Male haploid cells through direct spherification.

OBJECTIVE: To obtain de novo male gametes capable of inducing full preimplantation blastocyst development using a novel three-dimensional (3D) culture system. DESIGN: Mouse embryonic stem cells (mESCs) were spherified by plunging in sodium alginate followed by calcium chloride, delineating a 3D environment that simulates the seminiferous tubule. As a control, mESCs cultured on two-dimensional plates were used. Plates and spheres containing mESCs from both methods were exposed to Activin-A, bFGF, and KSR followed by exposure to BMP4, LIF, SCF, and EGF to promote differentiation into male germ-like cells. MAIN OUTCOME MEASURES: Cells were assessed for VASA, DAZL, and BOULE on days 3 and 10. Cells were later injected into activated oocytes and monitored using time-lapse imaging on days 15, 22, 29, and 36. Control conceptuses generated using mature epididymal spermatozoa were also monitored via time-lapse imaging. RESULTS: On day 3, cells differentiated on plates expressed VASA at 1% and DAZL at 29%. In spheres, VASA was expressed at a rate of 15% and DAZL at a rate of 45% (P<.001). On day 10, cells differentiated on plates had VASA expression of 7%, DAZL of 23%, and BOULE of only 0.5%. Cells differentiated into spheres expressed VASA at a rate of 20%, DAZL at 43%, and BOULE at 10% (P<.001). Subsequent differentiation in spheres on day 3 exhibited a DAZL (expressed in spermatogonia) expression of 43% and a VASA (further spermatogenesis progression) expression of 15%. On day 10, DAZL and VASA expressions were reassessed and increased to 45% and 18%, respectively. BOULE, a marker expressed solely in postmeiotic spermatocytes, was expressed at 8%, whereas acrosin was expressed in spermatids at 2%. On day 15, VASA expression plateaued at 17%, BOULE peaked at 10%, and acrosin reached 5%. On day 22, expression of VASA increased to 19%, BOULE decreased to 8%, and acrosin peaked at 7%. On day 29, VASA expression peaked at 20%, BOULE dropped to 2%, and acrosin remained stable at 7%. On day 36, VASA expression remained at 13%, whereas BOULE and acrosin expression decreased to 0% and 1%, respectively. The control cohort attained 88.4% fertilization and 76.9% blastocyst rates. De novo gametes achieved fertilization rates of 35.0%, 61.1%, 81.8%, and 50.0% on days 15, 22, 29, and 36, respectively. Neogametes-generated blastocyst rates were 5.0%, 16.7%, 36.4%, and 8.3% for days 15, 22, 29, and 36, respectively. CONCLUSION: Our novel 3D differentiation model can generate functional gametes and is aimed at obviating the need for allogeneic/xenogeneic transplantation. The decreased overall marker expression and the reduced blastocyst development indicated that intrasphere germ cell differentiation correlated with the length of mouse spermatogenesis at approximately 30 days. Future experiments will be conducted to confirm the reproducibility of our findings and the eventual generation of offspring.

Can a sperm selection technique improve embryo ploidy?

BACKGROUND: Spermatozoa with the highest motility retain a superior genomic integrity, and elevated sperm chromatin fragmentation (SCF) has been linked to a lower ability of the conceptus to develop and implant. Therefore, the utilization of a sperm selection method, such as microfluidic sperm selection (MFSS), is capable of reducing the SCF by yielding the most motile fraction of spermatozoa with the highest embryo developmental competence. What remains unclear, however, is the causal mechanism that links SCF to an impaired embryo development. OBJECTIVES: To identify a relationship between SCF and an unexpectedly high proportion of embryo aneuploidy, while addressing treatment options. MATERIALS AND METHODS: We identified couples with a high incidence of embryo aneuploidy in a previous intracytoplasmic sperm injection (ICSI) cycle with pre-implantation genetic testing for aneuploidy (PGT-A), utilizing spermatozoa selected by density gradient (DG). Terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) and neutral Comet assays were carried out on the semen specimens to assess total SCF and double-stranded DNA (dsDNA) fragmentation, respectively. These couples underwent subsequent ICSI/PGT-A cycles with MFSS. Total SCF and dsDNA fragmentation were compared between the two sperm selection methods. Embryo aneuploidy, implantation, clinical pregnancy, delivery, and pregnancy loss rates were compared between the couples' historical DG and subsequent MFSS cycles. RESULTS: In 57 couples undergoing 71 ICSI/PGT-A cycles, where DG sperm selection was carried out, a high incidence of aneuploid embryos (74.7%) resulted in poor implantation and no viable pregnancies. Testing for SCF, inclusive of dsDNA breaks, evidenced a SCF of 26.2% and dsDNA break of 3.6% in the raw specimen, that decreased to 18.0% (p < 0.001) and 3.1%, respectively, in the DG processed specimen. Following MFSS, total SCF and dsDNA fragmentation decreased to 1.9% and 0.3%, respectively (p < 0.001). The embryo euploidy rate remarkable improved from 25.3% in the DG cycles to 42.9% in the MFSS cycles (p < 0.001). The 6.7% implantation rate in the DG cycles increased to 65.5% in the MFSS cycles (p < 0.001). Similarly, the clinical pregnancy rate rose from 10.5% (DG) to 64.6% (MFSS), resulting in a 62.5% delivery rate (p < 0.001). DISCUSSION AND CONCLUSIONS: In couples with a relatively young female partner with a negative infertility workup, and a male partner with semen parameters adequate for ICSI, presenting with a high rate of embryo aneuploidy, an additional subtle male factor component may be the culprit. Thus, it is crucial to assess the SCF and test for the dsDNA breaks, which can eventually contribute to embryo chromosomal abnormalities. Given the inverse relationship between SCF and motility, a selection of the most motile gamete by MFSS enhanced the proportion of spermatozoa with an intact genome, contributing to the generation of more euploid embryos that are capable of implanting and yielding increased term pregnancies.

Keep an Eye Out for the Bifid Uvula: A Case Report.

The incidence of submucous cleft palate is between 1:1250-1:6000. It is important for this relatively silent deformity to be identified early and referred appropriately as a delay in diagnosis may affect the patient's quality of speech in the long-term. This paper presents a case of submucous cleft palate which was diagnosed late (age 14). Nevertheless, by the appropriate intervention of the multidisciplinary cleft team, the patient was treated successfully. Clinical Relevance. Dentists and doctors need to be aware of the signs and symptoms associated with submucous cleft palate so that they can refer the patient to an appropriate specialist centre for further investigation and treatment. Moreover, patients with this condition are shown to have a higher incidence of dental abnormalities, such as hypodontia and peg-shaped laterals.

How important are lateral cephalometric radiographs in orthodontic treatment planning?

INTRODUCTION: The purpose of this study was to investigate whether lateral cephalometric radiographs influence orthodontic treatment planning. It aimed to compare the odds of a change in treatment plan in three groups of orthodontists who treatment planned six cases on two occasions, T1 and T2, with the provision of a lateral cephalometric radiograph being varied. METHODS: The records of 6 orthodontic patients were copied onto compact discs and sent to the 199 participating orthodontists. The orthodontists were allocated to 3 groups, A, B, and C. Clinicians in group A were given all records except the lateral cephalometric radiographs at the T1 and T2 planning sessions. Clinicians in group B were given all records except the lateral cephalometric radiograph at T1 and all records including the lateral cephalometric radiograph and tracing at T2. Clinicians in group C were given all records including the lateral cephalometric radiographs and tracings at T1 and T2. All participants were sent records at T1; those who returned the treatment-planning questionnaire were sent the second set of records and questionnaire at T2, 8 weeks later. Invitations to participate were distributed to all specialist orthodontists who were members of the British Orthodontic Society (n = 950). Of these, 199 orthodontists agreed to take part, a response rate of 21%. Of the 199 who agreed to participate, 149 completed the first treatment-planning questionnaire (T1), for a response rate of 75%. Of the 149 who completed that questionaire, 114 completed the second treatment-planning questionnaire (T2), for a 77% response rate. RESULTS: The availability of a lateral cephalometric radiograph and its tracing did not make a significant difference to any treatment-planning decisions, with the exception of the decision to extract or not between groups B and C for all 6 patients combined, and between groups B and C and groups B and A for patient 4 (Class I incisor relationship on a Class II skeletal base). CONCLUSIONS: For most treatment-planning decisions in these 6 patients, the availability of a lateral cephalometric radiograph and its tracing did not make a significant difference to the treatment decisions. For 1 patient, there was a significant change in the extraction decision when a lateral cephalometric radiograph was provided. This highlights the uncertainty surrounding the necessity for lateral cephalometric radiographs in treatment planning. Further research in this area is encouraged to resolve this dichotomy.

Combined internal and external Le Fort III distraction osteogenesis--the "elusive vector".

This article describes a modified surgical technique using both internal and external distractors for distraction osteogenesis at the Le Fort III level. This technique optimizes vector control, superior to single-device techniques, resulting in excellent control and, ultimately, a functional occlusion.

Prospective randomized clinical trial to compare pain levels associated with 2 orthodontic fixed bracket systems.

INTRODUCTION: In this randomized clinical trial, we compared pain levels associated with 2 fixed appliance systems during initial orthodontic tooth movement. METHODS: Sixty-six patients (36 female, 30 male) were randomly allocated to either a conventional twin bracket (Tru Straight, Ormco Europe, Amersfoort, The Netherlands) or a self-ligating bracket (Damon 3, Ormco). Both arches were bonded, and a 0.014-in superelastic copper-nickel-titanium archwire was ligated in position. Pain intensity levels were recorded twice a day, over 7 days, by using 10-cm visual analog scales. Patients also recorded whether analgesia was taken. Contact point displacements were measured on study models to calculate Little's irregularity index. RESULTS: Fifty-two patients (78.8%) completed the trial (84.8% in the Tru Straight group and 72.7% in the Damon 3 group). Patients in the Damon 3 group reported lower mean maximum pain intensity (P = 0.053) and significantly lower mean pain intensity (P = 0.012) than did the Tru Straight patients. Patients who consumed analgesics reported significantly higher mean maximum pain intensity and mean pain intensity than those who did not take analgesia (P <0.001). The Damon 3 patients generally reported lower pain intensity than the Tru Straight patients. CONCLUSIONS: Although there were wide interindividual variations, in this study, the Damon 3 appliance resulted in lower pain intensity, on average, when compared with the Tru Straight appliance.