Preservation Of Embryos Research Articles

Malaria-Induced Murine Pregnancy Failure: Distinct Roles for IFN-γ and TNF

Although an important role for excessive proinflammatory cytokines in compromise of pregnancy has been established, an immunological basis for malaria-induced fetal loss remains to be demonstrated. In this study, the roles of IFN-gamma and TNF in Plasmodium chabaudi AS-induced fetal loss in mice were directly investigated. Pregnant IFN-gamma(-/-) mice experienced a more severe course of infection compared with intact C57BL/6 mice, characterized by high parasitemia, severe anemia, and marked weight loss. However, fetal loss was delayed in these mice relative to intact controls. Because IFN-gamma(-/-) mice exhibited sustained levels of plasma TNF, the role of this cytokine was examined. Whereas splenic tnf expression in C57BL/6 mice was highest 3 days before peak parasitemia, increased placental expression relative to uninfected mice was sustained, indicating that locally produced TNF may be important in malaria-induced pregnancy failure. Indeed, Ab neutralization of TNF resulted in preservation of embryos until day 12 of gestation, at which point all embryos were lost in untreated mice. Histological analysis revealed that TNF ablation preserved placental architecture whereas placentae from untreated infected mice had widespread hemorrhage and placental disruption, with fibrin thrombi in some maternal blood sinusoids. Consistent with a role for cytokine-driven thrombosis in fetal loss, expression of procoagulant tissue factor was significantly increased in the placentae of infected C57BL/6 mice but was reduced in mice treated with anti-TNF Ab. Together, these results suggest that IFN-gamma contributes to malaria-induced fetal loss and TNF is a critical factor that acts by inducing placental coagulopathy.

Economic downturn has resulted in a corresponding increase in patients choosing to discontinue frozen embryo storage

OBJECTIVE: Patients often maintain supernumerary cryopreserved embryos after reaching family building goals. Indeed, some purposely postpone the emotionally difficult decision to select a final disposition for these embryos, despite continued storage costs. In times of economic stress, preservation of embryos may be an unaffordable expense. This study was to evaluate the historic, worldwide economic downturn on local embryo storage demographics. DESIGN: Retrospective data analysis at a large academic IVF center. MATERIALS AND METHODS: Records of stored embryos from October 2006 to March 2009 were reviewed to determine the frequency of disposition options: donation for research or to another couple, or discard. Storage time was evaluated for those discontinuing storage. Comparisons were drawn using χ and Kruskal-Wallis tests. RESULTS: The number of patients discontinuing storage was similar when semiannual intervals were compared from October 2006 through September 2008 (p=0.08). Significantly more patients disposed of frozen embryos from October 2008 to March 2009 than preceding intervals. Duration of storage prior to removal did not differ among the periods analyzed.Tabled 1Storage PeriodsPatients Discontinuing Embryo StorageDays in Storage (median±SD)Oct 2006 - March 200732(3.9%)679 ± 371April - Sept 200730(3.4%)590 ± 494Oct 2007 - March 200841(4.3%)651 ± 549April - Sept 200824(2.3%)774 ± 627Oct 2008 - March 200978(7.0%)710 ± 589p<0.0001p=0.51 Open table in a new tab CONCLUSIONS: A sudden change in embryo storage habits is more likely the result of external influences rather than a paradigm shift in patient attitudes towards this emotionally charged issue. The historic economic crisis occurred simultaneously with an increased frequency of requests to dispose of cryopreserved embryos. Extreme economic conditions have forced patients to address an issue they have historically chosen to postpone when financially viable.

Exceptional preservation of embryos in the actinopterygian Saurichthys from the Middle Triassic of Monte San Giorgio, Switzerland

New excavations in the famous Monte San Giorgio area, close to the historical sites where the Cassina beds crop out (Lower Meride Limestone, Early Ladinian), revealed an interesting vertebrate fauna, including several specimens of the actinopterygian fish Saurichthys. Among the prepared specimens, three specimens of Saurichthys contain embryos, and in one example the embryos are preserved with traces of the soft parts, most probably phosphatized musculature. This discovery is of particular relevance because fossil embryos with preserved soft parts are extremely rare. In addition, although viviparity had previously been described for Saurichthys, the new specimens are rather better evidence and add further support to this reproductive mode.

Embryo fossilization is a biological process mediated by microbial biofilms

Fossilized embryos with extraordinary cellular preservation appear in the Late Neoproterozoic and Cambrian, coincident with the appearance of animal body fossils. It has been hypothesized that microbial processes are responsible for preservation and mineralization of organic tissues. However, the actions of microbes in preservation of embryos have not been demonstrated experimentally. Here, we show that bacterial biofilms assemble rapidly in dead marine embryos and form remarkable pseudomorphs in which the bacterial biofilm replaces and exquisitely models details of cellular organization and structure. The experimental model was the decay of cleavage stage embryos similar in size and morphology to fossil embryos. The data show that embryo preservation takes place in 3 distinct steps: (i) blockage of autolysis by reducing or anaerobic conditions, (ii) rapid formation of microbial biofilms that consume the embryo but form a replica that retains cell organization and morphology, and (iii) bacterially catalyzed mineralization. Major bacterial taxa in embryo decay biofilms were identified by using 16S rDNA sequencing. Decay processes were similar in different taphonomic conditions, but the composition of bacterial populations depended on specific conditions. Experimental taphonomy generates preservation states similar to those in fossil embryos. The data show how fossilization of soft tissues in sediments can be mediated by bacterial replacement and mineralization, providing a foundation for experimentally creating biofilms from defined microbial species to model fossilization as a biological process.

A comparison of slow freeze and vitrification in frozen embryo transfer in vitro fertilization

OBJECTIVE: Embryo cryopreservation enhances In Vitro Fertilization (IVF) success by allowing the preservation of additional embryos for use in subsequent cycles. Cryopreservation has previously utilized a slow freeze technique. We have used the rapid vitrification technique over the past three years. While vitrification is technically easier, there is limited data on its efficacy. Our objective was to determine whether vitrification is associated with equal or increased success rate in IVF.DESIGN: Retrospective study of Frozen Embryo Transfer (FET) cycle outcomes comparing slow freeze and vitrification techniques performed in a university hospital based IVF program.MATERIALS AND METHODS: We performed 158 cycles of FET between 2003-2007. We thawed 55 sets of slow freeze embryos and 103 sets of vitrified embryos. After a lupron induced or spontaneous menses, incremental dosing of estrace was used in our standard protocol, with progesterone supplementation added prior to day 3-5 of transfer. The slow freeze technique was performed with propylene glycol and sucrose solutions added incrementally. Embryos were incrementally cooled in the Biogenetic planar from 20°C to −30°C over 100 minutes and dropped in liquid nitrogen at −196° C. We thawed slow freeze embryos in a 37° C water bath for 5 minutes and rehydrated them incrementally in propylene glycol and sucrose solutions over 10 minutes. Vitrification technique was performed by dehydrating embryos in Vitrification Solution over 10 minutes. They were rapidly loaded into a straw and plunged into liquid nitrogen −196° C. Vitrified embryos were thawed by placing the straw in a 37° C water bath for 3 seconds followed by a series of post thaw solutions over 13 minutes. T-test and chi square analyses were performed with Systat 10.1.RESULTS: In slow freeze vs. vitrification; there were no differences in the patients' age (34.1 vs. 34.2), FSH (5.8 vs 5.5) or type of infertility. In slow freeze vs. vitrification, embryo survival was 73.1% vs. 88.8% (p< 0.001), chemical pregnancy rate was 32.7% vs. 52.4% (p= 0.018), clinical pregnancy rate was 27.2% vs. 46.6% (p= 0.018), and ongoing pregnancy rate was 20.0% vs. 36.9% (p=0.029). The spontaneous abortion rate was 33.3% vs. 26.0%, not significantly different.CONCLUSIONS: Vitrification was associated with a higher survival rate, and a higher clinical and ongoing pregnancy rate. This supports the continued use of the more rapid and simple vitrification technique. OBJECTIVE: Embryo cryopreservation enhances In Vitro Fertilization (IVF) success by allowing the preservation of additional embryos for use in subsequent cycles. Cryopreservation has previously utilized a slow freeze technique. We have used the rapid vitrification technique over the past three years. While vitrification is technically easier, there is limited data on its efficacy. Our objective was to determine whether vitrification is associated with equal or increased success rate in IVF. DESIGN: Retrospective study of Frozen Embryo Transfer (FET) cycle outcomes comparing slow freeze and vitrification techniques performed in a university hospital based IVF program. MATERIALS AND METHODS: We performed 158 cycles of FET between 2003-2007. We thawed 55 sets of slow freeze embryos and 103 sets of vitrified embryos. After a lupron induced or spontaneous menses, incremental dosing of estrace was used in our standard protocol, with progesterone supplementation added prior to day 3-5 of transfer. The slow freeze technique was performed with propylene glycol and sucrose solutions added incrementally. Embryos were incrementally cooled in the Biogenetic planar from 20°C to −30°C over 100 minutes and dropped in liquid nitrogen at −196° C. We thawed slow freeze embryos in a 37° C water bath for 5 minutes and rehydrated them incrementally in propylene glycol and sucrose solutions over 10 minutes. Vitrification technique was performed by dehydrating embryos in Vitrification Solution over 10 minutes. They were rapidly loaded into a straw and plunged into liquid nitrogen −196° C. Vitrified embryos were thawed by placing the straw in a 37° C water bath for 3 seconds followed by a series of post thaw solutions over 13 minutes. T-test and chi square analyses were performed with Systat 10.1. RESULTS: In slow freeze vs. vitrification; there were no differences in the patients' age (34.1 vs. 34.2), FSH (5.8 vs 5.5) or type of infertility. In slow freeze vs. vitrification, embryo survival was 73.1% vs. 88.8% (p< 0.001), chemical pregnancy rate was 32.7% vs. 52.4% (p= 0.018), clinical pregnancy rate was 27.2% vs. 46.6% (p= 0.018), and ongoing pregnancy rate was 20.0% vs. 36.9% (p=0.029). The spontaneous abortion rate was 33.3% vs. 26.0%, not significantly different. CONCLUSIONS: Vitrification was associated with a higher survival rate, and a higher clinical and ongoing pregnancy rate. This supports the continued use of the more rapid and simple vitrification technique.

Vitrification of in vitro matured oocytes of Mangalica and Large White pigs

The breeding of Mangalica, a native pig breed in Hungary, had been started in 1833, but this pig breed almost became extinct in Hungary in the past decades. In 1991, the number of sows was only 200. Although in these days the existing Mangalica population consists of more than 6000 animals representing different colour variations, the preservation of this traditional pig breed is still very important. Vitrification is a potential tool for the preservation of gametes and embryos of these animals. The aim of this study was to investigate the effects of vitrification on the developmental competence of Mangalica (M) and Large White (LW) oocytes following fertilisation. The oocytes were vitrified by the Open Pulled Straw (OPS) method using different concentrations of ethylene glycol and dimethyl sulphoxide as cryoprotectants. After rehydration the oocytes underwent in vitro fertilisation; the resultant zygotes were then cultured in vitro for four days to assess embryonic development. In the first experiment, in vitro maturation of M and LW oocytes was compared. No significant difference was observed in the nuclear maturation rate of LW and M oocytes. In the second experiment, the sensitivity of oocytes to vitrification was examined by evaluating oocyte morphology after thawing. A higher percentage of LW oocytes showed normal morphology compared to M oocytes, indicating that Mangalica oocytes are more sensitive to cryoprotectants than Large White oocytes. After warming and in vitro fertilisation, more than 50% of the oocytes started embryonic development and by the end of the incubation period morula stage embryos had developed in both groups. The results show that the OPS vitrification technique is well suited to preserve Mangalica oocytes and from these oocytes morula embryos can be produced.

Deciphering the fossil record of early bilaterian embryonic development in light of experimental taphonomy

Experimental analyses of decay in a tunicate deuterostome and three lophotrochozoans indicate that the controls on decay and preservation of embryos, identified previously based on echinoids, are more generally applicable. Four stages of decay are identified regardless of the environment of death and decay. Embryos decay rapidly in oxic and anoxic conditions, although the gross morphology of embryos is maintained for longer under anoxic conditions. Under anoxic reducing conditions, the gross morphology of the embryos is maintained for the longest period of time, compatible with the timescale required for bacterially mediated mineralization of soft tissues. All four stages of decay were encountered under all environmental conditions, matching the spectrum of preservational qualities encountered in all fossil embryo assemblages. The preservation potential of embryos of deuterostomes and lophotrochozoans is at odds with the lack of such embryos in the fossil record. Rather, the fossil record of embryos, as sparse as it is, is dominated by forms interpreted as ecdysozoans, cnidarians, and stem-metazoans. The dearth of deuterostome and lophotrochozoan embryos may be explained by the fact that ecdysozoans, at least, tend to deposit their eggs in the sediment rather than through broadcast spawning. However, fossil embryos remain very rare and the main controlling factor on their fossilization may be the unique conspiracy of environmental conditions at a couple of sites. The preponderance of fossilized embryos of direct developers should not be used in evidence against the existence of indirect development at this time in animal evolutionary history.

Cryopreservation and vitrification: recent advances in fertility preservation technologies

Over the last half the 20th Century, reproductive medicine has become a critically important branch of modern medical science. Fertility preservation is a vital branch of reproductive medicine and involves the preservation of gametes (sperm and oocytes), embryos, and reproductive tissues (ovarian and testicular tissues) for use in artificial reproduction. This technology gives millions of people suffering from reproductive ailments, cancer patients who have their reproductive functions destroyed by therapy (chemotherapy and radiation) and people undergoing sterilization, a chance to conceive. The most common fertility preservation technique is cryopreservation, which involves freezing cells and tissues at cryogenic temperatures. Cryopreserved cells and tissues can endure storage for centuries with almost no change in functionality or genetic information, making this storage method highly attractive. However, developing efficient cryopreservation techniques is challenging, as both freezing and thawing exposes cells to severe stresses, potentially causing cell death. There are two major techniques for cryopreservation: freeze–thaw processes and vitrification. The major difference between them is the total avoidance of ice formation in vitrification. The use of both theoretical models that describe cell response to freezing and thawing, and experimental investigations of freezing behavior, has led to the development of successful freeze–thaw and vitrification procedures for a number of cell types. Among reproductive cells, there exist efficient cryopreservation techniques for spermatozoa and embryos. Oocytes, however, present significant hurdles in achieving successful cryopreservation, primarily due to their sensitive microtubule structure. Recently, cryopreservation of ovarian and testicular tissues has been investigated with success reported. Ovarian cryopreservation can help circumvent many of the problems associated with oocyte cryopreservation, while testicular tissue preservation may be helpful when insufficient sperm counts are available for routine semen preservation.

Pregnancy Results Following Initiation of a Blastocyst Vitrification Program

Background: Preservation of blastocysts is a routine part of in vitro fertilization treatment and has traditionally been accomplished using slow freezing with low concentrations of cryoprotectants. Vitrification has recently emerged as an alternative procedure in the preservation of oocytes and embryos and it may confer some time-saving and survival advantages over the traditional procedure for blastocyst cryopreservation.

Written in stone: fossils, genes and evo–devo

Fossils give evo-devo a past. They inform phylogenetic trees to show the direction of evolution of developmental features, and they can reveal ancient body plans. Fossils also provide the primary data that are used to date past events, including divergence times needed to estimate molecular clocks, which provide rates of developmental evolution. Fossils can set boundaries for hypotheses that are generated from living developmental systems, and for predictions of ancestral development and morphologies. Finally, although fossils rarely yield data on developmental processes directly, informative examples occur of extraordinary preservation of soft body parts, embryos and genomic information.

Fresh Human Orthotopic Ovarian Cortex Transplantation: Long-term Results

An increasing number of young women are being diagnosed with cancer, and most cancer treatments have irreversibly negative effects on reproduction. Ovarian stimulation and preservation of oocytes or embryos is far from an ideal solution, in part because hormonal treatment may adversely affect some cancers. Another possibility is cryopreservation of ovarian tissue for later autotransplantation, but the ischemic damage caused by the procedure can lead to follicle loss. Using the ovarian medulla for orthotopic ovarian cortex transplantation might limit the risk of ischemia because the ovarian artery would assure a good blood supply. In addition, the medulla may have a role in follicular development. This prospective case-control study examined the effects of ischemia on long-term ovarian function in 12 premenopausal women who had abdominal hysterectomy for uterine disease and also bilateral fresh orthotopic transplantation of the entire ovarian cortex. Five control women had only hysterectomy. Serum levels of follicle-stimulating hormone (FSH) and anti-Müllerian hormone (AMH) were monitored over 2 years, and ovulatory cycles were identified by vaginal ultrasonography and estimating serum progesterone. Bilateral cortex resection and transplantation took about 30 minutes and caused no complications. Ovulation returned in 11 of the 12 grafted patients (91.7%). The mean number of ovulations was 9.3, compared to 12.0 in control women. Nine study patients remained ovulatory 2 years after surgery. Five of the 12 women in the study group had the same pattern of FSH secretion as the control women. In six cases the serum FSH increased postoperatively and then declined to less than 20 IU/liter after 5 months. Two patients ultimately had menopausal levels of FSH. In only one grafted patient did ovarian function cease after surgery. AMH levels correlated less closely with normal ovarian function than did FSH levels. Serum AMH tended to decline after surgery, but was higher in women whose FSH patterns suggested normal ovarian function. Although this is a small-scale study, the results indicate that fresh orthotopic ovarian cortex transplantation maintains normal ovarian function for at least 2 years in a majority of women, and also may protect against ovarian ischemia.

Current knowledge and future challenges in camelid reproduction

Reproductive biology research on camelids offers some interesting peculiarities and challenges to scientists and animal production specialists. The objective of this paper is to review camelid reproduction, advances in reproductive physiology and reproductive biotechnologies in camelids and discuss some areas for further research. In the female, the focus has been on understanding follicular dynamics. This has allowed development of synchronization and superovulation strategies to support embryo transfer technologies which are now commonly used in camels. Some advances have been achieved in preservation of embryos by vitrification. Fertilization, early embryo development and embryo signaling for maternal recognition of pregnancy are still not fully understood. New information on the interaction of the developing embryo and the endometrium may shed some light on this signaling as well as the mechanism of prevention of luteolysis. The presence of a seminal ovulation-inducing factor (OIF) was confirmed in llamas and alpacas. Chronology of oocytes maturation has been described. In vitro production of embryos has been achieved resulting in successful pregnancies and births in the dromedary. These techniques offer a new tool for the production and study of interspecies/cross-species embryos and their effect on pregnancy. Male reproductive function remains poorly studied. Semen preservation and artificial insemination still present many challenges and are not used in production at the moment. The involvement of climatic and nutritional conditions as well as the role of leptin in the regulation of reproductive function need to be evaluated.

Generation of viable fish from cryopreserved primordial germ cells

An increasing number of wild fish species are in danger of extinction, often as a result of human activities. The cryopreservation of gametes and embryos has great potential for maintaining and restoring threatened species. The conservation of both paternal and maternal genetic information is essential. However, although this technique has been successfully applied to the spermatozoa of many fish species, reliable methods are lacking for the long-term preservation of fish eggs and embryos. Here, we describe a protocol for use with rainbow trout (Oncorhynchus mykiss) primordial germ cells (PGCs) and document the restoration of live fish from gametes derived from these cryopreserved progenitors. Genital ridges (GRs), which are embryonic tissues containing PGCs, were successfully cryopreserved in a medium containing 1.8 M ethylene glycol (EG). The thawed PGCs that were transplanted into the peritoneal cavities of allogenic trout hatchlings differentiated into mature spermatozoa and eggs in the recipient gonads. Furthermore, the fertilization of eggs derived from cryopreserved PGCs by cryopreserved spermatozoa resulted in the development of fertile F1 fish. This PGC cryopreservation technique represents a promising tool in efforts to save threatened fish species. Moreover, this approach has significant potential for maintaining domesticated fish strains carrying commercially valuable traits for aquaculture purposes.

Avian Genetic Stock Preservation: An Industry Perspective

There are different types of poultry genetic resources including mutants, inbred lines, specialized/selected stocks, standard breeds, and elite commercial pure lines. These resources differ in their degree of value to the poultry industry. There is considerable concern within poultry breeding companies about the continuing losses of these genetic resources, particularly as this loss seems to have escalated over the past decade. Varied genetic stocks can provide fundamental information regarding gene function, genetic interactions, and genetic pathways. This information is important for efficient improvement of commercial poultry performance. Equally important is the role of these genetic resources in teaching and the education of students and future researchers. Currently, the only practical preservation method for birds in the poultry industry involves live bird conservation. Flocks of elite commercial stocks are maintained at multiple locations, providing insurance against disease outbreak and the possibility of quarantine restrictions. The current cryopreservation methods apply only to sperm. Thus, the W chromosome and the mitochondria, which are contributed by the female gamete, cannot be preserved. Cryopreserved semen shows considerable variation in both fertility and hatchability rates, not only among lines, but also among males within lines. The biological basis for this variation is unknown, and there is concern that the use of cryopreserved semen may result in unintended selection and loss of genetic variability. Cryopreservation cannot be applied in the poultry breeding industry until methodologies are developed that produce high viability for both male and female avian preserved gametes. More research is needed in the areas of sources of variation in viability following freeze/thaw, female gamete cryopreservation, and embryo preservation. Because there are currently no appropriate methods of cryopreservation available to the poultry industry, the long-term preservation of commercial elite stocks must continue to rely on live bird conservation. Because of the high costs of this live bird preservation, stocks with no perceived current economic value will not be maintained.

Avian Genetic Resource Banking: Can Fish Embryos Yield Any Clues for Bird Embryos?

Cryopreservation of avian germplasm is becoming better understood and more commonly practiced. However, one area that would be of great benefit for genome resource banking is the preservation of avian embryos. Little is know about the cryobiology of avian embryos, and they have never been successfully cryopreserved. However, it is likely that they share many of the challenges of other yolk-filled multicompartmental embryos. For example, the fish embryo has 1) a large overall size, resulting in a low surface-to-volume ratio, which retards water and cryoprotectant efflux/influx; 2) large-sized cells, such as the yolk, which could increase the likelihood of membrane disruption by intracellular ice formation; 3) compartments, such as the blastoderm and yolk, with differing permeability properties; and 4) susceptibility to chilling injury. Both the avian and fish systems share many physical and anatomical properties, and it is predicted that some of the same permeability barriers would exist in both as well. Although the systems are similar, some of the goals, and thus the practices, to protect the genome may be quite different. One of these major goals in avian developmental biology is to produce chicken:chicken transgenic animals, especially those with germ line transmission. Producing efficient germ line transmissions and being able to cryopreserve these transmissions would be extremely beneficial to both basic and agricultural science. This could be accomplished through the cryopreservation of embryonic gonadal tissue followed by grafting into a host. The gonadal/tail-graft system would provide an advantage for cryopreservation because it is small (in comparison with the whole embryo), has fairly uniform tissue, and contains the essential primordial germ line cells capable of recreating the genetic line of interest. Moreover, because the chicken is such a robust model for most other avian species, the cryopreservation of the gonadal/tail-graft may potentially open up similar treatments for other commercially important species.

Fertility options in women with gynecologic malignancies

AbstractReproductive aged women with gynecologic malignancies often desire future fertility when considering treatment options for their disease. In this review we will discuss the different surgical options available to patients with early stage cervical, ovarian and endometrial cancers. We will also review advances in reproductive technologies such as embryo preservation, oocyte preservation and ovarian tissue preservation and transplantation as potential fertility options for these patients. Recent data indicate that these modalities have efficacy without compromising cancer treatment. It is therefore important for gynecologic oncologists and fertility specialists to consider these strategies for women with early stage malignancies who desire fertility preservation.

Spontaneous Pregnancy and Fertility Preservation Program in Women Undergoing High Dose Chemotherapy for Hematological Malignancies.

Loss of fertility is a major concern in young women undergoing high dose chemotherapy (HDT). Although it is generally accepted that therapy of the myeloabelative range is related with a high rate of fertility loss, we observed during the last years eight spontaneous pregnancies with normal deliveries in young women after bone marrow transplantation. Seven patients (pt's) were with lymphoma and MM and were conditioned with BEAM regimen (n=6) and melphalan 200mg/sm (n=1) prior to an autologous SCT, while one patient had a secondary AML and underwent BEAM primed autologous SCT and Busulfex/FA primed allogeneic BMT. The median age at transplant of this group was 28y and median time from transplant to pregnancy was 25 months. More than 100 women of 18–40y.o were transplanted in our center during this period; however, obviously the fertility rate cannot be calculated as it is related with additional parameters including survival, post transplant complications and mainly patient's preferences. Naturally we observed during the same period many young patients with ovarian failure post transplant, as well as one successful pregnancy from a cryopreserved embryo.Methods. We Therefore initiated in October 2000 a fertility preservation program in which all women of 18 – 40y.o were offered a pretransplant IVF with embryo preservation, and/or ovarian tissue cryopreservation (OTC), according to their clinical status. 651 pt's were transplanted in our center in the last 44 months, of which 81 were women of 18–41y.o that were all enrolled in this program.Results. Seven pt's of this group (8.6%) underwent IVF. The major causes of denying IVF were the need to delay BMT for more than clinically accepted, prolonged preexisting ovarian failure, lack of a suitable partner and patient's preference. Seventeen pt's (21%) underwent OTC. The major causes of denying OTC were patient's preference (mainly due to no evidence of success with this method) and thrombocytopenia/neutropenia. During this period:One patient of this group was fertilized with her cryopreserved embryos 32 months after transplant and is at her 16 week of pregnancy.One patient underwent a successful transplantation of her cryopreserved ovarian tissue 2.5 years after HDC while in a documented ovarian failure, and gave birth to a healthy baby on June 2005. The OTC of this patient was performed after cis-platinum containing salvage therapy for relapsing NHL, prior to BEAM primed SCT, and immediately after a failure of hormonal stimulation for IVF.One patient underwent a cryopreserved ovarian tissue transplantation on July 2005Conclusions: 1. Spontanous pregnancy after HDT, mainly at the younger age, is not a rare phenomenon. 2. Most young patients prior to HDT are not eligible for IVF. 3. Pretransplant ovarian tissue cryopreservation is a feasible tool in this set-up. The first success with this method is promising.

Fertility options in women with gynecologic malignancies

Reproductive aged women with gynecologic malignancies often desire future fertility when considering treatment options for their disease. In this review we will discuss the different surgical options available to patients with early stage cervical, ovarian and endometrial cancers. We will also review advances in reproductive technologies such as embryo preservation, oocyte preservation and ovarian tissue preservation and transplantation as potential fertility options for these patients. Recent data indicate that these modalities have efficacy without compromising cancer treatment. It is therefore important for gynecologic oncologists and fertility specialists to consider these strategies for women with early stage malignancies who desire fertility preservation.

Preservation of whole sibling blastocyst stage embryos through vitrification for patients with the risk of OHSS in COH cycles

The present study was carried out to investigate the pregnancy outcome obtained from cryo-preservation of whole sibling blastocyst stage embryos for patients with the risk for severe OHSS as compared to those from transfers of fresh day 5 embryos or the surplus embryos frozen-thawed at the blastocyst stage for patients underwent conventional IVF-ET. Retrospective study. From May 1, 2001 to February 29, 2004, 411 patients participated in our thawing-ET program. During the same period, fifteen hundred and twenty-five patients were underwent freshly transfer of day 5 blastocyst stage embryos (n = 4286; group I) in our hospital. Three hundred and fifty-four patients who failed pregnancy in previous COH cycles received frozen-thawed supernumerary blastocyst stage embryos (n = 904; group II), while fifty-seven patients who did not undergo ET because of the risk of OHSS in previous COH cycles took frozen-thawed blastocyst stage embryos (n = 237; group III). Transferable embryos that developed to the expanded blastocyst stage on day 5 or 6 were vitrified by directly plunging EM-grid loading them into liquid nitrogen following artificial collapse of blastocoele. Vitrification solution was composed of DPBS containing 20% (v/v) hFF, 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, and 0.3 M sucrose. Thawing of vitrified blastocyst stage embryos was carried out by 2-steps on day 3 after ovulation. The transfer of vitrified-thawed embryos was performed on the next day. The viability of each group was assessed through the survival, hatching embryo, implantation, and clinical pregnancy rates as possible. The survival and hatching rates of group III were 84.0% (199/237) and 81.9% (163/199), respectively, which were similar to those 89.9% (956/1063) and 82.8% (792/956), respectively) of group II. However, significantly higher clinical pregnancy and implantation rates were obtained in group III (54.4%: 31/57 and 26.8%: 51/190, respectively) than those in group II (42.5%: 149/351; 19.6%: 177/904, respectively). Moreover, it was of significance to note that the clinical outcomes in group III were significantly higher than those in group I (44.6%: 680/1525; 23.4%:1002/4286, respectively). The present results suggest that when the risk for severe OHSS outbreaks suddenly after oocyte collection for COH cycles, cryo-preservation of whole sibling blastocyst stage embryos should have no hindrance to get conventional clinical outcome. Furthermore, we propose carefully a new program that cryo-preserving all sibling embryos obtained from COH cycle and transferring them into uterus of the next unstimulated cycle, probably improving the take-home baby rate by satisfying with both embryo quality and endometrial receptivity.

Microsatellite primers for Texas wild rice (Zizania texana), and a preliminary test of the impact of cryogenic storage on allele frequency at these loci

Seed collections in gene banks are useful for the preservation of wild germplasm, providing inexpensive insurance for species that survive in conventional cold storage (−18 °C). Seeds that cannot survive these conditions must be pretreated with cryoprotectants and stored at liquid nitrogen temperatures, which presents unique technical and methodological challenges. Implicit in this approach is the assumption that these added manipulations do not change the genetic diversity of the preserved collections. We used polymorphic microsatellite markers for an endangered aquatic grass, Texas wild rice (Zizania texana), to conduct a preliminary evaluation of the effects of cryogenic preservation of mature embryos on genetic diversity. Using several statistical approaches, we show that allele frequencies did not change in collections of seeds that underwent cryopreservation (cryoprotected) compared to those samples that was not exposed to cryopreservation (control). The retention of the allelic diversity at the five loci examined suggests that there were no significant changes in genetic diversity due to treatments and that these protocols may be appropriate for ex situ conservation of genetically diverse wild germplasm.