Bei Xu, Ph.D., Zhou Li, Ph.D., Hanwang Zhang, Ph.D., Lei Jin, Ph.D., Yufeng Li, Ph.D., Jihui Ai, Ph.D., and Guijin Zhu, M.D.

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1 Serum progesterone level effects on the outcome of in vitro fertilization in patients with different ovarian response: an analysis of more than 10,000 cycles Bei Xu, Ph.D., Zhou Li, Ph.D., Hanwang Zhang, Ph.D., Lei Jin, Ph.D., Yufeng Li, Ph.D., Jihui Ai, Ph.D., and Guijin Zhu, M.D. Reproductive Medicine Center, Tongji Hospital, Tongji Medicine College, Huazhong University of Science and Technology, Wuhan, People's Republic of China Objective: To investigate the relationship between serum P levels on the day of hcg administration and pregnancy outcomes in different responders undergoing IVF. Design: Retrospective study. Setting: Teaching hospital. Patient(s): A total of 11,055 women who underwent their first IVF/intracytoplasmic sperm injection cycles and a subgroup of 4,021 women undergoing frozen-embryo transfer (FET) cycles. Intervention(s): Patients underwent IVF-ET with the long GnRH agonist protocol. The ovarian response was classified as high (R20 oocytes; n ¼ 2,023), poor (%4 oocytes; n ¼ 827), or intermediate (remaining cases; n ¼ 8,205) according to the number of oocytes retrieved. Clinical outcomes of IVF-ET and FET cycles were analyzed according to plasma P levels. Main Outcome Measure(s): Ongoing pregnancy rates (PRs). Result(s): Ongoing PRs in fresh cycle were inversely associated with serum P levels on the day of hcg administration for all patients. Different P threshold concentrations were determined according to different ovarian response: We proposed a serum P level of 1.5 ng/ml as the threshold for poor responders, 1.75 ng/ml for intermediate responders, and 2.25 ng/ml for high responders. Our study does not show negative results for elevated P levels on oocyte performance in terms of fertilization, cleavage rate, or PR of FET cycles within different ovarian responses, offering no evidence for a detrimental effect of high P on oocyte quality. Conclusion(s): Elevated P levels on the day of hcg administration negatively influence PR regardless of different ovarian responses, although increased P threshold concentration is associated with better ovarian responses. The detrimental effect of P elevation on PR seems to be unrelated to oocyte quality in all responders. (Fertil Steril Ò 2012;97: Ó2012 by American Society for Reproductive Medicine.) Key Words: Progesterone, in vitro fertilization, ovarian response, ongoing pregnancy rate The introduction of GnRH agonists and antagonists for pituitary suppression during IVF significantly decreases the incidence of premature LH surge (1). Despite the use of GnRH analogues (GnRHa), however, several researchers have described a phenomenon reported as premature luteinization (2 4), which refers to a rise in serum P levels on the day of hcg administration above a threshold level which is usually arbitrarily defined. The term has since proven to be misleading, because subsequent research found that the rise in P levels seen during controlled ovarian hyperstimulation (COH) for IVF/intracytoplasmic sperm injection (ICSI) cycles cannot be Received February 6, 2012; revised March 11, 2012; accepted March 12, 2012; published online April 10, B.X. has nothing to disclose. Z.L. has nothing to disclose. H.Z. has nothing to disclose. L.J. has nothing to disclose. Y.L. has nothing to disclose. J.A. has nothing to disclose. G.Z. has nothing to disclose. This study was supported by grant from the National Natural Sciences Foundation of China and grant 2007CB from the 973 Program of China. Reprint requests: Guijin Zhu, M.D., Reproductive Medicine Center, Tongji Hospital, Tongji Medicine College, Huazhong University of Science and Technology, 1095 JieFang Avenue, Wuhan, , People's Republic of China ( zhu_guijin@sina.com). Fertility and Sterility Vol. 97, No. 6, June /$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert explained by luteinization of granulosa cells, which was not accompanied by increases in LH (5, 6). The question of whether the presence of these increased serum P levels on the day of hcg administration are associated with pregnancy rates (PRs) is a subject of much debate. At present, there is no consensus on whether P elevation is associated with the achievement of pregnancy. Several studies have denied the presence of such an association (7 15), othershaveconfirmed thepresenceofanegativeassociation between P elevation and PRs (16 20), and yet others have found a favorable effect on pregnancy outcome (21). Another question to address is the cause of the P rise during the follicular VOL. 97 NO. 6 / JUNE

2 ORIGINAL ARTICLE: ASSISTED REPRODUCTION phase of assisted reproduction cycles. It is more likely that the elevated P levels might be attributed to an excess number of follicles that each produces normal amounts of P (15). Yet in the late follicular phase, the secretion of P and the number of mature follicles correlate positively (22, 23). Therefore, it is reasonable to hypothesize that the P levels at the end of COH tend to be higher in patients with a good ovarian response (21). The raised peripheral concentrations of P in the late follicular phase are likely to influence the secretory changes of the endometrium, leading to impaired endometrial receptivity (24). On the other hand, patients who respond adequately to COH are more likely to produce better oocytes. Therefore, the consequences of elevated P level on IVF-ET outcome in patients with good response are likely to result from a balance between two antagonistic factors: the good embryo quality associated with a good ovarian response, and the impaired receptivity of the endometrium resulting from premature endometrial exposure to P (25). In low-ovarian-reserve infertile women that develop elevated P level on hcg day, their embryo quality does reduce PRs. This raises the question of whether the relationship between ongoing PRs and P level on hcg day found in poor responders is the result of endometrial receptivity, embryo quality, or both (26). Therefore, it seems necessary when investigating the topic of premature P elevation to differentiate the occurrence of this phenomenon according to different ovarian response. In the present study, we sought to investigate the relationship between serum P levels on the day of hcg administration and the probability of ongoing pregnancy categorized by the quality of the ovarian response to COH. MATERIALS AND METHODS Patients This was a noninterventional, retrospective, single-center cohort study of patients undergoing routine practice. To reflect the broad range of patients typically encountered in clinical practice, no inclusion/exclusion criteria were applied on baseline characteristics. Patients were treated at a single centre in the Tongji hospital between January 2002 and September A total of 11,055 patients undergoing IVF/ICSI treatment and a subgroup of 4,021 patients participating in an frozen-embryo transfer (FET) program were enrolled. All patients signed written informed consent. Institutional Review Board approval was not needed, because all women in the study underwent the routine long GnRHa IVF-ET clinical treatment performed in our unit and no additional intervention or sampling was performed. Protocol for COH Patients underwent COH with the use of a GnRH agonist long protocol. The pituitary suppression was achieved by subcutaneous injection of 0.1 mg triptorelin acetate (Decapeptyl; Ferring) daily starting in the midluteal phase of the preceding cycle, which was reduced to 0.05 mg once the downregulation was achieved. When complete pituitary desensitization was confirmed by a low plasma E 2 level of 30 pg/ml and an LH level of 2 miu/ml, ovarian stimulation was started with administration of IU/d recombinant FSH (Gonal-F; Serono) intramuscularly (IM). Recombinant hcg (250 mg; Ovidrel; Serono) was given to trigger ovulation when two leading follicles reached a mean diameter of 18 mm. Oocytes were retrieved transvaginally hours after hcg administration. Fewer than three embryos were transfered on day 2 or 3 after oocyte retrieval, and excess good-quality embryos were cryopreserved for subsequent FET cycles. The luteal phase was supported with 60 mg P injections IM from the day of oocyte retrieval. An ongoing pregnancy was defined as a pregnancy with a positive heartbeat by ultrasound after 12 weeks of gestation. Grouping of High, Intermediate, and Poor Ovarian Responders To get a first clinical impression of the results obtained, we categorized ovarian response into three arbitrary groups according to the number of oocytes retrieved: low ovarian response (%4 oocytes obtained), intermediate ovarian response (5 19 oocytes obtained), and high ovarian response (R20 oocytes obtained). We explored the relationship of serum P levels on the day of hcg administration and the ongoing PRs in different ovarian responses. Hormone Measurement Serum P and E 2 levels were measured on the day of hcg administration. Samples were tested with a microparticle enzyme immunoassay (Axsym System, Advia Centaur; Siemens), which had a sensitivity of 0.21 ng/ml. Intra- and interassay coefficients of variability were 7.2% and 5.7%, respectively, for P. The E 2 assay had a sensitivity of 7.0 pg/ ml, with intra- and interassay coefficients of variability of 11.3% and 5.0%, respectively. Statistical Analysis To avoid bias of the results by assuming that any relationship between serum P levels and ongoing PRs may be linear, patients were divided into eight distinct groups according to serum P levels on the day of hcg administration: <1.00, , , , , , , and R2.5 ng/ml (27). Ongoing PR was calculated for each P interval. Data were assessed for trend analysis with the use of a Mantel-Haenszel test. To identify the P threshold for a detrimental effect on cycle outcome, the odds ratio (OR) and 95% confidence interval (CI) of clinical PRs for each P interval, compared with the lowest P group, was calculated. Factors related to P elevation were assessed using a multivariate logistic regression analysis. Associations between demographic and clinical characteristics of the patients were assessed with the use of Student t test or Mann- Whitney U test for continuous variables and chi-square for categoric variables, as appropriate. RESULTS Patient Characteristics A total of 11,055 ovarian stimulation cycles (IVF; n ¼ 8,857; ICSI; n ¼ 2,198) and 4,021 FET treatment cycles were included 1322 VOL. 97 NO. 6 / JUNE 2012

3 Fertility and Sterility in the study. The average age of the participants was 30.5 years (range years). Clinical indications for IVF-ET included tubal-factor infertility (37%), male-factor infertility (26%), idiopathic infertility (15%), endometriosis (13%), and low ovarian reserve (9%). Overall Results The mean level of serum P on the day of hcg administration was ng/ml. When participants were analyzed according to the quality of the ovarian response, significantly higher serum P levels were observed with the high ovarian response group than with the intermediate and poor ovarian response groups ( , , and , respectively; P<.001). There were lowest P levels in the poor ovarian response group compared with the other groups (P<.001). Figure 1 shows the overall association between serum P levels and ongoing PRs in fresh IVF/ICSI cycles and FET cycles. There was a reduction in ongoing PRs with progressively greater concentrations of serum P in fresh IVF/ICSI cycles, although it does not appear to have a negative effect on ongoing PRs in FET cycles. On the contrary, the PRs seemed to be much better in FET cycles than in fresh cycles with greater concentrations of serum P. To analyze the association between variables involved in increased P levels, multivariate logistic regression was performed. The results showed that the number of oocytes retrieved, the total FSH dose, and the plasma E 2 values on the day of hcg administration were positively associated with increased P levels (Supplemental Table 1). FIGURE 1 High Ovarian Response Group Figure 2A shows the association between ongoing PRs and serum P levels in the high ovarian response group (n ¼ 2,023). Figure 2B shows the OR for ongoing PR in each of the serum P groups compared with the lowest P group. When serum P is in the interval >2.25 ng/ml, there are statistically significant reduced ongoing PRs. The serum P concentration of 2.25 ng/ml may represent the threshold concentration above which there is a negative effect of P on ongoing PR for high responders. Patients with low (<2.25 ng/ml) or high (R2.25 ng/ml) P levels were similar regarding age, basal FSH level, duration of infertility, amount of rfsh required for COH, duration of stimulation, plasma E 2 and P levels on the day of hcg administration, mean numbers of oocytes retrieved, mature oocytes, and embryos available, mean numbers of fertilized oocytes and cleavage embryos, fertilization rate, and cleavage rate (Supplemental Table 2). The results show that for the high ovarian response group, although the mean numbers of oocytes retrieved, mature oocytes, fertilized oocytes, and cleavage embryos were higher in the high P group than in the low P group, there were significantly poorer implantation rates (22% and 28%, respectively) and ongoing PRs (27.5% and 36.8%, respectively) in IVF/ICSI cycles, although the fertilization rates, cleavage rates, and ongoing PRs for FET cycles (56.2% and 57.6%, respectively) in patients with and without P elevation were similar. Intermediate Ovarian Response Group For the intermediate ovarian response group (n ¼ 8,205), Figure 3A shows the association between ongoing PRs and serum P levels. There was a reduction in ongoing PRs with progressively greater concentrations of serum P. Figure 3B shows the OR for ongoing PR for each of the serum P levels compared with the lowest P group. When serum P level is >1.75 ng/ml, there are statistically significantly reduced ongoing PRs. The serum P concentration of 1.75 ng/ml may represent the threshold concentration for intermediate ovarian response group. As in the high ovarian response group, COH and embryology data were compared in patients with low (<1.75 ng/ml) or high (R1.75 ng/ml) P levels (Supplemental Table 3). The results show that for the intermediate ovarian response group, although the mean numbers of oocytes retrieved and mature oocytes, the peak E 2 value, and the mean numbers of embryos available for ET, fertilized oocytes, and cleavage embryos were higher in the high P group than in the low P group, there were significantly poorer implantation rates (22% and 30%, respectively) and ongoing PRs in the high P group than in the low P group (29.3% and 39.3%, respectively), whereas the fertilization rates, cleavage rates, and ongoing PRs (39.1% and 36.8%, respectively) in FET cycles were not statistically different between the two groups. Relationship between serum P levels and ongoing pregnancy rates of fresh in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) and frozen embryo transfer (FET) cycles in the overall study population. Poor Ovarian Response Group In the poor ovarian response group (n ¼ 827), only very few patients (4.5%, 37/827) displayed a P level R2 ng/ml, so the intervals for P level R2 ng/ml ( , , and VOL. 97 NO. 6 / JUNE

4 ORIGINAL ARTICLE: ASSISTED REPRODUCTION FIGURE 2 As for the two previous groups, patient age, COH characteristics, and embryology data (Supplemental Table 4) were similar in patients with high (R1.5 ng/ml) and low (<1.5 ng/ml) P levels. Peak plasma E 2 values, the total FSH dose and duration were higher in the high P group than in the low P group, while the mean numbers of oocytes retrieved, mature oocytes, embryos available for ET, and fertilized oocytes and cleavage embryos were similar between the two groups. However, the ongoing PRs (10.6% and 20.8%, respectively) and implantation rates (10% and 17%, respectively) were lower in the high P group than in the low P group. In contrast to fresh cycles, elevated serum P levels did not significantly influence the ongoing PRs of FET cycles. (A) Relationship between ongoing pregnancy rates and serum P levels in the high ovarian response group. (B) Ongoing pregnancy rates according to serum P levels in the high ovarian response group. Data are expressed as odds ratio (95% confidence interval) for each of the serum P levels compared with the lowest P group (<1.0 ng/ ml). *P<.05 for comparison with the lowest P level interval. R 2.5 ng/ml) were merged. Figure 4A shows the association between ongoing PRs and serum P levels in the poor ovarian response group. There was a reduction in ongoing PRs when serum P was >1.5 ng/ml. Figure 4B shows the OR for ongoing PR for each of the serum P levels compared with the lowest P group. When serum P was >1.5 ng/ml, there were statistically significant reduced ongoing PRs. The serum P concentration of 1.5 ng/ml may therefore represent the threshold concentration for poor responders. DISCUSSION This retrospective study of more than 10,000 first IVF cycles revealed that elevated serum P levels on the day of hcg administration are associated with reduced implantation rates and ongoing PRs regardless of different ovarian responses. Different P threshold concentrations were determined according to the quality of the ovarian response by performing trend analysis: We propose that a serum P level of 1.5 ng/ml on the day of hcg administration can serve as the threshold for poor ovarian response, a serum P level of >1.75 ng/ml is associated with lower ongoing PRs for intermediate responders, and a P threshold of 2.25 ng/ml can be applied to high responders. These findings imply that increased P threshold concentration is associated with better ovarian responses. Our results are in contrast to the only meta-analysis performed to date, which found no relationship and dismissed the importance of raised follicular-phase P concentration on pregnancy outcomes (15). However, the value of that metaanalysis was thought to be limited because it defined an imprecise threshold for a detrimental serum P level, with most of the studies failing to demonstrate an association arbitrarily using a low threshold level of 0.9 ng/ml (28). Another point of difference between these studies is the variation in the statistical methods used to assess the specific cutoff value of circulating P:A some smaller studies used receiver operating characteristic (ROC) curve analysis to assess the impact of P levels on pregnancy outcome, although the area under the ROC curve may not be the most suitable test for this analysis, because the relationship between serum P levels and pregnancy outcomes is not linear (27, 29). The variation across these studies emphasizes the importance of using appropriate methodologic approaches (30). A trend analysis relating both variables would be of greater interest, which was available in a recent retrospective analysis of more than 4,000 IVF/ICSI ET cycles, the results showing that high serum P levels on the day of hcg administration was associated with a decreased PR (27). The pathogenesis of elevated P level in GnRHa cycles is more complicated and controversial. Because the presence of GnRHa assures the prevention of premature LH elevation in 95% 98% of patients (31, 32), it seems unsatisfactory to invariably define the increased preovulation LH levels as the sole pathogenic factor of premature luteinization. Recently, investigators have linked the appearance of 1324 VOL. 97 NO. 6 / JUNE 2012

5 Fertility and Sterility FIGURE 3 FIGURE 4 (A) Relationship between ongoing pregnancy rates and serum P levels in the intermediate ovarian response group. (B) Ongoing pregnancy rates according to serum P levels in the intermediate ovarian response group. Data are expressed as odds ratio (95% confidence interval) for each of the serum P levels compared with the lowest P group (<1.0 ng/ml). *P<.05 for comparison with the lowest P level interval. elevated P with multiple follicular development and increased ovarian steroidogenic activity occurring during COH (23, 27). According to the two-cell, two-gonadotropin theory of estrogen biosynthesis (33), FSH acts on granulose cells to promote conversion of cholesterol to P, which is passed to the thecal cells to be converted to androgens under the influence of LH. The androgens are then passed to the granulose cells to be converted to E 2. Differently from a single follicle in the natural cycle, during ovarian stimulation induced multiple follicle growth the P output to the periphery is magnified in accordance with the number of follicles and with the FSH drive, particularly if the FSH action is not balanced by LH (A) Relationship between ongoing pregnancy rates and increasing serum P levels in the poor ovarian response group. (B) Ongoing pregnancy rates according to serum P levels in the poor ovarian response group. Data are expressed as odds ratio (95% confidence interval) for each of the serum P levels compared with the lowest P group (<1.0 ng/ml). *P<.05 for comparison with the lowest P level interval. activity. Thus, the major components to the degree of P secretion from the ovaries were thought to be: the number of follicles; the degree of trophic stimulus (FSH drive to granulosa cells); and the degree of LH drive to thecal cells, which encourages conversion of P to androgens and estrogen (23). This hypothesis is supported by the results of a prospective trial showing that in COH, P peaks higher when FSH rather VOL. 97 NO. 6 / JUNE

6 ORIGINAL ARTICLE: ASSISTED REPRODUCTION than hmg is used (34). The result of our multivariate analysis showed that the number of oocytes retrieved, doses of FSH and serum E 2 levels on the day of hcg administration were the factors most related to the occurrence of serum P elevation, which also support the above mechanisms. In light of these observations, it would be better to take into account the ovarian response, rather than just the serum P level, when considering the reasons for this phenomenon (35). The results of the present study show that P elevation was associated with negative consequences on IVF outcome independently from whether it occurred in women whose COH yielded high, intermediate, or poor responses. The present series provided more cases to challenge the study by Fanchin et al. (25), which showed a modifying effect of ovarian response on the association between P elevation and the probability of pregnancy: elevated P level on hcg day adversely affected PR only in the poor responder group. In women with intermediate and high ovarian response, elevated P level on hcg day had no negative impact on IVF results. Again, however, the validity of that conclusion has been debated, because the association between serum P levels and PR was thought not to be identified by arbitrarily choosing a low threshold value of 0.9 ng/ml (27). Although a significant inverse relationship between serum P on the day of hcg and the success of IVF is established in many programs, the involved endocrinologic mechanism is unclear. It has been described in several studies that it may act as an ovarian event, with adverse effects on oocyte maturation, fertilization, or early cleavage (4, 5, 16, 19, 20). On the other hand, poorer embryo quality was not found in other studies (3, 9, 36, 37). These findings suggest that P elevation may influence the endometrium, leading to impaired endometrial receptivity. Melo et al. retrospectively analyzed 240 oocyte-donation cycles in which 120 women donated twice, with elevated P level in the first donation cycle and no P elevation in the following one. The result showed that P elevation did not have a negative impact on ongoing PRs in oocyte donation program (38), suggesting that the adverse effect of P production is not exerted at the level of the embryo. However, several clinical trials have been performed in which P supplementation for luteal phase support was started on the day of hcg administration, without any negative effect on PRs due to a deleterious effect on the endometrium (39 41). To discriminate the endometrial factor from the quality of the oocyte cohort, the clinical consequences of P elevation were analyzed within the context of different ovarian response. The results of our study show that although P elevation on the day of hcg was inversely associated with the probability of pregnancy, the numbers of total oocytes and mature oocytes retrieved were higher in the elevated P group. Moreover, it did not appear to have a negative effect on oocyte performance in terms of fertilization, cleavage rates, and ongoing PRs in FET cycles regardless of different ovarian responses, indicative of no detrimental effect of elevated P on oocyte/embryo quality. On the basis of the results that a significantly higher mean number of oocytes concurrent with an absence of detrimental effect on oocyte/embryo quality in the patients with elevated P suggests an alternative explanation: The raised concentrations of P results from multiple growing follicles are likely to influence endometrial development while having no influence on oocyte/embryo development. This can, in turn, lead to a change in the normal synchrony with the endometrium, when excessively disturbed; this asynchrony may reduce embryo implantation rates and PRs (24). In conclusion, the present study shows that P elevation leads to a significant decrease in implantation rates and ongoing PRs in all ovarian responses to COH. Increased P threshold concentration is associated with better ovarian responses. Though limited by the retrospective design, the negative association between P elevation and ongoing PRs and different P threshold concentration according to the quality of ovarian response proposed in this study could be used to optimize the treatment of different responders undergoing IVF/ICSI ET as a reference. For example, for patients with high ovarian response when the P concentration on the day of hcg administration is >2.25 ng/ml, it is suggested to freeze all the embryos and transfer in a subsequent FET cycle (15), because the FET result was much better than the fresh embryo transfer cycles. Whereas for intermediate responders, delaying ET by cryopreserving embryos might be advantageous with a P concentration >1.75 ng/ml. For poor responders, because there is a natural tendency to give higher doses of gonadotropins, which, as discussed above, were positively correlated with the occurrence of P elevation, it could be that less gonadotropin late in the follicular phase (42) or earlier hcg administration before P elevation (6) may be of benefit for patients with low ovarian reserve who have previously exhibited P levels >1.5 ng/ml. This assumption should be evaluated in more extensive studies. Acknowledgments: The authors acknowledge Dr. Dirk Geerts, Department of Pediatric Oncology/Hematology, Sophia Children's Hospital, Erasmus University Medical Center, Rotterdam, The Netherlands, for critical reading of the manuscript. REFERENCES 1. Smitz J, Ron-El R, Tarlatzis BC. The use of gonadotrophin releasing hormone agonists for in vitro fertilization and other assisted procreation techniques: experience from three centres. Hum Reprod 1992;7: Check JH, Chase JS, Nowroozi K, Dietterich CJ. Premature luteinization: treatment and incidence in natural cycles. Hum Reprod 1991;6: Hofmann GE, Bentzien F, Bergh PA, Garrisi GJ, Williams MC, Guzman I, et al. Premature luteinization in controlled ovarian hyperstimulation has no adverse effect on oocyte and embryo quality. Fertil Steril 1993;60: Bosch E, Valencia I, Escudero E, Crespo J, Simon C, Remohí J, et al. 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Implantation and P profiles in cycles with and without the use of gonadotropin releasing hormone agonist suppression. Hum Reprod 1990;5: Pal L, Jindal S, Witt BR, Santoro N. Less is more: increased gonadotropin use for ovarian stimulation adversely influences clinical pregnancy and live birth after in vitro fertilization. Fertil Steril 2008;89: VOL. 97 NO. 6 / JUNE

8 ORIGINAL ARTICLE: ASSISTED REPRODUCTION SUPPLEMENTAL TABLE 1 Multivariate analysis of factors related to P elevation. Parameter OR (95% CI) P value No. of oocytes ( ).0001 Total FSH dose ( ).001 E 2 on day of hcg administration ( ) e1 VOL. 97 NO. 6 / JUNE 2012

9 Fertility and Sterility SUPPLEMENTAL TABLE 2 Clinical features and cycle outcomes of ovarian stimulation between elevated and nonelevated P groups in patients with high ovarian response. Parameter Nonelevated P a Elevated P b P value No. of fresh cycles 1, Age (y) NS Basal FSH (IU/L) NS Duration of infertility (y) NS P value on hcg day (ng/ml) <.001 Peak E 2 value (pg/ml) 5,881 3,625 6,714 5, No. of rfsh vials NS rfsh duration (days) No. of oocytes retrieved <.001 No. of mature oocytes <.001 No. of fertilized oocytes No. of cleavage embryos No. of available embryos NS Fertilization rate (%) NS Cleavage rate (%) NS Implantation rate (%) <.001 Ongoing pregnancy rate (%) <.001 No. of FET cycles FET pregnancy rate (%) NS Note: Values are presented as mean SD. FET ¼ frozen embryo transfer; NS ¼ not significant. a P level <2.25 ng/ml. b P level R2.25 ng/ml. VOL. 97 NO. 6 / JUNE e2

10 ORIGINAL ARTICLE: ASSISTED REPRODUCTION SUPPLEMENTAL TABLE 3 Clinical features and cycle outcomes of ovarian stimulation between elevated and nonelevated P groups in patients with intermediate ovarian response. Parameter Nonelevated P a Elevated P b P value No. of fresh cycles 6,104 2,101 Age (y) NS Basal FSH (IU/L) NS Duration of infertility (y) NS P value on hcg day (ng/ml) <.001 Peak E 2 value (pg/ml) 3,535 1,447 4,620 2,762 <.001 No. of rfsh vials NS Duration of stimulation (d) <.001 No. of oocytes retrieved <.001 No. of mature oocytes <.001 No. of fertilized oocytes <.001 No. of cleavage embryos <.001 No. of available embryos <.001 Fertilization rate (%) NS Cleavage rates (%) NS Implantation rate (%) <.001 Ongoing pregnancy rate (%) <.001 Number of FET cycles 1, FET pregnancy rate (%) NS Note: Values are presented as mean SD. FET ¼ frozen embryo transfer; NS ¼ not significant. a P level <1.75 ng/ml. b P level R1.75 ng/ml e3 VOL. 97 NO. 6 / JUNE 2012

11 Fertility and Sterility SUPPLEMENTAL TABLE 4 Clinical features and cycle outcomes of ovarian stimulation between elevated and nonelevated P groups in patients with poor ovarian response. Parameter Nonelevated P a Elevated P b P value No. of fresh cycles Age (y) NS Basal FSH (IU/L) NS Duration of infertility (y) NS P value on hcg day (ng/ml) <.001 Peak E 2 value (pg/ml) 1,415 1,019 2,224 1,556 <.001 No. of rfsh vials rfsh duration (d) No. of oocytes retrieved NS No. of mature oocytes NS No. of fertilized oocytes NS No. of cleavage embryos NS Fertilization rate (%) NS Cleavage rate (%) NS No. of available embryos NS Implantation rate (%) Ongoing pregnancy rate (%) No. of FET cycles 27 9 FET pregnancy rate (%) 7 (2/27) 33.3 (3/9) NS Note: Values are presented as mean SD. FET ¼ frozen embryo transfer; NS ¼ not significant. a P level <1.50 ng/ml. b P level R1.50 ng/ml. VOL. 97 NO. 6 / JUNE e4