Successful pregnancies from cryopreserved human embryos produced by in vitro fertilization

Richard P. Marrs, M.D., Joanne Brown, Fumihiko Sato, M.D., Tetsuji Ogawa, M.D., Bill Yee, M.D., Richard Paulson, M.D., Paulo Serafini, M.D., and joyce M. Vargyas, M.D.

Los Angeles, California

Sixty-three embryos produced after in vitro fertilization in 30 infertile couples were frozen and stored. Dimethylsulfoxide was used as a cryoprotectant and embryos were frozen from the two-cell stage to early blastocyst development. Replacement occurred during spontaneous ovulatory cycles 2 to 15 months after embryo freezing. Embryo replacement was performed 3 to 6 days following identification of the luteinizing hormone surge in the spontaneous cycle. Thirty-five embryos were replaced into 25 women and two viable pregnancies resulted. (AM J 0BSTET GYNECOL 1987;156:1503-8.)

Key words: In vitro fertilization, cryopreservation, embryo freezing, embryo development

Live births after embryo freezing and storage have been demonstrated in several animal species. '-4 In 1985 the first live birth was reported after a human embryo fertilized in vitro was successfully frozen and stored prior to thawing and transfer back to the uterus of the egg donor.5 Since the delivery of the first baby, there have been several reports that described use of various types of freezing procedures as well as different stages of embryo development at the time of cryopreservation and storage.&-" This report will delineate the parameters used for cryopreservation of embryos produced by our human in vitro fertilization and embryo replacement program at the Hospital of the Good Samaritan, Los Angeles, California.

Material and methods

Patient groups. Thirty infertile women who had pre­viously completed one or more in vitro fertilization and embryo transfer cycles made up the study group. These individuals had embryo replacement performed with a maximum of four embryos; excess embryos (>4) were used for freezing and storage. The patient group ranged in age from 31 to 39 years and their infertility problems included tubal disease, endometriosis, and unexplained infertility. Prior to freezing, normal mor­phologic features and normal cleavage development were mandatory. Patients were counseled and signed a detailed informed consent statement that had been pre-

From the Women's Hospital, Los Angeles County-University of Southern California Medical Center, and the Hospital of the Good Samaritan.

Presented by invitation at the Fifty-third Annual Meeting of the Pa­cific Coast Obstetrical and Gynecological Society, Gleneden Beach, Oregon, September 21-25, 1986.

Reprint requests: Richard P. Marrs, M.D., Cedars Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048.

viously approved by our Ethics Review Committee and Institutional Review Board. A total of 63 cleaving em­bryos were frozen and then stored in these 30 indi­viduals.

Stage of embryonic development at the time of freezing. Oocytes were recovered after controlled ovar­ian stimulation9 and were inseminated according to their stage of maturation, as previously described. 10 Af­ter evidence of fertilization was visualized, cleavage de­velopment was observed and embryos were frozen at two-cell to early blastocyst development. The cryopro­tectant used for all embryos in this group was dimeth­ylsulfoxide.

Embryo freezing protocol. The cryoprotectant was prepared by making a stock solution of 2.34 ml of dimethylsulfoxide and 7.66 ml of 10% serum­supplemented Ham's F-10 medium (concentration of 3 mol/L). Ham's F-10 medium was a modification of stock Ham's F-10, which was made on a weekly basis for in vitro fertilization use.'' Four concentrations of dimethylsulfoxide (0.25 to 1.5 mol/L) were produced by use of the 3 mol/L stock solution with the addition of serum-supplemented Ham's F-10 to make the final concentrations. Aliquots of l ml of these four dilutions of dimethylsulfoxide were then added to organ culture plates (Falcon 303 7, Falcon Plastic, Becton-Dickinson, Oxnard, California). The embryos were placed at room temperature (24° C) into 0.25 mol/L dimethylsulfoxide and at 10-minute increments, they were slowly brought up to the 1.5 mol/L concentration at room temperature (24° C). After completion of equilibration at 1.5 mol/L dimethylsulfoxide, a plastic freezing straw was loaded by a flame-drawn pipette with 0.4 ml of the 1.5 mol/L dimethylsulfoxide/Ham's F-10 solution containing a single embryo. The straws were plugged, loaded into a specifically prepared freezing cane, and placed in a

1503

1504 Marrs et al.

Table I. Stage of embryo development at time of cryopreservation and viability after thaw

Stage of % Viable development after thaw

2-cell 1 2 0 4-cell 5 8 2 5-cell 2 3 0 6-cell 7 11 3 8-cell 27 43 18

10-cell 3 5 2 12-cell 7 11 2 16-cell 9 14 6

Early blastocyst 2 3 2

Total 63 100 35 (55.6%)

horizontal position m the freezing chamber of a CryoMed 1010 Programmable Freezing Unit (VBA Cryogenics, Santa Ana, California).

Cooling program. The staged cooling program was begun at room temperature and the chamber was cooled at 2° C per minute to -6° C. At this point, manual seeding was performed by touching the freez­ing straw with a supercooled instrument. Once seeding was visualized, the chamber was held at - 6° C for a total of 30 minutes, and from that point on, the tem­perature was reduced at 0.33° C per minute down to -45° C. The chamber was then cooled at 1 o C per minute from -45° to -75° C and the canes were placed into the liquid nitrogen refrigerator and stored at -196°C.

Embryo thawing. At the time of thaw, the cane was removed from the liquid nitrogen refrigerator and placed in a rack, where thawing occurred in an uncon­trolled fashion at room temperature. One~ complete thawing and liquification of the straw contents were observed, the end of the plastic straw was removed and the content of the straw was emptied into an organ culture plate containing 0.08 ml of 10% serum­supplemented Ham's F-10 medium. Initially, the em­bryos were visualized with a dissecting microscope (50 x magnification) and then observed with phase con­trast microscopy (400 x magnification). At 10-minute increments, 0.12, 0.2, 0.4, and 1.2 ml of Ham's F-10 were added to remove the cyroprotectant. Thereafter, the embryos were transferred to fresh Ham's F-10 sup­plemented with 10% serum and either cultured for 18 to 24 hours or transferred immediately.

Embryo replacement procedure. The embryo re­placement procedure was performed from 3 to 6 days after the luteinizing hormone surge was detected in serum. The procedure for embryo replacement was identical to the embryo replacement procedure used for routine in vitro fertilization and embryo transfer cydes.u

June 1987 Am J Obstet Gynecol

Table II. Embryo viability after thaw

No. of embryos frozen No. of embryos with 50% of

original blastomeres Incidence of pregnancy per embryo

frozen Incidence of pregnancy per embryo

replaced Incidence of pregnancy per patient

with replacement

Results

63 35 (55.6%)

2/63 (3.2%)

2/35 (5.7%)

2/25 (8%)

Thirty infertile couples made up the study group in which 63 embryos were cryopreserved and stored. The length of time that the embryos were stored in liquid nitrogen ranged from 2 to 15 months (7.1 ± 2.9 months, mean ± SD). The stage of development at the time of freezing ranged from two-cell development to early blastocyst. Seventy-six percent of the embryos were frozen at the eight-cell stage or beyond. The actual numbers of embryos frozen at each cell stage are shown in Table I. Of the original63 embryos that were frozen, 35 (55.6%) appeared viable after thawing. Embryos fro­zen at the eight-cell stage or beyond had a survival rate after thawing that was improved when compared with that of embryos frozen at the earlier cleavage stages (62.5% versus 33.3%). Viability was determined by the embryo having at least 50% of the original blastomeres intact after thawing. The 35 embryos that appeared viable were transferred back to 25 patients; five patients had no surviving embryos after freezing and thawing. Two pregnancies resulted in 25 transfer cycles (8% per transfer cycle). The embryo efficiency was determined by the number of pregnancies produced per number of embryos transferred, for a 5. 7% rate. The efficiency on a per-embryo-frozen basis resulted in a 3.2% effi­ciency rate (Table II).

Pregnancies established. Patient A had three pre­vious in vitro fertilization cycles that failed to produce a pregnancy. The first cycle, in 1984, resulted in the transfer of four embryos, and one embryo at the eight­cell stage was frozen and stored. In June of 1985, an­other in vitro fertilization cycle resulted in a transfer of four cleaving embryos and the freezing and storage of three additional embryos at six-cell, five-cell and four-cell stages. All embryos were frozen 48 hours after insemination. In September of 1985, all four embryos were thawed, and three viable embryos (eight-cell, six­cell, and five-cell development) were recovered and re­placed. A singleton pregnancy resulted. The pregnancy resulted either from an embryo that was stored for 11 months or from an embryo that was stored for 3 months. Replacement occurred 4 days after the lu­teinizing hormone surge was demonstrated in serum. A 9 pound, 10 ounce male infant was delivered at 39

Volume 156 Number 6

weeks' gestation by cesarean section and no abnormal­ities were observed.

Patient B had a single in vitro fertilization and em­bryo transfer cycle that resulted in the production of six embryos, four of which were replaced and two of which were frozen. The two embryos were frozen at 16-cell and eight-cell development, 72 hours after in­semination. After thawing, a morphologically normal eight-cell embryo and an abnormal-appearing 10-cell embryo were observed. The eight-cell embryo was transferred 3 days after the luteinizing hormone surge was detected and the patient is curtently close to term with a normal fetus shown by ultrasound and a 46,XY karyotype.

Extended culture after thaw. In four patients, em­bryos were thawed 18 hours prior to embryo replace­ment (Table III). Four of seven embryos demonstrated resumption of cleavage during this time interval. None of these patients attained a pregnancy following re­placement.

Comment This report illustrates the fact that a cleaving human

embryo developed by in vitro fertilization can produce live offspring after freezing and storage for an ex­tended period of time. The results of this work are similar to those of previous reports from Mohr et al.,5

who used 1.5 mol/L dimethylsulfoxide as a cryopi:o­tectant and a similar freezing program. However, in their report the embryo implantation rate after thawing was much higher than demonstrated in our results. In their report, the frequency of implantation of embryos surviving the freeze-thaw process was 16.2% (11 of 68 embryos that were transferred did implant). Our im­plantation rate after thawing was 5. 7%. However, if their data are broken down as to the number of suc­cessful implantations versus the number of embryos frozen, the actual embryo efficiency is at a level of 8% incidence of pregnancy per embryo frozen. Our data indicated a 3.2% incidence of pregnancy per embryo frozen. Our figures are similar to those in the report from Cohen et a!.,' where 80 embryos were frozen at the four- to 10-cell stage and four implantations oc­curred, for a 5% incidence of pregnancy per embryo frozen. When blastocyst stage was compared in their report, 44 blastocysts were frozen and 23 were found to be suitable for replacement. Of those 44, eight preg­nancies occurred, for a pregnancy rate of 18% per em­bryo frozen. The pregnancy rate per viabie embryo (after thawing) is even higher with implantation oc­curring in eight of 23 blastocysts replaced (34.8%). It is difficult to compare incidences of pregnancy on a per-embryo basis unless the cleavage stages are com­pared directly, and it appears that the embryo efficiency

Pregnancies from cryopreserved embryos 1505

Table III. Extended in vitro culture after embryo thawing

Embryo stage Embryo stage Embryo stage 18 hr at freeze at thaw after thaw

6-cell 4-cell 10-cell 6-cell 3-cell 3-cell 6-cell 3-cell 3-cell 8-cell 4-cell 6-cell 8-cell 6-cell 12-cell 8-cell 4-cell 4-cell

16-cell 16-ceiJ Morula

with less than a 16-cell development is fairly equal. It appears that there is a significantly increased chance for pregnancy if the blastocyst is transferred after thaw­ing.' However, when taken to the clinical setting, the number of oocytes that will develop to a blastocyst stage in culture is approximately 25% to 30% of eggs that are fertilized. 12 Therefore pregnancy outcome should be corrected to account for the 70% to 75% of patients in whom a blastocyst will not develop after fertilization in vii:ro. Thus the pregnancy incidence on a per-patient basis should be viewed as approximately one quarter of the incidence that Cohen et al.7 reported.

Another area of difficulty in comparing the freezing efficiency is the difficulty in analyzing embryos for vi­ability and implantation ability prior to freezing. The embryos that were frozen in our study group were those that were in excess of our optimal number (four em­bryos per replacement cycle). Moreover, the embryos with the best morphologic appearance were utilized for fresh replacement and those that were somewhat ir­regular or slower in development were used for freez­ing and storage. Therefore, in our population, the less than optimal embryos were held for freezing whereas the optimal einbryos were used for fresh replacement. It is not known in previous reports whether such a selection was performed prior to freezing and storage.

In an attempt to determine viability after thawing, four ofthe 25 patients with embryo replacement had an embryo culture time of 18 hours prior to replace­ment. In those individuals, four of the seven embryos that were cultured for 18 hours resumed cleavage, whereas the other three embryos did not. No preg­nancy occurred in these four individuals; therefore no statement can be made as to whether a delay in re­sumption of cleavage development during that period of time has any impact on pregnancy outcome. How­ever, it may be useful in the future to determine if ongoing cleavage development is present in embryos after freezing and thawing to determine whether there is a direct correlation with pregnancy establishment.

Areas of difficulty exist not only in the freezing and storage of the embryos but also in the determination

1506 Marrs et al.

of the optimal time for transfer. Three to six days fol­lowing the luteinizing hormone surge was used as the time interval for replacement, with the two pregnancies occurring at 3- and 4-day time intervals. However, enough clinical data are not yet available to determine whether this is a realistic window for embryo replace­ment. Another area of concern with the process is the cryoprotectant utilized. Various cryoprotectants (pro­pane glycol, dimethylsulfoxide, and glycerol) have been studied, and it appears that the selection of the cryo­protectant should be based on the embryonic cleavage stage."· 7•

13 However, at the present time there are not enough clinical data on human embryo freezing to fully determine this correlation.

In conclusion, the use of embryo freezing should become more efficient as more knowledge is obtained. In the future this process not only may be beneficial to the infertile couple but also might be used in situations for women facing chemotherapy for various malignan­cies during their reproductive years. These individuals can undergo stimulation, collection, fertilization, and storage of their embryos until such time that they have a disease-free interval and can then have an embryo replacement performed and carry their own pregnan­cies. Other useful areas for freezing may occur with genetic screening of embryos in a preimplantation phase where freezing for short periods of time will allow analysis of genetic competency prior to re­placement.

All in all, human embryo freezing at this point in time can be viewed only as a back-up system to fresh embryo replacement during in vitro fertilization cycles. As efficiency improves, it may be used more and more for storage of embryos to allow replacement during a spontaneous ovulatory cycle after egg collection occurs in a stimulated cycle. Evaluation of this type of process will determine whether its usefulness will be evident clinically.

REFERENCES

1. Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to -I96° C and 269° C. Science I972; I78:4Il.

2. Whittingham DB, Adams CE. Low temperature preser­vation of rabbit embryos.] Rerod Fertil 1976;47:269.

3. Trounson AO, Schein BF, Ollis GW,Jacobson ME. Frozen storage and transfer of bovine embryos. J Anim Sci I978;47:677.

4. Bilton RG, Moore NW. Factors affecting the viability of frozen stored cattle embroyos. Aust J Bioi Sci I979; 32:101.

5. Mohr LR, Trounson AO, Freeman L. Deep-freezing and transfer of human embryos. J In Vitro Fert Embryo Transfer 1985;2:1.

6. Zeilmaker GH, Alberda AT, van Gent I, Rifkmans CMPM, Drogendijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril I984;42:293.

7. Cohen], Simons RF, Edwards RG, Fehily CB, Fishel SB. Pregnancies following the frozen storage of expanding

June 1987 Am J Obstet Gynecol

human blastocysts. J In Vitro Fert Embryo Transfer I985;2:59.

8. Quinn P, KerinJPF. Experience with the cryopreservation of human embryos using the mouse as a model to establish successful techniques. J In Vitro Fert Embryo Transfer I986;3:40.

9. Vargyas JM, Morente C, Shangold G, Marrs RP. The ef­fect of different methods of ovarian stimulation for hu­man in vitro fertilization and embryo transfer. Fertil Steril I984;42:745.

IO. Marrs RP, Saito H, Yee B, Sato F, Brown]. Effect of variation of in vitro culture techniques upon oocyte fertilization-embryo development in human in vitro fer­tilization procedures. Fertil Steril I984;4I :5I9.

II. Marrs RP, Vargyas JM, Gibbons WE, Saito H, Mishell DR Jr. A modified technique of human in vitro fertilization and embryo transfer. AM j 0BSTET (;YNECOL I983; I47:3I8.

I2. Johnston I, Lopata A, Speirs A, Hoult I, Kellow G, duPlessis Y. In vitro fertilization: the challenge of the eighties. Fertil Steril I98I ;36:699.

I3. Frydman R, RainhornJD, Forman R, eta!. Programmed oocyte retreival during routine laparoscopy and embryo cryopreservation for later transfer. AMJ 0BSTET GYNECOL I986;155:II2.

Discussion DR. DONALD C. SMITH, Seattle, Washington. Before

I make any comments, I would like to acknowledge the tremendous contributions that Dr. Marrs and his group have made to the field of in vitro fertilization. As a measure of the magnitude of their present accomplish­ment, I know of no other reported ongoing pregnan­cies in the United States today achieved with cryopre­served embryos. Therefore we are privileged to hear the description of these historic gestations and the pro­cedures that made them possible.

Cryopreservation of human tissues for reproductive purposes and indications has gained tremendously in importance in recent years. Early work by the groups in Australia and Europe and now by Dr. Marrs and others in this country have advanced the technology to the level where it is now beginning to have clinical im­pact. As with any new procedure or technology, it is often difficult to predict what that impact will be, and in the instance of cryopreservation of human embryos and gamates, with the technical, legal, ethical, and moral considerations, the nonclinical issues assume even greater importance. In spite of these complexities, the role of freezing is beginning to evolve.

Necessity being the mother of invention, what were the needs that prompted the utilization of this tech­nology in human reproduction? The main impetus came from the in vitro fertilization programs address­ing the problem that Dr. Marrs illustrated with the two patients described, that is, what to do with extra em­bryos if they are obtained. Success rates reported from most in vitro fertilization programs are greatest when up to three or four embryos are replaced. Beyond that, the pregnancy rate does not significantly increase, but the rate of multiple gestation does. Using cryopreser­vation for storage of excess embryos will help alleviate this dilemma by allowing their replacement in some future nonstimulated cycle and hopefully increasing

Volume 156 Number 6

Table I. Embryo cryopreservation data

Patients Embryos frozen Patients with thawed embryos Thawed embryos Embryos with 50% blastomere

survival Patients with embryo replacement Chemical pregnancy Clinical pregnancy Ongoing pregnancy Rate of pregnancy per patient with

replacement Rate of pregnancy per embryo

thawed Rate of pregnancy per embryo

replaced

47 117

17 38 16 (42.1%)

13 1 1 1 7.7%

2.6%

6.3%

Data from Swedish Hospital Medical Center, Seattle, Washington.

the success rate of the entire in vitro fertilization un­dertaking.

This indication has now been extended to use in ga­mete intrafallopian transfer (GIFT), where the multiple gestation rate also increases significantly as increasing numbers of eggs, admixed with spermatozoa, are placed in the distal fallopian tubes. Sending extra ova to the laboratory, where they are fertilized and later frozen, will again allow the possibility of transferring embryos at some later date. Many of the gamete intra­fallopian transfer procedures performed are therefore combinations of in vitro fertilization and gamete intra­fallopian transfer with cryotechnology used in an at­tempt to increase the average success of a single stim­ulated laparoscopic cycle.

Looking to the future, if frozen embryo survival and implantation rates (embryo efficiency) can be brought nearer to those of fresh embryos, then the question may arise as to whether all embryos should be frozen and replaced during a subsequent natural cycle, when the uterine-hormonal environment of the recipient is more normal and perhaps more receptive. I would like to ask Dr. Marrs to comment on that possibility.

I would like to briefly present the experience with cryopreserved human embryos at the Swedish Hospital Medical Center in Seattle, Washington, which is shown in Table I. There has been one ongoing pregnancy achieved in 13 patients in whom at least one embryo has been replaced. Attention is directed to the preg­nancy incidence per patient, per embryo thawed, and per embryo replaced, and one should note the simi­larity to those reported by Dr. Marrs, although our numbers are smaller.

In summary, we are dealing with one of the newest of the recent advances in reproductive technologies. The role that it will ultimately play in clinical practice is yet to be determined. The American Fertility Society Committee on Ethics has recently recommended that "the use of human pre-embryo material for cryopres­ervation should be viewed as a clinical experiment until such time as the success rate and pre-embryo risks are

Pregnancies from cryopreserved embryos 1507

clearly defined:"' It will be through work from excellent programs such as the one directed by Dr. Marrs and others that these questions will eventually be answered. Again I would like to congratulate him on his achieve­ment and the fine presentation.

REFERENCE

l. Ethical considerations of the new reproductive technolo­gies. Fertil Steril 1986;46(suppl 1):535-55.

DR. DoN WoLF, Portland, Oregon. I would also like to recognize the contribution that Dr. Marrs has made in pioneering cryopreservation in this country. My question is focused on the anticipation that a number of the in vitro fertilization programs in this country will now begin to take on the responsibility of establishing cryopreservation programs. Given the information that Dr. Marrs provided, that is, that there are at least three different protocols that can be considered (freezing the early one- or two-cell embryo, the eight-cell stage em­bryo, and the late blastocyst stage embryo), I am won­dering how Dr. Marrs would advise these programs. Which of these "recipes" or protocols is optimal, or should all three be included? I was also very curi­ous that Dr. Marrs was using Hams F -10 instead of phosphate-buffered saline in cryopreservation. Are you working in a carbon dioxide-containing or an air atmosphere in your cryopreservation studies and how do you control pH during the introduction or removal of the cryoprotectant?

DR. IAN Ross DoNALD, Los Angeles, California. I would like to ask a question. Knowing the problems that we have with dimethylsulfoxide in patients with interstitial cystitis, I want to know if this baby had "bad breath"?

DR. CHARLES D. KIMBALL, Seattle, Washington. I would like to ask about the domesticated animals and the laboratory animals that have been used in working out this technique. What is the percentage of survival of frozen embryos in that group?

DR. MARRS (Closing). Dr. Smith asked a question that I think we have all thought about, that is, when do we start freezing all embryos? One advantage of the cryo­preserved embryo is to transfer it into the uterine cavity during a nonstimulated, nonoperative cycle. I think the answer to that question will come about when we qm show that our embryo freezing efficiency has improved to the point that we do not expect half of our embryos to be nonviable after the freezing and thawing. At that time, we will feel more comfortable in freezing the em­bryos as a first step and transferring later, not in the operative, stimulated cycle. However, there are two problems with thaL First of all, our ability to determine the optimal embryos is still very limited. We can take 10 fertilized oocytes that are produced by in vitro fer­tilization and let them go on to what we hope is a blas­tocyst stage and maybe two or three out of those 10 will actually undergo blastocyst development. Just be­cause they are cleaving in the early stages does not mean

1508 Marrs et al.

they are viable, pregnancy-producing embryos. I be­lieve that is a function of our culture conditions. Once we improve them, then our embryo quality will improve and our implantation success will improve. The other factor that adds bias to our freezing protocols is that we tend to use our best embryos during the fresh trans­fer, and we select out ollr "other embryos" or excess embryos for freezing. So we are biasing our results in freezing by this selection process, but I think the time will come when we feel comfortable enough to transfer all embryos after freezing and storage. I think we will see a better rate of implantation per patient and per embryo when we can transfer to the unstimulated uterus.

Dr. Wolf asked what freezing protocol in vitro fer­tilization programs should use. We really do not know yet. Testart's group in France has some excellent results with the freezing of one-cell, fertilized oocytes. This is at the pronuclear stage or the very early, two-cell de­veloping embryo. They use propanediol as a cryopro­tectant and a relatively rapid freezing program. Their success with those parameters has been shown to almost double the implantation and clinical pregnancy rates in comparison with results of freezing the four-cell or eight-cell embryo with dimethylsulfoxide. The group in England has frozen expanded blastocysts with glyc­erol as a cryoprotectant and they have found that 35% of the patients that undergo transfer of blastocysts that are frozen and thawed will experience implantation and pregnancy. However, embryos of only one in four patients will reach the blastocyst stage in culture. So the overall pregnancy rate is no different when one com­pares it to the rate for early cleaving embryos that are frozen and thawed. We are in a situation where you can change the stage of embryo development when freezing is performed, but the pregnancy rates are about equal, with the exception of the very early freez­ing of one-cell and two-cell embryos, which is what we

June 1987 Am J Obstet Gynecol

are looking at right now in our animal models. The other question that Dr. Wolf asked was about the use of Hams F -10 versus phosphate-buffered saline. We did use Hams F-10 in the 30 patients in this report. Since that time we have switched to phosphate-buffered sa­line, which allows a better buffering system when one is not working in a carbon dioxide-controlled environ­ment. We are not really sure whether that is going to make any difference.

The final question that was asked was what was the ability in an animal model to freeze and store and trans­fer and produce live offspring. Well, in a pure in vitro fertilization model, meaning that the oocytes are re­trieved and fertilized in vitro and the em"bryos are al­lowed to cleave and then be transferred or frozen, the animal system is just as inefficient as the human model. On the other hand, if you take an embryo that has already cleaved to a certain st~ge from the reproductive tract of an animal and then freeze it and transfer it to a recipient, the efficiency of implantation and preg­nancy is 50% to 60%, and that is based solely on the fact that fertilization has occurred in the reproductive tract and then the embryo was flushed and transferred or frozen . .1.\ut in a pure in vitro fertilization model, our animals function at the same level as humans, so we have to improve the in vitro fertilization parameters before we can improve the implantation success, in ei­ther an animal situation or the human. The final step or the final thing that is being looked at now with the group of scientists that recently joined us is that we are attempting to freeze the unfertilized oocyte. I think, once you can freeze and store an oocyte, then you can essentially "bank" eggs like you bank sperm and there is much more flexibility in the utilization of those ga­metes. I think that in the future, as far as cryopreser­vation techniques are concerned, we will be dealing with cryopreservation of the egg and the sperm in their separate states and then they can be used as needed.