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Effect of piroxicam administration before embryo transfer on IVF outcome: a randomized controlled trial
RBMOnline - Vol 19. No 4. 2009 604–609 Reproductive BioMedicine Online; www.rbmonline.com/Article/3800 on web 21 August 2009
Article Effect of piroxicam administration before embryo transfer on IVF outcome: a randomized controlled trial Luca Dal Prato was born in 1961. He studied medicine at the University of Bologna, Italy, and became MD in 1986. He completed his Obstetrics and Gynaecology residency at the University of Bologna under Professor Carlo Flamigni in 1990. From 1990 to 1996 he worked as a postgraduate fellow at the Reproductive Medicine Unit of the University of Bologna. Since 1996 he has been working as physician at Tecnobios Procreazione, Centre for Reproductive Health in Bologna. He has researched into the use of GnRH agonists and gonadotrophins for ovarian stimulation in assisted reproduction treatments.
Dr Luca Dal Prato Luca Dal Prato, Andrea Borini1 Tecnobios Procreazione, Centre for Reproductive Health, Via Dante 15, I-40125 Bologna, Italy 1 Correspondence: e-mail: [email protected]
Abstract Two hundred women aged between 28 and 43 years, with infertility from tubal, male, endometriosis or unexplained factor were randomly allocated into treatment (100 patients) and control (100 patients) groups. On the day after oocyte retrieval, each patient began supplementation with progesterone 8% vaginal gel, once daily. The patients in the treatment group received a single oral dose of 10 mg of the non-steroidal anti-inflammatory drug piroxicam 1–2 h before embryo transfer. No statistically significant difference was found between the two groups in any of the analysed endpoints. The rate of positive b-human chorionic gonadotrophin test per transfer was 37% in the women treated with piroxicam and 47% in controls. The clinical pregnancy rate per transfer and implantation rate were 34% and 19.2% with piroxicam, 38% and 21.9% in controls. The miscarriage rate was 11.8% and 13.2%. No beneficial effect of piroxicam on pregnancy rates was found evaluating either different infertility causes or different ages. This study shows that the administration of a single dose of piroxicam before embryo transfer has no additional effect on pregnancy outcome in patients receiving adequate doses of progesterone for luteal phase supplementation after IVF or ICSI. Keywords: embryo implantation, IVF embryo transfer, piroxicam, uterine contractility
Introduction
604
Embryo transfer, even when carried out with particular care, is an aggressive procedure that may induce endometrial inflammatory reaction and augmented myometrial contractility. Uterine contractions at the time of embryo transfer are detrimental for pregnancy rates after IVF (Fanchin et al., 1998). Since prostaglandin stimulates uterine contractions (Maslow and Lyons, 2004), the administration of a non-steroidal anti-inflammatory drug (NSAID) before transfer may improve uterine receptivity. The administration of a prostaglandin synthesis inhibitor at embryo transfer has been shown to improve pregnancy rates in cows (Elli et al., 2001; Scenna et al., 2005). This kind of adjuvant therapy seems to be promising in veterinary medicine. In humans, however, there are conflicting results.
A recent prospective, randomized, double-blind placebocontrolled study reported an increase in implantation and pregnancy rates after IVF with a single administration of 10 mg piroxicam orally 1–2 h before embryo transfer (Moon et al., 2004). The highest efficacy was found in young women with endometriosis. More recently, however, no benefit on implantation rates has been shown with the administration of indomethacin in oocyte recipients (Bernabeu et al., 2006). Piroxicam is an NSAID that exhibits anti-inflammatory, analgesic, and antipyretic activity. The mechanism of action, like that of other NSAIDs, is not completely understood but may be related to prostaglandin synthesis and release inhibition through a reversible inhibition of the cyclo-oxygenase enzyme.
Ó 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
Article - Piroxicam administration before embryo transfer - LD Prato & A Borini
Piroxicam is well absorbed following oral administration. Pharmacokinetic studies with piroxicam demonstrated a Cmax of 2.25 h. Drug plasma concentrations are proportional for 10 and 20 mg doses. After a single administration of 20 mg, concentrations reached 2.61 mg/ml. Concentrations generally peak within 3-h after medication, and the plasma half-life is approximately 50 h. In the presence of food, the piroxicam tmax was delayed to 4.3–4.6 h. The aim of the present randomized study was to evaluate the effect of piroxicam treatment on uterine receptivity and embryo implantation after IVF or ICSI procedures.
Materials and methods Protocol Between June 2005 and June 2007, a total of 200 women undergoing IVF or ICSI were enroled in the trial. The study was approved by the institutional review board. The main inclusion criteria were: age <44 years at the time of enrolment; regular ovulatory menstrual cycles of 25–33 days; infertility caused by tubal, idiopathic or male (exclusion of patients with cryptozoospermia requiring testicular sperm aspiration) factors or endometriosis; no more than two previous embryo transfers. Patients with FSH concentrations >15 IU/l on day 3 of the menstrual cycle, or who had previously shown poor response to gonadotrophins, were excluded from the study. Eligible patients who agreed to participate and gave their informed consent were randomized into two treatment groups. Pituitary desensitization was obtained with depot leuprorelin (Enantone 3.75; Takeda, Rome, Italy), half an ampoule (Dal Prato et al., 2004), in a long luteal protocol. At the onset of menses, patients began gonadotrophin stimulation as described elsewhere (Dal Prato et al., 2001). Briefly, they received 300 IU per day of r-hFSH (follitropin alfa, Gonal F; Serono, Rome, Italy) for 2 days and 150 IU per day for 4 days. In women aged 40 years or more, the FSH daily dose was increased by 75–150 IU according to basal FSH concentrations. The dose was then adjusted according to individual response as assessed by 17b-oestradiol assay and ultrasound scanning performed every other day. Recombinant human chorionic gonadotrophin (HCG) (Ovitrelle; Serono), 250 lg, was administered when at least three follicles reached a maximum diameter of 20 mm, of which at least one was >23 mm. Cycles in which fewer than three follicles developed were discontinued. Transvaginal oocyte retrieval was performed under ultrasound guidance 36 h after HCG administration. In compliance with Italian legislation (Law N. 40, March 2004), no more than three oocytes were inseminated per patient. On the day after oocyte retrieval, each patient began progesterone supplementation with Crinone 8% vaginal gel (Serono), 90 mg once daily. Supplementation continued until a pregnancy test was performed 15 days after embryo transfer or, in the event of pregnancy, until sonographic confirmation of embryonic viability (about 30–35 days after embryo transfer). RBMOnlineÒ
Embryo transfer procedure Two days after oocyte retrieval, all cleaved embryos were replaced into the uterine cavity via the transcervical route. Treatment group patients received a single oral dose of 10 mg of piroxicam (Brexin; Promedica Srl, Parma, Italy) 1–2 h before embryo transfer. All transfers were performed following the same protocol, in the lithotomy position with no ultrasound guidance, by one of the two authors and using the same catheter (Cook K-J-SMS-571500, USA; AB). The catheter consists of an outer sheet with a malleable obturator to be inserted just beyond the cervical internal os, and an inner soft delivery catheter containing the embryos. Transfers were classified as easy, intermediate or difficult according to the criteria reported by Tomas et al. (2002). After the transfer, patients had 10 min bed rest. Clinical pregnancy was defined as the presence of one or more gestational sacs detected on ultrasound scan performed at least 4 weeks after embryo transfer. Biochemical pregnancy loss was defined as a rise of b-HCG with no further evidence of the gestational sac on US scan. Miscarriage was defined as pregnancy loss after ultrasound confirmation of embryo implantation and before 12 weeks.
Parameters evaluated The primary endpoint, chosen according to previous studies (Moon et al., 2004), was the number of clinical pregnancies. The following secondary endpoints were also recorded: the number of patients with a positive b-HCG test, miscarriages, implantation rate. To evaluate the consistency between groups, evaluation was also made of: number of FSH ampoules, number of days of stimulation, 17b-oestradiol and number of follicles at HCG administration, total number of oocytes retrieved and mature oocytes, and number of transferred embryos.
Sample size Sample size was determined with Statistical Package for Social Sciences (SPSS) SamplePower (SPSS Inc., USA). The primary endpoint of the study was clinical pregnancy rate. A two-tailed test was used. According to previous studies (Moon et al., 2004), starting from an expected clinical pregnancy rate of 46.8% in the treated group and 27.6% in the control group, and assuming an alpha of 0.05 and a power of 0.8, a minimal sample size was estimated for each cohort of 100 patients.
Assignment Assignment to the different groups occurred on the day after oocyte retrieval, following assessment of oocyte fertilization. The randomization list was provided by an external
605
Article - Piroxicam administration before embryo transfer - LD Prato & A Borini
statistician, while the treatment sequence was delivered to the investigator in sealed envelopes. Dark envelopes were used so that their content could not be seen against bright light. Each envelope and allocation was sequentially numbered to prevent patients from being randomized out of sequence. Envelopes could not be opened in advance and were opened by a nurse not involved in the trial. No blinding was planned for this study because it was not possible to provide a placebo.
Statistical analysis Analysis was performed on an intention to treat (ITT) basis (all patients included in the study). Data are presented as mean and standard deviation (SD) or frequency. Normal distribution was evaluated using the Kolmogorov–Smirnov test. Means were compared by a two-sample t-test. Proportions for the two groups were compared using the chi-squared test and the Fisher’s exact test. A P-value <0.05 was considered statistically significant.
Results Participant flow and follow-up A total of 200 women were found eligible for protocol, had fertilized oocytes and were therefore randomized: 100 into group 1 (piroxicam 10 mg), 100 into group 2 (no treatment). All the 200 randomized patients underwent embryo transfer and completed the study. Figure 1 shows the participant flow and follow-up. IVF was performed in 61 patients in group 1 and in 58 patients in group 2, whereas ICSI was carried out in 39 patients in group 1 and in 42 patients in group 2. The two groups were homogeneous for age, duration of infertility, characteristics of ovarian stimulation and number of transferred embryos (2.5 ± 0.6 versus 2.6 ± 0.6) and of grade 1 (top quality) embryos (1.7 ± 0.8 versus 1.8 ± 0.8) (Table 1). There were no differences between groups in the quality of transfer. No transfer was considered as ‘difficult’; ‘intermediate’ transfers were equally distributed between groups and between the two operators. No statistically significant difference was found between the two groups in any of the analysed endpoints (Table 2). The rate of positive b-HCG per transfer was 37% in the women treated with piroxicam and 47% in controls. The clinical pregnancy rates per transfer and implantation rate were 34% and 19.2% with piroxicam, 38% and 21.9% in controls. The miscarriage rate was 11.8% and 13.2%.
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When clinical pregnancy rates were evaluated according to the causes of infertility, no significant differences were found between treatment and control groups (Table 3).
No beneficial effect of piroxicam on clinical pregnancy rates was found evaluating different ages (Table 4), particularly in patients under 40. In patients over 40 there was a trend toward higher clinical pregnancy rates in the treatment group compared with controls, but the difference was not statistically significant due to the small number of patients in this age group.
Discussion Embryo transfer is a key aspect of IVF procedures. Even when carried out very carefully, it is an aggressive procedure that may induce endometrial inflammatory reaction and augmented myometrial contractility, resulting in implantation failure even of very good quality embryos. The production of inflammatory cytokines is important for successful implantation: prostaglandins in the endometrium promote decidualization of the endometrium and implantation by increasing vascular permeability (Chakraborty et al., 1996). A low concentration of prostaglandins results in failure of ovulation, fertilization, decidualization and implantation in mice (Lim et al., 1997), but the exact role of prostaglandins in implantation in humans is not known. Prostaglandins in the myometrium increase during the late follicular phase, suddenly decrease after ovulation and reach a peak in the mid-luteal phase where the implantation window is situated (Vijayakumar and Walters, 1981). However, excessive production at the time of embryo transfer may be detrimental. Prostaglandin E stimulates midcycle uterine contraction, while ibuprofen, a prostaglandin inhibitor has shown a clear inhibitory action on mid-cycle uterine contractions (Maslow and Lyons, 2004). High frequency uterine contractions at the time of embryo transfer have been shown to affect embryo implantation and are associated with lower pregnancy rates after IVF (Fanchin et al., 1998). The administration of an NSAID before transfer may improve uterine receptivity. This kind of treatment showed good results in animal reproduction: a single dose of ibuprofen (Elli et al., 2001) or other prostaglandin synthesis inhibitors (Scenna et al., 2005) administered at the time of embryo transfer significantly improved pregnancy rates in cows. A prospective, randomized, double-blind placebo-controlled study investigated the efficacy of a single administration of 10 mg piroxicam orally 1–2 h before embryo transfer in women undergoing IVF (Moon et al., 2004) and reported an increase in implantation and pregnancy rates in both fresh and frozen-hawed cycles. The more favourable effect was seen in patients under 40 with tubal, male or endometriosis factors. Although of very similar design, this study showed quite different results and failed to provide evidence of any significant clinical effect of piroxicam on IVF outcome. Also, no beneficial effect of piroxicam on pregnancy rates was found RBMOnlineÒ
Article - Piroxicam administration before embryo transfer - LD Prato & A Borini
Figure 1. Participant flow through the trial. Table 1. Comparison of cycle characteristics in treatment (piroxicam) and control groups. Characteristic
Piroxicam
Control
No. of cycles (ITT) IVF/ICSI Age (years) Years of infertility Total FSH (IU) Oestradiol on HCG day (pg/ml) No. of follicles No. of oocytes No. of mature oocytes No. of inseminated oocytes No. of fertilized oocytes No. of transferred embryos No. of grade 1 embryos
100 61/39 35.7 ± 3.7 3.1 ± 2.3 2347 ± 896 1411 ± 925 15.4 ± 6.5 13.4 ± 6.1 8.4 ± 4.1 3.0 ± 0.2 2.6 ± 0.6 2.5 ± 0.6 1.7 ± 0.8
100 58/42 35.8 ± 3.4 2.7 ± 1.8 2299 ± 946 1291 ± 634 15.3 ± 5.9 12.7 ± 5.1 8.3 ± 3.7 3.0 ± 0.1 2.7 ± 0.7 2.6 ± 0.6 1.8 ± 0.8
Values are means ± SD, unless otherwise stated. There were no statistically significant differences between the two groups. HCG, human chorionic gonadotrophin; ICSI, intracytoplasmic sperm injection; ITT, intention to treat.
when evaluating different causes of infertility or age up to 40 years. A trend towards better outcome was found only RBMOnlineÒ
in patients aged over 40, where pregnancy rates in the treatment group were almost double that of controls. This
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Article - Piroxicam administration before embryo transfer - LD Prato & A Borini
Table 2. Comparisons of clinical outcomes in treatment (piroxicam) and control groups. Outcome
Piroxicam
Control
No. of ITT cycles (ET) Positive HCG (%/transfer) Biochemical pregnancy loss (%/HCG+) Clinical pregnancies (%/transfer) Implantation rate (%) Miscarriages (%/clinical pregnancy) Ectopic pregnancies (%/clinical pregnancy)
100 37 (37) 3 (8.1) 34 (34) 48/250 (19.2) 4 (11.8) 1 (2.9)
100 47 (47) 9 (19.1) 38 (38) 56/256 (21.9) 5 (13.2) 2 (5.3)
There were no statistically significant differences between the two groups (chi-squared test). ET, embryo transfer; HCG, human chorionic gonadotrophin; ITT, intention to treat.
Table 3. Clinical pregnancy rate according to cause of infertility. Aetiology
Piroxicam Control
Tubal
6/23 (26.1) 13/36 (36.1) 5/16 (31.3) 10/22 (45.5)
Male Endometriosis Unexplained
11/25 (44.0) 16/38 (42.1) 4/14 (28.6) 7/20 (35.0)
Values are n (%). There were no statistically significant differences between the two groups. Three patients in each group had a mixed cause of infertility (i.e. male plus endometriosis) and were excluded from the table.
Table 4. Clinical pregnancy rate according to patient age. Age (years)
Piroxicam Control n (%)
<35
17/42 (40.5) 11/44 (25.0) 28/86 (32.6) 6/14 (42.9)
35–39 Total <40 40
23/45 (51.1) 12/39 (30.8) 35/84 (41.7) 3/16 (18.8)
Values are n (%). There were no statistically significant differences between the two groups.
difference was not statistically significant, however, due to the small number of patients in this age group. The finding does not, however, provide sufficient evidence to conclude that piroxicam may be beneficial in older women.
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It has been demonstrated that progesterone is able to suppress uterine contractility in vivo and in vitro (Mueller et al., 2006b). Moreover, vaginal progesterone administra-
tion starting on the day of oocyte retrieval is able to relax uterine contractility at the time of embryo transfer (Fanchin et al., 2001). This rapid relaxation of the myometrium may prevent premature expulsion of embryos from the uterine cavity and improve pregnancy rates. Although the study by Fanchin was not powered to compare IVFembryo transfer outcome between the two groups, a trend for higher pregnancy and implantation rates was found in women beginning vaginal progesterone after retrieval as compared with controls beginning after transfer. No information on luteal phase supplementation was provided in the paper by Moon et al. (2004). A possible explanation for the different results of the present study may be vaginal P supplementation. Having recognized its effectiveness (Dal Prato et al., 2008), vaginal progesterone gel is routinely used for luteal phase supplementation from the day after oocyte retrieval. It may be supposed that, for the patients considered in the present study, starting vaginal progesterone supplementation from the day before transfer is enough to reduce uterine contractions at the time of embryo transfer. Any further effect of piroxicam on uterine environment is therefore negligible and unable to significantly influence the outcome of IVF or ICSI treatments. Otherwise, since it is known that different prostaglandins have different effects on uterine contractility (Mueller et al., 2006a), it might be that other drugs are more appropriate for regulation of uterine contractility. A recent paper (Bernabeu et al., 2006) investigated the effect of indomethacin treatment, started on the day before transfer, on implantation rate in oocyte recipients. As in the present study, this therapy did not lead to any improvement in outcome compared with standard endometrial preparations with transdermal oestradiol and vaginal progesterone only. It should be noted that recipients started vaginal progesterone about 2 days before transfer. The lack of blinding and the lack of a control arm treated with placebo may be a limitation of this study. However, since the protocol was strictly followed in the enrolment and assignment phases and both the laboratory team and physician performing the transfers were unaware of the treatment, it is unlikely that any study bias occurred. Moreover, in order to reduce any potential difference between RBMOnlineÒ
Article - Piroxicam administration before embryo transfer - LD Prato & A Borini
groups, transfers were performed by only two equally experienced physicians, following the same protocol and using the same catheter. The choice of drug type, and dose and time of administration for the trial was made according to the literature (Moon et al., 2004). It is possible that higher doses of the drug, the use of a different anti-inflammatory drug, or a different administration route (i.e. i.m.) may allow greater uptake and tissue distribution and so improve embryo implantation. This hypothesis requires further investigation. In conclusion, the trial shows that administration of a single dose of piroxicam before embryo transfer has no additional effect on pregnancy outcome in patients receiving adequate doses of progesterone for luteal phase supplementation after IVF or ICSI. Further studies are recommended to rule out any positive effect of different prostaglandin inhibitors or different doses on implantation and pregnancy rates.
References Bernabeu R, Roca M, Torres A et al. 2006 Indomethacin effect on implantation rates in oocyte recipients. Human Reproduction 21, 364–369. Chakraborty I, Das SK, Wang J et al. 1996 Developmental expression of the cyclo-oxygenase-1 and cyclo-oxygenase-2 genes in the peri-implantation mouse uterus and their differential regulation by the blastocyst and ovarian steroids. Journal of Molecular Endocrinology 16, 107–122. Dal Prato L, Bianchi L, Cattoli M et al. 2008 Vaginal gel versus intramuscular progesterone for luteal phase supplementation: a prospective randomized trial. Reproductive BioMedicine Online 16, 361–367. Dal Prato L, Borini A, Coticchio G et al. 2004 Half-dose depot triptorelin in pituitary suppression for multiple ovarian stimulation in assisted reproduction technology: a randomized study. Human Reproduction 19, 2200–2205. Dal Prato L, Borini A, Trevisi MR et al. 2001 Effect of reduced dose of triptorelin at the start of ovarian stimulation on the
outcome of IVF: a randomized study. Human Reproduction 16, 1409–1414. Elli M, Gaffuri B, Frigerio A et al. 2001 Effect of a single dose of ibuprofen lysinate before embryo transfer on pregnancy rates in cows. Reproduction 121, 151–154. Fanchin R, Righini C, de Ziegler D et al. 2001 Effects of vaginal progesterone administration on uterine contractility at the time of embryo transfer. Fertility and Sterility 75, 1136–1140. Fanchin R, Righini C, Olivennes F et al. 1998 Uterine contractions at the time of embryo transfer alter pregnancy rates after invitro fertilization. Human Reproduction 13, 1968–1974. Lim H, Paria BC, Das SK et al. 1997 Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 91, 197–208. Maslow KD, Lyons EA 2004 Effect of prostaglandin and antiprostaglandin on midcycle myometrial contractions. Fertility and Sterility 82, 511–513. Moon HS, Park SH, Lee JO et al. 2004 Treatment with piroxicam before embryo transfer increases the pregnancy rate after in vitro fertilization and embryo transfer. Fertility and Sterility 82, 816–820. Mueller A, Maltaris T, Siemer J et al. 2006 Uterine contractility in response to different prostaglandins: results from extracorporeally perfused non-pregnant swine uteri. Human Reproduction 21, 2000–2005. Mueller A, Siemer J, Schreiner S et al. 2006 Role of estrogen and progesterone in the regulation of uterine peristalsis: results from perfused non-pregnant swine uteri. Human Reproduction 21, 1863–1868. Scenna FN, Hockett ME, Towns TM et al. 2005 Influence of a prostaglandin synthesis inhibitor administered at embryo transfer on pregnancy rates of recipient cows. Prostaglandins and Other Lipid Mediators 78, 38–45. Tomas C, Tikkinen K, Tuomivaara L et al. 2002 The degree of difficulty of embryo transfer is an independent factor for predicting pregnancy. Human Reproduction 17, 2632–2635. Vijayakumar R, Walters WA 1981 Myometrial prostaglandins during the human menstrual cycle. American Journal of Obstetrics and Gynecology 141, 313–318.
Declaration: The authors report no financial or commercial conflicts of interest. Received 2 August 2008; refereed 29 January 2009; accepted 19 June 2009.
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Article Effect of piroxicam administration before embryo transfer on IVF outcome: a randomized controlled trial Luca Dal Prato was born in 1961. He studied medicine at the University of Bologna, Italy, and became MD in 1986. He completed his Obstetrics and Gynaecology residency at the University of Bologna under Professor Carlo Flamigni in 1990. From 1990 to 1996 he worked as a postgraduate fellow at the Reproductive Medicine Unit of the University of Bologna. Since 1996 he has been working as physician at Tecnobios Procreazione, Centre for Reproductive Health in Bologna. He has researched into the use of GnRH agonists and gonadotrophins for ovarian stimulation in assisted reproduction treatments.
Dr Luca Dal Prato Luca Dal Prato, Andrea Borini1 Tecnobios Procreazione, Centre for Reproductive Health, Via Dante 15, I-40125 Bologna, Italy 1 Correspondence: e-mail: [email protected]
Abstract Two hundred women aged between 28 and 43 years, with infertility from tubal, male, endometriosis or unexplained factor were randomly allocated into treatment (100 patients) and control (100 patients) groups. On the day after oocyte retrieval, each patient began supplementation with progesterone 8% vaginal gel, once daily. The patients in the treatment group received a single oral dose of 10 mg of the non-steroidal anti-inflammatory drug piroxicam 1–2 h before embryo transfer. No statistically significant difference was found between the two groups in any of the analysed endpoints. The rate of positive b-human chorionic gonadotrophin test per transfer was 37% in the women treated with piroxicam and 47% in controls. The clinical pregnancy rate per transfer and implantation rate were 34% and 19.2% with piroxicam, 38% and 21.9% in controls. The miscarriage rate was 11.8% and 13.2%. No beneficial effect of piroxicam on pregnancy rates was found evaluating either different infertility causes or different ages. This study shows that the administration of a single dose of piroxicam before embryo transfer has no additional effect on pregnancy outcome in patients receiving adequate doses of progesterone for luteal phase supplementation after IVF or ICSI. Keywords: embryo implantation, IVF embryo transfer, piroxicam, uterine contractility
Introduction
604
Embryo transfer, even when carried out with particular care, is an aggressive procedure that may induce endometrial inflammatory reaction and augmented myometrial contractility. Uterine contractions at the time of embryo transfer are detrimental for pregnancy rates after IVF (Fanchin et al., 1998). Since prostaglandin stimulates uterine contractions (Maslow and Lyons, 2004), the administration of a non-steroidal anti-inflammatory drug (NSAID) before transfer may improve uterine receptivity. The administration of a prostaglandin synthesis inhibitor at embryo transfer has been shown to improve pregnancy rates in cows (Elli et al., 2001; Scenna et al., 2005). This kind of adjuvant therapy seems to be promising in veterinary medicine. In humans, however, there are conflicting results.
A recent prospective, randomized, double-blind placebocontrolled study reported an increase in implantation and pregnancy rates after IVF with a single administration of 10 mg piroxicam orally 1–2 h before embryo transfer (Moon et al., 2004). The highest efficacy was found in young women with endometriosis. More recently, however, no benefit on implantation rates has been shown with the administration of indomethacin in oocyte recipients (Bernabeu et al., 2006). Piroxicam is an NSAID that exhibits anti-inflammatory, analgesic, and antipyretic activity. The mechanism of action, like that of other NSAIDs, is not completely understood but may be related to prostaglandin synthesis and release inhibition through a reversible inhibition of the cyclo-oxygenase enzyme.
Ó 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
Article - Piroxicam administration before embryo transfer - LD Prato & A Borini
Piroxicam is well absorbed following oral administration. Pharmacokinetic studies with piroxicam demonstrated a Cmax of 2.25 h. Drug plasma concentrations are proportional for 10 and 20 mg doses. After a single administration of 20 mg, concentrations reached 2.61 mg/ml. Concentrations generally peak within 3-h after medication, and the plasma half-life is approximately 50 h. In the presence of food, the piroxicam tmax was delayed to 4.3–4.6 h. The aim of the present randomized study was to evaluate the effect of piroxicam treatment on uterine receptivity and embryo implantation after IVF or ICSI procedures.
Materials and methods Protocol Between June 2005 and June 2007, a total of 200 women undergoing IVF or ICSI were enroled in the trial. The study was approved by the institutional review board. The main inclusion criteria were: age <44 years at the time of enrolment; regular ovulatory menstrual cycles of 25–33 days; infertility caused by tubal, idiopathic or male (exclusion of patients with cryptozoospermia requiring testicular sperm aspiration) factors or endometriosis; no more than two previous embryo transfers. Patients with FSH concentrations >15 IU/l on day 3 of the menstrual cycle, or who had previously shown poor response to gonadotrophins, were excluded from the study. Eligible patients who agreed to participate and gave their informed consent were randomized into two treatment groups. Pituitary desensitization was obtained with depot leuprorelin (Enantone 3.75; Takeda, Rome, Italy), half an ampoule (Dal Prato et al., 2004), in a long luteal protocol. At the onset of menses, patients began gonadotrophin stimulation as described elsewhere (Dal Prato et al., 2001). Briefly, they received 300 IU per day of r-hFSH (follitropin alfa, Gonal F; Serono, Rome, Italy) for 2 days and 150 IU per day for 4 days. In women aged 40 years or more, the FSH daily dose was increased by 75–150 IU according to basal FSH concentrations. The dose was then adjusted according to individual response as assessed by 17b-oestradiol assay and ultrasound scanning performed every other day. Recombinant human chorionic gonadotrophin (HCG) (Ovitrelle; Serono), 250 lg, was administered when at least three follicles reached a maximum diameter of 20 mm, of which at least one was >23 mm. Cycles in which fewer than three follicles developed were discontinued. Transvaginal oocyte retrieval was performed under ultrasound guidance 36 h after HCG administration. In compliance with Italian legislation (Law N. 40, March 2004), no more than three oocytes were inseminated per patient. On the day after oocyte retrieval, each patient began progesterone supplementation with Crinone 8% vaginal gel (Serono), 90 mg once daily. Supplementation continued until a pregnancy test was performed 15 days after embryo transfer or, in the event of pregnancy, until sonographic confirmation of embryonic viability (about 30–35 days after embryo transfer). RBMOnlineÒ
Embryo transfer procedure Two days after oocyte retrieval, all cleaved embryos were replaced into the uterine cavity via the transcervical route. Treatment group patients received a single oral dose of 10 mg of piroxicam (Brexin; Promedica Srl, Parma, Italy) 1–2 h before embryo transfer. All transfers were performed following the same protocol, in the lithotomy position with no ultrasound guidance, by one of the two authors and using the same catheter (Cook K-J-SMS-571500, USA; AB). The catheter consists of an outer sheet with a malleable obturator to be inserted just beyond the cervical internal os, and an inner soft delivery catheter containing the embryos. Transfers were classified as easy, intermediate or difficult according to the criteria reported by Tomas et al. (2002). After the transfer, patients had 10 min bed rest. Clinical pregnancy was defined as the presence of one or more gestational sacs detected on ultrasound scan performed at least 4 weeks after embryo transfer. Biochemical pregnancy loss was defined as a rise of b-HCG with no further evidence of the gestational sac on US scan. Miscarriage was defined as pregnancy loss after ultrasound confirmation of embryo implantation and before 12 weeks.
Parameters evaluated The primary endpoint, chosen according to previous studies (Moon et al., 2004), was the number of clinical pregnancies. The following secondary endpoints were also recorded: the number of patients with a positive b-HCG test, miscarriages, implantation rate. To evaluate the consistency between groups, evaluation was also made of: number of FSH ampoules, number of days of stimulation, 17b-oestradiol and number of follicles at HCG administration, total number of oocytes retrieved and mature oocytes, and number of transferred embryos.
Sample size Sample size was determined with Statistical Package for Social Sciences (SPSS) SamplePower (SPSS Inc., USA). The primary endpoint of the study was clinical pregnancy rate. A two-tailed test was used. According to previous studies (Moon et al., 2004), starting from an expected clinical pregnancy rate of 46.8% in the treated group and 27.6% in the control group, and assuming an alpha of 0.05 and a power of 0.8, a minimal sample size was estimated for each cohort of 100 patients.
Assignment Assignment to the different groups occurred on the day after oocyte retrieval, following assessment of oocyte fertilization. The randomization list was provided by an external
605
Article - Piroxicam administration before embryo transfer - LD Prato & A Borini
statistician, while the treatment sequence was delivered to the investigator in sealed envelopes. Dark envelopes were used so that their content could not be seen against bright light. Each envelope and allocation was sequentially numbered to prevent patients from being randomized out of sequence. Envelopes could not be opened in advance and were opened by a nurse not involved in the trial. No blinding was planned for this study because it was not possible to provide a placebo.
Statistical analysis Analysis was performed on an intention to treat (ITT) basis (all patients included in the study). Data are presented as mean and standard deviation (SD) or frequency. Normal distribution was evaluated using the Kolmogorov–Smirnov test. Means were compared by a two-sample t-test. Proportions for the two groups were compared using the chi-squared test and the Fisher’s exact test. A P-value <0.05 was considered statistically significant.
Results Participant flow and follow-up A total of 200 women were found eligible for protocol, had fertilized oocytes and were therefore randomized: 100 into group 1 (piroxicam 10 mg), 100 into group 2 (no treatment). All the 200 randomized patients underwent embryo transfer and completed the study. Figure 1 shows the participant flow and follow-up. IVF was performed in 61 patients in group 1 and in 58 patients in group 2, whereas ICSI was carried out in 39 patients in group 1 and in 42 patients in group 2. The two groups were homogeneous for age, duration of infertility, characteristics of ovarian stimulation and number of transferred embryos (2.5 ± 0.6 versus 2.6 ± 0.6) and of grade 1 (top quality) embryos (1.7 ± 0.8 versus 1.8 ± 0.8) (Table 1). There were no differences between groups in the quality of transfer. No transfer was considered as ‘difficult’; ‘intermediate’ transfers were equally distributed between groups and between the two operators. No statistically significant difference was found between the two groups in any of the analysed endpoints (Table 2). The rate of positive b-HCG per transfer was 37% in the women treated with piroxicam and 47% in controls. The clinical pregnancy rates per transfer and implantation rate were 34% and 19.2% with piroxicam, 38% and 21.9% in controls. The miscarriage rate was 11.8% and 13.2%.
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When clinical pregnancy rates were evaluated according to the causes of infertility, no significant differences were found between treatment and control groups (Table 3).
No beneficial effect of piroxicam on clinical pregnancy rates was found evaluating different ages (Table 4), particularly in patients under 40. In patients over 40 there was a trend toward higher clinical pregnancy rates in the treatment group compared with controls, but the difference was not statistically significant due to the small number of patients in this age group.
Discussion Embryo transfer is a key aspect of IVF procedures. Even when carried out very carefully, it is an aggressive procedure that may induce endometrial inflammatory reaction and augmented myometrial contractility, resulting in implantation failure even of very good quality embryos. The production of inflammatory cytokines is important for successful implantation: prostaglandins in the endometrium promote decidualization of the endometrium and implantation by increasing vascular permeability (Chakraborty et al., 1996). A low concentration of prostaglandins results in failure of ovulation, fertilization, decidualization and implantation in mice (Lim et al., 1997), but the exact role of prostaglandins in implantation in humans is not known. Prostaglandins in the myometrium increase during the late follicular phase, suddenly decrease after ovulation and reach a peak in the mid-luteal phase where the implantation window is situated (Vijayakumar and Walters, 1981). However, excessive production at the time of embryo transfer may be detrimental. Prostaglandin E stimulates midcycle uterine contraction, while ibuprofen, a prostaglandin inhibitor has shown a clear inhibitory action on mid-cycle uterine contractions (Maslow and Lyons, 2004). High frequency uterine contractions at the time of embryo transfer have been shown to affect embryo implantation and are associated with lower pregnancy rates after IVF (Fanchin et al., 1998). The administration of an NSAID before transfer may improve uterine receptivity. This kind of treatment showed good results in animal reproduction: a single dose of ibuprofen (Elli et al., 2001) or other prostaglandin synthesis inhibitors (Scenna et al., 2005) administered at the time of embryo transfer significantly improved pregnancy rates in cows. A prospective, randomized, double-blind placebo-controlled study investigated the efficacy of a single administration of 10 mg piroxicam orally 1–2 h before embryo transfer in women undergoing IVF (Moon et al., 2004) and reported an increase in implantation and pregnancy rates in both fresh and frozen-hawed cycles. The more favourable effect was seen in patients under 40 with tubal, male or endometriosis factors. Although of very similar design, this study showed quite different results and failed to provide evidence of any significant clinical effect of piroxicam on IVF outcome. Also, no beneficial effect of piroxicam on pregnancy rates was found RBMOnlineÒ
Article - Piroxicam administration before embryo transfer - LD Prato & A Borini
Figure 1. Participant flow through the trial. Table 1. Comparison of cycle characteristics in treatment (piroxicam) and control groups. Characteristic
Piroxicam
Control
No. of cycles (ITT) IVF/ICSI Age (years) Years of infertility Total FSH (IU) Oestradiol on HCG day (pg/ml) No. of follicles No. of oocytes No. of mature oocytes No. of inseminated oocytes No. of fertilized oocytes No. of transferred embryos No. of grade 1 embryos
100 61/39 35.7 ± 3.7 3.1 ± 2.3 2347 ± 896 1411 ± 925 15.4 ± 6.5 13.4 ± 6.1 8.4 ± 4.1 3.0 ± 0.2 2.6 ± 0.6 2.5 ± 0.6 1.7 ± 0.8
100 58/42 35.8 ± 3.4 2.7 ± 1.8 2299 ± 946 1291 ± 634 15.3 ± 5.9 12.7 ± 5.1 8.3 ± 3.7 3.0 ± 0.1 2.7 ± 0.7 2.6 ± 0.6 1.8 ± 0.8
Values are means ± SD, unless otherwise stated. There were no statistically significant differences between the two groups. HCG, human chorionic gonadotrophin; ICSI, intracytoplasmic sperm injection; ITT, intention to treat.
when evaluating different causes of infertility or age up to 40 years. A trend towards better outcome was found only RBMOnlineÒ
in patients aged over 40, where pregnancy rates in the treatment group were almost double that of controls. This
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Article - Piroxicam administration before embryo transfer - LD Prato & A Borini
Table 2. Comparisons of clinical outcomes in treatment (piroxicam) and control groups. Outcome
Piroxicam
Control
No. of ITT cycles (ET) Positive HCG (%/transfer) Biochemical pregnancy loss (%/HCG+) Clinical pregnancies (%/transfer) Implantation rate (%) Miscarriages (%/clinical pregnancy) Ectopic pregnancies (%/clinical pregnancy)
100 37 (37) 3 (8.1) 34 (34) 48/250 (19.2) 4 (11.8) 1 (2.9)
100 47 (47) 9 (19.1) 38 (38) 56/256 (21.9) 5 (13.2) 2 (5.3)
There were no statistically significant differences between the two groups (chi-squared test). ET, embryo transfer; HCG, human chorionic gonadotrophin; ITT, intention to treat.
Table 3. Clinical pregnancy rate according to cause of infertility. Aetiology
Piroxicam Control
Tubal
6/23 (26.1) 13/36 (36.1) 5/16 (31.3) 10/22 (45.5)
Male Endometriosis Unexplained
11/25 (44.0) 16/38 (42.1) 4/14 (28.6) 7/20 (35.0)
Values are n (%). There were no statistically significant differences between the two groups. Three patients in each group had a mixed cause of infertility (i.e. male plus endometriosis) and were excluded from the table.
Table 4. Clinical pregnancy rate according to patient age. Age (years)
Piroxicam Control n (%)
<35
17/42 (40.5) 11/44 (25.0) 28/86 (32.6) 6/14 (42.9)
35–39 Total <40 40
23/45 (51.1) 12/39 (30.8) 35/84 (41.7) 3/16 (18.8)
Values are n (%). There were no statistically significant differences between the two groups.
difference was not statistically significant, however, due to the small number of patients in this age group. The finding does not, however, provide sufficient evidence to conclude that piroxicam may be beneficial in older women.
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It has been demonstrated that progesterone is able to suppress uterine contractility in vivo and in vitro (Mueller et al., 2006b). Moreover, vaginal progesterone administra-
tion starting on the day of oocyte retrieval is able to relax uterine contractility at the time of embryo transfer (Fanchin et al., 2001). This rapid relaxation of the myometrium may prevent premature expulsion of embryos from the uterine cavity and improve pregnancy rates. Although the study by Fanchin was not powered to compare IVFembryo transfer outcome between the two groups, a trend for higher pregnancy and implantation rates was found in women beginning vaginal progesterone after retrieval as compared with controls beginning after transfer. No information on luteal phase supplementation was provided in the paper by Moon et al. (2004). A possible explanation for the different results of the present study may be vaginal P supplementation. Having recognized its effectiveness (Dal Prato et al., 2008), vaginal progesterone gel is routinely used for luteal phase supplementation from the day after oocyte retrieval. It may be supposed that, for the patients considered in the present study, starting vaginal progesterone supplementation from the day before transfer is enough to reduce uterine contractions at the time of embryo transfer. Any further effect of piroxicam on uterine environment is therefore negligible and unable to significantly influence the outcome of IVF or ICSI treatments. Otherwise, since it is known that different prostaglandins have different effects on uterine contractility (Mueller et al., 2006a), it might be that other drugs are more appropriate for regulation of uterine contractility. A recent paper (Bernabeu et al., 2006) investigated the effect of indomethacin treatment, started on the day before transfer, on implantation rate in oocyte recipients. As in the present study, this therapy did not lead to any improvement in outcome compared with standard endometrial preparations with transdermal oestradiol and vaginal progesterone only. It should be noted that recipients started vaginal progesterone about 2 days before transfer. The lack of blinding and the lack of a control arm treated with placebo may be a limitation of this study. However, since the protocol was strictly followed in the enrolment and assignment phases and both the laboratory team and physician performing the transfers were unaware of the treatment, it is unlikely that any study bias occurred. Moreover, in order to reduce any potential difference between RBMOnlineÒ
Article - Piroxicam administration before embryo transfer - LD Prato & A Borini
groups, transfers were performed by only two equally experienced physicians, following the same protocol and using the same catheter. The choice of drug type, and dose and time of administration for the trial was made according to the literature (Moon et al., 2004). It is possible that higher doses of the drug, the use of a different anti-inflammatory drug, or a different administration route (i.e. i.m.) may allow greater uptake and tissue distribution and so improve embryo implantation. This hypothesis requires further investigation. In conclusion, the trial shows that administration of a single dose of piroxicam before embryo transfer has no additional effect on pregnancy outcome in patients receiving adequate doses of progesterone for luteal phase supplementation after IVF or ICSI. Further studies are recommended to rule out any positive effect of different prostaglandin inhibitors or different doses on implantation and pregnancy rates.
References Bernabeu R, Roca M, Torres A et al. 2006 Indomethacin effect on implantation rates in oocyte recipients. Human Reproduction 21, 364–369. Chakraborty I, Das SK, Wang J et al. 1996 Developmental expression of the cyclo-oxygenase-1 and cyclo-oxygenase-2 genes in the peri-implantation mouse uterus and their differential regulation by the blastocyst and ovarian steroids. Journal of Molecular Endocrinology 16, 107–122. Dal Prato L, Bianchi L, Cattoli M et al. 2008 Vaginal gel versus intramuscular progesterone for luteal phase supplementation: a prospective randomized trial. Reproductive BioMedicine Online 16, 361–367. Dal Prato L, Borini A, Coticchio G et al. 2004 Half-dose depot triptorelin in pituitary suppression for multiple ovarian stimulation in assisted reproduction technology: a randomized study. Human Reproduction 19, 2200–2205. Dal Prato L, Borini A, Trevisi MR et al. 2001 Effect of reduced dose of triptorelin at the start of ovarian stimulation on the
outcome of IVF: a randomized study. Human Reproduction 16, 1409–1414. Elli M, Gaffuri B, Frigerio A et al. 2001 Effect of a single dose of ibuprofen lysinate before embryo transfer on pregnancy rates in cows. Reproduction 121, 151–154. Fanchin R, Righini C, de Ziegler D et al. 2001 Effects of vaginal progesterone administration on uterine contractility at the time of embryo transfer. Fertility and Sterility 75, 1136–1140. Fanchin R, Righini C, Olivennes F et al. 1998 Uterine contractions at the time of embryo transfer alter pregnancy rates after invitro fertilization. Human Reproduction 13, 1968–1974. Lim H, Paria BC, Das SK et al. 1997 Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 91, 197–208. Maslow KD, Lyons EA 2004 Effect of prostaglandin and antiprostaglandin on midcycle myometrial contractions. Fertility and Sterility 82, 511–513. Moon HS, Park SH, Lee JO et al. 2004 Treatment with piroxicam before embryo transfer increases the pregnancy rate after in vitro fertilization and embryo transfer. Fertility and Sterility 82, 816–820. Mueller A, Maltaris T, Siemer J et al. 2006 Uterine contractility in response to different prostaglandins: results from extracorporeally perfused non-pregnant swine uteri. Human Reproduction 21, 2000–2005. Mueller A, Siemer J, Schreiner S et al. 2006 Role of estrogen and progesterone in the regulation of uterine peristalsis: results from perfused non-pregnant swine uteri. Human Reproduction 21, 1863–1868. Scenna FN, Hockett ME, Towns TM et al. 2005 Influence of a prostaglandin synthesis inhibitor administered at embryo transfer on pregnancy rates of recipient cows. Prostaglandins and Other Lipid Mediators 78, 38–45. Tomas C, Tikkinen K, Tuomivaara L et al. 2002 The degree of difficulty of embryo transfer is an independent factor for predicting pregnancy. Human Reproduction 17, 2632–2635. Vijayakumar R, Walters WA 1981 Myometrial prostaglandins during the human menstrual cycle. American Journal of Obstetrics and Gynecology 141, 313–318.
Declaration: The authors report no financial or commercial conflicts of interest. Received 2 August 2008; refereed 29 January 2009; accepted 19 June 2009.
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