Small doses of LH activity are needed early in ovarian stimulation for better quality oocytes in IVF-ET

Study design: Forty-six infertile couples entering IVF-ET for the first time (either tubal or male factor) were divided after prospective randomization into two groups. In both groups the long protocol with GnRH-analogs was used. In group A, ovarian stimulation started with r-FSH (200 IU/day) for the first four days. In group B, the stimulation protocol started with one amp hMG (75 IU FSH + 75 IU LH activity) daily for four days, with simultaneous administration of r-FSH (150 IU/day). The outcome was compared. Results: Statistical difference was observed in the number of mature oocytes, the number of fertilized oocytes as well as the number of transferable embryos. In all cases, results were statistically significantly better ( p < 0.05) in group B. Clinical pregnancy rate, finally, was similar in the two groups. Conclusion: The relatively small sample size does not allow a definitive conclusion for the important role of LH during early oocyte maturation. Our results, however, indicate a beneficial effect when small doses of LH are used for ovarian stimulation in IVF-ET cycles. This effect may be more important in cases in which few embryos are available for transfer. Keywords Exogenous LH IVF-ET Oocyte quality Ovarian stimulation 1 Introduction The first recombinant DNA molecules were generated in 1972. Today, a variety of vectors are employed, the choice of which depends on the type of host cell to be used. Recently, follicle stimulating hormone (FSH) has been manufactured by means of recombinant DNA technology using a Chinese hamster ovary cell line transfected with the genes encoding human FSH [1] . The use of human menopausal gonadotrophin (HMG) in the treatment of human infertility and especially in IVF/ICSI procedures is well established. Luteinizing hormone (LH) is essential for the production of androgen precursors by theca cells. The latter are required for estradiol (E 2 ) biosynthesis. Although E 2 is not necessary for follicular development [2,3] , there are reports that it may be required for cytoplasm maturity of oocytes [4,5] . Inhibin, IGF-I, other growth factors, follistatin synergize with LH through autocrine and paracrine mechanisms in order to stimulate the theca cells to produce androgens [6,7] . In clinical practice, too much amount of LH during the time of follicular development and in the preovulatory phase may have detrimental effects on fertilization, cleavage and embryo quality [8,9] . On the other hand, it is obvious that LH is necessary for folliculogenesis, while the complete absence of LH has an adverse effect on the quality and maturity of oocytes [10,11] and particularly when GnRH-a are used for ovulation induction with several studies demonstrating the anti-gonadal effect of GnRH-a on the receptors of LH in granulosa and theca cells [12,13] . Since there is a tendency to use only recombinant FSH (r-FSH) for ovulation induction in the near future, exogenous LH administration for IVF treatment cycles may be necessary. It was suggested in a pilot study with a very small number of patients [14] that the amount of remaining endogenous LH, after GnRH-a suppression, is still sufficient to support FSH-induced oestrogen biosynthesis. Due to the lack of sufficient data in the literature and the fact that experience to ovulation induction regimens with new technology drugs, such as r-FSH, is still limited, the aim of this study was to examine whether exogenous LH administration has a beneficial effect on the quality of oocytes, fertilization potential, as well as pregnancy rate in IVF-ET cycles. A randomized trial comparing r-FSH versus r-FSH and LH was employed. 2 Materials and methods Forty-six infertile couples were divided after prospective randomization by closed envelopes into two groups (A = 22, B = 24). The project was approved by the Ethics Committee. All women were normogonadotropic. On cycle day 21, a baseline ultrasound scan was performed, followed by pituitary down regulation with GnRH-analogs (GnRH-a) (Suprefact (Hoechst, Germany) intranasal spray 100 μg five times daily). GnRH-a administration was continued until HCG administration. The extend of ovarian suppression was evaluated by ultrasound scan and serum E 2 (<40 pg/ml) before starting exogenous gonadotrophin administration. In group A, ovarian stimulation started with 200 IU/day r-FSH (Puregon, Organon, The Netherlands) for the first 4 days. In group B, the stimulation protocol started with one amp hMG (75 IU FSH + 75 IU LH activity) (Menogon, Ferring, Germany) daily for 4 days, with simultaneous administration of r-FSH (200 IU/day). The latter was continued without HMG administration. In both groups, the daily hormonal dose was individualized according to the ovarian response and GnRH-a was continued until hCG (10,000 IU, IM) was administered. Oocyte retrieval and fertilization was performed as previously described [15,16] . Embryos were scored and chosen for transfer based on more rapid cleavage, absence of fragmentation as well as size of blastomeres [17] . Results were analyzed using unpaired t -test. Clinical pregnancy rate was analyzed by the z -test for proportions. 3 Results The mean time of down regulation for group A was 18 days [13–27] and for group B 16 days [12–27] . Table 1 shows the outcome of IVF-ET in the patients of our study. Both groups consisted of women of similar age (33.0 ± 3.7 versus 32.4 ± 3.1). Serum E 2 concentration on the day of hCG administration was not significantly different (1638.1 ± 1001.1 versus 1782.0 ± 1028.4). The stimulation period was similar in both groups (9.7 ± 0.8 and 10.1 ± 1.4, respectively), as well as the number of ampules used (26.3 ± 7.9 versus 29.1 ± 6.7). The number of oocytes retrieved was also comparable (11.8 ± 4.4 versus 12.7 ± 6.4). Statistical difference was observed in the number of mature oocytes (7.3 ± 2.9 versus 10.7 ± 3.4), the number of fertilized oocytes (6.5 ± 1.8 versus 7.9 ± 3.0) as well as the number of transferable embryos (6.0 ± 2.8 versus 7.7 ± 3.1). In all cases, results were statistically significantly better ( p < 0.05) in group B. Clinical pregnancy rate, finally, was similar in the two groups (27.8% versus 20.8%). 4 Discussion The necessary and optimal dose of LH administration in IVF cycles for the achievement of good quality oocytes and embryos has not been determined yet. The two-cell theory suggests that both FSH and LH are needed for normal follicular growth and maturation, but until now the main role has been attributed to FSH. It is currently thought that only low concentrations of LH are needed in the normal ovulatory cycle [18] and human menopausal gonadotrophin (HMG) seems to offer advantages compared to purified FSH only in severe hypopituitary amenorrheic patients [10] , in whom there is almost complete absence of LH activity [11] . In our study, it was shown that LH administration in the early phase of folliculogenesis, early during the stimulation protocol, seems to have a beneficial effect on the maturity and fertilizability of oocytes, as well as on the number of transferable embryos ( Table 1 ), thus suggesting that the exogenous administration of LH is necessary for the correct development of selected follicles to preovulatory follicles. GnRH-a are world-wide employed in IVF-ET protocols as a part of controlled ovarian hyperstimulation. In these cases, the doses of HMG required are higher than in treatments that use solely HMG [19] . This lower responsiveness to HMG is probably due to a direct effect of GnRH-a on the ovary. Several studies have demonstrated the antigonadal effect of the in vivo administration of GnRH-a in rats [13,20] . Guerrero et al. have reported a decrease in the number of LH receptors of rat luteal cells cultured with GnRH-a for 48 h [13] . On the other hand, recombinant FSH preparation has become available (Puregon, Organon and Gonal-F, Serono) in order to eventually replace the existing derived compounds from the urine. Dosage regimens employed in IVF cycles, including gonadotrophin initial doses of either 150 IU or 225 IU, as well as r-FSH, have been based rather on empirical experience than on the results of randomized controlled trials. Our findings demonstrated a clinical improvement of the quality and fertilizability of oocytes when the patients are treated with r-FSH plus LH. These data seem to confirm the theory that LH (added in the first 4 days) stimulates the theca cells to produce increasing quantities of androgens, which are transformed to oestrogen by the granulosa cells, thus exhibiting increased aromatization capacity through FSH stimulation even when GnRH-a is used for ovulation induction [21] . The aim of every stimulation protocol is to yield as many mature and as few immature oocytes as possible. A major physiological function of LH throughout the menstrual cycle is the support of follicular growth induced by FSH by providing androgen substrate for the granulosa cell aromatase. For nuclear maturation, it has been formulated that gap junction-mediated transmission of follicle cell cAMP in the oocyte inhibits oocyte maturation. However, gonadotrophin stimulation ultimately terminates cumulous oocyte communication proposing the initiation of resumption of meiosis by interruption of direct transfer of cAMP to the oocytes [22,23] . The cytoplasmic maturational process is controlled by ovarian steroidogenesis [4] and steroids, such as E 2 , exert a significant role on the synthesis of cytoplasmic factors that induce decondensation of the sperm head and formation of male pronucleus [4] . Our previous study investigated the correlation of oocyte morphology with embryo quality after ICSI. We have demonstrated the importance of serum E 2 level during ovulation induction for cytoplasmic maturity and, consequently, for the quality of embryos. When E 2 was significantly lower during ovulation induction, the latter were of a worse quality [17] . Addition of HMG at the end of ovarian stimulation with r-FSH has also been shown to increase serum E 2 [24] . Although E 2 is not necessary for follicular development [2,3] , it seems that it is required for cytoplasmic maturation. Thus, LH plays a fundamental role in the process of oocyte maturation, since it is responsible for the production of androgen precursors by theca cells, which are required for E 2 biosynthesis. In addition, a recent study [25] has demonstrated that the concentration of LH plays an important role in the development of follicles. They showed that low amounts of LH (day 3 of the cycle, <3 mIU/l) reduces the activity of one or more intra-ovarian regulators and consequently it reduces significantly the number of follicles that become preovulatory. The intra-ovarian regulators are a group of substances, which have been considered important in the recent years. These factors have a regulatory effect on follicular growth and development and act through autocrine, paracrine and hormonal routes. They induce the mechanism for ovarian steroid production and stimulate ovarian proteins, growth factors, inhibin, activin, follistatin and IGF [6,7] . The secretion and regulation of these compounds is under gonadotrophic stimulation [6] . The previously mentioned observation by Noci confirms the important role of LH during ovulation induction and the beneficial effect of LH activity to the preovulatory growth of follicles. Indeed, a constant administration of LH throughout the whole ovarian stimulation has proven to have opposite effects [26] . The relatively small sample site does not allow a definitive conclusion for the important role of LH during early oocyte maturation. Our results, however, indicate the beneficial effect when small doses of LH activity are used for ovarian stimulation in IVF-ET cycles. More extensive studies on the pathophysiology of follicular growth and oocyte maturation will assist in the improvement of the quality of retrieved oocytes as well as that of embryos. References [1] P. Van Wezenbeek J. Draier F. Van Meel W. Olijve Recombinant follicle stimulating hormone. I. Construction, selection and characrization of a cell line D. Crommelin H. Schellekens Clone linic: developments in biotherapy vol. 1 1990 Kluwer Academic Publishers 251 254 [2] B. Couzinet N. Lestrat S. Brailly M. Forest G. Shaison Stimulation of ovarian follicular maturation with pure follicle stimulating hormone in women with gonadotropin deficiency J Clin Endocrinol Metab 66 1988 552 556 [3] J. Rabinovici J. Blankstein B. Goldman E. Rudakk Y. Dor C. Pariente In vitro fertilization and primary embryonic cleavage are possible in 17-hydroxylase deficiency despite extremely low intrafollicular 17 estradiol J Clin Endocrinol Metab 68 1989 693 697 [4] J.C. Osbon R.M. Moor The role of steroid signals in the maturation of mammalian oocytes J Steroid Biochem 19 1983 133 138 [5] Y. Yoshimura Y. Hosoi A. Bongivanni R. Santulli E.E. Wallach The effect of ovarian steroidogenesis on ovulation and ovum maturation in the in vitro perfused rabbit ovary Presented at 67th Ann Meeting of Endocrine Society Baltimore, June 19–21 1985 (Abstract #34) [6] D.T. Baird K.B. Smith Inhibin and related peptides in the regulation of reproduction Oxf Rev Reprod Biol 15 1992 191 232 [7] G.F. Erickson V.G. Garzo D.A. Mayoffin Insulin-like growth factor-1 regulates aromatase activity in human granulosa and granulosa luteal cells J Clin Endocrinol Metab 69 1989 716 724 [8] C. Howles M.C. Macnamee R.G. Edwards Follicular development and early luteal function of conception and non-conceptional cycles after human in vitro fertilization: endocrine correlates Hum Reprod 2 1987 17 21 [9] J. Stanger J.J. Yovich Reduced in vitro fertilization of human oocytes from patients with raised basal luteinizing hormone levels during the follicular phase Br J Obstet Gynaecol 92 1985 385 393 [10] B. Lunenfeld V. Inster Classification of anovulatory states P.G. Crosignani PS. Mishell Ovulation in the human 1995 Raven Press London 211 216 [11] W.J. Berger M.L. Taymor K. Karam F.N. Nudenberg The relative role of exogenous and endogenous follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in human follicular maturation and ovulation induction Fertil Steril 23 1972 783 790 [12] P.B.C. Jones A.J.W. Hsueh Direct effects of gonadotropin releasing hormone and its antagonist upon ovarian functions stimulated by FSH, prolactin and LH Biol Reprod 24 1981 747 759 [13] E.H. Guerrero P. Stein R.H. Asch E. Polak de Fried M. Tesone Effect of a gonadotropin-releasing hormone agonist on luteinizing hormone receptors and steroidogenesis in ovarian cells Fertil Steril 59 1993 803 809 [14] P. Devroey B. Mannaerts J. Smitz H. Coeling-Bennik Van Steirteghem Clinical outcome of a pilot efficacy study on recombinant human follicle-stimulating hormone (Org 32489) combined with various gonadotrophin-releasing hormone agonist regimens Hum Reprod 9 1994 1064 1069 [15] K. Kallianidis D. Loutradis P. Drakakis S. Milingos S. Michalas Comparison of the outcome of classical IVF and ICSI based on the quality of embryos V. Gomel P.C.K. Leung Proceedings of the 10th World Congress of IVF and Assisted Reproduction Monduzzi Editore 1997 715 719 [16] D. Loutradis A.A. Kiessling K. Kallianidis Human oocytes cultured in Ham's F-10 without Hypoxanthine J Assist Reprod Genet 4 1993 271 275 [17] D. Loutradis P. Drakakis K. Kallianidis S. Dendrinos S. Milingos S. Michalas Oocyte morphology correlates with embryo quality and pregnancy rate after intracytoplasmic sperm injection Fertil Steril 72 1999 240 244 [18] M.L. Taymor The regulation of follicle growth: Some clinical implications in reproductive endocrinology Fertil Steril 65 1996 235 241 [19] D. Lewinthal P.J. Taylor H.A. Pattison B. Coronblum Induction of ovulation with leuprolide acetate and human menopausal gonadotropin Fertil Steril 49 1995 585 588 [20] J. Testart B. Lefevre A. Bougeon Effects of gonadotrophin-releasing hormone agonist (GnRH-a) on follicle and oocyte quality Hum Reprod 8 1993 511 518 [21] A.J.W. Hsueh E.Y. Adashi P.B. Jones Welsh Hormonal regulation of the differentiation of cultured ovarian granulosa cells Endocrine Rev 5 1984 76 127 [22] N. Dekel W.H. Beers Rat oocyte maturation in vitro: relief of c-AMP inhibition with gonadotropins Proc Natl Acad Sci USA 75 1978 4369 4371 [23] N. Dekel W.H. Beers Development of the rat oocytes in vitro: inhibition and induction of maturation in the presence or absence of the cumulus oophorous Dev Biol 75 1980 247 251 [24] G. Adonakis N. Deshpande R.W. Yates R. Fleming Luteinizing hormone increases estradiol secretion but has no effect on progesterone concentrations in the late follicular phase of in vitro fertilization cycles in women treated with gonadotropin-releasing hormone agonist and follicle-stimulating hormone Fertil Steril 69 3 1998 450 453 [25] I. Noci R. Biagiotti M. Maggi F. Ricci A. Cinotti G. Scarselli Low day 3 luteinizing hormone values are predictive of reduced response to ovarian stimulation Hum Reprod 13 1998 531 534 [26] J. Balasch M. Creus F. Fabregues The effect of exogenous luteinizing hormone (LH) on oocyte viability: evidence from a comparative study using recombinant human follicle-stimulating hormone (FSH) alone or in combination with recombinant LH for ovarian stimulation in pituitary-suppressed women undergoing assisted reproduction J Assist Reprod Genet 18 5 2001 250 256