Detailed Morphological Analysis of Cryoinjury in Human Ovarian Tissue Following Vitrification or Slow Freezing.

Cryopreservation of human ovarian tissue represents a key procedure for fertility preservation. The two most widely used cryopreservation methods for human ovarian cortex samples are slow freezing\thawing (SF\T) and vitrification\warming (V\W). The aim of the present study was to analyze the effects of SF\T and V\W using a metal chamber, on specific follicle and oocyte structures and on the stromal organization post-cryopreservation. We did histology analysis of SF\T and V\W ovarian fragments from nine healthy subjects. Overall results showed that cryopreserved tissues presented significant rates of damage in primordial and primary follicles. Altered nuclear structure of primordial follicles and cell detachment from primordial and primary follicles were the main injuries observed after V/W and SF/T. The stromal components were similarly well preserved after cryopreservation. We conclude that both cryopreservation methods may be used for fertility preservation purposes with similar outcomes in terms of follicular and stromal integrity. Detachment of follicle cells from basal membrane represents an important cryoinjury that deserves further investigation.

Development of bone in chick embryos from Cobb 500 breeder hens fed diets supplemented with zinc, manganese, and copper from inorganic and amino acid-complexed sources.

Sources of Zn, Mn, and Cu (IZMC) as sulfates or as amino acid complexes (OZMC) were used to supplement Cobb 500 breeder hen diets. Experimental treatments consisted of diets supplemented with 1) 100, 100, and 10 mg/kg of Zn, Mn, and Cu, respectively, from IZMC (control); 2) 60, 60, and 3 mg/kg of Zn, Mn, and Cu, respectively, from IZMC plus 40, 40, and 7 mg/kg of Zn, Mn, and Cu, respectively, from OZMC (ISO); and 3) a diet with 100, 100, and 10 mg/kg of Zn, Mn, and Cu, respectively, from IZMC as in control plus 40, 40, and 7 mg/kg of supplemental Zn, Mn, and Cu from OZMC (on top). Ten replications of 20 females and 2 males were used per treatment. Eggs from breeders at 30, 40, 50 and 60 wk of age were incubated, and 5 embryos per replicate were collected at 10 (E10), 14 (E14), and 18 (E18) d of incubation. Midshaft width and calcification were measured for left tibia and femur stained with Alcian Blue and Alizarin Red S. At hatch, the left tibia of 5 chicks per replicate was sampled for histological evaluation of the diaphysis and distal epiphysis. Feeding the ISO treatment compared with the control diet increased the Zn (P < 0.05) but not Mn and Cu content of the yolk and albumen blend. At E14, the ISO and on-top treatments had a trend to increase tibia calcification at the rates of 1.6 and 1%, respectively (P < 0.1). The E18 ISO and on-top treatments had 2% thicker tibia compared with the control, regardless of hen age (P < 0.05). Also, at E18, calcification of tibia and femur was higher from hens fed the on-top treatment (P < 0.05). The chicks from the ISO and on-top groups had increased tibia moment of inertia (P < 0.01) at day of hatch. Broiler breeder hens consuming OZMC associated with IZMC produced embryos and hatching chicks with improvements in selected bone mineralization parameters.

The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions.

PURPOSES: Clinical application of human embryonic stem cells will be possible, when cell lines are created under xeno-free and defined conditions. We aimed to establish methodologies for parthenogenetic activation, culture to blastocyst and mechanical isolation of the inner cell mass (ICM) using bovine oocytes, as a model for derivation and proliferation of human embryonic stem cells under defined xeno-free culture conditions. METHODS: Cumulus-oocyte-complexes were in vitro matured and activated using Ca(2+)Ionophore and 6-DMAP or in vitro fertilized (IVF). Parthenotes and biparental embryos were cultured to blastocysts, when their ICM was mechanically isolated and placed onto a substrate of fibronectin in StemPro medium. After attachment, primary colonies were left to proliferate and stained for pluripotency markers, alkaline phosphatase and Oct-4. RESULTS: Parthenogenesis and fertilization presented significantly different success rates (91 and 79 %, respectively) and blastocyst formation (40 and 43 %, respectively). ICMs from parthenogenetic and IVF embryos formed primary and expanded colonies at similar rates (39 % and 33 %, respectively). Six out of eight parthenogenetic colonies tested positive for alkaline phosphatase. Three colonies were analyzed for Oct-4 and they all tested positive for this pluripotency marker. CONCLUSION: Our data show that Ca(2+) Ionophore, and 6-DMAP are efficient in creating large numbers of blastocysts to be employed as a model for human oocyte activation and embryo development. After mechanical isolation, parthenogetic derived ICMs showed a good rate of derivation in fibronectin and Stem-Pro forming primary and expanded colonies of putative embryonic stem cells. This methodology may be a good strategy for parthenogenetic activation of discarded human oocytes and derivation in defined conditions for future therapeutic interventions.

The effects of sample size on the outcome of ovarian tissue cryopreservation.

Cryopreservation of ovarian tissue is known to affect follicular survival. Several variables may be responsible for this. Little attention has focused on the effect of the size of the fragment to be cryopreserved. This study was conducted to assess the effect of the size of the tissue on follicular histology after freezing with 1,2-propanediol. Histological evaluations were performed of control and cryopreserved tissue. Fragments were cut 10 x 3 x 2 mm(3) (2 mm group) or 10 x 3 x 4 mm(3) (4 mm group). Percentages of normal follicles in control fragments cut into 2 and 4 mm slices were 56% and 34%, respectively. The relative risks to obtain normal follicles in the 2 mm and the 4 mm fragments after cryopreservation were 0.63 and 0.47, respectively. Freezing reduced follicle survival to a significantly greater extent in the larger tissue fragments. There is an increased risk of damage to primary and primordial follicles, when the tissue slices are cut with all dimensions larger than 2 mm.

Early cleavage of human embryos: an effective method for predicting successful IVF/ICSI outcome.

BACKGROUND: The need for effective parameters for selecting the best embryos is paramount when a large number of them are available for transfer. Other studies have reported that transfer of pre-selected embryos, based on cleavage to the 2-cell stage at 25 h and 27 h post-insemination/intracytoplasmic sperm injection (ICSI), increases implantation and pregnancy rates. We investigated whether extending the time for selection of cleaved embryos to 29 h post-insemination/ICSI had a similar effect on pregnancy and implantation rates. METHODS: Cleavage to the 2-cell stage was assessed at 25, 27 and 29 h post-insemination/ICSI. Embryos that had cleaved at any of these time points were designated as 'early cleavage' (EC), while others were designated as 'non-early cleavage' (NEC). EC embryos were selected and preferentially transferred. RESULTS: EC occurred in 57% of the cycles (61% IVF; 51% ICSI). Significantly (P = 0.02) more clinical pregnancies occurred in the EC group (23/42, 55%) compared with the group that had no embryo undergoing first cleavage up to 29 h post-insemination/ICSI (8/32, 25%). The EC group of patients was significantly younger than the NEC. CONCLUSION: Transfer of selected embryos that reached the 2-cell stage between 25 and 29 h post-insemination/ICSI is a reliable prognostic tool for patients undergoing assisted reproduction techniques.

Meiotic and mitotic Ca2+ oscillations affect cell composition in resulting blastocysts.

At fertilization periodic Ca2+ oscillations release oocytes from meiotic arrest. The present study examined whether these oscillations have a long-term role in pre- and postimplantation development, independent of their immediate effect. Sr(2+)-containing medium was used to induce oscillations during exit from meiosis and first embryonic mitosis and Sr(2+)-activated parthenotes were compared to ethanol-activated parthenotes and embryos generated by in vitro fertilization. After embryo culture, blastocysts were differentially stained for the inner cell mass and trophectoderm. It was found that oscillations both during exit from meiosis and during mitosis acted to increase the number of inner cell mass cells. In contrast, the trophectoderm cell number was largest in ethanol-activated parthenotes and smallest in fertilized embryos. Postimplantation development was also modestly improved by extending the time of exposure to Sr(2+)-containing medium. Together these data suggest that Ca2+ oscillations have a role in long-term embryonic events and that they provide more than merely a stimulus for meiotic resumption.

A cell cycle-associated change in Ca2+ releasing activity leads to the generation of Ca2+ transients in mouse embryos during the first mitotic division.

We have used Ca2+-sensitive fluorescent dyes to monitor intracellular Ca2+ during mitosis in one-cell mouse embryos. We find that fertilized embryos generate Ca2+ transients at nuclear envelope breakdown (NEBD) and during mitosis. In addition, fertilized embryos arrested in metaphase using colcemid continue to generate Ca2+ transients. In contrast, parthenogenetic embryos produced by a 2-h exposure to strontium containing medium do not generate detectable Ca2+ transients at NEBD or in mitosis. However, when parthenogenetic embryos are cultured continuously in strontium containing medium Ca2+ transients are detected in mitosis but not in interphase. This suggests that mitotic Ca2+ transients are detected in the presence of an appropriate stimulus such as fertilization or strontium. The Ca2+ transient detected in fertilized embryos is not necessary for inducing NEBD since parthenogenetic embryos undergo nuclear envelope breakdown (NEBD). Also the first sign that NEBD is imminent occurs several minutes before the Ca2+ transient. The Ca2+ transient at NEBD appears to be associated with the nucleus since nuclear transfer experiments show that the presence of a karyoplast from a fertilized embryo is essential. Finally, we show that the intracellular Ca2+ chelator Bapta inhibits NEBD in fertilized and parthenogenetic embryos in a dose-dependent manner. These studies show that during mitosis there is an endogenous increase in Ca2+ releasing activity that leads to the generation of Ca2+ transients specifically during mitosis. The ability of Ca2+ buffers to inhibit NEBD regardless of the presence of global Ca2+ transients suggests that the underlying cell cycle-associated Ca2+ releasing activity may take the form of localized Ca2+ transients.

Analysis of the chromosome complement of frozen-thawed mouse oocytes after parthenogenetic activation.

Frozen-thawed mouse oocytes were artificially activated with Sr2+ and analyzed cytogenetically at the first cleavage division to examine the behavior of the maternal chromosomes independently of the paternal complement. There was no significant difference in the rate of activation between frozen-thawed and freshly collected oocytes and the majority of oocytes (> 90%) had a normal haploid chromosome constitution. The incidence of second polar body retention in frozen-thawed oocytes was low and did not differ significantly from that observed in fresh oocytes and oocytes exposed to dimethylsulfoxide (DMSO) at 0 degree C or 37 degrees C for extended periods beyond those required for protection. The frequency of aneuploidy was similar for frozen-thawed and fresh oocytes but oocytes held at 0 degree C without DMSO or held at 37 degrees C with DMSO for 1 hr showed a 2.5 and 12-fold increase in the frequency of aneuploidy compared with oocytes subjected to a conventional oocyte/embryo freezing regime. It is concluded that the procedures used in successful oocyte cryopreservation do not increase the incidence of chromosomal abnormalities of maternal origin in the resulting embryos.

Cytogenetical analysis and developmental potential of vitrified mouse oocytes.

Mature mouse oocytes were cryopreserved by vitrification in 6 M dimethyl sulfoxide (VS). After warming they were either artificially activated with strontium (Sr2+), and the incidence of chromosome non-disjunction was assessed at first cleavage metaphase; or they were fertilized in vitro, and postimplantation survival was examined at Day 15 of gestation. Similar proportions of vitrified and freshly collected oocytes were activated with Sr2+ (75% and 82%, respectively). The majority of activated oocytes extruded the second polar body and formed a single pronucleus ( > 90%). When the exposure time to VS was increased from 90 to 110 sec without cooling, a significant proportion of activated oocytes arrested at the pronuclear stage (30%), and chromosome condensation did not occur. The frequency of aneuploidy in vitrified and control oocytes was similar, but when exposure to VS without cooling was extended, aneuploidy and second polar body retention were significantly higher than those of controls (p < 0.05). The rates of fertilization of vitrified (85%) and control oocytes (92%) did not differ. After transfer, similar proportions of vitrified and control embryos implanted (68-80%) and formed normal fetuses (38-49%). We conclude that vitrification in 6 M dimethyl sulfoxide is a simple and safe procedure for the preservation of mouse oocytes provided that the time of exposure to the cryoprotectant is carefully controlled.

Calcium oscillations and protein synthesis inhibition synergistically activate mouse oocytes.

We have examined the ability of the two parthenogenetic agents, strontium (Sr2+) and cycloheximide, to activate mouse oocytes. We demonstrate that Sr2+ and cycloheximide act synergistically to promote parthenogenetic activation up to the pronuclear stage in oocytes collected immediately after ovulation. These two agents appeared to act independently, since incubation in Sr2+ media triggered a series of intracellular Ca2+ rises without affecting protein synthesis and cycloheximide inhibited protein synthesis without causing any intracellular Ca2+ changes. In addition, cycloheximide did not alter the pattern of Ca2+ oscillations induced by Sr2+. In contrast, we show that another commonly used parthenogenetic activation treatment, the Ca2+ ionophore A23187, has dual effects. Exposure of oocytes to the Ca2+ ionophore, A 23187, in Ca(2+)- and Mg(2+)-free medium leads to the activation of young oocytes. However, as well as generating a Ca2+ increase, the treatment of mouse oocytes with A23187 and Ca(2+)- and Mg(2+)-free media led to a marked inhibition of protein synthesis. Our data show that parthenogenetic agents may have two important loci for activating mammalian oocytes and that the combined effect on Ca2+ release and protein synthesis is most effective.