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PubMed2026

IVM rescue: Effect of growth hormone supplementation combined to autologous cumulus co-culture on GV oocyte maturation and competency.

OBJECTIVE: IVM rescue is based on the in vitro maturation of mainly Germinal Vesicle (GV) oocytes collected from stimulated cycles. The objective was to investigate the effects of growth hormone (GH) and autologous cumulus cells co culture (CC) on oocyte meiosis resumption and maturation after 32 h post cumulus denudation, in order to obtain additional embryos for the couple as a rescue system to increase the changes of cumulative pregnancy. MATERIAL AND METHODS: Our study concerned 300 patients who underwent ICSI cycles, during which a total of 1940 cumulus-complex-oocytes were retrieved, giving 1260 metaphase II stage (MII), 200 at the metaphase I stage, and 480 at the Germinal Vesicle (GV) stage. Mature oocytes were microinjected on the same day of retrieval. Immature GV oocytes were divided into four groups, with the first undergoing in vitro maturation (IVM) without cumulus cells (group 1) and the second undergoing IVM with CC (group 2), the third undergoing IVM without CC and with GH (group 3), the fourth undergoing IVM with CC and with GH (group 4). After 32 h of IVM, the matured oocytes, underwent microinjection, followed by embryonic development monitoring. RESULTS: When comparing the IVM outcomes, we observed a significant increase in oocyte maturation, fertilization rates and the percentage of 8-cell embryos on day 3 across the different study groups (p < 0.001) (Figs. 2-5). Furthermore, all study groups (1-4) exhibited notably blastulation rates, with group 3 demonstrating the most promising clinical outcomes. A preliminary pregnancy rate of approximately 20% was recorded in group 3, suggesting a potential improvement in the developmental competence of oocytes matured under specific conditions. CONCLUSION: The IVM rescue of germinal vesicle oocyte could serve as an additional strategy to increase the chance getting extra embryos to patients. Autologous cumulus cells co-culture combined to GH supplementation to IVM media, appear to play a crucial role to enhance successful meiosis resumption, oocyte maturation and competency to support embryos development when the injected spermatozoa is not carrier of severe genome and epigenomic decays.

Humans↗
PubMed2004

Microinsemination with first-wave round spermatids from immature male mice.

In several mammalian species, including mice, round spermatids have been used to produce normal offspring by means of microinsemination techniques. In this study, we examined whether mouse round spermatids retrieved from immature testes undergoing the first wave of spermatogenesis had acquired fertilizing ability comparable to cells from mature adults. Microinsemination with round spermatids was performed by direct injection into preactivated oocytes, as previously reported. About 60-85% of the successfully injected oocytes developed to the morula/blastocyst stage after 72 h in culture, irrespective of the age of the males (17-25 days old). After embryo transfer, normal pups were obtained from all age groups, including the day-17 group, the stage at which the first round spermatids appeared. A high correlation (r=0.90) was found between the birth rate and male age (P<0.01, Spearman rank correlation), indicating that the efficiency of producing offspring was dependent on the age of the donor males. Imprinted genes (H19, Igf2, Meg3, and Igf2r) were expressed from the correct parental alleles (maternal, paternal, maternal, and maternal, respectively) in all (n=12) day-9.5 fetuses derived from day-20 spermatids. These results clearly indicate that at least some first-wave spermatogenic cells have a normal haploid genome with the correct paternal imprint and are capable of supporting full-term embryo development, as do mature spermatozoa from adults. The use of male germ cells from immature animals may save time in the production of inbred/congenic strains and rescue male-factor infertility of early onset.

Age Factors↗
PubMed2003

[High concentrations of serum estradiol in assisted reproduction].

OBJECTIVES: To analyze the influence of the levels of estradiol on the day of HCG in the pregnancy rate (PR) of ICSI and oocyte donation. STUDY DESIGN: Retrospective, comparative. MATERIAL AND METHODS: 333 patients underwent ICSI and 66 in oocyte donation were included dividing them according to the level of estradiol: a) < 1,000 pg/mL, b) 1,001-3,000 pg/mL and c) > 3,000 pg/mL. Therapeutic protocol: Down regulation with acetate leuprolide in late luteal phase, COH with FSHr and/or HMG, ultrasonographic monitoring and estradiol blood levels, HCG application with > 3 follicles > 18 mm, oocyte retrieval 34 hours later. We analyzed: PR, age (including receptors), FSH and LH. Number, mature grade and fertilized oocytes; luteal support, transfer quality and type of catheter. Statistical analysis (SPSS 11) with chi square, ANOVA and Kruskall-Wallis. RESULTS: ICSI: Older patients in group A (p < 0.001), but without difference between B and C groups (p = 0.08). Statistical difference in number of follicles, number of oocytes, fertilized oocytes and transferred embryos being less in the A group (p < 0.001). Statistical difference in PR 21.7, 35.6 and 25.7% in A, B and C groups respectively (p = 0.032). Oocyte donation: Group A has younger patients (p = 0.005), FSH and LH were similiar among groups. Major number of follicles were observed to increase estradiol levels, but major quantity of metaphase II and fertilized oocytes were observed in group B (p = 0.05). PR without significant differences: 50, 51.5 and 52.3% in groups A, B and C (p = 0.977). Without statistical difference in the age of receptors; transferred embryos, type of catheter and quality on ICSI and oocyte donation groups. CONCLUSION: High estradiol levels at the day of HCG application affect the PR in patients submitted to ICSI. The best results were obtained with estradiol levels between 1,000 and 3,000 pg/mL. In oocyte donation the high concentrations of estradiol do not affect the PR of the receptors.

Adult↗
PubMed2004

Neonatal outcome in a Danish national cohort of 3438 IVF/ICSI and 10,362 non-IVF/ICSI twins born between 1995 and 2000.

BACKGROUND: In Denmark, one-third of twin pregnancies are the result of IVF/ICSI treatment. Limited data on neonatal outcome in IVF/ICSI twins are available in the literature. METHODS: A register study was conducted on neonatal morbidity and mortality in a complete national twin cohort including all 3438 (3393 live-born) IVF/ICSI and 10,362 (10,239 live-born) non-IVF/ICSI twins born between 1995 and 2000. Twins were identified in the National Medical Birth Registry and dichotomized into IVF/ICSI and non-IVF/ICSI by cross-reference with the Danish IVF Registry. Data on neonatal morbidity and mortality were retrieved from the Danish Patient Registry and the Danish Registry of Causes of Deaths. In order to exclude monozygotic twins, sub-analyses on unlike-sex twins were conducted. RESULTS: A birth weight discordance of >20% was observed in 20.6% of IVF/ICSI versus 15.7% of control twin pairs (P < 0.001). The risk of discordant birth weight >20% was OR 1.29 (95% CI 1.04-1.58) in unlike-sex IVF/ICSI twins versus control twins. The risk of delivery at <37 completed weeks and birth weight <2500 g was similar in the two cohorts; however, in unlike-sex IVF/ICSI versus control twins the risk of delivery at <37 weeks and birth weight <2500 g was OR 1.22 (95% CI 1.09-1.38) and OR 1.25 (1.11-1.40) respectively. After stratification for maternal age and parity, these risks disappeared. IVF/ICSI twins carried a higher risk of admittance to a neonatal intensive care unit (NICU) than control twins (OR 1.18, 95% CI 1.09-1.27), and this was even more pronounced in unlike-sex twins [OR 1.34 (95% CI 1.19-1.51)]. No differences were observed in malformation or mortality rates between the two cohorts. CONCLUSIONS: Despite higher birth weight discordance and more NICU admissions among IVF/ICSI twins, neonatal outcome in IVF/ICSI twins seems to be comparable with that of non-IVF/ICSI twins, when only dizygotic twins were considered in the comparisons.

Birth Weight↗
PubMed2002

[First experiences with human blastocyst culture after IVF/ICSI under the conditions of the German embryo protection law].

OBJECTIVE: Due to the improvements in human embryo culture in the recent years, it is now possible to transfer embryos five days after oocyte retrieval and IVF or ICSI at the blastocyst stage with favorable implantation rates. In Germany it is illegal to cultivate more than 3 embryos, therefore the selection has to be done at the pronuclear stage. There we report our experiences of human blastocyst culture in a routine IVF/ICSI programme under the conditions of the German Embryo Protection Law. MATERIALS AND METHODS: The data of 100 couples undergoing the IVF-ICSI programme at the University Clinic of Würzburg were analysed prospectively. 14-18 hours after insemination or micro-injection two or three zygotes with the best pronuclear development were selected for further cultivation. Fertilized oocytes were cultured in sequential media and were then transferred into the uterus 5 days after oocyte recovery. The blastocysts were graded from 1-8. RESULTS: In 100 cycles a total of 859 oocytes were collected, of whom 663 were fertilized and reached the pronuclear stage (median fertilization rate 88.9 %). 251 zygotes were selected at the PN stage. 51 % of the selected zygotes achieved the blastocyst stage after 5 days (grade 1-5), 28 % were morulae (grade 6-7) and 21 % of the embryos arrested in their development (grade 8). The clinical pregnancy rate was 26 %. Women who conceived had a significant better development of blastocysts on day 5 (grade 4 versus grade 6, P < 0.01) than those not achieving pregnancy. CONCLUSIONS: In summary, under the current legal conditions in Germany, blastocyst culture cannot improve pregnancy rates as the rate of arrested embryos of over 20 % limits the chances of implantation.

Adult↗
PubMed2000

Progression to the blastocyst stage of embryos derived from testicular round spermatids.

Progression to the blastocyst stage of embryos derived from testicular round spermatids in men with non-obstructive azoospermia was studied. A total of 56 men were studied in whom partial spermatogenesis failure had occurred where only very few spermatozoa (fewer than the number of oocytes retrieved) were extracted from multiple testicular biopsy specimens. Oocytes remaining after intracytoplasmic injection of testicular spermatozoa (group 1) were injected with round spermatids (ROSI, group 2). Only embryos derived from group 1 were transferred. Remaining embryos were observed under culture for 8 days and their progression to the blastocyst stage was recorded. Of the 546 oocytes injected with testicular spermatozoa, 404 (73.9%) showed evidence of 2-pronuclear (2PN) fertilization. Injection of testicular round spermatids resulted in 2PN fertilization rate of 50% (P < 0.05). Using a four-point grading system, 53% of the good quality embryos (grade 1 or 2) in group 1 reached the blastocyst stage compared with 25% in group 2 (P < 0.05). The rate of progression to the blastocyst stage of grade 3 and grade 4 embryos was 46 and 8.5% in the two groups respectively (P < 0.05). Using a different three-point grading system for the blastocysts, 75.3% of the blastocysts in group 1 were either grade 1 or grade 2 and 24.7% were grade 3. However, in group 2 all blastocysts were grade 3. All embryos observed in group 1 reached the blastocyst stage by day 5 or 6 compared with 25% of the embryos reaching the blastocyst stage by this time in group 2. While 31.2% of the blastocysts in group 1 showed evidence of spontaneous hatching in vitro, none of the blastocysts in group 2 hatched. In conclusion, progression to the blastocyst stage occurred at a much lower and slower rate in embryos derived from testicular round spermatids. Furthermore, all blastocysts resulting from ROSI were of poor quality and none showed spontaneous hatching. These results may explain the dismal outcome associated with ROSI.

Blastocyst↗
PubMed2004

Hospital care utilization of IVF/ICSI twins followed until 2-7 years of age: a controlled Danish national cohort study.

BACKGROUND: IVF/ICSI twins are likely to have a higher risk of prematurity associated with higher morbidity. The aim of this study was to assess the use of hospital care resources in IVF/ICSI twins on data retrieved until 2-7 years of child age. METHODS: National controlled cohort study on hospital admissions and surgical interventions in 3393 IVF twins, 10,239 spontaneously conceived twins and 5130 IVF singletons born between 1995 and 2000 in Denmark. Cross-linkage of data from the Danish IVF Registry and the National Patient Registry enabled us to identify children who were admitted to hospital or underwent an operation. RESULTS: The frequency of hospitalized children was 69.8, 69.6 and 49.8%, and of children who underwent a surgical intervention 10.6, 11.2 and 8.5% in IVF/ICSI twins, control twins and IVF/ICSI singletons respectively. Odds ratios (OR) (95% confidence intervals) of hospitalization in IVF/ICSI twins versus control twins and IVF/ICSI singletons were 1.04 (0.96, 1.14) and 2.44 (2.22, 2.63) and OR adjusted for year of birth, maternal age and parity were 1.00 (0.91, 1.11) and 2.38 (2.17, 2.63) respectively. Also for term birth infants, IVF/ICSI twins were more likely to be hospitalized than IVF/ICSI singletons: adjusted OR 1.37 (1.22, 1.51). Similar risk of a surgical procedure was observed in IVF/ICSI versus control twins. However, IVF/ICSI twins more often underwent a surgical intervention than IVF/ICSI singletons: adjusted OR 1.26 (1.08, 1.47). This risk disappeared when restricted to term infants: adjusted OR 1.00 (0.81, 1.22). Different sex IVF/ICSI and control twins had equal risk of admissions and surgical interventions, and ICSI children had the same risk as children born after conventional IVF. CONCLUSIONS: Though the use of hospital care resources was similar in IVF/ICSI and control twins, the over-use in IVF/ICSI twins versus IVF/ICSI singletons adds to the arguments for implementing elective single embryo transfer as our standard procedure.

Child↗
PubMed2004

A successful model to assess embryo development after transplantation of prophase nuclei.

BACKGROUND: Germinal vesicle transplantation (GVT) provides a means of investigating interactions between karyoplasts and cytoplasts isolated from different cells. Technically, GVT can be accomplished with a high degree of efficiency without compromising the maturation of either the human or mouse oocyte nucleus. Although maturation, fertilization and preimplantation development have been established using GVT, full-term development has been reported only after supplementation with fresh mature ooplasm. In this study, we assess the ability of immature oocytes collected from gonadotrophin-primed ovaries to mature in vitro after GVT and develop to full-term. METHODS: GV oocytes were retrieved from either non-stimulated or pregnant mare's serum gonadotrophin (PMSG)-primed female mice. Microsurgically isolated GV karyoplasts were transplanted into previously enucleated oocytes. Oocytes successfully reconstituted by electrofusion were cultured for 14 h to allow nuclear maturation. Metaphase II oocytes were subjected to Piezo-ICSI, and those fertilized normally were cultured to the blastocyst stage. Some such embryos were transferred to pseudopregnant female mice to examine their potential for normal development. Cumulus-denuded non-manipulated oocytes that were matured in vitro served as controls. RESULTS: The reconstitution and maturation rates were comparable in oocytes isolated from PMSG-primed and from unstimulated ovaries. The rate of normal fertilization in oocytes from primed ovaries was significantly higher than that of their non-primed counterparts (63.5 versus 39.6%; P < 0.01). This difference was also confirmed in terms of blastocyst development (31.8 versus 7.9%; P < 0.01). Of a total of 70 embryos transferred to the oviduct of five recipient mice, 21.4% developed to normal live offspring. All developed as normal adults and proved to be fertile. The live birth rate was comparable to that obtained using non-manipulated control oocytes (22.3%). CONCLUSIONS: Higher rates of fertilization and blastocyst formation were obtained after GVT of mouse oocytes isolated from PMSG-primed ovaries compared with their non-primed counterparts. These represent the first mouse offspring derived from in vitro matured, cumulus-denuded oocytes treated by allo-GVT and fertilized by ICSI. Thus, GVT appears not to impair oocyte maturation, fertilization and pre- and post-implantation development and, after gonadotrophin priming, allows generation of healthy mouse offspring without mature ooplasm supplementation.

Animals↗
PubMed2002

The use of a detailed zygote score after IVF/ICSI to obtain good quality blastocysts: the German experience.

BACKGROUND: Due to improvements in embryo culture, it is now possible to transfer embryos 5 days after oocyte retrieval and IVF/ICSI at the blastocyst stage, giving a better synchronization with the female reproductive tract. In Germany it is illegal to culture more than three embryos. Therefore, there is need for a sufficient selection at the pronuclear (PN) stage to select the best zygotes and exclude those of poor quality. METHODS: A prospective trial was conducted in 168 IVF and ICSI cycles including the size, number and alignment of pronuclei and nucleoli, cytoplasmic halo effect, the presence of vacuoles and granularity of ooplasm. Based on the above criteria, the best zygotes were selected (score <15) for embryo transfer on day 5. Blastocysts were classified in eight grades based on the cleavage speed. RESULTS: A total of 1450 oocytes were collected, of which 1119 reached the pronuclear stage. Of the zygotes (n = 424) selected at the PN stage, 46% achieved the blastocyst stage after 5 days (grade 1-5), 26% the morula stage (grade 6-7) and 28% were arrested (grade 8). The mean zygote score showed a significant positive correlation with the mean blastocyst quality in ICSI, but not in IVF cycles. A cut-off of 15 was calculated for ICSI cycles giving the best discrimination with blastocyst grades (6 versus 7) and number of arrested embryos (23 versus 45%) below and above this cut-off. A total of 33 clinical pregnancies was achieved (20%). Women conceiving had a significantly better mean blastocyst development than those not conceiving. Strong cytoplasmic vacuolization and an extreme or no halo effect had a negative effect on blastocyst development. CONCLUSIONS: The data show that PN stage morphology is related to blastocyst development, but the rate of arrested embryos of almost 30% limits the chance of conception under the conditions of the German Embryo Protection Law.

Adult↗
PubMed2001

Genetic, andrological and clinical characteristics of patients with congenital bilateral absence of the vas deferens.

The possibility of retrieving spermatozoa from the epididymis allows patients with congenital bilateral absence of the vas deferens (CBAVD) to father a child by means of assisted reproduction techniques. This has, however, increased the chance of transmitting a mutated allele of the cystic fibrosis transmembrane conductance regulator (CFTR) gene which increases the risk of generating offspring with cystic fibrosis (CF). Because of the increased heterogeneity of the CFTR locus, the study of a discrete number of mutations, as usually carried out in a diagnostic work-up, is unable to ascertain the presence of a mutation in a relatively high proportion of the patients screened. In an attempt to increase the chance of detecting the presence of CFTR gene abnormalities, 37 patients with CBAVD and one patient with congenital unilateral agenesis of the vas deferens (CUAVD) underwent an enlarged diagnostic protocol, which included screening for the most expected mutations of the CFTR gene in our population, evaluation of the five thymidine (5T) allelic variant, sweat test, respiratory function tests, evaluation of steatocrit, and an accurate evaluation of the history of the patient to search for symptoms commonly found in patients with CF. A single CFTR gene mutation was found in 18 patients (48.6%) with CBAVD and in the patient with CUAVD. The most frequent mutation observed was the Delta F508. Eleven patients (45.8%) had the 5T variant and in five of them it was not associated with any detectable mutation of the CFTR gene. Two female partners were found to be carriers of a mutation, whereas 5 (18.5%) had the 5T variant. As many as 71% of CBVAD patients had the simultaneous presence of at least two signs and/or symptoms suggestive of CF, albeit they were of mild intensity and the patients felt fit and healthy. In conclusion, these results suggested that some patients with CBAVD without CFTR gene mutation or 5T variant, even when their sweat test is negative, may show clinical suspicion of carrying a CFTR gene mutation and therefore are at risk of generating children affected by CF if the partner carries a mutation as well. The screening for mutations and a careful clinical examination may contribute to better identification of patients with CFTR-related CBAVD.

Adult↗