Insulin secretion by pancreas of athymic mice injected with peripheral mononuclear cells from insulin-dependent diabetic patients.

We studied the effect of peripheral blood mononuclear cells (PBMNC) from insulin-dependent diabetic (IDDM) children on the insulin secretion pattern of the pancreas from recipient athymic mice. PBMNC from healthy controls or IDDM patients in different stages of disease were injected into athymic mice. PBMNC from newly diagnosed IDDM children elicited basal nonfasting hyperglycemia and in vitro inhibition of the first and second phases of glucose-stimulated insulin secretion in recipient mice. Animals injected with cells from chronically IDDM children showed normoglycemia, abnormal tolerance to glucose, and inhibition of first-phase insulin secretion. Mitomycin C treatment of MNC from IDDM patients abolished insulin secretion inhibition in recipient mice. PBMNC from newly diagnosed and chronically IDDM patients showed positive anti-beta-cell cellular immune aggression. Mice injected with cells from patients during the remission period showed normoglycemia and no alteration of insulin secretion patterns. When relapsed to their former clinical stage, injection of the cells significantly inhibited first-phase glucose-induced insulin secretion in recipients. PBMNC from newly diagnosed IDDM patients were found to migrate to the pancreas of recipient mice preferably as compared with cells from controls. Cells from chronically IDDM patients cultured with concanavalin A (Con A) increased insulin secretion inhibition; despite this, cells from children during the remission period cultured with Con A failed to modify insulin secretion in recipients. These results show that injection of PBMNC from diabetic patients leads to insulin secretion impairment in recipient mice pancreas, and provide a basis for the study of mechanisms involved in the onset and modulation of anti-beta-cell cellular immune aggression induced by human PBMNC.

[Pregnancy obtained by intracytoplasmic oocyte injection of spermatozoa retrieved from a cryopreserved testicular biopsy]. / Embarazo logrado por inyección intracitoplasmática de ovocitos con espermatozoides provenientes de una biopsia de testículo criopreservada.

We have obtained a clinical pregnancy in a 26 year old patient by means of in vitro oocyte fertilization by intracytoplasmic injection of spermatozoa obtained from a cryopreserved testicular biopsy. In a first attempt performed with fresh biopsy material, the woman became pregnant, but the pregnancy ended in a spontaneous abortion. In a second cycle, the spermatozoa were retrieved from a cryopreserved sample saved from the first attempt. Twelve metaphase II oocytes were collected and injected; from these, nine became fertilized, three preembryos were transferred at the eighth-cell stage and the other six were cryopreserved. An ongoing clinical pregnancy was obtained with two gestational sacs.

[Pregnancy with spermatozoa from the head of the epididymis in spermatic duct agenesis]. / Embarazo logrado con espermatozoides obtenidos de la cabeza epididimaria en una agenesia de vías espermáticas.

From a male affected of agenesis of corpus and cauda epididymis and vas deferens, sperms were surgically obtained aspirating epididymal content with the help of a surgical microscope. Motile sperms were separated after discontinuous Percoll gradient centrifugation and used to inseminate in vitro the spouse's oocytes 8 hrs. after aspiration according to the current techniques. Twenty hours later, oocytes were checked for fertilization and transferred to fresh culture medium. From 13 preovulatory oocytes only one was fertilized and transferred to the Fallopian tube at the 6 cells stage. Fourteen days after embryo transfer, serum HCG concentration was 320 mU/ml and on day 29th a 5mm fetal sac showing heart beats was detected by transvaginal sonography. After an uneventful 38 weeks gestation a normal baby girl weighing 2,800 g was delivered.

Clinical practice of embryo transfer.

In this review, several embryo transfer methods are considered, together with factors involved in achieving an effective transfer. The approach most used is transcervical intrauterine transfer. This is described in detail, together with the many variables influencing success, e.g. technical ability and training of personnel, catheter choice, value of a previous 'dummy transfer' and the need to minimize trauma during transfer and so prevent damage to the uterine lining, bleeding and uterine contractions. These factors can each negatively impact on pregnancy rates. Emphasis is put on quality, developmental stage and number of embryos to be transferred to limit multiple pregnancies and their unwanted side-effects. Culture to blastocyst stages and single embryo transfer when optimal quality embryos are available are discussed as means of avoiding multiple pregnancies. Reference is made to embryo cryopreservation and fertility following frozen embryo transfer. Other techniques, such as ultrasound-controlled transcervical intrauterine transfer, and ultrasound-controlled transmyometrial transfer, are reviewed. More invasive procedures, generically grouped as surgical embryo transfer, including gamete intra-Fallopian transfer (GIFT), zygote intra-Fallopian transfer (ZIFT), pronuclear stage transfer and embryo intra-Fallopian transfer (EIFT), are also described. These techniques had a place in IVF when the need to apply assisted reproductive techniques exceeded the capacity of most laboratories, but not today thanks to refined laboratory technology and improved understanding of implantation. Alternative assisted reproductive technologies, such as direct intra-follicular insemination (DIFI), Fallopian spermatic perfusion (FSP), peritoneal oocyte stage and sperm transfer and intra-vaginal culture (IVC), are mentioned briefly.

Empty follicle syndrome due to human errors: its occurrence in an in-vitro fertilization programme.

We report five cases in which no oocytes were retrieved after standard ovarian stimulation for in-vitro fertilization (IVF), and in which it was found that mistakes had been made at the time of human chorionic gonadotrophin (HCG) administration. In all five cases, oocyte retrieval was achieved after injecting HCG, when necessary, and reprogramming aspiration 24-36 h later. A mean of 7+/-3.2 MII oocytes were recovered per patient and 3.2+/-0.8 embryos were transferred. Three clinical pregnancies were obtained, and four healthy infants were born. In our programme, these were the only cases of empty follicle syndrome (EFS) that appeared over a total of 1118 cycles, and were all explained by human error in the administration of HCG. Our experience shows that human error could be considered a significant factor in the aetiology of empty follicle syndrome, and that EFS may be in part avoided by taking simple preventive measures.

Birth resulting from transfer of blastocysts cryopreserved with propanediol after spontaneous hatching.

This paper reports the birth of a healthy baby resulting from transfer of blastocysts that were cryopreserved using propanediol after spontaneous hatching. A young infertile couple underwent IVF treatment in the clinic. After several IVF attempts, two births resulted; the first one with fresh embryos in 1996 after three IVF cycles, and the second one in 1999 (after a new IVF cycle in 1998) with frozen blastocysts that had remained cryopreserved in 1.5 mol/l propanediol and 0.1 mol/l sucrose after spontaneous hatching. This report of a healthy baby following transfer of hatched blastocysts frozen in propanediol supports further exploration of this approach.

Mouse splenocyte transfer effect depends on donor's Junin virus infection stage.

Splenocytes from Junin-virus-persistently-infected euthymic mice taken at 45 days postinfection seemed unable to induce overt signs of disease, to cause death, or to modify brain viral levels when transferred to athymic Junin-virus-infected mice. Findings differed sharply when the same recipients were transferred with splenocytes taken at 6 or 30 days postinfection from immunocompetent mice infected in adult life, since mortality reached 80 or 50%, respectively, and brain viral titers were significantly lowered. Furthermore, splenocytes taken at 6 days postinfection from whole adult mice proved harmless to persistently infected euthymic mice. These findings strongly suggest the existence of an immune system alteration in the immunocompetent mouse, attributable to Junin virus persistence. This premise is based on the fact that splenocytes from persistently infected mice were unable to recognize viral antigen expressed on recipient-infected cells. The absence or impairment of a specific cytotoxic T cell population is hereby postulated.

Junin virus infection in genetically athymic mice.

The progression of Junin virus infection was studied in congenitally athymic mice. Immunocompetent littermates were used as infected controls. As expected, the latter developed lethal encephalitis, with viremia and considerable viral replication in the brain. The mortality rate was almost 100%; the few surviving controls exhibited high serum neutralizing antibody levels and a total absence of virus in blood and brain. In contrast, nude mice did not contract the disease; all survived with persistent viremia and virus in brain, but no serum neutralizing antibodies were detected. These results confirm previous research on thymectomized mice and those treated with anti-lymphocyte serum and tend to support the important role of cellular immunity in the pathogenesis of this viral disease.

Uses and limitations of two molecular cytogenetic techniques for the study of arrested embryos obtained through assisted reproduction technology

We studied chromosomal abnormalities in arrested embryos produced by assisted reproductive technology with fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) in order to determine the best technique for evaluating chromosomal aneusomies to be implemented in different situations. We examined individual blastomeres from arrested embryos by FISH and arrested whole embryos by CGH. All of the 10 FISH-analyzed embryos gave results, while only 7 of the 30 embryos analyzed by CGH were usable. Fifteen of the 17 embryos were chromosomally abnormal. CGH provided more accurate data for arrested embryos; however, FISH is the technique of choice for screening in preimplantation genetic diagnosis, because the results can be obtained within a day, while the embryos are still in culture.

Embarazo logrado con espermatozoides obtenidos de la cabeza epididimaria en una agenesia de vías espermáticas / Pregnancy with spermatozoa from the head of the epididymis in a spermatic ducts agenesis

De un hombre afectado de agenesia de cuerpo y cola de epidídimos y conductos deferentes, se obtuvieron espermatozoides mediante la aspiración microquirúrgica del contenido epididimario. Los espermatozoides móviles se separaron de las muestras aspiradas mediante centrifugación a través de un gradiente discontínuo de Percoll, y se procedió a la fertilización in vitro de los oocitos obtendios de la esposa con la técnica habitual. La inseminación se realizó a las 8 horas de la recolección de los oocitos. Veinte hora después se examinaron los oocitos para detectar signos de fertilización transferiéndolos a medio de cultivo fresco. De 23 oocitos preovulatorios, sólo fertilizó uno, el cual fue transferido a la trompa de Falopio al alcanzar el estadío de 6 células. A los 14 días de la transferencia se detectó en sangre una subunidad HCG de 320 mU/ml y el día 29 se visualizó por ecografía transvaginal un saco fetal de 5 mm con actividad cardíaca. Después de una gestación de 38 semanas se produjó el parto de una niña normal de 2 800 g de peso

Infeccion con Trypanosoma cruzi en ratones congenitamente atimicos. / Trypanosoma cruzi infection in congenitally athymic mice

En este trabajo se intento dilucidar el papel que juega la inmunidad celular en la enfermedad del raton infectado con dosis bajas T.cruzi, comparando el curso de la infeccion de ratones congenitamente atimicos (nu/nu) con sus hermanos de camada fenotipicamente normales (nu/+). Para esto se infectaron ratones de ambos grupos de 4 semanas de edad, por via intraperitoneal con 5000 o 5 tripomastigotes de sangre. El curso de la enfermedad fue mas severo en los ratones atimicos, con parasitemia significativamente mas alta y promedio de sobrevida menor que en los controles inmunocompetentes, sugiriendo que en este caso, los linfocitos timodependientes tienen un papel importante en la defensa contra la infeccion con T. cruzi