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Loro L Kujjo and Gloria I Perez

Maternal aging adversely affects oocyte quality (function and developmental potential) and consequently lowers pregnancy rates while increasing spontaneous abortions. Substantial evidence, especially from egg donation studies, implicates the decreased quality of an aging oocyte as a major factor in the etiology of female infertility. Nevertheless, the cellular and molecular mechanisms responsible for the decreased oocyte quality with advanced maternal aging are not fully characterized. Herein we present information in the published literature and our own data to support the hypothesis that during aging induced decreases in mitochondrial ceramide levels and associated alterations in mitochondrial structure and function are prominent elements contributing to reduced oocyte quality. Hence, by examining the molecular determinants that underlie impairments in oocyte mitochondria, we expect to sieve to a better understanding of the mechanistic anatomy of oocyte aging.

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B. A. Keel, B. W. Webster and D. K. Roberts

Summary. Ejaculates (164) were obtained from 17 donors serving on an artificial insemination by donor panel. Semen analysis was performed before and after freezing by an integrated microcomputerized system employing the multiple-exposure photography (MEP) method. Sperm count, motility, velocity, motility index (MI; product of the sperm velocity and percentage of motile spermatozoa) and motile density (MD) were determined for each ejaculate. After the initial evaluation the ejaculates were frozen in liquid nitrogen, thawed 24 h later, and assessed for post-thaw motility, velocity, MI and MD. The mean ± s.e. sperm count and volume for this group of donors was 148 ± 4 × 106/ml and 3·1 ± 0·1 ml, respectively. Mean ± s.e. values obtained from the prefreeze analysis were: motility = 64 ± 1%, velocity = 30 ± 0·4μm/sec, MI = 19 ± 0·5 μm/sec and MD = 94 ± 3 × 106/ml. Post-thaw analysis revealed a significant reduction (P < 0·01 in all values measured. Motility was reduced to 27 ± 1%, MI was reduced to 5 ± 0·3 μm/sec, and MD was reduced to 33 ± 1 × 106/ml Velocity was the least affected by cryopreservation, being reduced to 21 ± 0·5 μm/sec (P < 0·01). Cryopreservation resulted in a marked shift in the frequency distribution of sperm motility and motility index towards subnormal values while in the majority of ejaculates velocity and motile density were maintained in the normal range. Significant differences were noted amongst donors in the percentage change of the various semen measures as a result of cryopreservation. When within-subject coefficients of variation were calculated, velocity was the least variable parameter. These results indicate that, while cryopreservation results in significant reductions in the number of motile spermatozoa in the ejaculate, the velocity is only marginally reduced.

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E. A. McLaughlin, W. C. L. Ford and M. G. R. Hull

Summary. The motility characteristics of washed spermatozoa from 50 normal ejaculates were measured by time-lapse photography, before and after cryopreservation. Plasma membrane integrity was assessed by the hypo-osmotic swelling test and with the supravital fluorescent dye bisbenzimide (H33258).

There was a marked decline in the percentage of progressively motile spermatozoa after cryopreservation, the extent varying widely among donors. Results were, however, consistent between different ejaculates from the same individual. The ability of spermatozoa to survive cryopreservation could not be predicted from the properties of the semen beforehand.

The mean velocity of the spermatozoa was significantly reduced after freezing, but the lateral head displacement was unaltered. There was a significant reduction in the proportion of spermatozoa with intact plasma membranes after cryopreservation and the results of the hypo-osmotic swelling test and H33258 tests correlated closely. There was no correlation between the declines in the percentage of motile spermatozoa, or intact spermatozoa and the sperm velocity.

We conclude that membrane rupture is not the sole cause of loss of motile spermatozoa during freezing and that the decrease in the proportion of motile spermatozoa is caused, at least in part, by a separate process from that responsible for the decrease in the average swimming speed of spermatozoa.

Keywords: spermatozoa; cryopreservation; motility; membranes; semen; donor insemination; human

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A. A. Templeton, P. Van Look, R. E. Angell, R. J. Aitken, M. A. Lumsden and D. T. Baird

Summary. Volunteer women requesting laparoscopic sterilization were subjected to a fixed schedule of ovulation induction and oocyte recovery. Follicle aspiration was carried out in four groups: those to whom hCG was not administered and 12, 24 or 36 h respectively after the administration of hCG. For each group oocytes were cultured in vitro for 42 h, 30 h, 18 h and 6 h respectively, before insemination with donor spermatozoa. Oocyte recovery rates improved with longer hCG-to-recovery intervals (36% with no hCG to 81% 36 h after hCG). Although there was a slight reduction in fertilization rates when oocytes were not exposed to hCG in the follicle, normal cleavage was noted in more than 50% of oocytes in all four groups. It therefore appears that the final maturation stages of the human oocyte are not dependent on the midcycle gonadotrophin surge, provided the oocyte is matured in vitro before insemination. However, it was also evident that the fertilization rates were reduced when oocytes were removed from less mature follicles, as reflected by high androstenedione/ oestradiol ratios.

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E. A. McLaughlin, W. C. L. Ford and M. G. R. Hull

Summary. The contribution of the toxicity of glycerol–egg yolk–citrate (GEYC) cryopreservative medium to the loss of function of human spermatozoa during cryopreservation was determined by investigating the effect of mixing semen with the medium on sperm motility. The percentage of progressively motile spermatozoa, velocity (μm s−1) and lateral head displacement (μm) (mean ± sem, n = 28) were 55 ± 4·1, 47 ± 2·7, 4·4 ± 0·2 and 32 ± 3·8, 40 ± 2·5, 3·6 ± 0·25 and 15 ± 2·5, 28 ± 1·1, 2·8 ± 0·15 in suspensions of washed spermatozoa prepared from fresh, GEYC-treated and frozen–thawed semen, respectively. The variables changed only slightly after incubation for 3 h. The toxicity of GEYC did not vary significantly between samples which survived the complete freeze–thaw cycle well or very poorly. The toxicity of GEYC is responsible for about 50% of the loss of progressively motile spermatozoa during the complete cryopreservation process, but has little effect on the quality of motility. Susceptibility to GEYC does not explain observed differences in the ability of semen samples to survive freezing.

Keywords: cryopreservation; spermatozoa; motility; glycerol; donor insemination; human

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Anamaria C Herta, Francesca Lolicato and Johan E J Smitz

The currently available assisted reproduction techniques for fertility preservation (i.e. in vitro maturation (IVM) and in vitro fertilization) are insufficient as stand-alone procedures as only few reproductive cells can be conserved with these techniques. Oocytes in primordial follicles are well suited to survive the cryopreservation procedure and of use as valuable starting material for fertilization, on the condition that these could be grown up to fully matured oocytes. Our understanding of the biological mechanisms directing primordial follicle activation has increased over the last years and this knowledge has paved the way toward clinical applications. New multistep in vitro systems are making use of purified precursor cells and extracellular matrix components and by applying bio-printing technologies, an adequate follicular niche can be built. IVM of human oocytes is clinically applied in patients with polycystic ovary/polycystic ovary syndrome; related knowhow could become useful for fertility preservation and for patients with maturation failure and follicle-stimulating hormone resistance. The expectations from the research on human ovarian tissue and immature oocytes cultures, in combination with the improved vitrification methods, are high as these technologies can offer realistic potential for fertility preservation.

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Benjamin Fisch and Ronit Abir

Anti-cancer therapy, particularly chemotherapy, damages ovarian follicles and promotes ovarian failure. The only pharmacological means for protecting the ovaries from chemotherapy-induced injury is gonadotrophin-releasing hormone agonist, but its efficiency remains controversial; ovarian transposition is used to shield the ovary from radiation when indicated. Until the late 1990s, the only option for fertility preservation and restoration in women with cancer was embryo cryopreservation. The development of other assisted reproductive technologies such as mature oocyte cryopreservation and in vitro maturation of oocytes has contributed to fertility preservation. Treatment regimens to obtain mature oocytes/embryos have been modified to overcome various limitations of conventional ovarian stimulation protocols. In the last decades, several centres have begun cryopreserving ovarian samples containing primordial follicles from young patients before anti-cancer therapy. The first live birth following implantation of cryopreserved-thawed ovarian tissue was reported in 2004; since then, the number has risen to more than 130. Nowadays, ovarian tissue cryopreservation can be combined with in vitro maturation and vitrification of oocytes. The use of cryopreserved oocytes eliminates the risk posed by ovarian implantation of reseeding the cancer. Novel methods for enhancing follicular survival after implantation are presently being studied. In addition, researchers are currently investigating agents for ovarian protection. It is expected that the risk of reimplantation of malignant cells with ovarian grafts will be overcome with the putative development of an artificial ovary and an efficient follicle class- and species-dependent in vitro system for culturing primordial follicles.

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S. Samaké and L. C. Smith

The methods used to achieve blastomere cell cycle synchronization in embryos used as nuclear donors during embryo reconstruction have been largely unsuccessful. The aim of this study was to determine the reliability of 6-dimethylaminopurine (6-DMAP), an inhibitor of maturation promoting factor, to halt and to synchronize blastomere division in cleavage stage bovine embryos. A second goal was to assess its reversibility and toxicity in vitro. Eight-cell stage embryos obtained at 58 h after insemination were treated with several concentrations of 6-DMAP for 12 h. Treated embryos were assessed for cleavage arrest, chromatin morphology, DNA synthesis, histone H1 and scored for blastocyst formation and for hatching rate. They were subsequently fixed and the number of nuclei counted. Complete arrest of cell division was observed at concentrations of 3 mmol 6-DMAP l−1 and above. At these concentrations, interphase nuclei in arrest were noticeably larger compared with interphase nuclei of eight-cell control embryos. Removal from 6-DMAP led to release from cleavage arrest and was followed by synchronized mitosis, histone H1 kinase deactivation and re-entry into interphase within 4–5 h. Twenty-nine per cent of interphase nuclei were synthesizing DNA at the end of the 12 h treatment as indicated by BrdU analysis. At 2 h after removal from 6-DMAP, an abrupt decrease to 9% BrdU-positive nuclei was observed followed by an increase to 39% by 6 h and a decrease to 28% at 10 h. The ability of treated embryos to reach the blastocyst stage in vitro and the number of cells per blastocyst were reduced. These results indicate that 6-DMAP can reversibly arrest and synchronize cleavage to the fifth cell cycle in eight-cell bovine embryos. Although a decrease was observed in the proportion of blastocysts obtained after treatment, it is concluded that 6-DMAP is a useful tool for synchronization studies requiring donor nuclei at metaphase before fusion to recipient oocyte.

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J. M. Sreenan and T. McDonagh

Summary. In Exp. 1, embryo survival rates of 45 and 47% were recorded after artificial insemination and ipsilateral transfer respectively. In Exp. 2, pregnancy rates of 62 and 60% were recorded after artificial insemination and contralateral transfer to inseminated recipients respectively. In this experiment the contralateral transferred embryo survival rate was 44%. Transferred embryo survival was lower overall when donors and recipients were out of phase by 1 day than when exactly synchronous.

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K Hardy, C Wright, S Rice, M Tachataki, R Roberts, D Morgan, S Spanos and D Taylor

The advent of human in vitro fertilization (IVF) over 30 years ago has made the oocyte and preimplantation embryo uniquely accessible. This accessibility has given rise to new micromanipulation techniques, such as intracytoplasmic sperm injection for treatment of male infertility, as well as embryo biopsy for preimplantation diagnosis of both genetic disease and aneuploidy, a major cause of early embryo demise and miscarriage. In the UK, average pregnancy rates after IVF and embryo transfer are < 25%, even after transfer of several embryos. Unfortunately, a third of these pregnancies involve multiple gestations. Research is currently focusing on methods to improve IVF success rates while reducing twin and triplet pregnancies and their associated increased morbidity and mortality. One approach is to develop screening methods to identify the most viable embryos, so that transfer of fewer healthy embryos will result in a higher proportion of singleton pregnancies. Screening methods include optimizing culture conditions for prolonged culture and selection of viable blastocysts for transfer, or embryo biopsy and aneuploidy screening. Assisted reproduction is also increasingly important in other branches of medicine: survival rates for cancer sufferers are improving continually and there is now a significant need for approaches to preserve fertility after sterilizing chemo-and radiotherapy treatment. Techniques for cryopreserving male and female gametes or gonadal tissue are being developed, although systems to grow and mature these gametes are in their infancy. Finally, there are also concerns regarding the safety of these new assisted reproductive technologies.