Premature progesterone elevation impairs implantation and live birth rates in GnRH-agonist IVF/ICSI cycles

Arch Gynecol Obstet (2010) 281:747 752 DOI 10.1007/s00404-009-1248-0 REPRODUCTIVE MEDICINE Premature progesterone elevation impairs implantation and live birth rates in GnRH-agonist IVF/ICSI cycles Esra Bulgan Kiliçdag Bulent Haydardedeoglu Tayfun Cok Servet Ozden Hacivelioglu Tayfun Bagis Received: 15 July 2009 / Accepted: 29 September 2009 / Published online: 28 October 2009 Springer-Verlag 2009 Abstract Introduction Our aim was to investigate the clinical signiwcance of premature elevated progesterone levels in women with good ovarian response treated with long gonadotropin-releasing hormone agonist (GnRH-a) cycles and IVF. Premature elevated progesterone levels refer to a rise in serum progesterone levels on the day of human chorionic gonadotrophin (hcg) administration for Wnal oocyte maturation above a threshold level, which is usually arbitrarily dewned. Materials and methods This is a retrospective case study, which consists of 1,045 treatment cycles in women with low P/E2 [progesterone (ng/ml) 1,000/estradiol (pg/ml)] on the day of hcg. Elevated P levels on the day of hcg administration were dewned as >1.1 ng/ml. The data of the control group ( 1.1 ng/ml, n = 900 patients) were compared with those of the high P group (>1.1 ng/ml, n = 145). Results Compared with the control group, those in the high progesterone group had higher levels of estradiol on the day of hcg (3,143 vs. 2,382 pg/ml) (p: 0.000) and higher numbers of MII oocytes (14.0 and 12.9) (p: 0.001). The total number of embryos and the number of good-quality embryos transferred did not diver signiwcantly between the groups. Implantation rate (18.1 vs. 24.4%) (p: 0.008), and live birth rate (27.6 vs. 40%) (p: 0.004), were signiwcantly lower in patients with high progesterone levels. OR and 95% CI were 0.57 (0.39 0.84) for the live birth rate. Conclusion In women stimulated with GnRHa and hmg/ FSH for IVF, elevated serum progesterone levels on the day E. B. Kiliçdag (&) B. Haydardedeoglu T. Cok S. O. Hacivelioglu T. Bagis Division of Reproductive and Endocrinology Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Baskent University, Adana, Turkey e-mail: esrabulgan1972@mynet.com of hcg administration were associated with diminished implantation rates and live birth rates regardless of ovarian reserve. Keywords IVF Implantation GnRH agonist Progesterone Live birth rate Introduction Premature luteinization (PL) remains one of the most controversial topics in reproductive endocrinology. PL is usually dewned as subtle, premature increases in serum P concentrations on or before the day of hcg administration [1]. Although it is frequently encountered in superovulation protocols, the pathogenesis and etiology are still debatable. A GnRH agonist or a GnRH antagonist is frequently used in IVF protocols to prevent premature surges of LH resulting in luteinization of the follicle; however, elevated serum progesterone levels have been reported in 2 35% of controlled ovarian hyperstimulation cycles with GnRH agonist [2]. The pathogenesis of PL non-gnrh-a cycles is thought to be the result of increased preovulatory levels of LH. Most studies suggest that PL in non-gnrh-a cycles is associated with poor oocyte maturation, decreased fertilization and reduced embryo quality, and linked to low pregnancy (PRs) and high abortion rates [3, 4]. In contrast, the pathogenesis of PL in long GnRH-a cycles is more complicated and controversial [5 8]. Because the long GnRH-a protocol ensures the prevention of premature LH elevations in 95 98% of patients [9], it has been suggested that late follicular serum P levels are the result of the total amount of P secreted by a cohort of maturing follicles, consequent to the COH [4 10]. Investigators have linked the appearance of PL in long GnRH-a cycles with low [3, 4, 6, 7, 11, 12], high

748 Arch Gynecol Obstet (2010) 281:747 752 [10 13] or similar [2, 5, 14 16] oocyte and embryo qualities or pregnancy rates when compared to cycles with no PL. Younis et al. [17] have suggested another dewnition of PL, taking into account physiologic increases in late follicular P levels in women undergoing COH. PL was dewned as a P/E2 ratio [progesterone (ng/ml) 1,000/estradiol (pg/ml)] of >1 on the day of hcg administration and was suggested to be a manifestation of low ovarian reserve in women with unexplained infertility undergoing COH with hmg. The same investigators demonstrated that in long GnRH-a cycles, PL, dewned as late follicular P/E2 ratio >1, adversely avected clinical outcome and was related to low ovarian reserve [9]. The P/E2 >1 dewnition of PL could be used to diverentiate between distinct clinical situations of increased late follicular P levels. One situation is the result of P secretion from immature follicles as a product of poor ovarian reserve. The other is a consequence of physiologic P secretion from multiple healthy mature follicles in women with good ovarian reserve. The aim of the present study was to examine the clinical signiwcance of elevated progesterone levels in women with good ovarian reserve. For this purpose, we examined only patients with E2 >1,000 pg/ml on the day of hcg administration and P/E2 ratio <1. Primary outcome was live birth rate and secondary outcomes were fertilization, cleavage and implantation rates. Materials and methods This study was conducted from October 2004 to May 2008 in Baskent University Department of Gynecology and Obstetrics, Division of Reproductive and Endocrinology Unit. The study was approved by the Ethical Committee of Baskent University. Outcomes of patients with age <40, basal FSH <10, and treated with a long luteal GnRH-a protocol were evaluated. The Wrst two cycles were included in women with E2 >1,000 pg/ml and P/E2 ratio <1 on the day of hcg, resulting in a total of 1,045 cycles. Calculation of the progesterone/estradiol ratio was performed as follows: progesterone (ng/ml) 1,000/estradiol (pg/ml). In all cycles, ovarian stimulation was carried out with recombinant FSH/hMG after pituitary down-regulation with GnRH-a. The pituitary suppression was achieved with daily leuprolide acetate 1 mg (Lucrin, Abbott, France), beginning on the 21st day of the preceding menstruation. After ovarian suppression was achieved, the dose was reduced to 0.5 mg until the day of hcg. If there were no cysts 2 cm and the E2 was <50 pg/ml, gonadotropin stimulation with 150 300 IU of gonadotropins was performed, with E2 monitoring commencing on the morning of stimulation day 5. Ultrasound and blood E2 monitoring continued until the hcg administration criteria were met with at least three follicles having a maximum diameter of >17 mm. Transvaginal ultrasound-guided oocyte retrieval was performed 35 36 h after the hcg injection. Oocyte pickup was performed with a 17-gauge needle for oocyte retrieval under sedation with propofol (propofol 1% Fresenius Kabi R, Germany). The oocyte-corona complexes (OOC) were denuded, and ICSI was performed after 2 h of incubation and embryos were transferred on day 3. Our clinical policy is to use ICSI routinely in all patients. The embryo transfer policy depends on the number and quality of embryos developed. The transfer protocol was individualized for poor grade embryos up to four. All patients had luteal support with 90 mg of progesterone intravaginally (Crinone 8% gel, Serono) daily after oocyte retrieval. Clinical pregnancy was dewned as the presence of at least one gestational sac with detectable fetal cardiac activity by transvaginal ultrasonography. Live birth was dewned as delivery time after 27 weeks of gestation with at least one live baby. Hormone measurement Sera obtained for basal FSH and LH measurements were analyzed by microparticle enzyme immunoassay (IMx; Abbott, Abbott Park, IL). The intra-assay and interassay coeycients of variation were 5 and 11%, respectively, for FSH and 6 and 7%, respectively, for LH. Serum estradiol and progesterone levels were assayed by solid-phase iodine-125 RIA (Coat-A-Count; DPC, Los Angeles, CA). The intra-assay and interassay coeycients of variation were 7 and 9%, respectively, for estradiol and 9 and 10%, respectively, for progesterone. Progesterone levels Elevated P levels on the day of hcg administration were dewned as >1.1 ng/ml. The data of the control group ( 1.1 ng/ml, n = 900 patients) were compared with those of the high P group (>1.1 ng/ml, n = 145). This value had 90% speciwcity and 10% sensitivity for achieving pregnancy. This value was selected because the best property of a high P level for predicting no pregnancy was its speciwcity [18]. Baseline parameters compared among the study and control groups included the following: age; BMI; duration of infertility; serum levels of FSH, LH and E2 on day 3 of the menstrual cycle; serum levels of E2 and progesterone; numbers of retrieved oocytes and metaphase II oocytes; fertilization rate; cleavage rate and number of available embryos; rates of clinical pregnancies; and live birth rates.

Arch Gynecol Obstet (2010) 281:747 752 749 Statistical analysis Data are expressed as mean SD. Data were analyzed using Student s t test, χ 2 test and Mann Whitney two-sample test (unpaired, nonparametric) where appropriate. A value of P less than 0.05 was considered to be statistically signiwcant. The data were analyzed using SPSS for Windows (version 11.0; SPSS, Inc., Chicago, IL). Results In total 1,045 cycles were initially included in the study; 53 were canceled. In this study of 145 (13.9%) cycles, P levels were elevated on the day of hcg. Table 1 shows the characteristics of patients in the study and control groups. The mean age, BMI, infertility duration, and day 3 FSH, LH and E2 levels did not diver between the groups. Compared with control patients, those with high progesterone levels had higher levels of estradiol on the day of hcg (3,143 vs. 2,382 pg/ml; p: 0.000) and higher numbers of retrieved and MII oocytes (14.0 vs. 12.9; p: 0.001). FSH initiation dose (223.8 66.6 vs. 239.8 67.5) (p: 0.008) and total FSH dose (2,298.9 958.3 vs. 2,627.2 992.7) (p: 0.000) were signiwcantly higher in the high progesterone group. Stimulation days did not diver between the groups. Fertilization rate and cancelation rate were similar between the groups (Table 1). The total number of embryos, and the number of embryos and good quality embryos transferred were not signiwcantly diverent between the groups. Implantation rate (18.1 vs. 24.4%), positive hcg (58 vs. 44.1%), clinical pregnancy rate (48.7 vs. 35.9%), ongoing pregnancy rate (40.7 vs. 27.6%) and live birth rate (27.6 vs. 40%) were lower in patients with high progesterone levels. The odds ratio of live birth rate when elevated progesterone was present was 0.57 (95% CI, 0.39 0.84) compared with cycles in which elevated progesterone did not occur (Table 2). Discussion Our results indicate that in long GnRH-a cycles, premature elevation of progesterone on the day of hcg could adversely avect the clinical outcome of ICSI-ET. These study populations included only patients who had low P/E2 and normal or high E2 levels to exclude those with poor ovarian reserve. The cause of premature elevation of progesterone in GnRH agonist cycles remains unknown. Many researchers in the past have adopted the term premature luteinization for patients with progesterone elevation on the day of hcg administration for Wnal oocyte maturation [19]. This Table 1 Cycle characteristics of women with PL and control group on the day of hcg Control group PL group P Number of cycles that 900 (86.1%) 145 (13.9%) were started Age (year) 30.53 4.7 31.3 4.7 >0.05 BMI (kg/m 2 ) 25.12 4.43 25.40 3.88 >0.05 Infertility duration (year) 7.7 4.6 7.1 4.4 >0.05 Female infertility 152 (16.9) 22 (15.2%) >0.05 Male infertility 471 (52.3) 71 (49) >0.05 Female + male infertility 113 (12.6) 22 (15.2) >0.05 Unexplained infertility 164 (18.2) 30 (20.7) >0.05 Basal FSH (ml/iu) 5.8 1.5 5.7 1.7 >0.05 Basal LH (ml/iu) 5.1 3.4 5.0 2.4 >0.05 Basal E2 (pg/ml) 47.8 62.9 54.8 63 >0.05 Stimulation days 9.9 1.4 9.6 1.6 >0.05 First cycle number (%) 734 (81.6) (84.8) >0.05 FSH initiation dose (IU) 223.8 66.6 239.8 67.5 0.008 Total gonadotropin dose (IU) 2,298.9 958.3 2,627.2 992.7 0.000 Number of cycles 59 (6.6%) 13 (9%) >0.05 with hmg use hcg E2 (pg/ml) 2,382 1,212 3,143 1,313 0.000 hcg P (ng/ml) 0.7 0.2 1.5 0.3 0.000 Number of retrieved 16.9 7.1 20.7 9.4 0.08 oocytes MII 12.8 6.3 14.8 6.2 0.001 Fertilization rate (%) 67 21 64 21 >0.05 Cleavage rate (%) 65.8 20.4 63.6 21.3 >0.05 Canceled numbers (%) 47 (5.2) 6 (4.3) >0.05 suggests that the excessive amount of progesterone is produced by granulosa cells that have started the process of luteinization. However, the fact that a signiwcantly higher mean number of COCs, accompanied by a higher mean E2 level on the day of hcg administration, were present in the group of patients with elevated progesterone, when only the agonist studies were pooled, suggests an alternative explanation. It is likely, at least regarding the patients treated with GnRH agonists, that the elevated progesterone might be attributed to an excess number of follicles, each one producing a normal, for the late follicular phase, amount of progesterone. In this way, excess of proliferating granulosa cells leads to an increased progesterone production, independent of LH exposure. Thus, at least for the studies using GnRH agonists to inhibit LH surge, the use of the term premature luteinization in the presence of normal LH levels might not be appropriate [20]. So, we used the term of premature elevated progesterone levels instead of premature luteinization. Some authors have suggested that the urinary hcg or LH content of the menotropins is the cause of subtle elevations

750 Arch Gynecol Obstet (2010) 281:747 752 Table 2 Outcome of ET cycles in women with PL and control group Control group PL group P OR (95% CI) Number of started cycles 900 145 Live birth rate (%) 360 (40%) 40 (27.6) 0.004 0.57 (0.39 0.84) Implantation rate (%) 24.3 29.7 17.4 27.5 0.008 Positive hcg (%) 522 (58) 64 (44.1) 0.002 0.57 (0.40 0.82) Clinical pregnancy rate (%) 438 (48.7) 52 (35.9) 0.004 0.59 (0.41 0.85) Ongoing pregnancy rate (%) 366 (40.7) 40 (27.6) 0.002 0.56 (0.38 0.82) Total embryo numbers 8.3 4.5 9 5.2 >0.05 Number of transferred embryos 3.1 1.6 3.2 1.2 >0.05 Number of grade 1 embryos 1.5 1.2 1.7 1.3 >0.05 Percentage of freezing 122 (14.4%) 23 (11.7%) >0.05 in P levels [8]. However, even though follicles were not exposed to menotropins, some studies have found a high incidence of P elevation [18, 20]. In our study and control groups in most of the cycles rec-fsh was used. So we think P elevation may not be associated with the type of gonadotropin. Filicori et al. [21] found a strong positive correlation between the administered FSH dose and follicular P levels. Bosch et al. studied the prevalence and the evect of premature luteinization in GnRH-antagonist IVF-ET cycles. They found that total rec-fsh dose and stimulation days were signiwcantly higher in the premature luteinization group [18]. In our study, although stimulation days did not diver with the FSH initiation dose, the total FSH dose was signiwcantly higher in the high progesterone group. In the late follicular phase of COH, the elevation of P is the result of the total amount of P secreted by maturing follicles [4, 5]. Progesterone levels have been found to correlate positively with the number of mature follicles and with E2 levels on the day of hcg [5, 14, 19 22]. Younis et al. [9] dewned P: E2 >1 on the day of hcg as PL, which was suggested to be an important variable that could result in a manifestation of low ovarian reserve and low clinical PR. Recently, increased serum P: E2 ratio ( 1.2) on the day of hcg, as an indicator of PL, was reported to have poor predictive value for IVF outcomes in infertile women with normal ovarian reserve treated with a long GnRH-a protocol [23]. However, in that study only 41 PL patients and 98 non-pl patients were evaluated and the numbers of oocytes retrieved were signiwcantly lower in the PL group (4.2 2.8 vs. 12.7 6.6). Although the diverence was not signiwcant, the pregnancy rate was lower in the PL group (29.3 and 34.5%, respectively). We selected only patients who had E2 >1,000 pg/ml on the day of hcg and P/E2 <1 to exclude those with poor ovarian reserve. In our study population, the numbers of retrieved oocytes were similar between the groups (16.9 7.1 vs. 20.7 9.4). Many studies have described an adverse relationship between elevated circulating P and the occurrence of pregnancy [3, 4, 6, 12]. Although the precise mechanism is unclear, the evect can be attributed to a deleterious evect on either the endometrium or the oocyte. Ovarian stimulation is known to advance endometrial maturation and P itself may hasten closure of the window of implantation [24]. Premature luteinization of follicles also may have detrimental evects on oocyte quality as rexected in reduced rates of normal fertilization, cleavage, embryo morphology and implantation [3, 6]. In our study, fertilization rate, the total number of embryos, number of embryos transferred and numbers of good quality embryos transferred were not signiwcantly diverent between groups, making the proposed theory of impaired oocyte-embryo quality less likely. The diminished implantation rate in the high P group further supported reduced endometrial receptivity. Recently, Azem et al. demonstrated no deleterious evect of elevated P on embryo quality. However, high serum P adversely avects implantation and pregnancy rates. In cycles with elevated serum P, higher estradiol levels were noted, more oocytes were retrieved and manipulated, and more embryos were available for transfer. Embryo grading was comparable between the two groups. The average age was lower in the group with elevated P, but the pregnancy rate was signiwcantly lower (16.4 vs. 27.6%, p =0.03) [25]. Previously, Kolibianakis et al. [26] showed that advanced endometrium was present in all patients on the day of oocyte retrieval. The P receptors in IVF cycles were upregulated on the day of hcg. Thereby, even moderate increases of P, although still within normal limits, result in an ampliwcation of P action, leading to down-regulation of the ER, so-called luteinization [27]. By performing transfer on day 3, the embryo was exposed to an advanced endometrium and to P-induced uterine growth factor out of phase, leading to the establishment of an asynchronous embryo endometrium cross-dialogue [28]. PL during GnRH-antagonist IVF-ET cycles is a frequent event that is associated with lower pregnancy and implantation rates with day 3 ETs (18, 28). In addition, 482 patients

Arch Gynecol Obstet (2010) 281:747 752 751 undergoing elective single ET with GnRH-antagonist/rec- FSH protocol, patient s age <36 years and FSH <12 miu/l, and who were undergoing their Wrst or second IVF treatment, were evaluated prospectively by Papanikolaou [29]. They observed that patients with a rise in P of >1.5 ng/ml with day 3 single ETs had a signiwcantly lower probability of pregnancy compared to those in whom P was normal (OR: 0.39, 95% CI: 0.18 0.88). In contrast, on day 5 the PL had no evect on pregnancy outcome in the single blastocyst group (OR: 1.10, 95% CI: 0.53 2.27). They suggested that, when extending the culture to day 5, blastocyst transfer policy overcomes the detrimental association between the rise in P and the probability of pregnancy. In conclusion, the present study shows that premature elevated P levels in long GnRH-a cycles adversely avected clinical outcome regardless of ovarian reserve and did not seem to be linked to oocytes. 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752 Arch Gynecol Obstet (2010) 281:747 752 releasing hormone antagonists, and human chorionic gonadotropin on endometrial maturation on the day of oocyte pick-up. Fertil Steril 78(5):1025 1029 27. Papanikolaou EG, Bourgain C et al (2005) Steroid receptor expression in late follicular phase endometrium in GnRH-antagonist IVF cycles is already altered, indicating initiation of early luteal phase transformation in the absence of secretory changes. Hum Reprod 20(6):1541 1547 28. Barnes FL (2000) The evects of the early uterine environment on the subsequent development of embryo and fetus. Theriogenology 53(2):649 658 29. Papanikolaou EG, Kolibianakis EM et al (2009) Progesterone rise on the day of human chorionic gonadotropin administration impairs pregnancy outcome in day 3 single-embryo transfer, while has no evect on day 5 single blastocyst transfer. Fertil Steril 91(3):949 952