Pituitary suppression in ultrasound-monitored frozen embryo replacement cycles. A randomised study

T. El-Toukhy1, A. Taylor1,3, Y. Khalaf1, K. Al-Darazi1, P. Rowell1, P. Seed1,2 and P. Braude1,2

1 Assisted Conception Unit, Guy’s and St. Thomas’ Hospital NHS Trust, and 2 Department of Women’s Health, Guy’s, King’s and St. Thomas’ School of Medicine, St. Thomas Street, London SE1 9RT, UK.

3 Corresponding author at: Assisted Conception Unit, 4th Floor Thomas Guy House, Guy’s Hospital, St. Thomas Street, London SE1 9RT, UK. Tel.: 0207 955 5000 ext 3977; Fax: 0207 955 4430; e-mail: tarekeltoukhy{at}hotmail.com


    Abstract
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
BACKGROUND: This study was designed to assess the value of using a gonadotrophin-releasing hormone (GnRH) agonist prior to exogenous steroid supplementation for endometrial preparation in frozen-thawed embryo replacement (FER) cycles. METHODS: A prospective randomized trial of 234 patients undergoing FER cycles was conducted. The study population was randomly divided into two groups according to a computer-generated list. In group A (n = 117), a daily dose of 6 mg of oral estradiol valerate was initiated on menstrual day 1 following pituitary suppression using 400 mcg buserelin acetate daily. In group B (n = 117), the same dose of estradiol valerate was initiated on day 1 of bleeding without prior GnRH agonist therapy. In both groups, ovulation monitoring was not undertaken and progesterone pessaries (800 mg daily) were administrated when the endometrial thickness had reached 8 mm or more with embryo transfer taking place 2 days later. RESULTS: The two groups were comparable with respect to cause of infertility, age at stimulation (32.8 ± 4 vs 33.2 ± 3.9 years, P = 0.4), basal FSH level (6.3 ± 1.7 vs 6.4 ± 2 IU/l, P = 0.5), number of oocytes collected (16.9 ± 7.3 vs 16.5 ± 7.4, P = 0.7) and fertilized normally in the retrieval cycle (11.5 ± 4.9 vs 11 ± 4.9, P = 0.4) and number of embryos cryopreserved (6.6 ± 3.6 vs 6.2 ± 3.6, P = 0.3). There was no significant difference between the two groups in age at frozen replacement (33.6 ± 4.2 vs 34 ± 3.9 years, P = 0.4), duration of the proliferative phase (20.7 ± 8.6 vs 21 ± 9.2 days, P = 0.7) and number of thawed embryos replaced (2.3 ± 0.6 vs 2.2 ± 0.6, P = 0.2). However, compared with group B, group A achieved significantly higher pregnancy (37.6% vs 24%, OR 1.8, 95%CI 1.1–3.4), clinical pregnancy (24% vs 11.3%, OR 2.5, 95%CI 1.2–5.5) and live birth rates (20% vs 8.5%, OR 2.9, 95%CI 1.2–8). CONCLUSION: Medicated frozen embryo replacement cycles timed by endometrial thickness measurement alone without monitoring or suppression of ovarian activity are associated with reduced outcome.

Key words: cryo-thawed cycle outcome/embryo cryopreservation/pituitary suppression


    Introduction
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The transfer of cryopreserved embryos in women with functioning ovaries can be timed with ovulation in natural cycle or after artificially preparing the endometrium with exogenous hormones (Cohen et al., 1998Go; de Ziegler et al., 1991Go; Muasher et al., 1991Go; Oehninger et al., 2000Go). Although embryo transfer in a natural cycle is often simple and obviates the need for prolonged hormonal supplementation in early pregnancy, monitoring of ovulation can sometimes be pragmatically difficult, particularly in women with irregular menstrual cycles (Sathanandan et al., 1991Go).

Protocols for controlled endometrial preparation involving the use of gonadotrophin-releasing hormone (GnRH) agonists prior to steroid administration in order to suppress ovarian function and induce synchronization of endometrial and embryo development have been developed based on the experience gained from oocyte donation programmes (Salat-Baroux et al., 1988Go; Meldrum et al., 1989Go; Schmidt et al., 1989Go; Muasher et al., 1991Go; Younis et al., 1996Go). This strategy has allowed the achievement of pregnancy rates comparable to those obtained from fresh transfer (Queenan et al., 1995Go) as well as after cryo-thawed embryo replacement in a natural cycle (Al-Shawaf et al., 1993Go; Queennan et al., 1994). Moreover, attempts to simplify cycle programming have suggested that omitting the down-regulation phase does not adversely affect cycle outcome, provided ultrasound and/or endocrine monitoring of ovarian activity is performed (Lelaidier et al., 1992Go; Queenan et al., 1997Go; Simon et al., 1998Go; Simon et al., 1999Go; Dal Prato et al., 2002Go).

The aim of the current study was to test the hypothesis that there was no difference in pregnancy rates between the two regimes when employing a more simplified protocol using ultrasound measurement of endometrial thickness as the sole monitoring tool of cryo-thawed cycle preparation and without monitoring of ovarian activity.


    Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The study protocol was approved by our institutional ethics committee. All patients had previously undergone IVF with or without ICSI with embryo cryopreservation, had regular menstrual cycles and gave written informed consent prior to inclusion in the study. Patients using cryo-thawed embryos created from donated oocytes were not included.

Fresh embryo grading and cryopreservation
Fresh cleavage-stage embryos generated using IVF or ICSI were assigned grades according to strict criteria (Steer et al., 1992Go; Gerris et al., 1999Go; Van Royen et al., 1999). Embryos were selected for cryopreservation if they had reached 4 cells on day 2 (40–42 h) or 6–8 cells on day 3 (64–66 h) of in vitro culture, had symmetrical component blastomeres devoid of multinucleation and showed no more than 10% cytoplasmic fragmentation. A standard freezing protocol, employing 1,2-propanediol (PROH) and sucrose as cryoprotectants, was used (Lassalle et al., 1985Go; Edgar et al., 2000Go). Freezing and thawing solutions consisted of the cryoprotectants in a HEPES-buffered salt solution supplemented with 0.5% w/v human serum albumin (HSA). Cooling was performed using a programmable freezer (CryoLogic, CL-863, Victoria, Australia).

Endometrial preparation
Patients who volunteered to participate were randomly divided into two treatment groups between January 1998 and July 2001 using a computer-generated randomization list. Patient enrollment and assignment to their treatment groups was carried out at the time of participation by medical staff not involved in the study.

Group A (n = 117) had pituitary suppression prior to steroid hormone administration, while group B (n = 117) commenced steroid supplementation without prior pituitary desensitization. (Fig. 1) Pituitary suppression in group A patients was performed using buserelin nasal spray (Superfact, Hoechst UK Ltd., Hounslow, Middlesex, UK) starting in the mid-luteal phase (day 21) of the menstrual cycle. On day 1 of subsequent menstruation, estrogen stimulation was initiated using oral estradiol valerate 6 mg daily in two divided doses (Climaval, Novartis Pharmaceuticals, Surrey, UK). Group B patients started estrogen stimulation on day 1 of menstruation using the same dose of Climaval (6mg/day). In both groups, patients remained on this dose for 12–14 days, after which endometrial thickness was evaluated using an ultrasound scanner with a 6.5 MHz probe (Hitachi EUB 525, Tokyo, Japan). The dose of Climaval was increased to 8mg/day for a further 7–12 days if endometrial thickness was less than 8mm. When endometrial thickness had reached 8mm or more, micronized progesterone pessaries (Cyclogest, Shire Pharmaceuticals Ltd., Hants, UK) 400 mg twice daily were commenced and buserelin nasal spray stopped in group A patients. Ultrasound and/or endocrine monitoring of ovulation during treatment was not performed.



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Figure 1. Flow of participants through the stages of the randomized control trial.

 
Thawing
Embryos were thawed rapidly by removal from liquid nitrogen and exposure to room temperature for 45 s followed by immersion in a water bath at 30°C for 30 s. A three step process of propanediol removal in the presence of 0.2 M sucrose ensued at room temperature for 5 min in each step until final rehydration in a HEPES-buffered salt solution. Thawed embryos were then assessed for blastomere survival using an inverted microscope (Nikon UK Limited, Kingston, Surrey, UK). Embryos that had lost more than 50% of their original blastomeres were not transferred. A second evaluation was performed prior to transfer in order to record the resumption of mitosis as indicated by cleavage of at least one blastomere.

Frozen-thawed embryo transfer and hormonal support
Between one and three embryos were transferred to the uterus on the third day of progesterone administration using an Edwards–Wallace embryo transfer catheter (Sims Portex Ltd., Hythe, Kent, UK). After embryo replacement, hormonal supplementation was continued for 14 days until a urine pregnancy test was performed using commercially available kits. Patients with a positive test continued with estrogen and progesterone supplementation until they were 12 weeks pregnant.

Cycle outcome
Pregnancy was diagnosed by a positive urine test for hCG ~14 days after embryo transfer. 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. All pregnancies were followed to delivery. Implantation rate was defined as the number of gestational sacs observed on ultrasound compared with the number of embryos transferred.

Power calculation and statistical analysis
It was calculated that 235 frozen-thawed cycles were needed to detect a difference of 20% (between 40 and 20%) in the pregnancy rate per cycle between the two groups with 90% power at the 5% significance level.

Analysis was performed on an intention-to-treat basis (Daya, 2003Go). Outcome measures and the associated clinical variables were analysed and compared using two sample t-test, chi-square or Fisher’s exact test where appropriate. The odds ratios and confidence intervals (CI) were calculated using the exact method. Statview software package for Macintosh (Statview 4.1, Abacus Concepts Ltd., Berkeley, CA, USA) was used for statistical analysis.


    Results
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In total, 234 FER cycles were performed. In these cycles, 1054 frozen embryos were thawed, 655 embryos (62%) survived the process of thawing with 50% or more of their original blastomeres intact and 505 (48%) were replaced (mean 2.2 ± 0.6 embryo/transfer). The overall implantation, pregnancy and live birth rates per cycle started were 13%, 31% and 14.1%, respectively. The live birth rate was similar in cycles in which IVF or ICSI was used for oocytes insemination (13.8% vs 14.4%, respectively).

Out of the 234 cycles started, 11 cycles (4.7%) did not reach embryo transfer; four (3.4%) in group A (three for failed embryo thaw and one for poor endometrial development) and seven (5.9%) in group B (five for failed thaw, one for poor endometrial development and one for breakthrough bleeding).

Groups A and B were comparable with respect to their demographic and fresh cycle characteristics including age, previous pregnancy, basal FSH level, number of oocytes collected and fertilized normally and number of embryos replaced and cryopreserved per cycle (Table 1). In addition, the proportion of fresh cycles in which clinical pregnancy resulted was similar in the two groups (25.6% vs 23.5%, respectively).


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Table I. Patients' demographics and fresh cycle characteristics. Values are given as mean ± SD or as shown.
 
The mean duration between the original fresh and subsequent cryo-thawed cycles was similar in groups A and B [9.6 ± 9.9 (range 2–53) months vs 9.6 ± 9.3 (range 2–50) months, respectively]. No significant difference was observed between the two groups in relation to mean age at cryo-thawed replacement, number of embryos surviving the thawing process per cycle and proportion of cycles in which all embryos transferred had survived thawing with their original blastomeres fully intact and in which all embryos replaced had both survived intact and resumed cleavage prior to transfer. Patients in the two groups achieved similar mean endometrial thickness after a comparable length of the proliferative phase and subsequently had a similar mean number of thawed embryos replaced (Table 2).


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Table II. FER cycle characteristics and outcome. Values are given as mean ± SD or as shown.
 
Frozen-thawed cycle outcome is summarized in Table 2. Group A showed significantly higher pregnancy (OR 1.8, 95% CI 1.1–3.4, P = 0.02), clinical pregnancy (OR 2.5, 95% CI 1.2–5.5, P = 0.01) and live birth (OR 2.9, 95% CI 1.2–8, P = 0.01) rates compared with group B. The significant differences in outcome measures between the two groups persisted even after excluding from the analysis cycles in which patients did not reach embryo transfer (39% vs 25.5%, P = 0.03; 24.8% vs 11.8%, P = 0.01, and 20.4% vs 9.1%, P = 0.02, respectively). The rate of pregnancy loss in the first trimester was also significantly lower in group A compared with group B (38% vs 64%, P = 0.03). The number needed to treat (NNT) to achieve an additional pregnancy, clinical pregnancy or a live birth in group A was 7.5 (95% CI 4–67), 7.9 (95% CI 4.5–33.7) and 8.8 (95% CI 5–35) patients, respectively.


    Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The introduction of GnRH agonists in fresh IVF cycles has been associated with various benefits (Testart et al., 1993Go). However, their role in cryo-thawed embryo replacement cycles is less defined. There have been no randomized trials comparing the outcome of cryo-thawed embryo transfer in natural versus programmed cycles in women with regular menstrual cycles. The present study investigated the value of using a GnRH agonist in a simplified frozen embryo replacement programme using a fixed dose of estrogen and endometrial thickness measurement as the sole monitoring tool without attempting to monitor ovarian activity. Results of the study have shown that the addition of a GnRH agonist under such circumstances is associated with significantly higher pregnancy and live birth rates and a relatively small (7–9) NNT (Cook and Sackett, 1995Go).

Implantation and normal development of pregnancy primarily depend on interaction between embryo quality and endometrial receptivity. In the present study, an attempt was made to control for pre-freeze embryo quality. Additionally, markers of post-thaw embryo developmental potential; namely survival rate, absence of blastomere loss and post-thaw resumption of mitosis, were comparably distributed between the two studied groups and therefore could not account for the differences in outcome measures. Likewise, patients’ demographics as well as retrieval and cryo-thawed cycle characteristics were not different in the two groups (Tables 1 and 2).

The study results appear to contrast with those of a previous randomized study (Dal Prato et al., 2002), which concluded that endometrial preparation for frozen-thawed embryo transfer using steroid supplementation only is as effective as that involving preliminary pituitary desensitization. However, it is difficult to compare the two studies since the formulation and dosage of medication used were different. In addition, all women in the present study received the same dose of estrogen from onset of menses, whereas the starting dose of estrogen in the earlier study was lower in the down-regulated group. Furthermore, in the study of Dal Prato et al. (2002), cycles in which patients showed ultrasound evidence suggestive of ovulation were stopped and subsequently excluded from the analysis, whereas no ovulation monitoring was carried out in the present study.

The development of a receptive endometrium is a complex but indispensable process for successful implantation (Ubaldi et al., 1997Go). It has long been known that the endometrium undergoes certain alterations that prepare it to receive the developing embryo during a critical self-limited time known as the ‘implantation window’ (Bergh and Navot, 1992Go). The specific changes leading to acquisition of the receptive state of the endometrium are regulated by timely interaction of maternal hormones, cytokines and various growth factors (Psychoyos, 1986Go; Guidice, 1994Go; Sharkey, 1998Go; Sunder and Lenton, 2000Go). Although starting estradiol treatment in the early follicular phase inhibits ovulation (Lelaidier et al., 1992Go), complete pituitary suppression and effective cycle control may not be achieved and some follicular activity can still occur.

In women with functioning ovaries, exogenous estrogen supplementation from the early follicular phase without preliminary pituitary suppression can be associated with a rise in LH level similar to the pre-ovulatory surge, even in the absence of follicular recruitment (deZiegler et al., 1991; Speroff et al., 1994Go). LH receptors have been localized in the endometrium (Reshef et al., 1989Go; Ziecik et al., 1992Go), indicating that the endometrium is a target organ for LH. Unlike previous suggestions that LH can only indirectly affect the endometrium through ovarian steroid hormone production (deZiegler et al., 1991), recent studies have linked exposure to LH with various regulatory changes pertinent to the morphological and functional proliferation and differentiation of endometrial glands and stroma, mainly via activation of the adenylate cyclase and phospholipase C pathways and increasing the local synthesis of steroid hormones (Han et al., 1999Go; Zhou et al., 1999Go; Licht et al., 2001Go; Shemesh, 2001Go; Ku et al., 2002Go). As these changes are normally synchronized to follicular development in the natural cycle (Buffet and Bouchard, 2001Go), it is possible that untimely rises of LH levels during the course of hormonal supplementation could adversely interfere with the emergence of the receptive window and potentially cause defective implantation. In line with this theory is the decreased implantation rate and the considerably higher rate (64%) of early pregnancy loss encountered in the group of patients who did not receive the GnRH agonist. This finding accords with a similar observation in the study of Lelaidier et al. (1992Go), which reported a 50% early pregnancy loss rate and raised concerns that steroid supplementation without pituitary suppression could lead to improper endometrial development and therefore predispose to first trimester pregnancy loss.

Furthermore, ovulation can occur during hormonal supplementation (Gebbie et al., 1995Go; Elomaa and Lahteenmaki, 1999Go), leading to embryo–endometrial asynchrony and decreased pregnancy rates after embryo replacement (Ubaldi et al., 1997Go). Remohi et al., (1993Go) used increasing doses of oral estradiol valerate from as early as day 3 of menstruation in women with functioning ovaries receiving donated oocytes and demonstrated histological evidence of ovulation in almost 40% of those studied, proving that ovarian function continued despite steroid treatment. The same study showed that the clinical pregnancy rate was lowest when endometrial dating prior to progesterone supplementation revealed post-ovulatory changes in a medicated ‘mock’ cycle before the actual replacement cycle. This observation concords with the reduced pregnancy rates we achieved in the non-down regulated group, and highlights that when ovulation monitoring is not undertaken as in the present study, pituitary suppression via the use of a GnRH agonist allows the establishment of uniform conditions for exogenous steroids to work unopposed. It is to be noted, however, that although estrogen supplementation started on day 1 of menstruation, the mean duration of the proliferative phase in the current study was nearly 3 weeks, and that may have potentially predisposed patients in the non-down regulated group to a higher rate of ovulation compared with other regimens utilizing a fixed (14 day) regimen of estrogen supplementation prior to initiation of progesterone (3.2% in the study of Lelaidier et al., 1992Go).

In conclusion, the present study demonstrates that in regularly menstruating women undergoing controlled endometrial preparation using steroid hormones, timing of cryo-thawed embryo transfer with endometrial thickness measurement alone is associated with reduced outcome, probably due to incomplete pituitary suppression and/or undetected ovulation. In those circumstances, the physician can elect to either monitor or suppress ovarian activity, but should not omit both.


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 Introduction
 Materials and Methods
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Submitted 17 December, 2003 ; accepted: 5 January, 2004



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