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OBJECTIVE: To compare the degree of efficiency between density gradient centrifugation (DGC) method and an extended horizontal swim-up (SU) method. METHODS: A total of 97 couples undergoing in vitro fertilization were enrolled in the study. Semen samples were divided into three aliquots and treated using DGC, extended horizontal SU, and combined methods. DNA fragmentation and chromatin decondensation were detected in native semen samples and their three corresponding aliquots. The corresponding mature oocytes of each semen sample were divided into two sibling cultures. The first sibling culture was microinjected with semen pellets from DGC, and the second sibling culture was microinjected with semen pellets from the combination of both methods. Fertilization rate and embryonic development were assessed at day 3. RESULTS: DNA fragmentation and chromatin decondensation was significantly low in DGC and extended horizontal SU samples; however, the rates of DNA fragmentation and chromatin decondensation were significantly lower in extended horizontal SU samples than in DGC samples. The lowest rates of DNA fragmentation and chromatin decondensation corresponded to the samples treated with both methods. The highest rates of DNA fragmentation and chromatin decondensation corresponded to the samples treated with DGC. No significant difference was found in the fertilization rate or day 3 embryos between sibling cultures. CONCLUSION: The combination of DGC and the extended horizontal SU techniques is best for giving the lowest rates of sperm DNA fragmentation and chromatin decondensation.
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RESEARCH QUESTION: What are the main risk factors associated with ectopic pregnancy and what is the true incidence of ectopic pregnancies in an IVF programme? DESIGN: Retrospective single-centre study of 12,429 blastocyst transfers (8182 fresh and 4247 frozen embryo transfers) conducted between January 2010 and December 2017. IVF outcome was analysed, and ectopic pregnancy risk evaluated according to patient's characteristics and assisted reproductive technology treatment factors. RESULTS: Of 5061 patients reporting a positive pregnancy test, 43 were diagnosed with ectopic pregnancy (0.85%). Neither female age (36.7 versus 35.8 years), body mass index, quality of transfer nor stimulation protocol affected the ectopic pregnancy rate, but history of previous ectopic pregnancy (OR 3.26; Pâ¯=â¯0.0080), tubal surgery, or both (OR 6.20; P < 0.0001) did. The incidence of ectopic pregnancy was increased in women with uterine malformations (OR 3.85; Pâ¯=â¯0.0052), uterine pathologies (OR 5.35; Pâ¯=â¯0.0001), uterine surgeries (OR 2.29; Pâ¯=â¯0.0154) or sub-optimal endometrial build-up (OR 4.46 to 5.31; P < 0.0001). Transfer of slow-developing blastocysts (expressed by expansion) significantly increased the risk of ectopic pregnancy (OR 2.59; Pâ¯=â¯0.0102). CONCLUSIONS: Unfavourable uterine environment, including uterine pathologies, uterine or tubal surgery and suboptimal endometrial build-up were related to ectopic pregnancy. Low expansion grade of blastocysts was identified as an additional putative risk factor for ectopic pregnancy, indicating the importance of proper embryonal-maternal synchronization. The overall ectopic pregnancy rate after blastocyst transfer was low, comparable with reported ectopic pregnancy rates in spontaneous conceptions. Proper evaluation of tubal and uterine pathologies, optimizing endometrial preparation and the transfer of expanded blastocysts in a frozen embryo transfer cycle, might be beneficial.
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Transferencia de Embrión/efectos adversos , Inducción de la Ovulación/efectos adversos , Embarazo Ectópico/etiología , Técnicas Reproductivas Asistidas/efectos adversos , Adulto , Transferencia de Embrión/métodos , Femenino , Humanos , Embarazo , Índice de Embarazo , Estudios Retrospectivos , Factores de RiesgoRESUMEN
SummaryThe aim of this study was to investigate the effects of zona drilling and biopsy on day 3 followed by vitrification on day 5 on the cytoskeleton and development of human embryos, by analysing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy in human biopsied and non-biopsied embryos. In total, 98 human blastocysts (50 non-biopsied and 48 following biopsy on day 3) were vitrified on day 5 using either a commercial dimethyl sulphoxide (DMSO)-free vitrification kit or increasing concentrations of DMSO/EG (5%/5-10%/10-20%/20%). Following warming, the blastocysts were allowed to recover in culture for 24 h and were immunostained with α-tubulin, acetylated tubulin, and/or γ-tubulin antibodies in combination with 4',6-diamidino-2-phenylindole (DAPI). Labelled embryos were examined by both fluorescence and confocal laser scanning microscopy. The survival rates following warming (92% non-biopsied vs 83.3% biopsied) and the incidence of normal spindle chromosome configurations was not statistically different between the two groups (65.2% non-biopsied vs 59.2% biopsied, P>0.05). The incidence of spindle abnormalities including multipolarity, chromosome lagging, congression failure and chromosome bridging were also similar between the two groups (P>0.05). This study is the first to compare the incidence of cytoskeletal abnormalities in biopsied and non-biopsied human embryos following vitrification. We conclude that there was no significant difference in the survival rates and the incidence of spindle abnormalities between the two groups.
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Blastocisto/citología , Aberraciones Cromosómicas/embriología , Citoesqueleto/metabolismo , Embrión de Mamíferos/citología , Microscopía Confocal/métodos , Vitrificación , Biopsia , Blastocisto/metabolismo , Supervivencia Celular , Técnicas de Cultivo de Embriones , Transferencia de Embrión/estadística & datos numéricos , Embrión de Mamíferos/embriología , Desarrollo Embrionario , Humanos , Factores de Tiempo , Tubulina (Proteína)/metabolismoRESUMEN
PURPOSE: The aim of this study was to compare the effect of the deselection of spermatozoa presenting vacuole-like structures using IMSI (intracytoplasmic morphologically selected sperm injection) with ICSI (intracytoplasmic sperm injection) by means of neonatal outcomes. METHODS: In a retrospective two-center analysis, a total of 848 successful IMSI or ICSI cycles ending with a live birth, induced abortion, or intrauterine fetal death (IUFD) were included. RESULTS: The IMSI and ICSI groups included 332 and 655 babies or fetuses, respectively. The parents were older in the IMSI group than in the ICSI group (mothers were 35.1 vs 32.9 years, and fathers were 39.1 vs 36.2 years). The multiple pregnancy rate was higher in the IMSI group. The mean pregnancy duration and mean birth weight were almost identical in both groups. There was no significant difference in major congenital malformations between the two groups. However, this rate was decreased in the IMSI group compared to that in the ICSI group (1.8 vs 3.2%), the difference being mainly found in singletons (1.4 vs 3.3%). Boys were more often affected than girls in both groups. The percentages of chromosomal abnormalities did not differ between the IMSI and ICSI groups (0.6 and 0.8%). The reported congenital malformations mainly affected the heart, urogenital, and musculoskeletal systems. CONCLUSIONS: In the present study, the malformation rates observed in the IMSI and ICSI groups were not significantly different, even if slightly lower after IMSI. However, the observed difference followed the same trends observed in previous reports, indicating the possible impact of IMSI on decreasing congenital malformation occurrences. This highlights the necessity to prospectively evaluate the impact of IMSI on neonatal outcome after IVF treatment.
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Transferencia de Embrión , Infertilidad Masculina/terapia , Inyecciones de Esperma Intracitoplasmáticas/métodos , Espermatozoides/citología , Espermatozoides/fisiología , Adulto , Femenino , Humanos , Masculino , Embarazo , Resultado del Embarazo , Índice de Embarazo , Estudios RetrospectivosRESUMEN
OBJECTIVE: To evaluate whether is possible to vitrify oocytes in an aseptic (hermetically closed) fashion and maintain clinical results comparable with those of fresh oocytes. DESIGN: Prospective, observational, cohort, noninferiority trial. SETTING: Private in vitro fertilization center. PATIENT(S): One hundred eighty-four recipients of donated vitrified oocytes. INTERVENTION(S): Closed system vitrification. MAIN OUTCOME MEASURE(S): Pregnancy rate per cycle and clinical pregnancy rate per cycle. RESULT(S): No statistically significant differences were observed between two groups regarding the pregnancy rate per cycle (63.1% vs. 60.9%) or the clinical pregnancy rate per cycle (55.4% vs. 58.7%). Biochemical pregnancy rate was statistically significantly higher in the fresh group (7.6% vs. 2.2%). The mean number of embryos transferred was similar (2.0 ± 0.0 vs. 1.97 ± 0.3). Concerning embryologic data, there were no statistically significant differences regarding the fertilization, cleavage, top quality day-3 embryo, or blastocyst rates, whereas the top quality blastocyst rate on day 5 was statistically significantly higher in the fresh oocyte group (31.7% vs. 26.1%). CONCLUSION(S): Aseptically (in a closed system) vitrified oocytes show similar clinical efficiency compared with their sibling fresh oocytes.
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Asepsia/métodos , Criopreservación/métodos , Infertilidad/terapia , Donación de Oocito , Conservación de Tejido/métodos , Adulto , Asepsia/instrumentación , Criopreservación/instrumentación , Transferencia de Embrión , Femenino , Fertilidad , Fertilización In Vitro , Humanos , Infertilidad/diagnóstico , Infertilidad/fisiopatología , Nacimiento Vivo , Masculino , Persona de Mediana Edad , Embarazo , Índice de Embarazo , Estudios Prospectivos , Conservación de Tejido/instrumentación , Resultado del Tratamiento , VitrificaciónRESUMEN
PURPOSE: Various time-lapse studies have postulated embryo selection criteria based on early morphokinetic markers. However, late paternal effects are mostly not visible before embryonic genome activation. The primary objective of this retrospective study was to investigate whether those early morphokinetic algorithms investigated by time-lapse imaging are reliable enough to allow for the accurate selection of those embryos that develop into blastocysts, while of course taking into account the correlation with the type of injected spermatozoa. METHODS: During a period of 18 months, a total of 461 MII oocytes from 43 couples with severe male factor infertility and previous "external" IVF failures after cleavage-stage embryo transfer (ET) were fertilized by intracytoplasmic morphologically selected sperm injection (IMSI). Thereof, 373 embryos were monitored in a time-lapse incubator until ET on day 5. Blastocyst outcome in combination with three previously postulated MKc (cc2: t3-t2, 5-12 h; t3, 35-40 h; t5, 48-56 h) and the morphology of the selected sperm were analyzed. RESULTS: A significant increase in the rate of blastocysts (54.0 vs. 36.3 %; P < 0.01) and top blastocysts (25.3 vs. 10.8 %; P < 0.001) was observed in the group of those meeting all three morphokinetic criteria (MKc3). However, MKc3 were only met in 23.3 % of all embryos. Moreover, TBR was influenced by the type of injected spermatozoa. In both groups, TBR decreased dramatically (MKc3, 35.0 vs. 17.0 %; MKc < 3, 14.2 vs. 8.4 %) when class II/III sperm instead of class I were injected. CONCLUSION: Early morphokinetic parameters might give some predictive information but fail to serve as a feasible selective tool for the prediction of blastocyst development given the influence of the type of spermatozoa injected.
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Blastocisto/fisiología , Espermatozoides/fisiología , Imagen de Lapso de Tiempo/métodos , Adulto , Algoritmos , Blastocisto/citología , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Infertilidad Masculina/etiología , Masculino , Persona de Mediana Edad , Oocitos , Edad Paterna , Estudios Retrospectivos , Inyecciones de Esperma Intracitoplasmáticas/métodosRESUMEN
BACKGROUND: Successful embryo implantation depends on a well-timed maternal-embryonic crosstalk. Human chorionic gonadotropin (hCG) secreted by the embryo is known to play a key role in this process and to trigger a complex signal transduction cascade allowing the apposition, attachment, and invasion of the embryo into the decidualized uterus. Production of hCG was reported to be dependent on blastocyst quality and several articles suggested that intrauterine hCG injection increases pregnancy and implantation rates in IVF patients. However, no study has as yet analysed birth rates as final outcome. Our objective was to determine whether clinical outcome after blastocyst transfer can be improved by intrauterine injection of hCG and whether this is dependent on blastocyst quality. METHODS: A prospective randomised study was conducted in two settings. In cohort A, hCG application was performed two days before blastocyst transfer. In cohort B, the administration of hCG occurred just prior to embryo transfer on day 5. For both cohorts, patients were randomised to either intrauterine hCG application or to the control group that received culture medium. Clinical outcome was analysed according to blastocyst quality of transferred embryos. RESULTS: The outcome of 182 IVF-cycles (cohort A) and 1004 IVF-cycles (cohort B) was analysed. All patients received a fresh autologous blastocyst transfer on day five. Primary outcomes were pregnancy rates (PR), clinical pregnancy rates (cPR), miscarriage rates (MR), and live birth rates (LBR). No improvement of clinical outcome after intrauterine hCG administration on day 3 (cohort A) or day 5 (cohort B) was found, independently of blastocyst quality transferred. The final outcome in cohort A: LBR after transfer of top blastocysts was 50.0 % with hCG and 53.3 % in the control group. With non-top blastocysts, LBR of 17.1 % (hCG) and 18.2 % (control) were observed (n.s.). In cohort B, LBR with top blastocysts was 53.3 % (hCG) and 48.4 % (control), with non-top blastocysts it came to 28.7 % (hCG) and 35.0 % (control). The differences between the groups were statistically not significant. Furthermore, we investigated a possible benefit of hCG administration in correlation with female age. In both age groups (<38 years and ≥ 38 years) we found similar LBR after treatment with hCG vs. medium. A LBR of 47.1 % vs. 48.7 % was obtained in the younger group and 26.6 % vs. 30.8 % in the older group. CONCLUSIONS: In contrast to previous studies indicating a substantial benefit from intrauterine hCG application in cleavage stage embryo transfers, in our study we could not find any evidence for improvement of clinical outcome in blastocyst transfer cycles, neither with top nor with non-top quality morphology.
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Blastocisto/efectos de los fármacos , Gonadotropina Coriónica/uso terapéutico , Transferencia de Embrión/métodos , Índice de Embarazo , Adulto , Tasa de Natalidad , Gonadotropina Coriónica/farmacología , Femenino , Humanos , Nacimiento Vivo , Embarazo , Estudios Prospectivos , Resultado del Tratamiento , Adulto JovenRESUMEN
CONTEXT: Massage therapy is increasingly used to relieve physical and mental discomfort and is suggested as a safe therapeutic modality, without any significant risks or any known side effects. Although a multitude of complementary therapies, such as acupuncture, are applied in reproductive medicine, no information is available with regard to the application of massage as an adjuvant therapy in assisted-reproduction techniques (ARTs). OBJECTIVES: This study was intended to assess the effectiveness of a deep relaxation (andullation) therapy based on oscillating vibrations when used prior to embryo transfer (ET) in in vitro fertilization (IVF) cryo-cycles. DESIGN: The research team designed a retrospective, observational study. Participants willing to undergo the massage treatment were allocated to the intervention (andullation) group. SETTING: The study was performed at the IVF Centers Prof. Zech-Bregenz in Bregenz, Austria. PARTICIPANTS: A total of 267 IVF patients, with a mean age of 36.3 y, participated in this single-center study. INTERVENTION: All patients receiving a transfer of vitrified and warmed blastocysts between January and December 2012 were included in the evaluation. Prior to ET, the andullation group received a standardized program of therapy-a 30-min, deep relaxation massage on an oscillating (vibrating) device, whereas the control group did not. OUTCOME MEASURES: To determine efficacy, the primary outcomes that the study measured were (1) pregnancy rates (PRs), by testing urine and obtaining a positive ß-human chorionic gonadotropin (ß-hCG); and (2) ongoing, pregnancies (oPR), by observation of fetal heartbeat and birth rates (BR) as well as miscarriage rates. The patients' medical histories and types of infertility as well as the quality of the embryo transfers (ETs) were evaluated. RESULTS: In patients using the massage therapy prior to ET, significantly higher PRs, oPRs, and BRs were observed compared with the control group-PR: 58.9% vs 41.7%, P<.05; oPR: 53.6% vs 33.2%, P<.01; and BR: 32.0% vs 20.3%, P<.05. No differences were detected among groups for patients' ages, hormonal substitution protocols, endometrium structures and buildups, quality of transferred embryos, or quality of transfers. No adverse effects were noted in the massage group. CONCLUSIONS: The research team's results suggested that andullation therapy prior to blastocyst transfer in a cryo-cycle improves embryo implantation, most likely due to a reduction in stress (ie, a relaxation effect on patients), a reduction in uterine contractions, and, probably, an enhancement of the blood flow in the abdominal region. These findings provide support for use of andullation as a complementary therapy for ART.
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Transferencia de Embrión/estadística & datos numéricos , Fertilización In Vitro/estadística & datos numéricos , Masaje , Embarazo/estadística & datos numéricos , Adulto , Gonadotropina Coriónica/sangre , Femenino , Frecuencia Cardíaca Fetal/fisiología , Humanos , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Time-lapse imaging is increasingly applied as an adjunct to reproductive medicine. The gained information of the morphological and morphokinetic variables before the onset of transcription are supposed to be good predictors for the selection of the best embryo for transfer and are often seen in line with clinical outcomes. This retrospective case series investigated the outcome of transferred blastocysts that did not fulfil the proposed embryo scores at early cleavage or at later stages of development. The observations were made by time-lapse imaging. This study reports the birth of 16 healthy children after day-5 blastocyst transfer, of which at least one of the transferred embryos originated from deviant morphology and/or kinetic cleavage patterns. This case series suggests that some blastocysts derived from embryos with poor conventional morphological score and/or suboptimal morphokinetics can be successfully transferred and might result in live births. Such results might raise awareness that discarding embryos based only on early events is not a suitable approach to give patients the chance to conceive. In conclusion, to date only the transfer of viable embryos after culturing them until day 5 guarantees optimal embryo selection and helps to prevent embryo wastage.
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Blastocisto , Transferencia de Embrión , Oocitos , Adulto , Técnicas de Cultivo de Embriones , Implantación del Embrión , Retículo Endoplásmico Liso , Femenino , Humanos , Masculino , Oocitos/citología , Embarazo , Estudios Retrospectivos , Imagen de Lapso de Tiempo , Transferencia Intrafalopiana del CigotoRESUMEN
Since the introduction of the motile sperm organelle morphology examination, there has been increasing recognition of the fact that the presence of large nuclear vacuoles might have deleterious effects on embryo development. Nevertheless, one fundamental question still being debated is whether specific in-vitro conditions during the handling of semen have an impact on vacuole formation. This study's objective was to analyse whether incubation temperature (20, 37°C) or oxidative stress stimulates the formation of nuclear vacuoles. Furthermore, it examined whether vacuoles disappear in the presence of an acrosome reaction inducer. Therefore, a system of sperm-microcapture channels was developed to permit the observation of the same living spermatozoa over a period of 24h. Neither incubation at 37°C nor induction of oxidative stress led to de-novo formation of nuclear vacuoles. Induction of the acrosome reaction using calcium ionophore A23587 did not lead to any modifications in the proportion of spermatozoa with vacuoles or to the disappearance of pre-existing vacuoles. According to these observations, it is concluded that nuclear vacuoles on the sperm head are already produced at earlier stages of sperm maturation and are not induced or modulated by routine laboratory environments. The examination of spermatozoa at very high magnification has led to the increasingly widespread recognition that the presence of large vacuoles in the human sperm head has deleterious effects on embryo development. One fundamental question, however, still remains: do specific conditions in the laboratory during the preparation and the handling of semen have an impact on vacuole formation? Our initial objective was to analyse whether different incubation temperatures (20, 37°C) and the induction of oxidative stress lead to the formation of sperm head vacuoles. Furthermore, we examined whether vacuoles disappear in the presence of an acrosome reaction inducer. In order to do this we developed a system of sperm-microcapture channels, which permits the observation of the same living spermatozoa over a period of 24h. Incubation at 37°C or induction of oxidative stress did not lead to the formation of any new vacuoles. After inducing the acrosome reaction, we did not detect any modification in the proportion of vacuolated spermatozoa. According to our observations, different temperatures or environmental conditions in the laboratory have no impact on the formation or disappearance of vacuoles. We conclude that sperm head vacuoles are already produced at earlier stages of sperm maturation.
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Estrés Oxidativo , Cabeza del Espermatozoide/ultraestructura , Temperatura , Vacuolas/ultraestructura , Reacción Acrosómica/efectos de los fármacos , Adulto , Ionóforos de Calcio/efectos adversos , Ionóforos de Calcio/farmacología , Técnicas de Cultivo de Célula , Humanos , Masculino , Manejo de Especímenes/métodos , Cabeza del Espermatozoide/efectos de los fármacosRESUMEN
BACKGROUND: Poor sperm quality can negatively affect embryonic development and IVF outcome. This study is aimed at investigating the influence of various lifestyle factors on semen quality according to MSOME (motile sperm organelle morphology examination) criteria. METHODS: 1683 male patients undergoing assisted reproductive technologies (ART) in our clinic were surveyed about their age, BMI (body mass index), ejaculation frequency, nutrition, sports, sleeping habits and social behavior. Semen samples were collected and evaluation of semen parameters according to MSOME and WHO criteria was performed. Results were grouped and statistically analyzed. RESULTS: Although single parameters had minor effects on sperm parameter, the combination of age, BMI, coffee intake, ejaculatory frequency and duration of sexual abstinence were identified as factors having a negative effect on sperm motility. Additionally, we could demonstrate that MSOME quality was reduced. The negative impact of age, BMI and coffee intake on sperm quality could be compensated if patients had a high ejaculation frequency and shorter periods of sexual abstinence. CONCLUSIONS: Combinations of adverse lifestyle factors could have a detrimental impact on sperm, not only in terms of motility and sperm count but also in terms of sperm head vacuolization. This negative impact was shown to be compensated by higher ejaculation frequency and a shorter period of sexual abstinence. The compensation is most likely due to a shorter storage time in the male gonads, thus reducing the duration of sperms' exposure to reactive oxygen species (ROS).
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Estilo de Vida , Análisis de Semen , Motilidad Espermática , Espermatozoides/fisiología , Adulto , Factores de Edad , Índice de Masa Corporal , Café , Eyaculación , Ejercicio Físico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estado Nutricional , Estrés Oxidativo , Técnicas Reproductivas Asistidas , Abstinencia Sexual , Sueño , Conducta Social , Recuento de Espermatozoides , Cabeza del Espermatozoide/ultraestructura , Espermatozoides/ultraestructura , Factores de Tiempo , Vacuolas/ultraestructuraRESUMEN
In some IVF cycles, no fresh embryo transfer in the stimulated cycle is advisable. The cryopreservation of zygotes and the transfer of blastocysts in a cryo-embryo transfer is an option to circumvent an inadequate uterine environment due to risk of ovarian hyperstimulation syndrome, inappropriate endometrium build up, endometrial polyps or uterine myomas. For this strategy, highly secure and safe cryopreservation protocols are advisable. This study describes a protocol for aseptic vitrification of zygotes that results in high survival rates and minimizes the potential risk of contamination in liquid nitrogen during cooling and long-term storage. In mouse zygotes, there was no difference in efficiency as compared with a conventional open vitrification system. In IVF patients, aseptically vitrified zygotes showed no difference in blastocyst formation rate as compared with sibling zygotes kept in fresh culture. A clinical study comprising 173 cryo-cycles with a transfer of blastocysts originating from vitrified zygotes showed an ongoing pregnancy rate of 40.9%. The live birth rate per patient was 36.8%. A combination of good clinical results and increased safety conditions due to aseptic vitrification encourages the use of cryo-embryo transfer for patients with a suboptimal uterine environment in a fresh cycle. In stimulated IVF cycles, high doses of hormones are given to stimulate multifollicular growth. One drawback of the hormonal substitution is that the uterine environment is not at the same time optimally prepared for embryo implantation. A solution, which is increasingly under discussion, is to cryopreserve the embryos obtained in the stimulated cycle and to transfer them back into the optimal uterine environment in a subsequent cryo-cycle. This procedure requires highly secure and safe cryopreservation protocols in order to ensure benefits for both pregnancy and birth rates. We have established a protocol for the vitrification of zygote-stage embryos in aseptic devices, which minimize the potential risk of contamination during cooling and storage. The vitrified zygotes showed the same blastocyst development as compared with sibling zygotes in fresh culture. A clinical study comprising 173 cryo-cycles with transfer of blastocysts originating from vitrified zygotes shows an ongoing pregnancy rate of 40.9%. The live birth rate per patient was 36.8%. A combination of good clinical results and increased safety conditions due to aseptic vitrification conditions contributes to a change in transfer strategy and encourages us to increase the cryo-embryo transfer rate for an optimal uterine environment.
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Criopreservación/métodos , Transferencia de Embrión , Infertilidad Femenina/terapia , Enfermedades Uterinas/etiología , Vitrificación , Cigoto , Adulto , Animales , Bélgica/epidemiología , Tasa de Natalidad , Criopreservación/instrumentación , Ectogénesis , Femenino , Humanos , Infertilidad Femenina/complicaciones , Ratones , Ratones Endogámicos , Síndrome de Hiperestimulación Ovárica/epidemiología , Síndrome de Hiperestimulación Ovárica/fisiopatología , Embarazo , Índice de Embarazo , Distribución Aleatoria , Estudios Retrospectivos , Riesgo , Enfermedades Uterinas/complicaciones , Enfermedades Uterinas/epidemiología , Adulto JovenAsunto(s)
Enfermedades Gastrointestinales/fisiopatología , Infecciones/fisiopatología , Oocitos/fisiología , Índice de Embarazo , Adulto , Blastocisto/fisiología , Diarrea/microbiología , Diarrea/fisiopatología , Femenino , Fertilización In Vitro/métodos , Enfermedades Gastrointestinales/microbiología , Humanos , Infecciones/microbiología , Oocitos/patología , EmbarazoRESUMEN
BACKGROUND: Vitrification of human blastocysts is being used increasingly to cryopreserve supernumerary embryos following IVF. In this study, we investigate the effects of aseptic vitrification on the cytoskeleton and development of human blastocysts, by analysing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy. METHODS: A total of 55 fresh blastocysts and 55 day 5 dimethylsulphoxide/ethylene glycol vitrified blastocysts, which were allowed to remain in culture for 24 h post-warming, were rapidly fixed in ice cold methanol, and immunostained with an a-tubulin antibody to visualize microtubules in combination with antibodies against acetylated tubulin (to visualize spindles, poles and mid bodies), gamma tubulin (to identify spindle poles) and 4(6-diamidino-2-phenylindole) to visualize DNA. RESULTS: In total, 213 spindles were analysed in the control (fresh) group of which 183/213 (85.9%) were normal, 20/213 (9.4%) were abnormally shaped, 9/213 (4.2%) were multipolar and 1/213 (0.5%) was monopolar. A total of 175 spindles were analysed in the vitrified group, of which 120/175 (68.6%) were normal, 39/175 (22.3%) were abnormally shaped, 10/175 (5.7%) were multipolar and 6/175 (3.4%) were monopolar. The incidence of multipolar spindles was similar in the two groups, but the level of abnormally shaped spindles, often associated with chromosome lagging, or congression failure, was significantly higher in the vitrified group compared with the fresh group (P< 0.05). CONCLUSIONS: The high survival rate following thawing and the large proportion of normal spindle/chromosome configurations suggests that vitrification at the blastocyst stage on Day 5 does not adversely affect the development of human embryos and the ability of spindles to form and continue normal cell divisions. However, there was a significantly higher incidence of abnormal spindles in the vitrified group compared with the fresh group, notably of spindles with a focused and an unfocused pole as well as chromosome bridging and disorganized middle spindle fibres at telophase. Further investigation is warranted to elucidate the mitotic stages that are more vulnerable to damage during vitrification, the fate of the abnormal spindles and any potential effects that may be reflected on the chromosomal constitution of the developing blastocysts.
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Blastocisto/citología , Citoesqueleto/patología , Microscopía Confocal/métodos , Ciclo Celular , División Celular , Supervivencia Celular , Citoesqueleto/metabolismo , ADN/metabolismo , Dimetilsulfóxido/química , Glicol de Etileno/química , Femenino , Humanos , Oocitos/citología , Ovario/citología , Huso Acromático , Tubulina (Proteína)/metabolismo , VitrificaciónRESUMEN
BACKGROUND: This study aimed to investigate the influence of an oral antioxidative supplementation on sperm quality of in vitro fertilization (IVF) patients, as analyzed by sperm motility according to the WHO criteria and motile sperm organelle morphology examination (MSOME). METHODS: Semen samples were collected from 147 patients before undergoing an IVF/intracytoplasmic morphologically-selected sperm injection (IMSI) cycle and 2 - 12 months after an antioxidative supplementation. Semen analysis was evaluated according to WHO and MSOME criteria. Spermatozoa were grouped according to the size of nuclear vacuoles within the sperm's heads. Patients were divided into oligoasthenoteratozoospermic (OAT) and non-OAT men. Between first and second semen analysis, patients were supplemented orally with an antioxidative preparation. RESULTS: After the antioxidative therapy we observed a significant reduction in the percentage of immotile sperm cells in the patients. Additionally, the percentage of class I spermatozoa according to MSOME criteria was significantly higher after antioxidative supplementation. In OAT patients the percentage of class I sperm was found to be increased, although not significantly. However, we observed a drastic improvement in sperm motility as well as in total sperm count in this group. CONCLUSION: The results demonstrated a considerable improvement in semen quality, notably in OAT patients. Considering the putative relationship between semen quality on the one hand and reactive oxygen species on the other, the observed changes in the sperm parameters indicate that a decline in semen quality, and even subtle morphological changes, might be associated with oxidative stress. Our findings suggest that an antioxidative and micronutrient supplementation has a remarkable benefit for IVF patients having restricted sperm parameters, in particular.
Asunto(s)
Antioxidantes/administración & dosificación , Núcleo Celular/ultraestructura , Fertilización In Vitro , Análisis de Semen , Espermatozoides/ultraestructura , Vacuolas/diagnóstico por imagen , Suplementos Dietéticos , Humanos , Infertilidad Masculina/terapia , Masculino , Especies Reactivas de Oxígeno/metabolismo , Recuento de Espermatozoides , Motilidad Espermática , UltrasonografíaRESUMEN
OBJECTIVE: To understand the correlation between normalcy of the sperm, fertilization, and early embryo development, and to establish a detailed classification scoring scale for the individual spermatozoon with the highest predictive fertilizing potential in real time during intracytoplasmic sperm injection (ICSI). DESIGN: A retrospective and analysis. SETTING: Laboratory Drouot. PATIENT(S): 27 couples with male factor infertility referred for ICSI treatment. INTERVENTION(S): Before ICSI, motile spermatozoa were scored after aspiration. MAIN OUTCOME MEASURE(S): Oocyte fertilization, embryo development and morphology, outcome of scored motile injected spermatozoa. RESULT(S): Our suggested formula is (Normal head score = 2) + (Lack of vacuole score = 3) + (Normal base score = 1) = (Total score = 6) for a morphologic "normal top" spermatozoon, calculated with the major criteria affecting the outcome of ICSI. We take into account the normalcy of head size and shape, the base of the head, and the lack of vacuoles. Our scoring of three classes of injected spermatozoa revealed a statistically significant difference in fertilization rate: 39 out of 46 (84%), 94 out of 128 (73%), and 27 out of 44 (61%), respectively. Our examination of the contribution of maternal age in correlation to sperm score revealed a distinction between oocytes originating from women younger than 30 years and oocytes from women aged 30 years and older. CONCLUSION(S): Our suggested classification provides allows the best spermatozoon to be chosen for ICSI, particularly for oocytes from women aged 30 years and older.
Asunto(s)
Blastocisto/citología , Fertilización/fisiología , Análisis de Semen/métodos , Espermatozoides/clasificación , Espermatozoides/citología , Adulto , Blastocisto/fisiología , Forma de la Célula/fisiología , Células Cultivadas , Sistemas de Computación , Desarrollo Embrionario/fisiología , Femenino , Humanos , Infertilidad Masculina/terapia , Masculino , Inducción de la Ovulación , Embarazo , Índice de Embarazo , Control de Calidad , Estudios Retrospectivos , Inyecciones de Esperma Intracitoplasmáticas/métodosRESUMEN
Spermatozoa selection at high magnification before intracytoplasmic sperm injection seems to be positively associated with pregnancy rates after day 3 embryo transfers. The aim was to demonstrate an association between the presence of vacuoles in sperm nuclei and the competence of embryos to develop to day 5. Grading of spermatozoa at x 6000-x 12,500 magnification: grade I, no vacuoles; grade II,