Transplantation of intact rat gonads using vascular anastomosis: effects of cryopreservation,ischaemia and genotype

BACKGROUND: A limited store of ovarian follicles is present at birth and its progressive decline during ageing is hastened by alkylating agents and ionizing radiation during treatment for cancer or autoimmune disease. Oligo- or azoospermia can arise for similar reasons in men. There is some experimental evidence showing that targeted gene deletion or drugs to produce hypogonadotrophism can protect germ cells from wastage. Another strategy for conserving fertility is to cryopreserve ovarian or testicular tissue for subsequent transplantation. To maximize gonadal function, it is desirable to preserve whole gonads for transplantation using vascular anastomosis. METHODS AND RESULTS: We investigated this strategy in the rat model. All freshly isotransplanted ovaries (n = 8) survived and resumed follicle growth and secretion and, although ischaemia for 24 h at 4 degrees C did not disrupt ovarian function, the organs had fewer follicles. Four out of seven (57%) cryopreserved transplants survived for > or =60 days, were ovulatory and one pregnancy was established, but the ovarian reserve was compromised by fewer follicles. Ovarian allotransplants were vigorously rejected, even with moderate immunosuppression using cyclosporin A. On the other hand, only three out of seven (42%) fresh testicular isotransplants had active spermatogenesis, and none of the cryopreserved testes was functional. CONCLUSIONS: The effects of gonadectomy in rats can be reversed by isotransplants, but the results are more successful with ovaries than testes, and allotransplants were never successful. Intact cryopreserved ovaries can be restored to function after transplantation with vascular anastomoses.     

Effect of pentoxifylline on motility and membrane integrity of cryopreserved human spermatozoa

The purpose of this study was to examine the effects of pentoxifylline used before and after semen cryopreservation-thawing on sperm motility and membrane integrity. Twenty-four semen samples were split into four equal aliquots. Aliquots were incubated at 37 degrees C for 30 min, followed by cryopreservation with TEST-yolk freezing medium using slow programmable freezing protocol. After 2 weeks the sperm samples were thawed, washed twice in Quinn's Sperm Washing Medium (modified HTF with 5.0 mg/mL Human Albumin) and incubated at 37 degrees C for 30 min. Aliquots were treated by adding 3 mmol/L pentoxifylline to: (1) fresh sperm samples during incubation period prior to cryopreservation, (2) sperm samples as a supplement to the cryoprotectant prior to cryopreservation, and (3) thawed sperm samples during incubation period. One aliquot received no treatment (control group). The addition of 3 mmol/L pentoxifylline to fresh semen during incubation period prior to cryopreservation significantly decreased progressive and total motility compared with controls. However, the addition of 3 mmol/L pentoxifylline to cryopreserved semen after thawing significantly increased progressive and total motility compared with controls. After post-thaw, no differences in motion characteristics between sperm samples treated by adding 3 mmol/L pentoxifylline as a supplement to the cryoprotectant and control groups were observed. Post-thaw hypoosmotic swelling (HOS) test scores did not improve with the addition of pentoxifylline compared with the control group. It is concluded that pentoxifylline enhanced post-thaw motility of cryopreserved human spermatozoa when added after thawing. No improvement was found by freezing sperm with pentoxifylline.     

Creatine Kinase Level and Lipid Peroxidation Rate in Human Spermatozoa from Patients with Cancer

Purpose: The present study assessed whether the poor semen quality in patients with cancer results from the inhibition of sperm maturation as indicated by creatine kinase or from increased oxidative stress as assessed by lipid peroxidation of the sperm membrane. Methods: Cryopreserved semen specimens from patients with testicular (n = 10) and nontesticular (n = 12) cancer and normal healthy donors (n = 14) were analyzed for lipid peroxidation and creatine kinase levels. Results: The levels of creatine kinase and malonaldehyde did not differ among testicular or nontesticular patients with cancer or normal healthy donors. Conclusions: Poor semen quality in testicular and nontesticular patients with cancer is not related to creatine kinase or lipid peroxidation levels; it may be related to other factors.     

胎脑组织移植对额叶皮层损伤大鼠辨别学习,记忆之影响

为观察胎脑组织移植对双侧额叶皮层损伤大鼠学习、记忆功能的影响，对大鼠模型行胎脑组织移植。学习，记忆再现测验在Ｙ型迷宫中进行。大鼠学习和记忆成绩以测验时达至于是０次电击均为正确反应时所需的电击次数表示。结果表明，胎脑组织移植能显著减少达到上述标准所需的电击次数，且移植３个月较移植１０天的动物达到上次标准所需的训练次数减少更明显。     

Effect of coculture on subsequent survival and implantation of cryopreserved human embryos

Purpose This retrospective analysis was designed to assess the performance of human embryos following cryopreservation based on whether they were originally developed in standard culture medium (65 cycles, 223 embryos) or cocultured on partial monolayers of bovine oviductal epithelial cells (63 cycles, 198 embryos). Embryo cryosurvival and implantation were compared between the study group and the contemporaneously matched controls.Results During a 2-year period when no factors of the cryopreservation program were altered, 63 transfers of 159 surviving thawed control cleavage-stage embryos (71.3% survival) that were 54% intact gave rise to 11 viable pregnancies (17.5%/ET), to yield an implantation rate of 6.9% per embryo. Sixty-three transfers of 147 thawed cocultured embryos (74.2% survival) that were 61% intact gave rise to 17 viable pregnancies (27%/ET), which gave an implantation rate of 13.6% per embryo that was significantly higher than the control group (P< 0.05).Conclusion Coculture of embryos prior to cryopreservation does not appear to improve cryosurvival; however, it does improve implantation postthaw compared with embryos following standard culture prior to cryopreservation.Presented at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, Vienna, Austria, April 3–7, 1995.     

A Practical Procedure for the Cryopreservation of Marrow Cells Intended for Autotransplantation

A simple and practical method of unfractionated bone marrow processing and cryopreservation was studied. The date shows that RBCs can be rapidly sedimented by methylcellulose or sodium carboxymethyl starch within 15-45 min. The cells can be cryopreserved in a mixture consisting of 5% DMSO, 6% HES and 4% Albumin prepared in a Lactated Ringer solution which is widely used, and can be simply immersed into a -80dGC freezer and stored in liquid nitrogen until infusion. Recovery percentages of nucleated cell, cell viability and CFU-G were similar to those cryopreserved with the conventional method. Clinical toxicity was mild in 12 infused patients. Of them 8 patients had received high dose chemotherapy TBI and their peripheral WBC recovery was rapid. The recovery of WBC or Platelet (PLT) in the study group was similar to that of the control group whose marrow cells were cryopreserved in 10% DMSO. Therefore, cells cryopreserved with this method can also accelerate the hematopoietic recovery in myeloablatively treated patients.     

Successful ultrarapid freezing of unfertilized oocytes

Successful application of ultrarapid freezing techniques to unfertilized murine oocytes has not been reported. In an effort to improve results, preovulatory murine oocytes were exposed to three ultrarapid freezing protocols involving varying sucrose concentrations (0.25, 0.5, and 1.0M) and 3.5M dimethyl sulfoxide (DMSO) as cryoprotectants prior to direct immersion in liquid nitrogen. Postthaw morphology and rates of in vitro fertilization and embryo development were compared with those obtained after freezing oocytes employing two established programmed cooling techniques. The rates of fertilization and development to the blastocyst stage in vitro of oocytes undergoing ultrarapid freezing after exposure to 3.5M DMSO and 0.5M sucrose were similar or superior to those obtained with programmed cooling techniques. Of oocytes which appeared morphologically normal postthaw, only those which underwent ultrarapid freezing with 0.25 or 0.5M sucrose and 3.5M DMSO reached the blastocyst stage at rates similar to those of controls. Ultrarapid freezing may represent a viable option for successful murine oocyte cryopreservation.Presented in part at 44th Annual Meeting of the American Fertility Society, October 10–13, 1988, Atlanta, GA.     

Cryopreservation of in vitro-fertilized human embryos: Histologic and cytogenetic analysis of an ectopic conceptus

In this study, 39 embryos from 17 patients were cryopreserved in a Planer R204 cell freezer using the protocol of Mohret al. (J Vitro Fert Embryo Transfer 21-10, 1985). The procedure was modified by supplementing the cryoprotectant with 10% heat-inactivated and filtered (0.22 m) maternal serum instead of fetal calf serum, and embryos were frozen in 500-l plastic straws instead of glass ampoules. After 12–25 weeks of storage in liquid nitrogen, 12 embryos from six patients were thawed at 8.0°C min to room temperature, incubated in 75% maternal serum with Ham's F-10, and replaced in utero. One pregnancy occurred. The patient was a 34-year-old nulligravida with occluded fallopian tubes. A year prior, she conceived triplets from three embryos during an in vitro fertilization (IVF) cycle, but she delivered at 21 weeks and the infants did not survive. The second IVF attempt produced four embryos. Two were replaced during the IVF cycle, but they did not implant. Two were cryopreserved and replaced 25 weeks later. On day 28 after replacement, beta human chorionic gonadotropin (-hCG) was 4126 IU, but there was no gestational sac in utero on ultrasonographic examination. Laparoscopy disclosed a right tubal pregnancy which was removed with the fallopian tube. Histological examination demonstrated normal chorionic villi. The chromosomal pattern was 46 XX by direct analysis and cell culture.     

体外受精-胚胎移植周期中黄体期血清性激素水平与妊娠率的关系

目的探讨体外受精－胚胎移植（ＩＶＦ－ＥＴ）周期中黄体期血清性激素水平的变化及与妊娠率的关系。方法随机选取６２个采用卵泡刺激素／绝经期促性腺激素／绒毛膜促性腺激素（ＦＳＨ／ｈＭＧ／ｈＣＧ）促超排卵的ＩＶＦ－ＥＴ周期（６２例患者）,采用放射免疫测定技术,测定其自然周期与促超排卵周期中黄体期血清雌二醇（Ｅ２）、孕酮（Ｐ）、催乳素（ＰＲＬ）水平,观察其妊娠情况。结果促超排卵周期中黄体期血清性激素水平明显高于自然周期（Ｐ＜０．０５）。补充黄体酮者的Ｐ、Ｐ／Ｅ２值,明显高于未补充黄体酮者（Ｐ＜０．０５）。临床妊娠者的Ｅ２水平明显低于未妊娠者,而Ｐ／Ｅ２、ＰＲＬ值明显高于未妊娠者（Ｐ＜０．０５）,并且当Ｐ／Ｅ２值为３００～４００、ＰＲＬ值为６０～１００μｇ／Ｌ时妊娠率最高。结论ＩＶＦ－ＥＴ周期中黄体期血清性激素水平对妊娠有影响,其中Ｅ２、Ｐ协同发挥作用,ＰＲＬ在一定范围内有利于胚胎着床。在ＩＶＦ－ＥＴ中应适当补充黄体酮,调节性激素至最适水平,可提高临床妊娠率