Original Article
Pregnancy Potential of Human Oocytes – The Effect of Cryopreservation
N Engl J Med 1990; 323:1153-1156October 25, 1990
- Abstract
- Abstract
Background.
In vitro fertilization, sometimes involving the cryopreservation of human embryos, has become a routine procedure for the treatment of infertility. Even though there are embryos available for transfer in about 85 percent of the treatment cycles, the rate of pregnancy rarely exceeds 25 percent per cycle. We designed this study to investigate two questions: Does this high rate of failure result from inadequate technique, or does it simply reflect the maximal potential of a cohort of aspirated eggs to produce a pregnancy? And to what extent does cryopreservation affect the capacity for implantation of embryos?
Methods.
The study was conducted among patients enrolled in an egg-donation program. Aspirated eggs from a given cohort were distributed to the donor herself and a few recipients. The recipients were prepared by a standard protocol of hormone replacement and were assigned at random to the transfer of either fresh or frozen and thawed embryos. The donors received only fresh embryos.
Results.
Forty cycles of donation were studied. In 25 cycles (63 percent) pregnancy was established in the donor, in the recipient (or recipients), or in both. Of the fresh embryos that were transferred to the recipients, 24 percent were successfully implanted, as compared with only 7.7 percent of the frozen and thawed embryos (P<0.01). A pregnancy success rate of 37 percent per recipient cycle was observed in the recipients of fresh embryos, as compared with a rate of only 16 percent in those receiving frozen and thawed embryos (P<0.05).
Conclusions.
The majority of egg cohorts evidently possess the potential to produce a pregnancy, but cryopreservation of human embryos significantly reduces their capacity for implantation. (N Engl J Med 1990; 323: 1153–6.)
Article Activity
- Article
IN VITRO fertilization has become a routine procedure for the treatment of infertility, with a pregnancy success rate in many centers of 20 to 25 percent per egg collection. Considerable effort has been invested in improving laboratory procedures, methods of oocyte retrieval, and techniques of ovarian stimulation, without marked improvement in treatment results. It is not clear that a pregnancy success rate of 25 percent is the highest that can be expected. The rate depends on the potential of the retrieved eggs to produce pregnancy. In the framework of routine in vitro fertilization, it has been impossible to design and carry out an effective study of the pregnancy-producing potential of retrieved eggs, because it has been impossible to identify, isolate, and control for the numerous variables, including the different protocols used for ovarian stimulation, the quality of the sperm used, the receptivity of the uterus, and the use of cryopreservation.
An opportunity to control for some of these variables emerged from our program of egg donation, begun in 1985, in which a cohort of eggs retrieved from a donor is distributed among a group of recipients including the donor herself. Consequently, eggs from a single retrieval procedure are inseminated by different samples of semen, and the resulting embryos are transferred into different uteri. As a result, the eggs are subjected to various conditions, so that the possible effects of semen, endometrial environment, or both are controlled for. In this study, we investigated the effects of the various conditions on the rate of pregnancy, calculated per cohort of eggs. The rates we obtained may reflect the potential of a cohort to result in pregnancy.
Using the same model, we also studied the effect of cryopreservation on the implantation of embryos. The cryopreservation of human embryos is now a routine procedure in many programs of in vitro fertilization.1 2 3 4 Its reported benefits include an increased chance of pregnancy from a single oocyte-recovery procedure1 and a reduced risk of multiple pregnancy. Although this reduced risk would appear to be an obvious outcome of cryopreservation, it is debatable whether the chances of pregnancy per oocyte recovery are increased, especially since some embryos are likely to be damaged during cryopreservation itself. A correlation was recently reported between some morphologic features as seen on video recordings after cryopreserved embryos had been thawed and their capacity for implantation.5 Very little is known about the capacity for implantation and the extent to which it may depend on the quality of the embryos or the suitability of the uterine environment. Nor is it known whether freezing and thawing affect the capacity of embryos for implantation.
In a program of egg donation, cryopreservation offers a way to match the age of the embryo with the state of the recipient's endometrium.6 The same result can be achieved with fresh embryos, however, by applying various means of synchronization.7 , 8 Our model of egg donation, in which embryos are transferred under similar conditions — either fresh or after freezing and thawing — into similarly prepared uterine environments, provided an opportunity to examine the effect of cryopreservation on the rate of implantation.
Methods
Egg Donors
In Israel the donation of oocytes is permitted only by healthy patients who have no detectable genetic diseases and who are themselves undergoing in vitro fertilization because of infertility. For our study, potential donors who fulfilled these criteria were asked to donate one third of their aspirated eggs. It was explained before they signed a consent form that such a donation might diminish their own chances of conceiving to some extent. The donors were given no information about the identity of the recipients or the fate of the donated eggs, and the identity of the donors was not known to the recipients.
Recipients
The recipients were mainly women with primary ovarian failure, with a normal uterus and an adequate endometrial response to hormone replacement therapy (as judged by endometrial biopsies). Results of the husband's sperm analysis were normal in each case. All the recipients and their husbands gave written informed consent, including a statement that the newborn would be acknowledged as their own, regardless of any complications.
Hormone Replacement Therapy
Estradiol valerate was administered, initially at a dose of 1 mg per day. The dose was increased by 1 mg every day for the next five days. The dose was then reduced to 2 mg per day, and it was maintained at that level for the remainder of the cycle. On the second day of the maintenance dose of estradiol, 100 mg of progesterone per day was added, given in vaginal suppositories. This day was considered to correspond to the day of egg retrieval in the donors.
Ovarian Stimulation, Oocyte Retrieval, and Laboratory and Freezing Techniques
Two protocols of ovarian stimulation were used in the donors. In the first, 3.2 mg of a gonadotropin-releasing hormone analogue (D-Trp-6, decapeptyl depot controlled release, Ferring, Malmö, Sweden) was given on day 21 of the natural cycle, followed two weeks later by 225 IU of human menopausal gonadotropin (Teva, Kefar Sava, Israel) per day. In the second, 225 IU of human menopausal gonadotropin a day was given starting on the third day of the natural cycle. In both protocols, human chorionic gonadotropin (Chorigon, Teva) was injected after six days of increased serum estradiol levels,7 provided that the largest follicle reached 18 mm in diameter and estradiol levels were higher than 1835.5 pmol per liter (about 500 pg per milliliter). Oocytes were retrieved by ultrasonographically guided vaginal puncture and were cultured in modified Earle's medium. The method of freezing and thawing was that of Lassalle et al.9 At the stage of two to four cells, the embryos were dehydrated with propylene glycol and 0.1 M sucrose and then frozen in a programmed freezer (Planer, Sunbury on Thames, U.K.). Rapid thawing was performed in the presence of 0.2 M sucrose.
Synchronization of Cycles for the Transfer of Fresh Embryos
In cycles selected for the transfer of fresh embryos, we synchronized the cycles of the recipients with that of the donor, using the so-called six-day rule,7 which suggests that the median duration of the increase in estradiol during the follicular phase is six days. Hence, the recipients started to receive estradiol valerate on the day an increase was first noted in the donor. Exogenous progesterone was begun in the recipients on the day of egg retrieval.
Synchronization of Cycles for the Transfer of Cryopreserved Embryos
All embryos allocated for cryopreservation were frozen, regardless of their morphologic features. The recipients were treated with the same hormone-replacement protocol as those awaiting the transfer of fresh embryos. The cryopreserved embryos were thawed and transferred on the third day of progesterone therapy, which corresponded hormonally with 48 hours after egg retrieval in the donors.
Study Protocol
Thirty-two donors were treated in 40 donation cycles. The donated oocytes were distributed among 57 recipients treated in 84 cycles. Donation cycles were randomly assigned to the transfer of fresh embryos or cryopreservation. Two thirds of a donor's retrieved eggs were set aside for the donor herself, and the remaining one third was allocated to one or more recipients. The eggs thus allocated were then inseminated by the semen of the recipient's husband. Eggs originating from the same cohort were thus inseminated by different semen, and the resulting embryos, either fresh or after cryopreservation, were transferred into different uteri whose environments had all been prepared in the same way. There was no selection of embryos for freezing, and after thawing, all embryos that were not degenerating were transferred. In the donors all the available embryos were transferred fresh during the hyperstimulated cycles. In this study we defined pregnancy as any instance of established pregnancy, regardless of the number of embryos implanted. Implantation refers to the total number of implanted embryos. Embryos transferred were those that were judged suitable for replacement.
Results
Of 40 consecutive cycles of egg donation, pregnancy was established in 25 (63 percent). In 9 cycles (23 percent) either the donor or the donor plus the recipient (or recipients) conceived, and in 16 cycles (40 percent) only the recipient (or recipients) conceived. As shown in Table 1Table 1
Donor Age, Number of Aspirated Eggs per Cycle, and Fertilization Rate in Donor Cycles Resulting in Pregnancy as Compared with Those Failing to Produce Pregnancy.*, there were no differences in the mean age of the donors, mean number of eggs aspirated per retrieval procedure, or rate of fertilization between the donor cycles that resulted in pregnancy and those that did not. The donors were treated for mechanical infertility (26 cycles), unexplained infertility (9 cycles), endometriosis (3 cycles), and male infertility (3 cycles). Eggs from 18 cycles in women with mechanical infertility produced pregnancies (69 percent) in the donors, recipients, or both. Eggs from three cycles in women with unexplained infertility led to pregnancy (33 percent), and from one cycle in a woman with endometriosis (33 percent). All three cycles involving treatment for male infertility led to pregnancies in the recipients (100 percent).With respect to cryopreservation, of the 40 cycles of egg donation, 17 were synchronized for the transfer of fresh embryos to recipients other than the donor, and 13 of these (76 percent) resulted in pregnancy in the donors or the recipients (Table 2Table 2
Comparison of Donor Cycles According to Whether Embryos Were Transferred Fresh or Frozen and Thawed.*). Embryos from the other 23 cycles were cryopreserved, and they produced pregnancies in 12 cycles in the recipients (52 percent). (Donors received only fresh embryos.) The mean age of the donors was similar in the two groups.The results of treatment in the recipients are shown in Table 3Table 3
Treatment Results in Recipient Cycles Synchronized for the Transfer of Fresh Embryos, as Compared with Those Synchronized for the Transfer of Frozen and Thawed Embryos.*. The mean number of oocytes and the rate of fertilization were similar in the recipient cycles synchronized for the transfer of fresh embryos and those synchronized for the transfer of cryopreserved embryos. The implantation rate of fresh embryos, however, was significantly higher than that of frozen and thawed embryos (24 vs. 7.7 percent, P<0.01). Moreover, the rate of pregnancy in the group receiving fresh embryos was also significantly higher (37 vs. 15 percent, P<0.05).Discussion
The relatively low rate of implantation (9 to 11 percent) in most in vitro fertilization programs may be attributable to several factors associated with the procedure — the eggs, the sperm, the endometrium, and the laboratory techniques. Probably all these factors contribute in some measure to the unsatisfactory success rate of in vitro fertilization. In order to improve the rate of pregnancy, more information is needed about the contribution of each variable to the theoretical potential for pregnancy, but in the framework of a routine in vitro fertilization program it is not possible to separate the relative contributions. The use of cryopreservation permits some flexibility in the time between in vitro fertilization and the transfer of embryos, but it introduces new hazards to the embryo. Moreover, technical and ethical problems make it difficult to evaluate the results of cryopreservation of human embryos for the following reasons: embryos are in most cases preselected for cryopreservation, and normally only those with good morphologic features are considered; embryos damaged during cryopreservation are not transferred, so they are usually not included in the analysis of results; and there is no proper control group for studying the effect of cryopreservation on the implantation of embryos, because the embryos with the best morphologic features are chosen for fresh transfer during the stimulation cycle. In our study most of these difficulties were eliminated: eggs were randomly allocated for donation; there was no selection of embryos before freezing; endometria were prepared with identical protocols; and all frozen embryos were thawed and transferred regardless of morphologic features. The significantly lower rate of implantation in frozen and thawed embryos (7.7 percent) as compared with fresh embryos (24 percent) can thus be attributed to the use of cryopreservation.
Our experimental design made it possible to isolate the potential contributions of poor-quality sperm and nonreceptive endometria to the failure of implantation, because more than one sample of semen was used for each cohort of eggs, and the resulting embryos were transferred into more than one uterus. In this way we could come closer to determining the actual potential of a cohort of aspirated eggs to produce pregnancy. Our findings indicate that most (63 percent) of the cohorts aspirated in this study could result in pregnancy when provided with favorable conditions (sperm of good quality and a properly stimulated endometrium). It should be noted that the embryos from more than half the cohorts in this series were cryopreserved, resulting in a lower rate of pregnancy (52 percent). The true potential of these egg cohorts was thus probably higher than the rate of 63 percent we obtained. When only fresh embryos were transferred, 76 percent of the egg cohorts produced a pregnancy. This pregnancy rate of 76 percent is in accord with our findings in a previous series of egg-donation cycles, in which only fresh embryos were transferred and 84 percent of the cohorts produced a pregnancy.8 The high rate of pregnancy per cohort may be partly explained by the nature of egg donation itself: embryos that are foreign to the mother may be more effective in evoking an immune response required for implantation. Our finding that fresh embryos were implanted better in the recipients than in the donors may be also due to an immune phenomenon. A possible contribution of unrecognized male factors cannot be excluded, however, since simple semen analysis does not fully correlate with male fertility. It is also possible that an excess of embryos in the donors may actually decrease the chances of pregnancy, since multiple oocytes are most often the product of a cycle with high levels of estradiol and progesterone. The resulting overstimulation of the endometrium could be detrimental to implantation.
The findings of our study indicate that, at least in cases of egg donation, a better rate of pregnancy can be achieved using protocols of synchronization based on the transfer of fresh embryos. In contrast to this program, in which no more than three embryos were available for transfer to a recipient, a routine program of in vitro fertilization and embryo transfer might make more embryos available for transfer. The possible benefits of transferring fresh embryos could thus be complicated by the risks of multiple implantations, currently the main cause of perinatal morbidity in pregnancies involving in vitro fertilization. Acosta et al.10 studied the effect of the number of embryos transferred on the chances of multiple implantation. They reported that in 81 patients with multiple pregnancy resulting from in vitro fertilization, 2.2 embryos per cycle were able to establish a normal pregnancy, regardless of the number of "preembryos" transferred. These findings indicate that the transfer of five embryos rather than three or four does not necessarily increase the chance of multiple pregnancy. On the other hand, two thirds of the frozen and thawed embryos in our study lost their potential to establish a pregnancy. When the number of embryos to be used in fresh transfer is being determined, both points should be kept in mind. In the absence of more reliable ways to predict the potential of a given embryo for pregnancy, the morphologic features of the blastomeres could be taken as a possible guideline. Unless there are at least two or three morphologically sound embryos available for fresh transfer, we recommend that no embryos be allocated to cryopreservation.
Source Information
From the Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel Hashomer (D.L., J.D., E.R.), and the Department of Embryology and Teratology (D.L., L.N.), Sackler School of Medicine, Tel Aviv University, Tel Aviv (D.L., J.D., E.R., L.N., I.B.-S., Z.B.-R., S.M.), both in Israel. Address reprint requests to Dr. Levran at the Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel Hashomer 52621, Israel.
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- Citing Articles (10)
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Mahnaz Ashrafi, Nadia Jahangiri, Fatemeh Hassani, Mohammad Reza Akhoond, Tahereh Madani. (2011) The factors affecting the outcome of frozen–thawed embryo transfer cycle. Taiwanese Journal of Obstetrics and Gynecology 50:2, 159-164
CrossRef2
E.M. Kolibianakis, K. Zikopoulos, P. Devroey. (2003) Implantation Potential and Clinical Impact of Cryopreservation—A Review. Placenta 24, S27-S33
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E Geva, I Yovel, L Lerner-Geva, J.B Lessing, F Azem, A Amit. (2000) Intrauterine insemination before transfer of frozen-thawed embryos may improve the pregnancy rate for couples with unexplained infertility: preliminary results of a randomized prospective study. Fertility and Sterility 73:4, 755-760
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ANDREA BORINI, CARLO BULLETTI, MONICA CATTOLI, LUCIA SERRAO, VALERIA POLLI, STEFANIA ALFIERI, CARLO FLAMIGNI. (1997) Use of Recombinant Leukemia Inhibitory Factor in Embryo Implantation. Annals of the New York Academy of Sciences 828:1 Uterus, The, 157-161
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David Bider, Yehezkiel Menashe, Mordechai Goldenberg, Mordechai Dulitzky, Alexander Lifshitz, Jehoshua Dor. (1996) Dexamethasone as an adjuvant therapy for anovulatory, normoandrogenic patients during ovulation induction with exogenous gonadotropins. Journal of Assisted Reproduction and Genetics 13:8, 613-616
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David Levran, David Bider, Michal Yonesh, Ziva Yemini, Daniel S. Seidman, Shlomo Mashiach, Jehoshua Dor. (1995) A randomized study of intracytoplasmic sperm injection (ICSI) versus subzonal insemination (SUZI) for the management of severe male-factor infertility. Journal of Assisted Reproduction and Genetics 12:5, 319-321
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Neumann, Peter J.Gharib, Soheyla D.Weinstein, Milton C.. (1994) The Cost of a Successful Delivery with in Vitro Fertilization. New England Journal of Medicine 331:4, 239-243
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Annas, George J., . (1992) Using Genes to Define Motherhood — The California Solution. New England Journal of Medicine 326:6, 417-420
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(1991) New Reproductive Techniques. New England Journal of Medicine 325:14, 1043-1045
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Angell, Marcia, . (1990) New Ways to Get Pregnant. New England Journal of Medicine 323:17, 1200-1202
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