Artificial insemination and in-vitro fertilization using donor spermatozoa: a report on 15 years of experience

Donor insemination (DI) using cryopreserved semen commenced at The Royal Women's Hospital in 1976. Over the next 15 years we performed 5953 treatment cycles to achieve 816 pregnancies (13.7% per cycle) and 706 live births. In-vitro fertilization (IVF) using donor spermatozoa commenced in 1986. Over the next 5 years we performed 303 treatment cycles for 185 couples. Including subsequent transfer of cryopreserved embryos, a total of 33% of couples achieved a successful pregnancy by IVF. Statistical analysis indicated that, for DI pregnancies, the most important semen variable was the percentage post-thaw motility, whilst for normal fertilization in IVF it was the pre-freeze motility. These results may be explained by the compensatory effects of post-thaw processing of spermatozoa for IVF, but not for DI in our clinic.      

Engraftment of dogs with Ia-positive marrow cells isolated by avidin- biotin immunoadsorption

Previous work has shown failure of engraftment in lethally irradiated dogs when autologous marrow was depleted of Ia-positive cells with an anti-Ia antibody and complement before infusion. In the current study, we have utilized an avidin-biotin immunoadsorption procedure to obtain a population of highly enriched Ia-positive cells for autologous bone marrow transplantation in dogs given lethal irradiation. Dog marrow cells (2.4 to 7.0 X 10(9) cells) that contained 8.6% to 19.9% Ia- positive cells were treated successively with monoclonal antibody 7.2, which reacts with a framework determinant of Ia-antigen, and biotin- conjugated goat antimouse immunoglobulin. These treated cells were passed over a column of avidin-Biogel (polyacrylamide) and the adherent cells removed by mechanical agitation. Seven lethally irradiated dogs were transplanted with 5.9 to 33.4 X 10(6) recovered adherent cells per kilogram of which 69.0% to 88.0% were Ia-positive. All dogs had hematologic recovery; six are alive and well with durable engraftment and one died on day 15 posttransplant. They are immunologically normal as determined by lymph node and bone marrow biopsies, lymphocyte function, and immunophenotyping of peripheral blood and bone marrow cells. These data provide further evidence that canine hematopoietic stem cells express Ia-like antigens and that these cells are capable of complete hematopoietic and immunologic reconstitution in an autologous model.     

Cyclosporine as prophylaxis for graft-versus-host disease: a randomized study in patients undergoing marrow transplantation for acute nonlymphoblastic leukemia

Seventy-five patients, 13 to 49 years of age, with acute nonlymphoblastic leukemia in first remission were treated with cyclophosphamide, fractionated total body irradiation, and marrow transplantation from an HLA-identical sibling and randomized to receive either cyclosporine (CSP) (n = 36) or methotrexate (MTX) (n = 39) as prophylaxis for graft-v-host disease (GVHD). All patients engrafted, and 22 who were given CSP and 21 who were given MTX, are alive at 20 to 47 (median, 35) months (P = .5). Engraftment as assessed by granulocyte recovery (P less than .0005) and platelet transfusion requirement (P = .01) was faster in patients on CSP. Twelve patients (33%) on CSP and 22 (56%) on MTX developed acute GVHD of grades II through IV (P = .07) and 15 of 30 on CSP and 14 of 32 on MTX that were at risk developed chronic GVHD. The most frequent causes of death were interstitial pneumonitis and marrow relapse of leukemia, which occurred with similar frequency in both groups. Beneficial effects observed in patients on CSP included less severe mucositis and shorter duration of hospitalization; adverse effects included renal function impairment and hypertension. These data confirm that CSP is a useful immunosuppressant in patients undergoing marrow transplantation but fail to show a significant improvement in survival as compared with the standard regimen of MTX.     

Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor [see comments]

Peripheral blood stem cells (PBSCs) are widely used in autologous transplantation because of ease of collection and rapid hematopoietic reconstitution. However, PBSCs have rarely been used for allogeneic transplantation because of concerns about donor toxicities from cytokine administration and the theoretical increased risk of graft- versus-host-disease (GVHD) from the large number of T cells infused. Eight patients with advanced malignancies received allogeneic PBSC transplants from genotypically HLA-identical sibling donors. All donors received 5 days of recombinant human granulocyte colony-stimulating factor (rhG-CSF; 16 micrograms/kg/day) subcutaneously and were leukapheresed for 2 days. After treatment of the patient with total body irradiation and cyclophosphamide (n = 7) or etoposide, thiotepa, and cyclophosphamide (n = 1), PBSCs were infused immediately after collection and without modification. All patients received cyclosporine and either methotrexate (n = 6) or prednisone (n = 2) for GVHD prophylaxis, rhG-CSF was well tolerated with mild bone pain requiring acetaminophen occurring in two donors. All patients engrafted and in seven hematopoietic recovery was rapid, with 500 neutrophils/microL achieved by day 18 and 20,000 platelets/microL by day 12. Complete donor engraftment was documented by Y chromosome analysis in all four sex-mismatched donor-recipient pairs tested and by DNA analysis in two sex-matched pairs. One patient died on day 18 of veno-occlusive disease of the liver with engraftment but before chromosome analysis could be performed (results are pending in 1 patient). A second patient died of fungal infection 78 days after transplant. Grade 2 acute GVHD occurred in two patients and grade 3 GVHD occurred in one patient. One patient is 301 days from transplant in remission with chronic GVHD; the remaining five patients are alive and disease free 67 to 112 days after transplantation. Preliminary results indicate that allogeneic PBSCs mobilized by rhG-CSF can provide rapid hematologic recovery without an appreciably greater incidence of acute GVHD than would be expected with marrow. Further follow-up is required to determine the incidence of chronic GVHD and any potential beneficial effects on relapse after transplant.     

Allogeneic peripheral blood stem cell transplantation in patients with advanced hematologic malignancies: a retrospective comparison with marrow transplantation

Allogeneic peripheral blood stem cell (PBSC) transplants from HLA- identical siblings were performed in 37 patients with advanced hematologic malignancies. Outcomes were compared to a historical group of 37 similar patients with advanced hematologic malignancies receiving bone marrow (BM) transplants from HLA-identical donors. The PBSC group and historical BM group were well matched for diagnosis, disease stage, age, and graft-versus-host disease (GVHD) prophylaxis. Patients received PBSC transplants between 1993 to 1995 while BM patients were treated between 1989 to 1994. Engraftment, measured by the time to reach a peripheral neutrophil count > 500/L and platelet count > 20,000/microL without transfusions, occurred on days 14 and 11 in the patients transplanted with PBSC compared to days 16 and 15 in the patients receiving BM (P = .00063, .00014). The PBSC group required a median of 8 U of red blood cells and 24 U of platelets compared to 17 U of red blood cells and 118 U of platelets for BM transplant recipients (P = .0005, .0001). The estimated risks of developing grades 2 to 4 acute GVHD were 37% for the PBSC group and 56% for the BM group (P = .18), while the estimated risks of grades 3 to 4 acute GVHD were 14% for the PBSC group and 33% for the BM group, P = .05). Chronic GVHD occurred in 7 of 18 evaluable patients receiving PBSC and 6 of 23 evaluable patients receiving BM, P = .5. The estimated risks of transplant-related mortality at 200 days were 27% versus 45% (P = .33) relapse were 70% versus 53% (P = .27) and of overall survival were 50% and 41% (P = .39) for patients transplanted with PBSC or BM, respectively. This retrospective comparison suggests that compared to marrow transplantation from HLA-identical donors, allogeneic PBSC transplantation from HLA-identical donors is associated with faster engraftment, fewer transfusions, and no greater incidence of acute or chronic GVHD.     

Impact of the CHA2DS2-VASc score on anticoagulation recommendations for atrial fibrillation

BackgroundThe Congestive heart failure, Hypertension, Age ≥ 75 years, Diabetes mellitus, Stroke (CHADS₂) score is used to predict the need for oral anticoagulation for stroke prophylaxis in patients with atrial fibrillation. The Congestive heart failure, Hypertension, Age ≥ 75 years, Diabetes mellitus, Stroke, Vascular disease, Age 65-74 years, Sex category (CHA₂DS₂-VASc) schema has been proposed as an improvement. Our objective is to determine how adoption of the CHA₂DS₂-VASc score alters anticoagulation recommendations.MethodsBetween 2004 and 2008, 1664 patients were seen at the University of Virginia Atrial Fibrillation Center. We calculated the CHADS₂ and CHA₂DS₂-VASc scores for each patient. The 2006 American College of Cardiology/American Heart Association/Heart Rhythm Society guidelines for atrial fibrillation management were used to determine anticoagulation recommendations based on the CHADS₂ score, and the 2010 European Society of Cardiology guidelines were used to determine anticoagulation recommendations based on the CHA₂DS₂-VASc score.ResultsThe average age was 62 ± 13 years, and 34% were women. Average CHADS₂ and CHA₂DS₂-VASc scores were 1.1 ± 1.1 and 1.8 ± 1.5, respectively (P <.0001). The CHADS₂ score classified 33% as requiring oral anticoagulation. The CHA₂DS₂-VASc score classified 53% as requiring oral anticoagulation. For women, 31% had a CHADS₂ score ≥ 2, but 81% had a CHA₂DS₂-VASc score ≥ 2 (P =  .0001). Also, 32% of women with a CHADS₂ score of zero had a CHA₂DS₂-VASc score ≥ 2. For men, 25% had a CHADS₂ score ≥ 2, but 39% had a CHA₂DS₂-VASc score ≥ 2 (P <.0001).ConclusionCompared with the CHADS₂ score, the CHA₂DS₂-VASc score more clearly defines anticoagulation recommendations. Many patients, particularly older women, are redistributed from the low- to high-risk categories.     

急性下肢动脉缺血的临床研究

目的 探讨急性下肢动脉缺血(ALLI)的治疗策略和预后.方法 回顾性分析2003年1月至2009年12月收治的ALLI患者的临床资料.结果 ALLI 130例,包括急性动脉栓塞82例和急性动脉血栓形成48例.单纯全身溶栓和抗凝治疗12例,Fogarty导管取栓81例,行取栓及动脉旁路术15例,9例行导管性溶栓,骨筋膜室切开减压27例.一期截肢13例;死亡9例,死亡率为6.9％,二期截肢8例;总体截肢率为19.3％(21/109).吸烟史、合并糖尿病和起病时间超过24小时是影响保肢的独立危险因素.结论 尽早开始治疗ALLI和选择适当的治疗方法有助于提高疗效.     

A morphological classification of ganglion cells in the zebrafish retina

We examined the distribution and morphological types of ganglion cells in the retina of the zebrafish, a model vertebrate genetic organism. Using cresyl violet and methylene blue staining, a prominent central area was observed in the ventral temporal retina. The density of ganglion cell layer neurons averaged from approximately 12,000/mm2 in the dorsal-nasal retina to a peak of approximately 37,000/mm2 in the ventral-temporal retina. Individual zebrafish ganglion cells were labeled by backfilling with DiI through the optic nerve followed by reconstruction using confocal microscopy. The dendritic stratification and branching pattern of each labeled ganglion cell was examined in relation to the borders of the inner plexiform layer (IPL). We identified 11 different morphological types of ganglion cell. The most commonly labeled ganglion cells were two types termed Type III or IV, which displayed highly stratified dendritic arborizations in their respective ON-, OFF-sublaminae of the IPL. Their dendritic branching patterns were highly asymmetric with many thorn-like varicosities that profusely filled the area of arborization. In contrast, Type V cells formed a small simply branching dendritic field in the innermost portion of the ON-sublamina of the IPL. Two large ganglion cell types (Types I and II) with wide monostratified dendritic fields were found in both the ON- and OFF-sublamina of the IPL. Three different types of multistratified/bistratified ganglion cells were found (Types, IX, X, and XI.) whose dendrites occupied different regions of the IPL. The multistratified dendrites of IX cells occupied the whole width of the IPL, while the dendrites of Type XI cells formed vertical claw-like endings in only the ON-sublamina of the IPL. We conclude that zebrafish ganglion cells display a rich variety of types and branching patterns. This study establishes a series of baseline measurements of zebrafish ganglion cells to facilitate examination of genes playing a role in the specification and stratification of ganglion cell types.     

针刺对去卵巢大鼠脑内芳香化酶基因表达的影响

本研究旨在探讨雌激素对大鼠脑内芳香化酶（Atom）基因表达的影响和针刺“人中”、“内关”、“三阴交”穴对去卵巢大鼠脑内芳香化酶（Amm）表达的调整作用。实验选用成年4—5个月的SD雌性大鼠，将动物分为假手术组（Sham）、去卵巢组（Ovx）和去卵巢针刺组（Ovx＋Ac），3组动物都进行捆绑。用放射免疫分析方法测定血清中雌二醇、睾酮的含量，通过海马中心组织块的琼脂包被静止培养法转化睾酮，然后用放射免疫法测定雌二醇的量来计算组织块的Aran活性，使用原住杂交和免疫组织化学技术来观察海马AmmmRNA和蛋白的阳性神经元表达，然后使用计算机图像分析系统进行统计分析。实验结果显示：去卵巢大鼠体内雌二醇和睾酮的量明显降低，海马AmmmRNA和蛋白的阳性表达产物的平均面积与灰度值均明显减少，与假手术组相比均有显著性差异，脑内海马中心组织块的Amm活性稍微降低，与假手术组相比较不存在显著性差异；去卵巢针刺组与去卵巢组相比，血清雌二醇水平明显升高，海马AmmmRNA和蛋白的阳性表达产物的平均面积与灰度值均明显升高，血清睾酮水平出现降低，海马组织块的Atom活性出现升高，但没有达到显著性水平。以上结果表明：脑内Amm基因表达与血中雌激素水平密切相关，去卵巢大鼠针刺后脑部Arom基因表达量明显升高，使脑局部雌激素水平升高，对于预防及治疗老年脑部疾病有很好的作用。