bs_bs_banner doi:10.1111/jog.12328 J. Obstet. Gynaecol. Res. Vol. 40, No. 5: 1331 1337, May 2014 Prostaglandin therapy during the proliferative phase improves pregnancy rates following frozen embryo transfer in a hormone replacement cycle Koji Nakagawa 1, Yuko Ojiro 1, Hiroyasu Jyuen 1, Yayoi Nishi 1, Rie Sugiyama 2, Yasushi Kuribayashi 2 and Rikikazu Sugiyama 1 1 Division of Reproductive Medicine, Sugiyama Clinic, and 2 Center for Reproductive Medicine and Endoscopy, Sugiyama Clinic Marunouchi, Tokyo, Japan Abstract Aim: To determine the efficacy of prostaglandin administration during the proliferative phase in order to improve pregnancy rates following frozen embryo transfer during a hormone replacement cycle (HRC). Methods: From September 2010 through March 2012, patients (n = 135) were recruited who had undergone oocyte retrieval during a stimulation cycle with clomiphene and had deferred fresh embryo transfer (ET) due to a thin uterine endometrium. All patients were less than 40 years of age and underwent thawed ET following all embryo cryopreservation, and were randomly divided into two groups for thawed ET using a conventional hormone replacement cycle with or without prostaglandin derivatives (prostaglandin or conventional group). Prostaglandin derivatives were administrated during the proliferative phase. Pregnancy and implantation rates following frozen ET were compared between the two groups. Results: Although the endometrial thickness on the day of ET was similar for the prostaglandin and conventional groups, the pregnancy and implantation rates for the prostaglandin group were 40.0% and 22.0%, respectively, which was significantly higher than the rates for the conventional group (P < 0.01). Conclusion: Among patients who avoided fresh ET due to a thin endometrium, the pregnancy rate following a thawed cycle was low. However, it was improved when prostaglandin derivatives were used during the proliferative phase. Key words: clomiphene, prostaglandin, thawed embryo transfer, thin uterine endometrium. Introduction Recently, mild ovarian stimulation protocol using clomiphene citrate (CC) has gained in popularity 1 for use in assisted reproductive technology (ART) treatment, because this protocol does not require daily gonadotropin administration, 2 and it is less burdensome for patients compared with a conventional mild stimulation protocol using gonadotropin-releasing hormone antagonist. Although this protocol may make patients happy by reducing the number and times of injection, as well as reducing the cost, concerns have been raised about this protocol and its use of CC. One of their concerns is that CC sometimes thins the endometrium which can cause implantation failure. Indeed, when patients show a thin uterine endometrium on the day of embryo transfer (ET), they are forced to defer fresh ET, but they can undergo thawed ET in the following Received: January 16 2013. Accepted: October 11 2013. Reprint request to: Dr Koji Nakagawa, Division of Reproductive Medicine, Sugiyama Clinic, 1-53-1 Ohara, Setagaya, Tokyo 156-0041, Japan. Email: nakagawa-jiko@spice.ocn.ne.jp Conflict of interest: The authors have received no funding for this study, and they have no financial interest in any companies. 2014 The Authors 1331
K. Nakagawa et al. cycle. The uterine endometrium is known to thicken with graduated administrations of estrogen, and most of the patients deferring fresh ET due to a thin endometrium have been treated by a graduated estrogen administration protocol to prepare the uterine endometrium for thawed ET. Recently, an interesting paper was published that showed the pregnancy rates following frozen ET. 3 The authors of that report found that women who were forced to defer fresh ET due to a thin uterine endometrium experienced a pregnancy rate that was significantly lower than that for women who deferred fresh ET due to the risk of ovarian hyperstimulation syndrome (OHSS), and they suggested that the women of the former example may be failing to attain adequate endometrial thickness despite a graduated estrogen regimen. Therefore, an improvement is need in both the thickness and implantation ability of the uterine endometrium. Evidence suggests that prostaglandins (PG) play an important role in reproductive processes that include ovulation, implantation and menstruation. 4,5 A recent publication regarding the role of PG 6 found that patients with repeated in vitro fertilization (IVF) failure had very low levels of cytosolic phospholipase A 2, which is the rate-limiting enzyme in PG synthesis, and cyclooxygenase (COX)-2, possibly reducing the function of PG synthesis. The authors concluded that PG synthesis appears to be disrupted in patients with repeated IVF failure and they suggested that reduced PG synthesis in the human endometrium may lead to poor endometrial receptivity. Therefore, for the endometrial condition where reduced PG synthesis is thought to be a serious disadvantage for embryo implantation, the addition of PG during the proliferative phase may improve the implantation ability of the uterine endometrium. The purpose of the present study was to determine whether adding PG during the proliferative phase could improve the chance of successful embryo implantation because of the known role of PG in enhancing implantation. Methods From September 2010 through March 2012, we recruited patients (n = 135) who had undergone oocyte retrieval during a stimulation cycle with clomiphene and who had been forced to defer fresh ET due to a thin uterine endometrium. All patients were less than 40 years of age and had undergone thawed ET following all embryo cryopreservation. The patients were randomly divided into two groups for thawed ET using a conventional hormone replacement cycle (HRC) with or without PG derivatives (PG or conventional group). PG derivatives were administrated during the proliferative phase. Pregnancy and implantation rates following frozen ET were compared between the two groups. Patients and ovarian stimulation for oocyte retrieval (OPU) and patients conditions From September 2010 through March 2012, a total of 135 treatment cycles among 135 women who had undergone ART treatment at the Sugiyama Clinic were recruited for the present study. All patients were less than 40 years of age, and had been forced to defer ET due to a thin uterine endometrium on the day that fresh ET was available. Informed consent was obtained from them, and the institutional review board of the Sugiyama Clinic approved the study. All treatment cycles used our mild stimulation protocol with CC and recombinant-follicle stimulating hormone (rec-fsh). 1 Briefly, patients took 50 mg of clomiphene citrate (Serophen; Merck Serono, Tokyo, Japan) per day for 5 days between days 3 and 7 of the menstrual cycle, and 225 IU of rec-fsh (Gonal-F; Merck Serono) were administrated on days 3, 5 and 7 of the menstrual cycle. On day 9, when the dominant follicles reached 16 mm or more in diameter, 10 000 IU of hcg (Gonatropin, Mochida, Tokyo) or 600 μg of buserelin acetate (Buserequr; Fuji-Pharma, Tokyo, Japan) was nasally administrated, and OPU was performed 35 h later. Additional rec-fsh (150 IU/day) was administrated as needed, based on follicular growth. When the patients showed an endometrial thickness of less than 8 mm on the day of ET (day 3) and acquired a morphologically good embryo (MGE) on day 3 (72 h after OPU), they avoided fresh ET and all MGE were cryopreserved on day 3. Subsequently, they received a thawed ET during a different cycle. Vitrification and warming methods Embryos were cryopreserved via the vitrification method and were warmed using two previously published steps. 7 All solutions used in both vitrification and warming are commercially available (Vitrification kit; KITAZATO BioPhama, Sizuoka, Japan). Two hours after thawing, the appearance of the embryos was examined using an inverted microscope at 400 magnification. Embryos meeting the following criteria were defined as morphologically good: 1332 2014 The Authors
Prostaglandin therapy for thawed ET Figure 1 For the protocol of hormone replacement cycle (HRC), the uterine endometrium was prepared for embryo transfer (ET) using conjugated estrogens (Premarin 0.625 mg) and transdermal estradiol (Estrana TAPE 0.72 mg). These treatments were administrated from the third day of the menstrual cycle (first day without bleeding) until the day of the pregnancy test. Administration of progesterone (100 mg in oil; Progestone Depot-S) was initiated on the 12th day of the menstrual cycle. Three days after the initiation of progesterone treatment, the embryos were warmed and those that had survived and met the criteria mentioned below were used in embryo transfer. developed to at least the seven-cell stage with less than 10% fragmentation after warming. 8 This system of embryo assessment was based on the classification system described by Veeck. 9 Embryos that failed to meet the criteria mentioned above were not transferred to the uterine cavity. Preparation of uterine endometrium and ET All patients who could acquire MGE on day 3 underwent ET in the HRC. The conventional protocol for HRC involved a uterine endometrium that was prepared for ET using conjugated estrogens (Premarin 0.625 mg; Wyeth, Tokyo, Japan) and transdermal estradiol (Estrana TAPE 0.72 mg; Hisamitsu Pharmaceutical, Tokyo, Japan). These treatments were administrated from the third day of the menstrual cycle (first day without bleeding) until the day of the pregnancy test. The administration of progesterone (100 mg in oil; Progestone Depot-S; Fuji Pharmaceutical, Tokyo, Japan) was initiated on the 12th day of the menstrual cycle. Three days after the initiation of progesterone treatment, embryos were warmed and those that had survived and met the criteria mentioned below were used for ET. 8 All patients underwent thawed ET within the HRC with or without misoprostol, and they were randomized on the starting day of the HRC to one of the following two groups by a simple randomization method using the patient s identification number as follows: patients were assigned to the treatment groups when their identification number was odd, and to the control groups when it was even. The conventional protocol of the HRC (conventional group) is mentioned above, but the HRC with misoprostol, which is a PGE1 derivative, was defined as the PG-HRC protocol (PG group).the PG-HRC protocol was the same as the conventional HRC protocol except for the use of misoprostol, which is summarized in Figure 1. Briefly, on the day that the use of conjugated estrogens was begun, patients started to receive 200 μg of misoprostol (Cytotec; Kaken Phamaceutical, Tokyo, Japan) divided into two equal doses 12 h apart until the administration of progesterone (for 11 days). The conjugated estrogens and transdermal estradiol were administrated in the same manner as the conventional HRC protocol. Administration of progesterone (100 mg in oil; Progestone Depot-S, Fuji Pharmaceutical) was initiated on the 12th day of the menstrual cycle. Three days after the initiation of progesterone treatment, embryos were warmed and those that had survived and met the criteria mentioned above were used in ET. 8 Embryos were replaced into a patient s uterus transcervically using a soft catheter (Kitazato ET catheter; Kitazato Supply, Shizuoka, Japan). In all patients, either one or two embryos were transferred. 10 Assessment and data analysis A pregnancy was recognized when the development of a gestational sac was detected by transvaginal ultrasound imaging on the 21st day after the day of ET. The endometrial thickness on the day of ET, difference of endometrial thickness between on the ET cancellation day and the day of ET, pregnancy rate and implantation rate were compared between the conventional and PG groups. Statistical analysis was performed using an unpaired Student s t-test and a χ 2 -test. Statistical significance was set at P < 0.05. 2014 The Authors 1333
K. Nakagawa et al. Table 1 Backgrounds of patients in prostaglandin and conventional groups Prostaglandin group Conventional group No. of patients 67 68 No. of ET cycles 67 68 Age* 38.4 ± 4.1 39.0 ± 2.6 Endometrial thickness on the ET 7.1 ± 1.1 6.5 ± 1.1 cancellation day* No. of transferred embryos (mean) 1.5 1.4 Period between the last CC treatment and the day of embryo transfer* 54.4 ± 20.1 46.1 ± 18.6 *Values were mean ± standard deviation. CC, clomiphene citrate; ET, embryo transfer. Figure 2 (a) The endometrial thicknesses on the day of embryo transfer (ET) in the prostaglandin and conventional groups were 10.0 and 9.8 mm, respectively. (b) The change of endometrial thicknesses between the ET cancellation day and the day of ET in the prostaglandin and conventional groups were +3.0 and +2.8 mm, respectively, and there was no significant difference between the groups. Results Sixty-seven patients underwent thawed ET using PG-HRC (PG group) and 68 used conventional HRC (conventional group). The backgrounds of patients in both groups are summarized in Table 1. The average age (years) of the patients in the PG group was 38.4 ± 4.1, and it was similar to that in the conventional group (39.0 ± 2.6). Endometrial thicknesses on the ET cancellation day in the PG and conventional groups were 7.1 ± 1.1 and 6.5 ± 1.1 mm, respectively, and there were no significant differences. The endometrial thicknesses on the day of ET for the PG and conventional groups were 10.0 and 9.8 mm, respectively (Fig. 2a), and the changes in endometrial thicknesses between the ET cancellation day and the day of ET in the PG and conventional groups were +3.0 and +2.8 mm, respectively, and there were no significant differences between the groups (Fig. 2b). The pregnancy rate in the PG group was 40.0% which was significantly higher than that in the conventional group (25.0%, P < 0.01) (Fig. 3a). The implantation rate in the PG group (22.0%) was also significantly higher Figure 3 (a) The pregnancy rate in the prostaglandin group was 40.0%, which was significantly higher than that in the conventional group (25.0%, P < 0.01). (b) The implantation rate in the prostaglandin group (22.0%) was also significantly higher than that in the conventional group (13.0%, P < 0.01). than that in the conventional group (13.0%, P < 0.01) (Fig. 3b). The miscarriage rates for the PG and conventional groups were 25.1% and 28.6%, respectively, and there were no significant differences. Twelve patients (17.9%) of the PG group experienced side-effects from misoprostol that included either lower abdominal pain (including abdominal distension), nausea or discomfort in the stomach, but these were not severe and none of the patients stopped taking misoprostol due to these symptoms. Discussion Endometrial thickness on the day of ET is a good predictor of success for ART treatment, and the pregnancy and implantation rates for the patients who showed an endometrial thickness of between 7.1 and 14 mm were significantly higher than those of patients who showed a thin endometrium (<7 mm). 11 Therefore, the patients 1334 2014 The Authors
Prostaglandin therapy for thawed ET who showed a thin endometrium (<7 mm) on the day of ET deferred fresh ET. Cryopreservation was used for all embryos, and these patients had to undergo thawed ET in the following HRC with a graduated rising dose of estradiol. Recently, an interesting paper was published regarding pregnancy rates following frozen ET. 3 The authors of that paper compared the pregnancy rates following frozen ET based on the reasons for deferring fresh ET. In these cases, all embryos were cryopreserved due to the risk of OHSS or if there was inadequate endometrial thickness. In that report, the clinical and live delivered pregnancy rates were 42.8% and 31.9%, respectively, for women who deferred based on the risk of OHSS, and the rates for women who deferred due to inadequate endometrial thickness were significantly lower (28.7% and 21.8%, P < 0.05). The authors discussed why the women who deferred fresh ET due to thin uterine endometrium might have failed to attain adequate endometrial thickness despite a graduated estrogen regimen. According to their report, a particular group of patients following frozen ET from their OPU cycles had a lessened chance of pregnancy; therefore, that study provided the basis for choosing a comparison study group for the present study. In the present study, the endometrial thickness on the day of ET in the conventional group was improved only by the administration of estrogen and the change in endometrial thickness between the ET cancellation day and the day of ET in the conventional group was +2.8 mm. These data show that the condition of the uterine endometrium may be easily reversed by the graduated administration of estrogen. However, the pregnancy rate in the conventional group was significantly lower, which raised the question of why this low pregnancy rate occurred despite an improvement in endometrial thickness by graduated estrogen administration. We speculated that something had decreased the uterine receptivity because all patients had been stimulated with CC and rec-fsh in the previous cycle, and the CC might have caused the poor endometrial receptivity. Therefore, a solution to this problem is needed. The relationship between PG and the uterine endometrium has been studied for more than 20 years. PG dehydrogenase, which inactivates PG, plays a key role in controlling the effective local concentration of PG and is found in the human endometrium at high levels in the secretory phase of the menstrual cycle, but it is absent during the proliferative phase. 12 PG dehydrogenase is a critical enzyme in maintaining low PG levels within tissues. In the uterus, the enzyme is present in the myometrium, 12 and it is present in the endometrium during the higher activity of the secretory phase of the menstrual cycle compared with the activity during the proliferative phase. 13 A demonstration that raised PG dehydrogenase levels during the secretory phase indicated that inactivation of PG prevented myometrium contraction via PGE2. By contrast, a decrease in PG dehydrogenase levels in the endometrium during the proliferative phase maintained a high concentration of PG in the uterine endometrium, and contributed to a thickening of the uterine endometrium. However, Kelly and coworkers examined the PG dehydrogenase activity in the human endometrium from women who had been treated with CC at an early stage of the menstrual cycle, and they found high levels of PG dehydrogenase activity in the proliferative phase rather than in the secretory phase. 14 These results indicated that PG were lower in the proliferative phase than in the secretory phase. The PG, and particularly PGE2, may have an important role in developing the uterine endometrium, and subsequently in the development endometrial thickness. Recently, Achache and co-workers published interesting data. 6 According to their report, PG synthesis in the secretory endometrium appeared to be disrupted in patients with repeated IVF failure compared with fertile women, and they suggested that reduced PG synthesis, in particular PGE2, in the human endometrium may lead to poor endometrial receptivity. Therefore, PGE2 also plays an important role in the secretory phase, and a decrease in the PGE2 level must be avoided wherever possible. CC has induced low PG levels via a decrease in PG hydrogenase, and this adverse effect could remain in effect for the following cycles, so that addition of PGE1 during the proliferative phase may correct the PG levels, and, consequently, improve the pregnancy rate. Moreover, a report by Check et al. indicated that patients who took 81 mg of aspirin daily beginning on day 2 of the frozen ET cycle for 3 weeks showed significant lower pregnancy and implantation rates compared with patients who received no aspirin. 15 According to these reports, a decrease in PG levels in the uterine endometrium resulted in lower rates of pregnancy, even though in frozen ET cycles, the administration of PG may be a solution. There was a very interesting report concerning the characteristics of clomiphene citrate. This report 2014 The Authors 1335
K. Nakagawa et al. mentioned that clomiphene, possessing aspects as a weak estrogen and as an anti-estrogen, was a very potent PG synthesis inhibitor, being equal in this parameter to indomethacin. 16 From this report, CC acted as a PG synthesis inhibitor and raised PG dehydrogenase concentration in the human endometrium. Moreover, it is well known that the use of non-steroidal anti-inflammatory drugs (NSAIDs) inhibit both COX-1 and COX-2. COX-1 is responsible for the constitutive levels of PG, whereas COX-2 produces PG with proper stimulation. 17 Therefore, NSAIDs inhibit the downstream signaling pathways of PG, which impairs angiogenesis and cell adhesions, and consequently impairs embryo attachment to the uterine endometrium. The use of CC is also bad for implantation, and this effect may be retained. In the present study, we attempted to use PG derivatives for the groups who had limited use of frozen ET following a clomiphene controlled ovarian hyperstimulation (COH) protocol, but these products may only improve pregnancy rates following IVF-ET fresh cycles, with no improvement to the pregnancy rates following frozen ET cycles for embryos derived from COH without using CC, because some of the patients experienced recurrent implantation failure in ART treatment due to impaired PG levels in their uterine endometrium. 6 Several PG products are available in Japan such as alprostadil (PGE1) for the treatment of peripheral arterial occlusive diseases, or misoprostol (PGE1) for gastric ulcers or dinoprost (PGF2α) and dinoprostone betadex (PGE2) for the inducement of labor. Alprostadil is taken by i.v. injection and is expensive, which makes it difficult for patients to use, and dinoprost and dinoprostone betadex induce uterine contraction, and subsequently induce lower abdominal pain, which also makes this difficult for infertile patients to use. Misoprostol has a low price, and minimal side-effects. Of course, PGE2 products may be better theoretically, but we decided to use misoprostol as a PG product because it was regarded as safe (less burdensome than the use of PGE2 products), which is important. In conclusion, the patients who were forced to defer fresh ET cycles due to thin endometrium were treated with CC in ovarian stimulation, which improved the thickness of the uterine endometrium following a HRC that used the graduated administration of estrogen. However, the pregnancy rates following a thawed cycle were low. These lower pregnancy rates were improved when thawed ET was undergone in the HRC after the administration of PG derivatives during the proliferative phase. Acknowledgments The authors would like to thank Dr Toshifumi Takahashi (Department of Obstetrics and Gynecology, Yamagata University Faculty of Medicine) for his comments and advice, and the authors received no financial support. References 1. Nakagawa K, Nishi Y, Sugiyama R et al. A programmed schedule of oocyte retrieval using mild ovarian stimulation (clomiphene citrate and recombinant follicular stimulating hormone). Reprod Med Biol 2012; 11: 85 89. 2. Sugiyama R, Nakagawa K, Nishi Y, Sugiyama R, Ezaki K, Inoue M. The dilemma faced by patients who undergo single embryo transfer. Reprod Med Biol 2009; 8: 33 37. 3. Check LH, Choe JK, Brasile D, Cohen R, HOrwath D. Comparison of pregnancy rates following frozen embryo transfer according to the reason for freezing: Risk of ovarian hyperstimulationvs inadequate undometrial thickness. Clin Exp Obstet Gynecol 2012; 39: 434 435. 4. Jabbour HN, Sales KJ. Prostaglandin receptor signaling and function in human endometrial pathology. Trends Endocrinol Metab 2004; 15: 398 404. 5. Kang J, Chapdelaine P, Parent J, Madore E, Laberge PY, Fortier MA. Expression of human prostaglandin transporter in the human endometrium across the menstrual cycle. J Clin Endocrinol Metab 2005; 90: 2308 2313. 6. Achache H, Tsafrir A, Prus D, Reich R, Revel A. Defective endometrial prostaglandin synthesisidentified in patients with repeated implantationfailure undergoing in vitro fertilization. Fertil Steril 2010; 94: 1271 1278. 7. Sugiyama R, Nakagawa K, Shirai A et al. Clinical outcomes of resulting from transfer of vitrified human embryos using a new device for cryopreservation (plastic blade). J Assist Reprod Genet 2010; 27: 161 167. 8. Nakagawa K, Takahashi C, Nishi Y et al. Hyaluronanenriched transfer medium improves outcome in patients with multiple embryo transfer failures. J Assist Reprod Genet 2012; 29: 679 685. 9. Veeck LL. Atlas of the Human Oocyte and Early Conceptus. 2, Baltimore, MD: Williams & Wilkins Co, 1991. 10. Nakagawa K, Nishi Y, Sugiyama R, Kuribayashi Y, Sugiyama R, Inoue M. Elective single cleavage-stage embryo transfer need not result in lower pregnancy ratescompared to double cleavage-stage embryo transfer. J Obstet Gynocol Res 2010; 36: 777 782. 11. Amir W, Micha B, Ariel H, Liat L-G, Jehoshua D, Adrian S. Predicting factors for endometrial thickness during treatment with assisted reproductive technology. Fertil Steril 2007; 87: 799 804. 12. Casey ML, Hemsell DL, Johnston JM, Madonald PC. NAD dependent 15-hydroxyprostadlandin dehydrogenase activity in human endometrium. Prostaglandins 1980; 19: 115 122. 1336 2014 The Authors
Prostaglandin therapy for thawed ET 13. Abel MH, Kelly RW. Metabolism of prostaglandins by the non-pregnant human uterus. J Clin Endocrinol Metab 1983; 56: 678 685. 14. Kelly RW, Linan C, Thong J, Young EL, Baird DT. Prostaglandin inactivation is increased in endometrium after exposure to clomiphene. Prostaglandins Leukot Essent Fatty Acids 1994; 50: 235 238. 15. Check JH, Dietterich C, Lurie D, Nazari A, Chuong J. A matched study to determine whether low-dose aspirin without heparin improves pregnancy rates following frozen embryo transfer and/or affects endometrial sonographic parameters. J Assit Reprod Genet 1998; 15: 579 582. 16. Lerner LJ, Carminati P, Schiatti P. Correlation if antiinflammatory activity with inhibition of prostagrlandin synthesis activity of nonsteroidal anti-estrogens and esrofgens (38532). Proc Soc Exp Biol Med 1975; 148: 329 332. 17. Morita I. Distinct functions ofcox-1 andcox-2. Prostaglandins Other Lipid Mediat 2002; 68 69: 165 175. 2014 The Authors 1337