First delivery of healthy offspring after freezing and thawing of oocytes in Switzerland

The interest in long-term storage of uninseminated oocytes through cryopreservation has seen a recent upsurge, because it provides the potential to assist young women to postpone childbirth after having overcome a malignant disease or delaying childbirth until after management of a professional career. The low fertilisation rate of frozen/thawed oocytes in earlier feasibility trials can now be improved by using intracytoplasmic sperm injection (ICSI) for assisting the penetration of the spermatozoon through the oocyte’s hardened zona pellucida. Another reason for the reported low success rates of oocyte cryopreservation in earlier studies may have been the low developmental potential of spare oocytes, which were available for experimental cryopreservation. Oocytes retrieved from supernumerary follicles in women treated with gonadotropins for ovulation induction and intrauterine insemination can be used for the optimisation of cryostorage of uninseminated oocytes. We intended to investigate to what extent the well-established and successful cryopreservation protocols for pronucleate oocytes are also applicable for the cryopreservation of uninseminated oocytes. We herewith report the first successful pregnancy and delivery of frozen/thawed oocytes in Switzerland, which were inseminated with ICSI. In unbiased treatment groups the freezing and thawing of uninseminated oocytes and pronucleate oocytes give comparable results, if the additional manipulation during ICSI was taken into account.

The interest in long-term storage of uninseminated oocytes through cryopreservation has seen a recent upsurge, because it provides the potential to assist young women to postpone childbirth after having overcome a malignant disease or delaying childbirth until after management of a professional career.The low fertilisation rate of frozen/thawed oocytes in earlier feasibility trials can now be improved by using intracytoplasmic sperm injection (ICSI) for assisting the penetration of the spermatozoon through the oocyte's hardened zona pellucida.Another reason for the reported low success rates of oocyte cryopreservation in earlier studies may have been the low developmental potential of spare oocytes, which were available for experimental cryopreservation.Oocytes retrieved from supernumerary follicles in women treated with gonadotropins for ovulation induction and intrauterine insemination can be used for the optimisation of cryostorage of uninseminated oocytes.We intended to investigate to what extent the well-established and successful cryopreservation protocols for pronucleate oocytes are also applicable for the cryopreservation of uninseminated oocytes.We herewith report the first successful pregnancy and delivery of frozen/thawed oocytes in Switzerland, which were inseminated with ICSI.In unbiased treatment groups the freezing and thawing of uninseminated oocytes and pronucleate oocytes give comparable results, if the additional manipulation during ICSI was taken into account.

Summary
The treatment of both male and female infertility nowadays strongly relies on assisted reproductive technology, most notably in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI).However, consistently satisfying pregnancy rates can only be achieved within the context of controlled ovarian stimulation, particularly if performed by administrating gonadotropins throughout the follicular phase of the menstrual cycle.Through this hormonal pretreatment the reproductive biologist usually has access to more oocytes or embryos than can be subsequently replaced in a responsible way.In order to reduce the degree of multiple pregnancies and to enable the patient to have repeated embryo transfers in sequential menstrual cycles without having to undergo repeated ovarian stimulation, various strategies for the cryopreservation of supernumerary reproductive material have been developed over time.Not only the male and the female gonadal tissue can be cryopreserved (eg stored frozen), but also semen, uninseminated oocytes, oocytes at the pronucleate stage and embryos at various developmental stages.The freezing and thawing of uninseminated oocytes was actually one of the first techniques to be introduced successfully [1], but has found little application, mainly due to its low efficacy leading to inconsistent results and due to the availability of other, more efficient technologies, such as cryopreservation of embryos.In Switzerland, a restrictive legislation, as stipulated by the so-called "Fortpflanzungsmedizingesetz", has banned all freezing of embryos (unless in an emergency situation) since January 2001.Only a maximum of three embryos can be cultured and all viable embryos have to be transferred [2].Similar restrictive laws were issued in Germany and in Italy, the latter even banning the cryopreservation of oocytes at their pronucleate stage.Cryopreservation of embryos is being performed in most other countries worldwide.In Switzerland methods for freezing and thawing of oocytes in the pronucleate stage have been adopted successfully [3].Various studies have demonstrated that freezing and thawing of oocytes at the pronucleate stage is efficient and leads to acceptable pregnancy rates [4,5].
The discrepancy between the low efficacy of cryopreservation of oocytes and the much higher pregnancy rates after freezing and thawing of pronucleate oocytes is striking, because from the cryobiological point of view these structures are very similar.Both are developmentally separated by only a few hours, have the same size, a similar membrane and a similar cytoplasmic watery appearance.The difference between both structures relates to the status of the nucleus and the organelles associated with it, the meiotic spindle in particular.Having a well-established and successful cryostorage protocol for pronucleate oocyte at our disposal, we decided to evaluate the efficacy of freezing and thawing of uninseminated oocytes using our established freezing and thawing protocol for pronucleate oocytes.
When compared with pronucleate oocytes, the freezing and thawing of uninseminated oocytes have various potential advantages.Oocyte freezing could be used in women at risk of losing their reproductive potential, and in men in whom a malignant disease has been diagnosed and who will have to undergo either chemotherapy or radiation [6].In addition to this, the current trend of delaying family planning in women aiming at a professional career also entails the risk of losing most of their reproductive potential later, when a pregnancy would be feasible.Finally, prolonged cryostorage of uninseminated oocytes does not carry the burden of the many ethical concerns associated with the cryostorage of embryos or even of pronucleate oocytes.
The poor outcome of oocyte freezing and thawing has been partially attributed to the selective use of low quality oocytes in many of earlier feasibility trials, and to hardening of the zona pellucida during the freezing process, reducing its penetrability during subsequent in vitro fertilisation [7].Whereas the zona hardening can now be overcome with ICSI, we decided to use oocytes retrieved from infertile women previously stimulated with exogenous gonadotropins for intrauterine insemination, in whom supernumerary oocytes had to be removed in order to prevent multiple pregnancies [8].Herewith, we report the first pregnancy and delivery in Switzerland after freezing and thawing of uninseminated oocytes.

Material and methods
The pregnancy and delivery described in this report arose within the context of a prospective study, which was presented to and accepted by our local ethics committee (EKBB 141/04).This prospective study consisted of offering to infertile couples treated with gonadotropins for ovulation induction for IUI the option of freezing oocytes from supernumerary follicles, which were aspirated through transvaginal ultrasound-guided puncture, as described earlier [8].All couples signed an informed consent form prior to the cryopreservation of their oocytes.The cryopreservation protocol was identical to the protocol used for storage of pronucleate oocytes, which has been successfully used in our laboratory for many years.This protocol consisted of the stepwise addition of an increasingly concentrated cryoprotectant solution (0.25 M, 0.5 M, 0.75 M, 1.0 M, 1.25 M until the final concentration of 1.5 M each five minutes) containing both dimethylsulfoxide (DMSO) and propanediol (PROH) to the oocytes at room temperature.Over a period of 5.5 hours the cryoprotective solution containing the oocytes were then slowly cooled down using a computer-guided cooling system equipped with automatic seeding (CTE 920 Kryotechnik, Erlangen, Germany).Subsequently, the cryopreserved oocytes in the sealed straws were stored in liquid nitrogen.
If no pregnancy arose after IUI, the frozen oocytes were taken from the container and thawed rapidly at room temperature.The cryoprotectant was removed stepwise by transferring the oocytes into the cryoprotective solutions, as described above but in reverse order.Then, the oocytes were treated with ICSI and all fertilised oocytes with two pronuclei were replaced at the embryonic cleaving stage into the uterine cavity.The embryos were transferred to the recipient either during a natural menstrual cycle or after induction of follicular development with clomiphene citrate followed by ovulation induction with HCG.
The aim of this study was to examine both the feasibility and the efficacy of freezing and thawing of uninseminated oocytes under the conditions described above.The results of freezing and thawing of oocytes at the pronucleate stage of infertile women treated with either IVF or ICSI during the same time period, but not achieving pregnancy in their initial fresh treatment cycle, were selected as controls.

Case
A 29 year old patient was diagnosed in our service to have chronic anovulatory infertility due to the polycystic ovary syndrome as indicated by both elevated testosterone levels and the typical appearance of the ovaries during ultrasound scanning.Her husband had normal semen parameters according to the WHO-guidelines (1999) and the results of his physical exam were normal.The couple had a five year old daughter, but no other pregnancies.At the moment of the present treatment the patient had already undergone three treatments with clomiphene citrate and one ovulation induction with recombinant gonadotropins (Gonal F, Serono, Zug, Switzerland) following the chronic low dose protocol, but none of these treatments led to pregnancy.During the second treatment trial with recombinant gonadotropins a supernumerary cohort of mature ovarian follicles developed.Selective aspiration of some of these follicles had to be performed in order to prevent multiple pregnancy [8].A total of seven oocytes were aspirated through transvaginal, ultrasound-guided puncture of the follicles.These oocytes were cryopreserved following our cryostorage protocol for pronucleate oocytes (figure 1).This protocol consists of a slow cooling process of the denuded oocytes in a cryoprotectant solution containing DMSO and PROH.

Results of prospective cohort study
Between January 2005 and July 2007 20 cryopreservations of 102 uninseminated oocytes were performed originating from 19 women, whose oocytes were collected after treatments with gonadotropins for IUI in need of preovulatory aspiration of supernumerary follicles in order to prevent polyovulation and thereby multiple pregnancy [8].Of those women with unsuccessful IUI, 15 thawings of their frozen oocytes were performed (table 1).Of the 73 oocytes thawed, 33 survived (60.8%) and were treated with ICSI.24 of the 33 injected oocytes were fertilised with two visible pronuclei, developed to the cleaving embryo stage (72.7%) and replaced into the uterine cavity of nine recipients (transfer rate per oocyte freezing: 45.0%) leading to three pregnancies (pregnancy rate per freezing: 15.0%).One pregnancy ended as an early miscarriage and another pregnancy proved to be a vital tubal pregnancy as evidenced both by ultrasound and laparoscopy.A third pregnancy proved to be viable and is presented here as the first delivery after cryopreservation of an uninseminated oocyte in Switzerland.

Case
As the second ovulation induction with recombinant gonadotropins followed by IUI failed to produce a pregnancy, we offered the couple thawing of the frozen oocytes and insemination of the thawed oocytes with ICSI followed by transfer of the developing embryos into the uterine cavity.The couple agreed to participate in the trial and menstruation was induced with dydrogesteron (20 mg daily over 10 days, Duphaston, Solvay, Berne).The endometrium was then brought to proliferation by inducing follicular growth with clomiphene citrate (50 mg daily over five days starting on day five after menstruation, Serophene, Serono) and the ovulation of a 20 mm follicle was induced with human chorionic gonadotropins (10'000 international units of HCG, Pregnyl, Organon, Pfäffikon).Five days prior to ovulation induction the couple was instructed to refrain from sexual intercourse.After having confirmed ovulation with ultrasound the thawing of all seven oocytes was started in batches of up to three oocytes.Finally, all seven thawed oocytes were treated with ICSI, resulting in two embryos, which were replaced into the uterine cavity under transabdominal transvesical ultrasound guidance View of the two uninseminated oocytes (A), which after thawing were treated with ICSI.One of the resulting pronucleate oocytes (B) and the two embryos (C) were replaced approximately 48 hours after thawing.One of these embryos led to the pregnancy and delivery, reported in this communication.

Results
(figure 1).Six days after embryo transfer the luteal phase was supported with one single injection of 5000 international units of HCG (Pregnyl).An intact intrauterine singleton pregnancy was confirmed by serial measurements of human chorionic gonadotropin serum levels and vaginal ultrasound.The pregnancy evolved without complica-tions and the patient delivered a healthy boy in August 2006 in the University Women's Hospital of Basel.His neonatal birth weight was 3225 g with a length of 47 cm, and no malformations were detected.The child continued to develop well.

First delivery of healthy offspring after freezing and thawing of oocytes in Switzerland
Freezing and thawing of oocytes uninseminated pronucleate sign. 1   No No. of deliveries (% per freezing): 1 (5.0) 8 (6.7) n.s. 1 Chi-squared test with Yates' correction 2 As stipulated by Swiss legislation all cleaving embryos were transferred. 3One pregnancy ended in an early miscarriage, another pregnancy presented as a tubal pregnancy with embryonic cardiac activity, and one pregnancy proved to be intact leading to the delivery of a healthy child.

Discussion
Although the patient ovulated naturally during the replacement cycle, it is very likely that she conceived from the frozen-thawed oocyte, because she was asked to refrain from intercourse around the time of ovulation.This delivery is the first to be reported after freezing and thawing of an uninseminated, nonfertilised oocyte in Switzerland and fits well to the current renewed interest in oocyte cryopreservation [7].
The case described here is part of an ongoing prospective study, in which the feasibility of freezing and thawing of uninseminated oocytes is being examined systematically.It was thought that the low developmental quality of the oocytes used in previous cohort studies was a major determining factor in the unsatisfactory outcome of oocyte cryopreservation [7,9].The oocytes used in this study came from supernumerary ovarian follicles after ovarian stimulation with gonadotropins for scheduled treatments with intrauterine insemination [8].The published high pregnancy rate with this mode of therapy [10], the high pregnancy rate achieved in treatments, originally intended for ovulation induction but then converted into IVF due to the ovaries' overreaction to the applied gonadotropin dose [11], and the results of cytogenetic analysis of oocytes retrieved from supernumerary follicles [12] suggest that these oocytes possess a high developmental potential.For the cryopreservation of these good-quality oocytes we used the same protocol as for pronucleate oocytes, which has resulted in high pregnancy rates in our institution for many years.
In the present analysis we used the results of freezing and thawing of pronucleate oocytes collected from patients treated with IVF or ICSI during the same time period for control purposes.Because the thawings of the uninseminated oocytes were exclusively performed after unsuccessful IUI treatments, we excluded those thawings of pronucleate oocytes resulting from IVF or ICSI treatments, which had been successful in the fresh cycle.
Under those conditions we observed that the number of surviving oocytes after thawing (60.8%) was strikingly similar to the number of cleaving embryos resulting from thawing of frozen oocytes at the pronucleate stage (62.6%).However, the extra manipulation of the thawed oocytes with ICSI caused an additional progressive loss leading to significantly lower numbers of implanted embryos after thawing of uninseminated oocytes as compared to the thawed pronucleate oocytes (p <0.0001, table 1).Surprisingly, both the pregnancy rate and the delivery rate per freezing were not different among both groups, indicating the cryopreservation protocol per se is adequate for both cell types (table 1).Neverthe-less, the fertilisation and pregnancy rates presented in this prospective study are somewhat lower than those published in recent meta-analyses [13,14].Despite the successful pregnancy presented in this communication, the ongoing results of our freezing and thawing protocol of uninseminated oocytes using the protocols for pronucleate oocytes are somewhat disappointing.
The low success rate of freezing and thawing of uninseminated oocytes is thought to be caused by the disruption of the meiotic spindle apparatus during the slow cooling phase of conventional cryoconservation leading to aneuploidy in the resulting embryos [15].Better results have been published with conventional cryopreservation protocols based on other cryoprotective environments, particularly if sucrose is added to the cryoprotective solution [16,17].In recent years, much better results than with conventional cryopreservation were achieved with vitrification of uninseminated oocytes [18][19][20].Vitrification is a process of converting the oocyte and the cryoprotectant into a glass-like amorphous solid state, which is free of any crystalline structure.The formation of ice crystals is the major cause of cell damage through disruption of the membrane or of major organelles during conventional cryopreservation.Vitrification of uninseminated oocytes is achieved by adding the cryoprotectant at room temperature and by extreme supercooling into liquid nitrogen at -196 °C [21,22].At present there is still some concern with the longterm risk for the offspring, as toxic cryoprotec-tants are added to the oocytes at much higher concentrations (5 to 6 M) than in conventional slow-cooling cryopreservation protocols.Recent developments of vitrification, however, have enabled significant reductions of the freezing volume, which surrounds the oocytes, resulting in extremely rapid freezing rates [21,22].Speeding up freezing rates in vitrification allows reduction of the cryoprotectant concentrations resulting in similar freezing environments as in conventional freezing protocols, in which no malformations in the offspring have yet been described.
In conclusion, cryopreservation protocols, which have been shown to be adequate and successful for freezing and thawing of pronucleate oocytes, have now been shown to give rise to similar delivery rates with freezing and thawing of uninseminated oocytes.However, our experience also indicates that implantation and pregnancy rates of this freezing and thawing protocol are insufficient for its future use in cancer patients or in women aiming at preserving some of their oocytes for future family planning.