Molecular mechanisms associated with human germ cell aplasia in infertile men remain undefined. Here we perform single-cell transcriptome profiling to highlight differentially expressed genes and pathways in each somatic cell type in testes of men with idiopathic germ cell aplasia. We identify immaturity of Leydig cells, chronic tissue inflammation, fibrosis, and senescence phenotype of the somatic cells, as well markers of chronic inflammation in the blood. We find that deregulated expression of parentally imprinted genes in myoid and immature Leydig cells, with relevant changes in the ratio of Lamin A/C transcripts and an active DNA damage response in Leydig and peritubular myoid cells are also indicative of senescence of the testicular niche. This study offers molecular insights into the pathogenesis of idiopathic germ cell aplasia. Molecular mechanisms associated with human germ cell aplasia in infertile men remain undefined. Here the authors perform single-cell transcriptome profiling to highlight differentially expressed genes and pathways in each somatic cell type in testes of men with idiopathic germ cell aplasia. Germ cell aplasia (GCA), also known as Sertoli cell-only syndrome (SCOS) from a histological point of view, represents the most severe form of male infertility, clinically defined as non-obstructive azoospermia (NOA).Spermatogenesis depends on the presence of testicular spermatogonial stem cells, along with their amplification and transition into progenitor, differentiating spermatogonia and, eventually, spermatozoa2, in almost 80% of NOA cases it is not possible to identify a clear cause, thus including those cases in the wide group of idiopathic NOA (iNOA)3. In this context, several studies demonstrated the relevance of the somatic cells on stem cell number and male spermatogenesis in the testicular microenvironment7. Similarly, the testis microenvironment has a central role in GCA8, including an altered vitamin A and vitamin K signaling in Sertoli and Leydig cells, respectively9, and an enriched testicular bacterial load10.Although both monogenic and oligogenic mutations are associated with NOA12 with a relatively higher risk of death11 compared to fertile men. Overall, these data support the concept that male infertility may be considered as a proxy of overall men’s health13.Epidemiological data show that infertility status per se, and mostly in NOA cases, is associated with an increased risk of early onset of age-related chronic diseases Here, we leveraged the single-cell RNAseq to unveil the occurrence of dysregulated transcriptional pathways in testicular somatic cell populations in idiopathic GCA (iGCA). Findings from this study depict the contribution of 7 somatic cell populations to the complex framework of iGCA, and provide a list of defects potentially associated with an early onset of age-related pathological processes in men with iNOA. Moreover, most of the transcriptional signatures have been confirmed at tissue and systemic levels. Overall, the translational outcomes of this study have a relevant impact on the recognition of a number of biological processes and biomarkers associated i) with male infertility, and ii) early onset of aging processes in young men still in their reproductive age.In iNOA men with negative sperm retrieval at microdissection TEsticular Sperm Extraction (microTESE), the presence of seminiferous tubules lacking any germ cell configured the histological diagnosis of GCA Fig. . Convers14 plot  cluster to pericytes18 or vascular smooth muscle cells according to the expression of FABP420 on freshly isolated tissue specimens from microTESE. A total of 3880 cells, obtained after filtering out poor-quality cells from 3 iGCA donors  and SELENOH 30.Fifty-six downmodulated pathways were common to all somatic cell populations in iGCA, with the aforementioned exception of the TCL cluster Fig. –28. Mostpx4 Fig.  in SRT cpx4 Fig. , includiOther downregulated pathways of particular interest were the mitochondrial respiratory chain complex and the oxygen-dependent proline hydroxylation of hypoxia-inducible factor Fig. .24. To exclude the presence of potential artifacts due to different library preparations, we quantified the ribosomal genes expression global levels in samples from our cohort and from the Guo15 and Sohni24 datasets; overall, ribosomal protein genes were overexpressed in somatic cells compared to germ cells and the read coverage onto ribosomal protein genes was comparable in our OA sample and the ones from Guo and Sohni, thus pointing to a real biological difference rather than batch effects 32 and altered biochemical ECM composition associated with impaired metabolism of the retinoic acid9, we hypothesized that the somatic testicular niche of iGCA might be representative of a testis stuck at an immature stage. Here, we provided several observations to support our hypothesis. First, we examined the expression of the Delta Like Non-Canonical Notch Ligand 1 (DLK1) gene, which is considered a marker of the immaturity of LEY cells. DLK1 is expressed in the human testis during fetal, neonatal, and pre-pubertal stages33, is an inhibitory modulator of Notch signaling34, whose block determines the final maturation of fetal LEY cells35. Here, we found overexpression of DLK1 and NOTCH2 in LEY and MYD cells in iGCA vs. adult testis . By comparing the integrated datasets from the Guo15 and Sohni24 with ours, we found that imprinted genes are dramatically either over or under-expressed in LEY and MYD populations associated with iGCA, 47, whose secretion correlates with senescence. Somatic cells in three non-neoplastic testes  29–36), showed nuclear HMGB1 localization with a similar degree of staining. Conversely, HMGB1 was found in the cytoplasm of SRT, LEY, and MYD cells in all seven iGCA tested  proteinted Fig. . These rThe higher tumor frequency reported for men with azoospermia prompted us to proceed from chronic tissue inflammation to senescence and DNA damage as possible causes of genomic instability in iGCA.To support the senescent phenotype inferred by SASP detection, we looked for the presence of known indicators of cellular senescence-like cell cycle-related proteins. Indeed, although p16/CDKN2A (Cyclin-Dependent Kinase Inhibitor 2A) is not differentially expressed in iGCA somatic cells, the staining revealed a strong accumulation of p16 in iGCA LEY cells as opposed to control testis , thus pointing to an active DDR. In addition, by DAPI staining, we noted very distorted nuclei in the LEY and SRT cell population, reminiscent of nuclei in patients with nuclear envelopathies, a group of rare genetic disorders characterized by a variety of clinical symptoms connected with premature aging and caused by mutations in genes encoding for components of the nuclear lamina50. Indeed, the membrane of LEY nuclei from iGCA, but not SRT nuclei, were significantly more indented than the CTL cell counterparts (P < 0.001) as assessed by the nuclear circularity parameter, whereby the value 1 corresponds to a smooth nuclear surface and 0 to high levels of nuclear indentation  that is detected by the nuclear presence of phosphorylated histone H2AX (γH2AX), which is considered an early marker of DNA damage 49 Fig. . SRT, MYion Fig. . Likewision Fig. .lamin A/C gene  in our three iGCA patients which were in homozygosity (TT) for iGCA#2 and in heterozygosity (C/T) for iGCA #1 and #3; the same mutation was not observed in the 5 CTL datasets  site at the 3′ end of exon 10 , we hypothesized that the T substitution in the mutant alleles would (i) lead to a shorter U1 spliceosome complex half-life (1.12 a.u.), (ii) decrease the splicing events probability at that SD site, and (iii) both decrease the production of the full-length LMNA transcript and favor the truncated form LMNC. Indeed, the kallisto55 isoform analysis applied to the high-coverage RNA-seq on the LMNA/C locus (3000–6000 reads) identified five major LMNA/C transcript isoforms  while a significant fivefold enrichment of Variant 5 was detected (p = 0.02). The overexpression of Variant 5 functionally supports the hypothesis of the weak splice donor site: indeed, the transcript terminates with the exon10 sequence plus a short sequence from intron 10 (18 bp plus stop codon and 3′UTR in intron 10), which codes for the same 6aa present in Variant 2/LMN C. These data suggest that exon10-to-exon11 splicing events have lower odds in iGCA with rs4641 LMNA polymorphism than in CTLs with the wildtype gene splice sequence. In addition, the 5′end is transcribed from an alternative ex1, which excludes the first 100 aa in the protein that is then replaced by an alternative unrelated 20 aa sequence  37 (33–40) years) we observed that LH/tT ratio was independent of sperm parameters, whereas the same ratio was increased in an age-matched cohort of iNOA men with GCA  years)  in the 41 iNOA men with GCA vs. the 41 normozoospermic individuals with proven fertility Fig. . These drs) Fig. . Therefo58, associated with the cytoplasmic localization of HMGB1, the pro-inflammatory profile of MCR, and the presence of tissue-resident CD8+ TCL in iGCA patients, prompted us to investigate whether the inflammatory processes are only localized in the testis environment or are more systemic. Prompted by the overexpression of cytokines and chemokines in TCL, we quantified blood circulating CCL4 and CCL5, which belong to the SASP proteins secreted in inflammation and senescence. In iNOA men with iGCA vs. age-matched controls, levels of CCL4 and CCL5 chemokines are significantly higher  clearly revealed an overlap with all ribosomal genes, UBA52 and NACA reported being associated with aging, for all seven somatic iGCA clusters  is one of the principal components of the envelope and one of the most studied genes since it harbors hundreds of relevant mutations, many of which are linked to the Hutchinson–Gilford syndrome, an extremely rare, progressive genetic disorder that causes children to age rapidly.LMNA/C gene and results in a disproportionate meshwork of intermediate filament type V in the nuclear envelope. The lamin isoforms ratio is cell and tissue-specific and is designed to architecture chromatin in lamin-associated domains, which eventually governs cell and tissue-specific transcription60. In addition, the nuclear envelope also sustains nuclear mechanics and mechanotransduction61. Therefore, structural misshapen nuclei might be directly responsible for dysfunctional transcriptional landscapes in iGCA or, vice versa, harsh environments might induce structural damage on otherwise weak nuclei followed by dramatic transcriptional changes. In addition, prolonged mechanic and inflammatory stresses might sustain the overload of DNA damage, which eventually leads to genomic instability and cancer62. In addition, an aberrant nuclear organization can also lead to gene imprinting defects as recently demonstrated63. Our findings are just adding some more pieces to the puzzle of germ cell aplasia and will be worth deeper explorations in the future.From the molecular analyses in the three iGCA transcriptomes, we identified an excess of shorter LMNC-like transcripts that we attributed to a mild polymorphism in the 66 to a genomewide defect recalling multi-locus imprinting disturbances67 as opposed to faulty methylation of specific regions, worth to be characterized in the future. Any metabolic or environmental factors during embryo development might also account for imprinting alterations in iGCA somatic cells. Indeed, gestational hypertension/eclampsia is found associated with changes in the methylation levels of DLK168 and IGF270, which we also find substantially deregulated; moreover, offspring born from preeclamptic women are at higher risk for metabolic diseases in later life , conditions that apply also to iNOA men72. These findings provide hints on the epigenetic etiopathogenesis behind iGCA and offer relevant working hypotheses for an early onset of age-related chronic diseases in azoospermic men.During the development, the somatic cells reconstitute the genomic imprinting present in the zygote. Of note, several paternally and maternally imprinted genes are massively upregulated or downregulated in LEY and SRT cells in our iGCA patients as compared to CTLs Fig. . The obs73 to a pro-inflammatory testicular environment, eventually associated with GCA. In addition, the identified resident cytotoxic TCL, type 1 MCR, and the hyalinization of the seminiferous tubules support the hypothesis of a previous inflammatory or autoimmune event which turned into a chronic inflammatory status. However, the detection of cytoplasmic and extracellular HMGB1, a prototypical DAMP protein secreted upon inflammatory insults and inflammatory stimulus per se74, supports an active inflammatory status of the testes in iGCA patients.The increased signaling of IFNs in TCL and MCR cells, documents a shift from the immune-privileged status75. Several findings in this study corroborate the setting to a pre-neoplastic environment in iGCA tissues, such as the biallelic and deregulated expression of the imprinted gene IGF2, reported in more than 50% of human testicular germ cell tumors76 and the accumulation of γH2AX in MYD and LEY cells indicative of a sustained DDR.It is well-known that a chronic proinflammatory environment predisposes to increased incidence of cancer77. For instance, the oxygen-dependent proline hydroxylation downregulated pathway—a process that has been linked to stabilization and nuclear translocation of HIF1-alpha—and the downregulated transcription of selenium metabolism genes impair the redox control in tissues and blood (SELENOP). In addition, SELENOH proteins and the Gpx family of peroxireductases contribute to the redox status control and to the maintenance of genome stability78.Some deregulated metabolic pathways in iGCA testes might also contribute to preneoplastic environments and oncogenes activationUncontrolled redox stress in immature LEY cells, in the overall iGCA somatic compartment, and in blood hint to processes related to global early aging.This study unveils insights into idiopathic germ cell aplasia, such as the immature phenotype of LEY cells stuck at the prepuberal stage of development and expressing clear markers of senescence that might support the hypothesis of exhaustion in the somatic progenitor cell compartment, still needing an experimental validation.Pathways linked to mechanisms potentially responsible for the condition of iGCA open perspectives for a better understanding of the pathobiology behind the clinical condition of iNOA and the potential association with a number of clinical manifestations, such as chronic non-oncological diseases and higher rates of cancers.79. Men were classified as azoospermic when no spermatozoa were detected in at least two consecutive semen analyses80. Idiopathic non-obstructive azoospermia was defined after a comprehensive investigation of all known causes of azoospermia, according to the inclusion and exclusion criteria recently reported32.Specimens and complete clinical data were collected at one tertiary-referral center for reproductive medicine. According to the World Health Organization (WHO) criteria, infertility was defined as not conceiving a pregnancy after at least 12 months of unprotected intercoursesAdult human testicular samples: scRNA-seq was performed with fresh testicular tissue obtained from four azoospermic men during microTESE. Of those, three men were characterized by iNOA with negative sperm retrieval at microTESE and a histological diagnosis of SCOS; #1 , #2 , #3 . Testicular tissue specimen with normal spermatogenesis was obtained from 1 man with obstructive azoospermia (age = 37 years) associated with CFTR gene mutation, which was positive for sperm retrieval at surgery and depicted normal spermatogenesis at the histological analysis .81. Sperm retrieval was attempted on testis specimen, and patients were then dichotomized according to the surgical outcome ; tissue was delivered to the Unit of Obstetrics/Gynecology and processed for sperm retrieval as a part of in vitro fertilization program; sperm were counted, and sperm retrieval was expressed as the number of sperm/high power field (HPF)32. ScRNA-seq data from adult human testis were obtained from two previous studies24, which used testes removed from three deceased men , and from two vasectomies (age = 37 and 42) respectively, and proven to have normal spermatogenesis at the histological analysis15.All four azoospermic men underwent microTESE according to the original surgical techniqueTesticular specimens dedicated to this study were delivered from the operating theater to the laboratory on cold Krebs solution (Merck KGaA); after being split, part of the tissue was fixed for histological analysis, while the remaining part was processed for single cell dissociation. Normal maturation of the germline was defined according to a thorough histological analysis performed on formalin-fixed paraffin-embedded slides of the testis.82. Briefly, three μm paraffin sections from each sample were pretreated in a microwave oven, two cycles of 5 min each, at 780 W in 0.25 mM EDTA buffer, and then incubated for 2 h with specific antibodies against human antigens and counterstained with hematoxylin. The reactions were revealed by a non-biotin peroxidase detection system with 3,3-diaminobenzidine free base as the chromogen. Primary antibodies against human antigens were anti-Collagen I , anti-Collagen IV , anti-HMGB1 (Abcam 18256); negative control was obtained by omitting the primary Ab. Control tissues with normal spermatogenesis used for IHC analysis were obtained from the non-neoplastic tissue of men submitted to unilateral orchiectomy for nonmetastatic seminoma; the tissue was obtained from the most distant area from the tumor and identified as having normal germline cell maturation based on a thorough histological analysis. For each seminoma patient, preoperative semen cryopreservation excluded NOA in all cases9.The expression of collagen I, collagen IV, and HMGB1 were analyzed through immunohistochemistry (IHC) staining performed on FFPE slides of the testis according to a methodology previously describedn = 41) was used as a reference cohort for the hormonal profile.Previously collected formalin-fixed paraffin-embedded testis parenchyma, and venous blood from iNOA men was used for retrospective analysis and as the validation cohort. Peripheral blood from a cohort of men with proven fertility and normozoospermia , and was composed of 2 protocols for collecting data and using bio-specimens from infertile  and fertile men , and 1 protocol for biobanking . All methods were carried out in accordance with the approved guidelines.TM Tip strainers, Merck KGaA) to obtain a single cell. Cell numbers were counted in a Burker chamber and cell viability was evaluated by Trypan blue staining. Cells were suspended in 0.4% BSA/PBS at the concentration of 1000 cells/μl, ready for single-cell sequencing. The recovered cell number was in the range of 3–5 × 106 cells, with viability > 75%.Fresh testis parenchyma was washed twice in PBS and subjected to enzymatic digestion  for 10 min at 37 °C with gentle agitation, repeated twice (1 ml of digestion medium each time). The supernatants were unified and the cell suspension was filtered through strainers with mesh size 70 μm. The cells were pelleted down by centrifugation (450 g for 5 min), washed twice with 0.4% BSA/PBS, and filtered through a 40 μm cell strainer  according to manufacturer’s instructions.83. Briefly, 3–5 areas from the same histological section were evaluated for each donor, using 10× magnification acquired with Zeiss AxioImager M2M. Imagines from the histological section were acquired on brightfield to show tissue location, and on darkfield/polarized light to obtain collagen birefringence. Anisotropy was estimated by using the function “measure” and the plug-in FibrilTool84 in the Image J software. Anisotropy was measured on the basal membrane of the seminiferous tubules.The degree of organization of fibrils (anisotropy) was evaluated as previously reported–set-cell-number option). Reads were mapped to the reference genome using STAR v.2.5.3a and assigned to genes with featureCounts v.1.6.4. GRCh38 was used as the reference genome. Bam files were sorted with samtools (v1.9). Finally, Umi-Tools count was used to processing the UMIs aligned to each gene in each cell to find the number of distinct, error-corrected UMIs mapping to each gene. The UMI count tables of each cellular barcode were used for further analysis.For each experiment, 2500 single cells were processed on the Chromium platform (10×) using the Chromium Single Cell 3′ Library & Gel Bead Kit v3 kit (10×). After quality controls and quantification on TapeStation instrument (Agilent), libraries were sequenced on NextSeq500 or NovaSeq6000 platforms (Illumina) generating around 100'000 reads/cell. Raw sequencing data were demultiplexed with the mkfastq application (Cell Ranger v.3.0.2). UMI-Tools (v.1.0.0) whitelist and extract commands were used to identify and select the number of cell barcodes to use in downstream analysis, setting the cell number to 2000 for donor 1 and donor 2, 5000 for donor 3 and 1500 for donor OA , within the R environment (v3.6.1). Seurat objects were built from the UMI count tables from each individual experiment. We reported an average of 80,000 reads in each cell, and a median of 1560.0 genes per cell. We filtered out cells with less than 500 genes expressed and with more than 20% reads mapped to the mitochondrial genome (percent.mt). The retained cells were normalized and scaled, regressing out for number of RNA Features, percent.mt and cell cycle. PCA and unsupervised clustering were performed using the top 2000 highly variable genes and the first 20 PCs. Analyzing the resulting clusters in one of the replicas (ID 204—Donor1) we decided to further remove the cells forming one of the clusters presenting more than three mitochondrial genes among its marker genes and a low number of RNA features  and Sohni et al. (GSE124263). Annotation differences between these two datasets and our annotation (GENCODE v31 basic annotation) were corrected by updating the gene names from Guo et al. and Sohni et al. to their most recent gene alias in GENCODE v31. This conversion was performed using the Ensembl IDs, which remain comparable across different annotation versions. The data were then re-analyzed according to our pipeline, as described above. Guo et al. dataset was also used as a reference to characterize the cell identity of the OA and iNOA samples  in the immune cell clusters  in each cell cluster from the testis of iNOA men with negative sperm retrieval and of men with normal spermatogenesis was performed with the FindMarkers function in Seurat, using default values . We report all the resulting differential expressed genes in Supplementary Dataset P value < 0.05. We perform the enrichment analysis with the EnrichR package (v.2.1), which provides an R interface to the ‘Enrichr’ databases and statistics86. In the paper, we mainly described results obtained in the Reactome_2016 datasets and GO_Biological_Process_2018. However, in SI  and used to compute an adjacency matrix for all possible pairs of significant pathways, which was then represented with the CRAN package pheatmap (v.1.0.12).To reduce redundancy between genomics pathways and hence aid data interpretation, all the pathways of the Reactome_2016 and GO_Biological_Process_2018 datasets with adj. 28 using the statistical_analysis method, performing 100 iterations for the statistical analysis. We use as input-count-matrix the raw data extracted from the integrated Seurat object (RNA assay) and normalized as prescribed by CellPhoneDB authors (https://www.cellphonedb.org/faq-and-troubleshooting) for iGCA and control samples, separately. The metadata was produced from the Seurat metadata considering the defined cell type assigned after cell clustering. The interaction heatmaps were produced with the CellPhoneDB heatmap_plot function, which uses the pheatmap R library (v.1.0.12).The number of total interactions between cell types was assessed with CellPhoneDB v2.0We combined data from 3 datasets reporting single-cell RNA seq of neonatal, prepubertal, and adult human testes by Cairns group ) and analyzed those according to the standard Seurat workflow , built on 16 samples of CD3+ T cells isolated from tissues and blood, was used as a reference to characterize iGCA T cells  or Reveal Decloaker pH 6.0 , respectively. Next, tissue sections were allowed to cool and then kept at room temperature for 20 min. Tissue sections were washed twice in PBS and permeabilized in PBS containing 0.4% Triton  and 0.1% donkey serum  for 20 min at room temperature; the aspecific sites were blocked with blocking buffer (10% donkey serum in PBS-Triton 0.4%) for 1 h at room temperature. Tissue sections were incubated with primary antibodies in the humid box at 4 °C for 16 h. After two washes in washing buffer (PBS containing Triton 0.4%), secondary fluorescence-conjugated antibodies were added in a dark humid box and incubated for 1 h at room temperature. Slides were washed twice with washing buffer and nuclei were stained with NucBlue Live ReadyProbe Reagent diluted 1:10 in PBS for 30 min at room temperature. After removing the excess of this solution, tissue sections were rinsed in PBS and mounted with Fluorescence Mounting Medium . Images were acquired by using Olympus FluoVIEW FV3000RS Confocal, objective UPLXAPO 60XO (NA 1.42) Oil, and imaging analysis performed with the software ImageJ. Primary and secondary antibodies, including dilution of each antibody and the antigen retrieval used for each antibody, are reported in the Supplementary Dataset Supplementary Dataset 15 or collected after vasectomy24. The tissue specimens were frozen and then thawed for RNAseq.In our and other studies, similar procedures had been used for tissue dissociation, consisting of enzymatic digestion completed in 30 min. The cell processing was also similar, and the RNA-seq was performed with the 10× Genomic system on the Chromium platform. Collection and storage of the tissues were the main differences between our study and the others we used. In our study, we used fresh testicular tissue obtained during microTESE intervention, and the tissues were kept in cold Krebs solution and processed within 15 min from the collection in the surgery room (as reported above). In the studies from others, the authors used tissues from autoptic donorsTo overcome the potential bias in the DGE and modulated pathways analyses of comparing fresh vs. autoptic or frozen tissues, we have firstly combined control donors from multiple input datasets (from Cairns’ and Wilkinson’s groups). Secondly, we have used the integration method available in Seurat v3, which is aimed at correcting for technical differences between datasets , and performing comparative scRNA-seq analysis across different experimental conditions. Lastly, we added a comparative analysis of iNOA samples with the RNAseq dataset obtained from the testis of an OA man with normal spermatogenesis processed in our lab. We found similar DEG and deregulated pathways in LEY and MYD cells as in the comparison with others’ datasets , and significance was analyzed by two-tailed Mann–Whitney or Kruskal–Wallis test, with Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Supplementary Dataset 4Supplementary Dataset 5Supplementary Dataset 6Supplementary Dataset 7Supplementary Dataset 8Supplementary Dataset 9Supplementary Dataset 10Supplementary Dataset 11Supplementary Dataset 12Supplementary Dataset 13Supplementary Dataset 14Supplementary Dataset 15Supplementary Dataset 16Supplementary Dataset 17Supplementary Dataset 18Supplementary Dataset 19Supplementary Dataset 20Supplementary Dataset 21Supplementary Dataset 22Supplementary Dataset 23Supplementary Dataset 24Supplementary Dataset 25Supplementary Dataset 26Supplementary Dataset 27Supplementary Dataset 28Supplementary Dataset 29Supplementary Dataset 30Supplementary Dataset 31Supplementary Dataset 32Supplementary Dataset 33Supplementary Dataset 34Supplementary Dataset 35Supplementary Dataset 36Reporting Summary


Background and purpose — We noted several adverse events in patients in whom the first version of the STRYDE limb-lengthening nail  had been implanted. Pain, osteolysis, periosteal reactions, and cortical hypertrophy at the nail junction were noted. Here, we present the analysis of 23 retrieved STRYDE implants.Materials and methods — We undertook visual inspection of the retrieved nails and screws, mechanical evaluation of the junction, micro-CT analyses, microscopic inspection of the bushing, screws, screw holes, and separated parts of the implants. Positive material identification (PMI) and energy-dispersive X-ray spectroscopy (EDS) were used to analyze the chemical composition. The hardness of the material was also investigated.Results — 20/23 retrieved nails had visible signs of corrosion, i.e., discoloration at the telescopic junction. Micro-CT verified corrosion attacks in 12/12 scanned bushings. Corrosion, predominantly mechanically assisted crevice corrosion, was observed at the locking screws and screw holes in 20/23 nails. Biological material inside the nail was observed in addition to oozing from the junction of 2 nails during hardware removal, which was experimentally reproducible. Notably, the mechanical construction of the bushing changed from PRECICE P2 to STRYDE nails.Interpretation — STRYDE nails are not hermetically sealed, and liquid can pass the bushing. Biodur 108 itself is corrosion resistant; however, mechanically assisted crevice corrosion of the bushing, locking screws, and screw holes may be aggravated due to manufacturing aiming for increased strength and hardness of the alloy.Observing several adverse events, we recently published a nationwide cross-sectional analysis of all 30 STRYDE limb- lengthening nails  that were implanted in Denmark . 27/30 STRYDE nails have now been removed and we present data from metallurgical analysis of 23 of the retrieved implants. Supplementary data). Based on these initial observations, the engineers  decided together with JDR which analyses should be performed for the individual nails. Representative samples of “worst” and “best” cases in terms of discoloration at the telescoping nail junction were further analyzed by either FORCE Technology (FT) and/or the Technical University of Denmark (DTU).We performed an analysis of all STRYDE nails, removed either routinely, due to complications, or preemptively due to our recent discovery of adverse events with this implant . STRYDE nails from the 4 centers—Aarhus University Hospital, Aalborg University Hospital, Odense University Hospital, and Rigshospitalet, Copenhagen, Denmark—were visually inspected and photo documented .All nails were inspected and photo documented by the surgeons, and after FT and DTU received these. Handling of the implants was not uniform; some implants were wiped with an ethanol cloth only; others were also washed in a surgical instrument dishwasher before photo documentation. Movement of the telescoping nail parts was assessed with manual compression and distraction of the nail  with 20–100× magnification at DTU and a Leica MC 120 HD camera at FT. Microstructure examination was performed on cut sections of selected nails, followed by mounting in resin and subsequent grinding and polishing down to 1 µm diamond polishing. After etching in Vogels Sparbeize etchant, the microstructure was examined using a Leica DMI 5000 M metallographic microscope.Supplementary data).14 nails (12 STRYDE and 2 PRECICE P2.2 nails) were micro-CT scanned with a Nikon XT H 225 . Overview images of entire nails were recorded at 119.7 µm pixel size, while detailed scans were reconstructed at 19 or 23 µm isotropic resolution at the bushing for 12 nails, the O-rings for 5 nails and the ball bearing for 4 nails .The element weight percentage (wt.%) of the different components of the nail was assessed with X-ray fluorescence using a X-MET 8000 Optimum .SEM was performed using a ZEISS EVO MIK-015 BRUKER X-Flash DET—011 with 15 kV . Suspected biological material was evaluated with energy-dispersive X-ray spectroscopy (EDS) to verify high-levels of oxygen.Data are presented as median (range).The study was conducted with consent of the patients according to the Declaration of Helsinki and was approved by the local institutional review boards. The work conducted by FORCE Technology was financially supported by the Danish Agency for Higher Education and Science, Denmark. No other external financing was received. The authors declare no conflicts of interest.30 STRYDE limb-lengthening nails  were eligible for the study. The median lengthening of 30 bone segments of 27 patients, median age 20 (11–65) years was 35 (15–80) mm. For further details, please see Rölfing et al. (2021a).3 nails are still implanted. Median time from implantation to hardware removal of the 27 retrieved nails was 12 (3.5–20) months. 23/27 nails were available for analysis, because 1 implant was lost to sonication and 3 nails were removed and discarded before we became aware of the observed adverse events. 1 of the 23 nails was returned to the manufacturer for further analysis after visual inspection and photo documentation had been performed.2 surgeons observed “oozing” of a brown substance from the telescopic nail junction during hardware removal in addition to the observation of junctional discoloration .Moreover, 4 of the 27 retrieved STRYDE nails fractured, 3 before and 1 during hardware removal. All 4 patients were within the weight limit of the applied nail .Macroscopic inspection and photo documentation revealed discoloration at the telescoping junction in 20/23 implants  and 20/2Supplementary data).Manual compression and distraction of the devices showed that 13/20 nails could be telescoped several millimeters with little force . EDS analysis of the crystalloid structure showed high contents of fluorine 35 wt.% and aluminum 24 wt.%. The presence of biologic material within the nail was confirmed by visual inspection and a high oxygen content determined by EDS analysis . Moreover, the visible discoloration, i.e., corrosion products, contained the same elements.PMI showed that the housing body, bushing, and locking screws fulfilled the Biodur 108 alloy ASTM 2229 specifications (ASTM 2021), i.e., content of manganese (Mn), chromium (Cr), molybdenum (Mo), and iron (Fe) .Hardness analysis of the bushing, distraction rod, housing, and screws showed a hardness in the 410–445 HV range. This means that the manufactured nail has increased mechanical properties compared with the annealed conditions of the alloy .Distraction—retraction—distraction of 2 nails using the fast distractor showed that the nails were not hermetically sealed. Air was pressed out of the nail, forming bubbles during distraction underwater. After subsequent retraction underwater and drying of the nail, the nail was distracted, which led to brown liquid leaking out of the telescoping junction  before cortical hypertrophy occurs, which is likely the end result—unless the cortical destruction is substantial .Whereas MAGEC and PRECICE are made of a titanium alloy, STRYDE is made of stainless steel Biodur 108. In general, austenitic stainless steel (as in the AISI 300 series) is known to offer appropriate corrosion resistance for many applications including orthopedic implants. The resistance to uniform corrosion is explained by the existence of a dense chromium oxide forming a passive film on the surface. Nevertheless, the passive film can locally degrade, e.g., by small-amplitude wearing movements or due to the formation of a local aggressive environment in occluded regions prone to depletion of oxygen (crevice corrosion). High nitrogen containing austenitic stainless steel (HNS) such as Biodur 108 maintains the austenitic structure by the addition of nitrogen and manganese without having a major nickel content. Having nitrogen dissolved in the austenitic stainless steel results in higher strength compared with nickel containing austenitic stainless steel. HNS is also reported to offer improved pitting and crevice corrosion resistance in the solution-annealed state compared with the AISI 300 series . While our observational and experimental studies may not be able to determine a causal relation between corrosion leading to clinical symptoms and radiological findings, the covariation and temporal precedence make causality likely.


Phasic LA strains including reservoir, conduit, and booster pump strain were assessed by feature tracking. The outcome was a composite of hospital admissions for heart failure and all-cause mortality analyzed using Cox proportional hazard models. LAVmax and LAVmin were progressively larger while LAEF and LA strain measures were lower with worsening degree of DD . Among 132 patients with DD, 61 reached the composite outcome after on average 36-months of follow-up. Each of the LA parameters except for LA conduit strain was an independent predictor of the outcome in the adjusted Cox proportional hazard models . They remained significant outcome predictors after the model additionally adjusted for LV longitudinal strain. The AUC of outcome prediction was highest by LAEF (0.760) followed by LA reservoir strain (0.733) and LAVmin (0.725). Among all the LA features, increased LA volumes, reduced LAEF, reduced LA reservoir and booster pump strains were all associated with DD and DD severity. While LA strains are valuable, conventional parameters such as LAEF and LAVmin remain to be highly effective in outcome prediction with comparable performance.Left atrial (LA) features are altered when diastolic dysfunction (DD) is present. The relations of LA features to the DD severity and to adverse outcomes remain unclear using CMR images. We sought to compare LA features including volumes, emptying fraction, and strains as predictors of left ventricular (LV) DD and adverse outcomes. We compared four groups including normal controls (n = 32), grade I DD (n = 69), grade II DD (n = 42), and grade III DD (n = 21). DD was graded by echocardiography following the current ASE guidelines. Maximum LA volume (LAV The associated increase in LV filling pressures results in dilation of left atrial (LA) size and loss of LA compliance and contractility5, which can be assessed using LA wall strain and LA emptying fraction (LAEF). Therefore, features of the LA are regarded as the barometer of DD. To date, many publications have addressed some LA features and have established compelling evidence that LA parameters are essential in the evaluation of DD9. However, few have compared the relative importance of their associations with DD and DD-related adverse outcomes. In addition, most of the published work is based on echo parameters. As Cardiovascular Magnetic Resonance Imaging (CMR) becomes increasingly utilized in the evaluation of cardiomyopathy or heart failure there is a need to better understand CMR based LA indices. In this study we sought to compare the associations of four LA parameters: minimum and maximum LA volume (LAVmin and LAVmax), LAEF, and phasic LA strain  assessed by CMR, with DD, DD severity, and hazards of HF and mortality. We have also compared the hazards of LA features with that of LV longitudinal strain to demonstrate the relative importance of LA parameters.Diastolic dysfunction (DD), a condition of impaired left ventricular (LV) relaxation, is a risk factor for heart failure (HF) and cardiac mortality irrespective of LV ejection fraction (LVEF)10. The patient cohort (N = 132) was selected if there was evidence of DD, which was characterized by echocardiography based on the current ASE guidelines11. Of those, 66 were prospectively recruited for research and 66 were retrospectively analyzed from clinical studies. Patients were excluded if they were found to have history of atrial fibrillation, mitral stenosis, more than moderate mitral regurgitation, or prosthetic valve in the mitral position, which are conditions not supported by ASE guidelines for DD evaluation11. We also excluded subjects with indeterminate diastolic dysfunction grade. In addition to DD patients, normal controls were prospectively recruited who were free of cardiovascular history, major risk factors, and had both normal ECG and echocardiography. The vital signs and body surface area (BSA) for all subjects were collected at the time of CMR. Outcome data of hospitalized heart failure was extracted from electronic medical records of the health system consisting of 6 hospitals. The all-cause mortality information was obtained from National Death Index.This is a sub-study of a previously published larger study where participants underwent CMR and echocardiography within 7 days, between January 2007 and December 2015 at a single centerThis study was approved by the St. Francis Hospital Institutional Review Board. Informed written consent was obtained from all participants and research was performed in accordance with relevant guidelines and regulations.11 and indexed to body surface area. DD was assessed according to the algorithm from the recent ASE/EACI guidelines where mitral inflow early (E) and late (A) peak velocities, early diastolic annular myocardial longitudinal velocity (e′), tricuspid regurgitation and LA volume index were measured in order to determine the DD grade12.Patients were scanned with a multi-frequency transducer ultrasound system . Comprehensive echocardiographic examination was performed using standard views. From the apical window, pulsed wave Doppler was used to record mitral inflow for 3–5 cardiac cycles at the level of the mitral valve annulus and at the mitral leaflets’ tips. Tissue Doppler was applied to record mitral annular velocities at the septal and lateral corners of the annulus. The resulting annular velocities by pulsed wave Doppler were recorded for 3 to 5 cardiac cycles at a sweep speed of 100 mm/s. Tricuspid regurgitation signals were recorded by continuous wave Doppler from multiple windows. Two-dimensional measurements were performed according to recommendations of the American Society of EchocardiographyAll subjects underwent CMR on a 1.5 T scanner  with an 8-element phased array surface coil. Balanced ECG-gated cine imaging of the long axis planes  and a stack of 8–12 short axis planes , starting from the mitral annulus, was acquired using balanced steady state free precession sequence with 30 phases per cardiac cycle. The average temporal resolution was 50 ms, with a field of view of 240 mm, flip angle of 70 degrees, repetition time (TR) of 3.1 ms and echo time (TE) 1.3 ms. All patients were in normal sinus rhythm at the time of imaging.13 using 2- and 4-chamber long axis cine images with commercial software  and indexed to BSA. LA maximum volume (LAVmax) was assessed at LV end systole and LA minimum volume (LAVmin) at LV end diastole. Phasic LA strain was analyzed using feature tracking software . LA endocardial contours were first drawn manually and propagated through cardiac phases along the 2-, 3-, and 4- chamber views. Manual adjustment was made when needed. Peak LA strains were assessed as the average peak strain values of the 3 longitudinal planes . The association between our predictors and DD was assessed using area under the ROC curve (AUC) comparisons, and multinomial logistic regression. In these models, our covariates included age, gender, BSA, prevalent diabetes, hypertension, and smoking status chosen based on a disjunctive cause criterion14.Continuous variables were described as mean (standard deviation) and count data as frequency (percent). Our primary exposures of interest were phasic LA strain , LAEF, LAVmin and LAVmax), diabetes, hypertension, and smoking status. The associations of LA parameters with the composite outcome were also examined using AUC comparisons. In addition, the association between E/A and E/a′ with composite outcome was assessed using AUC curves. Statistical analyses were performed using SAS v. 9.4 . The association between our predictors of interest and a composite outcome of all-cause mortality and HF admission was assessed using Cox proportional hazards models. All-cause mortality data was obtained from the National Death Index and HF admissions based on electronic medical record review of the entire health care network. HF admissions were defined by an inpatient stay in a hospital setting with ICD 9 or ICD 10 diagnosis codes associated with congestive heart failure at the time of discharge. Individual Cox proportional hazards models were created for each primary exposure variable adjusting for age, gender, BSA (except for LAVp < 0.001)   consisted of four groups: normal controls n = 32), grade I DD (n = 69), grade II DD (n = 42), and grade III DD (n = 21). On average, patients with DD were 60 ± 14 years old and about half (N = 63) had advanced DD (grades II or III). Normal LVEF (> 50%) was present in 18 (14%) patients. Baseline characteristics of each DD grade are displayed in Table 64 consis, grade Imin, LAVmax, LAEF and LA reservoir strain respectively.A reproducibility analysis was performed on 15 randomly selected subjects. The Intra-observer reliability by ICC for LA indices were 0.99 , 0.96 , 0.92 , and 0.6  for LAVmin, LAVmax, LAEF, LA reservoir strain, and booster pump strain, but not in LA conduit strain. Using LA reservoir strain as an example, for every 1% decrease in LA strain the log-odds of a person having a higher grade of DD compared with normal control were 1.11 (CI 1.03–1.19), 1.25 (CI 1.14–1.38), and 1.50 (1.30–1.73) for grades I, II and III, respectively. In attempting to remove the potential confounding effect of LV strain on DD, we added LV longitudinal strain as a covariate to the previously described model and found that all exposures except LA conduit strain remained associated with worsening categories of DD . After adjusting for age, BSA, gender, prevalent diabetes, hypertension, and smoking status, all LA parameters with the exception of LA conduit strain were independently associated with the composite outcome, and remained independently associated when the models were additionally adjusted for LV longitudinal strain  patients reached the composite outcome due to 33 HF admissions and 28 deaths. No outcome events occurred in the normal controls. The outcome incidence was higher in more advanced DD: 45%, 63%, and 65% for grades I, II, and III, respectively  for LAVmax, 1.05 (1.00–1.10) for LA reservoir strain, 1.00 (0.95–1.05) for LA conduit strain and 1.07 (1.01–1.13) for LA booster pump strain. Overall, the results were similar to previously adjusted models.In a post-hoc analysis with an intention to remove potential confounding from LV abnormalities we tested the independent associations of LA parameters with the composite outcome in models adjusting for LVEF and indexed LV end diastolic volume in addition to age, gender, BSA, diabetes and hypertension. We found that LA parameters remained to be independent predictors of outcome with HR 1.05 (1.00–1.10) for LAVmin (0.725)  but combined LAVmax and LA reservoir strain did not . Furthermore, combined LAVmin and LAEF (0.76) or LAVmin and LA reservoir strain (0.73) did not improve outcome prediction from LAVmin alone .The associations of LA parameters with the composite outcome were additionally examined using AUC comparisons. The AUC was highest in LAEF (0.760) followed by LA reservoir strain 0.733) and LAV3 and LAVmin, LAVmax, LAEF, and phasic LA strains with DD. Each LA parameter except LA conduit strain was independently associated with DD in a dose–response manner. Furthermore, each of the LA parameters except LA conduit strain was independently associated with the composite outcome of HF admission and all-cause mortality in the adjusted model including LV longitudinal strain or LVEF as a covariate.In this study, we compared the relationships of LA parameters assessed by CMR including LAV18, we found dose-responses between LAV (both LAVmax and LAVmin) and the severity of DD. We have also confirmed prior reports that impaired LA function is closely associated with DD22 in a dose–response relationship in our study. Similarly, phasic LA strains also demonstrated similar dose–response relationships, confirming prior observations made from an echocardiography speckle-tracking study23.Consistent with prior observationsmin and LAEF in predicting adverse clinical outcomes. The outcome prediction by LAVmax alone is not as strong as LAVmin and LAEF. But by combining it with LAEF, the prediction is significantly enhanced. Our findings highlights the strength of LAVmin in DD evaluation which is superior to widely used LAVmax. Physiologically, LAVmin is directly modulated by LV filling pressure during LV end diastole as opposed to LAVmax which is predominantly influenced by the descending of mitral annulus thereby related to LV systolic function and LA compliance. Our collective findings suggest that LAVmin evaluation should be considered more favorably in the assessment of DD. It is also important to understand the relative importance of LA parameters in the context of other well-established imaging markers such as E/e′ and LV longitudinal strain in outcome prediction. We found that LAEF, the strongest outcome predictor of all LA indices including LA strain, has a comparable AUC from E/e′ and that from LV longitudinal strain. Given that LAEF correlates well with LV end diastolic pressure18 it is biologically plausible for it to predict clinical outcome effectively. Future research should examine the potential role of LAEF as an essential parameter in routine clinical evaluation.While LA reservoir and booster pump strain are valuable, we found it is comparable at best to LAV24 included a comprehensive list of studies that had investigated LAV and LA strain individually or collectively for their associations with DD. Most, if not all of the studies are echocardiography based and few, if any, use CMR. Additionally, we delineated the relative value of LAVmax, LAVmin, LAEF and phasic LA strain in DD and outcome prediction which has not been reported before. We have also established the associations of these LA indices with outcomes independent of LV longitudinal strain or LVEF. Of the LA parameters studied, LAVmin is easily attainable which allows LAEF to be calculated  from routine CMR or echocardiographic examination without the demand of additional post processing such as for LA strain. It is foreseeable to incorporate LAVmin and LAEF into future clinical algorithms for DD evaluation.It should be recognized that both LAV and LA strain evaluation are modality dependent. For instance, CMR has better spatial resolution than echocardiography rendering more accurate and reproducible LAV evaluation. In contrast, echocardiography has higher temporal resolution than CMR, hence providing a more favorable LA strain assessment. The technical differences may yield discrepancies in observations. That is why CMR specific investigations for LA in DD is essential despite the large body of literature using echocardiography. A recently published review article by Thomas et al.25. Therefore, the LA strain values generated from our normal subjects or from patients with DD cannot be directly compared with published studies using different vendors. Nonetheless, the relative importance of LA strain in our study that was compared with the normal controls is not subject to the variability of absolute value. It should also be noted that most of the feature tracking software is designed for LV strain evaluation and not for LA specifically. That is probably why the reproducibility of LA strain is only modest at best. However, our experience showed that the modern feature tracking program tracks the LA very well despite the thin wall. Tracking of the mobile mitral annulus is not always reproducible, and that seems to contribute to the variability. While the spatial resolution is excellent the temporal resolution of the CMR cine imaging is relatively low (about 50 ms) which may have compromised the accuracy of assessing the cardiac motion occurring within a short time frame such as LA boost pump strain. LA fibrosis evaluation by CMR can be valuable in patients with DD, but it was not performed in this study. While our electronic medical record allows us to capture HF admissions in the entire health system consisting of 6 hospitals, it is still possible that patients were admitted elsewhere, and therefore we may underestimate the scope of the outcomes. Lastly, this is a single center study and most of our patients are Caucasian. Future multicenter studies with diverse patient populations are warranted.We recognize the limitations of our study. This is a pooled sample of both prospective and retrospective cohorts. The demographic data from the retrospective cohort was collected from electronic medical records and thereby subject to availability. CMR and echocardiography were performed within the same day for half of the DD patients but within 7 days for the other half. As DD diagnosis and grading were based on echocardiographic criteria, CMR evaluation that followed echocardiography was subject to the interval variation of hemodynamics, although there was no significant change reported during the interval. The DD patients consisted of both reduced and preserved EF. While the sample size is powered for outcome risk assessment we acknowledge the relatively small sample size originating from a single center clinical cohort. In addition, female gender is under represented. Similar to LV strain assessment, the value of LA strain is likely vendor dependentmax is not a strong outcome predictor but the prediction can be enhanced significantly by combining it with LAEF. While LA strains are valuable, conventional parameters such as LAEF and LAVmin remain highly effective in outcome prediction with comparable performance.LA features including LAV, emptying fraction, as well as LA reservoir and booster pump strains have dose–response relationships with DD severity. They are also independent predictors of HF admission and all-cause mortality. LAV


Pregnancy luteomas are rare, benign, ovarian neoplasms resulting from increased androgenic activity during pregnancy. Often, they occur asymptomatically and are only diagnosed incidentally during imaging or surgery: cesarean section or postpartum tubal ligation. Most common symptoms associated with pregnancy luteoma include acne, deepening of voice, hirsutism, and clitoromegaly. Most pregnancy luteomas regress spontaneously postpartum. Thus, the management of pregnancy luteomas depends on the clinical situation.  We report a case of 28-year-old gravida 2, para 1 who presented at 39 + 1 weeks of gestation with prolonged labor and delivered by emergency cesarean. Intraoperatively, a huge left ovarian mass was identified and resected, and tissue was sent for histopathology and a diagnosis of pregnancy luteoma was made after the pathological report.  The present report emphasizes that pregnancy luteoma is a benign neoplasm and imprudent surgical intervention should be reserved. Proper imaging techniques, preferably MRI or ultrasonography that visualize the size of the ovary and reproductive hormonal profiles, would suffice for the diagnosis and management of pregnancy luteoma. Pregnancy luteoma is a rare nonneoplastic tumor-like lesion of the ovary that has an increased androgenic activity during pregnancy . It is ath year; and her menses were painless and of regular length, amount, and flow. During the index pregnancy, nothing remarkable was noted during her antenatal visits. She did two obstetric ultrasounds at 31 and 37 weeks, and both showed a viable fetus with no adnexal masses.A 28-year-old pregnant woman with BMI > 30, gravida 2, para 1, at 39 weeks and 1 day of gestation, was admitted to the antenatal ward presenting with lower abdominal pain and mucoid bloody stained discharge. Her current antenatal, medical, and gynecological history was unremarkable; menarche was during her 10th week of pregnancy and vaginal delivery of a premature male stillbirth of 1.5 kg about 2 years ago. She acknowledged having facial hirsutism that started from the 20th week of pregnancy and persisted postpartum but was not associated with deepening of the voice or other virilization symptoms.Past obstetric history is remarkable for preeclampsia from the 24On general examination, she had normal findings, with normal female hair distribution except for facial hair at the chin, and no other signs of virilization were noted. She reported that acne was present earlier in pregnancy but subsided as the pregnancy progressed.Her antenatal work-up revealed she was in the latent phase of labor and was kept for observation. Following an active onset of labor, she was transferred to the labor ward, but her labor progressed poorly and was prepared for emergency cesarean. Intraoperatively, a healthy female infant (with no virilization features) weighing 3.5 kg with Apgar scores of 9 and 10 at the first and fifth minutes, respectively, was extracted, and the uterus was then repaired accordingly. During the repair, a huge mass on the left adnexa measuring about 9 cm × 7 cm × 5.5 cm involving the left ovary was noted Figures , and uniThe gross pathological examination revealed a large ovarian tissue with two grossly visible yellowish tumor-like marked granular cysts measuring 1.5 cm by 1 cm and 1 cm by 1 cm. On histology, there were granular luteinized cells in both cell masses but were otherwise normal ovarian tissues with hemorrhages and two follicular cysts with 2-3 mitoses/10 HPF Figures . FinallyWe reviewed various pregnancy luteoma case reports published in English between 2000 and 2020. Our review included studies obtained from reference to published articles and literature search engines: PubMed or Google Scholar, using the terms “pregnancy luteoma.” A total of 205 case reports were reviewed from PubMed and 2,100 from Google Scholar. We identified and summarized 24 articles reporting 25 cases of pregnancy luteoma [β-hCG hormone levels favoring the proliferation of pregnancy luteoma [Pregnancy luteoma is a rare benign tumor-like enlargement of the ovary accompanying pregnancy . An occu luteoma , 26. The luteoma . Further luteoma , 27, a f luteoma .Patients with pregnancy luteoma have been reported to present with features of androgen excess, for instance, acne, deepening of the voice, facial and/or abdominal hirsutism, and clitoromegaly . These sThe diagnosis of pregnancy luteoma requires a high index of suspicion; once you suspect it based on the above-mentioned signs, perform detailed obstetric ultrasonography to visualize the size of the ovary. In addition, supportive investigations like hormonal assays should also be done to rule out other ovarian neoplasms .The manaBased on the presently described case, pregnancy luteoma is a benign neoplasm and imprudent surgical intervention should be reserved. A high index of suspicion is paramount to diagnose pregnancy luteoma, and once suspected, one should perform a detailed obstetric ultrasound visualizing the size of the ovary and hormonal assay for testosterone and its derivatives.


IntroductionAnesthesia induction and airway instrumentation are critical parts of anesthesia administration. Intravenous induction is time convenient but necessitates immediate commencement of ventilation. Inhalational sevoflurane induction takes longer but preserves spontaneous respiration. The primary aim of this study is to evaluate the intubation quality features achieved by sevoflurane as the sole induction agent compared with the standard intravenous induction, involving the use of muscle relaxants.MethodsSixty patients were randomly allocated into two groups: the Inhalational Vital Capacity Induction With Sevoflurane (IVCIS) group (n = 30) in which patients were intubated after sevoflurane inhalational anesthesia with the vital capacity technique and the Standard Intravenous Induction With Propofol, Fentanyl, and Rocuronium (SIPFR) group (n = 30) after propofol 1.5 mg/kg, fentanyl 2 μg/kg, and rocuronium 0.5 mg/kg administration intravenously. Group IVCIS patients were intubated when bispectral index (BIS) < 60 and end-expiratory sevoflurane ≥ 2 minimum alveolar concentration for > eight minutes. Scoring systems were used to evaluate induction and intubation conditions. The Statistical Package for the Social Sciences (SPSS) software version 25.0  was used for data analysis.ResultsIntubating and induction conditions were of equal quality in both groups. Sevoflurane induction duration was markedly prolonged. Heart rate was higher in IVCIS group patients throughout the induction, especially during laryngoscopy. Less blood pressure fluctuations were recorded in IVCIS group patients.ConclusionsInhalational vital capacity induction with sevoflurane provided acceptable intubating conditions and exhibited a safe hemodynamic profile, albeit the duration was more than 12 minutes. The combined administration of sedatives, opioids, and neuromuscular blocking agents constitutes a conventional practice of balanced anesthesia induction in both adults and children. Intravenous induction necessitates prompt and immediate airway manipulation maneuvers for the establishment of a patent airway . This maInhalational sevoflurane induction has been proposed as a favorable alternative in pediatric or non-compliant adult patients and in cases of difficult airways . Owing tSevoflurane induction has been extensively studied in the pediatric population, albeit only sparse data can be found in the literature that investigates the special features of sevoflurane induction until the establishment of a secure airway in adults. Over and above this, there is no consensus about the appropriate duration or other clinical features of inhalational induction that guarantee safe and effective intubating conditions since most studies investigate the administration of sevoflurane until the induction of anesthesia and not intubation .The present study was primarily conducted to investigate and compare the characteristics of intubating conditions achieved with inhalational or the intravenous induction of anesthesia in surgical patients, with the induction conditions and cardiac effects serving as secondary study outcomes.Study populationclinicaltrials.gov (identifier:NCT04802122).Written informed consent was obtained from participants after the nature of the procedure had been explained. This clinical study was performed according to The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The study protocol was approved by our Institutional Review Board  and registered on 2, presence of abdominal or intracranial hypertension, gastroesophageal reflux, severe hepatic or renal dysfunction, or pregnancy.All consecutive adult patients aged from 18 to 70 years, of both sexes, belonging to the American Society of Anaesthesiologists physical status classification I-III scheduled to undergo elective cholecystectomy under general anesthesia at our institution were eligible for enrolment in this prospective, randomized, and open-label study. Exclusion criteria included obesity defined as body mass index over 34.9 kg/mRandomizationhttps://sealedenvelope.com), the participants were randomly assigned into one of the two arms with a 1:1 ratio defined as the (a) Inhalational Vital Capacity Induction With Sevoflurane (IVCIS) and (b) Standard Intravenous Induction with Propofol, Fentanyl, and Rocuronium (SIPFR) groups. No use of blocks or stratification was applied to the randomization sequence while assignments specifying only the serial numbers were placed in a sealed envelop.Based on a computer-generated randomization table , and noninvasive blood pressure was instituted for each patient, and baseline recordings were obtained. Furthermore, an arterial cannula under local anesthesia was inserted and cardiac output monitoring  was initiated.Anesthesia induction2 100%, through a primed circuit, using the vital capacity technique. Priming of the circuit was done by connecting a 0.5L breathing bag and a gas sampling line at the Y piece of the breathing circuit while adjusting the sevoflurane vaporizer at 8%, fresh gas flow at 18L/min, and setting intermittent mandatory ventilation according to patients' age and body weight and waiting for end-tidal sevoflurane concentration to reach 2 minimum alveolar concentration (MAC) corrected by the patients' age. After priming, patients were asked to take a vital capacity breath from the residual lung volume, instructed to hold their breath as long as possible, and then perform the same maneuver again until loss of consciousness and return of spontaneous tidal breathing. The end-tidal sevoflurane concentration (SEVOet) was regulated, if needed, by continuous dial adjustments on the sevoflurane vaporizer, to ensure a steady state of two times its MAC. Endotracheal intubation with a proper-sized high-volume, low-pressure, cuffed endotracheal tube was attempted when the SEVOet was two times MAC (4%), for at least eight minutes and the BIS value < 60. In case of apnoea, we applied apnoeic oxygenation in combination with airway release manipulations, waiting for the reoccurrence of spontaneous breathing. The facemask was applied tightly to the patient's face and the adjusting pressure-limiting valve was open to spontaneous mode.In the IVCIS group, the induction to anesthesia was commenced by sevoflurane 8% (vaporizer setting) and OIn the SIPFR group, anesthesia was performed by bolus intravenous administration of propofol (1.5 mg/kg) and fentanyl (2 μg/kg) while rocuronium (0.5 mg/kg) was injected to facilitate endotracheal intubation after a three-minute time interval.2 absorber canister  attached at the anesthesia workstation.In both groups, anesthesia was provided with Leon Plus Anesthesia Workstation  with a sevoflurane vaporizer . The breathing circuit used was double limb adult expandable anesthesia circuits, 1.5L unstressed volume, 90" long, with a 2L breathing bag . There was a 1L COAnesthesia maintenance2O. Maintenance of anesthesia was ensured with sevoflurane in both groups, targeting a BIS value between 40 and 50. Additional bolus increments of rocuronium were administrated to maintain a post-tetanic count one to five until the cessation of pneumoperitoneum while intraoperative analgesic demands were covered by fentanyl (2 μg/kg) supplemented with remifentanil infusion (0.2-0.5 μg/Kg/min), as appropriate. At the end of the surgery, sugammadex was administered, aiming for a train-of-four ratio (TOFR) > 0.9.After intubation in both groups, mechanical ventilation was adjusted to a tidal volume of 8 mL/kg, plateau time of 30%, and a ventilatory frequency aiming to keep normocapnia with a fraction of inspired oxygen 40%. Positive end-expiratory pressure was applied to all cases being set at 5 cmHDefinitions of primary and secondary variablesThe quality of induction in both groups was assessed by the occurrence or not of limb movement (LM), cough (C), salivation (S), laryngospasm (L), and the documented response was graded on a two-point evaluation scale . Conventionally, scores ≤ 5 correspond to poor, 6-7 to acceptable, and 8 to excellent induction conditions. During intubation, mandible relaxation (MR), vocal cord position (VCP), airway reaction (AR), and limb movement (LM) were recorded and graded as appropriate , mean arterial blood pressure (MAP), cardiac output (CO), and stroke volume (SV) recordings were registered at baseline (before the anesthesia induction commencement), before and during the process of intubation, and 10 minutes thereafter.The primary end-point of the present study was the quality of intubating conditions while induction conditions and concomitant hemodynamic responses in each tested group served as secondary outcomes.Statistical analysisA sample size of 27 patients in each group was estimated for a two-sided alpha of 0.05 and power of 80%, assuming a minimum difference in the proportion of patients presenting excellent intubating conditions between groups as being clinically important. Allowing for a 10% drop-out rate, the final study population was set at 60 (30 patients per group). The normality of data was established by the Shapiro-Wilk test. The comparison between groups was performed by the independent T-test or Mann-Whitney test, as appropriate. Repeated-measures analyses of variance or Friedman’s test with Bonferroni adjustment for multiple comparisons were conducted to analyze normally distributed continuous variables over time, or nonparametric data, respectively. The chi-square or Fisher’s exact test was applied to compare the incidence of categorical data variables. Continuous data are presented as mean (standard deviation) or median (range) while nominal factors are reported as counts . For all statistical procedures, a P value of less than 0.05 was considered statistically significant. The Statistical Package for the Social Sciences (SPSS) software version 25.0  was used for all calculations.Although a total of 30 patients were recruited in each study arm to compensate for possible dropouts, data from all 60 patients enrolled in the study were available for the final analysis Figure . DemograThe classification of intubation conditions registered in both groups was equally identified as excellent or acceptable (P = 0.79). No case of poor intubation conditions was recorded among the study participants Table .Individual quality features of intubation conditions being achieved either by inhalational or intravenous induction to anesthesia could be regarded as equally satisfactory, considering that each component of the applied algorithm was graded as 3 in the vast majority of cases Table .Similarly, no significant difference in the laryngoscopic views, as assessed by the Cormack-Lehane classification system, was registered between the study groups (P = 0.85). In detail, 90% (n: 27) of the patients subjected to either practice were graded as I and II. Furthermore, the duration of intubation was similar between IVCIS and SIPFR groups .Overall, similar induction conditions were achieved between the two groups Table .Group SIPFR patients had lower MAP values throughout the induction, with the lowest MAP recorded before intubation. In group IVCIS, HR was higher throughout induction, intubation, and 10 minutes after intubation. Patients in group IVCIS underwent a significant increase in HR during intubation, compared to group SIPFR patients Table .There was no patient recorded having BIS <40 prior to intubation or BIS> 60 at any time after the induction of anesthesia.In this study, we provide evidence that inhalational vital capacity induction with sevoflurane provides acceptable induction and intubating conditions.The scoring systems used to grade induction and intubation conditions were a four-variable two and three-point system, respectively, which rated conditions as excellent, acceptable, or poor. Such grading systems have been used in the studies of Viby-Mongensen et al., Sivalingam et al., and Thwaites et al. -8.Most patients of the IVCIS group presented diaphragm contraction post-intubation and cuff inflation (slight airway reaction). This was a transient response probably elicited by the stimulus of intubation. All patients who presented this response immediately recommenced spontaneous breathing and there was no need for neuromuscular blocking agent administration. There was no failed intubation in either group. The intubating conditions in our study were better than those described in the study of Muzi et al. and similar to those described in the study of Khangwal et al. -10. The The most frequent undesirable condition occurring during induction in the IVCIS group was limb movement. The movement of the limbs or torso is related to Guedel anesthesia stage 2. This stage appears to be of prolonged duration during inhalational induction. The study of Hall et al. evinced that inhalational induction with sevoflurane 8% shortens the Guedel stage 2 duration, compared to sevoflurane 3% . The durThe administration of high sevoflurane concentration in the inhaled gas mixture has been associated with a more frequent occurrence of apnoea . TransieThe variation in HR during the induction of anesthesia followed the same course in both groups. It is noteworthy that in group IVCIS, HR was higher than in the SIPFR group, throughout the induction of anesthesia. At 10 min after intubation, in the SIPFR group, HR was lower than the HR of IVCIS group patients, nonetheless within normal range limits. These findings agree with the results of other studies on HR variation during sevoflurane administration, showing that by increasing sevoflurane concentration, the heart rate increases -16. The Inhalational vital capacity induction with sevoflurane provides acceptable induction and intubation conditions when targeting SEVOet 4% for more than 12 minutes. Breath-holding is a frequent complication of sevoflurane vital capacity induction, but it is transient; it does not cause hypoxemia and does not require manual support of breathing. Regarding hemodynamic parameters, MAP, SV, and CO are maintained within safe parameters during sevoflurane vital capacity induction. The major drawback of sevoflurane induction is its duration, in order to accomplish the perquisites for intubation used in this protocol. Eventually, the technique selected for induction of anesthesia and airway management is dictated by the practitioner's expertise, the patient's clinical status, and the available equipment.


Inguinal lymphadenectomy (iLAD) is effective for penile carcinoma treatment, but usually results in many complications. This study aims to clinically evaluate the feasibility and clinical significance of a laparoscopic radical iLAD approach partly preserving great saphenous vein branches for penile carcinoma patients.A total of 48 patients with penile cancer who underwent laparoscopic radical iLAD with retention of the great saphenous vein in Henan Cancer Hospital from 2012 Jan to 2020 Dec were included in this study. Sixteen penile carcinoma patients who underwent laparoscopic radical iLAD preserving parts of superficial branches of the great saphenous vein were identified as the sparing group, and the matched 32 patients who incised those branches were identified as control group. This new procedure was performed by laparoscopy, preserving parts of superficial branches of the great saphenous vein, superficial lateral and medial femoral veins. Clinicopathological features and perioperative variables were recorded. Postoperative complications, including skin flap necrosis, lymphorrhagia, and lower extremity edema were analyzed retrospectively.We found that the operative time of the sparing group is significantly longer than the control group (p = 0.011). There was no statistical difference in intraoperative blood loss, the lymph node number per side, average time to remove the drainage tube and postoperative hospital stay between the two groups. Compared to the control group, the sparing group showed a significantly decreased incidence of lower extremity edema (p = 0.018). The preservation of parts of superficial branches of the great saphenous vein was mainly decreased the incidence of edema below ankle (p = 0.034).This study demonstrated that the iLAD with preserving parts of superficial branches of the great saphenous vein, with a decreased incidence of postoperative complications, is a safe and feasible approach for penile cancer.The online version contains supplementary material available at 10.1186/s12893-022-01582-3. The traditional procedure of inguinal lymphadenectomy (iLAD) for penile carcinoma is associated with a high incidence of complications, including skin flap necrosis and edema of lower extremities. In 2003, Bissoff  firstly We retrospectively collected the records of 182 consecutive patients with penile carcinoma who received iLAD at Henan Cancer Hospital between 2012 Jan and 2020 Dec. Inclusion criteria: (1) Local lesions were pathologically confirmed as penile squamous cell carcinoma; (2) The local lesions were high-risk, with palpable active lymph nodes in the groin or with no palpable lymph nodes in the groin but positive by dynamic sentinel lymph node biopsy; (3) The local lesions were of medium and low risk, and there were palpable active lymph nodes in the groin. Lymph node metastasis was confirmed by inguinal lymph node resection biopsy or fine needle biopsy. Exclusion criteria: (1) Combined diseases of cardiovascular and cerebrovascular system or coagulation system, contraindicated with surgery; (2) Inguinal lymph node ulceration infection; (3) Preoperative radiotherapy in the inguinal region; (4) Distant metastases; (5) Preoperative ultrasound assessment of lower extremity blood vessels revealed deep venous diseases such as thrombosis and venous valve insufficiency. (6) Fixed lymph nodes in the groin. A total of 91 patients were performed laparoscopic iLAD with retention of the great saphenous vein, of which 16 patients underwent operation with retention of superficial branches of the great saphenous vein, superficial lateral and medial femoral veins (sparing group), and other 75 patients were included in control set. Then we matched 32 patients in a 1:2 ratio as control group according to clinicopathological features, including age, body mass index (BMI), smoking, American Society of Anesthesiologists (ASA) score, tumor stage, lymph node grading and history of pelvic lymph node dissections. In this study, only one senior surgeon (T.J. Y.) performed the vein preservation operation, and three senior surgeons  performed the laparoscopic iLAD with retention of the great saphenous vein. The flowchart of patient selection was showed in Fig. Enhanced CT or MRI and ultrasound were carried out to assess distant metastasis and the local lymph node status, and general condition and laboratory examination were performed to rule out surgical contraindications. The pathological grading and staging of penile biopsy or resection were performed based on UICC TNM grading system . Local ePatients were placed evenly in the supine position. The lower limbs were outreached at 30 degrees, and the knees bent at 120 degrees. One cm incision was first made 20 cm below the midpoint of the inguinal ligament. Skin was incised until the subcutaneous layer; the flap was separated bluntly from fat, with 12 cm half circle in direction to the groin, then inserting a 10 mm trocar for Surgical Cavity Mirror into the cavity. While connecting the pneumoperitoneum, the pressure was maintained at 10 mmHg. Second and third 1 cm incisions were made left and right to the first one, respectively, 5 cm close to the groin; 12 mm trocars were inserted into the cavity through the two cuts, respectively. Then, Separating the Camper’s fascia up to 3 cm above the inguinal ligament, the lateral margin to the Sartorius muscle, and the internal margin to adductor longus muscle. The great saphenous vein and its branches, superficial lateral- and medial- femoral veins were located, along the distal femoral triangle point. Then, vascular skeletonization of the great saphenous vein and its branches was performed by ultrasonic scalpel and self-made no damage rubber traction. Meanwhile, we tried to preserve the whole great saphenous vein, superficial medial and lateral femoral veins, unless they were too slim. The shallow level of superficial lymph nodes and fatty tissue located between fascia lata and Camper fascia was dissected. Along the 3 cm above the inguinal Canal level, the superficial epigastric vein was cut, and surrounding lymph nodes were harvested. All the tissues, such as adipose tissue and lymph nodes, between the flap and external oblique aponeurosis in this region were removed from top to the inguinal canal. The external pudendal vein was incised along the medial margin of adductor longus. The superficial iliac circumflex vein was cut along the lateral side of the sartorius muscle. The surrounding lymph nodes were removed along the inner side. Then, the other end of the superficial iliac circumflex vein, superficial epigastric vein, and external pudendal vein were cut at saphenous hiatus, close to the great saphenous vein. The femoral vascular sheath was opened in the femoral triangle. Then, vascular skeletonization of both femoral artery and femoral vein was performed. Lymph nodes surrounding deep femoral vessels were dissected. After the above steps, the tissues were removed in batches with a 12 mm Trocar. The surgical cavity was rinsed and soaked with sterile distilled water. Hemostasis was done perfectly. Drainage-tubes with negative pressure were inserted into the surgical cavity. Sometimes, refilling of the great saphenous vein could be observed. Wounds were sutured one by one. Elastic pressure bandages were used to fix flaps. After surgery, the superficial circumflex iliac vein, external pudendal vein, superficial epigastric vein surrounding lymph nodes , and femoral paraneoplastic oval fossa lymph node (deep lymph nodes) were separately sent for pathology. The trunk of the great saphenous vein was retained in control group, all branches were severed, and other steps were the same as sparing group  for 5 days post-surgery. Elastic stockings were applied to prevent venous thrombosis of lower limbs. Dressing was changed every 3 days post-operation, and the flap color and subcutaneous fluid were observed carefully. Vascular ultrasound of lower extremities was carried out 5 days after surgery in order to assess deep venous thrombosis. The negative pressure drainage tube was removed with drainage volume below 10 ml in 24 h. The elastic bandage, however, was maintained for 1 more week. The observed intraoperative complications included operative time, blood loss, number of dissected lymph nodes, and time of drainage tube removal, and the postoperative complications includes poor flap healing, lower limb edema, lymphocysts, venous thrombosis.All patients were followed in our department, their follow-up regimen included ultrasound, CT/MRI every 3 months within 2 years, and then were extended to 6 months thereafter.All statistical tests were performed with SPSS 22.0 , and two-side p < 0.05 was indicated as statistical significance. The chi-square test or Fisher’s exact test was used to determine the distribution of categorical variables, as indicated. The Student’s t tests were used for continuous variables.In 48 surgical procedures, a total of 96 limbs were successfully completed. Patient ages were 54(range: 36–65 years) years. There was no significant difference in age, BMI, smoking status, ASA score and TNM staging between the sparing group and control group Table . The meaNo skin flap necrosis and surgical intervention for flap healing was found in sparing group. Meanwhile, the incidence of subcutaneous infection incidence, lymphorrhagia, lymphocysts and venous thrombosis in sparing group and control group were 6.25% (1/16) and 9.37% (3/32), 18.75% (3/16) and 15.62% (5/32), 18.75% (3/16) and 18.75% (6/32), 12.5% (2/16) and 15.63% (5/32), respectively (Table Because of ≥ 2 inguinal lymph node metastases and enlarged pelvic lymph node, 3 patients underwent two pelvic lymph node dissections, with 1 patient displaying metastatic pelvic lymph nodes in control group (Table In traditional radical iLAD, the great saphenous vein and its branches should be cut, and the deep side and superficial inguinal lymph nodes are removed, transplanting tensor fascia lata to be used for covering femoral vessels. This procedure is the most effective treatment in penile carcinoma with lymph node metastasis. However, high incidence of complications, including skin flap infection, necrosis, edema of lower extremities, and lymphatic fistula, have been associated with this surgical method , 6. WithThere are five main great saphenous vein branches, including the superficial lateral femoral, superficial medial femoral, external pudendal, superficial epigastric, and superficial iliac circumflex veins. Lymphatic drainage flows from the lymph nodes surrounding the external pudendal vein to the perineal area close to the penis; meanwhile, studies have confirmed that part of the penis is affected by inguinal lymphatic drainage in the groin outside the area , as wellThe iLAD technique is crucial to clarify the patient’s pathological stage and assess survival . In a reThis study has some shortcomings. The results of this study should be further validated by multicenter, a large scale of sample size cohort and long-term studies because of a retrospective nature. Another shortcoming of the current study fails to collect the information of patients’ outcome. Even so, we firmly identified that the new procedure descripted in this study could benefit to decrease postoperative complications.Endoscopic iLAD retaining the great saphenous vein and parts of its branches is a safe and feasible technique. This approach especially suits early penile carcinoma patients with suspicious lymph node metastasis, with a decreased incidence of postoperative complications. For penile carcinoma patients with deep vein disease of lower limbs, preserving the great saphenous vein and its branches may be a good option.Additional file 1. Clinical information of 48 penile cancer patients.


Excessive use of chemical fertilizers has led to a reduction in the quality of arable land and environmental pollution. Using green manure to replace chemical fertilizers is one of the most effective solutions. To study the effect of green manure on the requirement for nitrogen fertilizer in oilseed rape, a field experiment with maize–oilseed rape rotation was conducted. Green manure was intercropped between rows of maize and returned after the maize harvest, with no green manure intercropped as control. Different nitrogen fertilizer treatments  were applied during the oilseed rape season. The results showed that with a 35% reduction in nitrogen application rate, the rapeseed grain yield was significantly higher with the maize intercropping with green manure returned to the field than with the maize monocropping treatment at the same nitrogen level. Under conditions of intercropping and return of green manure, compared with the full standard rate of nitrogen fertilizer treatment, a reduction in nitrogen application of 25–30% in the rape season had no significant effect on rape yield. The agronomic efficiency of nitrogen fertilizer on oilseed rape increased significantly, by 47.61–121%, with green manure incorporation. In addition, green manure incorporation significantly increased the soil organic matter content and the soil-available nitrogen content when chemical nitrogen fertilization was abandoned. Benefit analysis showed that a 25–35% reduction in chemical nitrogen fertilizer applied to oilseed rape crops could be achieved by intercropping green manure in the maize season before the sowing of rapeseed in the experimental area. In the long-term, this measure would increase nitrogen utility, reduce production costs, and have concomitant environmental benefits of improving the quality of cultivated land. Consequential to the continuous increase in the global population, the demand for agricultural products is increasing rapidly. In order to increase yields, the amount of chemical fertilizer used in agricultural production is escalating, while the application of organic fertilizer remains insufficient. The cultivability of arable land deteriorates under long-term chemical fertilizer application, and environmental pollution is prominent ,2,3. TheIntercropping green manure is a productive planting system that can make full use of sunlight and heat resources, soil moisture, and nutrients. It is a practical measure to produce and utilize green manure and contribute to sustainable agricultural production ,9,10. ItOilseed rape is the source of the world’s third most consumed edible oil after soybean and palm and is also the primary source of biodiesel . In 2018Maize–oilseed rape rotation is widely practiced as a crop rotation system worldwide ,21. In t(i)Lablab purpureus (L.) Sweet) facilitates reduction in the need for fertilizer-N in intercropping systems of maize and oilseed rape;legume Labadou ((ii)Brassica napus L.);growing Labadou in the summer-autumn growing season increases biomass production and fertilizer-N efficiency of winter oilseed rape ((iii)introducing an intercropping method to maize and oilseed rape rotation systems in dryland decreases N application rates.However, there are few reports of research on the potential reduction in the requirement for chemical fertilizer that may be achieved by intercropping with legumes in the preceding crop season and supplying green manure for the later season’s oilseed rape crop in the maize–oilseed rape rotation systems. We hypothesized that:−1).To test these hypotheses, a maize–oilseed rape rotation field trial was conducted to study the effects of different treatments on the yield of corn and rapeseed and basic soil properties. Maize intercropping with leguminous green manure (IC) and maize monocropping (MC) planting configurations were set up for the maize season, and different chemical nitrogen doses were set based on the standard local rapeseed nitrogen application rate , compared with MC + N, the rapeseed yield was significantly reduced by 20.31% in MC + 0.65N, while no significant differences were found under IC. In the second season (2019–2020), compared with MC + N, the rapeseed yield was significantly reduced by 20.12% and 14.51% in MC + 0.65N and MC + 0.75N, respectively . HoweverResults of the analysis of the nitrogen fertilizer agronomic efficiency of rapeseed under different treatments are shown in The effect of green manure under different nitrogen application rates on soil organic matter content is shown in The differences in the content of soil-available nitrogen between different treatments are shown in The differences in soil-available phosphorus content between different treatments are shown in The difference in soil-available potassium content between different treatments is shown in Existing studies have shown that rapeseed yield may vary according to the different effects of previous crop types ,26,27. IHowever, many researchers have found different results. In our study, even with a 25–35% reduction in nitrogen fertilizer, the rapeseed yield was not significantly different from the yield with the standard nitrogen dose when under the maize-intercropping mode, indicating that green manure in the previous maize intercropping could substitute 25–35% of nitrogen fertilizer for oilseed rape production in the experimental site.The amount of N transferred from a legume to associated crops is a subject of considerable controversy. It varies depending on conditions that impact legume N-fixation, such as legume species, symbiotic performance, and agronomic factors, such as weather conditions . In our The application rate of nitrogen fertilizer has a considerable influence on the yield and quality of rapeseed. Frugal application of nitrogen fertilizer has a critical role in saving production costs and improving the yield and quality of oilseed rape . At presIn this experiment, after two years of continuous planting and return of green manure, the soil organic matter significantly increased by 3.80% to 39.81%. The effect of planting and returning legume green manure on soil properties is closely related to the soil property background value . It has Studies have shown that green manure amendment can increase the content of available nitrogen , availabWe calculated the benefit under different treatments based on local maize and oilseed rape prices and labor costs. The results of the benefit analysis showed that although the intercropping would increase seed and labor costs, the overall benefit of intercropping would still be slightly higher than monocropping due to a reduction in the nitrogen fertilizer application dose by 25–35% . More im2O: soil = 5:1) was 7.53, alkali-hydrolyzable nitrogen was 61.43 mg kg−1, available phosphorus was 5.07 mg kg−1, and available potassium was 101.87 mg kg−1.The experimental site is located in Meijiadun Village, Ducheng Town, Huangzhou District, Huanggang City, Hubei Province, China. It is 114°88′ east longitude, 30°43′ north latitude, and 26.7 m altitude. It has a typical humid subtropical monsoon climate, with abundant water and heat resources. The rainfall is mainly concentrated from April to August. The annual average rainfall is (1287 ± 305) mm, the annual average temperature is (17.7 ± 0.5) °C, the annual average sunshine duration is 1900 h, and the frost-free period is 260 d. The soil texture is sandy loam and had the following chemical characteristics: SOM was 1.46%, pH (HLablab purpureus (L.) Sweet), leguminous green manure, was interplanted between the wide rows of maize (Zea mays L.), the variety of maize is zhengdan69, was grown for grain. The width between maize rows was 160 cm and between plants was 15 cm for the wide rows and 40 cm × 15 cm for the narrow rows. The row spacing for the maize monocropping treatment (MC) was 100 cm × 15 cm  was direct sown at a rate of 5.25 kg ha−1. The density was approximately 35,000 plants ha−1, and the variety of oilseed rape is yangguang 2009. The green manure was sown at the same time as the maize, with two rows of Labadou (40 cm × 8 cm) sown between the wide maize rows. The nutrient content of 100 kg Labadou was tested to be 2.33 kg N, 0.30 kg P2O5, and 2.53 kg K2O.The experimental design was based on the split zone test. The primary strategy was to compare the effect of green manure intercropping with monocropping, while the secondary strategy was to measure this interaction with varying fertilizer doses (only for oilseed rape). For the green manure intercropping treatment (IC), wide and narrow row spacings were adopted in the maize season, and Labadou ( × 15 cm . The sow2O5) and potassium chloride (60% K2O). The same amount of fertilizer was applied in all plots in the maize season (N:P2O5:K2O was 195:90:135 kg ha−1). In the oilseed rape season, the application rate of P2O5, K2O, and Boron fertilizer in all plots was 60, 75 and 7.5 kg ha−1, respectively. The application rate of nitrogen fertilizer was divided into four levels: full standard nitrogen application , 25% nitrogen reduction , 35% nitrogen reduction  and no nitrogen fertilizer  ; superphosphate (12% Pkg ha−1) . Three r2Cr2O7 oxidation method; available N using the Alkaline diffusion method; available phosphorus (AP) concentration using the Olsen method after extraction with 0.03 mol L−1 NH4F 0.025 mol L−1 HCL; available K using flame photometry after extraction with 1 mol L−1 NH4OAC, and pH  using a pH meter [Each year, mixed soil samples were collected from the soil surface layer (0–20 cm) at five points in each plot, using a soil borer, after the oilseed rape was harvested. After removing all animal and plant residues and pebbles, soil samples were air-dried and put through a 100 or 200 mesh sieve for further analysis. Soil properties were determined as follows: total soil organic matter (SOM) using the KpH meter . The agri represents the Agronomic efficiency of treatment i, Yi represents the yield of treatment i, YCK represents the yield of treatment CK, Ni represents the nitrogen fertilizer application dose of treatment i.AEBenefit analysis was calculated as follows: maize and oilseed rape production income, seed and fertilizer cost were calculated according to local agriculture market prices. Labor cost was calculated according to local labor market standards. Total income was the sum of maize and oilseed rape production income; the benefit is total income minus total cost.p = 0.05 was used to elucidate any significant differences.Experimental data were analyzed using the SPSS 16.0 software package and Microsoft Office Excel 2010. Analysis of variance (ANOVA) was carried out to determine the differences between the measured parameters for different treatments. The least significant difference (LSD) at −1 of legume green manure, a precondition ensuring the seed yield of rapeseed in the experimental site. Intercropping and returning can significantly increase the soil organic matter content of the experimental site and intercropping Labadou with maize is a promising sustainable and low-cost method to improve soil fertility.This study provided a better understanding of green manure utilization in the intercropping planting system to support more effective nitrogen fertilization in the maize–oilseed rape rotation system. Strategies that reduce fertilizer application while maintaining yield are vital for food production methodologies that guarantee agricultural sustainability, particularly in the drought rotation area. Intercropping legume green manure, Labadou, during the maize season and returning it to the field before rapeseed sowing showed obvious substitution effects for rapeseed nitrogen fertilizer. The results of two consecutive years of field trials showed that 25–35% of nitrogen fertilizers could be replaced by 19–24 t ha