Ovarian stimulation protocols based on follicle-stimulating hormone glycosylation pattern: impact on oocyte quality and clinical outcome

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1 Ovarian stimulation protocols based on follicle-stimulating hormone glycosylation pattern: impact on oocyte quality and clinical outcome Helmy Selman, Ph.D., a Arianna Pacchiarotti, M.D., a and Imam El-Danasouri, Ph.D. b a Praxi Provita, Rome, b Istituto Europeo Medicina della Riproduzione Abruzzese (I.E.M.A.), Chieti, Italy Objective: To evaluate the impact of follicle-stimulating hormone (FSH) with different glycosylation patterns on oocyte quality and clinical outcomes in an in vitro fertilization (IVF) treatment program. Design: Prospective randomized, open-label, clinical study. Setting: Assisted reproduction center. Patient(s): One hundred eighty-eight infertile couples undergoing assisted fertilization treatment. Intervention(s): All participants underwent a standard down-regulation with a gonadotropin-releasing hormone (GnRH) analogue. The patients were randomized into three groups: 63 patients received combined sequential acidic and less-acidic FSH, 65 patients received only less-acidic FSH (recombinant FSH), and 60 received only acidic FSH (human-derived FSH). Main Outcome Measure(s): Number of mature metaphase II oocytes, embryo quality, clinical pregnancy rates, and implantation rates. Result(s): The pregnancy and implantation rates were statistically significantly lower in the less-acidic recombinant FSH alone group than in the combined sequential acidic /less-acidic recombinant FSH, and acidic alone groups (33.3%, 43.5%, 39% and 17.3%, 24.5%, 20.4%, respectively). Metaphase II oocytes and grade 1 embryos were statistically significantly higher in favor of the combined sequential acidic /less-acidic recombinant FSH group compared with the less-acidic recombinant FSH alone group. Conclusion(s): The glycosylation patterns of the two types of FSH implemented for ovarian stimulation have different impacts on oocyte quality and clinical outcome. A sequential combined protocol using both acidic and lessacidic FSH for ovarian stimulation improves oocyte maturity and embryo cleavage, and increases pregnancy and implantation rates. (Fertil Steril Ò 2009;-:- -. Ó2009 by American Society for Reproductive Medicine.) Key Words: Acidic human FSH, embryo, FSH glycosylation, less acidic recombinant FSH, oocyte, ovarian stimulation Received June 20, 2009; revised September 19, 2009; accepted October 5, H.S. has nothing to disclose. A.P. has nothing to disclose. I.E. has nothing to disclose. Reprint requests: Helmy Selman, Ph.D., Praxi Provata Centro Fertilita, Via Magnagrecia, 117, Rome, Italy (FAX: þ ; helmy. selman@yahoo.com). Ovarian stimulation is an integral procedure in assisted reproduction treatment. It is achieved by the administration of exogenous gonadotropins to increase follicular recruitment and oocyte yield. At present, two follicle-stimulating hormone (FSH) preparations are commercially available for controlled ovarian hyperstimulation: human-derived FSH () and recombinant FSH. Human urinary FSH contains a higher proportion of acidic isoforms whereas recombinant FSH contains a higher proportion of less-acidic isoforms (1, 2). Less-acidic isoforms exhibit high in vitro bioactivity, but they have a faster clearance and thus a shorter circulatory half-life than the acidic FSH isoforms (3, 4). The slow clearance of the acidic isoforms results in more estrogenic follicles and follicular maturation and estradiol secretion (5). Clinical trials have shown that recombinant FSH is effective in terms of the number of oocytes retrieved, the number of embryos obtained, and the total gonadotropin dose needed, without increasing the risk of inducing ovarian hyperstimulation syndrome (OHSS) (6 8). Recently, the efficacy of recombinant FSH was compared with in terms of oocyte and embryo quality, and reported results were highly in favor of. Some investigators have attributed the difference to the presence of LH activity in the preparation, which has a positive effect on oocyte maturation and embryo quality (9 11), while others have assumed that the difference between recombinant FSH and may reside in the nature of FSH isoform activities (5, 11). Gonadotropin isoforms influence a variety of biological activities, including cellular growth and development, steroidogenesis, and protein synthesis (12 14). Additionally, follicle development patterns and oocyte quality are strongly influenced by the FSH glycoform range, and the requirement of the follicle may shift during progress through different stages of development (15). Several studies have documented the occurrence of significant changes in FSH heterogeneity during certain physiologic conditions, including puberty and the menstrual cycle (16 20). Acidic FSH isoforms are produced during the follicular and luteal phases when the estradiol level is low whereas less-acidic FSH isoforms are produced during the midcycle (i.e., when the estradiol level is high). This shift toward the production and secretion of less-acidic/sialylated FSH molecules in the midcycle and the preovulatory phase of the cycle may be an important mechanism to regulate the intensity of the FSH stimulus during the final steps of follicular maturation (21). Accordingly, we evaluated a combined sequential protocol using acidic and less-acidic FSH for ovarian stimulation compared with acidic or less-acidic FSH alone in patients undergoing in vitro fertilization (IVF) treatment. We evaluated the efficiency and efficacy of /09/$36.00 Fertility and Sterility â Vol. -, No. -, doi: /j.fertnstert Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 the three different stimulation protocols on oocyte and embryo quality and on pregnancy and implantation rates. MATERIALS AND METHODS In a prospective, open, randomized study, a total of 188 infertile couples undergoing their first IVF treatment were enrolled as participants from January 2008 to February The women, aged 27 to 38 years, were included if they fulfilled the following criteria: [1] infertility attributable to tubal factor, male factor, or idiopathic infertility; [2] serum hormonal profile within the normal range (FSH and LH < 12 miu/ml, estradiol <50 pg/ml, and prolactin <30 ng/ml); [3] regular ovulatory menstrual cycles; [4] presence of a normal uterine cavity; [5] body mass index (BMI) of 20 to 26 kg/m 2, and [6] first IVF treatment. Randomization was performed using a computer-generated random assignment schedule for each patient. Sealed and numbered envelopes were used to conceal the treatment allocation until randomization. The randomization took place after the confirmation of down-regulation and immediately before gonadotropin administration to minimize postrandomization withdrawals. All patients were counseled about the nature of the study and gave their written informed consent for their participation in the randomization procedure. Participating patients were registered in our local ethics committee register which approved the study. Only first-time intracytoplasmic sperm injection (ICSI) patients who satisfied the inclusion criteria were enrolled in the study to limit the heterogeneity of the patients and to minimize any confounding variables that may affect the results. The primary end points were as follows: oocyte maturity, embryo quality, and clinical pregnancy and implantation rates. The secondary end points were the total dose of FSH administered, total number of days of stimulation, serum estradiol levels and endometrial thickness on the day of human chorionic gonadotropin (hcg) administration, fertilization rate, embryo cleavage rate, cancellation rate, and incidence of moderate or severe OHSS. All end points except the cancellation rate and the incidence of OHSS were analyzed statistically. The participating patients underwent a standard down-regulation protocol with a gonadotropin-releasing hormone (GnRH) analogue hormone (triptorelin, Decapeptyl; Ipsen, Milan, Italy) at 0.1 mg/day on day 21 of their cycle. All patients were given a fixed dose of FSH from day 2 of their cycle until hcg administration. The patients were randomized into three groups: group A(n ¼ 63) received 225 IU of acidic (Fostimon; IBSA, Geneva, Switzerland) for the first 6 days starting from day 2 of the cycle and followed by 225 IU of less-acidic recombinant FSH (Gonal-F; Serono, Rome, Italy) until hcg administration; group B (n ¼ 65) received 225 IU of less-acidic recombinant FSH alone from day 2 of the cycle until hcg administration; and group C (n ¼ 60) received 225 IU of acidic (Fig. 1). The FSH dose was adjusted when necessary according to the follicular size and estradiol level. Fostimon is enriched with more acidic isoforms (isoelectric point [PI] range: ) whereas Gonal-F contains a high proportion of more basic isoforms (PI range: ) (2, 22). Patients with an excessive response to gonadotropins were counseled about the risk for OHSS and were advised to interrupt the stimulation cycle or to undergo oocyte retrieval with cryopreservation of the resultant embryos for replacement in the subsequent cycle. Patients were considered to be at high risk for developing OHSS if they had serum estradiol levels >4500 pg/ml on the day of hcg administration and >15 follicles ranging from intermediate size (<15 mm) to large size (>16 mm). Final oocyte maturation was triggered by the administration of 10,000 IU of hcg (Gonasi HP; IBSA). Retrieved oocytes were denuded immediately after retrieval and were assessed for their maturity. In observance of the current law in Italy, only three oocytes were then inseminated by ICSI, and surplus metaphase II (MII) oocytes were vitrified. The resultant embryos were scored according to established criteria (23, 24). Statistical analysis was performed using the JMP software (version 4.0.4; SAS, Inc., Cary, NC). For a desired statistical power of 90% based on an a level of 0.05, confidence intervals (CI) of 95%, and anticipated effective size (Cohen s D) of 0,5 (medium size), the minimum total sample size required according to two-tailed hypothesis was 174 patients at least 58 evaluable patients per group. The parameters were compared by use of an FIGURE 1 Scheme of stimulation protocols. Group A, n=63 Group B, n=65 Group C, n=60 Standard long down-regulation Stimulation FSH dose: 225 IU Day 6 Triggering hcg IU analysis of variance (ANOVA) two-way test to analyze continuous variables, including primary and secondary outcome parameters. P%05 was considered statistically significant. The data were analyzed using the two-tailed Student s t-test for independent data, Fisher s exact test, and a two by two table between groups where appropriate. All analyses were adjusted for age stratum in line with the study design. Correction for multiple comparison analysis was performed using either Bonferroni s or Sidak s adjustment methods. RESULTS Four patients cycles were canceled because of excessive ovarian response leading to high risk for OHSS (one woman in group A, two women in group B, and one in group C). No patients had poor ovarian response to gonadotropin treatment. Of the 188 studied patients, 184 underwent oocyte retrieval: 62 patients in group A, 63 in group B, and 59 in group C. As shown in Table 1, the three groups were comparable for demographic data and total FSH dose. There was no statistically significant difference observed among the three groups regarding the mean number of oocytes retrieved per patient. With respect to oocyte maturation, a statistically significantly higher proportion (P<.004) of MII oocytes was observed in group A compared with group B (64.1% vs. 45.5%), whereas no statistically significant differences were found between groups A and C (64.1% vs. 57.2%) or between groups B and C (45.5% vs 57.2%). A statistically significantly lower proportion (P<.001) of immature (GV) oocytes was found in groups A and C compared with group B (9.3% and 12.5% vs. 25.2%). Statistically significant differences (P<.003) were also found in favor of group A with respect to group B in terms of grade 1 embryos (55.2% vs. 39.3%) though no differences were detected between groups B and C and groups A and C regarding grade 1 embryos (39.3% vs. 49.2% and 55.2% vs. 49.2%, respectively). The number of grade 2 embryos was statistically significantly higher in group B. Statistically significantly higher implantation (P<.008; 24.5%, 20.4%, and 17.3%, respectively) and pregnancy (P<.009; 43.5%, 39%, 33.3%, respectively) rates were observed in favor of groups A and C compared with group B. No statistically significant differences were observed between groups A and C in terms of pregnancy or implantation rates (Table 2). DISCUSSION Although improved results and important innovations have occurred in assisted reproduction, the rate of pregnancy per retrieved oocyte 2 Selman et al. FSH glycosylation pattern and oocyte quality Vol. -, No. -,

3 TABLE 1 Demographic data and stimulation outcomes. /rfsh Group A rfsh Group B Group C P value Patients (n) Mean age (years) SD Mean BMI SD Mean duration of sterility (years) SD Cause of infertility Primary, % (n) 71.4 (45) 71.3 (46) 71.7 (43).321 Tubal factor, % (n) 42.8 (27) 43.0 (28) 43.4 (26).154 Male factor, % (n) 36.5 (23) 36.9 (24) 36.6 (22).469 Unexplained, % (n) 6.4 (4) 6.2 (4) 6.7 (4).443 Other 14.2 (9) 13.8 (9) 13.3 (8).411 Duration of stimulation (days) Estradiol level on hcg day (pg/ml) Total FSH dose Endometrial thickness on hcg day (mm) Notes: No statistically significant differences observed between the groups. BMI, body mass index; hcg, human chorionic gonadotropin;, human-derived follicle-stimulating hormone; SD, standard deviation; rfsh, recombinant follicle-stimulating hormone. remains far too low. A major limiting factor in assisted reproduction success rates is oocyte quality. In stimulated cycles, the achievement of oocytes with the proper maturation state or right development remains a difficult issue. The oocyte acquires its nuclear and cytoplasmic competence during folliculogenesis; consequently, controlled ovarian hyperstimulation or collection of immature oocytes for in vitro maturation perturbs this process, which may result in reduced developmental competence of oocytes. Exogenous ovarian stimulation increases oocyte yield but may compromise the developmental competence of the oocytes in stimulated cycles (25). Gonadotropin stimulation results in a modified steroid profile, thus altering the microenvironment of the developing follicles and their enclosed oocytes (26). There is some evidence that estradiol plays a key role in oocyte maturation (27 29). Tesarik and Mendoza (30, 31) reported that estradiol exerts a beneficial effect on cytoplasmic maturation via a nongenomic calcium-mediated mechanism, which contributes to oocyte capacitation for fertilization and early postfertilization development. Moreover, profound suppression of LH has been shown to be associated with a reduced cohort of embryos and a reduced estradiol/oocyte ratio (32, 33), and some investigators have suggested that, when using recombinant FSH only, it may be clinically beneficial to add LH in the late follicular phase or to further reduce the dose of GnRH analogue (34, 35). Additionally, oocyte maturity and development may be linked to the nature of FSH isoforms used for ovarian stimulation. Because of their structural differences, FSH isoforms differ in their ability to bind to the target cell receptors surviving in the circulation and to induce biological responses in vivo and in vitro (36 40). Evident differences between recombinant and urinary FSH have been recognized: recombinant FSH contains a higher proportion of less-acidic isoforms, whereas human urinary FSH contains a higher proportion of acidic forms. This difference is reflected in the biological bioactivity, rate of clearance, and biological function of these two different FSH preparations. It has been suggested that the less-acidic isoforms have a faster circulatory clearance and thus a shorter circulatory half-life (3) than the acidic isoforms (41, 42). However, a more recent study has shown that the slow clearance of the acidic isoform results in better follicular maturation and estradiol secretion than the less-acidic isoform produces (5). Additionally, it has been reported that the antral follicle threshold dose and the maximal tolerated dose are highly variable with different sources of FSH, which appears to be related to the combination of isoforms (43). The recombinant, less-acidic FSH isoforms fraction has been shown to induce antral follicles development in vitro at lower doses than pituitary FSH (44, 45); however, increasing doses of the less-acidic fraction coupled with a longer time period of follicle culturing may produce a more detrimental effect on embryo production than the higher acidic fraction. The combination of acidic and less-acidic isoform fractions in unfractionated FSH seems to provide protection against the negative effects of overdosing (15, 43). Obviously, these differences in the effect of FSH isoforms on follicular development patterns strongly suggest that oocyte development is also likely to be influenced, that normal follicle development and ultimately normal oocyte function depend on an appropriate balance of sequential differentiation, and that this balance is strongly influenced by FSH isoform distribution (15). A progressive shift toward more basic FSH isoforms occurs in vivo in the late follicular and periovolatory phase (20, 46), which may have important modulating effects on follicle and oocyte development; therefore, more attention should be paid to isoform distribution of exogenously applied gonadotropins used for ovarian stimulation. In our study, we attempted ovarian stimulation based on the natural menstrual cycle, starting with acidic human FSH then following it with the less-acidic recombinant FSH in a sequential protocol. Our results show that, although the number of retrieved oocytes did not statistically significantly differ among the stimulation protocols, a statistically significantly higher proportion of mature oocytes and grade 1 embryos occurred in the sequential protocol compared with the protocol using recombinant FSH alone. The sequential protocol did not statistically significantly differ from the protocol with human FSH alone. A statistically significantly higher proportion of immature oocytes was found in the recombinant FSH protocol compared with the other two protocols. Statistically significantly higher Fertility and Sterility â 3

4 TABLE 2 Embryologic characteristics and clinical outcomes. /rfsh Group A rfsh Group B Group C P value No. of patients undergoing egg retrieval No. of patients undergoing embryo transfer Mean no. of retrieved oocytes SD Mature MII oocytes, % 64.1 a 45.5 b 57.2 a.004 Mature MI oocytes, % Immature GV oocytes, % c Mean no. of embryos transferred/patient SD Grade 1 embryos, % 55.2 a 39.3 b 49.2 a.003 Grade 2 embryos, % c Grade 3 embryos, % Grade 4 embryos, % Pregnancy rate, % (n) 43.5 (27) a 33.3 (21) d 39 (23) a.009 Implantation rate, % d Abortion rate, % (n) 14.8 (4) 14.3 (3) 13 (3).776 Notes: GV, germinal vesicle;, human-derived follicle-stimulating hormone; MI, MII, metaphase I and II; rfsh, recombinant follicle-stimulating hormone; SD, standard deviation. a No statistically significant differences found between groups A and C. b Statistically significantly lower proportion of MII oocytes and grade 1 embryos in group B compared with group C. c Statistically significantly higher proportion of immature oocytes and grade 2 embryos in group B compared with groups A and C. d Statistically significantly lower pregnancy and implantation rates in group B compared with groups A and C. pregnancy and implantation rates were observed in the sequential protocol compared with the protocol with recombinant FSH alone; no statistically significant differences were found between the sequential protocol and that of human FSH alone, although the mean number of replaced embryos was similar in all groups. The miscarriage rates were also similar in all groups. These results support previous studies which have demonstrated that stimulation with the sequential protocol is highly efficacious in terms of oocyte quality, embryo quality, and pregnancy and implantation rates compared with stimulation with less-acidic recombinant FSH alone (47, 48). No statistically significant differences could be shown between the sequential protocol and the protocol with human FSH alone, although the trend was higher in favor of the sequential protocol. The higher proportion of mature oocytes and good quality embryos resulting from stimulation with the sequential protocol may reflect the positive effect of acidic FSH on oocyte follicular growth during recruitment and during the follicular growing phase. This observation might also be explained by the fact that the sequential protocol may mimic the physiologic cycle, where the more acidic FSH isoforms prevail during the follicular phase of the menstrual cycle and then shift to less-acidic FSH isoforms in the midcycle (i.e., periovulatory) phase. The shift toward the production and secretion of less-acidic/sialylated FSH molecules during this cycle phase may be an important mechanism for regulating the intensity of the FSH stimulus during the final steps of follicular maturation. Our results show that ovarian stimulation seems to be affected by FSH glycosylation patterns. Using a combination of acidic and less-acidic FSH appears to be a highly efficient protocol for ovarian stimulation as it significantly improves oocyte maturity and embryo quality as well as clinical outcome. However, additional investigation on a larger number of patients is needed to further characterize the impact of different FSH isoforms on oocyte follicular development and oocyte maturation, and to further assess the resulting implications for ovarian stimulation regimens. REFERENCES 1. Lambert A, Rodgers M, Mitchell R, Wood AM, Wardle C, Hilton B, et al. In-vitro biopotency and glycoform distribution of recombinant human follicle stimulating hormone (Org 32489), Metrodin and Metrodin-HP. Hum Reprod 1995;10: Lispi M, Bassett R, Crisci C, Mancinelli M, Martelli F, Ceccarelli D, et al. Comparative assessment of the consistency and quality of a highly purified FSH extracted from urine (urofollitropin) and a recombinant human FSH (follitropin a). Reprod Biomed Online 2006;13: Antonio MD, Borrelli F, Datola A, Bucci R, Mascia M, Polletta P, et al. Biological characterization of recombinant human follicle stimulating hormone isoforms. Hum Reprod 1999;14: Vitt UA, Kloosterboer HJ, Rose UM, Mulders JW, Kiesel PS, Bete S, et al. Isoforms of human recombinant follicle-stimulating hormone: comparison of effects on murine follicle development in vitro. Biol Reprod 1998;59: West CR, Carlson NE, Lee JS, McNeilly AS, Sharma TP, Ye W, et al. Acidic mix of FSH isoforms are better facilitators of ovarian follicular maturation and E 2 production than the less acidic. Endocrinology 2002;143: Daya S. Updated meta-analysis of recombinant follicle-stimulating hormone (FSH) versus urinary FSH for ovarian stimulation in assisted reproduction. Fertil Steril 2002;77: Al-Inany H, Aboulghar M, Mansour R, Gamal Serour G. Meta-analysis of recombinant versus urinary-derived FSH: an update. Hum Reprod 2003;18: Bergh C, Howles CM, Borg K, Hamberger L, Josefsson B, Nilsson L, et al. Recombinant human follicle stimulating hormone (, Gonal-F) vs. highly purified urinary FSH (Metrodin highly purified): results of a randomized comparative study in women undergoing assisted reproductive techniques. Hum Reprod 1997;12: Ng EHY, Lau EYL, Yeung WSBY, Ho PC. HMG is as good as recombinant human FSH in terms of oocyte and embryo quality: a prospective randomized trial. Hum Reprod 2001;2: Strehler E, Abt M, El-Danasouri I, De Santo M, Sterzik K. Impact of recombinant follicle-stimulating hormone and human menopausal gonadotrophins on 4 Selman et al. FSH glycosylation pattern and oocyte quality Vol. -, No. -,

5 in vitro fertilization outcome. Fertil Steril 2001;75: Selman HA, De Santo M, Sterzik K, Coccia E, El Danasouri I. Effect of highly purified urinary folliclestimulating hormone on oocyte and embryo quality. Fertil Steril 2002;78: Sairam MR, Bhargavi GN. A role for glycosylation of the alpha subunit in transduction of biological signal in glycoprotein hormones. Science 1985;229: Bishop LA, Robertson DM, Cahir N, Schofield PR. Specific roles for the asparagine-linked carbohydrate residues of recombinant human follicle-stimulating hormone in receptor binding and signal transduction. Mol Endocrinol 1994;8: Davis D, Liu X, Segaloff DL. Identification of the sites of N-linked glycosylation on the follicle-stimulating hormone (FSH) receptor and assessment of their role in receptor function. Mol Endocrinol 1995;9: Nayudu LP, Vitt UA, Barrios de Tomasi J, Pancharatna K, Ulloa-Aguirre A. Intact follicle culture: what it can tell us about the role of FSH glycoforms during follicle development. Reprod Biomed Online 2002;5: Padmanabhan V, Sairam MR, Hassing JM, Brown MB, Ridings JW, Beitins IZ. Follicle stimulating hormone signal transduction: role of carbohydrate in aromatase induction in immature Sertoli cells. Mol Cell Endocrinol 1991;79: Wide L. Follicle-stimulating hormones in anterior pituitary gland from children and adults differ in relation to sex and age. J Endocrinol 1989;123: Wide L, Bakos O. More basic forms of both human follicle stimulating hormone and luteinizing hormone in serum at mid cycle compared with the follicular and luteal phase. J Clin Endocrinol Metab 1993;76: Phillips DJ, Wide L. Serum gondotropin isoforms become more basic after an exogenous challenge of gonadotropin-releasing hormone in children undergoing pubertal development. J Clin Endocrinol Metab 1994;79: Zambrano E, Olivares A, Mendez JP, Guerrero L, Diaz-Cueto L, Veldhuis JD, et al. Dynamics of basal and gonadotropin-releasing hormone-releasable serum follicle-stimulating hormone charge isoform distribution throughout the human menstrual cycle. J Clin Endocrinol Metab 1995;80: Ulloa-Aguirre A, Damian-Matsumura P, Jimenez M, Zambarano E, Diaz-Sanchez V. Biological characterization of the isoforms of urinary human follicle stimulating hormone contained in a purified commercial preparation. Hum Reprod 1992;7: Caccia P. Comparative glycoforms distribution analysis of different FSH-based products present on the market. Conference on gonadotropins and receptors. Theobalds Park, Hertfordshire, United Kingdom, July 6 9, 2008, A3: Veeck LL. An atlas of human gametes and conception. London: Parthenon, Veeck LL. Oocyte assessment and biological performance. Ann NY Acad Sci 1988;541: Sundstr om P, Nilsson BO. Meiotic and cytoplasmic maturation of oocytes collected in stimulated cycles is asynchronous. Hum Reprod 1988;3: Moor RM, Osborn JC, Crosby IM. Gonadotrophin-induced abnormalities in sheep oocytes after superovulation. J Reprod Fertil 1985;74: Zelsinki-Wooten MB, Hess DL, Wolf D, Stouffer R. Steroid production during ovarian stimulation impairs oocyte fertilization but not folliculogenesis in rhesus monkey. Fertil Steril 1994;61: Wu TJ, Wang L, Wan YY. Detection of estrogen receptor messenger ribonucleic acid in human oocyte and cumulus oocyte complexes using reverse transcriptase polymerase chain reaction. Fertil Steril 1993;59: Hild-Petito S, Stouffer RL, Brenner RM. Immunocytochemical localization of estrogen and progesterone receptors in the monkey ovary throughout the menstrual cycle. Endocrinology 1988;123: Tesarik J, Mendoza C. Nongenomic effects of 17b-estradiol on maturing human oocytes: relationship to oocyte developmental potential. J Clin. Endocrino Metab 1995;80: Tesarik J, Mendoza C. Direct non-genomic effects of follicular steroids on maturing human oocytes: oestrogen versus androgen antagonism. Hum Reprod Update 1997;3: Westergaard LG, LaursenSB, Andersen CY. Increased risk of early pregnancy loss by profound suppression of luteinizing hormone during ovarian stimulation in normogonadotrophic women undergoing assisted reproduction. Hum Reprod 2000;15: Fleming R, Lloyd F, Herbert M, Fenwick J, Griffiths T, Murdoch A. Effects of profound suppression of luteinizing hormone during ovarian stimulation on follicular activity, oocyte and embryo function in cycles stimulated with purified follicle stimulating hormone. Hum Reprod 1998;13: Fleming R, Rehka P, Deshpande N, Jamieson ME, Yates RWS, Lyall HL. Suppression of LH during ovarian stimulation effects differ in cycles stimulated with purified urinary FSH and recombinant FSH. Hum Reprod 2000;15: Esposito MA, Barnhart KT, Coutifaris C, Patrizio P. Role of periovulatory luteinizing hormone concentrations during assisted reproductive technology cycles stimulated exclusively with recombinant follicle stimulating hormone. Fertil Steril 2001;75: Chappel SC, Ulloa-Aguirre A, Ramaley J. Sexual maturation in female rats: time-related changes in the isoelectric focusing pattern of anterior pituitary follicle-stimulating hormone. Biol Reprod 1983;28: Blum WF, Gupta D. Heterogeneity of rat FSH by chromatofocusing: studies on serum FSH, hormone released in vitro and metabolic clearance rates of its various forms. J Endocrinol 1985;105: Wide L. The regulation of metabolic clearance rate of human FSH in mice by variation of the molecular structure of the hormone. Endocrinology 1986;112: Wide L, Hobson BM. Influence of the assay method used on the selection of the most active forms of FSH from the human pituitary. Acta Endocrinol 1986;113: Ulloa-Aguirre A, Cravioto A, Damian-Matsumura P, Jimenez M, Zambrano E, Diaz-Sanchez V. Biological characterization of the naturally occurring analogues of intrapituitary human follicle-stimulating hormone. Hum. Reprod 1992;7: Flack MR, Bennet AP, Froehlich J, Anasti JN. Increased biological activity due to basic isoforms in recombinant human follicle stimulating hormone produced in a human cell line. J Clin Endocrinol Metab 1994;79: Galway AB, Hsueh AJ, Keene JL, Yamoto M, Fauser BC, Boime I. In vitro and in vivo bioactivity of recombinant human follicle-stimulating hormone and partially deglycosylated variants secreted by transfected eukaryotic cell lines. Endocrinology 1990;127: Vitt UA, Nayudu PL, Rose UM, Kloosterboer HJ. Embryonic development after follicle culture is influenced by follicle stimulating hormone isoelectric point charge. Biol Reprod 2001;65: Vitt UA, Kloosterboer HJ, Rose UM, Mulders JWM, Kiesel PS, Bete S, et al. Isoforms of human recombinant follicle-stimulating hormone: comparison of effects on murine follicle development in vitro. Biol Reprod 1998;59: Nayudu PL, Osborne SM. Factors influencing the rate of preantral and antral growth of mouse ovarian follicles in vitro. J Reprod Fertil 1992;95: Zambrano E, Barrios-de-Tomasi J, Cardenas M, Ulloa-Aguirre A. Studies on the relative in-vitro biological potency of the naturally-occurring isoforms of intrapituitary follicle stimulating hormone. Mol Hum Reprod 1996;2: Pacchiarotti A, Aragona C, Gaglione R, Selman H. Efficacy of a combined of urinary and recombinant follicle-stimulating hormone used for ovarian stimulation of patients undergoing ICSI cycle. J Assist Reprod Genet 2007;24: Selman H. Effect of two differently glycosylated FSHs on oocyte quality and clinical outcome. Gynecol Endocrinol 2008;24(Suppl 1):72. Fertility and Sterility â 5