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An obstetric analysis of fifty consecutive pregnancies after transfer of cryopreserved human embryos
An obstetric analysis of fifty consecutive pregnancies after transfer of cryopreserved human embryos Rene Frydman, MDt b Robert G. Forman, MD: J. Belaisch-Allart, MD: Andre Hazout, MD: Herve Fernandez, MD: and Jacques Testart, PhDb Clamart, France This report describes the obstetric outcome in 50 pregnancies resulting from the transfer of human embryos that had been cryopreserved for up to 2 years. The duration of cryopreservation did not influence the pregnancy rate after thawed embryo transfer. Thirty-one babies have been born from 28 pregnancies and a further seven pregnancies are currently in the second and third trimesters. Twenty-eight percent of the pregnancies failed to progress beyond the first trimester. Dne pregnancy was terminated at 22 weeks' gestation because of severe fetal malformation. Important antenatal events included premature uterine activity in six patients although only one patient with a singleton pregnancy ultimately was delivered prematurely. Retroplacental hemorrhage occurred in four patients but was of clinical consequence in only one. There was a high incidence of breech presentation at term in singleton pregnancies (12%). The cesarean section rate in this series was 21 %. An international registry of cryopreserved pregnancies would facilitate data collection in this relatively new clinical field. (AM J DesTET GYNECOL 1989;160:209-13.)
Key words: Perinatal outcome, in vitro fertilization, human embryo, cryopreservation
The first birth after transfer of cryopreserved human embryos was reported in 1984. 1 Since then, several methods have been described for the freezing and thawing of supernumerary human embryos.2.4 With the recent development of superovulation regimens comprising gonadotropins and agonists of gonadotropinreleasing hormone, it is probable that more oocytes and embryos will be obtained.' Embryo cryopreservation is therefore likely to play an increasing role in in vitro fertilization programs over the next few years. Several reports have described the obstetric outcome in pregnancies resulting from the transfer of "fresh" embryos:·lo but there has been no previous obstetric analysis of pregnancies arising after the transfer of frozenthawed human embryos. This article describes the pregnancy and obstetric factors relative to the first 50 consecutive pregnancies resulting from the transfer of cryopreserved embryos at Antoine Beclere Hospital, Clamart, France.
Patients and methods All 50 pregnancies in this series occurred in infertile patients being treated in the in vitro fertilization program at Antoine Beclere Hospital. Superovulation was induced in all patients in the in vitro fertilization cycle with combinations of gonadFrom the Department of Gynaecology and Obstetrus, Hopiial Antoine Beclere: and Unit 187, Institut National de la Sante et de la Recherche Meduale. b Received for publication February 17, 1988; revised June 2, 1988; accepted July 29, 1988. Reprint requests: Rene Frydman, MD, Sce de GynecologteObstetrique, Hopltal AntOine Beclere, 157, Rue de la Porte de Trivaux, 92141 Clamart, France.
otropins with or without clomiphene citrate. In some patients the day of oocyte retrieval was programmed in advance with progestogens or oral contraceptives as previously described." The number of embryos immediately transferred in the in vitro fertilization cycle varied between zero and three depending on the protocol. 12 13 The remaining embryos were frozen with 1.2 propanediol used as cryoprotectant. The cryopreservation technique has been described in detail elsewhere! The embryos in this series remained cryopreserved for between 1 month and 2 years. The thawed embryos were transferred in natural cycles in 41 patients and in stimulated cycles in 9 patients. Stimulation included the administration of one ampule of human menopausal gonadotropin (Neopergonal, Serono, Levallois, France) on the sixth, eighth, and tenth days of the cycle. Human chorionic gonadotropin occasionally was administered in both types of cycle, but usually a natural luteinizing hormone surge was documented. The day of luteinizing hormone or human chorionic gonadotropin administration was counted as day 1. The embryos were always transferred on day 4. One-cell zygotes frozen at the pronuclear stage were thawed 24 hours before the time of embryo transfer and subsequently cultured in vitro. Embryos at all other stages were thawed immediately before transfer. Details of embryo and culture factors that were thought to optimize the success of the cryopreservation procedure have been given elsewhere. I ' After embryo transfer, dydrogesterone (Duphaston, Duphar, Villeurbanne, France) was given to support the luteal phase. Patients submitted plasma samples for
209
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Frydman et al.
January 1989 Am J Obstet Gynecol
Table I. Duration of storage of cyropreserved embryos Cryostorage
Thawed embryos (n)
Transferred embryos
% of thawed embryos
Thawing cycles (n)
<3 4-6 7-12 13-24
341 185 72 19
239 137 48 16
70.1 74.1 66.7 84.2
245 109 41 12
31 13 4 2
12.6 11.9 9.8 16.7
617
440
71.3
387
50
12.9
TOTAL
% of thawmg cycles
Table II. Pregnancy rate and outcome related to number of embryos transferred Pregnancies Embryos transferred (n)
Transfer cycles (n)
n
2 3
I
204 85 22
TOTAL
311
Pregnancy outcome AbortIOn (n)
1
%
Live birth (n)
Ongoing* (n)
Preclinical
22 23 5
11 27 23
11 15t 2
2 3 2
4 2 1
3 2 0
50
16
28
7
7
5
First trimester
JElective I
0 0
Ectopic (n) I I
0 2
*Pregnancies > 12 weeks' gestation. tIncIudes three twin pregnancies.
estimation of the [3-subunit of human chorionic gonadotropin twice weekly commencing on the twelfth day after embryo transfer. Preclinical pregnancy was diagnosed by two assays of the [3-subunit of human chorionic gonadotropin >20 mIU/ml after the twelfth day following embryo transfer and the absence of a gestational sac at ultrasonography between the sixth week and the seventh week of amenorrhea. Of the 28 patients who have been delivered to date. 18 were delivered at Antoine Beclere Hospital and the remainder by a local obstetrician. After delivery at other units a questionnaire was completed by the attending obstetrician and returned to our unit for analysis and registration.
Results In this study cryopreserved embryos were thawed in 387 cycles in 320 patients. Table I relates the duration of embryo cryostorage to the results of embryo thawing and transfer. A total of 71.3% of frozen embryos were transferred. The remainder were unsuitable for transfer because of the degree of blastomere lysis. The pregnancy rate per thawing cycle was 12.9% in this series. Fifty-three amniotic sacs were recorded at early ultrasonography in the 50 pregnancies. The pregnancy rate per thawed embryo was 8.6% (53/617). The proportion of frozen embryos that were transferred and the pregnancy rate after transfer did not significantly vary with increasing duration of cryostorage.
Table II shows the pregnancy rate and outcome of pregnancy related to the number of frozen-thawed embryos transferred. The mean number of embryos transferred per transfer cycle was 1.42 ± 0.93 (± SD). Twenty-eight patients have been delivered to date. Twenty-five of the gestations were singleton and three were twins (31 babies). A further seven pregnancies are currently in the second and third trimesters. and these pregnancies are progressing normally. There were seven preclinical abortions and five first-trimester abortions. Elective abortion was performed at 22 weeks in one pregnancy because of severe fetal malformation consisting of homolateral upper- and lower-limb reduction deformity. Two tubal pregnancies were recorded, both of which were surgically treated. A total of 5.7% of thawed embryos produced a normal ongoing pregnancy (38/617). Antenatal factors. Forty-nine of the 50 patients were primigravid. The mean age was 34.1 ± 3.8 years (± SD). Antenatal factors were analyzed in those patients in whom pregnancy progressed beyond the first trimester and in whom the pregnancy outcome is known (28 deliveries and one induced abortion at 22 weeks). Three patients (l 0.3%) experienced first-trimester bleeding. In one of these patients the placenta was subsequently diagnosed to be low-lying and ultrasonographic evidence of retroplacental bleeding was obtained. This did not influence the obstetric outcome
Outcome of pregnancy after transfer of cryopreserved embryos
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211
Table III. Details of complicated deliveries Patient no.
Mode of delivery
Ge;tatzon (wh)
Comment
Emergency cesarean section
41
2 3 4 5 6 7
Emergency cesarean section Emergency cesarean section Emergency cesarean section Emergency cesarean section Elective cesarean section Forceps
38 40 34 35 39 39
8
Forceps
40
9 10
Forceps Forceps Forceps Forceps Forceps
34 38 36 40 41
Fetal distress. retroplacental hemorrhage Fetal distress Fetal distress Twins (breech) Twins Breech Poor second-stage progress and hypertension Breech (forceps to aftercoming head) Twins (prematurity) Poor second-stage progress Prematurity Poor second-stage progress Fetal distress
II
12 13
and an uncomplicated vaginal delivery occurred at term. The other two patients also subsequently had uncomplicated pregnancies and deliveries. Amniocentesis was performed in only two patients. one because of maternal age (40 years) and the second in the patient with the previously noted fetal abnormality. Chromosome analysis was normal in both cases. Pregnancy-induced hypertension occurred in three patients (10.3%). Six patients (20.7%) were hospitalized with premature labor. Three of these had twin pregnancies and all were delivered before the thirty-sixth week of pregnancy. The remaining three had singleton pregnancies. and spontaneous membrane rupture occurred prematurely in two of them. Only one of these patients ultimately was delivered before completion of the thirty-sixth week of pregnancy (premature delivery rate for singleton gestations, 4%). Retroplacental bleeding was diagnosed in four patients. In one, frank hemorrhage necessitated urgent cesarean section because of fetal distress. In the remainder bleeding was diagnosed incidentally at early ultrasonography or after delivery of the placenta and did not have adverse obstetric consequences. Delivery factors. Twenty-two of the 28 deliveries were vaginal and the remainder were by cesarean section (cesarean section rate 21.4%). The details of all instrumented and operative deliveries are shown in Table III. All cesarean sections were performed because of obstetric indications. Five of the six cesarean sections were emergency procedures. Three of the seven forceps deliveries were done because of slow progress in the second stage of labor. The remainder were done because of a variety of obstetric indications. The presentation was breech in five infants. Both infants in one of the twin pregnancies were breech. This patient was delivered by cesarean section. The other three breech presentations all occurred in singleton
gestations after the thirty-seventh week. Two of these were delivered vaginally and one by cesarean section. The incidence of breech delivery in singleton gestations at term in this series was 12%. Figs. 1 and 2 illustrate the birth weight and length of singleton infants as they relate to the normal range for gestational age. Four of the infants were outside the normal weight range. Two were at or below the tenth percentile for birth weight relative to gestational age (one associated with severe hypertension) and two were just above the ninetieth percentile. The length of these infants at birth also was outside the normal range. The Apgar scores at 1 minute after birth were ?7 in all infants except for one delivered by forceps and the twins that were in breech presentation and were delivered by cesarean section at 34 weeks. These infants responded to conventional resuscitation and the 10minute Apgar scores were normal. All 31 children delivered are currently alive and normal.
Comment This is the first report of the obstetric outcome of pregnancies resulting from the transfer of thawed crvopreserved human embryos after in vitro fertilization. It is not our purpose to discuss either the technique or the success rate of embryo cryopreservation. These factors have already been described in several publications. 14 • 15 However it is important to note that the duration of time of embryo cryostorage does not appear to influence the ability of the embryos to resist the cryopreservation procedures or the pregnancy potential of these embryos, at least in the first 2 years. In a previous publication we similarly noted no influence of increasing duration of cryostorage on embryo survival, although at that time it was reported that the survival of individual embryonic cells was adversely affected by longer periods of cryopreservation. l j
212
Frydman et al.
January 1989 Am .I Obstet Gynecol
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Fig. 1. Birth weight (in grams) related to gestational age of
neonates. Percentiles correspond to distribution of birth weights in spontaneously conceived singleton babies in the Paris region.
Fig. 2. Length at birth (in centimeters) related to gestational age of neonates. Percentiles correspond to distribution of birth lengths in spontaneously conceived singleton babies in the Paris region.
It is also relevant that, despite the accepted definition of surviving embryos as those that have retained 50% of the initial number of blastomeres after thawing, there have been two normal deliveries, one from our own unit" and one reported by Viega et al. '6 resulting from embryos with only one of four cells remaining after thawing. Clearly there is a need to review the definition of embryo survival, and it is our policy to transfer all embryos in which at least one blastomere remains intact after thawing. Although the number of pregnancies investigated in this study was limited to 50, the pregnancies all originated from one unit. Reviewing the world experience after embryo cryopreservation, Ashwood-Smith '7 reported 50 pregnancies (including 11 from our unit) leading to 23 births up to April 1986. More recently, Van Steirteghem and Van Den Abbeed 18 presented the results of a survey of > 100 centers in 18 countries dealing with the period up to the end of 1986. One hundred sixty-three pregnancies and 63 births were recorded. This also included some of our data. There have been several reports on the obstetric outcome of in vitro fertilization after fresh embryo transfer. 7• 11 In an analysis of 767 pregnancies, Steptoe et aJ.9 noted a 26.7% clinical abortion rate and an ectopic pregnancy rate of 2.1 %. The results of the Australian collaborative study showed an abortion rate of 21 % and a 5% incidence of ectopic pregnancies." In the present study only 11.6% (5 of 43) of the clinical pregnancies resulted in first-trimester abortion and the incidence of ectopic pregnancy was 4.7%.
The simultaneous replacement of multiple embryos leads to a high incidence of multiple gestation in in vitro fertilization. '9 Twins and other multiple pregnancies are known to be associated with an increased rate of prematurity.20 Recent evidence, however, has suggested that the incidence of premature delivery is elevated even in singleton gestations after in vitro fertilization. 8 The range in the published literature varies from 6.3% to 19%.6.8.10 Only one of 25 patients with a singleton pregnancy was delivered prematurely in this series. This is in agreement with our low rate of prematurity in pregnancies after fresh embryo transfer. 6 The discrepancy between our own results and those of others is probably more related to differences in obstetric practice than to in vitro fertilization technique because premature uterine activity is managed very actively in our unit. We previously reported a high incidence of breech presentations in in vitro fertilization. This finding appears to be confirmed by the present data. Furthermore, it does not seem to be related to multiple pregnancy or prematurity because the incidence in singleton pregnancies at term was 12%. In our maternity unit the incidence of breech presentation at delivery in spontaneously conceived pregnancies is 4.3%.6 Englert et aJ.2 ' suggested that placentation may be abnormal after embryo transfer and postulated that this was due to inadequate orientation of the blastocyst at the time of implantation. The placental site was not assessed in our work, but low placental implantation could be a factor in this high incidence of breech presentation.
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Outcome of
Alternatively, as in this series, there was a very high proportion of nulliparous patients. This factor also may be implicated. One major fetal limb malformation was detected in this study and the pregnancy was terminated at 22 weeks. No chromosomal abnormality was detected in the fetus. A fetal limb abnormality has also been reported by Trounson 22 after embryo cryopreservation. It cannot be stated if these abnormalities are related to the cryopreservation procedure. It is unlikely that the cryoprotectant is implicated because propanediol is used by our group and dimethyl sulfoxide by Trounson's group. Furthermore, freezing and thawing procedures have been documented to be free from genetic risks in many biologic systems. 17 The incidence of major congenital abnormality in in vitro fertilization pregnancies is not thought to be elevated when compared with spontaneously conceived pregnancies. 9 . JO Nevertheless, we would suggest a world registry for cryopreserved embryo pregnancies so that trends may be rapidly appreciated in large numbers of patients. The incidence of cesarean section in this series was only slightly raised compared with the cesarean section rate in spontaneous pregnancies 7 but is in contrast to the high rate in other series of in vitro fertilization pregnancies (33% to 56%7.9. 11). All operative procedures were done because of classic obstetric indications. It is probable that with increasing exposure to in vitro fertilization pregnancies, obstetricians are now more reassured and no longer regard a pregnancy conceived in vitro as inherently at risk, even in the absence of adverse obstetric features. In conclusion, this study describes the outcome of 50 pregnancies after the transfer of thawed cryopreserved embryos. In the 28 patients who have been delivered to date, there was a high incidence of breech presentation similar to that seen after in vitro fertilization with fresh embryos in our unit. There was a high incidence of premature uterine activity, although only one patient with a singleton gestation actually was delivered prematurely. Two small-for-dates infants were recorded. The cesarean section rate was only slightly higher than in the general population. The rate of early pregnancy loss after transfer of thawed cryopreserved embryos does not appear different from that of in vitro fertilization pregnancies with fresh embryo transfer. One major fetal abnormality was recorded. It is suggested that an international registry of pregnancies occurring after embryo cryopreservation should be established. REFERENCES I. Zeilmaker GH, Alberda AT, Van Gent I, Rijkmans CMPM, Drogendijk AC. Two pregnancies following transfer of intact frozen thawed embryos. Fertil Steril 1984;42: 293-6.
pre~nancy
after transfer of cryopreserved embryos
213
2. Freeman L, Trounson A, Kirby C. Cryopreservation of human embryos: progress on the clinical use of the technique in human in vitro fertilization. j In Vitro Fertil Embryo Transfer 1986;3:53-61. 3. Cohen J, Simons RS, Ferhilly CB, Edwards RG. Factors affecting survival and implantation of cryopreserved human embryos. j In Vitro Fertil Embryo Transfer 1986; 3:46-52. 4. Testartj, Lassalle B, Beiaisch-AlIartj, et al. High pregnancy rate after early human embryo freezing. Fertil Steril 1986;46:268-43. 5. Neveu S, Hedon B, Bringer j, et al. Ovarian stimulation by a combination of a gonadotrophin releasing hormone agonist and gonadotropins for in vitro fertilization 1987; 47:639-43. 6. Frydman R, Beiaisch-Allart j, Fries N, Hazout A, Glissant A, Testart j. An obstetric assessment of the first 100 births from the in vitro fertilization program at Clamart, France. AM j OBSTET GYNECOL 1986; 154:550-5. 7. Andrews MC, Muaster Sj, Levy DL, et al. An analysis of the obstetric outcome of 15 consecutive pregnancies conceived in vitro and resulting in 100 deliveries. AM j OSSTET GYNECOL 1986;154:848-54. 8. Australian in Vitro Fertilization Collaborative Group. High incidence of preterm births and early losses in pregnancy after in vitro fertilization. Br Med j 1985;291: 1160-3. 9. Steptoe PC, Edwards RG, Walters DE. Observations on 767 clinical pregnancies and 500 births after human in vitro fertilization. Hum Reprod 1986; I :89-94. 10. Diamond MP, Lavy G, Russell jB, Boyers SP, Nero F, Decherney AH. Weight of babies conceived in vitro. j In Vitro Fertil Embryo Transfer 1987;4:291-3. 11. Frydman R, Forman RG, Rainhorn jD, Beiaisch-Allart j, Hazout A, Testart j. A new approach to follicular stimulation for in vitro fertilization: programed oocyte retrieval. Fertil Steril 1986;46:657-62. 12. Frydman R, RainhornjD, Forman R, et al. Programmed oocyte retrieval during routine laparoscopy and embryo cryopreservation for later transfer. AM j Os STET GYNECOL 1986;155:112-7. 13. Frydman R, Forman RG, Beiaisch-Allart H, Hazout A, Testart j. An assessment of alternative policies for embryo transfer in an IVF-ET programme. Fertil Steril 1988;50: 466-70. 14. Testartj, Lassalle B, Forman R, et al. Factors influencing the success rate of human embryo freezing in an in vitro fertilization and embryo transfer program. Fertil Steril 1987;48:107-12. 15. Testartj, Lassalle B, Beiaisch-AlIartj, et al. Human embryo freezing. Ann NY Acad Sci [In press]. 16. Veiga A, Calderon G, Barri PN, Corolen B. Pregnancy after the replacement of frozen-thawed embryo with <50% intact blastomeres. Hum Reprod 1987;2:321-3. 17. Ashwood-Smith Mj. The cryopreservation of human embryos. Hum Reprod 1986; 1:319-32. 18. Van Steirteghem AC, Van Den Abbeed E. Survey on cryopreservation. Ann NY Acad Sci [In press]. 19. Muasher Sj, Wilkes C, Garcia jE, Rosenwaks Z, jones HWj. Benefits and risks of multiple transfer with in vitro fertilization. Lancet 1984; 1:570-1. 20. McCarthy Bj, Sachs BP, Layde PM, Burton A, Terry jS, Rochat R. The epidemiology of neonatal death in twins. AMj OSSTET GYNECOL 1981;141:252-6. 21. Englert Y, Imbert MC, Van Rosendaer E, et al. Morphological anomalies in the placentae of IVF pregnancies: preliminary report of a multicentric study. Hum Reprod 1987;2:155-7. 22. Trounson A. Preservation of human eggs and embryos. Fertil Steril 1986;46: 1-12.
Key words: Perinatal outcome, in vitro fertilization, human embryo, cryopreservation
The first birth after transfer of cryopreserved human embryos was reported in 1984. 1 Since then, several methods have been described for the freezing and thawing of supernumerary human embryos.2.4 With the recent development of superovulation regimens comprising gonadotropins and agonists of gonadotropinreleasing hormone, it is probable that more oocytes and embryos will be obtained.' Embryo cryopreservation is therefore likely to play an increasing role in in vitro fertilization programs over the next few years. Several reports have described the obstetric outcome in pregnancies resulting from the transfer of "fresh" embryos:·lo but there has been no previous obstetric analysis of pregnancies arising after the transfer of frozenthawed human embryos. This article describes the pregnancy and obstetric factors relative to the first 50 consecutive pregnancies resulting from the transfer of cryopreserved embryos at Antoine Beclere Hospital, Clamart, France.
Patients and methods All 50 pregnancies in this series occurred in infertile patients being treated in the in vitro fertilization program at Antoine Beclere Hospital. Superovulation was induced in all patients in the in vitro fertilization cycle with combinations of gonadFrom the Department of Gynaecology and Obstetrus, Hopiial Antoine Beclere: and Unit 187, Institut National de la Sante et de la Recherche Meduale. b Received for publication February 17, 1988; revised June 2, 1988; accepted July 29, 1988. Reprint requests: Rene Frydman, MD, Sce de GynecologteObstetrique, Hopltal AntOine Beclere, 157, Rue de la Porte de Trivaux, 92141 Clamart, France.
otropins with or without clomiphene citrate. In some patients the day of oocyte retrieval was programmed in advance with progestogens or oral contraceptives as previously described." The number of embryos immediately transferred in the in vitro fertilization cycle varied between zero and three depending on the protocol. 12 13 The remaining embryos were frozen with 1.2 propanediol used as cryoprotectant. The cryopreservation technique has been described in detail elsewhere! The embryos in this series remained cryopreserved for between 1 month and 2 years. The thawed embryos were transferred in natural cycles in 41 patients and in stimulated cycles in 9 patients. Stimulation included the administration of one ampule of human menopausal gonadotropin (Neopergonal, Serono, Levallois, France) on the sixth, eighth, and tenth days of the cycle. Human chorionic gonadotropin occasionally was administered in both types of cycle, but usually a natural luteinizing hormone surge was documented. The day of luteinizing hormone or human chorionic gonadotropin administration was counted as day 1. The embryos were always transferred on day 4. One-cell zygotes frozen at the pronuclear stage were thawed 24 hours before the time of embryo transfer and subsequently cultured in vitro. Embryos at all other stages were thawed immediately before transfer. Details of embryo and culture factors that were thought to optimize the success of the cryopreservation procedure have been given elsewhere. I ' After embryo transfer, dydrogesterone (Duphaston, Duphar, Villeurbanne, France) was given to support the luteal phase. Patients submitted plasma samples for
209
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Frydman et al.
January 1989 Am J Obstet Gynecol
Table I. Duration of storage of cyropreserved embryos Cryostorage
Thawed embryos (n)
Transferred embryos
% of thawed embryos
Thawing cycles (n)
<3 4-6 7-12 13-24
341 185 72 19
239 137 48 16
70.1 74.1 66.7 84.2
245 109 41 12
31 13 4 2
12.6 11.9 9.8 16.7
617
440
71.3
387
50
12.9
TOTAL
% of thawmg cycles
Table II. Pregnancy rate and outcome related to number of embryos transferred Pregnancies Embryos transferred (n)
Transfer cycles (n)
n
2 3
I
204 85 22
TOTAL
311
Pregnancy outcome AbortIOn (n)
1
%
Live birth (n)
Ongoing* (n)
Preclinical
22 23 5
11 27 23
11 15t 2
2 3 2
4 2 1
3 2 0
50
16
28
7
7
5
First trimester
JElective I
0 0
Ectopic (n) I I
0 2
*Pregnancies > 12 weeks' gestation. tIncIudes three twin pregnancies.
estimation of the [3-subunit of human chorionic gonadotropin twice weekly commencing on the twelfth day after embryo transfer. Preclinical pregnancy was diagnosed by two assays of the [3-subunit of human chorionic gonadotropin >20 mIU/ml after the twelfth day following embryo transfer and the absence of a gestational sac at ultrasonography between the sixth week and the seventh week of amenorrhea. Of the 28 patients who have been delivered to date. 18 were delivered at Antoine Beclere Hospital and the remainder by a local obstetrician. After delivery at other units a questionnaire was completed by the attending obstetrician and returned to our unit for analysis and registration.
Results In this study cryopreserved embryos were thawed in 387 cycles in 320 patients. Table I relates the duration of embryo cryostorage to the results of embryo thawing and transfer. A total of 71.3% of frozen embryos were transferred. The remainder were unsuitable for transfer because of the degree of blastomere lysis. The pregnancy rate per thawing cycle was 12.9% in this series. Fifty-three amniotic sacs were recorded at early ultrasonography in the 50 pregnancies. The pregnancy rate per thawed embryo was 8.6% (53/617). The proportion of frozen embryos that were transferred and the pregnancy rate after transfer did not significantly vary with increasing duration of cryostorage.
Table II shows the pregnancy rate and outcome of pregnancy related to the number of frozen-thawed embryos transferred. The mean number of embryos transferred per transfer cycle was 1.42 ± 0.93 (± SD). Twenty-eight patients have been delivered to date. Twenty-five of the gestations were singleton and three were twins (31 babies). A further seven pregnancies are currently in the second and third trimesters. and these pregnancies are progressing normally. There were seven preclinical abortions and five first-trimester abortions. Elective abortion was performed at 22 weeks in one pregnancy because of severe fetal malformation consisting of homolateral upper- and lower-limb reduction deformity. Two tubal pregnancies were recorded, both of which were surgically treated. A total of 5.7% of thawed embryos produced a normal ongoing pregnancy (38/617). Antenatal factors. Forty-nine of the 50 patients were primigravid. The mean age was 34.1 ± 3.8 years (± SD). Antenatal factors were analyzed in those patients in whom pregnancy progressed beyond the first trimester and in whom the pregnancy outcome is known (28 deliveries and one induced abortion at 22 weeks). Three patients (l 0.3%) experienced first-trimester bleeding. In one of these patients the placenta was subsequently diagnosed to be low-lying and ultrasonographic evidence of retroplacental bleeding was obtained. This did not influence the obstetric outcome
Outcome of pregnancy after transfer of cryopreserved embryos
Volume 160 Number 1
211
Table III. Details of complicated deliveries Patient no.
Mode of delivery
Ge;tatzon (wh)
Comment
Emergency cesarean section
41
2 3 4 5 6 7
Emergency cesarean section Emergency cesarean section Emergency cesarean section Emergency cesarean section Elective cesarean section Forceps
38 40 34 35 39 39
8
Forceps
40
9 10
Forceps Forceps Forceps Forceps Forceps
34 38 36 40 41
Fetal distress. retroplacental hemorrhage Fetal distress Fetal distress Twins (breech) Twins Breech Poor second-stage progress and hypertension Breech (forceps to aftercoming head) Twins (prematurity) Poor second-stage progress Prematurity Poor second-stage progress Fetal distress
II
12 13
and an uncomplicated vaginal delivery occurred at term. The other two patients also subsequently had uncomplicated pregnancies and deliveries. Amniocentesis was performed in only two patients. one because of maternal age (40 years) and the second in the patient with the previously noted fetal abnormality. Chromosome analysis was normal in both cases. Pregnancy-induced hypertension occurred in three patients (10.3%). Six patients (20.7%) were hospitalized with premature labor. Three of these had twin pregnancies and all were delivered before the thirty-sixth week of pregnancy. The remaining three had singleton pregnancies. and spontaneous membrane rupture occurred prematurely in two of them. Only one of these patients ultimately was delivered before completion of the thirty-sixth week of pregnancy (premature delivery rate for singleton gestations, 4%). Retroplacental bleeding was diagnosed in four patients. In one, frank hemorrhage necessitated urgent cesarean section because of fetal distress. In the remainder bleeding was diagnosed incidentally at early ultrasonography or after delivery of the placenta and did not have adverse obstetric consequences. Delivery factors. Twenty-two of the 28 deliveries were vaginal and the remainder were by cesarean section (cesarean section rate 21.4%). The details of all instrumented and operative deliveries are shown in Table III. All cesarean sections were performed because of obstetric indications. Five of the six cesarean sections were emergency procedures. Three of the seven forceps deliveries were done because of slow progress in the second stage of labor. The remainder were done because of a variety of obstetric indications. The presentation was breech in five infants. Both infants in one of the twin pregnancies were breech. This patient was delivered by cesarean section. The other three breech presentations all occurred in singleton
gestations after the thirty-seventh week. Two of these were delivered vaginally and one by cesarean section. The incidence of breech delivery in singleton gestations at term in this series was 12%. Figs. 1 and 2 illustrate the birth weight and length of singleton infants as they relate to the normal range for gestational age. Four of the infants were outside the normal weight range. Two were at or below the tenth percentile for birth weight relative to gestational age (one associated with severe hypertension) and two were just above the ninetieth percentile. The length of these infants at birth also was outside the normal range. The Apgar scores at 1 minute after birth were ?7 in all infants except for one delivered by forceps and the twins that were in breech presentation and were delivered by cesarean section at 34 weeks. These infants responded to conventional resuscitation and the 10minute Apgar scores were normal. All 31 children delivered are currently alive and normal.
Comment This is the first report of the obstetric outcome of pregnancies resulting from the transfer of thawed crvopreserved human embryos after in vitro fertilization. It is not our purpose to discuss either the technique or the success rate of embryo cryopreservation. These factors have already been described in several publications. 14 • 15 However it is important to note that the duration of time of embryo cryostorage does not appear to influence the ability of the embryos to resist the cryopreservation procedures or the pregnancy potential of these embryos, at least in the first 2 years. In a previous publication we similarly noted no influence of increasing duration of cryostorage on embryo survival, although at that time it was reported that the survival of individual embryonic cells was adversely affected by longer periods of cryopreservation. l j
212
Frydman et al.
January 1989 Am .I Obstet Gynecol
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Fig. 1. Birth weight (in grams) related to gestational age of
neonates. Percentiles correspond to distribution of birth weights in spontaneously conceived singleton babies in the Paris region.
Fig. 2. Length at birth (in centimeters) related to gestational age of neonates. Percentiles correspond to distribution of birth lengths in spontaneously conceived singleton babies in the Paris region.
It is also relevant that, despite the accepted definition of surviving embryos as those that have retained 50% of the initial number of blastomeres after thawing, there have been two normal deliveries, one from our own unit" and one reported by Viega et al. '6 resulting from embryos with only one of four cells remaining after thawing. Clearly there is a need to review the definition of embryo survival, and it is our policy to transfer all embryos in which at least one blastomere remains intact after thawing. Although the number of pregnancies investigated in this study was limited to 50, the pregnancies all originated from one unit. Reviewing the world experience after embryo cryopreservation, Ashwood-Smith '7 reported 50 pregnancies (including 11 from our unit) leading to 23 births up to April 1986. More recently, Van Steirteghem and Van Den Abbeed 18 presented the results of a survey of > 100 centers in 18 countries dealing with the period up to the end of 1986. One hundred sixty-three pregnancies and 63 births were recorded. This also included some of our data. There have been several reports on the obstetric outcome of in vitro fertilization after fresh embryo transfer. 7• 11 In an analysis of 767 pregnancies, Steptoe et aJ.9 noted a 26.7% clinical abortion rate and an ectopic pregnancy rate of 2.1 %. The results of the Australian collaborative study showed an abortion rate of 21 % and a 5% incidence of ectopic pregnancies." In the present study only 11.6% (5 of 43) of the clinical pregnancies resulted in first-trimester abortion and the incidence of ectopic pregnancy was 4.7%.
The simultaneous replacement of multiple embryos leads to a high incidence of multiple gestation in in vitro fertilization. '9 Twins and other multiple pregnancies are known to be associated with an increased rate of prematurity.20 Recent evidence, however, has suggested that the incidence of premature delivery is elevated even in singleton gestations after in vitro fertilization. 8 The range in the published literature varies from 6.3% to 19%.6.8.10 Only one of 25 patients with a singleton pregnancy was delivered prematurely in this series. This is in agreement with our low rate of prematurity in pregnancies after fresh embryo transfer. 6 The discrepancy between our own results and those of others is probably more related to differences in obstetric practice than to in vitro fertilization technique because premature uterine activity is managed very actively in our unit. We previously reported a high incidence of breech presentations in in vitro fertilization. This finding appears to be confirmed by the present data. Furthermore, it does not seem to be related to multiple pregnancy or prematurity because the incidence in singleton pregnancies at term was 12%. In our maternity unit the incidence of breech presentation at delivery in spontaneously conceived pregnancies is 4.3%.6 Englert et aJ.2 ' suggested that placentation may be abnormal after embryo transfer and postulated that this was due to inadequate orientation of the blastocyst at the time of implantation. The placental site was not assessed in our work, but low placental implantation could be a factor in this high incidence of breech presentation.
Volume 160 Number 1
Outcome of
Alternatively, as in this series, there was a very high proportion of nulliparous patients. This factor also may be implicated. One major fetal limb malformation was detected in this study and the pregnancy was terminated at 22 weeks. No chromosomal abnormality was detected in the fetus. A fetal limb abnormality has also been reported by Trounson 22 after embryo cryopreservation. It cannot be stated if these abnormalities are related to the cryopreservation procedure. It is unlikely that the cryoprotectant is implicated because propanediol is used by our group and dimethyl sulfoxide by Trounson's group. Furthermore, freezing and thawing procedures have been documented to be free from genetic risks in many biologic systems. 17 The incidence of major congenital abnormality in in vitro fertilization pregnancies is not thought to be elevated when compared with spontaneously conceived pregnancies. 9 . JO Nevertheless, we would suggest a world registry for cryopreserved embryo pregnancies so that trends may be rapidly appreciated in large numbers of patients. The incidence of cesarean section in this series was only slightly raised compared with the cesarean section rate in spontaneous pregnancies 7 but is in contrast to the high rate in other series of in vitro fertilization pregnancies (33% to 56%7.9. 11). All operative procedures were done because of classic obstetric indications. It is probable that with increasing exposure to in vitro fertilization pregnancies, obstetricians are now more reassured and no longer regard a pregnancy conceived in vitro as inherently at risk, even in the absence of adverse obstetric features. In conclusion, this study describes the outcome of 50 pregnancies after the transfer of thawed cryopreserved embryos. In the 28 patients who have been delivered to date, there was a high incidence of breech presentation similar to that seen after in vitro fertilization with fresh embryos in our unit. There was a high incidence of premature uterine activity, although only one patient with a singleton gestation actually was delivered prematurely. Two small-for-dates infants were recorded. The cesarean section rate was only slightly higher than in the general population. The rate of early pregnancy loss after transfer of thawed cryopreserved embryos does not appear different from that of in vitro fertilization pregnancies with fresh embryo transfer. One major fetal abnormality was recorded. It is suggested that an international registry of pregnancies occurring after embryo cryopreservation should be established. REFERENCES I. Zeilmaker GH, Alberda AT, Van Gent I, Rijkmans CMPM, Drogendijk AC. Two pregnancies following transfer of intact frozen thawed embryos. Fertil Steril 1984;42: 293-6.
pre~nancy
after transfer of cryopreserved embryos
213
2. Freeman L, Trounson A, Kirby C. Cryopreservation of human embryos: progress on the clinical use of the technique in human in vitro fertilization. j In Vitro Fertil Embryo Transfer 1986;3:53-61. 3. Cohen J, Simons RS, Ferhilly CB, Edwards RG. Factors affecting survival and implantation of cryopreserved human embryos. j In Vitro Fertil Embryo Transfer 1986; 3:46-52. 4. Testartj, Lassalle B, Beiaisch-AlIartj, et al. High pregnancy rate after early human embryo freezing. Fertil Steril 1986;46:268-43. 5. Neveu S, Hedon B, Bringer j, et al. Ovarian stimulation by a combination of a gonadotrophin releasing hormone agonist and gonadotropins for in vitro fertilization 1987; 47:639-43. 6. Frydman R, Beiaisch-Allart j, Fries N, Hazout A, Glissant A, Testart j. An obstetric assessment of the first 100 births from the in vitro fertilization program at Clamart, France. AM j OBSTET GYNECOL 1986; 154:550-5. 7. Andrews MC, Muaster Sj, Levy DL, et al. An analysis of the obstetric outcome of 15 consecutive pregnancies conceived in vitro and resulting in 100 deliveries. AM j OSSTET GYNECOL 1986;154:848-54. 8. Australian in Vitro Fertilization Collaborative Group. High incidence of preterm births and early losses in pregnancy after in vitro fertilization. Br Med j 1985;291: 1160-3. 9. Steptoe PC, Edwards RG, Walters DE. Observations on 767 clinical pregnancies and 500 births after human in vitro fertilization. Hum Reprod 1986; I :89-94. 10. Diamond MP, Lavy G, Russell jB, Boyers SP, Nero F, Decherney AH. Weight of babies conceived in vitro. j In Vitro Fertil Embryo Transfer 1987;4:291-3. 11. Frydman R, Forman RG, Rainhorn jD, Beiaisch-Allart j, Hazout A, Testart j. A new approach to follicular stimulation for in vitro fertilization: programed oocyte retrieval. Fertil Steril 1986;46:657-62. 12. Frydman R, RainhornjD, Forman R, et al. Programmed oocyte retrieval during routine laparoscopy and embryo cryopreservation for later transfer. AM j Os STET GYNECOL 1986;155:112-7. 13. Frydman R, Forman RG, Beiaisch-Allart H, Hazout A, Testart j. An assessment of alternative policies for embryo transfer in an IVF-ET programme. Fertil Steril 1988;50: 466-70. 14. Testartj, Lassalle B, Forman R, et al. Factors influencing the success rate of human embryo freezing in an in vitro fertilization and embryo transfer program. Fertil Steril 1987;48:107-12. 15. Testartj, Lassalle B, Beiaisch-AlIartj, et al. Human embryo freezing. Ann NY Acad Sci [In press]. 16. Veiga A, Calderon G, Barri PN, Corolen B. Pregnancy after the replacement of frozen-thawed embryo with <50% intact blastomeres. Hum Reprod 1987;2:321-3. 17. Ashwood-Smith Mj. The cryopreservation of human embryos. Hum Reprod 1986; 1:319-32. 18. Van Steirteghem AC, Van Den Abbeed E. Survey on cryopreservation. Ann NY Acad Sci [In press]. 19. Muasher Sj, Wilkes C, Garcia jE, Rosenwaks Z, jones HWj. Benefits and risks of multiple transfer with in vitro fertilization. Lancet 1984; 1:570-1. 20. McCarthy Bj, Sachs BP, Layde PM, Burton A, Terry jS, Rochat R. The epidemiology of neonatal death in twins. AMj OSSTET GYNECOL 1981;141:252-6. 21. Englert Y, Imbert MC, Van Rosendaer E, et al. Morphological anomalies in the placentae of IVF pregnancies: preliminary report of a multicentric study. Hum Reprod 1987;2:155-7. 22. Trounson A. Preservation of human eggs and embryos. Fertil Steril 1986;46: 1-12.