Oocyte biology and genetics revelations from polar bodies.

The enormous volume of the fertilized egg is attributable to the suppression of cleavage during oocyte growth and the unequal cleavages during the first and second meiotic divisions. The two products of these divisions are the diminutive polar bodies (PB), which contain a redundant set of chromosomes/chromatids plus cytoplasmic organelles. The PB have strictly limited but differential life spans; while viable they possess the genetic potential to support normal embryonic development after transfer to a cytoplast. In addition to the theoretical possibility of using this non-cloning technique to generate more embryos, polar bodies can be used for genetic testing. By cytogenetic analysis of both PB using fluorescent in-situ hybridization (FISH) or chromosome painting, partial or full chromosomal status in the oocyte can be predicted; this approach finds particular application for women of advanced reproductive age as well as with maternally inherited translocations and single gene defects. By studying both of the PB, potential problems of interpretation arising from allele dropout can be reduced; a heterozygous first polar body provides the least ambiguous result. Mitochondria segregate randomly during meiotic cleavages providing an opportunity also to use the PB to screen for mitochondrial mutations and deletions. Thus, the PB can serve useful diagnostic purposes, especially where pre-fertilization screening or avoidance of embryo biopsy is desirable.

Delaying the initiation of progesterone supplementation results in decreased pregnancy rates after in vitro fertilization: a randomized, prospective study.

OBJECTIVE: To compare IVF outcome between two protocols for luteal phase supplementation, one beginning on day 3 after oocyte retrieval and the other beginning on day 6 after retrieval. DESIGN: Prospective, randomized study. SETTING: University-based assisted reproductive technology center. PATIENT(S): One hundred twenty-six consecutive patients undergoing IVF between January and July 2000. INTERVENTION(S): Patients were randomized to begin luteal phase support using vaginal progesterone beginning either on day 3 after oocyte retrieval or on day 6 after oocyte retrieval. MAIN OUTCOME MEASURE(S): Clinical pregnancy rates and implantation rates. RESULT(S): All patients randomized underwent transfer. There were no differences in age, oocytes retrieved, or embryos transferred between the two groups. Those patients receiving luteal phase support with progesterone beginning on day 6 after retrieval had a significantly lower clinical pregnancy rate per transfer compared with those beginning support on day 3 after retrieval (44.8% vs. 61.0%, respectively). This difference in pregnancy rates was greater in those patients undergoing a luteal gonadotropin releasing hormone (GnRH) agonist down-regulation protocol (47.5% vs. 71.4%, day 6 vs. day 3, respectively). Beginning support on day 6 also significantly decreased implantation rates in the GnRH agonist group (21.0% vs. 34.0%, day 6 vs. day 3, respectively). CONCLUSION(S): Pregnancy rates are significantly decreased by initiating luteal-phase progesterone supplementation on day 6 after oocyte retrieval during in vitro fertilization cycles.

High FSH:LH ratio and low LH levels in basal cycle day 3: impact on follicular development and IVF outcome.

PURPOSE: To examine the impact of low basal cycle day 3 serum LH levels or a high FSH:LH ratio on IVF results. METHODS: A homogeneous group of patients was analyzed as identified by normal basal cycle of follicle stimulating hormone (FSH), Luteinizing hormone (LH), and estradiol (E2) levels. High responders (high LH:FSH ratio) and low responders (high FSH or E2 levels, and women > or = 42 years of age) were excluded from analysis. Only cycles stimulated with a combination of a GnRHa (luteal suppression) and pure FSH were studied. RESULTS: Patients with low basal LH levels (< 3 mIU/mL) did not differ significantly from controls in terms of response to controlled ovarian hyperstimulation but there was a clear trend toward poorer implantation and clinical pregnancy rates. On the other hand, patients with a high FSH:LH ratio (> 3) had significantly fewer mature oocytes aspirated, and lower implantation and clinical pregnancy rates than patients with gonadotropin ratio < or = 3. These negative effects were evident in the presence of normal basal FSH levels and after adequate matching of female's age and number of embryos transferred. CONCLUSIONS: These studies highlight a negative impact of a basal cycle high FSH:LH ratio (and possibly low LH levels) on follicular development and oocyte quality in these patients subjected to pituitary down-regulation followed by pure FSH administration. A high FSH:LH ratio may be therefore used as an early biomarker of poor ovarian response.

Characterization of telomerase activity in the human oocyte and preimplantation embryo.

Telomerase, a ribonucleoprotein, has been described as an essential component of highly proliferative cells as it stabilizes the telomeres and avoids cellular senescence. The objective of this study was to modify the polymerase chain reaction-based telomeric repeat amplification protocol to detect telomerase activity in the single cell and to characterize the activity expressed in the human oocyte through to the blastocyst stage embryo. A comparative evaluation of telomerase activity and developmental stage was conducted using discarded or donated human oocytes and embryos. Telomerase activity was detected in all developmental stages evaluated from immature oocytes through to blastocyst stage embryos. Immature oocytes and blastocysts had similar levels of telomerase activity; however, both groups had significantly (P < 0.05) higher activity than zygote through to pre-morula stage embryos. Seventy-five thawed zygotes were cultured to day 3, biopsied by removing 1-2 cells, and the biopsied embryos were cultured to blastocyst stage. There was no difference (P < 0.05) in telomerase activity between cells biopsied from embryos that reached the blastocyst stage and cells from those that arrested in growth. This study has shown that human oocytes through to blastocyst stage embryos express telomerase activity, but that the level of telomerase activity in biopsied blastomeres, of the day 3 cleavage stage embryo, is not predictive of embryonic growth potential.

Characterization of the biologic activities of a recombinant human zona pellucida protein 3 expressed in human ovarian teratocarcinoma (PA-1) cells.

OBJECTIVE: This study was undertaken to clone and express a recombinant human zona pellucida protein 3 and to characterize its biologic activities as a sperm ligand and an inducer of the acrosome reaction. STUDY DESIGN: Human ovarian teratocarcinoma (PA-1) cells were transfected with an expression vector containing human zona pellucida protein 3 complementary deoxyribonucleic acid with a sequence coding for a 6-histidine tail introduced into its 3' end. Purification of the secreted glycoprotein was performed by sequential affinity (lectin and nickel--nitrilotriacetic acid) and ion-exchange chromatography. RESULTS: Western blot analysis confirmed a molecular weight of approximately 65 kd for the purified product. A cell-free translation system revealed a correctly sized protein backbone of 47 kd. The recombinant human zona pellucida protein 3 demonstrated specific, potent, and dose-dependent competitive inhibition of sperm-zona pellucida binding in vitro under hemizona assay conditions. Recombinant human zona pellucida protein 3 also stimulated the acrosome reaction of live sperm. This effect was fast, dose dependent, and capacitation time dependent. Furthermore, advance incubation with pertussis toxin, an inactivator of heterotrimeric G proteins, blocked recombinant human zona pellucida protein 3--induced acrosomal exocytosis. CONCLUSION: The recombinant human zona pellucida protein 3 expressed in PA-1 cells manifested the full spectrum of expected biologic activities. It therefore represents a valuable tool for examination of human fertilization and the design of new strategies in diagnosis of male factor infertility and in contraception.

Vaginal misoprostol enhances intrauterine insemination.

This study examined whether the prostaglandin E(1) analogue misoprostol (400 microgram), when placed vaginally at the time of intrauterine insemination (IUI) improves pregnancy rates. A prospective, placebo-controlled, randomized and double-blind study involving 274 women in 494 IUI cycles resulted in a total of 64 pregnancies (13% per cycle). Misoprostol cycles totalled 253, with 43 pregnancies (17% per cycle), whereas placebo cycles totalled 241, with 21 pregnancies (9% per cycle). The cumulative pregnancy rate with misoprostol treatment was significantly greater than with placebo (P = 0.004, Cox proportional hazards regression). The benefit of misoprostol was seen in clomiphene cycles (14 versus 4%, P = 0.006), and was indicated in FSH cycles (33 versus 15%, borderline significance) and natural cycles (15.6 versus 7.7%, not significant), but was not seen in clomiphene/FSH cycles (18.2 versus 23.5%, not significant). Misoprostol treatment did not increase pain score on the day of IUI (1.1 versus 1.4) and at 1 day post IUI (0.6 versus 0.8). Complications were rare in both groups [six (2%) subject cycles in the misoprostol cycles compared with two (1%) in the placebo group]. It is concluded that the use of vaginal misoprostol may improve the chance for pregnancy in women having IUI in a wide variety of cycle types.

Is the timing of implantation affected by zona pellucida micromanipulation?

PURPOSE: Our purpose was to examine the timing of implantation and early embryo development following uterine transfer of oocytes/embryos previously subjected to zona pellucida micromanipulation. METHODS: A total of 68 singleton pregnancies resulting from IVF and embryo transfer with/without micromanipulation. Patients were divided into four groups according to the type of micromanipulation technique: assisted hatching, embryo biopsy, intracytoplasmic sperm injection, and no micromanipulation (control group). Serial serum beta-hCG levels were measured between 10 and 25 days after fertilization and log-transformed. Linear regression analyses were performed and extrapolated to hCG = 10 mIU/ml (hCG10) to estimate detectable implantation. The slopes of the regression lines were used to estimate the rising speed of hCG, an indirect sign of embryo development. RESULTS: There were no significant differences among groups with respect to hCG10, the slopes or intercepts of the regression lines. CONCLUSIONS: Various oocyte/embryo microsurgical procedures used in ART involving zona pellucida manipulation do not appear to affect the timing of implantation or early embryo development.

Crinone 8% (90 mg) given once daily for progesterone replacement therapy in donor egg cycles.

OBJECTIVE: To determine whether once-daily dosing of Crinone 8% (90-mg progesterone vaginal gel; Serono Laboratories, Inc., Norwell, MA) is sufficient for normal endometrial development and pregnancy support. DESIGN: Prospective cohort study. SETTING: Academic medical center. PATIENT(S): Eighty-six women who required complete progesterone replacement for a donor egg cycle. INTERVENTION(S): Crinone 8% (90 mg) once daily or IM progesterone (100 mg) once daily was administered from day 15 onward. Both groups underwent an endometrial biopsy on day 26 of a mock cycle, followed by a second cycle in which ET was performed. MAIN OUTCOME MEASURE(S): Endometrial development, serum progesterone levels, pregnancy rates, implantation rates, and bleeding patterns. RESULT(S): Mean (+/-SD) serum progesterone levels on day 26 were 11.3+/-6.5 ng/mL in the Crinone group and 65.2+/-12.5 ng/mL in the IM progesterone group. At histologic examination, endometrial biopsy specimens were found to be "in phase" for 100% (42/42) of women in the Crinone group and 95.5% (42/44) of women in the IM progesterone group. Although 8 of 42 patients had serum progesterone levels of <6 ng/mL, there was no correlation with endometrial development. Only 1 patient bled before the 14th day of progesterone therapy, and she went on to be delivered of twins. Clinical pregnancy, ongoing pregnancy, implantation, and miscarriage rates were not statistically different for the Crinone and IM progesterone groups: 45.6% (21/46) versus 52.3% (23/44); 39.1% (18/46) versus 40.9% (18/44); 21.5% (34/158) versus 19% (30/158), and 14.3% (3/21) versus 22% (5/23), respectively. Power was sufficient to detect a 25% difference in clinical pregnancy rates. CONCLUSION(S): Crinone 8% administered once daily appears to produce the same endometrial development and pregnancy rates as IM progesterone in women who require complete progesterone replacement, and it does not cause early bleeding.

Protecting the endometrium. Opposing the hyperplasia/malignancy potential of ERT.

Many trials have examined the clinical and histologic effects of various hormone replacement therapy combinations with the objective of minimizing the incidence of hyperplasia and the potential for subsequent development of adenocarcinoma. Reviewing the results of these trials, it appears that high-dose, long-term progestogen therapy is effective in protecting the endometrium, with duration having a greater impact than dose. Among women given 0.625 mg conjugated equine estrogen (CEE), sequential regimens should include 5 or 10 mg medroxyprogesterone acetate (MPA) or 200 mg micronized progesterone for 12 days or more. Continuous combined regimens require 2.5-5 mg MPA. With women who are taking 1.25 mg CEE the data are less clear, but recommendations include administration with 10 mg MPA for 12-14 days or 5 mg MPA continuous combined therapy.

Vaginal bleeding patterns in women receiving hormone replacement therapy. Impact of various progestogen regimens.

Postmenopausal bleeding is more prevalent than was previously thought, occurring in women regardless of whether or not they are on hormone replacement therapy. While estrogen-only regimens have been used for several beneficial effects, bleeding patterns associated with these regimens can be irregular and unpredictable, causing discomfort to the patient as well as increasing the risk of both endometrial hyperplasia and carcinoma. Studies in recent years have examined the effects of estrogen-only regimens as compared to different estrogen-plus-progestogen combination therapies to help regulate and minimize postmenopausal bleeding while providing endometrial protection.

The molecular mechanisms of oocyte maturation and early embryonic development are unveiling new insights into reproductive medicine.

The purpose of the present review is to outline the current understanding on the molecular mechanisms governing various stages of oocyte maturation, transition from maternal to embryonic control and the initial steps of pre-embryo development. The cytoplasmic and nuclear maturation of the oocyte during pre-ovulatory development can be viewed as separate entities. Cytoplasmic maturation and the acquisition of stores of RNA and protein dominates oocyte development between the premordial and pre-ovulatory stages of development. Initiation of nuclear maturation is marked by the breakdown of the nuclear envelope, or germinal vesicle and is triggered by the midcycle luteinizing hormone peak. In vitro, this is associated with a decrease in the intracellular concentrations of cAMP. This and several subsequent steps of meiosis are controlled by the M-phase promoting factor (MPF). While the constituents of MPF, p34cdc2 kinase and B-type cyclin, are also present in mitotically dividing cells, in meiotically dividing oocytes the regulation of MPF activity differs. An oocyte-specific protein kinase, c-mos, plays an important role in up-regulating the activity of MPF at various stages of final oocyte maturation. Several lines of evidence suggest that the proper function of the c-mos-MPF system is associated with important features of the last stages of oocyte maturation such as the resumption of meiotic maturation, inhibition of DNA replication between meiosis I and II, and the maintenance of the oocyte at metaphase II arrest until it is fertilized. Eventually the destruction of c-mos and active MPF following fertilization allows the initiation of mitotic cell division in the pre-embryo. The very first cell divisions of the human pre-embryo are still under the control of maternally inherited mRNA and protein. Several lines of evidence suggest that in humans, zygotic gene expression is initiated between the 4- and 8-cell stages, after which the pre-embryo begins to utilize its own genes. Some of the first genes to be expressed in the human pre-embryo encode proteins that are associated with cell division, extracellular growth modulatory signals as well as factors associated with implantation. We acknowledge that most of the data presented comes from species other than human, therefore at present the full biological role of the proposed regulatory pathways and control mechanisms for human biology remains speculative.

Experience with a novel vaginal progesterone preparation in a donor oocyte program.

OBJECTIVE: To compare the efficacy of a vaginal progesterone preparation with our standard IM preparation within a donor egg program. DESIGN: Prospective randomized trial. SETTING: Donor egg program at a university assisted reproductive therapy program (Jones Institute for Women's Health). PATIENT(S): Couples accepted into the donor egg program because of either premature ovarian failure or evidence of diminished ovarian reserve. INTERVENTION(S): Women were randomized into either a group receiving IM progesterone replacement or a group receiving vaginal progesterone replacement. Both groups underwent Estraderm patch/progesterone treatment in a mock cycle leading to an endometrial biopsy on day 26 followed by a second cycle in which ET was performed. Subjects with residual ovarian function received a GnRH agonist. In the IM treatment group, 100 mg was administered from cycle days 15 to 27. In the vaginal treatment group, Crinone 8%, a polycarbophil-based gel preparation containing 90 mg of micronized progesterone, was administered twice daily from the evening of day 14. MAIN OUTCOME MEASURE(S): Endometrial histology, serum levels of progesterone (on days 13, 17, 20, 24, and 26), the occurrence of pregnancy, implantation rate, and pregnancy outcome. RESULT(S): Fifty-four women randomized into the vaginal progesterone treatment group and 18 women in the IM treatment group achieved ET. Mean serum progesterone levels were higher in the IM treatment group than in the Crinone group. Endometrial histology was "in phase" for all subjects in both groups. Clinical pregnancies were observed in 26 of 54 women and 5 of 18 women in the Crinone and IM progesterone groups, respectively. The ongoing pregnancy rate (PR) of 31% (17/54) and implantation rate of 23% in the subjects receiving Crinone was not statistically different from the IM progesterone group's ongoing PR of 22% (4/18) and implantation rate of 18%. CONCLUSION: Vaginal progesterone replacement with the polycarbophil gel preparation was as effective as IM progesterone in producing clinical and ongoing pregnancies within our donor egg program in the dosages administered.

Endometrial effects of RU486 in primates--antiproliferative action despite signs of estrogen action and increased cyclin-B expression.

Continuous antiprogestin administration to hormone replaced, castrate monkeys inhibits estrogen-induced endometrial proliferation through mechanisms which remains unclear. To elucidate the molecular mechanisms of RU486-induced endometrial suppression, we treated six intact female cynomolgus monkeys on cycle days 2-22 sequentially with placebo, RU486 (1 mg/kg/day) and levonorgestrel (LNG) (2 microg/kg/day) intramuscularly (i.m.), with uterine wedge sections and endometrial biopsies collected on day 22 of each cycle. The uterine sections were evaluated for morphology, mitosis and proliferating cell nuclear antigen (PCNA) immunohistochemistry. Changes in the mRNA levels of ER, PR, cyclin-B and tumour suppressor gene p21 were assessed using co-amplification with beta-actin by reverse transcriptase-polymerase chain reaction (RT-PCR). Administration of RU486 uniformly resulted in characteristic suppression of endometrium with few mitosis, dense stroma and simple glands, whereas the effects of LNG were less uniform. Following RU486 administration, the levels of endometrial ER and PR mRNA were comparable to proliferative phase endometrium, and significantly higher than those seen in the secretory endometrium, indicating that some of the biological actions of E2 were not inhibited during RU486 treatment. Despite scarce mitosis, PCNA was readily detectable in all samples. Curiously, in comparison to secretory phase controls, the levels of cyclin-B, but not p21, mRNA were markedly increased following RU486. The effects of LNG on the levels of these mRNA species varied, with mean levels falling between those of the secretory phase controls, and RU486-treated specimens. The increase in cyclin-B mRNA and lack of mitosis suggests that anti-proliferative actions of RU486 in the primate endometrium might be associated with a cell-cycle block at the G2-M interphase. Whether mechanisms similar to these are associated with the beneficial clinical effects of RU486 seen in the treatment of various hormone dependent maladies remains to be determined.

Messenger ribonucleic acid kinetics in human oocytes--effects of in vitro culture and nuclear maturational status.

OBJECTIVE: To study the effects of different nuclear maturational status (prophase I [PI] versus metaphase II [MII]) and in vitro culture on the kinetics of maternal messenger ribonucleic acid (mRNA) in human oocytes. DESIGN: Molecular biology on excess oocytes obtained from our clinical IVF program. INTERVENTIONS: The oocytes, classified as either PI or MII at collection, were used as such or cultured in vitro for an additional 24 hours. The relative levels of c-mos and cyclin-B1 were measured using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: The mean levels of c-mos and cyclin-B1 transcripts were indistinguishable between the PI, MII, PI oocytes matured in vitro, PI oocytes failing to mature, and MII oocytes cultured for additional 24 hours. The variability in the levels of these transcripts increased during the vitro culture. CONCLUSIONS: The level of c-mos and cyclin-B1 transcripts were not different in PI versus MII oocytes, therefore, differences seen in the clinical outcome of PI and MII oocytes may be unrelated to levels of these gene products. C-mos and cyclin B1 mRNA were maintained in vitro, thus degradation of maternal RNA is not activated in excess during the 24-hour culture.

Polymerase chain reaction amplification specificity: incidence of allele dropout using different DNA preparation methods for heterozygous single cells.

PURPOSE: The purpose was to evaluate methods of DNA preparation in a single cell to determine the ability to amplify and correctly diagnose a targeted gene. METHODS: One- or two-cell lymphoblasts (n = 100/group), heterozygous for the normal and 4-base pair insertion on exon 11 of the beta-hexosaminidase A gene, were collected and prepared under the following conditions: (1) freeze-thaw liquid nitrogen, then boiling (LN2); (2) potassium hydroxide/dithiothreitol, heated to 65 degrees C, followed by acid neutralization (KOH); (3) boiling only (Bl); and (4) water only (H2O). Cells were analyzed by polymerase chain reaction using nested primers. RESULTS: The total number of cells amplifying [in brackets] and the cells with amplification for both alleles (heterozygous), the normal allele, or the mutant allele were as follows, respectively: LN2 [38], 11, 16, 11; KOH [97], 91, 5, 1; Bl [41], 17, 13, 11; and H2O [85], 41, 16, 28. With two cells per reaction tube the results were as follows: LN2 [85], 53, 14, 18; and KOH [97], 96, 1, 0. CONCLUSIONS: KOH lysis was significantly greater than with all other methods (P < 0.006) and should be used for single cells. This study also demonstrates the importance of using heterozygous cells to determine the ability to amplify both alleles as a method of quality control for single-cell analysis.

Primate reproductive organs reveal a novel pattern of proto-oncogene c-mos and transcription factor Oct-3 mRNA expression.

In mice, expression of the transcription factor Oct-3 and the proto-oncogene c-mos is limited to germ cells, suggesting a specific role for these factors in gamete physiology and early embryonic development. We have studied the expression pattern of Oct-3 and c-mos in various reproductive as well as control tissues in the cynomolgus monkey, using reverse transcriptase polymerase chain reaction (RT-PCR) and Northern analysis. Analogously with the data from the mouse model, strong expression of Oct-3 and c-mos could be detected in monkey ovary and oocytes. Unexpectedly, strong expression of c-mos was demonstrable in the pituitary gland and the amount of mRNA expression in the pituitary was roughly equal to that found in the ovary. Of the tissues examined, the testicular expression of c-mos was the most intense. Weak signal for c-mos mRNA was also seen in hypothalamus and brain; however, all other tissue types examined were negative for c-mos expression. In addition to the oocytes, expression of Oct-3 mRNA was detected in the ovarian granulosa cells, fallopian tube, myometrium, cervix, breast, liver, adrenal gland, pituitary, hypothalamus, brain cortex, prostate, and in testis. Thus, in the cynomolgus monkey, Oct-3 is predominantly, but not specifically, expressed in reproductive tissues. In the female monkey reproductive organs, the expression of c-mos seems to be germ cell specific. Therefore, further characterization of c-mos and Oct-3 functions in primate reproductive physiology, especially in gametogenesis and early embryonic development, is highly warranted.

Variations in bone status of contralateral and regional sites in young athletic women.

To determine if volleyball (VB), basketball (BB), soccer (SO) and swimming (SW) programs were associated with site-specific differences in contralateral, regional, and total body bone mineral density (BMD), 62 eumenorrheic female athletes [BB (N = 7), VB (N = 11), SO (N = 9), and SW (N = 7)] and controls participated in the study. The controls were categorized as either moderately active control (MOD) (N = 17) or sedentary control (SED) (N = 11) based on fitness and activity assessments. Contralateral, total body, lumbar (L2-L4), and femur BMD were measured (Lunar DPX). The between sport contralateral comparisons indicated that VB and BB had significantly greater leg and arm measurements than all other groups, while the within contralateral comparisons revealed significantly greater right arm measurements for all groups, except SW. No significant differences were found for the within group contralateral leg comparisons, except VB. VB and BB had significantly higher (P < or = 0.05) total body and lumbar BMD values than SW, MOD, and SED. At the femur neck, trochanter, and Ward's triangle, BB showed significantly higher BMD than SW, MOD, and SED. Only BB had significantly higher Ward's triangle BMD than SW, MOD, and SED. Our findings show site-specific differences in BMD associated with selected sports' programs.

Osmotic and physiological responses of mouse zygotes and human oocytes to mono- and disaccharides.

To survive cryopreservation, oocytes, zygotes and embryos must tolerate a sequence of volumetric contractions and expansions. These result as an egg or an embryo is exposed to a permeating cryoprotective additive, then to an increase followed by a decrease in the osmolality of its extracellular milieu as water freezes during cooling and then melts during warming, and finally to the dilution of the cryoprotective additive solution. Measurements of the extent to which mouse zygotes and human oocytes undergo osmotic contraction have been made by exposing them to solutions of monosaccharides (fructose, galactose, glucose) or disaccharides (maltose, sucrose, trehalose), ranging in concentration from 0.25 to 1.50 M. Mouse zygotes and human oocytes exhibit very similar responses to these solutions. Their volumes contract linearly as a function of 1/(osmolality) of the solutions, yielding estimates of non-osmotic volumes of 13-23%. Mouse zygotes exposed to 1.5 M concentrations of these solutions for 10 min lose 85% of their cell water. Yet > 75% of treated zygotes develop into hatching blastocysts. Human oocytes also appear to survive such extreme dehydration, based on a vital dye assay. These results suggest that solutions of various non-permeating saccharides can serve as osmotic buffers for the recovery of cryopreserved oocytes, zygotes and embryos.