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.

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine.

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.

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.

Cysteamine supplementation during in vitro maturation and embryo culture: a useful tool for increasing the efficiency of bovine in vitro embryo production.

Cysteamine when added during in vitro maturation (IVM) or in vitro embryo culture (IVC) stimulates glutathione (GSH) synthesis and improves embryo developmental rates. This suggests that GSH synthesis is decreased in the in vitro produced embryo. The present study was carried out to evaluate if addition of cysteamine to culture medium at the same time, during IVM and IVC of bovine oocytes, may promote an overall improvement on the developmental rate and embryo quality. Oocytes were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 or 100 microM of cysteamine for 24 hr. After IVM, the oocytes were fertilized (day 0). Day 2 embryos (2-8 cell) were washed and transferred to fresh IVC medium supplemented with 0, 25, 50, or 100 microM of cysteamine and cultured for 48 hr. After this, embryos were cultured in IVC medium without cysteamine until day 8 of IVC. In the present study, we confirmed our previous results by demonstrating that the percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.05) when 100 microM of cysteamine was added during IVM, and this was further improved when 100 and 50 microM of cysteamine where present during IVM and IVC, respectively (P < 0.05). After cryopreservation, no differences were observed on embryo development, but a significant increase on embryo hatching was found between unsupplemented and supplemented oocytes with 100 and 50 microM of cysteamine during IVM and IVC, respectively (P < 0.05). We can conclude that GSH synthesis stimulation during bovine IVM with cysteamine, concomitant with GSH stimulation during IVC, will be a useful and simple tool for increasing the efficiency of in vitro bovine embryo production.

[Treatment with enoxaparin adapted to the fertility programs in women with recurrent abortion and thrombophilia]. / Tratamiento con enoxaparina adaptado a los programas de fertilidad en mujeres con aborto recurrente y trombofilia.

Acquired and inherited thrombophilia are associated with recurrent pregnancy loss (RPL). Antithrombotic therapy could restore hemostatic balance and improve early placentation and gestational outcome. We evaluated the efficacy of enoxaparin adapted to the fertility program for prevention of pregnancy loss in 35 women (W) with early RPL and thrombophilia. Previous to the diagnosis of thrombophilia, they had had a total of 105 gestations of which 89 (85%) ended in early pregnancy loss. After diagnosis of thrombophilia, 35 subsequent pregnancies were treated with enoxaparin. In 5 cases assisted reproductive techniques were necessary to achieve pregnancy due to couple infertility. In 17 W who had had at least one preclinical pregnancy loss, enoxaparin (20 mg/d/s.c.) was started previous to conception and adapted to the fertility program. All the women continued with the gestational regime. Eighteen W with only clinical pregnancy loss started enoxaparin (20 mg twice per day s.c.) after biochemical pregnancy diagnosis. During gestations heparin dose was adjusted with anti Xa test, maintaining a range between 0.3 at 0.6 u/ml. With antithrombotic therapy, 30/35 (85%) of the pregnancies ended in live birth versus 16/105 (15%) of the pregnancies without treatment (p < 0.001). These results suggest that enoxaparin adapted to the fertility program can be effective in the prevention of preclinical and clinical abortion in women with thrombophilia.

Tratamiento con enoxaparina adaptado a los programas de fertilidad en mujeres con aborto recurrente y trombofilia. / [Treatment with enoxaparin adapted to the fertility programs in women with recurrent abortion and thrombophilia]

Acquired and inherited thrombophilia are associated with recurrent pregnancy loss (RPL). Antithrombotic therapy could restore hemostatic balance and improve early placentation and gestational outcome. We evaluated the efficacy of enoxaparin adapted to the fertility program for prevention of pregnancy loss in 35 women (W) with early RPL and thrombophilia. Previous to the diagnosis of thrombophilia, they had had a total of 105 gestations of which 89 (85

) ended in early pregnancy loss. After diagnosis of thrombophilia, 35 subsequent pregnancies were treated with enoxaparin. In 5 cases assisted reproductive techniques were necessary to achieve pregnancy due to couple infertility. In 17 W who had had at least one preclinical pregnancy loss, enoxaparin (20 mg/d/s.c.) was started previous to conception and adapted to the fertility program. All the women continued with the gestational regime. Eighteen W with only clinical pregnancy loss started enoxaparin (20 mg twice per day s.c.) after biochemical pregnancy diagnosis. During gestations heparin dose was adjusted with anti Xa test, maintaining a range between 0.3 at 0.6 u/ml. With antithrombotic therapy, 30/35 (85

) of the pregnancies ended in live birth versus 16/105 (15

) of the pregnancies without treatment (p < 0.001). These results suggest that enoxaparin adapted to the fertility program can be effective in the prevention of preclinical and clinical abortion in women with thrombophilia.

Efecto de la estimulación de la síntesis de glutation durante la maduración de oocitos de bovinos y cultivo de los embriones in vitro en la tasa de desarrollo y calidad de los embriones / Effect of stimulation of glutathione synthesis during in vitro maturation of bovine oocytes and in vitro embryo culture on the development rate and embryo quality

Objetivo: El desarrollo de un sistema de maduración (MIV) y cultivo (CIV) in vitro de oocitos humanos es importante. El uso de oocitos humanos en investigación es problemático. Los oocitos de bovinos han sido propuestos como el modelo más conveniente. Se llevaron a cabo experimentos donde se evaluó el efecto que tiene la estimulación de la síntesis de glutation (GSH) durante la MIV y el CIV sobre el desarrollo embrionario y la calidad de los mismos. Materiales y Métodos: Los oocitos provenientes de ovarios de matadero fueron madurados, fertilizados, cultivados y congelados in vitro. La síntesis de glutation fue estimulada con cisteamina. La tasa de desarrollo y calidad de los embriones se estudió en 5 grupos: MIV y CIV (Día 2, embriones de 2 a 6 células) sin suplementación de cisteamina (Cist) (Grupo Control) (A); MIV suplementado con 100 mM de Cist (MIV-100) y el CIV sin suplementación (B); MIV-100 y CIV suplementado con 25 AM de Cist (C); o 50 AM de Cist (D); o con 100 AM de Cist (E). Se realizaron 7 réplicas con 1.374 oocitos. Los datos transformados se analizaron mediante ANOVA y test de Tukey. Resultados: El desarrollo de los embriones en los grupos B, C y D fueron significativamente superiores al grupo control (A). El grupo D fue el mejor (P<0.05). Además, el grupo D presentó los mejores resultados de sobrevida y eclosión embrionaria luego del congelamiento, comparado con el grupo A. Discusión: Los resultados demuestran que la estimulación de la síntesis de GSH durante la MIV y el CIV de oocitos bovinos mejora las tasas de desarrollo de los embriones y su calidad. Estos resultados nos muestran el papel preponderante que cumple el metabolismo del GSH durante la maduración citoplasmática y el desarrollo de los embriones (AU)

Efecto de la estimulación de la síntesis de glutation durante la maduración de oocitos de bovinos y cultivo de los embriones in vitro en la tasa de desarrollo y calidad de los embriones / Effect of stimulation of glutathione synthesis during in vitro maturation of bovine oocytes and in vitro embryo culture on the development rate and embryo quality

Objetivo: El desarrollo de un sistema de maduración (MIV) y cultivo (CIV) in vitro de oocitos humanos es importante. El uso de oocitos humanos en investigación es problemático. Los oocitos de bovinos han sido propuestos como el modelo más conveniente. Se llevaron a cabo experimentos donde se evaluó el efecto que tiene la estimulación de la síntesis de glutation (GSH) durante la MIV y el CIV sobre el desarrollo embrionario y la calidad de los mismos. Materiales y Métodos: Los oocitos provenientes de ovarios de matadero fueron madurados, fertilizados, cultivados y congelados in vitro. La síntesis de glutation fue estimulada con cisteamina. La tasa de desarrollo y calidad de los embriones se estudió en 5 grupos: MIV y CIV (Día 2, embriones de 2 a 6 células) sin suplementación de cisteamina (Cist) (Grupo Control) (A); MIV suplementado con 100 mM de Cist (MIV-100) y el CIV sin suplementación (B); MIV-100 y CIV suplementado con 25 µM de Cist (C); o 50 µM de Cist (D); o con 100 µM de Cist (E). Se realizaron 7 réplicas con 1.374 oocitos. Los datos transformados se analizaron mediante ANOVA y test de Tukey. Resultados: El desarrollo de los embriones en los grupos B, C y D fueron significativamente superiores al grupo control (A). El grupo D fue el mejor (P<0.05). Además, el grupo D presentó los mejores resultados de sobrevida y eclosión embrionaria luego del congelamiento, comparado con el grupo A. Discusión: Los resultados demuestran que la estimulación de la síntesis de GSH durante la MIV y el CIV de oocitos bovinos mejora las tasas de desarrollo de los embriones y su calidad. Estos resultados nos muestran el papel preponderante que cumple el metabolismo del GSH durante la maduración citoplasmática y el desarrollo de los embriones