Journal of Assisted Reproduction and Genetics, Vol. 9, No. 4, 1992

NEW TECHNIQUES

Survival of Human Oocytes Cryopreserved with or Without the Cumulus in 1,2-Propanediol DANIEL G. IMOEDEMHE 1'2 and ALEJANDRO B. SIGUE 1

Submitted: December 11, 1991 Accepted: June 26, 1992

INTRODUCTION

Background: Although cryopreservation of human preembryos has been carried out with success, the cryostorage of oocytes, which pose fewer controversial moral, ethical, and legal problems has been much less successful. Various attempts to cryopreserve human oocytes have been mostly unsuccessful and the search for an optimal protocol for oocyte cryopreservation remains elusive. We therefore undertook this study to determine the effect of oocyte cryostorage in 1,2-propanediol. Method: Mature human oocytes with or without their cumuli were cryopreserved in precooled 1,2-propanediol, then thawed and inseminated with sperms for in vitro fertilization. The outcome of insemination and subsequent embryonic development were also recorded and compared. Results: Postthaw cryosurvival rate was significantly better when cryostorage was carried out with the oocyte cumulus intact as compared to those oocytes denuded of their cumuli (54 versus 27%, respectively; P < 0.05). Eight (44%) of 18 surviving postthaw oocytes with intact cumuli were fertilized normally, with cleavage in six, as compared to two (25%) and one, respectively, of those denuded of their cumulus prior to cryostorage. Development to the blastocyst stage was achieved in three embryos derived from oocytes with an intact cumulus at cryostorage. Conclusion: We conclude that 1,2-propanediol can be used with success in oocyte cryopreservation, although the issue of parthenogenecity is still to be resolved. Oocyte's with intact cumulus survive cryostorage better than those without it.

Whereas cryopreservation of human oocytes poses fewer legal and ethical problems, it has been employed with only little success in clinical practice. Cryopreservation of pronucleate and early cleavage embryos has been used with success (1--4) but only three successful pregnancies have been reported following uterine transfer of embryos derived from frozen human oocytes (5,6). Reduced survival following cryostorage (7-9) coupled with reduced fertilization rate and increased occurrence of polyploidy following fertilization of frozen-thawed oocytes has been reported (7, I0,11). In 1988 Kola et al. (12) also reported twoto threefold increase in the incidence of aneuploidy in mouse oocytes as a result of cooling. Cooling of oocytes leads to depolymerization of their meiotic spindles (13) with increased potential for aneuploidy. Dimethyl sulfoxide (DMSO), the commonly used cryoprotectant, causes spindle polymerization with increased potential for polyploidy (7,11-15). Therefore, a combination of cooling and DMSO, in theory, should produce a lesser adverse effect on the metaphase plate. However, DMSO has also been reported to cause disruption of the subcortical microfilament with release of cortical granules, leading to premature zona reaction and subsequent block to fertilization (16,17). Recent reports (9,1821) suggest that the thermo- and chemosensitivity of oocytes to cooling and DMSO, respectively, are dependent on the duration of and temperature at which exposure occurred, with a better outcome when oocytes are exposed to DMSO at lower temperatures. Although 1,2-propanediol (PROH) is regarded as a less toxic and more permeable cryoprotectant than DMSO, it has not been explored to the

KEY WORDS: cumulus; oocyte; cryopreservation; precooled; 1,2-propanediol.

1 Human Reproductive Biology Unit, Soliman Fakeeh Hospital, P.O. Box 2537, Jeddah 21461, Saudi Arabia. 2 To whom correspondence should be addressed.

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same extent as the latter in oocyte cryopreservation. This perhaps is due to its suggested ability to induce parthenogenecity (19,22). The effect of denuding oocytes of their cumulus cells prior to cryostorage is controversial in the few studies in which this has been evaluated. Trounson and Kirby (9) reported that removal of the cumulus adversely affected the outcome of cryopreservation, while other reports (23,24) have found no adverse effect. Johnson and Pickering (18) reported that the presence of the cumulus reduced the adverse effect of DMSO on the oocyte. In this study, we report our observations on cryopreservation of unfertilized human oocytes with or without their cumulus cells in PROH precooled to 4°C. The survival after thawing, in vitro fertilization, and subsequent embryonic development in vitro is also reported.

MATERIALS AND METHODS Sixty-three morphologically mature oocytes in excess of patients' therapeutic needs were obtained from 31 women undergoing in vitro fertilization (IVF) for tubal disease. All the couples were informed of the purpose of the study and they gave their consent for a maximum of 3 oocytes to be obtained from their ovaries after at least 10 mature oocytes had been aspirated for therapeutic purposes. They were informed that any embryos generated could be replaced in the uterine cavity only if no embryo was obtained from routine IVF of their therapeutic 10 or more oocytes. The study was carried out with the approval of the hospital internal regulatory body on the understanding that no thirdparty gametes would be involved. The mean age of the women was 31.7 years (range, 25-36 years). They had regular 26- to 32-day cycles and all the husbands had normal semen parameters. Controlled ovarian hyperstimulation (COH) was carried out in all the women with human menopausal gonadotropin (hMG; Pergonal; Serono, Rome, Italy) injections, 225 U daily from the second day of the menstrual cycle until the eighth day. Thereafter, the dose was varied according to individual response as evaluated by pelvic ultrasonography and serum estradiol from 150 to 225 U daily until three or more follicles of 19-mm mean diameter were present. The patients thereafter received 5,000 U of intramuscular human chorionic gonadotropin (hCG; Profasi; Serono, Rome, Italy) for fol-

IMOEDEMHE AND SIGUE

licular maturation 34--36 hr before oocyte recovery. Follicular monitoring by both ultrasound scan and serum luteinizing hormone (LH) and estradiol (E2) were carried out as described previously (25,26). Oocyte recovery was done in all the patients by transurethral ultrasound-guided oocyte recovery. Oocytes were scored for oocyte cumulus complex maturity as soon as they were recovered. Routine IVF laboratory techniques were as previously described (25) except for the oocyte freezing and thawing. Two mature oocytes were obtained from each patient excepting for one from whom three were obtained. Oocytes were allocated at random for freezing with their cumulus intact or with the cumulus and corona cells stripped. They were matched on a one-to-one basis except in the patient in whom three were obtained, two of which were allocated for freezing with the intact cumulus. Removal of the cumulus was achieved by exposure to 0.1% hyaluronidase (Sigma, UK) in Earle's medium at 37°C for 5 min. The corona cells were removed by gentle mechanical manipulation with a drawn-out pipette. The oocytes were then washed three times in Earle's medium supplemented with 10% maternal serum to rid them of any residual hyaluronidase. They were then incubated for 1 hr in the same medium in an atmosphere of 5% CO2, 5% 02, and 90% N 2 at 37°C prior to subjecting them to the freezing protocol. Oocytes with intact cumuli were incubated in the same environment for 4 hr following recovery prior to freezing. The freezing protocol is shown in Fig. 1. The oocytes were equilibrated stepwise for 5 min at a time in Earle's medium containing 0.5 M and then 1.0 M PROH. This was followed by equilibration for 5 min in 1.5 M PROH with 0.25 M sucrose and then 2.0 M PROH with 0.25 M sucrose. Equilibration cryoprotectant solutions were all precooled to 4°C prior to exposure of the oocytes. Oocytes were then loaded into plastic straws and transferred onto a computerized cell freezer (R 204; Planer Biomed, Middlesex, UK). Oocytes were cooled rapidly at 2°C/min to - 7 ° C when seeding was induced automatically and the temperature was held at this temperature for 5 min. Thereafter, a slow cooling rate of 0.3°C/rain was maintained until - 35°C, following which a rapid rate of 10°C/min was maintained up to 135°C, followed by plunging into liquid nitrogen. Oocytes were left stored in liquid nitrogen at - 196°C for 16-18 hr, after which they were thawed following the protocol shown in Fig. 2. Briefly, -

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straws were allocated to thaw in precooled medium at 4°C. Oocytes were then expelled into precooled Earle's medium at 4°C supplemented with 1.5 M PROH and 0.25 M sucrose. Stepwise removal of cryoprotectant in a reverse order to the equilibration procedure was carried out also at 4°C. Thereafter, oocytes were transferred to Earle's medium supplemented with 10% maternal serum at 37°C and washed three times to remove any residual cryoprotectant. They were then incubated for 2 hr at 37°C and inspected for intact survival prior to insemination with 50,000 to 100,000 washed and preincubated sperm from the patients' husbands. Oocytes were inspected for normal fertilization as evidenced by the presence of two pronuclei along with the presence of two polar bodies in the perivitelline space 16-18 hr later. Subsequent embryonic development of the fertilized oocytes was recorded. Statistical Analysis. Statistical analysis was carried out by chi-square test. A P value of less than 5% was regarded as significant.

/

MORPHOLOGICALLY MATURE OOCYTES

CUMULUS FREE

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0,1% HYALURONIDASE IN EARLE'S MEDIUM AT 37 °C

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INCUBATION (1 HR) EQUILIBRATION AT 4 °C

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2.0M

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PROH +

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CELL FREEZER (PLANER) 2,0 °ClmJn

-7 °C

SEEDING (5 mins)

-

196 °C]

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I STEPW!SE REMOVALOF CRYOPROTECTANT

I

i

INCUBATION FOR 2 HRS AT 37 "C ] (Inspection for Intact Survival)

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.L

1 OBSERVATION FOR FERTILIZATION AND EMBRYONICDEVELOPMENT

Fig. 2. Thawing and removal of cryoprotectant.

RESULTS

INTACT CUMULUS

[°l.

LIQUID NITROGEN

A total of 33 oocytes was frozen with their intact cumuli, whereas 30 had their cumulus stripped prior to freezing (Table I). Eighteen (54.5%) of oocytes frozen with an intact cumulus survived cryopreservation, vs eight (26.6%) of those without a cumulus. This difference was statistically significant (Xz = 5.039; P < 0.02). Eight (44.4%) of the surviving oocytes with an intact cumulus were successfully fertilized as demonstrated by the presence of two pronuclei and two polar bodies in the perivitelline space at 16--18 hr postinsemination. Six (75%) of these cleaved, of which three (50%) developed to fully expanded blastocysts at 130-140 hr postinsemination. Only two (25%) of the eight oocytes frozen without their cumulus that survived showed evidence of fertilization, and only one of these cleaved and did not develop beyond the two-cell stage. One Table I. Summary of the Outcome of Oocyte Cryopreservation

[

~0.3 ~C/min

J. lO °C/min

LIQUID NITROGEN AT -196 °C Fig. I. The freezing protocol.

Journal of Assisted Reproduction and Genetics, Vol. 9, No. 4, 1992

Postthaw survival No. fertilized Parthenogenetic activation No. cleaved Expanded blastocyst * X2 =

5.039; P < 0.02.

Cumulus intact (n = 33)

Cumulus denuded (n = 30)

18 (54.5%) 8 I (5.5%) 6 (75%) 3 (50%)

8 (26.6%) 2 (25%) -1 (50%)

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oocyte in the group with an intact cumulus demonstrated only one pronucleus at 16-18 hr postinsemination. It did not cleave and was excluded from the analysis as it was presumed to have been activated parthenogenetically.

DISCUSSION Cryopreservation of human embryos has been applied clinically with considerable success (1-4) but the same cannot be claimed for oocyte cryostorage. The chemosensitivity and cryosensitivity of oocytes during the process of cryopreservation have posed considerable difficulties, with disappointing results overall. Only three successfully completed pregnancies resulting from cryostored oocytes have been reported so far (5,6). In this study, we have tried to assess two factors, namely, the use of PROH and the effect of removal of the cumulus cells on cryostorage. The cryoprotectant PROH has been used widely with success in e m b r y o c r y o p r e s e r v a t i o n but has been only sparsely investigated for use in oocyte cryopreservationl Our observations in this study indicate that when PROH is precooled to 4°C prior to exposure of the oocyte to it, an acceptable rate of cryosurvival can be obtained. Cryosurvival under this condition is significantly improved by exposure of the intact oocyte with its cumulus to the cryoprotectant prior to freezing. Previous reports (19,21) have indicated that precooling of DMSO prior to oocyte exposure to it reduced the adverse effect of this cryoprotectant to the oocyte. Our report, while not testing this proposition, indicates that acceptable rates of intact cryosurvival of the oocyte can be obtained under the same conditions with PROH. PROH is a less toxic cryoprotectant than DMSO and also has a higher permeability coefficient than DMSO. These two characteristics may make it more acceptable than DMSO in oocyte cryopreservation. The effect of the removal of cumulus cells prior to oocyte exposure to cryoprotectant and cryostorage has been little studied. While no adverse effect has been reported following removal of the cumulus cells from mouse (23) and human (24) oocytes prior to cryopreservation, others (9,20) have found that this practice adversely affected the outcome. Our observation agrees with the latter observation, as only 27% of oocytes with their cumuli removed survived postthaw, compared to the 54% survival ob-

IMOEDEMHE AND SIGUE

served in those with an intact cumulus. This is further strengthened by our observations of a 44% fertilization rate in the same group and a 75% cleavage rate of fertilized oocytes. This indicates that the cumulus ceils may offer protection against the adverse effect of cryoprotectant and or cooling in a way yet unexplained. We can only hypothesize that perhaps the presence of the cumulus cells may offer some protection against sudden osmotic changes and stresses that could be induced by rapid influx and or efflux of cryoprotectant during the procedures of equilibration in and removal of cryoprotectant in the prefreeze and postthaw periods, respectively. Johnson (20) observed that the cumulus cells had to be removed after removal of cryoprotectant in order to optimize fertilization. We were unable to test this observation but our fertilization rate of 44% indicates that this may be unnecessary. Further studies would be required to resolve this. The sample presented here is small, thus limiting a definitive conclusion on such issues as the parthenogenecity of PROH. Nonetheless, our results indicate that PROH can be successfully employed in oocyte cryopreservation. They also indicate that oocytes may be better cryostored with their cumulus intact in order to optimize cryosurvival. We are of the opinion that these data give cause for optimism with regard to human oocyte cryopreservation and should encourage further studies in this area.

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8. Mandelbaum J, Junca AM, Plachot M, Alnot MO, SalatBaroux J, Alvarez S, Tibi C, Cohen J, Debache C, Tesquier: Cryopreservation of human embryos and oocytes. Hum Reprod 1988;3:117-119 9. Trounson A, Kirby C: Problems in cryopreservation of unfertilized eggs by slow cooling in dimethyl sulfoxide. Fertil Steril 1989;52:778-786 10. Feichtinger W, Benko I, Kameter P: Freezing human oocytes using rapid technique. In Future Aspects of Human in Vitro Fertilization. Feichtinger W, Kameter P (eds). Berlin, Springer-Verlag, 1987, pp 101-109 11. Mandelbaum J, Junca AM, Plachot M, Alnot MO, Tibi C, Cohen J, Salat-Baroux J: Cryopreservation of human embryos and oocytes using 1,2-propanediol with or without sucrose as cryoprotectant agents. In Workshop on Human Embryo and Oocyte Freezing. Menezo Y, Merieux C (eds). Annecy, France, 1986, pp 95-105 12. Kola I, Kirby C, Shaw J, Davey A, Trounson A: Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses. Teratology 1988;38:467 13. Sathananthan AH, Trounson A, Freeman L, Brady T: The effect of cooling human oocytes. Hum Reprod 1988;3:968977 14. A1-Hasani S, TolkdorfA, Diedrich K, Van der Ven H, Krebs D: Successful in vitro fertilization of frozen thawed rabbit oocytes. Hum Reprod 1986; 1:309-312 15. Glenister PH, Wood MJ, Kirby C, Whittingham DG: Incidence of chromosome anomalies in first cleavage mouse embryos obtained from frozen thawed oocytes fertilized in vitro. Gamete Res 1987;16:205-216 16. Vincent C, Picketing SJ, Johnson MH: The zona hardening effect of dimethyl sulfoxide requires the presence of an oocyte and is associated with reduction in the number of cortical granules present. J Reprod Fert 1990;89:253-259 17. Vincent C, Picketing SJ, Johnson MH, Quick SJ: Dimethyl

sulfoxide affects the organization of microfilaments in the mouse oocyte. Mol Reprod Dev 1990;26:227-235 Johnson MH, Picketing SJ: The effect of dimethylsulfoxide on the microtubular system of the mouse oocyte. Development 1987;100:313-324 Van der Eist J, Van der Abbeel E, Jacobs R, Wisse E, Van Steirteghem A: Effect of 1,2-propanediol and dimethyl sulfoxide on the meiotic spindle of mouse oocyte. Hum Reprod 1988 ;3:960-967 Johnson MH: The effect of fertilization of exposure of mouse oocytes to dimethyl sulfoxide: An optimal protocol. J Vitro Fert Embryo Transfer 1989;6:168-175 Picketing SJ, Braude PR, Johnson MH: Cryoprotection of human oocytes: Inappropriate exposure to DMSO reduces fertilization rates. Hum Reprod 1991 ;6,142-143 Shaw JM, Trounson AO: Parthenogenetic activation of unfertilized mouse oocyte by exposure to 1,2-propanediol is influenced by temperature, oocyte age and cumulus removal. Gamete Res 1989;24:11 Whittingham DG: Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at - 196°C. J Reprod Fertil 1977;49:89 Mandelbaum J, Junca AM, Tibi C, Plachot M, Alnot MO, Rim H, Salat-Baroux J, Cohen J: Cryopreservation of immature and mature hamster and human oocytes. In In Vitro Fertilization and Other Assisted Reproduction, HW Jones Jr, C Shrade (eds). Ann NY Acad Sci 1988;541:550 Imoedemhe DAG, Mahgoub OA, Wafik AH, Chan RCW, Sigue AB, Reyes VV: The human in vitro fertilization and embryo transfer program at the Soliman Fakeeh Hospital, Jeddah, Saudi Arabia. J Vitro Fert Embryo Transfer 1988; 5:52-53 Imoedemhe DAG, Wafik AH, Chan RCW: In vitro fertilization in women with "frozen pelvis": Clinical outcome of treatment. Fertil Steril 1988;49:268-271

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