S.I.S.ME.R., Reproductive Medicine Unit, Via Mazzini 12, 40138, Bologna, Italy
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: cryopreservation/embryo/ovarian hyperstimulation/thawing/zygotes
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ovarian stimulation protocol was similar for all patients. We used a down-regulation protocol with GnRH analogue and urinary gonadotrophin (Metrodin; Serono, Rome, Italy) as described in our previous work (Ferraretti et al., 1996). All the patients were administered 7500 UI of HCG before egg retrieval. On the day of oocyte recovery, 20 g of i.v. albumin (Immuno A.G., Vienna, Austria) was administered in all cases as previously described (Asch et al., 1993
). The oocytes were inseminated with the husband's spermatozoa and then incubated in the same condition as reported previously (Gianaroli et al., 1996b
). After 1418 h from insemination the fertilization was assessed as shown by the presence of two pronuclei. All the obtained zygotes from group B patients were immediately cryopreserved, while the zygotes of group A couples were kept in culture medium for a subsequent 48 h. Oocyte culture, insemination and embryo culture were carried out in 100 µl droplets of IVF medium (Scandinavian IVF Science AB, Goeteborg, Sweden) overlaid with pre-equilibrated mineral oil (Scandinavian IVF Sc.). Three or four fresh embryos, according to the patient's age, were transferred in group A, while the surplus viable embryos were cryopreserved. Pure progesterone in oil, 50 mg/day (Gestone; AMSA Laboratory, Florence, Italy), was given for luteal phase support from the day of embryo transfer.
All the studied patients were advised to rest at home and to monitor constantly the following parameters: weight, abdominal girth, fluid intake and urine output per 24 h. A controlled hyposodic and hyperproteic diet was also recommended. The patients were also asked to communicate the above parameters to the centre every 34 days or as soon as possible in cases of abnormal values or onset of symptoms such as nausea or vomiting.
Group A patients who failed the first fresh embryo replacement attempt and had the cryopreservation of surplus embryos, were submitted to a second attempt with thawed embryo transfer. These patients as well as the patients of group B, who had elective cryopreservation of all zygotes, underwent an appropriate hormonal replacement therapy (HRT) for the thawed cycle. The scheme included oral administration of oestradiol valerate (Progynova; Schering, Milan, Italy) 2 mg daily for the first 5 days of the cycle; 4 mg/day from day 6 to day 10; 6 mg/day from day 11 to day 13; then 4 mg/day from day 14 onward. On day 15 of the cycle, 50 mg of progesterone in oil was daily administered and on day 17 the dose was increased to 100 mg/day.
In group A patients, the cryopreserved embryos were thawed on day 17, and the viable embryos with 50% survived blastomeres were cultured in IVF medium for a few hours and then transferred immediately. Three or four cryopreserved pronucleate embryos of group B patients were thawed on the evening of the first day of progesterone administration and cultured for 3640 h in IVF medium before embryo transfer. If two or more zygotes did not cleave 24 h after being cultured, one or two additional zygotes were thawed. The cryopreservation and thawing of either zygotes or embryos were performed using 1,2-propanediol and sucrose in phosphate-buffered saline solution (PBS) following the procedure previously described (Lassalle et al., 1985
).
The patients who had frozenthawed embryos transferred with a positive test of HCG continued the HRT for a further 8 weeks. The patients in whom fresh embryos were replaced and had a positive HCG test continued the progesterone administration until the first ultrasound examination. Only clinical pregnancies detected by the presence of gestational sac and fetal heartbeat, by ultrasound examination, were recorded and included in this study.
Oestradiol blood analysis was performed using a radioimmunoassay procedure (Biochem Immunosystem, Rome, Italy).
For statistical analysis, independent Student's t-test and 2-test were used where appropriate.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
In group B, a total of 609 fertilized eggs from 58 patients were cryopreserved at the pronuclear stage (10.3 ± 5 zygotes/patient). No severe OHSS developed in any of the 58 patients. All patients underwent at least one thawed cycle. In 80 cycles (1.4 cycles/patient), 446 frozen zygotes were thawed and detected for their viability. After 3640 h of incubation, 301 out of 351 surviving zygotes had cleaved in culture. A total of 240 cleaving viable embryos was transferred in 79 cycles with an average of 3.1 ± 0.9 embryos/cycle. In one case the embryo transfer was cancelled because no thawed zygotes survived. Clinical pregnancies were established in 28 patients (35.4% per cycle: 48% per patient) with an implantation rate of 15%. Five pregnancies aborted during the first trimester, while the remaining pregnancies resulted in the delivery of 30 healthy babies. Most of the pregnancies were achieved during the first thawed cycle (20 out of 28), leading to a high number of spare cryopreserved zygotes. Table III shows the outcome of oocytes and embryos obtained from the two groups. Table IV
compares pregnancy, implantation and live birth rates achieved in both groups. The transfer of the surplus frozenthawed embryos in group A is also included as part of the final clinical outcome of the zygotes and embryo performance.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A retrospective analysis of our data revealed that the incidence of the severity and the duration of the syndrome was strictly related to the surge of the pregnancy. In cycles where no conception occurred the incidence of OHSS was 0.3% with a mean duration of 6.2 ± 1.7 days, whereas in pregnant cycles 3.1% had OHSS lasting 16.4 ± 3 days. Furthermore, in pregnant patients, the incidence of OHSS is directly related to the number of implanted embryos: 1.8% in singleton; 2.5% in twins; 21% in triplet and 33% in quadruplet (Gianaroli et al., 1996a). Recent studies have proposed the elective cryopreservation of all generated embryos from patients considered at risk of developing OHSS, but the results achieved were contradictory. Some investigators suggested that this procedure reduces the incidence of severe OHSS and does not influence significantly the pregnancy rate (Pattinson et al. 1994
). Other researchers argued that the elective cryopreservation of all embryos does not potentially prevent the development of OHSS, but reduces the pregnancy and live birth rates (Awonuga et al., 1996
). In an additional study, (Queenan et al., 1997
) it has been reported that despite the excellent pregnancy rate achieved with frozenthawed embryos, the onset of OHSS is not avoided. These controversies could be explained with the different freezing procedures adopted, the different criteria used to define which patient is at risk and which is not, and the lack of a prospective randomized study. These findings encouraged us to perform a prospective randomized study to evaluate the efficiency as well as the safety of the elective freezing of all embryos in the prevention of the development of OHSS in stimulated patients. Data regarding the survival rate of embryos thawed at the zygote or cleaving stage, highlight the difference in terms of implantation capability, which is superior in the case of pronucleate stage embryos (Demoulin et al., 1991
; Horne et al., 1997
). Based on these observations, we decided to favour the cryopreservation of the zygotes and the subsequent embryo culture after thawing, with the aim of maximizing the chances of pregnancy in the patients studied. The results of the present study suggest that cryopreservation of 2PN zygotes does not affect significantly the developing embryo rate and does not decrease the chance of pregnancy. By adopting low cut-off parameters for the risk of OHSS, the cryopreservation of all zygotes seems to reduce the incidence of OHSS. The discomfort for the group B patients perhaps is higher with respect to group A: they must undergo delayed embryo transfer and a slightly higher number of embryo replacements in order to achieve a pregnancy rate similar to group A. Additionally, they have to continue the HRT for 78 weeks in case of pregnancy. However, in our centre these patients did not have additional costs, and the different incidence of OHSS compared to the control group seems to justify all these inconveniences.
From the current results, we cannot confirm that the elective freezing of all zygotes could eliminate completely the risk of the syndrome, but it reduces the duration and the severity in the complete absence of pregnancy. If costs and benefits of this procedure are taken into consideration, they should include the number of patients undergoing increasing stress due to the policy of elective cryopreservation of all embryos. In our centre, the proportion of patients with the parameters for elective cryopreservation of all embryos is quite consistent (~15%). On the other hand we believe that, if this procedure avoids only one case of severe OHSS in young healthy women, it is a proper strategy to be adopted, especially when the possibility of taking home a baby is not decreased.
In conclusion, our prospective randomized study demonstrates that the elective cryopreservation of all zygotes in patients at risk of OHSS is safe, as it reduces the risk of developing the syndrome, and is efficient as it does not affect the live birth rate. According to this study, our ongoing strategy is to cryopreserve all the pre-embryos at the pronucleate stage in patients considered at risk of OHSS.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Araujo, E., Bernardini, L., Frederick, J.L. et al. (1995) Prospective randomized human chorionic gonadotrophin versus intramuscular progesterone for luteal-phase support in assisted reproduction. J. Assist. Reprod. Genet., 11, 7478.[ISI]
Asch, R.H., Ivery, G., Goldsman, M. et al. (1993) The use of intravenous albumin in patients at high risk for severe ovarian hyperstimulation syndrome. Hum. Reprod., 8, 10151020.[Abstract]
Awonuga, O.A., Pittrof, R.J., Zaidi, J. et al. (1996) Elective cryopreservation of all embryos in women at risk of developing ovarian hyperstimulation syndrome may not prevent the condition but reduces the live birth rate. J. Assist. Reprod. Genet., 13, 401406.[ISI][Medline]
Bassil, S., Godin, P.A., Stallaert, S. et al. (1995) Ovarian hyperstimulation syndrome: a review. Assist. Reprod. Rev., 5, 9096.
Demoulin, A., Jouan, C., Gerday, C., Dubois, M (1991) Pregnancy rates after transfer of embryos obtained from different stimulation protocols and frozen at either pronucleate or multicellular stages. Hum. Reprod., 6, 799804.[Abstract]
Friedman, C.I., Schmidt, G.E., Chang, F.E. and Kim, M.H. (1984) Severe ovarian hyperstimulation following follicular aspiration. Am. J. Obstet. Gynecol., 15, 436437.
Ferraretti, A.P., Gianaroli, L., Diotallevi, L. and Trounson, A.O. (1992) Dopamine treatment for severe ovarian hyperstimulation syndrome. Hum. Reprod., 7, 180183.[Abstract]
Ferraretti, A.P., Magli, C.M., Feliciani, E. et al. (1996) Relationship of timing of agonist administration in the cycle phase to the ovarian response to gonadotrophins in the long down-regulation protocols for assisted reproductive technologies. Fertil. Steril., 65, 114121.[ISI][Medline]
Gianaroli, L., Ferraretti, A.P. and Fiorentino, A. (1996a) The ovarian hyperstimulation syndrome. Reprod. Med. Rev., 5, 169184.
Gianaroli, L. Fiorentino, A., Magli, C.M. et al. (1996b) Prolonged spermoocyte exposure and high sperm concentration affect embryo viability and pregnancy rate. Hum. Reprod., 11, 25072511.[Abstract]
Gonen, Y., Balakier, H., Powell, W. et al. (1990) Use of gonadotrophin-releasing hormone agonist to trigger follicular maturation for in vitro fertilization. J. Clin. Endocrinol. Metab., 71, 918922.[Abstract]
Horne, G., Crtichlow, J.D., Newman, M.C. et al. (1997) A prospective evaluation of cryopreservation strategies in a two-embryo transfer programme. Hum. Reprod., 12, 542547.[ISI][Medline]
Koenig, E., Bussen, S., Suetterlin, M. and Steck, T. (1998) Prophylactic intravenous hydroxylethyl starch solution prevents moderatesevere ovarian hyperstimulation in in-vitro fertilization patients: a prospective, randomized, double-blind and placebo-controlled study. Hum. Reprod., 13, 24212424.[Abstract]
Lassalle, B., Testart, J. and Renard, J.P. (1985) Human embryo features that influence the success of cryopreservation with the use of 1,2-propanediol. Fertil. Steril., 44, 645651.[ISI][Medline]
Lee, C., Tummon, I., Martin, J. et al. (1998) Does withholding gonadotrophin administration prevent severe ovarian hyperstimulation syndrome? Hum. Reprod., 13, 11571158.[Abstract]
Mordel, N. and Schenker, J.G. (1993) Gonadotrophin-releasing hormone agonist and ovarian hyperstimulation syndrome in assisted reproduction. Hum. Reprod., 8, 20092014.[Abstract]
Navot, D., Bergh, P.A. and Laufer, N. (1992) Ovarian hyperstimulation syndrome in novel reproductive technologies: prevention and treatment. Fertil. Steril., 58, 249261.[ISI][Medline]
Pattinson, H.A., Hignett, M., Dunphy, B.C. and Fleetham, J.A. (1994) Outcome of thaw embryo transfer after cryopreservation of all embryos in patients at risk of ovarian hyperstimulation syndrome. Fertil. Steril., 62, 11921196.[ISI][Medline]
Queenan, Jr. J.T., Veek, L.L., Toner, J.P. et al. (1997) Cryopreservation of all prezygotes in patients at risk of severe hyperstimulation does not eliminate the syndrome, but the chances of pregnancy are excellent with subsequent frozenthawed transfers. Hum. Reprod., 12, 15731576.[Abstract]
Schenker, J.G. (1993) Prevention and treatment of ovarian hyperstimulation. Rev. Hum. Reprod., 8, 653659.
Sher, G., Salem, R., Feinman, M. et al. (1993) Eliminating the risk of life-endangering complications following overstimulation with menotropin fertility agents: a report on women undergoing in vitro fertilization and embryo transfer. Obstet. Gynecol., 81, 10091010.[Abstract]
Tomazevic, T. and Meden-Vrtovec, H. (1996) Early timed follicular aspiration prevents severe ovarian hyperstimulation syndrome. J. Assist. Reprod. Genet., 13, 282286.[ISI][Medline]
Submitted on November 2, 1998; accepted on March 1, 1999.