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PubMed1999

Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1, 2-propanediol.

Thin slices of human ovarian cortex were evaluated following cryopreservation in 1,2-propanediol (PROH)/sucrose under various conditions. Following rapid thawing, 1 microm sections were assessed by light microscopy and oocyte abnormalities were further examined by electron microscopy. Follicles (n = 503) were predominantly primordial (91%), with no follicles larger than the proliferating primary stage. Proportions of intact pre-granulosa cells and oocytes (expressed as percentages of the total numbers observed) were significantly reduced following cooling at three different rates with the highest levels of intactness (55 and 85% respectively) being achieved with slow cooling. The frequency of oocyte abnormalities [loss of organelles (mitochondria), organelle-free areas, and/or cytoplasmic vacuolation] was significantly increased at all cooling rates with slow cooling resulting in the highest proportion (56%) of normal oocytes. With slow cooling, increasing dehydration time increased the proportions of intact pre-granulosa cells and oocytes (maximum 74 and 91% respectively after 90 min dehydration). Under these conditions, the highest proportion of follicles with all pre-granulosa cells intact (44%) was observed, as was the highest proportion of 'normal' oocytes (85%). In this study, single step dehydration in PROH/sucrose for 90 min and slow cooling/rapid thawing results in the highest proportion of intact human primordial and primary follicles.

Adolescent↗
PubMed2000

Comparison of ethylene glycol, 1,2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts.

The aim of the study was to analyse the toxicity, the osmolar and cryoprotective activity of ethylene glycol (ETG) in terms of survival rate (SR), cleavage rate (CR) and expanded blastocysts percentage (EBP) of mouse embryos. Early mouse embryos and blastocysts were slowly cooled with ETG, 1,2-propanediol (PROH) or glycerol, and thawed. The Van t'Hoff curve for 1.5 mol/l ETG showed recovery of initial volume within 4 min. No differences were observed in CR and EBP of ETG-exposed compared with non-exposed mouse zygotes. The SR of zygotes frozen with PROH was significantly better than with ETG (92% and 60% respectively; P < 0.01), and a significantly better EBP was achieved for blastocysts frozen with glycerol compared with ETG (75% and 50% respectively; P < 0.05). For 4-cell stage embryos, no differences were observed in SR and EBP between ETG and PROH. Higher EBP was observed for 4-cell stage embryos (53%) frozen with ETG compared with pronucleate stage (19%) and blastocysts (48%). Low toxicity, good SR and EBP were observed for mouse embryos frozen with ETG, the best results being obtained at the 4-cell stage. At other embryonic stages, PROH and glycerol respectively seemed to provide better results.

Animals↗
PubMed2005

Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants.

BACKGROUND: Cryopreservation of testicular tissue is an option in fertility preservation for pre-pubertal boys who will lose spermatogenic cells as a result of chemotherapy. We compared three different protocols and cryoprotectants in cryopreservation of testicular tissue. METHODS: Testicular tissue obtained from 16 infertile men was evaluated by light microscopy(LM), immunostaining against MAGE-A4, transmission electron microscopy (TEM) and organ culture. Seminiferous tubules (1312) from non-frozen (n = 16) and frozen-thawed samples (n = 34) were studied following cryopreservation using protocols with either 1,2-propanediol (PrOH), glycerol or dimethylsulphoxide (DMSO) as cryoprotectants. RESULTS: Normal structure was seen in 86 +/- 6% (mean +/- SD) of the fresh tissue. After freezing with DMSO, 70 +/- 6% and after PrOH, 37+/-3% of the tubules were judged to be good. When glycerol was used, the structure of the basal compartment of the tubules was severely damaged. The ultrastructure of the cryopreserved samples as revealed by TEM and MAGE-positive spermatogonia confirmed the findings. Cryopreserved Leydig cells maintained their morphology and ability to release testosterone in culture. CONCLUSION: DMSO as a cryoprotectant (at a 0.7 mol/l concentration) proved to maintain the structure of testicular tissue, especially spermatogonia, after cryopreservation better than PrOH or glycerol.

Adult↗
PubMed2005

Observational clinical follow-up of oocyte cryopreservation using a slow-freezing method with 1,2-propanediol plus sucrose followed by ICSI.

BACKGROUND: The value of oocyte cryopreservation remains controversial. Two major problems exist: poor survival and injury to the oocyte meiotic spindle after freezing and thawing. METHODS: For slow oocyte cryopreservation, we used 1.5 mol/l 1,2-propanediol and 0.3 mol/l sucrose. We waited 3 h after thawing for possible recovery of the meiotic spindles before performing ICSI. RESULTS: Forty-three women undergoing IVF or ICSI cycles cryopreserved some or all of their harvested oocytes; of these, 20 thawed their cryopreserved oocytes for personal use and one for donation. The survival rate of oocytes after thawing was 75%, with 67% of oocytes fertilizing normally after ICSI. All 21 cycles (100%) resulted in fertilization and embryo transfers. Seven pregnancies (33%) resulted. Four women delivered five babies with normal karyotypes. Three conceptions are ongoing. Compared to 38 cycles of frozen-thawed embryos at the pronuclear stage in the same period, the percentages of survival, pregnancy and implantation were similar. Additionally, four unmarried women with white blood cell diseases underwent oocyte freezing before preconditioning treatment for haematopoietic stem cell transplantation. CONCLUSIONS: This protocol achieved reproducible success of survival, fertilization and pregnancy for freezing and thawing of human oocytes. The 3 h post-thaw incubation could permit restoration of the meiotic spindles, thus facilitating normal fertilization.

Adult↗
PubMed2006

1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology.

BACKGROUND: The aim of this work was to examine the effect of 1,2-propanediol (PrOH) and type of cryopreservation procedure (slow freezing and vitrification) on oocyte physiology. METHODS: Intracellular calcium of mouse metaphase II (MII) oocytes was quantified by fluorescence microscopy. The effect of PrOH on cell physiology was further assessed through analysis of zona pellucida hardening and cellular integrity. Protein profiles of cryopreserved oocytes were generated by time-of-flight mass spectrometry (TOF-MS). RESULTS: PrOH caused a protracted increase in calcium, which was sufficient to induce zona pellucida hardening and cellular degeneration. Using 'nominally calcium free' media during PrOH exposure significantly reduced the detrimental effects. Proteomic analysis identified numerous up- and down-regulated proteins after slow freezing when compared with control and vitrified oocytes. CONCLUSIONS: Using such approaches to assess effects on cellular physiology is fundamental to improving assisted reproduction techniques (ART). This study demonstrates that PrOH causes a significant rise in intracellular calcium. Using calcium-free media significantly reduced the increase in calcium and the associated detrimental physiological effects, suggesting that calcium-free media should be used with PrOH. In addition, analysis of the oocyte proteome following cryopreservation revealed that slow freezing has a significant effect on protein expression. In contrast, vitrification had a minimal impact, indicating that it has a fundamental advantage for the cryopreservation of oocytes.

Animals↗
PubMed1996

Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants.

Pieces of ovarian cortical tissue (0.3-2 mm in diameter) were obtained during gynaecological operations by biopsy or as a result of oophorectomy from 19 women aged 19-44 years. The tissue was frozen in a programmable freezer using one of two different cryoprotectants, either 1.5 M dimethylsulphoxide (DMSO), or a combination of 1,2-propanediol (1.5 M) and sucrose (0.1 M). After cryopreservation lasting from 24 h to 5 weeks, the ovarian pieces were thawed and studied histologically. Specimens taken before and after cryopreservation with either protectant showed no signs of tissue necrosis. Follicles at similar developmental stages were found before and after freezing. The proportions of follicles showing signs of atresia, 27% in the non-frozen tissue and 19% in the frozen-thawed tissue, were not significantly different. Oocytes, too, had the same appearance after freezing and thawing with both cryoprotectants as was seen in the specimens taken before freezing. These results suggest that cryopreservation of human ovarian tissue is feasible. However, the normality of the oocytes taken from tissue which has been frozen still needs to be established. Cryopreservation of ovarian tissue would be potentially an excellent method for storage of human oocytes once methods for their maturation in vitro have been developed.

Adult↗
PubMed1996

Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose.

Feasibility of cryopreservation of mature human oocytes using 1, 2-propanediol and sucrose was studied initially utilizing 1 and 2 day old unfertilized oocytes. Of these 285 aged oocytes 55% survived thawing, and 41% of 128 oocytes inseminated by single sperm intracytoplasmic injection (ICSI) fertilized normally. Limited embryonic development occurred in 51% of these embryos (n = 27) observed for the next 4 days. Cryosurvival of fresh donated oocytes (n = 81) was poorer (n = 20; 24.7%), while fertilization (n = 13; 65%) and embryo development (100%) was good prior to uterine transfer on day 3. Eight oocyte recipient cycles were undertaken, in which cryopreserved donated oocytes were thawed and inseminated by ICSI. Five of these cycles reached embryo transfer, and three pregnancies were initiated though none went successfully to term. Oocyte cryopreservation will ultimately facilitate oocyte donation procedures; however, cryosurvival of fresh frozen oocytes must be improved to at least the degree observed with aged unfertilized oocytes.

Cell Survival↗
PubMed1995

Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol.

This study reports the subsequent embryo development of cryopreserved mature human oocytes following insemination or intracytoplasmic sperm injection (ICSI). Metaphase II oocytes were cryopreserved using a slow freezing-rapid thawing procedure employing the cryoprotectant 1,2-propanediol. The study was conducted at two centres. The normal insemination of cryopreserved oocytes was undertaken in one centre, and ICSI of cryopreserved oocytes in the other. Both methods resulted in a 50% normal fertilization rate. A low rate of abnormal fertilization was observed in the inseminated group of oocytes (5%) compared with 21% for the ICSI oocytes; this was not significantly different. Embryo development was assessed daily for 7 days. All normal fertilized cryopreserved oocytes in both groups cleaved on day 2, with a similar appearance to in-vitro fertilization and ICSI embryos. In the normal inseminated oocytes, there was a significant decrease in the number of embryos cleaving on day 3 (33%) compared with the development of ICSI oocytes, with a subsequent gradual reduction over days 4 and 5 (22 and 11% respectively) resulting in one early blastocyst on day 7 (11%). In contrast, all ICSI-generated embryos continued to cleave on day 3, with a gradual reduction over subsequent days (day 4, 86%; day 5, 57%; day 6, 43%; day 7, 29%). By day 7, two of the blastocysts had started to hatch, resulting in a 66% hatching rate of blastocysts formed from ICSI of cryopreserved oocytes. This is the first study to show normal development to the hatching blastocyst stage following ICSI of cryopreserved human oocytes.

Blastocyst↗
PubMed1995

Parthenogenetic activation of human oocytes following cryopreservation using 1,2-propanediol.

Fresh and aged human oocytes were cryopreserved using 1,2-propanediol (PROH). After thawing, the oocytes were cultured for 20 h and examined for parthenogenetic activation using light microscopy and an ultraviolet DNA stain. Control fresh or aged oocytes and oocytes exposed to PROH without cryoperservation were also examined for activation. No control oocytes were observed to activate spontaneously (n = 43) and parthenogenetic activation was not induced by exposure to PROH alone (n = 26). In both fresh and aged cryopreserved oocytes, 27 and 29% of the oocytes respectively were activated, and these proportions were significantly elevated compared with the controls (P < 0.01). Although a similar rate of activation was observed for the cryopreserved fresh and aged oocytes, the form of parthenogenetic activation varied between these two types of oocyte. A single pronucleus was observed in 18% of the fresh and 5% of the aged cryopreserved oocytes. In contrast, the presence of two or more pronuclei was observed in 0% of the fresh and 19% of the aged cryopreserved oocytes.

Cell Nucleus↗
PubMed1989

Cryopreservation of human and rabbit oocytes and one-cell embryos: a comparison of DMSO and propanediol.

The aim of this study was to improve the cryopreservation of human oocytes and pronuclear embryos. One-step and multiple-step addition of dimethyl sulphoxide (DMSO) and 1,2-propanediol (PROH) and three different freezing protocols with intermediate temperatures of -35, -70 and -110 degrees C were investigated. This work was performed using rabbit oocytes as well as human oocytes and one-cell embryos from the routine IVF programme. Also, human polyploid pronucleate oocytes were used in controlled prospective studies of morphological intactness and development in vitro. Rabbit oocytes survived best (113/126) when PROH was added in one step and controlled freezing stopped at -110 degrees C. But the development was better (141/187) if DMSO was added in multiple steps and the oocytes were cooled to -70 degrees C before being plunged into liquid nitrogen. The mode of addition of the cryoprotectant influenced development only if slow freezing was stopped at -35 degrees C (51 versus 34%). Using PROH, the development after thawing was also better if cooling was stopped at -35 degrees C (51 versus 37%) and DMSO was superior to PROH when the oocytes were cooled slowly to -110 degrees C (66 versus 37%). In the human, significantly more pronucleated than unfertilized oocytes developed after freezing (92 versus 50%). The best results were achieved with pronuclear embryos using 1.5 M PROH and cooling to -110 degrees C, when 91.7% of the surviving oocytes developed further. This is a marked improvement of the development rate and comparable to embryo freezing.

Animals↗
PubMed1989

Propanediol-induced alterations in membrane integrity, metabolism and developmental potential of mouse zygotes.

The effects of 1,2-propanediol (PROH) on embryonic development, membrane integrity and metabolism on B6D2F1 mouse zygotes in the pronuclear stage were evaluated. In both the control and the group treated with 1.5 M PROH, 78% of the zygotes developed into 2-cell embryos. With 3 M PROH, the proportion of 2-cell embryos was only 7% (P less than 0.05). In a second series of experiments, pronuclear mouse eggs were incubated in either fluorescein diacetate (FDA) or 5 microM Acridine Orange (AO) then transferred to PROH. FDA-induced fluorescence, which is maintained until the cell membrane is damaged, was retained in 100% of the control and 98% of the zygotes treated with 1.5 M PROH. Exposure to 3.0 and 6.0 M PROH reduced the percentage of zygotes with FDA-induced fluorescence to 81% (P less than 0.05) and 5% (P less than 0.05) respectively. AO fluoresces yellow-green within the physiological pH range (7.4). After AO exposure, 95% of the control zygotes and 95% of the zygotes exposed to 1.5 M PROH possessed yellow-green fluorescence, indicating a normal cellular pH. Treatment with 3.0 and 6.0 M PROH caused a shift in the fluorescence such that 93% (P less than 0.05) and 100% (P less than 0.05) of the zygotes respectively no longer fluoresced yellow-green, indicating a lower pH. These results demonstrate that a 20-min exposure to 1.5 M PROH does not affect embryonic development, while PROH at greater than or equal to 3.0 M inhibits embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Propanediol alters intracellular pH and developmental potential of mouse zygotes independently of volume change.

Prior to treatment, mouse zygotes were incubated with acridine orange (AO) a fluorescent dye which fluoresces within a physiological pH range. The zygotes were then perifused with 1,2-propanediol (PROH) at rates of 0.18 mol/min, 0.36 mol/min and 1.0 mol/min and zygote volume and intracellular pH monitored. Zygotes perifused with phosphate-buffered saline maintained their initial volume and AO fluorescence. All of the zygotes exposed to PROH at a rate of 1 mol/min decreased in volume and lost their AO fluorescence by 10 min. The volume of the zygotes perifused at less than or equal to 0.36 mol/min was not altered. However, only 25% of the zygotes perifused at 0.36 mol/min maintained their fluorescence at 10 min and all lost their fluorescence by 15 min. At 0.18 mol/min 95% of the zygotes maintained their fluorescence at 10 min and 49% at 15 min. All of the PROH-exposed zygotes lost their fluorescence by 20 min. Although 2-cell development was not affected by 3.0 M PROH for 2.5 min, blastocyst development was reduced compared with controls (P less than 0.05). Longer exposures resulted in a significant decrease in both 2-cell and blastocyst development. These data demonstrate that a 2-7-min exposure to greater than or equal to 2.5 M PROH alters both the intracellular pH and developmental potential. Since these detrimental effects are independent of volume changes and therefore intracellular PROH concentrations, it is postulated that PROH mediates its toxic action by directly altering the cell membrane.

Acridine Orange↗
PubMed1993

Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle.

Human and mouse oocytes were cryopreserved by a slow freeze, rapid thaw method, using propanediol (PROH) as the cryoprotectant. A simulated cryopreservation was also included in the study to detect the level of damage attributable to the PROH alone. Comparison of the mouse and human oocytes cryopreserved by the same method showed opposing results, with a poor morphological survival rate of 4% observed for mouse oocytes and a subsequent normal fertilization rate of 0%. In 171 cryopreserved human oocytes a higher survival rate of 64% was achieved, and this showed more similarity to the mouse pronuclear oocytes survival of 53%. A comparison of human oocytes, cryopreserved within the cumulus and denuded of cumulus and corona prior to cryopreservation, demonstrated a higher survival rate in the denuded oocytes of 69% compared to 48%. A delay prior to cryopreservation of 1 or > or = 2 days had no effect on the immediate survival of oocytes, but culture for a further 24 h after thawing reduced survival, with the day 1 oocytes exhibiting the most dramatic reduction in survival (28%). Using a lectin binding method, abundant cortical granules were observed in all cryopreserved oocytes analysed. The meiotic spindle and chromosomes were examined in cryopreserved oocytes using fluorescence microscopy and 60% of the surviving oocytes had a normal spindle and chromosome configuration.

Animals↗
PubMed1978

Fractionation of unfixed chromatin by buoyant-density centrifugation in gradients containing 3-iodo-1,2-propanediol and metrizamide.

Buoyant-density centrifugation of unfixed chromatin has been performed in a newly devised medium containing 3-iodo-1,2-propanediol and metrizamide. Chromatins were obtained from isotopically labeled mouse hepatoma cells in suspension culture, either grown normally or density labeled in a medium containing bromodeoxyuridine, by mild digestion of isolated nuclei with micrococcal nuclease. When a mixture of normal and density labeled chromatin, marked with [14C]thymidine and [3H]bromodeoxyuridine, respectively, was centrifuged in the medium, chromatin peaks represented by labeled DNA were resolved to the extent expected from their separate banding profiles. Centrifugation of an equivalent chromatin mixture labeled with [14C] and [3H]lysine, respectively, also yielded resolution of chromatin peaks represented by labeled proteins. Only small amounts of labeled proteins were dissociated from chromatin in the gradient medium. Labeled proteins recovered from the gradient fractions were analyzed by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The results suggested that most of the histones remained associated with the original stretches of DNA during the centrifual fractionation period. Essentially all of the dissociated proteins were found to be non-histone proteins.

Bromodeoxyuridine↗
PubMed2004

Effect of 1,2-propanediol versus 1,2-ethanediol on subsequent oocyte maturation, spindle integrity, fertilization, and embryo development in vitro in the domestic cat.

This study assessed the impact of various cryoprotectant (CPA) exposures on nuclear and cytoplasmic maturation in the immature cat oocyte as a prerequisite to formulating a successful cryopreservation protocol. In experiment 1, immature oocytes were exposed to 0, 0.75, 1.5, or 3.0 M of 1,2-propanediol (PrOH) or 1,2-ethanediol (EG) at room temperature (25 degrees C) or 0 degrees C for 30 min. After CPA removal and in vitro maturation, percentage of oocytes reaching metaphase II (MII) was reduced after exposure to 3.0 M PrOH at 0 degrees C or 3.0 M EG at both temperatures. All CPA exposures increased MII spindle abnormalities compared to control, except 1.5 M PrOH at 25 degrees C. In experiments 2 and 3, immature oocytes were exposed to CPA conditions yielding optimal nuclear maturation that either had caused spindle damage (0.75 M PrOH, 1.5 M EG, and 3.0 M PrOH at 25 degrees C) or not (1.5 M PrOH at 25 degrees C). After maturation and insemination in vitro, oocytes were cultured for 7 days to assess treatment influence on developmental competence. CPA exposure did not affect fertilization, but the high incidence of MII spindle abnormalities resulted in a low percentage of cleaved embryos. Blastocyst formation and quality were influenced by both CPA types (EG was more detrimental than PrOH) and concentration (3.0 M was more detrimental than 1.5 M). Overall, cat oocytes appear to be highly sensitive to CPA except after exposure to 1.5 M PrOH at 25 degrees C, a treatment that still allowed approximately 60% of the oocytes to reach MII and approximately 20% to form blastocysts.

Animals↗
PubMed1997

1,2-propanediol-induced premature centromere separation in mouse oocytes and aneuploidy in one-cell zygotes.

Aneuploidy in germ cells results in reproductive failure and mental and physical disorders in humans. Unfortunately, little is known about the causes and mechanisms of aneuploidy induction. The objective of this study was to test the hypothesis that propylene glycol (1,2-propanediol; PG) induces cytogenetic aberrations in mouse metaphase II (MII) oocytes that predispose zygotes to aneuploidy. Female ICR mice received 7.5 IU eCG and 5.0 IU hCG 48 h later. PG doses of 1300, 2600, and 5200 mg/kg body weight were given 3 h post-hCG; controls received the solvent deionized water. Ovulated oocytes were collected 16 h after administration of PG and processed for cytogenetic analysis. For the one-cell zygote cytogenetic study, females were given PG and paired (1:1) with ICR males for 16 h. Females that mated were given 2 x 10(-3) M colchicine 22 h post-PG, and zygotes were collected 18 h later. PG significantly (p < 0.05) increased both the proportion of MII oocytes with premature centromere separation (PCS) and the proportion of aneuploid one-cell zygotes. These results support the hypothesis that PG-induced PCS in MII oocytes predisposes zygotes to aneuploidy.

Aneuploidy↗
PubMed2002

Multiple bridging modes in a novel trinuclear titanium 1,3-dioxypropane compound, Ti3(methyliminodiethanolate)2(1,3-propanediolate)4.

The title compound, bis(2,2'-methyliminodiethanolato)-1kappa(3)O,N,O';3kappa(3)O,N,O'-di-micro(3)-propane-1,3-diolato-1:2:3kappa(8)O:O,O':O'-micro-propane-1,3-diolato-1:3kappa(2)O:O'-propane-1,3-diolato-2kappa(2)O,O'-trititanium(IV), [Ti(3)(C(5)H(11)NO(2))(2)(C(3)H(6)O(2))(4)], has four 1,3-propanediolate ligands binding in three different modes. Two ligands chelate adjacent Ti atoms with normal micro(3)-O bridges, giving typical edge-sharing of the Ti distorted octahedra, one chelating to the central Ti atom with no micro-bridging, and the other spanning the cluster, binding only to the outermost Ti atoms. The two methyliminodiethanolate ligands each coordinate to the outer Ti atoms via their N and two O atoms. The Ti-O bond lengths range, in a self-consistent fashion, from 1.816 (2) to 2.082 (2) A, while the average Ti-N distance is 2.391 (3) A.

Journal Article↗
PubMed2002

Hydrogen bonding in 2-(hydroxymethyl)-1,3-propanediol and N,N'-bis[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]malonamide.

The molecule of 2-(hydroxymethyl)-1,3-propanediol, C(4)H(10)O(3), lies across a mirror plane in space group P2(1)/m, with disorder of both terminal hydroxyl H atoms. The molecules are linked by three O-H...O hydrogen bonds which combine to form sheets; in each O-H...O bond, the H atom resonates between the two O atoms. In the crystal structure of N,N'-bis[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]malonamide, C(11)H(22)N(2)O(8), the molecule lies about a twofold axis and has four strong hydrogen bonds which form a mixture of chains and dimers; these combine to give a three-dimensional supramolecular framework.

Journal Article↗