Human Reproduction, Vol. 18, No. 6, 1313-1318,
June 2003
© 2003 European Society of Human Reproduction and Embryology
Cryo-thawed embryos obtained from conception cycles have double the implantation and pregnancy potential of those from unsuccessful cycles
Assisted Conception Unit, Guy’s and St Thomas’ Hospital NHS Trust, St Thomas’ Street, London SE1 9RT, UK
1 To whom correspondence should be addressed. e-mail: tarekeltoukhy{at}hotmail.com
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Abstract |
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BACKGROUND: The purpose of this study was to evaluate the influence of fresh IVF/ICSI cycle outcome on the prognosis of the related frozen embryo replacement (FER) cycle. METHODS: 459 FER cycles, involving 2049 cleavage stage embryos with no or up to 10% fragmentation, were performed for which the outcome of the fresh cycle was recorded. The cycles were divided into two groups; group A included cycles in which cryopreserved embryos were obtained from fresh cycles in which conception occurred. Group B were cycles in which cryopreserved embryos originated from unsuccessful fresh cycles. RESULTS: Groups A and B were comparable with respect to mean (± SD) age at cryopreservation (33 ± 3.9 versus 33.2 ± 4 years, P = not significant), mean number of oocytes retrieved and fertilized normally in the fresh cycle (11 ± 5.2 versus 11.2 ± 4.8, P = not significant) and mean age at the cryo-thawed transfer (34.5 ± 4.2 versus 33.9 ± 4 years, P = not significant). No significant difference was found between the two groups with regard to mean number of embryos cryopreserved (6.5 ± 3.9 versus 6.2 ± 3.6) and subsequently thawed (4.5 ± 2.5 versus 4.5 ± 1.8) per cycle and number of cryo-thawed embryos transferred per cycle (2.0 ± 0.7 versus 2.1 ± 0.8). However, the implantation rate per transferred embryo in group A was double that in group B (23 versus 11.2%, P < 0.0001). Moreover, the clinical pregnancy and ongoing pregnancy rates per cycle were significantly higher in group A compared with group B (34.8 and 27.3% versus 15.6 and 13.1%, P < 0.0001 and P = 0.0003 respectively). The difference in FER cycle outcome could not be explained by confounding variables. CONCLUSIONS: After thawing, cryopreserved embryos originating from conception IVF/ICSI cycles achieve double the implantation and pregnancy rates of those obtained from unsuccessful cycles.
Key words: cryo-thawed cycle prognosis/embryo cryopreservation/IVF outcome
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The expectation of pregnancy is an important issue for infertile couples undergoing IVF. The ability to identify prognostic factors could help to reduce the considerable uncertainty that still surrounds the outcome of IVF treatment and provide more accurate outcome advice for couples.
With the current trend to avoid multiple pregnancy by electively reducing the number of embryos replaced per cycle (Martikainen et al., 2001
; Tiitinen et al., 2001) and saving surplus embryos for replacement later, the potential for embryo cryopreservation to augment the ultimate chance of pregnancy from a single attempt at oocyte retrieval is extended. Thus, the fresh and subsequent frozen–thawed transfers can be regarded as complementary stages of the same course of IVF treatment. Consequently, it would be helpful for patient counselling if prognosis of the latter stage could be related to the outcome of the initial (fresh) one.
Literature data which relate the fresh cycle outcome and prognosis of the subsequent frozen–thawed cycle are conflicting (Lin et al., 1995; Molloy et al., 1995). Studies which cited a favourable impact of conception in the fresh cycle (Toner et al., 1991; Lin et al., 1995; Wang et al., 2001) were retrospective, included heterogeneous groups of patients, used more than one regime during ovarian stimulation, did not account for embryo quality at cryopreservation, and as such could not be conclusive.
The aim of this study was to examine the influence of the fresh cycle outcome on the prognosis of the subsequent frozen–thawed transfer in patients who have similar embryo quality before cryopreservation.
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Materials and methods |
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Between February 1997 and July 2002, 405 consecutive couples underwent a total of 459 cleavage stage FER cycles for which the outcome of the fresh cycles was known. Cycles were excluded from the study if the embryos were derived from a fresh cycle that was stopped before embryo transfer (for example, to reduce the risk of the ovarian hyperstimulation syndrome), if embryos were cryopreserved at the pronuclear stage or if the cryo-thawed embryos transferred were derived from donated oocytes or from more than one fresh cycle.
Ovarian stimulation
Patients underwent pituitary down-regulation using buserelin (Suprefact; Hoechst UK Ltd, UK) in a mid-luteal start long protocol. Gonadotrophin treatment was initiated following satisfactory pituitary suppression as evidenced by a thin endometrium and absence of follicular activity or ovarian cysts. Ovarian stimulation was achieved using a daily FSH dose of 150–450 IU of highly purified urinary FSH (Metrodin HP; Serono Laboratories Ltd, UK) or recombinant FSH (Gonal-F; Serono or Puregon; Organon, UK) depending on age and previous response to ovarian stimulation. hCG 10 000 IU (Profasi; Serono or Pregnyl; Organon) was administered to induce oocyte maturation when at least three follicles had reached a mean diameter of 
18 mm.
IVF procedure
Transvaginal follicular aspiration was carried out 34–36 h after hCG injection using an ultrasound scanner with a 6.5 MHz probe (Hitachi EUB 525, Japan). Cumulus–oocyte complexes were isolated from follicular aspirates, washed and incubated at 37°C in an atmosphere of 5% CO2 in air. Oocytes were observed under an inverted microscope for evidence of fertilization 16–18 h after insemination. Fertilization was considered normal when two distinct pronuclei were visible.
ICSI
After removal of cumulus cells using hyaluronidase and mechanical pipetting, oocytes were examined for their nuclear maturity. ICSI was carried out on oocytes that had extruded the first polar body. Following sperm injection, the oocytes were transferred to 100 µl droplets of cleavage medium (Cook IVF, Australia) under oil and incubated overnight at 37°C in an atmosphere of 6% CO2 in air. The oocytes were examined for survival and fertilization on the following day (16–18 h after microinjection).
Embryo grading
Cleavage stage embryos were assigned grades according to strict morphological and developmental criteria (Steer et al., 1992; Gerris et al., 1999; Van Royen et al., 1999). Cycles were included in the study only if the fresh embryos selected for transfer and cryopreservation satisfied the following criteria: (i) embryos had reached the appropriate stage of development (i.e. 4 cells on day 2 (40–42 h) or 6–8 cells on day 3 (64–66 h) of in-vitro culture); (ii) component blastomeres were symmetrical in size, (iii) devoid of multinucleation and (iv) showed either no or 
10% cytoplasmic fragmentation.
Embryo transfer
Between one and three cleavage stage embryos were transferred to the uterus on day 2 or day 3 after insemination using an Edwards–Wallace embryo transfer catheter (Sims Portex Ltd, UK). All patients who underwent embryo transfer received supplemental progesterone pessaries (Cyclogest, Shire Pharmaceuticals Ltd, UK) 400 mg daily throughout the luteal phase.
FER cycle protocol
Cryopreservation
A standard freezing protocol, employing 1,2-propanediol (PROH) and sucrose as cryoprotectants, was used (Lassalle et al., 1985; Edgar et al., 2000). Freezing and thawing solutions consisted of the cryoprotectants in a HEPES-buffered salt solution supplemented with 0.5% w/v human serum albumin (HSA). Embryos were equilibrated in 1.5 mol/l propanediol solution for 10 min at room temperature before being transferred to 1.5 propanediol–0.1 mol/l sucrose and loaded individually into ministraws (Rocket Medical, UK). Cooling was performed using a programmable freezer (CryoLogic, CL-863, Australia) at a rate of –2°C/min to –7°C at which point manual seeding was performed. Cooling resumed at a rate of –0.3°C/min to –30°C before the ministraws were plunged and stored in liquid nitrogen.
Thawing
Embryos were thawed rapidly by removal from liquid nitrogen and exposure to air for 45 s followed by immersion in a water bath at 30°C for 30 s. A three-step process of PROH removal in the presence of 0.2 mol/l sucrose ensued at room temperature for 5 min in each step until final rehydration in a HEPES-buffered salt solution. Thawed embryos were then carefully assessed for blastomere survival using an inverted microscope (Nikon UK Ltd, UK) at a magnification of x200, before being transferred into culture medium at 37°C. Blastomeres were considered damaged when they were lysed, degenerated or dark. Embryos that had lost >50% of their original blastomeres were not transferred. A second evaluation was performed prior to transfer (usually 20 h after the initial evaluation) in order to record the resumption of mitosis as indicated by cleavage of at least one blastomere.
Endometrial preparation
Estradiol valerate 6 mg daily (Climaval; Novartis Pharmaceuticals, UK) was commenced orally on day 2 of the menstrual cycle and continued until endometrial thickness reached 8 mm. Progesterone supplementation (Cyclogest; Shire) 400 mg twice daily was commenced 48 h prior to transfer.
Frozen embryo replacement and hormonal support
As in the fresh transfer, one, two or three embryos were transferred to the uterus using an Edwards–Wallace catheter (Sims Portex Ltd). After embryo replacement, exogenous hormonal supplementation was continued for 14 days until a urine pregnancy test using commercially available kits was performed. Patients with a positive test continued with hormone supplementation until they were 12 weeks pregnant.
Cycle outcome
Pregnancy was diagnosed by a positive urine test for hCG 
14 days after embryo replacement. A clinical pregnancy was defined as the observation on ultrasound scanning of a gestational sac with fetal heart beat between 4 and 5 weeks after the positive pregnancy test. Ongoing pregnancy was defined as a viable intrauterine pregnancy beyond 16 weeks of gestation. Implantation rate was defined as the number of gestational sacs observed on ultrasound compared with the number of embryos transferred. A biochemical pregnancy was defined as a rise in serum hCG level in the absence of an intrauterine gestational sac 3 weeks after a positive urine pregnancy test. Such pregnancies were followed up using serum hCG levels until they declined to normal (non-pregnant) levels. The hCG levels were measured by a highly specific chemiluminescent immunoassay (Bayer Advia Analyser; Bayer Plc., UK) with <0.1% cross-reactivity with LH and <1 IU/l sensitivity.
Power calculation and statistical analysis
Assuming that 25% of cryo-thawed cycles follow an initial fresh cycle in which conception has occurred (Toner et al., 1991; Lin et al., 1995), we calculated that some 456 frozen–thawed cycles were needed to detect a difference of 15% (between 30 and 15%) in the clinical pregnancy rate between the two groups with 90% power, a two-sided alpha of 0.05 and an allocation ratio of 3:1 (Campbell et al., 1995).
Analysis of variance was performed for the different variables. Implantation, pregnancy and ongoing pregnancy rates per cycle and associated clinical variables were analysed and compared with Student’s t-test, 
2-test or Fisher’s exact test where appropriate. Statview software package for Macintosh (Statview 4.1; Abacus Concepts Ltd, USA) was used for statistical analysis. Statistical significance was set at P < 0.05.
Because the present work did not involve either therapeutic interventions or change to our routine IVF–embryo replacement protocols, we did not require additional approval from our institutional ethics committee. However, written informed consent was obtained from each couple upon entering our IVF programme and before starting a FER cycle.
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Results |
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A total of 405 couples (mean ± SD female age 33.2 ± 4 years, range 23–44) underwent 459 FER cycles and were included in the study. In these cycles, 2049 frozen embryos were thawed, 1331 embryos (65%) survived the process of thawing with
50% of their original blastomeres intact and 967 (47%) were replaced (mean 2.1 ± 0.7 embryo/cycle). The overall implantation and pregnancy rates per cycle were 14.8 and 32.6% respectively. Implantation and pregnancy rates were similar in cycles in which IVF or ICSI was used for oocyte insemination [15.8 versus 13.2%, P = not significant (NS) and 32.3 versus 31.4%, P = NS respectively] and in cycles in which thawed embryos were originally cryopreserved on day 2 or 3 of in-vitro culture (15.2 versus 12.7%, P = NS and 33.2 versus 32.6%, P = NS respectively). Among the original 405 retrieval IVF/ICSI cycles, 117 patients (28.6%) achieved a pregnancy (group A), while 288 cycles (71.4%) were not successful (group B). Groups A and B were comparable with respect to their demographic and basic characteristics, including age at the time of the fresh cycle, type and cause of infertility, basal (day 2–4) serum FSH level prior to ovarian stimulation and number of previous IVF/ICSI attempts (Table I).
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There was no statistically significant difference between the two groups with regard to fresh cycle characteristics including the daily FSH dose, mean number of oocytes collected and fertilized normally, proportion of patients receiving ICSI, oocyte fertilization rate and mean number of embryos replaced and cryopreserved per cycle. Additionally, the proportion of fresh cycles in which more than four embryos were cryopreserved was the same in the two groups (Table I).
Patients in group A returned for treatment using their cryopreserved embryos on average 9 months later than those in group B (17 ± 11 versus 8 ± 6 months, P < 0.001). In the frozen–thawed cycles (Table II), no significant difference was observed between groups A and B in relation to mean age at replacement, proportion of couples who underwent two treatment cycles, number of embryos thawed per cycle and percentage of embryos surviving the thawing process with 50% of their original blastomeres intact and of cycles in which embryos replaced had survived thawing with all their original blastomeres intact. Patients in the two groups achieved similar mean endometrial thickness following a comparable duration of exogenous estrogen stimulation and subsequently had a similar mean number of thawed embryos replaced. Significantly more replacements in group A included only embryos that showed post-thaw cleavage (P = 0.04).
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Frozen–thawed cycle outcome is summarized in Table II. Group A had significantly higher implantation, pregnancy, clinical pregnancy and ongoing pregnancy rates compared with group B. Patients in group A were twice as likely to achieve a pregnancy compared with group B. This difference persisted even after stratification of the two groups by female age at cryopreservation and by number of embryos cryopreserved (Table III). Likewise, when the analysis was limited to the first FER cycle only, the difference between group A and B in implantation and pregnancy rates remained highly significant (24.4 versus 12.3%, P = 0.0004; 53 versus 25.6%, P < 0.0001 respectively).
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Further analysis of group A failed to show a significant influence of the fresh cycle pregnancy outcome (i.e. biochemical versus clinical) on the occurrence of an ongoing pregnancy in the frozen–thawed cycle. Women who had a biochemical pregnancy in their fresh cycle achieved a similar ongoing pregnancy rate to those in whom the fresh cycle clinical pregnancy ended in a miscarriage before 24 weeks gestation or a live birth (33.3, 25.6 and 26.2% respectively, P = not significant).
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Discussion |
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Embryo cryopreservation provides additional opportunities for pregnancy beyond those achieved from the stimulated IVF cycle. Results of the present study support the hypothesis that fecundity of a fresh cycle is an independent factor that influences the success of the subsequent frozen–thawed cycle. The overall implantation rate per transferred embryo in the study was 14.5%, which compares favourably with other large series (Selick et al., 1995
; Edgar et al., 2000; Oehninger et al., 2000; Wang et al., 2001), and is similar to that reported in the study of Guerif et al. (2002), which used similar embryo selection criteria at cryopreservation. However, it reached 23% when the embryos were obtained from a conception cycle. Similarly, the overall clinical and ongoing pregnancy rates were 20.5 and 17%, but rose to 34.8 and 27.3% in the group of patients who conceived in their initial fresh cycle respectively.
Previous studies that looked at the influence of the fresh cycle outcome on success of the related cryo-thawed cycle (Toner et al., 1991; Lin et al., 1995; Wang et al., 2001) did not control for embryo quality. They also included patients treated with gamete intra-Fallopian transfer and IVF in the same series (Lin et al., 1995; Wang et al., 2001). In fact, when the analysis in one study (Lin et al., 1995) was limited to patients undergoing IVF alone in their initial cycles, the study failed to show a significant difference in the outcome of cryo-thawed cycles in favour of those who had a fresh attempt using fresh embryos. Moreover, although Toner et al. (1991) reported a higher implantation rate when the cryo-thawed embryos had originated from conception cycles, pregnancy rates were not different in the groups studied.
Unlike previous reports, we attempted to account for most of the confounding variables that might affect the outcome of fresh and frozen–thawed IVF cycles. Among those variables, embryo quality is one of the most important. Therefore, the present study has been restricted to cycles in which fresh embryos selected for replacement or cryopreservation had similar characteristics before cryopreservation, thus allowing for meaningful comparisons.
Our data could be explained by maternal influences, embryo-related differences or a combination of both factors. With regard to maternal influences, it has been demonstrated that women who had a previous pregnancy have a significantly higher live birth rate after IVF treatment than women with no previous pregnancies, even after adjusting for age (Templeton et al., 1996). Likewise, women who achieved a previous IVF pregnancy were also found to have a higher chance of pregnancy on subsequent attempts compared with those not conceiving (Tan et al., 1994; Molloy et al., 1995; Croucher et al., 1998; Templeton and Morris, 1998). The present study extends these observations and confirms that women who have achieved a pregnancy in a fresh IVF/ICSI cycle have a superior chance of conception in the related frozen–thawed cycle. These women are likely to be more fertile and possess more efficient reproductive mechanisms that facilitate implantation as evidenced by the occurrence of conception in their fresh attempt. This postulate is supported by the study of Simon et al. (1993) which suggested that patients who conceived in an IVF cycle have an enhanced endometrial receptivity that is resistant to the negative effects of superovulation, offering them an improved prognosis in subsequent attempts, compared with patients who conceived naturally but were subsequently infertile.
The present study results could also be explained on the basis of differences in embryo quality. There is considerable evidence that embryo morphology prior to cryopreservation impacts on the outcome of the cryo-thawed cycle (Mandelbaum et al., 1988; Schalkoff et al., 1993; Kondo et al., 1996; Karlstrom et al., 1997; Lightman et al., 1997). In this study, fresh embryos were selected according to defined morphological criteria (Staessen et al., 1992; Steer et al., 1992; Gerris et al., 1999; Van Royen et al., 1999); embryos that have reached 4 cells by day 2 or 6–8 cells by day 3 of in-vitro culture and showed symmetrical blastomeres with single nuclei and 10% fragmentation were considered of high quality and selected for either transfer or cryopreservation. This ensured a fairly similar quality among the embryo population studied. The study groups were also comparable with respect to post-thaw embryo survival and the proportion of thawed cycles in which all transferred embryos survived fully intact and showed no blastomere loss (Edgar et al., 2000). Nevertheless, cryo-thawed embryos derived from successful fresh cycles were more able to divide after thawing, leading to a significantly higher proportion of cycles in which only embryos that had divided after thawing were transferred (P = 0.04). This observation accords with previous studies (Van der Elst et al., 1997; Ziebe et al., 1998; Guerif et al., 2002), which demonstrated that post-thaw cleavage is a sign of superior implantation potential. On the other hand, limited capacity to resume cleavage after thawing has been reported to be associated with an increased prevalence of some numerical chromosomal abnormalities in blastomeres of cleavage stage embryos that survived cryopreservation, but did not cleave further after thawing (Laverge et al., 1998).
Further understanding of the superior developmental potential of embryos originating from conception cycles can be derived from the concept of cohort homogeneity (Trounson et al., 1986). It has been shown that embryo survival, cleavage and pregnancy rates of cryo-thawed embryos are very similar to those from their fresh sibling counterparts (Fugger et al., 1988). Selick et al. (1995) studied the implantation potential of sibling oocytes after fresh and frozen transfers using an ovum donation model and reported a similar chance of implantation among high quality embryos obtained from the same cohort of oocytes whether transferred fresh or in the cryo-thawed state. More recently, Fisch et al. (1999) demonstrated that cleavage stage embryo transfers were associated with higher pregnancy rates when their sibling embryos developed to the blastocyst stage during in-vitro culture. Our own data indicate a predictable pattern of performance within each cohort of embryos selected for transfer and cryopreservation that could not be explained by confounding variables and point to a homogeneous oocyte quality among the cohort of embryos selected for transfer and cryopreservation.
Finally, the present study shows that very early pregnancy failure in the stimulated IVF cycle does not impair the outlook of pregnancy achieved in the subsequent cryo-thawed cycle. Unlike the findings of Acosta et al. (1990) and Molloy et al. (1995), our results demonstrate that women who have a biochemical pregnancy in their fresh cycle achieve an ongoing similar pregnancy rate to those in whom the fresh cycle clinical pregnancy ended either in miscarriage or live birth. This is in agreement with previous investigators (Barlow et al., 1988; Levy et al., 1991; Croucher et al., 1998; Bates and Ginsburg, 2002) who reported that experiencing a biochemical pregnancy loss after IVF is a positive indicator for success in subsequent fresh attempts. Our study results broaden the limits of this observation to encompass the related cryo-thawed cycle.
In conclusion, the prognosis of cryo-thawed cycles should not be viewed in isolation from the outcome of its related fresh cycle. Although it is difficult to predict accurately those who will achieve a pregnancy when using their cryopreserved embryos, patients who conceive in their fresh cycle are twice as likely to have an ongoing pregnancy compared with those who were unsuccessful in their fresh attempt. Even a biochemical pregnancy loss in the fresh cycle carries a similarly favourable prognosis.
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Submitted on December 20, 2002; accepted on February 11, 2003.
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  S. Polinder, E.M.E.W. Heijnen, N.S. Macklon, J.D.F. Habbema, B.J.C.M. Fauser, and M.J.C. Eijkemans Cost-effectiveness of a mild compared with a standard strategy for IVF: a randomized comparison using cumulative term live birth as the primary endpoint Hum. Reprod., February 1, 2008; 23(2): 316 - 323. [Abstract] [Full Text] [PDF]
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