Percepciones y creencias sobre criopreservación embrionaria en mujeres y hombres que se realizan técnicas de reproducción asistida en Santiago, Chile / Perceptions and beliefs about embryo cryopreservation in women and men who have assisted reproductive techniques in Santiago, Chile

RESUMEN Introducción y objetivos: Aunque la criopreservación embrionaria es frecuentemente utilizada como parte de las técnicas de reproducción asistida, no existe información cuantitativa de cómo las parejas infértiles viven la experiencia de tener embriones criopreservados en Chile. El objetivo del estudio fue examinar las percepciones y creencias que tienen mujeres y hombres respecto de sus embriones criopreservados, sus perspectivas respecto de la donación reproductiva y destino de los embriones remanentes. Metodología: 153 mujeres y hombres con embriones criopreservados provenientes de un hospital público, Instituto de Investigaciones Materno Infantil y un centro privado, Clínica Las Condes, en Santiago, Chile, respondieron durante mayo 2015 a mayo 2016 un cuestionario en línea, anónimo, respecto de sus percepciones y creencias sobre criopreservación embrionaria. Resultados: Los encuestados reconocen a sus embriones criopreservados como un hijo (53.2%) o un proyecto de hijo (40.7%). Sólo 8% los considera un grupo organizado de células; sobre el 60% rechaza la opción de descartarlos o usarlos para investigación. Los participantes del hospital público tenían mayor disposición a donar sus embriones remanentes a otras parejas que aquellos del privado (61% vs 40%; P=0.016). Un 34% de las personas encuestadas estuvo de acuerdo con donar embriones a parejas de un mismo sexo. Conclusión: Este estudio muestra que las personas chilenas tienen un vínculo emocional con sus embriones criopreservados y no consideran descartarlos. Los resultados de este estudio pueden servir para dar adecuada consejería a las personas que se realizan técnicas de reproducción asistida, de tal modo de tomar decisiones informadas respecto de la criopreservación.

ABSTRACT Background and objetive: Although embryo cryopreservation is frequently used as part of assisted reproductive technology, quantitave information addressing how infertile couples live the experience of having cryopreserved embryos is lacking in Chile. The aim of this study is to examine men and women's perception and beliefs regarding their cryopreserved embryos, as well as their perspective on embryo donation and disposition. Methods: 153 women and men with frozen embryos from a public hospital, Instituto de Investigactiones Materno Infantil, and a private clinic, Clínica Las Condes, in Santiago, Chile, responded between May 2015 and May 2016 to an anonymous online survey addressing their perceptions and beliefs concerning their cryopreserved embryos. Results: Respondents considered their frozen embryos to be equivalent to a child (53.2%) or a potential child (40.7%). Only 8% regard them as an organized group of cells. Over 60% of respondents disagree with destroying surplus embryos or using them for research. Participants from the public hospital are more willing to donate their embryos to another couple than those from the private center (61% vs 40%; P=0.016); 34% of respondents agreed to donate surplus embryos to same sex couples. Conclusion: This study reveals that Chilean couples are emotionally bound to their frozen embryos, and that discarding them is not an option. The results from this survey will help strengthen counseling for couples to enable them to make informed decisions regarding their surplus embryos.

Evaluation of the clinical application efficacy of cryopreserved apheresis platelets and fresh apheresis platelets / 中国输血杂志

Objective To evaluate the clinical value of apheresis platelets throught heanalysisof case control on the clinical efficacy and safety of cryopreserved apheresis platelets and fresh apheresis platelets.Methods 2 035 clinical cases of platelet transfusion in August 2014 to December 2016 by Using the closed loop intelligent path management and evaluation information system,456 cases were selected as control cases.Platelets were divided into the cryopreserved apheresis platelets group (group A,n=199) and fresh apheresis platelets group (group B,n=257) according to the transfused platelet type.The clinical application value of cryopreserved single platelets was evaluated by comparing the basic data,the effective indexes and safety indexes of the two groups.Results 1) The cases were 43.6％ (199/456) in A groups,and 56.4％ (257/456) in B groups,there were no significant difference in gender,age and medical and surgical cases between A and B group (P＞0.05);2) 199 cases in group A were cryopreserved platelets of 2 275 U,including 121 medicine cases,the total amount of transfusion was about 60.9％ (1 385/2 275),78 surgical cases accounted for 39.1％ (890/2 275);In the distribution of diseases,the blood system diseases accounted for 49.2％ (1 120/2 275),the total amount of obstetrics and gynecology disease infusion accounted for 10.6％ (240/2 275),and the amount of tumor radiotherapy and chemotherapy accounted for 6.2％ (140/2 275);The proportion of ABO blood type distribution was O type 25.9％,A type 22.9％,Btype 20.7％,ABtype 30.5％,respectively;3) The Plt counts of group A and B were significantly different before and after transfusion (P ＜0.05).But there was no significant difference between the two groups of cases before transfusion and 24h Plt count after transfusion,the Plt counts difference,and 24 h CCI (P＞0.05);4) The effective rates of platelet transfusion in group A and B were 76.9％ and 76.7％,respectively.Which has no significant difference between the two groups (P＞0.05)).There was no significant difference between the two groups in medical and surgical cases (P＞0.05),but the effective rate of surgical cases in group A (84.6％) was higher than that in B group (75.3％).The difference effect of medicine and surgery cases in B group were not statistically significant (P＞0.05),but the difference effect of medicine and surgery cases in A group was statistically significant (P＜0.05),platelet transfusion inefficient in surgical cases (15.4％) was significantly lower than that of cases (28.1％);5) The incidence of adverse reactions of blood transfusion was 3.5％,4.7％ in group A and B,and the blood transfusion mortality rate was zero,the difference was not statistically significant (P＞ 0.05).Conclusion The clinical effectiveness and safety of cryopreserved apheresis platelets are similar to those of fresh apheresis platelets,and the former can be widely Used in clinic,in particular,it has certain advantages in the surgical hemostatic effect.but for repeated infusion cases or platelet transfusion ineffective cases should be given priority to fresh apheresis platelets.

C-type Anterior Lamellar Keratoplasty Using Cryopreserved Leftover Cornea for Terrien's Marginal Degeneration

PURPOSE: To report a case of Terrien's marginal degeneration treated with C-type anterior lamellar keratoplasty using cryopreserved leftover cornea. CASE SUMMARY: A 63-year-old female visited our clinic because of left ocular discomfort and visual deterioration over several years. The patient had +2.25 Dsph = -5.00 Dcyl × 111° of astigmatism, and best corrected visual acuity was 20/20. Microscopic slit lamp examinations revealed an approximately 10.0 mm width semilunar shaped stromal opacity with surrounding stromal lipid deposit, as well as superficial neovascularization with thinning at superior perilimbal cornea. Anterior segment optical coherence tomography confirmed extreme thinning at the opacified cornea. The patient was diagnosed with Terrien's marginal degeneration. To prevent corneal perforation, C-type anterior lamellar keratoplasty using cryopreserved leftover cornea was performed. After 18 months after operation, donor graft was successfully attached via the anterior segment optical coherence tomography and microscopic slit lamp examination and graft rejection was not observed. CONCLUSIONS: C-type anterior lamellar keratoplasty using a cryopreserved cornea can be an effective therapeutic strategy for Terrien's marginal degeneration.

A Case of Therapeutic Keratoplasty Using Cryo-preservative Cornea in Candida albicans Keratitis

PURPOSE: To report a case treated with therapeutic keratoplasty using a cryo-preserved cornea in a patient with Candida albicans keratitis. CASE SUMMARY: A 77-year-old female visited our clinic because of left ocular pain and visual disturbance for 3 days. Microscopic slit lamp examination revealed a 1.2 mm sized round corneal epithelial defect with deep stromal infiltration, brownish pigmentation and signs of inflammation with cyclitic membranes in the anterior chamber. On suspicion of Candida keratitis, we performed penetrating keratoplasty using a cryo-preserved donor cornea in Optisol-GS® (Bausch & Lomb, Irvine, CA, USA) solution with excision of the infected iris and colony of the anterior chamber. After the procedure, injection of intravitreal or intracameral amphotericin B and voriconazole were administered alternately. At 2 weeks after the second surgery, infection signs disappeared. At the follow-up in the outpatient clinic, signs of infection were not observed. CONCLUSIONS: Therapeutic keratoplasty using a cryo-preserved donor cornea can be an immediate and effective therapeutic strategy for Candida albicans keratitis.

Safety of the offspring conceived by assisted reproductive technology with cryopreserved donor sperm / 中华男科学杂志

<p><b>Objective</b>To investigate the pregnancy outcomes of assisted reproductive technology (ART) with cryopreserved donor sperm and the safety of the offspring thus conceived.</p><p><b>METHODS</b>The Human Sperm Bank of CITIC Xiangya Hospital provided cryopreserved donor semen to 31 reproductive centers in China between January 2006 and December 2012, with which 50247 ART cycles were accomplished. We compared the rates of birth defects and spontaneous abortion of intracervical insemination (ICI), intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI).</p><p><b>RESULTS</b>A total of 39 047 ART cycles were performed by artificial insemination with cryopreserved donor sperm, including 36 674 cycles of ICI and 2 372 cycles of IUI. Among the 8 612 clinical pregnancies achieved by ICI, there were 917 cases of spontaneous abortion (at <28 gestational wk) (10.6%) and 6133 live births, with 43 cases of birth defect (0.70%). Of the 547 clinical pregnancies achieved by IUI, there were 41 cases of spontaneous abortion (7.5%) and 426 live births, with 2 cases of birth defect (0.47%). Totally, 11 200 cycles of IVF and ICSI were accomplished with cryopreserved donor sperm. Of the 5 860 clinical pregnancies achieved by IVF, there were 456 cases of spontaneous abortion (7.8%) and 5089 live births, with 55 cases of birth defect (1.08%). Among the 350 clinical pregnancies achieved by ICSI, there were 30 cases of spontaneous abortion (8.6%) and 229 live births, with 3 cases of birth defect (1.31%). The birth defect rate of ART with cryopreserved donor sperm was significantly lower than that published by the Chinese Ministry of Health (0.86% vs 1.53%,P<0.01).</p><p><b>CONCLUSIONS</b>The safety of the offspring conceived by ART with cryopreserved donor sperm is controllable.</p>

Tectonic Lamellar Keratoplasty Using Cryopreserved Cornea in a Large Descemetocele

We describe herein a case of an impending corneal perforation with a large descemetocele in a patient with previous penetrating keratoplasty (PKP) that subsequently was treated with an emergent lamellar keratoplasty using frozen preserved cornea. A 76-year-old male patient, who had a PKP, presented with a completely whitish and edematous graft accompanied by large epithelial defects. Although antibiotics and antiviral agents were tried for three days, the corneal stroma abruptly melted, except for the Descemet's membrane and endothelium. Cryopreserved corneal tissue that was kept at -80degrees C was thawed and sutured on top of the remaining Descemet's membrane and endothelium. Pathological and microbiological tests were conducted using the remaining donor and recipient corneal tissues. After tectonic corneal transplantation on top of a large descemetocele, a healthy graft and relatively clear interfaces between graft-host junctions were maintained without serious adverse reactions throughout 6 month follow-up period. Microbiological evaluations of donor tissue at the time of thawing and tissue preparation were done, and the results were all negative. Tissue that was taken intraoperatively from the recipient cornea also showed negative microbiological results. In conclusion, tectonic lamellar keratoplasty, using cryopreserved corneal tissue, only onto the remaining Descemet's membrane and endothelium in an emergent condition, was a safe and effective treatment.

Sourcing human embryos for embryonic stem cell lines: Problems & perspectives.

The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ‘discarded’ or ‘spare’ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. in case a couple does not desire to ‘cryopreserve’ their embryos then all the embryos remaining following embryo transfer can be considered ‘spare’ or if a couple is no longer in need of the ‘cryopreserved’ embryos then these also can be considered as ‘spare’. But, the question raised by the ethicists is, “what about ‘slightly’ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ‘discarded’ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ‘discarding’ embryos. What would be the criteria for discarding embryos and the potential ‘use’ of ESC derived from the ‘abnormal appearing’ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

Tasa de embarazo de inseminación intrauterina heteróloga usando cánulas de transferencias de embriones

Objetivos: Conocer la tasa de embarazo de inseminación intrauterina (IIU) heteróloga, usando las cánulas de transferencia de embriones (TE). Diseño: Estudio retrospectivo. Institución: Centro de Fertilidad Procrear, Lima, Perú. Participantes: Parejas en tratamiento de infertilidad. Intervenciones: Se estudió todos los casos de procedimientos de IIU heteróloga en los que se usó semen de donante del banco de esperma, entre 2007 y septiembre de 2010. Todas fueron realizadas por el mismo ginecólogo y el mismo biólogo. Las IIU se realizaron utilizando cánulas de TE Frydman-Soft con guía, previa medición ecográfica del cérvix. Se analizó la tasa de embarazo por ciclo y por paciente, así como los factores asociados al embarazo. Para el análisis estadístico se usó Epi Info 3.5.1. Principales medidas de resultados: Tasa de embarazos. Resultados: Se realizó 30 procedimientos de IIU heteróloga, en 26 pacientes, con edad promedio 32,6 ± 5,5 años (rango 20 a 41 años), siendo el 40% mayor de 35 años. La tasa de embarazo fue 40% por ciclo y 46% por paciente. La mayoría (87%) tuvo solo un intento. La edad menor de 35 años y la respuesta folicular de 2 a 4 folículos mayores de 17 mm se asociaron a mejores resultados; mientras que tener una trompa obstruida, grosor endometrial ≤ 6 mm o inseminación después del día 15 del ciclo se asociaron a resultados negativos. Conclusiones: La IIU heteróloga con cánulas de transferencia de embriones ofrece excelentes resultados en tasa de gestación; además, sirve de entrenamiento para médicos que todavía no realizan procedimientos de alta complejidad.

Objectives: To determine the heterologous intrauterine insemination (H-IUI), rate of pregnancy by using embryo transfer cannula. Design: Retrospective study. Setting: Procrear Fertility Center, Lima, Peru. Participants: Couples in infertility treatment. Interventions: All H-IUI procedures using sperm bank donor semen from 2007 through September 2010 were studied, all performed by the same gynecologist and the same biologist. IUI was performed using Frydman-Soft embryo transfer cannula with guide following cervix measurement by ultrasound. Rate of pregnancy per cycle and per patient was analyzed as well as factors associated with pregnancy. Epi Info 3.5.1 was used for statistical analysis. Main outcome measures: Pregnancy rate. Results: Thirty H-IUI proceedings were performed in 26 patients with average age 32.6 ± 5.5 years (range: 20-41 years), 40% over 35 years. Pregnancy rate was 40% per cycle and 46% per patient. Most (87%) had only one attempt. Age less than 35 years and 2 to 4 follicles more than 17 mm were related to best results. Having an obstructed tube, endometrial thickness ≤ 6 mm, or insemination after cycle day 15 were associated to negative results. Conclusions: Heterologous IUI with Frydman-Soft embryo transfer cannula provided excellent results in conception and served for training physicians who still do not perform high-complexity procedures.

A Case of Ruptured Cryopreserved Homograft 7 Months after Implantation / 日本心臓血管外科学会雑誌

Infectious abdominal aortic aneurysm is a relatively rare disease, and there is no consensus regarding its surgical treatment. Medical infectious control should be concerned comparison with surgical treatment if there is sepsis, however we sometimes have no other choice but emergency operation for uncontrollable cases. In many reports, cryopreserved homografts were used as <i>in</i>-<i>situ </i>alternative grafts for infectious aortic aneurysms because they had some merits such as anti-infectious effects, suitability and so on. However the number of <i>in-situ </i>cryopreserved homograft replacement cases are few, and the long term result is unclear. We encountered a ruptured cropreserved homograft case 7 months after urgent <i>in-situ </i>cryopreserved homograft replacement. We report the case and refer to the relevans literature.

Study the expression of caspase-3 on trehalose as cryoprotectant for preserving aortic valve homograft in liquid nitrogen / 国际外科学杂志

Objective To observe the expression of caspase-3 on the trehalose as cryoprotectant for preserving aortic valve homograft in liquid nitrogen.Methods The aortic valve homograft was divided into 5groups,namely:0.1 mol/L DMSO(control group),0.1 mol/L trehalose(experimental group 1),0.1 mol/L trehalose+0.1 mol/L DMSO(experimental group 2),0.2 mol/L trehalose+0.1 mol/L DMSO(experimental group 3),0.3 mol/L trehalose+0.1 mol/L DMSO(experimental group4).At the time of 12 months,15 months and 18 months when preserved in liquid nitrogen,relative expression of caspase-3 of the aortic valve homograft was measured by RT-PCR and Western Blot.Fresh group was a negative control group.Results At the same time(P<0.05),the expression of caspase-3 of fresh aortic tissue was slightest.The experimental group 2 was in accord with the experiment group 3,which was of a sort compare with the fresh group.The experimental group 4,which was worse than the experimental group 2 and 3,ranked above the experimental group 1.The worst was the control group.Conclusions The joint use of trehalose and DMSO could well inhibit the expression of caspase-3.Moreover.0.1mol/L trehalose+0.1 mol/L DMSO and 0.2 mol/L trehalose +0.1 mol/L DMSO could maximize the inhibition of the expression of caspase-3.

Development of Cryopreserved Red Blood Cell Panels for Verifying ABO and D Blood Grouping Reagents / 대한수혈학회지

BACKGROUND: ABO blood grouping reagent verification is essential to ascertain safe blood transfusions. However, the research use of donated blood products has been hampered in Korea by the blood transfusion law and management policies. In this study, we developed cryopreserved red blood cell (RBC) panels utilizing the high glycerol method to verify the ABO and D blood grouping reagents. In addition, we evaluated the stability of ABO and D antigenicity. METHODS: Fresh blood was frozen by the high glycerol method, aliquoted and cryopreserved in 2 mL cryotubes. Twenty-four vials of bloods with types A (n=5), B (n=5), AB (n=4) and O (n=10) for ABO RBC panels, and eleven vials of blood types D positive (n=5), D negative (n=5) and D weak (n=1) for D RBC panels were established. Potency, avidity and specificity tests were carried out with four different commercial ABO and D blood grouping reagents. RESULTS: The potency of cryopreserved RBCs after thawing showed no statistical difference compared with pre-freezing RBCs. Avidity time measurements were 5 seconds in ABO blood and 20 seconds in D positive blood. Specificity test uniformly showed 100% specificity. When thawed RBCs were stored at 4degrees C for 7 days, the potency test measured at intervals of 2 days showed no variation. CONCLUSION: Cryopreserved RBC panels produced by the high glycerol method showed excellent results in stability test with reagents produced by manufacturers in Korea. Therefore, these panels can be utilized as a reliable method of verifying blood grouping reagents.

The experimental study of cryopreserved neural stem cells transplanting on Wistar rat spinal cord injury / 中华检验医学杂志

Objective To observe the therapeutic effect of spinal cord injury(SCI)by transplantation cryopreserved neural stem ceils(NSCs).Methods 45 Wistar rats were randomly divided into three groups.with 15 rats in each group.Transplantations were carried out two days after SCI,group A:0.9% saline water,group B:NSCs,group C:cryopreserved NSCs.After the transplantation,immunohistochemical and tissue incision were appeared to detect the survival and differentiation of the transplanted ceils in nivo,and the methods of BBB and oblique plate were used to estimate the recovery of function.Results Group B and C all had active NSCs except group A.The injuried spinal cord A[(3.9±0.1)mm2],B[(1.2±0.3)mm2]and C[(1.1±0.3)mm2],there was an variance in the three groups (F=423.949,P＜0.01),BBB test:group A[(2.0±0.5)mark],B[(16.4±0.8)mark]and C [(16.0±1.4)mark],there was an variance in the three groups(F=970.157,P＜0.01),the oblique plate test:group A[(31.3±2.9)degree],B[(46.8±2.1)degree]and C[(46.5.4-2.4)degree],there was an variance in the three groups(F=151.099,P＜0.01),the tests demonstrate there were no variance in group B and C(all P＞0.05),there was no variance in living rate between the cryopreserved NSCs group [(55.9±5.2)%]and no eryopreserved[(65.1±3.4)%,t=3.334,P＞0.05].Conclusions Cryopreserved NSCs keep the ability of reproducdon and differentiation.NSCs is a kind of valuable cell resource for the therapy of SCI.

Restoration of Reproductive Potential after Autotransplantation of Frozen Ovaries in Mice Pretreated with Cyclophosphamide / 한국유방암학회지

PURPOSE: Infertility due to ovarian failure that is caused by antineoplastic chemotherapeutic agents is one of the primary problems of female cancer atients who are in their reproductive years. It has become important to preserve the reproductive potential of female cancer patients. This study was conducted to determine whether autotransplantation of frozen ovaries can restore reproductive potential. METHODS: This study included 30 female mice that had normal reproductive potential. The mice were divided into 4 groups: the positive control, the negative control, the comparison group, and the experimental group. The positive control group received right total oophorectomy, and the negative control group received bilateral total oophorectomy. Greater than or equal to 90% of the left ovary was removed in the mice of the comparison group, and then cyclophosphamide was administered. In the experimental group, the right ovary taken out by right total oophorectomy, and this was crypreserved using the vitrification method. And then cyclophosphamide was administered. The cryopreserved ovary was autotransplanted to the left gonadal fat pad after greater than or equal to 90% of the left ovary was removed. The reproductive performance in each group was analyzed according to the pregnancy rate after mating. RESULTS: In the positive control group, all five mice became pregnant, and the number of fetuses was 4 to 5 (mean=4.60+/-0.55). In the comparison group, the pregnancy rate was 50%, and the mean number of fetuses was 1.40+/-0.55. In the experimental group, 7 of 10 (70%) mice became pregnant, and the mean number of fetuses was 4.71+/-2.56. There was no significant difference in the number of fetuses between the positive control and the experimental group (p=0.093), but there was a significant difference in the number of fetuses between the comparison group and the experimental group (p=0.019). CONCLUSION: The results of this study suggest that autotransplantation of frozen ovaries using the vitrification method may restore the impaired ovarian function induced by antineoplastic chemotherapeutic agents.

Comparative study between natural cycles and programmed cycles using exogenous steroid hormones for endometrial preparation in cryopreserved-thawed blastocyst transfer / 대한산부인과학회지

OBJECTIVE: The objective of this study was to compare the outcomes of cryopreserved-thawed blastocyst transfer (CT-BT) in natural or programmed cycles using exogenous steroid hormones. METHODS: A total of 221 CT-BT cycles were included and divided into two groups according to endometrial preparation protocols. In natural cycle group (n=116), monitoring was performed by transvaginal ultrasonography to detect ovulation. In programmed cycle group (n=105), oral estradiol valerate, 6 mg/day, was started on the third day of the menstrual cycle and administered continuously, and progesterone in oil 100 mg i.m. daily injection was started on cycle day 15. CT-BTs were performed on five days after ovulation in natural cycles and five days after the initiation of progesterone administration in programmed cycles. Pregnancy rates, implantation rates, and other clinical characteristics of the two groups were compared. RESULTS: Clinical characteristics of study subjects did not differ between the two groups. Post-thaw survival rates, number of transferred blastocysts, and number of good-quality blastocysts were not different. There were no statistically significant differences in implantation rates (21.1% vs. 19.4%), clinical pregnancy rates (36.2% vs. 36.2%), and ongoing pregnancy rates (28.4% vs. 27.6%) between the two groups. CONCLUSIONS: No statistically significant differences were found in pregnancy rates and implantation rates between the two protocols. Our results suggest that both protocols are equally effective for endometrial preparation in CT-BT cycles.

Outflow Reconstruction using Cryopreserved Aortic Patch in Right Lobe Living Donor Liver Transplantation

METHODS: Living donor liver transplantation (LDLT) using a right lobe graft has been widely used to compensate for the cadaveric organ shortage. Successful reconstruction of the middle hepatic vein (MHV) is required to provide an adequate functional volume in LDLT with using the right lobe. We describe herein a new technique using a cryo-preserved aortic patch for outflow reconstruction of the right lobe graft with or without MHV. METHODS: From November 2005 through March 2006, 20 adult patients who received a right lobe graft (n=10) or an extended right lobe graft (n=10) for LDLT were included. During the bench procedure of the right lobe graft, we reconstructed the new MHV with using cryopreserved veins just like the MHV of the extended right lobe graft, and we then made a venous pouch to form a common trunk between the MHV (or new MHV) and the RHV of the right lobe graft with using a cryopreserved aortic patch. During graft implantation, anastomosis of an outflow tract was made between the venous pouch of the graft and the common trunk of recipient's RHV-MHV-LHV. One week following the transplantation, measurement of the pressure gradient between the MHV and IVC was done, as well as performing regular follow-up 3D-CT scans and liver function tests. RESULTS: The mean pressure gradient between the reconstructed MHV and the recipient's IVC was 2.3+/-1.2mmHg, and in all cases, the serial liver function tests showed gradual improvement as the days progressed post-operatively. There was no evidence of hepatic venous congestion of the graft and/or obstruction of the reconstructed MHVs according to the serial postoperative follow-up images of the Doppler US and MD-CT. CONCLUSION: We suggest that reconstructing the outflow tract with a cryopreserved aortic patch is a good alternative technique for preventing anterior segment congestion in LDLT with using a right lobe graft with or without MHV.

Effect of Intrasplenic Transplantation of Cryopreserved Hepatocytes into Partially Hepatectomized Rats / 대한간학회지

BACKGROUND/AIMS: Various techniques of hepatocyte transplantation were actively studied as an alternative to liver transplantation, because of the difficulty of obtaining donor organ, technical difficulties, and high cost. Isolated hepatocytes could be appropriately banked and distributed on demand. We tried to investigate the effect of intrasplenic transplantation of allogenic cryopreserved hepatocytes, into spleen prior to 90% partial hepatectomy in rats, on the survival rate. METHODS: Cryopreserved hepatocytes, isolated by collagenase perfusion of the liver via the portal vein, were thawed and transplanted into the spleen of rats prior to induction of acute hepatic failure by resection of all lobes except caudate lobe (2.0x107 hepatocytes/rat). RESULTS: 1. The viability of freshly isolated hepatocyte was 70-5%, but cell viability after cryopreservation 30-0%. 2. Difference of survival in control and transplant group is not statistically significant. but the survival rate, 48 hours after 90% partial hepatectomy, for control (7) and transplanted group (11) were 0% and 18%, respectively. 3. Although the glucose reduction gradient was not significantly different between two groups, it was more prominent in the control group than in the transplanted group. 4. Engraftment and survival of transplanted hepatocytes were noted in the spleen 2 days after transplantation. CONCLUSIONS: We could not observe statistically significant improvement of survival with intrasplenic transplantation of cryopreserved hepatocytes in rats with 90% partial hepatectomy-nduced acute liver failure. However, 18% survival after 90% partial hepatectomy was noted in the transplanted group, compared to no survival in the control group. This suggests that intrasplenic transplantation of cryopreserved hepatocytes might be effective in the treatment of acute liver failure.

Clinical Significance of Endometrial Thickness and Pattern in Ovum Donation and Cryopreserved - Thawed Embryo Transfer Program / 대한산부인과학회잡지

OBJECTIVE: To investigate the clinical significance of endometrial thickness and pattan as a predictor of successful implantation of embryos in ovum donation and cryopreserved-thawed embryo transfer program. METHODS: From January, 1996 to March, 1998, 31 cycles of ovum donation and 31 cycles of cryopreserved-thawed embryo transfer were enrolled in this prospective study. Endometrial thickness was measured three times: prior to progesterone administration (P), 1 day and 3 days after P. In cryopreserved-thawed embryo transfer cycles, the measurement at 1 day after P was omitted. Endometrial pattern was observed prior to progesterone, and was considered meaningful when a multi-layered triple-line was seen with prominent outer and central hyperchogenic lines and inner hypoechogenic regions. RESULTS: There were no differences in embryo quality, dose or duration of estrogen, and endometrial thickness or pattern between conception and non-conception cycles in both ovum donation and cryapreserved-thawed embryo transfer pmgram. In ovum donation cycles, no cortelation was observed between estrogen dose and endometrial thickness or pattern. In cryopreserved-thawed embryo transfer cycles, total estrogen dose and endometral thickness at 3 days after P has a inverse correlation, and estrogen dose over 4.3 mg per day can predict expression of a multi-layered triple-line pattern, CONCLUSION: Endometrial thickness or pattern. cannot predict a successful implantaion of embryos in both ovum donation and cryopreserved-thawed embryo transfer cycles.

The Establishment of Sperm Penetration Assay Using Cryopreserved Hamster Oocyte / 대한비뇨기과학회지

To establish sperm penetrating assay (SPA) with using cryopreserved hamster oocyte, we performed the stepwise SPA with 1) fresh hamster oocyte and hamster sperm, 2) cryopreserved hamster oocyte and hamster sperm, 3) fresh hamster oocyte and human sperm, and 4) cryopreserved hamster oocyte and human sperm, in 4 cases of male hamster and 12 cases of fertile human. In SPA of hamster sperm with fresh hamster oocyte, the oocyte penetration rate (PR) were 100+0%, and the penetration index(mean penetration per oocyte, PI) was 22.4 +/- 1.8. In SPA of hamster sperm with cryopreserved hamster oocyte, the PR were 100 +/- 0%, and the Pl was 14.1 +/- 2.9 (p<0.01). When the oocytes were examined at 1, 2, 3, and 6 hour post insemination, hamster sperm penetration was 1 hour slower in cryopreserved oocytes than in fresh ones. In SPA of human sperm with fresh hamster oocyte, the PR was 79.5 +/- 10%, and the Pl was 2.78 +/- 2.6. In SPA of hamster sperm with cryopreserved hamster oocyte, the PR was 73.9+/- 16%, and the PI was 2.82 +/- 2.7. There`s no significant difference in SPA using human sperm. These results suggest that them may be some functional damages on cryopreserved oocyte, because Pl of fresh oocytes is higher than that of cryopreserved oocytes. However in sperm of human, it dose not make significant difference in Pl between fresh and cryopreserved oocytes. The SPA using cryopreserved hamster oocyte would appear to have wide application of the evaluation of infertility, the assessment of the treatment of infertility and the experiment in infertility field.

An Experimental Study of Cryopreservation of Aortic Valve Allograft with Maintenance of Cell Viability / 日本心臓血管外科学会雑誌

Aortic valve allografts have been used extensively for aortic valve replacement, aortic root replacement and relief of right ventricular outflow tract obstruction. Some investigators consider that the degree of cellular viability is important in determining allograft durability. In order to evaluate cell viability and histological changes of cryopreserved aortic valve allograft in a pig model, porcine aortic and pulmonary valves are subjected to cryopreservation. Porcine aortic valves were obtained from a slaughterhouse in a non-sterile condition. The dissected valves together with vascular walls were kept in a solution of antibiotics (CFX, IPM/CS, PCG, SM) for 6hr, at 37°C. After sterilization, no growth of aerobic and anaerobic bacteria, as well as fungi was seen in pieces of valves. For cryopreservation, the program freezing method (control freezing at a rate of -1°C/min) and the rapid freezing method (simple immersion in liquid nitrogen), with and without 10% dimethylsulfoxide (DMSO) for cryoprotective agents, were tested. Cell viability was assesed by cell growth from pieces of valves and vascular walls. Histological changes and cell viability were evaluated after storage periods of 1 week, 1 month and 3 months. By the program freezing method with 10% DMSO, cell viability was well preserved and no histological change was detected after 3 months storage. By the rapid freezing method with 10% DMSO, cell viability of valves and vascular walls, except for aorta, were preserved and histological changes were slight. The valves and vascular walls cryopreserved without DMSO showed no cell growth after storage of 1 week. The result suggests that the program freezing method with 10% DMSO is applicable in a clinical use.

Experimental Study on Reviability of Cryopreserved Human Spermatozoa / 대한비뇨기과학회지

In order to perform standardization in analytic methods of cryopreserved human semen, to investigate the differences of resistance to cryoinjury, to define the stage of critical cryoinjury during cryopreservation and to evaluate the quality change after thawing by time interval, the reviability of 30 normal and 30 abnormal semen were evaluated by the supravital stainings of spermatozoa using acridine orange and eosin yellow and the motility assay simultaneously according to the stages of freezing-thawing and the time interval after thawing. The results were summarized as follows: 1. Vitality was estimated higher than motility at all specimens and the gap between two became greater as motility decreased. 2. Reviability of abnormal semen was estimated lower than that of normal semen(p<0.05). 3. The critical cryoinjury to spermatozoa was noticed at the stage of freezing from 4 degrees C to -10 degrees C (p<0.05). 4. The significant decrease in quality of normal cryopreserved semen was noticed between 30 to 60 min. after thawing (p<0.05). These results suggest that the cryoinjury to human semen is different in nature, therefore it is advisable that the quality of cryopreserved human semen should be evaluated by vitality and motility assay simultaneously. And the resistance of abnormal semen to cryopreservation is so low that it would be difficult to be used clinically with satisfaction. Moreover the laboratory studies should be concentrated on freezing method to achieve better reviability and it is desirable in practice that post-thaw semen should be used within 30 min, after thawing.