1 Center For Reproductive Medicine and Infertility, Department of Obstetrics & Gynecology, The New York Presbyterian HospitalCornell Medical Center and 2 General Clinical Research Center, Rockefeller University, New York, New York, USA
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Abstract |
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Key words: FSH/IVF/ovulation induction/recombinant human luteinizing hormone
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Introduction |
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Since clinical conditions featuring absolute loss of endogenous LH and FSH are extremely rare, the pharmacological effects of these peptides have proven difficult to study. The recent commercial availability of highly purified FSH (FSH-HP) has offered an agent of substantially improved purity upon which important clinical observations may be made. Moreover, the synthesis of LH and FSH by recombinant-DNA technology (Hård et al., 1990; Hull et al., 1994
) has facilitated a still more precise in-vivo assessment of the two-cell, two-gonadotrophin hypothesis.
Comparisons between patients receiving stimulation protocols with relatively high LH content and relatively low LH content have been reported previously (Edelstein et al., 1990; Daya et al., 1995
; Fried et al., 1996
; Mercan et al., 1997
; Sills et al., 1998
). It was the purpose of this prospective, randomized study to compare cycle characteristics in women undergoing ovulation induction for in-vitro fertilization (IVF) using highly purified urinary FSH both with and without supplemental LH derived from recombinant DNA technology (rhLH), following luteal pituitary suppression with gonadotrophin-releasing hormone agonist (GnRHa).
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Materials and methods |
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Before treatment all patients were interviewed and examined, and the infertility duration and aetiology was ascertained. Using random number tables, study patients were randomized into one of two treatment groups. Group 1 received only FSH-HP (Fertinex®, Serono Laboratories, Norwall, MA, USA), administered s.c. Group 2 received FSH-HP plus recombinant human luteinizing hormone (rhLH, Lhadi®, Serono), administered as separate s.c. injections. The FSH-HP contained essentially no LH (0.1 IU LH/1000 IU FSH).
The supplemental LH used in this study was synthesized by transfection of a Chinese hamster ovary cell line with a plasmid containing the two subunit DNA sequences encoding for human LH. Using recombinant DNA techniques, a consistent LH preparation of ultra-high purity was produced; it may be considered to contain essentially no FSH or other undesired constituent proteins (Keene et al., 1989; Hård et al., 1990
; Mannaerts et al., 1991
). Recombinant human LH administered at doses of 75 IU has been shown to be equivalent to 75 IU of LH contained in human menopausal gonadotrophin (HMG), and has been well tolerated in doses up to 40 000 IU (Le Cotonnec et al., 1998
).
Ovarian stimulation
Pituitary down-regulation was achieved by administration of leuprolide acetate (1 or 0.5 mg/day, s.c.) commencing 8 days after ovulation in the cycle preceding gonadotrophin treatment. Serum samples for oestradiol, FSH, and LH concentration determinations were obtained by venepuncture from all study patients between 0700 and 0930 on menstrual cycle day 3. The GnRHa dose was reduced by one-half on the day gonadotrophin therapy began for all patients, and maintained at that dose until the day of human chorionic gonadotrophin (HCG) administration (Damario et al., 1995).
For all study patients, controlled ovulation induction was achieved with 26 ampoules of FSH-HP, beginning on cycle day 3. For patients receiving FSH-HP with supplemental LH, one ampoule (75 IU) of rhLH was administered s.c. each day. Daily FSH-HP dose was adjusted in response to follicular growth and serum oestradiol concentrations. In general, a `step-down' decremental pattern was followed for FSH-HP, as previously described (Damario et al., 1995). In this experimental protocol, the dose of rhLH remained constant throughout stimulation regardless of the FSH-HP dose. Up to 10 000 IU HCG (Profasi; Serono, West Orange, NJ, USA) was given i.m. when at least two follicles were
17 mm diameter.
Oocyte retrieval
Transvaginal ultrasound-guided needle aspiration of oocytes was performed 3435 h after HCG administration. Propofol (Zeneca USA, Wilmington, DE, USA; 1 mg/kg) with fentanyl citrate (Astra Pharmaceuticals, Westborough, MA, USA; 100 µg) was given i.v. for analgesia in all cases. Immediately following retrieval, oocytes were washed in HEPES buffered human tubal fluid (HTF) medium prepared in our laboratory and placed into droplets of HTF + 10% maternal serum under oil (Squibb, Princeton, NJ, USA). Cells were incubated in a humidified 5% CO2 atmosphere at 37°C.
Oocytes were graded for maturity using cumulus expansion criteria (Veeck, 1988). After 5 h, processed spermatozoa (total concentration = 1.5x105/ml) were added to droplets of medium with each oocyte. In cases of male factor infertility (defined as spermatozoa concentration <20x106/ml, motility <50%, and/or `strict' normal forms morphology <15%), intracytoplasmic sperm injection (ICSI) was performed as reported previously (Palermo et al., 1996
). Fertilization was considered successful after noting the presence of two pronuclei at 1218 h. Prezygotes were then transferred to droplets of fresh media with 15% maternal serum for continued culture.
Embryo transfer
Up to four embryos were transferred at 72 h following oocyte retrieval. One patient in each group (both age 40 years) had five embryos transferred. Embryo morphology immediately prior to transfer was scored according to standard criteria (Veeck, 1991
). Embryos were suspended in 2030 µl 75% maternal serum with HTF contained within a Wallace (Marlow Surgical Technology, Willoughby, OH, USA) or TomCat (Sherwood Medical, St Louis, MO, USA) catheter, and delivered approximately 1 cm inferior to the uterine fundus. Non-transferred embryos suitable for freezing were cryopreserved if requested by the patient.
Luteal support
All patients received i.m. progesterone (Schein Pharmaceutical, Florham Park, NJ, USA; 50 or 100 mg/day progesterone in oil) following fertilization. Progesterone was maintained at this dose either until a negative pregnancy test was obtained (ßHCG <5 IU by venepuncture, mean volume 7 ml) 2 weeks post-transfer, until clinical pregnancy was documented (defined as fetal cardiac activity seen via sonogram at cycle day 49), or until early pregnancy failure or demise was diagnosed.
Measured variables and statistical analysis
The following parameters were measured in this investigation: baseline serum oestradiol, LH, and FSH concentrations on menstrual cycle day 3, length of stimulation, serum oestradiol concentration on the day of HCG administration, total ampoules of gonadotrophin required for stimulation, total number of oocytes retrieved, and proportion of normally fertilized oocytes. Selected embryo morphological features, implantation and clinical pregnancy rates were also compared for both study groups.
Statistical analysis of values measured in both treatment cycles was by Student's t-test or Fisher's exact test, as appropriate, performed by computerized data program (Statsoft, Tulsa, OK, USA). A P < 0.05 was considered significant for all comparisons. Power analysis was performed by nQuery Advisor 2.0 (Statistical Solutions, Cork, Ireland).
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Results |
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Infertility aetiologies for the FSH-HP + rhLH treatment group included male factor (n = 9, 64%), tubal disease (n = 3, 18%), and idiopathic infertility (n = 2, 18%). For the FSH-HP only group, infertility diagnoses included male factor (n = 8, 47%), and tubal disease (n = 5, 29%); endometriosis and anovulation accounted for the remaining four patients. Patient age, BMI, and duration of infertility were similar for both treatment groups (Table I). Among patients randomized to receive FSH-HP, 11/17 (65%) had undergone prior gonadotrophin stimulation. Only one patient (7%) in the FSH-HP + rhLH group had any previous gonadotrophin treatment. While serum oestradiol and FSH obtained on cycle day 3 were not significantly different in the two treatment groups, patients randomized to the FSH-HP + rhLH group had higher serum LH concentrations on cycle day 3 (P < 0.05). The number of patients cancelled for poor follicular response (defined as serum oestradiol <100 pg/ml by the 5th day of gonadotrophin stimulation) was one (5%) in the FSH-HP group, and three (21.4%) in the FSH-HP + rhLH group.
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All patients tolerated their stimulation protocol well, and there were no cancellations due to adverse medication effects. There were no ectopic pregnancies in this study population.
The rate of implantation was not significantly different in the two groups, with 26.9 and 11.9% implantation achieved in the FSH-HP and FSH-HP + rhLH groups respectively (Table III). There was a trend toward improved clinical pregnancy/initiated cycle and clinical pregnancy/transfer results for the FSH-HP only group (Table III
). However, because of limited sampling, these differences between the study groups did not reach statistical significance. One miscarriage occurred in the FSH-HP + rhLH group; no spontaneous abortions occurred in the FSH-HP group.
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Discussion |
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This pilot study showed that oocyte yield, fertilization, and certain embryo morphological features are not substantially different between patients who received FSH-HP alone and those who received supplemental LH throughout follicular recruitment. The mean implantation rate and clinical pregnancy rates were apparently higher among patients receiving FSH-HP alone when compared to the rhLH group, although these differences were not statistically significant (P = 0.41 and 0.10 respectively). Such differences in implantation and clinical pregnancy rates in these patients should be interpreted with caution, for while these data may suggest a trend toward better outcomes following FSH-HP only treatment, a two-sided analysis established the detection of a significant difference (P < 0.05) in a study with a power of 80% would require a sample of 278 patients.
LH is a heterodimeric pituitary glycoprotein, with important roles in both ovarian steroid synthesis and ovulation (Channing et al., 1966; Armstrong et al., 1970
). While normal ovulation is impossible without LH (Shoham et al., 1993
), the preparations currently used in clinical infertility treatment have a broad range of LH content. The stereochemistry of LH is complex. The molecule's tertiary structure and electrochemical charge are maintained by considerable glycosylation with sialic-acid residues, determining metabolic clearance and antigenic properties of LH isoforms. Experimental evidence has shown that complete desialation of LH and related glycoproteins results in substantial reduction of bioactivity (Ross et al., 1972
), and can transform some gonadotrophin agonists into antagonists (Dunkel et al., 1993
). In addition to these structural features of LH, polymorphisms of LH have been identified both during the female reproductive cycle (Wide and Bakos, 1993
) and throughout life (Strollo et al., 1981
). Even in selected anovulatory patients, circulating LH seems to have some different physiochemical characteristics when compared to endogenous LH assayed in fertile controls (Ding and Huhtaniemi, 1991
). While valence and antigenic characteristics of LH appear to fluctuate over time, there is little doubt concerning the finding that less than 1% of follicular LH receptors need be bound to LH in order to facilitate normal steroidogenesis (Catt and Dufau, 1977
; Doerr, 1979
).
The up-regulation of LH-receptor (Ireland and Richards, 1978) and progesterone-receptor expression (Iwai et al., 1990
; Park and Mayo, 1991
) within peri-ovulatory follicles are among the numerous molecular events triggered by LH. However, elevations of serum LH concentrations during the follicular phase have been associated with impaired reproductive outcomes by several investigators (Sagle et al., 1988
; Regan et al., 1990
; Chappel and Howles, 1991
; Zelinski-Wooten et al., 1993
). Among maturing follicles in the later follicular phase, the appearance of granulosa cell LH-receptors may facilitate a decreased reliance on FSH-dependent development and potentiate equivalent responses from either FSH or LH for continued follicular growth (Zeleznik and Hillier, 1984
).
Earlier investigations showed that when recombinant FSH was administered alone in the setting of hypogonadotrophic hypogonadism, follicular growth was appropriate, although oestradiol production was low (Schoot et al., 1992). Both androgen and oestradiol concentrations were reduced in follicular fluid following such treatment, and blastocyst implantation was unsuccessful (Kousta et al., 1996
). However, when the hypogonadotrophic hypogonadal patient received recombinant FSH supplemented with LH, follicular fluid androgen and oestradiol concentrations were much higher, and pregnancy could be established (Balasch et al., 1995
).
How oestradiol might influence or reflect human oocyte health has not been fully elucidated, although preliminary research has suggested a potential role as a growth factor. Diminished serum oestradiol concentrations most probably represent impaired ovarian steroidogenesis after GnRHa down-regulation, and assessment of oestradiol response patterns have long been considered important markers for IVF cycle success (Liu et al., 1991; Davis and Rosenwaks, 1992
; Damario et al., 1995
). Nevertheless, oocytes have been recovered and successfully fertilized in the setting of extremely low oestradiol concentrations (i.e., 17
-hydroxylase deficiency) (Rabinovici et al., 1989
). Among study patients included here, steroidogenesis was apparently unaffected since oestradiol concentrations were not significantly reduced when FSH-HP was given without supplemental LH. Standard oestradiol response curves may not be applicable when FSH-HP is used for ovulation induction, however (Miller et al., 1998
).
Pharmacologically-induced hypo-oestrogenic states provide both a view to the oestrogenic contribution to reproduction, and approximate (through pituitary down-regulation) a hypogonadotrophic setting permitting assessment of treatment outcome following therapy with known gonadotrophins. Experimental results using GnRH antagonists, first in primates (Karnitis et al., 1994; Zelinski-Wooten et al., 1997
) and later in humans (Mannaerts, 1997
), further refined specific roles for FSH and LH by creating transient but profound arrest of endogenous gonadotrophin release.
The fact that pre-treatment serum LH concentrations differed in the two groups is notable, since those patients subsequently receiving supplemental rhLH already demonstrated a higher resting serum LH tone. It may be that subtle differences in reproductive outcome evidenced here are related to significant differences in baseline LH concentrations which could not be anticipated by randomization.
In summary, data from this pilot study support the concept that LH is needed only in trace amounts for follicular recruitment and development (Daya et al., 1995). The results of this study show that when a highly purified urinary FSH preparation is used after pituitary down-regulation with luteal GnRHa, the presence of extra (recombinant) LH throughout ovulation induction does not materially alter cycle performance. While such rhLH co-therapy appears to confer no benefit for implantation and clinical pregnancy rates, additional data are required to quantify the significance of this preliminary finding. Should clinical use of GnRH-antagonists (and rhFSH) become more prevalent, the resulting near complete loss of LH in the reproductive hormonal milieu may offer a compelling role for rhLH in selected patients. Other potentially important roles for rhLH might include its use as a substitute for HCG in oocyte maturation and/or ovulation. These applications represent topics of current research.
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Acknowledgments |
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Notes |
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References |
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Submitted on February 8, 1999; accepted on May 24, 1999.