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Follicle development is the most crucial step toward female fertility and is controlled mainly by follicle-stimulating hormone (FSH). In ovarian granulosa cells (GCs), FSH activates protein kinase A by increasing 3′,5′-cyclic adenosine 5′-monophosphate (cAMP). Since cAMP signaling is impinged in part by salt-inducible kinases (SIKs), we examined the role of SIKs on the regulation of FSH actions. Here, we report that SIKs are essential for normal ovarian function and female fertility. All SIK isoforms are expressed in human and rodent GCs at different levels (SIK3>SIK2>SIK1). Pharmacological inhibition of SIK activity potentiated the stimulatory effect of FSH on markers of GC differentiation in mouse, rat, and human GCs and estradiol production in rat GCs. In humans, SIK inhibition strongly enhanced FSH actions in GCs of patients with normal or abnormal ovarian function. The knockdown of SIK2, but not SIK1 or SIK3, synergized with FSH on the induction of markers of GC differentiation. SIK inhibition boosted gonadotropin-induced GC differentiation in vivo, while the genomic knockout of SIK2 led to a significant increase in the number of ovulated oocytes. Conversely, SIK3 knockout females were infertile, FSH insensitive, and had abnormal folliculogenesis. These findings reveal novel roles for SIKs in the regulation of GC differentiation and female fertility, and contribute to our understanding of the mechanisms regulated by FSH. Furthermore, these data suggest that specific pharmacological modulation of SIK2 activity could be of benefit to treat ovulatory defects in humans and to increase the propagation of endangered species and farm mammals.

Cryopreservation is one of the keystones in clinical infertility treatment. In particular vitrification has become a well-established and widely used routine procedure that has allowed important expansion of therapeutic strategies when IVF is used to treat infertility. Vitrification of human blastocysts allows us to maximize the potential for conception from any single in vitro fertilization cycle and prevents wastage of embryos. The technology may even be used to eliminate fresh embryo transfers for reasons of convenience, uterine receptivity, fertility preservation, preimplantation genetic diagnosis, or emergency management. In this chapter, the application of vitrification technology for cryopreserving human blastocysts will be revealed through step-by-step protocols. The results that are presented using the described protocols underscore the robustness of the vitrification technology for embryo cryopreservation.

Cryopreservation is one of the keystones in clinical infertility treatment. In particular vitrification has become a well-established and widely used routine procedure that has allowed important expansion of therapeutic strategies when IVF is used to treat infertility. Vitrification of human blastocysts allows us to maximize the potential for conception from any single in vitro fertilization cycle and prevents wastage of embryos. The technology may even be used to eliminate fresh embryo transfers for reasons of convenience, uterine receptivity, fertility preservation, preimplantation genetic diagnosis, or emergency management. In this chapter, the application of vitrification technology for cryopreserving human blastocysts will be revealed through step-by-step protocols. The results that are presented using the described protocols underscore the robustness of the vitrification technology for embryo cryopreservation.

To analyze cases in which no sperm could be identified after thawing among cryopreserved samples of rare or very low concentrations of sperm.Retrospective, single-institution, cross-sectional.Male infertility clinic.We identified couples that underwent intracytoplasmic sperm injection (ICSI) with the use of either ejaculated or testicular cryopreserved-thawed sperm. Inclusion criteria were men with100,000 total ejaculated sperm or men with azoospermia due to spermatogenic dysfunction who underwent microsurgical testicular sperm extraction with similarly low pre-cryopreservation sperm counts. Pre-cryopreservation specimens were categorized as "rare sperm only" (Group 1) or100,000 total sperm (group 2). "Rare sperm only" applied to cases in which only one to three sperm were identified in a search of20 high-power fields.None.Cases in which no sperm were able to be found post-thaw (i.e., complete cellular loss) for use at the time of a programmed IVF cycle.We analyzed 55 men (83 ICSI cycles). There were five ICSI cycles (6.0%) among five different couples in which no sperm could be identified post-thaw. Of these, four cases were from group 1 (8.5%) and one from group 2 (2.8%). Complete cellular loss occurred in 5.8% of testicular sperm samples and 7.1% of ejaculated sperm samples. There were no statistical associations between the ability to locate sperm post-thaw and the pre-cryopreservation parameters or sperm source.Failure to retrieve any sperm after thawing of rare or very low concentrations of cryopreserved sperm is an infrequent event and largely limited to those patients with rare quantities of sperm.

Transfer of blastocyst-stage embryos has been shown to increase pregnancy rates while allowing for improved selection of potentially viable embryos. At this late stage of development, lower numbers of embryos can be transferred, resulting in less high-order multiple pregnancies and increased implantation rates. Between January 2004 and February 2009, 8449 blastocysts from 2453 patients were vitrified. After 1398 vitrified embryo transfers (VET) of both day-5 and day-6 blastocysts with a mean patient age of 34.6 +/- 5.0 years, the study centre has seen a survival rate of 96.3% (2730/2835), an implantation rate of 29.4% and a clinical pregnancy rate per VET of 42.8% (599 pregnancies/1398 warmed embryo transfers). After more than 5 years of vitrifying blastocysts, the perinatal outcome was, from 348 deliveries with vitrified blastocysts, the births of 431 babies (202 boys and 229 girls). One of the benefits of blastocyst vitrification is that it can be undertaken on a more flexible basis by laboratory staff. Also, vitrification may allow individual blastocysts to be cryopreserved at their optimal stage of development and expansion.

Since its introduction in 1978, IVF has registered impressive gains in implantation rate and live birth rate. Some of these successes have been due to improvements in culture systems and in vitro culture media, as well as more advanced training and proficiency of embryologists. In the early days of IVF, it was common to transfer two or even more embryos per patient regardless of age or embryo quality. As a result, the rate of higher-order multiple gestation increased dramatically. Multiple gestations are high-risk pregnancies and should be avoided for several reasons. The only truly effective means to avoid multiple gestations is to transfer only one embryo at a time. However, patient autonomy represents a particular challenge especially given an overwhelming desire for twin pregnancies after IVF. Therefore, patient education is vital to encourage patients to find an elective single-embryo transfer (eSET), an acceptable treatment option. This decision can be supported by a having a successful cryopreservation program available for supernumerary embryos, as this approach can help maintain satisfactory reproductive outcomes. In this chapter, the application of vitrification technology for cryopreservation of human blastocysts (as a keystone in a successful single-embryo transfer program) is described in step-by-step protocols. The results here illustrate the robustness of current vitrification technology and can be persuasive in encouraging patients to transfer only one embryo at a time.

Objective To evaluate implantation of day 5 and day 6 vitrified and slow-frozen blastocysts. Design Retrospective analysis comparing two cryopreservation techniques. Setting Private IVF clinic. Patient(s) Five hundred eight cryopreserved embryo transfer candidates. Intervention(s) Supernumerary day 5 and day 6 blastocysts were vitrified or slow-frozen and transfered after warming or thawing. Main Outcome Measure(s) Comparison of two cryopreservation techniques with respect to survival rate, implantation, and pregnancy rates of day 5 and day 6 blastocysts. Result(s) In 254 vitrified transfer cycles, survival, embryonic implantation, and clinical pregnancy rates for day 5 blastocysts were 95.9%, 33.4%, 48.7%, respectively, and for day 6 blastocysts 97.5%, 25.9%, 42.8%. In 254 slow-frozen transfer cycles, survival, embryonic implantation, and clinical pregnancy rates for day 5 blastocysts were 91.4%, 29.6%, 42.8%, respectively, and for day 6 blastocysts 94.8%, 28.2%, 43.1%. Overall there was a slightly, but not significantly, higher outcome regarding implantation and clinical pregnancy with the use of day 5 blastocysts (31.3% and 45.4%, respectively) versus day 6 blastocysts (26.7, and 42.9%, respectively). Conclusion(s) Vitrification technique yields the same implantation and pregnancy rate as slow-frozen blastocyst transfers. Slow growing embryos can be cryopreserved on day 6, because they yield a satisfactory survival, implantation, and pregnancy rate.

Cryopreservation is one of the keystones in clinical infertility treatment. Especially vitrification has become a well-established and widely used routine procedure that allows important expansion of therapeutic strategies when in vitro fertilization (IVF) is used to treat infertility. Vitrification of human blastocysts allows us to maximize the potential for conception from any one in vitro fertilization cycle and prevents wastage of embryos. This goes even further toward to best utilize a patient's supernumerary oocytes after retrieval, maximizing the use of embryos from a single stimulation cycle. The technology may even be used to eliminate fresh embryo transfers for reasons of convenience, uterine receptivity, fertility preservation, preimplantation genetic diagnosis, or emergency management. In this chapter, the application of vitrification technology for cryopreserving human blastocyst will be revealed through step-by-step protocols. The results that are presented using the described protocols underscore the robustness of the vitrification technology for embryo cryopreservation.

Cryopreservation is one of the keystones in clinical infertility treatment. Especially vitrification has become a well-established and widely used routine procedure that allows important expansion of therapeutic strategies when in vitro fertilization (IVF) is used to treat infertility. Vitrification of human blastocysts allows us to maximize the potential for conception from any one in vitro fertilization cycle and prevents wastage of embryos. This goes even further toward to best utilize a patient's supernumerary oocytes after retrieval, maximizing the use of embryos from a single stimulation cycle. The technology may even be used to eliminate fresh embryo transfers for reasons of convenience, uterine receptivity, fertility preservation, preimplantation genetic diagnosis, or emergency management. In this chapter, the application of vitrification technology for cryopreserving human blastocyst will be revealed through step-by-step protocols. The results that are presented using the described protocols underscore the robustness of the vitrification technology for embryo cryopreservation.

During the last few years, cryopreservation has become a relevant addition to therapeutic concepts in reproductive medicine. New data and publications have made it difficult to maintain an overview of all of the new developments and their results. The focus of interest more recently, especially with the cryopreservation of human oocytes and human ovarian tissue, has been vitrification as an interesting alternative to slow freezing methods. Even though studies investigating the slow freezing of human mature oocytes have resulted in very different survival rates, it could be an option for donor oocyte programs, in the case of threatened ovarian loss or when there is an objection to embryo freezing. An optimal freezing protocol and later use of thawed human ovarian tissue is still a point of discussion. There are encouraging results regarding different kinds of autotransplantation, and recently the first birth after orthotopic autotransplantation of cryopreserved/thawed human ovarian tissue was described in the literature. Independent of any objections to cryopreservation in general, vitrification is a potential and effective alternative to conventional slow cryopreservation, especially for oocytes and embryos. Vitrification might be also be an option for human ovarian tissue; however this is only in its infancy and requires much additional investigation. Our article discusses new trends and results of actual studies regarding these issues.

Embryo cryopreservation has become an ethical necessity due to the way human in vitro fertilization (IVF) infertility therapy has developed. Limited embryonic implantation has by necessity driven IVF therapy to adopt ways to maximize the harvest of oocytes following ovarian hyperstimulation with its attendant risks. Collection of more oocytes has allowed more embryos to be generated to compensate for poor embryonic viability, often leading to transfer of multiple embryos to increase per transfer pregnancy rates. In an era of improving embryonic viability and prevailing trend toward single embryo transfers, production of excessive numbers of surplus embryos appears increasingly inappropriate. At which stage embryo cryopreservation can be undertaken most effectively remains controversial. Embryo cryopreservation nevertheless represents the current solution to the problem of excessive embryo production, but inherently raises ethical concerns for certain couples uncomfortable with what they might perceive to be "experimental" cryostorage, who in extreme circumstances may even choose to limit the number of oocytes inseminated to obviate the production of spare embryos. On a more practical level, cryostored embryos are co-owned by two people who may separate, and as such the embryos then face an uncertain fate, commonly decided in courts of law. Oocyte cryopreservation, if consistent and successful, offers a way to avoid the above complications of routine IVF therapy. Oocytes may need to be cryostored in the event of unforeseen non-production of sperm during IVF therapy, allowing a more measured consideration of donor sperm use or other means of sperm retrieval. Beyond IVF for infertility therapy using a couple's own gametes, oocyte cryopreservation provides a wonderful opportunity to optimize donor oocyte cryo-banking, reducing costs and improving convenience. Meanwhile, frozen embryo donation is an approach that many couples are uncomfortable with, and allows only for retrospective donor screening, and less gametic choice. Advances in ovarian tissue cryopreservation will probably provide the best approach for long term storage of female gametes for women wishing to elect to prolong their reproductive potential, or prior to cancer therapy. However, improved consistency with vitrification technology through standardization of protocols and cell-carrying systems is bringing routine single oocyte cryostorage, at all stages of egg maturity, closer to reality. This, coupled with in vitro maturation, will aid development of oocyte collection protocols using minimal amounts of gonadotropins. All of which will help drive IVF programs to consider cryostorage of excess oocytes and not embryos, inseminating post-thaw/warming only a limited number of oocytes at any one time, in anticipation of the need for only one or two embryos at transfer. The question then is how close are we to being able to provide routine clinical application of human oocyte cryostorage, and when will it be appropriate?

The target of any cryopreservation procedure should be to ensure high survival rates of living cells after thawing. Two important parameters determine the success of any cryopreservation protocol: the manner in which cells regain equilibrium in response to cooling, and the speed of freezing (cooling rate). Slow-rate freezing protocols result in the formation of ice crystals during cooling and warming. Vitrification, in which high cooling rates in combination with a high concentration of cryoprotectant are used, does not produce any ice crystals during cooling and warming. However, there is a practical limit to the attainable cooling speed, and also a biological limit to the concentration of cryoprotectant tolerated by the cells during vitrification. Although post-warming survival depends on the species, the developmental stage and the quality of the embryos being vitrified, it seems clear that vitrification methods are increasingly successful and might be a better method than slow cooling procedures in the field of cryobiology. Many of the potential problems and benefits underlying vitrification as a method of choice for embryo cryopreservation in clinical embryology will be discussed in this review.

Vitrification is the achievement of a “state of suspended animation” wherein molecular translational motions are arrested without structural reorganization of the liquid. Vitrification protocols are relatively simple for the practitioner, potentially faster, and inexpensive. The process relies on the placement of the cell in a very small volume of vitrification medium that must be cooled at extreme rates, which are difficult to obtain in regular enclosed cryostraws or vials. The growing number of reports of successfully completed pregnancies following vitrification makes for an extremely encouraging future for its wider adoption, the procedure appears safe, is easily assimilated into a busy IVF laboratory, and promises improved patient outcomes when used correctly. For blastocyst vitrification, a closed system is effective for achieving high implantation and pregnancy rates as seen with fresh embryo transfers. With this increased use of vitrification in human-assisted reproduction, we should see an evolution of the vitrification process as it is fine-tuned to clinical needs. Thus, the future should bring higher levels of clinical efficiency, utilization, and universal acceptance.

During embryo vitrification, it is advisable that cooling and storage should occur in a carrier device in which there is complete separation of the embryos from liquid nitrogen to ensure asepsis. The consequence of a reduction in the cooling rate resulting from the heat-insulating barrier aseptic devices has to be counteracted by gradually increasing intracellular concentrations of cryoprotectants without inducing a toxic effect. Blastocysts originating from couples with male and/or female factor infertility (group 1) or from oocyte donors (group 2) or from in-vitro matured oocytes (group 3) were gradually exposed to increasing concentrations of dimethylsulphoxide/ethylene glycol (5/5%, 10/10% and 20/20%) before aseptic vitrification using a specially designed carrier (VitriSafe), a modification of the open hemi-straw plug device. A total of 120 aseptic vitrification/warming cycles were performed in group 1, 91 in group 2 and 22 in group 3. Survival rates before embryo transfer, ongoing pregnancy and implantation rates were as follows: for group 1, 73, 43 and 26%; for group 2, 88, 53 and 34%; and for group 3, 69, 50 and 38%, respectively. In spite of reduced cooling rates due to aseptic vitrification conditions, a three-step exposure to cryoprotectant solutions protects the embryos effectively from cryo-injuries and guaranties high survival rates.

Vitrification in Assisted Human Reproduction: A Users Manual and Trouble-Shooting Guide - Libros de Medicina - Fertilidad y embarazo - 120,71

administered as a 4 hour prime-constant rate infusion. Blood samples were obtained at 3, 3.5, 3.75 and 4 hours. After sacrifice, germinal vesicle stage oocytes were isolated and denuded. The rate of appearance of each amino acid being traced was calculated using the tracer dilution method. RESULTS: Dietary protein restriction caused differential responses in the kinetics and concentrations of plasma serine and glycine, and folate supplementation did not ameliorate the observed responses. The total flux of serine, a methyl group donor, was higher in the LP and LPF groups (P

Objective To perform preimplantation genetic diagnosis (PGD) with 1.48-μm infrared diode laser assistance during embryo biopsy for two patients undergoing IVF. Design Case reports. Setting Private ART laboratory. Patient(s) Two couples undergoing IVF for infertility therapy, both of whom had previously delivered offspring afflicted with spinal muscular atrophy (type 1) after IVF therapy, and who underwent subsequent cycles of IVF coupled with PGD to screen for this disorder. Intervention(s) Two individual IVF cases involving intracytoplasmic sperm injection (ICSI), embryo biopsy with laser assistance, and PGD. Main outcome measure(s) The ease and apparent safety of human embryo biopsy using a 1.48-μm infrared laser for partial zona pellucida (ZP) dissection to assist with embryo blastomere biopsy was evaluated. Result(s) Both couples were deemed to have some unafflicted embryos for transfer on the fifth day of development after blastomere biopsy in conjunction with PGD. Patient A had a singleton pregnancy and delivered a healthy normal singleton male. Patient B had a twin pregnancy; however, one twin was spontaneously lost at 10 weeks but she ultimately delivered a healthy normal singleton male. Conclusion(s) These successful outcomes help to demonstrate the efficacy and safety of laser-assisted embryo biopsy to facilitate PGD screening.

The purpose of this study was to demonstrate the safety and efficacy of vitrification of human pronuclear stage (PN) embryos in the human assisted reproduction laboratory. Using single pronucleate (1PN) and three pronucleate (3PN) zygotes, the impact of vitrification in the Flexipet denuding pipette (FDP) as a carrier was assessed in terms of survival, embryonic development and blastocyst formation when compared according to the PN number, and unvitrified controls. A total of 65 1PN and 152 3PN zygotes were vitrified; after warming 82% (53/65) of 1PN and 90% (137/152) of 3PN survived. The overall percentage of warmed zygotes (1PN and 3PN) that cleaved and reached 2-cell stage did not differ (χ 2 ; P = 0.32) from the control groups (77%; 147/190 versus 85%; 115/136). In addition, when the cleavage behaviour was examined on day 3 for ≥4-cell stage, no significant differences (χ 2 ; P = 0.95) were observed between the vitrified group and the unvitrified control groups (74%; 109/147 versus 77%; 89/115). Comparing the developmental potential up to cavitation and blastocyst formation on day 5, the overall outcome of the vitrified PN was 31% compared with 33% for the controls (χ 2 ; P = 0.76). The simple vitrification protocol used in this study, and these data highlight the usefulness of vitrification using FDP as a consistent and effective cryopreservation method for pronuclear zygotes, and a suitable alternative to slow cryopreservation protocols.

As early as 1985, ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification was reported in an attempted alternative approach to cryostorage. Since then, vitrification techniques have entered more and more the mainstream of animal reproduction as an alternative cryopreservation method to traditional slow-cooling/rapid-thaw protocols. In addition, the last few years have seen a significant resurgence of interest in the potential benefits of vitrification protocols and techniques in human-assisted reproductive technologies. The radical strategy of vitrification results in the total elimination of ice crystal formation, both within the cells being vitrified (intracellular) and in the surrounding solution (extracellular). The protocols for vitrification are very simple. They allow cells and tissue to be placed directly into the cryoprotectant and then plunged directly into liquid nitrogen. To date, however, vitrification as a cryopreservation method has had very little practical impact on human-assisted reproduction, and human preimplantation embryo vitrification is still considered to be largely experimental. Besides the inconsistent survival rates that have been reported, another problem is the wide variety of different carriers and vessels that have been used for vitrification. Second, many different vitrification solutions have been formulated, which has not helped to focus efforts on perfecting a single approach. On the other hand, the reports of successfully completed pregnancies following vitrification at all preimplantation stages is encouraging for further research and clinical implementation. Clearly, however, attention needs to be paid to the inconsistent survival rates following vitrification.