A prospective, randomized, cross-over comparison of two methods of artificial insemination by donor on the incidence of conception: intracervical insemination by straw versus cervical cap

In a prospective, randomized study of insemination with donor semen, intracervical insemination by straw was compared with insemination using a cervical cap with an intracervical reservoir. A total of 91 patients completed 486 treatment cycles. There were no significant differences in age, parity, indication for insemination by donor, or method of cycle monitoring between women who became pregnant and those who did not conceive with either insemination method. In 236 standard intracervical insemination cycles, 14 patients became pregnant (5.9% per cycle), whereas 38 patients conceived in 250 cervical cap cycles (15.2% per cycle). Both the crude pregnancy rates and the cumulative pregnancy rates calculated by the Kaplan-Meier life-table method were significantly different (chi(2)-test, P < 0.001, and log-rank test, P < 0.005 respectively). Pregnancy rates in artificial insemination with cryopreserved donor semen may be improved by the use of a cervical cap when compared to cervical insemination by straw. The use of the cervical cap may prolong the exposure of the spermatozoa to the cervical mucus and prevent the backflow of semen into the vagina.      

Diagnosis of ectopic pregnancy after in vitro fertilization and embryo transfer

Objective: The combination of transvaginal sonography and serum hCG measurement is reliable in the diagnosis of ectopic pregnancy (EP) in spontaneous pregnancies. In patients who became pregnant through IVF-ET, transfer of multiple embryos after IVF could be responsible for the different performance of these tests. We evaluated the discriminative capacity of transvaginal sonography in combination with hCG measurement in the diagnosis of EP after IVF-ET.

Design: Prospective cohort study.

Setting and Patient(s): Consecutive patients, pregnant through IVF-ET, who presented with clinically suspected EP.

Intervention(s): Transvaginal sonography, serum hCG measurement at 6, 9, and 15 days after ET and after a negative transvaginal sonography.

Main Outcome Measure(s): Ectopic pregnancy confirmed at laparoscopy.

Result(s): Between September 1993 and May 1996, 86 women were included in the study, of whom 24 had an EP. Transvaginal sonography identified 46 intrauterine pregnancies and 5 EPs, but serum hCG could not diagnose EPs in patients in whom transvaginal sonography did not show a gestational sac. Serum hCG measurement 9 days after ET could identify pregnancy failure with 100% specificity at a cut-off value of 18 IU/L, but it could not identify patients with EP with enough certainty to justify immediate treatment.

Conclusion(s): We recommend single serum hCG measurement 9 days after ET to discriminate between viable and nonviable pregnancies. Transvaginal sonography can be postponed until 5 weeks after ET, except for patients with abdominal pain and/or vaginal bleeding, or patients with a serum hCG level of <18 IU/L.     

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombin-cleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors.     

Pre-operative sentinel lymph node localization in breast cancer with superparamagnetic iron oxide MRI: the SentiMAG Multicentre Trial imaging subprotocol

Objective:

Sentinel lymph node biopsy (SLNB) with a superparamagnetic iron oxide (SPIO) tracer was shown to be non-inferior to the standard combined technique in the SentiMAG Multicentre Trial. The MRI subprotocol of this trial aimed to develop a magnetic alternative for pre-operative lymphoscintigraphy (LS). We evaluated the feasibility of using MRI following the administration of magnetic tracer for pre-operative localization of sentinel lymph nodes (SLNs) and its potential for non-invasive identification of lymph node (LN) metastases.

Methods:

Patients with breast cancer scheduled to undergo SLNB were recruited for pre-operative LS, single photon emission CT (SPECT)-CT and SPIO MRI. T₁ weighted turbo spin echo and T₂ weighted gradient echo sequences were used before and after interstitial injection of magnetic tracer into the breast. SLNs on MRI were defined as LNs with signal drop and direct lymphatic drainage from the injection site. LNs showing inhomogeneous SPIO uptake were classified as metastatic. During surgery, a handheld magnetometer was used for SLNB. Blue or radioactive nodes were also excised. The number of SLNs and MR assessment of metastatic involvement were compared with surgical and histological outcomes.

Results:

11 patients were recruited. SPIO MRI successfully identified SLNs in 10 of 11 patients vs 11 of 11 patients with LS/SPECT-CT. One patient had metastatic involvement of four LNs, and this was identified in one node on pre-operative MRI.

Conclusion:

SPIO MRI is a feasible technique for pre-operative localization of SLNs and, in combination with intraoperative use of a handheld magnetometer, provides an entirely radioisotope-free technique for SLNB. Further research is needed for the evaluation of MRI characterization of LN involvement using subcutaneous injection of magnetic tracer.

Advances in knowledge:

This study is the first to demonstrate that an interstitially administered magnetic tracer can be used both for pre-operative imaging and intraoperative SLNB, with equal performance to imaging and localization with radioisotopes.     

Increased Coexpression of PD-1, TIGIT,and KLRG-1 on Tumor-Reactive CD8+ T Cells During Relapse after Allogeneic Stem Cell Transplantation

Allogeneic stem cell transplantation (allo-SCT) can be a curative treatment for patients with a hematologic malignancy due to alloreactive T cell responses recognizing minor histocompatibility antigens (MiHA). Yet tumor immune escape mechanisms can cause failure of T cell immunity, leading to relapse. Tumor cells display low expression of costimulatory molecules and can up-regulate coinhibitory molecules that inhibit T cell functionality on ligation with their counter-receptors on the tumor-reactive T cells. The aim of this explorative study was to evaluate immune checkpoint expression profiles on T cell subsets and on cytomegalovirus (CMV)- and/or MiHA-reactive CD8⁺ T cells of allo-SCT recipients using a 13-color flow cytometry panel, and to correlate these expression patterns to clinical outcomes. MiHA-reactive CD8⁺ T cells exhibited an early differentiated CD27⁺⁺/CD28⁺⁺ phenotype with low KLRG-1 and CD57 expression. These T cells also displayed increased expression of PD-1, TIM-3, and TIGIT compared with total effector memory T cells and CMV-specific CD8⁺ T cells in healthy donors and allo-SCT recipients. Remarkably, high coexpression of PD-1, TIGIT, and KLRG-1 on MiHA-reactive CD8⁺ T cells was associated with relapse after allo-SCT. Taken together, these findings indicate that MiHA-specific CD8⁺ T cells of relapsed patients have a distinctive coinhibitory expression signature compared with patients who stay in remission. This phenotype may serve as a potential monitoring tool in patients. Moreover, these findings suggest that PD-1 and TIGIT play important roles in regulating T cell-mediated tumor control, providing a rationale for immunotherapy with blocking antibodies to treat relapse after allo-SCT.     

A new CT scan method for measuring the tibial tubercle trochlear groove distance in patellar instability

Patellar malalignment leading to objective or potential patella instability can be caused by tibial tuberosity lateralisation. This can be treated with a tuberosity medialisation. CT scan measurements are needed to assess the tibial tubercle trochlear groove distance. When using the previously described methods to determine this distance it can be difficult to determine the anatomical structures on the maximum intensity projection images, and this can lead to measurement error. This study was designed to compare the reliability of a new computer based CT measurement to the previously described method to determine the tibial tubercle trochlear groove distance. For each method, four observers measured each of 50 knees twice. The inter- and intra-observer variability for the conventional method and a new method were determined. Using the conventional method, the number of knees for which the difference between the aggregate mean of all eight measurements and the mean of duplicate measurements per observer greater than 2 mm varied among the observers between 7 and 24 for the 50 knees, while this variation between four and seven for the same 50 knees using the new method. The limits of reproducibility based on measurements from the four different observers improved by 25%, indicating that the measurement error is considerably smaller with the new method. We advise using this more accurate method to improve the selection of patients for a tuberosity medialisation.     

Characterization of Giardia duodenalis by polymerase-chain-reaction fingerprinting

The development of a polymerase chain reaction (PCR) based fingerprinting method for the characterization of Giardia duodenalis isolates is described. The method uses three different PCRs; one is specific for the A (“Polish”) major group of G. duodenalis isolates, another is specific for the B (“Belgian”) group of isolates; and one amplifies a fragment of the glutamate dehydrogenase gene present in all G. duodenalis isolates. The PCRs perform highly sensitively on DNA from cultured trophozoites. Isolates were further characterized by restriction-fragment-length polymorphism (RFLP) analysis of the PCR products. In this way, representative isolates from the A and B groups could be grouped together into a number of subgroups. The stability of the genotypes with time and the reproducibility of the two methods were tested on cloned and subcloned lines from a number of isolates and proved to be highly satisfactory. The PCR/RFLP method was evaluated on cysts derived from a number of human patients. It is concluded that the PCR fingerprinting method described in this paper provides a reliable characterization method for Giardia isolates and has the potential to be used as a direct method of typing G. duodenalis cysts from feces. Received: 7 January 1998 / Accepted: 7 April 1998     

Characterization of Cryptosporidium parvum in human and animal feces by single-tube nested polymerase chain reaction and restriction analysis

A DNA isolation and purification method is described that produced DNA free of inhibitory substances in 148 of the 159 analyzed fecal samples. The polymerase chain reaction (PCR) product from a sensitive single-tube nested PCR that amplifies a part of an oocyst protein was used to characterize Cryptosporidium parvum genotypes by a simple restriction analysis. Genotype 1 was solely detected in human-derived oocysts, genotype 2 was present in both animal and human-derived oocysts. The ratio between both genotypes in humans in The Netherlands varied markedly between samples obtained during a period of augmented cases of cryptosporidiosis in the western part of the country and randomly selected samples from gastroenteritis patients. Sequence analysis of a 581-bp fragment from the nested PCR product revealed 12 nucleotide substitutions between the two genotypes. Sequences from isolates in each genotype group were identical. Received: 18 January 1999 / Accepted: 9 March 1999     

Structural insights into the Slit-Robo complex

Slits are large multidomain leucine-rich repeat (LRR)-containing proteins that provide crucial guidance cues in neuronal and vascular development. More recently, Slits have been implicated in heart morphogenesis, angiogenesis, and tumor metastasis. Slits are ligands for the Robo (Roundabout) receptors, which belong to the Ig superfamily of transmembrane signaling molecules. The Slit-Robo interaction is mediated by the second LRR domain of Slit and the two N-terminal Ig domains of Robo, but the molecular details of this interaction and how it induces signaling remain unclear. Here we describe the crystal structures of the second LRR domain of human Slit2 (Slit2 D2), the first two Ig domains of its receptor Robo1 (Ig1-2), and the minimal complex between these proteins (Slit2 D2-Robo1 Ig1). Slit2 D2 binds with its concave surface to the side of Ig1 with electrostatic and hydrophobic contact regions mediated by residues that are conserved in other family members. Surface plasmon resonance experiments and a mutational analysis of the interface confirm that Ig1 is the primary domain for binding Slit2. These structures provide molecular insight into Slit-Robo complex formation and will be important for the development of novel cancer therapeutics.