FOAD AZEM, JOSEPH B. LESSING, MIRA MALCOV, DALIT BEN-YOSEF, BENI ALMOG, & AMI AMIT

Gynecological Endocrinology, July 2008; 24(7): 368 372 ASSISTED REPRODUCTION Does high serum progesterone level on the day of human chorionic gonadotropin administration affect pregnancy rate after intracytoplasmic sperm injection and embryo transfer? FOAD AZEM, JOSEPH B. LESSING, MIRA MALCOV, DALIT BEN-YOSEF, BENI ALMOG, & AMI AMIT The Sara Racine IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (Received 28 May 2007; revised 10 January 2008; accepted 15 January 2008) Abstract Objective. The present study was conducted to evaluate the effect of serum progesterone (P) levels on the day of human chorionic gonadotropin (hcg) administration on embryo quality and pregnancy rate in intracytoplasmic sperm injection (ICSI) cycles. Design and setting. This was a retrospective analysis conducted in the in vitro fertilization (IVF) unit of a tertiary hospital. Patients. Two hundred and one patients who underwent a total of 280 IVF treatment cycles allocated to ICSI during routine IVF/embryo transfer treatment. Results. In cycles with elevated serum P, higher estradiol levels were noted (1915 pg/ml vs. 1256 pg/ml; p50.05), 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 significantly lower (16.4% vs. 27.6%, p ¼ 0.03). Conclusions. Our data demonstrate no deleterious effect of elevated P on embryo quality. However, high serum P adversely affects implantation and pregnancy rates. Keywords: Embryo quality, intracytoplasmic sperm injection, implantation, progesterone Introduction The effect of plasma progesterone (P) on pregnancy rates in in vitro fertilization (IVF) is a controversial issue [1]. Previously we and others reported that high P levels on the day of human chorionic gonadotropin (hcg) administration are associated with a decrease in pregnancy rate [1 3]. Conversely, other researchers have found no adverse effects of P on pregnancy rate [4,5]. The influence of P is related to adverse effects on endometrial receptivity, or unfavorable effects on oocyte or embryo quality [1,3]. Several studies demonstrated a deleterious effect of high levels of P on fertilization by increasing the tendency toward polyspermy. Furthermore, a higher fertilization rate was reported once P level was 50.9 ng/ml [6,7]. As sperm encounter with the oocyte is bypassed in intracytoplasmic sperm injection (ICSI), this will eliminate the effect of P on fertilization. Therefore the present retrospective study was conducted to examine the effect of elevated serum P on ICSI outcome. Materials and methods Patients Two hundred and one patients in our unit underwent a total of 280 IVF treatment cycles allocated to ICSI from April 2000 to December 2000. The indications for ICSI are shown in Table I. Ovulation induction Ovulation induction was performed using a routine protocol of gonadotropin-releasing hormone analog (buserelin acetate nasal spray, 900 mg/day) (D-Ser Correspondence: F. Azem, Sara Racine IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel. Tel: 972 3 6925604. Fax: 972 3 6925687. E-mail: azemf@tasmc.health.gov.il ISSN 0951-3590 print/issn 1473-0766 online ª 2008 Informa UK Ltd. DOI: 10.1080/09513590802196353

High P adversely affects ICSI outcome 369 Table I. Incidence of indications for intracytoplasmic sperm injection in the present study. Etiology Number of cycles Percentage Male factor only 194 69.3 Male and tubal factors 60 21.4 Unexplained infertility 23 8.1 Others 3 1.2 [TBU] 6-ethylamide-luteinizing hormone-releasing hormone, Suprefact 1 ; Hoechst AG, Frankfurt, Germany) and three ampoules of human menopausal gonadotropin (Pergonal 1 ; Teva Pharmaceutical Industries Ltd, Petah Tikva, Israel). When the follicles reached a mean diameter of 17 mm, 10 000 IU hcg (Chorigon 1 ; Teva Pharmaceutical Industries Ltd) was administered. Oocyte retrieval, using an ultrasound-guided transvaginal approach, was scheduled 34 36 h after hcg administration. Micromanipulation The cumulus oocyte complexes were isolated in IVF medium (Medi-Cult, Copenhagen, Denmark). Cumulus cells were removed with hyaluronidase (80 IU/ml, H-3757; Sigma, Rehovot, Israel) in flushing medium (Medi-Cult). Denuded oocytes were assessed for their specific meiotic stage according to the presence or absence of germinal vesicle and polar bodies, and metaphase II oocytes were prepared for injection. The ICSI procedure was performed as previously described [8]. Spermatozoa were assessed continuously for any indication of movement (i.e. twitching of head or tail) in the sperm medium droplet without polyvinylpyrrolidone (PVP). A single motile spermatozoon was aspirated from the separate sperm droplet into the injection pipette and then transferred to the 10% PVP droplet to separate it from attaching cells and debris. After the ICSI procedure, the oocytes were returned to the culture dish for further incubation, and were inspected under a stereomicroscope (Olympus; SZH, Tokyo, Japan) for survival and fertilization 16 20 h later. Embryo quality Embryo cleavage and quality were evaluated 46 48 h after ICSI. Embryo morphology was graded 1 4 according to the shape of the blastomeres and the amount of detached anuclear fragments: grade 1 ¼ excellent (embryos containing intact and symmetrical blastomeres with no anucleate fragments); grade 2 ¼ good (asymmetric cleavage or 520% embryo fragmentation); grade 3 ¼ fair (embryo fragmentation 20 50%); grade 4 ¼ poor (embryo fragmentation 4 50%) [9]. Clinical pregnancies were confirmed by sonographic demonstration of a gestational sac. Embryo transfer (ET) was performed 48 72 h after retrieval. A maximum of four embryos were transferred and the remaining ones were cryopreserved. A serum b-hcg pregnancy test was performed 12 days after ET. In the event of a positive result, vaginal ultrasonography was carried out 3 weeks later to determine the viability and number of gestational sacs. Progesterone assay Serum P was determined for three consecutive days before and on the day of hcg administration. Serum P was measured using ligand-labeled competitive chemiluminescent immunoassay (Diagnostic Products Corporation, Los Angeles, CA, USA). The intra- and inter-assay coefficient of variance for P at 0.81 ng/ml and 0.93 ng/ml (conversion factor to SI units, 3.185) was 16%. Statistical analysis An elevated P level was arbitrarily defined as 0.9 ng/ ml; this cut-off facilitated comparison with other reported data. Comparisons were made by Student s t test, w 2 analysis and Pearson s correlation coefficient test where applicable; p50.05 was considered statistically significant. Results Patients were divided into two groups according to the level of P on day of hcg administration. Group A comprised 92 (33%) cycles in which the P level was 0.9 ng/ml and group B comprised 188 (67%) cycles with P levels 4 0.9 ng/ml. Mean age was significantly lower in group B than in group A (31.3 years vs. 33.2 years; p ¼ 0.009). In cycles with elevated P levels, higher estradiol (E 2 ) levels were observed (1915 pg/ml vs. 1256 pg/ ml; p50.05), more oocytes were retrieved (12.8 vs. 8.7; p50.05), more oocytes were manipulated (9.4 vs. 6.9; p50.05), and more embryos were transferred (3.8 vs.3.4; p ¼ 0.02) (Table II). No differences were found between the two groups in the etiology of infertility, the protocol of controlled ovarian stimulation, fertilization rate (65% vs. 65.8%; p ¼ 0.78) or embryo quality (grade 1 and 2 embryos: 68.5% vs. 74.0%; p ¼ 0.27). In cycles with lower P levels, the clinical pregnancy rate was significantly higher (27.6% vs. 16.4%; p ¼ 0.03). Furthermore we found an inverse relationship between P level and pregnancy rate. Figure 1 shows the correlation between pregnancy rate per

370 F. Azem et al. Cycle characteristics Table II. Cycle characteristics of the study groups. Group A Group B (P 0.9 ng/ml) (P 4 0.9 ng/ml) p Value No. of embryo transfer cycles 92 188 Age (years) 33.2 + 3.6 31.3 + 3.4 0.009 Duration of infertility (years) 7.3 + 3.3 6.3 + 2.9 0.1 Peak E 2 (pg/ml) 1256 + 324 1915 + 647 50.05 No. of oocytes 8.7 + 4.6 12.8 + 6.5 50.05 No. of MII oocytes 6.9 + 2.9 9.4 + 3.7 50.05 Fertilization rate (%) 65.8 65 0.8 Grades 1-2 embryos (%) 74 68.5 0.27 No. of transferred embryos 3.4 + 1.2 3.8 + 1.2 0.02 Clinical pregnancy rate (%) 27.6 16.4 0.03 P, progesterone; E 2, estradiol; MII, metaphase II; data are presented as mean + standard deviation or %. Figure 1. Correlation between pregnancy rate per embryo transfer (PR/ET) and plasma progesterone (P) level on the day of human chorionic gonadotropin administration. embryo transfer and level of plasma P. Pregnancy rate consistently declined with the elevation of plasma P. No pregnancy was achieved in cycles with serum P level 4 4 ng/ml on the day of hcg administration. Contrary to our postulation we found no statistically significant difference in pregnancy rate in patients 35 years old (14.7%), patients aged 4 35 540 years (26.7%) and those 40 years old (20.0%; p ¼ 0.083). Plasma levels of P and E 2 tended to decline with age (p ¼ 0.053). Table III shows that 72.4% of patients aged 35 years had P levels 4 0.9 ng/ml, compared with 62.2% and 54.3% in patients aged 4 35 540 years or 40 years, respectively. Discussion Controversy continues to exist regarding the effect of elevated P on the day of hcg administration and IVF/ET and ICSI/ET outcome. Schoolcraft and colleagues [10] were the first to report low pregnancy rates in women with P40.5 ng/ml. These findings were subsequently supported by Mio and associates [6], who reported low fertilization rate, low rate of normal-morphology embryos and low pregnancy rate in oocytes exposed to P levels 4 1 ng/ml, compared with oocytes exposed to P levels 50.9 ng/ml. Furthermore, Fanhin and co-workers [2] reported low fertilization rate with P levels 4 0.9 ng/ml, but found no significant difference in fertilization rate, percentage of mature oocytes or rate of cleaved embryos. Hofmann and collaborators [11] found that oocytes exposed to different P levels, which were donated to women with comparable P levels, yielded no significant difference in pregnancy rate. These authors concluded that a high P level affects endometrial receptivity, rather than oocyte or embryo quality. Silverberg s group [1], who found similar pregnancy rates after freezing and thawing of embryos exposed to different levels of P in plasma, further supported this assumption. Previously our group reported that P levels 4 1.9 ng/ml resulted in lower pregnancy rate for the donors and significantly lower pregnancy rate for the recipients [3]. Because the endometria in the recipients were prepared uniformly, the authors concluded that this was the result of endometrial effects of P on oocyte or embryo quality. Conversely, the results of the present study demonstrated that high levels of P, prior to hcg administration, adversely affected the pregnancy rate in ICSI cycles. However, the embryo quality, as determined by embryo grading, appeared to be unaffected by elevated serum P levels. In addition, we found a linear decline in pregnancy rate concomitantly with rising P level, and with P44 ng/ml no pregnancy was attained. These findings are in contradiction to those of other authors [4,5] that showed no adverse effect of high serum P on the day of hcg administration on implantation rates after ICSI/ET. However, these authors did not examine the effect of high P on embryo quality. Theoretically, high levels of P may affect IVF/ET outcome at different stages: ovulation and oocyte

High P adversely affects ICSI outcome 371 Table III. Distribution (%) of progesterone (P) levels in correlation to age. P level 35 years old 435 540 years old 40 years old 50.9 ng/ml 27.6 37.8 45.7 0.9 ng/ml 72.4 62.2 54.3 quality, fertilization process, embryo quality or endometrial receptivity. Nagai and colleagues [12] showed that pig oocytes exposed to high levels of P demonstrated a significantly lower meiosis rate compared with oocytes cultured in low P concentration. Furthermore, Franchimont and associates [13] demonstrated that follicular-fluid P concentration was significantly lower in follicles that resulted in pregnancy. However, these findings were not supported by other studies [1,7,14] which demonstrated that although the number of retrieved oocytes was significantly higher in cycles with high P level, the percentages of atretic and mature oocytes were comparable. Similarly, in our study, we found that the number of retrieved oocytes was higher in cycles with raised P levels than in those with low P levels. Several studies have demonstrated a deleterious effect of P on fertilization. Hartshorne [15] confirmed high polyspermic fertilization in oocytes retrieved from follicles with high P levels. Givens [7] and Mio [6] and co-workers found a higher fertilization rate when P levels were 51 ng/ml and 0.9 ng/ml, respectively. No statistically significant difference in fertilization rate was found between our two study groups. In a previous study, Yovel and collaborators [3] postulated that high P levels may depreciate the quality of embryos. In the current study, we found no difference in embryo quality in the study groups. These findings confirm those of other authors [1,11,16] who found no deleterious effect of P on embryo quality. We examined embryo morphology around the fourto six-cell stage. However, high P levels may alter embryo morphology in later stages of cleavage [9]. The pregnancy rate was significantly lower in the group with high P levels, despite the fact that more ETs were performed in these patients. Furthermore, the average age of patients in this group was significantly lower than that of patients with low P levels. Based on this finding we conclude that high P levels adversely affect endometrial receptivity. It was suggested that elevated P levels might affect the synchrony of implantation processes, namely apposition and adhesion. In mice, P administration caused closure of the uterus with only primary apposition, and estrogen supplementation was essential for successful implantation [17,18]. In our study, we found lower E 2 and P levels and fewer oocytes and embryos in elderly patients compared with younger women. However, embryo quality, determined by morphological grading, was similar. Because high P levels were the only significant parameter, we concluded that these levels mask the effect of age and higher number of transferred embryos. In conclusion, although we found no effect of high levels of P on embryo morphology, the pregnancy rates were significantly lower in women with high P levels. A prospective, randomized study is warranted in order to clarify this controversial issue. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Silverberg KM, Burns WN, Olive DL, Riehl RM, Schenken RS. Serum progesterone levels predict success of in vitro fertilization/embryo transfer in patients stimulated with leuprolide acetate and human menopausal gonadotropins. J Clin Endocrinol Metab 1991;73:797 803. 2. Fanhin R, de Ziegler D, Taieb J, Hazout A, Frydman R. Premature elevation of plasma progesterone alters pregnancy rates of in vitro fertilization and embryo transfer. Fertil Steril 1993;59:1090 1094. 3. Yovel I, Yaron Y, Amit A, Peyser MA, David MP, Kogosowski A, Lessing JB. High progesterone levels adversely affect embryo quality and pregnancy rates in in vitro fertilization and oocyte donation programs. Fertil Steril 1995;64:128 131. 4. Martinez F, Coroleu B, Clua E, Tur R, Buxaderas R, Parera N, Barri PN, Balasch J. Serum progesterone concentrations on the day of HCG administration cannot predict pregnancy in assisted reproduction cycles. Reprod Biomed Online 2004;8:183 190. 5. Venetis CA, Kolibianakis EM, Papanikolaou E, Bontis J, Devroey P, Tarlatzis BC. Is progesterone elevation on the day of human chorionic gonadotrophin administration associated with the probability of pregnancy in in vitro fertilization? A systematic review and meta-analysis. Hum Reprod Update 2007;13:343 355. 6. Mio Y, Sekijima A, Iwabe T, Onohara Y, Harada T, Terakawa N. Subtle rise in serum progesterone during the follicular phase as a predictor of the outcome of in vitro fertilization. Fertil Steril 1992;58:159 166. 7. Givens CR, Schriock ED, Dandekar PV, Martin MC. Elevated serum progesterone levels on the day of human chorionic gonadotropin administration do not predict outcome in assisted reproduction cycles. Fertil Steril 1994;62:1011 1017. 8. Ben-Yosef D, Yogev L, Hauser R, Yavetz H, Azem F, Yovel I, Lessing JB, Amit A. Testicular sperm retrieval and cryopreservation prior to initiating ovarian stimulation as the first line approach in patients with non-obstructive azoospermia. Hum Reprod 1999;14:1794 1801. 9. Kondo I, Suganuma N. Embryo quality in failed in vitro fertilization patients. Hum Reprod 1994;4:80 84. 10. Schoolcraft W, Sinton E, Schlenker T, Huynh D, Hamilton F, Meldrum DR. Lower pregnancy rate with premature luteinization during pituitary suppression with leuprolide acetate. Fertil Steril 1991;55:563 566.

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