Assisted Conception Unit, Guys 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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Key words: cryo-thawed cycle prognosis/embryo cryopreservation/IVF outcome
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Introduction |
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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 frozenthawed 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 frozenthawed 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 frozenthawed transfer in patients who have similar embryo quality before cryopreservation.
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Materials and methods |
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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 150450 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 3436 h after hCG injection using an ultrasound scanner with a 6.5 MHz probe (Hitachi EUB 525, Japan). Cumulusoocyte 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 1618 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 (1618 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 (4042 h) or 68 cells on day 3 (6466 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 EdwardsWallace 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 propanediol0.1 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 EdwardsWallace 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 frozenthawed 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 Students t-test, 2-test or Fishers 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 IVFembryo 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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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 24) serum FSH level prior to ovarian stimulation and number of previous IVF/ICSI attempts (Table I).
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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 frozenthawed 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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Discussion |
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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 frozenthawed 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 frozenthawed 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 68 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.