Preservation Of Embryos Research Articles

51 ARTIFICIAL DORMANCY OF BOVINE EMBRYOS FOR A MAXIMUM OF 7 DAYS USING A SIMPLE MEDIUM

The worldwide pregnancy rate using cryopreserved mammalian embryos has not improved over the past 2 decades, probably because the freeze-thawing processes cause significant damage. Therefore, it is now relevant to examine the feasibility of short-term non-freezing preservation, and whether this could be applied to embryos that have high vitality and are to be transferred into recipients within several days. We introduce here an artificial dormancy fluid that can extend the hypothermic storage period of bovine embryos for a maximum of 7 days. First, to examine the effect of different basal media and the optimal concentration of fetal bovine serum (FBS) for hypothermic preservation, bovine blastocysts produced in vitro were stored at 4°C in a plastic ministraw in 1 of the following 3 media: PBS, medium 199, or Leibovitz L15 with various amount of FBS (0, 5, 20, 50, or 100%) for 3 days. Second, to examine the effect of Good's buffers, bovine embryos produced in vivo (morula to blastocyst stages) were stored at 4°C in a plastic ministraw in medium 199 plus 50% FBS supplemented with various Good's buffers [HEPES, TES, piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), MOPS, and 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS)] for 7 days. Following hypothermic preservation, the chilled embryos were squeezed out of the straw into PBS and washed 3 times in the same medium. Subsequently, the embryos were cultured in CR1aa medium supplemented with 5% FBS for 48 h at 38.5°C under 5% CO2 in air with high humidity. The viability rate of the embryos was assessed at the end of the culture period. Finally, to observe the pregnancy rate of chilled embryos, 32 embryos produced in vivo were stored at 4°C for 7 days in medium 199 plus 50% FBS supplemented with HEPES. Following hypothermic preservation, the chilled embryos were transferred into recipient heifers (1 embryo per recipient). Pregnancy was determined by real-time B-mode ultrasonography (Convex scanner HS-1500, Honda electronics Co. Ltd, Toyohashi, Japan) on Day 60 of gestation. Data were analysed using the chi-squared test. The viability rate of the embryos after hypothermic storage for 3 days was significantly increased for medium 199 plus 50% FBS [27/30 (90%)] compared with PBS [18/30 (60%)] or Leibovitz L15 [15/30 (50%)] plus 50% FBS (P < 0.05). Chilled embryos stored for 7 days in medium 199 plus 50% FBS supplemented with HEPES had much higher survival than embryos stored in the same medium with other Good's buffers. The pregnancy rate of the chilled embryos stored for 7 days was extremely high [24/32 (75%)] and normal live calves were delivered at term. In conclusion, maintaining artificial dormancy of bovine embryos for 7 days using a simple medium appears to be feasible. This is the first documented success of storing chilled mammalian embryos in a viable state for 7 days. To be of practical value, bovine embryo preservation at hypothermic temperatures must be able to maintain viability for periods longer than 7 days. This work was supported by the Program for Promotion of Basic and Applied Research for Innovations in Bio-Oriented Industry.

A catalyst for change in reproductive science: John D. Biggers as a mentor’s mentor

As we have done in the past, recognizing the true founders of reproductive medicine as we know it today is both an honor and responsibility that we provide to the JARG readership. In this issue of JARG, we take pride in honoring the lifelong achievements of Professor John D. Biggers on the occasion of his 90th birthday. For us personally, and for the many trainees and colleagues who have so generously contributed to this issue, assembling an appropriate tribute for Professor Biggers is a humbling task to say the least. Consider for example the wide-ranging career accomplishments that encompass, from the very beginning, over 60 years of a tireless, critical, and collegial unselfish commitment to so many that have conspired to make reproductive biology and medicine a prominent discipline in biomedical research today. We recognize him for the significance of his contributions to reproductive biology and medicine through the lens of a subset of scientists who have graciously made the following contributions. Hardly can a single journal issue or ASRM symposium achieve a thorough and balanced accounting of the ground-breaking and persistent influences Professor Biggers has had on so many. With this backdrop, we hope the JARG readership will appreciate our humble efforts at producing a glimpse of Professor Biggers’ career from which a sense of the compleat scientist will emerge. His accomplishments at the bench are far outweighed by the lasting legacy that lives on in his mentees and by his singular efforts to bring a scholarly interpretation of the evolution of ARTs to his peers, the public, and lawmakers [1]. In fact, it is fair to say that few amongst us can claim to have made inroads into the debates surrounding IVF, embryo culture, gamete and embryo preservation and cloning to the benefit of the scientific community as a whole and society more broadly. Intent is one thing-implementation quite another! What follows is fitting testimony to his career accomplishments and impacts built from personal recollections of his colleagues and friends. Next, several papers are contributed from collaborators are complemented by a series of original papers dealing with a range of topics for which he is known and that bear directly on the purview of JARG. Biggers is well-known for his travels around the world as a representative for the World Health Organization and as an invited speaker at meetings that cross many scientific disciplines. A partial list of his role in the Society for the Study of Reproduction (SSR) includes Founding Member of SSR, 1967 (Figs. 1, ​,22 and ​and33) Fig. 1 Copy of certificate noting John D Biggers, DSc, PhD as charter member of the Society for the Study of Reproduction (1967, SSR); Biggers also served as President of SSR 1968–69, Editor-In-Chief of the Societies’ journal Biology of Reproduction ... Fig. 2 Letter received from Dr Howard W. Johnson, Jr, MD recognizing Professor Biggers on the occasion of his 90th birthday Fig. 3 Newspaper article originally published November 1, 1979 in The Ledger-Star titled “Both sides swap barbs over test-tube babies.” This article represents one of the many times Biggers expert opinion was solicited from the media during the ... SSR President, 1968–69 Editor-in-Chief, Biology of Reproduction, 1970–1974 Carl G. Hartman Award Recipient, 1986 Fitting then that we recognize his accomplishments, which cover a span of 60 years, in this special issue of JARG given the influence Biggers’ has had on the discourse, basic science discoveries, and clinical impact of his contributions to the field of human ARTs. Congratulations John and thank you for enriching so many lives!

Evaluation of RNA quality in fixed and unembedded mouse embryos by different methods

Many miRNAs are highly expressed in spatiotemporal and precise tissue-specific patterns in development. Thus it is necessary to examine their expression pattern in mouse embryos. However, embryos from one pregnant mouse are more than enough for expression analysis such as RT-qPCR, which results in reluctant disposal of remaining embryos. Due to the limitation of short sampling time, it is vitally important to quickly preserve samples to ensure the RNA quality. Thus, it is necessary to develop appropriate methods to fix samples in advance. In this study, two fixatives [methanol/DMSO (4:1) and paraformaldehyde] were applied for embryo (12.5 dpc) fixation and two preservatives (methanol and 30% sucrose) were used for fixed embryo preservation. After storage for one month, the skin, skeletal muscle and brain tissues were dissected from the fixed and unembedded embryos. Total RNAs were extracted by TRIzol® reagent and measured by a spectrophotometer, then were subjected to amplify Actb, Hprt, Gapdh, Rnu6, Snord68 and miR-206-3p by RT-qPCR. Embryos fixed in methanol/DMSO and preserved in 100% methanol at −20°C were able to yield at least 349bp amplifiable RNA. Although paraformaldehyde fixation and 30% sucrose preservation method only yielded amplicons less than 156bp, it showed a remarkable ability in preserving small RNAs. Snord68 was expressed stably across skin, skeletal muscle and brain tissues like Rnu6, making its possibility as an internal control for qPCR data normalization. Using Snord68 and/or Rnu6 as internal control, we found that the miR-206-3p expression level in skin was about one quarter of its highest level in the skeletal muscle. Therefore, the techniques in this study would be useful for us to reasonably utilize and preserve precious samples.

Dynamics of morphophysiological charateristics in female water voles (Arvicola amphibius L.) during pregnancy

Pregnancy and lactation need increased energy consumption and are accompanied by morphological and physiological changes of various maternal organs and systems. In water voles, connection of female interior characteristics has been poorly studied. Under standard vivarium conditions, we evaluated morphophysiological parameters of non-reproducing females and of females at the early (1-7 days), intermediate (8-14 days), and late (15-21 days) stages of pregnancy. There were established effect of the reproductive state on mass of abdominal and brown fat, liver, adrenals, spleen and an increment of body mass (without uterus with embryos). In pregnant females, as compared with the non-pregnant ones, the blood thyroxin level was significantly lower (16.2 +/- 0.8 and 21.7 +/- 1.4 pmpl/l) and the blood testosterone level was higher (1.29 +/- 0.19 and 0.83 +/- 0.09 nmol/l). The blood glucose content fell in the end of pregnancy. Dynamics of morphophysiological parameters at the period of pregnancy reflects the compensatory body reactions providing preservation of embryos and preparation to breeding of offspring.

Contingent interactions among biofilm‐forming bacteria determine preservation or decay in the first steps toward fossilization of marine embryos

Fossils of soft tissues provide important records of early animals and embryos, and there is substantial evidence for a role for microbes in soft tissue fossilization. We are investigating the initial events in interactions of bacteria with freshly dead tissue, using marine embryos as a model system. We previously found that microbial invasion can stabilize embryo tissue that would otherwise disintegrate in hours or days by generating a bacterial pseudomorph, a three dimensional biofilm that both replaces the tissue and replicates its morphology. In this study, we sampled seawater at different times and places near Sydney, Australia, and determined the range and frequency of different taphonomic outcomes. Although destruction was most common, bacteria in 35% of seawater samples yielded morphology‐preserving biofilms. We could replicate the taphonomic pathways seen with seawater bacterial communities using single cultured strains of marine gammaproteobacteria. Each given species reproducibly generated a consistent taphonomic outcome and we identified species that yielded each of the distinct pathways produced by seawater bacterial communities. Once formed,bacterial pseudomorphs are stable for over a year and resist attack by other bacteria and destruction by proteases and other lytic enzymes. Competition studies showed that the initial action of a pseudomorphing strain can be blocked by a strain that destroys tissues. Thus embryo preservation in nature may depend on contingent interactions among bacterial species that determine if pseudomorphing occurs.We used Artemia nauplius larvae to show that bacterial biofilm replacement of tissue is not restricted to embryos, but is relevant for preservation of small multicellular organisms. We present a model for bacterial self‐assembly of large‐scale three‐dimensional tissue pseudomorphs, based on smallscaleinteractions among individual bacterial cells to form local biofilms at structural boundaries within the tissue. Localbiofilms then conjoin to generate the pseudomorph.

62 LONG-TERM STORAGE OF BOVINE EMBRYOS: BIRTH OF A CALF AFTER 22 YEARS OF CRYOPRESERVATION

After the pioneering work of Rostand (1946 CR Acad. Sci. III 222, 1524–1525) and Polge et al. (1949 Nature 164, 666–669) on semen cryopreservation and the reports on the first live young born after storage of mouse embryos for up to 8 days (Whittingham et al. 1972 Science 178, 411–414) or 8 months (Whittingham and Whitten 1974 J. Reprod. Fertil. 36, 433–435) in liquid nitrogen, the world’s first calf was obtained in Cambridge nearly 40 years ago, after freezing and thawing of a bovine embryo (Wilmut and Rowson 1973 Vet. Rec. 92, 686–690). Since then, especially in humans (case reports), the birth of healthy offspring after long-term storage of semen, zygotes, and embryos has been repeatedly reported (e.g. Dowling-Lacey et al. 2011 Fertil. Steril. 95, 1120.e1–3). But practical experiences in different other species such as mice or cattle show contradictory results on the fertility after transfer of long-term-stored gametes or embryos. Here, we report the birth of normal Holstein calves after long-term embryo preservation. After storage for 15 to 22 years, 4 embryos were thawed (air-thaw for 10 s and 25°C water bath for 20 s), which had been originally frozen in 10% glycerol (3 embryos) or in 1.5 M ethylenglycol (1 embryo) following a slow freezing protocol. Removal of glycerol was accomplished with a 4-step dilution procedure and 10.3% sucrose (EMCARETM Thawing System, ICPbio Reproduction, Glenfield, New Zealand), whereas ethylenglycol was removed with a washing step in EMCARETM (Holding Solution, ICPbio Reproduction). The embryos were transferred into the uterine horn ipsilateral to the corpus luteum of recipient cows at 7 days post-oestrus. One embryo was classified as expanded blastocyst grade 1 (7-1), 2 embryos were classified as morulae grade 1 (4-1), and the embryo frozen with ethyleneglycol was classified as a morula grade 2 (4-2) (IETS classification; 40× magnification). Three pregnancies were established (1 × 7-1; 1 × 4-1, and 1 × 4-2), and 3 healthy bull calves were born (18.1.2011, stored 15 years; 6.3.2012, stored 22 years; and 29.3.2012, stored 15 years). These case reports document that long-term-stored bovine embryos maintained developmental competence for up to 22 years of cryopreservation in liquid nitrogen. To our knowledge these cases represent the “oldest” cryopreserved bovine embryos resulting in healthy calves in Germany.

Uptake and experiences of breast cancer patients referred for fertility preservation.

86 Background: The 2006 ASCO guidelines suggest women of childbearing age receive counseling and referrals regarding the impact of treatment on fertility. Less is known about the use and uptake of FP in these women. The objective of this study was to evaluate the experience of patients referred for FP from a multidisciplinary breast cancer program. Methods: After IRB approval, patients diagnosed with a primary breast cancer between January 1, 2006 and June 30, 2011 and referred for FP counseling were identified via queries of institutional databases. Clinicopathologic, treatment, and outcome data were collected. Patients who received a consultation with a reproductive endocrinologist (REI) and did not pursue FP were selected for further contact via telephone survey. Results: Twenty-two patients had a consultation with REI; 10 were nulliparous. Fifteen of these saw an REI prior to any treatment. Thirteen (59%) had embryo or oocyte preservation. The majority (9) underwent consultation with both surgical and medical oncology prior to REI. Nine of the 13 had surgery prior to FP. Of the remaining 9, 7 (78%) responded to a telephone survey. Six (86%) recalled counseling at the initial visit or prior to chemotherapy. Barriers to FP included: cost (3), perceived delay in chemotherapy (2), being overwhelmed by cancer diagnosis (2), uncertainty of FP effects on cancer (1), and ethical concerns (1). Conclusions: Patients counseled by multiple oncology providers in a multidisciplinary setting prior to REI consultation were more likely to pursue FP. Those who did not cited a number of factors in their decision. Consultation with multiple oncology specialists may enhance decision-making regarding FP. [Table: see text]

Experimental mineralization of crustacean eggs: new implications for the fossilization of Precambrian–Cambrian embryos

Abstract. Phosphatized globular microfossils from the Ediacaran and lower Cambrian of South China represent an impressive record of early animal evolution and development. However, their phylogenetic affinity is strongly debated. Understanding key processes and conditions that cause exceptional egg and embryo preservation and fossilization are crucial for a reliable interpretation of their phylogenetic position. We conducted phosphatization experiments on eggs of the marbled crayfish Procambarus that indicate a close link between early mineralization and rapid anaerobic decay of the endochorional envelope. Our experiments replicated the different preservational stages of degradation observed in the fossil record. Stabilization of the spherical morphology was achieved by pre-heating of the eggs. Complete surface mineralization occurred under reduced conditions within one to two weeks, with fine-grained brushite (CaHPO4 · 2H2O) and calcite. The mechanisms of decay, preservation of surface structures, and mineral replacement in the experiment were likely similar during fossilization of Cambrian embryos.

La criopreservación embrionaria en estadios más avanzados de desarrollo disminuye la frecuencia de pacientes con embriones criopreservados y el número de embriones criopreservados

Multiple pregnancies are the main complication associated to assisted reproduction, due to the transfer of more than one embryo. Embryo cryopreservation allows the sequential transfer of all generated embryos, thus diminishing the risk of multiple pregnancies. However, it leads to accumulation of cryopreserved embryos. To reduce their accumulation in our unit, we started to preserve embryos as blastocysts, that have a rate of successful pregnancies of approximately 40%.To perform a sensitivity analysis of this change of policy on the accumulation of embryos.Records of 571 cycles of in vitro fertilization since 2007 were reviewed, assuming a transference rate of two embryos in women aged less than 35 years and three embryos in older women. The number of embryos that would be preserved as zygotes, eight cell stage or blastocysts, was analyzed.Multiple component logistic regression analysis showed a 20% reduction in the odds ratio of cryopreservation per year of age. There was a 95% reduction in the ratio, when comparing the preservation of third and first day embryos and a 99% reduction when comparing preservation of embryos in blastocyst stage and first day embryos.Cryopreservation at blastocyst stage effectively decreased the frequency of embryo cryopreservation.

Cryopreservation in blastocyst stage effectively reduce the number of embryos cryopreserved

Multiple pregnancies are the main complication associated to assisted reproduction, due to the transfer of more than one embryo. Embryo cryopreservation allows the sequential transfer of all generated embryos, thus diminishing the risk of multiple pregnancies. However, it leads to accumulation of cryopreserved embryos. To reduce their accumulation in our unit, we started to preserve embryos as blastocysts, that have a rate of successful pregnancies of approximately 40%. To perform a sensitivity analysis of this change of policy on the accumulation of embryos. Records of 571 cycles of in vitro fertilization since 2007 were reviewed, assuming a transference rate of two embryos in women aged less than 35 years and three embryos in older women. The number of embryos that would be preserved as zygotes, eight cell stage or blastocysts, was analyzed. Multiple component logistic regression analysis showed a 20% reduction in the odds ratio of cryopreservation per year of age. There was a 95% reduction in the ratio, when comparing the preservation of third and first day embryos and a 99% reduction when comparing preservation of embryos in blastocyst stage and first day embryos. Cryopreservation at blastocyst stage effectively decreased the frequency of embryo cryopreservation.

Vitrification of Mice Embryos in Different Developmental Stages Using Four Vitrification Methods

ABSTRACTThe aim of our study was to test four vitrification methods for preservation of mice embryos, in different developmental stages, using 6 mol/l glycerol and 20% sucrose as vitrification media. Morula stage embryos were vitrified with OPS straws or classic straws using two equilibration steps, with a hatching rate of 50% and 47.62% respectively. Blastocyst stage embryos survived better in 0.25 ml straws with three equilibration steps, 36.84% hatching rate. Two-cell embryos are sensitive to freezing, but satisfactory results could be obtained with OPS vitrification, hatching rates of 22.73%. The success of the vitrification technique depends on the developmental stage of the embryo and the method used.

Novel Approaches to the Cryopreservation of Human Spermatozoa: History and Development of the Spermatozoa Vitrification Technology

Cryobiology is very intensively applied in reproductive and veterinary medicine for preservation of gametes, embryos and reproductive tissues. Sub-zero temperatures combined with appropriate cryoprotective agents preserve the physiological and reproductive functions of the cells making long-term storage possible without loss of viability. With the use of cryoprotective agents it has become possible to develop cryopreservation techniques, such as the slow conventional freezing and vitrification that are in use in the present times. In slow controlled-rate conventional freezing extracellular ice crystals are formed whereas in vitrification no ice crystals are formed. Glass formation is compatible with the survival of the cell and the preservation of its intracellular structures provided the type(s) and concentrations of cryoprotectant used are not chemo- or osmotoxic. However, irrespective of the type of cooling method employed the cryosurvival of cells and tissues is influenced by the size and maturity of cells, amounts of intracellular water, quality and quantity of intracellular lipids, type of cells, their function and morphology. The intracellular milieu of cryopreserved cells and tissues remain less understood. The application of nanotechnology may help reveal and help advance our knowledge of the cryobiological principles involved in cryosurvival. At this moment the methods of cryopreservation that merit further investigation are vitrification and lyophilization. Vitrification is cheap if reagents are prepared in-house and the procedure can be performed rapidly. It has been successfully applied for gametes and embryos (of different stages of development), and reproductive cells/tissues, somatic cells and stem cells. However, vitrification is more demanding technically and requires operation and storage at sub-zero temperatures. On the other hand lyophilization deserves further investigation because it is a cheaper form of cryopreservation that may enable cryostorage at less demanding temperatures of 4°C and may even allow transport at ambient temperature. These possibilities are explored in this review.

Optimization of Blastocyst Vitrification May Require Artificial Collapse

INTRODUCTION: Vitrification and warming of blastocysts is quickly becoming established as a routine procedure for the preservation of embryos remaining after transfer. Further, reliable preservation and recovery of these embryos is becoming an important tool in allowing physicians and patients to consider elective single embryo transfer (eSET), since high pregnancy rates with warmed blastocysts negate the need to transfer more than one embryo in a fresh cycle. In the current study, we have looked at the impact of artificial collapse (AC) of blastocysts at the time of vitrification on pregnancy outcome after warming and transfer.

96 THE EFFECT OF CHEMICAL DELIPIDATION ON CRYOPRESERVABILITY OF CAT EMBRYOS

Embryo cryopreservation is a desired technique for long-term preservation of embryos. However, the success rate of cryopreserved in vitro produced cat embryos is currently poor. Until recently, the mechanism underlying the cause of cryoinjury that occurs during cooling and cryopreservation is not well understood. This study aimed to examine the effect of chemical delipidation (forskolin) before cryopreservation of 4- to 8-cell cat embryos on post-thaw embryo survival and in vitro developmental capability. A total of 333 cumulus oocyte complexes (COCs) were matured and fertilized in vitro. At 24 h post-IVF, the presumptive embryos were randomly assigned into one of the following groups: 1) non-frozen control (n = 63); 2) forskolin treatment without freezing (n = 52); 3) freezing without forskolin (n = 77); and 4) freezing after forskolin treatment (n = 89). The embryos were cryopreserved using a programmable controlled-rate freezer. After freezing and thawing, the embryos were subsequently cultured in vitro for a further 6 days. The development competence was assessed by morula and blastocyst rates on Days 5 and 8 of their development, respectively. Percentages of cleaved embryos on Day 2 (IVF = Day 0) did not significantly differ among groups, indicating that there was no adverse effect of forskolin on cleavage rates. Furthermore, blastocyst formation rates of cat embryos treated with forskolin (53.5 ± 3.1) did not significantly differ when compared with non-treated controls (54 ± 9.3). Forskolin-treated embryos survived after cryopreservation at a higher rate than non-forskolin treatment, in terms of survival (93.1 ± 2.6 v. 88.2 ± 1.4), morula (56.9 ± 7.6 v. 40.8 ± 5.7), and blastocyst formation (47.6 ± 6.4 v. 35.6 ± 3.6) rates. It is concluded that partial delipidation of cat embryos before cryopreservation improves the cryopreservability of cat embryos. This study demonstrates that intracellular lipid has an impact on cryopreservability of cat embryos. Further study is required to examine in utero development of these delipidated embryos after embryo transfer. This study was financially supported by the Zoo organisation of Thailand and CHE-TRF Senior Research Scholars RTA-5080010.

In Vitro Oocyte Maturation and Preantral Follicle Culture from the Luteal-Phase Baboon Ovary Produce Mature Oocytes

Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.

Organization, anatomy, and fungal endophytes of a Triassic conifer embryo

Much is known about the Paleozoic conifers and the conifers assignable to extant families that appear in the Jurassic; however, relatively little is known about the transitional conifers of the early Mesozoic, especially the Voltziales. To better understand the evolution of this group, we aim to increase knowledge of voltzialean anatomy and morphology. A fossil embryo of the group is described in this study. • Several permineralized seeds, one containing a well-preserved embryo, were collected from the early Middle Triassic Fremouw Peak locality in the Central Transantarctic Mountains. Samples were prepared using the standard acetate peel technique and studied in transmitted light. • The embryo belongs to Parasciadopitys aequata, a member of the Voltziales. The embryo and megagametophyte tissues are exquisitely preserved. The embryo is colonized by two distinct fungi. Sporocarps of the fungi are found in the megagametophyte and in the space between the megagametophyte and nucellus. The additions of embryo and megagametophyte characters to the description of P. aequata have made it one of the most completely known fossil taxa reproductively. • Preservation of fossil embryos, although rare, can expand the array of characters available in tracing the evolutionary history of plants. The embryo of P. aequata shares similarities with embryos of other extinct and extant conifer families. The association of the embryo with Combresomyces cornifer and Mycocarpon asterineum increases our understanding of the roles of microorganisms in Triassic ecosystems.

Cryopreservation of Human Embryos and Oocytes

Abstract: A procedure to cryopreserve mammalian embryos resulting in the birth of offspring was first described almost forty years ago. This procedure resulting in the birth of a child was reported 26 years ago. Since then, the preservation of human oocytes and embryos by cooling them to low subzero temperatures has become an integral part of Assisted Reproductive Technologies (ART). Hundreds of thousands of children have now been born after having been cryopreserved as oocytes or embryos. These results owe as much to the fundamental understanding of cryobiology as to the application of reproductive medicine. This brief review summarizes the history of embryo cryobiology, and presents a synopsis of basic cryobiology as it applies to present methods to improve the cryopreservation of human oocytes.

Dextran vitrification media prevents mucin coat and zona pellucida damage in rabbit embryo

Vitrification of embryos is being increasingly important for cryopreservation in mammals. However, damage and toxicity has to be reduced even more. The composition of cryoprotective medium used to immerse the embryos affects viability and developmental potential. The aim of this work was to assess the effect of the Polyvinylalcohol-PVA- and Dextran addition to vitrification media on the in vitro development of rabbit embryos from superovulated and non-superovulated females. Superovulation group were treated intramuscularly with 25 IU rhFSH. The vitrification media contained the same permeable cryoprotectans (Ethylene Glycol-ET- and Dimethyl Sulfoxide-Me 2SO-) and different macromolecules (PVA and Dextran) in different combinations. There was a significantly higher proportion of embryos without damages in mucin coat or zona pellucida after warming (undamaged embryos) in the control than in the superovulation group (95.8% vs. 83.2%, respectively). The proportion of undamaged embryos was significantly affected by the vitrification solution composition. The rate of undamaged embryos after warming in media containing 20% Me 2SO was significantly lower in media supplemented with PVA than in media with dextran (67.3 vs. 93.8, respectively). However, the proportion of undamaged embryos for the medium supplemented with dextran was similar for media with 15 or 20% Me 2SO. In conclusion, the addition of dextran to the vitrification media improve the preservation of rabbit embryos and permits to reduce the amount of Me 2SO for vitrification. Additionally, in vitro developmental ability of undamaged embryos were not affected by superovulation treatment nor vitrification media.

The legal status of in vitro embryos

Judicial approaches to stored (cryopreserved) human embryos, in western jurisprudence, tend not to reflect approaches within systems of moral ordering or particular religious traditions, which differ among themselves. The emerging judicial approach is pragmatic, protecting individuals’ rights of control and their interests in parenthood. Embryos are approached instrumentally, not by reference to any inherent characteristics that may be attributed to them outside the law. Political legislatures may adopt religious approaches, such as by prohibiting embryo preservation and limiting how many may be created in an IVF treatment cycle. Legislatures may alternatively set time limits on embryo preservation, however, on expiry of which they must be left to natural degeneration. In treating human embryos as property, courts recognize owners’ powers of voluntary disposition, for instance by gift, but have held back from making financial assessments of their value, for instance on loss, consistently with legislation prohibiting their exchange for payment.

First successful birth of twins in India following the transfer of vitrified oocytes

We report the first twin pregnancy and birth in India after the transfer of embryos generated from vitrified and thawed oocytes to a 41-year-old woman who was in a donor program. Embryos were generated from the microinjection of pre-prepared sperms into vitrified oocytes. Twin male babies weighing 750 and 860 g were born by emergency cesarean section. Vitrification is one of the most promising options in cryopreservation and preservation of embryos and oocytes.