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Effects of the gonadotropin-releasing hormone antagonist cetrorelix in the early postimplantation period on rat pregnancy
European Journal of Obstetrics & Gynecology and Reproductive Biology 155 (2011) 166–170
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Effects of the gonadotropin-releasing hormone antagonist cetrorelix in the early postimplantation period on rat pregnancy Niyazi Tug a,b,1,*, Unal Uslu c,1, Alev Cumbul c,1, Signem Eyuboglu d,1, Cetin Cam a,b,1, Ates Karateke e,1, Bayram Yilmaz d,1 a
Yeditepe University, Faculty of Medicine, Istanbul, Turkey Zeynep Kamil Hospital, Obstetrics and Gynecology Department, Istanbul, Turkey Histology and Embriology, Yeditepe University, Faculty of Medicine, Istanbul, Turkey d Medical Physiology, Yeditepe University, Faculty of Medicine, Istanbul, Turkey e Obstetrics and Gynecology, Yeditepe University, Faculty of Medicine, Istanbul, Turkey b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 March 2010 Received in revised form 19 October 2010 Accepted 15 December 2010
Objective: The effect of cetrorelix given in the early implantation period on rat pregnancy was investigated. Study design: Forty-nine virgin Sprague–Dawley rats were randomized into six groups. At the 4th or 8th days of sperm plug, groups received 15, 75, 150 mg/kg cetrorelix or saline. Three subjects were randomly selected from each group and sacrificed at 11th gestational day for histomorphometric analysis. The remaining subjects were allowed to complete their pregnancy period. Volumes of total conceptus, labyrinth zone, transitional zone, giant cell zone, and exocoelomic cavity were calculated according to Cavalieri’s principle. Results: Subjects receiving cetrorelix at 15 or 150 mg doses at the 4th day (D4) and those receiving cetrorelix at 150 mg dose at the 8th day (D8) of pregnancy delivered later than the controls. On necropsy examination at the 11th day, mean embryo weights of the cetrorelix 15 D4, 150 D4, 15 D8 and 75 D8 groups were found to be significantly lower than that of the controls (p < 0.05). On histomorphometric evaluation, volumes of the fetuses and the amniotic sacs were decreased by cetrorelix at all doses studied dose dependently. Gross congenital anomalies were observed in the pups of three rats of the cetrorelix 150 D4 and D8 groups. Conclusion: The results of this study suggest that cetrorelix in the early post-implantation period may lead to serious side effects in the rat. ß 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cetrorelix GnRH antagonist GnRH Cavalieri Placenta Stereology
1. Introduction Gonadotropin-releasing hormone (GnRH) is a hypothalamic secretory peptide which primarily acts on the gonadotropes of the anterior pituitary to synthesize and release follicle-stimulating hormone (FSH) and luteinizing hormone (LH). GnRH is synthesized in the human body as two isoforms: GnRH I/II. The mode of action of GnRH molecules on the target cells depends on the type of G protein coupling [1–3]. The GnRH receptor is a member of the rhodopsin-like family of G-protein-coupled receptors, mediating the regulation of GnRH to the reproductive hormonal cascades. In the human, there are two
* Corresponding author at: Yeditepe University, Faculty of Medicine, Physiology Department, I˙no¨nu¨ Mah. Kayıs¸dag˘ı C. 26 Ag˘ustos Yerles¸imi, 34755 Kadıko¨y, Istanbul, Turkey. Tel.: +90 505 514 35 41. E-mail addresses: [email protected], [email protected] (N. Tug). 1 These authors equally contributed to this work. 0301-2115/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2010.12.015
types of GnRH receptors (GnRH I/II) although some vertebrate species are known to contain more than two types of functional receptors [3,4]. GnRH receptors have also been found to be expressed in extra-pituitary tissues [5]. Female genital tissues like ovary, endometrium and placenta express GnRH receptors. GnRH plays essential roles in pregnancy. Embryo implantation and invasion of trophoblastic tissue are dependent on GnRH activity. Steroid synthesis in the placenta is also modulated by GnRH throughout pregnancy [2,3,5–8]. GnRH antagonists are competitive inhibitors of GnRH receptors, and are used for suppression of a premature LH surge in IVF–ICSI cycles. They are also used for the treatment of hormone-sensitive neoplasms [5], some benign diseases like endometriosis and leiomyomatosis, and ovarian hyperstimulation syndrome (OHSS) in some clinics [9–11]. Cetrorelix is a potent GnRH I antagonist and a reliable therapeutic agent for IVF–ICSI cycles. Its affinity for the GnRH I receptors has been found to be much higher than the original GnRH I [5].
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There are limited data in the literature about the effects of GnRH antagonists on pregnancy. In IVF–ICSI cycles, the use of GnRH antagonists is generally confined to the last few days of ovulation induction until the day of hCG injection. These agents, however, can also be used in the luteal phase in the management of OHSS if the patient is found not to be pregnant or embryo transfer is delayed [12]. Signs and symptoms of early severe OHSS have been shown to be resolved by daily GnRH antagonist treatment (0.25 mg/day) starting at the 3rd to 6th days after oocyte retrieval while all embryos were cryo-preserved [13,14]. It was reported, however, in a recently published study that three IVF–ICSI patients with early onset OHSS were treated with cetrorelix (0.25 mg/day) for four days starting on the 6th day after oocyte retrieval. All three patients were found to be pregnant and two of them delivered successfully without any complication. The clinical picture of OHSS regressed rapidly in these patients and the authors concluded that cetrorelix is a safe and effective drug in the management of early onset OHSS at 0.25 mg/day doses [11]. In this preliminary study, the effects of cetrorelix (Cetrotide, Merck-Serono, Istanbul, Turkey) at pharmacological doses given in the early implantation period on rat pregnancy were investigated. 2. Materials and methods 2.1. Subjects Subjects were virgin Sprague–Dawley rats of 4–6 months of age, weighing 200–300 g, raised with a 12 h light–dark cycle in a temperature-controlled environment (21–22 8C), with food and water available ad libitum. Observation of a vaginal plug was considered as the first day of gestation. Forty-nine pregnant rats were randomized into six groups: 1: control, 2: Ctx 15 D4, 3: Ctx 150 D4, 4: Ctx 15 D8, 5: Ctx 75 D8, 6: Ctx 150 D8 (Fig. 1).
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2.2. Experiments Experimental procedures were approved by local ethics comittee. Experiments were performed on 49 pregnant rats in six groups. Cetrorelix was administered as a single injection intraperitoneally to five groups of 41 subjects: the 1st and 2nd groups received 15 or 150 mg/kg cetrorelix at the 4th day of gestation (n = 8), while others received the drug at the 8th gestational day in 15, 75 (n = 8) or 150 mg/kg (n = 9) doses. The remaining eight subjects were given intraperitoneal saline at the 8th day of gestation and served as controls. Three subjects were randomly selected from each group (n = 18) and sacrificed on the 11th gestational day for histomorphometric analysis. The remaining subjects (n = 31) were allowed to complete their pregnancy period for 25 days or until labor. All delivered pups were sacrificed and examined for gross congenital anomalies. 2.3. Histology At the 11th day of gestation, subjects were anesthesized by intramuscular 80 mg/kg ketamine and 7 mg/kg xylazine. Transcardiac perfusion was performed using 2.5% glutaraldehyde in 0.5 M cacodilate solution. After perfusion, uterine tissues were excised and blocked as one embryo per paraffin block. Blocks were sliced into 10 mm slices using a microtome (HM 325) serially. Randomly selected 1/10 of the microscopic slides were stained by hematoxylin and eosin. 2.4. Stereologic evaluation A stereologic station composed of: CCD Digital camera (Optronics Microfire 1600 1200P, Goleta, CA, USA), snapshot card (ATI Fire GL, Advanced Micro Devices, Camberley, UK),
Fig. 1. Light microscopical appearance of rat gestation sites on day 11. Haematoxylin eosin stained paraffin sections at 4 objective magnification (scale bar 260 mm) showing details in the PC, placental cone; AS, amniotic sac and cavity; F, fetus. Groups (a), Ctx 15 D8 (b), Ctx 75 D8 (c), Ctx 150 D8 (d). Placental cone and amniotic sac were observed in all groups. Fetus was demonstrated in groups a, b and c but, not d (ctx150 D8).
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Table 1 Study outline illustrating experimental groups, doses of cetrorelix (Ctx) and termination days of the study. D4: beginning of injections on day 4; D8: beginning of injections on day 8. Numbers in brackets indicate the number of mothers that gave birth. Groups
Control
Ctx 15 D4
Ctx 150 D4
Ctx 15 D8
Ctx 75 D8
Ctx 150 D8
Total number of subjects Number of rats sacrificed on day 11 Number of rats allowed to deliver
8 3 5 (5)
8 3 5 (4)
8 3 5 (5)
8 3 5 (4)
8 3 5 (4)
9 3 6 (4)
computerized preparation table (Ludl XYZ Bioprecision, Hawthorne, NY, USA), microcutter (Heidenhain, Traunreut, Germany), light microscope (4000 magnification, Leica, Wetzlar, Germany) Stereo Investigator 7,5 software (Microbrightfield, Williston, VT, USA) installed computer was used for the stereologic evaluations. According to Cavalieri’s principle [15], a measurement scale (d = 100 mm) was used for the measurements. Volumes of total conceptus, labyrinth zone, transitional zone, giant cell zone, and exocoelomic cavity were calculated. 2.5. Statistical analysis Results were given as mean standard error of mean (SEM) and analyzed by one-way ANOVA, post hoc LSD, and Tukey’s tests and Kruskal–Wallis tests as appropriate, using SPSS 14 for Windows software. P < 0.05 was accepted as the degree of statistical significance. 3. Results All mated subjects were found to be pregnant at postmortem examination of the three randomly selected from each group at the 11th day of gestation, as placenta and decidua were observed (n = 18). No fetus or fetal membranes, however, were found in one from the Ctx 15 D8 and Ctx 150 D4, and all three from the Ctx 150 D8 groups. Of the remaining 31 subjects which were allowed to complete their normal pregnancy periods, normal vaginal delivery was observed in all 5 subjects of the control group and the Ctx 150 D4 group (n = 5). One subject of the Ctx 15 D4, Ctx 15 D8 and Ctx 75 D8 groups (n = 5) and two subjects of the Ctx 150 D8 group (n = 6) showed no sign or symptom of delivery. No significant difference was found in the mean rates of delivery among the groups (Kruskal–Wallis test, X2 = 4.373, p = 0.497). Numbers of pups per delivered subject in the groups were as follows: in the control and Ctx 15 D4 and Ctx 150 D4 groups, pup numbers were 11.80 0.76, 12.67 0.49 and 11.60 1.95, respectively, whereas these numbers were 13.25 1.40, 11.50 0.96 and 11.33 2.01 in the Ctx 15, 75 and 150 D8 groups, respectively. No significant difference was found in the numbers of pups per delivered rat between the groups (one-way ANOVA, f = 0.184, p = 0.965). Length of gestation (in days) of the delivered subjects in the groups were 21.20 0.19 of the controls (n = 5), 23.25 0.25 of the Ctx 15 D4 group (n = 4), 24.40 0.45 of the Ctx 150 D4 group (n = 5), 21.50 0.29 of the Ctx 15 D8 group (n = 4), 21.50 0.29 of the Ctx 75 D8 group (n = 4) and 23.25 0.48 of the Ctx 150 D8 group (n = 4). Length of gestation was significantly different between groups (oneway ANOVA, f = 15,667, p < 0.001). Compared with controls, duration of pregnancy was significantly longer in the Ctx 15 D4, Ctx 150 D4, and Ctx150 D8 groups (post hoc Tukey’s test, p < 0.01). No significant difference was found between the control group and the Ctx 15 and 75 D8 groups (post hoc LSD, p > 0.533). Gross anomalies were observed in the pups of three subjects. In one of the rats of the Ctx 150 D4 group, all pups delivered at the 25th day of gestation had had multiple organ anomalies; while pups of another rat of the same group delivered at the 24th day of gestation had lung anomaly. Petechial bleedings were observed in each lobe of the lungs and each lobe was noticeably hypoplasic. On the other hand, gastrointestinal system anomaly was observed in
all pups born at 22nd day of gestation of a mother in the Ctx 150 D8 group. The stomachs of these pups were hypoplasic and petechial bleedings were observed in the gastro-esophageal junction. All pups of the control group were grossly normal. On postmortem examinations performed at the 11th day, the mean weights of the fetuses were 0.243 0.005, 0.146 0.007, 0.181 0.006, 0.187 0.007, 0.142 0.009 g in the control (n = 45), Ctx 15 mg D4 (n = 61), Ctx 150 mg D4 (n = 27), Ctx 15 mg D8 (n = 29), Ctx 75 mg D8 (n = 27) groups, respectively. No fetus was obtained in the necropsy materials of the three sacrificed animals of the Ctx 150 mg D8 group, so this group was excluded from the calculation. Mean fetal weights in all drug groups on necropsy examination at the 11th day of gestation were significantly lower than those of the controls (one-way ANOVA, f = 38.704, p < 0.001; post hoc Tukey’s HSD test, p < 0.001). On postmortem examination at the 11th day of gestation, the volumes of the parts of amnion and placentas calculated according to the Cavelieri’s principle were given at Table 1. Mean fetal weights per rat values of the groups on postmortem examination performed at the 11th day of gestation were 0.242 0.006, 0.148 0.013 and 0.183 0.020 g in the control, Ctx 15 D4 and Ctx 150 D4 groups. The mean weights of Ctx 15 D8 and Ctx 75 D8 groups were 0.187 0.005 and 0.143 0.018, respectively. Cetrorelix administration at 15 and 150 mg at the 4th day and 15 and 75 mg at the 8th day of gestation decreased the weights of the embryos on the 11th day of gestation (one-way ANOVA, f = 10.856, p = 0.004, post hoc Tukey’s HSD test, p1–4 = 0.001, 0.015, 0.019, 0.001, respectively). 4. Comments The physiology of rat pregnancy has some similarities with that of humans. Events in the first week following ovulation to implantation of the blastocyst correlate well in both human and rat pregnancies. In the rat, the placental disc is formed at the 8th day of gestation and unlike that in the human, the placenta (socalled hemotrichorial) contains three layers of trophoblast. Human placenta has a villous structure whereas rat placenta is labyrinthine, like other rodents [16]. In IVF–ICSI cycles, cetrorelix is used for the suppression of a premature LH surge at doses of 0.25 mg daily or 3 mg as a single injection for three days per patient [5], which approximately equals to 5 and 60 mg/kg for a patient weighing 50 kg. In the present study, cetrorelix was administered as a single injection of 15, 75 and 150 mg/kg doses at the 4th and 8th day of sperm plug, and therefore the results are not comparable to humans. GnRH plays important roles in pregnancy, not only in the growth and differentiation of trophoblasts, but also in endometrial receptivity [1,8]. In a study conducted on primates, GnRH antagonist administration decreased placental steroid production and authors suggested that interference with GnRH activity during early pregnancy can result in stillbirths, possibly due to induction of placental insufficiency [17]. In another study on primates, longacting GnRH agonist (Zoladex 3.6 and 7.2 mg) or antagonist (Ganirelix 100 mg) administration starting at the 14th day of conception adversely affected pregnancy outcome [18]. In the present study, placental growth was impaired and the period of pregnancy was prolonged by cetrorelix administration (Tables 2 and 3).
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Table 2 Length of gestation (days), number of vaginal deliveries and delivered pups per subject. Subjects treated with cetrorelix 15, 75 and 150 mg/kg intraperitoneally at 4th and 8th gestational days and the controls (values are given as mean Standard Error of Mean; n = 25, one-way ANOVA, p < 0.01).
Number of delivered subjects Number of pups Length of gestation (days) *
ctControl
CTX 15 mg D8
CTX 75 mg D8
CTX 150 mg D8
CTX 15 mg D4
CTX 150 mg D4
5 11.80 0.76 21.20 0.19
4 13.25 1.40 21.50 0.29
4 11.50 0.81 21.50 0.29
4 12.50 2.01 23.25 0.48*
4 12.67 0.49 23.250.25*
5 11.60 1.95 24.40 0.45*
Tukey’s HSD, p < 0.01.
Table 3 Volume of the total conceptus, amniotic sac, fetus, fetal exocoelome, giant cell zone, transformation zone and labyrinth zone of the subjects treated with cetrorelix 15, 75 and 150 mg/kg intraperitoneally at 8th gestational day and the controls calculated by Cavelieri principle on the postmortem examination at 11th day of gestation. (Values are given as mean Standard Error of Mean; n = 12, one-way ANOVA, p < 0.01.). Postmortem examinations performed at 11th day In mm3
Total conceptus
Labyrinth zone
Giant cell zone
Transformation zone
Exocoelome
Amniotic sac
Fetus
Control Ctx 15 D8 Ctx 75 D8 Ctx 150 D8
15.323 1.080 14.688 0.632 13.326 0.526 14.534 0.516
9.590 0.805 8.708 0.367 8.175 0.225 9.199 0.368
0.371 0.055 0.724 0.079* 0.556 0.114 0.409 0.083
4.2050.168 3.638 0.256 3.778 0.196 4.847 0.286
1.157 0.237 1.618 0.191 0.817 0.126 0.080 0.049*
0.160 0.045* 0.026 0.014* 0.025 0.017* 0.000 0.000*
0,172 0,.058* 0,024 0.014* 0,024 0,012* 0,000 0,000*
*
Tukey’s HSD, p < 0.05.
Because of the relatively short half lives of the GnRH antagonists used in IVF clinics (cetrorelix, ganirelix) for suppression of the LH surge, their plasma activity is expected to have ceased at the time of implantation. Reports in the literature about the teratogenic potential of the GnRH antagonists used for suppression of the LH surge during IVF–ICSI cycles were analysed by Elizur et al. and it was concluded that GnRH antagonists did not increase the rates of congenital malformations when they were not used inadvertently [19–22]. Mais et al. showed that GnRH antagonist administration at any phase of the natural menstrual cycle disrupts normal ovarian function. Both gonadotropins and estradiol levels fell below the level of healthy controls; a luteolytic effect was only seen when antagonist was given during the mid-luteal phase and was negated by the administration of hCG [23]. Moreover, in a study on 23 donor cycles Tavaniotou et al. reported that ganirelix not only drops luteal phase serum LH concentrations, but also prolongs the length of the luteal phase compared to normal cycling controls [24]. The luteolytic effect of cetrorelix might be a reason for the adverse effects observed in the present study. However, as the placenta in early gestation is known to express both GnRH and GnRH receptors and depends upon normal GnRH physiology, a direct action on the placenta might also be another possible mechanism. GnRH antagonists are also used in many clinics, if the probability of pregnancy is ruled out, in the management of OHSS that develops in IVF–ICSI cycles [12]. Likewise, cryopreservation of all embryos is recommended for the management of early severe OHSS by GnRH antagonists [13,14]. This was not the case, however, in a recently conducted study: GnRH antagonist was administered to three patients who developed severe OHSS during IVF cycles. Injections were started at the 6th day after oocyte retrieval for four days. On day 6, the patients underwent blastocyst transfer and
received ganirelix (0.25 mg/day) for 4 days in combination with luteal phase support using exogenous oestradiol and progesterone. Two patients had successful pregnancies that resulted in the births of healthy infants, while one patient had a biochemical pregnancy. The authors concluded that this treatment was effective in the regression of established severe OHSS, and resulted in the birth of healthy infants. According to the results of that report, it seems that no impairment of embryo implantation was observed following administration of daily ganirelix for four consecutive days in the early implantation period as 2/3 of patients delivered healthy infants and the remaining one had biochemical pregnancy [11]. In the present study, however, administration of cetrorelix impaired placental development, prolonged pregnancy period, decreased embryo weights and caused congenital anomalies at high doses in the rat (Tables 2–4). GnRH plays an important role in the development of the placenta but the exact distribution of the expression and the entire function of GnRH subtypes are not fully understood yet [1,4,7,25– 27]. Placenta carries GnRH I and II receptors both in animal and human. In a study conducted by Chu et al., GnRH I and GnRH II were found to be co-expressed in villous and extravillous cytotrophoblasts, but terminally differentiated, multinucleated syncytial trophoblast expressed only GnRH I in vivo and in vitro [1,4]. Moreover, the effects of GnRH molecules in tissues are determined not only by the type of receptor, but also the coupled G-proteins on that receptor, which may result in a wide range of intracellular cascades [1–3]. Because of the differences in both structure and functions of rat and human placentas, a prediction of the effect of cetrorelix on human pregnancy is not possible from the results of the present study. In conclusion, the results of this study suggest that GnRH I plays an important role in the normal development of the rat placenta
Table 4 Number and mean weights of fetuses of the subjects treated with cetrorelix 15, 75 and 150 mg/kg intraperitoneally at 8th gestational day and the controls on the postmortem examination at 11th day of gestation. (Values are given as mean Standard Error of Mean; n = 12, one-way ANOVA, f = 38.704, p < 0.001; post hoc Tukey’s HSD test, p < 0.001.). Postmortem examinations performed at 11th day
Number of fetuses Mean weight (g) SEM *
p < 0.001.
Control
CTX 15 mg D8
CTX 75 mg D8
CTX 150 mg D8
CTX 15 mg D4
CTX 150 mg D4
45 0.243 0.005*
29 0.187 0.007*
27 0.142 0.009*
0 – –
61 0.146 0.007*
27 0.181 0.006*
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and fetus. Administration of the GnRH antagonist cetrorelix in the early post-implantation period suppresses feto-placental development, decreases embryo weight, delays delivery and causes serious fetal malformations at pharmacological doses in the rat. Our findings indicate that the effect is dose-dependent. Conflicts of interest Nothing to disclose. References [1] Wu HM, Wang HS, Huang HY, Soong YK, MacCalman CD, Leung PC. GnRH signaling in intrauterine tissues. Reproduction 2009;137(May (5)):769–77. [2] Stewart AJ, Sellar R, Wilson DJ, Millar RP, Lu ZL. Identification of a novel ligand binding residue Arg38(1.35) in the human gonadotropin-releasing hormone receptor. Mol Pharmacol 2008;73:75–81. [3] Millar RP, Lu ZL, Pawson AJ, Flanagan CA, Morgan K, Maudsley SR. Gonadotropin-releasing hormone receptors. Endocrinol Rev 2004;25(April (2)):235–75. [4] Chou CS, Beristain AG, Maccalman CD, Leung PCK. Cellular localization of gonadotropin-releasing hormone (GnRH) I and GnRH II in first-trimester human placenta and decidua. J Clin Endocrinol Metab 2004;89:1459–66. [5] Reissmann T, Schally AV, Bouchard P, Reithmu¨ller H, Engel J. The LHRH antagonist cetrorelix: a review. Hum Reprod Update 2000;6(July–August (4)):322–31. [6] Millar RP, Pawson AJ, Morgan K, Rissman EF, Lu ZL. Diversity of actions of GnRHs mediated by ligand-induced selective signaling. Front Neuroendocrinol 2008;29(January (1)):17–35. [7] Rama S, Petrusz P, Rao AJ. Hormonal regulation of human trophoblast differentiation: a possible role for 17beta-estradiol and GnRH. Mol Cell Endocrinol 2004;218(April (1–2)):79–94. [8] Rackow BW, Kliman HJ, Taylor HS. GnRH antagonists may affect endometrial receptivity. Fertil Steril 2008;89(May (5)):1234–9. [9] Griesinger G, Schultze-Mosgau A, Dafopoulos K. Recombinant luteinizing hormone supplementation to recombinant follicle-stimulating hormone induced ovarian hyperstimulation in the GnRH-antagonist multiple-dose protocol. Hum Reprod 2005;20(May (5)):1200–6. Epub 2005 January 21. [10] Olartecoechea B, Garcı´a Manero M, Royo P, Auba` M. Treatment for endometriosis. Rev Med Univ Navarra 2009;53(July–September (3)):12–4. [11] Lainas TG, Sfontouris IA, Zorzovilis IZ. Live births after management of severe OHSS by GnRH antagonist administration in the luteal phase. Reprod Biomed Online 2009;19(December (6)):789–95. [12] Rollene NL, Amols MH, Hudson SB, Coddington CC. Treatment of ovarian hyperstimulation syndrome using a dopamine agonist and gonadotropin releasing hormone antagonist: a case series. Fertil Steril 2009;92(September (3)). 1169.e15–17.
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Effects of the gonadotropin-releasing hormone antagonist cetrorelix in the early postimplantation period on rat pregnancy Niyazi Tug a,b,1,*, Unal Uslu c,1, Alev Cumbul c,1, Signem Eyuboglu d,1, Cetin Cam a,b,1, Ates Karateke e,1, Bayram Yilmaz d,1 a
Yeditepe University, Faculty of Medicine, Istanbul, Turkey Zeynep Kamil Hospital, Obstetrics and Gynecology Department, Istanbul, Turkey Histology and Embriology, Yeditepe University, Faculty of Medicine, Istanbul, Turkey d Medical Physiology, Yeditepe University, Faculty of Medicine, Istanbul, Turkey e Obstetrics and Gynecology, Yeditepe University, Faculty of Medicine, Istanbul, Turkey b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 March 2010 Received in revised form 19 October 2010 Accepted 15 December 2010
Objective: The effect of cetrorelix given in the early implantation period on rat pregnancy was investigated. Study design: Forty-nine virgin Sprague–Dawley rats were randomized into six groups. At the 4th or 8th days of sperm plug, groups received 15, 75, 150 mg/kg cetrorelix or saline. Three subjects were randomly selected from each group and sacrificed at 11th gestational day for histomorphometric analysis. The remaining subjects were allowed to complete their pregnancy period. Volumes of total conceptus, labyrinth zone, transitional zone, giant cell zone, and exocoelomic cavity were calculated according to Cavalieri’s principle. Results: Subjects receiving cetrorelix at 15 or 150 mg doses at the 4th day (D4) and those receiving cetrorelix at 150 mg dose at the 8th day (D8) of pregnancy delivered later than the controls. On necropsy examination at the 11th day, mean embryo weights of the cetrorelix 15 D4, 150 D4, 15 D8 and 75 D8 groups were found to be significantly lower than that of the controls (p < 0.05). On histomorphometric evaluation, volumes of the fetuses and the amniotic sacs were decreased by cetrorelix at all doses studied dose dependently. Gross congenital anomalies were observed in the pups of three rats of the cetrorelix 150 D4 and D8 groups. Conclusion: The results of this study suggest that cetrorelix in the early post-implantation period may lead to serious side effects in the rat. ß 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cetrorelix GnRH antagonist GnRH Cavalieri Placenta Stereology
1. Introduction Gonadotropin-releasing hormone (GnRH) is a hypothalamic secretory peptide which primarily acts on the gonadotropes of the anterior pituitary to synthesize and release follicle-stimulating hormone (FSH) and luteinizing hormone (LH). GnRH is synthesized in the human body as two isoforms: GnRH I/II. The mode of action of GnRH molecules on the target cells depends on the type of G protein coupling [1–3]. The GnRH receptor is a member of the rhodopsin-like family of G-protein-coupled receptors, mediating the regulation of GnRH to the reproductive hormonal cascades. In the human, there are two
* Corresponding author at: Yeditepe University, Faculty of Medicine, Physiology Department, I˙no¨nu¨ Mah. Kayıs¸dag˘ı C. 26 Ag˘ustos Yerles¸imi, 34755 Kadıko¨y, Istanbul, Turkey. Tel.: +90 505 514 35 41. E-mail addresses: [email protected], [email protected] (N. Tug). 1 These authors equally contributed to this work. 0301-2115/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2010.12.015
types of GnRH receptors (GnRH I/II) although some vertebrate species are known to contain more than two types of functional receptors [3,4]. GnRH receptors have also been found to be expressed in extra-pituitary tissues [5]. Female genital tissues like ovary, endometrium and placenta express GnRH receptors. GnRH plays essential roles in pregnancy. Embryo implantation and invasion of trophoblastic tissue are dependent on GnRH activity. Steroid synthesis in the placenta is also modulated by GnRH throughout pregnancy [2,3,5–8]. GnRH antagonists are competitive inhibitors of GnRH receptors, and are used for suppression of a premature LH surge in IVF–ICSI cycles. They are also used for the treatment of hormone-sensitive neoplasms [5], some benign diseases like endometriosis and leiomyomatosis, and ovarian hyperstimulation syndrome (OHSS) in some clinics [9–11]. Cetrorelix is a potent GnRH I antagonist and a reliable therapeutic agent for IVF–ICSI cycles. Its affinity for the GnRH I receptors has been found to be much higher than the original GnRH I [5].
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There are limited data in the literature about the effects of GnRH antagonists on pregnancy. In IVF–ICSI cycles, the use of GnRH antagonists is generally confined to the last few days of ovulation induction until the day of hCG injection. These agents, however, can also be used in the luteal phase in the management of OHSS if the patient is found not to be pregnant or embryo transfer is delayed [12]. Signs and symptoms of early severe OHSS have been shown to be resolved by daily GnRH antagonist treatment (0.25 mg/day) starting at the 3rd to 6th days after oocyte retrieval while all embryos were cryo-preserved [13,14]. It was reported, however, in a recently published study that three IVF–ICSI patients with early onset OHSS were treated with cetrorelix (0.25 mg/day) for four days starting on the 6th day after oocyte retrieval. All three patients were found to be pregnant and two of them delivered successfully without any complication. The clinical picture of OHSS regressed rapidly in these patients and the authors concluded that cetrorelix is a safe and effective drug in the management of early onset OHSS at 0.25 mg/day doses [11]. In this preliminary study, the effects of cetrorelix (Cetrotide, Merck-Serono, Istanbul, Turkey) at pharmacological doses given in the early implantation period on rat pregnancy were investigated. 2. Materials and methods 2.1. Subjects Subjects were virgin Sprague–Dawley rats of 4–6 months of age, weighing 200–300 g, raised with a 12 h light–dark cycle in a temperature-controlled environment (21–22 8C), with food and water available ad libitum. Observation of a vaginal plug was considered as the first day of gestation. Forty-nine pregnant rats were randomized into six groups: 1: control, 2: Ctx 15 D4, 3: Ctx 150 D4, 4: Ctx 15 D8, 5: Ctx 75 D8, 6: Ctx 150 D8 (Fig. 1).
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2.2. Experiments Experimental procedures were approved by local ethics comittee. Experiments were performed on 49 pregnant rats in six groups. Cetrorelix was administered as a single injection intraperitoneally to five groups of 41 subjects: the 1st and 2nd groups received 15 or 150 mg/kg cetrorelix at the 4th day of gestation (n = 8), while others received the drug at the 8th gestational day in 15, 75 (n = 8) or 150 mg/kg (n = 9) doses. The remaining eight subjects were given intraperitoneal saline at the 8th day of gestation and served as controls. Three subjects were randomly selected from each group (n = 18) and sacrificed on the 11th gestational day for histomorphometric analysis. The remaining subjects (n = 31) were allowed to complete their pregnancy period for 25 days or until labor. All delivered pups were sacrificed and examined for gross congenital anomalies. 2.3. Histology At the 11th day of gestation, subjects were anesthesized by intramuscular 80 mg/kg ketamine and 7 mg/kg xylazine. Transcardiac perfusion was performed using 2.5% glutaraldehyde in 0.5 M cacodilate solution. After perfusion, uterine tissues were excised and blocked as one embryo per paraffin block. Blocks were sliced into 10 mm slices using a microtome (HM 325) serially. Randomly selected 1/10 of the microscopic slides were stained by hematoxylin and eosin. 2.4. Stereologic evaluation A stereologic station composed of: CCD Digital camera (Optronics Microfire 1600 1200P, Goleta, CA, USA), snapshot card (ATI Fire GL, Advanced Micro Devices, Camberley, UK),
Fig. 1. Light microscopical appearance of rat gestation sites on day 11. Haematoxylin eosin stained paraffin sections at 4 objective magnification (scale bar 260 mm) showing details in the PC, placental cone; AS, amniotic sac and cavity; F, fetus. Groups (a), Ctx 15 D8 (b), Ctx 75 D8 (c), Ctx 150 D8 (d). Placental cone and amniotic sac were observed in all groups. Fetus was demonstrated in groups a, b and c but, not d (ctx150 D8).
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Table 1 Study outline illustrating experimental groups, doses of cetrorelix (Ctx) and termination days of the study. D4: beginning of injections on day 4; D8: beginning of injections on day 8. Numbers in brackets indicate the number of mothers that gave birth. Groups
Control
Ctx 15 D4
Ctx 150 D4
Ctx 15 D8
Ctx 75 D8
Ctx 150 D8
Total number of subjects Number of rats sacrificed on day 11 Number of rats allowed to deliver
8 3 5 (5)
8 3 5 (4)
8 3 5 (5)
8 3 5 (4)
8 3 5 (4)
9 3 6 (4)
computerized preparation table (Ludl XYZ Bioprecision, Hawthorne, NY, USA), microcutter (Heidenhain, Traunreut, Germany), light microscope (4000 magnification, Leica, Wetzlar, Germany) Stereo Investigator 7,5 software (Microbrightfield, Williston, VT, USA) installed computer was used for the stereologic evaluations. According to Cavalieri’s principle [15], a measurement scale (d = 100 mm) was used for the measurements. Volumes of total conceptus, labyrinth zone, transitional zone, giant cell zone, and exocoelomic cavity were calculated. 2.5. Statistical analysis Results were given as mean standard error of mean (SEM) and analyzed by one-way ANOVA, post hoc LSD, and Tukey’s tests and Kruskal–Wallis tests as appropriate, using SPSS 14 for Windows software. P < 0.05 was accepted as the degree of statistical significance. 3. Results All mated subjects were found to be pregnant at postmortem examination of the three randomly selected from each group at the 11th day of gestation, as placenta and decidua were observed (n = 18). No fetus or fetal membranes, however, were found in one from the Ctx 15 D8 and Ctx 150 D4, and all three from the Ctx 150 D8 groups. Of the remaining 31 subjects which were allowed to complete their normal pregnancy periods, normal vaginal delivery was observed in all 5 subjects of the control group and the Ctx 150 D4 group (n = 5). One subject of the Ctx 15 D4, Ctx 15 D8 and Ctx 75 D8 groups (n = 5) and two subjects of the Ctx 150 D8 group (n = 6) showed no sign or symptom of delivery. No significant difference was found in the mean rates of delivery among the groups (Kruskal–Wallis test, X2 = 4.373, p = 0.497). Numbers of pups per delivered subject in the groups were as follows: in the control and Ctx 15 D4 and Ctx 150 D4 groups, pup numbers were 11.80 0.76, 12.67 0.49 and 11.60 1.95, respectively, whereas these numbers were 13.25 1.40, 11.50 0.96 and 11.33 2.01 in the Ctx 15, 75 and 150 D8 groups, respectively. No significant difference was found in the numbers of pups per delivered rat between the groups (one-way ANOVA, f = 0.184, p = 0.965). Length of gestation (in days) of the delivered subjects in the groups were 21.20 0.19 of the controls (n = 5), 23.25 0.25 of the Ctx 15 D4 group (n = 4), 24.40 0.45 of the Ctx 150 D4 group (n = 5), 21.50 0.29 of the Ctx 15 D8 group (n = 4), 21.50 0.29 of the Ctx 75 D8 group (n = 4) and 23.25 0.48 of the Ctx 150 D8 group (n = 4). Length of gestation was significantly different between groups (oneway ANOVA, f = 15,667, p < 0.001). Compared with controls, duration of pregnancy was significantly longer in the Ctx 15 D4, Ctx 150 D4, and Ctx150 D8 groups (post hoc Tukey’s test, p < 0.01). No significant difference was found between the control group and the Ctx 15 and 75 D8 groups (post hoc LSD, p > 0.533). Gross anomalies were observed in the pups of three subjects. In one of the rats of the Ctx 150 D4 group, all pups delivered at the 25th day of gestation had had multiple organ anomalies; while pups of another rat of the same group delivered at the 24th day of gestation had lung anomaly. Petechial bleedings were observed in each lobe of the lungs and each lobe was noticeably hypoplasic. On the other hand, gastrointestinal system anomaly was observed in
all pups born at 22nd day of gestation of a mother in the Ctx 150 D8 group. The stomachs of these pups were hypoplasic and petechial bleedings were observed in the gastro-esophageal junction. All pups of the control group were grossly normal. On postmortem examinations performed at the 11th day, the mean weights of the fetuses were 0.243 0.005, 0.146 0.007, 0.181 0.006, 0.187 0.007, 0.142 0.009 g in the control (n = 45), Ctx 15 mg D4 (n = 61), Ctx 150 mg D4 (n = 27), Ctx 15 mg D8 (n = 29), Ctx 75 mg D8 (n = 27) groups, respectively. No fetus was obtained in the necropsy materials of the three sacrificed animals of the Ctx 150 mg D8 group, so this group was excluded from the calculation. Mean fetal weights in all drug groups on necropsy examination at the 11th day of gestation were significantly lower than those of the controls (one-way ANOVA, f = 38.704, p < 0.001; post hoc Tukey’s HSD test, p < 0.001). On postmortem examination at the 11th day of gestation, the volumes of the parts of amnion and placentas calculated according to the Cavelieri’s principle were given at Table 1. Mean fetal weights per rat values of the groups on postmortem examination performed at the 11th day of gestation were 0.242 0.006, 0.148 0.013 and 0.183 0.020 g in the control, Ctx 15 D4 and Ctx 150 D4 groups. The mean weights of Ctx 15 D8 and Ctx 75 D8 groups were 0.187 0.005 and 0.143 0.018, respectively. Cetrorelix administration at 15 and 150 mg at the 4th day and 15 and 75 mg at the 8th day of gestation decreased the weights of the embryos on the 11th day of gestation (one-way ANOVA, f = 10.856, p = 0.004, post hoc Tukey’s HSD test, p1–4 = 0.001, 0.015, 0.019, 0.001, respectively). 4. Comments The physiology of rat pregnancy has some similarities with that of humans. Events in the first week following ovulation to implantation of the blastocyst correlate well in both human and rat pregnancies. In the rat, the placental disc is formed at the 8th day of gestation and unlike that in the human, the placenta (socalled hemotrichorial) contains three layers of trophoblast. Human placenta has a villous structure whereas rat placenta is labyrinthine, like other rodents [16]. In IVF–ICSI cycles, cetrorelix is used for the suppression of a premature LH surge at doses of 0.25 mg daily or 3 mg as a single injection for three days per patient [5], which approximately equals to 5 and 60 mg/kg for a patient weighing 50 kg. In the present study, cetrorelix was administered as a single injection of 15, 75 and 150 mg/kg doses at the 4th and 8th day of sperm plug, and therefore the results are not comparable to humans. GnRH plays important roles in pregnancy, not only in the growth and differentiation of trophoblasts, but also in endometrial receptivity [1,8]. In a study conducted on primates, GnRH antagonist administration decreased placental steroid production and authors suggested that interference with GnRH activity during early pregnancy can result in stillbirths, possibly due to induction of placental insufficiency [17]. In another study on primates, longacting GnRH agonist (Zoladex 3.6 and 7.2 mg) or antagonist (Ganirelix 100 mg) administration starting at the 14th day of conception adversely affected pregnancy outcome [18]. In the present study, placental growth was impaired and the period of pregnancy was prolonged by cetrorelix administration (Tables 2 and 3).
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Table 2 Length of gestation (days), number of vaginal deliveries and delivered pups per subject. Subjects treated with cetrorelix 15, 75 and 150 mg/kg intraperitoneally at 4th and 8th gestational days and the controls (values are given as mean Standard Error of Mean; n = 25, one-way ANOVA, p < 0.01).
Number of delivered subjects Number of pups Length of gestation (days) *
ctControl
CTX 15 mg D8
CTX 75 mg D8
CTX 150 mg D8
CTX 15 mg D4
CTX 150 mg D4
5 11.80 0.76 21.20 0.19
4 13.25 1.40 21.50 0.29
4 11.50 0.81 21.50 0.29
4 12.50 2.01 23.25 0.48*
4 12.67 0.49 23.250.25*
5 11.60 1.95 24.40 0.45*
Tukey’s HSD, p < 0.01.
Table 3 Volume of the total conceptus, amniotic sac, fetus, fetal exocoelome, giant cell zone, transformation zone and labyrinth zone of the subjects treated with cetrorelix 15, 75 and 150 mg/kg intraperitoneally at 8th gestational day and the controls calculated by Cavelieri principle on the postmortem examination at 11th day of gestation. (Values are given as mean Standard Error of Mean; n = 12, one-way ANOVA, p < 0.01.). Postmortem examinations performed at 11th day In mm3
Total conceptus
Labyrinth zone
Giant cell zone
Transformation zone
Exocoelome
Amniotic sac
Fetus
Control Ctx 15 D8 Ctx 75 D8 Ctx 150 D8
15.323 1.080 14.688 0.632 13.326 0.526 14.534 0.516
9.590 0.805 8.708 0.367 8.175 0.225 9.199 0.368
0.371 0.055 0.724 0.079* 0.556 0.114 0.409 0.083
4.2050.168 3.638 0.256 3.778 0.196 4.847 0.286
1.157 0.237 1.618 0.191 0.817 0.126 0.080 0.049*
0.160 0.045* 0.026 0.014* 0.025 0.017* 0.000 0.000*
0,172 0,.058* 0,024 0.014* 0,024 0,012* 0,000 0,000*
*
Tukey’s HSD, p < 0.05.
Because of the relatively short half lives of the GnRH antagonists used in IVF clinics (cetrorelix, ganirelix) for suppression of the LH surge, their plasma activity is expected to have ceased at the time of implantation. Reports in the literature about the teratogenic potential of the GnRH antagonists used for suppression of the LH surge during IVF–ICSI cycles were analysed by Elizur et al. and it was concluded that GnRH antagonists did not increase the rates of congenital malformations when they were not used inadvertently [19–22]. Mais et al. showed that GnRH antagonist administration at any phase of the natural menstrual cycle disrupts normal ovarian function. Both gonadotropins and estradiol levels fell below the level of healthy controls; a luteolytic effect was only seen when antagonist was given during the mid-luteal phase and was negated by the administration of hCG [23]. Moreover, in a study on 23 donor cycles Tavaniotou et al. reported that ganirelix not only drops luteal phase serum LH concentrations, but also prolongs the length of the luteal phase compared to normal cycling controls [24]. The luteolytic effect of cetrorelix might be a reason for the adverse effects observed in the present study. However, as the placenta in early gestation is known to express both GnRH and GnRH receptors and depends upon normal GnRH physiology, a direct action on the placenta might also be another possible mechanism. GnRH antagonists are also used in many clinics, if the probability of pregnancy is ruled out, in the management of OHSS that develops in IVF–ICSI cycles [12]. Likewise, cryopreservation of all embryos is recommended for the management of early severe OHSS by GnRH antagonists [13,14]. This was not the case, however, in a recently conducted study: GnRH antagonist was administered to three patients who developed severe OHSS during IVF cycles. Injections were started at the 6th day after oocyte retrieval for four days. On day 6, the patients underwent blastocyst transfer and
received ganirelix (0.25 mg/day) for 4 days in combination with luteal phase support using exogenous oestradiol and progesterone. Two patients had successful pregnancies that resulted in the births of healthy infants, while one patient had a biochemical pregnancy. The authors concluded that this treatment was effective in the regression of established severe OHSS, and resulted in the birth of healthy infants. According to the results of that report, it seems that no impairment of embryo implantation was observed following administration of daily ganirelix for four consecutive days in the early implantation period as 2/3 of patients delivered healthy infants and the remaining one had biochemical pregnancy [11]. In the present study, however, administration of cetrorelix impaired placental development, prolonged pregnancy period, decreased embryo weights and caused congenital anomalies at high doses in the rat (Tables 2–4). GnRH plays an important role in the development of the placenta but the exact distribution of the expression and the entire function of GnRH subtypes are not fully understood yet [1,4,7,25– 27]. Placenta carries GnRH I and II receptors both in animal and human. In a study conducted by Chu et al., GnRH I and GnRH II were found to be co-expressed in villous and extravillous cytotrophoblasts, but terminally differentiated, multinucleated syncytial trophoblast expressed only GnRH I in vivo and in vitro [1,4]. Moreover, the effects of GnRH molecules in tissues are determined not only by the type of receptor, but also the coupled G-proteins on that receptor, which may result in a wide range of intracellular cascades [1–3]. Because of the differences in both structure and functions of rat and human placentas, a prediction of the effect of cetrorelix on human pregnancy is not possible from the results of the present study. In conclusion, the results of this study suggest that GnRH I plays an important role in the normal development of the rat placenta
Table 4 Number and mean weights of fetuses of the subjects treated with cetrorelix 15, 75 and 150 mg/kg intraperitoneally at 8th gestational day and the controls on the postmortem examination at 11th day of gestation. (Values are given as mean Standard Error of Mean; n = 12, one-way ANOVA, f = 38.704, p < 0.001; post hoc Tukey’s HSD test, p < 0.001.). Postmortem examinations performed at 11th day
Number of fetuses Mean weight (g) SEM *
p < 0.001.
Control
CTX 15 mg D8
CTX 75 mg D8
CTX 150 mg D8
CTX 15 mg D4
CTX 150 mg D4
45 0.243 0.005*
29 0.187 0.007*
27 0.142 0.009*
0 – –
61 0.146 0.007*
27 0.181 0.006*
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and fetus. Administration of the GnRH antagonist cetrorelix in the early post-implantation period suppresses feto-placental development, decreases embryo weight, delays delivery and causes serious fetal malformations at pharmacological doses in the rat. Our findings indicate that the effect is dose-dependent. Conflicts of interest Nothing to disclose. References [1] Wu HM, Wang HS, Huang HY, Soong YK, MacCalman CD, Leung PC. GnRH signaling in intrauterine tissues. Reproduction 2009;137(May (5)):769–77. [2] Stewart AJ, Sellar R, Wilson DJ, Millar RP, Lu ZL. Identification of a novel ligand binding residue Arg38(1.35) in the human gonadotropin-releasing hormone receptor. Mol Pharmacol 2008;73:75–81. [3] Millar RP, Lu ZL, Pawson AJ, Flanagan CA, Morgan K, Maudsley SR. Gonadotropin-releasing hormone receptors. Endocrinol Rev 2004;25(April (2)):235–75. [4] Chou CS, Beristain AG, Maccalman CD, Leung PCK. Cellular localization of gonadotropin-releasing hormone (GnRH) I and GnRH II in first-trimester human placenta and decidua. J Clin Endocrinol Metab 2004;89:1459–66. [5] Reissmann T, Schally AV, Bouchard P, Reithmu¨ller H, Engel J. The LHRH antagonist cetrorelix: a review. Hum Reprod Update 2000;6(July–August (4)):322–31. [6] Millar RP, Pawson AJ, Morgan K, Rissman EF, Lu ZL. Diversity of actions of GnRHs mediated by ligand-induced selective signaling. Front Neuroendocrinol 2008;29(January (1)):17–35. [7] Rama S, Petrusz P, Rao AJ. Hormonal regulation of human trophoblast differentiation: a possible role for 17beta-estradiol and GnRH. Mol Cell Endocrinol 2004;218(April (1–2)):79–94. [8] Rackow BW, Kliman HJ, Taylor HS. GnRH antagonists may affect endometrial receptivity. Fertil Steril 2008;89(May (5)):1234–9. [9] Griesinger G, Schultze-Mosgau A, Dafopoulos K. Recombinant luteinizing hormone supplementation to recombinant follicle-stimulating hormone induced ovarian hyperstimulation in the GnRH-antagonist multiple-dose protocol. Hum Reprod 2005;20(May (5)):1200–6. Epub 2005 January 21. [10] Olartecoechea B, Garcı´a Manero M, Royo P, Auba` M. Treatment for endometriosis. Rev Med Univ Navarra 2009;53(July–September (3)):12–4. [11] Lainas TG, Sfontouris IA, Zorzovilis IZ. Live births after management of severe OHSS by GnRH antagonist administration in the luteal phase. Reprod Biomed Online 2009;19(December (6)):789–95. [12] Rollene NL, Amols MH, Hudson SB, Coddington CC. Treatment of ovarian hyperstimulation syndrome using a dopamine agonist and gonadotropin releasing hormone antagonist: a case series. Fertil Steril 2009;92(September (3)). 1169.e15–17.
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