RESUMEN
In a preliminary, unpublished randomized study conducted in 2000 on 39 patients, including a placebo group, it was observed that the addition of growth hormone (GH) during ovarian stimulation in patients with poor-quality oocytes increased the pregnancy rate. However, the results were not statistically significant due to the small number of patients in each group. A protocol with 8 IU GH was tested in 291 patients with three or more previous failures of embryo transfer for no clearly identifiable reasons. The analysis was restricted to patients receiving either recombinant FSH or human menopausal gonadotrophin (HMG) (n = 245). They were compared retrospectively to all patients with three or more failures during the same period of time but stimulated only with recombinant FSH or HMG, without GH, in an observational study design. Co-stimulation with GH gave better results in terms of number of oocytes collected and embryos obtained. Pregnancy rate per retrieval was higher than in the control group (25.7% versus 18.2%, P < 0.01) and reached a level similar to the one observed in the study centre for the whole population. Ovarian stimulation associated with GH can be proposed for patients with a history of repeated assisted reproduction failures. An improvement of cytoplasmic competence is proposed as an explanation.
Asunto(s)
Fertilización In Vitro/métodos , Hormona del Crecimiento/farmacología , Oocitos/efectos de los fármacos , Análisis de Varianza , Gonadotropina Coriónica , Femenino , Hormona Liberadora de Gonadotropina/análogos & derivados , Humanos , Modelos Biológicos , Embarazo , Resultado del TratamientoRESUMEN
Until now, the morphological sperm analysis (spermocytogram) allows to define sperm normality, but the relationship between sperm morphology and fertility is not yet assessed. Although several studies do not report any relationship between abnormal sperm morphology and ICSI results, nevertheless, the success rate of ICSI sems to be dependent on injected sperm morphological aspect. Detailed morphological sperm examination (especially sperm head) at high magnification (from x 6600 to x 12500) (MSOME) in real time allows to select the best spermatozoa before oocyte injection (IMSI). In some cases, implantation and ongoing pregnancy rates were improved with this sperm selection method. Ultramorphologic criteria were established and the most predictive factor of sperm quality is the presence of vacuoles in the sperm head. Those vacuoles appear to be related to DNA damage (fragmentation and/or denaturation) and affect embryo development. To standardize those observations, several authors tried to establish sperm MSOME classifications in order to be used in routine and to replace the conventional spermocytogram in the next future.
Asunto(s)
Cabeza del Espermatozoide/patología , Cabeza del Espermatozoide/ultraestructura , Inyecciones de Esperma Intracitoplasmáticas/métodos , Espermatozoides/ultraestructura , Daño del ADN , Fragmentación del ADN , Femenino , Humanos , Masculino , Embarazo , Resultado del Embarazo , Índice de Embarazo , Motilidad Espermática/fisiología , Espermatozoides/anomalías , Espermatozoides/citología , Espermatozoides/fisiologíaRESUMEN
ICSI failures (essentially fertilization and implantation failures) can be due to the quality of the oocytes, the spermatozoon injected and its manipulation, uterine factors and paternal factors, such as DNA fragmentation of the spermatozoa. Pre-selection of the spermatozoon is the crucial phase for ensuring successful ICSI. The characteristics of the sperm must be checked under the microscope at a magnification of x 6600 as certain abnormalities, in particular nuclear vacuoles, cannot be seen at x 300. In our centre, we conducted a study on 72 patients which showed that with more than 30% of fragmented DNA, neither implantation or birth was achieved using ICSI. Performing IMSI on the same patients allowed us to obtain implantation rates of 17.4% with 30 to 40 % of fragmented DNA and of 33.3% above 40%. Births were achieved in 17.4% and 28.6% of cases respectively. IMSI has the advantage of allowing extremely careful selection of the spermatozoon microinjected, so that it is of normal shape, with the least vacuoles possible and a low DNA fragmentation rate. It is, however, a complicated technique that cannot be routinely performed.
Asunto(s)
Inyecciones de Esperma Intracitoplasmáticas , Humanos , Infertilidad Masculina/terapia , Masculino , Inyecciones de Esperma Intracitoplasmáticas/métodosRESUMEN
Embryos are conventionally selected for transfer based on the evaluation of the cleavage speed and extent of blastomere fragmentation. Here we examined whether the predictive value of these criteria, as indicators of the chance of embryo implantation, can be further potentiated by adding previously described criteria reflecting the regularity of pronuclear development. In a group of embryos selected for transfer in 380 fresh embryo transfer cycles according to the conventional criteria, the transfer of only those embryos that developed from zygotes judged normal at the pronuclear stage (pattern 0) gave significantly higher pregnancy (44.8%) and implantation (30.2%) rates compared with the pregnancy (22.1%; P < 0. 05) and implantation rates (11.2%; P < 0.001) for the transfers of only those embryos that developed from zygotes judged abnormal (non-pattern 0). The transfer of only one pattern 0 embryo was sufficient for the optimal chance of pregnancy (no differences in pregnancy rates after transfer of one, two or three pattern 0 embryos), whereas the transfer of two pattern 0 embryos mostly resulted in a twin pregnancy. The inclusion of the criteria based on pronuclear morphology can thus lead to the application of a single embryo transfer policy and optimize the selection of embryos for transfer and cryopreservation.
Asunto(s)
Núcleo Celular/ultraestructura , Implantación del Embrión , Embrión de Mamíferos/fisiología , Embrión de Mamíferos/ultraestructura , Inyecciones de Esperma Intracitoplasmáticas , Cigoto/ultraestructura , Fase de Segmentación del Huevo , Transferencia de Embrión , Femenino , Humanos , Embarazo , Índice de Embarazo , Embarazo MúltipleRESUMEN
Human embryo cryopreservation represents an indispensable extension of in-vitro fertilization (IVF) programmes as long as they are based upon the recovery of a large number of oocytes. The most widely used procedures include the cryopreservation of human zygotes or embryos in early cleavage, using 1,2-propanediol and sucrose as cryoprotectants. Our results over a 10 year period (1986-1995) on 5032 thawed cycles involving 14 222 stored embryos make it possible to appraise the results and the contribution of embryo freezing to assisted reproduction. Embryos survived the freeze-thaw process in 73% of cases leading to 4590 transfers of 2.2 embryos (91% of thawed cycles). The clinical pregnancy rate per transfer was 16%, the live birth rate 12%, and the rate of babies born alive per transferred embryo was 6%. Embryo freezing monitored 10 years later produced an average of 8% of additional births. By then, 86% of stored embryos had been thawed for transfer to patients. Destruction or donation were required for only 8% of all frozen embryos and there was no news from the parental couple in relation to almost 6% of embryos. The fate of the vast majority of embryos was decided during the first 5 years of storage. Blastocyst cryopreservation is making new strides, thanks to co-culture systems and embryo selection. Micromanipulation procedures seem to have little impact on the outcome of embryo freezing. Human oocyte freezing is again clinically applied. Indeed, much of the concern about injuries to the oocyte structures through the freeze-thaw process do not seem to be justified, and the problems with frozen-thawed oocyte fertilization has been overcome using intracytoplasmic sperm injection (ICSI). As long as oocyte in-vitro maturation is not well controlled, better results will probably be obtained with mature oocyte cryopreservation. Emerging methods include the freezing of immature oocytes, follicles and ovarian tissue.
Asunto(s)
Criopreservación , Transferencia de Embrión/métodos , Embrión de Mamíferos , Fertilización In Vitro , Oocitos , Femenino , Humanos , Masculino , Embarazo , Índice de EmbarazoRESUMEN
Intracytoplasmic sperm injection (ICSI), treatment of severe male infertility allows an accurate evaluation of oocyte maturity at recovery after corona-cell removal. In cycles comprising a GnRH analog desensitization and a stimulation by hMG or FSH, 12% of oocytes aspirated from follicles (> 14 mm), 34 hours post-hCG are still immature, in prophase or metaphase 1. They are able to achieve meiosis in vitro in 66% of the cases and will be fertilized (2 PN) by ICSI in 51% of the cases as the in vivo mature oocytes of the same cohort. Nevertheless, the quality of cytoplasmic maturation and consequently of embryonic viability remains to be assessed as there still are few pregnancies arising from in vitro matured oocytes. ICSI also represents the only way to obtain normal fertilization in some exceptional but observed anomalies of oocyte maturation, particularly when there is a lack of zona reaction leading to repetitive polyspermy in conventional IVF.
Asunto(s)
Fertilización In Vitro/métodos , Infertilidad Masculina/terapia , Microinyecciones/métodos , Oocitos/crecimiento & desarrollo , Espermatozoides , Adulto , Femenino , Humanos , Masculino , Meiosis , Inducción de la Ovulación/métodos , Embarazo , Resultado del EmbarazoRESUMEN
The expression of both components of the high-affinity leukaemia inhibitory factor receptor, LIFR beta and glycoprotein 130 (gp130), was investigated in human oocytes and individual in-vitro cultured preimplantation embryos by reverse transcription-polymerase chain reaction (RT-PCR). Messenger RNA of both LIFR beta and gp130 was detected in as little as 1/30 and 1/12 sample equivalents of cDNA respectively, in oocytes (n = 4), 4-cell and expanded, blastacyst stage embryos. LIFR beta but not gp130 transcripts were detected at the 2-, 8- and 10-cell stages, and in cavitating and hatched blastocysts. In order to exclude a simian origin of these PCR products resulting from the Vero cell line that was used as a feeder during culture to the blastocyst stage, they were digested with restriction endonucleases Taql (LIFR beta) or Kpnl (gp130). Their human origin was confirmed. The results support an earlier finding of LIFR beta mRNA expression in human blastocysts, and extend these results to earlier stages and oocytes. This is the first report of LIFR beta and gp130 transcription in human oocytes. Taken together these results demonstrate that transcription of LIFR beta and gp130 takes place throughout human preimplantation development, and suggest that functional LIF receptors might be present at these stages. These results further confirm the feasibility of performing mRNA phenotyping of multiple genes with RNA derived from a single preimplantation stage embryo.
Asunto(s)
Antígenos CD/biosíntesis , Blastocisto/metabolismo , Inhibidores de Crecimiento , Interleucina-6 , Linfocinas , Glicoproteínas de Membrana/biosíntesis , Oocitos/metabolismo , Receptores de Citocinas/biosíntesis , Receptor gp130 de Citocinas , ADN Complementario/análisis , Femenino , Humanos , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Reacción en Cadena de la Polimerasa , Embarazo , ARN Mensajero/análisis , Receptores OSM-LIFRESUMEN
Between 1990 and 1994, a clinical retrospective study has been carried out at Tenon Hospital on 1200 patients: during this periods. 4845 embryos have been cryopreserved and 31% of the patients had this procedure for their supranumerary embryos. The rate of implantation by embryo was 8% per transfer, comparable to those fresh embryos. Less than 1% of the embryos were abandoned. The contribution of cryopreservation to the IVF program is substantial, increasing pregnancy rate by 10%. Moreover, the rate of multiples pregnancies is significantly lower when implanting frozen thawed embryos (9.6%) vs fresh embryos (p < 0.001). There was no difference between frozen-thawed and fresh embryos, in the implantation rate by embryo, the mean gestational age, and birth weight of singleton, twin and triplet births. From a biological point de vue, a series of 3693 embryos, carried on the same period, in our center has showed that for the success of this procedure, the quality of the embryos was more important, than the duration of the storage. The incidence of major and minor congenital malformations was not different in the two groups of babies (less than 3%). But a retrospective analysis carried out on 84 children, showed 4 major abnormalities, after a follow up of 1 to 9 years. However these anomalies do not seem to have some evident correlation with the cryopreservation procedure; a larger series and a prospective study are needed to get significant results concerning the health of the children born after the procedure of cryopreserved embryos.
Asunto(s)
Criopreservación , Transferencia de Embrión , Embrión de Mamíferos , Fertilización In Vitro , Peso al Nacer , Anomalías Congénitas/epidemiología , Anomalías Congénitas/etiología , Femenino , Estudios de Seguimiento , Edad Gestacional , Humanos , Incidencia , Recién Nacido , Embarazo , Embarazo Múltiple , Estudios Retrospectivos , Factores de TiempoRESUMEN
Many studies in IVF practice, have tried to assess the maturity and quality of oocytes prior to insemination and relate it to IVF efficiency and to the pattern of ovarian stimulation. They were based on the indirect evaluation of the aspect of the cumulus-corona-cell complex (CCC) which was rapidly shown to be poorly correlated to the oocyte nuclear and cytoplasmic maturity in stimulated cycles. For sperm microinjection procedures, oocytes need to be peeled off from any follicular cell through hyaluronidase action in order to gain an easy access to the zona pellucida and the ooplasm. If therefore becomes possible to precisely known at recovery the nuclear status of the oocyte cohort as well as the rate of degenerative gametes. Immature oocytes can be further matured in vitro. Moreover, the ICSI procedure (intracytoplasmic sperm injection) allows a direct assessment of the cytoplasmic maturation, whatever the maturity of the ZP and its receptors and of the plasma membrane. On a preliminary evaluation of 70 ICSI cycles performed in our collaborative group from september to november 1994 and leading to a 24 % pregnancy rate per oocyte pick-up we focused on the true maturation of the oocyte cohort, its outcome and correlation with ICSI efficiency and stimulation protocol. On the 760 oocytes, 13.9% were atretic, 9% at the germinal vesicle stage (GV), 3.4% in metaphase 1 and 73.7% in metaphase 2 at recovery.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Fertilización In Vitro/métodos , Oocitos/crecimiento & desarrollo , Adulto , Estudios de Cohortes , Citoplasma , Femenino , Humanos , Metafase , Microinyecciones/métodos , Embarazo , Resultado del EmbarazoRESUMEN
Intracytoplasmic sperm injection is theoretically the first line treatment of many forms of severe male infertility, but the prognostic factors are to be determined according to the indications. Thus the results of two clinical teams working with the same biological Unit show statistically different pregnancy rates per oocyte pick-up (15.9% versus 35.7% - p < 0.02) and per transfer (16.9% versus 34.9% - p < 0.03). They could by explained by female factors such as age and ovarian response to stimulation, which probably have influence on embryonic quality.
Asunto(s)
Fertilización In Vitro/métodos , Microinyecciones , Oocitos/crecimiento & desarrollo , Citoplasma , Transferencia de Embrión , Femenino , Humanos , Masculino , Edad Materna , Microinyecciones/métodos , Inducción de la Ovulación , Embarazo , Resultado del Embarazo , PronósticoRESUMEN
A total of 17 couples with repetitive implantation failure after transfer of fresh or frozen-thawed embryos had half of their zygotes cultured in standard conditions and frozen at day 2 after insemination, and the other half cocultured with autologous granulosa cells and transferred at the morula or blastocyst stage at day 5 or 6 after oocyte retrieval. At the end of the culture period, supernatants of cocultures were recovered for steroid assays. Monolayers were stained for granulosa cell growth and morphological assessment. We observed that granulosa cells improve embryo development in vitro since 32 out of 60 (53%) reached the morula stage and 18 (30%) the blastocyst stage, leading to a total of 83% embryos available for transfer (compared with 3% without coculture). The ongoing pregnancy rate of these patients who were selected because they had at least three previous implantation failures, is only 5.9%, however, which is similar to the control group without coculture (6.3%). To conclude, granulosa cells improve embryo development but not the pregnancy rate after transfer of cocultured embryos in patients with multiple previous implantation failures.
Asunto(s)
Fase de Segmentación del Huevo/fisiología , Células de la Granulosa/fisiología , Cigoto/crecimiento & desarrollo , Adulto , Blastocisto/fisiología , Células Cultivadas , Transferencia de Embrión , Femenino , Fertilización In Vitro , Humanos , Persona de Mediana Edad , Mórula/fisiología , Esteroides/fisiologíaRESUMEN
Ten couples included in our in-vitro fertilization programme, selected because of one of more previous total fertilization failures or a low fertilization rate (< 20%), were entered in an experimental protocol of egg insemination on autologous granulosa cells. Half the oocytes from each patient were randomly assigned to either a control or a co-culture group. We observed no difference in the fertilization rate between the control (16.2%) and co-culture groups (12.1%). Only two couples benefited from this technique since fertilization was obtained only in co-culture. The poor efficiency of this protocol led us to propose the use of sperm micro-injection if all classical attempts to improve fertilization were unsuccessful.
Asunto(s)
Fertilización In Vitro , Células de la Granulosa/citología , Oocitos/citología , Células Cultivadas , Femenino , Humanos , Insuficiencia del TratamientoRESUMEN
Tests of sperm function are often considered to be able to predict the in vivo or in vitro fertility of a given patient although they only assess part of the process of sperm fertilizing ability. It appears from the literature, that the sperm penetration assay (SPA) which investigates the sperm fusiogenic and nuclear functions cannot accurately predict the in vivo fertility and there is still much debate on the reliability of its correlation with in vitro fertilization (IVF) principally in cases of severe sperm deficiency. With the development of new techniques of assisted fertilization, by-passing the zona pellucida, a test of sperm fusion, hetero or homospecific as well, could be interesting. Considering sperm ultrastructure and beyond some specific morphological alterations of the whole sperm population that can be responsible for the infertility, such studies were not able, as yet, to reveal significative differences between semen fertile in vitro or not.
Asunto(s)
Infertilidad Masculina/diagnóstico , Interacciones Espermatozoide-Óvulo , Espermatozoides/ultraestructura , Animales , Cricetinae , Femenino , Fertilización In Vitro , Infertilidad Masculina/epidemiología , Infertilidad Masculina/patología , Infertilidad Masculina/terapia , Masculino , Microscopía Electrónica , Oocitos , Valor Predictivo de las Pruebas , Embarazo , Resultado del Embarazo , Espermatozoides/patología , Zona PelúcidaAsunto(s)
Fertilización In Vitro , Infertilidad Femenina/diagnóstico , Infertilidad Masculina/diagnóstico , Oocitos/fisiología , Interacciones Espermatozoide-Óvulo , Espermatozoides/fisiología , Adulto , Sitios de Unión , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oligospermia , Insuficiencia del Tratamiento , Zona Pelúcida/fisiologíaRESUMEN
Chromosome abnormalities represent the major cause of pre- and post-implantation embryo wastage. Indeed, 29% of embryos produced by in-vitro fertilization have an abnormal karyotype. Parthenogenesis (1.6% of the cases) and triploidy (6.4%) are the only abnormalities detectable 17 h after insemination. A total of 23% of activated oocytes with a single pronucleus (1 PN) and 18% of fertilized eggs with three pronuclei (three PN) remained uncleaved, compared with 4% for diploid eggs. The rate of cleavage for parthenogenes is not different from normal eggs. On the contrary, we observed that 29% of three PN-eggs reached the 5- to 8-cell stage 42 h after insemination when compared to 15% for diploids mainly due to a direct division of 56% of three PN-eggs in three cells. A model of development for triploid eggs is proposed, taking into account physiological and cytogenetic observations. The quality of embryos expressed in terms of morphology did not show any difference between embryos proceeding from parthenogenes or diploid eggs. On the contrary, the rate of fragmentation is decreased for triploid (3%) compared with diploid (13%) or activated (17%) embryos. Embryos resulting from one, two, three or four pronucleate ova display different developmental capacities during 5 days' culture in vitro.
Asunto(s)
Aberraciones Cromosómicas , Trastornos de los Cromosomas , Desarrollo Embrionario y Fetal , Fertilización In Vitro , Diploidia , Femenino , Haploidia , Humanos , Cariotipificación , Partenogénesis , PoliploidíaRESUMEN
More than 60% of patients with polycystic ovary disease (PCO) cannot conceive after repeated ovulation inductions with Clomifene citrate although there is ovulation or more frequently follicle luteinization. Because of hyperstimulation, therapy with hMG has been superseded by low doses of purified FSH with variable results according to authors. It has been even claimed that there was no benefit to replace hMG with FSH. However, on the basis of the PCO physico-pathology, namely LH hypersecretion and androgen hyperproduction, it would be rational to associate the desensitization of the pituitary with LH-RH agonist and the ovary stimulation with variable doses of hMG or purified FSH. In the series where such therapy associating LH-RH agonists with purified FSH was applied, the results concerning suppression of LH and androgen secretion, and the occurrence of pregnancy were interesting. However, the risk of hyperstimulation still occurred. Thus, the first part concerns the critical review of these results while, in the second part, our experience in in vitro fecundation will be reported.
Asunto(s)
Menotropinas/uso terapéutico , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Andrógenos/metabolismo , Femenino , Hormona Folículo Estimulante/sangre , Hormona Folículo Estimulante/uso terapéutico , Humanos , Hormona Luteinizante/metabolismo , Menotropinas/sangreRESUMEN
Fertilization in-vitro offers the possibility of studying the karyotype of ovocytes obtained after superovulation, when they are not fertilized. Among 120 ovocytes, 30 p. cent presented a chromosomal anomaly. The same study was carried out on morphologically normal or abnormal embryos - and the percentage of chromosomal anomalies approximates here 27 p. cent. These studies offer a model of natural selection against chromosomal anomalies and confirm the limiting role of these anomalies in the success of FIV.
Asunto(s)
Fertilización In Vitro , Cariotipificación , Oocitos , Aneuploidia , Aberraciones Cromosómicas/diagnóstico , Trastornos de los Cromosomas , Femenino , Humanos , Selección Genética , SuperovulaciónRESUMEN
The major questions raised by frozen embryos are reviewed: how to freeze? which embryos to freeze? how to replace the frozen embryos? What is he contribution of frozen embryos in a program of fertilization in-vitro. The cryopreservation of human ovocytes is also discussed.
Asunto(s)
Transferencia de Embrión/métodos , Embrión de Mamíferos , Oocitos , Preservación de Órganos , Fertilización In Vitro/métodos , Congelación , HumanosRESUMEN
A multicentric study was carried out to analyse in a large series: (i) the chromosomal status of unfertilized oocytes, (ii) errors at fertilization and (iii) the chromosomal complement of cleaved embryos. Parameters such as type of sterility, maternal age, stimulation treatment, doses of gonadotrophins administered and oocyte preincubation time before insemination were studied in relation to the incidence of chromosome abnormalities. Twenty-six per cent of the unfertilized oocytes and 29.2% of the embryos had chromosome anomalies. Maternal age significantly increased the rate of aneuploidy in oocytes: 38% in patients over 35 years (versus 24% in younger patients). Fertilization-related abnormalities were significant, i.e. 1.6% parthenogenesis and 6.4% polyploidy. Unexplained infertility was correlated with an increase in the rate of parthenogenesis (4.2%) when compared with tubal infertility (1.2%). Triploidy was found to be correlated with three parameters. A lower rate of triploidy was observed in the group of couples referred because of male sterility (1.9% versus 6.3% for tubal sterility), in HMG-treated patients (2.4% versus 7% with analogues of LHRH/HMG) and with a short 2-h preincubation time before insemination (3% versus 7.2% for greater than 2 h). A general model for natural selection against embryos carrying a chromosome imbalance was proposed.