Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency.

Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research. VIDEO ABSTRACT.

Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Selective elimination of mitochondrial mutations in the germline by genome editing.

Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber's hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PAPERCLIP.

Exploration of the Cytoplasmic Function of Abnormally Fertilized Embryos via Novel Pronuclear-Stage Cytoplasmic Transfer.

In regular IVF, a portion of oocytes exhibit abnormal numbers of pronuclei (PN) that is considered as abnormal fertilization, and they are routinely discarded. However, it is known that abnormal ploidy still does not completely abandon embryo development and implantation. To explore the potential of cytoplasm from those abnormally fertilized oocytes, we developed a novel technique for the transfer of large cytoplasm between pronuclear-stage mouse embryos, and assessed its impact. A large volume of cytoplast could be efficiently transferred in the PN stage using a novel two-step method of pronuclear-stage cytoplasmic transfer (PNCT). PNCT revealed the difference in the cytoplasmic function among abnormally fertilized embryos where the cytoplasm of 3PN was developmentally more competent than 1PN, and the supplementing of fresh 3PN cytoplasm restored the impaired developmental potential of postovulatory "aged" oocytes. PNCT-derived embryos harbored significantly higher mitochondrial DNA copies, ATP content, oxygen consumption rate, and total cells. The difference in cytoplasmic function between 3PN and 1PN mouse oocytes probably attributed to the proper activation via sperm and may impact subsequent epigenetic events. These results imply that PNCT may serve as a potential alternative treatment to whole egg donation for patients with age-related recurrent IVF failure.

GNAI1 and GNAI3 Reduce Colitis-Associated Tumorigenesis in Mice by Blocking IL6 Signaling and Down-regulating Expression of GNAI2.

BACKGROUND & AIMS: Interleukin 6 (IL6) and tumor necrosis factor contribute to the development of colitis-associated cancer (CAC). We investigated these signaling pathways and the involvement of G protein subunit alpha i1 (GNAI1), GNAI2, and GNAI3 in the development of CAC in mice and humans. METHODS: B6;129 wild-type (control) or mice with disruption of Gnai1, Gnai2, and/or Gnai3 or conditional disruption of Gnai2 in CD11c+ or epithelial cells were given dextran sulfate sodium (DSS) to induce colitis followed by azoxymethane (AOM) to induce carcinogenesis; some mice were given an antibody against IL6. Feces were collected from mice, and the compositions of microbiomes were analyzed by polymerase chain reactions. Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) isolated from spleen and colon tissues were analyzed by flow cytometry. We performed immunoprecipitation and immunoblot analyses of colon tumor tissues, MDSCs, and mouse embryonic fibroblasts to study the expression levels of GNAI1, GNAI2, and GNAI3 and the interactions of GNAI1 and GNAI3 with proteins in the IL6 signaling pathway. We analyzed the expression of Gnai2 messenger RNA by CD11c+ cells in the colonic lamina propria by PrimeFlow, expression of IL6 in DCs by flow cytometry, and secretion of cytokines in sera and colon tissues by enzyme-linked immunosorbent assay. We obtained colon tumor and matched nontumor tissues from 83 patients with colorectal cancer having surgery in China and 35 patients with CAC in the United States. Mouse and human colon tissues were analyzed by histology, immunoblot, immunohistochemistry, and/or RNA-sequencing analyses. RESULTS: GNAI1 and GNAI3 (GNAI1;3) double-knockout (DKO) mice developed more severe colitis after administration of DSS and significantly more colonic tumors than control mice after administration of AOM plus DSS. Development of increased tumors in DKO mice was not associated with changes in fecal microbiomes but was associated with activation of nuclear factor (NF) κB and signal transducer and activator of transcription (STAT) 3; increased levels of GNAI2, nitric oxide synthase 2, and IL6; increased numbers of CD4+ DCs and MDSCs; and decreased numbers of CD8+ DCs. IL6 was mainly produced by CD4+/CD11b+, but not CD8+, DCs in DKO mice. Injection of DKO mice with a blocking antibody against IL6 reduced the expansion of MDSCs and the number of tumors that developed after CAC induction. Incubation of MDSCs or mouse embryonic fibroblasts with IL6 induced activation of either NF-κB by a JAK2-TRAF6-TAK1-CHUK/IKKB signaling pathway or STAT3 by JAK2. This activation resulted in expression of GNAI2, IL6 signal transducer (IL6ST, also called GP130) and nitric oxide synthase 2, and expansion of MDSCs; the expression levels of these proteins and expansion of MDSCs were further increased by the absence of GNAI1;3 in cells and mice. Conditional disruption of Gnai2 in CD11c+ cells of DKO mice prevented activation of NF-κB and STAT3 and changes in numbers of DCs and MDSCs. Colon tumor tissues from patients with CAC had reduced levels of GNAI1 and GNAI3 and increased levels of GNAI2 compared with normal tissues. Further analysis of a public human colorectal tumor DNA microarray database (GSE39582) showed that low Gani1 and Gnai3 messenger RNA expression and high Gnai2 messenger RNA expression were significantly associated with decreased relapse-free survival. CONCLUSIONS: GNAI1;3 suppresses DSS-plus-AOM-induced colon tumor development in mice, whereas expression of GNAI2 in CD11c+ cells and IL6 in CD4+/CD11b+ DCs appears to promote these effects. Strategies to induce GNAI1;3, or block GNAI2 and IL6, might be developed for the prevention or therapy of CAC in patients.

Abnormalities in human pluripotent cells due to reprogramming mechanisms.

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the 'gold standard', they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies.

Three-dimensional computed tomography confirmed that the meniscal root attachments and meniscofemoral ligaments are morphologically consistent.

PURPOSE: To clarify the characteristic features of the meniscal root attachments, meniscofemoral ligaments (MFLs), and related osseous landmarks on three-dimensional images using computed tomography. METHODS: Twenty-eight non-paired, formalin-fixed human cadaveric knees were evaluated in this study. The meniscal root attachments were identified and marked. Three-dimensional images were obtained after applying a contrast agent to the entire meniscal surfaces and MFLs, then the morphology of the meniscal root attachments and MFLs, and their positional relationships with osseous landmarks, were analyzed. RESULTS: Parsons' knob divided the medial meniscal anterior root attachment and lateral meniscal anterior root attachment on the anterior portion of the tibial plateau. The medial meniscal posterior root attachment was near the medial intercondylar tubercle. The lateral meniscal posterior root attachment (LMPRA) was closer to the lateral intercondylar tubercle. Both root attachments were near the posterior intercondylar fossa. The positional relationships between the meniscal root attachments and related osseous landmarks were consistent in all specimens. The MFLs originated from the lateral meniscus posterior horn, and the anterior MFL was closer to the LMPRA than the posterior MFL. The posterior MFL originated at approximately the midpoint between the LMPRA and the most posterior margin of the lateral meniscus. CONCLUSION: This study showed that the relationships between the characteristic features of the meniscal root attachments, MFLs, and related osseous landmarks were consistent. The clinical relevance of this study is that it improved understanding of the anatomy of the meniscal root attachments and MFLs.

The Coefficient of Variation of Step Time Can Overestimate Gait Abnormality: Test-Retest Reliability of Gait-Related Parameters Obtained with a Tri-Axial Accelerometer in Healthy Subjects.

The aim of this study was to investigate whether variation in gait-related parameters among healthy participants could help detect gait abnormalities. In total, 36 participants (21 men, 15 women; mean age, 35.7 ± 9.9 years) performed a 10-m walk six times while wearing a tri-axial accelerometer fixed at the L3 level. A second walk was performed ≥1 month after the first (mean interval, 49.6 ± 7.6 days). From each 10-m data set, the following nine gait-related parameters were automatically calculated: assessment time, number of steps, stride time, cadence, ground force reaction, step time, coefficient of variation (CV) of step time, velocity, and step length. Six repeated measurement values were averaged for each gait parameter. In addition, for each gait parameter, the difference between the first and second assessments was statistically examined, and the intraclass correlation coefficient (ICC) was calculated with the level of significance set at p < 0.05. Only the CV of step time showed a significant difference between the first and second assessments (p = 0.0188). The CV of step time also showed the lowest ICC, at <0.50 (0.425), among all parameters. Test-retest results of gait assessment using a tri-axial accelerometer showed sufficient reproducibility in terms of the clinical evaluation of all parameters except the CV of step time.

A microwell culture system that allows group culture and is compatible with human single media.

PURPOSE: A microwell culture system that facilitates group culture, such as well-of-the-well (WOW), improves embryonic development in an individual culture. We examined the effect of WOW on embryonic development in vitro with commercially available human single culture media. METHODS: Using four different commercial human single culture media, in vitro development and imprinted gene expression of bovine embryos cultured in WOW were compared to droplet culture (one zygote per drop). To determine the effects of microwell and group culture on embryonic development, different numbers of embryos were cultured in droplet or WOW. Diffusion simulation of accumulating metabolites was conducted using the finite volume method. RESULTS: WOW had a positive effect on bovine embryonic development, regardless of the type of single culture media. Imprinted gene expression was not different between droplet- and WOW-derived blastocysts. The microwell and group cultures in WOW showed a significant positive effect on the rate of total blastocysts and the rate of development to the expanded and hatching blastocyst stages. The assumed cumulative metabolite concentration of WOW with one embryo was 1.47 times higher than that of droplet culture with one embryo. Furthermore, the concentration of WOW with three embryos was 1.54 times higher than that of WOW with one embryo. CONCLUSIONS: In using human single culture media, a microwell culture system that allows group culture could be a powerful clinical tool for improving the success of assisted reproductive technologies.

Morphology of the Insertions of the Superficial Medial Collateral Ligament and Posterior Oblique Ligament Using 3-Dimensional Computed Tomography: A Cadaveric Study.

PURPOSE: To describe the insertions of the superficial medial collateral ligament (sMCL) and posterior oblique ligament (POL) and their related osseous landmarks. METHODS: Insertions of the sMCL and POL were identified and marked in 22 unpaired human cadaveric knees. The surface area, location, positional relations, and morphology of the sMCL and POL insertions and related osseous structures were analyzed on 3-dimensional images. RESULTS: The femoral insertion of the POL was located 18.3 mm distal to the apex of the adductor tubercle (AT). The femoral insertion of the sMCL was located 21.1 mm distal to the AT and 9.2 mm anterior to the POL. The angle between the femoral axis and femoral insertion of the sMCL was 18.6°, and that between the femoral axis and the POL insertion was 5.1°. The anterior portions of the distal fibers of the POL were attached to the fascia cruris and semimembranosus tendon, whereas the posterior fibers were attached to the posteromedial side of the tibia directly. The tibial insertion of the POL was located just proximal and medial to the superior edge of the semimembranosus groove. The tibial insertion of the sMCL was attached firmly and widely to the tibial crest. The mean linear distances between the tibial insertion of the POL or sMCL and joint line were 5.8 and 49.6 mm, respectively. CONCLUSIONS: This study used 3-dimensional images to assess the insertions of the sMCL and POL and their related osseous landmarks. The AT was identified clearly as an osseous landmark of the femoral insertions of the sMCL and POL. The tibial crest and semimembranosus groove served as osseous landmarks of the tibial insertions of the sMCL and POL. CLINICAL RELEVANCE: By showing further details of the anatomy of the knee, the described findings can assist surgeons in anatomic reconstruction of the sMCL and POL.

Morphology of insertion sites on patellar side of medial patellofemoral ligament.

PURPOSE: The purpose of this study was to clarify the insertion sites on the patellar side of the medial patellofemoral ligament (MPFL). METHODS: A total of 35 nonpaired human cadaveric knees were used in this study. After identification of the MPFL, the insertion sites on the patellar side of the MPFL were marked. Three-dimensional images were created, and the location and morphology of these insertion sites were analysed. RESULTS: The morphology of the insertion sites on the patellar side of the MPFL was consistent. The proximal fibres of the MPFL were inserted to the deep fascia of the vastus medialis obliquus (VMO) and medial margin of the vastus intermedius (VI). The distal fibres of the MPFL were inserted to the medial margin of the patella directly. The insertion lengths of the VMO, VI, and patella were 26.7 ± 5.0, 28.5 ± 4.4, and 18.5 ± 4.4 mm, respectively. The rate of the vertical distance from the superior pole of the patella to the superior edge of the MPFL in relation to the total patellar height was 12 ± 4.4 %. At the distal edge, the rate was 58 ± 9.6 %. CONCLUSION: The insertion sites on the patellar side of the MPFL were consistent. The MPFL inserted into the VMO and VI was significantly longer than into the patella. The clinical relevance of this study is to improve understanding of the anatomy of the insertion sites on the patellar side of the MPFL and the pathophysiology of patellar dislocation.

Morphology of the fibular insertion of the posterolateral corner and biceps femoris tendon.

PURPOSE: To clarify the fibular head insertion of the fibular collateral ligament (FCL), popliteofibular ligament (PFL), and biceps femoris tendon and related osseous landmarks on three-dimensional (3-D) images. METHODS: Twenty-one non-paired, formalin-fixed human cadaveric knees were evaluated in this study. The fibular head insertions of the FCL, PFL and biceps femoris tendon were identified and marked. 3-D images were created, and the surface area, location, positional relationships, and morphology of the fibular insertions of the FCL, PFL, and biceps femoris tendon and related osseous structures were analysed. RESULTS: The fibular head had a unique pyramidal shape, and the relationships of the fibular insertion of the FCL, PFL, and biceps femoris tendon were consistent. The fibular head consists of three aspects: lateral aspect, posterior aspect, and proximal tibiofibular facet. The insertions of the FCL, PFL, and biceps femoris tendon were attached to the centre from the distal side of the lateral aspects of the fibular head, posterior aspect of the fibular styloid process, and lateral aspect surrounding the FCL, respectively. The mean surface areas of the FCL and PFL fibular insertions were 100.1 ± 29.5 and 18.5 ± 7.2 mm2, respectively. CONCLUSION: This study showed that the relationships between the characteristic features of the fibular head and insertions of the FCL, PFL, and biceps femoris tendon were consistent. The clinical relevance of this study is that it improves understanding of the anatomy of the insertions of the PLC and biceps femoris tendon.

[Intramedullary Abscess of the Cervical Spinal Cord Caused by Advanced Periodontitis:Case Report].

We describe the case of a 60-year-old man with an intramedullary abscess of the cervical spinal cord caused by advanced periodontitis. He suddenly developed severe neck pain and rapidly progressive palsy of the left upper arm. T2-weighted sagittal magnetic resonance imaging(MRI)revealed a hyperintense area extending from C1 to C6. Gadolinium-enhanced T1-weighted MRI showed a ring-enhanced lesion at the C3-4 level that was hyperintense on diffusion-weighted MRI. The patient underwent drainage of the abscess through laminectomy. Cultures of the abscess contents revealed Fusobacterium nucleatum and Peptostreptococcus micros. Antibiotics administered to the patient to treat the infection with these anaerobic bacteria improved the neurological deficit eight weeks after surgery. The patient was also diagnosed with advanced periodontitis due to Fusobacterium nucleatum that might have caused the intramedullary abscess of the cervical spinal cord.

Deficiency of the multi-copy mouse Y gene Sly causes sperm DNA damage and abnormal chromatin packaging.

In mouse and man Y chromosome deletions are frequently associated with spermatogenic defects. Mice with extensive deletions of non-pairing Y chromosome long arm (NPYq) are infertile and produce sperm with grossly misshapen heads, abnormal chromatin packaging and DNA damage. The NPYq-encoded multi-copy gene Sly controls the expression of sex chromosome genes after meiosis and Sly deficiency results in a remarkable upregulation of sex chromosome genes. Sly deficiency has been shown to be the underlying cause of the sperm head anomalies and infertility associated with NPYq gene loss, but it was not known whether it recapitulates sperm DNA damage phenotype. We produced and examined mice with transgenically (RNAi) silenced Sly and demonstrated that these mice have increased incidence of sperm with DNA damage and poorly condensed and insufficiently protaminated chromatin. We also investigated the contribution of each of the two Sly-encoded transcript variants and noted that the phenotype was only observed when both variants were knocked down, and that the phenotype was intermediate in severity compared with mice with severe NPYq deficiency. Our data demonstrate that Sly deficiency is responsible for the sperm DNA damage/chromatin packaging defects observed in mice with NPYq deletions and point to SLY proteins involvement in chromatin reprogramming during spermiogenesis, probably through their effect on the post-meiotic expression of spermiogenic genes. Considering the importance of the sperm epigenome for embryonic and fetal development and the possibility of its inter-generational transmission, our results are important for future investigations of the molecular mechanisms of this biologically and clinically important process.

Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis.

We have developed a unique method for mouse transgenesis. The transposase-enhanced pronuclear microinjection (PNI) technique described herein uses the hyperactive piggyBac transposase to insert a large transgene into the mouse genome. This procedure increased transgene integration efficiency by fivefold compared with conventional PNI or intracytoplasmic sperm injection-mediated transgenesis. Our data indicate that the transposase-enhanced PNI technique additionally requires fewer embryos to be microinjected than traditional methods to obtain transgenic animals. This transposase-mediated approach is also very efficient for single-cell embryo cytoplasmic injections, offering an easy-to-implement transgenesis method to the scientific community.

Single blastomere removal from murine embryos is associated with activation of matrix metalloproteinases and Janus kinase/signal transducers and activators of transcription pathways of placental inflammation.

Single blastomere removal from cleavage-stage embryos, a common procedure used in conjunction with preimplantation genetic diagnosis (PGD), may affect reproductive outcomes. We hypothesized that negative pregnancy outcomes associated with PGD may be due to impairment of placental signaling pathways. The goal of this study was to determine the molecular mechanisms through which placental signaling is deregulated by blastomere removal. Four-cell stage murine embryos produced by in vitro fertilization were subjected to removal of a single blastomere (biopsied) or to the same manipulations without the blastomere removal (controls). Placental tissues from term (18.5 day) pregnancies obtained after embryo transfer were tested for levels of nitrosative species, interleukin 6, signal transducers and activators of transcription (STAT) 1 and 3, suppressors of cytokine signaling (SOCS) 1, 2 and 3 and matrix metalloproteinases (MMP) 1, 2, 3 and 9. Significant increases in nitrosative stress (P < 0.05), phosphorylative activation of STAT1 (P < 0.05) but not STAT3, lower levels of the inhibitors SOCS2 (P < 0.01) and SOCS3 (P < 0.001) and activation of MMP9 (P < 0.001) were observed in placentas derived from biopsied embryos, compared with controls. Such effects could contribute to greater levels of premature membrane rupture, incorrect parturition, preterm birth and intrauterine growth restriction associated with PGD. This work has determined signaling mechanisms that may be responsible for blastomere removal effects on placental function, with the potential to become targets for improving obstetric and neonatal outcomes in assisted reproduction.

Morphology of the popliteomeniscal fascicles around the popliteal hiatus on three-dimensional images reconstructed from 7 T magnetic resonance imaging: A cadaveric study.

BACKGROUND: This study aimed to clarify the characteristic features of the anteroinferior and posterosuperior popliteomeniscal fascicles (aiPMF and psPMF, respectively) and popliteal hiatus using three-dimensional (3D) reconstructions of 7 T magnetic resonance imaging (MRI) arthrography. METHODS: Six knees from human cadavers fixed using the Thiel embalming method were examined using 7 T MRI arthrography. 3D Images of the structures around the popliteal hiatus were reconstructed. Morphologies of the psPMF, aiPMF, and popliteal hiatus were investigated and their positional relationships analyzed. RESULTS: The PMFs attached to the periphery of the lateral meniscus (LM) to form the popliteal hiatus. Each coursed in an oblique direction. The mean length of the psPMF and aiPMF attachments to the LM were 6.8 and 21.6 mm, respectively; mean popliteal hiatus length was 12.8 mm. These lengths corresponded to 7.5%, 24.3%, and 14.5% of the total length of the LM, respectively. The aiPMF was thick near the lateral aspect of the articular capsule and became thinner towards the posteromedial aspect of the LM. The psPMF was thick near the posterior aspect of the articular capsule and became thinner towards the posterolateral aspect of the LM. CONCLUSION: Morphological properties of the aiPMF, psPMF, their attachments to the LM, and the popliteal hiatus were consistent across the cadaver specimens examined. Each PMF was thin near the popliteal hiatus and became thicker towards its attachment to the articular capsule. These findings may be useful for anatomical repair for the LM hypermobility.

Morphology of the proximal tibiofibular joint and ligaments using three-dimensional computed tomography: A cadaveric study.

BACKGROUND: The aim of our study was to clarify the morphology of the proximal tibiofibular joint (PTFJ), insertion sites of the proximal tibiofibular ligaments (PTFLs), and related osseous landmarks on three-dimensional (3D) computed tomography (CT) images. METHODS: Cadaveric knees were evaluated by dissection and 3D CT imaging. The anterior PTFL (A-PTFL) and posterior PTFL (P-PTFL) were isolated, and their tibial and fibular insertion sites were identified. The morphology and location of insertion sites and their positional relationships with osseous structures were analyzed on 3D CT images. RESULTS: The A-PTFL comprised up to four bundles, and the P-PTFL comprised two bundles. The mean length of the A-PTFL and P-PTFL was 11.3 mm and 10.3 mm, respectively. On the tibial side of the PTFJ, bony prominences were present at the A-PTFL and P-PTFL insertion sites and were clearly identified as osseous landmarks in all knees. On the fibular side, the A-PTFL and P-PTFL insertion sites were at the edge of the triangular pyramid of the fibular head. The mean PTFJ area was 198.8 mm2, and the mean inclination angle between PTFJ and tibial plane was 38.4°. There was an inverse correlation between the PTFJ surface area and the inclination angle. CONCLUSION: The present study clearly identified PTFL insertion sites on the tibia and fibula and showed the relationships between these insertions and osseous landmarks. These data improve our understanding of the anatomy of PTFL insertions, which may assist surgeons in performing anatomical reconstruction.

Morphology of the quadriceps tendon and its patella insertion site on three-dimensional computed tomography and magnetic resonance imaging: A cadaveric study.

AIM: This study was conducted to clarify the morphological properties of the quadriceps tendon (QT) and its patella insertion site using three-dimensional computed tomography and magnetic resonance imaging. METHODS: Twenty-one right knees from human cadavers were evaluated using three-dimensional computed tomography and magnetic resonance imaging. The morphologies of the QT and its patella insertion site were evaluated, along with intra-tendon differences in length, width, and thickness. RESULTS: The QT insertion site on the patella was dome-shaped without characteristic bony features. The mean surface area of the insertion site was 502.5 ± 68.5 mm2 (range, 336.0-610.7). The QT was longest 2.0 mm lateral to the central width of the insertion and gradually became shorter toward both edges (mean length, 59.7 ± 8.3 mm). The QT was widest at the insertion site (mean width, 39.1 ± 5.3 mm) and gradually became narrower toward the proximal side. The QT was thickest 2.0 mm medial to the center (mean thickness, 11.4 ± 1.9 mm). CONCLUSION: The morphological properties of the QT and its insertion site were consistent. The characteristics of the QT graft depend on the harvested region.

Blastomere removal from cleavage-stage mouse embryos alters steroid metabolism during pregnancy.

Preimplantation genetic diagnosis (PGD) is a genetic screening of embryos conceived with assisted reproduction technologies (ART). A single blastomere from an early-stage embryo is removed and molecular analyses follow to identify embryos carrying genetic defects. PGD is considered highly successful for detecting genetic anomalies, but the effects of blastomere biopsy on fetal development are understudied. We aimed to determine whether single blastomere removal affects steroid homeostasis in the maternal-placental-fetal unit during mouse pregnancy. Embryos generated by in vitro fertilization (IVF) were biopsied at the four-cell stage, cultured to morula/early blastocyst, and transplanted into the oviducts of surrogate mothers. Nonbiopsied embryos from the same IVF cohorts served as controls. Cesarean section was performed at term, and maternal and fetal tissues were collected. Embryo biopsy affected the levels of steroids (estradiol, estrone, and progesterone) in fetal and placental compartments but not in maternal tissues. Steroidogenic enzyme activities (3beta-hydroxysteroid dehydrogenase, cytochrome P450 17alpha-hydroxylase, and cytochrome P450 19) were unaffected but decreased activities of steroid clearance enzymes (uridine diphosphate-glucuronosyltransferase and sulfotransferase) were observed in placentas and fetal livers. Although maternal body, ovarian, and placental weights did not differ, the weights of fetuses derived from biopsied embryos were lower than those of their nonbiopsied counterparts. The data demonstrate that blastomere biopsy deregulates steroid metabolism during pregnancy. This may have profound effects on several aspects of fetal development, of which low birth weight is only one. If a similar phenomenon occurs in humans, it may explain low birth weights associated with PGD/ART and provide a plausible target for improving PGD outcomes.