of interest(ROIs)from heterogeneous tissues,with resolutions ranging from single cells to cell populations.Thus,LCM has been widely used for studying the cellular and molecular mechanisms of diseases.This review focuses on the differences among four types of commonly used LCM technologies and their applications in omics and disease research.Key attributes of application cases are also highlighted,such as throughput and spatial resolution.In addition,we comprehensively discuss the existing challenges and the great potential of LCM in biomedical research,disease diagnosis,and targeted therapy from the perspective of high-throughput,multi-omics,and single-cell resolution.展开更多
We performed this study to evaluate the clinical outcomes of microdissection testicular sperm extraction-intracytoplasmic sperm injection(micro-TESE-ICSI)treatment that used fresh or cryopreserved sperm in patients wi...We performed this study to evaluate the clinical outcomes of microdissection testicular sperm extraction-intracytoplasmic sperm injection(micro-TESE-ICSI)treatment that used fresh or cryopreserved sperm in patients with nonobstructive azoospermia(NOA).A total of 338 NOA patients with 344 consecutive cycles received treatment in the reproductive medicine center of Peking University Third Hospital in Beijing,China,from January 2014 to December 2017.Fresh oocytes and fresh sperm were used in 222 patients with 234 cycles(Group A).Fresh oocytes and cryopreserved sperm were used in 116 patients with 110 cycles(Group B).We compared patient characteristics,embryonic development,and pregnancy outcomes between Groups A and B.There was no statistical difference in the patient characteristics,and no differences were observed with fertilization or quality embryo rates between Groups A and B.The rates of clinical pregnancy and live birth were both higher for Group A than those for Group B(both P<0.05).In conclusion,fresh testicular sperm appears to produce better ICSI outcomes than cryopreserved testicular sperm in patients with NOA.展开更多
AIM： To examine the gene expression profile of gastric cancer （GC） by combination of laser capture microdissection （LCM） and microarray and to correlate the profiling with histological subtypes. METHODS： Using L...AIM： To examine the gene expression profile of gastric cancer （GC） by combination of laser capture microdissection （LCM） and microarray and to correlate the profiling with histological subtypes. METHODS： Using LCM, pure cancer cells were procured from 45 cancerous tissues. After procurement of about 5 000 cells, total RNA was extracted and the quality of RNA was determined before further amplification and hybridization. One microgram of amplified RNA was converted to cDNA and hybridized to cDNA microarray. RESULTS： Among 45 cases, only 21 were qualified for their RNAs. A total of 62 arrays were performed. These included 42 arrays for cancer （21 cases with dyeswab duplication） and 20 arrays for non-tumorous cells （10 cases with dye-swab duplication） with universal reference. Analyzed data showed 504 genes were differentially expressed and could distinguish cancerous and non-cancerous groups with more than 99% accuracy. Of the 504 genes, trefoil factors 1, 2, and 3 were in the list and their expression patterns were consistent with previous reports. Immunohistochemical staining of trefoil factor 1 was also consistent with the array data. Analyses of the tumor group with these 504 genes showed that there were 3 subgroups of GC that did not correspond to any current classification system, including Lauren＇s dassification. CONCLUSION： By using LCM, linear amplification of RNA, and cDNA microarray, we have identified a panel of genes that have the power to discriminate between GC and non-cancer groups. The new molecular classification and the identified novel genes in gastric carcinogenesis deserve further investigations to elucidate their clinicopathological significance.展开更多
The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed d...The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.展开更多
Since chromosome microdissection and microcloning was first developed by Scalenghe et al(1981)with poly-tene chromosome of Drosophila,it had been greatly im-proved and evolved to an efficient tool for generation of ch...Since chromosome microdissection and microcloning was first developed by Scalenghe et al(1981)with poly-tene chromosome of Drosophila,it had been greatly im-proved and evolved to an efficient tool for generation of chromosome-specific DNA libraries in human,animals and plants(Greenfield and Brown,1987;Ludecke et al,1989;Guan et al,1992;Jung et al,1992.展开更多
The key regulators and regeneration-associated genes involved in axonal regeneration of neurons after injury have not been clarified.In high-throughput sequencing,various factors influence the final sequencing results...The key regulators and regeneration-associated genes involved in axonal regeneration of neurons after injury have not been clarified.In high-throughput sequencing,various factors influence the final sequencing results,including the number and size of cells,the depth of sequencing,and the method of cell separation.There is still a lack of research on the detailed molecular expression profile during the regeneration of dorsal root ganglion neuron axon.In this study,we performed lase r-capture microdissection coupled with RNA sequencing on dorsal root ganglion neurons at 0,3,6,and 12 hours and 1,3,and 7 days after sciatic nerve crush in rats.We identified three stages after dorsal root ganglion injury:early(3-12 hours),pre-regeneration(1 day),and regeneration(3-7 days).Gene expression patterns and related function enrichment res ults showed that one module of genes was highly related to axonal regeneration.We verified the up-regulation of activating transcription factor 3(Atf3),Kruppel like factor 6(Klf6),AT-rich inte raction domain 5A(Arid5α),CAMP responsive element modulator(Crem),and FOS like 1,AP-1 transcription factor Subunit(Fosl1) in dorsal root ganglion neurons after injury.Suppressing these transcription factors(Crem,Arid5o,Fosl1 and Klf6) reduced axonal regrowth in vitro.As the hub transcription factor,Atf3 showed higher expression and activity at the preregeneration and regeneration stages.G protein-coupled estrogen receptor 1(Gper1),inte rleukin 12a(Il12α),estrogen receptor 1(ESR1),and interleukin 6(IL6) may be upstream factors that trigger the activation of Atf3 during the repair of axon injury in the early stage.Our study presents the detailed molecular expression profile during axonal regeneration of dorsal root ganglion neurons after peripheral nerve injury.These findings may provide reference for the clinical screening of molecular targets for the treatment of peripheral nerve injury.展开更多
This study aims to explore the factors influencing the success rate of the microdissection testicular sperm extraction(Micro-TESE)in patients with nonobstructive azoospermia(NOA)and cryptorchidism.Clinical data of 162...This study aims to explore the factors influencing the success rate of the microdissection testicular sperm extraction(Micro-TESE)in patients with nonobstructive azoospermia(NOA)and cryptorchidism.Clinical data of 162 patients with cryptorchidism who underwent Micro-TESE due to infertility from December 2015 to May 2020 in the First Affiliated Hospital of Nanjing Medical University were analyzed retrospectively.In the univariate analysis,significant differences in the age of patient at the time of orchidopexy(median[interquartile range,IQR]:7.0[4.0–11.0]years vs 11.5[9.0–14.5]years,P<0.001),interval between orchidopexy and Micro-TESE(mean±standard deviation:17.5±5.0 years vs 14.4±4.4 years,P<0.001),severity of cryptorchidism(unilateral[62.8%]vs bilateral[31.6%],P<0.001;location of cryptorchidism,intra-abdominal[27.3%]vs inguinal[44.8%]vs suprascrotal[66.7%],P<0.001),volume of the dominant testis(median[IQR]:17.00[15.00–19.00]ml vs 14.50[11.75–16.25]ml,P<0.001),and levels of follicle-stimulating hormone(FSH;P=0.004)and testosterone(P=0.006)were observed between the successful and failed sperm extraction groups.After conducting the multivariate analysis,four of these factors,including unilateral/bilateral cryptorchidism(P<0.001),location of cryptorchidism(P=0.032),age of orchidopexy(P<0.001),and dominant testicular volume,were adopted in the clinical prediction model to evaluate preoperatively the success rate of Micro-TESE for patients with NOA and cryptorchidism.The likelihood of successful sperm retrieval by Micro-TESE in men with NOA and cryptorchidism increased in patients with mild forms of cryptorchidism.展开更多
Sperm identification and selection is an essential task when processing human testicular samples for in vitro fertilization.Locating and identifying sperm cell(s)in human testicular biopsy samples is labor intensive a...Sperm identification and selection is an essential task when processing human testicular samples for in vitro fertilization.Locating and identifying sperm cell(s)in human testicular biopsy samples is labor intensive and time consuming.We developed a new computer-aided sperm analysis(CASA)system,which utilizes deep learning for near human-level performance on testicular sperm extraction(TESE),trained on a custom dataset.The system automates the identification of sperm in testicular biopsy samples.A dataset of 702 de-identified images from testicular biopsy samples of 30 patients was collected.Each image was normalized and passed through glare filters and diffraction correction.The data were split 80%,10%,and 10%into training,validation,and test sets,respectively.Then,a deep object detection network,composed of a feature extraction network and object detection network,was trained on this dataset.The model was benchmarked against embryologists’performance on the detection task.Our deep learning CASA system achieved a mean average precision(mAP)of 0.741,with an average recall(AR)of 0.376 on our dataset.Our proposed method can work in real time;its speed is effectively limited only by the imaging speed of the microscope.Our results indicate that deep learning-based technologies can improve the efficiency of finding sperm in testicular biopsy samples.展开更多
基金supported by the National Natural Science Foundation of China(No.82371633)Peking University Clinical Scientist Training Program and the Fundamental Research Funds for the Central University(BMU2023PYJ H012).
文摘Oncological microdissection testicular sperm extraction(onco-micro-TESE)represents a significant breakthrough for patients with nonobstructive azoospermia(NOA)and a concomitant in situ testicular tumor,to be managed at the time of sperm retrieval.Onco-micro-TESE addresses the dual objectives of treating both infertility and the testicular tumor simultaneously.The technique is intricate,necessitating a comprehensive understanding of testicular anatomy,physiology,tumor biology,and advanced microsurgical methods.It aims to carefully extract viable spermatozoa while minimizing the risk of tumor dissemination.This review encapsulates the procedural intricacies,evaluates success determinants,including tumor pathology and spermatogenic tissue health,and discusses the implementation of imaging techniques for enhanced surgical precision.Ethical considerations are paramount,as the procedure implicates complex decision-making that weighs the potential oncological risks against the profound desire for fatherhood using the male gametes.The review aims to provide a holistic overview of onco-micro-TESE,detailing methodological advances,clinical outcomes,and the ethical landscape,thus offering an indispensable resource for clinicians navigating this multifaceted clinical scenario.
基金国家"8 6 3"计划资助项目 (Z 17 0 4 0 1) 国家转基因植物研究与产业化资助项目 (J0 0 A 0 0 2 )~~
文摘The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the 'single tube', all the subsequence steps were conducted. After two round of LA (Linker adaptor)_PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600-1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with 32 P as probe. The PCR products were purified and ligated into clone vector-pGEM_T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl 2. It was estimated that there were more than 17 000 white clones in the library. The size of insert fragments distributed from 100-1 500 bp, with average of 600 bp. Using H. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H. villosa_specific clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.
文摘Objective： Laser capture microdisection has become indispensable to the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma （BTCC）. However, to obtain sufficient RNA from laser-capture microdissected cells is quite difficult. The study was designed to determinc a feasible technical routine to isolate transitional cells from bladder membrane, separate carcinoma cclls from stromal cells and to amplify the RNA isolated from laser-capture microdissected cells. Methods： Bladder transitional cell were obtained from frozen sections of bladder membrane applying LCM, by the same token, BTCC cells from frozen sections of BTCC tissue. Then RNA was extracted and linearly amplified in vitro. The expression levels of β-actin in primary total RNA and amplified RNA were detected using RT-PCR. Results： That RNA integrity was good after LCM was confirmed by control experiment Ⅰ; By control experiment Ⅱ, the correlation between the number of LCM-shooting and RNA quantity undcr arranged conditions was preliminarily confirmed. About 0.5-2.5kb RNA fragments were obtained after RNA amplification and β-actin levels were integral. Conclusion： Laser capture microdissection combined with RNA linear amplification in vitro can be successfully applied to obtain pure objective cells for research. The integrity of the amplified RNA is good and can be employed in further research.
文摘Background:Hormonal treatment and response as a predictor of sperm retrieval prior to microdissection testicular sperm extraction(micro-TESE)are not well established in the current literature.This study aimed to investigate the hormonal response as a predictor of sperm retrieval among men with nonobstructive azoospermia(NOA).Methods:Seventy-seven consecutive patients who had testosterone levels≤14 nmol/L were treated medically with an aromatase inhibitor or recombinant human chorionic gonadotropin(rec-hCG)prior to micro-TESE and were included.Thirty-four(44.2%)had unexplained NOA(UNEX),25(32.5%)had Klinefelter syndrome(KS),8(10.4%)had a history of cryptorchidism(UDT),4(5.2%)had microdeletion of the Azoospermia factor C(AZFc),and 6(7.8%)were treated previously with chemotherapy.Baseline and post-treatment serum hormonal levels were documented.Pre-op testosterone levels were entered into binary logistic regressions with age,Follicle-stimulating hormone(FSH),and Luteinizing hormone(LH)levels to test for significance with sperm retrieval.We then built logistic regression models to identify predictors of successful surgical sperm retrieval(SSR).Results:Forty-five patients(58%)had successful retrieval.In 32 patients(42%),no sperm was retrieved.Both the mean pre-op testosterone and the mean testosterone change between the two groups were significant(p=0.02 and p=0.011,respectively).Receiver operating characteristic(ROC)analysis demonstrated an area under the curve(AUC)of 0.785(95%CI=0.685-0.886,p<0.001).The Youden index coefficient was calculated for KS and UNEX.The cut-off point for KS was established at 0.764(sensitivity=0.875,false positive rate[FPR]=0.111),and 0.215 for UNEX(sensitivity=0.438,FPR=0.222).We also observed a correlation between age and SSR(p=0.05).In KS patients,SSR was determined by pre-op testosterone levels irrespective of age.Conclusion:Pre-operative hormonal response is a predictor for SSR in NOA patients who were treated medically.This data may help during pre-operative counselling.
基金This research was sponsored by the National Key Research and Development Project(SQ2018YFC100243)National Key Research and Development Project(2016YFC1000302)+4 种基金National Key Research and Developmental Program of China(2018YFC1003600)Young Scientists Fund of the National NaturalScience Foundation of China(Grant No.81601272)Clinical MedicinePlusX-Young Scholars Project,Peking University(Grant No.2102018237)Beijing Municipal Natural Science Foundation(7182177)National KeyResearch and Development Program of China(Grant No.2017YFC1002001).
文摘The aim of our study was to compare the sperm retrieval rates(SRRs)and clinical outcomes of patients with different causes of azoospermia who underwent microdissection testicular sperm extraction-intracytoplasmic sperm injection(micro-TESE-ICSI).We conducted a retrospective study at the Reproductive Medicine Center of Peking University Third Hospital in Beijing,China,from January 2014 to December 2017.This study examined 769 patients with nonobstructive azoospermia who underwent 347 cycles of micro-TESE-ICSI.Patients with azoospermia were classified into Group A(Klinefelter syndrome,n=284,125 cycles),Group B(azoospermia Y chromosome factor c[AZFc]microdeletion,n=91,64 cycles),Group C(cryptorchidism,n=52,39 cycles),Group D(previous mumps and bilateral orchitis,n=23,23 cycles),and Group E(idiopathic azoospermia,n=319,96 cycles).Clinical characteristics,SRR,embryonic development,and pregnancy outcomes of the patients were compared between all groups.Patients in Group D had the highest and most successful SRR.The average SRR for all patients was 46.0%.The rates of clinical pregnancy,implantation,and live birth in Group D were 78.3%,65.0%,and 74.0%,respectively,which were higher than those in all other groups(P<0.05).Group B patients had the lowest clinical pregnancy,implantation,and live birth rates of all groups(P<0.05).No differences were found in the miscarriage rate or birth defects among the groups(P>0.05).Patients with orchitis had the highest SRR and best clinical outcomes.Although AZFc microdeletion patients had a higher SRR,their clinical outcomes were worse.
文摘We performed this meta-analysis to evaluate the predictive value of different parameters in the sperm retrieval rate （SRR） of microdissection testicular sperm extraction （TESE） in patients with nonobstructive azoospermia （NOA）. All relevant studies were searched in PubMed, Web of Science, EMBASE, Cochrane Library, and EBSCO. We chose three parameters to perform the meta-analysis： follicle-stimulating hormone （FSH）, testicular volume, and testicular histopathological findings which included three patterns： hypospermatogenesis （HS）, maturation arrest （MA）, and Sertoli-cell-only syndrome （SCOS）. If there was a threshold effect, only the area under the summary receiver operating characteristic curve （AUSROC） was calculated. Otherwise, the pooled sensitivity, specificity, positive likelihood ratio （PLR）, negative likelihood ratio （NLR）, and the diagnostic odds ratio （DOR） were also calculated. Twenty-one articles were included in our study finally. There was a threshold effect among studies investigating FSH and SCOS. The AUSROCs of FSH, testicular volume, HS, MA, and SCOS were 0.6119, 0.6389, 0.6758, 0.5535, and 0.2763, respectively. The DORs of testicular volume, HS, and MA were 1.98, 16.49, and 1.26, respectively. The sensitivities of them were 0.80, 0.30, and 0.27, while the specificities of them were 0.35, 0.98, and 0.76, respectively. The PLRs of them were 1.49, 10.63, and 1.15, respectively. And NLRs were 0.73, 0.72, and 0.95, respectively. All the investigated factors in our study had limited predictive value. However, the histopathological findings were helpful to some extent. Most patients with HS could get sperm by microdissection TESE.
基金Supported by the Natural Science Foundation of Shanghai, No02ZB14072
文摘AIM. To study the differential gene expression profiles of target cells in primary gastric cancer and its metastatic lymph nodes using laser microdissection （LMD） in combination with cDNA microarray. METHODS： Normal gastric tissue samples from 30 healthy individuals, 36 cancer tissue samples from primary gastric carcinoma and lymph node metastasis tissue samples from 58 patients during gastric cancer resection were obtained using LMD in combination with cDNA microarray independently. After P27-based amplification, aRNA from 36 of 58 patients （group 1） with lymph node metastasis and metastatic tissue specimens from the remaining 22 patients （group 2） were applied to cDNA microarray. Semiquantitative reverse transcriptase polymerase chain reaction （RT-PCR） and imrnunohistochemical assay verified the results of microarray in group 2 and further identified genes differentially expressed in the progression of gastric cancer. RESULTS： The expression of 10 genes was up-regulated while the expression of 15 genes was down-regulated in 22 gastric carcinoma samples compared with that of genes in the normal controls. The results were confirmed at the level of mRNA and protein, and suggested that four genes （OPCML, RNASE1, YES1 and ACK1） could play a key role in the tumorigenesis and metastasis of gastric cancer. The expression pattern of 3 genes （OPCML, RNASE1 and YES1） was similar to tumor suppressor genes. For example, the expression level of these genes was the highest in normal gastric epithelium, which was decreased in primary carcinoma, and further decreased in metastatic lymph nodes. On the contrary, the expression pattern of gene ACK1 was similar to that of oncogene. Four genes were further identified as differentially expressed genes in the majority of the cases in the progression of gastric cancer. CONCLUSION： LMD in combination with cDNA microaro ray provides a unique support foe the identification of early expression profiles of differential genes and the expression pattern of 3 genes （OPCML, RNASE1 and YES1） associated with the progression of gastric cancer. Further study is needed to reveal the molecular mechanism of lymph node metastasis in patients with gastric cancer.
文摘In 220 consecutive patients with non-obstructive azoospermia, sperm retrieval was attempted by a combination of conventional and microdissection testicular sperm extraction （TESE）. For sperm retrieval, 2-3 conventional biopsies were performed followed by a microdissection TESE in cases of negative conventional biopsies. During the surgery, the vasculature of the testis was assessed using the operative microscope, and the location of positive biopsies was registered in relation to the blood supply. The overall sperm retrieval rate was 58.2%. From the initial conventional biopsies, sperm could be retrieved in 46.8% of the patients. With microdissection TESE, sperm could be retrieved from an additional 11.4% of the patients. The further use of microdissection TESE improved the sperm retrieval rate significantly （P=0.017）. No significant accumulation of positive biopsies was found towards the rete testis or the main testicular vessels.
基金supported by a grant from the Peking University Clinical Medicine Youth Special Fund(PKU20222LCXQ042).
文摘Microdissection testicular sperm extraction(micro-TESE)is widely used to treat nonobstructive azoospermia.However,a good prediction model is required to anticipate a successful sperm retrieval rate before performing micro-TESE.This retrospective study analyzed the clinical records of 200 nonobstructive azoospermia patients between January 2021 and December 2021.The backward method was used to perform binary logistic regression analysis and identify factors that predicted a successful micro-TESE sperm retrieval.The prediction model was constructed using acquired regression coefficients,and its predictive performance was assessed using the receiver operating characteristic curve.In all,67 patients(sperm retrieval rate:33.5%)underwent successful micro-TESE.Follicle-stimulating hormone,anti-Miillerian hormone,and inhibin B levels varied significantly between patients who underwent successful and unsuccessful micro-TESE.Binary logistic regression analysis yielded the following six predictors:anti-Mullerian hormone(odds ratio[OR]=0.902,95%confidence interval[Cl]:0.821-0.990),inhibin B(OR=1.012,95%Cl:1.001-1.024),Klinefelter’s syndrome(OR=0.022,95%Cl:0.002-0.243),Y chromosome microdeletion(OR=0.050,95%Cl:0.005-0.504),cryptorchidism with orchiopexy(OR=0.085,95%Cl:0.008-0.929),and idiopathic nonobstructive azoospermia(OR=0.031,95%Cl:0.003-0.277).The prediction model had an area under the curve of 0.720(95%Cl:0.645-0.794),sensitivity of 65.7%,specificity of 72.2%,Youden index of 0.379,and cut-off value of 0.305 overall,indicating good predictive value and accuracy.This model can assist clinicians and nonobstructive azoospermia patients in decision-making and avoiding negative micro-TESE results.
基金supported by the National Natural Science Foundation of China(81973701 and 82204772)the Natural Science Foundation of Zhejiang Province(LZ20H290002)+2 种基金the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine(ZYYCXTD-D-202002)the China Postdoctoral Science Foundation(2022M712811)Westlake Laboratory(Westlake Laboratory of Life Sciences and Biomedicine).
文摘Spatial omics technologies have become powerful methods to provide valuable insights into cells and tissues within a complex context,significantly enhancing our understanding of the intricate and multifaceted biological system.With an increasing focus on spatial heterogeneity,there is a growing need for unbiased,spatially resolved omics technologies.Laser capture microdissection(LCM)is a cutting-edge method for acquiring spatial information that can quickly collect regions of interest(ROIs)from heterogeneous tissues,with resolutions ranging from single cells to cell populations.Thus,LCM has been widely used for studying the cellular and molecular mechanisms of diseases.This review focuses on the differences among four types of commonly used LCM technologies and their applications in omics and disease research.Key attributes of application cases are also highlighted,such as throughput and spatial resolution.In addition,we comprehensively discuss the existing challenges and the great potential of LCM in biomedical research,disease diagnosis,and targeted therapy from the perspective of high-throughput,multi-omics,and single-cell resolution.
基金This research was sponsored by the National Key Research and Development Projects(No.2018YFC1003600,2016YFC1000302,2017YFC1002001 and SQ2018YFC100243)the Clinical Medicine PlusX Young Scholars Project,Peking University(No.2102018237)the Beijing Municipal Natural Science Foundation(No.7182177).
文摘We performed this study to evaluate the clinical outcomes of microdissection testicular sperm extraction-intracytoplasmic sperm injection(micro-TESE-ICSI)treatment that used fresh or cryopreserved sperm in patients with nonobstructive azoospermia(NOA).A total of 338 NOA patients with 344 consecutive cycles received treatment in the reproductive medicine center of Peking University Third Hospital in Beijing,China,from January 2014 to December 2017.Fresh oocytes and fresh sperm were used in 222 patients with 234 cycles(Group A).Fresh oocytes and cryopreserved sperm were used in 116 patients with 110 cycles(Group B).We compared patient characteristics,embryonic development,and pregnancy outcomes between Groups A and B.There was no statistical difference in the patient characteristics,and no differences were observed with fertilization or quality embryo rates between Groups A and B.The rates of clinical pregnancy and live birth were both higher for Group A than those for Group B(both P<0.05).In conclusion,fresh testicular sperm appears to produce better ICSI outcomes than cryopreserved testicular sperm in patients with NOA.
基金National Science Council, NSC-91-3112-B002-007, Taipei, Taiwan, China
文摘AIM： To examine the gene expression profile of gastric cancer （GC） by combination of laser capture microdissection （LCM） and microarray and to correlate the profiling with histological subtypes. METHODS： Using LCM, pure cancer cells were procured from 45 cancerous tissues. After procurement of about 5 000 cells, total RNA was extracted and the quality of RNA was determined before further amplification and hybridization. One microgram of amplified RNA was converted to cDNA and hybridized to cDNA microarray. RESULTS： Among 45 cases, only 21 were qualified for their RNAs. A total of 62 arrays were performed. These included 42 arrays for cancer （21 cases with dyeswab duplication） and 20 arrays for non-tumorous cells （10 cases with dye-swab duplication） with universal reference. Analyzed data showed 504 genes were differentially expressed and could distinguish cancerous and non-cancerous groups with more than 99% accuracy. Of the 504 genes, trefoil factors 1, 2, and 3 were in the list and their expression patterns were consistent with previous reports. Immunohistochemical staining of trefoil factor 1 was also consistent with the array data. Analyses of the tumor group with these 504 genes showed that there were 3 subgroups of GC that did not correspond to any current classification system, including Lauren＇s dassification. CONCLUSION： By using LCM, linear amplification of RNA, and cDNA microarray, we have identified a panel of genes that have the power to discriminate between GC and non-cancer groups. The new molecular classification and the identified novel genes in gastric carcinogenesis deserve further investigations to elucidate their clinicopathological significance.
文摘The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.
文摘Since chromosome microdissection and microcloning was first developed by Scalenghe et al(1981)with poly-tene chromosome of Drosophila,it had been greatly im-proved and evolved to an efficient tool for generation of chromosome-specific DNA libraries in human,animals and plants(Greenfield and Brown,1987;Ludecke et al,1989;Guan et al,1992;Jung et al,1992.
基金supported by the National Natural Science Foundation of China,Nos. 31730031 and 32130060the National Major Project of Research and Development,No. 2017YFA0104700the Natural Science Foundation of Jiangsu Province,No. BK20202013 (all to XSG)。
文摘The key regulators and regeneration-associated genes involved in axonal regeneration of neurons after injury have not been clarified.In high-throughput sequencing,various factors influence the final sequencing results,including the number and size of cells,the depth of sequencing,and the method of cell separation.There is still a lack of research on the detailed molecular expression profile during the regeneration of dorsal root ganglion neuron axon.In this study,we performed lase r-capture microdissection coupled with RNA sequencing on dorsal root ganglion neurons at 0,3,6,and 12 hours and 1,3,and 7 days after sciatic nerve crush in rats.We identified three stages after dorsal root ganglion injury:early(3-12 hours),pre-regeneration(1 day),and regeneration(3-7 days).Gene expression patterns and related function enrichment res ults showed that one module of genes was highly related to axonal regeneration.We verified the up-regulation of activating transcription factor 3(Atf3),Kruppel like factor 6(Klf6),AT-rich inte raction domain 5A(Arid5α),CAMP responsive element modulator(Crem),and FOS like 1,AP-1 transcription factor Subunit(Fosl1) in dorsal root ganglion neurons after injury.Suppressing these transcription factors(Crem,Arid5o,Fosl1 and Klf6) reduced axonal regrowth in vitro.As the hub transcription factor,Atf3 showed higher expression and activity at the preregeneration and regeneration stages.G protein-coupled estrogen receptor 1(Gper1),inte rleukin 12a(Il12α),estrogen receptor 1(ESR1),and interleukin 6(IL6) may be upstream factors that trigger the activation of Atf3 during the repair of axon injury in the early stage.Our study presents the detailed molecular expression profile during axonal regeneration of dorsal root ganglion neurons after peripheral nerve injury.These findings may provide reference for the clinical screening of molecular targets for the treatment of peripheral nerve injury.
文摘This study aims to explore the factors influencing the success rate of the microdissection testicular sperm extraction(Micro-TESE)in patients with nonobstructive azoospermia(NOA)and cryptorchidism.Clinical data of 162 patients with cryptorchidism who underwent Micro-TESE due to infertility from December 2015 to May 2020 in the First Affiliated Hospital of Nanjing Medical University were analyzed retrospectively.In the univariate analysis,significant differences in the age of patient at the time of orchidopexy(median[interquartile range,IQR]:7.0[4.0–11.0]years vs 11.5[9.0–14.5]years,P<0.001),interval between orchidopexy and Micro-TESE(mean±standard deviation:17.5±5.0 years vs 14.4±4.4 years,P<0.001),severity of cryptorchidism(unilateral[62.8%]vs bilateral[31.6%],P<0.001;location of cryptorchidism,intra-abdominal[27.3%]vs inguinal[44.8%]vs suprascrotal[66.7%],P<0.001),volume of the dominant testis(median[IQR]:17.00[15.00–19.00]ml vs 14.50[11.75–16.25]ml,P<0.001),and levels of follicle-stimulating hormone(FSH;P=0.004)and testosterone(P=0.006)were observed between the successful and failed sperm extraction groups.After conducting the multivariate analysis,four of these factors,including unilateral/bilateral cryptorchidism(P<0.001),location of cryptorchidism(P=0.032),age of orchidopexy(P<0.001),and dominant testicular volume,were adopted in the clinical prediction model to evaluate preoperatively the success rate of Micro-TESE for patients with NOA and cryptorchidism.The likelihood of successful sperm retrieval by Micro-TESE in men with NOA and cryptorchidism increased in patients with mild forms of cryptorchidism.
文摘Sperm identification and selection is an essential task when processing human testicular samples for in vitro fertilization.Locating and identifying sperm cell(s)in human testicular biopsy samples is labor intensive and time consuming.We developed a new computer-aided sperm analysis(CASA)system,which utilizes deep learning for near human-level performance on testicular sperm extraction(TESE),trained on a custom dataset.The system automates the identification of sperm in testicular biopsy samples.A dataset of 702 de-identified images from testicular biopsy samples of 30 patients was collected.Each image was normalized and passed through glare filters and diffraction correction.The data were split 80%,10%,and 10%into training,validation,and test sets,respectively.Then,a deep object detection network,composed of a feature extraction network and object detection network,was trained on this dataset.The model was benchmarked against embryologists’performance on the detection task.Our deep learning CASA system achieved a mean average precision(mAP)of 0.741,with an average recall(AR)of 0.376 on our dataset.Our proposed method can work in real time;its speed is effectively limited only by the imaging speed of the microscope.Our results indicate that deep learning-based technologies can improve the efficiency of finding sperm in testicular biopsy samples.
