Low dose prednisolone administration in routine ICSI patients does not improve pregnancy and implantation rates

F. Ubaldi1,3, L. Rienzi1, S. Ferrero1, R. Anniballo1, M. Iacobelli1, L. Cobellis2 and E. Greco1

1 Centre for Reproductive Medicine, European Hospital, Rome, 2 Department of Obstetrics and Gynaecology II, University of Naples, Naples, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Glucocorticoids have been used in conjunction with zona dissection to improve pregnancy and implantation rates in IVF patients. The aim of this prospective randomized study was to evaluate the effect of low-dose prednisolone in addition to the standard protocol, on pregnancy and implantation rates in routine ICSI patients before and after embryo replacement. METHODS: A total of 313 patients in 360 consecutive cycles (patients <39 years old and with three or less than three ICSI attempts) performed at our centre were randomly assigned by computer-generated list to receive either prednisolone (10 mg/day in two divided doses), starting on the first day of ovarian stimulation and continuing for 4 weeks (group A), or no treatment (group B). RESULTS: The mean age, number of previously failed IVF attempts, basal FSH levels and the mean rank of trials were comparable between groups A and B. The mean (± SD) number of metaphase II oocytes retrieved (11.9 ± 5.5 versus 12.0 ± 5.1), 2-pronuclei fertilization rate (67.2 versus 65.8%), the pregnancy and the implantation rates were not different between the study and control groups (49.0 and 23.6% versus 50.0 and 23.3% respectively). CONCLUSION: Low-dose prednisolone treatment in addition to the standard protocol before and after embryo replacement does not appear to have a significant effect on pregnancy or implantation rates.

Key words: assisted reproduction/glucocorticoids/implantation rate/prednisolone/pregnancy rate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the last decade, conflicting results have been published concerning the use of glucocorticoids during and after embryo transfer, administered with the aim of improving implantation and ongoing pregnancy rates (Kemeter and Feichtinger, 1986Go; Cohen et al., 1990; Polak de Fried et al., 1993Go; Lee et al., 1994Go; Moffitt et al., 1995Go; Bider et al., 1996aGo, bGo; Rein et al., 1996Go).

Glucocorticoids have been used in IVF patients who had zona-dissected embryo replacement (Cohen et al., 1990). These authors hypothesized that breaching the protective coating of the zona pellucida could expose the embryos to bacteria or leukocyte infiltration. The immunosuppression caused by glucocorticoid administration probably decreases the presence of uterine lymphocytes and of peripheral immune cells especially segmented neutrophils that could invade and destroy the zona-dissected embryos. Similar results were reported when assisted hatching was introduced (Cohen et al., 1990bGo). These results suggested that immunosuppression could have also improved pregnancy rates in routine IVF–embryo transfer patients. Several reasons could support the use of glucocorticoids in non-micromanipulated embryos. It was suggested that the use of glucocorticoids in normo-androgenic normo-ovulating women during a stressful condition such as the IVF–embryo transfer treatment could reduce adrenal gland activity, preventing hyperandrogenaemia (Howels et al., 1986Go; Kemeter and Feichtinger, 1986Go) and improving clinical results (Kemeter and Feichtinger, 1986Go). However, these results were not confirmed (Rein et al., 1996Go).

A complex interaction between the embryo and the endometrium involves hormones, growth factors, epithelial cells, stromal cells, leucocytes and the intervening extracellular matrix (Armant and Diaz, 1990Go). Glucocorticoids may have important effects on these components, modulating the early events of the implantation process (Finlay and Cristofalo, 1987Go; Dean et al., 1988Go; Durant et al., 1989Go; Simo et al., 1992Go).

Several investigators studied the role of natural killer (NK) cells in human implantation (Beer et al., 1996Go; Lachapelle et al., 1996Go). Women with recurrent abortion and infertile women with many previous failed IVF attempts, have elevated levels of peripheral and endometrial NK cells (Beer et al., 1996Go; Lachapelle et al., 1996Go). A reduction of peripheral NK cells after 3 days of 20 mg prednisolone administration has been reported (Pountain et al., 1993Go). Hasegawa et al. reported significantly increased implantation rates in women with autoimmune conditions undergoing IVF after administration of 10 mg/day prednisolone for 5 weeks. These authors suggested that since this dosage is too low to reduce autoantibody titres, the increased implantation rates observed could be derived from the anti-inflammatory action of the glucocorticoids through the reduction of NK cells (Hasegawa et al., 1998Go).

Another possible benefit of the use of glucocorticoids could be due to their anti-inflammatory action exerted at the level of the endometrium. The uterine environment could be compromised by the embryo transfer technique. The stimulus of an intrauterine catheter may initiate some inflammatory response that could be correlated to the technique of the embryo transfer itself (Hill, 1990Go).

In the present prospective randomized study, we evaluated the implantation and clinical pregnancy rates in routine ICSI patients treated with prednisolone in addition to the standard protocol before and after embryo replacement.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient population
From September 1999 to October 2000, 360 consecutive ICSI cycles in women referred to the Centre for Reproductive Medicine at the European Hospital, Rome, Italy, were evaluated prospectively according to the following criteria: female age <39 years, normal serum hormonal profile on day 3 of the cycle [including estradiol (E2), FSH, LH, prolactin, thyroid stimulating hormone, testosterone and dehydroepiandrosterone sulphate], normal uterine cavity diagnosed at hysteroscopy, three or less than three ICSI attempts performed at our centre, and at least two embryos transferred. Patients were excluded if they had contraindications for glucocorticoid therapy. All patients selected were randomized on the day of oocyte retrieval by a computer-generated randomization list to receive either prednisolone (Deltacortene 5 mg, Lepetit, Anagni, Frosinone, Italy) 10 mg/day in two divided doses (group A) or no treatment (group B). In group A, prednisolone was started on day 1 of controlled ovarian hyperstimulation and continued for 4 weeks. All patients were counselled regarding the ICSI procedure and they gave their informed consent to enter the study. This study was given approval by the ethical committee of the European Hospital, Rome.

Ovarian stimulation and oocyte retrieval
In all cycles ovarian stimulation was carried out by the association of GnRH agonist (s.c. buserelin acetate, Suprefact®; Hoechst, Marion Roussel, Milan, Italy), recombinant (r)FSH (Puregon®; Organon, Oss, The Netherlands; Gonal-F®; Serono, Rome, Italy) and HCG (Profasi®; Serono). Administration of the agonist was started on day 21 of the menstrual cycle with a s.c. dose of 0.2 ml administered twice daily. When serum E2 concentrations were <=40 pg/ml and no ovarian cystic structures were observed on ultrasound examination, ovarian stimulation was usually started with 200–225 IU/day of rFSH for four days. Thereafter, the rFSH dose was adapted individually according to the serum E2 increment and ultrasound measurements of follicular diameter. When serum E2 exceeded 1000 pg/ml and when at least three follicles >=18 mm in diameter were recorded by ultrasound, ovulation was induced with 10000 IU HCG. Oocyte retrieval was performed 36 hours after HCG administration under transvaginal ultrasound-guided puncture of the follicles. Oocyte and ejaculated semen preparation as well as the ICSI procedure have been extensively described elsewhere (Rienzi et al., 1998Go). Fertilization was considered normal when two clearly distinct pronuclei (PN) were present. Further embryonic development was assessed 24 hours later. The embryos were classified according to the following morphological criteria. Type A or excellent embryos are defined as embryos in which all blastomeres have an equal or non-equal size without fragmentation. The type B or good embryos have blastomeres of equal or non-equal size, with <20% of the volume of the embryo with anucleate fragments. The type C or fair embryos have anucleate fragments in 20–50% of the volume of the embryo (Rienzi et al., 1998Go). According to the number of good morphological quality embryos and to the age of the patient, up to four embryos (in most instances only two) were replaced into the uterine cavity ~70 h after the microinjection procedures.

All patients received also aspirin (Aspirinetta 0.1 g, Bayer, Milan, Italy) at the dose of 100 mg/day starting from day 1 of stimulation and continuing for at least four weeks (Rubinstein et al., 1999Go).

Whenever indicated, the {chi}2-test and the Student t-test were applied at the 5% level of significance.

Luteal-phase assessment and establishment of pregnancy
The luteal phase was supported by means of natural progesterone in oil, 50 mg/day i.m. (Prontogest 100 mg; Amsa, Barberino del Mugello, Italy). Treatment was started on the day of oocyte retrieval. Pregnancy was confirmed by a serial rise in serum HCG concentration on two consecutive occasions, 12 days after embryo replacement. Clinical pregnancy was determined by ultrasound demonstration of cardiac activity at 7 weeks. An abortion was considered preclinical when HCG levels did not reach 1000 mIU/ml and no gestational sac was detected at the ultrasound examination.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of a total of 313 patients selected who underwent 360 cycles, 289 patients in 315 cycles received at least two embryos and completed the study. A total of 45 cycles were excluded from the study: 10 cycles were cancelled because of a poor ovarian response, in three cycles HCG was not given to trigger ovulation because of the high risk of severe ovarian hyperstimulation and in 32 cycles less than two embryos were replaced. The treated patients reported no side-effects. Table IGo presents patient characteristics. There were no statistically significant differences between groups. The mean age was 33.1 ± 3.7 and 32.7 ± 3.4 years for group A and B respectively. Day 3 FSH serum level (6.8 ± 2.9 versus 7.3 ± 3.1 mIU/ml) and the mean number of prior IVF attempts (1.4 ± 0.5 versus 1.8 ± 0.7) were comparable between groups A and B respectively. ICSI results were also similar between groups with a mean number of metaphase II oocytes retrieved of 11.9 ± 5.5 versus 12.0 ± 5.1, a 2PN fertilization rate of 67.2 versus 65.8% and a mean number of embryos transferred per cycle of 2.8 ± 0.7 versus 2.7 ± 0.6 (groups A and B respectively). The clinical outcome of prednisolone treatment is given in Table IIGo. No significant differences were found between the two groups in any of the clinical outcome parameters.


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Table I. Comparison of cycle characteristics, hormonal profile and ICSI outcome between patients treated with and without prednisolone
 

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Table II. Comparison of the clinical outcome between patients treated with and without prednisolone
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of glucocorticoids before and after embryo transfer has been proposed with the aim of improving pregnancy and implantation. However, so far, conflicting data have been published (Kemeter and Feichtinger 1986Go; Cohen et al., 1990aGo; Polak de Fried et al., 1993Go; Lee et al., 1994Go; Moffitt et al., 1995Go; Bider et al., 1996aGo, bGo; Rein et al., 1996Go).

In this prospective randomized study we used a low dose of prednisolone for four weeks in order to induce and maintain an anti-inflammatory control according to the data previously reported (Birkenfeld et al., 1994Go; Hasegawa et al., 1998Go). It has been shown that low doses of glucocorticoids for prolonged treatment periods, similar to those used in our study, produce the same degree of neutrophilia and lymphocytopoenia as the higher doses (Claman, 1983Go) and similar pregnancy and implantation rates have been reported between patients receiving different doses of glucocorticoids (0, 16 or 60 mg 16-ß-methylprednisone) undergoing IVF–embryo transfer (Lee et al., 1994Go).

Only patients with a normal ovarian response were included in the current study. Poor responders (less than three follicles) were excluded because the clinical results could be impaired in these patients (Levi et al., 2001Go). All patients also received aspirin at the dose of 100 mg per day starting from day 1 of stimulation and continued for at least 4 weeks. Higher numbers of follicles and oocytes retrieved and higher implantation and pregnancy rates in patients treated with aspirin have been reported (Rubinstein et al., 1999Go). Low-dose aspirin inhibits the cyclo-oxygenase enzyme which converts arachinodate into thromboxane A2 (TXA2) avoiding vasoconstriction and platelet aggregation (Burch and Stanford, 1979Go). Moreover, the inhibition of the cyclo-oxygenase enzyme blocks the synthesis of prostaglandins which may stimulate inflammatory cells (monocytes, lymphocytes, neutrophils and macrophages) and uterine contractions (via prostaglandin F2{alpha}) affecting implantation (Vane, 1971Go).

We observed comparable clinical pregnancy (45.5 versus 46.1%) and implantation rates (23.6 versus 23.3%) between study and control groups respectively. Our data are in accordance with those recently reported (Bider et al., 1996aGo). These authors demonstrated in a randomized prospective non-placebo study that the administration of low-dose long-acting glucocorticoids (0.5 mg dexamethasone) with a high anti-inflammatory potency in a selected group of patients with tubal factor infertility did not improve the implantation or pregnancy rates. Even doubling the dose of glucocorticoids (1 mg dexamethasone) did not result in a statistically significant increase of the implantation or pregnancy rates in the group of glucocorticoids-treated patients (Bider et al., 1996bGo). Similar results had been already reported by other authors (Lee et al., 1994Go; Moffitt et al., 1995Go).

However, a few years earlier, Polak de Fried et al. performed an uncontrolled study in which a group of 33 patients, treated with high dose of glucocorticoids (60 mg 16-ß-methylprednisone), demonstrated a statistically significant difference in pregnancy (11 versus 42%) and implantation rates (3 versus 18% of patients, not treated, and treated with glucocorticoids respectively) (Polak de Fried et al., 1993Go). There are several differences between this latter study and our study or the above-mentioned studies (Lee et al., 1994Go; Moffitt et al., 1995Go; Bider et al., 1996aGo, bGo), namely, in the study design, the dosage of drug administration and the pregnancy and implantation rates in the control groups.

The study by Polak de Fried et al. was an uncontrolled study whereas our study and the others were prospective and randomized. With regard to the dosage administered, in our study we used a much lower dose of glucocorticoids compared with that used in the study carried out by Polak de Fried et al. However, low doses of glucocorticoids produce the same degree of neutrophilia and lymphocytopoenia as the higher doses (Claman, 1993). Moreover, similar pregnancy and implantation rates have been reported between patients receiving different doses (0, 16 or 60 mg) of the same drug (16-ß-methylprednisone) used in Polak de Fried's study (Lee et al., 1994Go).

The pregnancy and implantation rates reported by Polak de Fried et al. in the group of patients not treated with glucocorticoids was relatively low (11 and 3% respectively) whereas those obtained in patients treated with 16-ß-methylprednisone were comparable with the pregnancy and implantation rates observed in our control group and in the control groups of the other mentioned studies (Lee et al., 1994Go; Moffitt et al., 1995Go; Bider et al., 1996aGo,bGo). Therefore, it seems that the use of glucocorticoids may be useful in raising a low pregnancy and implantation rate rather than increasing standard clinical results.

In summary, in the present prospective randomized study we did not observe any differences with regard to the implantation and clinical pregnancy rates in routine ICSI patients treated with low-dose prednisolone in addition to the standard protocol before and after embryo replacement. The use of glucocorticoids maybe useful in the case of sub-optimal embryonic or uterine conditions, but it seems that they are not able to increase embryo development or uterine receptivity in standard laboratory and clinical conditions.


    Notes
 
3 To whom correspondence should be addressed at: Centre for Reproductive Medicine, European Hospital, Via Portuense 700–00148, Rome, Italy. E-mail: ubaldi.fm{at}tin.it Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on February 6, 2001; resubmitted on January 2, 2002; accepted on January 23, 2002.