Relationship of progesterone/estradiol ratio on day of hcg administration and pregnancy outcomes in high responders undergoing in vitro fertilization

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1 Relationship of progesterone/estradiol ratio on day of hcg administration and pregnancy outcomes in high responders undergoing in vitro fertilization Fa-Kung Lee, M.D., M.P.H., a,b Tsung-Hsuan Lai, M.D., c,d Tseng-Kai Lin, M.D., a,b Shang-Gwo Horng, M.D., M.P.H., a and Su-Chee Chen, M.D. c,d a Department of Obstetrics and Gynecology, Cathay General Hospital, HsinChu Branch, HsinChu; b Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsin Chu; c Fu Jen Catholic University School of Medicine, Taipei; and d Department of Obstetrics and Gynecology, Cathay General Hospital, Taipei, Taiwan Objective: To evaluate the predictive value of a serum P/E 2 ratio measured on the day of hcg administration regarding pregnancy outcomes in high responders undergoing IVF. Design: Retrospective study. Setting: Teaching hospital. Patient(s): Two hundred twenty-three infertile women classified as high responders in IVF-ET cycles. Intervention(s): Eligible infertile women undergoing IVF were assigned to four groups according to serum P levels on the day of hcg administration: group 1, P%0.9 ng/ml; group 2, 0.9 < P%1.4 ng/ml; group 3, 1.4 < P%2.0 ng/ml; group 4, P>2.0 ng/ml. The relationship of E 2 level and P/E 2 ratio on the day of hcg administration and pregnancy outcomes was analyzed. Main Outcome Measure(s): Implantation and pregnancy rate. Result(s): Patients in group 4 had highest E 2 level and P/E 2 ratio, as well as lowest implantation and pregnancy rates. Using P for grouping, the sensitivity/positive predictive values (%/%) of P/E 2 ratio in the four groups were 15/66, 30/65, 30/60, and 25/41, respectively. Conclusion(s): Using the level of a single sex hormone on hcg day to predict pregnancy outcome in high responders undergoing IVF is confounding, whereas using a P/E 2 ratio on hcg day is theoretically reasonable. However, the low sensitivity and positive predictive value make the use of P/E 2 clinically unfeasible. (Fertil Steril Ò 2009;92: Ó2009 by American Society for Reproductive Medicine.) Key Words: Receptivity, premature luteinization, P/E 2 ratio, IVF During in vitro fertilization (IVF) cycles, serum estradiol (E 2 ) levels and follicle growth monitored by ultrasound are used to determine adequate ovarian response during controlled ovarian hyperstimulation (COH). However, women with serum E 2 >3,000 pg/ml on the day of hcg administration (high responders) have been reported to have unfavorable pregnancy rates (PRs) due to the detrimental effects of high levels of E 2 on endometrial receptivity (1). Some studies, though, have failed to demonstrate a detrimental effect of high serum E 2 on PR (2 4). The effect of elevated progesterone (P) levels before administration of hcg, i.e., premature luteinization, on the pregnancy outcome in IVF programs is also a topic of research. Some studies have suggested that elevation of P on hcg day may lead to a poor pregnancy outcome due to Received February 3, 2008; revised July 9, 2008; accepted August 7, 2008; published online October 1, F.-K.L. has nothing to disclose. T.-H.L. has nothing to disclose. T.-K.L. has nothing to disclose. S.-G.H. has nothing to disclose. S.-C.C. has nothing to disclose. Reprint requests: Dr. Shang-Gwo Horng, M.D., Department of Obstetrics and Gynecology, Cathay General Hospital, HsinChu Branch, HsinChu, Taiwan, No 678, Section 2, Junghua Rd., HsinChu City, Taiwan (FAX: ; hsg@cgh.org.tw). impaired endometrial receptivity and poor oocyte quality (5 7). However, the unfavorable effect of elevated P was not found in other studies (8 12). In the late follicular phase of COH, P level reflects the total amount of P secreted by maturing follicles (5, 8, 13). Progesterone levels have been found to correlate positively with the number of mature follicles and with E 2 levels on the day of hcg administration (8, 14 17). Thus, high responders may have high levels of E 2 and P, and it is therefore difficult to use a single sex steroid hormone level on hcg day to predict pregnancy outcomes. Younis et al. (18, 19) have questioned the value of using absolute P levels on hcg day to predict pregnancy outcomes in IVF cycles. Instead, they proposed a P/E 2 ratio >1 as the definition of premature luteinization and demonstrated that a P/E 2 ratio >1 was associated with low ovarian reserve as well as poor pregnancy outcomes. Fanchin et al. (20) demonstrated that in the presence of an adequate response to COH, elevated P levels were not associated with lower PRs, but when the response to COH was weak, premature P elevation led to lower PRs. The influence of elevated P and E 2 on the day of hcg administration on pregnancy outcomes is a subject of debate Fertility and Sterility â Vol. 92, No. 4, October /09/$36.00 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 To the best of our knowledge, investigations regarding the effect of elevation of both P and E 2 on hcg day on pregnancy outcomes are lacking. Additionally, although well documented, studies regarding the relationship between the P/E 2 ratio and pregnancy outcome is limited to patients with low ovarian reserve and weak response (18 20). Whether the P/ E 2 ratio on hcg day can be used in high responders to predict pregnancy outcome remains unknown. Accordingly, the role of premature P elevation and P/E 2 ratio on PR in high responders should be explored. The aim of the present study was to analyze the relationship between these parameters on hcg day and PR in high responders undergoing IVF. MATERIALS AND METHODS Subjects The records of infertile women who were high responders (E 2 levels on the day of hcg administration >3,000 pg/ml) (1) undergoing IVF-ET treatment with the long GnRH agonist (GnRHa) protocol at Cathay General Hospital, Hsin Chu, from March 2003 to April 2007 were reviewed in this retrospective study. All patients registered for IVF or intracytoplasmic sperm injection (ICSI) were included, except for those with the following conditions: 1) infertility attributed to endocrine abnormalities, such as hyperprolactinemia, thyroid dysfunction, and Cushing syndrome; 2) previous COH with documented poor response resulting in a mature oocyte yield %3; 3) occult ovarian failure with day 3 basal FSH concentration R10 IU/L; or 4) using stimulation protocols other than GnRHa pituitary desensitization. All women were determined to have normal uterine cavities by hysterosalpingography or hysteroscopy. Institutional Review Board approval was obtained for this study. Protocols for COH In all cycles, ovarian stimulations were carried out with rfsh and hmg after pituitary down-regulation with GnRHa. The pituitary suppression was achieved by SC injection of leuprolide acetate (Lupron; Takeda Chemical Industries, Osaka, Japan) 0.2 mg daily, which was reduced to 0.1 mg after the down-regulation was achieved, starting in the midluteal phase of the preceding cycle. When a serum E 2 concentration of %50 pg/ml was confirmed on day 2 of the cycle, ovarian stimulation was started with administration of IU rfsh (Gonal-f; Serono, Aubonne, Switzerland) and/or IU hmg (Pergonal; Serono) IM per day. Gonadotropin dosage was adjusted by monitoring follicular size using transvaginal ultrasound (Acuson 128-P-10; Acuson, Mountain View, CA) after gonadotropin administration for 6 days. Human chorionic gonadotropin 10,000 IU IM (Profasi; Serono) was given to trigger ovulation when two leading follicles reached a mean diameter of 18 mm. Oocytes were retrieved transvaginally h after hcg administration. Evaluation of oocyte maturity was in accordance with the criteria established by Veeck (22). Routine sperm preparation and IVF/ICSI and embryo cultures were performed as in previously described protocols (23). Up to six embryos were transfered on day 2 or 3 after oocyte retrieval. The luteal phase was supported with 50 mg P in oil IM and 300 mg micronized P (Ultrogestan; Besins-Iscovesco Pharmaceuticals, Paris, France) administered daily (100 mg in the morning and 200 mg in the evening) vaginally starting on the day of ET. Hormone Measurement Basal hormonal ovarian reserve studies, which included serum FSH, LH, and E 2 levels were obtained on day 2 3 of the stimulation cycle with gonadotropin after pituitary suppression. The FSH, LH, and E 2 serum concentrations were measured using radioimmunoassays (RIA; Gambyt-CR; Diagnostic Products Corporation, Los Angeles, CA). The interassay coefficients of variation (CVs) for E 2, LH, and FSH were 5.4%, 4.5%, and 3.7%, respectively. The intra-assay CVs for E 2, LH, and FSH were 4.3%, 3.2%, and 4.7%, respectively. On the day of hcg administration, serum E 2 and P were also measured. Samples were assayed by RIA (Gambyt-CR). The intra-assay and interassay CVs were, respectively, 7.1% and 9.7% for E 2 and 9.6% and 4.9% for P. Grouping Patients were assigned to one of four groups according to the serum P levels, modified from the study conducted by Hofmann et al., on the day of hcg administration (21). Patients were assigned to groups as follows: group 1, P%0.9 ng/ml; group 2, 0.9 < P%1.4 ng/ml; group 3, 1.4 < P%2.0 ng/ml; group 4, P>2.0 ng/ml. The P/E 2 ratio was calculated by the following formula: P (in ng/ml) 1,000/E 2 (in pg/ml) (18, 19). Using P for grouping, the sensitivity/positive predictive value was also analyzed. Definitions of Clinical Outcomes Clinical pregnancy was defined as an intrauterine gestational sac and fetal heartbeat by ultrasound 3 weeks after ET. Statistics Statistical analysis was performed with the Statistical Package for Social Sciences (v 11.0 for Windows; SPSS, Chicago, IL). Pregnancy rate was analyzed by the c 2 test, whereas hormone levels, P/E 2 ratios, implantation rate, and pregnancy rate were analyzed by a one-way analysis of variance (ANOVA) with least significant difference post hoc test. A P value of <.05 was considered to be statistically significant. RESULTS After excluding patients who did not meet the eligibility criteria, we included 223 women in the study. Clinical indications for IVF-ET and the number of patients in each group are presented in Table 1. Fertility and Sterility â 1285

3 TABLE 1 Patient data. Group 1 P%0.9 ng/ml (n [ 29) Group < P%1.4 ng/ml (n [ 57) Group < P%2.0 ng/ml (n [ 62) Group 4 P>2.0 ng/ml (n [ 75) Male factors Normal sperm Azoospermia Oligospermia Asthenospermia Teratospermia Oligoasthenospermia Female factors Tubal obstruction Endometriosis Ovarian dysfunction Unexplained Multiple factors Lee. P/E 2 ratio in IVF high responders. Fertil Steril Hormone levels and pregnancy rates are presented in Table 2. Basal FSH, LH, and E 2 levels were not different between groups. The E 2 levels on the day of hcg administration were statistically different between groups, and were lowest in group 1 ( pg/ml) and highest in group 4 ( pg/ml) (one-way ANOVA: P<.001). Post-hoc analysis revealed that significant difference existed between groups 1 and 4 (P<.05), as well as between groups 2 and 4 (P<.05). The difference of P/E 2 ratio on hcg days of each group were also statistically significant (group 1, ; group 2, ; group 3, ; group 4, ; one-way ANOVA: P<.001). The P/E 2 ratio was lowest in group 1 and highest in group 4. A post hoc test revealed that significant difference existed between groups (P<.05 for all group-to-group comparisons). The PR in group 4 was significantly lower than in the other 3 groups (P<.05, c 2 test). It is of note that both E 2 and P/E 2 increased as P level increased. The number of oocytes retrieved and embryos transfered did not reach statistical significance. Implantation rates among the 4 groups reached statistical significance (group TABLE 2 Hormone data and pregnancy outcomes. Group 1 P%0.9 ng/ml (n [ 29) Group < P%1.4 ng/ml (n [ 57) Group < P%2.0 ng/ml (n [ 62) Group 4 P>2.0 ng/ml (n [ 75) P value Age (yrs) NS Day 3 FSH (miu/ml) NS Day 3 LH (miu/ml) NS Day 3 E 2 (pg/ml) NS hcg day E 2 (pg/ml) <.001 P/E 2 ratio <.001 Oocytes retrieved NS Embryos transfered NS Implantation rate (%) <.05 Pregnancy rate 19/29 (65.5%) 37/57 (64.9%) 37/62 (59.7%) 31/75 (41.3%) <.05 Note: Data are presented as mean SE. All data except pregnancy rate were analyzed by one-way analysis of variance with least significant difference post hoc analysis; pregnancy rate was analyzed by c 2 test. Lee. P/E 2 ratio in IVF high responders. Fertil Steril Lee et al. P/E 2 ratio in IVF high responders Vol. 92, No. 4, October 2009

4 1, %; group 2, %; group 3, %; group 4, %; one-way ANOVA: P<.05). Post hoc analysis revealed that women in group 4 had the lowest implantation rate, which was significantly lower than those of groups 1 and 2 (P<.05). Using P for grouping, the sensitivity/positive predictive values (%/%) of P/E 2 ratio in the four groups were 15/66, 30/65, 30/60, and 25/41, respectively. DISCUSSION The detrimental effect of high E 2 level on uterine receptivity has been discussed for many years (1, 24, 25). Simon et al. (1) have suggested that high responders (E 2 on hcg day >3,000 pg/ml) have significantly lower implantation and pregnancy rates and impaired endometrial receptivity. However, using the same definition for high responders, Sharara and McClamrock (2) failed to find a detrimental effect of high E 2 level on hcg day on pregnancy outcome. Although P levels were not analyzed in those studies, other studies proposed that not only E 2 level, but also P level on hcg day affects pregnancy outcome (5 7). However, the unfavorable effect of elevated P on pregnancy outcome was questioned by yet other studies (8 12). Decreased implantation and pregnancy rates may be caused by poor embryo quality, impaired endometrial receptivity, or both. Several studies investigating the oocyte and embryo quality in high responders have excluded the possibility that high E 2 levels on hcg day affect oocyte or embryo quality. Simon et al. (26) have shown that oocyte quality, fertilization rate, and embryo development until day 2 in high responders are similar to those in normal responders. In the same study, observations were made that pregnancy and implantation rates in recipients of embryos derived from high responders were similar to those in normal responders (26). Ng et al. (27) have demonstrated that embryo quality and implantation seem to be normal in subsequent frozen-thawed ETs. Likewise, that high E 2 level on hcg day might lead to poorer embryo quality has also been ruled out by a number of studies (7, 10, 13, 28, 29). Endometrial receptivity is determined by the complex interactions between E 2 and P. Nilsson et al. (30) found that the administration of levonorgestrel to fertile women resulted in a suppression of characteristic changes in the nonciliated cells, including centrally located microvilli, elongated glandular openings, and the appearance of pinopods. Additionally, they found that the addition of E 2 acted to restore most of the characteristic changes. Other studies also showed that supraphysiologic levels of steroid hormones not only induce morphologic alterations in endometrium (31, 32), but also alter endometrial E 2 /P ratios, which are associated with impaired endometrial receptivity (33). A significant reduction of nuclear receptors for P and E 2 has been demonstrated in both endometrial glands and stroma in women receiving COH (34). Recently, Pellicer et al. (35) have shown that high E 2 levels can directly inhibit adhesion of embryos to the endometrium. In contrast, COH may advance endometrium maturation (31), and P may hasten the closure of the implantation window (10). In the late follicular phase of COH, P level reflects the total amount of P secreted by maturing follicles (5, 8, 13). The P levels have been found to correlate positively with the number of mature follicles and with E 2 levels on the day of hcg administration (8, 14 17). Thus, using a single hormone level to predict pregnancy outcome is confounding and the influence of both E 2 and P should be taken into consideration. In the present study, the roles of P and the P/E 2 ratio in high responders were explored. The data showed that women with higher P levels had higher E 2 levels. This result is in agreement with the positive correlation between P and E 2 in the late follicular phase. However, the results also showed that women with a P level >2.0 ng/ml had the highest E 2 levels and lowest implantation and pregnancy rates. The suboptimal pregnancy outcomes in this group might be caused by the high level of E 2, P, or both. The uncertainty of the effects of high levels of E 2 and P on pregnancy outcomes, and the positive correlation between P and E 2 make it confounding to use a single sex steroid hormone level to predict the pregnancy outcome in high responders. Considering the secretion of P and E 2 from granulosa cells in late follicular phase and the interaction of both P and E 2 on endometrium, Younis et al. (18, 19) have suggested an elevation of P/E 2 ratio on hcg day be used as definition of premature luteinization. Younis et al. implied that defining premature luteinization as P/E 2 >1 could differentiate physiologic P secretion from multiple healthy mature follicles from that secreted from dysmature follicles; the latter could be related to low ovarian reserves and poor pregnancy outcome. Using P >0.9 ng/ml to define premature luteinization, Fanchin et al. (20) demonstrated that in the presence of an adequate response to COH, elevated P levels were not associated with lower PRs, but when the response to COH was weak, premature P elevation led to lower PRs. In high responders, we found that women with P >2.0 ng/ml have the highest P/E 2 ratio and lowest implantation and pregnancy rates, implying the association of high P/E 2 ratio with unfavorable pregnancy outcomes. Accordingly, it is reasonable to assume that an increased P/E 2 ratio on hcg day is more reliable in predicting pregnancy outcomes. Several theories have been suggested to explain the development of premature luteinization in long GnRHa cycles. Although hmg use was thought to be the cause, this was not confirmed by Younis et al. (19). Incomplete GnRHa pituitary down-regulation was also proposed; thus, increasing E 2 levels may induce LH secretion sufficient to stimulate granulosa cells to produce P (36 38). The FSH stimulation of LH receptors on granulosa cells leading to P production in the presence of low serum LH levels has also been proposed (36, 38 40). The present study also showed that high responders have high PRs but low implantation rates. The explanation for Fertility and Sterility â 1287

5 this finding is that higher numbers of embryos were transfered (12), because it has been suggested that impaired endometrial receptivity can be overcome by transfering more embryos (27) or better embryos (20). Our data is in good agreement with these observations and suggest that in the situation of high responders and high P/E 2 ratio, transfering a high number of embryos is an alternative method to overcome impaired endometrial receptivity. However, we did not actually use P/E 2 ratio to predict the pregnancy outcome, because of low sensitivity and positive predictive values (the sensitivity/positive predictive value [%/%] in the four groups were 15/66, 30/65, 30/60, and 25/41, respectively). It is well known that embryo quality is also an important factor that can influence pregnancy outcomes (41 43). It is of note that oocyte and embryo qualities are not affected by the elevations of ovarian steroid hormones (7, 10); therefore, using ovarian steroid hormones alone for predicting pregnancy outcome will yield inadequate or unsatisfactory sensitivity and positive predictive values. To clarify the influence of ovarian steroid on the pregnancy outcomes, the variable of embryo quality should be controlled, such as by using single embryo transfer or substratifying the embryo qualities. However, in the present retrospective study, we did not substratify embryo quality because we used to transfer embryos of different qualities back to the uterus. From the present data, we conclude that using the level of a single sex hormone on hcg day to predict pregnancy outcome in high responders undergoing IVF is confounding, whereas using a P/E 2 ratio on hcg day is theoretically reasonable. However, the low sensitivity and positive predictive value make the use of P/E 2 ratio on hcg day clinically unfeasible. Therefore, more studies are needed to confirm our observations and define the cut-off value of P/E 2 for high responders. REFERENCES 1. Simon C, Garcia Velasco JJ, Valbuena D, Peinado JA, Moreno C, Remohı J, et al. Increasing uterine receptivity by decreasing E 2 levels during the preimplantation period in high responders with the use of a follicle-stimulating hormone step-down regimen. Fertil Steril 1998;70: Sharara FI, McClamrock HD. High E 2 levels and high oocyte yield are not detrimental to in vitro fertilization outcome. Fertil Steril 1999;72: Levi LJ, Drews MR, Bergh PA, Miller BT, Scott RT. Controlled ovarian hyperstimulation does not adversely affect endometrial receptivity in in-vitro fertilization cycles. Fertil Steril 2001;76: Papageorgiou T, Guibert J, Goffinet F, Patrat C, Fulla Y, Janssens Y, et al. 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